Kinetic solvent deuterium isotope effect in transesterification of RNA models

Article abstract of DOI:10.1002/poc.883

2-Methylbenzimidazole ribonucleoside arylphosphates (1a,b) and alkylphosphates (2a,b) were synthesized as RNA model compounds containing a minimised number of exchangeable protons. Intramolecular transesterification of these substrates was studied in H₂O and D²O solutions over a wide pL range and apparent kinetic solvent deuterium isotope effects of the alkaline cleavage of both substrates and of the cleavage and isomerisation of 2a under neutral and acidic conditions were determined. The observed K _H2O/k_D2O of 4.9 obtained for the alkaline cleavage of the arylphosphate 1b can be primarily attributed to the ΔpK of the attacking nucleophile. The alkyl leaving group in 2a brings about an additional 1.5-fold isotope effect (k_H2O/k_D2O of 7.1 observed), which, considering the pL-dependence of the reaction, can not be explained by a process involving a proton transfer. Differences in solvation of the transition state are tentatively suggested as a source of the difference. In contrast to alkaline cleavage, under neutral and acidic conditions the cleavage and isomerisation of 2a showed no apparent solvent isotope effect. Several examples found in the literature show that intramolecular proton transfer from phosphate to the leaving group in pre-equilibria may not necessarily result in an observable solvent isotope effect. This may also explain the results obtained in the present work, since intramolecular proton transfer processes take place in transesterification reactions of 2a under neutral and acidic conditions. Relevance of the results obtained in the base catalysed cleavage to hammerhead ribozyme reaction is briefly discussed. Copyright

Full text of DOI:10.1002/poc.883

JOURNAL OF PHYSICAL ORGANIC CHEMISTRY
J. Phys. Org. Chem. 2005; 18: 385–397
Published online 20 December 2004 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/poc.883
Kinetic solvent deuterium isotope effect
in transesterification of RNA models
Noora Virtanen, Lauri Polari, Maria Va¨lila¨ and Satu Mikkola*
Department of Chemistry, University of Turku, FIN-20014 Turku, Finland
Received 25 June 2004; revised 28 September 2004; accepted 30 September 2004
ABSTRACT: 2-Methylbenzimidazole ribonucleoside arylphosphates (1a,b) and alkylphosphates (2a,b) were
synthesized as RNA model compounds containing a minimised number of exchangeable protons. Intramolecular
transesterification of these substrates was studied in H2O and D2O solutions over a wide pL range and apparent kinetic
solvent deuterium isotope effects of the alkaline cleavage of both substrates and of the cleavage and isomerisation of
2a under neutral and acidic conditions were determined. The observed kH O/kD O of 4.9 obtained for the alkaline
2
2
cleavage of the arylphosphate 1b can be primarily attributed to the ÁpK of the attacking nucleophile. The alkyl
leaving group in 2a brings about an additional 1.5-fold isotope effect (kH O/kD O of 7.1 observed), which, considering
2
2
the pL–dependence of the reaction, can not be explained by a process involving a proton transfer. Differences in
solvation of the transition state are tentatively suggested as a source of the difference. In contrast to alkaline cleavage,
under neutral and acidic conditions the cleavage and isomerisation of 2a showed no apparent solvent isotope effect.
Several examples found in the literature show that intramolecular proton transfer from phosphate to the leaving group
in pre-equilibria may not necessarily result in an observable solvent isotope effect. This may also explain the results
obtained in the present work, since intramolecular proton transfer processes take place in transesterification reactions
of 2a under neutral and acidic conditions. Relevance of the results obtained in the base catalysed cleavage to
hammerhead ribozyme reaction is briefly discussed. Copyright # 2004 John Wiley & Sons, Ltd.
Supplementary electronic material for this paper is available in Wiley Interscience at http://www.interscience.
wiley.com/jpages/0894-3230/suppmat/
KEYWORDS: RNA; phosphodiester bonds; transesterification; solvent isotope effect
INTRODUCTION
neutral conditions, a phosphorane intermediate is formed,
and isomerization of the natural 30,50-phosphodiester bond
to a 20,50-bond competes with cleavage (Scheme 1).
Under alkaline conditions the situation is not as clear.
Base-catalysed isomerization of the phosphodiester
bonds is not observed, so the reaction is believed to
proceed via a dianionic phosphorane-like transition state
or a marginally stable intermediate (Scheme 2). However,
a stepwise mechanism has also been recently proposed.4
Irrespective of the exact status of the dianionic species,
the departure of the alkyl leaving group takes place in the
rate-limiting step of the reaction.
The kinetic solvent deuterium isotope effect is a tool
utilized in mechanistic studies,5 which has been widely
applied to study phosphate transfer.6–8 Information on the
proton transfer processes in or before the rate-limiting
step of the reaction can be obtained, and the role of
solvent molecules as nucleophiles or general acid–base
catalysts can be deduced by comparing the rates of the
reaction in H2O and D2O solutions. Medium effects can
also contribute to the observed k(H2O)/k(D2O) values.5
Results of kinetic solvent deuterium isotope effect ex-
periments carried out with simple phosphate esters6 and
with ribozymes7 have been reported over the last few
decades. The mechanism of the metal ion-promoted
The mechanism of the cleavage of phosphodiesters has
been extensively studied1 ever since it was discovered
that DNA and RNA are polymers made up of nucleoside
units linked by phosphodiester bonds. Studies on the
cleavage of simple phosphodiesters in vitro have been
carried out to help to understand the mechanisms utilized
by enzymes involved in phosphate transfer processes.
More recently, the mechanism of autocatalytic cleavage
observed with ribozymes has attracted wide interest.2
Studies with RNA models have also been carried out to
define the parameters required of efficient catalysts for
RNA cleavage to develop artificial nucleases,3 chemical
catalysts that are hoped to permit the selective and
efficient cleavage of intracellular RNA molecules.
The phosphodiester bonds in RNA react via an intra-
molecular transesterification, where the 20-OH group
attacks the phosphate, resulting in the formation of a
pentacoordinated species.1c–e The status of this species
depends on the reaction conditions. Under acidic and
*Correspondence to: S. Mikkola, Department of Chemistry, University
of Turku, FIN-20014 Turku, Finland.
