Pyrene intercalating nucleic acids with a carbon linker

Article abstract of DOI:10.1080/15257770.2011.567202

We have synthesized a carbon linker analogue of INA (oligonucleotides containing insertions of 1-O-(1-pyrenylmethyl)glycerol). Thermal stability studies showed an increase in melting temperature in favor of the carbon linker analogue. We also synthesized a carbon linker analogue with two pyrenes geminally attached. Fluorescence studies of this intercalating nucleic acid with the pyrene moieties inserted as a bulge showed formation of an excimer band. When a mismatch was introduced at the site of the intercalator, an excimer band was formed for the destabilized duplexes whereas an exciplex band was observed when the stability of the duplex was retained. Copyright Taylor and Francis Group, LLC.

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PYRENE INTERCALATING NUCLEIC ACIDS
WITH A CARBON LINKER
Michael E. Østergaard a , Michael C. Wamberg a & Erik B. Pedersen a
a Nucleic Acid Center , Department of Physics and Chemistry,
University of Southern Denmark , Odense M, Denmark
Published online: 11 Apr 2011.
To cite this article: Michael E. Østergaard , Michael C. Wamberg & Erik B. Pedersen (2011) PYRENE
INTERCALATING NUCLEIC ACIDS WITH A CARBON LINKER, Nucleosides, Nucleotides and Nucleic Acids,
30:3, 210-226, DOI: 10.1080/15257770.2011.567202
To link to this article: http://dx.doi.org/10.1080/15257770.2011.567202
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Nucleosides, Nucleotides and Nucleic Acids, 30:210–226, 2011
Copyright ꢀC Taylor and Francis Group, LLC
ISSN: 1525-7770 print / 1532-2335 online
DOI: 10.1080/15257770.2011.567202
PYRENE INTERCALATING NUCLEIC ACIDS WITH A CARBON LINKER
Michael E. Østergaard, Michael C. Wamberg, and Erik B. Pedersen
Nucleic Acid Center, Department of Physics and Chemistry, University of Southern Denmark,
Odense M, Denmark
We have synthesized a carbon linker analogue of INA (oligonucleotides containing insertions of
2
1-O-(1-pyrenylmethyl)glycerol). Thermal stability studies showed an increase in melting temperature
in favor of the carbon linker analogue. We also synthesized a carbon linker analogue with two
pyrenes geminally attached. Fluorescence studies of this intercalating nucleic acid with the pyrene
moieties inserted as a bulge showed formation of an excimer band. When a mismatch was introduced
at the site of the intercalator, an excimer band was formed for the destabilized duplexes whereas an
exciplex band was observed when the stability of the duplex was retained.
Keywords Intercalating nucleic acids; pyrene intercalators; INA
INTRODUCTION
The use of intercalating nucleic acids to increase the stability of duplexes
has recently gained attention.[1] One of the most promising candidates for
the intercalating moiety is pyrene because it has almost the same surface
area as a regular Watson-Crick base pair, which makes a large stabilization
through base stacking with neighboring base pairs.[2] Another advantage is
that pyrene is a fluorescent molecule and if two pyrene residues come close
[3]
˚
together (3–5 A) the formation of an excimer band is possible.
When 1-O-(1-pyrenylmethyl)glycerol is inserted as a bulge into DNA
oligonucleotides (INA), a significant discrimination between DNA and RNA
is achieved,[4] as the DNA duplex is stabilized while the RNA duplex is desta-
bilized. Previously we have examined the stereochemistry of INA, showing
the R-isomer being better than the S-isomer.[5] We have also investigated
Received 30 September 2010; accepted 25 February 2011.
Dr. Vyacheslav Filichev is acknowledged for the preparation of ODN7.
The Nucleic Acid Center is a research center funded by The Danish National Research Foundation
for studies on nucleic acid chemical biology.
Address correspondence to Erik B. Pedersen, Nucleic Acid Center, Department of Physics and
Chemistry, University of Southern Denmark, Campusvej 55, DK-5230, Odense M, Denmark. E-mail:
ebp@ifk.sdu.dk
210

Pyrene Intercalating Nucleic Acids
211
FIGURE 1 Carbon linker analogues 1 and 2 of INA 3.
the influence of the linker length, showing that a longer linker will decrease
the stabilization of the duplex.[6]
In this article, we present a carbon linker analogue 1 to INA (3) and
an intercalating nucleic acid 2 having two pyrene residues attached via the
same carbon linker (Figure 1). The only difference between 1 and 3 is
the substitution of an oxygen atom with a methylene group. The methylene
group is often considered a bioisoster for oxygen, and we want to examine
how this substitution can affect duplex stability of the intercalating nucleic
acid.
Furthermore, we have investigated the behavior of 2 with two pyrenes in
close proximity of the intercalating site. There have been other reports about
synthetic oligonucleotides possessing two pyrenes,[7–9] but none with bond-
ing of two pyrenes to the same carbon atom where this has been reported
for masked polyols.[10, 11] The use of two fluorophores can be used to detect
single nucleotide polymorhism (SNP). Yamane et al.[12] used pyrene as a
quencer and fluorescein as a fluorophore to detect a mismatched base pair,
while Dioubankova et al.[13] and Christensen and Pedersen[14] have used
two pyrenes to detect point mutations. In this study we have investigated the
capability of bispyrene-labeled compound 2 to detect mutations.
RESULTS AND DISCUSSION
Enantiomerically pure (S)-γ -benzyloxymethyl-γ -butyrolactone (4) was
obtained from L-glutamic acid in four steps according to Taniguchi
et al.[15] L-glutamic acid was converted to (S)-γ -carboxy-γ -butyrolactone,
which was esterificated by reaction with EtOH using TsOH in cat-
alytic amount. The ester was reduced with NaBH4 in EtOH to (S)-γ -
hydroxymethyl-γ -butyrolactone which was O-benzyl protected using benzyl
bromide and Ag2O in DMF to obtain 4. The yield for the last reaction was
raised slightly in our hands by using NaH and microwave conditions instead
of Ag2O (Scheme 1).
1-Bromopyrene was synthesized from pyrene according to Lock[16] and
converted to pyren-1-yllithium using n-BuLi. Reaction of pyrenyllithium

