Exploring the role of the 5-substituent for the intrinsic fluorescence of 5-aryl and 5-heteroaryl uracil nucleotides: A systematic study

Article abstract of DOI:10.1039/c3ob40485d

Derivatives of UMP (uridine monophosphate) with a fluorogenic substituent in position 5 represent a small but unique class of fluorophores, which has found important applications in chemical biology and biomolecular chemistry. In this study, we have synth

Full text of DOI:10.1039/c3ob40485d

Organic &
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Exploring the role of the 5-substituent for the intrinsic
fluorescence of 5-aryl and 5-heteroaryl uracil
nucleotides: a systematic study†‡
Cite this: Org. Biomol. Chem., 2013, 11,
6357
Thomas Pesnot,a Lauren M. Tedaldi,b Pablo G. Jambrina,c Edina Rostac and
Gerd K. Wagner*b,c
Derivatives of UMP (uridine monophosphate) with a fluorogenic substituent in position 5 represent a
small but unique class of fluorophores, which has found important applications in chemical biology and
biomolecular chemistry. In this study, we have synthesised a series of derivatives of the uracil nucleotides
UMP, UDP and UTP with different aromatic and heteroaromatic substituents in position 5, in order to
systematically investigate the influence of the 5-substituent on fluorescence emission. We have
determined relevant photophysical parameters for all derivatives in this series, including quantum yields
for the best fluorophores. The strongest fluorescence emission was observed with a 5-formylthien-2-yl
substituent in position 5 of the uracil base, while the corresponding 3-formylthien-2-yl-substituted regio-
isomer was significantly less fluorescent. The 5-(5-formylthien-2-yl) uracil fluorophore was studied further
in solvents of different polarity and proticity. In conjunction with results from a conformational analysis
based on NMR data and computational experiments, these findings provide insights into the steric and
electronic factors that govern fluorescence emission in this class of fluorophores. In particular, they high-
light the interplay between fluorescence emission and conformation in this series. Finally, we carried
out ligand-binding experiments with the 5-(5-formylthien-2-yl) uracil fluorophore and a UDP-sugar-
dependent glycosyltransferase, demonstrating its utility for biological applications. The results from
our photophysical and biological studies suggest, for the first time, a structural explanation for the
fluorescence quenching effect that is observed upon binding of these fluorophores to a target protein.
Received 8th March 2013,
Accepted 5th August 2013
DOI: 10.1039/c3ob40485d
www.rsc.org/obc
main strategies have been pursued to generate such fluore-
scent nucleotides: (i) the replacement of the nucleobase with a
Introduction
Fluorescent nucleotides are highly useful as chemical tools for fluorescent isostere,6 (ii) the conjugation of a given nucleotide
numerous biological applications.1 While the natural nucleo- with an extrinsic fluorophore,5b,c,7 and (iii) the installation of a
tide building blocks of nucleic acids are largely non- fluorogenic substituent at the nucleobase.5a,8,9 In principle,
fluorescent, the incorporation of fluorescent derivatives has the latter approach is attractive for several reasons. Fluo-
been used to study, for example, DNA and RNA structure2 and rescence can be generated with a relatively small molecular
to detect oxidative lesions.3 Fluorescent nucleotides and change, minimising the potential for undesired interference
nucleotide conjugates have also been used successfully as with the biological target. In addition, the installation of the
probes in enzymological studies, e.g. in biochemical assays for fluorogenic substituent can often be achieved synthetically in
NAD-dependent enzymes4 and in ligand-displacement assays a relatively straightforward manner. This approach is therefore
for inhibitor screening against glycosyltransferases.5 Three finding increasing application for the development of fluore-
scent derivatives of both purine and pyrimidine nucleotides
and nucleotide conjugates.5a,8,9
aSchool of Pharmacy, University of East Anglia, Norwich, UK
An important example for intrinsically fluorescent nucleo-
tides are derivatives of uracil with a suitable, fluorogenic
substituent in position 5 (Fig. 1). Fluorescent nucleotides
and nucleotide conjugates incorporating such a 5-substituted
uracil fragment have been used successfully as probes for
nucleic acid structure9c–e and as fluorophores for glycosyltrans-
ferase ligand-displacement assays.5a To date, 5-substituents
bInstitute of Pharmaceutical Science, School of Biomedical Sciences, King’s College
London, UK
cDepartment of Chemistry, School of Biomedical Sciences, King’s College London,
Franklin-Wilkins Building, 150 Stamford Street, London, SE1 9NH, UK.
E-mail: gerd.wagner@kcl.ac.uk; Fax: +44 (0)20 7848 4045; Tel: +44 (0)20 7848 4747
†Dedicated to my father on the occasion of his 65th birthday (GKW).
‡Electronic supplementary information (ESI) available: Additional Fig. S1–S3;
NMR spectra for 2a, 2b and 2l–q. See DOI: 10.1039/c3ob40485d
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substituents in position 5 and systematically investigated the
effect of the 5-substituent on their photophysical properties.
Specific goals of this study included the identification of struc-
tural parameters that determine the quantum yield and exci-
tation/emission wavelength in this class of fluorophores in
aqueous media. The highest quantum yields were determined
for the thien-2-yl substituted derivatives 2p and 2q (Table 1),
which are 10-fold more fluorescent than any other nucleotide
analogues with an aryl- or heteroaryl substituent in position 5
that have been reported to date.1a An attractive feature of 2p
and 2q is their absorbance and fluorescence emission at rela-
tively long wavelengths, which makes these fluorophores suit-
able for analyses in biological environments. To illustrate such
applications, we have carried out ligand-binding experiments
with selected fluorophores and a UDP-sugar-dependent glycosyl-
transferase. In conjunction with results from solvatochromic
experiments, the results from these biological experiments
offer, for the first time, a structural explanation for the fluo-
rescence quenching effect that is observed upon binding of
these fluorophores to a target protein.
Fig. 1 Fluorescent 5-substituted uracil nucleotides and nucleotide conjugates.
that have been explored for the development of intrinsically
fluorescent uracil nucleosides and nucleotides have been
limited mainly to small heterocycles5a,9c–e or phenol and
anisol derivatives connected directly or via an alkenyl or
alkynyl linker to the uracil ring.9a,b While 5-substituted uracil
nucleotides, in which a known fluorophore is connected to the
uracil base via an electronically non-conjugating linker,
usually retain the photophysical features of the parent fluoro-
phore,1a,10 appending an additional aromatic or heteroaro-
matic moiety to the uracil base typically generates a new
fluorophore with unique and somewhat unpredictable photo-
physical characteristics.1a While it appears clear that the
fluorescence of 5-substituted uracil nucleotides derives from
the extended electron delocalisation across the uracil base and
the 5-substituent, the precise structural basis for the unique
fluorescence of these derivatives has not been systematically
investigated.
Results and discussion
Synthesis
To investigate the effect of the additional substituent on the
To address this question, we have prepared a series of uracil photophysical properties of 5-substituted uracil nucleotides,
nucleotides with different aromatic and heteroaromatic we prepared a series of uracil nucleotides with a broad range
Table 1 Yields and photophysical properties of 5-substituted UMP derivatives 2a–q
R
Code
Yield (%)
λmaxa (nm)
λemb (nm)
Stokes shiftc (cm−1
)
Fluorescence intensityb (a.u.)
Phenyl
4-Chlorophenyl
2a
2b
2c
2d
2e
2f
2g
2h
2i
2j
2k
2l
2m
2n
2o
2p
2q
71
66
79
69
52
33
47
60
63
45
58
57
57
56
25
31
61
21
19
278
281
279
281
285
273
221
283
280
289
278
279
314
348
267
348
351
341
341
403
398
434
427
11 157
10 462
12 801
12 168
40
78
4-Methyl-3-nitrophenyl
3-N-Boc-aminomethylphenyl
3-Hydroxyphenyl
2-Hydroxyphenyl
2-Naphthyl
4-Carboxyphenyl
4-Trifluoromethylphenyl
5-Methoxypyridin-3-yl
3-Mesylphenyl
6.7
11.6
d
d
d
—
—
—
415
450
411
383
414
382
444
437
431
453
433
434
459e
451e
12 534
23 027
11 005
9605
10 447
9793
13 320
8964
5534
15 378
5641
5449
7539
7153
0.9
32.2
17.6
5.3
74.8
5.3
4-Methoxyphenyl
Furan-2-yl
603
497
640
387
>650
>650
78e
5-Formylfuran-2-yl
3-Formylthien-2-yl
5-Acetylthien-2-yl
5-Formylthien-2-yl
5-Oximinothien-2-yl
5-Oximinothien-2-yl
Syn-2r
Anti-2r
105e
a Absorbance spectra recorded in H2O at 10 μM. b Fluorescence spectra recorded in H2O at 100 μM, unless otherwise stated. c Stokes shift =
(1/λexcitation − 1/λemission). d Not fluorescent. e In H2O at 20 μM.
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of electronically and sterically different 5-substituents. We
decided to target primarily the water-soluble uracil nucleotides
(i.e. UMP, UDP and UTP derivatives) rather than the corres-
ponding nucleosides in order to allow the investigation of the
target fluorophores in aqueous media, the relevant environ-
ment for any potential biological application. First, we pre-
pared 5-iodo UMP 1 either by direct iodination of UMP11 or by
phosphorylation of 5-iodouridine under Yoshikawa con-
ditions.12 The target 5-substituted UMP derivatives 2a–q were
obtained by cross-coupling of 1 to a set of aromatic and hetero-
aromatic boronic acids under our previously reported aqueous
Suzuki–Miyaura conditions (Scheme 1).5a,9b,11,13 Irrespective of
their steric and electronic properties, most boronic acids were
coupled readily to 5-iodo-UMP 1. This provided us with a
series of 17 different UMP analogues 2a–q bearing sterically
and electronically diverse 5-substituents as an ideal test set for
photophysical studies (Table 1). Further structural variation
was achieved by condensation of the 5-(5-formylthien-2-yl) sub-
stituted UMP derivative 2q with hydroxylamine (Scheme 2).
This transformation furnished the corresponding oximino
derivative 2r as a mixture of geometrical isomers, which were
readily separated by column chromatography. The configur-
ation of each isomer could be unambiguously assigned from
the chemical shift of the oxime proton in the 1H NMR
spectrum (anti-2r: 7.85 ppm; syn-2r: 8.35 ppm).
