Three pyrene-modified nucleotides: Synthesis and effects in secondary nucleic acid structures

Article abstract of DOI:10.1021/jo301571s

Synthesis of three pyrene-modified nucleosides was accomplished using the CuAAC reaction. Hereby, pyrene is attached either to the 5′-position of thymidine or to the 2′-position of 2′-deoxyuridine through triazolemethylene linkers, or to the 2′-position of 2′-deoxyuridine through a more rigid triazole linker. The three nucleosides were incorporated into oligonucleotides, and these were combined in different duplexes and other secondary structures, which were analyzed by thermal stability and fluorescence studies. The three monomers were found to have different impacts on the nucleic acid complexes. Hence, pyrene attached to the 5′-position shows a tendency for intercalation into the duplex as indicated by a general decrease in fluorescence intensity followed by an increase in duplex thermal stability. Pyrene attached to the 2′-position through a rigid triazole linker also shows a tendency for intercalation but with decrease in duplex stability, whereas the pyrene attached to the 2′-position through a triazolemethylene linker is primarily situated in the minor groove as indicated by an increase in fluorescence but here in most cases leading to increase in duplex stability. All three pyrene nucleotides lead to thermal stabilization of bulged duplexes and three-way junctions. In some cases when two pyrenes were introduced into the core of these complexes, the formation or disappearance of a fluorescence excimer band can be used to indicate the hybridization process. Hereby these oligonucleotides can act as specific recognition probes.

Full text of DOI:10.1021/jo301571s

Article
pubs.acs.org/joc
Three Pyrene-Modified Nucleotides: Synthesis and Effects in
Secondary Nucleic Acid Structures
Pawan Kumar, Khalil I. Shaikh, Anna S. Jørgensen, Surender Kumar,‡ and Poul Nielsen*
Nucleic Acid Center, Department of Physics, Chemistry and Pharmacy, University of Southern Denmark, 5230 Odense M, Denmark
S
* Supporting Information
ABSTRACT: Synthesis of three pyrene-modified nucleosides
was accomplished using the CuAAC reaction. Hereby, pyrene
is attached either to the 5′-position of thymidine or to the 2′-
position of 2′-deoxyuridine through triazolemethylene linkers,
or to the 2′-position of 2′-deoxyuridine through a more rigid
triazole linker. The three nucleosides were incorporated into
oligonucleotides, and these were combined in different
duplexes and other secondary structures, which were analyzed
by thermal stability and fluorescence studies. The three
monomers were found to have different impacts on the
nucleic acid complexes. Hence, pyrene attached to the 5′-
position shows a tendency for intercalation into the duplex as
indicated by a general decrease in fluorescence intensity
followed by an increase in duplex thermal stability. Pyrene attached to the 2′-position through a rigid triazole linker also shows a
tendency for intercalation but with decrease in duplex stability, whereas the pyrene attached to the 2′-position through a
triazolemethylene linker is primarily situated in the minor groove as indicated by an increase in fluorescence but here in most
cases leading to increase in duplex stability. All three pyrene nucleotides lead to thermal stabilization of bulged duplexes and
three-way junctions. In some cases when two pyrenes were introduced into the core of these complexes, the formation or
disappearance of a fluorescence excimer band can be used to indicate the hybridization process. Hereby these oligonucleotides
can act as specific recognition probes.
deoxyuridine and 2′-deoxycytidine.12 We have also recently
INTRODUCTION
■
applied this reaction to attach an additional nucleobase in the
The DNA double helix presents an excellent scaffold to attach
study of a series of double-headed nucleosides (C-H) with an
various entities in an organized manner to find applications in
diagnostics, DNA-mediated catalysis, nanotechnology, etc.1,2
A
additional nucleobase or aromatic moiety in the pro-S position
of the C5′-carbon of the thymidine (C-F)4,5 and with an
additional nucleobase (thymin-1-yl or adenin-9-yl) in the 2′-
position of the 2′-deoxyuridine (G and H).5 When
incorporated into oligonucleotides and mixed with comple-
mentary unmodified oligonucleotides containing a central
hairpin, G and H displayed stabilization of a three-way junction
(TWJ) in both dsDNA and DNA:RNA contexts. Cross-strand
stacking interactions between two additional bases from
opposite strands were also observed in a so-called (+1) zipper
motif for both G and H.5
series of nucleosides with additional nucleobases, commonly
termed double-headed nucleosides, has been presented by us
and others with the purpose of using the recognition potential
of the additional nucleobases in various nucleic acid
constructs.3−8 Recently, we have shown that a 2′-deoxyuridine
analogue (A) with an additional thymine in the 2′-position
attached through a methylene linker (Figure 1) behaves as a
compressed dinucleotide, recognizing nucleobases in the base
pair ladder in the center of a Watson−Crick duplex.6 On the
other hand, the double-headed nucleoside monomer B with a
methylene-linked thymine at the pro-S position of the C5′
carbon (Figure 1) when incorporated into an oligonucleotide
can interact with the thymine of another nucleotide monomer
B placed in the opposite strand in a so-called (−3) zipper
arrangement via stacking in the minor groove.3,4 The use of the
Cu(I)-catalyzed alkyne azide cycloaddition (CuAAC) reaction9
is well documented in the field of nucleoside/nucleic acid
chemistry.10,11 Recently, in our study of triazole stacking in the
major groove leading to highly stable DNA:RNA duplexes, we
have applied the CuAAC reaction to attach substituted and
unsubstituted phenyl groups in the 5-position of the 2′-
Because of the environment-dependent fluorescence, pyrene-
modified oligonucleotides have attracted considerable attention
as fluorescent probes for the detection of hybridization to
DNA/RNA complements.13,14 Hence, hybridization can lead to
modulation of the fluorescence intensity of the pyrene moieties.
Pyrene in an electronically excited-state forms a π-stacking
complex with a pyrene in the ground state when these two
pyrenes are separated by ∼3.4 Å to give an excimer
Received: July 24, 2012
Published: October 8, 2012
© 2012 American Chemical Society
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a
Scheme 1
Figure 1. Double-headed nucleosides. U = uracil-1-yl, Ψ = uracil-5-yl,
T = thymin-1-yl, A = adenin-9-yl.
fluorescence signal which is significantly red-shifted (with λmax
= ∼490 nm) relative to pyrene monomer fluorescence (with
λmax = ∼380−410 nm).15 The pyrene excimer signal has been
exploited for the detection of hybridization to DNA/RNA
complements,10,14,16 however not for the detection of
secondary nucleic acids structures such as a TWJ. Nevertheless,
pyrene-modified oligonucleotides have been used for the
stabilization of TWJs.17 In view of these observations, we
decided to replace the additional nucleobases in C and G with
pyrene, i.e., in the 2′-position of the 2′-deoxyuridine, and in the
5′(S)-C-position of the thymidine to investigate the stacking
interactions from the larger aromatic surface area of pyrene and
to study the effect of positioning pyrene at the branching point
of a TWJ as well as in bulged dsDNA and DNA:RNA duplexes.
Environment-dependent fluorescence properties of pyrene
present an additional tool to get further insight into these
interactions.
a
Reagents and conditions: (a) CuSO4·5H2O, TBTA, Na ascorbate,
THF:H2O:pyridine (5:5:2 or 6:3:2, v/v/v), 84% 2, 79% 6. (b) TBAF,
THF 82%. (c) NC(CH2)2OP(N(i-Pr)2)Cl, (i-Pr)2NEt, CH2Cl2, 69%
4, 48% 7, 64% 11. (d) DMTCl, pyridine, 79%. (e) CuSO4·5H2O, Na
ascorbate, THF:H2O:t-BuOH (3:1:1, v/v/v), 63%. TBTA = tris((1-
benzyl-1,2,3-triazol-4-yl)methyl)amine. DMT = 4,4′-dimethoxytrityl.
nucleoside 6. Subsequent phosphitylation of 6 afforded the
phosphoramidite 7, which can be used in standard automated
solid-phase oligonucleotide synthesis.19
Recently, we have shown the six-step synthesis from uridine
to the 2′-deoxy-2′-propargyluridine 8.20 This was reacted with
4,4′-dimethoxytrityl chloride in pyridine to afford the 5′-O-
DMT-protected nucleoside 9. Under CuAAC reaction
conditions, 9 was reacted with 1-azidopyrene21 to afford
pyrene-modified nucleoside 10. Phosphitylation of 10 afforded
phosphoramidite 11.
The three phosphoramidites 4, 7, and 11 were then
introduced into oligonucleotides (ON’s) to give the modified
nucleotide monomers K, L, and M (Figure 1), respectively, by
using standard solid-phase DNA synthesis with 1H-tetrazole as
activator. Extended coupling time of 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 duplexes3,4
(Table 1) and of bulged duplexes and TWJ’s3,7,22 (Tables 2 and
3).
