The synthesis of double-headed nucleosides by the CuAAC reaction and their effect in secondary nucleic acid structures

Article abstract of DOI:10.1039/c0ob00438c

Four double-headed nucleosides were prepared by the CuAAC reaction. Hereby, a triazole-containing linker connects an additional thymine or adenine to the 2′-position of 2′-deoxyuridine, a thymine to the 5′-position of thymidine and a thymine to the 6′-pos

Full text of DOI:10.1039/c0ob00438c

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PAPER
The synthesis of double-headed nucleosides by the CuAAC reaction and their
effect in secondary nucleic acid structures†
Anna S. Jørgensen, Khalil I. Shaikh, Gerald Enderlin, Elise Ivarsen, Surender Kumar and Poul Nielsen*
Received 16th July 2010, Accepted 2nd November 2010
DOI: 10.1039/c0ob00438c
Four double-headed nucleosides were prepared by the CuAAC reaction. Hereby, a triazole-containing
linker connects an additional thymine or adenine to the 2¢-position of 2¢-deoxyuridine, a thymine to the
5¢-position of thymidine and a thymine to the 6¢-position of an LNA-thymidine monomer. Whereas no
conclusive recognition effects of the additional thymines were found when introduced in LNA or at the
5¢-position, both thymine and adenine in the 2¢-position were found to stabilise three-way junctions in
both dsDNA and DNA : RNA contexts and to give cross-strand interactions in a DNA-duplex, when
specifically introduced in a so-called (+1)-zipper motif.
Introduction
Nucleic acids are intensively investigated as scaffolds for
supramolecular design for instance by organizing various entities
on the surface of the double helix.1–4 Double-headed nucleosides
are synthetic nucleoside analogues with two nucleobases. We
and others have recently prepared a series of double-headed
nucleosides in order to study the recognition effects of the
additional nucleobases in different nucleic acid constructs (Fig.
1).5–12 A uridine analogue with an additional thymine in the 2¢-
Fig. 1 Double-headed nucleosides. U = uracil-1-yl, T = thymin-1-yl. B =
various nucleobases.
interactions have been observed.8 The same has been observed for
double-headed amino-LNA nucleosides 4 (B = T or A).9
position, 1, demonstrated a stabilisation of a three-way junction
when incorporated in the center of the junction propably due to
Since the introduction of Click Chemistry in 2001,13 a lot of
the additional stacking introduced by the thymine.5 On the other
attention has been given to especially one reaction; the Cu(I)-
hand, a thymidine analogue with an additional thymine in the
5¢(S)-C-position, 3 (B = T), did not stabilize a three-way junction
catalyzed alkyne azide cycloaddition (CuAAC).14,15 This reaction
fulfils all the original demands for click chemistry13 including the
but demonstrated a very selective zipper-interaction in a duplex.
high efficiency and specificity, tolerancy of all other functional
With the monomer incorporated in each of the complementary
groups and no side products formed. It is now a well-established
reaction16,17 also in nucleoside/nucleic acid chemistry.2,18,19 We have
strands with two interspacing unmodified base-pairs (defined as a
(-3)-zipper), a large relative stabilisation was seen due to stacking
recently applied the CuAAC reaction in the study of triazoles
between the two additional thymines across the minor groove.6
stacking in the major groove leading to highly stable DNA : RNA
This was observed specifically in the (-3)-zipper motif, and an
duplexes.20,21 We have also recently studied a series of double-
even stronger contact was recently found when one (and only
functionalized nucleosides with triazoles replacing the additional
one) of the two thymines in the zipper was replaced with a phenyl
nucleobase in 3.7 It was found, however, that the (-3)-zipper
contact in the minor groove was smaller when one or both of
the additional thymines in the zipper were replaced by either
unsubstituted triazoles or triazoles connected directly to a phenyl
or a uracil. On the other hand, other contacts in the minor groove,
for instance hydrogen-bonds of the latter, were observed.7
Herein, we present four new double-headed nucleosides pre-
pared by the CuAAC reaction and placing the additional
nucleobases in three different positions; the 2¢-position of 2¢-
deoxyuridine, the 5¢(S)-C-position of thymidine and the 6¢(R)-
position of the LNA-thymidine monomer. All three positions
will place the additional nucleobases through a triazolemethylene
linker in the minor groove of a duplex.
group.7 Double-headed nucleosides with the additional nucleobase
in the 4¢-position, 2 (B = T or A) have been introduced into
each complementary strand of a duplex but only weak base-base
Nucleic Acid CenterbDepartment of Physics and Chemistry, University of
Southern Denmark, 5230 Odense M, Denmark. E-mail: pon@ifk.sdu.dk;
Fax: +45 66158780; Tel: +45 65502565
† Electronic supplementary information (ESI) available: Preparation of
compound 6, MALDI data for all oligonucleotides, a few more thermal
hybridisation data with M as well as selected NMR-spectra. See DOI:
10.1039/c0ob00438c
‡ Nucleic Acid Center is funded by the Danish National Research
Foundation for studies on nucleic acid chemical biology.
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93% yield. Phosphitylation afforded the phosphoramidite 11.
Recently, other examples of CuAAC reactions with 2¢-azido-
2¢-deoxyuridine have been presented.25,26 The protected 5¢(S)-C-
azidomethylthymidine 12 has been recently prepared by us and
used in CuAAC-reactions with a series of small alkynes.7 Here
the reaction with 5 gave the double-headed nucleoside 13 in 80%
yield. Desilylation gave 14 and phosphitylation gave the amidite
15. The branched LNA-derivative 16 has been recently presented
by us including also the CuAAC-reaction affording the double-
headed nucleoside 17.27 Protection with the DMT-group gave 18
and phosphitylation afforded the phosphoramidite 19.
Results and discussion
Chemical synthesis. The CuAAC reaction was used by the
combination of three different nucleoside azides and two propar-
gylated nucleobases (Scheme 1). 2¢-Azido-2¢-deoxyuridine was
prepared by a known method based on the nucleophilic opening
of 2,2¢-anhydrouridine,22 and protected with the DMT-group to
give 7,23 which was then reacted in two different cycloadditions.
With N1-propargylthymine 5,24 the CuAAC reaction afforded
the double-headed nucleoside 8 in a good yield. Standard
phosphitylation gave the phosphoramidite 9 as an appropriate
building block for automated oligonucleotide synthesis. With
the corresponding propargylated benzoyl-adenine 6, which was
obtained from the known N9-propargyladenine24 by benzoylation,
the other double-headed nucleoside 10 was obtained in a high
Preparation and evaluation of oligonucleotides. The four phos-
phoramidites were introduced into oligonucleotides by standard
methodology following the phosphoramidite approach. 1H-
Tetrazole was used as the coupling reagent for both modified
and unmodified 2¢-deoxynucleoside phosphoramidites. Extended
coupling time of 15–20 min. was applied for the modified
amidites affording >80% coupling yields. The oligonucleotide
(ON) sequences applied in this study are the same as used in
former studies of zipper contacts in duplexes6,7,12 (Table 1 and
2), and of bulged duplexes and three-way junctions5,6,28,29 (Table
3), respectively. The modified phosphoramidites, 9, 11, 15 and 19
were incorporated to give the modified monomers K, L, M and N,
respectively.
