Tethered dinuclear europium(III) macrocyclic catalysts for the cleavage of RNA

Article abstract of DOI:10.1021/ja8037799

Dinuclear europium(III) complexes of the macrocycles 1,3-bis[1-(4,7,10- tris(carbamoylmethyl)-1,4,7,10-tetraazacyclododecane]-m-xylene (1), 1,4-bis[1-(4,7,10-tris(carbamoylmethyl)-1,4,7,10-tetraazacyclododecane] -p-xylene (2), and mononuclear europium(III) complexes of macrocycles 1-methyl-,4,7,10-tris(carbamoylmethyl)-1,4,7,10-tetraazacyclododecane (3), 1-[3′-(N,N-diethylaminomethyl)benzyl]-4,7,10-tris(carbamoylmethyl)-1,4,7, 10-tetraazacyclododecane (4), and 1,4,7-tris(carbamoylmethyl)-1,4,7,10- tetraazacyclododecane (5) were prepared. Studies using direct excitation ( ⁷F₀ → ⁵D₀) europium(III) luminescence spectroscopy show that each Eu(III) center in the mononuclear and dinuclear complexes has two water ligands at pH 7.0, I = 0.10 M (NaNO ₃) and that there are no water ligand ionizations over the pH range of 7-9. All complexes promote cleavage of the RNA analogue 2-hydroxypropyl-4- nitrophenyl phosphate (HpPNP) at 25°C (I = 0.10 M (NaNO₃), 20 mM buffer). Second-order rate constants for the cleavage of HpPNP by the catalysts increase linearly with pH in the pH range of 7-9. The second-order rate constant for HpPNP cleavage by the dinuclear Eu(III) complex (Eu₂(1)) at pH 7 is 200 and 23-fold higher than that of Eu(5) and Eu(3), respectively, but only 7-fold higher than the mononuclear complex with an aryl pendent group, Eu(4). This shows that the macrocycle substituent modulates the efficiency of the Eu(III) catalysts. Eu₂(1) promotes cleavage of a dinucleoside, uridylyl-3′,5′-uridine (UpU) with a second-order rate constant at pH 7.6 (0.021 M^-1 s^-1) that is 46-fold higher than that of the mononuclear Eu(5) complex. Methyl phosphate binding to the Eu(III) complexes is energetically most favorable for the best catalysts, and this supports an important role for the catalyst in stabilization of the developing negative charge on the phosphorane transition state. Despite the formation of a bridging phosphate ester between the two Eu(III) centers in Eu₂(1) as shown by luminescence spectroscopy, the two metal ion centers are only weakly cooperative in cleavage of RNA and RNA analogues.

Full text of DOI:10.1021/ja8037799

Published on Web 10/10/2008
Tethered Dinuclear Europium(III) Macrocyclic Catalysts for the
Cleavage of RNA
Kido Nwe, Christopher M. Andolina, and Janet R. Morrow*
Department of Chemistry, UniVersity at Buffalo, State UniVersity of New York,
Amherst, New York 14260-3000
Received May 20, 2008; E-mail: jmorrow@buffalo.edu
Abstract: Dinuclear europium(III) complexes of the macrocycles 1,3-bis[1-(4,7,10-tris(carbamoylmethyl)-1,4,7,10-
tetraazacyclododecane]-m-xylene (1), 1,4-bis[1-(4,7,10-tris(carbamoylmethyl)-1,4,7,10-tetraazacyclododecane]-p-
xylene (2), and mononuclear europium(III) complexes of macrocycles 1-methyl-,4,7,10-tris(carbamoylmethyl)-1,4,7,10-
tetraazacyclododecane (3), 1-[3′-(N,N-diethylaminomethyl)benzyl]-4,7,10-tris(carbamoylmethyl)-1,4,7,10-tetraazacy-
clododecane (4), and 1,4,7-tris(carbamoylmethyl)-1,4,7,10-tetraazacyclododecane (5) were prepared. Studies using
direct excitation (7F0 f 5D0) europium(III) luminescence spectroscopy show that each Eu(III) center in the mononuclear
and dinuclear complexes has two water ligands at pH 7.0, I ) 0.10 M (NaNO3) and that there are no water ligand
ionizations over the pH range of 7-9. All complexes promote cleavage of the RNA analogue 2-hydroxypropyl-4-
nitrophenyl phosphate (HpPNP) at 25 °C (I ) 0.10 M (NaNO3), 20 mM buffer). Second-order rate constants for the
cleavage of HpPNP by the catalysts increase linearly with pH in the pH range of 7-9. The second-order rate constant
for HpPNP cleavage by the dinuclear Eu(III) complex (Eu2(1)) at pH 7 is 200 and 23-fold higher than that of Eu(5)
and Eu(3), respectively, but only 7-fold higher than the mononuclear complex with an aryl pendent group, Eu(4).
This shows that the macrocycle substituent modulates the efficiency of the Eu(III) catalysts. Eu2(1) promotes cleavage
of a dinucleoside, uridylyl-3′,5′-uridine (UpU) with a second-order rate constant at pH 7.6 (0.021 M-1 s-1) that is
46-fold higher than that of the mononuclear Eu(5) complex. Methyl phosphate binding to the Eu(III) complexes is
energetically most favorable for the best catalysts, and this supports an important role for the catalyst in stabilization
of the developing negative charge on the phosphorane transition state. Despite the formation of a bridging phosphate
ester between the two Eu(III) centers in Eu2(1) as shown by luminescence spectroscopy, the two metal ion centers
are only weakly cooperative in cleavage of RNA and RNA analogues.
Introduction
lanthanide ions in catalysis, however, have been hampered by
the complexity of lanthanide ion solution chemistry.1,10,13 We
It has been long suspected that the unique coordination
properties of lanthanide ions including their high charge, large
ionic radii, variable coordination geometry, and oxophilicity
give rise to their phenomenal efficiency as catalysts for the
cleavage of RNA.1-12 Efforts to unravel the mechanism of
have reported the use of direct excitation lanthanide lumines-
cence spectroscopy to characterize the solution speciation of
Eu(III) catalysts3,14-16 and to define the differences in coordina-
tion properties between lanthanide ion catalysts and the better
understood Zn(II) catalysts.17-21 Eu(III) excitation luminescence
spectroscopy is a powerful technique for the identification of
aqua and hydroxide Eu(III) complexes,14,22 their dimerization,23-25
and their binding constants to ligands and substrates.26,27
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N. H. Angew. Chem., Int. Ed. 2006, 45, 7056–7059.
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Soc. 2005, 127, 1064–1065.
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Soc. 1994, 116, 7439–7440.
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Int. Ed. Engl. 1994, 33, 773–775.
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1443–1451.
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9
10.1021/ja8037799 CCC: $40.75
2008 American Chemical Society
J. AM. CHEM. SOC. 2008, 130, 14861–14871 14861

A R T I C L E S
Nwe et al.
Syntheses. 1,3-Bis[1-(4,7,10-tris(carbamoylmethyl)-1,4,7,10-
tetraazacyclododecane]-m-xylene (1). To 0.500 g (1.25 mmol)
of compound 6 (1,3-bis-(1,4,7,10-tetraazacyclododecane))30 was
added 1.04 g (7.54 mmol) of K2CO3 and 1.04 g (7.60 mmol) of
bromoacetamide in 30 mL of absolute ethanol. The solution was
stirred at 80 °C overnight under nitrogen. Solid K2CO3 was filtered
off, and the solvent was removed under vacuum. The residue was
dissolved in a minimum amount of methanol, and chloroform was
added. The supernatant was filtered off,and the resulting white
precipitate was dried under vacuum. Yield: 90%. ESI m/z: 789 (M
+ 1), 827 (M + K), 811 (M + Na). 1H NMR (500 MHz, D2O): δ
) 2.55-3.25 (44 H, m, ring CH2, NCH2CONH2), 4.2 (4H, s,
ArCH2N), 7.54 (2H, m, Ph), 7.45 (2H, m, Ph). 13C NMR (300 MHz,
D2O): δ ) 49.50, 50.85, 51.21, 51.76, 55.78, 56.3 (ring CH2 and
NCH2CONH2), 68.9 (ArCH2N), 130.8, 132.1, 134.0 (Ph), 175.2,
176.4 (CdO).
