Hydrolytic stability of a phosphate-branched oligonucleotide incorporating a ribonucleoside 3′-phosphotriester unit

Article abstract of DOI:10.1080/15257770500544537

A phosphate-branched oligonucleotide has been prepared by using an appropriately protected trinucleoside phosphotriester building block in conventional solid-phase synthesis. Hydrolysis of the branched oligonucleotide has been followed over a wide pH range. Comparison of the present results with those previously obtained for simpler analogues indicates that a trinucleoside 3′,3′,5′-monophosphate, when embedded in an oligonucleotide structure, is stabilized toward hydroxide-ion catalyzed cleavage by more than one order of magnitude, lending some support to the feasibility of existence of phosphate-branched RNA X in biological systems. Copyright Taylor & Francis Group, LLC.

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Hydrolytic Stability of a Phosphate-
Branched Oligonucleotide Incorporating
a Ribonucleoside 3′-Phosphotriester Unit
Tuomas Lönnberg a
a Department of Chemistry , University of Turku , Turku, Finland
Published online: 23 Aug 2006.
To cite this article: Tuomas Lönnberg (2006) Hydrolytic Stability of a Phosphate-Branched
Oligonucleotide Incorporating a Ribonucleoside 3′-Phosphotriester Unit, Nucleosides, Nucleotides and
Nucleic Acids, 25:3, 315-323, DOI: 10.1080/15257770500544537
To link to this article: http://dx.doi.org/10.1080/15257770500544537
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Nucleosides, Nucleotides, and Nucleic Acids, 25:315–323, 2006
Copyright ꢀ Taylor & Francis Group, LLC
C
ISSN: 1525-7770 print / 1532-2335 online
DOI: 10.1080/15257770500544537
HYDROLYTIC STABILITY OF A PHOSPHATE-BRANCHED
OLIGONUCLEOTIDE INCORPORATING A RIBONUCLEOSIDE
3ꢀ-PHOSPHOTRIESTER UNIT
Tuomas Lo¨ nnberg
Department of Chemistry, University of Turku, Turku, Finland
2
A phosphate-branched oligonucleotide has been prepared by using an appropriately protected
2
trinucleoside phosphotriester building block in conventional solid-phase synthesis. Hydrolysis of the
branched oligonucleotide has been followed over a wide pH range. Comparison of the present re-
sults with those previously obtained for simpler analogues indicates that a trinucleoside 3 ꢁ,3 ꢁ,5 ꢁ-
monophosphate, when embedded in an oligonucleotide structure, is stabilized toward hydroxide-ion
catalyzed cleavage by more than one order of magnitude, lending some support to the feasibility of
existence of phosphate-branched RNA X in biological systems.
Keywords RNA; Hydrolysis; Mechanism; Phosphate-Branched Oligonucleotide; RNA X
INTRODUCTION
A branched RNAstructure containing a phosphotriester linkage, the
so-called RNA X (Figure 1), has been suggested to be formed in the cat-
alytic domain of human spliceosome.[1,2] The two residues flanking the pro-
posed phosphotriester linkage, viz. A53 and G54, are 2ꢁ-O-methylated,[3]
which greatly increases the stability of the phosphotriester toward hydrol-
ysis. On the basis of our previous studies,[4,5] methylation of the free 2ꢁ-
OH in a trinucleoside 3ꢁ,3ꢁ,5ꢁ-monophosphate mimic of RNA X, 2ꢁ,3ꢁ-O-
methyleneadenosin-5ꢁ-yl 2ꢁ,5ꢁ-di-O-methyluridin-3ꢁ-yl 5ꢁ-O-methyluridin-3ꢁ-yl
phosphate (1), increases the hydrolytic stability by at least three orders of
magnitude, resulting in a half-life of more than one day under physiological
conditions. However, the original observations on the existence of RNA X
also referred to in vitro transcribed snRNAs composed entirely of the four
native ribonucleosides[1,2] and such triester structures do not experience
Received 23 August 2005; accepted 29 December 2005.
The assistance of Dr. Petri Heinonen and Pa¨ivi Poija¨rvi with the oligonucleotide synthesis is gratefully
recognized.
Address correspondence to Tuomas Lo¨nnberg, Department of Chemistry, University of Turku,
Vatselankatu 2, FIN-20014 Turku, Finland. E-mail: tuanlo@utu.fi
315

