Analogues of uracil nucleosides with intrinsic fluorescence (NIF-analogues): Synthesis and photophysical properties

Article abstract of DOI:10.1039/c1ob06536j

Uridine cannot be utilized as fluorescent probe due to its extremely low quantum yield. For improving the uracil fluorescence characteristics we extended the natural chromophore at the C5 position by coupling substituted aromatic rings directly or via an alkenyl or alkynyl linker to create fluorophores. Extension of the uracil base was achieved by treating 5-I-uridine with the appropriate boronic acid under the Suzuki coupling conditions. Analogues containing an alkynyl linker were obtained from 5-I-uridine and the suitable boronic acid in a Sonogashira coupling reaction. The uracil fluorescent analogues proposed here were designed to satisfy the following requirements: a minimal chemical modification at a position not involved in base-pairing, resulting in relatively long absorption and emission wavelengths and high quantum yield. 5-((4-Methoxy-phenyl)-trans-vinyl)-2′-deoxy-uridine, 6b, was found to be a promising fluorescent probe. Probe 6b exhibits a quantum yield that is 3000-fold larger than that of the natural chromophore (Φ 0.12), maximum emission (478 nm) which is 170 nm red shifted as compared to uridine, and a Stokes shift of 143 nm. In addition, since probe 6b adopts the anti conformation and S sugar puckering favored by B-DNA, it makes a promising nucleoside analogue to be incorporated in an oligonucleotide probe for detection of genetic material.

Full text of DOI:10.1039/c1ob06536j

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PAPER
Analogues of uracil nucleosides with intrinsic fluorescence (NIF-analogues):
synthesis and photophysical properties†
Meirav Segal and Bilha Fischer*
Received 7th September 2011, Accepted 17th November 2011
DOI: 10.1039/c1ob06536j
Uridine cannot be utilized as fluorescent probe due to its extremely low quantum yield. For improving the
uracil fluorescence characteristics we extended the natural chromophore at the C5 position by coupling
substituted aromatic rings directly or via an alkenyl or alkynyl linker to create fluorophores. Extension of
the uracil base was achieved by treating 5-I-uridine with the appropriate boronic acid under the Suzuki
coupling conditions. Analogues containing an alkynyl linker were obtained from 5-I-uridine and the
suitable boronic acid in a Sonogashira coupling reaction. The uracil fluorescent analogues proposed here
were designed to satisfy the following requirements: a minimal chemical modification at a position not
involved in base-pairing, resulting in relatively long absorption and emission wavelengths and high
quantum yield. 5-((4-Methoxy-phenyl)-trans-vinyl)-2′-deoxy-uridine, 6b, was found to be a promising
fluorescent probe. Probe 6b exhibits a quantum yield that is 3000-fold larger than that of the natural
chromophore (Φ 0.12), maximum emission (478 nm) which is 170 nm red shifted as compared to uridine,
and a Stokes shift of 143 nm. In addition, since probe 6b adopts the anti conformation and S sugar
puckering favored by B-DNA, it makes a promising nucleoside analogue to be incorporated in an
oligonucleotide probe for detection of genetic material.
probes. These limitations include: experimental procedures
Introduction
required prior to detection, poor solubility in water/phosphate
buffer, toxicity of certain dyes,8 and background fluorescence of
The natural bases of DNA are not useful as fluorescent probes
because of their short absorption and emission wavelengths and
extremely low quantum yields (e.g., uridine exhibits λabs
260 nm, λem 308 nm, and Φ 4 × 10−5),1,2 and thus the use of
extrinsic probes is necessary. A common methodology for the
sensitive detection or quantification of RNA/DNA is the appli-
cation of fluorescent dyes.3 Currently marketed dyes4,5 for
nucleic acid staining and labeling include intercalating dyes that
are incorporated non-covalently to double stranded nucleic
acids,6 minor-groove binding dyes,7 and large hydrophobic fluor-
escent dyes8 that are incorporated covalently to nucleotide (oli-
gonucleotide) positions that do not interfere with base pairing.8
These dyes are used in various techniques for detecting genetic
material in DNA arrays,9 FISH,10 gels,11 in virus particles,12 and
in cells, by fluorescence microscopy, or in electrophoresed gels.13
Although the above-mentioned fluorescent dyes are useful for
nucleic acid detection and quantification, they suffer from limit-
ations regarding the preparation and application of nucleic acid
unreacted dye. In addition, the large hydrophobic dye attached to
a nucleotide alters the efficiency of enzymatic incorporation into
DNA/RNA, resulting in different levels of labeling and prohibits
quantification of nucleic acids.9,14 A two-step protocol involving
first the incorporation of a slightly modified nucleotide into
nucleic acid, followed by covalent binding of fluorescent dye,
also suffers from the limitation that a relatively small number of
dye molecules that can be incorporated.15,16
The limitations and complications of current methodologies
applying extrinsic dyes triggered the development of concep-
tually different fluorescent probes, formed by extension of the
natural chromophore.17–22
Such intrinsically fluorescent pyrimidine nucleosides include
5-furanyl-uracil,23,24 1 (λmax 316 nm, λem 431 nm, Φ 0.03 in
H2O), 5-pyrenyl-uracil,25,26 2 (λmax 350 nm, λem 447 nm, Φ
0.025 in MeOH), pyrrolo 2′-deoxy-cytosine,24,27–29 3 (λmax
353 nm, λem 448 nm, Φ 0.038 in tris buffer), phenyl pyrrolo
cytosine,30 4 (λmax 360, λem 465 nm, Φ 0.31 in phosphate
buffer) (Fig. 1). These fluorescent nucleosides were incorporated
into an oligonucleotide which, in turn, was used as a probe for
detection of genetic material in cells. The photophysical proper-
ties of the above mentioned fluorescent nucleosides were either
slightly changed or remained unchanged upon their incorpor-
ation into oligonucleotides.23,30,31
Department of Chemistry, Bar-Ilan University, Ramat-Gan, 52900,
Israel. E-mail: bilha.fischer@biu.ac.il; Fax: 972-3-6354907; Tel: 972-3-
5318303
†Electronic supplementary information (ESI) available: 13C and 1H
NMR, as well as absorption and emission spectra of reported com-
pounds. This material is available free of charge via the internet at http://
pubs.acs.org. See DOI: 10.1039/c1ob06536j
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Fig. 1 Previously reported fluorescent 5-substituted pyrimidine nucleoside analogues.
The limitations of current intrinsically fluorescent uridine
probes, namely, relatively short λem, low Φ values, and small
differences in emission wavelengths and quantum yields
between the single strand probe and the duplex, encouraged us
to develop novel uridine based fluorescent probes, with improved
photophysical properties.
form analogues 5a–e, 6a–c, 7a–c, and 8. We have studied the
contribution of both the type and position of the aromatic substi-
tuents on the fluorescence properties of the resulting uridine
derivatives. For comparison of electronic effects on the aromatic
ring, we also prepared the corresponding p-CF3- substituted ana-
logue, 6d.
Specifically, we targeted the development of fluorescent
uridine or 2′-deoxy-uridine analogues which can replace uridine
or 2′-deoxy-thymidine in RNA or DNA probes, respectively. We
focused on extending the uracil chromophore by minimal chemi-
cal modification leading to a satisfactory emission wavelength
and quantum yield without disturbing the H-bonding of the
uracil derivative with its complementary nucleobase.
