An uracil-linked hydroxyflavone probe for the recognition of ATP

Article abstract of DOI:10.3762/bjoc.14.63

Background: Nucleotides are essential molecules in living systems due to their paramount importance in various physiological processes. In the past years, numerous attempts were made to selectively recognize and detect these analytes, especially ATP using

Full text of DOI:10.3762/bjoc.14.63

An uracil-linked hydroxyflavone probe for the
recognition of ATP
Márton Bojtár1, Péter Zoltán Janzsó-Berend1, Dávid Mester2, Dóra Hessz3,
Mihály Kállay2, Miklós Kubinyi3,4 and István Bitter*1
Full Research Paper
Open Access
Address:
Department of Organic Chemistry and Technology, Budapest
University of Technology and Economics, 1521 Budapest, Hungary,
MTA-BME Lendület Quantum Chemistry Research Group,
Beilstein J. Org. Chem. 2018, 14, 747–755.
1
doi:10.3762/bjoc.14.63
2
Received: 20 November 2017
Accepted: 13 March 2018
Published: 03 April 2018
Department of Physical Chemistry and Materials Science, Budapest
University of Technology and Economics, 1521 Budapest, Hungary,
3
Institute of Materials and Environmental Chemistry, Research Center
for Natural Sciences, Hungarian Academy of Sciences, P. O. Box
86, 1519 Budapest, Hungary and 4Department of Physical
Associate Editor: N. Sewald
2
Chemistry and Materials Science, Budapest University of Technology
and Economics, 1521 Budapest, Hungary
© 2018 Bojtár et al.; licensee Beilstein-Institut.
License and terms: see end of document.
Email:
István Bitter* - bitter@oct.bme.hu
*
Corresponding author
Keywords:
ATP sensing; base-pairing; fluorescent probes; 3-hydroxyflavone;
nucleotide recognition
Abstract
Background: Nucleotides are essential molecules in living systems due to their paramount importance in various physiological
processes. In the past years, numerous attempts were made to selectively recognize and detect these analytes, especially ATP using
small-molecule fluorescent chemosensors. Despite the various solutions, the selective detection of ATP is still challenging due to
the structural similarity of various nucleotides. In this paper, we report the conjugation of a uracil nucleobase to the known
4
’-dimethylamino-hydroxyflavone fluorophore.
Results: The complexation of this scaffold with ATP is already known. The complex is held together by stacking and electrostatic
interactions. To achieve multi-point recognition, we designed the uracil-appended version of this probe to include complementary
base-pairing interactions. The theoretical calculations revealed the availability of multiple complex structures. The synthesis was
performed using click chemistry and the nucleotide recognition properties of the probe were evaluated using fluorescence spectros-
copy.
Conclusions: The first, uracil-containing fluorescent ATP probe based on a hydroxyflavone fluorophore was synthesized and eval-
uated. A selective complexation with ATP was observed and a ratiometric response in the excitation spectrum.
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Beilstein J. Org. Chem. 2018, 14, 747–755.
Introduction
Nucleotides play essential roles in various physiological pro- DMHF was found to form 1:1 and 1:2 complexes with ATP.
cesses, such as energy transportation [1], DNA synthesis [2] The two components were held together by π-stacking and by
and cell signaling events [3]. Especially, adenosine-5’-triphos- electrostatic interactions of the positively polarized dimethyl-
phate (ATP) is vital, since it is the main energy source in living amino group of DMHF and the negative charges of ATP. The
systems [4]. The recognition and sensing of ATP has therefore interaction of DMHF with nucleotides and a computer aided
paramount importance in the understanding of biological pro- simulation on the geometry of DMHF∙ATP complexes in vacuo
cesses. Amongst the numerous solutions [5-9], fluorescent was also reported. A recent study by the same authors describe
chemosensors using either indicator displacement assays further flavones with various association constants [45], howev-
[
10-15], cation-based recognition units [16-20], metal-com- er, none of them are the result of rational design.
