Synthesis and DNA binding properties of a new benzo[f]imidazo[1,5b]- isoquinoline-butan-1,2-diol derivative

Article abstract of DOI:10.1080/15257770600918524

A benzo[f]imidazo[1,5b]-isoquinoline derivative 4 with a 1,2-butandiol linker was prepared by reaction of a trimethylsilylated 5-naphthylidenehydantoin 3 with a 2,3-dideoxy-D-glycero-pentafuranoside 2 in 22% yield. After deprotection, the resulting compou

Full text of DOI:10.1080/15257770600918524

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Synthesis and DNA Binding Properties
of a New Benzo[f]Imidazo[1,5b]-
Isoquinoline-Butan-1,2-Diol Derivative
Youssef L. Aly a & Erik B. Pedersen a
a Nucleic Acid Center, Department of Chemistry , University of
Southern Denmark , Odense M, Denmark
Published online: 22 Dec 2010.
To cite this article: Youssef L. Aly & Erik B. Pedersen (2006) Synthesis and DNA Binding Properties of
a New Benzo[f]Imidazo[1,5b]-Isoquinoline-Butan-1,2-Diol Derivative, Nucleosides, Nucleotides and
Nucleic Acids, 25:12, 1335-1344, DOI: 10.1080/15257770600918524
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Nucleosides, Nucleotides, and Nucleic Acids, 25:1335–1344, 2006
Copyright ꢀC Taylor & Francis Group, LLC
ISSN: 1525-7770 print / 1532-2335 online
DOI: 10.1080/15257770600918524
SYNTHESIS AND DNA BINDING PROPERTIES OF A NEW
BENZO[f]IMIDAZO[1,5b]-ISOQUINOLINE-BUTAN-1,2-DIOL
DERIVATIVE
Youssef L. Aly and Erik B. Pedersen
Nucleic Acid Center, Department of
2
Chemistry, University of Southern Denmark, Odense M, Denmark
A benzo[f]imidazo[1,5b]-isoquinoline derivative 4 with a 1,2-butandiol linker was prepared
2
by reaction of a trimethylsilylated 5-naphthylidenehydantoin 3 with a 2,3-dideoxy-D-glycero-
pentafuranoside 2 in 22% yield. After deprotection, the resulting compound 5 was converted
to a DMT protected phosphoramidite building block 7 for standard DNA synthesis. DNA/DNA,
DNA/RNA duplexes with 5 inserted as bulges were destabilized, except when the new amidite was
used for the synthesis of a zipping duplex.
Keywords Intercalating nucleic acids; Benzo[f]imidazo[1,5b]-isoquinoline-butan-1,2-
diol; DNA duplex destabilization; Fluorescence
INTRODUCTION
A large number of nonnatural analogues of DNA have been synthesized
in recent years. Changing the structure of the base moiety has been a
useful strategy for improving the structure and the function in DNA.
For example, a number of bases have been used to test the importance
of specific hydrogen bonding interactions which may be important for
function of natural nucleic acid bases,[1–3] using this strategy, workers have
examined the importance of hydrogen bonding in stabilizing DNA and RNA
structure,[1] in protein-DNA interaction,[2] and in the fidelity of enzymatic
DNA and RNA synthesis.[3]
The presence of a large aromatic system can lead to a higher affinity for
single stranded DNA and double stranded DNA as it has been shown for
Received 20 December 2005; accepted 5 May 2006.
The Nucleic Acid Center is a research center funded by the Danish National Research Foundation
for studies on nucleic acid chemical biology.
Youssef L. Aly is on leave from Chemistry Department, Faculty of Education Kafr El-Sheikh Branch,
Tanta University, Kafr El-Sheikh, Egypt.
Mr. Michael Wamberg is gratefully acknowledged for his help on oligo synthesis.
Address correspondence to Erik B. Pedersen, Nucleic Acid Center, Department of Chemistry, Uni-
versity of Southern Denmark, Campusvej 55, DK-5230 Odense M, Denmark. E-mail: ebp@chem.sdu.dk.
1335

