Increased Affinity of 2′-O-(2-Methoxyethyl)-Modified Oligonucleotides to RNA through Conjugation of Spermine at Cytidines

Article abstract of DOI:10.1002/hlca.201900222

Structural modification at the 2′-O-position of riboses in oligonucleotide therapeutics is of critical importance for their use as drugs. To date, the methoxyethyl (MOE) substituent is the most important and features in dozens of antisense oligonucleotide

Full text of DOI:10.1002/hlca.201900222

HELVETICA
chimica acta
Accepted Article
Title: Increased Affinity of 2'-O-(2-methoxyethyl)-Modified
Oligonucleotides to RNA through Conjugation of Spermine at
Cytidines
Authors: Elodie Decuypere, Anastasiia Lepikhina, Jonathan Hall, and
François Halloy
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10.1002/hlca.201900222
Helvetica Chimica Acta
HELVETICA
Increased Affinity of 2'-O-(2-methoxyethyl)-Modified Oligonucleotides to
RNA through Conjugation of Spermine at Cytidines
Elodie Decuypere,a Anastasia Lepikhina,a François Halloy a and Jonathan Hall*,a
aETH Zürich, Department of Chemistry and Applied Biosciences, Vladimir Prelog Weg 1-5, CH–8093 Zürich, Switzerland (jonathan.hall@pharma.ethz.ch)
Dedicated to François Diederich on the occasion of his retirement
Structural modification at the 2'-O-position of riboses in oligonucleotide therapeutics is of critical importance for their use as drugs. To date, the
methoxyethyl (MOE) substituent is the most important and features in dozens of antisense oligonucleotides that have been tested in clinical trials. Yet,
the search for new improved modifications continues in a quest for increased oligonucleotide potency, improved transport in vivo and favorable
metabolism. Recently, we described how the conjugation of spermine groups to pyrimidines in oligonucleotides vastly increases their affinity for
complementary RNAs through accelerated binding kinetics. Here we describe how spermines can be linked to the exocyclic amino groups of cytidines in
MOE-oligonucleotides employing a straightforward "convertible nucleoside approach" during solid phase synthesis. Singly- or doubly-modified
oligonucleotides show greatly enhanced affinity for complementary RNA, with potential for a new generation of MOE-based oligonucleotide drugs.
Keywords: oligonucleotide conjugate • spermine • MOE oligonucleotide • hybridization • convertible nucleoside approach
the push for stereochemically-pure PS drugs has been boosted by
advances in phosphoramidite chemistry.[13] Both of these strategies are
Introduction
Single- and double-stranded oligonucleotides are a new class of
being applied to 2'-MOE- and LNA-oligonucleotides, since they may
therapeutics that have recently come of age.[1] They bind to their cellular
improve the pharmacokinetics properties and thereby show
RNA targets with high affinity and extraordinary selectivity according to
pharmacological activity in cell/tissue types where delivery is as yet
the rules of Watson-Crick (W-C) base-pairing, whereupon they elicit a
limiting.
variety of inhibitory mechanisms determined by their structural
Conjugated cationic oligospermines have been investigated extensively as
modifications and the class of RNA target. A large part of their current
carrier groups to improve the cellular delivery of oligonucleotides, and
success in the clinical setting is due to two decades of medicinal chemistry
found to an act in similar fashion to polyamine-type delivery vectors. [14, 15]
efforts. These have produced a handful of innovative nucleic acid analogs
[16]
We recently described how conjugation of polyamine chains derived
which satisfy the principal requirements for therapeutic use in man: a
from spermine to the C5 (but not to the 2’-hydroxyl) of uridines greatly
structure that maintains WC-binding affinity and selectivity, one that is
increased the binding affinity of 2’-O-methyl (OMe)-modified
stable to ubiquitous nucleases in vivo, one that tolerates the cellular factors
oligoribonucleotides for complementary RNAs.[17] We postulated that the
responsible for processing the RNA target (e.g. RNaseH enzymes) and one
polyamine likely positions itself in the duplex major groove, mimicking
that is accessible on large scale through solid-phase chemistry processes.
natural polyamines stabilizing cellular RNAs. This result was striking;
The mainstay of clinically-validated oligonucleotide modifications is the 2’-
however, the synthesis of these derivatives is rather tedious and the use of
O-(2-methoxyethyl) (2'-MOE)-ribose connected by backbone of
2'-OMe oligonucleotides as antisense agents is outdated. We therefore
phosphorothioate (PS) linkages.[2] Dozens of 2'-MOE-oligonucleotides
searched for an improved means to extend these findings to 2'-MOE
have shown useful activity in patients including the approved drugs
oligonucleotides.