plexes [21-27] and other direct sensing systems [28-30] have
significant advantages over the classical, separation-based Base pairing is a well-known phenomenon in the double helix
methods. The primary difficulties in the design of an ATP structure of DNA since the work of Watson and Crick. It is also
chemosensor are the structural similarity of ATP to other known that the cohesion of the double strand is provided by the
nucleotides (i.e., to guanosine-5’-triphosphate, GTP) and the efficient π-stacking interaction [46]. Adding additional recogni-
strong solvation of the chemosensor and the analyte in aqueous tion sites to existing nucleotide receptor molecules can lead to
media, reducing the association constant of their complex, and multi-point recognition and enhanced selectivity/sensitivity for
through that the sensitivity of the sensor [31,32]. The molecular ATP chemosensors. In our ongoing research, we are interested
recognition of nucleotides in most chemosensors is achieved by in the exploration of the function of complementary base-
charged recognition sites [16,18,19,33], or Zn-dipicolylamine pairing in ATP recognition as a possible way to enhance the
complexes [21,23,24] attracting the negatively charged phos- selectivity. Since ATP has one adenine nucleobase, a simple
phate units of ATP and by π-stacking between the fluorophores uracil/thymine unit appended to a neutral chemosensor oper-
of the sensors and the adenine moiety of ATP [29]. In aqueous ating mainly through π-interaction could be a good model for
solutions at physiological pH, the tetra-charged anionic ATP investigation. We selected DMHF as the fluorophore and core
consists of a hydrophilic (phosphate and ribose) and a more scaffold because of its easy synthesis and ratiometric fluores-
hydrophobic part (adenine). The former ensures a good solu- cent nature. First, we examined the possible structure and the
bility of ATP in water and generates an electrostatic field supramolecular interactions by quantum chemical calculations
around it while the latter is required for associations with simi- of our target compound, UHF (uracil-hydroxyflavone) and ATP
lar planar hydrophobic molecules involved in the biochemical (see Figure 1 for structures). The theoretical results indicated
processes of ATP [34]. The ideal ATP probe possessing all the the possibility of base-pairing interactions, which prompted us
prerequisites to bind ATP should be sensitive to electrostatic towards the synthesis of UHF by click chemistry. Fluorescence
fields in solution and in molecular assemblies as well. spectroscopy revealed a selective complexation with ATP with
3
-Hydroxyflavone (HF) fluorophores, especially the highly an association constant of around 2∙104 M−1 and a ratiometric
polarizable 4’-dialkylamino subfamily exhibit strong sensitivity response in the excitation spectrum.
to electric fields generated by ions and molecules in solution.
Results and Discussion
This property along the ESIPT process (excited state intramo-
lecular proton transfer) [35] makes them ideal for ratiometric Structure and calculations
environment-sensitive probes and sensors [36-43]. Among them Based on the detailed investigation of the supramolecular struc-
the 4’-dimethylamino derivative (DMHF, 4’-dimethylaminohy- ture of the DEHF∙ATP (4’-diethylaminohydroxyflavone) com-
droxyflavone) was utilized by Pivovarenko and co-workers in plex (see below), we envisioned the uracil group to be appended
ATP sensing in aqueous solution and in mitochondria [34,44]. on the A ring in close proximity to the nucleobase. In addition,
Figure 1: Structures of the studied hydroxyflavone derivatives.
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Beilstein J. Org. Chem. 2018, 14, 747–755.
some flexibility was required to obtain the proper conformation
for the possible H-bonding. From the synthetic point of view
the introduction of an uracil moiety can reasonably be accom-
plished at position 7 of DMHF with the appropriate C5-functio-
nalized uracil derivative. The synthetic accessibility and the
short spacer unit between the fluorophore and nucleobase were
the main aspects in the design of UHF.
The influence of the side arm with the uracil group on the
ability of UHF to act as an ATP sensor was investigated first by
theoretical calculations, in which the structures and energies for
the UHF∙ATP complex and – as a reference – for the
DEHF∙ATP complex were computed.
Foremost, a molecular mechanical (MM) conformation analy-
sis was performed for the individual molecules using the
MMFF force field [47]. The stable conformers were optimized
further at the density functional theory (DFT) level using the
PBE functional [48,49] with D3BJ dispersion correction [50,51]
and the 6-31G basis set [52]. Subsequently, the geometries ob-
tained were utilized for generating the initial structures of the
complexes, which were optimized with the same functional and
basis set. To mimic the experimental conditions all the DFT
calculations were performed using the polarized continuum
model (PCM) [53] with water as solvent. The Marvin [54],
ORCA [55], and MRCC [56] packages were used, respectively,
for the MM, DFT, and local CC calculations.
