One- and two-metal ion catalysis of the hydrolysis of adenosine 3'-alkyl phosphate esters. Models for one- and two-metal ion catalysis of RNA hydrolysis

Article abstract of DOI:10.1021/ja9607300

Adenosine 3'-O(PO₂^-)OCH₂R phosphate esters have been synthesized with R = 8-hydroxyquinol-2-yl (1a) and 8-(hydroxyquinolyl)-2-methylene (1b). The adenosine 3'-O(PO₂^-)OCH₂R structure has the essential features of an RNA dinucleotide. Equilibrium binding studies with metal ions Mg²⁺, Zn²⁺, Cu²⁺, and La³⁺ have been carried out with 1a, 1b, HOCH₂R (7a and 7b), and 8-hydroxyquinoline (8), and equilibrium constants (K(as)) have been determined for the formation of 1:1 (L)M(n+) complexes. The hydrolysis of 1a and 1b as well as (1a)M(n+) and (1b)M(n+) species are first order in HO^-. The rate enhancement for hydrolysis of 1a by complexation with metal ions is as follows: ~10⁵ with Zn²⁺, ~10³ with Mg²⁺, ~10⁵ with Cu²⁺, and ~10⁹ with La³⁺. Molecular modeling indicates that metal ions ligated to the 8-hydroxyquinoline moiety in the complexes (1a)M(n+) and (1b)M(n+) catalyze 1a and 1b hydrolysis by interacting as Lewis acid catalysts with the negatively charged oxygen atom of the phosphate group. In the instance of La³⁺ complexes, the ligated metal ion is within an interactive distance with both the negative phosphate oxygen and the leaving oxygen. This bimodal assistance by La³⁺ to the displacement reaction at phosphorus by the 2'-hydroxyl anion results in remarkable rate accelerations for the hydrolysis of (1a)La³⁺ and (1b)La³⁺ complexes. The complexes (1a)M(n+) and (1b)M(n+) are themselves hydrolyzed by metal ion catalysis in a reaction that is first order in HO^-, an observation consistent with a transition state composition of [(1a,b)M(n+)][M(n+)][HO^-]. We assume the kinetic equivalent [(1a,b)M(n+)][M(n+)OH] to represent the reacting species. In this catalysis the M(n+)OH is proposed to play the role of general base to deprotonate the 2'-OH while the metal in the complexes (1a,b)M(n+) is coordinated to a negative oxygen of the -(PO₂^-)- moiety. This double metal ion catalysis mimics a mechanism proposed for the ribozyme self-cleavage of RNA.

Full text of DOI:10.1021/ja9607300

J. Am. Chem. Soc. 1996, 118, 9867-9875
9867
One- and Two-Metal Ion Catalysis of the Hydrolysis of
Adenosine 3′-Alkyl Phosphate Esters. Models for One- and
Two-Metal Ion Catalysis of RNA Hydrolysis
Thomas C. Bruice,* Akira Tsubouchi, Robert O. Dempcy, and Leif P. Olson
Contribution from the Department of Chemistry, UniVersity of California at Santa Barbara,
Santa Barbara, California 93106
ReceiVed March 6, 1996X
Abstract: Adenosine 3′-O(PO2-)OCH2R phosphate esters have been synthesized with R ) 8-hydroxyquinol-2-yl
(1a) and 8-(hydroxyquinolyl)-2-methylene (1b). The adenosine 3′-O(PO2-)OCH2R structure has the essential features
of an RNA dinucleotide. Equilibrium binding studies with metal ions Mg2+, Zn2+, Cu2+, and La3+ have been carried
out with 1a, 1b, HOCH2R (7a and 7b), and 8-hydroxyquinoline (8), and equilibrium constants (Kas) have been
determined for the formation of 1:1 (L)Mn+ complexes. The hydrolysis of 1a and 1b as well as (1a)Mn+ and (1b)-
M
n+ species are first order in HO-. The rate enhancement for hydrolysis of 1a by complexation with metal ions is
as follows: ∼105 with Zn2+, ∼103 with Mg2+, ∼105 with Cu2+, and ∼109 with La3+. Molecular modeling indicates
that metal ions ligated to the 8-hydroxyquinoline moiety in the complexes (1a)Mn+ and (1b)Mn+ catalyze 1a and 1b
hydrolysis by interacting as Lewis acid catalysts with the negatively charged oxygen atom of the phosphate group.
In the instance of La3+ complexes, the ligated metal ion is within an interactive distance with both the negative
phosphate oxygen and the leaving oxygen. This bimodal assistance by La3+ to the displacement reaction at phosphorus
by the 2′-hydroxyl anion results in remarkable rate accelerations for the hydrolysis of (1a)La3+ and (1b)La3+ complexes.
The complexes (1a)Mn+ and (1b)Mn+ are themselves hydrolyzed by metal ion catalysis in a reaction that is first
order in HO-, an observation consistent with a transition state composition of [(1a,b)Mn+][Mn+][HO-]. We assume
the kinetic equivalent [(1a,b)Mn+][Mn+OH] to represent the reacting species. In this catalysis the Mn+OH is proposed
to play the role of general base to deprotonate the 2′-OH while the metal in the complexes (1a,b)Mn+ is coordinated
to a negative oxygen of the -(PO2-)- moiety. This double metal ion catalysis mimics a mechanism proposed for
the ribozyme self-cleavage of RNA.
Introduction
in the pKa of H2O upon binding to metal ions, metal bound
hydroxide ion is generated and acts as the nucleophile, or in
the case of RNA hydrolysis, acts as a base to generate 2′-O-,
which acts as the nucleophile; (ii) ligation of the metal ion at a
negative oxygen of the -(PO2-)- linker to increase the latter’s
electrophilic susceptibility to nucleophilic attack; and (iii)
assistance of departure by interaction of metal ion in the
transition state with the partial negative charge on leaving
oxygen.
The half-life for the hydrolysis of dialkyl phosphate esters at
neutrality exceeds the normal lifespan of a human.1 The mere
existence of enzymes capable of rapidly hydrolyzing DNA
establishes the existence of chemical mechanisms for facile
phosphodiester bond hydrolysis. Hydrolysis of RNA is much
more rapid in both chemical and enzymatic processess due to
anchimeric assistance of the ribose 2′-hydroxyl group. There
has been intensive interest in mechanisms of hydrolysis of
phosphodiesters owing to implications in enzyme hydrolysis of
DNA and RNA. A number of phosphoesterases and ribozymes
have been shown to require two or more metal ions for their
catality activity. Two metal ions have been shown to be
required for activity by the 3′,5′-exonuclease from the Klenow
fragment of Escherichia coli DNA polymerase I,2 E. coli
alkaline phosphatase,3 phospholipase C from Bacillus cereus,4
RNase H from HIV reverse transcriptase,5 and hammerhead
ribozyme.6 Metal ions have been proposed to play three
essential roles in the catalytic processes: (i) due to the decrease
It is appreciated that many metal ions promote the transes-
terification of RNA, for example, Zn2+ 7
,
Ni2+ 7c
La3+ 12
,
.
Co2+ 7f,g
There is
,
Cu2+ 7f,g,8
,
Mg2+,9 Mn2+ 7f,g,10
,
Pb2+ 11
,
Al3+ 7b
,
still a great deal to know about these reactions. Kinetic studies
directed at the determination of the mechanism of metal ion
(7) (a) Rorodorf, B. F.; Kearns, D. R.; Biopolymers 1976, 1491. (b)
Dimroth, K.; Witzel, H.; Hulsen, W.; Mirbach, H. Justus Liebigs Ann. Chem.