E-mail: satu.mikkola@utu.fi
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386
N. VIRTANEN ET AL.
We have therefore started a systematic study on the
kinetic solvent deuterium isotope effects in the transes-
terification of phosphodiester bonds of RNA. The under-
lying idea is to start the experiments using model systems
where the number of exchangeable protons has been
minimized so that any effects observed can be attributed
to only a few proton transfer processes, and then increase
the number of possible protolytic processes. As the first
part of the project, we report here the synthesis of model
compounds 1a,b [1-(50-O-methyl-ꢀ-D-ribofuranosyl)-2-
methylbenzimidazole 20- and 30-phenylphosphates] and
2a,b [1-(50-O-methyl-ꢀ-D-ribofuranosyl)-2-methylbenzi-
midazole 20-and 30-(2-methoxyethyl)phosphates] and the
results of studies on their intramolecular transesterifica-
tion reactions (Scheme 3) in H2O and D2O solutions over
a wide pL range. The 2-methylbenzimidazole base moi-
ety in 1 and 2 contains no exchangeable protons and the
proton transfer between the 50-OH and solvent has been
prevented by methylation. Compound 1 contains a good
leaving group, which is known to depart as an aryloxy ion
under neutral and alkaline conditions9 and, therefore, the
deprotonation of the 20-OH group is the only proton
transfer process involved in the alkaline cleavage. The
leaving group in 2 is poorer and its departure clearly
limits the observed reactivity.10 Under neutral and acidic
conditions, the alkyloxy leaving group of 2 becomes
protonated on departure. Furthermore, 2 also isomerises
under neutral and acidic conditions (Scheme 3), and the
kinetic solvent deuterium isotope effect on this reaction
was also determined. The mechanisms of the intramole-
cular transesterification reactions of RNA model com-
pounds are known and, therefore, the solvent isotope
effects obtained can be attributed to different acid- and
base-dependent reaction steps. The data obtained there-
fore provide useful background information to interpret
solvent isotope effects obtained with more complicated
systems such as metal ion-promoted, ribozyme and en-
zyme-catalysed reactions.
Scheme 1
Scheme 2
cleavage of phosphodiesters has also been studied by
applying kinetic solvent deuterium isotope effects.8
Although the solvent isotope effect is a powerful tool in
mechanistic studies, the results may be difficult to inter-
pret. With a simple well-defined system the interpretation
is often straightforward and unambiguous, but as the
systems studied grow more complicated, the number of
plausible mechanistic alternatives increase. The cleavage
of phosphodiester bonds of RNA is a very complicated
system owing to the number of potential ionic forms of
the ground and transition states. There are also a number
of exchangeable protons in the heterocyclic nucleic acid
bases, which may contribute to the overall isotope effect
observed. The situation becomes even more complicated
when reactions are carried out in buffer solutions and/or
in the presence of metal ions, which add to the number of
protolytic equilibria and proton transfer processes in the
reaction system.
RESULTS
Synthesis of 1a,b and 2a,b
Compounds 1a,b and 2a,b were synthesized by phos-
phorylation of protected 2-methylbenzimidazole nucleo-
sides 6a,b. The synthetic routes are shown in Schemes 4–
6 and a detailed description of the synthetic procedures is
given as electronic supplementary material (available in
Wiley Interscience). Two different routes were followed.
Initially, 6a,b were prepared from D-( ꢀ ) ribose, which
was cyclized in a reaction with benzyl alcohol in 1% HCl
solution to give 7. NMR analysis showed that the product
was predominantly in the ꢀ-configuration. The 2- and 3-
hydroxyl groups were protected with an isopropylidene
group and the 5-hydroxyl was methylated with methyl
iodide. The benzyl and 2,3-isopropylidene groups were
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SOLVENT ISOTOPE EFFECT IN RNA MODEL TRANSESTERIFICATION
387
Scheme 3
removed with trifluoroacetic acid. The free hydroxyl
groups were then acetylated with acetic anhydride in
pyridine and the fully protected sugar was reacted with
trimethylsilyl-protected 2-methylbenzimidazole (13)
using SnCl4 as a catalyst. After removal of the 20- and
30-acetyl protecting groups with NaOMe in methanol, the
nucleoside was reacted with TBDMSCl in pyridine with
diaminopyridine as a catalyst, which gave a mixture of 20-
and 30- protected nucleosides 6a,b. This route was initi-
ally chosen because all the reagents are inexpensive and
the methods well documented in the literature. However,
it is tedious owing to the large number of steps and the
overall yield was low. Therefore, an alternative route was
used to prepare another batch of the protected nucleoside,
and 6a,b were synthesized starting from 1-O-acetyl-
2,3,5-tri-O-benzoyl-ꢀ-D-ribofuranose (16), which was
reacted with silylated 2-methylbenzimidazole base 13
to prepare the corresponding nucleoside 17. Hydrolysis
of 17 in 0.4 M sodium methoxide in methanol yielded 18,
which was protected with a 20,30-isopropylidene group
and methylated at 50-OH. Acid-catalysed hydrolysis of
the isopropylidene group of 20 gave nucleoside 15, which
was reacted with TBDMSCl using Ag2NO3 as a catalyst
to give 6a,b.
Isomers 6a and 6b could not be separated by silica gel
chromatography so the phosphorylation reactions were
carried out with the isomer mixture. Compounds 1a,b
were prepared by phosphorylating 6a,b with phenyl
phosphorodichloridate using triazole as an activator
(Scheme 5). The TBDMS protecting group was removed
with tetrabutylammonium fluoride in anhydrous THF.
The deprotected phosphodiesters were purified by semi-
preparative reversed-phase high-performance liquid
chromatography (RP-HPLC). Compounds 2a,b were pre-
pared by phosphorylation of 6a,b with 2-cyanoethyl
N,N,N0,N0-tetraisopropylphosphoramidite in anhydrous
acetonitrile with tetrazole as an activator (Scheme 6).
