Synthesis and biophysical properties of oligodeoxynucleotides containing 2′-deoxy-5-(4-nitro-1H-imidazol-1-yl)-β-D-uridine and 2′-deoxy-5-(1,3-dioxo-1H-benzo[de]isoquinolin-2(3H)-yl)-β-D-uridine monomers

Article abstract of DOI:10.1002/cbdv.200900195

Detection of single-nucleotide polymorphisms (SNPs) of biologically relevant DNA and RNA samples remain a scientific and practical challenge. We have synthesized phosphoramidite building blocks and oligodeoxynucleotide probes containing novel 2′-deoxyuridine monomers modified by 5-(4-nitro-1H-imidazol-1-yl; (monomer X) or 2′-deoxy-5-(1,3-dioxo-1H- benzo[de]isoquinolin-2(3H)-yl; monomer Y) substituents. The effects of monomers X and Y on duplex thermal stability, and their capability towards discrimination of single-base mismatches were furthermore studied. Encouraging results were obtained with respect to thermal mismatch discrimination using oligodeoxynucleotides containing monomer X and fluorescence-based discrimination using oligodeoxynucleotides containing monomer Y.

Full text of DOI:10.1002/cbdv.200900195

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CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
Synthesis and Biophysical Properties of Oligodeoxynucleotides Containing
2’-Deoxy-5-(4-nitro-1H-imidazol-1-yl)-b-d-uridine and 2’-Deoxy-5-(1,3-dioxo-
1H-benzo[de]isoquinolin-2(3H)-yl)-b-d-uridine Monomers
by Andrzej Gondelaa)b), Thatikonda Santhosh Kumarb), Krzysztof Walczaka), and Jesper Wengel*b)
a) Department of Organic Chemistry, Biochemistry and Biotechnology, Selisian University of
Technology, Krzywoustego 4, PL-44-100 Gliwice
b) Nucleic Acid Center, Department of Physics and Chemistry, University of Southern Denmark,
Campusvej 55, DK-5230 Odense M (e-mail: jwe@ifk.sdu.dk)
Detection of single-nucleotide polymorphisms (SNPs) of biologically relevant DNA and RNA
samples remain a scientific and practical challenge. We have synthesized phosphoramidite building
blocks and oligodeoxynucleotide probes containing novel 2’-deoxyuridine monomers modified by 5-(4-
nitro-1H-imidazol-1-yl; (monomer X) or 2’-deoxy-5-(1,3-dioxo-1H-benzo[de]isoquinolin-2(3H)-yl;
monomer Y) substituents. The effects of monomers X and Y on duplex thermal stability, and their
capability towards discrimination of single-base mismatches were furthermore studied. Encouraging
results were obtained with respect to thermal mismatch discrimination using oligodeoxynucleotides
containing monomer X and fluorescence-based discrimination using oligodeoxynucleotides containing
monomer Y.
Introduction. – Detection of single-nucleotide polymorphisms (SNPs) has in the
last decade attracted much interest in the field of oligonucleotide chemistry [1].
Related hereto are base-pair mutations of gene sequences which may result in
modulation of gene expression and eventually disease [2]. Analysis of mutations and
SNPs may be performed by the use of oligonucleotide probes. The majority of existing
methods embark on differences in hybridization efficiency of matched and mismatched
oligonucleotide probes and the target sequence, and the use of a fluorescent reporter
group [3]. The reporter group can be attached directly to the sugar or the nucleobase,
or it can be a planar fluorophore which is able to structurally mimic the nucleobase [4].
Ideally, SNPs and mutations are detected by monitoring increased fluorescence of the
probe as read-out, either in case of fully matched hybridization between probe and
target [5], or if a mismatched base pair is present in the duplex [6]. Alternatively, if
hybridization, i.e., duplex formation, is dependent of a full match between probe and
target, the use of probes which fluoresce only when hybridized is an option [7]. Good
candidates for improved SNP probes are nucleotide monomers bearing modified
nucleobases which are directly conjugated to reporter groups [8]. Herein, we describe
synthesis, thermal denaturation, and fluorescence properties of novel oligodeoxynu-
cleotide (ODN) probes bearing a 4-nitro-1H-imidazol-1-yl or 1,3-dioxo-1H-benzo-
[de]isoquinolin-2(3H)-yl [9] substituent attached at C(5) of uracil base moieties.
Results and Discussion. – 5-Aminouracil (1) was used for synthesis of C(5)-
functionalized uracil derivatives 3a and 3b (Scheme 1). 5-(4-Nitro-1H-imidazol-1-
ꢀ 2010 Verlag Helvetica Chimica Acta AG, Zꢁrich

CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
351
yl)uracil (3a) was obtained in an Anrorc-type reaction of 1 with 1,4-dinitro-1H-
imidazole (2a) in DMSO as described in [10]. 5-(1,3-Dioxo-1H-benzo[de]isoquinolin-
2(3H)-yl)uracil (3b) was synthesized in 71% yield by reaction of 1 with naphthalene-
1,8-dicarboxylic anhydride 2b (Scheme 1). This reaction was performed in a mixture of
EtOH and DMSO in the presence of Et3N and DMAP. The use of the typical solvents
for such a transformation like glacial AcOH [11] or pyridine [12] were not efficient
because of poor solubility of 1 in these solvents.
Scheme 1
i) DMSO, r.t. ii) Et2N (TEA), 4-(dimethylamino)pyridine (DMAP), DMSO/EtOH 1:1 (v/v); 71%.
Coupling of C(5)-modified uracil 3a or 3b with 2-deoxy-1,3-di-O-acetyl-5-O-
propanoyl-d-ribofuranose (4) [13] was carried out in MeCN according to the
Vorbrꢀggen method [14] using TMSOTf as a catalyst (Scheme 2). The products were
isolated as inseparable anomeric mixtures of nucleoside derivatives 5a and 5b in 93 and
86% yield, respectively. Subsequent deacylation with MeONa in anhydrous MeOH,
followed by fractional crystallization from MeOH, afforded pure b-anomers 6a and 6b
in yields of 36 and 33%, respectively. The 5’-OH group of nucleosides 6a and 6b was
regioselectively protected using 4,4’-dimethoxytrityl chloride (DMTrCl) in anhydrous
pyridine. The appropriate nucleoside derivatives 7a and 7b were isolated in yields of 80
and 86%, respectively. Finally, O(3’)-phosphitylation using 2’-cyanoethyl N,N-diiso-
propylphosphoramidochloridite in the presence of Hꢀnigꢂs base furnished the
phosphoramidite building blocks 8a (83%) and 8b (73%) suitable for oligonucleotide
synthesis.
Synthesis of ODNs containing monomers X and Y (see Fig. 1 for structures, and
Tables 1, 2, and 3 for ODN sequences) was performed in 0.2 mmol scale using an
automated DNA synthesizer1). Standard reagents and procedures were used except for
extended coupling times (0.05m in MeCN, 15 min; 1H-tetrazole as a catalyst) for
phosphoramidites 8a and 8b. The stepwise coupling yields were >99% for unmodified
1
)
See Exper. Part.

