Synthesis of the Methyl Analog of 2′- O,4′- C-Ethylene-Bridged 5-Methyluridine via Intramolecular Radical Cyclization and Properties of Modified Oligonucleotides

Article abstract of DOI:10.1021/acs.joc.9b01035

The synthesis of 6′S-Me-2′-O,4′-C-ethylene-bridged 5-methyluridine (6′S-Me-ENA-T) was achieved using visible light-mediated stereoselective radical cyclization as a key step. This is the first example of a method for constructing a 2′,4′-bridged structure

Full text of DOI:10.1021/acs.joc.9b01035

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Article
Synthesis of the Methyl Analog of 2#-O,4#-C-Ethylene-
Bridged 5-Methyluridine via Intramolecular Radical
Cyclization and Properties of the Modified Oligonucleotides
Yuta Ito, Norika Tsutsui, Takashi Osawa, and Yoshiyuki Hari
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.9b01035 • Publication Date (Web): 26 Jun 2019
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Synthesis of the Methyl Analog of 2ʹ-O,4ʹ-C-Ethylene-Bridged 5-Methyluridine via
Intramolecular Radical Cyclization and Properties of the Modified
Oligonucleotides
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Yuta Ito, Norika Tsutsui, Takashi Osawa, and Yoshiyuki Hari*
Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Nishihama,
Yamashiro-cho, Tokushima 770-8514, Japan.
ABSTRACT: The synthesis of 6ʹS-Me-2ʹ-O,4ʹ-C-ethylene-bridged 5-methyluridine
(6ʹS-Me-ENA-T) was achieved using visible light-mediated stereoselective radical
cyclization as a key step. This is the first example of a method for constructing a
2ʹ,4ʹ-bridged structure from a 4ʹ-carbon radical intermediate. The 6ʹS-Me-ENA-T
monomer was successfully incorporated into oligonucleotides and their properties were
examined. The oligonucleotides containing 6ʹS-Me-ENA-T exhibited a highly selective
hybridization affinity toward single-stranded RNA and an excellent enzymatic stability,
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compared to the corresponding LNA- and ENA-modified oligonucleotides.
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INTRODUCTION
Oligonucleotides have recently received much attention as the next-generation drugs
with great potential to treat various diseases such a as spinal muscular atrophy,1 cancer,2
and other diseases.3 However, unmodified/natural oligonucleotides are not suitable for
use in oligonucleotide therapeutics due to their insufficient hybridization affinity with
the target nucleic acids and low resistance against nucleases. To improve these
properties of the oligonucleotides, a number of chemical modifications of the
nucleobase, sugar and phosphate moieties have been investigated.4 Among them, bridge
modification between the 2ʹ-oxygen atom and the 4ʹ-carbon atom of the ribose ring not
only increases the affinity of the oligonucleotides with single-stranded RNA (ssRNA)
and/or double-stranded DNA (dsDNA) but also improves their resistance to nuclease
degradation; therefore, a variety of bridged nucleic acids have been developed to date.5
In particular, LNA6/2ʹ,4ʹ-BNA,7 ENA,8 and S-cEt9 have already been used for clinical
trials (Figure 1). The bridge part of these 2ʹ-O,4ʹ-C-bridged nucleosides is generally
synthesized by an intramolecular SN2 reaction, namely ionic cyclization, in which the
2ʹ-oxygen atom attacks the 4ʹ-carbon atom bearing a leaving group (Scheme 1a). On the
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other hand, intramolecular radical cyclization is an attractive approach to construct a
bridged structure.10 For example, 2ʹ-C,4ʹ-C-bridged nucleic acids like 7ʹ-Me-carba-LNA
and 8ʹ-Me-carba-ENA are synthesized using intramolecular cyclization of the 2ʹ-carbon
radical (Figure 1 and Scheme 1b).11 However, this reaction cannot be applied to the
2ʹ-O,4ʹ-C-bridged nucleosides because the 2ʹ-oxygen atom is removed by the
Barton-McCombie deoxygenation, which creates the 2ʹ-carbon radical. In addition, the
position where these radical species are generated to construct the 2ʹ,4ʹ-bridged
structures via intramolecular radical cyclization has so far been limited to the 2ʹ-carbon
atom.11,12 With this background, we planned to synthesize new 2ʹ-O,4ʹ-C-bridged
nucleoside analogs through the generation of the radical species, via radical cyclization,
at different carbon atom positions.
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Figure 1. Structures of LNA and ENA analogs
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Scheme 1. Strategies for the construction of bridge structures
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As part of our ongoing research aimed at developing novel methods for the synthesis
of useful nucleoside analogs, we had recently reported the concise synthesis of 5ʹ-carba
analogs of nucleosides from the 2ʹ-deoxy-5ʹ-O-phthalimidonucleosides by the
photoredox-catalyzed deformylative 1,4-addition reaction (Scheme 2a).13 It is known
that oligonucleotides containing six-membered ENA exhibit higher nuclease resistance
than that containing five-membered LNA, without the loss of hybridization affinity
toward the ssRNA.8 Furthermore, oligonucleotides containing S-cEt, which possesses a
methyl group at the 6ʹ-position, not only increased the nuclease resistance but also
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reduced the risk of hepatotoxicity, compared to that containing LNA.9 These results
suggest that 6ʹ-Me-ENA can be expected as highly potential nucleic acids. Herein, we
report a novel approach to the synthesis of 6ʹ-Me-ENA-T by the intramolecular radical
cyclization through the generation of the 4ʹ-carbon radical. In addition, the hybridization
ability and nuclease resistance of the 6ʹ-Me-ENA-modified oligonucleotides were
evaluated.
