Synthesis of terminally modified oligonucleotides and their hybridization dependence on the size of the target RNAs

Article abstract of DOI:10.1039/b900301k

We have developed new artificial oligonucleotide probes that show selective recognition for short RNA targets over long RNA targets. Our results suggested that modification of the termini of the oligonucleotide probes by bulky substituents such as cyclohe

Full text of DOI:10.1039/b900301k

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
Synthesis of terminally modified oligonucleotides and their hybridization
dependence on the size of the target RNAs†
Kohji Seio,* Yusuke Takaku, Kazuya Miyazaki, Sayako Kurohagi, Yoshiaki Masaki, Akihiro Ohkubo and
Mitsuo Sekine*
Received 8th January 2009, Accepted 16th March 2009
First published as an Advance Article on the web 17th April 2009
DOI: 10.1039/b900301k
We have developed new artificial oligonucleotide probes that show selective recognition for
short RNA targets over long RNA targets. Our results suggested that modification of the
termini of the oligonucleotide probes by bulky substituents such as cyclohexyl and
4-(3,6,9-trioxaundecylenedioxy)phenyl (Bzcr) groups significantly improved the selectivity of the probes
toward the short RNA targets. The selectivity was further improved by the addition of a phosphate
group on the cyclohexane ring. Although much improved selectivity toward short RNA targets is
desirable in a general sense, it is particularly applicable to the selective detection of matured-miRNA
over pre-miRNAs.
Introduction
MicroRNAs are a group of noncoding RNAs having 18–25
nucleotide residues,1 which regulate a wide range of biological
functions such as development, differentiation, and metabolism.2–7
Several methods have been reported for the analysis of miRNAs
by use of microarray8–11 and PCR12–13 technologies. It is well
known that mature miRNAs are excised from longer pre-miRNAs
having stem–loop structures.1 Therefore, miRNA detection needs
to be performed in both a sequence- and size-selective manner.
Fig. 1 Schematic representation of the possible factors affecting the
In general, mature miRNAs can be detected by the above-
mentioned detection methods because of the lower abundance of
pre-miRNAs11,14 (the stem–loop structures of pre-miRNAs that
block the hybridization of the oligonucleotide probes), and the
inefficiency of the enzymatic labeling of the large pre-miRNAs.11
However, recent studies have revealed that the processing of pre-
miRNA and pri-miRNAs are regulated post-transcriptionally and
that the pre-miRNAs have become more abundant than the mature
miRNAs in some cases.15–16 Therefore, methods that can selectively
detect mature miRNAs are required for more detailed miRNA
analyses. Based on this consideration, we attempted to develop
new 2¢-O-methyl-RNA probes that could bind to short RNA
targets more tightly than long RNA targets, simply by adding
bulky or anionic substituents to the termini of the oligonucleotide
probes (Fig. 1). We methylated the 2¢-O-positions of the probes
considering the superiority of the 2¢-O-methyl-RNA to DNA and
RNA in terms of the strong affinity toward RNA targets.17 It
was expected that terminal modification would induce short-RNA
selectivity, either by lowering the stability of the duplexes with the
long RNA targets by steric and electrostatic interactions (Fig. 1a),
or by stabilizing the duplexes through terminal stacking effects
(Fig. 1b).
stability of duplexes of the terminally substituted oligonucleotide (blue)
with long RNA (red) and short RNA (green). a) The duplex with long
RNA can be destabilized by steric and electrostatic repulsion. b) The
duplex with short RNA can be stabilized by attractive interactions such as
stacking or van der Waals interactions at the termini.
As the substituents, we chose the N-carbamoylnucleosides hav-
ing bulky groups (Scheme 1A) and synthesized the corresponding
phosphoramidites 1a–1f, as shown in Schemes 1B, 3, 5 and 6, and
the oligonucleotides, as shown in Schemes 4 and 7. Previously,
incorporation of N-arylcarbamoylnucleosides into DNA duplexes
was reported to stabilize DNA duplexes by intercalation into the
opposite strands.18 We hypothesized that the replacement of the
intercalative aryl group to non-intercalative groups such as the
cyclohexyl and polyether rings might result in the de-stabilization
of duplexes containing long RNAs by steric interactions, while
the stability of duplexes containing short RNAs (Fig. 1) could
be maintained or increased. In addition, we studied the effects of
adding a phosphate group (carried out by incorporating dAChcmP
;
Scheme 1A) on the hybridization and size discrimination of the
target RNAs.
Results and discussion
Preparation of cytidine 3¢-phosphoramidite derivatives 1a–c and
oligonucleotides incorporating them
Department of Life Science, Tokyo Institute of Technology, 4259 Nagatsuka,
Midori-ku, Yokohama, 226-8501, Japan. E-mail: kseio@bio.titech.ac.jp,
msekine@bio.titech.ac.jp; Tel: +81 45 924 5706
† Electronic supplementary information (ESI) available: NMR spectra.
See DOI: 10.1039/b900301k
We first designed the deoxycytidine derivatives dCChcm, dCBzcr, and
their 3¢-phosphoramidite derivatives 1a and 1b shown in Scheme 1.
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which was subjected to the usual tritylation to give 3a. Subse-
quently, compound 3a was converted to the phosphoramidite 1a
in 41% yield, according to the usual procedure.19
Next, we synthesized the nucleoside precursor 3b containing
Bzcr, as shown in Scheme 3. 3¢,5¢-O-Bis(t-butyldimethylsilyl)-
deoxycytidine (4b)20 was treated with phenyl chloroformate in the
presence of pyridine to give the carbamate intermediate, which
was coupled with 4-(3,6,9-trioxaundecylenedioxy)phenylamine at
70 ◦C. Subsequently, the TBDMS groups were removed to give the
nucleoside 2b (dCBzcr) in 84% yield. Treatment of 2b with DMTrCl
gave the DMTr derivative 3b in 82% yield. The 5¢-protected
derivative 3b was then phosphitylated to give the phosphoramidite
unit 1b in 78% yield. Similarly, the 2¢-O-methyl derivatives 1c, 2c
(CmBzcr) and 3c were synthesized from 4c (Scheme 3).21
Scheme 1
Compounds 1a and 1b have an N-cyclohexylcarbamoyl group and
a 4-(3,6,9-trioxaundecylenedioxy)phenyl moiety (abbreviated as
Chcm and Bzcr (benzocrown ether) in this paper) on the amino
Scheme 3
By use of these phosphoramidites and commercially avail-
able universal support II,22 we synthesized the various 2¢-
O-methyl-RNA probes such as probes 2–4 having 5¢-Y-
CmAmAmCmCmUmAmCmUm-Z-3¢ sequences, where Y and Z rep-
resents the modified cytidine residue incorporated by the use of
1a–1c. The sequences and the structures of the probes are shown
in Table 1 and Scheme 4A, respectively.
group as a bulky substituent, chosen because of the commercial
Bzcr
availability of the reagents. We also designed Cm
and its
phosphoramidite compound 1c, the 2¢-O-methyl derivative of 1b,
to examine the effects of the sugar structure on the hybridization
properties.
The preparation of 1a is shown in Scheme 2. Deoxycytidine
hydrochloride was treated with cyclohexyl isocyanate to give 4-N-
(N-cyclohexylcarbamoyl)deoxycytidine (2a, dCChcm) in 89% yield,
Hybridization properties of probes 1–4 to the short and long RNAs
The hybridization properties of probes 2–4 toward short RNA and
long RNAs (Scheme 4D) were studied by measuring UV-melting
temperatures (Tm) and compared with those of probe 1 (Y, Z = dC)
without the modifications at the terminal deoxycitidine residues.
Here, short RNA is the 9-mer RNA having 3¢-GUUGGAUGA-
5¢ sequence complementary to the 5¢-CmAmAmCmCmUmAmCmUm-
3¢ sequence of probes 2–4, and long RNA is the 22-mer RNA
having additional 7-mer and 6-mer sequences at the 3¢- and
5¢-sites, respectively. As shown in Table 1, probe 1 (having unmodi-
fied deoxycytidines at both termini) bound to short and long RNAs
with identical affinities, both Tm values being 45 ◦C. In contrast,
the introduction of the N-cyclohexylcarbamoyl group (probe 2◦: Y,
Z = dCChcm) increased the Tm of the duplex with short RNA by 4 C,
keeping Tm of the duplex with long RNA unchanged. As the result,
Scheme 2
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Table 1 Tm (◦C) of the duplexes formed between the 2¢-O-methyl-RNA probes: 5¢-Y-CmAmAmCmCmUmAmCmUm-Z-3¢ and the short RNA or long RNA
targets
a
Short RNA (9-mer)
Long RNA (22-mer)
DTm
Probe 1 (Y, Z = dC)
45
49
57
58
53
50
45
45
53
55
49
47
43
40
42
0
+4
+4
+3
Probe 2 (Y, Z = dCChcm
)
Probe 3 (Y, Z = dCBzcr
)
)
Bzcr
Probe 4 (Y, Z = Cm
Probe 5 (Y, Z = dABzcr
)
+4
+3
Probe 6 (Y, Z = dAChcm
)
Probe 7 (Y = dAChcmP
)
52 (51)b
47
+9 (+8)b
+7
Probe 8 (Y = dAChcmP, Z = none)
Probe 9 (Y = none, Z = dAChcmP
)
45
+3
a DTm = (Tm of short-RNA) - (Tm of long-RNA). b The Tm and DTm of the experiment using short pRNA.
