Synthesis of a new intercalating nucleic acid analogue with pyrenol insertions and the thermal stability of the resulting oligonucleotides towards DNA over RNA

Article abstract of DOI:10.1007/s00706-010-0320-6

A new intercalating nucleic acid monomer Y was obtained via alkylation of pyren-1-ol with (S)-(+)-2-(2,2-dimethyl-1,3-dioxolan-4-yl)ethanol under Mitsunobu conditions followed by hydrolysis with 80% aqueous acetic acid to give a diol which was tritylated

Full text of DOI:10.1007/s00706-010-0320-6

Monatsh Chem (2010) 141:817–822
DOI 10.1007/s00706-010-0320-6
ORIGINAL PAPER
Synthesis of a new intercalating nucleic acid analogue
with pyrenol insertions and the thermal stability
of the resulting oligonucleotides towards DNA over RNA
•
Amany M. A. Osman Erik B. Pedersen
Received: 16 December 2009 / Accepted: 2 May 2010 / Published online: 26 May 2010
Ó Springer-Verlag 2010
Abstract A new intercalating nucleic acid monomer Y
was obtained via alkylation of pyren-1-ol with (S)-(?)-2-
(2,2-dimethyl-1,3-dioxolan-4-yl)ethanol under Mitsunobu
conditions followed by hydrolysis with 80% aqueous
acetic acid to give a diol which was tritylated with
4,40-dimethoxytrityl chloride followed by treatment with
2-cyanoethyltetraisopropylphosphordiamidite in the pres-
ence of N,N0-diisopropylammonium tetrazolide. In this way
the monomer Y was obtained as its dimethoxytrityl-pro-
tected phosphoramidite building block for standard DNA
synthesis. The corresponding oligonucleotides from Y have
nearly identical hybridization properties with those of
intercalating nucleic acid (INA) where neighboring oxygen
and carbon atoms are interchanged in the linker. The
synthesis of monomer Y avoids the use of allergic inter-
mediates which are a problem in the synthesis of INA.
Introduction
In recent years, our group has reported the synthesis of
intercalating nucleic acid INA (Z, Fig. 1) with bulge
insertions of 1-O-(pyrenylmethyl)glycerol. These oligos
have higher duplex stabilities and have high hybridization
specificity towards DNA over RNA [1–3]. Quadruplex-
forming INA has been designed and studied for its capacity
to inhibit the expression of the KRAS oncogene in pan-
creatic adenocarcinoma cells [4]. Nucleic acid duplexes are
stabilized both by hydrogen bonding interactions and by
base stacking [5–11]. Therefore polyaromatic units with
hydrophobicity in conjunction with a large surface area can
be used in place of the natural nucleic bases for mimicking
base stacking in Watson–Crick/Hoogsteen base pairing
[12–14]. The design and the synthesis of various oligonu-
cleotides containing planar polycyclic chromophores
such as pyrene, anthracene, dansyl, and others, which
have a long fluorescence lifetime and possibilities to form
p-stacking interactions in aqueous solutions, have been the
subject of active research during recent years [15, 16].
Pyrene has previously been tested as an intercalator cova-
lently attached to the O20 in a uracil nucleotide through a
methylene linker. The investigation showed a promising
stabilization when hybridized to DNA, whereas hybrid-
ization to RNA caused destabilization. Insertion in RNA
caused destabilization when hybridized to both RNA and
DNA [16–18]. Interestingly, it was discovered that a
deoxyoligonucleotide with two such neighboring modified
nucleotides is an excellent capture and detection probe
[19]. Furthermore, there are numerous studies of covalent
attachment of pyrene to glycerol for insertions in nucleic
acids [20–25]. In our ongoing study we are looking for a
new and safe method for the preparation of intercalating
nucleic acids. The problem in the reported synthesis of INA
Keywords Bioorganic chemistry ꢀ
Intercalating nucleic acid ꢀ DNA ꢀ Fluorescence ꢀ
Oligonucleotides ꢀ Pyren-1-ol
The Nucleic Acid Center is a research center funded by The Danish
National Research Foundation for studies on nucleic acid chemical
biology.
