Extended fluorescent uridine analogues: synthesis, photophysical properties and selective interaction with BSA protein

Article abstract of DOI:10.1039/d0nj02803g

The improvement in fluorescence properties of 2′-deoxyuridine was brought about by the introduction of (hetero)aromatic moieties at the C-5 position of uridine directly orviaalkenyl/phenyl/styryl linkers to create a library of biologically useful fluorescent nucleosides. The synthesis of these extended nucleoside analogues was carried out using Suzuki-Miyaura and Heck alkenylation reactions. A comparison of their photophysical properties allowed the identification of nucleosides1gand1hthat exhibit the highest quantum yields in an aqueous solution. Studies on the binding interaction of the most promising fluorescent analogues,1gand1h, with serum albumin proteins showed excellent selectivity towards BSA protein over α-amylase. Docking studies were also performed to predict the specific binding site of the nucleosides to BSA.

Full text of DOI:10.1039/d0nj02803g

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10.1039/D0NJ02803G.
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Extended fluorescent uridine analogues: Synthesis, photo-
physical properties and selective interaction with BSA protein
Received 00th January 20xx,
Accepted 00th January 20xx
Ajaykumar V. Ardhapurea, Vijay Gayakhea, Shatrughn Bhilarea, Anant R. Kapdia,*, Subhendu Sekhar
Bagb,* and Yogesh S. Sanghvic, Krishna Chaitanya Gunturud
DOI: 10.1039/x0xx00000x
The improvement in fluorescent property of 2’-deoxyuridine was brought about by the installation of (hetero)aromatic
moieties at the C–5 position of uridine directly or via alkenyl/phenyl/styryl linkers to create a library of biologically useful
fluorescent nucleosides. The synthesis of these extended nucleoside analogues was carried out using Suzuki-Miyaura and
Heck alkenylation reactions. A comparison of the photophysical properties allowed the identification of nucleosides 1g and
1h to exhibit the highest quantum yields in an aqueous solution. Studies on the binding interaction of the most promising
fluorescent analogues, 1g and 1h, with serum albumin proteins showed excellent selectivity towards BSA protein over α-
amylase. Docking studies were also performed to predict the specific binding site of the nucleosides to BSA.
www.rsc.org/
1. Introduction
materials etc.12–14 Several such probes have been known in the
Natural nucleosides are known to lack the ability to exhibit
practical fluorescence due to very short fluorescent decay.1It is
because of this reason that natural nucleosides fail to provide
structural information in DNA or RNA since signals are
normally averaged over all nucleobases in the DNA and RNA.2
Tthe lack of naturally occurring fluorescent bases have
therefore spurred the development of new fluorescent
nucleosides as possible probe molecule for DNA analysis.3,4
These nucleosides can be designed in such a way to get
optimized fluorescence properties, i.e., higher quantum yields,
longer lifetimes and can be utilized for the development of
nucleic acid-based diagnostics and sensing materials.5–7
Tremendous amount of research efforts have been put forth
over the past decade for the development of fluorescent
nucleosides by labelling the natural nucleosides with a
fluorophoric unit.8,9 Such modifications have been achieved in
a variety of different ways with transition-metal catalysed
processes as the most commonly applicable, efficient and
preferred pathway for achieving this goal.10,11 Palladium-
catalysed coupling processes amongst other catalytic reactions
allow easy introduction of fluorophoric groups on the
nucleoside substructure providing access to molecules with
promising fluorescent properties that could be employed as
nucleic acid sensors, protein sensors and DNA based light
literature that are obtained by the introduction of groups such
as benzofuranyl, alkenyl, alkynyl moiety on uridine base via
palladium-catalysed coupling reactions (Fig. 1).15–19
Noteworthy work done by Tor,17 Fischer20 and others need
a special mention as they have paved the way for the rapid
development of this area of research. Fischer and co-workers,
introduced the alkenyl and alkynyl extended nucleosides and
compared these analogues with directly attached uridine
analogues.21 These extended nucleosides showed efficient
charge transfer in the presence of alkenyl group, bringing
about an increase in the dipole moment. These molecules,
however, were only analysed for their photophysical properties
and could certainly have the potential to be utilised as
fluorescent probes.22–24
O
O
O
O
O
HN
HN
HN
O
O
N
O
N
N
1b
1c
1a
dR
dR
dR
Fischer et.al
Tor et.al
OCH3
O
N
O
HN
HN
O
O
N
1d
1e
Hudson et.al
dR
dR
Wagenknecht et.al
a. Department of Chemistry, Institute of Chemical Technology, Matunga, Mumbai-
400019, India (ar.kapdi@ictmumbai.edu.in)
dR = 2'-Deoxyribose sugar
Fig. 1: Fluorescent probes for different biological applications.
b. Bioorganic chemistry laboratory, Department of Chemistry, Indian Institute of
Technology, Guwahati 781039, Assam, India.
Modified nucleosides could also find application as protein
sensing probes due to their favourable interactions with serum
albumins that play an important role towards the transportation
of molecules (drugs or fatty acids) and could provide valuable
information about the absorption, distribution, metabolism and
cRasayan Inc.2802, Crystal Ridge Road, Encinitas, California, 92024-6615, United
States of America.