1336
Y. L. Aly and E. B. Pedersen
INA (intercalating nucleic acids)[4] and TINA (twisted intercalating nucleic
acids),[5] respectively. Moreover, the presence of the heterocycle may have
an effect on the electrostatic properties of the chromophore, which could
improve the affinity and sequence selectivity of this kind of intercalator.
Previously, we have designed a route for the synthesis of a condensed
isoquinoline derivatives, through a condensation reaction of a naphthyli-
deneimidazoledione with a deoxyribose derivative.[6] We now have devel-
oped this class of compounds for the insertion into DNA as a new example
of an intercalating nucleic acid.
RESULTS AND DISCUSSION
Methyl 2,3-dideoxy-5-O-(4-phenylbenzoyl)-D-glycero-pentafuranoside
(2) was prepared as described in the literature[7] by treatment of
2-deoxyribose (1) with HCl in methanol and subsequently with
4-phenyl benzoyl chloride in pyridine followed by acylation with phenyl
chlorothiocarbonate and deoxygenation by tri-n-butyltin hydride. The
trimethylsilylated naphthylidene-hydantoin derivative 3[6] was condensed
as devised by Vorbru¨ggen et al.[8,9] with the protected dideoxy sugar 2 in the
presence of trimethylsilyl trifluoromethanesulfonate (TMS triflate) at 5◦C
for 3 days. The reaction resulted in a ring closure reaction and the pseudo
nucleoside 4 was isolated in 22% yield as pure stereoisomer, however,
the configuration of C-7 is not known. The structure of compound 4 was
1
confirmed by H, 13 C-NMR, and mass spectroscopy and the spectroscopic
data are in agreement with analogous compound synthesized from a
2-deoxyribose derivative by the group of Pedersen et al.[6]
Removal of the protecting biphenylcarbonyl group from the glycon
moiety in 4 with methanolic ammonia at room temperature furnished the
diol derivative 5 (Scheme 1).
The primary hydroxy group of compound 5 was protected by
reaction with 4,4ꢁ-dimethoxytrityl chloride (DMTCl) in CH2Cl2 at
room temperature to give the DMT-protected compound 6, which
was converted to the phosphoramidite 7 with 2-cyanoethyl-N,N,N’,N’-
tetraisopropylphosphordiamidite (Scheme 2).
The amidite 7 was used for the synthesis of oligos, which all showed
correct masses in MALDI-TOF-MS analysis (Table 1).
The new modified oligos were hybridized toward complementary DNA
and RNA in such a way that the pseudonucleoside 5 formed bulges and
the duplexes were evaluated by thermal denaturation studies. The melting
temperatures (Tm) were determined as the maxima of the first derivatives of
the melting curve and they are listed in Tables 2 and 3.
The isoquinoline derivative 5 was found to destabilize the DNA/DNA
duplexes by −1.5◦C to −7◦C when inserted as a bulge into the central
region of the sequence (Table 2, entries 2 and 3). The stability was strongly
dependent on the sequence so that a pyrimidine rich neigborhood of the

Synthesis and DNA Binding Properties
1337
SCHEME 1 Synthesis of compound 5.
pseude nucleoside resulted in a substantially higher destabilization. The
destabilization was further enhanced for intercalating nucleic acid having
double insertions of 5 separated by 1–4 nucleobases as deduced from ꢀTm
−11.5◦C to –13◦C (Table 2, entries 4–6).
SCHEME 2 Synthesis of DMT-protected amidite 7.

