Rapid phosphodiester hydrolysis by an ammonium-functionalized copper(II) complex. A model for the cooperativity of metal ions and NH-acidic groups in phosphoryl transfer enzymes

Article abstract of DOI:10.1021/ja962806y

The copper(II) complexes (L(n))Cu(NO₃)₄·2(H₂O)] (n = 1: 1, n = 2: 2) of the ammonium-functionalized ligands [6,6'-(Me₂HNCH₂C≡-C)₂bpy]²⁺ (L¹) and [6,6'-(Me₃NCH₂C≡C)₂ bpy]²⁺ L² were prepared. Hydrolysis of the activated phosphodiester bis(p-nitrophenyl) phosphate (BNPP) by these complexes in ethanol-water 19:1 at 20°C was investigated. The rate constants for cleavage of the bound phosphodiester at pH 6.6 are k(cat) = 4.4(±0.4) x 10^-3 s^-1 for (L¹)Cu and k(cat) = 4(±1) x 10^-6 s^-1 for (L¹)Cu. (L¹)CU accelerates hydrolysis of BNPP 4 x 10⁷-fold and is 1000 times more reactive than (L²)Cu. This suggests that the high reactivity of (L¹)CU is related to the interaction of the acidic -NMe₂H⁺ group with the phosphodiester substrate. Bifunctional binding of a phosphate ester by metal coordination and hydrogen bonding with one ammonium group is observed in the crystallographically characterized complex [(L¹)₂Cu₂(1,3-μ-O₃POPh)₂(OH₂)₂](NO₃)₄ (3). A plausible mechanism of BNPP cleavage by (L¹)CU includes metal-hydroxide attack to the phosphodiester which is doubly activated by coordinative and hydrogen bonding. The copper(II) complex of L¹ represents a simple model for the efficient cooperativity of metal ions and NH-acidic amino acid side chains (Lys-ammonium, Arg-guanidinium, His-imidazolium) in enzymes that catalyze the cleavage of phosphate di- and monoesters.

Full text of DOI:10.1021/ja962806y

12704
J. Am. Chem. Soc. 1996, 118, 12704-12709
Rapid Phosphodiester Hydrolysis by an
Ammonium-Functionalized Copper(II) Complex. A Model for
the Cooperativity of Metal Ions and NH-Acidic Groups in
Phosphoryl Transfer Enzymes
Endre K o1 v a´ ri and Roland Kr a1 mer*
Contribution from the Anorganisch-Chemisches Institut der UniVersit a¨ t,
Wilhelm-Klemm-Strasse 8, D-48149 M u¨ nster, Germany
X
ReceiVed August 12, 1996
n
Abstract: The copper(II) complexes [(L )Cu(NO3)4‚2(H2O)] (n ) 1: 1, n ) 2: 2) of the ammonium-functionalized
ligands [6,6′-(Me2HNCH2CtC)2bpy]2 (L ) and [6,6′-(Me3NCH2CtC)2bpy] (L ) were prepared. Hydrolysis of
+
1
2+
2
the activated phosphodiester bis(p-nitrophenyl) phosphate (BNPP) by these complexes in ethanol-water 19:1 at 20
°
1
C was investigated. The rate constants for cleavage of the bound phosphodiester at pH 6.6 are kcat ) 4.4((0.4) ×
-
3
-1
1
-6 -1
2
1
7
0
s
for (L )Cu and kcat ) 4((1) × 10
s
for (L )Cu. (L )Cu accelerates hydrolysis of BNPP 4 × 10 -fold
2
1
and is 1000 times more reactive than (L )Cu. This suggests that the high reactivity of (L )Cu is related to the
interaction of the acidic -NMe2H group with the phosphodiester substrate. Bifunctional binding of a phosphate
ester by metal coordination and hydrogen bonding with one ammonium group is observed in the crystallographically
+
1
characterized complex [(L )2Cu2(1,3-µ-O3POPh)2(OH2)2](NO3)4 (3). A plausible mechanism of BNPP cleavage by
1
(L )Cu includes metal-hydroxide attack to the phosphodiester which is doubly activated by coordinative and hydrogen
1
bonding. The copper(II) complex of L represents a simple model for the efficient cooperativity of metal ions and
NH-acidic amino acid side chains (Lys-ammonium, Arg-guanidinium, His-imidazolium) in enzymes that catalyze
the cleavage of phosphate di- and monoesters.
4
5
Introduction
from E. coli, purple acid phosphatase from kidney beans and
6
in calcineurin. The tetrahedral transition state of metal-
mediated peptide bond cleavage is stabilized by a Lys-
ammonium group in bovine lens aminopeptidase.
Quite recently, several interesting studies have demonstrated
that the reactivity of simple metal complexes toward phos-
phodiesters can be increased substantially by attachment of basic
or nucleophilic auxiliary groups to the ligand. In contrast, the
highly efficient cooperativity of a metal ion and a NH-acidic
group has not yet been succesfully mimicked with a simple
model system. For this purpose we have designed the
ammonium-substituted 2,2′-bipyridine (bpy) ligand L . The
zinc(II) complex of this ligand is capable of cleaving bis(p-
4) Kim, E. E.; Wyckoff, H. W. J. Mol. Biol. 1991, 218, 449.
5) Str a¨ ter, N.; T. Klabunde, T.; Tucker, P.; Witzel, H.; Krebs, B. Science
995, 268, 1489.
Understanding the basic principles of enzymatic phosphoryl
transfer reactions is fundamental for the rational design of
efficient synthetic hydrolysis catalysts. A particular attractive
goal is the development of artificial phosphodiesterases for the
nonoxidative cleavage of the phosphodiester backbone in nucleic
acids. Such reagents might have important applications in future
7
8
1
biotechnology.
In many metal dependent phosphoryl transfer enzymes
catalysis is supported by amino acid side chains which act as
acids, bases or nucleophiles. The NH-acidic groups are
represented by the positively charged residues Arg-guanidinium,
His-imidazolium and Lys-ammonium. These groups stabilize
the pentacoordinate transition state (intermediate, respectively)
of the nucleophilic substitution reaction at phosphorus by
hydrogen bonding or proton transfer. In staphylococcal nuclease
9
1
(
(
1
(6) Griffith, J. P.; Kim, J. L.; Kim, E. E.; Sintchak, M.; Thomson, J. A.;
Fitzgibbon, M. J.; Fleming, M. A.; Caron, P. R.; Hsiao, K.; Navia, M. A.
