Oligodeoxynucleotides with extended zwitter-ionic internucleotide linkage

Article abstract of DOI:10.1016/S0040-4020(01)01034-1

Two non-natural nucleoside analogues, N-(2-hydroxyethyl)-2′,5′-dideoxy-5′-aminothymidine (dT^NH) and N-(2-hydroxyethyl)-N-methyl-2′,5′-dideoxy-5′-aminothymidine (dT^NMe), have been prepared and used in the synthesis of oligodeoxythymidilates and mixed-sequence oligodeoxynucleotides, modified at internucleotide linkages. Both modified oligodeoxythymidilates and mixed-sequence oligodeoxynucleotides have been shown to form zwitter-ionic phosphate-amine pairs as evidenced by their decreased electrophoretic mobility in denaturing polyacrylamide gel.

Full text of DOI:10.1016/S0040-4020(01)01034-1

TETRAHEDRON
Pergamon
Tetrahedron 57 +2001) 10287±10292
Oligodeoxynucleotides with extended zwitter-ionic
internucleotide linkage
Svetlana V. Kochetkova, Edward N. Timofeev, Ekaterina A. Korobeinikova,
p
Natalia A. Kolganova and Vladimir L. Florentiev
Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, B-334, 32 Vavilov Str., 117984 Moscow, Russian Federation
Received 21 June 2001; revised 13 September 2001; accepted 11 October 2001
0
0
0
NH
AbstractÐTwo non-natural nucleoside analogues, N-+2-hydroxyethyl)-2 ,5 -dideoxy-5 -aminothymidine +dT ) and N-+2-hydroxyethyl)-
0
0
0
NMe
N-methyl-2 ,5 -dideoxy-5 -aminothymidine +dT ), have been prepared and used in the synthesis of oligodeoxythymidilates and mixed-
sequence oligodeoxynucleotides, modi®ed at internucleotide linkages. Both modi®ed oligodeoxythymidilates and mixed-sequence oligo-
deoxynucleotides have been shown to form zwitter-ionic phosphate±amine pairs as evidenced by their decreased electrophoretic mobility in
denaturing polyacrylamide gel. q 2001Elsevier Science Ltd. All rights reserved.
1
. Introduction
be two methylenes. The results of conformational modeling
are shown in Fig. 2 and Table 1. The modi®ed site did not
disturb the original B-form, being arranged into the six-
membered quasicycle in a chair conformation with electro-
static interaction between charged phosphate oxygen and
protonated nitrogen. Some additional adjustment ¯exibility
was assumed for the hydroxyalkyl chain due to possible
inversions at nitrogen.
Synthetic oligonucleotide analogues represent signi®cant
interest in the development of new therapeutic agents and
1,2
biosensor probes. The enhancement of the speci®c af®nity
of complementary binding of modi®ed oligonucleotides to
target DNA or RNA represents the key task in the design of
modi®ed oligonucleotides. The substitution of charged
phosphodiester internucleotide linkage by uncharged or
positively charged fragments plays an important role in
the mechanism of duplex stabilization. Substitution of
phosphodiesters or entire sugar±phosphate backbone in
PNA, guanidinium analogues, phosphoramidates and
some other modi®cations has been shown to result in a
Calculated non-electrostatic components of potential energy
for modi®ed duplex differ insigni®cantly from those for
natural duplex, indicating negligible strand distortions. On
the other hand, considerable contribution of intramolecular
electrostatic interactions in modi®ed duplex results in lower
3
±5
considerable duplex stabilization effect.
Here, we report the synthesis of two new thymidine deriva-
0
tives with extended 5 -hydroxyl functionality and an amino
group available for charge interaction with the adjacent
phosphate in the oligodeoxynucleotide +Fig. 1).
2
. Results and discussion
NH
Modi®ed nucleosides dT and dT
NMe
were designed to
provide a possibility of charge interaction between the
0
5
-amino group of the nucleoside and the attached phospho-
diester group without considerable conformational changes
in the double helix. Model geometry optimization of the
modi®ed structure provided the suitable linker length
between amino- and hydroxyl groups, which appeared to
Keywords: nucleoside; oligodeoxynucleotide; modi®cation.
Corresponding author. Tel.: 17-95-1356591; fax: 17-95-1351405;
e-mail: ¯or@imb.imb.ac.ru
p
NH
NMe
Figure 1. Phosphodiester linkage formed by dT
+RˆMe) nucleoside analogues.