1338
Y. L. Aly and E. B. Pedersen
TABLE 1 Masses of the oligos
Entry
Sequence
Expected mass
MALDI TOF analysis
1
2
3
4
5
5ꢁ-CTCAAG5CAAGTC-3ꢁ
5ꢁ-AGCTTG5CTTGAG-3ꢁ
5ꢁ-CTCAA5G5CAAGCT-3ꢁ
5ꢁ-CTCA5AG5CAAGCT-3ꢁ
5ꢁ-CTCA5AGCA5AGCT-3ꢁ
4012.3
4074.3
4411.3
4411.3
4411.3
4010.0
4076.3
4415.5
4415.0
4414.3
For RNA a similar destabilization and sequence dependency was ob-
served (Table 3). A destabilization of the DNA/RNA duplex was only –1◦C
when 5 was inserted as a bulge in a purine rich region (Table 3,entry 2),
while large destabilizations (−10◦C to −14◦C) were observed in all cases for
double insertion (Table 3, entries 3–5).
In Table 4, it is investigated whether insertion of 5 opposite to a
bulge can compensate for the missing natural nucleobase. This often is
observed for pseudonucleotides with intercalating properties. However,
in this case absolutely no extra stabilization was achieved by inserting 5
opposite to bulging nucleobases. It seems that the structure of 5 is too
large and, therefore, causes steric interactions for intercalation. However, it
is interesting to note that the stabilization is maintained when 2 modified
nucleotides 5 are opposing each other in a zipping manner. In fact the
melting temperature was increased by 1◦C in this case. Besides this case, the
steric requirement of 5 only seems to allow intercalation in case of purine
rich oligos as shown for entries 2 in Table 2 and 3 as deduced from marginal
decreases in melting temperature.
TABLE 2 Melting temperatures of DNA/DNA duplexes with compound 5 inserted as a bulge at pH
= 7
Entry
Sequence
Tm (◦C)
ꢀ Tm (◦C)
1
2
3
4
5
6
5ꢁ-AGCTTG CTTGAG-3ꢁ
3ꢁ-TCGAAC GAACTC-5ꢁ
5ꢁ-AGCTTG CTTGAG-3ꢁ
3ꢁ-TCGAAC5GAACTC-5ꢁ
5ꢁ-AGCTTG5CTTGAG-3ꢁ
3ꢁ-TCGAAC GAACTC-5ꢁ
5ꢁ-AGCTTG C TTGAG-3ꢁ
3ꢁ-TCGAAC 5 G 5AACTC-5ꢁ
5ꢁ-AGCT TGCT TGAG-3ꢁ
3ꢁ-TCGA5ACGA5ACTC-5ꢁ
5ꢁ-AGCT TGCT TGAG-3ꢁ
3ꢁ-TCGA5ACGA5ACTC-5ꢁ
46
—
44.5
39
−1.5
−7
34.5
33.5
33.0
−11.5
−12.5
−13

Synthesis and DNA Binding Properties
1339
TABLE 3 Melting temperatures of DNA/RNA duplexes with 5 inserted as a bulge at pH = 7
Entry
Sequence
Tm (◦C)
ꢀ Tm (◦C)
—
1
2
3
4
5
5ꢁ-AGCUUG CUUGAG-3ꢁ
3ꢁ-TCGAAC GAACTC-5ꢁ
5ꢁ-AGCUUG CUUGAG-3ꢁ
3ꢁ-TCGAAC5GAACTC-5ꢁ
5ꢁ-AGCUUG C UUGAG-3ꢁ
3ꢁ-TCGAAC5G5AACTC-5ꢁ
5ꢁ-AGCUUG CU UGAG-3ꢁ
3ꢁ-TCGAAC5GA5ACTC-5ꢁ
5ꢁ-AGCU UGCU UGAG-3ꢁ
3ꢁ-TCGA5ACGA5ACTC-5ꢁ
40
39
−1
29.5
29
−10.5
−11
26
−14
FLUORESCENCE
The fluorescence emission upon excitation of 5 inserted into single
strands was found at ≈460 nm. The highest fluorescence intensity was
recorded for single strands when 5 was inserted 2 times as bulges, whereas
the lowest fluorescence intensity was observed for the corresponding du-
plexes (Figure 1). This can be taken as an evidence of intercalation of 5
on duplex formation. No eximer band was observed for 5 when inserted
1 or 2 times in the sequence as it was founded by Lewis and coworkers for
naphthalene base analogue in 1996.[10]
CONCLUSION
Through thermal melting temperature studies we have shown that
insertion of 5 in both strands of a DNA duplex in a zipping manner
TABLE 4 Melting temperature of DNA/DNA duplexes with compound 5 as a universal base or with
zipping of 5 at pH = 7
5ꢁ−AGCTTG Y CTTGAG −3ꢁ
3ꢁ−TCGAAC X GAACTC −5ꢁ
Entry
Y, X
Tm
ꢀTm
1
2
3
4
5
—, —
5, 5
A, —
A, 5
C, —
C, 5
G, —
G, 5
46.0
47.0
31.5
30.4
33.5
32.9
33.7
32.9
33.4
32.9
—
+ 1
—
− 1.1
—
− 0.6
—
− 0.8
T, —
T, 5
—
− 0.5