316
T. Lo¨nnberg
FIGURE 1 Structures of the branched building blocks and oligonucleotides 2a-e, their constituent pro-
tected nucleosides 3-5, the trinucleoside monophosphate 1, and RNA X.
a similar stabilization by 2ꢁ-O-alkylation. The results obtained with the
trinucleoside 3ꢁ,3ꢁ,5ꢁ-monophosphate model (1), when extrapolated to phys-
iological conditions, suggest a half-life of only 100 s for phosphate-branched
RNA,[4] hardly supporting the existence of the proposed species. It is, how-
ever, possible that in the actual RNA X the flanking oligonucleotide strands
stabilize the phosphotriester linkage by causing a conformational change
that retards the in-line displacement of the 2ꢁ- or 5ꢁ-linked nucleoside or by
altering the solvation of the initial and/or transition state.[6,7] To elucidate
whether 1 could really be expected to experience a marked stabilization when
embedded in an oligonucleotide structure, a small phosphate-branched
oligonucleotide (2a) has been synthesized and its hydrolytic reactions have
been followed over a wide pH range. Since the oligonucleotide arms consist
solely of thymidine residues (Figure 1), they are not expected to have any spe-
cific interactions with each other. Any changes in reactivity should, hence, be
due to steric or electrostatic repulsion between the oligonucleotide strands
and changes in solvation of the initial and/or transition state compared
to 1.

Hydrolysis of a Phosphate-Branched Oligonucleotide
317
EXPERIMENTAL
Oligonucleotide Sythesis
An appropriately protected trinucleoside phosphotriester (2b) was first
synthesized as previously described, viz. by tetrazole-promoted stepwise
displacement of the dimethylamino groups from tris(dimethylamino)-
phosphine with the appropriately protected nucleosides, added in the order:
(1) 3ꢁ-O-(tert-butyldimethylsilyl)thymidine, (2) 5ꢁ-O-(4,4ꢁ-dimethoxytrityl)-2ꢁ-
O-methyluridine, (3) 5ꢁ-O-(4,4ꢁ-dimethoxytrityl)-2ꢁ-O-levulinyluridine.[3,5,8]
The TBDMS protection was removed and the exposed 3ꢁ-hydroxy function
was phosphitylated to yield 2c. The oligonucleotide was then assembled on a
CPG-supported Q-linker at a loading of 28 µmol g−1 (Scheme 1). Standard
phosphoramidite strategy was utilized, except that a prolonged coupling
time (600 s) was applied to the branching building block 2c. The coupling
efficiency of the branching unit was 72%. The subsequent elongation of the
two 5ꢁ-branches then proceeded by normal efficiency. After completion of
the chain assembly, the cyanoethyl groups were removed by 3 min treatment
SCHEME 1 Synthesis of the protected branched oligonucleotide 2d.

