Synthesis and hybridization property of sugar and phosphate linkage modified oligonucleotides

Article abstract of DOI:10.1016/S0968-0896(99)00061-9

1-[3-Deoxy-5-O-(4,4'-dimethoxytriphenylmethyl)-3-C-hydroxymethyl-2-O-(2-methoxyethoxymethyl)-β-D-erythro-pentofuranosyl]thymine (13) was synthesized from 1,2-isopropylidene-D-xylose (1) as a building block of modified oligonucleotides. Three types of novel oligonucleotides were synthesized from 13 and their T(m)s were compared with those of the corresponding natural oligonucleotides. It was found that our synthesized oligomers had lower affinity to DNA and RNA than the natural oligomers. Copyright (C) 1999 Elsevier Science Ltd.

Full text of DOI:10.1016/S0968-0896(99)00061-9

Bioorganic & Medicinal Chemistry 7 (1999) 1467±1473
Synthesis and Hybridization Property of Sugar and Phosphate
Linkage Modi®ed Oligonucleotides
Lak Shin Jeong,a,* Ji Hea Lee,a Kyeong-Eun Jung,b Hyung Ryong Moon,a
Kichul Kimb and Hong Limb
aLaboratory of Medicinal Chemistry, College of Pharmacy, Ewha Womans University, Seoul 120-750, Korea
bDongbu Research Council, Agropharma Research Institute, 103-2 Moonjidong Yusunggu, Taejon, 305-380, Korea
Received 8 January 1999; accepted 4 March 1999
AbstractÐ1-[3-Deoxy-5-O-(4,40-dimethoxytriphenylmethyl)-3-C-hydroxymethyl-2-O-(2-methoxyethoxymethyl)-b-d-erythro-pento-
furanosyl]thymine (13) was synthesized from 1,2-isopropylidene-d-xylose (1) as a building block of modi®ed oligonucleotides.
Three types of novel oligonucleotides were synthesized from 13 and their Tms were compared with those of the corresponding
natural oligonucleotides. It was found that our synthesized oligomers had lower anity to DNA and RNA than the natural oli-
gomers. # 1999 Elsevier Science Ltd. All rights reserved.
Introduction
opportunity for the antisense agents in various diseases
and biological experiments is growing rapidly.
Since oligonucleotides complementary to messenger
RNA or viral RNA were proved to inhibit the produc-
tion of proteins and viral replications in the 1970s,1,2
and the natural gene regulation via antisense RNA was
discovered in the 1980s,3,4 antisense research for the
development of therapeutics has been explored exten-
sively. Many oligonucleotides also have been used as
important tools to study speci®c genes, proteins and
their functions in cells.
Vitravene, the ®rst antisense drug, was approved by the
Food and Drug Administration (FDA) for the treat-
ment of AIDS patients infected with cytomegalovirus
retinitis.11 The phosphorothioate oligomers, however,
have shown side e€ects, causing several immune
responses probably due to protein bindings.12±14 Since
phosphorothioate linkage is believed to cause these
undesired e€ects, several approaches which use non-
phosphorothioates or less phosphorothioate linkages
have been attempted. In 1997, the second generation
oligonucleotides consisted of mixed backbone of
phosphorothioate and 20-O-alkylated oligonucleotides
entered the human clinical trials.15
Antisense oligonucleotides bind to complementary mes-
senger RNA following Watson±Crick base pairing rules,
which result in the inhibition of protein synthesis.5±7
Natural oligonucleotides which are added from outside
of cells do not show enough antisense e€ects because of
rapid degradation by cell nucleases. In order to over-
come the degradation by nucleases and to improve a-
nity to DNA and RNA, base and sugar moieties of
nucleoside structures have been modi®ed extensively.
Recently, many sugar backbone modi®ed oligonuleo-
tides have been reported to increase resistance to
nucleases and to give better binding anities to
mRNA.6,16,17 When we recently started the antisense
research program and focused on the structure mod-
i®cation of nucleosides, oligonucleotide analogues with
electronegative moiety at the C20 position appeared to
have certain superior characteristics such as the pre-
ference of A-type geometry for duplex formation.18
Incorporation of methoxyethoxymethyl (MEM) group
into the 20 hydroxyl position of the sugar backbone
was chosen, expecting more 30-endo sugar conforma-
tion (A type duplex) due to the gauche e€ect19
between the furanose ring oxygen and the oxygen of
20-OMEM. In view of giving more ¯exibility of phos-
phate linkage which may induce A conformation with
Various conjugation approaches to improve delivery to
cells have also been reported, among which liposomes
are being used for many in vitro and in vivo experiments
for antisense screenings.8±10 The progress of the Human
Genome Project and other sequencing projects has led
to many gene sequences now being available, and the
Key words: Oligonucleotides; nuclosides; antiviral; hybridization.
