Synthesis and properties of a bridged nucleic acid with a perhydro-1,2-oxazin-3-one ring

Article abstract of DOI:10.1021/jo201597e

A novel derivative of 2′,4′-bridged nucleic acid, named hydroxamate-bridged nucleic acid (HxNA), containing a six-membered perhydro-1,2-oxazin-3-one ring, was designed and synthesized. The introduction of a carbonyl function along with an N-O linkage in the six-membered bridged structure is the unique structural feature of the novel 2′,4′- bridged nucleic acid analogue. The design was carried out to restrict the flexibility of the sugar moiety through the trigonal planarity of carbonyl function, which would improve the properties of the modification. The synthesized monomer was incorporated into oligonucleotides, and their properties were examined. The HxNA-modified oligonucleotides exhibited selectively high affinity toward complementary ssRNA. Furthermore, the nuclease resistance of the HxNA-modified oligonucleotide was found to be higher than that of the corresponding natural and 2′,4′-BNA/LNA-modified oligonucleotides. Interestingly, exposure of HxNA modified oligonucleotide to 3′-exonuclease resulted in gradual opening of the bridge, which stopped further digestion. Moreover, ring-opening of only one modification at the 3′-end of the oligonucleotides was observed, even if two or three HxNA modifications were present in the sequence. The results demonstrate the strong potential of the HxNA modification as a switch for the generation of highly nuclease-resistant RNA selective oligonucleotide in situ, which could have potential applications in antisense technology.

Full text of DOI:10.1021/jo201597e

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pubs.acs.org/joc
Synthesis and Properties of a Bridged Nucleic Acid with a Perhydro-
1,2-oxazin-3-one Ring
Ajaya R. Shrestha, Yoshiyuki Hari, Aiko Yahara, Takashi Osawa, and Satoshi Obika*
Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka, 565-0871, Japan
S
* Supporting Information
ABSTRACT: A novel derivative of 2′,4′-bridged nucleic acid, named hydroxamate-bridged nucleic acid (HxNA), containing a
six-membered perhydro-1,2-oxazin-3-one ring, was designed and synthesized. The introduction of a carbonyl function along with
an N−O linkage in the six-membered bridged structure is the unique structural feature of the novel 2′,4′-bridged nucleic acid
analogue. The design was carried out to restrict the flexibility of the sugar moiety through the trigonal planarity of carbonyl
function, which would improve the properties of the modification. The synthesized monomer was incorporated into
oligonucleotides, and their properties were examined. The HxNA-modified oligonucleotides exhibited selectively high affinity
toward complementary ssRNA. Furthermore, the nuclease resistance of the HxNA-modified oligonucleotide was found to be
higher than that of the corresponding natural and 2′,4′-BNA/LNA-modified oligonucleotides. Interestingly, exposure of HxNA
modified oligonucleotide to 3′-exonuclease resulted in gradual opening of the bridge, which stopped further digestion. Moreover,
ring-opening of only one modification at the 3′-end of the oligonucleotides was observed, even if two or three HxNA
modifications were present in the sequence. The results demonstrate the strong potential of the HxNA modification as a switch
for the generation of highly nuclease-resistant RNA selective oligonucleotide in situ, which could have potential applications in
antisense technology.
Recently, we have reported a modification with a six-
INTRODUCTION
■
27d
membered bridged structure, 2′,4′-BNANC
(NA-2), which
The approach of using chemically modified nucleic acids for the
selective control and regulation of gene expression has attracted
much attention because of its potential for the development of
highly potent therapeutics.1−10 Conformationally restricted
nucleic acids are one of the most interesting and promising
candidates which could exhibit many of the desired properties
of an ideal oligonucleotide.11−15 It is well-known that a nucleic
acid with its sugar conformation locked in N-type, termed 2′,4′-
bridged nucleic acid (2′,4′-BNA),16,17 or locked nucleic acid
(LNA)18 (NA-1, Figure 1) can exhibit unprecedented hybrid-
izing affinity to complementary strands (RNA and DNA),
sequence selectivity,19 aqueous solubility, and potential for in
vivo application.20,21 On the basis of the structure of 2′,4′-BNA/
LNA, many interesting modifications have been reported by
other laboratories22−26 and us27,28 in the search for bridged
nucleic acids with improved properties (Figure 1). It has been
found that the properties of the 2′,4′-bridged nucleic acids are
directly related to the size of the bridged structure and the
heteroatom in the bridge. Increasing the size of the bridge, in
general, increases the nuclease resistance at the expense of
hybridizing affinity,18,22,27 and the presence of heteroatom in
the bridge apparently improves binding affinity.22,25,27
exhibited interestingly high binding affinities toward ssRNA
and dsDNA along with high nuclease resistance. 2′,4′-BNANC
has the unique structural feature of N−O bond in the bridge of
the six-membered ring system, which is attributable for the
improved properties of the 2′,4′-BNANC-modified oligonucleo-
tides. Very recently, we have developed another bridged nucleic
acid with a seven-membered ring system having a cyclic urea
structure (NA-3).27e The modification was developed to
enhance the hydrophilicity of the modified oligonucleotide
through the introduction of N−H and CO groups in the
structure as a proton donor and acceptor, respectively. The
introduction of a carbonyl function in the bridge is an
interesting approach for restricting the flexibility of the sugar
conformation of a bridged nucleic acid with a larger ring
system, which could result improved properties of the nucleic
acid. In the case of the bridged nucleic acid with cyclic urea
structure, highly RNA selective binding affinity was obtained
along with promisingly high nuclease resistance.
Received: August 4, 2011
Published: November 7, 2011
© 2011 American Chemical Society
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six-membered bridged structure with hydroxamate (N−O
linkage and carbonyl function) moiety in the bridge. The
design thus includes the important structural features of 2′,4′-
BNANC and the bridged nucleic acid with cyclic urea structure,
which made it promising modification with potentially
improved properties. We have accomplished the synthesis of
the hydroxamate bridged nucleic acid (HxNA) with a thymine
nucleobase, HxNA-T, using a condensation reaction between
the aminooxy moiety at C2′ and the carboxyl moiety at C4′.
The synthesized HxNA was introduced into oligonucleotides,
and their hybridizing affinities and nuclease resistance were
investigated.
RESULTS AND DISCUSSION
■
Synthesis of HxNA. Considering the labile nature of N−O
moiety, the construction of the bridge of the target monomer
was strategically carried out at a late stage in the synthesis.
Starting from the common precursor of 2′,4′-BNA/LNA, i.e., 3-
O-benzyl-4-C-hydroxymethyl-1,2-O-isopropylidene-α-D-ribofur-
anose 1, the target monomers were synthesized in 14 and 16
synthetic steps. The regioselective benzylation of 1 afforded 2
in good yield.18b,27d The primary hydroxyl moiety of 2 was
protected by silylation using TBDPSCl as a protecting agent to
Figure 1. Structures of 2′,4′-bridged nucleic acids.
We herein report the design, synthesis, and properties of a
novel bridged nucleic acid analogue with a perhydro-1,2-oxazin-
3-one ring system (NA-4). The modification was designed as a
a
Scheme 1
a
Reagents and conditions: (a) NaH, BnBr, DMF, 0 °C to rt, 64%; (b) TBDPSCl, imidazole, DMF, rt, quant; (c) Ac2O, concd H2SO4, AcOH, rt; (d)
thymine, BSA, TMS-triflate, MeCN, reflux, 74% (two steps); (e) 40% MeNH2 (aq), THF, rt, quant; (f) TfCl, DMAP, CH2Cl2, rt, quant; (g) 1 N
NaOH (aq), THF, rt, 97%; (h) Tf2O, pyridine, CH2Cl2, 0 °C; (i) N-hydroxyphthalimide, DBU, MeCN, rt 79%, two steps; (j) 1 N TBAF/THF,
THF, reflux; (k) TEA·3HF, THF, reflux, 90%; (l) PDC/DMF, MS 4A, rt, 75%; (m) NH2NH2·H2O, EtOH, rt.