Here, we describe the design and synthesis of a novel series of
push–pull uridine-based fluorescent probes, 5–8 (Fig. 2), as well
as their spectroscopic and conformational properties. In addition,
we analyze the structure–activity relationships of probes 5–8,
and elucidate the requirements for obtaining probes with
enhanced fluorescence. Furthermore, we demonstrated the appli-
cation of a 2′-deoxy-uridine analogue, 6b, for the preparation of
labeled oligonucleotide probes useful for detection of genetic
material.32
We elected to couple the aromatic substituent at the uracil C5
position, since substitution at the C6 position will drive the
nucleoside into the undesired syn conformation.
Altogether, we prepared and characterized four types of C5
substituted uridine analogues where the aromatic systems are: a)
directly coupled (5a–e), b) coupled via an alkenyl linker (6a–c),
c) coupled via an alkynyl linker (7a–c), and d) coupled via a
dienyl linker (8) (Fig. 2).
We coupled aromatic or conjugated aromatic moieties to the C5
position of uracil by the Suzuki coupling reaction to provide
5a–e, 6a–c, and 8, and by the Sonogashira coupling to yield 7a–c.
Synthesis of 5-substituted-uridine derivatives
Suzuki coupling reactions are highly useful for the preparation
of biaryl compounds.35 The Pd-catalyzed Suzuki reaction proved
to be effective also for nucleoside cross-coupling,36 and was
applied here for the preparation of derivatives 5a–e, 6a,b and 8
from unprotected 5-iodo-uridine, 5-I-U, or 2′-deoxy-5-I-U, or 5-
(2-Br-vinyl)-uridine, in one-step and in reasonably good yields
(Schemes 1 and 2).37 Thus, 5-I-U, 9a/b, and the appropriate aryl
boronic acid derivative, e.g., 10a–d, in water : acetonitrile (2 : 1)
were treated with Na2CO3 and a catalyst system containing
Pd(OAc)2 and TPPTS.37,38 Products 5a–c were obtained in high
purity after silica gel chromatography.
Similarly, compounds 6c and 8 were prepared from 5-(2-bro-
movinyl)-uridine,39,40 13, and 3, 4, 5-trimethoxyphenylboronic
acid, 10c and trans-2-(4-methoxyphenyl)-vinylboronic acid,
11a, respectively (Scheme 2).
Products 7a–c, where the aromatic system is linked to the
uracil C5-position via an alkynyl moiety, were prepared by
Sonogashira coupling.41 Thus, 5-I-U, 9a/b, and the appropriate
alkynyl compound, 12a–c, in DMF, were treated with Pd(PPh3)4,
Results
Probe design
To design fluorescent probes based on suitable extensions of the
uracil chromophore, we applied the following strategies: 1) selec-
tion of minimal extensions of the natural nucleobase at positions
not involved in H-bonding with a complementary base. 2) Selec-
tion of probes that may be excited and may emit at the longest
possible wavelengths. 3) Selection of extensions of the uracil
base that can be achieved via a simple and short synthesis.
Specifically, the modifications we selected included coupling
uracil at the C5 position to various aromatic systems. As substi-
tuents that donate electrons to the π system enhance absorption
of light and increase fluorescence,33,34 we have coupled –OMe
and –OH substituted aromatic rings to the uracil scaffold, to
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Fig. 2 Fluorescent uridine analogues synthesized and explored here.
DIPEA and CuI in the presence of TEA to obtain 7a–c in yields
between 32–74% (Scheme 3). Like the above-mentioned Suzuki
reaction, the advantages of the Sonogashira reaction include the
avoidance of nucleoside protection and a facile one step reaction
which provides the product in a reasonable yield, upon an easy
purification.
sulphate). λabs and λem values of the selected reference com-
pounds were in the same range as those of the evaluated
compounds.
Absorption and emission of compounds 5a–e, 7a–c, and 8
were measured in methanol with tryptophan43 as a standard (Φ
0.14, λabs 280 nm, λem 350 nm). Analogues 6a–c were measured
in H2O (pH 7) with quinine sulphate as a standard 44(Φ 0.54,
λabs 350 nm, λem 446 nm) at 5 μM (OD < 0.05). The spectral
data for derivatives 5–8 are summarized in Table 1. All modified
nucleosides 5–8 were found to be significantly more emissive
than the uridine (Table 1).
Analogues 6a–c, in which the uracil chromophore is extended
by a cinnamyl moiety, exhibited the longest absorption and emis-
sion wavelengths and highest quantum yields as compared to
analogues 5a–e, 7a–c, and 8. Specifically, analogue 6c showed a
58 nm red shift of absorption wavelength as compared to uridine
(λabs 260 nm) and a relatively high quantum yield–6000-times
larger than that of uridine in water.1 Furthermore, analogue 6c
showed λem of 478 nm in water, representing a 170 nm longer
emission wavelength, as compared to uridine.1
Spectral properties of uridine derivatives 5–8
We measured the absorption and emission of analogues 5–8 in
MeOH or H2O and determined their quantum yields. The selec-
tion of solvent depended on the solubility of the analogue in
water. The analogues were measured preferentially in water (6a,
c, d) if soluble, and also in MeOH, if possible (5a, 6b, and 8).
The other analogues were measured in MeOH only (5b–e, 7a–c).
For accurate measurements of the fluorescence, the concen-
trations of the samples were in the linear range in which the
Beer–Lambert law applies, the optical density was less than 0.05
to avoid the inner filter effect.42 Quantum yield (ΦF) was calcu-
lated based on Φ of a known reference (tryptophan or quinine
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Table 1 Spectroscopic data for 5-substituted uridine analogues, 5–8
Compound
λabs (nm)
λem (nm)
Φa,b
Solvent
Uridine1
5a
5a
5b
5c
5d
5e
6a
6b
6b
6c
6d
7a
7b
7c
8
8
260
285
280
290
290
291
293
328
335
326
318
288
306
307
310
308
308
308
308
310
432
480
420
422
447
478
477
476
417
430
408
411
430
428
0.00004
0.020b
0.070b
0.020b
0.016b
0.030b
0.035b
0.100a
0.120a
0.036a
0.240a
0.014b
0.056b
0.032b
0.045b
0.045b
0.060b
H2O
MeOH
H2O
MeOH
MeOH
MeOH
MeOH
H2O
H2O
MeOH
H2O
H2O
MeOH
MeOH
MeOH
MeOH
H2O
a Data obtained with quinine sulphate as a reference b Data obtained
with tryptophan as a reference
Scheme 1 Reagents and reaction conditions: a. Ar-B(OH)2, 0.25 eq
TPPTS, 0.05 eq Pd(OAc)2, 3 eq Na2CO3, H2O:CH3CN (2 : 1), reflux,
3 h. Product yield: 5a: 50%; 5b: 64%; 5c: 42%; 5d: 13%; 5e: 50%; 8:
58%; 6a: 42%; 6b: 64% yield.
showed the least promising spectral properties with a red shift of
absorption wavelength of only 25–30 nm as compared to uridine
and quantum yield ≤ 0.06. However, the emission maximum
ranged from 308 nm (for analogue 5a), as for uridine, up to
478 nm (for analogue 5c), representing a 170 nm shift vs. the
emission maximum of uridine.
Conformational analysis of uridine derivatives 5–7
A common B-DNA-like duplex is formed from nucleotides pos-
sessing the anti conformation only.45 Therefore, fluorescent
nucleoside analogues possessing an anti conformation will be
favored for labeling DNA/RNA strands.
