Intercalating Nucleic Acids: The Influence of Linker Length and Intercalator Type on Their Duplex Stabilities

Article abstract of DOI:10.1081/NCN-120027829

Six new examples of intercalating nucleic acids were synthesized in order to evaluate the dependence of the length of the linker between oligo and intercalator on the thermal stability of their corresponding duplexes and triplexes.

Full text of DOI:10.1081/NCN-120027829

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Intercalating Nucleic Acids: The Influence of Linker
Length and Intercalator Type on Their Duplex
Stabilities
Ulf B. Christensen a b , Michael Wamberg a , Farag A. G. El‐Essawy a , Abd El‐Hamid
Ismail a , Christina B. Nielsen a , Vyacheslav V. Filichev a , Carsten H. Jessen a , Michael
Petersen a & Erik B. Pedersen a
a Nucleic Acid Center†† , Department of Chemistry , University of Southern Denmark ,
Campusvej 55, DK‐5230, Odense, Denmark
b Unest A/S , Lille Tornbjerg Vej 24 B, DK‐5220, Odense SØ, Denmark
Published online: 01 Jun 2007.
To cite this article: Ulf B. Christensen , Michael Wamberg , Farag A. G. El‐Essawy , Abd El‐Hamid Ismail , Christina B.
Nielsen , Vyacheslav V. Filichev , Carsten H. Jessen , Michael Petersen & Erik B. Pedersen (2004) Intercalating Nucleic
Acids: The Influence of Linker Length and Intercalator Type on Their Duplex Stabilities , Nucleosides, Nucleotides and
Nucleic Acids, 23:1-2, 207-225, DOI: 10.1081/NCN-120027829
To link to this article: http://dx.doi.org/10.1081/NCN-120027829
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NUCLEOSIDES, NUCLEOTIDES & NUCLEIC ACIDS
Vol. 23, Nos. 1 & 2, pp. 207–225, 2004
Intercalating Nucleic Acids: The Influence of Linker Length
and Intercalator Type on Their Duplex Stabilitiesy
#
Ulf B. Christensen, Michael Wamberg, Farag A. G. El-Essawy,
*
Abd El-Hamid Ismail, Christina B. Nielsen, Vyacheslav V. Filichev,
Carsten H. Jessen, Michael Petersen, and Erik B. Pedersen
Nucleic Acid Center,yy Department of Chemistry,
University of Southern Denmark, Odense, Denmark
ABSTRACT
Six new examples of intercalating nucleic acids were synthesized in order to evaluate
the dependence of the length of the linker between oligo and intercalator on the
thermal stability of their corresponding duplexes and triplexes.
Key Words: Intercalating nucleic acids; 7,9-Dimethylpyrido[3’,2’:4,5]thieno[3,2-d]
pyrimidine-4(1H)-one; Pyrene intercalators; DNA duplex stability of intercalating
nucleic acids; Oligonucleotides with covalently bound intercalators.
y
In honour and celebration of the 70th birthday of Professor Leroy B. Townsend.
#Current address: Ulf B. Christensen, Unest A/S, Lille Tornbjerg Vej 24 B, DK-5220 Odense
SØ, Denmark.
*
Correspondence: Michael Wamberg, Department of Chemistry, University of Southern Denmark,
Campusvej 55, DK-5230, Odense M, Denmark; Fax: +45-6615-8780; E-mail: mcw@chem.sdu.dk.
yy
acid chemical biology.
A research center funded by The National Danish Research Foundation for studies on nucleic
207
DOI: 10.1081/NCN-120027829
1525-7770 (Print); 1532-2335 (Online)
www.dekker.com
Copyright D 2004 by Marcel Dekker, Inc.

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Christensen et al.
INTRODUCTION
Intercalating nucleic acids (INAks) have recently gained commercial interest for
screening DNA methylation signatures in hundreds or thousands of tissue samples
when used in combination with the bisulphite DNA conversion technology.[1] The
INAks used for this purpose are synthesized by insertion of 1-O-(1-pyrenylmethyl)-
glycerol into DNA. This type of INAks shows a better binding in duplex formation
with a very high specificity and is innately fluorescent.[2,3]
We define an intercalating nucleic acid as an oligonucleotide with a covalently
bound intercalator. If the intercalator is linked to the backbone one has to consider the
structure of the backbone, linker and intercalator in order to evaluate the stability of
duplexes when using intercalating nucleic acids for hybridization.
Among others Ossipov et al.[4] are using long linker arms to provide maximum
flexibility and to prevent stereochemical alterations of the nucleoside conformation (for
hybridizations properties). Also it should be taken into account that the axial distance
[5]
˚
between two nucleobases are approximately 3.4 A. Hence it would be optimal if the
˚
backbone introduces additionally 3.4 A between the two neighbouring nucleobases of
the strand in which the intercalator is inserted. By introduction of additional flexibility
into the backbone of one strand the degree of rotational freedom of this strand is
enlarged and hence loss of entropy is larger upon hybridization to a complementary
strand. Therefore a minimum degree of flexibility should be introduced, but still
enough to support efficient intercalation. In order to fulfil these demands and to make
Figure 1. The seven intercalating nucleic acid monomers used in this investigation.

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209
an amidite that uses the same chemistry as normal commercial amidites, we chose the
vicinal dihydroxy system as the backbone.
If intercalation from an intercalator should increase affinity for the complementary
ssDNA, the intercalator has to interact with nucleobases from both the strand in which
it is incorporated and the opposite strand. It is therefore the purpose of the linker to
place the intercalator in an optimal position for p-interactions with the neighbouring
nucleobases, without disrupting the hydrogen bonding of the neighbouring base pairs.
The optimal linker length will most likely depend on the size of the intercalator.
The intercalator is the unit that introduces most of the novel features of INAk. It is
a compound which contains an essentially flat, conjugated aromatic or heteroaromatic
ring system that is able to co-stack with a DNA or RNA duplex. The intercalator is
chosen in consideration of several features. The size of the conjugated ring system and
the electron density are two influencing factors on the strength of the co-axial stacking.
The fluorescence properties of the intercalator are also important for the potential uses
of the INAks.
In order to gain more insight into the above mentioned criteria, seven different
intercalating nucleic acid monomers were synthesized (Figure 1) and incorporated
into oligonucleotides.
Besides thermal melting studies, we have made structural calculations (Macro-
Model) of INAks and NMR experiments to examine how the intercalator is situated in
the duplex.
RESULTS AND DISCUSSION
Chemistry
The synthetic routes towards the intercalating nucleic acid monomers 2–7 are
shown in Schemes 1–3, while the monomer 1 has previously been synthesized in our
group.[2] 3 and 7 were synthesized starting from chloromethylaryl compounds 8a,b. The
required alcohol (S)-2-(2,2-dimethyl-1,3-dioxolane)-4-ethanol was prepared from ma-
lic acid as described in the literature,[6,7] and reacted with 1-chloromethylpyrene or
9-chloromethylanthracene in the presence of potassium hydroxide yielding 9a,b.
Subsequent treatment with 80% aqueous acetic acid gave the diols 3 or 7, which were
protected with DMT-chloride to 10a,b and the products were converted to the
phosphoramidites 11a,b with 2-cyanoethyl N,N,N’,N’-tetraisopropylphosphorodiamidite.
The intercalating nucleic acid monomers 4 and 5 were synthesized as shown in
Scheme 2. 7,9-Dimethylpyrido[3’,2’:4,5]thieno[3,2-d]pyrimidine-4(1H)-one 12 was
prepared according to literature procedures.[8–10] The tosylates of (S)-(+)-2-(2,2-
dimethyl-1,3-dioxolane)methanol and (S)-(+)-2-(2,2-dimethyl-1,3-dioxolane)-4-ethanol
were used for the alkylation reaction to yield 13a,b. O-alkylation of 12 was also
observed. The subsequent three steps towards the phosphoramidites 15a,b were done as
described above for 11a,b.
For the synthesis of 2 and 6 (Scheme 3) a racemic mixture of 3-mercapto-1,2-
propanediol was used in the reaction with 1-chloromethylpyrene 8a and 2-bro-
momethylanthraquinone 16b, respectively. The amidites 18a,b were obtained by the
same protection and phophitylation reactions as in the previous examples, except that

