Characterization of the transition-state structures and mechanisms for the isomerization and cleavage reactions of uridine 3′-m-nitrobenzyl phosphate

Article abstract of DOI:10.1021/ja003400v

The transition-state structures and mechanisms of the isomerization to the 2′-isomer and cleavage reactions of uridine 3′-m-nitrobenzyl phosphate to m-nitrobenzyl alcohol and a 2′,3′-cyclic UMP at 86 °C and at pH 2.5, 5.5, and 10.5 have been characterized through kinetic isotope effects. The ¹⁸O primary isotope effect of 1.0019 ± 0.0007 and the secondary isotope effect of 0.9904 observed for the cleavage reaction at pH 2.5 are consistent with a neutral phosphorane-like transition-state structure. The cleavage and isomerization reactions at pH 2.5 proceed through a neutral phosphorane intermediate. The ¹⁸k_bridge and ¹⁸k_nonbridge of unity measured for the pH-independent isomerization reaction at neutral pH support a stepwise mechanism with a monoanionic phosphorane intermediate. The primary and secondary isotope effects of 1.009 ± 0.001 and of 0.9986 ± 0.0004 observed for the pH-independent cleavage reaction are consistent with either a stepwise mechanism through a monoanionic phosphorane intermediate or with an A_ND_N reaction with a transition state resembling a monoanionic phosphorane intermediate. The absolute requirement of a water-mediated proton transfer for the formation of a phosphorane intermediate is proven by the absence of the isomerization reaction in anhydrous tert-butyl alcohol. The primary isotope effect of 1.0272 ± 0.0001 for the cleavage reaction at pH 10.5 is consistent with a concerted reaction through a transition state in which the leaving group departs with almost a full negative charge.

Full text of DOI:10.1021/ja003400v

VOLUME 122, NUMBER 51
DECEMBER 27, 2000
© Copyright 2000 by the
American Chemical Society
Characterization of the Transition-State Structures and Mechanisms
for the Isomerization and Cleavage Reactions of Uridine
3′-m-Nitrobenzyl Phosphate
Barbara Gerratana, Gwendolyn A. Sowa, and W. W. Cleland*
Contribution from the Institute for Enzyme Research and Department of Biochemistry, UniVersity of
Wisconsin-Madison, Madison, Wisconsin 53705
ReceiVed September 15, 2000
Abstract: The transition-state structures and mechanisms of the isomerization to the 2′-isomer and cleavage
reactions of uridine 3′-m-nitrobenzyl phosphate to m-nitrobenzyl alcohol and a 2′,3′-cyclic UMP at 86 °C and
at pH 2.5, 5.5, and 10.5 have been characterized through kinetic isotope effects. The 18O primary isotope
effect of 1.0019 ( 0.0007 and the secondary isotope effect of 0.9904 observed for the cleavage reaction at pH
2.5 are consistent with a neutral phosphorane-like transition-state structure. The cleavage and isomerization
18
18
reactions at pH 2.5 proceed through a neutral phosphorane intermediate. The
k
and
k
of unity
bridge
nonbridge
measured for the pH-independent isomerization reaction at neutral pH support a stepwise mechanism with a
monoanionic phosphorane intermediate. The primary and secondary isotope effects of 1.009 ( 0.001 and of
0.9986 ( 0.0004 observed for the pH-independent cleavage reaction are consistent with either a stepwise
mechanism through a monoanionic phosphorane intermediate or with an ANDN reaction with a transition state
resembling a monoanionic phosphorane intermediate. The absolute requirement of a water-mediated proton
transfer for the formation of a phosphorane intermediate is proven by the absence of the isomerization reaction
in anhydrous tert-butyl alcohol. The primary isotope effect of 1.0272 ( 0.0001 for the cleavage reaction at pH
10.5 is consistent with a concerted reaction through a transition state in which the leaving group departs with
almost a full negative charge.
Introduction
resemble a trigonal bipyramid (TBP), assuming a phosphorane-
like structure. In the TBP geometry the ligands may occupy
two different positions, the apical or the equatorial. Both the
leaving-group departure and the nucleophile entry must occur
at the apical position. If the phosphorane intermediate formed
in the two-step mechanism (AN+DN) has a sufficient lifetime,
pseudorotation may occur to form a new TBP structure with
the ligand positions exchanged. This polytopal rearrangement
accounts for the different stereochemistry and product distribu-
tion of nucleophilic substitution at phosphorus.1
The central role played by phosphate esters as building blocks
of DNA and RNA requires a thorough understanding of the
mechanisms of their reactions. By studying the hydrolysis of
five-member cyclic and acyclic phosphates such as ethylene
phosphate and dimethyl phosphate, Westheimer set the founda-
tion for comprehending the reactions of phosphate esters.1
Phosphate diesters undergo nucleophilic substitution reactions
by an addition-elimination mechanism.2 In the concerted SN2(P)
(ANDN)3 reaction, the pentacoordinated transition state will
* To whom correspondence should be addressed.
(1) Westheimer, F. H. Acc. Chem. Res. 1968, 1, 70-78.
(2) Thatcher, G. R.; Kluger, R. AdV. Phys. Org. Chem. 1989, 25, 99-
265.
