Triazolyl C-nucleosides via the intermediacy of β-1′-ethynyl-2′-deoxyribose derived from a Nicholas reaction: Synthesis, photophysical properties and interaction with BSA

Article abstract of DOI:10.1016/j.tet.2019.04.038

We report the design and synthesis of triazolyl donor/acceptor unnatural C-nucleosides via alkyne (sugar)—azide (aromatic)1, 3-dipolar cyclo-addition reaction as a key step and studies on their photophysical properties. We have chosen β-1′-ethynyl-2′-deox

Full text of DOI:10.1016/j.tet.2019.04.038

Accepted Manuscript
Triazolyl C-nucleosides via the intermediacy of β-1′-ethynyl-2′-deoxyribose derived
from a Nicholas reaction: Synthesis, photophysical properties and interaction with
BSA
Subhendu Sekhar Bag, Suman Kalyan Das
PII:
S0040-4020(19)30450-8
https://doi.org/10.1016/j.tet.2019.04.038
TET 30283
DOI:
Reference:
To appear in: Tetrahedron
Received Date: 27 February 2019
Revised Date: 14 April 2019
Accepted Date: 16 April 2019
Please cite this article as: Bag SS, Das SK, Triazolyl C-nucleosides via the intermediacy of β-1′-
ethynyl-2′-deoxyribose derived from a Nicholas reaction: Synthesis, photophysical properties and
interaction with BSA, Tetrahedron (2019), doi: https://doi.org/10.1016/j.tet.2019.04.038.
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ACCEPTED MANUSCRIPT
Triazolyl C-Nucleosides via the Intermediacy of β-1'-ethynyl-2'-
deoxyribose Derived from a Nicholas Reaction: Synthesis,
Photophysical Properties and Interaction with BSA†
Subhendu Sekhar Bag* and Suman Kalyan Das
†
This work is dedicated to Professor Isao Saito (Kyoto University, Japan) and Amit Basak (IIT
Kharagpur, India) on their 79th and 68th, respectively, 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.ac.in
ABSTRACT: We report the design and synthesis of triazolyl donor/acceptor unnatural C-
nucleosides via alkyne (sugar)—azide (aromatic) 1, 3-dipolar cyclo-addition reaction as a key
1

ACCEPTED MANUSCRIPT
step and studies on their photophysical properties. We have chosen β-1'-ethynyl-2'-deoxyribose
as a precursor to synthesize triazolyl-C-nucleosides. Overcoming the difficulties, we obtain β-1'-
ethynyl-2'-deoxyribose as a major product following a Co (CO) catalyzed intramolecular
2
8
Nicholas reaction. The 1,3-diaxial interaction is the driving force for the α to β-anomeric
conversion while performing cobalt complexation followed by oxidation to afford β-1'- ethynyl-
2
'-deoxyribose as the major product. A Cu(I)-catalyzed click reaction between different aromatic
donor/acceptor azides and β-1'- ethynyl-2'-deoxyribose generates the desired unnatural triazolyl
cT
donor-acceptor aromatic C-nucleosides ( BDo/Ac) within 30 minutes. Single crystal X-ray
structure shows the puckered conformation of sugar as C3'-exo. Studies on the photophysical
properties suggests good fluorophoric as well as solvatochromic characteristics of these
cTAnth
cTPy
nucleosides. Two of the synthesized nucleosides,
B and
Do
B , are found to interact with
Do
BSA as the only tested protein via a generation of enhanced fluorescence signal. The designed
bases, thus, might find applications in stabilizing a DNA and in the biophysical study thereof, if
a pair of such donor acceptor C-nucleosides could be incorporated into a DNA sequence.
Key Words: Unnatural triazolyl-C-nucleosides; Nicholas reaction; α to β-anomeric conversion;
β-1'- ethynyl-2'-deoxyribose; click chemistry; triazolyl-donor/acceptor chromophores;
fluorescence.
1
. Introduction
The research toward the design and synthesis of non-natural nucleosides that are stable to
enzymatic and acid hydrolysis is a rapidly growing field as a part of expanding the genetic
alphabet, utilizing as therapeutic agents, biochemical probes and study of interactions involving
2

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1
-4
DNA. Benner’s idea of expansion of the genetic alphabet from four to six letters quickly
spreads, resulting in an increasing amount of research aimed at designing new base pairs having
4
f-g
hydrogen bonding patterns orthogonal to those in canonical Watson-Crick base pairs.
Later,
in 1995, Eric T. Kool has opened a new functional dimension with the creation of non-hydrogen
bonded unnatural nucleobase surrogates based on both N- and C-nucleosides. Inspired by Kool’s
work, much effort has been devoted to the development of hydrophobic functional base pairs by
5
a
several research groups. As a result, it is now clear that expansion of the genetic alphabet has
increased the functional potential of DNA. The non-H-bonded base pairs are found to stabilize
DNA via π-stacking and hydrophobic interactions among self-/hetero-pair. Considerable efforts
have thus been invested in delineating the impact of incorporation of non-H-bonded base pairs
such as C-aryl and/or N-aryl unnatural nucleosides into short oligonucleotide sequences either
via enzymatic or chemical means. Moreover, inclusion of unnatural base pairs greatly influences
the structure, stability of DNA duplex and on the efficiency and specificity in replication by
4
DNA polymerases.
In the design of non-hydrogen bonding base pairs, researchers have concentrated mainly on
the factors like, π-stacking, hydrophobicity, steric shape mimicry and in few cases the dipole
5
moment, etc., in the stabilization of DNA duplex. We are the first to uncover the charge transfer
6
a
(
CT) interaction as a novel force for DNA duplex stabilization. However, a limited or no efforts
have been paid for the synthesis of stable triazolyl-C-nucleosides. Though not direct nucleoside,
Nakahara et al., reported the synthesis of triazolyl-C-nucleoside containing DNA using click
7
chemistry via post synthetic modification.
To expand the set of unnatural triazolyl fluorescent nucleobases and keeping in mind the
importance of C-nucleosides, herein we want to report the synthesis of few fluorescent triazolyl-
3