Mipomersen[3] and Nusinersen.[4] Other prominent chemistries that have
In a previous work we applied the “convertible nucleoside” approach[18] to
reached clinical testing include the bicyclic ribonucleotides – the so-called
introduce methyl- and dimethylamine to the 4-position of cytidine by
locked nucleic acid (LNA)[5-7] and the constrained ethyl derivatives (cEt)[8]
–
nucleophilic displacement of triazole.[19] The triazole derivative is very
conveniently prepared just before or just after phosphitylation of the
protected nucleoside. Furthermore, this chemistry offers the possibility to
introduce the nucleophiles during solid-phase synthesis, i.e. before
protecting group removal and cleavage from the controlled pore glass
(CPG) solid support. Mono-substitution of the substituted amino group on
cytidine permits a regular G-C base-pair formation during hybridization if
the substituent – in our case, a spermine group – adopts a trans
conformation with cytidine N3.
and the structurally-distinct 6-ring morpholino phosphordiamidates
(PMO).[9]
The drive to develop new structural modifications for oligonucleotides in
industry and in academia continues, though priorities for medicinal
chemists have changed in line with evolving challenges in the field. For
instance, the development of carrier groups to deliver drugs more
efficiently and to a wider range of tissues has become a major focus since
the discovery that N-acetylgalactosamine (GalNac) conjugated groups are
able to increase 100-1000-fold delivery of siRNAs to hepatocytes.[10-12] Also,
1
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Here, we describe a convenient and straightforward synthesis of
polyamine-modified 2'-OMe- and 2'-MOE-oligonucleotides. Analogously
to polyamine modification at the uridine 5-position, spermine conjugation
to N4-of cytidine greatly increases hybridization affinities of modified
oligonucleotides to complementary RNAs.
the solvent was switched to dry isobutyronitrile at a concentration of
0.25 M for solid phase synthesis. After all bases were coupled,
spermine was added to the CPG for controlled displacement of the
triazole group. The reaction was assumed to occur at terminal
position of the spermine, as reported previously[21]. Deprotection,
cleavage and purification steps were then performed under standard
conditions to provide a library of purified spermine oligonucleotide
conjugates (Table 1). The 2'-MOE oligonucleotides were obtained
cleanly as shown by LCMS (entries 6-8, Table 1; supplementary
material); however, the three 2'-OMe oligonucleotides were
accompanied by small amounts of inseparable N-acetylated
byproducts, detected as shoulder peaks in the LCMS chromatagrams
(entries 2-4, Table 1).
Results and Discussion
The N4-triazole-modified 2’-OMe cytidine phosphoramidite
2 was
obtained in single step from commercially-available 5’-
a
dimethoxytrityl (DMT)-2’-OMe cytidine phosphoramidite
described previously.[20]
1, as
Melting temperature (TM) experiments were performed to study the
influence of the spermine on hybridization with complementary
RNAs and to compare the performance of 2'-OMe and 2'-MOE
analogs with their unmodified parent sequences (Table 1).
Previously, we showed that spermine conjugation through its
terminal amino group to C5 of uridine in 2'-OMe oligonucleotides
increases their TM's with complementary RNAs, by up to 12 0C per
modification. Moving the spermine fragment to the 4-position on the
nucleobase also boosted hybridization to RNA compared to the
parent oligonucleotide (entry 1, Table 1) for three sequences (Entries
2-4, Table 1) (TM's +4-6 0C per modification).
Scheme 1. Single-step preparation of N4-triazolo-2'-OMe cytidine.
2'-MOE-modified oligonucleotides are typically used with thymine
and 5-methyl cytosine pyrimidines because the 5-methyl group
increases hybridization affinity of the oligonucleotide to target
mRNAs. Efforts to prepare the 2'-MOE thymidine-convertible
nucleoside using a similar protocol to that employed for
2 failed,
Table 1. Composition and TM's of modified sequences bearing polyamine fragments.
possibly due to instability of the 2'-MOE phosphoramidite under
conditions of the reaction. This was remedied by beginning with
[b]
Chemistry
(ribose)
TM
ΔTM
( °C)
-
Entry
Sequence[a]
( °C)
55.5
58.5
61.0
63.8
58.3
62.4
52.9
49.8
62.2
64.4
64.7
65.4
74.9
commercially-available 5’-DMT-2'-MOE thymidine (3; Scheme 2).
1
2
CUUAGCAACCUG
2'-OMe, PS
2'-OMe, PS
2'-OMe, PS
2'-OMe, PS
2'-MOE, PS
2'-MOE, PS
2'-MOE, PS
2'-MOE, PS
2'-MOE, PS
2'-MOE, PS
2'-MOE, PS
2'-MOE, PS
2'-MOE, PS
Y
UUAGCAACCUG
+3.5
+5.5
+8.3
-
3
CUUAG
UUAG
Y
AACCUG
AACCUG
4
Y
Y
5
CTTAGCAACCTG
TTAGCAACCTG
6
X
+4.1
-5.4
-8.5
+3.9
+6.1
+6.4
+7.1
+16.6
Scheme
7
CTTAG
X
AACCTG
AACCTG
2. Synthesis
of N4-
8
X
TTAG
X
9
Y
TTAGCAACCTG
triazolo-5-
10
11
12
13
CTTAG
YAACCTG
Z
TTAGCAACCTG
CTTAG
Z
AACCTG
AACCTG
Z
TTAGZ
[a]
methylcytidine 2'-MOE phosphoramidite.