Table 2 Yields for 5-substituted UDP and UTP derivatives
Compound
Code
Yield (%)
5-Iodo UDP
5-Iodo UTP
3
4
66
60
73
50
60
77
85
53
74
67
5-Phenyl UDP
5a
5b
5c
5d
6a
6b
6c
6d
5-(4-Methoxyphenyl) UDP
5-(Furan-2-yl) UDP
5-(5-Formylthien-2-yl) UDP
5-Phenyl UTP
5-(4-Methoxyphenyl) UTP
5-(Furan-2-yl) UTP
5-(5-Formylthien-2-yl) UTP
derivatives (Scheme 1). Thus, 1 was converted into the corres-
ponding phosphoromorpholidate via a Mukaiyama redox
activation,14 followed by the reaction of the resulting morpholi-
date with a tributylammonium phosphate or pyrophosphate
salt to afford, respectively, 5-iodo-UDP 3 and 5-iodo-UTP 4.
Conveniently, our cross-coupling conditions could also be
applied to 3 and 4, as well as the nucleoside substrate
5-iodouridine. Thus, Suzuki–Miyaura cross-coupling of 3 and 4
to a selection of boronic acids provided 5-substituted deriva-
tives of UDP (5a–d) and UTP (6a–d) in moderate to high yields
(Table 2). The cross-coupling of 5-iodouridine with 5-for-
mylthien-2-ylboronic acid gave 5-(5-formylthien-2-yl) uridine 7,
the nucleoside analogue of UMP derivative 2q (Scheme 1).
5-Iodo-UMP 1 also provided an excellent starting point for
the synthesis of a selection of 5-substituted UDP and UTP
Photophysical characterisation
With a collection of 5-substituted uracil nucleotides in hand,
we set out to determine their photophysical properties. First,
we recorded the absorbance and fluorescence spectra of UMP
derivatives 2a–r at a defined concentration in water (Table 1).
This initial study showed that in this series, both the absor-
bance and emission maxima vary considerably, depending on
the nature of the 5-substituent. While most analogues carrying
a phenyl or substituted phenyl ring (2a–l) have an absorbance
maximum at around 280 nm, the absorbance maxima of most
analogues bearing a 5-membered heteroaromatic ring was, as
a general trend, significantly red-shifted (2m–r). This red-shift
is about 35 nm for the derivative with a simple, unsubstituted
furan substituent (2m), and about 70 nm for derivatives with
an additional formyl or acetyl group attached to a furan or
thiophene ring (2n, 2p, 2q). The one exception to this general
trend is the 5-(3-formylthien-2-yl) substituted derivative 2o,
whose absorbance maximum is slightly blue-shifted compared
to the phenyl-substituted derivatives. Interestingly, the UV
absorbance profile of 2o is in general very different from those
of its closest structural analogues 2n and 2q (Fig. 2).
Scheme 1 Synthesis of 5-substituted derivatives of UMP (2a–q), UDP (5a–d),
UTP (6a–d) and uridine (7). Reagents and conditions: (i) proton sponge, POCl3,
MeCN, −5 °C; (ii) R-B(OH)2, TPPTS, Na2Cl4Pd, Cs2CO3, H2O, 60 °C, 1 h; (iii) mor-
pholine, 2,2’-dipyridyldisulfide, PPh3, DMSO, rt, 1 h; (iv) KH2PO4 (3) or K4P2O7 (4),
tributylamine, tetrazole, MeCN, rt, 5 h. For substituents R and individual yields
see Tables 1 and 2.
UMP analogues with a six-membered 5-substituent (2a–l)
were relatively poor fluorescence emitters, with the exception
of 5-(4-methoxyphenyl)-UMP 2l, which was strongly fluorescent
under these conditions (Table 1). This is in agreement with
the pronounced fluorescence emission that has previously
been reported both for the corresponding, 5-methoxyphenyl-
substituted derivatives of uridine9a and UDP-galactose.5a In
Scheme 2 Synthesis of 5-(5-oximinothien-2-yl) UMP isomers syn-2r and
anti-2r.
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Table 3 Quantum yields for selected 5-substituted UMP fluorophores in water
R
Code
Q. Y.
Ha
UMP
2b
2l
2m
2n
2o
2p
2q
Syn-2r
Anti-2r
5 × 10–5
0.001
0.02
0.04
0.04
0.02
0.24
0.26
0.017
0.031
4-Chlorophenyl
4-Methoxyphenyl
Furan-2-yl
5-Formylfuran-2-yl
3-Formylthien-2-ylb
5-Acetylthien-2-ylb
5-Formylthien-2-ylb
5-Oximinothien-2-yl
5-Oximinothien-2-yl
a From ref. 29. b See ref. 9e for comparison with the unsubstituted
parent 5-(thien-2-yl) uridine: QY (H2O) 0.01, λex 314 nm, λem 434 nm.
measurements confirmed that 2p and 2q are the strongest
fluorescence emitters in this series. In particular, the
4-methoxyphenyl-substituted derivative 2l, which had shown
similar fluorescence intensity in the initial screen, had a more
than 10-fold lower quantum yield than 2p and 2q. To the best
of our knowledge, the quantum yields of 2p and 2q are the
highest for any 5-substituted uracil nucleotide and nucleoside
fluorophores that have been reported to date.9 Particularly
noteworthy is the 10-fold greater quantum yield for these sub-
stituted thiophene derivatives compared to the parent com-
pound bearing an unsubstituted thiophene ring in position 5
(Table 3). Interestingly, the same effect was not observed in the
furan series, where the same quantum yield was determined
for 2m, bearing an unsubstituted furan-2-yl ring in position 5,
and the 5-(5-formylfuran-2-yl) derivative 2n. Conversion of the
formyl group in 2q into the corresponding oximino group in
2r led to a significant drop in quantum yield. Interestingly,
however, the quantum yield of the anti-aldoxime (anti-2r) was
about double than that of its geometrical isomer (syn-2r).
Fig. 2 Absorbance (top) and fluorescence (bottom) spectra of selected UMP
derivatives in water at 10 μM (absorbance) and 100 μM (fluorescence). Fluo-
rescence emission was measured after excitation at λmax
.
contrast to most phenyl-substituted UMP derivatives, ana-
logues with a 5-membered heteroaromatic substituent (2m–q)
consistently displayed pronounced fluorescence emission.
5-Substituted uracil nucleosides with a simple thiophene or
furan substituent in position 5 have previously been reported
as fluorescence emitters.9d,e In our series, the strongest
fluorescence signal was observed for UMP derivatives with a
formyl- or acetyl-substituted thiophene or furan ring in posi-
tion 5 (2n, 2p, 2q). Indeed, two of these derivatives (2p, 2q)
showed saturating fluorescence at 100 μM (Table 1). These
three fluorophores also displayed the most pronounced
bathochromic shifts for their absorbance maxima, with λmax at
around 350 nm (Table 1). While the 5-formyl- or 5-acetyl-sub-
stituted furan and thiophene derivatives 2n, 2p, 2q all showed
qualitatively similar absorbance and fluorescence spectra,
stronger absorbance and weaker fluorescence emission was
observed for the furan derivative 2n than for the thiophene
derivatives 2p and 2q (Fig. 2). The intense fluorescence emis-
sion of 5-(5-formylthien-2-yl) UMP 2q was also preserved for
the corresponding 5-(5-formylthien-2-yl) derivative of UDP (5d)
and UTP (6d), indicating that, as expected, the presence of
the additional phosphate groups has little influence on
the absorbance and fluorescence emission of these 5-(5-for-
mylthien-2-yl) uracil nucleotides (Fig. S1†).
Conformational analysis
In order to better understand the interplay between structure
and fluorescence in this series, we next carried out an NMR-
based conformational analysis of those UMP derivatives, for
which quantum yields had been determined. Important con-
formational parameters in nucleosides and nucleotides are the
sugar pucker of the ribose (S-type vs. N-type) and the syn- or
anti-orientation of the nucleobase relative to the ribose ring
(Table 4). Both of these parameters can be extracted from
1H NMR data using established empirical approaches: the
coupling constant J1′,2′ is indicative of the ribose conformation,
while the chemical shift of the H-2′ signal, relative to the
5-unsubstituted parent UMP, is a good indicator for the position
of the syn/anti equilibrium.16 In aqueous solution, all 5-substituted
UMP derivatives that were included in this analysis displayed a
slight preference for the S-type conformation of the ribose,
similar to UMP itself (Table 4). In addition, the chemical shifts
for H-2′ did not deviate substantially from that of 5-unsubsti-
tuted UMP. This suggests that, like the parent nucleotide UMP,
all of these 5-substituted derivatives adopt preferentially the
anti conformation, in which the 5-substituent is oriented
In order to obtain a more quantitative measure of fluo-
rescence emission in this series, we next determined the
quantum yields for selected UMP derivatives (Table 3). These
measurements were performed in water following the pro-
cedure reported by Nighswander-Remper.15 Quantum yield
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Table 4 Conformational analysis of selected 5-substituted UMP derivatives
S-type conformer (%)
R
Code
(10× J1′,2′
)
H-2′ (ppm)
ΔH-2′ (ppm)
Syn/antia
H-6 (ppm)
H
UMP
2b
2l
2m
2n
2o
2p
51
55
56
53
4.41
4.45
4.42
4.45
4.51
4.42
4.46
4.45
4.46
4.46
0.00
0.04
0.01
0.04
0.1
0.01
0.05
0.04
0.05
0.05
anti
anti
anti
anti
anti
anti
anti
anti
anti
anti
8.13
7.90
7.76
8.15
8.40
8.13
8.35
8.38
8.17
8.17
4-Chlorophenyl
4-Methoxyphenyl
Furan-2-yl
5-Formylfuran-2-yl
3-Formylthien-2-yl
5-Acetylthien-2-yl
5-Formylthien-2-yl
5-Oximinothien-2-yl
5-Oximinothien-2-yl
53
n.d.b
52
2q
50
57
57
Syn-2ra
Anti-2ra
a The syn/anti nomenclature of oximes is not to be confused with the syn/anti equilibrium about the glycosidic bond. b Not determined due to
overlapping peaks in the 1H NMR spectrum.
Table 5 Calculated dihedral angle α for the relative orientation of the 5-substituent and the uracil ring in UMP derivatives 2o–2r
N-type
S-type
Energy difference: N-type vs.