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Scheme 2. Strategy for the Synthesis of 6ʹ-Me-ENA via Generation of 4ʹ-Carbon
Radical
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RESULTS AND DISCUSSION
The known 5-methyluridine derivative 114 was used as the starting material for the
synthesis of 6ʹ-Me-ENA-T (Scheme 3). Initially, BOM-protection of the nitrogen atom
at the 3-position of 1 followed by the deacetylation of 2 gave 3, the allylation of which
afforded 4 in good yield. The silyl protecting group of 4 was removed using TBAF to
give 5. Subsequently, the obtained alcohol 5 was treated with N-hydroxyphthalimide
(NHPI) under the Mitsunobu reaction conditions to provide the desired radical precursor
6.
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Scheme 3. Synthesis of N-Alkoxyphthalimide 6 from 1
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Next, the key radical cyclization was investigated using the visible light-mediated
photoredox reaction (Scheme 4). In the presence of fac-Ir(ppy)3 as a photocatalyst and
Hantzsch ester as a reductant and hydrogen source, N-alkoxyphthalimide 6 in
1,4-dioxane was irradiated with a 32 W compact fluorescent lamp (CFL) at 80 °C.
Interestingly, the desired six-membered 6ʹ-Me-ENA was not obtained, instead the
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compound (R)-7 with an eight-membered ring was obtained as a sole isomer in 83%
yield. This result indicates that the intramolecular 1,6-hydrogen atom transfer
(1,6-HAT) of the alkoxy radical I took precedence over the β-fragmentation (for the
generation of the 4ʹ-carbon radical), generating the benzyl radical II. In general, it is
know that 8-endo cyclization is fundamentally preferred over 7-exo cyclization.15
Therefore, this radical II exclusively underwent the 8-endo cyclization to form the
intermediate radical III and sequential hydrogen atom abstraction led to the
eight-membered compound (R)-7.
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Scheme 4. Radical Cyclization of N-Alkoxyphthalimide 6
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The stereoselective formation of (R)-7 could be explained as shown in Figure 2. To
avoid the steric repulsion between the phenyl group and hydrogen atom at 3ʹ-position in
the transition state IIa, the radical cyclization would proceed via the transition state IIb.
The stereochemistry of compound (R)-7 was confirmed by NOESY correlations
between 3ʹ-H and 7ʹ-H, 3ʹ-H and 9ʹ-Ha, 7ʹ-H and 9ʹ-Ha (Figures 2 and S1).
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Figure 2. Transition state models of 8-endo radical cyclization
As an alternative route for the synthesis of 6ʹ-Me-ENA, we focused on the generation
of the 4ʹ-carbon radical from N-acyloxyphthalimide by radical decarboxylation, which
is much faster than the deformylation in a radical reaction.16 The radical precursor,
N-acyloxyphthalimide 9, was prepared from alcohol 5 in two steps, by its oxidation to
carboxylic acid 8 followed by the condensation with NHPI (Scheme 5).
Scheme 5. Synthesis of N-Acyloxyphthalimide 9
The radical cyclization was performed with N-acyloxyphthalimide 9, as the radical
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precursor (Scheme 6). When the reaction of 9 was carried out under the same conditions
as Scheme 4, the desired radical decarboxylation and 6-exo cyclization proceeded
smoothly to produce the 6ʹ-Me-ENA-T monomer 10, with only the (S)-configuration at
the 6ʹ-position. Decreasing the reaction temperature to room temperature resulted in a
slight improvement of the yield. Meanwhile, the reaction without light irradiation at
80 °C resulted in the decomposition of 9 and the desired product was not obtained.
Under the classical free radical condition using Bu3SnH and AIBN, the unreacted 9 was
recovered in 52% yield without producing 10.17
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Scheme 6. Radical Cyclization of N-Acyloxyphthalimide 9
The observed stereoselectivity could be explained by the six-membered transition
states in the 6-exo cyclization as depicted in Figure 3. The reaction via conformation
IVa would be disfavored probably due to the 1,3-diaxial interaction between the olefin
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moiety and the 3ʹ-benzyloxy group. Therefore, the reaction should go through the
conformation IVb, with the less hindered olefin, to give (S)-10 selectively. The
stereochemistry of compound (S)-10 was confirmed by NOESY correlations between
1ʹ-H and 7ʹ-Ha, 6ʹ-Me and 7ʹ-Ha (Figures 3 and S2).
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Figure 3. Transition state models of 6-exo radical cyclization
For the incorporation of the 6ʹS-Me-ENA-T monomer into oligonucleotides,
simultaneous deprotection of the two benzyl and the BOM groups was required. This
afforded compound 11 at 93% yield (Scheme 7). Finally, the 4,4ʹ-dimethoxytrityl
(DMTr) protection of the primary alcohol group followed by the phosphitylation of the
secondary alcohol group gave phosphoramidite 13. The oligonucleotide synthesis was
performed in an automated DNA synthesizer using the common phosphoramidite
chemistry. The coupling efficiency for the incorporation of 13 was over 95% (estimated
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using the trityl monitor) with a prolonged coupling time from 25 s to 10 min.
