Photoinduced changes in hydrogen bonding patterns of 8-thiopurine nucleobase analogues in a DNA strand

Article abstract of DOI:10.1039/c3ob42427h

Hydrogen bonds (H-bonds) formed between nucleobases play an important role in the construction of various nucleic acid structures. The H-donor and H-acceptor pattern of a nucleobase is responsible for selective and correct base pair formation. Herein, we describe an 8-thioadenine nucleobase analogue and an 8-thiohypoxanthine nucleobase analogue with a photolabile 6-nitroveratryl (NV) group on the sulfur atom (SA^NV and SH^NV, respectively). Light-triggered removal of the NV group causes tautomerization and a change in the H-bonding pattern of SA^NV and SH^NV. This change in the H-bonding pattern has a strong effect on base recognition by 8-thiopurine nucleobase analogues. In particular, base recognition by SH^NV is clearly shifted from guanine to adenine upon photoirradiation. These results show that a photoinduced change in the H-bonding pattern is a unique strategy for manipulating nucleic acid assembly with spatiotemporal control. the Partner Organisations 2014.

Full text of DOI:10.1039/c3ob42427h

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Biomolecular Chemistry
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Photoinduced changes in hydrogen bonding
patterns of 8-thiopurine nucleobase analogues in
a DNA strand†
Cite this: Org. Biomol. Chem., 2014,
12, 2468
Kunihiko Morihiro,a,b Tetsuya Kodama,c Shohei Moria and Satoshi Obika*a,b
Hydrogen bonds (H-bonds) formed between nucleobases play an important role in the construction of
various nucleic acid structures. The H-donor and H-acceptor pattern of a nucleobase is responsible for
selective and correct base pair formation. Herein, we describe an 8-thioadenine nucleobase analogue
and an 8-thiohypoxanthine nucleobase analogue with a photolabile 6-nitroveratryl (NV) group on the
sulfur atom (SANV and SHNV, respectively). Light-triggered removal of the NV group causes tautomeriza-
tion and a change in the H-bonding pattern of SANV and SHNV. This change in the H-bonding pattern has
a strong effect on base recognition by 8-thiopurine nucleobase analogues. In particular, base recognition
by SHNV is clearly shifted from guanine to adenine upon photoirradiation. These results show that a
photoinduced change in the H-bonding pattern is a unique strategy for manipulating nucleic acid assem-
bly with spatiotemporal control.
Received 5th December 2013,
Accepted 13th February 2014
DOI: 10.1039/c3ob42427h
www.rsc.org/obc
nucleobases. Photoirradiation reinstates the H-bonding
capabilities and allows nucleobase interaction in the “OFF to
Introduction
The complementarity of natural A–T and G–C base pairs in ON” direction. Nucleobase-caged nucleosides can be widely
DNA is the principal mechanism for the preservation and flow used for the photoregulation of antisense oligodeoxynucleo-
of genetic information. The hydrogen-bonding (H-bonding) tides (ODNs),1,2 siRNAs,3,4 aptamers,5 ribozymes6,7 and deoxy-
patterns of the four natural nucleobases play an important ribozymes,8,9 diagnostic ODNs,10 DNA architectures,11 and
role in the selective and correct formation of base pairs. These DNA logic gates.12,13
H-bonding interactions can result in the formation of higher
Recently, we reported the synthesis and properties of a
order complexes of nucleic acids, depending on the sequence. unique light-responsive nucleobase analogue derived from
Therefore, the control of H-bonding interactions using external 2-mercaptobenzimidazole (SBNV) (Fig. 1a).14 SBNV is modified
stimuli is important for regulating biological processes and for with a photolabile 6-nitroveratryl (NV) group,15 and the nitro-
the possibility of developing unique DNA-based molecular gen at the 3-position serves as an H-acceptor (A). SBNV can
machines. Various external stimuli have been used to this end; selectively form a base pair with guanine even before photo-
light is an ideal trigger because the timing, location, and irradiation, unlike conventional caged nucleobases. Light-trig-
intensity of the irradiation can be easily controlled. Among gered removal of the NV group causes tautomerization of the
such strategies, nucleobase caging strategies involving the nucleobase, and changes the role of the 3-nitrogen atom from
installation of a photolabile group are very important. Photo- H-A to H-donor (D). Following this change in the H-bonding
labile caging groups perturb the H-bonding capabilities of the pattern, base recognition by SBNV can be shifted from guanine
to adenine. We also demonstrated that a light-triggered strand
exchange reaction targeting different mRNA fragment
aGraduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka,
sequences could be achieved using ODNs containing SBNV
.
