Syntheses and photophysical properties of 5′-6-locked fluorescent nucleosides

Article abstract of DOI:10.1039/c2ob26536b

Nine fluorescent 5′-6-locked nucleosides were synthesized by condensation of various 1,2-diketones with 5-amino-2′-deoxycytidine. The nucleosides have different substituents on the pyrazine core structure, ranging from two methyl groups to polyaromatic rings. The photophysical properties of each nucleoside were determined, with the nucleosides displaying diverse absorption and emission maxima, extinction coefficients and quantum yields. The nucleoside with the highest fluorescence brightness was phosphitylated and incorporated into an oligonucleotide by means of automated oligonucleotide synthesis. The labelled oligonucleotide in aqueous buffer exhibited a substantially lowered extinction coefficient and quantum yield compared to the nucleoside in THF. The photophysical properties of the nucleoside were also compared in different DNA structural contexts, a single strand, a 14-mer duplex, a 14-mer duplex with an 11-mer overhang, and a 25-mer nicked duplex labelled at the nick site. Circular dichroism and melting temperature studies verified that the nucleoside did not perturb or destabilize the DNA helixes. In fact, when incorporated at the nick site, the nucleoside was found to stabilize the nicked duplex notably compared to its unmodified counterpart. The brightness of the fluorescent nucleoside in DNA increased as the polarity of its surroundings decreased, being highest in the 25-mer nicked duplex where exposure to the polar solvent is minimized by stacking to the adjacent bases on both the 3′- and 5′-side. The nucleosides brightness in the nicked duplex was also found to increase with lowered temperature, in accordance with expected temperature-dependent changes in the stacked-unstacked equilibrium at the nick site. The Royal Society of Chemistry.

Full text of DOI:10.1039/c2ob26536b

Organic &
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Syntheses and photophysical properties of 5’–6-locked
fluorescent nucleosides†
Cite this: Org. Biomol. Chem., 2013, 11,
149
Kristmann Gislason, Dnyaneshwar B. Gophane and Snorri Th. Sigurdsson*
Nine fluorescent 5’–6-locked nucleosides were synthesized by condensation of various 1,2-diketones with
5-amino-2’-deoxycytidine. The nucleosides have different substituents on the pyrazine core structure,
ranging from two methyl groups to polyaromatic rings. The photophysical properties of each nucleoside
were determined, with the nucleosides displaying diverse absorption and emission maxima, extinction
coefficients and quantum yields. The nucleoside with the highest fluorescence brightness was phosphity-
lated and incorporated into an oligonucleotide by means of automated oligonucleotide synthesis. The
labelled oligonucleotide in aqueous buffer exhibited a substantially lowered extinction coefficient and
quantum yield compared to the nucleoside in THF. The photophysical properties of the nucleoside were
also compared in different DNA structural contexts, a single strand, a 14-mer duplex, a 14-mer duplex
with an 11-mer overhang, and a 25-mer nicked duplex labelled at the nick site. Circular dichroism and
melting temperature studies verified that the nucleoside did not perturb or destabilize the DNA helixes.
In fact, when incorporated at the nick site, the nucleoside was found to stabilize the nicked duplex
notably compared to its unmodified counterpart. The brightness of the fluorescent nucleoside in DNA
increased as the polarity of its surroundings decreased, being highest in the 25-mer nicked duplex where
exposure to the polar solvent is minimized by stacking to the adjacent bases on both the 3’- and 5’-side.
The nucleosides brightness in the nicked duplex was also found to increase with lowered temperature, in
accordance with expected temperature-dependent changes in the stacked–unstacked equilibrium at the
nick site.
Received 3rd August 2012,
Accepted 17th October 2012
DOI: 10.1039/c2ob26536b
www.rsc.org/obc
for conjugation to the 5-position of pyrimidines or the 7-posi-
tion of 7-deazapurines.5,6 However, this approach still allows
Introduction
Fluorescence spectroscopy is one of the most widely used spec- movements of the fluorophore independent of the nucleic
troscopic techniques in nucleic acid research.1–4 Applications acid, thus reducing the sensitivity of such a label for detection
range from visualizing the biomolecules, both in vitro and in of changes in the secondary or tertiary structure of the nucleic
vivo, to the study of intra- and intermolecular interactions. acid.
Due to the lack of emission from the natural nucleotides, flu-
An alternate approach for conjugating the fluorophore to
orescence studies of nucleic acids require incorporation of flu- the natural nucleotide is to replace a nucleotide base with a
orescent labels (fluorophores). For many applications, it is fluorescent polycyclic aromatic hydrocarbon.3 Despite being
sufficient to tether a fluorophore to an appropriate position in bulkier than the natural bases and lacking hydrogen bonding
the biomolecule with a flexible linker spanning a few atoms. capability, they have been applied successfully in a number of
However, the flexibility of such linkers limits the applicability biophysical studies.7–10 Nevertheless, structural studies of
for structural studies.3 To circumvent the limitation associated native nucleic acids require less intrusive labels; labels that
with flexible linkers, semi-rigid alkyne linkers have been used have larger bases than the natural nucleosides they replace,
and/or do not base-pair to opposite natural bases, can distort
the natural secondary structure of the biomolecule.