(4) (a) Anslyn, E.; Breslow, R. J. Am. Chem. Soc. 1989, 111, 4473-
4482. (b) Oivanen, M.; Schnell, R.; Pfleiderer, W.; Lo¨nnberg, H. J. Org.
Chem. 1991, 56, 5396-5401. (c) Kuusela, S.; Lo¨nnberg, H. J. Chem. Soc.,
Perkin Trans. 2 1994, 2109-2113. (d) Oivanen, M.; Schnell, R.; Pfleiderer,
W.; Lo¨nnberg, H. J. Org. Chem. 1991, 56, 3623-3628.
(3) Guthrie, J. P.; Jencks, W. P. Acc. Chem. Res. 1989, 22, 343-349.
10.1021/ja003400v CCC: $19.00 © 2000 American Chemical Society
Published on Web 12/06/2000

12616 J. Am. Chem. Soc., Vol. 122, No. 51, 2000
Gerratana et al.
Model compounds of RNA such as dinucleoside 3′,5′-
phosphates, ribonucleoside 3′-(alkyl phosphates) and polyribo-
nucleotides in acidic or neutral pH undergo two transesterifi-
cation reactions, a reversible one, which gives the 2′-isomer,
and the cyclization reaction with 2′,3′-cyclic phosphate as
product, which is then hydrolyzed to 2′- and 3′-phosphates.4
The similar kinetics of the cyclization and isomerization of
analogues of RNA under acidic conditions indicate that these
reactions proceed through the same neutral phosphorane inter-
mediate.4 The neutral phosphorane is sufficiently stable to
pseudorotate to give either the 2′,3′-cyclic monophosphate or
the 2′-isomer.5 Further evidence for the existence of a polytopal
rearrangement in the transesterification reaction is the overall
retention of configuration at the phosphorus in the reaction of
phosphoromonothioate analogues of 3′,5′-uridylyluridine.6
At neutral pH, although the isomerization and cleavage
reactions are of particular interest due to their biological
relevance, very little is known about their mechanisms. The
isomerization reaction is pH-independent from pH 4.0 to 8.0
and is believed to proceed through a phosphorane intermediate,
while the cyclization reaction shows pH-independence over a
smaller range around pH 5.0.4d The uncatalyzed cleavage
reaction may be concerted with a phosphorane-like transition
state or be stepwise with a phosphorane intermediate which may
or may not pseudorotate.4b,c In aqueous alkali, RNA molecules
are cleaved by intramolecular nucleophilic attack by the
2′-oxyanion. The 2′,3′-cyclic phosphate product is then hydro-
lyzed to 2′- and 3′-phosphates.7 Evidence for concerted cycliza-
tion is that no isomerization reaction has been observed with
model compounds of RNA,4b-d ab initio calculations indicate
that a dianionic phosphorane is too unstable to exist as an
intermediate,8 and the stereochemistry of the cleavage reaction
is inversion.6
Figure 1. Uridine 3′-m-nitrobenzyl phosphate, showing the positions
18
of the isotope effect measurements. (a)
k
measured at the
nonbridge
18
nonbridge oxygen atoms. (b)
atom.
k
measured at the bridge oxygen
bridge
The synthesis of uridine 3′-m-nitrobenzyl phophate was carried out
as previously reported.9 [14N], [15N, bridge-18O], and [15N, nonbridge-
18O2]-uridine 3′-m-nitrobenzyl phosphate compounds were synthesized
as previously described.10 Mass spectrometry (FAB) showed [15N,
nonbridge-18O2]-uridine 3′-m-nitrobenzyl phosphate to have 64%
incorporation of oxygen-18 at nonbridge oxygens. Mass spectrometry
(FAB) showed [15N, bridge-18O]-uridine 3′-m-nitrobenzyl phosphate to
have 93% incorporation of oxygen-18 at bridge oxygen. NMR
spectrometry was employed to check the purity and identity of the
compounds. 1H NMR spectra were recorded at 500 MHz in D2O with
TSP as an internal reference. 13C NMR spectra were recorded at 125
MHz in D2O with TSP as an internal reference. 31P NMR spectra (1H
decoupled) were recorded at 202 MHz in D2O unless otherwise specified
with 85% H3PO4 as an external reference.
Kinetic Measurements. Uridine 3′-m-nitrobenzyl phosphate sodium
salt (30 µmoles) was heated at 92 °C in a oil bath in 10 mL of 50 mM
buffer, and the temperature of the solution was 86 °C. Citric acid was
used as a buffer for the reaction run at pH 2.0, sodium acetate for pH
4.0 and 5.0, MES for pH 6.0, MOPS for pH 7.0, CHES for pH 9.0,and
CAPS for pH 11.5. Reactions were run at least in duplicate. Each
reaction was followed by periodically assaying withdrawn aliquots with
a Microsorb-MV C18 HPLC column (5 µm, 4.6 mm i.d. × 250 mm)
equilibrated with 6 mM NaH2PO4 pH 6.8, in 27% methanol. Detection
was accomplished by absorption at 254 nm. Uridine 3′-m-nitrobenzyl
phosphate elutes at 9.2 min, uridine 2′-m-nitrobenzyl phosphate at 4.6
min, UMP at 2.4 min, and m-nitrobenzyl alcohol at 18.2 min. The rates
were determined by integrating peak areas. The pH-rate profiles were
fitted to the following equations: kobs ) ko + [H+]/K1 for the
isomerization reaction and kobs ) ko + [H+]/K1+ K2/[H+] for the
cleavage reaction where ko is the pH independent rate.