+RˆH) or dT
0040±4020/01/$ - see front matter q 2001Elsevier Science Ltd. All rights reserved.
PII: S0040-4020+01)01034-1

10288
S.V.Kochetkova et al./ Tetrahedron 57 ꢀ2001) 10287±10292
An alternative synthetic scheme, that uses a temporary
3
0
ethyl)-2 ,5 -dideoxy-5 -aminothymidine. We attached a
-hydroxyl protecting group, was used for N-+2-hydroxy-
0
hydrophobic tBDMS tag to the 3 -hydroxyl of the thymidine
0
0
0
-tosylate +Scheme 2) in order to facilitate puri®cation
0
procedures for dT derivatives.
5
NH
0
cated by the formation of 2,5 -anhydro derivatives along
Substitution of 5 -p-toluenesulfonates 1 and 5 was compli-
0
with the target 5 -aminonucleosides dT
0
NMe
0
and 3 -tBDMS-
NH
dT , respectively. This effect appeared to be more
expressed in the case of ethanolamine, resulting in a
relatively low yield +26%) of compound 6. A three-fold
excess of either amine was suf®cient for complete reaction
and fast conversion of the tosylates, while larger excess
did not in¯uence the reaction ef®ciency and product
distribution.
Figure 2. Fragment of duplex, containing modi®ed internucleotide insert,
generated by conformational modeling. Spheres mark electrostatically
interacting atoms and phosphorus. Internucleotide insert collapses into
quasicycle with chair conformation.
total potential energy. These data allow one to assume
higher duplex stability for the modi®ed oligomer.
NH
The aliphatic amino group of dT analog was protected
by conversion to the corresponding tri¯uoroacetamide,
similarly to the commonly used aminomodi®ers.
NMe
The synthesis of the protected nucleoside derivative dT
started from deoxythymidine and was easily accomplished
6
0
group +Scheme 1). After substitution of the 5 -p-toluene-
in four steps without temporary protection of 3 -hydroxyl
0
sulfonate with N-methyl-ethanolamine, N-+2-hydro-
Phosphoramidites of protected nucleoside analogues were
7
prepared in situ following a known procedure and used
without isolation for the synthesis of modi®ed oligodeoxy-
nucleotides T1±T4 and M1±M2 +Table 2). We applied a
standard synthetic protocol for all the modi®ed nucleotides
and did not observe a decrease in coupling ef®ciency as
0
separated from the excess of amine on Dowex-50 +in
0
0
xyethyl)-N-methyl-2 ,5 -dideoxy-5 -aminothymidine was
1
NH4 form).
Table 1. Results of conformational modeling
21
Type of duplex
Energy components +kJ mol )
Total
Bond
Angle
Torsion
Van-der-Waals
Electro-static
Calculation without counter-ion
Unmodi®ed
Single modi®cation
22379.9
2266.3
6.3
5.4
306.9
61.1
1001.5
11.3
2780.5
37.7
22956.4
2381.9
a
Calculation with counter-ion
Unmodi®ed
Single modi®cation
25878.1
2150.3
45.6
9.6
360.1
90.4
1106.6
216.3
2729.8
72.9
26661.1
2308.6
a
a
For modi®ed duplex differences in energy between modi®ed and unmodi®ed duplexes are given.
Scheme 1. Reagents and conditions: +i) p-TsCl in Py, 08C; +ii) N-methyl ethanolamine in DMF, 1008C, 2 h; +iii) DMTCl in Py.

S.V.Kochetkova et al./ Tetrahedron 57 ꢀ2001) 10287±10292
10289
Scheme 2. Reagents and conditions: +i) tBDMSCl, Im in Py; +ii) ethanolamine in DMF, 1008C, 2 h; +iii) TFA anhydride, DMAP in acetonitrile, 2108C;
iv) DMTCl in Py; +v) 1M TBAF in THF.
+
Table 2. Oligonucleotide sequences and mass data
estimated by trityl monitoring. Post synthetic procedures
included usual ammonia deprotection +558C, 6 h) and
reverse phase HPLC. The structure of the oligonucleotides
was examined by MALDI TOF mass spectrometry +Fig. 3
and Table 2), con®rming the presence of modi®ed nucleo-
sides.
