DNSC: A fluorescent, environmentally sensitive cytidine derivative for the direct detection of GGG triad sequences

Article abstract of DOI:10.1039/c3ob41222a

With the goal of developing a fluorescent nucleoside sensitive to its environment, in this study we synthesized ^DNSC, a novel modified 2′-deoxycytidine bearing a 5-(dimethylamino)naphthalene-1-sulfonyl (dansyl) moiety at the N4 position, and tested its properties in monomeric and oligomeric states. ^DNSC undergoes intramolecular photoinduced electron transfer between its dansyl and cytosine units, resulting in remarkable changes in fluorescence that depend on the choice of solvent. In addition, the fluorescence behavior and thermal stability of oligonucleotides containing ^DNSC are dependent on the nature of the flanking and neighboring bases. Notably, ^DNSC exhibits fluorescence enhancement only in fully matched duplex DNA containing a GGG triad sequence. The environmental sensitivity of ^DNSC can be exploited as a fluorescence tool for monitoring the interactions of DNA with other biomolecules, including DNA, RNA, and proteins.

Full text of DOI:10.1039/c3ob41222a

Organic&
BiomolecularChemistry
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Volume 11 | Number 34 | 14 September 2013 | Pages 5549–5736
ISSN 1477-0520
PAPER
Byeang Hyean Kim et al.
DNSC: a fluorescent, environmentally sensitive cytidine derivative for
the direct detection of GGG triad sequences
1477-9226(2013)42:34;1-J

Organic &
Biomolecular Chemistry
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DNSC: a fluorescent, environmentally sensitive cytidine
derivative for the direct detection of GGG triad
sequences†
Cite this: Org. Biomol. Chem., 2013, 11,
5605
Ki Tae Kim,a Hyun Woo Kim,b Dohyun Moon,c Young Min Rheeb and
Byeang Hyean Kim*a
With the goal of developing a fluorescent nucleoside sensitive to its environment, in this study we syn-
thesized DNSC, a novel modified 2’-deoxycytidine bearing a 5-(dimethylamino)naphthalene-1-sulfonyl
(dansyl) moiety at the N4 position, and tested its properties in monomeric and oligomeric states. DNSC
undergoes intramolecular photoinduced electron transfer between its dansyl and cytosine units, result-
ing in remarkable changes in fluorescence that depend on the choice of solvent. In addition, the
fluorescence behavior and thermal stability of oligonucleotides containing DNSC are dependent on the
nature of the flanking and neighboring bases. Notably, DNSC exhibits fluorescence enhancement only in
fully matched duplex DNA containing a GGG triad sequence. The environmental sensitivity of DNSC can
be exploited as a fluorescence tool for monitoring the interactions of DNA with other biomolecules,
including DNA, RNA, and proteins.
Received 13th June 2013,
Accepted 25th June 2013
DOI: 10.1039/c3ob41222a
www.rsc.org/obc
application of these fluorescent nucleic acids remains limited
because of various drawbacks, which are expected to be over-
Introduction
The development of new fluorescent probes to investigate come through better design of novel types of fluorescent
specific DNA sequences or the interactions of DNA with other nucleosides.11
biomolecules remains a considerable challenge. Many fluo-
One of the general limitations of fluorescent DNA probes is
rescent nucleic acid probes have been designed over the years their susceptibility to quenching phenomena induced by
to detect specific sequences and single nucleotide polymorph- neighboring bases. In particular, fluorophores are commonly
isms (SNPs),1–3 to monitor the binding of targets to DNA,4 to quenched through photoinduced electron transfer (PET) to
detect specific enzymes,5,6 and to investigate nucleic acid guanine bases in DNA strands.12 Accordingly, previous fluo-
structures and functions.7,8 Commonly, fluorescent nucleo- rescence probes developed in our group, and in others, have
sides have been synthesized and incorporated into nucleic exhibited very low fluorescence intensity and sensitivity when
acid probes to impart them with unique properties, such as their target DNA strands have featured a series of guanine
site-specific responses (e.g., toward SNPs), micro-environ- (G)-rich sequences.13 Unfortunately, series of G bases appear
mental sensitivity, or high brightness.9–11 Nevertheless, the frequently in many biologically important DNA sequences,
including telomeric DNA strands,14 oncogene promoter
regions,15 codons for glycine, and CpG island regions.16 More-
over, mutations in consecutive G sequences have been an
important issue since the G-tract acts as a core sequence of the
binding site for transcription factors17 or is closely related to
HIV-1 reverse transcription.18 Therefore, the development of
novel fluorescent nucleic acids that exhibit higher fluorescence
in the presence of a G-rich sequence than in the presence of
other sequences will be necessary if we are to improve the
general applicability of fluorescent probes. Notably, to the best
of our knowledge there are no previously reported examples of
the direct sensing of the GGG triple sequence.
aDepartment of Chemistry, BK School of Molecular Science, Pohang University of
Science and Technology (POSTECH), Pohang 790-784, South Korea.
E-mail: bhkim@postech.ac.kr; Fax: +82 54-279-3399; Tel: +82 54-279-5856
bDepartment of Chemistry, Center for Self-assembly and Complexity, Institute for
Basic Science, Pohang University of Science and Technology (POSTECH),
Pohang 790-784, South Korea. E-mail: ymrhee@postech.ac.kr
cBeamline Division, Pohang Accelerator Laboratory, Pohang University of Science and
Technology (POSTECH), Pohang 790-784, South Korea.
