Synthesis and hybridization studies of α-configured arabino nucleic acids

Article abstract of DOI:10.1039/b905019c

Synthesis of α-L-arabino- and α-D-arabino-configured pentofuranosyl nucleosides of four of the natural bases [thymine (ara-T), adenine (ara-A), cytosine (ara-C) and guanine (ara-G)] is reported together with hybridization properties of oligonucleotides co

Full text of DOI:10.1039/b905019c

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www.rsc.org/obc | Organic & Biomolecular Chemistry
Synthesis and hybridization studies of a-configured arabino nucleic acids†
Pankaj Gupta,a,b Jyotirmoy Maity,b Gaurav Shakya,b Ashok K. Prasad,a,b Virinder S. Parmara,b and
Jesper Wengel*a
Received 11th March 2009, Accepted 11th March 2009
First published as an Advance Article on the web 15th April 2009
DOI: 10.1039/b905019c
Synthesis of a-L-arabino- and a-D-arabino-configured pentofuranosyl nucleosides of four of the
natural bases [thymine (ara-T), adenine (ara-A), cytosine (ara-C) and guanine (ara-G)] is reported
together with hybridization properties of oligonucleotides containing a-L-ara-T and -A, a-D-ara-T and
-A, and 2¢-amino-a-L-ara-T monomers. 2¢-O-Acetylated a-L-ara-T, -A, -C and -G, a-D-ara-T, -A, -C
and -G, and N2¢-acylated-a-L-ara-T phosphoramidite building blocks were synthesized and used
together with standard DNA phosphoramidites for solid-phase synthesis of 18-mer oligonucleotides.
Thermal denaturation experiments showed that incorporation of three or six of the arabino-configured
monomers into DNA-oligonucleotides reduced the binding affinity towards antiparallel DNA/RNA
complements. Fully modified a-L-ara-oligonucleotides did not hybridize with DNA/RNA
complements, whereas hybridization of fully modified a-D-ara-oligonucleotides with complementary
DNA/RNA in parallel strand orientation was confirmed.
Introduction
To understand why the nucleic acids in Nature are based
on b-D-pentofuranosyl ribonucleotide building blocks, numer-
ous alternative nucleic acid systems have been studied, in-
cluding ones based on pentopyranosyl or hexopyranosyl nu-
cleotide building blocks.1–10 Eschenmoser and co-workers have
found that pentopyranosyl-(4¢→2¢)-oligonucleotide systems com-
posed of b-ribopyranosyl, b-xylopyranosyl, a-lyxopyranosyl or
a-arabinopyranosyl units as repeating sugar building blocks
constitute stronger Watson–Crick base pairing systems than
RNA.9–12 The a-arabinopyranosyl system in fact belongs to the
strongest oligonucleotide base pairing systems known,12 which
signifies that maximization of base pairing strength within the
domain of pentose-derived oligonucleotide systems was not the
decisive driving force during the evolution of RNA.
Oligonucleotides (ONs) involving pentofuranosyl-based
systems, i.e. RNA stereoisomers with b-L-ribo-,13 a-D-ribo-,14
a-L-ribo-,15–17 b-D-arabino-,18–20 and a-D-arabino-21,22 configured
Fig. 1 Stereoisomers of RNA having ribo- and arabino-configuration.
nucleotide monomers have also been studied (Fig. 1).
Ashley13 did not observe any hybridization between b-L-ribo
special aspect of the a-anomeric ONs a-DNA and a-RNA14 is
ON L-r(U)12 and complementary DNA. In contrast, a mixture
their high resistance towards nucleases.26,27 Interestingly, Sawai
of L-r(U)12 and complementary RNA displayed a sharp hyper-
et al.28 showed by CD studies that single stranded a-DNA displays
chromic transition upon thermal heating indicative of hybridiza-
tion at room temperature. For a-D-RNA,14 hybridization was not
significant between a fully modified a-D-dodecaribonucleotide
and DNA in antiparallel orientation, whereas a clear transition
was observed in parallel orientation. These results were in
accordance with earlier reports for a-D-DNA,23–25 which showed
binding with complementary DNA in parallel orientation. A
weaker CD bands than those of corresponding b-oligomers, which
indicates weaker base-stacking interactions of the a-oligomers.
Incorporation of a single a-L-RNA15 monomer into a 9-mer DNA
strand induced a hybridization preference towards complementary
RNA as compared to DNA, a trend that was also observed upon
incorporation of three a-L-RNA monomers.
The 2¢-epimer of RNA, b-D-arabinofuranosyl nucleic acid
(ANA),18–20 exhibits preferential binding towards complementary
RNA over DNA, albeit in both cases with lower binding affinity
than the corresponding unmodified duplexes.26 Important for
potential antisense applications are the facts that ANA oligomers
are resistant to 3¢-exonucleases and that ANA/RNA duplexes are
substrates for the enzyme RNase H.26 Oligomers composed of
aNucleic Acid Center, Department of Physics and Chemistry, University of
Southern Denmark, 5230 Odense M, Denmark
bBioorganic Laboratory, Department of Chemistry, University of Delhi
(North Campus), Mall Road, Delhi 110 007, India
† Electronic supplementary information (ESI) available: Synthesis de-
scriptions of all a-D-arabino configured nucleosides. See DOI:
10.1039/b905019c
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monomers all having a-D-arabino configuration have also been
prepared. Such a-D-ara ONs preferentially form duplexes of
parallel strand orientation with DNA complements, but antipar-
allel duplexes with a-D-ara ON complements.21,22 A pentadeca
1-(a-D-arabinofuranosyl)thymine oligomer displayed stronger
RNA than DNA binding and also the capability to form a triplex
with a b-DNA duplex.22 Notably, the stability induced by an
a-D-ara nucleotide monomer against exonucleolytic degradation
was found to be significant.22 A recent report on a potentially
prebiotic synthesis of pyrimidine b-D-ribonucleosides by pho-
tochemically induced anomerization of a-D-ribonucleosides has
highlighted the relevance of studying a-configured nucleotides as
possible components of prebiotic nucleic acids.29
As illustrated by the selected literature results described above,
several ribo- and arabino-configured pentofuranosyl stereoiso-
mers of RNA have been synthesized and studied. To expand
these studies, we herein report synthesis of a-L-arabino- and
a-D-arabino-configured nucleosides of four of the natural bases,
oligomers thereof, and binding affinities of these towards DNA,
RNA and a-arabino-configured ONs. This study extends earlier
reports on a-D-ara ONs, and is the first study of a-L-ara ONs. We
report on fully a-D/L-ara modified ONs as well as stereoirregular
ONs, i.e. duplexes containing a-L-arabino-, a-D-arabino- and
b-D-ribo-configured nucleotide monomers.
Results and discussion
Synthesis
Nucleosides 1–4 (a-L-ara nucleosides) were synthesized from
L-arabinose by a known procedure.30 The nucleobase amino
groups of nucleosides 2–4 were acylated using the transient
protection method31 to give novel nucleosides 5 (76%) and 7 (78%),
and the known nucleoside 632 (92%). Regioselective O3¢- and
O5¢-protection of the nucleosides 1 and 5–7 was performed using
1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane (TIPDSCl2) in the
presence of pyridine furnishing nucleosides 833 (80%), 9 (81%),
10 (66%) and 11 (76%) which were next O2¢-acetylated by acetic
anhydride in pyridine to afford nucleoside 12 (86%), 13 (90%),
14 (88%) and 15 (80%), respectively. Removal of the TIPDS
group from nucleosides 12–15 by Et3N·3HF in acetonitrile yielded
O2¢-acylated nucleosides 16 (91%), 17 (93%), 18 (80%) and 19
(79%). Subsequent reaction with 4,4¢-dimethoxytrityl chloride
(DMTCl) in pyridine selectively afforded the O5¢-DMT protected
nucleosides 20 (84%), 21 (78%), 22 (81%) and 23 (45%), which
were O3¢-phoshphitylated by reaction with 2-cyanoethyl-N,N-
diisopropylchlorophosphoramidite to give the target nucleoside
phosphoramidite derivatives 24 (78%), 25 (79%), 26 (80%) and 27
(45%) (Scheme 1), respectively, suitable for automated solid phase
incorporation of a-L-ara monomers into ONs.
Scheme 1 Reagents and conditions: i) TMSCl, pyridine, BzCl; then aq. NH3 (for compounds 2–3); TMSCl, pyridine, isobutyric anhydride; then aq.
NH3 (for compound 4); ii) TIPDSCl2, pyridine; iii) Ac2O, pyridine; iv) Et3N·3HF, MeCN; v) DMTCl, pyridine, CH2Cl2; vi) NCCH2CH2OP(Cl)N(i-Pr)2,
DIPEA, CH2Cl2.
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Table 1 Oligonucleotides included in this study with MALDI-MS data
The enantiomeric a-D-ara nucleoside derivatives, i.e. eventually
the a-D-ara-T, -ABz, -CBz and -GiBu phosphoramidites correspond-
ing to 24–27, were synthesized from D-arabinose by identical
protocols as described above for the a-L-ara nucleosides. It
should be noted that we followed a different protocol than the
one published by Henke and Pfleiderer.21 In the experimental
descriptions found in the ESI,† all a-D-ara nucleoside derivatives
are denoted with the same number as the corresponding a-L-ara
nucleoside derivatives but followed by “D” (i.e. 20 and 20D for the
a-L-ara and a-D-ara nucleoside, respectively).
In order to evaluate a possible influence of an amino
functionality at the 2¢-position of these a-ara nucleoside
derivatives on hybridization capability, we also synthesized
a-L-ara phosphoramidite derivative 33 starting from a-L-
arabinofuranosylthymine (1). Nucleoside 1 was converted to 2,2¢-
anhydro-a-L-ribofuranosylthymine (28) in 32% yield by reaction
with diphenyl carbonate (DPC) in the presence of sodium
bicarbonate as catalyst and anhydrous N,N¢-dimethylformamide
as solvent. The resulting nucleoside 28 was further refluxed with
sodium azide in anhydrous N,N¢-dimethylformamide at 150 ◦C
for 9 h to yield 2¢-azido-2¢-deoxy-a-L-arabinofuranosylthymine
(29) in 61% yield. The azido group of nucleoside 29 was reduced
to an amino group by reaction with 10% Pd-C/H2 to yield 2¢-
amino-2¢-deoxy-a-L-arabinofuranosylthymine (30) in 82% yield.
The 2¢-amino group of nucleoside 30 was selectively protected
using ethyl trifluoroacetate and 4-(N,N-dimethyl)aminopyridine
in methanol at room temperature for 5 h to afford 1-(2¢-(N-
trifluoroacetyl)amino-a-L-arabinofuranosyl)thymine (31) in 87%
yield. Dimethoxytritylation was done using dimethoxytrityl chlo-
ride and pyridine to afford the corresponding nucleoside 32 in 78%
yield which on phosphitylation at the 3¢-position by reaction with
bis(N,N-diisopropylamino)-2-cyanoethoxyphosphine and N,N-
diisopropylammonium tetrazolide in anhydrous acetonitrile was
converted into the target phosphoramidite monomer 33 in 90%
yield (Scheme 2).
The oligomers (Table 1) used in this study were synthesized
on an automated DNA synthesizer using the phosphoramidite
approach (see experimental section for details).34 The influence of
the a-ara modifications on the thermal denaturation temperature
(Tm values) of duplexes was studied towards DNA and RNA
complements (Tables 2–6) by UV thermal denaturation experi-
ments using a medium salt buffer (100 mM NaCl, 0.1 mM EDTA,
10 mM NaH2PO4, 5 mM Na2HPO4, pH 7.0) at a concentration of
1.0 mM of each strand. The increase in absorbance at 260 nm as
a function of time was recorded while the temperature was raised
linearly from 5–80 ◦C at a rate of 1 ◦C/min. We decided to study
partly modified duplexes containing short segments of three or
six a-arabino configured nucleosides, and fully modified strands.
With this experimental design we enabled evaluation of both
stereoregular (a-ara:a-ara) and stereoirregular (a-ara:b-D-ribo;
a-D-ara:a-L-ara) hybridization. In all cases, the denaturation
curves displayed sigmoidal monophasic transitions.
shown for ara nucleotide-modified oligonucleotidesa
Mass Mass
found calcd.
