Bridged nucleic acid conjugates at 6′-thiol: Synthesis, hybridization properties and nuclease resistances

Article abstract of DOI:10.1039/c1ob05469d

The bridged nucleic acid (BNA) containing a thiol at the 6′-position in the bridged structure was synthesized from the disulfide-type BNA and conjugated with various functional molecules via the thioether or the disulfide linkage post-synthetically and ef

Full text of DOI:10.1039/c1ob05469d

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PAPER
Bridged nucleic acid conjugates at 6¢-thiol: synthesis, hybridization properties
and nuclease resistances†
Kazuto Mori, Tetsuya Kodama,* Takeshi Baba and Satoshi Obika*
Received 24th March 2011, Accepted 14th April 2011
DOI: 10.1039/c1ob05469d
The bridged nucleic acid (BNA) containing a thiol at the 6¢-position in the bridged structure was
synthesized from the disulfide-type BNA and conjugated with various functional molecules via the
thioether or the disulfide linkage post-synthetically and efficiently in solution phase. The
disulfide-linked conjugate was cleaved under reductive conditions derived from glutathione and an
oligonucleotide bearing a free thiol was released smoothly. Conjugated functional molecules had great
effects on duplex stability with the DNA complement. In contrast, the molecules little influenced the
stability with the RNA complement. Moreover, the oligonucleotides with functional groups at the
6¢-position had as high or higher resistances against 3¢-exonuclease than phosphorothioate
oligonucleotide (S-oligo).
has two major types of reaction, nucleophilic reaction and thiol–
disulfide exchange.8,10 As the disufide linkage is reversible, it is often
Introduction
used for DDS (Drug Delivery Systems).11 The disulfide linkage
is cleaved in the intracellular reducing environment and the ON
is selectively released into the cell.12 5¢- or 3¢-termini are mostly
selected as conjugation sites due to their easy accesibility.8 The
1¢-,13 2¢-14–16 and 4¢-positions17 of the sugar moiety, nucleobase,18
and internucleotidic phosphodiester bond19 are also used for con-
jugation. However, the three dimensional position of a functional
molecule varies depending on the conjugation site and this has an
important consequence for its effect.15,20
In this paper, we present the design and synthesis of a novel
BNA bearing a thiol group in the bridged structure, conjugation of
various molecules to the ON including the BNA, and evaluation of
their thermal stabilities and nuclease resistances. The C6¢-position
has not often been used as conjugation site21 and the effect of
conjugation at this position has not been investigated enough.
We report here the efficiency and the effect of conjugation at this
position. Post-synthetic conjugation (solution phase conjugation)
basically needs large equivalents of reagents because of the high
diluted conditions. In this study, ONs were conjugated via a thiol
group by a solution phase coupling approach because it is a
simple and rapid method. Owing to the bridged structure, the
three dimensional position of additional groups and molecules
will be highly controlled and ON conjugates will display functions
which are different from flexible analogs.
Selective inhibition of gene expression resulting from binding of
oligonucleotides (ONs) to the target sequences shows promise
as an attractive chemotherapy,1 but there have been few clinical
successes to date.2 This is mainly due to the fact that the natural
ONs do not have enough target specificity, resistance toward the
nucleases, and ability to penetrate the cell membrane.2 In order
to improve their properties, nucleic acids have been chemically
modified by a wide variety of approaches, for example, restriction
of sugar conformation to an appropriate form3,4 or conjugation
with functional groups and molecules.5 Numerous 2¢,4¢-bridged
nucleic acid (BNA)6/LNA7 analogues have been developed by
our group3 or other groups.4 Their sugar moieties are restricted
to North-type (N-Type) conformation by bridging between C2¢-
and C4¢-positions. They have high duplex forming ability for
complementary RNA because of the conformations similar to
the ribonucleotides of the RNA duplex, and have high resistance
against enzymatic degradation.
