Properties of oligonucleotide with phenyl-substituted carbocyclic nucleoside analogs for the formation of duplex and triplex DNA

Article abstract of DOI:10.1080/15257770.2012.737970

(1S,3S,4R)-1-Phenyl-1-thymidyl-3-hydroxy-4-hydroxymethylcyclopentane (10) and their analogs were synthesized, incorporated into the oligodeoxynucleotides, and their properties were evaluated for the formation of duplex and triplex DNA. The known chiral cyclopentanone derivative was converted into the corresponding ketimine sulfonamide derivative, which was subjected to a stereoselective PhLi addition. The formed sulfonamide was hydrolyzed to afford the primary amino group, on which the thymine moiety was built. The benzyl protecting groups were removed to form the nucleoside analog having a phenyl group and the thymine unit at the 1 position of a carbocyclic skeleton (10). In the estimation of the oligodeoxynucleotides incorporating 10 for duplex and triplex formation, the carbocyclic nucleoside analog 10 did not show the stabilizing effect for duplex formation; on the other hand, it stabilized the triplex. Therefore, the skeleton of the phenyl-substituted carbocyclic nucleoside analog 10 may be a platform for the formation of stable triplex DNA.

Full text of DOI:10.1080/15257770.2012.737970

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Properties of Oligonucleotide with
Phenyl-Substituted Carbocyclic
Nucleoside Analogs for the Formation of
Duplex and Triplex DNA
Tamer Nasr a b c , Yosuke Taniguchi a , Tomoko Takaki a , Hidenori
Okamura a & Shigeki Sasaki a
a Graduate School of Pharmaceutical Sciences, Kyushu University,
3-1-1 Maidashi, Higashi-ku, Fukuoka, Japan
b Department of Pharmaceutical Chemistry, Faculty of Pharmacy,
Helwan University, Helwan, Egypt
c Department of Pharmaceutical Chemistry, Faculty of Pharmacy,
King Khalid University, Kingdom of Saudi Arabia
Version of record first published: 05 Dec 2012.
To cite this article: Tamer Nasr , Yosuke Taniguchi , Tomoko Takaki , Hidenori Okamura & Shigeki
Sasaki (2012): Properties of Oligonucleotide with Phenyl-Substituted Carbocyclic Nucleoside Analogs
for the Formation of Duplex and Triplex DNA, Nucleosides, Nucleotides and Nucleic Acids, 31:12,
841-860
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Nucleosides, Nucleotides and Nucleic Acids, 31:841–860, 2012
Copyright ꢀC Taylor and Francis Group, LLC
ISSN: 1525-7770 print / 1532-2335 online
DOI: 10.1080/15257770.2012.737970
PROPERTIES OF OLIGONUCLEOTIDE WITH PHENYL-SUBSTITUTED
CARBOCYCLIC NUCLEOSIDE ANALOGS FOR THE FORMATION OF
DUPLEX AND TRIPLEX DNA
Tamer Nasr,1,2,3 Yosuke Taniguchi,1 To moko Takaki, 1 Hidenori Okamura,1 and
Shigeki Sasaki1
1Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi,
Higashi-ku, Fukuoka, Japan
2Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Helwan University,
Helwan, Egypt
3Department of Pharmaceutical Chemistry, Faculty of Pharmacy, King Khalid University,
Kingdom of Saudi Arabia
(1S,3S,4R)-1-Phenyl-1-thymidyl-3-hydroxy-4-hydroxymethylcyclopentane (10) and their analogs
2
were synthesized, incorporated into the oligodeoxynucleotides, and their properties were evaluated for
the formation of duplex and triplex DNA. The known chiral cyclopentanone derivative was con-
verted into the corresponding ketimine sulfonamide derivative, which was subjected to a stereoselec-
tive PhLi addition. The formed sulfonamide was hydrolyzed to afford the primary amino group, on
which the thymine moiety was built. The benzyl protecting groups were removed to form the nucleoside
analog having a phenyl group and the thymine unit at the 1ꢁ position of a carbocyclic skeleton (10).
In the estimation of the oligodeoxynucleotides incorporating 10 for duplex and triplex formation,
the carbocyclic nucleoside analog 10 did not show the stabilizing effect for duplex formation; on
the other hand, it stabilized the triplex. Therefore, the skeleton of the phenyl-substituted carbocyclic
nucleoside analog 10 may be a platform for the formation of stable triplex DNA.
Keywords Oligonucleotides; carbocyclic nucleoside analog; duplex DNA; triplex DNA;
asymmetric synthesis
INTRODUCTION
Carbocyclic nucleoside analogs have attracted interest because of their
potential biological activity. Also, a number of studies describe the prop-
erties and the applications of oligonucleotides incorporating carbocyclic
Received 3 September 2012; accepted 4 October 2012.
This work was supported by a Grant-in-Aid for Scientific Research (S) from the Japan Society for
Promotion of Science (JSPS) and CREST from the Japan Science and Technology Agency. Tamer Nasr
is grateful for the Scholarship from the Egypt Government.
Address correspondence to Shigeki Sasaki, Graduate School of Pharmaceutical Sciences, Kyushu
University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan. E-mail: sasaki@phar.kyushu-u.ac.jp
841

842
T. Nasr et al.
nucleoside analogs. For example, 2ꢁ-deoxyaristeromycin (dAr) is used as a
nucleoside analog that is resistant to DNA glycosylases. The 3D structure of
an 11-mer DNA duplex containing a single dAr-T base pair at the center part
was determined by NMR spectroscopy and MD simulations to be in a right-
handed conformation in solution.[1] It was also reported that the modified
oligonucleotides consisted of a carbocyclic analog formed a triplex DNA with
uridine oligoribonucleotide under physiological conditions.[2] In a series of
our studies on non-natural type oligonucleotides for the formation of the
duplex and triplex, we became interested in carbocyclic nucleoside analogs.
Triplex DNA has been investigated as a biological tool such as for inhibi-
tion[3] or activation[4] of gene expression, and gene recombination,[5] etc. In
an antiparallel triplex DNA, the purine-rich triplex forming oligonucleotide
(TFOs) binds the homopurine strand of the homopurine-homopyrimidine
duplex by two reverse Hoogsteen hydrogen bonds (G/G-C and A/A-T)
within the major groove of the duplex DNA in a sequence-specific man-
ner.[6] A pyrimidine nucleotide insertion into the purine strand causes
destabilization of the triplex. Previously, we developed nucleoside analogs
(WNA: W-shaped nucleoside analogs) having a nucleobase as a recogni-
tion part and an aromatic ring as a stacking part.[7,8] It was demonstrated
that the WNA-βT and WNA-βC exhibited selective recognition of a T-A or
a C-G interrupting site, respectively (Figure 1). However, it turned out in
the subsequent study that the recognition of the interrupting sites is de-
pendent on the neighboring nucleotides around the WNA analogs in the
TFOs.[9] This sequence dependency problem has been partially overcome
FIGURE 1 Speculated recognition model of WNA-βT/T-A and WNA-βC/C-G combinations (Color
figure available online).