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Christensen et al.
Scheme 1. (a) (S)-(+)-2,2-dimethyl-1,3-dioxolane-4-ethanol, KOH, toluene, 67–90%; (b) AcOH/
H2O (4:1); (c) DMT–Cl, pyr, 80–85%; (d) NC(CH2)2OP(NPr2i )2, N,N-diisopropylammonium
tetrazolide, CH2Cl2, 62–83%.
Scheme 2. (a) (S)-(+)-2,2-dimethyl-1,3-dioxolane-4-ethanoyl-O-para-toluenesulfonate, NaH,
DMF, 45%; (b) AcOH/H2O (4:1), 75%; (c) DMT–Cl, pyr, 65%; (d) NC(CH2)2OP(NPri2)Cl,
N,N-diisopropylammonium tetrazolide, CH2Cl2, 84%.

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Intercalating Nucleic Acids
211
Scheme 3. (a) HSCH2CHOHCH2OH, toluene or DMF, Et3N, 78–90%; (b) DMT–Cl, pyr, 66–
74%; (c) NC(CH2)2OP(NPri2)Cl, N,N-diisopropylammonium tetrazolide, CH2Cl2, 62–86%.
2-cyanoethyl N,N-diisopropylchlorophosphoramidite was used in the last step of the
reaction sequence.
The protected phosphoramidites of the intercalating nucleic acid monomers 1–6
were used for insertion of the corresponding monomers into a 12-mer oligonucleotide
using the same coupling times (2 min) in the oligo synthesis as was used for the
amidites of the natural nucleosides.
Thermal Melting Studies
Except from 3 and 4 all intercalating nucleic acid monomers stabilized the
corresponding duplexes when they were inserted as a bulge (Table 1). It is interesting
to observe the 6.8°C higher melting temperature for 5 than for 4 when inserted into
the duplex. These two compounds differ only in the length of the linker by one atom.
The linker seems too short in case of 4 which results in a lower duplex stability. On the
contrary, too long a linker seems also to be a disadvantage as deduced when comparing
Table 1. Melting temperatures of duplexes with monomers 1–6 inserted as a bulge or as an
end-positioned intercalating pseudonucleotide.
5’-AGCTTG–CTTGAG-3’
3’-TCGAACXGAACTC-5’
5’-GCTTGAG-3’
3’-CGAACTCX-5’
X
Tm (°C)
47.4
DTm (°C)
Tm (°C)
DTm (°C)
–
1
2
3
4
5
6
–
22.8
33.8
–
11.0
50.4
49.4
47.2
43.8
50.6
49.2
3.0
2.0
À0.2
À3.6
3.2
31.4
28.4
34.4
8.6
5.6
11.6
1.8

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Christensen et al.
the duplex stability for insertion 3 with the stability of the duplex from insertion of 1.
The oligo with insertion of the monomer 2, having a sulphur atom in the linker instead
of oxygen, showed almost the same thermal melting temperature as was observed for
the insertion of 1. The stabilization was only 1.0°C in favour of the oxygen analogue.
However, whereas the monomer 1 is an (R)-enantiomer, the monomer 2 is a racemic
mixture. This makes it difficult to draw conclusions for the duplex stabilities for
insertion of the individual enantiomers of 2 apart from assuming little dependence on
the duplex stability by exchanging oxygen with sulphur in the linker. Finally, the
anthraquinone 6 was stabilizing the duplex structure having a melting temperature of
49.2°C corresponding to an increase 1.8°C compared to the wild type duplex.
The dependence on the length of the linker becomes even clearer when
intercalating nucleic acid monomers are incorporated as an end-positioning intercalat-
ing pseudonucleotide (Table 1). An increase in melting temperature of 5.6°C is seen for
4 compared with the unmodified duplex, whereas 1 and 5 increases the melting
temperature with 11.0°C and 11.6°C, respectively. In between is the monomer 3 with
an increase of thermal melting of 8.6°C. Here the same trend as above is followed. The
intercalating nucleic acid monomers 1 and 5 result in nearly the same affinity for the
targets, while 3 with a longer linker is not as efficient as 1, and 4 having a shorter
linker than 5 is contributing with the lowest stabilization to the duplex.
To investigate the dependence on linker length further, some intercalating nucleic
acids with two intercalating moieties were prepared. The monomers were inserted with
varying distances as neighbours, next-nearest neighbours or with four nucleotides
between the monomers. Previously we have focused on the pyrene monomer 1 in the
same sequences[2] and its fluorescence properties which confirmed its intercalation
rather than groove binding in duplexes.[3] From Table 2 it is seen that it is sufficient for
two pyrene insertions to have only one natural base pair between the insertions to
improve the duplex stability. For sequences with the monomer 1, we have previously
shown that for this type of double insertions the improved stability was due to
intercalation rather than to stacking of the two intercalating agents on its self. This was
shown by quenching of a pyrene excimer band on duplex formation.[3] Although one
insertion of 3 lowered the duplex melting temperature, two insertions of 3 as next
nearest neighbours resulted in an oligo with nearly the same hybridization properties as
the wild type oligo. The monomer 5 with increased linker length due to an extra
methylene group also in this case with next nearest neighbours increased the melting
temperature in a comparable manner to what was observed for the pyrene monomer 1.
Table 2. Changes in melting temperatures of duplexes with 1[2], 3, 4 and 5 inserted as bulges.
X =
1 DTm (°C)
3 DTm (°C)
4 DTm (°C)
5 DTm (°C)
3’-TCG AAC XXG AAC TC-5’
3’-TCG AAC XGX AAC TC-5’
3’-TCG AXA CGA XAC TC-5’
3’-TCG AAC XG1 AAC TC-5’
À5.2
4.0
À5.0
0.4
À14.0
À9.6
À6.2
–
–
4.4
9.0
5.5
13.4
7.8
The target sequence was 5’-AGC TTG CTT GAG-3’ and the reference melting temperature for the
wild type duplex without insertions was 47.4°C.

