Solid-phase synthesis and hybrization behavior of partially 2′/3′-O-acetylated RNA oligonucleotides

Article abstract of DOI:10.1021/jo5002824

Synthesis of partially 2′/3′-O-acetylated oligoribonucleotides has been accomplished by using a 2′/3′-O-acetyl orthogonal protecting group strategy in which non-nucleophilic strong-base (DBU) labile nucleobase protecting groups and a UV-light cleavable linker were used. Strong-base stability of the photolabile linker allowed on-column nucleobase and phosphate deprotection, followed by a mild cleavage of the acetylated oligonucleotides from the solid support with UV light. Two 17nt oligonucleotides, which were synthesized possessing one specific internal 2′- or 3′-acetyl group, were used as synthetic standards in a recent report from this laboratory detailing the prebiotically plausible ligation of RNA oligonucleotides. In order to further investigate the effect of 2′/3′-O-acetyl groups on the stability of RNA duplex structure, two complementary bis-acetylated RNA oligonucleotides were also expediently obtained with the newly developed protocols. UV melting curves of 2′-O-acetylated RNA duplexes showed a consistent ～3.1 °C decrease in T_m per 2′-O-acetyl group.

Full text of DOI:10.1021/jo5002824

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pubs.acs.org/joc
Solid-Phase Synthesis and Hybrization Behavior of Partially
2′/3′‑O‑Acetylated RNA Oligonucleotides
Jianfeng Xu, Colm D. Duffy, Christopher K. W. Chan, and John D. Sutherland*
Medical Research Council Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge Biomedical Campus, Cambridge CB2
0QH, U.K.
S
* Supporting Information
ABSTRACT: Synthesis of partially 2′/3′-O-acetylated oligor-
ibonucleotides has been accomplished by using a 2′/3′-O-
acetyl orthogonal protecting group strategy in which non-
nucleophilic strong-base (DBU) labile nucleobase protecting
groups and a UV-light cleavable linker were used. Strong-base
stability of the photolabile linker allowed on-column
nucleobase and phosphate deprotection, followed by a mild
cleavage of the acetylated oligonucleotides from the solid
support with UV light. Two 17nt oligonucleotides, which were
synthesized possessing one specific internal 2′- or 3′-acetyl
group, were used as synthetic standards in a recent report from this laboratory detailing the prebiotically plausible ligation of
RNA oligonucleotides. In order to further investigate the effect of 2′/3′-O-acetyl groups on the stability of RNA duplex structure,
two complementary bis-acetylated RNA oligonucleotides were also expediently obtained with the newly developed protocols. UV
melting curves of 2′-O-acetylated RNA duplexes showed a consistent ∼3.1 °C decrease in Tm per 2′-O-acetyl group.
dites, formed by 2′−3′ migration of the acyl group, a 2′-acetal
levulinyl ester (ALE) protection strategy was used by the same
group in the synthesis of oligonucleotides microarrays.16 We
decided to adopt a complementary approach based on an
orthogonal protecting group strategy that we hoped would
streamline the automated solid-phase synthesis of partially 2′/
3′-O-acetylated oligoribonucleotides. It was further envisaged
that once an optimized synthesis of partially acetylated-RNA
was possible, the effect of this modification on secondary
structure adoption could be assessed and considered in relation
to an abiotic replication of RNA. In this paper, we describe the
design of a 2′/3′-O-acetyl orthogonal protecting group strategy,
synthesis of the required phosphoramidites and solid support,
and solid-phase synthesis of several partially acetylated
oligonucleotides.
INTRODUCTION
■
The case for ribonucleic acid (RNA) being involved in the
origin of life has been strengthened by the recent observation
that pyrimidine ribonucleoside-2′,3′-cyclic phosphates can be
assembled from simple building blocks under prebiotically
plausible conditions.1,2 Oligomerization of these activated
pyrimidine ribonucleotides to yield 3′,5′-linked RNA had
been a major challenge3−6 until our recent demonstration that
chemoselective acetylation of oligonucleotide-2′/3′-phosphates
facilitates templated ligation with preferential formation of the
native 3′,5′-phosphodiester bond.7 The first formed products of
this chemistry bear a 2′/3′-acetate ester adjacent to the 3′,5′/
2′,5′-phosphodiester bond formed during ligation. Standards
prepared using conventional synthetic chemistry were required
to probe ligation product linkage isomerism, but at the outset
of this work there was no literature procedure to prepare
oligoribonucleotides carrying acetyl groups at certain, prede-
termined 2′/3′-positions.
2′-Modification of oligonucleotides, for example, 2′-fluoro or
2′-O-alkyl, have been extensively investigated because of
potential applications such as antisense technologies.8−10
However, 2′-O-acylated RNA oligonucleotides have not found
widespread use perhaps due to the ease of 2′,3′-migration
during monomer synthesis and lack of compatibility with other
protecting groups.11−14 In a recent report, Damha and co-
workers re-evaluated 2′/3′-O-acyl protection and have
developed the levulinyl group as a 2′-OH protecting group in
oligonucleotide synthesis with on-column deprotection of RNA
being afforded by exposure to hydrazine.15 In order to avoid
contamination with isomeric 3′-O-levulinyl-2′-phosphorami-
RESULTS AND DISCUSSION
■
Protecting group strategies employed in oligonucleotide
chemistry commonly rely on acyl and formamidine protecting
groups for the nucleobases. However, removal of these
protecting groups requires treatment with K2CO3/MeOH or
heating in concentrated aqueous methylamine and/or
ammonia,17−21 conditions which would invariably cleave the
target 2′- or 3′-acetate esters. Merk et al. have pioneered the
use of (2-cyanoethyloxy)carbonyl (ceoc) protecting groups to
protect the exocyclic amino groups of adenosine, cytidine and
guanosine (A, C, and G).22 The non-nucleophilic strong base
Received: February 5, 2014
Published: March 25, 2014
© 2014 American Chemical Society
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1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) was used to remove
ceoc groups under nonprotic reaction conditions via a β-
elimination process that was thought to be compatible with the
maintenance of 2′/3′-O-acetylation.23 Moreover, another
advantage of this strategy is that the deprotection of
cyanoethyl-protected phosphate groups can be accomplished
in the same step.23 The synthesis of partially O-acetylated-RNA
also required an orthogonal protection for the 2′/3′-hydroxyl
groups where acetylation was not required. tert-Butyldimethyl-
silyl (TBS) ethers, frequently used for 2′-hydroxyl group
protection in conventional oligonucleotide synthesis, are easily
cleaved under mild conditions with triethylamine trihydro-
fluoride (TREAT·HF), which we also believed would not result
in the loss of a 2′/3′-O-acetyl group.24
Nucleobase Protection. In view of the previous results, it
was decided to protect the exocyclic amino groups of A, C, and
G with ceoc protecting group. In addition, the O6-position of G
was protected with a (4-nitrophenyl)ethyl (npe) group to
prevent nucleobase anion formation that might otherwise cause
greatly reduced deprotection kinetics of the N2-ceoc group.22
The (2-cyanoethoxy)carbonylation reactions were carried out
with either 2-cyanoethyl carbonochloridate (1) or 1-((2-
cyanoethoxy)carbonyl)-3-methyl-1H-imidazolium chloride (2)
(Scheme 1), which were synthesized by modified procedures of
Merk22 and Wielser.25 The protection of the exocyclic amino
groups of A and C was effected according to Pfleiderer’s
strategy.22
equiv of Ac2O, Et3N and catalytic DMAP for a maximum of 30
min to give 2′,3′,5′-tri-O-acetyl-guanosine 3.28 O6-Alkylation of
3 by Mitsunobu reaction gave a product that was inseparable
from triphenylphosphine(oxide) byproducts, and this material
was carried over to the next step. Deacetylation was
accomplished with concentrated aqueous ammonia, and the
limited exposure to basic conditions resulted in no loss of the
O6-npe protecting group. This three-step installation of the O6-
[2-(4-nitrophenyl)ethyl] protecting group gave 4 in 70% yield
from guanosine. Finally, the introduction of the N2-ceoc
protecting group was effected with 2-cyanoethyl carbon-
ochloridate 1 to afford the nucleobase-protected guanosine 5
in 96% yield.22
Synthesis of the 2′/3′-O-Acetyl RNA Phosphorami-
dites. With the nucleobase-protected materials in hand, we
envisaged two alternative ways to synthesize the 2′/3′-O-acetyl
RNA phosphoramidite monomers depending on the order of
the tritylation and acetylation steps. The first strategy had
tritylation before selective acetylation and was initially explored
starting with commercially available 5′-O-(4,4′-dimethoxytri-
tyl)-uridine 6. This was acetylated with 1 equiv of AcCl and
pyridine in THF to afford an inseparable regioisomeric mixture
of 2′/3′-O-acetyl-5′-O-(4,4′-dimethoxytrityl)uridine 7a and 7b
in a ratio of ca. 1:2.4 in favor of the 3′-O-acetyl regioisomer
(Scheme 3). Our inability to separate the regioisomers was not
Scheme 3. Monoacetylation of 5′-O-(4,4′-
Dimethoxytrityl)uridine 6
Scheme 1. (2-Cyanoethoxy)carbonylation Reagents
Nucleobase protection of G required several steps to install
both the O6-npe and the N2-ceoc protecting groups (Scheme
2). Preliminary experiments revealed that per-acylation of
guanosine was difficult because, while the hydroxyl groups
underwent smooth reaction, the acetylation of the N2-position
was very sluggish and did not proceed to completion. Thus,
rather than relying on temporary acylation of the hydroxyl
groups and the N2-position, we sought to only O-acylate,
reports from Pfleiderer and co-workers indicating that a free
N2-amine did not lead to extensive byproduct formation during
the subsequent O6-alkylation by Mitsunobu reaction.26,27 Thus,
the free hydroxyl groups of guanosine were protected with 3.6
seen as a problem at this stage as it was anticipated that 2′−3′-
migration of the acetyl groups would occur anyway during the
subsequent phosphitylation step.11
Next, we investigated the monoacetylation of the dimethox-
ytritylated base protected purine and cytidine nucleosides with
the same reagents and conditions as above. However, with
acetyl chloride it was not possible to bring about full conversion
of either starting material to monoacetylated products without
also forming significant quantities of the 2′,3′-O-bisacetylated
nucleosides. To overcome this problem, we considered the
alternative strategy of selective acetylation before tritylation,
encouraged by the prospect of being able to direct the
Scheme 2. Optimized Nucleobase Protection of Guanosine
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acetylation away from the 5′-position and toward the 2′/3′-
positions through the use of orthoester chemistry. Accordingly,
the base-protected nucleosides 8, 9, and 5 were first
quantitatively and selectively converted to the intermediate,
2′,3′-cyclic orthoesters 10 which were then hydrolyzed to
regioisomeric mixtures of 2′- and 3′-acetylated nucleosides 11−
13 (Scheme 4).29−31 Standard dimethoxytritylation of 11−13
would prevent us from using one route to make both
regioisomeric monomers. Phosphitylation with 17 required
Hunig’s base to neutralize in situ formed HCl; however, the
̈
base also promoted the migration of the acetyl group from the
2′-OH to the 3′-OH in line with our concerns (Table 1, entries
1 and 5).11 This result was not ideal, and so attention turned to
phosphitylating agent 18, which required acid catalysis (or
“activator”) to form the reactive phosphitylating species.32
Inspired by the literature,33 three different acidic activators of
varying pKa values were utilized in our investigation as follows,
4,5-dicyanoimidazole (DCI, pKa = 5.2),34 1H-tetrazole (pKa =
4.9), and 5-benzylthio-1H-tetrazole (BTT, pKa = 4.1) (Scheme
5b).35 Conducting phosphitylation reactions with 18 and the
three activators, it was found that the most acidic, BTT, gave
the most desirable results (Table 1). In the case of 14, the
product ratio was consistent with that of the starting material
(Table 1, entry 4). This suggested that the increased acidity of
the activator had accelerated the phosphitylation reaction and
minimized the migration of the acetyl group.32,36 Beyond our
expectations, the phosphitylation of 16 (Table 1, entry 6) and
pyrimidine nucleosides (15 and 9) with 18 and BTT gave a
more favorable 2′:3′-OAc regioisomer ratio compared to the
starting materials, but the reason for this effect remains unclear.
Thus, the 2′/3′-OAc regioisomeric mixtures 14, 15, 16, and
9 were all phosphitylated with 18 and BTT as the acidic
activator to give eight 2′/3′-O-acetyl RNA phosphoramidites
(19−22a,b, Scheme 5b), each as a pair of diastereoisomers due
to the presence of a stereogenic phosphorus center. Normal-
phase HPLC enabled separation of the regioisomers, and
although it was on occasion possible to separate the
diastereoisomers of a regioisomer, solid-phase synthesis used
the regioisomerically pure phosphoramidites as pairs of
diastereoisomers.
Scheme 4. Monoacetylation of the Base-Protected
Nucleosides 8, 9, and 5
then furnished the corresponding DMTr-nucleosides 14−16 in
good yield, albeit with unavoidable 2′-3′-migration of the acetyl
groups in favor of the 3′-O-acetyl isomer.
Synthesis of the 2′/3′-O-TBS RNA Phosphoramidites.
For those residues in the planned oligonucleotides where a 2′/
3′-O-acetate was not desired, 2′/3′-O-TBS-protected phosphor-
amidites were used during solid-phase synthesis. Beginning
with the nucleobase-protected nucleosides 8, 9, and 5, the 5′-
DMTr protecting group was introduced to give the 5′-DMTr
nucleosides 23, 24, and 25 (Scheme 6). To install the TBS
For the subsequent phosphitylation stage, we investigated
two commercially available reagents 2-cyanoethyl N,N-
diisopropyl phosphoramidochloridite (17) and 2-cyanoethyl
N,N,N′,N′-tetraisopropylphosphoramidite (18) (Scheme 5a).
We first investigated phosphitylation of the monoacetylated
purine nucleotides 14 and 16 as these had the most biased
2′:3′-OAc ratios, and we were concerned that further skewing
Scheme 5. (a) Phosphitylation Reagents. (b) Phosphitylation Reactions with 18 and BTT as Activator
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a
Table 1. Phosphitylation Reaction Conditions
isolated phosphoramidite ratio
entry
ribonucleoside mixture (1 equiv)
mixture ratio (2′-OAc:3′-OAc)
reaction conditions (equiv)
2′-OAc (a)
3′-OAc (b)
1
2
3
4
5
6
14a+14b
14a+14b
14a+14b
14a+14b
16a+16b
16a+16b
1:3
1:3
1:3
1:3
1:5
1:5
17 (1.3)
DIPEA (4)
DCI (2)
1
1
1
1
1
1
5
18 (2.0)
18 (1.2)
18 (1.2)
17 (1.3)
18 (1.2)
4
1H-Tetrazole (1)
BTT (1)
4
3
DIPEA (4)
BTT (1)
7
3.7
a
Reactions were conducted in anhydrous THF (0.1 M) at room temperature (rt) and reactions times within a range of 1−3 h. 1H-Tetrazole (0.45
M) and 5-benzylthio-1H-tetrazole (BTT, 0.3 M) were added to the reaction as a solution in anhydrous MeCN. DIPEA = N,N-diisopropylethylamine
(Hunig’s base), DCI = 4,5-dicyanoimidazole.
̈
Scheme 6. Synthesis of the 2′/3′-O-TBS RNA phosphoramidites
protecting group, we utilized the AgNO3-catalyzed method
developed by Hakimelahi et al. to bring about improved
selectivity for 2′-O-monosilylation of the 2′/3′-diol.37 Thus, the
nucleosides 23, 24, and 25 were treated with a slight excess of
TBS-Cl and AgNO3, in THF with pyridine as base, to give the
monosilylated nucleosides 26, 27, and 28 each as a
regioisomeric mixture with the ratio favoring the 2′-O-TBS
regioisomer. The regioisomers of 26 and 27 were easily
separated by silica gel flash column chromatography, but
separation of 28 required normal-phase HPLC. The separated
monosilylated-nucleoside derivatives were then phosphitylated
using 17 under basic conditions to give the final 2′/3′-O-TBS
adenosine 29a/b, 2′/3′-O-TBS cytidine 30a/b, and 2′/3′-O-
TBS guanosine 31a/b phosphoramidites in high yield. The 2′-
TBS uridine phosphoramidite was commercially available, and,
although the regioisomeric 3′-TBS phosphoramidite was not,
its precursor 32 was. Thus, 32 was also phosphitylated using
the same conditions to give the 3′-TBS uridine phosphor-
amidite 33.
the first protected nucleoside to the solid support were
considered. Typical commercial linkers such as succinate esters
34 are not compatible with the desired partially acetylated
oligoribonucleotides as they require nucleophilic cleavage
(Figure 1).18,38 The commercially available Q-linker 35 is
Synthesis of the Photolabile Linker. With the required
phosphoramidite monomers in hand, several linkers to attach
Figure 1. Structures of various solid-phase linkers.
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Scheme 7. Preparation of the Solid-Phase Support
cleaved by fluoride ions and was considered to be orthogonal to
2′/3′-O-acetyl groups.39 However, the Q-linker forms an ester
bond with the 2′/3′-hydroxyl of the first nucleotide that upon
cleavage yields a 2′,3′-diol-terminated oligoribonucleotide, and
the identity of the first nucleotide is also fixed. To obtain
maximum flexibility of chemistry at the 3′-oligonucleotide
terminus and allow any sequences of partially acetylated RNA
to be prepared, a universal linker that allowed the synthesis of
2′/3′-phosphate RNA oligonucleotides was sought. It was also
deemed to be desirable, prior to removal of the product from
the solid support, to be able to thoroughly remove excess DBU
to prevent hydroxide formation and deprotection byproducts
upon transfer to aqueous media. Hence, a strong-base-stable
linker that would allow on-column deprotection with DBU was
chosen as the ideal target.