Compounds 22a,b were reacted with anhydrous 2-meth-
oxyethanol in the presence of tetrazole under an N2
atmosphere to give 23a,b. The phosphoramidite group
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388
N. VIRTANEN ET AL.
Scheme 4
1
in the H NMR spectra. In the case of 1b this was also
was oxidized with iodine in a mixture of lutidine, water
and THF, the cyanoethyl group removed in methanolic
ammonia and the TBDMS group removed with tetrabu-
tylammonium fluoride. The products were purified by
RP-HPLC.
verified by the proton–phosphorus correlation spectrum
that showed coupling between the 20-H and phosphate. As
both isomers of the TBDMS protected nucleoside 6a,b
were present, it appears that the 20-isomer 6a is less
reactive in phosphorylation reactions than its 30-isomer
6b. In fact, practically no 30-isomer 2a was formed at all
and the yield of 1a was very low. Compound 2a was
prepared by isomerizing 2b in 0.1 M MES buffer at pH
5.5. Under these conditions the isomerization is the
predominant reaction of ribonucleoside alkylphospha-
tes,1d and the 30-isomer accumulates to a significant
The final products were analysed by high-resolution
1
mass spectrometry (HRMS) and H, 13C and 31P NMR
spectroscopy. Assignation of signals was supported by
proton–proton and proton–carbon correlation spectra.
The spectral data showed that in both cases the 20-isomer
was the predominant product. Clear splitting of the 20-H
signal due to the coupling with phosphate was observed
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SOLVENT ISOTOPE EFFECT IN RNA MODEL TRANSESTERIFICATION
389
Scheme 5
extent. This method could not be used to obtain 1a, since
the cleavage of ribonucleoside arylphosphates predomi-
nates over a wide pH range, and isomerization is only
observed under acidic conditions.11 For this reason, the
cleavage of 2-methylbenzimidazole arylphosphates was
studied with the 20-isomer 1b as the substrate, whereas
the 30-isomer 2a was used in kinetic studies of the
reactions of 2-methylbenzimidazole alkylphosphates.
Kinetic experiments
Cleavage of 1b was followed over a pL range of 7–13 and
the reactions of 2a from pL 1 to 13. Reactions of 2a and
the cleavage of 1b below pL 11 were followed by taking
aliquots from a reaction solution that was thermostated to
the appropriate temperature using a water bath. As is
explained in more detail in the Experimental section, the
aliquots were analysed by using RP-HPLC with a mixture
of acetic acid buffer and acetonitrile as an eluent. UV
detection at 245 nm was employed. At pL > 11 the
cleavage of 1b was too fast for HPLC analysis, and
the reaction was followed by UV spectrophotometry.
The reaction was carried out in the spectrophotometer
cell thermostated at 25.0 ꢁC and the absorbance at 287 nm
was recorded. At this wavelength the 2-methylbenzimi-
dazole base absorbs weakly, and the increase in absor-
bance due to the release of the phenolate product is
readily detected.
Scheme 6
formation of 3 and methoxyethanol, which, being non-
chromophoric, was not detected in the HPLC analysis.
Consistent with previous reports on the cleavage of
nucleoside alkyl phosphates,10–13 the cleavage of 2a is
slower than the subsequent hydrolysis of the cyclic
phosphate product 3, and 20- and 30-monophosphates of
2-methylbenzimidazole nucleoside (4a,b) were detected
The cleavage of 1b resulted in the formation of the
20,30-cyclic monophosphate of 2-methylbenzimidazole
nucleoside (3) and phenol (Scheme 3). Both products
were detected by HPLC and identified by spiking with
authentic samples. The cleavage of 2a resulted in the
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390
N. VIRTANEN ET AL.
Figure 1. pL–rate profiles of the cleavage of 1b to 3 and
phenolate at 25 ꢁC and I ¼ 1.0 M. Open circles, cleavage in
H2O solutions; closed circles, cleavage in D2O solutions
Scheme 7
assumption was verified by following the reaction spec-
trophotometrically at pH 3, where all the three processes
contribute to the overall disappearance of the starting
material. Less than 10% decrease in the total absorbance
at 250 nm was observed during three half-lives of the
reaction. As discussed below, the rate constants for
cleavage, isomerization and depurination match those
reported for similar substrates, which shows that the
calculation methods are correct.
The observed rate constants for the cleavage of 1b and
the cleavage and isomerization of 2a as a function of pL
are shown in Figs 1, 2(a) and 2(b), respectively. The data
in Fig. 1 were determined at 25 ꢁC and those in Fig. 2(a)
and (b) at 90 ꢁC. The observed rate constants of the
cleavage and isomerization of 2a in Fig. 2(a) and (b)
were fitted to Eqns (1) and (2), respectively.12
as the reaction products. Under neutral and acidic condi-
tions the monophosphate products were further depho-
sphorylated to yield the corresponding nucleoside 5.
Below pL 9, isomerization of 2a to its 20-isomer 2b
competes with the cleavage, and between pL 5 and 7
it is the faster process of the two, similarly to the reaction
of other ribonucleoside alkylphosphates.10–13 Compound
1b does not isomerize under neutral or slightly acidic
conditions. As was mentioned above, isomerization of
aryl phosphates has only been observed below pH 2.14
Acid-catalysed depurination of the 2-methylbenzimida-
zole nucleoside in 2a was detected between pL 2 and 7,
and 2-methylbenzimidazole 26 was formed as a product
(Scheme 7), consistent with results obtained previously
with benzimidazole nucleosides.15
2
kcobs ¼ ðka½Hþꢂ =Ka;1 þ kb½Hþꢂ=
Calculation of rate constants
Ka;1 þ kc þ kdKa;2=½HþꢂÞ=
ð1Þ
ð½Hþꢂ=Ka;1 þ 1 þ Ka;2=½HþꢂÞ
Rate constants for the cleavage of 1b between pL 7 and
11 were calculated from the decrease of the signal area of
1b in the chromatograms, and above pL 11 from the
increase of the absorbance at 287 nm detected spectro-
photometrically, by applying the integrated first-order
rate law. In the spectrophotometric analysis, the reaction
was followed for 5–10 half-lives to obtain the final
absorbance value. Rate constants for the cleavage and
isomerization of 2a were calculated on the basis of mole
fraction of the substrate remaining in the reaction solu-
tion using the Ufit program as described previously.16 In
cases where depurination was observed, the rate con-
stants of cleavage obtained from Ufit were divided into
contributions from phosphate cleavage and depurination
by using the ratio of the products formed. The mole
fractions based on signal areas were used as the basis of
the calculations, since the chromophoric group is the
same in the substrate and products, and the molar
absorptivity is not expected to vary significantly. This
2
kiobs ¼ ðke½Hþꢂ =Ka;1 þ kf½Hþꢂ=Ka;1 þ kgÞ=
ð2Þ
ð1 þ ½Hþꢂ=Ka;1Þ
In Eqn (1), kcobs, ka, kb, kc and kd are the observed first-
order rate constant for the cleavage of 2a, the second-
order rate constant for the acid-catalysed cleavage of
neutral 2a and the first-order rate constants for the
cleavage of neutral, monoanionic and dianionic form of
2a. In Eqn (2), kiobs, ke, kf and kg are the observed first-
order rate constant for isomerization of 2a to 2b, the
second-order rate constant for the acid-catalysed isomer-
ization of neutral 2a and the first-order rate constants for
isomerization of neutral and monoanionic form of 2a. Ka,1
and Ka,2 are the equilibrium constants for deprotonation of
the phosphate and 20-hydroxy groups, respectively.