Conjugation of functional groups and molecules to ONs is
frequently used for numerous applications, such as therapeutics,
diagnostics and nanotechnology, because it can easily improve
the existing ON’s properties; moreover, it can provide brand new
properties.8,9 ON conjugates are prepared by some condensation
reactions with an amino, a hydroxy, or a thiol group.10 Above all,
a thiol group is very useful since it reacts chemoselectively and
Results and discussion
Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yama-
daoka, Suita, Osaka 565-0871, Japan. E-mail: kodama@phs.osaka-
u.ac.jp, obika@phs.osaka-u.ac.jp; Fax: +81 6 6879 8204; Tel: +81 6 6879
8200
† Electronic supplementary information (ESI) available: HPLC and
MALDI data for ON conjugates, NMR spectra of new compounds, and
UV melting profiles. See DOI: 10.1039/c1ob05469d
Design and synthesis of phosphoramidite and preparation of
oligonucleotides
We previously synthesized a BNA monomer bearing a disulfide
bridged structure (disulfide-type BNA, 1).22 It was expected that
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this disulfide-type BNA with a 1,2-dithiane structure would
be converted to a BNA bearing a thiol at the 6¢-position by
photoirradiation,23 and various molecules were conjugated at the
6¢-position via the thiol. Since this thiol-containing BNA has the
skeleton of 2¢-thio-LNA, which has high duplex-forming ability
for complementary RNA and high enzymatic stability,24 the thiol-
containing BNA was also anticipated to have similar characters in
those regards.
The thiol-containing BNA was synthesized from disulfide-
type BNA monomer 122 as shown in Scheme 1. First, the
nucleoside 1, reported previously by us, was treated with tert-
butyldiphenylsilyl chloride (TBDPSCl) in the presence of N,N-
dimethyl-4-aminopyridine to protect the 3¢- and 5¢-hydroxy
groups. Then, the protected disulfide-type BNA monomer 2
was photoirradiated23 to yield a thiol-containing BNA monomer
3 effectively and diastereoselectively. The configuration at the
C6¢ atom was determined by NOE (nuclear Overhauser effect)
experiment. This diastereoselectivity is most likely explained by
steric hindrance between the thiol and bulky 3¢-O-TBDPS group.
Next, the crude product 3 was subjected to benzoyl chloride
in order to protect the chemically labile hemidithioacetal motif.
Subsequently, the 3¢- and 5¢-O-TBDPS groups were removed by
tetrabutylammonium fluoride to give nucleoside 5. Tritylation at
the 5¢-hydroxy group with 4,4¢-dimethoxytrityl chloride and phos-
phitylation at the 3¢-hydroxy group with N,N-diisopropylamino-
2-cyanoethylphosphino-chloridite afforded the desired phospho-
ramidite building block 7.
Compound 7 was introduced into ONs using an automated
DNA synthesizer. The sequences were the same as those of our
previous studies.25 The concentration of phosphoramidite 7 was
0.10 M and its coupling time was 20 min, while the concen-
tration of each unmodified phosphoramidite was 0.067 M and
their coupling times were 90 s. 5-[3,5-Bis(trifluoromethyl)phenyl]-
1H-tetrazole was used as an activator for both modified and
unmodified phosphoramidite. Coupling yields were checked by
trityl monitoring and were estimated to be over 95%. Synthesized
ONs were cleaved from the solid supports and deprotected in the
presence of 2,2¢-dithiodipyridine (DTDP) to prevent a thiol group
from reacting with released acrylonitrile and to preactivate the
thiol with the pyridinesulfenyl group, which acts as a good leaving
group in the presence of free thiol (Fig. 1).26 The obtained ON 8
and ON 9 were purified by reverse-phase HPLC (RP-HPLC) and
characterized by MALDI-TOF mass spectrometry.
Fig. 1 Conversion of phosphoramidite 7 to ON 8, 9.
Solution-phase conjugation
Various molecules were conjugated to ON 8 or 9 via thioether or
disulfide linkage. First, ON 8 or 9 was treated with dithiothreitol
(DTT) dissolved in sodium phosphate buffer (pH = 8.0) to give
ON 10 or 11, then without any purification of ON 10 or 11, acetic
acid was added to acidify the solution and avoid side reactions
(e.g., alkylation of amino groups on nucleobases). Subsequently,
1 M primary halogenoalkyl derivative in DMF was added to
the solution to afford thioether-linked conjugates (Fig. 2A). The
reactions were analyzed by RP-HPLC. After completing the
reactions, ONs were precipitated by adding 5 volumes of ethanol at
0 ◦C. The obtained ON conjugates were purified by RP-HPLC and
characterized by MALDI-TOF mass spectrometry. Conjugation
yields (HPLC yield and isolation yield) are shown in Fig. 2C. For
most modifiers, clean conversions to new ONs were observed. For
example, Fig. 3A shows the RP-HPLC profile of a representative
Scheme 1 Reagents and conditions: (a) TBDPSCl, DMAP, DMF, 100 ◦C,
15 h, 70%; (b) hn, CH2Cl2, rt, 30 min; (c) BzCl, pyridine, rt, 1 h, 81%
(over 2 steps); (d) TBAF, AcOH, THF, rt, 24 h, 70%; (e) DMTrCl,
pyridine, rt, 3 h, 79%; (f) iPr2NP(Cl)O(CH2)2CN, DIPEA, CH3CN, 0 ◦C,
1.5 h, 66%. TBDPS = tert-butyldiphenylsilyl, Bz = benzoyl, TBAF =
tetra-n-butylammonium fluoride, DMTr = 4,4¢-dimethoxytrityl, DIPEA =
N,N-diisopropylethylamine.