ONs
ON1 5¢-d(TTA TTA TTA TAA AAT TAT)
ON2 5¢-d(ATA ATT TTA TAA TAA TAA)
ON3 5¢-r(UUA UUA UUA UAA AAU UAU)
ON4 5¢-r(AUA AUU UUA UAA UAA UAA)
ON5 5¢-d(AAT AAT AAT ATT TTA ATA)
ON6 5¢-d(TAT TAA AAT ATT ATT ATT)
ON7 5¢-r(AAU AAU AAU AUU UUA AUA)
ON8 5¢-r(UAU UAA AAU AUU AUU AUU)
ON9 5¢-d(TTA TTA TT a-L-ara(A TA)A AAT TAT)
ON10 5¢-d(TTA TTA a-L-ara(TTA TAA) AAT TAT)
ON11 5¢-d(TTA TTA TT a-D-ara(A TA)A AAT TAT)
ON12 5¢-d(TTA TTA a-D-ara(TTA TAA) AAT TAT)
ON13 5¢-d(ATA ATT T a-L-ara(TA T)AA TAA TAA)
ON14 5¢-d(ATA ATT a-L-ara(TTA TAA) TAA TAA)
ON15 5¢-d(ATA ATT T a-D-ara(TA T)AA TAA TAA)
ON16 5¢-d(ATA ATT a-D-ara(TTA TAA) TAA TAA)
ON17 5¢-d(TTA TTA TT a-L-ara(A TNA)A AAT TAT)
5530 5537
5578 5581
5532 5537
5598 5581
5547 5551
5597 5599
5552 5551
5598 5599
5525 5532
ON18 5¢-d(TTA TTA a-L-ara(TNTNA TNAA)AAT TAT) 5573 5578
ON19 5¢-d(ATA ATT T a-L-ara(TNA TN)AA TAA TAA) 5542 5549
ON20 5¢-d(ATA ATT a-L-ara(TNTNA TNAA) TAA TAA) 5590 5596
ON21 5¢-a-L-ara-(TTA TTA TTA TAA AAT TAT)
ON22 5¢-a-L-ara-(ATA ATT TTA TAA TAA TAA)
ON23 5¢-a-D-ara-(TTA TTA TTA TAA AAT TAT)
ON24 5¢-a-D-ara-(ATA ATT TTA TAA TAA TAA)
5775 5774
5795 5792
5773 5774
5786 5792
a A = adenin-9-yl DNA/RNA monomer, T = thymin-1-yl DNA monomer;
U = uracil-1-yl RNA monomer; a-L-ara( ◊ ◊ ◊ ) indicates segments of the
sequences that are a-L-ara nucleotides; a-D-ara( ◊ ◊ ◊ ) indicates segments
of the sequences that are a-D-ara nucleotides; a-L-ara-TN = 2¢-amino-2¢-
deoxy-arabinofuranosyl thymin-1-yl monomer.
values are used below as reference values. In general, incorporation
of three a-L-ara-T/A nucleotides in the middle of duplexes
(entries 5–8), resulted in reduced thermal stabilities of the different
duplexes by at least 1 ◦C per a-L-ara nucleotide. Accordingly, no
duplex formation was observed when six a-L-ara-T/A nucleotides
were incorporated in one of the strands (entries 9–12), nor for
the fully modified a-L-ara oligonucleotides ON21 and ON22
(entries 13–16). The above data are based on Watson Crick
base pairing between stereosiomeric nucleotides, i.e. a-L-ara and
b-D-ribo (DNA/RNA) configured nucleotides. On the contrary,
the duplexes of entries 17 and 18 include segments of three
and six a-L-ara:a-L-ara base pairs, respectively. Interestingly,
a-L-ara:a-L-ara base pairing stabilizes the duplexes relative to the
duplexes having a-L-ara nucleotides in only one of the strands,
which is confirmed by the Tm value of 21 ◦C for the fully
a-L-ara modified duplex of entry 19. The latter result is in line
with earlier data reported for fully a-D-ara modified antiparallel
duplexes,21 but it is noteworthy that duplexes having segments
of both b-D-ribo:b-D-ribo base pairs and a-L-ara:a-L-ara base
pairs (entries 17 and 18) display thermal stabilities compara-
ble to those of duplexes formed between DNA/RNA strands
(entries 1–4).
a-L-ara oligomers—hybridization in anti-parallel orientation
2¢-Amino-2¢-deoxy-a-L-ara oligomers—hybridization in
anti-parallel orientation
Table 2 displays hybridization studies between two strands that
are complementary in an antiparallel binding mode. The first four
entries show the Tm values recorded for the different unmodified
duplexes containing DNA and/or RNA strands. These four Tm
To study the effect of 2¢-amino-a-L-ara nucleotides we evaluated
the duplexes depicted in Table 3 in the same sequence contexts as
those in Table 2 (except for the fully modified a-L-ara oligomers).
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Scheme 2 Reagents and conditions: i) Diphenyl carbonate, NaHCO3, DMF; ii) NaN3, DMF; iii) 10% Pd-C/H2, MeOH; iv) CF3COOC2H5, DMAP,
MeOH; v) DMTCl, pyridine, CH2Cl2; vi) bis(N,N-diisopropylamino)-2-cyanoethoxyphosphine, N,N-diisopropylammonium tetrazolide, MeCN.
We thus incorporated the a-L-ara-TN monomer together with
a-L-ara-T/A monomers, and obtained similar hybridization
results as for the a-L-ara-T/A containing duplexes (Table 2).
The duplexes containing ON19 with an a-L-ara-(TNATN) segment
(entries 22 and 23) are thermally more stable than those containing
ON13 with an a-L-ara-(TAT) segment (entries 7 and 8). 2¢-Amino
derivatives of ONs are interesting as possible conjugation or
protonation sites.35,36 In the case of 2¢-protonation one would
expect more favorable Tm values for the a-L-ara-TN containing
oligomers than the a-L-ara-T containing monomers and we
therefore evaluated hybridization at low salt conditions (as those
described in the caption below Table 3 but with only 10 mM
NaCl). Under these conditions, the duplexes of entries 22 and 28
in fact showed Tm values of 12.0 and 14.5 ◦C, respectively, whereas
no transition was observed for the duplexes of entries 8 and 17.
These results indicate that 2¢-amino-a-L-ara nucleotides, contrary
to 2¢-amino-DNA nucleotides,35 are at least in part protonated at
pH 7. It is furthermore interesting that whereas 2¢-amino-DNA
nucleotides, relative to DNA nucleotides, have a detrimental effect
on duplex stability,35 2¢-amino-a-L-ara and a-L-ara nucleotides
have a comparable effect.
recently reported the synthesis and hybridization studies towards
complementary DNA of a-D-ara ONs. They also studied fully
modified and segment-modified a-D-ara oligomers and found
that the presence of a-D-ara nucleotides was associated with
sequence dependent decreases in Tm values. Similarly, we found
(Table 4) that three a-D-ara-T/A nucleotides (entries 30–33)
decreased Tm values relative to unmodified control duplexes
(Table 2, entries 1, 3 and 4) to a similar extent as a-L-ara-
T/A nucleotides (Table 2). Six a-D-ara-T/A nucleotides strongly
reduced binding (entries 34–37) though stable duplexes were
formed with complementary RNA, and no duplex formation was
observed with the fully a-D-ara-T/A modified ON23 and ON24
(entries 38–41). As observed for the a-L-ara oligomers, a-D-ara:a-
D-ara base pairing is more favorable than a-D-ara:b-D-ribo base
pairing (entries 42–44), though it should be underlined that the
duplexes displayed in entries 17 and 42 and in entries 18 and
43 are diastereoisomeric whereas those of entries 19 and 44 are
enantiomeric.
a-D-ara oligomers—hybridization in parallel orientation
Fully modified a-L-ara or a-D-ara oligonucleotides (ON21–
ON24) showed no binding to complementary DNA/RNA in
antiparallel orientation. Earlier findings in the a-DNA series23–25
suggest preferential duplex formation between a-configured ONs
and DNA/RNA in parallel orientation. Similar results were
a-D-ara oligomers—hybridization in anti-parallel orientation
In order to directly compare the effect of D- vs. L-stereochemistry,
a-D-ara ONs were synthesized. Henke and Pfleiderer21 have
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Table 2 a-L-ara oligomers—hybridization in anti-parallel orientationa
Table 3 2¢-Amino-a-L-ara oligomers—hybridization in anti-parallel
orientationa
Entry Duplexes
Tm(◦C)
34.0
33.0
23.0
21.5
21.0
17.0
19.0
nt
Entry Duplexes
Tm(◦C)
ON17 5¢-d(TTA TTA TT a-L-ara(A TNA)A AAT TAT) 21.0
1.
2.
ON1 5¢-d(TTA TTA TTA TAA AAT TAT)
ON2 3¢-d(AAT AAT AAT ATT TTA ATA)
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
ON3 5¢-r(UUA UUA UUA UAA AAU UAU)
ON4 3¢-r(AAU AAU AAU AUU UUA AUA)
ON1 5¢-d(TTA TTA TTA TAA AAT TAT)
ON4 3¢-r(AAU AAU AAU AUU UUA AUA)
ON3 5¢-r(UUA UUA UUA UAA AAU UAU)
ON2 3¢-d(AAT AAT AAT ATT TTA ATA)
ON9 5¢-d(TTA TTA TTa-L-ara(A TA)A AAT TAT)
ON2 3¢-d(AAT AAT AAT ATT TTA ATA)
ON9 5¢-d(TTA TTA TT a-L-ara(A TA)A AAT TAT)
ON4 3¢-r(AAU AAU AAU AUU UUA AUA)
ON1 5¢-d(TTA TTA TTA TAA AAT TAT)
ON2 3¢-d(AAT AAT AAT ATT TTA ATA)
ON17 5¢-d(TTA TTA TT a-L-ara(A TNA)A AAT TAT) 15.5
ON4 3¢-r(AAU AAU AAU AUU UUA AUA)
3.
ON1 5¢-d(TTA TTA TTA TAA AAT TAT)
23.0
4.
ON19 3¢-d(AAT AAT AA a-L-ara(TNATN)T TTA ATA)
ON3 5¢-r(UUA UUA UUA UAA AAU UAU)
14.0
5.
ON19 3¢-d(AAT AAT AA a-L-ara(TNATN)T TTA ATA)
ON18 5¢-d(TTA TTA a-L-ara(TNTNA TNAA) AAT TAT) nt
ON2 3¢-d(AAT AAT AAT ATT TTA ATA)
6.
ON18 5¢-d(TTA TTA a-L-ara(TNTNA TNAA) AAT TAT) nt
ON4 3¢-r(AAU AAU AAU AUU UUA AUA)
7.
ON13 3¢-d(AAT AAT AA a-L-ara(T AT)T TTA ATA)
ON3 5¢-r(UUA UUA UUA UAA AAU UAU)
ON13 3¢-d(AAT AAT AA a-L-ara(T AT)T TTA ATA)
ON10 5¢-d(TTA TTA a-L-ara(TTA TAA) AAT TAT)
ON2 3¢-d(AAT AAT AAT ATT TTA ATA)
ON1 5¢-d(TTA TTA TTA TAA AAT TAT)
nt
8.
ON20 3¢-d(AAT AAT a-L-ara(AATN ATNTN) TTA ATA)
ON3 5¢-r(UUA UUA UUA UAA AAU UAU)
nt
9.
nt
ON20 3¢-d(AAT AAT a-L-ara(AATN ATNTN) TTA ATA)
ON17 5¢-d(TTA TTA TT a-L-ara(A TNA)A AAT TAT) 26.0
ON19 3¢-d(AAT AAT AA a-L-ara(TNATN)T TTA ATA)
ON18 5¢-d(TTA TTA a-L-ara(TNTNA TNAA) AAT TAT) 21.0
ON20 3¢-d(AAT AAT a-L-ara(AATN ATNTN)TTA ATA)
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
ON10 5¢-d(TTA TTA a-L-ara(TTA TAA) AAT TAT)
ON4 3¢-r(AAU AAU AAU AUU UUA AUA)
ON1 5¢-d(TTA TTA TTA TAA AAT TAT)
nt
nt
ON14 3¢-d(AAT AAT a-L-ara(AAT ATT) TTA ATA)
ON3 5¢-r(UUA UUA UUA UAA AAU UAU)
ON14 3¢-d(AAT AAT a-L-ara(AAT ATT) TTA ATA)
ON21 5¢-a-L-ara(TTA TTA TTA TAA AAT TAT)
ON2 3¢-d(AAT AAT AAT ATT TTA ATA)
ON21 5¢-a-L-ara(TTA TTA TTA TAA AAT TAT)
ON4 3¢-r(AAU AAU AAU AUU UUA AUA)
ON1 5¢-d(TTA TTA TTA TAA AAT TAT)
ON22 3¢-a-L-ara(AAT AAT AAT ATT TTA ATA)
ON13 5¢-r(UUA UUA UUA UAA AAU UAU)
ON22 3¢-a-L-ara(AAT AAT AAT ATT TTA ATA
ON9 5¢-d(TTA TTA TT a-L-ara(A TA)A AAT TAT)
ON13 3¢-d(AAT AAT AA a-L-ara(T AT)T TTA ATA)
ON10 5¢-d(TTA TTA a-L-ara(TTA TAA) AAT TAT)
ON14 3¢-d(AAT AAT a-L-ara(AAT ATT) TTA ATA)
ON21 5¢-a-L-ara(TTA TTA TTA TAA AAT TAT)
ON22 3¢-a-L-ara(AAT AAT AAT ATT TTA ATA)
a See captions below Table 1. Thermal denaturation temperatures (Tm/◦C)
of unmodified or modified duplexes measured as the maximum of the first
derivative of the melting curve (A260 vs temperature) recorded in medium
salt buffer (110 mM NaCl, 0.1 mM EDTA, 10 mM NaH2PO4, 5 mM
Na2HPO4, pH 7.0), using 1.0 mM concentration of the two complementary
strands. Tm values are average of at least two measurements; “nt” denotes
“no transition”.
nt
nt
nt
nt
nt
27.5
26.5
21.0
Table 4 a-D-ara oligomers—hybridization in anti-parallel orientationa
Entry Duplexes
Tm(◦C)
30.
