Chemical syntheses of oligodeoxyribonucleotides containing spore photoproduct

Article abstract of DOI:10.1021/jo400013n

5-(α-Thyminyl)-5,6-dihydrothymine, also called spore photoproduct or SP, is commonly found in the genomic DNA of UV-irradiated bacterial endospores. Despite the fact that SP was discovered nearly 50 years ago, its biochemical impact is still largely unclear due to the difficulty of preparing SP-containing oligonucleotide in high purity. Here, we report the first synthesis of the phosphoramidite derivative of dinucleotide SP TpT, which enables successful incorporation of SP TpT into oligodeoxyribonucleotides with high efficiency via standard solid-phase synthesis. This result provides the scientific community a reliable means to prepare SP-containing oligonucleotides, laying the foundation for future SP biochemical studies. Thermal denaturation studies of the SP-containing oligonucleotide found that SP destabilizes the duplex by 10-20 kJ/mol, suggesting that its presence in the spore-genomic DNA may alter the DNA local conformation.

Full text of DOI:10.1021/jo400013n

Article
pubs.acs.org/joc
Chemical Syntheses of Oligodeoxyribonucleotides Containing Spore
Photoproduct
Yajun Jian† and Lei Li*,†,‡
†Department of Chemistry and Chemical Biology, Indiana University−Purdue University Indianapolis (IUPUI), Indianapolis, Indiana
46202, United States
‡Department of Biochemistry and Molecular Biology, Indiana University School of Medicine (IUSM), 635 Barnhill Drive,
Indianapolis, Indiana 46202, United States
S
* Supporting Information
ABSTRACT: 5-(α-Thyminyl)-5,6-dihydrothymine, also
called spore photoproduct or SP, is commonly found in the
genomic DNA of UV-irradiated bacterial endospores. Despite
the fact that SP was discovered nearly 50 years ago, its
biochemical impact is still largely unclear due to the difficulty
of preparing SP-containing oligonucleotide in high purity.
Here, we report the first synthesis of the phosphoramidite
derivative of dinucleotide SP TpT, which enables successful incorporation of SP TpT into oligodeoxyribonucleotides with high
efficiency via standard solid-phase synthesis. This result provides the scientific community a reliable means to prepare SP-
containing oligonucleotides, laying the foundation for future SP biochemical studies. Thermal denaturation studies of the SP-
containing oligonucleotide found that SP destabilizes the duplex by 10−20 kJ/mol, suggesting that its presence in the spore-
genomic DNA may alter the DNA local conformation.
Different from CPD and (6−4) PD whose formations are
mediated by [2 + 2] photocyclization reaction, SP is formed via
an intramolecular H atom-transfer mechanism.30−32 Its
formation requires A-DNA, which is induced by the low
hydration level in endospores.11,27,33 This, coupled with other
key factors such as the presence of a group of DNA-binding
proteins named small acid soluble proteins to solidify the A-
conformation,34−36 determines SP to be the sole photolesion in
UV irradiated endospores. SP can be generated under in vitro
conditions via solid phase (dry film or ice) DNA photo-
reaction;15,31,37−39 however, its yield is very low (<1%).31 This
yield and the simultaneous formation of many other photo-
lesions determine that it is very difficult to obtain enough SP-
containing oligonucleotide with high purity for biological
studies. As a consequence, although SP has been discovered
for half a century,40 little is known about its impact to the
biological function of DNA.
The best means to prepare highly pure SP containing
oligonucleotide is via chemical synthesis using traditional
phosphoramidite chemistry, which requires SP phosphorami-
dite as a building block. Currently, an SP dinucleoside
phosphoramidite, which does not contain the phosphodiester
moiety, is available.41 However, the lack of the phosphodiester
linkage likely releases any distortion created by the methylene
bridge between the two thymine bases in SP. Thus, research
conducted using dinucleoside SP-containing oligomer may not
be truly biologically relevant. In this report, we describe the first
INTRODUCTION
■
Among the four nucleobases, thymine is most sensitive to UV
irradiation.1 In regular cells, the adjacent thymine residues in
genomic DNA will dimerize, resulting in cis-syn cyclobutane
dimer (CPD) and (6−4) photoproduct ((6−4) PD) as the
most common products (Figure 1).2−7 Further irradiation of
(6−4) PD under ∼310 nm UV light triggers isomerization of
the pyrimidinone ring to its Dewar valence isomer.8,9 In
addition to these three species, the fourth dimer, 5-(α-
thyminyl)-5,6-dihydrothymine, which is also called spore
photoproduct or SP, was identified as the sole photolesion in
the genomic DNA of UVC-irradiated bacterial endo-
spores.10−13
SPs are quickly repaired in germinating spores by a radical
SAM enzyme−spore photoproduct lyase,10,13−25 thus posing
little threat to spores’ survival. The unrepaired SPs, however,
prove lethal to germinating spores.26,27 It is currently unclear
whether the lethality of SP is due to its induction of
mutagenesis or due to its ability to halt polymerase. Moreover,
although SP was generally considered to exist only in bacterial
endospores, a recent study found it was the dominant DNA
photolesion in UV-irradiated airborne Mycobacterium parafor-
tuitum under 20−40% relative humidity (RH),28 suggesting
that SP may exist in other species as well. Its formation is likely
to be responsible for the UV-killing effect in the air sterilization
process. SP was also implied to be present in the UV-irradiated
frozen E. coli cells, and E. coli cells were more sensitive to SP
than to other dithymine photoproducts.29 These findings
suggest that SP may play a role in nature that is much bigger
than we currently think.
Received: January 5, 2013
© XXXX American Chemical Society
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Figure 1. Structures of the major type of thymine photoproducts.
Scheme 1. Synthesis of the Protected R Isomer of SP TpT
synthesis of the phosphoramidite for dinucleotide SP TpT,
which enables SP TpT incorporation into oligonucleotide with
high efficiency, making SP biological studies possible.
is thus impractical to generate enough SP via photoreaction. To
prepare SP phosphoramidite on a relatively large scale, new
strategies have to be developed.
Different from the other thymine dimers where chemical
syntheses are not available, synthesis of the dinucleotide SP
TpT was successfully achieved via a 14-step reaction by Begley
et al.53 The synthesis employed CC hydrogenation, methyl
bromination, and nucleophilic substitution to yield two
dinucleoside isomers and phosphodiester moiety insertion to
obtain the dinucleotide SP TpT. However, in Begley’s
synthesis, the two dinucleoside diastereoisomers were used as
intermediates and the R and S isomers were not separated by
HPLC until the very last step. As only the R isomer is formed in
endospores’ genomic DNA,54 it is desirable to separate these
isomers before the phosphodiester moiety is introduced.
Separation of the protected dinucleoside SP isomers was
achieved by Broderick et al.55 and Carell et al.,41 respectively,
using different deoxyribose protection reagents. Having these
previous works in mind, we decided to adopt a combined
approach to prepare the protected R isomer of SP TpT
(Scheme 1).
RESULTS AND DISCUSSION
■
The phosphoramidite derivatives of cis-syn CPD,42−49 (6−4)
PD,50 and Dewar PD51 have been available, which enabled
successful preparations of thymine dimer containing oligonu-
cleotides with high reaction efficiencies. All these derivatives
were generated via hybrid approaches where the corresponding
dimers were first produced by photochemistry using partially
protected dinucleotide TpT before the phosphoramidite
moiety was introduced to the 3′-OH group at the 3′-end of
the dimer via organic synthesis. Such an approach, however,
proves futile in preparing the SP phosphoramidite. As
mentioned above, the unprotected dinucleotide TpT generates
SP in ∼1% yield via solid-state photoreaction.31 Once the
phosphodiester moiety is protected by esterization with either
−CH3 or −CH2CH2CN, the resulting dinucleotide no longer
supports SP formation. Such a result agrees with our previous
finding that dinucleotide T(CH2)T which contains a neutral
formacetal linkage did not support SP photochemistry,52
suggesting the negative charge carried by the phosphodiester
linker is essential for the two thymine residues to adopt the
“right” stacking conformation to enable SP photochemistry. It
Fully protected thymidine 1 and dihydrothymidine 3 were
prepared from thymidine using Begley’s procedure.53 In that
approach, the methyl bromination reaction in the production of
2 was conducted via a photochemical assay using elemental
bromine. Carell et al. later employed N-bromosuccinimide
B
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Scheme 2. Synthesis of the SP TpT-Containing Oligonucleotide via Phosphoramidite P1
Scheme 3. Synthesis of the SP TpT-Containing Oligonucleotide via Phosphoramidite P2
(NBS) as a substitute of Br2 to brominate the methyl moiety
because NBS is easier to handle and reacts more mildly.56 We
further modified Carell’s method, using 1, 2-dichloroethane to
replace CCl4 as the solvent for the NBS reaction (Scheme 1).
Such a solvent change resulted in a slightly reduced reaction
yield (47% vs 60%); however, compared to CCl4, 1, 2-
dichloroethane is much cheaper and more environmentally
friendly. The coupling reaction between the enolate of 3 and
bromomethyl deoxyuridine 2 afforded 4 and 5 (1.2:1.0) in 53%
total yield. After separation via flash chromatography using a
literature procedure,55 the resulting 5 was treated with either
4% HF in pyridine53 or HCO2H in methanol55 to remove the
triethylsilyl (TES) protecting groups, generating 6. The yield of
6 is modest in either reaction due to the unexpected loss of the
tert-butyldimethylsilyl (TBS) moiety. We found that treating 5
with 4% HF·pyridine in acetonitrile drastically improved the
yield to 92%. TBS is very stable under this condition, and TES
can thus be selectively removed. Subsequent dimethoxytrityla-
tion of the 5′-OH followed by phosphorylation of the 3′-OH
afforded 8. After desilylation, the MSNT-mediated formation of
the phosphotriester yielded the protected SP 9 as a mixture of
two diastereomers in a ratio of 1:1.7 (Sp/Rp), as indicated by
LC−MS analysis.
