Synthesis of new triazole based imidazo[1,2-a]pyrazine-benzimidazole conjugates: H-bonding assisted FRET efficient ratiometric detection of pyrophosphate

Article abstract of DOI:10.1016/j.jphotochem.2017.08.009

Triazole tethered imidazo[1,2-a]pyrazine-benzimidazole conjugates 13-38 has been synthesized by click and Suzuki-Miyaura cross coupling reactions at C-8 and C-6 positions, respectively. The research findings clearly predicted that by modification of electronic structure of the receptor, the sensitivity of the recognition process could be modified. Compound 24 with hydroxyphenyl substituent, showed stronger binding to the pyrophosphate than other compounds. Compound 24 has been used as selective probe for ratiometric detection of pyrophosphate amongst the other anions. The binding event of compound 24 toward PPi has been successfully evaluated by absorption and emission spectroscopy as well as NMR titration method. The compound 24 showed H-bonding assisted facilitation of FRET phenomenon in the presence of PPi.

Full text of DOI:10.1016/j.jphotochem.2017.08.009

Accepted Manuscript
Title: Synthesis of new triazole based
imidazo[1,2-a]pyrazine-benzimidazole conjugates: H-bonding
assisted FRET efficient ratiometric detection of pyrophosphate
Authors: Richa Goel, Vijay Luxami, Kamaldeep Paul
PII:
S1010-6030(17)30666-4
DOI:
Reference:
http://dx.doi.org/doi:10.1016/j.jphotochem.2017.08.009
JPC 10784
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Journal of Photochemistry and Photobiology A: Chemistry
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Revised date:
Accepted date:
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4-8-2017
6-8-2017
Please cite this article as: Richa Goel, Vijay Luxami, Kamaldeep Paul, Synthesis of new
triazole based imidazo[1,2-a]pyrazine-benzimidazole conjugates: H-bonding assisted
FRET efficient ratiometric detection of pyrophosphate, Journal of Photochemistry and
Photobiology A: Chemistryhttp://dx.doi.org/10.1016/j.jphotochem.2017.08.009
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Synthesis of new triazole based imidazo[1,2-a]pyrazine-benzimidazole conjugates: H-
bonding assisted FRET efficient ratiometric detection of pyrophosphate
Richa Goel, Vijay Luxami* and Kamaldeep Paul*
School of Chemistry and Biochemistry, Thapar University, Patiala-147004, INDIA
*Corresponding Authors. Email Address: vluxami@thapar.edu (V. Luxami);
kpaul@thapar.edu (K. Paul)
Graphical abstract
1

Highlights
 A series of triazole based imidazo[1,2-a]pyrazine-benzimidazole analogs has been
synthesized.
 Compounds were synthesized through click and Suzuki reactions.
 Quantum yields of the synthesized compounds were determined.
 FRET efficiency of the molecule has been used for the ratiometric detection of
pyrophosphate.
Abstract
Triazole tethered imidazo[1,2-a]pyrazine-benzimidazole conjugates 13-38 has been synthesized
by click and Suzuki-Miyaura cross coupling reactions at C-8 and C-6 positions, respectively. The
research findings clearly predicted that by modification of electronic structure of the receptor, the
sensitivity of the recognition process could be modified. Compound 24 with hydroxyphenyl
substituent, showed stronger binding to the pyrophosphate than other compounds. Compound 24
has been used as selective probe for ratiometric detection of pyrophosphate amongst the other
anions. The binding event of compound 24 toward PPi has been successfully evaluated by
absorption and emission spectroscopy as well as NMR titration method. The compound 24
showed H-bonding assisted facilitation of FRET phenomenon in the presence of PPi.
Keywords: Imidazo[1,2-a]pyrazine, benzimidazole, click reaction, Suzuki reaction, FRET,
pyrophosphate.
1. Introduction
Phosphate-containing anionic species [1] are ubiquitous in biological systems and play important
mediatory roles in signal transduction pathways, and carrying genetic informations [2]. For
example, pyrophosphate ions (PPi) are involved in energy transduction in organisms, and
controlling metabolic processes via participation in enzymatic reactions [3]. Various important
biochemical reactions like DNA polymerization, synthesis of cyclic AMP and formation of
activated intermediates in protein synthesis, are catalyzed by DNA polymerase, adenylate
2