Photolysis was deemed to offer a mild acetyl-orthogonal
method for the cleavage of oligonucleotides from the solid
support. Photolabile linker 36 based on the o-nitrobenzyl group
has been developed by Greenberg and co-workers to allow the
synthesis of oligonucleotides with 3′-hydroxyl, 3′-phosphate,
and 3′-end-modified oligonucleotides.40−43 These linkers have
been modified by Damha and co-workers in order to improve
the photocleavage rates.44 The chain of the linker arm of 37 is
extended by one carbon and is branched at the benzyl position
to generate a tertiary carbon center. However, linker 37
possesses a hydrogen atom β-disposed to the oxygen atom of
the leaving group, and it was suspected that exposure to DBU
would cause a premature cleavage of the oligonucleotide via a
β-elimination process in the case of a phosphate linkage, as
suggested by analogy to the DBU cleavable o-nitrophenyl ethyl
carbonate linker 38 developed by Eritja and co-workers.23,45,46
Thus, it was thought that an alternative linker 39, which has
previously been utilized for the synthesis of peptides,47 and
shortened by one carbon relative to 37, would meet our
requirements. Thus, solid-phase synthesis would be used to
routinely generate 2′/3′-phosphate-terminated oligoribonucleo-
tides, and in the event that a diol-terminated oligoribonucleo-
tide was required, the terminal phosphate would be removed
with a phosphatase.
standard conditions, after which the DMTr-protected secon-
dary alcohol 43 was separated from 44 by normal-phase HPLC
with a 33% yield over two steps. The methyl ester of the
photolabile linker precursor 43 was hydrolyzed using LiOH in a
mixture of THF and water. The crude lithium benzoate salt was
reacted with isobutylchloroformate in anhydrous pyridine to
afford the mixed anhydride 45. LCAA-CPG was then
derivatized with 45 at a concentration of 200−1000 μmol g−1
of CPG under anhydrous conditions to give 46 with a loading
of 33.3−56.2 μmol g−1 after capping of unreacted amines
(Scheme 7).
To ensure the suitability of the solid support 46 for RNA
oligonucleotide synthesis, the photocleavage efficiency was
tested in the synthesis of an RNA oligoribonucleotide, of
sequence 5′-GCCGCCC-3′P (P = phosphate), using commer-
cially available nucleoside phosphoramidites on a 1 μmol scale.
After completion of the automated solid-phase synthesis,
without any deprotection of the oligonucleotide, the CPG
support was suspended in acetonitrile (1 mL) and exposed to
UV irradiation at λ = 365 nm. The cleavage was followed over
time by trityl assay of solubilized material (Figure 2), and this
Figure 2. Rate of oligonucleotide release from the solid-phase support
46 when it is irradiated at λ = 365 nm.
showed that photolysis was complete within 60 min with a
maximal 42.3 O.D. of crude oligonucleotide released. This
corresponded to approximately 683 nmol of oligonucleotide
and a 68% cleavage yield, thus indicating that the photolabile
linker was suitable for oligonucleotide synthesis as its cleavage
was both rapid and efficient.
Synthesis of Partially Acetylated-RNA Oligonucleo-
tides. Synthesis of the acetylated oligonucleotides was
conducted with a standard RNA synthesis cycle using a
Bioautomation MerMade 4 automated synthesis machine. In
The preparation of linker 39 on long-chain alkylamine
controlled-pore glass (LCAA-CPG) began with 3-formyl-4-
nitrobenzoate 40. Reaction of 40 with methylmagnesium
bromide at room temperature gave the desired α-methyl
alcohol 41 in admixture with the inseparable benzyl alcohol 42
as a minor byproduct (13%). The formation of 42 was
suspected to proceed by a radical pathway involving single-
electron transfer to the aromatic aldehyde from the Grignard
reagent.48 The alcohol mixture was dimethoxytritylated under
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Table 2. Yields of Oligonucleotides at Various Stages of Deprotection. Characterization Using MALDI-TOF Mass Analysis Is
Shown for Oligonucleotides That Were Purified
*
a
Subscript 2′-OAc indicates 3′,5′-linkage with 2′-OAc protection at the modification site while 3′-OAc indicates 2′,5′-linkage with 3′-OAc. This
b
oligonucleotide was synthesized using commercial amidites and was deprotected with 3:1 saturated ammonium hydroxide/EtOH. The overall
synthesis yield was obtained by comparing the total released amount of oligonucleotide from CPG to the synthesis cycle scale (1 μmol). The
c
cleavage yield was obtained by comparing the cleaved amount of oligonucleotide to the final trityl yield.
order to improve the coupling efficiency of relatively hindered
nucleoside phosphoramidites used in this work, an 8-fold excess
of phosphoramidite was added to the CPG solid support during
each coupling step in the automated synthesis cycle. Moreover,
the coupling time was increased to 20 min, and the activator
was changed to BTT from ETT (5-ethylthio-1H-tetrazole)
(pKa = 4.28) to improve the coupling yield. Although the
higher acidity of BTT (pKa = 4.10) helped to improve coupling
yields, its poor solubility in acetonitrile (0.35 M) caused it to
precipitate on ends of the reagent delivery lines used by the
automated synthesis machine. This led to blockage of reagent
line nozzles preventing reliable delivery of activator; this issue
became a significant problem during synthesis of longer
oligonucleotides as it led to the synthesis of mainly truncated
oligonucleotides. Therefore, the much more soluble activator
DCI (1.0 M) was employed, which did not crystallize on the
nozzles. Despite DCI (pKa = 5.2) being less acidic than BTT, it
has been shown to be an effective activator due to its greater
nucleophilicity.34
In preliminary experiments, the original capping reagents
CAP A (THF/2,6-lutidine/Ac2O) and CAP B (N-methylimi-
dazole) were utilized to synthesize an 8nt oligonucleotide with
a sequence of 5′-GCCG(2′OAc)GCCG-3′P. After deprotection
and photocleavage, the MALDI-TOF analysis of the free
oligonucleotide showed mass peaks corresponding to a mixture
of mono-, di-, and triacetylated 8nt oligonucleotides. The extra
acetyl groups were thought to result from N-acetylation on the
exocyclic amine of nucleobase during the capping procedure
with Ac2O, which has also been observed by Greenberg and co-
workers.20,49 As suggested by the same authors, the more
sterically hindered reagent, pivalic anhydride, was used in place
of Ac2O in the CAP A reagent (THF/2,6-lutidine/pivalic
anhydride, 4:1:1 v/v). Subsequently, the capping time was
increased from 1 to 5 min to obtain efficient capping with this
more sterically hindered reagent. The problem was also partly
attributable to the acetyl capped solid support, and so the CPG
was also capped with pivaloyl chloride in place of Ac2O to
eliminate this problem completely (Figure S1, Supporting
Information). At the end of the solid-phase synthesis cycle, the
final DMTr-group at the 5′-terminus was not removed since it
was thought that the exposed 5′-hydroxyl could act as a
nucleophile under basic conditions with the possible result of
acetyl migration from the 2′/3′-O-positions to the 5′-position
during the deprotection step with DBU.
Next, the CPG solid support with trityl-on oligonucleotides
was subjected to 0.5 M DBU in anhydrous acetonitrile at 40 °C
for 4 h to bring about the complete nucleobase deprotection.
Morpholine (10% v/v) was added to the deprotection solution
as an acrylonitrile scavenger to prevent the alkylation of the
deprotected nucleobases that is possible via a Michael-type
addition under strongly basic conditions.50 After on-column
deprotection, excess DBU and byproducts from the nucleobase
deprotection, such as acrylonitrile, p-nitrostyrene, and their
morpholinyl adducts, were washed away easily and thoroughly
with anhydrous acetonitrile. After on-column removal of the 5′-
terminal DMTr group with 3% TCA (trichloroacetic acid) in
CH2Cl2, an accurate trityl assay was used to calculate the overall
yield of full-length oligonucleotides (Table 2).
The next step was the UV-induced photocleavage of the
oligonucleotides from CPG solid support. The solid supports
were initially suspended in 3:1 mixture of H2O/MeCN and
subjected to UV irradiation at λ = 365 nm for 1 h. In
preliminary experiments, the sequences of 8nt polyU (Table 2,
entry 1), 8nt polyU with one internal 2′-O-acetylated uridine
(Table 2, entry 2), and 5′-GCCC(2′OAc)GCCC-3′P (Table 2,
entry 3) were synthesized to test the photocleavage after on-
column deprotection. The 8nt polyU modified with or without
internal 2′-acetylated uridine gave 80% cleavage (Table 2,
entries 1 and 2). However, a very poor photocleavage yield was
obtained for the 5′-GCCC(2′OAc)GCCC-3′P sequence (Table 2,
entry 3). The same sequence, synthesized from commercial
phosphoramidites, was previously tested (Figure 2) and showed
good photocleavage yield before deprotection. To investigate
the effect of oligoribonucleotide deprotection status upon
photocleavage, the same sequence was synthesized again with
commercial phosphoramidites (Table 2, entry 4) and then on-
column deprotected with 3:1 saturated ammonium hydroxide/
EtOH. Finally the photolabile CPG solid support was subjected
to UV light under the same conditions (3:1, H2O/MeCN).
This gave a much better photocleavage yield (65% cleavage)
compared to that obtained for the very close analogue with
DBU deprotection (Table 2, entry 3). The different
deprotection reagents of DBU or NH4OH, respectively,
resulted in the DBU or ammonium salts of the oligonucleo-
tides, and the different cleavage yields/recoveries suggested that
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Table 3. Tm and Thermodynamic Parameter Data for Partially Acetylated-RNA Duplexes
Experiments were performed in a 10 mM Na2HPO4, 0.5 mM Na2EDTA buffer (pH7) containing 1 M NaCl and using 2.5 μM of each
complementary strand over a temperature range of 30−90 °C. Data is an average of three heat−cool cycles. Error for Tm values represent standard
deviations of 6 values and are 0.8 °C. Errors for thermodynamic data represent standard deviations of 6 values and are 7.4% for ΔH°, within
8.5% for ΔS° and within 3.1 kJmol−1 for ΔG°37. Nonacetylated oligonucleotides were either synthesized using standard procedures or purchased
in HPLC-purified Na+ form. Underlined nucleotides denote overhanging sequence, subscripts denote site of acetylation or linkage isomerism, green
denotes site of 3′-5′ natural linkage and red denotes site of 2′-5′ unnatural linkage isomerism. See Figure S3 (Supporting Information) for UV
melting curves.
these salts had different solubility in H2O/MeCN mixtures with
the DBU-deprotected oligonucleotides having a lower
solubility. Therefore, pure acetonitrile and DMSO were chosen
as alternative solvents to carry out the photocleavage with the
same sequence (Table 2, entries 5 and 6). It was found that
DMSO was the ideal solvent and dramatically improved the
cleavage yield/recovery of oligonucleotides (Table 2, entries 6−
10).
partially acetylated oligoribonucleotides (Table 2, entries 7−
10) were characterized using MALDI-TOF mass analysis, and
their purity (>95%) was confirmed by analytical SAX-HPLC
(see Table S1 and Figure S2, Supporting Information).
Thermal Denaturation Studies. The structural effects of
partial acetylation on duplex stability were investigated using
UV-melting curves from which the melting temperature (Tm)
and other thermodynamic parameters could be extracted.51−53
The Tm values of duplexes that utilized a 17nt oligomer (Table
3, entries 3−6) were compared with their parent nonacetylated
duplexes (Table 3, entries 1 or 2); the duplexes that contained
the truncated 13nt oligonucleotides (without 5′-overhang)
(Table 3, entries 8−10) were compared with the nonacetylated
13nt duplex (Table 3, entry 7). Analysis of the thermal
denaturation data showed that 2′-O-acetylation, remarkably,
leads to a very consistent 3.1 °C decrease in duplex Tm per
acetyl group. Acetylation at the terminal positions was not
explored, but it is known that other modifications that decrease
duplex Tm give a less significant reduction at the strand ends.54
The destabilizing effect of 2′-O-acetylation is also evident in the
thermodynamic parameters where an increasing degree of
acetylation leads to a greater increase of ΔG°37 (e.g., Table 3,
entry 10 vs entry 7).
It is known that both the major and minor grooves of A-form
RNA duplexes are well hydrated with an extensive network of
hydrogen-bonded water molecules.55,56 In particular, the minor
groove hydration network is mediated by the 2′-hydroxyl
groups that, relative to a DNA duplex, serve to provide a greater
thermodynamic stabilization by acting as a scaffold to bridge
both strands of the duplex. Destabilization of the 2′-O-
acetylated duplexes is indicated by an increase in ΔH° and
ΔS°, which correspond to an unfavorable enthalpic and a
favorable entropic change. It is believed that 2′-O-acetylation
blocks the hydrogen bonding ability of the 2′-hydroxyl and this
reduces the degree of hydration in the minor groove.57
Reduction of the hydration network causes a loss of solvating
water molecules, which accounts for the favorable entropy gain.
Additionally, the number of hydrogen bonds is reduced and the
The final deprotection step to remove the 2′-TBS groups of
acetyl-RNA was carried out under standard conditions with
TREAT·HF in anhydrous DMSO. Finally, the deprotected
oligonucleotides were isolated by precipitation with sodium
acetate and 1-butanol, quantitated by UV absorption, and
analyzed by MALDI-TOF mass spectrometry. To obtain 2′,3′-
diol-terminated oligonucleotides the terminal 2′/3′-phosphate
was removed enzymatically with calf intestinal phosphatase
(CIP), after which the dephosphorylated oligonucleotides were
purified by strong anion exchange HPLC (SAX-HPLC). Failure
sequences and small amounts of deacetylated products were
efficiently separated from the desired acetylated oligonucleo-
tides (see Figure S3, Supporting Information, for HPLC trace).
With an optimized strategy developed for the synthesis of
partially acetylated oligonucleotides now available, authentic
standards were synthesized that corresponded to partially
acetylated products from the prebiotically plausible ligation of
oligoribonucleotides as previously described by our group
(Table 2, entries 7 and 8).7 Two complementary bisacetylated
13nt oligonucleotides were also made to test the effect on the
duplex stability by varying the degree of acetylation (Table 2,
entries 9 and 10). The first sequence was constructed from the
UGUG truncation of the 17nt oligonucleotide (Table 2, entry
7), and the second 13nt oligomer was the sequence
complement. Positions of acetylation were chosen so as to
utilize each of the four acetylated phosphoramidite monomers
and such that the resultant partially acetylated oligoribonucleo-
tides resembled products one might expect from the templated
ligation of tiled short oligoribonucleotides (3 < nt <6) under
prebiotic conditions previously described by our group. Several
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formation of water bridges is hindered, which both contribute
to the unfavorable enthalpy change.
CONCLUSION
■
An orthogonal protecting group strategy was successfully
employed for the synthesis of partially 2′/3′-O-acetylated
oligoribonucleotides. Key to the synthesis was the utilization of
non-nucleophilic strong base-labile nucleobase protecting
groups and a photocleavable linker. This strategy allowed on-
column deprotection of the nucleobases and phosphate groups
under non-nucleophilic conditions and also a mild photolytic
cleavage of RNA oligonucleotides from the solid support.
Partial 2′/3′-O-acetylation has been incorporated into several
oligonucleotides including a 17nt sequence and two comple-
mentary 13nt sequences. Their duplex-forming properties have
been investigated using UV-melting (thermal denaturation).
The results show that 2′-O-acetylation consistently destabilizes
the RNA duplex structure (ΔT = −3.1 °C per acetylation).
This effect was attributed to dehydration of the duplex minor
groove and subsequent disruption of the stabilizing hydrogen-
bonding network.
As expected, inclusion of an internal 2′,5′-linkage within a
duplex led to a reduction in the Tm (ΔTm = −6.9 °C) and
decreased the duplex stability (ΔΔG°37 = +19.4 kJmol−1, Table
3, entries 1 and 2).58 On acetylation of the 3′-hydroxyl at the
internal 2′,5′-linkage the Tm increased (ΔTm = +4.2 °C) and a
favorable decrease in ΔG°37 (ΔΔG°37 = −14.5 kJmol−1, Table
3, entries 2 and 4) was calculated that indicated a more stable
duplex. The reason for the stabilization effect of 3′-acetylation
on the RNA duplexes is not clear thus far. Szostak and co-
workers have shown that partially 2′,5′-linked RNA can form an
A type duplex with C2′-endo sugar puckering predominating at
the 2′,5′-linkage site.59 The C2′-endo (south) sugar pucker can
potentially be stabilized by acetylation of the 3′-hydroxyl group
by enhancing the σC−H2′ → σ*C−O3′ stereoelectronic effect. The
increased electronegativity of 3′-OAc relative to 3′-OH is
thought to further stabilize the already preferred C2-endo sugar
pucker (Figure 3). This could explain the relatively higher
stability of the duplex with 3′-O-acetylated RNA compared to
its 3′-nonacetylated counterpart.
EXPERIMENTAL SECTION
■
General Experimental Methods. Chemical shifts are reported in
ppm (δ). Coupling constants (J) are given in hertz, and the notations
s, d, t, and br represent the multiplicities singlet, doublet, triplet, and
broad signal. Assignment was based on H−1H COSY, HMBC, and
1
HMQC NMR spectra.
2-Cyanoethyl Carbonochloridate (1).22 Triphosgene (5.94 g, 20.0
mmol) was dissolved in anhydrous THF (50 mL) and cooled to 0 °C.