A. M. A. Osman ꢀ E. B. Pedersen (&)
Department of Physics and Chemistry, Nucleic Acid Center,
University of Southern Denmark, Campusvej 55,
5230 Odense M, Denmark
e-mail: ebp@ifk.sdu.dk
A. M. A. Osman
Department of Chemistry, Faculty of Science,
Menoufia University, Shebin El-Koam, Egypt
123

818
A. M. A. Osman, E. B. Pedersen
compound 4 in 60% yield. The secondary hydroxy group
was phosphitylated with 2-cyanoethyl N,N,N0,N0-tetra-
isopropylphosphordiamidite in anhydrous CH2Cl2 under
argon in the presence of N,N0-diisopropylammonium tet-
razolide as an activator to give the target amidite 5 in 88%
yield.
O
O
O
P
O
The DMT-protected phosphoramidite 5 was incorpo-
rated into DNA oligonucleotides by using normal
procedures. The coupling efficiency was over 96% as
determined by detritylation measurement. All modified
oligonucleotides were confirmed by matrix-assisted laser
desorption/ionization time-of-flight (MALDI-TOF) mass
spectrometry analysis (see ‘‘Experimental’’). The purity of
the final oligos was confirmed by ion-exchange HPLC.
DNA
O
DNA
P
O
O
O
O
O
DNA
DNA
Z
Y
Fig. 1 The synthesized intercalating nucleic acid Y and Z (INA)
Thermal stability studies
is the alkylation step of 2,2-dimethyl-1,3-dioxolan-4-
ylmethanol with 1-(chloromethyl)pyrene [1], which is one
of the strongest skin sensitizers ever reported [26–28] and
we have several times had severe problems in our labora-
tory due to improper handling of this compound.
Therefore, the major objective of this work was to explore
the intercalating effectiveness when interchanging the
oxygen atom and methylene group in the linker of INA so
that the oxygen atom is attached directly to the interca-
lating pyrene moiety instead of the methylene group. The
linker properties are not expected to be different, but the
stacking properties of the pyrene moiety cannot be pre-
dicted. Besides studying thermal stabilization properties of
corresponding oligonucleotides, the fluorescence properties
are considered interesting because the fluorescence of INA
shows a decrease in fluorescence emission upon excitation
at 340 nm upon hybridization to DNA [1–3] indicating that
the pyrene moiety intercalates into the DNA double helix
[29, 30].
The project was designed to study the effect of inter-
changing a carbon and an oxygen atom in the linker of INA
with the oxygen atom being attached directly to the inter-
calating pyrene moiety. When bulge insertion of the
monomer Y (Table 1, entry 2) is compared with the wild-
type oligo, an increase in melting temperature of the DNA/
(INA analogue) duplex (DTm = 4.6 °C) was observed.
This is a 1.6 °C increase in thermal melting temperature
when compared with the single insertion of the original
pyrene monomer Z. To investigate further the stabilization
phenomena, two monomers were inserted with varying
distances of one to four nucleobases between the two
insertions. The trend for insertions of pyrene monomer Y is
to give slightly higher thermal melting temperatures than
the pyrene monomer Z. The melting temperatures
increased steadily on increasing the number of bases
between the two inserted bases. When Y was inserted twice
with six nucleobases in between a stabilization of 15.7 °C
(7.9 °C per modification) was observed. When compared
with a single insertion with DTm = 4.6 °C (Table 1), the
results could also indicate a dependence on the insertion
sites. Destabilization of the pyrene monomer Y was
observed towards RNA when compared with the wild-type
DNA/RNA duplex. The difference in melting temperature
between the DNA/(INA analogue) duplex and the corre-
sponding RNA/(INA analogue) duplex is 14 °C when
pyrene monomer Y inserted as a bulge (Table 1, entry 2).
This difference is 8.8 °C larger than in the unmodified
oligo. This means that intercalating nucleic acids contain-
ing insertions of pyrene Y moiety are selective and
discriminate between DNA over RNA, as the DNA/DNA
duplexes were stabilized while DNA/RNA duplexes were
destabilized. For both stabilization and discrimination, the
difference in melting temperature between the DNA/(INA
analogue) duplex and the RNA/(INA analogue) is up to
27.7 °C when two pyrene Y moieties are inserted.