Scheme 4
probe 2 (Y, Z = dCChcm) showed higher affinity toward s◦hort RNA
than long RNA, as indicated by the DTm value of +4 C, which
was calculated by subtracting the Tm value with long RNA from
that with short RNA. These results indicated that the terminally
modified oligonucleotide probes showed selectivity toward short
RNA, and that the mechanism of the selectivity was not due to the
destabilization of the duplex with long RNA by steric hindrance,
but due to the stabilization of the duplex with short RNA by the
attractive interactions at the termini, as shown in Fig. 1b.
The steric effects of probe 3 (Y, Z = dCBzcr) with the larger Bzcr
groups were also examined. As shown in Table 1, the introduction
of the Bzcr group increased the affinity toward short and long
RNAs significantly by 12 ◦C and 8 ◦C, respectively, compared
with those of probe 1 (Y, Z = dC). This is probably because of the
stacking effects of the terminal benzene rings toward the opposite
bases. However, the DTm value did not improve compared with
that of the probe 2, which has smaller cyclohexane rings.
Next, the properties of probe 4 (Y, Z = CmBzcr) having
2¢-O-methylribose structure were examined to see the effects of
the conformational restraint on the sugar moiety.23 However, no
significant improvement was observed, either in the hybridization
property (Tm) or selectivity (DTm) relative to probe 3. Therefore,
we decided to optimize the structure of the modified nucleosides
at the terminus by using 2¢-deoxynucleotides.
Preparation of deoxyadnosine 3¢-phosphoramidite derivatives 1d
and 1e and oligonucleotides incorporating them
In order to increase the selectity toward short RNA of the 2¢-O-
methyl-RNA probes, we designed probes 5 (Y, Z = dABzcr) and 6
(Y, Z = dAChcm) with the adenine bases modified with the Bzcr
and cyclohexane rings, respectively, and probe 7 (Y, Z = dAChcmP
)
having the phosphate groups on the cyclohexane ring (Scheme 4B).
Probes 5 and 6 are designed in this manner since it was expected
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that the larger purine base could more effectively interact with the
opposite strands than the modified cytosine bases in probes 2 and
3. We also designed probe 7 with the terminal phosphate groups,
expecting that the anionic phosphate group could destabilize the
duplex with long RNA by the electrostatic interactions with the
phosphate backbone, as shown in Fig. 1a.
For these purposes, we prepared the phosphoramidites 1d and
1e, as shown in Scheme 5. Briefly, the protected deoxyadenosine
4d was converted to 2d and 2e according to the procedure
for 2b and 2c, respectively. Subsequently, 5¢-protection and
3¢-phosphitylation were performed in the usual manner to give
1d and 1e.
group (Lev) to give 6 in 64% yield. Desilylation followed by
tritylation gave the nucleoside 8, which was converted to the
phosphoramidite 1f in 77% yield by a similar procedure to that
used for 1a–c.19
Using phosphoramidites 1d, 1e and 1f, we prepared probes 5, 6,
and 7, respectively. Probes 5 (Y, Z = dABzcr) and 6 (Y, Z = dAChcm
)
were synthesized according to the standard phosphoramidite
chemistry protocol.
The phosphorylated oligonucleotide probe 7 (Y, Z = dAChcmP
)
was synthesized, as schematically shown in Scheme 7, using
the phosphoramidite 1f and 2-cyanoethyl 2-(4,4¢-dimethoxy-
trityloxyethylsulfonylethyl)
N,N-diisopropylphosphoramidite
(PhosphalinkTM) as the in situ phosphorylating agent during
the solid-phase DNA synthesis.24 First, the fully protected
oligonucleotide was synthesized on the CPG having the universal
linker. After the chain elongation, the Lev groups were removed
by treatment with hydrazine acetate, and the liberated hydroxyl
group was phosphorylated by treatment with Phosphalink.
Finally, the cleavage of the partially protected oligonucleotide
from the solid supports and the deprotection of the base and the
phosphate residues were performed by treatment with methanolic
ammonia. The following standard DMTr-ON purification gave
the desired probe 7.
Scheme 5 Reagents: i) PhOC(O)Cl, ii) cyclohexylamine or Bzcr-NH2,
iii) Et3N·3HF, Et3N.
We also prepared the phosphoramidite 1f for the synthesis of
probe 7 having dAChcmP at both ends (Scheme 6). Compound 4d was
converted to the N-(4-hydroxycyclohexyl)carbamoyl derivative 5
in 76% yield. The hydroxyl group of 5 was protected by a levulinoyl
Scheme 7
Hybridization properties of probes 5–7 to short and long RNAs
The Tm values of the duplex of probes 5–7 with short RNA and
long RNA were measured. The results are also shown in Table 1.
As shown in the table, Tm of the duplex probe 5/short RNA was
53 ◦C and that with long RNA was 49 ◦C, each of which was lower
than corresponding Tm of the cytidine derivative probe 3/short
RNA and probe 3/long RNA.◦Despite the decrease in these Tm
values, DTm of probe 5 was 4 C, which was identical to that of
probe 3. In the case of probe 6, the Tm values of the duplexes with
short RNA and long RNA were 50 ◦C and 47 ◦C, respectively.
The observed DTm (3 ◦C) was close to 4 ◦C (49–45 ◦C) of the
corresponding cytidine derivative probe 2. Although the modified
deoxyadenosine residues at the terminus of probe 5 and probe
Scheme 6
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6 could potentially form Watson–Crick hydrogen bonds with the
uridine resiudes at the opposite sites of long RNA, the lower affinity
of these probes toward long RNA indicated that these modified
adenosine residues did not form such hydrogen bonds probably
due to the steric hindrance by the substituents.
Interestingly, anionic modification of the oligonucleotide probe
improved the selectivity toward short RNA of probe 7 to give a
DTm value of +9 ◦C. The effect of the phosphate groups could be
extracted by comparing with the data of probe 6. Phosphorylation
slightly increased the stability with short RNA, as shown by Tm
values of probe 6/short RNA (50 ◦C) and probe 7/short RNA
(52 ◦C). On the other hand, the Tm for the duplex of probe
7/long RNA (43 ◦C) was destabilized by 4 ◦C by phosphorylation
relative to probe 6/long RNA (47 ◦C). These results indicated
that the increase in the selectivity toward short RNA could be
due to the destabilization of the duplex with long RNA probably
because of anionic repulsion between the phosphate groups and
the phosphodiester backbone of the long RNA target. Thus, it
was suggested that the presence of the anionic substituents at
the proper position of the probe’s termini could improve the size
selectivity of the oligonculeotide probes.
Fig. 2 Model structures of the (top) 5¢-terminal and (bottom) 3¢-terminal
regions of probe7 in probe7/short RNA duplex.
Considering the fact that the 5¢-hydroxyl group of matured
miRNA is generally phosphorylated, we also tested the hy-
bridization of probe 7 on short pRNA (Scheme 4D) with 5¢-
pAGUAGGUUG-3¢ sequence in which the 5¢-hydroxyl group was
phosphorylated. As a result, the Tm of probe 7/short pRNA duplex
the 3¢-terminal nucleobase of short RNA. These results clearly
show that substituents attached to the base moiety can effectively
interact with the opposite strand. In addition, the phosphate
◦
˚
was 51 C, which is almost identical to that of the probe 6/short
group on the cyclohexane ring is in close proximity (<4 A) to
RNA duplex. This result indicates that the presence of the 5¢-
phosphate on the target RNA does not affect the duplex stability.
the 3¢-hydroxyl group of short RNA, from which the additional
5¢-UAUAUUA-3¢ sequence would extend to long RNA. In con-
trast, the cyclohexane ring at the 3¢-terminus of probe 7 did not
stack on the 5¢-terminal nucleobase of short RNA, and the distance
between the phosphate group and the 5¢-hydroxyl group of short
Contribution of the 5¢ and 3¢ modifications to the DTm value
˚
As described above, our results suggested that the 11-mer
oligonucleotides with both terminal bases modified by bulky
anionic substituents could selectively recognize short 9-mer RNA
over long 22-mer RNA. In order to clarify the contribution of
5¢-terminal and 3¢-terminal modifications on the duplex stability
and the short RNA selectivity, we synthesized probes 8 (Y =
dAChcmP, Z = none) and 9 (Y = none, Z = dAChcmP) lacking the
3¢- and 5¢-terminal modifications, respectively (Scheme 4C). The
Tm values of the duplexes of probe 8 or 9 with short RNA and
long RNA are also shown in Table 1. The duplex of the probe 8
and short RNA was more stable (Tm 47 ◦C) than the duplex with
long RNA (Tm 40 ◦C), with a DTm value of +7 ◦C. In the case of
the 3¢-terminal-modified probe 9, the stability with short RNA was
lower (Tm 45 ◦C), while that with long RNA was higher (Tm 42 ◦C),
with a consequent decrease in selectivity (DTm +3 ◦C). This result
clearly shows that 5¢-terminal modification contributes more than
3¢-terminal modification to the short RNA selectivity. Interestingly,
the sum of DTm of the 5¢-modified probe 8 and the 3¢-modified
probe 9 was +10 ◦C, which is close to DTm = +9 ◦C of probe 7.