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a
afford 3. Acetolysis and acetylation of 3, followed by
stereoselective reaction with silylated thymine, afforded 4 in
74% yield. Subsequently, 4 was deacetylated by 40% methyl-
amine to afford 5 in quantitative yield. The configuration of the
2′-carbon of 5 was successfully inverted to afford 7 in excellent
yield by reacting 5 with TfCl to yield the 2,2′-anhydro
derivative 6, followed by alkaline hydrolysis. The N−O moiety
was introduced into the structure by an SN2 type substitution
reaction with N-hydroxyphthalimide in presence of 1,8-
diazabicyclo[5.4.0]undec-7-ene (DBU), yielding 9. Desilylation
of the TBDPS group was conducted using triethylamine
trihydrofluoride (TEA·3HF) yielded 12 in excellent yield. It has
been observed that the common desilylating agent, TBAF, was
not appropriate because ring-opening reaction occurred at the
base labile phthalimide moiety at 2′-position yielding 10 and 11.
Oxidation of the free hydroxyl moiety of 12 with PDC in DMF
afforded the carboxylic acid 13 in good yield. Reaction of 13
with hydrazine monohydrate in ethanol yielded 14 with a free
aminooxy moiety, which was used as the reactive key
intermediate for the synthesis of the HxNA monomers
(Scheme 1).
Scheme 2
The aminooxy and carboxyl moieties of the key intermediate
14 were coupled using 1-ethyl-3-(3-dimethylaminopropyl)-
carbodiimide hydrochloride (EDCI·HCl) in DMF as the
coupling agent and hydroxybenzotriazole monohydrate (HOB-
t·H2O) as the condensation additive to obtain the cyclized
1
product 15. The cyclized structure of 15 was confirmed by H
NMR spectroscopy where the 1′-H signal appeared as a singlet
(J1′,2′ = 0 Hz), similar to 2′,4′-BNA/LNA, suggesting that the
sugar moiety was in N-conformation.16a Debenzylation of 15 by
catalytic hydrogenolysis with Pearlman’s catalyst [20% Pd-
(OH)2/C] under a hydrogen atmosphere yielded the target
nucleoside monomer 16 (Scheme 2). It is noteworthy that
hydrogenolysis for debenzylation was not possible for the
synthesis of 2′,4′-BNANC, as bond cleavage occurred at the N−
O moiety,27d whereas the reaction occurred smoothly with no
cleavage reaction in the case of HxNA, presumably due to the
electron-withdrawing effect of the carbonyl function toward the
N−O moiety.
a
Reagents and conditions: (a) EDCI·HCl, HOBt·H2O, DMF, 77%,
two steps; (b) H2, Pd(OH)2/C, EtOH/CHCl3, 96%; (c) HCHO,
MeOH, rt, 65%, two steps; (d) NaBH3CN/1 M PPTS, MeOH, rt; (e)
EDCI·HCl, HOBt·H2O, DMF, rt, 80%, two steps; (f) H2, Pd(OH)2/
C, EtOH/CHCl3, rt, 90%.
group to give 21. The secondary hydroxyl group was then
phosphitylated with 2-cyanoethyl N,N,N′,N′-tetraisopropylphos-
phorodiamidite to yield the HxNA[NMe]-thymine phosphor-
amidite 22 as a mixture of two diastereoisomers (Scheme 3).
a
Scheme 3
Starting from the key intermediate 14, the synthesis of the N-
methyl congener of HxNA was accomplished in four
consecutive steps (Scheme 2). The free aminooxy moiety of
the key intermediate 7 was methylated by reductive amination;
reacting 14 with formaldehyde yielded the imine 17 which was
reduced by sodium cyanoborohydride in the presence of
pyridinium p-toluenesulfonate to afford 18. Cyclization of 18,
employing the coupling agent EDCI·HCl in DMF in the
presence of HOBt·H2O, yielded the product 19. Debenzylation
of 19 by catalytic hydrogenolysis afforded the target nucleoside
monomer 20.
a
The [NH] derivative of HxNA, having an unsubstituted
nitrogen in the bridge, was found to be less stable under basic
conditions, and it was difficult to synthesize desired
phosphoramidite monomer of the derivative. Therefore, we
have used the N-substituted HxNA, i.e., HxNA[NMe], for the
preparation of modified oligonucleotides in this study. The
synthesized HxNA monomer was incorporated into oligonu-
cleotides using the standard phosphoramidite protocol except
for a prolonged coupling time of 30−45 min with 5-[3,5-
bis(trifluoromethyl)phenyl]-1H-tetrazole (Activator 42: Act42)
as an activator. For the synthesis of the desired phosphor-
amidite 22, the primary hydroxyl group of the nucleoside 20
was selectively protected with the 4,4′-dimethoxytrityl (DMTr)
Reagents and conditions: (a) DMTrCl, TEA, pyridine, rt, 60%; (b) 2-
cyanoethyl N,N,N′,N′-tetraisopropyl phosphorodiamidite, 4,5-dicyanoi-
midazole, MeCN, rt, 70%.
During postsynthetic processing, it was found that ammonia
treatment for deprotection and removal of oligonucleotides
from the solid support was not possible because of
ammonolytic cleavage of the bridge. Therefore, the synthesized
oligonucleotides were treated with 50 mM potassium carbonate
in methanol to obtain the desired oligonucleotide without any
bond cleavage. The oligonucleotides were purified by reversed-
phase HPLC (RP-HPLC) and characterized by MALDI-TOF
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a
Table 1. Tm (°C) Values of Duplex Formed by HxNA-Modified Oligonucleotides with Complementary ssRNA and ssDNA
complementary RNA
complementary DNA
RNA selectivity
oligonucleotides
Tm (ΔTm/mod) (°C)
Tm (ΔTm/mod) (°C)
ΔTm(ssRNA−ssDNA) (°C)
5′-d(TTTTTTTTTT)-3′ (23)
5′-d(TTTTTXTTTT)-3′ (24)
5′-d(TTTXTXTTTT)-3′ (25)
5′-d(TTTXTXTXTT)-3′ (26)
18
19
−1
+8
b
20 (+2)
24 (+3)
29 (+3.7)
12 (−7)
14 (−2.5)
21 (+0.7)
+10
+8
a
Target ssRNA: 5′-r(AAAAAAAAAA)-3′(32). Target ssDNA: 5′-d(AAAAAAAAAA)-3′(33). Conditions: 10 mM sodium phosphate buffer (pH 7.2)
b
containing 100 mM NaCl; strand concentration = 4 μM. Tm values of 24/5′-r(AAAACAAAAA)-3′ and 24/5′-r(AAAAUAAAAA)-3′were less than
10 °C.
Figure 2. (A) Nuclease resistance of 5′-d(TTTTTTTTXT)-3′ against CAVP. X = HxNA (maroon solid diamond) (27a); natural DNA-T (red solid
square) (23); 2′,4′-BNA/LNA-T (green solid circle) (27b); phosphorothioate-T (blue solid triangle) (27c). Experiments were performed at 37 °C
in 100 μL of buffer containing 50 mM Tris-HCl (pH 8.0), 10 mM MgCl2, CAVP (0.175 μg), and 7.5 μM of oligonucleotide. (B) Nuclease resistance
of 27a with HxNA modified singly at 2nd position from the 3′-end. The T-10 mer 27a (maroon solid diamond) gradually degraded to the n − 1
fragment from the 3′-end, the bridge of the HxNA modification opened, and the resulting T-9 mer (green solid circle) was highly stable toward
nuclease degradation.
mass spectroscopy (mass spectral data and yields of the
oligonucleotides are provided in the Supporting Information).
Thermal Stability of the Duplex Formed by HxNA.
