Synthesis and hybridization properties of β- and α-oligodeoxynucleotides containing β- and α-1-(3-C-allyl-2-deoxy-D-erythro-pentofuranosyl)thymine and α-1-[3-C-(3-aminopropyl)-2-deoxy-D-erythro-pentofuranosyl]thymine

Article abstract of DOI:10.1039/a703173d

Convergent synthesis of β- and α-1-(3-C-allyl-2-deoxy-D-erythro-pentofuranosyl)thymine and their incorporation into β- and α-oligodeoxynucleotides (ODNs) is described. The thermal stabilities of duplexes formed between modified ODNs and complementary single-stranded DNA and RNA have been evaluated. In all cases stable duplexes are formed, but whereas β-ODNs containing β-3′-C-allylthymidine show moderately lowered thermal stability towards both DNA and RNA, α-ODNs containing α-3′-C-allylthymidine show significantly increased thermal stabilities compared with the corresponding β-ODN reference duplexes. Even more stable duplexes towards both DNA and RNA have been obtained using an α-ODN containing one α-1-[3-C-(3-aminopropyi)-2-deoxy-D-erythro-pentofuranosyl]thymine monomer.

Full text of DOI:10.1039/a703173d

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Synthesis and hybridization properties of â- and á-
oligodeoxynucleotides containing â- and á-1-(3-C-allyl-2-deoxy-D-
erythro-pentofuranosyl)thymine and á-1-[3-C-(3-aminopropyl)-2-
deoxy-D-erythro-pentofuranosyl]thymine
Pia N. Jørgensen,a Ulrik S. Sørensen,†,a Henrik M. Pfundheller,a
Carl E. Olsenb and Jesper Wengel*,c
a Department of Chemistry, Odense University, DK-5230 Odense M, Denmark
b Department of Chemistry, The Royal Veterinary and Agricultural University, DK-1871
Frederiksberg C, Denmark
c Department of Chemistry, Chemical Laboratory II, University of Copenhagen,
Universitetsparken 5, DK-2100 Copenhagen, Denmark
Convergent synthesis of â- and á-1-(3-C-allyl-2-deoxy-D-erythro-pentofuranosyl)thymine and their
incorporation into â- and á-oligodeoxynucleotides (ODNs) is described. The thermal stabilities of
duplexes formed between modified ODNs and complementary single-stranded DNA and RNA have been
evaluated. In all cases stable duplexes are formed, but whereas â-ODNs containing â-3Ј-C-allylthymidine
show moderately lowered thermal stability towards both DNA and RNA, á-ODNs containing á-3Ј-C-
allylthymidine show significantly increased thermal stabilities compared with the corresponding â-ODN
reference duplexes. Even more stable duplexes towards both DNA and RNA have been obtained using an
á-ODN containing one á-1-[3-C-(3-aminopropyl)-2-deoxy-D-erythro-pentofuranosyl]thymine monomer.
straightforward introduction of various nucleobases appealed
Introduction
to us. Recently, ODNs containing nucleoside monomers carry-
ing aminoalkyl linkers tethered at the 1Ј-C, 2Ј-O, 3Ј-O or 4Ј-C
positions of the carbohydrate moiety have been reported.7 Here
we introduce an ODN containing one 3Ј-C-aminopropyl-
derivatized monomer synthesized from the parent 3Ј-C-allyl
nucleoside.
Synthesis of modified oligodeoxynucleotides (ODNs) for use in
control of gene expression has been the subject of active
research during the last few years.1 In order to improve the
binding affinity, enzyme stability, cell-uptake, and other phar-
macokinetic properties, a variety of oligonucleotide analogues
have been synthesized and evaluated.2,3 In connection with a
project directed towards the synthesis of new ODN analogues
containing 3Ј-C-alkyl functionalities as attachment sites for
e.g. intercalating agents or lipophilic carriers, we decided to
synthesize 1-(3-C-allyl-2-deoxy-β--erythro-pentofuranosyl)-
thymine 13 (β) (Scheme 1). The allyl group is suitable for diverse
structural manipulations, and allows, e.g. easy access to both
the 3Ј-C-(2-hydroxyethyl)- and the 3Ј-C-(3-hydroxypropyl)-
modified nucleosides by either oxidative cleavage followed by
reduction or hydroboration followed by in situ oxidation. Add-
itionally, it would be interesting to evaluate 1-(3-C-allyl-2-
deoxy-β--erythro-pentofuranosyl)thymine as a novel mono-
meric substitute in modified ODNs. Until recently, only a few
examples of 3Ј-C-allyl-substituted nucleosides were known. 3Ј-
C-Allyl-2Ј,3Ј-deoxynucleosides were obtained by addition of a
3Ј-centred radical to allyltributyltin which proceeded in a stereo-
selective way to afford only the erythro-isomer corresponding
to addition from the less hindered α-face of the free-radical
intermediate.4 Recently, we have reported the synthesis of 3Ј-C-
allyluridines by cerium-assisted Grignard additions of allyl-
magnesium bromide to 3Ј-ketouridines.5 However, as in the
majority of Grignard reactions on ketonucleosides,6 the nucleo-
philic addition occurred preferentially from the α-face due to
steric hindrance from the base moiety. Based on these results
we decided to use a convergent synthetic strategy, hoping to
achieve a favourable ratio between the desired erythro- and the
threo-configurated products. In addition, the possibility of
BnO
T
O
OBn OH
1
T = thymin-1-yl
Results and discussion
1-(3-C-Allyl-3,5-di-O-benzyl-β--ribofuranosyl)thymine8
1
was prepared in six steps from 5Ј-O-silyl-protected 1,2-O-
isopropylidene-α--ribofuranosid-3-ulose in an overall yield of
47%. The reaction included a stereoselective Grignard addition
of allylmagnesium bromide and a 2Ј-O-acyl-assisted nitrogen
glycosylation to afford exclusively the β-nucleoside. Unfortun-
ately, free-radical deoxygenation9 of the 2Ј-hydroxy group of
compound 1 via the corresponding pentafluorophenyl thiono-
carbonate by Bu3SnH in the presence of azoisobutyronitrile
(AIBN) in hot benzene was unsuccessful in our hands, probably
due to uncontrolled intramolecular free-radical cyclizations. To
overcome this problem we decided to use 2-deoxy--ribose as
the starting compound for the synthesis of amidite 16a (and
16b). Conversion of 2-deoxy--ribose into an anomeric mixture
of methyl 2-deoxy--erythro-pentofuranoside was done under
kinetic control as previously described.10 Reaction of methyl 2-
deoxy--erythro-pentofuranoside with 1.03 mol equiv. of tert-
butyldimethylsilyl chloride (TBDMSCl) and imidazole in
dimethylformamide (DMF)11 afforded, after column chroma-
tography, pure 5-O-silylated α-anomer 2 (28% yield), the
† Present address: Department of Medicinal Chemistry, The Royal
Danish School of Pharmacy, Universitetsparken 2, DK-2100 Copen-
hagen, Denmark.
J. Chem. Soc., Perkin Trans. 1, 1997
3275

View Article Online
corresponding β-anomer 3 (19% yield) and a fraction contain-
ing an anomeric mixture of the disilylated glycosides (9% yield).
Assignment of the anomeric configuration was done by 1H–1H
2-D chemical-shift-correlation spectroscopy (COSY) and
nuclear Overhauser effect (NOE) difference experiments. In
an attempt to control the stereoselectivity of the nucleophilic
addition of allylmagnesium bromide to give the erythro-
configurated product, the α-anomer 2 was selected. Oxidation
of compound 2 with CrO3–pyridine–acetic anhydride reagent12
gave in 84% yield the corresponding 3-ulose 4, which was used
in the next step without further purification. Grignard addition
of 1.0 mol equiv. of allylmagnesium bromide afforded the 3-C-
allyl-α--erythro glycoside 5 in 16% yield, whereas the 3-C-allyl-
α--threo glycoside 6 was obtained in 50% yield. The stereo-
R1O
R1O
O
O
a
OCH3
OCH3
O
OH
2 R1 = TBDMS
3 R1 = TBDMS, β-anomer
4 R1 = TBDMS
b
R1O
R1O
O
O
+
OR2
OCH3
OR2
OCH3
1
chemistry of the Grignard products was confirmed by H–1H
5 R1 = TBDMS, R2 = H
7 R1 = R2 = H
9 R1 = R2 = Ac
6 R1 = TBDMS, R2 = H
8 R1 = R2 = H
10 R1 = R2 = Ac
COSY and NOE difference experiments. Thus, the α-erythro
configuration of product 5 was confirmed by observing NOEs
between H-1 and H-2β (~3%), 3-OH and H-4 (2%), and 3-OH
and H-2α (3%). Mutual NOEs between 3-OH and H-2β and
lack of an NOE between 3-OH and H-4 confirmed the α-threo
configuration of product 6. These results clearly show that the
stereochemical outcome of the Grignard addition was deter-
mined by the bulky group at the 5-position and that no
stereocontrolling effect was induced by the α-OMe substituent.
In addition, our experiments indicated that no more than 1.0
mol equiv. of allylmagnesium bromide should be used in the
Grignard reaction. Thus, reaction of 3-ulose 4 with an excess
of allylmagnesium bromide afforded a mixture of acyclic
derivatives as a result of additional nucleophilic attack at the
anomeric carbon atom. Deprotection of the furanosides 5 and
6 using tetrabutylammonium fluoride (TBAF) in tetrahydro-
furan (THF)13 afforded the diols 7 and 8 in 91 and 82% yield,
respectively. Subsequent acetylation of both the primary and
the tertiary hydroxy groups using acetic anhydride in anhydrous
pyridine in the presence of 4-(dimethylamino)pyridine
(DMAP) afforded compounds 9 and 10 in 77 and 45% yield,
respectively. Direct nitrogen glycosylation14 of 9 and 10 with
thymine using N,O-bis(trimethylsilyl)acetamide (BSA) as
silylating agent and trimethylsilyl trifluoromethanesulfonate
(TMS triflate) as a Lewis acid in anhydrous 1,2-dichloroethane
afforded inseparable anomeric mixtures of nucleosides 11
(β:α ~ 1:2.3) and 12 (β : α ~ 1:1.1) in 52 and 64% yield, respect-
ively. Exchange of 1,2-dichloroethane with the more polar solv-
ent CH3CN in the direct glycosylation reaction of compound 9
with thymine improved the yield to 74% and reversed the ratio
between the β- and α-anomer (β:α ~ 1.5:1). Contrary to the
alternative convergent strategy starting from 5Ј-O-silyl-
protected 1,2-O-isopropylidene-α--ribofuranosid-3-ulose,8 this
strategy gives access to both anomers which enables evaluation
of β- as well as α-ODN analogues. In this context it is notable
that α-DNA is interesting as a potential antisense agent as it
forms a more stable duplex with complementary RNA than
does the corresponding β-DNA strand,15 and is not cleaved by
many nucleases.16
c
c
d
d
e
e
R1O
R1O
O
O
OR2
T
T
+
OR2
11 R1 = R2 = Ac
13 R1 = R2 = H
12 R1 = R2 = Ac
g
HO
T
HO
O
OH
O
OH
+
f
12
T
14a
14b
DMTO
T
DMTO
O
O
+
h
i
13
T
OR1
OR1
15b R1 = H
15a R1 = H
16a R1 = NC[CH2]2OPNPri2
i
16b R1 = NC[CH2]2OPNPri2
O
N
CH3
HN
T =
O
Scheme 1 (a) CrO3, Pyridine, Ac2O, CH2Cl2; (b) 1.0 mol equiv.