dSchool of Chemical Sciences, SRTM University, Nanded 431606, India
Electronic Supplementary Information (ESI) available: [details of any
supplementary information available should be included here]. See
DOI: 10.1039/x0xx00000x
This journal is © The Royal Society of Chemistry 20xx
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excretion of the drug molecules.25–27 As serum albumins
represent the major protein of human blood plasma, the
interaction of the modified nucleosides would help assess their
potential as protein probes.28,29 It is to be noted that human
serum albumins (HSA) are highly expensive and therefore,
bovine serum albumin (BSA), a structural homolog of human
serum albumin, is extensively used as a model protein in
research.29
View Article Online
DOI: 10.1039/D0NJ02803G
(Hetero)arenes =
UdR
O
O
UdR
1b
1f
R'
UdR
HN
UdR
(Hetero)arenes
1g, 1h
O
N
1i, 1j
(Hetero)arenes
dR
(Hetero)arenes
1k, 1l
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UdR
The BSA comprise of three homologous domains (I–III) that
are divided into nine loops by 17 disulfide bonds.30–32 Studies
on the interaction of bioactive molecules with BSA has
revealed the presence of two major binding sites represented as
site-I and site-II located inside the hydrophobic cavities of sub-
domains IIA and IIIA, respectively.33 In literature, these sites
have been marked with small molecules that are termed as site
markers exhibiting specific binding locations in the albumin
structure.34 For site-I, small molecule markers such as
warfarin, phenylbutazone, dansylamide and iodipamide have
provided specific binding, while the common site-II markers
are ibuprofen, flufenamic acid and diazepam.35,36 A recent
study by Bag and co-workers also highlights the application of
modified nucleosides as probes for the easy detection of BSA
protein.37 This example along with many others suggests a
growing interest in the development of efficient nucleoside-
based probes for the specific detection of proteins with unique
biological functions.38,39
Given our interest in the development of modified nucleosides,
efficient palladium-catalysed coupling processes involving
water-soluble catalytic systems developed in-house have
allowed us to isolate the biologically relevant molecules
without any column purification.40 Therefore, we report herein
our studies on the synthesis, photophysical properties of
fluorescent uridine-based nucleosides molecules and
interaction of two (nucleosides, 1g and 1h) with BSA. Specific
interaction with BSA protein as compared to α-amylase was
confirmed not only on the basis of fluorescence studies but
also molecular docking studies, which were performed to
better understand the specific site binding events.
Fig. 2: Novel uridine-based fluorescent probes.
2.1 Synthesis of modified nucleosides:
At the outset of our studies, we set about synthesising the
proposed molecules by the conventional synthetic routes
involving palladium-catalysed cross-coupling reactions using
the catalytic systems developed in our research group.40,44–46
Compounds 1b and 1f were obtained by the palladium-
catalysed Suzuki-Miyaura cross-coupling of 5-iodo-2ꞌ-
deoxyuridine with 2-benzofuranyl boronic acid and 2-naphthyl
boronic acid, respectively in the presence of Pd(OAc)2/PTABS
catalyst system (Scheme 1).
O
O
Ar B(OH)2
Ar
A
I
NH
NH
Pd(OAc)2 (1.0 mol%)
PTABS (2.0 mol%)
HO
HO
N
O
N
O
NEt3, H2O (1.0 mL)
80 oC, 3.0 hr
O
O
Ar = 2-benzofuranyl &
2-naphthyl
HO
HO
1
1b
1f
= 2-benzofuranyl, 88%
= 2-naphthyl, 85%
O
N
O
O
N
B
HO
Ar B(OH)2
I
Br
HO
Ar
HO
NH
NH
NH
Pd(OAc)2 (1.0 mol%)
PTABS (2.0 mol%)
O
N
O
O
NEt3, H2O (2.0 mL)
80 oC, 6.0 hr
O
O
O
Ar = 2-benzofuranyl &
2-naphthyl
HO
HO
HO
1
1g
1h
= 2-benzofuranyl, 85%
= 2-naphthyl, 82%
Ar
Ar B(OH)2
Br
C
O
O
I
Pd(OAc)2 (1.0 mol%)
PTABS (2.0 mol%)
Pd(OAc)2 (2.0 mol%)
SPhos (4.0 mol%)
NH
NH
HO
HO
[A]
N
O
N
O
NEt3, CH3CN:H2O (2:1)
80 oC, 18 hr
NEt3, CH3CN:H2O (2:1)
80 oC, 20 hr
O
O
Ar = 2-benzofuranyl &
2-naphthyl
Br
O
N
HO
HO
1
1i
1j
= 2-benzofuranyl, 70%
= 2-naphthyl, 68%
NH
2. Results and Discussions
HO
O
Structural modification of the nucleobases via synthetic
processes has commonly been carried out to provide molecules
with diverse properties that prove to be useful in deciding the
type of applications they could be subjected to. Commonly,
researchers have fine-tuned the electronic properties of the
nucleobases by the introduction of functional groups such as
(hetero)arenes, unsaturation (alkenyl or alkynyl substituents)41–
[A]
=
O
HO
Br
B(OH)2
Ar
O
N
O
Ar B(OH)2
D
HO
I
Pd(OAc)2 (1.0 mol%)
PTABS (2.0 mol%)
Pd(OAc)2 (2.0 mol%)
SPhos (4.0 mol%)
NH
NH
[A]
HO
O
NEt3, H2O (3.0 mL)
80 oC, 18 hr
NEt3, CH3CN:H2O
80 oC, 20 hr
N
O
O
O
Ar = 2-benzofuranyl &
2-naphthyl
Br
O
N
HO
1
HO
43
NH
1k
1l
= 2-benzofuranyl, 86%
= 2-naphthyl, 75%
etc. Keeping this in mind, we envisaged the possibility of
HO
O
developing uridine-based fluorescent probes having key
structural features as depicted in Fig. 2. Extended
chromophores at the C–5 position of uridine could simply be a
(hetero)aromatic moiety attached directly or via an alkenyl or
phenyl or styryl linker to modulate the fluorescent properties.
Introduction of such diverse structural features would allow us
to investigate their effects on the photophysical properties as
well as to test their applicability towards BSA protein
interactions.
[A]
=
O
HO
Scheme: 1 Synthesis of fluorescent nucleosides.
It is to be noted that compound 1b is the same fluorescent
probe used by Tor and co-workers in their studies and would
serve as a reference point for the studies that we intend to
undertake on the different uridine-based analogues.
2 | J. Name., 2012, 00, 1-3
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Next, for the installation of alkenyl-conjugated (hetero)aryl
ARTICLE
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DOI: 10.1039/D0NJ02803G
analogues, we functionalised 5-iodo-2ꞌ-deoxyuridine in a series
of steps by first performing a Heck alkenylation procedure
with methylacrylate in the presence of Pd(OAc)2/PTABS
catalyst system and the obtained product was hydrolysed using
1M NaOH to provide 5-carboxyvinyl-2ꞌ-deoxyuridine. This
was further subjected to bromination using N-
bromosuccinimide to eventually furnish BVDU (Brivudine-
anti herpes agent).47 Suzuki-Miyaura coupling of the above
mentioned (hetero)arylboronic acids (2-benzofuranyl boronic
acid and 2-naphthyl boronic acid) with BVDU using
Pd(OAc)2/PTABS catalyst system provided the analogues 1g
and 1h in good yields (Scheme 1B, see SI, Scheme S1).