R
Code
S-type (kcal mol−1
)
α
“Out-of-plane”
α
“Out-of-plane”
3-Formyl
5-Acetyl
5-Formyl
5-Oximino
5-Oximino
2o
2p
2q
Syn-2r
−5.73
−2.80
−2.69
−2.70
−1.89
−135.3°
−161.4°
−162.2°
−159.7°
−165.3°
ca. 45°
ca. 20°
ca. 18°
ca. 20°
ca. 15°
−125.3°
162.7°
162.7°
161.5°
160.9°
ca. 55°
ca. 17°
ca. 17°
ca. 18°
ca. 19°
Anti-2r
towards the ribose ring. These conformational preferences are however, the dihedral angle α, which describes the relative
in agreement with those previously observed by X-ray crystallo- orientation of the 5-substituent and the uracil ring to each
graphy for related 5-(hetero)aryl uridine derivatives.9d,e,17
other, was very similar for both the N-type and S-type con-
To gain further insights into the conformational prefer- formation of each derivative. Thus, 2p, 2q and 2r show an
ences of these fluorophores, we calculated energy-minimized almost coplanar orientation of both rings, in both con-
geometries for the UMP derivatives 2o–2r. The absorbance formations (Table 5). In contrast, the calculated conformations
spectra obtained from these calculations were generally in of 2o suggest that in this case, the thiophene and uracil rings
good agreement with experimental results (ESI, Fig. S2†). In are oriented “out-of-plane” (Fig. 3).
agreement with the results from our NMR studies, which
suggested an almost 1 : 1 ratio of N-type and S-type confor-
Solvatochromism
mations, our calculations showed only very small energy differ-
ences between N-type and S-type conformations (Table 5). The
marginally higher stability of the N-type conformers in the
implicit solvent simulations may be due to an intramolecular
hydrogen bond between the phosphate group and the C3′ hydroxyl
group of the ribose. Interestingly, for 2o an additional intra-
molecular hydrogen bond was observed between the phos-
phate group and the 3-formyl substituent. Most importantly,
Next, we investigated the influence of different solvents on the
absorbance and fluorescence of the 5-(5-formylthien-2-yl)
uracil fluorophore, which gave the strongest emission in this
series. As the 5-(5-formylthien-2-yl)-substituted nucleotide 2q
has only limited solubility in organic solvents, we chose 5-(5-
formylthien-2-yl) uridine 7, the nucleoside analogue of 2q, for
these investigations. We prepared 200 nM solutions of 7 in
three solvents with a different polarity index (P): water (P = 9),
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Fig. 3 Chemical structures and calculated energy-minimised S-type conformers
of 2o (left) and 2q (right). The different orientations of the formylthiophene
substituent relative to the uracil ring in both fluorophores are clearly visible
(2o: out-of-plane, 2q: coplanar).
Fig. 5 Fluorescence emission of 7 in different solvent mixtures (λem 450 nm).
containing less than 70% of water, both in the case of
H2O–IPA and H2O–MeCN, whereas
a steep, non-linear
decrease in fluorescence was observed for solvent mixtures
containing between 85%–70% water. Such sudden variations
in fluorescence intensity are characteristic for non-specific
solvent effects mediated by hydrogen-bond interactions.18 The
observation that fluorescence emission was strongest in the
two protic solvents used in this study (H2O, IPA), and weaker
in the aprotic MeCN, may therefore suggest a possible role for
hydrogen bonding in the fluorescence of the 5-(5-formylthien-
2-yl) uracil fluorophore in solution.
Discussion of photophysical properties
Fig. 4 Absorbance (dashed lines) and fluorescence emission (full lines) of 5-(5-
formylthien-2-yl) uridine 7 at 200 nM in water (blue), isopropanol (red) and
acetonitrile (green).
In principle, various explanations are conceivable for the
differences in fluorescence emission that were observed
for the different 5-substituted UMP derivatives in this study,
including charge transfer contributions such as the twisted
intramolecular charge transfer (TICT) model,19 and confor-
mational effects.9c We reasoned that a key to understanding
the interplay of structure and fluorescence emission in this
series may be provided by the regioisomeric 5-formylthien-2-yl
fluorophores 2o and 2q, which differ markedly in their photo-
physical and conformational properties. Thus, 5-(3-formyl-
thien-2-yl) UMP 2o had a quantum yield more than 10-fold
lower than 5-(5-formylthien-2-yl) UMP 2q, the strongest
emitter in this series, while its λmax was blue-shifted by 84 nm
compared to 2q. In this context, it appeared significant that 2o
and 2q display different conformational preferences with
regard to the relative orientation of the formylthiophene and
uracil rings, the two constituent parts of the formylthien-2-yl
uracil fluorophore. While the uracil and thiophene rings adopt
a nearly coplanar orientation in 2q, they are forced out of
plane in the corresponding 5-(3-formylthien-2-yl) derivative 2o
(Fig. 3). As efficient electron delocalisation along π-bonds is
limited to planar systems, the non-coplanar orientation of the
two rings in 2o directly affects electron delocalisation across
the formylthien-2-yl uracil system. Experimentally, this is
reflected in the different chemical shifts for the H-6 proton,
which is shifted 0.25 ppm upfield for 5-(3-formylthien-2-yl)
acetonitrile (MeCN, P = 5.8) and isopropanol (IPA, P = 3.9) and
recorded their absorbance and fluorescence spectra (Fig. 4).
While the absorbance of 7 was largely unaffected by the
nature of the solvent, its fluorescence was strongly solvent-
dependent, with fluorescence emission about 20-fold stronger
in water than in MeCN. Interestingly, fluorescence and solvent
polarity were not directly correlated, since we observed higher
fluorescence in IPA than in MeCN. Moreover, if polarity was
the source of the variation in fluorescence, we would also have
expected to see a correlation with a hypsochromic shift in the
fluorescence spectra, which was not the case. In order to better
understand these variations in fluorescence, we carried out a
second set of experiments, recording the fluorescence emis-
sion of 7 in different solvent mixtures (H2O–IPA and H2O–
MeCN) at different ratios H2O : organic solvent (Fig. 5). These
experiments confirmed a complex correlation between solvent
polarity and fluorescence intensity for this fluorophore. As
expected from the preceding experiments, fluorescence emis-
sion is strongest in water and solvent mixtures containing
>85% water. An increase in the concentration of the organic
solvent led to a decrease in fluorescence intensity. However,
this decrease in fluorescence is linear only for solvent mixtures
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UMP 2o compared to its regioisomer 2q (Table 4), indicating
that the formyl group in 2q exerts a stronger electron-with-
drawing effect on the uracil ring than in 2o. Taken together,
these results suggest that electron delocalisation, possibly via
a push–pull system involving the nitrogen in position N1, may
play an important role for the strong fluorescence emission of
fluorophore 2q and closely related derivatives (e.g. 2p). Thus,
the fluorescence intensity of these fluorophores may be attrib-
uted to effective electron delocalisation across the entire
5-thienyl uracil fragment, resulting from both the presence of
the additional carbonyl group at the thiophene ring, and the
coplanar orientation of the substituted thiophene and uracil
rings.
Intriguingly, this interpretation would also offer an expla-
nation for the effects we have observed in our solvatochromic
experiments, which show increased fluorescence emission for
the 5-formylthien-2-yl fluorophore in protic solvents. Our com-
putational results indicate that hydrogen bonding plays a role
for the stabilisation of the preferred conformations of individ-
ual fluorophores. It can therefore be speculated that in the
case of strong emitters (2p, 2q), hydrogen bonding in protic
solvents may stabilise the fluorescent, coplanar conformation
e.g. through a bi- or multi-molecular network of solvent mole-
cules and fluorophore (Fig. S3, ESI†). This hypothesis, while
speculative at this point, is supported by the observation that
in the case of 2q and anti-2r, closely related regioisomers (2o,
syn-2r), for which this hydrogen bonding stabilisation is
not possible, due to their different geometries, are weaker
fluorophores.
Fig. 6 (a) Fluorescence quenching upon incubation of α-1,3-GalT with, respect-
ively, 5-(5-formylthien-2-yl) UDP-galactose 8, 5d, 2q and 7. (b) Chemical struc-
tures of fluorophores 8, 5d, 2q and 7.
Application of fluorophores in ligand-binding experiments
On the other hand, results from our photophysical studies
provide, for the first time, a structural explanation for the
strong fluorescence quenching observed for 5d and 8 upon
binding at B. taurus α-1,3-GalT. Our photophysical studies indi-
cate that a coplanar orientation of the 5-formylthien-2-yl and
uracil rings, possibly stabilised by hydrogen bonding, is a pre-
requisite for fluorescence emission in this series. The strong
fluorescence quenching observed for 5d and 8 may therefore
be caused by the binding of these GalT ligands in an orien-
tation that forces the fluorogenic 5-substituent out of the
plane of the uracil ring and/or disrupts hydrogen bonding to
O4 and the formyl group. Based on the results from our photo-
physical studies, such a non-coplanar orientation would be
expected to lead to a drop in fluorescence, as observed in our
experiments.
