Design of "click" Fluorescent Labeled 2′-deoxyuridines via C5-[4-(2-Propynyl(methyl)amino)]phenyl Acetylene as a Universal Linker: Synthesis, Photophysical Properties, and Interaction with BSA

Article abstract of DOI:10.1021/acs.joc.7b03097

Microenvironment-sensitive fluorescent nucleosides present attractive advantages over single-emitting dyes for sensing inter-biomolecular interactions involving DNA. Herein, we report the rational design and synthesis of triazolyl push-pull fluorophore-la

Full text of DOI:10.1021/acs.joc.7b03097

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Article
Design of “Click” Fluorescent Labelled 2#-deoxyuridines via C5-
4-(2-Propynyl(methyl)amino)}phenyl acetylene as a Universal
{
Linker: Synthesis, Photophysical Property and Interaction with BSA†
Subhendu Sekhar Bag, and Hiranya Gogoi
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.7b03097 • Publication Date (Web): 07 Jun 2018
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Design of “Click” Fluorescent Labelled 2′-deoxyuridines via C5-
{4-(2-Propynyl(methyl)amino)}phenyl acetylene as a Universal
Linker: Synthesis, Photophysical Property and Interaction with
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BSA†
Subhendu Sekhar Bag* and Hiranya Gogoi
th
†
This work is dedicated to Professor Amit Basak (IIT Kharagpur) on his 67 birth day
Bioorganic Chemistry Laboratory, Department of Chemistry, Indian Institute of Technology
Guwahati-781039, India. Fax: +91-258-2349; Te.: +91-258-2324;
Email: ssbag75@iitg.ernet.in.
ABSTRACT
Microenvironment-sensitive fluorescent nucleosides present attractive advantages over
single emitting dyes for sensing interbiomolecular interactions involving DNA. Herein, we
wanted to report the rational design and synthesis of triazolyl push-pull fluorophore labelled
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uridines via the intermediacy of C5-{4-(2-propynyl(methyl)amino)}phenyl acetylene as a
universal linker. The synthesised nucleosides showedinteresting solvatochromic characteristic
and/or intramolecular charge transfer (ICT) feature. Few of them also exhibited dual emitting
characteristics evidencing our designing concept. The HOMO-LUMO distribution showed that
the emissive states of these nucleosides were characterized with more significant electron
redistribution between the C5-{4-(2-propynyl(methyl)amino)}phenyl triazolyl donor moiety
and the aromatic chromophores linked to it leading to modulated emission property.The solvent
polarity sensitivity of these nucleosides was also tested. The synthesized triazolyl benzonitrile
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(10C), naphthyl (10E) and pyrenyl (10G), nucleosides were found to exhibit interesting
intramolecular charge transfer (ICT) and dual (LE/ICT) emission property. The dual emitting
pyrenyl-nucleoside maintained good ratiometric response in the BSA protein
microenvironment enabling the switch-on ratiometric sensing of BSA as the only protein
biomolecule. Thus, it is expected that the new fluorescent nucleoside analogues would be
useful in designing DNA probes for nucleic acids analysis or studying DNA-protein
interactions via a drastic change in fluorescence response due to change in micropolarity.
Key words: Fluorophore labelled uredines; triazolyl donor-acceptor; dual emitting
nucleosides; microenvironment-sensitive; ratiometric fluorescent nucleosides; sensing BSA.
INTRODUCTION
Polarity sensitive fluorescent molecules are ubiquitous for sensing of biomolecules and
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studying inter-biomolecular interactions inside a cell. In particular sensing of local
microenvironment of DNA is highly important in connection with the detection of DNA
mutations causing deleterious effect on cellular survival, high throughput screening and many
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other biotechnological applications. All these events in DNA rely on novel fluorescent probe-
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either as bare or unnatural fluorescent nucleosides or fluorescently labelled natural
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nucleosides. Though many such probe systems in relation to DNA have been reported but the
probes suffer from fluorescence quenching by neighbouring nucleobases or short wavelength
emission or poor microenvironment sensitivity.3a,4 Therefore, designing of novel emissive
probes, particularly, fluorescent nucleosides with unique fluorescence properties, extreme
sensitivity to change in DNA microenvironment and interactions are highly desirable. Among
the three approaches, linking a fluorophore in nucleoside bases is the major approach to
generate fluorescent nucleoside useable for DNA sensing.2-4, 5 As a result of tremendous
research efforts, a large number of fluorescently labelled nucleosides and corresponding
oligonucleotide probes have been designed and utilised to a variety of applications that include
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probing DNA hybridization, typing single nucleotide polymorphism (SNP), and monitoring
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the interbiomolecular interaction, to name a few. However, the majority of the reported
environmentally sensitive fluorescent nucleosides exhibited single band emission that sense
the differences in micropolarity either by change in emission intensity or wavelength.9-12
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Among these, Saito’s ESF nucleosides and fluorene-linked nucleoside by Hocek et al. are
highly attractive for monitoring the micropolarity changes within DNA. However, often
majority of such probes suffer from several shortcomings such as poor microenvironment
sensitivity and low quantum yields.2-5, 13-14
Therefore, to overcome these limitations, the concept of two-band emission would be more
advantageous over commonly utilised single-band fluorescent probes/nucleosides.15 Thus,
recording a ratio of the intensities at two wavelengths would allow ratiometric sensing which
is more advantageous than sensing based on single wavelength emission.16 Basically,
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ratiometric sensing results in an intrinsically calibrated emission response. Ratiometric
probing of DNA, though reported, but is based on labelling of DNA by two interacting dyes
such as FRET pair or excimer/exciplex pair.6a, 7b, 17 However, labelling with two dyes is
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difficult, time consuming as well as highly uneconomical.15-17 On the contrary, a single