Using 9 as precursor, an SP TpT phosphoramidite P1
(Scheme 2) was readily synthesized. P1 was stable enough to
survive the column purification; an 80% yield was obtained for
this reaction step. In another attempt, we removed the 2-
(trimethylsilyl)ethoxymethyl (SEM) protecting groups at-
tached to the N3 positions of the thymine ring in SP.
Surprisingly, deletion of SEM greatly destabilizes the
corresponding SP phosphoramidite. Although its formation
was indicated by ESI-MS analysis, this phosphoramidite
decomposed completely during purification via flash chroma-
tography. Such a result is surprising as phosphoramidites of
unprotected CPD and (6−4) PD species are all fairly
stable.42−44,46,50 We tentatively ascribe the instability of the
unprotected SP phosphoramidite to its unique chemical
structure. Moreover, in the previously synthesized dinucleoside
SP TT phosphoramidite, the N3 positions were also protected
by SEM.41 The N3 protection thus appears essential for the
production of a stable SP phosphoramidite, although it is
unclear why alteration at the remote N3 site can impact the
phosphoramidite moiety attached to the 3′-deoxyribose.
Subsequent oligonucleotide synthesis using P1 as a building
block afforded an SP TpT containing 10-mer d(CACC[SP
TpT]CATC) in a proof-of-concept experiment. However, the
coupling yield for the SP incorporation step was found to be
merely ∼15%, even after we extended the coupling time to 2 h
or repeated the coupling step twice. This result is in sharp
contrast to the >95% yield obtained in the previous preparation
of bent DNA, which used uracil alkylene cross-linked
phosphoramidites containing the 2-chlorophenyl protecting
group and a 20-min reaction time.57 Such a low coupling yield
determines that it is impractical to use P1 to synthesize SP-
containing oligonucleotide on a relatively large scale.
Analyzing the phosphoramidites of other thymine dimers
reveals that their phosphodiester moieties were all protected by
a small alkyl group,42−44,50,51 suggesting that the bulky 2-
chlorophenyl moiety in P1 may create some steric hindrance,
preventing an efficient coupling reaction from occurring during
the solid-phase synthesis. We therefore incubated 9 with
sodium methoxide to replace the 2-chlorophenyl moiety with a
−CH3 and prepared an SP phosphoramidite derivative P2
(Scheme 3). As expected, P2 fully supports the solid-state
synthesis, exhibiting a >90% coupling efficiency during the
preparation of the 10-mer d(CACC[SP TpT]CATC) (Figure
2A). During the synthesis, the reaction time was extended to 1
h for the SP incorporation step. In contrast, the coupling
reaction for a regular deoxyribonucleotide usually finishes
within 1 min. Such an elongated reaction time is necessary for
an efficient SP incorporation; similar strategies were utilized
previously to ensure the incorporation of other dithymine
photoproducts into oligonucleotides with satisfactory
yields.42,43 Product formation was confirmed by ESI-MS
analysis (Figure 2B). After purification by HPLC, the overall
yield for the SP containing 10-mer was found to be 63%, based
on the amount of resin used.
To demonstrate that long oligonucleotides can also be
prepared by this method, d(CTCGACACG[SP TpT]-
CGCATGCCA), a 20-mer, was synthesized. The SP TpT
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the 20-mer decreases the Tm by 5 °C (69.5 °C vs 74.5 °C)
(Figure 3B). Both measurements suggest that the presence of
SP significantly destabilizes the duplex oligonucleotide. It is
worth mentioning that the Tm difference between a modified
and the corresponding unmodified duplex is sensible to the
length of the oligonucleotide, and the difference is higher for a
shorter duplex. Therefore, the Tm difference (12 °C vs 5 °C)
observed between the 10- and the 20-mer is not surprising.
The crystal structure of the dinucleoside SP TT-containing
oligonucleotide was recently solved by Carell et al.41 The
structure reveals that the two thymines in SP TT hydrogen
bond with the two adenines on the complementary strand as if
they were undamaged. As the lack of the phosphodiester
linkage in dinucleoside SP TT (Scheme 4) could release the
potential conformational distortion created by the methylene
bridge between the two thymine bases of SP, we wonder if the
structure observed truly reflects that of the SP containing spore
genomic DNA.
We therefore synthesized the SP dinucleoside phosphor-
amidite P3 using a small acetyl group to replace the bulky tert-
butyldiphenylsilyl (TBDPS) moiety adopted previously to
protect the 3′-OH group of the 5′-thymidine (Scheme 4). P3
enables us to prepare the SP TT-containing 10-mer and 20-
mer, respectively, using sequences identical to those above. A
total yield of 60% (based on the amount of resin used) was
obtained in the 10-mer synthesis,30 which is comparable to the
63% yield found in the preparation of SP TpT containing 10-
mer, but much higher than the 15% total yield obtained
previously during a 12-mer preparation using the TBDPS-
protected SP TT phosphoramidite.41 This nearly 4-fold
improvement in yield is significant considering the cost of the
starting material and the 10+ synthetic steps to obtain the SP
TT-containing oligomer. This finding is consistent with the
observation above that P2 is a far better reagent than its bulkier
counterpart P1 for SP TpT incorporation. These results
indicate that the steric hindrance associated with the 3′-OH
moiety at the 5′-end of SP has a big impact to the coupling
reaction at the 3′-deoxyribose. Under conditions identical to
those above, the SP TT containing 10-mer duplex exhibits ∼25
°C Tm decrease compared to the undamaged parent 10-mer
strand (Figure 3A). The 20-mer is relatively stable, exhibiting a
Tm of 64.6 °C, which is still 10 °C lower than its parent strand
(Figure 3B).
Figure 2. (a) HPLC chromatograph (260 nm) of the crude reaction
mixture in the synthesis of 10-mer d(CACC[SP TpT]CATC). (b)
ESI-MS analysis of the resulting SP containing 10-mer oligonucleotide.
After deconvolution, the 10-mer exhibits a mass of 2921.51 (calcd
2921.53).
containing 20-mer was detected as the major product, as
proved by the HPLC analysis and confirmed by ESI-MS
spectrometry (Figure S3, Supporting Information). The overall
yield of the 20-mer was determined to be 30% relative to the
amount of resin used, again indicating P2 is a good building
block for SP incorporation. This method thus does not have
sequence limitation and can be readily used to prepare the long
oligonucleotides needed in typical biochemical studies such as
the polymerase extension experiments.
The successful preparation of SP TpT-containing oligonu-
cleotides provides us a good opportunity to study the influence
of SP to the stability of duplex oligonucleotide. Such
information is still unknown despite the fact that SP was
discovered nearly 50 years ago. We thus compared the thermal
denaturation curves of SP TpT-containing oligonucleotides
with the corresponding undamaged parent strands. Using the
10-mer sequence at 2.4 μM concentration in 10 mM phosphate
buffer at pH 7, which also contains 150 mM NaCl, 12 °C
difference (27.8 °C vs 39.5 °C) between the oligonucleotide
melting points (Tms) was observed (Figure 3A). Using the
same buffer and 1.6 μM oligomer, the presence of SP TpT in
To further reveal the impact of the dinucleotide SP TpT and
dinucleoside SP TT to the stability of the duplex oligonucleo-
tide, we measured the melting points (Tm’s) for the 10-mers
and 20-mers containing TpT, SP TpT, and SP TT, respectively,
as a function of concentration. For the 10-mer strands, using
oligomer concentrations ranging from 0.6 to 4.8 μM, the
thermodynamic parameters ΔH°, ΔS°, and ΔG° of the
dissociation process at 310 K were derived from concentration
dependent Tm investigations using van’t Hoff plots.58−60
Similarly, using oligomer concentrations ranging from 0.4 to
3.2 μM, the ΔH°, ΔS°, and ΔG° were determined for the
corresponding 20-mer strands as well.
As shown in Table 1, the calculated free energy change
(ΔΔG°) for the dinucleotide SP TpT-containing duplex was
found to be less negative than the native strand by 10 kJ/mol
for the 10-mer and 21 kJ/mol for the 20-mer, respectively, at 37
°C. We tentatively ascribe the positive ΔΔG° for the SP TpT-
containing duplexes to the poor H-bonding interaction with the
complementary adenines and/or the weak stacking interaction
between SP and its neighboring residues. This result suggests
Figure 3. (A) Thermal denaturation curves of the 10-mers
d(CACCTTCATC) (blue line), d(CACC[SP TpT]CATC (red
line), and d(CACC[SP TT]CATC) (black line) annealed with the
complementary strand d(GTGGAAGTAG), respectively. The buffer
contained 10 mM phosphate at pH 7 and 150 mM NaCl; the oligomer
concentration was 2.4 μM for all three samples. (B) Thermal
denaturation curves of the 20-mers d(CTCGACACGTTCGCATG-
CCA) (blue line), d(CTCGACACG[SP TpT]CGCATGCCA) (red
line), and d(CTCGACACG[SP TT]CGCATGCCA) (black line)
annealed with the complementary strand, respectively. The buffer
contained 10 mM phosphate at pH 7 and 150 mM NaCl; the oligomer
concentration was 1.6 μM for all three samples.
