Synthesis, pairing, and cellular uptake properties of C(6′)- functionalized tricyclo-DNA

Article abstract of DOI:10.1021/jo300648u

Tricyclo-DNA (tc-DNA) is a promising candidate for oligonucleotide-based therapeutic applications exhibiting increased affinity to RNA and increased resistance to nucleases. However, as many other oligonucleotide analogs, tc-DNA does not readily cross cell membranes. We wished to address this issue by preparing a prodrug of tc-DNA containing a metabolically labile group at C(6′) that promotes cellular uptake. Two monomeric nucleoside building blocks bearing an ester function at C(6′) (tc^ee-T and tc ^hd-T) were synthesized starting from a known C(6′) functionalized bicyclic sugar unit to which the cyclopropane ring was introduced via carbene addition. NIS-mediated nucleosidation of the corresponding glycal with in situ persilylated thymine afforded the β-iodonucleoside exclusively that was dehalogenated via radical reduction. Diversity in the ester function was obtained by hydrolysis and reesterification. The two nucleosides were subsequently incorporated into DNA or tc-DNA by standard phosphoramidite chemistry. The reactivity of the ester function during oligonucleotide deprotection was explored and the corresponding C(6′) amide, carboxylic acid, or unchanged ester functions were obtained, depending on the deprotection conditions. Compared to unmodified DNA, these tc-DNA derivatives increased the stability of duplexes investigated with ΔT_m/mod of +0.4 to +2.0 °C. The only destabilizing residue was tc^hd-T, most likely due to self-aggregation of the lipophilic side chains in the single stranded oligonucleotide. A decamer containing five tc^hd-T residues was readily taken up by HeLa and HEK 293T cells without the use of a transfection agent.

Full text of DOI:10.1021/jo300648u

Featured Article
pubs.acs.org/joc
Synthesis, Pairing, and Cellular Uptake Properties of C(6′)-
Functionalized Tricyclo-DNA
Jory Lietard and Christian J. Leumann*
Department of Chemistry and Biochemistry, University of Bern, Freiestrasse 3, CH-3012, Bern, Switzerland
S
* Supporting Information
ABSTRACT: Tricyclo-DNA (tc-DNA) is a promising
candidate for oligonucleotide-based therapeutic applications
exhibiting increased affinity to RNA and increased resistance
to nucleases. However, as many other oligonucleotide analogs,
tc-DNA does not readily cross cell membranes. We wished to
address this issue by preparing a prodrug of tc-DNA
containing a metabolically labile group at C(6′) that promotes
cellular uptake. Two monomeric nucleoside building blocks
bearing an ester function at C(6′) (tcee-T and tchd-T) were
synthesized starting from a known C(6′) functionalized
bicyclic sugar unit to which the cyclopropane ring was introduced via carbene addition. NIS-mediated nucleosidation of the
corresponding glycal with in situ persilylated thymine afforded the β-iodonucleoside exclusively that was dehalogenated via
radical reduction. Diversity in the ester function was obtained by hydrolysis and reesterification. The two nucleosides were
subsequently incorporated into DNA or tc-DNA by standard phosphoramidite chemistry. The reactivity of the ester function
during oligonucleotide deprotection was explored and the corresponding C(6′) amide, carboxylic acid, or unchanged ester
functions were obtained, depending on the deprotection conditions. Compared to unmodified DNA, these tc-DNA derivatives
increased the stability of duplexes investigated with ΔTm/mod of +0.4 to +2.0 °C. The only destabilizing residue was tchd-T, most
likely due to self-aggregation of the lipophilic side chains in the single stranded oligonucleotide. A decamer containing five tchd-T
residues was readily taken up by HeLa and HEK 293T cells without the use of a transfection agent.
INTRODUCTION
■
Control of translation by oligonucleotides was first described
more than three decades ago1 and has since evolved into
powerful therapeutic strategies2,3 among which the antisense,
the siRNA, and the anti micro-RNA approaches are most
promising.4,5 However, the barriers to the development of
nucleic acids as therapeutics are manifold and delivery into cells
is often recognized as being the most challenging one.6 Other
important parameters include resistance toward nuclease
degradation and affinity for the target RNA sequence. Chemical
modification has emerged as a powerful tool to tackle these
issues.2 In particular, nucleoside analogues with restricted
backbone flexibility have been shown to increase the affinity
toward complementary RNA and improve the stability of the
corresponding oligonucleotides.7 Such conformationally con-
strained nucleic acid structures include hexitol nucleic acids
(HNA),8 locked nucleic acids (LNA),9,10 and bicyclo- and
tricyclo-DNA (Figure 1).11−13
Tricyclo-DNA (tc-DNA) is an RNA structural mimic which
binds to complementary RNA with high affinity and high
selectivity.14 Furthermore, it is stable in serum, does not elicit
RNaseH cleavage and has recently been evaluated as antisense
and siRNA agent with promising results.15−17 However, like
most natural or modified oligonucleotides, tc-DNA also suffers
from poor cellular uptake and thus relies on the use of
transfection agents to help crossing of the cellular membrane.
Figure 1. Chemical structures of selected derivatives of the bicyclo-
and tricyclo-DNA family.
Transfection agents such as cationic lipids or polymers are
perhaps the most known carriers for oligonucleotide delivery,
Received: March 29, 2012
Published: May 2, 2012
© 2012 American Chemical Society
4566
dx.doi.org/10.1021/jo300648u | J. Org. Chem. 2012, 77, 4566−4577

The Journal of Organic Chemistry
Featured Article
Scheme 1. Synthesis of Tricyclo Compounds 3 and 4
Scheme 2. Synthesis of tcee-T Phosphoramidite 10
but their inherent toxicity has prevented their use in vivo.18
However, they represent only a fraction of a much broader
arsenal of strategies for improving the cellular uptake of
oligonucleotides. Such strategies include viral delivery meth-
ods,19 physical and mechanical techniques,20 and conjugation
with gold nanoparticles21 or with arginine-rich or cell-
penetrating peptides.22 Although they have been shown to
improve the uptake of nucleic acids, limitations such as toxicity
and immunogenicity have challenged their potential as
universal delivery systems. Therefore, important research efforts
were devoted to bypass the use of delivery agents.
Cationic or zwitterionic oligonucleotides have been shown to
promote cellular uptake in selected cases.23,24 In this context,
we have recently reported on bicyclo-DNA carrying a lysine
moiety able to cross the cell membrane without aid of
transfection agents.25 Bioconjugation to cholesterol or long
aliphatic chains has also proven successful in carrier-free
approaches.26,27 Recently, LNA gapmers with phosphoro-
thioates linkages have been evaluated in free cellular uptake
through a process called gymnosis which requires higher
oligonucleotide concentrations in the growth medium.28
Another promising concept consists in temporarily masking
the negative charge of the phosphates and has been coined the
“pro-oligonucleotide approach” in analogy to the prodrug
approach.29,30 The masked, charge neutral oligonucleotide can
more easily penetrate into the intracellular compartment where
endogenous enzymes convert the pro-oligonucleotide into the
biologically active nucleic acid. Variations of this theme include
modification of the O(2′) position of ribonucleotides to attach
biolabile groups in order to improve the cellular uptake of
RNA.31,32
In this work, we set out to synthesize “pro tricyclo-
oligonucleotides” bearing an ester function at position C(6′).
This position was selected since it can easily be synthetically
manipulated by virtue of the neighboring C(5′)-oxo function.
Moreover, this position is expected to be sufficiently distant
from the nucleobase to exclude interference with base pairing.
RESULTS AND DISCUSSION
■
Synthesis of tcee-T and tchd-T Building Blocks 10 and
15. The preparation of tcee-T and tchd-T phosphoramidites 10
and 15 started from the already known ketone 1 (mixture of
four diastereoisomers), which was previously used in the
synthesis of C(6′)-alkylated bicyclo nucleosides.33 Abstraction
of the α-hydrogen of the ketone with LiHMDS followed by
subsequent trapping of the enolate with TBS-Cl yielded
silylenol ethers 2 which were separated into the pure anomeric
forms (Scheme 1). The introduction of the cyclopropane ring
into 2 was first envisaged via the Simmons−Smith procedure
(CH2I2, Ag/Zn couple) but resulted in very low yields and
several undesired side products. When the Ag/Zn couple was
exchanged for ZnEt2 (Furukawa’s protocol34), the reaction
proceeded smoothly and in good yields. However, while
cyclopropanation of 2β afforded tricyclo compound 3β as the
only isomer, the same conditions applied to 2α led to a mixture
of two diastereoisomers that could easily be separated. The
major isomer 4α (62%) resulted from reaction at the concave
(endo) face of the bicyclic structure, while the desired isomer
3α with exo configuration of the cyclopropane ring was only
obtained in 30% yield. The stereochemical outcome of the
reaction with 2β can be explained by the directing effect of the
homoallylic hydroxy group and steric hindrance at the
alternative endo face.35 In 2α, the methoxy substituent might
4567
dx.doi.org/10.1021/jo300648u | J. Org. Chem. 2012, 77, 4566−4577

The Journal of Organic Chemistry
Featured Article
Scheme 3. Synthesis of tchd-T Phosphoramidite 15
Figure 2. X-ray structures of nucleoside: 8A (left), and 8B (right).
engage in hydrogen bonding to the tertiary hydroxy group
preventing it from coordinating to the reactive metal species,
thus giving rise to a mixture of isomers. Furthermore, the ester
function at C(6′) might also play a directing role in the
cyclopropanation reaction.36 The configuration at the cyclo-
With nucleoside 8 in hand, access to the phosphoramidite 10
was straightforward. Tritylation of the 5′-OH group with
DMTr-Cl afforded intermediate 9 which was subsequently
phosphitylated under standard conditions to give phosphor-
amidite 10.
To obtain the long-chain alkyl ester derivative tchd-T, ester 7
was first saponified to afford the 3′-desilylated carboxylic acid
11 in good yield, which was used in the next step without
further purification (Scheme 3). Condensation of 11 with 1-
hexadecanol mediated by EDC·HCl and DMAP, followed by
desilylation with HF·pyridine led to the free nucleoside 13 in
65% yield over two steps. Tritylation of O(5′) and
phosphitylation of O(3′) to give 14 and 15, respectively,
proceeded under similar conditions as for building block 10 but
required somewhat longer reaction times.
propane ring was assigned by H NMR NOE, H−1H COSY,
and 2D NOESY experiments (see the Supporting Information).
For the nucleosidation step, we considered the N-
iodosuccinimide induced addition of persilylated thymine to
the corresponding glycal 5 (Scheme 2). Indeed, this strategy
has been successfully applied to unmodified tricyclo pyrimidine
nucleosides, yielding only β-nucleosides.37 Therefore, the
combined anomers of 3 were converted into glycal 5 by
treatment with TMSOTf, affording only one product and
thereby verifying the cyclopropyl ring configuration (Scheme
2). The crude, 3′-silylated glycal 5 was then subjected to a NIS-
mediated nucleosidation with persilylated thymine which
pleasingly yielded only the β-nucleoside 6. Without further
purification, the C(2′)-iodonucleoside 6 was subjected to
radical reduction with Bu3SnH and AIBN, providing nucleoside
7 in 67% yield over three steps. Removal of the O(5′)- and
O(3′)-silyl protecting groups with HF·pyridine gave compound
8, the relative configuration at the anomeric center of which
was assessed by 1H NMR difference NOE experiments.
Irradiation of H(1′) led to strongly enhanced signals at the
H(4′) (4%) and H(2′) (5.9%) (see the Supporting
Information). These observations confirmed the β-configu-
ration at C(1′). The structure of nucleoside 8 was also
confirmed independently by X-ray crystallography.
1
1
X-ray Structure of Nucleoside 8. To gain information on
the conformational preferences of the new tricyclonucleoside
derivatives, the solid-state structure of nucleoside 8 was solved
by X-ray crystallography. The asymmetric unit contains two
symmetry-unrelated molecules 8A and 8B (Figure 2). The
main structural difference between 8A and 8B lies in the
furanose substructure which belongs to the C(2′)-exo (2E)
conformation in 8A and to the O(4′)-endo (0E) conformation
in 8B, with calculated pseudorotation phase angles (P) of
342.8° (8A) and 85.2° (8B), respectively. Although the
glycosidic torsion angles χ are slightly different (Table 1),
both structures have the thymine moiety in the anti
conformation. In the asymmetric unit, there exists a intricate
pattern of intermolecular H-bonds as well as stacking contacts
of the bases which probably accounts for the differences in χ
4568
dx.doi.org/10.1021/jo300648u | J. Org. Chem. 2012, 77, 4566−4577