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fluorophore with dual emission property would be much more beneficial. Highly increased
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dipole moment and dipole–dipole interactions in the excited state allow such fluorophores to
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be able to sense the changes in local micropolarity within a biomolecular microenvironment or
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in cell. Therefore, dual emissive fluorophores are very useful as ratiometric probe because
they offer facile and straightforward quantification of a biomolecular event through the ratio
of their two bands. However, due to the scarcity of such fluorophores that display dual
emissions and the difficulties in their syntheses, the phenomena of dual emission based sensing
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of biomolecular events are poorly explored, especially, in the field of DNA analysis. With a
poor literature reports and the unique ability for sensing the change in the microenvironment
of DNA biomolecules, the design of dual emissive modified nucleosides that can control the
equilibrium between two excited states at ambient temperature without changing the solvent
properties is an unavoidable research area.
As a part of our continuous research efforts in the design of solvofluorochromic
molecules/biomolecular building blocks, 20b, 21 we thought that it would be worthwhile to
design dual emissive modified nucleosides. Based on our experience, literature reports and
wider applicability we considered design of C-5 labeled uridines as model nucleoside probes
useable for DNA analysis in future.3a, 20, 22 However, there is no report wherein C5-position of
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′-deoxyuridine is linked by an electron donor unit as a post-synthetically modifiable functional
group which effectively can generate a modulated fluorescence property of a fluorophore if
attached at the terminus or the terminal alkyne can be reacted with a fluorophoric azide
functionality. Previously, we have shown the “installation/modulation of fluorescence
response” of various small fluorescent molecules and an interesting dual emission behaviour
from pyrene when attached to N,N-dimethylanilino triazole donor unit.23 Inspired by our
previous result and motivated by the importance of dual emitting probe for DNA analysis,19
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we thought that it would be worthwhile to generate a set of fluorescent 2'-deoxyuridine
nucleosides which could show interesting intramolecular charge transfer property or dual
emission. We further thought that attaching an electron donor phenylacetylene unit as a post-
synthetically modifiable functional group at the C5-position of 2'-deoxyuridine would be
beneficial to generate a set of fluorescent 2'-deoxyuridines with modulated fluorescence
property of a fluorophore via azide-alkyne cycloaddition reaction.21-22 Furthermore, the same
nucleoside if incorporated into DNA can offer the opportunity of generating fluorescent
oligonucleotide probes via post synthetic click reaction with modulated fluorescence
property.Following the aforementioned design logics herein we report the synthesis of 5-(3-
((4-ethynylphenyl)(methyl)amino)propynyl)-2'-deoxyuridineas a possible post-synthetically
modifiable nucleoside and its application to generate a set of triazolyl fluorescent 2'-
deoxyuridine. Study of photophysical properties of few such fluorescent 2'-deoxy uridines
revealed interesting solvatochromic photophysical properties corroborating our design
concept. We also tried to support the experimental photophysical properties of three fluorescent
uridines by TDDFT calculation.
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RESULTS and DISCUSSION
The Design Concept
Our design involves the synthesis of
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universal linker, 4-
(Propynyl(methyl)amino)phenyl acetylene and its incorporation into C5-position of 2’-
deoxyuridine. The universal linker containing 2’-deoxy uridine can then undergo Huisgen 1,
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-dipolar cycloaddition reaction with donor-acceptor chromophore containing fluorogenic
azides to afford the target fluorescent uridines. The donor aromatic substituted triazole moiety
was thought to allow an intramolecular charge transfer (ICT) process from triazolo-linked
moiety to the fluorophoric units leading to solvatochromic fluorescence at a longer wavelength.
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Moreover, the fluorophores, such as pyrene, coupled electronically with donor aryltriazoles,
could show dual fluorescence property or interesting modulated solvatochromic emission
response (Figure 1). Thus, our design would ultimately lead to predetermined photophysical
properties of the fluorophores and hence of the nucleoside. The dual fluorescent nucleosides
having ratiomatric fluorescence property could be utilised for DNA analysis if incorporated in
a DNA for the generation of fluorescent oligonucleotide probes.
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Figure 1. The design concept of dual emitting nucleoside.
Synthesis of the Fluorescently Labelled 2'-deoxy Uridines
To generate a series of target fluorescent uredines, we first synthesised the universal linker
containing uridine 8. Thus, the synthesis was started from N-methyl aniline (1) which was
converted first to its p-iodo derivative 2. A Sonogashira coupling with trimethylsilylacetylene
affording compound 3 underwent propargylation to yield TMS-protected linker unit 4. The
TMS-protected donor acetylene linker was then coupled with bis-TBDMS protected 5-iodo-2'-
deoxyuridine through its free propargyl end via a Sonogashira coupling to get compound 7.21
The deprotection of TMS group ultimately afforded the bis-protected 2'-deoxy uridine
containing donor substituted phenylacetylene as universal linker 8 (Scheme 1). Finally, the
universal linker containing uridine was allowed to react with various donor-acceptor
substituted fluorogenic aromatic azides (A-G, Figure 2) synthesised from the corresponding
amines (H-N; See SI Figure S1a) under click reaction condition to afford the target fluorescent
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uredines 9A-G, in diprotected form, in very good yields (Scheme 1). The final products (See
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SI, Figure S1b) were obtained in purity via a silica-gel column chromatography and
characterized by NMR and mass spectrometry.