Results and discussion
Chemistry
The synthetic route for obtaining the acyclic amidite 5 is
shown in Scheme 1. The synthesis of (S)-4-(pyren-1-
yloxy)butane-1,2-diol (3) was achieved under Mitsunobu
conditions. In this way (S)-2-(2,2-dimethyl-1,3-dioxolan-4-
yl)ethanol (1), diethyl azodicarboxylate (DEAD), tri-
phenylphosphine, and 1-pyrenol (2) in dry ice-cooled
tetrahydrofuran (THF) were reacted overnight [31, 32].
Subsequent treatment with 80% aqueous acetic acid gave
the diol 3 in 87% yield after purification by silica column
chromatography. The primary hydroxy group was protected
with 4,40-dimethoxytrityl chloride (DMT-Cl) and NEt3
in CH2Cl2 under nitrogen to give the O-DMT-protected
123

Synthesis of a new intercalating nucleic acid analogue
819
Scheme 1
1. DEAD/THF/Ph3P
2. 80% aq. AcOH
O
DMT-Cl
+
O
OH
CH2Cl2, Et3N
O
OH
O
2
1
HO
HO
HO
DMTO
4
3
NC(CH2)2OP(NiPr2)2
Diisopropylammonium
tetrazolide, CH2Cl2
O
NC(CH2)2O(NiPr2)PO
DMTO
5
Table 1 Melting temperatures of DNA/DNA and DNA/RNA duplexes with X = Y inserted as a bulge
Target DNA 50-AGCTTGCTTGAG-30 Target RNA 50-AGCUUGCUUGAG-30 Discrimination Tm(DNA)-Tm(RNA)
Entry Oligo
Tm (°C)
Tm (°C)
DTm (°C)
1
30-TCGAACG 47.4
AACTC-50
42.2
5.2
Entry Oligo
X = Y
X = Z
X = Y
X = Z
X = Y
X = Z
Tm (°C) DTm (°C) Tm (°C) DTm (°C) Tm (°C) DTm (°C) Tm (°C) DTm (°C) DTm (°C) DTm (°C)
2
3
4
5
6
30-TCGAACXGAACTC-50
52.0
4.6
4.3
50.4
51.4
55.6
60.8
3.0
4.0
38.0
35.1
36.1
33.3
41.7
-4.2
-7.1
-6.1
-8.9
-0.5
37.8
34.2
32.6
35.0
-4.4
-8.0
-9.6
-7.2
14.0
16.6
22.0
27.7
21.4
12.6
17.2
23.0
25.8
30-TCGAACXGXAACTC-50 51.7
30-TCGAACXGAXACTC-50 58.1
30-TCGAXACGAXACTC-50 61.0
30-TCGXAACGAAXCTC-50 63.1
10.7
13.6
15.7
8.2
13.4
a
-
a
a
a
a
-
-
-
-
Data of Tm values X = Z were taken from a previous study for comparison [1]
T
m (°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 (1.5 lM of each strand) in 140 mM NaCl, 10 mM sodium phosphate buffer, 1 mM EDTA, pH 7.0
a
Not determined
Fluorescence properties
neighbors. This is in contrast to INA in a previous study
which showed an excimer band at 480 nm [1] and this is
also in contrast with other examples of excimer bands
where perylene and pyrene moieties were inserted as next
nearest neighbors in peptide nucleic acid (PNA) analogues
of oligonucleotides [33, 34]. The missing excimer bands
may be an advantage because of reduced risk of interference
with fluorescence markers that are routinely conjugated to
oligos for detection purposes.