The Journal of Organic Chemistry
Featured Article
required in order to achieve coupling efficiencies of 94% for
tcee-T 10 and 97% for tchd-T 15, as judged from the trityl assay.
A set of conditions for deprotection and detachment from
solid support has been elaborated that either allows to conserve
the ester functions in the oligonucleotides or to transform them
into amide or carboxy functions (Scheme 4).33 Treatment with
concd NH3 (55 °C, 16 h) converted all ethyl ester groups into
the corresponding amide functions yielding oligonucleotides
ON 1, 3, 5, 7−9 (Table 2). Treatment with potassium
hydroxide led to hydrolysis of the ester function (→ ON 10),
while deprotection with 25% benzylamine left the ester groups
untouched (→ ON 11).
Table 1. Selected Torsion Angles and Furanose Puckers of
Nucleoside 8 in Comparison to bc-, tc-, and Natural
Deoxynucleosides
nucleoside
furanose pucker
γ (deg)
δ (deg)
χ (deg)
8A
8B
C(2′)-exo
O(4′)-endo
C(2′)-exo
C(1′)-exo
C(2′)-endo
149.9
158.3
152
100.8
99.4
107
−170
−125.9
−164
−107.6
−119
a
tc-dA
b
bc-T
149.3
57
133.5
122
c
dN
a
Conformation of a tricyclodeoxyadenosine unit in a DNA duplex (ref
38). Data taken from ref 11. Average deoxynucleotide conformation
b
c
in B-DNA (ref 39).
For oligonucleotides containing tchd-T units we selected
ethanolic ammonia (concd NH3/EtOH 1:3) as a deprotection
mixture. While after 24 h at 40 °C no trace of aminolysis of the
ester function was observed by ESI−-MS for singly modified
sequences (ON 2 and 4), some aminolysis was observed with
ON 6 containing two hexadecyl ester moieties. Deprotection in
concd NH3/EtOH 1:3 at 40 °C for only 2 h was sufficient to
produce the oligothymidylates containing five tcee-T or tchd-T
units (ON 12 and 13). Indeed, no hydrolyzed ester in the
crude material was detected by mass spectrometry.
All oligonucleotides were purified by ion-exchange (IE)
HPLC using standard methods. Purification of ON 13 bearing
five C16 chains, however, was not as straightforward and
required the use of 1 M NaClO4 as eluent and organic
cosolvents. Under these conditions, ON 13 eluted, somewhat
surprisingly, as a broad peak with multiple shoulders, most
likely arising from oligonucleotide aggregates. Reversed-phase
HPLC on C4 or C18 columns was impossible due to very strong
retention of the oligonucleotide on the solid phase. After
purification and subsequent desalting, the integrity of all
oligonucleotides was routinely verified either by ESI− or
and P values. In both molecules 8A and 8B the torsion angle γ
falls into the antiperiplanar range, which is consistent with the
values found for unmodified members of the bc-DNA and tc-
DNA families.11,38 In addition, the torsion angle δ is in a +ac
orientation as observed in tc-DNA residues. Summarizing all
the structural features, it appears that the addition of an alkyl
group at position C(6′) does not significantly change the
intrinsic conformational preferences of the tricyclic core unit.
Oligonucleotide Synthesis and Deprotection. The
previously prepared tcee-T and tchd-T phosphoramidites 10
and 15 were incorporated into a series of mixed-base decamer
oligodeoxynucleotides as single or double substitutions by
standard solid-phase synthesis on a 1.3 μmol scale (ON 1−11,
Table 2). The modified units were introduced into either
natural DNA (ON 1−6) or tc-DNA (ON 7−11). Additionally,
decaoligothymidylates containing five alternating tcee-T, tchd-T,
or tc-T substitutions and labeled with 6-carboxyfluorescein
(FAM) at their 3′-end were prepared for cellular uptake
experiments (ON 12−14). An increased concentration (0.15 M
in CH3CN) and an extended coupling time of 12 min were
a
Table 2. Oligonucleotides Prepared with tcee-T and tchd-T Amidites 10 and 15
a
Capital letters: natural DNA nucleotides. Capital italic letters: 6′-unfunctionalized tricyclodeoxynucleotides tc-A, tc-C, tc-G, tc-T (structure, see
b
Figure 1, top right). Lowercase letter: 6′-functionalized tc-T units. FAM = 6-carboxyfluorescein.
4569
dx.doi.org/10.1021/jo300648u | J. Org. Chem. 2012, 77, 4566−4577

The Journal of Organic Chemistry
Featured Article
a
complement, ON 5 exhibits again stabilization of the hybrid
duplex with Tm‘s comparable to those of single-modified
oligonucleotides. This is also a feature found for unmodified tc-
units in the same sequence context14 and confirms again that
the grafting of a substituent at C(6′) on the tricyclo scaffold
does not affect the pairing efficiency.
Scheme 4
On the other hand, upon replacement of an ethyl by a
hexadecyl group, all duplexes were destabilized (Table 3).
Moreover, this destabilization was particularly pronounced with
DNA as a complement, where ΔTms of −9.0 and −8.0 °C were
measured with ON 2 and 4, respectively. When paired with
complementary RNA, oligonucleotides containing one tchd-T
unit were less destabilizing (−5.2 and −4.7 °C). Such a loss in
affinity was cumulative, as a double-modified oligonucleotide
(ON 6) gave Tms of about half of that of the control (20.1 °C
vs 44.9 °C; 23.1 °C vs 43.6 °C). Furthermore, this
destabilization was associated with a lower hyperchromicity
upon duplex melting, suggesting imperfect base-pairing (see the
Supporting Information).
UV−melting experiments were also performed in the series
of decathymidylates alternately substituted with either tcee-T,
tchd-T, or tc-T units and carrying a 3′-FAM label (Table 4).
Table 4. Tm Data (°C) from UV−melting Curves (260 nm)
of Modified Decathymidylates Sequences with
a
Complementary DNA and RNA
a
Oligonucleotide deprotection conditions: (a) concd NH3, 55 °C, 16
b
c
entry
Tm (°C) vs DNA (ΔTm/mod) Tm (°C) vs RNA (ΔTm/mod)
h; (b) 25% benzylamine in MeOH/H2O 1:2, 65 °C, 8 h; (c) 0.1 M
KOH, 55 °C, overnight.
ON 12
ON 13
ON 14
18.3 (−1.1)
n.d.
20.6 (−0.2)
n.d.
MALDI-TOF mass spectrometry (see the Supporting In-
formation).
Hybridization Properties of Substituted Oligodeox-
ynucleotides. The affinities of the modified oligodeoxynu-
cleotides to their DNA and RNA complements was assessed by
UV melting curves at 260 nm in standard saline buffer (10 mM
Na2HPO4, 150 mM NaCl, pH 7.0) (Table 3). Introduction of a
19.2 (−1.0)
19.9 (−0.3)
a
Conditions: 2 μM single strands in 150 mM NaCl and 10 mM
NaH2PO4 at pH 7.0. Tm of the FAM-labeled unmodified duplex: 24.0
°C; Tm of the FAM-labeled unmodified duplex: 21.6 °C. n.d.: not
b
c
detected.
Analysis of the Tm data revealed that discontinuous tc-T
incorporations (ON 14) were destabilizing when paired to dA10
and rA10 as a complement (−1.0 and −0.3 °C/mod,
respectively), a result that was observed similarly also for ON
12 containing five alternative tcee-T units. However, the
presence of five tchd-T units (ON 13) made the determination
of a Tm value impossible. Indeed, no sigmoidal curves and thus
no duplex formation was observed upon melting of ON 13·with
cDNA or RNA (see the Supporting Information). We
hypothesize that this is the result of a competition between
base pairing and hydrophobic interactions of the C16 side
chains in ON 13, organizing it in structures such as micelles or
vesicles where the oligonucleotide is less accessible for
hybridization.
Table 3. Tm data (°C) from UV−melting Curves (260 nm) of
Modified Decamer Sequences with Complementary DNA
and RNA
a
b
c
entry
Tm (°C) vs DNA (ΔTm/mod)
Tm (°C) vs RNA (ΔTm/mod)
ON 1
ON 2
ON 3
ON 4
ON 5
ON 6
45.7 (+0.8)
37.0 (−9.0)
45.3 (+0.4)
38.0 (−8.0)
42.9 (−1.0)
20.1 (−13.0)
45.7 (+2.1)
38.4 (−5.2)
45.6 (+2.0)
38.9 (−4.7)
46.1 (+1.3)
23.1 (−10.2)
a
Conditions: 2 μM single strands in 150 mM NaCl and 10 mM
b
c
NaH2PO4 at pH 7.0. Tm of the unmodified duplex: 44.9 °C; Tm of
the unmodified duplex: 43.6 °C
Hybridization Properties of Substituted Tricyclo-
oligonucleotides. The tc-T derivatives were also incorpo-
rated into tc-oligonucleotides to study the influence of a
modified tc-unit on the pairing properties of tc-DNA (Table 5).
UV−melting curve analysis showed that the strong stabilization
brought about by the fully modified tc-DNA (+13.5 °C and
+20.5 °C with cDNA and RNA, respectively) is conserved
upon incorporation of the functionalized tc-units, with ΔTms
ranging from −1.6 to +2.0 °C overall. This indicates that short
alkyl chains at position C(6′) do not interfere with the pairing
properties of tc-DNA, suggesting again a smooth accommoda-
tion of all substituents in the duplexes. The amide and ethyl
single tc-T unit bearing an amide group at position C(6′) (ON
1 and 3) led to an increase in Tm of +0.4 to +0.8 °C with a
DNA complement and to +2.0 °C with an RNA complement.
This clearly indicates that the amide substituent at C(6′) does
not perturb duplex formation. Furthermore, the degree of
stabilization is comparable to the single incorporation of an
unmodified tc-T unit (−0.4 °C) in the same sequence.14 Two
modifications separated by a natural dG unit (ON 5) slightly
decreased the Tm of a DNA duplex when compared to the
unmodified duplex (−1.0 °C/mod.). However, with RNA as a
4570
dx.doi.org/10.1021/jo300648u | J. Org. Chem. 2012, 77, 4566−4577