1340
Y. L. Aly and E. B. Pedersen
FIGURE 1 Fluorescence measurement of single strand DNA and its duplexes with complementary DNA.
Numbering refers to entries in Table 1. The duplexes are found in entries 4 and 5 in Table 2.
stabilized the duplex whereas this was not the case on insertion of 5 as bulges
in DNA/DNA or DNA/RNA duplexes.
EXPERIMENTAL
NMR Spectra were recorded on a Varian Gemini 2000 spectrometer
(1 H 300 MHz, 13 C 75.5 MHz, 31P 121.5 MHz); δ values in ppm relative to
Me4Si as internal standard (1 H NMR); for 13 C NMR: CDCl3 (δ 77.0), DMSO
(δ 39.44). Accurate-ion mass determinations were performed using the 4.7 T
Ultima Fourier transform (FT) mass spectrometer (IonSpec, Irvine, CA,
USA). All modified oligos were confirmed by MALDI-TOF MS analysis on a
Voyager Elite Biospectrometry Research Station from PerSeptive Biosystems.
The (M+H)+ and (M+Na)+ ions were peak matched using ions derived
from the 2,5-dihydroxybenzoic acid matrix. The progresses of reactions were
monitored by TLC (analytical silica gel plates 60 F 254 Merck), visualized by
UV light (254 nm). Column chromatography (CC) silica-gel-packed column
(silica gel 60,0.040–0.063 mm, Merck, Darmstacht, Germany). Solvents used
for column chromatography were distilled prior to use, while reagents were
used as purchased.
Silylated of 5-naphthylidenehydantoin (3). A mixture of the 5-naphthylidene-
hydantoin (1.9 g, 8.0 mmol), anhydrous (NH4)2SO4 (0.005 g, 0.04 mmol),
and 1,1,1,3,3,3-hexamethyldisilazane (60 mL) was refluxed overnight. The

