Intercalating nucleic acids: The inversion of the stereocenter in 1-O-(pyren-1-ylmethyl)glycerol from R to S. Thermal stability towards ssDNA, ssRNA and its own type of oligodeoxynucleotides

Article abstract of DOI:10.1016/j.tetlet.2004.04.124

The synthesis and insertions of (S)-1-O-(pyren-1-ylmethyl)glycerol into intercalating nucleic acids is described. Insertions of this S-isomer as a bulge lead to reduced binding affinity towards complementary ssDNA compared to intercalating nucleic acids possessing (R)-1-O-(pyren-1-ylmethyl)glycerol in the same positions. Insertions of both (R) or (S) 1-O-(pyren-1-ylmethyl)glycerols as bulges into two complementary strands decreased the stability of the complex compared to dsDNA possessing the pyrene pseudo-nucleotide in one of the strands.

Full text of DOI:10.1016/j.tetlet.2004.04.124

Tetrahedron
Letters
Tetrahedron Letters 45 (2004) 4907–4910
Intercalating nucleic acids: the inversion of the stereocentre in 1-O-
(pyren-1-ylmethyl)glycerol from R to S. Thermal stability towards
ssDNA, ssRNA and its own type of oligodeoxynucleotides
Vyacheslav V. Filichev,a Khalid M. H. Hilmy,a Ulf B. Christensenb and Erik B. Pedersena,*
aDepartment of Chemistry, Nucleic Acid Center, University of Southern Denmark, DK-5230 Odense M, Denmark
bUnest A/S, DK-5220 Odense SØ, Denmark
Received 19 March 2004; revised 14 April 2004; accepted 21 April 2004
Available online 10 May 2004
Abstract—The synthesis and insertions of (S)-1-O-(pyren-1-ylmethyl)glycerol into intercalating nucleic acids is described. Insertions
of this S-isomer as a bulge lead to reduced binding affinity towards complementary ssDNA compared to intercalating nucleic acids
possessing (R)-1-O-(pyren-1-ylmethyl)glycerol in the same positions. Insertions of both (R) or (S) 1-O-(pyren-1-ylmethyl)glycerols
as bulges into two complementary strands decreased the stability of the complex compared to dsDNA possessing the pyrene pseudo-
nucleotide in one of the strands.
ꢀ 2004 Elsevier Ltd. All rights reserved.
Synthetic oligonucleotides possessing fluorescence
5'
5'
3'
O
O
intercalator moieties have found wide applications in
molecular biology and medicine.1 Recently our group
has reported the synthesis, hybridization and fluores-
cence properties of nucleic acids called INATM, which
contained bulge- and end-insertions of (R)-1-O-(pyren-
1-ylmethyl)glycerol (1, Fig. 1).2 Intercalating nucleic
acids showed greatly increased affinity for complemen-
tary single-stranded DNA (ssDNA), but reduced affinity
for an identical sequence of single-stranded RNA
(ssRNA).2
O
n
Int
Int
N
3'
O
O
1 n = 1;
2 n = 2
3
5'
O
Int
5'
R
O
N
O
Int
R
O
O
3'
O
3'
An excimer band at 480 nm generated by two pyrene
pseudo-nucleotides inserted as next-nearest neighbors as
bulges was quenched upon hybridization to a fully
complementary strand. However, no or very little
quenching was found when there were mismatch
nucleobases presented between, or next to the interca-
lators. This allows one to detect single nucleotide poly-
morphisms (SNPs) without depending on differences in
4 R = H or Me
Int = Pyren-1- yl
5
Figure 1.
the thermal melting temperatures of duplexes.3 These
unprecedented properties of INATM could be explored
for diagnostic purposes identifying ‘profiles’ or ‘signa-
tures’ of the human genome.4 In depth structural studies
of INATM and analogues is needed in order to under-
stand the nature of the phenomena and to discover new
applications.
Keywords: Intercalating nucleic acids; Pyrene intercalator; Thermal
stability; Discrimination of DNA over RNA; Self-complexation.
* Corresponding author. Tel.: +45-6550-2555; fax: +45-6615-8780;
e-mail: ebp@chem.sdu.dk
From NMR and molecular modeling studies of INATM
DNA duplexes it was concluded that pyrene intercalated
/
A research center funded by The Danish National Research
Foundation for studies on nucleic acid chemical biology.
0040-4039/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2004.04.124