cyclase and aminoacyl-tRNA synthetase, respectively. While the hydrolysis of ATP with
concomitant release of PPi is an important factor in biochemical pathways [4-6]. Therefore,
several research groups have focused on the detection of this biological anion. The design of
fluorescent chemosensor for detection of pyrophosphate ion remains a challenge [7-9]. Most of
the developed phosphate anion sensors are based on transition metal complexes where the cavity
formed by the metal ion with the receptor provides a cooperative binding site for phosphate
derivative [10]. In this context, Zn(II) complex has been frequently used for detection of PPi [11-
16]. But complexes with other metal ions like Tb(III) [17], Cd(II) [10], Mn(II) [18], Cu(II) [19-
25] and Eu(III) [26] have also been employed. Some of these sensors displayed remarkable
selectivity and sensitivity. However, considerable synthetic efforts are required for their
preparation. Moreover, sensing molecules for detection of PPi are limited in literature and the
potential for the development of these PPi sensors are at the primitive stage for bioanalytical
applications. Until now, few chemosensors are reported for detection of PPi in the absence of
metal ions [27-30].
Amongst different approaches proposed for ratiometric ion sensing, Förster resonance energy
transfer (FRET) is a non-radiative energy transfer process in which the excitation energy of the
donor is transferred to nearby acceptor via long-range dipole–dipole interaction and/or short-
range multipolar interaction. FRET is generally designed as fluorescence sensor to adopt the
photophysical changes produced on complexation [31-33]. Recently, FRET based probes [34-35]
have been used in cell physiology, optical therapy and selective as well as specific sensing
toward target analytes [36-38]. However, despite many advantages, ratiometric sensing of PPi
using FRET phenomenon is not known. Imidazo[1,2-a]pyrazine is known to exhibit fluorescence
properties such as chemiluminescence [39] and bioluminescence [40], found in the scaffold of
the Coelenterazine and a bioluminescent compound isolated from the Jellyfish Aequorea
Vistoria. Benzimidazole moiety has been commonly utilized as the molecular recognition site for
cation, anion and neutral molecules due to its unique spectral properties. Besides their medicinal
uses, imidazopyrazine and benzimidazole derivatives have found technical applications as dyes,
electroluminescent materials, organic semiconductors and as suitable ligand in coordination
chemistry [41-42]. Keeping above points in consideration, in the present manuscript the two
fluorescent moieties have been combined through a spacer as imidazo[1,2-a]pyrazine-
benzimidazole conjugates for FRET based ratiometric determination of pyrophosphate (PPi).
3

Moreover, conjugates based on imidazo[1,2-a]pyrazine and benzimidazole moieties as sensors
have not been reported so far. Herein, we have synthesized these conjugates by implementing
click reaction and Suzuki-Miyaura cross coupling reaction at respective C-8 and C-6 positions of
imidazo[1,2-a]pyrazine and studied their photophysical properties to determine the FRET based
ratiometric detection of pyrophosphate. The motivation behind the synthesis of these compounds
was to synthesize sensor for biologically important anions. Thus, criterion for choosing the
organic moiety was fluorescent electron deficient moiety having nitro group so that it can
interact with electron rich anions. In continuation to search for an efficient compound, a series of
organic compounds were synthesized to fine tune the FRET efficiency. The compound 24
showed the best quantum yield and FRET efficiency and was chosen for the further study.
2. Experimental
2.1 General Experimental Conditions
All commercially available compounds (Avra, Spectrochem, Aldrich, Merck etc.) were used
without further purification. Final reactions were carried\ out in an oil bath using Microwave
1
Vials (10-15 ml). Melting points were determined in open capillaries and were uncorrected. H
and 13C NMR spectra were performed on Jeol ECS 400 NMR spectrometer, which was operated
1
at 400 MHz for H nuclei and 100 MHz for 13C nuclei, using CDCl3, DMSO-d6 and
trifluoroacetic acid (TFA) as solvents. Chemical shifts are reported in parts per million (ppm)
with TMS as internal reference and J values are given in hertz. 2D NOE studies were performed
on same instrument. Mass Spectra of the synthesized compounds were recorded at Water
Micromass-Q-T of Micro. Reactions were monitored by thin layer chromatography (TLC) with
silica plate coated with silica gel HF-254 and column chromatography was performed with silica
gel 60-120/100-200 mesh. Ethylacetate and methanol were adopted solvent systems.
2.1.1 General procedure for synthesis of compounds 4 and 5:
2-Bromoethylamine hydrobromide / 3-bromopropylamine hydrobromide (4.90 mmol) was
dissolved in distilled water with stirring. Sodium azide was added (13.84 mmol) carefully to this
solution in succession with continuous stirring. The reaction mixture was refluxed for 12 h. After
completion of reaction, reaction mixture was cooled to room temperature and was quenched by
4

addition of sodium hydroxide (17.5 mmol) and further stirred for 30 min at room temperature.
Thereafter, mixture was extracted with diethyl ether and dried over sodium sulphate to obtain
ether extract and stored at low temperature. The ether extract being volatile in nature, was used
as such for next reaction.
2.1.2 General procedure for synthesis of compounds 6 and 7:
To the ether extract containing 4 or 5 was added to the solution of 6,8-dibromoimidazo[1,2-
a]pyrazine 1 (1.80 mmol) in acetonitrile in the presence of diisopropylethylamine (DIPEA). The
reaction mixture was refluxed for 24 h. After completion of reaction, the mixture was extracted
with chloroform and water. Organic layer was separated, dried over sodium sulphate, filtered and
concentrated under vacuum. The crude mixture was then purified by silica gel chromatography
60-120 mesh using hexane: ethyl acetate (8:2) as eluents.
2.1.3 Synthesis of 2-(3-nitrophenyl)-1-(prop-2-ynyl)-1H-benzo[d]imidazole (9):
To 2-(3-nitrophenyl)-1H-benzo[d]imidazole 8 (4.08 mmol) was added 80% solution of propargyl
bromide in toluene (8.40 mmol) in the presence of potassium carbonate (4.08 mmol) and DMF.
The mixture was stirred at room temperature for 12 h. Completion of reaction was monitored by
TLC. Reaction was quenched by addition of ice cold water. The solid product was filtered to
obtain pure off white solid 9.
2.1.4 General procedure for synthesis of compounds 10 and 11:
To a stirred solution of 2-(3-nitrophenyl)-1-(prop-2-ynyl)-1H-benzo[d]imidazole 9 (3.6 mmol)
and 6 or 7 (3.6 mmol) in 50 ml ethanol:water (8:2), copper sulphate pentahydrate (5 mol%) and
sodium ascorbate (10 mol%) were added and stirred at room temperature for 2 h. After
completion of reaction, water was added and extracted with chloroform. Chloroform layer was
dried over anhydrous sodium sulphate, filtered and concentrated in vacuum to obtain pure solid
10 or 11.
2.1.5 General procedure for synthesis of compounds 13-38:
To a solution of 10 or 11 (0.178 mmol) in mixture of 1,4-dioxane : water (9:1) in a sealed tube,
boronic acid (0.178 mmol) and K2CO3 (0.178 mmol) were added under inert atmosphere. Then,
5