Synthesis and DNA Binding Properties
1341
clear solution obtained was cooled and the excess of solvent was evaporated
in vacuo to give the silylated compound as pale yellow oil.
7-[(S)-4-(p-Biphenylcarbonyloxy)-3-hydroxybutyl]-7 H-7 a, 9-diazacyclopenta-[b]-
phenanthrene-8,10-dione (4). A solution of the sugar 2 (1.68 g, 5.4 mmol)
in dry MeCN (20 mL) was added to a stirred solution of the silylated
5-naphthylidenehydantoin 3 in dry MeCN (30 mL) and the mixture was
cooled to −50◦C. A solution of trimethylsilyl trifluoromethanesulfonate
(1.08 mL, 6 mmol) in dry MeCN (10 mL) was added dropwise during 5 min
at −50◦C and the mixture was stirred at −30◦C for 4 hours and then at
5◦C for 2 days. The reaction mixture was diluted with CH2Cl2 (200 mL),
washed with cold saturated aqueous NaHCO3 (200 mL) and water (2 ×
100 mL), and dried over anhydrous MgSO4. The mixture was concentrated
in vacuo and the residue was chromatographed on a silica gel column with
CHCl3/MeOH (95:5, v/v) to afford compound 4 (0.8 g, 22%) as a yellow
foam. 1 H-NMR (DMSO-d6) δ 1.25−1.14 (m, 2 H, H-2ꢁ), 1.42−1.44 (m, 1 H,
H-1ꢁ), 1.97−2.01 (m, 1 H, H-1ꢁ), 4.01−4.02 (br, s, 1 H, OH), 4.22−4.42 (m,
3 H, H-3ꢁ, H-4ꢁ), 6.75−6.79 (m, 1 H, H-7), 7.17 (s, 1 H, H-11), 7.45−8.09 (m,
15 H, Harom), 9.35 (s, 1 H, NH); 13 C-NMR (DMSO-d6) δ 16.33 (C-1ꢁ), 21.97
(C-2ꢁ), 58.22 (C-7), 68.70 (C-4ꢁ), 74.14 (C-3ꢁ), 115.20 (C-11), 118.75, 123.53,
125.68, 126.17, 126.54, 126.95, 127.17, 127.38, 127.84, 128.39, 128.49,
128.64, 128.82, 129.06, 129.32, 129.53, 129.49, 130.216, 131.02, 138.71,
138.79, 144.63 (Carom), 157.26, 163.53, 165.17 (3 × C = O).
7-[(S)-3,4-dihydroxybutyl]-7 H-7 a,
9-diazacyclopenta[b]phenanthrene-8,10-
dione (5). Compound 4 (0.67 g, 1.3 mmol) was stirred with saturated
ammonia in methanol (100 mL) for 3 days at room temperature. The
solvent was evaporated in vacuo and the residue was chromatographed
on a silica gel with CHCl3/MeOH (95:5, v/v) to afford compound 5.
Yield: 0.2 g (44%); yellow foam; 1 H-NMR (DMSO-d6) δ 1.06−1.14 (m,
2 H, H-2ꢁ), 1.44−1.44 (m, 1 H, H-1ꢁ), 1.99−2.03 (m, 2 H, H-1ꢁ), 3.04−3.16
(m, 3 H, H-3ꢁ, H-4ꢁ), 4.42−4.52 (br, s, 2 H, 2 OH), 5.46−5.51 (m, 1 H,
H-7), 6.88 (s, 1 H, H-11), 7.56−8.01 (m, 6 H, Harom), 11.57 (s, 1 H, NH);
13 C-NMR (DMSO-d6) δ 30.07 (C-1ꢁ), 31.11 (C-2ꢁ), 58.43 (C-7), 65.60
(C-4ꢁ), 70.42 (C-3ꢁ), 112.20 (C-11), 125.88, 125.98, 127.82, 128.52, 129.41,
129.80, 130.45, 133.66, 135.16, 135.25 (Carom), 154.41, 163.63 (2 × C = O).
HRMS (MALDI) m/z Calcd for C19H18N2O4 [M+ + Na] 361.1162, found
361.1159.
7-[(S)-4-(4,4’-Dimethoxytrityloxy) 3-hydroxybutyl]-7 H-7 a, 9-diazacyclopenta[b]-
phenanthrene-8,10-dione (6). To compound 5 (0.915 g, 2.71 mmol) in dry
CH2Cl2 (40 mL) under nitrogen was added dry Et3 N (0.35 mL, 3.26 mmol),
followed by addition of 4,4ꢁ-dimethoxytrityl chloride (1.1 g, 3.26 mmol).
The reaction mixture was stirred at room temperature under nitrogen for
48 hours. The solvent was removed in vacuo, and the residue was purified
by column chromatography using 4:5:1 EtOAc-petroleum ether (60–80◦C)-
Et3 N to give compound 6. Yield 1.18 g (67%); yellow foam; 1 H-NMR

