Intramolecular acid-base catalysis of a phosphate diester: Modeling the ribonuclease mechanism

Article abstract of DOI:10.1021/ja710693x

The hydrolysis of the phosphate diester bis(2-(1-methyl-1H-imidazolyl)phenyl) phosphate BMIPP was evaluated as an intramolecular model for the ribonuclease A mechanism. Kinetic and thermodynamic data, isotope effects and ESI-MS and ESI-MS/MS analysis supp

Full text of DOI:10.1021/ja710693x

Published on Web 02/05/2008
Intramolecular Acid-Base Catalysis of a Phosphate Diester: Modeling the
Ribonuclease Mechanism
Elisa S. Orth,† Tiago A. S. Branda˜o,† Humberto M.S. Milagre,‡ Marcos N. Eberlin,‡ and
Faruk Nome*,†
UniVersidade Federal de Santa Catarina, Floriano´polis, SC 88040-900, Brazil. ThoMSon Mass Spectrometry
Laboratory, Institute of Chemistry, State UniVersity of Campinas, Campinas, SP, Brazil
Received November 29, 2007; E-mail: faruk@qmc.ufsc.br
Scheme 1
The mechanism of ribonuclease A (RNase A) activity has been
widely studied by evaluating different aspects such as roles of the
catalytic amino acids, of different substrates, of thio effects, of
organic solvents, and pH and temperature dependences.1 The usually
accepted mechanism is the general acid-base pathway where a
deprotonated imidazole group acts as a general base and another
protonated imidazole group acts as a general acid interacting with
the leaving group. This “classical” mechanism has been questioned
and a triester-like route has been considered with protonation of a
nonbridging phosphoryl oxygen leading to a transition state
resembling those typical of phosphate triester reactions.2 Many
bifunctional mimics have been studied, including oligonucleotides
with imidazole residues3 and cyclodextrins with imidazole groups
attached to the sugar residues.4 In the particular case of cyclodextrin
mimics of RNase A, their catalytic efficiency is attributed not only
to the imidazole groups but also to their hydrophobic binding to
the substrate,5 although the importance of the hydrophobic effects
has been questioned, because catalytic effects are small for reactions
of a variety of substrates with polymers with hydrophobic centers.6
Although all these models resemble some aspects of RNase A
reactions, they do not support fully the classical mechanism. We
report the hydrolysis kinetics of a phosphate diester, (bis(2-(1-
ESI-MS/MS data support the reaction pathway as depicted in
Scheme 1.
methyl-1H-imidazolyl)phenyl) phosphate) BMIPP, which bears two
imidazole groups that may potentially act as the general acid-
base catalysts conserved in the active site of RNase A (Scheme 1).
To probe the mechanism of BMIPP hydrolysis, we used our
knowledge in similar7 and related8 studies and applied ESI-MS-
(/MS) in the negative ion mode to monitor the course of reaction.
Reagents, intermediates, and products in anionic forms were trans-
ferred directly from the reaction solution to the gas phase, detected
by ESI-MS, and then characterized by ESI-MS/MS via their
unimolecular dissociation chemistry. After 25 min of hydrolysis
of BMIPP in aqueous solution at pH 6.5 and 60 °C, a character-
istic ESI-MS (Figure 1) was recorded. In this spectrum, a series of
major anions are detected and identified as the anionic form of the
reactant BMIPP of m/z 409, the monoester Me-IMPP of m/z 253,
and the final phenolic product IMP of m/z 173, as well as inorganic
Kinetics as a function of pH are shown in Figure 4, and reactions
were followed spectrophotometrically at 290 nm with 7.0 × 10-5
M BMIPP. Figure 4 shows a bell-shaped pH-rate profile indicating
bifunctional catalysis with a rate maximum at pH 6.5-6.8, where
one imidazole group is protonated and another is deprotonated. The
pH profile indicates that the monocationic (BMIPPH+) and
monoanionic species (BMIPP-) are unreactive under our experi-
mental conditions and hydrolysis of Me-IMPP- is sufficiently slow
that it is the product at pH g 4 (Scheme 1).
The data in Figure 4 were fitted with eq 1, derived from Scheme
1, giving kzd ) 1.98 × 10-3 s-1 for reaction of the zwitterionic
species, with kinetic pKa values of 6.10 and 7.20 at 60 °C.
kobs ) kzdøBMIPP
(1)
-
-
P in the form of PO3 of m/z 79 and H2PO4 of m/z 97. Note the
accurate mass measurements that corroborate the composition
assignments. ESI-MS/MS was then used to characterize these im-
portant species via collision-induced dissociation. The resulting
tandem mass spectra also supported the identifications: the Me-
IMPP anion of m/z 253 is found to dissociate nearly exclusively to
PO3- of m/z 79 (Figure 2), deprotonated IMP of m/z 173 dissociates
mainly by two routes that lead either to the fragment ion of m/z
158 by the loss of a methyl radical or the fragment of m/z 118
by likely the loss of 1-methyl-1H-azirine (C3H5N) thus forming
the 2-cyanophenoxy anion (Figure 3). Therefore, ESI-MS and
Titration measurements at 25 °C confirmed this assumption, with
pKa values of 6.2 and 7.8 for the acid dissociation of BMIPPH+-
† Universidade Federal de Santa Catarina.
‡ State University of Campinas.
Figure 1. ESI-MS after 25 min of hydrolysis of BMIPP in aqueous solution
at pH 6.5 and 60 °C.
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10.1021/ja710693x CCC: $40.75 © 2008 American Chemical Society