In the of case of the DEHF∙ATP complex one stable structure
was found, while regarding the UHF∙ATP complex there were
two. The optimized geometries are presented in Figure 2.
The investigation of the DEHF∙ATP 1:1 complex revealed a
sandwich structure, which is held together by a π–π stacking-
and an H-bond interaction. The triphosphate group is posi-
tioned near ring C of the DEHF and the π-stacking occurs be-
tween the adenine group and rings A and B of the flavone. The
Figure 2: Optimized geometries for (a) DEHF∙ATP, (b) UHF∙ATP with
the adenine of ATP “sandwiched” between the uracil and flavone units
and (c) UHF∙ATP with hydrogen bonds between the uracil and the
adenine moieties.
alignment of the flavones and ATP components is similar as in energetically more favorable, high accuracy local coupled-
DMHF∙ATP, obtained by a computer aided simulation [34].
cluster (CC) calculations were performed. The complexation
energies were computed using the local CC singles and doubles
For the UHF∙ATP complex, two structurally rather different with perturbative triples [CCSD(T)] method [57] and the aug-
stable conformers were found in the calculations. In the first cc-pVTZ basis set [58]. In the matter of the “double-sandwich”
case (Figure 2b), the structure of the complex is similar to the complex the complexation energy is 39 kcal/mol, while
DEHF∙ATP complex. However, an additional π-stacking inter- regarding the complex which is stabilized through the base-pair
action is formed between the adenine and the uracil groups interactions 42 kcal/mol is obtained. The energies mentioned
creating a so-called “double-sandwich” structure. In the other above are in accordance with the number and strength of the
case, the π–π interactions vanish and the complex is stabilized H-bonding and π-stacking interactions. Based on the numerical
through the base-pair interactions (Figure 2c). Two H-bonds are results, it can be stated that the formation of the base-pair inter-
formed between the adenine and the uracil groups, and two ad- actions further stabilizes the complex, and this structure is ener-
ditional H-bonds between ring B of the flavone and the adenine getically more favorable. These results prompted us towards the
group also stabilize the structure. To decide which structure is synthesis and evaluation of this promising molecule.
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Beilstein J. Org. Chem. 2018, 14, 747–755.
Synthesis
The synthesis of UHF is depicted in Scheme 1.
did not alter the spectra significantly, but provided a stable solu-
tion suitable for absorption and fluorescence titration experi-
ments. The fluorescence spectra of UHF in the presence of ATP
UHF was synthesized by the CuAAC (click) reaction of in different concentrations are shown in Figure 3.
-propargyloxy-3-hydroxyflavone 3 and 5-azidomethyluracil
4) [59]. The hydroxyflavone was prepared according to the Upon addition of ATP, a new band appears at 440 nm in the ex-
7
(
standard literature process for the preparation of these com- citation spectra. This feature can be attributed to the specific
pounds [60]: the substituted hydroxyacetophenone 1 [61] was intermolecular proton transfer from the hydroxy group of the
condensed to the corresponding chalcone using strongly basic flavone to the phosphate moiety of the ATP [34]. The fluores-
conditions and reacted with alkaline hydrogen peroxide to cence enhancement is remarkable upon excitation at 470 nm –
obtain the clickable fluorophore. All new compounds were a 7-fold increase can be observed using this excitation wave-
characterized by NMR and high-resolution mass spectrometry. length. No significant changes were recorded in the absorption
spectra (Figure S15 in the Supporting Information File 1) using
Optical spectroscopy
a 10 cm path length cuvette. The association constant was
The solubility of the UHF probe was very poor in water which calculated for 1:1 and 1:2 complexes, a better fit was obtained
resulted in the decrease of fluorescence over time upon dilution for the unimolar complexation corroborating the theoretical
from the stock solution in DMSO. The addition of γ-cyclo- results. The value was determined to be 2.3 ± 0.2∙104 M−1 using
dextrin as a solubilizer to the samples (HEPES buffer, pH 7.4) non-linear curve fitting analysis from multiple titration experi-
Scheme 1: Synthesis of UHF. (i) 4-Dimethylaminobenzaldehyde, DMF, NaOMe, rt, 17 h, (ii) hydrogen peroxide, NaOH, ethanol, rt, 24 h,
(iii) 5-azidomethyluracil, [Cu(MeCN)4]BF4, THF, rt, 24 h.