ID
Sequence
M
found +Mcalc.)
0
T0
T15
T2
T3
T4
M0
M15
M2
5 -d+T)
9
±
0
NH
-d+TTTTT TTTT)
2718 +2721)
2734 +2735)
2852 +2850)
2910 +2906)
±
0
NMe
5 -d+TTTTT TTTT)
NH
0
5 -d+TT TT TT TT T)
NMe NMe NMe NMe
NH
NH
NH
0
0
0
0
5 -d+TT TT TT TT T)
NH
Protonation of the secondary amino-group in either dT - or
5 -d+GATATCTTC)
NMe
NMe
dT -modi®ed oligonucleotides results in the zwitter-ionic
-d+GATAT CTTC)
NMe
2751+2747)
2867 +2861)
NMe
NMe
5 -d+GAT AT CT TC)
structures that affect the chain charge. Electrophoretic
mobility of the modi®ed oligonucleotides in denaturing
polyacrylamide gel re¯ects the effect of added positive
charges +Fig. 4). The higher the content of modi®ed
nucleotides was, the lower was the mobility of zwitter-ionic
Figure 4. Denaturing polyacrylamide gel-electrophoresis of the unmodi®ed
oligomer T0 in lane 1and oligonucleotides T 1± T4 in lanes 2±5 +A).
Electrophoresis of mixed-sequence oligonucleotides M0, M1and M2 in
lanes 1, 2 and 3, respectively +B).
Figure 3. MALDI spectrum of the oligonucleotide T1.

10290
S.V.Kochetkova et al./ Tetrahedron 57 ꢀ2001) 10287±10292
oligomers in the electric ®eld. Differences in the electro-
phoretic mobility between oligothymidilates T3 and T4
are most probably related to the differences in hydro-
dynamic volume of the molecules +Fig. 4+A)). This effect
does not appear at the level of single replacement, while
tetra-substituted oligomers differ enough to be separated
in the gel given the same chain charge. Mixed-sequence
oligodeoxynucleotides M1±M2 exhibit generally the same
behavior as follows from Fig. 4+B).
by distant dependence of dielectric constant. Atom charges
were calculated by ab initio. +6-31G basis set).
p
0
0
4.1.1. 5 -O-p-Toluenesulfonyl-2 -deoxythymidine *1).
p-Toluenesulfonyl chloride +2.45 g, 12.9 mmol) was added
0
to the cooled +08C) solution of 2 -deoxythymidine +3.12 g,
12.9 mmol) in 50 mL of dry pyridine. The reaction mixture
was stirred for 2 h at 08C and left for slow warming up to
room temperature and then concentrated in vacuo. Residue
was recrystallized from ethanol, yielding 2.27 g +44%) of
white solid, mp 174±1768C; +Found: C, 51.4; H, 5.08.
C H N O S requires C, 51.51; H, 5.09; N, 7.07%]; R
Physico-chemical studies of the modi®ed oligothymidilates,
including thermal denaturation and circular dichroism
experiments are currently being conducted.
1
7
20
2
7
f
+10% aq. NH /dioxan) 0.70; n
3
1
+KBr) 3190, 2990, 1720,
max
2
670, 1440, 1320, 1270, 1210, 1140, 1020, 980 cm ; d
1
H
We also note that despite the observed ability to induce
intramolecular charge interaction, the thymidine derivative
dT provides an additional possibility for oligonucleotide
functionalization. Secondary amino-group inserted into
the phosphodiester backbone may be used for side-chain
modi®cations of the oligonucleotide.
+400 MHz, DMSO-d ) 1.75 +s, 3H, thymine CH ), 2.02±
6 3
0
2.20 +m, 2H, 2 CH ), 2.40 +s, 3H, CH ±Ph), 3.85±3.90
2
3
NH
0 0
+m, 1H, 3 CH), 4.12±4.22 +m, 2H, 5 CH ), 4.23±4.30 +m,
2
0
0
1H, 4 CH), 5.38 +d, 1H, 3 OH, Jˆ4.5 Hz), 6.15 +t, 1H,
0
1 CH, J
0
0
ˆ7 Hz), 7.36 +s, 1H, 6CH), 7.47 and 7.79 +d,
1
,2
2H, C H , Jˆ8 Hz), 11.22 +s, 1H, NH); d +100.6 MHz,
6
4
C
DMSO-d ) 165.1, 151.9, 151.8, 146.6, 145.5, 137.6,
6
1
1
2
37.3, 133.7, 131.6, 129.5, 129.0, 127.8, 127.0, 111.2,
10.8, 88.7, 85.5, 85.3, 84.7, 71.9, 71.6, 71.4, 62.8, 39.9,
2.5, 13.7, 13.5.