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CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
Scheme 2
i) 1,1,1,3,3,3-Hexamethyldisilazane (HMDS), (NH4)2SO4, then MeCN, trimethylsilyl triflate (Tf¼
trifluoromethylsulfonyl; TMSOTf), ꢀ15 to 08; 5a: 93%, 5b: 86%. ii) a) MeONa/MeOH, 90 min, r.t.; b)
Amberlist IRC 120; 6a: 36%, 6b: 33%). iii) Bis(4-methoxyphenyl)(phenyl)methyl chloride (DMTrCl),
pyridine, r.t.; 7a: 80%, 7b: 86%. iv) NC(CH2)2OP(Cl)N(i-Pr)2, MeCN, EtN(i-Pr)2, r.t.; 8a: 83%, 8b:
73%. v) DNA Synthesizer.
DNA phosphoramidites, and ca. 99% for 8a and 8b. Removal of nucleobase-protecting
groups and cleavage from the solid support for ODNs containing monomer X (ON2,
ON3, and ON11) were achieved using standard conditions (32% aq. NH3 for 12 h at
558). In the case of ODNs containing monomer Y (ON4 and ON12), application of the
standard deprotection conditions resulted in substantial cleavage of the 1,3-dioxo-1H-
benzo[de]isoquinolin-2(3H)-yl moiety (up to 40% cleavage)2), resulting in formation
of by-products containing C(5)-amino-2’-deoxyuridine monomers. When a shorter
deprotection time was employed (up to maximum 6 h at 558), the undesired cleavage
product was still isolated (in up to a yield of 30%)2). The best results were obtained
when the deprotection was carried out at room temperature for 24 h leading to benzo-
isoquinolin-2-yl cleavage to an extent of only 5–10%. Previously, when 1,3-dioxo-1H-
benzo[de]isoquinolin-2(3H)-yl-protected dA, dG, and dC monomers were used for
2
)
Reported percentages are based on ion-exchange chromatography.

CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
353
synthesis of nucleotide dimers, the benzoisoquinolinyl moieties were cleaved com-
pletely using 40% aq. NH3 (508 for 4 h) [12]. Our results showed that the 1,3-dioxo-1H-
benzo[de]isoquinolin-2(3H)-yl moiety formed on 5-aminouracil is relatively stable and
can be successfully installed on ODN derivatives when using mild deprotection
conditions. The crude O5’-DMTr protected ODNs were purified by RP-HPLC,
followed by detritylation using 80% aq. AcOH for 20 min at room temperature and
EtOH precipitation, to afford the pure ODNs. The composition and purity of the
ODNs was confirmed by HR-MALDI-MS and ion-exchange HPLC, respectively1).
Fig. 1. Structures of monomers X and Y
Table 1. Thermal Denaturation Studiesa) of Nonamer ODNs Containing Monomer Xb) or Yb) towards DNA
and RNA Complements, and Singly Mismatched DNA Targetsc). Tm Valuesc) in 8 (DTm values in parentheses).
DNA: 3’-CAC TBT ACG
RNA
ON9
A
ON5
A
ON6
C
ON7
G
ON8
T
B:
ON1 5’-GTG ATA TGC
ON2 5’-GTG AXA TGC
ON3 5’-GXG AXA XGC
ON4 5’-GTG AYA TGC
28.5
12.0 (ꢀ 16.5)d) 19.0 (ꢀ 9.5)d) 11.5 (ꢀ 17.0)d) 26.5
23.0 (ꢀ 5.5)d) n.t.e)
n.t.e)
–f)
n.t.e)
–f)
19.0 (ꢀ 7.5)
n.t.e)
14.0 (ꢀ 14.5)f) –f)
–f)
n.t.e)
n.t.e)
–f)
–f)
a) Thermal denaturation temperatures were measured as the maximum of the first derivative of the melting
curve (A260 vs. temperature) recorded in medium salt buffer ([Naþ ]¼110 mm, [Clꢀ ]¼100 mm, pH 7.0
(NaH2PO4/Na2HPO4)), using 1.0 mm concentrations of the two complementary strands. b) See Fig. 1 for
structures of monomer X and Y. c) Tm Values are average values of at least two measurements. d) DTm Value
e
calculated relative to the DNA:DNA (ON1 :ON5) or DNA :RNA (ON1 :ON9) reference duplexes. ) No clear
f
transition observed above 108. ) Denaturation experiment not performed.
The effect of monomers X or Y on duplex stabilities under medium salt conditions
was evaluated by UV thermal denaturation experiments in nonamer (ON2–ON4,
Table 1) and 21-mer sequence contexts (ON11 and ON12; Tables 2 and 3). The thermal
denaturation curves of all modified duplexes displaying a thermal denaturation
temperature (Tm value; Tables 1–3) exhibited sigmoidal monophasic transition curves