Similarly, for the synthesis of styryl-conjugated (hetero)aryl
analogues (1i and 1j) of 2ꞌ-deoxyuridine, BVDU was subjected
to one-pot tandem Heck alkenylation/Suzuki-Miyaura coupling
reaction with the first step carried out using 4-bromostyrene
(Heck reaction) and Pd(OAc)2/PTABS catalyst system while,
for the second step involving the activation of C–Br bond, a
more electron-rich phosphine S-Phos was added and a second
coupling involving the Suzuki-Miyaura cross-coupling with
different boronic acids (2-benzofuranyl boronic acid and 2-
naphthyl boronic acid), furnished the desired products in good
yields as a part of a sequential one-pot tandem catalytic
procedure (Scheme 1C). Finally, phenyl-conjugated analogues
(1k and 1l) were obtained directly from 5-iodo-2ꞌ-deoxyuridine
via the one-pot double tandemcatalytic protocol involving two
simultaneous Suzuki-Miyaura coupling reactions48 in a similar
fashion to that described above. The beauty of our
methogdology lies in the fact that all the compounds were
purified by filtration followed by recrystallization without any
tedious column purification. The synthetic analougs were
thoroughly characterized by 1HNMR, 13C NMR and HRMS.
2.2. Photophysical studies of modified nucleosides:
With the synthesised analogues in hand, we next turned our
attention towards studying the photophysical properties of all
the molecules. The spectral properties of nucleoside analogues
were analysed in different solvent of varying polarity (dioxane-
non polar to buffer-polar). With the natural uridine base
absorbing at 262 nm, most of the synthesized analogues
showed large red shifted absorbance. Interestingly, some of the
nucleosides, such as 1g, 1h & 1i showed red shift in the
absorbance values ranging from 80-100 nm. This observation
reflected a strong electronic coupling among the substituent
aryl/heteroaryl moiety and the π-cloud of the uridine nucleus.
From the table 1, it could also be observed that the analogues
containing benzofuranyl moiety shows further enhancement in
the absorbance as well as emission intensities (Fig. 3).
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Fig.3: Representative emission spectra’s of 1g and 1h.
While all the synthesized nucleosides showed marginal shift in
absorbance, a considerable red shift of about 40-50 nm in
fluorescence was observed with increase in the solvent
polarity. In particular, nucleoside 1g and 1h exhibited higher
solvatochromicity in fluorescence and extremly large Stoke’s
shift in buffer (129-134 nm). We also determined the quantum
yield of fluorescence for all nucleosides in various solvents
and compared them with quantum yield in buffer solution.
Quantum yield (ΦF) was calculated based on ΦF of a known
reference (quinine sulphate or 2-aminopyridine).49
Table 1: Photophysical data for modified nucleosides (A).
Photophysical data for nucleosides (A)
λmax
(nm)
323
321
321
321
326
324
324
322
306
300
301
305
305
308
306
295
343
342
342
340
343
343
345
337
333
330
332
333
331
335
337
327
360
356
354
352
358
357
352
є/ cm-
λem
(nm)
385
421
418
381
408
386
389
443
383
402
387
389
397
386
389
441
424
446
444
431
438
431
430
471
406
426
428
413
420
411
412
456
415
432
417
414
427
419
423
472
404
427
420
406
418
413
Stokes
shift/nm
62
100
97
60
82
62
65
121
77
102
86
84
94
є x ФF
Solvent
ΦF
1M-1
Dioxan
MeOH
EtOH
CH3CN
CHCl3
DMF
DMSO
Buffer
Dioxan
MeOH
EtOH
CH3CN
CHCl3
DMF
DMSO
Buffer
Dioxan
MeOH
EtOH
CH3CN
CHCl3
DMF
DMSO
Buffer
Dioxan
MeOH
EtOH
CH3CN
CHCl3
DMF
DMSO
Buffer
Dioxan
MeOH
EtOH
CH3CN
CHCl3
DMF
DMSO
Buffer
Dioxan
MeOH
EtOH
CH3CN
CHCl3
DMF
8500
0.12
0.10
0.16
1020
1332
1724
13320
10780
4790
1b
3550
15870
15970
2980
10090
9670
8420
3550
5890
8180
0.08
0.11
1269
1756
0.02
0.02
0.05
0.02
0.04
0.02
0.04
0.04
0.313
0.15
0.19
0.17
0.21
0.19
0.21
0.05
0.14
0.07
0.11
0.06
0.06
0.10
0.11
0.09
0.36
0.37
0.47
0.16
0.42
0.23
0.22
0.04
0.25
0.14
0.20
0.13
0.15
0.19
201
193
421
71
235
163
324
281
1f
76
83
8110
7040
146
79
104
102
91
95
88
85
134
73
96
96
80
89
76
75
129
55
76
63
62
69
62
71
-
65
94
86
71
81
77
22700
26025
26600
27550
22275
20900
18250
24000
15300
24400
24250
25200
26550
22475
17825
8050
45050
45400
47700
40900
44450
22400
24000
7105
3903
5054
4683
4667
3971
3832
1200
2142
1708
2667
1512
1593
2247
1960
724
16218
16798
22419
6544
18669
5152
5280
1g
1h
1i
339
333
334
335
337
336
24550
27025
24975
23825
15350
23075
6137
3783
4995
3097
2302
4384
1j
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DMSO
336
328
331
324
325
327
331
334
329
322
313
310
309
309
309
316
314
305
21000
5150
417
444
408
430
428
414
418
414
414
440
393
416
413
400
412
395
398
422
81
116
77
106
103
87
87
80
75
118
80
106
104
91
103
79
0.17
0.06
0.46
0.34
0.45
0.41
0.62
0.76
0.75
0.02
0.06
0.07
3570
309
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Buffer
Dioxan
MeOH
EtOH
CH3CN
CHCl3
DMF
DMSO
Buffer
Dioxan
MeOH
EtOH
CH3CN
CHCl3
DMF
46200
41700
43900
41900
46400
38250
33050
26700
30200
30450
71650
26950
71600
19725
70325
18850
21252
14178
19755
17179
28768
29070
24787
534
LUMO
HLG
1k
(1h) -1.65
(1g) -1.75
3.59
3.76
1812
2131
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2.3 Dioxane-Water titration studies:
Based on the previous investigation of various nucleoside
analogues, we identified molecules with promising
1l
0.03
0.08
0.01
808
1578
188
2
DMSO
Buffer
84
117
fluorescence properties in different solvent environments,
namely 1g and 1h. It is also interesting to note that the
difference in emission wavelengths from non-polar (dioxane)
environment to polar (buffer) underwent approximately 50 nm
red shift in both the cases (1g = 45 nm and 1h = 50 nm). This
property combined with large extinction coefficients, high
quantum yields and long excitation wavelengths make 1g and
1h excellent candidates as solvatochromic fluorophores.52
To test their solvatochromocity, we subjected 1g and 1h to the
dioxane-water titration studies that involved the gradual
increase in the percentage of water (polar environment) in
dioxane (non-polar environment). Thus, a decreasing pattern of
absorbance with a little blue shift in wavelength was observed
as the molecules underwent transition from the non-polar
environment to the polar environment (Fig. 4). The
fluorescence spectra of both these molecules showed
significant red shift (50 nm for 1g and 52 nm for 1h), which
implicates that the nucleosides 1g and 1h could potentially act
as solvatochromic environmentally sensitive fluorescent
(ESF)62 nucleosides and are capable of exhibiting broad ICT
emissions in low viscosity environments too. Based on these
results, we decided to explore the potential of the two
vinyl(hetero)aryl-extended uridine analogues 1g and 1h for
protein sensing applications.