3-Hydroxypropionitrile (2.73 mL, 40.0 mmol) was diluted with
anhydrous THF (17 mL) and was added dropwise to the solution of
triphosgene over a 2 h period. The resultant mixture was warmed to rt
and stirred overnight. Anhydrous pyridine (4.84 mL, 60.0 mmol) was
diluted with anhydrous THF (5 mL) and added dropwise to the
solution at 0 °C. The resultant mixture was warmed to rt and stirred
for a further 1 h. The precipitated pyridinium hydrochloride salt was
removed by filtration, and the supernatant was evaporated under
vacuum. The title compound was isolated as a pale yellow viscous oil
in quantitative yield. The product was used immediately without
further purification or stored at −30 °C under argon until required: 1H
NMR (400 MHz, CDCl3) δ 4.52 (t, J = 6.3 Hz, 2H, −OCH2CH2CN),
2.84 (t, J = 6.3 Hz, 2H, −OCH2CH2CN); 13C NMR (100 MHz,
CDCl3) δ 151.7 (CO), 115.3 (CN), 65.0 (−OCH2CH2CN), 17.8
(−OCH2CH2CN).
Figure 3. Sugar pucker at a 2′,5′-linkage site (in otherwise 3′,5′-linked
RNA) can be C2′-endo or C3′-endo, although the former is preferred
in A-form duplex. It is thought that the increase in Tm that is observed
on acetylation of the 3′-hydroxyl of a 2′,5′-linkage site is caused by an
increase in C2′-endo sugar pucker preference due to an increase in the
magnitude of the σC−H2′ → σ*C−O3′ stereoelectronic effect as the
antibonding orbital becomes a better acceptor due to the increased
electron withdrawal.
O6-[2-(4-nitrophenyl)ethyl]guanosine (4). 2′,3′,5′-Triacetylguano-
sine 3 (10.8 g, 26.3 mmol) was suspended in anhydrous dioxane (100
mL). p-Nitrophenylethanol (5.27 g, 31.5 mmol) and triphenylphos-
phine (8.27 g, 31.5 mmol) were added, and the resultant mixture was
heated at 80 °C for 45 min. Diisopropyl azodicarboxylate (6.20 mL,
31.5 mmol) was added dropwise, upon which the solution began to
boil and then the solution was stirred at 60 °C for a further 1 h. The
solution was cooled to rt and evaporated to dryness under vacuum to
give an oil, from which the intermediate 2′,3′,5′-triacetyl-O6-[2-(4-
nitrophenyl)ethyl]guanosine mixed with triphenylphosphine oxide was
isolated by flash column chromatography (60:35:5, EtOAc/n-hexane/
MeOH). The mixture was taken up in MeOH (200 mL) and cooled to
0 °C. To the solution was added saturated aq NH3 (200 mL) and the
solution stirred in a sealed vessel at rt overnight. The solution was
degassed, and then the solvent was removed under vacuum to give an
orange oil. The oil was taken up in MeOH (∼80 mL). and on
concentration by evaporation under vacuum a yellow solid
precipitated. The mixture was cooled at 4 °C overnight and the
resultant solid precipitate collected by filtration and washed with cold
MeOH (3 × 20 mL). The solid contained acetamide and so was
suspended in H2O (100 mL) and heated at 90 °C for 15 min. Once
cooled to rt, the insoluble material was collected by filtration and
washed with H2O (3 × 20 mL). The solid was air-dried and then dried
under high vacuum to give the title compound over two steps as a
The data obtained through these thermal denaturation
experiments have interesting implications. The copying of a
long single-strand of RNA would create an RNA duplex that,
because of its inordinately high Tm, would be difficult to
denature, and this poses a formidable challenge for the
nonenzymatic replication of RNA at the origin of life.60 The
stability of these “dead-end” duplexes can be reduced by
incorporation of a small proportion of 2′,5′-linkages. However,
2′,5′-linkages are known to be hydrolyzed more rapidly than
3′,5′-linkages in the context of a duplex and could lead to
premature chain cleavage and degradation of oligonucleo-
tides.61 The (temporary) reduction of Tm and duplex stability
by partial acetylation has the distinct potential advantage of
allowing the nonenzymatic template-directed synthesis of
longer oligonucleotides than is possible with native RNA,
while only forming native 3′,5′-phosphodiester bonds. The
more facile strand separation of partially acetylated RNA under
prebiotically plausible conditions would expedite its replication
relative to the replication of native RNA. After replication in
partially acetylated “genotypic” form, unmodified “phenotypic”
RNA could emerge simply through subsequent hydrolysis.
1
yellow amorphous solid (8.45 g, 76%): H NMR (400 MHz, DMSO-
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d6) δ 8.18 (d, J = 8.2 Hz, 2H, aromatic), 8.09 (s, 1H, H8), 7.64 (d, J =
8.2 Hz, 2H, aromatic), 6.44 (s, 2H, NH), 5.78 (d, J = 5.9 Hz, 1H,
H1′), 5.37 (d, J = 6.0 Hz, 1H, 2′-OH), 5.12−5.06 (m, 2H, 3′-OH and
5′-OH), 4.68 (t, J = 6.8 Hz, 2H, −OCH2−), 4.46 (q, J = 5.7 Hz, 1H,
H2′), 4.10 (q, J = 4.3 Hz, 1H, H3′), 3.89 (q, J = 3.8 Hz, 1H, H4′),
3.65−3.50 (m, 2H, H5′), 3.25 (t, J = 6.8 Hz, 2H, −CH2ph); 13C NMR
(100 MHz, DMSO-d6) δ 160.1 (C6), 159.7 (C2), 154.3 (C4), 146.7,
146.3 (aromatic), 138.1 (C8), 130.3, 123.4 (aromatic), 113.8 (C5),
86.6 (C1′), 85.3 (C4′), 73.5 (C2′), 70.4 (C3′), 65.5 (−OCH2−), 61.4
(C5′), 34.4 (−CH2ph); HRMS (ESI-TOF) m/z [M + H]+ calcd for
C18H21N6O7 433.1472, found 433.1462.
(acetyl CO), 163.7, 163.4 (C4), 158.8, 158.7 (aromatic), 151.2 (C2,
7b), 150.6 (C2, 7a), 147.4, 144.2 (aromatic), 140.2, 140.0, 139.6 (C6,
7a+7b), 135.3, 135.2, 135.1, 130.3, 130.2, 130.2, 129.2, 129.1, 128.3,
128.3, 128.2, 128.2, 127.9, 127.9, 127.3, 127.2, 113.4, 113.2 (aromatic),
102.8, 102.8 (C5, 7a+7b), 89.5 (C1′, 7b), 87.4, 87.3 (DMTr-C), 86.8
(C1′, 7a), 83.7 (C4′, 7a), 81.7 (C4′, 7b), 76.0 (C2′, 7a), 74.1 (C2′,
7b), 71.9 (C3′, 7b), 69.9 (C3′, 7a), 62.4, 62.2 (C5′, 7a+7b), 55.4
(OCH3, 7a+7b), 21.6, 20.9 (acetyl CH3, 7a+7b). HRMS (ESI-TOF)
m/z [M + H]+ calcd for C32H32N2O9Na 611.2000, found 611.1979.
N6-[(2-Cyanoethoxy)carbonyl]-2′/3′-O-acetyladenosine (11a
and 11b). To a suspension of 8 (7.00 g, 19.2 mmol) in anhydrous
dioxane (280 mL) were added trimethyl orthoacetate (2.07 mL, 16.4
mmol) and TFA (8.4 μL, 0.11 mmol) and the mixture stirred at 50 °C
for 3 h. Water (70 mL) was added to the mixture, which was then
stirred for a further 1 h at 50 °C. The solvent was removed, and the
residue was purified by flash column chromatography (2−10%
methanol in DCM) to give the title products as an off-white solid
mixture of regioisomers (7.30 g, 94%) (note: 11b: 11a in a ratio of ca.
2.3: 1, calculated by integrations of both H-1′ of 11a and 11b from 1H
N2-[(2-Cyanoethoxy)carbonyl]-O6-[2-(4-nitrophenyl)ethyl]-
guanosine (5). Compound 4 (4.32 g, 10.0 mmol) was coevaporated
with anhydrous pyridine (3 × 20 mL). The residue was dissolved in
anhydrous pyridine (40 mL) and anhydrous CH2Cl2 (55 mL). To the
solution was added dropwise Me3Si-Cl (7.61 mL, 60.0 mmol), and the
resultant mixture was stirred at rt for 20 min. 2-Cyanoethyl
carbonochloridate (1) (2.00 g, 15.0 mmol) diluted in anhydrous
CH2Cl2 (10 mL) was added dropwise and the mixture stirred for a
further 3 h. MeOH (30 mL) was added to quench the reaction and
remove the Me3Si groups. The solvent was removed under vacuum,
and the resultant oil was coevaporated with 1:1 MeOH/toluene (3 ×
30 mL). The residue was taken up in MeOH (15 mL), and H2O was
added dropwise until precipitation of a solid began. The solution was
kept at 4 °C overnight to afford a slightly pink precipitate that was
collected by filtration and washed with cold MeOH (3 × 20 mL). The
solid contained pyridinium·HCl, and this was removed by boiling the
solid as a suspension in H2O (50 mL) for 15 min. After the suspension
was cooled to rt, the solid was collected, washed with H2O (3 × 20
mL), and dried under high vacuum to yield the title compound as a
slightly off-white amorphous solid (5.08 g, 96%): 1H NMR (400 MHz,
DMSO-d6) δ 10.54 (s, 1H, NH), 8.43 (s, 1H, H8), 8.18 (d, J = 8.7 Hz,
2H, aromatic), 7.66 (d, J = 8.7 Hz, 2H, aromatic), 5.89 (d, J = 5.9 Hz,
1H, H1′), 5.45 (d, J = 5.9 Hz, 1H, 2′-OH), 5.16 (d, J = 4.7 Hz, 1H, 3′-
OH), 4.93 (t, J = 5.5 Hz, 1H, 5′-OH), 4.79 (t, J = 6.9 Hz, 2H,
−OCH2−), 4.61 (q, J = 5.7 Hz, 1H, H2′), 4.31 (t, J = 6.0 Hz, 2H,
−COOCH2−), 4.19 (td, J = 4.8 Hz, 3.3 Hz, 1H, H3′), 3.92 (q, J = 4.2
Hz, 1H, H4′), 3.65 (ABX, JAB = 11.8, JAX = 4.9 Hz, 1H, H-5′), 3.54
(ABX, JBA = 11.8, JBX = 4.9 Hz, 1H, H5″), 3.34−3.31 (m, 2H,
−CH2ph), 2.94 (t, J = 6.0 Hz, 2H, −CH2CN); 13C NMR (100 MHz,
DMSO-d6) δ 159.7 (C6), 153.1 (C4), 151.9 (C2), 151.5 (CO),
146.4, 146.3 (aromatic), 141.4 (C8), 130.4, 123.4 (aromatic), 118.6
(CN), 117.3 (C5), 87.1 (C1′), 85.7 (C4′), 73.4 (C2′), 70.4 (C3′),
66.4 (−OCH2−), 61.4 (C5′), 59.4 (−COOCH2−), 34.2 (−CH2ph),
17.7 (−CH2CN); HRMS (ESI-TOF) m/z [M + H]+ calcd for
C22H24N7O9 530.1636, found 530.1620.
2′/3′-O-Acetyl-5′-O-(4,4′-dimethoxytrityl)uridine (7a and 7b). To
a solution of commercially available 5′-O-(4,4′-dimethoxytrityl)uridine
(3.00 g, 5.49 mmol) in anhydrous THF (20 mL) was added anhydrous
pyridine (0.44 mL, 5.49 mmol), followed by acetyl chloride (0.39 mL,
5.49 mmol) at 0 °C. The mixture was warmed to rt, stirred for 3 h, and
then quenched by addition of saturated aq NaHCO3. The organics
were extracted with CH2Cl2 (3 × 20 mL). The combined organic
layers were dried over MgSO4 and finally concentrated under vacuum.
The residue was purified by flash column chromatography (50:50:1:1,
EtOAc/Tol/MeOH/Et3N) to give the title compounds 7a and 7b
(2.20 g, 68%) as a regioisomeric mixture (note: the isomers were
isolated as a mixture in a ratio of ca. 2.4:1 7b:7a calculated by
integrations of both H1′ of 7a and 7b): 1H NMR (400 MHz, CDCl3)
δ 9.40 (s, 0.7H, NH, b), 8.66 (d, J = 1.4 Hz, 0.3H, NH, 7a), 7.84 (d, J
= 8.1 Hz, 0.7H, H6, 7b), 7.79 (d, J = 8.2 Hz, 0.3H, H6, 7a), 7.44−7.18
(m, 9H, aromatic, 7a+7b), 6.84 (d, J = 8.8 Hz, 4H, aromatic, 7a+7b),
6.14 (d, J = 4.5 Hz, 0.3H, H1′, 7a), 6.00 (d, J = 4.9 Hz, 0.7H, H1′, 7b),
5.45−5.33 (m, 1.3H, H5 and H2′), 5.26 (t, J = 4.9 Hz, 0.7H, H3′, 7b),
4.59 (q, J = 4.8 Hz, 0.3H, H3′, 7a), 4.53 (q, J = 5.4 Hz, 0.7H, H2′, 7b),
4.28 (d, J = 4.4 Hz, 0.7H, H4′, 7b), 4.14 (dt, J = 4.9, 2.3 Hz, 0.3H, H4′,
7a), 3.91 (d, J = 6.5 Hz, 0.7H, 2′-OH, 7b), 3.79 (s, 6H, OCH3, 7a+7b),
3.55 (app. dd, J = 11.0, 2.3 Hz, 1H, H5′, 7a+7b), 3.49−3.43 (m, 1H,
H5″, 7a+7b), 2.48 (d, J = 3.9 Hz, 0.3H, 3′-OH, 7a) 2.18, 2.14 (2 × s,
3H, acetyl CH3, 7a+7b); 13C NMR (CDCl3, 101 MHz): δ 170.7, 170.5
1
NMR): H NMR (400 MHz, CDCl3) δ 9.92 (s, 0.70H, NH, 11b),
9.79 (s, 0.30H, NH, 11a), 8.62 (s, 0.30H, H8, 11a), 8.40 (s, 0.70H,
H8, 11b), 8.27 (s, 0.30H, H2, 11a), 8.21 (s, 0.70H, H2, 11b), 6.17 (d,
J = 5.7 Hz, 0.30H, H1′, 11a), 6.00 (broad, 0.40H, 5′-OH), 5.92 (d, J =
7.8 Hz, 0.70H, H1′, 11b), 5.74 (t, J = 5.5 Hz, 0.30H, H2′, 11a), 5.58
(d, J = 5.2 Hz, 0.70H, H3′, 11b), 5.14 (dd, J = 7.9, 5.3 Hz, 0.70H, H2′,
11b), 4.85 (t, J = 4.3 Hz, 0.30H, H3′, 11a), 4.64 (s, 0.60H, OH),
4.49−4.38 (m, 2H, −OCH2−, 11a+11b), 4.35 (s, 0.70H, H-4′, 11b),
4.31 (q, J = 2.4 Hz, 0.30H, H4′, 11a), 4.07−3.74 (m, 2H, H5′, 11a
+11b), 2.82 (t, J = 6.1 Hz, 2H, −CH2CN, 11a+11b), 2.21 (s, 2.10H,
acetyl CH3, 11b), 2.08 (s, 0.90H, acetyl CH3, 11a); 13C NMR (CDCl3,
101 MHz) δ 170.7 (acetyl CO, 11b), 170.1 (acetyl CO, 11a),
152.5 (C8, 11a), 151.9 (C8, 11b), 150.8 (CO, 11b), 150.7 (CO,
11a), 150.3 (C4, 11a+11b), 149.8 (C6, 11b), 149.6 (C6, 11a), 143.7
(C2, 11b), 143.1 (C2, 11a), 123.4 (C5, 11b), 123.0 (C5, 11a), 117.5
(CN, 11b), 117.4 (CN, 11a), 90.8 (C1′, 11b), 88.3 (C1′, 11a), 87.0
(C4′, 11a), 85.8 (C4′, 11b), 75.8 (C2′, 11a), 74.5 (C3′, 11b), 72.7
(C2′, 11b), 70.1 (C3′, 11a), 62.9 (C5′, 11b), 62.0 (C5′, 11a), 60.4
(−OCH2−, 11b), 60.4 (−OCH2−, 11a), 21.1 (acetyl CH3, 11b), 20.8
(acetyl CH3, 11a), 18.4 (−CH2CN, 11a+11b); HRMS (ESI-TOF) m/
z [M + H]+ calcd for C16H19N6O7 407.1315, found 407.1310.
N4-[(2-Cyanoethyloxy)carbonyl]-2′/3′-O-acetylcytidine (12a and
12b). Compound 12a/b (6.46 g, 82% yield, 12b:12a in a ratio of ca.
2.5:1) was obtained as a regioisomeric mixture from 9 after following
the same reaction and workup procedures as for 11a/b: 1H NMR (400
MHz, DMSO-d6) δ 10.96 (s, 1H, NH), 8.39−8.34 (m, 1H, H6, 12a
+12b), 7.01 (s, 1H, H5, 12a+12b), 5.95 (d, J = 3.9 Hz, 0.30H, H1′,
12a), 5.86 (d, J = 4.9 Hz, 0.70H, H1′, 12b), 5.79 (d, J = 5.7 Hz, 0.70H,
2′-OH, 12b), 5.50 (d, J = 5.6, 0.30H, 3′-OH, 12a), 5.39, 5.19 (2 ×
broad, 1H, 5′-OH, 12a+12b), 5.17 (t, J = 4.5 Hz, 0.30H, H2′, 12a),
5.02 (t, J = 5.0 Hz, 0.70H, H3′, 12b), 4.30 (m, 3H, H2′ and
−OCH2−), 4.22 (q, J = 5.4 Hz, 0.30H, H3′, 12a), 4.13−4.10 (m,
0.70H, H4′, 12b), 3.92 (dt, J = 5.6, 2.7 Hz, 0.70H, H4′, 12a), 3.76−
3.58 (m, 2H, H5′, 12a+12b), 2.93 (t, J = 6.0 Hz, 2H, −CH2CN, 12a
+12b), 2.08 (s, 3H, acetyl CH3, 12a+12b); 13C NMR (101 MHz,
DMSO-d6) δ 169.8 (acetyl CO, 12b), 169.3 (acetyl CO, 12a),
162.9, 162.8 (C4, 12a+12b), 154.5, 154.2 (C2, 12a+12b), 152.9 (C
O, 12a+12b), 145.0, 145.0 (C6, 12a+12b), 118.4 (CN, 12a+12b),
94.9, 94.8 (C5, 12a+12b), 89.6 (C1′, 12b), 87.7 (C1′, 12a), 85.0 (C4′,
12a), 82.4 (C4′, 12b), 75.8 (C2′, 12a), 72.6 (C2′, 12b), 71.9 (C3′,
12b), 67.8 (C3′, 12a), 60.3, 60.2, 60.0 (C5′ and −OCH2−, 12a+12b),
20.8, 20.7 (acetyl CH3, 12a+12b), 17.6 (−CH2CN, 12a+12b); HRMS
(ESI-TOF) m/z [M + H]+ calcd for C15H19N4O8 383.1203, found
383.1213.