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Fig. 2 Synthesis of thioether-linked conjugates and disulfide-linked conjugates. (A) Thioether-linked conjugation by S-alkylation, (B) disulfide-linked
conjugation by disulfide exchange reaction, and (C) their conjugation yields. HPLC yield in parenthesis was determined by RP-HPLC of reaction solution,
and the isolation yield was based on the UV absorption at 260 nm of purified ON.
synthesis of ON 13 from ON 8 via ON 10. When treating ON
8 with DTT for 2 h, the peak corresponding to ON 8 (retention
time = 28 min) disappeared, and ON 10 (retention time = 20 min)
was generated. Furthermore, 24 h after adding 2-bromoacetamide,
ON 10 was transformed into ON 13 (retention time = 16 min).
For further synthetic details, see experimental section and the
ESI.† These nucleophilic conjugations proceed in good yields,
but the yield of ON 15 was slightly lower because it was more
lipophilic than other ONs and was lost to some extent in ethanol
precipitation.
The disulfide-linked conjugates were generally prepared by
disulfide exchange reactions between a free thiol in reaction
partners and a pyridinesulfenyl-activated ON 8 or 9 (Fig. 2B).
In disulfide-linked conjugation, as in the case of thioether-
linked conjugation, good conversions to ON conjugates were
observed. For example, Fig. 3B shows the RP-HPLC profile of
a typical synthesis of ON 16 from ON 8. Twenty four hours after
treating ON 8 with L-cysteine, the peak corresponding to ON 8
diminished and ON 16 (retention time = 14 min) appeared (see
the experimental section and ESI for further synthesis).† Each
conjugation yield was good, except ON 19. When ON 8 was treated
with glutathione, the yield of ON 17 was very low owing to the
reducing ability of glutathione (ON 17 was reduced by glutathione
to give ON 10). In this case, the yield was improved by treating
ON 10 with S-(2-thiopyridyl)glutathione.27 Since thiocholesterol
was not reacted with ON 8 due to its insolubility, the conjugation
was performed by mixing 2-pyridyl-3-cholesteryl disulfide28 with
ON 10. Nonetheless, the yield of ON 19 was still low because of
its low solubility.
These data show that the C6¢ thiol group has high reactivity
and chemoselectivity except for the conjugation with a por-
tion of highly lipophilic molecules. This BNA is expected to
conjugate with a huge variety of molecules easily in solution
phase.
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Fig. 3 RP-HPLC analyses of solution phase conjugations with a linear gradient of MeCN 6 to 12% over 30 min. (A) Thioether-linked conjugation with
acetamide and (B) disulfide-linked conjugation with L-cysteine.
Evaluation of thermal stabilities
nucleobases31 (DTm = +6 ◦C, DDTm = +9 ◦C). These data indicate
that in DNA–DNA duplexes, conjugated groups and molecules
directed toward the minor groove of duplexes and influence those
hybridization properties.
We evaluated the affinity of the synthesized ONs with complemen-
tary single-stranded RNA (ssRNA) and DNA (ssDNA) through
UV melting experiments. The UV melting profiles are shown in
Fig. S1 and S2 (ESI†), and the thermal denaturation temperatures
(Tm values) are summarized in Table 1.
ON 10 formed a stable duplex with complementary ssRNA
◦
(DTm = +3 C) in a similar manner to previous BNAs3 because
the bridged structure restricted the sugar conformation to N-
type.24 Surprisingly, in the DNA–RNA hybrid duplex, almost all
conjugated molecules had no effect on the hybridization properties
(DDTm = 0 ◦C to -1 ◦C) unlike double stranded DNA. This
suggests that attached molecules are directed toward the outside of
the helix in DNA–RNA hybrid duplexes and locked the positions
by the rigid sugar conformation. The results have exhibited that,
using this BNA, a wide variety of functional molecules can be
attached to the center of the sequence, not 5¢- or 3¢-termini, without
taking into account the affinity for target RNA. Generally, this
characteristic is advantageous for antisense delivery technology.