1,4-Bis[1-(4,7,10-tris(carbamoylmethyl)-1,4,7,10-tetraazacy-
clododecane]-p-xylene (2). A procedure similar to that described
for 1 was used to produce a white solid in 90% yield from
compound 7 (1,4-bis-(1,4,7,10-tetraazacyclododecane)).30 ESI m/z:
789 (M + 1), 827 (M + K). 1H NMR (500 MHz, D2O): δ )
2.45-3.15 (40 H, m, ring CH2, NCH2CONH2), 3.22 (s, 4H,
NCH2CONH2), 3.85 (s, 4H, NCH2Ar), 7.30 (s, 4H, Ph). 13C NMR
(300 MHz, D2O): δ ) 50.5, 51.3, 51.5, 52.3, 56.3, 57.0, 58.3 (ring
CH2 and NCH2CONH2 and ArCH2N), 130.8, 163.8 (Ph), 176.2,
177.2 (CdO).
1-[3′-(N,N-Diethylaminomethyl)benzyl]-4,7,10-tris(ter-butoxy-
carbonyl)-1,4,7,10-tetraazacyclododecane (9). An acetonitrile
solution containing 8 (1-[3′-(bromomethyl)benzyl]-4,7,10-tris(ter-
butoxycarbonyl)-1,4,7,10-tetraazacyclododecane)31 (1.00 g, 1.53
mmol) and diethylamine (200 µL, 2.08 mmol) was stirred at room
temperature for 6 h. The solvent was removed under vacuum. The
resulting crude product was washed with hexanes and dried twice
to obtain a pale yellow solid which was used without further
purification. Yield 90%. ESI m/z: 648 (M + 1). 1H NMR (500
MHz, CDCl3): δ ) 1.20 (6H, t, CH2CH3), 1.42 (18 H, m, CH3),
1.48 (9H, m, CH3), 2.65 (8H, m, ring CH2, CH2CH3), 3.2-3.81
(28H, m, ring CH2, ArCH2N, ArCH2N(CH2CH3)2), 7.22-7.4 (4H,
m, Ph). 13C NMR (300 MHz, CDCl3): δ ) 11.0 (CH2CH3) 29.0,
29.4 (CH3; t-but), 47.2 (CH2CH3), 48.4, 50.1, 55.1, 56.2, 57.5, 65.7,
72.8 (ring CH2 and ArCH2N(CH2CH3)2 and ArCH2N), 80.1
(C(CH3)3), 128.1, 129.3, 129.8, 130.4, 132.0, 137.5 (Ph), 156.0,
156.4, 156.8 (CdO).
Even more challenging than the study of mononuclear Eu(III)
complexes is the design and characterization of effective
dinuclear lanthanide ion catalysts. Our past work with self-
assembled dinuclear lanthanide catalysts containing a bridging
hydroxide ligand showed that there was little cooperativity
between metal ion centers for the cleavage of phosphate
diesters.28 Other reported self-assembled multinuclear lanthanide
complexes contain weakly bound ligands and bridging hydrox-
ides that lead to cooperative behavior of the two metal ion
centers toward phosphate diester cleavage.1,5 The fragile nature
of these highly reactive complexes makes it difficult to define
the solution speciation of these systems and to probe the
mechanism of catalytic cleavage.
Here we present studies of two dinuclear Eu(III) macrocyclic
complexes that have two coordination sites on each Eu(III) ion
center for catalysis. Cleavage of an RNA model, 2-hydroxypro-
pyl-4-nitrophenyl phosphate (HpPNP), and a dinucleoside,
uridylyl-3′,5′-uridine (UpU), by these dinuclear catalysts is
compared to their mononuclear analogues to gauge the extent
of lanthanide ion cooperativity in the dinuclear catalysts. These
comparisons are further illuminated by characterization of
solution speciation and Eu(III) complex binding to carbonate
and to phosphate esters that are substrate and transition state
analogues. Despite the lack of Ln(III) ion cooperativity in
catalysis, the tethered dinuclear lanthanide ion complexes
presented here are among the most active metal ion macrocyclic
catalysts for dinucleoside and RNA analogue cleavage in water
at 25 °C that have been reported to date. Direct excitation
luminescence spectroscopy provides a unique opportunity to
fully characterize the coordination properties of these active
catalysts including the surprising absence of hydroxide ligands
and the formation of bridging phosphate ester complexes.
Experimental Section
Cyclen (1,4,7,10-tetraazacyclododecane) was purchased from
Strem chemicals. Acetonitrile and methanol were dried over calcium
hydride. HpPNP,29 1,4,7-tris(carbamoylmethyl)-1,4,7,10-tetraza-
cyclododecane (5), and Eu(5) were prepared as reported earlier.16
1-[3′-(N,N-Diethylaminomethyl)benzyl]-1,4,7,10-tetraazacy-
clododecane (10). Compound 9 (1.00 g, 1.55 mmol) was dissolved
in 20 mL of ethanol, and 7 mL of concentrated hydrochloric acid
was slowly added. The solution was stirred at room temperature
for 8 h during which time a precipitate formed. The precipitate
was filtered, washed with ethanol, and dried. The dried hydrochlo-
ride salt was then dissolved in a minimum amount of water, and
the pH was adjusted to 12.5. The solution was extracted with
dichloromethane, and the organic layer was dried over anhydrous
sodium sulfate. The solvent was removed to yield a pale yellow
+
Uridylyl(3′,5′)uridine (UpU, NH4 salt), uridine (2′,3′)-cyclic
monophosphate (2′,3′-cUMP), uridine 3′-monophosphate (3′-UMP),
uridine 2′-monophosphate (2′-UMP), and uridine were reagent grade
from Sigma-Aldrich. Milli-Q purified water was boiled and bubbled
with nitrogen gas for 1 h prior to use in kinetic experiments or in
luminescence measurements to minimize carbonate concentrations.
An Orion research digital ionalyzer, model 510, equipped with a
temperature compensation probe was used for all pH measurements.
All 1H and 13C NMR were recorded on either a Gemini-300, Inova-
400, or Inova-500 spectrometer.
1
solid. (yield, 85%). ESI m/z: 348 (M + 1). H NMR (500 MHz,
D2O): δ ) 1.2 (6H, t, CH2CH3), 2.8-3.7 (24H, m, ring CH2,
CH2CH3), 3.9 (2H, s, ArCH2N), 4.2 (2H, s, ArCH2N(CH2CH3)2),
7.2-7.6 (4H, m, Ph). 13C NMR (300 MHz, D2O): δ ) 8.10
(NCH2CH3), 41.9 (NCH2CH3), 42.2, 43.8, 44.3, 46.9 (ring CH2),
55.7 56.5 (ArCH2N and ArCH2N(CH2CH3)2), 129.8, 130.1, 130.9,
131.7, 132.7, 135.3 (Ph).
(23) Brittain, H. G.; Mantha, S.; Tweedle, M. F. J. Less-Common Met.
1986, 126, 339–342.
(24) Hernandez, G.; Brittain, H. G.; Tweedle, M. F.; Bryant, R. G. Inorg.
Chem. 1990, 29, 985–988.
1-[3′-(N,N-Diethylaminomethyl)benzyl]-4,7,10-tris(carbam-
oylmethyl)-1,4,7,10-tetraazacyclododecane (4). Bromoacetamide
(0.60 g, 4.32 mmol), compound 10 (0.50 g, 1.44 mmol) and K2CO3
(25) Zhang, X.; Chang, C. A.; Brittain, H. G.; Garrison, J. M.; Telser, J.;
Tweedle, M. F. Inorg. Chem. 1992, 31, 5597–5600.
(26) Epstein, D. M.; Chappell, L. L.; Khalili, H.; Supkowski, R. M.;
Horrocks, W. D., Jr.; Morrow, J. R. Inorg. Chem. 2000, 39, 2130–
2134.
(27) Supkowski, R. M.; Horrocks, W. D. Inorg. Chem. 1999, 38, 5616–
5619.
(30) Le Baccon, M.; Chuburu, F.; Toupet, L.; Handel, H.; Soibinet, M.;
Dechamps-Olivier, I.; Barbier, J.-P.; Aplincourt, M. New J. Chem.
2001, 25, 1168–1174.
(31) Xia, C.-Q.; Jiang, N.; Zhang, J.; Chen, S.-Y.; Lin, H.-H.; Tan, X.-Y.;
Yue, Y.; Yu, X.-Q. Bioorg. Med. Chem. 2006, 14, 5756–5764.
