Donor/acceptor chromophores-decorated triazolyl unnatural nucleosides: Synthesis, photophysical properties and study of interaction with BSA

Organic &

Biomolecular Chemistry

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Donor/acceptor chromophores-decorated

triazolyl unnatural nucleosides: synthesis,

photophysical properties and study of interaction

with BSA†

Cite this: Org. Biomol. Chem., 2016,

14, 5088

Subhendu Sekhar Bag,* Sangita Talukdar, Suman Kalyan Das, Manoj Kumar Pradhan

and Soumen Mukherjee

Much eﬀort has been put forth to develop unnatural, stable, hydrophobic base pairs with orthogonal

recognition properties and study their eﬀect on DNA duplex stabilisation. Our continuous eﬀorts on the

design of ﬂuorescent unnatural biomolecular building blocks lead us to the synthesis of some triazolyl

donor/acceptor unnatural nucleosides via an azide–alkyne 1,3-dipolar cycloaddition reaction as a key

step, which we want to report herein. We have studied their photophysical properties and found interest-

ing solvatochromic ﬂuorescence for two of the nucleosides. Photophysical interactions among two

donor–acceptor β-nucleosides as well as a pair of α/β-nucleosides have also been evaluated. Further-

more, we have exploited one of the ﬂuorescent nucleosides in studying its interaction with BSA with the

help of UV-visible and steady state ﬂuorescence techniques. Our design concept is based on the hypoth-

esis that a pair of such donor/acceptor nucleosides might be involved in π-stacking as well as in photo-

physical interactions, leading to stabilization of the DNA duplex if such nucleosides can be incorporated

into short oligonucleotide sequences. Therefore, the designed bases may ﬁnd application in biophysical

studies in the context of DNA.

Received 5th March 2016,

Accepted 21st April 2016

DOI: 10.1039/c6ob00500d

www.rsc.org/obc

opened a new dimension in the design of hydrophobic un-

natural DNA base analogues considering possible aromatic

Introduction

The concept of expanding the genetic alphabet was pioneered stacking, hydrophobic or CH–π interactions between the base

with the development of orthogonal base pairing between iso- pairs to stabilize DNA duplexes.⁶Triggered by Kool’s work,

G and iso-C by Prof. Alex Rich¹in 1962, with an expectation to much eﬀort has been put forth to develop unnatural, stable,

generate DNA systems with enhanced functional and informa- hydrophobic base pairs of orthogonal recognition properties

tional potential. Inspired by this, Prof. Steven A. Benner in the towards expanding the genetic alphabet. Scientists are now able

late 1980s succeeded in expanding the genetic alphabet from to show the expanded informational and functional potential of

four to six letters.^2a–iFollowing Benner’s work, many research- DNA with the expanded genetic alphabet.⁷Therefore, not only

ers have contributed to the field of expansion of the genetic is the design and synthesis of eﬃcient new base pairs an excit-

alphabet.^3–5As a result, a large number of unnatural nucleo- ing research area, but also the application of these artificial

sides capable of showing H-bonding/π-stacking interaction base pairs to drive the synthesis of unnatural proteins is cur-

properties have been developed and their biophysical pro- rently an attractive research field.⁸Thus, the eﬀorts toward

perties in the context of DNA have been vigorously developing a third base pair have mainly focused on the design

investigated.^3–5Later on in 1994, the creation of non-H- of nucleobase analogues to pair via orthogonal hydrogen

bonding unnatural nucleobase surrogates by Kool et al. has bonding, based on the work of the Benner group, and on predo-

minantly non-H-bonding analogues that pair via hydrophobic

interactions, based on the work of the Kool group. Recent

eﬀorts have resulted in the design and construction of a

number of such base pairs that includes stable H-bonded pairs⁹

and unnatural, stable, hydrophobic base pairs.¹⁰

However, the rational design of non-hydrogen bonding base

pairs remains a challenge. In most of the design of non-hydro-

gen bonding base pairs, such as C-aryl and/or N-aryl unnatural

Bio-organic Chemistry Laboratory, Department of Chemistry, Indian Institute of

Technology Guwahati-781039, India. E-mail: ssbag75@iitg.ernet.in;

Fax: +91-258-2349; Tel: +91-258-2324

†Electronic supplementary information (ESI) available: Photophysical spectra,

NMR spectra, and crystallographic data. CCDC 995716 and 995717. For ESI and

crystallographic data in CIF or other electronic format see DOI: 10.1039/

c6ob00500d

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nucleosides, researchers have concentrated mainly on factors like aﬀord good stabilization to the duplex DNA. We are the first to

π-stacking, hydrophobicity, steric shape mimicry and in a few introduce the concept of charge transfer-mediated DNA duplex

cases the dipole moment, etc., in the stabilization of DNA stabilization by a pair of unnatural donor–acceptor triazolyl

duplexes.^6–11However, because of the challenging problem of bases. While the strong duplex stabilization of the self-pair is

enzymatic replication of such base pairs, only very few of these the result of π–π-stacking interactions, the hetero-pair stabiliz-

artificial base pairs have been eﬃciently and selectively replicated. ation has been explained on the basis of charge transfer com-

Furthermore, the lack of naturally occurring fluorescent plexation between the donor and the acceptor bases.²⁰

bases has spurred the development of artificial nucleosides Moreover, the nucleosides were found to stabilize an abasic site

with interesting photophysical properties which could be used to an extent that is comparable to that of the stability of a

as probes for DNA analysis, to develop nucleic acid-based diag- natural A–T pair.²¹Inspired by our first development and result,

nostics and sensing materials.^12,13As a result of tremendous we thought that it would be worthwhile to synthesize more tria-

research eﬀorts, from various scientific corners out of inspi- zolyl nucleosides containing donor/acceptor aromatics so as to

ration from nature or a rational designing approach, several produce a greater number of such exciting pairs with interesting

unnatural fluorescent nucleobases have been designed for the photophysical properties. The basis of our triazolyl nucleoside

development of functional nucleic acids, such as nucleic acid- design was our previous study wherein we showed that the tria-

based sensors, DNA-based light harvesting materials and oligo- zolyl units are capable of installing fluorescence properties into

nucleotides with novel electronic or magnetic properties.^14–17a non-fluorescent precursor, as well as electronic coupling

Considering the drawback of structural perturbation caused by between a triazole and a fluorophore giving rise to a modulated

extrinsic fluorescent labels, enzymatic replication as well as emission response to the fluorophore.¹⁹

the application as functional DNA materials, the generation of

There exist numerous examples of natural/unnatural

ideal fluorescent DNA nucleoside base analogues/base surro- nucleosides decorated with the triazole moiety, however in

gates would be much more beneficial. Thus, the field of many of the examples, the triazole moiety has been utilized as

designing fluorescent nucleoside base analogues of high solvo- a linker either to link a fluorophore with the natural bases or

fluorochromicity is flourishing, and many more advancements as a replacement of the phosphodiester linkage. Moreover, the

in the field are expected in the near future. Therefore, the focus of the synthesis of the few existing triazolyl nucleoside

rapidly growing research toward the expansion of the genetic bases wherein the nucleobases were replaced by the triazole

alphabet as well as the growing demand of nucleic acid-based units was mainly of synthetic interest or to generate a set of

diagnostics and sensing materials necessitates the design of biologically active nucleosides. However, there are no reports

fluorescent unnatural nucleobases with tuned photophysical of fluorescent triazolyl nucleosides which stabilise DNA

properties, a pair of which could impart high duplex stability duplexes to a good extent.

and polymerase replication fidelity.

Inspired by our recent result and in order to generate a new

While in the design of unnatural nucleobases/fluorescent dataset of donor–acceptor triazolyl nucleoside/fluorescent tria-

nucleoside base surrogates the concept of H-bonding mimicry zolyl base surrogates, we report herein the synthesis and

or forces like π–π stacking/hydrophobic interactions were the photophysical properties of some triazolyl donor/acceptor

central consideration, forces like charge transfer complexation nucleosides (Fig. 1). The synthesized new triazolyl nucleosides

have not been considered in the context of fluorescent DNA showed interesting photophysical properties and are expected to

base design or duplex stabilisation. Only recently, Iverson stabilize DNA duplexes via π-stacking and/or charge transfer

et al.¹⁸have demonstrated that the DNA duplex stability and interactions if incorporated in DNA in the future. As an appli-

specificity can be driven by the electrostatic complementarity cation we studied one nucleoside for its interaction with a

between an electron-rich and an electron deficient non-nucleo- protein (BSA) biomolecule and found that it could sense proteins

sidic base pair. However, interactions among the unnatural with a switch-on fluorescence response with high binding associ-

donor–acceptor nucleobases via charge transfer complexation ation. Previously, we focused on the synthesis and isolation of

have not been considered, which might have a potential only β-nucleosides, however in this report we focus on the syn-

impact on DNA duplex stabilization.

thesis of both α- and β-nucleosides. The exploration of both the

Our continuous research eﬀorts towards the generation of α- and β-nucleosides in the DNA context is our future focus.

fluorescent biomolecular building blocks with tuned photo-

physical properties via click reactions¹⁹has inspired us to

think of an alternative force for base pairing which could

stabilize the DNA duplex as well as aﬀord fluorescent oligonu-

cleotides with interesting photophysical properties. Thus,

Results and discussion

Synthesis of unnatural triazolyl donor–acceptor nucleosides

inspired by Mulliken’s proposal, we took a project for the syn- In order to generate a new dataset of nucleoside base surro-

thesis of donor–acceptor nucleobases and studied whether the gates, we report here the synthesis and phophysical properties

charge transfer complexation (CT) force among a pair of of some more triazolyl donor/acceptor nucleosides. We have

donor–acceptor nucleobases would stabilize the duplex DNA. chosen various possible combinations of donor/acceptor aro-

We ultimately succeeded to show that a donor–acceptor fluo- matics to generate triazolyl donor/acceptor nucleosides with a

rescent triazolyl nucleobase pair formed a CT complex and hope that several donor–acceptor combinations of such

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Fig. 1 Schematic presentation for the synthesis of triazolyl unnatural nucleosides (UNN) via a click reaction along with the structures of donor–

acceptor alkynes used in the study.

nucleosides might show interesting photophysical properties the synthesis of the β-epimer (11) with 90% yield after purify-

and aﬀord new pairs of unnatural base pairs for incorporation ing by silica gel column chromatography (230–400 mesh)

into DNA, which might stabilize DNA duplexes via π-stacking using a hexane : EtOAc solvent system with a gradient of =

and/or charge transfer interactions. The synthesis of the new 30 : 1 to 20 : 1. The TMS–N₃and Lewis acid-mediated protocol

class of nucleosides involves the popular 1,3-dipolar azide– (Method B) was adopted to aﬀord the bis-toluoyl-protected

alkyne cyclization pathway as a key step in a similar way as we α-azidonucleosides (12) in 59% yield starting from Hoﬀer’s

described previously, and is shown in Scheme 1.²⁰

chlorosugar (Scheme 1).^20–23Our journey of synthesizing

We first synthesized the nucleosides in a similar manner to several triazolyl nucleosides started from both the α- and

a procedure we reported previously.^20,21Thus, the synthesis β-azides. Thus, both the epimers were reacted separately with

started with Hoﬀer’s chlorosugar (10)^22awhich was syn- various aromatic alkynes (A–K, Fig. 1) containing donor/accep-

thesised from 2-deoxyribose sugar following our earlier pub- tor substituents in the presence of click reagents at 80 °C in

lished literature. Hoﬀer’s chlorosugar was then reacted with THF to aﬀord triazolyl donor/acceptor nucleosides (13β–23β,

CsN₃in DMSO which aﬀorded a 1 : 10 mixture of α- and 13α–17α and 20α–23α) in the bis-toluoyl protected form in

β-anomers in excellent yield (Method A), which was utilized for good yields (80–90%, Table 1). Next, methoxide/methanol-

Scheme 1 Synthesis of unnatural donor–acceptor triazolyl α- and β-nucleosides.

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Table 1 Alkynes used, short structures of the nucleosides, and yields

Yield^a(%)

Entry

1

Alkyne

Nucleosides [R = toluoyl or H]

α-Isomer (nucleoside no.)

β-Isomer (nucleoside no.)