ACCEPTED MANUSCRIPT
C-nucleoside containing donor-acceptor aromatics as nucleobases. The basis of our design
remains in the fact that a pair of donor-acceptor triazolyl-C-nucleosides could form a strong
charge transfer complex similar to our earlier reported triazolyl-N-nucleoside pair. Thus, we
expect that these new sets would impart good duplex stabilization if incorporated into DNA via
charge transfer interaction force which is our future target. We also report the results of
photophysical study and the binding interaction with protein BSA.
2
. Results and Discussion
2
.1.The Concept
As a part of our ongoing research effort towards generation of molecules with tuned
6
b
photophysical properties via click reaction and the recent result on DNA duplex stabilization
6
a
by a pair of triazolyl-N-nucleobases via CT interaction, we thought that it would be worthwhile
to synthesize triazolyl-C-nucleosides containing donor/acceptor aromatics to produce modulated
photophysical response into the unnatural nucleosides (UNNs) and to explore our earlier idea
further. The logic behind the C-nucleoside as target is as follows- (a) C-glycosides are highly
8
stable with respect to enzymatic and chemical degradation , (b) C-nucleosides analogs such as
8
b
pseudo-thymidine or formycin A are useful to create more robust DNA structures, (c) various
useful synthetic strategies are available to expand the genetic alphabet via the C-nucleoside
8
b
generation, (d) many more non-standard hydrogen bonding pattern can be implemented on C-
8a, 9
nucleoside in compared to the N-nucleosides.
Because of all these above reasons C-
nucleosides are being utilized in decorating DNA and RNA both via chemicals as well as
1
0
enzymatic synthesis. Overall, as the triazoles are also metabolically inert and can readily
associate with biological targets through hydrogen bonding and dipole interactions, we thought
4

ACCEPTED MANUSCRIPT
that triazolyl-C-nucleosides if incorporated into a DNA sequence, the triazole as well as aromatic
6
a
units might play an important role in stabilizing a DNA duplex via π-stacking interaction.
Furthermore, we envisaged that the pseudoaromatic 1,2,3-triazole can modulate the electronic
characteristics of the aromatic bases and endow new properties to the unnatural nucleosides.
Moreover, the triazolyl isosteres can also improve charge transfer interaction properties if
incorporated into a DNA.
Utilizing this concept, we framed our objective for the synthesis of unnatural C-triazolyl
nucleosides with donor-acceptor property via alkyne-azide cycloaddition chemistry and study the
photophysical properties of some of them. We also interested to test whether the fluorescent C-
nucleoside could interact with a model protein, such as BSA. For that purpose, we took the help
of fluorescence techniques. The logic behind our aim to study the interaction of nucleoside with
BSA lies in the following facts. Since last 50 years, nucleoside analogues have become the
corner stone for the drug development relating to the treatment for cancer or viral infections and
till the date about 40 nucleoside analogues have been approved for clinical use while many more
1
1
are in clinical trials or at the development stage. Moreover, the metabolism of nucleosides is
vital to a cell’s survival and about half of all enzymes (cellular protein) are nucleoside-dependent
1
2
proteins. Therefore, structural genomics of proteins need understanding the structure–function
relationships of nucleoside-binding proteins. As for an example, nucleotide-protein interactions
are considered critical in the action of some purine nucleoside analogs or isoniazid drugs that
1
3
have recently been utilized for the treatment of Mycobacterium tuberculosis (Mtb) infections.
Thus, functions of many proteins are mostly dependent on selective binding of nucleosides
which participate in processes such as catalysis and signal transduction. Therefore, study of
specific interaction with nucleoside/ nucleotide is central to understand the function of many
5

ACCEPTED MANUSCRIPT
1
2
proteins. In the circulatory system the most abundant protein, Human serum albumin (HSA),
plays a crucial role in multiple biological processes such as it maintains plasma pressure and
balances nutrition in the human body. The structural homolog of it, the Bovine serum albumin
(BSA), is, therefore, extensively used as a model protein in research involving study of drug-
protein interaction. This study would offer insight into protein-small molecules/nucleosides
binding events and to understand primarily the structure, dynamics and functions that ultimately
help in designing newer small molecular drug candidates for biological and clinical applications.
Therefore, we are also interested in bovine serum albumin (BSA) as a model protein for studying
the interaction with new unnatural fluorescent triazolyl-C-nucleosides 1D and 1E (Scheme 1,
Figure 1).
2
.2.Synthesis
There exists several methodologies for the generation of novel C-nucleosides and interesting
8
b,14
nucleobase pairs usable for various DNA based applications.
Despite of that, the Cu(I)-
1
5a, 16
catalyzed azide-alkyne cycloaddition reaction
has got attention only in recent years in the
field of nucleic acid chemistry for the design of artificial nucleobases with interesting properties,
functional nucleic acids of potential value in biology/medicine/material sciences and in
1
5
nanotechnology. As a part of our earlier study we also adopted the click chemistry protocol
utilizing alkynyl sugar and chromophoric aromatic azides for the synthesis of C-triazolyl
nucleoside with interesting photophysical property.
The synthesis of the key intermediate, the alkynyl sugar 1 was achieved starting from
6
a, 17
Hoffer’s chlorosugar and following a modified literature procedure.
Thus, ethynyl
magnesium chloride was allowed to react with bis-toluoylated Hoffers cholorosugar 2 to afford
6