212
M. E. Østergaard et al.
SCHEME 1 Reagents and conditions: i: a) HCl(Conc.), NaNO2, H2O; ii: EtOH, TsOH, 57%; iii: a) NaBH4,
EtOH, 64%, b) NaH, DMF, MW, 62%.
reagent with (S)-γ -benzyloxymethyl-γ -butyrolactone (4) gave a lactole,
which, without further purification, was reduced using boron trifluoride
etherate and triethylsilane to (S)-1-benzyloxy-5-pyren-1-ylpentan-2-ol (5) in
35% overall yield (Scheme 2). To prevent pyren-1-yllithium from reacting
twice with the same molecule, it was added slowly to (S)-γ -benzyloxymethyl-
γ -butyrolactone (4). The O-benzyl protection group at (S)-1-benzyloxy-5-
pyren-1-ylpentan-2-ol (5) was removed by catalytic hydrogenation using pal-
ladium on charcoal to give (S)-5-pyren-1-ylpentan-1,2-diol (1) in 22% yield.
Unfortunately, it could also partly reduce the pyrene which explains the
low yield. The diol was made ready for oligonucleotide synthesis using stan-
dard chemistry. The primary alcohol was protected using 4,4ꢁ-dimethoxytrityl
chloride in dry pyridine to give the O-5-(4,4ꢁ-dimethoxytrityl) protected com-
pound 6 in 63% yield. Finally, the secondary alcohol was phosphitylated
using 2-cyanoethyl-N ,N ,N ꢁ,N ꢁ-tetraisopropylphosphordiamidite to give 7 in
82% yield.
To add two pyrene residues to the same carbon atom, pyren-1-yllithium
was prepared as described above and then (S)-γ -benzyloxymethyl-γ -
butyrolactone (4) was added to give (S)-5-benzyloxy-1,1-dipyren-1-ylpentan-
1,4-diol (8) in 56% yield. This compound was reduced with boron trifluoride
etherate and triethylsilane to give 9 in 55% yield and deprotected with cat-
alytic hydrogenation giving (S)-5,5-dipyren-1-ylpentan-1,2-diol (2) in 56%
.
SCHEME 2 Reagents and conditions: i: a) 1-Bromopyrene, n-BuLi, THF, added slowly to 4, b) BF3 Et2O,
Et3SiH, CH2Cl2, 35%; ii: H2, Pd on charcoal, CH2Cl2:MeOH (1:9), 22%; iii: DMT-Cl, pyridine, 63%; iv:
P(NPri2)2O(CH2)2CN, diisopropylamine tetrazolide, CH2Cl2, 82%.

Pyrene Intercalating Nucleic Acids
213
.
SCHEME 3 Reagents and conditions: i: 1-Bromopyrene, n-BuLi, THF, 56%; ii. BF3 Et2O, Et3SiH,
CH2Cl2, 55%; iii: H2, Pd on charcoal, CH2Cl2:MeOH (1:9), 22%; iv: DMT-Cl, pyridine, 60%; v:
P(NPri2)2O(CH2)2CN, diisopropylamine tetrazolide, CH2Cl2, 80%.
yield. This diol was protected with 4,4ꢁ-dimethoxytrityl chloride in 60% yield
and phosphitylated to give 11 in 80% yield (Scheme 3).
THERMAL MELTING STUDIES
Seven different oligonucleotides were synthesized, four incorporating 1
and three incorporating 2. The sequence was chosen in order to compare
the properties of 1 with 3. The latter has earlier been incorporated into
a highly conserved HIV-1 long terminal repeat region which is the same
sequence as used herein.[4,17]
DNA duplex stability was increased by 3–4◦C when 1 was incorporated as
a bulge (Tables 1 and 2), and when two incorporations were inserted four
nucleotides apart, a stabilization of 15.3◦C was observed. This means that
there is a cooperative stabilization because otherwise only a stabilization of
6–8◦C would be observed. However, if two incorporations were made too
close to its other, this cooperative stabilization was lost (Table 1, ODN 3).
The pyrene intercalator showed discrimination between DNA and RNA,
as 1 in all cases destabilized the RNA duplexes (Table 1). These results