A

87 (13α)

—

97 (13β)

91 (1β)

2^b

B

C

95 (14α)

95 (2α)

99 (14β)

95 (2β)

3

52 (15α)

—

90 (15β)

91 (3β)

4

D

E

98 (16α)

86 (4α)

99 (16β)

88 (4β)

5^b

96 (17α)

86 (5α)

95 (17β)

92 (5β)

6

7

F

—

85 (18β)

81 (6β)

G

—

95 (19β)

83 (7β)

8^b

H

I

98 (20α)

82 (8α)

93 (20β)

91 (8β)

9^b

95 (21α)

94 (9α)

90 (21β)

91 (9β)

10

11

J

79 (22α)

97 (22β)

—

K

99 (23α)

—

86 (23β)

—

^aYields in the top row for a particular isomer are for R = toluoyl and for the bottom row are for R = H. ^bSee ref. 20 for the β-isomers of entries 2,

5, 8 and 9.

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mediated deprotection of the toluoyl groups ultimately between H1′–H2′β, H1′–H_Triazoleand H_Triazole–H_Arylbut the

aﬀorded the desired α- and β-triazolyl nucleosides (1β–9β, 2α, absence of a cross peak between H1′–H4′ which was present in

4α–5α and 8α–9α) with excellent yields. All the protected and the β-isomer.

deprotected nucleosides were characterized by NMR, mass

We have recently reported the crystal structure of β-^TPhenB_Do

spectrometry, IR, melting temperature and in three cases by (5β), which showed an intermolecular π-stacked and H-bonded

single crystal X-ray analysis. The α-epimers were synthesized helical layer network.²⁰On the other hand, both the packing

with the aim to incorporate them in future into short oligo- diagram and crystal arrangement of β-^TNBB_Ac(8β) show a

nucleotide sequences to check the thermal stability of the H-bonded helical layer chain-like structure. Molecules of a

α-DNA²²as well for the β-DNA context. β-Epimers are the build- single layer are held by H-bonding between sugar

ing blocks of natural DNA. Table 1 represents the isolated CH₂OH⋯ONO– of nitrobenzene. Another ONO– of one layer is

yields and short structures of the nucleosides. Fig. 2 represents involved in H-bonding with the sugar CH₂OH of the second

the full structures of all deprotected triazolyl nucleosides.

layer. The two adjacent layers are also involved in stabilized

π-stacking interactions between the phenyl ring of a nitro-

benzene of one layer and a closely spaced triazole ring of

Structural studies

After getting all the nucleosides in hand we next studied the another layer (Fig. 4).

conformation. The conformation of the β-nucleosides was

The crystal structure of the β-^TPhenB_Do(5β) nucleoside

established via NOESY spectra as well as via the single crystal shows a twist between the triazole and the phenanthrene,

TPhen

TNB

X-ray structure of two β-nucleosides,

B

(5β) and

B

Ac

suggesting that the phenanthrene unit might be able to

Do

(8β). The NOESY spectra of two representative nucleosides, 5β engage in a partial stacking interaction upon incorporation

and 9β (Fig. 3), showed the presence of cross peaks between into DNA, which was shown in our earlier report in the context

H1′–H4′, H1′–H2′α, H1′–H_Triazoleand H_Triazole–H_Aryl, indicating of DNA duplex stabilization in the self-pair duplex containing

their β-conformation in solution. The α-conformation of the the β-^TPhenB_Do(5β) nucleoside.²⁰However, the nitrobenzene

nucleosides was also established via the NOESY spectrum of and the triazole moiety of β-^TNBB_Ac(8β) are in the same plane,

α-^TPhenB_Do(5α) as well as via the single crystal X-ray structure which would allow the triazolylnitrobenzene unit to take part

of α-^TMNapB_Ac(4α). The NOESY spectrum of the representative in intercalation or in a full stacking interaction within the

α-nucleoside, 5α (Fig. 3) showed the presence of cross peaks nucleobases inside a DNA duplex after incorporation into

Fig. 2 Structures of the synthesized triazolyl donor/acceptor triazolyl α-/β-nucleosides.

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Fig. 3 Presentation of NOESY cross peaks in two representative β-nucleosides and in one α-nucleoside.

Fig. 4 (a) ORTEP (50% thermal ellipsoid) diagram, (b) crystal packing and (c) π-stacked and H-bonded helical layer network of β-^TNBB_Ac(8β) nucleo-

sides [CCDC 995717].

DNA. These all suggested that the triazolyl phenanthrene/ H-bonding interactions (2.836 Å and 2.826 Å, respectively)

nitrobenzene nucleoside can indeed engage in H-bonding as between the 2′-OH of the sugar of one layer and the pyranose

well as π-stacking interactions, and the eﬀect has been shown ring –O– of the sugar of the second layer. Interestingly, each

in the DNA designed in our earlier study.²⁰

plane runs anti-parallel with respect to the pyranose sugar

The nucleoside α-configuration was also established via the unit. Most importantly, the crystal arrangement follows uni-

single crystal X-ray structure of α-^TMNapB_Do(4α). The crystal directional growth along the c-axis (Fig. 5).

packing and arrangements of α-^TMNapB_Do(4α) showed a

Study of photophysical properties

H-bonded corrugated sheet-like layered structure. The two cor-

rugated planes (each plane containing two layers making a After getting all the nucleosides in hand, we studied their UV-

sheet) are held together via van der Waals and H-bonding visible and fluorescence photophysical properties. Previously

interactions. The first layers of each plane are held by van der we and subsequently others have shown that the linking of

Waals (2.387 Å) interactions between the naphthylmethoxy-H fluorescent/non-fluorescent units with a triazole moiety can

of the first plane and the 5′-CH₂–H of the sugar unit of the lead to the installation of fluorescence emission properties to

second plane. Similarly, the second layers of each plane are the non-fluorescent molecules and/or modulation of the pro-

held by weak H-bonding (2.657 Å) interactions between the perties of a fluorescent molecule.¹⁹The synthesized nucleic

naphthylmethoxy-O of the first plane and the 3′-CH–H of the acid building blocks (2′-deoxyribosides) also behaved in a

sugar unit of the second plane. Each layer of a plane is held similar way with respect to their photophysical properties. We

together via both intramolecular H-bonding (2.030 Å, between studied the photophysical properties of a few of our syn-

the triazolyl N₂- and the 3′-OH–H) and intermolecular thesized β-nucleosides in various solvents. The nucleoside 1β

TNDMB

H-bonding (2.714 Å, between the triazolyl 3′-OH and the 5′-OH

(

B ) containing a N,N-dimethylaminobenzene group

Do

of sugar units). The two layers of a plane are held via π–π stack- exhibits very strong absorption maxima at around 270–280 nm

ing interactions in a T-shaped fashion between two aromatic in various organic solvents with ∼5 nm solvatochromicity.

units of each layer via aromatic CH–π-interactions and weak Excitation at the absorption maximum of each solvent shows

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Fig. 5 (a) ORTEP (taking one molecule and 50% thermal ellipsoid) diagram, (b) crystal packing and various interactions and (c) π-stacked and

H-bonded corrugated sheet-like network of α-^TMNapB_Do(4α) nucleosides [CCDC 995716].

an emission at around 365 nm with a red-shift of 10 nm as the shows a structureless emission at around 333 nm with a

solvent polarity increases. While the nucleoside exhibits a long quenched incidence of fluorescence as the polarity of the

wavelength emission, most probably an intramolecular charge solvent increases (Fig. 7a and b).

TMNap

transfer (ICT)^24,25band at 485 nm in polar solvents like DMF,

The nucleoside 4β

(

B ) containing a methoxy-

Do

DMSO and in acetonitrile, the fluorescence intensity becomes naphthalene aromatic unit exhibits very strong absorption

negligible in polar protic solvents like EtOH and MeOH. The with vibronic structures at 285, 290 and 302 nm in the least

quenched incidence of fluorescence can be attributed to the polar solvent, toluene. The absorbance is characterized by a

H-bonding-mediated radiationless decay of the chromophore hypsochromic shift (2–5 nm) and little hyperchromism as the

in polar protic solvents (Fig. 6a and b).²⁶The nucleoside 2β polarity of the solvent increases. Excitation at the absorption

TDMB

(

B ) containing a 3,5-dimethoxybenzene group exhibits a maximum (290–300 nm) of each solvent shows an emission at

Do

very strong absorption maximum at around 252–270 nm and a around 365 nm with an increase in the intensity as the solvent

weak band at around 290 nm in various organic solvents. Exci- polarity increases (Fig. 7c and d).

TPhen

tation at the absorption maximum of each solvent shows emis-

The

B

(5β) nucleoside shows blue-shifted absor-

Do

sion at around 320 nm with an irregular trend in the intensity bance as the polarity of the solvent increases (λ_max= 302 nm in

as the solvent polarity increases (Fig. 6c and d).

dioxane → 298 nm in MeOH). Upon excitation at 300 nm,

TPhen

The nucleoside 3β (^TPhOBB_Do), which is a phenoxyphenyl-

B

Do

shows structured bands at 363 and 380 nm with

triazole nucleobase, possesses

a

structureless absorption similar intensities and low quantum yields in all solvents.²⁰

maximum at around 257–260 nm with little or no solvato- The triazolylpyrene nucleoside 6β (^TPyB_Do) shows structureless

chromicity or change in absorbance in all organic solvents absorption at 355 nm in toluene which is blue-shifted to

tested. Excitation at the absorption maximum of each solvent

Fig. 6 UV-visible and ﬂuorescence spectra of nucleoside 1β (a–b) and

nucleoside 2β (c–d) in various organic solvents (concentration of each

nucleoside was 10 μM).

Fig. 7 UV-visible and ﬂuorescence spectra of nucleoside 3β (a–b) and

nucleoside 4β (c–d) in various organic solvents (concentration of each

nucleoside was 10 μM).

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Fig. 9 (a) UV-visible and (b) ﬂuorescence spectra of nucleoside 7β in

various organic solvents (concentration of each nucleoside was 10 μM).

Fig. 8 (a) UV-visible and (b) ﬂuorescence spectra of nucleoside 6β in

various organic solvents (concentration of each nucleoside was 10 μM).

absorbance is characterized by a hypsochromic shift (2–5 nm)

and hyperchromism as the polarity of the solvent increases.

Excitation at 280 nm shows an emission at around 315 nm in

toluene which becomes broad with an appearance of a red-

shifted hump (at 335 nm in EtOAc → 353 nm in MeOH) of

increased intensity as the solvent polarity increases. This

broadened band is most probably the ICT emission. In

summary all the triazolyl aromatic nucleosides show signifi-

cantly red-shifted absorption and emission properties from

those of the parent aromatics. It is also obvious that the emis-

sion intensity, wavelength and quantum yields are higher than

any of the natural nucleosides.²⁰

As representative examples, we have also studied the photo-

physical properties, in various organic solvents, of two repre-

sentative α-triazolyl nucleosides, namely, 4α and 9α (Fig. 10a–

b and c–d, respectively). Thus, the UV-visible spectra of the

α-^TMNapB_Do(4α) nucleoside shows a slightly blue-shifted

(3–4 nm) absorbance for all the vibronic bands at 284, 290 and

300 nm as the polarity of the solvent increases (Fig. 10a),

which is similar to the corresponding β-anomer, the 4β-nucleo-

348 nm with the increase in absorbance as the solvent polarity

increases, showing an electronic coupling of the pyrenyl π-elec-

tron cloud with the triazole unit. However, it shows a struc-

tured emission when excited at 350 nm with the appearance of

prominent maxima at 387 and 408 nm of similar intensities

and quantum yields as the polarity of the solvent increases

(Fig. 8a, b and Table 2). The fluorescence life time data also

supports this observation (see ESI, Table S4†). On the other

hand, the nucleoside 7β (^TBPyB_Do) containing a butylpyrene tri-

azole exhibits very strong absorption with vibronic structures

characteristic of butylpyrene at 314, 329 and 346 nm in all sol-

vents tested. The absorbance is characterized by a hypsochro-

mic shift (2–5 nm) and little hyperchromism as the polarity of

the solvent increases. Excitation at the absorption maximum

(∼350 nm) in each solvent shows structured emissions at 380,

401, and 421 nm with an increase in intensity as the solvent

polarity increases. The fluorescence life time data also sup-

ports this observation (Fig. 9a, b and Table 2).

The nucleoside 9β (^TCNBB_Ac) containing a cyanobenzene

acceptor aromatic unit possesses a very strong structureless

absorption at 287 nm in the least polar solvent, toluene.²⁰The

Table 2 Summary of the photophysical properties of nucleosides 6β

and 7β

UV-vis. and fluorescence properties

Entry

Solvent

λ_abs(nm)

ε_max× 10³

λ_fl(nm)

Φ_f

TPy

B

Do

(6β)

Toluene

Dioxane

THF

EtOAc

CHCl₃

DMF

DMSO

ACN

EtOH

353

351

349

351

348

347

346

32.6

41.0

40.7

41.2

38.6

38.7

39.1

40.8

39.6

40.8

408

407

408

406

405

0.13

0.22

0.13

0.08

0.12

0.39

0.58

0.09

0.10

0.09

MeOH

TBPy

B

Do

(7β)

Toluene

THF

EtOAc

CHCl₃

ACN

EtOH

MeOH

345

344

343

345

343

342

34.2

45.2

42.7

38.8

40.3

45.6

41.3

417

0.07

0.09

0.06

0.08

0.07

0.08

Fig. 10 UV-visible and ﬂuorescence spectra of nucleosides 4α (a–b)

and 9α (c–d) in various organic solvents (concentration of each nucleo-

0.08 side was 10 μM).