allylMgBr, Et2O; (c) TBAF, THF; (d) Ac2O, DMAP, pyridine; (e) A:
thymine, BSA, TMS triflate, CH2ClCH2Cl; B: thymine, BSA, TMS
triflate, CH3CN; (f) NH3 in CH3OH; (g) CH3ONa in CH3OH; (h)
DMTCl, AgNO3, pyridine, THF; (i) NC(CH2)2OP(Cl)NPri2, DIPEA,
CH2Cl2. T = thymin-1-yl
Deacetylation of the threo-anomers 12 using methanolic
ammonia afforded the β-anomer 14a in 39% yield and the α-
anomer 14b in 44% yield after column chromatographic separ-
ation. These two novel nucleosides are currently being evaluated
for biological activity. Treatment of the erythro anomers 11
with CH3ONa–CH3OH afforded an inseparable anomeric mix-
ture 13 in 81% yield. Dimethoxytritylation of mixture 13 in
anhydrous THF–anhydrous pyridine, with AgNO3 as catalyst,17
afforded 1-[3-C-allyl-2-deoxy-5-O-(4,4Ј-dimethoxytrityl)-β--
erythro-pentofuranosyl]thymine 15a in 47% yield, and 1-[3-C-
allyl-2-deoxy-5-O-(4,4Ј-dimethoxytrityl)-α--erythro-pentofur-
anosyl]thymine 15b in 26% yield. The structural assignment of
the β and α nucleosides was done by NOE difference experi-
irradiation of H-4Ј gave an NOE-effect in H-1Ј (7%)] was not
seen for the α-anomer 15b for which an NOE between H-4Ј and
H-6 (6%) was observed. These results were further supported by
the relative chemical shifts of H-5Јa,b and H-4Ј. The H-4Ј sig-
nal of the α-anomer is shifted downfield relative to the signal of
the β-anomer, causing the difference in the chemical shifts
between the H-5Јa,b and the H-4Ј to be greater in the α-anomer
than in the β-anomer.18–19 In the H NMR spectrum of the β-
1
anomer 15a, the H-5Јa,b and the H-4Ј signals appeared with a
chemical-shift difference of 1.04 ppm which was extended to
1.29 ppm for the α-anomer 15b, caused by a downfield shift of
the H-4Ј. Phosphitylation20 of compound 15a and 15b by
reaction with 2-cyanoethyl N,N-diisopropylphosphoramido-
1
ments and one-dimensional H NMR spectroscopy. The key
mutual NOE between H-1Ј and H-4Ј observed for the β-anomer
15a [irradiation of H-1Ј gave an NOE-effect in H-4Ј (3%), while
3276
J. Chem. Soc., Perkin Trans. 1, 1997

View Article Online
chloridite in the presence of N,N-diisopropylethylamine
(DIPEA) in anhydrous CH2Cl2 afforded the nucleoside
phosphoramidites 16a in 75% yield and 16b in 77% yield,
respectively, after column chromatographic purification and
precipitation from light petroleum.
TBDMSO
TBDMSO
O
O
a
T
OCH3
OH
OTMS
5
21
With the aim of evaluating the properties of 3Ј-C-allyl β--
ribo ODN-analogues, the phosphoramidite derivative 20 was
synthesized (Scheme 2) from 1-(3-C-allyl-3,5-di-O-benzyl-β--
ribofuranosyl)thymine 1.8 Debenzylation of compound 1 using
BCl3 in CH2Cl2 at Ϫ78 ЊC afforded nucleoside 17 in 73% yield.
As expected, removal of the benzyl groups using H2 and
Pd(OH)2–C as catalyst in absolute EtOH at room temperature
caused concomitant reduction or the allylic double bond.
Dimethoxytritylation of triol 17 using 1.2 mol equiv. of 4,4Ј-
dimethoxytrityl chloride (DMTCl) in anhydrous pyridine
afforded nucleoside 18 in 82% yield. This was followed by silyl-
ation of the free 2Ј-hydroxy group using TBDMSCl in
anhydrous DMF and imidazole as catalyst to afford compound
19 in 82% yield. Subsequent phosphitylation, as described
above for the preparation of compound 16a, afforded the phos-
phoramidite 20 in 78% yield after column chromatographic
purification and precipitation from light petroleum.
b
R1O
TBDMSO
HO
O
O
c
T
T
OR2
OTMS
PhthN
23 R1 = TBDMS, R2 = TMS
24 R1 = R2 = H
22
d
e
R1O
O
+ β-anomer
T
OR2
25 R1 = DMT, R2 = H
26 R1 = DMT, R2 = NC[CH2]2OPNPri2
PhthN
f
BnO
T
R1O
a
T
DMTO
d
T
O
O
O
Scheme 3 (a) Thymine, BSA, TMS triflate, CH3CN; (b) BH3ؒ1,4-
oxathiane, THF; NaOH, 30% H2O2; (c) pthalimide, Ph3P, DEAD,
THF; (d) TBAF, THF; (e) DMTCl, AgNO3, pyridine, THF; (f)
NC(CH2)2OP(Cl)NPri2,
DIPEA,
CH2Cl2.
T = thymin-1-yl.
OBn OH
OH OR2
O
OTBDMS
PhthN = phthalimido.
NCCH2CH2OPNPri2
17 R1 = R2 = H
18 R1 = DMT, R2 = H
19 R1 = DMT, R2 = TBDMS
20
Synthesis of DMT-on ODNs A–L (Table 1) was performed
by use of standard phosphoramidite methodology on an auto-
mated DNA-synthesizer using the appropriate building blocks
[16a, 16b, 26, α-thymidine 3Ј-O-2-(cyanoethyl)phosphoramidite
and commercial thymidine 3Ј-O-2-(cyanoethyl)phosphor-
amidite]. The coupling efficiency of the modified phosphor-
amidites 16a, 16b, and 26 were approximately 40% (two times
24 min coupling) compared with >99% for α-thymidine and
standard phosphoramidites (2 min coupling). The coupling
efficiency of the building block 20 was in all experiments below
5%. The low coupling yield obtained with compound 20 can be
explained by the tertiary nature of the phosphitylated hydroxy
group in connection with steric hindrance from the 2Ј-O-(tert-
butyldimethylsilyl) group (compare with structures 16a, 16b
and 26). No ODNs containing compound 20 could therefore be
obtained. The 5Ј-O-dimethoxytrityl-protected oligomers were
removed from the solid support by treatment with conc.
ammonia at room temperature for 72 h, which also removed the
phosphate and nucleobase-protecting groups and the phthaloyl
group in species L. Purification using disposable reversed-phase
chromatography cartridges (which includes detritylation)
afforded the unprotected oligomers A–L. The purity of the
modified oligomers was confirmed by anion-exchange high-
performance liquid chromatography (HPLC) analysis. The
composition of the oligomers was verified by matrix-assisted
laser desorption mass spectrometry (MALDI-MS), a powerful
method for mass analysis of ligomers.22,23 The observed relative
molecular masses correspond within experimental error to
those calculated (Table 2).
The hybridization properties of the modified oligomers
towards their complementary DNA and RNA strands were
checked by UV melting point (Tm) measurement as described.24
The results are given in Table 1. Incorporation of 16a (mono-
mer X) once or twice in the middle of a 14-mer (ODNs A–D)
induces a minor destabilization of the duplex formed with
complementary DNA (∆Tm ~Ϫ1 ЊC modification). However,
when RNA is used as the complementary strand, the resulting
duplexes are destabilized by Ϫ2.8 to Ϫ4.0 ЊC per modification.
These results support the assumption that the monomer X
adopts a 2Ј-endo conformation, as reported earlier for a similar
nucleoside,25 which is favourable for DNA–DNA duplex form-
1
b
c
Scheme 2 (a) BCl3, CH2Cl2, hexane; (b) DMTCl, pyridine; (c)
TBDMSCl, imidazole, DMF; (d) NC(CH2)2OP(Cl)NPri2, DIPEA,
CH2Cl2. T = thymin-1-yl
The chemistry used to synthesize the phosphoramidite
monomer 26, which was used on an automated DNA syn-
thesizer to introduce a 3Ј-C-(3-aminopropyl) linker into an oli-
gonucleotide, is shown in Scheme 3. Direct nitrogen glycosyl-
ation14 of furanoside 5 with thymine using BSA as silylating
agent and TMS triflate as Lewis acid in CH3CN afforded an
inseparable anomeric mixture 21 of the α- and β-nucleoside
(α:β ~ 3:1) in 83% yield. Hydroboration of this mixture with
BH3ؒ1,4-oxathiane in THF followed by in situ oxidation with
alkaline hydrogen peroxide gave 3Ј-C-(3-hydroxypropyl)
nucleosides 22 in 63% yield. Mitsunobu21 reaction of alcohol
22 with phthalimide afforded the phthaloyl-protected primary
amines 23 in 86% yield. Treatment of the protected bis-ether 23
with TBAF in THF removed the silyl groups in 33% yield and
the deprotected nucleosides 24 were allowed to react with
DMTCl in anhydrous THF containing anhydrous pyridine,
with AgNO3 as catalyst, to protect the 5Ј-hydroxy groups. At this
stage, the α- and β-nucleosides were easily separated by silica
gel chromatography to give 1-[2-deoxy-5-O-(4,4Ј-dimethoxy-
trityl)-3-C-(phthalimidopropyl)-α--erythro-pentofuranosyl]-
thymine 25 in 43% yield and the corresponding β-anomer in
15% yield. The structural assignment of the α-and β-nucleoside
was based on one-dimensional 1H NMR analysis. Thus, for the
β-anomer, the H-5Јa,b and the H-4Ј signals appeared with a
chemical-shift difference of 0.91 ppm which was extended to
1.36 ppm for the α-anomer 25. The configuration of the α-
anomer was further confirmed by a significant NOE of H-4Ј
(5%) by irradiation of H-6. The α-anomer 25 was treated with
2-cyanoethyl N,N-diisopropylphosphoramidochloridite in the
presence of DIPEA in anhydrous CH2Cl2 to give the phospho-
ramidite 26 in 41% yield. Owing to the low yield obtained for
the β-anomer corresponding to compound 25 incorporation
of this anomer awaits completion of an alternative synthetic
strategy.
J. Chem. Soc., Perkin Trans. 1, 1997
3277

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Table 1 Sequences and melting experiments of synthesized β- and α-ODNs.
Sequence
Tm ( ЊC)a
∆Tm ( ЊC)a Tm ( ЊC)b
∆Tm ( ЊC)b
5Ј-TTTTTTTTTTTTTT-3Ј
5Ј-TTTTTTTXTTTTTT-3Ј
5Ј-TTTTTTXXTTTTTT-3Ј
5Ј-TTTTTXTTTXTTTT-3Ј
5Ј-TTTTTTTYTTTTTT-3Ј
5Ј-TTTTTTYYTTTTTT-3Ј
5Ј-TTTTTYTTTYTTTT-3Ј
5Ј-α-(TTTTTTTTTTTTTT)C-3Ј
5Ј-α-(TTTTTTTYTTTTTT)C-3Ј
5Ј-α-(TTTTTTYYTTTTTT)C-3Ј
5Ј-α-(TTTTYTTTYTTTTT)C-3Ј
5Ј-α-(TTTTTTZTTTTTT)C-3Ј
A
B
C
D
E
F
G
H
I
35.5
34.5
34.0
33.0
18.5
15.0
14.5
38.0
37.0
37.0
37.0
38.0
29.0
Ϫ1.0
Ϫ0.8
Ϫ1.3
Ϫ17.0
Ϫ10.3
Ϫ10.5
25.0
23.5
23.0
Ϫ4.0
Ϫ2.8
Ϫ3.0
43.5
40.0
37.0
36.0
41.0
Ϫ1.0
Ϫ0.5
Ϫ0.5
0.0
Ϫ3.5
Ϫ3.3
Ϫ3.8
Ϫ2.5
J
K
L
T = thymidine monomer; C = 2Ј-deoxycytidine monomer; X = β-monomer derived from amidite 16a; Y = α-monomer derived from amidite 16b;
Z = α-3-C-3-aminopropyl monomer derived from amidite 26. Tm = melting temperature; ∆Tm = change in Tm per modification compared with the
corresponding reference duplex. a Complexed with dA14. b Complexed with rA14.
