Hydrolytic stability of 2′,3′-O-methyleneadenos-5′-yl 2′,5′-di-O-methylurid-3′-yl 5′-O-methylurid- 3′(2′)-yl phosphate: Implications to feasibility of existence of phosphate-branched RNA under physiological conditions

Article abstract of DOI:10.1039/b500054h

Hydrolytic reactions of 2′,3′-O-methyleneadenos-5′-yl 2′,5′-di-O-methylurid-3′-yl 5′-O-methylurid- 3′(2′)-yl phosphate (1a,b) have been followed by RP-HPLC over a wide pH range to evaluate the feasibility of occurrence of phosphate-branched RNA under phys

Full text of DOI:10.1039/b500054h

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A R T I C L E
Hydrolytic stability of 2ꢀ,3ꢀ-O-methyleneadenos-5ꢀ-yl 2ꢀ,5ꢀ-di-O-
methylurid-3ꢀ-yl 5ꢀ-O-methylurid-3ꢀ(2ꢀ)-yl phosphate: implications to
feasibility of existence of phosphate-branched RNA under
physiological conditions†
Tuomas Lo¨nnberg,* Johanna Kiiski and Satu Mikkola
Department of Chemistry, University of Turku, FIN-20014, Turku, Finland.
E-mail: tuanlo@utu.fi
Received 6th January 2005, Accepted 24th January 2005
First published as an Advance Article on the web 16th February 2005
Hydrolytic reactions of 2ꢀ,3ꢀ-O-methyleneadenos-5ꢀ-yl 2ꢀ,5ꢀ-di-O-methylurid-3ꢀ-yl 5ꢀ-O-methylurid-3ꢀ(2ꢀ)-yl phosphate
(1a,b) have been followed by RP-HPLC over a wide pH range to evaluate the feasibility of occurrence of
phosphate-branched RNA under physiological conditions. At pH <2, where the decomposition of 1a,b is first order
in [H3O+], the P–O5ꢀ bond is cleaved 1.5 times as rapidly as the P–O3ꢀ bond. Under these conditions, the reaction
probably proceeds by an attack of the 2ꢀ-OH on the phosphotriester monocation. Over a relatively wide range from
pH 2 to 5, the hydrolysis is pH-independent, referring to rapid initial deprotonation of the attacking 2ꢀ-OH followed
by general acid catalyzed departure of the leaving nucleoside. The P–O5ꢀ bond is cleaved 3 times as rapidly as the
P–O3ꢀ bond. At pH 6, the reaction becomes first order in [HO−], consistent with an attack of the 2ꢀ-oxyanion on
neutral phosphate. The product distribution is gradually inversed: in 10 mmol L−1 aqueous sodium hydroxide,
cleavage of the P–O3ꢀ bond is favored over P–O5ꢀ by a factor of 7.3. The results of the present study suggest that the
half-life for the cleavage of 1a,b under physiological conditions is only 100 s. Even at pH 2, where 1a,b is most stable,
the half-life for its cleavage is less than one hour and the isomerization between 1a and 1b is even more rapid than
cleavage. The mechanisms of the partial reactions are discussed.
likelihood is of minor importance, since the reactions of small
Introduction
molecule ribonucleoside 3ꢀ-phosphodiesters and triesters are not
It has been suggested that two small nuclear RNAs, viz. U2
sensitive to the identity of base moieties. The results also shed
and U6 snRNA of human spliceosome, form a phosphotriester
some light on the transesterification reaction catalyzed by the
structure by an attack of the 2ꢀ-OH group of adenosine
self-splicing group II introns, which occurs by a mechanism
21 of U2 snRNA on an A53pG54 phosphodiester bond of
similar to that proposed for the RNA X formation.
U6 snRNA.1,2 This finding is somewhat unexpected because
ribonucleoside 3ꢀ-dialkylphosphates are known to undergo a
Results
facile nucleophilic attack of the neighboring 2ꢀ-OH function on
Preparation of fully protected trinucleoside 3ꢀ(2ꢀ),3ꢀ,5ꢀ-
monophosphates (2a,b)
the phosphotriester group.3,4 To evaluate the hydrolytic stability
of the proposed species by a model compound that mimics
phosphate-branched RNA (RNA X) more closely than simple
3ꢀ-dialkylphosphates, a trinucleoside-2ꢀ,3ꢀ,5ꢀ-monophosphate,
2ꢀ,3ꢀ-O-methyleneadenos-5ꢀ-yl 2ꢀ,5ꢀ-di-O-methylurid-3ꢀ-yl 5ꢀ-O-
methylurid-3ꢀ-yl phosphate (1a), has been synthesized and its
decomposition studied over a wide pH range. While in RNA X
(Fig. 1) the 5ꢀ-OH of A21, 3ꢀ-and 5ꢀ-OH of A53 and 3ꢀ-OH of G54
are involved in phosphodiester linkages, in the model compound
(1a) the respective sites are alkylated. The base moieties of 1a
differ from those present in the RNA X structure, but this in all
N6-Benzoyl-2ꢀ,3ꢀ-O-methyleneadenos-5ꢀ-yl 2ꢀ,5ꢀ-di-O-methyl-
urid-3ꢀ-yl 2ꢀ(3ꢀ)-O-(4,4ꢀ-dimethoxytrityl)-5ꢀ-O-methylurid-3ꢀ(2ꢀ)-
yl phosphates (3a,b) were obtained by 4,5-dicyanoimidazole-
(3a) or tetrazole-(3b) promoted stepwise displacement of the
dimethylamino groups from tris(dimethylamino)phosphine
with the appropriately protected nucleosides (4, 5, 6) (Schemes 1
and 2). The nucleosides were prepared as described in the
literature.5–9 Removal of the N6-benzoyl protection with
methanolic ammonia, followed by RP-HPLC purification,
then gave the desired triesters 2a,b. Attempts to separate the
two diastereomers of 2a were unsuccessful. In contrast, a
good separation of the diastereomers of 2b was achieved by
RP-HPLC.