In recent years, a number of fluorescent nucleoside analogs
that contain nucleobases in which the aromatic ring system
has been extended to provide a fluorescent base, while retain-
ing the hydrogen bonding face of the base, have been syn-
thesized and applied in various biophysical studies.2,11 For
example, our group has previously reported the detection and
University of Iceland, Department of Chemistry, Science Institute, Dunhagi 3, 107
Reykjavik, Iceland. E-mail: snorrisi@hi.is; Fax: +354 5528911; Tel: +354 5254801
†Electronic supplementary information (ESI) available: Structures for diketones
3a–11a. Temperature dependence spectra for II and IV in 30% ethylene glycol in
aqueous buffer. CD spectra for DNAs II and IV and their unmodified counter-
parts. HPLC traces for nucleosides 3–11. NMR spectra for nucleosides 3–11 and
phosphoramidite 12. See DOI: 10.1039/c2ob26536b
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Scheme 1 General reaction scheme for synthesis of 5’–6-locked nucleosides.
Detailed structures of diketones are shown in ESI (Fig. S1†) and of the product
in Fig. 2. The yields of the reaction are listed in Table 1.
Fig. 1 Previously reported 5’–6-locked, 1,10-phenanthroline-containing
nucleoside 1, shown base-paired to A.
Results and discussion
Synthesis and characterization of fluorescent nucleosides
identification of single nucleotide mismatches employing a
cytidine analog (Çf), where a tetramethylisoindoline is fused to
the cytosine base to form a phenoxazine analog.12–14 Addition-
An advantage of the synthetic approach is that a variety of 1,2-
ally, two nucleosides with similar structures were recently diketones can be condensed with the diamino nucleoside 2
applied as the first nucleoside analog fluorescence resonance (Scheme 1). Eight of the nine diketones used here are commer-
energy transfer (FRET)-pair.15 Although a number of different
cially available while one (9a, Fig. S1†) was obtained in a single
nucleoside analogs containing extended fluorescent bases step from a commercially available precursor (8a).17 Conden-
have been synthesized and applied in different nucleic acid sation of the diketones used here (3a–11a, Fig. S1†) yielded the
5′–6-locked nucleosides 3–11 (Fig. 2), that can be cast into four
studies, the search for new nucleosides with advantageous
properties is still an active area of research. It is of interest to categories. Nucleoside 3 contains two methyl substituents on
discover fluorescent probes that have increased brightness the core base structure, while the second group (4–7) contains
a pair of aromatic rings. The third group contains the ace-
(increased absorption coefficient and/or quantum yield),
increased Stokes shift, emission at higher wavelengths (in the naphthylene nucleosides 8 and 9, and the fourth group is
near infrared range) and increased sensitivity to its microenvir- comprised of nucleosides where the aromatic system of the
onment (base pairing, stacking, etc.).1
core base structure has been extended with three and four
An important aspect in the design of new fluorescent benzene rings (nucleosides 10 and 11).
analogs, like other spectroscopic probes, is their ease of syn-
Nucleosides 3, 8, 10 and 11 were prepared in excellent
thesis. We have previously reported the 1,10-phenanthroline- yields (Table 1) by simply heating the corresponding 1,2-dike-
containing 5′–6-locked nucleoside 1 (Fig. 1) which was readily tone with 218–20 in a mixture of ethanol and water.16 However,
accommodated at the end of DNA duplexes.16 Nucleoside 1 is nucleosides 4–7 and 9 could not be synthesized by this
a T-analog, forming a base-pair with A, where one of the nitro- approach, even after prolonged heating, presumably due to the
gens in the pyrazine ring replaces the 4-carboxy oxygen of T as limited solubility of the corresponding 1,2-diketones in the
a hydrogen bond acceptor (Fig. 1). Nucleoside 1 was simply polar solvent system. Indeed, replacing ethanol with aceto-
prepared by the reaction of a 1,2-diketone with 5-amino-2′- nitrile yielded the remaining nucleosides in moderate yields
deoxycytidine (2) and was directly phosphitylated for incorpor- (Table 1). The stereochemistry of the chiral centers formed by
ation of 1 into nucleic acids by solid-phase synthesis. The phe- the condensation was assumed to be the same as determined
nanthroline nucleoside displayed fluorescence both before by Kalman and co-workers using NOE-experiments.19 UV-Vis
and after incorporation into oligonucleotides.
and fluorescence spectra were recorded for all nucleosides in
To investigate if the readily-prepared 5′–6 ring-closed struc- tetrahydrofuran (Fig. 3). The excitation wavelength for fluor-
tural scaffold could yield fluorescent nucleosides with escence measurements was chosen just below the maximum of
improved spectroscopic properties and to demonstrate the the highest absorption peak to avoid scattering of the excitation
generality of the synthetic approach, we have prepared nine light interfering with the measurements. The photophysical
fluorescent 5′–6-locked nucleosides. The structure of the pyra- properties for nucleosides 3–11 are listed in Table 1. Nucleoside
zine ring extension ranges from a dimethyl substituted pyra- 3 was fluorescent with an absorption maximum at 319 nm and
zine ring to pyrazine rings conjugated to polyaromatic a Stokes shift of 36 nm. Its moderate extinction coefficient and
moieties. The nucleosides exhibited a wide range of absorp- quantum yield results in a fluorescence brightness (FB) of 3240.
tion maxima, emission maxima, extinction coefficients and Thus, the core structure is moderately fluorescent and its photo-
quantum yields. Additionally, one nucleoside was incorporated physical properties might be tunable by substituents.
into an oligonucleotide and its photophysical properties
In compounds 4–7, both the methyl groups of nucleoside 3
studied, both at the end of a DNA double helix and at an have been substituted with phenyl, p-fluorophenyl, p-methoxy-
internal nick site in a DNA duplex.
phenyl or thiophene rings, respectively. The ortho-terphenyl-
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like structure of these nucleosides allows only partial conju- Nucleosides 4–7 had absorption and emission maxima that
gation of the aromatic rings with the central pyrazine ring due are red-shifted compared to those of nucleoside 3 and
to steric hindrance. Not unexpectedly, extending the aromatic increased Stokes shifts. Although nucleosides 4–7 had similar
system yielded higher extinction coefficients but the quantum photophysical properties, the thiophene-containing nucleoside
yields for 4–7 were considerably lower than that of 3. 7 is the most notable with the highest emission wavelength
and Stokes shift.