3. Conclusions
We have synthesized two new thymidine analogues that
allow intramolecular charge interaction in the zwitter-
ionic oligonucleotides. Corresponding phosphoramidites
have been prepared and used in the synthesis of modi®ed
oligonucleotides. Denaturing gel-electrophoresis experi-
ments provided evidence for the intramolecular zwitter-
ionic interactions.
0 0 0 0
.1.2. 5 -N-Methyl,N-*2-hydroxyethyl)-2 ,5 -dideoxy-5 -
aminothymidine *2). The solution of 1 +0.37 g, 0.94
mmol) and N-methyl-ethanolamine +0.21g 2.82 mmol) in
4
5 mL DMF was heated at 1008C for 2 h. Then, DMF was
removed in vacuo. Residue was dissolved in water +100 mL)
1
and applied to a column with Dowex 50 in NH4 form.
Excess of amine was washed off with water. Aminonucleo-
side 2 was eluted with 1.5% aqueous ammonia. Correspond-
ing fraction was evaporated to dryness to give 0.18 g +67%)
as a white solid foam; [Found: C, 52.1; H, 7.1; N, 14.0.
C H N O requires C, 52.16; H, 7.07; N, 14.04%]; R
4. Experimental
1
3
21
3
5
f
4
.1. General
+10% aq. NH
1
+400 MHz, DMSO-d
3
/dioxan) 0.25; nmax +KBr) 3290, 3010, 1710,
2
1
680, 1440, 1270, 1210, 1150, 1020, 980 cm ; d
H
1
13
H and C NMR spectra were recorded on a Brucker
) 1.78 +s, 3H, thymine CH
), 2.00±
3
6
0
4
00 MHz spectrometer. IR spectra were obtained from
Specord M80 spectrophotometer. MALDI TOF analysis of
modi®ed oligonucleotides was performed on a Kratos
Kompact MALDI IV mass spectrometer. TLC analysis
was carried out on the Whatman AL SIL G/UV TLC plates.
2.14 +m, 2H, 2 CH
2H, HOCH CH N), 2.55±2.64 +m, 2H, 5 CH
2
), 2.25 +s, 3H, NCH ), 2.43±2.51+m,
3
0
), 3.42±3.50
2
2
2
0
+m, 2H, HOCH CH
2
N), 3.75±3.79 +m, 1H, 3 CH), 4.10±4.12
2
0
0
+m, 1H, 4 CH), 6.15 +t, 1H, 1 CH, J
0
0
ˆ7 Hz), 7.40 +s, 1 H,
) 167.2, 153.4, 137.1,
10.9, 85.7, 85.2, 73.4, 61.5, 61.2, 60.4, 44.61, 39.9, 13.9.
1
,2
6CH); d
1
+100.6 MHz, DMSO-d
C 6
The synthesis of oligodeoxynucleotides was carried out on
automatic DNA synthesizer ASM-102U +Biosset, Russia)
using custom protocol. Reverse phase HPLC puri®cation
0
0
4.1.3. 5 -N-Methyl,N-*2-*O-4,4 -dimethoxytrityl)ethyl),-
0
0
0
0
2 ,5 -dideoxy-5 -aminothymidine *3). 4,4 -Dimethoxy-
trityl chloride +0.27 g, 0.81mmol) was added to a stirred
solution of 2 +0.22 g, 0.77 mmol) in 2 mL of dry pyridine.