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213
Interestingly, the combination of 1 and 5 as next nearest neighbours in the same strand
increases the melting of the duplex with 5.5°C, which is nearly the same as, found for
same type of double insertion with 1. The highest stabilizations of the duplexes as
determined from increases in melting temperatures were found in case of four natural
base pairs between two insertions. In fact the same dependence of duplex stabilities on
linker length as for the next-nearest neighbours was found in this case. Again the
monomers 1 and 5 show similarity in being the best stabilizers of the duplex in
comparison with the monomers 3 and 4, respectively, which seem to have less
appropriate linker lengths. When the monomers 1, 3 and 4 were double inserted as
neighbours a considerable drop in melting temperature was observed. One can imagine
that this is due to increased flexibility of the strand with double insertions and
difficulties in accommodating the two intercalating ring inside the helix structure.
From molecular calculations (MacroModel) on the DNA/INAk duplex, we
anticipated that the neighbouring nucleobase in the 3’ position of the intercalator (in
the same strand) is more crucial for the ability of stacking than the nucleobases of the
opposite strand. Therefore the sequence was changed, so the neighbouring bases to the
intercalating monomer were either both cytosine or both guanine (Table 3). In both
cases the intercalator was placed between two C–G base pairs like in the original
sequence from Table 1.
Without intercalators the melting temperature for this new sequence was higher
than for the original wild type sequence from Table 1. When the pyrene INAk 1 was
inserted between two cytosines, DTm was marginally higher than for the original
sequence (3.3°C compared to 3.0°C). Shifting the intercalator to the complementary
strand leads to an increase in melting temperature (DTm = 7.6°C), indicating better
stacking when the intercalator is neighbouring two guanines in the same strand. For 3 the
DTm was 2.5°C in case of two neighbouring cytosines, which is also an improvement
from the corresponding sequence with 3 from Table 1 (DTm = À0.2°C). However, when
the intercalator was shifted to the complementary strand, the increase in melting
temperature was only 3.6°C compared to the wild type duplex. Thus for this sequence the
longer linker in 3 is also a disadvantage when compared with the length of the linker in
1, showing the necessity for the intercalator to be able to interact with both strands in
order to bring about an efficient stabilization effect. We also investigated anthracene 7 as
an intercalator in this context. However, anthracene destabilized the duplex and was
therefore not interesting for further experiments.
Furthermore we decided to investigate the affinity for triplex formation of
intercalating nucleic acids containing the monomers 1 and 3, as there is a particular
Table 3. Melting temperatures of duplexes X = 1, 3 and 7 inserted as a bulge.
Sequences
–(Ref) Tm (°C)
1 DTm (°C)
3 DTm (°C)
7 DTm (°C)
À1.3
5’-AGCTTG–GTTGAG-3’
3’-TCGAACXCAACTC-5’
49.6
3.3
2.5
5’-AGCTTGXGTTGAG-3’
3’-TCGAAC–CAACTC-5’
49.6
7.6
3.6
–

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Christensen et al.
interest in the development of DNA intercalators, which can efficiently promote triple
helix formation. Usually the stabilization of the triplex structure is observed upon direct
addition of the heterocyclic compounds sometimes possessing positively charged side
chain to a solution containing all three oligonucleotide sequences.[11,12] Intercalators
linked by a polymethylene linker to the 5’-end of the triplex forming oligonucleotide
(TFO) have been described.[13–16] Recently we investigated the effects of insertion of
5-methyl-N4-(1-pyrenylmethyl)-2’-deoxycytidine as a bulge into DNA towards DNA/
DNA duplex, three-way junction and triplex structures.[17] In the latter case its insertion
as a bulge in the middle of the triplex sequence afforded a 10.4°C increase in the
melting temperature compared with the wild type triplex. Therefore it is obvious to
suppose a stabilization of the triplex with an intercalating nucleic acid. The in-
tercalating nucleic acid monomers 1 and 3 were inserted as a bulge, as next-nearest
neighbours in the middle, and at the 5’-end of the TFO (Table 4). The single insertion
in the middle of TFO had no effect on the Tm value in the case of 3 but a drop in Tm of
5°C was observed for 1 compared to the reference TFO system. The insertions of both
intercalating nucleic acid monomers as the next-nearest neighbours led to an even more
dramatic decrease in melting temperature, which is contrary to the results obtained with
duplexes. However, the monomers 1 and 3 linked to the 5’-end of TFO resulted in
stabilization of triplexes (DTm of 9.0°C for 1 and 11.0°C for 3). When the effects of
bulge insertions of these monomers into TFO are investigated, it can be concluded that
3 (containing the longer linker) stabilizes the triplex better than does 1. This is contrary
to what is seen for duplexes. Furthermore it could be speculated that further
stabilization of the triplexes by the pyrene moiety could be achieved by further
increasing the length of the linker to allow more favorable stacking interactions into the
duplex strands. This could be observed in the case of 5-methyl-N4-(1-pyrenylmethyl)-
cytidine[17] where the nucleotide was also involved in base stacking and allowed the
pyrene moiety being in stronger interactions with neighbouring nucleobases. On the
other hand, the considerable stabilization by TFO possessing 1 and 3 at the 5’-end is
believed due to the stacking interactions only (the effect as a lid) without disturbing of
triple helixes.
To exclude that we were measuring a duplex formation between the T rich TFO
and the A rich purine strand, we also determined the melting temperature of the 14-mer
TFO hybridized to the 17-mer purine strand. As this mixture had a Tm of 20°C, there
was clearly no interference with the triplex measurements in Table 4, except for the
examples with next nearest neighbours. However, in this case the conclusion about the
dramatically lowering of the triplex melting temperatures is still correct.
Table 4. Melting temperatures of triplex studies with 1 and 3. The target duplex of 5’-GAC GGG
GAA AGA AAA AA-3’ and 3’-CTG CCC CTT TCT TTT TT-5’ showed thermal melting at 58°C.
X =
Tm (°C)
1 Tm (°C)
3 Tm (°C)
5’-CCC CTT–TCT TTT TT-3’
5’-CCC CTTXTCT TTT TT-3’
5’-CCC CTTXTXCT TTT TT-3’
5’-XCCC CTT TCT TTT TT-3’
36
31
<20
45
36
23
47

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215
NMR STUDIES
In order to determine exactly how INAk intercalate and what consequences the
intercalation has for the structure of the duplex, NMR experiments were performed and
compared with thermal stability measurements, using 1 as an example. Unfortunately, it
turned out that the chosen ATXAT duplex possessed some characteristics that made
good quality NMR spectra difficult to obtain and structure determination impossible to
perform. The results gained from NMR studies therefore became more vague and
quantitative than expected. For the NMR studies we had chosen a 9-mer oli-
gonucleotide, for which NMR data for the DNA:DNA duplex structure was
available.[18] 1 was inserted as a bulge in the middle of the duplex in an A–T region.
The numbering assignment of the nucleobases in this duplex is shown in Figure 2.
Incorporation of INAk 1 in the strand gives an increase in melting temperature of
10.3°C compared to the unmodified duplex, which melted at 30.9°C.
The first spectra obtained for the ATXAT duplex showed some very broad lines,
especially in the aromatic region, therefore a series of dilution experiments were
performed to reveal whether this was a concentration dependent characteristic of the
sample. From these experiments we realized that there is a dependency of the sample
concentration on the line shape of the aromatic protons with lines becoming sharper at
low duplex concentration. In order to circumvent this problem another approach was
taken, and instead of diluting the sample, DMSO-d6 was added. This was based on the
idea that a change in polarity would decrease the aggregation of the duplex, which was
probably the problem at higher concentrations. However, the amount of DMSO added
had to be moderate since the addition pushes the stability equilibrium of a DNA duplex
towards ssDNA.
The spectra acquired after addition of DMSO displayed cross peaks that could be
assigned to the pyrene moiety, but no specific assignment could be performed,
suggesting that the pyrene is intercalated as expected but not restrained to one site,
thus inducing high mobility and a very flexible core. This theory is supported by the
fact that no cross peaks between T13 and A14 can be observed in the spectra,
indicating intercalation between these residues or a major disturbance of the normal
helical structure. Comparison of the chemical shift changes observed relative to the
unmodified duplex[18] support the theory of intercalation. Especially A6:H2 draws
attention as this proton is shifted from 7.21 ppm in the unmodified duplex to 6.64
ppm in the ATXAT duplex, a pronounced shift probably due to ring current effects
from the intercalating pyrene moiety. Examination of the aromatic protons in the
insertion area reveals that A14:H8 and A6:H8 shift downfield 0.44 and 0.26 ppm
respectively, whereas no significant shift could be observed for T5:H6 and T13:H6.
It is intriguing that the effect of INAk is more pronounced for the aromatic protons
—
Figure 2. The numbering assignment of the nucleobases in the ATXAT duplex used for NMR
studies. The intercalator 1 is denoted X.

