Modulation of pyrene fluorescence in DNA probes depends upon the nature of the conformationally restricted nucleotide

Article abstract of DOI:10.1021/jo702747w

(Graph Presented) The DNA probes (ODNs) containing a 2′-N-(pyren-1- yl)-group on the conformationally locked nucleosides [2′-N-(pyren-1-yl) carbonyl-azetidine thymidine, Aze-pyr (X), and 2′-N-(pyren-1-yl)carbonyl- aza-ENA thymidine, Aza-ENA-pyr (Y)], show

Full text of DOI:10.1021/jo702747w

Modulation of Pyrene Fluorescence in DNA Probes Depends upon
the Nature of the Conformationally Restricted Nucleotide
Dmytro Honcharenko, Chuanzheng Zhou, and Jyoti Chattopadhyaya*
Department of Bioorganic Chemistry, Box 581, Biomedical Center, Uppsala UniVersity,
SE-75123 Uppsala, Sweden
jyoti@boc.uu.se
ReceiVed December 25, 2007
The DNA probes (ODNs) containing a 2′-N-(pyren-1-yl)-group on the conformationally locked nucleosides
[2′-N-(pyren-1-yl)carbonyl-azetidine thymidine, Aze-pyr (X), and 2′-N-(pyren-1-yl)carbonyl-aza-ENA
thymidine, Aza-ENA-pyr (Y)], show that they can bind to complementary RNA more strongly than to the
DNA. The Aze-pyr (X) containing ODNs with the complementary DNA and RNA duplexes showed an
increase in the fluorescence intensity (measured at λem ≈ 376 nm) depending upon the nearest neighbor at
the 3′-end to X [dA (∼12-20-fold) > dG (∼9-20-fold) > dT (∼2.5-20-fold) > dC (∼6-13-fold)]. They
give high fluorescence quantum yields (ΦF ) 0.13-0.89) as compared to those of the single-stranded ODNs.
The Aza-ENA-pyr (Y)-modified ODNs, on the other hand, showed an enhancement of the fluorescence
intensity only with the complementary DNA (1.4-3.9-fold, ΦF ) 0.16-0.47); a very small increase in
fluorescence is also observed with the complementary RNA (1.1-1.7-fold, ΦF ) 0.17-0.22), depending
both upon the site of the Y modification introduced as well as on the chemical nature of the nucleobase
adjacent to the modification site into the ODN. The fluorescence properties, thermal denaturation experiments,
absorption, and circular dichroism (CD) studies with the X- and Y-modified ODNs in the form of matched
homo- and heteroduplexes consistently suggested (i) that the orientation of the pyrene moiety is outside the
helix of the nucleic acid duplexes containing a dT-d/rA base pair at the 3′-end of the modification site for
both X and Y types of modifications, and (ii) that the microenvironment around the pyrene moiety in the
ODN/DNA and ODN/RNA duplexes is dictated by the chemical nature of the conformational constraint in
the sugar moiety, as well as by the nature of neighboring nucleobases. The pyrene fluorescence emission
in both X and Y types of the conformationally restricted nucleotides is found to be sensitive to a mismatched
base present in the target RNA: (i) The X-modified ODN showed a decrease (∼37-fold) in the fluorescence
intensity (measured at λem ≈ 376 nm) upon duplex formation with RNA containing a G nucleobase mismatch
(dT-rG pair instead of dT-rA) opposite to the modification site. (ii) In contrast, the Y-modified ODN in
the heteroduplex resulted in a ∼3-fold increase in the fluorescence intensity upon dT-rG mismatch, instead
of matched dT-rA pair, in the RNA strand. Our data corroborate that the pyrene moiety is intercalated in
the X-modified mismatched ODN/RNA (G mismatch) heteroduplex as compared to that of the Y-modified
ODN/RNA (G mismatch) heteroduplex, in which it is located outside the helix.
1. Introduction
binding dyes, or on the fluorescently labeled oligonucleotide
(ON) probes, which hybridize specifically to the target DNA
or RNA by taking advantage of the Watson-Crick base-pairing
The fluorescence assays used in nucleic acid detection are
either based on nonspecific double-stranded DNA (dsDNA)
10.1021/jo702747w CCC: $40.75 © 2008 American Chemical Society
Published on Web 03/11/2008
J. Org. Chem. 2008, 73, 2829-2842
2829

Honcharenko et al.
FIGURE 1. Structures of azetidine thymidine [Azetidine (M)], 2′-N-(pyren-1-yl)carbonyl-azetidine thymidine [Aze-pyr (X)], aza-ENA thymidine
[Aza-ENA (N)], and 2′-N-(pyren-1-yl)carbonyl-aza-ENA thymidine [Aza-ENA-pyr (Y)] monomers are shown. Modified oligodeoxynucleotide
sequences (ODNs 1-14) used in this study are also listed. For a complete list of oligonucleotides, see Table S1 in the Supporting Information.
rules. The nonspecific probes, such as ethidium bromide1 or
oxazole yellow dye (YO-PRO 1),2 intercalate into double-
stranded DNA with a dramatic increase in fluorescence and are
capable of detecting amplification and product accumulation
but are unable to provide unambiguous verification of the
identity of the amplified product.3 Specific detection of nucleic
acids based on fluorescence increase upon hybridization to DNA
or RNA of cyanine dyes conjugated to ONs4,5 or peptide nucleic
acids (PNAs)6,7 has some disadvantages: (i) low fluorescence
quantum yields of the hybridized duplexes,5,6 (ii) sequence- and
temperature-dependent high levels of fluorescence of the single-
stranded probes,4,6,8 and (iii) small increases in fluorescence
intensity upon hybridization with complements.5 Another type
of approach based on the increase of the distance between
fluorophore and neighboring nucleobases upon binding to a
complementary ON has been used in such probes as molecular
beacons,9 TaqMan probes,10,11 Scorpion primers,12 or polya-
mide-fluorophore conjugates.13 However, the sensitivity of
assays with such probes may be limited by sequence-dependent
nucleobase-fluorophore quenching.13 Among several modifica-
tions, the pyrene-modified ONs have been synthesized and
exploited as fluorescent probes of DNA and RNA in hybridiza-
tion assays by utilizing the pyrene monomer14-20 or excimer21-26
fluorescence emission. The fact that pyrene fluorescence is
largely affected by environmental factors, such as the nature of
the solvent (fluorescence increases in the polar medium, whereas
a fluorescence decrease is observed in the hydrophobic medium)
or by the neighboring nucleobases (which often decrease
fluorescence by quenching), gives possibility for one to employ
its properties in the development of potential probes for the
investigation of nucleic acid structures,27-32 detection of single
nucleotide polymorphisms,17,19,33 or real-time PCR techniques.34
In this Article, we report on the synthesis and fluorescence
properties of DNA probes containing novel sugar-conformation
constrained 2′-N-pyrene-functionalized nucleosides [2′-N-(pyren-
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2830 J. Org. Chem., Vol. 73, No. 7, 2008

Modulation of Pyrene Fluorescence in DNA Probes
SCHEME 1a,b
a Reagents and conditions: (i) pyrenecarboxylic acid, PyBOP, DIPEA, DMF, rt, 1.5 h, 87%; (ii) ammonium formate, 20% Pd(OH)2/C, methanol, reflux,
36 h, 78%; (iii) DMTrCl, pyridine, rt, 12 h, 82%; (iv) NC(CH2)2OP(Cl)N(iPr)2, DIPEA, THF, 0 °C, 30 min, then rt, 1.5 h, 85%. b Abbreviations: Thy,
thymin-1-yl, Bn, Benzyl; DMTr, 4,4′-dimethoxytrityl; PyBOP, (benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate; DIPEA, diisopropyl-
ethylamine; DMF, N,N-dimethylformamide; rt, room temperature; THF, tetrahydrofuran.
a
SCHEME 2
a Reagents and conditions: (i) pyrenecarboxylic acid, PyBOP, DIPEA, DMF, rt, 2 h, 90%; (ii) ammonium formate, 20% Pd(OH)2/C, methanol, reflux,
36 h, 82%; (iii) DMTrCl, pyridine, rt, 32 h, 76%; (iv) NC(CH2)2OP(Cl)N(iPr)2, DIPEA, THF, 0 °C, 30 min, then rt, overnight, 73%.
1-yl)carbonyl-azetidine thymidine, Aze-pyr (X), and 2′-N-
(pyren-1-yl)carbonyl-aza-ENA thymidine, Aza-ENA-pyr (Y) in
Figure 1]. It has been found that the chemical structure of
different conformationally constrained units (Azetidine35 vs Aza-
ENA36) on the sugar moiety imparts different fluorescence
properties of N-conjugated pyren-1-yl moiety in the ODNs/DNA
versus ODNs/RNA duplexes. Unlike previously reported DNA
probes containing 2′-N-(pyren-1-yl)carbonyl-2′-amino-LNA modi-
fication,20 the X-modified ODNs showed DNA/RNA comple-
ments discrimination depending on the sequence context with
high fluorescence quantum yields (ΦF ) 0.13-0.89). Also, in
contrast to the 2′-O-(1-pyrenylmethyl)uridine14,15 or 2′-O-(pyren-
1-ylmethylcarbamoyl)uridine16-modified ONs, which display
significant fluorescence increase upon hybridization with RNA,
the Y-modified probes show an enhancement of the fluorescence
intensity only with the complementary DNA. The Aze-pyr (X)
versus Aza-ENA-pyr (Y) can successfully distinguish between
the matched nucleobase, A, and the mismatched nucleobase,
G, in the complementary RNA. Thus, this constitutes the
example of RNA base-mismatch recognition (dT-rA vs dT-
rG pair) by fluorescence emission using pyrene-modified DNA
in the DNA/RNA heteroduplex. It is noteworthy that earlier
mismatch discrimination studies have been performed in pyrene-
modified DNA/DNA17,19,26 or RNA/RNA15 homoduplexes. Only
2′-N-(pyren-1-yl)acetyl-2′-amino-R-L-LNA probes37 have been
recently used for detection of single nucleotide mismatches in
DNA/RNA heteroduplex, however, utilizing the mismatch
detection mechanism based on differences in fluorescence
intensity between duplexes with matched or mismatched targets
upon excimer formation.
2. Results
2.1. Synthesis of Pyrene-Modified ODNs. Synthesis of the
novel Aze-pyr (X) and Aza-ENA-pyr (Y) nucleosides is shown
in Schemes 1 and 2. Nucleosides 1 and 6 were prepared
(33) Nakatani, K. ChemBioChem 2004, 5, 1623-1633.
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Chattopadhyaya, J. J. Org. Chem. 2006, 71, 299-314.
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Honcharenko, D.; Chattopadhyaya, J. J. Am. Chem. Soc. 2006, 128, 15173-
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1122-1125.
J. Org. Chem, Vol. 73, No. 7, 2008 2831