Table 2 Mass analysis of synthesized ODNs.
suggest them to be promising as bioactive molecules. The most
likely application of 3Ј-C-allylnucleosides, however, will be as a
precursor for the synthesis of 3Ј-C-(2-hydroxyethyl)- or 3Ј-C-(3-
hydroxypropyl)nucleosides and their amine analogues. Interest-
ing properties of the latter were demonstrated (ODN L) and
further research in this direction seems justified.
ODN
Calc. [M ϩ H]ϩ (m/z)
Found [M ϩ H]ϩ (m/z)
A
B
C
D
E
F
G
H
I
4198
4238
4278
4278
4238
4278
4278
4487
4527
4567
4567
4544
4200
4241
4280
4280
4240
4280
4280
4487
4529
4565
4566
4545
Experimental
NMR spectra were recorded at 250 MHz for 1H NMR and 62.9
MHz for 13C NMR on a Bruker AC-250 spectrometer or at 500
MHz for 1H NMR, 125 MHz for 13C NMR and 202.3 MHz for
31P NMR on a Varian Unity 500 spectrometer. Chemical shifts
are in ppm relative to tetramethylsilane as internal standard (1H
NMR and 13C NMR) and relative to 85% H3PO4 as external
standard (31P NMR). J Values are given in Hz. 1H NMR peak
assignments for compounds 2, 3, 5, 6, 14a, 14b, 15a, 15b, 17–19
and 21–25 were derived from 1H–1H COSY and/or NOE differ-
ence experiments. 13C NMR peak assignments for compounds
3, 5, 6 and 17 were derived from intensive nuclei enhancement
J
K
L
ation whereas the conformation of the carbohydrate moiety in
a DNA–RNA duplex is known to be 3Ј-endo.26 As expected,
incorporation of the α-monomer Y derived from compound
16b into a β-ODN (E–G) results in a large destabilization of the
duplex formed with complementary DNA. This further con-
firms the assigned configuration of the nucleosides. As men-
tioned, α-ODNs form thermally more stable duplexes with
RNA than do β-ODNs. The melting experiments depicted in
Table 1 show the same tendency as α-ODN values are on average
14.5 ЊC higher towards complementary RNA than are those for
the corresponding β-ODN–RNA duplexes. However, incorpor-
ation of α-monomer Y once or twice in the middle of a α-T14
(ODNs H–K) induces a similar destabilizing effect (∆Tm ~
Ϫ3.5 ЊC/modification) on a duplex formed with complementary
RNA as seen above for X when incorporated in a β-strand.
When DNA is used as the complementary strand, incorpor-
ation of Y has only a minor effect on the hybridization proper-
ties of the modified α-ODNs. The hybridization obtained
by modified β-ODNs with complementary DNA is similar to
that obtained earlier for the corresponding 3Ј-C-(hydroxy-
methyl)thymidine.27
1
by polarization transfer (INEPT) and H–13C COSY experi-
ments. Fast-atom bombardment (FAB) mass spectra were
recorded on a Kratos MS 50 RF spectrometer. Microanalyses
were performed at the Department of Chemistry, University of
Copenhagen. The silica gel used for column chromatography
(0.040–0.063 mm) was purchased from Merck. ODNs were
synthesized on an Assembler Gene Special DNA-Synthesizer
(Pharmacia Biotech). Purification of 5Ј-O-DMT-on ODNs
was accomplished using disposable Oligopurification Cart-
ridges (COP, Cruachem). MALDI-MS was performed in
positive mode on a Micromass TofSpec E mass spectrometer
using
a
matrix of diammonium citrate and 2,6-di-
hydroxyacetophenone. Analytical anion-exchange HPLC
(RESOURCETM Q, 1 cm3, Pharmacia Biotech) was performed
on a Waters Delta Prep 3000 Preparative Chromatography
System. Melting profiles of duplexes were obtained on a
Perkin-Elmer UV/VIS spectrometer fitted with a PTP-6 Peltiér
temperature-programming element. The reference oligonucle-
otide (rA14) was purchased from DNA Technology ApS,
Aarhus, Denmark. Light petroleum refers to the fraction with
distillation range 60–80 ЊC.
As depicted in Table 1, the hybridization properties of the
ODN L containing one 3Ј-C-aminopropyl α-monomer Z in the
middle were the most promising obtained towards both com-
plementary DNA and RNA as no (towards DNA) or only
minor (towards RNA) destabilizing effect was observed. These
results could originate from the cationic nature of the amino
group possibly leading to favourable partial neutralization of
the phosphate backbone.
Methyl 5-O-(tert-butyldimethylsilyl)-2-deoxy-á-D-erythro-
pentofuranoside 2 and methyl 5-O-(tert-butyldimethylsilyl)-2-
deoxy-â-D-erythro-pentofuranoside 3
In summary, synthesis of the novel β- and α-anomers of 3Ј-C-
allylthymidine has been accomplished and these nucleosides
have been incorporated into β- and α-ODNs. Incorporation of
the β-anomer into β-ODNs and the α-anomer into α-ODNs
have a similar impact on duplex formation with complementary
DNA and RNA. The increased thermal stabilities observed for
all the modified α-ODNs, compared with the unmodified T14,
An anomeric mixture of methyl 2-deoxy--erythro-pentofur-
anoside10 (51.32 g, 0.35 mmol) and imidazole (57.06 g, 0.84
mol) was dissolved in anhydrous DMF (400 cm3) under argon.
TBDMSCl (58.02 g, 0.36 mol) as a solution in anhydrous DMF
(100 cm3) was added dropwise over a period of 1 h. After 5 h,
the reaction mixture was concentrated under reduced pressure.
Purification by silica gel column chromatography (0–25%
3278
J. Chem. Soc., Perkin Trans. 1, 1997

View Article Online
EtOAc in light petroleum) afforded α-anomer 2 (25.83 g, 28%)
as the less polar compound, stereoisomer 3 (17.84 g, 19%) and a
fraction of the diprotected glycoside (12.29 g, 9%).
118.24 (᎐CH ) and 133.80 (᎐CH); δ (CDCl ) 0.11 [s, 6 H,
᎐ ᎐
2 H 3
Si(CH3)2], 0.91 [s, 9 H, C(CH3)3], 2.01 (dd, J 2.9 and 13.8, 1 H,
Ha-2), 2.17 (dd, J 5.6 and 13.8, 1 H, Hb-2), 2.37–2.50 (m, 2 H,
CH2), 3.35 (s, 3 H, OCH3), 3.66 (s, 1 H, OH), 3.78–3.81 (m, 1 H,
H-4), 3.93–3.95 (m, 2 H, H2-5), 5.07–5.11 (m, 2 H, H-1 and
Compound 2: δC(CDCl3) Ϫ5.64 and Ϫ5.42 [Si(CH3)2],
18.13 [C(CH3)3], 25.72 [C(CH3)3], 40.96 (C-2), 54.60 (OCH3),
63.63 (C-5), 73.03 (C-3), 87.69 (C-4) and 105.50 (C-1);
δH(CDCl3) 0.02 [s, 6 H, Si(CH3)2], 0.85 [s, 9 H, C(CH3)3], 1.91–
2.15 (m, 2 H, H2-2), 2.84 (d, J 10.4, 1 H, OH), 3.34 (s, 3 H,
OCH3), 3.53 (dd, J 4.8 and 10.9, 1 H, Ha-5), 3.70 (dd, J 3.6 and
10.9, 1 H, Hb-5), 4.05–4.09 (m, 1 H, H-4), 4.11–4.18 (m, 1 H, H-
3) and 5.04 (m, 1 H, H-1).
a
b
᎐CH ), 5.15–5.16 (m, 1 H, ᎐CH ) and 5.85–5.99 (m, 1 H,
᎐
᎐
᎐
2
2
᎐CH); FAB-MS m/z 303 [M ϩ H]ϩ (Found: C, 59.37; H,
9.60%).
Methyl 3-C-allyl-2-deoxy-á-D-erythro-pentofuranoside 7
Compound 5 (1.08 g, 3.57 mmol) was dissolved in anhydrous
THF (15 cm3)under argon. 1.1  TBAF in THF (3.2 cm3, 3.57
mmol) was added and the mixture was stirred for 15 min. EtOAc
(70 cm3) was added and the organic phase was washed with
saturated aq. NaHCO3 (3 × 30 cm3). The water phase was
extracted with EtOAc (60 cm3), and the organic phases were
combined, dried (Na2SO4) and concentrated under reduced
pressure. Purification using silica gel column chromatography
(20% EtOAc in light petroleum) afforded title diol 7 as an oil
(612 mg, 91%); δC(CDCl3) 38.76 (C-2), 44.95 (CH2), 54.92
(OCH3), 61.99 (C-5), 79.41 (C-3), 89.20 (C-4), 104.53 (C-1),
Compound 3: δC(CDCl3) Ϫ5.46 and Ϫ5.41 [Si(CH3)2], 18.27
[C(CH3)3], 25.88 [C(CH3)3], 40.93 (C-2), 54.98 (OCH3), 65.01
(C-5), 73.63 (C-3), 85.66 (C-4) and 105.02 (C-1); δH(CDCl3)
0.08 [s, 6 H, Si(CH3)2], 0.91 [s, 9 H, C(CH3)3], 1.99 (s, 1 H, OH),
2.06 (ddd, J 5.3, 6.6 and 13.3, 1 H, Ha-2), 2.22 (ddd, J 2.1, 6.8
and 13.3, 1 H, Hb-2), 3.32 (s, 3 H, OCH3), 3.54 (dd, J 8.0 and
9.7, 1 H, Ha-5), 3.75–3.90 (m, 2 H, Hb-5 and H-4), 4.42–4.44 (m,
1 H, H-3) and 5.06 (dd, J 2.1 and 5.3, 1 H, H-1).
Anomeric mixture of compounds 2 and 3: FAB-MS m/z 261
[M Ϫ H]Ϫ (Found: C, 54.74; H, 9.64. Calc. for C12H26O4Si: C,
54.92; H, 9.99%).