1342
Y. L. Aly and E. B. Pedersen
(CDCl3) δ 1.22−1.28 (m, 2 H, H-2ꢁ), 2.74−2.77 (m, 1 H, H-1ꢁ), 2.83−2.85
(m, 1 H, H-1ꢁ), 3.52 (s,1 H, OH), 3.71, 3.72 (2 s, 6 H, 2 × OCH3), 3.77−3.78
(m, 2 H, H-4ꢁ), 4.10−4.13 (m, 1 H, H-3ꢁ), 5.48 (m, 1 H, H-7), 7.18−7.92 (m,
20 H, Harom), 7.82 (s, 1 H, NH); 13 C-NMR (CDCl3) δ 29.66 (C-1ꢁ), 30.34
(C-2ꢁ), 55.65 (OCH3), 59.13 (C-7), 69.42 (C-4ꢁ), 74.98 (C-3ꢁ), 85.91 (C-Ar3),
112.81 (C-11), 113.08, 126.72, 127.80, 128.17, 129.77, 135.80, 144.69, 158.01
(DMT), 125.78, 126.42, 127.47, 127.96, 129.15, 129.86, 129.93, 130.62,
134.51, 142.69 (Carom), 158.51, 163.35 (2 × C = O). HRMS (MALDI) m/z
Calcd for C40H36N2O6 [M+ + Na] 663.2466, found 663.2463.
7-[(S)-3-O-(2-Cyanoethoxy(diisopropylamino)phosphino)-4-(4,4ꢁ-dimethoxytrityl-
oxy)butyl]-7 H-7 a, 9-diazacyclopenta[b]phenanthrene-8,10-dione (7). Compound
6 (0.17 g, 0.27 mmol) was dissolved under nitrogen in dry CH2Cl2
(10 mL). N,N -Diisopropylammonium tetrazolide (97 mg, 0.56 mmol)
was added, followed by dropwise addition of 2-cyanoethyltetraisopropyl-
phosphordiamidite (190 mg, 0.59 mmol). After 30 hours analytical TLC
showed the absence of starting material, and the reaction was quenched
with H2O (1 mL), followed by addition of CH2Cl2 (10 mL). The mixture was
washed with saturated. aqueous. NaHCO3 (2 × 10 mL). The organic phase
was dried over MgSO4, and filtered, and the solvent was evaporated under
reduced pressure. The residue was purified by column chromatography
with 4:5:1 EtOAc-petroleum ether (60–80◦C)-Et3 N to afford compound 7.
1
Yield: 120 mg (53%); yellow foam; H-NMR (CDCl3) δ 0.91–0.93 (m,
2 H, H-2ꢁ), 1.06–1.08 (m, 12 H, 4 × CH3[Pri]), 1.23–1.25 (m, 2 H, H-1ꢁ),
2.03–2.04 (m, 2 H, CH2CN), 3.36–3.41 (m, 2 H, OCH2CH2CN), 3.47−3.54
(m, 2 H, 2 × CH(CH3)2), 3.72 (s, 6 H, 2 × OCH3), 3.74–3.75 (m, 2 H,
H-4ꢁ), 4.10–4.12 (m, 1 H, H-3ꢁ), 5.44 (m, 1 H, H-7), 6.72–7.81 (m, 20 H,
Harom), 8.68 (s, 1 H, NH); 13 C-NMR (CDCl3) δ 14.15 (CH2CN), 19.97
(CH2CH2), 24.31, 24.40, 24.49, 24.55 (4 × CH3), 29.59, 29.85 (C-1ꢁ, C-2ꢁ),
55.10 (OCH3), 59.51 (C-7), 65.24 (C-4ꢁ), 73.51(C-3ꢁ), 86.08 (C-Ar3), 112.78
(C-11), 117.54 (CN), 112.98, 126.57, 127.87, 128.44, 129.91, 135.88, 144.94,
158.03, (DMT), 125.69, 126.40, 127.45, 127.70, 128.60, 129.85, 130.53,
133.55, 136.04, 142.80 (Carom), 158.24, 163.24 (2 × C = O); 31P-NMR
(CDCl3) δ 148.65 (s), 149.03 (s) in ratio 5:4, respectively. HRMS (MALDI)
m/z Calcd for C49H52N4O7P [M+ + K] 879.3288, found 879.3663.
Synthesis and Purification of Oligos
Oligodeoxynucleotides (ODN) were synthesized on an Expedite Nucleic
Acid Synthesis System model 8909 from Applied Biosystems (Foster City,
CA, USA). The phosphoramidite was dissolved in dry MeCN, as a 0.75 M
solution and incorporated into the growing oligonucleotide chain using
elongated coupling times (10 minutes coupling versus 2 minutes for nor-
mal nucleotide couplings). After the completed DNA syntheses, the 5ꢁ-O-
DMT-on oligonucleotides were cleaved off from the solid support (room