N2-[(2-Cyanoethoxy)carbonyl]-O6-[2-(4-nitrophenyl)ethyl]-2′/3′-
O-acetyl-guanosine (13a and 13b). Compound 13a/b (3.23 g, 99%
yield, 13b: 13a in a ratio of ca. 1.5:1) was obtained as a regioisomeric
mixture from 5 after following the same reaction and workup
1
procedures as for 11a/b: H NMR (400 MHz, DMSO-d6) δ 10.58−
10.57 (m, 1H, NH, 13a+13b), 8.46−8.45 (m, 1H, H-8, 13a+13b),
8.18, 7.66 (d, J = 8.6 Hz, 4H, aromatic, 13a+13b), 6.14 (d, J = 5.9 Hz,
3319
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The Journal of Organic Chemistry
Featured Article
0.40H, H1′, 13a), 5.89 (d, J = 7.0 Hz, 0.60H, H1′, 13b), 5.81 (d, J =
6.1 Hz, 0.60H, 2′-OH, 13b), 5.59−5.55 (m, 0.80H, H2′ and 3′-OH,
13a), 5.31 (dd, J = 5.4, 2.3 Hz, 0.60H, H3′, 13b), 5.12 (t, J = 5.6 Hz,
0.60H, 5′-OH, 13b), 5.02 (t, J = 5.4 Hz, 0.40H, 5′-OH, 13a), 4.91 (q, J
= 6.2 Hz, 0.6H, H-2′, 13b), 4.82−4.77 (m, 2H, −OCH2−, 13a+13b),
4.53 (td, J = 5.3, 3.7 Hz, 0.40H, H3′, 13a), 4.31 (t, J = 6.0 Hz, 2H,
−COOCH2−, 13a+13b), 4.10 (q, J = 4.1 Hz, 0.60H, H4′, 13b), 3.97
(q, J = 4.1 Hz, 0.40H, H4′, 13a), 3.73−3.56 (m, 2H, H5′, 13a+13b),
3.34−3.31 (m, 2H, −CH2ph, 13a+13b), 2.95 (t, J = 6.0 Hz, 2H,
−CH2CN, 13a+13b), 2.12 (s, 1.80H, acetyl CH3, 13b), 2.03 (s, 1.20H,
acetyl CH3, 13a); 13C NMR (101 MHz, DMSO-d6) δ 169.6 (acetyl
CO, 13a+13b), 159.8 (C6, 13a+13b), 153.2 (C4, 13b), 152.9 (C4,
13a), 152.0 (C2, 13a+13b), 151.5 (CO, 13a+13b), 146.5, 146.4,
146.3 (aromatic), 141.4 (C8, 13a), 141.2 (C8, 13b), 130.4, 123.4
(aromatic), 118.6 (CN, 13a+13b), 117.3 (C5, 13a+13b), 86.9 (C1′,
13b), 86.1 (C4′, 13a), 84.8 (C1′, 13a), 83.5 (C4′, 13b), 75.3 (C2′,
13a), 73.2 (C3′, 13b), 71.8 (C2′, 13b), 68.8 (C3′, 13a), 66.5
(−OCH2−, 13a+13b), 61.3, 61.3 (C5′, 13a+13b), 59.5, 59.3
(−COOCH2−, 13a+13b), 34.3, 34.2 (−CH2ph, 13a+13b), 20.8
(acetyl CH3, 13b), 20.6 (acetyl CH3, 13a), 17.8, 17.7 (−CH2CN, 13a
+13b); HRMS (ESI-TOF) m/z [M + H]+ calcd for C24H26N7O10
572.1741, found 572.1757.
(m, 2.75H, H4′, 15b and −OCH2−, 15a+15b), 4.20−4.16 (m, 0.25H,
H4′, 15a), 3.79−3.78 (m, 6H,-OCH3, 15a+15b), 3.61−3.49 (m,
1.25H, H5′, 15a+15b), 3.39 (dd, J = 11.2, 2.7 Hz, 0.75H, H5″, 15b),
2.72 (m, 2H, −CH2CN, 15a+15b), 2.14 (s, 0.75H, acetyl CH3, 15a),
2.11 (s, 2.25H, acetyl CH3, 15b); 13C NMR (101 MHz, CDCl3) δ
170.6 (acetyl CO, 15a), 170.4 (acetyl CO, 15b), 162.9 (C4, 15a
+15b), 158.8 (aromatic, 15a+15b), 155.9 (C2, 15a+15b), 152.1 (C
O, 15a+15b), 144.4, 144.2, 144.1 (C6 and aromatic, 15a+15b), 135.6,
135.4, 135.2 (aromatic, 15a+15b), 130.1, 130.1, 128.3, 128.1, 127.2
(aromatic, 15a+15b), 117.0, 116.9 (CN, 15a+15b), 113.4 (aromatic,
15a+15b), 95.6 (C5, 15a+15b), 92.7 (C1′, 15b), 89.0 (C1′, 15a),
87.3, 87.2 (DMTr-C, 15a+15b), 82.8 (C4′, 15a), 81.8 (C1′, 15b),
76.7 (C2′, 15a), 74.5 (C2′, 15b), 71.5 (C3′, 15b), 68.5 (C3′, 15a),
61.6 (C5′, 15b), 61.4 (C5′, 15a), 60.2 (−OCH2−, 15a+15b), 55.3
(−OCH3, 15a+15b), 20.8 (acetyl CH3, 15a+15b), 18.1 (−CH2CN,
15a+15b); HRMS (ESI-TOF) m/z [M + H]+ calcd for C36H37N4O10
685.2510, found 685.2528.
N2-[(2-Cyanoethoxy)carbonyl]-O6-[2-(4-nitrophenyl)ethyl]-2′/3′-
O-acetyl-5′-O-(4,4′-dimethoxytrityl)guanosine (16a and 16b).
Compound 16a/b (3.63 g, 74% yield, 16b:16a in a ratio of ca. 5:1)
was obtained as a regioisomeric mixture from 12a/b after following the
1
same reaction and workup procedures as for 14a/b: H NMR (400
N6-[(2-Cyanoethoxy)carbonyl]-2′/3′-O-acetyl-5′-O-(4,4′-dimeth-
oxytrityl)adenosine (14a and 14b). Compound 13a/b (1.50 g, 3.69
mmol) was coevaporated with anhydrous pyridine (3 × 20 mL). The
residue was taken up in anhydrous pyridine (35 mL). DMTr-Cl (2.50
g, 7.38 mmol) was added and the mixture stirred for 3 h. MeOH (20
mL) was added and the mixture stirred for 10 min. The solvent was
removed under vacuum, and the residue was taken up in CH2Cl2 (30
mL). The organics were washed with saturated aq NaHCO3 (3 × 50
mL). The organics were separated, dried over Na2SO4, and evaporated
to dryness. The residue was coevaporated with toluene (3 × 20 mL),
followed by CH2Cl2 (3 × 20 mL), after which point the residue was
purified by flash column chromatography (0−5% MeOH in 50%
toluene in EtOAc with 1% Et3N) to give a regioisomeric mixture (1.64
g, 63%) (note: 14b:14a in a ratio of ca. 3.2:1, calculated by
integrations of both H-1′ in 1H NMR): 1H NMR (400 MHz, CDCl3)
δ 9.75, 9.63 (2 × s, 1H, NH, 14a+14b), 8.69, 8.67 (2 × s, 1H, H8, 14a
+14b), 8.26, 8.24 (2 × s, 1H, H2, 14a+14b), 7.39−6.74 (m, 13H,
aromatic, 14a+14b), 6.27 (d, J = 4.5 Hz, 0.24H, H1′, 14a), 6.10 (d, J =
6.5 Hz, 0.76H, H1′, 14b), 5.87 (t, J = 4.9 Hz, 0.24H, H2′, 14a), 5.47
(dd, J = 5.4, 2.4 Hz, 0.76H, H3′, 14b), 5.14 (t, J = 6.0 Hz, 0.76H, H2′,
14b), 4.88 (t, J = 5.2 Hz, 0.24H, H3′, 14a), 4.40−4.33 (m, 2.76H,
−OCH2− and H-4′), 4.27 (q, J = 3.9 Hz, 0.24H, H4′, 14a), 3.74−3.73
(m, 6H,-OCH3, 14a+14b), 3.56−3.35 (m, 2H, H5′, 14a+14b), 2.72−
2.68 (m, 2H, −CH2CN, 14a+14b), 2.15 (s, 2.28H, acetyl CH3, 14b),
2.08 (s, 0.72H, acetyl CH3, 14a); 13C NMR (CDCl3, 101 MHz) δ
170.5 (acetyl CH3, 14b), 170.2 (acetyl CH3, 14a), 158.6 (aromatic,
14a+14b), 152.9 (C8, 14a), 152.6 (C8, 14b), 151.3 (C4, 14a+14b),
150.6 (−OCH2−, 14a+14b), 149.4 (C6, 14b), 149.3 (C6, 14a), 144.4,
144.3 (aromatic, 14a+14b), 142.1 (C2, 14a), 141.8 (C2, 14b), 135.6,
135.5, 135.4, 135.4, 130.1, 130.1, 128.2, 128.1, 128.0, 127.1 (aromatic,
14a+14b), 122.5 (C5, 14a+14b), 117.0 (CN, 14a+14b), 113.3
(aromatic, 14a+14b), 89.2 (C1′, 14b), 86.9 (DMTr-C, 14b), 86.7
(DMTr-C, 14a), 86.6 (C1′, 14a), 83.9 (C4′, 14a), 83.4 (C4′, 14b),
75.8 (C2′, 14a), 73.8, 73.7 (C2′, C3′, 14b), 70.2 (C3′, 14a), 63.3
(C5′, 14a), 63.0 (C5′, 14b), 60.1 (−OCH2−, 14a+14b), 55.3
(−OCH3, 14a+14b), 21.0 (acetyl CH3, 14b), 20.8 (acetyl CH3,
14a), 18.2 (−CH2CN, 14a+14b); HRMS (ESI-TOF) m/z [M + H]+
calcd for C37H37N6O9 709.2610, found 709.2617.
MHz, CDCl3) δ 8.17−8.13 (m, 2H, aromatic, 16a+16b), 8.08 (s,
0.83H, H8, 16b), 7.94 (s, 0.17H, H8, 16a), 7.64 (s, 1H, NH), 7.51−
7.46 (m, 2H, aromatic, 16a+16b), 7.39−7.09 (m, 9H, aromatic, 16a
+16b), 6.77−6.68 (m, 4H, aromatic, 16a+16b), 6.42 (s, 0.75H, 2′-OH,
16b), 6.09 (d, J = 4.0 Hz, 0.17H, H1′, 16a), 5.92−5.89 (m, 1H, H1′
and H2′), 5.48 (d, J = 5.5 Hz, 0.83H, H3′, 16b), 5.18 (t, J = 5.3 Hz,
0.83H, H2′, 16b), 5.11 (t, J = 5.6 Hz, 0.17H, H3′, 16a), 4.78 (t, J = 6.8
Hz, 2H, −OCH2−, 16a+16b), 4.47−4.32 (m, 2.83H, H4′ and
−COOCH2−), 4.19 (q, J = 4.5 Hz, 0.17H, H4′, 16a), 3.75, 3.74 (2 ×
s, 6H, OCH3, 16a+16b), 3.48−3.23 (m, 4H, H5′ and −CH2ph, 16a
+16b), 2.78 (t, J = 6.1 Hz, 1.67H, −CH2CN, 16b), 2.69−2.66 (m,
0.33H, −CH2CN, 16a), 2.18 (s, 2.50H, acetyl CH3, 16b), 2.15 (s,
0.50H, acetyl CH3, 16a); 13C NMR (CDCl3, 101 MHz) δ 170.5
(acetyl CO, 16b), 170.2 (acetyl CO, 16a), 161.0, 160.8 (C6, 16a
+16b), 158.6 (aromatic), 152.7 (C4, 16a), 151.8 (C4, 16b), (C2,
16b), 151.4 (C2, 16a) 150.9, 150.4 (CO, 16a+16b), 147.0, 145.8,
145.6, 144.6, 144.2 (aromatic), 141.0 (C8, 16a), 140.5 (C8, 16b),
135.8, 135.4, 135.3, 130.2, 130.1, 130.0, 128.3, 128.0, 127.9, 126.9,
123.9 (aromatic), 118.5 (C5, 16a+16b), 116.8 (CN, 16a+16b), 113.2
(aromatic), 91.6 (C1′, 16b), 86.9 (DMTr-C), 86.6 (C1′, 16a), 85.4
(C4′, 16b), 83.7 (C4′, 16a), 75.8 (C2′, 16a), 75.2, 75.1 (C2′, C3′,
16b), 70.1 (C3′, 16a), 67.2 (−OCH2−, 16a+16b), 63.7 (C5′, 16b),
63.3 (C5′, 16a), 60.1, 59.7 (−COOCH2−, 16a+16b), 55.3 (OCH3),
35.1 (−CH2ph, 16a+16b), 21.2, 20.8 (acetyl CH3, 16a+16b), 18.4,
18.3 (−CH2CN, 16a+16b); HRMS (ESI-TOF) m/z [M + H]+ calcd
for C45H44N7O12 874.3048; Found 874.3030.
N6-[(2-Cyanoethoxy)carbonyl]-5′-O-(4,4′-dimethoxytrityl)-
adenosine (23). Compound 8 (0.92 g, 2.52 mmol) was coevaporated
with anhydrous pyridine (3 × 20 mL). The residue was taken up in
anhydrous pyridine (20 mL). DMTr-Cl (1.03 g, 3.4 mmol) was added,
and the mixture was stirred for 3 h. The solvent was removed under
vacuum, and the residue was taken up in CH2Cl2 (20 mL). The
organic layer was washed with saturated aq NaHCO3 (3 × 30 mL) and
dried over Na2SO4, and the solvent was removed under vacuum. The
crude residue was coevaporated with toluene (3 × 20 mL), followed
by CH2Cl2 (2 × 20 mL) to remove residual pyridine, and finally
purified by flash column chromatography (75:25:2, EtOAc/Tol/Et3N
→ 40:2:2:2, EtOAc/Tol/MeOH/Et3N) to give the title compound as
1
N4-[(2-Cyanoethyloxy)carbonyl]-2′/3′-O-acetyl-5′-O-(4,4′-di-
methoxytrityl)cytidine (15a and 15b). Compound 15a/b (11 g, 96%
yield, 15b:15a in a ratio of ca. 3:1) was obtained as a regioisomeric
mixture from 12a/b after following the same reaction and workup
a slightly yellow foam (1.41 g, 83%): H NMR (400 MHz, CDCl3) δ
8.67 (s, 1H, H8), 8.28 (s, 1H, H2), 7.31−7.14 (m, 9H, aromatic), 6.74
(d, J = 8.8 Hz, 4H, aromatic), 6.11 (d, J = 5.5 Hz, 1H, H1′), 4.90 (t, J
= 5.4 Hz, 1H, H-2′), 4.51 (dd, J = 5.0, 2.8 Hz, 1H, H3′), 4.42−4.36
1
procedures as for 14a/b: H NMR (400 MHz, CDCl3) δ 8.34 (d, J =
(m, 3H, H4′ and −OCH2−), 3.74 (s, 6H, OCH3), 3.46 (ABX, JAB =
7.5 Hz, 1H, H6, 15a+15b), 7.43−7.21 (m, 9H, aromatic, 15a+15b),
7.03 (s, 1H, H5, 15a+15b), 6.86−6.83 (m, 4H, aromatic, 15a+15b),
6.05 (d, J = 1.7 Hz, 0.25H, H1′, 15a), 5.92 (d, J = 2.7 Hz, 0.75H, H1′,
15b), 5.47 (dd, J = 4.8, 1.9 Hz, 0.25H, H2′, 15a), 5.21 (t, J = 5.7 Hz,
0.75H, H3′, 15b), 4.67−4.63 (m, 1H, H3′, 15a and H2′, 15b), 4.33
10.6, JAX = 3.3 Hz, 1H, H5′), 3.34 (ABX, JBA = 10.6, JBX = 3.8 Hz, 1H,
H5″), 2.71 (t, J = 6.2 Hz, 2H, −CH2CN); 13C NMR (CDCl3, 101
MHz) δ 158.5 (aromatic), 152.3 (C8), 150.9 (C4), 150.3 (CO),
149.1 (C6), 144.3 (aromatic), 141.8 (C2), 135.4, 135.4, 129.9, 127.9,
127.8, 126.9 (aromatic), 122.3 (C5), 116.8 (CN), 113.1 (aromatic),
3320
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Featured Article
90.0 (C1′), 86.5 (DMTr-C), 85.5 (C4′), 75.3 (C2′), 72.1 (C3′), 63.4
(C5′), 60.0 (−OCH2−), 55.2 (OCH3), 18.1 (−CH2CN); HRMS
(ESI-TOF) m/z [M + H]+ calcd for C35H35N6O8 667.2511, found
667.2533.