Suita, Osaka 565-0871, Japan. E-mail: obika@phs.osaka-u,ac.jp;
Fax: +81 6-6879-8204; Tel: +81 6-6879-8200
These results indicate that a photoinduced change in the
H-bonding pattern of a nucleobase is a good strategy for
manipulating nucleic acid assemblies in a spatially and tem-
porally controlled manner. In this paper, to further investigate
the effect of this change in H-bonding pattern of nucleobases
on the base recognition ability, we designed new light-respon-
sive nucleoside analogues bearing the NV group: 8-thioade-
nine and 8-thiohypoxanthine (SANV and SHNV, respectively;
bNational Institute of Biomedical Innovation (NIBIO), 7-6-8 Saito-Asagi, Ibaraki,
Osaka 567-0085, Japan
cGraduate School of Pharmaceutical Sciences, Nagoya University, Furo-cho, Chikusa-
ku, Nagoya, Aichi 464-8601, Japan
†Electronic supplementary information (ESI) available: NMR spectra of new
compounds, HPLC and MALDI-TOF MS analysis of modified ODNs, photoreac-
tion of modified ODNs and UV melting curves for modified duplexes. See DOI:
10.1039/c3ob42427h
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Scheme 1 Preparation of 6-nitroveratrylthiol 2. Reagents and con-
ditions: (a) KSAc, THF, rt; (b) conc. HCl aq., MeOH, 60 °C, 94% over two
steps.
from [D, A] to [D, D] and [A, A] to [A, D], respectively (Fig. 1).
Tm evaluation of modified ODNs revealed that photoinduced
changes in H-bonding patterns of 8-thiopurine nucleobase
analogues have a pronounced effect on base recognition
abilities.
Results and discussion
The syntheses of the phosphoramidites bearing SANV and
SHNV as a nucleobase are summarized in Scheme 1. 6-Nitrover-
atrylthiol (2) was prepared from 6-nitroveratrylbromide (1)
(Scheme 1) and subjected to reaction with 8-bromo-2′-deoxy-
adenosine derivative (3)18 to afford 4 (Scheme 2). Phosphityla-
tion at the 3′-hydroxyl group provided SANV-phosphoramidite
5. For the preparation of SHNV-phosphoramidite 9, 8-bromo-
inosine (6)19 was treated with 2 to give 7 (Scheme 3). Tritylation
of the primary hydroxyl group in 7 and phosphitylation of the
secondary hydroxyl group provided phosphoramidite 9.
Amidite blocks 5 and 9 were applied to an automated DNA
synthesizer to incorporate SANV and SHNV into ODNs. SANV
and SHNV were incorporated into the middle of the pyrimidine
Fig. 1 (a) Change in base recognition by SBNV upon photoirradiation.
(b) Photoinduced changes in hydrogen bonding patterns of SANV and
(c) SHNV
.
Fig. 1b and c). 8-Thiopurine analogues should preferentially
adopt the syn conformation about the glycosidic bond due to
steric repulsion between the C8-sulfur atom and the 4′-oxygen
atom in the anti conformer.16,17 SANV and SHNV should also
adopt the syn conformation and use the H-A and H-D Hoogs-
teen face to contact the target base. The H-bonding pattern of
SANV and SHNV at the Hoogsteen face would thus be changed
Scheme 2 Preparation of the phosphoramidites bearing SANV. Reagents and conditions: (a) 2, K2CO3, DMF, rt, 52%; (b) (iPr2N)P(Cl)O(CH2)2CN,
iPr2NEt, MeCN, rt, 77%.