* Corresponding author. Tel.: 82-2-3277-3466; fax: 82-2-3277-2851;
e-mail: lakjeong@mm.ewha.ac.kr
0968-0896/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(99)00061-9

1468
L. S. Jeong et al. / Bioorg. Med. Chem. 7 (1999) 1467±1473
RNA, the 30-hydroxymethyl group was also designed
with 20-O-MEM substituent. Although several syntheses
of 20,30-dideoxy-30-C-hydroxymethyl nucleosides have
been published,20,21 30-deoxy-20-O-substituted-30-C-
hydroxymethyl nucleoside has not been reported. Thus,
we report the synthesis of 30-deoxy-20-O-substituted-30-
C-hydroxymethyl nucleoside and its oligomers and
hybridization study.
as a good glycosyl donor for the synthesis of the desired
nucleoside.
Synthesis of 1-[3-deoxy-3-C-hydroxymethyl-2-O-(2-meth-
oxyethoxymethyl)-5-O-(4,40 -dimethoxytrityl)-b-d-ery-
thro-pentofuranosyl]thymine (13) from the acetate 6 is
illustrated in Scheme 2. Condensation of the acetate 6
with silylated thymine in the presence of TMSOTf (tri-
methylsilyl tri¯uoromethanesulfonate) gave 7 (62%)
which was treated with benzoyl chloride in pyridine at
80 ꢀC to produce the N3-benzoylated derivative 8 in
70% yield. Selective deacetylation of 8 in the presence of
benzoyl group was achieved with K2CO3 in MeOH at
5 ꢀC to a€ord the diol 9. It has to be mentioned that
200-O-acetyl group of 8 could be selectively removed over
5 -O-acetyl or N3-benzoyl group in small scale (30 mg)
reaction, but in large scale (1.61 g) reaction, both acetyl
groups were cleaved under the same reaction conditions.
Thus, 50-hydroxyl group of 9 had to be protected to the
benzoate 10 by treating with benzoyl chloride in pyri-
dine at 5 ꢀC. The remaining 20-hydroxyl group of 10
was reacted with 2-methoxyethoxymethyl chloride
(MEM-Cl) and N,N-diisopropylethylamine in methyl-
ene chloride at 50 ꢀC for two days to give 11, which was
treated with methanolic ammonia to a€ord 50-O- and
N3-deprotected nucleoside 12. Compound 12 was trea-
ted with 4,40-dimethoxytrityl chloride in pyridine at
room temperature for 24 h followed by the treatment
with tetra-n-butylammonium ¯uoride in THF to yield
the desired nucleoside 13. The nucleoside 13 was con-
verted to the phosphoramidite 14, which was used for
the synthesis of the desired oligonucleotides A±C.
Results and Discussion
Nucleoside synthesis
To synthesize the desired oligonucleotides, the parent
nucleoside was ®rst synthesized from the condensation
of the silylated thymine with the appropriate 3-deoxy-3-
C-hydroxymethyl sugar derivative whose synthesis is
shown in Scheme 1.
1,2-O-Isopropylidene-a-d-xylofuranose (1) was con-
verted to 2 by the known method reported by Lin and
his co-workers.21 Treatment of 2 with benzyl bromide in
DMF at 0 ꢀC gave the benzylate 3. Deprotection of the
1,2-O-isopropylidene and 5-O-TBDMS groups of 3 with
50% acetic acid at 100 ꢀC for 3 h followed by acetyla-
tion with acetic anhydride in pyridine a€orded the ace-
tate 4 as an anomeric mixture. We ®rst tried to
synthesize the desired target nucleoside from compound
4, but in the last step, the 4,40-dimethoxytrityl (DMT)
group of the 50-position was too unstable under the
reaction conditions that remove the benzyl protecting
group to obtain the desired product. Therefore, benzyl
protecting group had to be changed to the tert-butyl-
diphenylsilyl group which could be easily deprotected
under the mild conditions.
Oligonucleotide synthesis and Tm studies
Assembly of oligonucleotides A±C was accomplished
by using ABI 392 DNA/RNA synthesizer on a 1 mmol
scale from 14 and commercial 20-deoxynucleoside phos-
phoramidites. Standard protocols were used except
10 min coupling time for the incorporation of modi®ed
nucleotide. All oligomers were characterized by digestion
to constituent bases followed by HPLC analysis and
mass spectrometric analysis. Their duplex melting tem-
peratures with complementary DNA or RNA sequences
Thus, benzyl group of the compound 4 was removed by
the catalytic hydrogen transfer reaction (palladium hy-
droxide on carbon and cyclohexene in ethanol) to yield
the 3-C-hydroxymethyl derivative 5, which was treated
with tert-butyldiphenylsilyl chloride and imidazole in
DMF to give 1,2,5-tri-O-acetyl-3-deoxy-3-C-[(tert-butyl-
diphenyl)silyloxymethyl]-d-ribofuranose (6) which serves
Scheme 1. Reagents: (a) BnBr, NaH, n-Bu4NI, DMF, 0ꢀC. (b) i. 50% AcOH, 100ꢀC; ii. Ac2O, pyridine. (c) Pd(OH)2, cyclohexene, EtOH, 80 ꢀC. (d)
TBDPSCl, imidazole, DMF, rt.