117.79 (᎐CH ) and 133.72 (᎐CH); δ (CDCl ) 2.00–2.03 (m, 2 H,
᎐
᎐
2
H
3
H2-2), 2.27–2.47 (m, 2 H, CH2), 2.65 (br s, 1 H, OH), 3.40 (s, 3
H, OCH3), 3.52–3.73 (m, 2 H, H2-5), 4.14 (dd, J 3.7 and 4.9, 1
Methyl 5-O-(tert-butyldimethylsilyl)-2-deoxy-á-D-glycero-pento-
furanoside-3-ulose 4
H, H-4), 5.09–5.17 (m, 3 H, H-1 and ᎐CH ) and 5.89–6.06 (m, 1
᎐
2
To a suspension of CrO3 (5.00 g, 50.0 mmol) in anhydrous
CH2Cl2 (150 cm3) were added anhydrous pyridine (8.1 cm3, 0.10
mol) and methyl glycoside 2 (5.86 g, 22.3 mmol) followed by
Ac2O (4.7 cm3, 49.7 mmol). The reaction mixture was stirred
under argon for 4 h at room temp. EtOAc (500 cm3) was added
and the mixture was filtered through a silica gel column. Con-
centration of the organic phase under reduced pressure
afforded title keto glycoside 4 as a pale yellow oil (4.77 g, 82%)
which was used without further purification in the next step;
δC(CDCl3) Ϫ5.70 and 5.51 [Si(CH3)2], 18.17 [C(CH3)3], 25.70
[C(CH3)3], 44.18 (C-2), 54.82 (OCH3), 62.25 (C-5), 78.81 (C-4),
104.80 (C-1) and 212.28 (C-3); δH(CDCl3) 0.04 and 0.05
[2 × s, 6 H, Si(CH3)2], 0.86 [s, 9 H, C(CH3)3], 2.33–2.60 (m, 2
H, H2-2), 3.44 (s, 3 H, OCH3), 3.83–3.98 (m, 3 H, H2-5 and H-4)
and 5.36 (m, 1 H, H-1).
H, ᎐CH) (Found: C, 57.60; H, 8.47. Calc. for C H O : C, 57.43;
H, 8.57%).
᎐
9
16
4
Methyl 3-C-allyl-2-deoxy-á-D-threo-pentofuranoside 8
Compound 6 (739 mg, 2.44 mmol) was dissolved in anhydrous
THF (10 cm3) under argon. 1.1  TBAF in THF (2.2 cm3, 2.4
mmol) was added and the mixture was stirred for 15 min.
EtOAc (40 cm3) was added and the organic phase was washed
with saturated aq. NaHCO3 (3 × 20 cm3). The water phase was
extracted with EtOAc (40 cm3), and the organic phases were
combined, dried (Na2SO4) and concentrated under reduced
pressure. Purification using silica gel column chromatography
(20% EtOAc in light petroleum) afforded title diol 8 as an oil
(377 mg, 82%); δC(CDCl3) 43.77 (CH2), 47.49 (C-2), 55.23
(OCH3), 61.23 (C-5), 80.82 (C-3), 81.87 (C-4), 103.81 (C-1),
Methyl 3-C-allyl-5-O-(tert-butyldimethylsilyl)-2-deoxy-á-D-
erythro-pentofuranoside 5 and methyl 3-C-allyl-5-O-(tert-butyl-
dimethylsilyl)-2-deoxy-á-D-threo-pentofuranoside 6
119.18 (᎐CH ) and 133.11 (᎐CH); δ (CDCl ) 2.04 (dd, J 2.9 and
᎐
᎐
2
H
3
14.0, 1 H, Ha-2), 2.20 (dd, J 5.64 and 14.0, 1 H, Hb-2), 2.43–2.47
(m, 2 H, CH2), 2.70 (br s, 1 H, OH), 3.37 (s, 3 H, OCH3), 3.80–
3.82 (m, 1 H, H-4), 3.93–3.94 (m, 2 H, H2-5), 5.13–5.17 (m, 2 H,
Compound 4 (4.29 g, 16.46 mmol) was coevaporated with
anhydrous toluene (2 × 25 cm3) and dissolved under argon in
anhydrous Et2O (150 cm3). The mixture was cooled to 0 ЊC, a 1
 solution of allylmagnesium bromide in anhydrous Et2O (16.5
cm3, 16.5 mmol) was added dropwise and the mixture was
stirred for 16 h at room temp. The reaction was quenched by
addition of saturated aq. NH4Cl (400 cm3) and the aqueous
phase was extracted with Et2O (3 × 250 cm3). The organic
phase was dried (Na2SO4) and evaporated. Purification by silica
gel column chromatography (0–3% EtOAc in light petroleum,
v/v) afforded erythro product 5 (789 mg, 16%) as the less polar
compound and its threo stereoisomer 6 (2.51 g, 50%).
a
b
H-1 and ᎐CH ), 5.20–5.21 (m, 1 H, ᎐CH ) and 5.83–6.00 (m, 1
᎐
᎐
2
2
H, ᎐CH); FAB-MS m/z 187 [M Ϫ H]Ϫ.
᎐
Methyl 3,5-di-O-acetyl-3-C-allyl-2-deoxy-á-D-erythro-
pentofuranoside 9
To a solution of compound 7 (1.71 g, 9.09 mmol) in anhydrous
pyridine (30 cm3) under argon were added DMAP (1.11 g, 9.10
mmol) and Ac2O (6.01 cm3, 54.54 mmol). After stirring of the
mixture for 48 h at room temperature, the solvent was evapor-
ated off and saturated aq. NaHCO3 (80 cm3) was added. The
aqueous phase was extracted with CH2Cl2 (3 × 125 cm3), and
the organic phases were combined, dried (Na2SO4) and concen-
trated under reduced pressure. Silica gel column chrom-
atography (10–30% EtOAc in light petroleum) gave title diac-
etate 9 (1.90 g, 77%); δC(CDCl3) 20.75 and 21.53 (2 × CH3),
37.23 (CH2), 44.66 (C-2), 54.98 (OCH3), 63.22 (C-5), 81.69 (C-
Compound 5: δC(CDCl3) Ϫ5.65 and Ϫ5.49 [Si(CH3)2], 18.10
[C(CH3)3], 25.81 [C(CH3)3], 38.67 (C-2), 44.71 (CH2), 54.90
(OCH3), 62.57 (C-5), 79.88 (C-3), 89.20 (C-4), 104.93 (C-1),
117.30 (᎐CH ) and 134.49 (᎐CH); δ (CDCl ) 0.06 and 0.07
᎐
᎐
2
H
3
[2 × s, 6 H, Si(CH3)2], 0.89 [s, 9 H, C(CH3)3], 1.88–1.93 (m, 1 H,
Ha-2), 2.14 (dd, J 5.2 and 13.2, 1 H, Hb-2), 2.36–2.55 (m, 2 H,
CH2), 3.39 (s, 3 H, OCH3), 3.59 (s, 1 H, OH), 3.66–3.69 (m, 2 H,
H2-5), 4.05–4.08 (m, 1 H, H-4), 5.05–5.08 (m, 2 H, H-1 and
4), 87.25 (C-3), 103.35 (C-1), 118.87 (᎐CH ), 131.90 (᎐CH) and
᎐
᎐
2
170.24 and 170.49 (2 × COCH3); δH(CDCl3) 2.02 and 2.09 (2 s,
a
b
a
᎐CH ), 5.12–5.15 (m, 1 H, ᎐CH ) and 5.93–6.10 (m, 1 H,
6 H, 2 × CH3), 2.25–2.45 (m, 3 H, H2-2 and CH2 ), 2.99 (dd, J
᎐
᎐
2
2
b
᎐CH) (Found: C, 59.71; H, 9.88. Calc. for C H O Si: C, 59.56;
7.4 and 14.5, 1 H, CH2 ), 3.37 (s, 3 H, OCH3), 4.20 (dd, J 7.0
᎐
15 30
4
H, 10.00%).
and 11.6, 1 H, Ha-5), 4.44 (dd, J 2.9 and 7.0, 1 H, H-4), 4.57 (dd,
J 2.9 and 11.6, 1 H, Hb-5), 5.03 (dd, J 2.3 and 4.9, 1 H, H-1),
Compound 6: δC(CDCl3) Ϫ5.61 and 5.49 [Si(CH3)2], 18.12
[C(CH3)3], 25.75 [C(CH3)3], 44.27 (CH2), 47.10 (C-2), 55.09
(OCH3), 62.42 (C-5), 80.64 (C-3), 81.69 (C-4), 103.93 (C-1),
a
b
5.07–5.10 (m, 1 H, ᎐CH ), 5.14–5.15 (m, 1 H, ᎐CH ) and 5.64–
᎐
᎐
2
2
5.80 (m, 1 H, ᎐CH); FAB-MS m/z 272 [M ϩ H]ϩ.
᎐
J. Chem. Soc., Perkin Trans. 1, 1997
3279

View Article Online
Methyl 3,5-di-O-acetyl-3-C-allyl-2-deoxy-á-D-threo-
pentofuranoside 10
1-(3,5-Di-O-acetyl-3-C-allyl-2-deoxy-á,â-D-threo-
pentofuranosyl)thymine 12
To a solution of compound 8 (2.92 g, 1.55 mmol) in anhydrous
pyridine (5 cm3) under argon were added DMAP (195 mg, 1.6
mmol) and Ac2O (0.68 cm3, 6.20 mmol). After stirring of the
mixture for 48 h at room temperature, additional Ac2O (0.68
cm3, 6.20 mmol) was added. The reaction mixture was stirred
for another 72 h, the solvent was evaporated off, and saturated
aq. NaHCO3 (15 cm3) was added. The aqueous phase was
extracted with CH2Cl2 (3 × 25 cm3), and the organic phases
were combined, dried (Na2SO4) and concentrated under
reduced pressure. Silica gel column chromatography (30%
EtOAc in light petroleum, v/v) gave title diacetate 10 (191 mg,
45%); δC(CDCl3) 20.81 and 21.58 (2 × CH3), 40.05 and 44.74
(CH2 and C-2), 54.93 (OCH3), 63.61 (C-5), 81.06 (C-4), 87.82
Method A. Used amounts. Methyl glycoside 10 (129 mg, 0.47
mmol), thymine (120 mg, 0.95 mmol), anhydrous CH2ClCH2Cl
(7 cm3), BSA (0.70 cm3, 2.84 mmol) and TMS triflate (0.13 cm3,
0.65 mmol). Purification using preparative TLC (PLC) (2%
CH3OH in CH2Cl2, double run) afforded an anomeric mixture
of the β- and α-nucleoside 12 (112 mg, 64%); δC(CDCl3) 12.33
and 12.44 (2 × CH3), 20.66, 20.68, 21.46 and 21.66
(4 × COCH3), 37.99, 38.03, 40.52and41.65(2 × C-2ЈandCH2Ј),
62.64 and 63.37 (2 × C-5Ј), 83.13, 83.59, 84.08, 86.38, 86.81 and
88.39 (2 × C-1Ј, -3Ј and -4Ј), 110.46 and 110.95 (2 × C-5),
120.31 and 120.67 (2 × ᎐CH Ј), 130.55 and 131.01 (2 ×
᎐
2
᎐CHЈ), 134.63 and 136.00 (2 × C-6), 150.31 and 150.34 (2 ×
᎐
C-2), 163.84 and 164.01 (2 × C-4) and 169.23, 169.74, 170.43
and 170.60 (4 × COCH3); δH(CDCl3) 1.86, 1.87, 1.93, 2.00, 2.04
and 2.05 (6 s, 2 × CH3, 4 × COCH3), 2.35 (dd, J 7.6 and 15.0,
CH2Јa), 2.62–2.95 (m, 2 × H2-2Ј and CH2Јb), 3.16 (dd, J 6.6 and
15.0, CH2Јb), 4.07–4.15 (m, H2-4Ј), 4.38–4.43 (m, H2-5Ј), 5.10–
(C-3), 103.59 (C-1), 119.34 (᎐CH ), 131.75 (᎐CH) and 169.65
᎐
᎐
2
and 170.72 (2 × COCH3); δH(CDCl3) 2.00 and 2.10 (2 s, 6 H,
2 × CH3), 2.33 (dd, J 1.7 and 14.9, 1 H, Ha-2), 2.65 (dd, J 5.7
a
and 15.0, 1 H, Hb-2), 2.65–2.73 (m, 1 H, CH2 ), 3.05–3.13 (m, 1
b
H, CH2 ), 3.36 (s, 3 H, OCH3), 4.12–4.21 (m, 2 H, H2-5), 4.38–
5.25 (m, 2 × ᎐CH Ј), 5.61–5.77 (m, 2 × ᎐CHЈ), 5.99–6.06 (m,
᎐ ᎐
2
4.47 (m, 1 H, H-4), 5.05–5.17 (m, 3 H, H-1 and ᎐CH ) and 5.67–
2 × H-1Ј), 7.04 and 7.35 (2 s, 2 × H-6) and 9.89 and 9.93 (2 s,
2 × NH); FAB-MS m/z 367 [M ϩ H]ϩ (Found: C, 54.99; H,
6.00; N, 7.53. Calc. for C17H22N2O7ؒ0.25H2O: C, 55.06; H, 6.12;
N, 7.55%).