The concentration of the nucleosides 1b and 1f is 10μM; 1g, 1h, 1j, and 1l is 4 μM
and 1k, 1i is 2 μM, l ex = lmax abs in each solvent.
Stokes shift for all the analogues in different solvents also has
been presented in Table 1. Analogues 1g and 1h in which the
2ꞌ-deoxyuridine chromophore has been extended by
a
conjugated vinyl(hetero)aryl moiety exhibited longer
absorption and emission wavelengths than most other
nucleoside analogues as well as higher quantum yields in
buffer solution (Figure 3). An appreciable Stokes shift was also
observed in both the cases providing further support to our
proposal for the enhancement in photophysical properties
brought about by the introduction of extended conjugation on
the 2ꞌ-deoxyuridine moiety.
To better understand the photophysical property we also
performed theoretical calculations on the two most promising
analogues namely, 1g and 1h, using Gaussian 09 program
package at B3LYP/6-31G(d,p) level of theory.51 The
theoretical and observed values of the absorbance and emission
maxima are tabulated in the Table 2 and the HOMO-LUMO
energy levels have been shown in the table 3.
Table 2:Comparison between theoretical and practical
absorbance and emissions for 1g and 1h (B).
Comparison of theoretical and practical absorbance and emissions values for 1g
and 1h. (B)
λmax/nm
observed
λmax/nm
theoretical
λem./nm
observed
λem./nm
theoretical
414
Comp
1g
1h
343
333
359
347
424
406
404
The inspection of frontier orbital pictures show that the
HOMO and LUMO levels are slightly stabilized and
destabilized, respectively in 1h as compared to 1g (Table 3).
This can be attributed to the increased bonding and anti-
bonding interactions within the naphthalene moiety (1h) rather
than in benzofuranyl moiety (1g). LUMO suggested a strong
electronic coupling among the uridine nucleus and the attached
chromophore.37,50
Emission spectra’s for Dioxane-water titration for 1g
Table 3: Frontier orbital levels (in eV) and HOMO-LUMO gap
(in eV) of molecules 1g and 1h obtained at B3LYP/6-31G(d,p)
level.51
HOMO
(1g) -5.34
(1h) -5.41
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1g and 1h (47 and 50 nm, respectively) when excited at 340
View Article Online
DOI: 10.1039/D0NJ02803G
nm (Fig. 5 & 6).
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Emission spectra’s for Dioxane-water titration for 1h
Fig. 4: Dioxane-water titration for 1g and 1h.
2.4. Protein interaction studies
Finally, the excellent solvatochromocity of 1g and 1h has
prompted us to employ these molecules for sensing the protein
micro-environment. To determine whether the modified nucleosides
could find application in protein sensing, we decided to employ
Bovine Serum Albumin32,53 and α-Amylase8 proteins as the readily
available and the most commonly studied proteins in literature.
Interaction studies of the modified nucleosides with the proteins
could be performed using fluorescence spectroscopy as any change
in the fluorescence intensity as well as emission wavelength would
signify positive interactions between the substrate and the target.
For the development of chemotherapeutic agents, the understanding
of drug (small molecules)-protein interactions is of principle
significance.25 In last few decades, nucleoside analogs have been
reported for their potential antiviral and anticancer activity and
more than 40 nucleoside analogues have been approved as active
drug molecules and many are in clinical trials.54 However, many of
the nucleoside analogs show weak cell membrane penetrability and
therefore these molecules failed as potential drug candidates.54–56 In
addition to this, metabolic process of nucleosides is crucial at
cellular level and many enzymes and their functions are known to
rely on nucleosides. Hence, the study of nucleoside analogues
interaction with protein became essential, which can help to
understand structure-function relationship and to decipher the
molecular mechanism involving interbiomolar interactions.53,54,56 It
can also provide essential knowledge about the transportation,
metabolism and cell penetration of nucleoside analogues that can
help in designing and synthesis of a new class of nucleoside-based
drugs.57 In this regard, we decided to test the interaction of
synthesized nucleoside analogues with protein and selected a model
protein, bovine serum albumin (BSA). BSA is almost 76% identical
to human serum albumin (HSA) protein and is commonly used as a
model to examine the interaction with drugs.28,58,59
Fig. 5: (a) Fluorescence titration of 1g (10μM) with various
concentration of BSA in water (λexc.= 280 nm) (b) Fluorescence
titration of 1g with various concentrations of BSA. (c) Fluorescence
titration of 1g (10μM) with various concentration of α-Amylase in
water (λexc.= 280 nm) (d)Plot of (Imin− I)/(Imin− Imax) vs.
log[Vol.BSA] of 1g for calculating the detection limit.