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SOLVENT ISOTOPE EFFECT IN RNA MODEL TRANSESTERIFICATION
391
Figure 2. (a) pL–rate profiles of the cleavage of 2a to 3 and methoxyethanol at 90 ꢁC and I ¼ 1.0 M. Open circles, cleavage in
H2O solutions; closed circles, cleavage in D2O solutions. (b). pL–rate profiles of the isomerization of 2a to 2b at 90 ꢁC and
I ¼ 1.0 M. Open circles, isomerization in H2O solutions; closed circles, isomerization in D2O solutions
Adjusting the pL of the reaction solutions
tions are independent of the buffer used, suggest that the
pK values calculated are reliable.
Under acidic conditions, pL was adjusted with HCl or
DCl, and under alkaline conditions with NaOH or NaOD.
Between pL 3 and 10 the lyonium ion concentration was
maintained with an appropriate buffer. Varying the buffer
concentration did not have any effect on the observed rate
constants, and the rate constants refer to a total buffer
concentration of 0.1 M. The ionic strength was maintained
at 1.0 M with NaCl or NaNO3. The pL values under the
experimental conditions were calculated using the data
available for the effects of temperature and ionic strength
on the pKa values of the buffer acids and on the water
autoprotolysis constant pKw in H2O and D2O. The
calculations performed are explained in detail in the
Experimental section and the pKa and pKw values used
are given in Table 1. Even though in some cases the
calculation of the pK values is based on rather extensive
extrapolation, the facts that the rate constants obtained in
different buffers are consistent with each other, both in
H2O and D2O, with those reported before for other
ribonucleoside alkylphosphates, and that the solvent iso-
tope effects calculated under acidic and neutral condi-
DISCUSSION
The rate constants shown in Figs 1, 2(a) and 2(b) are
consistent with previous data on the reactivity of ribonu-
cleoside aryl and alkyl phosphates. The pL–rate profiles
of the cleavage and isomerization of 2a are similar to
those obtained previously with 30,50-UpU12 and with
adenosine11 and uridine10,13 30-alkylphosphates. The
cleavage of 2a shows a first-order dependence on hydro-
xide ion concentration above pL 7 and on hydronium ion
concentration below pL 4. Between pL 1 and 2, a second-
order dependence on hydronium ion concentration is
observed. Consistent with previous reports, isomerization
is not base-catalysed, but this reaction is independent of
pL above pL 5. Above pL 9, the cleavage is much faster
than isomerization and the rate constants for isomerisa-
tion cannot be obtained. The cleavage of 1b is much
faster owing to the presence of a good aryloxy leaving
Table 1. pKa values of buffers and values of autoprotolysis
constants of water used to calculate the pL values of
reaction solution (all values refer to I ¼ 1.0 M)
H2O,
H2O,
D2O,
D2O,
Parameter
25 ꢁCa
90 ꢁCa
25 ꢁCa
90 ꢁCa
pKa formate
pKa acetate
pKa MOBS
pKa HEPES
pKa CHES
pKa glycine
pKa Et3N
3.74
4.67
6.82
4.13
5.19
7.44
7.45
9.75
8.07
10.43
8.33
8.96
10.99
13.727
11.58
14.683
pKw
12.102
12.984
Figure 3. pL–rate profiles of the depurination of 2a at 90 ꢁC
and I ¼ 1.0 M. Open circles, depurination in H2O solutions;
closed circles, depurination in D2O solutions
a
References and detailed descriptions of the calculations performed can be
found in the text.
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392
N. VIRTANEN ET AL.
group, and isomerization of this compound cannot be
detected under the experimental conditions.9,14,17 The
rate constants of acid-catalysed depurination of 2a
(Fig. 3) are fully consistent with data on the depurination
of other benzimidazole nucleosides.15 The observed rate
constants can be fitted to a reaction scheme where the
only active ionic form is the protonated base. The kinetic
pKa is 4.6, which in good agreement with the pKa of 4.0
at 393 K.15
and 1.04 with dialkyl 2-carboxyphenyl phosphates. If the
OH(HOꢀ)/kOH(DOꢀ) value of 4.9 is completely attrib-
k
uted to the deprotonation of the attacking nucleophile, a
ÁpKa of 0.69 can be calculated, which is consistent with
the ÁpKa value of 0.63 estimated for the cis-hydroxyl
group of cis-4-hydroxytetrahydrofuran 3-phosphate.6c
The alkaline cleavage of nucleoside alkylphosphates
differs from the cleavage of corresponding arylpho-
sphates in that the departure of the leaving group more
clearly determines the rate of the reaction. The ꢀlg values
of the alkaline cleavage of uridine 30-aryl and alkylpho-
sphates are ꢀ0.549 and ꢀ1.10,10 respectively. The highly
negative value obtained with alkylphosphates has been
suggested to support a mechanism where the departure of
the alkyloxy leaving group is far advanced in the transi-
tion state.1e,10 In contrast, the modestly negative ꢀ values
obtained with uridine 30-arylphosphates are consistent
with a reaction proceeding through a symmetric transi-
tion state with bond formation to the incoming nucleo-
phile and the bond cleavage of departing aryloxyion
being similarly advanced.9 Alternatively, these data
have been interpreted in terms of a change in rate-limiting
step, with the formation of a dianionic phosphorane rate
limiting for aryloxy leaving groups and its breakdown for
alkoxy leaving groups.4 In either case, the transition
states will be substantially different and the mechanistic
interpretation does not change the conclusions about the
origin of the solvent isotope effects observed. As the
solvent isotope effect on the pre-equilibrium deprotona-
tion of the attacking OH group can be assumed to be
similar in 1b and 2a, the difference in the apparent
Solvent isotope effects on the alkaline
cleavage of 1b and 2a
The solvent isotope effects on the alkaline cleavage of 1b
and 2a were first calculated on the basis of the second-
order rate constants of hydroxide/deuteroxide ion-catalys-
ed cleavage (kOL) obtained fromthe slopes of kobs vs [LOꢀ]
plots using the linear part of the pL–rate profiles above
pL 7. The kOH(HOꢀ) and kOD(DOꢀ) values for the cleav-
age of 1b were 10.4 ꢃ 0.2 and 2.1 ꢃ 0.1 mol dmꢀ3 sꢀ1
;
¼
for the cleavage of 2a, kOH ¼ 1.61 ꢃ 0.03 and kOD
0.22 ꢃ 0.01 mol dmꢀ3 sꢀ1. These data give solvent iso-
tope effects of 4.9 ꢃ 0.2 for 1b and 7.2 ꢃ 0.2 for 2a.
