Synthesis of thymidine dimer containing novel (N-acetyl)imino linkage

Article abstract of DOI:10.1002/jccs.200100139

By starting with 3′-keto-5′-O-protected thymidine, 3′-O-amino-5′-t-butyl-diphenylsilyl thymidine (9) was prepared and coupled with 3′-O-t-butyl-diphenylsilyl-5′-formyl thymidine to form a nucleoside dimer (11) containing oxime linkage. The backbone of thi

Full text of DOI:10.1002/jccs.200100139

JournaloftheChineseChemicalSociety, 2001, 48, 949-952
949
Note
Synthesis of Thymidine Dimer ContainingNovel(N-Acetyl)iminoLinkage
Te-Fang Yang* (
), Fang-Chi Chien (
) and Fen-Yu Chung (
)
DepartmentofAppliedChemistry,ChaoyangUniversityofTechnology,Wufeng413,Taichung,Taiwan,R.O.C.
By start ing with 3 -keto-5 -O-protected thymidine, 3 -O-amino-5 -t-butyl-diphenylsilyl thymidine (9)
was pre pared and cou pled with 3 -O-t-butyl-diphenylsilyl-5 -formyl thymidine to form a nucleoside dimer
(11) con tain ing oxime link age. The back bone of this dimer, then, un der went re duc tion fol lowed by acetyl a-
tion to give an (N-acetyl)imino link age, and a novel dinucleotide (13) was ob tained.
INTRODUCTION
Remarkableprogressonthesearchforpromisingoligo-
Scheme I
T
U
HO
TBDPSO
O
O
nucleotides as po ten tial antisense agents has been made in the
lastdecade.1-5 Es pe cially, some prob lems such as chirality,2
nucleolyticdegradation4 and low bind ing af fin ity found with
oligonucleotides con tain ing phos pho rus link ages have al-
most been over come by adopt ing those con tain ing var i ous
non-ionic, hydrolyticallystableandnon-chiralbackbones.
Forexample,amide-linkedoligomershaveprovedtobefully
resistanttocleavagebyribozyme6 or have shown good bind-
ing af fin ity to com ple men tary RNA.7 On the other hand,
meth y lene(methylimino) (MMI) linked tetramer8 was found
to be ex tremely sta ble in the pres ence of nu cleases. These
phe nom ena seem to im ply that an internucleosidic link age
con tain ing both imino and am ide groups might be a use ful
mod i fi ca tion of the back bone in antisense oligonucleotides.
Al though most of the re ported non-natural back bones con-
tained in dinucleotides, so far, are four-atom moi eties, a few
nucleoside dimers such as 1,9 2,10 310 and 4,11 in which the
link ages are com posed of a three-atom chain (Scheme I),
have also been in ves ti gated. It is note wor thy that the oligo-
nucleotide into which the oxyamide-linked dimer11 had been
incorporated was shown to affiliate with complementary
DNAaswellasthesequencewhichpossessesnaturallinkage
only. This promising result attracted our attention and
prompted us to study the syn the sis of some oxyamide-like
linkednucleotidedimers.
S
T
T
S
L
O
O
HO
HO
2, L : S
3 , L : S=O
1
T
HO
U
O
DMTO
O
N
O
O
O
T
N
T
O
O
O
H
Me
HO
TBDPSO
5
4
Compound 5is ex pected to in cor po rate into an oligo nucleo-
tide, andthenewstrandcouldbeappliedtoantisensetherapy.
RESULTS AND DISCUSSION
Ini tially, 5 -O-protected keto thymidine 6 wassimply
re duced with NaBH4 to predominantlyprovide (3 - - hy-
droxy)thymidine (7) (Scheme II) as the ma jor prod uct. A
Mitsunobu re ac tion of 7 with N-hydroxyphthalimide2 gave
3 -O-phthalimidothymidine (8), which was then treated with
methyl hydrazine to af ford 3 -O-aminothymidine (9). Cou-
pling re ac tion of9 with 5 -formyl thymidine (10)7 proceeded
smoothlyatroomtemperatureinaceticacidsolution(Scheme
III), and the oxime linked thymidine dimer ( 11)wasobtained
in 85% yield. Very re cently, Gotor and co work ers13 have just
Thus, we are interested in (N-acetyl)imino linkage
since it is achiral, non-ionic12 and nearly isosteric to the
oxyamide group. Fur ther more, it is rea son able to an tic i pate
that the (N-acetyl)imino group would also pro ject it self away
from the im ped ing ef fects of the sugar moi ety,11 and the
conformationalrigidity9 of the group could be in fa vor of the
du plex he lix. Herein we re port the syn the sis of thymidine
dimer an a log which con tains 3 -( )-O-N(Ac)-5 link age (5).