Honcharenko et al.
TABLE 1. Thermal Denaturation Studiesa of Modified ODNs in Duplexes with Complementary DNA and RNA
Tm/°C
duplex with
duplex with
complementary
RNA 1d
complementary
no.
ODNsb
ODN sequences
DNA 2c
1
2
3
4
5
6
7
8
9
10
11
12
13
DNA 1 (native)
ODN 1 (Aze-pyr)
ODN 2 (Aze-pyr)
ODN 3 (Aze-pyr)
ODN 4 (Aza-ENA-pyr)
ODN 5 (Aza-ENA-pyr)
ODN 6 (Aza-ENA-pyr)
ODN 1a (Azetidine)
ODN 2a (Azetidine)
ODN 3a (Azetidine)
ODN 1b (Aza-ENA)
ODN 2b (Aza-ENA)
ODN 3b (Aza-ENA)
5′-dCTTCATTTTTTCTTC
5′-dCTTCAXTTTTTCTTC
5′-dCTTCATTXTTTCTTC
5′-dCTTCATTTTXTCTTC
5′-dCTTCAYTTTTTCTTC
5′-dCTTCATTYTTTCTTC
5′-dCTTCATTTTYTCTTC
5′-dCTTCAMTTTTTCTTC
5′-dCTTCATTMTTTCTTC
5′-dCTTCATTTTMTCTTC
5′-dCTTCANTTTTTCTTC
5′-dCTTCATTNTTTCTTC
5′-dCTTCATTTTNTCTTC
43.5
32.5
31
30.5
34
43
37.5
38
37
42
34
34
40.5
40
41e
39e
39.5e
42e
42e
42.5e
40e
40e
40e
48e
47.5e
46.5e
a Tm values measured as the maximum of the first derivative of the melting curve (A260 vs temperature) recorded in medium salt buffer ([Na+] ) 110 mM,
[Cl-] ) 100 mM, pH 7.0 (NaH2PO4/Na2HPO4)), using 1.0 µM concentrations of the two complementary strands. b See Figure 1 for all abbreviations. c DNA
2: 5′-dGAAGAAAAAATGAAG. d RNA 1: 5′-rGAAGAAAAAAUGAAG. e Tm’s were taken from ref 39, where Tm’s of the corresponding native DNA
1/DNA 2 and DNA 1/RNA 1 were found to be 45 and 44 °C, respectively.
according to the literature methods.35,36 Appropriately pyrene-
functionalized units 2 and 7 were synthesized via coupling of
pyrenecarboxylic acid24 with corresponding nucleosides 1 and
6. In both cases, the reaction afforded a mixture of rotamers
(87% and 90%, respectively) because of the restricted rotation
of the amide bond of the (pyren-1-yl)carbonyl unit. Fully
deprotected nucleosides 3 (78%) and 8 (82%) were prepared
by debenzylation of corresponding derivatives 2 and 7, respec-
tively, using Pd(OH)2/ammonium formate in methanol. Treat-
ment of unprotected nucleosides 3 and 8 with 4,4′-dimethox-
ytrityl chloride in pyridine afforded 4 and 9 in 82% and 76%,
respectively, which were transformed to their corresponding
phosphoramidites 5 (85%) and 10 (73%) for solid-phase
oligonucleotide synthesis.
ODNs 1-14 containing X and Y monomers (Figure 1) were
synthesized according to conventional DNA synthesis38 except
for extended coupling time for phosphoramidites 5 [10 min,
4,5-dicarbonitrile-1H-imidazole (DCI) as an activator] and 10
[10 min, 5-(ethylthio)-1H-tetrazole (ETT) as an activator]. ODNs
were deprotected using 32% aqueous ammonia at room tem-
perature during 24 h, purified by polyacrylamide gel electro-
phoresis (PAGE), and their composition was verified by
MALDI-TOF MS analysis [Table S1 in the Supporting Infor-
mation]. Single modifications (monomers X or Y) were placed
one at a time at three different positions in the central part of
the non-self-complementary oligodeoxynucleotide 15-mers with
the same nucleobase context at the 3′-end of the modification
site (Figure 1, ODNs 1-6) as well as with different nucleobase
neighbors at both sides (Figure 1, ODNs 1, 4, 7-14).
the corresponding unmodified duplexes. The binding properties
for both X-modified ODNs 1-3 and Y-modified ODNs 4-6
are summarized in Table 1.
The X-modified ODNs 1-3/RNA hybrid duplexes (entries
2-4 in Table 1) were found to be less stable by 2-3 °C than
the corresponding Azetidine-modified ODNs/RNA duplexes35
(entries 8-10 in Table 1); they also have a 5-6 °C drop in
melting temperatures (Tm’s) as compared to the native coun-
terpart. The duplexes of Y-modified ODNs 4-6 with comple-
mentary RNA (entries 5-7 in Table 1) showed relatively higher
target binding affinity (∆Tm ) +2.5 to +4.5 °C) as compared
to the isosequential X-modified ODNs 1-3/RNA hybrids
(entries 2-4 in Table 1). However, incorporation of the pyren-
1-yl moiety to the Aza-ENA unit leads to a ∼5-6 °C drop in
Tm with respect to the corresponding Aza-ENA-modified ODNs/
RNA hybrid duplexes36 (entries 11-13 in Table 1) and ∼1-3
°C drop as compared to the native counterpart. With comple-
mentary DNA 2, all pyrene-modified ODNs showed a signifi-
cant drop in duplex melting with respect to the native counterpart
and a similar destabilizing effect of pyren-1-yl moiety (∆Tm )
-6 to -7 °C) as compared to the corresponding isosequential
M- and N-modified duplexes.
Binding affinity of pyrene-modified ODNs 1 and 4 (entries
2-4 for X and entries 6-8 for Y in Table 2) to the RNA
possessing a single base mismatch (RNAs 2-4, Table 1 in
Supporting Information) appeared to be quite similar to the Tm
profile of the ODNs/DNA 2 duplexes.
2.3. Absorption Spectra of the Pyrene-Modified ODNs and
Their Duplexes with DNA and RNA. The pyrene absorption
maxima were found to be at 341 nm for both Aze-pyr (X)-
modified nucleoside 3 and Aza-ENA-pyr (Y) nucleoside 8
(Schemes 1 and 2) in sodium phosphate buffer (pH 7.0) (Figure
S1 in the Supporting Information), which is characteristic for
the pyrene chromophore. Figure 2 shows, as an example, the
absorption spectra of two isosequential (i) X-modified ODN 1,
its duplexes with complementary DNA 2 and RNA 1 (inset A
in Figure 2), and (ii) Y-modified ODN 4 and its corresponding
DNA 2 and RNA 1 duplexes (inset B in Figure 2) in the region
between 300 and 400 nm.
2.2. Thermal Denaturation Studies of Pyrene-Modified
ODNs. The binding of pyrene-modified ODNs 1-6 containing
monomers X or Y to their complementary DNA 2 and RNA 1
was investigated by UV thermal denaturation experiments in
medium salt neutral buffer, and compared to the corresponding
unmodified native, Azetidine35 (M)-, and Aza-ENA36,39 (N)-
modified duplexes. In all cases, melting profiles at 260 nm
displayed sigmoidal curves with a shape similar to those for
(38) Caruthers, M. H.; Barone, A. D.; Beaucage, S. L.; Dodds, D. R.;
Fisher, E. F.; McBride, L. J.; Matteucci, M.; Stabinsky, Z.; Tang, J. Y.
Methods Enzymol. 1987, 154, 287-313.
(39) Honcharenko, D.; Barman, J.; Varghese, O. P.; Chattopadhyaya, J.
Biochemistry 2007, 46, 5635-5646.
For the X-modified ODN 1, the pyrene absorption band at
347 nm was found to be shifted to a shorter wavelength upon
duplex formation with the complementary DNA 2 (343 nm) or
2832 J. Org. Chem., Vol. 73, No. 7, 2008

Modulation of Pyrene Fluorescence in DNA Probes
FIGURE 2. Absorption spectra of (A) Aze-pyr (X)-modified ODN 1 and (B) Aza-ENA-pyr (Y)-modified ODN 4 hybridized to the complementary
DNA 2, complementary RNA 1, and single base mismatch (G, U, C) RNA 2, RNA 3, and RNA 4, respectively. Spectra were recorded at a total
strand concentration of 19.5 µM at 25 °C.
TABLE 2. Thermal Denaturation Studiesa of Modified ODNs in
Duplexes with RNA Possessing a Single Base Mismatch
molecular properties of the modified nucleosides (including the
pKa of the nucleobase)40 depending on whether the modified
Tm/°C
duplex with
mismatched
RNA
sugar ring is a bicyclic 1′,2′-fused35 or a 2′,4′-fused.36 High
amplitude and range of dynamics of the important backbone
torsion (δ) in Azetidine versus Aza-ENA indicate that 1′,2′-
fused nucleosides have higher flexibility of the backbone as
compared to that of the 2′,4′-fused counterpart.40 A small shift
in pyrene maximum for the X-modified ODN 1/RNA 1 hybrid
with respect to the ODN 1/DNA 2 duplex also indicates some
differences in microenvironment around the pyrene unit. In
contrast, these changes were not observed in case of Y-modified
duplexes upon hybridization with the complementary DNA 2
or RNA 1. Figure 2 also shows the absorption spectra profile
for the X-modified ODN 1 and Y-modified ODN 4 hybridized
with RNAs 2, 3, and 4 containing single base mismatch (G, U,
C, respectively) opposite to the modification site in ODN strand.
As for the hybrid duplex formed by X-modified ODN 1 and
RNA 2 (G mismatch) as well as for Y-modified ODN 4/RNA
2 (G mismatch) duplex, UV spectra showed uniquely different
profiles. In the Aze-pyr-modified ODN 1/RNA 2 (G mismatch)
duplex, pyrene absorption band showed a red shift from 347 to
351 nm, whereas for the Aza-ENA-pyr-modified ODN 4/RNA
2 (G mismatch) duplex, pyrene maximum shifted to the shorter
wavelength, from 345 to 342 nm. In X-modified ODN 1/RNA
4 (C mismatch) duplex, pyrene absorption band had a red shift
of 2 nm. Other ODN 4/RNA 3 (U mismatch), ODN 4/RNA 4
(C mismatch), and ODN 1/RNA 3 (U mismatch) showed the
same wavelength of 344 nm for the pyrene absorption band.
2.4. Fluorescence Properties of Pyrene-Modified ODNs
and Their Duplexes with DNA and RNA. Figure 3 shows
the fluorescence emission spectra of Aze-pyr (X)-modified ODN
1 (inset A in Figure 3) and Aza-ENA-pyr (Y)-modified ODN
4 (inset B in Figure 3) and their corresponding duplexes with
complementary DNA 2 or RNA 1 targets. Single-stranded ODN
1 containing X modification (Figure 1) exhibits an unstructured
band at λmax ≈ 412 nm (inset A in Figure 3). Upon hybridization
no.
ODNsb
ODN sequences
1
2
3
4
5
6
7
8
ODN 1 (Aze-pyr)
ODN 1 (Aze-pyr)
ODN 1 (Aze-pyr)
ODN 1 (Aze-pyr)
ODN 4 (Aza-ENA-pyr) 5′-dCTTCAYTTTTTCTTC
ODN 4 (Aza-ENA-pyr) 5′-dCTTCAYTTTTTCTTC
ODN 4 (Aza-ENA-pyr) 5′-dCTTCAYTTTTTCTTC
ODN 4 (Aza-ENA-pyr) 5′-dCTTCAYTTTTTCTTC
5′-dCTTCAXTTTTTCTTC
5′-dCTTCAXTTTTTCTTC
5′-dCTTCAXTTTTTCTTC
5′-dCTTCAXTTTTTCTTC
37.5c
33.5d
33e
31f
42c
34d
32e
30.5f
a Tm values measured as the maximum of the first derivative of the
melting curve (A260 vs temperature) recorded in medium salt buffer ([Na+]
) 110 mM, [Cl-] ) 100 mM, pH 7.0 (NaH2PO4/Na2HPO4)), using 1.0
µM concentrations of ODN and target RNA. b See Figure 1 for all
abbreviations. c RNA ) RNA 1: 5′-rGAAGAAAAAAUGAAG. d RNA )
RNA 2: 5′-rGAAGAAAAAGUGAAG. e RNA ) RNA 3: 5′-rGAA-
GAAAAAUUGAAG. f RNA ) RNA 4: 5′-rGAAGAAAAACUGAAG, the
mismatch bases in RNA sequences are indicated by the bold and underline
fonts.
RNA 1 (342 nm), which were close to the absorption maximum
observed for the corresponding Aze-pyr-modified nucleoside
3. However, the pyrene absorption band at 345 nm for the
Y-modified ODN 4 showed very small changes (344 nm) in
the corresponding DNA 2 or RNA 1 duplexes, as compared to
the 341 nm for the corresponding Aza-ENA-pyr-modified
nucleoside 8. The difference in UV spectra between X-modified
ODN 1 and Y-modified ODN 4 suggests that incorporation of
either X or Y modification into oligonucleotide with the identical
sequence motif changes the structure of the corresponding
duplexes differently, which also changes the environment around
the pyrene moiety in a different manner. Conformationally
preorganized North-East (Azetidine, 44° < P < 54°, 29° < φm
< 33°)35 or North (Aza-ENA, 7° < P < 27°, 44° < φm < 52°)36
(M and N in Figure 1) should have significant contribution in
the location and interaction of the pyren-1-yl moiety in the ODN
1 or ODN 4. It has been shown that the type of conformational
constrains in the sugar moiety has varied influence on the
(40) Plashkevych, O.; Chatterjee, S.; Honcharenko, D.; Pathmasiri, W.;
Chattopadhyaya, J. J. Org. Chem. 2007, 72, 4716-4726.
J. Org. Chem, Vol. 73, No. 7, 2008 2833