Figure 3: Variation of fluorescence spectra of UHF (1.0 μM) upon addition of increasing amounts of ATP in 0.02 M HEPES buffer which also contains
.1 mM γ-cyclodextrin. Left: excitation spectra, detection wavelength: 540 nm; right: emission spectra, excitation wavelength: 470 nm. The inset
shows the emission at 540 nm as a function of the ATP concentration, whereas the curve represents the result of a non-linear fitting to the spectra.
0
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Beilstein J. Org. Chem. 2018, 14, 747–755.
ments. The various flavones tested in [45] had association con-
stants in the range of 0.3–3∙103 M−1, considerably lower values.
The effect of γ-cyclodextrin was examined in some preliminary
experiments. Lowering the concentration to 0.05 mM resulted
in unreliable spectra due to possible precipitation of the com-
plex. A higher cyclodextrin concentration resulted in the same
fluorescence response with an association constant of
5
.7∙104 M−1, however, the exact effect of various sized cyclo-
dextrins on the complexation of flavones with ATP is currently
unknown and will be the subject of an upcoming study by our
group.
To ascertain this method of signal transduction, the spectra of
UHF and UHF∙ATP were recorded at different pH values
(
Figure 4). Since the pKa value of the hydroxy group of the
flavones is around 9 [54], no significant deprotonation should
occur at pH 7.8. As can be seen in Figure 4, the excitation band
of UHF around 400 nm is the same at each pH value. Upon ad-
dition of ATP, the most significant enhancement was recorded
at pH 7.8, the pronounced band around 450 nm indicates the
Figure 5: Fluorescence enhancement (F/F0) values of UHF (1.0 μM)
upon addition of different nucleotides at 540 nm (excitation: 470 nm),
in 0.02 M HEPES buffer in the presence of 0.1 mM γ-cyclodextrin. The
analytes were added in 0.3 mM concentration.
beneficial effect of this minor increase in the basicity of the ability by their inherent self-calibration nature [62]. Most
media to the proton tranfer.
3-hydroxyflavone-based probes exploit the ESIPT nature of
these fluorophores to generate multiple emission bands [39,41-
The selectivity of the flavone probe has been investigated in a 43]. In this case, however, a new fluorescence band appears in
screening experiment; the results are summarized in Figure 5. the excitation spectra due to an intermolecular proton transfer
As can be seen in Figure 5, among the different nucleotides, from the flavone to the phosphate chain of the nucleotide.
only ADP generated a slight fluorescence enhancement at Therefore, UHF can be applied as a ratiometric probe, setting
5
40 nm. According to [34], GTP and AMP caused a slight fluo- different excitation wavelengths and measuring the fluores-
rescence enhancement in the case of DMHF; this was not cence intensity at a selected wavelength. Figure 6 shows the in-
detected in the case of UHF which might be the result tensity ratio F470/F400 (the subscript indicates two different
of the complementary nucleobase attached to the flavone scaf- excitation wavelengths) of UHF upon addition of ATP at
fold.
540 nm.
Ratiometric fluorescence measurements received particular To the best of our knowledge, 3 is the first „clickable” hydroxy-
attention in the past decade due to their high sensitivity and reli- flavone. Considering the high interest in ratiometric fluorescent
Figure 4: Excitation spectra of UHF (dark green line, 1 μM) and UHF + 300 equiv ATP (red line), measured at different pH values. The fluorescence
enhancement values at 470 nm are also noted. The spectra were detected at 540 nm in 0.02 M HEPES buffer, in the presence of 0.1 mM γ-cyclo-
dextrin.
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Synthetic procedures
1
-(2-Hydroxy-4-(prop-2-yn-1-yloxy)phenyl)ethanone (1)
[
61]: The propargyl compound 1 was synthesized as described
with a modified purification method. 1-(2,4-dihydroxyphenyl)-
ethanone (6.00 g, 39.4 mmol, Sigma) was dissolved in acetone
(
90 mL). Potassium carbonate (6.54 g, 47.3 mmol, 1.2 equiv)
and tetrabutylammonium bromide (2.54 g, 7.89 mmol,
.2 equiv) were added and the mixture was cooled in an ice
bath. Subsequently, propargyl bromide (80% in toluene,
.83 mL, 43.3 mmol, 1.1 equiv) was added dropwise. The reac-
0
4
tion mixture was stirred for 14 hours at ambient temperature.