1959, 620, 94. (c) Stern, M. K.; Bashkin, J. K.; Sall, E. D. J. Am. Chem.
Soc. 1990, 112, 5357. (d) Eichhorn, G. L.; Butzow, J. J. Biochemistry 1971,
10, 2014. (e) Butzow, J. J.; Eichhorn, G. L. Biochemistry 1971, 10, 2019.
(f) Butzow, J. J.; Eichhorn, G. L. Biopolymers 1965, 3, 95. (g) Ikenaga,
H.; Inoue, Y. Biochemistry 1975, 13, 577.
(8) 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.
(9) Wintermeyer, W.; Zachau, H. G. Biochim. Biophys. Acta 1973, 299,
82.
(10) Dange, V.; Van Atta, R. B.; Hecht, S. M. Science 1990, 248, 585.
(11) (a) Werner, C.; Krebs, B.; Keith, G.; Dieheimer, G. Biochim.
Biophys. Acta 1976, 432, 161. (b) Brown, R. S.; Hingerty, B. E.; Dewan,
J. C. Nature 1983, 303, 543. (c) Brown, R. S.; Dewan, J. C.; Klug, A.
Biochemistry 1985, 24, 4785. (d) Behlen, L. S.; Sampson, J. R.; Direnzo,
A. B.; Uhlenbeck, O. C. Biochemistry 1990, 29, 2515. (e) Farkas, W. R.
Biochim. Biophys. Acta 1967, 155, 401. (f) Morrow, J. R.; Buttrey, L. A.;
Shelton, V. M.; Berback, K. A. J. Am. Chem. Soc. 1992, 114, 1903.
(12) (a) Eichhorn, G. L.; Butzow, J. J. Biopolymers 1965, 3, 79. (b)
Baumann, E.; Trapman, H.; Fischler, F. Biochem. Z. 1954, 328.
X Abstract published in AdVance ACS Abstracts, October 1, 1996.
(1) Westheimer, F. H. In Phosphorus Chemistry, DeVelopment in
American Science; Walsh, E. N., Griffith, E. J., Parry, R. W., Quin, L. D.,
Eds.; ACS Symposium Series; American Chemical Society: New York,
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(2) Beese, L. S.; Steiz, T. A. EMBO J. 1991, 10, 25.
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1991, 252, 88.
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S0002-7863(96)00730-5 CCC: $12.00 © 1996 American Chemical Society

9868 J. Am. Chem. Soc., Vol. 118, No. 41, 1996
Bruice et al.
Scheme 1
Experimental Section
Materials. 8-[(tert-Butyldiphenylsilyl)oxy]-2-methylquinoline (2)
and [8-[(tert-butyldiphenylsilyl)oxy]quinolyl]methanol (3a) were pre-
pared according to the method previously reported.15b All reagents for
syntheses were purchased from commercial vendors and used without
further purification. All solvents were dried over proper drying agents
and distilled before use. Buffers and metal chlorides were of highest
purity available and used as received from the suppliers. Solutions of
KCl, used to maintain constant ionic strength, were demetalated by
filtration through a Chelex 100 resin (Bio-Rad) column. Glass-distilled
deionized water was used for all kinetic and equilibrium studies. 1H
NMR spectra were recorded on a Varian Gemini-200 or a General
Electric GN-500 spectrometer with tetramethylsilane as an internal
standard. When deuterium oxide (D2O) or deuterated dimethyl
sulfoxide (DMSO-d6) was used as a solvent, the chemical shifts were
reported relative to the signal of the solvent (D2O, 4.63 ppm; DMSO-
d6, 2.50 ppm). IR spectra were obtained on a Perkin-Elmer 1330
spectrophotometer. High-resolution mass spectrometry was performed
at the University of California, Los Angeles, Mass Facility.
Preparation of Phosphodiesters. Adenosine 3′-[(8-Hydroxy-
quinolyl)methyl phosphate] (1a). Tetrazole (125 mg, 1.78 mmol) was
added to a solution of N6-benzoyl-5′-(4, 4′-dimethoxytrityl)-2′-(tert-
butyldimethylsilyl)adenosine-3′-[â-(cyanoethyl)-N,N-diisopropylphos-
phoramidite] (4) (500 mg, 0.508 mmol) in dry acetonitrile (5 mL) under
argon, and the solution was stirred at room temperature for 20 min. To
this solution was added a solution of the alcohol 3a (214 mg, 0.518
mmol) in dry acetonitrile (5 mL). The resulting solution was stirred
for 90 min at 25 °C and then evaporated. The residue, containing the
coupling product, was dissolved in methylene chloride (4 mL) and
cooled to 0 °C. A solution of 3 M tert-butyl hydroperoxide in 2,4,4-
trimethylpentane (0.65 mL, 1.95 mmol) was added, and the resulting
solution was stirred for 30 min at 0 °C. To this solution was added 50
mL of 2% dichloroacetic acid in methylene chloride. The solution was
stirred for 2 min at room temperature and then poured into 50 mL of
5% sodium bicarbonate solution. The organic layer was separated, and
the aqueous layer was extracted with methylene chloride (2 × 50 mL).
The organic extracts were combined, dried over sodium sulfate, and
concentrated. The residue was chromatographed through a silica gel
column eluting with 5% methanol in ethyl acetate to afford the
diastereomers of 6a. To a solution of the crude product in ethanol (4
mL) was added 40 mL of ammonium hydroxide, and the mixture was
heated at 60 °C for 1 h. After the mixture was cooled to room
temperature, the reaction solution was evaporated by codistillation with
absolute ethanol. Addition of ethyl acetate to the residue afforded the
2′-(tert-butyldimethylsilyl)-protected nucleotide as a white solid (188
mg, 54% for the four step process): mp >120 °C (slow dec); IR (KBr)
3162, 1687, 1649, 1604, 1475, 1432, 1214, 1076, 838 cm-1; 1H NMR
(DMSO-d6) δ 9.10 (s, 1H, phenol), 8.37 and 8.13 (2 × s, 2H, purine
ArH), 8.22 (d, J ) 8.5 Hz, 1H, quinoline ArH), 7.65 (d, J ) 8.5 Hz,
1H, quinoline ArH), 7.42 (br s, 2H, -NH2), 7.38 (m, 2H, quinoline
ArH), 7.07 (dd, J ) 2.4, 6.7 Hz, 1H, quinoline ArH), 5.91 (d, J ) 5.2
Hz, 1H, 1′-H), 5.06 (d, JPH ) 7.6 Hz, 2H, -POCH2-), 4.69 (m, 1H,
2′-H), 4.64 (m, 1H, 3′-H), 4.30 (m, 1H, 4′-H), 3.7-3.56 (m, 2H, 5′-
H), 3.11 and 2.86 (2 × t, 4H, J ) 6.7 Hz, NCCH2CH2NH3+), 0.68 (s,
9H, -SiC(CH3)3), -0.12 and -0.20 (2 × s, 6H, -SiCH3); HRMS
(FAB) m/z 619.2102 (calcd for C26H36N6O8PSi (M + H)+ 619.2104).