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Scheme 1. (a) Synthesis of TMS-protected-{4-(2-Propynyl(methyl)amino)}phenyl acetylene
linker (4). (b) Synthesis of diprotected C5-(3-((4-ethynylphenyl)(methyl)amino)propynyl)-2'-
deoxyuridine as a possible modifiable universal linker nucleoside (8) and its various
fluorescent analogous 2'-deoxyuridine nucleosides in diprotected (9A-G) and in deprotected
(10C, E, G) form.
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Figure 2. Chemical structures of the synthesised aromatic azides used.
Studies on the Photophysical Properties
The fluorescent nucleosides possessing good solvofluorochromicity are the ideal candidates
for utilisation in monitoring change in micropolarity inside and outside a DNA duplex.
Therefore, to test whether the synthesised nucleoside would exhibit solvofluorochromic
property for future application in generating fluorescent oligonucleotide probe, we next studied
their photophysical properties in various organic solvents. We also tried to correlate the
solvatochromicity with their donor-acceptor characteristic features. Therefore, after having all
the nucleosides in very pure form via silica-gel (60-120 mesh) column chromatography
followed by recrystalisation from CHCl /MeOH solvent mixture, we proceeded to record the
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UV-visible and fluorescence photophysical properties of few selected synthesised nucleosides.
In general, we observed that the solvent polarity had only a minor influence on the
absorption properties of most of the fluorescent nucleosides. However, the fluorescent emission
property was modulated enormously upon changing the polarity of the solvents. 23-24 Thus, the
analysis of absorption spectra of nitrobenzene containing nucleoside (9B) revealed absorptions
at 290 and 365 nm in dioxane with very little solvatochromism (by 2-4 nm blue shift). Upon
excitation at 365 nm, it showed weak structured emission at around 390 nm with almost similar
intensity in all solvents except for EtOH, DMF, CHCl wherein enhanced intensity was
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observed (Fig. 3a-b; SI, Fig S2 and Table S1). A similar absorption pattern with less
solvatochromicity was shown by the trifluoromethyl coumarin containing nucleoside (9D) with
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absorption at 291 and 375 nm. Very weak and broad emission at 445 nm was its characteristic
when excited at long wavelength absorption band (Fig. 3c-d; SI, Fig S3 and Table S1).
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Figure 3. The UV-visible and fluorescence emission spectra of nucleosides 9B (a-b); 9D (c-d)
and 9F (e-f) in different solvents (10 M).
The nucleoside 9F containing triazolylanthracene exhibited vibronic absorption,
characteristic of anthracene at 386, 367, 348 nm, with negligible solvatochromicity. However,
it showed interesting emissions when excited at 365 nm. In highly polar and protic solvents it
only showed structured emission characteristic of anthracene centered at 415 nm which we
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characterized as emission from a locally excited (LE) state. However, in nonpolar solvents such
as in dioxane and in moderately polar solvents such as CHCl , EtOAc and THF the nucleoside
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exhibited dual emission. It possessed LE emission bands at around 415-420 nm and
solvatochromic ICT bands with decreased intensity at 490, 493, 534 and 543 nm in dioxane,
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CHCl , EtOAc and THF, respectively (Fig. 3e-f; SI, Fig S4 and Table S1).
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Benzonitrile chromophore containing nucleoside 10C is an interesting fluorescent
nucleoside showing highly solvatochromic ICT emission. Thus, the UV-visible spectra showed
short wavelength absorption at 288 and long wavelength weak absorption centered at 346 nm
in almost all solvents (Figure 4a). Upon excitation at long wavelength absorption band, we
observed structureless, broad and strong solvent polarity dependent emission (Figure 4b). It
showed a strong emission in dioxane at 471 nm which experienced 80 nm red shift when the
polarity of the solvent increased to ACN/DMF (551 nm) with gradual decrease in intensity. In
polar protic solvent like methanol, H-bond mediated quenching event resulted in almost no
emission.24a, 25a Upon titration of a solution of 10C in dioxane by water a red shift in emission
of about 72 nm was observed in 30% water while decreased intensity along with negligible
shift in wavelength was the result observed from its absorption spectra in various solvents.
Beyond this, addition of further water led to almost full quenching of fluorescence (Figure 4c-
d). These features indicated an emission from an ICT state.23-24, 25b-g Fluorescence quantum
yields are also found to follow the similar trend as solvent polarity was increased (SI, Table
S1-4).We measured the fluorescence lifetime for this nucleoside which showed biexponential
decay in all solvents following a similar trend as that was observed in steady state emission
upon changing the solvent polarity. As for an example, in acetonitrile the average life time and
quantum yield were 1.75 ns and 0.04 ns, respectively, while in dioxane the corresponding
values were 2.8 ns and 0.34 ns, respectively (Table 1; SI, Fig. S10-S11). This may be because
of the fluorescence quenching through non-radiative pathway via hydrogen bonding which is
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again supported from an increased non-radiative rate constant (Knr) from 2.31 in dioxane to
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.46 in ACN (Table 1).24a
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1
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1
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1
2
2
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2
2
2
2
2
2
2
3
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3
3
3
3
3
3
3
4
4
4
4
4
4
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4
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5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 4. (a) UV-visible, (b) fluorescence emission spectra in various organic solvents for
compound 10C. Dioxane-water titration: (c) UV-visible, (d) fluorescence emission spectra for
same. Concentration of the nucleoside 10C was 10 M.
Table 1. Summary table of fluorescence lifetimes of the fluorophore 10C.
Solvents
Dioxane
THF
ACN
D
95
W
5
D
85
W
15
D
80
W
20
70 30
D W
1
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6
Properties