Uracil nucleosides are expected to possess conformational
flexibility, due to possible rotations around the glycosidic bond
and pseudorotation of the ribose ring as well as possible rotations
around the C4′–C5′ bond angle (γ). However, it is well-estab-
lished that most nucleosides adopt a predominant conformation
in solution (which is nevertheless in equilibrium with other con-
formations).46 Thus, it has been shown that a majority of purine
and pyrimidine nucleosides favor an anti conformation of the
base ring with respect to the sugar ring.47 Likewise, the ribose
ring exhibits a puckered conformation in which either the C2′ or
C3′ atom is furthest from the plane of the other atoms of the
ribose ring, named as south (S) and north (N) conformations,
respectively.48 Finally, it has been shown that the ribose exocyc-
lic group exists predominantly in a gauche–gauche (gg) confor-
mation about the C4′–C5′ bond with the O5′ atom projecting
over the furanose ring.
Scheme 2 Reagents and reaction conditions: a. 0.25 eq TPPTS, 0.05 eq
Pd(OAc)2,3eqNa2CO3,H2O : CH3CN(2 : 1),reflux,3h.6c:21%;8:42%.
1
Here, we employed H, and 13C NMR spectra to analyze the
solution conformation of selected nucleoside analogues, 5d, 6a
and 7a.
Scheme 3 Reagents and reaction conditions: a. alkyne 12a–c, 0.2 eq
CuI, 0.1 eq Pd(PPh3)4, 3 eq DIEAP, DMF, 18 h, RT. Product yield: 7a:
32%; 7b: 61%; 7c: 74% yield.
Conformation around the glycosidic bond
Analogues 5a–e, 7a–c, and 8 showed relatively low quantum
yields, ranging from 0.016–0.060. Analogues 5a–e in which the
aromatic system is directly linked to the uracil C5-position
Pyrimidine nucleosides can adopt two main conformations, syn
or anti, in which the uracil O2 points above or away from the
sugar ring. The quantitative determination of the conformation
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Fig. 3 A,B) NOE interactions within compound 7a. C) Structure of compound 6a.
around the glycosidic bond can be obtained by monitoring the
Table 2 Conformational parameters of analogues 5d, 6a and 7b in
DMSO-d6 solution
3
3
vicinal coupling constants JC6–H1′ and JC2–H1′, which are
extracted from 13C NMR spectra for each nucleoside. A practical
rule for the orientation of the base relative to the ribose was for-
Conformer population
around C4′–C5′ bond
Sugar
Range of
3
3
mulated: a value of JC2–H1′ < JC6–H1′ indicates that χ is in the
anti conformation, whereas the reverse indicates that χ is in the
syn conformation.49
Compound
puckering %S
χ angle
% gg
% tg
% gt
5d
6a
7a
54
52
51
n. d.
80
75
n. d.
10
12
n. d.
10
13
anti
anti
anti
Ippel et al. reparametrized and generalized the Karplus
equations for the glycosidic bond conformation of purine and
pyrimidine nucleosides and nucleotides.49 Eqn (1) and (2)49
were used in this study to calculate the glycosidic bond angle χ
(O4′–C1′–N1–C2) in nucleosides 5d, 6a and 7a based on 3JC6–H1′
and 3JC2–H1′ values.50,51
n.d.: not determined
were within the range defined as anti (Table 2), which concurs
with previously published data.53
3JC6–H1′ ¼ 4:5cos2 ðχ ꢀ 60Þ ꢀ 0:6cosðχ ꢀ 60Þ þ 0:1 ð1Þ
Sugar puckering
3JC2–H1′ ¼ 4:7cos2 ðχ ꢀ 60Þ þ 2:4cosðχ ꢀ 60Þ þ 0:1 ð2Þ
The conformation of the D-ribose ring of nucleosides 5d, 6a and
7a was analyzed in terms of a dynamic equilibrium between two
favored puckered conformations: a type N conformer and a type
S conformer.54 N and S equilibrium populations were calculated
from observed J1′2′ and J3′4′ couplings as reported previously.
According to this method, the observed vicinal couplings are
related to the relative proportion of conformers by eqn (3)–(5):
These calculations give several possible χ values. In some
cases we had to base our choice of χ values on additional confor-
mational evidence. For instance, a cross peak between H-6 and
H-2′/3′ in NOESY spectrum indicates an anti nucleotide confor-
mation (Fig. 3).52
NOE interactions in compounds 5d, 6a, and 7b were observed
between H-6 and H-1′/2′/3′ e.g., (Fig. 3A). These interactions
suggest that the conformation of these compounds is anti,
similar to the conformation of natural uridine. Moreover, all cal-
culated χ values for the C5-substitued nucleosides 5d, 6a and 7b
J1′2′ ¼ 9:3ð1 ꢀ XNÞ ¼ 9:3XS
J2′3′ ¼ 4:6XN þ 5:3ð1 ꢀ XNÞ
ð3Þ
ð4Þ
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J3′4′ ¼ 9:3XN
ð5Þ
substitution of H by an OH group. Similarly, when H was substi-
tuted by OMe as in 5d, the quantum yield shifts only slightly
from 0.02 to 0.03. I.e., an EDG on the scaffold of nucleosides 5
does not increase quantum yield. Furthermore, even the substi-
tution of three OMe groups as in 5c, does not change the
quantum yield (0.016).
Unlike nucleoside 5, in the nucleoside 6 series, a significant
additive effect of the phenyl ring substituents, in 6c vs. 6a, was
observed where both Φ (0.24) and λem (476 nm) were increased
for 6c. Moreover, analogue 6c was found to be 15-times more
emissive than the related analogue 5c (Φ 0.24 vs. 0.016). Appar-
ently, it is not the aryl substituent affecting the quantum yield,
but rather the fluorophore, i.e. 5-cinnamyl-U vs. 5-phenyl-U.
The effect of an EDG at the meta position of the aromatic ring
on the fluorescence of the nucleoside analogue was studied for
the alkynyl-linked analogues 7a–c.
The mole fraction of conformers S and N can be calculated
directly from the observed values of J1′2′ and J3′4′. Using the
assigned J-coupling constants and the above equations, the mole
fractions of conformers S and N for nucleoside 5d, 6a and 7a
were calculated.51,54 Analogues 5d, 6a and 7d showed a minute
preference for the S conformer of the ribose ring (Table 2).
Conformations of the exocyclic CH2OH group
The nucleoside coupling constants J4′5′ and J4′5′′ can be inter-
preted in terms of three classical staggered rotamers, with a pre-
ferred gauche-gauche conformation.53 The mole fractions of
each staggered rotamer of C4′–C5′ were calculated from the fol-
lowing expressions (eqn (6)–(8)):
Compound 7a bearing OMe at the para position was com-
pared with two alkynyl analogues, 7b, R1 = R3 = OMe and 7c,
R3 = NH2 at the meta positions. An EDG at the meta position
resulted in less emissive nucleosides 7b,c as compared to 7a
where R2 = OMe (Φ 0.032 and 0.045 vs. 0.056). Namely, an
EDG at the meta position(s) does not contribute significantly to
the enhancement of the Φ value. Furthermore, nucleoside 7a
exhibits slightly improved fluorescence (Φ 0.056) vs. 5d (Φ
0.030) where the aryl moiety is directly linked to uridine and
also has OMe group at para position, probably since this ana-
logue has a longer conjugated π-system. In the series of ana-
logues 5d, 6a and 7a which contain a p-OMe-phenyl group that
is directly-, alkenyl-, or alkynyl- linked, respectively, the cinna-
myl analogue 6a proved to have the highest quantum yield (0.1)
and longest emission wavelength (447 nm), again indicating that
extending the uracil base with a cinnamyl moiety, rather than
phenyl or phenalkynyl, resulted in a favored fluorophore. Sur-
prisingly, analogue 8, bearing a dienyl linker and a longer fluoro-
phore, was less emissive than 6a (see discussion below).