To a solution of the above nucleotide (188 mg, 0.273 mmol) in THF
(6 mL) was added 2.7 mL of 1 M tetrabutylammonium fluoride in
THF. After the solution was stirred for 1 h at room temperature, the
solvent was evaporated. The residue was dissolved in a minimum
amount of water and filtered through a column of Bio-Rex 70 cation
exchange resin (sodium form) eluting with water. The fraction
containing the nucleotide was concentrated, and the residue was
chromatographed through a column of Bakerbond phenyl reversed-
phase gel eluting with 50% methanol in water. Nucleotide 1a was
isolated and precipitated from ethanol-hexane (108 mg, 75%): mp
>165 °C (slow dec); IR (KBr) 3390, 3218, 1649, 1604, 1238, 1081
catalysis are often compromised by the formation of insoluble
nucleotide metal ion complexes, the precipitation of metal
hydroxides, formation of metal hydroxide gels, and the com-
plexity of the reaction system. Substrates that have been
employed as RNA models to test the catalytic activities of
various metal ions13 include dinucleotides such as UpU and
ApA. Recently, several designed ligands capable of complexing
two metals have been reported to catalyze the transesterification
of dinucleotides by cooperation of both metal ions.14 Lantha-
num ion and its complexes have attracted attention as catalysts
for phosphate ester hydrolysis in this15 and many other
laboratories16 because of the high efficiency of La3+ catalysis.
We have now synthesized phosphate diesters 1a and 1b
(Scheme 1) and studied the influence of Cu2+, Zn2+, Mg2+, and
La3+ on the rates of their hydrolyses. Phosphate esters 1a and
1b were designed to allow modeling of the influence of metal
ions on RNA hydrolysis. The 8-hydroxyquinoline moieties of
1a and 1b serve to place a metal ion at variable distance to the
-(PO2-)- function, the leaving oxygen, and nucleophilic 2′-
hydroxyl group.
A part of this work has been reported in our preliminary
paper.17
(13) (a) Linkletter, B.; Chin, J. Angew. Chem., Int. Ed. Engl. 1995, 34,
472. (b) Breslow, R.; Huang, D.-L.; Anslyn, E. Proc. Natl. Acad. Sci. U.S.A.
1989, 86, 1746. (c) Breslow, R.; Huang, D.-L. Proc. Natl. Acad. Sci. U.S.A.
1991, 88, 4080. (d) Shelton, V. M.; Morrow, J. R. Inorg. Chem. 1991, 30,
4295. (e) Irisawa, M.; Takeda, N.; Komiyama, M. J. Chem. Soc., Chem.
Commun. 1995, 1221. (f) Kuusela, S.; Rantanen, M.; Lo¨nnberg, H. J. Chem.
Soc., Perkin Trans. 2 1995, 2269.
(14) (a) Yashiro, M.; Ishikubo, A.; Komiyama, M. J. Chem. Soc., Chem.
Commun. 1995, 1793. (b) Young, M. J.; Chin, J. J. Am. Chem. Soc. 1995,
117, 10577. (c) Chapman, W. H., Jr.; Breslow, R. J. Am. Chem. Soc. 1995,
117, 5462.
(15) (a) Tsubouchi, A.; Bruice, T. C. J. Am. Chem. Soc. 1994, 116, 11614.
(b) Tsubouchi, A.; Bruice, T. C. J. Am. Chem. Soc. 1995, 117, 7399.
(16) (a) Komiyama, M.; Matsumura, K.; Matsumoto, Y. J. Chem. Soc.,
Chem. Commun. 1992, 640. (b) Takasaki, B. K.; Chin, J. J. Am. Chem.
Soc. 1993, 115, 9337. (c) Morrow, J. R.; Buttrey, L. A.; Berback, K. A.
Inorg. Chem. 1992, 31, 16. (d) Breslow, R.; Zhang, B. J. Am. Chem. Soc.
1994, 116, 7894.
1
cm-1; H NMR (DMSO-d6) δ 9.64 (br s, 1H, phenol), 8.36 and 8.11
(2 × s, 2H, purine ArH), 8.30 (d, J ) 8.6 Hz, 1H, quinoline ArH),
7.70 (d, J ) 8.6 Hz, 1H, quinoline ArH), 7.41 (m, 2H, quinoline ArH),
(17) Dempcy, R. O.; Bruice, T. C. J. Am. Chem. Soc. 1994, 116, 4511.

Metal Ion Catalysis of RNA Hydrolysis
J. Am. Chem. Soc., Vol. 118, No. 41, 1996 9869
7.07 (dd, J ) 2.8, 6.0 Hz, 1H, quinoline ArH), 5.88 (d, J ) 6.4 Hz,
1H, 1′-H), 5.59 (br s, 1H, 2′-hydroxyl), 5.05 (d, JPH ) 7.7 Hz, 2H,
-POCH2-), 4.64 (m, 2H, 2′- and 3′-H), 4.04 (m, 1H, 4′-H), 3.48 (m,
2H, 5′-H); HRMS (FAB) m/z 505.1237 (calcd for C20H22N6O8P (M +
H)+ 505.1239).
the integrated area (At) of the nucleotide peak vs time (t) where A0 is
the integrated peak area at t ) 0.
The rates of the hydrolysis of 1a in the presence of La3+ were
measured by the stopped-flow method using an OLIS RMS-1000 rapid-
scanning stopped-flow system thermostated at 30 °C. One driving
syringe contained LaCl3 in buffer solution (0.1 M), and the other driving
syringe contained the substrate (20 µM) in the same buffer solution
(0.1 M). Ionic strength was held constant at 1.0 with KCl. The
reactions were followed by monitoring the change of the absorbance
at 260 nm. The collected data points were fit to theoretical curves by
software written for OLIS RSM (rapid-scanning monochromator) to
give the pseudo-first-order rate constants.
Adenosine 3′-[2-(8-Hydroxyquinolyl)ethyl phosphate] (1b). To
a solution of 8-[(tert-butyldiphenylsilyl)oxy]-2-methylquinoline (2) (3.0
g, 7.5 mmol) in dry THF (30 mL) was slowly added n-butyllithium
(10 mmol, 1.6 M in hexane) at -20 °C. After being stirred for 1 min,
the reaction mixture was warmed quickly to 0 °C, and gaseous
formaldehyde (generated by thermal cracking of paraformaldehyde at
∼200 °C and entrained in a flow of nitrogen) was bubbled rapidly into
the solution until the red color of the carbanion was discharged, ca. 5
min. The mixture was then poured into 100 mL of 5% aqueous
NaHCO3, and the product was extracted with ether. The separated
organic phase was dried over Na2SO4 and the solvent removed by rotary
evaporation. The crude product was purified by column chromatog-
raphy on silica gel (75:25 hexane/ethyl acetate) to give 2-[8-[(tert-
butyldiphenylsilyl)oxy]quinolyl]ethanol (3b) as a viscous yellow oil:
1H NMR (CDCl3) δ 8.08 (d, J ) 8.5 Hz, 1H, quinoline ArH), 7.79 (d,
J ) 7.8 Hz, 4H, phenyl), 7.42-7.27 (m, 8H, phenyl and quinoline
ArH), 7.90 (t, J ) 7.9 Hz, 1H, quinoline ArH), 6.75 (d, J ) 8.5 Hz,
1H, quinoline ArH), 5.10 (br s, 1H, OH), 4.12 (t, J ) 5.3 Hz, 2H,
CH2CH2OH), 3.16 (t, J ) 5.3 Hz, 2H, CH2CH2OH), 1.18 (s, 9H, -SiC-
(CH3)3); 13C NMR (CDCl3) δ 160.12, 151.05, 140.30, 136.40, 135.36,
132.70, 129.75, 128.15, 127.68, 125.80, 121.97, 119.84, 117.22, 61.03,
60.33, 38.80, 26.30, 20.98, 19.56, 14.12, 0.97; IR (KBr) 3350 (OH),
3100, 3035, 3025, 2985, 2960, 2845, 1820, 1785, 1750, 1675, 1670,
For the hydrolysis of 1b in the presence of La3+, the reactions were
performed in quartz cuvettes (1 cm path length), thermostated at 30
°C, containing solutions (3 mL) of buffer and LaCl3. The reactions
were initiated by adding the stock solution of 1b to the buffer solution
to give the final concentration of 10 µM in 1b. The change of the
absorbance (A) at 260 nm was followed by a Perkin-Elmer 553
spectrophotometer. The pseudo-first-order rate constants were obtained
by fitting the theoretical equation for a first-order process to the data
points of plots of A vs time.