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Scheme 7. Synthesis of Phosphoramidite 13
The duplex-forming ability of the 6ʹS-Me-ENA-modified oligonucleotides with the
complementary ssRNA and ssDNA was evaluated using the UV-melting experiments
and compared with those of the corresponding natural, LNA- and ENA-modified
oligonucleotides (Table 1). The melting temperatures (Tms) of the duplexes of the
6ʹS-Me-ENA-modified oligonucleotides ON2–4 with the ssRNA were higher than that
of the natural oligonucleotide ON1 and comparable to that of the oligonucleotides
containing LNA ON5–7 and ENA ON8–10. As for ssDNA, ON2–4 showed almost no
stabilization or a slight destabilization (ΔTm/mod. = –3.0 to +0.3 °C), though ON5–7
and ON8–10 showed slight stabilization (ΔTm/mod. = 0 to +2.0 °C). The differences in
the Tm values with the ssRNA and ssDNA are summarized in Figure 4. For example, in
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comparison with singly modified oligonucleotides, 6ʹS-Me-ENA-modified ON2 (ΔTm = +6 °C)
displayed larger ΔTm than LNA-modified ON5 (ΔTm = +3 °C) and ENA-modified ON8 (ΔTm =
+4 °C). These results suggested that the 6ʹS-Me-ENA-modified oligonucleotides could
have the ssRNA-selective hybridization ability compared to the corresponding LNA-
and ENA-modified oligonucleotides.
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Table 1. Duplex-Forming Ability of Modified Oligonucleotides with Complementary
ssRNA and ssDNAa
Tm (ΔTm/mod.) (°C)
oligonucleotides
ssRNA
ssDNA
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d(TCTTCTTTTTCTCT) (ON1)
d(TCTTCTTXTTCTCT) (ON2)
d(TCTTCTXXXTCTCT) (ON3)
d(TCTTCXTXTXCTCT) (ON4)
d(TCTTCTTYTTCTCT) (ON5)
d(TCTTCTYYYTCTCT) (ON6)
d(TCTTCYTYTYCTCT) (ON7)
d(TCTTCTTZTTCTCT) (ON8)
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43 (–3.0)
45 (–0.3)
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48 (+2.0)
49 (+1.0)
51 (+1.7)
46 (0)
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d(TCTTCTZZZTCTCT) (ON9)
61 (+4.7)
49 (+1.0)
48 (+0.7)
d(TCTTCZTZTZCTCT) (ON10) 64 (+5.7)
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aConditions: 10 mM sodium cacodylate buffer (pH 7.4), 100 mM KCl and 2.5 μM of
each oligonucleotide. X = 6ʹS-Me-ENA-T, Y = LNA-T, Z = ENA-T, C =
2ʹ-deoxy-5-methylcytidine. The sequence of target RNA and DNA complements are
5ʹ-r(AGAGAAAAAGAAGA)-3ʹ and 5ʹ-d(AGAGAAAAAGAAGA)-3ʹ. ΔTm/mod.: the
change in the Tm value (ΔTm) per modification compared with the unmodified
oligonucleotide ON1.
Figure 4. Differences in Tm values with ssRNA and ssDNA. T = 6ʹS-Me-ENA, LNA or
ENA.
The triplex-forming ability of the 6ʹS-Me-ENA-modified oligonucleotides with
dsDNA was also investigated (Figure 5). Although the ΔTm/mod. values of
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oligonucleotides containing the 6ʹS-Me-ENA-T slightly decreased by 0–2 °C compared
to those of the oligonucleotides containing LNA and ENA, the 6ʹS-Me-ENA-modified
oligonucleotides could sufficiently stabilize the triplex formed with the dsDNA.
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Figure 5. ΔTm per modification values of the triplex formed by modified
oligonucleotides with dsDNA. Conditions: 10 mM sodium cacodylate buffer (pH 7.4),
100 mM KCl, 10 mM MgCl2 and 1.5 μM of each oligonucleotide. T = 6ʹS-Me-ENA,
LNA or ENA.
Finally, the nuclease resistance of the T10-mer containing the 6ʹS-Me-ENA-T at the
second position from the 3ʹ-terminus was evaluated using the 3ʹ-exonuclease (Crotalus
adamanteus venom phosphodiesterase, CAVP) and compared to those of the natural,
LNA- and ENA-modified T10-mers. As shown in Figure 6, the natural and
LNA-modified T10-mers (O11 and ON13, respectively) were completely degraded
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within 5 and 10 min, respectively. Although the ENA-modified T10-mer ON14 was
more stable than ON11 and ON13, the ON14 that resisted degradation was less than 5%,
after 60 min. In contrast, more than 50% of the 6ʹS-Me-ENA-modified T10-mer ON12
resisted degradation even after 60 min. This excellent nuclease stability of the
6ʹS-Me-ENA-modified nucleotide was probably due to the steric hindrance of the
6ʹ-methyl group.18
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Figure 6. Nuclease degradation experiments. Conditions: 0.1 unit/mL Crotalus
adamanteus venom phosphodiesterase (CAVP), 10 mM MgCl2, 50 mM Tris-HCl (pH =
8.0), 7.5 μM of each oligonucleotide at 37 °C. Sequence: 5ʹ-d(TTTTTTTTTT)-3ʹ, T =
natural (black, ON11), 6ʹS-Me-ENA (red, ON12), LNA (blue, ON13), ENA (green,
ON14).