6.88−6.79 (m, 4H, aromatic), 6.00 (d, J = 5.1 Hz, 1H, H1′), 5.47 (d, J
= 6.0 Hz, 1H, 2′-OH), 4.88 (q, J = 5.4 Hz, 1H, H2′), 4.49 (t, J = 4.6
Hz, 1H, H3′), 4.32 (t, J = 6.0 Hz, 2H, −OCH2−), 4.06 (q, J = 4.5 Hz,
1H, H4′), 3.72 (s, 6H, OCH3), 3.36 (m, 1H, H5′), 3.15 (ABX, JBA
=
N4-[(2-Cyanoethyloxy)carbonyl]-5′-O-(4,4′-dimethoxytrityl)-
cytidine (24). Compound 24 (15 g, 89% yield) was obtained from 9 g
of 9 after following the same reaction and workup procedure as for 23:
1H NMR (400 MHz, CDCl3) δ 8.32 (d, J = 7.5 Hz, 1H, H6), 7.39−
10.5, JBX = 4.9 Hz, 1H, H5″), 2.93 (t, J = 6.0 Hz, 2H, −CH2CN), 0.84
(s, 9H, Si(C(CH3)3), 0.08 (s, 3H, Si(CH3)2), 0.05 (s, 3H, Si(CH3)2);
13C NMR (DMSO, 101 MHz) δ = 158.1 (aromatic), 151.7, 151.6,
151.5 (CO, C8, C4), 149.6 (C6), 144.7 (aromatic), 143.7 (C2),
135.4, 129.6, 129.6, 127.7, 127.6, 126.7 (aromatic), 124.2 (C5), 118.5
(CN), 113.1 (aromatic), 88.3 (C1′), 85.6 (DMTr-C), 83.6 (C4′), 72.2
(C3′), 72.0 (C2′), 63.0 (C5′), 59.9 (−OCH2−), 55.0 (OCH3), 25.8
(SiC(CH3)3), 18.0 (−CH2CN), 17.6 (SiC(CH3)3), −4.5 (Si(CH3)2),
−5.1 (Si(CH3)2); HRMS (ESI-TOF) m/z [M + H]+ calcd for
C41H49N6O8Si 781.3381, found 781.3380.
7.16 (m, 9H, aromatic), 6.97 (s, 1H, H5), 6.82 (dd, J = 9.0, 2.3 Hz,
4H, aromatic), 5.90 (d, J = 1.8 Hz, 1H, H1′), 4.48−4.24 (m, 5H, H2′,
H3′, H4′, and −OCH2−), 3.77 (s, 6H, OCH3), 3.52−3.39 (m, 2H,
H5′), 2.74 (t, J = 6.3 Hz, 2H, −CH2CN); 13C NMR (CDCl3, 100
MHz) δ 162.8 (C4), 158.7, 158.7 (aromatic), 156.3 (C2), 152.0 (C
O), 144.8 (C6), 144.2, 135.6, 135.3, 130.2, 130.1, 128.2, 128.1, 127.2
(aromatic), 116.8 (CN), 113.4 (aromatic), 95.6 (C5), 92.9 (C1′), 87.1
(DMTr-C), 84.9, 76.5, 70.7 (C2′, C3′, C4′), 62.3 (C5′), 60.3
(−OCH2−), 55.3 (OCH3), 18.1 (−CH2CN); HRMS (ESI-TOF) m/z
[M + H]+ calcd for C34H34N4O9Na 665.2218, found 665.2233.
N2-[(2-Cyanoethoxy)carbonyl]-O6-[2-(4-nitrophenyl)ethyl]-5′-O-
(4,4′-dimethoxytrityl)guanosine (25). Compound 25 (4.27 g, 91%
yield) was obtained from 2.97 g of 9 after following the same reaction
N4-[(2-Cyanoethyloxy)carbonyl]-2′-O-(tert-butyldimethylsilyl)-5′-
O-(4,4′-dimethoxytrityl)cytidine (27a) and N4-[(2-Cyanoethyloxy)-
carbonyl]-3′-O-(tert-butyldimethylsilyl)-5′-O-(4,4′-dimethoxytrityl)-
cytidine (27b). Compounds 27a (8.12 g, 46%) and 27b (4.26 g, 24%)
were obtained as separated isomers from 15 g of 24 after following the
1
same reaction and workup procedures as for 26a/b. 27a: H NMR
(400 MHz, CDCl3) δ 9.10 (s, 1H, NH), 8.52 (s, 1H, H6), 7.49−7.21
(m, 9H, aromatic), 6.86 (m, 5H, aromatic and H5), 5.89 (s, 1H, H1′),
4.45−4.25 (m, 4H, H2′, H3′ and −OCH2−), 4.10 (d, J = 7.9 Hz, 1H,
H−C4′), 3.80 (s, 6H, OCH3), 3.65−3.50 (m, 2H, H5′), 2.83−2.67
(m, 2H, −CH2CN), 2.43 (d, J = 8.1 Hz, 1H, 3′-OH), 0.93 (s, 9H,
SiC(CH3)3), 0.31 (s, 3H, Si(CH3)2), 0.19 (s, 3H, Si(CH3)2); 13C
NMR (CDCl3, 100 MHz) δ 162.6 (C4), 158.8 (aromatic), 154.7 (C2),
151.9 (CO), 145.1 (C6), 144.2, 135.6, 135.3, 130.2, 130.2, 128.3,
128.1, 127.2 (aromatic), 116.6 (CN), 113.4 (aromaic), 94.8 (C5), 90.8
(C1′), 87.2 (aromatic), 83.1 (C4′), 76.6, 69.1 (C2′ and C3′), 61.4
(C5′), 60.1 (−OCH2−), 55.3 (OCH3), 25.9 (SiC(CH3)3), 18.1
(SiC(CH3)3 and -CH2CN), −4.2 (Si(CH3)2), −5.3 (Si(CH3)2);
HRMS (ESI-TOF) m/z [M + H]+ calcd for C40H49N4O9Si
757.3269, found 757.3281.
1
and workup procedure as for 23: H NMR (400 MHz, CDCl3) δ
8.15−8.12 (m, 3H, H8 and aromatic), 7.81 (s, 1H, NH), 7.47 (d, J =
8.7 Hz, 2H, aromatic), 7.18−7.08 (m, 9H, aromatic), 6.99 (br s, 1H,
2′-OH), 6.69 (2 × d, J = 8.8 Hz, 4H, aromatic), 5.90 (d, J = 6.2 Hz,
1H, H1′), 4.93 (t, J = 5.7 Hz, 1H, H2′), 4.79 (t, J = 6.7 Hz, 2H,
−OCH2−), 4.49−4.38 (m, 4H, H3′, H4′, −COOCH2−), 3.74 (s, 6H,
OCH3), 3.38 (ABX, JAB = 10.6, JAX = 3.2 Hz, 1H, H5′), 3.29 (t, J = 6.8
Hz, 2H, −CH2ph), 3.17 (ABX, JBA = 10.6, JBX = 3.2 Hz, 1H, H5″), 2.77
1
(t, J = 6.1 Hz, 2H, −CH2CN); H NMR (100 MHz, CDCl3) δ 161.0
(C6), 158.6 (aromatic), 151.8 (C4), 151.1 (C2), 150.7 (CO), 147.0,
145.6, 144.3 (aromatic), 140.3 (C8), 135.5, 135.3, 130.0, 128.0, 127.8,
126.9, 123.9 (aromatic), 118.6 (C5), 116.7 (CN), 113.2 (aromatic),
92.1 (C1′), 87.1 (C4′), 86.7 (DMTr-C), 76.7 (C2′) 74.0 (C3′), 67.2
(−OCH2−), 63.9 (C5′), 60.1 (−COOCH2−), 55.3 (OCH3), 35.1
(−CH2ph), 18.4 (−CH2CN); HRMS (ESI-TOF) m/z [M + H]+ calcd
for C43H42N7O11 832.2942, found 832.2941.
N2-[(2-Cyanoethoxy)carbonyl]-O6-[2-(4-nitrophenyl)ethyl]-2′-O-
(tert-butyldimethylsilyl)-5′-O-(4,4′-dimethoxytrityl)guanosine (28a)
and N2-[(2-Cyanoethoxy)carbonyl]-O6-[2-(4-nitrophenyl)ethyl]-3′-
O-(tert-butyldimethylsilyl)-5′-O-(4,4′-dimethoxytrityl)guanosine
(28b). The separated regioisomers 28a (1.64 g, 44%) and 28b (1.29 g,
35%) were obtained as colorless foams from 3.2 g of 25 after following
the same reaction and workup procedures as for 26a/b. 28a: 1H NMR
(400 MHz, CDCl3) δ 8.16 (d, J = 8.6 Hz, 2H, aromatic), 7.99 (s, 1H,
H8), 7.53 (d, J = 8.6 Hz, 2H, aromatic), 7.44 (d, J = 7.0 Hz, 2H,
aromatic), 7.33 (2 × d, J = 8.9 Hz, 4H, aromatic), 7.25−7.17 (m, 3H,
aromatic), 6.78 (2 × d, J = 8.7 Hz, 4H, aromatic), 5.93 (d, J = 5.7 Hz,
1H, H1′), 5.03 (t, J = 5.4 Hz, 1H, H2′), 4.84 (t, J = 6.6 Hz, 2H,
−OCH2−), 4.42 (q, J = 3.5 Hz, 1H, H3′), 4.30 (t, J = 6.2 Hz, 2H,
−COOCH2−), 4.23 (q, J = 3.1 Hz, 1H, H4′), 3.77 (m, 6H, OCH3),
3.50 (ABX, JAB = 10.6, JAX = 2.6 Hz, 1H, H5′), 3.40 - 3.32 (m, 3H, H5″
and −CH2ph), 2.72 (d, J = 3.7 Hz, 1H, 3′-OH), 2.66 (t, J = 6.2 Hz,
2H, −CH2CN), 0.84 (s, 9H, SiC(CH3)3), 0.00 (s, 3H, Si(CH3)2),
−0.18 (s, 3H, Si(CH3)2); 13C NMR (100 MHz, CDCl3) δ 160.9 (C6),
158.7 (aromatic), 153.0 (C4), 151.5 (C2), 150.4 (CO), 147.0,
145.8, 144.8 (aromatic), 141.0 (C8), 135.9, 135.8, 130.2, 128.2, 128.0,
127.1, 123.9 (aromatic), 118.8 (C5), 116.8 (CN), 113.3 (aromatic),
88.5 (C1′), 86.7 (DMTr-C), 84.5 (C4′), 75.4 (C2′), 71.6 (C3′), 67.1
(−OCH2−), 63.8 (C5′), 59.6 (−COOCH2−), 55.4 (2 × OCH3), 35.2
(−CH2ph), 25.7 (SiC(CH3)3), 18.3 (−CH2CN), 18.0 (SiC(CH3)3),
−4.9, −5.0 (Si(CH3)2); HRMS (ESI-TOF) m/z [M + H]+ calcd for
C49H56N7O11Si 946.3807, found 946.3785. 28b: 1H NMR (400 MHz,
CDCl3) δ 8.15 (d, J = 8.7 Hz, 2H, aromatic), 8.05 (s, 1H, H8), 7.50 (d,
J = 8.7 Hz, 2H, aromatic), 7.46 (s, 1H, NH), 7.34−7.32 (m, 2H,
aromatic), 7.24−7.14 (m, 7H, aromatic), 6.74 (d, J = 8.6 Hz, 4H,
aromatic), 5.93 (d, J = 5.2 Hz, 1H, H1′), 4.80 (t, J = 6.8 Hz, 2H,
−OCH2−), 4.69 (q, J = 5.3 Hz, 1H, H2′), 4.56 (dd, J = 5.2, 3.3 Hz,
1H, H3′), 4.42−4.33 (m, 2H, −COOCH2−), 4.23−4.15 (m, 2H, H4′,
2′-OH), 3.76 (s, 6H, OCH3), 3.43 - 3.21 (m, 4H, H5′ and −CH2ph),
2.75 (t, J = 6.2 Hz, 2H, −CH2CN), 0.89 (s, 9H, SiC(CH3)3), 0.11 (s,
3H, Si(CH3)2), 0.04 (s, 3H, Si(CH3)2); 13C NMR (101 MHz, CDCl3)
δ 160.8 (C6), 158.7 (aromatic), 152.5 (C4), 151.1 (C2), 150.5 (C
N6-[(2-Cyanoethoxy)carbonyl]-2′-O-(tert-butyldimethylsilyl)-5′-
O-(4,4′-dimethoxytrityl)adenosine (26a) and N6-[(2-Cyanoethoxy)-
carbonyl]-3′-O-(tert-butyldimethylsilyl)-5′-O-(4,4′-dimethoxytrityl)-
adenosine (26b). Compound 23 (4.00 g, 6.00 mmol) was dissolved in
anhydrous THF (50 mL). Anhydrous pyridine (1.80 mL, 22.2 mmol)
and AgNO3 (1.22 g, 7.20 mmol) were added. The mixture was
warmed until the AgNO3 was fully dissolved. While the mixture was
still warm, TBDMS-Cl (1.18 g, 7.80 mmol) was added, resulting in a
colorless precipitate. The mixture was stirred in the dark for 5 h. The
solid was removed by filtration and the supernatant immediately
filtered into saturated aq NaHCO3 (50 mL). The aqueous phase was
extracted with EtOAc (3 × 50 mL). The combined organic phases
were dried over MgSO4. Finally, the solvent was removed under
vacuum. The crude residue was purified, and the regioisomers were
separated by flash column chromatography (3:1 → 2:1 → 1:1, Et2O/
EtOAc) to give 26a (2.50 g, 53%) and 26b (0.91 g, 20%) both as
1
colorless foams. 26a: H NMR (400 MHz, DMSO) δ 10.80 (s, 1H,
NH), 8.58, 8.57 (2 × s, 2H, H2 and H8), 7.43−7.35 (m, 2H,
aromatic), 7.31−7.16 (m, 7H, aromatic), 6.85 (dd, J = 9.0, 3.1 Hz, 4H,
aromatic), 6.05 (d, J = 4.8 Hz, 1H, H-1′), 5.18 (d, J = 5.9 Hz, 1H, 3′-
OH), 4.86 (t, J = 4.9 Hz, 1H, H2′), 4.29 (m, 3H, −OCH2− and H3′),
4.13 (q, J = 4.5 Hz, 1H, H4′), 3.73 (s, 6H, OCH3), 3.29 (m, 2H, H5′),
2.93 (t, J = 6.0 Hz, 2H, −CH2CN), 0.75 (s, 9H, SiC(CH3)3), −0.04 (s,
3H, Si(CH3)2), −0.14 (s, 3H, Si(CH3)2); 13C NMR (DMSO, 101
MHz) δ 158.0 (aromatic), 151.6, 151.6 (C8, CO), 149.6 (C4, C6),
144.8 (aromatic), 142.9 (C2), 135.5, 135.4, 129.7, 127.8, 127.6, 126.7
(aromatic), 124.0 (C5), 118.5 (CN), 113.1 (aromatic), 88.2 (C1′),
85.5 (DMTr-C), 83.5 (C4′), 74.8 (C2′), 70.1 (C3′), 63.4 (C5′), 59.9
(−OCH2−), 55.0, 54.9 (OCH3), 25.5 (SiC(CH3)3), 17.8, 17.6
(−CH2CN, SiC(CH3)3), −4.8 (Si(CH3)2), −5.3 (Si(CH3)2); HRMS
(ESI-TOF) m/z [M + H]+ calcd for C41H49N6O8Si 781.3381, found
1
781.3354. 26b: H NMR (400 MHz, DMSO) δ 10.81 (s, 1H, NH),
8.63 (s, 1H, H2), 8.56 (s, 1H, H8), 7.39−7.17 (m, 9H, aromatic),
3321
dx.doi.org/10.1021/jo5002824 | J. Org. Chem. 2014, 79, 3311−3326

The Journal of Organic Chemistry
Featured Article
O), 147.0, 145.8, 144.5 (aromatic), 140.9 (C8), 135.8, 135.7, 130.1,
128.2, 127.9, 127.0, 123.9 (aromatic), 118.8 (C5), 116.8 (CN), 113.2
(aromatic), 90.3 (C1′), 86.7 (DMTr-C), 85.9 (C4′), 75.2 (C2′), 73.1
(C3′), 67.1 (−OCH2−), 63.5 (C5′), 59.8 (−COOCH2−), 55.3
(OCH3), 35.2 (−CH2Ar), 25.9 (SiC(CH3)3), 18.37 (−CH2CN), 18.27
(SiC(CH3)3), −4.56, −4.71 (Si(CH3)2); HRMS (ESI-TOF) m/z [M +
H]+ calcd for C49H56N7O11Si 946.3807, found 946.3787.
74.2, 74.0 (C2′), 72.2, 72.1 (C3′), 63.0 (C5′), 60.3 (−OCH2−), 58.8,
58.6, 58.1, 57.9 (−OCH2−), 55.4 (OCH3), 43.5, 43.4 (iPr CH), 24.8,
24.7, 24.6, 24.5, 24.4, 24.3 (iPr CH3), 21.1, 21.0 (acetyl CH3), 20.3,
20.2, 20.1, 20.0 (CH2CN), 18.3 (CH2CN); 31P NMR (162 MHz,
CDCl3) δ 151.56 (s), 151.18 (s); HRMS (ESI-TOF) m/z [M + H]+
calcd for C46H54N8O10P 909.3701, found 909.3699.
N4-[(2-Cyanoethyloxy)carbonyl]-2′-O-acetyl-5′-O-(4,4′-dimeth-
oxytrityl)cytidine-3′-O-(2-cyanoethyl-N,N-diisopropyl)phosphor-
General Procedure for the Synthesis of 2′/3′-O-Acetylated
RNA Phosphoramidites. To a solution of substrate (1 equiv) and 2-
cyanoethyl N,N,N′,N′-tetraisopropylphosphoramidite 18 (2 equiv) in
dry THF was added a solution of 5-benzylthio-1H-tetrazole (BTT) in
anhydrous MeCN (0.35 M, 1 equiv) dropwise. The reaction mixture
was stirred at rt for 3 h, after which saturated aq NaHCO3 was added
to quench the reaction. The organics were extracted with EtOAc (3 ×
10 mL), combined, and dried over MgSO4. The solvent was removed,
and the residue was applied to a short flash chromatography column.