᎐
2
5.81 (m, 1 H, ᎐CH).
᎐
1-(3,5-Di-O-acetyl-3-C-allyl-2-deoxy-á,â-D-erythro-
pentofuranosyl)thymine 11
1-(3-C-Allyl-2-deoxy-á,â-D-erythro-pentofuranosyl)thymine 13
The anomeric mixture of nucleosides 11 (894 mg, 2.41 mmol)
was dissolved in anhydrous CH3OH (25 cm3) under argon and
CH3ONa (660 mg, 12.2 mmol) was added. After stirring of the
mixture for 12 h at room temp., water (25 cm3) was added and
the mixture was neutralized with 4  HCl. The aqueous phase
was extracted with 3-methylbutan-1-ol (3 × 100 cm3) and the
organic phase was concentrated under reduced pressure. Purifi-
cation using silica gel column chromatography (0–5% CH3OH
in CH2Cl2) afforded an anomeric mixture of β- and α-
nucleoside 13 (β:α; 1.5:1) (569 mg, 81%); δC(CD3OD) 12.50
and 12.57 (2 × CH3), 40.94, 41.13, 44.32 and 45.76 (2 × C-2Ј,
2 × CH2Ј), 62.20 and 62.37 (2 × C-5Ј), 80.48, 81.51, 85.97,
87.64, 90.00 and 91.87 (2 × C-1Ј, -3Ј, -4Ј), 110.02 and 111.34
Method A. Methyl glycoside 9 (1.79 g, 6.57 mmol) and thy-
mine (1.66 g, 13.14 mmol) were dissolved in anhydrous
CH2ClCH2Cl (100 cm3) under argon and BSA (9.75 cm3, 39.45
mmol) was added. The mixture was refluxed for 15 min at
78 ЊC. The resulting clear mixture was allowed to cool to room
temperature and TMS triflate (1.83 cm3, 9.20 mmol) was added
dropwise over a period of 10 min. After being stirred for 48 h at
room temperature, the mixture was diluted with CH2Cl2 (100
cm3) and poured into ice–water (50 cm3) saturated with
NaHCO3. The aqueous phase was extracted with CH2Cl2
(3 × 150 cm3), and the organic phase was combined, dried
(Na2SO4) and concentrated under reduced pressure. Purific-
ation using silica gel column chromatography (10–30% EtOAc
in light petroleum) afforded an anomeric mixture of the β- and
α-nucleoside 11 (β:α; 1:2.3) (1.25 g, 52%).
(2 × C-5), 118.67 and 118.85 (2 × ᎐CH Ј), 134.64 and 134.72
᎐
2
(2 × ᎐CHЈ), 138.61 and 139.26 (2 × C-6), 152.32 and 152.44
᎐
Method B. To a stirred suspension of the methyl glycoside 9
(901 mg, 3.32 mmol) and thymine (843 mg, 6.68 mmol) in
anhydrous CH3CN (30 cm3) under argon at room temperature
was dropwise added BSA (5 cm3, 20.23 mmol). The mixture was
stirred for 1 h until clearness was attained. The reaction mixture
was cooled to Ϫ30 ЊC, and TMS triflate (0.95 cm3, 5.25 mmol)
was added dropwise. The reaction mixture was stirred for 72 h at
room temperature, then was diluted with CH2Cl2 (50 cm3), and
the reaction was quenched with saturated aq. NaHCO3 (2 × 50
cm3). After being washed with water (2 × 50 cm3), the organic
phase was dried (Na2SO4) and concentrated under reduced
pressure. Purification using silica gel column chromatography
(0–1% MeOH in CH2Cl2) afforded an anomeric mixture of the
β- and α-nucleoside 11 (β:α; 1.5:1) (894 mg, 74%); δC(CDCl3)
12.41 and 12.45 (2 × CH3), 20.70, 21.52 and 21.59
(4 × COCH3), 35.75 and 36.05 (2 × C-2Ј), 42.85 and 43.62
(2 × CH2Ј), 62.79 and 63.05 (2 × C-5Ј), 82.24, 83.92, 84.04,
85.76, 88.49 and 88.68 (2 × C-1Ј, -3Ј and -4Ј), 109.55 and 111.04
(2 × C-2) and 166.36 and 166.61 (2 × C-4); δH(CD3OD) 2.02
and 2.08 (2 s, 2 × CH3), 2.14–2.38 (m, 2 × Ha-2Ј and Hb-2Ј),
2.57–2.73 (m, 2 × CH2Ј and Hb-2Ј), 3.73–3.98 (m, 2 × H-4Ј and
-5Ј), 4.05 (m, OH), 4.34 (m, OH), 5.25–5.33 (m, 2 × ᎐CH Ј),
᎐
2
6.00–6.16 (m, 2 × ᎐CHЈ), 6.33 (dd, J 2.1 and 7.7, H-1Ј), 6.46
᎐
(dd, J 5.4 and 9.3, H-1Ј), 8.21 and 8.22 (2 s, 2 × H-6) and 9.97
and 10.03 (2 s, 2 × NH); EI-MS m/z 282 (Mϩ, 9%); HR-MS
(Found: Mϩ, 282.1210. Calc. for C13H18N2O5: M, 282.1216)
(Found: C, 53.20; H, 6.31; N, 9.94. Calc. for C13H18N2O5ؒ
0.25H2O: C, 53.60; H, 6.57; N, 9.62%).
1-(3-C-Allyl-2-deoxy-â-D-threo-pentofuranosyl)thymine 14a and
1-(3-C-allyl-2-deoxy-á-D-threo-pentofuranosyl)thymine 14b
An anomeric mixture of nucleosides 12 (79 mg, 0.22 mmol) was
dissolved in a saturated solution of NH3 in methanol (10 cm3).
After stirring of the mixture for 15 h at room temp., the solvent
was removed under reduced pressure and the crude product was
purified by silica gel column chromatography (3% CH3OH in
CH2Cl2) to give anomers 14a (23 mg, 39%) and 14b (26 mg,
44%).
(2 × C-5), 119.41 and 119.53 (2 × ᎐CH Ј), 131.03 and 131.16
᎐
2
(2 × ᎐CHЈ), 134.48 and 135.17 (2 × C-6), 150.39 and 150.48
᎐
(2 × C-2), 163.81 and 164.12 (2 × C-4) and 169.71, 169.92 and
170.10 (4 × COCH3); δH(CDCl3) 1.94, 1.96, 2.07 and 2.13 (4 s,
2 × CH3, 4 × COCH3), 2.36–2.85 (m, 2 × H2-2 and 2 × CH2Јa),
3.08–3.29 (m, 2 × CH2Јb), 4.11–4.19 (m, 2 × H-4Ј), 4.33–4.41
β-Isomer 14a: δC(CDCl3) 12.36 (CH3), 42.56 and 44.63 (C-2Ј,
CH2Ј), 60.87 (C-5Ј), 79.61 (C-4Ј), 84.48, 85.11 (C-1Ј and -3Ј),
109.47 (C-5), 119.43 (᎐CH Ј), 132.57 (᎐CHЈ), 138.08 (C-6),
᎐
᎐
2
150.83 (C-2) and 164.50 (C-4); δH(CDCl3) 1.81 (s, 3 H, CH3),
2.32–2.58 (m, 4 H, H2-2Ј, CH2Ј), 3.74 (m, 1 H, H-4Ј), 3.84 (br s,
1 H, OH), 4.08–4.16 (m, 2 H, H2-5Ј), 4.78 (s, 1 H, OH), 5.15–
5.22 (m, 2 H, ᎐CH Ј), 5.84–6.00 (m, 1 H, ᎐CHЈ), 6.08 (dd, J 2.9
and 4.69–4.72 (2 m, 2 × H -5Ј), 5.00–5.17 (m, 2 × ᎐CH Ј), 5.63–
᎐
2
2
5.75 (m, 2 × ᎐CHЈ), 6.18–6.27 (m, 2 × H-1Ј), 7.36 and 7.39 (2 s,
᎐
2 × H-6) and 9.99 and 10.03 (2 s, 2 × NH); EI-MS m/z 366 [Mϩ,
25%] (Found: C, 55.54; H, 6.17; N, 7.31. Calc. for C17H22N2O7:
C, 55.73; H, 6.05; N, 7.65%).
᎐
᎐
2
and 7.5, 1 H, H-1Ј), 7.99 (s, 1 H, H-6) and 9.93 (s, 1 H, NH).
α-Isomer 14b: δC(CDCl3) 12.46 (CH3), 42.05 and 45.01 (C-2Ј,
3280
J. Chem. Soc., Perkin Trans. 1, 1997

View Article Online
CH2Ј), 60.87 (C-5Ј), 81.01 (C-4Ј), 85.28 and 86.09 (C-1Ј and
-3Ј), 111.14 (C-5), 119.28 (᎐ CH Ј), 132.69 (᎐CHЈ), 135.74 (C-6),
150.74 (C-2) and 164.39 (C-4); δH(CDCl3) 2.00 (s, 3 H, CH3),
2.36–2.45 (m, 4 H, H-2Ј, CH2Ј), 3.46 (s, 1 H, OH), 3.96–4.02 (m,
2 H, H2-5Ј), 4.10 (m, 1 H, H-4Ј), 4.60 (br s, 1 H, OH), 5.13–5.18
1-{3-C-Allyl-3-O-[cyanoethoxy(diisopropylamino)phosphino]-2-
deoxy-5-O-(4,4Ј-dimethoxytrityl)-á-D-erythro-pentofuranosyl}-
thymine 16b
᎐
᎐
2
Method C. Used amounts. Nucleoside 15b (304 mg, 0.52
mmol), anhydrous CH2Cl2 (2.1 cm3), DIPEA (0.57 cm3, 3.33
mmol) and 2-cyanoethyl N,N-diisopropylphosphoramido-
chloridite (0.25 cm3, 1.06 mmol) as above. Purification using
silica gel column chromatography (EtOAc–CH2Cl2–Et3N–light
petroleum, 15:30:5:50, v/v/v/v) followed by precipitation in
light petroleum (150 cm3) at Ϫ20 ЊC [after re-dissolution in
anhydrous toluene (1.5 cm3)] afforded compound 16b as a solid
(306 mg, 77%); δP(CDCl3) 143.94.
(m, 2 H, ᎐CH Ј), 5.82–5.98 (m, 1 H, ᎐CHЈ), 6.28 (dd, J 6.1 and
᎐
᎐
2
8.1, 1 H, H-1Ј), 7.19 (s, 1 H, H-6) and 10.05 (br s, 1 H, NH).