† Electronic supplementary information (ESI) available: 13C NMR
spectra of compounds 2a, 2b, 4a, 4b, 10a and 10b. See http://
www.rsc.org/suppdata/ob/b5/b500054h/
Fig. 1 Trinucleoside monophosphate 1a as a mimic for the proposed RNA X structure.
T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 5 O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 1 0 8 9 – 1 0 9 6
T h i s j o u r n a l i s
1 0 8 9
©

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Scheme 1 Preparation of the trinucleoside 3ꢀ,3ꢀ,5ꢀ-monophosphate 2a‡.
Scheme 2 Preparation of the trinucleoside 2ꢀ,3ꢀ,5ꢀ-monophosphate 2b§.
Product distribution and reaction pathways
from the reaction mixture at appropriate time intervals by RP-
HPLC. The 2ꢀ-O-(4,4ꢀ-dimethoxytrityl) group of 2a,b was first
removed with 99 mmol L−1 HCl in 1,4-dioxane to give 1a,b,
after which the appropriate aqueous solution was created in
the reaction vessel so that the final amount of 1,4-dioxane was
The hydrolysis of 1a,b was followed over a wide pH range (0–9)
at 25 ◦C by analyzing the composition of the aliquots withdrawn
‡ Reagents and conditions: (a) DMTrCl, Py, (b) chromatographic
separation, P(NMe2)3, DCI, MeCN, (c) 2ꢀ,5ꢀ-di-O-methyluridine (5),
DCI, MeCN, (d) 6, DCI, MeCN, (e) I2, H2O, THF, 2,6-lutidine, (f)
NH3, MeOH.
§ Reagents and conditions: (a) P(NMe2)3, tetrazole, MeCN, (b) 5,
tetrazole, MeCN, (c) 4b, tetrazole, MeCN, (d) I2, H2O, THF, 2,6-lutidine,
(e) NH3, MeOH.
1 0 9 0
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 1 0 8 9 – 1 0 9 6

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Scheme 3 Hydrolytic reaction pathways of 1a,b.
5% (v/v). The aliquots were quenched by cooling to −16 ◦C
and adjusting their pH to approximately 3 with a formic acid
buffer when necessary. The products were identified by spiking
with authentic samples and the peak areas were converted to
relative concentrations by calibrating the system with uridine
and adenosine solutions of known concentration.
from 0.04 to 0.24 mol L−1 and extrapolating the observed pseudo
first-order rate constants to zero buffer concentration by linear
regression.
pH–Rate profiles
Fig. 2 shows the pH–rate profile for the overall disappearance
of 1. At pH <2, the overall reaction is first-order in [H3O+]. The
predominant reaction is cleavage of the P–O5ꢀ bond (Scheme 3,
route A), accounting for 60% of the hydrolysis of 1. Over a
relatively wide range from pH 2 to 5, the overall reaction is
pH-independent and cleavage of the P–O5ꢀ bond (route A)
predominates, accounting for 75% of the overall hydrolysis. At
pH 6, the overall reaction becomes first-order in [HO−] and
cleavage of the P–O3ꢀ bond (route B) gradually starts to take
over (Fig. 3). In 10 mmol L−1 aqueous sodium hydroxide, 88%
of the overall hydrolysis results from cleavage of the P–O3ꢀ bond.
Two distinct sets of products were observed in the entire pH
range (Scheme 3). Release of 2ꢀ,3ꢀ-O-methyleneadenosine (m <
A, 8) by P–O5ꢀ bond fission produced the isomeric diesters 5ꢀ-O-
methyluridylyl-3ꢀ,3ꢀ-(2ꢀ,5ꢀ-di-O-methyluridine) [mU(3ꢀ,3ꢀ)m2U,
9a] and 5ꢀ-O-methyluridylyl-2ꢀ,3ꢀ-(2ꢀ,5ꢀ-di-O-methyluridine)
[mU(2ꢀ,3ꢀ)m2U, 9b]. Cleavage of one of the P–O3ꢀ bonds, in
turn, resulted in the formation of 2ꢀ,5ꢀ-di-O-methyluridine
(m2U, 5) and the isomeric diesters 5ꢀ-O-methyluridylyl-
3ꢀ,5ꢀ-(2ꢀ,3ꢀ-O-methyleneadenosine) [mU(3ꢀ,3ꢀ)m
and
5ꢀ-O-methyluridylyl-2ꢀ,5ꢀ-(2ꢀ,3ꢀ-O-methyleneadenosine)
< A, 10a]
[mU(2ꢀ,3ꢀ)m < A, 10b]. In acidic solutions, formation of the
former set predominated, in alkaline solutions the latter. The
product distribution was not dependent on the regioisomer
(1a,b) or the diastereomer of 1b used as a starting material.
No nucleoside monophosphates were formed regardless of pH,
indicating that the diester products were not hydrolysed under
the experimental conditions.
The pseudo first-order rate constants for the decomposition
of 1 were obtained by applying the integrated first-order rate
equation to the time-dependent diminution of the concentration
of the starting material. Between pH 3 and 6, considerable
buffer catalysis was observed (Table 1). Buffer-independent rate
constants were obtained by varying the buffer concentration
Table 1 Second-order rate constants for the buffer-catalyzed cleavage
of 1a,b
Buffer acid
Formic acid
Acetic acid
[HA]/[A−]
kclbuf /10−4 L mol−1 s−1
Fig. 2 pH–Rate profile for the decomposition (eqn. (1)) and mutual
isomerization (eqn. (2)) of 1a,b at 25 ◦C, I(NaNO3) = 1.0 mol L−1: (᭹)
cleavage of the diastereomeric mixture of 1a, (ꢀ) cleavage of the first
diastereomer of 1b, (ꢁ) cleavage of the second isomer of 1b, (ꢂ) mutual
isomerization of 1a and 1b.
3 : 1
1 : 3
1 : 1
1 : 3
6.3 0.8
21
29
47
2
3
3
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 1 0 8 9 – 1 0 9 6
1 0 9 1

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Table 2 Rate constants for the partial reactions of cleavage and
isomerization of 1a,b
kH/10−3 L mol−1 s−1
kW/10−5 s−1
k
OH/103 L mol−1 s−1
kcl
kis
k1
k2
1.6 0.3
5.96 0.07
1.0 0.2
0.6 0.1
9.0 0.9
—
6.8 0.7
2.3 0.2
7.5 0.9
50 000 10 000
0.9 0.1
6.6 0.8
concentrations of the monomeric products of these competing
reactions (eqns. (3) and (4)). The calculated rate constants are
presented in Table 2.