Honcharenko et al.
FIGURE 3. Fluorescence emission spectra of (A) Aze-pyr (X)-modified ODN 1 and (B) Aza-ENA-pyr (Y)-modified ODN 4 and the corresponding
duplexes with complementary DNA 2 or RNA 1 targets. The temperature-dependent fluorescence changes (A) at 376 nm for the X-modified ODN
1/RNA 1 duplex and (B) at 385 nm for the Y-modified ODN 4/DNA 2 duplex are shown in the inset. Spectra were recorded at a total strand
concentration of 0.8 µM and with an excitation wavelength of 340 nm.
of ODN 1 with complementary DNA 2 or RNA 1, two vibronic
bands appear at λmax ≈ 376 nm (band I) and λmax ≈ 396 nm
(band III). Interestingly, fluorescence intensity (measured at λem
≈ 376 nm) increased 18.5-fold in ODN 1/RNA 1 hybrid and
only 6.4-fold in ODN 1/DNA 2 duplex (inset A in Figure 3).
Other X-modified ODNs 2 and 3, which have the same sequence
motif at the 3′-end of the modification site, exhibited similar
fluorescence properties (inset A in Figures S2 and S3 in the
Supporting Information).
Introduction of Y modification into the isosequential ODN
4 (Figure 1) resulted in remarkably different fluorescence
properties. Single-stranded ODN 4 displays structure involving
two bands at λmax ≈ 385 nm and λmax ≈ 399 nm (inset B in
Figure 3). The Y-modified ODN 4/RNA 1 duplex showed
similar but less structured shape with the same λmax for bands
I and III without significant changes in the fluorescence intensity
(measured at λem ≈ 385 nm). In contrast, a 3.9-fold increase in
fluorescence intensity and formation of two vibronic bands at
λmax ≈ 382 nm (band I) and λmax ≈ 399 nm (band III) resulted
upon duplex formation with complementary DNA 2 (inset B in
Figure 3). However, other Y-modified ODNs 5 and 6 containing
dT at the 3′-end of the modification site did not show a
significant difference in fluorescence intensity upon hybridiza-
tion with complementary DNA 2 or RNA 1 (inset B in Figures
S2 and S3 in the Supporting Information).
Fluorescence quantum yields (ΦF) of the single-stranded
probes containing X or Y modifications and their corresponding
duplexes with DNA or RNA were measured41 in sodium
phosphate buffer (pH 7.0) at 20 °C relative to the pyrenebutanoic
acid (PBA) in methanol (ΦF ) 0.065)42,43 (see Experimental
Section). The quantum yields of fully deprotected Aze-pyr
nucleoside 3 and Aza-ENA-pyr nucleoside 8 (Schemes 1 and
2) were determined to be ΦF ) 0.83 and ΦF ) 0.28, respectively
(Figures S4 and S5 in the Supporting Information). ODNs 1-6
containing modification at different positions in the sequence
with the same nucleotide sequence at the 3′-end of the
modification site showed different fluorescence properties
depending on the type of modification. Duplexes of X-modified
ODNs 1-3 (Figure 1) with complementary RNA 1 exhibit the
same high fluorescence quantum yields (ΦF ) 0.80-0.85)
(entries 3, 9, and 12 in Table 3) as the corresponding unprotected
nucleoside 3, showing higher quantum yields as compared to
the state-of-the-art pyrene-labeled probes,17,19 and were found
to be comparable to ΦF of probes containing pyrene-function-
alized 2′-amino-LNA.20 X-modified ODNs 1-3/DNA 2 homo-
duplexes display a decrease in fluorescence (ΦF ) 0.36-0.40)
(Table 3). However, fluorescence of duplexes formed by
Y-modified ODNs 4-6 (Figure 1) with complementary DNA
2 or RNA 1 (ΦF ) 0.19-0.22) did not change much as
compared to the corresponding single-stranded probes (ΦF )
0.16-0.17), except the homoduplex of Y-modified ODN 4 with
DNA 2, which showed an enhancement of fluorescence quantum
yield (ΦF ) 0.47) (Table 3), probably related to the site-
dependency of incorporation of Aza-ENA-pyr modification to
the ODN.
The emission intensities at λem ≈ 376 nm for the X-modified
ODN 1/RNA (inset A in Figure 3) or at λem ≈ 382 nm for the
Y-modified ODN 4/DNA 2 (inset B in Figure 3) duplexes were
decreased with melting of the corresponding duplexes (Figures
S6 and S7 in the Supporting Information), and only the
emissions for the corresponding modified ODN 1 or ODN 4
were observed at high temperature. However, a 2-fold decrease
in intensity for the pyrene monomer emission does not cor-
respond to the melting Tm of the duplex in both cases (Table
1). The Tm-like curve shows a pseudomelting behavior ap-
proximately ∼10-14 °C lower than the actual melting observed
at 260 nm (UV). This suggests that the pyren-1-yl moiety does
not contribute in the duplex stabilization through stacking or/
and Watson-Crick hydrogen bonding; its temperature-depend-
ent fluorescence changes, on the other hand, suggest that its
exposure to the immediate microenvironment changes in a
systematic manner during melting of the duplex.
Aze-pyr (X)-modified ODNs containing dC or dA at the 3′-
end of the modification site in duplexes with complementary
DNA or RNA display high fluorescence quantum yields up to
0.59 or 0.89, respectively (ODNs 7 and 8 in Table 3, Figure S8
in the Supporting Information), without discrimination between
DNA and RNA. The X-modified ODNs containing dG only at
the 3′-end of the modification site only, or at both 3′- and 5′-
ends of the X modification in ODN/DNA or ODN/RNA
duplexes, showed relatively poorer fluorescence quantum yields
(ΦF ) 0.13-0.21) (ODNs 9 and 10 in Table 3, Figure S9 in
the Supporting Information). However, Aza-ENA-pyr (Y)-
(41) Fery-Forgues, S.; Lavabre, D. J. Chem. Educ. 1999, 76, 1260-
1264.
(42) Manoharan, M.; Tivel, K. L.; Zhao, M.; Nafisi, K.; Netzel, T. L. J.
Phys. Chem. 1995, 99, 17461-17472.
(43) Netzel, T. L.; Nafisi, K.; Headrick, J.; Eaton, B. E. J. Phys. Chem.
1995, 99, 17948-17955.
2834 J. Org. Chem., Vol. 73, No. 7, 2008