RESULTS AND DISCUSSION
■
Chemical Synthesis. The three nucleosides included in the
present study were synthesized using the copper-catalyzed
alkyne azide cycloaddition (CuAAC) reaction9 (Scheme 1).
Nucleoside 2 was synthesized by the reaction of commercially
available 1-ethynylpyrene with appropriately protected 5′(S)-C-
azidomethylthymidine 1, which in turn was prepared by using
our previously reported seven-step procedure from thymidine.4
The reaction went smoothly by the use of standard conditions
for the CuAAC reaction.9 Desilylation of 2 using TBAF gave 3,
which on phosphitylation under standard conditions afforded
the corresponding phosphoramidite 4 as an appropriate
building block for automated oligonucleotide synthesis.
Uridine was converted in three high-yielding steps into the
known intermediate 5′-O-dimethoxytrityl-2′-azido-2′-deoxyur-
idine 5,18 which was then reacted with 1-ethynylpyrene under
the same CuAAC reaction conditions as before to give
Hybridization Studies. At first, an 11-mer duplex was
studied, in which five centrally placed thymidines can be
replaced systematically with the modified monomers (Table 1).
One or two incorporation(s) of either of the monomers K, L,
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Table 1. Hybridization Data for Modified Duplexes
a
b
c
Tm/°C, (ΔTm/°C ), [ΔΔTm/°C ]
entry
1
ON
duplex
zipper
X = K
X = L
X = M
T1
T2
T1
X1
T1
X2
X3
T2
X4
T2
X5
T2
T1
X6
X7
T2
X5
X2
X5
X1
X4
X2
X3
X2
X4
X1
X3
X1
5′-d(CGC ATA TTC GC)-3′
3′-d(GCG TAT AAG CG)-5′
5′-d(CGC ATA TTC GC)-3′
3-′d(GCG XAT AAG CG)-5′
5′-d(CGC ATA TTC GC)-3′
3′-d(GCG TAX AAG CG)-5′
5′-d(CGC ATA TXC GC)-3′
3′-d(GCG TAT AAG CG)-5′
5′-d(CGC ATA XTC GC)-3′
3′-d(GCG TAT AAG CG)-5′
5′-d(CGC AXA TTC GC)-3′
3′-d(GCG TAT AAG CG)-5′
5′-d(CGC ATA TTC GC)-3′
3′-d(GCG XAX AAG CG)-5′
5′-d(CGC AXA XTC GC)-3′
3′-d(GCG TAT AAG CG)-5′
5′-d(CGC AXA TTC GC)-3′
3′-d(GCG TAX AAG CG)-5′
5′-d(CGC AXA TTC GC)-3′
3′-d(GCG XAT AAG CG)-5′
5′-d(CGC ATA XTC GC)-3′
3′-d(GCG TAX AAG CG)-5′
5′-d(CGC ATA TXC GC)-3′
3′-d(GCG TAX AAG CG)-5′
5′-d(CGC ATA XTC GC)-3′
3′-d(GCG XAT AAG CG)-5′
5′-d(CGC ATA TXC GC)-3′
3′-d(GCG XAT AAG CG)-5′
45.5
45.5
45.5
45.5
(0.0)
37.0
(−8.5)
45.5
45.5
(0.0)
2
3
45.5
50.5
(0.0)
(0.0)
(+5.0)
47.0
38.5
43.5
4
(+1.5)
(−7.0)
41.0
(−2.0)
46.5
45.5
5
(0.0)
(−4.5)
40.5
(+1.0)
46.0
50.5
6
(+0.5)
(−5.0)
27.5
(+5.0)
46.0
50.5
7
(+0.5)
(−18.0)
26.5
(+5.0)
44.5
51.0
8
(−1.0)
53.0
(−19.0)
33.5
(+5.5)
39.5
9
(+1)
(−1)
(−1)
(−2)
(−3)
(−4)
(+7.5) [+7.0]
43.5
(−12.0) [−6.5]
40.5
(−6.0) [−16.0]
47.5
10
11
12
13
14
(−2.0) [−2.5]
44.5
(−5.0) [+9.0]
48.5
(+2.0) [−3.0]
51.0
(−1.0) [−1.0]
50.5
(+3.0) [+7.5]
40.5
(+5.5) [−0.5]
50.0
(+5.0) [+3.5]
37.5
(−5.0) [+2.5]
17.5
(+4.5) [+1.5]
47.0
(−8.0) [−8.0]
46.0
(−28.0) [−14.5]
19.0
(+1.5) [+0.5]
44.0
(+0.5) [−1.0]
(−26.5) [−10.5]
(−1.5) [+0.5]
a
Melting temperatures obtained from the maxima of the first derivatives of the melting curves (A260 vs temperature) recorded in a buffer containing
2.5 mM Na2HPO4, 5.0 mM NaH2PO4, 100 mM NaCl, 0.1 mM EDTA, pH 7.0, using 1.0 μM concentrations of each strand. K, L, and M correspond
b
to the incorporation of the amidites 4, 7, and 11, respectively. Differences in melting temperatures as compared to the unmodified duplexes; ΔTm =
c
Tm(duplex) − Tm(T1:T2). Differences in melting temperatures as compared to singly modified duplexes; ΔΔTm = ΔTm(a:b) − (ΔTm(a:T2) + ΔTm(T1:b)).
Table 2. Hybridization Data for Regular and Bulged DNA:DNA and DNA:RNA Duplexes
a
b
c
X8 = 5′- d(GCT CAC XCT CCC A), Tm/°C, (ΔTm1/°C ), [ΔTm2/°C ]
complement
X = T
X = TT
X = K
X = L
X = M
DNA
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)
52.5
61.0
43.0
50.5
43.5
50.5
Nd
Nd
54.5 (+2.0)
48.5 (−4.0)
56.5 (−4.5)
43.5 (+0.5) [−10.0]
54.0 (+3.5) [−6.5]
43.0 (−0.5) [+0.5]
52.5 (+2.0) [+2.0]
56.5 (+3.5)
60.0 (−1.0)
55.0 (+12.0) [ +2.0]
55.0 (+5.0) [−5.0]
47.0 (+3.5) [+4.5]
52.0 (+2.0) [+2.0]
RNA
58.0 (−3.0)
DNA, A-bulge
RNA, A-bulge
DNA, GA-bulge
RNA, GA-bulge
53.5
60.5
42.5
50.5
52.5 (+9.5) [−1.0]
54.5 (+4.0) [−6.0]
48.5 (+5.0) [+6.0]
51.0 (+0.5) [+0.5]
a
Melting temperatures obtained from the maxima of the first derivatives of the melting curves (A260 vs temperature) recorded in a buffer containing
2.5 mM Na2HPO4, 5.0 mM NaH2PO4, 100 mM NaCl, 0.1 mM EDTA, pH 7.0, using 1.0 μM concentrations of each strand. K, L, and M correspond
b
c
to the incorporation of the amidites 4, 7, and 11, respectively. Differences in melting temperatures as compared to X = T. Differences in melting
temperatures as compared to X = TT.
and M in the same ON’s gave the sequences K1−K7, L1−L7,
and M1−M7 (Table 1). These sequences were first mixed with
unmodified complementary sequences, and the melting
temperatures (Tm) of these modified duplexes were recorded
by UV spectroscopy (Table 1, entries 2−8) and compared with
the Tm of the corresponding unmodified duplex (entry 1).