At first, an 11-mer duplex was studied, in which five centrally
placed thymidines can be replaced systematically with the mod-
ified monomers (Table 1). When either of the two 2¢-modified
monomers K and L was incorporated, this gives the sequences
K1–K5 and L1–L5. These sequences were mixed with unmodified
complementary sequences and the melting temperatures (Tm) of
these modified duplexes were measured in thermal denaturation
experiments. These were compared with the Tm of the correspond-
ing unmodified duplex, and the differences in melting temperature
(DTms) are shown in Table 1. As evident from the data, all single
incorporations of K or L demonstrated a rather uniform and large
decrease in duplex stability of 8–11 ◦C. In other words, neither
the adenine nor the thymine substituents are very well adapted in
the minor groove of the duplex indicating that the 2¢-C-triazole is
unfavourable for duplex formation. A small part of the decrease,
however, might be deduced to the uracil replacing the thymine in
the core of the duplex.
Hereafter, the modified sequences where mixed with comple-
ments also containing the same modification (K or L) in order
to study the possibility for zipper contacts in the minor groove.
Hence, the two modifications in each of the complementary
strands were placed with 0–3 interspacing base-pairs revealing
possible (+1) to (-4)-zippers as shown in Table 1. In the case of
(-2) to (-4)-zippers, all melting temperatures were decreased with
19–23 ◦C indicating a fully additive negative effect for each of the
two modifications. Hence, when the changes in Tm were compared
to the changes obtained for each of the corresponding single
modified duplexes, the changes (defined as DDTms) were between
0 and -5 ◦C indicating absolutely no positive contacts between the
modifications in the minor groove. When the two modifications
were placed on two neighbouring base-pairs giving a possible (-1)-
zipper contact, the picture varies between the studied duplexes.
Hence the duplex formed between K5 and K1 was somewhat more
stable than the K4 : K2 duplex (with a 5.5 ◦C difference in Tm), and
Scheme 1 Reagents and conditions: a, Na ascorbate, CuSO4·5H2O,
tBuOH, H2O, pyridine, 72%; b, NC(CH2)2OP(Cl)N(iPr)2, DIPEA,
DCE, 70%; c, Na ascorbate, CuSO4·5H2O, THF, H2O, pyridine,
93%; d, NC(CH2)2OP(Cl)N(iPr)2, DIPEA, CH2Cl2, 62%; e, same as
a, 80%; f, TBAF, THF, 91%; g, same as d, 90%; h, ref. 27 (i)
Na ascorbate, CuSO4·5H2O, tBuOH, H2O, MW, 75%, (ii) H2, Pd/C,
EtOH, 67%; i, DMT-Cl, CH3CN, pyridine, 60%; j, same as d, 41%;
TBS = tert-butyldimethylsilyl, Pixyl = 9-phenylxanthen-9-yl, DMT =
4,4¢-dimethoxytrityl.
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Table 1 Hybridisation data for incorporations of monomer K, L, M and N
DTm/◦Ca[DDTm]/◦Cb
X =
X =
ON Duplex
K
L
M
N
Zipper ON Duplex
K
L
M
N
ref.c 5¢-d(CGC ATA TTC GC)
ref.c 3¢-d(GCG TAT AAG CG)
(+1)
X5
X2
5¢-d(CGC AXA TTC GC) -8.0
3¢-d(GCG TAX AAG CG) [+7.5]
-5.5
n.dd
n.dd
[+11.0]
X1
X2
X3
X4
X5
5¢-d(CGC ATA TTC GC)
3¢-d(GCG XAT AAG CG)
X5
X1
5¢-d(CGC AXA TTC GC) -14.5
3¢-d(GCG XAT AAG CG) [+1.5]
-14.5 n.dd
[+2.0]
n.dd
-8.0
-7.5
-8.5
-8.5
-6.0 +3.0
-6.0 +5.0
(-1)
(-1)
(-2)
(-3)
(-4)
5¢-d(CGC ATA TTC GC)
3¢-d(GCG TAX AAG CG)
X4
X2
5¢-d(CGC ATA XTC GC) -20.0
3¢-d(GCG TAX AAG CG) [-2.5]
-17.0 -14.0 +6.0
[0.0] [-1.5] [+0.5]
5¢-d(CGC ATA TXC GC)
3¢-d(GCG TAT AAG CG)
X3
X2
5¢-d(CGC ATA TXC GC) -21.5
3¢-d(GCG TAX AAG CG) [-3.0]
-19.0 -14.0 +9.0
[-0.5] [-1.5] [+2.5]
-11.0 -10.0 -6.5 +1.5
5¢-d(CGC ATA XTC GC)
3¢-d(GCG TAT AAG CG)
X4
X1
5¢-d(CGC ATA XTC GC) -21.0
3¢-d(GCG XAT AAG CG) [-3.0]
-22.0 -12.0 +4.5
[-5.0] [+0.5] [+1.0]
-10.0 -8.5
-8.0 -8.0
-6.5 +0.5
5¢-d(CGC AXA TTC GC)
3¢-d(GCG TAT AAG CG)
X3
X1
5¢-d(CGC ATA TXC GC) -22.0
3¢-d(GCG XAT AAG CG) [-3.0]
-22.5 -12.0 +2.5
[-4.0] [+0.5] [-2.0]
n.dd n.dd
a Differences in melting temperatures as compared to the unmodified duplex; DTm = Tm(duplex) - Tm(ref), Tm(ref) = 46.0 ◦C. Melting temperatures (Tm
values/◦C) were obtained from the maxima of the first derivatives of the melting curves (A260 vs. temperature) recorded in a medium salt buffer (Na2HPO4
(2.5 mM), NaH2PO4 (5 mM), NaCl (100 mM), EDTA (0.1 mM), pH 7.0) using 1.0 mM concentrations of each strand. K, L, M and N correspond to
the incorporation of the amidites, 9, 11, 15 and 19, respectively. b Differences in melting temperatures as compared to singly modified duplexes; DDTm
DTm(a : b) - (DTm(a : ref) + DTm(ref : b)). c Reference sample. d Not determined.
=
Table 2 Hybridisation data for crossed zippers with monomer K and L
that a larger compensation in the duplex stability was obtained
with the adenines of L (DDTm = +11 ◦C) than with the thymines
of K (DDTm = +7.5 ◦C), as the adenine has a larger surface for
stacking than the thymine. In order to investigate the possibility
for mixed base-contacts the sequences containing K and L were
also mixed (Table 2). The (+1)-zipper contacts of the K5 : L2 and
L5 : K2 duplexes were comparable to the contacts found for the
two adenines with DDTms of +11.5 and +9.5 ◦C, respectively. This
indicates that the zipper effect is based on stacking and not on any
Watson-Crick base-pair formation between A and T. In order to
get the full picture of possible A-T contacts, all other combinations
of K and L sequences were also investigated (Table 2). The results
were in all cases fully comparable to the results found with either
two K’s or two L’s, that is all DDTms being negative, despite in one
(-1)-zipper being slightly positive, indicating in general no positive
base-base contacts in the minor groove.