[m < A]
k1 =
k2 =
kcl
(3)
[m < A] + [m2U]
[m2U]
Fig. 3 Ratio of k1 (route A) to k1 + k2 (overall cleavage) as a function
of pH (T = 25 ◦C, I(NaNO3) = 1.0 mol L−1).
kcl
(4)
[m < A] + [m2U]
Discussion
Mutual isomerization of 1a and 1b is rapid and it can only
be followed at pH <3.3,4 At this pH, the reaction is already
first-order in [HO−]. In other words, isomers 1a and 1b are
rapidly equilibrated except under markedly acidic conditions.
Essentially identical rate constants were observed regardless of
the regioisomer (1a,b) or the diastereomer of 1b used as a starting
material.
Hydronium-ion catalyzed cleavage
At pH <2, the overall disappearance of 1 is first-order in [H+].
In this acid-catalyzed region, the leaving group departs as an
alcohol (Scheme 4) and the rate of the reaction is only moderately
sensitive to the acidity of the leaving group, the blg value being
−0.40.3 The ratio of the second-order rate constant for the
hydronium-ion catalyzed cleavage of the P–O5ꢀ bond (route A),
Under conditions where the overall cleavage is pH-
independent, considerable buffer catalysis was observed. Only
the basic buffer constituent, viz. the carboxylate anion, con-
tributed to the catalysis. Table 1 summarizes the second-order
rate constants for the catalysis by formic and acetic acid buffers
at two different ratios of [HA]/[A−].
k1 (0.96 × 10−3 L mol−1 s−1), to the respective rate constant
H
for the cleavage of the P–O3ꢀ bond (route B), k2 (0.64 ×
H
10−3 L mol−1 s−1), is 1.5, whereas the previously reported blg value
of −0.40 and the approximate DpKa of 1.0 between 8 and 510
would predict a ratio of 0.4. This discrepancy might be attributed
to the fact that the 2ꢀ,5ꢀ-di-O-methylurid-3ꢀ-yl group as a steri-
cally demanding substituent prefers an equatorial position in the
pentacoordinated phosphorane intermediate, thus making its
departure less probable.11 For comparison, a similar, albeit some-
what smaller, effect has been observed with the hydronium-ion
catalyzed hydrolysis of uridine-3ꢀ-alkylphosphates: the rate cons-
tant for the isopropyl derivative is only 40% of the value pre-
dicted by the results obtained with other alkyl groups.12 Since
2ꢀ,5ꢀ-di-O-methylurid-3ꢀ-yl is even bulkier than isopropyl, the
observed rate retardation is of expected magnitude. Taking this
The observed rate constant for the cleavage of 1, kclobs, may be
expressed by eqn. (1). A similar equation may be written for the
isomerization (eqn. (2)).
OH
cl
k
KW
obs
cl
k
= kHaH
+
+ kWcl
+
+
(1)
cl
aH
+
OH
is
k
KW
obs
is
k
= kHaH
+
+ kWis
(2)
is
aH
+
W
OH
kclH, kcl and kcl are the rate constants for the hydronium-
ion catalyzed, pH-independent and hydroxide-ion catalyzed
steric effect into account, the ratio of rate constants k1H and k2
agrees fairly well with the previously reported blg value of −0.40.
H
W
OH
cleavage, respectively. Similarly, kisH, kis and kis
are the
respective rate constants for isomerization. These rate constants
were obtained by fitting the experimental values to eqns. (1)
and (2) by a nonlinear least-squares method. The rate constants
for the overall cleavage of 1, kcl, can then be bisected to the
rate constants for the partial reactions, viz. cleavage of P–O5ꢀ
(route A) and P–O3ꢀ (route B) bonds, on the basis of the relative
Hydroxide-ion catalyzed cleavage
At pH >6, the overall disappearance of 1 is first-order in [OH−]
and the departure of 5 (route B) is favored over that of 8 (route
A). This change in the product distribution is expected, since in
Scheme 4 Mechanism of the hydronium-ion catalyzed cleavage of 1a,b.
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 1 0 8 9 – 1 0 9 6
1 0 9 2

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Scheme 5 Mechanism of the hydroxide-ion catalyzed cleavage of 1a,b.
acid-catalyzed hydrolysis the leaving group departs as an alcohol pH-Independent and buffer catalyzed cleavage
and in base-catalyzed hydrolysis as an alkoxide ion (Scheme 5).
The base-catalyzed cleavage is thus considerably more sensitive
to the pKa of the conjugate acid of the leaving group, the blg
value being −1.38.3 Evidently, isomerization is also first-order
in [OH−] and too rapid to be followed. The ratio of the second-
order rate constant for the hydroxide-ion catalyzed cleavage of
In the relatively wide range from pH 2 to 5, where isomerization
is much faster than cleavage, the cleavage is pH-independent
but susceptible to general base catalysis by carboxylate buffer
anions. The latter reaction may be interpreted to be a sequential
specific base/general acid catalysis, where the attacking 2ꢀ-OH is
deprotonated in a pre-equilibrium step and proton transfer from
the general acid to the leaving oxyanion occurs concerted with
the rate-limiting P–O bond scission (Scheme 6).3 The second-
order rate constant for the buffer catalyzed cleavage is nearly
independent of the leaving group (8 vs. 5), probably because
the polar effect of the departing nucleoside on the rate of proton
transfer and bond cleavage are opposite. In striking contrast, the
first-order rate constants for the pH- and buffer-independent
cleavage differ considerably, the departure of 8 being favored
over that of 5 by a factor of 3. This behavior, albeit surprising,
may tentatively be explained as follows. In all likelihood the
the P–O5ꢀ bond (route A), k1 (0.9 × 103 L mol−1 s−1), to the
OH
respective rate constant for the cleavage of the P–O3ꢀ bond (route
OH
B), k2 (6.6 × 103 L mol−1 s−1), is 0.14, while a ratio of 0.042
would be expected on the basis of the previously reported blg
value and the difference between pKa values of 8 and 5. As in
the case of the hydronium-ion catalyzed cleavage, this probably
results from the fact that the bulky 2ꢀ,5ꢀ-di-O-methylurid-3ꢀ-yl
group prefers equatorial orientation in the pentacoordinated
phosphorane intermediate. Taking this into account, the ratio
OH
OH
of the observed rate constants k1 and k2 agrees reasonably
well with the known blg value of −1.38.