CONCLUSIONS
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We developed a novel synthetic method for the construction of a 2ʹ-O,4ʹ-C-bridged
nucleoside, 6ʹS-Me-ENA, by intramolecular 4ʹ-carbon radical cyclization. Although a
4ʹ-carbon radical was not generated from N-alkoxyphthalimide because the 1,6-HAT of
the alkoxy radical rather than radical deformylation occurred preferentially,
N-acyloxyphthalimide could successfully generate the 4ʹ-carbon radical, followed by
6-exo cyclization to selectively afford the desired 6ʹS-Me-ENA-T monomer. The
6ʹS-Me-ENA-modified oligonucleotides showed selective hybridizing affinity toward
ssRNA and high nuclease resistance compared to the LNA- and ENA-modified
oligonucleotides. These results suggest that the 6ʹS-Me-ENA can be a useful material
for practical applications such as antisense therapy. In addition, we believe that the
proposed radical cyclization method can be a powerful tool for synthesis of new nucleic
acid analogs and will contribute to development of promising materials of
oligonucleotides.
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EXPERIMENTAL SECTION
General Information. All moisture-sensitive reactions were conducted in well-dried
glassware under an Ar atmosphere. Anhydrous benzene, DCM, 1,4-dioxane, MeCN,
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MeOH, pyridine and THF were used as purchased. H NMR, C{1H} NMR, P{1H}
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NMR spectra were recorded on a Bruker AVANCE III HD 500 equipped with a BBO
cryoprobe or Varian MERCURY plus 300. Chemical shift values were reported in ppm,
relative to internal tetramethylsilane ( = 0.00 ppm) or solvent residual signals ( = 3.31
ppm for CD3OD) for 1H NMR, solvent residual signals ( = 77.0 ppm for CDCl3 and 
= 49.0 ppm for CD3OD) for 13C{1H} NMR, and external 5% H3PO4 ( = 0.00 ppm) for
31P{1H} NMR. IR spectra were recorded on a JASCO FT/IR-4200 spectrometer.
High-resolution mass spectrometry was performed on a Waters SYNAPT G2-Si
(Quadrupole/TOF). For column chromatography, silica gel PSQ-60B (Fuji Silysia) was
used. The progress of the reaction was monitored by analytical thin-layer
chromatography (TLC) on pre-coated aluminum sheets (Silica gel 60 F254 by Merck).
For HPLC, a JASCO EXTREMA (PU-4180, CO-4060 or CO-4061, UV-4075 and
AS-4050) instrument with CHF122SC (ADVANTEC) fraction collector was used.
UV-melting experiments were carried out using JASCO V-730 UV/VIS
spectrophotometer equipped with a Tm analysis accessory. Synthesis of oligonucleotides
was performed on an automated DNA synthesizer (Gene Design nS-8II).
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Synthesis of 6ʹS-Me-ENA-T phosphoramidite 13
1-(2-O-Acetyl-4-C-tert-butyldiphenylsilyloxymethyl-3,5-di-O-benzyl--D-ribofuranosyl)
-3-(benzyloxymethyl)thymine (2)
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To a solution of 1 (5.63 g, 7.52 mmol) and DBU (2.47 mL, 16.5 mmol) in THF (50
mL), BOMCl (2.08 mL, 15.0 mmol) was added at 0 °C. After being stirred for 27.5 h at
room temperature, the reaction mixture was quenched with sat. NaHCO3 aq. and
extracted with AcOEt. The combined organic layer was washed with water and brine,
dried over Na2SO4, and concentrated in vacuo. The crude residue was purified by
column chromatography (hexane/AcOEt = 2:1) to give compound 2 as a colorless oil
(5.61 g, 86%). 1H NMR (500 MHz, CDCl3): δ 7.62 (dd, J = 8.0, 1.0 Hz, 2H), 7.59 (dd, J
= 8.0, 1.0 Hz, 2H), 7.50 (s, 1H), 7.43-7.20 (m, 21H), 6.16 (d, J = 4.5 Hz, 1H), 5.48 and
5.44 (ABq, J = 9.5 Hz, 2H), 5.38 (dd, J = 6.0, 5.5 Hz, 1H), 4.66 (s, 1H), 4.57-4.50 (m,
4H), 4.41 (d, J = 5.5 Hz, 1H), 3.94 and 3.73 (ABq, J = 11.0 Hz, 2H), 3.82 and 3.70
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(ABq, J = 10.0 Hz, 2H), 1.93 (s, 3H), 1.52 (s, 3H), 1.04 (s, 9H). C{1H} NMR (126
MHz, CDCl3): δ 170.0, 163.3, 151.2, 138.0, 137.5, 137.3, 135.7, 135.5, 134.5, 133.0,
132.7, 129.9, 129.7, 128.6, 128.4, 128.2, 128.1, 127.9, 127.7, 127.7, 127.7, 127.7, 127.6,
127.5, 110.5, 87.9, 86.5, 77.4, 74.9, 74.5, 73.7, 72.0, 70.5, 63.9, 26.9, 20.6, 19.2, 12.7.