The Journal of Organic Chemistry
Featured Article
unmodified dT10 with a fluorescein label as a control. The
experiments were performed in pure water and in standard
saline buffer (Figure 3). In water, low emission of fluorescence
was obtained for all FAM-labeled oligonucleotides ON 12−14,
suggesting a substantial degree of self-association under these
conditions (Figure 3A). Upon addition of the detergent Triton
x-100 above its critical micelle concentration, a large restoration
of fluorescence emission was observed for ON 12 and 14 and
dT10-FAM. However, the increase of fluorescence emission for
ON 13 was limited (Figure 3B). In standard saline, the
intensity of fluorescence emission for ON 12 and 14 and dT10-
FAM reached a maximum and did not vary upon addition of
Triton x-100 (Figures 3C,D). On the other hand, under these
conditions, a substantial quenching of fluorescence for ON 13
was still detected and was only partially restored after addition
of Triton x-100. Taken together, these results indicate that ON
13 self-associates even in buffered saline. The limited
restoration of fluorescence emission for ON 13 after addition
of a detergent implies that self-quenching is not entirely
eliminated. It is therefore reasonable that this amphiphilic
oligonucleotide still exists in an aggregated state even in the
presence of large quantities of a detergent.
Cellular Uptake. The previously prepared fluorescein-
labeled oligonucleotides were then evaluated for their ability to
cross the cellular membrane in the absence of a transfection
agent. Two human cell lines, HeLa and HEK 293T, were
transfected with ON 12, 13 and 14 at 10 μM concentration
each, and the unmodified dT10-FAM was used as a control.
After 2 days of incubation at 37 °C, the cells were fixed and
analyzed by confocal fluorescence microscopy (Figure 4). No
visible internalization of the control oligonucleotide dT10-FAM
was detected in any cell line. Similarly, the decathymidylates
modified with either tc-T or tcee-T (ON 12 and 14) were not
Table 5. Tm Data (°C) from UV−melting Curves (260 nm)
of Modified Decamer Sequences in the Context of a tc-DNA
Backbone with Complementary DNA and RNA
a
b
c
entry
Tm (°C) vs DNA (ΔTm/mod) Tm (°C) vs RNA (ΔTm/mod)
ON 7
ON 8
ON 9
ON 10
ON 11
57.8 (−0.7)
57.1 (−1.4)
57.3 (−0.6)
56.9 (−0.8)
57.2 (−0.6)
65.0 (+0.7)
64.4 (+0.1)
63.9 (−0.2)
66.3 (+1.0)
63.9 (−0.2)
a
Conditions: 2 μM single strands in 150 mM NaCl and 10 mM
b
NaH2PO4 at pH 7.0. Tm of fully modified tricyclo-DNA/DNA
duplex: 58.5 °C; Tm of fully modified tricyclo-DNA/RNA duplex:
c
64.3 °C.
ester groups were both found to have negligible effects on the
thermal stability (ON 9 and 11).
Interestingly, additional carboxylate groups (as in ON 10)
led to an increase in Tm with RNA as a complement, implying
that additional negative charges do not perturb the pairing
affinity of tc-DNA. This is an important observation as
according to the prodrug approach the carboxylic acid function
is expected to be the functional group after enzymatic ester
hydrolysis. It therefore appears that ester hydrolysis will be
required to functionally activate the oligonucleotide.
Fluorescence Emission Measurements. The analysis of
the UV−melting curves of ON 13 led us to propose that self-
association of the single strands via their hydrophobic
hexadecyl ester functions might occur in water. Consequently,
this should result in significant self-quenching of fluorescein
fluorescence by proximity effects.40 We measured the emission
of fluorescence of all FAM-labeled oligonucleotides after
excitation at 480 nm. In addition, we used the corresponding
Figure 3. Fluorescence emission spectra of FAM-labeled oligonucleotides: (A) in H2O; (B) in H2O after addition of 0.36 mM Triton x-100; (C) in
150 mM NaCl, 10 mM NaH2PO4, pH 7.0; (D) in 150 mM NaCl, 10 mM NaH2PO4, pH 7.0 after addition of 0.36 mM Triton x-100. Conditions: 2
μM single strand, excitation at 480 nm, 20 °C.
4571
dx.doi.org/10.1021/jo300648u | J. Org. Chem. 2012, 77, 4566−4577

The Journal of Organic Chemistry
Featured Article
Figure 4. Fluorescence microscopy analysis of HeLa (top row) and HEK 293T cells (bottom row) treated with: (A, E) dT10-FAM; (B, F) ON 12,
(C, G) ON 14, (D, H) ON 13. Conditions: 10 μM ODN concentration, 2 days incubation at 37 °C, cells are then fixed with 3.7% paraformaldehyde
and their nuclei stained with DAPI (blue channel).
able to penetrate through the cellular membrane. On the other
hand, ON 13 was readily taken up (Figure 4D,H) in both cell
lines, thus highlighting the role of the side chains attached to
the tricyclo skeleton. The distribution pattern was mainly
cytoplasmic with a uniform rather than a granular localization.
In HeLa cells, fluorescence was also visible on the contours of
the cells, suggesting that some material remained associated
with the cell membrane. In contrast, the contours of HEK cells
were not fluorescent and ON 13 was more homogeneously
distributed throughout the cytosol. These observations raise
questions about the mechanism of internalization and the
subsequent trafficking. Indeed, the uniform cytosolic distribu-
tion implies that if endocytosis was the pathway for uptake,
then most of the material has leaked out of the endosomal
compartment. Furthermore, the presence of membrane-bound
oligonucleotide suggests that endocytosis may not be the only
route of internalization. As an alternative pathway, the fusion of
ON 13 with the cell surface can be assumed which has
previously been shown to occur with cationic lipids as delivery
agents.41 Clearly, additional experiments will be required in
order to identify the mechanism of uptake for ON 13.
releasing the corresponding carboxylic acid which was shown to
increase DNA and RNA affinity. Furthermore, the cellular
uptake of oligonucleotides containing either tc-T, tcee-T, or
tchd-T units showed that a C16 side chain is capable of helping
to cross the cellular membrane of two different cell lines.
Fluorescence microscopy revealed a rather uniform cytosolic
distribution of ON 13 together with surface-bound material.
The exact mechanism by which ON 13 enters cells remains
elusive at this point. Taken together, these results represent an
important step toward the preparation of a tc-DNA prodrug.
Further studies on the enzymatic processing of the hexadecyl
ester and preliminary tests on the antisense activities of tc-
oligonucleotides containing tchd-T units are underway.
EXPERIMENTAL SECTION
■
General Methods. All reactions were performed under Ar and in
dried glassware. Anhydrous solvents for reactions were obtained by
filtration through activated alumina or by storage over molecular sieves
(4 Å). Column chromatography (CC) was performed on silica gel
with an average particle size of 40 μm. All solvents for column
chromatography were of technical grade and distilled prior to use.
Thin-layer chromatography was performed on silica gel plates.
Visualization was achieved either under UV light or by dipping in
staining solution [CerIV-sulfate (10.5 g), phosphormolybdenic acid (21
g), conc. H2SO4 (60 mL), H2O (900 mL) or p-anisaldehyde (3.7 mL),
concd H2SO4 (5 mL), glacial acetic acid (1.5 mL), ethanol (135 mL)]
followed by heating with a heat gun. NMR spectra were recorded at
400 or 300 MHz field width (1H) in either CDCl3 or CD3OD. δ in
ppm relative to residual undeuterated solvent [CHCl3: 7.26 ppm (1H)
and 77.0 ppm (13C); CHD2OD: 3.35 ppm (1H) and 49.3 ppm (13C)],
CONCLUSIONS
■
In this work, we have presented the synthesis of tc-T derivatives
with functionalities connected to C(6′) of the tricyclic sugar
structure with the aim of improving the cellular uptake of tc-
DNA. Two phosphoramidites, tcee-T 10 and tchd-T 15, were
prepared in 8 and 10 steps, respectively, from ketone 1. The
key synthetic steps are the β-selective NIS-mediated
nucleosidation and hydrolysis/re-esterification that allows for
the preparation of a variety of tc-T esters without altering the
major part of the synthetic scheme. Both phosphoramidites
were successfully incorporated into oligonucleotides and the
reactivity of the ester group was investigated under various
deprotection conditions. Hydrolysis and aminolysis afforded
the corresponding carboxylic acid and amide forms, whereas
mild ethanolic ammonia treatment left the ethyl and hexadecyl
esters untouched in most cases. Amide, acid or ethyl ester
substituted oligonucleotides led to a significant stabilization
when paired to cDNA, and this effect was even more expressed
with RNA as a complement. With tchd-T however, all duplexes
were destabilized and it appears that there exists a competition
between base pairing and self-aggregation of the long aliphatic
chains as evidenced by UV-melting experiments and
fluorescence emission spectroscopy. Nevertheless, tchd-T
maintains its potential as a nucleoside analogue for
oligonucleotide-based therapies. Indeed, the hexadecyl ester is
expected to be hydrolyzed after internalization into cells, thus
1
J in Hz. Signal assignments are based on DEPT and on H−1H and
1H−13C correlation experiments (COSY/HSQC). High-resolution
mass spectra were recorded on an ion-trap instrument in the ESI+
mode.
(1S,5S)-8-[(tert-Butyldimethylsilyl)oxy]-7-(ethoxycarbonyl)-
methyl-5-hydroxy-3-methoxy-2-oxybicyclo[3.3.0]oct-7-ene
(2). To a solution of 1 (1.0 g, 3.87 mmol)33 in dry THF (39 mL) was
slowly added LiHMDS (842 mg, 5.03 mmol) 0.75 M THF at −78 °C.
A solution of TBDMS-Cl (875 mg, 5.81 mmol) and Et3N (0.22 mL,
1.54 mmol) in dry THF (14 mL) was added dropwise after 20 min.
The resulting clear yellow solution was allowed to warm to rt after 50
min and was stirred for an additional 2 h. The mixture was then
diluted with EtOAc (150 mL) and washed with satd NaHCO3 (2 ×
125 mL) and brine (1 × 125 mL). The aqueous phase was extracted
with EtOAc (2 × 400 mL), and the combined organic phases were
dried over MgSO4, filtered, and evaporated. The crude orange oil was
purified by CC with EtOAc/hexane (1:5 → 1:1), affording 2α (1.03 g,
72%) and 2β (211 mg, 15%) as colorless oils.
Data for 2α: Rf = 0.51 (EtOAc/hexane 2:1); 1H NMR (300 MHz,
CDCl3) δ 5.00 (d, J = 4.1 Hz, 1H, H−C(3)), 4.65 (s, 1H, H−C(1)),
4.09 (2q, J = 7.8, 4.0 Hz, 2H, CH3CH2O), 3.35 (s, 3H, MeO), 3.10 (s,
1H, OH), 3.02 (s, 2H, H−C(6)), 2.47 (d, J = 0.8 Hz, 2H, H−C(9)),
4572
dx.doi.org/10.1021/jo300648u | J. Org. Chem. 2012, 77, 4566−4577