[Pd(PPh3)4] (5mol%) was added with continued nitrogen purging. Sealed the tube and refluxed
the reaction mixture for 6-8 h. Completion of reaction was determined by TLC. The mixture was
extracted with chloroform and water. Organic layer was dried over sodium sulphate to obtain
crude product which was further purified by column chromatography to get pure products 13-38.
2.2 Photophysical measurements
All photophysical measurements were performed in acetonitrile. Absorption spectra were
measured with a UV-2500, Shimadzu spectrophotometer. Fluorescent measurements were
performed with a Carry Eclipse spectrophotometer. The quantum yields of the imidazo[1,2-
a]pyrazine analogues were determined relative to anthracene.
2.3 Fluorescence quantum yield
The fluorescence quantum yield Φfs for all compounds was determined at room temperature in
analytical grade CH3CN using anthracene (Φfr = 0.22) in acetonitrile as the standard. The
quantum yield was calculated by using eqn--1, in which Φfs is the radiative quantum yield of the
sample, Φfr is the radiative quantum yield of reference, As and Ar are the absorbance of the
sample and the reference, respectively, Ds and Dr are the areas of emission for the sample and
reference respectively, Ls and Lr are the lengths of the absorption cells, and Ns and Nr are the
refractive indices of the respective sample and reference solutions (pure solvents were assumed).
Ns2 Ds
110A L
r
r
fs  fr 


--------1
110A L
Nr2
Dr
s
r
3. Results and discussion
3.1 Chemistry
Imidazo[1,2-a]pyrazine-benzimidazole conjugates have been synthesized by the reaction of 2-
aminopyrazine and N-bromosuccinimide at room temperature followed by cyclization with
o
chloroacetaldehyde at 110 C for 72 h to obtain 6,8-dibromoimidazo[1,2-a]pyrazine 1 in 80%
yield. Compound 1 was then reacted with 2-azidoethanamine 4 and 3-azidopropanamine 5 [43-
44] (obtained from reaction of 2-bromoethylamine hydrobromide 2 and 3-bromopropylamine
hydrobromide 3 with sodium azide for 12 h at reflux temperature followed by stirring with
6

sodium hydroxide), to afford respective N-(2-azidoethyl)-6-bromoimidazo[1,2-a]pyrazin-8-
amine 6 in 72% yield and N-(3-azidopropyl)-6-bromoimidazo[1,2-a]pyrazin-8-amine 7 in 65%
yield (Scheme 1). These azides have low thermal stability, so, these were stored in ether or
hexane at low temperature.
On the other hand, reaction of 2-(3-nitrophenyl)-1H-benzo[d]imidazole 8 with propargyl
bromide, in the presence of potassium carbonate and DMF at room temperature for 12 h obtained
2-(3-nitrophenyl)-1-(prop-2-ynyl)-1H-benzo[d]imidazole 9 in 95% yield (Scheme 2).
Keeping both the precursors, copper catalyzed azide-alkyne cycloaddition (CuAAC) reaction
was performed by reacting azides 6 and 7 with alkyne 9 in the presence of copper sulphate and
sodium ascorbate at room temperature for 2 h to afford imidazo[1,2-a]pyrazine-benzimidazole
conjugates 10 and 11 in 81% and 85% yields, respectively (Scheme 3).
However, a mixture of 1,4-disubstituted triazole 10 and 1,5-disubstituted triazole 12 in 2:1
ratio was obtained with reaction of azide 6 and alkyne 9 on heating without using any catalyst. It
has been observed that thermal [2+3] dipolar cycloaddition of alkyne to azide is not a
regiospecific reaction [45]. NOESY experiment was carried out to confirm the formation of 1,4
and 1,5-disubstituted products. Positive NOE was observed between the protons of triazole at C-
5’ and alkyl groups at N-1’ and C-4’ positions in compound 10, suggesting that the triazole
proton and both alkyl groups are in close proximity. On the other hand, No NOE signal was
observed between the C-4’ proton of triazole and alkyl group at N-1’ position in 1,5-
disubstituted triazole 12. However, positive NOE signal was observed between triazole proton at
C-4’ and C-5’ substituted alkyl group (Figure 1).
Conjugate 10 was further reacted with various of aryl boronic acids through Suzuki-Miyaura
cross coupling at C-6 position of imidazo[1,2-a]pyrazine using Pd(PPh3)4 and K2CO3, providing
library of arylated compounds 13-28 (Scheme 4, Table 1). Several electron donating and electron
withdrawing groups were introduced on aryl group of imidazo[1,2-a]pyrazine. The reactions
were well tolerated with five member rings viz., thiophen-2-yl (13), thiophene-3-yl (14) and
furan-2-yl (15). Substitution with electron withdrawing groups on phenyl ring such as 4-
fluorophenyl (17), 4-chlorophenyl (18), 4-bromophenyl (19), 3-trifluoromethylphenyl (20), 4-
formylphenyl (27) and 4-acetylphenyl (28) gave good yields of products while electron donating
groups, 3-methylphenyl (21) and 2-methoxy phenyl (26) were obtained in moderate yields.
Increase in yield was also observed with methoxy substitution from ortho (26) to para position
7