E-mail: dmoon@postech.ac.kr; Tel: +82 54-279-1547
†Electronic supplementary information (ESI) available: Supporting figures and
graphs, additional UV absorption and fluorescence emission spectra, NMR
titration results, crystallographic data and MALDI-TOF MS analysis of ODNs.
CCDC 930728. For ESI and crystallographic data in CIF or other electronic
format see DOI: 10.1039/c3ob41222a
The 5-(dimethylamino)naphthalene-1-sulfonyl (dansyl) group
is a practical fluorophore used widely in protein sequencing
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and amino acid analysis. In addition, the dansyl fluorophore
has also been used as the reporting part of fluorescent DNA
systems because of its large Stokes shifts, intense absorption
bands, strong fluorescence, and high sensitivity to the hydro-
phobicity of its environment.19 In previous examples of chemi-
cal conjugation to nucleosides, dansyl groups have been
attached to the 2′-OH, C-5, and C-8 positions of the
nucleosides20–22 to obtain probes for monitoring the inter-
actions between DNA strands, between DNA and ligands, or to
rationally design multiple fluorescent DNA probes. Notably,
the fluorescence of the dansyl group is very sensitive to its
position in the nucleoside or DNA strand and to the polarity of
its environment. For example, oligonucleotides with dansyl
linked by a relatively short linker at C-5 position of pyrimidine
exhibited little or no change in fluorescence by duplex for-
mation while the dansyl group at the C-8 position of purine exhibi-
ted an enhancement of fluorescence intensity.20 Based on these
previous reports, we expected that direct conjugation of a
dansyl group in the region of a nucleobase that undergoes
hydrogen bonding might lead to greater fluorescence sensi-
tivity toward changes in single nucleotides or structural
changes of DNA (e.g., duplex formation). Furthermore, proxi-
mity of the base and the dansyl group may induce unique elec-
tronic interaction in the excited state.
In this study, we easily synthesized the nucleoside DNSC,
which features a dansyl group attached directly at the N4 posi-
tion – a hydrogen bonding unit and a major groove site – of cyto-
sine and then we monitored its fluorescence properties as well
as those of oligonucleotides incorporating it. We found that
DNSC exhibits unique properties for a dansyl-derivative, allow-
ing its use as a fluorescence-on system for the GGG triplet
sequence.
Scheme 1 Synthesis of DNSC and 3. Reagents and conditions: (a) tert-butyl-
dimethylsilyl chloride, imidazole, DMF, r.t., 12 h, 88%; (b) dansyl chloride, DIPEA,
CH2Cl2, 50 °C, 24 h, 70%; (c) tetrabutylammonium fluoride, THF, r.t., 6 h, 97%;
(d) 4,4’-dimethoxytrityl chloride (DMT-Cl), DIPEA, DMF, 55 °C, 24 h, 80%.
TBDMS, tert-butyldimethylsilyl; DIPEA, N,N-diisopropylethylamine; DMF, N,N-
dimethylformamide; THF, tetrahydrofuran.
Results and discussion
Synthesis and photophysical properties of DNS
C
To directly conjugate a dansyl group to a nucleoside through
its amino group, we chose 2′-deoxycytidine as the starting
material. Protecting the 3′- and 5′-OH groups of 2′-deoxycyti- ring). The planes of the cytosine and dansyl groups were
dine through reactions with tert-butyldimethylsilyl chloride aligned almost orthogonally because of the sulfonyl group. In
(TBDMS-Cl), followed by reaction with dansyl chloride and the packing structure, we observe anti-parallel π–π interactions
tetrabutylammonium fluoride-mediated deprotection of the between the dansyl groups of pairs of DNSC units (Fig. 1b);
TBDMS groups, we obtained compound DNSC. To incorporate in addition, one DNSC unit recognized another through O(5)–
DNSC into oligonucleotides, we protected DNSC through reac- H(5)⋯O(6) hydrogen bonding.
tion with dimethoxytrityl chloride (DMT-Cl) to obtain 3, the
precursor of DNA phosphoramidite. Scheme 1 summarizes the chemical calculations to study the photophysical behavior of
synthetic process.
DNSC, analyzing three of its excited states with a developmental
Encouraged by its solid state structure, we used quantum
We confirmed the identities of these compounds using H version of the Q-Chem software package23 (see Table 1 and
and 13C NMR spectroscopy, mass spectrometry, and melting Fig. S3 in ESI†). We optimized the geometries at the B3LYP/
point measurements. In addition, we obtained single crystals 6-31G(d) computational level24 and determined the properties
of DNSC through slow evaporation of MeCN from a 20 mM solu- of the excited states using the SOS-CIS(D0)/6-31G(d) method.25
tion in a sealed tube. Fig. 1a displays the structure of DNSC as To incorporate an approximate solvent effect, we employed the
determined using X-ray crystallography (CCDC registry conductor-like screening model (COSMO) by treating the
number: 930728). The individual units of DNSC featured a syn solvent as a continuum having a dielectric constant ε of 80.26
configuration (oxygen on C-2 is projecting toward the furanose In Table 1, all computed energies are listed with respect to the
1
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Table 2 Photophysical properties of DNSC
λabsa (nm)
λemia (nm)
ε
a,c (cm−1 M−1
)
Φf
a,b
MeOH
MeCN
DMF
DMSO
Water
295
298
290
290
295
490
474
470
480
520
48 200
44 700
31 000
29 600
33 700
0.0090
0.010
0.54
0.74
0.0028
a Measured at 3.3 μM at 298 K. b Fluorescence quantum yield measured
relative to fluorescein (Φf
coefficient.