(28) Farquhar, E. R.; Richard, J. P.; Morrow, J. R. Inorg. Chem. 2007, 46,
7169–7177.
(29) Brown, D. M.; Usher, D. A. J. Chem. Soc. 1965, 6558–6564.
9
14862 J. AM. CHEM. SOC. VOL. 130, NO. 44, 2008

Dinuclear Europium(III) Macrocyclic Catalysts
A R T I C L E S
(0.60 g, 4.38 mmol) were added to absolute ethanol and the solution
was heated to reflux for 4 h. The solution was filtered to remove
inorganic salts, the supernatant was dried and the solvent was
removed under vacuum. The resulting solid residue was washed
with ethyl acetate and dried under high vacuum to obtain 4 as white
diethyl phosphate (DEP), 20 mM buffer, and 0.10 M NaNO3. The
data were fit to eq 1.32 Here kobsd and k0 are observed rate constants
of cleavage in the presence and absence of phosphate ester,
respectively, [A]t is the total concentration of phosphate ester, [Eu]t
is total concentration of the catalyst, and Ki is the inhibition constant.
1
solid (yield, 80%). ESI m/z: 541 (M + Na). H NMR (500 MHz,
kobsd
CD3OD): δ ) 1.1 (6H, t, CH2CH3), 2.2-2.8 (16H, m, ring CH2,
N(CH2CH3)2), 3.15 (4H, m, ring CH2), 3.41 (2H, s, ArCH2N), 3.46
(2H, s, ArCH2N(CH2CH3)2), 3.98, 4.08 (6H, s, NCH2CONH2),
7.22-7.4 (4H, m, Ph). 13C NMR (300 MHz, CD3OD): δ ) 11.8
(CH2CH3), 47.5 (CH2CH3), 51.5, 52.5, 56.8, 58.5 (ring CH2), 62.4,
68.6, 70.4 (ArCH2N(CH2CH3)2 and ArCH2N and NCH2CONH2),
129.6, 130.2, 130.6, 133.1, 137.6, 139.0 (Ph), 178.2 (CdO).
)
k0
[Eu] - [A] - K + [Eu] 2 + [A]t2 + Ki2 - 2[Eu]t[A]t + 2[Eu]tKi + 2Ki[A]t
√
t
t
i
t
(
)
2[Eu]t
(1)
Cleavage of UpU. The cleavage of UpU was monitored by
separating the reactant and the products over a C18 Varian
microsorb-mv column (250 mm × 4.6 mm; 5 µm) using a Beckman
System Gold high-performance liquid chromatograph (HPLC)
equipped with 168 UV-vis detector with peak detection at 260
nm. The elution was isocratic with a pH 4.3 buffer (20 mM
ammonium acetate) with the flow rate of 1 mL/min from t ) 0
min to t ) 7 min. Methanol was increased from 0 to 20% from t
) 7 min to t ) 16 min. 4-Nitrobenzenesulfonic acid (NBS) was
used as an internal standard. The reactant UpU and products 2′:
3′-cUMP, 3′-UMP, 2′-UMP, and uridine were identified by
comparison of retention time with those of authentic materials. The
retention times for the reactant, products, and internal standard under
these conditions are as follows: 2′:3′-cUMP, 5.4 min; 3′-UMP 6.9
min; 2′-UMP 7.9 min; uridine 13.3 min; UpU 17.9 min; NBS 16.0
min. In a typical experiment, solutions contained Eu2(1) (0.5-2.0
mM), NBS (0.20 mM), Hepes (20 mM), and NaNO3 (0.10 M).
The pH of the solution was adjusted to 7.6, and the solution was
incubated at 25 °C. Experiments were initiated by addition of UpU
(0.040 mM), and the sample was kept at 25 °C throughout the
experiment. The percent conversion of reactant (f) was calculated
as the ratio of the substrate concentrations at time ) t and time )
0 (eq 2).
1-Methyl-,4,7,10-tris(carbamoylmethyl)-1,4,7,10-tetraazacy-
clododecane (3). Compound 516 (0.140 g, 0.410 mmol), K2CO3
(0.410 g, 2.97 mmol), and 31 µL of CH3I solution (99.8%) were
added to absolute ethanol and heated to 75 °C overnight. The solid
K2CO3 base was filtered, and the solvent was removed. The
resulting solid residue was washed with ethyl acetate and dried to
obtain a white solid (yield 90%). ESI m/z: 358 (M + 1). 1H NMR
(400 MHz, D2O): δ ) 2.58 (3H, s, CH3), 2.50-2.81 (16H, m, ring
CH2), 3.02, 3.12 (2H, s, NCH2CONH2). 13C NMR (300 MHz, D2O):
δ ) 45.7(CH3), 49.7, 52.0, 53.3, 59.5 56.4, 57.2 (ring CH2 and
NCH2CONH2), 176.9, 177.4 (CdO).
Eu(III) Complexes. Eu(III) complexes were prepared by re-
fluxing Eu(CF3SO3)3 and the macrocyclic ligands in dry methanol
for 8 h. In a typical preparation, 0.200 g (0.254 mmol) of ligand 1
or 2 and 0.312 g (0.521 mmol) of Eu(CF3SO3)3 were refluxed in
20 mL of dry methanol for 8 h. The same procedure was followed
for the monomers except only 1.05 equiv of metal salt was utilized.
For the complexes with ligand 1, 2, and 4, the solvent was reduced
to 1 mL and chloroform was slowly added until a precipitate formed
at the bottom of the flask. The supernatant was decanted, and the
solid was dried under vacuum to give a white solid. For the Eu(III)
complex of ligand 3, the solvent was reduced to 1 mL and
chloroform was added until an oil formed. The oil was separated
and dried under vacuum to give a solid. Eu2(1): Yield 90%. Anal.
Calcd for C42H64N14O24F18S6Eu2 ·(CHCl3): C 24.48, H 3.08, N 9.30.
Found C 24.33, H 2.96, N 9.15. Eu2(2): Yield 90%. Anal. Calcd
for C42H64N14O24F18S6Eu2 ·(3CHCl3): C 23.74, H 2.99, N 8.81.
Found C 23.72, H 2.80, N 8.65. Eu(3): Yield 90%. ESI m/e: 806,
808 ([Eu(3)](CF3SO3)3 - CF3SO3-). Anal. Calcd for C18H31-
N7O12F9S3Eu1 ·(3CHCl3)(H2O): C 19.80, H 2.91, N 8.08. Found C
19.87, H 2.91, N 7.96. Eu(4): Yield 90%. ESI m/z: 967, 969
[S]t
ln(f) ) ln
) -kobsd
t
(2)
(
)
[S]t)0
For the monomeric catalyst, the concentration of catalyst ranged
from 1.0 to 4.0 mM and the UpU concentration was 0.080 mM.
At t ) 0 and at appropriate time intervals, 35 µL aliquots were
withdrawn, mixed with the same volume of quenching solution,
and stored in a freezer for HPLC analysis. The percent conversion
of product (f) was calculated as the ratio of the uridine concentration
at time ) t and the concentration of reactant at time ) 0 (eq 3).
([Eu(4)](CF3SO3)3
N8O12F9S3Eu1 ·(2CHCl3)(2H2O): C 26.74, H 3.76, N 8.05. Found
C 26.85, H 3.86, N 7.88.
-
CF3SO3-). Anal. Calcd for C29H46-
[uridine]
[UpU]0
) kobsd
t
(3)
Kinetic Measurements. A Beckman Coulter DU 640 UV-vis
spectrophotometer with thermostatted cell compartment was utilized
for kinetic measurements. Cleavage of HpPNP at 25.0 °C was
followed by monitoring the increase in absorbance at 400 nm due
to the formation of 4-nitrophenoxide anion. The following con-
centrations were used for the catalysts: Eu2(1), Eu2(2), and Eu(3)
(0.20-1.0 mM), Eu(4) (0.10-0.50 mM), Eu(5) (0.20-2.0 mM).
The pseudo-first-order rate constants kobsd (s-1) for these reactions
were determined as the slopes of semilogarithmic plots of reaction
progress (A∞ - At) against time, where At is the observed absorbance
at 400 nm and A∞ is the final absorbance at the end of the reaction.