(25) of phenyl ring. Similarly, compounds 29-38 were obtained by the reaction of compound 11
with different aryl boronic acids (Scheme 4, Table 1). The chemical structures were fully
1
13
characterized by H and C NMR as well as mass spectrometry (Figures S1-S64, Supporting
Information, File 1).
In case of halogen containing compounds, the electron withdrawing group such as 3-
(trifluoromethyl)phenyl moiety in 20 and 33 gave good quantum yields (0.53 and 0.62,
respectively) in comparison to other halogenated compounds 17 (0.14), 18 (0.38) and 19 (0.38).
On increasing linker length from ethyl to propyl as exemplified by compounds 20 and 27 with 33
and 38, increase in quantum yields were observed. FRET efficiency based upon the fluorescence
quantum yield has also been determined. The FRET efficiency (η) was evaluated using the
equation η = 1 − ΦF(donor in dyad )/ΦF(free donor). Here, ΦF(donor in dyad) is the fluorescence quantum yield
of donor part in dyad and ΦF(free donor) is the fluorescence quantum yield of donor when not
connected to acceptor. It has been observed that presence of some electron donating substituents
showed lower FRET efficiencies in comparison to electron withdrawing substituents. However,
the maximum FRET efficiency (92%) from donor part of dyad to acceptor part was displayed by
compound 24 with 2-hydroxyphenyl substituent attached at the acceptor part.
Imidazo[1,2-a]pyrazine behaved as an acceptor and showed absorption band at 360 nm. There
was a significant spectral overlap between absorption spectra of the imidazo[1,2-a]pyrazine
(acceptor) and emission spectra of the benzimidazole (donor) (Figures 2, S65 and S66). Dyad 24
showed the emission band at 365 nm with Φf = 0.95 and η = 92%. Keeping in view, due to
maximum quantum yield and FRET efficiency of compound 24 amongst other series of
-
-
compounds, the effect of various anions like F-, Cl-, Br-, I-, AcO-, H2PO4 , NO3 , CN-, SCN-,
-
2-
HSO4 S2-, CO3 , SO32- and PPi was studied on compound 24.
-
-
-
The addition of various anions (100 µM) viz., F-, Cl-, Br-, I-, CN-, H2PO4 , NO3 , AcO-, HSO4 ,
2-
2-
SCN-, S2-, CO3 , SO3 , etc. and metal ions such as Cu2+, Zn2+, Co2+, Ni2+, Al3+, Fe3+, Cr3+ etc.
caused insignificant change in absorption spectrum of dyad 24 (Figures S67, S68). However, the
presence of PPi ions induced the formation of new absorption band. The titration of solution of
dyad 24 (20 µM, CH3CN) with PPi showed a gradual decrease in absorbance at 286 nm
associated with an increase in absorbance at 370 nm with an isobestic point at 340 nm.
Appearance of absorption band at 370 nm in the presence of PPi could be attributed due to
intramolecular charge transfer (ICT).
8

A plot of log [PPi] concentration Vs Ao/A showed a linear increase in absorbance between 10-6 -
10-5 M PPi. The “On-Off-On” behavior at 286 nm and 370 nm has been used for the ratiometric
estimation of PPi (Figure 3, S69) [46]. The time dependence response of the sensor was checked
over the varied time. The absorption changes attained by the sensor were intact over the period of
24 hrs (Figure S70).
The solution of dyad 24 (CH3CN, 20 µM) upon excitation at λex 280 nm exhibited emission
maxima at 365 nm. Upon addition of aliquot of PPi, the fluorescence intensity at 365 nm was
gradually quenched and associated with red shift of emission maxima to 430 nm (Figure 4). The
dyad 24 initially showed the emission at 365 nm typically due to benzimidazole moiety. In the
presence of PPi, new emission band appeared at 430 nm typically due to imidazopyrazine which
clearly showed that in the presence of PPi excited benzimidazole moiety transfers its energy to
imidazopyrazine through FRET and started giving imidazopyrazine emission at 430 nm.The
-
-
-
addition of all other anions (100 µM) viz., F-, Cl-, Br-, I-, CN-, H2PO4 , NO3 , AcO-, HSO4 , SCN-
etc. caused insignificant change in the emission of dyad 24 (Figure S71).
The selectivity of dyad 24 towards PPi was further ascertained by the competition experiment
-
-
-
where competing anions viz., Cl-, Br-, I-, CN-, H2PO4 , NO3 , AcO-, HSO4 , SCN- etc. were added to
the solution of dyad 24 before addition of PPi. As predicted from the results, the FRET efficiency
in the presence of PPi remained by and large unaffected in the presence of several interfering
anions. This indicates that dyad 24 is selective for recognition of PPi even in the presence of other
anions (Figure S72) The lowest detection limit for PPi estimation was found to be 0.2 µM..
Further insight and quantitative evaluation of the sensing capability of compounds 10, 11 and
13-38 (20 µM, CH3CN) with pyrophosphate was obtained from UV-vis and fluorescence
experiments (Figures S73 and S74, Supporting information). It was surprising that only compound
24 with 2-hydroxylphenyl ring at C6 position of imidazo[1,2-a]pyrazine, showed significant
change with pyrophosphate indicating that 2-hydroxyphenyl moiety is responsible for binding with
PPi. The formation of new emission band could be attributed to intramolecular interaction in the
excited state of the hydroxyphenyl and benzimidazole moieties facilitated in the presence of
coordinated PPi. The distance between the hydroxyphenyl and benzimidazole moieties in absence
of PPi was closer to Förster distance and thus showed 92% FRET efficiency, whereas presence of
PPi, decrease the Förster distance and hence FRET efficiency is increased to 98%.
9