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Scheme 4. Synthesis of the Dinucleoside SP TT-Containing Oligonucleotide via Phosphoramidite P3
(Dynamic Adsorbents, Inc., 32−63 μm). For TLC analysis, precoated
Table 1. Thermodynamic Parameters for Duplex Formation
in Native and SP-Containing Oligonucleotides
plates (w/h F254, Dynamic Adsorbents Inc., 0.25 mm thick) were
1
used. The H, 13C, and 31P NMR spectra were obtained on a Bruker
ΔG°
ΔΔG°
(kJ/mol)
(37 °C)
500 MHz NMR Fourier transform spectrometer. NMR spectra were
recorded in sample solutions in deuterated chloroform (CDCl3), with
residual chloroform (Δ 77.0 ppm for 13C NMR) and TMS (Δ 0 ppm
for 1H NMR), deuterated methanol (Δ 3.31 ppm for 1H NMR and Δ
49.1 ppm for 13C NMR), or deuterated methyl sulfoxide (DMSO-d6),
ΔH°
ΔS°
(kJ/mol)
(37 °C)
(kJ/mol) (kJ/mol/K)
10-mer
20-mer
native
−278
−259
−240
−778
−670
−618
−0.763
−0.735
−0.709
−2.11
−1.825
−1.701
−41
−31
−20
−124
−103
−90
SP TpT
SP TT
native
+10
+21
1
with residual methyl sulfoxide (Δ 2.50 ppm for H NMR and Δ 39.5
ppm for 13C NMR) taken as the standard. The chemical shifts in NMR
spectra were reported in parts per million (ppm). Mass (MS) analysis
was obtained via ESI with an ion-trap mass analyzer. The HR-MS was
performed with Q-TOF LC/MS spectrometer; the data were acquired
via Agilent MassHunter Workstation Data Acquisition (B.03.00) and
analyzed via Qualitative Analysis of MassHunter Acquisition Data
(B.03.00) software.
SP TpT
SP TT
+21
+34
that similar to cis-syn CPD,61,62 SP also destabilizes the duplex
oligonucleotide. As shown by Taylor et al., the presence of cis-
syn CPD destabilizes the duplex strands by 5.7−8.4 kJ/mol
depending on the oligonucleotide sequence studied.62 Barring
the sequence difference, our data implies that the SP TpT may
cause more local distortion to the duplex structure than the cis-
syn CPD in aqueous solution. Moreover, the presence of
dinucleoside SP TT destabilizes the duplex strands even further
(Table 1). Although Carell’s work implies that the presence of
SP TT induces little conformational change; such a structure
may only reflect the DNA conformation in the crystal state and
is unlikely to represent that of the SP TpT containing oligomer
in solution.
α-Bromo-3′,5′-O-bis(tert-butyldimethylsilyl)-3-[(trimethyl-
silylethoxy)methyl]thymidine (2). Benzoyl peroxide (168 mg, 0.69
mmol) and NBS (8.71 g, 48.95 mmol) were added in turn to a
solution of 1 (13.90 g, 23.13 mmol) in anhydrous 1,2-dichloroethane
(168 mL). The reaction mixture was stirred at 80 °C for 1 h, and the
solvent was removed by rotary evaporation. The resulting residue was
purified via column chromatography (eluent: hexane/EtOAc = 7:1) to
afford 2 as a pale yellow oil (8.18 g, 12.03 mmol, 52% yield): Rf = 0.60
(hexane/ethyl acetate = 4:1); 1H NMR (500 MHz, CDCl3) δ 0.01 (s,
9H), 0.08 (s, 3H), 0.09 (s, 3H), 0.137 (s, 3H), 0.144 (s, 3H), 0.87−
1.08 (m, 20H), 1.92−2.04 (m, 1H), 2.30−2.39 (m, 1H), 3.64−3.72
(m, 2H), 3.74−3.81 (m, 1H), 3.86−3.92 (m, 1H), 3.95−4.01 (m, 1H),
4.24 (d, J = 10.4 Hz, 1H), 4.31 (d, J = 10.7 Hz, 1H), 4.36−4.41 (m,
1H), 5.38−5.45 (m, 2H), 6.28−6.34 (m, 1H), 7.89 (s, 1H); 13C NMR
(126 MHz, CDCl3) δ −5.35, −5.38, −4.9, −4.7, −1.5, 18.0, 18.1, 18.4,
25.7, 26.0, 26.2, 41.9, 63.0, 67.7, 70.2, 72.2, 86.2, 88.2, 110.9, 137.8,
150.4, 161.3.
(5S)- and (5R)-α-[3′,5′-O-Bis(tert-butyldimethylsilyl)-3-
[(trimethylsilylethoxy)methyl]thymidyl]-5,6-dihydro-3′,5′-O-
bis(triethylsilyl)-3-[(trimethylsilylethoxy)methyl]thymidine
(4,5). LDA (5.40 mL, 10.80 mmol, 2 M solution in hexane/THF/
ethylbenzene) was slowly added to a solution of 353 (4.36 g, 7.23
mmol) in THF (60 mL) at −78 °C. The reaction mixture was stirred
at the same temperature for 2 h before addition of 2 (6.14 g, 9.04
mmol) in THF (15 mL). The resulting mixture was stirred at −78 °C
for 30 min and then slowly warmed to room temperature over 3 h.
After being stirred at room temperature overnight, the mixture was
diluted with EtOAc, washed with NH4Cl, water, and brine
successively, and dried over anhydrous Na2SO4. After the solvent
was removed via rotary evaporation, the resulting residue was purified
CONCLUSION
■
Here we report the first synthesis of SP TpT phosphoramidite,
which allows us to prepare SP TpT-containing oligonucleotide
with high purity and high efficiency. This work clears the major
obstacle in studying the impact of SP to the biofunction of
DNA.
EXPERIMENTAL SECTION
■
General Methods. dA-, dT-, dC-, and dG-phosphoramidites were
purchased from Glen Research (Sterling, VA). CPG Nucleosides
carriers were obtained from 3-Prime (3-prime, Aston, PA). All
reactions were carried out using oven or flame-dried glassware under a
nitrogen atmosphere in distilled solvents. Dichloromethane and
pyridine were distilled over calcium hydride. Purification of reaction
products was carried out by flash chromatography using silica gel
E
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via column chromatography (eluent: hexane/EtOAc = 7:1) to afford 4
(S-isomer, 2.43 g, 2.02 mmol, 28% yield) and 5 (R-isomer, 2.00 g, 1.66
mmol, 23% yield) as colorless oils. S-Isomer: Rf = 0.62 (hexane/ethyl
1H), 2.02−2.09 (m, 2 H), 2.11−2.19 (m, 1 H), 2.24 (ddd, J = 3.0, 6.1,
13.3 Hz, 1H), 2.61 (d, J = 14.0 Hz, 1H), 2.68 (d, J = 14.2 Hz, 1H),
3.16 (d, J = 13.1 Hz, 1H), 3.19 (d, J = 12.9 Hz, 1H), 3.32 (dd, J = 4.8,
10.7 Hz, 1H), 3.34 (dd, J = 4.6, 10.3 Hz, 1H), 3.56 (t, J = 8.1 Hz, 2H),
3.63−3.68 (m, 2H), 3.72−3.78 (m, 2H), 3.79 (s, 6H), 3.82 (dd, J =
3.9, 11.0 Hz, 1H), 3.92 (td, J = 3.5, 8.8 Hz, 1H), 4.36−4.42 (m, 1H),
4.43−4.47 (m, 1H), 5.16 (d, J = 9.7 Hz, 1H), 5.18 (d, J = 9.6 Hz, 1H),
5.30 (d, J = 9.8 Hz, 1H), 5.32 (d, J = 9.7 Hz, 1H), 6.33 (t, J = 7.1 Hz,
1H), 6.34 (t, J = 6.9 Hz, 1H), 6.83−6.87 (m, 4H), 7.19−7.24 (m, 1H),
7.28−7.32 (m, 2H), 7.32−7.37 (m, 4 H), 7.44−7.47 (m, 2H), 7.51 (s,
1H); 13C NMR (126 MHz, CDCl3) δ −5.4, −5.3, −4.8, −4.7, −1.4,
17.9, 18.1, 18.2, 18.4, 22.3, 25.7, 25.9, 32.3, 36.5, 40.1, 43.0, 44.6, 55.2,
62.8, 63.5, 66.7, 67.5, 69.9, 70.4, 71.9, 72.0, 83.2, 83.5, 85.4, 86.4, 87.5,
108.4, 113.2, 126.8, 127.9, 128.1, 130.01, 130.03, 135.80, 135.84,
139.4, 144.7, 150.6, 152.3, 158.5, 163.5, 173.4; ESI-HRMS m/z calcd
1
acetate = 4:1); H NMR (500 MHz, CDCl3) δ −0.02 (s, 9H), −0.01
(s, 9H), 0.083 (s, 3H), 0.087 (s, 3H), 0.097 (s, 3H), 0.100 (s, 3H),
0.60 (q, J = 8.0 Hz, 6H), 0.63 (q, J = 7.9 Hz, 6H), 0.89 (s, 9H), 0.90
(s, 9H), 0.92−1.00 (m, 22H), 1.17 (s, 3H), 1.91−2.02 (m, 3H), 2.31
(ddd, J = 2.2, 5.7, 13.2 Hz, 1H), 2.53 (d, J = 14.2 Hz, 1H), 2.80 (d, J =
14.2 Hz, 1H), 3.05 (d, J = 13.1 Hz, 1H), 3.32 (d, J = 13.1 Hz, 1H),
3.54−3.61 (m, 3H), 3.61−3.66 (m, 2H), 3.68 (dd, J = 3.8, 10.9 Hz,
1H), 3.73 (dd, J = 5.0, 10.9 Hz, 1H), 3.78 (dd, J = 3.5, 10.9 Hz, 1H),
3.81−3.86 (m, 1H), 3.92−3.96 (m, 1H), 4.30−4.34 (m, 1H), 4.38 (td,
J = 2.4, 5.5 Hz, 1H), 5.14 (d, J = 9.4 Hz, 1H), 5.21 (d, J = 9.6 Hz, 1H),
5.34 (s, 2H), 6.24 (dd, J = 5.6, 8.0 Hz, 1H), 6.37 (dd, J = 6.3, 8.1 Hz,
1H), 7.46 (s, 1H); 13C NMR (126 MHz, CDCl3) −5.5, −5.3, −4.9,
−4.7, −1.5, −1.4, 4.3, 4.7, 6.7, 6.8, 17.9, 18.0, 18.1, 18.3, 20.7, 25.7,
25.9, 32.3, 37.0, 40.8, 42.2, 44.9, 63.2, 63.3, 66.7, 67.3, 70.0, 70.2,
72.67, 72.73, 84.9, 86.2, 86.7, 88.0, 108.4, 138.0, 150.6, 152.5, 163.3,
+
+
for C65H106N5O14Si4 (M + NH4 ) 1292.6813, found 1292.6811.