Scheme 6 Mechanism of the pH-independent and buffer-catalyzed cleavage of 1a,b.
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 1 0 8 9 – 1 0 9 6
1 0 9 3

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reaction involves a rate-limiting water-mediated proton transfer
from the phosphorane hydroxyl ligand to the departing oxygen
(Scheme 6).4,13 The ease of this kind of intramolecular proton
transfer is possibly sensitive to the structure of the solvation
sphere around the reaction center, and this, in turn, depends on
the detailed geometry of the phosphorane intermediate. The
Brønsted b value for the observed general base catalysis is
approximately 0.5, which means that the a value for the general
acid catalyzed step of the sequential specific base/general acid
catalysis is 0.5. The previously reported blg value for the reaction
is −0.73. Taking these results together, the proton transfer and
P–O bond fission appear to be more or less equally advanced in
the transition state.
On the basis of these results one may estimate the half-life for
the cleavage of 1a,b under physiological conditions to be only
100 s. Even at pH 2, where 1a,b is most stable, the half-life for
its cleavage is less than one hour and the isomerization between
1a and 1b is even more rapid than cleavage. The existence of
RNA phosphotriester linkages such as the one in RNA X in
biological systems seems, hence, doubtful at best. It should be
noted, though, that while the observations on RNA X formation
refer to in vitro transcribed snRNAs composed entirely of the
four native ribonucleotides,1,2 in nature, the two residues flanking
the proposed phosphotriester linkage, A53 and G54, are 2ꢀ-O-
methylated14, which undoubtedly stabilizes the phosphotriester
toward hydrolysis. On the basis of our previous studies on
the hydrolytic stability of a fully alkylated trinucleoside-3ꢀ,3ꢀ,5ꢀ-
monophosphate, 2ꢀ,3ꢀ-O-methyleneadenos-5ꢀ-yl bis(2ꢀ,5ꢀ-di-O-
methylurid-3ꢀ-yl) phosphate, this stabilization may be estimated
to be at least three orders of magnitude at neutral pH, resulting
in a half-life of more than one day.10 The possible shielding of
the phosphotriester linkage from solvent by the folded RNA
strands remains a topic for further experiments. Alternatively,
the conformation of the folded RNA structure might cause an
unfavorable orientation of the 2ꢀ-OH nucleophile with respect
to the phosphate moiety, thus inhibiting the cleavage.11,15 It is
also evident on the basis of this study that steric demands of the
pentacoordinated phosphorane intermediate can be a significant
factor in determining the course of splicing reactions catalyzed
by large ribozymes.
2ꢀ,3ꢀ-O-Methyleneadenos-5ꢀ-yl 2ꢀ,5ꢀ-di-O-methylurid-3ꢀ-yl 2ꢀ-O-
(4,4ꢀ-dimethoxytrityl)-5ꢀ-O-methylurid-3ꢀ-yl phosphate (2a). 2ꢀ-
O-(4,4ꢀ-Dimethoxytrityl)-5ꢀ-O-methyluridine (4a, 0.299 mmol,
0.1678 g) was coevaporated twice from anhydrous pyridine and
once from anhydrous MeCN. The residue was dissolved in
anhydrous MeCN (10 mL) and tris(dimethylamino)phosphine
(0.33 mmol, 0.06 mL) was added. A solution of 4,5-
dicyanoimidazole (0.36 mmol, 0.0425 g) in MeCN (1.44 mL) was
added and the reaction mixture was stirred at room temperature
for 20 h. 2ꢀ,5ꢀ-Di-O-methyluridine5–7 (5, 0.3012 mmol, 0.082 g)
was coevaporated twice from anhydrous pyridine and once from
anhydrous MeCN, and the residue was added to the reaction
mixture. A solution of 4,5-dicyanoimidazole (0.36 mmol, 0.0425 g)
in MeCN (1.44 mL) was added and the reaction mixture
was stirred at room temperature for 24 h. N6-Benzoyl-2ꢀ,3ꢀ-O-
methyleneadenosine8,9 (6, 0.334 mmol, 0.1282 g) was coevapo-
rated twice from anhydrous pyridine and once from anhydrous
MeCN, and the residue was added to the reaction mixture.
A solution of 4,5-dicyanoimidazole (0.36 mmol, 0.0425 g) in
MeCN (1.44 mL) was added and the reaction mixture was stirred
at room temperature for 24 h, followed by an additional 2 h at
45 ◦C. Iodine (0.47 mmol, 0.12 g) in a mixture of water (2.1 mL),
THF (4.2 mL) and 2,6-lutidine (1.1 mL) was added and the
reaction mixture was stirred for 90 min at room temperature.
CH2Cl2 (70 mL) was added and the resulting mixture was washed
with saturated aqueous NaHSO3. The aqueous phase was back
extracted with CH2Cl2 and the product was purified on a silica
gel column eluting with a mixture of MeOH, CH2Cl2 and Et3N
(5 : 94 : 1, v/v). The crude product thus obtained was dissolved in
saturated methanolic ammonia (20 mL). After being stirred for
5 h at room temperature, the reaction mixture was evaporated to
dryness. The product was purified by HPLC on a LiChrospher
RP-18 column (10 × 250 mm, 5 lm) eluting with a mixture of
0.05 mol L−1 of aqueous NH4OAc and MeCN (53 : 47, v/v).
Finally the buffer salts were removed on the same column by
eluting with a mixture of water and MeCN. Compound 2a was
obtained as two diastereomers in an approximately 1 : 2 ratio.