DTm/◦Ca [DDTm]/◦Cb
X/Y =
Zipper
(+1)
(-1)
ON
Duplex
K/L
L/K
X5
X2
5¢-d(CGC AXA TTC GC)
3¢-d(GCG TAY AAG CG)
-5.0
[+11.5]
-6.0
[+9.5]
X5
X1
5¢-d(CGC AXA TTC GC)
3¢-d(GCG YAT AAG CG)
-15.5
[+1.0]
-14.5
[+1.5]
X4
X2
5¢-d(CGC ATA XTC GC)
3¢-d(GCG TAY AAG CG)
-19.0
[-0.5]
-19.5
[-3.5]
(-1)
X3
X2
5¢-d(CGC ATA TXC GC)
3¢-d(GCG TAY AAG CG)
-21.5
[-2.0]
-20.0
[-2.5]
(-2)
X4
X1
5¢-d(CGC ATA XTC GC)
3¢-d(GCG YAT AAG CG)
-23.5
[-5.0]
-18.5
[-2.0]
(-3)
X3
X1
5¢-d(CGC ATA TXC GC)
3¢-d(GCG YAT AAG CG)
-23.5
[-4.0]
-20.5
[-2.5]
Hereafter, the monomer M containing a CH2-triazole-CH2
linked thymine at the 5¢(S)-C-position was incorporated in all the
same sequences despite one (M1–M4, Table 1). When these were
mixed with unmodified complements, the decreases in stability for
the single modified duplexes were very uniform with a decrease
in Tm of 6–6.5 ◦C. Hereby, the 5¢-substituent is slightly better
accommodated in the duplex as compared to the corresponding
2¢-substituent, in line with the 5¢-position being closer to the rim
of the minor groove and away from the duplex core. Compared to
other 5¢-substituents (analogues of 3, Fig. 1), the thermal penalty
paid for the thymine-methylene-triazole substituent is comparable
to other substituted triazoles7 and to the thymine as introduced
by our first monomer 3 (B = T), which gave Tms of around -5 ◦C.6
When the modified sequences M1–M4 were mixed, no zipper
contacts were found, as indicated by very neutral DDTms of -1.5 or
+0.5 ◦C. This is completely opposite the situation found for 3 (B =
T), for which a very strong (-3)-zipper was found (DDTms around
(-4)
a
,b See Table 1.
the same, though less pronounced, was valid for the L5 : L1 duplex
compared to L4 : L2. Nevertheless, the relative gains in Tm (as valid
from the DDTms of up to +2.0 ◦C) were still too small to conclude
for a contact between the 2¢-substituents in the duplex, and they
might just reflect other sequence specific variations related to, for
instance, duplex hydration.
When the possible (+1)-zipper contact was studied, interesting
results were revealed. Hence the duplexes K5 : K2 and L5 : L2 were
equally or more stable than either of the corresponding single
modified duplexes indicating a stabilising contact between the
2¢-substituents. Based on simple modeling showing the two 2¢-
carbons in opposite positions relative to the duplex core, this
contact could be the additional nucleobases partly intercalating
in the duplex opposing each other. This is supported by the fact
◦
+6–7 C),6 but parallel to the results found for an unsubstituted
triazole7 or a thymine on an extended linker 3 (B = T-CH2).12
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Table 3 Hybridization data for bulged duplexes and three-way junctionsa
Tm/◦C (DTm)/◦C
X =
X6
5¢- d(GCT CAC XCT CCC A),
T
TT
K
L
M
DNA
RNA
DNA, A-bulge
RNA, A-bulge
DNA, GA -bulge
RNA, GA-bulge
DNA hairpin
3¢- d(CGA GTG AGA GGG T)
3¢- r(CGA GUG AGA GGG U)
3¢- d(CGA GTG AAG AGG GT)
3¢- r(CGA GUG AAG AGG GU)
3¢- d(CGA GTG AGA GAG GGT)
3¢- r(CGA GUG AGA GAG GGU)
3¢- d(CGA GTG ACC CGC GTT
TTC GCG AGA GGG T)
52.5
61.0
43.0
50.5
43.5
50.5
24.5
34.5
n.d.b 47.0 (-5.5)
n.d.b 60.0 (-1.0)
47.5 (-5.0)
48.0 (-4.5)
59.5 (-1.5)
58.0 (-3.0)
53.5
60.5
42.5
50.5
26.0
35.5
38.5
37.5
43.0 (0.0/-10.5)
46.0 (+3.0/-7.5)
50.5 (0.0/-10.0)
41.5 (-2.0/-1.0)
49.5 (-1.0/-1.0)
39.5 (-3.5/-14.0)
47.5 (-3.0/-13.0)
39.0 (-4.5/-3.5)
48.0 (-2.5/-2.5)
49.5 (-1.0/-11.0)
40.0 (-3.5/-2.5)
49.5 (-1.0/-1.0)
28.0(+3.5/+2.0)
35.0 (+0.5/-0.5)
37.5 (-0.5/-1.0)
41.5 (+4.5/+4.0)
0 mM Mg2+
5 mM Mg2+
28.5 (+4.0/+2.5) 24.0 (-0.5/-2.0)
36.0 (+1.5/+0.5) 32.0 (-2.5/-3.5)
10 mM Mg2+ 38.0
38.0 (0.0/-0.5)
34.5 (-3.5/-4.0)
RNA haripin
3¢- r(CGA GUG ACG CGU UUU
CGC GAG AGG GU)
0 mM Mg2+
5 mM Mg2+
37.0
44.5
39.5 (+2.5/+2.0) 38.0 (+1.0/+0.5)
n.d.b n.d.b
47.5 49.0 (+2.0/+1.5)
46.0 (+1.5)
47.5 (+0.5/0.0)
n.d.b
47.0 (0.0/-0.5)
10 mM Mg2+ 47.0
a Melting temperatures (Tm values/◦C) obtained from the maxima of the first derivatives of the melting curves (A260 vs. temperature) recorded in a medium
salt buffer (Na2HPO4 (2.5 mM), NaH2PO4 (5 mM), NaCl (100 mM), EDTA (0.1 mM), pH 7.0) using 1.0 mM concentrations of each strand. In brackets,
differences in melting temperatures as compared to X = T/TT. K, L and M correspond to the incorporation of the amidites, 9, 11 and 15, respectively.
b Not determined.
Apparently, the triazole itself cannot induce any positive stacking
contact, and with a methylene in the linker, the flexibility
removes any significant effect of the thymines. Like with other
5¢-functionalized nucleosides,6,7,12 we also studied multiple incor-
porations of M but found no melting temperatures indicating any
compensating effects of having more than one monomer in the
strand (see ESI†).
were smaller with DTms of only -1, -1.5, and -3 ◦C for K, L and
M, respectively. Hence, especially the 2¢-modifications are much
better accommodated in a DNA : RNA duplex than in dsDNA.