The Tm value of the ssDNA–ON 10 (containing naked thiol)
duplex was lower than that of the natural DNA duplex (DTm
=
-3 ◦C), but conjugation with a cationic group (ON 14) led to
improvement of the duplex stability (DTm = -1 ◦C, DDTm = +2 ◦C).
On the contrary, in the case of an anionic molecule conjugation,
negative charge of carboxylic acid (ON 17) destabilized its duplex
with ssDNA (DTm = -8 ◦C, DDTm = -5 ◦C). For the cationic
group conjugate,29 it reduced the electrostatic repulsion between
the phosphodiester anions in contrast to the anionic group
conjugate which increased it.30 A pyrene conjugate also showed
high duplex forming ability for ssDNA because of stacking
effects or interaction between the pyrene moiety and hydrophobic
Effect of functional groups on nuclease resistances
Table 1 Evaluation of thermal denaturation temperatures (Tm values)
We examined the resistance of decathymidilate derivatives involv-
ing a single thiol-containing BNA structure or its conjugates
toward 3¢-exonuclease (Crotalus Adamanteus Venom Phosphodi-
esterase, CAVP)32 degradation and compared those with natural
ssDNA and a phosphorothioate-modified ON (S-oligo). After
incubation of each ON solution at 37 ◦C in the presence of CAVP,
the ratio of intact ONs was analyzed at several time points by
RP-HPLC (Fig. 4). The natural ssDNA was completely digested
within 2 min, whereas ON 11 bearing a free thiol and its conjugates
were considerably stable under these conditions; 90% of intact ON
11 survived after 40 min. In the same manner as other BNAs,
3¢-phosphodiester linkages of these BNAs were hardly digested
by the 3¢-exonuclease, probably due to the sterically hindered
environment around the 3¢-phosphodiester linkage.25 Although
the nuclease resistance of natural DNA was the same either in
the presence or absence of DTT (0.75 mM) and DTDP (3.8 mM),
the reducing agent could not be excluded as the cause of the high
enzymatic stability of ON 11 (free thiol).32 Conjugation of some
functional groups significantly enhanced the stability: acetamide-
conjugate (ON 21) and ethylamine-conjugate (ON 22) were more
stable than phosphorothioate-modified ON. This result is opposed
DNA complement
RNA complement
a
a
ON
Tm
DTm
DDTmb/◦C
Tm
DTm
DDTmb/◦C
natural
10
51
48c
47
48
50
57
47
43
—
-3
-4
-3
-1
+6
-4
-8
—
—
-1
0
+2
+9
-1
-5
47
50c
50
50
49
47
49
49
—
+3
+3
+3
+2
0
—
—
0
12
13
0
14
-1
-3
-1
-1
15
16
+2
+2
17
Each strand sequence: 5¢-GCGTTXTTTGCT-3¢ (X = modified monomer),
target strand sequence: 5¢-AGCAAAAAACGC-3¢. Thermal denaturation
studies’ conditions: 10 mM sodium phosphate buffer (pH 7.2) containing
100 mM NaCl; each strand concentration = 4 mM; scan rate of 0.5 ◦C
min-1 at 260 nm. The number is the average of three independent
measurements.a DTms are calculated relative to Tm values of unmodified
DNA–DNA and DNA–RNA duplexes. b DDTms are calculated relative
to Tm values of ON 10–DNA and ON 10–RNA duplexes. c Thermal
denaturation study’s condition: 10 mM sodium phosphate buffer (pH = 7.4)
containing 100 mM NaCl and 400 mM TCEP; each strand concentration =
4 mM; scan rate of 0.5 ◦C min-1 at 260 nm.
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Conclusions
We successfully synthesized a novel BNA bearing a thiol group
in its bridged structure and conjugated functional molecules
to it at the 6¢-thiol group post-synthetically and efficiently in
solution phase. Conjugated functional molecules had great effects
on duplex stability with the DNA complement. In contrast,
the molecules did not influence the stability with the RNA
complement so much. Moreover, the ONs have as high or higher
resistances against 3¢-exonuclease than phosphorothioate ON (S-
oligo).