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Fig. 11 Diagram of the HOMO–LUMO of nucleosides 4α, 4β, 9α and 9β calculated at the B3LYP//6-31G* level of theory using the Gaussian 03

program package.

side. Surprisingly, the change in both the absorbance and four triazolyl unnatural nucleosides. The redistribution of elec-

wavelength in the highest aprotic polar solvent, DMSO, is tronic charge density between HOMO–LUMO supports the

found to be more drastic for 4α compared to 4β. An examin- observed emission properties of the fluorophores (Fig. 11).²⁸

ation of the emission spectra of 4α reveals the opposite

For example, the TDDFT calculations show the dominant

vibronic structure to that of 4β in all the solvents tested. transition S₀→ S₁(HOMO → LUMO) in the lowest-lying singlet

However, the relative intensities of the emissions at 357, excited state of both the donor and acceptor triazolyl α/β-

368 nm of the 4α-nucleoside remains almost similar to that nucleosides. The prominent transition is found to be at

observed for the 4β anomer when excited at 300 nm (Fig. 10b). 270 nm (4.59 eV; HOMO → LUMO) with f = 0.78 and CI = 0.69

On the other hand, the nucleoside α-^TCNBB_Do(9α) shows for 4β, and for 4α the transitions occur at 267 nm (4.6389 eV;

similar absorption behavior to 9β with more or less similar HOMO → LUMO and HOMO−1 → LUMO) with f = 0.75 and CI

but slight solvatochromicity. On excitation at 280 nm, the = 0.63013 and 0.11, respectively. Similarly, for 9α/9β the promi-

nucleoside 9α shows a broad emission band at around 316 nm nent transition is found to be at 313–314 nm, f = 0.12, CI = 0.6

in toluene, similar to the corresponding 9β-anomer. As the (3.95 eV; HOMO → LUMO). While the transitions HOMO−1 →

solvent polarity increases the spectral feature becomes broader LUMO (293 nm, 4.22 eV, f = 0.08, CI = 0.45) from the second,

and shifts to a longer wavelength region. In MeOH the emis- HOMO → LUMO+1 (273 nm, 4.53 eV, f = 0.04, CI = 0.56) from

sion band of 9α appears at 327 nm with maximum intensity the third and HOMO−1 → LUMO (247 nm, 5.03 eV, f = 1.11,

(Fig. 10c and d) indicating a similar fluorophoric nature as CI = 0.43) from the fourth singlet excited states are also found

that of the 9β-anomer. Thus, both the α- and β-anomers for 9α, the 9β-nucleoside shows the transitions HOMO →

behave similarly with respect to their photophysical properties. LUMO+1 (291 nm, 4.25 eV, f = 0.10, CI = 0.46) from the second,

HOMO → LUMO+2 (262 nm, 4.72 eV, f = 0.02, CI = 0.68) from

Theoretical calculation

the third and HOMO → LUMO+1 (246 nm, 5.03 eV, f = 1.25,

CI = 0.40) from the fourth singlet excited state. The transitions

in the gas phase are in close agreement with the experimental

results of 287, 301 nm (in toluene) for 4α, 285, 290 and 302 (in

toluene) for 4β, 272, 292 nm (in dioxane) for 9α and 287 nm

(in toluene) for 9β.

The emission spectra of the donor–acceptor triazolyl un-

natural nucleosides 4α, 4β, 9α and 9β show a slight solvato-

chromic eﬀect which is due to electronic charge redistribution

between the triazole moiety and the fluorophoric unit which is

Next, the absorption and emission properties of four represen-

tative donor/acceptor aromatic triazolyl nucleosides (4α/4β

and 9α/9β) were studied theoretically (TDDFT calculation)

using the Gaussian 03 program package.²⁷The calculations

show that the electronic transition from S_oto S₁or other poss-

ible electronic transitions are feasible, as is revealed from the

HOMO–LUMO overlap and the transition oscillator strength

( f ). This indicates that the reverse transition, i.e., S₀← S₁, is

also fully allowed, suggesting the fluorophoric nature of all

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also reflected from their individual HOMO–LUMO diagrams

(Fig. 11). The emissive states are characterized by more

significant electron redistribution, i.e., ICT feature, as is

suggested by the HOMO–LUMO distributions.²⁸

Study of photophysical interaction

Next, the possible photophysical interaction properties among

the Do/Ac nucleoside pairs have been evaluated in diﬀerent

solvents. For this purpose, we have chosen two triazolylpyrene

nucleosides 6β and 7β as donor nucleosides and the triazolyl-

cyanobenzene nucleoside 9β as the acceptor nucleoside. We

first have examined the interaction of the donor nucleoside 6β

with an increasing concentration of acceptor nucleoside 9β in

dioxane by recording the UV-visible and fluorescence spectra.

Thus, titration of a solution of the triazolylpyrene nucleoside

(6β, 10 μM) in 1,4-dioxane with an increasing concentration of

triazolyl cyanobenzene nucleoside 9β shows an isosbestic

point at 305 nm in the UV-visible spectra, indicating that the

two nucleosides are in equilibrium. The mixture shows absorp-

tions with vibronic maxima at 247 nm and 280 nm wherein

the absorption of triazolylcyanobenzene 9β is buried, and a

structureless absorption band at 351 nm that is due to triazo-

lylpyrene absorption. With increasing the concentration of the

acceptor nucleoside 9β, the extinction coeﬃcient of the

absorption maxima at 247 and 280 nm are increased while the

absorption at 351 nm is decreased with very little blue-shift

(Fig. 8).

Fig. 12 (a) UV-visible and ﬂuorescence spectra of the triazolylpyrene

TPy

nucleoside,

B

(6β, 10 μM) and triazolyl cyanobenzene nucleoside,

Do

TCNB

B

Ac

(9β, 10 μM) in dioxane. (b–c) UV-visible and ﬂuorescence titra-

tion spectra of a solution of 6β (10 μM) in 1,4-dioxane with an increasing

concentration of nucleoside 9β. (d) Change in the emission intensity of

triazolylpyrene upon addition of the acceptor nucleoside triazolylcyano-

benzene indicating a quenching of ﬂuorescence incidence.

The fluorescence excitation spectra (superimposable to the

absorption spectra of the monomer) monitored at 386 nm also

shows an isosbestic point at 291 nm with three maxima at 248,

281 and 353 nm. The band at 353 nm shows a decreased inten-

sity along with a little blue-shift indicating that the donor–

acceptor nucleosides interact with each other. When excited at

the pyrene absorption maximum (λ_ex= 350 nm) we found no

change in either the emission intensity or the wavelength.

However, the emission spectra (λ_ex= 280 nm) of the mixture

generate two main emission bands at 320 nm characteristic of

triazolylcyanobenzene and at 406 nm corresponding to the

emission of triazolylpyrene with an isosbestic point at 370 nm.

The decreased intensity of the emission band at 406 nm indi-

cates the quenching of the fluorescence of triazolylpyrene by

the increased concentration of triazolylcyanobenzene (Fig. 12

and ESI, Table S5†).

TBPy

Fig. 13 (a) UV-visible and ﬂuorescence spectra of nucleosides

B

Do

TCNB

(7β, 10 μM) and

B

Ac

(9β, 10 μM) in dioxane. (b–c) UV-visible and

On the other hand, the titration of a solution of butylpyre-

nyltriazolyl nucleoside (7β, 10 μM) in 1,4-dioxane with increas-

ing concentration of the triazolylcyanobenzene nucleoside (9β)

shows isosbestic points at 247 and 307 nm in the UV-visible

spectra, indicating that the two nucleosides are in equilibrium.

The mixture shows absorptions with vibronic maxima at

244 nm and 278 nm wherein the absorption of triazolylcyano-

benzene is buried, and a structureless absorption band at

344 nm along with small bands at 312 and 323 nm that are

due to triazolylpyrene absorption. With increasing the concen-

tration of the acceptor nucleoside 9β, the extinction coeﬃ-

cients of the absorption maxima at 244 and 278 nm are found

to be increased, while the absorption at 344 nm is decreased

ﬂuorescence titration spectra of a solution of 7β (10 μM) in 1,4-dioxane

with an increasing concentration of nucleoside 9β. (d) Change in the

emission intensity of triazolylpyrene upon addition of the acceptor

nucleoside triazolylcyanobenzene.

with a very small blue-shift (2 nm). A peak at 290 nm also

appears, which is the characteristic band of the triazolylcyano-

benzene nucleoside (Fig. 13).

The fluorescence excitation spectra (superimposable to

the absorption spectra of the monomer) monitored at 386 nm

also show an isosbestic point at 299 nm with three maxima at

245, 277 and 344 nm. The band at 344 nm shows a decreased

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intensity along with a little blue-shift, indicating that the two

donor–acceptor nucleosides interact with each other. The

emission spectra (λ_ex= 275 nm) of the mixture features two

main emission bands at around 319 nm, characteristic of tri-

azolylcyanobenzene, and at 386 nm, along with other struc-

tured bands at 376, 413 and 444 nm corresponding to the

emission of the butylpyrenyltriazolyl nucleoside with an iso-

sbestic point at 372 nm. The decreased intensity of the emis-

sion band at 376/386 nm indicates the quenching of the

fluorescence of butylpyrenyltriazole by the increased concen-

tration of triazolylcyanobenzene (Fig. 13 and ESI, Table S5†).

A closer look at the UV-visible spectra of the nucleoside 6β

and/or 7β and the fluorescence emission spectra of nucleoside

9β indicates that there might be a possibility of a FRET

process occurring between the donor–acceptor nucleoside

pairs leading to quenching of the fluorescence (Fig. 12a and

13a).²⁹We have calculated the Stern–Volmer quenching con-

stant for each pair of donor–acceptor nucleosides from the

slope of the plot of F_o/F vs. conc. of the quencher, which is

found to be 5 × 10⁻²for the 6β/9β pair and 3.3 × 10⁻²for the

7β/9β pair (see ESI, Table S5†). However, at this stage we are

unable to conclude the exact mechanism of fluorescence

quenching and it needs further investigation. Moreover, exci-

tation at the absorption maxima of either nucleoside 6β or 7β

does not change the fluorescence intensity of the pyrenyl fluo-

rescence in both cases, even with increasing the concentration

of the donor nucleoside 9β. Surprisingly, decreased fluo-

rescence of triazolylcyanobenzene has been observed upon

increasing the concentration of triazolylpyrenyl nucleosides 6β

or 7β when excited at 280 nm (absorption of triazolylcyano-

benzene) indicating non-FRET quenching of the fluorescence

of the nucleoside 9β by 6β and/or 7β, wherein the nucleosides

6β and 7β, containing pyrenyltriazole and butylpyrenyltriazole

respectively, act as quenchers of triazolylcyanobenzene (see

ESI, Fig. S1–2a†). This reverse quenching is most probably

because of an electron transfer process from pyrenyltriazole

and/or butylpyrenyltriazole to the acceptor cyanobenzene unit

of the nucleoside 9β.

Similarly, a titration experiment has been carried out

between a representative α-acceptor nucleoside, α-^TCNBB_Ac(9α)

and the same β-donor nucleoside, ^TBPyB_Do(7β), to examine the

possible photophysical interaction properties, if any, in

dioxane. We, thus, have titrated a solution of the donor nucleo-

side 7β with an increasing concentration of the acceptor

nucleoside 9α in dioxane by recording the UV-visible and fluo-

rescence spectra. The UV-visible titration spectra show an iso-

sbestic point at around 307 nm indicating that the two

interacting nucleosides are in equilibrium. The mixture shows

increasing absorptions of the vibronic maxima at 245, 264, 275

and 285 nm wherein the absorption of triazolylcyanobenzene

is buried, and unchanged vibronic absorption bands corres-

ponding to triazolylpyrene at 310, 325 and 344 nm. The

absorption band of triazolylcyanobenzene also appears with

increasing absorptivity at 288–292 as the concentration of 9α

increases (Fig. 14a). Thus, the UV-visible titrations indicate a

similar result to the titration experiment between 7β and 9β.

