Naphthyridine-based symmetrical and unsymmetrical pyridinium amides in sensing of biotin salt

Article abstract of DOI:10.1080/10610270903089738

Two naphthyridine-based receptors have been designed and synthesised for biotin salt. The compounds serve as good hosts for the detection of biotin carboxylate rather than biotin ester. The correct dispositions of the binding groups under an isophthaloyl

Full text of DOI:10.1080/10610270903089738

Supramolecular Chemistry
Vol. 22, No. 2, February 2010, 81–94
Naphthyridine-based symmetrical and unsymmetrical pyridinium amides in sensing of biotin salt
Kumaresh Ghosh*, Avik Ranjan Sarkar and Tanushree Sen
Department of Chemistry, University of Kalyani, Kalyani, Nadia 741 235, India
(Received 26 September 2008; final version received 22 May 2009)
Two naphthyridine-based receptors have been designed and synthesised for biotin salt. The compounds serve as good hosts
for the detection of biotin carboxylate rather than biotin ester. The correct dispositions of the binding groups under an
isophthaloyl spacer enable the receptors to bind both the cyclic urea and the carboxylate ends simultaneously with moderate
binding constant values. The receptors are effective for the binding of tetrabutylammonium salt of biotin with a concomitant
increase in the fluorescence of naphthyridine and show appreciable binding of biotin salt in CH3CN containing 1.2% DMSO.
1
The binding was monitored in CH3CN containing 1.2% DMSO and DMSO using H NMR, UV–vis and fluorescence
spectroscopic methods.
Keywords: naphthyridine-based receptors; fluorimetric detection; biotin salt; 3-aminopyridinium salt
Introduction
of the other biotin molecule. The valeryl chain is severely
twisted from the maximally extended all trans-confor-
mation. To bind and sense this interesting biological
substrate of defined stereochemistry, considerable effort
was directed with a limited number of designed receptors.
In this aspect, the use of Troger’s base receptor, as reported
by Wilcox and co-workers (9), was noteworthy. Herranz
et al. (10) reported the recognition of biotin methyl ester.
Goswami and Dey (11) designed pyridine-based simple
receptors for complexation of biotin itself. In their
approach, both the carboxylic acid functional group and
the cyclic urea part of the bicyclic unit were simultaneously
complexed by pyridine amide motifs. In relation to this, the
fluorometric detection of biotin carboxylate anion is
unknown in the literature. As part of our ongoing research
in molecular recognition of carboxylic acids and carbox-
ylates by artificial synthetic receptors (12), here we report
two new bis-amide salts 1 and 2 for detection and sensing of
biotin salt in solvents such as DMSO and CH3CN
containing 1.2% DMSO.
Design and synthesis of fluorescent receptors with less
complicated structures for substrates of biological signifi-
cance are very appealing in the area of molecular
recognition research (1). In this context, suitable hetero-
cycles with a number of hydrogen-bonding atoms are
considered to be useful for providing a set of hydrogen
bonds for complexation of complementary guest molecules.
Naphthyridine motif in this capacity is a well-known
heterocycle, which has been used as a supramolecular
synthon in the area of supramolecular chemistry (2). In our
earlier report, we have shown its use in fluorimetric
detection of tartaric acid, citric acid, etc. (3). Goswami and
Mukherjee (4) devised a dinaphthyridine receptor, which
was excellent in solubilising urea in the less polar solvent
chloroform. Zimmerman and co-workers (5) reported its
enormous uses in different contexts in supramolecular
chemistry. The relative background of this simple hetero-
cycle synthon was thus encouraging and we thought it
would be interesting to design a receptor for sensing and
selective recognition of biotin and its derivatives.
Biotin (referred to vitamin H in humans) is an essential
cofactor for a number of enzymes that have diverse
metabolic functions (6). It is involved in carbon dioxide
uptake, transfer and removal reactions (7). Structurally, it
consists of a pentanoic acid side chain and a cis-fused
bicyclic moiety with a sulphur atom in the ring. Crystal-
lographic studies have confirmed the relative stereochem-
istry at the asymmetric carbon (8). The crystal structure of
biotin shows that the carboxyl group of one biotin molecule
is intermolecularly hydrogen bonded to the urea linkage
Results and discussion
Synthesis
Receptors 1 and 2 were synthesised by following the
procedures given in Schemes 1 and 2. High dilution
coupling of 3-aminopyridine and n-propylamine with
isophthaloyl diacid chloride in the dry CH2Cl2 solvent gave
the hetero-bis-amide receptor 4, which was further reacted
with 2-N-acetyl-7-bromomethyl-1,8-naphthyridine (13) in
CH3CN under refluxing condition to afford the bromide
*Corresponding author. Email: ghosh_k2003@yahoo.co.in
ISSN 1061-0278 print/ISSN 1029-0478 online
q 2010 Taylor & Francis
DOI: 10.1080/10610270903089738
http://www.informaworld.com