Nucleosides 8 and 9 both contain naphthalene moieties
connected to the pyrazine ring through a five membered ring.
Nucleoside 9 contains two nitro substituents on the naphtha-
lene ring that increase the π-electron delocalization in the aro-
matic ring and thereby affect the absorption and emission of
the fluorophores. For this reason, nitro groups are often incor-
porated into fluorophores in an attempt to obtain beneficial
changes in photophysical properties.21 In nucleoside 9,
however, the effect was a dramatic quenching of its emission
with a maximum at 590 nm. Nucleoside 8, on the other hand,
exhibited quantifiable emission although the quantum yield
was low. The absorption spectrum of 8 (Fig. 3F) has an inter-
esting profile; it has a weak absorption peak at 366 nm and an
intense one at 329 nm (a similar absorption structure was seen
for nucleoside 9). Excitation at either maximum resulted in
emission with a maximum at 517 nm. Excitation at both
bands leads to similar quantum yields but the much higher
extinction coefficient at the lower wavelength resulted in con-
siderably higher FB. Nucleoside 8 displayed the highest Stokes
shift of the nucleosides in this study (∼150 nm) and the
highest emission maximum wavelength (517 nm).
In contrast to the other nucleosides in this study, nucleo-
sides 10 and 11 contain fully fused six-membered aromatic
rings where the π-electrons can resonate throughout the whole
structure. Nucleosides 10 and 11 displayed red shifts in both
absorption (67 nm and 96 nm, respectively) and emission
(44 nm and 97 nm, respectively) compared to nucleoside 3.
While nucleoside 11 has a similar extinction coefficient as 3
and a higher quantum yield, nucleoside 10 has a similar
quantum yield but a much higher extinction coefficient, yield-
ing the brightest nucleoside in this study. Nucleosides 10 and
11 have rather small Stokes shifts, similar to nucleoside 3.
Photophysical studies of DNA constructs containing
nucleoside 10
The brightest nucleoside in this study, nucleoside 10, was
Fig. 2 Structures of 5’–6-locked nucleosides 3–11.
chosen to investigate the photophysical properties of
Table 1 Synthetic yields and photophysical properties for nucleosides 3–11 in THF
a
Compound
Yield
λab
ε
λem
ΦF
FBb
3
4
5
6
7
8
9
10
11
84%
47%
50%
20%
28%
70%
10%
95%
91%
319
340
342
355
8000
355
403
406
428
459
517
—
0.405
0.005
0.007
0.019
0.012
0.054, 0.047
—
3240
52.3
73.3
191
133
920, 2410
—
7790
6460
11 000
11 200
10 200
11 200
17 000, 50 900
—
363
329, 366
344, 383
386
21 800
8380
399
452
0.358
0.771
415
a Fluorescence quantum yield. b Fluorescence brightness in M−1 cm−1, calculated as FB = ΦF × ε.
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Fig. 3 (A–I) UV-Vis and fluorescence spectra for nucleosides 3–11 in THF, shown as solid and broken lines, respectively. Y-axes are omitted for clarity, spectra with y-
axes can be seen in Fig. S6.†
fluorescent 5′–6-locked nucleosides in DNA. Thus, phosphora- in an aqueous solution. DNA I absorbs at approximately the
midite 12 was synthesized in one step from nucleoside 10 same wavelength as nucleoside 10 in THF (385 nm). The emis-
using standard reaction conditions, except that a few drops of sion maximum is, however, red-shifted by ∼20 nm (420 nm).
dry DMSO were added to the reaction mixture due to limited The extinction coefficient and quantum yield of DNA I were
solubility of 10 in CH2Cl2 (Scheme 2). Although the lack of a lowered, when compared to 10 in THF, leading to a consider-
5′-OH group restricts the use of 5′–6-locked nucleosides to ably lower fluorescence brightness (Table 2). It is not uncom-
5′-end labelling of nucleic acids, the 5′–6-locked nucleosides mon for the photophysical properties of fluorescent
have the advantage that preparation of their phosphoramidites nucleosides to change upon incorporation into oligonucleo-
for oligonucleotide synthesis does not require the usual tides. Such changes can be attributed to several factors, such
5′-trityl protection.
as base-stacking and the polarity of the solvent.2,22
Nucleoside 10 was incorporated into a 14-mer oligonucleo-
To further investigate the effect of the solvent exposure on
tide (I, Table 2) using standard solid-phase DNA synthesis and the emission of I, we designed and prepared DNA structures
its incorporation was verified by mass spectrometry. The II–IV: the fully complementary 14-mer duplex (II), a 14-mer
photophysical properties of DNA I were subsequently studied duplex containing an 11-mer overhang (III) and a 25-mer
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interactions and base-pairing have little effect on the bright-
ness of 10.22 The increase in brightness for III and IV also indi-
cates that exposure of nucleoside 10 to the solvent has a
significant effect on its brightness. In duplex II, one side of
the nucleoside is still exposed to the solvent, while the over-
hang in III could, in principle, provide stacking interactions.