Hereby, the differences in melting temperature (ΔTm’s) were
determined (Table 1). For the monomer K, ΔTm’s in the range
of −1.0 to +1.5 °C were observed, indicating that all single and
double incorporations of monomer K are well accommodated
in dsDNA. For the monomer L, it is clear from the observed
ΔTm values (Table 1) that single or double incorporation gives
a significant destabilization of the duplexes formed, and the
largest decreases were seen for the modifications toward the 3′-
end of the duplexes (entries 2 and 4). However, when
monomer L was incorporated exactly in the middle of the
sequence, no destabilization of the duplex was observed (entry
3). These results are well in accordance with the independent
study on monomer L.19 Similar destabilization of modified
duplexes was observed with monomers G and H having an
additional nucleobase on the triazole ring in place of the
pyrene,5 suggesting the triazole attached directly to the 2′-
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Table 3. Hybridization Data for Three-Way Junctions
a
b
Tm /°C (ΔTm /°C )
DNA hairpin: 3′- d(CGA GTG A CC CGC
GTT TTC GCG AGA GGG T)
RNA hairpin: 3′- r(CGA GUG A CG CGU
UUU CGC G AG AGG GU)
ON
sequence
[Mg2+], 0 mM
[Mg2+], 10 mM
[Mg2+], 0 mM
[Mg2+], 10 mM
T
K8
5′-d(GCT CAC TCT CCC A)-3′
5′-d(GCT CAC KCT CCC A)-3′
5′-d(GCT CAC LCT CCC A)-3′
5′-d(GCT CAC MCT CCC A)-3′
5′-d(GCT CAC TTC TCC CA)-3
5′-d(GCT CAC KKC TCC CA)-3′
5′-d(GCT CAC LLC TCC CA)-3′
5′-d(GCT CAC MMC TCC CA)-3′
5′-d(GCT CAC KLC TCC CA)-3′
5′-d(GCT CAC LKC TCC CA)-3′
5′-d(GCT CAC MKC TCC CA)-3′
24.5
38.0
37.0
47.0
43.5 (+19.0)
33.5 (+9.0)
38.0 (+14.0)
26.0
52.5 (+14.5)
43.0 (+5.0)
47.5 (+9.0)
38.5
43.5 (+6.5)
44.0 (+7.0)
48.5 (+11.5)
37.5
51.0 (+4.0)
52.5 (+5.5)
57.5 (+10.5)
47.5
L8
M8
TT
KK
LL
47.5 (+21.5)
37.0 (+11.0)
39.5 (+13.5)
36.0 (+10.0)
47.0 (+21.0)
48.0 (+22.0)
54.5 (+16.0)
46.0 (+7.5)
49.5 (+10.5)
45.0 (+6.5)
54.0 (+15.5)
54.5 (+16.0)
46.0 (+8.5)
48.5 (+11.0)
49.5 (+12.0)
43.0 (+5.5)
49.0 (+11.5)
50.5 (+13.0)
54.0 (+6.5)
56.5 (+9.0)
59.0 (+11.5)
53.5 (+6.0)
58.0 (+10.5)
59.0 (+11.5)
MM
KL
LK
MK
a
Melting temperatures obtained from the maxima of the first derivatives of the melting curves (A260 vs temperature) recorded in a buffer containing
2.5 mM Na2HPO4, 5.0 mM NaH2PO4, 100 mM NaCl, 0.1 mM EDTA, pH 7.0, using 1.0 μM concentrations of each strand. K, L, and M correspond
b
to the incorporation of the amidites 4, 7, and 11, respectively. Differences in melting temperatures as compared to X = T.
position of the 2′-deoxyuridine as the primary destabilizing
factor. Monomer M, in general, induced an increase in the
duplex stability, suggesting that a flexible methylene linker
between the 2′-position of the 2′-deoxyuridine and triazole ring
is crucial for the stability of the modified duplex (compare data
for monomers L and M). Nevertheless, neutral or negative
influence on the duplex stability was observed when monomer
M was placed near to the 3′-end in an ON sequence (entries 2
and 4).
sterical interaction, for instance a simultaneous intercalation of
the two pyrene units leading to the local unwinding of the
duplex, thereby causing a decrease in thermal stability as
reported previously for other ON’s modified with intercalating
pyrene moieties in a (+1) zipper arrangement.23 However,
another possibility could be a contact between two pyrene units
in the minor groove away from the duplex core thereby
disturbing hydration of the duplex leading to a thermally
unstable duplex. In all other cases (entries 10−14) very stable
duplexes were formed, but no zipper contacts were observed for
monomer M as indicated by very neutral values of ΔΔTm in the
range of −3.0 °C to +1.5 °C.
Hereafter, the modified sequences were mixed to study the
possibility for zipper contacts between the same modifications
(K, L, or M) in the minor groove or to reveal the possibility for
intercalation of the pyrene into the duplex. 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. When the modified
sequences K2 and K5 were mixed, to form a so-called (+1)
zipper arrangement, the resulting duplex was found to be
thermally more stable than either of the two duplexes with just
one modification (compare entry 9 with entries 3 and 6),
suggesting a favorable zipper contact (ΔΔTm = +7.0 °C)
between two pyrenes from opposite strands. An energetically
unfavorable contact between the pyrene moieties with two
modifications in a (−3) zipper motif (entry 13, ΔΔTm = −8.0
°C) was observed, which is completely opposite to what was
found for monomer B in the same arrangement.3,4 For the
remaining zippers (entries 10, 11, 12, and 14) no specific
contacts were indicated, as the ΔΔTm values are small.
For monomer L, a relative increase in the thermal stability of
the (−1) zipper duplexes (Table 1, entries 10 and 11, large
positive ΔΔTm’s) suggested a stabilizing contact between 2′-
substitiuents. An energetically unfavorable zipper interaction
was observed for monomer L in a (+1) zipper contact (entry 9,
ΔΔTm = −6.5 °C). Highly unstable duplexes were observed in
the (−3) and (−4) zipper motifs with monomer L (entries 13
and 14). However, it is important to note that these duplexes
are modified with monomer L close to 3′-end in each strand,
where monomer L induced largest destabilization of the duplex
(entries 2 and 4).
Finally, also the zipper arrangements with more than two
pyrenes in the duplex as obtained by using the sequences X6−
X7 were studied. For results and discussion, see Supporting
Information.
To study the effect of the monomers K, L, and M in different
bulged duplexes and three-way junctions, three 13-mer
sequences with the monomer placed in the central position,
K8, L8, and M8, were prepared and mixed with different
complementary strands (Table 2). First, a standard duplex
formed with a fully matched DNA sequence was studied. An
increase in the thermal stability of the modified duplex was
observed for monomers K and M compared to the unmodified
duplex (ΔTm = 2.0 and 3.5 °C, respectively), whereas
monomer L led to the destabilization of the modified duplex
by 4.0 °C compared to the unmodified duplex. These data are
in line with the observations from Table 1. Upon hybridization
with an RNA complement, all the monomers led to a
destabilization of the corresponding DNA:RNA duplex.
However, monomer M was found to be the best tolerated
with a ΔTm of −1.0 °C.
Hereafter, we investigated the hybridization of the modified
13-mer sequences with complementary bulged DNA and RNA
sequences, that is 14- or 15-mer sequences with one or two
unpaired nucleotides. When placed opposite to the bulged
adenine monomers, K and M induced large increases in the
thermal stability of a DNA:DNA duplex compared to an
unmodified bulged duplex (ΔTm = +9.5 °C and +12.0 °C,
respectively, Table 2). Moreover, thermal stability of the
modifed duplexes was found to be comparable to that of a
regular duplex (i.e., with two thymidines instead of K or M).
Monomer M in a (+1) zipper arrangement induced
significant destabilization of the zipper duplex (ΔTm = −6.0
°C and ΔΔTm = −16.0 °C, entry 9). This could indicate a
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Figure 2. Representative steady-state fluorescence emission spectra for modified ON’s (K2, K6, L1, L2, M2, and M3) in the absence (red) and
presence (blue) of complementary DNA (λex = 350 nm; T = 10 °C; [ON] = 1.0 μM). Different scaling of the Y-axes is used. For the remaining
ON’s, and full spectra of T1-M2 ([ON] = 0.25 μM), see Figures S1−S4 (Supporting Information).
This suggests a favorable stacking interaction of the additional
pyrene from monomer K or M into the bulge. However, for a
DNA:DNA duplex having an adenine bulge opposite to
monomer L, no change in the thermal stability was observed
when compared to an unmodified A-bulge DNA duplex. A
significant but smaller stabilization of the corresponding
DNA:RNA bulged duplex (ΔTm = +4.0 °C and +5.0 °C,
respectively) was also observed for monomer K or M.
Interestingly, for a DNA:RNA duplex, monomer L was found
to stabilize the modified duplex by 3.5 °C. When studied in
GA-bulged duplexes, the monomers, in general, were found to
induce smaller stabilizations of the modified duplexes compared
to the unmodified duplex.
Next, ON’s K8, L8, and M8 were mixed with DNA and RNA
strands with standard stable hairpin sequences flanked by
single-stranded regions being complementary to the modified
sequences. Hereby, the effect of additional pyrenes at the
branching point of TWJ’s was studied (Table 3). The DNA
hairpin complement used in this study contains an additional
CC-bulge compared to the RNA complement to form a well-
defined secondary structure.24 As evident from Table 3,
monomer K was found to have a very large stabilizing effect
on the modified TWJ with ΔTm = +19.0 °C as compared to
that of the unmodified TWJ with thymidine in place of
monomer K, suggesting favorable stacking interactions from the
large aromatic surface of pyrene with nucleobases from one of
the three stems of the TWJ. Monomers L and M demonstrated
smaller but significant increases in the thermal stability of the
corresponding TWJ (ΔTm = +9.0 °C and +14.0 °C,
respectively). A comparison of monomers L and M revealed
that introduction of the flexible methylene linker between the
triazole moiety and the 2′-carbon has a favorable effect on the
stability of the TWJ in accordance with that observed for the
regular duplexes (Table 1). Nevertheless, the results for
monomer K suggested that the 5′-(S)-C position is placing
pyrene in a better position for stacking interactions with
neighboring nucleobases than the 2′-position. In comparison,
monomer M was found to have the largest stabilizing effect in
regular duplexes. The stabilizing effect of monomer K was
found to be significantly reduced when studied in a DNA:RNA
TWJ. However, for monomers L and M, the effect on the
stability of the DNA:RNA TWJ was found to be comparable to
that on the stability of the DNA:DNA TWJ. Upon addition of
Mg2+ to the complexes, the stability of the TWJ increases
significantly in all the studied cases, but the relative stabilization
effect of the modifications decreases slightly.