P-(2-Chlorophenyl) (5R)-α-[3′,5′-O-Bis(tert-butyldimethyl-
silyl)-3-[(trimethylsilylethoxy)methyl]thymidyl]-5,6-dihydro-
5′-O-(4,4′-dimethoxytrityl)-3′,5′-O-bis(triethylsilyl)-3-
[(trimethylsilylethoxy)methyl]-3′-thymidylate (8). 2-Chloro-
phenyl dichlorophosphate (0.93 mL, 5.74 mmol) was added dropwise
to a solution of 1,2,4-triazole (792 mg, 11.47 mmol) and triethylamine
(1.60 mL, 11.47 mmol) in THF (80 mL). The reaction was stirred for
40 min at room temperature. In a separate flask, dinucleotides 7 (1.46
g, 1.15 mmol) were dissolved in pyridine (80 mL). The solution of
phosphoryl triazole was transferred into a flask by cannula through a
sintered glass funnel. The reaction mixture was stirred for 1 h at room
temperature and quenched by the addition of saturated NaHCO3 and
the solvent removed under reduced pressure. The crude product was
dissolved in dichloromethane, extracted with saturated aq NaHCO3,
and dried over Na2SO4. After the solvent was removed via rotary
evaporation, the resulting residue was purified via column chromatog-
raphy (eluent: dichloromethane/methanol = 15:1) to afford 8 as a
colorless oil (1.69 g, 1.14 mmol, 99% yield): Rf = 0.50 (dichloro-
1
173.5. R-Isomer: Rf = 0.65 (hexane/ethyl acetate = 4:1); H NMR
(500 MHz, CDCl3) δ −0.02 (s, 9H), 0.00 (s, 9H), 0.10 (s, 3H), 0.11
(s, 3H), 0.118 (s, 3H), 0.123 (s, 3H), 0.61 (q, J = 7.9 Hz, 6H), 0.65 (q,
J = 7.9 Hz, 6H), 0.90 (s, 9H), 0.93 (s, 9H), 0.94−1.01 (m, 22H), 1.22
(s, 3H), 1.86−2.04 (m, 3H), 2.24 (ddd, J = 3.1, 6.1, 13.3 Hz, 1H), 2.65
(d, J = 13.5 Hz, 1H), 2.74 (d, J = 13.5 Hz, 1H), 3.09 (d, J = 13.1 Hz,
1H), 3.21 (d, J = 12.9 Hz, 1H), 3.53−3.71 (m, 6H), 3.75 (dd, J = 5.3,
11.9 Hz, 1H), 3.78−3.84 (m, 2H), 3.92 (dd, J = 3.9, 8.2 Hz, 1H),
4.28−4.35 (m, 1H), 4.41−4.46 (m, 1H), 5.13−5.23 (m, 2H), 5.30−
5.40 (m, 2H), 6.33 (dd, J = 6.2, 7.5 Hz, 1H), 6.35 (dd, J = 6.1, 8.1 Hz,
1H), 7.52 (s, 1H); 13C NMR (126 MHz, CDCl3) δ −5.4, −5.3, −4.9,
−4.7, −1.45, −1.42, 4.2, 4.6, 6.7, 6.8, 17.9, 18.08, 18.11, 18.4, 22.0,
25.7, 25.9, 32.3, 36.8, 40.2, 42.9, 44.2, 62.9, 63.0, 66.7, 67.4, 69.9, 70.3,
72.0, 72.1, 84.4, 85.3, 86.3, 87.5, 108.5, 139.1, 150.6, 152.3, 163.5,
173.6.
(5R)-α-[3′,5′-O-Bis(tert-butyldimethylsilyl)-3-[(trimethylsilyl-
ethoxy)methyl]thymidyl]-5,6-dihydro-3-[(trimethylsilyl-
ethoxy)methyl]thymidine (6). HF·Py (28.7 mL, 1.7 M in
acetonitrile) was slowly added to a solution of 5 (4.88 g, 4.06
mmol) in acetonitrile (80 mL) at −20 °C. After being stirred at the
same temperature for 6 h, the reaction mixture was diluted with
EtOAc, washed with NaHCO3, water, and brine, and dried over
anhydrous Na2SO4. After the solvent was removed via rotary
evaporation, the resulting residue was purified via column chromatog-
raphy (eluent: hexane/EtOAc/MeOH = 1:1:0.1) to afford 6 as a white
solid (3.64 g, 3.74 mmol, 92% yield): Rf = 0.20 (hexane/ethyl acetate =
4:1); 1H NMR (500 MHz, CDCl3) δ −0.02 (s, 9H), 0.01 (s, 9H), 0.09
(s, 6H), 0.11 (s, 6H), 0.89 (s, 9H), 0.92 (s, 9H), 0.94−0.99 (m, 2H),
1.16 (s, 3H), 1.93−2.02 (m, 3H), 2.07 (ddd, J = 3.1, 6.3, 13.5 Hz, 1H),
2.19−2.28 (m, 2H), 2.61 (d, J = 3.9 Hz, 1H), 2.72 (d, J = 14.3 Hz,
1H), 2.79 (d, J = 14.5 Hz, 1H), 3.13 (d, J = 4.8 Hz, 1H), 3.19 (d, J =
13.1 Hz, 1H), 3.50 (d, J = 13.1 Hz, 1H), 3.57 (t, J = 8.2 Hz, 2H),
3.65−3.82 (m, 6H), 3.88 (dd, J = 3.1, 6.0 Hz, 1H), 3.95 (dd, J = 3.8,
10.0 Hz, 1H), 4.39−4.46 (m, 2H), 5.14 (d, J = 9.5 Hz, 1H), 5.23 (d, J
= 9.4 Hz, 1H), 5.27 (d, J = 9.1 Hz, 1H), 5.41 (d, J = 9.2 Hz, 1H), 6.23
(t, J = 7.1 Hz, 1H), 6.32 (dd, J = 2.0, 8.0 Hz, 1H), 7.57 (s, 1H); 13C
NMR (126 MHz, CDCl3) δ −5.5, −5.3, −4.9, −4.7, −1.5, −1.4, 17.9,
18.0, 18.2, 18.4, 20.8, 25.7, 25.9, 31.9, 37.6, 40.7, 42.2, 45.0, 62.5, 63.1,
66.7, 67.8, 69.9, 70.2, 72.2, 72.3, 85.6, 85.7, 86.1, 87.9, 108.6, 139.0,
150.6, 152.3, 163.5, 174.3.
1
methane/methanol = 9:1); H NMR (500 MHz, acetone-d6) δ −0.02
(s, 9H), −0.01 (s, 9H), 0.13 (s, 3H), 0.14 (s, 3H), 0.167 (s, 3H), 0.174
(s, 3H), 0.80−0.90 (m, 4H), 0.94 (s, 9H), 0.95 (s, 9H), 1.01 (s, 3H),
2.00−2.10 (m, 1 H), 2.14−2.25 (m, 2 H), 2.26−2.50 (m, 3 H), 2.67
(d, J = 13.7 Hz, 1H), 3.11−3.24 (m, 4 H), 3.57 (t, J = 7.9 Hz, 2H),
3.60−3.66 (m, 2H), 3.757 (s, 3 H), 3.761 (s, 3 H), 3.78−3.84 (m, 1
H), 3.90 (dd, J = 4.3, 10.9 Hz, 1H), 3.95−4.00 (m, 1H), 4.14 (s, 1H),
4.54−4.62 (m, 1H), 5.01 (s, 1H), 5.14 (d, J = 9.9 Hz, 1H), 5.16 (d, J =
10.0 Hz, 1H), 5.23 (d, J = 9.4 Hz, 1H), 5.28 (d, J = 9.8 Hz, 1H), 6.29
(t, J = 7.0 Hz, 1H), 6.32−6.38 (m, 1H), 6.83−6.93 (m, 4H), 7.01−
7.03 (m, 1H), 7.17−7.26 (m, 2H), 7.27−7.37 (m, 6 H), 7.41−7.48 (m,
2H), 7.51 (s, 1H), 7.87 (s, 1H); 31P NMR (202 MHz, acetone-d6) δ
−14.09 - −10.93 (broad multiple); ESI-HRMS m/z calcd for
−
C71H105ClN4O17PSi4 (M − H)− 1463.5978, found 1463.6009.