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Christensen et al.
of A6 and A14 than for T5 and T13, but it is presumably because of the larger
ring system of adenine, and thereby the bigger influence from the stacking with the
pyrene moiety.
For the changes observed for the sugar protons the picture become more unclear.
Here the observed changes are greatest for T5 and T13, whereas A6 and A14 only
seem to be under little influence, except for A14:H2’. For the two adenines all the
observed shifts are downfield, but for the thymines both upfield and downfield
shifts are observed, making predictions of how the pyrene moiety is situated in the
duplex difficult.
Exchange peaks for T5 and T7 indicates that the duplex has more than one
conformation. This is supported by the fact that neither the aromatic nor the aliphatic
pyrene protons can be assigned, indicating that the pyrene moiety may have more than
one orientation.
It was peculiar to us that lines in the spectra of the ATXAT were so broad, when
insertion of INAk creates a thermal stabilization of 10.3°C. The answer to this might
be the concentration of the duplex. In the measurements of the thermal stability, the
concentrations used were approximately 500 times lower (2.5–3.5 mM), than in the
NMR experiments (1.36 mM). This suggests that a destabilization might be observed if
the concentration had been higher, which is supported by the fact that dilution of the
NMR sample gave sharper lines in the spectra.
MOLECULAR MODELING
To learn more about the binding conformations of the intercalating nucleic acid
monomers, when incorporated into oligos, conformational searches using Macromodel
8.1[19] were performed. The conformational searches were based on stochastic dynamics
simulations followed by multiple minimizations of snapshots collected every 5 pico-
seconds. In both cases the Macromodel implementation of the Amber force field[20,21]
and the GB/SA water model[22] were used.
The most extensive study was performed on a 12-mer duplex having 1 as a bulge.
A 2 nanoseconds stochastic dynamics simulation[23] at 300 K yielded 400 structures,
which were minimized. The 400 minimized structures showed two main conforma-
tions of the pyrene moiety. INAk monomer 1 and the two neighbouring base pairs for
the two main conformations are shown in Figure 3. The calculation shows that the
pyrene monomer preferentially stacks to a base pair to the 5’-side and a purine to the
3’-side (upper Figure 3) or to a base pair to the 3’-side and a purine to the 5’-side
(lower Figure 3). These findings support the results of the NMR study described above,
about a conformational equilibrium.
A smaller study contained four 12-mer duplexes containing intercalating nucleic
acid monomers 2–5 as bulges. The duplexes were all subject to a 500 picoseconds
stochastic simulation at 300 K leading to 100 structures of each duplex that were
minimized. In all four cases only one binding conformation of the intercalating group
was observed. Comparison of the binding modes of monomer 2–5 with that of 1
revealed some interesting features. For monomers 1, 2, 3 and 5 the aromatic ring
system occupied the same space inside the duplex showing that a longer linker like
in the case of 3 does not lead to a displacement of the intercalator, but rather a

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217
Figure 3. A slice of a calculated structure of the same duplex in two conformations. The top base
pair is kept in the same position for the upper and lower figure, showing the pyrene monomer
stacking in two different conformations.
change in the backbone to adopt the extra atoms. The calculated binding con-
formation of monomer 4 shows that the intercalator is not able to reach the same
position in the duplex as the four other monomers, leading to a position partially
outside the duplex. These results support the findings in the thermal studies where a
shortening of the linker (5 ! 4) was more destabilizing than a lengthening of the
linker (1 ! 3).
CONCLUSION
Through melting temperature studies we have shown dependence on linker length
for intercalating nucleic acid monomers exemplified by intercalators pyrene and 7,9-
dimethylpyrido[3’,2’:4,5]thieno[3,2-d]pyrimidine-4(1H)-one. For the latter the differ-
ence from one carbon more in the linker increased the affinity to the target strand,
while the opposite was the case for pyrene intercalators. In some cases 4 was seen to
destabilize the duplex, while 5 was strongly stabilizing the duplex. Hence it is
important that the combined length of linker and intercalator is optimal, to obtain a
large increase in affinity between the intercalating nucleic acid and the target DNA
sequences. Intercalating nucleic acids with the monomers 1 and 5 have nearly the
same affinity for their target sequence, even though the intercalating moieties are
very different.
The results from molecular calculations and NMR studies support the possibility of
a rapid equilibrium between at least two conformations around the site of intercalation
of the pyrene monomer 1. The implication of this equilibrium is that the optimal linker
and intercalator combinations have not yet been found and that higher thermal
stabilizations are to be reported in the future.