2+
Ca and Arg-guanidinium electrostatically activate the substrate
and facilitate attack of a nucleophile, possibly a calcium-
coordinated hydroxide.2 Active site cooperativity of Zn and
Lys-ammonium was proposed for phosphodiester hydrolysis by
nuclease S1 from Aspergillus orycae.3 Metal ion promoted
phosphate monoester hydrolysis is supported by Arg-guani-
dinium or His-imidazolium residues in alkaline phosphatase
Cell 1995, 82, 507-522.
(
7) (a) Burley, S. K.; David, P. R.; Taylor, A.; Lipscomb, W. N. Proc.
2+
Natl. Acad. Sci. U.S.A. 1990, 87, 6878-6882. (b) Burley, S. K.; David, P.
R.; Sweet, R. M.; Taylor, A; Lipscomb, W. N. J. Mol. Biol. 1992, 224,
113-140.
(
8) (a) Breslow, R.; Berger, D.; Huang, D.-L. J. Am. Chem. Soc. 1990,
1
12, 3686-3687. (b) Kimura, E.; Kodama, Y.; Koike, T.; Shiro, M. J. Am.
Chem. Soc. 1995, 117, 8304-8311. (c) Morrow, J. R.; Aures, H.; Epstein,
D. J. Chem. Soc., Chem. Commun. 1995, 2431-2432. (d) Young, M. J.;
Wahnon, D.; Hynes, R. C.; Chin, J. J. Am. Chem. Soc., 1995, 117, 9441-
9447. (e) Kimura, E.; Kodama, M.; Koike, T.; Shiro, M. J. Am. Chem.
Soc., 1995, 117, 8304-8311. (f) Chu, F.; Smith, J.; Lynch, V. M.; Anslyn,
E. V. Inorg. Chem. 1995, 34, 5689-5690 (the authors propose that in a Zn
complex either imidazole or imidazolium might act as an auxiliary group,
providing an 1.5-fold increase in RNA cleavage rate). See, also: (g) Hsu,
C.-M.; Cooperman, B. S. J. Am. Chem. Soc. 1976, 98, 5657-5663. (h)
Koike, T.; Inoue, M.; Kimura, E.; Shiro, M. J. Am. Chem. Soc. 1996, 118,
3091-3099.
X
Abstract published in AdVance ACS Abstracts, December 1, 1996.
(1) (a) Bashkin, J. K. Bioinorganic Chemistry of Copper; Chapman &
Hall: New York, 1993; pp 132-139. (b) Hall, J.; H u¨ sken, D.; Pieles, U.;
Moser, H. E.; H a¨ ner, R. Chemistry Biology 1994, 1, 185. (c) Bashkin, J.
K., Frolova, E. I.; Sampath, S. U. J. Am. Chem. Soc. 1994, 116, 5981-
5
982. (d) Magda, D., Miller, R. A.; Sessler, J. L.; Iverson, B. J. Am. Chem.
Soc. 1994, 116, 7439-7440.
(2) (a) Anfinsen, C. B. Science 1973, 181, 223. (b) Cotton, F. A.; Hazen,
E. E., Jr.; Legg, M. J. Proc. Natl. Acad. Sci. U.S.A. 1979, 76, 2551. (c)
Weber, D. J.; Meeker, A. K.; Mildvan, A. S. Biochemistry 1991, 30, 6103-
(9) A weak metal-ammonium synergism was observed for the Mg2
promoted hydrolysis of ATP which proceeds 1.4 times faster in the presence
of 1 M NH4 ions: Sigel, H.; Tribolet, R. J. Inorg. Biochem. 1990, 40,
+
6
114.
+
(3) Witzel, H.; Berg, W.; Creutzenberg, O.; Karreh, A. Zinc Enzymes;
Birkh a¨ user: Boston, 1986; pp 295-306.
173-179.
S0002-7863(96)02806-5 CCC: $12.00 © 1996 American Chemical Society

An Ammonium-Functionalized Cu(II) Complex
J. Am. Chem. Soc., Vol. 118, No. 50, 1996 12705
(ꢀ ) 1.21 × 10 M cm-1), 333 (ꢀ ) 1.27 × 10 ). Anal. Calcd for
4
-1
4
nitrophenyl) phosphate (BNPP), but it is only about 5 times
more reactive than related zinc complexes that do not carry
20 8
C H28CuN O14 (668.0): C, 35.91; H, 4.36; N, 16.75. Found C, 35.67;
H, 4.32; N, 16.18.
10
11
functional groups. A preliminary study revealed that the
2
2
1
[(L )Cu(NO
3
)
4
2 3 2
‚2(H O)] (2). L (NO ) (23 mg, 49 µmol) was
copper(II) compound of L has a much higher reactivity than
1
dissolved in 250 µL of water. Five hundred microliters of a Cu-
NO O stock solution (97 mM in ethanol) and 4.2 mL of dry
ethanol were added subsequently with stirring. Cooling to 5 °C for 1
(
L )Zn. Although copper(II) is not a natural cofactor of
(
3
)
2
‚3H
2
phosphodiesterase enzymes, various studies on the hydrolysis
of activated phosphodiesters and of RNA by copper(II) com-
plexes have contributed to the general understanding of metal
promoted phosphodiester hydrolysis.12,8d
h yielded green crystals which are isolated and air dried (yield 11 mg,
4
3
M
2%). UV (ethanol-water 19:1): λmax ) 321 nm (ꢀ ) 1.26 × 10
-1
-1
4
cm ), 333 (ꢀ ) 1.30 × 10 ). Anal. Calcd for C22
8
H32CuN O
14
In this paper we present a detailed kinetic study of BNPP
(696.1): C, 37.96; H, 4.63; N, 16.10. Found C, 37.79; H, 4.51; N,
15.80.
1
hydrolysis by (L )Cu together with crystallographic evidence
[(L1)
a solution of L (NO
phosphate (15 mg, 50 µmol) in 3 mL of ethanol-water (19:1) was
added with stirring Cu(NO ‚3H O (12 mg, 50 µmol). The solution
(1,3-µ-O
(OH
)
2 2
](NO
)
3 4
for bifunctional interaction of the complex with a phosphate
ester.