2.4.1 BSA protein interaction studies
Strong solvatochromic properties of nucleoside analogues, 1g
& 1h provided us with an opportunity to test their possibility
of sensing BSA as these could be predicted to bind at the
hydrophobic pocket of BSA.
Thus, we investigated the effect of BSA protein on the UV
visible and emission wavelength of the modified nucleosides
by regulating the concentration of BSA, which was added
slowly to the aqueous solutions of the respective nucleosides
1g and 1h. The fluorescence intensity was found to gradually
increase with the subsequent increase in the concentration of
BSA although, a blue shift was observed for the nucleosides,
Fig. 6: (a) Fluorescence titration of 1h (10μM) with various concentration of
BSA in water (λexc.= 280 nm) (b) Fluorescence titration of 1h with various
concentrations of BSA. (c) Fluorescence titration of 1h (10μM) with various
concentration of α-Amylase in water (λexc.= 280 nm) (d)Plot of (Imin−
I)/(Imin− Imax) vs. log[Vol.BSA] of 1h for calculating the detection limit.
A blue shift in the emission wavelength of the two nucleosides
signifies a change in the environment of the nucleosides from
the hydrophilic (aqueous) to the hydrophobic (BSA protein
cavity) environment. On closer inspection of the BSA protein
structure, it is evident that site-I and site-II, which are the sites
of interaction with drug molecules are located in the
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hydrophobic cavities of subdomains IIA and IIIA, respectively
(Fig 7).60 The possibility of the modified nucleosides
interacting with the BSA protein in these domains could
therefore explain the blue shift observed in the above study.
amylase to the probes solution did not shift the emission spectra to
DOI: 10.1039/D0NJ02803G
shorter wavelength region (no blue shift was observed) indicating a
View Article Online
very weak association between the probes and α-amylase protein.
The absence of suitable hydrophobic cleft in α-amylase to bind the
modified nucleosidesis would be the main reason for α-amylase to
show weak interactions with the probes as compared to BSA.
Fluorescence experiments performed so far suggest that the probes
could preferentially sense BSA with much larger enhancement in
fluorescence intensity compared to α-amylase. It could be observed
that the probes 1g and 1h are able to sense BSA with 196% and
312% enhancement in fluorescence intensity, while a mere 36% and
45% enhancement could be observed in the case of α-amylase. This
large amplification in fluorescence intensity could therefore be
useful as a suitable technique for sensing BSA protein and
distinguish it from α-amylase (Fig 9).
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Fig.7: Permission obtained from RSC for reuse.60
The association constants of probes, 1g and 1h with BSA was next
determined fluorimetrically by a Benesi−Hildebrand plot, which
were found to be 2.51 × 104 and 4.08 × 104 M−1, respectively (Fig
8). Association constants also reflect the strong binding of both
probes, 1g and 1h with BSA. Therefore, it is clear from the above
findings that both the molecules are efficient in sensing the BSA
protein via the generation of enhanced fluorescence intensity.
Fig.9: Comparison of fluorescence intensity changes [(Imax-
Imin)/Imin X 100%] of probes 1g and 1h upon addition of BSA and
alfa-amylase, respectively. Imin is fluorescent emission intensity of
free probe and Imax is the maximized fluorescence intensity after
adding BSA/ α-amylase.
2.4.3 Molecular Docking studies
The preferential interaction with the protein could be related to
the structure of BSA as the nucleosides could bind strongly
inside the two possible hydrophobic cavities in the sub-
domains IIA and IIIA. To verify this assumption and to
ascertain the preferable site for interaction between the
modified nucleosides 1g and 1h with the BSA protein, mocular
docking studies were performed using AutoDock 4.1.52 The
docking analysis shows that both 1g and 1h exhibit strong
interactions with sub-domain IIIA than in IIA. In addition to
hydrophobic interactions posed within the cavity, the
molecules could be held into the cavities by strong hydrogen-
bonding (HB) with the different amino acid residues as
highlighted in Table 4. Moreover, sub-domain IIIA has more
affinity towards 1h than for 1g. This also suggests that the
modified nucleosides could potentially be categorised as site-
II markers (strong binding interaction with the IIIA sub-
domain) as compared to site-I markers (having interactions
with sub-domain IIA). In order to understand the sensing
selectivity of synthesized nucleosides towards BSA over α-
amylase, the docking studies were extended accordingly with
α-amylase and the results are given in supporting information.
In line with the experimental findngs, lesser binding
efficiencies were observed for 1g and 1h compared to BSA
case. Noteworthy to mention here is that, tryptophan residues
have shown negligible role in nucleoside-protien interactions
whereas glutamine, arginine, serine, phenylalanine and lysine
Fig. 8: Benesi-Hildebrand Plot of 1g (left) and 1h (right) with BSA protein
showing association constant of BSA protein.
2.4.2 α-Amylase interaction studies
To know the selectivity in interaction with protein, we next studied
the binding interaction with α-amylase, which is a globular protein
similar to BSA. While a single polypeptide chain BSA consists of
582 amino acids with two tryptophan residues, α-amylase contains
496 amino acids and 17 tryptophan residues in a single polypeptide
chain. Thus, both the proteins are utilized as models for studying
drug-protein interaction. The BSA sensing selectivity of our
nucleosides 1g and 1h was compared with that of α-amylase sensing
ability using spectroscopic technique.
Fluorescence titration experiment was therefore conducted using
aqueous solutions of nucleosides 1g and 1h. The experiment was
carried out by exciting both the probes at 340 nm and 330 nm,
respectively with an increase in α-amylase concentration , however
no change in the fluorescence intensity of the probes was observed.
The result reflected that both the probes failed to show any specific
interaction with α-amylase (See spectra’s (c) in Figures 5 and 6).
Moreover, as compared to the BSA, the gradual addition of α-
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residues have been found for HB interactions in BSA. At the
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shown HB interactions in α-amylase with nucleosides.