950 J. Chin. Chem. Soc., Vol. 48, No. 5, 2001
Yang et al.
ticacidatroomtemperature.
Scheme II
Reductionoftheoximelinkagein11 by dimethyl phen-
ylsilane14,15 led to the imino link age con tained in thymidine
dimer 12.Wethenintendedtocarriedoutacetylationreaction
of the imino link age with acetyl chlo ride but failed. The re ac-
tion wasfi nally achieved by using ace tic an hy dride asthe re-
source of acetyl moi ety, and this re sulted in the for ma tion of
dimeranalogue13(81%). Re moval of TBDPS group in13 by
TBAFfurnishedthetargetproduct(5). The N-O bond in com-
pound 5 is sta ble in trifluoroacetic acid and is ex pected to be
as sta ble as the N-O bond in com pound 4.11
T
R O
T
1
R O
1
O
O
PhthNOH, PPh3
DEAD, DMF
NaBH4 , MeOH
r.t.; 10 min.
O
HO
6, R1 = TBDPS
7
T = thymine
T
T
R O
1
R O
1
O
O
CH3NHNH2
CH2 Cl2
H NO
PhthNO
2
Inconclusion,wehavesynthesizedaseriesofthymidine
dimeranalogues(11 , 12, 5)containing3-atomlinkages. The
method is fac ile and ef fi cient, and the re agents adopted are
inexpensiveandreadilyavailable.Incorporationofthetitle
dinucleotide into DNA se quences is be ing in ves ti gated and
theduplexmeltingtemperatureswillbemeasured.
9, R1 = TBDPS
8
reported the cou pling re ac tion of 3 -carbazoyl thymidine
with 5 -formyl-3 -O-t-butyl-dimethyl thymidine at 40 C us-
ing meth a nol as the sol vent and for mic acid as the cat a lyst.
We did adopt these con di tions for the re ac tion com pound9
withcompound10. However, product11 was ob tained in low
yield. It is also note wor thy that the tert-butyl-diphenyl-silyl
(TBDPS) group was a better one, com pared with the trityl
group or tert-butyl-dimethyl-silyl (TBDMS) group, for the
pro tec tion of both 5 -O on com pound 7 and 3 -O on com-
pound 10, since the for mer (TBDPS) is com pat i ble with ace-
EXPERIMENTAL SECTION
General
Melting points were determined on a Fargo MP-10
melt ing point ap pa ra tus and are un cor rected. 1H- and 13C-
NMR spec tra were re corded with a Varian-200 MHz in stru-
ment,usingtetramethylsilaneasinternalstandard.Chemical
shifts are given in val ues/ppm and cou pling con stants (J)
are given in hertz (Hz). TLC was per formed on pre-coated
glass plates of Sil ica Gel 60 F254 (0.25 mm, E. Merck). Col-
umnchromatographywasperformedusingMercksilicagel
60 (230-400 mesh). High-resolution mass spec tra (HRMS)
were de ter mined on a JEOL JMX-SX/SX 102 A mass spec-
trometer at NationalChung-HsingUniversity.Elemental
analyseswereperformedintheMicroanalyticalLaboratory
atNationalTaiwanUniversity.
Scheme III
T
TBD PSO
O
O
T
O
O
H
AcOH
T
9
+
N
r.t., 16 h.
O
TB DP SO
10
TBD PS O
11
T
TB DP SO
O
1-[5-O-(tert-Butyldiphenylsilyl)-2-deoxy- -D-erythro -
pentofuranosyl]thy mine (7)
O
HSiMe Ph
2
Ac2 O
T
H N
To a so lu tion of6 (3.00 g, 6.27 mmol) in meth a nol (100
mL), NaBH4 (316 mg, 20.71 mmol) was added. The re ac tion
mixturewasstirredatroomtemperaturefor10min.Afterthe
solventwasremovedunderreducedpressure,theresiduewas
ex tracted with ethyl ac e tate (30 mL 3). The ex tracts were
com bined and washed with wa ter (50 mL). The or ganic layer
was further washed with 20% acetic acid (50 mL), 5%
NaHCO3 (50 mL), dried (MgSO4)andconcentrated. Theresi-
due was purified with silica gel columnchromatography
(19:1, CH2Cl2/MeOH) to give a 4:1 mix ture of 7 (2.18 g,
72%) and its stereoisomer, 5-O-tert-butyldiphenylsilyl
Py, r.t.