318
T. Lo¨nnberg
with 1.5% (v/v) DBU in MeCN and the linker was cleaved by 1 min treatment
with 50 mmol L−1 methanolic K2CO3. Finally, the released product 2d was
purified by RP HPLC and characterized by ESI-MS.
Kinetics of the Hydrolysis of 2a
Because 2a is an unstable compound, the 2ꢁ-levulinyl protection of 2d
has to be removed immediately before each kinetic run. The deprotection
conditions were optimized by using 2ꢁ-O-levulinyluridine as a model com-
pound. In a mixture of hydrazine, acetic acid, and DMSO (1:7:404, v/v) at
25◦C, total conversion of 2ꢁ-O-levulinyluridine to uridine was achieved in 15
min. The hydrolysis of 2a was followed over a wide pH range (0–9) at 25◦C
and a constant ionic strength of 1.0 mol L−1 (NaNO3) by analyzing the com-
position of the aliquots withdrawn from the reaction mixture at appropriate
time intervals by capillary electrophoresis. Prior to each kinetic run, the 2ꢁ-
O-levulinyl group of 2d was removed by 20-min treatment with hydrazinium
acetate in DMSO, as described above, to yield 2a. The deprotection reaction
was then quenched with acetone and the appropriate aqueous solution was
created in the reaction vessel so that the final amounts of DMSO and acetone
were 3% and 1% (v/v), respectively. The aliquots withdrawn were, in turn,
quenched by cooling to 0◦C and adjusting their pH to approximately 3 with
a citric acid buffer.
3ꢁ-O-(tert-Butyldimethylsilyl)thymidine (3).
The compound was pre-
pared by using established synthetic procedures.1H NMR (δH) (500 MHz,
DMSO-d6) 11.30 (s, 1H), 7.67 (s, 1H), 6.15 (dd, J1 = 6.9 Hz, J2 = 7.0 Hz),
5.08 (s, 1H), 4.14 (m, 1H), 3.76 (m, 1H), 3.57 (m, 2H), 2.19 (m, 1H), 2.04
(m, 1H), 1.77 (s, 3H), 0.88 (s, 9H), 0.09 (s, 6H). HRMS (FAB) M+ calcd
357.1855, obsd 357.1846.
5ꢁ-O-(4,4ꢁ-Dimethoxytrityl)-2ꢁ-O-methyluridine (4).
2ꢁ-O-Methyluridine
was prepared as desribed in the literature,[9] after which the 5ꢁ-O-
1
dimethoxytrityl protection was attached by the conventional method. H
NMR (δH) (500 MHz, DMSO-d6) 11.42 (s, 1H), 7.74 (d, 1H, J = 8.1 Hz),
7.39–7.14 (m, 13H), 5.30 (d, 1H, J = 8.1 Hz), 5.24 (d, 1H, J = 7.0 Hz), 4.21
(m, 1H), 3.95 (m, 1H), 3.82 (m, 1H), 3.75 (s, 6H), 3.42 (s, 3H), 3.29–3.23
(m, 2H). ESI−-MS m/z 559.1 [M-H]−.
5ꢁ-O-(4,4ꢁ-Dimethoxytrityl)-2ꢁ(3ꢁ)-O-levulinyluridine (5a,b).
5ꢁ-O-(4,4ꢁ-
Dimethoxytrityl)uridine (10.8 mmol, 5.87 g) was dissolved in pyridine
(50 mL). A solution of levulinic anhydride (11.9 mmol, 2.54 g) in 1,4-dioxane
(50 mL) was added, followed by a catalytic amount of DMAP. After stirring
for 4 h at room temperature, the reaction mixture was evaporated to dryness