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Christensen et al.
EXPERIMENTAL
General
NMR spectra were recorded on a Varian Gemini 2000 NMR spectrometer at
1
300 MHz for H and 75 MHz for 13C with TMS as internal standard. EI mass spectra
were recorded on a Finnigan Mat SSQ 701 spectrometer, MALDI spectra were recorded
on a Fourier Transform Ion Cyclotron Resonance Mass Spectrometer. Elementary
analyses were performed at H.C. Ørsted Institute, University of Copenhagen. Silica gel
(0.040–0.063 mm) used for column chromatography and analytical silica gel TLC plates
60 F254 were purchased from Merck. UV-light or a solution of (NH4)6Mo7O24 4H2O
ꢀ
(50 g) and Ce2(SO4)3 (1 g) in 5% sulphuric acid were used for visualization of TLC.
Solvents used for column chromatography were distilled prior to use, while reagents were
used as purchased.
General procedure for synthesis of (S)-2,2-dimethyl-4-[2-(pyren-1-ylmethoxy)-
ethyl]-[1,3]dioxolane (9a) and (S)-4-[2-(anthracen-9-ylmethoxy)-ethyl]-2,2-dimeth-
yl-1,3-dioxolane (9b). To a solution of (S)-(+)-2,2-dimethyl-1,3-dioxolane-4-ethanol
(442 mg, 3 mmol or 1.02 g, 7 mmol for reaction with 8a, 8b, respectively) in dry
toluene (100 mL) was added pulverized KOH (3.82 g, 68 mmol) followed by
1-chloromethylpyrene 8a (900 mg, 3.6 mmol) or 9-chloromethylanthracene 8b (1.90 g,
8.4 mmol). A Dean-Starck apparatus was used, while refluxing the reaction mixture
for 24 h. H2O (50 mL) was added and the organic phase was washed with H2O
(3 ꢁ 50 mL), dried (MgSO4) and evaporated under reduced pressure giving a brown
oil, which was purified by silica gel column chromatography (EtOAc/petroleum ether
25:75, v/v).
1
9a. Yield: 734 mg (67%) as a yellow oil. H NMR (CDCl3): d 7.96–8.33 (m, 9H,
arom), 5.18 (s, 2H, CH2), 4.22 (m, 1H, H-2’), 4.01 (m, 1H, H-1’), 3.71 (t, 2H, J = 5.4
Hz, H-4’), 3.55 (m, 1H, H-1’), 1.61–1.97 (m, 2H, H-3’), 1.39, 1.33 (2 ꢁ s, 6H, 2 ꢁ
CH3). 13C NMR (DMSO-d6): d 131.35, 131.25, 131.19, 130.76, 129.26, 127.64, 127.36,
126.84, 125.89, 125.18, 124.43, 123.34 (C-arom), 108.50 (C(CH3)2), 73.82, 71.70,
69.61, 67.13 (C-1’, C-2’, C-4’, CH2), 33.94 (C-3’), 26.91, 25.73 (C(CH3)2). MS
(MALDI): m/z = 383 (MNa+).
1
9b. Yield: 2.12 g (90%) as an oil. H NMR (CDCl3): d 8.43 (s, 1H, arom), 8.35
(d, 2H, J = 8.5 Hz, arom), 7.99 (d, 2H, J = 7.9 Hz, arom), 7.42–7.55 (m, 4H, arom),
5.44 (s, 2H, CH2), 3.88–3.93 (m,1H, H-1’), 3.73–3.78 (m, 2H, H-1’ and H-2’), 3.46 (t,
1H, J = 7.8 Hz, H-4’), 3.03–3.09 (m, 1H, H-1’), 1.81–1.92 (m, 2H, H-3’), 1.37 (s, 3H,
CH3), 1.32 (s, 3H, CH3). 13C NMR (CDCl3): d 131.41, 130.93, 129.30, 128.99, 128.32,
126.12, 124.91, 124.25 (C-arom), 108.46 (C(CH3)2), 73.70, 69.49, 67.17, 65.14 (C-1’,
C-2’, C-4’, CH2), 33.89 (C-3’), 26.89, 25.73 (2 ꢁ CH3).
(S)-4-(Pyren-1-ylmethoxy)-butane-1,2-diol (3). Compound 9a (633 mg, 1.8 mmol)
was stirred in 80% acetic acid (100 mL) for 42 h at room temperature. The solvent was
1
evaporated in vacuo to give 3 as a yellow oil in quantitative yield. H NMR (CDCl3):
d 7.92–8.28 (m, 9H, arom), 5.15 (s, 2H, CH2), 4.54 (br s, 2H, 2 ꢁ OH), 3.85–3.90 (m, 1H,
H-2’), 3.64–3.75 (m, 2H, H-4’), 3.40–3.59 (m, 2H, H-1’), 1.69–1.81 (m, 2H, H-3’). 13C
NMR (CDCl3): d 131.34, 131.13, 130.67, 129.23, 127.84, 127.46, 127.28, 126.96, 125.92,

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219
125.25, 124.41, 122.98 (C-arom) 71.72, 71.15, 68.08, 66.41 (C-1’, C-2’, C-4’, CH2), 32.74
(C-3’). MS (MALDI): m/z = 343 (MNa+).
(S)-4-(Anthracen-9-ylmethoxy)-butane-1,2-diol (7). Quantitative yield. 1H
NMR (DMSO-d6): d 8.63 (s, 1H, arom), 8.43 (d, 2H, J = 8.5 Hz, arom), 8.11 (d,
2H, J = 7.9 Hz, arom), 7.51–7.63 (m, 4H, arom), 5.42 (s, 2H, CH2), 4.51 (br s, 2H,
2 ꢁ OH), 3.78 (m, 1H, H-2’), 3.19–3.57 (m, 4H, H-1’, H-4’), 1.48–1.82 (m, 2H, H-3’).
13C NMR (DMSO-d6): d 130.85, 130.28, 129.35, 128.69, 127.70, 126.11, 125.04,
124.46 (C-arom), 68.43, 67.19, 65.93, 64.04 (C-1’, C-2’, C-4’, CH2), 33.72 (C-3’).
(S)-1-(4,4’-Dimethoxytriphenylmethyloxy)-4-(pyren-1-ylmethoxy)-butan-2-ol
(10a). Compound 3 (515 mg, 1.6 mmol) was dissolved in dry pyridine (10 mL) and
dimethoxytrityl chloride (DMT–Cl) (652 mg, 1.9 mmol) was added. The reaction mixture
was stirred for 42 h at room temperature. The solvent was evaporated off under reduced
pressure, and the residue was purified by silica gel column chromatography (EtOAc/
cyclohexane/triethylamine 24:74:2, v/v/v). Yield: 800 mg (80%) as a yellow oil. 1H NMR
(CDCl3): d 7.95–8.33 (m, 9H, arom), 7.17–7.41 (m, 9H, arom), 6.76 (d, 4H, arom), 5.16
(s, 2H, CH2), 4.01 (m, 1H, H-2’), 3.70–3.76 (m, 2H, H-4’), 3.72 (s, 6H, 2 ꢁ CH3O), 3.08–
3.11 (m, 2H, H-1’), 2.78 (br s, 1H, OH), 1.82 (m, 2H, H-3’). 13C NMR (CDCl3): d 158.38,
149.80, 144.88, 136.03, 131.24, 130.76, 129.99, 128.09, 127.73, 127.37, 126.88, 126.68,
125.89, 125.20, 125.18, 124.86, 124.69, 124.46, 123.24, 113.02 (C-arom) 85.91 (C-trityl),
71.68, 69.46, 67.94, 67.23 (C-1’, C-2’, C-4’, CH2), 55.11 (2 ꢁCH3O), 33.55 (C-3’). HRMS
(MALDI): m/z calcd for C42H38O5Na+ (MNa+): 645.2644, found 645.2621.
(S)-4-(Anthracen-9-ylmethoxy)-1-(4,4’ -dimethoxytriphenylmethyloxy)-butan-2-
1
ol (10b). Yield: 210 mg (85%) as yellow foam. H NMR (CDCl3): d 6.77–8.45 (m,
22H, arom), 5.44 (s, 2H, CH2), 3.82–4.14 (m, 1H, H-2’), 3.79 (s, 6H, 2 ꢁ OCH3),
3.05–3.09 (m, 2H, H-4’ ), 1.72–2.06 (m, 2H, H-3’ ). 13C NMR (CDCl3): d 158.58,
158.38, 147.31, 144.86, 139.44, 136.05, 134.08, 131.38, 130.91, 129.98, 129.10,
128.98, 128.09, 127.74, 127.03, 126.69, 126.20, 124.93, 124.17 (C-arom), 113.13,
113.03 (4 ꢁ CH-DMT), 85.89 (C-trityl), 69.41, 68.10, 67.17, 65.20 (C-1’, C-2’, C-4’,
CH2), 55.21, 55.16 (2 ꢁ OCH3), 33.55 (C-3’).
Phosphoramidite of (S)-1-(4,4’ -dimethoxytriphenylmethyloxy)-4-(pyren-1-
ylmethoxy)-butan-2-ol (11a). Compound 10a (305 mg, 0.5 mmol) was dissolved
under N2 in dry CH2Cl2 (7 mL). N,N-Diisopropylammonium tetrazolide (124 mg,
0.7 mmol) was added followed by dropwise addition of 2-cyanoethyl-N,N,N’,-
N’-tetraisopropylphosphorodiamidite (302 mg, 1 mmol). The reaction mixture was
stirred at room temperature for 24 h. The solvent was evaporated under reduced
pressure and the residue was purified by silica gel column chromatography (EtOAc/
cyclohexane/triethylamine 49:49:2, v/v/v). Yield: 250 mg (62%) as a colourless oil. 31P
(CDCl3): d 150.25, 149.89. HRMS (MALDI): m/z calcd for C51H55N2O6PNa+ (MNa+):
845.3689, found 845.3705.
Phosphoramidite of (S)-4-(Anthracen-9-ylmethoxy)-1-(4,4’ -dimethoxytriphenyl-
methyloxy)-butan-2-ol (11b). Yield: 206 mg (83%) as yellow foam. 1H NMR
(CDCl3): d 6.75–8.45 (m, 22H, arom), 5.43 (2 ꢁ s, 2H, CH2), 4.12 (m, 1H, H-2’), 3.76