Thus, O4 of
3
was exchanged for 1,2,4-triazole providing 5 via
temporary silyl-protection of the 3'-OH. Following deprotection, the
nucleoside was directly phosphitylated using 2-cyanoethyl-N,N-
diisopropylchlorophosphoramidite to provide
6. The triazole-
bearing phosphoramidites were then employed for the solid-phase
synthesis of PS oligonucleotides on controlled pore glass (CPG). Due
[b] Complementary phosphodiester RNA strand (CAGGUUGCUAAG)
to poor solubility of the modified phosphoramidite
6 in acetonitrile,
2
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Next, we performed the melting experiments under the same
conditions for the isosequential 2'-MOE oligonucleotides. The
terminally-modified sequence showed a TM increase of 4.1 0C over the
parent sequence (Entries 6 and 5, respectively, Table 1). However,
modification at an internal position dramatically lowered the
hybridization affinity (TM's of -5.4 and -8.5 0C, Entries 7 and 8,
respectively; Table 1). It seemed most likely that this undesired effect
derived from interference of the spermine fragment with the
neighboring ring methyl group during hybridization. This finding
was consistent with the work of Prakash et al., who reported
decreased binding for an N4-spermine group on thymidine in DNA
conjugates.[22] To clarify this, we synthesized two 2'-MOE sequences
containing spermine-substituted 2'-OMe uridines (Entries 9 and 10,
Table 1). Indeed, these showed increased binding affinity to the
target RNA with TM's of +3.9 and +6.1 0C, respectively.
Increased binding affinity from chemical modification of therapeutic
oligoribonucleotides has been a major goal of the RNA field for decades,
since it produces greater inhibition of target mRNAs at lower
concentrations and with less frequent dosing in vivo. In this work, we
introduced
a
single modification to the cytidines of 2'-MOE
oligonucleotides, and increased its binding affinity for complementary
RNAs. Two spermine additions on a 12-mer 2'-MOE PS oligonucleotide
increased its TM to complementary RNA by more than 16 oC. The precedent
for this work was our earlier observation that positioning a polyamine in
the major groove increases dramatically the rate of association of the
antisense sequence for its target.[17] In that case the polyamine was
conjugated through several synthetic steps to the 5-position of uracils. The
chemistry in this work is especially attractive since the polyamine fragment
is relatively easily introduced to the clinically-proven 2'-MOE modification
by shifting the substituent around the pyrimidine by one position to N4.
Our investigation confirmed that attachment of affinity-enhancing groups
to N4 is permitted if the exocyclic amino group can adopt a trans
conformation with N3 during base-pairing with guanidine, and there is no
C5-methyl group.
Hence, we prepared the 2'-MOE analog beginning from uracil, rather
than from thymine. We synthesized the required DMT-protected 2'-
MOE uridine phosphoramidite in five steps from the anhydro-
derivative
anhydro ring (to
produced the nucleoside precursor
7
using literature protocols. Thus, opening of the bicyclic
) and dimethoxytritylation at the 5’-hydroxyl group
. The triazole group was
Most chemical modifications to oligonucleotides are made on the
ribose and protrude into the minor groove.[24] This usually attenuates
the ability of the oligonucleotide to induce RNase H effector
enzymes, but it offers an improved protection against ubiquitous
nucleases in vivo; PS 2'-MOE oligonucleotides are highly resistant to
nucleases.
8
9
subsequently introduced after temporary silylation of the 3'-hydroxyl
group (Scheme 3). Phosphitylation then yielded the
phosphoramidite 12 ready for oligonucleotide synthesis.
Scheme 3. Synthesis of N4-triazolo-cytidine 2'-MOE phosphoramidite.
We synthesized three full PS 2'-MOE sequences using 12 (Entries 11,
12, 13; Table 1) and measured their melting temperatures after
hybridization to complementary RNAs. We observed increase in TM of
On the other hand, modifications of nucleobases that protrude into
the major groove of an 2'-MOE-mRNA duplex are useful because of
the possibility to raise the hybridization affinity without adversely
affecting RNase H activity. Previous efforts to achieve this for 2'-MOE
oligonucleotides include alkyne and heterocycle modifications
fusions to the pyrimidines.[25] Although they raised binding affinity in
vitro, they did not improve cellular activity unless delivered with
o
6.4 C and 7.1 oC for the terminally-modified and internally-modified
sequences (Entries 11 and 12), respectively. With
a double
incorporation of the building block (entry 13), the TM was increased
by 16.6 oC.
Conclusions
cationic lipids. Incorporation
of
this
chemistry into
The 2'-MOE alkylation of ribose is inarguably the most important structural
modification to single-stranded oligonucleotide drugs.2 It led to the
clinical success of Nusinersen4 and Mipomersen,3 as well as several other
2'-MOE-based drugs that are undergoing late-stage clinical testing.[23]
pharmacologically-interesting 2'-MOE oligonucleotides with the aim
to improve cellular activity is ongoing.