L. S. Jeong et al. / Bioorg. Med. Chem. 7 (1999) 1467±1473
1469
Scheme 2. Reagents: (a) Silylated thymine, TMSOTf, CH2Cl2, rt to 50ꢀC. (b) BzCl, pyridine, 80ꢀC. (c) K2CO3, MeOH, 5 ꢀC. (d) BzCl, pyridine, 5 ꢀC.
(e) MEMCl, CH2Cl2, i-Pr2NEt. (f) NH3, MeOH, rt. (g) i. DMTCl, pyridine, rt; ii. n-Bu4NF, THF, rt. (h) (i-Pr)2NPCl(O)CH2CH2CN, i-Pr2NEt, rt.
(50-AGGGAGAGAAAG-30) under low (100 mM NaCl)
and high (1 M NaCl) ionic strength condition are sum-
marized in Table 1. The result of Tm studies with oligo-
mers A±C containing one to four modi®ed nucleosides
showed that introduction of 20-methoxyethoxymethyl
group to extended phosphate linkage decreased melting
temperature signi®cantly, indicating destabilization with
DNA ( 4ꢁ 8 ꢀC/mod.).
and reported to have lower anity to DNA and
RNA.22 When A±C dodecamers were compared to
these oligomers in hybridization property, the presence
of 20-OMEM substituent in 30-extended oligonucleotide
appeared to destabilize DNA duplex more. However,
the duplex with RNA showed the same degree of Tm
decrease. Therefore, it can be concluded that the pres-
ence of the 20-OMEM group interrupts the hybridiza-
tion with DNA because of the steric hindrance of the 30-
extended linkage. However, it has the positive e€ect
with RNA probably due to the electronegative e€ect of
substituents giving same degree of Tm decrease in com-
parison to oligonucleotides of 30-a-C-methylene phos-
phodiester linkage.
It was observed that the ionic strength of bu€er for Tm
measurement did not a€ect Tm changes since the same
degree of Tm drop was observed in 100 mM NaCl and 1
M NaCl bu€er solution. On the other hand, Tm was
less decreased with RNA complementary sequence
( 1ꢁ 2.5 ꢀC/mod.). The better anity of the modi®ed
oligonucleotides A±C for RNA target than DNA target
might be caused from the preference of 30-endo form (A
type duplex) due to the gauche e€ect19 between the fur-
anose ring oxygen and the electronegative 20-OMEM
group.
Conclusions
The introduction of the electronegative OMEM group
at the 20-position of the 30-extended oligonucleotide did
not give better hybridization property with DNA as well
as RNA than the corresponding natural oligomers.
Even though application of these oligomers to antisense
Recently, oligonucleotides (18-mer) containing a 30-a-C-
methylene phosphodiester linkage have been synthesized

1470
L. S. Jeong et al. / Bioorg. Med. Chem. 7 (1999) 1467±1473
Table 1. Tm, ÁTm, and ÁTm/mod. values of synthesized oligomers and natural oligomers
Condition
Oligomer
Sequence
Tm
ÁTm
ÁTm/mod.
DNA 1M NaCl
Natural
50-CTTTCTCTCCCT-30
50-CTTTCTCTCCCT-30
50-CTTTCTCTCCCT-30
50-CTTTCTCTCCCT-30
54.8
48.8
38.9
30.0
Ð
6
15.9
24.8
Ð
6
7.95
6.2
A
B
C
DNA 100 mM NaCl
RNA 100 mM NaCl
Natural
50-CTTTCTCTCCCT-30
50-CTTTCTCTCCCT-30
50-CTTTCTCTCCCT-30
50-CTTTCTCTCCCT-30
42.9
38.8
27.9
18.1
Ð
4.1
15
Ð
A
B
C
4.1
7.5
6.2
24.8
Natural
50-CTTTCTCTCCCT-30
50-CTTTCTCTCCCT-30
50-CTTTCTCTCCCT-30
50-CTTTCTCTCCCT-30
53.8
52.8
49.8
43.8
Ð
1
4
10
Ð
1
2
2.5
A
B
C
T indicates 30-deoxy-30-C-hydroxymethyl-20-O-methoxyethoxymethyl-5-methyluridine.
technology does not seem to be promising, they may
still ®nd their uses in other areas like protein studies.