IR (ATR) cm-1: 3068, 3030, 2930, 2858, 1747, 1713, 1666, 1453, 1363, 1228. HRMS
(ESI-TOF): calcd for C51H56N2NaO9Si [M + Na]+ 891.3653, found 891.3661.
3-Benzyloxymethyl-1-(4-C-tert-butyldiphenylsilyloxymethyl-3,5-di-O-benzyl--D-ribofur
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anosyl)thymine (3)
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To a solution of 2 (12.5 g, 14.4 mmol) in THF (150 mL), 40% MeNH2 aqueous
solution (37 mL, 0.43 mol) was added at room temperature. After being stirred for 1 h,
the reaction mixture was concentrated in vacuo and diluted with AcOEt. The AcOEt
solution was washed with water and brine, dried over Na2SO4, and concentrated in
vacuo. The crude residue was purified by column chromatography (hexane/AcOEt =
2:1) to give compound 3 as a colorless foam (10.8 g, 91%). All spectral properties were
identical to those reported in the literature.19
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1-(2-O-Allyl-4-C-tert-butyldiphenylsilyloxymethyl-3,5-di-O-benzyl--D-ribofuranosyl)-3
-(benzyloxymethyl)thymine (4)
To a solution of 3 (3.0 g, 3.63 mmol) in THF (36 mL), NaH (218 mg, 5.45 mmol, 60%
dispersion in mineral oil) was added at 0 °C, and the mixture was stirred for 30 min at
room temperature. After addition of allyl bromide (0.94 mL, 10.9 mmol) at 0 °C, the
reaction mixture was stirred for 1 h. After addition of water, the reaction mixture was
extracted with AcOEt. The combined organic layer was washed with water and brine,
dried over Na2SO4, and concentrated in vacuo. The crude residue was purified by
column chromatography (hexane/AcOEt = 4:1 to 2:1) to give compound 4 as a colorless
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oil (2.97 g, 95%). 1H NMR (500 MHz, CDCl3): δ 7.71 (d, J = 1.0 Hz, 1H), 7.65 (dd, J =
8.0, 1.5 Hz, 2H), 7.62 (dd, J = 8.0, 1.5 Hz, 2H), 7.43-7.22 (m, 21H), 5.85 (d, J = 3.0 Hz,
1H), 5.75-5.67 (m, 1H), 5.50 and 5.47 (ABq, J = 9.5 Hz, 2H), 5.15 (dd, J = 17.0, 1.5 Hz,
1H), 5.07 (dd, J = 10.5, 1.5 Hz, 1H), 4.68 (s, 2H), 4.67 and 4.43 (ABq, J = 12.0 Hz, 2H),
4.58 and 4.52 (ABq, J = 11.5 Hz, 2H), 4.26 (d, J = 5.5 Hz, 1H), 4.11 (dd, J = 13.0, 6.5
Hz, 1H), 4.10 and 3.71 (ABq, J = 10.5 Hz, 2H), 4.05 and 3.95 (ABq, J = 11.5 Hz, 2H),
4.01 (dd, J = 13.0, 6.0 Hz, 1H), 3.93 (dd, J = 5.5, 3.0 Hz, 1H), 1.45 (s, 3H), 1.05 (s, 9H).
13C{1H} NMR (126 MHz, CDCl3): δ 163.5, 150.8, 138.0, 137.7, 137.5, 135.8, 135.6,
134.8, 133.8, 133.4, 133.0, 129.7, 129.6, 128.5, 128.3, 128.2, 128.0, 127.8, 127.7, 127.6,
127.6, 127.5, 117.9, 109.6, 88.1, 87.9, 80.1, 75.8, 73.6, 72.9, 72.0, 71.0, 71.0, 70.4, 64.6,
26.9, 19.3, 12.5. IR (ATR) cm-1: 3070, 3029, 2929, 2857, 1707, 1661, 1454, 1361, 1265.
HRMS (ESI-TOF): calcd for C52H58N2NaO8Si [M + Na]+ 889.3860, found 889.3864.
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1-(2-O-Allyl-4-C-hydroxymethyl-3,5-di-O-benzyl--D-ribofuranosyl)-3-(benzyloxymethy
l)thymine (5)
To a solution of 4 (2.94 g, 3.39 mmol) in THF (34 mL), TBAF (1 M solution in THF,
6.78 mL, 6.78 mmol) was added at room temperature. After being stirred for 3 h, the
reaction mixture was concentrated in vacuo. The crude residue was purified by column
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chromatography (hexane/AcOEt = 3:2 to 1:2) to give compound 5 as a colorless oil
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(2.08 g, 98%). H NMR (300 MHz, CDCl3): δ 7.66 (d, J = 1.0 Hz, 1H), 7.40-7.20 (m,
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15H), 6.10 (d, J = 2.5 Hz, 1H), 5.96-5.83 (m, 1H), 5.47 (s, 2H), 5.32 (dd, J = 17.0, 1.5
Hz, 1H), 5.21 (dd, J = 10.5, 1.5 Hz, 1H), 4.77 and 4.44 (ABq, J = 11.5 Hz, 2H), 4.68 (s,
2H), 4.55 and 4.49 (ABq, J = 11.5 Hz, 2H), 4.44-4.37 (m, 1H), 4.40 (d, J = 6.0 Hz, 1H),
4.19 (dd, J = 13.0, 6.0 Hz, 1H), 4.05 (dd, J = 6.0, 2.5 Hz, 1H), 3.83-3.70 (m, 4H), 2.71
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(br s, 1H), 1.48 (d, J = 1.0 Hz, 3H). C{1H} NMR (75 MHz, CDCl3): δ 163.2, 150.6,
137.8, 137.1, 137.1, 134.5, 133.4, 128.4, 128.4, 128.1, 128.1, 127.9, 127.6, 127.5, 127.5,
127.4, 118.3, 109.7, 89.3, 87.2, 80.3, 75.8, 73.4, 72.8, 71.9, 71.5, 70.5, 70.2, 63.6, 12.4.