= 0.79) in 0.1 M
NaOH. c Extinction
270 nm; in contrast, DNSC exhibits its value of λex near 295 nm
in aqueous solution. This bathochromic shift presumably
resulted from electron withdrawal by the sulfonyl group at the
N4 position. We also confirmed that the UV absorption of the
original dansyl moiety was maintained for the solution of DNS
C
in water (see Fig. S1a in ESI†). In the normalized UV absorp-
tion spectra of DNSC, we observed an absorption band at
290–298 nm for all solvents, with a shoulder at 330 nm only
for the solutions in the polar aprotic solvents DMF and DMSO
(Fig. 2a). Interestingly, the fluorescence of DNSC exhibited a
Fig. 1 (a) Solid state structure of DNSC (30% probability ellipsoids) and (b) its
packing in the crystal.
Table 1 Calculated excited state energies and transition characters of DNSC
Energy (kcal mol−1
)
Statea
Vacuum
ε = 80
Transition character
S1
S2
S3
98.94
100.98
107.5
96.71
101.02
108.72
Local transition at the dansyl moiety
Local transition at the dansyl moiety
Local transition at the base
a Three lowest-energy singlet excited states (S1, S2, S3); see the
Experimental section for computational details.
ground state energies under vacuum and in a continuum
solvent. By investigating the character of each excited state, we
found that the dansyl moiety was excited mainly in the S1 and
S2 states, while an electronic transition at cytosine contributed
significantly to the S3 state. Importantly, the energy differ-
ences among S1, S2, and S3 (4.3–12.0 kcal mol−1) in the polar
solvent were sufficiently small to allow internal conversions
between these excited states under ambient conditions.
Because excited DNA bases can dissipate energy non-radia-
tively,27 these results implied that the fluorescence of DNSC
would likely be quenched through internal conversions to the
state with the local electronic transition at the cytosine unit.
Next, we checked the photophysical properties of DNSC as solu-
tions in CH2Cl2, THF, MeOH, MeCN, DMF, DMSO, and water
Fig. 2 (a) UV absorption and (b) fluorescence emission spectra of DNSC in
various solvents (concentration, 3.3 μM; total volume, 1 mL; excitation, 295 nm,
(Table 2). Typically, cytosine exhibits UV absorbance at excitation and emission slit: 5 nm).
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significant solvent-dependency. Firstly, we confirmed solvent-
dependency of fluorescence emission by using ET(30) values
(Fig. S1b in ESI†). In MeOH, MeCN, and water, DNSC displayed
very low fluorescence quantum yields, whereas it provided very
strong fluorescence in DMF and DMSO; for example, the fluo-
rescence quantum yield of DNSC in DMSO was 264-fold greater
than that in water (Table 2, Fig. 2b). Typically, dansylamide
derivatives lacking a quencher have moderate quantum yields
(ca. 0.26–0.53) in other solvents.28 Accordingly, the very low
quantum yield of DNSC in MeCN presumably arose as a result
of quenching by the nucleobase. Moreover, dansylamide is a
solvatochromic fluorophore which exhibits large Stokes shifts
in polar solvent,29 while DNSC does not exhibit significant
Stokes shift according to solvent change. Thus, we expected
that the properties of dansyl were significantly changed by
direct conjugation with cytosine. Next, we needed to investi-
gate the properties of DNSC under a variety of pH conditions
since cytosine is a pH sensitive molecule and can be proto-
nated at low pH in order to form a biologically important
structure, i-motif. As a result, we observed that the fluo-
rescence of DNSC exhibited a high pH-dependency, with the
inflection point at pH 6.9 (Fig. 3). Through NMR spectroscopic
titration experiments (see Fig. S2 in ESI†),30 we found that the
protonation or deprotonation of DNSC underwent a transition
at pH 6.8, consistent with the fluorescence data. We speculated
that the pH-dependent fluorescence change resulted from
deprotonation of DNSC that occurred at sulfonamide, the N4
position of the cytosine unit. Previous reports confirmed the
decrease of the pKa value of dansylamide (from 10.6 to 6.3) in
the presence of the carbonyl group, which can stabilize the
Fig. 4 Cyclic voltammogram of DNSC (3.4 mM aqueous solution containing
50% MeOH; supporting electrolyte, 100 mM KCl; reference electrode, Ag/AgCl;
scan rate, 50 mV s−1).