Reaction in tert-Butyl Alcohol. The sodium salt of uridine 3′-m-
nitrobenzyl phosphate (10 µmol) was loaded on a Dowex 50W-X8
column (1.2 × 27 cm) in the proton form. The column was eluted
with distilled H2O, and the pooled fractions were concentrated to 5
mL and titrated to pH 6.5 with 1.0 M tetrabutylammonium hydroxide
in water. The solution was lyophilized and then dried over P2O5 in
vacuo for 2 days. The tetrabutylammonium salt of uridine 3′-m-
nitrobenzyl phosphate was dissolved in 5 mL of anhydrous tert-butyl
alcohol in a glovebox. The boiling point of tert-butyl alcohol is 83 °C,
and its melting point is 25 °C; thus, the reaction needed to be run with
a condenser with running water at 37 °C to prevent crystallization of
the alcohol on the coils of the condenser. The reaction was run at 86
°C under nitrogen for 5 days. After removal of tert-butyl alcohol, each
time point aliquot was analyzed by HPLC as already described. The
solution mixture was analyzed after 5 days by 31P NMR (80% tert-
butyl alcohol and 20% acetone-d6).
Heavy atom kinetic isotope effects are a powerful method to
study the mechanism and the transition-state structure of
enzymatic and nonenzymatic reactions of phosphate diesters.9
18
The magnitude of the primary isotope effect,
k
, is an
bridge
indication of the degree of bond breaking measured at the
leaving group oxygen atom in the transition state. The partial
loss or increase of bond order between the nonbridge oxygen
atoms and the phosphorus atom in the transition state is revealed
18
by the secondary kinetic isotope effects,
k
(Figure 1).
nonbridge
We have measured the heavy atom kinetic isotope effects in
the cleavage and isomerization reactions of uridine 3′-m-
nitrobenzyl phosphate at different pHs. The isotope effects
reported shed light on the mechanism of the isomerization and
cyclization reactions of RNA and of model compounds of RNA
in acidic, neutral, and basic conditions and fully characterize
their phosphorane-like transition states.
Experimental Section
Materials and Methods. Methanol-18O, H218O,14NH414NO3, and
15NH415NO3 were purchased from Isotec. All synthetic reagents and
anhydrous tert-butyl alcohol were from Aldrich. Buffers were from
Sigma. The Microsorb-MV C18 HPLC column was purchased from
Varian Analytical Instruments. Econosil C18 HPLC column was from
Alltech.
(5) Uchimaru, T.; Uebayasi, M.; Hirose, T.; Tsuzuki, S.; Yliniemela,
A.; Tanabe, K.; Taira, K. J. Org. Chem. 1996, 61, 1599-1608.
(6) Oivanen, M.; Ora, M.; Almer, H.; Stro¨mberg, R.; Lo¨nnberg, H. J.
Org. Chem. 1995, 60, 5620-5627.
(7) Brown, D. M.; Magrath, D. I.; Neilson, A. H.; Todd, A. R. Nature
1956, 177, 1124.
Kinetic Isotope Effect Measurements. The kinetic isotope effects
were measured using the remote label method.11 In this method substrate
labeled at both the position of interest and the remote label position is
(8) Lim, C.; Karplus, M. J. Am. Chem. Soc. 1990, 112, 5872-5873.
(9) (a) Sowa, G. A.; Hengge, A. C.; Cleland, W. W. J. Am. Chem. Soc.
1997, 119, 2319-2320. (b) Hengge, A. C.; Tobin, A. E.; Cleland, W. W.
J. Am. Chem. Soc. 1995, 117, 5919-5926. (c) Hengge, A. C.; Cleland, W.
W. J. Am. Chem. Soc. 1991, 113, 5835-5841.
(10) Sowa, G. A. Ph.D. Thesis, University of Wisconsin-Madison,
Madison, WI, 1997.
(11) O’Leary, M. H.; Marlier, J. F. J. Am. Chem. Soc. 1979, 101, 3300-
3306.

Isomerization and CleaVage Reactions of RNA
J. Am. Chem. Soc., Vol. 122, No. 51, 2000 12617
mixed with the substrate depleted in the heavy atom at the remote label
position to reconstitute the natural abundance at the remote position.
In the reactions of uridine 3′-m-nitrobenzyl phosphate the nitrogen atom
of the nitro group is the remote label. A 4 mM solution of the
appropriate substrate mixture (80 µmol) was prepared in 20 mL of 50
mM acetate buffer for reaction at pH 5.5, 50 mM of citric acid for pH
2.5, and 50 mM of CAPS for pH 10.5. Reactions run at 86 °C were
stopped by cooling on ice before the isomerization reaction of the
uridine 3′- and uridine 2′-m-nitrobenzyl phosphate reached equilibrium.