82
K. Ghosh et al.
Scheme 1. (i) Addition of n-propylamine and 3-aminopyridine at high dilution condition, Et3N, dry DCM; (ii) 2-N-acetyl-7-
bromomethyl-1,8-naphthyridine in dry CH3CN, heating with stirring for 48 h and (iii) NH4PF6–MeOH.
salt 5. Anion exchange of the bromide salt 5 using NH4PF6
yielded the desired salt 1 in 67% yield. Similarly, the
symmetrical bis-amide 6, obtained from the reaction
between 3-aminopyridine and isophthaloyl diacid chloride
in dry CH2Cl2 according to Scheme 2, was reacted with 2-
N-acetyl-7-bromomethyl-1,8-naphthyridine in dry CH3CN
to give compound 7. Subsequent replacement of the Br2
ions by PF26 ions in 7 afforded the salt 2 in 13% yield.
To gain an insight into the binding and sensing of
tetrabutylammonium salt of biotin by the designed
synthetic receptors 1 and 2, UV–vis, fluorescence and
1H NMR spectroscopic techniques were employed. In this
study, biotin methyl ester and tetrabutylammonium acetate
were also considered to establish the mode of binding of
carboxylate ion of biotin salt into the open clefts of the
receptors.
Scheme 2. (i) 3-Aminopyridine, triethylamine in dry DCM; (ii) 2-N-acetyl-7-bromomethyl-1,8-naphthyridine in dry CH3CN, heating
with stirring for 48 h and (iii) NH4PF6 in MeOH.