Finally, six 14-mer sequences with two consecutive
incorporations of the same or different monomers (KK, LL,
MM, KL, LK, and MK) were synthesized to study the presence
of an additional pyrene in the branching point of the TWJ
(Table 3). The idea was that the two pyrenes might interact to
stabilize the complexes and/or form fluorescent probes (vide
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infra). Before studying TWJ’s, the modified sequences were
mixed with regularly matched DNA or RNA strands and, in all
cases, small to medium decreases in the thermal stability were
observed (Table S3, Supporting Information), ΔTm’s between
−1.5 °C and −8.5 °C). Next, these modified ON’s were mixed
with DNA and RNA strands with the standard stable hairpin
sequences (Table 3). The two adjacent incorporations of either
of the monomers K, L, or M in the branching points induced
large increases in the thermal stability of the resulting duplex
compared to the unmodified sequence with two thymidines in
place of the modified monomers. The highest increase (ΔTm =
+21.5 °C) was observed for monomer K (ON KK) in line with
the single incorporation of monomer K (ON K8). However,
when compared to the effect of the first modification in a 13-
mer sequence (ON's K8, L8, and M8), the effect of introducing
an extra pyrene monomer into the TWJ was found to be small.
Interestingly, with two different monomers at the branching
point, thermal stability of the complex with monomer L toward
the 5′-end and monomer K toward the 3′-end (ON LK) was
found to be significantly higher than that of the opposite
arrangement of monomers (ON KL), indicating possible
favorable contact between the two pyrenes from the different
monomers in the former arrangement. This is in line with the
large stabilizing effect observed with ON MK in the TWJ (ΔTm
= +22.0 °C). In the DNA:RNA TWJ, the relative stability
induced by two K monomers was found to be significantly less
than that observed in DNA:DNA TWJ context while
monomers L and M do not show any preference for a DNA
hairpin complement over a RNA hairpin complement in
parallel to data obtained for ON's L8 and M8. ON’s LK and
MK also showed a relative preference for a DNA hairpin over
an RNA hairpin complement similar to ON KK. As observed
earlier for ON's K8, L8, and M8, the addition of Mg2+ led to
increases of the TWJ stability in all cases, but the relative
stabilization effect of the modifications decreases slightly.
Fluorescence Studies. We recorded the steady-state
fluorescence emission spectra of all the 11-mer sequences
modified with monomers K, L, and M in the absence and
presence of unmodified and modified complementary DNA
(see sequences in Table 1) at 10 °C by exciting pyrene at 350
nm. For all single-stranded ON’s containing monomer K, two
well-defined bands for the pyrene monomer fluorescence (λmax
∼387 and ∼406 nm) were observed (Figures 2 and S1
(Supporting Information)). Upon hybridization with the
complementary DNA sequence, a decrease in the fluorescence
emission intensity was observed, indicating an intercalation of
the pyrene into the duplex where its fluorescence is quenched
by the neighboring bases. A broad pyrene−pyrene excimer
band at λmax = ∼490 nm was obtained for the ON’s K6 and K7
with two incorporations of the pyrenes at alternate positions
(Figure 2 and S1), suggesting the close proximity of two
pyrenes in the single-stranded probes. Near complete
disappearance of the excimer signal along with the decrease
in the intensity of pyrene monomer fluorescence upon
hybridization with the DNA complement indicated the
intercalation of one or both of the pyrene units.
to all other ON’s incorporated with monomer L (Table 1). In a
different sequence context studying monomer L, a decrease in
the fluorescence quantum yield upon hybridization with
complementary DNA has been observed,19 suggesting the
intercalation of the pyrene into the duplex. Thermal
denaturation data (Table 1) in combination with steady-state
fluorescence spectra for monomer L suggested the intercalation
of the pyrene toward the 3′-direction, which may cause local
unwinding of the duplex when present near the 3′-end,
accounting for the destabilization of the duplex in such cases.
Fluorescence emission intensity for ON's M1−M5 was also
found to be dependent on the sequence (Figures 2 and S3
(Supporting Information)). Single-stranded ON’s with an AMA
context (M2 and M5) were found to be highly fluorescent as
compared to the other ON’s (M1, M3, and M4), strengthening
the fact that adenine is the weakest quencher of the pyrene
fluorescence.25,26 In general, a large increase in the fluorescence
intensity upon hybridization with a complementary DNA
strand was observed for ON’s modified with monomer M,
which indicates the positioning of the pyrene unit into the
minor groove away from the duplex core. However, when
placed close to the 3′-end (ON's M1 and M3), the
corresponding modified duplexes (T1:M1 and M3:T2) were
found to be significantly less fluorescent.
Hereafter, steady-state fluorescence emission spectra of all
interstrand zippers (see Table 1) were recorded. The emission
spectra of all the studied zipper duplexes for monomers K, L,
and M displayed structured pyrene emission bands with λmax in
the range of 385−391 nm and 404−409 nm (Figure 3). When
monomer K was placed in a (−1) zipper arrangement in
modified duplexes (K5:K1 and K4:K2), a pyrene excimer signal
with λmax = ∼490 nm was observed as a broad shoulder,
suggesting the close proximity of two pyrene units from
monomer K in opposite strands. DNA duplexes with more than
two incorporations of monomer K in general displayed broad
bands corresponding to the pyrene excimer fluorescence signal
(Figure S6 (Supporting Information)).
An excimer band with only very low fluorescence intensity
was observed when monomer L was placed in a (−1) zipper
arrangement in modified duplexes (L5:L1 and L4:L2) (Figure
3). This supports the Tm data for this arrangement, which
showed favorable interaction between two additional pyrene
units from opposite strands (Table 1), but it is not clear how
this is structured. The intensity of this excimer signal increases
in the presence of an additional pyrene in the modified
duplexes (Figure S6).
Zippers of monomer M (Figures 3 and S6) showed the same
very strong intensities as observed for the single modified
duplexes (Figures 2 and S3). For monomer M, the presence of
an excimer band in a (+1) zipper arrangement (M5:M2)
further strengthens our hypothesis of close contact between
two pyrene units as the cause for relatively low thermal stability
(Table 1). This specific (+1) zipper contact between two
pyrene units of monomer M from opposite strands was found
to be undisturbed by the presence of additional pyrene units in
the modified duplex as indicated by the steady-state
fluorescence spectra of multizippers (Figure S6).
For monomer L (Figures 2 and S2 (Supporting Informa-
tion)), no significant change in the fluorescence intensity upon
hybridization was observed except when placed centrally in the
duplex (ON L2), which displayed a large decrease in the
fluorescence intensity (compare L2 and T1:L2, Figure 2). It is
important to note that ON L2 induced no thermal
destabilization of the corresponding duplex T1:L2 in contrast
Steady-state fluorescence spectra of 13-mer sequences (K8,
L8, and M8) were measured in the absence and presence of
matched DNA or RNA targets (Figure S8 (Supporting
Information)). A hybridization-induced decrease in pyrene
monomer fluorescence was observed for monomer K upon
mixing ON K8 with DNA or RNA complement, strengthening
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increase in the thermal stability of these modified duplexes
compared to unmodified bulged duplexes (Table 2). When
introduced into TWJ’s, all pyrene monomers (K, L, and M)
displayed only small changes in the fluorescence intensity.
Finally, the six 14-mer sequences with two adjacent
incorporations of the same or different monomers in the
middle of the sequence were investigated (Figure 4). Steady-
state fluorescence spectra of the single-stranded 14-mer
sequence with two adjacent incorporations of monomer K or
L (ON's KK and LL, Table 3) displayed the typical two bands
(λmax = ∼386 nm and ∼406 nm) for pyrene monomer
fluorescence along with a pyrene−pyrene excimer band with
λmax = ∼490 nm of low intensity. However, the corresponding
sequence MM displayed an intense unstructured band with λmax
= ∼480 nm for the excimer signal along with very intense bands
for pyrene monomer fluorescence (λmax = ∼384 nm and ∼403
nm). Upon hybridization with fully matched DNA or RNA
sequences, ON KK, and to some extent LL, displayed increases
in the fluorescence bands as well as a significant increase of the
excimer signal (Figure 4). From these results it can be
concluded that both pyrenes are pointing into the minor groove
away from the duplex core and are able to interact with each
other. ON MM displayed a decrease in the fluorescence
emission intensity of the pyrene monomer bands and a
complete disappearance of the excimer signal upon hybrid-
ization with the DNA complement (Figure 4). This indicates
intercalation of at least one of the pyrenes into the duplex.