TFA
O
TBD PSO
12
T
TBD PSO
O
TBAF
O
N
5
THF, r.t.
T
O
O
Me
TBD PS O
25
thymidine (0.55 g, 18%). Prop erties of 7: [ ]D +10.6 (c
13

SynthesisofThymidineDimer
J. Chin. Chem. Soc., Vol. 48, No. 5, 2001 951
0.35, CHCl3), mp 73-74 C; IR: 3396 (br), 2932, 1691, 1470,
1280, 1109, 1070 cm-1; Rf 0.67 (19:1, CH2Cl2/MeOH);
1H-NMR (CDCl3, 200 MHz) 1.08 (s, 9H), 1.75 (s, 3H), 2.24
(dd, J = 14.8 Hz, 1.8 Hz), 2.55 (m, 1H), 3.59 (bs, 1H), 3.92
(dd, J = 7.6 Hz, 4.8 Hz), 4.04-4.13 (m, 2H), 4.51 (m, 1H),
6.20 (dd, J = 8.0 Hz, 2.4 Hz), 7.25-7.73 (m, 11H), 9.86 (bs,
1H); 13C-NMR (CDCl3, 50 MHz) 12.39, 19.06, 26.71,
40.88, 62.48, 70.88, 83.37, 84.79, 110.01, 127.83, 130.00,
132.47, 135.47, 137.29, 150.91, 164.24.
7.36-7.69 (m, 11H), 9.08 (bs, 1H); 13C-NMR (CDCl3, 50
MHz) 11.93, 19.36, 26.90, 38.67, 64.84, 83.68, 84.22,
84.68, 111.20, 127.95, 130.01, 132.27, 135.22, 135.54,
150.42, 163.80; HRMS (FAB, MH+) calcd for C26H34N3O5Si:
496.2267; found: 496.2272.
Compound 11
To a so lu tion of9 (0.45 g, 0.91 mmol) in ace tic acid (15
mL) was added10(0.45 g, 0.95 mmol). Af ter be ing stirred for
3 hours at room tem per a ture, the re ac tion mix ture was con-
centratedunderreducedpressure.Theresiduewasextracted
with CH2Cl2 (30 mL). The or ganic layer was washed with sat-
uratedNaHCO3 so lu tion (30 mL), dried (MgSO4), and con-
centrated. Thecrudeproductwasthenpurifiedwithsilicagel
columnchromatography(24:1,CH2Cl2/MeOH) to give 11
(0.73 g, 85%) as a white solid. [ ]D25 +47.8 (c 0.46, CHCl3),
mp 98-99 C; IR: 3377 (br), 2937, 1690, 1471, 1109 cm-1; Rf
0.57 (24:1, CH2Cl2/MeOH); 1H-NMR (CDCl3, 200 MHz)
1.10 (s, 18H), 1.86 (s, 6H), 1.96 (m, 1H), 2.08-2.25 (m, 2H),
2.48 (m, 1H), 3.20-4.08 (m, 4H), 4.44-4.88 (m, 2H), 6.20-
6.41 (m, 2H), 7.19 (s, 1H), 7.30 (s, 1H), 7.34-7.68 (m, 21H),
8.82 (bs, 1H), 8.91 (bs, 1H); Anal. Calcd for C52H61N5O9Si2·
0.5 H2O: C, 64.71; H, 7.30; N, 7.26; Found: C, 64.71; H, 7.05;
N, 7.17.