View Article Online
DOI: 10.1039/D0NJ02803G
same time, threonine, glycine, arginine and asparagine residues
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Table 4: Docking studies for establishing the exact site of interaction for nucleosides with BSA protein.
Molecule
Sub-domain
Ribbon
H-bonding
Affinity
(kcal/mol)
-8.2
8
9
1g
IIA
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IIIA
-9.6
1h
IIA
-8.7
IIIA
-10.0
General remarks: C, H and N analyses were carried out locally.
NMR data (1H or 31P) were recorded locally on 400 and 500
spectrometers. HPCL-MS analyses were performed locally. The
ionization mechanism used was electrospray in positive and
negative ion full scan mode using acetonitrile as solvent and
nitrogen gas for desolvation. Imides and other commercially
available chemicals were purchased locally and were used without
further purification. All the solvents were dried by standard
methods before use. UV-absorptions study was performed locally.
Emission wavelengths were measured with 300 watt Xenon lamp.
All other characterizations details have been provided to
conclusively to ascertain the structure of all the complexes (see
Supporting information for NMR spectra).
In conclusion, we have been successful in synthesising and
demonstrating the positive effect of introducing extended
conjugation on the uridine nucleoside substructure providing
enhancement in fluorescence properties. These modified
fluorescent nucleosides have further been analysed towards their
interaction with two different proteins namely, Bovine Serum
Albumin and α-Amylase. Both the compounds showed specific
binding ability with BSA over α-Amylase that was evident from
the change in fluorescence intensity. Docking studies have also
been undertaken to ascertain the site of interaction in BSA for
the modified nucleosides, while α-Amylase at the same time
showed lower binding to the nucleoside analogues
Computational Details
Experimental Section
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All the geometry optimizations at ground and first excited states
128.1, 127.4, 126.5, 126.3, 126.2, 126.1, 113.3, 87.5, 84.6, 70.1,
View Article Online
have been performed at B3LYP/6-31G(d,p) and TD-B3LYP/6-
31G(d,p) level of theories respectively with Gaussian 09 suite of
program.51 Second order gradients were verified to assure the
obtained geometries were global minima on the respective potential
energy surfaces. The spontaneous radiative decay rate kr from
60.9, 40.1. ESI-MS (m/z) = 355 [M + H+], 377 [M + Na+].
DOI: 10.1039/D0NJ02803G
5-(E)-(2-benzofuranylvinyl)-2ꞌ-deoxyuridine (1g):44,45
O
O
N
excited state to ground state has been calculated by using equation
NH
E3
푘푟 = 4∆ 3 휇2
∆
푐
HO
E
where is excitation energy, is the speed of light and
O
3
퐶
O
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휇
is the transition dipole moment in atomic units. The radiative life
time 푟is obtained as,
푘1 . Molecular docking studies were
HO
휏
휏푟 =
푟
performed using AutoDockVina algorithm [35]. BSA (PDB: 3V03)
structural information was obtained from Protein Data Bank.All the
heteroatoms and water molecules were removed from the PDB
structure.
Representative procedure was followed from previously published
manuscript: Yield 88%.1H NMR (400 MHz, DMSO-d6) δ 11.56
(s, 1H), 8.24 (s, 1H), 7.59 – 7.46 (m, 3H), 7.21 (dtd, J = 25.9, 7.4,
1.1 Hz, 2H), 6.96 (d, J = 16.1 Hz, 1H), 6.85 (s, 1H), 6.15 (t, J =
6.6 Hz, 1H), 5.27 (d, J = 4.2 Hz, 1H), 5.19 (t, J = 5.2 Hz, 1H),
4.28 – 4.23 (m, 1H), 3.78 (dd, J = 7.0, 3.6 Hz, 1H), 3.68 – 3.55
(m, 2H), 2.21 – 2.08 (m, 2H). 13C NMR (101 MHz, DMSO-d6) δ
162.4, 155.5, 154.5, 149.7, 140.1, 129.3,125.0, 123.5, 121.3,
116.2, 111.0, 110.3, 105.2, 87.9, 84.8, 70.4, 61.4, 46.1. ESI-MS
(m/z) = 331 (M+ + H+). Anal. Calcd for C17H18N2O5: C, 61.81; H,
5.49; N, 8.48. Found: C, 61.87; H, 5.59; N, 8.51.
AutoDock Tools was used to prepare the ligand (1g and 1h) and
proteins for docking. Polar hydrogen atoms and Gasteiger charges
were added to the protein. The DFT optimized ligand structures
were considered in docking studies. All the rotatable bonds in the
ligand were allowed for free rotation. The whole protein was
placed in the center of a simulation box. The box dimensions 23 ×
24.5 × 37 and 36 × 24 × 52 cubic angstroms were chosen
sub-domains IIA and IIIA of BSA respectively. Grid point
spacing of 1 Å was used. AutoDockVina results were
rendered in PyMOL. The hydrogen bonds and
hydrophobiccontacts between the ligands and BSA were
analyzed and represented with LigPlot+ version v.1.4.5
5-(E)-(2-naphthylvinyl)-2ꞌ-deoxyuridine (1h):44,45
O
NH
HO
5-(2-Benzofuranyl)-2'-deoxyuridine(1b):44,45
N
O
O
O
HO
O
N
Representative procedure was followed from previously
published manuscript: Yield 86%.1H NMR (400 MHz, DMSO-d6) δ
11.50 (s, 1H), 8.25 (s, 1H), 7.84 (dd, J = 7.1, 5.6 Hz, 4H), 7.70 (d, J =
8.7 Hz, 1H), 7.55 (d, J = 16.3 Hz, 1H), 7.48 – 7.40 (m, 2H), 7.01 (d, J
= 16.4 Hz, 1H), 6.17 (t, J = 6.4 Hz, 1H), 5.29 – 5.17 (m, 2H), 4.31 –
4.24 (m, 1H), 3.83 – 3.76 (m, 1H), 3.71 – 3.56 (m, 2H), 2.22 – 2.10
(m, 2H).13C NMR (101 MHz, DMSO-d6) δ 162.6, 149.8, 138.6,
135.4, 133.7, 132.8, 128.6, 128.2, 128.0, 127.9, 126.8, 126.2,
126.2, 123.5, 122.1, 111.2, 87.8, 84.8, 70.3, 61.4, 46.1. ESI-MS
(m/z) = 381 [M + H+], 404 [M + Na+].