The thermal stability of the duplex formed by HxNA with
complementary RNA, r(A10) (32), and single-stranded DNA,
d(A10) (33), was monitored by UV melting experiments (Tm
experiments). The results were compared with those obtained
with natural d(T10) oligonucleotide (23) and summarized in
Table 1. The Tm value of the duplex formed between r(A10)
and singly modified oligonucleotide (24) was 2 °C higher than
compared to that of natural d(T10)/r(A10) duplex. An increase
in the number of modifications increased the Tm value. For
doubly (25) and triply (26) modified oligonucleotides, the
increase in Tm was +3 and +3.7 °C per modification,
respectively. In summary, the increase in Tm value per
modification (ΔTm/mod) of the HxNA ranged from 2.0 to
3.7 °C. Thus, the RNA binding affinity of HxNA was
sufficiently higher than that of natural oligonucleotide and
nucleic acid modified oligonucleotides. The Tm value of the
duplex formed by singly modified HxNA oligonucleotide (24)
with complementary ssRNA was +8 °C higher than that
obtained with complementary ssDNA. The difference in Tm
value increased to +10 °C when the oligonucleotide was
modified doubly with HxNA (25) and +8 °C higher in the case
of triply modified oligonucleotide (26) (Table 1). This result
showed that the RNA selectivity of HxNA was more or less
constant, in contrast with the urea-type bridged nucleic acid
where the RNA selectivity increased as the number of
modifications increased.27e
Nuclease Resistance of HxNA. The resistance of
oligonucleotides modified with HxNA toward 3′-exonuclease
(Crotalus adamanteus venom phosphodiesterase, CAVP,
Pharmacia) was examined and compared with that of natural
oligonucleotide, 2′,4′-BNA/LNA, and phosphorothioate (PS)-
modified oligonucleotides, respectively. All of the oligonucleo-
tides used in this study were 10-mers; those bearing
modifications were modified singly at the second position
from the 3′-end. The 10-mer HxNA-modified oligonucleotide
(27a) was found to be less resistant to enzymatic degradation
than the PS-modified oligonucleotide (27c) but exhibited
better resistance than 2′,4′-BNA/LNA (27b) and natural d(T10)
oligonucleotide (23) (Figure 2A).
comparable to that of ENA;22 however, it was lower than that
27d
of 2′,4′-BNANC
(Table SI-3, Supporting Information). As
regards RNA binding affinity, the effect of the hydroxamate
bridge in HxNA was limited and less than what we expected in
this case. In the case of duplex formation with complementary
10-mer DNA [d(A10)] the Tm values for singly (24) and doubly
(25) modified oligonucleotides decreased by 7 and 2.5 °C per
modification, respectively. Increasing the number of modifica-
tions to three (26) increased the Tm value to 0.7 °C per
modification compared to the natural oligonucleotide.
Thus, the HxNA-modified oligonucleotides exhibited very
high selectivitiy toward RNA similar to the urea-type bridged
nucleic acid,27e indicating that introduction of a carbonyl
function may help increase RNA selectivity of the bridged
Interestingly, following the enzymatic removal of the first
nucleotide, enzymatic digestion completely stopped. The
progress of the enzymatic reaction was monitored by RP-
HPLC; as the peak of 27a, [5′-d(TTTTTTTTXT)-3′, where X
= HxNA[NMe]-T], slowly diminished in size (peak A) a new
peak (peak B) appeared and increased in size. The intensity of
peak B increased with reaction time, and no other peaks
appeared except C which is the peak of the T1 monomer
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(Figure 2B and 3). This result showed the high resistance of the
degraded oligomer 28 toward enzymatic digestion. The
experiments. Gratifyingly, no further digestion was observed,
and the remaining modifications were not affected by the
nuclease. MALDI-TOF mass analysis confirmed that the rest of
the bridged structures remained intact (Figure SI-45 and Figure
SI-46, Supporting Information). The HPLC profiles obtained
during these experiments are shown in Figures SI-36−SI-38 in
the Supporting Information. The MALDI-TOF masses of the
degraded oligonucleotides with an unlocked HxNA modifica-
tion are summarized in Table SI-2 in the Supporting
Information. These results demonstrate the significant potential
of the HxNA modification to act as a switch for generating
highly nuclease-resistant monomer in situ of the enzymatic
reaction, which may have interesting applications in the
development of antisense oligonucleotides.
CONCLUSION
■
We have designed and synthesized a novel 2′,4′-bridged nucleic
acid, HxNA, which contains a six-membered perhydro-1,2-
oxazin-3-one ring system. The synthesis was accomplished in
14 and 16 synthetic steps using a condensation reaction
between an aminooxy and a carboxyl moiety to close the ring.
The synthesized HxNA[NMe]-T monomer was incorporated
into oligonucleotides, and their properties were investigated.
The HxNA[NH] monomer was not used in this study because
of its instability under basic conditions and the difficulty of
synthesizing its phosphoramidite derivative. The HxNA[NMe]-
modified oligonucleotides exhibited highly selective hybridizing
affinity toward complementary ssRNA with an increase in Tm
values of 2−3.7 °C per modification. The nuclease resistance of
HxNA-modified oligomer was higher than that of 2′,4′-BNA/
LNA. Most interestingly, oligomers containing the unlocked
derivative of the modification were found to be highly resistant
toward enzymatic digestion: exposure of the HxNA-modified
oligonucleotide to nuclease resulted in opening of the ring, with
the ring-opened derivative resisting further degradation.
Furthermore, only the modification located at the extreme 3′-
end was affected by the nuclease; the other modifications
remained unaffected. Thus, this modification has considerable
potential to be developed into an in situ switch for generating
highly nuclease-resistant antisense oligonucleotides with high
RNA selectivity.
Figure 3. HPLC profile of the enzymatic degradation of HxNA-
modified oligonucleotide 27a with respect to time. Column: XBridge
RP18 (3.0 × 50 mm). Mobile phase: Linear gradient of CH3CN (7 to
14% over 20 min) in 0.1 M triethylammonium acetate (pH 7.0). Flow
rate: 0.8 mL/min. Detection: absorbance at 260 nm.
degraded oligomers were isolated by RP-HPLC, and analyzed
by MALDI-TOF mass spectrometry. Mass analysis confirmed
that the degraded fragment 28 was the 9-mer oligonucleotide,
but the bridged structure of the modification opened (Figure
SI-43, Supporting Information). To check whether the buffer
solution contributed to the ring-cleavage reaction, dT8XT
HxNA-modified oligonucleotide (27a) was incubated in the
buffer solution for same length of time as in the enzymatic
degradation experiment (40 min). This control experiment
confirmed that the buffer solution played no role in either the
degradation or the ring-opening of the modification.
The nuclease digestion experiment was conducted on three
other 10-mer HxNA-modified oligonucleotides: modified singly
at the fifth position [5′-d(TTTTTXTTTT)-3′ (24)], doubly at
the fifth and seventh positions [5′-d(TTTXTXTTTT)-3′ (25)],
and triply at the third, fifth, and seventh positions [5′-
d(TTTXTXTXTT)-3′ (26)] from their 3′-end. In the case of
the singly modified oligomer 24, enzymatic digestion continued
up to the fifth position from the 3′-end, as evidenced by the
appearance of five peaks in the RP-HPLC profile. The fifth
peak of these peaks, peak B, intensified with respect to time.
Isolation and analysis (Figure SI-44, Supporting Information)
of the degraded oligomer confirmed that the sequence of the
highly nuclease resistant oligomer fragment was 29 [5′-
d(TTTTTX′)-3′, where X′ = unlocked HxNA] with the ring
of the modification opened. This result suggested that the
HxNA modification has potential for generating highly
nuclease-resistant oligomers in situ of the enzymatic reaction.
If the enzyme cleaves only the modified moiety located at the
extreme 3′-end of an oligonucleotide with multiple HxNA
modifications, the modification could be developed into a
switch to generate highly nuclease-resistant RNA selective
oligonucleotides. To study the effect of the enzyme on the
HxNA modification at positions other than the 3′-end, doubly
(25) and triply (26) modified oligonucleotides were exposed to
the 3′-exonuclease. As expected, degradation stopped at the first
modified site from the 3′-end of both 25 and 26, after opening
of the modification, similar to the results of the previous
EXPERIMENTAL SECTION
■
General Methods. All moisture-sensitive reactions were carried
out in well-dried glassware under a N2 atmosphere. Anhydrous
dichloromethane, DMF, MeCN, and pyridine were used as purchased.