ACCEPTED MANUSCRIPT
bis-toluoylated 1'-ethynyl-2'-deoxy C-nucleoside 3 as an anomeric mixture (β/α) in 92 % overall
1
7a, d
isolated yield.
As the separation by open column chromatography was a tedious job we
1
8
followed intramolecular Nicholas reaction to achieve the separation as well as the β-anomer in
high yield. Thus, the treatment of ethynyl-C-nucleoside 3 with dicobaltoctacarbonyl (Co (CO) )
2
8
afforded the cobalt complex as a 3 :1 mixture of α : β-anomers 4 in 80 % overall isolated yield.
o
Next, the mixture 4 was treated with BF .OEt /Et N at -78 C to get β-anomer as a major
3
2
3
product with a 1:2 mixture of α : β-anomers 5. At this stage we separated the anomeric mixture
via silica-gel column chromatography to afford the desired β-anomer as a cobalt complex 6 in 64
o
%
yield. Finally, the decomplexation was done by treatment with iodine in THF solution at 0 C
and the crude material was subjected to silica-gel column chromatography to get yield of
1
7
bistoluoylated 1'-ethynyl-2'- deoxy-β-C-nucleoside 1 in 80 % yield. This intermediate has been
1
9
utilized mostly in Sonogashira coupling for the synthesis of alkynylated nucleosides. We
wanted to utilize it for the wide synthesis of triazolyl unnatural C-nucleosides in connection to
6
a
our earlier report of synthesis of triazolyl-N-nucleoside.
Thus, after having the key intermediate 1, various donor-acceptor aromatic azides (A-G)
were allowed to undergo copper (I) catalyzed Huisgen 1,3-dipolar azide-alkyne cycloaddition
reaction followed by deprotection with sodium methoxide in methanol to afford triazolyl donor-
acceptor aromatic C-nucleosides 1A-G in 60-96% yields (Scheme1, Figure 1). All the
compounds were characterized by NMR, IR and mass spectrometry.
7

ACCEPTED MANUSCRIPT
Scheme 1. Synthesis of unnatural triazolyl donor-acceptor C-nucleosides.
Aromatic azides used A-G
Synthesized triazolyl C-nucleoside 1A-G
N
A
N3
N
OMe
OMe
N
N
N
N
N
N
cTNDBBDo
HO
cTPyBDo
HO
O
O
(85%)
(61%)
1E
B
C
N3
N3
1A
OH
OH
OMe
N
N
N
N
N
N
OMe
CN
HO
cTDMBBDo
83%)
cTCNBB
Ac
HO
O
O
(
(85%)
1F
OH
1B
OH
D
E
N3
N3
NO2
N
N
N
N
N
N
HO
cTNBB
HO
Ac
cTNapBDo
96%)
O
O
(
86%)
(
OH
OH
1G
1
C
F
N3
N
N
N
NC
cTAnthB
(78%)
1D
HO
Do
O
G
N3
NO2
OH
8

ACCEPTED MANUSCRIPT
Figure 1. Structures of the synthesized unnatural C-triazolyl nucleosides (yields are in
parentheses).
2
.3.Anomeric Conversion and the Mechanism
It is to be noted that the use of Nicholas Reaction to thermodynamically equilibrate the
isomers and to obtain the preferred diastereomer in C-nucleoside is interesting. Below is a
possible mechanism which showed that both the cobalt and the Lewis acid played a crucial role
to drive the anomeric conversion in favour of β-anomer irreversibly (Scheme 2). Once cobalt
complex of anomeric mixture 3 or for an example 3α (α-anomer) was formed, the alkyne moiety
became sterically crowded because of steric interaction between the cobalt complex held at C1'
3
position with the toluoyl group situated at C ' position as shown in 4α. Therefore, the complex
4
α-anomer would always try to get relieve of such strain. Addition of Lewis acid, BF .Et O, the
3
2
sugar “O” got coordinated (A) and formation of B
immediately relieve the steric strain by adopting the planar form B2 and thereby the cobalt
complex at C ' moved from pseudoaxial to pseudoequatorial position. Furthermore, the planar
1
-carbocation was the result which
1
carbocation was also stabilized by cobalt via coordination through the pseudoequatorial lobe of
the sp2 orbital. The oxygen then had the opportunity to close the ring from downside whenever
triethylamine or aqueous solution of sodium bicarbonate was added to quench BF .OEt (Scheme
3
2
2
). This led to the removal of steric strain (as was in 4α conformation) by locating cobalt in the
pseudoequatorial site as shown in 4β. Moreover, the role of anchimeric assistance by the
neighboring >C=O of toluoyl protecting group at C-3 of sugar to stabilize the plannar
carbocation B2 from the α-face and thus, to pull the cobalt complex at C1' to the
pseudoequitorial side could not be ruled out. This would lead to ring closer by the sugar-O to
9

ACCEPTED MANUSCRIPT
afford β-anomer (SI, Scheme S1). Thus, the Lewis acid quenching led to generate β-isomer
which is free from any steric strain (4β). Ultimately, the α:β ratio in nucleoside 4 changed from
3
:1 to 1:2 in nucleoside 5 (Scheme 1-2).
Scheme 2. The possible mechanism of anomeric conversion of 4α to 4β.
2
.4.Structural Studies
Next, we determined the structure of the deprotected nucleoside from NOESY spectra from
which it was found that there was an interaction between 1'H-2'H while 1'H-3'H interaction was
absent in case of triazolylanthracene nucleoside, 1D, suggesting its β-orientation. The β-
1
0

ACCEPTED MANUSCRIPT
conformation of the C-triazolyl nucleoside was further confirmed by single crystal X-Ray
cTDMB
6a
structure of nucleoside 1B (
B ) (Figure2a). The X-ray single crystal analysis showed an
Do
unit cell consisting of two C-triazolyldimethoxybenzene nucleoside with space group P 21
o
Monoclinic, cell length, a = 12.19, b = 4.96, c = 12.46Å; cell angles
α = 90, β = 93.59, γ = 90
3
and cell volume 752.37Å (Figure 2b). The molecules inside the unit cell oriented in an angle to
each other in respect of H-bond acceptor/donor. The triazolyl-N3, 3'-OH and 5'-OH were
involved in H-bonding with other molecule. In particular, triazolyl-N3 was involved in H-
bonding with 3'-OH of another molecule (2.93 Å). The 5'-OH involved in two H-bonding
interactions with 5'- OHs of two other molecules (2.76/1.94 Å).
1
1