RNA was rather long (ca. 10 A). Thus, effects arising from the
weak steric and electrostatic repulsion were expected even when
the additional 5¢-AUUGGUA-3¢ sequence was extended from the
5¢-hydroxyl group. This structural model obtained by the MD
calculation suggests a possible explanation for the contribution of
the 5¢-substituent to the short RNA selectivity being larger than
the 3¢-substituent.
Conclusions
In this paper, we have proposed a new design for artificial
oligonucleotide probes, enabling them to selectively recognize
a shorter RNA target over a longer RNA target. Our results
suggest that the modification of the terminus of the oligonucleotide
probes by bulky substituents such as cyclohexane and Bzcr
groups improved the selectivity of the probes toward short RNA
targets. The selectivity was further improved by the addition of
the phosphate group to the cyclohexane ring. We compared the
contribution of the 5¢- and 3¢-terminal modifications toward the
short RNA selectivity and proved that 5¢-modification contributed
more.Similar selectivity over the longer RNA target was also
observed even when the 5¢-end of the target RNA was phospho-
rylated.
Molecular modeling of the duplex
In order to visualize the positions of the phophorylated cyclohex-
ane ring at the 5¢- and 3¢-termini, we performed 15 ns molecular
dynamic simulation of the probe 7/short RNA duplex. Shown in
Fig. 2 is the average structure for the last 5 ns. As shown in Fig. 2,
the cyclohexane ring at the 5¢-terminus of probe 7 stacked with
The maximum selectivity toward the short RNA target (as
measured by the DTm value) was 9 ◦C in the case of the probe
modified at the both ends. However, because we used very short
9-mer RNA as the model compound of matured miRNA, this
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◦
value of 9 C would not be sufficient for real ~25-mer miRNAs
dine (28 mL). To this solution, 4,4¢-dimethoxytrityl chloride
(1.15 g, 3.41 mmol) was added and the resulting solution was
stirred at ambient temperature for 3 h. After the reaction was
quenched by addition of methanol (3 mL), the solvents were
removed under reduced pressure. The residue was dissolved in
ethyl acetate (30 mL) and the solution was washed three times
with saturated aq. NaHCO3 (30 mL each), dried over Na2SO4,
filtered and concentrated under reduced pressure. The residue was
chromatographed on silica gel column with chloroform-methanol
(10:1, v/v) containing 0.5% pyridine to give 3a (1.45 g, 78%); dH
(CDCl3, 500 MHz) 1.18–1.33 (5H, br), 1,57–1.59 (1H, br), 1.72
(2H, br), 1.90 (2H, br), 2.16–2.21 (1H, m), 2.57–2.60 (1H, br),
3.39 (1H, dd, J = 3.2, 10.6), 3.47 (1H, dd, J = 3.7 Hz, 10.6 Hz),
3.62 (1H, br), 3.79 (6H, s), 4.09 (1H, d, J = 3.9 Hz), 4.44 (1H,
br), 6.23 (1H, t, J = 5.7 Hz), 6.85 (4H, d, J = 8.3 Hz), 7.21–
7.44 (9H, m), 8.01 (1H, d, J = 7.6 Hz), 8.57 (1H, br), 10.86 (1H,
br); dC (CDCl3) 25.2, 25.7, 33.0, 41.9, 49.4, 55.4, 62.9, 71.3, 86.2,
86.7, 87.1, 113.5, 127.3, 128.2, 128.3, 129.2, 130.1, 135.5, 135.6,
targets. Interestingly, we also found that probe 8 lacking 3¢-
modification showed a rather large DTm of 7 ◦C. This result
indicated the possibility of using 5¢-modified probes such as
probe 8 to discriminate the 3¢-end of matured miRNA from the
unprocessed pre-miRNAs. Although improved selectivity toward
short RNA targets might be necessary, such short-RNA selective
binding of terminally modified oligonucleotides seems to be
applicable for the selective detection of matured-miRNA over
pre-miRNAs. Currently, such discrimination is performed using
hairpin-type oligonucleotide probes in combination with PCR12
and oligonucleotide microarray25 technologies. Although such a
hairpin design has been reported to be effective to discriminate
matured miRNAs from pre-miRNAs, the formation of the hairpin
structures required rather long stem–loop regions in the probes,
which are not necessary if size discrimination could be achieved
by use of simple 5¢- and 3¢-terminal modifications. Improvement
in the selectivity toward short RNA targets of terminally modified
oligonucleotides and development of applications to miRNA
detection and microarray techniques are underway, and will be
reported elsewhere.
+
142.3, 144.3, 158.8; HRMS (ESI) m/z calcd for C37H42N4NaO7 :
677.2946, found 677.2994.
Experimental section
4-N-[N-[3,4-(3,6,9-Trioxaundecylenedioxy)phenyl]carb-
amoyl]deoxycytidine (2b)
General
3¢,5¢-O-Bis(t-butyldimethylsilyl)deoxycytidine
(4b:
1.0
g,
1H, 13C and 31P NMR spectra were obtained at 500, 126 and
203 MHz, respectively. The chemical shifts were measured relative
2.20 mmol) was dissolved in dichloromethane (22 mL). To
this solution, pyridine (266 mL, 3.30 mmol) and phenyl
chloroformate (330 mL, 2.64 mmol) were added, and the resulting
solution was stirred at ambient temperature for 30 min. The
reaction mixture was diluted with chloroform (30 mL) and washed
three times with saturated NaHCO3 (30 mL each). The organic
layer was dried over Na2SO4, filtered and concentrated under
reduced pressure. The residue was co-evaporated three times with
anhydrous pyridine and then dissolved in anhydrous pyridine
(22 mL). To this solution, 4¢-aminobenzo-15-crown[5]ether
(1.87 g, 6.60 mmol) was added and the resulting mixture was
stirred 3 h at 70 ◦C. After being cooled to room temperature,
the solvent was removed under reduced pressure and the residue
was diluted with chloroform (30 mL). The solution was washed
three times with saturated aq. NaHCO3 solution (30 mL each),
dried over Na2SO4, filtered and concentrated under reduced
pressure. The residue was co-evaporated with three times with
anhydrous pyridine and then dissolved in anhydrous pyridine
(22 mL). To this solution, 3HF·NEt3 (1.8 mL, 11 mmol) and
NEt3 (1.5 mL, 11 mmol) were added. The solution was stirred
at ambient temperature for 1 d and the solvent removed under
reduced pressure. The residue was triturated with a mixture of
chloroform (10 mL) and ethyl acetate (30 mL) to give 2b (992 mg,
84%). dH (DMSO, 500 MHz) 2.02–2.04 (1H, m), 2.25–2.28 (1H,
m), 3.54–3.61 (10H, m), 3.74–3.78 (4H, m), 3.84–3.85 (1H, br),
4.01–4.04 (4H, br), 4.21–4.22 (1H, br), 5.07 (1H, t, J = 5.2 Hz),
5.30 (1H, d, J = 4.2 Hz), 6.12 (1H, t, J = 6.3 Hz), 6.36–6.37 (1H,
br), 6.91–6.93 (2H, br), 7.18 (1H, s), 8.23 (1H, d, J = 7.6 Hz),
10.08 (1H, s), 11.16 (1H, br); dC (DMSO) d 40.8, 45.8, 61.0,
68.5, 68.8, 69.0, 69.8, 69.9, 70.0, 70.4, 86.1, 87.9, 94.9, 106.6,
112.0, 114.6, 131.9, 143.9, 144.9, 148.8, 151.2, 153.5, 162.3;
HRMS (ESI) m/z calcd for C24H32N4NaO10+: 559.2011, found
559.2006.
1
to tetramethylsilane (0.0 ppm) and DMSO-d6 (2.49 ppm) for H
NMR, CDCl3 (77.0 ppm) and DMSO-d6 (39.7 ppm) for 13C NMR
and 85% phosphoric acid (0.0 ppm) for 31P NMR.