1H NMR spectra were recorded at 300 and 400 MHz, 13C NMR were
recorded at 75 and 100 MHz, and the 31P spectrum was recorded at
161 MHz. Chemical shift values are expressed in δ values (ppm)
relative to tetramethylsilane (TMS) as internal standard and residual
1
solvents for H NMR, and CHCl3 (δ = 77.00 ppm), methanol (δ =
49.00 ppm), and DMSO (39.50 ppm) for 13C NMR, and 85% H3PO4
(δ = 0 ppm) for 31P NMR. Fast atom bombardment mass spectra
(FAB-MS) were recorded in positive-ion mode. For column
chromatography, silica gel PSQ 100B was used. The progress of the
reaction was monitored by analytical thin-layer chromatography
(TLC) on precoated aluminum sheets.
3,5-Di-O-benzyl-4-C-tert-butyldiphenylsiloxymethyl-1,2-O-iso-
propylidene-α-D-ribofuranose (3). To a solution of compound 2
(15.0 g, 37.4 mmol) in DMF (150 mL) were added imidazole (5.8 g,
85.1 mmol) and TBDPSCl (15.0 mL, 57.3 mmol) at 0 °C and the
mixture stirred for 15 h at room temperature. After completion of the
reaction, ice−water was added, and the product was extracted with
diethyl ether. The organic phase was washed with water and brine
solution and dried (Na2SO4). The product was purified by column
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chromatography (n-hexane/ethyl acetate = 9:1) to afford 3 as a clear
95.7 mmol). The reaction mixture was placed in an ice bath, TfCl (7.6
mL, 71.2 mmol) was added dropwise, and the reaction mixture was
stirred at room temperature for 1 h. After completion of the reaction,
the reaction was quenched with ice-cold water, and the product was
extracted with dichloromethane. The organic phase was washed with
water and brine solution, and dried (Na2SO4). The product was
purified by column chromatography (n-hexane/ethyl acetate = 1:1) to
afford 6 as white solid (16.5 g, quant.). Mp: 51−54 °C. [α]26D = −33.5
oil (23.8 g, quant). [α]25 = +84.8 (c 1.00, CHCl3). IR (KBr): 1457,
D
1
1372, 1105, 1025, cm−1. H NMR (300 MHz, CDCl3): δH 1.07 (9H,
s), 1.29 (6H, s), 3.67 (1H, d, J = 10.2 Hz), 3.76 (1H, d, J = 10.2 Hz),
4.09 (1H, d, J = 11.1 Hz), 4.14 (1H, d, J = 11.1 Hz), 4.33 (1H, d, J =
5.1 Hz), 4.46−4.59 (4H, m), 4.79 (1H, d, J = 12.3 Hz), 5.78 (1H, d, J
= 3.9 Hz), 7.27−7.76 (20H, m). 13C NMR (75 MHz, CDCl3): δC 19.2,
26.2, 26.5, 26.7, 64.6, 71.9, 72.2, 73.5, 76.6, 78.1, 79.5, 87.5, 104.1,
113.1, 127.4, 127.5, 127.5, 127.5, 127.6, 128.1, 128.2, 129.4, 133.1,
133.4, 134.7, 135.6, 135.7, 137.8, 138.0. MS (FAB): m/z 661 (M +
Na+). Anal. Calcd for C39H46O6Si: C, 73.32; H, 7.26. Found: C, 73.44;
H, 7.32.
2′-O-Acetyl-3′,5′-di-O-benzyl-4′-C-tert-butyldiphenylsiloxymeth-
yl-5-methyluridine (4). To a stirring solution of compound 3 (23.0 g,
36.0 mmol) in acetic acid (26.0 mL, 460 mmol) were added acetic
anhydride (45.0 mL, 480 mmol) and concd sulfuric acid (200 μL) at 0
°C. The reaction mixture was stirred at room temperature for 5 h.
After completion of the reaction, the solution was neutralized with satd
NaHCO3, and the product was extracted with ethyl acetate. The
organic phase was washed with water and brine solution and dried
(Na2SO4). After concentration, the crude product (25.7 g) was
obtained as yellow syrup which was used for the next reaction without
purification.
1
(c 1.00, CHCl3). IR (KBr): 1667, 1650, 1563, 1482, 1112 cm−1. H
NMR (300 MHz, CDCl3) δ: 1.04 (9H, s), 1.98 (3H, s), 3.33 (2H, m),
3.70 (1H, d, J = 10.8 Hz), 3.83 (1H, d, J = 10.8 Hz), 4.31 (1H, d, J =
12 Hz), 4.32 (1H, d, J = 8.8 Hz), 4.38 (1H, d, J = 12 Hz), 4.60 (1H, d,
J = 11.5 Hz), 4.60 (1H, d, J = 11.5 Hz), 5.52 (1H, dd, J = 5.2, 8 Hz),
6.27 (1H, d, J = 6.0 Hz), 7.09−7.67 (21H, m). 13C NMR (75 MHz,
CDCl3): 13.9, 18.9, 26.7, 63.9, 69.4, 73.4, 83.9, 87.1, 88.7, 89.9, 118.9,
127.4, 127.6 127.7, 127.8, 128.1, 128.3, 128.4, 128.5, 129.8, 130.1,
131.9, 132.3, 132.3, 135.3, 135.5, 136.4 136.9, 159.1, 172.3. MS (FAB):
m/z 689 (M + H+). Anal. Calcd for C41H44N2O6Si: C, 71.48; H, 6.44;
N, 4.07. Found: C, 71.38; H, 6.49; N, 4.08.
3′,5′-Di-O-benzyl-4′-C-tert-butyldiphenylsiloxymethyl-5-methyl-
arabinouridine (7). To a solution of compound 6 (16.5 g, 23.4
mmol) in THF (200 mL) was added 1 N NaOH solution (70.0 mL,
70.0 mmol), and the mixture was stirred at room temperature for 16.5
h. The solution was neutralized with NH4Cl solution, and the product
was extracted with dichloromethane. The organic phase was washed
with water and brine solution, dried (Na2SO4), and concentrated. The
crude product thus obtained was purified by column chromatography
(n-hexane/ethyl acetate = 1:1) to afford 7 as white solid (16.4 g, 97%).
Mp: 67−70 °C. [α]26 = +24.5 (c 0.840, CHCl3). IR (KBr): 3347,
3184, 1690, 1471 cm−1D. 1H NMR (400 MHz, CDCl3) δ: 0.99 (9H, s),
1.54 (3H, s), 3.42 (1H, d, J = 10.0 Hz), 3.52 (1H, d, J = 10.5 Hz), 3.60
(1H, d, J = 10.0 Hz), 3.79 (1H, d, J = 10.5 Hz), 4. 34 (1H, d, J = 6.4
Hz), 4.43 (1H, d, J = 11.6 Hz) 4.51 (1H, d, J = 11.6 Hz), 4.62 (1H, d, J
= 11.6 Hz), 4.90 (1H, d, J = 6.4 Hz), 4.93 (1H, d, J = 11.6 Hz), 5.10
(1H, dd, J = 6.4, 12.4 Hz), 6.36 (1H, d, J = 6.0 Hz), 7.19−7.68 (20H,
m), 7.77 (1H,s), 9.95 (1H, s). 13C NMR (100 MHz, CDCl3): 12.1,
18.9, 26.5, 64.1, 69.6, 72.6, 73.7, 75.4, 81.7, 85.4, 85.5, 109.7, 127.6,
127.8, 127.8, 127.8, 127.9, 128.1, 128.3, 128.5, 129.6, 129.7 132.5,
135.6, 135.7, 137.1, 137.4, 138.2, 151.4, 164.7. MS (FAB): m/z 707
(M + H+). Anal. Calcd for C41H46N2O7Si: C, 69.66; H, 6.56; N, 3.96.