ACCEPTED MANUSCRIPT
cTNMB
Figure 2. (a) ORTEP diagram of
B
Do (1B) with thermal ellipsoid at 50% probability. (b)
cTNMB
Unit cell packing of
BDo (1B) viewed from crystal axis “b”. (c) Overall packing
arrangement leading to helical layer like architecture viewed along crystal axis “a”.
o
The CH-π interaction (with a molecular plane angle of 90 ; 2.87/3.37Å) was also found to be
prominent between Hs of -OCH of one C-triazolyldimethoxybenzene nucleoside and π-
3
aromatics from another C-triazolyldimethoxybenzene nucleoside. The sugar’s 2'-H is involved in
interaction with 3'-OH of another molecule (2.56 Å). One of the 5'-H and the 4'-H of another
molecule were also involved in short interaction (2.36Å). The second 5'-H was involed in
interaction with aryl-OMe (2.40Å). The overall crystal structure was found to be similar to that
6
a
of our reported N-triazolyl nucleosides and indicated the ability of the C-triazolyl nucleoside,
1
B, to be engaged in H-bonding as well π-stacking interaction (Figure 2c). Overall a helical
layer like architecture was the result while looking the expanded form from crystal axis “a”
cTDMB
(Figure 2c). Furthermore, the crystal structure of the
BDo nucleoside showed a twist only
o
by 8-9 between the triazole and the dimethoxy phenyl ring which suggested that aromatic ring
unit might involve in stacking interaction with the bases in a duplex DNA if incorporated into a
DNA strand.
2
.5.The Sugar and N2-Glycosidic Conformation
The glycosyl torsion angle in natural nucleosides is weakly associated with a specific sugar
pucker. While a syn-conformation is generally associated with S-sugar pucker, the anti-
conformation can be adopted by both N- and S-sugar puckers. Thus, we calculated the phase
cTNMB
o
angle of pseudo rotation (P) for the nucleoside
B (1B) which was found to be 227.49
Do
1
2

ACCEPTED MANUSCRIPT
0 2 2
and the value of torsion angle about C (1')-C (2')-C (3')-C (4') i.e. θ which is equal to ʋ (= τ )
o
was obtained as -31.18 . The maximum angle of torsion [τ = ʋ = ʋ /cosP] was thus calculated
m
max
2
o
to be 46.145 . From these parameters it was evident that the nucleoside adopted a sugar pucker
as South (S) with C(3') exo-conformation which often found in B-DNA because of its flexible
2
0
20c
nature. It is thus similar to our reported tetrazolylnucleoside
but opposite to the N-
6
a, 20d
triazolylnucleosides
which adopted C(3') endo-, North conformation. In the C-triazolyl
nucleosides, the –Ns of triazole ring remained opposite to the sugar O4' making the system of no
repulsion [among the lone pair of triazolyl-Ns and O4'] and stable anti-conformation for
adopting S-sugar pucker. Because of repulsion between lone pairs of triazolyl-Ns and O4', the
base adopted a syn-disposition of the C1/C2-of triazole ring and an anti-disposition of triazole’s
cTNMB
–
Ns. Thus, the disposition of base in
BDo (1B) from the crystal structure revealed its
6
a, 20d
similarity with that in our earlier published N-triazolyl nucleosides.
2
.6.Study of Photophysical Properties
After having all the nucleosides in hand we studied the photophysical properties of few
selective nucleosides in various organic solvents and in buffer. All the UV-visible spectra were
o
recorded (with a 10 µM sample concentration) at 25 C with a cell of 1cm path length and 1nm
slit width. The same sample was used to measure its florescence emission property with a cell of
o
1
cm path length and 3nm emission/excitation slit width at 25 C. Thus, the UV visible spectra of
cTNDB
triazolyl N,N-dimethylamino phenyl nucleoside 1A (
B ) showed structureless absorption
Do
band at around 285-300 nm in various organic solvents. With the increase in the polarity of the
solvent the absorption band shifted towards higher wavelength in the case of ethyl acetate,
acetonitrile. However, in buffer the absorption band experienced a 4-5 nm blue shift (Figure 3a).
1
3

ACCEPTED MANUSCRIPT
Upon excitation at absorption maxima, structureless emission band appeared in each solvent.
With increasing polarity of the solvent the emission maxima experienced a large red shift started
from 380 to 432 nm. In buffer (sodium phosphate buffer, pH 7.0) the emission intensity was
almost appeared to be negligible in comparison to other solvents which might be because of H-
6
b, 20d
bond mediated quenching (Figure 3b and See SI, Fig. S1a, Table S2).
Figure 3. (a) UV-visible absorption and (b) fluorescence emission spectra of C-triazolyl
cTNDB Do
nucleoside, 1A (
B ), in various organic solvents [concentration of the nucleoside 10 µM].
cTDMB
The UV visible spectra of triazolyl-3,5-dimethoxy benzene 1B (
B ) nucleoside
Do
exhibited structureless broad absorption band at around 255-258 nm. With increasing polarity of
the organic solvent the absorption band shifted towards higher wavelength except in buffer
where no shift in wavelength was observed. Absorption bands also experienced a hyperchromic
shift with an increase in the polarity of the solvents (See SI, Fig. S1b). The emission spectra
recorded by excitation at UV maxima at each solvent exhibited structureless emission bands. In
the least polar solvent, 1,4-dioxane, the emission band appeared at 284 nm and in aqueous buffer
it appeared at 410 nm. Overall, the emission band experienced a large red shift of 124 nm with
1
4