In 8 h, the reaction was completed and reaction mixture was
concentrated in vacuo. Residue was dissolved in chloro-
2
was performed using 25£4 mm Hypersil C18 column in
0
.05 M TEAA buffer. DMT-protected oligomers were
puri®ed in the gradient of acetonitrile from 10 to 50%
over 30 min. Completely deprotected oligonucleotides
were additionally puri®ed in the gradient from 0 to 25%
of acetonitrile +over 30 min) in the above buffer.
form. Solution was washed with water, dried over Na SO
2 4
and evaporated to dryness. Residue was puri®ed by column
chromatography on silicagel. Column was eluted conse-
quently with 20% hexane in chloroform, chloroform and
2% ethyl alcohol in chloroform. Appropriate fractions
were evaporated to give 0.33 g +75%) as a white dry
Denaturing gel-electrophoresis of the radioactively labeled
+
3
2
P) oligonucleotides was run in a 20% +19:1) polyacryl-
amide matrix and 100 mM tris-borate buffer, pH 7, and 7 M
urea as the running buffer.
foam; [Found: C, 68.0; H, 6.6; N, 6.9. C34H N O requires
39 3 7
C, 67.87; H, 6.53; N, 6.98%]; R 0.4 +10% methanol/chloro-
f
Conformational calculations were performed by molecular
mechanic method with Hyper Chem 6.0 +AMBER second
generation parameters ). Effect of water was approximated
form); nmax +KBr) 3290, 3000, 1720, 1690, 1440, 1270,
2
1
1210, 1140, 1020, 980 cm ; d
1.69 +s, 3H, thymine CH ), 2.22 +s, 3H, NCH
3
+400 MHz, DMSO-d
)
6
H
8
), 2.03±2.12
3

S.V.Kochetkova et al./ Tetrahedron 57 ꢀ2001) 10287±10292
10291
0
0
+
3
3
m, 2H, 2 CH ), 2.50±2.65 +m, 4H, OCH CH N and 5 CH ),
2
at 2108C +0.25 g, 0.63 mmol) and N,N-dimethylaminopyr-
idine +0.08 g, 0.65 mmol) in 3 mL of dry acetonitrile. In 2 h,
the reaction mixture was diluted by chloroform and washed
with water. Organic layer was dried over Na SO and
2
2
2
.08 +t, 2H, OCH CH N, Jˆ6 Hz), 3.73 +s, 6H, OCH ),
0 0
.78±3.82 +m, 1H, 3 CH), 4.06±4.10 +m, 1H, 4 CH), 5.16
2
2
3
0
0
+
d, 1H, 3 OH, Jˆ4.5 Hz), 6.12 +t, 1H, 1 CH, Jˆ7 Hz), 7.18±
2
4
0
CH), 11.20 +s, 1H, NH); d +100.6 MHz, DMSO-d ) 163.7,
7
6
1
1
5
.39 +m, 13H, 4,4 -dimethoxytrityl aromatic), 7.45 +s, 1H,
evaporated to dryness, yielding 0.28 g +89%) of a colorless
gum. Product was used without additional puri®cation.
[Found: C, 48.3; H, 6.7; N, 13.9. C H F N O Si requires
C
6
58.0, 150.4, 145.1, 136.0, 135.9, 129.6, 127.8, 127.7,
26.6, 113.1, 109.4, 85.5, 84.4, 83.9, 71.8, 61.6, 59.8,
7.2, 55.0, 43.1, 38.5, 12.1.
2
0
32
3
3
6
C, 48.47; H, 6.51; N, 14.04%]; R +10% methanol/chloro-
f
form) 0.5, nmax +KBr) 3330, 2990, 1720, 1690, 1630, 1440,
1
2
270, 1250, 1210, 1150, 1070, 970 cm ; d +400 MHz,
1
H
0
-deoxythymidine *5). t-Butyldimethylsilyl chloride
0
4
2
+
.1.4. 5 -O-p-Toluenesulfonyl-3 -O-t-butyldimethylsilyl-
DMSO-d ) 0.08 +s, 6H, +CH ) Si), 0.87 +s, 9H, tBu), 1.80
6 3 2
+s, 3H, thymine CH ), 2.06±2.13 and 2.33±2.41 +m, 2H,
3
0
0.86 g, 5.73 mmol) and imidazole +0.39 g, 5.73 mmol)
0
2 CH ), 3.30±3.60 +m, 6H, HOCH CH , CH CH N and
2
2
2
2
2
0
0
were added to a stirred solution of 1 +2.27 g, 5.73 mmol)
in 5 mL of dry pyridine. In 8 h, the reaction was completed
and pyridine was evaporated. Residue was dissolved in
chloroform; solution was washed with water, dried over
5 CH ), 3.85±4.92 +m, 1H, 3 CH), 4.55±4.63 +m, 1H,
2
0
0
dC +100.6 MHz, DMSO-d ) 165.1, 151.9, 138.3, 137.8,
4 CH), 6.11 +t, 1H, 1 CH, J
0
0
ˆ7 Hz), 7.48 +s, 1H, 6CH);
1
2
6
130.2, 111.3, 85.8, 84.6, 74.8, 73.8, 60.0, 58.8, 51.1, 50.1,
27.0, 19.1, 13.4, 23.3, 23.5.
