TT dinucleotides containing an isoxazoline moiety: Synthesis and binding affinity study

Article abstract of DOI:10.1081/NCN-100107187

We have prepared four diastereomers of TT dinucleotides containing an isoxazoline moiety and four dodecanucleotides incorporating these TT dinucleotides in the middle of the DNA sequence. We also have determined the melting temperatures of these modified oligonucleotides (Tm values) by measuring change in UV absorbance.

Full text of DOI:10.1081/NCN-100107187

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TT DINUCLEOTIDES CONTAINING AN ISOXAZOLINE
MOIETY: SYNTHESIS AND BINDING AFFINITY STUDY
Jong Rock Kong a , Sang Kook Kim a , Byung Jo Moon b , Su Jeong Kim a & Byeang Hyean Kim c
a Department of Chemistry , Center for Integrated Molecular Systems, Pohang University of
Science and Technology , Pohang, 790-784, Korea
b Department of Biochemistry , College of Natural Sciences, Kyungpook National University ,
Taegu, 702-701, Korea
c Department of Chemistry , Center for Integrated Molecular Systems, Pohang University of
Science and Technology , Pohang, 790-784, Korea
Published online: 19 Aug 2006.
To cite this article: Jong Rock Kong , Sang Kook Kim , Byung Jo Moon , Su Jeong Kim & Byeang Hyean Kim (2001) TT
DINUCLEOTIDES CONTAINING AN ISOXAZOLINE MOIETY: SYNTHESIS AND BINDING AFFINITY STUDY, Nucleosides, Nucleotides and
Nucleic Acids, 20:10-11, 1751-1760, DOI: 10.1081/NCN-100107187
To link to this article: http://dx.doi.org/10.1081/NCN-100107187
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NUCLEOSIDES, NUCLEOTIDES & NUCLEIC ACIDS, 20(10&11), 175171760 (2001)
TT DINUCLEOTIDES CONTAINING
AN ISOXAZOLINE MOIETY: SYNTHESIS
AND BINDING AFFINITY STUDY
Jong Rock Kong,1 Sang Kook Kim,1 Byung Jo Moon,2
Su Jeong Kim,1 and Byeang Hyean Kim1,*
1Department of Chemistry, Center for Integrated Molecular
Systems, Pohang University of Science and Technology,
Pohang 7907784, Korea
2Department of Biochemistry, College of Natural Sciences,
Kyungpook National University, Taegu 7027701, Korea
ABSTRACT
We have prepared four diastereomers of TT dinucleotides containing an
isoxazoline moiety and four dodecanucleotides incorporating these TT
dinucleotides in the middle of the DNA sequence. We also have de-
termined the melting temperatures of these modified oligonucleotides
(Tm values) by measuring change in UV absorbance.
INTRODUCTION
Synthesis of modified oligonucleotides is very important especially in
the field of antisense and antigene based drug discovery1. We have been in-
terested in the replacement of the phosphodiester linkages of the natural
oligonucleotides with novel, neutral, and heterocyclic linkages based on their
stability and rigidity. Recently we have reported the design and synthesis of
the isoxazoline linkage for replacement of nucleotide phosphodiester2. We
further developed and elaborated the idea for the modification of the natural
*Corresponding author.
1751
Copyright # 2001 by Marcel Dekker, Inc.
www.dekker.com

1752
KONG ET AL.
phosphodiester linkage and report here a facile synthesis of TT dinucleotides
with modified linkages and determination of the Tm values of DNA dode-
canucleotides with modified dinucleotides linkages.
RESULTS AND DISCUSSION
Synthetic routes for four diastereomers are summarized in Scheme 1.
Selective protection of 50-hydroxyl group of thymidine with 4,40-dimethoxy-
Scheme 1.

TT DINUCLEOTIDES
1753
trityl (DMTr) group followed by p-tolylthionocarbonation of 30-hydroxyl
group of thymidine with p-tolylchlorothionoformate provided compound 2 in
good yield. Radical allylation with allyltributyltin and AIBN afforded the
desired dipolarophiles 33. Nitrile oxide cycloaddition between the precursor
42 and the dipolarophile 3 gave a 1:1 diastereomeric mixture of TT dinu-
cleotide 5. We isolated the less polar diastereomer 5a (Rf ¼ 0.30 in the eluent
of ethyl acetate:hexane ¼ 2:1) in 31% yield and the more polar one 5b (Rf ¼
0.26 in the eluent of ethyl acetate:hexane ¼ 2:1) in 30% yield. At this moment
we cannot determine the absolute configuration of the isoxazoline stereogenic
center in each diastereomer decisively and tentatively assign both diaster-
eomers based on their polarity.