2
Cu
2
3
2
2
POPh) ‚2 EtOH‚2H O (3). To
(22 mg, 50 µmol) and disodium phenyl
1
3 2
)
)
3 2
2
Experimental Section
was allowed to stand for 2 h. Bright blue X-ray quality crystals were
obtained. The solvent was decanted, and the product was washed with
few diethyl ether and air dried (yield 19.6 mg, 52%). Anal. Calcd for
General Information. All reagents unless otherwise indicated were
of analytical grade and were used without further purification. Etha-
nol-water solvent mixtures were prepared from absolute ethanol and
ultrapure water. 2,4,6-Trimethylpyridine was distilled before use. pH
measurements in ethanol-water 19:1 (v/v) were performed with a
combined glass electrode calibrated with aqueous buffers and were
reproducible within (0.05 units. The pH values given in this work
correspond to pH meter reading + 0.8. This correction value was
obtained by extrapolation of literature data for ethanol-water mix-
C
56
H78Cu
N
2 12
O
26
P
2
(1524.3): C, 44.16; H, 5.16; N, 11.03. Found C,
43.21; H, 4.85; N, 10.95.
3 2
Spectrophotometric Titration. A solution of the ligand (L (NO )
1
2
3 2
or L (NO ) , respectively, 50 µM) and sodium diphenyl phosphate (100
mM) was prepared in ethanol-water 19:1 (v/v). The pH of the solution
was adjusted to 6.6 with NaOH in ethanol-water 19:1. Appropriate
amounts of a Cu(NO
19:1) were added with stirring. Immediately after addition of metal
salt a UV spectrum of the solution was taken at 20 °C, and the pH was
readjusted to 6.6 when necessary. The absorbance at 333 nm was
3
)
2
‚3H O stock solution (5 mM in ethanol-water
2
1
3
tures. EPR spectra were recorded on a Bruker ESP 300 X-band
spectrometer using ethanol-water 19:1 glasses at 77 K. UV-vis spectra
were measured on a Shimadzu UV 3100 spectrometer.
1
2+
6
,6′-Bis(3-dimethylaminopropynyl)-2,2′-bipyridine (L -2 H) and
corrected for the weak absorbance of Cu ions in the absence of ligand
1
were prepared by previously reported methods.10
2
-1
-1
L (NO
3
)
2
under the same conditions (ꢀ333 ) 3.6 × 10 M cm ). An isosbestic
point at ca. 313 nm was found when data were corrected for the
6
,6′-Bis(3-trimethylammoniopropynyl)-2,2′-bipyridine dinitrate
2
-1
-1
2
). 6,6′-Bis(3-dimethylaminopropynyl)-2,2′-bipyridine10 (200
3 2
)
absorbance of excess Cu (ꢀ313 ) 7.0 × 10
constants were calculated by fitting the increase in 333 nm absorbance
for Cu:L ratios > 1 to a 1:1 complex formation model, using ꢀ333
M
cm ). Stability
(
L (NO
mg, 0.63 mmol) was dissolved in 50 mL of acetonitrile. A solution
containing CH I (0.39 mL, 6.3 mmol) in 5 mL of acetonitrile was added
)
3
4
1
4
2
1
.27 × 10 for (L )Cu and ꢀ333 ) 1.30 × 10 for (L )Cu (the free
dropwise with stirring. After 15 min the white precipitate was
centrifuged off and washed with 20 mL of acetonitrile. The product
was suspended in 50 mL of ethanol, and 12.6 mL of a 0.10 M aqueous
ligands do not absorb at this wavelength).
1
pH Titration. A solution of L (NO
3
)
2
or 1 (5 mM), respectively,
AgNO
3
solution was added with stirring. After 0.5 h the precipitate
containing sodium diphenyl phosphate (100 mM), was prepared in
ethanol-water 19:1 (v/v). Aliquots (50 µL) of a CO -free NaOH
was removed by filtration and washed with 20 mL of ethanol. Filtrate
2
and washing solution were combined and reduced to dryness in Vacuo.
solution (50 mM) in ethanol-water 19:1 were added with stirring. The
pH was measured with a glass electrode and read after 30 s equilibration
time.
1
The white residue was dried in vacuo for 5 h (172 mg, yield 58%). H
NMR (D
2
O, 300 MHz): δ 3.51 (18 H, s, CH
.98 (2 H, d, J ) 7.6 Hz, bpy-H), 8.26 (2 H, t, J ) 7.9 Hz, bpy-H),
.33 (2 H, d, J ) 8.1 Hz, bpy-H). UV (ethanol-water 19:1): λmax
3 2
), 4.76 (4 H, s, CH ),
7
8
2
Kinetics. Reaction solutions were prepared by combining appropri-
1
2
)
ate amounts of ligand (L (NO
3
)
2
, L (NO
3
)
2
, or bpy), Cu(NO
)
3 2
2
‚3H O
4
-1
-1
97 nm (ꢀ ) 1.76 × 10 M cm ). Anal. Calcd for C22
H
28
N
6
O
6
(stock solution in ethanol), 2,4,6-trimethylpyridinium bis(p-nitrophenyl)-
phosphate (200 mM stock solution in ethanol, 1:1 mixture of 2,4,6-
trimethylpyridine and bis(p-nitrophenyl)phosphoric acid) and 2,4,6-
trimethylpyridinium nitrate. The final composition of solvent mixture
was ethanol-water 19:1 (v/v). It was not possible to perform the kinetic
experiments in the presence of excess (>0.3 M) standard electrolytes
(472.5): C, 55.92; H, 5.97; N, 17.78. Found: C, 55.30; H, 6.11; N,
1
8.20.
[
1
1
(L )Cu(NO
dissolved in a mixture of 250 µL of water and 3.6 mL of dry ethanol.
A stock solution (1.18 mL) of Cu(NO ‚3H O (40 mM in ethanol)
)
3 4
‚2(H
2 3 2
O)] (1). L (NO ) (22 mg, 50 µmol) was
3
)
2
2
was added with stirring. The green solution was allowed to stand at
room temperature for 2 h. Green crystals of 1 were isolated and air
dried (yield 19 mg, 56%). UV (ethanol-water 19:1): λmax ) 322 nm
(e.g., NaClO
4 3
, NaNO ) owing to precipitate formation. To achieve a
constant ionic strength (I ≈ 0.1 M) the 2,4,6-trimethylpyridinium salts
of bis(p-nitrophenyl)phosphate and nitrate, respectively, were combined
such that the total salt concentration was 100 mM in all reaction
solutions. The reaction was initiated by adjusting the desired pH by
addition of NaOH solution in ethanol-water 19:1. This does not result
in a change of ionic strength since the 2,4,6-trimethylpyridinium cation
(
(
10) K o¨ v a´ ri, E.; Kr a¨ mer, R. Chem. Ber. 1994, 127, 2151-2157.