Previously, we have shown that the fluorescent, 5-(5-for-
mylthien-2-yl)-substituted UDP-galactose derivative 8 (Fig. 6)
can be used as a fluorescent probe in ligand displacement
assays with bacterial and mammalian galactosyltransferases
(GalTs), including B. taurus α-1,3-GalT.5a This application was
based on the quenching of the fluorescence of sugar-nucleo-
tide 8 upon the specific binding to the respective target
enzyme. With the corresponding 5-(5-formylthien-2-yl) uridine
(7), UMP (2q) and UDP (5d) derivatives in hand, we investi-
gated if a similar quenching effect would be observed with
these alternative, structurally simplified ligands for B. taurus
α-1,3-GalT. Interestingly, in these experiments, efficient
quenching was still observed with 5-(5-formylthien-2-yl) UDP
5d, while the effect was less pronounced with 5-(5-formylthien-
2-yl) UMP 2q, and disappeared altogether with 5-(5-for-
mylthien-2-yl) uridine 7 (Fig. 6). This rank order corresponds
to the known binding affinities of the 5-unsubstituted parent
molecules UDP, UMP and uridine for UDP-galactose-depen-
Conclusion
dent GalTs.20 While UDP-galactose and UDP often have similar In this study, we have developed a rapid and convenient syn-
binding constants for a given GalT, removal of one or both thesis of 5-aryl and 5-heteroaryl-substituted UMP, UDP and
phosphates invariably leads to a drop in affinity.20 The same UTP derivatives. This has allowed the systematic investigation
correlation holds for our series of fluorophores. Thus, the lack of the photophysical properties of these non-natural nucleo-
of fluorescence quenching in the case of uridine 7 can very tides. Two novel UMP derivatives bearing, respectively, a
likely be attributed to the lack of binding affinity of this fluoro- 5-acetylthien-2yl (2p) and 5-formylthien-2yl (2q) group in posi-
phore for B. taurus α-1,3-GalT.
tion 5 stood out as the strongest fluorescence emitters in this
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series, with quantum yields of around 0.25. This represents a in ppm and referenced to methanol (δH 3.34, δC 49.50) for solu-
10-fold improvement compared to previously reported 5-substi- tions in D2O, to DMSO (δH 2.50, δC 39.52) or to CDCl3 (δH 7.26,
tuted uracil fluorophores. The superiority of a 5-formyl- or δC 77.16). Coupling constants (J) are reported in Hz. Resonance
5-acetyl thiophene ring as a fluorogenic substituent, compared allocations were made with the aid of COSY and HSQC experi-
to both 5-unsubstituted heterocycles and various substituted ments. NOESY measurements were carried out with a relax-
phenyl substituents, appears to be due to two factors: first, the ation delay of 1.000 s and a mixing time of 0.200 s. Accurate
extended delocalised π electron system in the case of the electrospray ionisation mass spectra (HR ESI-MS) were
5-formyl- or 5-acetylthienyl uracil fluorophore, and second obtained on a Finnigan MAT 900 XLT mass spectrometer at
the coplanar orientation of the fluorogenic 5-substituent and the EPSRC National Mass Spectrometry Service Centre in
the uracil ring. Importantly, the conformational preferences of Swansea. Preparative chromatography was performed on a Bio-
5-(5-formylthien-2-yl) UMP 2q, the strongest fluorescence logic LP chromatography system equipped with a peristaltic
emitter in this series, allow such a coplanar orientation, which pump and a 254 nm UV Optics Module under the following
is conducive to efficient electron delocalisation across the two conditions.
ring systems. In contrast, in the regioisomer 5-(3-formylthien-
2-yl) UMP 2o, the presence of the formyl group in position 3 of
Purification method 1
the thiophene ring prevents the coplanar orientation of the Ion-pair chromatography was performed using Lichroprep
fluorogenic 5-substituent and the uracil ring and, possibly as a RP-18 resin gradient 0–10% acetonitrile (or methanol) against
direct consequence, leads to a markedly reduced quantum 0.05 M TEAB (triethylammonium bicarbonate: prepared by
yield.
bubbling CO2 gas through a mixture of Et3N in water until
This interpretation may also provide an explanation both saturation was achieved) over 480 mL, flow rate 5 mL min−1
.
for the solvent dependency of fluorescence emission in this Product-containing fractions were combined and reduced to
series of fluorophores, and for the fluorescence quenching dryness. The residue was co-evaporated repeatedly with metha-
which we have observed in ligand binding experiments. The nol to remove residual TEAB.
strongest fluorescence emission of the 5-(5-formylthien-2-yl)
Purification method 2
uracil fluorophores was observed in water. This finding raises
the possibility that in protic solvents the fluorescent, coplanar Anion-exchange chromatography was performed using
a
orientation of the 5-formylthien-2-yl and uracil rings may be Macro prep 25Q resin, gradient 0–100% 1 M TEAB (pH 7.3)
stabilised through hydrogen bonding. The role of hydrogen against H2O over 480 mL, flow rate 5 mL min−1. Product-
bonding for fluorescence in this series is further illustrated by containing fractions were combined and reduced to dryness.
results for the oximino-substituted fluorophore 2r, where the The residue was co-evaporated repeatedly with methanol to
anti-isomer (which can engage in hydrogen bonding) has a remove residual TEAB.
greater quantum yield than the syn-isomer (which cannot).
General method for the Suzuki–Miyaura cross-coupling of
Finally, it can be speculated that this stabilising interaction, 5-iodouridine, 5-iodo UMP (1), 5-iodo UDP (3) and 5-iodo UTP
and consequently the coplanar orientation of the fluorogenic (4). A 2-necked round bottom flask with 5-iodouridine or the
substituent and the uracil ring, may be disrupted upon respective 5-iodouridine nucleotide 1, 3 or 4 (1 equiv.), Cs2CO3
binding of these fluorophores to a target protein, leading to (2 equiv.) and the requisite boronic acid (1.5 equiv.) was
the strong fluorescence quenching observed in our ligand purged with N2. TPPTS (0.0625 equiv.), Na2Cl4Pd (0.025 equiv.)
binding experiments. Taken together, these findings provide and degassed H2O (4 mL) were added, and the reaction was
new insights into the structural basis for fluorescence and stirred under N2 for 1 h at 60 °C. Upon completion, the reac-
fluorescence quenching in this series of 5-substituted uracil tion was cooled to room temperature. The black suspension
nucleotides. The results from this study therefore provide a was concentrated under reduced pressure, and the residue was
basis for the further rational optimisation of this important taken up in MeOH. The methanolic suspension was filtered
class of fluorophores, and for their application as sensors in through celite, and the residue was washed with methanol.
biological assays.
The combined filtrates were evaporated under reduced
pressure and the residue was purified using purification
method 1.
5-Iodouridine-5′-monophosphate (1).21
A
suspension of
Experimental section
5-iodouridine (480 mg, 1.3 mmol)22 and proton sponge (1.7 g,
All chemicals were obtained commercially and used as 7.8 mmol) in dry acetonitrile (20 mL) was cooled to −5 °C and
received unless stated otherwise. Fluorophore 8 was syn- stirred under N2. POCl3 (58 μL, 0.6 mmol) was added dropwise
thesised as previously reported.5a TLC was performed on pre- to this suspension. The orange-coloured reaction was stirred at
coated aluminium plates (Silica Gel 60 F254, Merck). −5 °C for 4 h, at which time TLC indicated near complete con-
Compounds were visualized by exposure to UV light (254 and version (IPA–H2O–NH3 6 : 3 : 1; Rf 0.31; Rf 0.71). The reaction
SM
365 nm). NMR spectra were recorded at 298 K on a Varian VXR was quenched with 250 mL of ice cold 0.2 M TEAB buffer. The
400 S spectrometer, a Bruker Avance DRX-300 or a Bruker pale orange solution was stirred for 1 h at 0 °C. After allowing
Avance DPX-400 spectrometer. Chemical shifts (δ) are reported to reach 25 °C, the aqueous layer was washed with ethyl
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acetate (3×) and concentrated under reduced pressure. The H-1′), 7.30–7.48 (4H, m, Ph), 7.86 (1H, s, H-6); δC (75.5 MHz,
crude residue was purified sequentially by purification D2O) 20.4 (CH2), 28.2 (tBu), 65.1 (d, JC,P = 6.0 Hz, C-5′), 70.7
methods 1 and 2 to provide 1 in its triethylammonium salt (C-3′), 74.1 (C-2′), 84.3 (d, JC,P = 8.3 Hz, C-4′), 84.9 (tBu), 89.3
form (1.7 equiv.) as a colourless, glassy solid in 53% yield (C-1′), 101.4 (C-Ph), 116.7 (C-5), 127.4, 127.7, 128.2, 129.7,
(472 mg). δH (400 MHz, D2O) 3.96–4.10 (2H, m, H-5′), 4.22–4.26 132.7 (C-Ph), 139.2 (C-6), 152.2 (C-2), 159.1 (CHO), 165.4 (C-4);
(1H, m, H-4′), 4.31 (1H, t, J = 4.7 Hz, H-3′), 4.38 (1H, t, J = 5.3 δP (121.5 MHz, D2O) 7.6. m/z (ESI) 528.1384 [M − H]−,
Hz, H-2′), 5.93 (1H, d, J = 5.3 Hz, H-1′), 8.27 (1H, s, H-6); δC C21H27N3O11P requires 528.1389.
(75.5 MHz, D2O) 64.8 (d, JC,P = 4.6 Hz, C-5′), 69.6 (C-5), 70.8
5-(3-Hydroxyphenyl)-uridine-5′-monophosphate
(2e). The
(C-3′), 74.6 (s, C-2′), 84.8 (d, JC,P = 8.8 Hz, C-4′), 89.6 (C-1′), triethylammonium salt of the title compound was obtained as
147.1 (C-6), 152.8 (C-2), 164.3 (C-4); δP (121.5 MHz, D2O) a glassy solid in 52% yield (6.8 mg) from 1 (10 mg, 22.2 μmol)
7.6. m/z (ESI) 448.9255 [M − H]−, C9H11IN2O9P requires and 3-hydroxyphenylboronic acid according to the general
448.9252.
method. δH (400 MHz, D2O) 4.05–4.12 (2H, m, H-5′), 4.27–4.31
5-Phenyluridine-5′-monophosphate (2a). The triethylammo- (1H, m, H-4′), 4.40 (1H, t, J = 4.7 Hz, H-3′), 4.47 (1H, t, J =
nium salt of the title compound was obtained as a glassy 4.9 Hz, H-2′), 6.01 (1H, d, J = 4.9 Hz, H-1′), 6.86 (1H, d, J =
solid in 71% yield (13.5 mg) from 1 (16.5 mg, 36.7 μmol) and 7.3 Hz, Ph), 7.23 (1H, s, Ph), 7.25–7.27 (1H, m, Ph), 7.33 (1H, t,
phenylboronic acid according to the general method. δH J = 7.8 Hz, Ph), 8.09 (1H, s, H-6); δC (75.5 MHz, D2O) 64.3 (d, JC,
(400 MHz, D2O) 4.01–4.15 (2H, m, H-5′), 4.24–4.30 (1H, m,
= 3.8 Hz, C-5′), 70.2 (C-3′), 74.8 (C-2′), 84.5 (d, JC,P = 9.8 Hz,
P
H-4′), 4.32–4.40 (1H, m, H-3′), 4.45 (1H, t, J = 5.5 Hz, H-2′), 5.98 C-4′), 89.4 (C-1′), 115.4, 115.6, 116.0, 120.5, 120.7, 130.7,
(1H, d, J = 5.6 Hz, H-1′), 7.38–7.51 (5H, m, Ph), 7.82 (1H, s, 133.9 (C-5 + C-Ph), 139.3 (C-6), 152.0 (C-2), 156.9 (C-4);
H-6); δC (75.5 MHz, D2O) 65.0 (d, JC,P = 4.5 Hz, C-5′), 70.6 (C-3′), δP (121.5 MHz, D2O) 7.6. m/z (ESI) 415.0540 [M − H]−,
74.1 (C-2′), 84.3 (d, JC,P = 8.3 Hz, C-4′), 89.2 (C-1′), 116.9 (C-5), C15H16N2O10P requires 415.0548.