Properties of Oligonucleotide with Phenyl-Substituted Carbocyclic Nucleoside Analogs 843
FIGURE 2 Structure modification of WNA to 1ꢁ-β-phenyl-α-thymidine (A). Structure modification of
1ꢁ-β-phenyl-α-thymidine to 1-phenyl-1-thymidyl-3-hydroxy-4-hydroxymethyl-cyclopentane (10) (B).
by selecting modified WNA analogs depending on the TFO sequence.[10] In
an attempt to develop new nucleoside analogs that may be generally used
in the TFO, we designed analogs having both the phenyl and the nucle-
obase parts at the 1ꢁ-position of the sugar part (Figure 2A). However, it
turned out that the 1ꢁ-phenyl-substituted ribonucleoside analogs were unsta-
ble under acidic condition and not suitable for incorporation into ODNs.[11]
Thus, in this study, we focused on the carbocyclic nucleoside analogs as the
more stable 1,1ꢁ-disubstituted one (Figure 2B). Figure 3 illustrates the ex-
pected triplex structure and the recognition mode of 1-phenyl-1-thymidyl-
3-hydroxy-4-hydroxymethyl-cyclopentane (PTC) regarding the phenyl and
thymine moieties. As it is incorporated in oligodeoxynucleotides, it should
be obtained in an optically pure form. Here, we describe the synthesis of
10, its incorporation into the oligodeoxynucleotides and evaluation of their
properties for duplex and triplex DNA.
FIGURE 3 The predicted structure of triplex formation with TFO having 10, which is depicted in the
Space-Filling Model in the TFO shown in green color (A). An expected base-triplet between 10 and a TA
base pair (B) (Color figure available online).

844
T. Nasr et al.
RESULTS AND DISCUSSION
Synthesis of (1S,3S,4R)-1-Phenyl-1-Thymidyl-3-Hydroxy-4-
Hydroxymethylcyclopentane (10)
The synthesis of 10 was started with chiral (1S,2R)-2-
benzyloxymethylcyclopent-3-enol 1, which was obtained in more than
96% e.e. by a sequence of reactions according to the reported procedure
involving the alkylation of cyclopentadiene using benzylchloromethyl
ether and NaH and the subsequent asymmetric hydroboration with (-)-
diisopinocamphenylborane[12] (Scheme 1). Next, 1 was benzylated to give
2 in 90% yield, followed by hydroboration of 2 with 9-BBN to produce
SCHEME 1 (1S,3S,4R)-1-Phenyl-1-thymidyl-3-hydroxy-4-hydroxymethylcyclopentane. (a)(1) NaH, THF,
0◦C, 1 hour; (2) benzylchloromethylether, THF, –60◦C, 2 hours; (3) –(Ipc)2BH, THF, –60◦C, 1 hour
then 0◦C, 16 hours; (4) 3 M NaOH, 30% H2O2, 0◦C, 12 hours (25% for four steps). (b) benzyl bromide,
Nah, DMF, 2 hours, r.t. (90%). (c) (1) 9-BBN, THF, r.t., 12 hours; (2) 3 M NaOH, 30% H2O2, 0◦C,
12 hours (79% for two steps). (d) PCC, CH2Cl2, Celite, 12 hours, r.t. (81%). (e) R(+)-2-methyl-2-
propane-sulfinamide, Ti(OEt)4, THF, 70◦C, 4 hours (94%). (f) PhLi, AlMe3, toluene,-78◦C, 3 hours
(46%). (g) 4M HCL in dioxane, MeOH, r.t., 1 hour (83%). (h) 3-Methoxy-2-methyl-acryloyl isocyanate,
dry benzene, r.t., 1 hour (81%) (i) 1 M aq. H2SO4, DMF, 80–100◦C, 5 hour (60%). (j) H2, Pd(OH)2/C,
MeOH, 2M HCl (cat.) (98%). (k) H2, Pd(OH)2/C, MeOH, HCl (cat), r.t., 1 hour (56%). (l) (1)N3-
benzoyl thymine, PPh3, DIAD, CH3CN, –40◦C to r.t. 16 hours. (2) 1% NaOH in MeOH, r.t., 16 hours (3)
Pd(OH)2/C, H2, MeOH, HCl (cat.), r.t., 1 hour.(m) (1) PhLi, THF, –78◦C, 3 hour; (2) H2, Pd(OH)2/C,
EtOAc, 20 min, r.t.

Properties of Oligonucleotide with Phenyl-Substituted Carbocyclic Nucleoside Analogs 845
TABLE 1 1,2-addition of PhLi to tert-butane sulfinylketimine derivative
Entry
Reagent
Catalyst
Solvent
Temperature
Time (hour)
Yield
1
2
3
4
5
6
7
8
PhLi (1.1 eq.)
PhLi (3 eq.)
PhLi (3 eq.)
PhLi (3 eq.)
PhLi (3 eq.)
PhLi (3 eq.)
PhLi (3 eq.)
PhLi (2 eq.)
Me3Al (1.1 eq.)
Me3Al (1.1 eq.)
CsF (1.1 eq.)
CsF + Me3Al d
CuI (1.1 eq.)
CuI + Me3Al d
–
Toluene
Toluene
Toluene
Toluene
Toluene
Toluene
THF
–78◦C→0◦Ca
–78◦C→0◦Ca
–78◦C→0◦Ca
–78◦C→0◦Ca
–78◦C→0◦Ca
–78◦C→0◦Ca
–78◦C→0◦Cg
–78oC
11
11
11
11
11
11
4.5
19
23%b
46%b
Tracec
n. d.e
Tracec
n. d.f
Traceh
n. d. j
BF3.Et2O (2 eq.)
THF
aThe reaction mixture was stirred at –78◦C for 3 hours and warmed up gradually to 0◦C and stirred
at that temperature for more 1 hour. bThe target compound was obtained as two isomers in the ratio
(9:1). cAs indicated by TLC. dEach is a 1.1 equivalent. eSame TLC result as entry 2. fTLC showed that the
target amount is less than entry 2. gThe reaction was stirred at –78◦C for 2.5 hours, then allowed to warm
gradually to 0◦C within 2 hours. hTLC shows that the target compound was obtained as a trace, while the
by-product was the major part. jTLC shows that the reaction was complicated and the desired product
was obtained in low yield.
the known compound 3 as the major product in 79% yield in 2 steps.[13]
The compound 3 was oxidized with PCC to afford the cyclopentanone 4
in 81% yield. The control compounds (11, 12, and 13) were synthesized
from 1, 3, and 4, respectively. In order to build the thymine moiety of 10,
the compound 7 was designed as the intermediate. In order to obtain the
primary amine at the quaternary carbon, we adopted the method involving
1,2-addition of the phenyl group to the ketimine 5.
For the addition of nucleophiles to ketimine, selection of the proper
protecting group of the ketimine was a key issue, and the sulfonamide group
was used in this study. Thus, a THF solution of the ketone 4 was refluxed
with tetraethylorthotitanate and R-(+)-2-methyl-2-propane sulfonamide to
afford the desired ketimine 5 in total yield 94%.[14] The crude compound
5 was subjected to flash chromatography in order to avoid hydrolysis on a
silica gel column, and the purified 5 was kept under argon at –30◦C. The
addition of PhLi to 5 was investigated under different conditions, and the
results are summarized in Table 1.
When a toluene solution of the ketimine 5 was reacted with trimethylalu-
minum and phenyllithium at –78◦C, the desired product 6 was obtained in
23% yield as two isomers in the ratio of 9:1(Table 1, entry 1). The two isomers
were easily separated by column chromatography, but it was not possible to
determine their stereochemistry by 2D 1H-NMR Noesy measurements. The
yield was increased to 46% when the amount of PhLi was increased to 3
equivalents (Table 1, entry 2). The other additives, CsF, CuI, or BF3.Et2O
did not produce a better result than the conditions of entry 2. The tert-
butylsulfinyl group of the major isomer of 6 was easily cleaved by 4 M HCl
in dioxane and methanol at room temperature[14a] to afford the primary
amine 7 in 83% yield (Scheme 1). The stereochemistry at the 1’-position was
not determined at this step. A benzene solution of the primary amine 7 was