Scheme 3 Preparation of the phosphoramidites bearing SHNV. Reagents and conditions: (a) 2, K2CO3, DMF, rt, 25%; (b) DMTrCl, pyridine, rt, 85%; (c)
(iPr2N)P(Cl)O(CH2)2CN, iPr2NEt, MeCN, rt, 74%.
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Table 1 Tm values of DNA duplexesa
Tm (°C)
UV (−)
Tm b (°C)
UV (+)
Duplex
X : Y
10 : 14
10 : 15
10 : 16
10 : 17
11 : 14
11 : 15
11 : 16
11 : 17
12 : 17
13 : 16
12 : 15
12 : 16
13 : 14
13 : 17
SANV : A
26
35
24
32
31
35
30
26
41
43
33
29
32
35
24
28
26
32
39
26
31
28
41
43
33
29
32
35
SANV : G
Fig. 2 ODN sequences used in this study.
SANV : C
SANV : T
SHNV : A
SHNV : G
SHNV : C
SHNV : T
A : T
G : C
A : G (mismatch)
A : C (mismatch)
G : A (mismatch)
G : T (wobble)
a Conditions: each ODN (4.0 μM), NaCl (20 mM), sodium phosphate
buffer (10 mM, pH 7.2). b Tm values of DNA duplexes after irradiation
(365 nm) at 37 °C for 5 min.
SANV : G pair was slightly less stable than natural base pairs,
and its stability was similar to the stability of the G : T wobble
base pair. After photoirradiation at 365 nm for 5 min, SA
showed the highest affinity towards thymine (ΔTm ≧ 4 °C);
however, the Tm values of duplexes containing SA are, on the
whole, low (Tm ≦ 32 °C). SHNV in ODN 11 showed the highest
affinity towards guanine, similar to SANV (ΔTm ≧ 4 °C). The Tm
Fig. 3 Time course conversion of (a) SANV to SA in ODN 10 and (b)
SHNV to SH in ODN 11 by photoirradiation. Conditions: each ODN
(0.1 nmol, 10 μM), sodium phosphate buffer (pH 7.2, 25 mM). Irradiation
(365 nm) was performed at rt. Error bars indicate standard deviation
(n = 3).
(T) strand of ODN 10 and ODN 11. After cleavage from the value of the duplex containing the SHNV : G base pair was also
resin and purification by reversed-phase (RP) HPLC, the struc- slightly lower than that of natural duplexes. In contrast, after
ture of each ODN was confirmed by MALDI-TOF MS analysis. irradiation, the preferred base-pairing partner for SHNV clearly
The sequence of each ODN used in this study is shown in changed to adenine (ΔTm ≧ 8 °C). Notably, the stability of
Fig. 2.
SH : A was comparable to that of the natural A : T base pair.
These results suggest that photoirradiation induces a change
The photoreactivity of SANV and SHNV in a DNA strand was
investigated by RP-HPLC analysis using ODN 10 and ODN 11. in base recognition by SHNV from guanine to adenine. Fig. 4
When irradiated at 365 nm at 37 °C, ODN 10 and ODN 11 illustrates the changes in the UV melting profiles of ODN-
11-formed DNA duplexes and clearly indicates that the change
gradually disappeared. MALDI-TOF MS showed that the result-
ing ODNs were SA-/SH-ODNs and confirmed that the NV group in base recognition by SHNV is triggered by photoirradiation.
of SANV and SHNV was efficiently removed. Fig. 3 shows the
percentage of the remaining SANV-/SHNV- and resulting SA-/SH-
Although further conclusive experiments such as NMR or
X-ray structural analysis are needed to elucidate the precise
ODNs at several irradiation time points. The photoreaction base pair structures, the results of the Tm measurements
was complete within 60 s for both ODNs, and the yield of suggest that SANV and SHNV recognize guanine via two
H bonds on the Hoogsteen face, as shown in Fig. 5. The stabi-
NV-removed ODNs was estimated from the HPLC peak area to
be about 80%.