In the 25-mer nicked duplex IV, the shielding effect is most
pronounced, indicating that the 5′-flanking T–A base-pair is
stacking on top of the 10-A pair despite the nick in the duplex.
Crystal structures of nicked DNAs have shown that the helix
adopts a B-DNA conformation over the nick site indicating
that base-pairing and stacking interactions overcome the loss
of the covalent phosphodiester connection.23 Additionally,
nicked duplexes have been shown to have mobilities similar to
their un-nicked counterparts in polyacrylamide gels.24 Thus,
our data indicate that 5′–6-locked nucleosides can be incorpor-
ated into internal positions in duplex DNAs for spectrophoto-
metric studies or to investigate electron transport through a
three component system as in IV.
The equilibrium between un-stacked and stacked confor-
mations in nicked DNAs follows the Boltzmann distribution,24
resulting in an increasing population of the un-stacked confor-
mation with rising temperature. Thus, if the brightness of
nucleoside 10 in duplex IV increased due to stacking with the
base-pair on the other side of the nick, the brightness should
also be temperature-dependent. Therefore, the photophysical
properties of duplexes II and IV were compared at three
different temperatures (Fig. 4). Indeed, while the fluorescence
brightness of II showed virtually no temperature dependence,
the brightness of IV showed a marked decrease as the tempera-
ture was raised from 4 °C to 30 °C. Hence, the enhancement in
the brightness of 10 in IV most likely stems from stacking at
the nick site, which shields the nucleoside from the solvent.
Further evidence for the sensitivity of nucleoside 10 to the
polarity of its surroundings was obtained by repeating the
aforementioned experiments in 30% aqueous ethylene glycol,
commonly used as a cryoprotectant in biological samples
(Table 2, Fig. S1†). The lower polarity of the solvent resulted in
an increase in the brightness of 10 in all DNA structures. The
difference in brightness between DNAs where the nucleoside
is exposed to (I and II) or shielded from (III and IV) the solvent
was lower for the ethylene glycol solution (two-fold) than water
Scheme 2 Synthesis of phosphoramidite 12 from nucleoside 10 (DIPAT: di-
isopropylammonium tetrazolide).
nicked duplex (IV) to examine how the fluoroside behaves at a
nick site in duplex DNA (Table 2). To verify that the nucleo-
sides did not significantly perturb or destabilize the DNA
duplexes, circular dichroism (CD) spectra and melting temp-
eratures (Tms) were recorded for duplexes II and IV along with
their unmodified counterparts, where 10 was replaced with
T. CD-spectra of modified and unmodified duplexes displayed
the same structures with the spectra showing the characteristic
B-DNA molar ellipticities at ca. 250 nm (negative) and 280 nm
(positive) (ESI†). DNA II displayed a minor increase in stability
over its unmodified duplex (ΔTm = 0.7 °C). The three strand
duplexes (DNA IV) displayed two Tms, with the lower Tms pre-
sumably reflecting the melting of the 11-mer duplex region.
The stabilizing effect of 10 was more pronounced in the three
strand system IV when compared to its unmodified counter-
part (ΔTm1 = 5.3 °C and ΔTm2 = 2.8 °C), presumably due to
stronger stacking interactions of 10 to the flanking thymine
base at the 3′-end of the 11-mer oligonucleotide.
Having established that nucleoside 10 does not signifi-
cantly distort B-form DNA duplexes, the photophysical proper-
ties of 10 in structures II–IV were determined. While the
14-mer duplex II displayed approximately the same brightness
as the single strand I, the FB increased by ∼35% by addition of
the 11-mer overhang in III and by ∼65% in the nicked duplex
IV, the similar FBs obtained for I and II indicate that stacking
Table 2 Structures and photophysical quantities for 10 in DNA structures I–IV at 20 °C
DNA
I
Structure
Tm (°C)a
Solventb
ε (M−1 cm−1
)
ΦF
FBc
3′-GTGCTACGCTCCG10-5′
A
B
A
B
A
B
A
7760
7220
6830
7580
7770
6850
5770
0.008
0.018
0.010
0.017
0.013
0.023
0.031
62.8
131
65.9
125
99.4
160
179
II
5′-CACGATGCGAGGCA-3′, 3′-GTGCTACGCTCCG10-5′
5′-CACGATGCGAGGCA-AGAGGACTCGC-3′, 3′-GTGCTACGCTCCG10-5′
66.2 (65.5)
III
IV
5′-CACGATGCGAGGCA–AGAGGACTCGC-3′, 3′-GTGCTACGCTCCG10
TCTCCTGAGCG-5′
58.6 (53.3),
69.7 (66.9)
B
5720
0.044
251
a Values in parentheses are for unmodified duplexes where 10 is replaced by T. b A = phosphate buffer, B = 30% ethylene glycol in phosphate
buffer. c Fluorescence brightness in M−1 cm−1, calculated as FB = ΦF × ε.
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brightness should be readily accessible, for example by incor-
porating chromophores that contain both electron-withdraw-
ing and electron-donating groups, which is known to polarize
the π-system and thereby increase the brightness.25,26
Experimental section
Chemicals were purchased from Sigma Aldrich, Acros and
Apollo Scientific Ltd and used without further purification.