The regioisomeric mixture thus obtained was dissolved in DCM
(∼200 mg/mL), purified, and separated by normal-phase HPLC.
General Procedure for the Synthesis of 2′/3′-O-TBS RNA
Phosphoramidites. To a solution of substrate (1 equiv) in
anhydrous THF was added N,N-diisopropylethylamine (3.5 equiv)
and 2-cyanoethyl N,N-diisopropyl phosphoamidochloridite 17 (1.4
equiv) at 0 °C. The mixture was warmed to rt and stirred for 5 h.
Anhydrous methanol was added to quench the reaction, and the
mixture was stirred for a further 30 min. The reaction was diluted with
EtOAc and washed with saturated aq NaHCO3 (3 × 20 mL). The
combined organic layers were dried over MgSO4, and the solvent was
evaporated under vacuum. The crude product was purified by flash
column chromatography to give a mixture of two diastereoisomers.
NMR and MS Data for Phosphoramidites. N6-[(2-Cyanoeth-
oxy)carbonyl]-2′-O-acetyl-5′-O-(4,4′-dimethoxyltrityl)adenosine-3′-
1
amidite (20a): H NMR (400 MHz, CDCl3) δ 8.28 (s, 1H, H6),
7.51−7.21 (m, 9H, aromatic), 6.96−6.72 (m, 5H, aromatic and H5),
6.16 (2 × d, J = 2.9 Hz, 1H, H1′), 5.56 (dd, J = 4.9, 3.5 Hz, 0.44H,
H2′), 5.52−5.45 (m, 0.56H, H2′), 4.72−4.58 (m, 1H, H3′), 4.37 (t, J
= 6.4 Hz, 2H, −OCH2−), 4.32−4.22 (m, 1H, H4′), 3.92−3.35 (m,
i
12H, −OCH2−, OCH3, H5′ and Pr CH), 2.77 (m, 2H, −CH2CN),
2.64 (q, J = 6.4 Hz, 0.86H, −CH2CN), 2.36 (q, J = 6.0 Hz, 1.14H,
−CH2CN), 2.14, 2.11 (2 × s, 3H, acetyl CH3), 1.34−1.00 (m, 12H, iPr
CH3); 13C NMR (CDCl3, 101 MHz) δ 169.2, 169.1 (acetyl CO),
162.9 (C4), 158.8 (aromatic), 154.8 (C2), 152.1 (CO), 144.7 (C6),
144.1, 144.0, 135.5, 135.3, 135.1, 130.3, 128.5, 128.4, 128.1, 127.3
(aromatic), 117.9, 117.5, 117.1, 116.9, 116.8 (CN), 113.4 (aromatic),
95.4 (C5), 88.8 (C1′), 87.2 (DMTr-C), 83.2, 82.9 (C4′), 75.3, 74.8
(C2′), 69.8 (C3′), 61.4 (C5′), 60.1 (−OCH2−), 58.5, 58.3, 58.2, 58.0
(−OCH2−), 55.3 (OCH3), 45.7, 45.6, 45.4, 43.4, 43.3, 43.2 (iPr CH),
24.7, 24.6, 24.5, 23.2, 23.1, 23.1, 23.0 (iPr CH3), 21.1, 20.9 (acetyl
CH3), 20.4, 20.3, 20.2, 20.1 (−CH2CN), 18.1 (−CH2CN); 31P NMR
(162 MHz, CDCl3) δ 150.62 (s), 150.07 (s); HRMS (ESI-TOF) m/z
[M + H]+ calcd for C45H54N6O11P 885.3588, found 885.3617.
N4-[(2-Cyanoethyloxy)carbonyl]-3′-O-acetyl-5′-O-(4,4′-dimeth-
oxytrityl)cytidine-2′-O-(2-cyanoethyl-N,N-diisopropyl)phosphor-
amidite (20b): 1H NMR (400 MHz, CDCl3) δ 8.52 (s, 1H, H6), 8.13
(br s, 1H, NH), 7.42−7.21 (m, 9H, aromatic), 6.85 (m, 5H, H5 and
aromatic), 6.14 (d, J = 1.8 Hz, 0.54H, H1′), 6.06 (s, 0.46H, H1′),
5.25−5.06 (m, 1H, H3′), 4.74−4.66 (m, 0.54H, H2′), 4.65−4.56 (m,
0.46H, H2′), 4.42−4.29 (m, 3H, −OCH2−, H4′), 4.09−3.50 (m, 11H,
1
O-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite (19a): H NMR
(400 MHz, CDCl3) δ 8.71, 8.69 (2 × s, 2H, H8, NH), 8.19 (s, 1H,
H2), 7.49−7.12 (m, 9H, aromatic), 6.79 (dd, J = 8.6, 4.8 Hz, 4H,
aromatic), 6.29 (2 × d, J = 5.9 Hz, 1H, H1′), 6.00 (t, J = 5.4 Hz, 0.5H,
H2′), 5.90 (t, J = 5.7 Hz, 0.5H, H2′), 5.01−4.78 (m, 1H, H3′), 4.52−
4.38 (m, 2.5H, −OCH2−, H4′), 4.35 (q, J = 3.6 Hz, 0.5H, H4′), 3.98−
3.46 (m, 11H, −OCH2−, OCH3, iPr CH, H5′), 3.38 (ABX, JBA = 10.7
Hz, JBX = 4.0 Hz, 1H, H5″), 2.79 (t, J = 6.3 Hz, 2H, −CH2CN), 2.64
(t, J = 6.4 Hz, 1H, −CH2CN), 2.36 (m, 1H, −CH2CN), 2.12, 2.08 (2
i
−OCH2−, OCH3, Pr CH and H5′), 3.41 (d, J = 11.4 Hz, 1H, H5″),
2.77 (t, J = 6.5 Hz, 2.50H, −CH2CN), 2.64 (dt, J = 16.6, 5.9 Hz,
1.50H, −CH2CN), 2.06 (m, 3H, acetyl CH3), 1.23−1.11 (m, 12H, iPr
CH3); 13C NMR (CDCl3, 101 MHz) δ 169.9, 169.8 (acetyl CO),
162.2 (C4), 158.8 (aromatic), 154.7 (C2), 151.6 (CO), 144.9 (C6),
144.1, 135.4, 135.3, 130.2, 128.3, 128.2, 127.3 (aromatic), 118.2, 117.8
(−CH2CN), 116.5 (−CH2CN), 113.5 (aromatic), 94.9 (C5), 90.7,
90.3 (C1′), 87.5 (DMTr-C), 80.8, 80.7, 80.5 (C4′), 75.7, 75.5, 74.5,
74.4 (C2′), 69.9, 69.5, 69.4 (C3′), 61.0, 60.7 (C5′), 60.3 (−OCH2−),
59.0, 58.8, 58.6 (−OCH2−), 55.3 (OCH3), 43.6, 43.5 (iPr-CH), 24.8,
24.7, 24.6 (iPr-CH3), 20.9, 20.4, 20.3 (acetyl CH3), 18.2 (−CH2CN);
31P NMR (162 MHz, CDCl3) δ 152.32 (s), 150.29 (s); HRMS (ESI-
i
× s, 3H, acetyl CH3), 1.23−1.00 (m, 12H, Pr CH3); 13C NMR
(CDCl3, 101 MHz) δ 169.9 (acetyl CO), 158.7 (aromatic), 153.0
(C8), 151.6 (C4), 150.3 (CO), 149.1 (C6), 144.4, 144.3 (aromatic),
141.9 (C2), 135.6, 135.5, 135.4, 130.3, 130.2, 128.4, 128.2, 128.0,
127.2, 127.1 (aromatic), 122.6 (C5), 117.7, 117.4 (CN), 116.8 (CN),
113.3 (aromatic), 86.9 (DMTr-C), 86.1 (C1′), 84.7, 84.4 (C4′), 74.8,
74.7 (C2′), 71.5, 71.3, 71.0, 70.8 (C3′), 63.0 (C5′), 60.3 (−OCH2−),
59.0, 58.8, 58.3, 58.1 (−OCH2−), 55.4 (OCH3), 43.5, 43.4, 43.3 (iPr
CH), 24.9, 24.8, 24.7, 24.6 (iPr CH3), 21.0, 20.9 (acetyl CH3), 20.3,
20.2 (−CH2CN), 18.3 (−CH2CN); 31P NMR (162 MHz, CDCl3) δ
151.17 (s), 150.22 (s); HRMS (ESI-TOF) m/z [M + H]+ calcd for
C46H54N8O10P 909.3701, found 909.3665.
TOF) m/z [M + H]+ calcd for C45H53N6O11NaP 907.3402, found
907.3374.
N2-[(2-Cyanoethoxy)carbonyl]-O6-[2-(4-nitrophenyl)ethyl]-2′-O-
acetyl-5′-O-(4,4′-dimethoxytrityl)guanosine-3′-O-(2-cyanoethyl-
N,N-diisopropyl)phosphoramidite (21a): 1H NMR (400 MHz,
CDCl3) δ 8.16 (d, J = 8.6 Hz, 2H, aromatic), 7.96, 7.95 (2 × s, 1H,
H8), 7.52 (2 × d, J = 8.6 Hz, 2H, aromatic), 7.47−7.15 (m, 9H,
aromatic), 6.77 (d, J = 8.8 Hz, 4H, aromatic), 6.16 (d, J = 6.5 Hz,
0.40H, H1′), 6.09 (d, J = 6.2 Hz, 0.60H, H1′), 6.04 (t, J = 5.7 Hz,
0.45H, H2′), 5.95 (t, J = 5.9 Hz, 0.55H, H2′), 4.90−4.78 (m, 3H, H3′
and −OCH2−), 4.41−4.26 (m, 3H, H4′, −COOCH2−), 3.97−3.25
N6-[(2-cyanoethoxy)carbonyl]-3′-O-acetyl-5′-O-(4,4′-dimethoxy-
ltrityl)adenosine-2′-O-(2-cyanoethyl-N,N-diisopropyl)phosphor-
amidite (19b): 1H NMR (400 MHz, CDCl3) δ 8.74, 8.71, 8.69 (3 × s,
2H, NH, H8), 8.26, 8.23 (2 × s, 1H, H2), 7.47−7.13 (m, 9H,
aromatic), 6.88−6.73 (m, 4H, aromatic), 6.26 (d, J = 5.3 Hz, 0.5H,
H1′), 6.22 (d, J = 5.7 Hz, 0.5H, H1′), 5.57, 5.52 (t, J = 4.4 Hz, 1H,
H3′), 5.21, 5.15 (2 × dt, J = 10.4, 5.4 Hz, 1H, H2′), 4.46 (t, J = 6.2 Hz,
2H, −OCH2−), 4.33 (q, J = 3.7 Hz, 1H, H4′), 3.88−3.64 (m, 7H,
i
(m, 14H, −OCH2−, OCH3, Pr CH, H5′ and −CH2ph), 2.76−2.62
(m, 3H, −CH2CN), 2.31 (t, J = 6.4 Hz, 1H, −CH2CN), 2.09 (m, 3H,
i
acetyl CH3), 1.21−1.02 (m, 12H, Pr CH3); 13C NMR (CDCl3, 101
i
OCH3, −OCH2−), 3.63−3.36 (m, 5H, H5′, −OCH2−, Pr CH), 2.80
MHz) δ 169.8 (acetyl CO), 160.8 (C6), 158.7 (aromartic) 153.1,
152.9 (C4), 151.6 (C2), 150.5, 150.4 (CO), 147.0, 145.9, 144.5,
144.3 (aromatic), 141.0, 140.7 (C8), 135.8, 135.7, 135.6, 135.6, 130.3,
130.2, 128.5, 128.3, 128.0, 127.1, 123.9 (aromatic), 118.8, 118.7 (C5),
117.7, 117.4, 116.9 (CN), 113.3, 113.2 (aromatic), 86.8, 86.7 (DMTr-
C), 86.3, 85.8 (C1′), 84.8, 84.5 (C4′), 74.3, 74.1, 74.0 (C2′), 71.4,
71.3, 70.9, 70.7 (C3′), 67.1 (−OCH2−), 63.4, 63.3 (C5′), 59.6, 59.5
(−COOCH2−), 59.0, 58.8, 58.1, 57.8 (−OCH2−), 55.4 (OCH3), 43.5,
43.4, 43.2 (iPr CH), 35.2 (−CH2ph), 24.8, 24.7, 24.6 (iPr CH3), 21.1,
20.9 (acetyl CH3), 20.5, 20.4, 20.2, 20.1, 18.3 (−CH2CN); 31P NMR
(t, J = 6.2 Hz, 2H, −CH2CN), 2.55 (td, J = 6.4, 2.9 Hz, 1H,
−CH2CN), 2.33 (t, J = 6.4 Hz, 1H, −CH2CN), 2.14, 2.10 (2 × s, 3H,
acetyl CH3), 1.20−1.00 (m, 9H, iPr CH3), 0.88 (d, J = 6.7 Hz, 3H, iPr
CH3); 13C NMR (CDCl3, 101 MHz) δ 169.9, 169.8 (acetyl CO),
158.7 (aromatic), 153.0, 152.8 (C8), 151.7, 151.6 (C4), 150.4, 150.3
(CO), 149.1, 149.0 (C6), 144.5 (aromatic), 142.0, 141.8 (C2),
135.6, 135.5, 130.2, 128.3, 128.2, 128.1, 128.0, 127.2 (aromatic), 122.6
(C5), 117.5, 117.4 (CN), 116.8 (CN), 113.4, 113.3 (aromatic), 87.7,
87.6, 87.5 (C1′), 87.0, 87.0 (DMTr-C), 82.5, 82.1 (C4′), 74.7, 74.5,
3322
dx.doi.org/10.1021/jo5002824 | J. Org. Chem. 2014, 79, 3311−3326

The Journal of Organic Chemistry
Featured Article
(162 MHz, CDCl3) δ 150.90 (s), 150.28 (s); HRMS (ESI-TOF) m/z
[M + H]+ calcd for C54H61N9O13P 1074.4126, found 1074.4131.
N2-[(2-Cyanoethoxy)carbonyl]-O6-[2-(4-nitrophenyl)ethyl]-3′-O-
acetyl-5′-O-(4,4′-dimethoxytrityl)guanosine-2′-O-(2-cyanoethyl-
N,N-diisopropyl)phosphoramidite (21b): 1H NMR (400 MHz,
CDCl3) δ 8.17 (2 × d, J = 8.7 Hz, 2H, aromatic), 8.02 (s, 0.5H,
H8), 7.97 (s, 0.5H, H8), 7.53 (d, J = 8.7 Hz, 2H, aromatic), 7.40−7.16
(m, 9H, aromatic), 6.79−6.74 (m, 4H, aromatic), 6.10 (d, J = 5.5 Hz,
0.5H, H1′), 6.02 (d, J = 5.9 Hz, 0.5H, H1′), 5.63−5.60 (m, 1H, H3′),
5.37 (dt, J = 10.9, 5.6 Hz, 0.5H, H2′), 5.19 (dt, J = 10.6, 5.5 Hz, 0.5H,
H2′), 4.87−4.82 (m, 2H, −OCH2−), 4.34 (t, J = 6.2 Hz, 2H,
−COOCH2−), 4.29−4.25 (m, 1H, H4′), 3.84−3.66 (m, 7H, OCH3,
and −OCH2−), 3.58−3.41 (m, 5H, H5′, −OCH2−, and iPr CH), 3.34
(t, J = 6.9 Hz, 2H, −CH2CN), 2.75−2.70 (m, 2H, −CH2CN), 2.58 (t,
J = 6.3 Hz, 1H, −CH2CN), 2.38−2.25 (m, 1H, −CH2CN), 2.14−2.14,
2.11 (2 × s, 3H, acetyl CH3), 1.14−1.08 (m, 9H, iPr CH3), 0.90 (d, J =
N6-[(2-Cyanoethoxy)carbonyl]-2′-O-(tert-butyldimethylsilyl)-5′-
O-(4,4′-dimethoxytrityl)adenosine-3′-O-(2-cyanoethyl-N,N-diiso-
propyl)phosphoramidite (29a): 1H NMR (400 MHz, CDCl3) δ 8.67,
8.65 (s, 1H, H8), 8.49 (s, 1H, NH), 8.24, 8.21 (s, 1H, H2), 7.51−7.19
(m, 9H, aromatic), 6.92−6.73 (m, 4H, aromatic), 6.08 (d, J = 6.3 Hz,
0.55H, H1′), 6.03 (d, J = 6.1 Hz, 0.45H, H1′), 5.12−4.98 (m, 1H,
H2′), 4.52−4.32 (m, 4H, −OCH2−, H3′ and H4′), 4.03−3.52 (m,
i
11H, −OCH2−, OCH3, Pr CH and H5′), 3.41−3.27 (m, 1H, H5″),
2.81 (m, 2H, −CH2CN), 2.73−2.57 (m, 1H, −CH2CN), 2.44−2.22
(m, 1H, −CH2CN), 1.23−1.11 (m, 9H, iPr CH3), 1.05 (d, J = 6.8 Hz,
i
3H, Pr CH3), 0.75 (s, 9H, SiC(CH3)3), −0.03, −0.06 (2 × s, 3H,
Si(CH3)2), −0.22, −0.23 (2 × s, 3H, Si(CH3)2); 13C NMR (CDCl3,
101 MHz) δ 158.7 (aromatic), 152.9 (C8), 151.5 (C4), 150.2 (C
O), 148.9 (C6), 144.7, 144.6 (aromatic), 142.3 (C2), 135.8, 135.8,
135.6, 135.6, 130.3, 130.3, 130.2, 128.4, 128.3, 128.1, 128.0, 127.1
(aromatic), 122.7, 122.6 (C5), 117.7, 117.4 (CN), 116.8 (CN), 113.3,
113.3 (aromatic), 88.6, 88.4 (C1′), 86.9, 86.8 (aromatic), 84.4, 84.1,
84.1 (C4′), 75.4, 74.8, 74.8 (C2′), 73.5, 73.4, 72.9, 72.7 (C3′), 63.4,
63.2 (C5′), 60.2 (−OCH2−), 59.0, 58.8, 57.8, 57.6 (−OCH2−), 55.4
(OCH3), 43.6, 43.5, 43.2, 43.0 (iPr CH), 25.7 (SiC(CH3)3), 24.9, 24.8,
24.7 (iPr CH3), 20.6, 20.3, 20.2, 18.3 (−CH2CN), 18.1, 18.0
(SiC(CH3)3), −4.5, −4.6, −5.0 (Si(CH3)2); 31P NMR (162 MHz,
CDCl3) δ 151.04 (s), 149.06 (s); HRMS (ESI-TOF) m/z [M + H]+
calcd for C50H66N8O9SiP 981.4460, found 981.4433
i
6.8 Hz, 3H, Pr CH3); 13C NMR (101 MHz, CDCl3) δ 169.9 (acetyl
CO), 160.8 (C6), 158.7 (aromatic), 153.1, 152.9 (C4), 151.5 (C2),
150.4, 150.3 (CO), 147.0, 145.8, 144.7, 144.6 (aromatic), 141.0,
140.9 (C8), 135.8, 135.7, 135.6, 130.2, 130.1, 128.2, 128.0, 127.9,
127.1, 127.0, 123.8 (aromatic), 118.8, 118.6 (C5), 117.6, 117.3 (CN),
116.9 (CN), 113.3, 113.2 (aromatic), 87.9, 87.8 (C1′), 86.8, 86.7
(DMTr-C), 82.6, 82.1 (C4′), 74.4, 74.2, 73.4, 73.2 (C2′), 72.4, 72.3
(C3′), 67.1 (−OCH2−), 63.4, 63.3 (C5′), 59.6 (−COOCH2−), 58.6,
58.4, 58.1, 58.0 (−OCH2−), 55.3 (OCH3), 43.5, 43.4, 43.3 (iPr CH),
35.2 (−CH2ph), 24.8, 24.7, 24.6, 24.4 (iPr CH3), 21.1, 21.0 (acetyl
CH3), 20.3, 20.0, 19.9, 18.3 (−CH2CN); 31P NMR (162 MHz,
CDCl3) δ 151.62, 151.01; HRMS (ESI-TOF) m/z [M + H]+ calcd for
C54H61N9O13P 1074.4126, found 1074.4169.