129.1, 129.4, 129.5, 132.4 (C-Ph), 139.2 (C-6), 152.3 (C-2), 165.5
5-(2-Hydroxyphenyl)-uridine-5′-monophosphate
(2f). The
(C-4); δP (121.5 MHz, D2O) 7.6. m/z (ESI) 399.0593 [M − H]−, triethylammonium salt of the title compound was obtained as
C15H16N2O9P requires 399.0599.
a glassy solid in 33% yield (4.4 mg) from 1 (10 mg, 22.2 μmol)
5-(4-Chlorophenyl)-uridine-5′-monophosphate
(2b). The and 2-hydroxyphenylboronic acid according to the general
triethylammonium salt of the title compound was obtained as method. δH (400 MHz, D2O) 3.98–4.01 (2H, m, H-5′), 4.23–4.27
a glassy solid in 20% yield (5.5 mg) from 1 (20 mg, 49.6 μmol) (1H, m, H-4′), 4.30–4.34 (1H, m, H-3′), 4.42 (1H, t, J = 5.4 Hz,
and 4-chlorophenylboronic acid according to the general H-2′), 6.04 (1H, d, J = 5.5 Hz, H-1′), 7.02 (2H, dd, J = 7.6
method. δH (400 MHz, D2O) 4.00–4.12 (2H, m, H-5′), 4.22–4.29 and 11.5 Hz, Ph), 7.30–7.35 (2H, m, Ph), 7.98 (1H, s, H-6); δC
(1H, m, H-4′), 4.30–4.344 (1H, m, H-3′), 4.45 (1H, d, J = 5.4 Hz, (150 MHz, D2O) 64.3, 70.6, 74.3, 84.4 (d, JC,P = 8.6 Hz), 89.0,
H-2′), 6.01 (1H, d, J = 5.5 Hz, H-1′), 7.46–7.58 (4H, m, Ph), 7.90 112.7, 117.7, 120.2, 121.4, 130.7, 132.0, 141.2, 152.1, 154.0,
(1H, s, H-6); δC (75.5 MHz, D2O), 65.0, 70.9, 74.3, 82.5, 89.6, 165.4; δP (121.5 MHz, D2O) 7.6. m/z (ESI) 415.0546 [M − H]−,
111.6, 129.6, 129.9, 131.2, 131.4, 139.6, 152.4, 165.8; δP C15H16N2O10P requires 415.0548.
(121.5 MHz, D2O) 10.4. m/z (ESI) 433.0210 [M
−
H]−,
5-(2-Naphthyl)-uridine-5′-monophosphate (2g). The triethyl-
ammonium salt of the title compound was obtained as a
C15H15Cl35N2O9P requires 433.0209.
5-(3-Nitro-(4-methylphenyl))-uridine-5′-monophosphate (2c). glassy solid in 47% yield (6.3 mg) from 1 (10 mg, 22.2 μmol)
The triethylammonium salt of the title compound was and 1-naphthylboronic acid according to the general method.
obtained as a glassy solid in 79% yield (10.9 mg) from 1 δH (400 MHz, D2O) 3.90–4.03 (2H, m, H-5′), 4.22–4.49 (4H, m,
(10 mg, 22.2 μmol) and 4-methyl-3-nitrophenylboronic acid H-2′, H-3′, H-4′), 6.07 (1H, d, J = 5.6 Hz, H-1′), 7.49–7.65 (4H,
according to the general method. δH (400 MHz, D2O) 2.57 (3H, m, napht), 7.72–7.78 (1H, m, napht), 7.93 (1H, s, H-6), 8.01
s, Me), 4.01–4.12 (2H, m, H-5′), 4.26–4.28 (1H, m, H-4′), 4.33 (2H, m, napht); δC (75.5 MHz, D2O) 64.6 (C-5′), 70.9 (C-3′), 73.8
(1H, t, J = 4.7 Hz, H-3′), 4.45 (1H, t, J = 5.4 Hz, H-2′), 6.00 (1H, (C-2′), 83.7 (C-4′), 89.3 (C-1′), 113.2 (C-5), 117.6, 125.8, 126.5,
d, J = 5.5 Hz, H-1′), 7.46–7.71 (2H, 2d, J = 7.9 and 8.0 Hz, Ph), 127.1, 127.4, 129.1, 129.5, 130.0, 130.1, 133.9 (C-naphthyl),
7.99 (1H, s, H-6) 8.16 (1H, s, Ph); δC (75.5 MHz, D2O) 19.8 140.9 (C-6), 154.6 (C-2), 166.0 (C-4); δP (121.5 MHz, D2O) 7.6.
(Me), 64.9 (C-5′), 70.5 (C-3′), 74.3 (C-2′), 84.3 (d, J = 9.1 Hz, m/z (ESI) 449.0760 [M − H]−, C19H18N2O9P requires 449.0755.
C-4′), 89.4 (C-1′), 114.4, 115.8, 125.2, 131.3, 133.9, 134.0
5-(4-Carboxyphenyl)-uridine-5′-monophosphate
(2h). The
(C-5 + C-Ph), 139.7 (C-6), 149.2 (C-Ph), 152.0 (C-2), 164.9 (C-4); triethylammonium salt of the title compound was obtained as
δP (121.5 MHz, D2O) 7.6. m/z (ESI) 458.0607 [M − H]−, a glassy solid in 60% yield (9.6 mg) from 1 (10 mg, 22.2 μmol)
C16H17N3O11P requires 458.0606.
and 4-carboxyphenylboronic acid according to the general
5-(3-(N-Boc-methylamine)-phenyl)-uridine-5′-monophosphate method. δH (400 MHz, D2O) 4.00–4.08 (2H, m, H-5′), 4.25–4.30
(2d). The triethylammonium salt of the title compound was (1H, m, H-4′), 4.31–4.35 (1H, m, H-3′), 4.48 (1H, t, J = 5.6 Hz,
obtained as a glassy solid in 69% yield (10.2 mg) from 1 H-2′), 6.00 (1H, d, J = 5.6 Hz, H-1′), 7.38, 7.69 (4H, 2d, J =
(10 mg, 22.2 μmol) and 3-N-Boc-aminomethylphenylboronic 8.1 Hz, Ph), 7.73 (1H, s, H-6); δC (75.5 MHz, D2O) 64.7 (d, JC,P
=
acid according to the general method. δH (400 MHz, D2O) 1.42 5.3 Hz, C-5′), 70.6 (C-3′), 73.8 (C-2′), 84.3 (d, JC,P = 8.3 Hz, C-4′),
t
(9H, s, Bu), 4.00–4.10 (2H, m, H-5′), 4.20–4.35 (4H, m, H-4′, 89.7 (C-1′), 116.3 (C-5), 129.2, 129.7, 135.2, 136.7 (C-Ph), 139.8
H-3′, CH2), 4.47 (1H, t, J = 5.5 Hz, H-2′), 6.01 (1H, d, J = 5.7 Hz, (C-6), 152.2 (C-2), 165.3 (C-4), 171.1 (COOH); δP (121.5 MHz,
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D2O) 7.6. m/z (ESI) 443.0498 [M − H]−, C16H16N2O11P requires (400 MHz, D2O) 4.05–4.15 (2H, m, H-5′), 4.25–4.31 (1H, m,
443.0497. H-4′), 4.31–4.36 (1H, m, H-3′), 4.45 (1H, t, J = 5.3 Hz, H-2′), 6.00
5-(4-Trifluoromethylphenyl)-uridine-5′-monophosphate (2i). (1H, d, J = 5.3 Hz, H-1′), 6.52 (1H, dd, J = 1.8 and 3.4 Hz,
The triethylammonium salt of the title compound was H-fur), 6.85 (1H, d, J = 3.4 Hz, H-fur), 7.55 (1H, d, J = 1.7 Hz,
obtained as a glassy solid in 63% yield (9.2 mg) from 1 (10 mg, H-fur), 8.15 (1H, s, H-6); δC (100.6 MHz, D2O) 65.0 (d, JC,P
=
22.2 μmol) and 4-trifluoromethylphenylboronic acid according 4.3 Hz), 70.5, 74.3, 84.1 (d, JC,P = 8.4 Hz), 89.4, 108.0, 109.5,
to the general method. δH (400 MHz, D2O) 4.01–4.06 (2H, m, 112.0, 136.0, 143.1, 145.8, 151.3, 162.8; δP (121.5 MHz, D2O) 7.6.
H-5′), 4.24–4.28 (1H, m, H-4′), 4.30–4.36 (1H, m, H-3′), 4.46 m/z (ESI) 389.0397 [M − H]−, C13H14N2O10P requires 389.0392.
(1H, t, J = 5.6 Hz, H-2′), 6.02 (1H, d, J = 5.7 Hz, H-1′), 7.68–7.80
(4H, 2d, J = 8.3 Hz, Ph), 7.98 (1H, s, H-6); δC (75.5 MHz, D2O) triethylammonium salt of the title compound was obtained as
64.6 (d, JC,P = 3.8 Hz, C-5′), 70.6 (C-3′), 74.1 (C-2′), 84.5 (d, JC,P a glassy solid in 56% yield (8.8 mg) from 1 (10 mg, 22.2 μmol)
5-(5-Formylfuran-2-yl)-uridine-5′-monophosphate (2n). The
=
8.3 Hz, C-4′), 89.4 (C-1′), 115.6 (C-5), 126.1 (q, J = 3.8 Hz, C-F3), and 5-formyl-2-furanboronic acid according to the general
129.8, 130.2, 136.3 (C-Ph), 140.1 (C-6), 152.2 (C-2), 157.7 method. δH (400 MHz, D2O) 4.00–4.15 (2H, m, H-5′), 4.24–4.26
(C-Ph), 165.1 (C-4); δP (121.5 MHz, D2O) 7.6. m/z (ESI) 467.0473 (1H, m, H-4′), 4.30–4.38 (1H, m, H-3′), 4.49–4.53 (1H, m, H-2′),
[M − H]−, C16H15F3N2O9P requires 467.0473.