The overall yield starting from 4a was 4.3% (15 mg). Attempts
to separate the two diastereomers of 2a were unsuccessful but
because of the biased product distribution, the 1H NMR could
1
nevertheless be resolved. H NMR (dH) (500 MHz, DMSO-d6,
major diastereomer) 8.13 (s, 1H), 8.10 (s, 1H), 7.68 (d, 1H, J =
8.2 Hz), 7.60–6.60 (m, 13H), 7.15 (d, 1H, J = 8.2 Hz), 6.25 (d,
1H, J = 8.2 Hz), 6.13 (d, 1H, J = 2.4 Hz), 5.94 (d, 1H, J =
6.1 Hz), 5.55 (d, 1H, J = 8.2 Hz), 5.33 (dd, 1H, J1 = 2.4 Hz,
J2 = 6.4 Hz), 5.25 (d, 1H, J = 8.2 Hz), 5.13 (s, 1H), 5.07 (s,
1H), 5.06 (dd, 1H, J1 = 3.7 Hz, J2 = 6.6 Hz), 4.87 (m, 1H),
4.33 (m, 1H), 4.23 (m, 2H), 4.08 (m, 1H), 3.98 (m, 1H), 3.64 (s,
3H), 3.63 (s, 3H), 3.57 (m, 1H), 3.39 (m, 2H), 3.32–3.10 (m, 4H),
3.25 (s, 3H), 3.18 (s, 3H), 3.15 (s, 3H). 1H NMR (dH) (500 MHz,
DMSO-d6, minor diastereomer) 8.15 (s, 1H), 8.10 (s, 1H), 7.64
(d, 1H, J = 8.2 Hz), 7.60–6.60 (m, 13H), 7.20 (d, 1H, J =
8.2 Hz), 6.33 (d, 1H, J = 8.1 Hz), 6.14 (d, 1H, J = 2.4 Hz), 5.86
(d, 1H, J = 6.3 Hz), 5.55 (d, 1H, J = 8.2 Hz), 5.34 (dd, 1H, J1 =
2.4 Hz, J2 = 6.4 Hz), 5.25 (d, 1H, J = 8.2 Hz), 5.17 (s, 1H),
5.09 (s, 1H), 5.08 (dd, 1H, J1 = 3.1 Hz, J2 = 6.6 Hz), 4.84 (m,
1H), 4.36 (m, 1H), 4.23 (m, 2H), 4.08 (m, 1H), 3.95 (m, 1H),
3.62 (s, 3H), 3.61 (s, 3H), 3.60 (m, 1H), 3.40 (m, 2H), 3.32–3.10
(m, 4H), 3.26 (s, 3H), 3.23 (s, 3H), 3.14 (s, 3H). 31P NMR (dP)
(202 MHz, DMSO-d6, major diastereomer) −2.97. 31P NMR
(dP) (202 MHz, DMSO-d6, minor diastereomer) −2.07. HRMS
(FAB) M− calcd 1154.3508, obsd 1154.3497.
Experimental
Materials
Nucleosides were commercial products and they were used
as received after checking the purity by HPLC. The buffer
constituents were of reagent grade.
2ꢀ(3ꢀ)-O-(4,4ꢀ-Dimethoxytrityl)-5ꢀ-O-methyluridine (4a,b). 5ꢀ-O-
Methyluridine5 (7, 1.93 mmol, 0.4977 g) was coevaporated
three times from anhydrous pyridine. The residue was dissolved
in anhydrous pyridine (50 mL) and 4,4ꢀ-dimethoxytrityl chloride
(2.62 mmol, 0.8844 g) was added. After being stirred for 15 h at
room temperature and 24 h at 45 ◦C, the reaction mixture was
evaporated to dryness. A conventional CH2Cl2–aq. NaHCO3
workup was carried out and the product was purified on a silica
gel column eluting with a mixture of MeOH, CH2Cl2 and Et3N
(2 : 97 : 1, v/v). Yields were 51% (0.55 g) and 13% (0.14 g) for 4a
1
and 4b, respectively. H NMR (dH) (500 MHz, DMSO-d6, 4a)
11.29 (s, 1H), 7.16 (d, 1H, J = 8.1 Hz), 6.7–7.5 (m, 13H), 6.06
(d, 1H, J = 7.5 Hz), 5.38 (d, 1H, J = 8.1 Hz), 5.00 (d, 1H, J =
5.3 Hz), 4.13 (dd, 1H, J1 = 4.8 Hz, J2 = 7.4 Hz), 3.86 (m, 1H),
3.71 (s, 3H), 3.70 (s, 3H), 3.32 (m, 1H), 3.18 (m, 2H). 1H NMR
(dH) (500 MHz, DMSO-d6, 4b) 11.37 (s, 1H), 7.25 (d, 1H, J =
8.1 Hz), 6.8–7.7 (m, 13H), 5.99 (d, 1H, J = 7.0 Hz), 5.84 (d, 1H,
J = 6.5 Hz) 5.69 (d, 1H, J = 8.1 Hz), 4.05 (dd, 1H, J1 = 4.8 Hz,
J2 = 7.4 Hz), 3.93 (m, 1H), 3.73 (m, 1H), 3.35 (s, 3H), 3.10 (s,
3H), 2.89 (m, 2H). HRMS (FAB, 4a) M− calcd 559.2080, obsd
559.2089. HRMS (FAB, 4b) M− calcd 559.2080, obsd 559.2074.
2ꢀ,3ꢀ-O-Methyleneadenos-5ꢀ-yl 2ꢀ,5ꢀ-di-O-methylurid-3ꢀ-yl 3ꢀ-
O-(4,4ꢀ-dimethoxytrityl)-5ꢀ-O-methylurid-2ꢀ-yl phosphate (2b).