Metal Binding Equilibra. The equilibrium constants for binding
of the nucleotides 1a and 1b, corresponding leaving alcohols 7a and
7b, and 8-hydroxy-2-methylquinoline (8) with metal ions were spec-
trophotometrically determined at 30 °C (µ ) 1.0 with KCl). Buffer
solutions (3 mL, 0.1 M) of substrates (10 µM) in thermostated cuvettes
were titrated with metal chloride solutions of known concentration and
the absorbances (A) at 260 and 243 nm were recorded on a Perkin-
Elmer 553 spectrophotometer. The volume of titrant added was 50
µL, less than 2% of that of the buffered solutions of 1a, 1b, 7a, 7b,
and 8.
1590, 1510, 1480, 1450, 1370, 1310, 1265, 1200, 1105, 945 cm-1
;
HRMS (CI; NH3) m/z 428.2046 (calcd for C27H30NO2Si (M + H+)
428.2048). The desired nucleotide 1b was prepared from the alcohol
3b (225 mg, 0.53 mol) as described for 1a. The pure nucleotide 1b
(110 mg, 38% based on 3b for the whole process) was obtained as a
white solid: dec >140 °C; IR (KBr) 3363, 3210, 1649, 1602, 1243,
Spectrophotometric pH Titration. The pKa (phenolic proton
ionization) of the nucleotides 1a and 1b and corresponding leaving
alcohols 7a and 7b and phenol 8 were determined spectrophotometri-
cally at 30 °C (µ ) 1.0 with KCl). The absorbances of the solutions
of substrates (20 µM) were recorded at 234 and 260 nm with the pH
varied from 8.3 to 11.3 and also at pH 7 and 12. All solutions were
buffered with CAPS (0.05 M) except for those with pH 7 (HEPES)
and pH 12 (KOH). In order to determine pKa values, plots of the
absorbances at two wavelengths were fit by the theoretical equation
for the dissociation of a monoprotic acid. The values obtained at 234
and 260 nm were averaged.
1
1087 cm-1; H NMR δ 8.36 and 8.13 (2 × s, 1H, purine ArH), 8.13
(d, J ) 8.4 Hz, 1H, quinoline ArH), 7.38 (t, J ) 7.4 Hz, 1H, quinoline
ArH), 7.38 (br s, 2H, NH2), 7.30 (d, J ) 7.9 Hz, 1H, quinoline ArH),
7.15 (d, J ) 8.0 Hz, 1H, quinoline ArH), 6.91 (d, J ) 7.3 Hz, 1H,
quinoline ArH), 5.86 (d, J ) 6.9 Hz, 1H, 1′-H), 4.60 (m, 2H, 2′- and
3′-H), 4.25 (m, 2H, POCH2CH2), 4.03 (m, 1H, 4′-H), 3.56 (m, 2H,
5′-H), 3.21 (t, J ) 6.0 Hz, 2H, POCH2CH2); LRMS (FAB) m/z 518.3
(calcd for C21H23N6O8P (M + H+) 518.4).
Kinetic Measurements. The ionic strength of the reaction solutions
was maintained at 1.0 with KCl. Buffers (0.1 M) were used within 1
pH unit of their pKa values to maintain constant pH; the buffers
employed were MES (2-(N-morpholino)ethanesulfonic acid), HEPES
(N-(2-hydroxyethyl)piperazine-N′-3-propanesulfonic acid), CHES ((2-
cyclohexylamino)ethanesulfonic asid), CAPS (3-(cyclohexylamino)-1-
propanesulfonic asid), phosphate, with pH adjusted by addition of KOH.
EDTA (10 mM) was incorporated into buffer solutions for nonmetal
ion-assisted reactions to sequester trace metal ion impurities. The pH
values of the buffer solutions were measured with a Radiometer Model
26 pH meter and a combination glass electrode.
Results
The preparation of adenosine (8-hydroxyquinolyl)methyl
phosphate (1a) and adenosine 2-(8-hydroxyquinolyl)ethyl phos-
phate (1b) is illustrated in Scheme 1. Coupling of the alcohols
3a,b with N6-benzoyl-5′-(4, 4′-dimethoxytrityl)-2′-(tert-bu-
tyldimethylsilyl)adenosine 3′-(â-cyanoethyl N,N-diisopropyl-
phosphoramidite) (4) in the presence of triazole afforded the
intermediates 5a,b. Oxidation of 5a,b with tert-butyl hydro-
peroxide followed by removal of the 5′-dimethoxytrityl protect-
ing group with 2% dichloroacetic acid in methylene chloride
yielded the diasteromers of 6a,b. These diastereomers were
converted into the tetrabutylammonium salts of target nucle-
otides 1a and 1b by cleavage of the cyanoethyl, the N6-benzoyl,
and the tert-butyldiphenylsilyl groups with ammonium hydrox-
ide, followed by deprotection of the 2′-tert-butyldimethylsilyl
group with tetrabutylammonium fluoride (TBAF). The sodium
salts of 1a and 1b were obtained by cation-exchange chroma-
tography.
The hydrolysis of 1a and 1b in the presence and absence of metal
ions, except for hydrolysis promoted by La3+, was followed by HPLC.
A Perkin-Elmer series 100 pump module equipped with an Alltima
C18 analytical column (Alltech) was used, and chromatograms were
recorded on a Hewlett-Packard HP 3392A integrator connected to a
Hewlett-Packard HP 1050 variable-wavelength detector set at 260 nm.
Kinetic runs were initiated by adding 5-10 µL of the nucleotide stock
solution into 1.5 mL of the buffer solution thermally equilibrated at 30
°C to give the final concentration of 10 µM in 1a,b. Aliquots of the
reaction solutions were periodically injected (50 µL) on the HPLC
column. The samples were eluted with 20% acetonitrile for 1a and
15% acetonitrile for 1b in 50 mM potassium phosphate buffer (pH
3.4) at a flow rate of 1.0 mL/min. Disappearance of the nucleotides
1a,b was monitored. The reactions followed pseudo-first-order kinetics
Metal Binding Studies. Association constants for the metal
ion complexes of 1a and 1b, in addition to their corresponding
leaving alcohols 7a and 7b, with Zn2+, Mg2+, Cu2+, and La3+
were determined at constant pH by spectrophotometric titration
(30 °C; µ ) 1.0 with KCl). As a control, complexation of
8-hydroxy-2-methylquinoline (8) was also examined. Typical
for at least 4 half-lives. The pseudo-first-order rate constants (kobsd
)
were evaluated by fitting the first-order-rate law (eq 1) to the plots of
obsdt
A ) A0ek
(1)

9870 J. Am. Chem. Soc., Vol. 118, No. 41, 1996
Bruice et al.