N6-[(2-Cyanoethoxy)carbonyl]-3′-O-(tert-butyldimethylsilyl)-5′-
O-(4,4′-dimethoxytrityl)adenosine-2′-O-(2-cyanoethyl-N,N-diiso-
propyl)phosphoramidite (29b): 1H NMR (400 MHz, CDCl3) δ 8.70,
8.68 (s, 1H, H8), 8.56 (s, 1H, NH), 8.30, 8.27 (s, 1H, H-2), 7.44−7.14
(m, 9H, aromatic), 6.79 (m, 4H, aromatic), 6.27 (d, J = 4.3 Hz, 0.62H,
H1′), 6.18 (d, J = 4.7 Hz, 0.38H, H1′), 5.01 (dt, J = 11.3, 4.6 Hz,
0.38H, H2′), 4.82 (dt, J = 9.5, 4.5 Hz, 0.62H, H2′), 4.57−4.49 (m, 1H,
H3′), 4.45 (t, J = 6.2 Hz, 2H, −OCH2−), 4.29−4.13 (m, 1H, H4′),
2′-O-Acetyl-5′-O-(4,4′-dimethoxytrityl)uridine-3′-O-(2-cyanoeth-
yl-N,N-diisopropyl)phosphoramidite (22a): 1H NMR (400 MHz,
CDCl3) δ 8.28 (s, 1H, NH), 7.81−7.69 (m, 1H, H6), 7.45−7.20 (m,
9H, aromatic), 6.90−6.78 (m, 4H, aromatic), 6.24−6.12 (m, 1H, H−
C1′), 5.53 (t, J = 5.3 Hz, 0.4H, H2′), 5.38 (t, J = 5.7 Hz, 0.6H, H2′),
5.36−5.27 (m, 1H, H5), 4.76−4.61 (m, 1H, H3′), 4.30 (d, J = 2.6 Hz,
0.5H, H4′), 4.20 (d, J = 3.4 Hz, 0.5H, H4′), 3.98−3.85 (m, 0.5H,
−OCH2−), 3.84−3.74 (m, 6H, OCH3), 3.73−3.39 (m, 5.5H,
i
3.87−3.42 (m, 11H, −OCH2−, OCH3, Pr CH and H5′), 3.36−3.20
(m, 1H, H5″), 2.83−2.76 (m, 2H, −CH2CN), 2.50 (t, J = 6.4 Hz, 1H,
i
−CH2CN), 2.38 (t, J = 6.3, 1H, −CH2CN), 1.15−1.02 (m, 9H, Pr
CH3), 0.95−0.79 (m, 12H, iPr CH3 and SiC(CH3)3), 0.09, 0.06 (2 × s,
3H, Si(CH3)2), 0.01, −0.00 (2 × s, 3H, Si(CH3)2); 13C NMR (CDCl3,
101 MHz) δ 158.7, 158.7 (aromatic), 152.7, 152.6 (C8), 151.4, 151.4
(C4), 150.3, 150.3 (CO), 149.0, 149.0 (C6), 144.6 (aromatic),
142.6, 142.4 (C2), 135.7, 135.7, 130.2, 130.2, 130.2, 128.3, 128.3,
128.0, 128.0, 127.1, 127.1 (aromatic), 122.7 (C5), 117.6 (CN), 116.8
(CN), 113.3, 113.3 (aromatic), 88.1, 88.0, 87.9 (C1′), 86.9, 86.7
(DMTr-C), 84.8, 84.4 (C4′), 76.0, 75.9, 75.6, 75.4 (C2′), 71.9, 71.6,
71.5 (C3′), 63.1, 62.8 (C5′), 60.3, 60.2 (−OCH2−), 58.6, 58.4, 57.9,
57.7 (−OCH2−), 55.4 (OCH3), 43.5, 43.3, 43.2 (iPr CH), 25.9
(SiC(CH3)3), 24.8, 24.7, 24.4 (iPr CH3), 20.4, 20.3, 20.2, 20.1, 18.3
(−CH2CN), 18.2 (SiC(CH3)3), −4.2, −4.3, −4.7, −4.8 (Si(CH3)2);
31P NMR (162 MHz, CDCl3) δ 150.44 (s), 150.15 (s); HRMS (ESI-
i
−OCH2−, H5′ and Pr CH), 2.66 (td, J = 6.2, 1.9 Hz, 0.8H,
−CH2CN), 2.46−2.31 (m, 1.2H, CH2CN), 2.20−2.07 (m, 3H, acetyl
CH3), 1.32−1.01 (m, 12H, iPr CH3); 13C NMR (101 MHz, CDCl3) δ
169.9, 169.7 (acetyl CO), 162.7 (C4), 158.9 (aromatic), 150.3 (C2),
144.2, 144.1 (aromatic), 140.1 (C6), 135.3, 135.2, 135.1, 135.0, 134.4,
130.4, 130.3, 128.5, 128.4, 128.2, 127.4 (aromatic), 117.8, 117.4 (CN),
113.5, 113.4 (aromatic), 102.9 (C5), 87.5, 87.4 (DMTr-C), 86.5, 86.3
(C1′), 84.2, 83.8, 83.7 (C4′), 74.8, 74.5 (C2′), 71.5, 71.3, 70.7, 70.5
(C3′), 62.7, 62.5 (C5′), 58.9, 58.7, 58.3, 58.1, 57.9 (−OCH2−), 55.4
(OCH3), 43.5, 43.4, 43.3 (iPr CH), 24.8, 24.7 (iPr CH3), 21.1, 20.9
(acetyl CH3), 20.5, 20.3 (−CH2CN); 31P NMR (162 MHz, CDCl3) δ
151.04 (s), 150.25 (s); HRMS (ESI-TOF) m/z [M + H]+ calcd for
C41H49N4O10PNa 811.3084, found 811.3083.
TOF) m/z [M + H]+ calcd for C50H66N8O9SiP 981.4460, found
981.4424.
N4-[(2-Cyanoethyloxy)carbonyl]-2′-O-(tert-butyldimethylsilyl)-5′-
O-(4,4′-dimethoxytrityl)cytidine-3′-O-(2-cyanoethyl-N,N-diiso-
propyl)phosphoramidite (30a): 1H NMR (400 MHz, CDCl3) δ
8.70−8.11 (m, 2H, H6, NH), 7.50−7.21 (m, 9H, aromatic), 6.92−6.59
(m, 5H, aromatic and H5), 5.92 (d, J = 2.3 Hz, 0.32H, H1′), 5.82 (s,
0.68H, H1′), 4.45−4.21 (m, 5H, H2′, H3′, H4′ and −OCH2−), 3.92−
3.38 (m, 12H, OCH3, H5′, iPr CH and −OCH2−), 2.76 (t, J = 6.5 Hz,
2H, −CH2CN), 2.59 (t, J = 6.3 Hz, 0.68H, −CH2CN), 2.41 (t, J = 6.4
3′-O-Acetyl-5′-O-(4,4′-dimethoxytrityl)uridine-2′-O-(2-cyanoeth-
yl-N,N-diisopropyl)phosphoramidite (22b): 1H NMR (400 MHz,
CDCl3) δ 8.57 (br s, 1H, NH), 7.86 (dd, J = 10.1, 8.1 Hz, 1H, H6),
7.38−7.22 (m, 9H, aromatic), 6.85−6.83 (m, 4H, aromatic), 6.13, 6.09
(2 × d, J = 4.4 Hz, 1H, H1′), 5.39−5.27 (m, 2H, H5 and H3′), 4.63−
4.56 (m, 1H, H2′) 4.23−4.22 (m, 1H, H4′), 3.89−3.55 (m, 11H,
i
−OCH2−, OCH3, Pr CH and H5′), 3.46−3.41 (m, 1H, H5″), 2.67−
i
2.53 (m, 2H, −CH2CN), 2.10 (2 × s, 3H, acetyl CH3), 1.18−1.14 (m,
Hz, 1.32H, −CH2CN), 1.31−0.97 (m, 12H, Pr CH3), 0.94−0.86 (m,
i
12H, Pr CH3); 13C NMR (CDCl3, 101 MHz) δ 170.0, 169.9 (acetyl
9H, SiC(CH3)3), 0.25 (s, 3H, Si(CH3)2), 0.17−0.09 (m, 3H,
Si(CH3)2); 13C NMR (CDCl3, 101 MHz) δ 162.5, 162.3 (C4),
158.8 (aromatic), 154.7 (C2), 151.9 (CO), 145.2 (C6), 144.2,
144.1, 135.5, 135.4, 135.2, 130.4, 130.3, 128.5, 128.0, 127.3 (aromatic),
117.6, 117.5, 116.6 (CN), 113.4, 113.3 (aromatic), 94.7 (C5), 91.5
(C1′), 87.3, 87.2 (DMTr-C), 81.7, 81.5, 75.9, 75.3, 71.5, 69.6 (C2′/
C3′/C4′), 61.6, 61.0 (C5′/−OCH2−), 60.1 (−OCH2−), 58.4, 58.3,
58.2 (C(5′)/−OCH2−), 55.3, 55.3 (OCH3), 45.5, 45.4, 43.4, 43.2,
43.1 (iPr CH), 26.0, 25.9 (SiC(CH3)3), 25.0, 24.9, 24.9, 24.8, 24.7,
24.6, 23.1, 23.0 (iPr CH3), 20.6, 20.5, 20.3, 20.3, 20.2 (−CH2CN),
18.2, 18.1 (−CH2CN/SiC(CH3)3), −4.2, −4.3, −4.9, −5.0 (Si-
CO), 162.9 (C4), 158.9 (aromatic), 150.3, 150.2 (C2), 144.3
(aromatic), 140.3, 140.1 (C6), 135.2, 135.1, 130.3, 130.2, 128.2,
128.2, 127.4 (aromatic), 117.8, 117.6 (CN), 113.5 (aromatic), 102.6
(C5), 88.3, 88.2 (C1′), 87.6, 87.5 (DMTr-C), 81.5 (C4′), 74.9, 74.8,
74.6, 74.5 (C2′), 71.3, 71.1 (C3′), 62.2, 62.0 (C5′), 58.8, 58.6, 58.4
(−OCH2−), 55.4 (OCH3), 43.7, 43.6, 43.5 (iPr CH), 24.9, 24.8, 24.7,
24.6 (iPr CH3), 21.1, 21.0 (acetyl CH3), 20.4, 20.3 (−CH2CN); 31P
NMR (162 MHz, CDCl3) δ 151.80 (s), 151.11 (s); HRMS (ESI-
TOF) m/z [M + H]+ calcd for C41H49N4O10PNa 811.3084, found
811.3072.
3323
dx.doi.org/10.1021/jo5002824 | J. Org. Chem. 2014, 79, 3311−3326

The Journal of Organic Chemistry
Featured Article
(CH3)2); 31P NMR (162 MHz, CDCl3) δ 150.30 (s), 149.10 (s);
HRMS (ESI-TOF) m/z [M + H]+ calcd for C49H66N6O10PSi
957.4347, found 957.4380.
s, 3H, Si(CH3)2); 13C NMR (CDCl3, 101 MHz) δ 160.7 (C6), 158.6
(aromatic), 153.1, 153.0 (C4), 151.4 (C2), 150.5, 150.4 (CO),
147.0, 145.9, 144.7, 144.7 (aromatic), 141.2, 141.1 (C8), 135.9, 135.8,
130.2, 128.3, 128.0, 127.0, 123.8 (aromatic), 118.8, 118.7 (C5), 117.6,
117.6 (CN), 116.9 (CN), 113.3 (aromatic), 87.7, 87.6 (C1′), 86.8,
86.7 (DMTr-C), 85.3, 84.8 (C4′), 75.8, 75.7, 75.4, 75.2 (C2′), 72.2,
71.9 (C3′), 67.1 (−OCH2−), 63.5, 63.3 (C5′), 59.6, 59.6
N4-[(2-Cyanoethyloxy)carbonyl]-3′-O-(tert-butyldimethylsilyl)-5′-
O-(4,4′-dimethoxytrityl)cytidine-2′-O-(2-cyanoethyl-N,N-diiso-
1
propyl)phosphoramidite (30b): H NMR (400 MHz, CDCl3) δ 8.68
(br s, 1H, H6), 7.46−7.17 (m, 9H, aromatic), 6.86−6.65 (m, 5H,
aromatic and H5), 6.17−6.13 (m, 1H, H1′), 4.36−4.24 (m, 4H, H2′,
H3′ and −OCH2−), 4.21−4.09 (m, 1H, H4′), 4.08−3.93 (m, 1H,
−OCH2−), 3.87−3.70 (m, 8H, OCH3, H5′ and −OCH2−), 3.69−
(−COOCH2−), 58.3, 58.1, 58.0, 57.8 (−OCH −), 55.3 (OCH3),
2
43.4, 43.3, 43.1 (iPr CH), 35.2 (−CH2ph), 25.9 (SiC(CH3)3), 24.8,
24.8, 24.7, 24.6, 24.4, 24.3 (iPr CH3), 20.4, 20.4, 20.1, 20.0, 18.4
(−CH2CN), 18.2 (SiC(CH3)3), −4.3, −4.7, −4.8 (Si(CH3)2); 31P
NMR (162 MHz, CDCl3) δ 150.24 (s), 149.97 (s); HRMS (ESI-
TOF) m/z [M + H]+ calcd for C58H72N 9O12NaSiP 1168.4700, found
1168.4669.
i
3.57 (m, 2H, Pr CH), 3.35−3.29 (m, 1H, H5″), 2.83−2.54 (m, 4H,
i
−CH2CN), 1.21−1.01 (m, 12H, Pr CH3), 0.73, 0.72 (2 × s, 9H,
SiC(CH3)3), 0.03, −0.02 (2 × s, 3H, Si(CH3)2), −0.09, −0.10 (2 × s,
3H, Si(CH3)2); 13C NMR (CDCl3, 101 MHz) δ 162.4, 162.3 (C4),
158.7, 158.7 (aromatic), 154.4 (C2), 151.9 (CO), 144.9 (C6),
143.6, 143.6, 135.0, 135.0, 130.3, 130.2, 130.2, 130.2, 128.5, 128.5,
127.9, 127.3, 127.2 (aromatic), 118.3, 117.9, 117.1, 116.6 (CN), 113.2
(aromatic), 94.9 (C5), 90.0 (C1′), 87.1, 87.1 (DMTr-C), 82.3, 82.1
(C4′), 76.0, 75.9 (C2′), 69.0, 68.9 (C3′), 60.6, 60.5 (C5′), 60.0
(−OCH2−), 58.6, 58.4, 58.3, 58.2, 58.2, 57.9 (−OCH2−), 55.2
(OCH3), 43.5, 43.3, 43.2, 43.0 (iPr CH), 25.7, 25.6, 25.5 (SiC(CH3)3),
24.7, 24.7, 24.6, 24.6, 24.6, 24.5, 24.4 (iPr CH3), 20.1, 20.1, 20.0, 18.0
(−CH2CN), 17.9 (SiC(CH3)3), −4.3, −5.2 (Si(CH3)2); 31P NMR
(162 MHz, CDCl3) δ 151.23 (s), 147.97 (s); HRMS (ESI-TOF) m/z
[M + H]+ calcd for C49H65N6O10PSiNa 979.4161, found 979.4132.