1,2,5-Tri-O-acetyl-3-C-(benzyloxymethyl)-3-deoxy-D-ribo-
furanose (4). A solution of 3 (7.77 g, 0.02 mol) in 50%
AcOH (155 mL) was heated at 100 ꢀC for 1 h. The reac-
tion mixture was allowed to cool and evaporated to give
a syrup, which was treated with pyridine (28 mL) and
acetic anhydride (28 mL) and the solution was stirred at
room temperature for 48 h. The solvent was removed
under vacuum and the residue was dissolved in EtOAc
(200 mL). The organic layer was washed with H2O
(50 mL) and brine (50 mL), dried over anhydrous
MgSO4 and ®ltered. The ®ltrate was evaporated and the
residue was puri®ed by silica gel column chromato-
graphy (hexane:EtOAc=4:1) to give 4 (5.83 g, 81%, a/
b=1/9) as a syrup: 1H NMR (CDCl3) (b-anomer) d 2.01
(s, 3H, CH3), 2.06 (s, 3H, CH3), 2.07 (s, 3H, CH3), 2.65±
2.72 (m, 1H, 3-H), 3.51 (dd, 1H, J=6.8, 9.1 Hz, 3-CHa),
3.65 (dd, 1H, J=7.6, 9.1 Hz, 3-CHb), 4.10 (dd, 1H,
J=6.3, 11.4 Hz, 5-CHa), 4.23±4.29 (m, 1H, 4-H), 4.34
(dd, 1H, J=2.9, 11.4 Hz, 5-CHb), 4.45 (d, 1H, J=
11.5 Hz, CHa-Ph), 4.54 (d, 1H, J=11.5 Hz, CHb-Ph),
5.30 (d, 1H, J=4.6 Hz, 2-H), 6.09 (s, 1H, 1-H), 7.26±7.39
(m, 5H, Ph). Anal. calcd for C19H24O8: C, 59.99; H, 6.36.
Found: C, 59.59; H, 6.71.
Experimental
Ultra violet (UV) spectra were recorded on a Beckman
1
DU-68 spectrophotometer and H, 13C and 31P NMR
spectra were recorded on a Bruker DPX (250 or
300 MHz) spectrometer using CDCl3 or DMSO-d6 and
chemical shifts are reported in parts per million (d)
down®eld from tetramethylsilane as internal standard
for 1H and 13C NMR and from H3PO4 as external
standard for 31P NMR; coupling constants are in hertz.
FAB mass spectra were recorded on Jeol HX 110 spec-
trometer. Laser desorption mass spectra for modi®ed
oligonucleotides were obtained using Voyage MALDI
Workstation at Korea Basic Science Center (Taejon,
Korea). Elemental analyses were performed by the
general instrument laboratory of Ewha Womans Uni-
versity, Korea. TLC was performed on Merck pre-
coated 60F254 plates. Column chromatography was
performed using silica gel 60 (230±400 mesh, Merck).
All the anhydrous solvents were distilled over CaH2 or
P2O5 or Na/benzophenone prior to the reaction.
1,2,5-Tri-O-acetyl-3-deoxy-3-C-hydroxymethyl-D-ribo-
furanose (5). To a solution of 4 (4.42 g, 12 mmol) in
ethanol was added cyclohexene (50 mL) and palladium
hydroxide on carbon (1.52 g) and the reaction mixture
was heated at 80 ꢀC for 5 h. The solution was ®ltered
through a Celite and the ®ltrate was evaporated to give
a syrup, which was puri®ed by silica gel column chro-
matography (hexane:EtOAc=1:1) to yield 5 (3.09 g,
3-C-(Benzyloxymethyl)-3-deoxy-5-O-[(tert-butyldimethyl)-
silyl]-1,2-O-isopropylidene-ꢀ-D-ribofuranose (3). To a
solution of 221 (10.26 g, 0.03 mol) in DMF (40 mL) was
added sodium hydride (60% dispersion in mineral oil,
1.93 g, 0.05 mol) at 0 ꢀC and benzyl bromide (5.75 mL,
0.05 mol) and tetra-n-butylammonium iodide (1.18 g,
3.00 mmol) were successively added to the mixture.
After being stirred at room temperature for 3 h, the
reaction mixture was neutralized with acetic acid and
extracted with EtOAc (250 mL). The organic layer was
dried over anhydrous MgSO4 and evaporated to give a
syrup, which was puri®ed by silica gel column chroma-
tography (hexane:EtOAc=20:1) to a€ord 3 (7.77 g,
1
89%) as a syrup: H NMR (CDCl3) (b-anomer) d 2.14
(s, 3H, CH3), 2.15 (s, 3H, CH3), 2.17 (s, 3H, CH3), 2.43±
2.48 (m, 1H, 3-H), 3.64±3.86 (m, 2H, 3-CH2), 4.11±4.32
(m, 4H, OH, 4-H and 5-H), 5.35 (d, 1H, J=2.5 Hz, 2-
H), 6.08 (s, 1H, 1-H). Anal. calcd for C12H18O8: C,
49.65; H, 6.25. Found: C, 49.99; H, 6.70.