abs
f
0.021
81,338
0.21
281,341
0.305
282,33
1
0.186
281,
336
0.249
282,
334
0.262
284, 335
0.277
283,
339
2


(nm)
max
fl
max
4
72
507
546
510
530
534
539
(nm)

[ns]
f
0.345
0.72
(33%)
3.87
(67%)
1.22
2.31
0.291
0.18
(33%)
4.35
(67%)
0.98
2.38
0.044
0.82
(31%)
2.17
(69%)
0.25
5.46
0.259
0.30
(53%)
4.73
(47%)
1.08
3.08
0.055
1.55
(73%)
4.27
(27%)
0.24
4.13
0.039
1.06
(82%)
4.49
(18%)
0.23
5.72
0.019
0.82
(78%)
3.78
(22%)
0.13
6.76
0
1
2
3
4
5
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7
8
9
0
1
2
3
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8
9
0
1
2
3
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
τ
1
τ
2
[ns]
8
−1
f
k [10 s ]
k
8
−1
nr [10 s ]
p q
D W = Dioxane (D) and water (W) solvent mixture; p and q are volume of each solvent.
The naphthalene containing nucleoside (10E) showed structureless absorption at around
2
88 nm with little solvatochromicity as was revealed both from the spectra in various organic
solvents as well as dioxane-water titration (Figure 5a-b). As the polarity increases only a slight
increase in intensity was observed. However, strong solvent polarity dependent emission was
shown by it that was centered at around 449 nm in dioxane upon excitation at 290 nm. A
spectral shift of 80 nm was observed with increase in intensity as we move from dioxane to
ACN (529 nm). However, in polar protic solvent, such as MeOH, the intensity and hence the
quantum yields were decreased which might be due to H-bonding effect (Figure 5c; Table S1-
S3, S5). A dioxane-water titration experiment revealed a red shift of 83 nm (449-532 nm) upto
50% dioxane-water mixture. These results were clearly an indication of an ICT emission.
However, addition of 30-60% water revealed another band at 388 nm with increase in intensity.
Beyond 70% water the single band at 388 nm was prominent which might be the LE emission
(Figure 5d).
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0
1
2
3
4
5
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 5. (a-b) UV-visible spectra in various organic solvents and in dioxane-water solvent
system, respectively, and (c-d) corresponding fluorescence emission spectra for the nucleoside
1
0E. Concentration of the nucleoside 10E was 10 M.
Even more interesting photophysics was observed in case of pyrene labeled nucleoside
(10G) which showed slight blue shifted (by 2-4 nm) solvatochromic absorption of the
structured bands at 328 and 344 nm when changing the polarity from dioxane to methanol
(Figure 6a). The nucleoside 10G showed dual emission behavior in low polar solvents like,
toluene, dioxane, chloroform, ethylacetate and THF. As the solvent polarity was increased the
long-wavelength ICT band at 453 nm in toluene shifted to 537 nm in THF with decreased in
intensity. The LE band in these solvents consists of structured pyrene like emission at 385 and
4
03 nm. However, in highly polar solvent ACN and in polar protic solvent MeOH, EtOH
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structured band characteristic of a pyrene emission (only LE emission) had been observed
(
Figure 6b). A solvatochromicity test in dioxane-water mixture also supported this observation
(Figure 6c-d). Thus, the ICT emission at 487 nm was highly dominated over the LE emission
in pure dioxane. As the % of H O was increased upto 40%, the intensity of ICT band decreased
gradually with strong red shift of 89 nm while the intensity of LE emission increased gradually.
However, in 50% H O in dioxane and beyond we observed pure LE emission (Figure 6d). The
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
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8
9
0
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0
1
2
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7
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9
0
1
2
3
4
5
6
7
8
9
0
2
2
fluorescence quantum yield of 10G also followed the similar trend (SI, Table S1-3, S6-S7).
Figure 6. (a) UV-visible, (b) fluorescence emission spectra in various organic solvents for
compound 10G. Dioxane-water titration: (c) UV-visible, (d) fluorescence emission spectra for
same. Concentration of the nucleoside 10G was 10 M.
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To interpret the photophysical properties in a more intuitive manner, the time resolved
fluorescence spectra was recorded for pyrenyl nucleoside (10G) in different solvents which
also supported observations of steady state fluorescence. Thus, we collected the lifetime data
by monitoring both at LE and ICT bands with an excitation light of 337 nm which displayed
biexponential decays (Table 2; SI, Table S6-7). The relative contribution of the longer lifetime
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
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7
8
9
0
1
2
3
4
5
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7
8
9
0
component (
polarity was increased (in THF, 
.99 ns ) to 61% ( = 0.66 ns) for the case of shorter life time component when the decay was
monitored at LE emission (400 nm). In methanol, the component contributing 49% has decay
time  = 7.64 ns while the other component having decay time  = 0.59 ns contribute 51%
2
= 10.88 ns) in dioxane was found to decrease from 49% to 39% as the solvent
1
= 7.13 ns) while the same was increased from 51% (
1
=
0
1
2
1
suggesting only a weak emission from LE state and the chromophoric unit followed a non-
radiative pathway producing no ICT emission in methanol (Table 2). Furthermore, the value
of 
according to the observed wavelength in dioxane and THF. Thus, the contribution from longer
lifetime component  increased from 49% to 57% as the monitoring wavelength was changed
from 400 nm to 495 nm in dioxane solvent. Similar behavior was observed in THF solvent.
The decreased intensity of ICT band was also reflected by the decreased lifetime from  = 0.13
in dioxane) to  = 0.06 ns in THF (Table 2; SI, Fig. S12-13). Furthermore, the value of  and
remain consistent while the relative contributions of the two lifetimes vary according to the
1
and 
2
remain consistent while the relative contributions of the two lifetimes vary
2
1
(
1
1

2
observed wavelength in dioxane-water solvent mixture (Table 2).
Table2. Summary table of fluorescence property of nucleoside 10G in various solvents.
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6
Solvents
Properties
Dioxane
THF
ACN
D
95
W
5
D
85
W
15
D
70
W
30
50 50
D W

f
0.021
28, 343
0.21
343
0.305
327, 341
0.186
328 , 343
0.249
328 , 343
0.277
328 , 343
0.294
328, 343
abs
max
3


(nm)
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
fl
max
3
87, 400,
94
0.007
386, 403,
533
0.002
384, 400, 386, 401,
590
0.003
386, 401,
550
0.022
386, 400,
570
0.038
385, 399,
580
0.038
(nm)
4
526
0.024
1.5(32%)

f
τ
1
[ns]
0.9(51%)
0.6(61%)
1.0 (42%)
1.4(29%)
1.8(26%)
2.2(21%)
τ
2
[ns]
10.8(49%)
0.012
1.68
0.054
0.13(43%) 0.06(49%) 0.1(40%)
1.3 (57%)
0.71
7.1(39%) 11.7(58%) 23.4(68%) 26.8(71%) 28.4(74%) 29.3(79%)
0.006
3.13
LE
k
f
0.004
1.37
0.002
0.015
0.60
0.027
0.3(49%)
3.4(51%)
0.14
0.011
0.50
0.01
0.2(56%)
1.3(44%)
0.15
0.017
0.44
0.003
0.1(56%)
1.3(44%)
0.05
0.016
0.41
0.0005
0.3(92%)
1.31(8%)
0.013
k
nr

f
0.035
τ
τ
1
[ns]
ICT
2
[ns]
1.9 (51%)
0.35
1.4 (60%)
0.021
10.73
k
f
k
nr
12.44
9.55
5.23
15
15.82
25.63
8
-1
f p q
k and knr in 10 s ; D W = Dioxane (D) and water (W) solvent mixture; p and q are volume of each solvent.
To get an insight into the different solvatochromic behaviours of benzonitrile (10C),
naphthyl (10E) and pyrenyl (10G) nucleoside, the spectral dependency on solvent polarity was
2
6a
studied on the basis of Lipert-Mataga model. The polarity parameter of various solvents were
calculated from the following equation 1.26
2