5 -O-tert-Butyldiphenylsilyl-3 -O-phthalimido-thymidine
(8)
Diethyl azodicarboxylate (0.79 g, 4.54 mmol) was
added dropwise to a mix ture of 7 (1.9 g, 3.95 mmol), tri-
phenylphosphine (1.2 g, 4.58 mmol) and N-hydroxy phth-
alimide (0.74 g, 4.54 mmol) in THF at 0 C. The re ac tion
mix ture was stirred for 3 hours and then con cen trated un der
re duced pres sure. The yel low ish syrup was ex tracted with
CH2Cl2 (50 mL 3), washed with brine, dried (MgSO4) and
concentrated.Theresiduewaspurifiedwithsilicagelcolumn
chromatography(3:2:2,EtOAc/n-hexane/CH2Cl2) to give 8
as a white solid (2.18 g, 88%). [ ]D25 +102.1 (c 1.0, CHCl3),
mp 69-70 C; IR: 3387 (br), 2937, 1733, 1690, 1466, 1366,
1276, 1180, 1109, 1080 cm-1; Rf 0.53 (3:2:2, EtOAc/n-
hexane/CH2Cl2); 1H-NMR (CDCl3, 200 MHz) 1.05 (s, 9H),
1.63 (s, 3H), 2.25 (m, 1H), 2.85 (dd, J = 12.6 Hz, 5.2 Hz, 1H),
3.90 (dd, J = 12 Hz, 3Hz, 1H), 4.02 (dd, J = 12 Hz, 3Hz, 1H),
4.47 (m, 1H), 5.05 (d, J = 6 Hz, 1H), 6.58 (dd, J = 9.0 Hz, 5.2
Hz, 1H), 7.32-7.65 (m, 11H), 7.76-7.89 (m, 4H), 9.19 (br s,
1H); 13C-NMR (CDCl3, 50 MHz) 12.00, 19.20, 26.85,
37.18, 64.08, 76.36, 76.99, 77.63, 83.15, 84.58, 88.02,
111.17, 123.75, 127.91, 128.70, 130.08, 132.01, 132.56,
134.72, 135.03, 135.17, 135.39, 150.11, 163.7; HRMS (FAB,
MH+) calcd for C34H36N3O7Si: 626.2322; found: 626.2328.
Compound 12
Dimethylphenylsilane (0.13 g, 0.95 mmol) was added
dropwise to a solution of 11 (0.60 g, 0.52 mmol) in tri-
fluoroacetic acid (5 mL) at 0 C. The re ac tion mix ture was
stirredfor3hours, thenconcentratedunderreducedpressure.
TheresiduewasextractedwithCH2Cl2 (20 mL). The or ganic
layer was washed with sat u rated NaHCO3 solution(50mL),
dried (MgSO4), and con cen trated. The crude prod uct was pu-
rifiedwithsilicagelcolumnchromatography(3:2:1,EtOAc/
n-hexane/CH2Cl2) to give 12 as a white solid (0.35 g, 55%).
25
[ ]D +84.5 (c 0.47, CHCl3), mp 93-94 C; IR: 3377 (br),
3 -O-Amino-5 -O-tert-butyldiphenylsilyl-thymidine (9)
To a so lu tion of8 (2.0 g, 3.20 mmol) in CH2Cl2 (30 mL)
at -10 C was added methyl hydrazine (0.18 g, 3.84 mmol).
Thereactionmixturewasstirredat-10 C for 30 min. and at 0
C for 1.5 hours. Sat u rated brine (30 mL) was then added to
wash the re ac tion mix ture. The or ganic layer was col lected,
dried (MgSO4), and con cen trated to give a white solid. The
crude prod uct was recrystallized (CH2Cl2/cyclohexane)to
2928, 1690, 1466, 1428, 1109 cm-1; Rf 0.64 (3:2:1, EtOAc/n-
1
hexane/CH2Cl2); H-NMR (CDCl3, 200 MHz) 1.09 (s,
18H), 1.56 (s, 3H), 1.86 (s, 3H), 2.02-2.13 (m, 2H), 2.33 (ddd,
J = 13.6 Hz, 6.0 Hz, 3.0 Hz, 1H), 2.50 (dd, J = 13.6 Hz, 5.0
Hz, 1H), 2.64-2.83 (m, 2H), 3.75 (dd, J = 12 Hz, 3 Hz, 1H),
3.95-4.0 (m, 2H), 4.08 (m, 1H), 4.50 (m, 1H), 6.17-6.34 (m,
2H), 6.99 (bs, 1H), 7.34-7.67 (m, 22H), 9.05 (bs, 1H), 9.07
(bs, 1H); 13C-NMR (CDCl3, 50 MHz) 11.94, 12.52, 19.00,
19.36, 26.85, 26.99, 37.33, 39.39, 53.39, 64.76, 74.32, 83.20,