1
59%) as a syrup: H NMR (DMSO-d6) d 0.13 (s, 6H,
2ÂCH3), 0.97 (s, 9H, t-butyl), 1.35 (s, 3H, CH3), 1.48 (s,
3H, CH3), 2.12±2.19 (m, 1H, 3-H), 3.52±3.97 (m, 5H, 3-
CH2, 3-H, and 5-H), 4.46 (d, 1H, J=11.8 Hz, CHa-Ph),
4.56 (d, 1H, J=11.8 Hz, CHb-Ph), 4.76 (t, 1H,
J=4.1 Hz, 2-H), 5.83 (d, 1H, J=4.1 Hz, 1-H), 7.32±7.39
(m, 5H, Ph). Anal. calcd for C22H36O5Si: C, 64.67; H,
8.88. Found: C, 64.88; H, 8.90.
1,2,5-Tri-O-acetyl-3-C-[(tert-butyldiphenyl)silyloxymeth-
yl]-3-deoxy-D-ribofuranose (6). To a solution of 5
(3.09 g, 10.65 mmol) and imidazole (2.18 g, 32.01 mmol)
in DMF (30 mL) was added tert-butyldiphenylsi-
lylchloride (4.16 mL, 16 mmol). After being stirred at
room temperature for 2 h, the reaction mixture was
evaporated and extracted with EtOAc (150 mL). The

L. S. Jeong et al. / Bioorg. Med. Chem. 7 (1999) 1467±1473
1471
organic layer was washed with brine (50 mL), dried
(MgSO4) and evaporated. The residue was puri®ed by
silica gel column chromatography (hexane:EtOAc=
silica gel pad and then evaporated. The residue was
puri®ed by silica gel column chromatography (hexane:
EtOAc=2:1) to a€ord 9 (1.05 g, 74%): 1H NMR
(CDCl3) d 1.12 (s, 9H, t-butyl), 2.00 (s, 3H, 5-CH3), 2.12
(t, 1H, J=5.3 Hz, 50-OH, D2O exchangeable), 2.43±2.49
(m, 1H, 30-H), 3.17 (d, 1H, J=4.0 Hz, 20-OH, D2O
exchangeable), 3.68±3.76 (m, 1H, 50-Ha), 3.96 (d, 2H,
J=4.0 Hz, 30-CH2), 3.97±4.04 (m, 1H, 50-Hb), 4.32 (td,
1H, J=2.8, 9.3 Hz, 40-H), 4.48±4.52 (m, 1H, 20-H), 5.68
(d, 1H, J=1.9 Hz, 10-H), 7.44±7.96 (m, 16H, 3ÂPh and
H-6). Anal. calcd for C34H38 N2O7Si: C, 66.43; H, 6.23;
N, 4.56. Found: C, 66.77; H, 6.01; N, 4.43.
1
1.5:1) to give 6 (4.73 g, 84%). Major anomer: H NMR
(CDCl3) d 1.04 (s, 9H, t-butyl), 1.98 (s, 3H, CH3), 2.02
(s, 3H, CH3), 2.09 (s, 3H, CH3), 2.56±2.67 (m, 1H, 3-H),
3.64±3.86 (m, 3H, 3-CH2 and 4-H), 4.07±4.27 (m, 2H, 5-
H), 5.30 (d, 1H, J=4.9 Hz, 2-H), 6.08 (s, 1H, 1-H), 7.40±
7.68 (m, 10H, 2ÂPh). Anal. calcd for C28H36O8Si: C,
63.61; H, 6.86. Found: C, 63.97; H, 7.26.
1-[2,5-Di-O-acetyl-3-C-(tert-butyldiphenyl)silyloxymethyl-
3-deoxy-ꢁ-D-erythro-pentofuranosyl]thymine (7). A solu-
tion of thymine (0.83 g, 6.61 mmol) in 1,1,1,3,3,3-
hexamethyldisilazane (HMDS, 10 mL) and catalytic
amount of ammonium sulfate was re¯uxed overnight
and concentrated to dryness under nitrogen. To this
residue was added a solution of 6 (2.69 g, 5.09 mmol) in
CH2Cl2 followed by TMSOTf (1.38 mL, 6.61 mmol) and
the mixture was stirred overnight at room temperature
and heated at 50 ꢀC for 24 h. The reaction mixture was
diluted with saturated NaHCO3 solution (20 mL) and
extracted with CH2Cl2 (70 mL) and the organic phase
was washed with H2O (20 mL) and brine (20 mL), dried
(MgSO4) and evaporated. The residue was puri®ed by
silica gel column chromatography (CHCl3:MeOH=
1-[5-O-Benzoyl-3-C-(tert-butyldiphenyl)silyloxymethyl-3-
deoxy-ꢁ-D-erythro-pentofuranosyl]-N3-benzoylthymine
(10). To a solution of 9 (0.839 g, 1.37 mmol) in pyridine
(20 mL) was added benzoyl chloride (0.24 mL,
2.05 mmol) at 5 ꢀC and the mixture was stirred for
40 min. After MeOH (5 mL) was added to the mixture,
the whole mixture was stirred for 20 min. The solvent
was removed under vacuum. The residue was dissolved
in EtOAc (100 mL) and the organic layer was washed
with H2O (20 mL) and brine (20 mL), dried (MgSO4)
and ®ltered. The ®ltrate was evaporated and the residue
was puri®ed by silica gel column chromatography
(hexane:EtOAc=3:1) to give 10 (0.62 g, 63%) as a white
1
1
50:1) to give 7 (1.87 g, 62%) as a white foam: H NMR
foam: H NMR (CDCl3) d 1.09 (s, 9H, t-butyl), 1.72 (s,
(CDCl3) d 1.06 (s, 9H, t-butyl), 1.76 (s, 3H, 5-CH3), 2.01
(s, 3H, CH3), 2.10 (s, 3H, CH3), 2.52±3.01 (m, 1H, 30-
H), 3.71 (dd, 1H, J=6.0, 11.7 Hz, 30-CHa), 3.76 (dd, 1H,
J=5.6, 11.7 Hz, 30-CHb), 4.25±4.42 (m, 3H, 40-H and 50-
H), 5.36 (dd, 1H, J=3.8, 7.3 Hz, 20-H), 5.91 (d, 1H,
J=3.8 Hz, 10-H), 7.38±7.67 (m, 11H, 2ÂPh and H-6).