11) K o¨ v a´ ri, E.; Heitker, J.; Kr a¨ mer, R. J. Chem. Soc., Chem. Commun.
1
995, 1205-1206.
(
12) (a) Morrow, J. R., Trogler, W. C. Inorg. Chem. 1988, 27, 3387-
+
1
is replaced by Na . For the pH-rate-profiles shown for (L )Cu and
(
3
394. (b) Stern, M. K.; Bashkin, J. K.; Sall, E. D. J. Am. Chem. Soc. 1990,
2
+
L )Cu (Figure 6) the Na /2,4,6-trimethylpyridinium ratio increases with
1
12, 5357-5359. (c) Modak, A. S.; Gard, J. K.; Merriman, M. C.; Winkeler,
K. A.; Bashkin, J. K.; Stern, M. K. J. Am. Chem. Soc. 1991, 113, 283-
2
increasing pH. In a control experiment 2,4,6-trimethylpyridine was
used instead of NaOH to adjust pH 6.3; the same hydrolysis rates were
determined. This observation rules out that reactivity is significantly
affected by medium effects. In the absence of buffer, initial rates
appeared to be similar to those in the buffered reaction solutions, but
accurate data were difficult to obtain since the rate readily decreases
owing to a decrease in pH.
At pH < 6.6 in ethanol-water 19:1 (v/v) p-nitrophenol does in effect
not dissociate into p-nitrophenolate and protons. To detect the increase
in p-nitrophenol concentration in the reaction solutions (10 mL), at
least five 0.1 ml samples were taken in appropriate time intervals and
mixed with 3 mL of 40 mM NaOH solution in ethanol-water (2:3)
91. (d) Bashkin, J. K.; Jenkins, L. A. J. Chem. Soc., Dalton Trans. 1993,
631-3632. (e) Burstyn, J. N.; Deal, K. A. Inorg. Chem. 1993, 32, 3585.
3
(f) Wall, M.; Hynes, R. C.; Chin, J. Angew. Chem. 1993, 105, 1696-1697;
Angew. Chem., Int. Ed. Engl. 1993, 32, 1633-1634. (g) Wahnon, D.; Hynes,
R. C.; Chin, J. J. Chem. Soc., Chem. Commun. 1994, 1441-1442. (h)
Linkletter, B.; Chin, J. Angew. Chem. 1995, 107, 529-531; Angew. Chem.,
Int. Ed. Engl. 1995, 34, 472-474. (i) Young, M. J.; Chin, J. J. Am. Chem.
Soc. 1995, 117, 10577-10578. (j) Deal, K. A., Burstyn, J. N. Inorg. Chem.
1
996, 35, 2792-2798. (k) Deal, K. A.; Hengge, A. C.; Burstyn, J. N. J.
Am. Chem. Soc. 1996, 118, 1713-1718.
13) (a) Bates, R. G. Determination of pH, 2nd ed.; John Wiley & Sons:
(
New York, 1964; pp 226-227. (b) Galster, H. pH Messung; VCH:
Weinheim, 1990; pp 60-61, 243-244.

12706 J. Am. Chem. Soc., Vol. 118, No. 50, 1996
K o¨ V a´ ri and Kr a¨ mer
containing 2 mM EDTA. The produced p-nitrophenolate (ꢀ400 ) 19 100
-
1
-1
M
cm in ethanol-water (2:3)) was detected photometrically. For
2% conversion of BNPP an approximately linear increase of
p-nitrophenolate concentration with time was observed in all cases
correlation coefficients > 0.99). Control experiments in which BNPP
<
(
was replaced by diphenyl phosphate show that the complexes them-
selves do not develop undesirable absorbances at 400 nm in course of
the kinetic experiments.
To characterize reaction products of BNPP hydrolysis selected
solutions were treated with EDTA (2 equiv per equiv Cu) and reduced
to dryness. The residue was taken up in a minimum amount of
31
ethanol-water (1:1). P NMR spectra displayed signals for unreacted
BNPP (-11.2 ppm vs external H PO ) and for the monoester nitro-
3
4
phenyl phosphate (1.7 ppm) but not for phosphate (expected at 6.8
ppm).
To determine the spontaneous hydrolysis of BNPP a 100 mM
solution of 2,4,6-trimethylpyridinium bis(p-nitrophenyl)phosphate was
prepared in ethanol-water 19:1 and adjusted to pH 6.6 as described
above. The kinetic measurements were performed at 20((0.5) °C. All
data are average values of three kinetic runs.
1
2
Reaction solutions containing (L )Cu and (L )Cu turned reddish
brown on prolonged standing. This is attributed to an intense 485 nm
+
CT-band of [(L)
2
Cu] complexes formed by Cu(II)-Cu(I) reduction.
It was estimated that in the kinetic experiments <5% of the initially
1
present Cu(II) complex is reduced. A 1:1 mixture of L and copper(I)
perchlorate is not reactive toward BNPP. Furthermore, we can rule
1
out that the high reactivity of (L )Cu is related to the cooperativity of
copper(II) and peroxide which eventually might form by reaction of
Cu(I) with dioxygen. The same rate enhancements are observed when
the kinetic experiments are run on air or strictly under oxygen free
conditions. It was reported that cleavage of amides and phosphodiesters
1
4
3 2 2 2
OPh) (OH )
]4+ (top)
is rapidly promoted by metal-peroxide cooperativity.
X-ray Diffraction Studies. X-ray quality crystals of [(L1)
µ-O POPh) (OH ](NO ‚2EtOH‚2H O (3) were obtained during the
Figure 1. Crystal structure of [Cu
and of the mononuclear [Cu(1)(1,3-µ-PO OPh)(OH )] subunit of the
centrosymmetric complex. Hydrogen atoms at N(3) and O(1) were
located in the difference Fourier map and refined with variable Uiso
2
(1)
2
(1,3-µ-PO
2 2
Cu (1,3-
3
2
3
2
2
)
2
3
)
4
2
.
preparation of the complex. An appropriate single crystal was fixed
Other H-atoms are omitted for clarity. Selected bond distances [Å] and
angles [deg]: Cu(1)-O(1) 1.982(3), Cu(1)-O(2) 1.937(2), Cu(1)-
O(3a) 1.932(2), Cu(1)-N(1) 2.021(3), Cu(1)-N(2) 2.273(3); O(1)-
Cu(1)-O(2) 175.8(1), O(3a)-Cu(1)-N(1) 167.5(1), O(3a)-Cu(1)-
O(1) 88.5(1), O(3a)-Cu(1)-O(2) 90.1(1), N(1)-Cu(1)-N(2) 76.2(1);
N(3)sO(4a) 2.666(5), O(1)sO(4a) 2.735(4), N(3)-H-O(4a) 176(5),
O(1)-H-O(4a) 153(5).
on a glass fiber with grease and cooled to 170 K.