N6-Benzoyl-2ꢀ,3ꢀ-O-methyleneadenosine (6, 0.81 mmol, 0.31 g)
was coevaporated three times from anhydrous pyridine and
once from anhydrous MeCN. The residue was dissolved in
anhydrous MeCN (3.0 mL) and tris(dimethylamino)phosphine
(0.99 mmol, 0.180 mL), and tetrazole (0.97 mmol, 0.068 g) were
1 0 9 4
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 1 0 8 9 – 1 0 9 6

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added. The reaction mixture was stirred for 220 min at room
temperature. 2ꢀ,5ꢀ-Di-O-methyluridine (5, 0.81 mmol, 0.22 g)
was coevaporated three times from anhydrous pyridine and
once from anhydrous MeCN, and the residue was added to the
reaction mixture, together with anhydrous MeCN (1.0 mL) and
tetrazole (1.43 mmol, 0.1 g). The reaction mixture was stirred for
temperature, the reaction mixture was evaporated to dryness.
A conventional CH2Cl2–aq. NaHCO3 workup was carried out
and the combined organic phases were evaporated to dryness.
Approximately half of the residue (1.12 mmol, 0.6264 g)
and 2ꢀ,5ꢀ-di-O-methyluridine (5, 1.23 mmol, 0.3355 g) were
coevaporated once from anhydrous MeCN and the residue
was dissolved in anhydrous MeCN. A solution of tetrazole
(1.26 mmol, 0.0882 g) in MeCN (2.8 mL) was added and
the reaction mixture was stirred for 4 h at room temperature.
Iodine (2.0 mmol, 0.52 g) in a mixture of THF (9.2 mL),
water (4.6 mL) and 2,6-lutidine (2.3 mL) was added and the
reaction mixture was stirred for an additional 2 h at room
temperature, after which it was concentrated under reduced
pressure. A CH2Cl2–aq. NaHSO3 workup was carried out and
the combined organic phases were evaporated to dryness. The
product (a mixture of isomers) was purified on a silica gel
column eluting with a mixture of CH2Cl2 and MeOH (95 : 5,
v/v). The crude product mixture thus obtained was dissolved in
saturated methanolic ammonia (15 mL). After being stirred for
4 h at room temperature, the reaction mixture was evaporated
to dryness. The residue was dissolved in a mixture of acetic
acid (6 mL), water (2 mL) and THF (6 mL) and the reaction
mixture was stirred for 23 h at room temperature, after which it
was reduced to dryness. The products were purified by HPLC
on a Supelcosil LC-18 column (25 cm × 21.2 mm, 12 lm)
eluting with a mixture of 0.06 mol L−1 of aqueous NaOAc and
MeCN (89 : 11, v/v). Finally the buffer salts were removed
on the same column by eluting with a mixture of water and
MeCN (87 : 13, v/v). The overall yields starting from 2ꢀ(3ꢀ)-
O-isopropyldimethylsilyl-5ꢀ-O-methyluridine 3ꢀ(2ꢀ)-(2-cyano-
ethyl-N,N-diisopropylphosphoramidite) were 2.4% (18.6 mg)
◦
25 h at 50 C. 3ꢀ-O-(4,4ꢀ-Dimethoxytrityl)-5ꢀ-O-methyluridine
(4b, 1.04 mmol, 0.58 g) was coevaporated three times from
anhydrous pyridine and once from anhydrous MeCN, and
the residue was added to the reaction mixture, together with
tetrazole (1.14 mmol, 0.080 g). The reaction mixture was stirred
for 24 h at 40 ◦C, after which iodine (0.51 mmol, 0.13 g)
in a mixture of water (2.4 mL), THF (4.8 mL) and 2,6-
lutidine (1.2 mL) was added. After being stirred for 90 min at
room temperature the reaction mixture was concentrated under
reduced pressure and the residue was dissolved in saturated aq.
NaHSO3 (40 mL). The mixture was extracted with CH2Cl2 and
the organic phase was dried with Na2SO4 and evaporated to
dryness. The product was purified on a silica gel column eluting
with a mixture of MeOH, Et3N and CH2Cl2 (2 : 1 : 97, v/v).
The crude material thus obtained was dissolved in saturated
methanolic ammonia (10 mL). The reaction mixture was stirred
for 6 h at room temperature, after which it was evaporated to
dryness. The product was purified first on a silica gel column
eluting with a mixture of MeOH, Et3N and CH2Cl2 (4 : 1 : 95,
v/v), then by HPLC on a Supelcosil LC-18 column (25 cm ×
21.2 mm, 12 lm) eluting with a mixture of 0.05 mol L−1 of
aqueous NH4OAc and MeCN (53 : 47, v/v). Finally the buffer
salts were removed on the same column by eluting with a mixture
of water and MeCN (45 : 55, v/v). Compound 2b was obtained
as two diastereomers in approximately 1 : 1 ratio. A good
separation of the diastereomers was achieved by HPLC. The
1
and 4.5% (34.9 mg) for 9a and 9b, respectively. H NMR (dH)
1
overall yield starting from 6 was 11% (103 mg). H NMR (dH)
(400 MHz, DMSO-d6, 9a) 7.72 (d, 1H, J = 8.2 Hz), 7.66 (d, 1H,
J = 8.1 Hz), 5.85 (d, 1H, J = 5.8 Hz), 5.81 (d, 1H, J = 6.9 Hz),
5.70 (d, 1H, J = 8.1 Hz), 5.69 (d, 1H, J = 8.1 Hz), 4.56 (m, 1H),
4.54 (s, 1H), 4.38 (m, 1H), 4.18 (m, 1H), 4.13–4.08 (m, 2H),
3.90 (dd, 1H, J1 = 5.2 Hz, J2 = 5.9 Hz), 3.59–3.44 (m, 4H),
3.34 (s, 3H), 3.32 (s, 3H), 3.30 (s, 3H). 1H NMR (dH) (400 MHz,
DMSO-d6, 9b) 7.68 (d, 1H, J = 8.2 Hz), 7.67 (d, 1H, J = 8.2 Hz),
5.86 (d, 1H, J = 4.5 Hz), 5.85 (d, 1H, J = 6.3 Hz), 5.71 (d, 1H,
J = 8.1 Hz), 5.66 (d, 1H, J = 8.0 Hz), 4.54 (s, 1H), 4.52 (m,
1H), 4.41 (m, 1H), 4.18 (m, 1H), 4.10 (dd, 1H, J1 = 5.6 Hz, J2 =
5.6 Hz), 3.55–3.38 (m, 4H), 3.30 (s, 3H), 3.28 (s, 3H), 3.25 (s,
3H). 31P NMR (dP) (202 MHz, DMSO-d6, 9a) −0.55. 31P NMR
(dP) (202 MHz, DMSO-d6, 9b) −0.82. ESI−-MS (9a): m/z 591.5
[M − H]−. ESI−-MS (9b): m/z 591.2 [M − H]−.