Found: C, 69.42; H, 6.54; N, 3.97.
The crude product (25.7 g, 37.6 mmol) was dissolved in acetonitrile
(200 mL), and thymine (14.2 g, 110 mmol) and bis(trimethylsilyl)-
acetamide (BSA) (47.0 mL, 190 mmol) were added. The solution was
heated at 40 °C until all the substrate dissolved and then was cooled to
0 °C. TMS-triflate (10.5 mL, 57.7 mmol) was added dropwise, and the
reaction mixture was stirred at room temperature for 1 h. After
completion of the reaction, ice−water was added, and the product was
extracted with dichloromethane. The organic phase was washed with
water and brine solution, and dried (Na2SO4). The solution was
concentrated to afford the crude product which was purified by
column chromatography (n-hexane/ethyl acetate = 1:1) to afford 4 as
a white solid (20.0 g, 74%, two steps). Mp: 55−59 °C. [α]D24 = −11.7
1
(c 0.800, CHCl3). IR (KBr): 1747, 1693, 1232, 1113 cm−1. H NMR
(300 MHz, CDCl3): δH 1.03 (9H, s), 1.52 (3H, s), 1.96 (3H, s), 3.68
(1H, d, J = 10.8 Hz), 3.71 (1H, d, J = 10.5 Hz), 3.75 (1H, d, J = 10.5
Hz), 3.94 (2H, d, J = 10.8 Hz), 4.40 (1H, d, J = 5.7 Hz), 4.55 (2H, m),
5.37 (1H, t, J = 6.0 Hz), 6.15 (1H, d, J = 6.0 Hz), 7.18−7.62 (20H, m),
7.87 (1H, s). 13C NMR (75 MHz, CDCl3): 12.0, 19.2, 20.6, 26.9, 63.8,
72.2, 73.7, 74.6, 74.9, 77.7, 85.5, 87.8, 111.3, 127.6, 127.7, 127.7, 127.7,
127.8, 128.1, 128.3, 128.6, 129.7, 129.8, 132.6, 132.9, 135.5, 135.7,
135.7, 137.2, 137.5, 150.4, 163.6, 170.2. MS (FAB): m/z 749 (M +
H+). Anal. Calcd for C43H48N2O8Si: C, 68.96; H, 6.46; N, 3.74. Found:
C, 68.92; H, 6.45; N, 3.74.
3′,5′-Di-O-benzyl-4′-C-tert-butyldiphenylsiloxymethyl-2′-O-(1,3-
dihyro-1,3-dioxo-2H-isoindol-2-yl)-5-methyluridine (9). To a solu-
tion of compound 7 (3.00 g, 4.24 mmol) in dichloromethane (15 mL)
were added pyridine (1.70 mL, 21.2 mmol) and trifluoromethane-
sulfonic anhydride (1.50 mL, 8.91 mmol) at 0 °C. The reaction
mixture was stirred in an ice bath for 40 min. After completion of the
reaction, ice-cold water was added, and the product was extracted with
dichloromethane. The organic phase was washed with water and brine,
dried (Na2SO4), and concentrated. The crude product 8 (4.10 g) was
used for the next reaction without further purification.
3′,5′-Di-O-benzyl-4′-C-tert-butyldiphenylsiloxymethyl-5-methyl-
uridine (5). To a solution of compound 4 (20.0 g, 26.7 mmol) in
THF (100 mL) was added 40% aqueous methylamine solution (62.1
mL, 800 mmol) and stirred for 30 min at room temperature. After
completion of the reaction, the product was extracted with ethyl
acetate. The organic phase was washed with water and brine solution
and dried (Na2SO4). The product was purified by column
chromatography (n-hexane/ethyl acetate = 1:1) to afford 5 as a
The crude triflate 8 (4.60 g. 5.48 mmol) was dissolved in
acetonitrile (20 mL), and N-hydroxyphthalimide (4.00 mg, 24.5
mmol) and DBU (3.70 mL, 24.7 mmol) were added. The reaction
mixture was stirred at room temperature for 24 h. After completion of
the reaction, the solution was diluted with dichloromethane, and water
was added. The product was extracted with dichloromethane. The
organic phase was washed with water and brine, dried (Na2SO4), and
concentrated. The product was purified by column chromatography
(n-hexane/ethyl acetate = 2:1) to produce 9 as a white amorphous
solid (2.80 g, 79% two steps). Mp: 78−80 °C. [α]25D = +43.0 (c 1.00,
CHCl3). IR: νmax (KBr): 3188, 3067, 2934, 2862, 1791, 1730, 1692,
white solid (18.3 g, quant). Mp: 61−63 °C. [α]25 = −12.2 (c 0.750,
D
1
CHCl3). IR (KBr): 3403, 3175, 1688, 1468, 1272, 1113 cm−1. H
NMR (400 MHz, CDCl3): δH 1.05 (9H, s), 1.60 (3H, s), 3.55 (1H, d,
J = 10.4 Hz), 3.63 (1H, d, J = 10.4 Hz), 3.75 (1H, d, J = 10.8 Hz), 3.81
(1H, d, J = 10.8 Hz), 3.84 (1H, d, J = 10.4 Hz), 4.30 (1H, d, J = 5.6
Hz,) 4.41 (1H, ddd, J = 4.8 Hz, 5.6 Hz, 10.8 Hz), 4.49 (2H, s), 4.64
(1H, d, J = 11.2 Hz), 4.75 (1H, d, J = 11.2 Hz), 5.95 (1H, d, J = 5.0
Hz), 7.21−7.66 (20H, m), 9.04 (1H, s). 13C NMR (100 MHz,
CDCl3): 12.1, 19.0, 26.8, 64.2, 72.2, 73.6, 74.1, 74.5, 78.5, 87.9, 90.9,
110.9, 127.6, 127.8, 127.8, 127.8, 128.9, 128.0, 128.0, 128.5, 129.9,
132.2, 132.2, 135.6, 136.5, 137.2, 137.2, 150.5, 163.8. MS (FAB): m/z
707 (M + H+). Anal. Calcd for C41H46N2O7Si: C, 69.66; H, 6.56; N,
3.96. Found: C, 69.59; H, 6.59; N, 3.93.
1
1465, 1427, 1421, 1366, 1267, 1189, 1106, 973 cm−1. H NMR (400
MHz, CDCl3): δH 1.08 (9H, s), 1.38 (3H, s), 3.65 (1H, d, J = 10.4
Hz), 4.02 (1H, d, J = 11.6 Hz), 4.09 (1H, d, J = 10.4 Hz), 4.22 (1H, d,
J = 11.6 Hz), 4.48 (1H, d, J = 11.2 Hz), 4.52 (1H, d, J = 5.2 Hz), 4.55
(1H, d, J = 11.2 Hz), 4.73 (1H, d, J = 11.2 Hz), 4.85 (1H, dd, J = 3.2
Hz, 2.8 Hz), 5.13 (1H, d, J = 11.2 Hz), 6.40 (1H, d, J = 3.2 Hz), 7.16
− 7.86 (26H, m), 8.37 (1H, br s). 13C NMR (100 MHz, CDCl3): δC
11.8, 19.3, 26.9, 64.6, 70.5, 73.1, 73.6, 75.1, 87.2, 88.3, 88.5, 110.4,
2,2′-Anhydro-3′,5′-di-O-benzyl-4′-C-tert-butyldiphenylsiloxy-
methyl-5-methyluridine (6). To a solution of compound 5 (17.0 g,
24.0 mmol) in dichloromethane (250 mL) was added DMAP (11.7 g,
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123.6, 127.6, 127.7, 127.8, 128.3, 128.5, 129.6, 129.6, 132.9, 133.3,
134.5, 135.6, 135.7, 137.2, 137.4, 150.0, 163.1. MS (FAB) m/z 852 (M
+ H+). HRMS (FAB) calcd for C49H49N3O9Si (M + H+): 852.3316,
found 852.3284.