1-[3-C-Allyl-2-deoxy-5-O-(4,4Ј-dimethoxytrityl)-â-D-erythro-
pentofuranosyl]thymine 15a and 1-[3-C-allyl-2-deoxy-5-O-(4,4Ј-
dimethoxytrityl)-á-D-erythro-pentofuranosyl]thymine 15b
An anomeric mixture of nucleoside 13 (569 mg, 2.11 mmol),
AgNO3 (358 mg, 2.11 mmol) and DMTCl (1.785 g, 5.27 mmol)
were dissolved in anhydrous THF (50 cm3), and anhydrous
pyridine (0.85 cm3, 10.5 mmol) was added. The mixture was
stirred at room temp. for 72 h. The mixture was filtered into 5%
aq. sodium hydrogen carbonate (100 cm3). The product was
extracted into CH2Cl2 (4 × 100 cm3), and the combined extract
was dried (Na2SO4), and concentrated under reduced pressure.
Purification by PLC (1% pyridine, 3% CH3OH in CH2Cl2, v/v/v,
3 runs) gave the α-anomer 15b (304 mg, 26%) as the less polar
isomer and the β-anomer 15a (545 mg, 47%).
1-(3-C-Allyl-â-D-ribofuranosyl)thymine 17
To a solution of compound 18 (2.16 g, 4.51 mmol) in anhydrous
CH2Cl2 (70 cm3) under argon at Ϫ78 ЊC was added dropwise
BCl3 (1  solution in hexane; 18.1 cm3, 18.1 mmol). The mix-
ture was stirred for 3.5 h at Ϫ78 ЊC, additional BCl3 was added
(1  hexane solution; 4.0 cm3, 4.0 mmol), and the mixture was
stirred for a further 2 h. MeOH (50 cm3) was added to the
mixture, which was then stirred overnight at room temp. After
concentration under reduced pressure and coevaporation with
MeOH (3 × 5 cm3), the residue was purified using silica gel col-
umn chromatography (3–6% MeOH in CH2Cl2) to give title
compound 17 (977 mg, 73%), which was used in the next step
without further purification. An analytical sample was
obtained by recrystallization from MeOH; δC(CD3OD) 12.68
(CH3), 39.65 (CH2Ј), 62.22 (C-5Ј), 78.62 (C-2Ј), 79.91 (C-3Ј),
β-Isomer 15a. δC(CDCl3) 11.22 (CH3), 39.89 (C-2Ј), 44.16
(CH2Ј), 55.22 (OCH3), 62.46 (C-5Ј), 80.13 (C-3Ј), 83.97 (C-1Ј),
87.34 (CPh3, C-4Ј), 111.21 (C-5), 113.18, 127.92, 128.01, 128.44,
129.92, 130.25, 130.28, 134.71, 134.91, 143.64 and 158.82
(Carom), 119.84 (᎐CH Ј), 132.50 (᎐CHЈ), 136.12 (C-6), 150.59
᎐
᎐
2
(C-2) and 163.87 (C-4); δH(CDCl3) 1.22 (s, 3 H, CH3), 2.12–2.41
(m, 4 H, CH2Ј, H2-2Ј), 3.02 (dd, J 2.4 and 10.8, 1 H, Ha-5Ј), 3.67
(dd, J 3.5 and 10.8, 1 H, Hb-5Ј), 3.79 (s, 6 H, 2 × OCH3), 4.06
(m, 1 H, H-4Ј), 4.90 (dd, J 1.4 and 17.1, 1 H, ᎐CH Јa), 5.09 (dd,
88.78 (C-4Ј), 89.05 (C-1Ј), 112.14 (C-5), 118.91 (᎐CH Ј), 135.11
᎐
2
(᎐CHЈ), 139.63 (C-6), 153.68 (C-2) and 166.90 (C-4);
᎐
δH(CD3OD) 1.88 (s, 3 H, CH3), 2.49–2.56 (dd, J 8.3 and 14.8, 1
H, CH2Јa), 2.56–2.63 (dd, J 6.2 and 14.8, 1 H, CH2Јb), 3.72–3.77
(dd, J 2.6 and 12.1, 1 H, Ha-5Ј), 3.80–3.84 (dd, J 2.3 and 12.2, 1
H, Ha-5Ј), 3.94–3.96 (t, J 2.6, 1 H, H-4Ј), 4.16 (d, J 7.9, 1 H, H-
᎐
2
J 1.4 and 10.2, 1 H, ᎐CH Јb), 5.68–5.82 (m, 1 H, ᎐CHЈ), 6.51
᎐
᎐
2
(dd, J 5.1 and 9.4, 1 H, H-1Ј), 6.84 (m, 4 H, ArH), 7.21–7.45 (m,
9 H, ArH), 7.86 (s, 1 H, H-6) and 9.20 (s, 1 H, NH).
2Ј), 5.12–5.21 (m, 2 H, ᎐CH Ј), 5.99–6.05 (m, 1 H, ᎐CHЈ), 6.00
᎐
᎐
2
α-Isomer 15b: δC(CDCl3) 12.50 (CH3), 39.93 (C-2Ј), 45.43
(CH2Ј), 55.19 (OCH3), 63.17 (C-5Ј), 79.57 (C-3Ј), 86.96 (C-4Ј),
87.20 (CPh3), 89.32 (C-1Ј), 109.15 (C-5), 113.27, 126.91, 127.96,
128.04, 129.91, 129.93, 135.43, 135.72, 144.37 and 158.57
(d, J 7.9, 1 H, H-1Ј) and 8.05 (s, 1 H, H-6); FAB-MS m/z 299
[M ϩ H]ϩ (Found: C, 52.39; H, 5.84; N, 9.26. Calc. for
C13H18N2O6: C, 52.34; H, 6.08; N, 9.39%).
1-[3-C-Allyl-5-O-(4,4Ј-dimethoxytrityl)-â-D-ribofuranosyl]-
thymine 18
(Carom), 120.02 (᎐CH Ј), 132.63 (᎐CHЈ), 137.58 (C-6), 150.52
᎐
᎐
2
(C-2) and 164.21 (C-4); δH(CDCl3) 1.90 (s, 3 H, CH3), 2.17–2.34
(m, 3 H, CH2Ј, Hα-2Ј), 2.79 (dd, J 7.7 and 14.3, 1 H, Hβ-2Ј), 3.02
(dd, J 2.6 and 10.8, 1 H, Ha-5Ј), 3.44 (dd, J 3.7 and 10.8, 1 H,
Hb-5Ј), 3.78 (s, 6 H, 2 × OCH3), 4.31 (m, 1 H, H-4Ј), 4.98 (dd,
J 1.3 and 17.0, 1 H, ᎐CH Јa), 5.10 (dd, J 1.3 and 10.1, 1 H,
Nucleoside 17 (977 mg, 3.28 mmol) was coevaporated with
anhydrous pyridine (3 × 10 cm3) and re-dissolved in anhydrous
pyridine (8 cm3). DMTCl (1.33 g, 3.93 mmol) was added and
the mixture was stirred for 16 h under argon at room temp. The
solution was evaporated under reduced pressure, the residue
was re-dissolved in CH2Cl2 (40 cm3), and the solution was
washed with saturated aq. NaCl (3 × 30 cm3). The water phase
was extracted with CH2Cl2 (2 × 10 cm3). The combined organic
phases were dried (Na2SO4), and concentrated under reduced
pressure. Purification using silica gel column chromatography
(20–60% EtOAc in light petroleum, 0.5% pyridine, v/v/v) gave
title compound 18 (1.66 g, 82%); δC(CDCl3), 11.19 (CH3), 38.25
(CH2Ј), 55.18 (OCH3), 62.04 (C-5Ј), 77.83 and 78.31 (C-2Ј and
-3Ј), 86.00, 87.39 and 87.58 (C-1Ј, -4Ј and CPh3), 111.28 (C-5),
113.26, 113.30, 127.38, 128.07, 128.61, 132.50, 134.80, 134.90,
᎐
2
᎐CH Јb), 5.69–5.80 (m, 1 H, ᎐CHЈ), 6.41 (m, 1 H, H-1Ј), 6.85
᎐
᎐
2
(m, 4 H, ArH), 7.16–7.45 (m, 9 H, ArH), 7.71 (s, 1 H, H-6) and
9.13 (s, 1 H, NH); FAB-MS m/z 584 [M]ϩ (Found: C, 69.28; H,
6.20; N, 5.19. Calc. for C34H36N2O7ؒ0.1H2O: C, 69.63; H, 6.22;
N, 4.78%).
1-{3-C-Allyl-3-O-[cyanoethoxy(diisopropylamino)phosphino]-2-
deoxy-5-O-(4,4Ј-dimethoxytrityl)-â-D-erythro-pentofuranosyl}-
thymine 16a
Method C. General method for phosphitylation. Nucleoside
15a (545 mg, 0.93 mmol) was coevaporated with anhydrous
CH3CN (3 × 2 cm3) and was then dissolved under argon in
anhydrous CH2Cl2 (3.7 cm3). DIPEA (1.02 cm3, 5.97 mmol) was
added followed by dropwise addition of 2-cyanoethyl N,N-
diisopropylphosphoramidochloridite (0.44 cm3, 1.86 mmol).
After 5 h, CH3OH (1 cm3) was added and the reaction mixture
was diluted with EtOAc (20 cm3) containing triethylamine (0.2
cm3), washed successively with saturated aq. NaHCO3 (3 × 30
cm3) and saturated aq. NaCl (2 × 30 cm3), dried (Na2SO4) and
evaporated under reduced pressure. Purification using silica gel
column chromatography (EtOAc–CH2Cl2–Et3N–light petrol-
eum, 15:30:5:50, v/v/v/v) followed by precipitation in light
petroleum (200 cm3) at Ϫ20 ЊC [after re-dissolution in
anhydrous toluene (2 cm3)] afforded compound 16a as a solid
(534 mg, 75%); δP(CDCl3) 138.88 and 138.28.
136.70, 143.80 and 158.96 (᎐CHЈ, C-6 and Carom), 118.75
᎐
(᎐CH Ј), 152.01 (C-2) and 164.37 (C-4); δ (CDCl ) 1.13 (s, 3 H,
᎐
2
H
3
CH3), 2.21–2.29 (dd, J 8.3 and 14.5, 1 H, CH2Јa), 2.46–2.53 (dd,
J 5.6 and 14.5, 1 H, CH2Јb), 3.24–3.29 (dd, J 2.2 and 10.7, 1 H,
Ha-5Ј), 3.64–3.68 (dd, J 2.9 and 10.9, 1 H, Hb-5Ј), 3.76 (s, 6 H,
2 × OCH3), 4.17 (m, 1 H, H-4Ј), 4.28 (d, 1 H, J 7.2, H-2Ј), 4.49
(d, 1 H, J 17.1, ᎐CH Јa), 4.90 (d, 1 H, J 10.3, ᎐CH Јb), 5.75–5.83
᎐
᎐
2
2
(m, 1 H, ᎐CHЈ), 6.15 (d, 1 H, J 7.2, H-1Ј), 6.82–6.86 (m, 4 H,
᎐
ArH), 7.23–7.38 (m, 9 H, ArH) and 7.79 (s, 1 H, H-6); FAB-MS
m/z 600 [M]ϩ (Found: C, 67.88; H, 5.86; N, 5.13. Calc. for
C34H36N2O8ؒ0.2C5H5N: C, 68.19; H, 6.05; N, 5.00%).