Upon single insertion of Y the fluorescence spectrum bands
with maxima at 390 and 410 nm upon excitation at 340 nm
(Fig. 2) were quenched when hybridized to the comple-
mentary single-stranded (ss) DNA, indicating that the
bulging pyrene moiety Y interacts strongly with the double
helix by intercalation. The quenching effect upon hybrid-
ization with complementary ssDNA is more pronounced
than for ssRNA (Fig. 2), which was expected due to lower
stability of RNA/INA-type duplexes. Double pyrene
insertions as next nearest neighbors in the oligonucleotide
gave the same results as for the single insertion (data not
shown). Surprisingly, no excimer formation was observed
for ssDNA with two insertions of Y as next nearest
Experimental
NMR spectra were recorded on a Varian Gemini 2000
1
NMR spectrometer at 300 MHz for H, 75 MHz for 13C,
123

820
A. M. A. Osman, E. B. Pedersen
and the residue was coevaporated with 2 9 30 cm3
toluene/EtOH (5:1, v/v). The residue was dried under
reduced pressure. The residue was purified by silica gel
column chromatography (5% MeOH/CH2Cl2, v/v) to
1
afford 0.261 g 3 (87%) as a clear colorless oil. H NMR
(300 MHz, DMSO-d6): d = 1.88–2.19 (m, 2H, CH2
CH2O), 3.45 (m, 2H, CH2OH, CHOH), 3.87 (m, 1H, CH2
OH), 4.48 (m, 2H, CH2O), 4.64, 4.74 (2 9 s, 2H, 2 9 OH),
7.77 (d, 1H, J = 8.7 Hz, Ar), 7.94–8.26 (m, 7H, Ar), 8.39
(d, 1H, J = 9.3 Hz, Ar) ppm; 13C NMR (75 MHz, DMSO-
d6): d = 33.28 (CH2CH2O), 65.55 (CH2O), 66.05 (CH2
OH), 68.12 (CHOH), 109.71, 119.31, 120.85, 124.02,
124.24, 124.55, 125.98, 126.14, 126.31, 127.24, 128.60,
128.76, 131.04, 131.19, 131.47, 152.70 (Carom) ppm;
HRMS (ESI): m/z = 329.1145 ([M ? Na]?, calcd.:
329.1150).
Fig. 2 Fluorescence measurements (X = Y) of a 13mer, single-
stranded oligo (INA analogue) with pyrene insertion (top), its duplex
with complementary 12mer RNA (middle), and its duplex with
complementary 12mer DNA (bottom). The sequences are as shown in
Table 1, entry 2
(S)-1-(Bis(4-methoxyphenyl)phenylmethoxy)-4-(pyren-1-
yloxy)butan-2-ol (4, C41H36O5)
Compound 3 (0.200 g, 0.653 mmol) and 0.5 cm3 anhy-
drous NEt3 were dissolved in 10 cm3 anhydrous CH2Cl2
and 0.265 g 4,40-dimethoxytrityl chloride (0.784 mmol)
was added under nitrogen at room temperature overnight.
When TLC showed no more starting material, the reaction
mixture was quenched with 2 cm3 MeOH. Ethyl acetate
(100 cm3) was added, and the mixture was extracted with
saturated aqueous NaHCO3 (2 9 40 cm3). The water phase
was extracted with ethyl acetate (2 9 20 cm3). The
combined organic phases were dried (Na2SO4), filtered,
and evaporated under reduced pressure. The residue was
coevaporated with toluene/EtOH (1:1, v/v, 2 9 25 cm3).
The residue was purified by silica gel column chromatog-
raphy (ethyl acetate/cyclohexane/NEt3 30:70:1, v/v/v),
and 121.5 MHz for 31P with TMS as an internal standard
for 1H, deuterated solvents CDCl3 (d = 77.00 ppm),
DMSO-d6 (d = 39.44 ppm) for 13C, and 85% H3PO4 as an
external standard for 31P. Chemical shifts are reported in
parts per million (d). Electrospray ionization mass spectra
(ESI–MS) were recorded on a 4.7 T HiResESI Uitima (FT)
mass spectrometer. This spectrometer is controlled by the
OMEGA Data System. All modified oligonucleotides were
confirmed by MALDI-TOF MS analysis on a Voyager
Elite Biospectrometry research station from PerSeptive
Biosystems. Analytical silica gel TLC plates 60 F254 were
purchased from Merck and were visualized in UV light
(254 nm). The silica gel (0.040–0.063 mm) used for
column chromatography was purchased from Merck.
Solvents used for column chromatography were distilled
prior to use. THF was distilled from Na/benzophenone.