lities of the SANV and SHNV : G base pairs were slightly lower
The effects of photoinduced changes in H-bonding patterns than that of the natural base pairs. It would appear that steric
repulsion between the 8-sulfur atom in SANV and the 2-amino
of SANV and SHNV on their base recognition ability were exam-
ined by measuring the Tm values of DNA duplexes containing group in guanine decreases the stability of the SANV : G pair
ODN 10 and ODN 11 (Table 1). ODN 10 and ODN 11 were indi- (Fig. 5a). This observation is consistent with previous reports
showing that the base pair between 2-thiouracil and 2,6-diami-
vidually hybridized to four ODNs, generating eight distinct
duplexes in which each nucleobase analogue was paired with nopurine is significantly destabilized because of steric hin-
all possible natural nucleobases. For comparison, naturally drance.20,21 Also, SHNV may form a wobble pair with guanine
similar to the U : G mismatch pair commonly found in RNA22
matched duplexes containing the A : T and G : C base pairs in
the same position were also examined. The duplex containing (Fig. 5b); therefore, the SHNV : G pair is less stable than natural
the SANV : G pair showed the highest Tm value of all the combi- base pairs. Light-induced changes in H-bonding patterns have
nations of SANV with other nucleobases (ΔTm ≧ 3 °C). The
profound effects on the base recognition abilities of 8-
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interact with thymine using its Watson–Crick face in the anti-
conformation; however, the resulting poor base-recognition-
ability indicated that SA still adopted a syn conformation after
photoirradiation due to the steric bulk around the 8-thio
group. On the other hand, SH can interact with adenine using
an [A, D] H-bonding pattern on the Hoogsteen face (Fig. S5†).
Conclusions
In conclusion, we have developed 8-thiopurine nucleobase
analogues bearing a photolabile NV group on the sulfur atom.
The H-bonding patterns of the Hoogsteen face in SANV
and SHNV could be changed by photoirradiation. Tm analysis
indicated that light-induced changes in the H-bonding pattern
profoundly influence the base recognition ability of the
nucleobase in duplex DNA. In particular, base recognition by
SHNV is efficiently shifted from guanine to adenine upon
photoirradiation. We believe that these unique light-responsive
nucleobase analogues could be powerful tools for the spatio-
temporal control of DNA assembly.
Experimental
General
Reagents and solvents were purchased from commercial sup-
pliers and were used without purification unless otherwise
specified. All experiments involving air- and/or moisture-sensi-
tive compounds were carried out under an N2 atmosphere. All
reactions were monitored with analytical TLC (Merck Kieselgel
60 F254). Column chromatography was carried out using Fuji
Silysia FL-100D. Physical data were measured as follows: NMR
spectra were recorded on a JEOL JNM-ECS-400 spectrometer
using CDCl3 or DMSO-d6 as the solvent with tetramethylsilane
as an internal standard. IR spectra were recorded on a JASCO
FT/IR-4200 spectrometer. Optical rotations were recorded on a
JASCO P-2200 instrument. FAB mass spectra were measured on
a JEOL JMS-700 mass spectrometer. MALDI-TOF mass spectra
were recorded on a Bruker Daltonics Autoflex II TOF/TOF mass
spectrometer.
Fig. 4 Light-triggered changes in the denaturation profiles of duplexes
containing ODN 11 determined by correlating the absorbance at
260 nm vs. temperature. Conditions as given in Table 1.
Synthesis of the phosphoramidite-bearing SANV and SHNV
nucleobase analogues
6-Nitroveratrylthiol (2). To
a solution of 6-nitroveratryl
bromide (1.47 g, 5.36 mmol) in dry THF (54.0 mL) was added
potassium thioacetate (734 mg, 6.43 mmol) and the reaction
mixture was stirred for 5 h at room temperature. The solvent
was removed in vacuo and the residue was partitioned between
AcOEt and H2O. The separated organic layer was washed with
brine and then dried (Na2SO4) and concentrated in vacuo. The
resulting residue (1.52 g) was dissolved in MeOH (51.0 mL),
and 35% aqueous HCl (3.20 mL) was added. After being
stirred for 12 h at 60 °C, the solvent was removed in vacuo. The
residue was purified on a silica gel column eluted with
hexane–AcOEt (4 : 1 to 1 : 1) to give 2 (1.15 g, 94%) as a yellow
Fig. 5 Plausible change in base recognition by SANV and SHNV upon
photoirradiation.