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V. V. Filichev et al. / Tetrahedron Letters 45 (2004) 4907–4910
as expected but was not restrained to one site, thus
inducing high mobility and a very flexible core.5;6 Sev-
eral different pyrene pseudo-nucleotides inserted as
bulges have been published and their hybridization and
fluorescence properties have been studied. The inser-
tions via three-carbon moieties in the phosphate back-
bone (structures 37 and 4,8 Fig. 1) or using a longer
linker between the backbone and the pyrene (structure
2,5 Fig. 1) decreased the thermal duplex stability due to
loss in entropy compared to the less flexible (R)-1-O-
methyleneglycerol. It was also shown that the aromatic
moiety could not reach the same position in the duplex if
a shorter linker was used thus destabilizing the duplex.5
O
O
HO
HO
O
c, d
a, b
OH
6
7
DMTO
i
O
NC(CH2)2O(NPr 2)PO
8
Scheme 1. Reagents and conditions: (a) 1-(chloromethyl)pyrene,
KOH, toluene; (b) 80% aq CH3COOH, rt, overnight, 75% over
two steps; (c) DMTCl, pyridine, rt, overnight, 85%; (d)
NC(CH2)2OP(NPri2)2, diisopropylammonium tetrazolide, CH2Cl2,
0 ꢁC to rt, overnight, 95%.
Using cheap, racemic 2,2-dimethyl-1,3-dioxalane-4-
methanol as a starting compound for the synthesis of
intercalating nucleic acids could be a competitive
advantage of INATM. To evaluate the influence of the
other stereoisomer of INATM on the hybridization
affinity towards ssDNA and ssRNA we report here
the synthesis and insertion of (S)-1-O-(pyren-1-yl-
methyl)glycerol into oligodeoxynucleotides as a bulge.
ODNs derived from compound 8 were synthesized by
the solid phase phosphoramidite method on an auto-
mated ExpediteTM Nucleic Acid Synthesis System
Model 8909 (Applied Biosystems) in 0.2 lmol-scale
using standard nucleotide coupling conditions (2 min
coupling), but increased deprotection time (100 s) com-
pared to the standard (50 s) was applied to achieve
better release of the DMT-group after coupling of
INATM. The coupling efficiency using 4,5-dicyanoimi-
dazole as an activator was estimated to be over 99%.
ODNs were worked up as described before5 and con-
firmed by MALDI-TOF analysis.12 The results of
thermal denaturation studies of intercalating nucleic
acids possessing either R or newly synthesized S isomers
of 1-O-methyleneglycerol in the same sequences and
performed under the same conditions are listed in
Tables 1 and 2.
A range of synthetic oligonucleotides such as Peptide
Nucleic Acid (PNA)9 and Locked Nucleic acid (LNA)10
having increased affinity for DNA and RNA bind even
stronger to a complementary sequence of their own
type. Such a high self-affinity strongly limits the number
of sequences of DNA and RNA that can be targeted
with the synthetic oligonucleotides, due to the formation
of stable intra- and inter-molecular secondary struc-
tures. Therefore we would like to determine the binding
affinity of intercalating nucleic acids to sequences with
the same type of modifications possessing either R or S
stereoisomers of 1-O-(pyren-1-ylmethyl)glycerol.
The target amidite 8 needed for oligodeoxynucleotide
(ODN) synthesis was obtained applying the same se-
quence of reactions as described for INATM, but using
the opposite stereoisomer of protected glycerol. We
started from (R)-(+)-2,2-dimethyl-1,3-dioxolane-4-
methanol (6) and 1-(chloromethyl)pyrene (Scheme 1).
The latter compound was found to provoke an allergic
reaction upon contact with the skin and therefore should
be kept in the closed vessel or used immediately after its
preparation from pyren-1-ylmethanol and thionyl chlo-
ride. The alkylation of the dioxolane 6 in the presence of
KOH under Dean–Stark conditions followed by depro-
tection in 80% aq acetic acid gave (S)-1-O-(pyren-1-
ylmethyl)glycerol (7) in 75% yield. Compound 7 was
treated with 4,40-dimethoxytrityl chloride (DMTCl) in
dry pyridine affording (R)-1-O-(4,40-dimethoxytriphen-
ylmethyl)-3-O-(pyren-1-ylmethyl)glycerol in 85% yield
after purification on a silica gel column. The phospho-
ramidite 8 was obtained in 95% yield after treatment of
the latter compound with 2-cyanoethyl-N,N,N0,N0-tet-
raisopropylphosphordiamidite in the presence of diiso-
propylammonium tetrazolide in dry CH2Cl2 overnight
followed by silica gel purification. Before ODN synthesis
the phosphoramidite 811 was co-evaporated twice with
CH3CN and a 0.075 M solution of 8 in 1:1 mixture of dry
CH3CN and CH2Cl2 was prepared for the oligo syn-
thesis.
The comparison of hybridization affinities showed that 5
with inverted stereochemistry at the secondary OH-
group of the 1-O-methyleneglycerol was less potent in
increasing affinity for complementary ssDNA than 1.
The difference in Tm of these two isomers towards ssDNA
varied from 3.0 ꢁC for a single insertion (Table 1, entry 2)
and up to 3.5 ꢁC for double insertions separated by four
nucleobases (Table 1, entry 4). Nevertheless the melting
temperature of intercalating nucleic acids containing
insertions of 1or 5 with complementary ssRNA were
nearly the same, but the discrimination of ssDNA over
ssRNA was reduced in the case of 5 (Table 1).
The decreased hybridization affinity towards ssDNA
when using 5 instead of 1 could be explained by the lack
of flexibility of the (S)-1-O-methylglycerol linker, which
cannot compensate for the inversion of the stereocentre
thus decreasing the efficiency of the pyrene intercalation.
Due to differences in affinity for the two enantiomers, it
is obvious that using racemic 2,2-dimethyl-1,3-dioxa-
lane-4-methanol as a starting compound for the syn-
thesis of intercalating nucleic acids could lead to poorly
defined melting temperatures.