Synthesis of 9. A solution of the phosphorylated dinucleotides 8
(1.69 g, 1.14 mmol) and TBAF (11.4 mL, 1 M THF solution) in THF
(115 mL) was stirred for 6 h at room temperature. The solvent was
replaced with dichloromethane, and then the resulting solution was
washed with NaHCO3 and dried over anhydrous Na2SO4. After the
solvent was removed via rotary evaporation, the resulting residue was
purified via column chromatography (eluent: dichloromethane/
methanol = 15:1) to afford a reaction intermediate as a yellow oil.
The product was azotropically dried with pyridine and dissolved in
anhydrous pyridine (330 mL), and then MSNT (1.69 g, 5.70 mmol)
was added. The reaction mixture was stirred at room temperature
overnight. After being quenched with water, the solvent was removed
under reduced pressure and then extracted with dichloromethane. The
extracts were dried over anhydrous Na2SO4. After concentration under
reduced pressure, the residue was purified via column chromatography
(eluent: dichloromethane/methanol/thiethylamine = 20:1:0.025) to
afford 9, a mixture of 2 diastereoisomers in a ratio of 1:1.7 (Sp/Rp
mixtures), as indicated by LC−MS, as a white solid (1.13 g, 0.92
mmol, 81% yield for two steps): Rf = 0.66−0.67 (dichloromethane/
methanol = 9:1); 1H NMR (500 MHz, acetone-d6) δ −0.07−0.05 (m,
18H), 0.84−0.97 (m, 4H), 1.14 (s, 1.11H), 1.23 (s, 1.89H), 1.94−2.02
(m, 0.37H), 2.16−2.37 (m, 1.63 H), 2.49−2.75 (m, 3 H), 2.77−2.92
(5R)-α-[3′,5′-O-Di(tert-butyldimethylsilyl)-3-[(trimethylsilyl-
ethoxymethyl)]thymidyl]-5,6-dihydro-5′-O-(4,4′-dimethoxytri-
tyl)-3′,5′-O-bis(triethylsilyl)-3-[(trimethylsilylethoxy)methyl]-
thymidine (7). DMTrCl (937 mg, 2.77 mmol) was added to a
solution of 6 (2.25 g, 2.31 mmol) in pyridine (13 mL). After being
stirred at room temperature overnight, the solvent was removed under
reduced pressure. The resulting residue was purified via column
chromatography (eluent: hexane/EtOAc = 2:1) to afford 7 as a white
solid (2.69 g, 2.11 mmol, 80% yield): Rf = 0.50 (hexane/ethyl acetate =
1
1:1); H NMR (500 MHz, CDCl3) δ −0.02 (s, 9H), 0.00 (s, 9H),
0.109 (s, 3H), 0.113 (s, 3H), 0.116 (s, 3H), 0.119 (s, 3H), 0.91 (s,
9H), 0.93 (s, 9H), 0.85−0.97 (m, 4H), 1.16 (s, 3H), 1.95−2.02 (m,
F
dx.doi.org/10.1021/jo400013n | J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry
Article
(m, 1 H), 3.14−3.55 (m, 4H), 3.56−3.69 (m, 4H), 3.71−3.81 (m, 6
H), 3.85−4.13 (m, 2H), 4.33−4.54 (m, 3H), 4.57−4.72 (m, 1H),
5.04−5.35 (m, 5H), 6.09 (dd, J = 5.5, 8.5 Hz, 0.37H), 6.28−6.44 (m,
1.63H), 6.71−6.88 (m, 4H), 7.14−7.53 (m, 13H), 7.71 (s, 0.37H),
7.94 (s, 0.63H); 13C NMR (126 MHz, acetone-d6) δ −2.1, −2.0,
17.62, 17.64, 17.7, 17.2, 21.5, 24.1, 34.0, 34.8, 37.2, 39.2, 40.1, 40.6,
41.2, 44.4, 44.6, 54.6, 62.2, 62.9, 66.0, 66.1, 68.59, 68.63, 69.5, 69.7,
70.13, 70.15, 70.25, 70.34, 75.4, 75.5, 77.5, 77.6, 80.6, 80.7, 83.4, 83.7,
83.9, 84.0, 84.3, 85.2, 86.1, 86.2, 108.8, 110.3, 112.96, 113.02, 121.44,
121.45, 121.50, 121.52, 124.77, 124.82, 125.02, 125.1, 126.5, 126.6,
126.7, 127.65, 127.69, 128.0, 128.1, 128.3, 128.6, 130.02, 130.07,
130.08, 130.6, 130.7, 135.57, 135.63, 135.69, 135.74, 137.2, 137.4,
145.10, 145.14, 146.15, 146.19, 146.39, 146.44, 150.6, 150.7, 153.4,
153.5, 158.7, 162.6, 162.9, 173.0, 173.3; 31P NMR (202 MHz, acetone-
Rf = 0.63−0.71 (dichloromethane/ethyl acetate/triethyl amine =
4.5:4.5:1); 1H NMR (500 MHz, acetone-d6) δ −0.06−0.08 (m, 18H),
0.82−0.99 (m, 4H), 1.08−1.43 (m, 21H), 2.23−2.65 (m, 4H), 2.65−
2.81 (m, 2H), 3.18−3.54 (m, 4H), 3.55−3.73 (m, 9H), 3.74−4.01 (m,
9H), 4.07−4.46 (m, 3H), 4.53−5.06 (m, 2H), 5.09−5.35 (m, 4H),
6.11−6.47 (m, 2H), 6.81−6.97 (m, 4H), 7.16−7.54 (m, 9H), 7.69−
7.75 (m, 0.5H), 7.88−7.96 (m, 0.5H); 31P NMR (202 MHz, acetone-
d6) δ −2.14, −2.11, −0.10, 148.55, 148.66, 148.68, 148.72; ESI-HRMS
+
+
m/z calcd for C63H96N7O17P2Si2 (M + NH4 ) 1340.5877, found
1340.5861.
(5R)-3′-O-Acetyl-α-[3′,5′-O-bis(tert-butyldimethylsilyl)-3-
[(trimethylsilylethoxy)methyl]thymidyl]-5,6-dihydro-5′-O-
(4,4′-dimethoxytrityl)-3-[(trimethylsilylethoxy)methyl]-
thymidine (11). To a solution of 7 (861 mg, 0.67 mmol) in dry
pyridine (8 mL) was added Ac2O (684 mg, 6.70 mmol). After the
reaction was complete (12 h), the solvent was removed via rotary
evaporation and the resulting residue was purified via column
chromatography (eluent: hexane/ethyl acetate = 4:1) to afford 11 as
a white solid (883 mg, 0.67 mmol, 100% yield): Rf = 0.50 (hexane/
+
d6) δ −9.39, −6.44; ESI-HRMS m/z calcd for C59H80ClN5O16PSi2
+
(M + NH4 ) 1236.4565, found 1236.4556.
Synthesis of SP Phosphoramidite P1. To a solution of 9 (75
mg, 61 μmol)[1:1.7 (Sp/Rp mixtures)] in dry CH2Cl2 (4 mL) under
argon atmosphere were added DIPEA (54 μL, 310 μmol) and 2-
cyanoethyl-N,N-diisopropylchlorophosphoramidite (42 μL, 180
μmol). After 1 h of stirring, the solvent was removed by rotary
evaporation, and the resulting residue was purified via column
chromatography (eluent: dichloromethane/ethyl acetate/triethylamine
= 5:5:1) to yield P1, a mixture of 4 diastereoisomers in a ratio of
1:1:1.7:1.7, as indicated by LC−-MS, as a white solid (69 mg, 49 μmol,
80% yield): Rf = 0.20−0.30 (dichloromethane/ethyl acetate/triethyl-
1
ethyl acetate = 3:1); H NMR (500 MHz, CDCl3) δ −0.01 (s, 9H),
0.00 (s, 9H), 0.105 (s, 3H), 0.107 (s, 3H), 0.112 (s, 6H), 0.87−0.97
(m, 4H), 0.91 (s, 9H), 0.92 (s, 9H), 1.14 (s, 3H), 1.96−2.07 (m, 2H),
2.06 (s, 3H), 2.20−2.29 (m, 2H), 2.57 (d, J = 14.0 Hz, 1H), 2.66 (d, J
= 13.8 Hz, 1H), 3.21 (d, J = 12.9 Hz, 1H), 3.27 (d, J = 13.2 Hz, 1H),
3.29 (dd, J = 4.1, 10.0 Hz, 1H), 3.35 (dd, J = 3.9, 10.2 Hz, 1H), 3.58 (t,
J = 8.2 Hz, 2H), 3.61−3.67 (m, 2H), 3.73 (dd, J = 5.5, 10.7 Hz, 1H),
3.79 (s, 6H), 3.81 (dd, J = 4.2, 11.4 Hz, 1H), 3.92−3.95 (m, 1H), 3.97
(dd, J = 3.9, 7.0 Hz, 1H), 4.42−4.46 (m, 1H), 5.16 (d, J = 9.5 Hz, 1H),
5.20 (d, J = 9.4 Hz, 1H), 5.23−5.32 (m, 3H), 6.30 (dd, J = 6.2, 7.4 Hz,
1H), 6.34 (dd, J = 5.4, 9.5 Hz, 1H), 6.84−6.89 (m, 4H), 7.19−7.24
(m, 1H), 7.29−7.37 (m, 6H), 7.43−7.48 (m, 3H); 13C NMR (126
MHz, CDCl3) δ −5.4, −5.3 −4.8, −4.7, −1.42, −1.41, 17.9, 18.09,
18.14, 18.4, 21.0, 21.9, 25.7, 25.9, 32.5, 34.2, 40.2, 42.8, 44.8, 55.2(2C),
62.9, 63.5, 66.8, 67.4, 70.1, 70.3, 72.1, 74.5, 81.8, 83.9, 85.6, 86.3, 87.5,
108.3, 113.2, 126.8, 127.9, 128.1, 130.0, 130.1, 135.74, 135.75, 135.9,
139.0, 144.7, 150.6, 152.6, 158.5, 163.3, 170.3, 173.4; ESI-MS m/z
1
amine = 4.5:4.5:1); H NMR (500 MHz, acetone-d6) δ −0.07−0.05
(m, 18H), 0.89−0.97 (m, 4H), 1.09−1.27 (m, 21H), 2.37−2.80 (m,
6H), 3.15−3.37 (m, 4H), 3.43−3.54 (m, 1H), 3.55−3.97 (m, 15H),
4.01−4.70 (m, 5H), 5.07−5.32 (m, 5H), 6.07−6.46 (m, 2H), 6.76−
6.88 (m, 4H), 7.16−7.60 (m, 13H), 7.65−7.98 (m, 1H); 31P NMR
(acetone-d6) δ −9.36, −9.23, −6.66, 148.72, 148.88, 149.31; ESI-
+
+
HRMS m/z calcd for C68H97ClN7O17P2Si2 (M + NH4 ) 1436.5643,
found 1436.5628.