ρgg ¼ ½ðJt þ JgÞ ꢀ ðJ4′5′ þ J4′5′′Þꢁ=ðJt ꢀ JgÞ
ρtg ¼ ðJ4′5′ ꢀ JgÞ=ðJt ꢀ JgÞ
ð6Þ
ð7Þ
ð8Þ
ρgt ¼ ðJ4′5′′ ꢀ JgÞ=ðJt ꢀ JgÞ
The observed proton signals, labeled H5′ and H5′′, refer to
downfield and upfield signals, respectively. The percentages of
gg, gt, and tg conformers are presented in Table 2. For analogues
6a and 7a there is a clear preference for the gg rotamer around
the C4′–C5′ bond, 75 and 80%, respectively (Fig. 3C). For com-
pound 5d the relative rotamer populations could not be deter-
mined, due to second order spectra.
Discussion
5-Substituted uridine derivatives exhibit a significant
enhancement of the fluorescent properties of uridine
The presence of a rigid, planar aromatic ring system in a com-
pound turns it to a potentially fluorescent molecule. The size of
the aromatic system also affects the excitation–emission wave-
length and fluorescence intensity. A few functional groups can
affect the wavelength and fluorescence intensity when substituted
into aromatic systems. The influence of the substitutions on the
fluorescence depends not only on their location in the aromatic
system but also on their tendency to donate electrons or to with-
draw electrons.55
In conclusion, all tested uridine analogues 5–8 showed signifi-
cantly improved spectral properties as compared to uridine. Ana-
logues in which the aromatic moiety is directly coupled to uracil
showed the smallest spectral changes as compared to uridine,
with up to 500-fold enhancement of the quantum yield of
uridine, (Φ 0.02 and λem 308 nm), while analogues where uracil
was extended by a cinnamyl moiety, e.g., 6c, exhibited the most
promising spectral data, up to 6000-fold enhancement of the
quantum yield of uridine, (Φ 0.24 and λem 476 nm).
We targeted the preparation of push–pull uridine-containing
systems to achieve a maximal enhancement of fluorescent prop-
erties triggered by a minimal chemical modification. Such mol-
ecules contain both electron rich and electron poor moieties.
Since we did not know whether uracil plays the role of an elec-
tron rich or poor moiety, we synthesized analogue 6a and 6d, p-
substituted by OCH3 and CF3, respectively. Compound 6a
proved to be a significantly more promising probe with Φ ca. 7-
fold higher than that of 6d and λem 30 nm longer than that of 6d
(Table 1). This observation implies that uracil plays the role of
an electron poor moiety in those push–pull systems.
The enhanced fluorescence (bathochromic shift) of analogues
5–8 vs. uridine is due to an intramolecular charge transfer
mechanism
Fluorescent uridine analogues 5–8 were designed as push–pull
probes to enhance the fluorescent properties of uridine. Specifi-
cally, these analogues are composed of three moieties—an elec-
tron donor (D, an electron rich aryl group); an electron acceptor
(A, uracil ring), and an electron rich linker which is a double/
triple bond (as in analogues 6 and 7), a diene (as in 8) or a
single bond (as in 5). Analogues 5–7 belong to a group of fluor-
escent molecules termed ‘molecular rotors’, which includes 9-
(dicyanovinyl) julolidine (DCVJ)56 and p-(dimethylamino) stil-
bazolium (p-DASPMI).56
To understand the effect of electron donating groups (EDGs)
on the quantum yield of nucleosides 5–7, we first compared ana-
logues 5a and 5b, where R2 = H and OH, respectively. Appar-
ently, no change in quantum yield (Φ 0.02) was due to
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Molecular rotors respond to photoexcitation with an intramo-
lecular charge transfer (ICT) from the donor to the acceptor unit.
The electrostatic forces induce an intramolecular twisting motion
of D and A units relative to each other.42,56,57
group of these analogues exists predominantly in a gauche–
gauche conformation about the C4′–C5′ bond (Fig. 3C).
Conclusions
In addition, the charge transfer occurring in the excited state
(D+–π–A−) results in increased dipole moment. Polar solvent
molecules orient themselves along the fluorophore dipole by
aligning their electric fields. Upon relaxation the solvent mol-
ecules return to the ground-state orientation resulting in a batho-
chromic shift of the emitted light. This shift reflects the energy
expended for the reorientation of the solvent molecules, which in
turn is dependent on the solvent polarity.30,56,58 Generally, polar
solvents stabilize ICT states56 and thus increase relaxation from
the ICT state. Specifically, MeOH and water form H-bonds with
the molecular rotor and increase ICT formation rate.
In summary, based on the relatively large quantum yields, up to
6000-fold more than uridine, and long emission wavelengths of
6b, and 6c (Φ 0.12 and 478 nm; 0.24 and 476 nm), in addition
to the uridine-like conformation of these analogues, we suggest
6b and 6c, as promising fluorescent probes involving an ICT
mechanism. We will report shortly the incorporation of analogue
6b into oligonucleotides and the fluorescent properties of the
resulting DNA single strands and duplexes, towards the develop-
ment of a new diagnostic tool.32
Since uridine does not posses a typical molecular rotor struc-
ture, it does not undergo an ICT mechanism. Yet, push–pull ana-
logues 5–8 exhibit strong ICT as reflected by a bathochromic
shift of up to 170 nm (e.g. analogues 5c and 6b,c), as compared
to uridine.
Experimental
General
Compounds were characterized by nuclear magnetic resonance
using Bruker AC-200, DPX-300 or DMX-600 spectrometers. 1H
NMR spectra were measured at 200, 300 or 600 MHz. For con-
formational analysis 1H-NMR and NOESY spectra were per-
formed with DMSO-d6 as solvent. Chemical shifts are expressed
in ppm. Mass spectra were obtained on a MicroMass QTOF
(Waters, UK) spectrometer (MS ESI). Progress of reactions was
monitored by TLC on precoated Merck silica gel plates
(60F-254). Visualization was accomplished by UV light.
Medium pressure chromatography was carried out using auto-
mated flash purification system (Biotage SP1 separation system,
Uppsala, Sweden). All moisture sensitive reactions were carried
out in flame-dried reactions flasks with rubber septa, and the
reagents were introduced with a syringe. All reactants in moist-
ure sensitive reactions were dried overnight in a vacuum oven.
Absorption spectra were measured on a UV-2401PC UV-VIS
recording spectrophotometer (Shimadzu, Kyoto, Japan). Emis-
sion spectra were measured using Aminco–Bowman series 2
(AB2) Luminescence Spectrometer (Thermo electron corpor-
ation, Markham, Ontario, Canada). Samples with maximum
absorbance of less than 0.05 were used to avoid inner filter
distortion.