Hereafter, the hybridisation ofthe modified sequences with com-
plementary bulged DNA and RNA sequences were investigated.
With an additional adenine opposite K in a DNA-duplex, no
change in thermal stability was observed when compared to an
unmodified A-bulged DNA duplex. This indicates that the 2¢-
substituent is accommodated into the bulged region but that no
positive influence from the additional thymine is obtained. With
monomer L in the bulge, a stabilisation of the bulge of 3.0 ◦C was
observed indicating some effect of the additional adenine. This
could again be due to a stacking effect from the larger purine. In
contrary, monomer M demonstrated a decrease in thermal stabil-
Finally, the monomer N with the same triazole-thymine sub-
stituent in the 6¢(R)-position of LNA was incorporated in four
sequences N1–N4. LNA (locked nucleic acid) monomers induce a
large increase in thermal stability when introduced in t◦he nucleic
acid duplexes,30 and in general increases in Tm of 2–5 C can be
expected in a DNA-duplex, though both larger and smaller values
have been reported.31 With monomer N incorporated, increases in
◦
T
m were indeed observed with DTms of +0.5–5.0 ◦C (Table 1). The
ity of the modified A-bulge (DTm = -3.5 C) in accordance with
variation, however, indicates some sequence specific interactions
of the 6¢-substituent. When monomer N was incorporated twice
in the duplex as in the four combinations of the four single
modified sequences, the increases in Tm were, as expected, of +2.5–
9.0 ◦C corresponding to +1.3–4.5 ◦C for each monomer. When
recalculated as DDTm values between -2 and +2.5 ◦C, however,
these indicated no important contacts in the minor groove. On the
other hand, the large substituent at the 6¢-position was generally
well accommodated in a duplex showing that the azide 16 can be
a key compound in new series of derivatised LNA’s.
The monomers K, L and M were also investigated in another
sequence context in order to investigate the effect of the additional
nucleobase in bulged duplexes and three-way junctions (TWJs).
Hence, three 13-mer sequences with the monomer placed in
the central position, K6, L6 and M6 were prepared and mixed
with different complementary strands (Table 3). First, a fully
complementary DNA-sequence was applied and the effects on the
standard duplex were hereby investigated. The three monomers
led to decreases in thermal stability compared to the unmodified
duplex of 5.5, 5.0 and 4.5 ◦C, respectively, which were somewhat
smaller decreases as compared to the other sequences studied
(Table 1). However, this is expectable due to the difference in
sequence length. With an RNA complement, the decreases in Tm
results of other nucleoside analogues with 5¢-C-substituents.6,12
When comparing the melting temperatures of the three bulged
duplexes with regular duplexes, that is with two thymidines instead
of K, L or M, however, large decreases of 10.5, 7.5 and 13 ◦C were
seen. This demonstrates that these double-headed nucleosides, like
1 and all studied analogues of 3, do not behave as dinucleotides.
With complementary RNA, the results were similar with decreases
in thermal stability, DTms, of the modified A-bulged duplexes of
0 ◦C for L, -1 ◦C for K and -3 ◦C for M. With larger GA bulges,
slightly larger decreases in thermal stability were observed with
DTms varying between -1 and -4.5 ◦C, the decreases being larger
with a DNA than with an RNA complement, and with monomer
M as compared to monomers K and L.
Finally, the effect of double-headed nucleosides in a TWJ was
studied by mixing the modified sequences K6, L6 and M6 with
DNA and RNA strands with standard stable hairpin sequences
flanked by single stranded regions being complementary to the
modified sequences. As in our former studies,6,12 the DNA strand
contains an additional CC-bulge as compared to the RNA strand
in order to form a more well-defined secondary structure. In the
DNA : DNA TWJ, both monomers K and L were found to induce
increases in thermal stability of around 4 ◦C when compared
to the unmodified TWJ with a thymidine in the same position.
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Compared to a larger TWJ with two centrally placed thymidines,
the increases with K and L were only slightly smaller (DTms of
+2–2.5 ◦C). These results indicated a substantial stabilising effect
from the 2¢-substituents (the triazole, the additional base or the
combination of the two) most probably from a stacking on one of
the three stems in the TWJ. By the addition of Mg2+, the stability
of the TWJ increased but the relative stabilising effect of the
modification decreased to give neutral Tms with a 10 mM Mg2+
concentration. With monomer M in the TWJ, no stabilisation was
found without Mg2+ in the solution and relative destabilisation
was observed by the addition of Mg2+. In the DNA : RNA TWJ,
the stabilisation was even higher with DTms of +4.5 ◦C for K,
+2.5 ◦C for L and +1 ◦C for M, decreasing to DTms of +2 ◦C for
K, +0.5 ◦C for L and a neutral 0 ◦C for monomer M by a 10 mM
Mg2+ concentration. The stabilisation of the DNA : RNA TWJ
found with the double-headed nucleoside monomer K is hereby
more pronounced than earlier found for any other of the double-
headed nucleosides studied in this sequence, including the other
2¢-modified monomer 1.5
in DNA duplex stabiliy due to the conformationally restricted
LNA-monomer30,31 was found as expected, and apparently, the
substituent is well-accommodated in the minor groove.
Concerning the study of zipper contacts in the minor groove,
that is the (-1) to (-4)-zippers of Table 1 and 2, no strong
and specific contacts were detected for any of the monomers
K–N. Thus, the monomer 3 (B = T)6 is still the only of our
5¢-functionalized nucleoside analogues that is able to form a
strong and very specific (-3)-zipper contact as indicated by large
compensations of the drop in Tm otherwise induced by the
modifications with DDTms of 6–7 ◦C. On the other hand, the
(+1)-zipper contacts obtained with K and L are very interesting
indicating a large compensation for the decrease in duplex stability
otherwise obtained with single modifications. Hence, the overall
drop in Tm of duplexes with (+1)-zippers of L and K or 2xL is
only around 5 ◦C. From simple modelling, this effect must be due
to an intercalation of the substituents into the duplex core and
eventually an extension of the duplex. Apparantly, this extension
gives room for the 2¢-connected triazoles in the minor groove. The
zipper contact is, on the other hand apparently not a hydrogen-
bonding interaction as two adenines form a contact that is equally
strong as a cross-zipper with an adenine and a thymine. The effect,
therefore, seems to be due to stacking alone. This result shows a
short way to an extended nucleic acid duplex structure with a
normal back-bone made from simple 2¢-modifications. This can
be important in future nanoscale design of artificial nucleic acid
structures.
For the stabilisation of a TWJ, it is clear, that the 2¢-modified
monomers K and L demonstrate a significantly larger effect than
the 5¢-modified monomer M. This is in line with the positioning
of the 2¢-substituents pointing towards the core of the complexes
and in line with our earlier results demonstrating stabilisations
of a TWJ with 1 but not with 3 (B = T or T-CH2).5,6,12 Most
interestingly, and in contrary to 1,5 the stabilisaton of a TWJ is
most pronounced when the target is an RNA-sequence. This gives
the method and the monomers K and L a therapeutic potential
following antisense strategies33 with oligonucleotides containing
these double-headed nucleosides targeting RNA with a preference
for secondary structures. Finally, it is also interesting that the
double-headed nucleoside with an adenine, L, in contrary to K,
is able to stabilise an A-bulged duplex. This indicates that the
additional adenine is stacking in the complex.