846
T. Nasr et al.
reacted with freshly prepared 3-methoxy-2-methyl-acryloyl isocyanate[15] to
afford compound 8 in 81% yield. Compound 8 was subjected to cyclization
by refluxing with 15 eq. of 1 M aqueous H2SO4 in DMSO for 5 hours to afford
compound 9 in 60% yield. The stereochemistry was determined as shown
1
in 9 by 2D H-NMR NOESY measurement. The benzyl protecting groups
of 9 were removed by hydrogenolysis with H2-Pd(OH)2/C in the presence
of HCl as a catalyst to afford the desired 1ꢁ-phenyl substituted carbocyclic
thymidine 10 (PTC) in 98% yield.
In order to evaluate the effect of both the phenyl and the thymine moi-
eties at the 1ꢁ-position of the compound 10, we synthesized the control com-
pounds 11 (ABC), 12 (TC), and 13 (PC) (Scheme 1). The carbocyclic abasic
control 11 was obtained in 56% yield from the optically active compound
1 upon treatment with hydrogen gas in the presence of Pd(OH)2/C and
2 M aqueous HCl as a catalyst, and its spectral data were comparable to the
reported data.[16] The compound 12 was synthesized from the compound 3
according to the reported procedure, and its spectral data were comparable
to the reported data.[13] The cyclopentanone compound 4 was reacted with
phenyllithium at –78◦C to afford the alcohol as a mixture of two isomers.
Two isomers were not separated by column chromatography. A solution of
the racemic mixture in ethyl acetate was reacted with H2 gas in the presence
of Pd(OH)2/C and 2 M aq. HCl to afford the compound 13 together with
1
its α-isomer in the ratio (2:1) as confirmed by H-NMR. Because these two
isomers could not be separated by column chromatography, the mixture was
used for the next reaction without separation.
Synthesis of Oligonucleotides
The diol compounds 10, 11, 12, and 13 were transformed to the corre-
sponding 5ꢁ-dimethoxytrityl (DMTr)-protected 3ꢁ-phosphoramidite precur-
sors 14, 15, 16, and 17, respectively[10d] (Scheme 2). The DMTr-protected 13
was separated from its α-isomer by column chromatography, and its stereo-
chemistry was determined by proton 2D-NMR spectra. The β-isomer was con-
verted to the corresponding amidite precursor 17. These DMTr-protected
3ꢁ-phosphoramidite precursors were incorporated into ODNs by automated
DNA synthesizer by the conventional method. After purification of these
ODNs by reverse-phase HPLC, the structures of the synthesized ODNs were
confirmed by the MALDI-TOF mass measurement. DNA 1 and 2 were used
to evaluate the duplex formations, and TFO (1–4) was used to evaluate the
ability for triplex formations (Scheme 2).
Measurement of UV Melting Temperature (Tm Values)
The Tm values of DNA 1 and 2 having the carbocyclic compound or
2ꢁ-deoxy-thymidine were measured using the DNA with complementary se-
quences having dC, dT, dA, and dG at the opposite site for Z and are

Properties of Oligonucleotide with Phenyl-Substituted Carbocyclic Nucleoside Analogs 847
SCHEME 2 Sequences of DNA 1–2 and TOF 1–4 incorporating 10, and the control compounds 11, 12,
and 13. (a) (1) DMTrCl, dry pyridine, r.t., 1.5 hours; (2) iPr2NP(Cl)OCH2CH2CN, DIPEA, CH2Cl2, 0◦C,
1 hour. (b) (1) DNA synthesizer (2) 28% NH3 aq., 55◦C, 6 hour. (3) HPLC purification (4) 10% aq.
Acetic acid, r.t., 30 min.
summarized in Figure 4. It was clearly shown that these carbocyclic com-
pounds did not show the stabilizing effect for the duplex formation. These
results indicated that the stereochemistry of the thymine base is not suitable
and that the phenyl moiety has no stabilizing effect for the formation of the
FIGURE 4 UV-melting temperature of the duplex formed with DNA 1 (A) or DNA 2 (B). The nucleotide
at the complementary position to Z is shown in color bars. Melting profiles were measured using 2 μM
each of the DNA strand in 10 mM sodium phosphate buffer (pH 7.0) containing 100 mM NaCl and
10 mM MgCl2 at the scan rate of 1.0◦C/min at 260 nm (Color figure available online).

848
T. Nasr et al.
duplex DNA. Compared to the Tm values of the mismatched duplexes with
Z = T, all carbocyclic nucleoside analogs destabilized the duplex. This may
be attributed to the difference in the sugar puckering and base configuration
of carbocyclic nucleoside analogs.[17]
Evaluation of Triplex Formation by Gel-Shift Assay
TFO (1–4) was used to evaluate the ability for triplex formation by gel-
shift assay and the equilibrium association constants (Ks) for the formation
of triplex formation were calculated from the radioactive intensity of the
bands according to the procedure described in our previous report.[8,9] All
association constants (Ks) are summarized in Figure 5.
In the case of TFO 1, 10 produced relatively high stability for the C-G,
T-A, and GC sites with small selectivity (Figure 5A). Either 11 or 12 did not
afford stable triplexes. Interestingly, highly stable triplex was formed with
13. In contrast, highly stable triplexes were formed only with TFO 2 (Z = 13)
with selectivity for a GC site (Figure 5B). In the sequence of TFO 3, 10, 11,
and 12 showed nonselective stabilizing effects except for a TA interrupting
site (Figure 5C). Selective stabilization for the CG and TA interrupting site
was found with 13. No stable triplexes were formed with TFO 4 (Figure 5D).
FIGURE 5 Equilibrium association constants (Ks) of TFOs containing carbocyclic nucleoside analogs
(Color figure available online).

Properties of Oligonucleotide with Phenyl-Substituted Carbocyclic Nucleoside Analogs 849
As 13 provided the highest stabilizing effect in TFO 1–3, the phenyl
ring of 13 might function as a hydrophobic and/or stacking unit, whereas
the thymine unit of 10 cancelled its stabilizing effect probably due to the
steric repulsion. In our previous study with the W-shaped nucleoside analogs
(WNA), a strong sequence dependency was observed. Although the origin
of sequence dependency could not be analyzed in detail, the room for
the formation of the base triplet in the antiparallel triplex seems to be
restricted and its shape to be determined by the surrounding bases. This
study showed that the replacement of O-4 by the CH2 group might produce
a more hindered room for the base triplet.
CONCLUSIONS
This study has shown that the carbocyclic nucleoside analog is a potential
candidate as a new platform structure for the synthesis of non-natural analogs
for the formation of the triple helix DNA. Considering that the phenyl-
substituted carbocyclic compound 13 showed a relatively high stabilizing
effect and that the additional thymine substitution of 10 diminished the
stabilizing effect, a proper heterocycle might provide selective interaction
with a T-A or a C-G interrupting site.
EXPERIMENTAL
1
Melting points are uncorrected. The H NMR (400, 500 MHz) and 13C
NMR (100, 125 MHz) spectra were recorded with Varian UNITY-400 and
INOVA-500 spectrometers, respectively. IR spectra were obtained using a
PerkinElmer FTIR-SpectrumOne. ESI-Mass spectra (ESI-MS) were taken
in either positive or negative mode using methanol and formic acid or
methanol only as a solvent respectively. High-resolution mass spectra were
recorded on an Applied Biosystems Mariner System 5299 spectrometer. Col-
umn chromatography was carried out on 60N or FL60D silica gel. Thin
layer chromatography (TLC) was performed on precoated silica gel 60F245
plates. Reactions below –40◦C were carried out using an Eyela low tempera-
ture pairstirrer PSL-1800. DNA was synthesized by a DNA/RNA synthesizer
(Applied Biosystems 394 or Nihon Techno Service NTS-H6 DNA/RNA syn-
thesizer) and purified by HPLC. The HPLC column was a Nacalai Tesque
COSMOSIL 10 × 250 mm 5C18-MS-II). HPLC buffers were prepared from
high quality reagents and filtered through Milipore, MillicupꢀR -LH. All DNA
samples were freeze-dried. MALDI-TOF mass spectra were obtained by a
Microflex-KS Bruker Daltonios. UV spectra and Tm values were determined
by a Beckman Coulter DU 800 spectrophotometer linked to Beckman Coul-
ter high performance temperature controller. Fluorescence spectra were
obtained with a Jasco FP-750 spectrofluorometer linked to Jasco ETC-272T