Modulation of Pyrene Fluorescence in DNA Probes
TABLE 3. Relative Fluorescence Emission Intensities and Fluorescence Quantum Yields for the Single-Stranded Pyrene-Modified ODNs and
Their Duplexes with DNA or RNA
relative fluorescence
intensityc (λmax
)
single-stranded ODNs and duplexes with
complementary DNA or RNAb
378 nm
(band I)
391 nm
(band III)
ratio of intensity
(band III/band I)
Fluorescent
no.
ODNsa
ODN 1
Φ
1
2
3
4
5
6
7
8
9
5′-dCTTCAXTTTTTCTTC
1.0d
6.4d
18.5d
0.5d
4.0d
0.9d
1.0d
5.3d
19.9d
1.0
7.0e
7.1e
16.5e
2.0e
8.2e
5.1e
10.5f
7.8f
7.0
1.1
0.9
4.0
2.0
5.7
10.5
1.5
1.0
5.0
1.5
1.2
1.0
0.9
1.3
0.9
1.2
1.1
1.0
1.1
1.2
1.0
1.1
1.2
5.4
0.9
1.5
8.7
1.1
0.9
4.0
0.9
1.1
3.8
0.9
0.9
1.0
1.1
1.0
1.0
1.1
1.1
0.8
1.1
1.0
0.8
1.1
1.0
0.63
0.40
0.83
0.21
0.75
0.79
0.69
0.39
0.85
0.60
0.36
0.80
0.16
0.47
0.19
0.27
0.34
0.43
0.16
0.21
0.21
0.17
0.21
0.22
0.53
0.59
0.57
0.73
0.85
0.89
0.08
0.19
0.13
0.06
0.21
0.18
0.13
0.25
0.17
0.33
0.21
0.22
0.06
0.16
0.10
0.04
0.16
0.09
(Aze-pyr)
5′-dCTTCAXTTTTTCTTC-DNA 2
5′-dCTTCAXTTTTTCTTC-RNA 1
5′-dCTTCAXTTTTTCTTC-RNA 2 (G mismatch)
5′-dCTTCAXTTTTTCTTC-RNA 3 (U mismatch)
5′-dCTTCAXTTTTTCTTC-RNA 4 (C mismatch)
5′-dCTTCATTXTTTCTTC
5′-dCTTCATTXTTTCTTC-DNA 2
5′-dCTTCATTXTTTCTTC-RNA 1
5′-dCTTCATTTTXTCTTC
5′-dCTTCATTTTXTCTTC-DNA 2
5′-dCTTCATTTTXTCTTC-RNA 1
5′-dCTTCAYTTTTTCTTC
5′-dCTTCAYTTTTTCTTC-DNA 2
5′-dCTTCAYTTTTTCTTC-RNA 1
5′-dCTTCAYTTTTTCTTC-RNA 2 (G mismatch)
5′-dCTTCAYTTTTTCTTC-RNA 3 (U mismatch)
5′-dCTTCAYTTTTTCTTC-RNA 4 (C mismatch)
5′-dCTTCATTYTTTCTTC
5′-dCTTCATTYTTTCTTC-DNA 2
5′-dCTTCATTYTTTCTTC-RNA 1
5′-dCTTCATTTTYTCTTC
5′-dCTTCATTTTYTCTTC-DNA 2
5′-dCTTCATTTTYTCTTC-RNA 1
5′-dCTTCAXCTTTTCTTC
5′-dCTTCAXCTTTTCTTC-DNA 3
5′-dCTTCAXCTTTTCTTC-RNA 5
5′-dCTTCAXATTTTCTTC
5′-dCTTCAXATTTTCTTC-DNA 4
5′-dCTTCAXATTTTCTTC-RNA 6
5′-dCTTCAXGTTTTCTTC
5′-dCTTCAXGTTTTCTTC-DNA 5
5′-dCTTCAXGTTTTCTTC-RNA 7
5′-dCTTCGXGTTTTCTTC
5′-dCTTCGXGTTTTCTTC-DNA 6
5′-dCTTCGXGTTTTCTTC-RNA 8
5′-dCTTCAYCTTTTCTTC
5′-dCTTCAYCTTTTCTTC-DNA 3
5′-dCTTCAYCTTTTCTTC-RNA 5
5′-dCTTCAYATTTTCTTC
5′-dCTTCAYATTTTCTTC-DNA 4
5′-dCTTCAYATTTTCTTC-RNA 6
5′-dCTTCAYGTTTTCTTC
5′-dCTTCAYGTTTTCTTC-DNA 5
5′-dCTTCAYGTTTTCTTC-RNA 7
5′-dCTTCGYGTTTTCTTC
5′-dCTTCGYGTTTTCTTC-DNA 6
5′-dCTTCGYGTTTTCTTC-RNA 8
ODN 2
(Aze-pyr)
19.5f
5.0f
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
ODN 3
(Aze-pyr)
2.7
8.8
4.1f
10.6f
1.0h
3.7h
1.4h
2.6h
2.2h
2.6h
1.0h
1.5h
1.5h
1.0h
1.6h
1.7h
5.4e
11.9e
8.3e
8.7e
13.9e
18.8e
4.0e
17.7e
10.0e
3.8e
23.6e
21.6e
1.0h
1.7h
1.2h
1.0h
0.8h
0.8h
0.8h
3.4h
1.6h
0.8h
3.0h
1.7h
ODN 4
(Aza-ENA-pyr)
1.0g
3.9g
1.1g
3.0g
1.9g
2.3g
1.0g
1.4g
1.3g
1.0g
1.4g
1.4g
1.0d
13.6d
5.7d
1.0d
12.3d
20.2d
1.0d
19.7d
9.2d
1.0d
26.1d
23.3d
1.0g
1.5g
1.2g
1.0g
0.7g
0.7g
1.0g
3.0g
1.6g
1.0g
2.7g
1.7g
ODN 5
(Aza-ENA-pyr)
ODN 6
(Aza-ENA-pyr)
ODN 7
(Aze-pyr)
ODN 8
(Aze-pyr)
ODN 9
(Aze-pyr)
ODN 10
(Aze-pyr)
ODN 11
(Aza-ENA-pyr)
ODN 12
(Aza-ENA-pyr)
ODN 13
(Aza-ENA-pyr)
ODN 14
(Aza-ENA-pyr)
a See Figure 1 for all abbreviations. b DNA 2: 5′-dGAAGAAAAAATGAAG. DNA 3: 5′-dGAAGAAAAGATGAAG. DNA 4: 5′-dGAAGAAAATAT-
GAAG. DNA 5: 5′-dGAAGAAAACATGAAG. DNA 6: 5′-dGAAGAAAACACGAAG. RNA 1: 5′-rGAAGAAAAAAUGAAG. RNA 2: 5′-rGAA-
GAAAAAGUGAAG. RNA 3: 5′-rGAAGAAAAAUUGAAG. RNA 4: 5′-rGAAGAAAAACUGAAG. RNA 5: 5′-rGAAGAAAAGAUGAAG. RNA 6:
5′-rGAAGAAAAUAUGAAG. RNA 7: 5′-rGAAGAAAACAUGAAG. RNA 8: 5′-rGAAGAAAACACGAAG. c Relative fluorescence intensities were obtained
at 20 °C based on the single-stranded ODNs 1-6. d Measured at λmax 376 nm (band I). e Measured at λmax 396 nm (band III). f Measured at λmax 397 nm
(band III). g Measured at λmax 385 nm (band I). h Measured at λmax 399 nm (band III).
modified ODNs 11, 13, and 14 with different nucleobase
adjacent to the modification site, upon hybridization with
complementary DNA or RNA, reveal fluorescence (ΦF ) 0.09-
0.25, Table 3, inset A in Figure S10, and Figure S11 in the
Supporting Information) similar to that of Y-modified ODNs
4-6. The Y-modified ODN 12/DNA 4 and ODN 12/RNA 6
duplexes showed a small decrease in fluorescence as compared
to the single-strand ODN 12 containing dA at the 3′-end of the
modification site (Table 3, inset B in Figure S10 in the
Supporting Information).
ODNs and their duplexes with DNA or RNA. The modulation
of intensity of these two pyrene bands as a function of their
exposure to the microenvironment is well known,44,45 when
pyrene-1-yl chromophore is acting as a reporter group for the
polarity changes in the microenvironment. The incorporation
of different pyrene-modified units into isosequential ODNs
resulted in different fluorescence intensity ratios of band III/
band I for the corresponding Aze-pyr (X)-modified ODN 1
(44) Nakajima, A. Bull. Chem. Soc. Jpn. 1971, 44, 3272-3277.
(45) Kalyanasundaram, K.; Thomas, J. K. J. Am. Chem. Soc. 1977, 99,
2039-2044.
In Table 3 are shown the relative fluorescence emission
intensities (bands I and III) for the single-stranded modified
J. Org. Chem, Vol. 73, No. 7, 2008 2835

Honcharenko et al.
FIGURE 4. Fluorescence emission spectra of (A) Aze-pyr (X)-modified ODN 1 and (B) Aza-ENA-pyr (Y)-modified ODN 4 hybridized to the
complementary RNA 1 and single base mismatch (G, U, or C) RNA 2, RNA 3, and RNA 4, respectively. Spectra were recorded at a total strand
concentration of 0.8 µM and with an excitation wavelength of 340 nm.
(band III/band I ≈ 7.0) (entry 1 in Table 3) and Aza-ENA-pyr
(Y)-modified ODN 4 (band III/band I ≈ 1.0) (entry 13 in Table
3), which is to be compared to fluorescence intensity ratio of
∼0.6 for band III (λem ≈ 396 nm)/band I (λem ≈ 376 nm) for
the PBA, as a reference, in sodium phosphate buffer (pH 7.0),
thereby suggesting that the pyrene-1-yl moiety in the ODNs 1
and 4 is experiencing two different microenvironments. Similar
properties were found for other X-modified ODNs 2, 3, 7-10
and Y-modified ODNs 5, 6, 11-14 (entries 7, 10, 25, 28, 31,
34 and 19, 22, 37, 40, 43, 46, respectively, in Table 3) with a
small decrease in the intensity ratio of band III/band I for
modified ODNs containing dG at the 3′-end of the modification
site or dG at both sides of modification. Binding of the
X-modified ODNs 1-3 to the complementary DNA or RNA
(entries 2, 3, 8, 9, 11, 12, 26, 27, 29, 30, 32, 33, 35, and 36 in
Table 3) resulted in a decrease in the fluorescence intensity ratio
of band III/band I ≈ 0.9-1.5, with respect to the corresponding
single-stranded ODNs. The intensity ratio of band III/band I
for the corresponding isosequential ODNs 4-6, 11-14 contain-
ing Y modification in duplexes with complementary DNA or
RNA was found to be different from those of corresponding
isosequential X-modified ODNs, and showed a small increase
in the fluorescence intensity ratio of band III/band I ≈ 1.0-1.3
(entries 15, 20, 21, 23, 24, 38, 41, 42, 44, 45, 47, and 48 in
Table 3) in comparison to the corresponding single-stranded
ODNs. Only Y-modified ODN 4 containing dT at the 3′-end of
the modification site in duplex with complementary DNA
showed a small increase in the fluorescence intensity ratio of
band III/band I ≈ 0.9 (entry 14 in Table 3) with respect to the
corresponding single-stranded ODN 4.
with fully match RNA. The fluorescence intensity ratio of band
III/band I increased from 0.9 for fully matched ODN 1/RNA 1
duplex to 2.0-5.7 in the mismatched ODN 1/RNAs duplexes
(entries 4-6 in Table 3).
The Y-modified ODN 4, on the other hand, showed different
fluorescent properties upon hybridization with RNA involving
G, U, C mismatch opposite to the modification site (entries 16-
18 in Table 3). The emission spectra display structure with two
vibronic bands, especially in case of G mismatch (inset B in
Figure 4), and an increase in fluorescence intensities (measured
at λem ≈ 385 nm) from ∼1.7-fold (U mismatch) to ∼2.1-fold
(C mismatch) and ∼2.7-fold (G mismatch) with ΦF ) 0.21-
0.79 (entries 16-18 in Table 3) as compared to the fully match
ODN 4/RNA duplex. The fluorescence intensity ratio of band
III/band I decreased from 1.3 for fully matched ODN 4/RNA 1
duplex to 0.9-1.2 in the mismatched ODN 4/RNAs duplexes
(entries 16-18 in Table 3).
Fluorescence excitation spectra of the Aze-pyr (X)-modified
ODN 1 and its duplexes with complementary DNA 2, RNA 1,
and mismatched RNAs 2-4 were monitored at 376 nm (inset
A in Figure 5). Spectral patterns for the single-stranded ODN
1 and corresponding duplexes with targets were different from
each other, indicating different emitting species. Also, only
X-modified ODN 1/RNA 1 heteroduplex showed a spectral
profile similar to those of free pyrene in aqueous solution.46
Spectral patterns for the excitation spectra of Aza-ENA-pyr (Y)-
modified ODN 4 and its duplexes with complementary DNA
2, RNA 1, and mismatched RNAs 2-4 at 380 nm (inset B in
Figure 5), as well as those of the corresponding duplexes with
X-modified analogue ODN 1, were substantially different
(Figure 5). The large decreases in the intensities are observed
for the X-modified ODN 1 hybridized with mismatched RNAs
2-4 as compared to the matched ODN 1/DNA 2 and ODN
1/RNA 1 duplexes. The excitation spectra of Y-modified
analogues also displayed a change in the fluorescence intensities
depending on the nature of target DNA or RNA, showing the
strongest peaks for the ODN 4/DNA 2 homoduplex. These
results reflect the changes in the microenvironment around the
pyrene moiety brought about by the nature of the conformational
The fluorescence emission spectra for the X-modified ODN
1 (inset A) and Y-modified ODN 4 (inset B) hybridized with
RNAs 2, 3, and 4 possessing a single base mismatch (G, U, C,
respectively) opposite the modification site in the ODN strand
are shown in Figure 4.
The emission spectra of all X-modified ODN 1/RNAs
heteroduplexes (entries 4-6 in Table 3) showed unstructured
bands with a decrease in fluorescence intensities (measured at
λem ≈ 376 nm) from ∼4.6-fold for (U mismatch) to ∼20-fold
for (C mismatch) and ∼37-fold for (G mismatch) with ΦF )
0.21-0.79 (entries 4-6 in Table 3) with respect to the duplex
(46) Cho, N.; Asher, S. A. J. Am. Chem. Soc. 1993, 115, 6349-6356.
2836 J. Org. Chem., Vol. 73, No. 7, 2008