31.
32.
33.
34.
35.
36.
37.
38.
39.
40.
41.
42.
43.
44.
ON11 5¢-d(TTA TTA TTa-D-ara(A TA)A AAT TAT) 21.5
a See caption below Table 1. Thermal denaturation temperatures (Tm/◦C)
of unmodified or modified duplexes measured as the maximum of the first
derivative of the melting curve (A260 vs temperature) recorded in medium
salt buffer (100 mM NaCl, 0.1 mM EDTA, 10 mM NaH2PO4, 5 mM
Na2HPO4, pH 7.0), using 1.0 mM concentration of the two complementary
strands. Tm values are average of at least two measurements; “nt” denotes
“no transition”.
ON2 3¢-d(AAT AAT AAT ATT TTA ATA)
ON11 5¢-d(TTA TTA TTa-D-ara(A TA)A AAT TAT) 21.0
ON4 3¢-r(AAU AAU AAU AUU UUA AUA)
ON1 5¢-d(TTA TTA TTA TAA AAT TAT)
19.0
17.5
nt
ON15 3¢-d(AAT AAT AAa-D-ara(T AT)T TTA ATA)
ON3 5¢-r(UUA UUA UUA UAA AAU UAU)
ON15 3¢-d(AAT AAT AAa-D-ara(T AT)T TTA ATA)
ON12 5¢-d(TTA TTA a-D-ara(TTA TAA) AAT TAT)
ON2 3¢-d(AAT AAT AAT ATT TTA ATA)
ON12 5¢-d(TTA TTA a-D-ara(TTA TAA) AAT TAT)
ON4 3¢-r(AAU AAU AAU AUU UUA AUA)
ON1 5¢-d(TTA TTA TTA TAA AAT TAT)
13.0
nt
reported by Henke and Pfleiderer21 for fully modified a-D-ara
ONs which were shown to form parallel duplexes with com-
plementary DNA of only slightly lower stabilities than the
unmodified reference duplexes. We confirmed duplex formation
in parallel orientation between fully modified a-D-ara ONs
and complementary DNA (Table 5, entries 51 and 53) and
also showed for the first time duplex formation between fully
modified a-D-ara ONs and complementary RNA (entries 52
and 54). Parallel duplex formation neither between the unmod-
ified reference DNA/RNA strands (entries 45–50) nor the fully
modified a-L-ara ONs (ON21 and ON22) and DNA/RNA were
observed.
ON16 3¢-d(AAT AAT a-D-ara(AAT ATT) TTA ATA)
ON3 5¢-r(UUA UUA UUA UAA AAU UAU)
ON16 3¢-d(AAT AAT a-D-ara(AAT ATT) TTA ATA)
ON23 5¢-a-D-ara(TTA TTA TTA TAA AAT TAT)
ON2 3¢-d(AAT AAT AAT ATT TTA ATA)
ON23 5¢-a-D-ara(TTA TTA TTA TAA AAT TAT)
ON4 3¢-r(AAU AAU AAU AUU UUA AUA)
ON1 5¢-d(TTA TTA TTA TAA AAT TAT)
16.0
nt
nt
nt
ON24 3¢-a-D-ara(AAT AAT AAT ATT TTA ATA)
ON3 5¢-r(UUA UUA UUA UAA AAU UAU)
ON24 3¢-a-D-ara(AAT AAT AAT ATT TTA ATA)
nt
ON11 5¢-d(TTA TTA TTa-D-ara(A TA)A AAT TAT) 26.5
ON15 3¢-d(AAT AAT AAa-D-ara(T AT)T TTA ATA)
ON12 5¢-d(TTA TTA a-D-ara(TTA TAA) AAT TAT)
ON16 3¢-d(AAT AAT a-D-ara(AAT ATT) TTA ATA)
ON23 5¢-a-D-ara(TTA TTA TTA TAA AAT TAT)
ON24 3¢-a-D-ara(AAT AAT AAT ATT TTA ATA)
16.0
a-L-ara vs a-D-ara hybridization in anti-parallel orientation
20.0
We have shown above that a-D-ara:a-D-ara base pairing and
a-L-ara:a-L-ara base pairing in central segments flanked by
b-D-ribo:b-D-ribo base paired segments enable stable duplex
a See captions below Table 1 and Table 2.
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Table 5 a-D-ara oligomers—hybridization in parallel orientationa
a-D-ara:a-D-ara or a-L-ara:a-D-ara base pairs). Fully modified
a-L-ara oligonucleotides, neither in antiparallel nor in a parallel
orientation were able to form duplexes towards DNA or RNA
complement. We have confirmed reports21 that fully modified a-D-
ara oligonucleotides are able to form rather stable parallel duplexes
with complementary a-D-ara oligonucleotides (also shown herein
in the enantiomeric a-L-ara series). We have shown that fully
modified a-L-ara oligonucleotides are unable to form parallel
or antiparallel duplexes with complementary DNA/RNA, and
confirmed parallel duplex formation of fully modified a-D-ara
oligonucleotides with a DNA complement. Furthermore, we have
for the first time shown parallel duplex formation between fully
modified a-D-ara oligonucleotides and RNA complements. In
summary, our results contribute to expanding the remarkable
stereochemical space available for nucleic acid duplex formation
which underlines the opportunity that prebiotic nucleic acid chem-
istry could have involved stereoirregular nucleic acid components.
Entry
45.
46.
47.
48.
49.
50.
51.
52.
53.
54.
Duplexes
Tm(◦C)
nt
ON1 5¢-d(TTA TTA TTA TAA AAT TAT)
ON5 5¢-d(AAT AAT AAT ATT TTA ATA)
ON2 5¢-d(ATA ATT TTA TAA TAA TAA)
ON6 5¢-d(TAT TAA AAT ATT ATT ATT)
ON3 5¢-r(UUA UUA UUA UAA AAU UAU)
ON7 5¢-r(AAU AAU AAU AUU UUA AUA)
ON4 5¢-r(AUA AUU UUA UAA UAA UAA)
ON8 5¢-r(UAU UAA AAU AUU AUU AUU)
ON1 5¢-d(TTA TTA TTA TAA AAT TAT)
ON7 5¢-r(AAU AAU AAU AUU UUA AUA)
ON2 5¢-d(ATA ATT TTA TAA TAA TAA)
ON8 5¢-r(UAU UAA AAU AUU AUU AUU)
ON23 5¢-a-D-ara(TTA TTA TTA TAA AAT TAT)
ON5 5¢-d(AAT AAT AAT ATT TTA ATA)
ON23 5¢-a-D-ara(TTA TTA TTA TAA AAT TAT)
ON7 5¢-r(AAU AAU AAU AUU UUA AUA)
ON6 5¢-d(TAT TAA AAT ATT ATT ATT)
ON24 5¢-a-D-ara (ATA ATT TTA TAA TAA TAA)
ON8 5¢-r(UAU UAA AAU AUU AUU AUU)
ON24 5¢-a-D-ara(AAT AAT AAT ATT TTA ATA)
nt
nt
nt
nt
nt
17.5
18.0
17.0
16.5
Experimental
a See captions below Table 1 and Table 2.
General
Reactions under anhydrous conditions were carried out under
an atmosphere of nitrogen. After drying an organic phase over
Na2SO4, filtration was performed. Solvents were of HPLC grade,
of which DMF, pyridine, acetonitrile and dichloromethane were
Table 6 a-L-ara vs a-D-ara hybridization in anti-parallel orientationa
Tm(◦C)
Entry Duplexes
˚
dried over molecular sieves (4 A). TLC was run on Merck silica
55.
56.
57.
58.
59.
60.
ON9 5¢-d(TTA TTA TT a-L-ara(A TA)A AAT TAT)
25.0
27.0
17.0
16.0
nt
60 F254 aluminium sheets. 1H NMR spectra were recorded at
300 MHz, 13C NMR spectra at 75.5 MHz and 31P NMR spectra
at 121.5 MHz (dH: CDCl3 7.26 ppm, DMSO-d6 2.50; dC: CDCl3
77.0 ppm, DMSO-d6 39.4). Chemical shifts are reported in ppm
relative to either tetramethylsilane or the deuterated solvent as
ON15 3¢-d(AAT AAT AAa-D-ara(T AT)T TTA ATA)
ON11 5¢-d(TTA TTA TTa-D-ara(A TA)A AAT TAT)
ON13 3¢-d(AAT AAT AAa-L-ara(T AT)T TTA ATA)
ON10 5¢-d(TTA TTA a-L-ara(TTA TAA) AAT TAT)
ON16 3¢-d(AAT AAT a-D-ara(AAT ATT) TTA ATA)
ON12 5¢-d(TTA TTA a-D-ara(TTA TAA) AAT TAT)
ON14 3¢-d(AAT AAT a-L-ara(AAT ATT) TTA ATA)
ON21 5¢-a-L-ara(TTA TTA TTA TAA AAT TAT)
ON24 3¢-a-D-ara(AAT AAT AAT ATT TTA ATA)
ON23 5¢-a-D-ara(TTA TTA TTA TAA AAT TAT)
ON22 3¢-a-L-ara(AAT AAT AAT ATT TTA ATA)
a internal standard for H NMR and 13C NMR, and relative to
1
85% H3PO4 as external standard for 31P NMR. Assignments of
NMR spectra, when given, are based on 2D NMR experiments
(the assignments of methylene protons/methylene carbons may
be interchanged). Coupling constants (J values) are given in
Hz. MALDI-HRMS were recorded in positive ion mode on an
nt
a See captions below Table 1 and Table 2.
Ion Spec Fourier transform mass spectrometer. The H and 13C
1
NMR data of the a-D-arabino-nucleosides were identical with
the corresponding enantiomeric a-L-arabino-nucleosides, and as
these nucleosides were synthesized by the same synthetic route
in similar scales (but starting from enantiomeric starting mate-
rials), we report only yield, optical rotation and high resolution
mass spectral data for the a-D-arabino-nucleosides; only if the
experimental procedure has been changed relative to the literature
procedure have synthetic details been added. All the a-D-arabino-
nucleoside triols 1D-4D were synthesized from D-arabinose and
their structural assignments were confirmed by comparison of the
NMR data with those reported in the literature.21,22
formation. As an expansion we also studied segments of
three, six and fully modified a-L-ara:a-D-ara base pairs (Table 6,
entries 55–60). Interestingly, duplexes of three and six a-L-ara/
a-D-ara in central segments (entries 55–58) showed similar
thermal stabilities as the duplexes with stereoregular central
segments (entries 17, 18, 42 and 43) demonstrating a remarkable
stereochemical space available for nucleic acid duplex formation.
In contrast, no hybridization between fully modified a-L-ara and
fully modified a-D-ara ONs was observed (entries 59–60).