Fig. 14 (a–b) UV-visible and ﬂuorescence titration spectra of a solution

of 7β (10 μM) in 1,4-dioxane with an increasing concentration of nucleo-

TBPy

side 9α. (c) Change in the emission intensity of the pyrene of

B

Do

(7β) upon addition of the acceptor nucleoside α-triazolylcyanobenzene

(α-^TCNBB_Ac, 9α). (d) Overlap of the UV-visible and ﬂuorescence spectra

TBPy

TCNB

of the nucleosides

B

(7β, 10 μM) and α-

B

Ac

(9α, 10 μM),

Do

respectively, in dioxane, showing a possibility of FRET.

The fluorescence excitation spectra monitored at 386 nm

show an isosbestic point at 280 nm with decreased intensities

of the bands at 241, 264 and 275 nm, and an obviously

increased intensity of the buried triazolylcyanobenzene band

at around 285 nm. Interestingly an increase in the intensity of

all the vibronic bands of pyrene is observed as the concen-

tration of 9α increases, which is contrary to the titration

between 7β and 9β wherein the opposite result is observed.

This observation indicates a strong interaction among the 9α/

7β donor–acceptor nucleosides. The emission spectra (λ_ex

=

280 nm) of the mixture show two main structured emission

bands at around 325 nm characteristic of triazolylcyanoben-

zene and centered at 405 nm corresponding to the emission of

the butylpyrenyltriazolyl nucleoside (Fig. 14b). A closer look at

the emission spectra of only 7β and 9α and their 1 : 1 mixture

reveals a decrease in the intensity of 9α and an increase in the

intensity of 7β emission, indicating a FRET process from 9α as

an energy donor to 7β as the energy acceptor, and is supported

by overlapped emission spectra of the donor and absorbance

spectra of the acceptor nucleoside (Fig. 14c and d). Therefore,

while interaction between the donor–acceptor β-nucleosides

(9β/7β) reveals non-FRET quenching of the acceptor β-butyro-

pyrenyltriazole nucleoside (7β), a FRET-based enhanced emis-

sion of the β-nucleoside (7β) is observed while titrating with an

acceptor α-nucleoside (9α).

We next explored the novel triazolyl butyramidopyrene

nucleoside 7β (^TBPyB_Do) for studying interactions with BSA

protein. The UV-visible absorption of nucleoside 7β in phos-

phate buﬀer showed a structured absorption characteristic of

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pyrene at around 313, 326 and 342 nm. Addition of an increas- sity is not so drastic, indicating a weak interaction between the

ing concentration of BSA to the probe solution resulted in a probe nucleoside and BSA inside the hydrophobic pocket in

structured absorption that experienced strong hyperchromicity an excited state. That the probe nucleoside 7β (^TBPyB_Do

)

along with a bathochromic shift of 2–4 nm of all the bands. resided on the hydrophobic pocket and experienced highly

The BSA absorption at 280 nm also shifted to 285 nm. The restricted rotational motion was evident from an enhancement

bathochromic shift indicated that pyrene was accommodated of the fluorescence anisotropy from 0.003 to 0.02 (ESI,

in the hydrophobic region, which was also supported by the Fig. S2†).³⁰Finally, we thought that a mixture of the donor–

absorption of the pyrene moiety in various organic solvents acceptor nucleosides could interact with BSA diﬀerently. For

(Fig. 9). These observations indicated a ground state binding that purpose a 1 : 1 mixture of the donor nucleoside 7β and

interaction between BSA and nucleoside 7β, most probably in the acceptor nucleoside 9α was considered as a probe, and the

the hydrophobic region (Fig. 15a). We next carried out fluo- probe solution was titrated with an increasing concentration of

rescence titration experiments. In phosphate buﬀer the nucleo- BSA. The UV-visible spectra with pyrene absorption experience

side 7β showed strong emission bands at 376, 397 and 414 nm a drastic hyperchromicity along with a red-shift of 3–5 nm of

which correspond to pyrene-structured emissions. A compari- the vibronic bands of pyrene at 326 and 342 nm as the concen-

son of the BSA emission and the absorption spectra of nucleo- tration of BSA increases. Moreover, the buried absorption of

side 7β revealed that there is a possibility of a FRET process the acceptor nucleoside also is found to increase as the BSA

that occurs from BSA to the TBPy moiety of nucleoside 7β (ESI, concentration increases (Fig. 15c). This observation indicates a

Fig. S2B†). Thus, when excited at the absorption maximum of strong interaction between BSA and the donor–acceptor

BSA (280 nm) wherein there was very weak absorption of mixture in the hydrophobic pocket of BSA. Upon excitation at

nucleoside 7β, and comparing the emission spectrum of only the pyrene absorption at 342 nm and increasing the concen-

BSA with that of only the nucleoside and a 1 : 1 mixture of tration of BSA led to a little or almost no quenching of the

BSA : nucleoside, it was observed that the emission intensity of pyrene emission. However, on excitation at the BSA absorption

the nucleoside 7β increased while that of BSA decreased of 280 nm, an overall quenching of the emission of the probe,

(Fig. 15b and ESI, Fig. S2†). This observation clearly suggests both in the pyrene or in the triazolylcyanobenzene region, is

that the probe nucleoside is accommodated inside the hydro- observed (Fig. 15d). This might be because of FRET quenching

phobic pocket of BSA and the TBPy moiety of nucleoside 7β in the hydrophobic pocket. That the probe resided in the

faces closer to one of the tryptophans of BSA, leading to a hydrophobic pocket of BSA was evident from an increased an-

FRET process from tryptophan to TBPy. Upon gradual addition isotropy from 0.006 to 0.06.³⁰Therefore, the fluorescence

of an increasing amount of BSA, the emission from the TBPy results show that the interaction of the 1 : 1 mixture of the 7β/

moiety also increased gradually. However, the change in inten- 9α nucleosides with BSA is the opposite to the interaction

between nucleoside 7β only and BSA. All the results suggested

that both hydrophobic as well as electrostatic interactions

played an important role in the present interaction study

TBPy

between the probe nucleoside 7β

1 : 1 mixture 7β/9α and BSA.

(

B )/BSA and a

Do

Conclusion

We have synthesized some new triazolyl nucleosides via an

azide–alkyne cycloaddition reaction as a key step of the syn-

thesis with very good yields, and their photophysical pro-

perties in various organic solvents have been evaluated. The

alkynes are commercially available and cheap and the azides

can easily be accessed from the chlorosugar. These nucleo-

sides, particularly as donor–acceptor pairs in DNA, are

expected to show interesting π–π stacking and photophysical

properties. Biophysical studies in the DNA context with the

unexplored bases are our current research target. We also

exploited one of the triazolyl nucleosides in studying the inter-

action process with BSA and found that hydrophobic as well as

electrostatic interactions played an important role in the

sensing of BSA by the probe nucleoside 7β (^TBPyB_Do). Also,

under study are the noncovalent interactions of pyrenyl nucleo-

sides, in particular, with proteins.

TBPy

Fig. 15 (a) Absorption titration of the probe nucleoside 7β (

B

Do

)

upon addition of a gradually increasing concentration of BSA. (b) Steady

state emission of the probe nucleoside 7β (^TBPyB_Do) in the absence or in

the presence of BSA showing a FRET process and gradual enhancement

of the probe emission. (c–d) Absorption and ﬂuorescence titration of a

1 : 1 mixture of 7β/9α [10 μM] with an increasing concentration of BSA

(at λ_ex= 280 nm; probe concentration is 10 µM).

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TLC, the reaction mixture was evaporated and partitioned

between water and ethyl acetate. The organic layer was washed

with water twice, then with brine solution twice and finally

Experimental section

General experimental and materials

¹H NMR spectra were recorded on a 400 MHz machine; ¹³C kept over Na₂SO₄for drying. Next the reaction mixture was fil-

NMR spectra were measured at 100 MHz. The coupling con- tered and concentrated by evaporation. The products were

stant (J value) is reported in hertz. The chemical shifts are then separated by column chromatography and characterized.

shown in ppm downfield from tetramethylsilane, using The average isolated yields were between 90–99%.

residual chloroform (δ = 7.24 in ¹H NMR, δ = 77.0 in ¹³C

Synthesis of 3′,5′-bis{O-(p-toluoyl)}-2′-deoxy-1′-β-triazolyl-N,

1

NMR), dimethyl sulfoxide (δ = 2.48 in H NMR, δ = 39.5 in ¹³C N-dimethylaminobenzene nucleoside (13β, bis-toluoyl-

1

NMR) and/or methanol (δ = 3.30 in H NMR, δ = 49.0 in ¹³C β-^TNDMBB_Do). Using the general procedure, starting from 60 mg

NMR) as an internal standard. Melting point (mp) was of β-azido-deoxyribose sugar (11, 0.15 mmol) and 26.14 mg of

recorded (in some cases) on a microscopic melting point 1-ethynyl-N,N-dimethylaniline (alkyne A, 0.18 mmol), 78.26 mg

apparatus. Fast atom bombardment (FAB) masses and/or (0.145 mmol) of the triazolyl-N,N-dimethylaminobenzene

HRMS were recorded on a high resolution mass spectrometer. nucleoside 13β was isolated as a yellow solid. Yield 96.6%; mp

BSA, Na₂HPO₄and NaH₂PO₄·H₂O (for preparation of the phos- 185–188 °C; IR (KBr) 1721, 1612, 1508, 1296, 1279, 1109 cm⁻¹

;

phate buﬀer) were purchased from Merck, India and used ¹H NMR (CDCl₃, 400 MHz) δ 2.38 (3H, s), 2.44 (3H, s), 2.99

without further purification. Water was taken from a Milli-Q (6H, s), 3.12–3.18 (2H, m), 4.57 (1H, dd, J = 3.6, 11.6 Hz),

purification system. All solutions were prepared 1 hour before 4.65–4.72 (2H, m), 5.77–5.79 (1H, m), 6.54 (1H, t, J = 6.8 Hz),

the experiments were done. The probe molecules [nucleoside 6.7 (2H, d, J = 8.8 Hz), 7.2 (2H, d, J = 7.6 Hz), 7.28 (2H, d, J =

TBPy

B

(7β)] were synthesized and purified according to the 8.0 Hz), 7.54 (2H, d, J = 8.8 Hz), 7.79 (1H, s), 7.89 (2H, d, J =

Do

procedure described.

8.0 Hz), 7.96 (2H, d, J = 8.4 Hz); ¹³C NMR (CDCl₃, 100 MHz)

Synthesis of 2-deoxy-3,5-di-O-p-toluoyl-α-^D-ribofuranosyl δ 21.8, 38.6, 40.52, 64.0, 74.9, 83.6, 88.9, 112.5, 116.6, 118.6,

chloride (10).²⁰The synthesis of bis-toluoyl-protected Hoﬀer’s 126.6, 126.8, 129.4, 129.8, 129.9, 144.2, 144.6, 148.8, 150.6,

chlorosugar 10 was achieved starting from 2-deoxyribose sugar 166.0, 166.3; HRMS calcd for C₃₁H₃₃N₄O₅[M + H]⁺541.2445,

(4 g, 29.8 mmol) with an overall yield of 4.48 g (59%, 0.01 mol) found 541.2485.

following our published protocol.²⁰

Synthesis of 3′,5′-bis{O-(p-toluoyl)}-2′-deoxy-1′-α-triazolyl-N,

Synthesis of 2-deoxy-3,5-bis[O-(p-toluoyl)]-β-D-ribofuranosyl- N-dimethylaminobenzene nucleoside (13α, bis-toluoyl-

azide (11, Method A).³¹The β-azido sugar was synthesised via α-^TNDMBB_Do). Using the general procedure, starting from

the reaction of toluoyl-protected chloro-deoxyribose sugar (10, 50 mg of α-azido-deoxyribose sugar (12, 0.126 mmol) and

1 eq., 1.5 g, 3.86 mmol) and cesium azide (CsN₃) (1.2 eq., 22.071 mg of 1-ethynyl-N,N-dimethylaniline (alkyne A,

4.63 mmol, 0.81 g) in dry DMSO following our literature prode- 0.607 mmol), 58.9 mg (0.109 mmol) of the triazolyl-N,N-di-

cure with 90% yield (1.37 g).³¹

methylaminobenzene nucleoside 13α was isolated as a yellow

Synthesis of 2-deoxy-3,5-bis[O-(p-toluoyl)]-α-D-ribofuranosyl- solid. Yield 86.5%; mp 158–160 °C; IR (KBr) 1718, 1611, 1377,

azide (12) (Method B).^20,22,23,31The α-azido sugar (12) was syn- 1309, 1102 cm⁻¹; ¹H NMR (CDCl₃, 400 MHz) δ 2.33 (3H, s), 2.4

thesised following our earlier report utilising a BF₃·Et₂O (0.1 (3H, s), 2.96 (6H, s), 2.99–3.14 (2H, m), 4.55–4.64 (2H, m),

eq., 0.386 mmol, 4.76 ml)-mediated reaction of toluoyl-pro- 4.8–4.84 (1H, m), 5.65 (1H, d, J = 5.6 Hz), 6.53 (1H, d, J =

tected chloro-deoxyribose sugar (10, 1.5 g, 3.86 mmol) and tri- 6.4 Hz), 6.73 (2H, d, J = 8.8 Hz), 7.09 (2H, d, J = 7.6 Hz), 7.24

methylsilylazide (TMS–N₃) (1.2 eq., 4.63 mmol, 0.61 ml) at (2H, d, J = 8 Hz), 7.66 (2H, d, J = 8 Hz), 7.7 (2H, d, J = 8 Hz),

0 °C in dry DCM to aﬀord the α-azido sugar (12) in 59% (0.9 g) 7.94 (2H, d, J = 8.0 Hz), 7.98 (1H, s); ¹³C NMR (CDCl₃,

yield.^20,22,23,31

100 MHz) δ 21.7, 38.7, 40.5, 64.1, 74.7, 84.8, 89.9, 112.5, 116.5,

118.9, 126.3, 126.8, 129.4, 129.8, 144.2, 144.3, 148.4, 150.5,

165.9, 166.2; HRMS calcd for C₃₁H₃₃N₄O₅[M + H]⁺541.2445,

found 541.2440.