In an effort to gain more detailed information of the interaction between compound 24 and PPi,
1H NMR titration study was carried out in CD3CN (Figure 5). The assignments of protons of
compound 24 have been confirmed by 2D NOESY and COSY NMR experiments (Figures S35-
S36). The proton signal at δ 8.1 was attributed to the 2-hydroxyphenyl proton Ha. Signal at δ 7.2
was due to overlapping triplets from the characteristic protons Hb and Hc, and signal at δ 7.5 was
due to proton Hd of hydroxyphenyl moiety. Protons of OH at δ 12.8 and NHc at δ 6.8 disappeared
on the addition of 0.5 equivalent of PPi. Proton Ha adjacent to the hydroxyl group broadened and
shifted downfield by 1.2 ppm (8.1-9.3 ppm) until the end of the titration experiment (over two
equivalents of PPi added). The large shifting as well as broadening of signal might be due to
hydrogen bonding of compound 24 with pyrophosphate (Figure 5). The signal of the other proton
Hd is separated out and shifted downfield by 0.4 ppm (7.5-7.9 ppm) upon addition of
pyrophosphate ion. Moreover, two double doublets at δ 8.2 and 8.3 of nitrobenzene in
benzimidazole are also approaching towards each other. This interesting behavior of compound 24
could be explained by considering the important contribution given by the benzimidazole and 2-
hydroxylphenyl in assisting the interaction of the receptor and pyrophosphate anion. On the basis
1
of H NMR studies of compound 24 with pyrophosphate, the proposed binding modes for
compound 24 with PPi is shown in Figure 6.
4. Conclusions
We have synthesized a series of imidazo[1,2-a]pyrazine-benzimidazole conjugates, based upon
“click” and “Suzuki-Miyaura cross coupling” reactions. It has been observed that by simply
changing the substituents in the acceptor part of conjugate, FRET efficiency of the compound
can be tuned. In particular, the substitution with 2-hydroxy phenyl group at C-6 position of
imidazo[1,2-a]pyrazine (24), showed the best FRET efficiency and used for ratiometric detection
of pyrophosphate. The fluorescent electron deficient moiety on the benzimidazole ring due to
nitro group was also interacted with electron rich anions. Thus, the compound 24 showed the
best quantum yield as well as FRET efficiency. Only few organic compounds in the absence of
metal complexes have been reported in literature for the detection of PPi. The present manuscript
described the first organic compound for the PPi sensing with FRET phenomenon and having
good detection limit (Table S1).
10

Supporting Information
Additional information, characterization methods, experimental, analytical data, and copies of
NMR spectra.
Acknowledgements
KP thanks DST, New Delhi (EMR/2014/000669) and UGC, New Delhi (41-322/2012, SR) for
providing funds. RG is indebted to CSIR for SRF (09/677(0020)/2013.EMR-I). SAI labs, Thapar
University is also acknowledged for recording NMR spectra.
11

Notes and References
1. J.K. Heinonen, Biological role of inorganic pyrophosphate, Kluwer Academic Publishers,
Boston, 2001.
2. J.A. Drewry, S. Fletcher, H. Hassan, P.T. Gunning, Novel asymmetrically functionalized bis-
dipicolylamine metal complexes: peripheral decoration of a potent anion recognition scaffold,
Org. Biomol. Chem. 7 (2009) 5074-5077.
3. C.P. Mathews, K.E. van Hold, Biochemistry, The Benjamin/Cummings Publishing Company,
Inc., Redwood City, CA, 1990.
4. W.N. Limpcombe, N. Sträter, Recent advances in zinc enzymology, Chem. Rev. 96 (1996)
2375-2434.
5. P. Nyrén, Enzymatic method for continuous monitoring of DNA polymerase activity, Anal.
Biochem. 167 (1987) 235-238.
6. T. Tabary, L.Y. Ju, J.H. Cohen, Homogeneous phase pyrophosphate (PPi) measurement
(H3PIM). A non-radioactive, quantitative detection system for nucleic acid specific
hybridization methodologies including gene amplification, J. Immunol. Methods, 156 (1992)
55-60.
7. E.J. O'Neil, B.D. Smith, Anion recognition using dimetallic coordination complexes, Coord.
Chem. Rev. 250 (2006) 3068-3080.
8. R. Marťınez-Ḿanez, F. Sancenon, Fluorogenic and chromogenic chemosensors and reagents
for anions, Chem. Rev. 103 (2003) 4419-4476.
9. P.D. Beer, P.A. Gale, Anion recognition and sensing: The state of the art and future
perspectives, Angew. Chem. Int. Ed. 40 (2001) 486-516.
10. S. Mizukami, T. Nagano, Y. Urano, A. Odani, K. Kikuchi, A fluorescent anion sensor that
works in neutral aqueous solution for bioanalytical application, J. Am. Chem. Soc. 124
(2002) 3920-3925.
11. C. Park, J.-I. Hong, A new fluorescent sensor for the detection of pyrophosphate based on a
tetraphenylethylene moiety, Tetrahedron Lett. 51 (2010) 1960–1962.
12