850
T. Nasr et al.
temperature controller. CD spectra were measured with a Jasco J-720 W
spectropolarimeter.
(1S,2R)-1-Benzyloxy-2-Benzyloxymethylcyclopent-4-one (4)
In a dry flask, Celite (1.5 g) was suspended in dichloromethane (6 mL)
at room temperature. In to the above suspension, PCC (1.034 g, 4.8 mmol)
was added to afford an orange suspension. A solution of 3[13] (azeotropically
dried with dry dichloromethane three times, 500 mg, 1.6 mmol) in dry
dichloromethane (2 mL) was added into the above mixture, and the reaction
mixture was stirred at room temperature for 12 hours to afford a deep brown
suspension. The reaction mixture was diluted with diethyl ether (30 mL) and
filtered through a bad of Celite and silica gel. The filtrate was evaporated
under vacuum to afford a crude pale yellow oil, which was purified by column
chromatography (60 N silica gel and hexane/ ethyl acetate 3:1) to afford 4 as
a pure pale yellow oil (81% yield). IR (cm−1) 3030, 2858, 1744, 1496, 1454.
1H-NMR (400 MHz, CDCl3) δ 7.35–7.24 (10 H, m), 4.55–4.47 (4 H, m), 4.14
(1H, m), 3.55 (1 H, dd, J = 4.8 Hz), 3.50–3.44 (1 H, m), 2.68–2.52 (3H, m),
2.32 (1H, dd, J = 4.8 and 4.4 Hz), 2.18 (1H, dd, J = 6.0 and 6.4 Hz). 13C-NMR
(125.68 MHz, CDCl3) 215.9, 138.0, 128.4-127.5, 78.0, 73.2, 71.3, 70.4, 44.4,
42.4, 40.0. ESI-HRMS (m/z) calcd. for C20H22O3 [M+H]+ 311.1642, found
311.1622.
(1S,2R)-1-Benzyloxy-2-Benzyloxymethylcyclopent-4-N-tert-
Butanesulfinyl Imine (5)
The ketone (4) (133 mg, 0.429 mmol) was azeotropically dried with dry
acetonitrile three times (3 × 3 mL) and then dissolved in 0.6 mL dry THF
under argon. Into the above solution, a solution of tetraethylorthotitanate
(208 μL, 0.943 mmol, >95% W) in 1.2 mL dry THF was added dropwise
at room temperature. To this reaction mixture, R-(+)-2-methyl-2-propane
sulfonamide (64 mg, 0.514 mmol) was added and the reaction mixture was
immediately heated at 70◦C for 4 hours. After cooling to room temperature,
the mixture was added rapidly into a volume of brine (1.75 mL) to form
a white solid. The mixture was filtered through a Celite pad and the col-
lected paste was washed with ethyl acetate. The filtrate was transferred to a
separating funnel and washed with brine (15 mL). The separated brine was
extracted with ethylacetate (30 mL). The combined organic solvents were
dried over anhydrous sodium sulfate and evaporated under vacuum to afford
a crude dark yellow oil, which was purified by flash column chromatography
(60 N silica gel, hexane/ ethylacetate 4:1→3:1→2:1) to afford the target as a
yellow oil (166 mg, 94% yield). The target should be eluted within 15 minutes
as it slowly hydrolyzes on silica gel. IR (cm−1) 2917.0, 2861.0, 1637.5, 1496.2,
1
1454.1, 1361.3. H-NMR (400 MHz, CDCl3) 7.33–7.24 (m, 10H), 4.45-4.42

Properties of Oligonucleotide with Phenyl-Substituted Carbocyclic Nucleoside Analogs 851
(m, 4H), 4.06–4.11 (m, 1H), 3.50–3.23 (m, 3H), 2.89–2.80 (m, 1H), 2.68
(m, 1H), 2.58–2.46 (m, 1H), 1.20 (s, 9H). ESI-MS for C24H31NO3S [M+H]+
calcd. 414.21 found 414.24.
(1S,2R)-1-Benzyloxy-2-Benzyloxymethyl-4-Phenylcyclopent-4-N-
tert-butanesulfinyl Amine (6)
In a very dry flask, compound (5) (30 mg, 0.0726 mmol) was dried
azeotropically in acetonitrile (3 × 2 mL), then dissolved in 0.3 mL dry
toluene under argon atmosphere and cooled to –78◦C. Trimethylaluminum
(2M solution in toluene, 40 μL, 0.0799 mmol) was then added and the reac-
tion mixture was stirred at –78◦C for 30 minutes. Phenyllithium (1.09 M/L in
cyclohexane/diethyl ether, 200 μL, 0.2179 mmol) was added dropwise, and
the reaction mixture was stirred at –78◦C for 3 hours, then allowed to warm
gradually to 0◦C over 7 hours. After stirring for 1 hour at 0◦C, TLC indicated
the disappearance of the starting material. The mixture was quenched by
the slow addition of a saturated aqueous solution of sodium sulfate until gas
evolution stopped, then warmed to room temperature, filtered and diluted
with ethyl acetate (10 mL). The organic layer was separated and washed with
water (10 mL) and brine (10 mL), dried over sodium sulfate, and evapo-
rated to afford a crude yellowish brown oil. The crude product was purified
by short flash column chromatography (FL60D silica gel, hexane/ ethyl ac-
etate 3:1) to afford the target as a yellowish brown oil (46% total yield for 2
isomers in the ratio 9:1).
Physical Data for the Major Isomer
IR (cm−1) 3265.3, 3029.0, 2959.6, 2862.0, 1743.9, 1603.1, 1496.0, 1453.8.
1H-NMR (400 MHz, CDCl3) 7.58–7.08 (15 H, m, aromatic), 5.22 (1H, s),
4.51 (2H, d, J = 3.4 Hz), 4.47 (2H, d, J = 12.0 Hz), 4.05 (1H, d, J = 4.5 Hz),
3.60 (1H, dd, J = 4.5 and 3.4 Hz), 3.44–3.36 (1H, m), 3.12–2.86 (2H, m),
2.40 (1H, d, J = 14.8 Hz), 2.03 (1H, t, J = 10.8 Hz), 1.9 (1H, dd, J = 6.4 and
6.8 Hz), 0.97 (9H, s). 13C-NMR (125.68 MHz, CDCl3) 144.5, 138.5, 138.5,
128.4, 128.3, 128.3, 128.3, 128.3, 128.36, 128.0, 127.8, 127.5, 127.5, 127.5,
127.5, 127.2, 126.9, 126.7, 82.9, 73.0, 72.0, 70.8, 68.9, 55.6, 48.0, 44.8, 39.3,
22.7, 22.5, 22.3. ESI-HRMS (m/z) for C30H37NO3S [M+H]+ calcd. 492.2567
found 492.2595.
Physical Data for the Minor Isomer
1
IR (cm−1) 2918.7, 2859.4, 1604.0, 1495.7, 1474.0, 1454.3, 1361.6. H-
NMR (400 MHz, CDCl3) 7.36–7.08 (m, 15 H), 6.34 (1H, NH), 4.49 (s, 4H),
3.51–3.40 (m, 4 H), 3.38–3.10 (m, 2 H), 2.86-2.43 (m, 2H), 1.21 (s, 9H).
C30H37NO3S (M.W. 491.25). ESI-MS for C30H37NO3S [M+H]+ calcd. 492.25
found 492.31.