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Christensen et al.
(s, 6H, 2 ꢁ OCH3), 2.53–3.68 (m, 8H, H-1’, H-4’, OCH2CH2CN, 2 ꢁ CH(CH3)), 2.31
(t, 2H, J = 6.8 Hz, CH2), 1.89–2.26 (m, 2H, H-3’), 1.10–1.18 (m, 12H, 4 ꢁ CH3). 31P
NMR (CDCl3): d 149.85, 149.55.
3-N-(S)-2@,2@-Dimethyl-1@,3@-dioxalane-4@-ethanyl)-7,9-dimethylpyrido-
[3’,2’:4,5]thieno[3,2-d]pyrimidine-4(1H)-one (13b). 7,9-Dimethylpyrido-[3’,-
2’:4,5]thieno[3,2-d]pyrimidine-4(1H)-one 12 (1.16 g, 5.0 mmol) was suspended in
anhydrous DMF (20 mL) and NaH (0.2 g, 5.0 mmol, 60% dispersion in oil) was added.
The mixture was stirred for 2h until all H2 evolving ceased. Then (S)-2,2-dimethyl-1,-
3-dioxolane-4-ethanoyl-O-para-toluenesulfonate (0.78 g, 5.1 mmol) was added and the
mixture was stirred for 24h at 80°C. The mixture was evaporated to dryness in vacuo,
co-evaporated with dry toluene (3 ꢁ 10 mL) in vacuo and the residue was purified by
silica gel column chromatography (5% EtOAc in CHCl3) to get a colourless product.
1
Yield: 0.81 g (45%); mp. 119–121°C; IR (KBr): 1676 (CO) cmÀ1. H NMR (CDCl3):
d 7.22, 7.07 (2 ꢁ s, 2H, arom), 4.05–4.36 (m, 4H, 2 ꢁ CH2), 3.61 (m, 1H, CH), 2.91,
2.67 (2 ꢁ s, 6H, 2 ꢁ CH3), 1.92–2.20 (m, 2H, CH2), 1.47, 1.39 (2 ꢁ s, 6H, C(CH3)2).
13C NMR (CDCl3): d 162.84, 159.87, 157.56, 152.10, 149.56, 146.89, 124.43, 122.59,
122.28 (C-arom), 109.33 (C(CH3)2), 72.55 (CH), 68.96, 44.51, 32.77 (3 ꢁ CH2), 26.97,
25.51 (C(CH3)2), 24.51, 19.29 (2 ꢁ CH3). HRMS (MALDI): m/z calcd for
C18H21O3N3SNa+ (MNa+): 382.1190, found 382.1199. Anal. Calcd for C18H21O3N3S:
C, 60.15; H, 5.89; N, 11.69. Found: C, 60.26; H, 5.90; N, 11.57.
(S)-4-(7,9-Dimethylpyrido[3’ ,2’ :4,5]thieno[3,2-d]pyrimidine-4(1H)-one)-butane-
1,2-diol (5). 3-N-((S)-2@,2@-dimethyl-1@,3@-dioxalane-4@-ethanoyl)-7,9-dimethylpyri-
do[3’,2’:4,5]thieno[3,2-d]pyrimidine-4(1H)-one 13b (0.75 g, 2.1 mmol) was stirred in
80% AcOH (20 mL) for 24h at room temperature. The product was obtained by
concentration in vacuo and co-evaporation with EtOH. The residue was purified by
silica gel column chromatography (5% MeOH in CHCl3) to get the colourless product.
1
Yield: 0.50 g (75%); mp. 104–106°C. H NMR (DMSO): d 8.58, 7.28 (2 ꢁ s, 2H,
arom), 4.52–4.76 (m, 2H, 2 ꢁ OH), 4.08–4.25 (m, 2H, CH2), 3.26–3.49 (m, 3H, CH2
and CH), 2.87, 2.59 (2 ꢁ s, 6H, 2 ꢁ CH3), 1.69–1.95 (m, 2H, CH2). 13C NMR
(DMSO): d 160.96, 159.16, 156.26, 151.16, 149.25, 145.90, 123.42, 122.19, 120.03 (C-
arom), 68.19 (CH), 65.29, 43.50, 32.02 (3 ꢁ CH2), 23.29, 18.27 (2 ꢁ CH3). MS–EI:
m/z = 319 (M+, 10), 301 (20), 232 (100). Anal. Calcd for C15H17N3O3S: C, 56.41; H,
5.37; N, 13.16. Found: C, 55.84; H, 5.38; N, 12.77.
(S)-1-(4,4’ -Dimethoxytriphenylmethyloxy)-4-(7,9-dimethylpyrido[3’ ,2’ :4,5]
thieno[3,2-d]pyrimidine-4(1H)-one)-2-butanol (14b). (S)-4-(7,9-Dimethylpyri-
do[3’,2’:4,5]thieno[3,2-d]pyrimidine-4(1H)-one)-butan-1,2-diol 5 (0.6 g, 1.9 mmol)
was dissolved in dry pyridine (5 mL) and DMT–Cl (0.71 g, 2.1 mmol) was added.
The reaction mixture was stirred at room temperature overnight. It was concentrated in
vacuo and co-evaporated with dry toluene (3 ꢁ 10 mL). The residue was purified by
silica gel column chromatography (EtOAc/cyclohexane/triethylamine 49:49:2, v/v/v) to
1
yield a white foam. Yield: 0.77 g (65%); mp. 134–136°C. H NMR (CDCl3): d 8.22 (s,
1H, arom), 6.78–7.40 (m, 14H, arom), 4.21–4.24 (m, 2H, CH2), 3.75 (s, 6H,
2 ꢁ OCH3), 3.61–3.69 (m, 2H, CH and OH), 3.11–3.14 (m, 2H, CH2), 2.93, 2.67,
(2 ꢁ s, 6H, 2 ꢁ CH3), 1.79–2.45 (m, 2H, CH2). 13C NMR (CDCl3): d 162.93, 159.94,
158.48, 157.93, 152.25, 147.77, 146.97, 144.57, 135.74, 135.33, 129.92, 128.02,