For all reactions, the values of kobsd were found to be reproducible
to (5%. Solutions contained 0.0200 M of the following buffers:
MES (6.2-7.2), Hepes (6.8-7.7), EPPS (8.0-8.8), and CHES
(8.8-10) with 0.10 M (NaNO3) to maintain ionic strength. Cleavage
of HpPNP (1.0 mM) in the presence of the Eu(III) catalysts at pH
8.0 gave rise to a single peak in the 31P NMR spectrum at 18.6
ppm for the cyclic phosphate ester.
Luminescence Spectroscopy. Eu(III) excitation spectra and
excited-state lifetimes were obtained using a Spectra-Physics Quanta
Ray PRO-270-10 Q-switched Nd:YAG pump laser (10 Hz, 60-70
mJ/pulse) and a MOPO SL for all luminescence measurements.
7
5
Briefly, the F0 f D0 transition of the Eu(III) ion was scanned
between 578 and 582 nm while the 5D0 f 7F2 emission band was
monitored by using a band pass filter (628 ( 27 nm). Excitation
spectra were fit by using the program Peak Fit 4.12 (Jandel). Time-
resolved luminescence measurements were collected by using a
digital Tektronix TDS 3034B oscilloscope. Data were fit to a single
exponential decay by using Sigmaplot version 9.01 (Systat).
Solutions contained 0.10 M NaNO3 and 20 mM buffer with Eu(III)
complex concentrations varying from 0.10 to 1.0 mM.
The binding constants for Eu2(1) and Eu(4) to MP, DEP, UpU,
or NBS were determined by following the decrease in luminescence
intensity, and the data were fit to eq 4.27 Here C is the Eu(III)
macrocyclic complex, CA is Eu(III) macrocyclic anion complex,
For inhibition studies, the same procedure was followed to
prepare the samples and the concentration of the inhibitor was varied
accordingly. The solution contained 0.10-0.50 mM Eu2(1) or 0.10
mM Eu(3), 0-5.0 mM methyl phosphate (MP) or 0-20 mM
(32) Yang, M.-Y.; Morrow, J. R.; Richard, J. P. Bioorg. Chem. 2007, 35,
366–374.
9
J. AM. CHEM. SOC. VOL. 130, NO. 44, 2008 14863

A R T I C L E S
Nwe et al.
Scheme 1
Scheme 2a
Solution Chemistry of Eu(III) Complexes. Direct excitation
Eu(III) luminescence spectroscopy22,35-37 was used to charac-
terize the solution speciation of the Eu(III) complexes over the
pH range of 7-9 and to monitor binding of phosphate esters.
7
5
In these studies, the F0 f D0 transition was excited and the
intense luminescence emission band (5D0 f 7F2) was monitored.
7
5
The F0 f D0 transition occurs between two electronic states
that are nondegenerate and thus not split by ligand fields,
leading to a single excitation peak for each Eu(III) complex
species. The Eu(III) complexes were further characterized by
time-resolved luminescence experiments. Luminescence life-
times of Eu(III) complexes are strongly affected by nonradiative
processes such as vibronic coupling to ligand groups, especially
the OH groups of water or alcohol ligands. The number of bound
waters (q) are estimated from measurements of the luminescence
lifetimes of the complex in H2O and D2O.35,38 This data is used
in eq 5 to calculate q where kH2O and kD2O are the rate constants
for luminescence decay in H2O and D2O, respectively.35
Additional contributions to quenching arise from ligand NH
groups and from outer-sphere water molecules. These terms are
a Boc ) t-butoxycarbonyl.
A is the anionic ligand concentration, X is mole fraction, and I is
luminescence intensity at a chosen wavelength. The solutions
contained 0.10 mM Eu(4) or 0.033-0.50 mM Eu2(1) complex, 20
mM buffer, 0.10 M NaNO3, and 0-2.0 mM MP, 0-20 mM DEP,
0-4.0 mM UpU, or 3.0 mM NBS.
grouped into kXH
.
q ) A[kH2O - kD2O - kXH
]
(5)
(6)
kXH ) R + δnOdCNH
F ) [C]tot + [A]tot + Kd
Quenching constants vary slightly for different classes of
Eu(III) complexes, so we chose values (R ) 0.25 ms-1 for outer-
sphere contribution, δ ) 0.075 ms-1 for amide NH, A ) 1.2
ms) that were reported for Eu(III) complexes with similar
structures.35,38,39 In particular, the quenching value for N-H
in the macrocyclic ring (i.e., Eu(5)) has not been extensively
studied, and we use a value determined recently for a similar
class of complexes.16 Outer-sphere quenching contributions (R)
vary from 0.15 to 0.31 ms-1 depending on the overall charge
on the Eu(III) complexes and the hydrophilic character of the
pendent groups.39 Outer-sphere quenching contributions from
weakly coordinating anions present at high concentrations may
have a small contribution as well.40-42
F - F2 - 4[C] [A]
√
tot
tot
[CA] )
2
[CA]
[C]tot
XCA
)
XC ) 1 - XCA
Iobsd ) XCIC + XCA CA
I
(4)
Results
Synthesis of Ligands and Complexes. Dinuclear ligands 1 and
2 were prepared by alkylation of compounds containing two
cyclen macrocycles connected through xylyl linkers (cyclen )
1,4,7,10-tetraazacyclododecane, Scheme 1, compound 6).30 This
method proved to be superior to alternative synthetic routes33,34
that link macrocycles already containing three pendent amide
groups. Two new mononuclear ligands were also prepared.
Macrocycle 3 was prepared by methylation of ligand 5 with
methyl iodide. Macrocycle 4 was prepared from 831 by treatment
with diethylamine (Scheme 2). Subsequently, the amine groups
of 9 were deprotected and pendent amide groups were added
to give macrocycle 4. All four macrocyclic ligands were treated
with Eu(CF3SO3)3 in methanol to give the Eu(III) complexes
in good yield.
Luminescence spectroscopy studies of the four Eu(III)
complexes of 1-4 show evidence of the stereoisomerism that
(35) Supkowski, R. M.; Horrocks, W. D. Inorg. Chim. Acta 2002, 340,
44–48.
(36) Latva, M.; Takalo, H.; Mukkala, V.-M.; Kankare, J. Inorg. Chim. Acta
1998, 267, 63–72.
(37) Choppin, G. R.; Wang, Z. M. Inorg. Chem. 1997, 36, 249–252.
(38) Parker, D.; Dickins, R. S.; Puschmann, H.; Crossland, C.; Howard,
J. A. K. Chem. ReV. 2002, 102, 1977–2010.
(39) Beeby, A.; Clarkson, I. M.; Dickins, R. S.; Faulkner, S.; Parker, D.;
Royle, L.; de Sousa, A. S.; Williams, J. A. G.; Woods, M. J. Chem.
Soc., Perkin Trans. 2 1999, 493–503.
(40) Billard, I.; Rustenholtz, A.; Semon, L.; Lutzenkirchen, K. Chem. Phys.
2001, 270, 345–354.
(33) Harte, A. J.; Jensen, P.; Plush, S. E.; Kruger, P. E.; Gunnlaugsson, T.
Inorg. Chem. 2006, 45, 9465–9474.
(41) Nehlig, A.; Elhabiri, M.; Billard, I.; Albrecht-Gary, A.-M.; Lutzen-
kirchen, K. Radiochim. Acta 2003, 91, 37–43.
(34) Pope, S. J. A.; Kenwright, A. M.; Boote, V. A.; Faulkner, S. Dalton
Trans. 2003, 3780–3784.
(42) Rustenholtz, A.; Billard, I.; Duplatre, G.; Lutzenkirchen, K.; Semon,
L. Radiochim. Acta 2001, 89, 83–89.
9
14864 J. AM. CHEM. SOC. VOL. 130, NO. 44, 2008

Dinuclear Europium(III) Macrocyclic Catalysts
A R T I C L E S
5
7
Figure 1. 7F0 f D0 excitation spectra (5D0 f F2 emission) of solutions of (a) Eu2(1), (b) Eu2(2), (c) Eu(3), and (d) Eu(4), 1.0 mM complex at pH 7.0,
buffer ) 20 mM, I ) 0.10 M NaNO3.