3
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mmol, 1g) was co-evaporated three times in dry acetonitrile (10 ml). TMOE
was redissolved in dry acetonitrile (25 ml) under argon, to which first dry
triethylamine (35.5 mmol, 5 ml) and then trimethylsilyl chloride (8.1 mmol,
1 ml) were added dropwise. The reaction mixture was stirred for 30 min at
room temperature. Then 1,2,4-triazole (38.7 mmol, 2.677 g) was added
with further stirring. After 10 min the reaction mixture was cooled to 0 °C
and phosphorus oxychloride (4.8 mmol, 0.5 ml) was added drop wise. After
15 min the ice bath was removed and the reaction was stirred for 4 h. The
solvent was removed under vacuum and the obtained crude material was
partitioned between aqueous sodium bicarbonate and ethylacetate (30
ml) and washed with brine (30 ml). The organic layer was dried over
sodium sulphate. Solvents were then removed under reduced pressure.
The crude product was purified by column chromatography
(EtOAc/Hexane 80:20). Compound was obtained as a white foam: 1.03 g,
87% overall yield. 1H NMR (MeOD4): δ (ppm) 9.25 (s, 1H), 8.54 (s, 1H), 8.13 (s,
1H), 7.41–7.37 (m, 2H), 7.29–7.19 (m, 7H), 6.82–6.79 (m, 4H), 5.91 (d, J =
16Hz, 1H), 4.49–4.45 (m, 1H), 4.17–4.13 (m, 1H), 4.12–4.03 (m, 2H), 3.88–3.79
(m, 1H), 3.70 (s, 6H), 3.64 (dd, J = 11Hz, 2Hz, 1H), 3.60–3.44 (m, 3H), 3.29 (s,
3H), 1.69 (s, 3H), 0.01 (s, 9H); 13C NMR (MeOD4): δ (ppm) 159.11, 158.53,
154.7, 152.7, 147.2, 145.0, 144.3, 135.2, 130.1 (4C), 128.2 (2C), 127.6 (2C),
126.8, 112.9 (4C), 106.6, 90.6, 86.7, 83.0, 82.4, 71.6, 69.9, 68.8, 60.7, 57.8, 57.4
(4C), 15.2, -1.27 (3C). ESI‐HRMS: m/z calcd for C39H47N5O8Si [(M+Na)+]
764.3086, found 764.3078.
Experimental Section
General information.
All NMR spectra were recorded on a Bruker AV400. H NMR, 13C NMR and
31P NMR were measured in deuterated solvents. ESI Mass Spectra were
recorded on a Bruker’s solariX (ESI/MALDI‐FTICR‐MS). All glassware was
dried thoroughly prior to use. Triethylamine was distilled from CaH2, and
stored over KOH pellets under argon. N‐Methyl morpholine was distilled
from BaO, and stored over KOH pellets under argon. Phosphorus
trichloride was purchased from Aldrich and used as received.
Tetrahydrofuran and toluene were purchased from Aldrich and stored over
molecular sieves. The other organic solvents were reagent grade and used
as received. Silica Gel column chromatography was carried out using
Merck Silica Gel 40-60 µm (230–400 mesh). Analytical TLC was performed
on Merck Kieselgel 60 F 254 Aluminium sheet.
1
Synthesis of compounds.
(2R,3R,4R,5R)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-
methoxy-5-(2-oxo-4-(1H-1,2,4-triazol-1-yl)pyrimidin-1(2H)-yl)
tetrahydrofuran-3-yl (2-cyanoethyl) diisopropylphosphoramidite (2).
U-OMe (0.64 mmol, 500 mg) was co-evaporated three times in dry
acetonitrile (5 ml). In the meantime, 1,2,4-triazole (12.86 mmol, 1.39 g) was
dissolved in dry acetonitrile (10 ml), to which dry triethylamine (13.2 mmol,
2 ml) was added subsequently. U-OMe was redissolved in dry acetonitrile
(5 ml) and was added to the 1,2,4-triazole solution via syringe. The reaction
was then stirred for 10 min at room temperature. An ice bath was used to
cool the reaction to 0 °C. Then phosphorus oxychloride (1.3 mmol, 0.1 ml)
was added dropwise. The reaction was stirred for 4 h at 0 °C. The reaction
mixture was then directly partitioned between ethylacetate and aqueous
sodium bicarbonate and washed twice with brine. The organic layer was
dried over sodium sulphate. Solvents were then removed under reduced
pressure. The crude product was further purified by column
chromatography (EtOAc/Hexane 80:20, 2% TEA). Compound was obtained
as a white foam: 296 mg, 58% overall yield. 1H NMR (CDCl3): δ (ppm) 9.17 (s,
1H), 8.83 (dd, J = 8Hz, 1H), 8.01 (s, 1H), 7.31–7.20 (m, 9H), 6.83–6.76 (m, 4H),
6.38 (d, J = 8Hz, 1H), 5.97 (s, 1H), 4.54 (td, J = 8Hz, 4Hz, 1H), 4.25–4.22 (m,
1H) 3.94 (d, J = 4Hz, 1H), 3.86–3.77 (m, 2H), 3.75 (s, 6H), 3.65 (s, 3H) 3.64–
3.40 (m, 4H), 2.54 (t, J = 8Hz, 1H), 2.34 (t, J = 8Hz, 1H), 1.43 (d, J = 8Hz, 1H),
1.10 (dd, J = 12Hz, 8Hz, 9H), 0.96 (d, J = 8Hz, 2H); 31P NMR (CDCl3): δ (ppm)
150.51; 13C NMR (CDCl3): δ (ppm) 159.27, 158.82 (2C), 153.82, 147.42,
1433.99, 143.2, 135.4, 137.07, 130.42, 128.50, 128.05 (2C), 127.32, 117.50,
11.32 (4C), 94.75, 89.87, 87.25, 83.65, 82.66, 81.92, 69.06, 68.55, 60.42, 60.11,
59.80, 58.89, 58.61, 58.17, 57.95, 55.28, 43.24, 24.77, 24.71, 24.59, 24.50,
20.40, 14.22. ESI‐HRMS: m/z calcd for C42H50N7O8P [(M+H)+] 812.3531, found
812.3520.