V. V. Filichev et al. / Tetrahedron Letters 45 (2004) 4907–4910
4909
Table 1. Melting temperaturesa of intercalating nucleic acids derived from 1 and 5 in duplexes with DNA and RNA
Entry Oligo
Target DNA 50-AGCTTGCTTGAG
Target RNA 50-AGCUUGCUUGAG
Discrimination
TmðDNAÞ ꢀ TmðRNAÞ
1
30-TCGAACGAACTC
48.0 ꢁC
40.5 ꢁC
7.5 ꢁC
X ¼
1
5
1
5
1
5
Tm (ꢁC) DTm (ꢁC) Tm (ꢁC) DTm (ꢁC) Tm (ꢁC) DTm (ꢁC) Tm (ꢁC) DTm (ꢁC) DTm (ꢁC) DTm (ꢁC)
2
3
4
30-TCGAACXGA-
ACTC
30-TCGAACXG-
XAACTC
30-TCGAXACGAX-
ACTC
51.5
52.5
61.0
+3.5
48.5
49.3
57.5
+0.5
+1.3
+9.5
38.0
34.5
34.8
)2.5
)6.0
)5.7
37.0
33.5
36.5
)3.5
)7.0
)4.0
13.5
18.0
26.2
11.5
15.8
21.0
+4.5
+13.0
a Tm (ꢁC) was determined as the first derivative of melting curves by measuring the absorbance at 260 nm against increasing temperature (1.0 ꢁC/min)
on equimolar mixtures (1.0 lM of each strand) in 140 mM NaCl, 10 mM sodium phosphate buffer, 1 mM EDTA, pH 7.0.
Table 2. Melting temperaturesa of INATM/DNA and INATM/INATM duplexes
Entry
Oligo
Target INA
X ¼ 1
5
50-AGCTTGXCTTGAG
1
2
3
4
5
30-TCGAACGAACTC
50.3
43.6
41.0
58.0
47.5
41.8
41.0
30-TCGAACXGAACTC
1
5
1
5
30-TCGAXACGAXACTC
54.0
50-AGCTTGXCXTTGAG
6
7
8
30-TCGAACGAACTC
30-TCGAACXGXAACTC
49.7
45.0
39.0
45.0
39.7
37.5
1
5
50-AGCTXTGCTXTGAG
9
30-TCGAACGAACTC
30-TCGAXACGAXACTC
50.0
32.0
30.0
47.1
30.0
29.5
10
11
1
5
a Tm (ꢁC) was determined as in Table 1.
In other studies of INATM we found that DTm can vary
in the range of +1.0 to +11.0 ꢁC per modification
(unpublished data). The sequence dependence is clearly
illustrated by the fact that the double insertion of 1
separated by four base pairs in one strand gave only a
DTm of +2.0 ꢁC (Table 2, entry 9) in comparison with
+13.0 ꢁC (Table 1, entry 4) when 1 was placed in the
other strand.
strand (Table 2, entries 4 and 5) when compared to
hybridization with unmodified ssDNA (Table 1, entry 4).
When two insertions of 1 or 5 were separated by four
nucleobase pairs and placed opposite to each other in
both strands (Table 2, entries 10 and 11), the change of
hypochromicity in melting transitions was considerably
lower than in all other cases. This could be due to partly
interrupted base-stacking in the duplex and/or because
of the formation of different secondary structures having
small changes in hypochromicity at their transition
states.
It could be expected that the incorporation of 1 or 5 in
two complementary strands, opposite to each other
could increase the hybridization affinity, as it allows the
large pyrene moieties to co-axially stack with each other.
However in all cases a decrease in melting temperatures
was observed (Table 2, entries 2, 3, 7, 8, 10, 11) when
compared to the wild-type duplex (Table 1, entry 1). This
means that coaxial stacking of two pyrene moieties is not
large enough to compensate for a distortion of the
double helix by two ethylene glycol linkers situated in
opposite DNA strands. Decreased melting temperatures
were also detected when intercalating nucleic acids hav-
ing a double insertion of either 1 or 5, separated by four
nucleobases, hybridized to complementary DNA pos-
sessing a pyrene pseudo-nucleotide in the middle of the
From this study it can be concluded that intercalating
nucleic acids with bulge insertions of (R)-1-O-(pyren-1-
ylmethyl)glycerol (1) have higher affinity for comple-
mentary ssDNAs compared to insertions of (S)-1-O-
(pyren-1-ylmethyl)glycerol (5). When intercalating
pseudo-nucleotides 1 or 5 are positioned opposite each
other, the corresponding duplexes have lower thermal
stability than wild-type complexes. We believe that
INATM is the first group of synthetic DNAs having a
high affinity for the natural DNA and not for its own
type of oligodeoxynucleotides.