Conclusion
9-(a-L-Arabinofuranosyl)-6-N-benzoyladenine (5). The nucle-
oside 2 (2.00 g, 7.49 mmol) was co-evaporated with pyridine (2 ¥
50 mL) and redissolved in pyridine (50 mL). Trimethylsilyl chloride
(TMSCl) (3.70 mL, 29.24 mmol) was added and the mixture
was stirred at RT for 1 h whereupon benzoyl chloride (1.16 mL,
8.98 mmol) was added dropwise. The resulting mixture was stirred
for 4 h at RT whereupon water (10 mL) was added. After stirring
for 5 min at RT, aqueous ammonia (18 mL, ~29% v/v) was
added and the resulting mixture was stirred for 15 min at RT and
We have successfully synthesized a-L-ara and a-D-ara phos-
phoramidite monomers and prepared fully modified as well
as partially modified oligonucleotides thereof. Reduced duplex
thermal stabilities relative to unmodified reference duplexes were
observed for oligonucleotides containing a segment of three and
six modifications of ara-T or ara-A monomers, whereas more
stable duplexes were obtained when the central segment included
exclusively a-ara-configured nucleotides (a-L-ara:a-L-ara or
2394 | Org. Biomol. Chem., 2009, 7, 2389–2401
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then evaporated to dryness under reduced pressure. The residue
was purified by column chromatography over silica gel (3–4%
methanol in ethyl acetate, v/v) to give nucleoside 5 as a white
solid material (2.30 g, 76%). Rf = 0.2 (10% MeOH in CH2Cl2,
reduced pressure and the residue diluted with dichloromethane
(200 mL). The organic phase was washed successively with
saturated aqueous solutions of NaHCO3 (2 ¥ 200 mL) and brine
(2 ¥ 200 mL) and was dried over Na2SO4. The solvent was removed
under reduced pressure and the residue was purified by column
chromatography over silica gel (80% ethyl acetate in petroleum
v/v). [a]D = -3.8 (c 0.1, MeOH). 1H NMR (DMSO-d6) d 3.40–
32
3.69 (2H, m, H-5¢), 4.13 (1H, q, J = 5.4 Hz, H-3¢), 4.24–4.29 (1H,
m, H-4¢), 4.75 (1H, q, J = 5.4 Hz, H-2¢), 4.96 (1H, t, J = 5.7 Hz,
OH-5¢), 5.69 (1H, d, J = 4.8 Hz, OH-3¢), 5.88 (1H, d, J = 5.1 Hz,
OH-2¢), 6.02 (1H, d, J = 5.1 Hz, H-1¢), 7.52–7.69 (3H, m, H-3¢¢,
H-4¢¢ and H-5¢¢), 8.04–8.07 (2H, m, H-2¢¢ and H-6¢¢), 8.70 (1H, s,
H-2), 8.78 (1H, s, H-8) and 11.2 (1H, br s, NH). 13C NMR (DMSO-
d6) d 61.1 (C-5¢), 75.9 (C-3¢), 79.2 (C-2¢), 85.6 (C-4¢), 88.6 (C-1¢),
125.9 (C-5), 128.5, 132.5, 133.4 (Ar-Bz), 143.6 (C-8), 150.3 (C-4),
151.6 (C-2), 152.1 (C-6) and 165.6 (COPh). MALDI-HRMS m/z
394.1124 ([M + Na]+, calculated for C17H17N5O5Na 394.1121).
ether, v/v) to afford nucleoside 9 as a white solid material (3.06 g,
32
81%). Rf = 0.28 (75% EtOAc in petroleum ether, v/v). [a]D
=
-22.5 (c 0.1, MeOH). 1H NMR (CDCl3) d 1.05–1.11 (28H, m, 4 ¥
CH(CH3)2), 3.94 (2H, br s, H-5¢), 4.39 (2H, br s, H-3¢ and H-4¢),
5.14 (1H, br s, H-2¢), 5.99 (2H, d, J = 6.0 Hz, H-1¢ and OH-2¢),
7.52–7.63 (3H, m, H-3¢¢, H-4¢¢ and H-5¢¢), 8.07 (2H, d, J = 7.3 Hz,
H-2¢¢ and H-6¢¢), 8.78 (2H, s, H-2 and H-8) 11.19 (1H, s, NH). 13
C
NMR (CDCl3) d 12.0, 12.2, 12.5, 12,9 (4 ¥ CH(CH3)2), 16.8, 16.90,
17.1, 17.2, (4 ¥ CH(CH3)2), 60.9 (C-5¢), 75.0 (C-3¢), 76.8 (C-2¢),
81.3 (C-4¢), 87.0 (C-1¢), 126.0 (C-5), 128.3 (C-3¢¢ and C-5¢¢), 128.4
(C-2¢¢ and C-6¢¢), 132.2 (C-4¢¢), 133.3 (C-1¢¢), 143.9 (C-8), 150.4 (C-
6), 151.6 (C-4), 152.2 (C-2), 165.5 (COPh). MALDI-HRMS m/z
636.2624 ([M + Na]+, calculated for C29H43N5O6Si2Na 636.2644).
1-(a-L-Arabinofuranosyl)-4-N-benzoylcytosine (6)32. The nu-
cleoside 6 was obtained as a white solid material (92% yield).
MALDI-HRMS m/z 370.1043 ([M + Na]+, calculated for
C16H17N3O6Na 370.1009). NMR data were identical with the
reported data.32
1-(3¢,5¢-O-(1,1,3,3-Tetraisopropylidisiloxane-1,3-diyl)-a-L-ara-
binofuranosyl)-4-N-benzoylcytosine (10). The nucleoside
6
9-(a-L-Arabinofuranosyl)-2-N-isobutyrylguanine (7). The nu-
cleoside 4 (3.20 g, 11.30 mmol) was dissolved in anhydrous
pyridine (100 mL) and TMSCl (7.17 mL, 56.53 mmol) was added.
After stirring for 30 min at RT, isobutyric anhydride (2.25 mL,
13.56 mmol) was added and the resulting mixture was stirred
for 2 h at RT. H2O (16 mL) was added and after stirring for
5 min at RT, aqueous ammonia (32 mL, ~29%) was added and the
resulting mixture was stirred at RT for another 15 min whereupon
it was evaporated to dryness under reduced pressure. The resulting
residue was purified by silica gel column chromatography (8–11%
methanol in chloroform, v/v) to afford nucleoside 7 as a white
solid material (3.12 g, 78%). Rf = 0.51 (15% MeOH in CH2Cl2,
(2.50 g, 7.20 mmol) was co-evaporated with pyridine and dissolved
in anhydrous pyridine (100 mL) under stirring. 1,3-Dichloro-
1,1,3,3-tetraisopropyldisiloxane (2.48 mL, 7.92 mmol) was added
and the resulting mixture was stirred for 3 h at RT under an
atmosphere of nitrogen. The solvent was removed under reduced
pressure and the residue diluted with dichloromethane (200 mL).
The organic phase was washed successively with saturated aqueous
solutions of NaHCO3 (2 ¥ 200 mL) and brine (2 ¥ 200 mL)
and dried over Na2SO4. The solvent was removed under reduced
pressure and the residue was purified by column chromatography
over silica gel (80% ethyl acetate in petroleum ether, v/v) to afford
nucleoside 10 as a white solid material (2.77 g, 66%). Rf = 0.6 (30%
v/v). [a]D = -16.5 (c 0.1, MeOH). 1H NMR (DMSO-d6) d 1.11
32
EtOAc in petroleum ether, v/v). [a]D = +14.5 (c 0.1, MeOH). 1H
32
(6H, d, J = 6.7 Hz, CH(CH3)2), 2.50 (1H, sept, J = 6.7 Hz,
CH(CH3)2), 3.48 (1H, dd, J = 11.7 and 4.8 Hz, H-5¢a), 3.60 (1H,
dd, J = 11.7 and 3.2 Hz, H-5¢b), 4.01 (1H, t, J = 5.6 Hz, H-3¢),
4.15 (1H, q, J = 3.9 Hz, H-4¢), 4.53 (1H, t, J = 5.0 Hz, H-2¢), 5.76
(1H, d, J = 4.8 Hz, H-1¢), 7.11–7.45 (3H, 3 s, OH-5¢, OH-3¢ and
OH -2¢), 8.21 (1H, s, H-8), 11.69 (1H, br s, NH), 12.09 (1H, br s,
NHCOCH(CH3)2). 13C NMR (DMSO-d6) d 19.5 (CH(CH3)2),
35.4 (CH(CH3)2), 61.8 (C-5¢), 75.9 (C-3¢), 80.3 (C-2¢), 86.0
(C-4¢), 88.7 (C-1¢), 120.9 (C-5), 139.0 (C-8), 148.7 (C-4), 149.3
(C-2), 155.6 (C-6), 180.8 (COCH(CH3)2). MALDI-HRMS m/z
376.1257 ([M + Na]+, calculated for C14H19N5O6Na 376.1227).
NMR (CDCl3) d 1.00–1.12 (28H, m, 4 ¥ CH(CH3)2), 3.91 (1H, m,
H-4¢), 4.03 (1H, dd, J = 13.0 and 2.5 Hz, H-5¢a), 4.13 (1H, dd, J =
13.0 and 3.2 Hz, H-5¢b), 4.24 (1H, m, H-3¢), 4.47 (1H, t, J = 7.6 Hz,
H-2¢), 4.64 (1H, s, OH-2¢), 5.58 (1H, d, J = 4.3 Hz, H-1¢), 7.52 (2H,
t, J = 7.1 Hz, H-3¢¢ and H-5¢¢), 7.60–7.67 (2H, m, H-5 and H-4¢¢),
7.89 (1H, d, J = 7.2 Hz, H-2¢¢ and H-6¢¢), 8.03 (1H, d, J = 7.2 Hz,
H-6), 8.70 (1H, br s, NH). 13C NMR (CDCl3) d 12.8, 13.2, 13.4,
13,8 (4 ¥ CH(CH3)2), 17.2, 17.4, 17.6, 17.8 (4 ¥ CH(CH3)2), 61.1
(C-5¢), 75.8 (C-3¢), 82.8 (C-2¢), 83.1 (C-4¢), 94.2 (C-1¢), 96.8 (C-5),
127.8 (C-3¢¢ and C-5¢¢), 129.5 (C-2¢¢ and C-6¢¢), 133.7 (C-6 and C-
4¢¢), 143.0 (C-2), 162.9 (C-4), 166.1 (COPh). MALDI-HRMS m/z
612.2507 ([M + Na]+, calculated for C28H43N3O7Si2Na 612.2531).
1-(3¢,5¢-O-(1,1,3,3-Tetraisopropyldisiloxane-1,3-diyl)-a-L-ara-
binofuranosyl)thymine (8)33. The nucleoside 8 was obtained as a
9-(3¢,5¢-O-(1,1,3,3-Tetraisopropyldisiloxane-1,3-diyl)-a-L-ara-
binofuranosyl)-2-N-isobutyrylguanine (11). The nucleoside 7
(2.90 g, 8.21 mmol) was co-evaporated with pyridine (2 ¥ 20 mL)
and dissolved in anhydrous pyridine (100 mL) under stirring. 1,3-
Dichloro-1,1,3,3-tetraisopropyldisiloxane (2.83 mL, 9.03 mmol)
was added dropwise and the resulting mixture was stirred for
12 h at RT under an atmosphere of nitrogen. The solvent
was removed under reduced pressure and the residue diluted
with dichloromethane (120 mL). The organic phase was washed
successively with saturated aqueous solutions of NaHCO3 (2 ¥
120 mL) and brine (2 ¥ 120 mL) and dried over Na2SO4. The
solvent was removed under reduced pressure and the residue was
32
white amorphous solid material (80% yield). [a]D = -12.6 (c 0.1,
MeOH). MALDI-HRMS m/z 523.2255 ([M + Na]+, calculated
for C22H40N2O7Si2Na 523.2266). NMR data were identical with
the reported data.33
9-(3¢,5¢-O-(1,1,3,3-Tetraisopropylidisiloxane-1,3-diyl)-a-L-ara-
binofuranosyl)-6-N-benzoyladenine (9). The nucleoside 5 (2.30 g,
6.19 mmol) was co-evaporated with pyridine and dis-
solved in anhydrous pyridine (100 mL). 1,3-Dichloro-1,1,3,3-
tetraisopropyldisiloxane (2.13 mL, 6.81 mmol) was added drop-
wise under stirring at RT and the mixture was stirred for 5 h at RT
under an atmosphere of nitrogen. The solvent was removed under
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purified by column chromatography over silica gel (35% ethyl
acetate in petroleum ether, v/v) to afford nucleoside 11 as a white
solid material (3.36 g, 76%). Rf = 0.45 (30% MeOH in CH2Cl2,
(1H, s, H-8), 9.41 (1H, s, NH). 13C NMR (CDCl3) d 12.8, 12.9,
13.2, 13.4, 134, 13.8 (4 ¥ -CH(CH3)2), 17.2, 17.2, 17.3, 17.4, 17.6,
17.6, 17.7 (4 ¥ -CH(CH3)2), 20.8 (COCH3), 61.7 (C-5¢), 74.9 (C-3¢),
80.9 (C-2¢), 83.7 (C-4¢), 86.7 (C-1¢), 123.6 (C-5), 128.3 (C-3¢¢ and
C-5¢¢), 128.85 (C-2¢¢ and C-6¢¢), 133.0 (C-4¢¢), 134.1 (C-1¢¢), 142.1
(C-8), 150.1 (C-6), 152.1 (C-4), 153.2 (C-2), 165.7 (COPh), 170.3
(COCH3). MALDI-HRMS m/z 678.2724 ([M + Na]+, calculated
for C31H45N5O7Si2Na 678.2749).
v/v). [a]D = +14.0 (c 0.1, MeOH). 1H NMR (CDCl3) d 1.04–1.22
32
(28H, m, 4 ¥ CH(CH3)2), 1.28 (6H, d, J = 6.6 Hz, COCH(CH3)2),
2.78 (1H, sept, J = 6.6 Hz, COCH(CH3)2), 3.96 (2H, br s, H-5¢),
4.21 (1H, br s, H-4¢), 4.45 (1H, t, J = 8.2 Hz, H-3¢), 5.13 (1H, br s,
H-2¢), 5.78 (1H, d, J = 5.3 Hz, H-1¢), 6.30 (1H, s, OH-2¢), 7.73 (1H,
s, H-8), 10.36 (1H, s, NH), 12.0 (1H, br s, NHCOCH(CH3)2). 13
C
1-(2¢-O-Acetyl-3¢,5¢-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-
diyl)-a-L-arabinofuranosyl)-4-N-benzoylcytosine (14). The nu-
cleoside 10 (2.60 g, 4.42 mmol) was co-evaporated with pyridine
and dissolved in anhydrous pyridine (30 mL) under stirring. Acetic
anhydride (0.67 mL, 7.07 mmol) was added and the resulting
mixture was stirred at RT under an atmosphere of nitrogen for 5 h.