However, against the RNA complement, ON MM displayed an
intense but somewhat red-shifted excimer signal. This indicated
that in a DNA:RNA duplex, the two pyrenes are in close
proximity to each other to form an excimer signal, making this
probe a promising candidate for the selective detection of
single-stranded RNA over single-stranded DNA. This difference
in excimer formation between dsDNA and DNA:RNA might
be due to structurally different minor grooves.
Next, the 14-mer sequences with two adjacent incorporations
of different monomers were studied. ON LK (see Table 3)
displayed a very weak and broad band for pyrene excimer
fluorescence (Figure 4). When mixed with the RNA comple-
ment, ON LK displayed an intense band at λmax = ∼450 nm
with a broad shoulder starting at ∼475 nm, which can be
assigned to the pyrene−pyrene excimer signal. However,
against the matched DNA sequence, a very broad band with
center at λmax = ∼480 nm was found. ON KL failed to display
the excimer signal in its steady-state fluorescence spectrum also
upon hybridization with complementary DNA. Upon hybrid-
ization with the RNA complement, a very weak excimer signal
was observed. This could be attributed to the large distance
between the two pyrenes (pointing in opposite directions) in
the single-stranded probe and in the corresponding modified
duplexes. For sequence MK, an intense excimer signal was
observed in the single-stranded form as well as upon
hybridization with DNA or RNA sequences.
Finally, steady-state fluorescence spectra of all the 14-mer
sequences were recorded in the presence of standard stable
hairpin sequences forming TWJ’s (Figure 4). ON KK displayed
a weak and broad excimer signal upon mixing with the RNA
hairpin complement. However, no such excimer formation was
observed against the DNA hairpin complement, suggesting that
the formation of excimer is selective for the RNA hairpin
complement, but the very low intensity of this signal probably
makes it unsuitable for any diagnostic application. ON LL
displayed a weak excimer signal upon hybridization with the
Figure 3. Steady-state fluorescence emission spectra of interstrand
zippers (Table 1) (λex = 350 nm; T = 10 °C; [ON] = 1.0 μM).
Different scaling of the Y-axes is used. For full spectra of duplexes M5-
M2 and M4-M2 ([ON] = 0.25 μM) see Figure S5 (Supporting
Information).
our hypothesis of intercalation of pyrene from monomer K into
the duplex. Monomer L displayed a large increase in the
fluorescence intensity upon hybridization with the matched
RNA target compared to single-stranded probe L8, while
against complementary DNA no significant change in the
fluorescence intensity was observed upon hybridization. This
suggested that pyrene is pointing away from the base stack into
the minor groove of the DNA:RNA duplex while it is at least
partly intercalated with neighboring bases in the DNA:DNA
duplex. For monomer M (ON M8), no significant change in
the steady-state fluorescence spectra was observed upon
hybridization with complementary RNA, whereas an increase
in fluorescence intensity was again seen with DNA target.
Thereafter, steady-state fluorescence spectra of sequences
K8, L8, and M8 were recorded in the presence of
complementary bulged DNA or RNA sequences, or DNA or
RNA strands with hairpin sequences forming TWJ’s (Figure
S8). In general, relatively low fluorescence intensity of the
pyrene in the corresponding bulged duplexes with additional
adenine in the complementary strand was observed, indicating
intercalation of the pyrene. This is in line with the large
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Figure 4. Steady-state fluorescence emission spectra of modified single-stranded 14-mer ON’s (KK, LL, MM, KL, LK, MK) and their duplexes with
complementary DNA or RNA sequence, and standard stable hairpin sequences flanked by single-stranded regions being complementary to the
modified sequences (λex = 350 nm; T = 10 °C ; [ON] = 1.0 μM). Different scaling of Y-axes is used.
DNA or RNA hairpin complements. ON MM displayed an
excimer formation upon mixing with DNA or RNA hairpin
complements, however, with decreased intensity as compared
to the single-stranded probe MM. As no excimer formation was
observed for ON MM against a regular DNA complement, ON
MM has a potential to discriminate between a DNA hairpin and
a regular DNA complement. Interestingly, ON LK displayed an
intense unstructured excimer signal against the DNA/RNA
hairpin complements, whereas in the opposite arrangement of
the monomers, ON KL failed to show any excimer signal. ON
MK also displayed excimer signals against the hairpin
complements, however, also in the single-stranded states and
in the duplexes.
the (−3) zipper orientation of two monomers in the
complementary strands leads to a significant decrease in duplex
stability. Hence, the two pyrenes cannot intercalate into the
same space in the duplex, and indeed no excimer band has been
observed. A decrease in fluorescence intensity indicated
intercalation of the pyrene also in the DNA:RNA duplex.
The introduction of monomer K into bulged duplexes or TWJ’s
leads to strong increases in stability, more so in DNA:DNA
than in DNA:RNA complexes, probably in connection to
aromatic stacking in the complexes as validated by small
decreases in fluorescent intensity upon hybridization.
Monomer L. In general, incorporation of this monomer
into duplexes leads to decreases in thermal stability as
compared to unmodified duplexes. The general destabilization
might be a result of the rather inflexible structure with the
triazole connected directly to the C-2′ of the ribose and thereby
a sterical conflict in the duplex core. In another study similar
decreases in duplex stability have been observed for L.19 In
general, fluorescence intensity decreases upon hybridization,
indicating that the pyrene moiety at least partly intercalates into
the duplex. In contrast to the DNA:DNA duplexes, the pyrene
of L seems not to intercalate into the DNA:RNA duplex, as a
huge increase in fluorescence is seen upon hybridization. In
bulged duplexes, this picture changes as L leads to an increase
DISCUSSION
■
In the following, the hybridization and subsequent fluorescence
properties of the three monomers are summarized:
Monomer K. On the basis of the general decrease in
fluorescence intensity upon hybridization, the 5′-position for
the pyrene moiety of K seems in favor of intercalation into the
duplex. The intercalation might be situated between the first
and the second unmodified base-pair in the 5′-direction to the
modified nucleotide as justified by the fact that specifically only
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in stability, especially of the bulged DNA:RNA complex
followed by a small decrease in fluorescence, indicating
intercalation into the bulge. This stabilizing effect is not seen
with the bulged DNA:DNA. Also the TWJ’s were stabilized by
L, but whereas the effect is smaller than with K for the
DNA:DNA complex, it is slightly larger for the DNA:RNA
complex.
the constitution, for instance the aromatic surface, rather than
the position of the substituent dictates the behavior in duplexes
and TWJ’s. Compared to other pyrene-containing nucleotides
described in the literature, there are differences and similarities.
Monomer M behaves differently when compared to other 2′-
modified analogues, such as 2′-OCH2Pyr or 2′-N(CH3)-
CH2Pyr.14,27 These have shown a strong tendency to intercalate
in DNA:DNA duplexes, whereas this does not seem to be the
case for M. This might be due to local conformational
differences around the modified monomers, as also indicated by
the fact that monomer L is more prone to intercalation than M.
Monomer M. In contrast to monomer L, the incorporation
of M into duplexes generally leads to stabilized duplexes. This
might be due to the flexibility inserted by a methylene linker
between the triazole and C2′. In general, the stabilized duplexes
are followed by very large increases in fluorescence intensity,
whereas the few neutral or destabilized duplexes are followed
by decreases in fluorescence intensity. This clearly indicates
that the sequence contexts have a large influence and that the
pyrene moiety in general seems to be positioned in the minor
groove without intercalation. Two or more incorporations of
monomer M were found to be well tolerated in an 11-mer
dsDNA, suggesting that formation of heavily modified duplexes
is a possibility with monomer M. Unfavorable interaction
between the substituents in the (+1) zipper structure is
indicated by fluorescence as well as by Tm. Monomer M is also
found to stabilize the bulged duplexes, and in the case of a
single nucleotide A-bulge, this is followed by relative decreases
in fluorescence intensity, indicating that intercalation takes
place in the bulges. Also the TWJ’s are significantly stabilized,
apparently as a consequence of stacking as justified from
decreases in fluorescence.
Fluorescent Probes for Hybridization. The double-
modified sequences (Table 3) are all found to stabilize the
TWJ’s significantly, which is of course interesting from a
targeting aspect. Nevertheless, equally interesting is the
diversity in fluorescence, and especially excimer formation,
upon formation of the complexes (duplexes or TWJ’s),
indicating the potential of these ON’s as recognition probes.
Hence, MM displays a strong excimer band as a single strand
and in a DNA:RNA duplex but not in a DNA:DNA duplex.
This makes MM a potential probe for RNA over DNA
complements. As might be expected from the structures, LK
with the two pyrenes positioned toward each other is much
more interesting than KL. LK demonstrates large excimers
when complexed with hairpins as compared to its duplexes, and
it might function as a probe for hairpins. Finally, KK forms
large excimers in both DNA:DNA and DNA:RNA duplexes
and much smaller excimers with hairpins. Hereby, KK acts
exactly opposite to that of LK as a probe for single-stranded
complements over hairpins. MK does not show the same
potential as LK because excimer bands are formed in all
complexes as well as in the single strand.