214
M. E. Østergaard et al.
TABLE 1 Melting temperatures of duplexes with monomers 1–3 inserted as a bulge and theoretical
and experimental masses
DNA
RNA
5ꢁ-AGCTTGCTTGAG-3ꢁ
5ꢁ-AGCUUGCUUGAG-3ꢁ
Mass
ODN
Sequence
Tm/◦C
ꢀTm
Tm/◦C
ꢀTm
Calc. Found
1
2
3
4
5ꢁ-CTCAAGCAAGCT-3ꢁ
5ꢁ-CTCAAG1CAAGCT-3ꢁ
5ꢁ-CTCAA1G1CAAGCT-3ꢁ
5ꢁ-CTCA1AGCA1AGCT-3ꢁ
5ꢁ-CTCAAG2CAAGCT-3ꢁ
5ꢁ-2CTCAAGCAAGCT-3ꢁ
5ꢁ-CTCAAG3CAAGCT-3ꢁ
47.4
51.7
53.6
62.7
39.7
56.3
50.1
—
42.4
37.9
35.5
34.6
30.6
45.9
37.8
—
—
—
+4.3
+6.2
+15.3
−7.7
+8.9
+2.7
−4.5
−6.9
−7.8
−11.8
+3.5
−4.6
3979 3979
4344 4345
4344 4345
4179 4178
4179 4180
5
6
74
show that 1 can only make optimal base stacking in a B-type helix. Earlier
reported results show that 3 (INA) has the same properties as 1 and desta-
bilizes a RNA duplex to the same extent,[4] but the stabilization of a DNA
duplex is lower (+2.7◦C) compared to 1 (+4.3◦C). The reason for this dif-
ference could be the slight change in the dihedral angle from a C-O group
to a methylene group and/or because of a conformational preference of the
methylene group. It is known that 5-alkyl-1,3-dioxanes have lower conforma-
tional preferences than the corresponding cycloalkanes.[18] Apparently, the
substitution of the oxygen atom with a less electronegative methylene group
does not give a negative stabilization effect in the aqueous surroundings of
the DNA strand.
Shifting the intercalator to the complementary strand (Table 2, ODNs
8–9) gave a slightly lower stabilization for 1 and a higher destabilization for
2. For 2 the change in ꢀTm was from −7.7◦C to −10.3◦C.
Furthermore we have investigated the pyrene intercalator as a universal
base. Results are presented in Table 3. Positions 7 in both strands were
varied with all four natural bases, 1, and 2. INA (3) was inserted in one of the
strands for comparison. Incorporation of the pyrene intercalators 1–3 in the
13-mer duplex resulted in a lowering of the melting temperature (Tm) no
matter the opposite base compared to the matched base pair. However, the
TABLE 2 Melting temperatures of duplexes with the intercalator shifted to the complementary stranda
Mass
ODN
Sequence
Tm/◦C
ꢀTm
Calc.
Found
8
9
5ꢁ-AGCTTG1CTTGAG-3ꢁ
5ꢁ-AGCTTG2CTTGAG-3ꢁ
50.8
37.1
+3.4
4040
4241
4040
4241
−10.3
aThe target sequence was 3ꢁ-TCGAACGAACTC-5ꢁ and the reference melting temperature for wildtype
duplex without insertions was 47.4◦C.

Pyrene Intercalating Nucleic Acids
215
TABLE 3 Melting temperatures of duplexes where a natural nucleotide was replaced either with
another natural nucleotide or with monomers 1–3. The melting temperature of 3 versus 3 was taken
from the literature[5]
5ꢁ-AGCTTGXCTTGAG-3ꢁ
3ꢁ-TCGAACYGAACTC-5ꢁ
X\Y
A
C
G
T
1
2
3
A
C
G
T
1
39.4
35.5
45.9
48.9
42.0
34.8
37.4
23.6
52.4
32.5
44.2
36.1
45.8
54.3
46.7
36.1
41.4
33.7
50.5
35.1
43.1
35.8
42.9
33.8
46.8
47.3
44.1
46.4
44.0
36.4
41.0
41.1
37.4
40.4
37.0
34.8
43.1
44.1
41.5
43.1
2
3
43.6
intercalators have a universal base effect as the Tm varied only 3.2◦C for 1 and
3.6◦C for 2. For INA (3), the narrow range is 2.6◦C. Shifting the intercalator
to the opposite strand resulted in a more narrow range. Not surprisingly, the
highest Tm is achieved when the intercalator is placed opposite a cytosine
and the lowest Tm when the intercalator is facing a guanosine.
Previously we have shown that two intercalating pyrene moieties inserted
in complementary strands directly opposite each other will lead to destabi-
lization of the duplex.[5,19] This was also the case in this study. While destabi-
lizing, the carbon analogue 1 gave a marginally higher thermal melting than
is reported for INA.[5] When 2 was inserted opposite either 1 or 2, it lead to
a further destabilization.
From Table 1, can be seen that 2 is actually destabilizing the DNA du-
plex by −7.7◦C, when inserted as a bulge in the middle of the sequence.
Nearly the same value was obtained when ethylene glycol was inserted in-
stead of an intercalator.[4] This might indicate that the two pyrene moieties
do not intercalate, probably due to steric crowding of the geminal bonded
pyrenes. For the RNA duplex, the destabilization is −11.8◦C, which implies
a further disturbance of the duplex structure than when ethylene glycol was
inserted as a bulge.[4] When 2 was inserted as an end-positioned intercalating
pseudonucleotide (ODN 6), a stabilization of both DNA and RNA duplexes
was achieved due to the lid effect. For further examination on whether 2 is
intercalating or not, we carried out fluorescence experiments on ODN5.
FLUORESCENCE STUDIES
While 1 gave standard pyrene fluorescence emission spectra, 2 gave rise
to an excimer band with a maximum at 480 nm, when ODN5 was hybridized
to the complementary strand, upon excitation at 340 nm. The single strand
(Figure 2A, ODN5) led to the formation of a band with a maximum at 445
nm. Yamana et al.[20] have made similar observations of band formation

216
M. E. Østergaard et al.
FIGURE 2 Fluorescence emission spectra of (A and B) ODN5 hybridized with mutant 1–6 and wildtype
strands; (C) single stranded ODN5 and hybridized with mutant 5 at increasing temperatures; and (D)
ODN6 hybridized with mutant 7–9 and with wildtype strands.
at 450 nm, when studying fluorescence of oligonucleotides containing 2ꢁ-
O-(1-pyrenylmethyl)uridine. They ascribe this fluorescence emission to an
exciplex of the pyrene and an adjacent base in the duplex. In our case, the
greater flexibility of the nucleobases in the single strand might allow these to
come in close proximity of one of the pyrenes, which leads to the formation
of the exciplex band. For the hybridized ODN5, the destabilization implies
that the pyrenes do not intercalate. However, base paring still occurs, which
might leave the pyrenes outside the duplex and unable to interact with the
nucleobases. Instead the excimer band formation is seen.
To investigate this further, a study of mismatched sequences was per-
formed. The thermal melting studies of these sequences can be seen in
Table 4. When the mismatch was introduced at the 3ꢁ position of the inter-
calator, the duplex stability was not altered much, while a destabilization of
4–6◦C was seen, when the mismatch was at the 5ꢁ position of the intercala-
tor. Fluorescence spectra of these sequences revealed an exciplex band with
maximum at 445 nm for mismatch at the 3ꢁ–site of the intercalator (mutant
1–3) and an excimer band maximum at 480 nm for the sequences with the
mismatch at the 5ꢁ–site of the intercalator (mutant 4–6) (Figures 2A and
4B).
As no destabilization was observed for the mutant 1–3 even though a
mismatch was introduced, the pyrene must interact with the duplex and it
is likely to believe it occurs through intercalation. Hereby one of the pyrene