5.97 (1H, d, J = 5.3 Hz, H-1′), 7.16 (1H, d, J = 3.7 Hz, fur), 7.58
5-(5-Methoxy-(3-pyridyl))-uridine-5′-monophosphate (2j). (1H, d, J = 3.7 Hz, fur), 8.40 (1H, s, H-6) 9.47 (1H, s, CHO);
The triethylammonium salt of the title compound was δC (75.5 MHz, D2O) 64.9 (C-5′), 70.4 (C-3′), 74.0 (C-2′), 84.3 (d,
obtained as a glassy solid in 45% yield (6.6 mg) from 1 (10 mg, JC,P = 8.5 Hz, C-4′), 90.1 (C-1′), 106.4, 112.6, 119.0, 138.7 (C-5,
22.2 μmol) and 5-methoxypyridine-3-boronic acid according to C-fur), 140.1 (C-6), 151.4 (C-2), 153.7 (C-fur), 172.3 (C-4), 181.1
the general method. δH (400 MHz, D2O) 3.92 (3H, s, MeO), (CHO); δP (121.5 MHz, D2O) 7.6. m/z (ESI) 417.0336 [M − H]−,
4.01–4.07 (2H, m, H-5′), 4.26–4.28 (1H, m, H-4′), 4.33 (1H, t, J = C14H14N2O11P requires 417.0341.
4.7 Hz, H-3′), 4.46 (1H, t, J = 5.4 Hz, H-2′), 6.01 (1H, d, J =
5-(3-Formylthien-2-yl)-uridine-5′-monophosphate (2o). The
5.5 Hz, H-1′), 7.59 (1H, s, pyr), 7.68–8.12 (2H, m, pyr), 8.00 triethylammonium salt of the title compound was obtained as
(1H, s, H-6); δC (150 MHz, D2O) 56.5, 64.7, 70.5, 74.1, 84.3, a glassy solid in 25% yield (3.5 mg) from 1 (10 mg, 22.2 μmol)
89.5, 129.2, 131.1, 130.8, 135.4, 136.5, 140.1, 144.2, 152.0, and 3-formyl-2-thiopheneboronic acid according to the general
164.9; δP (121.5 MHz, D2O) 7.6. m/z (ESI) 430.0658 [M − H]−, method. δH (400 MHz, D2O) 3.98–4.06 (2H, m, H-5′), 4.24–4.27
C15H17N3O10P requires 430.0657.
(1H, m, H-4′), 4.27–4.32 (1H, m, H-3′), 4.40–4.45 (1H, m, H-2′),
5-(3-Methanesulfonylphenyl)-uridine-5′-monophosphate (2k). 6.00 (1H, m, H-1′), 7.55 (2H, m, Th), 8.13 (1H, s, H-6) 9.67 (1H,
The triethylammonium salt of the title compound was s, CHO); δC (150 MHz, D2O) 64.4 (C-5′), 70.4 (C-3′), 74.2 (C-2′),
obtained as a glassy solid in 58% yield (8.8 mg) from 1 (10 mg, 84.3 (C-4′), 89.6 (C-1′), 107.6 (C-5), 127.0, 128.5, 138.9, 142.3
22.2 μmol) and 3-(methylsulfonyl)phenylboronic acid accord- (C-Th), 144.7 (C-6), 151.8 (C-2), 164.7 (C-4), 188.9 (CHO); δP
ing to the general method. δH (400 MHz, D2O) 3.30 (3H, s, Me), (121.5 MHz, D2O) 7.6. m/z (ESI) 433.0117 [M
−
H]−,
4.00–4.06 (2H, m, H-5′), 4.25–4.27 (1H, m, H-4′), 4.32 (1H, t, J = C14H14N2O10PS requires 433.0112.
4.8 Hz, H-3′), 4.48 (1H, t, J = 5.5 Hz, H-2′), 6.02 (1H, d, J =
5-(5-Acetylthien-2-yl)-uridine-5′-monophosphate (2p). The
5.6 Hz, H-1′), 7.70–8.13 (4H, m, Ph), 8.03 (1H, s, H-6); δC triethylammonium salt of the title compound was obtained as
(75.5 MHz, D2O) 43.8 (Me), 64.6 (C-5′), 70.5 (C-3′), 74.1 (C-2′), a glassy solid in 31% yield (4.1 mg) from 1 (10 mg, 22.2 μmol)
84.4 (d, JC,P = 7.5 Hz, C-4′), 89.5 (C-1′), 115.0 (C-5), 127.3, 127.9, and 5-acetyl-2-thiopheneboronic acid according to the general
130.9, 134.0, 139.4, 139.5 (C-Ph), 140.2 (C-6), 152.1 (C-2), 165.1 method. δH (400 MHz, D2O) 2.60 (3H, s, Me), 4.10–4.18 (2H, m,
(C-4); δP (121.5 MHz, D2O) 7.6. m/z (ESI) 477.0380 [M − H]−, H-5′), 4.29–4.34 (1H, m, H-4′), 4.38 (1H, t, J = 5.7 Hz, H-3′), 4.46
C16H18N2O11PS requires 477.0374.
(1H, t, J = 5.2 Hz, H-2′), 6.00 (1H, d, J = 5.2 Hz, H-1′), 7.60 (1H,
5-(4-Methoxyphenyl)-uridine-5′-monophosphate
(2l). The d, J = 4.2 Hz, Th), 7.90 (1H, d, J = 4.2 Hz, Th), 8.35 (1H, s,
triethylammonium salt of the title compound was obtained as H-6); δC (150 MHz, D2O) 26.5, 64.5, 70.6, 74.7, 84.7,
a glassy solid in 57% yield (15.3 mg) from 1 (20 mg, 49.6 μmol) 89.5, 109.7, 126.0, 136.1, 138.8, 142.4, 143.1, 151.2, 163.5,
and 4-methoxyphenylboronic acid according to the general 196.7; δP (121.5 MHz, D2O) 7.6. m/z (ESI) 447.0263 [M − H]−,
method. δH (300 MHz, D2O) 4.02–4.14 (2H, m, H-5′), 4.23–4.28 C15H16N2O10PS requires 447.0269.
(1H, m, H-4′), 4.32 (1H, t, J = 4.6, H-3′), 4.42 (1H, t, J = 5.1,
5-(5-Formylthien-2-yl)-uridine-5′-monophosphate (2q). The
H-2′), 5.98 (1H, d, J = 5.6 Hz, H-1′), 7.02, 7.44 (4H, 2d, J = 8.0 triethylammonium salt of the title compound was obtained as
and 8.0 Hz, Ph), 7.76 (1H, s, H-6); δC (100.6 MHz, D2O) 55.9, a glassy solid in 61% yield (8.6 mg) from 1 (10 mg, 22.2 μmol)
65.1 (d, JC,P = 4.7 Hz), 70.6, 74.1, 84.1 (d, JC,P = 8.6 Hz), 89.2, and 5-formyl-2-thiopheneboronic acid according to the general
114.6, 116.1, 124.9, 130.5, 138.2, 151.9, 159.3, 165.2; δP method. δH (400 MHz, D2O) 4.12–4.20 (2H, m, H-5′), 4.29–4.31
(121.5 MHz, D2O) 3.9. m/z (ESI) 429.0701 [M
C16H18N2O10P requires 429.0705.
−
H]−, (1H, m, H-4′), 4.38 (1H, t, J = 4.7 Hz, H-3′), 4.45 (1H, t, J =
5.0 Hz, H-2′), 5.98 (1H, d, J = 5.0 Hz, H-1′), 7.66 (1H, d, J =
5-(Furan-2-yl)-uridine-5′-monophosphate (2m). The triethyl- 4.0 Hz, Th), 7.94 (1H, d, J = 4.0 Hz, Th), 8.38 (1H, s, H-6) 9.75
ammonium salt of the title compound was obtained as a (1H, s, CHO); δC (75.5 MHz, D2O) 64.7 (d, JC,P = 4.5 Hz, C-5′),
glassy solid in 57% yield (14.6 mg) from 1 (20 mg, 49.6 μmol) 70.4 (C-3′), 74.9 (C-2′), 84.4 (C-4′), 89.7 (C-1′), 110.0, 125.9,
and 2-furanboronic acid according to the general method. δH 139.1, 140.1, 142.0 (C5 + C-Th), 144.9 (C-6), 152.7 (C-2), 163.5
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(C-4), 187.8 (CHO); δP (121.5 MHz, D2O) 7.6. m/z (ESI) 433.0107 d, J = 4.6 Hz, H-1′), 8.08 (1H, s, H-6); δC (75.5 MHz, D2O) 65.5
[M − H]−, C14H14N2O10PS requires 433.0112.