12. X.-h. Huang, Y. Lu, Y.-b. He, Z.-h. Chen, A metal–macrocycle complex as a fluorescent
sensor for biological phosphate ions in aqueous solution, Eur. J. Org. Chem. (2010) 1921–
1927.
13. J.H. Lee, J. Park, M.S. Lah, J. Chin, J.-I. Hong, High-affinity pyrophosphate receptor by a
synergistic effect between metal coordination and hydrogen bonding in water, Org. Lett. 9
(2007) 3729–3731.
14. H.N. Lee, Z. Xu, S.K. Kim, K.M.K. Swamy, Y. Kim, S.-J. Kim, J. Yoon, Pyrophosphate-
selective fluorescent chemosensor at physiological pH:ꢀ Formation of a unique excimer upon
addition of pyrophosphate, J. Am. Chem. Soc. 129 (2007) 3828–3829.
15. H.N. Lee, K.M.K. Swamy, S.K. Kim, J.-Y. Kwon, Y. Kim, S.-J. Kim, Y.J. Yoon, J. Yoon,
Simple but effective way to sense pyrophosphate and inorganic phosphate by fluorescence
changes, Org. Lett. 9 (2007) 243–246.
16. A. Ojida, H. Nonaka, Y. Miyahara, S. Tamaru, K. Sada, I. Hamachi, Bis(Dpa-ZnII) appended
xanthone: Excitation ratiometric chemosensor for phosphate anions, Angew. Chem. Int. Ed.
45 (2006) 5518-5521.
17. C.M.G. dos Santos, T. Gunnlaugsson, The recognition of anions using delayed lanthanide
luminescence: The use of Tb(III) based urea functionalised cyclen complexes, Dalton Trans.
(2009) 4712–4721.
18. K.M.K. Swamy, S.K. Kwon, H.N. Lee, S.M.S. Kumar, J.S. Kim, J. Yoon, Fluorescent
sensing of pyrophosphate and ATP in 100% aqueous solution using a fluorescein derivative
and Mn2+, Tetrahedron Lett. 48 (2007) 8683–8686.
19. S. Watchasit, A. Kaowliew, C. Suksai, T. Tuntulani, W. Ngeontae, C. Pakawatchai, Selective
detection of pyrophosphate by new tripodal amine calix[4]arene-based Cu(II) complexes
using indicator displacement strategy, Tetrahedron Lett. 51 (2010) 3398–3402.
20. Z. Guo, W. Zhu, H. Tian, Hydrophilic copolymer bearing dicyanomethylene-4h-pyran moiety
as fluorescent film sensor for Cu2+ and pyrophosphate anion, Macromolecules, 43 (2010)
739–744.
21. M.J. Kim, K.M.K. Swamy, K.M. Lee, A.R. Jagdale, Y. Kim, S.-J. Kim, K.H. Yoo, J. Yoon,
Pyrophosphate selective fluorescent chemosensors based on coumarin–DPA–Cu(II)
complexes, Chem. Commun. (2009) 7215–7217.
13

22. S.Y. Kim, J.-I. Hong, Dual signal (color change and fluorescence ON–OFF) ensemble system
based on bis(Dpa-CuII) complex for detection of PPi in water, Tetrahedron Lett. 50 (2009)
1951–1953.
23. X. Huang, Z. Guo, W. Zhu, Y. Xie, H. Tian, A colorimetric and fluorescent turn-on sensor
for pyrophosphate anion based on a dicyanomethylene-4H-chromene framework, Chem.
Commun. (2008) 5143–5145.
24. Y.J. Jang, E.J. Jun, Y.J. Lee, Y.S. Kim, J.S. Kim, J. Yoon, Highly effective fluorescent and
-
colorimetric sensors for pyrophosphate over H2PO4 in 100% aqueous solution, J. Org. Chem.
70 (2005) 9603–9606.
25. L. Fabbrizzi, N. Marcotte, F. Stomeo, A. Taglietti, Pyrophosphate detection in water by
fluorescence competition assays: Inducing selectivity through the choice of the indicator,
Angew. Chem. Int. Ed. 41 (2002) 3811–3814.
26. N. Shao, J. Jin, G. Wang, Y. Zhang, R. Yang, J. Yuan, Europium(III) complex-based
luminescent sensing probes for multi-phosphate anions: modulating selectivity by ligand
choice, Chem. Commun. (2008) 1127–1129.
27. T.-M. Fu, C.-Y. Wu, C.-C. Cheng, C.-R. Yang, Y.-P. Yen, A novel highly selectively
colorimetric and fluorescent chemosensor for pyrophosphate ion, Sens. Actuators B, 146
(2010) 171–176.
28. Z. Xu, J.-Y. Choi, J. Yoon, Fluorescence sensing of dihydrogen phosphate and
pyrophosphate using imidazolium anthracene derivatives, Bull. Korean Chem. Soc. 32 (2011)
1371-1374.
29. C. Caltagirone, C. Bazzicalupi, F. Isaia, M.E. Light, V. Lippolis, R. Montis, S. Murgia, M.
Olivari, G. Picci, A new family of bis-ureidic receptors for pyrophosphate optical sensing,
Org. Biomol. Chem. 11 (2013) 2445-2451.
30. P. Sokkalingam, D. S. Kim, H. Hwang, J.L. Sessler, C.-H. Lee, A dicationic calix[4]pyrrole
derivative and its use for the selective recognition and displacement-based sensing of
pyrophosphate, Chem. Sci. 3 (2012) 1819-1824.
31. T. Förster, Zwischenmolekulare energiewanderung und fluoreszenz, Ann. Phys. 2 (1948) 55-
75.
32. B. W. vander Meer, G. Coker III, S.-Y. Simon Chen, VCH, Weinheim, Germany, 1994
14