Hydrolysis of a Phosphate-Branched Oligonucleotide
319
and a conventional aq. NaHCO3/CH2Cl2 was carried out. The organic phase
was evaporated to dryness and the residue was purified on a silica gel col-
umn eluting with a mixture of Et3N, MeOH, and CH2Cl2 (1:4:95, v/v). The
product was obtained as a mixture of 5a and 5b (2:1) in an overall yield of
1
78% (5.41 g). H NMR (δH) (500 MHz, DMSO-d6, 5a) 11.43 (s, 1H), 7.69
(d, 1H, J = 8.1 Hz), 7.41–7.23 (m, 13H), 5.81 (d, 1H, J = 5.9 Hz), 5.78 (d,
1H, J = 5.8 Hz), 5.45 (d, 1H, J = 8.1 Hz), 5.14 (m, 1H), 4.41 (m, 1H), 4.10
(m, 1H), 3.75 (s, 6H), 3.34–3.19 (m, 2H), 2.74 (m, 2H), 2.55 (m, 2H), 2.11
(s, 3H). 1H NMR (δH) (500 MHz, DMSO-d6, 5b) 11.43 (s, 1H), 7.67 (d, 1H,
J = 8.1 Hz), 7.41–7.23 (m, 13H), 5.91 (d, 1H, J = 5.7 Hz), 5.55 (d, 1H, J =
5.8 Hz), 5.42 (d, 1H, J = 8.1 Hz), 5.26 (m, 1H), 4.35 (m, 1H), 3.99 (m, 1H),
3.75 (s, 6H), 3.34–3.19 (m, 2H), 2.74 (m, 2H), 2.55 (m, 2H), 2.11 (s, 3H).
HRMS (FAB) M− calcd 643.2314, obsd 643.2292.
3ꢁ-O-(tert-Butyldimethylsilyl)thymidin-5ꢁ -yl 5ꢁ-O-(4,4ꢁ-dimethoxytrityl)-
2ꢁ-O-methyluridin-3ꢁ-yl 5ꢁ -O-(4,4ꢁ -dimethoxytrityl)-2ꢁ-O-levulinyluridin-3ꢁ-yl
phosphate (2b). 3ꢁ-O-(tert-Butyldimethylsilyl)thymidine (3, 4.95 mmol,
1.77 g) was dissolved in anhydrous MeCN (5 mL) and tris(dimethyl-
amino)phosphine (5.11 mmol, 0.928 mL) and 1H-tetrazole (5.94 mmol,
0.416 g) in anhydrous MeCN (13.2 mL) were added. After stirring
for 1 h at room temperature under inert atmosphere, 5ꢁ-O-methyl-
(4,4ꢁ-dimethoxytrityl)-2ꢁ-O-methyluridine (4, 4.95 mmol, 2.78 g) and 1H-
tetrazole (5.94 mmol, 0.416 g) in anhydrous MeCN (13.2 mL) were
added. The reaction mixture was stirred for 22 h, after which 5ꢁ-O-(4,4ꢁ-
dimethoxytrityl)-2ꢁ(3ꢁ)-O-levulinyluridine (5a,b, 8.39 mmol, 5.41 g) and 1H-
tetrazole (5.94 mmol, 0.416 g) in anhydrous MeCN (13.2 mL) were added.
After stirring for 24 h, iodine (5.50 mmol, 1.41 g) in a mixture of water
(8.6 mL), THF (17.2 mL), and 2,6-lutidine (4.3 mL) was added. The reaction
mixture was stirred for 1 h, after which it was concentrated under reduced
pressure. The residue was dissolved in CH2Cl2 and washed with saturated
aqueous NaHSO3. The aqueous phase was back-extracted with CH2Cl2. The
combined organic phases were evaporated to dryness and the residue was
purified on a silica gel column eluting first with a mixture of Et3N, MeOH,
and CH2Cl2 (1:6:93, v/v), then with a mixture of Et3N, MeOH, CH2Cl2,
and EtOAc (1:3:18:78, v/v). Yield 34.5% (1.81 g). 1H NMR (δH) (500 MHz,
DMSO-d6) 11.48 (s, 1H), 11.45 (s, 1H), 11.33 (s, 1H), 7.66 (d, 1H, J = 8.1
Hz), 7.61 (d, 1H, J = 8.1 Hz), 7.43 (s, 1H), 7.41–7.19 (m, 26H), 6.17 (dd,
1H, J1 = 6.9 Hz, J2 = 7.0 Hz), 5.92 (d, 1H, J = 5.5 Hz), 5.73 (d, 1H, J =
3.0 Hz), 5.50 (dd, 1H, J1 = 8.1 Hz, J2 = 1.4 Hz), 5.39 (dd, 1H, J1 = 8.1 Hz,
J2 = 1.6 Hz), 5.35 (m, 1H), 5.16 (m, 1H), 5.07 (m, 1H), 4.38 (m, 1H), 4.29
(m, 1H), 4.21–4.11 (m, 4H), 3.95 (m, 1H), 3.74 (s, 6H), 3.71 (s, 6H), 3.40
(s, 3H), 3.39–3.24 (m, 4H), 2.75–2.55 (m, 4H), 2.22 (m, 1H), 2.07 (m, 1H),
2.05 (s, 3H), 1.71 (s, 3H), 0.85 (s, 9H), 0.07 (s, 3H), 0.06 (s, 3H). 31P NMR