Table 1. Association Constants (30 °C, µ ) 1.0) of Complexes of
the Nucleotides, Their Leaving Alcohols, and 8-Hydroxy-2-
methylquinolinea and pKa values of Phenolic Proton Ionization
Zn2+ c
Mg2+ d
La3+
b
pKa
log Kas (1aH-1M)
log Kas (1bH-1M)
log Kas (7aH-1M)
log Kas (7bH-1M)
log Kas (8H-1M)
9.73
9.92
7.04
7.27
7.81
7.83
7.60
3.03
2.60
2.52
3.60
3.09
NDe
NDe
9.61
7.35f
9.82
10.04
5.18g
<4.00h
a Kas (LH-1) ) [LH-1M]/[LH-1][M]. b Acid dissociation constants
(30 °C, µ ) 1.0) of the phenolic protons of the hydroxyquinoline
moieties, determined by spectrophotometric pH titration (see Experi-
mental Section). c Averaged values from the titration at pH 6.17 and
6.47. d Values from the titration at pH 8.8, except for 1bH-1Mg (pH
9.33). e Not determined. f Averaged value from the titration at pH 5.59,
6.05, and 6.47. g Value at pH 6.52. h From ref 15b.
Figure 1. Plots of absorbance of 1a (0), 7a (4), and 8 (O) at 260 nm
vs [Zn2+] at 30 °C (µ ) 1.0) and pH 6.17. The solid lines best fit to
the data points were generated by use of eq 3.
plots of A260 vs metal ion concentration are provided in Figure
1 for titration of 1a, 7a, and 8 with ZnCl2 at pH 6.17. Apparent
association constants (Km), defined in eq 2, were obtained from
Km
L + MH-1 y z (LH-1)M + H+
(2)
Km ) [(LH-1)M]aH/[L][M]
Figure 2. Dependence of the pseudo-order-rate constant (kobsd) for
hydrolysis of 1a (b) and 1b (O) on [Zn2+] (pH 6.50) and [Mg2+] (pH
9.83 with 1a and pH 9.56 with 1b) at 30 °C . The solid lines were
created by using eq 6.
L ) 1a, 1b, 7a, 7b, or 8, M ) Zn2+, Mg2+, Cu2+, or La3+
the nonlinear least-squares fit of A260 vs [Mn+] by use of eq 3.
In eq 3, A0 and A1 refer to the absorbances of the solution at
[Mn+] ) 0 and at saturation upon complexation, respectively,
[Mn+] the concentration of unbound metal ion, and aH proton
activity. Equation 3 was derived by combination of eq 2 and
mass balance in terms of the ligands.
saturated at a concentration of Cu2+ four times that of [L] with
1a, 7a, and 8 at pH 5.54 and for 1b and 7b at pH 6.2. Thus,
we are unable to state with any certainty that complexes with
a 1:1 stoicheometry are formed with Cu2+
.
Metal Ion-Promoted Hydrolysis. Hydrolyses of 1a and 1b
(A0aH + A1Km[Mn+])
(aH + Km[Mn+])
in the presence of increasing concentrations of Zn2+, Mg2+
,
A )
(3)
La3+, and Cu2+ were investigated at 30 °C (µ ) 1.0 with KCl)
and constant pH. Rates of the metal ion-promoted hydrolysis
of 1a and 1b were essentially independent of the concentrations
of the buffers employed (MES, HEPES, and CHES) when varied
In the curve fitting, [Mn+] can be replaced by the total
concentration of metal ion ([Mn+]T) due to [Mn+]T . [L]. The
lines in Figure 1 are the best fits of A260 vs [ZnCl2] using eq 3.
The values of Km providing the best fit were converted into
those of Kas by dividing by the acid dissociation constants Ka
(Table 1) of the phenolic proton of the 1a, 7a, and 8 ligands
(eq 4).
over a 10-fold change. The reactions involving Zn2+, Mg2+
,
and Cu2+ were followed by HPLC. All the reactions produced
(8-hydroxyquinolyl)methanol (7a) or -ethanol (7b), depending
on which nucleotide was used, and 2′,3′-cyclic AMP together
with minor products 2′-AMP and 3′-AMP. The identity of these
products was established by comparison of their retention times
with those of authentic samples. The minor species 2′- and
3′-AMP were produced in roughly equal amounts and presum-
ably result from the subsequent hydrolysis of 2′,3′-cyclic AMP.
For La3+-promoted hydrolysis of 1a and 1b, the reactions were
so fast that stopped-flow spectrophotometry was required in
order to follow the time course of the reactions (see Experi-
mental Section). In what follows, the term “hydrolysis” or
“hydrolytic cleavage” refers to rate-determining intramolecular
transesterification by the 2′-OH group to provide directly the
2′,3′-cyclic AMP intermediate.
Kas ) Km/Ka ) [(LH-1)M]/[LH-1][M]
(4)
The values of log Kas for other metal ions were calculated in
the same manner used for Zn2+ complexes (plots of A260 vs
[M] are not shown). The Kas values calculated from the plots
of A260 vs [M] were consistent with those evaluated from the
plots of the absorbance at another wavelength (243 nm). The
values of log Kas in Table 1 are the average of numbers
determined at two wavelengths. Due to the rapid hydrolysis
of 1a and 1b in the presence of La3+, the thermodynamic values
of the association constants for formation of La3+ complexes
could not be determined.
Because the Km values for the copper complexes are so large,
they could not be determined Via our spectrophotometric titration
procedure. This is because the condition [Cu2+]T . [L] required
for fitting eq 3 to the experimental values of A vs [Mn+] cannot
be used. The increase of absorbance at 260 nm was almost
Plots of the pseudo-first-order rate constants (kobsd) vs metal
ion concentration are shown in Figure 2 for hydrolysis of 1a
and 1b in the presence of Zn2+ (pH 6.50) and Mg2+ (pH 9.83
for 1a, pH 9.56 for 1b) and in Figure 3 for La3+ (pH 7.43 for
1a, pH 7.50 for 1b). Examination of Figures 2 and 3 shows
that hydrolysis of 1a and 1b exhibit a biphasic dependence on
metal ion concentration. Both phases, at a given constant pH,

Metal Ion Catalysis of RNA Hydrolysis
J. Am. Chem. Soc., Vol. 118, No. 41, 1996 9871
Figure 3. Influence of [La3+] on the pseudo-first-order rate constant
(kobsd) for hydrolysis of 1a (b) and 1b (O) at 30 °C (µ ) 1.0). The
lines are nonlinear least-squares fits to the data points by use of eq 6.
The constriction kM ) 0 was used for 1b.
Figure 4. Dependence of the pseudo-first-order rate constant (kobsd
)
for hydrolysis of 1a on [Cu2+] (b, inset) and 1b (O) at 30 °C (µ )
1.0).
are linearly dependent upon metal ion concentration. The
dependence of kobsd on metal ion concentration can be described
by preequilibrium formation of a reactive 1:1 metal-ligand
complex that undergoes both spontaneous (kO) and metal ion-
catalyzed (kM[M]) hydrolysis (eq 5).