N2-[(2-Cyanoethoxy)carbonyl]-O6-[2-(4-nitrophenyl)ethyl]-2′-O-
(tert-butyldimethylsilyl)-5′-O-(4,4′-dimethoxytrityl)guanosine-3′-O-
(2-cyanoethyl-N,N-diisopropyl)phosphoramidite (31a): 1H NMR
(400 MHz, CDCl3) δ 8.16 (d, J = 8.7 Hz, 2H, aromatic), 8.04, 8.02
(2 × s, 1H, H8), 7.58−7.30 (m, 9H, aromatic), 7.30−7.17 (m, 2H,
aromatic), 7.09 (s, 1H, NH), 6.80 (m, 4H, aromatic), 6.01 (d, J = 7.2
Hz, 0.40H, H1′), 5.86 (d, J = 6.9 Hz, 0.60H, H1′), 5.12 (dd, J = 6.9,
5.2 Hz, 0.60H, H2′), 5.00 (dd, J = 7.2, 4.5 Hz, 0.40H, H2′), 4.86 (t, J =
6.8 Hz, 2H, −OCH2−), 4.43−4.19 (m, 4H, H3′, H4′ and
−COOCH2−), 3.98−3.87 (m, 0.47H, −OCH2−), 3.98−3.87 (m,
0.53H, −OCH2−), 3.81−3.75 (m, 6H, OCH3), 3.68−3.46 (m, 4H, iPr
CH, H5′ and −OCH2−), 3.39−3.22 (m, 3H, −CH2ph and H5″),
2.34−2.16 (m, 3H, −CH2CN), 2.34−2.16 (m, 1H, −CH2CN), 1.23−
1.13 (m, 9H, iPr CH3), 1.01 (d, J = 6.7 Hz, 3H, iPr CH3), 0.75 (s, 9H,
SiC(CH3)3), −0.01, −0.06 (2 × s, 3H, Si(CH3)2), −0.25, −0.26 (2 ×
s, 3H, Si(CH3)2); 13C NMR (100 MHz, CDCl3) δ 160.8 (C6), 158.8,
158.5 (aromatic), 153.3, 153.1 (C4), 151.6, 151.4 (C2), 150.5, 150.4
(CO), 147.0, 146.8, 145.9, 145.7, 144.8, 144.6 (aromatic), 141.4,
140.6 (C8), 136.0, 135.8, 135.7, 135.6 (aromatic), 130.2, 130.2, 130.1,
130.0, 129.9, 128.3, 128.2, 128.1, 128.0, 127.2, 126.9, 123.8, 123.6
(aromatic), 118.9, 118.6 (C5), 118.0, 117.4, 116.9, 116.8 (CN), 113.4,
113.3, 113.2, 113.1 (aromatic), 88.4, 87.5 (C1′), 86.9, 86.6 (DMTr-C),
84.7, 84.3 (C4′), 75.8, 74.1, 74.0 (C2′), 73.6, 73.5, 72.7, 72.6 (C3′),
67.1, 67.0 (−OCH2−), 63.6, 63.5 (C5′), 59.5, 59.7 (−OCH2−,
−COOCH2−), 59.1, 57.8, 57.6 (−OCH2−), 55.4, 55.1 (OCH3), 43.6,
43.5, 43.1, 43.0 (iPr CH), 35.2, 35.0 (−CH2ph), 25.7, 25.5, 25.4
(SiC(CH3)3), 24.9, 24.8, 24.7, 24.5 (iPr CH3), 20.6, 20.5, 20.2, 20.1
(−CH2CN), 18.3, 18.2, 18.1, 18.0 (−CH2CN and SiC(CH3)3), −4.5,
−4.6, −5.1, −5.3 (Si(CH3)2); 31P NMR (162 MHz, CDCl3) δ 151.07
(s), 149.03 (s); HRMS (ESI-TOF) m/z [M + H]+ calcd for
C58H73N9O12SiP 1146.4886, found 1146.4889.
3′-O-(tert-Butyldimethylsilyl)-5′-O-(4,4′-dimethoxytrityl)uridine-
2′-O-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite (33): 1H
NMR (400 MHz, CDCl3) δ 8.72, 8.53 (2 × s, 1H, NH), 8.14, 8.06
(2 × d, J = 8.2 Hz, 1H, H6), 7.38−7.23 (m, 9H, aromatic), 6.87−6.81
(m, 4H, aromatic), 6.15 (d, J = 3.2 Hz, 0.4H, H1′), 6.06 (d, J = 2.2 Hz,
0.6H, H1′), 5.35, 5.27 (2 × d, J = 8.1 Hz, 1H, H5), 4.37−4.19 (m, 2H,
H2′, H3′), 4.14−4.06 (m, 1H, H4′), 3.99−3.85 (m, 1H, −OCH2−),
i
3.84−3.54 (m, 10H, OCH3, −OCH2−, Pr CH, H5′), 3.37−3.29 (m,
i
1H, H5″), 2.72−2.47 (m, 2H, −CH2CN), 1.18−1.15 (m, 12H, Pr
CH3), 0.80−0.78 (m, 9H, SiC(CH3)3), 0.09, 0.04 (2 × s, 3H,
Si(CH3)2), −0.01, −0.03 (2 × s, 3H, Si(CH3)2); 13C NMR (CDCl3,
101 MHz) δ 163.2, 163.1 (C4), 158.9, 158.9 (aomatic), 150.3, 150.1
(C2), 144.2, 144.2 (aromatic), 140.6, 140.4 (C6), 135.2, 135.1, 135.0,
130.4, 130.4, 128.5, 128.4, 128.1, 127.4, 127.4 (aromatic), 118.1, 117.8
(CN), 113.4, 113.4 (aromatic), 102.2, 102.1 (C5), 88.7, 88.6, 88.5
(C1′), 87.4, 87.3 (DMTr-C), 83.6, 83.5 (C4′), 76.6, 76.5 (C2′), 70.5,
70.5, 70.4 (C3′), 61.7, 61.6 (C5′), 58.5, 58.3, 58.3, 58.1 (−OCH2−),),
55.4, 55.4 (OCH3), 43.5, 43.4, 43.3 (iPr CH), 25.9 (SiC(CH3)3), 24.9,
24.9, 24.8, 24.8, 24.6, 24.5 (iPr CH3), 20.5, 20.4, 20.4, 20.3
(−CH2CN), 18.2, 18.1 (SiC(CH3)3), −4.1, −4.2, −4.8, −4.9
(Si(CH3)2); 31P NMR (162 MHz, CDCl3) δ 150.42 (s), 149.37 (s);
HRMS (ESI-TOF) m/z [M + H]+ calcd for C45H61O9N4PSiNa
883.3838, found 883.3816.
Procedure for the Synthesis of Photolabile Linker. Methyl 4-
(1-(Bis(4-methoxyphenyl)(phenyl)methoxy)ethyl)-3-nitrobenzoate
(43). Methyl 3-formyl-4-nitrobenzoate 40 (purchased from Sigma)
(5.00 g, 23.9 mmmol) was dissolved in anhydrous diethyl ether (150
mL), and methylmagnesium bromide (1 M in dibutyl ether, 59.7 mL,
59.7 mmol) was added dropwise slowly, maintaining the reaction
vessel at rt. The reaction mixture was stirred for 4 h and quenched by
the addition of saturated ammonium chloride (50 mL). The organics
were extracted with Et2O (3 × 50 mL). The combined organic layers
were washed with water (50 mL) and brine (50 mL) and dried over
MgSO4. The solvent was removed under vacuum, and the residue was
purified by flash column chromatography. The product was found to
also contain a benzyl alcohol side product 42 that could not be
separated by flash column chromatography. The material was used in
the next step without further purification. Alcohol 41 containing 42
(2.60 g, 11.5 mmol) was coevaporated with anhydrous pyridine (3 ×
10 mL). The residue and DMTr-Cl (3.90 g, 11.5 mmol) were
dissolved in anhydrous pyridine (20 mL) and anhydrous CH2Cl2 (10
mL), and the reaction mixture was stirred at rt overnight. MeOH (20
mL) was added, and the mixture was stirred for a further 10 min
before the solvent was removed under vacuum. The residue was
dissolved in CHCl3 (50 mL) and washed with saturated aq NaHCO3
(2 × 40 mL). The organic layer was dried over MgSO4 and evaporated
under vacuum. The residue was purified by flash column
chromatography (99:1, toluene/Et3N) to afford a yellow oil, which
was further purified by normal-phase HLPC give the title product 43
N2-[(2-Cyanoethoxy)carbonyl]-O6-[2-(4-nitrophenyl)ethyl]-3′-O-
(tert-butyldimethylsilyl)-5′-O-(4,4′-dimethoxytrityl)guanosine-2′-O-
(2-cyanoethyl-N,N-diisopropyl)phosphoramidite (31b): 1H NMR
(400 MHz, CDCl3) δ 8.15 (d, J = 7.6 Hz, 2H, aromatic), 8.07, 8.05
(2 × s, 1H, H8), 7.52 (d, J = 8.6 Hz, 2H, aromatic), 7.45−7.17 (m, 9H,
aromatic), 6.78 (d, J = 8.6 Hz, 4H, aromatic), 6.14 (d, J = 5.2 Hz,
0.51H, H1′), 6.04 (d, J = 5.6 Hz, 0.49H, H1′), 5.08 (dt, J = 10.8, 5.1
Hz, 0.50H, H2′), 4.86−4.76 (m, 2.50H, −OCH2−, H2′), 4.47 (t, J =
4.1 Hz, 1H, H3′), 4.36 (q, J = 6.0 Hz, 2H, −COOCH2−), 4.17−4.13
1
(4.10 g, 33% for two steps) as a colorless solid: H NMR (400 MHz,
CDCl3) δ 8.21 (d, J = 1.7 Hz, 1H, H2), 7.97 (dd, J = 8.2, 1.7 Hz, 1H,
H6), 7.82 (d, J = 8.2 Hz, 1H, H5), 7.48−7.43 (m, 2H, DMTr), 7.29−
7.10 (m, 7H, DMTr), 6.68−6.59 (m, 4H, DMTr), 5.23 (q, J = 6.2 Hz,
1H, H7), 3.92 (s, 3H, COOCH3), 3.74, 3.73 (2 × s, 6H, DMTr
OCH3), 1.65 (d, J = 6.2 Hz, 3H, H8); 13C NMR (CDCl3, 101 MHz) δ
165.0, 158.5, 158.4, 147.6, 146.0, 145.3, 136.2, 135.7, 132.0, 130.1,
i
(m, 1H, H4′), 3.84−3.45 (m, 11H, −OCH2−, OCH3, Pr CH and
H5′), 3.35−3.27 (m, 3H, −CH2ph and H5″), 2.74 (q, J = 6.2 Hz, 2H,
−CH2CN), 2.51 (t, J = 6.3 Hz, 1H, −CH2CN), 2.40−2.32 (m, 1H,
−CH2CN), 1.14−1.03 (m, 9H, iPr CH3), 0.91−0.85 (m, 12H, iPr CH3
and SiC(CH3)3), 0.11−0.07 (2 × s, 3H, Si(CH3)2), 0.00−−0.01 (2 ×
3324
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The Journal of Organic Chemistry
Featured Article
130.02, 130.0, 128.7, 127.8, 127.8, 126.8, 124.7, 113.1, 113.0, 68.0,
55.2, 55.2, 52.5, 24.8; HRMS (ESI-TOF) m/z [M + H]+ calcd for
C31H29O7N1Na 550.1836, found 550.1827.
decanted, followed by washing of the pellet with ethanol (2 × 0.75
mL) and finally drying the pellet in a SpeedVac at 65 °C for 1 h. The
dried pellet was dissolved in RNAase-free water (0.5−1 mL), and the
RNA oligomer was quantified by UV absorbance at λ 260 nm and
analyzed by MALDI-TOF MS to assess the synthesis. Dephosphor-
ylation (if required) of the acetylated RNA oligonucleotide began by
dilution to a concentration of 1 μg/10 μL with PBS buffer (0.01 M
phosphate, 0.138 M NaCl, 0.0027 M KCl, 1 mM MgCl2, pH 7.4). To
the dissolved oligoribonucleotide calf intestinal alkaline phosphatase
(0.5u/μg) was added, and the mixture was heated at 37 °C for 2 h. To
the solution was added one volume of 1 M TEAA buffer (pH 7.0), and
the oligonucleotide was desalted using a Waters C18 Sep-Pak column
prior to HPLC purification of the oligonucleotide. Acetylated RNA
oligomers were purified by preparative SAX-HPLC. The target
fractions were combined and desalted by dialysis against 10 mM
TEAA buffer (pH 7.0) at 4 °C. Finally, the purified oligonucleotides
were quantified by UV absorbance at 260 nm, and characterized by
MALDI-TOF MS.
Photolabile-CPG (45). Compound 43 (500 mg, 0.94 mmol) was
dissolved in THF (1.50 mL) and water (0.50 mL). LiOH (24.9 mg,
1.04 mmol) was added, and the mixture was stirred at rt for 24 h, the
reaction being monitored by TLC until no starting material was
observable. The solvent was removed to give a white solid, and this
material was used in the next step without further purification. The
residue was coevaporated with anhydrous pyridine (3 × 4 mL) and
redissolved in anhydrous pyridine (4 mL). Isobutyl chloroformate
(72.0 mg, 0.53 mmol) was added, and the formation of a white
precipitate was observed. The reaction mixture was stirred for 30 min
before the precipitate was removed by filtration under argon, and the
supernatant was filtered directly into oven-dried glassware. The solvent
was removed under vacuum and the resultant crude mixed anhydride
was dissolved in anhydrous CH2Cl2 (4 mL), followed by the addition
of N,N-diisopropylethylamine (68 mg, 0.53 mmol) and long-chain
alkylamine controlled pore glass (250 mg, 120−200 mesh, nominal
diameter 500 Å) (Sigma). The suspension was rotated gently under
argon at rt for 24 h. The CPG was filtered and washed with CH2Cl2,
MeCN, water, MeCN, and finally CH2Cl2 (15 mL each). The
modified CPG was dried overnight under vacuum and then treated
with CAP A (80:10:10, THF/2,6-lutidine/pivaloyl chloride) (2 mL)
and commercially available CAP B (Link Technologies) (90:10, THF/
N-methylimidazole) (2 mL) solution, and the mixture was rotated
gently for 1.5 h. The CPG was filtered, washed with CH2Cl2 (15 mL),
and dried under vacuum. The loading was determined by Trityl assay,
and loading values of 33.3−56.2 μmol/g were obtained. The prepared
CPG was stored in the dark at 4 °C.
Automated Synthesis of Acetylated RNA Oligonucleotides.
RNA oligonucleotide synthesis was performed on 1 μmol scale with
modified monomers (0.1 M in 1:1 CH2Cl2/MeCN) and utilized the
photolabile-CPG 46. The synthesis cycle began with detritylation
using 3% dichloroacetic acid (DCA) in CH2Cl2. The coupling step
utilized 80 μL of each amidite (0.1 M, 1:1, MeCN/CH2Cl2) and 1 M
4,5-dicyanoimidazole (DCI) as the activator with a single 20 min
coupling, with the exception that the acetylated amidites were given a
15 min double coupling step. A 5 min capping step was carried out
with CAP A (80:10:10, THF/2,6-lutidine/pivalic anhydride), and CAP
B (90:10, THF/N-methylimidazole) solutions. The oxidation of the
phosphites was carried out with 0.02 M iodine (THF/pyridine/water,
7:2:1). The automation program was set as DMTr-ON such that the
final DMTr group was not removed.
Deprotection, Cleavage, and Purification of the Acetylated-
RNA Oligonucleotides. Without removal of the CPG from the
synthesis column, the CPG was dried under vacuum for 15 min. A
solution of 0.5 M DBU in anhydrous MeCN (3.5 mL, 10%
morpholine) was initially passed through the column for 5 min, and
then the column and CPG were immersed in the DBU solution under
an atmosphere of argon for 6 h at 40 °C with sonication every hour.
The column was washed with anhydrous acetonitrile (10 mL) and
CH2Cl2 (10 mL). The final DMTr group was removed by passing 3%
TCA in CH2Cl2 through the column until the washings became
colorless. The collected orange solution was diluted to 50 mL with
CH2Cl2, and the yield of full-length product was calculated by trityl
assay. At this point, the CPG was placed into a 4 mL UV transparent
vessel and suspended in DMSO (0.5 mL). A single wavelength LED
light (λ = 365) was placed above the UV transparent vessel, focusing
on the region containing CPG, in a black box to prevent extra UV light
escaping into the environment. The CPG was irradiated at 365 nm
(max = 34.5 mW/cm2, Prizmatix Mic-LED-365) for 1 h. The CPG was
removed by filtration and washed with DMSO (2 × 0.5 mL), and the
fractions were combined. The DMSO was removed by lyophilization,
and the residue was redissolved in anhydrous DMSO (200 μL). To the
solution was added Et3N·3HF (125 μL), the mixture was thoroughly
mixed and then heated at 65 °C for 3 h. Sodium acetate (3 M, 25 μL,
pH 7) was added, followed by thorough mixing. After the addition of
n-butanol (1 mL) the mixture was cooled at −80 °C for 30 min and
then centrifuged for 10 min at 13200 rpm. The n-butanol was
ASSOCIATED CONTENT
* Supporting Information
■
S
1H and 13C NMR spectra for all new compounds, MADLI-
TOF mass spectra, and HPLC traces of synthesized partially 2′
or 3′-O-acetylated RNA oligonucleotides and UV melting
curves of duplexes. This material is available free of charge via
the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*E-mail: johns@mrc-lmb.ac.uk.
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by the MRC (Project No.
MC_UP_A024_1009) and the EPSRC. We thank Drs. Stefan
Freund and Trevor Rutherford for assistance with NMR
spectroscopy.
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