Similar solvent and pH-dependent fluorescence behavior
has been observed previously for various other fluorescent
nucleosides. For example, the fluorescence of 5-(1-pyrenyl)-
modified pyrimidine exhibits significant pH-dependence.33–35
In particular, Py-dC displays very low fluorescence over the
entire pH range, much like DNSC.12,33,35–37 Because the fluo-
rescence of Py-dC can be quenched through proton-coupled
electron transfer, we suspected that the luminescence of DNSC
could also be quenched through a similar mechanism. To
obtain evidence for this hypothesis, we measured the electro-
chemical potential of DNSC (Fig. 4), observing the oxidation
potential of the dansyl group at +0.81 V and the reduction
potential of the cytosine unit at −1.08 V, using a Ag/AgCl elec-
trode. Accordingly, the dansyl moiety exhibited almost the
same oxidation potential as that determined previously
(+0.9 V),28a,38 whereas the reduction potential of the dansyl-
modified cytosine unit was slightly lower than that of natural
cytosine.39 According to the Rehm–Weller equation,40 ΔE(0,0)
was equal to 2.96 eV (Fig. S4 in ESI†); together with the
redox potential of DNSC, we determined the value of ΔGeT to be
−1.07 eV (−103 kJ mol−1), implying that electron transfer would
be energetically favored.
anionic form of dansylamide.31 Likewise, dansylamide in DNS
C
seemed to have a low pKa value of 6.8 because of the stabiliz-
ing effect of the cytosine ring. We also observed a fluorescence
enhancement on deprotonation of sulfonamide.31a,32 Accord-
ingly, we concluded that the degree of deprotonation influ-
ences the fluorescence of DNSC.
Study of the fluorescence behavior of DNSC in oligonucleotides
Next, we introduced DNSC into four types of 15-mer oligo-
nucleotide (ODN) sequences containing different flanking bases.
In previous studies, dansyl moieties have been conjugated to
nucleosides in a variety of positions, including the C5 and C8
positions of the nucleobases and 2′-O position of the sugar
ring.20–22 The same dansyl moiety can readily modify the fluo-
rescence properties of ODNs merely by changing the position
and linker for conjugation. We suspected that ODNs contain-
ing a DNSC residue might exhibit significant changes in fluo-
rescence upon duplex formation, as a result of changes in the
protonation state of its cytosine unit; in addition, we expected
different neighboring bases to provide different environments
Fig. 3 Fluorescence intensity of DNSC plotted with respect to pH (concen-
tration, 3 μM; total volume, 1 mL; excitation, 295 nm).
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Table 3 ODN sequences investigated in this study
ODN
Sequence (5′ to 3′)
DNS-Na
Nat-Na
GCT CTT NDNSCN TTC TCG
GCT CTT NCN TTC TCG
ODN-X-Yb
DNS-Probe
Htelo
CGA GAA YXY AAG AGC
A CCC TAA CDNSCC TAA CDNSCC TAA CCC T
A(GGGTTA)3GGGT
a N = A, T, C, G. b X, Y = A, T, C, G.
for the DNSC residue in the ODNs, further influencing the fluo-
rescence behavior.
We synthesized twenty-four 15-mer ODNs, including four
modified sequences, and confirmed their masses using MALDI-
TOF mass spectrometry (see Table 3 and Table S1 in ESI†).
The four DNS-N strands (N = A, T, C, G) had the DNSC residue in
the middle of the ODN with different flanking bases. We
checked the basic UV absorptions and fluorescence emissions
of the single- and double-strand forms of the DNS-N ODNs.
Each DNS-N had 17 different states—including the single strand
and fully matched and singly and triply mismatched double
strands—when combined with the Nat-N sequences. We
recorded all of these UV absorption and fluorescence emission
spectra in a buffer solution at pH 9 in the absence of any salt.
First, we checked the CD spectra of all the strands mixed
with DNS-C to confirm duplex formation. We observed positive
and negative CD signatures for duplexes at 275 and 250 nm,
respectively, even for those containing single or triple mis-
matches; in contrast, the single-stranded DNS-C exhibited very
weak CD signals at these wavelengths (see Fig. S5 in ESI†). In
the UV absorption spectra of these ODNs, we observed the
same patterns, with an intense absorption at 260 nm, resulting
from the absorptions of the natural nucleobases (see Fig. S6 in
ESI†). Because DNSC absorbs at wavelengths of 295 and
330 nm, its absorption bands overlapped with those of the
other residues in the ODNs. We could not detect any specific
differences in the UV spectroscopic behavior of these ODNs;
we did, however, observe unique phenomena in their fluo-
rescence spectra. For DNS-C, we observed a three-fold enhance-
Fig. 5 Fluorescence emission spectra of DNS-C in duplexes with different triad
sequences (concentration, 1.5 μM; total volume, 1 mL; buffer, 100 mM Tris,
pH 9.0; excitation, 295 nm, excitation and emission slit: 10 nm).
Fig. 6 Relative fluorescence emission intensities of DNS-N strands in the pres-
ence of various complementary triad sequences.
that the fluorescence of the DNSC residue is dependent on the
ment in fluorescence only for its duplex with ODN-G-G, which nature of its complementary and flanking bases.