IR (ATR) cm-1: 3470, 3067, 3030, 2930, 2870, 1707, 1661, 1465, 1453, 1364, 1271.
HRMS (ESI-TOF): calcd for C36H40N2NaO8 [M + Na]+ 651.2682, found 651.2681.
1-[2-O-Allyl-4-C-[O-(1,3-dihydro-1,3-dioxo-2H-isoindol-2-yl)hydroxymethyl]-3,5-di-O-
benzyl--D-ribofuranosyl]-3-(benzyloxymethyl)thymine (6)
To a mixture of 5 (78.7 mg, 0.13 mmol), NHPI (53.1 mg, 0.33 mmol) and Ph3P (85.4
mg, 0.33 mmol) in THF (2 mL), DIAD (73.8 μL, 0.38 mmol) was added dropwise at
0 °C. After being stirred at reflux for 1.5 h, the reaction mixture was concentrated in
vacuo. The crude residue was purified by column chromatography (CHCl3/AcOEt =
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5:1) to give compound 6 as a colorless oil (52.6 mg, 54%). H NMR (500 MHz,
CDCl3): δ 7.81 (dd, J = 5.5, 3.0 Hz, 2H), 7.73 (dd, J = 5.5, 3.0 Hz, 2H), 7.71 (s, 1H),
7.36-7.23 (m, 15H), 6.13 (d, J = 2.5 Hz, 1H), 5.89-5.81 (m, 1H), 5.48 and 5.46 (ABq, J
= 10.0 Hz, 2H), 5.28 (dd, J = 17.0, 1.0 Hz, 1H), 5.16 (d, J = 10.5, 1H), 4.77 and 3.96
(ABq, J = 10.5 Hz, 2H), 4.71 and 4.47 (ABq, J = 11.5 Hz, 2H), 4.67 (s, 2H), 4.64 and
4.60 (ABq, J = 11.5 Hz, 2H), 4.43-4.39 (m, 3H), 4.34 (dd, J = 13.0, 5.0 Hz, 1H), 4.18
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(dd, J = 13.0, 6.5 Hz, 1H), 4.06 (dd, J = 5.5, 2.5 Hz, 1H), 1.44 (s, 3H). C{1H} NMR
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(126 MHz, CDCl3): δ 163.4, 163.0, 150.8, 138.0, 137.4, 137.3, 134.7, 134.4, 133.8,
128.8, 128.6, 128.4, 128.2, 128.0, 128.0, 127.7, 127.7, 127.6, 123.5, 118.3, 109.7, 89.2,
86.5, 80.0, 78.7, 75.9, 73.7, 72.9, 72.1, 71.5, 70.4, 69.8, 12.5. IR (ATR) cm-1: 3064,
3030, 2926, 2871, 1790, 1732, 1707, 1659, 1453, 1363, 1274. HRMS (ESI-TOF): calcd
for C44H43N3NaO10 [M + Na]+ 796.2846, found 796.2846.
(1R,6R,8S,9R,11R)-3-benzyloxymethyl-1-[9-benzyloxymethyl-9-hydroxymethyl-6-phenyl
-2,7,10-trioxabicyclo[6.3.0]undecane-11-yl]thymine (7)
A solution of 6 (63.0 mg, 0.081 mmol), Hantzsch ester (30.9 mg, 0.12 mmol) and
fac-Ir(ppy)3 (0.5 mg, 0.81 μmol) in 1,4-dioxane (2 mL) was deaerated by Ar bubbling
for 0.5 h. The mixture was stirred and then irradiated with a 32 W CFL at 80 °C. After
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stirring for 1 h, the crude solution was concentrated in vacuo and purified by flash
column chromatography (hexane/AcOEt = 3:1 to 1:1) to give compound 7 as a colorless
oil (42.7 mg, 83%). 1H NMR (500 MHz, CDCl3): δ 7.40-7.24 (m, 16H), 6.12 (d, J = 5.0
Hz, 1H), 5.51 and 5.47 (ABq, J = 10.0 Hz, 2H), 4.71 (s, 2H), 4.58 (s, 2H), 4.52 (dd, J =
6.5, 4.0 Hz, 1H), 4.33 (d, J = 6.0 Hz, 1H), 4.11-4.06 (m, 2H), 3.77-3.65 (m, 4H), 3.56
(ddd, J = 12.0, 6.0, 4.0 Hz, 1H), 2.37 (dd, J = 9.0, 6.0 Hz, 1H), 2.10-1.99 (m, 2H),
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1.91-1.84 (m, 1H), 1.75-1.67 (m, 1H), 1.73 (d, J = 1.0 Hz, 3H). C{1H} NMR (126
MHz, CDCl3): δ 163.5, 151.1, 142.3, 138.0, 137.5, 135.6, 128.6, 128.5, 128.3, 128.0,
127.7, 127.6, 127.6, 127.5, 125.6, 110.4, 90.8, 88.4, 84.4, 82.7, 80.2, 73.7, 72.4, 72.2,
71.8, 70.6, 63.8, 34.5, 26.2, 12.9. IR (ATR) cm-1: 3495, 3062, 3030, 2926, 2864, 1710,
1666, 1654, 1451, 1363, 1273. HRMS (ESI-TOF): calcd for C36H40N2NaO8 [M + Na]+
651.2682, found 651.2686.