Upon completition, water was added (70 mL) and the pH was
set to 5 using dilute hydrochloric acid. The mixture was
extracted using ethyl acetate (3 × 70 mL), the organic phase was
washed with water (3 × 50 mL) and brine (50 mL) and dried on
MgSO4. The solvent was removed and the remaining off-white
solid was recrystallized from 10 mL boiling ethanol to remove
the unwanted dialkylated product and residual starting material.
Yield: 4.21 g (56%) white crystals. 1H NMR (500 MHz,
CDCl3) δ 12.70 (s, 1H, OH), 7.66 (m, 1H, 6-ArH), 6.51 (m, 2H,
Figure 6: Ratio of the fluorescence intensities at 540 nm, the samples
were excited at 470 and 400 nm. The red curve represents the result
of a non-linear fitting.
3
-,5-ArH), 4.72 (d, J = 2.5 Hz, 2H, OCH2), 2.57 (s, 3H, CH3),
markers, we believe that this compound might be applicable in 2.56 (m, 1H, CH); 13C NMR (75 MHz, CDCl3) δ 202.84
bioconjugate chemistry and related fields.
(C=O), 165.12 (C2-Ar-OH), 163.94 (C4-Ar-O), 132.50 (CH6-Ar),
14.64 (C1-Ar), 107.99 (CH3-Ar), 102.20 (CH2-Ar), 77.58 (over-
lapping with CDCl3, Calkyne) 76.47 (CHalkyne), 56.06 (CH2),
1
Conclusion
In conclusion, we have designed a uracil-conjugated, 4’-amino- 26.42 (CH3).
3
-hydroxyflavone-based fluorescent probe (UHF) for the selec-
tive recognition of ATP. The theoretical results showed that the (E)-3-(4-(Dimethylamino)phenyl)-1-(2-hydroxy-4-(propar-
base-pairing interactions are feasible in the supramolecular gyloxy)phenyl)prop-2-en-1-one (2): To a solution of 1 (1.50 g,
structre of UHF∙ATP. The synthesized probe showed large fluo- 7.89 mmol) and 4-dimethylaminobenzaldehyde (1.18 g,
rescence enhancement and a ratiometric response towards ATP 7.89 mmol, 1 equiv) in 15 mL anhydrous dimethylformamide
with an association constant of 2.3∙104 M−1. Excellent selec- was added sodium methoxide (1.53 g, 28.4 mmol, 3.6 equiv)
tivity was observed with other nucleotides that might be and the resulting mixture was stirred at room temperature for
the result of the beneficial effect of the complementary nucleo- 17 hours under an argon atmosphere. The deep red solution was
base.
poured into ice water (80 mL) and the pH was set to 5 using
dilute hydrochloric acid. The mixture was extracted with ethyl
acetate (3 × 30 mL), the organic phase was washed with water
(3 × 30 mL) and brine (30 mL) and dried on MgSO4. The sol-
Experimental
General
Solvents, reagents and starting materials were obtained from vent was evaporated and the oily residue was crystallized from
commercial suppliers and used without further purification. diethyl ether. The precipitate was collected by filtration and
5
-Chloromethyluracil [63] was synthesized as described previ- dried in vacuo to obtain 1.55 g (61%) of orange crystals.
ously. The fluorescence spectra were measured on an Edin- 1H NMR (500 MHz, CDCl3) δ 13.74 (s, 1H, OH), 7.90–7.82
burgh Instruments FLSP 920 fluorescence spectrometer. The (m, 2H, 6-ArH, CH=), 7.55 (d, J = 8.9 Hz, 2H, 2’-ArH), 7.36
1
H NMR spectra were taken on a Bruker Avance DRX-500 or (d, J = 15.2 Hz, 1H, CH=), 6.69 (d, J = 8.9 Hz, 2H, 3’-ArH),
DRX-300 spectrometer with chemical shifts reported in ppm 6.57–6.48 (m, 2H, 5-ArH, 3-ArH), 4.73 (d, J = 2.4 Hz, 2H,
TMS in in the case of CDCl3 and the residual DMSO in OCH2), 3.05 (s, 6H, CH3), 2.57 (t, J = 2.4 Hz, 1H, alkyne);
(
the case of DMSO-d6 was used as internal standard). The 13C NMR (126 MHz, CDCl3) δ 192.13 (C=O), 166.32
exact mass measurements were performed using a Q-TOF (C2Ar-OH), 163.52 (C4Ar-O), 152.37 (C4’-Ar-N), 145.85
Premier mass spectrometer (Waters Corporation, 34 Maple St, (CHdouble bond, Ar-C), 131.15 (CH6-Ar), 130.81 (2CH2’-Ar),
Milford, MA, USA) using electrospray ionization in positive 122.64 (CAr or double bond), 115.15 (CAr or double bond), 114.61
mode.