To enhance ICT, and hence bathochromic shift, we substituted
analogue 5a with an EDG at the para position (5c) or at the para
and meta positions (5d). Indeed the strong ICT in 5c resulted in
170 nm red shift as compared to uridine. A similar behavior was
observed for analogues 6b and 6c. A strong ICT is observed
with an EDG at para position as opposed to meta positions, as
demonstrated by analogue 7a (p-OMe; λem 430 nm) vs. 7b (m-
di-OMe; λem 408 nm). Moreover, an EWG at the para position
(CF3) (6d), resulted in a low ICT as compared to 6a–c, substi-
tuted with an EDG. Compound 8, bearing a dienyl linker,
showed a shorter emission wavelength and a lower quantum
yield as compared to 6a. This is possibly due to the diene
moiety between the acceptor unit (uracil) and the donor unit (p-
methoxyphenyl), which enables the distribution of the negative
charge in the excited state on the diene and less on the uracil
acceptor, unlike in the vinyl analogue, 6a, thus reducing ICT.
The higher quantum yields found for analogues 6a–c, measured
in water, as compared to analogues 5 and 7, measured in MeOH,
is possibly due to the higher viscosity of water (0.897 cP) vs.
MeOH (0.544 cP). For molecular rotors the solvent viscosity is
the primary determinant of fluorescent quantum yield.42,56
C5-Substituents have no influence on the conformation of
uridine analogues
UV measurements
Absorption spectra were determined in H2O (pH 7) for com-
pounds 6a–c, and MeOH for analogues 5a–c, 7a–c, and 8. The
concentration of the samples was 6 μM. Absorbance was kept
less than 0.05 AU in order to avoid inner filter distortion.
Samples were measured in a 10 mm quartz cell.
The conformation of pyrimidine nucleosides required for the for-
mation of a B-DNA-like duplex, includes an anti nucleoside
with S sugar puckering and gg conformer of the ribose exocyclic
group.
We found that substituents at the C5 position of the uracil base
did not influence the nucleoside standard conformation, probably
since they exert no steric hindrance.
Fluorescent measurements
All χ values calculated for the C5-substituted uracil nucleo-
sides, 5–7, were within the range defined as anti, consistent with
previous data that pyrimidine analogues substituted at positions
other than C2 and C6 exist in a predominantly anti confor-
mation.53 Analogues 5–7 showed a slight preference for the S
conformer of the ribose ring. Moreover, the ribose exocyclic
Emission spectra of compounds 6a–c were determined in H2O
(pH 7) and in MeOH for compounds 5a–c, 7a–c, and 8.
Measurement conditions of analogues 5–8 included 740 V sensi-
tivity and a 4 nm slit. The concentration of the samples was
6 μM. Samples were measured in a 10 mm quartz cell.
This journal is © The Royal Society of Chemistry 2012
Org. Biomol. Chem., 2012, 10, 1571–1580 | 1577

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1
Quantum yield measurements
solid in a 42% yield (42.7 mg). H NMR (300 MHz , DMSO
d6): δ 8.14 (s, 1H), 6.85 (s, 2H), 6.24 (t, J = 7.43 Hz, 1H), 5.76
(d, J = 4.9,1H), 4.39 (m, 1H), 3.80 (s, 6H), 3.72 (s, 3H), 2.27
(m, 2H) ppm. 13C NMR (00 MHz , DMSO d6) δ 162.01,
152.51, 149.82, 137.92, 136.92, 113.26, 105.37, 87.67, 84.61,
70.24, 61.02, 60.02, 55.85. ESI+ MS m/z: 410 (MH+). Analysis
calculated for C18H22N2O9: C 52.68, H 5.36, N 6.83, O 35.12.
Found: C 52.92, H 5.22, N 7.23, O 35.41
The quantum yield of each compound was calculated from the
observed absorbance and the area of the fluorescence emission
band. The fluorescence quantum yield of all nucleosides was
determined relative to the quantum yield of quinine sulfate
(0.58)59 or tryptophan (0.14)59 in H2O (pH 7) or MeOH,
respectively, according to eqn (9).
2
Φ I
ODR
OD
η
η
R
Φ
¼
ꢂ
ꢂ
ð9Þ
F
IR
R
1-((2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydro-
furan-2-yl)-5-(4-methoxyphenyl)pyrimidine-2,4(1H,3H)-
dione (5d)
Where R = reference, I = integration of the peak and η =
refractive index of the solvent.
Product 5d was obtained from 9a (100 mg, 0.27 mmol) and 4-
methoxy-phenylboronic acid (53 mg, 0.35 mmol) following the
above procedure. Product 5d was obtained as a white solid in a
1-((2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydro-
furan-2-yl)-5- phenylpyrimidine-2,4(1H,3H)-dione (5a) - A typical
procedure
1
13% yield (12.5 mg). H NMR (300 MHz , DMSO d6): δ 11.39
(br. s, 1H), 8.15 (s, 1H), 7.49 (d, J = 9 Hz, 2H) 6.93 (d, J = 8.9
Hz, 2H), 5.86 (d, J = 4.8 Hz, 1H), 5.86 (br. s, 1H), 5.34 (br. s,
1H), 5.12 (br. s, 1H), 4.14 (m, 1H), 4.04 (m, 1H), 3.89 (m, 1H),
3.76 (s, 3H), 3.66 (m, 2H) ppm. 13C NMR (600 MHz , DMSO
d6) δ 162.22, 158.51, 150.17, 137.03, 129.06, 125.33, 113.55,
113.20, 88.16, 84.72, 73.88, 69.67, 60.44, 55.09. ESI+ MS m/z:
351 (MH+). Analysis calculated for C16H18N2O7: C 54.86, H
5.18, N 8.00, O 31.97. Found: C 54.51, H 5.36, N 8.25, O
31.65.
Water : acetonitrile (2 : 1, 1.6 mL) was added through a septum
to a nitrogen-purged round bottom flask containing 9a (100 mg,
0.27 mmol), phenylboronic acid (41.15 mg, 0.33 mmol),
Pd(OAc)2 (3 mg, 0.01 mmol), TPPTS (38.37 mg, 0.06 mmol)
and Na2CO3 (85.85 mg, 0.81 mmol). The mixture was stirred
under reflux for 3 h and monitored by TLC (8 : 2 CHCl3 :
MeOH). The solvent was evaporated and water was added. The
resulting solution was freeze dried. The residue was separated on
a silica gel column (90 : 10 CHCl3 : MeOH). Product 5a was
1
obtained as a white solid in a 50% yield (43.4 mg). H NMR
(300 MHz, CD3OD): δ 8.34 (s, 1H) 7.56–7.53 (m, 2H),
7.38–7.31 (m, 4 H), 6.50 (m, 1H), 5.99 (d, J = 4.2 Hz, 1H),
4.23–4.21 (m, 2H), 4.03 (m, 1H), 3.89–3.71 (m, 2H) ppm. 13C
NMR (600 MHz , DMSO d6) δ 164.62, 141.48, 139.93, 129.54,
129.39, 129.30, 128.64, 116.08, 90.92, 86.26, 80.15, 76.08,
71.12, 63.00, 61.85. ESI+ MS m/z: 343 (MNa+). Analysis calcu-
lated for C15H16N2O6: C 56.37, H 5.01, N 8.77, O 30.06.
Found: C 55.97, H 4.68, N 8.57, O 29.65.