83.71, 84.63, 111.28, 127.95, 127.99, 130.06, 130.18,
132.18, 132.84, 132.88, 132.97, 135.20, 135.54, 135.66,
135.72, 136.11, 150.27, 150.36, 163.52, 163.76; HRMS
25
provide 9 (1.38 g, 87%). [ ]D +5.1 (c 0.73, CHCl3), mp
85-86 C; IR: 3387 (br), 2937, 1690, 1466, 1276, 1109, 1080
cm-1; Rf 0.53 (2:1, EtOAc/n-hexane); 1H-NMR (CDCl3, 200
MHz) 1.10 (s, 9H), 1.57 (s, 3H), 2.06 (ddd,J = 13.8 Hz, 9.2
Hz, 6.0 Hz, 1H), 2.58 (dd, J = 13.8 Hz, 5.2 Hz, 1H), 3.85 (dd,
J = 11.4 Hz, 2.2 Hz, 1H), 4.04 (dd, J = 11.4 Hz, 2.2 Hz, 1H),
4.16 (m, 1H), 4.46 (m, 1H), 6.36 (dd, J = 9.2 Hz, 5.2 Hz, 1H),
(FAB, MH+) calcd for C H63N5O9Si2: 957.4164, found:
52
957.4181; Anal. Calcd for C52H63N5O9Si2·1.5 H2O: C, 63.40;

952 J. Chin. Chem. Soc., Vol. 48, No. 5, 2001
Yang et al.
H, 6.71; N, 7.11. Found: C, 63.17; H, 6.31; N, 6.99.
Coun cil of Tai wan, R.O.C. (Grant NSC 88-2113-M- 324-
005), which is grate fully ac knowl edged. The au thors thank
the In stru men tal Cen ter at Na tional Tai wan Uni ver sity for
theelementalanalysesandtheRegionalInstrumentsCenter
atNationalChung-HsingUniversityforhighresolutionmass
spectra.
Com pound 13
To a so lu tion of12 (0.33 g, 0.35 mmol) in pyridine (5
mL) was added ace tic an hy dride (0.1 g, 1.05 mmol). The re-
ac tion mix ture was stirred at room tem per a ture for 2 hours
andthenconcentratedunderreducedpressure. Theresidue
was ex tracted with CH2Cl2 (20 mL), washed with sat u rated
NaHCO3 so lu tion (20 mL), dried (MgSO4,andconcentrated.
The crude prod uct was pu ri fied with sil ica gel col umn chro-
matography (3:2:1, EtOAc/n-hexane/CH2Cl2) to give 13
(0.28 g, 81%) as a white solid. [ ]D25 +54.8 (c 0.98, CHCl3),
mp 89-90 C; IR: 3377, 2937, 1690, 1466, 1109, 1071 cm-1;
Received March 9, 2001.
Key Words
Thymidine dimers; Nucleotides; Antisense agents;
Acetylimino linkage.
1
Rf 0.65 (3:2:1, EtOAc/n-hexane/CH2Cl2); H-NMR (CDCl3,
200 MHz) 1.08 (s, 18H), 1.67 (s, 3H), 1.90 (s, 3H), 1.98 (s,
3H), 1.95-2.55 (m, 4H), 3.23 (m, 1H), 3.53-3.72 (m, 2H),
3.90-4.48 (m, 5H), 6.31 (m, 2H), 7.38-7.63 (m, 21H), 9.33
(bs, 1H), 9.55 (bs, 1H). 13C-NMR (DMSO-d6, 50 MHz)
12.08, 12.19, 18.93, 19.22, 20.91, 26.74, 26.91, 37.03, 39.25,
64.40, 74.39, 82.59, 83.07, 84.26, 84.87, 85.64, 112.90,
127.92, 127.98, 128.05, 130.13, 130.20, 130.34, 131.94,
132.35, 132.59, 132.88, 134.70, 135.20, 135.39, 135.61,
150.21, 150.27, 163.85. Anal. Calcd for C54H65N5O10Si2: C,
64.84; H, 6.55; N, 7.00; Found: C, 64.73; H, 6.89; N, 6.73.
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A mix ture of 13 (0.20 g, 0.20 mmol) and tetrabutyl am-
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[ ]D25 +65.0 (c 0.26, MeOH), mp 128-129 C; IR: 3396 (br),
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2H), 3.82-4.16 (m, 5H), 4.70 (m, 1H), 5.17 (t, J = 5 Hz, 1H,
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ACKNOWLEDGEMENTS
This work was supported by the National Science