4-N-(N-Cyclohexylcarbamoyl)deoxycytidine (2a)
Deoxycytidine hydrochloride (5.0 g, 19.0 mmol) was dissolved in
anhydrous N,N-dimethylformamide (190 mL). To this solution,
triethylamine (2.6 mL, 19.0 mmol) and cyclohexyl isocyanate
(2.7 mL, 21 mmol) were added, and the resulting solution was
stirred at 50 ◦C for 1 d. After the completion of the reaction, the
solvents were removed under reduced pressure and the residue
was diluted with a mixture of chloroform (150 mL) and isopropyl
alcohol (50 mL). The organic layer was washed three times with
water (200 mL each), dried over MgSO4, and concentrated under
reduced pressure. The residue was chromatographed on a silica gel
column (100 g) by use of chloroform-methanol (8:1, v/v) to give
2a (5.94 g, 89%); dH (DMSO, 500 MHz) 1.21–1.34 (5H, m), 1.50–
1.52 (1H, br), 1.63–1.66 (2H, br), 1.78–1.81 (2H, br), 1.96–2.01
(1H, m), 2.20–2.25 (1H, m), 3.52–3.62 (3H, m), 3.82 (1H, dd, J =
3.7 Hz, J = 7.3 Hz), 4.18–4.22 (1H, m), 5.02 (1H, t, J = 5.2 Hz),
5.25 (1H, d, J = 4.2 Hz), 6.09 (1H, t, J = 6.3 Hz), 6.24 (1H, br),
8.15 (1H, d, J = 7.3 Hz), 9.79 (1H, br); dC (DMSO) 23.9, 25.2, 23.4,
40.7, 47.7, 61.0, 70.0, 85.9, 87.8, 94.6, 143.3, 152.7, 153.4, 162.3;
+
HRMS (ESI) m/z calcd for C16H24N4NaO5 : 375.1639, found
375.1663.
4-N-(N-Cyclohexylcarbamoyl)-5¢-O-
(4,4¢-dimethoxytrityl)deoxycytidine (3a)
Coumpound 2a (1.0 g, 2.84 mmol) was co-evaporated three
times with anhydrous pyridine and dissolved in anhydrous pyri-
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5¢-O-(4,4¢-Dimethoxytrityl)-4-N-[N-[3,4-(3,6,9-
trioxaundecylenedioxy)phenyl]carbamoyl]deoxycytidine (3b)
5¢-O-(4,4¢-Dimethoxytrityl)-2¢-O-methyl-4-N-[N-[3,4-(3,6,9-
trioxaundecylenedioxy)phenyl]carbamoyl]-deoxycytidine (3c)
Compound 2b (1.2 g, 2.24 mmol) was co-evaporated three times
with anhydrous pyridine and then dissolved in anhydrous pyridine
(22 mL). To this solution, 4,4¢-dimethoxytrityl chloride (909 mg,
2.68 mmol) was added and the solution was stirred at ambient
temperature for 3 h. After the reaction was quenched by the
addition of methanol (3 mL), the solution was diluted with ethyl
acetate (20 mL), washed three times with saturated aq. NaHCO3
(20 mL each), dried over Na2SO4, filtered and concentrated under
reduced pressure. The residue was chromatographed on a silica
gel column (16 g) with chloroform-methanol (99:1, v/v) to give
3b (1.15 g, 82%). dH (CDCl3, 500 MHz) 2.13 (1H, br), 2.51 (1H,
br), 3.29–3.32 (1H, dd, J = 3.2 Hz, J = 10.6 Hz), 3.39–3.42 (1H,
dd, J = 3.7 Hz, J = 10.6 Hz), 3.64–3.71 (8H, m), 3.73 (6H, s),
3.78–3.84 (4H, m), 3.98–4.13 (5H, m), 4.38 (1H, br), 6.19 (1H, t,
J = 5.9 Hz), 6.73–6.80 (4H, m), 7.16- 7.40 (11H, m), 8.01 (1H, d,
J = 7.3 Hz), 10.91–10.95 (2H, br); dC (CDCl3) 41.8, 55.4, 63.1,
68.5, 69.5, 69.6, 69.8, 70.2, 70.4, 70.7, 70.8, 86.0, 86.8, 97.6, 106.0,
111.7, 113.4, 114.9, 127.1, 128.1, 128.3, 130.1, 133.9, 135.7, 135.8,
142.4, 144.4, 144.5, 149.1, 151.5, 156.5, 158.7, 164.8; HRMS (ESI)
m/z calcd for C45H50N4NaO12+: 861.3317, found 861.3315.
Compound 2c (450 mg, 0.79 mmol) was co-evaporated three
times with anhydrous pyridine and then dissolved in anhydrous
pyridine (7.9 mL). To this solution, 4,4¢-dimethoxytrityl chloride
(323 mg, 0.95 mmol) was added and the solution was stirred at
ambient temperature for 2.5 h. After the reaction was quenched
by the addition of methanol (1 mL), the solution was diluted
with ethyl acetate (10 mL), washed three times with saturated aq.
NaHCO3 (10 mL), dried over Na2SO4, filtered and concentrated
under reduced pressure. The residue was chromatographed on
a silica gel column (30 g) with chloroform-hexane (5:1, v/v) to
give 3c (655 mg, 95%). dH (CDCl3, 500 MHz) 2.51 (1H, d, J =
10.0 Hz), 3.53–3.55 (1H, m), 3.61–3.63 (1H, m), 3.70 (3H, s), 3.76–
3.83 (18H, m), 3.90–3.92 (4H, m), 4.03–4.05 (1H, m), 4.12 (2H, t,
J = 4.5 Hz), 4.18 (2H, t, J = 4.5 Hz), 4.43–4.48 (1H, m), 6.07 (1H,
s), 6.78 (1H, d, J = 8.8 Hz), 6.86–6.88 (4H, m), 7.05- 7.08 (1H,
m), 7.24–7.42 (10H, m), 8.48 (1H, d, J = 7.8 Hz), 10.86 (1H, s),
11.26 (1H, s); dC (CDCl3) 55.4, 58.9, 59.0, 61.1, 68.1, 69.3, 69.8,
69.9, 70.7, 70.8, 71.1, 71.2, 83.3, 84.2, 87.3, 88.1, 88.2, 98.2, 107.9,
112.9, 113.5, 114.9, 127.3, 128.2, 128.5, 130.1, 130.3, 133.3, 135.5,
135.8, 143.2, 143.9, 145.5, 149.4, 151.6, 156.7, 158.8, 158.9, 165.2.
HRMS (ESI) m/z calcd for C46H52N4NaO13+: 891.3423, found
891.3406.
2¢-O-Methyl-4-N-[N-[3,4-(3,6,9-trioxaundecylenedioxy)-
phenyl]carbamoyl]cytidine (2c)
6-N-[N-[3,4-(3,6,9-Trioxaundecylenedioxy)phenyl]carbamoyl]-
deoxyadenosine (2d)
3¢,5¢-O-Bis(t-butyldimethylsilyl)-2¢-O-methylcytidine (4c: 1.0 g,
2.1 mmol) was dissolved in dichloromethane (21 mL). To this
solution, pyridine (250 mL, 3.1 mmol) and phenyl chloroformate
(309 mL, 2.5 mmol) were added, and the resulting solution was
stirred at ambient temperature for 30 min. The reaction mixture
was diluted with chloroform (20 mL) and washed three times with
saturated NaHCO3 (20 mL each). The organic layer was dried over
Na2SO4, filtered and concentrated under reduced pressure. The
residue was co-evaporated three times with anhydrous pyridine
and then dissolved in anhydrous pyridine (21 mL). To this solution,
4¢-aminobenzo-15-crown[5]ether (2.9 g, 10.3 mmol) was added
and the resulting mixture was stirred 3 h at 70 ◦C. After being
cooled to room temperature, the solvent was removed under
reduced pressure and the residue was diluted with chloroform
(20 mL). The solution was washed three times with saturated
aq. NaHCO3 solution (20 mL each), dried over Na2SO4, filtered
and concentrated under reduced pressure. The residue was co-
evaporated three times with anhydrous pyridine and then dissolved
in anhydrous pyridine (21 mL). To this solution was added
3HF·NEt3 (1.7 mL, 10.3 mmol) and NEt3 (1.0 mL, 10.3 mmol).
The solution was stirred at ambient temperature for 1 d and
the solvent removed under reduced pressure. The residue was
triturated with a mixture of chloroform (10 mL) and ethyl acetate
(30 mL) to give 2b (503 mg, 43%). dH (DMSO, 500 MHz) 3.45 (3H,
s), 3.59–3.63 (9H, m), 3.72–3.79 (6H, m), 3.88–3.89 (1H, m), 4.01–
4.07 (5H, m), 5.11 (1H, d, J = 6.8 Hz), 5.20 (1H, t, J = 5.0 Hz),
5.86 (1H, d, J = 2.7 Hz), 6.36 (1H, br), 6.90–6.94 (2H, m), 7.16
(1H, s), 8.37 (1H, d, J = 7.6 Hz), 10.10 (1H, br), 11.17 (1H, br);
dC (DMSO) 57.8, 59.5, 67.5, 68.5, 68.8, 68.9, 69.8, 69.9, 70.4, 83,6,
3¢,5¢-O-Bis-(t-butyldimethylsilyl)deoxyadenosine
(100
mg,
0.208 mmol) was dissolved in pyridine (2.1 mL). To this solution
phenyl chloroformate (57 mL, 0.458 mmol) was added and the
resulting solution was stirred at ambient temperature for 2 h.
4¢-Aminobenzo-15-crown[5]ether (295 mg, 1.04 mmol) was added
and the solution was stirred at 85◦ for 30 min. After being cooled
to room temperature, the solvent was removed under reduced and
the residue was dissolved in chloroform (5 mL) and washed three
times with saturated aq. NaHCO3 (5 mL each). The organic layer
was dried over Na2SO4, filtered and concentrated under reduced
pressure. The residue was co-evaporated twice with anhydrous
pyridine and then dissolved in anhydrous pyridine (2.1 mL).