852
T. Nasr et al.
(1S,2R)-1-Benzyloxy-2-Benzyloxymethyl-4-Phenylcyclopent-4-
Amine (7)
The major isomer of compound (6) (13 mg, 0.0264 mmol) was dissolved
in methanol (0.25 mL), then 4 M HCl in dioxane (0.132 mmol, 35 μL) was
added and the reaction mixture was stirred at room temperature for 30 min-
utes until TLC indicated the disappearance of all the starting material. The
reaction mixture was diluted with methanol (5 mL) and neutralized with
solid sodium bicarbonate until effervescence stopped. The mixture was fil-
tered and diluted with ethyl acetate (10 mL). The separated organic layer was
washed with water (5 mL), dried over anhydrous sodium sulfate and evapo-
rated to afford a crude yellow oil (10 mg). The crude product was purified
by flash column chromatography (FL60D silica gel, chloroform/methanol
50:1→40:1) to afford the desired amine as a yellowish brown oil (83% yield).
1
IR (cm−1) 3059.9, 3028.4, 2924.5, 2854.1, 1602.4, 1495.3, 1453.2. H-NMR
(400 MHz, CDCl3) 7.47–7.16 (15H, m, aromatic), 4.56 (2H, d, J = 4.4 Hz),
4.49 (2H, s), 4.01–3.99 (1H, m), 3.48 (1H, d, J = 6.5 Hz), 3.43 (1H, d, J =
6.5 Hz), 2.85–2.76 (1H, m), 2.53–2.29 (3H, br m), 2.25 (1H, d, J = 13.6 Hz),
2.17 (1H, dd, J = 6.5 Hz), 1.88 (1H, dd, J = 10.4 Hz). ESI-HRMS (m/z) for
C26H29NO2 [M+H]+ calcd. 388.2271 found 388.2270.
The Acyclic Intermediate (8)
Freshly prepared 3-methoxy-2-methyl-acryloyl isocyanate (121.82 mg,
0.864 mmol) was added to a solution of the amine (7) (85 mg, 0.217 mmol)
in dry benzene (2.2 mL) under argon, and the reaction mixture was stirred
at room temperature for 45 minutes. The solvents were evaporated to afford
a crude brown oil (257 mg), which was purified by column chromatography
(60N silica gel, hexane ethyl acetate 4:1→3:1→2.5:1) to produce 8 as a pale
brown oil (81% yield). IR (cm−1) 3242.5, 2917.2, 2850.2, 1689.3, 1660.9,
1
1616.9, 1642.1, 1494.4, 1453.1. H-NMR (400 MHz, CDCl3) 9.35 (1H, s,
NH), 7.49–7.15 (17 H, m), 4.59 (1H, s), 4.48 (3H, m), 4.01-3.91 (1H, m),
3.78 (3H, s), 3.55–3.47 (2H, m), 2.78–2.68 (3H, m), 2.21 (1H, dd, J = 7.4
and 7.4 Hz), 1.96 (1H, t), 1.68 (3H, s, CH3). 13C-NMR (125.68 MHz, CDCl3)
168.8, 158.4, 152.3, 144.5, 138.5, 138.5, 128.3, 128.3, 128.1, 128.1, 128.1,
127.6, 127.6, 127.5, 127.2, 126.6, 125.7, 125.7, 124.5, 110.7, 81.5, 73.1, 71.6,
71.1, 65.0, 61.4, 45.9, 45.0, 40.8, 8.7. ESI-HRMS for C32H36N2O5 [M+H]+
calcd. 529.2697 found 529.2713.
The Benzyl Protected Derivative (9)
The acyclic intermediate (8) (7 mg, 0.0132 mmol) was dissolved in 1M,
aq. H2SO4 (198 μL) and the mixture was heated at 100◦C. DMSO was then
added dropwise until a clear yellow solution was obtained. The reaction mix-
ture was further heated at the same temperature for 5 hours. After cooling

Properties of Oligonucleotide with Phenyl-Substituted Carbocyclic Nucleoside Analogs 853
to room temperature, the reaction mixture was neutralized by the addition
of solid sodium bicarbonate until effervescence stopped. The mixture was
filtered and the collected solid was washed with ethyl acetate. The combined
filtrates were washed with water (10 mL), and the separated water was ex-
tracted with ethyl acetate (2 × 20 mL). The combined organic solvents were
dried over sodium sulfate and evaporated to afford a crude brown oil, which
was purified by column chromatography (FL60D silica gel, hexane/ethyl
acetate 4:1→3:1→2:1→3:2) to produce 9 as a colorless oil (4 mg, 60%
yield). IR (cm−1) 3026.1, 2924.6, 1684.5, 1453.8, 1364.9. 1H-NMR (400 MHz,
CDCl3) 7.77 (1H, s), 7.61 (1H, s), 7.32–7.19 (15H, m), 4.47 (2H, s,), 4.46
(2H, s), 4.09–3.98 (1H, m), 3.46 (2H, d, J = 5.2 Hz), 2.96–2.93 (1H, m), 2.87
(1H, m), 2.51 (1H, dd, J = 7.2 Hz, 8.0 Hz), 2.48–2.46 (1H, m), 2.27 (1H, dd,
10.6 Hz), 1.86 (3H, s). 13C-NMR (125.68 MHz, CDCl3) 163.7, 150.1, 143.2,
138.8, 138.1, 128.4–125.7, 110.5, 80.5), 73.4, 73.3, 71.5, 71.2, 45.5, 43.2, 39.8,
12.7. ESI-HRMS for C31H32N2O4 [M+H]+ calcd. 497.2435 found 497.2478.
(1R,3S,4R)-1-Phenyl-1-Thymidyl-3-Hydroxy-4-
Hydroxymethylcyclopentane (10)
Pd(OH)2/C (22 mg) was added to a solution of 9 (22 mg, 0.443 mmol)
in methanol (6 mL). The flask was purged with hydrogen gas and the
mixture was stirred for 20 minutes at room temperature after the addi-
tion of two drops of 2 M aqueous HCl. The reaction mixture was filtered
through a basic alumina pad to remove Pd(OH)2/C and HCl, and the pad
was eluted with 10 mL methanol. The combined methanol was evaporated
to afford a colorless crude oil, which was purified by flash column chro-
matography (FL60D silica gel, HPLC grade chloroform/methanol 80:1→
50:1→30:1→20:1→8:1) to afford the desired material as a pure colorless
25
oil (14.4 mg, 98% yield). [α] D + 16.4◦ (c 0.45, EtOH). IR (cm−1) 3373.1,
1
3029.8, 2926.7, 1666.4, 1496.3, 1369.1. H-NMR (400 MHz, CD3OD) 7.95
(1H, s), 7.32–7.18 (5H, m), 4.063 (1H, q, J = 6.0 Hz), 3.67 (1H, dd, J =
5.0 and 5.0 Hz), 3.57 (1H, ddd, J = 7.2, 6.8, and 6.8 Hz), 2.81 (1H, dd, J
= 8.8 and 8.0 Hz), 2.75–2.67 (2H, m), 2.34–2.28 (1H, m), 2.20–2.11 (1H,
m), 1.95 (3H, s). 13C-NMR (125.68 MHz, CD3OD) 166.8, 152.3, 145.5, 141.1,
129.4-126.2, 109.8, 73.9, 73.6, 63.9), 50.1, 47.3, 41.2), 12.1. ESI-HRMS for
C17H20N2O4 [M+Na]+ calcd. 339.1315 found 339.1317.
(1RS,3S,4R)-3-Hydroxy-4-Hydroxymethyl-1-Phenylcyclopentane
(13)
To a solution of the ketone 4 (100 mg, 0.322 mmol, dried by co-
evaporation three times with acetonitrile) in anhydrous THF (1 mL) at
–78◦C, PhLi (1.14 M/L, 422 μL, 0.483 mmol) was added dropwise, and the