1-[3-C-Allyl-2-O-(tert-butyldimethylsilyl)-5-O-(4,4Ј-dimethoxy-
trityl)-â-D-ribofuranosyl]thymine 19
Nucleoside 18 (1.61 g, 2.68 mmol) was coevaporated with
J. Chem. Soc., Perkin Trans. 1, 1997
3281

View Article Online
anhydrous CH3CN (3 × 10 cm3) and re-dissolved in anhydrous
DMF (10 cm3). Imidazole (1.46 g, 21.4 mmol) was added fol-
lowed by the addition of TBDMSCl (1.61 g, 10.7 mmol). The
mixture was stirred at room temp. under argon for 72 h. MeOH
(3 cm3) was added and the solution was concentrated under
reduced pressure. The residue was re-dissolved in CH2Cl2 (30
cm3) and this solution was washed with water (3 × 20 cm3). The
water phase was extracted with CH2Cl2 (20 cm3) and the com-
bined organic phases were dried (Na2SO4), and concentrated
under reduced pressure. Purification using silica gel column
chromatography (20–30% EtOAc in light petroleum, 0.5%
pyridine, v/v/v) gave title compound 19 (1.54 g, 82%);
δC(CDCl3) Ϫ4.62 and Ϫ4.56 (2 × SiCH3), 10.43 (CH3), 17.70
[C(CH3)3], 25.52 [C(CH3)3], 38.40 (CH2Ј), 55.19 and 55.28
(2 × OCH3), 61.68 (C-5Ј), 78.00 and 78.46 (C-2Ј and -3Ј), 84.57,
85.93 and 87.66 (C-1Ј, -4Ј and CPh3), 111.83 (C-5), 113.26,
113.30, 127.68, 128.04, 128.93, 130.66, 132.34, 134.41, 134.44,
[s, 2 × C(CH3)3], 1.93 (s, 2 × CH3), 1.98–2.11 (m, 2 × Ha-2Ј),
2.40–2.65 (m, 2 × Hb-2Ј, 2 × CH2Ј), 3.73–3.98 (m, 2 × H2-5Ј),
4.04‡ (m, H-4Ј), 4.22 (m, H-4Ј), 4.94–5.17 (m, 2 × ᎐CH Ј),
᎐
2
5.74–5.99 (m, 2 × ᎐CHЈ), 6.15‡ (dd, J 4.9 and 9.2, H-1Ј), 6.27
᎐
(dd, J 2.5 and 8.1 H-1Ј), 7.61‡ and 7.64 (2 s, 2 × H-6) and 9.22
and 9.34‡ (2 s, 2 × NH); FAB-MS m/z 469 [M ϩ H]ϩ (Found:
C, 56.47; H, 8.20; N, 5.78. Calc. for C22H40N2O5Si2: C, 56.37; H,
8.60; N, 5.98%).
1-[5-O-(tert-Butyldimethylsilyl)-2-deoxy-3-C-(3-hydroxy-
propyl)-3-O-trimethylsilyl-á,â-D-erythro-pentofuranosyl]-
thymine 22
To a stirred solution of allyl compound 21 (918 mg, 1.96 mmol)
in anhydrous THF (4 cm3) under argon at room temp. was
added BH3ؒ1,4-oxathiane (0.27 cm3 of a 7.8  solution in 1,4-
oxathiane, 2.11 mmol). The mixture was cooled to 0 ЊC and 2 
aq. NaOH (1.1 cm3) was slowly added followed by the dropwise
addition of 30% aq. H2O2 (0.28 cm3); stirring was continued for
60 min at room temp. The reaction mixture was poured into
ice–water (50 cm3) and extracted with Et2O. The organic phase
was washed successively with saturated aq. NaHCO3 (2 × 50
cm3) and water (50 cm3), dried (Na2SO4), and evaporated under
reduced pressure. The crude product was purified by silica gel
column chromatography (0–40% EtOAc in light petroleum, v/v)
to give title compound 22 as a solid (602 mg, 63%); δC(CDCl3)
Ϫ5.75, Ϫ5.61 and Ϫ5.44 [2 × Si(CH3)2], 2.00 and 2.27
[2 × Si(CH3)3], 12.50 and 12.56‡ (2 × CH3), 18.07 and 18.17‡
[2 × C(CH3)3], 25.78 and 25.90‡ [2 × C(CH3)3], 27.71‡ and
27.82 (2 × C-2Љ), 31.49‡ and 31.81 (2 × C-1Љ), 44.67‡ and
46.01 (2 × C-2Ј), 62.79, 62.94 and 63.19 (2 × C-5Ј and 2 × C-
3Љ), 84.38 and 84.48‡ (2 × C-3Ј), 85.79 (2 × C-1Ј), 88.61‡ and
89.93 (2 × C-4Ј), 109.69 and 109.97‡ (2 × C-5), 135.88‡ and
137.30 (2 × C-6), 150.44‡ and 150.65 (2 × C-2) and 164.15‡
and 164.26 (2 × C-4); δH(CDCl3) 0.01–0.24 [2 × Si(CH3)2 and
2 × Si(CH3)3], 0.91 [s, 2 × C(CH3)3], 1.59–2.04 (m, 2 × CH3, Ha-
2Ј‡, H2-1Љ), 2.10 (dd, J 1.7 and 14.1, Ha-2Ј), 2.48‡ (m, Hb-2Ј),
2.53 (dd, J 8.3 and 14.1, Hb-2Ј), 3.67–3.90 (m, 2 × H2-5Ј and H2-
3Љ), 4.07‡ (m, H-4Ј), 4.23 (m, H-4Ј), 6.18‡ (dd, J 4.9 and 9.2, H-
1Ј), 6.27 (dd, J 2.5 and 8.2, H-1Ј), 7.63‡ and 7.69 (2 s, 2 × H-6)
and 9.00 (br s, 2 × NH); FAB-MS m/z 487 [M ϩ H]ϩ (Found:
C, 54.34; H, 8.44; N, 5.59. Calc. for C22H42N2O6Si2: C, 54.29; H,
8.70; N, 5.76%).
136.44, 143.29 and 159.15 (᎐CHЈ, C-6 and Carom), 118.37
᎐
(᎐CH ), 150.94 (C-2) and 163.77 (C-4); δ (CDCl ) 0.00 and 0.19
᎐
2
H
3
(2 s, 6 H, 2 × SiCH3), 0.92 [s, 9 H, C(CH3)3], 0.95 (s, 3 H, CH3),
2.32 (d, J 6.8, 2 H, CH2Ј), 3.42 (d, J 10.6, 1 H, Ha-5Ј), 3.72–3.76
(dd, J 3.1 and 10.1, 1 H, Hb-5Ј), 3.79 (s, 6 H, 2 × OCH3), 4.03–
4.10 (m, 2 H, ᎐CH Јa, H-4Ј), 4.38 (d, J 7.5, 1 H, H-2Ј), 4.78 (d,
᎐
2
J 10.7, 1 H, ᎐CH Јb), 5.71–5.77 (m, 1 H, ᎐CHЈ), 6.22 (d, J 7.4,
᎐
᎐
2
1 H, H-1Ј), 6.84 (m, 4 H, ArH), 7.20–7.34 (m, 9 H, ArH), 7.83
(s, 1 H, H-6) and 8.76 (s, 1 H, NH); FAB-MS m/z 714
[M]ϩ(Found: C, 66.78; H, 6.90; N, 3.99. Calc. for C40H50-
N2O8Siؒ0.25 H2O: C, 66.78; H, 7.08; N, 3.89%).
1-{3-C-Allyl-2-O-(tert-butyldimethylsilyl)-3-O-[2-cyanoethoxy-
(diisopropylamino)phosphino]-5-O-(4,4Ј-dimethoxytrityl)-
â-D-ribofuranosyl]thymine 20
Method C. Used amounts. Nucleoside 19 (252 mg, 0.35
mmol), anhydrous CH2Cl2 (3 cm3), DIPEA (2.63 mmol, 0.45
cm3), 2-cyanoethyl N,N-diisopropylphosphoramidochloridite
(1.4 mmol, 0.33 cm3) as above. After 24 h, the reaction was
quenched by addition of CH3OH (1 cm3). Purification using
silica gel column chromatography (0.5–1% Et3N in CH2Cl2, v/v)
followed by precipitation in light petroleum (250 cm3) at Ϫ40 ЊC
after re-dissolution in anhydrous toluene (2 cm3) afforded title
compound 20 (249 mg, 78%); δP(CDCl3) 139.50 and 140.75.
1-[3-C-Allyl-5-O-(tert-butyldimethylsilyl)-2-deoxy-3-O-
trimethylsilyl-á,â-D-erythro-pentofuranosyl]thymine 21
1-[5-O-(tert-Butyldimethylsilyl)-2-deoxy-3-C-(3-phthalimido-
propyl)-3-O-trimethylsilyl-á,â-D-erythro-pentofuranosyl]-
thymine 23
To a stirred suspension of the methyl glycoside 5 (720 mg, 2.38
mmol) and thymine (604 mg, 4.79 mmol) in anhydrous CH3CN
(25 cm3) under argon at room temp. was added dropwise BSA
(4.7 cm3, 19.01 mmol). The mixture was stirred for 1 h until
clearness. The reaction mixture was then cooled to Ϫ30 ЊC, and
TMS triflate (0.86 cm3, 4.75 mmol) was dropwise added. The
reaction mixture was stirred for 7 days at room temp. before
being diluted with CH2Cl2 (50 cm3) and the reaction quenched
with saturated aq. NaHCO3 (2 × 50 cm3). After being washed
with water (2 × 50 cm3) the organic phase was dried (Na2SO4),
and concentrated under reduced pressure. Purification using
silica gel column chromatography (0–1% MeOH in CH2Cl2,
v/v) afforded a (1:2) anomeric mixture of nucleosides 21
(926 mg, 83%); δC(CDCl3) Ϫ5.92, Ϫ5.81, Ϫ5.76 and Ϫ5.61
[2 × Si(CH3)2], 1.92, 2.03 and 2.17 [2 × Si(CH3)3], 12.39 and
12.45‡ (2 × CH3), 17.92 and 18.01‡ [2 × C(CH3)3], 25.65 and
25.78 [2 × C(CH3)3], 39.41‡ and 39.84 (2 × C-2Ј), 44.74‡ and
46.16 (2 × 62.56‡ and 62.93 (2 × C-5Ј) 83.85 and 83.92‡
(2 × C-3Ј), 85.44‡ and 85.92 (2 × C-1Ј), 88.62‡ and 89.99
(2 × C-4Ј), 109.66 and 109.99‡ (2 × C-5), 117.83‡ and 118.18
Compound 22 (602 mg, 1.24 mmol), phthalimide (237 mg, 1.61
mmol) and triphenylphosphine (444 mg, 1.69 mmol) were dis-
solved in anhydrous THF (1.3 cm3) under argon. The mixture
was cooled to 0 ЊC and a solution of diethyl azodicarboxylate
(DEAD) in anhydrous THF (0.62 cm3 THF; 1.59 mmol) was
added dropwise. After 24 h at room temperature, the solvent
was evaporated off under reduced pressure. The crude product
was dissolved in CH2Cl2 (50 cm3) and washed successively with
saturated aq. NaHCO3 (2 × 40 cm3) and water (2 × 40 cm3).