Analytical HPLC was used to determine the fraction of the cyclization
and isomerization reactions as already described. The solution was
titrated to pH 7.0 with 1 N KOH. m-Nitrobenzyl alcohol, the product
of the cleavage reaction, was separated by extraction of the solution
three times with freshly distilled diethyl ether. The ether layers were
washed once with 10 mM NaH2PO4, pH 4.5, and the acidic aqueous
layer was back-extracted three times with diethyl ether. The combined
ether layers were dried over anhydrous Mg2SO4 and concentrated by
rotary evaporation. The residue was sublimed at 95 °C for 20 min.9
After removal of the diethyl ether, the aqueous layer containing the 2′
and 3′ isomers was concentrated to 3 mL, filtered with 0.45 µm filters,
and chromatographed on an Econosil C18 HPLC column (22 mm i.d.
× 250 mm) equilibrated with 6 mM NaH2PO4, pH 6.8, in 15%
methanol. Detection was accomplished by absorption at 254 nm. Two
major peaks with retention times of 28-35 min, corresponding to the
3′ isomer, and of 13-16 min, corresponding to the 2′ isomer, eluted
with a flow rate of 14 mL/min. After removal of the methanol, each
peak was concentrated to ∼25 mL. Each solution was made 1 N in
HCl, and the uridine 3′- and uridine 2′-m-nitrobenzyl phosphate were
hydrolyzed to m-nitrobenzyl alcohol at 95 °C for 48-72 h. Hydrolysis
was followed by analytical HPLC as described. When the hydrolysis
was completed, the cooled solution was titrated to pH 12.5 with 1 N
KOH. The m-nitrobenzyl alcohol was extracted and treated as described
above. Control experiments were run to ensure that no fractionation
occurred in the separation of the two isomers by preparative HPLC.
An elemental analyzer (Carlo Erba-NA 1500 Series 2) coupled with a
Europa Tracermass 20-20 isotope ratio mass spectrometer was used
to analyze the isotopic composition of each sample.
Figure 2. pH-rate profile for the isomerization (0) and cleavage (9)
reactions of 3 mM uridine 3′-m-nitrobenzyl phosphate in 50 mM buffer
at 86 °C.
Figure 3. 31P NMR spectrum of the reaction mixture of 2 mM uridine
3′-m-nitrobenzyl phosphate in anhydrous tert-butyl alcohol after 5 days
at 86 °C. The 31P NMR spectrum (1H decoupled) was recorded at 202
MHz in 80% tert-butyl alcohol and 20% acetone-d6 with 85% H3PO4
as an external reference.
Data Analysis. The isotope effects were calculated from the
following equations:
Table 1. Kinetic Isotope Effects of the Cleavage and Isomerization
Reaction of Uridine 3′-m-Nitrobenzyl Phosphate at 86 °C
18
18
18
a
18
b
k
) ln(1 - fc)/ln[1 - (Rc fc/Ro)]
(1)
pH
reaction
k
k
k
nonbridge
cleavage
bridge
nonbridge
2.5 cleavage
5.5 cleavage
1.0019 ( 0.0007 0.9904
0.9885
18
k
) ln[(1 - fc - 2fi)/(1 - fc)]/
1.009 ( 0.001 0.9986 ( 0.0004 0.9983
isomerization
5.5 isomerization 1.0004 ( 0.0002 0.9990 ( 0.0007 0.9988
10.5 cleavage 1.0272 ( 0.0001
ln{1 - [(2Ri fi/Ro)/(1 - Rc fc/Ro)]} (2)
a Kinetic isotope effect for 18O in both nonbridge oxygen atoms.
b Isotope effects corrected to 27 °C using the equation: ln(IE at 27
°C) ) (359 K/300 K) ln(IE at 86 °C).
where fc is the fraction of the cleavage reaction, fi is the fraction of the
isomerization reaction, Ro is the isotopic ratio in the starting material,
Rc is the isotopic ratio in the cleavage product m-nitrobenzyl alcohol,
and Ri is the isotopic ratio in the isomerization product uridine 2′-m-
nitrobenzyl phosphate. These equations were derived as described in
the appendix on the assumption that the Keq of the isomerization reaction
is unity, as it is for uridylyl (3′,5′)uridine,4d and that the rates of cleavage
of the 3′ isomer and the 2′ isomer are the same.12
phosphate at 86 °C is shown in Figure 2. In the acidic region
the rates of isomerization and cleavage reactions are very similar.
The isomerization reaction is pH-independent from pH 5.0 to
9.0. The cleavage reaction is pH-independent in a smaller range
of pHs from 5.0 to 7.0. In this region the rates of isomerization
and cleavage reactions are almost identical. Under alkaline
conditions the cleavage reaction becomes first order with respect
to hydroxide ion and the isomerization product uridine 2′-m-
nitrobenzyl phosphate is not formed at pHs higher than 9.0.
The kinetic isotope effects calculated with eqs 1 and 2 were corrected
for incomplete isotopic incorporation in the starting material using the
remote label equations.11
Results
Uridine 3′-m-nitrobenzyl phosphate at 86 °C undergoes a
reversible isomerization to uridine 2′-m-nitrobenzyl phosphate
and a cleavage reaction with m-nitrobenzyl alcohol and 2′,3′-
cyclic UMP as products. The 2′,3′-cyclic UMP is readily
hydrolyzed to 2′-UMP and 3′-UMP. The pH rate profile of the
isomerization and cleavage reaction of uridine 3′-m-nitrobenzyl
Kinetic isotope effects have been measured with the remote
label method11 at pH 2.5, 10.5, and 5.5 to study the transition-
state structures of the hydronium- and hydroxide-dependent
reactions and of the pH-independent reactions, respectively. The
isotope effects are shown in Table 1 with their standard errors.