(C-5′), 69.2 (C-5), 70.3 (C-3′), 74.4 (s, C-2′), 84.1 (d, JC,P = 8.5 Hz,
5-[(5-Formaldoximyl)-thien-2-yl]-uridine-5′-monophosphate C-4′), 89.3 (C-1′), 146.7 (C-6), 152.4 (C-2), 163.9 (C-4); δp
(2r). To an aqueous solution (1 mL) of 2q (7 mg, 13 μmol) and (121.5 MHz, D2O) −6.6 (d, JP,P = 23.1 Hz), −11.2 (d, JP,P = 23.1
hydroxylamine hydrochloride (1.1 mg, 16 μmol), a solution of Hz). m/z (ESI) 528.8912 [M − H]−, C9H12IN2O12P2 requires
sodium bicarbonate (1.4 mg, 0.16 mmol) in water (1 mL) was 528.8916.
added dropwise over 1 minute with stirring. The reaction was
5-Iodouridine-5′-triphosphate (4).24 5-Iodouridine-5′-mono-
stirred at room temperature for 3 h, and the solvent was phosphomorpholidate (81 mg, 0.156 mmol) was prepared as
removed in vacuo. The residue was purified by ion-pair chrom- described for 3 and repeatedly (3×) co-evaporated with pyri-
atography on a Lichroprep RP-18 resin, using a gradient of dine. To the white solid was added potassium pyrophosphate
aqueous TEAB (0.05 M, pH 7.3) against 0–25% methanol over (280 mg, 0.628 mmol) and tributylamine (303 µL, 1.26 mmol)
a total volume of 400 mL (flow rate: 2 mL min−1). Anti-2r and the mixture was further co-evaporated (3×) with pyridine.
eluted first, followed closely by syn-2r. The respective product- Tetrazole (55 mg, 0.785 mmol) and dry DMF (5 mL) were
containing fractions were combined and freeze-dried at least added to the dry mixture and the reaction was left stirring for
twice to remove excess TEAB. Thus, each isomer was obtained 5 h at room temperature. The crude mixture was concentrated
as a yellow residue in yields of, respectively, 19% (1.5 mg, under reduced pressure and isolated using purification
2 μmol, anti-2r) and 21% (3.4 mg, 3 μmol, syn-2r). Anti-2r: δH method 1. The triethylammonium salt of the title compound
(400 MHz, D2O) 3.98 (2H, t, J = 4.6 Hz, H-5′); 4.20–4.23 (1H, m, (3.2 equiv.) was obtained as a glassy solid in 60% yield
H-4′), 4.33 (1H, dd, J = 4.4 and 5.3 Hz, H-3′), 4.46 (1H, t, J = (86.8 mg). δH (400 MHz, D2O) 4.20–4.26 (2H, m, H-5′),
5.7 Hz, H-2′), 6.04 (1H, d, J = 5.8 Hz, H-1′), 7.52 (1H, d, J = 4.26–4.30 (1H, m, H-4′), 4.37–4.44 (2H, m, H-3′, H-2′), 5.94 (1H,
4.1 Hz, Th), 7.54 (1H, d, J = 4.1 Hz, Th), 7.85 (1H, s, H-oxime), d, J = 4.9 Hz, H-1′), 8.27 (1H, s, H-6); δC (75.5 MHz, D2O) 65.7
8.17 (1H, s, H-6); δP (162 MHz, D2O) 0.45. Syn-2r: δH (400 MHz, (d, JC,P = 6.1 Hz, C-5′), 69.2 (C-5), 70.4 (C-3′), 74.4 (s, C-2′), 84.3
D2O) 3.98 (2H, t, J = 4.6 Hz, H-5′); 4.20–4.23 (1H, m, H-4′), 4.33 (d, J = 9.1 Hz, C-4′), 89.1 (C-1′), 146.7 (C-6), 152.4 (C-2), 164.0
(1H, dd, J = 4.4 and 5.3 Hz, H-3′), 4.46 (1H, t, J = 5.7 Hz, H-2′), (C-4); δp (121.5 MHz, D2O) −6.5 (d, JP,P = 20.7 Hz), −11.6 (d,
6.03 (1H, d, J = 5.7 Hz, H-1′), 7.31 (1H, d, J = 4.0 Hz, Th), 7.49 JP,P = 19.4 Hz), −22.6 (d, JP,P = 20.7 Hz). m/z (ESI) 608.8588
(1H, d, J = 4.0 Hz, Th), 8.17 (1H, s, H-6), 8.35 (1H, s, H-oxime); [M − H]−, C9H13IN2O15P3 requires 608.8579.
δP (162 MHz, D2O) 0.45. Anti-2r/syn-2r: δC (100 MHz, D2O) 59.0
5-Phenyluridine-5′-diphosphate (5a). The triethylammo-
(CH2), 63.7 (CH2), 70.2 (CH), 73.5 (CH), 83.6 (CH), 83.7 (CH), nium salt of the title compound (1.9 equiv.) was obtained as a
88.9 (CH), 110.3, 118.4, 123.3, 130.9, 133.4, 136.8, 142.6, 147.2, glassy solid in 73% yield (10.6 mg) from 3 (11.6 mg, 20.8 μmol)
161.2, 163.0, 163.5, 186.3, 190.3, 198.4. m/z (ESI) 448.0222 and phenylboronic acid according to the general method. δH
[M − H]−, C14H15N3O10PS requires 448.0221.
(400 MHz, D2O) 4.15–4.19 (2H, m, H-5′), 4.27–4.31 (1H, m,
5-Iodouridine-5′-diphosphate (3).23 1 (292 mg, 0.65 mmol) H-4′), 4.38–4.43 (1H, m, H-3′), 4.47 (1H, t, J = 5.6 Hz, H-2′), 6.05
was converted into the corresponding phosphoromorpholidate (1H, d, J = 5.8 Hz, H-1′), 7.40–7.58 (5H, m, phenyl), 7.88 (1H, s,
as previously described,11 affording 354 mg of 5-iodo UMP H-6); δC (75.4 MHz, D2O) 65.7 (d, JC,P = 3.7 Hz, C-5′), 70.5 (C-3′),
phosphoromorpholidate as a colourless powder (99% yield). δH 73.9 (C-2′), 84.1 (d, JC,P = 5.0 Hz, C-4′), 89.1 (C-1′), 118.9 (C-5),
(400 MHz, D2O) 3.04–3.16 (4H, m, morph), 3.63–3.73 (4H; m, 129.1, 129.4, 129.5, 133.8 (C-Ph), 139.2 (C-6), 152.3 (C-2), 165.5
morph), 3.98–4.15 (2H, m, H-5′), 4.24–4.26 (1H, m, H-4′), (C-4); δP (121 MHz, D2O) −6.6 (d, JP,P = 23.1 Hz), −11.1 (d, JP,P
=
4.27–4.32 (1H, m, H-3′), 4.37 (1H, t, J = 5.3 Hz, H-2′), 5.94 (1H, 23.1 Hz). m/z (ESI) 239.0096 [M − 2H]2−, C15H16N2O12P2
d, J = 5.3 Hz, H-1′), 8.18 (1H, s, H-6); δC (75.5 MHz, D2O) 44.2 requires 239.0095.
(morph), 63.4 (d, JC,P = 5.3 Hz, C-5′), 66.3 (morph), 68.0 (C-5),
5-(4-Methoxyphenyl)uridine-5′-diphosphate
(5b). The
68.6 (C-3′), 73.2 (C-2′), 83.0 (d, JC,P = 8.5 Hz, C-4′), 88.2 (C-1′), triethylammonium salt of the title compound (1.9 equiv.) was
145.0 (C-6), 153.0 (C-2), 161.9 (C-4); δP (121.5 MHz, D2O) 11.0. obtained as a glassy solid in 50% yield (7.2 mg) from 3
m/z (ESI) 519.9968 [M + H]+, C13H19IN3O9P requires 519.9976. (11.0 mg, 20.8 μmol) and 4-methoxyphenylboronic acid accord-
5-Iodo UMP phosphoromorpholidate (50 mg, 96 µmol, sodium ing to the general method. δH (400 MHz, D2O) 3.87 (3H, s,
salt form) was repeatedly (3×) co-evaporated with pyridine. MeO), 4.16–4.22 (2H, m, H-5′), 4.26–4.31 (1H, m, H-4′), 4.41
KH2PO4 (34 mg, 191 µmol) and tributylamine (92 µL, (1H, m, H-3′), 4.47 (1H, t, J = 5.6 Hz, H-2′), 6.04 (1H, d, J =
382 mmol) were added, and the mixture was repeatedly (3×) 5.8 Hz, H-1′), 7.07 (2H, d, J = 8.8 Hz, Ph), 7.49 (2H, d, J =
co-evaporated with pyridine to remove excess tributylamine. 8.8 Hz, Ph), 7.83 (1H, s, H-6); δC (125 MHz, D2O) 55.9 (MeO),
Tetrazole (33 mg, 470 µmol) and dry DMF (5 mL) were added 65.7 (d, JC,P = 3.0 Hz, C-5′), 70.5 (C-3′), 73.9 (C-2′), 82.6 (C-4′),
to the dry mixture. The reaction was left stirring for 5 h at 89.0 (C-1′), 114.9, 116.5, 125.1, 130.8 (C-5 + C-Ph), 138.5 (C-6),
room temperature. The crude mixture was concentrated under 152.3 (C-2), 159.6 (C-Ph), 166.1 (C-4); δP (121 MHz, D2O) −6.6
reduced pressure and isolated using purification method (d, JP,P = 23.1 Hz), −11.1 (d, JP,P = 23.1 Hz). m/z (ESI) 254.0149
1. The triethylammonium salt of the title compound [M − 2H]2−, C16H18N2O13P2 requires 254.0148.
(1.0 equiv.) was obtained as a glassy solid in 66% yield
5-(Furan-2-yl)uridine-5′-diphosphate
(5c). The
triethyl-
(40.2 mg). δH (400 MHz, D2O) 4.00–4.05 (2H, m, H-5′), ammonium salt of the title compound (1.9 equiv.) was obtained
4.10–4.15 (1H, m, H-4′), 4.20–4.25 (2H, m, H-3′,H-2′), 5.77 (1H, as a glassy solid in 60% yield (8.6 mg) from 3 (11.0 mg,
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20.8 μmol) and 2-furanboronic acid according to the general method A. δH (400 MHz, D2O) 4.23–4.30 (2H, m, H-5′),
method. δH (400 MHz, D2O) 4.20–4.26 (2H, m, H-5′), 4.29–4.33 4.31–4.33 (1H, m, H-4′), 4.45–4.52 (2H, m, H-2′ and H-3′), 6.06
(1H, m, H-4′), 4.44 (1H, t, J = 5.4 Hz, H-3′), 4.49 (1H, t, J = (1H, d, J = 5.2 Hz, H-1′), 6.54 (1H, d, J = 1.5 Hz, fur), 6.89 (1H,
5.4 Hz, H-2′), 6.04 (1H, d, J = 5.5 Hz, H-1′), 6.54 (1H, dd, J = d, J = 3.1 Hz, fur), 7.59 (1H, s, fur), 8.23 (1H, s, H-6); δC
1.8 and 3.4 Hz, fur), 6.89 (1H, d, J = 3.3 Hz, fur), 7.59 (1H, d, (125 MHz, D2O) 65.9 (d, JC,P = 4.3 Hz, C-5′), 70.4 (C-3′),
J = 1.1 Hz, fur), 8.21 (1H, s, H-6); δC (75.5 MHz, D2O) 65.9 74.2 (C-2′), 84.4 (d, JC,P = 7.6 Hz, C-4′), 89.0 (C-1′), 108.2
(C-5′), 70.7 (C-3′), 74.5 (C-2′), 84.4 (C-4′), 89.5 (C-1′), 108.4 (C-5), (C-5), 109.6 (fur4), 112.1 (fur3), 136.4 (C-6), 143.5 (fur2),
109.9 (fur4), 112.4 (fur3), 136.6 (C-6), 143.7 (fur2), 146.3 (fur1), 146.0 (fur1), 151.7 (C-2), 163.3 (C-4); δP (121 MHz, D2O) −6.5
152.0 (C-2), 163.6 (C-4); δP (121 MHz, D2O) −6.5 (d, JP,P = 23.1 (d, JP,P = 20.7 Hz), −11.5 (d, JP,P = 19.7 Hz), −22.6 (d, JP,P
=
Hz), −11.0 (d, JP,P = 23.1 Hz). m/z (ESI) 233.9991 [M − 2H]2−
,
20.1 Hz). m/z (ESI) 548.9728 [M − H]−, C13H16N2O16P3 requires
C13H14N2O13P2 requires 233.9991.