Thin layer chromatography (TLC) was carried out using glass
plates pre-coated with silica gel (0.25 mm, F-254) from Sili-
cycle. Nucleosides were identified using staining with p-anisal-
dehyde. Flash column chromatography was performed using
ultra pure flash silica gel from Silicycle (40–63 μm, 60 Å).
Acetonitrile, dichloromethane and pyridine were freshly dis-
tilled from calcium hydride prior to use. DMSO was dried by
vacuum distillation and stored over activated 3 Å molecular
sieves under argon until used. All moisture- and air sensitive
reactions were carried out in oven- or flame-dried glassware
under an inert argon atmosphere. The phosphate buffer used
contained 10 mM phosphate, 100 mM NaCl, 0.1 mM EDTA,
pH 7. NMR spectra were recorded on a Bruker Advance 400
spectrometer. 1H NMR chemical shifts are reported in refer-
ence to undeuterated residual solvent (D2O (4.60 ppm), CDCl3
(7.26 ppm), d6-DMSO (2.50 ppm)). 13C NMR chemical shifts
are reported in reference to undeuterated residual solvent
(CDCl3 (77.0 ppm), d6-DMSO (39.43 ppm)). 31P NMR chemical
shifts were reported relative to 85% H3PO4 as an external stan-
dard. UV-Vis spectra were recorded on a Varian Cary 4000
UV-Vis Spectrometer. Fluorescence spectra were recorded on a
JY Horiba SPEX Fluorolog τ-3 spectrofluorimeter. CD and Tm
measurements were performed on a Jasco J-810 spectropolari-
meter. MALDI-ToF-MS measurements were performed on a
Bruker Autoflex III and ESI-MS measurements were performed
on a Bruker MicroTOF Q.
Fig. 4 Temperature dependence of the fluorescence brightness of nucleoside
10 in II and IV.
(three-fold). This was expected, because of a smaller change in
polarity of the fluorophore’s microenvironment upon shield-
ing from a less polar solvent.
Conclusions
Nine 5′–6-locked nucleosides have been prepared and their
photophysical properties determined. The core structure of
nucleoside 3 had a moderate extinction coefficient and a
quantum yield with absorption and emission maxima at
rather low wavelengths. While additions to the core structure
in all cases red-shifted both maxima, the effect on fluor-
escence brightness varied from almost quantitative quenching
in 9 to a significant increase in nucleosides 10 and 11. The
absorption and emission of nucleoside 10 were significantly
quenched upon incorporation into DNA, but to a smaller
extent when placed at a nick in a DNA duplex. The emissive
strength of 10 in the nicked helix also showed correlation with
temperature in agreement with the expected changes in the
ratio between stacking–unstacking at the nick site. Tm
measurements showed that nucleoside 10 stabilized the
nicked duplex when compared to thymidine. Since previous
work has shown that natural helixes mostly retain their struc-
ture despite a single nick in one strand, the Tm results indicate
that 5′–6-locked nucleosides might be applicable for internal
labelling of duplexes through three strand systems as in DNA
IV. It should be mentioned that, although all the diketones in
this study are symmetrical, the reactivity of the amino groups
of 2 differs enough for unsymmetrical diketones to be con-
densed, yielding one product, as long as one of the ketone
groups is more reactive.19 A clear advantage of the strategy
described here is that the preparation of labelled oligonucleo-
tides only requires three synthetic steps from the correspond-
Nucleoside 3
5-Amino-2′-deoxycytidine 2 (100 mg, 0.413 mmol) and biacetyl
3a (36 mg, 0.413 mmol) were suspended in 75% aq. ethanol
(2 mL) and heated at 83 °C for 4 hours. The reaction mixture
was allowed to cool, the solvent removed under vacuum. The
compound was purified on preparative TLC (MeOH : CH2Cl2,
1
10 : 90) to yield 3 as a white solid (102 mg or 84%). H NMR
(CDCl3) δ 2.40–2.46 (1H, m, H2′), 2.48 (3H, s, CH3), 2.50 (3H, s,
CH3), 2.76–2.82 (1H, m, H2′′), 4.04 (1H, d, J = 12.6, H5′), 4.10
(1H, dd, J1 = 12.7, J2 = 2.3, H5′′), 4.48 (1H, d, J = 2.6, H4′), 4.50
(1H, d, J = 3.7, H3′), 6.09 (1H, s, H6), 6.13 (1H, d, J = 6.4, H1′),
8.26 (1H, s, H3). 13C NMR (CDCl3) δ 21.18, 46.43, 50.78, 72.35,
73.26, 85.22, 88.77, 90.35, 128.08, 141.43, 146.87, 149.48,
152.68. ESI-MS: [M + H]+: calc. 293.12, found 293.12.
Nucleoside 4
ing diketone, which should facilitate the preparation of DNAs 5-Amino-2′-deoxycytidine 2 (100 mg, 0.413 mmol) and benzil
modified with new 5′–6-locked nucleotides. Thus, nucleosides 4a (104 mg, 0.496 mmol) were suspended in acetonitrile
within this 5′–6-locked family that have higher fluorescence (5.5 mL) and water (1.5 mL) and heated at 45–50 °C for 16 h.