Pyrene Intercalating Nucleic Acids
217
TABLE 4 Tm measurements on mismatches toward ODN5
5ꢁ-AGCTTX–YTTGAG-3ꢁ
3ꢁ-TCGAAC2GAACTC-5ꢁ
Target
X
Y
Tm/◦C
ꢀTm
Wildtype
Mutant1
Mutant2
Mutant3
Mutant4
Mutant5
Mutant6
G
C
A
T
G
G
G
C
C
C
C
G
A
T
39.7
43.6
38.3
40.2
35.4
33.5
32.9
–
+3.9
−1.4
+0.5
−4.3
−6.2
−6.8
moieties would be close to a nucleobase, and exciplex band formation is
seen. This might not be the case, when the mismatch is on the 5ꢁ-site of
the intercalator. The stability of the duplex is lower, which implies less
interaction from/of the pyrenes with the nucleobases. The pyrenes are more
free and therefore in positions to form excimer band, which we expect to
happen outside the duplex. As no interaction with the nucleobases is possible
from there, the possibility of exciplex band formation is ruled out. We have
made examples with other sequences for mismatch studies which all resulted
in lower duplex stabilities and in all those cases an excimer band was observed
in the fluorescence spectrum (data not shown).
A molecular modelling study confirms the possibility of these duplexes
(Figure 3). Despite the bulkiness of the intercalating pyrene moieties, there
FIGURE 3 Molecular modelling of ODN5 with mutant 2 (left) showing the possible structure of inter-
calation and ODN5 with wildtype (right) showing the possible structure of minor groove binding.

218
M. E. Østergaard et al.
TABLE 5 Melting temperatures of mismatched duplexes with 2 inserted as an
end-positioned intercalating pseudonucleotide (ODN6); the reference melting
temperature for the wildtype duplex without insertions was 46.6◦C
5ꢁ-AGCTTGCTTGAXTTTT-3ꢁ
3ꢁ-TCGAACGAACTC2–5ꢁ
Target
X
Tm
ꢀTm
Wildtype
Mutant7
Mutant8
Mutant9
G
C
A
T
60.0
53.2
52.3
52.9
+13.4
+6.6
+5.7
+6.3
is room for it inside the duplex with no major disturbance of the base pairing.
The right side of Figure 3 shows the duplex with two pyrenes in the minor
groove.
Fluorescence experiments were carried out at 20◦C. Control experiments
at 10◦C and 30◦C, respectively, did not show any difference. First, when the
Tm was exceeded, did the excimer band profile shift toward the profile of
the single strand (Figure 2C).
Finally, a mismatch study with ODN6 was performed. As described
above, stabilization was achieved when the intercalator inserted as an end-
positioned pseudonucleotide. The target sequence was extended with four
thymine bases and a mismatch was introduced at the 3ꢁ–site of the interca-
lator (Table 5), and this resulted in a lower stabilization than observed for
the matched oligonucleotide.
Fluorescence studies of ODN6 revealed exciplex band formation (Figure
2D). As the intensity is the same in all four cases, the intercalator 2 must be
placed in the same position in the duplex in all four cases. Hybridized to the
complementary DNA or RNA, ODN6 gives the same fluorescence spectre.
CONCLUSION
We have synthesized a carbon linker analogue to INA and evaluated
the duplex thermal stability which showed an improvement in favor of the
carbon linker analogue toward DNA duplexes, while no significant change
was observed for RNA duplexes. For an intercalating nucleic acid containing
a bulge insertion of two pyrene moieties in a geminal position, we observed
in the fluorescence spectra an excimer band whereas for a mismatched
duplex an exciplex band was observed when the duplex was not substantially
destabilized.
EXPERIMENTAL
NMR spectra were recorded on a Varian Gemini 2000 NMR spectrometer
at 300 MHz for 1H, 75 MHz for 13C and 121.5 MHz for 31P. δ values are in ppm