thiopurine nucleobase analogues. ODN containing SA showed
low recognition ability toward any nucleobase; this can be
explained by the fact that SA has an H-bonding [D, D] pattern
on the Hoogsteen face, but no natural nucleobase has an [A, A]
pattern for base pair formation with SA (Fig. 5a). SA can
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solid; mp 86–87 °C; H NMR (400 MHz, CDCl3) δ 7.66 (1H, s), 13.5 Hz, SCH2Ar), 4.37 (1H, brs, H-3′), 3.86 (3H, s, Ar-OCH3),
6.86 (1H, s, H-2 or H-5), 4.03 (2H, d, J = 8.5 Hz, SCH2Ar), 3.99 3.85 (3H, s, Ar-OCH3), 3.81–3.77 (1H, m, H-4′), 3.60–3.57 (1H,
(3H, s, Ar-OCH3), 3.95 (3H, s, Ar-OCH3), 2.23 (1H, t, J = 8.5 Hz, m, H-5′a), 3.46–3.44 (1H, m, H-5′b), 2.98–2.91 (1H, m, H-2′a),
SH); 13C NMR (100 MHz, CDCl3) δ 153.3, 147.8, 139.6, 132.0, 2.12–2.06 (1H, m, H-2′b); 13C NMR (100 MHz, DMSO-d6)
112.5, 108.2, 56.3, 56.2, 27.0; IR (KBr) 2577, 1520, 1273 cm−1
FAB-LRMS m/z
C9H11NNaO4S 252.0306, found 252.0306.
6-N-Benzoyl-5′-O-(4,4′-dimethoxytrityl)-8-(6-nitroveratrylthio)- (MH+); FAB-HRMS calcd for C19H22N5O8S 480.1189, found
2′-deoxyadenosine (4). To
solution of 318 (300 mg, 480.1207.
0.408 mmol) in dry DMF (4.10 mL) were added K2CO3 5′-O-(4,4′-Dimethoxytrityl)-8-(6-nitroveratrylthio)-2′-deoxyino-
;
δ 155.6, 152.6, 149.5, 147.9, 146.5, 145.2, 139.7, 127.4, 124.7,
252 (MNa+); FAB-HRMS calcd for 115.3, 108.3, 88.0, 84.3, 70.9, 61.9, 56.1, 56.1, 37.2, 34.1; IR
(KBr) 3366, 1686, 1523, 1275 cm−1; FAB-LRMS m/z = 480
=
a
(169 mg, 1.22 mmol) and 2 (103 mg, 0.449 mmol) at room sine (8). To a solution of 7 (560 mg, 1.17 mmol) in dry pyri-
temperature. After being stirred for 1 h at room temperature, dine (12.0 mL) was added 4,4′-dimethoxytrityl chloride
the resulting mixture was partitioned between Et2O and H2O. (474 mg, 1.40 mmol) at room temperature. After being stirred
The separated organic layer was washed with brine, dried for 5 h at room temperature, the reaction was quenched by
(Na2SO4) and concentrated in vacuo. The resulting residue was addition of MeOH. The resulting mixture was partitioned
purified on a silica gel column eluted with hexane–AcOEt (2 : 3 between AcOEt and H2O. The separated organic layer was