1-((2R,3R,4R,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)
-4-hydroxy-3-(2-methoxyethoxy)tetrahydrofuran-2-yl)-5-methyl-4-
(1H-1,2,4-triazol-1-yl)pyrimidin-2(1H)-one (5). TMOE-C4-1,2,4-triazole 4
(0.9 mmol, 657 mg) was dissolved in dry tetrahydrofurane (10 ml). Then dry
triethylamine (0.45 mmol, 4.5 ml) was added followed by slowly adding
triethylamineŸ3 hydrofluoric acid (9.0 mmol, 1.3 ml). The reaction was
stirred at room temperature for 2 h. The reaction mixture was directly
partitioned between ethylacetate and aqueous sodium bicarbonate and
washed twice with brine. The organic layer was dried over sodium sulphate.
Solvents were then removed under reduced pressure to yield the final
product: 536 mg, 89% overall yield. 1H NMR (MeOD4): δ (ppm) 9.21 (s, 1H),
8.40 (s, 1H), 8.09 (s, 1H), 7.37–7.34 (m, 2H), 7.26–7.13 (m, 7H), 6.78-6.74 (m,
4H), 5.85 (s, 1H), 4.43 (dd, J = 8.8Hz, 4.8Hz, 1H), 4.11–4.05 (m, 3H), 3.85–3.80
(m, 1H), 3.66 (s, 6H), 3.56–3.54 (m, 2H), 3.44 (dq, J = 10.4Hz, 2.4Hz, 2H), 3.28
(s, 3H), 1.68 (s, 3H); 13C NMR (CDCl3): δ (ppm) 158.9, 158.4, 154.7, 152.7, 147.3,
145.0, 144.5, 135.5, 135.2, 130.0 (2C), 128.1 (2C), 127.6 (2C), 126.7 (2C), 112.9
(4C), 106.5, 90.3, 86.7, 82.9, 82.6, 71.6, 69.7, 68.1, 61.1, 57.8, 54.3 (4C), 15.2.
ESI‐HRMS: m/z calcd for C36H39N5O8 [(M+Na)+] 692.2691, found 692.2677.
(2R,3R,4R,5R)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-
(2-methoxyethoxy)-5-(5-methyl-2-oxo-4-(1H-1,2,4-triazol-1-
yl)pyrimidin-1(2H)-yl)
tetrahydrofuran-3-yl
(2-cyanoethyl)
diisopropylphosphoramidite (6). Compound 5 (0.3 mmol, 200 mg) was
co-evaporated three times in dry acetonitrile (5 ml) and dissolved in dry
dichlormethane (5 ml); the solvent was subsequently degased for 2 min.
Then, first dry diisopropylethylamine (0.45 mmol, 0.08 ml) was added
1-((2R,3R,4R,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)
-3-(2-methoxyethoxy)-4-((trimethylsilyl)oxy)tetrahydrofuran-2-yl)-5-
methyl-4-(1H-1,2,4-triazol-1-yl)pyrimidin-2(1H)-one (4). TMOE (1.6
4
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followed by the dropwise addition of
2-cyanoethyl-N,N-
and the mixture was then partitioned between ethylacetate and aqueous
sodium bicarbonate and washed twice with brine. The organic layer was
dried in sodium sulphate. Solvents were then removed under reduced
pressure. The crude product was purified by column chromatography
(DCM/MeOH 95:5). Compound was obtained as a yellow foam: 1.6 g, 77%
overall yield. 1H NMR (MeOD4): δ (ppm) 8.03 ( d, J = 8Hz, 1H); 7.49–7.41 (m,
2H); 7.31–7.23 (m, 5H); 7.12–7.10 (m, 2H); 6.87–6.80 (m, 4H); 5.91 (d, J =
2.4Hz, 1H); 5.21 (d, J = 8Hz, 1H); 4.44 (dd, J = 7.2Hz, 5.2Hz, 1H); 4.07–4.05 (m,
2H); 3.98–3.93 (m, 1H); 3.84–3.81 (m, 1H); 3.77 (s, 6H); 3.61–3.58 (m, 2H);
3.48–3.47 (m, 2H), 3.36 (s, 3H); 13C NMR (MeOD4): δ (ppm) 166.15; 160.29;
151.96; 150.04; 145.98; 142.28; 141.22; 136.88; 136.57; 131.44; 130.45;
129.46; 128.95; 128.08; 114.25; 113.86; 102.28; 89.31; 88.22; 84.32; 84.04;
73.09; 71.00; 70.07; 59.19; 55.74; 55.66. ESI‐HRMS: m/z calcd for C33H36N2O9
[(M+Na)+] 627.2313, found 627.2310.