Na SO4 and evaporated to dryness in vacuo. Product,
2
2
used without additional puri®cation; [Found: C, 54.0; H,
.61g +90%), was obtained as a white solid foam and
0
0
4.1.7. 5 -N-Tri¯uoroacetyl-N-*2-*O-4,4 -dimethoxytrityl)-
0
0
0
0
6
5
3
9
.7; N, 5.5. C H N O SSi requires C, 54.10; H, 6.71; N,
2
ethyl)-3 -O-t-butyldimethylsilyl-2 ,5 -dideoxy-5 -amino-
thymidine *8). The compound was synthesized as described
above for 3 starting from 0.25 g of 7. The product was
puri®ed by column chromatography on silicagel using,
consequently, 50% hexane in chloroform, chloroform and
1% methyl alcohol in chloroform. Appropriate fractions
were evaporated to a yellow gum. Yield 0.33 g +83%);
[Found: C, 61.6; H, 6.4; N, 5.2. C41
C, 61.72; H, 6.32; N, 5.27%]; R +10% methanol/chloro-
form) 0.8; nmax +KBr) 3000, 1720, 1690, 1640, 1440,
2
3
34
7
.49%]; R +10% methanol/chloroform) 0.8, n
f
+KBr)
max
180, 3000, 1720, 1690, 1440, 1270, 1210, 1150, 980,
00 cm ; d +400 MHz, DMSO-d ) 0±0.1+m, 6H,
2
1
H
6
+
CH ) Si), 0.84±0.92 +m, 9H, tBu), 1.95 +s, 3H, thymine
3 2
0
CH ), 2.02±2.28 +m, 2H, 2 CH ), 2.45 +s, 3H, CH ±Ph),
3
4
3
2
3
0 0
.95±3.97 +m, 1H, 3 CH), 4.11±4.24 +m, 2H, 5 CH ),
2
0
0
.32±4.34 +m, 1H, 4 CH), 6.28 +t, 1H, 1 CH,
ˆ6.5 Hz), 7.35 and 7.77 +d, 2H, C H , Jˆ7 Hz), 7.35
H F N O Si requires
50 3 3 8
J1
0
0
f
2
6
4
+
s, 1H, 6CH); d +100.6 MHz, DMSO-d ) 165.1, 151.7,
6
C
2
1
1
1
8
2
51.0, 146.7, 146.5, 137.6, 137.4, 133.5, 131.8, 131.6,
29.5, 129.4, 129.2, 129.0, 125.3, 111.2, 86.9, 85.7, 85.6,
4.7, 83.6, 74.3, 73.0, 71.6, 71.4, 70.9, 45.5, 39.7, 36.8,
7.3, 27.0, 22.5, 19.0, 13.5, 21.8, 23.4, 23.7.
1270, 1260, 1210, 1160, 1070, 980 cm ; d
DMSO-d ) 0.03±0.09 +m, 6H, +CH Si), 0.82±0.91+m,
), 2.00±2.15 and 2.30±
H
+400 MHz,
)
3 2
6
9H, tBu), 1.75 +s, 3H, thymine CH
3
0
2.42 +m, 2H, 2 CH
3.65±3.80 +m, 10H, OCH , CH CH N and 5 CH ), 3.85±
), 3.30±3.35 +m, 2H, OCH
CH
2
N),
2
2
0
3.95 +m, 1H, 3 CH), 4.25±4.43 +m, 1H, 4 CH), 6.07±6.14
3
2
2
2
0
0
,5 -dideoxy-5 -aminothymidine *6). The solution of 5
0
0
0
4
2
+
.1.5. 5 -N-*2-Hydroxyethyl)-3 -O-t-butyldimethylsilyl-
0
2.55 g, 5.00 mmol) and ethanolamine +0.92 g, 15.00
0
0
0
+m, 1H, 1 CH), 6.75±7.52 +m, 14H, 4,4 -dimethoxytrityl
aromatic and 6CH); d +100.6 MHz, DMSO-d ) 161.8,
C
6
mmol) in 5 mL DMF was heated at 1008C for 2 h. Then,
DMF was removed in vacuo. Residue was dissolved in
diethyl ether; solution was washed twice with water, dried
over Na SO and evaporated to dryness. Compound 6 was
puri®ed by column chromatography on silicagel using,
consequently 20% hexane in chloroform, chloroform and
158.1, 148.9, 145.2, 136.9, 129.5, 127.7, 127.9, 126.6,
113.0, 109.8, 85.6, 83.8, 73.2, 72.1, 59.8, 58.4, 55.1, 50.0,
26.9, 19.0, 12.0, 23.3, 23.5.