854
T. Nasr et al.
reaction mixture was stirred at the same temperature for 3 hours. After warm-
ing to room temperature, the reaction mixture was quenched with saturated
aqueous ammonium chloride (5 mL) and extracted with ethyl acetate (2 ×
30 mL). The combined organic solvents were washed with water (30 mL)
and brine (30 mL), dried over anhydrous sodium sulfate, and evaporated
to yield a crude brownish yellow oil, which was purified by column chro-
matography (60 N silica gel, hexane/ethyl acetate 5:1) to afford the benzyl
protected derivative of 13 as an oil. 71 mg (57% total yield of two isomers).
1
IR (cm−1) 3441, 3040, 2923, 2854, 1495, 1452, 1361. H-NMR (400 MHz,
CDCl3) δ 7.51–7.46 (2 H, m), 7.34–7.20 (13 H, m), 4.58–4.44 (4 H, m), 4.13
(1 H, d, J = 5.4 Hz), 3.52 (1 H, dd, J = 5.4 Hz), 3.40–3.36 (1 H, m), 2.82–2.78
(1H, m), 2.39–2.33 (1H, m), 2.27 (1H, d, J = 14.4 Hz), 2.15 (1H, dd, J =
5.4 and 6.0 Hz), 1.86 (1H, dd, J = 8.8 and 8.4 Hz), 1.23 (1 H, s). ESI-HRMS
(m/z) calcd. for C26H28O3 [M+K]+ 427.1670, found 427.1715.
The above oil (10 mg, 0.025 mmol) was dissolved in EtOAc (0.5 mL),
followed by the addition of Pd(OH)2/C (20%, 5 mg) and one drop of 2 M
aqueous HCl. The reaction flask was purged with hydrogen gas, and the
mixture was stirred at room temperature for one hour. The reaction mix-
ture was filtered through a basic alumina pad to remove Pd(OH)2/C and
HCl. The filtrate was dried over sodium sulfate and evaporated to afford a
crude yellowish brown oil, which was purified by column chromatography
(60 N silica gel, chloroform/methanol 40:1→30:1) to afford 13 as a colorless
oil (mixture of 2 isomers, 1:1.75 as determined by H1-NMR) and the total
yield was 83%. IR (cm−1) 3342.5, 3025.3, 2932.9, 2869.5, 1601.7, 1494.0,
1449.1, 1350.8. 1H-NMR (400 MHz, CDCl3) 7.29–7.15 (5H, m, aromatic pro-
tons), 4.14–4.12 (1H, m), 3.78–3.76 (1H, m), 3.64–3.59 (1H, m), 3.59–3.06
(3H, m), 2.43–2.23 (1H, m), 2.20 (1H, br s), 1.96–1.81 (1H, m), 1.79–1.76
(2H, m). ESI-HRMS for C12H16O2 [M+Na]+ calcd. 215.1042 found
215.1016.
Synthesis of DMTr-Protected Amidite Precursor 14
To a solution of compound 10 (24 mg, 0.075 mmol, dried azeotropically
with acetonitrile) in dry pyridine (0.34 mL), was added 4,4-dimethoxytrityl
chloride (39 mg, 0.114 mmol) under argon atmosphere. The reaction was
stirred at room temperature for 1.5 hours. The reaction was quenched with
saturated aqueous sodium bicarbonate (5 mL), extracted with chloroform
(10 mL). The organic layer was dried over anhydrous sodium sulfate. The
organic layer was evaporated to yield a crude yellow oil, which was purified by
column chromatography (60 N silica gel, chloroform/methanol 100:1 and
changed gradually to 20:1 containing 0.5% pyridine) to afford the DMTr-
protected derivative of 10 as an oil in 87% yield as a colorless oil. IR (cm−1)
1
3032.9, 2927.6, 2868.0, 1686.2, 1625.7, 1607.3, 1552.1, 1508.7, 1445.7. H-
NMR (400 MHz, CDCl3) 8.02 (1H, bs), 7.58 (1H, s), 7.42-7.12 (14H, m,

Properties of Oligonucleotide with Phenyl-Substituted Carbocyclic Nucleoside Analogs 855
aromatic), 6.80 (4H, d, J = 8.2 Hz), 4.10 (1H, q, J = 6.2 Hz), 3.77 (6H, s,
2× OCH3), 3.38 (1H, m), 3.18–3.05 (1H, m), 2.74–2.64 (2H, m), 2.55–2.40
(2H, m), 2.35–2.22 (2H, m), 1.96 (3H, s). ESI-MS for C38H38N2O6 [M–H]−
calcd 617.26 found 617.27
The above oil (21 mg, 0.034 mmol, dried azeotripically with acetoni-
trile three times) was dissolved in dry dichloromethane (0.43 mL) con-
taining N,N -diisoproopylethylamine (34 μL, 0.204 mmol), followed by the
addition of 2-cyanoethyl-N,N -diisopropylchlorophosphoramidite (46 μL,
0.204 mmol) at 0◦C. The reaction mixture was stirred at 0◦C for 1 hour. The
reaction mixture was quenched with saturated aqueous sodium bicarbonate
(5 mL) and extracted with ethyl acetate (10 mL). The organic layer was dried
over anhydrous sodium sulfate and evaporated to yield a crude yellow oil,
which was purified by flash column chromatography (FL60D silica gel, HPLC
grade hexane/ ethylacetate 1:2) to afford 14 in 83% yield as pale yellow oil.
IR (cm−1) 2966.9, 2930.4, 1868.6, 1607.9, 1508.7, 1463.8, 1446.7, 1364.4.
1H-NMR (400 MHz, CDCl3) 7.84 (1H, bs), 7.71–7.10 (15H, m), 6.80–6.71
(4H, m), 4.41–4.26 (1H, m), 3.77 (3H, s), 3.76 (3H, s), 3.73–3.46 (4H, m),
3.28–3.20 (1H, m), 3.18–3.13 (1H, m), 3.08–2.92 (1H, m), 2.90–2.78 (1H, m),
2.74–2.62 (1H, m), 2.58 (1H, t, J = 11.2 Hz), 2.52–2.44 (1H, m), 2.44–2.29
(2H, m), 2.02 (1.5H, s), 1.96 (1.5H, s), 1.32–1.22 (4H, m), 1.20–1.04 (6H,
m), 0.98 (2H, d, J = 6.8 Hz). 31P-NMR (161.9 MHz, CDCl3) δ 150.3, 148.9.
ESI-MS for C47H55N4O7P [M+H]+ calcd. 819.38 found 819.42.
Synthesis of DMTr-Protected Amidite Precursor 15
To a solution of 11 (50 mg, 0.431 mmol, dried azeotropically three times
with dry pyridine) in dry pyridine (1.93 mL), was added 4,4-dimethoxytrityl
chloride (219 mg, 0.646 mmol) under argon atmosphere. The reaction
mixture was stirred at room temperature for 45 minutes and quenched with
saturated aqueous sodium bicarbonate (5 mL). The mixture was extracted
with chloroform (10 mL), and the organic layer was dried over anhydrous
sodium sulfate, evaporated to yield a crude yellow oil, which was purified
by column chromatography (60 N silica gel, chloroform/methanol 100:1
containing 0.5% pyridine) to afford the DMTr-protected derivative of 11
in 90% yield as a colorless oil. IR (cm−1) 3439.3, 2955.5, 2835.2, 1607.4,
1
1582.9, 1508.1, 1463.1, 1445.3. H-NMR (500 MHz, CDCl3) 7.44–7.19 (9H,
m, arom.), 8.81 (4H, d, J = 8.8 Hz), 3.92 (1H, q, J = 7.2 Hz), 3.77 (6H,
s, 2× OCH3), 3.29 (1H, dd, J = 5.2 and 5.2 Hz), 2.94 (1H, t, J = 9.0 Hz),
2.49 (1H, bs, OH), 2.11–2.02 (1H, m), 1.94–1.84 (1H, m), 1.82–1.64 (2H,
m), 1.61–1.46 (3H, m). ESI-MS for C27H30O4 [M+Na]+ calcd. 441.20 found
441.19
To the above oil (106 mg, 0.253 mmol, dried azeotropically with
acetonitrile three times) in dry dichloromethane (3.2 mL) containing