The reactions at pH 5.5 were run in triplicate, while the
measurements at pH 2.5 and 10.5 were run in duplicate.
Although the 18knonbridge reported at pH 2.5 has been calculated
from a single measurement, the calculated Ro value from the
experimental Ri, Rc, and fc values agrees with the measured Ro.
(12) While the pK’s of the 2′- and 3′-hydroxyl groups differ by 0.29 pH
units, this will cause little difference in the reaction rates of the 3′- and
2′-phosphodiesters because both participate as protonated species. The
Brønsted value is ∼0.3 for such reactions,13 which would cause only a ∼20%
difference in the rates of cleavage of the 3′- and 2′-isomers.

12618 J. Am. Chem. Soc., Vol. 122, No. 51, 2000
Gerratana et al.
the observed isotope effect is described by eq 4.
Scheme 1
18
k
)
18k1[(18k3/18k2 + k3/k2)/(1 + k3/k2)]
(4)
obs
18
This equation is simplified to
k
)
18k1 by assuming that
obs
The 18O isotope effects have been corrected for the isotopic
incorporation and for the 15k effect (1.0004 ( 0.0007).9a,11
The reaction in anhydrous tert-butyl alcohol was run at the
same temperature as the aqueous reactions. Only the cleavage
18k3 and 18k2 are the same due to the similarity of the chemical
steps and of the pK’s of the leaving groups. Thus, the observed
isotopic discrimination is determined by the isotope effect on
the rate of formation of the phosphorane intermediate. This
secondary kinetic isotope effect gives information on the
transition-state structure for the formation of the phosphorane
intermediate.
reaction was observed with a rate constant of 1.11 × 10-6 sec-1
.
The presence of 2′,3′-cyclic UMP and not its hydrolytic products
2′- and 3′-UMP was shown by the existence of a peak at 17.01
ppm in the 31P spectrum of the reaction mixture (Figure 3). This
observation is consistent with the 31P spectrum of cyclic
phosphates such as ethyl ethylene phosphate which show a peak
in the 15 ppm region.14 The peak at -0.24 ppm is assigned to
the tetrabutylammonium salt of uridine 3′-m-nitrobenzyl phos-
phate. The tetrabutylammonium salt of 3′-UMP in 80% tert-
butanol and 20% acetone-d6 shows a peak at 2.4 ppm (data not
shown).
Hydronium Ion-Catalyzed Reactions.15 The primary and
secondary 18O isotope effects were measured for the cleavage
reaction at pH 2.5 and at 86 °C. The primary isotope effect
represents the loss of bond order with the m-nitrobenzyl group
during the breakdown of the phosphorane intermediate. The
magnitude of the observed isotope effect is decreased by the
presence of the forward commitment (eq 3). The observed
primary isotope effect is 0.19%.16 Calculation of the intrinsic
primary isotope effect requires assumption of a value for the
forward commitment in eq 3. The neutral phosphorane inter-
mediate can break down in three ways to give cleavage to
m-nitrobenzyl alcohol and a 2′,3′-cyclic phosphate, or to the
2′- or 3′-isomers of uridine m-nitrobenzyl monophosphate. If
these rates are equal because of the similarity of pK’s of the
leaving groups,12 the forward commitment would be 0.5, and
the intrinsic primary isotope effect at 86 °C is 1.0028. The
isomerization reactions produce single products, while the
cleavage reactions produce two products but one of these is a
less stable cyclic phosphate. The pKa of the m-nitrobenzyl
alcohol is also 2 pH units greater than those of the 2′- and 3′-
hydroxyl groups, which might suggest a faster rate of cleavage
than that of isomerization if the proton shift to the leaving group
determines the rate. The balance between these factors is not
clear, and thus we can only say that the primary 18O isotope
effect on cleavage is greater than 0.19% at 86 °C. The small
normal intrinsic primary isotope effect results from the com-
bination of two opposite effects, the loss of the P-O stretch
and the formation of an O-H bond. If protonation of the leaving
group is so far advanced that it largely compensates for the loss
of the P-O stretch, a slightly normal primary isotope effect
would be expected.