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CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
with shapes similar to those obtained for the unmodified reference duplexes. A single
incorporation of monomer X into a nonamer ON (ON2; Table 1) resulted in a
substantial decrease in thermal stability of duplexes with complementary DNA or
RNA (ꢀ 5.5 and ꢀ7.58, respectively, when compared to the unmodified reference
duplexes formed with ON1). Incorporation of monomer X induces a less pronounced
stability-decreasing effect when incorporated into a 21-mer ODN (i.e., ON11; Tables 2
and 3). The affinity-decreasing effect of monomer X was further confirmed in the
nonamer context, as three incorporations of monomer X (i.e., ON3; Table 1) prevented
duplex formation towards both complementary DNA and RNA. Single incorporation
of monomer Y induced an even stronger affinity-decreasing effect for a nonamer
duplex with the DNA complement (ꢀ14.58 compared to the unmodified DNA
duplex), whereas no duplex was formed with complementary RNA (Table 1). A single
incorporation of monomer Y into the 21-mer ON, ON12, accordingly induced
substantial decreases in the thermal stabilities of the duplexes formed (ꢀ 5.58 against
DNA and ꢀ6.08 against RNA; Tables 2 and 3).
Table 2. Thermal Denaturation Studiesa) of 21-mer ODNs Containing Monomers X or Y towards Complementary
DNA and Singly Mismatched DNA Targets. Tm Valuesc) in 8C (DTm values in parentheses).
DNA: 3’-ACG TGA CAT BCA GAC ATG GTA
ON13
A
ON14
C
ON15
G
ON16
T
B:
ON10 5’-TGC ACT GTA TGT CTG TAC CAT
ON11 5’-TGC ACT GTA XGT CTG TAC CAT
ON12 5’-TGC ACT GTAYGT CTG TAC CAT
61.0
53.0 (ꢀ 8.0)b) 57.5 (ꢀ 3.5)b) 55.5 (ꢀ 5.5)b)
58.5 (ꢀ 2.5)b) 52.5 (ꢀ 6.0)c) 55.0 (ꢀ 3.5)c) 54.0 (ꢀ 4.5)c)
55.5 (ꢀ 5.5)b) 54.0 (ꢀ 1.5)d) 55.5 (0.0)d)
54.5 (ꢀ 1.0)d)
a) For conditions of thermal denaturation experiments, see legend of Table 1. b) DTm Values calculated relative
to duplex ON10 :ON13. c) DTm Values calculated relative to duplex ON11 :ON13. d) DTm Values calculated
relative to that of duplex ON12 :ON13.
Watson–Crick base-pairing selectivity of ONs containing a single incorporation of
monomers X or Y was studied by evaluating duplex formation with DNA and RNA
targets containing a singly mismatched nucleotide positioned directly opposite to the
centrally positioned X and Y monomers. It is noteworthy that ON2 containing
monomer X did not form duplexes with singly mismatched DNA target strands
(Table 1), and that the 21-mer ON with a single monomer X (ON11) displayed
satisfactory base-pairing selectivity approaching that of the unmodified ON10 against
both the DNA and RNA complements (Table 2 and 3). Incorporation of a single
monomer Y into the nonamer or 21-mer sequences induced poor base-pairing
selectivity based on the thermal denaturation effects observed (Tables 1–3). The
ability to discriminate a single base-mismatch target strands is one of the desirable
characteristics of nucleic acid probes for potential applications in therapeutics and
diagnostics [15]. The results obtained for monomer X suggest that ODNs containing
monomer X are promising probe candidates, and confirm the suitability of the 5-
position of uracil bases as a site of attachment of aromatic moieties.

CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
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Table 3. Thermal Denaturation Studiesa) of 21-mer ODNs Containing Monomers X or Y towards Complementary
RNA and Singly Mismatched RNA Targets. Tm Valuesc) in 8C (DTm values in parentheses).
RNA: 3’-ACG UGA CAU BCA GAC AUG GUA
ON17
A
ON18
C
ON19
G
ON20
U
B:
ON10 5’-TGC ACT GTA TGT CTG TAC CAT
ON11 5’-TGC ACT GTA XGT CTG TAC CAT
ON12 5’-TGC ACT GTAYGT CTG TAC CAT
63.0
54.5 (ꢀ 8.5)b) 60.5 (ꢀ 2.5)b) 54.0 (ꢀ 9.0)b)
59.0 (ꢀ 4.0)b) 53.0 (ꢀ 6.0)c) 57.0 (ꢀ 2.0)c) 54.0 (ꢀ 5.0)c)
57.0 (ꢀ 6.0)b) 54.0 (ꢀ 3.0)d) 55.5 (ꢀ 1.5)d) 54.5 (ꢀ 2.5)d)
a) For conditions of thermal denaturation experiments, see legend of Table 1. b) DTm Values calculated relative
to duplex ON10 :ON17. c) DTm Values calculated relative to duplex ON11 :ON17. d) DTm Values calculated
relative to that of duplex ON12 :ON17.
Monomer Y was selected for this study because of its fluorescence properties [9].
Steady-state fluorescence emission spectra1) of ON12 and the corresponding 21-mer
duplexes with matched and mismatched DNA or RNA targets were recorded in the Tm
buffer at 1083) using 1.0 mm of each ODN strand and an excitation wavelength of
343 nm (Fig. 2). The fluorescence-emission spectrum of single-stranded ON12 is
characterized by a broad unstructured band (lmax ca. 415 nm). Interestingly, increases
in the fluorescence emission intensity were observed upon hybridization with
complementary DNA and RNA. In addition, the duplex with DNA, i.e., ON12 :ON13,
displayed a significant red shift (ca. 15 nm) of its fluorescence emission maxima
compared to that of the duplex formed with complementary RNA, i.e., ON12 :ON17.
Fluorescence emission spectra of duplexes formed between the probe ON12, and
singly mismatched DNA and RNA targets are also included in Fig. 2. Duplexes with
Y:C and Y:T/U mismatches display moderately increased fluorescence intensity
relative to that of the single stranded ON12, but decreased fluorescence intensities
relative to the matched duplexes. Interestingly, Y:G mismatches constitute a special
case, as these duplexes displayed significant increases in the fluorescence intensities
(3.5- and 7.5-fold, resp.) compared to the single stranded ON12. In addition, it is
noteworthy that these Y:G mismatched duplexes, i.e., ON12 :ON15 and ON12 :ON19,
are characterized by having fluorescence maxima at 393 nm. Such a distinctive
fluorescence behavior reveals that monomer Y may render such ODN probes useful for
nucleic acid-based applications in line with other base-discriminating fluorescent
(BDF) probes [4].
Conclusions. – We have successfully synthesized 2’-deoxy-5-(nitroimidazolyl)- and
2’-deoxy-5-benzoisoquinolinyl)uridine phosphoramidite building blocks in four steps
starting from 1,3-O-diacetyl-2-deoxy-5-O-propanoyl-d-ribofuranose and 5-functional-
ized uracil derivatives. The obtained monomers X and Y were incorporated into
oligodeoxynucleotides according to standard procedures. Incorporation of these
monomers into nonamer and 21-mer oligodeoxynucleotides resulted in considerable
decreases of the thermal stability of duplexes with DNA or RNA complements.
Oligodeoxynucleotides containing the C(5)-(nitroimidazolyl)-ribofuranosyl monomer
3
)
Similar results were observed at 208.