Pyrene Intercalating Nucleic Acids
219
with tetramethylsilane as internal standard for 1H NMR, deuterated solvents
(CDCl3 (δ77.00), DMSO (δ39.44)) for 13C NMR, and 85% H3PO4 as external
standard for 31P NMR. Accurate ion mass determinations were performed
using an Ionspec 4.7 T Ultima Fourier transform mass spectrometer (Irvine,
CA, USA). The M+ or [M+Na]+ ions were peak matched using ions derived
from the 2,5-dihydroxybenzoic acid matrix. Silica gel (0.040–0.063 mm) used
for column chromatography and analytical silica gel TLC plates 60 F254 were
purchased from Merck (Darmstadt, Germany). Ultraviolet (UV)-light or a
stain of (NH4)6Mo7O24·4H2O/Ce2(SO4)3 (50:1) in 5% sulfuric acid were
used for visualization. Reagents were used as purchased.
Preparation of (S)-1-(benzyloxy)-5-(pyren-1-yl)pentan-2-ol (5)
1-Bromopyrene (3.05 g, 10.7 mmol) was dissolved in anhydrous THF
(70 mL) under N2 and the temperature was lowered to −78◦C. 2.4 M n-BuLi
in hexane (5.13 mL, 12.3 mmol) was added within 20 minutes. In another
flask (S)-γ -benzyloxymethyl-γ -butyrolactone[15] (4) (2.00 g, 9.70 mmol) was
dissolved in anhydrous THF (20 mL) under N2 and the temperature is
lowered to −78◦C. After 30 minutes, the lithium reagent was cannulated
slowly to the lactone over 2 hours and the temperature was raised to −30◦C
over 2 hours. The reaction was quenched by addition of water (70 mL),
the mixture was extracted with Et2O (5 × 50 mL), and the organic phase
dried (MgSO4) and evaporated in vacuo to afford an oil. The residue was
dissolved in anhydrous CH2Cl2 (70 mL) and treated at −78◦C with BF3·Et2O
(2.54 mL, 24.3 mmol) and Et3SiH (3.36 mL, 24.3 mmol) and the mixture
was stirred overnight while the temperature was allowed to reach room
temperature. The reaction was quenched by addition of saturated aqueous
NaHCO3 solution (80 mL), the mixture was extracted with CH2Cl2 (3 ×
30 mL), and the organic phase dried (MgSO4) and evaporated in vacuo.
The residue was purified by silica gel column chromatography (petroleum
ether/EtOAc, 4:1). The product was obtained as a yellow foam (1.35 g, 35%).
1
Rf = 0.24 (petroleum ether/EtOAc, 3:1). H NMR (300 MHz, CDCl3) δ
1.44–1.64 (2H, m, H3), 1.76–2.03 (2H, m, H4), 2.43 (1H, brs, OH), 3.20–3.40
(4H, m, H1, H5), 3.78–3.83 (1H, m, H2), 4.43 (2H, s, OCH2Ph), 7.20–7.28
(5H, m, phenyl), 7.75–8.21 (m, 9H, pyrene). 13C NMR (75 MHz, CDCl3) δ
27.63 (C4), 32.94 (C5), 33.29 (C3), 70.21 (C1), 73.21 (OCH2Ph), 74.48 (C2),
122.85, 123.33, 124.58, 124.69, 124.73, 124.94, 125.69, 126.47, 127.12, 127.15,
127.43, 127.63, 127.69, 128.37, 128.54, 129.70, 130.82, 131.33, 136.52, 137.86
(Ar). HRMS (MALDI): m/z calcd for C28H26O2Na+ (MNa+): 417.1825, found
417.1818.
Preparation of (S)-5-(benzyloxy)-1,1-(dipyren-1-yl)pentan-1,4-diol
(8)
1-Bromopyrene (3.05 g, 10.7 mmol) was dissolved in anhydrous THF
(25 mL) under N2 and the temperature was lowered to −78◦C. 2.4 M n-BuLi

220
M. E. Østergaard et al.
in hexane (5.13 mL, 12.3 mmol) was added within 20 minutes. After
20 minutes, a solution of (S)-γ -benzyloxymethyl-γ -butyrolactone (4) (1.10
g, 5.33 mmol) dissolved in anhydrous THF (15 mL) under N2, was added
over 30 minutes and the mixture was stirred for an additional hour before the
temperature was raised −30◦C within 2 hours. The reaction was quenched by
addition of H2O (50 mL), the mixture was extracted with Et2O (3 × 40 mL),
and the organic phase was dried (MgSO4) and evaporated in vacuo. The
residue was purified by silica gel column chromatography (petroleum
ether/EtOAc, 2:1) and the product was obtained as a yellow foam (1.81
1
g, 56%). Rf = 0.47 (petroleum ether/EtOAc, 1:1). H NMR (300 MHz,
CDCl3) δ 1.25–1.55 (2H, m, H3), 2.77 (1H, brs, OH), 2.96–3.20 (4H, m, H2,
H5), 3.70–3.75 (1H, m, H4), 3.81 (1H, brs, OH), 4.25 (2H, s, OCH2Ph),
7.08–7.20 (5H, m, phenyl), 7.49–7.52, 7.73–7.82, 7.91–8.00, 8.14–8.35,
8.58–8.65 (18H, m, pyrene). 13C NMR (75 MHz, CDCl3) δ 27.88 (C3),
39.41 (C2), 70.67 (C5), 73.04 (OCH2Ph), 74.29 (C4), 79.86 (C1), 124.30,
124.59, 124.68, 124.70, 124.77, 124.79, 124.92, 125.11, 125.31, 125.68,
125.80, 125.85, 126.52, 127.24, 127.28, 127.32, 127.38, 127.63, 128.30,
130.11, 130.15, 130.80, 130.87, 131.14, 137.73, 140.96, 141.06 (Ar). HRMS
(MALDI): m/z calcd for C44H34O3Na+ (MNa+): 633.2400, found 633.2411.
Preparation of (S)-1-(benzyloxy)-5,5-(dipyren-1-yl)pentan-2-ol (9)
To a solution of (S)-5-(benzyloxy)-1,1-di(pyren-1-yl)pentan-1,4-diol (8)
(1.50 g, 2.45 mmol) in CH2Cl2 (20 mL) at −78◦C under N2 was added
BF3·Et2O (0.27 mL, 2.70 mmol) and Et3SiH (0.35 mL, 2.70 mmol). The
mixture was stirred overnight while the temperature was allowed to reach
room temperature. The reaction was quenched by addition of saturated
aqueous NaHCO3 solution (30 mL), the mixture was extracted with CH2Cl2
(3 × 15 mL), and the organic phase dried (MgSO4) and evaporated in vacuo.
The residue was purified by silica gel column chromatography (petroleum
ether/EtOAc, 3:1) and the product was obtained as a yellow foam (0.801 g,
55%). Rf = 0.22 (petroleum ether/EtOAc, 3:1). 1H NMR (300 MHz, CDCl3)
δ 1.22–1.26 (2H, m, H3), 2.44–2.57 (1H, m, H4), 2.73–2.76 (1H, m, H4),
3.19–3.25 (1H, m, H1), 3.34–3.37 (1H, m, H1), 3.89 (1H, brs, H2), 4.40 (2H,
s, OCH2Ph), 6.13 (1H, t, J 7.4, H5), 7.20–7.23 (5H, m, phenyl), 7.86–8.09,
8.39–8.43, 8.50–8.53 (18H, m, pyrene). 13C NMR (75 MHz, CDCl3) δ 31.69
(C3), 32.78 (C4), 41.88 (C5), 70.43 (C2), 73.22 (OCH2Ph), 74.46 (C1),
122.87, 124.80, 124.90, 124.94, 125.04, 125.17, 125.21, 125.36, 125.39, 125.77,
126.84, 127.41, 127.64, 127.68, 127.84, 127.89, 128.35, 128.69, 128.87, 129.76,
129.81, 130.64, 130.69, 131.31, 131.34, 137.80, 138.60, 139.17 (Ar). HRMS
(MALDI): m/z calcd for C44H34O2Na+ (MNa+): 617.2451, found 617.2443.
General Procedure for the Deprotection of a Benzyl Ether
The benzyl ether was dissolved in a mixture of CH2Cl2 and methanol
(1:9 v/v) and Pd on charcoal (100 mg pr. 1 mmol) was added. N2 was let