X-ray data were collected on a Siemens P3 diffractometer using
MoKR-radiation (0.71073 Å). The structure was solved using direct
15
methods (SHELXS-86) and refined by full-matrix least-squares against
2
16
F (SHELXL 93). Anisotropic thermal parameters were used for all
non-hydrogen atoms. H(N3) and H(O1) were localized in the final
difference Fourier map and were refined with variable Uiso. Other
hydrogen atom parameters follow a riding model. The molecular sites
of solvate water and ethanol and of one nitrate counter ion are partially
occupied. During refinement the positional occupancies were fixed at
a value of 0.5.
hydrolysis.17 L carries two -NMe2H group linked to a metal
binding 2,2′-bipyridine (bpy) unit by alkyne spacers. In the
complexes the pendant ammonium groups are perfectly posi-
tioned to form hydrogen bonds with a phosphodiester coordi-
nated to the metal. At the same time, the spacers prevent
intramolecular metal chelation by -NMe2.
1
+
2 12 26 2
Formula C56H78Cu N O P , formula weight 1524.3, crystal system
triclinic, space group P 1h ; light blue single crystal with dimensions 0.1
×
0.2 × 0.15 mm; a ) 11.718(2), b ) 12.121(2), c ) 13.037(3) Å, R
3
)
74.90(3), â ) 85.40(3), γ ) 75.25(3)°, V ) 1728.7(6) Å , Z ) 1;
Structure of Complexes. The green crystalline complexes
ω-scan, no absorption correction, 4° < 2θ < 54°, 7949 reflections
measured, 7575 independent, 5493 observed with I > 2σ(I), 469
1
2
[
(L )Cu(NO3)4‚2H2O] (1) and [(L )Cu(NO3)4‚2H2O] (2) were
2
obtained from the ligands and Cu(NO3)2‚3H2O in ethanol-water
parameters; R (on F) ) 0.0496, wR (on F ) ) 0.1417, GOF 0.749;
1
-3
19:1. Reaction of L (NO ) with Cu(NO ) ‚3H O and the
max./min. residual electron density: 1.507/-0.47 e Å (the maximum
is localized at a disordered nitrate counterion).
3 2
3 2
2
monoester disodium phenyl phosphate yielded bright blue
1
crystals of the dimeric complex [(L )2Cu2(1,3-µ-O3POPh)2-
(
OH2)2](NO3)4‚2EtOH‚2 H2O (3) which has a crystallographic
Results and Discussion
centre of symmetry (Figure 1). The copper ions are bridged
by two phenyl phosphate molecules. Each copper is coordinated
by the bpy-unit of L , two phosphate oxygens and one water
We have communicated a preliminary kinetic study on
hydrolysis of the activated phosphodiester BNPP by the
copper(II) complex of L . The p-nitrophenyl substrate was
1
1
11
molecule. The N O coordination polyhedron is a square based
2
3
used since unactivated phosphodiesters are highly resistant to
pyramid. Four short bonds are formed in the basal plane with
O(1), O(2), O(3a) and N(1). The bond with the second bpy
nitrogen N(2) in the apical position is significantly longer. This
(
14) (a) B. K. Takasaki, B. K.; Chin, J. J. Am. Chem. Soc. 1995, 117,
582. (b) Murthy, N. N.; Mahroof-Tahir, M.; Karlin, K. D. J. Am. Chem.
Soc. 1993, 115, 10404.
15) Sheldrick, G. M. SHELXS-86, Program for Crystal Structure
Solution; Universit a¨ t G o¨ ttingen: 1986.
16) Sheldrick, G. M. SHELXL-93, Program for the Refinement of Crystal
Structures; Universit a¨ t G o¨ ttingen: 1993.
8
(
(17) For a critical discussion on the use of activated p-nitrophenyl esters
see (a) Menger, F. M.; Ladika, M. J. Am. Chem. Soc. 1987, 109, 3145-
3146. (b) Breslow, R.; Singh, S. Bioorg. Chem. 1988, 16, 408, and references
8d and 12d.
(

An Ammonium-Functionalized Cu(II) Complex
J. Am. Chem. Soc., Vol. 118, No. 50, 1996 12707
1
Figure 3. Titration of L , 1, and 2 with NaOH. Conditions: ethanol-
water 19:1, [sodium diphenyl phosphate] ) 100 mM, T ) 20((0.5)
Figure 2. EPR spectrum of a glassy ethanol-water 19:1 solution at
°
C.
7
7 K, containing 1 (5 mM), diphenyl phosphate (70 mM), and 2,4,6-
trimethylpyridine buffer (100 mM) at pH 6.3.
Complex Formation in Solution. Whereas bpy forms a
2
+
rather stable 1:1 complex with Cu in aqueous solution
1
8
particular coordination geometry has also been observed in 1
6.3
-1
23
(
formation constant K ) 10
M
at 25 °C), the copper
19
and in [{6,6′-(pentyne-1-yl)2bpy}2Cu2(µ-Cl)2(Cl)2] and appears
to be a consequence of the steric influence of the alkyne
substituents. In copper(II) complexes of 2,2′-bipyridine usually
both Cu-Nbpy bonds are short in-plane interactions. Thus, the
related complex [(bpy)2Cu2(1,3-µ-O3POR)2(OH2)2](NO3)2 (R )
adenosyl) displays a square-pyramidal geometry with in-plane
O2N2 coordination and the apical position occupied by a water
1
2
complexes of L and L have surprisingly low stability in water.
To achieve complex formation an ethanol-water 19:1 solvent
mixture was used for the kinetic experiments.
Formation constants of the 1:1 complexes in this medium
were obtained by spectrophotometric titration of L with cop-
per(II) nitrate in the presence of DPP. The latter phosphodiester
is much less reactive than BNPP and is not hydrolyzed during
the manipulations.
2
0
molecule.
Coordination of copper to the bpy moiety of the ligands is
easily monitored by the shift of the bpy π-π* band from λmax
Another interesting feature of the structure in Figure 1 is
hydrogen bonding of the phosphate monoester with one am-
monium group, as indicated by the interatomic distance N(3)-
≈
298 nm in the free ligands to λmax ) 333 nm in the complexes.