to 1 : 2 with 0.5% Et3N) to give 4 (187 mg, 52%) as a yellow washed with saturated aqueous NaHCO3, followed by brine,
foam; 1H NMR (400 MHz, CDCl3) δ 8.93 (1H, brs, NH), 8.43 and then dried (Na2SO4) and concentrated in vacuo. The
(1H, s, H-2), 8.04 (2H, d, J = 7.5 Hz), 7.64–7.16 (14H, m), residue was purified on a silica gel column eluted with CHCl3–
6.77–6.73 (4H, m), 6.27 (1H, t, J = 7.0 Hz, H-1′), 4.99–4.85 (3H, MeOH (50 : 1 with 0.5% Et3N) to give 8 (770 mg, 85%) as a
m, H-3′ and SCH2Ar), 4.06 (1H, dd, J = 10.0 and 6.0 Hz, H-4′), yellow foam; 1H NMR (400 MHz, CDCl3) δ 7.70 (1H, s), 7.67
3.88 (3H, s, Ar-OCH3), 3.76 (3H, s, Ar-OCH3), 3.75 (3H, s, Ar- (1H, s), 7.53 (1H, s), 7.39 (1H, d, J = 7.5 Hz), 7.29–7.18 (1H, m),
OCH3), 3.71 (3H, s, Ar-OCH3), 3.40–3.34 (3H, m, H-2′a and 6.78 (4H, dd, J = 8.5 and 3.0 Hz), 6.20 (1H, t, J = 6.5 Hz, H-1′),
H-5′), 2.38 (1H, brs, OH), 2.33–2.26 (1H, m, H-2′b); 13C NMR 4.95 and 4.91 (each 1H, each d, J = 13.5 Hz, SCH2Ar), 4.76–4.74
(100 MHz, CDCl3) δ 164.7, 158.4, 158.4, 154.1, 153.2, 150.7, (1H, m, H-3′), 4.02–3.99 (1H, m, H-4′), 3.97 (3H, s, Ar-OCH3),
148.4, 146.5, 144.7, 140.8, 135.9, 135.8, 133.8, 132.8, 130.0, 3.92 (3H, s, Ar-OCH3), 3.77 (6H, s, 2 × Ar-OCH3), 3.45–3.42 (1H,
130.0, 128.9, 128.1, 128.0, 127.9, 127.8, 126.8, 123.7, 114.1, m, H-5′a), 3.35–3.31 (1H, m, H-5′b), 3.18–3.11 (1H, m, H-2′a),
113.0, 113.0, 108.0, 86.3, 85.7, 84.3, 72.8, 63.6, 56.4, 56.3, 55.2, 2.30–2.23 (1H, m, H-2′b); 13C NMR (100 MHz, CDCl3) δ 158.4,
37.1, 33.6; IR (KBr) 1721 (CvO), 1521 (NO2 as), 1274 (NO2 sy) 158.0, 153.1, 150.6, 150.0, 148.3, 144.6, 142.7, 139.9, 135.9,
cm−1; [α]D22–64.8 (c 1.00, CHCl3); FAB-LRMS m/z = 885 (MH+); 130.0, 130.0, 128.2, 128.1, 127.7, 126.8, 124.9, 115.0, 113.0,
FAB-HRMS calcd for C47H45N6O10S 885.2918, found 885.2928.
108.2, 86.3, 85.5, 84.2, 72.7, 63.8, 56.6, 56.3, 55.2, 37.5, 34.1; IR
6-N-Benzoyl-5′-O-(4,4′-dimethoxytrityl)-3′-O-(N,N-diisopropyl- (KBr) 3007, 1678, 1519, 1276 cm−1; FAB-LRMS m/z = 782
β-cyanoethylphosphoramidyl)-8-(6-nitroveratrylthio)-2′-deoxy- (MH+); FAB-HRMS calcd for C40H40N5O10S 782.2496, found
adenosine (5). To a suspension of 4 (150 mg, 0.17 mmol) in 782.2531.