Pyrene Intercalating Nucleic Acids
221
through the solution for 30 minutes, and then the reaction was evacuated
twice and a hydrogen atmosphere at 1 atm was established. The catalyst was
filtered off after 48 hours and washed with EtOAc. The organic phase was
evaporated and the residue purified by silica gel column chromatography.
Preparation of (S)-5-(pyren-1-yl)pentan-1,2-diol (1)
From (S)-1-(benzyloxy)-5-(pyren-1-yl)pentan-2-ol (5) (1.03 g, 2.61
mmol) the product was obtained as a light yellow foam (178 mg, 22%).
1
Rf = 0.14 (petroleum ether/EtOAc, 2:1). H NMR (300 MHz, DMSO-d6)
δ 1.42–1.45, 1.63–1.65 (2H, m, H3), 1.83–1.97 (2H, m, H4), 2.52 (1H,
brs, OH), 3.22 (1H, brs, OH), 3.31–3.34 (4H, m, H1, H5), 3.54 (1H, m,
H2), 7.93–8.39 (9H, m, pyrene). 13C NMR (75 MHz, DMSO-d6) δ 27.77
(C4), 32.79, 33.32 (C3, C5), 65.99 (C1), 70.97 (C2), 123.47, 124.12, 124.17,
124.63, 124.78, 124.80, 125.99, 126.31, 127.03, 127.37, 127.39, 127.99, 129.09,
130.35, 130.82, 137.13 (Ar). HRMS (MALDI): m/z calcd for C21H20O2 (M+):
304.1458, found 304.1461.
Preparation of (S)-5,5-di(pyren-1-yl)pentan-1,2-diol (2)
From (S)-1-(benzyloxy)-5,5-di(pyren-1-yl)pentan-2-ol (8) (1.83 g, 3.07
mmol) the product was obtained as a light yellow foam (345 mg, 22%). Rf =
1
0.18 (petroleum ether/EtOAc, 1:1). H NMR (300 MHz, DMSO-d6) δ
1.53–1.63, 1.74–1.84 (2H, m, H3), 2.52 (1H, brs, OH), 2.52–2.78 (2H, m,
H4), 3.20 (1H, brs, OH), 3.23–3.35 (2H, m, H1), 3.58–3.63 (1H, m, H2),
6.28 (1H, t, J = 6.8 Hz, H5), 8.00–8.27, 8.62–8.73 (18H, m, pyrene). 13C
NMR (75 MHz, DMSO-d6) δ 31.99 (C3), 32.88 (C4), 41.21 (C5), 65.95 (C1),
71.29 (C2), 122.98, 124.03, 124.27, 124.84, 125.00, 125.13, 125.49, 125.58,
126.12, 126.75, 127.31, 127.68, 128.10, 128.21, 129.12, 129.15, 130.14, 130.17,
130.81, 139.28, 139.67 (Ar). HRMS (MALDI): m/z calcd for C37H28O2Na+
(MNa+): 527.1982, found 527.2004.
General Procedure for the DMT Protection of Alcohols
The alcohol was dissolved in anhydrous pyridine under N2. DMT-Cl was
added and the mixture was stirred for 4 hours. The solvent was evaporated
in vacuo and co-evaporated with anhydrous xylene and dried on a desiccator
overnight. The residue was purified by silica gel column chromatography.
Preparation of (S)-1-[bis(4-methoxyphenyl)phenylmethoxy]-5-
(pyren-1-yl)pentan-2-ol (6)
From (S)-5-(pyren-1-yl)pentan-1,2-diol (1) (178 mg, 0.58 mmol) the
product was obtained as a light yellow foam (170 mg, 63%). Rf = 0.44
1
(petroleum ether/EtOAc, 2:1). H NMR (300 MHz, CDCl3) δ 1.53–1.63,