The value obtained with 1b is reasonably consistent with
the value of 4.01 reported for the alkaline cleavage of
2-hydroxypropyl-p-nitrophenyl phosphate.8d As the alka-
line cleavage of RNA involves deprotonation of the
nucleophilic OH group in a pre-equilibrium before attack
at the phosphate, the values obtained consist of contribu-
tions from solvent isotope effects on the pKa of the
attacking nucleophile and on the cleavage reaction of
the dianionic substrate.
k
OH(HOꢀ)/kOH(DOꢀ) values show that a reaction invol-
ving a release of an alkyloxy leaving group is more
sensitive to the isotopic composition of the solvent and
an additional isotope effect of 1.5 needs to be accounted
for in the reaction involving an alkyl leaving group.
It must be noted that the attacking nucleophiles in 1b
and 2a are not in the same position and their pKa values
are expected to be different.12 When the reactivities of 1a
and 1b are compared, it is observed that the cleavage of
1a is approximately four times faster than that of 1b (data
not shown), suggesting that the pKa of the 20-OH is lower
than that of 30-OH by ꢄ0.6 pKa units (assuming that the
fully ionized forms have identical reactivity). Differences
of the same magnitude have also been obtained with
dinucleoside monophosphates.12 However, as the reac-
tivity difference between 1a and 1b is the same in H2O
and D2O, the observed solvent isotope effects must also
be the same. Therefore, even though different absolute
values of kOH(HOꢀ) and kOH(DOꢀ) would have been
obtained with 1a, the ÁpKa value obtained by the
equivalent analysis would have been the same as those
obtained with 1b.
pKa values for similar OH nucleophiles have been
determined kinetically by measuring the rate constants
for the cleavage at pHs where the deprotonation of the
hydroxy group approaches completion.12 Saturation is
not observed with either of the substrates studied in the
present work, suggesting that the 20/30-OH groups of
the benzimidazole nucleoside are more basic than
those in natural nucleosides, and consequently the
ÁpKa [pKa(D2O) ꢀ pKa(H2O)] could not be reliably
determined in the present case. An estimate was obtained
from the non-linear fit according to Eqn (1) as discussed
below. The kOH(HOꢀ)/kOH(DOꢀ) value of 4.9 obtained
with 1b is probably entirely due to this difference in the
deprotonation of the attacking OH group of the 20-
methylbenzimidazole nucleoside. This suggestion is
based on the fact that the cleavage of nucleoside arylpho-
sphates under neutral and alkaline conditions results in a
release of aryl oxyanion.9 Once the attacking OH group is
deprotonated, there is no other proton transfer between
the solvent and the substrate and, consequently, no other
source of primary isotope effect. The solvent isotope
effect for the attack of a fully ionized oxyanion on a
diester is 0.9 with the p-nitrophenyl ester of 30-TMP,6i 0.8
with cis-4-hydroxytetrahydrofuran 3-phenylphosphate6c
The rate constants obtained for the reaction of dianion
2a from the non-linear fits are not sufficiently accurately
defined owing to the lack of data at very high pL to draw
firm conclusions, but the same 1.5-fold isotope effect on
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SOLVENT ISOTOPE EFFECT IN RNA MODEL TRANSESTERIFICATION
393
the cleavage is observed. Similarly, the ÁpKa value
(0.54) obtained from the fits is consistent with the value
obtained as the ratio of the second-order rate constants of
the alkaline cleavage of 1b (0.69). The pKa,2 values
obtained are 12.06 and 12.60 in H2O and D2O, respec-
tively, and as discussed above, the pKa value of the 20-OH
group of 2a in H2O is slightly higher than the correspond-
ing pKa value in 30,50-UpU (11.5 at 90 ꢁC12).
work with 2a, the NHþ4 ion might not enhance the
hammerhead reaction by proton transfer to the leaving
group, but let it depart without catalysis.
Solvent isotope effects under neutral
and acidic conditions
The origin of the additional solvent isotope effect of
1.5 observed in the cleavage of 2a is not clear. As shown
in Fig. 2(a) and reported by several authors previously,
the cleavage of nucleoside 30-alkylphosphates shows a
first-order dependence on hydroxide ion concentration.
No kinetically significant protonation/deprotonation step,
other than the deprotonation of the attacking nucleophile,
can take place in the cleavage reaction, since this would
result in a different dependence on hydroxide ion con-
centration. Therefore, the additional effect has to be
attributed to the properties of the phosphorane intermedi-
ate/transition state. Medium effects, such as differences
in solvation, can contribute significantly to observed
solvent isotope effect5,18 and the 1.5-fold difference
could result from a poorer hydrogen bond stabilization
of the polar transition state in D2O than in H2O. Con-
sistent with this suggestion, k(H2O)/k(D2O) values from
1.1 to 1.8, which have been attributed to medium effects,
have been reported for the nucleophilic reactions of
carboxylic acid esters.18 The fact that such an effect is
reduced in the cleavage of the arylphosphate 1b can be
explained by the lower polarity of the transition state: as
the charge on the leaving group in the transition state for
the cleavage of an alkylphosphate is more developed, the
charge is less dispersed than in the case of arylpho-
sphates, and so hydrogen bonding with solvent may be
more important than in the case of the arylphosphate 1b.