1-((2R,4S,5R)-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-
yl)-5-(4-methoxyphenyl)pyrimidine-2,4(1H,3H)-dione (5e)
Product 5e was obtained from 9b (100 mg, 0.27 mmol) and 4-
methoxy-phenylboronic acid (53 mg, 0.35 mmol) following the
above procedure. Product 5e was obtained as a white solid in a
1
50% yield (47.6 mg). H NMR (300 MHz , DMSO d6): δ 8.16
(s, 1H), 7.47 (d, J = 9 Hz, 2H), 6.91 (d, J = 8.9 Hz, 2H), 6.33 (t,
J = 6.31 Hz, 1H), 4.42 (m, 1H), 3.95 (m, 1H), 3.78 (m, 5H),
2.28 (m, 2H) ppm. 13C NMR (600 MHz , DMSO d6) δ 162.24,
158.54, 149.93, 136.93, 125.44, 113.57, 87.48, 84.41, 70.29,
61.04, 55.11. ESI+ MS m/z: 335 (MH+). Analysis calculated for
C16H18N2O6: C 57.48, H 5.43, N 8.38, O 28.71. Found: C
56.98, H 5.12, N 9.02, O 28.35.
1-((2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydro-
furan-2-yl)-5-(4-hydroxyphenyl)pyrimidine-2,4(1H,3H)-
dione (5b)
Product 5b was obtained from 9a (100 mg, 0.27 mmol) and 4-
hydroxyphenylboronic acid (58.38 mg, 0.33 mmol) following
the above procedure. Product 5b was obtained as a white solid in
1
1-((2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydro-
furan-2-yl)-5-(4-methoxystyryl)pyrimidine-2,4(1H,3H)-
dione (6a)
a 64% yield (58 mg). H NMR (300 MHz, CD3OD): δ 8.61 (s,
1H), 7.4 (d, J = 4.4, 2H), 6.8 (d, J = 4.2 Hz 2H), 6.5 (m, 1H),
5.99 (d, J = 4.75, 1H), 4.25–4.16 (m, 2H), 4.0 (m, 1H),
3.85–3.76 (m, 2H) ppm. ESI+ MS m/z: 359 (MNa+). Analysis
calculated for C15H16N2O7: C 53.57, H 4.80, N 8.33, O 33.30.
Found: C 53.29, H 4.51, N 7.97, O 32.9.
Product 6a was obtained from 9a (100 mg, 0.27 mmol) and 2-
((4-methoxy-phenyl)-trans-vinyl-boronic acid (62.75 mg,
0.35 mmol) following the above procedure. Product 6a was
1
obtained as a white solid in a 42% yield (42.7 mg). H NMR
(300 MHz, CD3OD): δ 8.32 (s, 1H), 7.4 (d, J = 8.7 Hz, 2H), 7.3
(d, J = 16 Hz, 1 H), 6.87 (d, J = 8.7 Hz, 2H), 6.71 (d, J = 16 Hz,
1H), 5.94 (d, J = 3.94, 1H), 4.23–4.22 (m, 2H), 4.06–4.04 (m,
1H), 3.93–3.76 (m, 2H), 3.82 (s, 3H) ppm. 13C NMR (600 MHz
, DMSO d6) δ 162.21, 158.76, 149.74, 137.07, 130.03, 127.32,
127.27, 118.247, 111.05, 88.18, 84.58, 73.82, 69.39, 60.41,
55.13. ESI+ MS m/z: 377 (MH+). Analysis calculated for
1-((2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydro-
furan-2-yl)-5-(3,4,5-trimethoxyphenyl)pyrimidine-2,4(1H,3H)-
dione (5c)
Product 5c was obtained from 9b (100 mg, 0.27 mmol) and 3, 4,
5- trimethoxybenzeneboronic acid (74.2 mg, 0.35 mmol) follow-
ing the above procedure. Product 5c was obtained as a white
1578 | Org. Biomol. Chem., 2012, 10, 1571–1580
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C18H20N2O7: C 57.44, H 5.36, N 7.44, O 29.76. Found: C
57.24, H 5.20, N 7.60, O 29.35.
d6): δ 8.14 (s, 1H), 7.41 (m, 2H), 7.39 (d, J = 16.7 Hz, 1H), 6.86
(m, 2H), 6.61 (d, J = 15.3 Hz, 1H), 6.33 (d, J = 15.3 Hz, 1H),
6.65 (d, J = 16.8 Hz, 1H), 6.74 (s, 2H), 5.86 (d, J = 3.0 Hz, 1H),
4.22 (m, 2H), 4.01 (m, 1H), 3.83–3.72 (m, 5H) ppm. ESI+ MS
m/z: 425 (MNa+).
1-((2R,4S,5R)-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-
yl)-5-(4-methoxystyryl)pyrimidine-2,4(1H,3H)-dione (6b)
Product 6b was obtained from 9b (100 mg, 0.27 mmol) and 2-
((4-methoxy-phenyl)-trans-vinyl-boronic acid (62.74 mg,
0.35 mmol) following the above procedure. Product 6b was
obtained as a white solid in a 64%yield (65.6 mg).1H NMR
(300 MHz, DMSO d6): δ 11.47 (bs, 1H), 8.17 (s, 1H), 7.41 (d, J
= 2 Hz, 2 H), 7.35 (d, J = 16.8 Hz, 1H), 6.93 (d, J = 2 Hz, 2H),
6.76 (d, J = 16.8 Hz, 1H), 6.19 (t, J = 3.5 Hz, 1H), 5.28 (d, 1H),
5.19 (t, 1H), 4.29 (m, 1H), 4.11 (m, 1H), 3.76 (s, 3H), 3.7–3.6
(m, 2H), 2.17 (m, 2H) ppm. 13C NMR (600 MHz , DMSO d6) δ
162.70, 159.11, 149.93, 137.12, 130.46, 127.58, 127.46, 118.91,
117.70, 87.68, 84.73, 70.13, 61.14, 55.07, 40.42. ESI+ MS m/z:
361 (MH+). Analysis calculated for C18H20N2O6: C 59.83, H
5.54, N 7.75, O 26.59. Found: C 59.43, H 5.19, N 7.5, O 26.43.
1-((2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydro-
furan-2-yl)-5-((4-methoxyphenyl)ethynyl)pyrimidine-2,4(1H,3H)-
dione (7a) - A typical procedure
Tetrakis(triphenylphosphine)palladium(0) (62 mg, 0.05 mmol),
copper(I) iodide (2.6 mg, 0.11 mmol), diisopropylethylamine
(0.2 mL) and 4-ethynyl anisole (0.21 mL, 1.62 mmol) were
added to a solution of 9a (200 mg, 0.54 mmol) in anhydrous
DMF (16 mL). The reaction mixture was stirred at room temp-
erature overnight under nitrogen atmosphere. After 18 h no start-
ing material was detected by TLC (8 : 2 CH2Cl2 : MeOH). The
solvent was evaporated and the residue was purified on a silica
gel column using CH2Cl2 : MeOH (80 : 20) yielding 7a as a pale
yellow solid in a 32% yield (66 mg). 1H NMR (300 MHz,
DMSO-d6): δ 11.723 (br. s, 1H) 8.40 (s, 1H), 7.42 (d, J = 8.4
Hz, 2 H), 6.97 (d, J = 8.7 Hz, 2H), 5.78 (d, J = 4.8 Hz, 1H),
5.44 (d, J = 5.1 Hz, 1H), 5.25 (t, J = 4.4 Hz, 1H), 5.08 (d, J =
5.4 Hz, 1H), 4.08 (m, 1H), 4.06 (m, 1H), 3.87 (m, 1H), 3.78 (s,
3H), 3.68–3.60 (m, 2H) ppm. 13C NMR (600 MHz , DMSO d6)
δ 167.22, 158.98, 153.92 142.7480, 132.60, 132.53, 115.35,
114.28, 98.45, 90.39, 88.66, 84.31, 73.82, 69.55, 60.64, 55.24.