TT dinucleotide diastereomers with sulfur heteroatom were synthesized
by using a cycloaddition with the sulfur containing dipolarophile 10. DMTr
protection of 50-hydroxyl group of thymidine followed by mesylation of
30-hydroxyl group provided compound 6. Treatment of compound 6 with
LiOH Á H2O and another mesylation afforded compound 8 in 77% overall
yield. Three-step sequence (displacement with mercaptoethanol, mesylation,
and elimination) of 8 gave the desired dipolarophile 10. Nitrile oxide
cycloaddition between the precursor 42 and the dipolarophile 10 provided a
3:1 diastereomeric mixture of TT dinucleotide 11. We isolated by column
chromatography the less polar compound 11a (Rf ¼ 0.25 in the eluent of
ethyl acetate:hexane ¼ 2:1) as the major product in 59% yield and the more
polar one 11b (Rf ¼ 0.23 in the eluent of ethyl acetate:hexane ¼ 2:1) in 20%
yield.
With four diastereomers (5a, 5b, 11a, and 11b) in hand, we next carried
out measurement of the Tm values of the DNA dodecanucleotides containing
these dinucleotidic fragments. We prepared 2-cyanoethyl phosphoramidite
building blocks of TT dinucleosides and directly applied them to solid phase
oligonucleotide synthesis protocols4 on the ABI 391 DNA synthesizer. The
sequences and Tm values of DNA dodecanucleotides are described in Table 1,
and Fig. 1 shows the typical curves of relative absorbance for Tm value
determination.
Compared to unmodified duplex, incorporation of one isoxazoline
backbone (5a) little decreased the duplex stability by a DTm of À3.3 ꢀC, and
another type of duplex with 5b more decreased (À16.5 ꢀC) duplex stability.
On the other hand, the duplexes containing both the higher and lower Rf’s
sulfur-isoxazoline backbone slightly more decreased (À5.5 ꢀC and À5.0 ꢀC)
than the duplex containing higher Rf’s isoxazoline backbone (5a). Substi-
tution of a phosphodiester linkage with isoxazoline or sulfur-isoxazoline
backbone reduced the contribution of the base hydrogen bonding of the
oligonucleotides slightly. These results suggest that the isoxazoline (5a and
5b) and sulfur-isoxazoline (11a) backbone modified nucleotides can be useful
antisense oligonucleotides candidates for antiviral agents. Furthermore more
substitutions with these TT dinucleotides containing an isoxazoline moiety

1754
KONG ET AL.
Table 1. Tm Values of Dodecanucleotides Containing One Isoxazoline and Sulfur-isoxazo-
line Backbone
Oligo name
Sequence
Tm (ꢀC)a
DTm (ꢀC)
A12
50–AAAAAAAAAAAA–30
50–TTTTTTTTTTTT–30
50–TTTTTTTTTTTT–30
50–TTTTTTTTTTTT–30
50–TTTTTTTTTTTT–30
50–TTTTTTTTTTTT–30
50–TTTTTTTTTTTT–30
T12b
38.1
34.8
21.6
38.5
33.0
33.5
0
À3.3
À16.5
0
T12-(5a)b
T12-(5b)b
T12c
T12-(11a)c
T12-(11b)c
À5.5
À5.0
a
Melting temperatures were determined by measuring change in absorbance at 260 nm
(cuvette, 1 cm path length) as a function of temperature in Tris–HCl buffer (10 mM, pH 7.0)
containing 100 mM NaCl and 20 mM MgCl2. Temperature was raised 0.5 ꢀC=min. All the
values are averaged from at least three experiments.
b
Total strands concentration was 2.5 mM.
Total strands concentration was 3.0 mM.
c
may provide positive hybridization data and RNA hybridization may afford
positive Tm changes.
EXPERIMENTAL
1H NMR and 13C NMR spectra were obtained on FT-300 Mhz Bruker
Apect 3000 spectrometer. The molecular mass was determined by using Jeol
JMS-AX505WA (FAB). The IR spectra were obtained on Bruker FT-IR
Figure 1. Thermal denaturation curves of duplex oligonucleotides.