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The reaction mixture was allowed to cool, the solvent evapor- column chromatography (CH2Cl2 : MeOH; 98 : 2 to 95 : 5) to
ated and the residue purified by column chromatography yield 7 as a white solid (50 mg, 28%). 1H-NMR (400 MHz,
(CH2Cl2 : MeOH; 98 : 2 to 95 : 5) to yield 4 as a white solid DMSO-d6): δ 2.18 (1H, ddd, J = 14.1, 6.5, 3.8, C2′H), 2.47 (1H,
1
(80 mg, 47%). H-NMR (400 MHz, DMSO-d6): δ 2.20 (1H, ddd, dd, J = 10.2, 4.4, C2′H), 3.90 (2H, dt, J = 12.5, 7.4, C5′H), 4.20
J = 14.2, 6.2, 3.7, C2′H), 2.47 (1H, d, J = 7.8, C2′H), 3.95 (2H, dd, (1H, m, C4′H), 4.33 (1H, s, OH), 5.16 (1H, d, J = 4.3, C3′H), 5.93
J = 17.4, 6.9, C5′H), 4.22–4.31 (1H, m, C4′H), 4.35 (1H, s, OH), (1H, d, J = 5.7, C1′H), 6.00 (1H, s, C6H), 7.00–7.08 (2H, m, ArH),
5.17 (1H, d, J = 4.2, C3′H), 5.95 (1H, d, J = 6.2 Hz, C1′H), 6.09 7.08–7.14 (2H, m, ArH), 7.71 (2H, ddd, J = 9.1, 4.1, 2.2, ArH),
(1H, s, C6H), 7.31 (5H, dd, J = 7.0, 2.0 Hz, ArH), 7.34 (5H, d, J = 10.83 (1H, s, NH). 13C-NMR (400 MHz, DMSO-d6): δ 45.67,
3.9 Hz, ArH), 10.81 (1H, s, NH). 13C-NMR (400 MHz, DMSO-d6): 71.27, 71.83, 84.24, 87.89, 89.98, 127.53, 127.76, 128.01,
δ 45.70, 71.28, 71.86, 84.52, 87.92, 90.02, 127.97, 128.66, 128.16, 128.88, 129.89, 129.93, 138.12, 139.64, 139.82, 142.65,
129.28, 129.38, 130.10, 137.69, 138.00, 142.75, 145.54, 148.85, 144.68, 148.67. ESI-MS: [M + H]+: calc. 429.06, found 429.07.
151.13. ESI-MS: [M + H]+: calc. 417.15, found 417.16.
Nucleoside 8
Nucleoside 5
5-Amino-2′-deoxycytidine 2 (100 mg, 0.413 mmol) and dike-
5-Amino-2′-deoxycytidine 2 (150 mg, 0.620 mmol) and 1,2-bis- tone 8a (75 mg, 0.413 mmol) were suspended in 75% ethanol
(4-fluorophenyl)ethane-1,2-dione 5a (183 mg, 0.743 mmol) (2 mL) and heated at 95 °C overnight. The reaction mixture
were suspended in acetonitrile (8 mL) and water (2 mL) and was allowed to cool and the solid formed collected by fil-
heated at 40–45 °C for 16 h. The reaction mixture was allowed tration, washed with ethanol and ether and dried. Column
to cool, the solvent evaporated and the residue purified by chromatography (CH2Cl2 : MeOH; 98 : 1 to 90 : 10) yielded 8 as
column chromatography (CH2Cl2 : MeOH; 98 : 2 to 95 : 5) to a pale yellow solid (113 mg, 70%). 1H NMR (DMSO-d6) δ
yield 5 as a white solid (140 mg, 50%). 1H-NMR (400 MHz, 2.22–2.28 (1H, m, H2′), 2.54–2.60 (1H, m, H2′′), 4.10 (2H, s,
DMSO-d6): δ 2.19 (1H, ddd, J = 14.1, 6.5, 3.7 Hz, C2′H), 2.47 H5′), 4.31–4.33 (1H, m, H4′), 4.43 (1H, s, H3′), 6.05 (1H, d, J =
(1H, d, J = 7.8, C2′H), 3.94 (2H, dd, J = 16.8, 7.3, C5′H), 6.2, H1′), 6.25 (1H, s, H6), 8.13–8.19 (4H, m, Ar-H), 8.37 (1H, d,
4.19–4.30 (1H, m, C4′H), 4.34 (1H, s, OH), 5.16 (1H, d, J = 4.3, J = 7.4, Ar-H), 8.44 (1H, d, J = 7.5 Hz, Ar-H), 9.11 (1H, d, J = 7.6,
C3′H), 5.94 (1H, d, J = 5.8, C1′H), 6.07 (1H, s, C6H), 7.18 (4H, q, Ar-H), 9.14 (1H, d, J = 7.5, Ar-H). 13C NMR (DMSO-d6) δ 152.33,
J = 9.1, ArH), 7.35 (4H, ddd, J = 21.4, 8.8, 5.5, ArH), 10.82 (1H, 148.88, 146.51, 143.22, 131.58, 131.13, 130.59, 130.42, 129.19,
s, NH). 13C-NMR (400 MHz, DMSO-d6): δ 45.70, 71.28, 71.88, 128.85, 128.78, 128.67, 122.97, 121.64, 90.19, 88.01, 85.02,
84.45, 87.92, 90.01, 114.95, 115.01, 115.17, 115.22, 130.23, 71.87, 71.36, 45.79. ESI-MS: [M + H]+: calc. 439.12, found
131.40, 131.48, 131.63, 131.72, 133.98, 134.01, 134.31, 134.34, 439.12.
144.51, 148.81, 150.13, 160.57, 160.97, 163.01, 163.42. ESI-MS:
Nucleoside 9
[M + H]+: calc. 453.14, found 453.14.