Thus, this thiol-containing BNA is expected to have a wide field
of application, for example, ON labeling, DNA detection, RNA-
directed therapeutics, and DDS.
Fig. 4 Nuclease resistance of ONs against Crotalus Adamanteus Venom
Phosphodiesterase (CAVP). Hydrolysis of the ONs (7.5 mM) was carried
out at 37 ◦C in buffer (100 mL) containing 50 mM Tris-HCl (pH 8.5),
10 mM MgCl2, and CAVP (0.35 mg). ON 11 was prepared by treating ON
9 with DTT and used without any purification.
Experimental section
General
to Chattopadhyaya’s work;33 introduction of amide or amine
substitution lead to lowered enzymatic stability. Even though ON
20 (–Me) and ON 23 (–S-cationic tripeptide) were more degraded
than ON 11, these ONs were significantly more stable than natural
ON. Since the enzymatic stabilities were not reflected in bulkiness
of the ON conjugates, these results are attributed to other effects,
not only to the steric effect.34 However, the conjugation effects
regarding the resistance to enzymatic degradation are not clear
yet. We will soon elucidate the relevance between substituents at
the C6¢ position and nuclease resistances.
Dichloromethane, DMF and pyridine were distilled from CaH2.
1H NMR (400 and 300 MHz), 13C NMR (100.5 and 75.5 MHz)
and 31P NMR (161.8 MHz) were recorded on JEOL JNM-ECS-
400 or JNM-AL-300 spectrometers. Chemical shifts are reported
in parts per million referenced to internal tetramethylsilane (0.00
ppm) and residual CHCl3 (7.26 ppm) and methanol (3.31 ppm) for
1H NMR, and chloroform-d1 (77.0 ppm) and methanol-d4 (49.0
ppm) for 13C NMR. Relative to 85% H3PO4 as external standard
for 31P NMR. IR spectra were recorded on a JASCO FT/IR-4200
spectrometers. Optical rotations were recorded on a JASCO DIP-
370 instrument. Mass spectra were measured on JEOL JMS-700
mass spectrometers. MALDI-TOF mass spectra were recorded
on a Bruker Daltonics Autoflex II TOF/TOF mass spectrometer.
For column chromatography, Fuji Silysia PSQ-100B or FL-100D
silica gel was used. For high performance liquid chromatography
(HPLC), SHIMADZU LC-6AD, SPD-10AVVP and CTO-10AVP
were used.
Response of disulfide linkage to glutathione
In order to make the best use of the disulfide linkage, it is necessary
that the disulfide bond is cleaved. It is known that the disulfide
bond is cleaved under intracellular reductive conditions.12 But,
it is possible that the disulfide bond is not cleaved because of
the sterically hindered environment around the linkage. Thus,
we assessed the response of the disulfide linkage in ON 18 to
glutathione. Fig. 5 shows the RP-HPLC profile of conversion from
ON 18 (disulfide form) to ON 10 (free thiol form). When treating
ON 18 with glutathione (1 mM to 10 mM) for 30 min, the peak
corresponding to ON 18 disappeared, and ON 10 was generated.
This result demonstrates that the disulfide-linked conjugates will
be cleaved on intracellular reductive conditions and the ONs with
free thiol-containing BNA monomers be released in cells.
1-{2-Thio-3,5-di-O-(tert-butyldiphenylsilyl)-2-S,4-C-(1-thia-
ethylene)-b-D-ribofuranosyl}thymine (2). To a solution of com-
pound 122 (50 mg, 0.16 mmol) and N,N-dimethyl-4-aminopyridine
(96 mg, 0.79 mmol) in DMF (0.20 mL) was added tert-
butyldiphenylsilyl chloride (0.14 mL, 0.55 mmol) and the resultant
mixture was stirred at 100 ◦C for 15 h under a N2 atmosphere.
After addition of CH3OH, the reaction mixture was diluted with
Et2O, washed with H2O, dried over MgSO4, and concentrated.