Discussion
The preparation of four new double-headed nucleosides by a
CuAAC reaction demonstrated the convenience of this method
for introducing new functionalities, herein additional nucle-
obases, into nucleosides. Especially, the use of the 2¢-azido-2¢-
deoxyruridine is convenient as the double-headed nucleosides were
obtained in high yields in a small number of steps. Thus, the
phosphoramidite 9 is obtained in 25% overall yield in only 5 linear
steps from uridine. Also the 5¢-modified double-headed nucleoside
phosphoramidite 15 was conveniently obtained through the azide
12, which itself needed 7 synthetic steps from thymidine.7,32 The
LNA-azide derivative 16 was more difficult to obtain using 15
linear steps,27 but in all cases, the CuAAC reaction was very
efficient in introducing the additional nucleobase.
A consequence of using the CuAAC method over other
ways of introducing the additional nucleobases is, of course,
the 1,4-disubstituted 1,2,3-triazole moiety, which should also be
accomodated in the nucleic acid secondary structures. Our recent
studies on unsubstituted 5¢(S)-C-triazolemethyl-thymidines have
shown relatively low decreases in duplex stability (DTms between
-1 ◦C and -5.8 ◦C in sequences as found in Table 1),7 indicating
that the triazole itself is reasonably well accommodated in the
5¢-position. Therefore, the overall decreases in duplex stability
induced by monomer M is due to the entire substituent, probably
by disturbance of hydration of the duplex, and not specifically
due to the triazole. On the other hand, it seems obvious that a
2¢-C-triazole substituent is having a much more detrimental effect
on duplex stability due to its position deep in the minor groove.
This is evident from the loss in thermal stability of 5–11 ◦C with
K and L incorporated in DNA duplexes. On the other hand, the
double-headed nucleoside 1 also gave a drop in Tm of 4 ◦C for the
DNA duplex, when incorporated into the X6 sequence,5 indicating
a smaller negative contribution from the triazole itself, and in the
corresponding DNA : RNA duplex, monomer 1 gave a DTm of
-1 ◦C,5 which is the same as obtained herein with K and L. Hence
the triazole as well as the additional nucleobase is much better
accomodated in a DNA : RNA duplex. Concerning the behaviour
of the LNA double-headed nucleoside monomer N, increases
Conclusion
The CuAAC is a very convenient method for introducing
additional nucleobases into nucleosides and oligonucleotides.
The triazole groups are not hampering the duplex structures
significantly, and the additional nucleobases have been found
to increase the affinity of the oligonucleotides for secondary
nucleic acid structures like for instance an RNA-hairpin structure.
Furthermore, an extended duplex with additional nucleobases
stacking has been found. Hereby, the use of double-headed
nucleosides can be an important tool in the construction of
artificial nucleic acid structures.
Experimental section
All commercial reagents were used as supplied. Reactions were
carried out under argon or nitrogen when anhydrous solvents
This journal is
The Royal Society of Chemistry 2011
Org. Biomol. Chem., 2011, 9, 1381–1388 | 1385
©

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were used. Column chromatography was performed with Silica
gel 60 (particle size 0.040–0.063 mm, Merck). NMR spectra were
recorded on a Varian Gemini 2000 spectrometer or a Bruker
Advance III 400 spectrometer. Values for d are in ppm relative
to tetramethylsilane as an internal standard or 85% H3PO4 as
an external standard. Assignments of NMR-signals are based
on 2D spectra and follow standard nucleoside convention. ESI
mass spectra were performed on an Thermo Finnigan TSQ 700
spectrometer.
Preparation of 2¢-(4-(N6-benzoyladenine-9-ylmethyl)-1,2,3-
triazole-1-yl)-2¢-deoxy-5¢-O-(4,4¢-dimethoxytrityl)uridine
(10).
A solution of N6-benzoyl-N9-propargyladenine 624 (120 mg,
0.43 mmol) in a mixture of THF, H2O and pyridine (6 : 3 : 1
v/v, 6.0 mL) was stirred at room temperature. A solution of
sodium ascorbate (43 mg, 0.22 mmol) and CuSO4·5H2O (15 mg,
0.06 mmol) in H2O (0.25 mL) was added to the reaction mixture.
Nucleoside 723 (206 mg, 0.36 mmol) was added and the reaction
mixture was stirred at room temperature for 16 h. CH2Cl2 (15 mL)
was added and the resulting mixture was washed with a saturated
aqueous solution of NaHCO3 (15 mL), brine (15 mL), H2O
(15 mL) and aqueous EDTA [a suspension of EDTA (208 mg,
0.71 mmol) in H2O (16 mL) neutralized with 25% aqueous NH3
(0.35 mL)]. The combined aqueous phases were extracted with
CH2Cl2 (2 ¥ 10 mL) and the combined organic phases were dried
(MgSO4) and concentrated under reduced pressure. The residue
was purified by silica gel column chromatography (0 – 6% MeOH
and 0.5% pyridine in CH2Cl2) to afford the product 10 (285 mg,
93%) as a white foam; Rf 0.34 (10% MeOH in CH2Cl2); 1H NMR
(400 MHz, DMSO-d6) d 11.43 (s, 1H, NH(U)), 11.18 (s, 1H,
NH(A)), 8.76 (s, 1H, H-2(A)), 8.55 (s, 1H, H-8(A)), 8.24 (s, 1H,
H-5(triazole)), 8.05 (d, 2H, J = 7.4 Hz, Bz), 7.81 (d, 1H, J = 8.1
Hz, H-6(U)), 7.64 (t, 1H, J = 7.3 Hz, Bz), 7.55 (t, 2H, J = 7.6 Hz,
Bz), 7.44 – 7.21 (m, 9H, Ar), 6.93 – 6.88 (m, 4H, Ar), 6.41 (d, 1H,
J = 4.5 Hz, H-1¢), 5.79 (d, 1H, J = 5.7 Hz, 3¢-OH), 5.63 (s, 2H,
CH2N), 5.52 (dd, 1H, J = 4.7, 6.7 Hz, H-2¢), 5.46 (dd, 1H, J = 1.9,
8.1, H-5(U)), 4.52 (q, 1H, J = 6.5 Hz, H-3¢), 4.22 (m, 1H, H-4¢),
3.74 (s, 6H, 2 ¥ OCH3), 3.38 – 3.26 (m, 2H, H-5¢); 13C NMR (101
MHz, DMSO-d6) d 165.6 (C O(Bz)), 162.9 (C-4(U)), 158.1 (Ar),
152.2, 151.6, 150.1 (C-2(A), C-4(A), C-6(A)), 150.2 (C-2(U)),
144.7 (Ar), 144.5 (C-8(A)), 141.7 (C-4(triazole)), 140.7 (C-6(U)),
135.4, 135.2 (Ar), 133.4, 132.4 (Bz), 129.8 (Ar), 128.4 (Bz), 127.9,
127.7, 126.8 (Ar), 125.2 (C-5(A), C-5(triazole)), 113.3 (Ar), 102.0
(C-5(U)), 87.3 (C-1¢), 85.9 (CAr3), 83.3 (C-4¢), 68.9 (C-3¢), 64.7
(C-2¢), 62.9 (C-5¢), 55.0 (OCH3), 38.2 (CH2N); HRMS-ESI m/z
849.3093 [MH]+: calcd (C45H40N10O8H+) 849.3103.