Discussion
The pK of the hydroxyl group of the leaving group m-
nitrobenzyl alcohol is 14.9,9a close to the pK’s of both the 3′-
and 2′-hydroxyl groups of the ribose ring which are respectively
12.84 and 12.55.4b The similarity in the leaving groups pK’s is
reflected in the almost identical pH rate profiles for the cleavage
and isomerization reactions of polyuridylic acid, uridylyl (3′,5′)-
uridine4b,c and uridine 3′-m-nitrobenzyl phosphate. Thus uridine
3′-m-nitrobenzyl phosphate can be considered a good model
compound to study the kinetics and mechanism of the isomer-
ization and cleavage reactions of RNA. In addition there is only
one nitrogen atom in the leaving group, m-nitrobenzyl alcohol,
making uridine 3′-m-nitrobenzyl phosphate an ideal candidate
to measure the kinetic isotope effects of the isomerization and
cleavage reactions with the remote isotope-labeling method.11
The minimal mechanism for the cleavage and isomerization
reactions is shown in Scheme 1, where k3 is the irreversible
step for the cleavage reaction and k1 and k2 represent the rates
of formation from and breakdown of a phosphorane to a
phosphodiester (uridine 3′- or 2′-m-nitrobenzyl phosphate). X,
S, and P represent, respectively, all of the possible phosphorane
intermediates that can be formed through pseudorotation, the
sum of the 3′- and 2′-isomers, and the cleavage products. In
the reaction with the bridge-labeled uridine 3′-m-nitrobenzyl
phosphate, the only isotope-sensitive step is k3 because no
significant change in bond order occurs at the bridge oxygen
in the previous steps. Therefore, the apparent 18k isotope effect
is given by eq 3 where 18k3 is the intrinsic isotope effect and
k3/k2 is the forward commitment. The forward commitment
represents the propensity of the phosphorane to go forward to
the cleavage products as opposed to reforming one of the
phosphodiesters.
The inverse secondary 18O isotope effect of 1.15% (corrected
to 27 °C) indicates an increase in bond strength with the
(15) An inverse secondary isotope effect of ∼1.5% can be expected from
the protonation of the second nonbridge oxygen when forming a neutral
phosphorane from a monoanionic phosphorane (Scheme 2) based on
secondary isotope effects of 1.54 and of 1.9%, respectively, measured for
the deprotonation of glycerol-3-phosphate and of phosphoric acid (see ref
17). If we assume that the strength of a PdO and a P-OH bond are similar,
based on the 18O fractionation factors of 1.033 for the hydroxyl of L-malate
and of 1.031 for the carbonyl group of ketoglutarate (see ref 18), then the
secondary isotope effect for the reaction from an anionic phosphodiester to
a monoanionic phosphorane can be predicted to be close to unity and that
for forming a neutral phosphorane should be ∼1.5% inverse. A second
model uses the normal secondary isotope effect of 2.5% measured for the
base-catalyzed hydrolysis of the triester diethyl(4-carbamoylphenyl) phos-
phate (see ref 19) to determine the change from a PdO to a P-O- bond.
With this model a secondary isotope effect of ∼0.7% inverse is predicted
in the formation of a neutral phosphorane and of ∼0.9% normal for the
formation of a monoanionic phosphorane from an anionic phosphodiester.
It is not clear which model is more correct, but the isotope effects we
observed are closer to those predicted by the first model.
18
k
) (18k3 + k3/k2)/(1 + k3/k2)
(3)
obs
An approximate value of the intrinsic primary isotope effect
can be calculated from this equation by assuming a reasonable
value for the forward commitment as described later.
Both the formation and breakdown of the phosphorane
intermediates are isotope-sensitive steps in the reaction with the
nonbridge labeled uridine 3′-m-nitrobenzyl phosphate, and thus
(16) The value of the intrinsic primary isotope effect calculated at 86
°C cannot be corrected to room temperature without knowing how much
of the value is temperature-independent, but it cannot be larger than 1.0033
at 27 °C.
(17) Knight, W. B.; Weiss, P. M.; Cleland, W. W. J. Am. Chem. Soc.
1986, 108, 2759-2761.
(18) Cleland, W. W. Methods Enzymol. 1980, 64, 104-125.
(13) Kirby, A. J.; Varvoglis, A. G. J. Am. Chem. Soc. 1967, 89, 415-
423.
(14) Gorenstein, D. G. Phosphorus-31 NMR: Principles and Applica-
tions; Academic Press: Orlando, Fla, 1984; pp 7-36.

Isomerization and CleaVage Reactions of RNA
J. Am. Chem. Soc., Vol. 122, No. 51, 2000 12619
Scheme 2
nonbridge oxygens in the formation of the phosphorane
intermediate. The inverse effect is due to the formation of a
neutral phosphorane which has an additional O-H stretch and
bending motions with respect to the monoanionic phosphodi-
esters. Since the 18O equilibrium isotope effect for protonation
of a phosphate nonbridge oxygen is 1.6% inverse,17 the
magnitude of the secondary 18O isotope effect represents a
somewhat late transition state, which is not surprising, consider-
ing the relative instability of the phosphorane intermediate.
The primary and secondary kinetic isotope effects observed
are consistent with the mechanism described in Scheme 2. An
initial preequilibrium protonation step is followed by the
formation of a neutral phosphorane intermediate with a con-
comitant water-mediated proton transfer from the 2′-hydroxyl
group to the second nonbridge oxygen. Models suggest the
number of water molecules needed to mediate this proton
transfer is two or three. After the nucleophile 2′-hydroxyl attack
at an apical position, the phosphorane intermediate formed is
in fast equilibrium with all of the possible phosphoranes formed
by polytopal rearrangements. The m-nitrobenzyl alcohol departs
from a phosphorane in which it is located in the apical position.