Table 1. Luminescence Lifetime Data and Number of Bound
is characteristic of cyclen-based macrocycles. The stereoisom-
erism displayed by monomeric Ln(III) complexes of cyclen
Waters (q) for Eu(III) Complexes
complexa
λ (nm)
τH2O (ms)
kH2O (ms-1
)
τD2O (ms)
kD2O (ms-1
)
q
derivatives with pendent groups involves two elements of
chirality. One arises from the pendent group orientation
(clockwise or counterclockwise) and the other from the two
conformations of the tetraazamacrocyclic ring.38,43 This gives
rise to two different diastereomers including one with twisted
square antiprismatic (TSAP) geometry and the other with square
antiprismatic (SAP) geometry.44,45 In solution, it is common to
observe both isomers although generally one predominates in
solution. Previous work in our laboratory showed that the two
diastereomers of the monomeric diaqua complex Eu(5)(H2O)2
each gave rise to a separate 7F0 f 5D0 excitation peak14,16 and
that one of the diastereomeric forms predominated in solution.44
The excitation luminescence spectra of Eu2(1), Eu2(2), Eu(3),
and Eu(4) contain peaks that we assign to the two diastereomeric
forms of their aqua complexes in solution, analogous to
Eu(5)(H2O)2. These Eu(III) complexes of 1-4 also contain one
to two red-shifted peaks in their excitation spectrum that we
attribute to carbonate complexes as discussed below. The two
peaks assigned as the SAP and TSAP diastereomers are at
similar wavelengths for all complexes: Eu2(1), 579.82, 579.50
nm; Eu2(2) 579.84, 579.55 nm; Eu(3), 579.83, 579.62 nm; Eu(4),
579.82, 579.54 nm, as shown in Figure 1. The pH independence
of these peaks for Eu(III) complexes of 1 and 3 (Supporting
Information Figures S1 and S3) and the similarity of peak
position to analogous complexes16 are consistent with their
assignment as the two diastereomers. Further support is given
by the invariability of the luminescence lifetimes for the major
species of the Eu(III) complexes of 1-3 over the pH range of
Eu2(1)
Eu2(2)
Eu(3)
Eu(4)
Eu(5)b
579.82
579.84
579.83
579.82
580.08
0.31
0.30
0.32
0.33
0.26
3.2
3.3
3.1
3.0
3.9
1.7
1.7
1.6
1.5
1.6
0.60
0.60
0.61
0.66
0.64
1.8
1.9
2.1
1.9
2.0
a 0.50 mM complex, 20 mM Hepes, pH 7.0, I ) 0.10 M (NaNO3).
b Ref 16.
7.0-9.0, consistent with a single species in solution under these
conditions (data not shown). Luminescence lifetime data for
Eu(III) complexes of 1-4 taken at the wavelength maximum
of the major peak shows that the major species for all complexes
has approximately two bound waters for each Eu(III) center
(Table 1). This is consistent with septadentate macrocyclic
ligands and nine-coordinate Eu(III) complexes.
The small red-shifted peak at 580.55 nm for Eu2(1) and at
580.62 nm for Eu(3) grows in over a period of several days
and is more intense at high pH (Supporting Information Figures
S1-S3). Similarly, Eu2(2) and Eu(4) have two minor red-shifted
peaks at 580.22 and 580.55 nm and 580.12 and 580.48 nm,
respectively. We assign these peaks to carbonate complexes
based on the increase in intensity of these peaks upon addition
of sodium carbonate to a solution of Eu2(1), Eu(3), Eu(4), or
Eu2(2) at pH 7.0 (Supporting Information Figures S4-S7).
Excitation at the wavelength maximum of these peaks gives
luminescence lifetimes that correspond to an absence of bound
water ligands (Supporting Information Table S1). Thus, the two
peaks likely arise from two different diastereomeric complexes
with a bidentate carbonate ligand, similar to observations made
by Bruce et al. with monomeric Eu(III) complexes.46 All
attempts to eliminate contamination with carbonate in solutions
failed to further decrease the intensity of these peaks, presumably
because carbonate binds avidly to these complexes. The
(43) Aime, S.; Botta, M.; Ermondi, G. Inorg. Chem. 1992, 31, 4291–4299.
(44) Aime, S.; Barge, A.; Bruce, J. I.; Botta, M.; Howard, J. A. K.; Moloney,
J. M.; Parker, D.; de Sousa, A. S.; Woods, M. J. Am. Chem. Soc.
1999, 121, 5762–5771.
(45) Aime, S.; Barge, A.; Botta, M.; De Sousa, A. S.; Parker, D. Angew.
Chem., Int. Ed. 1998, 37, 2673–2675.
9
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A R T I C L E S
Nwe et al.
Chart 1
Table 2. Dissociation Constants at 25 °C, I ) 0.10 M (NaNO3), pH
7.6, 20 mM Hepes for Eu(III) and Zn(II) Complexes with
Phosphate Estersa
formation of carbonate complexes and precipitates at high pH
(>9.5) precluded the analysis of water ligand ionization
constants.
complex
K(MP, mM)
∆GMP (kcal/mol)
K(DEP, mM)
∆GDEP (kcal/mol)
Binding constants of phosphate esters to the Eu(III) com-
plexes were measured to give insight into the role of these
catalysts in RNA cleavage. Binding of a phosphate diester, DEP
(Chart 1), and a phosphate monoester, MP, to dinuclear and
mononuclear Eu(III) complexes was studied by luminescence
spectroscopy and by kinetic inhibition studies. Luminescence
binding titrations were carried out on Eu2(1), because there is
less interference from carbonate adducts and on Eu(4) and Eu(5)
as examples of mononuclear complexes.
Europium excitation spectra of Eu(4) (Supporting Information
Figure S8) titrated with MP show a decrease in the intensity of
the original major excitation peak as MP binds to the Eu(III)
complex. This decrease in luminescence intensity is used to
determine a dissociation constant by fitting the data to eq 4 for
formation of a 1:1 complex between Eu(4) and MP. Dissociation
constants are given in Table 2.
Eu2(1)
0.060
(0.025)b
0.28
5.8
(6.3)b
4.9
0.43
4.6
Eu(5)
Eu(4)
7.5
2.9
0.018b
0.18
6.5b
5.1
Eu(3)
2.7
94
16
3.5
1.4
2.5
Zn(12)c
Zn2(13)c
14
0.010
2.5
6.9
a Obtained from kinetic inhibition experiments except where noted.
b From luminescence titration, average of three measurements. c From
ref 20 and 32.
The excitation spectra of Eu2(1) titrated with MP is shown
in Figure 2. The peak-fitted excitation spectra of Eu2(1) with
bound MP (Supporting Information Figure S10) shows that there
are two distinct Eu(III) centers. The small frequency separation
between the peaks suggests that these species are isomers of
each other, possibly arising from the different diastereomeric
forms imparted by the macrocyclic ligand. Eu2(1) binds to a
single equivalent of MP (Supporting Information Figure S11),
and the data was accordingly fit to eq 4 for 1:1 binding27 with
the binding constant given in Table 2 (Figure 3).
To determine whether a single or both Eu(III) centers in
Eu2(1) interact with MP, and whether MP replaces a water
ligand upon binding to Eu(III) complexes, luminescence life-
times as a function of MP concentration were measured for
Figure 2. 7F0 f 5D0 excitation spectra (5D0 f 7F2 emission) of solutions
of 3.30 µM Eu2(1) at pH 7.6 titrated with 25-380 µM MP showing a
decrease in the intensity of peaks at 579.82 and 580.55 nm.
Eu2(1), and values of bound water molecules (q) were calculated.
These data show that one bound water per Eu(III) ion in Eu2(1)
is replaced by the phosphate ester (q ∼ 1) (Supporting
Information Table S1). Luminescence lifetime data at all points
in the titration fit to a single exponential, consistent with rapid
(46) Bruce, J. I.; Dickins, R. S.; Govenlock, L. J.; Gunnlaugsson, T.;
Lopinski, S.; Lowe, M. P.; Parker, D.; Peacock, R. D.; Perry, J. J. B.;
Aime, S.; Botta, M. J. Am. Chem. Soc. 2000, 122, 9674–9684.