The Journal of Organic Chemistry
Featured Article
2.19 (d, J = 13.4 Hz, 1H, H−C(4)), 2.01 (dd, J = 13.4, 4.1 Hz, 1H, H−
C(4)), 1.22 (t, J = 7.1 Hz, 3H, CH3CH2O), 0.93 (s, 9H, (CH3)3C−Si),
0.18 (s, 3H, (CH3)2Si), 0.14 (s, 3H, (CH3)2Si); 13C NMR (75 MHz,
CDCl3) δ 171.1 (CO2Et), 147.5 (C(8)), 110.73 (C(7)), 105.5 (C(3)),
92.0 (C(1)), 83.7 (C(5)), 60.8 (CH3CH2O), 54.7 (MeO), 47.2
(C(4)), 40.9 (C(6)), 32.4 (C(9)), 25.92 ((CH3)3C−Si), 18.5
((CH3)3C-Si), 14.5 (CH3CH2O), −3.9, −4.2 ((CH3)2Si); ESI+-
HRMS m/z calcd for C18H32O6NaSi ([M + Na]+) 395.1860, found
395.1847.
CH3CH2O, H−C(1)), 3.36 (s, 3H, MeO), 2.52 (d, J = 15.8 Hz, 1H,
H−C(10)), 2.43 (dd, J = 13.8, 5.3 Hz, 1H, H−C(7)), 2.32 (d, J = 15.8
Hz, 1H, H−C(10)), 2.14 (dd, J = 13.8, 1.2 Hz, 1H, H−C(5)), 2.00
(dd, J = 14.0, 1.6 Hz, 1H, H−C(7)), 1.96 (d, J = 13.9 Hz, 1H, H−
C(5)), 1.88 (s, 1H, O-H), 1.24 (t, J = 7.1 Hz, 3H, CH3CH2O), 1.02
(dd, J = 6.1, 0.8 Hz, 1H, H−C(3)), 0.89 (s, 9H, (CH3)3C−Si), 0.79
(dd, J = 6.1, 1.6 Hz, 1H, H−C(3)), 0.19 (s, 3H, (CH3)3C−Si), 0.13 (s,
1
3H, (CH3)3C−Si); H NMR difference NOE (400 MHz, CDCl3) δ
5.07 (H−C(8)) → 3.36 (4.7%, O-Me), 2.43 (4.8%, H−C(7)); 0.79
(Hβ-C(3)) → 4.10 (3.9%, H−C(1)), 1.96 (1.7%, H−C(5)), 0.85
(25%, Hα-C(3)), 0.85 (Hα-C(3)) → 2.32 (2.9%, H−C(10)), 0.79
(20.3%, Hα-C(3)); 13C NMR (100 MHz, CDCl3) δ 173.0 (CO2Et),
105.7 (C(8)), 89.5 (C(1)), 86.1 (C(6)), 66.7 (C(2)), 60.5
(CH3CH2O), 55.0 (MeO), 50.0 (C(7)), 46.9 (C(5)), 37.1 (C(10)),
29.9 (C(4)), 26.0 ((CH3)3C−Si), 22.9 (C(3)), 18.2 ((CH3)3C-Si),
14.5 (CH3CH2O), −3.4, −3.8 ((CH3)2Si); ESI+-HRMS m/z calcd for
C19H34O6SiNa ([M + Na]+) 409.2017, found 409.2008.
1
Data for 2β: Rf = 0.29 (EtOAc/hexane 2:1); H NMR (300 MHz,
CDCl3) δ 5.15 (dd, J = 5.4, 2.7 Hz, 1H, H−C(3)), 4.53 (s, 1H, H−
C(1)), 4.10 (q, J = 7.1 Hz, 2H, CH3CH2O), 3.30 (s, 3H, MeO), 3.16
(d, J = 15.8, 1H, H−C(6)), 2.89 (dd, J = 15.7, 0.8 Hz, 1H, H−C(6)),
2.74 (d, J = 15.6 Hz, 1H, H−C(9)), 2.48 (d, J = 15.6 Hz, 1H, H−
C(9)), 2.32 (dd, J = 13.6, 5.4 Hz, 1H, H−C(4)), 2.16 (dd, J = 13.6, 2.7
Hz, 1H, H−C(4)), 1.99 (s, 1H, OH), 1.23 (t, J = 7.1 Hz, 3H,
CH3CH2O), 0.94 (s, 9H, (CH3)3C−Si), 0.19, 0.18 (2s, 6H, (CH3)2Si).
13C NMR (75 MHz, CDCl3) δ 171.2 (CO2Et), 148.3 (C(8)), 110.4
(C(7)), 106.5 (C(3)), 92.2 (C(1)), 84.3 (C(5)), 60.8 (CH3CH2O),
55.3 (MeO), 48.8 (C(4)), 45.2 (C(9)), 32.6 (C(6)), 25.8 ((CH3)3C−
Si), 18.39 ((CH3)3C-Si), 14.5 (CH3CH2O), −3.9, −4.1 ((CH3)2Si);
ESI+-HRMS m/z calcd for C18H32O6NaSi ([M + Na]+) 395.1860,
found 395.1858.
Data for 4α: Rf = 0.73 (hexane/EtOAc 3:2); 1H NMR (400 MHz,
CDCl3) δ 5.10 (d, J = 4.2 Hz, 1H, H−C(8)), 4.70 (d, J = 1.5 Hz, 1H,
H−C(1)), 4.12 (qd, J = 7.1, 1.6 Hz, 2H, CH3CH2O), 3.34 (s, 3H,
MeO), 3.21 (s, 1H, O-H), 2.50 (d, J = 16.3 Hz, 1H, H−C(10)), 2.34
(d, J = 16.3 Hz, 1H, H−C(10)), 2.17−2.07 (m, 2H, H−C(5)), 1.95 (d,
J = 13.9 Hz, 1H, H−C(7)), 1.79 (dd, J = 13.9, 4.3 Hz, 1H, H−C(7)),
1.24 (t, J = 7.1 Hz, 3H, CH3CH2O), 0.85 (s, 9H, (CH3)3C−Si), 0.85−
0.82 (m, 1H, H−C(3)), 0.62 (d, J = 6.5 Hz, 1H, H−C(3)), 0.17 (s,
(1S,2R,4R,6S,8R)-2-[(tert-Butyldimethylsilyl)oxy]-4-
(ethoxycarbonyl)methyl-6-hydroxy-8-methoxy-9-oxytricyclo-
[4.3.01,6.02,4]nonane (3β). To a solution of 2β (0.35 mg, 0.95 mmol)
in dry CH2Cl2 (10 mL) was added diethylzinc (1 M in hexane, 5.7 mL,
5.7 mmol) dropwise at 0 °C, followed by diiodomethane (0.76 mL, 9.5
mmol) after 15 min. A white precipitate formed, and the resulting
suspension was allowed to warm to rt. After 5 h, the mixture was
diluted with EtOAc (100 mL) and washed with satd NH4Cl (1 × 50
mL) and 10% Na2S2O3 (1 × 50 mL). The aqueous phases were
combined and extracted with EtOAc (2 × 100 mL). The organic
phases were dried over MgSO4, filtered, and evaporated. The crude
dark yellow solid was purified by CC with EtOAc/hexane (1:5 → 1:3)
to give the title compound 3β (0.30 g, 81%) as a colorless oil.
1
3H, (CH3)3C−Si), 0.08 (s, 3H, (CH3)3C−Si); H NMR difference
NOE (400 MHz, CDCl3) δ 5.10 (H−C(8)) → 3.34 (5.7%, O-Me),
1.79 (3.7%, H−C(7)), 4.70 (H−C(1)) → 3.21 (2.7%, O-H); 0.62
(Hβ-C(3)) → 2.32 (2.9%, H−C(5)), 0.83 (19.7%, Hα-C(3)); 13C
NMR (75 MHz, CDCl3) δ 173.0 (CO2Et), 106.9 (C(8)), 98.2 (C(1)),
87.1 (C(6)), 68.3 (C(2)), 60.6 (CH3CH2O), 54.5 (MeO), 46.6
(C(7)), 44.0 (C(10)), 38.0 (C(5)), 32.0 (C(4)), 25.9 ((CH3)3C−Si),
22.3 (C(3)), 18.0 ((CH3)3C-Si), 14.5 (CH3CH2O), −4.0, −4.3
((CH3)2Si); ESI+-HRMS m/z calcd for C19H35O6Si ([M + H]+)
387.2197, found 387.2198.
(1S,2R,4R,6S)-2-[(tert-Butyldimethylsilyl)oxy]-4-
[(ethoxycarbonyl)methyl]-6-hydroxy-9-oxytricyclo-
[4.3.01,6.02,4]non-7-ene (5). To a solution of 3 (323 mg, 0.84 mmol)
in dry CH2Cl2 (6.1 mL) was added 2,6-lutidine (0.485 mL, 4.18
mmol) at 0 °C, followed by TMSOTf (0.43 mL, 2.51 mmol) after15
min. After an additional 30 min at 0 °C, the resulting yellow solution
was warmed to rt and stirred for another 2 h. The reaction was then
quenched by the dropwise addition of satd NaHCO3 (10 mL) and
diluted with EtOAc (30 mL). The organic phase was washed with satd
NaHCO3 (2 × 30 mL), and the aqueous phase was extracted with
EtOAc (3 × 60 mL). The combined organic phases were then dried
over MgSO4, filtered, and concentrated to yield crude 5 (385 mg) that
was used without further purification in the next step.
1
Data for 3β: Rf = 0.43 (hexane/EtOAc 2:1); H NMR (300 MHz,
CDCl3) δ 5.03 (d, J = 4.8 Hz, 1H, H−C(8)), 4.18−4.09 (m, 3H,
CH3CH2O, H−C(1)), 3.31 (s, 3H, MeO), 2.55 (m, 2H, H−C(10),
H−C(5)), 2.23 (m, 2H, H−C(7), H−C(10)), 2.11 (dd, J = 12.4, 4.9
Hz, 1H, H−C(7)), 1.92 (d, J = 13.7 Hz, 1H, H−C(5)), 1.24 (t, J = 7.1
Hz, 3H, CH3CH2O), 0.88 (s, 9H, (CH3)3C−Si), 0.85−0.80 (m, 2H,
1
H−C(3)), 0.18 (s, 3H, (CH3)2Si), 0.09 (s, 3H, (CH3)2Si); H NMR
difference NOE (400 MHz, CDCl3) δ 5.03 (H−C(8)) → 3.31 (5.5%,
O-Me), 2.11 (3.7%, H−C(7)), 0.85 (H−C(3)) → 4.12 (2.2%, H−
C(1)), 2.23 (1.9%, H−C(10)), 1.92 (1.7%, H−C(5)); 13C NMR (75
MHz, CDCl3) δ 173.3 (CO2Et), 105.6 (C(8)), 91.9 (C(1)), 86.8
(C(6)), 68.2 (C(2)), 60.5 (CH3CH2O), 54.5 (MeO), 49.4, 49.2
(C(7), C(5)), 37.3 (C(10)), 30.7 (C(4)), 25.9 ((CH3)3C−Si), 22.3
(C(3)), 18.1 ((CH3)3C-Si), 14.2 (CH3CH2O), −3.57, −3.62
((CH3)2Si); ESI+-HRMS m/z calcd for C19H35O6Si ([M + H]+)
387.2197, found 387.2205.
1
Data for 5: Rf = 0.88 (EtOAc/hexane 2:1); H NMR (300 MHz,
CDCl3) δ 6.30 (d, J = 2.7 Hz, 1H, H−C(8)), 5.05 (d, J = 2.7 Hz, 1H,
H−C(7)), 4.45 (s, 1H, H−C(1)), 4.11 (q, J = 7.1 Hz, 3H,
CH3CH2O), 2.47 (d, J = 15.8 Hz, 1H, H−C(5)), 2.31 (d, J = 15.8
Hz, 1H, H−C(5)), 2.17 (dd, J = 13.4, 1.7 Hz, 1H, H−C(10)), 2.02 (d,
J = 13.3 Hz, 1H, H−C(10)), 1.23 (t, J = 7.1 Hz, 3H, CH3CH2O), 0.90
(dd, J = 5.9, 1.8 Hz, 1H, H−C(3)), 0.87 (s, 9H, (CH3)3C−Si), 0.81 (d,
J = 5.9 Hz, 1H, H−C(3)), 0.17 (s, 3H, ((CH3)2Si), 0.13 (s, 3H,
((CH3)2Si), 0.07 (s, 9H, ((CH3)3Si); 13C NMR (75 MHz, CDCl3) δ
173.0 (CO2Et), 146.4 (C(8)), 108.6 (C(7)), 95.0 (C(1)), 91.6
(C(6)), 67.3 (C(2)), 60.5 (CH3CH2O), 50.8 (C(5)), 37.1 (C(10)),
30.7 (C(4)), 26.0 ((CH3)3C−Si), 23.9 (C(3)), 18.2 ((CH3)3C-Si),
14.5 (CH3CH2O), 2.1 ((CH3)3Si), −3.5, −3.6 ((CH3)2Si); ESI+-
HRMS m/z calcd for C21H39O5Si2 ([M + H]+) 427.2331, found
427.2331.
1-[(3′S,5′R,6′R)-3′-O-Trimethylsilyl-5′-O-(tert-butyldimethyl-
silyl)-6′-[(ethoxycarbonyl)methyl]-2′-deoxy-2′-iodo-3′,5′-etha-
no-5′,6′-methano-β-D-ribofuranosyl]thymine (6). To a mixture
of thymine (530 mg, 4.2 mmol) in dry CH2Cl2 (6 mL) was added BSA
(1.53 mL, 6.3 mmol) at rt. It was left for 1 h until a fine suspension
had formed. A solution of 5 (668 mg, 1.4 mmol) in dry CH2Cl2 (6
mL) was then slowly added. After being cooled to 0 °C, N-
(1S,2R,4R,6S,8S)-2-[(tert-Butyldimethylsilyl)oxy]-4-
[(ethoxycarbonyl)methyl]-6-hydroxy-8-methoxy-9-
oxytricyclo[4.3.01,6.02,4]nonane (3α) and (1S,2S,4S,6S,8S)-2-
[(tert-butyldimethylsilyl)oxy]-4-[(ethoxycarbonyl)methyl]-6-
hydroxy-8-methoxy-9-oxytricyclo[4.3.01,6.02,4]nonane (4α). To
a heavily stirred solution of 2α (1.01 g, 2.68 mmol) in dry CH2Cl2 (45
mL) was slowly added diethylzinc (1 M in hexane, 16.1 mL, 16.1
mmol) at 5−10 °C. After fuming had ceased, diiodomethane (2.15
mL, 2.68 mmol) was added dropwise and the mixture allowed to reach
rt. After being stirred overnight, the reaction mixture was diluted with
EtOAc (300 mL) and washed with satd NH4Cl (1 × 200 mL) and
10% Na2S2O3 (1 × 200 mL). The aqueous phases were extracted with
EtOAc (2 × 400 mL) and the combined organic phases dried over
MgSO4, filtered, and evaporated. Purification of the resulting yellow oil
by CC (hexane/EtOAc 5:1 → 2:1) afforded 3α (0.31 g, 30%) and 4α
(0.65 g, 62%) both as colorless oils.
1
Data for 3α: Rf = 0.58 (hexane/EtOAc 3:2); H NMR (400 MHz,
CDCl3) δ 5.07 (dd, J = 5.3, 1.6 Hz, 1H, H−C(8)), 4.15−4.09 (m, 3H,
4573
dx.doi.org/10.1021/jo300648u | J. Org. Chem. 2012, 77, 4566−4577