The solvent isotope effect values reported for the clea-
vage of arylphosphates6c,d,i by fully ionized intramole-
cular nucleophiles are close to unity, which supports the
suggestion that these medium effects play a minor role in
the case of arylphosphates.
The apparent solvent isotope effects obtained with 1b
and 2a in this work are very similar to those obtained in
hammerhead ribozyme reaction in the presence of Mg2 þ
and NHþ4 ions (4.47a and 7.69,7e respectively). Therefore,
it is tempting to speculate about the mechanism of the
hammerhead cleavage on the basis of the present results.
Since the kobs(H2O)/kobs(D2O) value is similar to that
obtained with 1b, it may be that Mg2 þ ions promote the
hammerhead reaction mainly by stabilizing the phosphor-
ane and/or the leaving group, thereby making the phos-
phorane resemble that in the cleavage of an
arylphosphate. The solvent isotope effects obtained do
not need to be explained by catalysis of the deprotonation
of the 20-OH by Mg2 þ , but could simply reflect the
spontaneous deprotonation of the attacking 20-nucleo-
phile. As the kobs(H2O)/kobs(D2O) value obtained in the
presence of NHþ4 ions is very close to that obtained in this
In contrast to the alkaline cleavage, no significant solvent
isotope effect was observed in the cleavage and isomer-
ization of 2a under acidic conditions, as is shown by the
results in Fig. 2(a) and (b) and Table 2. The second-order
rate constants of the acid-catalysed cleavage (kcH in
Table 2) and isomerization (kiH) of 2a give isotope effects
of 0.9 and 1.0, respectively. Similar values are obtained
from the rate constants of different partial reactions
obtained from the non-linear fit according to Eqns (1)
and (2) collected in Table 2.
Practically no solvent isotope effect on the sponta-
neous cleavage and isomerization of monoanionic 2a was
observed either. The rate constants of spontaneous reac-
tions (kc and kg in Table 2) obtained by the non-linear fits
of Eqns (1) and (2) give solvent isotope effects of 1.3 and
1.2 for cleavage and isomerization, respectively.
The similarity of the k(H2O)/k(D2O) values for the
cleavage and isomerization reactions obtained under
neutral conditions is slightly surprising, since the me-
chanisms of these reactions are different. Both reactions
take place via the same monoanionic phosphorane inter-
mediate,1d,e which is formed via a minor tautomer with a
deprotonated 20-oxy group and a protonated phosphate
(Scheme 8).14 However, the second steps, which are rate-
limiting, at least in the cleavage reaction, involve differ-
ing proton transfers. The second step of the isomerization
does not require protonation of the leaving group as the
endocyclic leaving group can depart as an oxyanion,
whereas exocyclic cleavage requires protonation of the
leaving group oxygen (Scheme 8).1e Therefore, the clea-
vage reaction involves a proton transfer from the phos-
phorane to the leaving group, which probably takes place
simultaneously with the departure of the leaving group,
while isomerization involves no such protonation. Hence,
different solvent isotope effects may be expected. It is
possible that the intramolecular proton transfer proposed
to take place in the second step of the cleavage reaction,
even if it is concerted with the departure of the leaving
group, need not result in an observable solvent isotope
effect, but that the effects of proton transfer from the
phosphate and to the leaving group compensate each
other. Supporting this contention are several reports of
k(H2O)/k(D2O) values close to zero for the dephosphor-
ylation of various different phosphate esters.19 All these
reactions involve a proton transfer from a monoanionic
phosphate to the leaving group in a pre-equilibrium or
concertedly to form a zwitterionic species with a dianio-
nic phosphate and a protonated leaving group. Therefore,
the similar and small k(H2O)/k(D2O) values of the
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394
N. VIRTANEN ET AL.
Table 2. Kinetic solvent deuterium isotope effects of intramolecular transesterification of the phosphodiester bond of 1b
and 2a
Parameter
In H2O
In D2O
k(H2O)/k(D2O)
k
HO(1b) (dm3 molꢀ1 sꢀ1 a
)
10.4 ꢃ 0.2
1.61 ꢃ 0.03
2.1 ꢃ 0.1
0.223 ꢃ 0.005ꢀ3
(7.3 ꢃ 0.4) ꢅ 10
(2.40 ꢃ 0.07) ꢅ 10ꢀ3
0.030 ꢃ 0.034ꢀ7
(1.3 ꢃ 0.8) ꢅ 10
0.16 ꢃ 0.06
4.94
7.24
0.92
1.00
1.5
1.3
1.0
1.0
1.2
k
HO(2a) (dm3 molꢀꢀ11 sꢀꢀ11 cb
)
)
)
kcH(2a) (dm3 mol sꢀ1 d
(6.7 ꢃ 0.4) ꢅ 10ꢀ3
(2.30 ꢃ 0.07) ꢅ 10ꢀ3
0.045 ꢃ 0.037ꢀ7
(1.7 ꢃ 0.8) ꢅ 10
0.16 ꢃ 0.06
kiH(2a) (dm3 molꢀ1 s
kd(2a, dianion) (sꢀ1 e
kc(2a, monoanion) (sꢀ1 f
kb(2a, neutral) (sꢀ1 g
ka(2a, neutral) (dm3 molꢀ1 sꢀ1 h
kg(2a, monoanion) (sꢀ1 i
kf(2a, neutral) (sꢀ1 j
ke(2a, neutral) (dm3 molꢀ1 sꢀ1 k
)
)
)
)
3 ꢃ 2
3 ꢃ 2
)
(1.2 ꢃ 0.3) ꢅ 10ꢀ6
(1.0 ꢃ 0.3) ꢅ 10ꢀ6
)
0.1 ꢃ 0.1
0.1 ꢃ 0.1
1.0
1.0
)
1 ꢃ 2
2 ꢃ 4
a
b
c
d
e
f
Second-order rate constant of base-catalysed cleavage of 1b at 25 ꢁC. Obtained as a slope of plot kobs vs [LOꢀ].
Second-order rate constant of base-catalysed cleavage of 2a at 90 ꢁC. Obtained as a slope of plot kobs vs [LOꢀ].