5ꢀ-O-Methyluridylyl-(3ꢀ,5ꢀ)-(2ꢀ,3ꢀ-O-methyleneadenosine), 5ꢀ-
O-methyluridylyl-(2ꢀ,5ꢀ)-(2ꢀ,3ꢀ -O-methyleneadenosine) (10a,b).
A mixture of 2ꢀ-O-isopropyldimethylsilyl-5ꢀ-O-methyluridine
3ꢀ-(2-cyanoethyl-N,N-diisopropylphosphoramidite) and 3ꢀ -O-
isopropyldimethylsilyl-5ꢀ -O-methyluridine 2ꢀ -(2-cyanoethyl-
N,N-diisopropylphosphoramidite) (see the synthesis of 9a,b
above, 1.01 mmol, 0.6072 g) and N6-benzoyl-2ꢀ,3ꢀ-O-methylene-
adenosine (6, 1.21 mmol, 0.4671 g) were coevaporated once
from anhydrous MeCN. To the residue, a solution of tetrazole
(1.22 mmol, 0.085 g) in MeCN (5.7 mL) was added and the
reaction mixture was stirred for 4 h at room temperature.
Iodine (2.0 mmol, 0.52 g) in a mixture of THF (9.2 mL),
water (4.6 mL) and 2,6-lutidine (2.3 mL) was added and the
reaction mixture was stirred for an additional 2 h at room
temperature, after which it was concentrated under reduced
pressure. A CH2Cl2–aq. NaHSO3 workup was carried out and
the combined organic phases were evaporated to dryness. The
product (a mixture of isomers) was purified on a silica gel
column eluting with a mixture of CH2Cl2 and MeOH (95 : 5,
v/v). The crude product mixture thus obtained was dissolved in
saturated methanolic ammonia (15 mL). After being stirred for
5 h at room temperature the reaction mixture was evaporated
to dryness. The residue was dissolved in a mixture of acetic
(500 MHz, DMSO-d6, first diastereomer) 8.35 (s, 1H), 8.13
(s, 1H), 7.64 (d, 1H, J = 8.2 Hz), 7.54 (d, 1H, J = 8.2 Hz), 7.48–
6.87 (m, 15H), 6.28 (d, 1H, J = 7.0 Hz), 6.21 (d, 1H, J = 2.8 Hz),
5.79 (d, 1H, J = 6.5 Hz), 5.73 (d, 1H, J = 8.1 Hz), 5.71 (d, 1H,
J = 8.1 Hz), 5.28 (dd, 1H, J1 = 2.9 Hz, J2 = 6.7 Hz), 5.17
(s, 1H), 5.13 (s, 1H), 4.99 (dd, 1H, J1 = 4.0 Hz, J2 = 6.6 Hz),
4.85 (m, 1H), 4.78 (m, 1H), 4.35 (m, 1H), 4.27 (m, 2H), 4.12
(m, 1H), 4.04 (m, 1H), 3.73 (s, 3H), 3.72 (s, 3H), 3.45–3.30
1
(m, 6H), 3.29 (s, 3H), 3.24 (s, 3H), 3.03 (s, 3H). H NMR (dH)
(500 MHz, DMSO-d6, second diastereomer) 8.29 (s, 1H), 8.08 (s,
1H), 7.64 (d, 1H, J = 8.2 Hz), 7.56 (d, 1H, J = 8.2 Hz), 7.53–6.87
(m, 15H), 6.24 (d, 1H, J = 2.6 Hz), 6.20 (d, 1H, J = 6.0 Hz), 5.88
(d, 1H, J = 6.4 Hz), 5.73 (dd, 1H, J1 = 2.1 Hz, J2 = 8.1 Hz), 5.69
(dd, 1H, J1 = 2.0 Hz, J2 = 8.0 Hz), 5.27 (dd, 1H, J1 = 2.7 Hz, J2 =
6.5 Hz), 5.17 (s, 1H), 5.14 (s, 1H), 5.03 (dd, 1H, J1 = 3.8 Hz, J2 =
6.4 Hz), 4.89 (m, 1H), 4.49 (m, 1H), 4.33 (m, 1H), 4.31–4.24
(m, 2H), 4.17 (m, 1H), 4.04 (m, 1H), 3.73 (s, 3H), 3.72 (s, 3H),
3.46–3.23 (m, 6H), 3.31 (s, 3H), 3.25 (s, 3H), 3.06 (s, 3H). 31P
NMR (dP) (202 MHz, DMSO-d6, first diastereomer) −1.84. 31
HRMS (FAB) M− calcd 1154.3508, obsd 1154.3540.
P
NMR (dP) (202 MHz, DMSO-d6, second diastereomer) −2.07.
5ꢀ-O-Methyluridylyl-(3ꢀ,3ꢀ)-(2ꢀ,5ꢀ-di-O-methyluridine), 5ꢀ-O-
methyluridylyl-(2ꢀ,3ꢀ)-(2ꢀ,5ꢀ-di-O-methyluridine) (9a,b). 5ꢀ-O-
Methyluridine (7, 26.0 mmol, 6.7 g) was dissolved in anhydrous
pyridine (100 mL) and chlorodimethylisopropylsilane
(26.7 mmol, 4.2 L) was added. The reaction mixture was
stirred for 2 h at 40 ◦C, after which it was evaporated to dryness.
A conventional aq. NaHCO3–CH2Cl2 workup was carried out
and the product was purified on a silica gel column eluting
with a mixture of MeOH, Et3N and CH2Cl2 (5 : 1 : 94, v/v).