320
T. Lo¨nnberg
(δP) (202 MHz, DMSO-d6) −2.46. HRMS (FAB) M− calcd 1603.5589, obsd
1603.5670.
Thymidin-5ꢁ-yl 5ꢁ-O-(4,4ꢁ-dimethoxytrityl)-2ꢁO-methyluridin-3ꢁ-yl 5ꢁ-O-
(4,4ꢁ-dimethoxytrityl)-2ꢁ-O-levulinyluridin-3ꢁ-yl phosphate (2e).
3ꢁ-O-(tert-
Butyldimethylsilyl)thymidin-5ꢁ-yl 5ꢁ-O-(4,4ꢁ-dimethoxytrityl)-2ꢁ-O-methyluri-
din-3ꢁ-yl 5ꢁ-O-(4,4ꢁ-dimethoxytrityl)-2ꢁ-O-levulinyluridin-3ꢁ-yl phosphate (2b,
0.458 mmol, 0.735 g) was dissolved in THF (3.14 mL) and Et3N·3HF
(112 µL) was added. After being stirred for 18 h at room temperature, the
reaction mixture was purified on a silica gel column eluting with a mixture
of Et3N, MeOH, and CH2Cl2 (1:6:93, v/v). Yield 65.7% (0.449 g). 1H NMR
(δH) (500 MHz, DMSO-d6) 11.45 (m, 2H), 11.33 (s, 1H), 7.67 (d, 1H, J =
8.1 Hz), 7.60 (d, 1H, J = 8.1 Hz), 7.39 (s, 1H), 7.37–7.19 (m, 26 H), 6.18
(dd, 1H, J1 = 6.9 Hz, J2 = 7.0 Hz), 5.89 (d, 1H, J = 5.5 Hz), 5.72 (d, 1H, J =
3.0 Hz), 5.47 (d, 1H, J = 8.1 Hz), 5.36 (d, 1H, J = 8.1 Hz), 5.35 (m, 1H),
5.26 (m, 1H), 5.15 (m, 1H), 4.28 (m, 2H), 4.25–4.08 (m, 4H), 3.93 (m, 1H),
3.74 (s, 6H), 3.70 (s, 6H), 3.38 (s, 3H), 3.37–3.22 (m, 4H), 2.75–2.55 (m,
4H), 2.45 (m, 2H), 2.05 (s, 3H), 1.70 (s, 3H). MS (FAB) M− calcd 1489.5,
obsd 1490.
OP -[5ꢁ -O-(4,4ꢁ-Dimethoxytrityl)-2ꢁ-O-methyluridin-3ꢁ-yl]-[5ꢁ-O-(4,4ꢁ-dimet-
hoxytrityl)-2ꢁ-O-levulinyluridylyl-(3ꢁ,5ꢁ)-thymidine] 3ꢁ-(2-cyanoethyl-N,N-diis-
opropylphosphoramidite) (2c). Thymidin-5ꢁ-yl 5ꢁ-O-(4,4ꢁ-dimethoxytrityl)-
2ꢁ-O-methyluridin-3ꢁ-yl 5ꢁ-O-(4,4ꢁ-dimethoxytrityl)-2ꢁ-O-levulinyluridin-3ꢁ-yl
phosphate (2e, 0.259 mmol, 0.449 g) was dissolved in anhydrous MeCN
(11 mL) and 2-cyanoethyl-N,N,N’,N’-tetraisopropylphosphorodiamidite
(0.472 mmol, 150 µL) and 1H-tetrazole (0.36 mmol, 0.0252 g) in anhydrous
MeCN (800 µL) were added. The reaction mixture was stirred for 17 h at
room temperature, after which CH2Cl2 (50 mL) and 5% (m/m) aqueous
NaHCO3 (30 mL) were added. The phases were separated and the organic
phase was dried (Na2SO4) and evaporated to dryness. On the basis of
the 31P NMR spectrum the yield was estimated to be 76%. 31P NMR (δP)
(202 MHz, DMSO-d6) −148.15 (s, 1P), −2.47 (s, 1P). HRMS (FAB) M−
calcd 1689.5835, obsd 1689.5884.
RESULTS AND DISCUSSION
At pH < 6, disappearance of the starting material is accompanied by for-
mation of four product peaks at approximately 2:1:2:2 ratio. At pH > 6, only
the peaks of the two faster migrating products are observed at approximately
2:1 ratio. Previous studies with trinucleoside 3ꢁ,3ꢁ,5ꢁ-monophosphates have
revealed a similar change in product distribution: under acidic conditions,
the P-O3ꢁ and P-O5ꢁ bonds are cleaved at approximately equal rates, whereas