The crude product was purified by column chromatography (n-
hexane/AcOEt = 3/1) to give compound 2 (88 mg, 70%) as a
white foam; [a]D25 -21.2 (c 1.0, CH2Cl2); IR nmax (KBr): 1690 cm-1;
1H NMR (300 MHz, CDCl3) d 1.00 (9 H, s), 1.10 (9 H, s), 1.43 (3
H, s), 2.77 (1 H, d, J = 13 Hz), 2.81 (1 H, d, J = 6 Hz), 3.38 (1 H, d,
J = 12 Hz), 3.60 (1 H, d, J = 13 Hz), 3.71 (1 H, d, J = 12 Hz), 3.89
(1 H, d, J = 6 Hz), 6.58 (1 H, s), 6.77 (1 H, s), 7.16–7.63 (20 H, m),
7.98 (1 H, brs); 13C NMR (75.5 MHz, CDCl3) d 12.2, 19.3, 19.3,
26.8, 27.0, 31.6, 51.6, 66.7, 68.3, 84.9, 89.2, 110.2, 127.8, 127.8,
127.9, 130.0, 130.2, 130.4, 132.2, 132.3, 132.6, 134.4, 135.4, 135.4,
135.6, 135.8, 149.6, 163.6; MS (FAB) m/z 795 (M + H+); HRMS
(FAB): Calcd for C43H51N2O5S2Si2 (M + H+): 795.2778. Found:
795.2782.
Fig. 5 RP-HPLC analysis of conversion from ON 18 to ON 10. ON 18
in the absence of glutathione (dashed line) and in the presence of 10 mM
glutathione (solid line).
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1-[2-Thio-3,5-di-O-(tert-butyldiphenylsilyl)-2-S,4-C-{(S)-sul-
1-[2-Thio-5-O-(4,4¢-dimethoxytrityl)-2-S,4-C -{(R)-(benzoyl-
sulfanyl)methylene}-b-D-ribofuranosyl]thymine (6). To a solution
of compound 5 (260 mg, 0.61 mmol) in pyridine (3.1 mL)
was added 4,4¢-dimethoxytrityl chloride (310 mg, 0.92 mmol)
and the resultant mixture was stirred at room temperature for
2.5 h under a N2 atmosphere. Furthermore 4,4¢-dimethoxytrityl
chloride (210 mg, 0.61 mmol) was added to the mixture and
the mixture was stirred at room temperature for 30 min. After
addition of H2O, the reaction mixture was diluted with CH2Cl2,
dried over Na2SO4, and concentrated. The crude product was
purified by column chromatography (0.5% triethylamine in n-
hexane/AcOEt = 1/1 to AcOEt only) to give compound 6 (350 mg,
79%) as a white foam; [a]2D4 -100.4 (c 1.0, CH2Cl2); IR nmax (KBr):
1508, 1580, 1608, 1685, 1973, 2044, 2321, 2835, 2933, 3066, 3412
fanylmethylene}-b-D-ribofuranosyl]thymine (3). Compound
2
(200 mg, 0.25 mmol) was dissolved in CH2Cl2 (100 mL) at room
temperature. The solution was stirred and photoirradiated by a
high pressure mercury lamp at ambient temperature for 30 min.
The solvent was removed to give compound 3 as a white foam.
The product was used without purification; [a]2D5 +21.2 (c 1.0,
CH2Cl2); IR nmax (KBr): 1589, 1686, 1898, 1961, 2857, 2930, 3071,
1
3178 cm-1; H NMR (400 MHz, CDCl3) d 1.08–1.12 (21 H, m),
2.23 (1 H, d, J = 12 Hz), 3.01 (1 H, d, J = 2 Hz), 4.02 (1 H, d, J =
11 Hz), 4.45 (1 H, d, J = 12 Hz), 4.48 (1 H, d, J = 2 Hz), 4.57 (1
H, d, J = 11 Hz), 5.80 (1 H, s), 7.26–7.70 (20 H, m), 8.27 (1 H,
brs); 13C NMR (100.5 MHz, CDCl3) d 11.7, 19.5, 20.2, 27.2, 27.8,
49.8, 54.2, 60.9, 74.1, 90.3, 91.3, 110.1, 128.1, 128.3, 128.4, 130.4,
130.4, 130.6, 130.8, 132.5, 133.3, 134.4, 135.6, 135.9, 136.2, 149.8,
164.0; MS (FAB) m/z 795 (M + H+); HRMS (FAB): Calcd for
C43H51N2O5S2Si2 (M + H+): 795.2778. Found: 795.2776.