1-(2-O-Allyl-4-C-carboxy-3,5-di-O-benzyl--D-ribofuranosyl)-3-(benzyloxymethyl)thym
ine (8)
To a solution of 5 (613 mg, 0.97 mmol) in MeCN-H2O (1:1, 10 mL), PhI(OAc)2 (691
mg, 2.14 mmol) and TEMPO (30.4 mg, 0.19 mmol) were added at room temperature.
After being stirred for 21 h, the reaction mixture was quenched with sat. NaHCO3 aq.
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and extracted with AcOEt. The combined organic layer was washed with water and
brine, dried over Na2SO4, and concentrated in vacuo. The crude residue was purified by
column chromatography (CHCl3 to CHCl3/MeOH = 20:1) to give compound 8 as a
colorless oil (564 mg, 90%). 1H NMR (500 MHz, CDCl3): δ 7.39 (s, 1H), 7.34-7.20 (m,
15H), 6.34 (d, J = 5.5 Hz, 1H), 5.75-5.68 (m, 1H), 5.43 (s, 2H), 5.12 (d, J = 17.5, 1H),
5.10 (d, J = 10.5 Hz, 1H), 4.73 and 4.68 (ABq, J = 11.5 Hz, 2H), 4.63 (s, 2H), 4.52 and
4.47 (ABq, J = 11.5 Hz, 2H), 4.37 (d, J = 5.0 Hz, 1H), 4.18 (t, J = 5.5 Hz, 1H), 4.05 and
3.78 (ABq, J = 10.5 Hz, 2H), 3.98 (dd, J = 13.0, 5.5 Hz, 1H), 3.93 (dd, J = 12.5, 5.5 Hz,
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1H), 1.52 (s, 3H). C{1H} NMR (126 MHz, CDCl3): δ 171.8, 163.3, 151.3, 137.8,
137.0, 134.6, 133.5, 128.6, 128.4, 128.2, 128.2, 128.0, 127.7, 127.6, 127.6, 118.6, 110.6,
89.3, 88.1, 79.4, 77.8, 74.3, 73.8, 72.1, 71.7, 71.5, 70.5, 12.6. IR (ATR) cm-1: 3065,
3030, 2926, 2868, 1712, 1668, 1655, 1453, 1363, 1276. HRMS (ESI-TOF): calcd for
C36H38N2NaO9 [M + Na]+ 665.2475, found 665.2474.
1-[2-O-Allyl-4-C-[O-(1,3-dihydro-1,3-dioxo-2H-isoindol-2-yl)carboxy]-3,5-di-O-benzyl
--D-ribofuranosyl]-3-(benzyloxymethyl)thymine (9)
To a solution of 8 (8.2 g, 12.8 mmol) in DCM (100 mL), NHPI (3.12 g, 19.1 mmol)
and EDCI·HCl (3.67 g, 19.1 mmol) were added at room temperature. After being stirred
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for 4.5 h, the reaction mixture was diluted with water and extracted with AcOEt. The
combined organic layer was washed with water and brine, dried over Na2SO4, and
concentrated in vacuo. The crude residue was purified by column chromatography
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NMR (500 MHz, CDCl3): δ 7.89 (dd, J = 5.5, 3.0 Hz, 2H), 7.79 (dd, J = 5.5, 3.0 Hz,
2H), 7.45-7.23 (m, 16H), 6.44 (d, J = 5.5 Hz, 1H), 5.79 (ddt, J = 17.0, 10.5, 5.5 Hz, 1H),
5.49 and 5.47 (ABq, J = 9.5 Hz, 2H), 5.18 (dd, J = 17.0, 1.5 Hz, 1H), 5.13 (dd, J = 10.5,
1.5 Hz, 1H), 4.86 and 4.81 (ABq, J = 12.0 Hz, 2H), 4.68 (s, 2H), 4.62 (s, 2H), 4.42 (d, J
= 4.5 Hz, 1H), 4.20 (dd, J = 5.0, 4.5 Hz, 1H), 4.13 and 4.05 (ABq, J = 10.5 Hz, 2H),
4.11-4.03 (m, 2H), 1.56 (d, J = 1.0 Hz, 3H). 13C{1H} NMR (126 MHz, CDCl3): δ 165.7,
163.3, 161.2, 151.0, 137.9, 137.1, 136.7, 134.7, 134.6, 133.8, 128.9, 128.7, 128.3, 128.3,
128.3, 128.3, 127.9, 127.7, 127.6, 124.0, 118.2, 110.5, 88.6, 88.6, 79.3, 77.5, 74.1, 73.8,
72.1, 71.5, 71.2, 70.5, 12.7. IR (ATR) cm-1: 3064, 3032, 2926, 2868, 1788, 1743, 1713,
1670, 1657, 1453, 1362, 1277. HRMS (ESI-TOF): calcd for C44H41N3NaO11 [M + Na]+
810.2639, found 810.2639.