Preparation
of
2¢-deoxy-5¢-O-(4,4¢-dimethoxytrityl)-2¢-(4-
solution
(thymin-1-ylmethyl)-1,2,3-triazole-1-yl)uridine (8).
A
of nucleoside 723 (191 mg, 0.33 mmol), N1-propargylthymine 524
(75 mg, 0.46 mmol), sodium ascorbate (42 mg, 0.21 mmol) and
CuSO4·5H2O (17 mg, 0.07 mmol) in a mixture of t-BuOH, H2O
and pyridine (5 : 5 : 1 v/v, 5.5 mL) was stirred at room temperature
for 19 h and a color change from yellow to green was observed.
CH2Cl2 (15 mL) was added and the resulting mixture was washed
with a saturated aqueous solution of NaHCO3 (10 mL). The
aqueous phase was extracted with CH2Cl2 (2 ¥ 10 mL), and the
combined organic phases were dried (MgSO4) and concentrated
under reduced pressure. The residue was purified by flash column
chromatography (0–10% MeOH and 1% Et3N in CH2Cl2) to
afford the product 8 (176 mg, 72%) as a white solid; Rf 0.36 (10%
1
MeOH in CH2Cl2); H NMR (400 MHz, CDCl3) d 10.50–10.35
(br m, 2H, NH), 8.31 (s, 1H, H-5(triazole)), 7.84 (d, 1H, J = 8.2
Hz, H-6(U)), 7.42 – 7.17 (m, 10H, Ar, H-6(T)), 6.83 (d, 4H, J =
8.6 Hz, Ar), 6.57 (d, 1H, J = 6.1 Hz, H-1¢), 5.46 (t, 1H, J = 5.8
Hz, H-2¢), 5.34 (d, 1H, J = 8.1 Hz, H-5(U)), 5.00–4.75 (m, 2H,
CH2N), 4.79 (t, 1H, J = 4.6 Hz, H-3¢), 4.46 (m, 1H, H-4¢), 3.76 (s,
6H, 2 ¥ OCH3), 3.53 (s, 2H, H-5¢), 1.79 (s, 3H, CH3); 13C NMR
(101 MHz, CDCl3) d 165.1, 163.6 (C-4(U), C-4(T)), 158.9 (Ar),
151.6, 151.0 (C-2(U), C-2(T)), 144.3 (Ar), 142.0 (C-4(triazole)),
141.0 (C-6(T)), 139.6 (C-6(U)), 135.3, 135.1, 130.3, 128.3, 128.2,
127.4 (Ar), 126.4 (C-5(triazole)), 113.5 (Ar), 111.1 (C-5(T)), 103.3
(C-5(U)), 87.5 (CAr3), 86.1 (C-1¢), 85.3 (C-4¢), 71.2 (C-3¢), 66.5
(C-2¢), 63.0 (C-5¢), 55.4 (OCH3), 43.5 (CH2N), 12.2 (CH3(T));
HRMS-ESI m/z 758.2565 [MNa]+: calcd (C38H37N7O9Na+)
758.2545.
Preparation of 2¢-(4-(N6-benzoyladenine-9-ylmethyl)-1,2,3-
triazole-1-yl)-3¢-O-(P-2-cyanoethyl-N,N -diisopropylaminopho-
sphinyl)-2¢-deoxy-5¢-O-(4,4¢-dimethoxytrityl)
uridine
(11).
Nucleoside 10 (255 mg, 0.30 mmol) was coevaporated with
anhydrous CH2Cl2 (2 ¥ 8 mL) and dissolved in the same solvent
(4.0 mL). N,N-diisopropylethylamine (0.52 mL, 2.99 mmol)
Preparation of 3¢-O-(P-(2-cyanoethoxy)-N,N-diisopropyl-
aminophosphinyl)-2¢-deoxy-5¢-O-(4,4¢-dimethoxytrityl)-2¢-(4-(thy-
min-1-ylmethyl)-1,2,3-triazole-1-yl)uridine
(9). Nucleoside
and
N,N-diisopropylamino-2-cyanoethylphosphinochloridite
8 (144 mg, 0.20 mmol) was coevaporated with anhydrous
DCE (3 ¥ 4 mL) and dissolved in the same solvent (2.0 mL).
N,N-diisopropylethylamine (0.40 mL, 2.30 mmol) and N,N-
diisopropylamino-2-cyanoethylphosphinochloridite (0.09 mL,
0.40 mmol) were added and the reaction mixture was stirred
at room temperature for 2 h. Ethanol (1.0 mL) and CH2Cl2
(15 mL) were added and the resulting solution was washed with
a saturated aqueous solution of NaHCO3 (10 mL). The aqueous
phase was extracted with CH2Cl2 (3 ¥ 8 mL), and the combined
organic phases were dried (MgSO4) and concentrated under
reduced pressure. The residue was purified by silica gel column
chromatography (0 – 5% MeOH and 0.5% pyridine in CH2Cl2)
to afford the product 9 (129 mg, 70%) as a white foam; Rf 0.54
(10% MeOH in CH2Cl2); 31P NMR (162 MHz, CDCl3) d 151.9,
149.4 (Ratio 5 : 3); HRMS-ESI m/z 958.3623 [MNa]+: calcd
(C47H54N9O10PNa+) 958.3624.
(0.10 mL, 0.45 mmol) were added and the reaction mixture was
stirred at room temperature for 4 h. Ethanol (1.5 mL) and CH2Cl2
(15 mL) were added and the resulting solution was washed with
a saturated aqueous solution of NaHCO3 (10 mL). The aqueous
phase was extracted with CH2Cl2 (3 ¥ 8 mL), and the combined
organic phases were dried (Na2SO4) and concentrated under
reduced pressure. The residue was purified by silica gel column
chromatography (0–5% MeOH and 0.5% pyridine in CH2Cl2)
to afford the product 11 (195 mg, 62%) as a white foam; Rf
0.47 (10% MeOH in CH2Cl2); 31P NMR (162 MHz, DMSO-d6)
d 149.9, 148.2 (Ratio 3 : 4); HRMS-ESI m/z 1049.4174 [MH]+:
calcd (C54H57N12O9PH+) 1049.4182.
Preparation of 3¢-O-(tert-butyldimethylsilyl)-5¢-O-pixyl-5¢(S)-
C-(4-(thymin-1-ylmethyl)-1,2,3-triazol-1-yl)methylthymidine (13).