CONCLUSION
■
We have described efficient synthetic access to three new
pyrene-modified nucleotides. These have shown interesting
properties in the formation of synthetic nucleic acid complexes
with varying fluorescence emission. Monomer K forms stable
duplexes with the pyrene moiety intercalating in a predictable
way. Monomer L leads to somewhat distorted and destabilized
duplexes with the pyrene intercalated, whereas monomer M
leads to stabilized duplexes with the pyrene situated in the
minor groove. All three monomers can lead to significant
stabilization of bulged duplexes and TWJ’s, overall with K > M
> L in DNA:DNA complexes and M > K > L in DNA:RNA
complexes. Interesting excimer formation in combination with
stabilized TWJ’s is observed with two adjacent monomers LK,
whereas interesting excimer formation in duplexes is observed
for KK and for MM, the latter for DNA:DNA duplexes making
it only a probe for DNA over RNA. The study demonstrates
that pyrene-modified nucleotides can have various applications
as diagnostic probes and in nucleic acid targeting and that the
positioning of the pyrene moiety is delicate and crucial for the
structural output.
EXPERIMENTAL SECTION
■
General. All commercial reagents were used as supplied, except
CH2Cl2 which was distilled prior to use. Anhydrous solvents were
dried over 4 Å activated molecular sieves (CH2Cl2, DMF, pyridine,
and DCE) or 3 Å activated molecular sieves (CH3CN). All reactions
were carried out under an atmosphere of argon or nitrogen, when
using anhydrous solvents. Reactions were monitored using TLC
analysis with Merck silica gel plates (60 F254). To visualize the plates,
they were exposed to UV light (254 nm) and/or immersed in a
solution of 5% H2SO4 in methanol followed by charring. Standard
column chromatography was performed using silica gel 60 (0.040−
0.063 mm). 1H NMR, 13C NMR, and 31P NMR spectra were recorded
at 400, 101, and 162 MHz, respectively. Chemical shift values (δ) are
reported in ppm relative to either tetramethylsilane (1H NMR) or the
deuterated solvents as internal standard for 13C NMR (δ: CDCl3 77.16
ppm, DMSO-d6 39.52 ppm) and relative to 85% H3PO4 as external
standard for 31P NMR. 2D spectra (1H−1H COSY and 1H−13C
In summary, interesting properties have been found for all
the three monomers K, L, and M, and all have the potential to
find application in the targeting of nucleic acid sequences or in
diagnostic probes. From a perspective of targeting DNA or
RNA, it is very interesting that the pyrene monomers can lead
to the formation of very stable TWJ’s due to aromatic stacking.
When compared to the corresponding double-headed nucleo-
sides (Figure 1), it is clear that the pyrene-modified monomers
K, L, and M demonstrate completely different properties. For
instance, K has the opposite tendency to that of monomer B, as
K leads to a destabilized (−3) zipper complex. Whereas the
pyrene of K intercalates into the duplex, the thymine of B (and
of C) interacts in the minor groove. In general, this study of
pyrene analogues of double-headed nucleotides has shown that
1
HSQC) have been used in assigning H and 13C NMR signals. High
resolution ESI (quadrupole) mass spectra were recorded in positive
mode.
3′-O-(tert-Butyldimethylsilyl)-5′-O-pixyl-5′(S)-C-[(4-(pyren-1-
yl)-1,2,3-triazol-1-yl)methyl]thymidine (2). To a solution of
compound 14 (500 mg, 0.75 mmol) and 1-ethynylpyrene (226 mg,
1.00 mmol) in THF:H2O:pyridine (12 mL, 5:5:2 v/v) were added
sodium ascorbate (37 mg, 0.19 mmol) and CuSO4·5H2O (25 mg, 0.10
mmol), and the solution was stirred at room temperature overnight.
Additional sodium ascorbate (37 mg, 0.19 mmol) and CuSO4·5H2O
(25 mg, 0.10 mmol) were added, and the reaction mixture was stirred
for another 3 h at room temperature. To the mixture was added
CH2Cl2 (40 mL), and it was washed with a 10% aqueous solution of
NaHCO3 (30 mL). The aqueous phase was extracted with CH2Cl2 (3
× 40 mL), and the combined organic phase was dried (Na2SO4) and
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concentrated under reduced pressure. The residue was purified by
column chromatography (1−3% MeOH in CH2Cl2), affording the
product 2 (564 mg, 84%) as a yellow foam. Rf 0.36 (5% MeOH in
CH2Cl2); 1H NMR (300 MHz, CDCl3) δ 8.55 (d, J = 9.6 Hz, 2H, Ar),
8.21−7.99 (m, 8H, Ar, NH), 7.82 (d, 1H, J = 0.6 Hz, H6), 7.67 (s, 1H,
H5-triazole), 7.52−7.33 (m, 9H, Ar), 7.25−7.03 (m, 4H, Ar), 6.24 (dd,
1H, J = 5.1, 9.6 Hz, H1′), 4.16 (dd, 1H, J = 8.8, 13.0 Hz, H6′), 3.91−
3.80 (m, 2H, H5′, H6′), 3.48 (s, 1H, H4′), 3.35 (d, J = 4.8 Hz, 1H,
H3′), 2.05 (s, 3H, CH3), 2.00 (m, 1H, H2′), 1.91 (m, 1H, H2′), 0.68
(s, 9H, (CH3)3), −0.19 (s, 3H, CH3Si), −0.29 (s, 3H, CH3Si); 13C
NMR (75 MHz, CDCl3) δ 163.7 (C4), 152.2, 152.0 (Ar), 150.3 (C2),
147.4 (C4-triazole), 146.2 (Ar), 135.7 (C6), 131.5, 131.4, 131.0, 130.9,
130.7, 130.5, 128.2, 128.1, 128.0, 127.8, 127.7, 127.4, 127.1, 126.2,
125.5, 125.5, 125.2, 125.1, 124.9, 124.8, 124.7, 124.2, 124.1, 124.0,
122.9, 122.5, 117.6, 117.2 (Ar + C5-triazole), 111.2 (C5), 86.8 (C4′),
84.9 (C1′), 78.9 (C-Ar3), 73.2 (C3′), 72.2 (C5′), 50.3 (C6′), 41.0
(C2′), 25.7 ((CH3)3), 17.7 (CSi), 12.8 (CH3), −4.5, −4.7 (CH3Si);
HR-ESI MS m/z 916.3504 ([M + Na]+, C54H51N5O6SiNa+ calcd
916.3501).
CH2Cl2 (3 × 10 mL). The combined organic phases were dried
(Na2SO4) and concentrated under reduced pressure. The residue was
purified by column chromatography (50−100% EtOAc, 0.5% pyridine
in petroleum ether) to afford the product 6 (112 mg, 79%) as a yellow
foam; Rf 0.36 (5% MeOH in CH2Cl2); 1H NMR (400 MHz, CDCl3) δ
8.78 (s, 1H, NH), 8.32 (d, 1H, J = 9.3 Hz, pyrene), 8.16 (s, 1H, H5-
triazole), 8.10−7.74 (m, 9H, pyrene, H6), 7.44−7.13 (m, 9H, DMT),
6.84 (d, 4H, J = 8.6 Hz, DMT), 6.43 (d, 1H, J = 5.3 Hz, H1′), 5.36 (d,
1H, J = 8.2 Hz, H5), 5.25 (t, 1H, J = 5.7 Hz, H2′), 4.90 (dd, 1H, J =
5.9, 11.8 Hz, H3′), 4.53 (m, 1H, H4′), 4.21 (3′−OH), 3.78 (s, 6H,
OCH3), 3.56 (m, 2H, H5′); 13C NMR (101 MHz, CDCl3) δ 162.7
(C4), 158.99, 158.97 (DMT), 150.4 (C2), 147.4 (C4-triazole), 144.4
(DMT), 139.4 (C6), 135.3, 135.1 (DMT), 131.5, 131.3, 130.7, 130.32,
130.27, 128.42, 128.38, 128.1, 127.4, 127.3, 127.0, 126.1, 125.5, 125.3,
124.94, 124.90, 124.87, 124.5, 124.2, 124.1 (DMT, pyrene, C5-
triazole), 113.6 (DMT), 103.2 (C5), 87.6 (C-Ph3), 87.0 (C1′), 84.8
(C4′), 71.0 (C3′), 66.6 (C2′), 62.4 (C5′), 55.4 (OCH3). HRMS-ESI:
m/z 798.2948 ([M + H]+, C48H39N5O7H+ calcd 798.2922).