856
T. Nasr et al.
N,N -diisopropylethylamine (265 μL, 1.521 mmol) was added 2-cyanoethyl-
N,N -diisoprpylchlorophosphoramidite (339 μL, 1.521 mmol) at 0◦C, and
the reaction mixture was stirred at the same temperature for 1 hour. The
reaction mixture was quenched with saturated aqueous sodium bicarbonate
(5 mL), extracted with ethyl acetate (10 mL). The organic layer was dried
over anhydrous sodium sulfate, filtered and evaporated to yield a crude
yellow oil, which was purified by flash column chromatography (FL60D sil-
ica gel, HPLC grade hexane/ ethyl acetate 4:1) to afford 15 in 80% yield
1
as a pale yellow oil. IR (cm−1) 2964.0, 1607.6, 1508.5. H-NMR (500 MHz,
CDCl3) 7.46–7.10 (9H, m), 6.81–6.77 (4H, m), 4.15–4.08 (1H, m), 3.77 (6H,
s), 3.71–3.51 (4H, m), 3.09–3.02 (1H, m), 3.00–2.89 (1H, m), 2.56 (0.5H,
t, J = 6.6 Hz), 2.49–2.36 (1.5H, m), 2.24–2.15 (1H, m), 2.02–1.91 (1H, m),
1.78–1.55 (4H, m), 1.42–1.88 (1H, m), 1.17–1.08 (10H, m), 1.07–1.03 (2H,
d, J = 6.6 Hz). 31P-NMR (161.9 MHz, CDCl3) δ 148.0, 147.4. ESI-MS for
C36H47N2O5P [M+H]+ calcd 619.32 found 619.38.
Synthesis of DMTr-Protected Amidite Precursor 16
To a solution of compound 12 (24 mg, 0.099 mmol, dried azeotropically
three times with dry pyridine) in dry pyridine (0.45 mL), was added 4,4-
dimethoxytrityl chloride (51 mg, 0.149 mmol) under argon atmosphere. The
reaction mixture was stirred at room temperature for 1 hour and quenched
with saturated aqueous sodium bicarbonate (5 mL). The mixture was ex-
tracted with chloroform (10 mL), and the organic layer was dried over
anhydrous sodium sulfate, evaporated to yield a crude yellow oil, which was
purified by column chromatography (60 N silica gel, chloroform/methanol
100:1 and changed gradually to 10:1 containing 0.5% pyridine) to afford the
DMTr-protected 12 in 93% yield as a colorless oil. IR (cm−1) 3418.3, 3191.6,
1
3054.0, 2907.6, 1679.7, 1607.6, 1580.6, 1508.7, 1464.6. H-NMR (400 MHz,
CDCl3) 8.18 (1H, bs), 7.34–7.14 (10H, m), 6.77 (4H, d, J = 7.6 Hz), 4.80
(1H, quin., J = 7.6 Hz), 4.14–4.06 (1H, m), 3.73 (6H, s), 3.19–3.13 (1H, m),
3.00–2.88 (1H, m), 2.70 (1H, bs), 2.38–2.26 (2H, m), 1.87 (3H, s), 1.91–1.83
(1H, m), 1.83–1.76 (1H, m), 1.76–1.66 (1H, m). ESI-MS for C32H34N2O6
[M+Na]+ calcd. 565.23 found 565.21.
To the above oil (50 mg, 0.092 mmol, dried azeotropically with ace-
tonitrile three times) in dry dichloromethane (1.16 mL) containing N,N -
diisopropylethylamine (97 μL, 0.553 mmol), was added 2-cyanoethyl-N,N -
diisopropylchlorophosphoramidite (123 μL, 0.553 mmol) at 0◦C, and the
reaction mixture was stirred at the same temperature for 1 hour. The re-
action mixture was quenched with saturated aqueous sodium bicarbonate
(5 mL), and extracted with ethyl acetate (10 mL). The organic layer was
dried over anhydrous sodium sulfate and evaporated to yield a crude yel-
low oil, which was purified by flash column chromatography (FL60D silica
gel, HPLC grade hexane/ethylacetate 1:2) to afford 16 in 64% yield as a

Properties of Oligonucleotide with Phenyl-Substituted Carbocyclic Nucleoside Analogs 857
1
colorless oil. IR (cm−1) 2967.3, 2200.0, 1783.0, 1608.0, 1508.9, 1464.6. H-
NMR (400 MHz, CDCl3) 8.16 (1H, bs, NH), 7.47 (0.5H, s), 7.44 (0.5 H, s),
7.40-7.01 (9H, m), 6.82–6.79 (4H, m), 5.12–5.03 (1H, m), 4.43–4.26 (1H, m),
4.15–4.07 (1H, m), 3.92–3.79 (1H, m), 3.775 (3H, s), 3.773 (3H, s), 3.76–3.44
(5H, m), 3.09–2.94 (1H, m), 2.86–2.29 (3H, m), 2.03 (1.5H, s), 1.93 (1.5H,
s), 1.86–1.52 (2H, m), 1.31–1.10 (12H, m). 31P-NMR (161.9 MHz, CDCl3) δ
149.4, 148.7. ESI-MS for C41H51N4O7P [M+H]+ calcd. 743.35 found 743.38.
Synthesis of DMTr-Protected Amidite Precursor 17
To a solution of 13 (38 mg, 0.197 mmol, dried azeotropically three times
with dry pyridine) in dry pyridine (0.88 mL), was added 4,4-dimethoxytrityl
chloride (100 mg, 0.296 mmol) under argon atmosphere, and the reaction
mixture was stirred at room temperature for 1 h, then quenched with satu-
rated aqueous sodium bicarbonate (5 mL). The mixture was extracted with
chloroform (10 mL), and the organic layer was dried over anhydrous sodium
sulfate, evaporated to yield a crude yellow oil, which was purified by open
column chromatography (FL60D silica gel, hexane/ethylacetate 4:1) to pro-
duce the DMTr protected derivative of 13 as a mixture of two isomers is
89% yield. The isomers were separated by repetition of column chromatog-
raphy to produce the α-isomer as a major isomer and the β-isomer as a minor
isomer. The stereochemistry was determined by 2D NMR COSY and NOESY.
Physical Data for α-Phenyl Isomer
IR (cm−1) 3452.2, 3060.9, 3026.6, 2931.3, 2862.20, 2835.5, 1607.0, 1582.4,
1508.1, 1462.0, 1445.3. 1H-NMR (400 MHz, CD3OD) 7.44 (2H, d, aromatic,
J = 8.0 Hz), 7.32–7.01 (12H, m), 6.84 (4H, d, J = 8.8 Hz), 4.03 (1H, q, J =
6.4 Hz), 3.76 (6H, s), 3.22 (1H, dd, J = 5.4, 5.4 Hz), 3.12–308 (1H, m), 3.00
(1H, quin, J = 8.8 Hz), 2.31 (1H, dd, J = 6.4 and 6.4 Hz), 2.27–2.14 (1H,
m), 1.99–1.91 (2H, m), 1.67 (1H, ddd, J = 8.4, 8.8, and 8.4 Hz). 13C-NMR
(125.68 MHz, CD3OD) 160.0-108.1, 87.2, 76.5, 66.2, 55.7, 48.9, 44.5, 42.7,
37.1. ESI-MS for C33H34O4 [M+Na]+ calcd. 517.23 found 517.35.
Physical Data for β-Phenyl Isomer
IR (cm−1) 3352.0, 3023.8, 2924.8, 2853.5, 1607.1, 1582.2, 1508.1, 1446.2.
NMR (400 MHz, CD3OD) 7.43 (2H, d, aromatic, J = 7.6 Hz), 7.32–7.10
(12H, m, aromatic), 6.83 (4H, d, J = 8.4 Hz), 4.17–4.07 (1H, m), 3.76 (6H,
s, 2× CH3O), 3.38–3.08 (3H, m), 2.35–2.19 (2H, m), 2.02–1.91 (2H, m),
1.81 (1H, ddd, J = 6.4, 6.4, 6.8 Hz). ESI-MS for C33H34O4 [M+K]+ calcd.
533.20 found 533.38.
To a solution of the β-isomer (30 mg, 0.06 mmol, dried azeotropically
with acetonitrile three times) in dry dichloromethane (0.75 mL) containing