For Cu:L ratios > 1 an isosbestic point at 313 nm was observed,
1
O(4a) of 2.67 Å. This clearly shows that the (L )Cu fragment
and the increase in 333 nm absorbance could be fitted to a 1:1
can provide double electrostatic activation of a phosphate ester
by both metal coordination and hydrogen bonding. A second
hydrogen bond is formed between O(4a) and coordinated water,
O(4a)-O(1) being 2.74 Å. O(1) is located only 3.2 Å away
from the phosphorus atom. A metal hydroxide which is
generated by deprotonation of coordinated water might well act
as a nucleophile and attack the phosphorus atom of the
coordinated substrate.
4
-1
model. Equilibrium constants K ) 1.8((0.1) × 10 M for
1
1
2+
the formation of (L )Cu from L and Cu and K ) 2.1((0.1)
4
-1
2
2
2+
×
10 M for the formation (L )Cu from L and Cu were
calculated (pH 6.6, 20 °C and 100 mM DPP concentration).
1
pH Titration. Ethanol-water 19:1 solutions of L (NO3)2
and of complexes 1 and 2 in the presence of 100 mM sodium
diphenyl phosphate were titrated with NaOH (Figure 3).
Titration of the copper complexes was limited to pH < 6.8
because of precipitate formation at higher pH. The limited data
available for the complexes and the low accuracy of pH
measurements in ethanol-water 19:1 did not allow a detailed
analysis of the titration curves. We have estimated protonation
constants using the equation pKa ) pH + log ([acid]/[base])
Information on the copper coordination geometry in an
ethanol-water 19:1 glass is obtained from EPR spectroscopy.
EPR solutions had the same composition as those used for
kinetic experiments (see below), but BNPP was replaced by
the less reactive diphenyl phosphate (DPP) (Figure 2). The
spectrum is typical for Cu(II) complexes with approximate
tetragonal symmetry, and according to empirical plots of Peisach
13b
which is applicable to pKa determination of weak acids. For
1
the ammonium groups of L (NO3)2 pKa ) 7.0 is derived from
the pH after addition of 1.0 equiv of NaOH. For 2 pKa ) 6.6
is estimated for Cu-coordinated water from the pH at 50%
neutralization. This value should correspond to the acidity
-
1
and Blumberg the values g∫ ) 2.35 and A∫ ) 0.0158 cm are
in agreement with an in-plane O3N donor set.21 Thus, the
coordination geometry found in the crystalline state appears to
be retained in glassy solution. A very similar EPR spectrum is
2
constant of H2O in (L )Cu(OH2)[O2P(OR)2] since kinetic data
(
see below) indicate substrate saturation at 100 mM phosphodi-
2
obtained for (L )Cu under the same conditions, g∫ ) 2.36 and
1
ester concentration. (L )Cu is expected to have three titratable
protons (two -NMe2H and Cu-coordinated H2O). If we
speculate that all three acidic groups have the same pK a best
-
1
A∫ ) 0.0156 cm . In contrast, the literature EPR parameters
+
for [(bpy)Cu(OH2)2]2 with equatorial N2O2-coordination, g∫
+
a
-
1
)
2.29-2.31 and A∫ ) 0.0166-0.0169 cm , are significantly
fit of experimental and theoretical pH values at 20, 27 and 33%
neutralization (addition of 0.6, 0.8 and 1.0 equiv NaOH) is
2
2
different.
EPR spectra show that in 1:1 metal-to-ligand
mixtures (L )Cu and (L )Cu are the major species. Signal
patterns for (L)2Cu and for Cu2 ions are not detectable.
1
2
obtained for pKa ≈ 7.0.
+
1
2
Kinetics. Reactivities of (L )Cu, (L )Cu and (bpy)Cu toward
1
the activated phosphodiester BNPP were compared. (L )Cu and
2
(
(
(
(
18) K o¨ vari, E.; Kr a¨ mer, R., unpublished results.
(L )Cu were used in 5 mM concentration. Extrapolation of the
19) K o¨ v a´ ri, E.; Kr a¨ mer, R. Z. Naturforsch. 1994, 49b, 1324-1328.
20) Aoki, K. J. Am. Chem. Soc. 1978, 100, 7106-7107.
21) Peisach, J.; Blumberg, W. E. Arch. Biochem. Biophys. 1974, 165,
stability data obtained by spectrophotometric titration reveals
that under these conditions < 10% dissociation of the complexes
into free ligand and metal ion is expected. Initial rates (<2%
6
91.
(22) (a) Walker, F. A.; Sigel, H.; McCormick, D. B. Inorg. Chem. 1972,
1
3
1, 2756-2763. (b) Siddiqui, S.; Shepherd, R. E. Inorg. Chem. 1986, 25,
(23) Smith, R. M.; Martell, A. E. Critical Stability Constants, Vol. 4;
Plenum: New York, 1974.
869-3876.

12708 J. Am. Chem. Soc., Vol. 118, No. 50, 1996
K o¨ V a´ ri and Kr a¨ mer
1
4+
Figure 4. Saturation kinetics for the hydrolysis BNPP by (L )Cu (5
mM) in ethanol-water 19:1. Conditions: pH 6.6((0.1), 20((0.5) °C,
I ≈ 100 mM, buffer: 2,4,6-trimethylpyridine (100 mM).
Figure 5. Saturation kinetics for the hydrolysis BNPP by (bpy)Cu2+
(
°
1 mM) in ethanol-water 19:1. Conditions: pH 6.6((0.1), 20((0.5)
C, I ≈ 100 mM, buffer: 2,4,6-trimethylpyridine (100 mM).
conversion of BNPP) were determined by spectrophotometric
Table 1. Observed and Relative Values of k for BNPP
cat
3
1
Hydrolysis by Copper(II) Complexesa
determination of the released p-nitrophenolate. By P NMR
analysis of the reaction solutions only signals of the monoester
nitrophenyl phosphate but not for phosphate could be detected.
In a previous communication11 we have shown that BNPP
cat (s-1
catalyst
k
)
k
rel
1
-3
7
4
(
(
L )Cu
4.4((0.4) × 10
4 × 10
4 × 10
2
-6
L )Cu
4((1) × 10
1
-6
4
cleavage rate is linearly dependent on (L )Cu concentration.