3′-O-(2-Cyanoethoxy-N,N-diisopropylaminophosphinyl)-5′-
O-(4,4′-dimethoxytrityl)-2′-(4-(pyren-1-yl)-1,2,3-triazole-1-yl)-
2′-deoxyuridine (7). Nucleoside 6 (184 mg, 0.23 mmol) was
coevaporated twice with anhydrous DCE and dissolved in the same
solvent (4.0 mL). N,N-Diisopropylethylamine (0.41 mL, 2.35 mmol)
and 2-cyanoethyl-N,N-diisopropylamino chlorophosphite (0.11 mL,
0.49 mmol) were added, and the reaction mixture was stirred at room
temperature for 4 h. Ethanol (1.5 mL) and CH2Cl2 (20 mL) were
added, and the solution was washed with a saturated aqueous solution
of NaHCO3 (15 mL). The aqueous phase was extracted with CH2Cl2
(2 × 10 mL), and the combined organic phases were dried (Na2SO4)
and concentrated under reduced pressure. The residue was purified by
column chromatography (0−100% EtOAc, 0.5% pyridine in petroleum
ether). The residue was precipitated three times from a solution in
EtOAc (1.5 mL) poured into cold petroleum ether (70 mL) to afford
the product 7 (111 mg, 48%) as a light yellow foam; Rf 0.59 (5%
MeOH in CH2Cl2); 31P NMR (162 MHz, CDCl3) δ 151.8, 150.2
(3:2). HRMS-ESI: m/z: 1020.3857 ([M + Na]+, C57H56N7O8PNa+
calcd 1020.3821).
5′-O-Pixyl-5′(S)-C-[(4-(pyren-1-yl)-1,2,3-triazol-1-yl)methyl]-
thymidine (3). Nucleoside 2 (373 mg, 0.42 mmol) was dissolved in
anhydrous THF (6.0 mL), and a 1.0 M solution of TBAF in THF
(0.42 mL, 0.42 mmol) was added. The reaction mixture was stirred for
3 h at room temperature. CH2Cl2 (20 mL) was added, and the
resulting solution was washed with a saturated aqueous solution of
NaHCO3 (15 mL). The aqueous phase was extracted with CH2Cl2 (3
× 10 mL), and the combined organic phases were washed with water
(20 mL), dried (Na2SO4), and concentrated under reduced pressure.
The residue was purified by column chromatography (0−10% MeOH,
0.5% pyridine in CH2Cl2) to afford the product 3 (267 mg, 82%) as a
1
light yellow foam; Rf 0.56 (10% MeOH in CH2Cl2); H NMR (400
MHz, CDCl3) δ 8.59 (d, 1H. J = 9.3 Hz, Ar), 8.51 (s, 1H, NH), 8.24−
7.99 (m, 8H, Ar), 7.66 (s, 1H, H5-triazole), 7.56−7.00 (m, 14H, H6,
Ar), 6.13 (t, 1H, J = 6.6 Hz, H1′), 4.19 (m, 1H, H6′), 4.03 (m, 1H,
H6′), 3.92 (m, 1H, H5′), 3.73 (m, 1H, H3′), 3.50 (t, 1H, J = 4.2 Hz,
1H, H4′), 2.26−2.06 (m, 3H, 3′−OH, H2′), 2.00 (s, 3H, CH3); 13C
NMR (101 MHz, CDCl3) δ 163.6 (C4), 152.2, 152.1 (Ar), 150.2
(C2), 147.6 (C4-triazole), 146.5 (Ar), 136.1 (C6), 131.6, 131.40,
131.38 131.0, 130.8, 130.5, 128.6, 128.4, 128.2, 128.1, 127.7, 127.5,
127.2, 126.3, 125.7, 125.4, 125.2, 125.1, 125.0, 124.9, 124.7, 124.4,
124.2, 123.8, 122.8, 122.7, 117.5, 117.0 (Ar, C5-triazole), 111.4 (C5),
85.0 (C4′), 84.2 (C1′), 78.4 (C-Ar3), 71.4 (C4′), 70.2 (C3′), 50.5
(C6′), 40.1 (C2′), 12.9 (CH3). HR-ESI MS m/z 802.2642 ([M +
Na]+, C48H37N5O6Na+ calcd 802.2637).
3′-O-(2-Cyanoethoxy-N,N-diisopropylaminophosphinyl)-5′-
O-pixyl-5′(S)-C-[(4-(pyren-1-yl)-1,2,3-triazol-1-yl)methyl]-
thymidine (4). To a solution of 3 (300 mg, 039 mmol) in anhydrous
CH2Cl2 (4 mL) were added N,N-diisopropylethylamine (0.26 mL,
1.50 mmol) and 2-cyanoethyl-N,N-diisopropylamino chlorophosphite
(0.20 mL, 0.90 mmol) under Ar atmosphere. The reaction mixture was
stirred at room temperature for 2 h. CH2Cl2 (30 mL) was added, and
the mixture was washed with a 10% aqueous solution of NaHCO3.
The aqueous phase was extracted with CH2Cl2 (3 × 30 mL), and the
combined organic phase was dried (Na2SO4) and concentrated under
reduced pressure. The residue was purified by column chromatog-
raphy (0−20% (CH3)2CO, 0.25% pyridine in CH2Cl2) to give the
product 4 (260 mg, 69%) as a yellow foam: Rf 0.27, 0.36 (10%
(CH3)2CO in CH2Cl2); 31P NMR (162 MHz, CDCl3) δ 151.61,
151.47; HR-ESI MS m/z 1002.3718 ([M + Na]+, C57H54N7O7PNa+
calcd 1002.3714).
5′-O-(4,4′-Dimethoxytrityl)-2′-C-(prop-2-yn-1-yl)-2′-deoxy-
uridine (9). Nucleoside 820 (165 mg, 0.62 mmol) was coevaporated
with anhydrous pyridine (2 × 5 mL) and redissolved in the same
solvent (10 mL). 4,4′-Dimethoxytrityl chloride (270 mg, 0.80 mmol)
was added, and the reaction mixture was stirred at room temperature
for 16 h. Ethanol (1.0 mL) was added, and the mixture was
concentrated under reduced pressure. The residue was dissolved in
CH2Cl2 (20 mL), and the mixture was washed with a saturated
aqueous solution of NaHCO3 (2 × 15 mL). The combined aqueous
phase was extracted with CH2Cl2 (2 × 15 mL), and the combined
organic phase was dried (Na2SO4), concentrated under reduced
pressure, and coevaporated with a mixture of toluene and EtOH (15
mL, 1:2, v/v). The residue was purified by column chromatography
(0−5% MeOH in CH2Cl2, v/v) to afford the product 9 (280 mg, 79%)
1
as a slightly yellow solid. Rf 0.5 (5% MeOH in CH2Cl2); H NMR
(400 MHz, DMSO-d6) δ 11.32 (br s, 1H, NH), 7.53 (d, 1H, J = 8.4
Hz, H6), 7.40−7.24 (m, 9H, Ar), 6.92−6.89 (m, 4H, Ar), 5.92 (d, 1H,
J = 8.0 Hz, H1′), 5.50 (d, 1H, J = 5.2 Hz, 3′−OH), 5.47 (d, 1H, J = 8.4
Hz, H5), 4.19 (m, 1H, H3′), 4.14 (m, 1H, H4′), 3.74 (s, 6H, OCH3),
3.25 (dd, 1H, J = 4.4, 10.0 Hz, H5′), 3.15 (dd, 1H, J = 4.4, 10.0 Hz,
H5′), 2.78 (t, 1H, J = 2.4 Hz, CCH), 2.48−2.41 (m, 2H, H2′, CH2),
2.23 (m, 1H, CH2); 13C NMR (100 MHz, DMSO-d6) δ 162.8 (C4),
158.0 (DMT), 150.6 (C2), 144.5 (C6), 140.2, 135.2, 135.1, 129.6,
130.2, 127.8, 127.5, 126.6, 113.1 (DMT), 101.8 (C5), 86.8 (C1′), 85.8
(CPh3), 84.9 (C4′), 81.7 (CCH), 71.5 (C3′), 71.4 (CCH), 63.7
(C5′), 54.9 (OCH3), 45.9 (C2′), 13.6 (CH2CCH); HRMS-ESI: m/
z: 591.2094 ([M + Na]+, C33H32N2O7Na+ calcd 591.2107).