diisopropylaminophosphite (0.45 mmol, 0.1 ml). The reaction was stirred
for 2 h at room temperature. The reaction mixture was then directly
partitioned between ethylacetate and aqueous sodium bicarbonate and
washed twice with brine. The organic layer was dried in sodium sulphate.
Solvents were then removed under reduced pressure. The crude product
was purified by column chromatography (EtOAc/Hexane 80:20, 1% TEA).
1
Compound was obtained as a white foam: 112 mg, 43% overall yield H
NMR (MeOD4): δ (ppm) 9.22 (s, 1H); 8.54 (d, J = 4Hz, 1H); 8.11 (s, 1H), 7.44–
7.39 (m, 2H); 7.32–7.18 (m, 7H); 6.83 (m, 4H); 5.90 (d, J = 4 Hz, 1H); 4.72–4.60
(m, 1H), 4.33–4.20 (m, 2H); 4.17–4.03 (m, 1H), 3.31 (s, 3H); 2.65 (t, J = 5.6 Hz,
1H); 2.53–2.46 (m, 1H); 1.57 (s, 3H); 1.12–1.05 (m, 9H); 0.96 (d, J = 6.8Hz, 3H);
31P NMR (CDCl3): δ (ppm) 150.50, 149.71; 13C NMR (MeOD4): δ (ppm) 159.04;
158.40; 154.82; 152.59; 147.20; 144.96; 144.32; 135.23; 130.27; 128.36;
127.66 (2C); 126.92; 118.46; 112.89 (2C); 106.86; 91.14; 86.78; 82.44; 82.06;
80.89; 71.74; 70.17; 69.82; 69.52; 68.95; 60.24; 58.00; 57.92; 57.83; 57.69;
54.34; 43.10; 42.96; 23.82; 23.75; 23.62; 19.74; 19.61; 15.23; 14.96. ESI‐HRMS:
m/z calcd for C42H56N7O9P [(M+Na)+] 892.3769, found 892.3755.
1-((2R,3R,4R,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)
-3-(2-methoxyethoxy)-4-((trimethylsilyl)oxy)tetrahydrofuran-2-yl)-4-
(1H-1,2,4-triazol-1-yl)pyrimidin-2(1H)-one (10). Compound
8 (1.6
mmol, 1.0 g) was co-evaporated three times in dry acetonitrile (15 ml). UMOE
was redissolved in dry acetonitrile (40 ml), to which first dry triethylamine
(35.5 mmol, 5 ml) and then trimethylsilyl chloride (8.1 mmol, 1 ml) was
added dropwise. The reaction mixture was stirred for 30 min at room
temperature. Then 1,2,4-triazole (38.7 mmol, 2.677 g) was added. After 10
min the reaction mixture was cooled down to 0 °C where phosphorus
oxychloride (4.8 mmol, 0.5 ml) was added dropwise. After 15 min the ice
bath was removed and the reaction was stirred for 4h at room temperature.
The solvent was removed under vacuum and the obtained crude was
partitioned between aqueous sodium bicarbonate and ethylacetate (30
ml) and washed twice with brine (30 ml). The organic layer was dried in
sodium sulphate. Solvents were then removed under reduced pressure.
The crude product was purified by column chromatography
(EtOAc/Hexane 80:20). Compound was obtained as a white foam: 784 mg,
75% overall yield. 1H NMR (MeOD4): δ (ppm) 9.37 (s, 1H), 9.03 (d, J = 7.2Hz,
1H); 8.23 (s, 1H); 7.43–7.40 (m, 2H); 7.36–7.28 (m, 7H); 6.91–6.89 (m, 4H);
6.55 (d, J = 7.2Hz, 1H); 6.00 (s, 1H); 4.53 (dd, J = 9.2Hz, 4.8 Hz, 1H); 4.23 (dt, J
= 9.1Hz, 2.2Hz, 1H); 4.13–4.12 (m, 1H); 4.08–4.06 (m, 2H); 3.90 (ddd, J =
11.1Hz, 5.0Hz, 3.8Hz, 1H); 3.81 (s, 6H); 3.75 (dd, J = 11.1Hz, 2.3Hz, 1H); 3.63
(ddd, J = 5.0Hz, 3.8Hz, 1.0Hz, 1H); 3.49 (dd, J = 11.1Hz, 2.3Hz, 1H); 3.38 (s,
3H); 0.06 (s, 9H); 13C NMR (CDCl3): δ (ppm) 159.38, 158.89 (2C), 154.56,
153.95, 147.56, 143.79, 143.32, 135.33, 135.01, 130.33 (2C), 130.26 (2C),
128.48 (2C), 128.09, (2C), 127.42, 113.37 (4C), 94.93, 90.17, 87.17, 82.63,
82.40, 71.81, 70.00, 68.29, 59.88, 58.97, 55.32, 0.00. ESI‐HRMS: m/z calcd for
C38H45N5O8Si [(M+Na)+] 750.2930, found 750.2929.