2
4
0 0
4.1.8. 5 -N-Tri¯uoroacetyl-N-*2-*O-4,4 -dimethoxytrityl)-
0
pound was prepared by standard desilylation procedure
0
0
ethyl)-2 ,5 -dideoxy-5 -aminothymidine *9). The com-
9
5
% methyl alcohol in chloroform. Appropriate fractions
were evaporated to a colorless gum. Yield 0.52 g +26%);
Found: C, 53.9; H, 8.4; N, 10.4. C H N O Si requires
from 8 with a yield of 87% as a white solid foam; [Found:
[
C, 61.6; H, 5.2; N, 6.1. C35
5.31; N, 6.15%]; R +10% methanol/chloroform) 0.6; nmax
+KBr) 3330, 3000, 1710, 1640, 1440, 1260, 1210, 1150,
H F N O requires C, 61.49; H,
36 3 3 8
1
8
33
3
5
C, 54.11; H, 8.32; N, 10.52%]; R +10% methanol/chloro-
f
f
form) 0.25; nmax +KBr) 3300, 3000, 1720, 1680, 1440, 1260,
1
2
210, 1150, 1020, 980, 900 cm ; d +400 MHz, DMSO-d )
1
21
1060, 980 cm ; d
thymine CH
3.37 +t, 2H, OCH CH
+400 MHz, DMSO-d
) 1.87 +s, 3H,
0
H
6
H
6
0
thymine CH ), 1.95±2.07 and 2.18±2.33 +m, 2H, 2 CH ),
.07 +s, 6H, +CH ) Si), 0.86 +s, 9H, tBu), 1.78 +s, 3H,
0
3
), 2.15±2.22 and 2.29±2.38 +m, 2H, 2 CH
),
2
3
2
N, Jˆ5.5 Hz), 3.50±3.95 +m, 11H,
3
2
2
2
0
0
.39±3.50 +m, 2H, HOCH CH ), 3.71±3.79 +m, 1H, 3 CH),
0
2
3
4
7
1
2
.60 +t, 2H, CH CH N, Jˆ6 Hz), 2.70±2.75 +m, 2H, 5 CH ),
OCH
CH
2
2
N, 5 CH
2
, 3 CH and OCH
0
), 4.15±4.21 +m, 1H,
3
2
2
2
0
0
4,4 -dimethoxytrityl aromatic and 6CH); d
4 CH), 6.16 +t, 1H, 1 CH, Jˆ6.0 Hz), 6.78±7.39 +m, 14H,
2
2
0
.56 +s, 1H, 6CH); d +100.6 MHz, DMSO-d ) 205.5, 165.1,
0
0
.33±4.36 +m, 1H, 4 CH), 6.11 +t, 1H, 1 CH, J
0
0
ˆ7 Hz),
C
+100.6 MHz,
1
2
DMSO-d ) 163.7, 160.0, 150.6, 147.2, 138.3, 130.9,
6
129.1, 127.9, 114.1, 110.8, 86.4, 74.4, 73.2, 60.7, 59.5,
56.0, 50.9, 27.8, 12.1.
C
6
51.9, 137.7, 111.1, 87.2, 85.2, 74.4, 61.6, 53.3, 52.2, 47.2,
7.1, 19.1, 13.6, 12.3, 23.3, 23.4.
0 0
.1.6. 5 -N-Tri¯uoroacetyl-N-*2-hydroxyethyl)-3 -O-t-
butyldimethylsilyl-2 ,5 -dideoxy-5 -aminothymidine *7).
Tri¯uoroacetic anhydride +0.09 mL, 0.63 mmol) in 2 mL
of acetonitrile was added slowly to a stirred solution of 6
4
0
0
0
References
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