NH
HO
O
O
HO
Representative procedure was followed from previously published
manuscript. Yield 88 %. H NMR (400 MHz, DMSO-d6) δ 11.72
1
(s, 1H), 8.70 (s, 1H), 7.58 (d, J = 7.4 Hz, 1H), 7.50 (d, J = 8.1 Hz,
1H), 7.30 (s, 1H), 7.24 (t, J = 7.5 Hz, 1H), 7.17 (t, J = 7.4 Hz, 1H),
6.19 (t, J = 6.3 Hz, 1H), 5.28 (d, J = 4.2 Hz, 1H), 5.20 (t, J = 4.1
Hz, 1H), 4.30 (s, 1H), 3.84 (d, J = 2.8 Hz, 1H), 3.74 – 3.59 (m,
2H), 2.25 – 2.15 (m, 2H).13C NMR (101 MHz, DMSO-d6) δ
160.7, 153.4, 149.7, 149.4, 137.5, 129.1, 124.7, 123.4, 121.4,
111.2, 105.1, 104.3, 87.9, 85.5, 70.4, 61.2, 40.8. ESI-MS (m/z) =
345 (M+ + H+).
5-(E)-(4-(2-benzofuranyl) styryl)-2'-deoxyuridine(1i):44,45
O
O
5-(2-Naphthyl)-2'-deoxyuridine (1f):44,45
NH
HO
N
O
O
O
HO
NH
HO
N
O
Representative procedure followed from previously published
manuscript:
O
A solution of Palladium acetate (1.12 mg, 1.0 mol %) and PTABS
ligand (2.93 mg, 2.0 mol %) in degassed CH3CN:H2O (2:1 v/v, 1.0
mL) at ambient temperature under N2 added 5-IdU (0.5 mmol) and
the solution stirred for 5 min at 80°C. After that reaction mixture
allowed to cool at room temperature and then added 4-
bromostyrene acid (0.75 mmol) along with triethylamine (0.14 ml,
1 mmol) and degassed CH3CN:H2O (2:1 v/v, 2.0 mL). The
resulting solution was then stirred at 80°C for 18 hrs. The reaction
progress was observed by TLC analysis, after the completion of
the reaction, palladium acetate (2.24 mg, 2 mol %) and S-phos
HO
Representative procedure was followed from previously published
manuscript. Yield 85 %. 1H NMR (400 MHz, DMSO-d6) δ=
11.59 (s, 1H), 8.37 (s, 1H), 8.14 (s, 1H), 7.93–7.88 (m, 3H), 7.71-
7.68 (m, 1H), 7.54-7.48 (m, 2H), 6.27 (t, 1H, J= 6.2 Hz), 5.28 (d,
1H, J= 1.9 Hz), 5.19-5.17 (m, 1H), 4.32 (s, 1H), 3.84 (d, 1H, J=
2.8 Hz), 3.67-3.58 (m, 2H), 2.33–2.15 (m, 2H). 13C NMR (101
MHz, DMSO-d6) d = 162.4, 150.0, 138.6, 132.9, 132.1, 130.9,
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ligand (5.8 mg, 4.0 mol %) alongwith (hetero)aryl boronic acid
View Article Online
DOI: 10.1039/D0NJ02803G
(1.0 mmol) followed by addition of degassed CH3CN:H2O (1.0
ml) at ambient temperature under N2 added and the reaction
mixture was stirred at 80 °C for further 20 hrs. After the
completion of the reaction, the solvent was removed invacuo and
the resultant residue obtained was purified using column
chromatography in the CH2Cl2/MeOH solvent system (98:2) to
afford the desired product as a white solid compound 1i (70%).
O
O
N
NH
HO
O
O
HO
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1H NMR (500 MHz, DMSO-d6) δ 11.55 (s, 1H), 8.32 (s, 1H), 7.88
(d, J = 8.4 Hz, 2H), 7.69 (d, J = 8.4 Hz, 2H), 7.62 (dd, J = 16.1, 7.9
Hz, 2H), 7.42 (s, 1H), 7.32 – 7.21 (m, 2H), 6.21 (t, J = 6.5 Hz, 1H),
5.26 (d, J = 4.3 Hz, 1H), 5.17 (t, J = 4.8 Hz, 1H), 4.31 – 4.25 (m,
1H), 3.80 (d, J = 3.1 Hz, 1H), 3.60 (m, 2H), 2.27 – 2.13 (m, 2H).13C
NMR (125 MHz, DMSO-d6) δ 162.5, 155.4, 154.6, 150.2, 138.8,
134.1, 129.3, 128.7, 128.7, 125.1, 124.8, 123.7, 121.6, 113.1, 111.5,
102.4, 87.9, 85.0, 70.5, 61.3, 40.6. ESI-MS (m/z) = 421 [M + H+],
444 [M + Na+].