To a stirred solution of compound 127 (500 mg, 0.75 mmol)
1386 | Org. Biomol. Chem., 2011, 9, 1381–1388
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and N1-propargylthymine 524 (164 mg, 1.00 mmol) in a mixture
of t-BuOH, H2O and pyridine (5 : 5 : 2 v/v, 12 mL) was added
sodium ascorbate (37 mg, 0.19 mmol) and CuSO4·5H2O (25 mg,
0.10 mmol) and the mixture was stirred at room temperature for 18
h. A second portion of N1-propargylthymine (164 mg, 1.00 mmol),
sodium ascorbate (37 mg, 0.19 mmol) and CuSO4·5H2O (25 mg,
0.10 mmol) was added and the mixture was stirred for another 3
h. Ethyl acetate (30 mL) was added and the mixture was washed
with water (30 mL). The aqueous phase was extracted with ethyl
acetate (3 ¥ 30 mL), and the combined organic phases were dried
(Na2SO4) and concentrated under reduced pressure. The residue
was purified by column chromatography (33–100% ethyl acetate
and 0.2% pyridine in petrolium ether) affording the product 13
nucleoside 14 (300 mg, 0.42 mmol) in anhydrous CH2Cl2 (5 mL)
was added N,N-diisopropylethylamine (350 mL, 2.00 mmol) and
2-cyanoethyl-N,N-diisopropylphosphoramidochloridite (300 mL,
1.35 mmol) and the mixture was stirred at room temperature
for 3 h. CH2Cl2 (30 ml) was added and the mixture was washed
with a saturated aqueous solution of NaHCO3 (30 mL). The
aqueous phase was extracted with CH2Cl2 (3 ¥ 30 mL) and the
combined organic phases were dried (Na2SO4) and concentrated
under reduced pressure. The residue was purified by column
chromatography (0-25% acetone and 0.2% pyridine in CH2Cl2)
to afford the product 15 (345 mg, 90%) as a colorless foam;
Rf 0.28 (25% acetone in CH2Cl2); 31P NMR (75 MHz, CDCl3)
d 152.07, 151.13; HRMS-ESI m/z 940.3541 [MNa]+: calcd
(C47H52N9O9PNa)+ 940.3518.
1
(500 mg, 80%) as a colorless foam; Rf 0.26 (ethyl acetate); H
NMR (300 MHz, CDCl3) d 9.60–9.30 (br s, 2H, NH), 7.72 (s, 1H,
H-6), 7.48–7.30 (m, 10H, H-5(triazole), Ar), 7.26 (s, 1H, H-6),
7.13–6.98 (m, 4H, Ar), 6.23 (dd, 1H, J = 4.8, 9.6 Hz, H-1¢), 4.95,
4.73 (AB, 2H, J = 15.0 Hz, CH2N), 4.15 (m, 1H, H-6¢), 3.74 (m, 1H,
H-5¢), 3.52 (dd, 1H, J = 3.9, 13.5 Hz, H-6¢) 3.25 (d, 1H, J = 4.5 Hz,
H-3¢), 3.19 (br s, 1H, H-4¢), 2.06 (s, 3H, CH3), 2.04–1.75 (m, 5H,
H-2¢, CH3), 0.66 (s, 9H, C(CH3)3), -0.22 (s, 3H, Si(CH3)2), -0.36 (s,
3H, Si(CH3)2); 13C NMR (75 MHz, CDCl3) d 164.3, 164.1 (C-4),
152.2, 152.0 (Ar), 150.9, 150.4 (C-2), 146.1, 141.9, 140.1, 135.5 (C-
6, C-4(triazole), Ar), 131.3, 130.9, 130.8, 130.6, 128.1, 127.7, 127.6,
124.8, 124.1, 124.0, 122.9, 122.3, 117.6, 117.2 (Ar, C-5(triazole)),
111.3, 111.2 (C-5), 86.4 (C-4¢), 84.8 (C-1¢), 78.8 (CAr3), 73.3 (C-3¢),
71.9 (C-5¢), 50.1 (C-6¢), 42.7 (CH2N), 40.9 (C-2¢), 25.6 (C(CH3)3),
17.7 (C(CH3)3), 12.9, 12.4 (CH3), -4.7, -4.8 (Si(CH3)2); HRMS-
ESI m/z 854.3324 [MNa]+: calcd (C44H49N7O8SiNa+) 854.3304.
Preparation of (1S,3R,4R,6R,7S)-1-(4,4¢-dimethoxytrityl-
oxymethyl)-7-hydroxy-3-(thymin-1-yl)-6-(4-(thymin-1-ylmethyl)-
1,2,3-triazol-1-yl)methyl-2,5-dioxabicyclo[2.2.1]heptane (18). To
a stirred solution of nucleoside 1727 (100 mg, 0.2 mmol) in a
mixture of anhydrous CH3CN and pyridine (1 : 1 v/v, 4 mL),
was added 4,4¢-dimethoxytrityl chloride (104 mg, 0.31 mmol).
The mixture was stirred at room temperature for 16 h and then
quenched by the addition of methanol (1 mL). The mixture
was concentrated under reduced pressure, and the residue was
dissolved in CH2Cl2 (40 mL). The organic phase was washed
with water (8 mL) and a saturated aqueous solution of NaHCO3
(8 mL). The organic phase was dried (MgSO4) and concentrated
under reduced pressure. The residue was purified by silica gel
column chromatography (3–5% EtOH in chloroform) to afford
the product 18 (97 mg, 60%) as a white solid; Rf 0.39 (10%
CH3OH in CH2Cl2); 1H NMR (300 MHz, CDCl3) d 10.30–10.10
(br m, 2H, NH), 8.00 (s, 1H, H-5(triazole)), 7.60 (s, 1H, H-6),
7.42–7.17 (m, 10H, Ar, H-6), 6.85–6.77 (m, 4H, Ar), 5.73 (s, 1H,
H-1¢), 4.82–4.47 (m, 7H, H-2¢, H-3¢, H-6¢, 2 ¥ CH2N), 3.74 (s, 6H,
2 ¥ OCH3), 3.55 (AB, 2H, J = 8.4, 10.2 Hz, H-5¢), 2.15 (br s, 1H,
OH), 1.75 (s, 3H, CH3), 1.59 (s, 3H, CH3); 13C NMR (CDCl3,
75 MHz) d 164.8, 164.5 (C-4), 158.8 (Ar), 151.4, 150.5 (C-2),
144.4, 142.2, 140.6 (C-6, C-4(triazole)), 135.3, 135.2, 134.6, 130.2,
128.2, 127.3 (Ar), 125.5 (C-5(triazole)), 113.5 (Ar), 111.2, 110.8
(C-5), 88.2, 87.2, 86.8, (CAr3, C-1¢, C-4¢), 79.8 (C-6¢), 79.1 (C-3¢),
72.1 (C-2¢), 58.4 (C-5¢), 55.4, 55.3 (OCH3), 51.0 (CH2N), 42.8
(CH2N), 12.6, 12.2 (CH3); HRMS-ESI m/z 814.2261 [MNa]+:
calcd (C42H43N7O10Na)+ 814.2807.