The solvent was removed under reduced pressure and the residue
was dissolved in dichloromethane and was washed successively
with saturated aqueous solutions of NaHCO3 (2 ¥ 50 mL) and
brine (2 ¥ 50 mL) and dried over Na2SO4. The solvent was removed
under reduced pressure and the residue was purified by column
chromatography over silica gel (60% ethyl acetate in petroleum
NMR (CDCl3) d 12.7, 13.1, 13.4, 13.8 [4 ¥ CH(CH3)2], 17.2, 17.3,
17.3, 17.4, 17.6, 17.6, 17.8 (COCH(CH3)2 and 4 ¥ CH(CH3)2),
36.6 (COCH(CH3)2), 61.5 (C-5¢), 74.9 (C-3¢), 78.9 (C-2¢), 82.8 (C-
4¢), 89.8 (C-1¢), 120.6 (C-5), 138.8 (C-8), 148.1 (C-4), 148.5 (C-2),
155.6 (C-6), 179.9 [COCH(CH3)2]. MALDI-HRMS m/z 596.2993
([M + H]+, calculated for C26H46N5O7Si2 596.2930).
1-(2¢-O-Acetyl-3¢,5¢-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-
diyl)-a-L-arabinofuranosyl)thymine (12). The nucleoside
8
(3.35 g, 6.69 mmol) was co-evaporated with pyridine and dissolved
in anhydrous pyridine (65 ml) under stirring. Acetic anhydride
(0.94 ml, 9.91 mmol) was added and the resulting mixture was
stirred at RT under an atmosphere of nitrogen for 5 h. The solvent
was removed under reduced pressure and the residue was dissolved
in dichloromethane and was washed successively with saturated
aqueous solutions of NaHCO3 (2 ¥ 180 ml) and brine (2 ¥ 180 ml)
and dried over Na2SO4. The solvent was removed under reduced
pressure and the residue was purified by column chromatography
over silica gel (20–25% ethyl acetate in petroleum ether, v/v) to
afford nucleoside 12 as a white solid material (3.11 g, 86%). Rf =
ether, v/v) to afford nucleoside 14 as a white solid material (2.45 g,
32
88%). Rf = 0.51 (30% EtOAc in petroleum ether, v/v). [a]D
=
1
-22.6 (c 0.1, MeOH). H NMR (CDCl3) d 1.01–1.12 (28H, m,
4 ¥ CH(CH3)2), 2.11 (3H, s, COCH3), 3.97–4.03 (2H, m, H-5¢),
4.29–4.34 (1H, m, H-4¢), 4.59 (1H, t, J = 7.4 Hz, H-3¢), 5.61 (1H,
t, J = 5.2 Hz, H-2¢), 5.98 (1H, d, J = 5.2 Hz, H-1¢), 7.48–7.53 (3H,
m, H-5, H-3¢¢ and H-5¢¢), 7.61 (1H, m, H-4¢¢), 7.79 (2H, d, J =
7.4 Hz, H-2¢¢ and H-6¢¢), 7.89 (1H, d, J = 7.3 Hz, H-6), 8.70 (1H,
s, NH). 13C NMR (CDCl3) d 14.5, 15.0, 15.3, 15.5 (4 ¥ CH(CH3)2),
18.9, 19.0, 19.3, 19.4, 19.5 (4 ¥ CH(CH3)2), 22.7 ((COCH3), 63.9
(C-5¢), 76.2 (C-3¢), 82.5 (C-2¢), 86.0 (C-4¢), 91.5 (C-1¢), 99.4 (C-
5), 129.9 (C-2¢¢ and C-6¢¢), 131.0 (C-3¢¢ and C-5¢¢), 135.1 (C-4¢¢
and C-6), 146.5 (C-2 & C-1¢¢), 148.2 (C-4), 164.9 (COPh), 172.2
(COCH3). MALDI-HRMS m/z 654.2629 ([M + Na]+, calculated
for C30H45N3O8Si2Na 654.2637).
32
0.7 (5% MeOH in CH2Cl2, v/v). [a]D = -8.9 (c 0.1, MeOH). 1H
NMR (CDCl3) d 1.02–1.11 (28H, m, 4 ¥ CH(CH3)2), 1.95 (3H,
s, CH3-5), 2.09 (3H, s, COCH3), 3.97 (2H, d, J = 4.2 Hz, H-5¢),
4.21 (1H, m, H-4¢), 4. 56 (1H, t, J = 7.8 Hz, H-3¢), 5.55 (1H, t,
J = 6.8 Hz, H-2¢), 5.87 (1H, d, J = 5.7 Hz, H-1¢), 7.15 (1H, s,
H-6) and 9.01 (1H, br s, NH). 13C NMR (CDCl3) d 12.8, 12.9,
13.2, 13.5, 13.7 (4 ¥ CH(CH3)2), 17.1, 17.2, 17.29, 17.6, 17.7 (4 ¥
CH(CH3)2), 20.9 (COCH3), 62.2 (C-5¢), 74.2 (C-3¢), 80.1 (C-2¢),
83.5 (C-4¢), 87.5 (C-1¢), 111.9 (C-6), 136.0 (C-5), 150.9 (C-2), 163.9
(C-4) and 170.5 (COCH3). MALDI-HRMS m/z 565.2372 ([M +
Na]+, calculated for C24H42N2O8Si2Na 565.2371).
9-[2¢-O-Acetyl-3¢,5¢-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-
diyl)-a-L-arabinofuranosyl)-2-N-isobutyrylguanine (15). The nu-
cleoside 11 (3.20 g, 5.93 mmol) was co-evaporated with pyridine
and dissolved in anhydrous pyridine (60 mL) under stirring. Acetic
anhydride (0.9 mL, 9.49 mmol) was added and the resulting
mixture was stirred at RT under an atmosphere of nitrogen for 5 h.
The solvent was removed under reduced pressure and the residue
was dissolved in dichloromethane (50 mL) and was washed with
saturated aqueous solutions of NaHCO3 (2 ¥ 60 mL) and brine
(2 ¥ 60 mL) and dried over Na2SO4. The solvent was removed
under reduced pressure and the residue was purified by column
chromatography over silica gel (40% ethyl acetate in petroleum
9-(2¢-O-Acetyl-3¢,5¢-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-
diyl)-a-L-arabinofuranosyl)-6-N-benzoyladenine (13). The nucle-
oside 9 (3.00 g, 4.89 mmol) was co-evaporated with pyridine and
dissolved in anhydrous pyridine (30 ml) under stirring. Acetic
anhydride (0.74 ml, 7.83 mmol) was added and the resulting
mixture was stirred at RT under an atmosphere of nitrogen for 5 h.
The solvent was removed under reduced pressure and the residue
was dissolved in dichloromethane and was washed successively
with saturated aqueous solutions of NaHCO3 (2 ¥ 150 ml) and
brine (2 ¥ 150 ml) and dried over Na2SO4. The solvent was removed
under reduced pressure and the residue was purified by column
chromatography over silica gel (45–60% ethyl acetate in petroleum
ether, v/v) to afford nucleoside 15 as a white solid material (2.75 g,
32
80%). Rf = 0.35 (30% EtOAc in petroleum ether, v/v). [a]D
=
1
-38.9 (c 0.1, MeOH). H NMR (CDCl3) d 1.03–1.12 (28H, m,
4 ¥ CH(CH3)2), 1.24 (6H, d, J = 5.7 Hz COCH(CH3)2), 2.10
(3H, s, COCH3), 2.69 (1H, sept, J = 5.7 Hz, COCH(CH3)2),
3.91–4.05 (2H, m, H-5¢), 4.31 (1H, br s, H-4¢), 4.58 (1H, t, J =
6.9 Hz, H-3¢), 5.85 (1H, d, J = 3.2 Hz, H-2¢), 6.05 (1H, br s,
H-1¢), 7.93 (1H, s, H-8), 9.20 (1H, br s, NH), 12.09 (1H, br s,
NHCOCH(CH3)2). 13C NMR (CDCl3) d 12.7, 13.2, 13.5, 13.7 (4 ¥
CH(CH3)2), 17.2, 17.3, 17.5, 17.6, 17.6, 17.7, 19.3 (4 ¥ CH(CH3)2
and COCH(CH3)2), 20.9 (COCH3), 36.7 (COCH(CH3)2), 62.1
ether, v/v) to give nucleoside 13 as a white solid material (2.87 g,
32
90%). Rf = 0.44 (75% EtOAc in petroleum ether, v/v). [a]D
=
-14.6 (c 0.1, MeOH). 1H NMR (CDCl3) d 0.91–1.14 (28H, m, 4 ¥
CH(CH3)2), 2.08 (3H, s, COCH3), 4.0–4.05 (2H, m, H-5¢), 4.53
(1H, q, J = 3.5 Hz, H-4¢), 4.67 (1H, t, J= 8.0 Hz, H-3¢), 6.07–6.13
(2H, m, H-1¢ and H-2¢), 7.46–7.57 (3H, m, H-3¢¢, H-4¢¢ and H-5¢¢),
8.03 (2H, d, J = 7.3 Hz, H-2¢¢ and H-6¢¢), 8.19 (1H, s, H-2), 8.80
2396 | Org. Biomol. Chem., 2009, 7, 2389–2401
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(C-5¢), 75.8 (C-3¢), 80.9 (C-2¢), 83.9 (C-4¢), 86.5 (C-1¢), 122.0 (C-5),
138.3 (C-8), 148.1 (C-4), 148.6 (C-2), 155.9 (C-6), 170.3 (COCH3)
179.0 (COCH(CH3)2). MALDI-HRMS m/z 638.3109 ([M + H]+,
calculated for C28H48N5O8Si2 638.3035).
pressure and the residue was co-evaporated with toluene (2 ¥
30 mL) and purified by column chromatography over silica gel
(70–80% ethyl acetate in petroleum ether, v/v) to afford nucleoside
18 as a white solid material (1.13 g, 80%). Rf = 0.52 (10% MeOH
in CH2Cl2, v/v). [a]D = -31.6 (c 0.1, MeOH). 1H NMR (DMSO-
32
1-(2¢-O-Acetyl-a-L-arabinofuranosyl)thymine (16). The nucle-
oside 12 (2.40 g, 4.42 mmol) was co-evaporated with anhydrous
acetonitrile (2 ¥ 50 mL) and dissolved in anhydrous acetonitrile
(80 mL). To this solution was added Et3N·3HF dropwise (1.62 mL,
9.95 mmol) dissolved in acetonitrile (3.0 mL). The mixture was
stirred at RT under an atmosphere of nitrogen for 5 h whereupon
the solvent was evaporated under reduced pressure. The residue
was co-evaporated with toluene (2 ¥ 50 mL) and purified
by column chromatography over silica gel (2–5% methanol in
chloroform, v/v) to afford the nucleoside 16 as a white solid
material (1.21 g, 91%). Rf = 0.2 (10% MeOH in CH2Cl2, v/v).
d6) d 2.08 (3H, s, COCH3), 3.54–3.56 (2H, m, H-5¢), 4.16 (1H, q,
J = 3.6 Hz, H-4¢), 4.38 (1H, t, J = 4.3 Hz, H-3¢), 4.96 (1H, t, J =
5.6 Hz, OH-5¢), 5.29 (1H, br s, H-2¢), 5.74 (1H, d, J = 4.1 Hz, OH-
3¢), 5.85 (1H, d, J = 2.1 Hz, H-1¢), 7.36 (1H, d, J = 5.8 Hz, H-5),
7.51 (2H, t, J = 7.3 Hz, H-3¢¢ and H-5¢¢), 7.62 (1H, t, J = 7.1 Hz,
H-4¢¢), 8.01 (2H, d, J = 7.4 Hz, H-2¢¢ and H-6¢¢), 8.15 (1H, d, J =
7.2 Hz, H-6), 11.25 (1H, s, NH). 13C NMR (DMSO-d6) d 20.6
(COCH3), 60.8 (C-5¢), 73.0 (C-3¢), 81.3 (C-2¢), 88.2 (C-4¢), 90.8
(C-1¢), 95.9 (C-5), 128.1 (C-2¢¢ and C-6¢¢), 128.4 (C-3¢¢ and C-5¢¢),
132.7 (C-4¢¢), 133.1 (C-1¢¢), 146.1 (C-6), 154.4 (C-4), 163.3 (C-2),
167.3 (COPh), 169.5 (COCH3). MALDI-HRMS m/z 412.1120
([M + Na]+, calculated for C18H19N3O7Na 412.1115).