The Journal of Organic Chemistry
Featured Article
iodosuccinimide (504 mg, 2.24 mmol) was added portionwise over 1.5
h. The mixture was stirred for an additional 30 min at 0 °C and for 1.5
h at rt. The clear, brown solution was diluted with EtOAc (50 mL) and
washed with satd NaHCO3 (2 × 50 mL) and satd Na2CO3 (1 × 50
mL). The aqueous phase was extracted with EtOAc (3 × 200 mL), and
the combined organic phases were dried over MgSO4, filtered, and
evaporated to give 1.2 g (>100%) of a viscous orange oil that was
subjected to the next reaction without further purification.
CD3OD) δ 174.4 (CO2Et), 166.7 (C(4)), 152.3 (C(2)), 138.4 (C(6)),
111.2 (C(5)), 92.8 (C(4′)), 88.0 (C(1′)), 85.2 (C(3′)), 66.6 (C(5′)),
61.7 (CH3CH2O), 49.2 (C(2′)), 47.8 (C(7′)), 37.2 (C(9′)), 31.2
(C(6′)), 22.4 (C(8′)), 14.7 (CH3CH2O), 12.6 (Me-C(5)); ESI+-
HRMS m/z calcd for C17H23O7N2 ([M + H]+) 367.1500, found
367.1507.
1-[(3′S,5′R,6′R)-5′-O-[(4,4′-Dimethoxytriphenyl)methyl]-6′-
[(ethoxycarbonyl)methyl]-2′-deoxy-3′,5′-ethano-5′,6′-metha-
no-β-D-ribofuranosyl]thymine (9). To a colorless solution of 8
(257 mg, 0.7 mmol) in dry pyridine (3.4 mL) was added DMTr-Cl
(548 mg, 1.6 mmol) in four equal portions over a time range of 2.5 h
at rt. The resulting orange solution was stirred for 8.5 h before
addition of another 0.5 eq. of DMTr-Cl. After reaction overnight the
mixture was diluted with EtOAc (30 mL) and subsequently washed
with satd NaHCO3 (2 × 25 mL). The aqueous phase was extracted
with EtOAc (3 × 50 mL). The combined organic phases were dried
over MgSO4, filtered and evaporated. The resulting orange foam was
purified by CC with EtOAc/hexane (1:1 → 4:1) containing 0.1% Et3N
to give DMTr-protected nucleoside 9 (366 mg, 78%) as a white foam.
1
Data for 6: Rf = 0.4 (EtOAc/hexane 2:1); H NMR (300 MHz,
CDCl3) δ 8.48 (br, 1H, H−N(3)), 7.69 (d, J = 1.2 Hz, 1H, H−C(6)),
6.35 (d, J = 5.9 Hz, 1H, H−C(1′)), 4.95 (d, J = 5.9 Hz, 1H, H−C(2′)),
4.30 (s, 1H, H−C(4′)), 4.11 (q, 2H, CH3CH2O), 2.56 (d, J = 16.9 Hz,
1H, H−C(7′)), 2.22 (d, J = 16.9 Hz, 1H, H−C(7′)), 1.96 (m, 2H, H−
C(9′)), 1.90 (d, J = 1.1 Hz, 3H, Me-C(5)), 1.24 (t, J = 7.1, 0.8 Hz, 3H,
CH3CH2O), 0.94 (d, J = 6.3 Hz, 1H, H−C(8′)), 0.85 (s, 10H,
(CH3)3C−Si, H−C(8′)), 0.19 (s, 9H, (CH3)3Si), 0.12 (d, J = 0.8 Hz,
6H, (CH3)2Si); 13C NMR (75 MHz, CDCl3) δ 172.4 (CO2Et), 164.0
(C(4)), 150.6 (C(2)), 136.3 (C(6)), 110.7 (C(5)), 93.9 (C(1′)), 91.5
(C(4′)), 88.8 (C(3′)), 66.3 (C(5′)), 60.9 (CH3CH2O), 42.8 (C(9′)),
40.4 (C(2′)), 36.4 (C(7′)), 29.9 (C(6′)), 25.9 ((CH3)3C−Si), 25.7
(C(8′)), 18.1 ((CH3)3C-Si), 14.5 (CH3CH2O), 12.8 (Me-C(5)), 2.3
((CH3)3Si), −3.5, −3.9 ((CH3)2Si); ESI+-HRMS m/z calcd for
C26H43O7N2INaSi2 ([M + Na]+) 701.1546, found 701.1557.
1
Data for 9: Rf = 0.58 (EtOAc/EtOH 95:5); H NMR (300 MHz,
CDCl3) δ 8.42 (br, 1H, H−N(3)), 8.21 (d, J = 1.0 Hz, 1H, H−C(6)),
7.44−7.41 (m, 2H, H-arom), 7.33 (dd, J = 8.9, 6.8 Hz, 1H, H-arom),
7.22−7.19 (m, 3H, H-arom), 6.77 (t, J = 8.9 Hz, 1H, H-arom), 5.83
(dd, 1H, H−C(1′)), 4.16 (m, 2H, CH3CH2O), 3.76, 3.75 (2s, 6H,
MeO), 2.86 (d, J = 16.6 Hz, 1H, H−C(9′)), 2.51 (d, J = 16.6 Hz, 1H,
H−C(9′)), 2.30 (dd, J = 11.4, 5.9 Hz, 2H, H−C(2′)), 2.19 (d, J = 14.2
Hz, 1H, H−C(7′)), 2.11 (s, 3H, Me-C(5)), 1.90−1.87 (m, 2H, H−
C(7′), O-H), 1.21 (t, J = 7.1 Hz, 1H, CH3CH2O), 0.98 (d, J = 5.8 Hz,
1H, H−C(8′)), 0.73 (d, J = 6.4 Hz, 1H, H−C(8′)); 13C NMR (75
MHz, CDCl3) δ 172.8 (CO2Et), 164.0 (C(4)), 159.1 (MeO-C-arom),
150.3 (C(2)), 146.3, 136.9, 136.8 (C-arom), 136.7 (C(6)), 131.7,
131.6, 129.3, 127.7, 127.3, 113.0, 112.9 (CH-arom), 110.5 (C(5)),
91.3 (C(4′)), 86.4 (C(1′)), 88.1, 85.0 (C(3′)), C(Ph)3), 68.7 (C(5′)),
60.7 (CH3CH2O), 55.5 (MeO−DMTr), 49.4 (C(2′)), 47.5 (C(7′)),
36.7 (C(9′)), 31.4 (C(6′)), 22.7 (C(8′)), 14.5 (CH3CH2O), 12.6 (Me-
C(5)); ESI+-HRMS m/z calcd for C38H40O9N2Na ([M + Na]+)
691.2626, found 691.2640.
1-[(3′S,5′R,6′R)-3′-O-Trimethylsilyl-5′-O-(tert-butyldimethyl-
silyl)-6′-[(ethoxycarbonyl)methyl]-2′-deoxy-3′,5′-ethano-5′,6′-
methano-β-D-ribofuranosyl]thymine (7). To an orange suspension
of 6 (1.17 g, 1.4 mmol) and Bu3SnH (0.56 mL, 2.1 mmol) in dry
toluene (12 mL) was added AIBN (115 mg, 0.7 mmol) at rt. The
mixture was heated to 95 °C for 1 h. The yellow solution was then
evaporated and the residue purified by CC with hexane/EtOAc (6:1
→ 1:1) + 1% Et3N. Nucleoside 7 (515 mg, 67% from 3) was isolated
as a white foam.
1
Data for 7: Rf = 0.65 (EtOAc/hexane 1:1); H NMR (300 MHz,
CDCl3) δ 8.58 (br, 1H, H−N(3)), 7.79 (d, J = 1.1 Hz, 1H, H−C(6)),
5.97 (dd, J = 5.3, 3.4 Hz, 1H, H−C(1′)), 4.26 (s, 1H, H−C(4′)), 4.11
(q, J = 7.1 Hz, 2H, CH3CH2O), 2.56 (m, 2H, H−C(2′)), 2.23−2.20
(m, 2H, H−C(9′)), 2.15 (d, J = 14.2 Hz, 1H, H−C(7′)), 1.91 (d, J =
0.9 Hz, 3H, Me-C(5)), 1.75 (d, J = 14.1 Hz, 1H, H−C(7′)), 1.23 (t, J =
7.1 Hz, 3H, CH3CH2O), 1.01 (d, J = 6.2 Hz, 1H, H−C(8′)), 0.90 (s,
9H, (CH3)3C−Si), 0.88−0.81 (m, 2H, H−C(8′)), 0.19 (s, 3H,
(CH3)2Si), 0.14 (s, 3H, (CH3)2Si), 0.10 (s, 9H, (CH3)3Si); 13C NMR
(100 MHz, CDCl3) δ 172.4 (CO2Et), 164.2 (C(4)), 150.2 (C(2)),
136.4 (C(6)), 109.6 (C(5)), 94.2 (C(4′)), 89.2 (C(1′)), 86.7 (C(3′)),
66.9 (C(5′)), 60.7 (CH3CH2O), 47.9 (C(2′)), 47.2 (C(7′)), 36.6
(C(9′)), 29.6 (C(6′)), 25.9 ((CH3)3C−Si), 21.7 (C(8′)), 18.1
((CH3)3C-Si), 14.5 (CH3CH2O), 12.8 (Me-C(5)), 2.1 ((CH3)3Si),
−3.6, −3.7 ((CH3)2Si); ESI+-HRMS m/z calcd for C26H45O7N2Si2
([M + H]+) 553.2760, found 553.2769.
1-[(3′S,5′R,6′R)-5′-O-[(4,4′-Dimethoxytriphenyl)methyl]-6′-
[(ethoxycarbonyl)methyl]-3′-O-(2-cyanoethoxy)-
diisopropylaminophosphanyl-2′-deoxy-3′,5′-ethano-5′,6′-
methano-β-d-ribofuranosyl]thymine (10). To a solution of 9 (40
mg, 0.06 mmol) and N-ethyldiisopropylamine (0.04 mL, 0.24 mmol)
in dry THF (0.4 mL) was carefully added 2-cyanoethyl N,N-
diisopropylchlorophosphoramidite (0.03 mL, 0.12 mmol) at rt. After
the slightly yellow solution was stirred for 1 h, the mixture was diluted
with EtOAc (20 mL) and washed with satd NaHCO3 (2 × 10 mL).
The aqueous phase was extracted with EtOAc (3 × 20 mL), and the
combined organic phases were dried over MgSO4. After filtration and
evaporation, the residual oil was purified by CC (EtOAc/Hexane 1:1
→ 4:1 + 0.1% Et3N) to give the title compound 10 (45 mg, 87%) as a
white foam.
1-[(3′S,5′R,6′R)-6′-[(Ethoxycarbonyl)methyl]-2′-deoxy-3′,5′-
ethano-5′,6′-methano-β-D-ribofuranosyl]thymine (8). To a
solution of 7 (510 mg, 0.92 mmol) in dry pyridine (6 mL) was
carefully added HF (70% in pyridine, 0.34 mL, 18.4 mmol) at 0 °C.
After 20 min of stirring, the reaction mixture was allowed to warm to
rt. After a total of 32 h, the yellow reaction mixture was diluted with
EtOAc (40 mL) and silica gel (4 g) was added. The mixture was
concentrated, coevaporated with toluene (2 × 10 mL) and purified by
CC (CH2Cl2/EtOH 95:5 to 9:1) to give 8 (290 mg, 86%) as white
crystals.
1
Data for 10: Rf = 0.57, 0.63 (EtOAc/hexane 4:1); H NMR (300
MHz, CDCl3) δ 8.24 (br, 1H, H−N(3)), 8.17 (d, J = 1.2 Hz, 1H),
7.45−7.42 (m, 2H, H-arom), 7.33 (dd, J = 8.7, 5.8 Hz, 4H, H-arom),
7.23−7.21 (m, 3H, H-arom), 6.77 (dd, J = 8.9, 7.7 Hz, 4H, H-arom),
5.83 (dd, J = 6.8, 4.4 Hz, 1H, H−C(1′)), 4.18−4.09 (m, 2H,
CH3CH2O), 3.78, 3.77, 3.76 (3s, 6H, MeO), 3.71−3.38 (m, 4H,
OCH2CH2CN, (Me2CH)2N), 2.89−2.82 (2d, 1H, H−C(9′)), 2.72
(dd, J = 14.3, 6.9 Hz, 1H, H−C(2′)), 2.58−2.51 (m, 3H,
OCH2CH2CN, H−C(9′)), 2.48−2.44 (m, 2H, H−C(7′), H−C(4′)),
2.32 (dd, J = 13.6, 4.9 Hz, 0.5H, H−C(2′)), 2.25 (dd, J = 14.1, 4.2 Hz,
0.5H, H−C(2′)), 2.10 (br, 3H, Me-C(5)), 2.05−1.99 (m, 1H,), 1.21,
1.20 (2t, J = 7.0 Hz, 3H, CH3CH2O), 1.10−1.05 (m, 12H,
(Me2CH)2N), 0.95 (m, 1H, H−C(8′)), 0.85 (d, J = 6.5 Hz, 0.5H,
H−C(8′)), 0.77 (d, J = 6.3 Hz, 0.5H, H−C(8′)); 13C NMR (75 MHz,
CDCl3) δ 172.7, 172.5 (CO2Et), 164.1 (C(4)), 159.0 (MeO-C-arom),
150.2 (C(2)), 146.3, 146.2, 136.9, 136.8 (C-arom), 136.74, 136.66
(C(6)), 131.7, 131.6, 129.3, 127.7, 127.3, 127.2 (CH-arom), 117.77,
117.7 (CN), 113.0, 112.9 (CH-arom), 110.32, 110.26 (C(5)), 91.8,
91.2 (C(4′)), 88.3, 88.2, 88.1 (C(3′)), C(Ph)3), 87.2, 86.9 (C(1′)),
1
Data for 8: Rf = 0.31 (CH2Cl2/EtOH 9:1); H NMR (400 MHz,
CD3OD) δ 7.94 (d, J = 1.2 Hz, 1H, H−C(6)), 6.16 (dd, J = 6.7, 5.7
Hz, 1H, H−C(1′)), 4.19−4.12 (m, 3H, CH3CH2O, H−C(4′)), 2.59 (d,
J = 16.4 Hz, 1H, H−C(9′)), 2.49 (dd, J = 13.9, 6.8 Hz, 1H, H−C(2′)),
2.43−2.37 (m, 2H, H−C(2′), H−C(9′)), 2.15 (dd, J = 13.9, 1.4 Hz,
1H, H−C(7′)), 1.94 (d, J = 1.1 Hz, 3H, Me-C(5)), 1.92 (d, 1H, H−
C(7′)), 1.26 (t, J = 7.1 Hz, 3H, CH3CH2O), 1.01 (d, J = 5.9 Hz, 1H,
H−C(8′)), 0.82 (dd, J = 5.9, 1.6 Hz, 1H, H−C(8′)). 1H NMR
difference NOE (400 MHz, CD3OD) δ 7.95 (H−C(6)) →6.17 (2.5%,
H−C(1′)), 2.41 (2.9%, H−C(2′), H−C(9′)), 2.16 (1.2%, H−C(7′)),
1.95 (5%, Me-C(5)); 6.17 (H−C(1′)) →7.94 (2.1%, H−C(6)), 4.14
(4%, H−C(4′)), 2.49 (5.9%, H−C(2′)); 13C NMR (100 MHz,
4574
dx.doi.org/10.1021/jo300648u | J. Org. Chem. 2012, 77, 4566−4577