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14866 J. AM. CHEM. SOC. VOL. 130, NO. 44, 2008

Dinuclear Europium(III) Macrocyclic Catalysts
A R T I C L E S
Figure 3. Plot of observed luminescent intensity (579.82 nm) of Eu2(1)
(3.30 µM) as a function of total added MP concentration at 25 °C, I )
0.10 M NaNO3, pH 7.6, 20 mM Hepes. The solid curve represents the best
fit to eq 4 with Kd ) 23 µM.
Figure 4. pH rate profile of second-order rate constants for cleavage of
HpPNP by Eu(5) (b), Eu(3) (O), Eu2(1) (1), and Eu2(2) (4). The solid
lines through the values for second-order rate constants (kEu) show the
theoretical fits of these data to the logarithmic form of eq 8.
Scheme 3
phosphate ester exchange on the luminescence time scale. For
comparison, luminescence lifetime data on Eu(5) (Supporting
Information Table S2) also showed that a single water molecule
was displaced upon formation of Eu(5)(MP), similar to phos-
phate binding to analogous Eu(III) mononuclear macrocyclic
complexes.47,48 Binding of MP to Eu2(1) was studied over a
range of concentrations of the complex including 3.30, 16.7,
and 50.0 µM. The similarity in binding constant values (Kd )
23, 29, 23 µM, respectively) shows that binding is independent
of catalyst concentration and thus does not involve the formation
of a higher aggregate. Taken together, these data show that the
MP bridges the two Eu(III) centers in Eu2(1).
Binding of DEP to Eu2(1) was studied by excitation
luminescence spectroscopy (Supporting Information Figures S12
and S13). The peak-fitted excitation spectrum (Supporting
Information Figure S14) shows two new peaks similar to that
observed upon MP binding to Eu2(1) and consistent with two
isomeric forms of the complex. Luminescence lifetimes taken
as a function of DEP concentration show that the number of
bound water molecules (q in Supporting Information Table S1)
per Eu(III) ion in the complex is reduced to a single bound
water upon binding of the phosphate diester, signifying that both
Eu(III) centers are bound to DEP. We favor binding of a single
DEP to the dinuclear complexes based on the similarity of the
excitation spectrum of the Eu2(1)(DEP) complex (Supporting
Information Figure S14) to that of the Eu2(1)(MP) complex
(Supporting Information Figure S10) that shows two closely
spaced Eu(III) excitation peaks. However, a 2:1 complex
(Eu2(1)(DEP)2) cannot be ruled out from our data. Titration of
the mononuclear complex, Eu(5), with DEP also leads to
displacement of a single water ligand (Supporting Information
Table S2).
Pseudo-first-order rate constants (kobsd, s-1) for the cleavage
of HpPNP were determined for a range of catalyst concentra-
tions. The second-order rate constants (kEu, M-1 s-1) at 25 °C,
I ) 0.10 M (NaNO3) for the cleavage of HpPNP catalyzed by
the Eu(III) complexes were determined as the slopes of the plots
of kobsd against catalyst by fitting to eq 7 (as shown in Supporting
Information Figure S15), and the values are listed in Supporting
Information Table S3. Second-order rate constants were deter-
mined over a pH range that was limited by the solubility
properties of each catalyst. Only one pH value was studied for
Eu(4) because of the low solubility of the complex at higher
pH values. For the other four complexes, plots of the log of the
second-order rate constants as a function of pH are shown in
Figure 4 along with the data for complex Eu(5), as determined
previously.16 Eu(III) macrocyclic complexes (1-3 and 5)
showed a linear dependence of second-order rate constant on
pH, and the data was fit to eq 8 derived for Scheme 3.
kobs)kEu[Eu(L)]
kEu)kT[OH-]
(7)
(8)
Cleavage of UpU. The progress of the cleavage reaction of
UpU catalyzed by Eu2(1) at pH 7.6 and 25 °C was monitored
by HPLC. Each substrate peak area was normalized to that of
the internal standard. The observed first-order rate constant (kobsd
,
s-1) was determined as the negative slope of logarithmic fraction
(ln(f)) against time (eq 2). Supporting Information Figure S16
shows representative plots of logarithmic fractional conversion
of UpU to products over time. Supporting Information Figure
S17 shows a plot of first-order rate constant as a function of
Eu2(1) concentration (1-4 mM), whose slope is the second-
order rate constant (0.021 M-1 s-1). A complicating feature of
these reactions is that the HPLC internal standard, NBS, binds
to Eu2(1) nearly as strongly as does UpU (Kd ) 1.4 mM, NBS;
Kd ) 0.72 mM, UpU) (Supporting Information Figures S18-21)
and is present in excess over UpU, serving to weaken the
effective binding constant of the catalyst to UpU substrate.
Under these conditions with 2.0 mM catalyst, we calculate that
only 20% is complexed to UpU, in contrast to the 80%
Cleavage of HpPNP. Eu(III) complexes of 1-5 catalyze the
cleavage of HpPNP as followed by monitoring the increase in
absorbance at 400 nm due to formation of 4-nitrophenolate
anion. Cleavage occurs by an intermolecular transesterification
process as shown by identification of the cyclic phosphate ester
as the sole phosphorus-containing product by using 31P NMR
spectroscopy.
(47) Atkinson, P.; Bretonnie`re, Y.; Parker, D.; Muller, G. HelV. Chim. Acta
2005, 88, 391–405.
(48) Atkinson, P.; Bretonniere, Y.; Parker, D.; Muller, G. HelV. Chim. Acta
2005, 88, 391–405.
9
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A R T I C L E S
Nwe et al.
Scheme 4
Chart 2
Figure 5. Diethyl phosphate (DEP) and monomethyl phosphate (MP)
inhibition of the Eu2(1) complex (MP (b), DEP (O)) catalyzed cleavage of
HpPNP at 25 °C, I ) 0.10 M (NaNO3), pH 7.6, 20 mM Hepes buffer.
Data is fit to eq 1 with Ki ) 0.060 mM for MP and 0.43 mM for DEP.
and for a dimeric complex studied previously (Eu2(11)2, Chart
2)28 show strikingly similar characteristics. For all complexes,
the log of the second-order rate constants plotted as a function
of pH shows a simple linear dependence on pH for the pH range
examined, typically 7-9. This data is consistent with the
relatively simple solution speciation behavior of these Eu(III)
catalysts over a broad pH range including no water ligand
ionization under these conditions. This contrasts to the pH-rate
profile observed for metal ion complexes that have water ligand
ionizations within the studied pH range. For example, second-
order rate constants for the cleavage of HpPNP catalyzed by
Zn(II) complexes (Zn(12) and Zn2(13) in Chart 2) are linear as
a function of pH at low pH but demonstrate a downward break
at the pKa value of the Zn-H2O where pH > pKa.18,20,32,49,50
The pH rate profile of HpPNP cleavage for these Eu(III) and
Zn(II) complexes is consistent with a proton being lost from
the substrate or catalyst on going to the transition state. Two
mechanistic pathways by which this loss of proton may
proceed19,21,32 include (1) loss of the proton from the catalyst
to form a M(L)(OH) complex that interacts with the substrate
as a general base catalyst in the rate-limiting step or (2) loss of
the proton from the substrate 2′-hydroxyl in a pre-equilibrium
step followed by interaction of M(L)(OH2) with the deprotonated
substrate in the rate-limiting step (Scheme 5, left and right side,
respectively). Our work with deuterium solvent isotope studies
of the cleavage of an RNA analogue shows that there no
Brønsted general base catalysis19 in the presence of metal ion
Figure 6. Diethyl phosphate (DEP, O) and monomethyl phosphate (MP,
b) inhibition of the Eu(3) (0.2 mM) catalyzed cleavage of HpPNP at 25
°C, I ) 0.10 M (NaNO3), pH 7.6, 20 mM Hepes. Data is fit to eq 1 with
Ki ) 2.7 mM for DEP and 0.18 mM for MP.
complexed in the absence of the sulfonate internal standard.
This leads to the observed linear dependence of the reaction
rate constant on catalyst concentration.
The progress of the cleavage reaction of UpU catalyzed by
Eu(5) at pH 7.6 and 25 °C was monitored by HPLC for the
appearance of uridine. The observed first-order rate constant
(kobsd, s-1) was determined as the slope of fraction (f) against
time over the first 2-6% of reaction (eq 3; Supporting
Information Figure S22). A plot of first-order rate constant as
a function of Eu(5) concentrations, whose slope is the second-
order rate constant (4.4 × 10-4 M-1 s-1), is shown in
Supporting Information Figure S23.