Anal. calcd for C31H37N2O8Si: C, 62.71; H, 6.28; N,
4.72. Found: C, 62.96; H, 6.26; N, 4.98.
3H, 5-CH3), 2.51±2.58 (m, 1H, 30-H), 3.17 (br s, 1H, 20-
OH), 4.01 (s, 1H, 30-CHa), 4.04 (s, 1H, 30-CHb), 4.46
(dd, 1H, J=4.1, 12.5 Hz, 50-Ha), 4.56 (dd, 1H, J=1.9,
6.1 Hz, 20-H), 4.59±4.65 (m, 1H, 40-H), 4.77 (dd, 1H,
J=2.1, 12.5 Hz, 50-Hb), 5.72 (d, 1H, J=1.9 Hz, 10-H),
7.40±8.04 (m, 21H, 4ÂPh and H-6). Anal. calcd for
C41H42 N2O8Si: C, 68.50; H, 5.89; N, 3.90. Found: C,
68.91; H, 6.03; N, 3.54.
1-[2,5-Di-O-acetyl-3-C-(tert-butyldiphenyl)silyloxymethyl-
3-deoxy-ꢁ-D-erythro-pentofuranosyl]-N3-benzoylthymine
(8). To a solution of 7 (1.87 g, 3.14 mmol) in pyridine
was added benzoyl chloride (1.46 mL, 12.57 mmol) and
the reaction mixture was stirred at 80 ꢀC overnight and
diluted with CH2Cl2 (40 mL). MeOH (5 mL) was added
to the mixture and stirred for 30 min. The solvent was
evaporated and the residue was extracted with EtOAc
(150 mL) and the organic layer was washed with H2O
(30 mL) and brine (30 mL), dried (MgSO4) and evapo-
rated. The residue was puri®ed by silica gel column
1-[5-O-Benzoyl-3-C-(tert-butyldiphenyl)silyloxymethyl-3-
deoxy-2-O-(2-methoxyethoxymethyl)-ꢁ-D-erythro-pento-
furanosyl]-N3-benzoylthymine (11). To a solution of 10
(0.70 g, 0.97 mmol) in CH2Cl2 (10 mL) added N,N-dii-
sopropylethylamine (0.51 mL, 2.92 mmol) and 2-meth-
oxyethoxymethyl chloride (0.33 mL, 2.92 mmol) and the
reaction mixture was stirred at 50 ꢀC for two days. The
solvent was removed under vacuum and the residue was
puri®ed by silica gel column chromatography (hexane:
EtOAc=2.5:1) to give 11 (0.586 g, 75%) as a white
1
foam: H NMR (CDCl3) d 1.06 (s, 9H, t-butyl), 1.67 (s,
chromatography (hexane:EtOAc=2.5:1) to give
8
3H, 5-CH3), 2.54±2.69 (m, 1H, 30-H), 3.28 (s, 3H,
OCH3), 3.37 (t, 2H, J=4.3 Hz, CH2OCH3), 3.45±3.61
(m, 2H, CH2CH2OCH3), 3.89 (dd, 1H, J=7.9, 10.7 Hz,
30-Ha), 4.03 (dd, 1H, J=5.3, 10.7 Hz, 30-Hb), 4.44 (dd,
1H, J=1.3, 5.9 Hz, 20-H), 4.52±4.61 (m, 2H, 40-H and
50-Ha), 4.72 (d, 1H, J=6.9 Hz, OCHaO), 4.88±4.93 (m,
2H, 50-Hb and OCHbO), 5.86 (d, 1H, J=1.3 Hz, 10-H),
7.39±8.08 (m, 21H, 4ÂPh and H-6). Anal. calcd for
C45H50 N2O10Si: C, 66.98; H, 6.25; N, 3.47. Found: C,
67.04; H, 6.35; N, 3.41.
(1.54 g, 70%): 1H NMR (CDCl3) d 1.06 (s, 9H, t-butyl),
1.95 (s, 3H, 5-CH3), 2.00 (s, 3H, CH3), 2.12 (s, 3H,
CH3), 2.53±2.64 (m, 1H, 30-H), 3.68±3.78 (m, 2H, 30-
CH2,), 4.24±4.43 (m, 3H, 40-H and 50-H), 5.36 (dd, 1H,
J=3.9, 7.3 Hz, 20-H), 5.94 (d, 1H, J=3.9 Hz, 10-H),
7.38±7.67 (m, 15H, 3ÂPh), 8.04 (s, 1H, H-6). Anal.
calcd for C38H42N2O9Si: C, 65.31; H, 6.06; N, 4.01.
Found: C, 65.71; H, 5.91; N, 4.28.