1-((2R,3R,4R,5R)-4-hydroxy-5-(hydroxymethyl)-3-(2-methoxyethoxy)
tetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione (8). BH3 THF (5.5
mmol, 5.5 ml) was added to an ice-cooled flask. Then 2’-methoxyethanol
(24 mmol, 1.9 ml) was added dropwise. After a few minutes the flask was
allowed to warm up to room temperature, and it was stirred for 30 min.
Tetrahydrofuran was then evaporated under reduced pressure.
2’methoxyethanol (7 ml) was added as a solvent followed by starting
compound 7 (4.4 mmol, 1.0 g) and sodium bicarbonate (26 mg). The
reaction was stirred at 150 °C under reflux for 48 h. The reaction mixture
was directly prepared for column chromatography for purification
(DCM/MeOH 95:5). Compound was obtained as a dark yellow foam: 1.1 g,
85% overall yield. 1H NMR (MeOD4): δ (ppm) 8.07 (d, J = 8Hz, 1H); 5.95 (d, J
= 4Hz, 1H); 5.69 (d, J = 8Hz, 1H); 4.23 (t, J = 5.6 Hz, 1H); 4.05 (dd, J = 5.2 Hz,
4 Hz, 1H); 3.99 (dt, J = 5.6Hz, 2.8Hz, 1H); 3.89–3.84 (m, 2H); 3.81–3.72 (m,
2H); 3.59 (ddd, J = 5.2Hz, 3.6Hz, 1.6Hz, 2H); 3.37 (s, 3H); 13C NMR (MeOD4):
δ (ppm) 166.18; 152.18; 142.55; 102.49; 89.12; 86.06; 83.78; 73.01; 70.87;
70.04; 61.75; 59.15. ESI‐HRMS: m/z calcd for C12H18N2O7 [(M+Na)+] 325.1006,
found 325.1011.
1-((2R,3R,4R,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)
-4-hydroxy-3-(2-methoxyethoxy)tetrahydrofuran-2-yl)pyrimidine-
2,4(1H,3H)-dione (9). Compound 8 (3.71 mmol, 1.12 g) was co-
evaporated three times in dry pyridine (10 ml). The flask was cooled to 0 °C
in an ice bath and redissolved in dry pyridine (10 ml).
Dimethyoxytriphenylmethylchloride (2.41mmol, 0.71 g) was added to the
reaction. After a few minutes the ice bath was removed and the reaction
was stirred overnight at room temperature. Another portion of
dimethyoxytriphenylmethylchloride (2.41 mmol, 0.71 g) was added and
the reaction was further stirred at room temperature for 4 hours. Methanol
(1 ml) was finally added and the reaction was again stirred at room
temperature for 30 min. Solvents were removed under reduced pressure
1-((2R,3R,4R,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)
-4-hydroxy-3-(2-methoxyethoxy)tetrahydrofuran-2-yl)-4-(1H-1,2,4-
triazol-1-yl)pyrimidin-2(1H)-one (11). Compound 10 (0.9 mmol, 650 mg)
was dissolved in dry tetrahydrofuran (10 ml). Then dry triethylamine (0.45
mmol, 4.5 ml) was added followed by slow addition of triethylamineŸ3
hydrofluoric acid (9.0 mmol, 1.3 ml). The reaction was stirred at room
5
This article is protected by copyright. All rights reserved.

10.1002/hlca.201900222
Helvetica Chimica Acta
HELVETICA
temperature for 2 h. The reaction mixture was directly partitioned between
ethylacetate and aqueous sodium bicarbonate and washed with brine. The
organic layer was dried in sodium sulphate. Solvents were then removed
under reduced pressure to yield the final product: 609 mg, 93 % overall
yield. 1H NMR (MeOD4): δ (ppm) 9.34 (s, 1H); 8.93 (d, J = 7.2Hz, 1H); 8.22 (s,
1H); 7.45–7.43 (m, 2H); 7.33–7.23 (m, 7H), 6.89–6.86 (m, 4H); 6.58 (d, J =
7.2Hz, 1H); 5.94 (s, 1H); 4.54 (dd, J = 9.4Hz, 4.8Hz, 1H); 4.19–4.14 (m, 2H);
4.09 (d, J = 4.8Hz, 1H); 3.92 (ddd, J = 11.3Hz, 5.7Hz, 3.9Hz, 1H); 3.78 (s, 6H);
3.74–3.70 (m, 1H); 3.67–3.60 (m, 2H); 3.57–3.53 (m, 1H); 3.39 (s, 3H); 13C NMR
(MeOD4): δ (ppm) 160.75, 160.35, 156.51, 154.75, 149.27, 145.65, 144.60,
137.05, 136.51, 131.53 (2C), 131.37 (2C), 129.57 (2C), 129.05 (2C), 128.20,
114.34 (4C), 96.06, 91.45, 88.39, 84.07, 83.98, 72.92, 71.03, 69.09, 61.98,
59.16, 55.77 (2C). (1C is missing).