ACCEPTED MANUSCRIPT
increasing polarity of the solvents from dioxane through methanol to buffer (See SI, Fig. S1b).
Dioxane-water titration experiment also supported the high solvatochromicity in absorption
cTDMB
shown by the nucleoside. Thus, in dioxane, nucleoside 1B (
B ) showed a structureless
Do
absorption band at 261 nm. With increasing the volume of water the absorption at 261 nm was
found to shift gradually toward lower wavelength region and at 100% water the absorption
maxima ultimately appeared at 253 nm. Thus hypochromicity with 8 nm blue shift was observed
in absorption spectra of the nucleoside as the volume of water was increased (See SI, Fig. S1c-d,
Table S2). This indicated a possible electronic redistribution between triazole ring and
dimethoxy benzene ring. Upon excitation at 260 nm again a structureless broad emission band
was observed at 358 nm in dioxane. With increasing volume of water, the emission maxima
shifted towards higher wavelength region and in 100% water the emission maxima appeared at
4
14 nm. About 56 nm red shift of the emission maxima with the increase of the volume of water
was observed probably due to strong electronic coupling between the triazole and trimethoxy
benzene ring. Thus, the strong electronic coupling decreased the HOMO-LUMO energy gap
which allows the emission maxima shifted to the higher wavelength region.
cTNap
The UV visible spectra of C-triazolylnapthalene nucleoside 1C (
BDo) in various organic
solvents showed structureless broad absorption bands centred at 280-293 nm. With increasing
polarity of the solvents the absorption bands experienced hyperchromic effect. Excitation at
absorption maxima, the emission spectra in various solvents showed emission band at around
3
45 nm whereas in buffer it experienced a 25 nm red shift. This shift in wavelength in polar
buffer is possibly due to polar solvent dipole-solute dipole interaction. This red shift indicated
that the excited state is more polar as compared to ground state. Thus, the polar solvents
stabilized excited state more than the ground state. Therefore, there was an overall decrease in
1
5

ACCEPTED MANUSCRIPT
2
0e
the energy gap between excited and ground state resulting in red shift. With increasing polarity
of the solvents the intensity of the emission spectra decreased indicating a solvent-solute
6
b, 20d
interaction mediated fluorescence quenching (See SI, Fig. S2a-b, Table S2).
cTAnth
The UV visible spectra of triazolylanthracene nucleoside 1D (
B ) exhibited structured
Do
absorption bands at around 346, 364 and 385 nm in various organic solvents. As the polarity of
the solvent increased characteristic blue shift of 5 nm and slight hyperchromicity were observed
(Table 1 and See SI, Fig. S2a). Upon excitation at absorption maxima of 365 nm, the nucleoside
1
D showed structured emission at 392, 417 and 442 nm, characteristics of modulated anthracene
fluorescence. Both the UV-visible and emission spectroscopic properties reflected the
modulation of anthracene photophysics upon electronically coupling of π-electron cloud with
triazole unit. The emission spectra experienced a gradual decrease in intensity as the solvent
polarity increased indicating a solvent-solute or hydrogen bonding interaction mediated
6
b, 20d
fluorescence quenching (Table 1 and See SI, Fig. S2a).
Figure 4. (a) UV-visible absorption and (b) fluorescence emission spectra of C-triazolyl
cTPy Do
nucleoside 1E ( B ) in various organic solvents [concentration of each nucleoside 10 µM].
cTPy
In case of triazolylpyrene nucleoside, 1E ( B ), the UV visible spectra displayed
Do
structured absorption bands, characteristics of pyrene, at around 275, 330 and 345 nm in various
1
6

ACCEPTED MANUSCRIPT
polar and nonpolar organic solvents (Figure 4a, Table 1, and see SI Fig. S2b). The UV-visible
spectra experienced 4-5 nm blue shift with increasing solvent polarity similar to what was
observed in case of triazolylanthracene nucleoside, 1D. Upon excitation at absorption maxima,
the nucleoside displayed structured-emission characteristic of electronically coupled pyrene
emission bands at around 380, 400 and 423 nm. Both the spectroscopic properties reflected the
modulation of pyrene photophysics via electronically coupling of π-electron cloud with triazolyl
ring. However, the electronic coupling between aromatic π-cloud and the triazolyl ring is less in
6
a, 20d
C-nucleoside compared to what we observed in our earlier reported N-nucleoside.
Both the
results followed our initial finding of modulation of fluorescence property via triazole ring
6
b
formation. The emission spectra revealed that with increase in solvent polarity, except in buffer
and DMSO, the nucleoside 1E experienced a hypochromic effect with no promising shift in
wavelength of the emission maxima. Interestingly, in buffer (sodium phosphate buffer, pH 7.0) it
experienced about 4-5 nm blue shift with much enhanced fluorescence intensity (Figure 4b and
Table 1 and see SI Fig. S2b). The blue shift in emission might be the result of dipolar interaction
cTPy
between the triazolyl pyrene unit of
BDo and the polar buffer solvent. This also indicated
more polar ground state compared to its excited state. As the ground state became more polar
than excited state, the ground state would be more stabilized by the polar buffer compared to
the excited state leading to overall increase in the energy gap between excited and ground state
2
0e
and hence the result of blue shift.
Table 1. Summary of UV-visible and fluorescence properties of C-nucleoside 1D and 1E.
Solvents
ꢀ
Dioxane CHCl3
EtOAC DMF DMSO ACN
1D
MeOH
Buffer
Entry
ꢀ
abs
3
3
46,
65,
347,
365,
345,
363,
346,
366,
348,
368,
345,
362,
344,
362,
345,
363,
λ
max
(
nm)
1
7