Table 2. Kinetic Constants (30 °C, µ ) 1.0) for the Hydrolysis of
1a and 1b with Various Metal Ions V ) k0HO[(1a or 1b)Mn+][HO-]
+ kMHO[(1a or 1b)Mn+][Mn+][HO-]
a
nucleotide metal log Kas
k0HO b (s-1 M-1
)
kMHO b (s-1 M-2
)
1a
1a
1a
1a
1b
1b
1b
1b
Zn2+
Mg2+
Cu2+
La3+
Zn2+
Mg2+
Cu2+
La3+
7.56
2.88
NDc
5.21
7.51
2.61
NDc
3.78
5.01 × 102
1.73 × 104
1.28 × 10-5
∼0
Km
2.19
k0 + kM[Mn+
8.49 × 101
7.51 × 105
1.87 × 102
1
-1 + M
w
-H x (1H-1)M(n-2)+
]8 P
(5)
+
n+
+H+
2.87 × 107
7.92 × 103
∼0
The pseudo-first-order rate expression of eq 6 follows from
eq 5. In eq 6, k0 denotes the first-order rate constant for
8.62 × 10-1
∼101 d
2.29 × 107
1.16 × 105
NDc
kobsd ) (k0 + kM[Mn+])Km[Mn+]/(aH + Km[Mn+]) (6)
a Kinetically determined by use of eq 6. b Determined by use of eqs
7 and 8. c Not determined. d Roughly estimated value. See text.
spontaneous hydrolysis of (1H-1)M(n-2)+, kM the second-order
rate constant for metal ion-catalyzed hydrolysis of the complex,
[Mn+] the total concentration of the metal ion, Km association
constant for formation of the 1:1 metal ligand complex, and aH
the proton activity of the reaction solution. This equation was
used to fit the data points of the plots of kobsd vs [Mn+] in order
to obtain the kinetic constants Km, k0, and kM (Figures 2 and
3). For the Mg2+-promoted hydrolysis of 1b (Figure 2, right
side, open circle), the constriction of kM ) 0 was applied for
The dependence of kobsd on [Mn+] seen in Figures 2-4 is
typical of the plots of kobsd vs [Mn+] for each metal ion catalysis
of the hydrolysis of 1a over the pH ranges studied. The values
of k0 and kM are obtained from plots of kobsd vs [Mn+] at several
different pH. Plots of the log of kM and kO vs pH are shown in
Figure 5 (closed symbols) for hydrolysis of 1a by each metal
ion. These pH-rate profiles show a linear relationship with a
slope of +1. The lines in Figure 5 show best fits to the data
points with use of eqs 7 and 8 on the basis of the first-order
dependence on [HO-] (Kw ) 10-13.8326).
the curve fitting, since the slope of the plots of kobsd vs [Mg2+
]
is essentially zero at the higher metal ion concentrations. The
satisfactory fit with this constriction indicates that the hydrolysis
of the species (1bH-1)Mg is not measurably catalyzed by free
Mg2+. The same constriction was also applied to the fitting
for the La3+-promoted hydrolysis of 1b (Figure 3, open circle),
though little can be made of this since the magnitude of the
rate constants restricts the concentration range of La3+ that can
be used.
k0HOKw
k0 ) k0HO[HO-] )
(7)
(8)
aH
kMHOKw
kM ) kMHO[HO-] )
aH
Plots of kobsd vs metal ion concentration for Cu2+-promoted
hydrolysis (pH 6.05) of 1a and 1b are shown in Figure 4. At
the concentrations of Cu2+ employed, 1a and 1b are entirely
converted into copper complexes (see binding studies). No
influence of the metal ion concentration on kobsd was seen in
the hydrolysis of the 1a complex with Cu2+ (see inset in Figure
4), whereas the pseudo-first-order rate constant for hydrolysis
of the 1b complex exhibits a linear dependence on [Cu2+]. Thus,
the hydrolysis of a copper complex of 1a is not catalyzed by
additional Cu2+ while the hydrolysis of the copper complex of
HO
The values of k0 and kMHO, used to create the best fit, are
listed in Table 2. The kinetically determined values of Kas
(Table 2)sobtained from the kinetic values of Km by dividing
by Kasare roughly consistent with those determined thermo-
dynamically. For the metal ion-promoted hydrolysis of 1b a
kinetic study was carried out, except with Cu2+, at only a single
pH. The points of the log of k0 and kM for the metal
ion-promoted hydrolysis of 1b are included in Figure 5 (open
symbols). Assuming the same pH dependence of k0 and kM
for metal ion-promoted hydrolysis of 1b as determined for 1a,
1b is catalyzed by addition of Cu2+
. The concentration-
HO
HO
independent value of kobsd for 1a and intercept of the line in
the plots of kobsd vs [Cu2+] for 1b represent the first-order rate
constants (k0) for spontaneous hydrolysis of copper complexes
of 1a and 1b, respectively. The slope of the line gives the
second-order rate constant (kM; eq 5).
we calculated the values of k0 and kM using eqs 7 and 8.
The results are also included in Table 2.
Hydrolysis of the Nucleotides 1a and 1b in the Absence
of Metal Ions. EDTA (1 mM) was incorporated into the
reaction solution to sequester traces of metal ion impurities, and

9872 J. Am. Chem. Soc., Vol. 118, No. 41, 1996
Bruice et al.
Scheme 3
such complexes the metal ions are ligated by the 8-hydroxy-
quinoline moiety of 1a and 1b. With the exception of Cu2+
(see Results), the similarity of thermodynamically and kinetically
determined association constants assures that reactive species
are complexes with a 1:1 stoichiometry. In the second path
the hydrolytic reaction is first order in the complexes (1a)Mn+
or (1b)Mn+ and first order in free metal ion. Thus, metal ion
promotes hydrolysis (kM[Mn+]) of the 1:1 metal ion:ligand
complexes.
The Structure of LMn+ Complexes and the Mechanism
of Their Hydrolysis by Lyate Species. In the ground state
structure of the 1:1 complexes of metal ions with 1a and 1b,
the negatively charged phosphodiester -(PO2-)- moiety is
much favored as a ligand, compared to the neutral oxygen,
which becomes a leaving group. The metal ion in such 1:1
complexes can act as a Lewis acid catalyst, neutralizing the
charge on the phosphate ester by ligation to one of the two
(PO2-) oxygens and thereby activating the phosphorus atom
toward nucleophilic attack (Scheme 2). In the single case of
La3+ (Vida infra), the metal ion may ligate to both oxygens of
the (PO2-) entity as well as to the leaving oxygen (Scheme 3).
Due to the formation of 2′,3′-cyclic phosphate and the first-
order dependence of the reaction rate on [HO-], the nucleophilic
entity must be the anion of 2′-OH, which presumably attacks
phosphate phosphorus inline with the leaving oxygen atom.
Metal ion ligation to the oxygen of alkyl-OH lowers the pKa
of the latter thereby increasing the concentration of alkyl-O-.