Synthesis of 10. NaOMe (118 mg, 2.12 mmol) was added to a
solution of 9 (861 mg, 0.71 mmol) [1:1.7 (Sp/Rp mixtures)] in
MeOH (10 mL). After being stirred at room temperature for 1 h, the
reaction mixture was diluted with EtOAc, washed with NH4Cl, water
and brine successively, and dried over anhydrous Na2SO4. After the
solvent was removed via rotary evaporation, the resulting residue was
purified via column chromatography (eluent: dichloromethane/
methanol/triethylamine = 20:1:0.025) to afford 10, a mixture of two
diastereoisomers in a ratio of 1:1.2 (Sp/Rp mixtures), as indicated by
LC−MS, as a white solid (719 mg, 0.64 mmol, 90% yield): Rf = 0.25
(hexane/ethyl acetate/methanol = 5:5:1); 1H NMR (500 MHz,
acetone-d6) δ −0.05−0.03 (m, 18H), 0.85−0.96 (m, 4H), 1.15 (s,
1.35H), 1.23 (s, 1.65H), 2.11−2.38 (m, 2H), 2.43−2.82 (m, 4H),
3.13−3.34 (m, 3H), 3.37−3.55 (m, 1H), 3.57−3.72 (m, 7H), 3.74−
3.81 (m, 6H), 3.85−4.06 (m, 2H), 4.26−4.57 (m, 3H), 4.66−5.02 (m,
2H), 5.11−5.34 (m, 4H), 6.10−6.18 (m, 0.45H), 6.28−6.42 (m,
1.55H), 6.84−6.95 (m, 4H), 7.19−7.40 (m, 7H), 7.44−7.50 (m, 2H),
7.74 (s, 0.45H), 7.92 (s, 0.55H); 13C NMR (126 MHz, acetone-d6) δ
−2.08, −2.06, −2.04, 17.6, 17.7, 21.9, 24.1, 34.1, 34.7, 37.3, 39.2, 39.8,
40.6, 41.0, 44.5, 44.6, 46.0, 53.7, 53.8, 53.9, 54.59, 54.62, 62.3, 62.8,
65.69, 65.73, 65.96, 66.01, 66.77, 66.83, 75.9, 76.0, 80.8, 80.9, 81.0,
83.4, 83.7, 84.0, 84.1, 84.7, 86.08, 86.11, 109.2, 110.3, 113.0, 126.6,
126.7, 127.7, 128.0, 128.1, 130.00, 130.01, 130.1, 135.65, 135.70,
135.75, 137.17, 137.23, 145.1, 145.2, 150.6, 150.7, 153.2, 153.5,
158.68, 158.74, 162.6, 162.8, 173.0, 173.3; 31P NMR (202 MHz,
+
calcd for C67H108N5O15Si4+ (M + NH4 ) 1334.6919, found 1334.6914.
(5R)-3′-O-Acetyl-α-[3-[(trimethylsilylethoxy)methyl]-
thymidyl]-5,6-dihydro-5′-O-(4,4′-dimethoxytrityl)-3-
[(trimethylsilylethoxy)methyl]thymidine (12). To a solution of
11 (907 mg, 0.69 mmol) in THF (10 mL) was added TBAF (2.1 mL,
2.10 mmol). After the reaction was complete (2 h), the reaction
mixture was diluted with EtOAc, washed with saturated aq NaHCO3,
and dried over anhydrous Na2SO4. After removal of the solvent via
rotary evaporation, the resulting residue was purified via column
chromatography (eluent: dichloromethane/methanol = 15:1) to afford
12 as a white solid (708 mg, 0.65 mmol, 95% yield): Rf = 0.25
1
(dichloromethane/methanol = 9:1); H NMR (500 MHz, CDCl3) δ
−0.003 (s, 9H), 0.003 (s, 9H), 0.88−0.99 (m, 4H), 1.11 (s, 3H), 2.07
(s, 3H), 2.07−2.12 (m, 1H), 2.22−2.32 (m, 2H), 2.42 (dd, J = 3.4, 6.3
Hz, 1H), 2.45 (dd, J = 3.3, 6.2 Hz, 1H), 2.58 (d, J = 14.3 Hz, 1H), 2.64
(d, J = 14.3 Hz, 1H), 3.22 (d, J = 13.2 Hz, 1H), 3.29 (d, J = 13.4 Hz,
1H), 3.32−3.39 (m, 3 H), 3.59−3.69 (m, 4 H), 3.80 (s, 6H), 3.82−
3.85 (m, 1H), 3.98−4.04 (m, 2H), 4.09 (dd, J = 2.9, 5.6 Hz, 1H),
4.52−4.58 (m, 1H), 5.14 (d, J = 9.8 Hz, 1H), 5.23 (d, J = 9.6 Hz, 1H),
5.28−5.31 (m, 1H), 5.32 (d, J = 9.4 Hz, 1H), 5.36 (d, J = 9.4 Hz, 1H),
6.22 (t, J = 6.5 Hz, 1H), 6.35 (dd, J = 5.4, 9.5 Hz, 1H), 6.85−6.90 (m,
4H), 7.20−7.25 (m, 1H), 7.29−7.38 (m, 6H), 7.43−7.47 (m, 2H),
7.84 (s, 1H); 13C NMR (126 MHz, CDCl3) δ −1.5, −1.4, 18.05,
18.13, 21.0, 21.4, 32.2, 34.3, 41.5, 42.6, 45.1, 55.2 (2C), 62.1, 63.5,
67.2, 67.5, 70.2, 71.8, 74.6, 82.0, 84.0, 86.4, 87.3, 87.6, 107.7, 113.2,
126.9, 127.9, 128.2, 130.01, 130.03, 135.9, 139.3, 144.4, 150.6, 152.6,
acetone-d6)
δ −2.27, 0.03; ESI-HRMS m/z calcd for
+ +
C54H79N5O16PSi2 (M + NH4 ) 1140.4798, found 1140.4785.
+
Synthesis of P2. To a solution of 10 (138 mg, 123 μmol) [1:1.2
(Sp/Rp mixtures)] in dry CH2Cl2 (9 mL) under argon atmosphere
were added DIPEA (106 μL, 615 μmol) and 2-cyanoethyl-N,N-
diisopropylchloro phosphoramidite (84 μL, 369 μmol). After the
solution was stirred for 1 h, the solvent was removed via rotary
evaporation, and the residue was purified via column chromatography
(eluent: dichloromethane/ethyl acetate/triethylamine = 5:5:1) to
afford P2, a mixture of four diastereoisomers in a ratio of 1:1:1.1:1.1, as
indicated by LC−MS, as a white solid (138 mg, 104 μmol, 85% yield):
158.5, 163.4, 170.4, 174.0; ESI-HRMS m/z calcd for C55H80N5O15Si2
(M + NH4 ) 1106.5190, found 1106.5195.