General procedure for the synthesis of the triazolyl donor/

acceptor aromatic nucleosides via “Click” reaction

In a two necked round bottomed flask, α- and/or β-azido-deoxy-

Synthesis of 3′,5′-bis{O-(p-toluoyl)}-2′-deoxy-1′-β-triazolyldi-

ribose sugar (1.0 equiv.) was taken and dry THF was added. methoxyphenyl nucleoside (14β, bis-toluoyl-β-^TDMBB_Do). This

The solution was then degassed by purging with N₂gas for was synthesized and characterized according to our published

10 min. While continuing degassing, the aromatic alkyne (1.5 protocol.²⁰

equiv.) was added through the side neck followed by addition

Synthesis of 3′,5′-bis{O-(p-toluoyl)}-2′-deoxy-1′-α-triazolyldi-

of 6 mol% of sodium ascorbate dissolved in a small quantity methoxyphenyl nucleoside (14α, bis-toluoyl-α-^TDMBB_Do). Using

of water. After that 1 mol% of copper sulphate dissolved in a the general procedure, starting from 200 mg (12, 0.506 mmol)

small quantity of water was added through the side neck. The of α-azido-deoxyribose sugar and 98.45 mg (alkyne B,

reaction mixture was further degassed for another 5 min. The 0.607 mmol) of 1-ethynyl-3,5-dimethoxybenzene, 267.6 mg

final ratio of THF : H₂O in the reaction mixture was main- (0.480 mmol) of the triazolyldimethoxyphenyl nucleoside 14α

tained at 3 : 1. Finally, diisopropylethylamine (DIPEA) was was isolated as a colourless gel. Yield 95%; IR (KBr) 3145,

added to the reaction mixture and was refluxed at 75–80 °C for 1720, 1611, 1269 cm⁻¹. H NMR (CDCl₃, 400 MHz) δ 2.21 (3H,

1

about 12 hours. After completion of the reaction monitored by s), 2.29 (3H, s), 2.9–3.01 (2H, m), 3.68 (6H, s), 4.45–4.52 (2H,

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m), 4.71–4.74 (1H, m), 5.54–5.55 (1H, m), 6.33 (1H, t, J = Using the general procedure, starting from 50 mg (12,

2.4 Hz), 6.45–6.47 (1H, m), 6.86 (2H, d, J = 2.4 Hz), 6.98 (2H, d, 0.126 mmol) of α-azido-deoxyribose sugar and 27.52 mg

J = 8 Hz), 7.13 (2H, d, J = 8 Hz), 7.56 (2H, d, J = 8 Hz), 7.83 (2H, (alkyne D, 0.151 mmol) of 2-ethynyl-6-methoxynaphthalene,

d, J = 8 Hz), 8.01 (1H, s); ¹³C NMR (CDCl₃, 100 MHz) δ 21.6, 71.3 mg (0.123 mmol) of the triazolylmethoxynaphthalene

21.7, 38.8, 55.4, 64.0, 74.7, 85.1, 90.2, 100.7, 103.7, 118.4, nucleoside 16α was isolated as a white solid. Yield 98%; mp

126.2, 126.7, 129.3, 129.4, 129.6, 129.7, 132.3, 144.3, 144.5, 144–148 °C; IR (KBr) 3154, 1717, 1611, 1270, 851, 819, 750,

1

147.7, 161.2, 165.9, 166.2; HRMS calcd for C₃₁H₃₂N₃O₇690 cm⁻¹; H NMR (CDCl₃, 400 MHz) δ 2.27 (3H, s), 2.41 (3H,

[M + H]⁺558.2235, found 558.2232.

s), 3.03–3.16 (2H, m), 3.94 (3H, s), 4.59–4.68 (2H, m), 4.87–4.88

Synthesis of 3′,5′-bis{O-(p-toluoyl)}-2′-deoxy-1′-β-triazolyl- (1H, m), 5.69 (1H, d, J = 6.4 Hz), 6.62 (1H, d, J = 6.8 Hz), 7.05

phenoxyphenyl nucleoside (15β, bis-toluoyl-β-^TPhOBB_Do). Using (2H, d, J = 8 Hz), 7.13–7.17 (2H, m), 7.26 (2H, d, J = 8 Hz),

the general procedure, starting from 60 mg (11, 0.152 mmol) 7.68–7.71 (3H, m), 7.75 (1H, d, J = 8.4 Hz), 7.86–7.88 (1H, m),

of β-azido-deoxyribose sugar and 35.4 mg (alkyne C, 7.96 (2H, d, J = 8 Hz), 8.17 (1H, s), 8.19 (1H, s); ¹³C NMR

0.182 mmol) of 1-ethynyl-phenoxybenzene, 80.60 mg (CDCl₃, 100 MHz) δ 21.7, 21.8, 38.9, 55.4, 64.1, 74.9, 85.1, 90.3,

(0.137 mmol) of the triazolylphenoxyphenyl nucleoside 15β 105.9, 117.9, 119.4, 124.5, 125.9, 126.3, 126.8, 127.4, 129.1,

was isolated as a white solid. Yield 90%; IR (KBr) 1721, 1612, 129.5, 129.7, 129.8, 134.5, 144.4, 144.6, 148.2, 158.1, 165.9,

1508, 1296, 1279, 1109 cm⁻¹; ¹H NMR (CDCl₃, 400 MHz) δ 2.36 166.3; HRMS calcd for C₃₄H₃₂N₃O₆[M + H]⁺578.2286, found

(3H, s), 2.44 (3H, s), 2.89–2.93 (1H, m), 3.13–3.19 (1H, m), 4.56 578.2296.

(1H, dd, J = 4, 12 Hz), 4.68–4.78 (2H, m), 5.79–5.80 (1H, m),

Synthesis of 3′,5′-bis{O-(p-toluoyl)}-2′-deoxy-1′-β-triazolyl-

6.56 (1H, t, J = 6.2 Hz), 6.99 (2H, d, J = 8.4 Hz), 7.04 (2H, d, J = phenanthrene nucleoside (17β, bis-toluoyl-β-^TPhenB_Do). This

8.4 Hz), 7.13–7.15 (1H, m), 7.19 (2H, d, J = 8 Hz), 7.26–7.29 was synthesized and characterized according to our published

(3H, m), 7.34–7.38 (2H, m), 7.59 (2H, d, J = 8.8 Hz), 7.86–7.88 protocol.²⁰

(2H, m), 7.96 (2H, d, J = 8 Hz); ¹³C NMR (CDCl₃, 100 MHz)

Synthesis of 3′,5′-bis{O-(p-toluoyl)}-2′-deoxy-1′-α-triazolyl-

δ 21.81, 38.8, 63.9, 74.9, 83.8, 89.2, 117.7, 119.1, 119.2, 123.7, phenanthrene nucleoside (17α, bis-toluoyl-α-^TPhenB_Do). Using

125.5, 126.6, 126.8, 127.4, 129.1, 129.5, 129.8, 129.9, 144.4, the general procedure, starting from 200 mg (0.506 mmol) of

144.7, 147.9, 157.1, 157.5, 166.1, 166.3; HRMS calcd for α-azido-deoxyribose sugar (12) and 122.77 mg (alkyne E,

C₃₅H₃₂N₃O₆[M + H]⁺590.2286, found 590.2287.

0.607 mmol) of 9-ethynylphenanthrene, 288.7 mg

Synthesis of 3′,5′-bis{O-(p-toluoyl)}-2′-deoxy-1′-α-triazolyl- (0.483 mmol) of the triazolylphenanthrene nucleoside 17α was

phenoxyphenyl nucleoside (15α, bis-toluoyl-α-^TPhOBB_Do). Using isolated as a yellow gel. Yield 95.5%; IR (KBr) 1719, 1611,

the general procedure, starting from 50 mg (12, 0.126 mmol) 1270, 1100, 752, 728 cm⁻^{1 1}H NMR (CDCl₃, 400 MHz) δ ¹H

;

of α-azido-deoxyribose sugar and 29.3 mg (alkyne C, NMR (CDCl₃, 400 MHz) δ 2.24 (3H, s), 2.44 (3H, s), 3.07–3.14

0.152 mmol) of ethynylphenoxybenzene, 38.5 mg (1H, m), 3.29–3.3 (1H, m), 4.61–4.71 (2H, m), 4.9–4.91 (1H, m),

(0.065 mmol) of the triazolylphenoxyphenyl nucleoside 15α 5.70 (1H, d, J = 6.8 Hz), 6.69 (1H, d, J = 6 Hz), 7.00 (2H, d, J =

was isolated as a white solid. Yield 51.85%; mp 129–130 °C; IR 8 Hz), 7.29 (2H, d, J = 7.6 Hz), 7.51–7.55 (1H, m), 7.59–7.71 (5H,

(KBr) 1718, 1699, 1278, 1244, 1178, 1109 cm⁻^{1 1}H NMR m), 7.81 (1H, d, J = 7.6 Hz), 7.88 (1H, s), 7.97 (2H, d, J = 8 Hz),

;

(CDCl₃, 400 MHz) δ 2.26 (3H, s), 2.35 (3H, s), 2.93–3.10 (2H, 8.22 (1H, s), 8.38 (1H, d, J = 8 Hz), 8.71 (1H, d, J = 8 Hz), 8.77

m), 4.49–4.59 (2H, m), 4.74–4.77 (1H, m), 5.59 (1H, d, J = 6.4 (1H, d, J = 8 Hz); ¹³C NMR (CDCl₃, 100 MHz) δ 21.6, 21.7, 39.1,

Hz), 6.49 (1H, d, J = 6 Hz), 6.95–7.07 (7H, m), 7.19 (2H, d, J = 64.1, 74.8, 85.2, 90.4, 121.2, 122.6, 122.9, 126.2, 126.4, 126.8,

8 Hz), 7.28 (2H, t, J = 8 Hz), 7.59 (2H, d, J = 8 Hz), 7.68 (2H, d, J 126.9, 126.9, 127.2, 128.5, 128.8, 129.4, 129.7, 129.8, 130.1,

= 8.8 Hz), 7.87 (2H, d, J = 8.4 Hz), 7.99 (1H, s); ¹³C NMR (CDCl₃, 130.5, 130.7, 131.3, 144.3, 144.4, 146.9, 165.9, 166.2; HRMS

100 MHz) δ 21.9, 38.9, 64.1, 74.8, 74.8, 85.2, 90.3, 117.7, 119.2, calcd for C₃₇H₃₂N₃O₅[M + H]⁺598.2336, found 598.2333.

123.7, 125.9, 126.4, 126.9, 127.5, 129.4, 129.5, 129.8, 129.9,

130.0, 144.5, 144.6, 147.6, 157.2, 157.6, 166.1, 166.3; HRMS pyrene nucleoside (18β, bis-toluoyl-β-^TPyB_Do). Using the general

calcd for C₃₄H₃₂N₃O₆[M + H]⁺590.2286, found 590.2283.