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221
127.83, 126.84, 124.52, 122.66, 113.13 (C-arom), 86.26 (C-trityl), 67.22 (CH), 55.15
(2 ꢁ OCH3), 66.72, 43.92, 32.37 (3 ꢁ CH2), 24.55, 19.33 (2 ꢁ CH3). HRMS
(MALDI) m/z calcd for C36H35N3O5SNa+ (MNa+): 644.2190, found 644.2190.
(S)-1-(4,4’-Dimethoxytriphenylmethyloxy)-3-(7,9-dimethylpyrido[3’,2’:4,5]-
thieno[3,2-d]pyrimidine-4(1H)-one)-2-propanol (14a). Yield: 0.78 g (65%); mp.
1
80–83°C. H NMR (CDCl3): d 8.13 (s, 1H, arom), 6.80–7.45 (m, 14H, arom), 4.31–
4.42 (m, 1H, CH), 4.25 (br s, 1H, OH), 3.75 (s, 6H, 2 ꢁ OCH3), 3.46–3.49 (m, 2H,
CH2), 3.23–3.25 (m, 2H, CH2), 2.87, 2.66 (2 ꢁ s, 6H, 2 ꢁ CH3). 13C NMR (CDCl3): d
162.94, 160.02, 158.64, 158.50, 152.36, 148.35, 147.15, 144.58, 135.67, 129.35,
128.23, 128.08, 126.99, 124.50, 122.72, 121.91, 113.31 (C-arom), 86.56 (C-trityl),
69.15 (CH), 55.26 (2 ꢁ OCH3), 64.66, 50.61 (2 ꢁ CH2), 24.61, 19.41 (2 ꢁ CH3).
MS–EI: m/z = 304 (M+-DMT, 30); 303 (DMT+, 100). Anal. Calcd for C35H33N3O5S:
C, 69.17; H, 5.47; N, 6.91. Found: C, 68.44; H, 5.41; N, 6.73.
Phosphoramidite of (S)-1-(4,4’-dimethoxytriphenylmethyloxy)-4-(7,9-dimeth-
ylpyrido[3’,2’:4,5]thieno[3,2-d]pyrimidine-4(1H)-one)-2-butanol (15b). (S)-1-(4,-
4’-Dimethoxytriphenylmethyloxy)-4-(7,9-dimethylpyrido[3’,2’:4,5]thieno[3,2-d]pyrimi-
dine-4(1H)-one)-2-butanol 14b (310 mg, 0.5 mmol) was dissolved under N2 in
anhydrous CH2Cl2 (10 mL). N,N-Diisopropylammonium tetrazolide (0.11 g, 0.67
mmol) was added followed by dropwise addition of 2-cyanoethyl-N,N,N’,N’-
tetraisopropylphophorodiamidite (0.3 g, 1.0 mmol). The reaction was stirred overnight
under N2, concentrated in vacuo and purified by silica gel column chromatography
(EtOAc/cyclohexane/triethylamine 49:45:12, v/v/v) to give a white foam. Yield: 345 mg
1
(84%). H NMR (CDCl3): d 8.14 (s, 1H, arom), 7.16–7.39 (m, 10H, arom), 6.73–6.80
(m, 4H, arom), 4.43–4.55 (m, 1H, CH), 3.57 (s, 6H, 2 ꢁ OCH3), 3.18–3.56 (m, 10H,
4 ꢁ CH2, 2 ꢁ NCH), 2.66, 2.79 (2 ꢁ s, 6H, 2 ꢁ CH3), 2.42–2.47 (m, 2H, CH2CN),
0.86–1.34 (m, 12H, 4 ꢁ CH3). 31P NMR (CDCl3): d 149.78, 149.39.
Phosphoramidite of (S)-1-(4,4’-dimethoxytriphenylmethyloxy)-3-(7,9-dimethyl-
pyrido[3’,2’:4,5]thieno[3,2-d]pyrimidine-4(1H)-one)-2-propanol (15a). Yield: 323
1
mg (80%). H NMR (CDCl3): d 8.12 (s, 1H, arom), 7.16–7.39 (m, 10H, arom), 6.77–
6.80 (m, 4H, arom), 4.52–4.57 (m, 1H, OCH), 4.32–4.43 (m, 2H, OCH2), 3.98–4.02 (m,
2H, 2 ꢁ NCH), 3.77 (s, 6H, 2 ꢁ OCH3), 3.50–3.54 (m, 2H, CH2), 3.15–3.33 (m, 2H,
CH2), 2.89, 2.67 (2 ꢁ s, 6H, 2 ꢁ CH3), 2.42–2.49 (m, 2H, CH2CN), 0.84–1.33 (m, 12H,
4 ꢁ CH3). 31P NMR (CDCl3): d 150.73, 150.45.
3-(Pyren-2-ylmethylthio)-propane-1,2-diol (2). 1-Chlormethylpyrene 8a (269 mg,
1 mmol), 3-mercapto-1,2-propanediol (108 mg, 1 mmol) and triethylamine (200 mg,
2 mmol) were dissolved in dry toluene (20 mL) under N2. The reaction mixture was
stirred at room temperature for 20 h. The pale yellow powder was filtered off and
recrystallized from EtOAc. Yield: 90% as yellow crystals; m.p. 90°C. 1H NMR (DMSO-
d6): d 8.09–8.55 (m, 9H, arom), 4.94 (s, 2H, CH2), 4.65 (m, 1H, H-2’), 3.48 (m, 2H,
H-1’), 2.59 (m, 2H, H-3’).
2-(2,3-Dihydroxy-propylthiomethyl)-anthraquinone (6). 3-Mercapto-1,2-propa-
nediol (108 mg, 1 mmol), 2-bromomethylanthraquinone 16b (301 mg, 1 mmol) and
triethylamine (200 mg, 2 mmol) were dissolved in dry DMF (10 mL) and stirred at