In our model, however, metal ions Mg2+, Zn2+, and Cu2+
complexed to the 8-hydroxyquinoline moiety of 1a and 1b
cannot easily ligate with the 2′-OH. This would require
formation of 12- and 13-membered rings with proper orientation
about six or seven single bonds in the metal complexes of 1a
and 1b, respectively. Molecular modeling suggests that not only
are these metal ions incapable of ligating with the nucleophile
or the leaving group when complexed to the phosphodiester,
but also water or hydroxide ligands in the metal ion’s immediate
coordination sphere cannot directly interact with the nucleophilic
or leaving alcohols. For example, it is not possible for OH-
attached to Zn2+ in the (1a)Zn2+ or (1b)Zn2+ complexes to
deprotonate the 2′-OH while retaining the correct geometry for
inline attack on the phosphate, nor is it possible for H2O attached
to Zn2+ in the (1a)Zn2+ or (1b)Zn2+ complexes to protonate
the leaving oxygen without disrupting phosphate coordination
to the Zn2+. This is so regardless of whether the metal ion is
four-, five-, or six-coordinate.
Figure 5. pH-rate profiles of the rate constant k0 (top) and kM (bottom)
at 30 °C (µ ) 1.0) for hydrolysis of 1a (closed symbols) and 1b (open
symbols) promoted by Zn2+ ([), Mg2+ (2), Cu2+(9), and La3+ (b).
The lines are drawn by use of eqs 7 and 8.
Scheme 2
the hydrolysis of 1a was studied at pH 7.0 and 30 °C (µ )
1.0). The concentration of 1a remained unchanged for 6
months. To estimate the reaction rate of hydrolysis of 1a and
1b without metal ions, we carried out the hydrolysis in 0.2 M
KOH solution (µ ) 1.0 with KCl) at 30 °C. The second-order
rate constants for reaction with HO- were calculated as 7.5 ×
10-4 s-1 M-1 for 1a and 3.1 × 10-4 s-1 M-1 for 1b.
Discussion
All of the metal ions (Mg2+, Zn2+, Cu2+, and La3+) tested
promoted hydrolysis of the nucleotides 1a and 1b Via intramo-
lecular transesterification with formation of 2′,3′-cyclic adeno-
sine monophosphate and alcohols 7a and 7b, respectively
(Scheme 2). The dependence of the pseudo-first-order rate
constants (kobsd) on metal ion concentration establishes involve-
ment of two catalytic pathways (eq 5).18 The first is spontaneous
hydrolysis (k0) of the 1:1 metal ion complexes of 1a and 1b. In
(18) In a preliminary study,17 employing cacodylate buffer, it was found
that (1a)Zn was not hydrolyzed by a mechanism involving two metals. This
is due to the strong interaction of Zn2+ with this buffer species. Such an
interaction would decreases the free metal ion concentration, inhibiting the
second catalytic pathway. On the other hand, MES, HEPES, CHES, and
CAPS buffers used in the present study are known to minimize the
interaction with metal ions.

Metal Ion Catalysis of RNA Hydrolysis
J. Am. Chem. Soc., Vol. 118, No. 41, 1996 9873
Figure 6. (Top) stereoview of the structure of (1a)Zn with the phosphate oxyanion ligated to the zinc at a bond length of 1.98 Å. The distance
between Zn2+ and the leaving oxygen (3.36 Å) is too far for interaction, and any bonding would require distortion of the zinc tetrahedral structure.
(Bottom) Stereoview of the structure of ((1a)La)2+. The distances between La3+ and both the leaving oxygen and phosphate oxyanion are suitable
for interaction. In both the top and bottom structures, the 2′-OH is in position for an inline displacement of the leaving oxygen. The structures were
built as follows: the X-ray structure of the complex of 8-hydroxyquinoline with Zn2+ (ref 29) was transferred from the Cambridge structural data
base and linked at the 2-position of the quinoline ring to a energy-minimized structure of a Quanta-generated adenine-ribose-O(PO2-)OCH2H with
the elimination of H2. For the La3+ complex, the La-O and La-N bond distances in 8-hydroxyquinoline chelation were adjusted to 2.55 and 2.67
Å, respectively. The torsion angles of the adenine-ribose-O(PO2-)OCH2R portion were manipulated to analyze the possible interactions.
Computer molecular modeling of the metal ion complexes
has been carried out with the aid of the known structure of AMP
esters and the X-ray structures of metal ion complexes of
8-hydroxyquinoline (Figure 6). Molecular modeling of the 1:1
complexes of 1a and 1b with divalent metal ions shows that
interaction between the negative charge of the -(PO2-)-
oxygen and the metal ions ligated to the 8-hydroxyquinoline
moiety is favored. This is so regardless of metal ions being
geometry of these metal complexes. The same may be said
about the possibility of pentameric Zn2+ ligating with an oxygen
of >PO2 as well as the leaving oxygen.
-
The situation is quite different in the case of the 1:1
complexes of 1a and 1b with La3+. Molecular modeling of
the 1:1 complex of La3+ with 1a shows the ease in which this
metal can simultaneously interact with both the -(PO2-)- and
leaving oxygen moieties (Figure 6 (bottom)). This is due to
the greater La3+-O bond length (∼2.60 Å)19 and, unlike
transition metal ions, a lack of directionality of bonding by La3+
due to filled d-orbitals.20
tetrahedral Zn2+, octahedral Mg2+, or square planar Cu2+
.
Interaction of metal ions with the leaving oxygen is also possible
for 1:1 complexes of Mg2+ and Cu2+ but not with tetrahedral
Zn2+ (Figure 6 (top)). Simultaneous interaction in the ground
state, however, of Mg2+ or Cu2+ with both >PO2- oxygen and
leaving oxygen is impossible due to the rigid and well-oriented
The spontaneous hydrolysis of the 1:1 complex of 1a, and
probably 1b, with metal ions is first-order in [HO-] (eq 7). The
bimolecular rate constants k0HO (eq 7) are sensitive to the identity

9874 J. Am. Chem. Soc., Vol. 118, No. 41, 1996
Bruice et al.
of the metal ion, varying by a factor of 106; the order of activity
is La3+. Zn2+ > Cu2+ . Mg2+ in hydrolysis of the 1:1 metal
complex of both 1a and 1b (Table 2). In general, Cu2+ is the
most effective Lewis acid catalyst among divalent transition
metal ions in the hydrolysis of carboxylic acid esters21 or
pentavalent phosphorus compounds.22 This is due, at least in
part, to the high association constants of Cu2+ complexes.
7b are greater than the dissociation constant of La3+ with 8.
This observation establishes that the lanthanum of the 8-hy-
droxyquinoline La3+ complexes in 1a and 1b can interact
productively with the leaving oxygen during hydrolysis. Ca-
talysis of hydrolysis of phosphate esters by metal ion-assisted
departure of the leaving oxygen is well documented.23
Rate enhancements for hydrolysis of the phosphodiesters 1a
and 1b brought about by metal ion complexation can be obtained
from the ratios of the rate constants for hydroxide ion hydrolysis
(k0HO) of metal complexed and non complexed esters. The rate
enhancement for hydrolysis of 1a,b by complexation with metal
ions is as follows: ∼105 with Zn2+, ∼103 with Mg2+, ∼105
with Cu2+, and ∼109 with La3+. A rate acceleration of 105 for
Zn2+ complexes is comparable to that proposed by Westheimer1
in Lewis acid catalysis by cancellation of negative charge on
oxygen atom of the phosphodiesters. For the La3+ complexes
the remarkable rate enhancement exceeding 105 results from
the additional interaction of La3+ with the leaving oxygen, as
described above.