dithiazole‐5‐thione (DDTT; Sulfurizing Reagent II; Glen Research, Virginia)
in dry pyridine/ACN (60:40) for sulfurization. Unmodified 2’-OMe and 2’-
MOE phosphoramidites were prepared at 0.1 M in anhydrous acetonitrile
and triazole-modofied 2’-OMe and 2’-MOE phosphoramidites were
prepared at 0.25 M in anhydrous isobutyronitrile. The CPG support was
suspended in water and 8 mg per modification of neat spermine was
directly added. The suspension was shaken gently overnight. A complete
deprotection was carried out by treatment of 30% aqueous ammonium
hydroxide (200 µl) for 5 h at 55 °C. Further work-up was identical to that of
unmodified sequences. The reaction was cooled to room temperature and
the solid support was filtered and washed with 2x200 µl of EtOH and H2O
1:1 (v/v). The solvents were removed under reduced pressure and the
residue was dissolved in 200 µl water. The crude material was purified by
RP-HPLC. Running buffer: buffer A (0.1 M triethylammonium acetate),
buffer B (methanol); gradient for the DMT‐on purification: 5–80% buffer B
over 6 min. DMT-deprotection was performed in 40% of acetic acid (200 µl)
for 45 min at room temperature. A final RP-HPLC purification (gradient for
the DMT‐off purification: 5 –35% buffer B over 5 min) yielded the final
products.
(2R,3R,4R,5R)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-
(2-methoxyethoxy)-5-(2-oxo-4-(1H-1,2,4-triazol-1-yl)pyrimidin-
1(2H)-yl)
tetrahydrofuran-3-yl
(2-cyanoethyl)
diisopropylphosphoramidite (12). Compound 11 (1.79 mmol, 1.17 g)
was co-evaporated three times in dry acetonitrile (15ml). Then it was
dissolved in dry dichlormethane (13 ml), and the solvent was degased for
two minutes. Then dry diisopropylethylamine (2.68 mmol, 467 µl) was
added followed by the dropwise addition of 2-cyanoethyl N,N-diisopropyl-
chlorophosphoramidite (2.15 mmol, 478 µl). The reaction was stirred for 2
h at room temperature. The reaction mixture was then directly partitioned
between ethylacetate (25 mL) and aqueous sodium bicarbonate (25 mL)
and washed with brine. The organic layer was dried over sodium sulphate
and evaporated under vacuum. The crude product was purified by column
chromatography (EtOAc/Hexane 80:20, 1% TEA) Compound was obtained
as a white foam: 1.3 g, 85% overall yield. 1H NMR (CDCl3): δ (ppm) 9.23 (s,
1H); 8.89 (d, J = 8Hz, 1H); 8.07 (s, 1H); 7.46–7.27 (m, 9H); 6.87–6.74 (m, 4H);
6.46 (d, J = 8Hz, 1H); 6.02 (s. br., 1H); 4.57 (td, J = 12Hz, 8Hz, 1H); 4.39–4.33
(m, 1H); 4.24–4.13 (m, 2H); 3.97–3.89 (m, 1H); 3.81 (s, 6H); 3.78–3.69 (m, 2H);
3.66–3.51 (m, 6H); 3.38 (s, 3H); 2.51 (t, J = 4Hz, 1H); 1.28 (t, J = 8Hz, 3H); 1.19–
1.14 (m, 6H); 1.02 (d, J = 8Hz, 3H); 31P NMR (MeOD4): δ (ppm) 150.53, 148.32;
13C NMR (CDCl3): δ (ppm) 160.68, 160.40, 156.67, 149.16, 145.55, 145.43,
144.50, 136.83, 136.57, 136.35, 131.71, 131.63, 129.78, 129.07, 128.30,
119.84, 114.32, 96.34, 92.31, 88.50, 83.77, 83.27, 82.24, 73.03, 71.45, 71.18,
70.66, 70.02, 60.95, 59.37, 59.22, 59.16, 55.74, 44.51, 44.38, 25.14, 24.95,
23.19, 21.17, 20.97. ESI‐HRMS: m/z calcd for C44H54N7O9P [(M+H)+] 856.3793,
found 856.3782.
Supplementary Material
Supporting information for this article (solid-synthesis LCMS profiles, UV
melting curves, and NMR spectra of compounds) is available at
http://dx.doi.org/10.1002/MS-number.
Acknowledgements
This works was partly funded by the NCCR RNA and Disease from the Swiss
National Science Foundation.
Author Contribution Statement
ED, AL, FH contributed to study design, synthesis and testing of
oligonucleotides, and writing the manuscript. JH contributed to study
design and wrote the paper.
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8
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