procedure, starting from 100 mg (11, 0.253 mmol) of β-azido-

Synthesis of 3′,5′-bis{O-(p-toluoyl)}-2′-deoxy-1′-β-triazolyl- deoxyribose sugar and 68.59 mg (alkyne F, 0.304 mmol) of

Synthesis of 3′,5′-bis{O-(p-toluoyl)}-2′-deoxy-1′-α-triazolyl-

methoxynaphthalene

nucleoside

(16β,

bis-toluoyl- 1-ethynylpyrene, 133.63 mg (0.215 mmol) of the triazolylpyrene

β-^TMNapB_Do).³¹This was synthesized and characterized accord- nucleoside 18β was isolated as a yellow solid. Yield 85%; IR

ing to our published protocol.³¹Thus, using the general pro- (KBr) 2926, 1719, 1610, 1278 cm⁻¹; H NMR (CDCl₃, 400 MHz)

1

cedure, starting from 60 mg (0.152 mmol) of δ 2.16 (3H, s), 2.45 (3H, s), 2.98–3.04 (1H, m), 3.29–3.36 (1H,

β-azidodeoxyribose sugar (11) and 33.16 mg (alkyne D, m), 4.63 (1H, dd, J = 3.2, 11.2 Hz), 4.74–4.79 (2H, m), 5.86–5.87

0.182 mmol) of 2-ethynyl-6-methoxynaphthalene, 87.04 mg (1H, m), 6.69 (1H, t, J = 6.4 Hz), 7.03 (1H, d, J = 8 Hz), 7.29 (2H,

(0.151 mmol) of the triazolylmethoxynaphthalene nucleoside d, J = 7.6 Hz), 7.89 (2H, d, J = 8 Hz), 7.98–8.11 (8H, m),

16β was isolated as

197–200 °C; R_f= 0.45 in 2 : 1 (v/v) hexane–ethyl acetate; HRMS 100 MHz) δ 21.9, 38.9, 64.0, 74.9, 83.9, 89.3, 121.6, 124.9, 125.3,

calcd for C₃₄H₃₂N₃O₆[M + H]⁺578.2286, found 578.2365.²⁹

125.6, 126.3, 126.6, 127.3, 127.5, 128.1, 128.4, 128.8, 129.4,

a

brown solid. Yield 99.3%; mp 8.16–8.22 (4H, m), 8.63 (1H, d, J = 8 Hz); ¹³C NMR (CDCl₃,

Synthesis of 3′,5′-bis{O-(p-toluoyl)}-2′-deoxy-1′-α-triazolyl- 129.5, 129.8, 130.0, 144.3, 144.7, 148.1, 166.1, 166.4; HRMS

methoxynaphthalene nucleoside (16α, bis-toluoyl-α-^TMNapB_Do). calcd for C₃₉H₃₃N₃O₅[M + 2H]⁺623.2420, found 623.2454.

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Organic & Biomolecular Chemistry

Synthesis of 3′,5′-bis{O-(p-toluoyl)}-2′-deoxy-1′-α-triazolyl- s); ¹³C NMR (CDCl₃, 100 MHz) δ 21.9, 39.0, 64.0, 74.8, 85.4,

butylpyrene nucleoside (19β, bis-toluoyl-β-^TBPyB_Do). Using the 90.5, 118.9, 119.4, 126.2, 129.4, 129.5, 129.7, 129.9, 132.9,

general procedure, starting from 50 mg (11, 0.126 mmol) of 144.5, 144.8, 146.1, 165.9, 166.3; HRMS calcd for C₃₀H₂₇N₄O₅

β-azido-deoxyribose sugar and 47.06 mg (alkyne G, [M + H]⁺523.1976, found 523.1974.

0.151 mmol) of pyrenebutyric acid N-propynyl amide,

Synthesis of 3′,5′-bis{O-(p-toluoyl)}-2′-deoxy-1′-β-triazolyl-

86.00 mg (0.122 mmol) of the triazolylbutylpyrene nucleoside formylbenzene nucleoside (22β, bis-toluoyl-β-^TBenzalB_Ac). Using

19β was isolated as a dirty white solid. Yield 94.58%; IR (KBr) the general procedure, starting from 60 mg (0.152 mmol) of

3334.6, 2942.2, 1726.1, 1650.5, 1610, 1278 cm⁻^{1 1}H NMR β-azido-deoxyribose sugar (11) and 23.69 mg (0.182 mmol) of

;

(CDCl₃, 400 MHz) δ 2.16–2.22 (2H, m), 2.27 (1H, d, J = 8 Hz), 4-ethynylbenzaldehyde, 77.75 mg (0.148 mmol) of compound

2.33 (3H, s), 2.42 (3H, s), 2.76–2.82 (1H, m), 3.17–3.24 (1H, m), 26β was isolated as a white solid. Yield 97.4%; mp 188–190 °C;

3.35 (1H, t, J = 8 Hz), 4.43–4.63 (5H, m), 4.53–4.55 (1H, m), IR (KBr) 2852, 1727, 1714, 1695, 1610, 1280, 1112cm⁻^{1 1}H

;

5.71–5.74 (1H, m), 6.14–6.16 (1H, m), 6.38 (1H, t, J = 6 Hz), NMR (CDCl₃, 400 MHz) δ 2.36 (3H, s), 2.45 (3H, s), 2.94–2.96

7.15 (2H, d, J = 8 Hz), 7.25 (2H, d, J = 8 Hz), 7.69 (1H, s), (1H, m), 3.16–3.19 (1H, m), 4.54–4.57 (1H, m), 4.70 (1H, s),

7.79–7.83 (3H, m), 7.91–8.00 (6H, m), 8.08 (2H, d, J = 8 Hz), 4.76–4.79 (1H, m), 5.80–5.82 (1H, d, J = 2.4 Hz), 6.58 (1H, t, J =

8.13–8.16 (2H, m), 8.26 (1H, d, J = 8.8 Hz); ¹³C NMR (CDCl₃, 6 Hz), 7.18 (1H, d, J = 7.2 Hz), 7.29 (2H, d, J = 8 Hz), 7.79 (2H,

100 MHz) δ 20.0, 20.2, 27.9, 31.3, 33.1, 33.8, 35.8, 62.7, 73.3, d, J = 7.6 Hz), 7.84–7.88 (4H, m), 7.96 (2H, d, J = 8 Hz), 8.05

81.3, 86.6, 120.8, 122.2, 123.3, 124.6, 125.1, 125.3, 125.8, 126.1, (1H, s), 10.02 (1H, s); ¹³C NMR (DMSO-d₆, 100 MHz) δ 21.9,

127.1, 127.7, 127.8, 128.1, 128.2, 129.3, 129.7, 135.1, 142.3, 38.0, 64.8, 75.5, 83.7, 89.4, 122.9, 126.7, 127.6, 127.7, 130.2,

142.7, 144.2, 164.1, 164.2, 171.1; HRMS calcd for C₄₄H₄₂N₄O₆130.2, 130.4, 130.6, 131.1, 136.8, 137.2, 144.8, 145.2, 146.9,

[M + 2H]⁺722.3104, found 722.3107.

166.4, 166.5, 193.1; HRMS calcd for C₃₀H₂₈N₃O₆[M + 2H]⁺

Synthesis of 3′,5′-bis{O-(p-toluoyl)}-2′-deoxy-1′-β-triazolylni- 526.1973, found 526.1982.

trophenyl nucleoside (20β, bis-toluoyl-β-^TNBB_Ac). This was syn-

Synthesis of 3′,5′-bis{O-(p-toluoyl)}-2′-deoxy-1′-α-triazolyl-

thesized and characterized according to our published formylbenzene nucleoside (22α, bis-toluoyl-α-^TBenzalB_Ac). Using

protocol.²⁰

the general procedure, starting from 50 mg (0.126 mmol) of

Synthesis of 3′,5′-bis{O-(p-toluoyl)}-2′-deoxy-1′-α-triazolylni- α-azido-deoxyribose sugar (12) and 19.65 mg (0.151 mmol) of

trophenyl nucleoside (20α, bis-toluoyl-α-^TNBB_Ac). Using the 4-ethynylbenzaldehyde, 52 mg (0.099 mmol) of compound 22α

general procedure, starting from 200 mg (0.506 mmol) of was isolated as a white solid. Yield 78.5%; mp 142–145 °C; IR

azido-deoxyribose sugar (12) and 89.31 mg (0.607 mmol) of (KBr) 1717, 1694, 1610, 1286, 1107 cm⁻^{1 1}H NMR (CDCl₃,

;

1-ethynyl-4-nitrobenzene, 268.8 mg (0.496 mmol) of com- 400 MHz) δ 2.23 (3H, s), 2.33 (3H, s), 2.94–3.11 (2H, m),

pound 20α was isolated as a yellow solid. Yield 98%; mp 4.49–4.58 (2H, m), 4.78 (1H, s), 5.59 (1H, d, J = 5.6 Hz), 6.51

180–182 °C; IR (KBr) 1716, 1610, 1514, 1339 cm⁻^{1 1}H NMR (1H, d, J = 6.8 Hz), 6.96 (2H, d, J = 7.6 Hz), 7.17 (2H, d, J =

;

(CDCl₃, 400 MHz) δ 2.34 (3H, s), 2.43 (3H, s), 3.04–3.23 (2H, 8 Hz), 7.54 (2H, d, J = 7.6 Hz), 7.8–7.88 (6H, m), 8.16 (1H, s),

m), 4.59–4.69 (2H, m), 4.86–4.89 (1H, m), 5.68 (1H, d, J = 9.92 (1H, s); ¹³C NMR (CDCl₃, 100 MHz) δ 21.7, 21.8, 38.9,

6 Hz), 6.6 (1H, d, J = 5.6 Hz), 7.07 (2H, d, J = 8 Hz), 7.28 (2H, d, 64.0, 74.8, 85.3, 90.4, 119.7, 126.1, 126.2, 126.8, 129.4, 129.5,

J = 8 Hz), 7.63 (2H, d, J = 8.0 Hz), 7.95 (4H, d, J = 8.4 Hz), 8.24 129.7, 129.8, 130.4, 135.9, 136.4, 144.4, 144.7, 146.6, 165.9,

(1H, s), 8.26 (1H, s), 8.27 (1H, s); ¹³C NMR (CDCl₃, 100 MHz) 166.2, 191.8; HRMS calcd for C₃₀H₂₈N₃O₆[M + H]⁺526.1973,

δ 21.9, 38.9, 64.0, 74.8, 85.4, 90.5, 119.7, 124.4, 126.3, 126.8, found 526.1973.

129.4, 129.5, 129.7, 129.9, 136.9, 144.5, 144.8, 145.8, 147.5,

165.9, 166.3; HRMS calcd for C₂₉H₂₇N₄O₇[M + H]⁺543.1874, fluorophenyl nucleoside (23β, bis-toluoyl-β-^TDFBB_Ac). Using the

found 543.1879. general procedure, starting from 60 mg (0.152 mmol) of

Synthesis of 3′,5′-bis{O-(p-toluoyl)}-2′-deoxy-1′-β-triazolyldi-

Synthesis of 3′,5′-bis{O-(p-toluoyl)}-2′-deoxy-1′-β-triazolyl- β-azido-deoxyribose sugar (11) and 25 mg (0.182 mmol) of

cyanophenyl nucleoside (21β, bis-toluoyl-β-^TCNBB_Ac). This was 1-ethynyl-2,4-difluorobenzene, 69.6 mg (0.130 mmol) of com-

synthesized and characterized according to our published pound 23β was isolated as a white solid. Yield 86%; mp

protocol.²⁰

165–170 °C; IR (KBr) 1729, 1709, 1610, 1281, 1112 cm⁻^{1 1}H

;

Synthesis of 3′,5′-bis{O-(p-toluoyl)}-2′-deoxy-1′-α-triazolyl- NMR (CDCl₃, 400 MHz) δ 2.36 (3H, s), 2.44 (3H, s), 2.88–2.92

cyanophenyl nucleoside (21α, bis-toluoyl-α-^TCNBB_Ac). Using the (1H, m), 3.26–3.29 (1H, m), 4.56 (1H, dd, J = 4, 12 Hz),

general procedure, starting from 200 mg (0.506 mmol) of 4.65–4.69 (2H, m), 5.8–5.81 (1H, m), 6.54 (1H, t, J = 6 Hz),

α-azido-deoxyribose sugar (12) and 77.199 mg (0.607 mmol) of 6.82–6.88 (1H, m), 6.98 (1H, t, J = 8 Hz), 7.16 (2H, d, J = 8 Hz),

4-ethynylbenzonitrile, 250.65 mg (0.480 mmol) of compound 7.28 (2H, d, J = 8 Hz), 7.84 (2H, d, J = 8 Hz), 7.96 (2H, d, J = 8

21α was isolated as a white solid. Yield 95%; mp 160–163 °C; Hz), 8.08–8.09 (1H, m), 8.18–8.25 (1H, m); ¹³C NMR (CDCl₃,

IR (KBr) 2226, 1717, 1611, 1104 cm⁻^{1 1}H NMR (CDCl₃, 100 MHz) δ 21.8, 21.9, 38.4, 63.9, 74.9, 83.8, 89.1, 104.2, 111.9,

;

400 MHz) δ 2.35 (3H, s), 2.43 (3H, s), 3.03–3.1 (1H, m), 112.1, 121.3, 126.6, 126.8, 129.3, 129.4, 129.8, 129.95, 141.1,

3.17–3.2 (1H, m), 4.58–4.68 (2H, m), 4.84–4.86 (1H, m), 5.67 144.1, 144.6, 166.01, 166.29; HRMS calcd for C₂₉H₂₆F₂N₃O₅

(1H, d, J = 8 Hz), 6.59 (1H, d, J = 6.8 Hz), 7.06 (2H, d, J = 8 Hz), [M + H]⁺534.1835, found 534.1886.