To this solution 3HF·NEt3 (169 mL, 1.04 mmol) and NEt3
(144 ml, 1.04 mmol) were added. After being stirred at ambient
temperature for 1 d, the solvent was evaporated under reduced
pressure. The residue was dissolved in chloroform (5 mL) and
the solution was washed three times with saturated aq. NaHCO3
(5 mL each), dried over Na2SO4, filtered and concentrated under
reduced pressure. The residue was chromatographed on a silica
gel column with chloroform-methanol (200:1.5, v/v) to give 2d
(63 mg, 54%). dH (DMSO, 500 MHz) 2.35–2.37 (1H, m), 2.74–2.77
(1H, m), 3.54–3.56 (1H, m), 3.62–3.64 (9H, m), 3.76–3.80 (4H,
m), 3.90 (1H, dd, J = 4.4 Hz, J = 7.6 Hz), 4.03–4.08 (4H, m),
4.43–4.45 (1H, m), 5.02 (1H, t, J = 5.6 Hz), 5.35 (1H, d, J =
4.1 Hz), 6.45 (1H, t, J = 6.8 Hz), 6.94 (1H, d, J = 8.5 Hz), 7.10
(1H, dd, J = 2.3 Hz, J = 8.5 Hz), 7.29 (1H, d, J = 2.3 Hz), 8.67
(2H, s), 10.03 (1H, s), 11.61 (1H, s); dC (DMSO) 61.6, 68.5, 68.8,
69.0, 69.8, 70.4, 70.6, 83.8, 88.0, 106.7, 112.0, 114.6, 120.5, 132.2,
142.2, 144.7, 148.8, 150.0, 150.3, 150.8. 150.9; HRMS (ESI) m/z
84.2, 87.8, 94.7, 106.5, 112.0, 114.6, 131.7, 143.9, 144.9, 148.8,
+
151.2, 153.2, 162.3; HRMS (ESI) m/z calcd for C25H34N4NaO11
:
+
589.2116, found 589.2122.
calcd for C25H32N6NaO9 : 583.2123, found 583.2107.
2446 | Org. Biomol. Chem., 2009, 7, 2440–2451
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5¢-O-(4,4¢-Dimethoxytrityl)-6-N-[N-[3,4-(3,6,9-
trioxaundecylenedioxy)phenyl]carbamoyl]deoxyadenosine (3d)
stirred at ambient temperature for 2 h. To this solution trans-4-
aminocyclohexanol (3.6 g, 31.3 mmol) was added. After being
stirred at 85 ◦C for 30 min, the mixture was cooled to ambient
temperature, diluted with chloroform (50 mL), washed three
times with saturated NaHCO3 (50 mL), diried over Na2SO4,
filtered, and concentrated under reduced pressure. The residue
was chromatographed on an NH-silica gel column (60 g) with
hexane-chloroform (12 : 5, v/v) to give the title compound in 76%
yield (3.0 g). dH (DMSO, 500 MHz) 0.08–0.11 (12H, m), 0.79–0.93
(18H, m), 1.26–1.34 (4H, m), 1.82–1.84 (2H, br), 1.94–1.96 (2H,
br), 2.33–2.38 (1H, m), 2.91–2.96 (1H, m), 3.46–3.48 (1H, m),
3.55–3.57 (1H, m), 3.65 (1H, dd, J = 4.4 Hz, J = 11.0 Hz), 3.79
(1H, dd, J = 5.9 Hz, J = 11.0 Hz), 3.85–3.88 (1H, m), 4.57 (1H,
dd, J = 4.4 Hz), 4.63–4.66 (1H, m), 6.40 (1H, t, J = 6.6 Hz);
8.52 (1H, s), 8.57 (1H, s), 9.35 (1H, d, J = 7.6 Hz), 9.54 (1H,
s); dC (DMSO) -5.5, -5.0, -4.8, 17.7, 17.9, 25.7, 30.4, 33.5, 38.5,
48.0, 62.4, 67.8, 71.8, 83.5, 87.1, 120.3, 142.1, 150.0, 150.3, 150.7,
Compound 2d (500 mg, 0.892 mmol) was co-evaporated three
times with anhydrous pyridine and then dissolved in anhydrous
pyridine (8.9 mL). To this solution DMTrCl (363 mg, 1.07 mmol)
was added and the resulting solution was stirred at ambient
temperature for 1 d. After the reaction was quenched by addition
of methanol (1 mL), the solution was diluted with chloroform
(10 mL). The solution was washed three times with saturated aq.
NaHCO3 (10 mL each), dried over Na2SO4, filtered and concen-
trated under reduced pressure. The residue was chromatographed
on an NH–silica gel column (50 g) with chloroform-methanol
(100:1, v/v) to give 3d (547 mg, 71%). dH (CDCl3, 500 MHz) 2.56–
2.61 (1H, m), 2.83–2.88 (1H, m), 3.36–3.44 (2H, m), 3.77 (14H,
s), 3.90–3.92 (4H, m), 4.12–4.19 (5H, m), 4.69–4.70 (1H, br), 6.45
(1H, t, J = 6.3 Hz), 6.78–6.85 (5H, m), 6.97 (1H, dd, J = 2.4 Hz,
J = 8.5 Hz), 7.20–7.39 (10H, m), 8.07 (1H, br), 8.11 (1H, s), 8.51
(1H, s), 11.55 (1H, s); dC (CDCl3) 40.1, 45.7, 55.3, 63.9, 68.8, 69.5,
69.7, 70.4, 70.5, 70.9, 72.2, 84.6, 86.3, 106.9, 112.5, 113.2, 114.8,
120.9, 127.0, 127.9, 128.2, 130.1, 130.2, 132.3, 135.8, 141.7, 144.7,
145.3, 149.3, 149.9, 150.2, 150.8, 151.4, 158.6; HRMS (ESI) m/z
calcd for C46H50N6NaO11+: 885.3430, found 885.3413.
+
152.6; HRMS (ESI) m/z calcd for C29H52N6NaO5Si2 : 643.3430,
found 643.3407.
3¢,5¢-O-Bis(tert-butyldimethylsilyl)-6-N-[N-(trans-4-
levulinyloxycyclohexyl)carbamoyl]deoxyadenosine (6)
Compound 5 (2.2 g, 3.55 mmol) was rendered anhydrous by
repeated coevaporation with anhydrous pyridine and finally dis-
solved in anhydrous dichloromethane (36 mL). To this solution le-
vulinic acid (729 mg, 7.09 mmol), N,N¢-dicyclohexylcarbodiimide
(1.46 g, 7.09 mmol) and 4-dimethylaminopyridine (43 mg,
0.34 mmol) were added, and the resulting solution was stirred
at ambient temperature for 1 h. The precipitate was removed by
filtration and washed with dichloromethane (50 mL). The filtrate
was washed three times with saturated aq. NaHCO3 (50 mL
each), dried over Na2SO4, filtered and concentrated under reduced
pressure. The residue was chromatographed on an NH-silica gel
column (50 g) with hexane-chloroform (2:1, v/v) to give 6 (1.62 g,
64%). dH (CDCl3, 500 MHz) 0.08–0.10 (12H, m), 0.90–0.91 (18H,
m), 1.47–1.57 (4H, m), 2.00–2.03 (2H, br), 2.14–2.17 (2H, br), 2.19
(3H, m), 2.43–2.48 (1H, m), 2.54–2.58 (2H, m), 2.60–2.65 (1H, m),
2.73–2.76 (2H, m), 3.76 (1H, dd, J = 3.2 Hz, J = 11.2 Hz), 3.84–
3.88 (2H, m), 4.02 (1H, dd, J = 3.4 Hz, J = 7.1 Hz), 4.60–4.63
(1H, m), 4.76–4.80 (1H, m), 6.47 (1H, t, J = 6.3 Hz), 7.88 (1H, s),
8.27 (1H, s), 8.50 (1H, s), 9.40 (1H, d, J = 7.6 Hz); dC (CDCl3)
-5.4, -4.7, -4.6, 18.1, 18.5, 25.1, 25.7, 25.8, 26.0, 28.4, 29.8, 30.0,
34.0, 38.1, 41.1, 48.0, 62.8, 71.9, 72.1, 84.5, 88.1, 120.8, 141.5,
150.0, 150.4, 151.0, 153.4, 172.3, 206.8; HRMS (ESI) m/z calcd
6-N-(N-Cyclohexylcarbamoyl)deoxyadenosine (2e)
Compound 2e was synthesized from 4d (2.9 g, 4.2 mmol) in
55% yield (864 mg) according to the procedure for 2d by use
of cyclohexylamine (2.4 mL, 21 mmol) in place of 4¢-aminobenzo-
15-crown[5]ether. dH (DMSO, 500 MHz) 1.25–1.38 (5H, br), 1.54–
1.56 (1H, br), 1.67–1.69 (2H, br), 1.87–1.89 (2H, br), 2.31–2.35
(1H, m), 2.71–2.76 (1H, m), 3.51–3.53 (1H, br), 3.60–3.66 (2H,
br), 3.88 (1H, dd, J = 4.4 Hz, J = 7.6 Hz), 4.42–4.43 (1H, m),
5.01 (1H, br), 5.35 (1H, br), 6.42 (1H, t, J = 6.7 Hz), 8.55 (1H, s),
8.61 (1H, s), 9.41 (1H, d, J = 7.6 Hz), 9.50 (1H, s); dC (DMSO)
24.2, 25.3, 32.6, 48.1, 61.6, 70.7, 83.8, 88.1, 120.2, 142.1, 150.0,
+
150.4, 150.9, 152.6; HRMS (ESI) m/z calcd for C17H24N6NaO4 :
399.1751, found 399.1786.