ESI+ MS m/z: 375 (MH+). Analysis calculated for C18H18N2O7:
C 57.75, H 4.85, N 7.48, O29.92. Found: C 57.42, H 5.03, N
7.12, O 29.52.
1-((2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydro-
furan-2-yl)-5-(3,4,5-trimethoxystyryl)pyrimidine-2,4(1H,3H)-
dione (6c)
Product 6c was obtained from 13 (100 mg, 0.28 mmol) and 2-
((4-methoxy-phenyl)-trans-vinyl-boronic acid (62.7 mg,
0.35 mmol) following the above procedure. Product 6c was
obtained as a white solid in a 21% yield (27 mg). 1H NMR
(300 MHz, DMSO d6): δ 8.18 (s, 1H), 7.29 (d, J = 16.74 Hz,
1H), 6.74 (d, J = 16.8 Hz, 1H), 6.74 (s, 2H), 5.83 (d, 3.3 Hz,
1H), 4.17 (m, 2H), 4.05 (m, 1H), 3.98 (m, 8H), ppm. ESI+ MS
m/z: 459 (MNa+).
1-((2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydro-
furan-2-yl)-5-((3,5-dimethoxyphenyl)ethynyl)pyrimidine-2,4
(1H,3H)-dione (7b)
1-((2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydro-
furan-2-yl)-5-((E)-4-(trifluoromethyl)styryl)pyrimidine-2,4
(1H,3H)-dione (6d)
Product 7b was obtained from 9a (200 mg, 0.54 mmol) and 1-
ethynyl-3,5-dimethoxybenzene (262.7 mg, 1.62 mmol) follow-
ing the above procedure. Product 7b was obtained as a brown
solid in a 61% yield (133 mg).1H NMR (300 MHz, DMSO-d6):
δ 11.723 (br. s, 1H), 8.48 (s, 1H), 6.6 (m, 1 H), 6.5 (m, 1H),
5.76 (d, J = 4.2 Hz, 1H), 5.46 (d, J = 5.1 Hz, 1H), 5.29 (t, J =
6.75 Hz, 1H), 5.10 (d, J = 5.4 Hz, 1H), 4.07 (m, 2H), 3.88 (m,
1H), 3.75 (s, 6H), 3.6 (m, 2H) ppm. 13C NMR (600 MHz ,
DMSO d6) δ 161.35, 160.40, 149.65, 144.24, 123.83, 108.87,
101.65, 98.04, 88.50, 84.69, 82.04, 73.95, 69.21, 60.14, 55.93.
ESI+ MS m/z: 405 (MH+). Analysis calculated for C19H20N2O8:
C 56.43, H 4.99, N 6.93, O 31.65. Found: C 56.03, H 4.80, N
7.03, O 31.33.
Product 6d was obtained from 9b (100 mg, 0.27 mmol) and 2-
((4-trifluoromethyl-phenyl)-trans-vinylboronic acid (75 mg,
0.35 mmol) following the above procedure. Product 6b was
obtained as a white solid in a 27% yield (30 mg). 1H NMR
(300 MHz, DMSO d6): δ 11.56 (br. s, 1H), 8.28 (s, 1H), 7.68 (s,
4H), 7.52 (J = 16.2 Hz, 1H), 7.08 (d, J = 14.7 Hz, 1H), 6.20 (t,
J = 6.3 Hz, 1H), 5.28 (d, J = 3.6 Hz, 1H), 5.20 (t, 1H, J = 3.6
Hz), 4.29 (m, 1H), 4.10 (m, 1H), 3.82 (m, 2H), 3.32 (s, 3H),
2.18 (m, 2H) ppm. NMR (600 MHz , DMSO d6) δ 162.04,
149.40, 141.68, 139.36, 129.11, 126.82, 126.45, 125.82, 125.53,
124.36, 110.24, 87.50, 84.53, 69.92, 60.96. ESI+ MS m/z: 397
(MH+).
5-((3-aminophenyl)ethynyl)-1-((2R,3R,4S,5R)-3,4-dihydroxy-5-
(hydroxymethyl)tetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-
dione (7c)
1-((2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydro-
furan-2-yl)-5 ((1E,3E)-4-(4-methoxyphenyl)buta-1,3-dien-1-yl)
pyrimidine-2,4(1H,3H)-dione (8)
Product 7b was obtained from 9a (200 mg, 0.54 mmol) and 3-
ethynylaniline (0.17 ml, 1.62 mmol) following the above pro-
cedure. Product 7b was obtained as a brown solid in a 74% yield
Product 8 was obtained from 13 (63 mg, 0.18 mmol) and 2-((4-
methoxy-phenyl)-trans-vinyl-boronic acid (40 mg, 0.25 mmol)
following the above procedure. Product 8 was obtained as a
white solid in a 48% yield (35 mg). 1H NMR (300 MHz, DMSO
1
(144 mg). H NMR (300 MHz, DMSO-d6): δ 11.68 (br. s, 1H),
8.39 (s, 1H), 7.01 (t, J = 3.2 Hz, 1 H), 6.5 (m, 1H), 6.65 (m, 1 H),
Org. Biomol. Chem., 2012, 10, 1571–1580 | 1579
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28 D. A. Berry, K. Y. Jung, D. S. Wise, A. D. Sercel, W. H. Pearson,
H. Mackie, J. B. Randolph and R. L. Somers, Tetrahedron Lett., 2004,
45, 2457–2461.
29 C. Liu and C. T. Martin, J. Biol. Chem., 2002, 277, 2725–2731.
30 A. S. Wahba, A. Esmaeili, M. J. Damha and R. Hudson, Nucleic Acids
Res., 2010, 38, 1048–1056.
31 C. Wanninger-Weiss and H. A. Wagenknecht, Eur. J. Org. Chem., 2008,
64–71.
32 M. Segal, E. Yavin, P. Kafri, Y. Shav-Tal, and B. Fischer, unpublished
results
6.59 (m, 1H) 5.7 (d, J = 4.8 Hz, 1H), 5.45 (d, J = 6 Hz, 1H),
5.25 (m, 1H), 5.09 (d, J = 5.4 Hz, 1H), 4.11–4.07 (m, 2H),
4.04–43.99 (m, 1H), 3.87–3.86 (s, 1H), 3.69–3.56 (m, 2H) ppm.
13C NMR (600 MHz , DMSO d6) δ 161.48, 149.73, 148.78,
143.58, 129.18, 122.60, 118.69, 116.07, 114.52, 98.59, 92.89,
88.40, 84.81, 80.86, 73.93, 69.39, 60.31. ESI+ MS m/z: 360
(MH+). Analysis calculated for C17H17N3O6: C 56.82, H 4.77, N
11.69, O 26.72. Found: C 56.46, H 4.55, N 11.21, O 26.32.