5-Amino-2′-deoxycytidine 2 (100 mg, 0.413 mmol) and 5,6-dini-
troacenaphthylene-1,2-dione 9a (90 mg, 0.496 mmol) were sus-
Nucleoside 6
5-Amino-2′-deoxycytidine 2 (150 mg, 0.619 mmol) and dike- pended in acetonitrile (8 mL) and water (2 mL) and heated at
tone 6a (200 mg, 0.743 mmol) were suspended in acetonitrile 45 °C for 16 h. The reaction mixture was allowed to cool, the
(7.5 mL) and water (1.5 mL) and heated at 50 °C for 16 h. The solvent evaporated and the residue purified by preparative TLC
reaction mixture was allowed to cool, the solvent evaporated (CH2Cl2 : MeOH, 90 : 10) to yield 9 as a yellow solid (18 mg,
1
and the residue purified by column chromatography (CH2Cl2 : 10%). H-NMR (400 MHz, DMSO-d6): δ 2.20 (1H, ddd, J = 14.1,
MeOH; 98 : 2 to 95 : 5) to yield 6 as a white solid (60 mg, 20%). 6.5, 3.7, 1H, C2′H), 2.51–2.60 (1H, m, C2′H), 3.99 (2H, dd, J =
1H-NMR (400 MHz, DMSO-d6): δ 2.19 (1H, ddd, J = 14.0, 6.4, 19.0, 7.2, C5′H), 4.24–4.32 (1H, m, C4′H), 4.38 (1H, s, OH), 5.20
3.7, C2′H), 2.47 (1H, d, J = 7.6, C2′H), δ 3.75 (6H, s, OCH3), (1H, d, J = 3.8, C3′H), 5.95 (1H, d, J = 6.0, C1′H), 6.05 (1H, s,
3.84–4.03 (2H, m, C5′H), 4.20–4.28 (1H, m, C4′H), 4.34 (1H, s, C6H), 8.23 (2H, dd, J = 7.5, 4.5, ArH), 8.51 (2H, dd, J = 7.2, 4.5,
OH), 5.15 (1H, d, J = 4.2, C3′H), 5.94 (1H, d, J = 6.2, C1′H), 6.04 ArH), 11.12 (1H, s, NH). 13C-NMR (400 MHz, DMSO-d6): δ
(1H, s, C6H), 6.88 (4H, dd, J = 8.8, 3.4, ArH), 7.26 (4H, dd, J = 45.69, 71.33, 72.05, 84.60, 88.06, 90.16, 113.11, 121.96, 123.80,
25.1, 8.7, ArH), 10.69 (1H, s, NH). 13C-NMR (400 MHz, DMSO- 129.50, 130.08, 131.65, 132.59, 136.06, 139.92, 144.49, 144.89,
d6): δ 45.73, 55.01, 55.05, 71.30, 71.80, 84.57, 87.91, 90.02, 145.46, 145.76, 148.44, 152.22. ESI-MS: [M + H]+: calc. 479.09,
113.45, 113.48, 129.23, 130.02, 130.47, 130.57, 130.78, 142.24, found 479.09.
145.09, 148.92, 150.42, 158.92, 159.51. ESI-MS: [M + H]+: calc.
477.17, found 477.17.
Nucleoside 10
5-Amino-2′-deoxycytidine 2 (100 mg, 0.413 mmol) and phenan-
threne-9,10-dione 10a (103 mg, 0.496 mmol) were suspended
Nucleoside 7
5-Amino-2′-deoxycytidine 2 (100 mg, 0.413 mmol) and 1,2-di- in 75% ethanol (2 mL) and heated at 83 °C for 16 h. The reac-
(thiophen-2-yl)ethane-1,2-dione 7a (111 mg, 0.496 mmol) were tion mixture was allowed to cool and the solid formed col-
suspended in acetonitrile (5.5 mL) and water (1.5 mL) and lected by filtration, washed with ethanol and ether and dried
heated at 45–50 °C for 16 h. The reaction mixture was allowed under suction. Crude product was purified by trituration from
to cool, the solvent evaporated and the residue purified by THF with ether to yield 10 as a pale yellow solid (162 mg,
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1
95%). H NMR (d6-DMSO) δ 2.19–2.26 (1H, m, H2′), 2.52–2.58 Oligonucleotide synthesis
(1H, m, H2′′), 4.07 (2H, s, H5′), 4.29–4.32 (1H, m, H4′), 4.41
Oligonucleotides were synthesized on a 1000 nmol scale by
using an ASM 800 DNA/RNA synthesizer with the manufac-
turer’s standard protocols. The modified nucleotide was intro-
duced by pausing the synthesizer program after completion of
the prior cycle, removing the column from the synthesizer and
running 200 μL of standard activator solution and 200 μL of a
0.05 M solution of 12 in CH3CN back and forth through the
column for 15 min. Then the column was re-mounted on the
synthesizer to complete the cycle. The oligonucleotides were
cleaved from the solid support, deprotected by using standard
(1H, s, H3′), 6.02 (1H, d, J = 6.0, H1′), 6.24 (1H, s, H6),
7.76–7.82 (4H, m, ArH), 8.80–8.85 (2H, m, ArH), 8.94–8.98 (2H,
m, ArH), 11.12(1H, s, NH). 13C NMR (DMSO-d6) δ 149.04,
143.71, 139.38, 135.08, 133.89, 131.38, 130.06, 129.34, 128.95,
128.67, 128.45, 127.95, 127.74, 124.99, 123.82, 123.44, 123.35,
90.16, 88.02, 84.82, 72.20, 71.45, 45.81. ESI-MS: [M + H]+: calc.