5¢-O-(4,4¢-Dimethoxytrityl)-6-N-(cyclohexylcarbamoyl)-
deoxyadenosine (3e)
Compound 3e was synthesized from 2e (300 mg, 0.80 mmol) in
60% yield (324 mg) according to the procedure for 3d. dH (CDCl3,
500 MHz) 1.26–1.44 (5H, br), 1.60–1.63 (1H, br), 1.73 (2H, br),
2.00–2.02 (2H, br), 2.55–2.59 (1H, m), 2.82–2.87 (1H, m), 3.36–
3.43 (2H, m), 3.75 (6H, s), 3.82–3.84 (1H, br), 4.18–4.20 (1H, m),
4.68–4.69 (1H, br), 6.47 (1H, t, J = 6.4 Hz), 6.77 (4H, d, J =
8.1 Hz), 7.16–7.38 (9H, m), 8.10 (1H, s), 8.23 (1H, s), 8.43 (1H, s),
9.44 (1H, d, J = 7.8 Hz); dC (CDCl3) 24.8, 25.8, 33.2, 40.3, 49.0,
55.3, 63.8, 72.4, 84.6, 86.3, 86.7, 113.3, 120.9, 127.0, 128.0, 130.1,
135.7, 135.8, 141.1, 144.6, 149.9, 150.5, 151.1, 153.2, 158.7; HRMS
+
for C34H59N6O7Si2 : 719.3978, found 741.3752.
6-N-[N-(trans-4-Levulinyloxycyclohexyl)-
carbamoyl]deoxyadenosine (7)
Compound 6 (1.5 g, 2.08 mmol) was rendered anhydrous by
repeated coevaporation with anhydrous pyridine and finally dis-
solved in anhydrous pyridine (21 mL). To this solution 3HF·NEt3
(1.70 mL, 10.4 mmol) and triethylamine (1.44 mL, 10.4 mmol)
were added and the resulting solution was stirred at ambient tem-
perature for 12 h. The reaction was quenched by addition of water
(5 mL), the solution was concentrated under reduced pressure.
The residue was dissolved in chloroform (20 mL) and washed three
times with saturated aq. NaHCO3 (20 mL each). The organic layer
+
(ESI) m/z calcd for C38H42N6NaO6 : 701.3058, found 701.3044.
6-N-[N-(trans-4-Hydroxycyclohexyl)carbamoyl]-3¢,5¢-O-bis(tert-
butyldimethylsilyl)deoxyadenosine (5)
3¢,5¢-O-Bis(tert-butyldimethylsilyl)deoxyadenosine
6.25 mmol) and phenyl chloroformate (1.72 mL 13.8 mmol)
were dissolved in pyridine (63 mL) and the solution was
(3.0
g,
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was dried over Na2SO4, filtered and concentrated under reduced
pressure. The residue was chromatographed on a NH-silica gel
column (60 g) with chloroform-methanol (200:3, v/v) to give 7
(730 mg, 72%); dH (CDCl3, 500 MHz) 1.48–1.55 (4H, m), 2.01–
2.04 (2H, br), 2.15–2.17 (2H, br), 2.20 (3H, s), 2.35–2.38 (1H, m),
2.56–2.58 (2H, t, J = 6.6 Hz), 2.74–2.77 (2H, t, J = 6.6 Hz), 3.02–
3.08 (1H, m), 3.79–3.86 (2H, m), 3.96–3.99 (1H, m), 4.24 (1H, s),
4.76–4.82 (2H, m), 5.80 (1H, dd, J = 2.2 Hz, J = 11.2 Hz), 6.38
(1H, dd, J = 5.6 Hz, J = 9.3), 8.05 (1H, s), 8.09 (1H, s), 8.49
(1H, s), 9.30 (1H, d, J = 7.3 Hz); dC (CDCl3) 28.4, 29.8, 30.0,
30.4, 38.1, 41.1, 48.3, 63.3, 72.1, 73.1, 87.5, 89.5, 121.9, 142.8,
149.4, 150.7, 151.0, 153.5, 172.4, 207.0; HRMS (ESI) m/z calcd
(CDCl3, 500 MHz) 1.12–1.21 (12H, m), 2.46- 2.49 (1H, m), 2.61–
2.64 (1H, m), 2.70–2.74 (1H, m), 2.90–2.94 (1H, m), 3.33–3.44 (2H,
m), 3.57–3.74 (4H, m), 3.77 (14H, m), 3.91–3.92 (4H, m), 4.15–
4.21 (4H, m), 4.30–4.33 (1H, m), 4.76–4.81 (1H, m), 6.46–6.49
(1H, m), 6.77–6.80 (4H, m), 6.88 (1H, d, J = 8.5 Hz), 7.02–7.04
(1H, m), 7.20–7.40 (10H, m), 8.14 (1H, d, J = 12.0 Hz), 8.53
(1H, s), 11.55 (1H, br); dC (CDCl3) 20.3, 20.4, 20.5, 20.6, 24.6,
24.7, 39.6, 43.3, 43.4, 55.3, 58.2, 58.4, 58.5, 63.4, 63.5, 69.1, 69.6,
69.8, 70.6, 70.7, 71.1, 71.2, 73.4, 73.5, 74.1, 74.2, 84.9, 86.0, 86.2,
86.6, 107.6, 113.1, 113.2, 115.1, 117.6, 117.7, 121.1, 127.0, 127.9,
128.2, 128.3, 130.2, 132.3, 135.7, 141.7, 144.6, 145.7, 149.6, 150.0,
150.2, 150.3, 150.9, 151.4, 158.6; 31P NMR (CDCl3) d 149.9, 150.0.
HRMS (ESI) m/z calcd for C55H67N8NaO12P+: 1085.4508, found
1085.4510.
+
for C22H30N6NaO7 : 513.2068, found 513.2011.
5¢-O-(4,4¢-Dimethoxytrityl)-6-N-[N-(trans-4-
levulinyloxycyclohexyl)carbamoyl]deoxyadenosine (8)
4-N-(N-Cyclohexylcarbamoyl)-5¢-O-(4,4¢-
dimethoxytrityl)deoxycytidine 3¢-(2-cyanoethyl
N,N-diisopropylphophoramidite) (1a)
Compound 7 (600 mg, 1.22 mmol) was rendered anhydrous
by repeated co-evaporation with anhydrous pyridine and finally
dissolved in anhydrous pyridine (12 mL). To this solution 4,4¢-
dimethoxytrityl chloride (497 mg, 1.47 mmol) was added and the
solution was stirred at ambient temperature for 4 h. Methanol
(3 mL) was added and the mixture was diluted with chloroform
(10 mL) and washed three times with saturated aq. NaHCO3
(10 mL each). The organic layer was dried over Na2SO4, fil-
tered and concentrated under reduced pressure. The residue was
chromatographed on a NH-silica gel column (10 g) with hexane-
chloroform (1:1, v/v) to give 8 (741 mg, 77%); dH (CDCl3,
500 MHz) 1.45–1.60 (4H, m), 2.01–2.03 (2H, br), 2.14–2.18 (2H,
br), 2.20 (3H, s), 2.54–2.58 (3H, m), 2.75 (2H, t, J = 6.5 Hz),
2.82–2.87 (1H, m), 3.36–3.43 (2H, m), 3.76 (6H, s), 3.84–3.86
(1H, m), 4.15–4.18 (1H, dd, J = 4.4 Hz, J = 8.3 Hz), 4.68–4.71
(1H, m), 4.77–4.81 (1H, m), 6.47 (1H, t, J = 6.3 Hz), 6.78 (4H,
d, J = 8.5 Hz), 7.19–7.39 (9H, m), 8.09 (1H, s), 8.13 (1H, br),
8.43 (1H, s), 9.38 (1H, d J = 7.6 Hz); dC (CDCl3) 28.5, 29.8,
30.0, 30.4, 38.1, 40.3, 48.2, 55.3, 63.8, 72.2, 72.5, 84.6, 86.3, 86.7,
113.3, 120.9, 127.1, 128.0, 128.2, 130.1, 135.7, 141.1, 144.6, 150.0,
150.4, 151.1, 153.3, 158.7, 172.4, 206.9; HRMS (ESI) m/z calcd
The title compound was synthesized according to the same
procedure as that used for 1d, using 3a (200 mg, 0.31 mmol), 1H-
tetrazole (13 mg, 0.18 mol), diisopropylamine (26 mL, 0.18 mmol)
and 2-cyanoethyl N,N,N¢,N¢-tetraisopropylphosphordiamidite
(107 mL, 0.34 mmol), to give the product in 41% yield (106 mg). dH
(CDCl3, 500 MHz) 1.08–1.18 (12H, m), 1.23–1.35 (5H, br), 1,59–
1.61 (1H, br), 1.72 (2H, br), 1.92 (2H, br), 2.17–2.23 (1H, m),
2.44–2.46 (1H, m), 2.59–2.74 (2H, m), 3.34–3.37 (1H, m), 3.43–
3.49 (1H, m), 3.55–3.66 (4H, m), 3.75–3.77 (1H, m), 3.81 (6H, s),
4.24 (1H, br), 4.56–4.61 (1H, m), 6.23–6.28 (1H, m), 6.83–6.86
(4H, m), 7.22–7.42 (9H, m), 7.98–8.07 (1H, m), 8.79 (1H, br),
10.88 (1H, br); dC (CDCl3) 20.3, 20.4, 20.5, 24.6, 24.7, 24.8, 25.3,
25.8, 29.8, 33.0, 41.1, 41.3, 43.3, 43.4, 43.5, 49.3, 55.4, 58.3, 58.4,
58.5, 62.5, 62.9, 72.8, 73.2, 85.7, 85.9, 86.8, 86.9, 97.5, 113.4, 117.5,
127.2, 127.3, 128.1, 128.3, 130.1, 130.2, 130.3, 135.4, 135.5, 135.6,
142.1, 144.3, 153.5, 156.4, 158.8, 165.0; 31P NMR (CDCl3) d 149.2,
150.2. HRMS (ESI) m/z calcd for C46H59N6NaO8P+: 877.4024,
found 877.4079.