1-[3-C-(tert-Butyldiphenyl)silyloxymethyl-3-deoxy-ꢁ-D-
erythro-pentofuranosyl]-N3-benzoylthymine (9). To
solution of 8 (1.61 g 2.30 mmol) in MeOH (50 mL) was
added K2CO3 (1.27 g, 9.19 mmol) at 5 ꢀC and the
mixture was stirred at 5 ꢀC for 30 min. The mixture
was neutralized with acetic acid, ®ltered through a short
a
1-[3-C-(tert-Butyldiphenyl)silyloxymethyl-3-deoxy-2-O-
(2-methoxyethoxymethyl)-ꢁ-D-erythro-pentofuranosyl]-
thymine (12). A solution of 11 (0.58 g, 0.72 mmol)
in methanolic ammonia (50 mL) was stirred at room
temperature for two days. The solvent was evaporated

1472
L. S. Jeong et al. / Bioorg. Med. Chem. 7 (1999) 1467±1473
and the residue was puri®ed by silica gel column chro-
matography (hexane:EtOAc=1:1) to a€ord 12 (0.49 g,
100%): H NMR (CDCl3) d 1.11 (s, 9H, t-butyl), 1.96
3.30 (m, 2 H), 3.37 (s, 3H, OCH3), 3.38 (s, OCH3), 3.76±
3.50 (m, 2 H), 3.81 (s, 6H, OCH3), 4.24 (m, 2 H), 4.49
(m, 2 H), 4.87 (m, 2 H), 4.97 (m, 2 H), 6.00 (dd, 1H, 10-
H), 6.86±6.83 (m, 8 H), 7.45±7.23 (m, 18 H), 7.62 (s, 1H,
H-6), 7.69 (s, 1H, H-6), 8.19 (s, 1H, NH); 31P NMR
(CDCl3) d 147.1, 149.6; FAB-MS (m/e) 863 (M+H)+.
1
(s, 3H, 5-CH3), 2.32 (t, 1H, J=5.8 Hz, 50-OH, D2O
exchangeable), 2.44±2.53 (m, 1H, 30-H), 3.31 (s, 3H,
OCH3), 3.41 (t, 2H, J=4.5 Hz, CH2OCH3), 3.50±3.65
(m, 2H, CH2CH2OCH3), 3.79±4.09 (m, 4H, 30-CH2 and
50-H), 4.25 (td, 1H, J=2.8, 9.1 Hz, 40-H), 4.39 (dd, 1H,
J=2.1, 6.2 Hz, 20-H), 4.72 (d, 1H, J=6.9 Hz, OCHaO),
4.90 (d, 1H, J=6.9 Hz, OCHbO), 5.81 (d, 1H,
J=2.1 Hz, 10-H), 7.43±7.87 (m, 11H, 2ÂPh and H-6),
8.13 (br s, 1H, NH, D2O exchangeable). Anal. calcd for
C31H42 N2O8Si: C, 62.18; H, 7.07; N, 4.68. Found: C,
62.47; H, 7.09; N, 4.65.
Oligonucleotide synthesis and puri®cation
All oligonucleotides were synthesized using ABI 392
DNA/RNA synthesizer on a 1 mmol scale. For the
modi®ed phosphoramidite the coupling was extended to
10 min. The solid supports and protecting groups were
cleaved by the treatment of concentrated NH4OH at
55 ꢀC for 17 h. The solution was lyophilized with addi-
tion of triethylamine every hour to prevent detrityla-
tion. Triethylammonium acetate solution (TEAA
100 mM, 0.5 mLꢁ1 mL, pH 7) was added and the resi-
due was puri®ed by reverse phase HPLC (Hamilton
PRP-1, 300 mmÂ7 mm, 18ꢁ28% CH3CN/100 mM
TEAA in 20 min, pH 7, monitored 260 nM). The desired
fractions were lyophilized to dryness and water
(2Â1 mL) was added and lyophilized again to remove
TEAA salt. The treatment of remaining residue with
0.3 mL of AcOH for 20 min cleaved trityl groups. After
lyophilization with ethanol (0.3 mL), the residue was
taken up in 1 mL of H2O, extracted with diethyl ether
(3Â1 mL), and then lyophilized to dryness. After the
addition of 1 mL of water to the dry DNA pellet the
solution was quanti®ed by UV absorbance at 260 nM at
70 ꢀC. The extinction coecients of the natural nucleo-
side used for calculation were as follows: dAMP, 15200:
dCMP, 7700: TMP, 8830: dGMP, 11500. The extinction
coecients of 20-methoxyethoxymethylthymidine at
260 nM was presumed same as TMP. The composition
of oligonucleotides was con®rmed by enzyme hydrolysis
followed by HPLC analysis (Hewlett±Packard, ODS
hypersil, C-18; 20 mM K2HPO4, pH 5.6 (A), MeOH (B),
100% A to 40% B, 20 min) and mass spectrometric
data. The purity of the oligomers A±C was found to be
more than 99%. RNA (50-AGGGAGAGAAAG-30)
was purchased from the Midland Certi®ed Reagent
Company (Midland, TX, USA) on 1 mmol scale.