858
T. Nasr et al.
TABLE 2 MALDI TOF-MS analysis [M-H]− of synthesized, modified (ODNs) incorporating compound
10-13.
DNA(Z) TFO(Z)
Nucleotide for Z
Single Stranded DNA
Calcd
Found
DNA 1(10)
DNA 2(10)
TFO1(10)
TFO 2(10)
TFO3(10)
TFO4(10)
DNA 1(11)
DNA 2(11)
TFO1(11)
TFO 2(11)
TFO3(11)
TFO4(11)
DNA 1(12)
DNA 2(12)
TFO1(12)
TFO 2(12)
TFO3(12)
TFO4(12)
DNA 1(13)
DNA 2(13)
TFO1(13)
TFO 2(13)
TFO3(13)
TFO4(13)
10
10
10
10
10
10
11
11
11
11
11
11
12
12
12
12
12
12
13
13
13
13
13
13
5ꢁ CTT TCT TZT CCT TTC T 3ꢁ
3ꢁ GAA AGA AZA GGA AAGA 5ꢁ
4800.60
5090.85
5812.91
5828.90
5812.91
5796.92
4600.60
4890.85
5612.91
5628.90
5612.91
5596.92
4724.60
5014.85
5736.91
5752.90
5736.91
5720.92
4677.72
4967.97
5690.03
5706.02
5690.03
5674.04
4800.78
5091.85
5810.09
5828.16
5811.34
5793.00
4598.30
4887.94
5613.12
5627.31
5610.56
5597.40
4721.01
5010.12
5734.19
5747.22
5733.85
5717.10
4677.90
4965.36
5690.87
5705.62
5686.17
5674.54
3ꢁ GGA AGG AZG GAG GAG GGA 5ꢁ
3ꢁ GGA AGG GZG GAG GAG GGA 5ꢁ
3ꢁ GGA AGG GZA GAG GAG GGA 5ꢁ
3ꢁ GGA AGG AZA GAG GAG GGA 5ꢁ
5ꢁ CTT TCT TZT CCT TTC T 3ꢁ
3ꢁ GAA AGA AZA GGA AAGA 5ꢁ
3ꢁ GGA AGG AZG GAG GAG GGA 5ꢁ
3ꢁ GGA AGG GZG GAG GAG GGA 5ꢁ
3ꢁ GGA AGG GZA GAG GAG GGA 5ꢁ
3ꢁ GGA AGG AZA GAG GAG GGA 5ꢁ
5ꢁ CTT TCT TZT CCT TTC T 3ꢁ
3ꢁ GAA AGA AZA GGA AAGA 5ꢁ
3ꢁ GGA AGG AZG GAG GAG GGA 5ꢁ
3ꢁ GGA AGG GZG GAG GAG GGA 5ꢁ
3ꢁ GGA AGG GZA GAG GAG GGA 5ꢁ
3ꢁ GGA AGG AZA GAG GAG GGA 5ꢁ
5ꢁ CTT TCT TZT CCT TTC T 3ꢁ
3ꢁ GAA AGA AZA GGA AAGA 5ꢁ
3ꢁ GGA AGG AZG GAG GAG GGA 5ꢁ
3ꢁ GGA AGG GZG GAG GAG GGA 5ꢁ
3ꢁ GGA AGG GZA GAG GAG GGA 5ꢁ
3ꢁ GGA AGG AZA GAG GAG GGA 5ꢁ
N,N -diisopropylethylamine (63 μL, 0.364 mmol), was added 2-cyanoethyl-
N,N -diisopropylchlorophosphoramidite (81 μL, 0.364 mmol) at 0◦C, and
the reaction mixture was stirred at the same temperature for 1 hour. The
reaction mixture was quenched with saturated aqueous sodium bicarbonate
(5 mL), and extracted with ethyl acetate (10 mL). The organic layer was
dried over anhydrous sodium sulfate, evaporated, and purified by flash col-
umn chromatography (FL60D silica gel, HPLC grade hexane/ethyl acetate
4:1) to afford 17 in 69% yield as a colorless oil. IR (cm−1) 2963.5, 2929.0,
1
1607.4, 1508.3. H-NMR (400 MHz, CDCl3) 7.49–7.40 (2H, m, aromatic),
7.32–7.16 (12H, m, aromatic), 6.81–6.72 (4H, m, aromatic), 4.32–4.25 (1H,
m), 3.77 (3H, s, OCH3), 3.76 (3H, s, OCH3), 3.72–3.51 (3H, m), 3.37–3.36
(1H, m), 3.19–3.16 (1H, m), 3.14–3.03 (1H, m), 2.56 (1H, t, J = 6.8 Hz),
2.49–2.21 (3H, m), 2.17-2.01 (1H, m), 1.88–1.80 (1H, m), 1.55–1.39 (2H, m),
1.23–0.85 (12H, m). 31P-NMR (161.9 MHz, CDCl3) δ 148.20, 147.48. ESI-MS
for C42H51N2O5P [M+H]+ calcd 695.36 found 695.24.
Synthesis and Purification of ODN
All oligonucleotides were synthesized by the standard DNA synthesis
procedures. The synthesized ODNs were cleaved from the CPG column in

Properties of Oligonucleotide with Phenyl-Substituted Carbocyclic Nucleoside Analogs 859
28% ammonium solution at 55◦C for 5 hours and were purified by HPLC
(Nacalai Tesque COSMOSIL C18-ARII) using a linear gradient (A: 0.1 M
TEAA buffer, B: CH3CN, B conc. 10% to 40%/20 min). The DMTr-group was
cleaved in 10% aqueous acetic acid at room temperature and the resulting
DMTr-OH was removed by washing with ether. The purities and structures
of the synthesized TFOs and labeled-ODNs were confirmed by MALDI-TOF
Mass measurement (Table 2).
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