1H NMR (400 MHz, DMSO-d6) δ 11.51 (s, 1H), 8.23 (s, 1H),
7.86 (d, J = 8.3 Hz, 2H), 7.67 – 7.50 (m, 4H), 7.46 – 7.38 (m, 2H),
7.31 – 7.20 (m, 2H), 6.96 (d, J = 16.5 Hz, 1H), 6.15 (t, J = 6.6 Hz,
1H), 5.29 – 5.18 (m, 2H), 4.29 – 4.22 (m, 1H), 3.81 – 3.75 (m,
1H), 3.69 – 3.55 (m, 2H), 2.20 – 2.09 (m, 2H).13C NMR (100
MHz, DMSO-d6) δ 162.5, 155.4, 149.8, 138.8, 138.4, 132.9,
132.7, 129.3, 128.7, 127.2, 127.0, 125.4, 125.0, 122.4, 121.5,
111.4, 111.1, 102.3, 87.8, 84.8, 70.3, 61.4, 35.4.ESI-MS (m/z) =
447 (M+ + H+), 470 (M+ + Na+)
5-(E)-(4-(2-Naphthyl)phenyl)-2ꞌ-deoxyuridine (1l):44,45
5-(E)-(4-(2-naphthyl) styryl)-2'-deoxyuridine(1j):44,45
O
O
NH
HO
NH
N
O
HO
O
N
O
O
HO
HO
Representative procedure followed from previously published
manuscript:- a solution of Palladium acetate (1.12 mg, 1.0 mol %)
and PTABS ligand (2.93 mg, 2.0 mol %) in degassed water (1.0
mL) at ambient temperature under N2 added 5-IdU (0.5 mmol) and
the solution stirred for 5 min at 80°C. After that reaction mixture
allowed to cool at room temperature and then added 4-bromophenyl
boronic acid (0.75 mmol) along with triethylamine (0.14 mL, 1
mmol) and degassed water (2.0 mL). The resulting solution was
then stirred at 80°C for 18 hrs. The reaction progress was observed
by TLC analysis, after the completion of the reaction, palladium
acetate (2.24 mg, 2 mol %) and S-phos ligand (5.8 mg, 4.0 mol %)
alongwith 2-naphthyl boronic acid (1.0 mmol) followed by addition
of degassed acetonitrile (1.0 mL) at ambient temperature under N2
Representative procedure was followed by using the
previously published procedure: Yield 68%.1H NMR (400
MHz, DMSO-d6) δ 11.50 (s, 1H), 8.21 (d, J = 14.9 Hz, 1H),
7.86 (d, J = 7.4 Hz, 2H), 7.70 – 7.08 (m, 9H), 6.92 (dd, 16.4
Hz, 2H), 6.18 – 6.11 (m, 1H), 5.28 – 5.16 (m, 2H), 4.29 – 4.22
(m, 1H), 3.81 – 3.75 (m, 1H), 3.68 – 3.55 (m, 2H), 2.14 (m,
2H). 13C NMR (100 MHz, DMSO-d6) δ 162.5, 154.6, 149.8,
138.8, 138.4, 129.3, 128.7, 127.2, 127.0, 125.4, 125.1, 125.0,
123.6, 122.4, 121.5, 111.4, 111.1, 110.4, 108.9, 102.3, 87.8,
84.8, 70.3, 61.4, 40.5. ESI-MS (m/z) = 457 (M+ + H+), 480
(M+ + Na+).
°
added and the reaction mixture was stirred at 80 C for further 20
hrs. After the completion of the reaction, the solvent was removed
in vacuo and the resultant residue obtained was purified using
column chromatography in the CH2Cl2/MeOH solvent system
(98:2) to afford the desired product as a white solid compound 1l
5-(4-(2-benzofuranyl)phenyl)-2ꞌ-deoxyuridine (1k):44,45
Representative procedure followed from previously published
manuscript:
1
(75 %). H NMR (500 MHz, DMSO-d6) δ 11.59 (s, 1H), 8.33 (s,
1H), 8.26 (s, 1H), 8.02 (t, J = 7.8 Hz, 2H), 7.96 (d, J = 7.8 Hz, 1H),
7.89 (dd, J = 8.6, 1.5 Hz, 1H), 7.84 (d, J = 8.4 Hz, 2H), 7.72 (d, J =
8.3 Hz, 2H), 7.59 – 7.50 (m, 2H), 6.27 (t, J = 6.6 Hz, 1H), 5.31 (d, J
= 4.2 Hz, 1H), 5.20 (t, J = 4.9 Hz, 1H), 4.32 (td, J = 7.3, 3.7 Hz,
1H), 3.85 (dd, J = 5.9, 2.9 Hz, 1H), 3.64 (m, 2H), 2.29 (dt, J = 13.1,
6.4 Hz, 1H), 2.19 (ddd, J = 13.2, 6.1, 3.6 Hz, 1H).13C NMR (125
MHz, DMSO-d6) δ 162.6, 150.3, 139.0, 138.5, 137.4, 133.7, 132.8,
132.6, 128.9, 128.8, 128.6, 127.9, 127.0, 126.9, 126.6, 125.4, 125.3,
113.4, 87.9, 85.0, 70.6, 61.4, 40.5. ESI-MS (m/z) =431[M + H+],
454 [M + Na+].
A solution of Palladium acetate (1.12 mg, 1.0 mol %) and PTABS
ligand (2.93 mg, 2.0 mol %) in degassed water (1.0 mL) at ambient
temperature under N2 added 5-IdU (0.5 mmol) and the solution
stirred for 5 min at 80°C. After that reaction mixture allowed to cool
at room temperature and then added 4-bromophenyl boronic acid
(0.75 mmol) along with triethylamine (0.14 mL, 1 mmol) and
degassed water (2.0 mL). The resulting solution was then stirred at
80°C for 18 hrs. The reaction progress was observed by TLC
analysis, after the completion of the reaction, palladium acetate
(2.24 mg, 2 mol %) and S-phos ligand (5.8 mg, 4.0 mol %)
alongwith 2-benzofuranyl boronic acid (1.0 mmol) followed by
addition of degassed acetonitrile (1.0 mL) at ambient temperature
Acknowledgements: A.R.K. would like to acknowledge the
Department of Science and Technology for the DST SERB EMR
project (EMR/2016/005439). Alexander von Humboldt
Foundation is also thanked for the equipment grant to A. R. K. The
authors also would like to thank University Grants Commission
India for UGC-SAP fellowship for V. G.; S. B and UGC-CSIR
fellowship for A.V.A.
°
under N2 added and the reaction mixture was stirred at 80 C for
further 20 hrs. After the completion of the reaction, the solvent was
removed invacuo and the resultant residue obtained was purified
using column chromatography in the CH2Cl2/MeOH solvent system
(98:2) to afford the desired product as a white solid compound 1k
(86 %).
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Extended fluorescent uridine analogues: Synthesis, photo-physical
properties and selective interaction with BSA protein
Ajaykumar V. Ardhapurea, Vijay Gayakhea, Shatrughn Bhilarea, Anant R. Kapdia,*, Subhendu Sekhar Bagb,* and
9
Yogesh S. Sanghvic, Krishna Chaitanya Gunturud
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UdR
(Hetero)arenes
O
R'
Fluorescent Protein Sensing nucleosides
HN
UdR
(Hetero)arenes
O
N
dR
(Hetero)arenes
Bovine Serum albumin
UdR