5′-O-(4,4′-Dimethoxytrityl)-2′-(1-(pyren-1-yl)-1,2,3-triazole-
4-yl)methyl)-2′-deoxyuridine (10). Nucleoside 9 (100 mg, 0.17
mmol) was dissolved in THF:H2O:tBuOH (10 mL, 3:1:1, v/v/v). To
the solution was added an aqueous solution of sodium ascorbate (1 M,
0.6 mL, 0.6 mmol), an aqueous solution of CuSO4 (0.6 mL, 7.5% w/v,
0.17 mmol), and 1-azidopyrene (100 mg, 0.40 mmol). The mixture
5′-O-(4,4′-Dimethoxytrityl)-2′-(4-(pyren-1-yl)-1,2,3-triazole-
1-yl)-2′-deoxyuridine (6). Nucleoside 5 (102 mg, 0.18 mmol), 1-
ethynylpyrene (51 mg, 0.23 mmol), and TBTA (17 mg, 0.05 mmol)
were dissolved in a mixture of THF:H2O:pyridine (6:3:2, 1.7 mL), and
the solution was degassed under argon. A solution of sodium ascorbate
(14 mg, 0.07 mmol) and CuSO4·5H2O (9 mg, 0.04 mmol) in water
(0.25 mL) was added, and the reaction mixture was stirred at room
temperature for 24 h. The reaction mixture was diluted with CH2Cl2
(15 mL), and the mixture was washed with a saturated aqueous
solution of NaHCO3 (10 mL). The aqueous phase was extracted with
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Article
was stirred at room temperature for 2 h was and then diluted with
EtOAc (20 mL) and brine (20 mL). The phases were separated, and
the organic phase was washed with a saturated aqueous solution of
NaHCO3 (20 mL). The combined aqueous phase was extracted with
EtOAc (2 × 10 mL), and the combined organic phase was dried
(Na2SO4) and concentrated under reduced pressure. The residue was
purified by column chromatography (0−4% MeOH in CH2Cl2) to
afford the product 10 (90 mg, 63%) as a yellowish green solid. Rf 0.4
(80% EtOAc in petroleum ether); 1H NMR (400 MHz, CDCl3) δ 8.88
(br s, 1H, NH), 8.27 (d, J = 7.8 Hz, 1H, Ar), 8.24 (d, J = 8.2 Hz, 1H,
Ar), 8.21 (d, J = 7.8 Hz, 1H, Ar), 8.17 (d, J = 8.8 Hz, 1H, Ar), 8.11 (d,
J = 8.8 Hz, 1H, Ar), 8.10−8.03 (m, 2H, Ar), 8.02 (d, J = 8.0 Hz, 1H,
Ar).7.92 (s, 1H, H5-triazole), 7.77 (d, 1H, J = 8.0 Hz, Ar), 7.75 (d, 1H,
J = 8.0 Hz, Ar), 7.41−7.39 (m, 2H, Ar), 7.31−7.28 (m, 6H, Ar, H6),
7.23−7.20 (m, 2H, Ar, H6), 6.83 (d, 4H, J = 8.8 Hz, Ar), 6.25 (d, 1H, J
= 8.0 Hz, H1′), 5.85 (d, 1H, J = 8.8 Hz, H5), 4.64−4.61 (m, 2H, OH,
H3′), 4.29 (m, 1H, H4′), 3.75 (s, 6H, OCH3), 3.53−3.50 (m, 2H,
H5′), 3.35 (dd, 1H, J = 14.6, 10.0 Hz, CH2), 3.07 (dd, 1H, J = 14.6, 4.4
Hz, CH2), 2.72 (m, 1H, H2′); 13C NMR (100 MHz, CDCl3) δ 162.8
(C4), 158.7 (DMT), 150.6 (C2), 145.0, 144.4, 140.2, 135.4, 135.3,
132.3, 131.1, 130.5, 130.1, 130.0, 129.9, 129.0, 128.1, 128.0, 127.1,
126.9, 126.8, 126.5, 126.1, 126.0, 125.0, 124.8, 124.7, 124.1, 123.2,
120.7, 113.3 (DMT, pyrene, C6), 102.7 (C5), 88.2, 86.9 (C-Ph3, C1′),
85.6 (C4′), 73.1 (C3′), 64.0 (C5′), 55.2 (OCH3), 51.0 (C2′), 20.9
(CH2); HR ESI MS m/z 834.2902 ([M + Na]+, C49H41N5O7Na+ calcd
834.2898).
3′-O-(2-Cyanoethyl-N,N-diisopropylaminophosphinyl)-5′-O-
(4,4′-dimethoxytrityl)-2′-(1-(pyren-1-yl)-1,2,3-triazole-4-yl)-
methyl)-2′-deoxyuridine (11). Nucleoside 10 (180 mg, 0.22 mmol)
was coevaporated twice with anhydrous DCE and dissolved in
anhydrous CH2Cl2 (5.0 mL). N,N-Diisopropylethylamine (0.20 mL,
1.14 mmol) and 2-cyanoethyl-N,N-diisopropylamino chlorophosphite
(80 μL, 0.33 mmol) were added, and the reaction mixture was stirred
at room temperature for 1 h. Ethanol (0.2 mL) and CH2Cl2 (15 mL)
were added, and the resulting solution was washed with a saturated
aqueous solution of NaHCO3 (15 mL). The aqueous phase was
extracted with CH2Cl2 (2 × 10 mL), and the combined organic phases
were dried (Na2SO4) and concentrated under reduced pressure. The
residue was purified by column chromatography (0−2% MeOH in
CH2Cl2) to afford the product 11 (145 mg, 64%) as a light yellow
foam; Rf 0.50 (3% MeOH in CH2Cl2); 31P NMR (162 MHz, CDCl3)
δ 150.5, 149.3. HRMS-ESI m/z 1034.3953 ([M + Na]+
C58H58N7O8PNa+ calcd 1034.3977).
Oligonucleotide Synthesis. Oligonucleotide synthesis was
carried out on an automated DNA synthesizer following the
phosphoramidite approach. Synthesis of oligonucleotides K1−8,
L1−8, M1−8, KK, LL, MM, MK, LK, and KL were performed on a
0.2 μmol scale (CPG support) by using the modified nucleoside
phosphoramidites 4, 7, and 11 to introduce monomers K, L, and M,
respectively, 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 the
modified phosphoramidites, a prolonged coupling time of 20 min was
used. 1H-Tetrazole was used as activator. In general, coupling yields
for all 2-cyanoethyl phosphoramidites were >80%. The 5′-O-DMT-
ON oligonucleotides were removed from the solid support by
treatment with concentrated aqueous ammonia at 55 °C for 12 h.
The oligonucleotides were purified by reversed-phase HPLC on a C18
10 μm; 7.8 × 150 mm column + precolumn: C18 10 μm, 7.8 × 10
mm. Buffer A: 0.05 M triethyl ammonium acetate, pH 7.4. Buffer B:
75% MeCN/H2O (3:1, v/v). 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 80% aqueous acetic acid for 20
min and neutralized by addition of sodium acetate (3 M, 15 μL), and
then sodium perchlorate (5 M, 15 μL) was added followed by acetone
(1 mL). The pure oligonucleotides precipitated overnight at −20 °C.
The mixture was then placed in a centrifuge and subjected to 12 000
rpm for 10 min at 4 °C. The supernatant was removed and the pellet
washed with cold acetone (2 × 1 mL). The pellet was then dried for
30 min under reduced pressure and dissolved in pure water (1 mL),
and the concentration was measured as OD 260 nm. The purity and
constitution of the ON’s were confirmed by IC analysis and MALDI-
TOF MS [M − H]+, respectively (Table S1).
Thermal Denaturation Experiments. Samples were dissolved in
a medium salt buffer containing 2.5 mM Na2HPO4, 5 mM NaH2PO4,
100 mM NaCl, and 0.1 mM EDTA at pH = 7.0 with 1.0 μM
concentrations of the two complementary oligonucleotide sequences.
The increase in UV absorbance at 260 nm as a function of time was
recorded while the temperature was increased linearly from 10 °C to
80 °C at a rate of 1.0 °C/min by means of a Peltier temperature
programmer. The melting curves were found to be reversible. All
determinations are averages of at least duplicates within 0.5 °C. For
melting experiments with Mg2+ concentrations of 10 mM, MgCl2 (2.0
μL of a 5.0 M solution) was added to the samples.
Fluorescence Experiments. Fluorescence spectra (360−600 nm)
were recorded using quartz optical cells with a path length of 1.00 cm.
The samples from the thermal denaturation experiments were reused
for the fluorescence experiments. All samples were annealed at 75 °C
prior to measurements. Background spectra were recorded and
subtracted from all measurements. The spectra were recorded at 10
°C.
ASSOCIATED CONTENT
* Supporting Information
■
S
MALDI-TOF data for oligonucleotides. Further thermal
denaturation data and fluorescence spectra. Selected NMR
spectra. This material is available free of charge via the Internet
at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*Fax: +45 66158780. E-mail: pouln@sdu.dk.
■
Present Address
‡Department of Chemistry, University College, Kurukshetra
University 136119, India.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The project was supported by The Danish Research Agency’s
Programme for Young Researchers, Nucleic Acid Center and
The Danish National Research Foundation, The Danish
Councils for Independent Research | Technology and
Production Sciences (FTP) and Natural Science (FNU), and
The Villum Kann Rasmussen Foundation.
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