548.9718.
5-(5-Formylthien-2-yl)uridine-5′-diphosphate (5d). The
5-(5-Formylthien-2-yl)uridine-5′-triphosphate (6d). The
triethylammonium salt of the title compound (1.6 equiv.) was triethylammonium salt of the title compound (3.4 equiv.) was
obtained as a glassy solid in 77% yield (8.2 mg) from 3 obtained as a glassy solid in 67% yield (16.9 mg) from 4
(14.0 mg, 26.4 μmol) and 5-formyl-2-thiopheneboronic acid (16 mg, 26.3 μmol) and 5-formyl-2-thiopheneboronic acid
according to the general method. δH (400 MHz, D2O) 4.26–4.32 according to the general method. δH (400 MHz, D2O) 4.30–4.44
(2H, m, H-5′), 4.32–4.35 (1H, m, H-4′), 4.43–4.50 (2H, m, H-2′ (3H, m, H-5′, H-4′), 4.44–4.51 (2H, m, H-2′ and H-3′), 6.04 (1H,
and H-3′), 6.01–6.07 (1H, m, H-1′), 7.71–7.75 (1H, m, Th), s, H-1′), 7.73 (1H, s, Th), 8.01 (1H, s, Th), 8.45 (1H, s, H-6); 9.78
7.99–8.01 (1H, m, Th), 8.45 (1H, s, H-6); 9.78 (1H, s, CHO); δC (1H, s, CHO); δC (75.5 MHz, D2O) 65.9 (C-5′), 70.4 (C-3′), 75.0
(75.5 MHz, D2O) 65.5 (C-5′), 70.2 (C-3′), 74.9 (C-2′), 84.2 (C-4′), (C-2′), 84.5 (C-4′), 89.5 (C-1′), 101.5, 109.7, 126.1, 139.2, 140.6,
89.7 (C-1′), 109.3 (C-5), 125.8, 138.9, 140.3, 142.0, 144.8 (C-6 + 142.2, 145.0 (C-Th + C6 + C-5 + C-2), 163.6 (C-4), 188.0 (CHO);
C-Th), 151.1 (C-2), 163.3 (C-4), 187.8 (CHO); δP (121 MHz, D2O) δP (121 MHz, D2O) −6.5 (d, JP,P = 20.7 Hz), −11.5 (d, JP,P
=
−6.5 (d, JP,P = 23.1 Hz), −11.0 (d, JP,P = 23.1 Hz). m/z (ESI) 19.7 Hz), −22.6 (d, JP,P = 20.1 Hz). m/z (ESI) 592.9448 [M − H]−,
512.9776 [M − H]−, C14H15N2O13P2S1 requires 512.9776.
5-Phenyluridine-5′-triphosphate (6a). The triethylammo-
C14H16N2O16P3 requires 592.9439.
5-(5-Formylthien-2-yl)-uridine (7). The title compound was
nium salt of the title compound (3.2 equiv.) was obtained as a obtained from 5-iodouridine (100 mg, 270 μmol)22 and
glassy solid in 85% yield (34.2 mg) from 4 (20 mg, 33.8 μmol) 5-formyl-2-thiopheneboronic acid according to the general
and phenylboronic acid according to the general method. δH method in 23% yield (16 mg). δH (400 MHz, DMSO-d6)
(400 MHz, D2O) 4.17–4.27 (2H, m, H-5′), 4.29–4.31 (1H, m, 3.65–3.85 (2H, m, H-5′), 3.90–3.95 (1H, m, H-4′), 4.02–4.17 (2H,
H-4′), 4.43–4.52 (2H, m, H-2′ and H-3′), 6.06 (1H, d, J = 5.5 Hz, m, H-2′ and H-3′), 5.11 (1H, d, J = 5.9 Hz, OH-3′), 5.54 (1H, d,
H-1′), 7.40–7.57 (5H, m, Ph), 7.92 (1H, s, H-6); δC (125 MHz, J = 5.0 Hz, OH-2′), 5.60 (1H, t, J = 4.3 Hz, OH-5′), 5.80 (1H, d,
D2O) 66.0 (C-5′), 70.7 (C-3′), 74.1 (C-2′), 84.3 (C-4′), 88.9 J = 3.0 Hz, H-1′), 7.56 (1H, d, J = 4.1 Hz, Th), 7.93 (1H, d, J =
(C-1′), 117.0 (C-5), 129.2, 129.5, 129.6, 132.4, (C-Ph), 4.0 Hz, Th), 9.02 (1H, s, H-6), 9.87 (1H, s, CHO), 11.9 (1H, s,
139.2 (C-6), 152.4 (C-2), 165.5 (C-4) ; δP (121 MHz, D2O) −6.5 NH); δC (75.5 MHz, DMSO-d6) 59.6 (C-5′), 68.7 (C-3′), 74.5
(d, JP,P = 20.7 Hz), −11.1 (d, JP,P = 19.7 Hz), −22.6 (d, JP,P
=
(C-2′), 84.4 (C-4′), 89.5 (C-1′), 106.9 (C-5), 122.9, 137.3, 138.7,
20.7 Hz). m/z (ESI) 558.9935 [M − H]−, C15H18N2O15P3 requires 141.6, 144.4, 149.4 (C-2 + C-6 + C-Th), 161.4 (C-4), 184.3 (CHO).
558.9926.
m/z (ESI) 353.0448 [M − H]−, C14H13N2O7S requires 353.0449.
5-(4-Methoxyphenyl)uridine-5′-triphosphate
triethylammonium salt of the title compound (3.0 equiv.) was
(6b). The
Photophysical characterisation
obtained as a glassy solid in 53% yield (12.1 mg) from 4
Absorbance and fluorescence spectra. UV absorbance
(10.9 mg, 17.9 μmol) and 4-methoxyphenylboronic acid accord- spectra were recorded in H2O on a PerkinElmer Lambda
ing to the general method. δH (400 MHz, D2O) 3.87 (3H, s, 25 UV-Vis spectrometer at ambient temperature in FarUV
MeO), 4.16–4.28 (2H, m, H-5′), 4.29–4.32 (1H, m, H-4′), 4.44 quartz cells (path length 1.0 cm). Fluorescence spectra were
(1H, t, J = 5.4 Hz, H-3′), 4.49 (1H, t, J = 5.4, H-2′), 6.06 (1H, d, recorded in H2O on a PerkinElmer LS-45 spectrometer at
J = 6.0 Hz, H-1′), 7.07 (2H, d, J = 8.9 Hz, Ph), 7.50 (2H, d, J = ambient temperature in a quartz micro fluorescence cell (path
8.8 Hz, Ph), 7.86 (1H, s, H-6); δC (75.5 MHz, D2O) 55.9 length 1.0 cm).
(MeO), 65.9 (C-5′), 70.6 (C-3′), 73.9 (C-2′), 84.2 (d, JC,P = 8.8 Hz,
Quantum yields. Nucleotide derivatives were serially diluted
C-4′), 88.7 (C-1′), 114.9, 116.6, 125.1, 130.8, 138.4, 152.3, in H2O (10, 20, 30, 40 and 50 μM for absorbance measure-
159.6, 165.6 (C-2, C-4, C-6, C-5, C-Ph); δP (121 MHz, D2O) −6.5 ments, and 0.2, 0.4, 0.6, 0.8, 1 μM for fluorescence measure-
(d, JP,P = 20.7 Hz), −11.5 (d, JP,P = 19.7 Hz), −22.7 (d, JP,P = 20.1 ments), and UV absorbance and fluorescence emission (with
Hz). m/z (ESI) 589.0040 [M − H]−, C16H20N2O16P3 requires λmax absorbance = λem fluorescence) were recorded for all
589.0031.
samples. To determine quantum yields, for each absorbance
5-(Furan-2-yl)uridine-5′-triphosphate
(6c). The
triethyl- and fluorescence spectrum the area under the curve (AUC) was
ammonium salt of the title compound (3.0 equiv.) was obtained calculated by numerical integration, applying the mid-point
as a glassy solid in 74% yield (11.4 mg) from 4 (10.9 mg, rule. For each compound, AUCabs and AUCem were then
17.9 μmol) and 2-furanboronic acid according to general plotted over compound concentration according to AUCabs
=
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A × [conc] + B and AUCem = A′ × [conc] + B′. From these were provided by the Imperial College High Performance
linear plots, the gradients A and A′ were extracted, and for Computing Service. We are grateful to Prof. Monica Palcic
each compound the specific quantum yield Φs, under these (Copenhagen) for making the α-1,3-GalT clone available, and
experimental conditions, was calculated as the ratio A′/A. for help with enzyme expression.
Quantum yields determined with this protocol for two refer-
ence compounds, 2-aminopyridine (Φs 0.60) and L-tryptophan
(Φs 0.14) were in agreement with literature values.25 The
quantum yields for reference compounds were used to calcu-
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Acknowledgements
We thank the EPSRC (First Grant EP/D059186/1) and the MRC
(Grant no. 0901746), for funding and the EPSRC National
Mass Spectrometry Facility, Swansea, for the recording of mass
spectra. The computational resources for the G09 calculations
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