Modulation of Pyrene Fluorescence in DNA Probes
FIGURE 5. Fluorescence excitation spectra of (A) Aze-pyr (X)-modified ODN 1 and (B) Aza-ENA-pyr (Y)-modified ODN 4 and the corresponding
duplexes with complementary DNA 2, complementary RNA 1, and single base mismatched (G, U or C) RNA 2, RNA 3, and RNA 4, respectively,
obtained by monitoring wavelengths at (A) 376 nm and (B) 380 nm. Spectra were recorded at a total strand concentration of 0.8 µM.
FIGURE 6. (A) CD spectra of duplexes formed by native DNA 1 with complementary DNA 2 and Aze-pyr (X)-modified ODN 1 and Aza-ENA-
pyr (Y)-modified ODN 4 with complementary DNA 2. (B) CD spectra of duplexes formed by native DNA 1 with complementary RNA 1 and
pyrene-modified ODNs 1 and 4 with complementary RNA 1 and with RNA 2 containing single base (G) mismatch. The total strand concentration
was 10 µM.
constrain imposed at the pyrene chromophore as well as by
different nucleobases in the complementary strand opposite to
the X or Y.
cases, as for the X-modified ODN 1/DNA 2 as well as for the
Y-modified ODN 4/DNA 2 duplexes, CD profiles around 260
nm (which reflect the interaction between the transition moments
of nucleobases) were slightly modified from that of the native
duplex and have a B-like structures.
2.5. CD Spectra of Pyrene-Modified ODNs and Their
Duplexes with DNA and RNA. CD spectra for the X-modified
ODN 1 and Y-modified ODN 4 in duplexes with complementary
DNA 2 (inset A in Figure 6) and RNA 1 (inset B in Figure 6)
were recorded to examine the detailed structures of the corre-
sponding duplexes. The Aze-pyr-modified ODN 1/DNA 2
duplex exhibited a CD profile that is similar to that of the
unmodified DNA 1/DNA 2 homoduplex, albeit with an increase
of the intensity for the induced CD (ICD) peak at ∼278 nm.
No ICD absorption was however found at the region between
300 and 380 nm corresponding to the pyrene-chromophore (inset
A in Figure 6, Figure S12 in the Supporting Information). In
contrast, homoduplex formed by Aza-ENA-pyr-modified ODN
4 and complementary DNA 2 showed relatively weak ICD with
a small shift in Cotton peaks to the shorter wavelength. In this
case, a positive-induced CD resulting from the pyrene-1-yl unit
at the region between 300 and 380 nm was detected (inset A in
Figure 6, Figure S12 in the Supporting Information). In both
Upon binding of the X-modified ODN 1 to complementary
RNA 1, the CD profile was found to be close to the unmodified
heteroduplex DNA 1/RNA 1 with change of the intensity for
the ICD peak at ∼278 nm, in the same manner as for the
X-modified ODN 1/DNA 2 duplex. Also, no detectable peaks
were found in the region corresponding to the pyrene chro-
mophore (inset B in Figure 6, Figure S12 in the Supporting
Information). The CD profile for the Y-modified ODN 4/RNA
1 heteroduplex showed ICD similar to the native counterpart,
however, with peaks shifted to the shorter wavelength (blue
shift) and with positive ICD around 340 nm (inset B in Figure
6, Figure S12 in the Supporting Information). The X-modified
ODN 1/RNA 1 duplex and the Y-modified ODN 4 in duplex
with complementary RNA 1 showed relatively similar global
helical conformation with respect to the unmodified hybrid
duplex, however differing from each other.
J. Org. Chem, Vol. 73, No. 7, 2008 2837

Honcharenko et al.
Similar changes in CD profile were detected for other Aze-
pyr-modified ODNs 2-3 and Aza-ENA-pyr-modified ODNs
5-6 upon hybridization with complementary DNA 2 or RNA
1 (Figures S13 and S14 in the Supporting Information). Notably,
single-stranded X-modified ODN 1 showed negative ICD
around 340 nm, whereas no detectable peaks were observed in
the same region for the single-stranded Y-modified ODN 4
(Figure S15 in the Supporting Information).
Also, we analyzed the CD profiles of two isosequential
X-modified ODN 1 and Y-modified ODNs 4 hybridized with
RNA 2 possessing single base (G) mismatch opposite to the
modification site, which showed contrasting fluorescence prop-
erties (Figure 4). Thus, CD spectra indicate that both hetero-
duplexes have structures very closely similar to the native double
helix of the DNA/RNA heteroduplex (inset B in Figure 6).
However, at the region between 300 and 380 nm, the negative
ICD was observed for the X-modified ODN 1/RNA 2 duplex
and absence of ICD band for the Y-modified ODN 4/RNA 2
duplex (inset B in Figure 6, Figure S12 in the Supporting
Information), indicating different stereochemical location and
interaction of the pyren-1-yl moiety around the chromophores
in the microenvironment in the ODN 1/RNA 2 or ODN 4/RNA
2 heteroduplexes.
excited pyrene to nucleobases.42,50 Also, the red shift of
absorption maxima for the X-modified ODNs 1 (6 nm) and
Y-modified ODNs 4 (4 nm) (Figure 2) can be consistent with
π-stacking involving pyrene and nucleobases, which is known
to produce a shift of the absorption to a longer wavelength.18,46
Only Y-modified ODN 12 containing dA at the 3′-end of the
modification site displays fluorescence with ΦF ) 0.33 similar
to that of Aza-ENA-pyr-modified nucleoside 8. Upon hybridiza-
tion with complementary RNA 1, the X-modified ODNs 1-3
containing dT at the 3′-end of the modification site displayed
structured shape of fluorescence emission with a large increase
(up to ∼20-fold) in fluorescence intensity and high quantum
yield (ΦF ) 0.8-0.85) (inset A in Figure 3, Table 3). However,
only ∼2.5-6-fold increase in fluorescence intensity with ΦF
) 0.36-0.40 appeared for X-modified homoduplexes formed
with the complementary DNA 2 (Table 3). Blue shift of pyrene
absorption band (from 347 to 342 nm for Aze-pyr-modified
ODN 1/RNA 1 and from 347 to 343 nm for ODN 1/DNA 2
duplexes) (Figure 2) and no ICD in the region between 300
and 380 nm (Figures 6 and S12 in the Supporting Information),
in comparison with a negative ICD for the single-stranded
X-modified ODN (Figure S15 in the Supporting Information),
were found for the X-modified ODNs 1-3 in duplexes with
complementary DNA 2 and RNA 1. It has been shown that a
correlation exists between the positive versus negative mode
of the ICD signal around 340 nm and the groove binding mode
of benzo[a]pyrene;51 thus, a negative ICD is correlated only
with an intercalation mode, whereas the positive ICD is
correlated with both an external binding and an intercalation
mode. This suggests that pyrene-1-yl moiety in our duplexes
with complementary DNA 2 or RNA 1 is relatively freer from
the π-stacking interaction with the neighboring nucleobases
rather than the stacked or intercalation mode found in the single-
stranded ODN. The observed decrease in fluorescence intensity
ratio of band III/band I from 7.0 for single-stranded ODN 1 to
0.9-1.1 for Aze-pyr-modified ODN 1/RNA 1 or ODN 1/DNA
2 duplexes (Table 3) indicates that pyrene-1-yl moiety in these
duplexes is located in more hydrophilic environment in com-
parison to the hydrophobic mode in the single-stranded X-
modified ODN 1. Therefore, change of the microenvironment
around pyrene-1-yl residue is crucial for the increase in
fluorescence intensity in the duplexes. Even though the CD
spectra of the DNA and RNA duplexes containing X modifica-
tion did not show a significant difference at the pyrene
absorption band, the fluorescence properties and absorption
spectra of the corresponding duplexes indicate some difference
in pyrene location or interaction mode.
3. Discussion
3.1. Modulation of Pyrene Fluorescence in the Matched
ODN/DNA and ODN/RNA Duplexes by the Nature of
Conformational Constraint in the Sugar Moiety. The incor-
poration of pyrene unit into the specified position of ODNs by
covalent attachment to the 2′-O-center,14-16,18,31,32 2′-N-
center47,48 of the sugar, or 2′-N-center of the 2′,4′-conforma-
tionally constrained residue (such as 2′-amino-LNA)20,49 has
provided fluorescent probes that display strong fluorescence
upon hybridization to the target RNA14-16,18 only, or to both
RNA and DNA.20 In this Article, we have explored the
fluorescence properties of ODNs containing conformationally
constrained 2′-N-(pyren-1-yl)carbonyl-azetidine thymidine, Aze-
pyr (X), and 2′-N-(pyren-1-yl)carbonyl-aza-ENA thymidine,
Aza-ENA-pyr (Y), modifications (Figure 1) in duplexes with
target DNA or RNA.
It has been found that the chemical structure of different
conformationally constrained units (Azetidine35 vs Aza-ENA36
as in Figure 1) on the sugar moiety brought about a significant
difference in fluorescence properties even at the nucleoside level
in that the fluorescent quantum yields (ΦF) of unprotected Aze-
pyr-modified nucleoside 3 and Aza-ENA-pyr-modified nucleo-
side 8 were found to be 0.83 and 0.28, respectively. In both
cases, absorption maxima were observed at 341 nm, character-
istic of the pyrene chromophore. The incorporation of the
corresponding modifications into ODNs (Figure 1) resulted in
a fluorescence decrease with ΦF ) 0.06-0.73 for the X-
modified ODNs 1-3, 7-10 and 0.04-0.17 for the Y-modified
ODNs 4-6, 11, 13, and 14 (Table 3), suggesting that the
emission of the pyrene in modified ODNs was quenched by
the neighboring nucleobases, which are known to be efficient
quenchers for pyrene fluorescence via electron transfer from
The Aza-ENA-pyr-modified ODN 4 containing dT at the 3′-
end of the modification site shows an enhancement of the
fluorescence intensity only with the complementary DNA 2 (up
to 3.9-fold, ΦF ) 0.47), not with RNA 1 (1.1-fold increase in
fluorescence intensity with ΦF ) 0.19) (inset B in Figure 3,
Table 3). However, other Y-modified ODNs 5-6 with the same
nucleobase context at the 3′-end of the modification site in
duplexes with complementary DNA 2 or RNA 1 displayed
similar fluorescence properties (inset B in Figures S2 and S3
in the Supporting Information, Table 3). It is likely that this
observation is related to the site-dependency of incorporation
of Y modification to the ODN, when the modulation of the
(47) Kalra, N.; Babu, B. R.; Parmar, V. S.; Wengel, J. Org. Biomol.
Chem. 2004, 2, 2885-2887.
(48) Kalra, N.; Parlato, M. C.; Parmar, V. S.; Wengel, J. Bioorg. Med.
Chem. Lett. 2006, 16, 3166-3169.
(50) Netzel, T. L.; Zhao, M.; Nafisi, K.; Headrick, J.; Sigman, M. S.;
Eaton, B. E. J. Am. Chem. Soc. 1995, 117, 9119-9128.
(51) Pradhan, P.; Jernstrom, B.; Seidel, A.; Norden, B.; Graslund, A.
Biochemistry 1998, 37, 4664-4673.
(49) Hrdlicka, P. J.; Babu, B. R.; Sorensen, M. D.; Wengel, J. Chem.
Commun. 2004, 1478-1479.
2838 J. Org. Chem., Vol. 73, No. 7, 2008