C O M M U N I C A T I O N S
lecular general acid catalysis by the dimethylammonium center in
8-dimethylamino-1-naphthyl phosphates 2a and 2b, the hydrolysis
of the monoester 2a is 18 times faster than that of the diester 2b.10
Figure 2. ESI-MS/MS of the Me-IMPP anion of m/z 253.
Conversely, hydrolysis of the BMIPP diester is ∼540 times more
reactive than the zwitterionic Me-IMPP monoester. The result
indicates important intramolecular general base catalysis as shown
in Scheme 2. Similarly to the reaction of 2a, we have no evidence
to implicate a pentacovalent intermediate, and any such species
would be very short-lived.10
The intramolecular catalytic efficiency of BMIPP is even more
significant in comparisons with reactions involving cleavage of
RNA and various derivatives by imidazole buffers that show
enhancements of up to 3-fold,11b or the intramolecular hydrolysis
of bis-(2-carboxyphenyl) phosphate, where was observed an
unexpectedly low general acid catalysis, of only 4-fold.11c
In BMIPP, the distance between proton-accepting and -donating
nitrogen atoms is ca. 7 Å (corresponding distances in RNase are
∼6.5 Å). Molecular models of the ground state of BMIPP show
that the general acid is hydrogen bonded to both, the aryl oxygen
and to one of the negatively charged nonbridging phosphoester
oxygens. This initial state is consistent with reports of Anslyn et
al.12 for the guanidinium substituent as intramolecular general-acid
catalyst in phosphoryl transfer reactions and observed a 40-fold
rate enhancement relative to a reaction with proton transfer to the
nonbridging phosphoester oxygen. The catalytic effect in BMIPP
results from the combination of (i) favorable activation of a water
molecule by general base catalysis and (ii) the concerted proton
transfer from the general acid catalyst to the bridge oxygen atom,
which requires simultaneous rotation of the imidazolium to approach
planarity with the phenyl ring in the transition state. The detailed
mechanism of the observed effect will be discussed in our full paper
including density functional theory calculations.
Figure 3. ESI-MS/MS of deprotonated IMP of m/z 173.
Figure 4. pH-rate profiles for the hydrolysis of BMIPP (2) and Me-
IMPP (9), at 60 °C, µ ) 1.0 (KCl). The solid lines for BMIPP and Me-
IMPP represent fits with eqs 1 and 2, respectively. The black solid line is
the fit for hydrolysis of 2-(2′-imidazolium)phenyl phosphate (IMPP).9
Scheme 2. Mechanism Proposed for the Hydrolysis of BMIPP
Acknowledgment. We thank the Brazilian foundations CNPq,
FAPESC, and FAPESP for financial assistance.
Supporting Information Available: Synthesis and characterization
of bis(2-(1-methyl-1H-imidazolyl)phenyl)phosphate, BMIPP; tables of
kinetic data. This material is available free of charge via the Internet
at http://pubs.acs.org.
and BMIPP, respectively. The hydrolysis of BMIPPH+ is slower
than the hydrolysis of the zwitterionic form of Me-IMPP, and the
data for hydrolysis of the monoester in Figure 4 were fitted with
eq 2, giving kzm ) 3.65 × 10-6 s-1 and pKa ) 4.52. The calculated
rate and acid dissociation constant are similar to those reported for
the hydrolysis of 2-(2′imidazolium)phenyl phosphate (IMPP, solid
line in Figure 4), indicating that the methyl group decreases slightly
the catalytic efficiency of the imidazolium group.9
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kobs ) kzmøMe-IMPP
(2)
The observed isotope kinetic effect, kH2O/kD2O, was 1.42, consistent
with proton transfer in transition state formation and similar with
values reported for other intramolecularly catalyzed phosphodiester
hydrolysis reactions.9,10 The activation parameters ∆Hq )19.0 (
1.2 kcal/mol and ∆Sq ) -9.05 ( 0.5 eu calculated for the
zwitterionic species of BMIPP, at pH 5.5-7.0 at each temperature,
indicate a bimolecular hydrolysis mechanism, involving a water
molecule in the transition state.9
The hydrolysis of BMIPP is considerably faster by a factor of
106, than those of diphenyl phosphates with leaving groups of
similar pKa (∼7.85),11a which is attributed to intramolecular general
acid-base catalysis. It is important to notice that in the intramo-
(12) Anslyn, E. V.; Piatek, A. M.; Gray, M. J. Am. Chem. Soc. 2004, 126,
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