(1R,2S,5R,6R,8S)-1-[8-benzyloxy-1-(benzyloxymethyl)-2-methyl-4,7-dioxabicyclo[3.2.1]
octane-6-yl]-3-(benzyloxymethyl)thymine (10)
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A suspension of 9 (1.5 g, 1.9 mmol), Hantzsch ester (723 mg, 2.86 mmol) and
fac-Ir(ppy)3 (12.4 mg, 0.02 mmol) in 1,4-dioxane (19 mL) was deaerated by Ar
bubbling for 0.5 h. The mixture was stirred and then irradiated with a 32 W compact
fluorescent lamp at room temperature. After stirring for 2 h, a yellow homogeneous
solution was obtained. The crude solution was concentrated in vacuo and purified by
flash column chromatography (hexane/AcOEt = 5:1 to 3:1) to give compound 10 as a
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white foam (668 mg, 59%). H NMR (500 MHz, CDCl3): δ 7.89 (s, 1H), 7.38-7.23 (m,
15H), 6.02 (s, 1H), 5.47 and 5.45 (ABq, J = 9.5 Hz, 2H), 4.74 and 4.50 (ABq, J = 11.5
Hz, 2H), 4.70 (s, 2H), 4.61 and 4.55 (ABq, J = 11.5 Hz, 2H), 4.29 (d, J = 3.0 Hz, 1H),
4.02 (d, J = 3.0 Hz, 1H), 3.95 and 3.58 (ABq, J = 11.0 Hz, 2H), 3.87 (dd, J = 11.5, 6.5
Hz, 1H), 3.49 (dd, J = 11.5, 11.0 Hz, 1H), 2.54-2.47 (m, 1H), 1.41 (s, 3H), 0.73 (d, J =
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7.0 Hz, 3H). C{1H} NMR (126 MHz, CDCl3): δ 163.5, 150.6, 138.0, 137.5, 137.3,
134.3, 128.6, 128.4, 128.3, 128.1, 127.9, 127.7, 127.7, 127.6, 109.4, 86.4, 85.6, 75.5,
73.6, 72.2, 71.8, 71.4, 70.3, 68.1, 66.8, 30.6, 12.5, 9.9. IR (ATR) cm-1: 3065, 3030, 2961,
2926, 2868, 1704, 1659, 1452, 1363, 1278, 1213. HRMS (ESI-TOF): calcd for
C35H38N2NaO7 [M + Na]+ 621.2577, found 621.2574.
(1R,2S,5R,6R,8S)-1-[8-hydroxy-1-hydroxymethyl-2-methyl-4,7-dioxabicyclo[3.2.1]octa
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Compound 10 (169 mg, 0.28 mmol) was treated in MeOH (5 mL) at room temperature
under a hydrogen atmosphere in the presence of Pd(OH)2/C (170 mg). After stirring for
20 h, the reaction mixture was filtered and concentrated in vacuo. The crude residue was
purified by column chromatography (CHCl3/MeOH = 10:1) to give compound 11 as a
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white foam (78.4 mg, 93%). H NMR (500 MHz, CD3OD): δ 8.34 (d, J = 1.0 Hz, 1H),
5.96 (s, 1H), 4.16 (d, J = 3.0 Hz, 1H), 4.07 (d, J = 3.0 Hz, 1H), 3.98 and 3.64 (ABq, J =
12.0 Hz, 2H), 3.79 (dd, J = 11.5, 6.5 Hz, 1H), 3.46 (dd, J = 11.5, 11.5 Hz, 1H),
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2.40-2.32 (m, 1H), 1.86 (s, 3H), 0.74 (d, J = 6.5 Hz, 3H). C{1H} NMR (126 MHz,
CD3OD): δ 166.7, 152.0, 137.9, 110.2, 87.5, 86.6, 79.9, 67.7, 65.2, 60.4, 31.0, 12.6,
10.0. IR (ATR) cm-1: 3395, 3073, 2963, 2878, 1682, 1659, 1653, 1468, 1274. HRMS
(ESI-TOF): calcd for C13H18N2NaO6 [M + Na]+ 321.1063, found 321.1064.
(1R,2S,5R,6R,8S)-1-[8-hydroxy-1-(4,4ʹ-dimethoxytrityloxymethyl)-2-methyl-4,7-dioxabi
cyclo[3.2.1]octane-6-yl]thymine (12)
To a solution of 11 (180 mg, 0.60 mmol) in pyridine (6 mL), DMTrCl (306 mg, 0.90
mmol) was added at 0 °C. After being stirred for 18 h at room temperature, the reaction
mixture was quenched with sat. NaHCO3 aq. and extracted with AcOEt. The combined
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