Modulation of Pyrene Fluorescence in DNA Probes
microenvironment of the pyrene-1-yl moiety in DNA or RNA
duplexes is possible. In contrast to the isosequential X-modified
ODN 1, the intensity ratio of band III/band I for the Y-modified
ODN 4 was found to be similar to the fluorescence intensity
ratio of band III/band I for the corresponding Y-modified ODN
4/DNA 2 and ODN 4/RNA 1 duplexes (Table 3). Also, a small
decrease of fluorescence intensity ratio of band III/band I for
the Aza-ENA-pyr-modified ODN 4 upon hybridization with
complementary DNA 2 suggests that the pyrene was relocated
to more hydrophilic area, whereas in heteroduplex with comple-
mentary RNA 1 (increase of fluorescence intensity ratio of band
III/band I) it was positioned into a more hydrophobic environ-
ment. However, the absorption spectra for both Y-modified
ODN 4/DNA 2 and ODN 4/RNA 1 duplexes showed the same
small shift of the pyrene absorption maxima (1 nm), with respect
to the single-stranded ODN 4, to the shorter wavelength (Figure
2). In contrast to the X-modified duplexes, the CD profile of
the duplexes formed by Y-modified ODN 4 with complementary
DNA 2 or RNA 1 indicates positive ICD in the region between
300 and 380 nm (Figures 6 and S12 in the Supporting
Information). This observation can be correlated with the
external binding mode or groove-localized pyrene chromophore
in ODN 4/DNA 2 homoduplex (increased fluorescence intensity
and decreased intensity ratio of band III/band I). The pyrene-
1-yl moiety in ODN 4/RNA 1 heteroduplex is also localized
presumably outside the helix, but has a more hydrophobic
environment, as compared to that of ODN 4/DNA 2 homodu-
plex, with fluorescence intensity close to the single-strand level
and increased fluorescence intensity ratio of band III/band I.
fluorescence quenching can take place through electron transfer
or complex formation with G.42,52-55 This G-specific quenching
has been used in the development of BDF nucleoside (4′PyT),
which exhibits intense fluorescence when the 4′PyT is involved
in a complementary base pair with A.19 The Aze-pyr (X) and
Aza-ENA-pyr (Y) modifications (Figure 1) showed extraordi-
nary fluorescence properties in DNA/RNA heteroduplexes
possessing single G mismatch in complementary RNA opposite
to the modification site. In X-modified ODN 1/RNA 2 hetero-
duplex, fluorescence intensity was reduced by ∼37-fold (ΦF )
0.21) with respect to the duplex with fully matched RNA 1,
when Aze-pyr-modified nucleotide in ODN 1 was involved in
a base pair with A (inset A in Figure 4, entries 3 and 4 in Table
3). In contrast, the nucleobase mismatch by G instead of A in
the RNA strand upon hybridization with Y-modified ODN 4
resulted by a ∼3-fold increase in fluorescence intensity with
ΦF ) 0.27 (inset B in Figure 4, entries 15 and 16 in Table 3).
Other heteroduplexes, involving U or C mismatches, displayed
a relatively smaller decrease (for X-modified duplexes) or
increase (for Y-modified duplexes) in fluorescence intensity
(Figure 4, Table 3). The fluorescence intensity ratio of band
III/band I, which is known to be affected by local environmental
polarity,44,45 was increased (from 0.9 for fully matched to 4.0
for G mismatched) for the X-modified ODN 1/RNA 2 hetero-
duplex and decreased (from 1.3 for fully matched to 0.9 for G
mismatched) for the Y-modified ODN 4/RNA 2 heteroduplex
(Table 3). It is suggested that the mode of interaction or location
of the pyrene in these heteroduplexes is different. The red shift
(from 347 to 351 nm) of the pyrene absorption maximum for
the X-modified ODN 1 upon hybridization with RNA 2 (G
mismatch) (inset A in Figure 2) indicates the stacking interaction
between pyrene and nucleobases, when the pyrene-1-yl moiety
was in the more hydrophobic environment, whereas a shift to
the shorter wavelength of the pyrene absorption maximum (from
345 to 342 nm) appeared for the Y-modified ODN 4/RNA 2
heteroduplex (inset B in Figure 2), indicating relatively poorer
stacking interaction as compared to that of the single-stranded
ODN 4, when pyrene chromophore is located in the more
hydrophilic environment. These conclusions are in agreement
with CD profiles for the corresponding ODN 1/RNA 2 and ODN
4/RNA 2 duplexes. The negative ICD at pyrene absorption
region was observed for the X-modified ODN 1/RNA 2 (G
mismatch) heteroduplex (inset B in Figure 6, Figure S8 in the
Supporting Information), suggesting that the pyrene was located
in the chiral environment and intercalated; therefore, its absorp-
tion was significantly quenched by neighboring G sites. In
contrast, no ICD signal at the same region was detected for the
Y-modified ODN 4/RNA 2 (G mismatch) heteroduplex (inset
B in Figure 6, Figure S8 in the Supporting Information), when,
most likely, pyrene was positioned away from the helix, and
therefore exhibits enhanced fluorescence.
3.2. Modulation of Pyrene Fluorescence in the Matched
ODN/DNA and ODN/RNA Duplexes by the Nature of the
Neighboring Nucleobase. The other Aze-pyr-modified ODNs
7-10 in duplexes with complementary DNA or RNA showed
various emission enhancements depending on the nucleobase
adjacent to the X modification in the ODN (from 5.7-fold
increase in fluorescence intensity for ODN 7/RNA 5 hetero-
duplex containing dC-rG pair at the 3′-end of the modification
site to 26.1-fold increase for the ODN 10/DNA 6 homoduplex
containing dG-dC pair at both sides) with ΦF ) 0.13-0.89
(Table 3). Notably, smaller fluorescence quantum yields (ΦF
) 0.13-0.21) of X-modified ODNs 9 and 10 containing dG at
the 3′-end of the modification site or at both 3′- and 5′-ends in
the duplexes with complementary DNA or RNA, as compared
to other ODNs 1, 7, and 8, which have dT, dC, and dA
neighbors, respectively, may be correlated with the intercalation
mode of pyrene. However, these probes still demonstrate an
increase in fluorescence intensity (∼9-26-fold, measured at λem
≈ 376 nm) upon hybridization with complementary target with
respect to the single-stranded ODNs (Table 3). The fluorescent
properties of Aza-ENA-pyr-modified ODNs 11-14 upon hy-
bridization with complementary DNA or RNA were found to
be similar to those of the Y-modified ODN 4 containing dT at
the 3′-end of the modification site, except ODN 12 (dA at the
3′-end of the modification site), which showed a small drop in
fluorescence intensity (Table 3).
4. Conclusions
(1) The impact of chemical nature of the conformational
constraint in the sugar unit (1′,2′-Azetidine lock vs 2′,4′-Aza-
3.3. Modulation of Pyrene Fluorescence in the Mis-
matched ODNs/RNA Duplexes. Recently, studies on fluores-
cently labeled nucleic acids probes capable of detecting single-
base mismatches have been performed in DNA/DNA17,19,26,37
as well as in RNA/RNA15 homoduplexes. Among several
nucleobases, guanine exhibits exceptionally high quenching
efficiency because it is the most electron-donating base, and
(52) Knapp, C.; Lecomte, J. P.; Mesmaeker, A. K.; Orellana, G. J.
Photochem. Photobiol., B 1996, 36, 67-76.
(53) Kawai, K.; Takada, T.; Tojo, S.; Ichinose, N.; Majima, T. J. Am.
Chem. Soc. 2001, 123, 12688-12689.
(54) Marras, S. A. E.; Kramer, F. R.; Tyagi, S. Nucleic Acids Res. 2002,
30, e122/1-e122/8.
(55) Kawai, K.; Yokoohji, A.; Tojo, S.; Majima, T. Chem. Commun.
2003, 2840-2841.
J. Org. Chem, Vol. 73, No. 7, 2008 2839