TT DINUCLEOTIDES
1755
PS55þ and melting point was determined by Electrothermal 1A9000
instrument. Silica gel column chromatography was performed on Merck,
Kiesel gel 60 (707230 mesh).
50-O-(4,40-Dimethoxytrityl)-30-O-(4-methylphenoxythiocarbonyl)thymidine (2)
A solution of 1 (1.1 g, 1.95 mmol) in dichloromethane (20 mL) was
cooled in an ice-water bath and p-tolylchlorothionoformate (318 mL,
2.11 mmol) was added. After the reaction mixture was stirred for 5 min,
triethylamine (326 mL, 2.34 mmol) and 4-dimethylaminopyridine (286 mg,
2.34 mmol) were added. The resulting reaction mixture was allowed to warm
to room temperature and then stirred for 2 h. Distilled water (30 mL) was
added and the organic layer was separated off. The aqueous layer was ex-
tracted two times with dichloromethane (20 ml). The organic layer was dried
over MgSO4 and evaporated in vacuo. The residue was purified by silica gel
column chromatography to give 2 as a white solid (1.14 g, 1.64 mmol, 84%):
Rf ¼ 0.85 (EA:MC ¼ 1:2); IR, n(CHCl3, cmÀ1) 3182, 3059, 2931, 2836, 1694,
1
1508, 1252, 1224, 1193; H NMR(CDCl3) 10.03(br, 1H, NH), 7.69(d, 1H,
J ¼ 0.8 Hz, C6-H), 7.43(d, 2H, J ¼ 7.2 Hz, phH), 7.3477.18(m, 10H, phH),
6.97(d, 2H, J ¼ 8.5 Hz, phH), 6.84(d, 4H, J ¼ 8.2 Hz, phH), 6.55(m, 1H, H10),
6.01(m, 1H, H30), 4.44(s, 1H, H40), 3.75(s, 6H, OMe), 3.6873.48(2 m, H50),
2.35(m, 2H, H20), 2.34(s, 3H, phMe), 1.49(s, 3H, C5-Me); 13C NMR(CDCl3)
195.0, 164.7, 159.2, 159.2, 151.7, 151.2, 144.7, 137.0, 135.7, 135.6, 130.6,
130.5, 128.5, 127.6, 121.8, 113.8, 112.2, 87.8, 85.0, 84.8, 84.3, 78.1, 77.6, 77.3,
20
55.7, 21.5, 21.4, 14.6, 12.2; MS(FABþ, m=z) 694.2(Mþ); [a]D ¼ À3.43(c 1.0,
CH2Cl2); mp 101.57103.9 ꢀC.
50-O-(4,40-Dimethoxytrityl)-30-C-allyl-30-deoxy-thymidine (3)
A mixture of 2 (2.21 g, 3.21 mmole), allyltributyltin (5 mL, 16.0 mmole),
and AIBN (102 mg, 62 mmole) in toluene (32 mL) was refluxed for 15 h.
The reaction mixture was concentrated invacuo. The residue was purified by
silica gel column chromatography to give 3 as a white solid (1.19g,
2.09mmol, 65%): R f ¼ 0.80 (Hex:EA ¼ 1:2); IR, n(CHCl3, cmÀ1 ) 3182.5,
3058.7, 2930.6, 2835.9, 1693.8, 1507.9, 1251.6, 1224.0, 1192.6; 1H
NMR(CDCl3) 10.19(br, 1H, NH), 7.76(s, 1H, C6-H), 7.4777.21(m, 9H,
phH), 6.84(d, 4H, J ¼ 8.46 Hz, phH), 6.09(t, 1H, J ¼ 3.45 Hz, H10), 5.73(m,
1H, CH ¼ CH2), 5.06(dd, 2H, J ¼ 35 Hz, 13.1 Hz, CH ¼ CH2), 3.86(m, 1H,
H40), 3.75(s, 6H, OMe), 3.59(m, 1H, H50), 3.26(m, 1H, H50), 2.5572.47(m,
1H, H20), 2.3372.25(m, 1H, H20), 2.2072.03(m, 2H, CH2), 1.51(s, 3H, C5-
Me); 13C NMR(CDCl3) 1164.74, 158.08, 150.99, 144.85, 136.22, 135.98,
135.67, 130.74, 130.54, 128.94, 128.62, 128.34, 127.45, 117.44, 113.63, 110.76,
86.95, 85.58, 85.50, 55.63, 37.60, 36.32, 21.41, 14.60, 12.45; MS(FABþ, m=z)
568.43(Mþ); [a]D ¼ þ8.7 (c 1.0, CH2Cl2); mp 78.4779.0 ꢀC; Anal. Calcd. for
23
C34H36O6N2: C, 71.81; H, 6.38; N, 4.93. Found: C, 72.18; H, 6.53; N, 4.69.