ACCEPTED MANUSCRIPT
3
85
386
384
386
0.047 0.042
394,
419,
445
388
383
382
384
5
ε
max X 10
0.032
0.031
393,
417,
441
0.037
391,
414,
438
0.031
392,
414,
436
0.049
390,
413,
436
0.006
395,
415,
438
fl
3
4
4
93,
15,
40
398,
420,
443
1E
279,
332,
346
λ
max
(
nm)
Entry
ꢀ
abs
2
3
3
78,
28,
43
278,
330,
344
0.125
383,
400
275,
326,
341
0.129
383,
400
278,
330,
344
0.128 0.115
382,
400
275,
327,
341
0.129
381,
399
275,
327,
341
0.129
380,
399
275,
325,
341
0.079
376,
396
λ
max
(
nm)
5
ε
10
0.125
3
4
max X
fl
82,
00
381,
400
λ
max
(
nm)
On the other hand, the acceptor nucleoside C-triazolyl-o-cyanobenzene nucleoside 1F
cTCNB
(
B ) exhibited structureless broad absorption band at around 279-286 nm experiencing
Ac
blue shift (by 5-6 nm) along with a hypochromic effect as the polarity of the solvent increased.
This property indicated the modulation of photophysical property via electronically coupling
6
b
between π-electron cloud of o-cyanobenzene and the linked triazolyl ring. The excitation
spectra contained band at 366 nm with much higher in intensity than the band appeared at 276
nm (Figure 5a and SI, Fig. S2c and Table S3). Upon excitation at maxima of each solvent (340-
3
73 nm), the nucleoside showed strong emission at around 427-439 nm in organic solvents. The
emission spectra experienced 12 nm red shift as the polarity of the organic solvents increased
which was possibly because of internal charge transfer from donor triazole to acceptor cyano
benzene moiety (Figure 5b and SI, Fig. S2c and Table S3). However, a strongly blue shifted
emission at 366 nm was observed in highly polar aqueous buffer. The blue shift in emission
might be attributed if we consider the ground state of the triazolyl-o-cyanobenzene chromophoric
cTCNB
unit of the nucleoside 1F (
B ) to be more polar compared to the excited state. Therefore,
Ac
the polar buffer solvent would stabilize more the ground state than the excited state leading to
overall increase in the energy gap between excited and ground state. Ultimately, the blue shift in
1
8

ACCEPTED MANUSCRIPT
2
0e
emission was the result. Moreover, the solvent sodium phosphate buffer could play a role in
the excited-state solvent relaxation process. Thus, in such a case the emission might come only
2
0e-f
from the high energy partially relaxed excited-states leading to a blue shift in emission.
Figure 5.(a) UV-visible absorption and (b) fluorescence emission spectra of C-triazolyl
cTCNB
nucleoside1F (
B ) in various organic solvents [concentration of each nucleoside 10 µM].
Ac
2
.7.Theoretical Calculation
To get an idea on how the variation of solvent polarity could affect the absorption and
emission property of our C-triazolyl nucleosides, we carried out TDDFT calculation using G09
2
1
program package. It was clear from the HOMO-LUMO orbital overlap and transition oscillator
strength (f) of the donor/acceptor triazolyl nucleosides that the S and S electronic transition or
0
1
the other possible electronic transitions were quite feasible. So the feasibility of the reverse
transition S to S transition was revealing the fluorophoric nature of our unnatural triazolyl
1
0
nucleosides. The redistribution of the electronic charge density among the possible energy states
certainly supported the solvatochromic nature and the intramolecular charge transfer
1
9

ACCEPTED MANUSCRIPT
characteristic of few of our synthesized unnatural C-triazolyl donor/acceptor nucleosides (Figure
6
b, 20c-d
S6-7 and SI, Section 3).
cTNDB
Thus, the experimental UV-absorption for
BDo (1A) in least polar solvent dioxane was
found to be 290 nm which is close to the theoretical value of 289 nm with a prominent
HOMO→LUMO (S →S ) transition with CI value of 0.55and oscillator strength of 0.37 (See SI,
0
1
Fig. S3, Table S4). Similarly, the calculated excitation energies for S
0
→S
1
transition of donor
cTDMB
cTNap
cTAnth
cTPy
aryl triazolyl nucleosides,
B
Do (1B),
B
Do (1C),
B
Do (1D), and
BDo (1E) were
found to be 281 nm (f = 0.03, CI = 0.66); 300 nm (f = 0.15, CI = 0.69); 398 nm (f = 0.09; CI =
.70) and 352 nm (f = 0.42; CI = 0.68) (in vacuum), respectively (Figure 6 for ID-E and See SI
0
Fig. S3 for 1A-C, Table S4). These values are in close agreement with the experimental values in
least polar solvent dioxane in each cases of 255 nm; 283 nm; 346, 365, 385 nm; and 278, 328,
3
43 nm, respectively.
cTAnth
cTPy
Figure 6. (a) HOMO-LUMO energy diagram for nucleoside 1D(
B
Do) and 1E (
BDo).
2
0

ACCEPTED MANUSCRIPT
0 1
For nucleoside 1B, the S →S transition in lowest excited state appeared at much longer
wavelength of 281 nm (f = 0.03) which was far from the experimental value of 255 nm in
dioxane. However, the transitions at 259 nm (f = 0.29) in vacuum [HOMO-1→LUMO; CI =
0
.68] correlated well with the observed absorbance at 255 nm in dioxane (See SI Fig. S3, Table
6
b, 20c-d
S4).
Similarly, the experimental UV-absorption value for 1E in dioxane (278 nm) was
well correlated with the theoretical transition value of 274 nm (f = 0.20) corresponding to the
transition HOMO→LUMO+1 (S →S ) with CI value of 0.33.
0
2
All these observations reflected in the emission spectra of the donor aryl triazolyl C-
nucleosides which showed solvatochromic effect. This was possibly due to electronic charge
redistribution between triazole moiety and fluorophoric unit which was also clear from their
individual HOMO-LUMO diagrams (See SI, Section 3). The HOMO−LUMO distributions
suggested that the emissive states of all the donor aryl-triazolyl nucleosides were locally excited
(
LE) states except for the case of nucleosides, 1A and 1B. These two nucleosides (1A and 1B)
having donor substituents, -N(Me ) and 3, 5-dimethoxy groups on the aromatic cores,
2
respectively, exhibited emissive states with more significant electron redistribution, i.e.,
intramolecular charge transfer transition (ICT) feature which was also supported from the
6
b, 20c-d
calculated results of HOMO−LUMO distribution.
2
1