222
M. E. Østergaard et al.
1.77–1.87, 1.93–2.03 (4H, m, H3, H4), 2.51 (1H, brs, OH), 2.97–3,04,
3.13–3.16 (2H, m, H1), 3.30 (2H, t, J = 7.7 Hz, H5), 3.72 (6H, s, 2 ×
OCH3), 3.83 (1H, m, H2), 6.75–6.78, 7.15–7.29, 7.39–7.41 (13H, m, phenyl),
7.78–8.07, 7.92–8.23 (9H, m, pyrene). 13C NMR (75 MHz, CDCl3) δ 27.68
(C4), 33.25, 33.33 (C3, C5), 55.13, 55.14 (2 × OCH3), 67.44 (C1), 70.83
(C2), 86.03 (CPh3), 113.08, 123.40, 124.62, 124.72, 124.76, 124.99, 125.02,
125.73, 126.51, 126.76, 127.15, 127.24, 127.47, 127.79, 128.07, 128.59, 129.73,
129.99, 130.88, 131.39, 135.91, 135.98, 136.64, 144.80 158.43 (Ar). HRMS
(MALDI): m/z calcd for C42H38O4Na+ (MNa+): 629.2662, found 629.2675.
Preparation of (S)-1-[bis(4-methoxyphenyl)phenylmethoxy]-5,5-di
(pyren-1-yl)pentan-2-ol (10)
From (S)-5,5-di(pyren-1-yl)pentan-1,2-diol (2) (315 mg, 0.62 mmol) the
product was obtained as a light yellow foam (304 mg, 60%). Rf = 0.44
1
(petroleum ether/EtOAc, 2:1). H NMR (300 MHz, CDCl3) δ 1.72–1.77
(2H, m, H3), 2.37 (1H, brs, OH), 2.46–2.74 (2H, m, H4), 2.97–3.14 (2H,
m, H1), 3.65 (6H, s, 2 × OCH3), 3.87–3.94 (1H, m, H2), 6.13 (1H, t, J
= 7.5 Hz, H5), 6.67–6.70, 7.14–7.37 (13H, m, phenyl), 7.85–8.12, 8.39–8.53
(18H, m, pyrene). 13C NMR (75 MHz, CDCl3) δ 32.14 (C3), 32.78 (C4), 41.92
(C5), 55.06, 55.08 (2 × OCH3), 67.25 (C2), 71.07 (C1), 85.98 (CPh3), 113.03,
122.90, 124.83, 124.92, 125.04, 125.17, 125.21, 125.42, 125.80, 126.73, 126.85,
127.44, 127.77, 127.85, 127.90, 128.04, 128.69, 128.86, 129.75, 129.81, 129.96,
130.67, 130.72, 131.34, 131.36, 135.80, 135.88, 138.68, 139.20, 144.75, 158.38
(Ar). HRMS (MALDI): m/z calcd for C58H46O4Na+ (MNa+): 829.3288, found
829.3301.
General Procedure for the Synthesis of a Phosphoramidite
The DMT protected alcohol was dissolved in anhydrous CH2Cl2 under N2
and N ,N -diisopropylammoniumtetrazolide (2.1 eqiv.) was added. Then 2-
cyanoethyl-N ,N ,N ꢁ,N ꢁ-tetraisopropylphosphoramidite (2.2 eqiv.) was added
dropwise and the mixture was stirred for 24 hours. The reaction was
quenched by addition of water (1 mL). The amount of CH2Cl2 solvent was
doubled and washed twice with saturated NaHCO3 solution, dried (MgSO4)
and the solvent evaporated in vacuo. The product was purified by silica gel
column chromatography.
Preparation of the Phosphoramidite of (S)-1-[bis(4-methoxyphenyl)
phenylmethoxy]-5-(pyren-1-yl)pentan-2-ol (7)
From (S)-1-[bis(4-methoxyphenyl)phenylmethoxy]-5-(pyren-1-yl)pen-
tan-2-ol (6) (170 mg, 0.28 mmol) the product was obtained as a white foam
(182 mg, 82%). Rf = 0.47 (petroleum ether/EtOAc, 2:1 and traces of Et3N).
1H NMR (300 MHz, CDCl3) δ 0.99–1.14 (14H, m, 2 × CH(CH3)2), 1.77–1.91

Pyrene Intercalating Nucleic Acids
223
(4H, m, H3, H4), 2.34 (2H, t, J = 6.6 Hz, OCH2CH2CN), 2.94–3.18
(2H, m, H1), 3.29–3.36 (2H, m, H5), 3.44–3.55 (2H, m, OCH2CH2CN),
3.70 (6H, s, 2 × OCH3), 3.99–4.06 (1H, m, H2), 6.70–6.75, 7.15–7.29,
7.39–7.43 (13H, m, phenyl), 7.79–8.27 (9H, m, pyrene). 13C NMR (75
MHz, CDCl3) δ 20.11, 20.19 (OCH2CH2CN), 24.37, 24.39, 24.46, 24.48,
24.54, 24.57, 24.64, 24.66 (2 × CH(CH3)2), 26.79, 27.11 (C4), 33.27, 33.34
(C3, C5), 42.86, 43.03, 43.15 (2 × CH(CH3)2), 55.07 (2 × OCH3), 57.88,
58.14 (OCH2CH2CN), 72.89, 73.09 (C1), 73.77, 74.00 (C2), 85.77, 85.82
(CPh3), 112.91 (Ar-C), 117.60 (CN), 123.42, 123.56, 124.59, 124.71, 124.96,
125.03, 125.70, 125.73, 126.47, 126.54, 126.60, 127.03, 127.07, 127.22,
127.39, 127.47, 127.63, 128.11, 128.18, 128.57, 129.67, 129.98, 130.02,
130.06, 130.85, 131.36, 136.11, 136.22, 136.63, 136.87, 144.99, 158.27, 158.30
(Ar). 31P NMR (121.5 MHz, CDCl3) δ 149.07, 149.59. HRMS (ESI): m/z calcd
for C51H55N2O5PK+ (MK+): 845.3480 found 845.3503.
Preparation of the phosphoramidite of (S)-1-[bis(4-methoxyphenyl)
phenylmethoxy]-5,5-di(pyren-1-yl)pentan-2-ol (11)
From (S)-1-[bis(4-methoxyphenyl)phenylmethoxy]-5,5-di(pyren-1-yl)pen
tan-2-ol (10) (253 mg, 0.25 mmol) the product was obtained as a yellow
foam (251 mg, 80%). Rf = 0.44 (petroleum ether/EtOAc, 2:1 and traces of
1
Et3N). H NMR (300 MHz, CDCl3) δ 0.99–1.05 (14H, m, 2 × CH(CH3)2),
2.02–2.03 (2H, m, H3), 2.18–2.23 (2H, m, H4), 2.34 (2H, t, J = 6.6 Hz,
OCH2CH2CN), 2.99–3.23 (2H, m, H1), 3.48–3.56 (2H, m, OCH2CH2CN),
3.62 (3H, s, OCH3), 3.63 (3H, s, OCH3), 4.05–4.15 (1H, m, H2), 6.10–6.12
(1H, m, H5), 6.62–6.65, 7.15–7.41 (13H, m, phenyl), 7.91–8.12, 8.44–8.52
(18H, m, pyrene). 13C NMR (75 MHz, CDCl3) δ 19.99, 20.06 (OCH2CH2CN),
24.44, 24.53, 24.60, 24.69 (2 × CH(CH3)2), 31.23, 31.82, 31.91, 32.24, 32.40
(C3, C4), 42.19 (C5), 42.96, 43.13 (2 × CH(CH3)2), 55.02 (2 × OCH3),
57.93, 58.18 (OCH2CH2CN), 73.10, 73.31 (C2), 73.77, 73.99 (C1), 85.83
(CPh3), 112.91 (Ar-C), 117.57 (CN), 122.93, 122.99, 123.03, 124.85, 124.95,
125.05, 125.18, 125.41, 125.47, 125.52, 125.81, 126.55, 126.60, 126.84, 127.45,
127.67, 127.84, 128.08, 128.14, 128.77, 129.77, 129.97, 130.04, 130.70, 131.36,
136.03, 136.14, 139.00, 139.14, 139.25, 144.96, 145.01, 158.28 (Ar). 31P
NMR (121.5 MHz; CDCl3) δ 149.09, 149.46. HRMS (ESI): m/z calcd for
C67H63N2O5PH+ (MH+): 1007.4547, found 1007.4575.
Molecular Modelling
The molecular modelling was performed with MacroModel 8.1 from
Schro¨dinger Inc. (Cambridge, MA, USA)[21] All calculations were con-
ducted with the AMBER∗ force field[22,23] and the GB/SA water model.[24]
The dynamics simulation was performed with stochastic dynamics,[25]
a
SHAKE algorithm to constrain bonds to hydrogen,[26] a time step of 1.5
fs, and a simulation temperature of 300 K. Simulation for 1.0 ns generated