1756
KONG ET AL.
TT Dinucleotide 5
To a solution of 3 (370 mg, 0.67 mmol) and 4 (490 mg, 1.33 mmol) in
dichloromethane (12 mL) was added NaOCl (1.8 mL, 4% aqueous solution)
over a period of 48 hours. Methyl sulfide (200 mL) was added and then the
reaction mixture was stirred for 1 hours. Distilled water (30 mL) was added
and the organic layer was separated off. The organic residue was dried over
MgSO4 and evaporated in vacuo. The residue was purified by silica gel col-
umn chromatography to give 5a and 5b as a white solid (5a ¼ 31%;
5b ¼ 30%); 5a: Rf ¼ 0.30 (EA:Hex ¼ 2:1); IR, n(neat, cmÀ 1) 3188, 3059,
2953, 2930, 2856, 1691, 1509, 1469, 1253, 835; 1H NMR (300 MHz, Acetone-
d6) d 10.28710.24 (2s, 2H, NH), 7.72 (s, 1H, C6-H), 7.59(s, 1H, C6-H),
7.5277.23 (m, 9H, phH), 6.89 (d, 4H, J ¼ 7.15 Hz, phH), 6.32 (d, 1H, H10),
6.11 (d, 1H, H10), 4.82 (s, 1H, isoxazoline-H), 4.59(s, 2H, isoxazoline-CH 2),
3.90 (d, 1H, J ¼ 8.0 Hz, H40), 3.77 (s, 6H, OMe), 3.56 (d, 1H, J ¼ 9.75 Hz,
H40), 3.33 (m, 1H, H30), 3.21 (m, 1H, H50), 2.46 (m, 2H, H20), 2.32 (2 m, 2H,
H20), 1.80 (s, 3H, C5-Me), 1.70 (m, 2H, CH2), 1.54 (s, 3H, C5-Me), 0.91 (s,
9H, tert-Bu), 0.12 (d, 6H, J ¼ 5.55 Hz, 2Me; 13C NMR (75.5 MHz, acetone-
d6) d 164.3, 164.0, 159.2, 157.7, 150.9, 145.5, 137.2, 136.3, 136.2, 136.1, 130.6,
128.6, 128.3, 127.3, 113.5, 110.7, 110.0, 86.7, 85.3, 82.7, 80.3, 74.1, 63.5, 55.1,
40.6, 39.6, 39.5, 37.7, 36.1, 30.3, 30.0, 29.7, 29.5, 29.2, 29.0, 28.7, 25.7, 18.0,
16
12.3, 12.1, À 5.0; MS (FAB, m=z) 958.29 (MþþNa); [a]D ¼ À 3.1 (c ¼ 0.96,
CH2Cl2); mp: 123.67124.2 ꢀC; 5b: Rf ¼ 0.26 (EA:Hex ¼ 2:1); IR, n(neat,
1
cmÀ1) 3201, 3061, 2953, 2930, 2857, 1690, 1509, 1469, 1253, 836; H NMR
(300 MHz, acetone-d6) d 10.22, 10.17 (2s, 2H, NH), 7.69(s, 1H, C6-H),
7.5077.19(m, 10H, phH þ C6-H), 6.86 (d, 4H, J ¼ 8.5 Hz, phH), 6.29(m,
1H, H10), 6.06 (d, 1H, J ¼ 4.6 Hz, H10), 4.80 (m, 1H, H40), 4.60 (m, 1H,
isoxazoline-H), 4.59(m, 1H, H4 0), 3.74 (m, 1H, H30), 3.34 (m, 1H, H50), 3.31
(m, 2H, H50þH30), 2.74 (m, 2H, isoxazoline CH2), 2.43 (m, 2H, H20),
2.3572.21 (2 m, 2H, H20), 1.76 (s, 3H, C5-Me), 1.51 (s, 3H, C5-Me), 0.88 (s,
9H, tert-Bu), 0.08 (d, 6H, 2Me); 13C NMR (75.7 MHz, acetone-d6) d 164.3,
164.1, 159.2, 159.2, 157.8, 151.9, 145.45, 137.1, 136.2, 136.1, 136.0, 130.5,
128.7, 128.3, 127.2, 126.3, 113.6, 110.8, 110.5, 109.9, 109.1, 86.8, 86.4, 85.4,
85.3, 82.5, 79.9, 78.8, 78.5, 78.1, 63.1, 55.1, 40.2, 39.6, 40.0, 37.7, 35.2, 28.9,
28.7, 22.9, 12.1, 12.1, À 5.1; MS (FAB, m=z) 958.20 (MþþNa);
16
[a]D ¼ þ52.6 (c ¼ 1.01, CH2Cl2); mp: 114.17114.6 ꢀC.