1
cm-1; H NMR (400 MHz, CDCl3) d 1.01–1.14 (21 H, m), 3.08
(1 H, d, J = 2 Hz), 4.22 (2 H, s), 4.48 (1 H, d, J = 2 Hz), 5.45 (1
H, s), 5.83 (1 H, s), 7.26–7.96 (26 H, m); 13C NMR (100.5 MHz,
CDCl3) d 12.3, 54.0, 54.0, 55.4, 59.8, 73.3, 87.5, 90.0, 91.1, 110.5,
113.5, 127.3, 127.5, 128.2, 128.3, 128.9, 130.2, 134.2, 134.8, 135.0,
135.1, 136.1, 144.1, 150.2, 158.8, 158.8, 164.1, 191.0; MS (FAB)
m/z 747 (M + Na+); HRMS (FAB): Calcd for C39H36N2NaO8S2
(M + Na+): 747.1811. Found: 747.1821.
1-[2-Thio-3,5-di-O-(tert-butyldiphenylsilyl)-2-S,4-C-{(R)-(ben-
zoylsulfanyl)methylene}-b-D-ribofuranosyl]thymine (4). To a so-
lution of compound 3 (200 mg, 0.25 mmol) in pyridine (2.5 mL)
was added benzoyl chloride (0.088 mL, 0.76 mmol) and the
resultant mixture was stirred at room temperature for 1 h under
a N2 atmosphere. After addition of H2O, the reaction mixture
was diluted with AcOEt, dried over Na2SO4, and concentrated.
The crude product was purified by column chromatography
(CHCl3/CH3OH = 60/1) to give compound 4 (184 mg, 81%, over 2
steps) as a white foam; [a]2D3 +43.4 (c 1.0, CH2Cl2); IR nmax (KBr):
1-[2-Thio-3-O-{2-cyanoethoxy(diisopropylamino)phosphino}-
5-O-(4,4¢-dimethoxytriryl)-2-S,4-C-{(R)-(benzoylsulfanyl)methy-
lene}-b-D-ribofuranosyl]thymine (7). To a solution of compound
6 (350 mg, 0.48 mmol) in acetonitrile (8.8 mL) was added
N,N-diisopropylethylamine (0.34 mL, 1.9 mmol) and N,N-
diisopropylamino-2-cyanoethylphosphino chloridite (0.22 mL,
0.97 mmol) and the resultant mixture was stirred at 0 ◦C for 2
h under a N2 atmosphere. The reaction mixture was concentrated
and the crude product was purified by column chromatography
(0.5% triethylamine in n-hexane/AcOEt = 1/1 to 1/2) to give
compound 7 (290 mg, 66%) as a white foam; 31P NMR (161.8
MHz, CDCl3) d 150.0, 150.4; MS (FAB) m/z 925 (M + H+);
HRMS (FAB): Calcd for C48H54N4O9PS2 (M + H+): 925.3070.
Found: 925.3076.
1
1689, 2858, 2891, 2933, 3050, 3069 cm-1; H NMR (400 MHz,
CDCl3) d 1.05– 1.14 (21 H, m), 3.07 (1 H, d, J = 2 Hz), 4.22 (2 H,
s), 4.48 (1 H, s), 5.45 (1 H, s), 5.83 (1 H, s), 7.23–7.90 (26 H, m);
13C NMR (100.5 MHz, CDCl3) d 11.4, 19.4, 20.0, 27.0, 27.4, 53.6,
54.0, 60.8, 74.0, 90.3, 91.7, 109.9, 127.5, 128.0, 128.0, 128.1, 128.1,
128.5, 128.9, 130.1, 130.2, 130.3, 130.4, 130.6, 132.1, 132.6, 133.1,
134.0, 134.3, 135.4, 135.8, 136.1, 149.6, 164.0, 191.2; MS (FAB)
m/z 899 (M + H+); HRMS (FAB): Calcd for C50H55N2O6S2Si2
(M + H+): 899.3040. Found: 899.3073.