Binding constants of Eu2(1) and Eu(3) to MP and DEP were
obtained by competitive inhibition of HpPNP cleavage. Nor-
malized plots of the first-order rate constants at varying
concentrations of phosphate ester as an inhibitor are shown in
Figures 5 and 6. Data for MP binding was fit to eq 1, derived
from Scheme 4 for 1:1 binding of MP to the Eu(III) complexes.
Equation 1 is used to accommodate the tight binding of the
complexes to the phosphate esters. Inhibition constants are given
in Table 2.
(49) Yang, M.-Y.; Richard, J. P.; Morrow, J. R. Chem. Commun. 2003,
2832–2833.
(50) Mathews, R. A.; Rossiter, C. S.; Morrow, J. R.; Richard, J. P. Dalton
Trans. 2007, 3804–3811.
Discussion
(51) Morrow, J. R.; Amyes, T. L.; Richard, J. P. Acc. Chem. Res. 2008,
41, 539–548.
Reaction Mechanism. The kinetic data for HpPNP cleavage
by the complexes examined here, Eu2(1), Eu2(2), Eu(3), Eu(5),
(52) Rossiter, C. S.; Mathews, R. A.; Morrow, J. R. J. Inorg. Biochem.
2007, 101, 925–934.
9
14868 J. AM. CHEM. SOC. VOL. 130, NO. 44, 2008

Dinuclear Europium(III) Macrocyclic Catalysts
A R T I C L E S
Scheme 5
Scheme 6
Another type of comparison is provided by the division of
the apparent third-order rate constant for cleavage of HpPNP
by the second-order rate constant for hydroxide promoted
cleavage of HpPNP (kT/kOH in Table 3). This gives a rate
constant that expresses the magnitude of the binding interaction
of the catalyst with the transition state.16,28,50,51 The free energies
derived from this interaction (Scheme 6) represent the transition
state stabilization of the reaction by the catalyst (∆Gsq in eq 9).
This analysis shows that the dinuclear catalysts have a transition
state binding energy of nearly 11 kcal/mol. Yet, there is only a
1.2 kcal/mol difference between the alkylated mononuclear
catalyst Eu(3) and the least reactive of the dinuclear catalysts.
These small free energy differences suggest that the two Eu(III)
centers in the dinuclear catalysts lack strong cooperativity in
the cleavage of HpPNP. By contrast, there is a 3.4 kcal/mol
difference in the Zn(II) mononuclear compared to dinuclear
complex in Table 3. The dinuclear Zn(II) complex (Zn2(13))
has a bridging alkoxide group that brings together the two Zn(II)
centers to interact with the substrate in a cooperative manner,
whereas the dinuclear Eu(III) complexes lack a bridging donor
group. To further elucidate the catalytic properties of these
complexes, binding to the phosphate esters DEP, as a substrate
analogue, and MP, as a transition state analogue, were
analyzed.21,32,50
Binding of the Eu(III) complexes to DEP is weak. There is
little discrimination between the different Eu(III) complexes as
there is only a 1.5 kcal/mol free energy difference between the
strongest and weakest Eu(III) complex interactions with DEP.
DEP replaces one water ligand on each Eu(III) center in both
dinuclear and mononuclear complexes, Eu2(1) and Eu(5). This
suggests that the phosphate diester substrates studied here (UpU
and HpPNP) bind directly to the Eu(III) centers. DEP most
likely binds by bridging the two Eu(III) centers in Eu2(1), but
this interaction through both Eu(III) centers does not markedly
increase the binding constant compared to a mononuclear
complex.
Table 3. Rate Constants for HpPNP Cleavage by Eu(III) and Zn(II)
Complexes at 25 °C, 20 mM buffer, I ) 0.10 M (NaNO3)
complex
k2 (M-1 s-1 a
)
kT (M-2 s-1 b
)
kT/kOH (M-1 c
)
∆Gsq (kcal/mol)d
Eu2(1)
Eu2(2)
Eu(3)
5.5
3.5
8.7 × 106
6.4 × 106
7.0 × 105
1.2 × 105
3.8 × 103
1.1 × 106
4.0 × 104
8.7 × 107
6.4 × 107
7.0 × 106
1.2 × 106
3.8 × 104
3.8 × 107
4.0 × 105
-10.8
-10.7
-9.4
0.33
0.32
0.042
0.0013
0.25
Eu(4)e
Eu(5)f
-8.3
-6.2
-9.6
-7.6
Zn(12)g
Zn2(13)g
Eu2(11)2
h
a Second-order rate constant at pH 7.6. b Apparent third-order rate
constant for the metal complex catalyzed reaction in the pH range where
there is a first-order dependence on hydroxide. c Rate acceleration in the
pH range where both metal complex catalyzed and background reaction
are first order in hydroxide for kOH ) 0.099 M-1 s-1 d Stabilization of
.
the transition state from eq 9. e At pH 7.0. f From ref 16. g From ref 20.
h From ref 28.
catalysts, in support of the pathway shown on the right of
Scheme 5. In addition, experiments show that the M(L)(OH2)
species but not the M(L)(OH) species binds strongly to dianionic
phosphate esters such as MP. The interaction of the dianionic
MP with a metal complex represents the electrostatic component
of metal ion stabilization of the dianionic phosphorane transition
state.21,32
Metal Ion Cooperativity. Two metal ion cooperativity in RNA
cleavage is desirable for the formation of highly effective
catalysts. Yet, the extent of metal ion cooperativity is difficult
to identify because dinuclear catalysts must be compared to
mononuclear catalysts20 that have subtle differences in donor
groups. To make rigorous comparisons, we have advocated the
use of apparent third-order rate constants for cleavage, calculated
as the slope of a plot of second-order rate constants as a function
of [OH-] at pH values less than the pKa of the metal water
ligand.18,28,50 This facilitates comparisons between catalysts with
different water ligand ionization constants. Comparison of third-
order rate constants for Eu(III) complexes of 1-3 and 5 (kT in
Table 3) shows that the most active Eu(III) dinuclear catalyst
is 70-fold better than the mononuclear catalyst Eu(5), but only
12-fold better than the methylated version, Eu(3). Eu(4), which
has an aromatic pendent group most closely related to that in
the dinuclear complexes, is the best choice of a mononuclear
catalyst for comparison. We could not obtain a pH dependence
for this complex, but its second-order rate constant at pH 7.0 is
3-fold higher than that of Eu(3) and only 7-fold less than that
of the best dinuclear catalyst under these conditions (Supporting
Information Table S3). This shows that the ancillary pendent
group is a dominant factor in enhancing the reactivity of the
Eu(III) center and that the analysis of cooperativity between
two metal ion centers in a dinuclear complex is dependent on
the choice of appropriate mononuclear catalyst.
Eu(III) complexes of 1-4 bind to MP more tightly than DEP
with dissociation constants ranging from 0.018 to 0.28 mM.
This 2 kcal increase in binding strength over DEP is attributed
to the higher anionic charge on MP that leads to a stronger
interaction with the cationic Eu(III) complexes. Similar to DEP,
MP displaces a water ligand on each Eu(III) center. Binding is
strongest for the Eu(III) complexes that contain an aromatic
pendent group (Eu(4) and Eu2(1)). This shows that the electron-
withdrawing aromatic group increases the Lewis acidity of the
Eu(III) center to form stronger interactions with anionic ligands.
Even an alkyl substituent in Eu(3) leads to slightly stronger
binding than for the unsubstituted macrocyclic complex, Eu(5).