Second-order rate constant of acid-catalysed cleavage of 2a at 90 ꢁC. Obtained as a slope of plot kobs vs [L3Oþ].
Second-order rate constant of acid-catalysed isomerization of 2a at 90 ꢁC. Obtained as a slope of plot kobs vs [L3Oþ].
Rate constant of the cleavage of dianionic 2a at 90 ꢁC. Obtained by a non-linear fit of kobs vs [Hþ] according to Eqn (1).
Rate constant of the spontaneous cleavage of monoanionic 2a at 90 ꢁC. Obtained by a non-linear fit of kobs vs [Hþ] according to Eqn (1).
Rate constant of the cleavage of neutral 2a at 90 ꢁC. Obtained by a non-linear fit of kobs vs [Hþ] according to Eqn (1).
g
h
i
Second-order rate constant of acid-catalysed cleavage of neutral 2a. Obtained by a non-linear fit of kobs vs [Hþ] according to Eqn (1).
Rate constant of the spontaneous isomerization of monoanionic 2a at 90 ꢁC. Obtained by a non-linear fit of kobs vs [Hþ] according to Eqn (2).
Rate constant of the isomerization of neutral 2a at 90 ꢁC. Obtained by a non-linear fit of kobs vs [Hþ] according to Eqn (2).
Second-order rate constant of the acid-catalysed isomerization of neutral 2a at 90 ꢁC. Obtained by a non-linear fit of kobs vs [Hþ] according to Eqn (2).
j
k
Scheme 8
Scheme 9
cleavage and isomerization observed under neutral con-
ditions can be explained most plausibly by suggesting
that neither step of the two reactions is characterized by
any significant solvent isotope effect.
The results obtained under acidic conditions are
slightly more difficult to explain. The cleavage and
isomerization occur via two different routes, showing
second- and first-order dependences on hydronium ion
concentration. The second-order dependence refers to the
reaction that proceeds via a cationic phosphorane formed
by a nucleophilic attack of a neutral 20-OH on the doubly
protonated phosphate (rate constants ka for cleavage and
ke for isomerization in Table 2) (Scheme 9). In the
reaction showing a first-order dependence, the phosphate
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SOLVENT ISOTOPE EFFECT IN RNA MODEL TRANSESTERIFICATION
395
apparent k(H2O)/k(D2O) of 0.32 is consistent with the
mechanism of the acid-catalysed hydrolysis of the N-
glycosidic bond (Scheme 7). The rate-limiting step is the
unimolecular bond rupture of the base-protonated sub-
strate, which results in the formation of 2-methylbenzi-
midazole base and ribosyl carbocation.14 Water attacks
the carbocation in a subsequent step, and the nucleophilic
attack does not contribute to the observed solvent isotope
effect. The clear inverse solvent isotope effect is most
simply attributed to the pre-equilibrium protonation of
the substrate, which is favoured in D2O.
CONCLUSIONS
The alkaline cleavage of nucleoside phosphodiesters is
characterized by large apparent solvent deuterium isotope
effect values. In the case of phosphodiesters with an aryl
leaving group, the effect observed can be accounted for
satisfactorily by the equilibrium effect on the ÁpKa of the
attacking nucleophile. When the leaving group is an
alkyloxy function, an additional 1.5-fold isotope effect
is observed, which may be due to the requirement for
better solvation of the more highly polarized transition
state. In contrast to alkaline cleavage, cleavage and
isomerization under neutral and acidic conditions showed
no significant solvent isotope effects which probably
result from very small effects and from opposite effects
involved in different steps of the reaction that compensate
each other, respectively.
Scheme 10
and the phosphorane are neutral (kb and kf) (Scheme 10).
In both cases, a prototropic arrangement that places a
proton on the leaving group oxygen takes place in the
second step of the reaction, and the leaving group departs
as an alcohol. As can be seen in Schemes 9 and 10, the
reactions involve protonation of the phosphate. As
the pKa of the phosphate is higher in D2O than in
H2O,20 the proportion of the protonated form of the
phosphate is higher and, therefore, D2O solution favours
the formation of the intermediate. If, as above, the
intramolecular proton transfer processes taking place in
the second step of isomerization and cleavage do not
result in an observable solvent isotope effect, then an
inverse solvent isotope effect would have been expected.
No such effects are observed and the k(H2O)/k(D2O)
values obtained under acidic conditions were close to
unity. However, the suggestion that the intramolecular
proton transfer does not result in a solvent isotope
effect is based on observations obtained with phospho-
monoesters, where a species with a dianionic phosphate
and a protonated leaving group is formed. A similar
species is formed with 2a under neutral conditions, but
it may be that these results cannot be extrapolated to
reactions where the proton transfer takes place between
initially neutral species. Such a proton transfer might
be characterized by a k(H2O)/k(D2O) value higher than
unity which compensates for any effects on ground-state
protonation.
EXPERIMENTAL
General
Acetonitrile, pyridine and THF were refluxed with NaH,
and acetone with Na2SO4, and then distilled. 1,2-Dichlor-
oethane was refluxed with PO5. DMF and xylene were
dried with molecular sieves. Triazole was purified by
recrystallization from ethanol. D2O (99.97% D) and
D3COOD (99.5% D) were products of Eurisotop and
DCOOD (98% D), NaOD (40 wt% in D2O, 99% D)
and DCl (20% in D2O, 99.5% D) of Aldrich. Quarz-
distilled water was used to prepare the reaction solutions.
Buffer constituents were of analytical grade and other
reagents of at least reagent grade. HPLC-grade acetoni-
trile was used in HPLC eluents. Details of the syntheses
of 1a,b and 2a,b are given in the electronic supplemen-
tary material available in Wiley Interscience.
Preparation of buffers and calculation
of pKa values
Plots of observed rate constants of depurination vs
[Hþ] give second-order rate constants of 0.020 and
0.062 mol dmꢀ3 sꢀ1 in H2O and D2O, respectively. The
All the reaction solutions were in freshly distilled water
and buffers were prepared just before the experiments
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J. Phys. Org. Chem. 2005; 18: 385–397

396
N. VIRTANEN ET AL.
to avoid CO2 absorption. The pH of acidic solutions
was adjusted with a known concentration of HCl or
DCl. Under alkaline conditions, NaOH or NaOD was
used. pH values between 2 and 10 were obtained with
appropriate buffer systems. Formate, acetate, HEPES
[N-(2-hydroxyethyl)piperazine-N0-(ethanesulfonic
acid), 27], MOBS [4-(N-morpholine)butanesulfonic
acid, 28], CHES [2-(N-cyclohexylamino)ethanesulfo-
nic acid, 29], glycine, triethylamine were employed.