7.28 (2H, d J = 8.4 Hz), 7.62 (2H, d, J = 8 Hz), 7.69 (2H, d, J =

Synthesis of 3′,5′-bis{O-(p-toluoyl)}-2′-deoxy-1′-α-triazolyldi-

8.8 Hz), 7.9 (2H, d, J = 8.6 Hz), 7.95 (2H, d, J = 8 Hz), 8.21 (1H, fluorophenyl nucleoside (23α, bis-toluoyl-α-^TDFBB_Ac). Using the

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general procedure, starting from 50 mg (0.126 mmol) of

Synthesis of 2′-deoxy-1′-β-triazolyl-phenoxyphenyl nucleoside

α-azido-deoxyribose sugar (12) and 20.88 mg (0.151 mmol) of (3β, β-^TPhOBB_Do). Using the general procedure for deprotection

1-ethynyl-2,4-difluorobenzene, 66.65 mg (0.125 mmol) of com- starting from 100.00 mg (0.169 mmol) of compound 15β,

pound 23α was isolated as a white solid. Yield 99.2%; mp 54.1 mg (0.153 mmol) of compound 3β was isolated as a white

117–120 °C; IR (KBr) 1716, 1610, 1281, 1270, 1101 cm⁻^{1 1}H solid. Yield 90.67%; mp 143 °C; IR (KBr) 3399, 1488, 1241,

;

NMR (CDCl₃, 400 MHz) δ 2.24 (3H, s), 2.32 (3H, s), 2.96–3.02 1090, 1067, 1039 cm⁻¹

;

¹H NMR (CD₃OD, 400 MHz)

(1H, m), 3.06 (1H, d, J = 14.8 Hz), 4.49–4.58 (2H, m), 4.74–4.77 δ 2.52–2.55 (1H, m), 2.79–2.83 (1H, m), 3.64–3.77 (2H, m),

(1H, m), 5.59 (1H, d, J = 6.4 Hz), 6.51 (1H, d, J = 5.2 Hz), 4.04–4.07 (1H, m), 4.56–4.59 (1H, m), 6.43–6.45 (1H, m), 7.01

6.75–6.81 (1H, m), 6.88–6.93 (1H, m), 6.99 (2H, d, J = 8 Hz), (4H, d, J = 6.8 Hz), 7.13 (1H, t, J = 6.0 Hz.), 7.36 (2H, t, J =

7.17 (2H, d, J = 8 Hz), 7.58 (2H, d, J = 8 Hz), 7.86 (2H, d, J = 6.4 Hz), 7.78 (2H, d, J = 7.2 Hz), 8.45 (1H, s); ¹³C NMR (CD₃OD,

8 Hz), 8.14 (1H, d, J = 3.6 Hz), 8.15–8.21 (1H, m); ¹³C NMR 100 MHz) δ 41.9, 63.4, 72.4, 89.9, 90.5, 120.1, 120.4, 120.6,

(CDCl₃, 100 MHz) δ 21.8, 38.9, 64.1, 74.7, 85.1, 90.3, 103.9, 124.9, 126.8, 128.5, 131.2, 148.7, 158.4, 159.2; HRMS calcd for

104.2, 104.4, 111.9, 112.2, 120.6, 120.7, 126.2, 126.8, 129.0, C₁₉H₂₀O₄N₃[M + H]⁺354.1453, found 354.1480.

129.1, 129.3, 129.5, 129.8, 140.8, 144.4, 144.5, 166.1, 166.3;

Synthesis

of

2′-deoxy-1′-β-triazolylmethoxynaphthalene

HRMS calcd for C₂₉H₂₆F₂N₃O₅[M + H]⁺534.1835, found nucleoside (4β, β-^MNapB_Do).³¹This was synthesized in a similar

534.1837.

way as we reported previously.³¹Thus, using the general pro-

cedure for deprotection starting from 85 mg (0.147 mmol) of

compound 16β, 52.92 mg (0.153 mmol) of compound 4β was

isolated as a white solid. Yield 88%; R_f= 0.5 in 100% ethyl

acetate; mp 141–145 °C; HRMS calcd for C₁₈H₂₀N₃O₄[M + H]⁺

General procedure for toluoyl deprotection of the triazolyl

donor/acceptor aromatic nucleosides

The bis-toluoylated nucleoside (1 equiv.) was dissolved in dry 342.1453, found 342.1479.²⁹

methanol. Sodium methoxide (3.5 equiv.) was subsequently Synthesis of

2′-deoxy-1′-α-triazolylmethoxynaphthalene

added. The solution was left stirring at room temperature over- nucleoside (4α, α-^MNapB_Do). Using the general procedure for

night. The solution was evaporated and the deprotected pro- deprotection starting from 280 mg (0.48 mmol) of compound

ducts were separated by column chromatography. All the 16α, 141.1 mg (0.413 mmol) of compound 4α was isolated as a

β-epimers except 22β (^TBenzalB_Ac) and 23β (^TDFBB_Ac) were depro- white solid. Yield 86%; mp 183–185 °C; IR (KBr) 3353, 1611,

tected. None of the α-epimers were deprotected.

1502, 1261, 1218, 1142, 1106, 861, 815 cm⁻¹; ¹H NMR (CD₃OD,

Synthesis of

2′-deoxy-1′-β-triazolyl-N,N-dimethylanilino 400 MHz) δ 2.42 (1H, td, J = 2.4, 14.4 Hz), 2.75–2.82 (1H, m),

nucleoside (1β, β-^TNDMBB_Do). Using the general procedure for 3.51–3.61 (2H, m), 3.79 (3H, s), 4.18 (1H, q, J = 3.6, 4.0 Hz),

deprotection starting from 98.2 mg (0.182 mmol) of com- 4.34–4.37 (1H, m), 6.37 (1H, dd, J = 2.4, 7.6 Hz), 7.03 (1H, dd,

pound 13β, 50.4 mg (0.165 mmol) of compound 1β was iso- J = 2.4, 9.2 Hz), 7.13 (1H, d, J = 2.4 Hz), 7.67–7.77 (3H, m), 8.10

lated as a yellow solid. Yield 91.11%; mp 168 °C; IR (KBr) (1H, s), 8.56 (1H, s); ¹³C NMR (DMSO, 100 MHz) δ 55.3, 61.4,

3330, 1617, 1507, 1201, 1105, 1051, 1013 cm⁻^{1 1}H NMR 70.4, 88.5, 88.9, 106.0, 119.2, 119.5, 123.5, 124.1, 125.9, 127.4,

;

(CD₃OD, 400 MHz) δ 2.36–2.42 (1H, m), 2.64–2.71 (1H, m), 128.6, 129.6, 133.9, 146.7, 157.5; HRMS calcd for C₁₈H₂₀O₄N₃

2.84 (6H, s), 3.49–3.54 (1H, m), 3.59–3.64 (1H, m), 3.90 (1H, q, [M + H]⁺342.1453, found 342.1466.

J = 4.2, 4.8 Hz), 4.43 (1H, q, J = 4.6, 6 Hz), 6.29 (1H, t, J = 6 Hz),

Synthesis of 2′-deoxy-1′-β-triazolylphenanthrene nucleoside

6.68 (2H, d, J = 8.4 Hz), 7.51 (2H, d, J = 8.8 Hz), 8.21 (1H, s); (5β, β-^TPhenB_Do). This was synthesized and characterized

¹³C NMR (CD₃OD, 100 MHz) δ 40.9, 41.9, 63.5, 72.5, 89.9, 90.4, according to our published protocol.²⁰

113.9, 119.3, 119.8, 127.8, 149.8, 152.4; HRMS calcd for

C₁₅H₂₁N₄O₃[M + H]⁺305.1608, found 305.1643.

Synthesis of 2′-deoxy-1′-α-triazolylphenanthrene nucleoside

(5α, α-^TPhenB_Do). Using the general procedure for deprotection

Synthesis of 2′-deoxy-1′-β-triazolyl-dimethoxyphenyl nucleo- starting from 280 mg (0.486 mmol) of compound 17α,

TDMB

side (2β,

B ). This was synthesized and characterized 145.9 mg (0.404 mmol) of compound 5α was isolated as a pale

Do

according to our published protocol.²⁰

yellow solid. Yield 83%; IR (KBr) 3364, 1121 1056, 857, 841,

Synthesis of 2′-deoxy-1′-α-triazolyl-dimethoxyphenyl nucleo- 746, 728 cm⁻¹; H NMR (CD₃OD, 400 MHz) δ 2.50 (1H, td, J =

side (2α, α-^TDMBB_Do). Using the general procedure for de- 2.4, 14.4 Hz), 2.79–2.87 (1H, m), 3.52–3.63 (2H, m), 4.23 (1H,

protection starting from 260 mg (0.47 mmol) of compound q, J = 3.8, 3.2 Hz), 4.36–4.39 (1H, m), 6.47 (1H, dd, J = 2,

14α, 144.0 mg (0.448 mmol) of compound 2α was isolated as a 7.4 Hz), 7.48–7.53 (2H, m), 7.56–7.61 (2H, m), 7.83 (1H, d, J =

white solid. Yield 95.4%; IR (KBr) 3340, 1208, 1082, 8.0 Hz), 7.85 (1H, s), 8.11 (1H, d, J = 8.4 Hz), 8.52 (1H, s), 8.66

1

1053 cm⁻¹; H NMR (CD₃OD, 400 MHz) δ 2.40 (1H, td, J = 2.4, (1H, d, J = 8.4 Hz), 8.73 (1H, d, J = 8.0 Hz); ¹³C NMR (CD₃OD,

14.4 Hz), 2.73–2.80 (1H, m), 3.49–3.53 (1H, m), 3.56–3.60 (1H, 100 MHz) δ 42.4, 63.6, 73.0, 91.2, 92.0, 124.1, 124.4, 124.6,

m), 3.70 (6H, s), 4.16 (1H, q, J = 3.6 Hz), 4.32–4.35 (1H, m), 127.4, 128.4, 128.5, 128.9, 129.9, 130.3, 131.7, 132.1, 132.4,

6.33 (1H, d, J = 2.4 Hz), 6.35 (1H, d, J = 2.0 Hz), 6.87 (2H, d, J = 132.9, 148.0; HRMS calcd for C₂₁H₂₀N₃O₃[M + H]⁺362.1499,

2.4 Hz), 8.47 (1H, s); ¹³C NMR (CD₃OD, 100 MHz) δ 42.0, 56.0, found 362.1498.

63.3, 72.7, 90.7, 91.5, 101.6, 104.8, 121.22, 130.3, 130.9, 133.5,

Synthesis of 2′-deoxy-1′-β-triazolylpyrene nucleoside (6β,

148.9, 162.9; HRMS calcd for C₁₅H₂₀O₅N₃[M + H]⁺322.1402, β-^TPyB_Do).³²Using the general procedure for deprotection start-

found 322.1416.

ing from 60 mg (0.097 mmol) of compound 18β, 30.61 mg

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Organic & Biomolecular Chemistry

(0.080 mmol) of compound 6β was isolated as a white Crystallographic description and ORTEP diagram

solid. Yield 81.4%; IR (KBr) 3376.8, 2942.5, 1068 cm⁻¹

;

Crystal data were collected with a CCD diﬀractometer using

graphite monochromated MoKα radiation (λ = 0.71073 Å) at

298 K. Cell parameters were retrieved using and refined with

softwares on all observed reflections.³³Data reduction was per-

formed with the software and corrected for Lorentz and polar-

ization eﬀects.³³Absorption corrections were applied with the

program.³⁴The structure was solved by direct methods

implemented in a program and refined by full-matrix least-

squares methods on F².³⁵All non-hydrogen atomic positions

were located in diﬀerence Fourier maps and refined anisotro-

pically. The hydrogen atoms were placed in their geometrically

generated positions. Colourless crystals were isolated, of rec-

tangular shape, from acetonitrile at room temperature.