contains a GGG triad in the middle of its sequence (Fig. 5). For
a comparison of the fluorescence intensity, we estimated the
The unique fluorescence enhancement of the DNSC residue
appears to be closely related to the stability of its duplex. In
fluorescence quantum yield of oligonucleotides by using the the presence of a salt, we observed a slightly increased fluo-
extinction coefficient. We assumed that DNSC have the same rescence for all of the fully matched duplexes of DNS-A, -T, and
extinction coefficient even in oligonucleotides and calculated
-G (see Fig. S8 in ESI†). These results meant that the formation
the absorbance from the concentration of oligonucleotides. of a stable duplex might facilitate deprotonation of N4 posi-
We finally obtained 0.020 for duplex having GGG triad and tion. Actually, according to previous studies, proton transfer
from cytosine to guanine can occur in the G/C pair.41 Thus, we
0.0056–0.0092 for the others. Compared to the nucleoside in
aqueous solution, DNSC in oligonucleotides have similar expected that this process may be favoured in the GGG triad
quantum yields, which means that it was exposed to water. because of the neighbouring strong G/C pair holding the DNSC/
G pair together and the low pKa value of DNSC leading to easy
None of the duplexes containing mismatched sequences
exhibited any enhancement of fluorescence. Notably, DNS-A, deprotection and fluorescence enhancement. From measure-
-T, and -G exhibited no fluorescence enhancements even in ments of melting temperatures (Tm), we also observed the
duplexes with their fully matched sequences in the absence of
highest thermal stability for the duplex of DNS-C and ODN-
a salt (see Fig. 6 and Fig. S7 in ESI†). These findings confirmed G-G in the presence of salts (Table 4).
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Table 4 Values of Tm of fully matched and single-base-mismatched duplexes of DNS-N strands
Tm (°C)
–NAN–
–CDNSCC– (DNS-C)
–GDNSCG– (DNS-G)
–ADNSCA– (DNS-A)
–TDNSCT– (DNS-T)
21.0a (41.0)b
−3.0 (−4.0)
17.2 (39.2)
−7.0 (−6.0)
15.4 (36.4)
−4.0 (−5.0)
23.1 (44.6)
−16.5 (−14.0)
26.0 (43.4)
−1.0 (−3.6)
20.2 (42.2)
−5.0 (−3.0)
23.4 (44.3)
1.0 (−0.1)
19.0 (36.7)
1.0 (−1.3)
17.7 (37.6)
−1.5 (−2.6)
20.3 (39.3)
5.0 (3.0)
21.0 (40.1)
6.0 (1.5)
c
ΔTm
–NTN–
ΔTm
13.1 (34.1)
−5.0 (−6.1)
13.3 (34.4)
−1.0 (−3.0)
13.6 (35.6)
−19.4 (−18.9)
–NCN–
ΔTm
–NGN–
ΔTm
22.5 (42.5)
−20.1 (−18.0)
17.4 (39.5)
−18.0 (−14.0)
a Sample concentration, 1.5 μM; buffer, 100 mM Tris (pH 9.0); without salt. b Sample concentration, 1.5 μM; buffer, 100 mM Tris (pH 9.0),
100 mM NaCl, 20 mM MgCl2. c Thermal stability differences with natural sequences (Tm of modified duplex − Tm of natural duplex).
with their complementary sequences because the electron-with-
drawing sulfonyl group and aromatic ring of the dansyl moiety
of the DNSC residue would modify the strength of the hydrogen
bond between the cytosine and guanine bases. Indeed, DNSC
exhibited different properties that differentiated it from natural
deoxycytidine—primarily through disruption of the original
hydrogen bonding pattern with guanine. We observed a highly
destabilizing effect of the DNSC residue for the fully matched
duplexes of the DNS-N strands in the presence of a salt
(Table 4; ΔTm = –14.0, −18.0, −14.0, −18.9 °C, relative to the
Nat-N strands). This destabilizing effect of DNSC was still main-
tained in single-mismatched duplexes. According to previous
reports, exocyclic amine42 and exocyclic adduct43 were deleter-
ious to duplex stability due to steric hindrance of the bulky exo-
cyclic group and the weakened hydrogen bond. Similarly, we
Fig. 7 Fluorescence enhancement upon duplex formation between DNS-Probe
suspected that steric hindrance existed between the dansyl unit
and the flanking bases as a result of the almost perpendicular
and Htelo. Inset: photograph of the fluorescence of (a) DNS-Probe and (b)
DNS-Probe + Htelo (sample concentration, 1.5 μM; total volume, 1 mL; buffer,
arrangement (the angle of N(2)–S(1)–C(12) is 106.72°, see
100 mM Tris, pH 9.0; excitation, 295 nm, excitation and emission slit: 10 nm).
Table S4 in ESI†) of the cytosine and dansyl moieties. As a
result of this effect, the thermal stabilities of the fully matched
Direct detection of the GGG triad sequence by DNS
C
and single-base-mismatched duplexes were similar, allowing
DNSC to be used as a universal base. On the other hand, the
destabilizing effect of DNSC was diminished in single-adenine-
mismatched duplex (–NAN–) of DNS-C and -T (Table 4). For
DNS-T, in particular, the single-adenine-mismatched duplex
had a higher stability than the natural duplex (ΔTm = +1.5 °C)
while the other single mismatched duplex exhibited a lower
stability than the natural one (ΔTm = −6.1, −3.0, −18.9 °C,
respectively). There should be interactions between adenine and
DNSC bases, inducing a new type of weak DNSC/dA pair. To sum
up, the dansyl group of DNSC seemed to distort the stable ori-
ginal G/C pair and to destabilize the entire duplex. However,
DNSC exhibits the properties of a universal base, featuring even
stability for all fully matched and single-base-matched duplexes.
We also confirmed the unique stability of single-adenine-
mismatched duplexes, although the stability of these duplexes
was susceptible to the nature of the flanking bases.