(CAr or double bond), 111.97 (2C3’-Ar), 107.67 (CH3-Ar), 102.39
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Beilstein J. Org. Chem. 2018, 14, 747–755.
(
CH2-Ar), 77.80 (Calkyne), 76.35 (CHalkyne), 56.05 (CH2), (500 MHz, DMSO-d6) δ 11.16 (s, 1H, 3-uracil NH), 10.94 (br s,
4
3
0.24 (CH3); HRMS calcd. for [M + H+]: 322.1443; found: 1H, 1-uracil NH), 8.79 (s, 1H, OH), 8.21 (s, 1H, ArH, triazole),
22.1443. 8.10 (d, J = 8.5 Hz, 2H, 2’-ArH, aniline), 7.97 (d, J = 8,8 Hz,
H, ArH, 5-chromone), 7.72 (s 1H, 6-uracil), 7.39 (s, 1H, ArH,
1
2
4
-(4-(Dimethylamino)phenyl)-3-hydroxy-7-propargyloxy- 8-chromone), 7.07 (d, J = 9,3 Hz, 1H, ArH, 6-chromone), 6.85
H-chromen-4-one (3): Chalcone 2 (500 mg, 1.56 mmol) was (d, J = 8.6 Hz, 2H, 3’-ArH, aniline), 5.30 (s, 2H, CH2), 5.19 (s,
dissolved in ethanol (25 mL) and sodium hydroxide (700 mg, 2H, CH2), 3.02 (s, 6H, CH3); 13C NMR (126 MHz, DMSO)
7.6 mmol, 11 equiv), dissolved in water (12.5 mL), was added. δ 171.46 (C=Ochromone), 163.64 (C=O6-uracill), 161.90 (CAr-O),
1
To the deep red solution was added 0.75 mL 30% hydrogen 155.98 (CAr-O), 151.16 (C=O2-uracil), 150.85 (C4’-Ar, aniline),
peroxide and the mixture was stirred at room temperature. After 146.17 (CAr-O), 142.98 (CHAr, triazole), 141.70 (CH4-uracil),
2
4 hours, the yellow solution was poured into ice water and 136.82 (C-OHchromone), 128.64 (2CH2’-Ar, aniline), 125.96
pH 5 was set by concentrated acetic acid. The pure product pre- (CHAr, chromone), 124.83 (CHAr, triazole), 118.06 (CAr, chromone),
cipitated as yellow crystals, filtered, washed with water and 115.42 (CAr, chromone), 114.46 (CHAr, chromone), 111.37
dried in vacuo to yield 401 mg (77%) product. 1H NMR (2CH3’-Ar, aniline), 106.18 (C5-uracil), 101.15 (CHAr, chromone),
(
500 MHz, DMSO-d6) δ 9.09 (s, 1H, OH), 8.10 (d, J = 8.8 Hz, 61.75 (CH2-O), 45.90 (CH2-N), 40.02 (CH3); HRMS calcd. for
H, 2’-ArH), 7.99 (d, J = 8.8 Hz, 1H, 5-ArH), 7.30 (d, J = 2.4 [M + H+]: 503.1679; found: 503.1675.