The Journal of Organic Chemistry
Featured Article
68.5 (C(5′)), 60.7 (CH3CH2O), 58.1, 57.9 (JC,P = 15.5 Hz,
OCH2CH2CN), 55.43, 55.40 (MeO−DMTr), 47.1, 47.0 (C(2′)),
45.2, 45.1 (C(7′)), 43.6, 43.5, 43.3 (JC,P = 12.7 Hz, (Me2CH)2N), 36.8,
36.7 (C(9′)), 31.33, 31.26, 24.7, 24.6, 24.5, 24.4 ((Me2CH)2N), 22.3
(C(8′)), 20.5, 20.4 (JC,P = 8.1 Hz, OCH2CH2CN), 14.5 (CH3CH2O),
12.7 (Me-C(5)). 31P NMR (122 MHz, CDCl3) δ 144.0, 142.3; ESI+-
HRMS m/z calcd for C47H58O10N4P ([M + H]+) 869.3885, found
869.3896.
being stirred overnight, the mixture was heated to 45 °C for 4 h to
complete conversion. The resulting turbid solution was diluted with
EtOAc (20 mL) and the product adsorbed on silica gel (3 g).
Evaporation followed by purification of the adsorbed material by CC
(CH2Cl2/EtOH 9:1) afforded 13 (192 mg, 65% over two steps) as a
white foam.
1
Data for 13: Rf = 0.60 (CH2Cl2/EtOH 9:1); H NMR (300 MHz,
MeOD) δ 7.93 (d, J = 1.1 Hz, 1H, H−C(6)), 6.15 (dd, J = 6.6, 5.7 Hz,
1H, H−C(1′)), 4.14 (s, 1H, H−C(4′)), 4.11 (td, J = 6.6, 1.6 Hz, 2H,
CH2O), 2.59 (d, J = 16.3 Hz, 1H, H−C(9′)), 2.49 (dd, J = 13.9, 6.8
Hz, 1H, H−C(2′)), 2.44−2.35 (m, 2H, H−C(2′), H−C(9′)), 2.13 (dd,
J = 13.9, 1.1 Hz, 1H, H−C(7′)), 1.94 (d, J = 0.9 Hz, 3H, Me-C(5)),
1.94−1.89 (m, 1H, H−C(7′)), 1.70−1.59 (m, 2H, CH2CH2O), 1.33−
1.29 (m, 26H, CH2-alk), 1.01 (d, J = 5.9 Hz, 1H, H−C(8′)), 0.90 (t, J
1-[(3′S,5′R,6′R)-5′-O-(tert-Butyldimethylsilyl)-6′-(carboxy-
methyl)-2′-deoxy-3′,5′-ethano-5′,6′-methano-β-D -
ribofuranosyl]thymine (11). To a solution of 7 (520 mg, 0.94
mmol) in EtOH (2 mL) was added 4 N KOH (1.06 mL, 4.23 mmol)
at rt. The resulting yellow solution was stirred for 2.5 h and was then
diluted with H2O (20 mL). Neutralization was performed by 1 M
KHSO4 (until pH 3 was reached, 12 mL). Extraction of the aqueous
phase with CH2Cl2 (4 × 40 mL), followed by drying of the organic
phase over MgSO4, filtration, and evaporation yielded 11 (391 mg,
92%) as a yellow foam that was used without further purification in the
next step.
= 6.7 Hz, 3H, CH3-alk), 0.82 (dd, J = 5.9, 1.4 Hz, 1H, H−C(8′)); 13
C
NMR (101 MHz, MeOD) δ 174.5 (CO2R), 166.8 (C(4)), 152.4
(C(2)), 138.4 (C(6)), 111.2 (C(5)), 92.8 (C(4′)), 88.0 (C(1′)), 85.1
(C(3′)), 66.7 (C(5′)), 65.9 (CH2O), 49.8, 49.6, 49.4, 49.3, 49.2, 49.0,
48.7, 48.5 (CHD2OD, C(2′)), 47.8 (C(7′)), 37.2 (C(9′)), 33.2
(C(6′)), 31.2, 31.0, 30.91, 30.89, 30.87, 30.81, 30.78, 30.6, 30.5 (CH2-
alk), 29.9 (CH2CH2O), 27.2, 23.9 (CH2-alk), 22.4 (C(8′)), 14.6 (CH3-
alk), 12.7 (Me-C(5)); ESI+-HRMS m/z calcd for C31H51O7N2 ([M +
H]+) 563.3691, found 563.3678.
1
Data for 11: Rf = 0.18 (CH2Cl2/EtOH 9:1); H NMR (300 MHz,
MeOD) δ 7.91 (d, J = 1.0 Hz, 1H, H−C(6)), 6.06 (dd, J = 6.4, 3.4 Hz,
1H, H−C(1′)), 4.25 (s, 1H, H−C(4′)), 2.60 (dd, J = 13.9, 3.4 Hz, 1H,
H−C(2′)), 2.49 (dd, J = 13.9, 6.5 Hz, 1H, H−C(2′)), 2.35 (q, J = 15.9
Hz, 2H, H−C(9′)), 1.96 (s, 2H, H−C(7′)), 1.90 (d, J = 0.9 Hz, 3H,
Me-C(5)), 1.02 (d, J = 6.2 Hz, 1H, H−C(8′)), 0.94 (s, 9H, (CH3)3C−
Si), 0.91 (d, J = 4.4 Hz, 1H, H−C(8′)), 0.23 (s, 3H, (CH3)2Si), 0.19 (s,
3H, (CH3)2Si); 13C NMR (75 MHz, MeOD) δ 176.0 (CO2H), 166.8
(C(4)), 152.3 (C(2)), 138.2 (C(6)), 110.5 (C(5)), 94.1 (C(4′)), 89.8
(C(1′)), 86.3 (C(3′)), 68.6 (C(5′)), 48.8 (C(2′)), 47.7 (C(7′)), 37.5
(C(9′)), 31.1 (C(6′)), 26.4 ((CH3)3C−Si), 23.4 (C(8′)), 19.0
((CH3)3C-Si), 13.0 (Me-C(5)), −3.4, −3.6 ((CH3)2Si); ESI+-HRMS
m/z calcd for C21H33O7N2Si ([M + H]+) 453.2052, found 453.2059.
1-[(3′S,5′R,6′R)-5′-O-(tert-Butyldimethylsilyl)-6′-
[(hexadecoxycarbonyl)methyl]-2′-deoxy-3′,5′-ethano-5′,6′-
methano-β-D-ribofuranosyl]thymine (12). To a slightly turbid
solution of 11 (50 mg, 0.11 mmol) in dry CH2Cl2 (1.5 mL) was added
1-hexadecanol (32 mg, 0.13 mmol) followed by DMAP (7 mg, 0.056
mmol) at rt. The resulting mixture was cooled in an ice bath, and a
solution of EDC·HCl (32 mg, 0.17 mmol) in dry CH2Cl2 (1.5 mL)
was added dropwise. The mixture was allowed to warm to rt and was
stirred for another 1 h. The clear solution was then diluted with EtOAc
(25 mL) and washed with satd NaHCO3 (2 × 15 mL) and H2O (1 ×
15 mL). The aqueous phase was extracted with EtOAc (3 × 45 mL),
and the combined organic phases were dried over MgSO4, filtered, and
concentrated. The crude material was used without further purification
in the next step. For characterization, a sample of the crude material
was purified by column chromatography (5% EtOH in CH2Cl2).
1-[(3′S,5′R,6′R)-5′-O-[(4,4′-Dimethoxytriphenyl)methyl]-6′-
[(hexadecoxycarbonyl)methyl]-2′-deoxy-3′,5′-ethano-5′,6′-
methano-β-D-ribofuranosyl]thymine (14). To a colorless solution
of 13 (190 mg, 0.34 mmol) in dry pyridine (2.5 mL) was added
DMTr-Cl (343 mg, 1.01 mmol) in four equal portions over 1.5 h. The
resulting orange solution was stirred for 22 h before being diluted with
EtOAc (30 mL). The organic phase was washed with satd NaHCO3 (2
× 20 mL) and the aqueous phase extracted with EtOAc (3 × 50 mL).
The combined organic phases were dried over MgSO4, filtered, and
concentrated. The crude orange foam was purified by CC (hexane/
EtOAc 1:1 + 0.1% Et3N, then hexane/EtOAc 1:4) to yield 14 (200
mg, 69%) as a slightly yellow foam.
1
Data for 14: Rf = 0.48 (EtOAc/hexane 4:1); H NMR (300 MHz,
CDCl3) δ 8.89 (s, 1H, H−N(3)), 8.24 (d, J = 0.8 Hz, 1H, H−C(6)),
7.42 (d, J = 9.1 Hz, 2H, H-arom), 7.32 (t, J = 8.3 Hz, 4H, H-arom),
7.23−7.12 (m, 3H, H-arom), 6.79−6.70 (m, 4H, H-arom), 5.84 (t, J =
6.1 Hz, 1H, H−C(1′)), 4.13−4.03 (m, 2H, CH2O), 3.73 (s, 3H, MeO),
3.71 (s, 3H, MeO), 2.88 (d, J = 16.7 Hz, 1H, H−C(9′)), 2.52 (d, J =
16.7 Hz, 1H, H−C(9′)), 2.46 (s, 1H, OH), 2.33 (dd, J = 14.0, 6.7 Hz,
1H, H−C(2′)), 2.29−2.18 (m, 3H, H−C(7′), H−C(4′)), 2.12 (s, 3H,
Me-C(5)), 1.90 (d, J = 14.0 Hz, 1H, H−C(7′)), 1.61−1.50 (m, 2H,
CH2CH2O), 1.29−1.15 (m, 26H, CH2-alk), 0.95 (d, J = 6.1 Hz, 1H,
H−C(8′)), 0.86 (t, J = 6.7 Hz, 3H, CH3-alk), 0.73 (d, J = 6.3 Hz, 1H,
H−C(8′)); 13C NMR (101 MHz, CDCl3) δ 173.0 (CO2R), 164.2
(C(4)), 159.1 (MeO-C-arom), 150.5 (C(2)), 146.4, 136.9 (C-arom),
136.7 (C(6)), 131.7, 131.6, 129.2, 127.6, 127.1, 112.9, 112.8 (CH-
arom), 110.8 (C(5)), 91.0 (C(4′)), 87.9 (C(3′)), 86.0 (C(1′)), 85.2
(C(Ph)3), 68.5 (C(5′)), 64.9 (CH2O), 55.4 (MeO), 49.5 (C(2′)), 47.4
(C(7′)), 36.6 (C(9′)), 32.1 (C(6′)), 31.3, 29.91, 29.87, 29.77, 29.75,
29.6, 29.4 (CH2-alk), 28.8 (CH2CH2O), 26.2, 22.9 (CH2-alk), 22.8
(C(8′)), 14.3 (CH3-alk), 12.6 (Me-C(5)); ESI+-HRMS m/z calcd for
C52H68O9N2Na ([M + Na]+) 887.4817, found 887.4814.
1-[(3′S,5′R,6′R)-5′-O-[(4,4′-Dimethoxytriphenyl)methyl]-6′-
[(hexadecoxycarbonyl)methyl]-3′-O-[(2-cyanoethoxy)-
diisopropylaminophosphanyl]-2′-deoxy-3′,5′-ethano-5′,6′-
methano-β-D-ribofuranosyl]thymine (15). To a colorless solution
of N-ethyldiisopropylamine (0.16 mL, 0.93 mmol, 4 equiv) and
nucleoside 15 (200 mg, 0.23 mmol) in 2.5 mL of dry THF (2.5 mL)
was added 2-cyanoethyl N,N-diisopropylchlorophosphoramidite (0.1
mL, 0.45 mmol) at rt. The resulting yellow solution was stirred for 1 h
and gradually turned into a suspension. The reaction mixture was then
diluted with EtOAc (40 mL) and washed with satd NaHCO3 (2 × 25
mL). The aqueous phase was extracted with EtOAc (3 × 50 mL), and
the combined organic phases were dried over MgSO4, filtered, and
evaporated. The crude yellow oil was purified by CC (hexane/EtOAc
1:1 +0.1% Et3N) to give 15 (220 mg, 89%, mixture of isomers) as a
white foam.
1
Data for 12: Rf = 0.51 (CH2Cl2/EtOH 9:1); H NMR (300 MHz,
CDCl3) δ 8.60 (s, 1H, H−N(3)), 7.82 (d, J = 1.1 Hz, 1H, H−C(6′)),
6.03 (dd, J = 6.1, 3.1 Hz, 1H, H−C(1′)), 4.27 (s, 1H, H−C(4′)), 4.03
(t, J = 6.8 Hz, 2H, CH2O), 2.63 (dd, J = 14.0, 3.2 Hz, 1H, H−C(2′)),
2.56 (dd, J = 14.1, 6.2 Hz, 1H, H−C(2′)), 2.30 (q, J = 16.1 Hz, 2H,
H−C(9′)), 2.13 (s, 1H, OH), 2.04−1.90 (m, 2H, H−C(7′)), 1.91 (d, J
= 0.8 Hz, 3H, Me-C(5)), 1.63−1.52 (m, 2H, CH2CH2O), 1.28−1.23
(m, 29H, CH2-alk), 1.00 (d, J = 6.4 Hz, 1H, H−C(8′)), 0.89 (s, 10H,
(CH3)3C−Si, H−C(8′)), 0.88−0.83 (m, 3H, CH3-alk), 0.18 (s, 3H,
(CH3)2Si), 0.13 (s, 3H, (CH3)2Si); 13C NMR (101 MHz, CDCl3) δ
172.6 (CO2R), 164.2 (C(4)), 150.3 (C(2)), 136.4 (C(6)), 109.9
(C(5)), 92.9 (C(4′)), 88.8 (C(1′)), 85.4 (C(3′)), 67.2 (C(5′)), 65.1
(CH2O), 48.4 (C(2′)), 47.7 (C(7′)), 36.5 (C(9′)), 32.1 (C(6′)), 29.91,
29.87, 29.84, 29.81, 29.75, 29.6, 29.5 (CH2-alk), 28.8 (CH2CH2O),
26.1 (CH2-alk), 25.9 ((CH3)3C−Si), 22.9 (CH2-alk), 22.3 (C(8′)),
18.1 ((CH3)C-Si), 14.3 (CH3-alk), 12.8 (Me-C(5)), −3.7, −3.6
((CH3)2Si); ESI+-HRMS m/z calcd for C37H65O7N2Si ([M + H]+)
677.4556, found 677.4556.
1-[(3′S,5′R,6′R)-6′-[(Hexadecoxycarbonyl)methyl]-2′-deoxy-
3′,5′-ethano-5′,6′-methano-β-D-ribofuranosyl]thymine (13).
HF·pyridine (0.19 mL, 10.4 mmol) was slowly added at 0 °C to a
solution of 12 (374 mg, 0.52 mmol) in pyridine (4 mL), and the
mixture was left for 20 min before it was allowed to warm to rt. After
4575
dx.doi.org/10.1021/jo300648u | J. Org. Chem. 2012, 77, 4566−4577