4910
V. V. Filichev et al. / Tetrahedron Letters 45 (2004) 4907–4910
6. Nielsen, C. B.; Petersen, M.; Pedersen, E. B.; Hansen, P.
E.; Christensen, U. B. Bioconjugate Chem. 2004, 15, 260.
7. Filichev, V. V.; Pedersen, E. B. Org. Biomol. Chem. 2003,
1, 100.
8. Dioubankova, N. N.; Malakhov, A. D.; Stetsenko, D. A.;
Korshun, V. A.; Gait, M. J. Org. Lett. 2002, 4, 4607.
9. Wittung, P.; Nielsen, P. E.; Buchardt, O.; Egholm, M.;
Norden, B. Nature 1994, 368, 561.
Insertions of 5 into intercalating nucleic acids can be
used to level a melting temperature if required, while still
maintaining its reduced affinity for ssRNA. We believe
that intercalating nucleic acids possessing 1 or 5 as
bulges can become a powerful tool for demanding
hybridizations and can substitute normal DNA oligos
when they form secondary structures or have high self-
affinity.
10. Koshkin, A. A.; Nielsen, P.; Meldgaard, M.; Rajwanshi,
V. K.; Singh, S. K.; Wengel, J. J. Am. Chem. Soc. 1998,
120, 13252.
11. Compound 8: 1H NMR (CDCl3) d 1.20 (m, 12H, 4 · Me),
2.42 (m, 2H, CH2CN), 3.22 (m, 2H, OCH2CH), 3.50–3.90
(m, 12H, CH2ODMT, OCH2CH2CN, 2 · CH [Pri],
2 · OCH3), 4.18 (m, 1H, CH2CHCH2), 5.25 (m, 2H,
CH2–pyrene), 6.60–6.70, 7.10–7.45 (m, 13H, DMT), 7.80–
8.40 (m, 9H, pyrene); 31P NMR (CDCl3) d 150.20, 150.24
in ratio 3:2, respectively; HRMS, found m=z 831.357, calcd
for C50H53N2O6P [M+Na]þ 831.353.
Acknowledgement
We are grateful to Dr. K. B. Jensen for mass-spectro-
metric analysis of oligonucleotides.
12. MALDI-TOF analysis of intercalating nucleic acids:
References and notes
1. (a) Niemeyer, C. M.; Blohm, D. Angew. Chem., Int. Ed.
1999, 111, 3039; (b) Beaucage, S. L.; Iyer, R. P. Tetrahe-
dron 1993, 49, 6123.
Oligo
X ¼
m=z,
Calcd.
(Da)
m=z,
Found
(Da)
2. Christensen, U. B.; Pedersen, E. B. Nucleic Acids Res.
2002, 30, 4918, INATM is commercially available from
UNEST A/S.
3. Christensen, U. B.; Pedersen, E. B. Helv. Chim. Acta 2003,
86, 2090.
30-TCGAACXGAACTC
30-TCGAACXGXAACTC
30-TCGAXACGAXACTC
50-AGCTTGXCTTGAG
5
5
5
1
5
1
5
1
5
3981
4348
4348
4042
4042
4409
4409
4409
4409
3977
4349
4350
4043
4041
4407
4409
4410
4408
€
4. Louet, S. Bioentrepreneur 23 June 2003 http://dx.doi.org/
10.1038/bioent748.
50-AGCTTGXCXTTGAG
50-AGCTXTGCTXTGAG
5. Christensen, U. B.; Wamberg, M.; El-Essawy, F. A. G.;
Ismail, A. E.-H.; Nielsen, C. B.; Filichev, V. V.; Jessen, C.
H.; Petersen, M.; Pedersen, E. B. Nucleosides Nucleotides
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