50-O-(4,40-Dimethoxytrityl)-30-O-mesyl-30-deoxy-thymidine (6)
A solution of 1 (3.0 g, 5.31 mmol) in pyridine (70 mL) was cooled in an
ice-water bath and methane sulfonylchloride (1.23 mL, 15.9mmol) was ad-
ded. After the reaction mixture was stirred for 5 h at 0ꢀ, 5% NaHCO3
aqueous solution (100 ml) was added and extracted with dichloromethane.
The organic residue was dried over MgSO4 and evaporated in vacuo. The

TT DINUCLEOTIDES
1757
residue was purified by silica gel column chromatography to give 6 as a white
solid (3.10 g, 4.99 mmol, 94%): Rf ¼ 0.55 (EA:MC=1:1); IR, n(CHCl3,
1
cmÀ 1) 3186, 3058, 2934, 2837, 1692, 1509, 1252, 1110; H NMR(aceton-d6)
10.24(br, 1H, NH), 7.60(s, 1H, C6-H), 7.5177.25(2 m, 9H, phH), 6.90(d, 4H,
J ¼ 8.5 Hz, phH), 6.37(m, 1H, H10), 5.51(s, 1H, H30), 4.34(s, 1H, H40), 3.77(s,
6H, OMe), 3.49(m, 2H, H50), 3.16(s, 3H, SO2CH3), 2.69(m, 2H, H20), 1.49(s,
9H, C5-Me); 13C NMR(Aceton-d6) 163.92, 159.31, 150.87, 145.20, 135.89,
130.56, 128.36, 127.41, 113.61, 110.10, 87.36, 84.75, 83.99, 81.17, 63.52,
55.13, 38.13, 37.10, 30.26, 30.00, 29.74, 29.49, 29.23, 28.98, 28.72, 11.72;
20
MS(FABþ, m=z) 622.0(Mþ); [a]D ¼ þ15.0 (c 1.0, CH2Cl2); mp
81.8782.2 ꢀC.
1-(20-Deoxy-50-O-4,40-dimethoxytrityl-b-D-threo-penosyl)thymidine (7)
To a solution of 6 (2.0 g, 3.23 mmol) in ethanol (20 mL) and distilled
water (12 mL) was added LiOH Á H2O (406 mg, 9.69 mmole). After the reac-
tion mixture was refluxed for 3 h, the ethanol was evaporated and extracted
with dichloromethane. The organic residue was dried over MgSO4 and
evaporated in vacuo. The residue was purified by silica gel column chroma-
tography to give 7 as a white solid (1.47 g, 2.71 mmol, 84%): Rf ¼ 0.45
(EA:MC ¼ 1:1); IR, n(CHCl3, cmÀ 1) 360073300, 3165, 3036, 2933, 2836,
1
1697, 1509, 1251, 1069; H NMR(aceton-d6) 10.36(br, 1H, NH), 7.75(s, 1H,
C6-H), 7.5577.19(3 m, 9H, phH), 6.86(d, 4H, J ¼ 8.7 Hz, phH), 6.20(d,
J ¼ 6.7 Hz, 1H, H10), 4.47(s, 1H, OH), 4.41(m, 1H, H30), 4.17(m, 1H, H40),
3.73(s, 6H, OMe), 3.62(t, 1H, J ¼ 8.7 Hz, H50), 3.42(m, 1H, H50), 2.62(m, 1H,
H20), 2.08(m, 1H, H20), 1.72(s, 3H, C5-Me); 13C NMR(aceton-d6) 164.7,
160.0, 151.2, 145.8, 137.98, 136.4, 136.4, 130.6, 128.6, 128.2, 121.1, 113.5,
109.4, 86.1, 85.6, 84.3, 70.5, 63.3, 55.2, 41.6, 12.5; MS(FABþ, m=z)
20
544.1(Mþ); [a]D ¼ À22.6 (c 1.0, CH2Cl2); mp 82.2782.6 ꢀC.