The Journal of Organic Chemistry
Featured Article
1
Data for 15: Rf = 0.77, 0.70 (EtOAc/hexane 7:3); H NMR (300
MHz, CDCl3) δ 8.31 (br, 1H, H-N(3)), 8.21−8.15 (m, 1H, H−C(6)),
7.47−7.38 (m, 2H, H-arom), 7.33 (dd, J = 8.8, 6.3 Hz, 4H, H-arom),
7.23−7.18 (m, 3H, H-arom), 6.76 (dd, J = 8.9, 7.8 Hz, 4H, H-arom),
5.83 (dd, J = 6.7, 4.4 Hz, 1H, H−C(1′)), 4.10−4.02 (m, 2H, CH2O),
3.79−3.75 (m, 6H, MeO), 3.71−3.37 (m, 4H, OCH2CH2CN,
(Me2CH)2N), 2.86 (dd, J = 16.6, 3.2 Hz, 1H, H−C(9′)), 2.76−2.67
(m, 1H, H−C(2′)), 2.59−2.41 (m, 4.5H, OCH2CH2CN, H−C(9′),
H−C(4′), H−C(7′)), 2.36−2.20 (m, 1.4H, H−C(7′), H−C(2′)),
2.12−2.08 (m, 3H, Me-C(5)), 2.07−2.00 (m, 1H, H−C(7′), H−
C(2′)), 1.58−1.49 (m, 2H, CH2CH2O)), 1.32−1.16 (m, 26H, CH2-
alk), 1.12−1.03 (m, 12H, (Me2CH)2N), 0.94 (dd, J = 10.0, 6.5 Hz, 1H,
H−C(8′)), 0.85 (t, J = 6.7 Hz, 3H, CH3-alk), 0.76 (d, J = 6.3 Hz, 1H,
H−C(8′)); 13C NMR (101 MHz, CDCl3) δ 172.9, 172.7 (CO2R),
164.1 (C(4)), 159.04, 159.01 (MeO-C-arom), 150.2 (C(2)), 146.3,
146.2 (C-arom), 136.9, 136.8, 136.7 (C-arom, C(6)), 131.7, 131.6,
129.3, 127.7, 127.24, 127.17 (CH-arom), 117.8, 117.7 (CN), 112.94,
112.87 (CH-arom), 110.4, 110.3 (C(5)), 91.71, 91.67, 91.2, 91.1
(C(4′)), 88.5, 88.4, 88.3, 88.2, 88.1 (C(3′), C(Ph)3), 87.1, 86.8
(C(1′)), 68.4 (C(5′)), 65.0 (CH2O), 58.1, 58.0, 57.9, 57.8 (JC,P = 18.8
Hz, 19.5 Hz, OCH2CH2CN), 55.41, 55.38 (MeO), 47.2, 47.1 (C(2′)),
45.3, 45.1 (C(7′)), 43.6, 43.47, 43.45, 43.3 (JC,P = 12.9 Hz, 12.7 Hz,
(Me2CH)2N), 36.7, 36.6 (C(9′)), 32.1 (C(6′)), 31.4, 31.3, 30.0, 29.9,
29.8, 29.7, 29.6, 29.5 (CH2-alk), 28.8 (CH2CH2O), 26.2 (CH2-alk),
24.7, 24.6, 24.54, 24.47, 24.4 ((Me2CH)2N), 22.9 (CH2-alk), 22.24,
22.20 (C(8′)), 20.51, 20.49, 20.44, 20.41 (JC,P = 8.0 Hz, 7.3 Hz,
OCH2CH2CN), 14.33(CH3-alk), 12.7 (Me-C(5)). 31P NMR (162
MHz, CDCl3) δ 143.7, 142.2; ESI+-HRMS m/z calcd for
C61H86O10N4P ([M + H]+) 1065.6076, found 1065.6113.
Oligonucleotide Synthesis. Syntheses of oligonucleotides were
performed on the 1.3 μmol scale of a DNA synthesizer using standard
solid-phase phosphoramidite chemistry. Oligomers were assembled
using the manufacturer’s protocols on nucleoside preloaded CPG or
fluorescein-labeled (FAM) solid supports (Roche Diagnostics).
Natural phosphoramidites (dT, dC4Bz, dA6Bz,dG2dmf, Vivotide) were
coupled as a 0.1 M solution in CH3CN, tricyclophosphoramidites (tc-
T, tc-C4Bz, tc-A6Bz, tc-G2dmf) as 0.15 M solution in CH3CN (0.15 M in
dichloroethane for tc-A6Bz phosphoramidite). The coupling step was
90 s for natural phosphoramidites. An extended coupling time of 12
min for tricyclonucleosides was necessary to achieve average coupling
efficiencies of 94% for tcee-T 10 and 97% for tchd-T 15 (trityl assay).
As a coupling reagent, 5-(ethylthio)-1H-tetrazole (0.25 M in CH3CN)
was used. Capping was performed with a solution of DMAP (0.5 M in
CH3CN, Cap A) and a solution of 25% Ac2O and 12.5% sym-collidine
in CH3CN (Cap B). Oxidation was performed with a solution of 20
mM I2 and 0.45 M sym-collidine in 2.1:1 CH3CN/H2O. Detritylation
was carried out using a solution of 3% dichloroacetic acid in
dichloroethane.
mL/min flow rate and detection at 260 nm. Purified oligonucleotides
were desalted over Sep-Pak cartridges, quantified at 260 nm with a
Nanodrop spectrophotometer, and analyzed by ESI− mass spectrom-
etry. Oligonucleotides were then stored at −18 °C.
UV−Melting Curves. UV−melting curves were recorded on a
Varian Cary Bio100 UV/vis spectrophotometer. Absorbances were
monitored at 260 nm, and the heating rate was set to 0.5 °C/min. A
cooling−heating−cooling cycle in the temperature range 15−80 °C
was applied. Tm values were obtained from the maximum of the first
derivative curves and reported as the average of at least three ramps
( 1 °C error). To avoid evaporation of the solution, the sample
solutions were covered with a layer of dimethylpolysiloxane. All
measurements were carried out in NaCl (150 mM), Na2HPO4 (10
mM) buffer at pH 7.0 with a duplex concentration of 2 μM.
Fluorescence Spectroscopy. The solutions were prepared in
NaCl (150 mM), Na2HPO4 (10 mM) buffer at pH 7.0 with a duplex
concentration of 2 μM. Measurements were performed on a Varian
Cary Eclipse fluorescence spectrophotomer in quartz cuvettes with a
path length of 1 cm. The photomultiplier voltage was set to 500 V, and
the solutions were excited at 480 nm. The emission spectra were
recorded at 20 °C from 490 to 600 nm.
Cell Culture and Transfection. HeLa and HEK 293T cells were
grown at 37 °C in Dulbecco’s Modified Eagle’s Medium (DMEM)
supplemented with 10% (v/v) fetal calf serum (FCS), 100 units/mL of
penicillin and 100 μg/mL of streptomycin (P/S). Cells were split 1:10
every 2 or 3 days. For cellular uptake experiments, 1 × 105 HeLa and 2
× 105 HEK 293T cells were seeded in duplicate in six-well plates
containing coverslips 24 h before treatment with oligonucleotides.
Then, the medium was replaced by a 1 mL solution of fluorescein-
labeled oligonucleotide (10 μM final concentration) in DMEM + /+
(FCS, P/S). The medium was removed after 48 h at 37 °C and the
cells were washed with 2 × 1 mL PBS. They were finally suspended in
1 mL fresh DMEM +/+.
Cell Imaging. Fixation of the cells was carried out using a 1 mL
solution of paraformaldehyde (3.7% PFA in PBS) for 10 min, after
which the cells were washed with 2 × 1 mL PBS. After
permeabilization of the cell membrane with 0.2% Triton x-100 for
10 min and washing with 2 × 1 mL PBS the cells were mounted on
coverslips and treated with a few drops of polyvinyl alcohol (Mowiol)
containing nuclear stain 4′,6′-diamidino-2-phenylindole (DAPI) and
analyzed on a fluorescence microscope (Leica DMI6000 B, Leica
Microsystems with Leica Application Suite software).
ASSOCIATED CONTENT
■
S
* Supporting Information
1H and 13C NMR spectra of all compounds, 31P NMR spectra
of phosphoramidites 10 and 15. 1H−1H COSY and 2D
1
NOESY spectra of compounds 3α and 4α, H NMR NOE
Oligonucleotide Deprotection and Purification. Deprotection
of the oligonucleotides containing amide-tc-T units (ON 1, 3, 5, 7−9),
and detachment from the solid support was carried out using standard
conditions (concd aq NH3 for 16 h at 55 °C). To maintain the ethyl
and hexadecyl ester functions, ON 2, 4, 6, 12, and 13 were treated
with a 1:3 NH3/EtOH solution at 40 °C for 24 h. ON 11 was
deprotected in 25% benzylamine (EtOH/H2O 1:2) at 65 °C for 8 h.
In all cases, the solutions were centrifuged after deprotection, the
supernatants were removed and the remaining beads washed with 0.25
mL of H2O. The combined supernatants were then concentrated to
dryness. To perform hydrolysis of the ethyl ester (ON 10), the solid
phase was treated with a 0.1 M KOH solution. After an overnight
shaking at 55 °C, the solution was neutralized with 1 M HCl and
desalted (see below). Crude oligomers were purified by ion-exchange
HPLC. As mobile phases, the following buffers were prepared: (A) 25
mM Trizma (2-amino-2-hydroxymethyl-1,3-propanediol) in H2O, pH
8.0; (B) 25 mM Trizma, 1.25 M NaCl in H2O, pH 8.0. For oligos
containing tchd-T building blocks, ON 6: (A) 25 mM Trizma in H2O,
pH 8.0; (B) 25 mM Trizma, 1 M NaClO4 in H2O, pH 8.0. ON 13:
(A) 25 mM Trizma in H2O/ACN 4:1, pH 8.0; (B) 25 mM Trizma, 1
M NaClO4 in H2O/ACN 4:1, pH 8.0. Linear gradients of B in A were
used (typically 0 to 40% or 0 to 50% B in A over 30 min), with a 1
spectrum and crystal data of compound 8. Characterization of
oligonucleotides. Representative melting curves of ON 2, 4, 6,
and 12−14. HPLC trace and mass spectrum of ON12 as a
representative example. This material is available free of charge
via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*Phone: +41-31-631-4355. Fax: +41-31-631-3422. E-mail:
leumann@ioc.unibe.ch.
■
ACKNOWLEDGMENTS
Financial support of this work by the Swiss National Science
Foundation (Grant No. 200020-115913) and by the Associa-
■
́
tion Monegasque contre les Myopathies (AMM) is gratefully
acknowledged. We also thank the group of Chemical
Crystallography of the University of Bern (PD Dr. P. Macchi)
for the X-ray structure solution and the Swiss National Science
Foundation (R’equip project 206021_128724) for cofunding
4576
dx.doi.org/10.1021/jo300648u | J. Org. Chem. 2012, 77, 4566−4577