33. P. Mahato, S. Saha, E. Suresh, R. DiLiddo, P.P. Parnigotto, M.T. M.K. Conconi,
Kesharwani, B. Ganguly, A. Das, Ratiometric detection of Cr3+ and Hg2+ by a naphthalimide-
rhodamine based fluorescent probe,Inorg. Chem. 51 (2012) 1769-1777.
34. J. Wu, W. Liu, J. Ge, H. Zhang, P. Wang, New sensing mechanisms for design of fluorescent
chemosensors emerging in recent years, Chem. Soc. Rev. 40 (2011) 3483-3495.
35. X. Chen, T. Pradhan, F. Wang, J.S. Kim, J. Yoon, Fluorescent chemosensors based on
spiroring-opening of xanthenes and related derivatives, Chem. Rev. 112 (2012) 1910-1956.
36. C. Ma, F. Zeng, L. Huang, S. Wu, FRET-based ratiometric detection system for mercury ions
in water with polymeric particles as scaffolds, J. Phys. Chem. B, 115 (2011) 874-882.
37. B.R. White, H.M. Liljestrand, J.A. Holcombe, A ‘turn-on’ FRET peptide sensor based on the
mercury bindingprotein MerP, Analyst, 133 (2008) 65-70.
38. G. Fang, M. Xu, F. Zeng, S. Wu, β-cyclodextrin as the vehicle for forming ratiometric
mercury ion sensor usable in aqueous media, biological fluids, and live cells, Langmuir, 26
(2010) 17764-17771.
39. K. Teranishi, T. Nishiguchi, H. Ueda, Enhanced chemiluminescence of 6-(4-
methoxyphenyl)imidazo[1,2-a]pyrazin-3(7H)-one
by
attachment
of
a
cyclomaltooligosaccharide (cyclodextrin). Attachment of cyclomaltononaose (δ-
cyclodextrin), Carbohydr. Res., 338 (2003) 987-993.
40. K. Teranishi, Luminescence of imidazo[1,2-a]pyrazin-3(7H)-one compounds, Bioorg. Chem.,
35 (2007) 82-111.
41. P. Molina, A. Tarraga, F. Oton, Imidazole derivatives: A comprehensive survey of their
recognition properties, Org. Biomol. Chem. 10 (2012) 1711-1724.
42. Z. Xu, S.K. Kim, J. Yoon, Revisit to imidazolium receptors for the recognition of anions:
highlighted research during 2006–2009, Chem. Soc. Rev. 39 (2010) 1457-1466.
43. N. Song, M. Ding, Z. Pan, J. Li, L. Zhou, H. Tan, Q. Fu, construction of targeting-clickable
and tumor-cleavable polyurethane nanomicelles for multifunctional intracellular drug
delivery, Biomacromolecules, 14 (2013) 4407−4419.
44. S. Angelos, Y.-W. Yang, K. Patel, J.F. Stoddart, J.I. Zink, pH-Responsive supramolecular
nanovalves based on cucurbit[6]uril pseudorotaxanes, Angew. Chem. Int. Ed. 47 (2008)
2222–2226.
15

45. C.W. Tornøe, C. Christensen, M. Meldal, Peptidotriazoles on solid phase:ꢀ [1,2,3]-Triazoles
by regiospecific copper(i)-catalyzed 1,3-dipolar cycloadditions of terminal alkynes to azides,
J. Org. Chem. 67 (2002) 3057-3064.
46. H.A. Benesi, J.H. Hildebrand, A spectrophotometric investigation of the interaction of iodine
with aromatic hydrocarbons, J. Am. Chem. Soc. 71 (1949) 2703-2707.
16

NO NOE
3’
2’
4’
5’
NOE
1’
2
5
1
2
3
3
3’
8
1
2’
1’
4
4
5
4’
7
8
6
5’
6
NOE
7
NOE
Figure 1 NOE correlation experiments of 1,4-(10) and 1,5-(12) disubstituted triazoles
Figure 2: Spectral overlap between absorption spectrum of imidazopyrazine (acceptor,
compound 6 (Φf = 0.08) and emission spectrum of benzimidazole (donor, compound 9 (Φf =
0.1)).
17

Figure 3: (a) Changes in absorption spectrum of compound 24 (20 µM in CH3CN) upon addition
of tris(tetrabutylammonium) hydrogen pyrophosphate (PPi); (b) Plot of A370/A286 vs log [PPi] for
the ratiometric response of compound 24 towards PPi.
Figure 4: (a) Changes in emission spectrum of compound 24 (20 µM in CH3CN) upon addition
of PPi (0.2 µM to 1000 µM); (b) Plot of I430/I365 vs log [PPi] for the ratiometric response of
compound 24 towards PPi.
18