1-(20-Deoxy-30-O-mesyl-50-O-4,40-dimethoxytrityl-b-D-lyxosyl)thymidine (8)
A solution of 7 (9.0 g, 15.9 mmol) in pyridine (160 mL) was cooled in an
ice-water bath and methane sulfonylchloride (3.7 mL, 47.9mmol) was added.
After the reaction mixture was stirred for 8 h at 0ꢀ, 5% NaHCO3 aqueous
solution was added and extracted with dichloromethane. The organic residue
was dried over MgSO4 and evaporated invacuo. The residue was purified by
silica gel column chromatography to give 6 as a white solid (9.10 g,
14.6 mmol, 92%): Rf ¼ 0.55 (EA:MC=1:1); IR, n(CHCl3, cmÀ 1) 3181, 3054,
3034, 2934, 2837, 2551, 1698, 1508, 1250, 1142; 1H NMR(aceton-d6)
10.07(br, 1H, NH), 8.54(t, 1H, J ¼ 2.7 Hz, C6-H), 7.3977.22(m, 9H, phH),
6.87(d, 4H, J ¼ 8.9Hz, phH), 6.27(t, 1H, J ¼ 4.0 Hz, H10), 5.40(d, 1H,
J ¼ 3.8 Hz, H30), 4.42(t, 1H, J ¼ 1.7 Hz, H40), 3.76(s, 6H, OMe), 3.58(m, 1H,

1758
KONG ET AL.
H50), 3.37(m, 1H, H50), 3.00(s, 3H, SO2Me), 2.99(m, 1H, H20), 2.45(m, 1H,
H20), 1.70(s, 3H, C5-Me); 13C NMR(Aceton-d6) 163.4, 163.4, 158.9, 150.6,
150.5, 149.8, 135.8, 135.7, 135.4, 130.2, 128.2, 127.9, 113.2, 113.2, 110.0, 86.7,
23
83.8, 81.3, 79.6, 61.7, 54.7, 39.2, 11.9; MS(FABþ, m=z) 622.0(Mþ); [a]D
¼
À31.5 (c 1.0, CH2Cl2); mp 85.4786.7 ꢀC.
30-Deoxy-30-S-(2-hydroxyethylthio)-50-O-(4,40-dimethoxytrityl)thymidine (9)
A solution of 8 (9.08 g, 14.6 mmole) in DMF (40 mL) was heated at
700 ꢀC and a solution of DBU (3.5 mL, 25.4 mmole) and mercaptoethanol
(7.2 mL, 101.8 mmole) in DMF (100 mL) was added. After the reaction
mixture was stirred for 3 h at 70 ꢀC, distilled water (100 mL) was added and
the reaction mixture was extracted with dichloromethane. The organic re-
sidue was dried over MgSO4 and evaporated in vacuo. The residue was
purified by silica gel column chromatography to give 10 as a white solid
(6.53 g, 10.8 mmol, 74%): Rf ¼ 0.30 (EA:MC ¼ 1:1); IR, n(CHCl3, cmÀ1
)
375073300, 3182, 3056, 2931, 1688, 1252, 1035; 1H NMR(aceton-d6)
10.31(br, 1H, NH), 7.78(s, 1H, C6-H), 7.5677.22(2 m, 9H, phH), 6.91(d, 4H,
0
J ¼ 8.7 Hz, phH), 6.20(dd, 1H, J ¼ 6.9Hz, 6.9Hz, H1 ), 3.99(m, 2H, H30-
þH40), 3.70(s, 6H, OMe), 3.58(m, 2H, CH2), 3.56(m, 1H, H50), 3.41(m, 1H,
H50), 2.70(m, 2Hþ1H, CH2þH20), 2.44(m, 1H, H20), 1.29(s, 3H, C6-Me); 13C
NMR(aceton-d6) 164.2, 159.3, 150.9, 145.4, 136.3, 136.1, 130.6, 128.6, 128.3,
127.3, 113.6, 110.2, 86.8, 85.5, 84.9, 63.0, 62.2, 55.1, 41.8, 40.6, 34.3, 31.8,
20
12.0; MS(FABþ, m=z) 604.09(Mþ); [a]D ¼ þ22.7 (c 1.0, CH2Cl2); mp
77.4778.1 ꢀC; Anal. Calcd. for C33H36O7N2S: C, 65.55; H, 6.00; N, 4.63.