2
Hz, 1H, 8-ArH), 7.06 (dd, J = 8.8, 2.4 Hz, 1H, 6-ArH), 6.85 (d,
J = 8.8 Hz, 2H, 3’-ArH), 4.99 (d, J = 2.5 Hz, 2H, OCH2), 3.69 Fluorescence measurements
(
(
(
t, J = 2.4 Hz, 1H, alkyne), 3.02 (s, 6H, CH3); 13C NMR All the spectroscopic experiments were carried out at 25 °C. In
75 MHz, CDCl3) δ 171.47 (C=O), 160.96 (CAr-O), 155.78 all experiments, 0.02 M HEPES was used as buffer solution.
CAr-O), 150.86 (C4’-Ar-N), 146.27 (CAr-O), 136.89 (C-OH), Since the solubility of UHF in pure water is negligible, a stock
1
1
28.66 (2CH2’-Ar), 126.08 (CHAr), 118.01 (CAr), 115.75 (CAr), solution of 1.0 mM was prepared in DMSO which was diluted
14.38 (CHAr), 111.37 (2CH3’-Ar), 101.47 (CHAr), 78.99 with the buffered solution of γ-cyclodextrin (0.1 mM) and the
(
Calkyne), 78.52 (CHalkyne), 56.18 (CH2), 39.64 (CH3); HRMS analyte. The DMSO content in these samples was well below
calcd. for [M + H+]: 336.1236; found: 336.1232.
1%. Each spectrum was measured after reaching the equilib-
rium (5 minutes), and by using γ-cyclodextrin, it was stable
5
-Azidomethyluracil (4) [59]: The azido compound was syn- over a longer period of time. Since the spectra of UHF and
thesized as previously described. To a solution of UHF∙ATP did not change upon addition of γ-cyclodextrin in
-chloromethyluracil [63] (1.00 g, 6.23 mmol) in dimethylform- pure HEPES buffer (measurement performed before precipita-
5
amide (24 mL), sodium azide (0.81 g, 12.5 mmol, 2 equiv) was tion), it is unlikely that they form any type of inclusion com-
added. The mixture was stirred at ambient temperature for 1 h, plexes disturbing the recognition of ATP.
then poured to 50 mL of water. The resulting solution was
extracted with ethyl acetate (5 × 30 mL), the organic phase was Association constant determination
washed with water (40 mL) and dried on MgSO4. After evapo- The association constant has been obtained from the emission
ration, the oily residue was crystallized from diethyl ether, spectra using standard methods for non-linear curve fitting [65].
filtered and dried to give 0.46 g (44%) product as white crys- The best fit was obtained using 1:1 stoichiometry which con-
tals. 1H NMR (500 MHz, DMSO-d6) δ 11.29 (s, 1H, 3-uracil firmed our model of complexation [66].
NH), 10.99 (s, 1H, 1-uracil NH), 7.64 (s, 1H, 6-uracil CH), 4.02
(
(
s, 2H, CH2); 13C NMR (75 MHz, DMSO-d6) δ 163.97
Supporting Information
C=O6-uracil), 151.23, 141.94 (CH4-uracil), 106.72 (C5-uracil),
4
6.56 (CH2).
Supporting Information File 1
NMR spectra and additional figures.
Uracil-hydroxyflavone probe (UHF): The click reaction of 3
and 4 was performed as follows. Propargyl derivative 3
[
https://www.beilstein-journals.org/bjoc/content/
supplementary/1860-5397-14-63-S1.pdf]
(
200 mg, 0.596 mmol) and azide compound 4 (100 mg,
.596 mmol, 1 equiv) was dissolved in tetrahydrofuran (25 mL),
0
and TBTA [64] (32 mg, 0.1 equiv) and [Cu(MeCN)4]BF4 Acknowledgements
(
14 mg, 0.075 equiv) were added. The reaction mixture was We thank the Hungarian Research Foundation for the financial
stirred for 24 h, and the product precipitated from the solution. support of this work (Grant No. K108752). P. Z. J.-B. and D.
The precipitate was filtered, washed with THF thoroughly and M. are thankful to the Ministry of Human Capacities for the
dried to yield 264 mg (88%) product as a yellow solid. 1H NMR New National Excellence Program financial support. The
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Beilstein J. Org. Chem. 2018, 14, 747–755.
2
2
2
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ORCID® iDs
Márton Bojtár - https://orcid.org/0000-0001-8459-4659
Mihály Kállay - https://orcid.org/0000-0003-1080-6625
Miklós Kubinyi - https://orcid.org/0000-0002-6343-0820
István Bitter - https://orcid.org/0000-0002-2664-896X
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