Based on previous results, we suggest that the fluorescence
enhancement of a duplex is sensitive to its stability and to the
nature of the bases neighboring the DNSC moiety; further
studies will be necessary, however, to confirm the utility of
DNSC for detection of GGG sequences. For that, we designed a
simple fluorescent probe, DNS-Probe, for sensing the GGG
triad sequence (Table 3). DNS-Probe contains two DNSC resi-
dues in the middle of its sequence to detect each GGG triad
position in complementary Htelo, a human G-quadruplex-
forming sequence. As expected, DNS-Probe exhibited a three-
fold enhancement of fluorescence in its duplex form (Fig. 7)
and we could observe
a fluorescence enhancement of
DNS-Probe with the naked eye in the presence of Htelo.
Accordingly, DNSC appears to be useful for the detection of bio-
logically relevant sequences containing the GGG triad.
Study of the thermal stability of duplex containing DNS
C
Furthermore, we checked the duplex stabilities of the DNS-N
and Nat-N strands from their melting temperature curves
Conclusions
recorded in the absence and presence of a salt. We expected The development of novel fluorescent nucleosides remains a
that the DNS-N strands would exhibit novel types of interactions challenging task for the sensitive detection of specific DNA
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sequences and for monitoring the interactions between DNA equiv.) in dry DMF and then the mixture was stirred at room
strands and other biomolecules. The dansyl group is used temperature for 10 h. After concentrating, the residue was par-
widely as a fluorophore for nucleosides because it is sensitive titioned between CH2Cl2 (100 mL) and water (100 mL). The
to hydrophobic/hydrophilic conditions. Nevertheless, the organic phase was washed with saturated NaCl (2 × 100 mL),
quest remains for new dansyl-conjugated nucleosides that dried (Na2SO4), and filtered. The solvent was evaporated and
can provide more sensitive fluorescence detection of DNA the residue was dried under reduced pressure. The crude
systems. For that reason, we synthesized DNSC and character- product was purified through flash chromatography (SiO2,
ized its photophysical properties. Relative to general dansyl- EtOAc) to give a white solid 1 (800 mg, 88%). TLC: Rf = 0.18
amides, DNSC exhibits more significant enhancements in (EtOAc); m.p. = 178–180 °C; 1H NMR (300 MHz, DMSO-d6,
fluorescence in response to changes in the polarity of the 25 °C): δ = 0.07 (12H, s, SiCH3), 0.87 (9H, s, t-Bu), 0.88 (9H, s,
solvent, presumably resulting from PET from the dansyl group t-Bu), 1.97–2.06 (1H, m, 2′-H), 2.10–2.18 (1H, m, 2′-H),
to the cytosine unit. Finally, we incorporated DNSC also into a 3.67–3.81 (3H, m, 5′-H, 3′-H), 4.32–4.36 (1H, m, 4′-H), 5.70 (1H,
series of 15-mer ODNs. Most dramatically, the DNS-C sequence d, J = 7.5 Hz, 5-H), 6.15 (1H, t, J = 6.3 Hz, 1′-H), 7.16 (2H, br,
exhibited a threefold enhancement in fluorescence after N4-H), 7.70 (1H, d, J = 7.2 Hz, 6-H); 13C NMR (75 MHz; DMSO-
forming a duplex with a sequence containing a GGG triad d6, 25 °C): δ = −5.7, −5.6, −5.0, −4.8, 17.6, 17.9, 25.6, 25.7,
sequence.
62.3, 71.4, 84.5, 86.4, 93.8, 140.3, 154.9, 165.5; HRMS-FAB MS
Clearly, DNSC exhibits properties quite different from those (m/z): calcd for C21H41N3O4Si2 [M + Na]+: 478.2534; found:
of the natural cytosine and dansyl group. Accordingly, we 478.2536.
expect that the unique properties of fluorescent DNSC could be
applied in several fluorescent systems for studying GGG triads,
Synthesis of 4-N-dansyl-5′-O, 3′-O-di-tert-butyldimethylsilyl-2′-
deoxycytidine (2)
G-quadruplex or i-motif sequences, and DNA–protein inter-
actions in due course.
Compound 1 (517 mg, 1.13 mmol) and dansyl chloride
(457 mg, 1.5 equiv.) were dried under vacuum for 2 h and then
they were dissolved in dry CH2Cl2 (20 mL) and stirred under
Ar. DIPEA (0.3 mL, 2.0 equiv.) was added and then the mixture
was stirred for 24 h. The solvent was evaporated under vacuum
and the residue was partitioned between CH2Cl2 (100 mL) and
water (100 mL). The organic phase was washed with saturated
NaCl (2 × 100 mL), dried (Na2SO4), and filtered. The product
was purified through column chromatography (SiO2, CH2Cl2 to
CH2Cl2–EtOAc = 1 : 1) to yield a pale-yellow product 2 (546 mg,
70%). TLC: Rf = 0.88 (CH2Cl2–EtOAc = 4 : 1); m.p. = 103–105 °C;
1H NMR (300 MHz, DMSO-d6, 25 °C): δ = 0.03 (12H, s, SiCH3),
0.83 (18H, s, SiCH3), 2.11–2.29 (2H, m, 2′-H), 2.81 (6H, s,
N–CH3), 3.65–3.80 (3H, m, 5′-H, 3′-H), 4.30–4.31 (1H, m, 4′-H),
5.96 (1H, t, J = 5.4 Hz, 1′-H), 6.38 (1H, d, J = 8.1 Hz, 5-H),
7.23 (1H, d, J = 7.2 Hz, PhH), 7.56 (1H, t, J = 8.7 Hz, PhH), 7.62
(1H, t, J = 7.5 Hz, PhH), 7.89 (1H, d, J = 8.1 Hz, 6-H), 8.20 (1H,
d, J = 8.4 Hz, PhH), 8.32 (1H, d, J = 8.7 Hz, PhH), 8.43 (1H, d, J
= 8.4 Hz, PhH), 12.22 (1H, s, N4-H); 13C NMR (75 MHz; DMSO-
d6, 25 °C): δ = −5.7, −5.1, −4.8, 17.6, 17.9, 25.6, 45.0, 61.7,
70.3, 85.2, 86.8, 95.4, 115.0, 120.2, 123.5, 126.8, 127.5, 129.0,
129.1, 129.3, 138.3, 142.3, 148.3, 151.1, 159.8; HRMS-FAB
MS (m/z): calcd for C33H52N4O6SSi [M+]: 688.3146; found:
688.3149.