1
which afforded 0.239 g 4 (60%) as a clear oil. H NMR
(300 MHz, CDCl3): d = 2.10–2.15 (m, 2H, CH2CH2O),
3.24–3.27 (m, 1H, CHHODMT), 3.36–3.40 (m, 1H,
CHOH), 3.71 (s, 6H, 2 9 OCH3), 3.79 (s, 1H, OH),
4.19–4.23 (m, 1H, CHHODMT), 4.38–4.44 (m, 2H,
CH2O), 6.77 (d, 4H, J = 8.7 Hz, DMT), 7.20–7.33 (m,
7H, arom), 7.43–7.51 (m, 3H, arom), 7.86–7.99 (m, 4H,
arom), 8.05–8.11 (m, 3H, arom), 8.34 (d, 1H, J = 9 Hz,
arom) ppm; 13C NMR (75 MHz, CDCl3): d = 33.31
(CH2CH2O), 55.12 (2 9 OCH3), 65.65 (CH2O), 67.52
(CHOH), 68.67 (CH2ODMT), 86.26 (OCPh3), 109.19,
113.12, 121.10, 124.14–131.71, 135.91, 144.83, 152.81,
158.49 (arom, DMT) ppm; HRMS (ESI): m/z = 631.2474
([M ? Na]?, calcd.: 631.2455).
˚
Triethylamine and CH2Cl2 were dried over 4 A molecular
sieves.
(S)-4-(Pyren-1-yloxy)butane-1,2-diol (3, C20H18O3)
An ice-cooled solution of 0.197 cm3 diethyl azodicarbox-
ylate (1.268 mmol) in 28 cm3 dry THF was treated with
0.150 cm3 (S)-2-(2,2-dimethyl-1,3-dioxolan-4-yl)ethanol
(1.056 mmol) for 25 min. Then 0.3 g 1-pyrenol (1.375
mmol) and 0.332 g triphenylphosphine (1.268 mmol) were
added to the mixture. The resultant mixture was stirred in
an ice bath for 30 min and then allowed to warm to room
temperature overnight. The mixture was quenched with
18.5 cm3 saturated NH4OH and extracted with ethyl
acetate. The organic layer was washed with water, dried
(MgSO4), and concentrated under reduced pressure. With-
out further purification the residue was added to 25 cm3
aqueous solution of acetic acid (80%) and stirred at room
temperature for 24 h. The solvent was removed in vacuo
(S)-1-(Bis(4-methoxyphenyl)phenylmethoxy)-4-(pyren-1-
yloxy)butan-2-yl 2-cyanoethyl diisopropylphosphoramidite
(5, C50H53N2O6P)
Compound 4 (0.170 g, 0.279 mmol) was dissolved under
argon in 20 cm3 anhydrous CH2Cl2 and 72 mg N,N0-
diisopropylammonium tetrazolide (0.42 mmol) was added
123

Synthesis of a new intercalating nucleic acid analogue
821
followed by dropwise addition of 0.266 cm3 2-cyanoethyl
N,N,N0,N0-tetraisopropylphosphordiamidite (0.837 mmol)
under external cooling with an ice–water bath. After
24 h, analytical TLC showed no more starting material and
the reaction was quenched with 15 cm3 water. Layers were
separated and the organic phase was washed with 15 cm3
water. The combined water layers were washed with
25 cm3 CH2Cl2. The combined organic phases were dried
(Na2SO4) and filtered, and the solvent was removed under
reduced pressure. The residue was purified by silica gel
column chromatography (ethyl acetate/cyclohexane/NEt3
30:70:1, v/v/v), which afforded 0.20 g 5 (88%) as clear
100 mm3 water and 50 mm3 aqueous AcONa (3 M) were
added, and 15 mm3 sodium perchlorate and the ONs were
precipitated from 500 mm3 acetone. All modified ODNs
were confirmed by MALDI-TOF analysis on a Voyager
Elite Biospectroscopy research station from PerSeptive
Biosystems. ODN found m/z (calculated m/z) as follows:
oligo entry 2: 3,981.6 (3,981.8); oligo entry 3: 4,351.3
(4,349.1); oligo entry 4: 4,351.8 (4,349.1); oligo entry 5:
4,351.6 (4,349.1); oligo entry 6: 4,351.0 (4,349.1). The
purity of the final ODNs was found to be over 90%,
checked by ion-exchange chromatography using
a
LaChrom system from Merck Hitachi on Genpak-Fax
column (Waters). Melting temperature measurements were
performed on a Perkin-Elmer UV–Vis spectrometer
Lambda 35 fitted with a PTP-6 temperature programmer.