−1 n −1

f =
−
(1)
2
2 +1 2n +1
abs
max
fl
max
We tried to correlate the absorption and fluorescence maxima (% and % , respectively,
-1
in cm ) with the solvent polarity parameter Δf for the nucleoside 10C, 10E and 10G (Figure
a-c). Thus, from the plots it was clear that for all the cases, the νabs values apparently correlate
linearly with Δf values. This indicated that the ground state of these fluorophoric nucleoside
were moderately polar in nature. However, the νfl values for all the nucleosides showed a very
7
̃
̃
good linear correlation with Δf for the whole range of the solvent polarity tested indicating that
the nature of the fluorescent states remained essentially unchanged in all the solvents for all
cases. However, due to the modulation by the solvents, the spectral feature changes
significantly. The highly polar nature of the fluorescence states of these nucleosides were
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evident from the high slopes of the ν
̃fl vs Δf plots indicating their possible intramolecular charge
transfer (ICT) character.23-25, 27
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
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4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 7. Plots of ν
10E (b) and 10G (c) (ν
f in different solvents for 10C, 10E and 10G.
̃fl and ν̃
̃
̃
̃

Next, the Stokes’ shift (Δν
̃
2
6
equation 2 where μ
e
and μ
g
are the excited (fluorescent) state and the ground state dipole
moments of the fluorophore, h is Planck’s constant, c is the velocity of light and r is the Onsager
radius of the dipole-solvent interaction sphere.
2
2
( −  )
e
g