The Journal of Organic Chemistry
Featured Article
(31) Lavergne, T.; Baraguey, C.; Dupouy, C.; Parey, N.; Wuensche,
W.; Sczakiel, G.; Vasseur, J.-J.; Debart, F. J. Org. Chem. 2011, 76,
5719−5731.
the single-crystal X-ray diffractometer at the department of
Chemistry and Biochemistry of the University of Bern. We
thank Prof. Oliver Muhlemann for providing access to the cell
̈
(32) Parey, N.; Baraguey, C.; Vasseur, J.-J.; Debart, F. Org. Lett. 2006,
8, 3869−3872.
culture facility and equipment and Karin Schranz for technical
assistance.
(33) Luisier, S.; Leumann, C. J. Heterocycles 2010, 82, 775−790.
(34) Furukawa, J.; Kawabata, N.; Nishimura, J. Tetrahedron 1968, 24,
53−58.
REFERENCES
■
(35) Staroscik, J. A.; Rickborn, B. J. Org. Chem. 1972, 37, 738−740.
(36) Hoveyda, A. H.; Evans, D. A.; Fu, G. C. Chem. Rev. 1993, 93,
1307−1370.
(1) Zamecnik, P. C.; Stephenson, M. L. Proc. Natl. Acad. Sci. U.S.A.
1978, 75, 280−284.
(2) Watts, J. K.; Corey, D. R. J. Pathol. 2012, 226, 365−379.
(3) Kole, R.; Krainer, A. R.; Altman, S. Nat. Rev. Drug Discov. 2012,
11, 125−140.
(37) Luisier, S.; Silhar, P.; Leumann, C. J. Nucleic Acids Symp. Ser.
2008, 52, 581−582.
(38) Pallan, P. S.; Ittig, D.; Heroux, A.; Wawrzak, Z.; Leumann, C. J.;
Egli, M. Chem. Commun. 2008, 883−885.
(4) Bennett, C. F.; Swayze, E. E. Annu. Rev. Pharmacol. Toxicol. 2010,
50, 259−293.
(39) Saenger, W. Principles of Nucleic Acid Structure; Springer-Verlag:
New York, 1984.
(5) Kurreck, J. Angew. Chem., Int. Ed. 2009, 48, 1378−1398.
(6) Juliano, R.; Bauman, J.; Kang, H.; Ming, X. Mol. Pharmaceutics
2009, 6, 686−695.
(40) Cuppoletti, A.; Cho, Y.; Park, J.-S.; Strassler, C.; Kool, E. T.
̈
Bioconjugate Chem. 2005, 16, 528−534.
(7) Herdewijn, P. Biochim. Biophys. Acta 1999, 1489, 167−179.
(8) Hendrix, C.; Rosemeyer, H.; Verheggen, I.; Van Aerschot, A.;
Seela, F.; Herdewijn, P. Chem.Eur. J. 1997, 3, 110−120.
(9) Koshkin, A. A.; Singh, S. K.; Nielsen, P.; Rajwanshi, V. K.; Kumar,
R.; Meldgaard, M.; Olsen, C. E.; Wengel, J. Tetrahedron 1998, 54,
3607−3630.
(41) Ming, X.; Sato, K.; Juliano, R. L. J. Controlled Release 2011, 153,
83−92.
(10) Obika, S.; Nanbu, D.; Hari, Y.; Morio, K.-i.; In, Y.; Ishida, T.;
Imanishi, T. Tetrahedron Lett. 1997, 38, 8735−8738.
(11) Tarkoy, M.; Bolli, M.; Schweizer, B.; Leumann, C. Helv. Chim.
̈
Acta 1993, 76, 481−510.
(12) Steffens, R.; Leumann, C. Helv. Chim. Acta 1997, 80, 2426−
2439.
(13) Renneberg, D.; Leumann, C. J. J. Am. Chem. Soc. 2002, 124,
5993−6002.
(14) Ittig, D.; Gerber, A.-B.; Leumann, C. J. Nucleic Acids Res. 2011,
39, 373−380.
(15) Renneberg, D.; Bouliong, E.; Reber, U.; Schumperli, D.;
̈
Leumann, C. J. Nucleic Acids Res. 2002, 30, 2751−2757.
(16) Ittig, D.; Liu, S.; Renneberg, D.; Schumperli, D.; Leumann, C. J.
̈
Nucleic Acids Res. 2004, 32, 346−353.
(17) Ittig, D.; Luisier, S.; Weiler, J.; Schumperli, D.; Leumann, C.
̈
Artif. DNA PNA XNA 2010, 1, 9−16.
(18) Lv, H.; Zhang, S.; Wang, B.; Cui, S.; Yan, J. J. Controlled Release
2006, 114, 100−109.
(19) Luo, D.; Saltzman, W. M. Nat. Biotechnol. 2000, 18, 33−37.
(20) Hahn, P.; Scanlan, E. Top. Curr. Chem. 2010, 296, 1−13.
(21) Rosi, N. L.; Giljohann, D. A.; Thaxton, C. S.; Lytton-Jean, A. K.
R.; Han, M. S.; Mirkin, C. A. Science 2006, 312, 1027−1030.
(22) Morris, M. C.; Deshayes, S.; Heitz, F.; Divita, G. Biol. Cell 2008,
100, 201−217.
(23) Debart, F.; Abes, S.; Deglane, G.; Moulton, H. M.; Clair, P.;
Gait, M. J.; Vasseur, J. J.; Lebleu, B. Curr. Topics Med. Chem. 2007, 7,
727−737.
(24) Nothisen, M.; Kotera, M.; Voirin, E.; Remy, J.-S.; Behr, J.-P. J.
Am. Chem. Soc. 2009, 131, 17730−17731.
(25) Lietard, J.; Ittig, D.; Leumann, C. J. Bioorg. Med. Chem. 2011, 19,
5869−5875.
(26) Wolfrum, C.; Shi, S.; Jayaprakash, K. N.; Jayaraman, M.; Wang,
G.; Pandey, R. K.; Rajeev, K. G.; Nakayama, T.; Charrise, K.; Ndungo,
E. M.; Zimmermann, T.; Koteliansky, V.; Manoharan, M.; Stoffel, M.
Nat. Biotechnol. 2007, 25, 1149−1157.
(27) Krutzfeldt, J.; Rajewsky, N.; Braich, R.; Rajeev, K. G.; Tuschl, T.;
Manoharan, M.; Stoffel, M. Nature 2005, 438, 685−689.
(28) Stein, C. A.; Hansen, J. B.; Lai, J.; Wu, S.; Voskresenskiy, A.;
Høg, A.; Worm, J.; Hedtjarn, M.; Souleimanian, N.; Miller, P.; Soifer,
̈
H. S.; Castanotto, D.; Benimetskaya, L.; Ørum, H.; Koch, T. Nucleic
Acids Res. 2010, 38, e3−e3.
(29) Tosquellas, G.; Alvarez, K.; Dell’Aquila, C.; Morvan, F.; Vasseur,
J.-J.; Imbach, J.-L.; Rayner, B. Nucleic Acids Res. 1998, 26, 2069−2074.
(30) Bologna, J. C.; Vives, E.; Imbach, J. L.; Morvan, F. Antisense
Nucleic Acid Drug Dev. 2002, 12, 33−41.
4577
dx.doi.org/10.1021/jo300648u | J. Org. Chem. 2012, 77, 4566−4577