[a]D = -7.0 (c 0.1, MeOH). 1H NMR (DMSO-d6) d 1.79 (3H, s,
32
C-5CH3), 2.04 (3H, s, COCH3), 3.46 (1H, d, J = 11.6 Hz, H-5¢a),
3.56 (1H, d, J = 11.6 Hz, H-5¢b), 4.20 (2H, br s, H-3¢ and H-4¢),
4.91 (1H, br s, OH-5¢), 5.25 (1H, t, J = 4.9 Hz, H-2¢), 5.73 (2H,
d, J = 4.1 Hz, H-1¢), 5.86 (1H, br s, OH-3¢), 7.64 (1H, s, H-6)
and 11.25 (1H, br s, NH). 13C NMR (DMSO-d6) d 12.1 (C-5CH3),
20.5 (COCH3), 62.1 (C-5¢), 75.2 (C-3¢), 83.6 (C-2¢), 87.7 (C-4¢),
89.2 (C-1¢), 125.1 (C-5), 129.4 (C-3¢¢ and C-5¢¢), 129.7 (C-2¢¢ and
C-6¢¢), 133.9 (C-4¢¢), 134.9 (C-1¢¢), 144.8 (C-8), 151.2 (C-6), 153.1
(C-4), 153.2 (C-2), 165.5 (COPh) and 171.9 (COCH3). MALDI-
HRMS m/z 323.0850 ([M + Na]+, calculated for C12H16N2O7Na
323.0849).
9-(2¢-O-Acetyl-a-L-arabinofuranosyl)-2-N -isobutyrylguanine
(19). The nucleoside 15 (2.60 g, 4.47 mmol) was co-evaporated
with anhydrous acetonitrile (2 ¥ 30 mL) and dissolved in
anhydrous acetonitrile (60 mL). To this solution was dropwise
added Et3N·3HF (1.64 mL, 10.06 mmol) dissolved in acetonitrile
(3.0 mL) and the resulting mixture was stirred at RT under an
atmosphere of nitrogen for 5 h. The solvent was evaporated under
reduced pressure and the residue was coevaporated with toluene
(2 ¥ 50 mL) and purified with column chromatography (4–5%
methanol in chloroform, v/v) over silica gel to afford nucleoside
19 as a white solid material (1.14 g, 79%). Rf = 0.35 (70% EtOAc
32
in petroleum ether, v/v). [a]D = -27.0 (c 0.1, MeOH). 1H NMR
9-(2¢-O-Acetyl-a-L-arabinofuranosyl)-6-N-benzoyladenine (17).
The nucleoside 13 (2.20 g, 3.35 mmol) was co-evaporated with
anhydrous acetonitrile (2 ¥ 50 mL) and dissolved in anhydrous ace-
tonitrile (55 ml). To this solution was dropwise added Et3N·3HF
(1.23 mL, 7.55 mmol) dissolved in acetonitrile (3.0 mL). The
mixture was stirred at RT under an atmosphere of nitrogen for 5 h
whereupon the solvent was evaporated under reduced pressure.
The residue was co-evaporated with toluene (2 ¥ 50 mL) and
purified by column chromatography over silica gel (70–80% ethyl
acetate in petroleum ether, v/v) to afford nucleoside 17 as a white
solid material (1.28 g, 93%). Rf = 0.35 (10% MeOH in CH2Cl2,
(DMSO-d6) d 1.12 (6H, d, J = 6.6 Hz, COCH(CH3)2), 2.06 (3H,
s, COCH3), 2.78 (1H, sept, J = 6.6 Hz, COCH(CH3)2), 3.52–3.63
(2H, m, H-5¢), 4.21–4.24 (2H, m, H-3¢ and H-4¢), 4.94 (1H, t, J =
5.2 Hz, OH-5¢), 5.58 (1H, t, J = 4.5 Hz, H-2¢), 5.89 (1H, d, J =
4.8 Hz, OH-3¢), 5.95 (1H, d, J = 3.8 Hz, H-1¢), 8.22 (1H, s, H-
8), 11.59 (1H, br s, NH), 12.12 (1H, br s, NHCOCH(CH3)2).
13C NMR (DMSO-d6) d 18.8 (COCH(CH3)2), 20.5 (COCH3),
34.7 (COCH(CH3)2), 60.4 (C-5¢), 73.0 (C-3¢), 81.5 (C-2¢), 85.3
(C-4¢), 85.6 (C-1¢), 120.1 (C-5), 138.1 (C-8), 148.1 (C-4), 148.4 (C-
2), 154.8 (C-6), 169.8 (COCH3) 180.1 [COCH(CH3)2]. MALDI-
HRMS m/z 418.1313 ([M + Na]+, calculated for C16H21N5O7Na
418.1333).
v/v). [a]D = -38.6 (c 0.1, MeOH). 1H NMR (DMSO-d6) d 2.06
32
(3H, s, COCH3), 3.60 (2H, m, H-5¢), 4.29–4.35 (1H, m, H-4¢),
4.37–4.39 (1H, m, H-3¢), 4.94 (1H, t, J = 5.6 Hz, OH-5¢), 5.85
(1H, t, J = 4.7 Hz, H-2¢), 5.95 (1H, d, J = 4.9 Hz, OH-3¢), 6.21
(1H, d, J = 4.2 Hz, H-1¢), 7.53–7.67 (3H, m, H-3¢¢, H-4¢¢, H-5¢¢),
8.05 (2H, d, J = 7.3 Hz, H-2¢¢ and H-6¢¢), 8.65 (1H, s, H-2), 8.78
(1H, s, H-8). 13C NMR (DMSO-d6) d 19.0 (COCH3), 58.9 (C-5¢),
71.6 (C-3¢), 79.7 (C-2¢), 83.9 (C-4¢), 84.9 (C-1¢), 124.2 (C-5), 126.9
(C-3¢¢, C-5¢¢, C-2¢¢ and C-6¢¢), 130.9 (C-4¢¢), 131.8 (C-1¢¢), 141.9
(C-8), 148.9 (C-6), 150.2 (C-4), 150.4 (C-2), 164.1 (COPh), 168.4
(COCH3). MALDI-HRMS m/z 436.1213 ([M + Na]+, calculated
for C19H19N5O6Na 436.1227).
1-(2¢-O-Acetyl-5¢-O-(4,4¢-dimethoxytrityl)-a-L-arabinofura-
nosyl)thymine (20). The nucleoside 16 (0.60 g, 1.99 mmol) was
coevaporated with anhydrous pyridine (2 ¥ 5 mL) and then
dissolved in a mixture of dichloromethane and pyridine (30 mL,
4:1). 4,4¢-Dimethoxytrityl chloride (DMTCl; 0.74 g, 2.19 mmol)
was added under an atmosphere of nitrogen and the resulting
mixture was stirred at RT for 10 h. Additional DMTCl (0.075 g,
0.22 mmol) was added after 10 h and reaction mixture was
stirred for another 2 h. The reaction mixture was evaporated to
dryness and the residue was dissolved in dichloromethane (60 mL)
whereupon washing was performed using a saturated aqueous
solution of NaHCO3 (2 ¥ 20 mL). The organic phase was dried over
Na2SO4, filtered and evaporated to dryness under reduced pressure
to get a residue which was purified by column chromatography
over silica (60–70% ethyl acetate in petroleum ether, v/v) to afford
nucleoside 20 (1.01 g, 84%) as a white solid material. Rf = 0.4
(5% MeOH in CH2Cl2, v/v). 1H NMR (DMSO-d6) d 1.81 (3H, d,
1-(2¢-O-Acetyl-a-L-arabinofuranosyl)-4-N -benzoylcytosine
(18). The nucleoside 14 (2.30 g, 3.65 mmol) was co-evaporated
with anhydrous acetonitrile (2 ¥ 30 mL) and dissolved in anhy-
drous acetonitrile (60 mL). To this solution was dropwise added
Et3N·3HF (1.33 mL, 8.21 mmol) dissolved in acetonitrile (3.0 mL)
and the resulting mixture was stirred at RT under an atmosphere
of nitrogen for 5 h. The solvent was evaporated under reduced
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Org. Biomol. Chem., 2009, 7, 2389–2401 | 2397
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J = 0.9 Hz, CH3-5), 1.99 (3H, s, COCH3), 3.12 (2H, d, J = 4.5 Hz,
H-5¢), 3.72 (6H, s, 2 ¥ OCH3), 4.22 (1H, q, J = 5.1 Hz, H-3¢), 4.46
(1H, q, J = 4.5 Hz, H-4¢), 5.24 (1H, t, J = 4.5 Hz, H-2¢), 5.84–5.88
(2H, m, H-1¢ and 3¢-OH), 6.91 (4H, d, J = 8.4 Hz, ArH), 7.23–
7.42 (9H, m, ArH), 7.67 (1H, d, J = 0.9 Hz, H-6) and 11.35 (1H,
s, NH). 13C NMR (DMSO-d6) d 12.1 (C-5CH3), 20.4 (COCH3),
55.0 (2 ¥ OCH3), 62.8 (C-5¢), 73.2 (C-3¢), 80.7 (C-2¢), 83.9 (C-4¢),
85.4 (C-1¢), 87.8 (C(Ph)3), 109.3 (C-5), 113.2, 126.7, 127.6, 127.8,
129.6, 135.4, 135.4, 136.9, 144.7, 150.4, 158.0 (Ar-DMT, C-6 and
C-2), 163.8 (C-4), 169.6 (COCH3).MALDI-HRMS m/z 625.2146
([M + Na]+, calculated for C33H34N2O9Na 625.2156).
OH-3¢), 6.91 (4H, d, J = 7.8 Hz, Ar-DMT), 7.22–7.65 (13H, m,
H-5, Ar-DMT and Ar-Bz), 8.01 (2H, d, J = 7.5 Hz, Ar-Bz), 8.22
(1H, d, J = 7.5 Hz, H-6) and 9.21 (1H, br s, NHCOPh). 13C NMR
(CDCl3) d 20.4 (COCH3) 55.0 (2 ¥ OCH3), 63.0 (C-5¢), 73.9 (C-3¢),
81.0 (C-2¢), 85.4 (C-4¢), 86.5 (C-1¢), 91.0 (C(Ph)3), 95.8 (C-5), 113.2
(Ar-DMT), 126.7, 127.6, 127.8, 128.4, 129.6, 132.7, 133.1, 135.3,
135.4, 144.7 (Ar-DMT and Ar-Bz), 146.2 (C-6), 154.4 (C-2), 158.1
(Ar-DMT), 163.4 (C-4), 167.2 (COPh), 169.1 (COCH3). MALDI-
HRMS m/z 714.2395 ([M + Na]+, calculated for C39H37N3O9Na
714.2421).
9-(2¢-O-Acetyl-5¢-O-(4,4¢-dimethoxytrityl)-a-L-arabinofura-
nosyl)-2-N-isobutyrylguanine (23). The nucleoside 19 (0.50 g,
1.20 mmol) was co-evaporated with pyridine (2 ¥ 10 mL) and then
dissolved in a mixture of dichloromethane and pyridine (30 mL,
4:1). DMTCl (0.48 g, 1.43 mmol) was added and the resulting
mixture was stirred for 12 h under an atmosphere of nitrogen.
The reaction mixture was evaporated to dryness under reduced
pressure and the residue was dissolved in dichloromethane (60 mL)
whereupon washing was performed using a saturated aqueous
solution of NaHCO3 (2 ¥ 20 mL). The organic phase was dried
over Na2SO4 and evaporated to dryness under reduced pressure.