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CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
Fig. 2. Steady-state fluorescence emission spectra of ON12 and its duplexes with complementary and
single-base-mismatched DNA (left) and RNA (right) targets
X show satisfactory thermal mismatch discrimination against single base-mismatched
DNA or RNA targets. Oligonucleotides containing the C(5)-(benzoisoquinolinyl)-
ribofuranosyl monomer Y display interesting mismatch discrimination based on
fluorescence emission, in particular against guanine mismatches in both DNA and
RNA targets. The straightforward synthesis and interesting single base mismatch-
discriminative power make oligodeoxynucleotides containing monomers X and Y
promising candidates for in vitro and in vivo applications.
Experimental Part
General. All reagents and solvents were of anal. grade, obtained from commercial suppliers and used
without further purification except for CH2Cl2, which was distilled prior to use. Anh. pyridine was used as
obtained from commercial suppliers. MeCN was dried through storage over activated 3-ꢃ molecular

CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
357
sieves. Anh. CH2Cl2, ClCHCH2Cl, EtN(i-Pr)2, 1,4-dioxane, and Et3N were dried through storage over
activated 4-ꢃ molecular sieves. Reactions were conducted under Ar, whenever anh. solvents were used.
All reactions were monitored by TLC using silica-gel-coated plates with a fluorescence indicator (SiO2
60, F-254), which were visualized a) by UV light and b) by dipping in 5% conc. H2SO4 in abs. EtOH (v/v),
followed by heating. Column chromatography (CC) was performed using silica gel 60-packed columns
(particle size 0.040–0.063 mm, Merck) and moderate pressure (pressure ball). Evaporation of solvents
was carried out under reduced pressure at a temp. below 508. After CC, appropriate fractions were
1
pooled, evaporated, and dried at high vacuum for at least 12 h. H-, 13C-, and 31P-NMR spectra were
recorded at 300, 75.5, and 121.5 MHz, resp., chemical shifts d are reported in ppm relative to Me4Si or
deuterated solvent as internal standard (d(H) CDCl3 7.26 ppm, (D6)DMSO 2.50 ppm; d(C) CDCl3
77.16 ppm, (D6)DMSO 39.52 ppm; 85% d(P) H3PO4 0 ppm); coupling constants Jare given in Hz; traces
of solvents in NMR spectra were identified by reference to published data [16]. HR-ESI-Mass spectra
were recorded in positive-ion mode on an IonSpec Fourier transform mass spectrometer with 2,5-
dihydroxybenzoic acid as a matrix; in m/z.
1-(3’-O-Acetyl-2’-deoxy-5’-O-propanoyl-d-ribofuranos-1’-yl)-5-(4’’-nitro-1H-imidazol-1’’-yl)-pyrimi-
dine-2,4-(1H,3H)-dione (5a). A mixture of 5-(4-nitro-1H-imidazol-1-yl)uracil (3a [10]; 0.45 g,
2.00 mmol), (NH4)2SO4 (20 mg, 0.15 mmol), and hexamethyldisilazane (HMDS; 10.0 ml, 47.0 mmol)
was heated under reflux with stirring for 14 h. The excess of HMDS was evaporated under reduced
pressure, and the resulting oily residue was dissolved in anh. MeCN (25 ml) and cooled to ꢀ158. 1,3-Di-
O-acetyl-2-deoxy-5-O-propanoyl-d-ribofuranose (4 [13]; 0.70 g, 2.50 mmol) was added, followed by