Complex stability also explains the usual advantage of Zn2+
,
as a Lewis acid catalyst compared to Mg2+. For spontaneous
hydrolysis of the complexes (1)Mn+, the Zn2+ complexes are
more active than the Mg2+ complexes by a factor of 200 for
both 1a and 1b and the Cu2+ complexes by a factor of 40 for
1a and 10 for 1b. The Zn2+ 1:1 complexes of 1a and 1b are
more susceptible to lyate catalysis of hydrolysis than the
corresponding Cu2+ species by ca. 1 kcal/mol. Rationalization
of differences in ∆Gq amounting to a kcal or less are seldom
assured. In the present case the differences in strain for the
8-hydroxyquinoline-complexed tetrahedral Zn2+ and square
planar Cu2+ reaching the negative oxygen of -(PO2-)- may
be brought into consideration. For Cu2+ the square planar
geometry requires a N-Cu2+-O angle of 90°. The angle and
bond length for interaction of Zn2+ with negative -(PO2-)- is
exactly that required by the tetrahedral metal ion (Figure 6 (top)).
In our designs, we extended the methylene linker between
the phosphate group and leaving oxygen of 1a to an ethylene
linker in 1b to ascertain what would happen if the leaving
oxygen could more closely approach the metal ion. In the
hydrolysis of the metal complexes of 1b, the interaction of the
metal ions with the incipiently negatively charged leaving
oxygen involves a six-membered cyclic transition state. The
values of the rate constants (k0HO) for hydrolysis of (Mn+)1a
and (Mn+)1b differ by less than 1 order of magnitude for any
given Mn+. This shows that there is no significant difference
in the transition states. Metal ion catalysis of hydrolysis of 1a
and 1b is due, therefore, to interaction of metal ion with the
negative charge of the phophodiester. The small reduction of
the reactivity on going from 1a to 1b is probably due to an
increase in the flexibility of the (PO2-)-ligated cyclic 1:1
complex. On addition of a rotatable bond to (1a)Mn+ there is
an extension of the seven-membered ring to the eight-membered
The Mechanism of Hydrolysis of LMn+ Complexes by
M
n+. The complexes (1a)Mn+ and (1b)Mn+ are themselves
hydrolyzed by metal ion catalysis (kM[Mn+] in eq 5). The pH-
rate profile for this catalytic pathway establishes the reaction
to be first-order in [HO-] indicating a transition state composi-
tion of [(1a,b)Mn+][Mn+][HO-]. We assume the kinetic
equivalent [(1a,b)Mn+][Mn+OH] to represent the reacting spe-
HO
cies and derive the rate constants (eq 8) as kM (Table 2).
Values of the rate constants for hydrolysis of (1a)Mn+ and (1b)-
Mn+ are strongly dependent upon the nature of Mn+. Thus,
kMOH for Zn2+(HO)-catalyzed hydrolysis of (1a)Zn2+ and (1b)-
OH
Zn2+ are quite comparable, whereas the values for kM for
catalysis of hydrolysis of (1a)Mg2+ and (1b)Mg2+ by a second
Mg2+ are both barely or not at all detectable. This difference
in catalytic activity of Zn2+ and Mg2+ can be explained by the
difference in the values of pKa for water ligated to each metal
ion; pKa for Zn(H2O)2+ is 9.524 and that for Mg(H2O)2+ is 11.4.25
These results suggest that metal hydroxide is a general base
deprotonating the nucleophilic 2′-OH, while the substrate bound
metal ion associates with the phosphate oxyanion. A combina-
tion of Lewis acid/general base catalysis is known in the
intramolecular transesterification of phosphate esters26
ring of (1b)Mn+
.
In comparison with the divalent metal ions studied, the
In contrast to the similarities in the reactivity of (1a)Mn+ and
(1b)Mn+, when Mn+ represents Zn2+ and Mg2+, the hydrolysis
of the complex (1a)Cu2+ is not catalyzed by a second Cu2+
ligation of La3+ has a more pronounced positive effect on the
HO
rates of hydrolysis of 1a and 1b. The values of k0 for the
hydrolysis of the complexes of 1a and 1b with La3+ are 103
HO
while the value of kM for the reaction of (1b)Cu2+ with a
HO
times greater than k0 of the Zn2+ complexes, whereas the
second Cu2+ is ca. 107 M-2 s-1
.
Hence, some sort of
geometrical feature of the substrate is important in determining
the facility of second metal ion catalysis in the instance of Cu2+
kinetically determined association constants of La3+ complexes
(1a,b)La3+ are smaller than those of the (1a,b)Zn2+ complexes
by a factor of 102. As supported by molecular modeling (Figure
6 (bottom)), this remarkable activity must originate from a
combination of Lewis acid catalysis by coordination to the
phosphate oxyanion and by interaction with the putative charge
on the leaving oxygen in the transition state associated with
departure of the leaving group. The interaction between La3+
and the leaving oxygen can be appreciated from the following
observations. The association constants of La3+ with 7a and
.
It has been known that Cu(HO)+ exists in polymeric species27
at neutral pH due to a pKa value (7.22)28 considerably lower
than those for other metal ions tested here. Accordingly, the
concentration of monomeric copper hydroxide Cu(HO)+ is
decreased below that anticipated from the reported value of pKa
(23) (a) Murakami, Y.; Takagi, M. J. Am. Chem. Soc. 1969, 91, 5131.
(b) Murakami, Y.; Sunamoto, J. Bull. Chem. Soc. Jpn. 1971, 44, 1827. (c)
Fife, T. H.; Pujari, M. P. J. Am. Chem. Soc. 1988, 110, 7790. (d) De Rosch,
M. A.; Trogler, W. C. Inorg. Chem. 1990, 29, 2409.
(24) Rabenstein, D. L.; Blakney, G. Inorg. Chem. 1973, 12, 128.
(25) Basolo, F.; Pearson, R. G. Mechanisms of Inorganic Reactions;
Wiley; New York, 1967.
(19) Guerriero, P.; Casellato, U.; Sitran, S.; Vigato, P. A.; Graziani, R.
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Metal Ion Catalysis of RNA Hydrolysis
J. Am. Chem. Soc., Vol. 118, No. 41, 1996 9875
(7.22) of H2O ligated to Cu2+ at a given pH. On the assumption
that the catalytic species is Cu(HO)+, therefore, the determined
values of kMHO would be incorrectly small. On the other hand,
the actual catalyst may well be the polymeric species of copper
hydroxide. If so, the difference in catalytic activity of Cu2+ in
the hydrolysis of (1a)Cu+ and (1b)Cu+ might be explained by
the structure of a cupric oxide gel? Because of the facility of
La3+-catalyzed hydrolysis of 1a,b we were not able to inves-
proposed that magnesium hydroxide acts as a base to deproto-
nate the 2′-OH group at a ribose ring for nucleophilic attack on
the phosphorus atom. Another Mg2+ coordinates both to the
negative charge on -(PO2-)- and to the leaving oxygen such
that this Mg2+ facilitates both 2′-O- attack and departure of
the leaving oxygen.6 This two-metal ion catalysis, which is a
combination of Lewis acid/general base catalysis, is essentially
identical to our proposed mechanism for the two La3+-mediated
tigate the dependence of kobsd over a sufficient range of [La3+
]
hydrolysis of (1)M2+
.
HO
to allow comparison of kM (Table 2) for (1a)La2+ and (1b)-
La2+. La3+ catalysis is only reported for (1a)La2+
.
Acknowledgment. We thank the office of Naval Research
and the National Institutes of Health for financial support.
Biological Implications. A two-metal ion mechanism has
recently been proposed6a for ribozyme-mediated hydrolysis of
RNA. In the instance of hammerhead ribozyme, it has been
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