+
for C43H48N6NaO9 : 815.3375, found 815.3365.
5¢-O-(4,4¢-Dimethoxytrityl)-6-N-[N-[3,4-(3,6,9-
trioxaundecylenedioxy)phenyl]carbamoyl]-deoxycytidine
General procedure for the synthesis of the phosphoramidite
compounds: 5¢-O-(4,4¢-Dimethoxytrityl)-6-N-[N-[3,4-(3,6,9-
trioxaundecylenedioxy)phenyl]carbamoyl]deoxyadenosine
3¢-(2-cyanoethyl N,N-diisopropylphophoramidite) (1d)
3¢-(2-cyanoethyl N,N-diisopropylphophoramidite) (1b)
The title compound was synthesized according to the same
procedure as that used for 1d, using 3c (600 mg, 0.72 mmol), 1H-
tetrazole (30 mg, 0.43 mol), diisopropylamine (61 mL, 0.43 mmol)
and 2-cyanoethyl N,N,N¢,N¢-tetraisopropylphosphordiamidite
(250 mL, 0.79 mmol) to give the product in 78% yield (580 mg). dH
(CDCl3, 500 MHz) 1.07–1.19 (12H, m), 2.31- 2.35 (1H, m), 2.44–
2.47 (1H, m), 2.56 (1H, br), 2.66–2.70 (1H, m), 3.39–3.42 (1H, m),
3.53–3.66 (5H, m), 3.72–3.80 (14H, m), 3.91 (4H, m), 4.11–4.22
(5H, m), 4.64–4.67 (1H, m), 6.31 (1H,s), 6.81–6.87 (5H, m), 7.03-
7.08 (1H, m), 7.23–7.42 (9H, m), 7.49–7.52 (1H, m), 8.18–8.25
(1H, m), 10.93 (1H, s), 11.29 (1H, s); dC (CDCl3) 20.3, 20.4, 24.7,
24.8, 41.1, 43.4, 43.5, 55.4, 58.4, 58.5, 62.1, 68.9, 69.7, 69.9, 70.6,
70.8, 71.1, 71.2, 71.8, 71.9, 85.9, 86.8, 87.1, 97.9, 107.1, 112.4,
113.5, 115.2, 117.5, 117.7, 127.3, 128.2, 128.4, 130.2, 130.3, 133.7,
135.4, 135.6, 137.2, 142.9, 144.1, 145.1, 149.4, 151.7, 156.7, 158.8,
158.9, 165.0; 31P NMR (CDCl3) d 149.6, 150.0. HRMS (ESI) m/z
calcd for C54H67N6NaO13P+: 1061.4396, found 1061.4366.
Compound 3d (547 mg, 0.63 mmol) was rendered anhydrous by
co-evaporation with anhydrous pyridine (3 ¥), anhydrous toluene
(3 ¥) and anhydrous CH2Cl2 (3 ¥). The residue was then dissolved
in anhydrous CH2Cl2 (6.3 mL). To this solution 1H-tetrazole
(27 mg, 0.38 mol), diisopropylamine (54 mL, 0.380 mmol) and 2-
cyanoethyl N,N,N¢,N¢-tetraisopropylphosphordiamidite (221 mL,
0.97 mmol) were added. The solution was stirred at ambient
temperature for 5 h. After the solvent was removed under reduced
pressure, the residue was dissolved in ethyl acetate-diethyl ether
(10 mL, 1:10, v/v) and washed five times with 0.2 M aq.
NaOH (10 mL each). The organic layer was dried over Na2SO4,
filtered and concentrated under reduced pressure. The residue
was chromatographed on an NH-silica gel column (20 g) with
chloroform-methanol (200:1, v/v) to give 3d (517 mg, 77%). dH
2448 | Org. Biomol. Chem., 2009, 7, 2440–2451
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5¢-O-(4,4¢-Dimethoxytrityl)-2¢-O-methyl-6-N-[N-[3,4-(3,6,9-
trioxaundecylenedioxy)phenyl]carbamoyl]deoxycytidine
3¢-(2-cyanoethyl N,N-diisopropylphophoramidite) (1c)
4.78–4.81 (2H, m), 6.43–6.46 (1H, m), 6.76–6.79 (4H, m), 7.18–
7.40 (9H, m), 8.21–8.24 (1H, m), 8.34–8.36 (1H, br), 8.43 (1H,
s), 9.48 (1H, d, J = 7.6 Hz); dC (CDCl3) 20.2, 20.3, 20.4, 20.5,
24.5, 24.6, 24.7, 28.4, 29.7, 29.9, 30.3, 38.0, 39.3, 43.3, 43.4, 48.0,
48.2, 55.2, 55.3, 58.2, 58.3, 58.5, 63.3, 63.5, 72.1, 73.3, 73.4, 74.0,
84.8, 86.0, 86.5, 113.1, 117.5, 117.6, 121.0, 126.9, 127.9, 128.1,
128.2, 130.1, 135.7, 141.6, 144.6, 150.0, 150.4, 151.0, 153.3, 158.6,
172.3, 206.7; dP (CDCl3) 149.7, 149.8; HRMS (ESI) m/z calcd for
C52H65N8NaO10P+: 1015.4453, found 1015.4482.
The title compound was synthesized according to the same
procedure as that used for 1d, using 3c (600 mg, 0.69 mmol), 1H-
tetrazole (29 mg, 0.41 mol), diisopropylamne (59 mL, 0.41 mmol)
and 2-cyanoethyl N,N,N¢,N¢-tetraisopropylphosphordiamidite
(241 mL, 0.76 mmol) to give the product in 66% yield (485 mg).
dH (CDCl3, 500 MHz) 1.00–1.20 (12H, m), 2.39- 2.41 (1H, m),
2.59–2.61 (1H, m), 3.46–3.70 (8H, m), 3.76–3.87 (16H, m), 3.90–
3.94 (5H, m), 4.12–4.18 (4H, m), 4.28–4.29 (1H, m), 4.40–4.62
(1H, m), 6.07–6.10 (1H, m), 6.77–6.80 (1H, m), 6.84–6.88 (4H,
m), 7.09- 7.18 (1H, m), 7.23–7.47 (10H, m), 8.47–8.58 (1H, m),
10.90 (1H, s), 11.29 (1H, s); dC (CDCl3) 20.3, 20.4, 24.5, 24.7,
43.3, 43.4, 55.4, 58.2, 58.4, 58.6, 58.9, 60.1, 60.8, 68.9, 69.1, 69.3,
69.5, 69.7, 69.8, 70.6, 70.7, 71.1, 81.8, 81.9, 83.2, 83.9, 87.1, 87.2,
89.1, 89.4, 98.0, 107.4, 112.5, 113.4, 114.8, 117.6, 117.7, 127.4,
128.1, 128.7, 130.3, 133.5, 135.3, 135.4, 135.6, 143.1, 143.2, 143.7,
145.2, 149.3, 151.5, 156.6, 158.8, 165.0; 31P NMR (CDCl3) d 151.5,
151.8. HRMS (ESI) m/z calcd for C55H69N6NaO14P+: 1091.4502,
found 1091.4510.