1
Figure 5: H NMR stack plot of a CD3CN solution of compound 24 (5 mM) upon addition of
tris(tetrabutylammonium)hydrogen pyrophosphate (0.5-1.0 eq.).
FRET
N
N
N
N
N
N
N
N
N
N
PPi
N
N
N
N
N
N
N
N
H
H
H
H
O
O
O-
P
O-
Hb
Hb
O-
O
O
Ha
Ha
O
O
N
P
N
O
H
O
O
O
Large Forster Distance
between donor Acceptor
Decreased Forster Distance
between donor Acceptor
Figure 6: Suggested binding structure for 24 with PPi
N
N
NH2
N
N
NH2
Br
N
N
N
Chloroacetaldehyde
IPA, 110 0C, 72 h
NBS, DMSO:H2O
rt, 6 h.
Br
Br
Br
1 (80% yield)
(i) NaN3, H2O, reflux, 12 h.
(ii) NaOH
1
NH2
N
N
NH2.HBr
N3
n
Br
n
DIPEA, acetonitrile,
reflux, 24 h.
N3
2; n = 1
3; n = 2
4; n = 1
5; n = 2
Br
N
NH
n
6; n = 1 (72% yield)
7; n = 2 (65% yield)
19

Scheme 1 Synthesis of imidazo[1,2 a]pyrazinamine ethyl 6 and propyl 7 azide
NO2
NO2
N
N
Br
K2CO3, DMF,
rt, 12 h.
N
N
H
8
9 (95% yield)
Scheme 2 Synthesis of N-propargylated benzimidazole 9
NO2
N
N
N
N
N
N
N
N
N
N
N
N
CuSO4.5H2O,
N
N3
sodium ascorbate,
ethanol:water,
rt, 2 h.
Br
NH
n
Br
NH
n
9
6; n = 1
7; n = 2
NO2
10; n = 1, 81% yield
11; n = 2, 85% yield
;
Scheme 3 Synthesis of triazole tethered imidazo[1,2-a]pyrazine-benzimidazole conjugates 10
and 11
N
N
N
N
N
N
N
N
N
N
N
N
N
R1B(OH)2, K2CO3,
Pd(PPh3)4,
N
N
N
R1
NH
n
Br
NH
n
dioxane:water,
NO2
sealed tube, reflux, 6-8 h.
NO2
10; n = 1
11, n = 2
13-38
Scheme 4 Suzuki-Miyaura coupling of ethyl and propyl triazole tethered imidazo[1,2-
a]pyrazine-benzimidazole conjugates
20

Table 1 Suzuki-Miyaura coupling of ethyl 10, 13-28 and propyl triazole 11, 29-38 bridged
imidazo[1,2-a]pyrazine-benzimidazole conjugates and their physiochemical parameters
3.2 Photophysical properties
Compounds 10, 11 and 13-38 were further studied for their photophysical properties (Table 1).
Quantum yields of all compounds were calculated with respect to anthracene in acetonitrile. It
has been revealed that presence of electron withdrawing groups viz., 4-formyl phenyl (27 and
38) and 4-acetyl phenyl (28) gave excellent quantum yields (0.90, 0.92 and 0.85, respectively).
abs
emi
Compound Time
(h)
R1
bromo
thiophen-2-yl
thiophen-3-yl
n
Yields
(%)
81
73
81
62
80
73
79
84
69
61
77
55
43
91
58
88
71
85
68
67
76
78
53
57
62
81
83
80
λmax
λmax
€
Φf
FRET
efficiency
(nm)
287
286
288
288
(nm)
375
420
366
375
4
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
2
36600
42250
46250
36200
53800
47250
56050
50450
50400
50250
57700
55050
26600
66950
37550
47300
59750
28100
30300
30400
28350
32550
17800
22750
37000
31800
34950
31250
0.22 85.3
0.19 87.3
0.13 91.3
0.26 82.6
0.52 65.3
0.14 90.6
0.38 72.6
0.38 74.6
0.53 64.6
0.32 62.6
0.28 81.3
0.11 25.3
0.95 92.0
0.15 90.0
0.48 45.3
0.90 ND
0.85 ND
0.15 90.3
0.28 81.3
0.70 53.3
0.14 90.6
0.41 74.6
0.62 18.6
0.56 78.6
0.36 76.0
0.43 71.3
0.82 71.0
0.92 ND
10
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
11
29
30
31
32
33
34
35
36
37
38
8
8
8
6
7
7
7
6
6
6
8
8
7
6
6
8
furan-2-yl
phenyl
291, 280 408
4-fluorophenyl
4-chlorophenyl
4-bromophenyl
3-(trifluoromethyl)phenyl
3-methylphenyl
4-ethylphenyl
naphthalen-1-yl
2-hydroxyphenyl
4-methoxyphenyl
2-methoxyphenyl
4-formylphenyl
4-acetylphenyl
bromo
282
284
283
283
285
283
285
286
365
380
380
395
406
385
420
365
286, 258 373
289 407
340, 274 496
340, 272 475
6
287
287
288
282
284
283
284
283
365
368
405
370
390
392
372
371
8
8
8
6
7
7
8
6
6
6
thiophen-3-yl
phenyl
4-fluorophenyl
4-chlorophenyl
3-(trifluoromethyl)phenyl
3-methylphenyl
4-ethylphenyl
4-methoxyphenyl
2-methoxyphenyl
4-formylphenyl
286, 258 372
289 370
341, 274 505
21