1-[3-Deoxy-3-C-hydroxymethyl-2-O-(2-methoxyethoxy-
methyl)-5-O-(4,40-dimethoxytriphenylmethyl)-ꢁ-D-erythro-
pentofuranosyl]thymine (13). A solution of 12 (0.485 g,
0.81 mmol) and 4,40-dimethoxytrityl chloride (0.550 g,
1.22 mmol) in pyridine (15 mL) was stirred at room
temperature for 24 h. MeOH (1 mL) was added to the
mixture and stirred for 30 min. The solvent was
removed under reduced pressure. The residue was dis-
solved in EtOAc (100 mL). The organic layer was
washed with H2O (15 mL) and brine (15 mL), dried
(MgSO4) and ®ltered. The ®ltrate was evaporated to
give the residue, which was treated with tetra-n-butyl-
ammonium ¯uoride (1.0
M
solution, 0.97 mL,
0.97 mmol) in THF (10 mL) at 0 ꢀC. After being stirred
at 0 ꢀC for 1 h, the reaction mixture was stirred at room
temperature for 3 h. The solvent was removed under
reduced pressure and the residue was puri®ed by silica
gel column chromatography (hexane:EtOAc=1:1 to
CHCl3:MeOH=30:1) to give 13 (0.48 g, 89%) as a pale
1
yellow foam: H NMR (CDCl3) d 1.42 (s, 3H, 5-CH3),
2.68±2.79 (m, 1H, 30-H), 3.25 (dd, 1H, J=3.0, 11.1 Hz,
50-Ha), 3.42±3.46 (m, 4H, OCH3 and OH), 3.49±3.91 (m,
13H, 30-CH2, 2ÂOCH3, CH2CH2 and 50-Hb), 4.21±4.26
(m, 1H, 40-H), 4.52 (d, 1H, J=5.3 Hz, 20-H), 4.85 (d,
1H, J=6.7 Hz, OCHaO), 5.23 (d, 1H, J=6.7 Hz,
OCHbO), 5.86 (s, 1H, 10-H), 6.85±7.83 (m, 14H, C6H5,
2ÂC6H4 and H-6), 8.41 (br s, 1H, 3-NH, D2O
exchangeable). Anal. calcd for C36H42 N2O10: C, 65.24;
H, 6.39; N, 4.23. Found: C, 65.64; H, 6.64; N, 4.63.
Laser desorption mass spectral data for oligonucleotides
A±C
1-[3-Deoxy-5-O-dimethoxytrityl-3-C-hydroxymethyl-2-
O-(2-methoxyethoxymethyl)-ꢁ-D-erythro-pentofuranosyl]-
thymine 2-cyanoethyl N,N-diisopropylphosphor amidite
(14). A solution of 13 (0.23 g, 0.35 mmol) in freshly dis-
tilled dichloromethane (5 mL) containing N,N-diiso-
propylchlorophosphoramidite (160 mL, 0.7 mmol) and
N,N-diisopropylethylamine (183 mL, 1.05 mmol) was
stirred at room temperature for 2 h under nitrogen.
Saturated NaHCO3 solution (20 mL) was added to the
reaction mixture. The organic layer was separated and
washed with brine (10 mL) and water (10 mL), dried
over anhydrous Na2SO4 and evaporated. The residue
was puri®ed by silica gel column chromatography
(Et3N(0.5%)/EtOAc (25%)/CH2Cl2) to give 14 (0.18 g,
50-d(CTTTCTCTCCCT)-30 (A): calculated mass, 3615;
observed mass, 3619: 50-d(CTTTCTCTCCCT)-30 (B):
calculated mass, 3734; observed mass, 3737: 50-
d(CTTTCTCTCCCT)-30 (C): calculated mass, 3972;
observed mass, 3974.
Tm measurement
Melting temperatures were determined using Beckmann
DU 650 spectrometer equipped with Beckmann High
Performance Temperature Controller. Nitrogen gas was
passed over the cell at lower temperature than room
temperature to prevent the condensation of moisture.
The temperature was increased from 5 ꢀC to 90 ꢀC in
1 ꢀC steps (1 ꢀC/min). The modi®ed oligonucleotides
were hybridized with complementary DNA or RNA
1
61%) as a diastereomeric mixture: H NMR (CDCl3) d
1.11 (d, 3H, NHCHCH3), 1.13 (d, 3H, NHCHCH3),
1.28 (d, 3H, NHCHCH3), 1.30 (d, 3H, NHCHCH3),),
1.40 (s, 3H, 5-CH3), 1.42 (s, 3H, 5-CH3), 2.77 (m, 2 H),

L. S. Jeong et al. / Bioorg. Med. Chem. 7 (1999) 1467±1473
1473
sequences in a medium salt bu€er containing 100 mM
NaCl or 1 M NaCl, 10 mM sodium phosphate, 0.1 mM
EDTA, pH 7.0 and 2.5 mM of each oligonucleotide.
Melting temperatures were evaluated by taking the ®rst
derivative of the absorbance versus temperature curve.
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