ACCEPTED MANUSCRIPT
cTCNB
Figure7. HOMO-LUMO energy diagram for nucleoside 1F (
BAc).
cTCNB
On the other hand, in the case of acceptor aryl triazolyl unnatural nucleoside
BAc (1F)
having –CN as acceptor substituent the dominant orbital transitions in the low-lying singlet
excited states were found to be S →S (HOMO→LUMO) with transition wavelength of 284 nm
0
1
(f = 0.06; CI = 0.68) in vacuum (Figure 7, and See SI, Table S4). The experimental UV-
absorption values in dioxane was found to be 287 for 1F which is close to the calculated value.
These observations supported the potential fluorophoric nature of the acceptor nucleoside. The
emissive state of the acceptor nucleoside was well characterized with more significant electron
redistribution, i.e., ICT feature which was again supported by the HOMO−LUMO distributions.
6
b, 20c-d
cTAnthB
cTPy
2
.8.Study of Interaction of 1D (
Do) and 1E ( B ) with BSA Protein
Do
cTAnth
Inspired by the interesting photophysical properties of nucleoside 1D (
B ) and 1E
Do
cTPy
(
B ) we next studied their interaction behaviour with BSA protein. The study of interaction
Do
2
2

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between a drug and plasma proteins is an interesting research field in chemistry, biology and
1
1a
medicine. In this respect serum albumin protein is as models for gaining fundamental insights
1
1b
into drug-protein interactions. The increasing number of clinically utilized nucleosides
demonstrates the strong potentiality of nucleoside family as drug candidates. Many nucleosides
could not find clinical use despite their broad range of biological activity which is because of
1
1b, 22a-b
poor cell membrane penetrability.
Therefore, study of specific interaction with nucleoside/
nucleotide and plasma proteins is highly important.
Human serum albumin (HSA) is a multifunctional plasma protein which maintains plasma
pressure, balances nutrition in the human body. Most importantly, it is the most abundant protein
in the circulatory system. Bovine serum albumin (BSA) is a structural homolog (76% sequence
homology) of human serum albumin and is experimentally well accessible. Because of that
Bovine serum albumin (BSA) is widely used as a model serum albumin protein to investigate
2
3
protein–drug interactions in detail. Therefore, we become interested in bovine serum albumin
(BSA) as a model protein for studying the interaction with new unnatural fluorescent nucleoside,
cTAnth
cTPy
1
D (
B
Do) and 1E (
BDo).
2
3

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Figure 8. (a) UV-visible and (b) fluorescence titration spectra of 1D in absence or in presence of
BSA [λex = 280 nm]. The same spectra, (c) and (d), respectively, are for nucleoside 1E in
presence or in absence of increasing concentration of BSA [λ = 280 nm]. For all cases pH 7.0;
ex
probe conc. = 10 µM.
cTAnth
Thus, triazolyl C-nucleoside
BDo (1D) showed structured band in sodium phosphate
aqueous buffer, pH 7.0, at 345 nm, 364, 385 and 414 nm in the UV-visible spectra. With
increasing concentration of BSA the absorptions at 345, 364 and 385 nm experienced prominent
hyperchromism with slight blue shift while the absorption band at 414 nm exhibited
hypochromism with appearance of a clear isobestic point at 395 nm (Figure 8a). All these
observations clearly suggested a strong interaction between nucleoside 1D with BSA. Excitation
at 280 nm, a gradual decrease in emission intensity of the probe at 418 nm and an iso-emissive
point at 395 nm were the results of titration with increasing amount of BSA (Figure 8b). The
2
4

ACCEPTED MANUSCRIPT
similar result was observed when the probe-BSA solution was excited at probe absorption
wavelength i.e. at 363 nm (See SI, Fig. S4d). This observation indicated that the chromophoric
moiety, triazolylanthracene (TAnth), of the probe nucleoside resided in the hydrophobic pocket
and interacting with BSA. A gradual enhancement of steady state fluorescence
anisotropy/polarisation with increasing concentration of BSA finally supported the above
2
3
conclusion (Figure 9a). The Stern-Volmer quenching constant was calculated and found to be
3
-1
24
1
.0x10 LMol (See SI, Fig. S4f).
Figure 9. Steady-state anisotropy and polarisation plot of (a) 1D (λem = 420) and (b) 1E (λem
=
3
80) in presence of increasing BSA concentration.
cTPy
Next, we studied the interaction of nucleoside 1E with BSA. In absence of BSA,
B (1E)
Do
showed characteristic pyrene absorption band at 325 and 341 nm in phosphate buffer. Upon
gradual addition of BSA both the bands experienced hyperchromic effect with slight blue shift (2
cTPy
nm) (Figure 8c). This observation indicated a strong interaction of the
B nucleoside with
Do
the hydrophobic pocket of BSA. This was also supported from the emission spectra. Thus, upon
cTPy
excitation at 345 nm the
BDo nucleoside showed structured emission bands at 378, 397and
2
5

ACCEPTED MANUSCRIPT
4
17 nm (See SI, Fig. S5d). With gradual addition of increasing amount of BSA the band at 417
nm experienced a red shift of 2 nm with quenching of fluorescence (See SI, Fig. S5d). The
similar quenching of fluorescence intensity at 417 nm was also observed when the probe-BSA
solution was excited at 280 nm which is the absorption maxima of BSA (Figure 8d). This
cTPy
phenomenon was supported by the restricted motion of
BDo in the hydrophobic pocket of
BSA which was again supported from the enhanced fluorescence anisotropy/polarisation as the
2
3
concentration of BSA was increased gradually (Figure 9b). Stern-Volmer quenching constant
3
-1
24
was found to be 6.6 x10 LMol (See SI, Fig. S5f).
Overall, both the nucleosides interacted with BSA in the hydrophobic pocket which was
2
3c
supported from a fluorescence anisotropy study. Small molecules experienced no restricted
rotation in a free state or in less viscous environment. However, upon binding with a large
molecule such as protein, the movement of small molecules gets restricted. Thus, the
fluorescence anisotropy/polarisation is widely used to study the microenvironment around a
small molecular probe under interaction with proteins. Both the nucleoside 1D and 1E in buffer
showed very low anisotropy/polarisation. However, as the increasing amount of BSA was added
to their individual solutions, the fluorescence anisotropy/polarisation of both the nucleosides
increased and gradually reached the saturation (Figure 9a-b). Thus, the anisotropy/polarisation
experiment supported that both the nucleosides went inside the hydrophobic binding pocket of
2
3c
BSA, thus, free rotation of the probes become restricted. From the binding constants, it was
also revealed that the nucleoside 1E bound more tightly inside the binding pocket of BSA
compared to nucleoside 1D which was also reflected in the fluorescence. Thus, the binding event
was associated with stronger quenching of fluorescence in case of 1E compared to 1D. The
2
6