The isotope effects measured also agree with a mechanism of
the cleavage reaction where the neutral phosphorane formed
with both the m-nitrobenzyl group and the 2′-hydroxyl in apical
positions. This phosphorane may give the cleavage products
without pseudorotating.
nitrobenzyl phosphate are similarly dependent on the hydronium
ion concentration (Figure 2) and the cleavage reaction proceeds
through a neutral phosphorane intermediate, it is likely that the
isomerization reaction in acidic pHs proceeds through the same
neutral phosphorane intermediate which after pseudorotation
may undergo cleavage of the apical bond with the m-nitrobenzyl
moiety (cleavage reaction) or of the apical bond with the 3′-
hydroxyl of the ribose ring (isomerization reaction) as shown
in Scheme 2.
pH-Independent Reactions.15 The isomerization reaction
must proceed through a phosphorane intermediate, while the
cleavage reaction may be either concerted or stepwise. The
stepwise mechanisms for the isomerization and cleavage reac-
tions are shown in Scheme 3. The putative phosphorane may
exist in several ionization states. At acidic pH an inverse
secondary isotope effect was observed, indicating the formation
of a neutral phosphorane. A monoanionic phosphorane inter-
mediate is proposed for the isomerization reaction at neutral
pH based on the secondary isotope effect close to unity and the
pH-independent rate (Figure 2). A secondary isotope effect close
to unity was also measured for the cleavage reaction, indicating
that the two reactions may proceed through the same mono-
anionic phosphorane intermediate as illustrated in Scheme 3.
In the breakdown of the phosphorane intermediate to give the
2′-isomer, the bond order with the bridge oxygen should remain
essentially unchanged, which is consistent with the observed
bridge oxygen isotope effect close to unity. In the cleavage
reaction the bond between the bridge oxygen and the phosphorus
is broken in the degradation of the phosphorane intermediate.
The observed primary 18O isotope effect is 0.9%. Calculation
of the intrinsic primary isotope effect requires assumption of a
value for the forward commitment in eq 3. If we assume a
forward commitment of 0.5, as we did earlier for the hydronium-
catalyzed reaction, the intrinsic primary isotope effect is 1.35%.
The same uncertainties in the forward commitment value apply
here, and thus we can only say that the primary 18O isotope
Westheimer’s rules dictate that both the leaving group and
the nucleophile must respectively depart or enter in the apical
position of a phosphorane.1 It is thus impossible for the 2′-
hydroxyl to attack in a concerted manner with the departure of
the 3′-hydroxyl of the ribose moiety. Thus, the isomerization
of uridine 3′-m-nitrobenzyl phosphate to uridine 2′-m-nitrobenzyl
phosphate must occur through a phosphorane intermediate. Since
the rates of isomerization and cleavage of uridine 3′-m-
(19) Caldwell, S. R.; Raushel, F. M.; Weiss, P. M.; Cleland, W. W. J.
Am. Chem. Soc. 1991, 30, 7444-7450.

12620 J. Am. Chem. Soc., Vol. 122, No. 51, 2000
Gerratana et al.
Scheme 3
Figure 4. Proposed transition-state structure for the concerted cleavage
reaction at neutral pH.
Figure 5. Proposed transition-state structure for the concerted cleavage
reaction in alkaline conditions.
effect on cleavage is greater than 0.9%. The size of the primary
effect indicates that the bond between the phosphorus and the
bridge oxygen is considerably cleaved in the transition state.
Thus, the secondary and primary isotope effects measured for
the cleavage reaction are consistent with a stepwise mechanism
with a monoanionic phosphorane intermediate. As for the
reaction in acidic conditions, a water-mediated proton transfer
from the 2′-hydroxyl group to the second nonbridge oxygen is
required for the formation of the phosphorane intermediate as
shown by the absence of the isomerization reaction in anhydrous
conditions in tert-butanol.
The isotope effects observed for the cleavage reaction do not
invalidate an ANDN mechanism with a transition state resembling
a phosphorane (Figure 4) in which the bond with the m-
nitrobenzyl group has been weakened and the strength of the
bonds between the phosphorus and the nonbridge oxygens has
not changed significantly from the ground state. This transition-
state structure differs from the structure of the transition state
for the ribonuclease A reaction in which the secondary isotope
effect of 0.5% and the primary isotope effect of 1.6% were
consistent with a concerted mechanism with a slightly associa-
tive transition state.9a
a phosphorane intermediate, did not occur in anhydrous con-
ditions. (In aqueous solution at pH 9.0, the isomerization
was readily detected under conditions where the cleavage
reaction was three times faster than in tert-butyl alcohol.)
This result proves the absolute requirement of a water-
mediated proton transfer for the formation of a phosphorane
intermediate. The cleavage reaction was observed in anhydrous
conditions with a rate 3-fold faster than the cleavage rate at
pH 6.0. We believe that a small level of base present in the
solution catalyzed the cleavage reaction. This base-catalyzed
cleavage is an ANDN reaction and does not proceed through a
phosphorane intermediate.
Hydroxide Ion-Catalyzed Reactions. The transition-state
structure for the alkaline hydrolysis of uridine 3′-m-nitrobenzyl
phosphate is shown in Figure 5. The primary isotope effect of
2.72% suggests that the P-Obridge bond is largely broken in the
transition state and that the m-nitrobenzyl group mainly leaves
as an alkoxide. A mechanism with a phosphorane intermediate
is unlikely based on ab initio calculations suggesting that a
dianionic phosphorane is too unstable to exist as an intermediate8
and considering that the isomerization reaction which requires
a phosphorane intermediate is not observed. The hydroxide ion-
catalyzed cleavage reaction is thus a concerted SN2 reaction.