These macrocyclic substitutent effects are reminiscent of those
(53) Canary, J. W.; Xu, J.; Castagnetto, J. M.; Rentzeperis, D.; Marky,
L. A. J. Am. Chem. Soc. 1995, 117, 11545–11547.
(54) Cacciapaglia, R.; Casnati, A.; Mandolini, L.; Peracchi, A.; Reinhoudt,
D. N.; Salvio, R.; Sartori, A.; Ungaro, R. J. Am. Chem. Soc. 2007,
129, 12512–12520.
kEuKa/Kw
∆Gsq ) ∆GEq - ∆GNq ) RT ln
(9)
[
]
kOH
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J. AM. CHEM. SOC. VOL. 130, NO. 44, 2008 14869

A R T I C L E S
Nwe et al.
catalyst in water at 25 °C.50 These complexes show a greater
than 3 kcal/mol stabilization compared to the dinuclear complex
Eu2(11)2. This large stabilization energy is anticipated because
macrocycles 1 and 2 bind to Eu(III) to leave open two
coordination sites for interaction with substrate and avoid the
use of negatively charged carboxylate pendent groups that
generally decrease catalytic efficiency. The influence of the
ligand donor groups is one of the most important factors in
lanthanide catalysis. For example, dinuclear lanthanide catalysts
with bridging hydroxyl groups and negatively charge carboxylate
groups of amino acids presumably have many more open
coordination sites than Eu(III) complexes of 1 or 2 but still are
less active than the catalysts here.1
Scheme 7
observed for Zn(II) macrocyclic complexes.32,52,53 For Zn(II)
complexes, the decreased interaction of the alkylated macro-
cyclic amines of the complex with water leads to poor solvation
of highly charged cationic complexes and stronger anion binding
affinity. Despite these trends, the free energy differences between
the mononuclear and dinuclear Eu(III) complexes for binding
of MP (1.4 kcal) are not very large. By comparison, the free
energy for interaction of MP to a dinuclear complex (Zn2(13)
that likely binds MP through a bridging interaction is 4.4 kcal/
mol greater than for a related mononuclear Zn(II) catalyst.32
The difference is that the bridging alkoxide group in Zn2(13)
facilitates strong interaction with MP through both Zn(II)
centers. The dinuclear Eu(III) catalyst Eu2(1) also binds a single
MP through an intramolecular bridging interaction (Scheme 7),
as shown by luminescence spectroscopy experiments. However,
the two metal ion centers are not anchored together through a
bridging group prior to binding MP as they are for the dinuclear
Zn(II) catalyst. This lack of preorganization for Eu2(1) may lead
to a less favorable interaction with MP and, by analogy, a less
favorable interaction with the phosphorane that is the likely form
of the transition state.
There is a weak correlation between the transition state
stabilization energy for these complexes for the cleavage of
HpPNP (Table 1) and the free energy of binding to the transition
state analogue, MP (Table 3). Eu2(1) has one of the strongest
binding interactions with MP and the largest transition state
stabilization of Eu(III) complexes 1-5. The poorest catalyst,
Eu(5), binds to MP the most weakly. However, as discussed
above, neither the MP binding energy difference between mono
and dinuclear catalysts Eu(3) and Eu2(1) (-5.1 vs -6.3 kcal/
mol) nor the difference in free energy for binding the transition
state (-9.4 vs -10.8 kcal/mol) are large. This suggests that,
despite the interaction of both Eu(III) centers in binding MP,
DEP, and presumably the transition state phosphorane, the
second loosely tethered metal ion center is not effective at
enhancing binding strength (Scheme 7). Despite the similar
trends in strength of MP and transition state interactions of the
Eu(III) complexes, it is clear that there are other factors
important in catalysis that are not expressed in MP binding.
The 6 kcal/mol free energy of binding of Eu2(1) to MP is a
fraction of the nearly 11 kcal/mol stabilization of the transition
state energy for cleavage of HpPNP. Factors not represented
include differences in the phosphate ester versus phosphorane
geometry and the extent equilibrium solvation of charge.32
Although there is little cooperativity between the two metal
centers, the dinuclear Eu(III) complexes of 1 and 2 have the
largest transition state stabilization energy reported to date by
our laboratory for cleavage of HpPNP by a simple metal ion
Cleavage of UpU. Second-order rate constants for the cleavage
of UpU at pH 7.0 for Eu2(1) and Eu(5) are 0.021 and 4.4 ×
10-4 M-1 s-1, respectively. This shows that cleavage of UpU
by Eu2(1) at pH 7.6 is 46-fold higher than for Eu(5), a little
less than the 70-fold difference observed for HpPNP cleavage.
Comparison to the rate constant for cleavage of UpU18 by
hydroxide at pH 7.0 gives transition state stabilization energies
(kT/kOH) of 2.0 × 107 (10 kcal/mol) and 3.9 × 105 M-1 (7.6
kcal/mol), respectively, slightly less than that observed for the
cleavage of HpPNP by these catalysts. On the basis of the
analysis with HpPNP and MP binding studies, the larger
reactivity for the dinuclear complex in the cleavage of UpU is
attributed to an increase in anion binding affinity of the Eu(III)
center by the aryl pendent group and possibly to a small extent
of cooperativity between the two Eu(III) centers.
The second-order rate constant for cleavage of UpU catalyzed
by Eu2(1) is 26-fold larger than Zn2(10) under similar condi-
tions18 and 2-fold larger than a dinuclear Zn(II) catalyst that
has both a bridging alkoxide donor group to promote cooper-
ativity of metal ions and hydrogen bond donors for interactions
with the phosphate diester linkage.52 Comparison with additional
multinuclear Cu(II) and Zn(II) catalysts is complicated by the
high temperatures generally used in dinucleotide cleavage
experiments and by the addition of organic cosolvents to
solubilize the complexes.18,54-56 Recent work on water-soluble
dinuclear Cu(II) catalysts shows that first-order rate constants
for cleavage of UpU at neutral pH at 1.0 mM catalyst and 50
°C are comparable53 to those observed here for Eu2(1) at the
lower temperature of 25 °C.
Apart from the multinuclear metal ion complex catalysts with
relatively well-defined solution chemistry, there are examples
of metal ion complex catalysts that have poor ligands and form
difficult to characterize yet highly active aggregates.1,5 The most
efficient metal ion catalyst for RNA cleavage reported to date
is in this category and is a multinuclear species formed at high
pH in solutions containing 2 mM LaCl3 and buffer. This
complex promotes the cleavage of ApA with a half-life of 13 s.5
Characterization of this labile catalyst is difficult but is thought
to contain multinuclear La(III) centers that form at pH > 8 to
produce a catalyst that promotes cleavage optimally at pH 9.
In comparison, Eu2(1) cleaves UpU at near neutral pH with a
half-life of 4 h for 2 mM catalyst, a 104-fold acceleration over
cleavage of UpU with no catalyst under these conditions.18
A
challenge to inorganic chemists is to identify the features of
(55) Cacciapaglia, R.; Casnati, A.; Mandolini, L.; Reinhoudt, D. N.; Salvio,
R.; Sartori, A.; Ungaro, R. Inorg. Chim. Acta 2007, 360, 981–986.
(56) Yashiro, M.; Kaneiwa, H.; Onaka, K.; Komiyama, M. Dalton Trans.
2004, 605–610.
(57) Iranzo, O.; Richard, J. P.; Morrow, J. R. Inorg. Chem. 2004, 43, 1743–
1750.
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Dinuclear Europium(III) Macrocyclic Catalysts
A R T I C L E S
these aggregates that lead to effective catalysis and to transfer
these features to catalysts in well-defined complexes.
groups form Eu(III) complexes that are among the most effective
reported to date for cleavage of HpPNP and for a dinucleoside,
UpU, in water at 25 °C. Further improvements may be possible
through choice of a linker that has a donor group that bridges
the two lanthanide ion centers to preorganize them for interaction
with substrate.
Conclusion
Lanthanide ion complexes with neutral ligands are highly
effective catalysts for the cleavage of phosphate esters and RNA.
Anionic phosphate esters bind strongly to these complexes by
replacing a water ligand, and this strong inner-sphere interaction
is undoubtedly an important one in catalysis. Importantly, at
near neutral pH, these lanthanide complexes do not form
hydroxide complexes but exist as the aqua complexes that are
generally the more reactive form.16,32,49 This stands in contrast
to complexes of Zn(II) and Cu(II) which readily form hydroxide
complexes at neutral pH values.20,50,57 Thus, the selectivity of
these lanthanide ion complexes for binding phosphate esters over
hydroxide is one important feature of these catalysts. Studies
here show that macrocyclic ligands with aromatic pendent
Acknowledgment. We gratefully acknowledge the National
Science Foundation (CHE0415356) for support of this work and
for a major instrumentation award (CHE-0321058) to build the laser
system. We thank John Richard for helpful discussions.
Supporting Information Available: Europium(III) excitation
luminescence data, binding isotherms for ligands with eu-
ropium(III) complexes, and kinetic data. This material is
available free of charge via the Internet at http://pubs.acs.org.
JA8037799
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