%= %+
f
(2)
0
2
hcr
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6
The linear plot with large slopes suggested a good correlation between Δν̃ values for all the
three nucleosides, 10C, 10E and 10G (long wavelength emissive band) in different solvents
(Figure 7d) indicating the fluorescence states are highly polar in nature.
0
1
2
3
4
5
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8
9
0
1
2
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4
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9
0
Theoretical Calculation
We next carried out theoretical calculation to support the observed polarity-dependent
2
8
emission and the ICT feature using Gaussian 09 program package. The possible transitions
obtained from a TDDFT calculation, were, thus, analysed (Figure 8; SI, section 5). From the
HOMO-LUMO overlap and transition oscillator strength, it is clear that the S
transitions are fully allowed for all the three representative nucleosides indicating the reverse
transition, i.e., S ←S , as fully allowed. Redistribution of electronic charge density was
0
→S electronic
1
0
1
reflected from an overlap in HOMO-LUMO supporting the solvatochromicity and
intramolecular charge transfer emissions (Figure 8).23, 29 As for example, except for coumarin
(9D), the TDDFT calculations suggested the S
0
→S transitions with high configuration
1
interaction (CI) values as the dominant orbital transitions in the low-lying singlet excited states
of the studied nucleosides. Interestingly, the universal linker unit with the triazole at C5-
position of 2’-deoxyuridine comprised the HOMO, while the aromatic fluorophoric unit
irrespective of their substituents formed the LUMO in all the studied nucleosides supporting
our designing concept. Except for the case of coumarin (9D), the triazole unit overlapped with
the LUMO of the chromophore indicating a good electronic redistribution and ICT character
(SI, section 5).
Thus, the calculated excitation energy for the transition S
0
→S for pyrene (10G) and
1
benzonitrile (9C) containing fluorescent nucleosides were found to be 436 nm (2.84 eV, f =
.1299; CI = 0.704); 407 nm (3.05ev, f = 0.217, CI = 0.705) (in vacuum), respectively. These
values co-related with the experimental results of 343 nm (THF) and 346 nm (THF)
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respectively. Other two prominent transitions are at 414.8 nm (2.98 eV; f = 0.0006; CI = 0.703)
and 348.8 nm (3.56 eV; f = 0.508; CI = 0.641) for pyrenyl nucleoside and at 376.4 nm (3.29
eV; f = 0.0010; CI = 0.705) and 315 nm (3.93 eV; f = 0.0551; CI = 0.702) for benzonitrile
nucleoside (Figure 8a-b). For the case of naphthalene (10E) and coumarin (9D) labelled
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
nucleosides, along with weak S
are observed from S →S and S
of these nucleosides as LE state.
0
→S
1
transition (f = 0.0046/0.0008), the prominent transitions
0
2
0
→S
3
, respectively (SI, section 5) indicating the emissive states
From the strong HOMO-LUMO mixing it is clear that the emissive state of bezonitrile (10C)
is characterized with more significant electron redistribution, i.e., ICT feature. The calculations
rationalized the explanation of ICT origin of the solvent polarity dependency of the
nucleosides’ emission (Figure 8a). The pyrene nucleoside (10G) showed dual emission. The
long wavelength band is highly sensitive to polarity of the solvent i.e., ICT origin. Thus the
dual emission comes from both LE and CT states. The transition from HOMO-1 to LUMO is
closer to the experimental value with higher f value (f = 0.508) indicating that the S state is
2
populated well (Figure 8b; SI, Section 5.3).
Figure 8. The possible transitions from TD-DFT calculation for the representative nucleosides
containing (a) benzonitrile (10C) and (b) pyrene (10G).
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Study of Interaction with BSA
Finally, the dual emitting nucleosides, in particular the pyrenyl nucleoside, 10G, is both
fundamentally and conceptually new and novel which could serve as sensitive probe for
0
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0
1
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0
studying nucleoside-protein/nucleoside-DNA interaction and in other wider applications.30
Understanding the nature of drug-protein interactions or small molecule-protein interaction is
of paramount importance for the development of new chemotherapeutics. Since last 50 years,
the nucleosides analogues have become the corners stone for clinical use for the treatment of
cancer or viral infections. However, only about 40 nucleoside analogues have been approved
3
1
for clinical use till the date and few are in clinical trials. Despite their broad range of biological
activity, many nucleosides could not find applications as drugs mainly due to poor cell
membrane penetrability.31-32 Furthermore, the nucleoside metabolism is vital to cellular
survival. About half of all enzymes are nucleoside-dependent. Therefore, study of nucleoside-
protein interaction and understanding the structure–function relationships of nucleoside-
binding proteins are highly important to get functional insights for many proteins.31-32
Therefore, immense clinical importance of nucleosides and their analogues put in force to
accumulate knowledge how they are being recognized and transported into the cell.
Understanding such events could not only provide knowledge about nucleoside-related
biological processes but also help designing of new nucleoside derivatives as possible drugs.33
Furthermore,the biological roles of nucleosides are dependent on specific interaction with
cellular proteins. As for an example, nucleotide-protein interactions are considered critical in
the action of some purine nucleoside analogs or isoniazid drugs that have recently been for the
treatment of Mycobacterium tuberculosis (Mtb) infections.34
Selective binding of small molecules/nucleosides have great impact on the functions of
many proteins, especially with plasma proteins. Therefore, study of specific interaction with
nucleoside/ nucleotide and plasma proteins is highly important. In this view, we, therefore,
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thought that the dual emission from the new nucleoside 10G might offer some interesting
insight into the interaction with a biomolecule such as a model protein bovine serum albumin
(BSA). With 76% sequence identity to human serum albumin (HSA), BSA is widely used as a
model protein in investigating protein-drug interaction in detail.35 Moreover, BSA is
experimentally well accessible. Similar to HAS, BSA contain three linearly arranged domains
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(
I–III). It has two major drug binding sites situated in the subdomains IIA (site I) and IIIA (site
II).36 The two tryptophan (Trp) residues, Trp134 and Trp 212, are located in the outer
hydrophilic environment and in the hydrophobic binding pocket of site I of BSA which is
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widely used to study drug-BSA interaction fluorimetrically. Thus, we finally decided to study
the interaction of 10G with model protein BSA using UV-visible and fluorescence
spectroscopy. Our main aim was to test whether the pyrenyl fluorescent nucleoside 10G could
interact with any biomolecule, such as a protein biomolecule. Moreover, we wanted to know
that in protein’s microenvironment the nucleoside probe whether could retain its dual emission
property. Therefore, at this stage, we did not consider the probes for studying interaction with
other proteins to test their protein binding specificity. This extended study along with the study
with DNA biomolecule is under consideration of our laboratory.