ACCEPTED MANUSCRIPT
cTAnth
cTPy
binding of nucleosides 1D (
B
Do) and 1E (
BDo) with BSA did not perturb the
conformation of BSA as was revealed from CD spectra (See SI, Fig. S6).
Figure 10. Docking possess and the interactions of nucleoside (a) 1D and (b) 1E in the
hydrophobic pocket of BSA.
2
5
Next, docking calculations were carried out using Autodock 4. The amino acid sequence of
BSA protein was observed from the NCBI website,
http://www.ncbi.nlm.nih.gov/protein/CAA76847.1 The 3D model of the BSA protein was built
using the 3D structure 1AO6 chain 'A' as template using the ESyPred3D11 web server. This
template shares 72.4% identities with the BSA sequence. Binding energies from docking study
were found to be -9.11 kcal/mol and -9.43 kcal/mol for 1D and 1E, respectively. Further, the
docking study was also supported the binding of the fluorophoric moieties, (TAnth for 1D and
TPy for 1E) of the probe nucleosides 1D and 1E in the hydrophobic pocket of BSA as was
1
1a, 26
revealed from their corresponding docking poses (Figure 10a-b and See SI, Fig. S7-S10).
3
. CONCLUSION
In conclusion, we successfully synthesised few triazolyl donor-acceptor C-nucleosides. The
Nicholas reaction was adopted as the key step for the synthesis of the intermediate β-sugar
2
7

ACCEPTED MANUSCRIPT
alkyne. It was interesting that the Nicholas reaction helped in thermodynamical equilibration of
the isomers and to obtain the preferred β-diastereomer in C-nucleoside in presence of Lewis acid.
We showed that both the cobalt and the Lewis acid played a crucial role to drive the anomeric
equilibrium in favour of β-anomer irreversibly. Finally, 1,3-dipolar azide –alkyne cycloaddition
reaction with various donor-acceptor aromatics afforded the target C-nucleosides in very good
yield. Few of the synthesised nucleosides showed interesting photophysical property. All the
experimental results supported that the nucleosides adopted a sugar pucker as South(S) with
C(2') endo-conformation and found similar to our earlier reported tetrazolyl nucleoside but
opposite to the N-triazolyl nucleosides which adopted C(3') endo, North conformation. However,
in all the cases, because of repulsion between lone pairs of C/N-triazolyl/tetrazolyl-Ns and O4',
the base adopted a syn-disposition of the Cs of the triazoles/tetrazole rings and an anti-
disposition of triazole’s/tetrazole’s –Ns. Therefore, the C-nucleosides with locked-S(anti)
conformations would help understanding the impact of conformational restrictions in DNA if
incorporated. Our current research focus is on the same direction. Two of the fluorescent
nucleosides were used to study interaction with BSA. Thus, we found a moderate to strong
interaction within hydrophobic pocket of BSA as was indicated from quenching incidence of
fluorescence emission.
4
4
. EXPERIMENTAL SECTION
.1.General Experimental
2
8

ACCEPTED MANUSCRIPT
All reactions were carried out under a nitrogen atmosphere. Organic extracts were dried over
anhydrous sodium sulphate. Solvents were removed in a rotary evaporator under reduced
pressure. Silica gel (60-120 mesh size) was used for the column chromatography. Reactions were
1
monitored by TLC on silica gel 60 F254 (0.25mm). H NMR spectra were measured with Bruker
1
3
6
00 (600 MHz) and C NMR spectra were measured with Bruker 600 (150 MHz) spectrometer.
Coupling constant (J value) was reported in hertz. The chemical shifts were shown in ppm
1
downfield from tetramethylsilane, using residual chloroform (
δ
= 7.24 in H NMR,
δ
= 77.23 in
= 2.48
1
3
1
13
C NMR), methanol (
δ
= 3.34 in H NMR,
δ
= 49.2 in C NMR), dimethyl sulfoxide (
δ
1
13
in H NMR,
δ
= 39.5 in C NMR), as an internal standard. Mass spectra were recorded using
WATERS MS system, Q-tof premier and data analyzed using Mass Lynx 4.1. IR spectra were
recorded in KBr or neat on a Perkin Elmer Spectrum one FT-IR spectrometer.
4
.2.Synthesis and characterization
Synthesis of 2 -deoxy-3, 5-bistoluoyl-D-ribofuranosyl-1'-ethyne
1
7a, d
'
α
/β-mixture (3):
In
a nitrogen flushed 2-neck round bottom flask fitted with magnetic stirring bar and septum,
Hoffer’s chlorosugar (2, 1.8 g, 4.64 mmol) was charged and THF was added. A 0.5 M solution
of ethynyl magnesium chloride (13 ml, 6.5 mmol) was added to the reaction mixture and allowed
to stir for 3h to achieve complete conversion of the starting material which was checked by TLC.
After the completion of the reaction, it was diluted with ethyl acetate and the combined organic
fraction was washed with aqueous solution of ammonium chloride and brine. The organic
fraction was then evaporated and concentrated in vacuo and the residue was purified by column
chromatography using hexane: ethyl acetate (25:1) to get a mixture of
α and β anomer 3. Overall
2
9