Hz), 4.51 (1H, d, J = 11 Hz), 4.57 (1H, s), 4.58 (1H, d, J = 11 Hz),
4.73 (1H, d, J = 12.4 Hz), 4.88 (1H, d, J = 12.4 Hz), 5.18 (1H, s), 6.25
(1H, s), 7.21−7.38 (10 H, m), 7.57 (1H, s), 7.75 (1H, br s). 13C NMR
(100 MHz, CDCl3): δC 11.8, 64.4, 69.3, 72.2, 73.7, 78.2, 80.1, 87.6,
110.3, 127.8, 127.9, 127.9, 128.0, 128.1, 128.2, 128.3, 128.4, 135.1,
137.5, 137.6 150.8, 163.3, 167.9. MS (FAB): m/z 480 (M + H+).
HRMS (FAB): calcd for C25H25N3O7 (M + H+) 480.1771, found
480.1779.
3′,5′-Di-O-benzyl-2′-O-(1,3-dihyro-1,3-dioxo-2H-isoindol-2yl)-4′-
C-hydroxymethyl-5-methyluridine (12). To a solution of compound
9 (2.80 g, 3.28 mmol) in THF (15 mL) was added TEA·3HF (5.60
mL, 34.3 mmol), and the solution was refluxed for 18 h. The reaction
mixture was then cooled and ice-cold water was added. The product
was extracted with ethyl acetate, washed with satd sodium hydro-
gencarbonate and brine, dried (Na2SO4), and concentrated. The
product was purified by column chromatography (n-hexane/ethyl
acetate = 1:1) to produce 12 as white amorphous solid (1.80 g, 90%).
Mp: 96−98 °C. [α]25D = +39.1 (c 1.00, CHCl3). IR νmax (KBr): 3504,
3181, 3062, 2881, 1789, 1733, 1689, 1466, 1375, 1272, 1187, 1105,
(1S,5R,6R,8S)-3-Aza-8-hydroxy-1-hydroxymethyl-6-(thymin-1-yl)-
4,7-dioxabicyclo[3.2.1]octan-2-one (16). To a solution of compound
15 (110 mg, 0.229 mmol) in ethanol/chloroform 5:1 (5 mL) was
added palladium hydroxide on carbon (110 mg). The reaction vessel
was degassed several times with hydrogen gas, and the reaction
mixture was stirred at room temperature under a hydrogen
atmosphere overnight. After completion of the reaction, the solution
was filtered, and the filtrate was concentrated. The product was further
purified by column chromatography (n-hexane/ethyl acetate = 1:1 to
100% ethyl acetate) to produce 16 as a white solid (55.0 mg, 80%).
1
1057, 974 cm−1. H NMR (400 MHz, CDCl3): δH 1.36 (3H, s), 2.64
(1H, br s), 3.83 (1H, d, J = 10.4 Hz), 3.92 (1H, d, J = 7.2 Hz), 3.96
(1H, d, J = 10.4 Hz), 4.12 (1H, d, J = 7.2 Hz), 4.49 (1H, d, J = 11.2
Hz), 4.54 (1H, d, J = 11.2 Hz), 4.63 (1H, d, J = 6 Hz), 4.74 (1H, d, J =
12 Hz), 4.93 (1H, dd, J = 6 Hz, 1.6 Hz), 5.16 (1H, d, J = 11.6 Hz),
6.34 (1H, d, J = 1.2 Hz), 7.15−7.84 (15H, m), 8.59 (1H, br s). 13C
NMR (100 MHz, CDCl3): δC 11.7, 64.1, 70.0, 73.0, 73.6, 75.3, 87.2,
87.8, 88.6, 110.4, 123.7, 127.7, 128.0, 128.1, 128.3, 128.5, 128.6, 128.6,
134.6, 135.5, 136.8, 137.2, 149.8, 163.4. MS (FAB): m/z 614 (M +
H+). HRMS (FAB): calcd for C33H31N3O9 (M + H+) 614.2138, found
614.2155.
Mp: 261−263 °C dec. [α]24 = +31.81 (c 1.00, MeOH). IR νmax
D
(KBr): 3474, 3406, 3237, 3056, 2979, 2932, 2819, 1693, 1481, 1422,
1
1386, 1359, 1282, 1207, 1104, 1052, 997, 967, 916 cm−1. H NMR
(400 MHz, CD3OD): δH 1.86 (3H, s), 3.88 (1H, d, J = 12.8 Hz), 4.20
(1H, d, J = 12.8 Hz), 4.54 (1H, J = 3.6 Hz), 4.67 (1H, J = 3.6 Hz),
6.07 (1H, s), 7.94 (1H, d, J = 1.4 Hz). 13C NMR (100 MHz, DMSO):
δC 12.4, 48.6, 55.3, 61.9, 79.8, 80.5, 86.9, 108.6, 135.0, 149.9, 163.8,
167.1. MS (FAB): m/z 300 (M + H+). HRMS (FAB): calcd for
C11H13N3O7 (M + H+) 300.0831, found 300.0830.
3′,5′-Di-O-benzyl-4′-carboxyl-2′-O-(1,3-dihyro-1,3-dioxo-2H-iso-
indol-2yl)-5-methyluridine (13). To a solution of compound 12 (1.80
g, 2.93 mmol) in DMF (20 mL) was added 4A molecular sieves (2.00
g), and the mixture was stirred for 10 min. To the solution was added
PDC (11.6 g, 30.8 mmol), and the mixture was stirred at room
temperature for 16 h. The reaction was quenched with ice-cold water.
The product was extracted with ethyl acetate, washed with water, and
purified by column chromatography (n-hexane/ethyl acetate = 1:1 to
ethyl acetate/methanol 10:1). The product 13 was obtained as white
3′,5′-Di-O-benzyl-4′-carboxyl-2′-O-(N-methyleneamino)-5-meth-
yluridine (17). Formalin (37 wt % in H2O, 80.0 μL, 0.986 mmol) was
added to a stirring solution of compound 14 (380 mg, 0.763 mmol) in
methanol (5 mL). The reaction mixture was stirred at room
temperature for 2 h. The solution was concentrated, and then water
was added. The product was extracted with ethyl acetate, washed with
water and brine, dried (Na2SO4), and concentrated. The product was
purified by column chromatography (n-hexane/ethyl acetate = 1:1 to
1:2) to produce 17 as a white solid (250 mg, 65% two steps). Mp: 81−
amorphous solid (1.40 g, 75%). Mp: 137−139 °C. [α]26 = +23.9 (c
D
83 °C. [α]26 = −29.3 (c 1.00, CHCl3). IR νmax (KBr): 3172, 3064,
1.00, CHCl3). IR νmax (KBr): 3178, 3066, 3032, 2873, 1790, 1736,
D
1468, 1376, 1275, 1187, 1125, 967 cm−1 1H NMR (400 MHz,
.
1
2944, 2872, 1699, 1469, 1366, 1274, 1127, 1070, 916 cm−1. H NMR
CDCl3): δH 1.59 (3H, s), 3.90 (1H, d, J = 10.4 Hz), 4.10 (1H, d, J =
10.4 Hz), 4.59 (1H, d, J = 11.6 Hz), 4.63 (1H, d, J = 11.6 H z), 4.68
(1H, d, J = 4.4 Hz), 4.91 (1H, d, J = 11.2 Hz), 5.06 (1H, t, J = 5.6 Hz)
5.27 (1H, d, J = 11.2 Hz), 6.70 (1H, d, J = 6.4 Hz), 7.27 - 7.86 (16 H,
m), 9.16 (1H, s). 13C NMR (100 MHz, CDCl3): δC 11.9, 71.4, 73.9,
74.6, 74.6, 74.7, 78.1, 87.7, 88.4, 89.0, 111.4, 123.7, 127.6, 127.7, 127.8,
128.1, 128.2, 128.3, 128.4, 128.5, 134.8, 136.2, 136.3, 136.7, 150.6,
162.9, 164.0, 170.6. MS (FAB) m/z 628 (M + H+). HRMS (FAB):
calcd for C33H29N3O10 (M + H+) 628.1931, found 628.1938.