+
(5R)-3′-O-Acetyl-α-[5′-O-(tert-butyldiphenylsilyl)-3-
[(trimethylsilylethoxy)methyl]thymidyl]-5,6-dihydro-5′-O-
(4,4′-dimethoxytrityl)-3-[(trimethylsilylethoxy)methyl]-
thymidine (13). Imidazole (93 mg, 1.36 mmol) and TBDPSCl (0.23
mL, 0.88 mmol) were added in turn to a solution of 12 (746 mg, 0.68
mmol) in dry DMF (3 mL) at 0 °C. The reaction mixture was stirred
at room temperature overnight before being quenched with aq
G
dx.doi.org/10.1021/jo400013n | J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry
Article
NaHCO3. The mixture was diluted with EtOAc, washed with water
and brine successively, and dried over anhydrous Na2SO4. After the
solvent was removed via rotary evaporation, the resulting residue was
purified via column chromatography (eluent: hexane/EtOAc = 1:1) to
afford 13 as a white solid (677 mg, 0.51 mmol, 75% yield): Rf = 0.33
(hexane/ethyl acetate =1:1); 1H NMR (500 MHz, CDCl3) δ −0.01 (s,
9H), 0.00 (s, 9H), 0.84−0.98 (m, 4H), 1.03 (s, 3H), 1.09 (s, 9H),
2.01−2.05 (m, 1H), 2.06 (s, 3H), 2.11 (d, J = 3.5 Hz, 1H), 2.12−2.25
(m, 2H), 2.33−2.40 (m, 2H), 2.60 (d, J = 14.4 Hz, 1H), 3.13 (d, J =
13.1 Hz, 1H), 3.23 (d, J = 13.4 Hz, 1H), 3.31 (dd, J = 4.2, 10.3 Hz,
1H), 3.35 (dd, J = 4.0, 10.5 Hz, 1H), 3.58 (t, J = 8.3 Hz, 2 H), 3.62−
3.68 (m, 2 H), 3.79 (s, 6H), 3.88−4.03 (m, 4H), 4.47−4.57 (m, 1H),
5.14 (d, J = 9.7 Hz, 1H), 5.19 (d, J = 9.6 Hz, 1H), 5.25−5.32 (m, 3H),
6.27 (t, J = 6.4 Hz, 1H), 6.34 (dd, J = 5.4, 9.5 Hz, 1H), 6.83−6.89 (m,
4H), 7.15−7.24 (m, 1H), 7.26−7.48 (m, 15H), 7.67−7.73 (m, 4H);
13C NMR (126 MHz, CDCl3) δ −1.4 (2 C), 18.10, 18.13, 19.2, 21.0,
21.6, 26.9, 32.3, 34.2, 39.8, 42.7, 44.6, 55.2 (2 C), 63.5, 64.0, 66.9, 67.5,
70.1, 70.3, 71.7, 74.5, 81.8, 83.9, 85.2, 85.7, 86.4, 108.4, 113.2, 126.8,
127.8, 127.9, 128.1, 129.89, 129.94, 130.0, 130.1, 132.92, 132.93,
135.50, 135.53, 135.7, 135.8, 138.8, 144.7, 150.5, 152.5, 158.5, 163.2,
SP TT reaction, an additional step is needed to remove the TBDPS
protecting group, which was achieved by the following procedure: the
resulting cleavage solution was dried by lyophilization, heated at 65 °C
in a mixture of anhydrous DMSO and triethylamine trihydrofluoride
(TEA·(HF)3) for 1 h, and precipitated by addition of butanol. After
HPLC purification, the yields for the 10-mer and 20-mer sequence
were found to be 63% and 30%, respectively, in the dinucleotide SP
TpT incorporation and 60% and 23%, respectively, in the dinucleoside
SP TT incorporation. The yields were calculated based on the amount
of resin used.
HPLC Assay for Product Purification. HPLC chromatography
was performed at room temperature with a Waters (Milford, MA)
breeze HPLC system with a 2489 UV/Visible detector at 260 nm. An
Waters XBridge OST C18 column (2.5 μm, 4.6 × 50 mm) was
equilibrated with 5% CH3CN in 0.1 M TEAA buffer at pH 7.0 (buffer
A), and compounds were eluted with an ascending gradient (0−35%)
of buffer B in 15 min which is composed of 70% buffer A and 30%
acetonitrile at a flow rate of 1 mL/min. The SP containing
oligonucleotides were collected using 1.5 mL Eppendorf tubes that
were lyophilized and redissolved in 10 μL of dd H2O. The resulting
solution (0.5 μL) was then injected into the Agilent 6520 Accurate-
Mass Q-TOF LC/MS spectrometer, and the data was acquired and
analyzed via Agilent MassHunter software.
Measurement of Oligonucleotide Melting Point (Tm). UV
melting curves of oligonucleotide duplexes were obtained with a
Perkin-Elmer UV/vis/NIR spectrometer (Lambda 19) equipped with
a PTP-1 peltier temperature programmer. Quarts cuvettes with 1 cm
optical path length were employed. The variation of UV absorbance
with temperature was monitored at λ = 260 nm. The temperature was
scanned between 4 and 94 °C from both directions, and the rate of
temperature change was 0.4 °C/min. The experiments were carried
out in 10 mM sodium phosphate buffer at pH 7.0, which contains 150
mM NaCl, and duplex concentrations of 0.4, 0.8, 1.6, and 3.2 μM for
20-mer oligonucleotide and 0.6, 1.2, 2.4, and 4.8 μM for 10-mer
oligonucleotide. Melting points were obtained from the inflection
points of the baseline corrected averaged melting curves. Thermody-
namic parameters were calculated using the published protocol.58−61
+
170.3, 173.3; ESI-HRMS m/z calcd for C71H98N5O15Si3+ (M + NH4 )
1344.6367, found 1344.6360.
(5R)-3′-O-Acetyl-α-[3′-O-(2-cyanoethyl-N,Ndiisopropyl)-
phosphoramidite (5′-O-(tert-butyldiphenylsilyl)-3-
[(trimethylsilylethoxy)methyl]thymidyl]-5,6-dihydro-5′-O-
(4,4′-dimethoxytrityl)-3-[(trimethylsilylethoxy)methyl]-
thymidine, (P3). To a solution of 13 (102 mg, 77 μmol) in dry
CH2Cl2 (7 mL) under argon atmosphere were added DIPEA (67 μL,
384 μmol) and 2-cyanoethyl-N,N-diisopropylchlorophosphoramidite
(51 μL, 230 μmol). After 1 h of stirring, the solvent was removed via
rotary evaporation, and the residue was purified via column
chromatography (eluent: hexane/ethyl acetate/triethylamine =
2:1:0.01) to afford P3, a mixture of two diastereoisomers in a ratio
of 1.3:1 (Sp/Rp mixtures, by 31P NMR), as a white solid (94 mg, 62
1
μmol, 81% yield): Rf = 0.50 (hexane/ethyl acetate = 2:1); H NMR
(500 MHz, CDCl3) δ −0.01−0.03 (m, 18H), 0.87−1.06 (m, 8H),
1.07−1.12 (m, 9H), 1.17 (s, 1.5H), 1.18 (s, 1.5H), 1.19−1.24 (m, 9H),
2.02−2.09 (m, 4H), 2.10−2.30 (m, 3H), 2.43−2.57 (m, 2H), 2.59−
2.67 (m, 2H), 3.13 (d, J = 13.0 Hz, 1H), 3.19−3.27 (m, 1H), 3.29−
3.38 (m, 2H), 3.55−3.67 (m, 2H), 3.61−3.68 (m, 4H), 3.80 (s, 6H),
3.69−3.87 (m, 2H), 3.88−3.95 (m, 2H), 3.97−4.01 (m, 1H), 4.11−
4.18 (m, 1H), 4.60−4.71 (m, 1H), 5.11−5.20 (m, 2H), 5.25−5.33 (m,
3H), 6.28−6.41 (m, 2H), 6.82−6.90 (m, 4H), 7.19−7.24 (m, 1H),
7.27−7.49 (m, 15H), 7.65−7.73 (m, 4H); 31P NMR (202 MHz,
ASSOCIATED CONTENT
* Supporting Information
■
S
Spectroscopic characterization of the new compounds. This
information is available free of charge via the Internet at http://
pubs.acs.org.
CDCl3)
δ 148.76, 149.25; ESI-HRMS m/z calcd for
+
C80H112N6O16PSi3 (M + H+) 1527.7180, found 1527.7172.
P-Methylthymidylyl-(5′→3′)-thymidine (14) and P-(2-
Cyanoethyl)thymidylyl-(5′→3′)-thymidine (15). Syntheses of
these compounds were conducted according to previously published
procedures, respectively.43,63
AUTHOR INFORMATION
Corresponding Author
*Tel: 317-278-2202. E-mail: lilei@iupui.edu.
■
Notes
The authors declare no competing financial interest.
Solid-State Photoreaction of 14 and 15. The photoreaction
was carried out using a Spectroline germicidal UV sterilizing lamp
(Dual-tube, 15 w, intensity: 1550 μw cm−2) with the samples ∼9 cm to
the lamp using the protocol described in our previous publication.31
Product analyses by LC−MS found no formation of the corresponding
SP products.
Oligonucleotide Synthesis. The oligonucleotides was synthe-
sized using standard solid-phase synthesis conditions, in a column type
reactor fitted with a sintered glass frit which could be maintained
airtight with a serum cap.64−68 All of the synthetic cycles were
performed on a 2 μmol scale. The average coupling yield for SP
incorporation was >90%, as estimated by HPLC analysis.
After the solid-phase synthesis, the SEM- and DMTr-protecting
groups were removed by stirring with 1 M SnCl4 in CH2Cl2 for 0.5 h
at room temperature under anhydrous conditions. The oligonucleo-
tides were cleaved from resin with concentrated aq. NH3·H2O at 55
°C for 18 h in a sealed tube. The methyl group from the SP
phosphotriester moiety was also removed by this ammonium
hydroxide treatment.42−44 The resins were then washed with H2O
for 3 times and the washing solutions combined. For the dinucleoside
ACKNOWLEDGMENTS
■
We thank the National Institutes of Health (R00ES017177),
the IUPUI RSFG, IUCRG, and the IUPUI startup fund for
their generous support. The NMR and MS facilities are
supported by National Science Foundation MRI grants CHE-
0619254 and DBI-0821661, respectively.
REFERENCES
■
(1) Cadet, J.; Vigny, P. Photochemistry and the Nucleic Acids; Wiley:
New York, 1990; Vol. 1.
(2) Douki, T.; Cadet, J. Biochemistry 1994, 33, 11942.
(3) Douki, T.; Cadet, J. Biochemistry 2001, 40, 2495.
(4) Douki, T.; Reynaud-Angelin, A.; Cadet, J.; Sage, E. Biochemistry
2003, 42, 9221.
(5) Courdavault, S.; Baudouin, C.; Sauvaigo, S.; Mouret, S.; Candeias,
́
S.; Charveron, M.; Favier, A.; Cadet, J.; Douki, T. Photochem.