Found: C, 65.50; H, 6.12; N, 4.52.
20-Deoxy-30-S-(1-vinylsulfinyl)-50-O-(4,40-dimethoxytrityl)thymidine (10)
A solution of 9 (840 mg, 1.38 mmol) in pyridine (26 mL) was cooled in
an ice-water bath and methane sulfonylchloride (322 mL, 4.16 mmol) was
added. After the reaction mixture was stirred for 8 h at 0 ꢀC, 5% NaHCO3
aqueous solution was added and extracted with dichloromethane. The or-
ganic residue was dried over MgSO4 and evaporated in vacuo. The residue
t
was dissolved in dry THF (26 mL) and cooled at 0 ꢀC. BuOK (467 mg,
4.16 mmol) was added and stirred for 10 min at 0 ꢀC. Distilled water (30 mL)
was added and extracted with dichloromethane. The organic residue was
dried over MgSO4 and evaporated invacuo. The residue was purified by silica
gel column chromatography to give 10 as a white solid (679mg, 1.16 mmol,
84%): Rf ¼ 0.70 (EA:MC ¼ 1:1); IR, n(CHCl3, cmÀ 1) 3182, 3056, 2930, 2885,
1689, 1509, 1001; 1H NMR(aceton-d6) 10.17(br, 1H, NH), 7.77(s, 1H, C6-H),
7.7777.24(2 m, 9H, phH), 6.89(d, 4H, J ¼ 9.0 Hz, phH), 6.39(m, 1H, CH¼
CH2), 6.22(m, 1H, H10), 5.18(m, 2H, CH¼CH2), 4.00(m, 2H, H30þH40),

TT DINUCLEOTIDES
1759
3.78(s, 6H, OMe), 3.56(m, 1H, H50), 3.36(d, 1H, J ¼ 10.8 Hz, H50),
2.7072.44(2 m, 2H, H20), 1.54(s, 3H, C5-Me); 13C NMR(aceton-d6) 174.8,
159.2, 151.0, 144.6, 135.9, 135.7, 135.7, 130.2, 128.6, 128.4, 127.6, 115.6,
113.7, 113.7, 113.4, 87.2, 86.0, 85.1, 78.0, 77.8, 77.6, 77.2, 62.5, 55.7, 41.5,
23
40.0, 14.6, 12.4; MS(FABþ, m=z) 586.06(Mþ); [a]D ¼ þ8.7 (c 0.1, CH2Cl2);
mp 73.2773.7 ꢀC; Anal. Calcd. for C33H34O6N2S: C, 67.56; H, 5.84; N, 4.77.
Found: C, 67.25; H, 6.04; N, 4.61.
TT Dinucleotide 11
To a solution of 10 (1.00 g, 11.7 mmol) and 4 (945 mg, 2.56 mmol) in
dichloromethane (34 mL) was added NaOCl (4.7 mL, 4% aqueous solution)
over a period of 48 h. Methyl sulfide (200 mL) was added and then the re-
action mixture was stirred for 1 h. Distilled water (50 mL) was added and the
organic layer was separated off. The organic residue was dried over MgSO4
and evaporated in vacuo. The residue was purified by silica gel column
chromatography to give 11a and 11b as a white solid (11a ¼ 59%; 11b ¼ 20%);
11a Rf ¼ 0.25 (EA:Hex ¼ 2:1); IR, n(neat, cmÀ 1) 3186, 3062, 2930, 2856,
1695, 1509, 1470, 1253, 834; 1H NMR (300 MHz, acetone-d6) d 10.30 (s, 2H,
NH), 7.76 (s, 1H, C6-), 7.52-.76-.22 (2 m, 10H, C60-þphH), 6.88 (m, 4H,
phH), 6.36 (m, 1H, H10), 6.19(d, 1H, J ¼ 3.57 Hz, isoxazoline-), 5.86 (d, 1H,