Honcharenko et al.
ENA lock) on the modulation of the fluorescence properties of
oligodeoxynucleotides containing 2′-N-(pyren-1-yl)carbonyl-
azetidine thymidine, Aze-pyr (X), and 2′-N-(pyren-1-yl)carbon-
yl-aza-ENA thymidine, Aza-ENA-pyr (Y), in the duplexes with
complementary DNA or matched/mismatched RNA has been
described.
fully match RNA duplex], and large increase in fluorescence
intensity (∼6-23-fold) for the singly Aze-pyr-modified probes
upon hybridization with complementary RNA, as compared to
the ∼2.5-fold increase for the singly modified probes containing
pyrene-functionalized 2′-amino-LNA,20 can be exploited for
monitoring RNA hybridization and RNA folding in general. The
fluorescence increase for the pyrene emission of Aza-ENA-pyr
(1.4-3.9-fold) as well as Aze-pyr-modified probes (2.7-26.1-
fold) in the duplexes with DNA complements and base-
mismatch (dT-rA vs dT-rG pair) recognition in the hetero-
duplexes with RNA [increase in fluorescence intensities for the
singly Aza-ENA-pyr-modified ODN from ∼1.7-fold (U mis-
match) to ∼2.1-fold (C mismatch) and ∼2.7-fold (G mismatch)
with respect to the fully match RNA duplex] can open the way
for the design of new base-discriminating fluorescent DNA
probes. However, Y-modified probes will have limitation if dA
is present at the 3′-end of the modification site in the sequence.
Also, the destabilizing effect of the X-modified probes on the
duplex with complementary targets as well as relatively long
synthetic pathways for both modifications in general may
influence their potential application.
(2) The pyrene moiety is presumably outside the helix of the
matched nucleic acid duplexes containing dT-d/rA pair at the
3′-end of the modification site for both types of modifications,
which can be concluded from the following observations: (i)
enhanced fluorescence emission, (ii) small band III/band I
intensity ratio, (iii) absence of ICD band or presence of positive
ICD band at the pyrene region (340 nm), (iv) blue shift in pyrene
absorption maxima with respect to the single-stranded probes,
and (v) decrease in the thermal stability of the duplexes.
(3) Pyrene probes display high fluorescence quantum yields
for the Aze-pyr-modified ODNs in duplexes with complemen-
tary DNA and RNA from 0.13 to 0.89 depending on the
nucleobase adjacent to the X modification in the ODN.
Relatively poorer quantum yields (ΦF ) 0.09-0.47) are
however found for the Aza-ENA-pyr-modified ODNs in the
DNA and RNA duplexes as compared to conformationally
constrained 2′-N-(pyren-1-yl)carbonyl conjugated 2′-amino-
LNA20 (ΦF ) 0.34-0.91) for singly pyrene-modified DNA and
RNA duplexes.
(4) The specific hybridization-induced increase in fluores-
cence intensities is from ∼13-fold for ODN/DNA and ∼6-fold
for ODN/RNA duplexes for the X-modified ODN containing
dC at the 3′-end of the modification site and up to ∼26-fold
upon hybridization with complementary DNA or ∼23-fold with
RNA for the X-modified ODN containing dG at both sides of
modification. On the other hand, for the Y-modified ODNs the
fluorescence intensities increase up to 3.9-fold upon hybridiza-
tion with complementary DNA and only 1.1-1.7-fold with
RNA; however, Aza-ENA-pyr probe containing dA at the 3′-
end of the modification site showed ∼1.4-fold decrease in
fluorescence intensities in ODN/DNA and ODN/RNA duplexes.
The above results reflect the relative changes of the microen-
vironment of pyrene moiety brought about by the chemical
nature of the conformationally restricted nucleotide, as well as
different structures of the DNA and RNA duplexes.
6. Experimental Section
(1R,3R,4S,5R)-3-Benzyloxymethyl-4-benzyloxy-6-N-(pyren-1-
ylcarbonyl)-1-(thymine-1-yl)-6-aza-2-oxabicyclo[3.2.0]heptane
(2). To a stirred solution of pyrenecarboxylic acid (0.016 g, 0.064
mmol) in N,N-dimethylformamide (2 mL) were added (benzotriazol-
1-yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyBOP)
(0.035 g, 0.067 mmol), diisopropylethylamine (0.01 mL, 0.062
mmol), and nucleoside 1 (0.02 g, 0.044 mmol). The reaction mixture
was stirred for 1.5 h at room temperature. Saturated aqueous
NaHCO3 solution (5 mL) was added, and the mixture was extracted
with dichloromethane (3 × 20 mL). The organic layer was dried
over anhydrous MgSO4, filtered, and concentrated in vacuo. The
crude product was purified by silica gel column chromatography
(0-4% methanol in dichloromethane, v/v) to give 2 (0.026 g, 0.038
mmol, 87%) as a mixture of diastereomers. Rf ) 0.39 [96:4 CH2-
Cl2/CH3OH (v/v)]. MALDI-TOF m/z: [M + H]+ found, 678.3;
calcd, 678.2. 13C NMR (67.9 MHz, CDCl3) δ: 172.6, 163.6, 149.0,
137.6, 135.2, 132.8, 131.0, 130.5, 129.3, 128.9, 128.4, 128.3, 127.7,
127.3, 127.0, 126.3, 126.0, 125.8, 125.4, 124.6, 124.5, 124.2, 123.9,
111.6, 90.0, 82.4, 73.5, 73.2, 69.5, 68.1, 61.3, 12.1.
(1R,3R,4S,5R)-3,4-Hydroxy-6-N-(pyren-1-ylcarbonyl)-1-(thy-
mine-1-yl)-6-aza-2-oxabicyclo[3.2.0]heptane (3). Compound 2
(0.72 g, 1.06 mmol) dissolved in 20 mL of methanol, Pd(OH)2 on
charcoal (20% moist, 0.22 g), and ammonium formate (0.86 g, 13.6
mmol) were added to the solution of nucleoside. The resulting
suspension was heated under reflux for 36 h. The reaction mixture
was filtered through silica gel bed and washed with hot methanol
(40 mL). The filtrate was concentrated to dryness in vacuo and
purified by silica gel column chromatography (0-8% methanol in
dichloromethane, v/v) to afford 3 (0.41 g, 0.82 mmol, 78%). Rf )
0.34 [90:10 CH2Cl2/CH3OH (v/v)]. MALDI-TOF m/z: [M + H]+
found, 498.1; calcd, 498.2. 13C NMR (67.9 MHz, CD3OD) δ: 175.2,
166.7, 151.7, 138.4, 134.5, 132.8, 132.4, 130.6, 130.3, 129.7, 129.4,
128.5, 128.0, 127.6, 127.4, 126.5, 126.1, 125.8, 125.4, 112.3, 91.0,
86.0, 85.0, 74.5, 73.1, 72.0, 70.4, 63.2, 62.6, 62.2, 60.2, 12.4.
(1R,3R,4S,5R)-3-(4,4′-Dimethoxytrityloxymethyl)-4-hydroxy-
6-N-(pyren-1-ylcarbonyl)-1-(thymine-1-yl)-6-aza-2-oxabicyclo-
[3.2.0]heptane (4). Nucleoside 3 (0.265 g, 0.53 mmol) was co-
evaporated with anhydrous pyridine (3 × 5 mL) and dissolved in
6 mL of the same solvent, and 4,4′-dimethoxytrityl chloride (0.19
g, 0.56 mmol) was added and stirred at room temperature for 12 h
under nitrogen atmosphere. The reaction mixture was poured into
cold saturated aqueous NaHCO3 solution (10 mL) and extracted
with dichloromethane (3 × 30 mL). The organic layer was dried
(5) The intercalation mode of pyrene moiety in the Aze-pyr-
modified ODN hybridized with RNA possessing G mismatch
is consistent with the following observations: (i) significant
decrease in fluorescence intensity (∼37-fold, measured at λem
≈ 376 nm), (ii) red shift in pyrene absorption maxima with
respect to the single-stranded probes, (iii) strong negative ICD
at the pyrene region (340 nm), and (iv) large intensity ratio of
band III/band I of the emission. In the Aza-ENA-pyr-modified
ODN/RNA (with G mismatch in the RNA strand) heteroduplex,
the pyrene-chromophore is positioned away from the helix,
which is consistent with the following observations: (i)
enhanced fluorescence emission, (ii) blue shift in pyrene
absorption maxima with respect to the single-stranded probes,
(iii) absence of ICD band at the pyrene region, and (iv) decrease
in fluorescence intensity ratio of band III/band I.
5. Implication
High quantum efficiency (ΦF ) 0.13-0.89), mismatch
discrimination [decrease in fluorescence intensities (measured
at λem ≈ 376 nm) from ∼4.6-fold (U mismatch) to ∼20-fold
(C mismatch) and ∼37-fold (G mismatch) with respect to the
2840 J. Org. Chem., Vol. 73, No. 7, 2008