Reactions in Anhydrous tert-Butanol. The need for water
catalysis of the pH-independent isomerization and cleavage
reactions was tested by running the reaction in anhydrous
tert-butanol. The isomerization, which must proceed through

Isomerization and CleaVage Reactions of RNA
J. Am. Chem. Soc., Vol. 122, No. 51, 2000 12621
Rc ) P*/P
Conclusions
The kinetic isotope effects measured at pH 2.5 and the pH
rate profile in the acidic range support a mechanism involving
a neutral phosphorane intermediate for the isomerization and
cleavage reactions of uridine 3′-m-nitrobenzyl phosphate. Both
a concerted transition state with very little change in bond order
with the nonbridge oxygens and a stepwise mechanism employ-
ing a monoanionic phosphorane intermediate are consistent with
the reported isotope effects for the pH-independent cleavage
reaction. The pH-independent isomerization reaction proceeds
through the monoanionic phosphorane. We have shown the
absolute requirement of water molecules to mediate a concerted
proton transfer for the formation of the phosphorane intermediate
by running the reaction in anhydrous tert-butyl alcohol. The
alkaline hydrolysis of uridine 3′-m-nitrobenzyl phosphate is a
concerted reaction with a transition state in which the leaving
group departs with almost a full negative charge.
The ratio of Rc/Ro is then:
18k3
-k3t
Rc/Ro ) (1 - e-k t/ )/(1 - e
)
(8)
3
The fraction of cleavage is given by:
fc ) P/Ao ) (1 - e-k t
)
3
and:
so:
(1 - fc) ) -e-k t
3
ln(1 - fc) ) -k3t
Replacing -k3t in eq 8:
Acknowledgment. This work was supported by NIH Grant
GM18938 to W.W.C. This study made use of the National
Magnetic Research Facility at Madison, which is supported by
NIH Grant RR02301 from the Biomedical Research Technology
Program, National Center for Research Resources. Equipment
in the facility was purchased with funds from the University of
Wisconsin, the NSF Biological Instrumentation Program (Grant
DMB-8415048), NIH Biomedical Research Technology Pro-
gram (Grant RR02301), NIH Shared Instrumentation Program
(Grant RR02781) and the U.S. Department of Agriculture. We
thank Armand R. Krueger of the Horticulture Department at
the University of Wisconsin-Madison for use of the Europa
Tracermass 20-20 isotope ratio mass spectrometer. We thank
Dr. Perry A. Frey for helpful comments.
18k3
Rc/Ro ) (1 - e-k t/ )/fc
3
or:
18k3
1 - (Rc fc/Ro) ) e-k t/
3
Then:
ln[1 - (Rc fc/Ro)] ) -k3t/18k3
and, replacing -k3t
18k3 ) ln(1 - fc)/(ln[1 - (Rc fc/Ro)])
(1)
For the isotope effect on isomerization, we define the mass ratio
in B as:
Appendix
The scheme for reaction of uridine 3′-m-nitrobenzyl mono-
phosphate (A) to give the 2′-isomer (B) or to give the cleavage
products m-nitrobenzyl alcohol and 2′,3′-cyclic UMP (P) can
be diagrammed:
Ri ) B*/B
and the fraction of isomerization as:
fi ) B/Ao ) (1/2)(e-k t - e-(2k +k )t
)
3
1
3
Then:
18k3
-[(2k1/18k1)+(k3/18k3)]t
Ri/Ro ) (1/2)(e-k t/ - e
)
3
When the differential equations for this scheme are solved,
the concentrations of A, B, and C are given by:
and:
18k3
-2k1t/18k1
-k3t/18k3
Ri fi/Ro ) (1/2)[e-k t/ - (e
)(e
(1/2)(1 - Rc fc/Ro)(1 - e-2k t/
)] )
3
A ) (Ao/2)(e-(2k +k )t + e-k t
)
)
(5)
(6)
(7)
1
3
3
18k1
1
)
B ) (Ao/2)(e-k t - e-(2k +k )t
3
1
3
Rearranging and taking logs:
P ) Ao(1 - e-k t
)
3
18k1 ) -2k1t/ln[1 - (2Ri fi/Ro)/[1 - (Rc fc/Ro)]]
(9)
Rearranging the equation for fi and substituting for e-k t gives:
To derive the isotope effects for cleavage (k3) and isomer-
ization (k1), one first writes a similar set of equations for the
18O-labeled species, adding an asterisk to A, B, P, and Ao to
indicate the label, and dividing each rate constant by the isotope
effect on this rate constant. Thus k1 is divided by 18k1 and k3 by
18k3. The mass ratio in the initial 3′-isomer is then:
3
1
e
-2k t ) (1 - fc - 2fi)/(1 - fc)
Taking logs and replacing -2k1t in eq 9:
18k1 ) ln[(1 - fc - 2fi)/(1 - fc)]/
Ro ) A/o/Ao
ln{1 - [(2Ri fi/Ro)/[1 - (Rc fc/Ro)]]} (2)
while the mass ratio in P at a fraction of cleavage, fc, is:
JA003400V