J ¼ 5.6 Hz, H10), 4.88 (m, 1H, H30), 4.63 (d, 1H, J ¼ 3.4 Hz, H40), 4.04-3.98
(m, 2H, H40þH30), 3.75 (s, 6H, OMe), 3.60 (m,1H, H50), 3.41 (m, 2H, iso-
xazoline-CH2), 2.95 (m, 1H, H50), 2.6572.30 (3 m, 4H, H20), 1.49(s, 3H, C5-
e), 1.09(s, 3H, C5-Me), 0.89(s, 9H, tert-Bu), 0.12 (d, 6H, 2Me); 13C NMR
(75.5 MHz, acetone-d6) d 164.2, 164.1, 159.3, 159.0, 150.9, 145.2, 136.8, 136.2,
135.9, 135.8, 130.6, 128.6, 128.3, 127.4, 113.6, 111.0, 110.3, 86.9, 86.4, 85.5,
84.7, 84.6, 82.1, 74.2, 62.5, 55.1, 41.9, 41.2, 40.3, 39.6, 18.1, 12.3, 12.0, À 5.0,
23
À 5.0; MS (FAB, m=z) 976.27 (MþþNa) ; [a]D ¼ À 133 (c ¼ 1.0, CH2Cl2);
mp: 123.67124.2 ꢀC. 11b: Rf ¼ 0.23 (EA:Hex ¼ 2:1); IR, n (neat, cmÀ 1 ) 3194,
1
3062, 2953, 2930, 2856, 1693, 1447, 1363, 1252, 779; H NMR (300 MHz,
acetone-d6) d 10.40, 10.38 (2 s, 2H, NH), 7.74 (s, 1H, C6-H), 7.7477.24 (2 m,
10H, phHþ C6-H), 6.90 (d, 4H, J ¼ 8.40 Hz, phH), 6.31 (t, 1H, J ¼ 6.6 Hz,
H10), 6.22 (d, 1H, J ¼ 6.3 Hz, H10), 6.14 (dd, 1H, J ¼ 9.6 Hz, 9.9 Hz, iso-
xazoline-H), 4.87 (dd, 1H, J ¼ 3.9Hz, J ¼ 2.4 Hz, H30), 4.70 (d, 1H,
J ¼ 3.9Hz, H4 0), 4.07 (d, 2H, H30þH40), 3.7873.71 (sþm, 7H, OMeþH50),
3.51 (s, 3H, isoxazoline-CH2), 3.03 (m, 1H, H50), 2.8572.41 (3 m, 4H, H20),
1.78 (s, 3H, C6-Me), 1.52 (s, 3H, C6-Me), 0.92 (s, 9H, tert-Bu), 0.14 (d, 6H,
2Me); 13C NMR(75.5 MHz, acetone-d6) d 164.2, 164.1, 159.2, 159.2, 159.1,
151.0, 145.5, 137.4, 136.3, 136.1, 136.1, 130.6, 128.6, 128.3, 127.2, 126.3,
113.6, 110.8, 110.4, 87.0, 85.1, 84.6, 84.4, 82.1, 74.2, 62.9, 55.1, 42.1, 41.1,
40.6, 39.5, 25.7, 18.1, 12.1, 11.9, À 5.0; MS (FAB, m=z) 976.23 (MþþNa);
16
[a]D ¼ þ174 (c ¼ 0.96, CH2Cl2); mp: 113.57114.6 ꢀC.

1760
KONG ET AL.
ACKNOWLEDGMENTS
This work was financially supported by the grants (HMP-00-B-21500-0112,
1CB0009201) and BK21 program. BHK is grateful to Basic Science Research In-
stitute, POSTECH for the generous support.
REFERENCES
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A.; Haner, R.; Martin, P.; Moser, H.E. Acc. Chem. Res. 1995, 28, 3667374.
c) Egli, M. Angew. Chem. Int. Ed. Engl. 1996, 35, 189471909.
2. Kim, S.J.; Lee, J.Y.; Kim, B.H. Bioorg. Med. Chem. Lett. 1998, 19, 131371316.
3. a) Kim, J.Y.; Kim, B.H. Nucleosides, Nucleotides & Nucleic Acids 2000, 19,
6377650. b) Chu, C.K.; Walter, S. J. Org. Chem. 1989, 54, 276772769.
4. Gait, M.J. Oligonucleotide Synthesis. A Practical Approach; IRL Press: Oxford,
1984: Chapter 4.
Received October 9, 2000
Accepted May 14, 2001