224
M. E. Østergaard et al.
250 structures, which were all minimized using a Polak-Ribier conjugated
gradient method with a convergence threshold of 0.005 kJ/mol. The
minimized structures were examined with XCluster from Schro¨dinger, and
a representative structure for the lowest-energy cluster was selected. The
starting structure[19] was taken from the Brookhaven Protein Database and
modified in MacroModel. The base pair next to the intercalating site was
changed to obtain the desired mismatch.
Oligonucleotide Synthesis and Purification
ODNs were synthesized on a DNA 3400 Synthesizer from Applied Biosys-
tems (Foster City, CA, USA). The phosphoramidites 7 and 11 were dissolved
in a 1:1 mixture of dry CH2Cl2 and dry MeCN, as a 0.75 M solution and
incorporated into the growing oligonucleotide chain using elongated cou-
pling times (10-minute coupling versus 2-minute for normal nucleotide cou-
plings). The yield of the oligo synthesis varied from 61–74% corresponding
to 96–98% coupling efficiency pr step. The 5ꢁ-O-DMT-on oligonucleotides
were cleaved off the solid support (room temperature, 2 hours) and depro-
tected (55◦C, overnight) using 32% aqueous ammonia. Purification of the
5ꢁ-O-DMT-on ODNs was accomplished using a Waters Model 7956 HPLC
(Milford, MA, USA) with a Waters 600 controller, a Waters 2996 detector,
and a Waters 717 autosampler on a Waters Xterra MS C18; 10 µm, 7.8 ×
150 mm column. Buffer A: 0.05M triethyl ammonium acetate in H2O, pH =
7.4; Buffer B: 75% CH3CN in H2O; flow 2.5 mL/min. Gradients: 2 minutes
100% A, linear gradient to 70% B in 38 minutes, linear gradient to 100%
B in 7 minutes, 100% B in 3 minutes, linear gradient to 100% A in 1 min-
utes, and then 100% A in 9 minutes. Product peak at ∼25 minutes. The
ODNs were DMT deprotected in 100 µL 80% AcOH (20 minutes), diluted
with 1M aqueous NaOAc (150µL) and precipitated from ethanol (600 µL).
The ODNs were kept at −18◦C for 1 hour followed by centrifugation for
20 minutes upon cooling. The supernatant was decanted off and the ODNs
were dried in a vacuum centrifuge. All modified ODNs were confirmed by
HiRes-ESI analyses on the Ionspec 4.7 T Ultima Fourier transform mass spec-
trometer (Irvine, CA, USA) or by MALDI-TOF analysis on a Voyager Elite
Bio Spectrometry Research Station from Perceptive Biosystems (Lancashire,
UK). The purity of the ODNs was analyzed on an ion exchange LaChrom
HPLC (Pleasaton, CA, USA) and found to be over 96%.
Melting Temperature Measurements
The thermal stability studies were performed on a Perkin-Elmer Lambda
20 UV/VIS spectrometer (Beaconsfield, UK) fitted with a PTP-6 tempera-
ture programmer. Melting temperature (Tm) measurements for DNA/DNA
and DNA/RNA duplex studies were conducted in a 1 mM EDTA, 10 mM
Na2HPO4·2H2O, 140 mM NaCl buffer at pH 7.0 for 1.0 µM of each strand.

Pyrene Intercalating Nucleic Acids
225
The melting temperature was determined as the maximum of the first deriva-
tive plots of the melting curves obtained by measuring absorbance at 260 nm
against increasing temperature (1◦C/min) and is with an uncertainty of
1.0◦C as determined by repetitive experiments.
Fluorescence Measurements
The fluorescence measurements were performed on a Perkin-Elmer LS-
55 luminescence spectrometer (Beaconsfield, UK) fitted with a Julabo F25
temperature controller (Seelbach, Germany) with excitation at 340 nm and
detection at 360–600 nm. All measurements were conducted at 20◦C using
the same concentrations and buffer conditions as for the melting studies.
Control measurements conducted at either 10◦C or 30◦C did not reveal any
difference in emission band profiles.
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