(bpy)Cu
1.5((0.2) × 10
1.2((0.3) × 10
1.1((0.3) × 10
1.4 × 10
1
-10
Dependence of hydrolysis rate on the concentration of BNPP
is shown in Figure 4. Saturation kinetics with good Michaelis-
Menten behaviour is observed at pH 6.6. A plot of 1/V vs 1/c
L
1.1
1
-
10
none (kuncat
)
a
Error limits are based on the reproducibility of kinetic measure-
ments. For reaction conditions see Figure 5.
-
3
-1
gave kcat ) 4.4 × 10
s
and KM ) 0.013 M. BNPP bound
1
7
to (L )Cu hydrolyzes 4 × 10 times faster than free BNPP [kuncat
-
10 -1
1
)
1.1 × 10 s ] under the same conditions (Table 1). L in
the absence of copper has no significant effect on BNPP
hydrolysis.
2
For (L )Cu kinetic measurements were complicated by
progressive Cu(II)-Cu(I) reduction on prolonged reaction times.
Reliable kinetic data were obtained for pH > 6.3 and for high
BNPP concentrations. Initial reaction rates (dc/dt) in the
2
2
presence of 2.5 mM (L )Cu and 5 mM (L )Cu were 0.9((0.2)
-
8
-8
-1
×
10 and 2.0((0.5) × 10 M s , respectively, at pH 6.6,
2
0 °C and 100 mM BNPP concentration. This is indicative of
2
a first order dependence of rate on (L )Cu concentration (at
lower complex concentration data were not well reproducible,
2
and precipitates formed at higher concentration of (L )Cu). No
1
4+
2
4+
Figure 6. Dependence of BNPP hydrolysis by (L )Cu and (L )Cu
5 mM) on pH. Conditions: ethanol-water 19:1, [BNPP] ) 100 mM,
further increase of rate was observed when the BNPP concen-
tration was increased from 70 to 100 mM, indicating substrate
(
-6
-1
2
20((0.5) °C, I ≈ 100 mM, buffer: 2,4,6-trimethylpyridine (100 mM).
saturation of the complex. Thus, kcat ) 4 × 10
s
for (L )Cu
at pH 6.6 was derived directly from Vmax.
pKa ≈ 6.8 of coordinated water can be roughly estimated for
Trogler and Morrow previously described a detailed kinetic
study on the hydrolysis of BNPP and the related substrate
p-nitrophenyl ethyl phosphate by (bpy)Cu in aqueous solution
2
both complexes from the kinetic data. For (L )Cu this is in
reasonable agreement with the value obtained by pH titration,
at 75 °C.12a These data are not directly comparable to our results
pKa ≈ 6.6. The pH-rate profiles show that the very different
1
2
reactivities of (L )Cu and (L )Cu at pH 6.6 cannot be attributed
to large differences in the pKa of copper coordinated water. At
in ethanol-water 19:1. Therefore, we have reinvestigated
BNPP cleavage by (bpy)Cu in the ethanolic medium at 20 °C.
Rate is linearly dependent on complex concentration for 0.1
1
higher pH values one would expect for (L )Cu a decrease of
2
+
reactivity and a bell shaped pH-rate profile owing to deproto-
nation of the ammonium groups. However, this effect is not
yet significant for pH < 6.6. We believe that only one of the
two ammonium groups is involved in catalysis. Statistically,
at pH 6.6 at least one ammonium group is protonated in 92%
mM < [(bpy)Cu ] < 1 mM. A plot of rate Vs BNPP
concentration at pH 6.7 and 1 mM (bpy)Cu is shown in Figure
5
1
; Michaelis-Menten analysis of these data gave kcat ) 1.5 ×
-
6
-1
0
s
and KM ) 0.0083 M. The smaller value of KM for
1
(bpy)Cu compared to (L )Cu indicates that BNPP has a higher
1
-
1
of the (L )Cu molecules if both ammonium groups have pKa )
affinity to the former complex since KM corresponds to the
substrate binding constant of the complexes. Possibly this is a
consequence of some steric hindrance by the 6,6′-substituents
7
.0.
In case of (bpy)Cu rate increases with pH and reaches a
1
plateau at pH ≈ 6.7. At pH > 7.2 reactivity decreases
in (L )Cu.
12a
significantly. Trogler and Morrow attributed a decrease of
(bpy)Cu activity in alkaline aqueous solution to the formation
of inactive hydroxide-bridged dimers (bpy)Cu(µ-OH)2Cu(bpy).
A comparison of the complexes based on kcat reveals that
1
2
(
L )Cu is 1000 times more reactive toward BNPP than (L )Cu
and 2900 times more reactive than (bpy)Cu under the same
conditions (Table 1).
1
The reactivity of (L )Cu toward BNPP decreases more than
10-fold when the water content of the ethanol-water mixture
is increased from 5% to 15%. This cannot be attributed
1
2
(
L )Cu and (L )Cu display a similar increase of rate with
pH (Figure 6). Provided that Cu-OH is the reactive nucleophile,

An Ammonium-Functionalized Cu(II) Complex
J. Am. Chem. Soc., Vol. 118, No. 50, 1996 12709
Chart 1. Plausible Mechanism of BNPP Hydrolysis by
expected to be quite similar. The large difference in reactivity
is attributed to stabilization of the phosphorane intermediate by
1
(L )Cu (R ) p-nitrophenyl).
hydrogen bonding or proton transfer, respectively, in case of
1
(
L )Cu. Additionally, the X-ray crystal structure of 3 indicates
that hydrogen bonding in the ground state might fix the substrate
in a suitable orientation for the attack of copper coordinated
hydroxide at phosphorus.
Molecular models based on the crystallographically deter-
1
mined square-pyramidal geometry of (L )Cu reveal no obvious
possibility how both ammonium groups might simultaneously
stabilize a coordinated phosphorane intermediate by hydrogen
exclusively to enhanced dissociation of the complex into free
ligand and metal ion, as observed by spectrophotometry. Rather,
+
+
interaction of -NMe2H with the phosphodiester substrate is
bonding. In our view participation of two -NMe2H groups
2
9
disfavored by H2O which is a highly competitive solvent for
is unlikely but cannot be completely ruled out.
hydrogen bonding interactions.24
An alternative mechanistic pathway that includes nucleophilic
attack of -NMe2 at phosphorus should be discussed. Simple
tertiary amines do not accelerate BNPP hydrolysis, but it was
proposed that the diamines Me2N(CH2)nNMe2 (n ) 1-3)
The Substrate ApA. Linkletter and Chin have discovered
that (2,9-dimethyl-1,10-o-phenanthroline)Cu2+ rapidly cleaves
the RNA dinucleotide ApA in aqueous solution.1 Interestingly,
in crystals of [(2,9-dimethyl-1,10-o-phenanthroline)2Cu2(µ-Cl)2-
2e
2
7a
25
1
catalyze BNPP cleavage by nucleophilic attack of -NMe2.