415.13, found 415.13.
Nucleoside 11
5-Amino-2′-deoxycytidine 2 (100 mg, 0.413 mmol) and pyrene- conditions (conc. aq. NH3, 55 °C, 12 h) and purified by 20%
4,5-dione 11a (96 mg, 0.413 mmol) were suspended in 95% denaturing polyacrylamide gel electrophoresis. The modified
ethanol (4 mL) and heated at 83 °C overnight. The reaction oligonucleotide was characterized by MALDI-ToF mass spec-
mixture was allowed to cool and the solid formed collected by trometry: 5′-d(10GC CTC GCA TCG TG) (I) calc. 4404, found
filtration, washed with ethanol and ether. Crude product was 4404.
purified by trituration from THF with ether to yield 11 as a
yellow solid (165 mg or 91%). H NMR (DMSO-d6) δ 2.22–2.28
1
UV-Vis and fluorescence studies
(1H, m, H2′), 2.54–2.60 (1H, m, H2′′), 4.10 (2H, s, H5′),
4.31–4.33 (1H, m, H4′), 4.43 (1H, s, H3′), 6.05 (1H, d, J = 6.2,
H1′), 6.25 (1H, s, H6), 8.13–8.19 (4H, m, Ar-H), 8.37 (1H, d, J =
7.4, Ar-H), 8.44 (1H, d, J = 7.5, Ar-H), 9.11 (1H, d, J = 7.6, Ar-H),
9.14 (1H, d, J = 7.5, Ar-H), 11.12(1H, s, NH). 13C NMR (DMSO-
d6) δ 149.03, 134.84, 130.85, 128.54, 128.19, 127.85, 127.77,
127.71, 127.41, 127.15, 127.07, 126.96, 126.73, 124.85, 122.65,
121.32, 90.17, 88.05, 84.84, 72.24, 71.46, 45.82. ESI-MS:
[M + H]+: calc. 439.13, found 439.15.
Solutions of nucleosides were prepared by dissolving a known
amount of each nucleoside in 10 mL of THF. Solutions of oli-
gonucleotides were prepared by dissolving 3 nmol of each
strand in 100 μL phosphate buffer, annealing and diluting to
1 mL with phosphate buffer. UV-Vis and fluorescence spectra
were then recorded of each sample at three different concen-
trations by diluting the original sample with phosphate buffer.
Quantum yields were determined using the equation Φx
=
Φst(Gradx/Gradst)(ηx/ηst), where the subscripts st and x denote
standard and sample respectively, Φ is the fluorescence
quantum yield, Grad the gradient from the plot of integrated
fluorescence intensity vs. absorbance and η the refractive index
of the solvent. Anthracene in ethanol was used as a standard
(ΦF = 0.27).27,28
Phosphoramidite 12
Nucleoside 10 (25 mg, 0.060 mmol) and diisopropyl
ammonium tetrazolide (25 mg, 0.144 mmol) were suspended
in CH2Cl2 (2 mL) and anhydrous DMSO added dropwise until
the suspension went into solution. NC(CH2)2OP[N(iPr)2]2
(47 mg, 0.156 mmol) was then added and the solution stirred
for 2 hours. CH2Cl2 (3 mL) was added and the organic layer
washed with sat. NaHCO3(aq) (3 × 5 mL) and brine (5 mL). The
organic phase was dried with anhydrous Na2SO4 and the
solvent removed in vacuo. The residue was dissolved in a
minimum amount of dry CH2Cl2 and precipitated with pet-
roleum ether. Precipitation was repeated twice and the residue
dried under vacuum to yield 12 as a pale yellow solid (23 mg,
62%). 1H NMR (400 MHz, CDCl3) δ 0.86 (2H, t, J = 6.9),
1.33–1.08 (14H, m), 2.60–2.50 (1H, m), 2.62 (2H, t, J = 6.6),
2.81–2.69 (1H, m), 2.95–2.83 (1H, m), 3.66–3.54 (1H, m),
3.90–3.70 (1H, m), 4.29–4.08 (2H, m), 4.74–4.53 (1H, m),
6.21–6.15 (1H, m), 6.29 (1H, s), 7.80–7.61 (3H, m), 8.60–8.52
(1H, m), 8.94 (1H, d, J = 7.9), 9.10–9.02 (1H, m). 31P NMR
(CDCl3) δ 148.78, 148.52.
Tm and CD measurements
Solutions of oligonucleotides were prepared by dissolving
3 nmol of each strand in 100 μL phosphate buffer, annealing
and diluting to 0.5 mL with phosphate buffer. Samples for Tm
studies were heated at a rate of 5 °C min−1
.
Acknowledgements
We thank the Icelandic Research Fund (070600007) and the
Science Institute, University of Iceland for financial support,
Prof. Fredrik Westerlund for assistance with UV-Vis and fluor-
escence measurements and members of the Sigurdsson
research group for helpful discussions.
Notes and references
HPLC analysis
Analytical HPLC chromatograms were obtained on a Beckman
Coulter Gold HPLC system, using a GL Sciences Inertsustain
C18 4.6 × 150 mm analytical column with UV detection at
260 nm. The method was a linear gradient from 96 : 4,
H2O : CH3CN to 100% CH3CN.
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