2′-O-Amino-3′,5′-di-O-benzyl-4′-carboxyl-5-methyluridine (14).
To a solution of compound 13 (400 mg, 0.637 mmol) in ethanol (2
mL) was added hydrazine monohydrate (40.0 μL, 0.823 mmol), and
the mixture was stirred at room temperature for 10 min. After
completion of the reaction, the reaction solution was concentrated and
ethyl acetate was added. The precipitate was filtered, and the filtrate
was extracted with ethyl acetate, washed with water and brine, and
dried (Na2SO4). The crude compound 14 (380 mg) was used for the
next reaction without further purification.
(400 MHz, CDCl3): δH 1.53 (3H, s), 3.77 (1H, d, J = 10.8 Hz), 4.07
(1H, d, J = 10.8 Hz), 4.45 (1H, d, J = 4.8 Hz), 4.54 (1H, d, J = 11.2
Hz), 4.60 (1H, d, J = 12 Hz), 4.63 (1H, d, J = 12 Hz), 4.69 (1H, J =
11.2 Hz), 5.04 (1H, dd, J = 4.4 Hz, 3.2 Hz), 6.44 (1H, d, J = 7.2 Hz),
6.53 (1H, d, J = 8 Hz), 7.03 (1H, d, J = 7.2 Hz), 7.19−7.34 (10H, m),
7.52 (1H, s), 9.56 (1H, br s). 13C NMR (100 MHz, CDCl3): δC 12.0,
72.0, 73.9, 74.7, 79.8, 83.6, 86.2, 89.7, 111.8, 125.2, 127.7, 127.7, 127.7,
127.8, 128.2, 128.3, 128.7, 128.9, 136.2, 136.6, 137.2, 139.9, 150.7,
164.2, 170.8. MS (FAB): m/z 510 (M + H+). HRMS (FAB): calcd for
C26H27N3O8 (M + H+) 510.1876. found 510.1880.
3′,5′-Di-O-benzyl-4′-carboxyl-2′-O-(N-methylamino)-5-methylur-
idine (18). To a solution of compound 17 (250 mg, 0.488 mmol) in a
methanolic solution of pyridinium p-toluenesulfonate (1M, 4.90 mL,
4.89 mmol) was added sodium cyanoborohydride (62.0 mg, 0.986
mmol) at 0 °C, and the mixture was stirred for 10 min. Then the
reaction mixture was allowed to come to room temperature and stirred
for 2 h. After completion of the reaction, the solvent was evaporated,
and the product was diluted with ethyl acetate. The product was
washed with water and brine, dried (Na2SO4), and concentrated. The
crude product 18 (235 mg) was used for the next reactions without
further purification.
(1S,5R,6R,8S)-3-Aza-8-benzyloxy-1-benzyloxymethyl-3-methyl-6-
(thymin-1-yl)-4,7-dioxabicyclo[3.2.1]octan-2-one (19). To a solu-
tion of compound 18 (235 mg, 0.459 mmol) in dimethylformamide (4
mL) were added EDCI·HCl (105 mg, 0.547 mmol) and HOBt·H2O
(75.0 mg, 0.555 mmol). The reaction mixture was stirred at room
temperature overnight. After completion of the reaction, water was
added, and the product was extracted with ethyl acetate, washed with
water and brine, dried (Na2SO4), and concentrated. The compound
was purified by column chromatography (n-hexane/ethyl acetate = 4:1
to 1:1) to produce 19 as a white solid (190 mg, 80% two steps). Mp:
90−92 °C. [α]26D = +62.3 (c 1.00, CHCl3). IR νmax (KBr): 3164, 3029,
(1S,5R,6R,8S)-3-Aza-8-benzyloxy-1-benzyloxymethyl-6-(thymin-
1-yl)-4,7-dioxabicyclo[3.2.1]octan-2-one. (15). The crude key inter-
mediate 14 (320 mg, 0.643 mmol) was dissolved in DMF (5 mL), and
EDCI·HCl (150 mg, 0.782 mmol) and HOBt·H2O (106 mg, 0.784
mmol) were added at room temperature. The reaction mixture was
stirred at room temperature for 11 h, and then ice-cold water was
added. The product was extracted with ethyl acetate. The organic
phase was washed with water and brine and dried (Na2SO4). The
product was purified by column chromatography (n-hexane/ethyl
acetate = 2:1 to 1:1) to yield 15 as a white solid (240 mg, 77% two
steps). Mp: 96−98 °C. [α]23 = +89.53 (c 1.00, CHCl3). IR νmax
D
(KBr): 3190, 3064, 3033, 2926, 2877, 1699, 1494, 1455, 1392, 1362,
1
1268, 1203, 1109, 1053, 1018, 984, 916 cm−1. H NMR (400 MHz,
CDCl3): δH 1.19 (3H, s), 3.98 (1H, d, J = 11 Hz), 4.28 (1H, d, J = 11
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2926, 2878, 1698, 1456, 1392, 1362, 1274, 1215, 1155, 1094, 1065,
983 cm−1. 1H NMR (400 MHz, CDCl3): δH 1.41 (3H, s), 3.25 (3H, s),
3.95 (1H, d, J = 11.6 Hz), 4.25 (1H, d, J = 11.6 Hz), 4.34 (1H, d, J =
3.2 Hz), 4.56 (1H, d, J = 10.8 Hz), 4.59−4.64 (3H, m), 4.73 (1H, d, J
= 10.8 Hz), 6.08 (1H, s), 7.23−7.37 (10 H, m), 7.62 (1H, d, J = 1.6
Hz), 9.22 (1H, br s). 13C NMR (100 MHz, CDCl3): δC 11.9, 33.7,
64.7, 69.5, 72.6, 73.9, 78.5, 80.2, 87.9, 110.8, 127.6, 127.9, 128.2, 128.3,
128.5, 134.5, 136.5, 137.2, 149.9, 163.9. MS (FAB): m/z 494 (M +
H+). HRMS (FAB): calcd for C26H27N3O7 (M + H+) 494.1927, found
494.1931.
Synthesis System according to the standard phosphoramidite protocol.
5-[3,5-Bis(trifluoromethyl)phenyl]-1H-tetrazole (Activator 42: Act42)
was used as the activator, and Cap mix A (10% acetic acid in
tetrahydrofuran) and Cap mix B (10% 1-methylimidazole in
tetrahydrofuran/pyridine) were used as the capping agents. The
standard synthesis cycle (trityl off mode) was used for assembly of the
reagents and synthesis of the oligonucleotides, except that the coupling
time was extended to 30−45 min for the HxNA monomers. The
synthesized HxNA-phosphoramidite was dissolved in anhydrous
acetonitrile. Standard CPG-solid supports from Glen Research were
used. After synthesis, the synthesized oligonucleotides were cleaved
from the solid support by treatment with 50 mM K2CO3 in methanol
solution at room temperature for 90 min. The extract was treated with
1 M triethylamine acetic acid (TEAA), and the oligonucleotides were
purified by Nap-10 column and reversed-phase HPLC (RP-HPLC)
and then characterized by MALDI-TOF mass spectrometry.
UV Melting Experiments. The UV melting experiments were
carried out on Shimadzu UV-1800 and Shimadzu UV-1650 instru-
ments. To determine the Tm of the duplexes, equimolar amounts of
target RNA/DNA strands and modified oligonucleotides were
dissolved in 10 mM sodium phosphate buffer (pH 7.2) containing
100 mM NaCl to provide a final strand concentration of 4 μM. The
samples were annealed at 90 °C and slowly cooled to room
temperature. The melting experiment was monitored at 260 nm
from 0 to 80 °C at a scan rate of 0.5 °C/min. Tm was calculated as the
temperature at which the duplexes were half dissociated, determined
by taking the first derivative of the melting curve.