dropwise addition of trimethylsilyl triflate (TMSOTf; 0.17 ml, 1.0 mmol). The mixture was stirred at ꢀ
158 for 1 h and warmed to 08 during 1.5 h. Then, 5% aq. NaHCO3 (25 ml) was added, and the resulting
mixture was extracted with CH2Cl2 (3ꢁ25 ml). The combined org. phase was dried (Na2SO4),
evaporated to dryness, and the resulting brownish solid was purified by CC (0–5% MeOH in CHCl3 (v/v))
to furnish an anomeric mixture of nucleosides 5a (a/b 1:1 based on 1H-NMR signals). White solid
(0.82 g, 93%). Rf (10% MeOH in CH2Cl2 (v/v)) 0.44. 1H-NMR (CDCl3, Me4Si): 1.08 (t, J¼7.5, Me); 1.16
(t, J¼7.5, Me); 2.00 (s, Me); 2.12 (s, Me); 2.25–2.43 (m, CH2, HaꢀC(2’)); 2.56–2.64 (m, 1 HbꢀC(2’));
2.80–2.91 (m, 1 HbꢀC(2’)); 4.21 (d, J¼4.2, CH2(5’)); 4.26–4.31 (m, CH2(5’)); 4.48–4.54 (m, 1 HꢀC(4’));
4.74–4.78 (m, 1 HꢀC(4’)); 5.17–5.25 (m, 1 HꢀC(3’)); 5.25–5.29 (m, 1 HꢀC(3’)); 6.21–6.30 (m,
2 HꢀC(1’)); 7.72 (d, J¼1.5, 1 HꢀC(2’’)); 7.84 (d, J¼1.5, 1 HꢀC(2’’)); 8.03 (s, 1 HꢀC(6)); 8.08 (d, J¼1.5,
1 HꢀC(5’’)); 8.12 (s, 1 HꢀC(6)); 8.21 (d, J¼1.5, 1 HꢀC(5’’)); 11.57 (br. s, 2 NH). 13C-NMR (CDCl3,
Me4Si): 8.9; 8.9; 20.8; 21.0; 27.3 (2 C); 37.8; 38.5; 63.3; 63.4; 73.7; 74.1; 83.1; 84.9; 86.0; 87.9; 111.9; 112.7;
120.2; 120.3; 134.9; 135.4; 136.4 (2C); 147.3 148.9; 149.0; 158.6; 158.9; 169.7; 170.3; 173.7; 174.1. HR-ESI-
MS: 460.1080 ([MþNa]þ , C17H19N5NaO9þ ; calc. 460.1075).
1-(2’-Deoxy-b-d-ribofuranosyl-1’-yl)-5-(4’’-nitro-1H-imidazol-1’’-yl)-pyrimidine-2,4-(1H,3H)-dione
(6a). Nucleoside 5a (0.73 g, 1.73 mmol) was suspended in anh. MeOH (25 ml), followed by addition of
MeONa (0.40 g, 7.40 mmol). The mixture was stirred for 1.5 h, whereupon H2O (5 ml) and Amberlist IRC
120 (ca. 1 g) were added to obtain neutral pH. The resulting suspension was decanted, and the resin was
washed with MeOH (10 ml). The combined MeOH phase was evaporated to dryness. Fractional
crystallization of the residue from MeOH gave pure b-anomer 6a (168 mg). An additional portion of 6a
(40 mg) was isolated from the evaporated filtrate by CC (10% MeOH/CH2Cl2). Compound 6a was
obtained as a white solid material in a combined yield of 208 mg (36%). Rf (5% MeOH in CH2Cl2 (v/v))
0.11. 1H- and 13C-NMR spectra were identical to published data [10]. HR-ESI-MS: 362.0713 ([MþNa]þ
,
C12H13N5NaOþ7 ; calc. 362.0707).
1-{5’-O-[Bis(4-methoxyphenyl)(phenyl)methyl]-2’-deoxy-b-d-ribofuranosyl-1’-yl}-5-(4’’-nitro-1H-
imidazol-1’’-yl)-pyrimidine-2,4-(1H,3H)-dione (7a). Compound 6a (0.08 g, 0.23 mmol) was co-evapo-
rated with anh. pyridine (2ꢁ5 ml) and dissolved in anh. pyridine (1 ml). 4,4’-Dimethoxytrityl chloride
(DMTrCl; 100 mg, 0.3 mmol) was added, and the mixture was stirred at r.t. for 3 h, whereupon MeOH
(0.2 ml) was added, and stirring was continued for 20 min. The mixture was diluted with CH2Cl2 (10 ml)
and washed with 5% aq. NaHCO3 (1ꢁ5 ml). The aq. phase was extracted with CH2Cl2 (2ꢁ10 ml), and
the combined org. phase was dried (Na2SO4) and evaporated to dryness. The residue was purified by
silica-gel CC (0–5% MeOH in CH2Cl2, containing 0.5% Et3N (v/v/v)) to obtain 7a (111 mg, 80%). White
1
solid. Rf (5% MeOH in CH2Cl2 (v/v)) 0.21. H-NMR (CDCl3, Me4Si): 2.35–2.47 (m, HaꢀC(2’)); 2.55–