The residue was purified by column chromatography over silica
gel (70–75% ethyl acetate in petroleum ether) to give nucleoside
23 as a white solid material (0.39 g, 45%). Rf = 0.4 (5% MeOH
in CH2Cl2, v/v). 1H NMR (DMSO-d6) d 1.12 (6H, d, J = 6.6 Hz,
COCH(CH3)2), 2.00 (3H, s, COCH3), 2.78 (1H, sept, J = 6.6 Hz,
COCH(CH3)2), 3.11–3.21 (2H, m, H-5¢), 3.74 (6H, 2 s, 2 ¥ OCH3),
4.34 (1H, dd, J = 10.2 Hz and 4.8 Hz, H-3¢), 4.53 (1H, dd, J = 9.6
and 5.1 Hz, H-4¢), 5.54 (1H, t, J = 4.2 Hz, H-2¢), 6.00–6.02 (1H,
m, OH-1¢ and OH-3¢), 6.90 (4H, d, J = 8.4 Hz, Ar-DMT), 7.23–
7.42 (9H, m, Ar-DMT), 8.24 (1H, s, H-8), 11.59 (1H, br s, NH),
12.12 (1H, br s, NHCOCH(CH3)2). 13C NMR (DMSO-d6) d 18.8
(COCH(CH3)2), 20.4 (COCH3), 34.7 (COCH(CH3)2), 55.0 (2 ¥
OCH3), 62.5 (C-5¢), 73.7 (C-3¢), 81.6 (C-2¢), 83.8 (C-4¢), 85.4 (C-1¢),
85.8 (C-Ph3), 113.2 (Ar-DMT), 120.2 (C-5), 126.7, 127.6, 127.8,
129.7, 135.4, 135.4 (Ar-DMT), 138.2 (C-8), 144.7 (Ar-DMT),
148.1 (C-4), 148.3 (C-2), 154.7 (C-6), 158.1 (Ar-DMT), 169.6
(COCH3), 180.1 (COCH(CH3)2). MALDI-HRMS m/z 720.2625
([M + Na]+, calculated for C37H39N5O9Na 720.2639).
9-(2¢-O-Acetyl-5¢-O-(4,4¢-dimethoxytrityl)-a-L-arabinofura-
nosyl)-6-N-benzoyladenine (21). The nucleoside 17 (0.65 g,
1.57 mmol) was co-evaporated with pyridine (2 ¥ 15 mL) and
then dissolved in a mixture of dichloromethane and pyridine
(50 mL, 4:1). DMTCl (0.586 g, 1.72 mmol) was added and the
resulting mixture was stirred for 12 h under an atmosphere of
nitrogen. The reaction mixture was evaporated to dryness under
reduced pressure and the residue was dissolved in dichloromethane
(100 ml) whereupon washing was performed using a saturated
aqueous solution of NaHCO3 (2 ¥ 30 mL). The organic phase
was dried over Na2SO4 and evaporated to dryness under reduced
pressure. The residue was purified by column chromatography
over silica gel using (75–80% ethyl acetate in petroleum ether,
containing 0.5% Et3N, v/v/v) to afford nucleoside 21 as a white
solid material (0.87 g, 78%). Rf = 0.36 (5% MeOH in CH2Cl2,
1
v/v). H NMR (CDCl3) d 2.06 (3H, s, COCH3), 3.34 (2H, m,
H-5¢), 3.78 (6H, s, 2 ¥ OCH3), 4.49–4.53 (1H, m, H-4¢), 4.57 (1H,
q, J = 4.8 Hz, H-3¢), 5.52 (1H, s, H-2¢), 5.99 (1H, d, J = 1.5 Hz,
H-1¢), 6.10 (1H, d, J = 9.6 Hz, OH-3¢), 6.88 (4H, d, J = 8.7 Hz, Ar-
H), 7.18–7.62 (12H, m, Ar-H), 8.01 (2H, d, J = 7.2 Hz, Ar-H), 8.14
(1H, s, C-2H), 8.77 (1H, s, C-8H) and 9.21 (1H, br s, NHCOPh).
13C NMR (CDCl3) d 20.7 (COCH3) 55.3 (2 ¥ OCH3), 62.8 (C-5¢),
77.0 (C-3¢), 85.1 (C-2¢), 86.3 (C(Ph)3), 87.9 (C-4¢), 90.2 (C-1¢), 113.2
(Ar-DMT), 123.7 (C-5), 126.9, 127.9, 128.0, 128.2, 129.0, 130.1,
133.1, 133.5, 135.8, 135.9 (Ar-Bz, Ar-DMT), 143.2 (C-8), 144.7
(Ar-DMT), 150.2 (C-6), 150.5 (C-4), 152.3 (C-2), 158.6 (OCH3),
164.6 (COPh), 170.6 (COCH3). MALDI-HRMS m/z 738.2582
([M + Na]+, calculated for C40H37N5O8Na 738.2534).
9-(2¢-O-Acetyl-5¢-O-(4,4¢-dimethoxytrityl)-a-L-arabinofura-
nosyl)-4-N-benzoylcytosine (22). The nucleoside 18 (0.50 g,
1.29 mmol) was co-evaporated with pyridine (2 ¥ 10 mL) and then
dissolved in a mixture of dichloromethane and pyridine (30 mL,
4:1). DMTCl (0.48 g, 1.41 mmol) was added and the resulting
mixture was stirred for 12 h under an atmosphere of nitrogen.
The reaction mixture was evaporated to dryness under reduced
pressure and the residue was dissolved in dichloromethane (60 mL)
whereupon washing was performed using a saturated aqueous
solution of NaHCO3 (2 ¥ 20 mL). The organic phase was dried
over Na2SO4 and evaporated to dryness under reduced pressure.
The residue was purified by column chromatography over silica gel
(75–80% ethyl acetate in petroleum ether, v/v) to give nucleoside
22 as a white solid material (0.72 g, 81%). Rf = 0.7 (10% MeOH in
CH2Cl2, v/v). 1H NMR (CDCl3) d 1.90 (3H, s, COCH3), 3.07 (1H,
dd, J = 9.9 Hz and 5.7 Hz, H-5¢a), 3.19 (1H, dd, J = 9.9 Hz and
5.7 Hz, H-5¢b), 3.74, 3.81 (6H, s, 2 ¥ OCH3), 4.15 (1H, q, J = 3.3 Hz,
H-4¢), 4.67 (1H, q, J = 5.4 Hz, H-3¢), 5.25 (1H, d, J = 2.4 Hz,
H-2¢), 5.85 (1H, d, J = 2.4 Hz, H-1¢), 5.87 (1H, d, J = 4.5 Hz,
1-(2¢-O-Acetyl-3¢-O-(2-cyanoethoxy(diisopropylamino)phosphi-
no)-5-O-(4,4¢-dimethoxytrityl)-a-L-arabinofuranosyl)thymine
(24). 2-Cyanoethyl-N,N-diisopropylphosphoramidochloridite
(0.36 mL, 0.16 mmol) was added dropwise to a stirred solu-
tion of the nucleoside 20 (0.90 g, 1.49 mmol) and anhydrous
N,N-diisopropylethylamine (1.2 mL, 6.88 mmol) in anhydrous
dichloromethane (40 mL) and the resulting mixture was stirred at
RT for 5 h under an atmosphere of nitrogen. Methanol (2 mL),
followed by dichloromethane (25 mL) were added and washing
was performed using saturated aqueous NaHCO3 (2 ¥ 20 mL). The
organic phase was dried over Na2SO4 and evaporated to dryness
under reduced pressure. The residue was purified by column chro-
matography (35–40% ethyl acetate in petroleum ether, containing
0.5% Et3N, v/v/v) over silica gel to give phosphoramidite 24 as
a white solid material (0.93 g, 78%). Rf = 0.51, 0.53 (75% EtOAc
in petroleum ether, v/v). 31P NMR (121.5 MHz, CDCl3) d 152.44
and 153.08. MALDI-HRMS m/z 825.3216 ([M + Na]+, calculated
for C42H51N4O10PNa 825.3235).
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9-(2¢-O-Acetyl-3¢-O-(2-cyanoethoxy(diisopropylamino)phosphi-
no)-5¢-O-(4,4¢-dimethoxytrityl)-a-L-arabinofuranosyl)-6-N-ben-
zoyladenine (25). The nucleoside 21 (0.80 g, 1.11 mmol) was co-
evaporated with anhydrous acetonitrile (3 ¥ 15 mL) and then
dissolved in anhydrous dichloromethane (50 mL). Anhydrous
N,N ¢-diisopropylethyl amine (1.6 mL, 9.18 mmol) was added fol-
lowed by 2-cyanoethyl-N,N ¢-diisopropylphosphoamidochlorodite
(0.30 mL, 1.34 mmol), and the resulting mixture was stirred for 3 h
at RT under an atmosphere of nitrogen. Methanol (3 mL) followed
by dichloromethane (35 mL) were added and successive washings
were performed using saturated aqueous solutions of NaHCO3
(2 ¥ 35 mL) and brine (2 ¥ 35 mL). The organic phase was dried
over Na2SO4 and evaporated to dryness under reduced pressure.
The residue was purified by column chromatography over silica gel
(50–60% ethyl acetate in petroleum ether, containing 0.5% Et3N,
v/v/v) to afford nucleoside 25 as a white solid material (0.78 g,
79%). Rf = 0.50, 0.55 (50% ethyl acetate in petroleum ether, v/v).
31P NMR (CDCl3) d 151.95 and 152.76. MALDI-HRMS m/z
938.3582 ([M + Na]+, calculated for C49H54N7O9PNa 938.3612).
0.5% Et3N, v/v/v) to afford nucleoside 27 as a white solid material
(0.24 g, 50%). Rf = 0.45, 0.50 (50% EtOAc in petroleum ether, v/v).
31P NMR (CDCl3) d 151.49 and 151.78. MALDI-HRMS m/z
920.3762 ([M + Na]+, calculated for C46H56N7O10PNa 920.3718).
2,2¢-Anhydro-a-L-ribofuranosylthymine (28). The nucleoside
1 (2.00 g, 7.74 mmol) was dissolved in anhydrous N,N ¢-
dimethylformamide (8 mL) followed by addition of diphenyl
carbonate (4.14 g, 19.36 mmol) and sodium carbonate (0.19 g,
2.32 mmol), and the resulting reaction mixture was refluxed for
3 h whereupon it was cooled to RT and poured slowly into diethyl
ether (200 mL) with stirring. The dark brown residue thus obtained
was washed twice with diethyl ether (2 ¥ 100 mL) and subjected
to column chromatography on silica gel (14–18% methanol in
chloroform, v/v) to afford nucleoside 28 as a white solid material
(0.69 g, 32%). Rf = 0.51 (20% MeOH in CHCl3, v/v). 1H-NMR
(DMSO-d6): d 1.95 (s, 3H, CH3), 3.63–3.68 (m, 2H, H-5¢a+b), 3.85
(br s, 1H, H-4¢), 4.22 (m, 1H, H-3¢), 5.28 (t, 1H, J = 5.3 Hz, H-
2¢), 6.24 (d, 1H, J = 5.2 Hz, H-1¢), 7.70 (s, 1H, H-6). 13C-NMR
(75.5 MHz): d 12.8 (CH3), 59.9 (C-5¢), 70.6 (C-3¢), 81.2 (C-4¢), 82.4
(C-2¢), 90.0 (C-1¢), 118.0 (C-5), 133.1 (C-6), 162.0 (C-4). MALDI-
HRMS m/z 263.0641 ([M + Na]+, calculated for C10H12N2O5Na
263.0638).
9-(2¢-O-Acetyl-3¢-O-(2-cyanoethoxy(diisopropylamino)phosphi-
no)-5¢-O-(4,4¢-dimethoxytrityl)-a-L-arabinofuranosyl)-4-N-ben-
zoylcytosine (26). The nucleoside 22 (0.60 g, 0.87 mmol) was
co-evaporated with anhydrous acetonitrile (3 ¥ 10 mL) and then
dissolved in anhydrous dichloromethane (30 mL). Anhydrous
N,N ¢-diisopropylethyl amine (1.0 mL, 5.74 mmol) was added fol-
lowed by 2-cyanoethyl-N,N ¢-diisopropylphosphoamidochlorodite
(0.23 mL, 0.95 mmol) and the resulting mixture was stirred for 12 h
at RT under an atmosphere of nitrogen. Additional 2-cyanoethyl-
N,N ¢-diisopropylphosphoamidochlorodite (0.1 mL, 0.42 mmol)
was added and stirring was continued for 5 h. Methanol (2 mL)
followed by dichloromethane (20 mL) were added and successive
washings were performed using saturated aqueous solutions of
NaHCO3 (2 ¥ 20 mL) and brine (2 ¥ 20 mL). The organic phase
was dried over Na2SO4 and evaporated to dryness under reduced
pressure. The residue was purified by column chromatography over
silica gel (50–60% ethyl acetate in petroleum ether, containing
0.5% Et3N, v/v/v) to afford nucleoside 26 as a white solid
material (0.415 g, 80%). Rf = 0.50, 0.55 (50% EtOAc in petroleum
ether, v/v). 31P NMR (CDCl3) d 151.95 and 151.96. MALDI-
HRMS m/z 914.3481 ([M + Na]+, calculated for C48H54N5O10PNa
914.3500).