Synthesis and DNA Binding Properties
1343
temperature, 2 hours) and deprotected (55 ◦C, overnight) using 32% aque-
ous 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, and a Waters 717 autosampler on a Waters Xterra MS C18
column. Buffer A, 0.05 M triethyl ammonium acetate in H2 O, pH 7.4; buffer
B, 75% CH3CN in H2 O; flow 2.5 ml/minutes. Gradients, 2 minutes 100%
A, linear gradient to 100% B in 28 minutes, 100% B in 10 minutes, linear
gradient to 100% A in 1 minute, and then 100% A in 9 minutes. The ODNs
were DMT deprotected in 100 µl 80% aq. AcOH (20 minutes), diluted with
1 M aq. NaOAc (150 µl) and precipitated from ethanol (600 µl).
Melting Temperature Measurements
The thermal stability studies were performed on a Perkin-Elmer UV/VIS
spectrometer Lambda 20 (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
Na2HPO4·2H2 O, 140 mM NaCl buffer at pH 7.0 for 1.0 µM of each strand.
The melting temperature was determined as the maximum of the first
derivative plots of the melting curves obtained by measuring absorbance
at 260 nm against increasing temperature (1.0◦C/minute) and is with an
uncertainty +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 389 nm and
detection at 395–595 nm. All measurements were conducted at 10◦C using
the same concentrations and buffer conditions as for the melting studies.
REFERENCES
1. SantaLucia, J. Jr.; Kierzek, R.; Turner, D.H. Context dependence of hydrogen bond free energy
revealed by substitutions in an RNA hairpin. Science 1992, 256, 217–219.
2. (a) Smith, S.A.; Rajur, S.B.; McLaughlin, L.W. Specific purine N7-nitrogens are critical for high
affinity binding by the Trp repressor. Nature Struct. Biol. 1994, 1, 198. (b) Lesser, D.R.; Kurpiewski,
M.R.; Jen-Jacobson, L. The energetic basis of specificity in the Eco RI endonuclease-DNA interaction.
Science 1990, 250, 776–786.
3. (a) Kornberg, A.; Baker, T.A. DNA Replication, (W.H. Freeman, New York, 1992), 2nd ed. (b) Echols,
H.; Goodman, M.F. Fidelity mechanisms in DNA replication. Annu. Rev. Biochem. 1991, 60, 477–511.
(c) Strazewski, P.; Tamm, C. Replication experiments with nucleotide base analogs. Angew. Chem.
1990, 102, 37–59.
4. Christensen, U.B.; Pedersen, E.B. Intercalating nucleic acids containing insertions of 1-O-(1-
pyrenylmethyl)glycerol: stabilisation of dsDNA and discrimination of DNA over RNA. Nucl. Acids
Res. 2002, 30, 4918–4925.

1344
Y. L. Aly and E. B. Pedersen
5. Filichev, V.V.; Pedersen, E.B. Stable and selective formation of Hoogsteen-type triplexes and
duplexes using Twisted Intercalating Nucleic Acids (TINA) prepared via postsynthetic Sonogashira
solid-phase coupling reactions. J. Am. Chem. Soc. 2005, 127, 14849–14858.
6. El-Barbary, A.A.; Khodair, A.I.; Pedersen, E.B. Hydantoin analogs of thymidine. J. Org. Chem. 1993,
58, 5994–5999.
7. Motawia, M.S.; Pedersen, E.B. A new route to 2ꢁ,3ꢁ-dideoxycytidine. Liebigs Ann. Chem. 1990, 6, 599–
602.
8. Vorbru¨ggen, H.; Krolikiewicz, K.; Bennua, B. Nucleosides syntheses 22. Nucleosides syntheses with
trimethylsilyl triflate and perchlorate as catalysts. Chem. Ber. 1981, 114, 1234–1235.
9. Vorbru¨ggen, H.; Bennua, B. Nucleosides syntheses 25. a new simplified nucleosides syntheses. Chem
Ber. 1981, 114, 1279–1286.
10. Lewis, F.; Zhang, Y.; Liu, Xiaoyang.; Xu, Ning.; Letsinger, R. Naphthalenedicarboxamides as
fluorescent probes of inter- and intramolecular electron transfer in single strand, hairpin, and
duplex DNA. J. Phys. Chem. B 1999, 103, 2570–2578.