Their pKa values under the experimental conditions
were calculated as explained below. The values used
are given in Table 1. The pH of the reaction solutions
was checked with a pH meter, but the lyonium ion
concentrations given are based on calculated pKa va-
lues. Formate and acetate buffers were available in a
deuterated form. In other cases, the deuterated form
was prepared by evaporating the protiated form three
times from D2O. D2O solutions were kept in a desic-
cator after preparation.
assumed to be similar to that on the pKa of HEPES. This
was considered a valid approximation, since HEPES (27)
and MOBS (28) are structurally very similar. MOBS was
the only sulfonic acid derivative used at 90 ꢁC, and the
pKa value at 90 ꢁC of 6.82 was calculated using the
temperature dependence reported.30 pKa values of
MOBS and CHES in D2O were calculated by using
ÁpKa values reported for morpholine and cyclohexy-
lammonium, respectively.31 The approximation is as-
sumed to be valid since the deprotonation equilibria in
MOBS (28) and and CHES (29) take place at the
nitrogen atom of the zwitterionic form. The ÁpKa
reported for morpholine was used also to calculate the
pKa of HEPES in D2O. Values of 7.44 (90 ꢁC), 10.43
(25 ꢁC) and 8.07 (25 ꢁC) were calculated for the pKa of
MOBS, CHES and HEPES, respectively, in D2O at
I ¼ 1.0 M. The effects of temperature32 and ionic
strength32,33 on the pKa of glycine are known, and a
value of 8.33 was calculated at 90 ꢁC and I ¼ 1.0 M. A
ÁpKa of 0.63 has been reported for glycine34 and
assuming that it is independent of temperature and ionic
strength, a pKa of 8.96 was calculated for glycine in D2O
at 90 ꢁC and I ¼ 1.0 M. The pKa of triethylamine at
I ¼ 1.0 M at 25 ꢁC has been reported (10.9929). The pKa
in D2O at 25 ꢁC (11.58) was calculated using ÁpKa
reported for trimethylammonium ion.35
HPLC and spectrophotometric methods
All reactions, except for those followed spectrophoto-
metrically, were carried out in stoppered tubes that were
thermostated to 90.0 ꢃ 0.1 or 25.0 ꢃ 0.1 ꢁC in a water-
bath. Reactions were initiated by adding a small volume
of substrate stock solution to the thermostated reaction
solution to make a final concentration of 0.1 mM or less.
The 10–15 aliquots of 150 ml were withdrawn to cover 1–
3 half-lives of the reaction, and the reactions were
quenched by keeping the samples in an ice-bath. Acidic
samples were neutralized with a small volume of sodium
acetate solution and basic samples with acetic acid
solution. In cases where the samples were not analysed
immediately, they were kept at ꢀ20 ꢁC until the analysis.
The samples were analysed by RP-HPLC using a Hy-
persil ODS C18 (reactions of 1b) or Aquasil C18 column
(reactions of 2a). Both columns were obtained from
ThermoHypersil-Keystone. The dimensions of the col-
umns were 150 ꢅ 4 mm i.d., 5 mm particle size. The
eluents were mixtures of 0.06 M acetic acid buffer (pH
4.3, containing 0.1 M NH4Cl) and acetonitrile. The acet-
onitrile content was 13 and 8% in the reactions of 1b
and 2a, respectively. Signals were detected by a UV
detector at 245 nm. Cleavage of 1b at pH >11 was
carried out in the spectrophotometer cell thermostated
to 25.0 ꢃ 0.1 ꢁC. The absorbance at 287 was detected and
the reaction was followed until the absorbance no longer
increased.
The pL values of reaction solution were calculated
using pKw and pKa values referring to the experimental
conditions. Using the data on the effects of temperature
and ionic strength on the activity coefficients,21 pKw
values of H2O at I ¼ 1.0 M of 13.727 and 12.102 were
calculated at 25 and 90 ꢁC. The ionic product of D2O and
its temperature dependence at I ¼ 0 M are known.22 As-
suming that the ÁpKw value pKw(D2O) ꢀ pKw(H2O) is
independent of ionic strength, pKw values of D2O at
I ¼ 1.0 M at 25 and 90 ꢁC of 14.683 and 12.984, respec-
tively, were calculated.
The effects of temperature23and ionic strength24 on the
pKa of formic acid are known, and a value of 3.74 at
90 ꢁC and at ionic strength of 1.0 M was calculated. A
ÁpKa [pKa(D2O) ꢀ pKa(H2O)] of 0.394 has been re-
ported25 and assuming that the effects of temperature
and I are the same as in H2O, a pKa of 4.13 can be
calculated for DCOOD in D2O at 90 ꢁC and at ionic
strength of 1.0 M. The pKa values of acetic acid and
CD3COOD at 90 ꢁC were calculated by using the known
temperature dependence26 and the effect of the ionic
strength was calculated by using the effect reported at
20 ꢁC,27 assuming that it is the same in both cases. pKa
Values of CHES (9.7528) and HEPES (7.4529) at
I ¼ 1.0 M were calculated using data found in the litera-
ture. The effect of I on the pKa value of MOBS was
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SOLVENT ISOTOPE EFFECT IN RNA MODEL TRANSESTERIFICATION
397
Shiiba T, Tsuzuki H, Yajima H, Sumaoka J. J. Chem. Soc., Perkin
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Bonfa L, Gatos M, Mancin F, Tecilla P, Tonellato U. Inorg. Chem.
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Acknowledgements
¨
The authors thank Professor Harri Lonnberg and Profes-
sor Jari Hovinen for their help with the synthesis,
Dr Nick Williams for his help with the manuscript and
Dr Jari Sinkkonen for his help with NMR analysis and
assignments.
9. Davis AM, Hall AD, Williams A. J. Am. Chem. Soc. 1988; 110:
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