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83
Figure 1. Changes in UV–vis spectra for (a) 1 ðc ¼ 6:37 £ 1025 MÞ in DMSO upon addition of tetrabutylammonium salt of biotin
carboxylate, (b) 1 ðc ¼ 6:54 £ 1025 MÞ in DMSO upon addition of biotin methyl ester and (c) 1 ðc ¼ 6:54 £ 1025 MÞ in DMSO upon
addition of tetrabutylammonium acetate.
UV–vis experiment
of biotin salt to a DMSO solution of receptor 2, the change
in absorbance of the peaks for naphthyridine was more
significant and comparable to receptor 1. In this aspect,
Figure 2(a) demonstrates the UV–vis titration spectra of 2
in the presence of increasing amounts of biotin salt.
In contrast to this, the titration spectra of 2 in the presence
of biotin methyl ester and tetrabutylammonium acetate are
indicated in Figure 2(b) and (c), respectively. During
titration with biotin salt, biotin methyl ester and
tetrabutylammonium acetate, there was no red or blue
shift of the absorption peaks of receptor 2.
Continuous variation methods were used to determine
the stoichiometric ratios of the receptors and the guests (14).
Figures 3 and 4 show the Job plots of the product of the
concentration of the host and the relative change in
absorbance due to complexation with the mole fraction of
the guests for a series of solutions in which the total
concentration of the receptor and the guest, in each case,
was constant. In all the cases, the crossing of the curves at
0.5 illustrates that both the receptors formed 1:1 complexes
with the biotin salt as well as biotin methyl ester.
The spectral features of 1 and 2 in UV–vis were also
collected in CH3CN containing 1.2% DMSO. To have
stronger hydrogen-bonding interactions of receptors 1 and 2
The UV–vis experiments on both receptors 1 and 2 were
performed in solvents of different polarities (DMSO and
CH3CN containing 1.2% DMSO). Initially, the charac-
teristic UV–vis changes were investigated upon addition
of tetrabutylammonium salt of biotin to a DMSO solution
of 1. Upon addition of biotin salt, the intensities of the
absorption peaks at 312, 325 and 337 nm for naphthyr-
idine were decreased in a regular fashion (Figure 1(a)).
There was no red or blue shift of the absorption peaks
during complexation. UV–vis titration of receptor 1 was
also carried out in DMSO on addition of varying amounts
of biotin methyl ester. As can be seen from Figure 1(b),
the change in absorbance of 1 is less compared to
the case of 1 with biotin salt. The change in absorbance
of 1 was further tested with tetrabutylammonium acetate
(Figure 1(c)) to establish the binding feature of biotin
salt. In this aspect, it is seen that the change in
absorbance at 312, 325 and 337 nm for naphthyridine is
less and irregular in nature compared to the case of biotin
salt in Figure 1(a).
To get more hydrogen-bonding arrangements around
the functionalities of biotin salt, the symmetrical
bis-amide 2 was interesting. Upon successive addition
Figure 2. Changes in UV–vis spectra for 2 ðc ¼ 6:54 £ 1025 MÞ in DMSO upon addition of (a) tetrabutylammonium salt of biotin
carboxylate, (b) biotin methyl ester and (c) tetrabutylammonium acetate.

84
K. Ghosh et al.
Figure 3. (a) Job plot of receptor 1 (at 337 nm) with tetrabutylammonium salt of biotin; the total concentration of the receptor and the
guest is 6:37 £ 1026 M. (b) Job plot of receptor 1 (at 337 nm) with biotin methyl ester; the total concentration of the receptor and the guest
is 6:37 £ 1026 M.
with the substrates, the polarity of the medium was tuned.
DMSO is a competitive hydrogen-bonding partner that
reduces the desired host–guest interactions to a significant
extent. Considering the solubility of the receptors, we took
CH3CN containing 1.2% DMSO as a medium where the
hydrogen-bonding interactions between the receptors and
substrates were expected to be favourable and little stronger
than the case in pure DMSO. In this connection, the changes
in absorbances of 1 and 2 in the presence of tetrabuty-
lammonium acetate, tetrabutylammonium salt of biotin and
biotin methyl ester were monitored in the CH3CN
containing 1.2% DMSO solvent.
Fluorescence experiment
Simultaneous fluorescence study of 1 and 2 both in the
presence and absence of biotin salt was performed in the
solvents DMSO and CH3CN containing 1.2% DMSO. For
example, upon addition of tetrabutylammonium salt of
biotin to the DMSO solution of 1, the emission at 353 nm for
naphthyridine was increased gradually (Figure 6(a)),
whereas the emission of 1 was marginally perturbed in the
presence of biotin methyl ester (Figure 6(b)). Figure 6(c)
shows the comparison of emission of 1 in the presence of
10 equiv. amounts of biotin salt and biotin methyl ester.
Similar experiment on receptor 2 was carried out on
excitation of the receptor solution at 315 nm. The emission
spectrum of 2 in DMSO showed characteristic peaks at 371
and 453 nm (Figure 7). The peak at longer wavelength is
presumably due to the complex formed at the excited state
Figure 5 corroborates the UV–vis spectral changes of
both 1 and 2 in the presence of biotin salt and biotin methyl
ester in CH3CN containing 1.2% DMSO. The change in
absorbance of 2 is considerable than 1.
Figure 4. (a) Job plot of receptor 2 (at 337 nm) with tetrabutylammonium salt of biotin; the total concentration of the receptor and the
guest is 6:37 £ 1026 M. (b) Job plot of receptor 2 (at 337 nm) with biotin methyl ester; the total concentration of the receptor and the guest
is 6:37 £ 1026 M.