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Christensen et al.
room temperature for 16 h. The solvent was evaporated off under reduced pressure. The
pale yellow powder was recrystallized from EtOAc. Yield: 78% as yellow powder; m.p.
1
110°C. H NMR (DMSO-d6): d 7.84–8.15 (m, 7H, arom), 3.99 (m, 2H, CH2), 3.66 (m,
1H, H-2’), 3.39 (m, 2H, H-3’), 2.55 (m, 2H, H-1’).
1-(4,4’-Dimethoxytriphenylmethyloxy)-3-(pyren-2-ylmethylthio)-propan-2-ol
(17a). Compound 2 (100 mg, 0.31 mmol) and dimethoxytrityl chloride (140 mg,
0.36 mmol) were dissolved in dry pyridine (5 mL). The reaction mixture was stirred 24
h at room temperature. The solvent was evaporated off under reduced pressure and the
residue was purified by silica gel column chromatography (EtOAc/cyclohexane/
triethylamine 49:49:2, v/v/v). Yield: 66% as a yellow oil. 1H NMR (DMSO-d6): d
6.76–8.35 (m, 22 H, arom), 4.40 (s, 2H, CH2), 3.78 (m, 1H, H-2’), 3.74 (s, 6H,
2 ꢁ OCH3), 3.20 (m, 2H, H-1’) 2.87 (m, 2H, H-3’). 13C NMR (DMSO-d6): d 158.46,
144.70, 135.82, 130.67, 130.00, 129.37, 129.11, 128.06, 127.82, 127.39, 126.80,
126.02, 125.27, 129.54, 123.26 (C-arom), 86.20 (C-trityl), 69.73 (C-2’), 66.31, 66.17
(C-1’, C-3’), 55.18 (CH2), 36.00, 34.54 (2 ꢁ CH3O).
2-[3-(4,4’-Dimethoxytriphenylmethyloxy)-2-hydroxy-propylthiomethyl]-anthra-
quinone (17b). Compound 16b (170 mg, 0.5 mmol) and dimethoxytrityl chloride
(213 mg, 0.55 mmol) were dissolved in dry pyridine (10 mL). The reaction mixture
was stirred at room temperature for 16 h. The solvent was evaporated off under reduced
pressure, and the residue was purified by silica gel column chromalography (EtOAc/
cyclohexane/triethylamine 49:49:2, v/v/v). Yield: 74% as a colourless oil. 1H-NMR
(DMSO-d6): d 6.97–8.29 (m, 17H, arom) 3.82 (m, 2H, CH2), 3.78 (s, 6H, 2 ꢁ CH3O),
3.26 (m, 1H, H-2’), 2.63 (m, 2H, H-1’). 13C NMR (DMSO-d6): d 182.83, 188.64
(2 ꢁ C O), 158.43, 145.41, 144.57, 135.72, 134.48, 134.06, 133.99, 133.41, 129.39,
128.96, 128.15, 128.01, 127.79, 127.64, 127.41, 127.14, 126.81, 113.09 (C-arom),
86.26 (C-trityl), 72.12 (C-2’), 69.86, 66.02 (C-1’, C-3’), 55.12 (CH2), 36.29, 35.26
(2 ꢁ CH3O).
Phosphoramidite of 1-(4,4’-dimethoxytriphenylmethyloxy)-3-(pyren-2-
ylmethylthio)-propan-2-ol (18a). Compound 17a (100 mg, 0.33 mmol) and N,N-
diisopropylammonium tetrazolide (180 mg, 0.50 mmol) were dissolved in 10 mL dry
CH2Cl2 under N2. 2-Cyanoethyl-N,N-diisopropylchlorophosphoramidite (133 mg, 0.6
mmol) was added dropwise. The reaction mixture was stirred 48 h at room temperature.
The reaction mixture was quenched by addition of methanol (0.5 mL) and washed with
sat. NaHCO3 (3 ꢁ 15 mL). The organic layer was dried over Na2SO4 and evaporated
in vacuo. The product was purified by silica gel column chromatography (cyclohexane/
EtOAc/triethylamine 69:29:2, v/v/v). Yield: 62% as a colourless oil. 13P NMR (CDCl3):
d 150.33, 149.94.
Phosphoramidite of 2-[3-(4,4’-dimethoxytriphenylmethyloxy)-2-hydroxy-pro-
pylthiomethyl]-anthraquinone (18b). Compound 17b (165 mg, 0.25 mmol), 2-
cyanoethyl-N,N-diisopropylchlorophosphoramidite (111 mg, 0.50 mmol) and N,N-
diisopropylammonium tetrazolide were dissolved in dry CH2Cl2 (10 mL). The reaction
mixture was stirred at room temperature for 48 h. The reaction mixture was quenched
by adding methanol (0.5 mL) and H2O (150 mL). The organic layer was separated,

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Intercalating Nucleic Acids
223
dried (Na2SO4) and the solvent was evaporated in vacuo. The product was purified by
silica gel column chromatography (cyclohexane/EtOAc/triethylamine 69:29:2, v/v/v).
Yield: 86% as a colourless oil. 13P NMR (CDCl3): d 150.10, 149.75.
NMR Experiments
The NMR experiments were performed on a Varian INOVA 600 spectrometer at
25°C. NOESY spectra in D2O were acquired using 2048 complex points in t2 and a
spectral width of 6000 Hz. A total of 512 t1 experiments were recorded using the States
phase cycling scheme. The residual signal of HOD was removed by low-power
presaturation. NOESY spectra in H2O at 5°C and 25°C were obtained using the
watergate pulse sequence. The spectra were acquired with 2048 complex points in t2
and a spectral width of 12000 Hz. A total of 512 t1 experiments were recorded using
the States phase cycling. TOCSY spectra with mixing times of 30 and 70 ms and
DQF–COSY spectra were obtained using the States phase cycling scheme. Dilution
and DMSO titration 1D experiments were performed on a Varian Unity 500
spectrometer at 25°C with a spectral width of 5000 Hz. All the acquired data were
processed using FELIX (Version 98, MSI, San Diego, CA). All spectra were apodized
by a skewed sine bell squared in F1 and F2.
Oligonucleotide Synthesis, Purification and Melting
Temperature Determination
The ODN syntheses were carried out on an Expeditek Nucleic Acid Synthesis
System Model 8909 from Applied Biosystems. The appropriate amidite was dissolved
in a 1:1 mixture of dry MeCN and CH2Cl2, as a 0.1 M solution, and inserted into the
growing oligonucleotides chain using the same conditions as for normal nucleotide
couplings (2 min coupling time). The ODNs were synthesized with DMT on and
purified on a Waters Prep LC 4000 HPLC with a Waters Prep LC controller and a
Waters 2487 Dual l Absorbance detector on a Waters XterrakMS C18 column. Buffer
A [950 ml of 0.1 M NH4HCO3 and 50 ml of MeCN, (pH = 9.0)] and buffer B [250 ml
of 0.1 NH4HCO3 and 750 ml of MeCN, (pH = 9.0)] Gradients: 5 min. 100% A, linear
gradient to 70% B in 30 min., 2 min. with 70% B, linear gradient to 100% B in 8 min.
and then 100% A in 15 min (product peak at ꢂ 37 min). The ODNs were DMT
deprotected in 100 mL 80% acetic acid and precipitated from ethanol.
All modified ODNs were confirmed by MALDI–TOF analysis on a Voyager Elite
Bio spectrometry Research Station from Perceptive Biosystems. Melting temperature
measurements were performed on a Perkin–Elmer UV/VIS spectrometer fitted with a
PTP-6 temperature programmer. Melting temperature (Tm) measurements for duplex
studies were determined a 1 mM EDTA, 10 mM Na2HPO4 2H2O, 140 mM NaCl buffer
ꢀ
at pH = 7.0 for 1.5 mM of each strand. Melting temperature measurements for triplex
studies were determined in a 10 mM NaOAc, 0.5 M NaCl buffer at pH = 5.5. The
triplexes were formed by first mixing the two strands of the Watson-Crick duplex, each
at a concentration of 1.0 mM. The solution was heated to 80°C, cooled to room
temperature where the third (TFO) strand was added and then kept at 15°C for 30 min.
The melting temperature was determined as the maximum of the first derivative

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224
Christensen et al.
plots of the melting curve and is with an uncertainty 1.0°C as determined by
repetitive experiments.
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Received August 26, 2003
Accepted October 6, 2003

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