(1S,5R,6R,8S)-3-Aza-8-hydroxy-1-hydroxymethyl-3-methyl-6-
(thymin-1-yl)-4,7-dioxabicyclo[3.2.1]octan-2-one (20). To a solu-
tion of compound 19 (190 mg, 0.385 mmol) in ethanol/chloroform
5:1 (5 mL) was added palladium hydroxide on carbon (190 mg). The
reaction vessel was degassed several times with hydrogen gas, and the
reaction mixture was stirred at room temperature under a hydrogen
atmosphere overnight. After completion of the reaction, the solution
was filtered, and the filtrate was concentrated. The product was further
purified by column chromatography (n-hexane/ethyl acetate = 1:1 to
100% ethyl acetate) to produce 20 as a white solid (105 mg, 90%).
Mp: 232−234 °C dec; [α]26D = +14.9 (c 1.00, EtOH). IR νmax (KBr):
3444, 3226, 3070, 2941, 1678, 1469, 1412, 1281, 1199, 1078,988 cm−1
.
1H NMR (400 MHz, CD3OD): δH 1.86 (3H, s), 3.22 (3H, s), 3.87
(1H, d, J = 12.8 Hz), 4.18 (1H, d, J = 12.8 Hz), 4.53 (1H, J = 3.6 Hz),
4.71 (1H, J = 3.6 Hz), 6.04 (1H, s), 7.95 (1H, d, J = 1.2 Hz). 13C
NMR (100 MHz, CD3OD): δC 12.6, 33.7, 57.3, 63.9, 82.1, 82.8, 89.1,
111.0, 136.9, 151.8, 166.4. MS (FAB): m/z 314 (M + H+). HRMS
(FAB): calcd for C12H15N3O7 (M + H+) 314.0988, found 314.0981.
(1S,5R,6R,8S)-3-Aza-1-(4,4′-dimethoxytrityloxymethyl)-8-hy-
droxy-3-methyl-6-(thymin-1-yl)-4,7-dioxabicyclo[3.2.1]octan-2-one
(21). To a stirring solution of compound 20 (50.0 mg, 0.159 mmol)
in pyridine (3 mL) were added DMTrCl (65 mg, 0.191 mmol) and
triethylamine (100 μL, 0.727 mmol). The reaction mixture was stirred
at room temperature for 8 h. Saturated NaHCO3 was added, and the
product was extracted with ethyl acetate. The organic layer was washed
with water and brine, dried (Na2SO4), and concentrated. The product
was purified by column chromatography (1% triethylamine in n-
hexane/ethyl acetate = 2:1 to 100% ethyl acetate) to produce 21 as a
Nuclease Resistance Study. The sample solutions were prepared
by dissolving 0.75 μmol of oligonucleotides in 50 mM Tris-HCl buffer
(pH 8.0) containing 10 mM MgCl2. In each sample solutions, 0.175
μL CAVP was added and the cleavage reaction was carried out at 37
°C. A portion of each reaction mixture was removed at timed intervals
and heated to 90 °C for 5 min to deactivate the nuclease. Aliquots of
the timed samples were analyzed by RP-HPLC to evaluate the amount
of intact oligonucleotides remaining. The percentage of intact
oligonucleotide in each sample was calculated and plotted against
the digestion time to obtain a degradation curve with time.
yellowish white solid (58.0 mg, 60%). Mp: 136−138 °C. [α]28
=
D
ASSOCIATED CONTENT
■
−21.1 (c 1.00, CHCl3). IR νmax (KBr): 3339, 3189, 3062, 2926, 2850,
1693, 1608, 1509, 1464, 1395, 1253, 1177, 1080, 1033, 978 cm−1. H
1
S
* Supporting Information
NMR (400 MHz, CDCl3): δH 1.36 (3H, s), 3.18 (3H, s), 3.72 (1H, d,
J = 12 Hz), 3.76 (6H, s), 3.95 (1H, d, J = 12 Hz), 4.70 (1H, d, J = 3.6
Hz), 4.76 (1H, d, J = 3.6 Hz), 6.03 (1H, s), 6.81−6.84 (4H, m), 7.21 −
7.42 (9H, m), 7.71 (1H, s). 13C NMR (100 MHz, CDCl3): δC 12.0,
33.7, 55.2, 57.9, 64.5, 81.0, 87.3, 87.5, 111.3, 113.4, 127.1, 127.3, 127.7,
127.8, 128.0, 128.1, 129.1, 130.1, 130.1, 134.2, 135.0, 139.4, 144.1,
149.7, 158.7, 163.7. MS (FAB): m/z 638 (M + Na+). HRMS (FAB):
calcd for C33H33N3O9Na (M + Na+) 638.2109, found 638.2097.
(1S,5R,6R,8S)-3-Aza-8-(2-cyanoethoxy(diisopropylamino)-
phosphinoxy)-1-(4,4′-dimethoxytrityloxymethyl)-3-methyl-6-(thy-
min-1-yl)-4,7-dioxabicyclo[3.2.1]octan-2-one (22). To a stirring
solution of compound 21 (50.0 mg, 0.081 mmol) in anhydrous
acetonitrile were added 2-cyanoethyl-N,N,N′,N′-tetraisopropylphos-
phorodiamidite (30.0 μL, 0.094 mmol) and 4,5-dicyanoimidazole
(10.0 mg, 0.058 mmol). The reaction mixture was stirred at room
temperature for 7 h. After completion of the reaction, satd NaHCO3
was added, and the product was extracted with ethyl acetate. The
organic layer was washed with water and brine, dried (Na2SO4), and
concentrated. The product was purified by column chromatography
(1% triethylamine in n-hexane/ethyl acetate = 2:1 to 1:1) to produce
22 as a white solid (46.0 mg, 70%). Mp: 117−119 °C. 1H NMR (400
MHz, CDCl3): δH 0.97−1.26 (15H, m), 2.39 (1H, t, J = 6 Hz), 2.40−
2.63 (1H, m), 3.18 (3H, s), 3.53−3.62 (4H, m), 3.79 (6H, s), 3.95−
4.04 (1H, m), 4.88−4.99 (2H, m), 6.08 (1H, s), 6.80−6.85 (4H, m),
7.23−7.43 (9H, m), 7.78 (1H, s), 8.66 (1H, br s). 31P NMR (161
MHz, CDCl3): δ 150.9, 151.5. MS (FAB): m/z 816 (M + H+). HRMS
(FAB): calcd for C42H50N5O10P (M + H+) 816.3373, found 816.3376.
Oligonucleotide Synthesis. Oligonucleotides 23−27 were synthe-
sized on a 0.2 μmol scale using an Expedite 8909 Nucleic Acid
MALDI-TOF-MS data and yields of oligonucleotides 23 to
27a, and MALDI-TOF-MS data of oligonucleotides 28 to 31
1
with an unlocked modification, H and 13C spectra of all new
compounds (3−7, 9, 12, 13, 15−17, 19−21), 1H and 31P NMR
spectrum of 22, UV melting curves for the duplexes between
oligonucleotides 23−26 and DNA or RNA complement, CD
spectra of duplexes formed by HxNA-modified oligonucleo-
tides, Job plot experiment of 24 and 32, HPLC profiles of
enzymatic degradation of singly (24), doubly (25), and triply
(26) modified HxNA oligonucleotides with respect to time, and
MALDI-TOF-MS spectra for 24−31. This material is available
free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*E-mail: obika@phs.osaka-u.ac.jp.
ACKNOWLEDGMENTS
■
A part of this work was supported by the Program for
Promotion of Fundamental Studies in Health Sciences of the
National Institute of Biomedical Innovation (NIBIO), Program
to Disseminate Tenure Tracking System of the Ministry of
Education, Culture, Sports, Science and Technology, Japan
(MEXT) and a Grant-in-Aid for Scientific Research on
Innovative Areas (No. 22136006) from MEXT.
9898
dx.doi.org/10.1021/jo201597e | J. Org. Chem. 2011, 76, 9891−9899

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