dry MeCN (1.7 mL) were added N,N-diisopropylethylamine
5′-O-(4,4′-Dimethoxytrityl)-3′-O-(N,N-diisopropyl-β-cyanoethyl-
(0.089 mL, 0.51 mmol) and 2-cyanoethyl-N,N′-diisopropylchloro- phosphoramidyl)-8-(6-nitroveratrylthio)-2′-deoxyinosine (9). To
phosphoramidite (0.057 mL, 0.26 mmol) at room tempera- a solution of 8 (690 mg, 0.88 mmol) in dry MeCN (8.8 mL) was
ture. After being stirred for 30 min, the resulting mixture was added N,N-diisopropylethylamine (0.46 mL, 2.7 mmol) and
partitioned between AcOEt and H2O. The separated organic 2-cyanoethyl-N,N′-diisopropylchlorophosphoramidite (0.29 mL,
layer was washed with saturated aqueous NaHCO3, followed by 1.3 mmol) at room temperature. After being stirred for 30 min
brine, then dried (Na2SO4) and concentrated in vacuo. The at room temperature, the resulting mixture was partitioned
residue was purified on a silica gel column eluted with hexane– between AcOEt and H2O. The separated organic layer was
AcOEt (3 : 2 with 0.5% Et3N) to give 5 (142 mg, 77%) as a yellow washed with saturated aqueous NaHCO3, followed by brine,
foam; 31P NMR δ 141.4, 141.1; FAB-LRMS m/z = 1085 (MH+); then dried (Na2SO4) and concentrated in vacuo. The residue
FAB-HRMS calcd for C56H62N8O11PS 1085.3996, found was purified on a silica gel column eluted with hexane–AcOEt
1085.4053.
(1 : 4 with 0.5% Et3N) to AcOEt : MeOH (10 : 1 with 0.5% Et3N)
8-(6-Nitroveratrylthio)-2′-deoxyinosine (7). To a solution of to give 9 (600 mg, 74%) as a yellow foam; 31P NMR δ 148.6,
619 (1.65 g, 5.00 mmol) in dry DMF (50.0 mL) were added 148.4; FAB-LRMS m/z = 982 (MH+); FAB-HRMS calcd for
K2CO3 (829 mg, 6.00 mmol) and 2 (1.37 g, 6.00 mmol) at room C49H57N7O11PS 982.3574, found 982.3625.
temperature. After being stirred for 24 h at room temperature,
Oligonucleotide synthesis
the solvent was removed in vacuo. The residue was purified on
a silica gel column eluted with AcOEt to AcOEt–MeOH (10 : 1) Solid-phase oligonucleotide synthesis was performed on an
to give 7 (596 mg, 25%) as a yellow powder; 1H NMR nS-8 Oligonucleotides Synthesizer (GeneDesign, Inc.) using
(400 MHz, DMSO-d6) δ 12.5 (1H, brs, NH), 8.02 (1H, s), 7.68 commercially available reagents and phosphoramidites. The
(1H, s), 7.47 (1H, s), 6.14 (1H, t, J = 6.5 Hz, H-1′), 5.33 (1H, brs, modified phosphoramidite was incorporated into the oligo-
OH), 4.91 (1H, brs, OH), 4.79 and 4.75 (each 1H, each d, J = nucleotide with a coupling efficiency comparable to that
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of commercially available phosphoramidites without any
modifications to the coupling conditions. Oligonucleotides
were synthesized (with trityl-off) on a 500 Å CPG solid support
column (0.2 μmol scale) using 5-(bis-3,5-trifluoromethylphe-
nyl)-1H-tetrazole (0.25 M in MeCN) as the activator. Cleavage
from the solid support and deprotection were accomplished
with concentrated ammonium hydroxide solution at 55 °C for
12 h. The crude oligonucleotides were purified on a Nap 10
column (GE Healthcare) followed by RP-HPLC on a XBridge™
OST C18 column, 2.5 μm, 10 × 50 mm (Waters) using MeCN in
0.1 M triethylammonium acetate buffer (pH 7.0). The purified
oligonucleotides were quantified by UV absorbance at 260 nm
and confirmed by MALDI-TOF mass spectrometry.
Notes and references
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Acknowledgements
This work was supported by the Japan Society for the Pro-
motion of Science (JSPS), the Ministry of Education, Culture,
Sports, Science and Technology (MEXT), and the Advanced
Research for Medical Products Mining Programme of the
National Institute of Biomedical Innovation (NIBIO), Japan.
This journal is © The Royal Society of Chemistry 2014
Org. Biomol. Chem., 2014, 12, 2468–2473 | 2473