The UV-visible spectra in phosphate buffer revealed that the probe nucleoside 10G
exhibited very weak, broad and structureless short and long wavelength absorptions at around
2
83 and 351 nm. The characteristic pyrenyl absorption was not present in contrary to that in
the organic solvent. Upon gradual addition of BSA to a solution of the probe, the absorption
bands experienced strong hyperchromicity and hypsochromic shift of 5-7 nm along with
appearance of characteristic pyrenyl bands at around 345 and 329 nm. This indicated a strong
binding interaction of the probe in the hydrophobic region of BSA (Figure 9a; SI, Section 6).
The Job’s plots suggested a 1:1 probe: BSA binding events (Figure 9b). The association
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constant was also evaluated from a Benesi-Hildebrand plot showing value of 1.0 x 10 M and
-1
30, 32
binding free energy -5.5 kcal/mole (Figure 9c; SI, Section 6).
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Figure 9. (a) UV-visible spectra of 10G in absence or in presence of BSA. (b) Absorption
Job’s plot of probe, 10G in presence of BSA protein indicates a 1:1 stoichiometry of the probe
to BSA in the complex. (c) Benesi-Hildebrand plot. (d) Fluorescence emission titration spectra
of 10G in absence or in presence of BSA (λex = 342 nm).The probe concentration [10G]
=10µM.
Next, a fluorescence titration experiment was carried out to investigate the BSA sensing
ability. In buffer, the probe showed dual emission, LE (at 381, 398 and 427) band characteristic
of pyrene and ICT band at 501 nm. Interestingly, the nucleoside 10G retained its dual emitting
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property in all the concentration of BSA. It showed a regularly increased in both the LE and
ICT bands when excited at 343 nm upon gradual addition of an increasing amount of BSA
(Figure 9d). The quantum yield variations also supported the same. Thus, the quantum yields
of only probe, 1:1 Probe: BSA, 1:22 and 1:30 probe (10G): BSA in 2% DMF in buffer were
found to be 0.0025 (LE), 0.003 (ICT); 0.006 (LE), 0.006 (ICT); and 0.009 (LE), 0.014 (ICT),
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.007(LE), 0.015 (ICT); respectively (SI, Table S8). While position and shape of the LE band
remained unaltered, peak broadenings and blue shits were the major results of ICT bands with
increasing concentration of BSA similar to what was observed in organic solvents or in
dioxane-water titration experiments of the probe (SI, Figure S7). All these observations clearly
suggested the accommodation of the fluorophoric pyrenyl moiety of the probe inside the
hydrophobic pocket of BSA. Therefore, even in the BSA microenvironment, the probe 10G
behaved as a system of dual emitting switch-on fluorescent probe.
Figure 10. (a) Plot of intensity of LE or ICT and of (b) LE/ICT vs. equivalent conc. of BSA
added. The probe concentration [10G] =10µM.
The LE, ICT fluorescence intensity and the ILE/IICT ratio (I398/I500) were plotted against the
equivalent concentration of BSA added to clearly demonstrate the fluorescence behaviour of
the ratiometric fluorescent pyrenyl nucleoside probe. Thus, from the plot in figure 10a it is
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clear that the intensity of both the LE and ICT bands increased upto 14 equivalent of BSA with
respect to 10 μM probe concentration. Further addition of BSA does not induce notable change
in both LE and ICT bands. The same plot for ratio LE/ICT vs. equivalent concentration of BSA
added shows that the LE/ICT values decreased or ICT/LE increases exponentially indicating
that the probe nucleoside 10G functions as a good ratiometric BSA sensor (Figure 10b).38
Ratiomatric fluorescence sensing offers increased signal-to-noise ratio leading to more reliable
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quantification compared to that by using a probe of emission at a wavelength. Furthermore,
ratiometric fluorescence is more reliable compared to absolute fluorescence intensity in
demonstrating sensing events because the ratios do not suffer from simultaneous drifts or
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fluctuations of individual signals. Therefore, the probe nucleoside 10G could find wide
applications in ratiometric sensing in chemistry, biology and in materials sciences.38-40
Figure 11. (a) Docking pose and (b) various interactions of the nucleoside 10G inside the
hydrophobic pocket of BSA.
Finally, we carried out molecular docking calculation to get insight into the binding site of
4
1
the probe nucleoside using Autodock 4.2. From the docking study, it is clear that the dual
emitting pyrenyl nucleoside 10G binds to both the chains of BSA. As revealed from the
docking pose in figure 11a, the Leu115, Pro117, Asp118, Leu122, Lys136, Glu 140, Ile141
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and Tyr137 residues of chain B was involved in hydrophobic interactions mostly with the
chromophoric phenyl triazolyl pyrenyl unit.30, 42 The π-stacking between pyrenyl unit and the
closely spaced Tyr made the association strong. On the other hand, the uridine ring and the
sugar-OH functionalities are involved in strong H-bonding interactions with Leu112, Lys114
and Glu519 of chain A and Glu125 of chain B of BSA leading to increased association (Figure
1
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1b). The overall free energy change associated with the interaction of the probe nucleoside
with BSA obtained from molecular docking was found to be -9.2 kcal/mol. The α-helicity of
the BSA upon binding to the probe remained unperturbed as was evident from a CD spectral
titration. The slight increase in intensity and % α-helicity of BSA was possibly because of
conformational adjustment of BSA upon association with the probe nucleoside (SI, Section
6
.5).
SUMMARY and CONCLUSION
Therefore, all the experimental and theoretical results clearly showed that the fluorogenic
aromatic azides having no substituent or electron withdrawing substituent, such as –CN, upon
reaction with the universal donor phenylacetylene linker of the 2’-deoxyuridine under “click”
reaction condition would able to generate interesting fluorescent nucleoside. The main
importance of our work is that we utilized a simple donor phenylacelene as linker to generate
solvatochromic fluorescent nucleoside via click reaction with readily available fluorogenic aryl
azides. The linker labelled nucleoside would be useful in generating oligonucleotide which can
be made fluorescent post-synthetically and can then be utilised as probe for DNA analysis.
Furthermore, the fluorophoric moieties in these nucleosides showed a direct correlation
between the fluorescence intensities and the solvent polarity. All these results along with the
fluorescence band-shape and quantum yields revealed a correlation between the D-A structure
and the emissive states. That the emissions from the nucleoside 10C, 10E and 10G were
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originating from ICT states, indicated by their strong fluorescence emission with broad band-
shapes, high quantum yield, high solvofluorochromicity. In addition to that, the nucleoside
1
10G exhibited solvent polarity independent LE emission originated from a non-polar −*
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state. Therefore, the dual emission as was observed for nucleoside 10Gindicated the presence
of a mixed LE and CT state wherein the switching between these two states depends on the
structure and the solvent polarity.
The strong polarity sensitive ICT emission from these nucleosides can be utilized in
monitoring the change in micropolarity inside and outside a DNA duplex. Thus, these