Experimental
General experimental details
Each reaction was performed under anhydrous conditions
under Ar or N2. All reagents were purchased from Sigma-
Aldrich, Fluka, Proligo, and Glen Research and were used
without additional purification. Most solvents were used
without distillation, except for those for phosphoramidite syn-
thesis. High-resolution fast atom bombardment (HRMS-FAB)
mass spectra were recorded using a Jeol JMS700 HR mass
spectrometer at the Korea Basic Science Center, Daegu, Korea.
MALDI-TOF mass spectra were recorded at Bioneer, Taejon,
Korea. 1H NMR spectra were recorded using a FT-300 MHz
Bruker Aspect 3000 spectrometer. UV and fluorescence spectra
and Tm of oligonucleotides were recorded using Cary 100 and
Eclipse spectrometers (Varian). Samples for UV/fluorescence
spectroscopy were prepared in a quartz cell (path length:
1 cm). DNA synthesis was performed using a Polygen 12
synthesizer. An Agilent high-performance liquid chromato-
graphy system (1100 Series) was used to purify the synthesized
ODNs; Agilent, ZORBA X Eclipse XDB-C18, 9.4 × 250 mm; gra-
dient elution: 0 min, A : B = 90 : 10; 10 min, A : B = 90 : 10;
20 min, A : B = 0 : 100; 25 min, A : B = 0 : 100; 30 min, A : B =
90 : 10; solution A, 0.1 M triethylammonium acetate (TEAA)
buffer (pH 7.2)–MeCN, 95 : 5; solution B, 0.1 M TEAA buffer
(pH 7.2)–MeCN, 1 : 1; flow rate: 2.5 mL min−1; UV detection:
254 nm.
Synthesis of 4-N-dansyl-2′-deoxycytidine (DNSC)
THF (10 mL) and 1 M tetrabutylammonium fluoride (TBAF,
2.1 mL, 4 equiv.) were added to compound 2 (360 mg,
0.52 mmol) and then the mixture was stirred at room tempera-
ture for 6 h. The solvent was evaporated and then CH2Cl2 was
added to the residue, which was purified through column
chromatography (SiO2; CH2Cl2–MeOH, from 20 : 1 to 10 : 1) to
obtain a yellow compound DNSC (233 mg, 97%). TLC: Rf = 0.41
Synthesis of 5′-O,3′-O-di-tert-butyldimethylsilyl-2′-
deoxycytidine (1)
TBDMS-Cl (602 mg, 4.0 equiv.) was added to a solution of (CH2Cl2–MeOH = 9 : 1); m.p. = 128–130 °C; 1H NMR (300 MHz,
2′-deoxycytidine (450 mg, 2 mmol) and imidazole (817 mg, 6.0 DMSO-d6, 25 °C): δ = 2.05–2.11 (2H, m, 2′-H), 2.80 (6H, s,
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NCH3), 3.50–3.55 (2H, m, 5′-H), 3.75–3.78 (1H, m, 3′-H), coordinates were allowed to ride on their respective atoms.
4.15–4.18 (1H, m, 4′-H), 5.00 (1H, t, J = 5.4 Hz, 5′-OH), 5.23 None of the hydrogen atoms on carbon atoms were included
(1H, d, J = 4.5 Hz, 3′-OH), 6.02 (1H, t, J = 6.3 Hz, 1′-H), 6.39 during the least-squares refinement. Refinement of the struc-
(1H, d, J = 8.1 Hz, 5-H), 7.22 (1H, d, J = 7.5 Hz, PhH), 7.56 (1H, ture converged at a final value of R1 of 0.0385 and R2 of 0.1072
t, J = 8.4 Hz, PhH), 7.63 (1H, t, J = 8.1 Hz, PhH), 8.03 (1H, d, J = for 4600 reflections [with I > 2σ(I)] (R1 = 0.0434 and wR2
=
7.8 Hz, 6-H), 8.24 (1H, d, J = 7.2 Hz, PhH), 8.34 (1H, d, J = 0.1019 for all reflections). The largest difference peak and hole
8.7 Hz, PhH), 8.43 (1H, d, J = 8.4 Hz, PhH), 12.19 (1H, br, were 0.886 and −0.439 e Å−3, respectively. A summary of
N4-H); 13C NMR (75 MHz; DMSO-d6, 25 °C): δ = 45.1, 48.6, the crystallographic data and refinement parameters is given
61.0, 70.0, 85.3, 87.8, 95.9, 115.1, 120.3, 123.6, 126.9, 127.6, in the ESI (Table S2–S6, and Fig. S9 in ESI†). CCDC 930728
129.1, 129.2, 129.4, 138.3, 142.8, 148.6, 151.2, 160.0; contains the supplementary crystallographic data for this
HRMS-FAB MS (m/z): calcd for C21H24N4O6S [M+]: 460.1417; paper.
found: 460.1420.