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CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
2.67 (m, HbꢀC(2’)); 3.30–3.40 (m, 2 CH2(5’)); 3.719 (s, 2 MeO); 3.12–3.17 (m, HꢀC(4’)); 4.66–4.72 (m,
HꢀC(3’)); 6.29 (t, J¼6.3, HꢀC(1’)); 6.70–6.74 (m, 4 arom. H); 7.00–7.23 (m, 11 arom. H, HꢀC(2’’));
7.48 (s, HꢀC(5’’)); 8.21 (s, HꢀC(6)). 13C-NMR (CDCl3, Me4Si): 44.9; 55.3; 55.4; 63.4; 72.2; 86.7; 87.7
(2 C); 112.4; 113.3 (2 C); 113.4 (2 C); 120.7; 127.4; 127.9 (2 C); 128.0 (2 C); 129.9 (4 C); 134.9; 135.1;
136.7; 143.9; 147.6; 149.7; 158.7 (2 C); 159.9. HR-ESI-MS: 664.2019 ([MþNa]þ , C33H31N5NaO9þ ; calc.
664.2014).
1-{5-O-[Bis(4-methoxyphenyl)(phenyl)methyl]-3’-O-(2-cyanoethoxy(diisopropylamino)phosphi-
no)-2’-deoxy-b-d-ribofuranosyl-1’-yl}-5-(4’’-nitro-1H-imidazol-1’’-yl)-pyrimidine-2,4-(1H,3H)-dione
(8a). Nucleoside 7b (110 mg, 0.165 mmol) was co-evaporated with anh. MeCN (2ꢁ5 ml) and dissolved
in anh. MeCN (2 ml). To the obtained stirred soln. at r.t. was added EtN(i-Pr)2 (170 ml, 9.9 mmol),
followed by 2-cyanoethyl N,N’-(diisopropyl)phosphoramidochloridite (50 ml, 0.22 mmol). After 60 min,
the mixture was diluted with CH2Cl2 (20 ml) and washed with 5% aq. NaHCO3 (1ꢁ10 ml), and the aq.
phase was extracted with CH2Cl2 (2ꢁ10 ml). The combined org. phase was dried (Na2SO4) and
evaporated to dryness under reduced pressure. The residue was purified by silica-gel CC (0–2.5% MeOH
in CH2Cl2 (v/v)) to afford 8a (116 mg, 83%). White solid. Rf (50% AcOEt in petroleum ether (v/v)) 0.33.
31P-NMR (CDCl3, H3PO4): 149.52; 149.60. HR-ESI-MS: 864.3098 ([MþNa]þ , C42H48N7NaO10Pþ ; calc.
864.3092).
2-(2’,4’-Dioxo-1H,3H-pyrimidin-5’-yl)-1H-benzo[de]isoquinoline-1,3(2H)-dione (3b). A suspension
of 1 (0.127 g, 1.00 mmol), naphthalene-1,8-dicarboxylic anhydride (2b; 0.200 g, 0.10 mmol), Et3N
(1.00 ml, 7.20 mmol), and DMAP (50.0 mg, 0.41 mmol) was heated under reflux in a mixture of DMSO
(5.0 ml) and EtOH (5.0 ml) for 96 h. The mixture was then poured onto ice (25 g), and the precipitate
formed was filtered off, washed successively with H2O and MeOH, and dried in the air to afford 3b
(0.218 g, 71%). Rf (15% MeOH in CH2Cl2 (v/v)) 0.47. 1H-NMR ((D6)DMSO, Me4Si): 7.82 (s, HꢀC(6’));
7.91 (t, J¼7.7, H ꢀC(6), HꢀC(7)); 8.53 (d, J¼7.7, H ꢀC(4), HꢀC(5), HꢀC(8), HꢀC(9)); 11.30 (s, NH);
11.59 (s, NH). 13C-NMR ((D6)DMSO, Me4Si): 109.5; 121.8; 127.4; 127.7; 131.2; 131.4; 134.9; 142.3; 151.0;
160.8; 163.2. EI-MS: 307 (Mþ, C16H9N3O4þ ).
2-[1’-(3’’-O-Acetyl-2’’-deoxy-5’’-O-propanoyl-d-ribofuranosyl-1’’-yl)-2’,4’-dioxo-1H,3H-pyrimidin-
5’-yl]-1H-benzo[de]isoquinoline-1,3(2H)-dione (5b). A mixture of 3b (0.614 g, 2.00 mmol), (NH4)2SO4
(20 mg, 0.15 mmol), and HMDS (10.0 ml, 47.0 mmol) was heated under reflux with stirring for 14 h. The
excess of HMDS was evaporated and the resulting oily residue was dissolved in anh. MeCN (25 ml).
Compound 4 [13]; 0.70 g, 2.50 mmol) was added, and the mixture was cooled to ꢀ158 whereupon
TMSOTf (0.17 ml, 1.00 mmol) was added dropwise. The mixture was stirred at ꢀ158 for 1 h and then
stirred for 1.5 h, while the temp. was allowed to reach 08. Then, 5% aq. NaHCO3 (25 ml) was added, and
the resulting mixture was extracted with CH2Cl2 (3ꢁ25 ml). The combined org. phase was dried
(Na2SO4) and evaporated to dryness. The resulting residue was purified by CC (0–5% MeOH in CHCl3
(v/v)) to yield an anomeric mixture of nucleosides 5b (a/b 1:1 based on 1H-NMR signals) as a white solid
material (0.89 g, 86%). Rf (5% MeOH in CHCl3 (v/v)) 0.23. 1H-NMR ((D6)DMSO, Me4Si): 0.84 (t, J¼
7.5, Me); 1.02 (t, J¼7.5, Me); 1.97 (s, Me); 2.07 (s, Me); 2.20–2.42 (m, 7 H, CH2, HaꢀC(2’’), HbꢀC(2’’));
2.73–2.83 (m, 1 HbꢀC(2’’)); 4.10–4.24 (m, 5 H, CH2(5’’), HꢀC(4’’)); 4.72 (t, J¼4.8, 1 HꢀC(4’’)); 5.08 (d,
J¼5.7, 1 HꢀC(3’’)); 5.17–5.22 (m, 1 HꢀC(3’’)); 6.16 (d, J¼6.6, 1 HꢀC(1’’)); 6.24 (t, J¼7.1, 1 H ꢀC(1’’));
7.90–7.95 (m, 4 arom. H); 8.09 (s, 1 HꢀC(6’)); 8.10 (s, 1 HꢀC(6’)); 8.48–8.56 (m, 8 arom. H); 11.86 (br. s,
NH); 11.99 (br. s, NH). 13C-NMR ((D6)DMSO, Me4Si): 8.6; 8.8; 20.6; 20.7; 26.5; 26.6; 36.5; 37.6; 63.3;
63.5; 73.8; 79.2; 81.5; 84.5; 85.1; 87.8; 109.6; 110.9; 121.7 (2 C); 122.0; 122.1; 127.4 (2 C); 127.4 (2 C); 127.7;
127.7; 130.9; 131.1; 131.2; 131.3; 131.5; 131.5; 134.8; 134.8; 135.1 (2 C); 140.5; 140.8; 149.7; 149.8; 159.7;
160.2; 163.1; 163.1; 163.2; 163.3; 170.0; 170.5; 173.4 (2C). HR-ESI-MS: 544.1332 ([MþNa]þ
,
C26H23N3NaOþ9 ; calc. 544.1326).
2-[1’-(2’’-Deoxy-b-d-ribofuranosyl-1’’-yl)-2’,4’-dioxo-1H,3H-pyrimidin-5’-yl]-1H-benzo[de]isoqui-
noline-1,3(2H)-dione (6b). Nucleoside 5b (0.80 g, 1.53 mmol) was suspended in anh. MeOH (25 ml), and
MeONa (0.5 g, 9.25 mmol) was added under stirring. After stirring for 100 min, H2O (5 ml) and
Amberlist IRC 120 (ca. 1 g) were added to reach neutral pH. The resulting suspension was decanted, and
the resin was washed with MeOH (3ꢁ10 ml). The combined MeOH phase was evaporated to dryness.
Fractional crystallization from MeOH gave pure b-anomer 6b (0.218 g, 33%). White crystalline material.
Rf (10% MeOH in CH2Cl2 (v/v)) 0.20. 1H-NMR ((D6)DMSO, Me4Si): 2.03–2.12 (m, HaꢀC(2’’)); 2.21–

CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
359
2.28 (m, HbꢀC(2’’)); 3.44–3.57 (m, CH2(5’’)); 3.80 (q, J¼3.8, HꢀC(4’’)); 4.19–4.24 (m, HꢀC(3’’)); 4.39
(t, J¼5.3, OHꢀC(5’’)); 5.31 (d, J¼4.3, OHꢀC(3’’)); 6.23 (t, J¼6.6, HꢀC(1’’)); 7.92, (t, J¼7.8, 2 arom.
H); 8.26, (s, HꢀC(6’)); 8.51–8.55 (m, 4 arom. H); 11.90 (br. s, NH). 13C-NMR ((D6)DMSO, Me4Si): 40.1;
61.2; 70.1; 84.6; 87.6; 110.5; 121.7; 127.4 (2 C); 127.7; 131.3 (2 C); 131.6; 135.0 (2 C); 140.6; 149.9; 159.8;
163.0; 163.1. HR-ESI-MS: 446.0964 ([MþNa]þ , C21H17N3NaO7þ ; calc. 446.0959).
2-[1’-{5-O-[Bis(4-methoxyphenyl)(phenyl)methyl]-2’’-deoxy-b-d-ribofuranosyl-1’’-yl}-2’,4’-dioxo-
1H,3H-pyrimidine-5’-yl)-1H-benz[de]isoquinoline-1,3(2H)-dione (7b). Compound 6b (60 mg,
0.188 mmol) was co-evaporated with anh. pyridine (2ꢁ5 ml) and dissolved in anh. pyridine (2 ml).
DMTrCl (72.0 mg, 0.283 mmol) was added, and the resulting mixture was stirred at r.t. for 20 h and then
evaporated to dryness. The residue was dissolved in AcOEt (20 ml), and the resulting org. phase was
successively washed with sat. aq. NaHCO3 (10 ml) and brine (2ꢁ10 ml). The org. phase was evaporated
to dryness, and the residue was purified by CC (0–5% MeOH in CH2Cl2 containing 0.5% Et3N (v/v/v)) to
furnish 7b (87 mg, 86%). White solid. Rf (10% MeOH in CH2Cl2 (v/v)) 0.38. 1H-NMR (CDCl3, Me4Si):
2.32–2.55 (m, CH2(2’’)); 3.26–3.38 (m, CH2(5’’)); 3.65 (s, MeO); 3.65 (s, MeO); 3.99–4.04 (m,
HꢀC(4’’)); 4.53–4.59 (m, HꢀC(3’’)); 5.5 (br. s, OH, NH); 6.43 (t, J¼6.5, HꢀC(1’’)); 6.58–6.69 (m, 5
arom. H); 6.92 (t, J¼7.8, 2 arom. H); 7.07 (dd, J¼1.1, 8.9, 4 arom. H); 7.14 (dd, J¼1.1, 8.3, 2 arom. H);
7.61–7.73 (m, 2 arom. H); 8.01 (s, HꢀC(6’)); 8.14–8.20 (m, 2 arom. H); 8.39 (dd, J¼1.1, 7.3, 1 arom. H);
8.46 (dd, J¼1.1, 7.3, 1 arom. H). 13C-NMR (CDCl3; Me4Si): 41.6; 55.2; 55.2; 63.4; 72.0; 85.4; 86.3; 86.8;
113.2 (4 C); 122.4; 122.4; 126.6; 126.8 (2 C); 127.7 (4 C); 128.7; 129.9 (2 C); 130.0 (2 C); 131.6; 131.7;
131.9; 134.3; 134.4; 135.1; 135.3; 140.4; 144.5; 150.0; 158.4; 158.5; 159.5; 163.3; 163.6. HR-ESI-MS:
748.2271 ([MþNa]þ , C42H35N3NaOþ9 ; calc. 748.2265).
2-(1’-{5’’-O-[Bis(4-methoxyphenyl)(phenyl)methyl]-3’’-O-[(2-cyanoethoxy)(diisopropylamino)-
phosphino]-2’’-deoxy-b-d-ribofuranosyl-1’’-yl}-2’,4’-dioxo-1H,3H-pyrimidine-5’-yl)-1H-benz[de]isoqui-
noline-1,3(2H)-dione (8b). Nucleoside 7b (110 g, 0.151 mmol) was co-evaporated with anh. MeCN (2ꢁ
5 ml) and dissolved in anh. MeCN (2 ml) containing 20% of EtNiPr2 (v/v). To this soln. was added 2-
cyanoethyl N,N’-(diisopropyl)phosphoramidochloridite (67 ml, 0.303 mmol), and the resulting mixture
was stirred at r.t. for 4 h, whereupon abs. EtOH (1 ml) was added. The resulting mixture was diluted with
CH2Cl2 (25 ml) and then successively washed with sat. aq. NaHCO3 (10 ml) and brine (10 ml). The
combined aq. phase was extracted with CH2Cl2 (20 ml) and evaporated to dryness. The residue was
purified by silica-gel CC (0–50% acetone in petroleum ether (v/v)) to afford 8b (103 mg, 73%). White
solid material. Rf (50% acetone in petroleum ether (v/v)) 0.4. 31P-NMR (CDCl3; H3PO4): 149.71; 149.16.
HR-ESI-MS: 948.3344 ([MþNa]þ , C51H52N5NaO10Pþ ; calc. 948.3344).
Synthesis of Oligodeoxynucleotides. Oligodeoxynucleotides (ODNs) containing monomers X and Y
(see Scheme 1 for structures) were synthesized in 0.2-mmol scale on an automated DNA synthesizer using
succinoyl linked LCAA-CPG (long-chain alkylamine-controlled pore glass) columns with a pore size of
500 ꢃ. Standard procedures were used for DNA amidites, i.e., Cl3CCOOH in CH2Cl2 as detritylation
reagent, 0.25m 4,5-dicyano-1H-imidazole (DCI) in MeCN as activator, Ac2O in THF as cap A soln., 1-
methyl-1H-imidazole in THF as cap B soln., and 0.02m I2 in H2O/pyridine/THF as oxidizing soln. For the
amidites 8a and 8b, extended coupling times (0.05m phosphoramidites in MeCN, 15 min) and 1H-
tetrazole as catalyst were used, resulting in stepwise coupling yields of ca. 99%. Removal of nucleobase-
protecting groups and cleavage from the solid support for ODNs containing monomer X (i.e., ON2, ON3,
and ON11) was achieved using standard conditions (32% aq. NH3 for 12 h at 558), whereas ONs
containing monomer Y (i.e., ON4 and ON12) were deprotected using 32% aq. NH3 at r.t. for 24 h.
Unmodified DNA and RNA strands were obtained from commercial suppliers. Purification to at least
80% purity of all modified ODNs was performed by RP-HPLC (using the system described below) on
O(5’)-DMTr-containing ODNs, followed by detritylation (80% aq. AcOH, 20 min, r.t.) and precipitation
(abs. EtOH), followed by washing with abs. EtOH (2ꢁ300 ml). The system used for purification was a
Waters 600 system equipped with an Xterra MS C18 (10 mm, 7.8ꢁ10 mm) pre-column and an Xterra MS
C18 (10 mm, 7.8ꢁ150 mm) column using the representative gradient protocol depicted in Table 4. The
composition of all synthesized ODNs was verified by MALDI-MS analysis (cf. Table 6) recorded in
negative-ion mode on a PerSeptive Voyager STR using 3-hydroxypicolinic acid as a matrix, whereas the
purity (> 80%) was verified by ion-exchange HPLC using an a LaChrom L-7000 system equipped with a
Dionex PA100 column (4ꢁ250 mm) at pH 8 using the protocol shown in Table 5.