Preparation of 5¢-O-pixyl-5¢(S)-C-(4-(thymin-1-ylmethyl)-1,2,3-
triazol-1-yl)methylthymidine (14). A solution of compound 13
(450 mg, 0.54 mmol) dissolved in anhydrous THF (6 mL) was
added a 1.0 M solution of TBAF in THF (1 mL, 1.00 mmol) and
stirred at room temperature for 20 h. The mixture was poured into
ethyl acetate (40 mL) and the mixture was washed with a 10%
aqueous solution of NaHCO3 (40 mL). The aqueous phase was
extracted with ethyl acetate (3 ¥ 4 mL), and the combined organic
phases were dried (Na2SO4) and concentrated under reduced
pressure. The residue was purified by column chromatography
(10% MeOH and 0.2% pyridine in CH2Cl2) to afford the product
14 (353 mg, 91%) as a colorless foam; Rf 0.32 (10% MeOH in
1
CH2Cl2); H NMR (300 MHz, DMSO-d6) d 11.37 (s, H, NH),
11.30 (s, H, NH), 7.76–7.27 (m, 16H, H-6, H-5(triazole), Ar),
7.11–6.88 (m, 4H, Ar), 5.93 (t, 1H, J = 7.2 Hz, H-1¢), 5.00 (d,
1H, J = 4.5 Hz, OH-3¢), 4.83 (s, 2H, CH2N), 4.25 (dd, 1H, J =
9.3, 15.3 Hz, H-6¢), 3.77–3.69 (m, 2H, H-5¢, H-6¢), 3.49 (m, 1H,
H-3¢), 3.17 (1H, t, J = 2.7 Hz, H-4¢), 1.93–1.86 (m, 5H, H-2¢, CH3),
1.73 (s, 3H, CH3); 13C NMR (75 MHz, DMSO-d6) d 164.2, 163.7
(C-4), 151.0, 150.9, 150.6, 150.2, 147.2, 142.3, 140.9, 135.4 (Ar,
C-2, C-4(triazole), C-6), 130.9, 130.8, 130.3, 130.1, 127.8, 127.0,
124.2, 123.7, 123.6, 122.5, 122.0, 116.6, 116.2 (Ar, C-5(triazole)),
109.6, 108.8 (C-5), 85.0 (C-1¢), 83.6 (C-4¢), 77.1 (CAr3), 71.9 (C-
5¢), 70.0 (C-3¢), 49.6 (C-6¢), 42.0 (CH2N), 39.7 (C-2¢), 12.3, 11.9
(CH3); HRMS-ESI m/z 740.2437 [MNa]+: calcd (C38H35N7O8Na)+
740.2439.
Preparation
of
(1S,3R,4R,6R,7S)-7-(P-2-cyanoethoxy-
N,N - diisopropylaminophosphinyl)oxy - 1 - (4,4¢ - dimethoxytrityl) -
oxymethyl-3-(thymin-1-yl)-6-(4-(thymin-1-ylmethyl)-1,2,3-tri-
azol-1-yl)methyl-2,5-dioxabicyclo[2.2.1]heptane
(19). To
a
stirred solution of nucleoside 18 (58 mg, 0.073 mmol)
in anhydrous CH2Cl2 (1.0 mL) was added N,N-
diisopropylethylamine (55 mL, 0.31 mmol) and 2-cyanoethyl-
N,N-diisopropylphosphoramidochloridite (49 mL, 0.22 mmol),
and the mixture was stirred at room temperature for 112 h. The
mixture was diluted with CH2Cl2 (30 mL) and washed with a
saturated aqueous solution of NaHCO3 (5 mL), water (5 mL)
and brine (5 mL). The organic phase was dried (Na2SO4) and
concentrated under reduced pressure. The residue was purified by
column chromatography (30–50% acetone and 0.5% pyridine in
CH2Cl2) to give the product 19 (30 mg, 41%) as a white foam;
Rf 0.68 (10% CH3OH in CH2Cl2); 31P NMR (CDCl3, 300 MHz)
Preparation
of
3¢-O-(P-2-cyanoethoxy-N,N-diisopropyl-
aminophosphinyl)-5¢-O-pixyl-5¢(S)-C-(4-(thymin-1-ylmethyl)-1,
2,3-triazol-1-yl)methylthymidine (15). To a stirred solution of
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d 151.08, 150.96; HRMS-ESI m/z 1014.3904 [MNa]+: calcd
(Nucleic Acid Based Drug Design Training Center) in the Sixth
Framework Programme Marie Curie Host Fellowships for Early
Stage Research Training under contract no. MEST-CT-2004-
504018, The Danish Natural Science Research Council and
Møllerens Fond.
(C51H60N9O11Na)+ 1014.3886.
Synthesis of oligodeoxynucleotides
Oligonucleotide synthesis was carried out on an automated DNA
synthesizer following the phosphoramidite approach. Synthesis of
oligonucleotides was performed on a 0.2 mmol scale by using the
phosphoramidites 9, 11, 15 and 19 as well as the corresponding
commercial 2-cyanoethyl phosphoramidites of the natural 2¢-
deoxynucleosides. The synthesis followed the regular protocol for
the DNA synthesizer. For modified phosphoramidites, however, a
prolonged coupling time of 15 or 20 min was used. 1H-Tetrazole
was used as the activator and coupling yields for 2-cyanoethyl
phosphoramidites were >98% for 11, >90% for 9, >85% for 15 and
>60% for 19. The 5¢-O-DMT-ON oligonucleotides were removed
from the solid support by treatment with concentrated aqueous
ammonia at 55 ◦C for 16 h, which also removed the protecting
groups. The oligonucleotides were purified by reversed-phase
HPLC on a Waters 600 system using a Xterra prep MS C18; 10 mm;
7.8 ¥ 150 mm column; Buffer A: 0.05 M Triethyl Ammonium
Acetate pH 7.4. Buffer B: MeCN–H2O (1 : 1). Program used: 2
min 100% A, 100–30% A over 38 min, 10 min 100% B, 10 min
100% A. All oligonucleotides were detritylated by treatment with
an 80% aqueous solution of acetic acid for 20 min, quenched with
a aqueous solution of sodium acetate (3 M, 15 mL) and then added
sodium perchlorate (5 M, 15 mL) followed by acetone (1 mL). The
resulting mixture was precipitated over night at -20 ◦C. After
centrifugation 12000 rpm, 10 min at 4 ◦C, the supernatant was
removed and the pellet washed with cold acetone (2 ¥ 1 mL) and
dried for 30 min under reduced pressure, and dissolved in pure
water (500 or 1000 mL). The concentration was determined by
UV at 260 nm, and the purity confirmed by IC analysis. MALDI-
TOF-MS [M - H]- confirmed the constitution (see ESI†).
Notes and references
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Acknowledgements
The project was supported by The Danish Research Agency’s
Programme for Young Researchers, Nucleic Acid Center and
The Danish National Research Foundation, The NAC-DRUG
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