Modulation of Pyrene Fluorescence in DNA Probes
over anhydrous MgSO4, filtered, and evaporated under reduced
pressure followed by co-evaporation with toluene (2 × 20 mL) to
remove pyridine partially. The crude product was purified by silica
gel column chromatography (0-4% methanol in dichlorometh-
ane (v/v), containing 1% pyridine) to give 4 (0.35 g, 0.44 mmol,
82%). Rf ) 0.27 [96:4 CH2Cl2/CH3OH (v/v)]. MALDI-TOF
m/z: [M + H]+ found, 800.2; calcd, 800.3. 13C NMR (67.9 MHz,
CDCl3 plus DABCO) δ: 173.0, 163.8, 158.4, 149.7, 149.6, 144.7,
135.8, 134.8, 132.8, 131.0, 130.5, 130.0, 129.4, 129.0, 128.1, 127.7,
127.0, 126.7, 126.5, 126.1, 126.0, 124.6, 124.5, 124.4, 124.2, 123.6,
113.0, 111.8, 88.6, 86.2, 84.3, 72.9, 71.9, 62.5, 62.1, 55.1, 46.9,
29.6, 12.3.
(1R,3R,4S,5R)-4-(2-Cyanoethoxy(diisopropylamino)-phosphi-
noxy)-3-(4,4′-dimethoxytrityloxymethyl)-6-N-(pyren-1-ylcarbo-
nyl)-1-(thymine-1-yl)-6-aza-2-oxabicyclo[3.2.0]heptane (5). Nu-
cleoside 4 (0.17 g, 0.21 mmol) was dissolved in 3 mL of dry THF,
and diisopropylethylamine (0.19 mL, 1.09 mmol) was added at 0
°C under a nitrogen atmosphere followed by 2-cyanoethyl-N,N-
diisopropylphosphoramidochloridite (0.1 mL, 0.45 mmol). After
30 min the reaction was warmed to room temperature and stirred
for 1.5 h. Methanol (0.2 mL) was added, and stirring was continued
for 10 min, after which the reaction mixture was poured into
saturated aqueous NaHCO3 solution (10 mL) and extracted with
freshly distilled dichloromethane (3 × 30 mL). The organic layer
was dried over anhydrous MgSO4, filtered, and concentrated in
vacuo. The crude residue was purified by silica gel column
chromatography (20-70% CH2Cl2 in cyclohexane containing 2%
Et3N) to afford 5 (0.18 g, 0.18 mmol, 85%) as a mixture of isomers.
Rf ) 0.39 [96:4 CH2Cl2/CH3OH (v/v)]. MALDI-TOF m/z: [M +
H]+ found, 1000.4; calcd, 1000.4. 31P NMR (67.9 MHz, CDCl3)
δ: 149.9, 148.6.
(1R,5R,7R,8S)-5-Benzyloxymethyl-8-benzyloxy-2-N-(pyren-1-
ylcarbonyl)-7-(thymin-1-yl)-2-aza-6-oxabicyclo[3.2.1]octane (7).
Chromatography (0-4% methanol in dichloromethane, v/v).
Yield: 0.134 g (0.19 mmol, 90%) as a mixture of diastereomers.
Rf ) 0.35 [96:4 CH2Cl2/CH3OH (v/v)]. MALDI-TOF m/z: [M +
H]+ found, 692.4; calcd, 692.3. 13C NMR (67.9 MHz, CDCl3) δ:
170.8, 170.2, 164.1, 163.5, 162.5, 150.0, 149.9, 137.1, 137.0, 135.4,
134.9, 131.6, 131.5, 130.9, 130.8, 130.5, 130.4, 129.3, 129.2, 128.8,
128.5, 128.4, 128.0, 127.9, 127.7, 127.6, 127.3, 126.9, 126.8, 126.1,
126.0, 125.5, 125.3, 125.1, 124.7, 124.5, 124.2, 124.1, 124.0, 123.7,
123.5, 123.4, 109.9, 107.8, 86.1, 84.6, 84.2, 73.4, 73.2, 72.9, 72.4,
72.2, 71.6, 71.3, 69.6, 69.3, 67.3, 62.4, 56.1, 56.0, 48.1, 48.0, 41.6,
41.1, 36.3, 35.9, 31.3, 29.5, 27.3, 26.8, 26.3, 25.8, 25.7, 11.9, 11.5.
(1R,5R,7R,8S)-5,8-Hydroxy-2-N-(pyren-1-ylcarbonyl)-7-
(thymin-1-yl)-2-aza-6-oxabicyclo[3.2.1]octane (8). Chromatog-
raphy (0-8% methanol in dichloromethane, v/v). Yield: 0.081 g
(0.16 mmol, 82%). Rf ) 0.35 [90:10 CH2Cl2/CH3OH (v/v)].
MALDI-TOF m/z: [M + H]+ found, 512.4; calcd, 512.2. 13C NMR
(67.9 MHz, DMSO-d6) δ: 169.7, 168.8, 164.3, 163.6, 150.3, 149.6,
135.7, 135.2, 131.0, 130.9, 130.7, 130.6, 130.4, 130.2, 128.3, 128.0,
127.8, 127.6, 127.3, 127.2, 126.8, 126.6, 126.4, 126.3, 125.8, 125.7,
125.6, 125.4, 125.1, 125.0, 124.9, 124.7, 124.6, 124.0, 123.9, 123.8,
123.7, 108.0, 107.8, 107.6, 85.6, 85.3, 85.2, 64.3, 64.0, 63.4, 63.2,
63.0, 60.9, 58.5, 54.9, 42.0, 41.5, 36.1, 35.6, 34.7, 27.3, 26.0, 25.3,
25.0, 12.5, 12.3.
(1R,5R,7R,8S)-5-(4,4′-Dimethoxytrityloxymethyl)-8-hydroxy-
2-N-(pyren-1-ylcarbonyl)-7-(thymin-1-yl)-2-aza-6-oxabicyclo-
[3.2.1]octane (9). Chromatography (0-4% methanol in dichlo-
romethane (v/v), containing 1% pyridine). Yield: 0.06 g (0.074
mmol, 76%). Rf ) 0.22 [96:4 CH2Cl2/CH3OH (v/v)]. MALDI-TOF
m/z: [M + H]+ found, 814.2; calcd, 814.3. 13C NMR (67.9 MHz,
CDCl3 plus DABCO) δ: 171.3, 171.2, 164.7, 163.3, 158.5, 150.2,
149.7, 144.3, 144.0, 135.5, 135.2, 134.8, 131.7, 131.1, 130.8, 130.6,
130.0, 129.1, 128.9, 128.1, 127.9, 127.7, 127.0, 126.8, 126.3, 126.2,
125.7, 125.5, 124.6, 124.3, 123.5, 113.2, 110.3, 110.0, 86.7, 86.4,
85.9, 85.7, 85.4, 85.1, 68.0, 66.6, 65.2, 64.1, 63.5, 63.3, 59.4, 59.3,
55.1, 51.2, 46.1, 43.2, 42.2, 41.8, 40.6, 36.0, 34.9, 29.6, 27.1, 26.7,
26.3, 25.5, 12.1, 11.7.
(1R,5R,7R,8S)-8-(2-Cyanoethoxy(diisopropylamino)-phosphi-
noxy)-5-(4,4′-dimethoxytrityloxymethyl)-2-N-(pyren-1-ylcarbo-
nyl)-7-(thymin-1-yl)-2-aza-6-oxabicyclo[3.2.1]octane (10). Chro-
matography (20-70% CH2Cl2 in cyclohexane containing 2% Et3N).
Yield: 0.11 g (0.11 mmol, 73%) as a mixture of isomers. Rf )
0.34 [96:4 CH2Cl2/CH3OH (v/v)]. MALDI-TOF m/z: [M + H]+
found, 1014.4; calcd, 1014.4. 31P NMR (67.9 MHz, CDCl3) δ:
153.3, 151.6, 150.1, 149.5.
Oligonucleotide Synthesis. All ODNs were synthesized by the
conventional phosphoramidite method38 by using a DNA/RNA
synthesizer. Standard procedures were used to synthesize ODNs
except for extended coupling time (10 min, DCI as an activator)
for phosphoramidite containing 2′-N-(pyren-1-yl)carbonyl-azetidine
unit and 2′-N-(pyren-1-yl)carbonyl-aza-ENA unit (coupling time
10 min, ETT as an activator). RNA synthesis was performed by
standard procedures56,57 using 2′-O-tBDMS as 2′-OH protecting
group and ETT as an activator with coupling time of 120 s. The
ODNs were cleaved from solid support and deprotected from
nucleobase protecting group using 32% aqueous ammonia at room
temperature for 24 h. Deprotection of the oligo-RNAs was carried
out using anhydrous methanolic ammonia (25% NH3/MeOH) at
55 °C for 16 h followed by 1 M TBAF/THF treatment. All ONs
were purified by PAGE (20% polyacrylamide/7 M urea), extracted
with 0.3 M NaOAc, and desalted with C18-reverse phase cartridges.
Purity of products was confirmed by PAGE and was greater than
95%. Each ON was verified by MALDI-TOF MS (Table S1 in the
Supporting Information). Concentration of ODNs containing 2′-N-
(pyren-1-yl)carbonyl-azetidine unit or 2′-N-(pyren-1-yl)carbonyl-
aza-ENA unit was determined accounting for the contribution to
the absorbance at 260 nm from the (pyren-1-yl)carbonyl- moiety.
The molar extinction coefficient (ꢀ*) for modified ODNs 1-14
was calculated from the extinction coefficient (ꢀ) for the corre-
sponding ODNs using the equation:
A260/A340
A260/A340 - A260*/A340
ꢀ* ) ꢀ ×
*
where A260 and A340 are the absorbance of Aze-pyr (X)- or Aza-
ENA-pyr (Y)-modified ODNs at 260 and 340 nm, respectively;
A260* and A340* are the absorbance of pyrenecarboxylic acid at 260
and 340 nm, respectively.
UV Melting Experiments. Determination of the Tm’s of the
ODNs/RNA or ODNs/DNA hybrid duplexes was carried out in
medium salt buffer, containing 100 mM NaCl (pH 7.0, adjusted
with 10 mM NaH2PO4/5 mM Na2HPO4) and 0.1 mM EDTA.
Absorbance was monitored at 260 nm in the temperature range
from 15 to 70 °C with ramp of 1 °C per min. Samples (mixture of
1.0 µM ODN and 1.0 µM RNA or DNA) were denatured at 80 °C
for 4 min followed by slow cooling to 15 °C prior to the
measurements. Tm values were obtained from the maxima of the
first derivatives of the melting curves. All thermal denaturation
temperatures given are the averages of at least of two independent
sets of experiments and are within (0.2 °C error range.
UV Absorption and Fluorescence Measurements, and CD
Experiments. Oligonucleotides solutions were prepared as de-
scribed in UV melting experiments (19.5 µM for ODNs 1-6 and
9.75 µM for ODNs 7-14, final strand concentration). Absorption
spectra were obtained at 25 °C using 1.0 cm path length cell. For
the fluorescence measurements above, samples were diluted with
the same buffer to the concentration of 0.8 µM of strands, and
spectra were obtained at 20 °C ((0.1 °C) using quartz optical cell
with a path length of 1.0 cm. Fluorescence emission spectra
(excitation wavelength of 340 nm) and excitation spectra (monitor-
ing wavelength at 376 and 380 nm) were obtained as an average
(56) Sproat, B.; Colonna, F.; Mullah, B.; Tsou, D.; Andrus, A.; Hampel,
A.; Vinayak, R. Nucleosides Nucleotides 1995, 14, 255-273.
(57) Milecki, J.; Zamaratski, E.; Maltseva, T. V.; Foldesi, A.; Adamiak,
R. W.; Chattopadhyaya, J. Tetrahedron 1999, 55, 6603-6622.
J. Org. Chem, Vol. 73, No. 7, 2008 2841

Honcharenko et al.
of five scans using an excitation slit of 4.0 nm, emission slit of 2.5
nm, and scan speed of 500 nm/min. Corrections were made for
solvent background, but no attempts were made to eliminate
dissolved oxygen in the buffer solution for the fluorescence
measurements. The fluorescence quantum yields [ΦF(ODN)] of
pyrene-labeled ODNs were determined according to:41
the Swedish Foundation for Strategic Research (Stiftelsen fo¨r
Strategisk Forskning), and the EU-FP6 funded RIGHT project
(project no. LSHB-CT-2004-005276) is gratefully acknowl-
edged. We would like to thank Dr. J. Barman for MALDI-TOF
MS analysis.
Supporting Information Available: General experimental
methods; experimental conditions for the preparation of 2′-N-(pyren-
1-yl)carbonyl-aza-ENA thymidine and its phosphoramidite building
block; MALDI-TOF MS of synthesized ONs; absorption spectra
of fully deprotected nucleosides 3 and 8; fluorescence emission
spectra of X-modified ODN 2-3 and Y-modified ODN 5-6; area
versus A340 curves for the determination of the emission quantum
yields of fully deprotected nucleosides 3 and 8; fluorescence
emission spectra for the temperature-dependent fluorescence changes
of the duplexes formed by ODN 1 or ODN 4 with complementary
target; fluorescence emission spectra of ODNs 7-14; expanded
CD spectra of the duplexes formed by ODNs 1 or 4 with
complementary DNA, RNA, and mismatched (G) RNA; CD spectra
of the native, X-modified, and Y-modified DNA/DNA and DNA/
ΦF(ODN) ) [A340(PBA)/A340(ODN)] × [F(ODN)/F(PBA)] ×
[n(H2O)/n(MeOH)]2 × ΦF(PBA)
where ΦF(PBA) is the fluorescence quantum yield of PBA as a
reference with a known ΦF of 0.065;42,43 A340 is the absorbance of
the sample at the excitation wavelength (340 nm); F is the area of
the fluorescence emission spectra of the sample from 360 to 590
nm; and n(H2O) and n(MeOH) are the refractive indexes of water
(1.3328) and methanol (1.3288). Fluorescence quantum yields were
determined as an average of minimum two measurements within
(10% error range. For CD experiments, oligonucleotide solutions
were prepared as described in UV melting experiments (10 µM,
final strand concentration). CD spectra were recorded from 380 to
220 nm at 25 °C using 0.2 cm path length cell. Spectra were
obtained as an average of five scans from which the CD spectrum
of buffer was subtracted.
1
RNA duplexes; H NMR spectra of compounds 2-4 and 7; 13C
NMR spectra of compounds 1-4, 6-9; and 31P NMR spectra of
compounds 5 and 10. This material is available free of charge via
the Internet at http://pubs.acs.org.
Acknowledgment. Generous financial support from the
Swedish Natural Science Research Council (Vetenskapsrådet),
JO702747W
2842 J. Org. Chem., Vol. 73, No. 7, 2008