Computational details
Synthesis of 4-N-dansyl-5′-O-dimethoxytrityl-2′-
All quantum chemical calculations were performed with a
deoxycytidine (3)
developmental version of Q-Chem.23 Geometry optimization of
DIPEA (0.89 mL, 10 equiv.) was added to a solution of DNSC
DNSC was undertaken at the B3LYP/6-31G(d) level.24 The
(233 mg, 0.51 mmol) in dry DMF. After 1 h, DMT-Cl (260 mg,
obtained geometry was utilized for calculations of excited
1.5 equiv.) was added and then the mixture was stirred at
state energies. The SOS-CIS(D0) method with the 6-31G(d)
55–60 °C for 10 h. After quenching the reaction with MeOH
basis set was adopted for computing the excited state pro-
(2 mL), the mixture was stirred for 1 h. After concentrating, the
perties because this method provides a reliable behavior
residue was partitioned between CH2Cl2 (100 mL) and
for charge-transfer excited states and quasi-degenerate excited
NaHCO3 (100 mL). The organic layer was washed with satu-
states.25 The solvent effect was considered by adopting
rated NaCl (2 × 100 mL), dried (Na2SO4), and filtered. The
COSMO.26
solvent was evaporated and the residue was dried under
reduced pressure. The crude product was purified through
flash chromatography (SiO2; hexane–EtOAc, from 3 : 1 to 1 : 1)
Cyclic voltammetry
A solution of DNSC (3.4 mM) was prepared in 50% aqueous
to give a yellow solid 3 (311 mg, 80%). TLC: Rf = 0.21 (hexane–
1
MeOH. The electrolyte was 100 mM KCl. The redox potential
of DNSC was measured using a Model 263A potentiostat/
galvanostat (Princeton Applied Research) at a scan rate of
100 mV s−1 and over a scan range of 1.5 V. Two carbon elec-
trodes were employed as the working and counter electrodes;
a saturated Ag/AgCl electrode was used as the reference
electrode.
EtOAc = 1 : 1); m.p. = 132–134 °C; H NMR (300 MHz, DMSO-
d6, 25 °C): δ = 2.05–2.14 (2H, m, 2′-H), 2.80 (6H, s, NCH3),
3.25–3.30 (2H, m, 5′-H), 3.75 (6H, s, OCH3), 3.85–3.87 (1H, m,
3′-H), 4.27–4.30 (1H, m, 4′-H), 5.31 (1H, m, 3′-OH), 6.00 (1H, t,
J = 5.4 Hz, 1′-H), 6.23 (1H, d, J = 7.8 Hz, 5-H), 6.89 (4H, d, J =
6.6 Hz, PhH), 7.22–7.37 (10H, m, PhH), 7.52–7.58 (2H, m,
PhH), 7.89 (1H, d, J = 8.1 Hz, 6-H), 8.11 (1H, d, J = 6.9 Hz,
PhH), 8.32 (1H, d, J = 8.4 Hz, PhH), 8.42 (1H, d, J = 8.4 Hz,
PhH), 12.21 (1H, br, N4-H); 13C NMR (75 MHz; DMSO-d6,
25 °C): δ = 45.0, 55.0, 62.6, 69.0, 85.0, 85.5, 85.9, 95.7, 113.2,
115.0, 120.2, 123.4, 126.8, 127.5, 127.7, 127.9, 129.3, 129.6,
129.8, 135.1, 135.5, 138.2, 142.5, 144.5, 148.3, 151.2, 158.1,
159.8; HRMS-FAB MS (m/z): calcd for C42H42N4O8S [M+]:
762.2723; found: 762.2720.
Acknowledgements
We thank the EPB Center Program of MEST/NRF
(20120000528) and the NRF of Korea (2012R1A2A2A01047069)
for financial support and H. C. Jeon for help with the cyclic
voltammetry experiments.
Crystal data for DNSC (C21H24N4O6S)
A crystal of DNSC was coated with paratone-N oil and its diffrac-
tion data measured at 95 K with synchrotron radiation (λ =
0.72999 Å) on an ADSC Quantum-210 detector at 2D SMC with
a Si (111) double crystal monochromator (DCM) at the Pohang
Accelerator Laboratory, Korea. The ADSC Q210 ADX program44
was used for data collection and HKL3000sm (v. 703r) soft-
ware45 was used for cell refinement, reduction, and absorption
correction. The crystal structure of DNSC was solved using the
direct method and refined through full-matrix least-squares
calculations with the SHELX-TL (v. 2008) program package.46
All non-hydrogen atoms were refined anisotropically. The
hydrogen atoms were assigned isotropic displacement coeffi-
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