Synthesis of probes 2–6
The terminally modified 2¢-O-methyloligoribonucleotides were
synthesized on a DNA/RNA synthesizer on a 1 mmol scale by
using the commercially available deoxycytidine phosphoramidite,
2¢-O-methyl-RNA phosphoramidites, the universal supports II
(Glen Research Inc.) and phosphoramidites 1a–1e. The standard
RNA synthesis protocol implemented in the DNA synthesizer was
used. The cleavage of the synthesized 5¢-DMTr-oligonucleotide
from the solid support and the deprotection of the nucleobases
were carried out by treatment with 28% aqueous ammonia at room
temperature for 8 h. The solution was evaporated under reduced
pressure at room temperature to remove ammonia, and the residue
was diluted with 0.1 M ammonium acetate (50 mL). The solution
was placed on the C18 cartridge column and the failure sequences
were eluted by use of 10% CH3CN/0.1 M ammonium acetate.
After being washed with 0.1 M ammonium acetate and water, the
column was treated with aqueous 2% TFA to remove the DMTr
group, washed with 0.1 M ammonium acetate and water. The
target oligonucleotide was eluted by use of 30% CH3CN/water
and the fractions containing the target were lyophilized to give
the crude oligonucleotide. Pure material was obtained by being
purified on an anion-exchange HPLC by use of 0–50% gradient
of 1 M NaCl in 25 mM sodium phosphate–10% CH3CN. The
salts were removed by use of the C18 cartridge column to give the
pure oligonucleotide after being lyophilized to dryness. The yields
of the pure materials were 26% for the DNA and 33% for the
2¢-O-methyl-RNA. These yields were calculated by assuming the
molar extinction coefficients of dCchcm, dCbzcr, dAchcm and dAbzcr
to be identical to those of deoxycytidine and deoxyadenosine,
respectively.
5¢-O-(4,4¢-Dimethoxytrityl) -6-N-[N-(cyclohexylcarbamoyl]-
deoxyadenosine 3¢-(2-cyanoethyl N,N-
diisopropylphosphoramidite) (1e)
The title compound was synthesized according to the
same procedure as that used for 1d, using 3e (250 mg,
0.37 mmol), 1H-tetrazole (16 mg, 0.22 mmol), diisopropy-
lamine (31 mL, 0.22 mmol) and 2-cyanoethyl N,N,N¢,N¢-
tetraisopropylphosphordiamidite (129 mL, 0.41 mmol) to give the
product in 45% yield (146 mg). dH (CDCl3, 500 MHz) 1.11–1.20
(12H, m), 1.25–1.48 (5H, br), 1.61–1.64 (1H, br), 1.71–1.76 (2H,
br), 2.02–2.04 (2H, br), 2.45–2.48 (1H, m), 2.59–2.70 (2H, m),
2.89–2.93 (1H, m), 3.31–3.43 (2H, m), 3.57–3.77 (4H, m), 3.78
(6H, s), 3.84–3.89 (1H, m), 4.28–4.32 (1H, m), 4.76–4.78 (1H, m),
6.44–6.47 (1H, m), 6.76–6.80 (4H, m), 7.19–7.40 (9H, m), 7.86 (1H,
br), 8.09 (1H, s), 8.11 (1H, s), 8.45 (1H, s), 9.38 (1H, d, J = 7.6 Hz);
dC (CDCl3) 20.2, 20.4, 24.6, 24.7, 25.7, 29.7, 33.2, 39.2, 43.3, 43.4,
48.9, 55.2, 58.3, 58.5, 63.3, 63.5, 73.3, 73.5, 74.2, 84.8, 85.8, 86.0,
86.5, 113.1, 117.5, 121.0, 126.9, 127.8, 128.2, 130.1, 135.7, 141.6,
144.6, 150.0, 150.5, 151.0, 153.1, 158.5; dP (CDCl3) 149.4, 149.5;
HRMS (ESI) m/z calcd for C47H59N8NaO7P+: 901.4137, found
901.4109.
Probe2: e260 = 98220, yield: 11%, MALDI-TOF mass [M + H+]
calcd 3740.9, found 3738.1.
Probe3: e260 = 98220, yield: 9%, MALDI-TOF mass [M + H+]
calcd 4080.9, found 4081.3.
Probe4: e260 = 98220, yield: 1%, MALDI-TOF mass [M + H+]
5¢-O-(4,4¢-Dimethoxytrityl)-6-N-[N-(trans-4-
levurlnyloxycyclohexyl)carbamoyl]-deoxyadenosine
3¢-(2-cyanoethyl N,N-diisopropylphosphoramidite) (1f)
calcd 4140.9, found 4138.1.
Probe5: e260 = 112400, yield: 24%, MALDI-TOF mass [M +
H+] calcd 4128.9, found 4134.8.
The title compound was synthesized according to the
Probe6: e260 = 112400, yield: 24%, MALDI-TOF mass [M +
same procedure as that used for 1d, using
8 (350 mg,
H+] calcd 3760.8, found 3766.0.
0.441 mmol), 1H-tetrazole (19 mg, 0.264 mmol), diisopropy-
lamine (37 mL, 0.264 mmol) and 2-cyanoethyl N,N,N¢,N¢-
tetraisopropylphosphordiamidite (154 mL, 0.486 mmol), to give
the product in 77% yield (303 mg). dH (CDCl3, 500 MHz) 1.12–
1.21 (12H, m), 1.48–1.60 (4H, m), 2.02–2.04 (2H, br), 2.15–2.17
(2H, br), 2.20 (3H, s), 2.46–2.48 (1H, m), 2.56–2.63 (3H, m), 2.74–
2.77 (2H, m), 2.93–2.98 (1H, m), 3.35–3.43 (2H, m), 3.60–3.75
(4H, m), 3.77 (6H, s), 3.80–3.86 (1H, m), 4.29–4.32 (1H, m),
Synthesis of probes 7–9
By use of phosphoramidite 1e, 2¢-O-methyl-RNA phospho-
ramidites and universal support II, the fully protected oligonu-
cleotide XCAACCUACUX, where X represents the Lev-protected
nucleoside residue introduced by using 1f, was elongated on the
CPG supports. The CPG supports were dried under reduced
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pressure and then transferred to a glass syringe equipped
with a glass filter. 0.5 M NH2NH2 in a mixture of pyridine
(60 mL) and acetic acid (40 mL) was added. After 15 min,
the solution was eluted and the CPG supports were washed
three times with anhydrous acetonitrile and then dried un-
der reduced pressure. After 10 min argon gas was intro-
duced to the syringe, and 1H-tetrazole (3.5 mg, 50 mmol),
2-cyanoethyl [2-(4,4¢-dimethoxytrityloxy)ethyl]sulfonylethyl N,N-
diisopropylphosphoramidite (32 mg, 50 mmol) and anhydrous
acetonitrile (100 mL) was added. After 1 min, the solution
was eluted and the CPG supports were washed three times
with acetonitrile. Subsequently, the phosphite intermediate was
oxidized by treatment with 0.1 M I2 in a mixture of pyridine
(450 mL) and H2O (50 mL) for 2 min. The solution was eluted and
the CPG supports were washed three times with acetonitrile, dried
under reduced pressure and then treated with 2.0 M ammonia
in methanol (2.0 ml) for 2 h. The solution was eluted and the
eluent was concentrated under reduced pressure. The residue was
dissolved in 28% aq. NH3 and the reaction mixture was left to
stand for 18 h. Ammonia was removed under reduced pressure
and the material was purified on a C18 cartridge column with the
standard DMTr-ON procedure. The material was further purified
by an anion-exchange HPLC to give probe7. Probes 8 and 9 were
also synthesized by use of phosphoramidite 1f for either the first
or the last coupling.
treated with the PME method.29 After a 5000-step minimization,
the systems were heated to 300 K within 0.1 ns with a positional
2
˚
restraint of 10 kcal/mol/A on the heavy atoms during which
time the volume was kept constant. Afterwards, the system was
equilibrated under a constant pressure of 1 atm for 0.2 ns at 300 K
without any positional constraints. The simulation was continued
for additional 10 ns at 300 K. The data of the last 5 ns were
used for the analyses. The atomic charges of the aglycon part of
dAchcmp were calculated by using the Gaussian03 program30 at the
HF/6-31G* level.
Acknowledgements
This study was supported by a grant-in-aid from the Industrial
Technology Research Grant Program in 2005 of the New Energy
and Industrial Technology Development Organization (NEDO)
of Japan. This study was also supported in part by a KAKENHI
20350074 grant from the Ministry of Education, Culture, Sports,
Science and Technology (Japan) and the ‘Research for Promoting
Technological Seeds’ program of JST.
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calcd 3950.8, found 3953.3.
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calcd 3417.7, found 3419.8.
Tm measurement
Each oligonucleotide was dissolved in 10 mM sodium phosphate
(pH 7.0) containing 0.1 M NaCl and 0.1 mM EDTA so that
the final concentration of each oligonucleotide was 1.0 mM. The
solution was separated into quartz cells (10 mm) and incubated
at 85 ◦C. After 800 s, the solution was cooled to 5 ◦C at a rate of
0.5 ◦C/min and then heated to 85 ◦C at the same rate. During this
annealing and melting, the absorption at 260 nm was recorded
every 1 ◦C and used to draw UV–melting curves. The Tm value
was calculated as the temperature that gave the maximum of the
first derivative of the UV–melting curve.
MD simulation
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)
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