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CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
Table 4. RP-HPLC Gradient Protocola)
Buffer A [vol.-%]
Time [min]
Buffer B [vol.-%]
0
2
100
100
30
0
0
0
0
70
100
100
0
40
47
50
51
60
100
100
0
a) Buffer A: 0.05m TEAA (triethylammonium acetate), pH 7.4; Buffer B: 75% MeCN in H2O (v/v);
flow rate 2.5 ml/min.
Table 5. Ion-Exchange HPLC Gradient Protocola)
Time [min]
H2O [vol.-%]
2m NaCl [vol.-%]
0.25m Tris·Cl [vol.-%]
0
2
88
88
55
10
10
88
88
2
2
35
80
80
2
10
10
10
10
10
10
10
20
25
30
35
45
2
a) Flow rate 1 ml/min.
Table 6. MALDI-MS Data of Synthesized ODNsa)
Sequence m/z
ON
Found
Calc.
ON2
ON3
ON4
ON11
ON12
5’-GTG AXA TGC
5’-GXG AXA XGC
5’-GTG AYA TGC
5’-TGC ACT GTA XGT CTG TAC CAT
5’-TGC ACT GTAYGT CTG TAC CAT
2752
3050
2934
6482
6568
2850
3044
2935
6484
6567
a) For structures of monomer X and Y, see Scheme 1.
Thermal Denaturation Studies. Concentrations of ODNs were estimated using the following
extinction coefficients (OD/mmol): for DNA: G (12.01), A (15.20), T (8.40), and C (7.05); for RNA: G
(13.70), A (15.40), U (10.00), and C (9.00); for modified monomers: X (8.44) and Y (16.80). The ODNs
(1.0 mm each strand) were thoroughly mixed, denatured by heating, and subsequently cooled to the
starting temp. of the experiment. Quartz optical cells with a path length of 1.0 cm were used. Thermal
denaturation temps. (Tm values/8C) were measured on a Perkin-Elmer Lambda 35 UV/VIS spectrometer
equipped with a PTP-6 Peltier temp. programmer and determined as the maximum of the first derivative
of the thermal denaturation curve (A260 vs. temp.) recorded in medium salt buffer (Tm buffer; 100 mm
NaCl, 0.1 mm EDTA, and pH 7.0 adjusted with 10 mm NaH2PO4/5 mm Na2HPO4). A temp. ramp of 1.08/
min was used in all experiments. Reported thermal denaturation temps. are an average of two
measurements within ꢂ1.08.

CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
361
Protocol for Steady-State Fluorescence Studies. Fluorescence measurements were performed on a
Perkin-Elmer LS 55 luminescence spectrometer equipped with a temp. controller. Quartz optical cells
with a path length of 1.0 cm were used. Measurements were conducted using 1.0 mm of strands in Tm
buffer. Corrections were made for solvent background, but no attempts were made to eliminate dissolved
oxygen in the buffer soln. Solns. were heated to 70–808 prior to measurements, followed by cooling to the
temp. of the experiment. Steady-state fluorescence emission spectra (360–600 nm) were obtained at 108
(ꢂ0.18) as an average of five scans at an excitation wavelength of 343 nm using an excitation slit of
10 nm, emission slit of 10 nm, and scan speed of 120 nm/min.
The Nucleic Acid Center is a research center of excellence founded by the Danish National Research
Foundation. We thank the Danish National Research Foundation for financial support.
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Received June 14, 2009