33 L. A. Strait, R. Ketcham, D. Jambotkar and V. P. Shah, J. Am. Chem.
Soc., 1964, 86, 4628–4630.
34 G. Liu, S. Pu and X. Wang, Tetrahedron Lett., 2010, 66, 8862–8871.
35 L. Yin and J. Liebscher, Chem. Rev., 2007, 107, 133–173.
36 A. G. Firth, I. J. Fairlamb, K. Darley and C. G. Baumann, Tetrahedron
Lett., 2006, 47, 3529–3533.
37 A. Collier and G. K. Wagner, Synth. Commun., 2006, 36, 3713–3721.
38 P. Capek, R. Pohl and M. Hocek, Org. Biomol. Chem., 2006, 4, 2278–
2284.
References
1 S. Udenfriend and P. Zaltzman, Anal. Biochem., 1962, 3, 49–59.
2 P. R. Callis, J. Chem. Soc., Chem. Commun., 1983, 34, 329–357.
3 X. Fei and Y. Gu, Prog. Nat. Sci., 2009, 19, 1–7.
4 R. P. Haugland, Handbook of fluorescent probes and research products;
Molecular probes, 2002.
39 E. De Clercq, C. Desgranges, P. Herdewijn, I. S. Sim, A. S. Jones,
M. J. McLean and R. T. Walker, J. Med. Chem., 1986, 29, 213–
217.
40 R. Kumar, L. Xu, E. E. Knaus, L. I. Wiebe, D. R. Tovell, D. L. Tyrrell
and T. M. Allen, J. Med. Chem., 1990, 33, 717–723.
41 J. H. Cho, C. D. Prickett and K. H. Shaughnessy, J. Org. Chem., 2010,
3678–3683.
5 R. F. Delgadillo and L. J. Parkhurst, Photochem. Photobiol., 86, 261–
272.
6 L. J. L. Shi, Mao Min, Li Gang, Li Cheng Yao and Zhang Jin Gang,
Chinese Journal of Agricultural Biotechnology, 2009, 6, 55–59.
7 M. Munde, M. Ismail, R. Arafa, P. Peixoto, C. J. Colla, Y. L. Liu,
A. Lansiaux, C. Bailly, D. W. Boykin and W. D. Wilson, J. Am. Chem.
Soc., 2007, 129, 13732–13743.
42 J. R. Lakowicz, Principles of fluorescent spectroscopy, Baltimore, Mary-
land, Second Edition edn, 1986.
43 E. P. Kirby and R. F. Steiner, J. Phys. Chem., 1970, 74, 4480–4490.
44 W. H. Melhuish, J. Phys. Chem., 1961, 65, 229–235.
45 K. Umemoto, M. H. Sarma, G. Gupta and R. H. Sarma, Biochemistry,
1990, 29, 4714–4722.
8 R. T. Ranasinghe and T. Brown, Chem. Commun., 2005, 5487–5502.
9 R. Pribylova, P. Kralik and I. Pavlik, Mol. Biotechnol., 2009, 42, 30–40.
10 A. Arvey, A. Hermann, C. C. Hsia, E. Ie, Y. Freund and W. McGinnis,
Nucleic Acids Res., 2010, 38, e115-111–e115-117.
11 M. B. Wabuyele and S. A. Soper, Single Mol., 2001, 2, 13–21.
12 C. Beer and M. Wirth, Anim. Cell Technol., 2005, 321–324.
13 R. Venkat and S. Knisley, J. Biomed. Opt., 2006, 11, 024019.
14 J. Ibach and S. Brakmann, Angew. Chem., Int. Ed., 2009, 48, 4683–4685.
15 W. Lv, X. D. Chen, D. Aumiler and A. D. Xia, Sci. China, Ser. B: Chem.,
2009, 52, 1148–1153.
16 M. E. Sanborn, B. K. Connolly, K. Gurunathan and M. Levitus, J. Phys.
Chem. B, 2007, 111, 11064–11074.
17 D. W. Dodd and R. H. E. Hudson, J. Org. Chem., 2009, 6, 378–391.
18 R. H. E. Hudson and A. Ghorbani-Choghamarani, Org. Biomol. Chem.,
2007, 5, 1845–1848.
46 W. Saenger, in Principles of Nucleic Acid Structure, Springer-Verlag,
New York, 1984, pp. 107–110.
47 J. T. Donohue and N. Kenneth, Chem. Rev., 1960, 2, 363–371.
48 M. Sundaralingam, Biopolymers, 1969, 7, 821–860.
49 J. H. Ippel, S. S. Wijmenga, R. Jong, H. A. Heus, C. W. Hilbers,
E. Vroom, G. A. Marel and J. H. Van Boom, Magn. Reson. Chem., 1996,
34, S156–S176.
50 C. Altona and M. Sundaralingam, J. Am. Chem. Soc., 1972, 94, 8205–
8212.
51 D. B. Davies and S. S. Danyluk, Biochemistry, 1974, 13, 4417–4434.
52 F. R. Prado, P. C. Giessner and B. Pullman, J. Theor. Biol., 1978, 74,
259–277.
19 R. H. E. Hudson and J. M. Moszynski, Synlett, 2006, 2997–3000.
20 R. H. E. Hudson and A. K. Dambenieks, Heterocycles, 2006, 68, 1325–
1328.
53 W. Uhl, J. Reiner and H. G. Gassen, Nucleic Acids Res., 1983, 11, 1167–
1180.
21 L. M. Wilhelmsson, A. Holmen, P. Lincoln, P. E. Nielsen and B. Norden,
J. Am. Chem. Soc., 2001, 123, 2434–2435.
54 C. Altona and M. Sundaralingam, J. Am. Chem. Soc., 1973, 95, 2333–
2344.
22 R. Sirivolu Venkata, P. Chittepu and F. Seela, Chem. biochem., 2008, 9,
2305–2316.
55 M. Furst, H. Kallmann and F. H. Brown, J. Chem. Phys., 1957, 26,
1321–1332.
56 M. A. Haidekker and E. A. Theodorakis, J. Biol. Eng., 2010, 4, 1754–
1611.
57 H.-A. Wagenknecht, Nat. Prod. Rep., 2006, 23, 973–1006.
58 R. Nandy and S. Sankararaman, Beilstein J. Org. Chem., 2010, 6, 992–
1001 No 1112
23 N. J. Greco and Y. Tor, Tetrahedron, 2007, 63, 3515–3527.
24 R. H. E. Hudson, A. K. Dambenieks and J. M. Moszynski, Proc.
SPIE–Int. Soc. Opt. Eng., 2005, 5969, 59690J-59691–59690J-59610.
25 T. L. Netzel, M. Zhao, K. Nafisi, J. Headrick, M. S. Sigman and
B. E. Eaton, J. Am. Chem. Soc., 1995, 117, 9119–9128.
26 A. Cuppoletti, Y. Cho, J. S. Park, C. Strassler and E. T. Kool, Assay Drug
Dev. Technol., 2005, 3, 449–450.
59 A Guide to recording fluorescence quantum yields, Jobin Yvon Ltd.,
Dalston Gardens, Stanmore, UK.
27 R. H. E. Hudson, A. K. Dambenieks and R. D. Viirre, Nucleosides,
Nucleotides Nucleic Acids, 2005, 24, 581–584.
1580 | Org. Biomol. Chem., 2012, 10, 1571–1580
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