The organic phase was dried (Na2SO4), and concentrated under
reduced pressure. Silica gel column chromatography (CH2Cl2)
afforded title compound 23 as a solid (656 mg, 86%);
δC(CDCl3) Ϫ6.07 and Ϫ5.86 [2 × Si(CH3)2], 1.81 and 2.05
[2 × Si(CH3)3], 12.42 and 14.28‡ (2 × CH3), 17.83 and 17.95‡
[2 × C(CH3)3], 25.57 and 25.72‡ [2 × C(CH3)3], 23.63‡ and
23.90 (2 × C-2Љ), 32.40‡ and 32.81 (2 × C-1Љ), 37.88 and 37.97‡
(2 × C-3Љ), 45.94 (2 × C-2Ј), 61.98 and 63.05‡ (2 × C-5Ј),
84.18‡ and 84.20 (2 × C-3Ј), 85.61‡ and 85.77 (2 × C-1Ј),
88.45‡ and 89.76 (2 × C-4Ј), 109.71 and 110.00‡ (2 × C-5),
123.30‡, 123.33, 132.10, 132.15‡, 134.06‡ and 134.09
(2 × Carom), 135.82‡ and 137.30 (2 × C-6), 150.28‡ and 150.50
(2 × C-2), 164.05 (2 × C-4) and 168.40‡ and 168.43 (2 × CON);
δH(CDCl3) 0.01–0.18 [2 × Si(CH3)2 and 2 × Si(CH3)3], 0.81
(2 × ᎐CH Љ), 133.63 (2 × ᎐CHЉ), 135.76‡ and 137.12 (2 × C-6),
᎐
᎐
2
150.47‡ and 150.65 (2 × C-2) and 164.28‡ and 164.38 (2 × C-
4); δH(CDCl3) 0.01–0.20 [2 × Si(CH3)2 and 2 × Si(CH3)3], 0.92
‡ Minor anomer.
3282
J. Chem. Soc., Perkin Trans. 1, 1997

View Article Online
[s, 2 × C(CH3)3], 1.71–1.96 (m, 2 × CH3, Ha-2Ј‡, H2-1Љ and
H-2Љ), 2.05 (dd, J 14.3, Ha-2Ј), 2.40‡ (dd, J 4.8 and 12.5, Hb-2Ј),
2.45 (dd, J 8.2 and 14.2, Hb-2Ј), 3.60–3.71 (m, Ha-5Ј‡, H-5Јa
and H2-3Љ), 3.81‡ (m, Hb-5Ј), 4.01‡ (m, H-4Ј), 4.16–4.23 (m,
H-4Ј and Hb-5Ј), 6.15‡ (dd, J 4.8 and 9.2, H-1Ј), 6.24 (dd, J 2.0
and 8.2, H-1Ј), 7.58‡ and 7.65 (2 s, H2-6), 7.66–7.81 (2 × ArH)
and 8.58 and 8.69‡ (2 s, 2 × NH); FAB-MS m/z 616 [M ϩ H]ϩ.
1-{3-O-[2-Cyanoethoxy(diisopropylamino)phosphino]-2-deoxy-
5-O-(4,4Ј-dimethoxytrityl)-3-C-phthalimidopropyl-á-D-erythro-
pentofuranosyl}thymine 26
Nucleoside 25 (267 mg, 0.37 mmol) was coevaporated with
anhydrous CH3CN (3 × 2 cm3) and dissolved under argon in
anhydrous CH2Cl2 (2 cm3). DIPEA (0.4 cm3, 2.34 mmol) was
added followed by dropwise addition of 2-cyanoethyl N,N-
diisopropylphosphoramidochloridite (0.17 cm3, 0.72 mmol).
After stirring of the mixture for 5 h, CH3OH (0.5 cm3) was
added and the reaction mixture was diluted with EtOAc (20
cm3) containing triethylamine (0.2 cm3), washed successively
with saturated aq. NaHCO3 (3 × 30 cm3) and saturated aq.
NaCl (2 × 30 cm3), dried (Na2SO4), and evaporated under
reduced pressure. Purification using silica gel column chrom-
atography (EtOAc–CH2Cl2–Et3N–petroleum ether, 15:30:5:
50, v/v/v/v) followed by precipitation in light petroleum (30
cm3) at Ϫ20 ЊC [after re-dissolution in anhydrous toluene (1
cm3)] afforded compound 26 as a solid (141 mg, 41%);
δP(CDCl3) 140.78 and 140.90.
1-[2-Deoxy-3-C-(3-phthalimidopropyl)-á,â-D-erythro-pento-
furanosyl]thymine 24
To a solution of nucleoside 23 (668 mg, 1.09 mmol) in
anhydrous THF (27 cm3) was added 1.1  TBAF in THF (2.2
cm3, 2.42 mmol). The reaction mixture was stirred under argon
for 20 min and then was concentrated to dryness. Purification
using silica gel column chromatography (20% EtOAc in light
petroleum, v/v) afforded title compound 24 as solid (154 mg,
33%); δC(CDCl3) 12.12 and 12.22‡ (2 × CH3), 23.37‡ and 23.57
(2 × C-2Љ), 32.21‡ and 32.80 (2 × C-1Љ). 38.09‡ and 38.24
(2 × C-3Љ), 42.60‡ and 44.56 (2 × C-2Ј), 58.17 and 62.02‡
(2 × C-5Ј), 79.60, 80.78‡, 85.78‡, 87.32, 88.93‡ and 91.51 (C-
1Ј, -3Ј and -4Ј), 108.43 and 110.74‡ (2 × C-5), 123.29, 131.96
and 134.06 (2 × Carom), 137.29‡ and 137.93 (2 × C-6), 151.06‡
and 151.32 (2 × C-2), 164.34‡ and 164.72 (2 × C-4) and
168.72‡ and 168.83 (CON); δH(CDCl3) 1.75–2.62 (m, 2 × CH3,
H2-2Ј, H2-1Љ and H2-2Љ), 3.67–4.55 (m, 2 × H-4Ј, H2-5Ј and H2-
3Љ), 6.17 (d, J 5.0, H-1Ј), 6.27‡ (dd, J 5.2 and 9.2, H-1Ј), 7.66–
7.81 (m, 2 × H-6 and ArH) and 10.90 (br s, 2 × NH); FAB-MS
m/z 430 [M ϩ H]ϩ.
Synthesis of oligonucleotides
The synthesis of ODNs was carried out on a 0.2 µmol scale (5
µmol amidite per cycle, Pharmacia Primer SupportTM) using
compounds 16a, 16b, 26, αT-cyanoethylphosphoramidite and
commercial βT-cyanoethylphosphoramidite. The synthesis fol-
lowed the regular protocol for the DNA synthesizer. For the
modified phosphoramidites the coupling time was increased
from 2 to 24 min and the cycle was repeated twice. The ODNs
were removed from the support and deblocked by treatment
with conc. ammonia at room temp. for 72 h. Purification and
detritylation was achieved on Cruachem oligonucleotide purifi-
cation cartridges using the standard procedure. The purity of
the ODNs was confirmed by analytical anion-exchange HPLC.
The solvent systems consisting of 10 m NaOH (A) and 10 m
NaOH ϩ 1.8  NaCl (B) were used in the following order: 10
min linear gradient of 25–30% B in A, 40 min linear gradient of
30–45% B in A, 1 min 45–100% B in A, 1 min 100% B, 1 min
1-[2-Deoxy-5-O-(4,4Ј-dimethoxytrityl)-3-C-(3-phthalimido-
propyl)-á-D-erythro-pentofuranosyl]thymine 25
An anomeric mixture of nucleoside 24 (368 mg, 0.86 mmol),
AgNO3 (174 mg, 1.02 mmol) and DMTCl (1.45 g, 4.28 mmol)
were dissolved in anhydrous THF (35 cm3), and pyridine (0.35
cm3, 4.33 mmol) was added. The mixture was stirred at room
temp. for 72 h with exclusion of light. The mixture was filtered
into 5% aq. NaHCO3 (50 cm3). The product was extracted into
CH2Cl2 (4 × 100 cm3), and the organic phase was dried
(Na2SO4), and concentrated under reduced pressure. Purific-
ation by PLC (2% pyridine, 5% CH3OH in CH2Cl2, v/v/v, two
runs) gave the α-anomer 25 (267 mg, 43%) as the less polar
isomer and the corresponding β-anomer (94 mg, 15%).
β-Anomer 25: δC(CDCl3) 11.18 (CH3), 23.36 (C-2Љ), 31.81
(C-1Љ), 37.92 (C-3Љ), 43.99 (C-2Ј), 55.12 (OCH3), 62.69 (C-5Ј),
80.98 (C-3Ј), 84.15 (C-1Ј), 87.32 (C-4Ј), 87.84 (CPh3), 111.33
(C-5), 113.24, 127.35, 128.01, 130.28, 130.34, 134.78, 134.99,
143.75 and 158.88 (DMT), 123.31, 131.99 and 134.06 (Phth),
136.58 (C-6), 150.76 (C-2), 163.96 (C-4) and 168.62 (CON);
δH(CDCl3) 1.22 (s, 3 H, CH3), 1.45–1.76 (m, 4 H, H2-1Љ and -2Љ),
2.09 (dd, J 9.5 and 12.4, 2 H, Ha-2Ј), 2.40 (dd, J 4.8 and 12.4, 1
H, Hb-2Ј), 3.15 (dd, J 2.2 and 10.8, 1 H, Ha-5Ј), 3.53 (m, 2 H,
H2-3Љ), 3.62 (dd, J 3.3, 10.8, 1 H, Hb-5Ј), 3.79 (s, 6 H, 2 × OCH3),
4.06 (m, 1 H, H-4Ј), 6.49 (dd, J 4.8 and 9.5, 1 H, H-1Ј), 6.84
(m, 4 H, DMT), 7.23–7.38 (m, 9 H, DMT), 7.65–7.85 (m, 5 H,
Phth and H-6) and 9.18 (s, 1 H, NH).
linear gradient of 100–25% B in A. Flow rate 1.67 cm3 minϪ1
The purified ODNs eluted as one peak after approximately 14
min for products H–L and after approximately 20 min for
products A–G.
.
Melting experiments
The melting experiments were carried out in medium salt
buffer, 1 m ethylenediaminetetra-acetic acid (EDTA), 10 m
Na2HPO4, 140 m NaCl, pH 7.2 at a concentration of 2 nmol
for each strand. The increase in absorbance at 260 nm as a
function of time was recorded while the temperature was raised
from 10 to 60 ЊC at a rate of 1 ЊC per min. The melting temper-
ature was determined as the maximum of the first-derivative
plot of the 260 nm transition.
Acknowledgements
The Danish Natural Science Research Council is thanked for
financial support.
α-Anomer 25: δC(CDCl3) 12.26 (CH3), 23.56 (C-2Љ), 32.05
(C-1Љ), 37.82 (C-3Љ), 44.94 (C-2Ј), 54.88 (OCH3), 63.28 (C-5Ј),
80.08 (C-3Ј), 86.74 (C-4Ј), 87.36 (CPh3), 89.86 (C-1Ј), 108.64
(C-5), 113.19, 126.79, 127.91, 129.81, 129.88, 135.46, 135.79,
144.46 and 158.47 (DMT), 123.75, 131.85 and 133.87 (Phth),
137.92 (C-6), 150.66 (C-2), 164.71 (C-4) and 168.37 (CON);
δH(CDCl3) 1.24–1.79 (m, 4 H, H2-1Љ and -2Љ), 1.84 (s, 3 H, CH3),
2.32 (d, J 14.0, 1 H, Hα-2Ј), 2.72 (dd, J 7.6 and 14.4, 1 H, Hβ-2Ј),
2.99 (dd, J 2.0 and 10.7, 1 H, Ha-5Ј), 3.41 (dd, J 3.6 and 10.7, 1
H, Hb-5Ј), 3.59 (m, 2 H, H2-3Љ), 3.78 (s, 6 H, 2 × OCH3), 4.35
(m, 1 H, H-4Ј), 6.36 (d, J 7.0, 1 H, H-1Ј), 6.84 (m, 4 H, DMT),
7.17–7.43 (m, 9 H, DMT), 7.64–7.77 (m, 5 H, Phth, H-6) and
10.03 (s, 1 H, NH); FAB-MS m/z 731 [M]ϩ.
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Paper 7/03173D
Received 8th May 1997
Accepted 25th June 1997
3284
J. Chem. Soc., Perkin Trans. 1, 1997