(
Cl)2] and [(L )2Cu2(1,3-µ-O3POPh)2(OH2)2](NO3)4 the copper
1
The pH-rate-profile of (L )Cu does not exclude this possibility.
However, molecular models of a (L )Cu-BNPP complex show
ions have a very similar square-pyramidal geometry with one
longer, apical bond to a nitrogen atom of the bpy or phen ligand,
1
1
that formation of the pentavalent phosphorane by intramolecular
nucleophilic attack of -NMe2 at the phosphorus atom of
coordinated BNPP is highly disfavored for steric reasons.
respectively. We therefore have speculated that (L )Cu and
(
2,9-dimethyl-1,10-o-phenanthroline)Cu2+ might have similar
reactivities toward ApA. Although we could readily reproduce
the results of Linkletter and Chin in aqueous solution, in an
ethanol-water 19:1 medium at pH 6.6 neither (2,9-dimethyl-
1
In summary, the ammonium-functionalized complex (L )Cu
7
accelerates hydrolysis of BNPP 4 × 10 -fold and issto our
+
1
1
,10-o-phenanthroline)Cu2 nor (L )Cu (5 mM) cleave detect-
knowledgesthe most reactive complex of a divalent metal ion
able amounts of ApA (0.1 mM) after 5 h according to reversed
phase HPLC analysis.
1
for the cleavage of this phosphodiester. (L )Cu is 1000 times
2
+
more reactive than (L )Cu in which -NMe2H groups are
Mechanism. A plausible reaction mechanism for the hy-
+
1
replaced by -NMe3 and 2900 times more reactive than (bpy)-
Cu. The crystal structure of [(L )2Cu2(1,3-µ-O3POPh)2-
drolysis of BNPP by (L )Cu includes double electrostatic
1
activation of the substrate by metal coordination and interaction
with one of the ammonium groups, followed by nucleophilic
attack of Cu coordinated hydroxide at phosphorus (Chart 1).
Attack of coordinated hydroxide in a template reaction is a
generally accepted mechanism of phosphodiester cleavage by
4
+
(OH2)2]
gives direct proof for intramolecular hydrogen
+
bonding of one -NMe2H group with a coordinated phosphate
ester. Based on pH rate profiles we propose that nucleophilic
attack of Cu-OH to coordinated BNPP is operative in both
1
2
1
metal complexes in which two cis-orientated coordination sites
(L )Cu and (L )Cu. The outstanding reactivity of (L )Cu is
are available.2
6,12a
Furthermore, it is known that ammonium
explained by hydrogen bonding of the coordinated phosphodi-
groups facilitate phosphodiester cleavage by general acid
catalysis.27 Chart 1 combines these two features in the same
molecule.
+
ester with -NMe2H which provides an additional electrostatic
activation and possibly fixes the substrate in close proximity to
1
the nucleophile. (L )Cu may be regarded as a simple model
1
Although (L )Cu displays a square-pyramidal Cu geometry
for the highly efficient active site cooperativity of metal ions
and NH-acidic amino acid side chains, a common functional
motif of phosphoryl transfer enzymes.
the metal-hydroxide mechanism in chart 1 is comparable to that
proposed for square planar (bpy)Cu since an in-plane coordina-
1
tion of both phosphodiester and hydroxide to (L )Cu is obvious
according to crystallographic and EPR spectroscopic results. The
in-plane Cu-O bond lengths of 6 (1.96 (0.04 Å) are very
similar to the Cu-O distances of (bpy)Cu-complexes with in-
plane N2O2-coordination.20 In (trpy)Cu (trpy ) 2,2′:6′,2′-
terpyridine) only one in-plane coordination site is available for
catalysis, and this complex is inactive for the hydrolysis of
Acknowledgment. We thank M. L a¨ ge for the crystal-
lographic data collection and M. Bode and Prof. W. M u¨ ller-
Warmuth for the EPR spectra. Financial support was provided
by the Deutsche Forschungsgemeinschaft and the Fonds der
Chemischen Industrie.
1
2a,28
BNPP.
1
2
For (L )Cu and (L )Cu the Lewis acidity and the coordination
geometry of the metal and the pKa of coordinated water are
Supporting Information Available: X-ray structural infor-
mation for 3 including positional and thermal parameters and
bonding parameters (4 pages). See any current masthead page
for ordering and Internet access instructions.
(24) Kneeland, D. M.; Ariga, K.; Lynch, V. M.; Huang, C.-Y.; Anslyn,
E. V. J. Am. Chem. Soc. 1993, 115, 10042-10055.
(
(
25) Preston, H. S.; Kennard, C. H. L. J. Chem. Soc. (A) 1989, 1880.
26) (a) Hendry, P., Sargeson, A. M. Prog. Inorg. Chem. 1990, 38, 201.
(
b) Chin, J. Acc. Chem. Res. 1991, 24, 145.
27) (a) Komiyama, M.; Yoshinari, K. J. Chem. Soc., Chem. Commun.
989, 1880. (b) Yoshinari, K.; Yamazaki, K.; Komiyama, M. J. Am. Chem.
JA962806Y
(
1
Soc. 1991, 113, 5899. (c) Dalby, K. N.; Kirby, A. J.; Hollfelder, F. Pure
Appl. Chem. 1994, 66, 687.
(29) To settle this question we have prepared [6-(Me2HNCH2CtC)-6′-
(CH3CH2CH2CtC)bpy](NO3) which carries only one ammonium group.
The copper(II) complex of this ligand appeared to be much more reactive
than (L )Cu, but a detailed kinetic analysis was complicated by rapid
Cu(II)-Cu(I) reduction. The reason for the reduction sensitivity of this
complex is not clear.
(28) Interestingly, (trpy)Cu efficiently hydrolyses RNA and 2′,3′-cAMP.
2
The reaction mechanism is essentially different from that of BNPP
1
2b,d
hydrolysis by (bpy)Cu.
For a discussion, see: Bashkin, J. K.; Jenkins,
L. A. Comments Inorg. Chem. 1994, 16, 77-93.