The isomers were not separated at this point. The isomeric
mixture (2.52 mmol, 0.9015 g) was coevaporated once from
anhydrous pyridine. To the residue 2-cyanoethyl-N,N,Nꢀ,Nꢀ-
tetraisopropylphosphorodiamidite (3.15 mmol, 1.0 mL)
and a solution of tetrazole (2.79 mmol, 0.195 g) in MeCN
(6.2 mL) were added. After being stirred for 90 min at room
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 1 0 8 9 – 1 0 9 6
1 0 9 5

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acid (6 mL), water (2 mL) and THF (6 mL) and the reaction
mixture was stirred for 23 h at room temperature, after which
it was evaporated to dryness. The products were purified by
HPLC on a Supelcosil LC-18 column (25 cm × 21.2 mm, 12
lm) eluting with a mixture of 0.06 mol L−1 of aqueous NaOAc
and MeCN (89 : 11, v/v). Finally the buffer salts were removed
on the same column by eluting with a mixture of water and
MeCN (87 : 13, v/v). The overall yields starting from 2ꢀ(3ꢀ)-O-
isopropyldimethylsilyl-5ꢀ-O-methyluridine 3ꢀ(2ꢀ)-(2-cyanoethyl-
N,N-diisopropylphosphoramidite) were 8.5% (26.5 mg) and
The initial substrate concentration in the kinetic runs was ca.
10−4 mol L−1. The composition of the samples withdrawn at
appropriate intervals was analyzed by HPLC on a Hypersil-
Keystone Aquasil C18 column (4 × 150 mm, 5 lm) using
0.06 mol L−1 formic acid buffer (pH = 3) and MeCN as an
eluent. The amount of MeCN was 7% for the first 15 min,
after which it was increased linearly to 27% during 20 min. The
observed retention times (tR, min) for the hydrolytic products
of 1 (the flow rate was 1.0 mL min−1) were as follows: 33.6
(1), 25.1 (9a), 23.1 (9b), 20.0 (10a), 14.5 (10b), 8.9 (5), 7.2 (8).
The products were identified by spiking with authentic samples
and the peak areas were converted to relative concentrations by
calibrating the system with uridine and adenosine solutions of
known concentration. The pseudo first-order rate constants for
the decomposition of 1 were obtained by applying the integrated
first-order rate equation to the time-dependent diminution of the
concentration of the starting material.
1
12.2% (38.1 mg) for 10a and 10b, respectively. H NMR (dH)
(400 MHz, DMSO-d6, 10a) 8.40 (s, 1H), 8.32 (s, 1H), 7.60 (d,
1H, J = 8.2 Hz), 7.35 (s, 2H), 6.15 (d, 1H, J = 3.2 Hz), 5.78
(d, 1H, J = 6.9 Hz), 5.67 (d, 1H, J = 8.0 Hz), 5.28 (dd, 1H,
J1 = 3.0 Hz, J2 = 6.4 Hz), 5.17 (s, 1H), 5.14 (s, 1H), 4.94 (dd,
1H, J1 = 3.6 Hz, J2 = 6.6 Hz), 4.28 (m, 1H), 4.22 (m, H), 4.03
(dd, 1H, J1 = 5.1 Hz, J2 = 6.8 Hz), 3.91 (m, 1H), 3.90–3.76 (m,
2H), 3.35–3.17 (m, 2H), 3.18 (s, 3H). 1H NMR (dH) (400 MHz,
DMSO-d6, 10b) 8.40 (s, 1H), 8.21 (s, 1H), 7.65 (d, 1H, J = 8.0
Hz), 7.33 (s, 2H), 6.11 (d, 1H, J = 3.0 Hz), 5.81 (d, 1H, J = 4.7
Hz), 5.60 (d, 1H, J = 8.1 Hz), 5.29 (dd, 1H, J1 = 3.2 Hz, J2 = 5.8
Hz), 5.13 (s, 1H), 5.11 (s, 1H), 4.88 (dd, 1H, J1 = 2.9 Hz, J2 =
6.5 Hz), 4.37 (m, 1H), 4.23 (m, 1H), 3.99 (m, 1H), 3.91 (m,
1H), 3.83 (m, 2H), 3.55–3.42 (m, 2H), 3.29 (s, 3H). 31P NMR
(dP) (202 MHz, DMSO-d6, 10a) 0.29. 31P NMR (dP) (202 MHz,
DMSO-d6, 10b) −0.92. ESI−-MS (10a): m/z 598.2 [M − H]−.
ESI−-MS (10b): m/z 598.3 [M − H]−.
Acknowledgements
We thank Prof. Harri Lo¨nnberg for fruitful discussions.
References
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2 S. Valadkhan and J. L. Manley, Nature, 2001, 413, 701.
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Kinetic measurements. Reactions were carried out in sealed
tubes immersed in a thermos◦tated water bath, the temperature
◦
of which was adjusted to 25 C within 0.1 C. The reactions
were started by adding the starting material in DMSO (20 lL)
to 80 lL of 99 mmol L−1 HCl in 1,4-dioxane. After 70 min,
1520 lL of the desired reaction solution (prethermostated to
25 ◦C) were added. The hydronium ion concentration of the
reaction solutions was adjusted with nitric acid and formate,
acetate, MES, HEPES, triethanolamine and glycine buffers. The
pH values of the buffers were checked with a pH meter. The
ionic strength of the solutions was adjusted to 1.0 mol L−1
with NaNO3. Between pH 3 and 6, considerable buffer catalysis
was observed. In the case of acetate, for example, the second-
order rate constant for the buffer-catalyzed cleavage was ca.
1.8 × 10−3 L mol−1 s−1. Buffer-independent rate constants were
obtained by varying the buffer concentration from 0.05 to
0.25 mol L−1 and extrapolating the observed pseudo first-order
rate constants to zero buffer concentration by linear regression.
14 S. Massenet, A. Mougin and C. Branlant, in The Modification
and Editing of RNA, ed. H. Grosjean and R. Benne, ASM Press,
Washington, D. C., 1998, 201.
15 S. Mikkola, U. Kaukinen and H. Lo¨nnberg, Cell Biochem. Biophys.,
2001, 34, 95.
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O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 1 0 8 9 – 1 0 9 6