nucleosides would be useful in generating fluorescent oligonucleotide probes for DNA
analysis. Furthermore, the sensing index derived from the intensity ratio between ICT and LE
emission could be employed for the same. The dual emitting fluorescent pyrene containing
nucleoside would impact greatly in nucleoside research as rare examples of such nucleosides
exist in literature. Further, the donor phenylacetylene linker reported here is interesting and
would find wide applications in generating fluorescent nucleosides or post synthetically
derived fluorescent oligonucleotide probes. Our current research activity focuses in this
direction. Finally, the preliminary study suggested that the dual emitting nucleoside was
efficient to interact with BSA via a switch on fluorescence and was able to retain its
photophysical property in the biomolecular microenvironment. As the ratiometric fluorescence
sensing is highly advantageous compared to a single wavelength emission, the ratiometric
fluorescent nucleoside probe 10G would find wide future application in chemistry, biology and
in material sciences.
EXPERIMENTAL SECTION
Materials and Methods
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All reactions were carried out under inert atmosphere using flame-dried glassware.
Combined organic layers were dried over anhydrous sodium sulphate. After work up solvents
were removed in a rotary evaporator under reduced pressure. For column chromatography
Silica gel (60- 120 mesh) was used. Reactions were monitored by TLC on silica gel 60 F254
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13
(
0.25). H NMR spectra were recorded either at 400MHz or at 600 MHz and C NMR spectra
were recorded either at 100 MHz or at 125 MHz (mentioned accordingly). Coupling constants
J value) were reported in hertz (Hz). The chemical shifts were shown in ppm downfield form
(
1
13
tetramethylsilane, using residual chloroform (δ = 7.26 in H NMR, δ = 77.23 in C NMR) or
1
13
DMSO (δ = 2.5 in H NMR, δ = 39.5 in C NMR) as an internal standard. All the NMR-FID
has processed in MestReNova v6.0.2 software. High-resolution mass spectra (HRMS) were
recorded on a Mass spectrometer in positive mode using electrospray ionization-time of flight
(ESI-TOF) reflection experiments. IR spectra were recorded on KBr plate in a FT-IR
-1
spectrophotometer and reported in frequency of absorption (cm ).
Characterisation
Synthesis of 4-iodo-N-methylaniline (2): This compound was synthesised following a
modified literature procedure. 43a N-methylaniline (1, 3.95 g, 13.84 mmol) was taken in a round
bottom (R.B.) flask and was dissolved in 20 ml of pyridine: dioxane (1:1). The resulting
o
reaction mixture was cooled to 0 C in an ice bath and half of the total 5.6 g (22.14 mmol) of
I
2
was added to it. While maintaining the ice cooled condition, remaining half amount of I was
2
added after 2 hours. After stirring for about 3 h, ice bath was removed and the reaction mixture
was stirred at room temperature for another 22 h. After completion of reaction, monitored by
TLC, the reaction mixture was partitioned between ethyl acetate and water. Collected organic
layer was washed with saturated sodium thiosulfate solution, water and brine solution. After
evaporation, the crude product was purified by column chromatography (Si-gel, Hex: EtOAc
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6
1
=
30:1) to obtain the 4-iodo-N-methylaniline2 as dark yellow liquid. Yield 92 % (7.97 g). H
NMR (CDCl , 400 MHz):  7.4 (d, J = 8.4 Hz, 2H), 6.36 (d, J = 8.4 Hz, 2H), 3.71 (s, 1H),
3
1
3
2
.767 (s, 3H); C NMR (CDCl
3
, 100MHz):  148.9, 137.8, 114.7, 77.7, 30.6.
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Synthesis of N-methyl-4-((trimethylsilyl)ethynyl)aniline (3): This compound was
synthesised following a modified literature procedure. 5 g (21.45 mmol) of 4-iodo-N-
methylaniline (2) was taken in a dry R.B. and dissolved in 20 ml of dry benzene. To the above
solution 10 ml of dry n-butylamine was added and the resulting mixture was degassed for 10
minutes by bubbling N
2
through it. PdCl
2
(PPh
3
) (0.45 g, 0.03 mmol) followed by CuI (0.04 g,
2
0.01 mmol), were added to the reaction mixture while continuing the degassing. Finally,
trimethylsilylacetylene (3.16 g, 32.17 mmol) was added to the reaction mixture and it was
refluxed (70-80 °C) under N atmosphere for 7 hours. After completion of reaction, the reaction
2
mixture was partitioned between ethyl acetate and water. Collected organic layer was washed
with aqueous ammonium chloride solution, water, brine solution and dried over anhydrous
Na
2
SO . After evaporation, the crude product was purified by column chromatography (Si-gel,
4
Hex: EtOAc = 30:1) to obtain product 3 as reddish brown liquid. Yield: 83% (3.62 g). IR
-1 1
(KBr) ̃ 3421, 2147, 1609, 1521cm ; H NMR (CDCl
3
, 400 MHz):  7.06 (d, J = 8.8 Hz, 2H),
13
6
.23 (d, J = 8.8 Hz, 2H), 3.64 (s, 1H), 2.56 (s, 3H), 0.00 (s, 9H); C NMR (CDCl , 100
3
MHz):          +ESI-HRMS calculated for C12H18NSi
+
[M+H] 204.1203, found 204.1198.
Synthesis of N-methyl-N-(prop-2-yn-1-yl)-4-((trimethylsilyl)ethynyl)aniline (4): This
compound was synthesised following a modified literature procedure.43b 2.5 g (12.29 mmol)of
N-methyl-4-((trimethylsilyl)ethynyl)aniline (3) was taken in a R.B. and dissolved in 8 ml of
dry DMF. Anhydrous K
2
CO
3
(2.55 g, 18.44 mmol) was added to the mixture followed by
o
propargylbromide (2.19 g, 18.44 mmol). The resulting reaction mixture was stirred at 50 C for
1
5 hours under N atmosphere. After completion of reaction, the reaction mixture was
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partitioned between ethyl acetate and water. Collected organic layer was washed with water,
brine solution and dried over anhydrous Na
2
SO . After evaporation, the product was purified
4
by column chromatography (Si-gel, Hex: EtOAc = 30:1) and obtained as brown semi solid.
1
1
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-1 1
Yield: 79 %( 2.36g). IR (KBr) ̃ 3279, 2147, 1883, 1605, 1517cm ; H NMR (CDCl , 400
3
MHz):  7.36 (d, J = 8.4 Hz, 2H), 6.71 (d, J = 8.4 Hz, 2H), 4.05 (d, J = 1.6 Hz, 2H), 2.98 (s,
13C
100
3H),
2.17
(s,
2H),
0.2
(s,
9H);
NMR
(CDCl
3
,
MHz):            +ESI-HRMS calculated
+
for C15
H
20NSi [M+H] 242.1360, found 242.1356.
Synthesis of3’,5’-di-O-tert-butyldimethylsilyl-5-iodo-2’-deoxyuridine (6):This compound
was synthesised following a modified literature procedure. 44a 4 g (11.23 mmol) of 5-iodo-2'-
deoxyuridine (5) was taken in a dry R.B. and dissolved in dry DMF. The reaction mixture was
vacuumed and filled with N . Imidazole (3.85 g, 56.49 mmol) was added to the reaction mixture
2
and the clear reaction mixture was vacuumed one more time and filled with N . The above
2
o
mixture was cooled to 0 C in an ice bath and t-butyldimethylsilylchloride (5.08 g, 33.69 mmol)
was added to the reaction mixture. The ice bath was removed after 1 hour and the resulting
mixture was stirred at room temperature under N
the reaction mixture was partitioned between ethyl acetate and water. Collected organic layer
was washed with water, brine solution and dried over anhydrous Na SO . After evaporation,
2
for 18 hours. After completion of reaction,
2
4
the product was purified by column chromatography (Si-gel, Hex: EtOAc = 5:1) and obtained
as white foam. Yield: 96% (6.35 g). IR (KBr) ̃ 3457, 3184, 3062, 2954, 2931, 2857,1694,
-
1 1
1
4
1
607cm ; H NMR (CDCl , 600 MHz):  8.96 (s, 1H), 8.07 (s, 1H), 6.26 (t, J = 6.6 Hz, 1H),
3
.38 (s, 1H), 3.97 (s, 1H), 3.88 (d, J = 11.4 Hz, 1H), 3.75 (d, J = 10.8 Hz, 1H), 2.31-2.28 (m,
H), 2.00-1.96 (m, 1H), 0.93 (s, 9H), 0.88 (s, 9H), 0.14 (d, J = 6.6 Hz, 6H), (0.07) (d, J = 4.8
13
Hz, 6H); C NMR (CDCl , 150 MHz):  160.2, 150.0, 144.6, 88.6, 86.0, 72.7, 68.5, 63.2, 42.2,
3
2
9
ACS Paragon Plus Environment