2¢-Azido-2¢-deoxy-a-L-arabinofuranosylthymine (29). To
a
stirred solution of nucleoside 28 (0.80 g, 3.33 mmol) in anhydrous
N,N ¢-dimethylformamide (16 mL) was added sodium azide (1.73 g,
26.7 mmol) and the resulting reaction mixture was heated under
reflux for 9 h and then cooled to RT. The solvent was removed
under reduced pressure and the residue was purified by silica gel
column chromatography (4–5% methanol in chloroform, v/v) to
afford the nucleoside 29 as a yellowish oil (0.58 g, 61%). Rf =
1
0.25 (5% MeOH in CHCl3, v/v). H-NMR (DMSO-d6) d 1.90
(s, 3H, CH3), 3.62–3.76 (m, 2H, H-5¢), 4.18–4.22 (m, 2H, H-2¢ &
H-3¢), 4.31 (m, 1H, H-4¢), 5.79 (br s, 1H, H-1¢), 7.54 (s, 1H, H-
6). 13C-NMR (DMSO-d6) d 12.8 (CH3), 62.6 (C-5¢), 72.1 (C-2¢),
75.6 (C-3¢), 87.7 (C-4¢), 90.5 (C-1¢), 112.1 (C-5), 138.5 (C-6), 152.7
(C-4), 166.7 (C-2). MALDI-HRMS m/z 306,0795 ([M + Na]+,
calculated for C10H13N5O5Na 306.0808).
2¢-Amino-2¢-deoxy-a-L-arabinofuranosylthymine (30). To a so-
lution of nucleoside 29 (0.10 g, 0.35 mmol) in anhydrous methanol
(4 mL) was added 10% Pd/C (10 mg) and the resulting reaction
mixture was stirred for 8 h under an atmosphere of hydrogen.
The mixture was filtered on a small bed of Celite and the bed was
washed with 20 mL of hot methanol. The combined fractions after
washing and filtration was evaporated to dryness under reduced
pressure to afford nucleoside 4 as an off-white solid material
(74 mg, 82%). Rf = 0.32 (20% MeOH in CHCl3, v/v). 1H-NMR
(DMSO-d6) d 1.90 (s, 3H, CH3), 3.62 (m, 1H, H-2¢), 3.66–3.71 (m,
2H, H-5¢), 4.13–4.17 (m, 1H, H-3¢), 4.28 (br s, 1H, H-4¢), 5.88 (d,
1H, J = 3.6 Hz, H-1¢), 7.59 (s, 1H, H-6). 13C-NMR (DMSO-d6) d
12.1 (CH3), 61.4 (C-2¢), 62.7 (C-5¢), 75.8 (C-3¢), 85.5 (C-4¢), 89.4
(C-1¢), 109.2 (C-5), 136.7 (C-6), 150.8 (C-4), 163.8 (C-2). MALDI-
HRMS m/z 258.1090 ([M + H]+, calculated for C10H16N3O5Na
258.1084).
9-(2¢-O-Acetyl-3¢-O-(2-cyanoethoxy(diisopropylamino)phosphi-
no)-5¢-O-(4,4¢-dimethoxytrityl)-a-L-arabinofuranosyl)-2-N -iso-
butyrylguanine (27). The nucleoside 23 (0.35 g, 0.49 mmol) was
co-evaporated with anhydrous acetonitrile (3 ¥ 10 mL) and then
dissolved in anhydrous dichloromethane (30 mL). Anhydrous
N,N ¢-diisopropylethyl amine (1.0 mL, 5.74 mmol) was added fol-
lowed by 2-cyanoethyl-N,N ¢-diisopropylphosphoamidochlorodite
(0.144 g, 0.6 mmol) and the resulting mixture was stirred for 12 h
at RT under an atmosphere of nitrogen. Additional 2-cyanoethyl-
N,N ¢-diisopropylphosphoamidochlorodite (0.1 mL, 0.42 mmol)
was added and stirring was continued for 5 h. Methanol (2 mL)
followed by dichloromethane (20 mL) were added and successive
washings were performed using saturated aqueous solutions of
NaHCO3 (2 ¥ 20 mL) and brine (2 ¥ 20 mL). The organic phase
was dried over Na2SO4 and evaporated to dryness under reduced
pressure. The residue was purified by column chromatography
over silica gel (50–60% ethyl acetate in petroleum ether, containing
1-(2¢-N-Trifluoroacetyl-a-L-arabinofuranosyl)thymine
(31).
To a suspension of nucleoside 30 (0.60 g, 2.33 mmol) in methanol
(20 mL) was added 4-(N,N-dimethylamino)pyrimidine (0.16 g,
1.30 mmol) and ethyl trifluoroacetate (0.43 g, 3.03 mmol), and
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the resulting reaction mixture was stirred for 5 h at RT. The
solvent was removed under reduced pressure, and the residue
was subjected to column chromatography on silica gel (0–10%
methanol in dichloromethane, v/v) to afford nucleoside 31 as
a white solid material (0.71 g, 87%). Rf = 0.5 (15% MeOH in
Synthesis of oligonucleotides containing a-ara nucleotide
monomers. The oligomers (Table 1) were synthesized on an auto-
mated DNA synthesizer using the phosphoramidite approach. The
synthesis of ON9–ON20 containing three or six a-ara nucleotide
monomers was performed in 0.2 mmol scale whereas the fully
modified a-L-ara oligomers ON21–ON24 were synthesized in
1.0 mmol scale on a universal support. Standard conditions of
the synthesizer were used for incorporation of DNA monomers.
The a-L-ara-, a-D-ara- and 2¢-amino-a-L-ara phosphoramidite
building blocks were used for the synthesis of modified oligomers
ON9–ON24. The stepwise coupling yields for amidites 24–27
and 33 (10 min coupling time, 1H-tetrazole as activator for
a-L-ara-T and a-D-ara-T monomers; 15 min coupling time,
pyridinium hydrochloride as activator for a-L-ara-A and a-D-
ara-A monomers) were 98–100% based on the absorbance of the
dimethoxytriyl cation released after each coupling step. After
detritylation, cleavage from the solid support and deacylation
was carried out by using 30% aqueous ammonia solution (12 h,
55 ◦C). For fully modified oligomers, 2M methanolic ammonia
1
CH2Cl2, v/v). H NMR (DMSO-d6) d 1.82 (3H, s, CH3), 3.45
(1H, d, J = 11.4 Hz, H-5¢a), 3.63 (1H, d, J = 11.7 Hz, H-5¢b),
4.17 (1H, m, H-4¢), 4.30 (1H, m, H-3¢), 4.49 (1H, q, J = 6.3 Hz,
H-2¢), 4.96 (1H, br s, OH-5¢), 5.68 (1H, d, J = 5.7 Hz, OH-3¢),
5.91 (1H, d, J = 7.8 Hz, H-1¢), 7.73 (1H, s, H-6), 7.79 (1H, d, J =
5.4 Hz, NH-2¢) and 11.29 (1H, s, NH). 13C NMR (DMSO-d6) d
12.0 (CH3), 59.9 (C-2¢), 60.4 (C-5¢), 70.7 (C-3¢), 84.1 (C-4¢), 84.8
(C-1¢), 110.1 (C-5), 117.6 (CF3), 136.1 (C-6), 150.6 (C-2), 156.5
(COCF3) and 163.6 (C-4). MALDI-HRMS m/z 376.0727 ([M +
Na]+, calculated for C12H14F3N3O6Na 376.0726).
1-(2¢-N -Trifluoroacetyl-5¢-O-(4,4¢-dimethoxytrityl)-a-L-ara-
binofuranosyl)thymine (32). The nucleoside 31 (0.63 g,
1.78 mmol) was co-evaporated with anhydrous pyridine (2 ¥
5 mL) and then dissolved in a mixture of dichloromethane and
pyridine (24 mL, 2:1) whereupon DMTCl (0.69 g, 2.05 mmol) was
added. The resulting mixture was stirred for 14 h at RT under
an atmosphere of nitrogen whereupon dichloromethane (100 mL)
was added. The resulting mixture was washed with a saturated
aqueous solution of NaHCO3 (2 ¥ 50 mL), dried over Na2SO4 and
evaporated to dryness under reduced pressure. The residue was
purified by column chromatography over silica gel (20–80% ethyl
acetate in petroleum ether, v/v) to afford nucleoside 32 (0.91 g,
78%) as a white solid material. Rf = 0.4 (5% MeOH in CH2Cl2,
◦
was applied (30 min, 20 C) followed by 30% aqueous ammonia
(12 h, 55 ◦C). Analysis by ion-exchange HPLC verified the purity
of all a-ara modified oligomers to be >80%, and their composition
was verified by MALDI-TOF mass spectrometry (Table 1).
Thermal denaturation experiments. UV-based thermal denat-
uration experiments were performed on a Perkin-Elmer Lambda
35 UV/vis spectrometer equipped with PTP 6 (Peltier Temperature
Programmer) in a medium or low salt buffer (see captions below
tables for details). The solution of the two oligonucleotides (1.0 mM
of each strand) were thoroughly mixed, heated to 90 ◦C and
subsequently cooled to the starting temperature of the experiment
(5 ◦C). Thermal denaturation temperatures (Tm values, ◦C) were
determined as the maximum of the first derivative of the thermal
denaturation curve (A260 vs temperature; reported Tm values are
an average of two measurements within 1.0 ◦C).
1
v/v). H NMR (DMSO-d6) d 1.84 (3H, s, CH3), 3.05 (1H, dd,
J = 4.5 Hz, H-5¢a), 3.16 (1H, br s, H-5¢b), 3.73 (6H, s, 2 ¥ OCH3),
4.38 (2H, m, H-3¢ and H4¢), 4.51 (1H, q, J = 7.5 Hz, H-2¢), 5.77
(1H, d, J = 5.1 Hz, OH-3¢), 5.99 (1H, d, J = 7.5 Hz, H-1¢), 6.91
(4H, d, J = 8.7 Hz, ArH), 7.20–7.44 (9H, m, ArH), 7.78 (1H, s,
H-6), 9.78 (1H, d, J = 7.8 Hz, NH-2¢) and 11.35 (1H, s, NH).
13C NMR (DMSO-d6) d 12.0 (CH3), 55.0 (2 ¥ OCH3), 59.7 (C-
2¢), 62.7 (C-4¢), 71.2 (C-3¢), 81.8 (C-4¢), 84.4 (C-1¢), 85.3 (C(Ph)3),
110.3 (C-5), 113.2 (Ar-DMT), 117.6 (CF3), 126.6, 127.7, 127.8,
129.7, 135.5, 136.1, 144.8, 150.7, 158.0 (Ar-DMT, C-6 and C-2),
158.0 (COCF3) and 163.6 (C-4). MALDI-HRMS m/z 678.2034
([M + Na]+, calculated for C33H32F3N3O8Na 678.2033).
Acknowledgements
The Nucleic Acid Center is a research center of excellence
funded by the Danish National Research Foundation for studies
on nucleic acid chemical biology. We gratefully acknowledge
financial support by The Danish National Research Foundation,
The Danish Research Agency and Department of Biotechnology
(DBT, New Delhi). J.M. thanks the Council of Scientific and
Industrial Research (CSIR, New Delhi) for the award of senior
research fellowships.
1-(2¢-N -Trifluoroacetyl-3¢-O-(2-cyanoethoxy(diisopropylam-
ino)phosphino)-5-O-(4,4¢-dimethoxytrityl)-a-L-arabinofuranosyl)-
thymine (33). The nucleoside 32 (0. 51 g, 0.77 mmol)
was dissolved in anhydrous acetonitrile (20 ml) and N,N-
diisopropylammonium tetrazolide (0.20 g, 1.16 mmol) and
bis(N,N-diisopropylamino)-2-cyanoethoxyphosphine (0.61 ml,
1.94 mmol) were added. The resulting reaction mixture was stirred
for 2 h at RT under an atmosphere of nitrogen whereupon it
was diluted with EtOAc (100 mL), washed with H2O (75 mL),
washed with a 5% aqueous solution of NaHCO3 (75 mL), washed
with H2O (75 ml), dried over Na2SO4 and evaporated to dryness
under reduced pressure. The residue was purified by silica gel
column chromatography (20–60% EtOAc in petroleum ether, v/v)
to afford nucleoside 33 (0.60 g, 90%) as a white solid material. Rf =
0.65 (80% EtOAc in petroleum ether, v/v); 31P NMR (CDCl3)
d 150.8 and 151.2. MALDI-HRMS m/z 878.3111 ([M + Na]+,
calculated for C42H49F3N5O9PNa 878.3112).
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The Royal Society of Chemistry 2009
Org. Biomol. Chem., 2009, 7, 2389–2401 | 2401
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