Crystallographic description for 2′-deoxy-1′-β-triazolylnitro-

benezene nucleoside (8β, β-^TNBB_Ac). Crystal dimension (mm):

0.30 × 0.26 × 0.17. C₁₃H₁₄N₄O₅, Mr = 306.28; orthorhombic,

space group P212121; a = 6.4456(7) Å, b = 9.8102(8) Å, c =

21.8210(19) Å; α = 90.00°, β = 90.00°, γ = 90.00°, V = 1379.8(2)

Å³; Z = 4; ρ_cal= 1.474 g cm⁻³; μ (mm⁻¹) = 0.116; F(000) =

640.00; refinement method = full-matrix least-squares on F²;

final R indices [I > 2σ_l] R(reflections) = 0.0509(1701), wR₂(re-

flections) = 0.1143(3180); goodness of fit = 0.933.

Crystallographic description for 2′-deoxy-1′-α-triazolyl-

methoxynaphthalene nucleoside (4α, α-^MNapB_Do). Crystal

dimension (mm): 0.29 × 0.22 × 0.15. C₁₈H₁₉N₃O₄, Mr = 341.36;

monoclinic, space group P21; a = 5.808(7) Å, b = 29.856(4) Å,

c = 9.539(11) Å; α = 90.00°, β = 90.185°, γ = 90.00°, V = 1654.2(4)

Å³; Z = 4; ρ_cal= 1.371 g cm⁻³; μ (mm⁻¹) = 0.099; F(000) =

720.00; refinement method = full-matrix least-squares on F²;

final R indices [I > 2σ_l] R(reflections) = 0.0478(2923), wR₂(re-

flections) = 0.1066(4410); goodness of fit = 0.989.

¹H NMR (CD₃OD + CDCl₃, 400 MHz) δ 2.64–2.68 (1H, m),

2.95–2.99 (1H, m), 3.73–3.86 (2H, m), 4.13 (1H, s), 4.55 (1H, s),

6.60 (1H, t, J = 5.6 Hz), 8.03–8.29 (8H, m), 8.56 (1H, d, J =

8 Hz), 8.67 (1H, s); ¹³C NMR (CD₃OD, 100 MHz) δ 38.88, 41.39,

62.65, 71.59, 89.41, 89.79, 123.69, 125.56, 125.79, 126.04,

126.38, 127.23, 128.13, 128.68, 128.92, 129.06, 131.65, 132.19,

147.69; HRMS calcd for C₂₃H₂₁N₃O₃[M + 2H]⁺387.1583, found

387.1532.

Synthesis of 2′-deoxy-1′-β-triazolylbutylpyrene nucleoside

(7β, β-^TBPyB_Do). Using the general procedure for deprotection

starting from 50 mg (0.069 mmol) of compound 19β, 27.88 mg

(0.058 mmol) of compound 7β was isolated as a white solid.

Yield 83%; IR (KBr) 3432, 3356.8, 2926.8, 1715 cm⁻¹; H NMR

1

(CD₃OD, 400 MHz) δ 1.99–2.07 (2H, m), 2.25 (2H, t, J = 7.2 Hz),

2.29–2.35 (1H, m), 2.55–2.61 (1H, m), 3.19–3.24 (2H, m)

(merged with solvent peak), 3.24–3.58 (2H, m), 3.89 (1H, q, J =

4.4 Hz), 4.27–4.39 (3H, m), 6.24 (1H, t, J = 6 Hz), 7.74 (1H, d,

J = 7.6 Hz), 7.84–8.06 (8H, m), 8.16 (1H, d, J = 9.2 Hz); ¹³C

NMR (CD₃OD, 100 MHz) δ 30.9, 33.8, 35.8, 36.7, 41.9, 63.4,

72.4, 89.8, 90.3, 123.1, 124.5, 125.9, 126.0, 126.2, 126.3, 127.1,

127.8, 128.4, 128.5, 128.6, 129.9, 131.4, 132.4, 132.9, 137.4,

146.6, 175.93; HRMS calcd for C₂₈H₃₀N₄O₄[M + 2H]⁺486.2267,

found 486.2230.

Synthesis of 2′-deoxy-1′-β-triazolylnitrophenyl nucleoside

(8β, β-^TNBB_Ac). This was synthesized and characterized accord-

ing to our published protocol.²⁰

Synthesis of 2′-deoxy-1′-α-triazolylnitrophenyl nucleoside

(8α, α-^TNBB_Ac). Using the general procedure for deprotection

starting from 290 mg (0.54 mmol) of compound 20α, 135.5 mg

(0.442 mmol) of compound 8α was isolated as a yellow solid.

Yield 82%; mp 147–150 °C; IR (KBr) 3523, 3473, 1513, 1341,

857 cm⁻^{1 1}H NMR (CD₃OD, 400 MHz) δ 2.52–2.57 (1H, m),

;

2.87–2.94 (1H, m), 3.63–3.74 (2H, m), 4.30–4.33 (1H, m),

4.46–4.49 (1H, m), 6.5–6.5 (1H, dd, J = 2, 7.6 Hz), 8.07 (2H, d,

Photophysical studies of the nucleosides

UV-visible measurements. All the UV–visible spectra of the

J = 8.8 Hz), 8.3 (2H, d, J = 8.8 Hz), 8.8 (1H, s); ¹³C NMR compounds (10 μM) were measured in diﬀerent solvents using

(CD₃OD, 100 MHz) δ 42.2, 63.4, 72.7, 91.0, 91.8, 122.7, 125.5, a UV-visible spectrophotometer with a cell of 1 cm path

127.5, 138.4, 146.9, 148.9; ESI calcd for C₁₃H₁₅N₄O₅[M + H]⁺length. The measurements were carried out in the absorbance

307.1042, found 307.1376.

mode. The absorbance values of the sample solutions were

Synthesis of 2′-deoxy-1′-β-triazolylcyanophenyl nucleoside measured in the wavelength regime of 200–550 nm. All the

(9β, β-^TCNBB_Ac). This was synthesized and characterized accord- sample solutions were prepared just before doing the

ing to our published protocol.²⁰

experiment.

Synthesis of 2′-deoxy-1′-α-triazolylcyanophenyl nucleoside

Fluorescence experiments. All the sample solutions were

(9α, α-^TCNBB_Ac). Using the general procedure for deprotection prepared as described in the UV measurement experiments.

starting from 240 mg (0.46 mmol) of compound 21α, 123.5 mg Fluorescence spectra were obtained using a fluorescence

(0.431 mmol) of compound 9α was isolated as a white solid. spectrophotometer at 25 °C using a 1 cm path length cell. The

Yield 93.8%; mp 124–129 °C; IR (KBr) 3450, 3403, 2232, excitation wavelengths for all the cases were set at the exci-

1

1085 cm⁻¹; H NMR (CD₃OD, 400 MHz) δ 2.40 (1H, td, J = 2, tation maxima of each sample in each solvent, and emission

14.8 Hz), 2.73–2.80 (1H, m), 3.49–3.54 (1H, m), 3.56–3.59 spectra were measured in the wavelength regime of

(1H, m), 4.17 (1H, q, J = 3.8, 7.0 Hz), 4.32–4.35 (1H, m), 300–700 nm with an integration time of 0.2 s. All the sample

6.37 (1H, dd, J = 2, 7.6 Hz), 7.67 (2H, d, J = 8.4 Hz), 7.89 (2H, d, solutions were prepared just before doing the experiment.

J = 8.4 Hz), 8.64 (1H, s); ¹³C NMR (CD₃OD, 100 MHz) δ 42.1, A total volume of 1.0 ml from a stock solution of 2 ml of 10 μM

63.3, 72.9, 90.9, 91.8, 112.6, 122.4, 127.2, 127.4, 136.5, 147.1; concentration for each case was used for the fluorescence

HRMS calcd for C₁₄H₁₅N₄O₃[M + H]⁺287.1144, found experiments in a 1 ml cell. Fluorescence emissions were col-

287.1147.

lected by exciting the samples at the wavelength corresponding

5104 | Org. Biomol. Chem., 2016, 14, 5088–5108

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Paper

to their absorption maximum. Steady-state fluorescence emis- of the sample solutions were measured in the wavelength

sion spectra were recorded at room temperature as an average regime of 200–700 nm. All the experiments were carried out

of five scans using an excitation slit of 3.0 nm, emission slit of with freshly prepared sample solutions.

3.0 nm, and a scan speed of 120 nm min⁻¹. The fluorescence

Fluorescence study with BSA. All fluorescence and steady

quantum yields (Φ_f) were determined using quinine sulphate state anisotropy experiments were performed using a Fluoro-

as a reference with the known Φ_f(0.55) in 0.1 molar solution max 4 spectrophotometer with a cell of 1 cm path length at

in sulphuric acid. The following equation was used to calculate 298 K. The excitation wavelength for the probe (nucleoside

TBPy

the quantum yield:

B

Do

(7β) or 1 : 1 mixture of 7β : 9α as the probe) was set at

280 nm and 350 nm, and emission spectra were measured

in the wavelength regime of 290–650 nm. Steady state

anisotropy of the solutions was measured using Fluoromax

4 spectrophotometer.

Fl^A_S^reaAbs_Rn_S

2

Φ_S¼ Φ_R

^AreaAbs_Sn_R

Fl_R

where Φ_Ris the quantum yield of a standard reference,

Fl^A_S^rea(sample) and Fl_R^Area(reference) are the integrated emis-

sion peak areas, Abs_S(sample) and Abs_R(reference) are the

absorbances at the excitation wavelength, and n_S(sample) and

n_R(reference) are the refractive indices of the solutions.

The fluorescence lifetime experiments were carried out

using a time resolved fluorescence spectrophotometer at 25 °C

using a 1 cm path length cell. A 375 nm laser was used as the

excitation light source. The lifetime data were calculated by

software with a fixed fitting range. The time correlated single

photon counting (TCSPC) method was used to calculate the

lifetime data. The lifetime data (Global Analysis) were calcu-

lated by the software package with a fitting range of 205–4000

channels.

Acknowledgements

This work is financially supported by the Department of

Science and Technology [DST: SR/SI/OC-69/2008] and the

Department of Biotechnology [DBT: BT/PR5169/BRB/10/1065/

2012], Govt. of India.

Notes and references

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Studies on the interaction of nucleoside ^TBPyB_Do(7β) with BSA

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Preparation of BSA solution. Phosphate buﬀer of pH 7.0 was

used to prepare the solution of BSA (Merck). A 250 μM of stock

BSA solution was prepared by dissolving 0.0222 g of BSA in

1.28 mL phosphate buﬀer (20 mM) of pH 7.0. From that stock

solution a sub-stock of 1000 μM BSA was prepared. The com-

pound stock solution was prepared in DMF because of the

TBPy

poor solubility in water. 0.6 mg of nucleoside

B

(7β) or

Do

1 : 1 mixture of 7β : 9α as the probe was dissolved in 1 mL DMF

to make a stock probe solution of concentration 1092.4 μM.

General experimental on interaction studies of BSA by

photophysical studies. All the spectral measurements were

carried out at room temperature. To study the interaction of

compounds with BSA, an aqueous solution of the nucleoside

TBPy

B

Do

(7β) or 1 : 1 mixture of 7β : 9α as the probe (10 μM for

nucleoside) was titrated with diﬀerent concentrations of BSA

(ranging from 0, 0.2, 0.4, 0.6, 0.8, 1.0, 1.4, 1.8, 2.2, 2.6, 3.0

equivalents). The total volume of the final solution for each

sample was 3 mL. The % of DMF content was 2%. The pres-

ence of 2% DMF does not induce structural changes to bio-

molecules. Each sample solution was mixed well before

spectral measurements.

UV-visible study with BSA. The UV-visible absorbance

measurements were performed using a Shimadzu UV-2550 UV-

visible spectrophotometer with a cell of 1 cm path length at

298 K. All the UV-visible studies were carried out in 20 mM

phosphate buﬀer of pH 7.02 containing solution at 298 K. 2%

DMF was used to solubilize the probe. The measurements

were taken in the absorbance mode and the absorbance values

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Donor/acceptor chromophores-decorated triazolyl unnatural nucleosides: Synthesis, photophysical properties and study of interaction with BSA

DOI: 10.1039/c6ob00500d

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