Photobiol. 2004, 79, 145.
H
dx.doi.org/10.1021/jo400013n | J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry
Article
(6) Mouret, S. p.; Baudouin, C.; Charveron, M.; Favier, A.; Cadet, J.;
Douki, T. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 13765.
(7) Park, H.; Zhang, K.; Ren, Y.; Nadji, S.; Sinha, N.; Taylor, J.-S.;
Kang, C. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 15965.
(8) Taylor, J. S.; Cohrs, M. P. J. Am. Chem. Soc. 1987, 109, 2834.
(9) Taylor, J.-S.; Lu, H.-F.; Kotyk, J. J. Photochem. Photobiol. 1990, 51,
161.
(46) Murata, T.; Iwai, S.; Ohtsuka, E. Nucleic Acids Res. 1990, 18,
7279.
(47) Tommasi, S.; Swiderski, P. M.; Tu, Y.; Kaplan, B. E.; Pfeifer, G.
P. Biochemistry 1996, 35, 15693.
(48) Kosmoski, J. V.; Smerdon, M. J. Biochemistry 1999, 38, 9485.
(49) Butenandt, J.; Burgdorf, L. T.; Carell, T. SynthesisStuttgart
1999, 1085.
(50) Iwai, S.; Shimizu, M.; Kamiya, H.; Ohtsuka, E. J. Am. Chem. Soc.
1996, 118, 7642.
(10) Desnous, C. l.; Guillaume, D.; Clivio, P. Chem. Rev. 2010, 110,
1213.
(51) Yamamoto, J.; Hitomi, K.; Todo, T.; Iwai, S. Nucleic Acids Res.
2006, 34, 4406.
(11) Setlow, P. Environ. Mol. Mutagen. 2001, 38, 97.
(12) Nicholson, W.; Schuerger, A.; Setlow, P. Mutat. Res. Fundam.
Mol. Mech. Mugag. 2005, 571, 249.
(52) Lin, G.; Chen, C.-H.; Pink, M.; Pu, J.; Li, L. Chem.Eur. J.
2011, 17, 9658.
(13) Li, L. Biochim. Biophys. Acta, Proteins Proteomics 2012, 1824,
(53) Kim, S. J.; Lester, C.; Begley, T. P. J. Org. Chem. 1995, 60, 6256.
(54) Mantel, C.; Chandor, A.; Gasparutto, D.; Douki, T.; Atta, M.;
Fontecave, M.; Bayle, P. A.; Mouesca, J. M.; Bardet, M. J. Am. Chem.
Soc. 2008, 130, 16978.
(55) Chandra, T.; Broderick, W. E.; Broderick, J. B. Nucleosides
Nucleotides Nucleic Acids 2009, 28, 1016.
1264.
(14) Yang, L.; Lin, G.; Liu, D.; Dria, K. J.; Telser, J.; Li, L. J. Am.
Chem. Soc. 2011, 133, 10434.
(15) Cheek, J.; Broderick, J. J. Am. Chem. Soc. 2002, 124, 2860.
(16) Chandra, T.; Silver, S. C.; Zilinskas, E.; Shepard, E. M.;
Broderick, W. E.; Broderick, J. B. J. Am. Chem. Soc. 2009, 131, 2420.
(17) Silver, S.; Chandra, T.; Zilinskas, E.; Ghose, S.; Broderick, W.;
Broderick, J. J. Biol. Inorg. Chem. 2010, 15, 943.
(18) Chandor, A.; Berteau, O.; Douki, T.; Gasparutto, D.; Sanakis, Y.;
Ollagnier-De-Choudens, S.; Atta, M.; Fontecave, M. J. Biol. Chem.
2006, 281, 26922.
(19) Chandor-Proust, A.; Berteau, O.; Douki, T.; Gasparutto, D.;
Ollagnier-de-Choudens, S.; Fontecave, M.; Atta, M. J. Biol. Chem.
2008, 283, 36361.
(20) Benjdia, A.; Heil, K.; Barends, T. R. M.; Carell, T.; Schlichting, I.
Nucleic Acids Res. 2012, 40, 9308.
(21) Pieck, J.; Hennecke, U.; Pierik, A.; Friedel, M.; Carell, T. J. Biol.
Chem. 2006, 281, 36317.
(22) Rebeil, R.; Sun, Y.; Chooback, L.; Pedraza-Reyes, M.; Kinsland,
C.; Begley, T. P.; Nicholson, W. L. J. Bacteriol. 1998, 180, 4879.
(23) Rebeil, R.; Nicholson, W. L. Proc. Natl. Acad. Sci. U.S.A. 2001,
98, 9038.
(56) Friedel, M.; Pieck, J.; Klages, J.; Dauth, C.; Kessler, H.; Carell, T.
Chem.Eur. J. 2006, 12, 6081.
(57) Murata, S.; Mizumura, Y.; Hino, K.; Ueno, Y.; Ichikawa, S.;
Matsuda, A. J. Am. Chem. Soc. 2007, 129, 10300.
(58) Kibbe, W. A. Nucleic Acids Res. 2007, 35, W43.
(59) Marky, L. A.; Breslauer, K. J. Biopolymers 1987, 26, 1601.
(60) Ober, M.; Muller, H.; Pieck, C.; Gierlich, J.; Carell, T. J. Am.
̈
Chem. Soc. 2005, 127, 18143.
(61) Taylor, J. S.; Garrett, D. S.; Brockie, I. R.; Svoboda, D. L.;
Telser, J. Biochemistry 1990, 29, 8858.
(62) McAteer, K.; Jing, Y.; Kao, J.; Taylor, J. S.; Kennedy, M. A. J.
Mol. Biol. 1998, 282, 1013.
(63) Nicholson, W. L.; Galeano, B. Appl. Environ. Microbiol. 2003, 69,
1327.
(64) Beaucage, S. L.; Caruthers, M. H. Synthetic Strategies and
Parameters Involved in the Synthesis of Oligodeoxyribonucleotides
According to the Phosphoramidite Method; John Wiley & Sons, Inc.:
New York, 2001.
(65) Vargeese, C.; Carter, J.; Yegge, J.; Krivjansky, S.; Settle, A.;
Kropp, E.; Peterson, K.; Pieken, W. Nucleic Acids Res. 1998, 26, 1046.
(66) Sinha, N. D.; Biernat, J.; McManus, J.; Koster, H. Nucleic Acids
Res. 1984, 12, 4539.
(67) Koster, H.; Stumpe, A.; Wolter, A. Tetrahedron Lett. 1983, 24,
747.
(24) Fajardo-Cavazos, P.; Rebeil, R.; Nicholson, W. Curr. Microbiol.
2005, 51, 331.
(25) Yang, L.; Lin, G.; Nelson, R. S.; Jian, Y.; Telser, J.; Li, L.
Biochemistry 2012, 51, 7173.
(26) Setlow, B.; Setlow, P. Appl. Environ. Microbiol. 1988, 54, 1275.
(27) Setlow, P. Annu. Rev. Microbiol. 1995, 49, 29.
(28) Peccia, J.; Hernandez, M. Appl. Environ. Microbiol. 2001, 67,
4225.
(68) Matteucci, M. D.; Caruthers, M. H. Tetrahedron Lett. 1980, 21,
719.
(29) Smith, K. C.; Yoshikawa, H. Photochem. Photobiol. 1966, 5, 777.
(30) See the Supporting Information.
(31) Lin, G.; Li, L. Angew. Chem., Int. Ed. 2010, 49, 9926.
(32) Du, Q.; Zhao, H.; Song, D.; Liu, K.; Su, H. J. Phys. Chem. B
2012, 116, 11117.
(33) Setlow, B.; Setlow, P. Proc. Natl. Acad. Sci. U.S.A. 1987, 84, 421.
(34) Griffith, J.; Makhov, A.; Santiagolara, L.; Setlow, P. Proc. Natl.
Acad. Sci. U.S.A. 1994, 91, 8224.
(35) Mohr, S. C.; Sokolov, N. V.; He, C. M.; Setlow, P. Proc. Natl.
Acad. Sci. U.S.A. 1991, 88, 77.
(36) Nicholson, W. L.; Setlow, B.; Setlow, P. Proc. Natl. Acad. Sci.
U.S.A. 1991, 88, 8288.
(37) Slieman, T. A.; Rebeil, R.; Nicholson, W. L. J. Bacteriol. 2000,
182, 6412.
(38) Varghese, A. J. Biochemistry 1970, 9, 4781.
(39) Varghese, A. J. Photochem. Photobiol. 1971, 13, 357.
(40) Donnellan, J. E., Jr.; Setlow, R. B. Science 1965, 149, 308.
(41) Heil, K.; Kneuttinger, A. C.; Schneider, S.; Lischke, U.; Carell,
T. Chem.Eur. J. 2011, 17, 9651.
(42) Taylor, J.-S.; Brockie, I. R. Nucleic Acids Res. 1988, 16, 5123.
(43) Taylor, J. S.; Brockie, I. R.; O’Day, C. L. J. Am. Chem. Soc. 1987,
109, 6735.
(44) Ortiz Mayo, J. U.; Thomas, M.; Saintome,
Tetrahedron 2003, 59, 7377.
́
C.; Clivio, P.
(45) Friedel, M. G.; Gierlich, J.; Carell, T. In The Chemistry of
Cyclobutanes; John Wiley & Sons, Ltd.: New York, 2006; p 1031.
I
dx.doi.org/10.1021/jo400013n | J. Org. Chem. XXXX, XXX, XXX−XXX