All ODNs were measured in a buffer consisting of 140 mM
NaCl, 10 mM sodium phosphate, 1 mM EDTA, pH 7.0,
with a concentration of 1.5 mm3 each strand. The solutions
were heated to 80 °C for 5 min and cooled to 5 °C and
were then kept at this temperature for 10 min. The melting
temperature (Tm, °C) was determined as the maximum of the
first-derivative plots of the melting curves obtained by absor-
bance at 260 nmagainst increasingtemperature (1.0 °C/min).
All melting temperatures are within the uncertainly 1.0 °C
as determined by repetitive experiments.
1
colorless oil. H NMR (300 MHz, CDCl3): d = 1.08–1.17
(m, 12H, 2 9 CH(CH3)2), 2.20–2.30 (m, 2H, CH2CH2O),
2.40 (t, 2H, J = 6.6 Hz, CH2CN), 3.15–3.20 (m, 1H,
CHHODMT), 3.33–3.40 (m, 1H, CHOP), 3.54–3.80 (m,
10H, 2 9 OCH3, 2 9 CH(CH3)2, CH2CH2OP), 4.30–4.50
(m, 3H, CHHODMT, CH2O), 6.73–6.85 (m, 4H, DMT),
7.15–7.40 (m, 7H, arom), 7.45–7.57 (m, 3H, arom), 7.86–
8.15 (m, 7H, arom), 8.37–8.45 (m, 1H, arom) ppm; 13C
NMR (75 MHz, CDCl3): d = 20.10 (CH2CN), 24.49,
24.61, 24.70 (2 9 CH(CH3)2), 33.51 (CH2CH2O), 43.07,
43.23 (2 9 C(CH3)2), 55.12 (2 9 OCH3), 58.04
(OCH2CH2CN), 64.98 (CH2O), 66.50 (CHOP), 71.15
(CH2ODMT), 86.02 (OCPh3), 109.04, 113.00, 121.27,
124.10–130.13, 136.08, 144.96, 158.37 (arom, DMT) ppm;
31P NMR (121.5 MHz, CDCl3): d = 149.71, 150.18 ppm
Fluorescence measurements
in
a ratio of 5:4; HRMS (ESI): m/z = 831.3542
([M ? Na]?, calcd.: 831.3533).
The fluorescence measurements were measured on a Per-
kin-Elmer LS-55 luminescence spectrometer fitted with a
Julabo F25 temperature controller set at 10 °C in a buffer
consisting of 140 mM NaCl, 10 mM sodium phosphate,
1 mM EDTA, pH 7.0, with a concentration of 1.5 mm3
each strand. Excitation wavelength was set to 340 nm and
detection at 360–600 nm. The excitation and emission slits
were set to 4 and 2.5 nm, respectively.
Oligonucleotide synthesis, purification, and melting
temperature determination
DMT-on oligodeoxynucleotides (ODNs) were prepared at
˚
0.2 lmol scales on 500 A CPG supports with an Expe-
diteTM Nucleic Acid Synthesis System Model 8909 from
Applied Biosystems, using 1H-tetrazole as an activator for
the coupling reaction. A 0.05 mM solution of the phos-
phoramidite 5 in anhydrous CH2Cl2 was used and inserted
into the growing oligonucleotides by using an extended
coupling time of 10 min. DMT-on oligonucleotides bound
to CPG supports were treated with 1 cm3 aqueous ammo-
nia (32%) at room temperature and then at 55 °C
overnight. Purification of 50-O-DMT-on ONs was accom-
plished by reversed-phase semipreparative HPLC on a
Waters Xterra MS C18 column with a Waters Delta Prep
4000 Preparative Chromatography System; buffer A =
0.05 M triethylammonium acetate in water (pH 7.4), buffer
B = 75% CH3CN in water, flow = 2.5 cm3 min-1, using the
following gradient: 2 min 100% A, linear gradient to
70% B in 38 min, linear gradient to 100% B in 3 min,
and then 100% A in 10 min. ODNs were DMT-deprotected
in 100 mm3 80% acetic acid for 20 min. Afterwards,
Acknowledgments This work was supported by the Sixth Frame-
work Program Marie Curie Host Fellowships for Early Stage
Research Training under contract number MEST-CT-2004-504018
and by the Nucleic Acid Center, which is funded by The Danish
National Research Foundation for studies on nucleic acid chemical
biology.
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