Hydrolysis of a Phosphate-Branched Oligonucleotide
321
SCHEME 2 Hydrolytic reaction pathways of the branched oligonucleotide 2a.
in aquous alkali the P-O3ꢁ bond cleavage accounts for almost 90% of the
overall reaction.[3,8] Assuming that hypochromicity is negligible, the poten-
tial product oligonucleotides 6, 7, 8, and 9 (Scheme 2) should have relative
molar absorptivities of 7, 8, 4, and 11, respectively. Accordingly, the faster
migrating pair of product peaks was assigned as 9 and 8, respectively, and
the slower migrating pair as 6 and 7, respectively. Mass spectrometric analy-
sis of reaction solutions lacking the background electrolyte tends to support
this interpretation. Accordingly, in 10 mmol L−1 HNO3, the following ions
were observed (m/z): 2a: 887.0 (M5−), 738.8 (M6−); 6: 1032.8 (M2−), 688.4
(M3−), 412.4 (M5−); 7: 1194.5 (M2−), 596.6 (M4−), 396.2 (M6−), 340.1 (M7−);
8: 584.3 (M2−), 389.9 (M3−); 9: 1164.2 (M2−), 468.2 (M7−). In 10 mmol L−1
NaOH, 2a is decomposed within seconds and only product ions were, hence,
detected (m/z): 8: 389.9 (M3−), 9: 547.1 (M6−). Undoubtedly, 2a also un-
dergoes a facile isomerization to its 2ꢁ,3ꢁ,5ꢁ-analogue 10 (Scheme 1, Route
C), although only one peak corresponding to starting material is observed.
Apparently the two isomers migrate equally rapidly, hence preventing their
separation by capillary electrophoresis.
The pH-rate profile for the disappearance of 2a is depicted in Figure 2.
At pH < 2, the overall disappearance of 2a is first-order in [H3O+]. On the
basis of the observed product ratio (2:1:2:2) and relative molar absorptivities

322
T. Lo¨nnberg
FIGURE 2 pH-Rate profiles for the cleavage of the branched oligonucleotide 2a ( ) and trinucleoside
ꢀ
monophosphate 1 (dashed line); T = 25◦C, I(NaNO3) = 1.0 mol L−1
.
(11:4:7:8), the reaction proceeds approximately as rapidly by P-O5ꢁ (Route
A) and P-O3ꢁ (Route B) bond cleavage. Over a wide range from pH 2 to 5, the
overall reaction is pH-independent and a largely similar product distribution
is observed as with the hydronium ion catalyzed cleavage. At pH > 6, the over-
all reaction is first-order in [HO−]. Only the P-O3ꢁ cleavage products (8 and
9) can be detected, indicating that the P-O3ꢁ cleavage (Route B) predomi-
nates under alkaline conditions, as with 1. Rate constants for the hydronium
ion–catalyzed, pH-independent, and hydroxide ion–catalyzed cleavage, kcHl ,
W
OH
kcl , and k , were obtained by fitting the observed pseudo first-order rate
cl
obs
cl
constants, k , to Eq. (1) by a nonlinear least-squares method. The calculated
rate constants, together with those previously obtained for 1, are presented
in Table 1.
OH
k KW
cl
obs
cl
W
k
= kcHl aH + kcl +
(1)
+
+
aH
The rate constants for the hydronium ion–catalyzed and pH-
independent cleavage are largely similar with 2a and 1, suggesting that the
TABLE 1 Rate Constants for the Partial Reactions of Cleavage of
the Phosphate-Branched Oligonucleotide 2a and the Trinucleoside
Monophosphate 1
kcHl /10−3 L mol−1 s−1
kcWl /10−5 s−1
k
/103 Lmol−1 s−1
OH
cl
2a
1
3.9 0.5
1.6 0.3
6
3
0.62 0.06
7.5 0.9
9.0 0.9

Hydrolysis of a Phosphate-Branched Oligonucleotide
323
short oligonucleotide arms play no major role under acidic conditions. Hy-
droxide ion–catalyzed cleavage of 2a, however, is 12 times slower than the
respective reaction of 1. For comparison, hydroxide ion–catalyzed cleavage
of an individual phosphodiester linkage within poly(U) is as rapid as the
respective reaction of UpU.[10] Accordingly, the increasing number of nega-
tively charged phosphodiester bonds is not as such a sufficient explanation
for the observed retardation of hydroxide ion–catalyzed cleavage. In any
case, it seems, on the basis of these results, that even short oligonucleotide
chains with no tendency to fold into specific tertiary structures may signifi-
cantly alter the rate of cleavage of ribonucleoside phosphotriester linkages.
In the case of a more complex structure, such as RNA X, the stabilization
may be even more marked, possibly sufficient to make RNA X a species
that can actually be detected under physiological conditions. The present
data additionally serves as a necessary background for further studies with
phosphate-branched oligonucleotides, the arms of which may have specific
interactions with one another.
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