1 - [2 - Thio - 2 - S,4 - C - {(R) - (benzoylsulfanyl)methylene}-b-D-
ribofuranosyl]thymine (5). To a solution of compound 4 (64 mg,
0.071 mmol) in THF (0.71 mL) was added tetrabutylammonium
fluoride (1.0 M in THF, 0.21 mL, 0.21 mmol) and acetic acid
(0.061 mL, 1.1 mmol) and the resultant mixture was stirred at
room temperature for 23 h. Furthermore, tetrabutylammonium
fluoride (1.0 M in THF, 0.14 mL, 0.14 mmol) and acetic acid
(0.041 mL, 0.7 mmol) was added to the mixture and the mixture
was stirred at room temperature for 25 h. The reaction mixture
was concentrated and the crude product was purified by column
chromatography (CHCl3/CH3OH = 20/1 to 10/1) and purified
further by column chromatography (AcOEt/CH3OH = 30/1) to
give compound 5 (21 mg, 70%) as a white foam; [a]2D5 +192.9 (c
1.0, MeOH); IR nmax (KBr): 1580, 1597, 1685, 2099, 2320, 2925,
ON synthesis
Synthesis of the thiol-containing BNA-modified ONs was per-
formed on an Applied Biosystems ExpediteTM 8909 Nucleic
Acid Synthesis System on a 1.0 mmol scale using a phospho-
ramidite coupling protocol and 5-[3,5-bis(trifluoromethyl)phenyl]-
1H-tetrazole as the activator. The concentration of phospo-
ramidite 7 was 0.10 M and its coupling time was 20 min, while
the concentration of each unmodified phosporamidite was 0.067
M and their coupling times were 90 s. The solid-supported ONs
(DMTr-off) were treated with 50 mM K2CO3 and 100 mM 2,2¢-
dithiodipyridine in CH3OH at room temperature for 2.5 h, then
neutralized by 5% acetic acid and concentrated. The crude ONs
were roughly purified with a GE Healthcare Nap 10 column, and
then carefully by RP-HPLC on a Waters XBridgeTM OST C18
2.5 mm (10 ¥ 50 mm) using MeCN in 0.1 M triethylammonium
acetate buffer (pH = 7.0). The purity of the ONs was analyzed
by RP-HPLC on a Waters XBridgeTM Shield RP 18 2.5 mm
(4.6 ¥ 50 mm) and characterized by MALDI-TOF mass spec-
trometry. The overall yields were 8.4% for ON 8 and 16.6% for
ON 9 calculated from the UV absorbance at 260 nm.
1
3063, 3373 cm-1; H NMR (400 MHz, CD3OD) d 1.85 (3 H, s),
3.65 (1 H, s), 3.85 (1 H, d, J = 12 Hz), 3.91 (1 H, d, J = 12 Hz), 4.59
(1 H, d, J = 3 Hz), 5.33 (1 H, s), 5.88 (1 H, s), 7.47– 7.51 (2 H, m),
7.61– 7.65 (1 H, m), 7.92– 7.94 (2 H, m), 8.12 (1 H, s); 13C NMR
(100.5 MHz, CD3OD) d 12.7, 54.7, 58.5, 72.6, 91.4, 92.8, 110.1,
128.3, 130.0, 135.1, 137.3, 137.6, 152.0, 166.5, 192.3; MS (FAB)
m/z 423 (M + H+); HRMS (FAB): Calcd for C18H19N2O6S2 (M +
H+): 423.0685. Found: 423.0681.
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General procedure for solution-phase conjugation of ONs
Research on Innovative Area (22136006) from the Ministry
of Education, Culture, Sports, Science and Technology, Japan
(MEXT), and the Program for Promotion of Fundamental Studies
in Health Sciences of the National Institute of Biomedical
Innovation (NIBIO).
Thioether-linked conjugation. 500 mM ON 8 or 9 (12 mL,
6 nmol) was treated with 10 mM dithiothreitol (DTT) dissolved in
100 mM sodium phosphate buffer (pH = 8.0) (60 mL, 0.6 mmol) at
room temperature for 2 h to give ON 10 or 11, then, 5% acetic acid
(3.4 mL) was added, and 1 M primary halogenoalkyl derivative
in DMF (1.8 mL, 1.8 mmol) was added to the solution to afford
a thioether-linked conjugate. The reaction was analyzed by RP-
HPLC. After completing the reaction, the ON was precipitated
by adding 5 volumes of ethanol. The mixture was kept at 0 ◦C
for 30 min, centrifuged at 13 200 rpm for 15 min at 4 ◦C, and
the resulting supernatant solution was removed. The obtained
ON conjugates were purified by RP-HPLC and characterized
by MALDI-TOF mass spectrometry. Each conjugation yield
was calculated from UV absorption at 260 nm of the ON
conjugate.
Notes and references
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Acknowledgements
This work was supported by a Grant-in-Aid for Challenging
Exploratory Research (22651076) from the Japan Society for
the Promotion of Science (JSPS), a Grant-in-Aid for Scientific
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experiment were equal to the other experiments.
This journal is
The Royal Society of Chemistry 2011
Org. Biomol. Chem., 2011, 9, 5272–5279 | 5279
©