Supramolecular Chemistry
85
Figure 5. Changes in UV–vis spectra for (a) 1 with biotin salt, (b) 1 with biotin methyl ester, (c) 2 with biotin salt and (d) 2 with biotin
methyl ester in CH3CN containing 1.2% DMSO.
either between the pendant naphthyridines (excimer) or
between naphthyridine and pyridinium moieties (exciplex).
In the presence of biotin salt, the monomer emission
intensity at 371 nm is greatly increased compared to the
emission at 453 nm (Figure 7(a)). The gradual increase in
intensity of the monomer emission of the naphthyridine
motifinboth1and2during complexationisattributedtothe
hydrogen bond-mediated perturbation of the excited np*
state in a destabilising manner for which the pp* state
becomes the lowest energy singlet excited state (1c, 15).
However, the change in monomer emission of 2 is
considerable and distinguishable from 1. In comparison,
the change in emission of 2, in the presence of biotin methyl
ester, isinsignificant(Figure7(c)). Inan efforttounderstand
the nature of the peak at 453 nm in 2, we thoroughly
investigated its interaction with all the guests (tetrabuty-
lammonium acetate, tetrabutylammonium salt of biotin and
biotin methyl ester) in the solvents such as MeOH and
CH3CN containing 1.2% DMSO. The results were
compared with the findings obtained in pure DMSO.
Figure 8(a) shows the change in fluorescence of 2 in
different solvents. With the change in polarity of the
solvents, the monomer emission peak shifts to either
direction (Figure 8(a)). But under identical condition, the
position of absorption peaks of 2 was almost unaltered
(Figure 8(b)). It is mentionable that, while the peak for
excimer at longer wavelength due to closely spaced
naphthyridines in 2 was noticed in both DMSO and CH3CN
containing 1.2% DMSO, the same was not found in pure
MeOH. We, therefore, investigated the change in monomer
versusexcimeremissionof 2with guest concentrationin the
DMSO and CH3CN containing 1.2% DMSO solvents only.
It is mentionable that the emission characteristic of 2 in
CH3CN containing 1.2% DMSO is concentration depen-
dent (Figure 8(c)). Almost negligible monomer emission
of 2 in concentrated solution indicated its strong self-
association.
In CH3CN containing 1.2% DMSO, the excimer in 2 is
exclusively destroyed at high concentration of biotin salt
(Figure 9(a)) and the monomer emission is smoothly
increased to a considerable extent. In comparison, the
emission of 2 in the same solvent is hardly changed in
the presence of biotin methyl ester (Figure 9(b)). But the
change in emission of 2 is quite appreciable in the presence

86
K. Ghosh et al.
Figure 6. (a) Changes in fluorescence spectra for 1 ðc ¼ 6:37 £ 1025 MÞ in DMSO upon addition of tetrabutylammonium salt of biotin. (b)
Changes in fluorescence spectra for 1 ðc ¼ 6:37 £ 1025 MÞ in DMSO upon addition of biotin methyl ester. (c) Comparison in emission of 1 in
the presence of 10 equiv. biotin salt and biotin methyl ester. (d) Titration curves for biotin salt and biotin methyl ester with 1.
of acetate (Figure 9(c)). In this connection, Figure 10
describes the plot of IM/IEx (IM and IEx indicate the
intensities of the monomer and the excimer, respectively)
versus guest concentration in CH3CN containing 1.2%
DMSO (Figure 10(a)) and DMSO (Figure 10(b)). In pure
DMSO, while the emissions of both the monomer and the
excimer in 2 are increased considerably in the presence of
biotin salt (Figure 10(b)), the same are decreased in the
presence of acetate and the excimer still persisted. These
findings thus clearly underline the fact that, in DMSO, the
isophthalamide core of 2 is effectively involved in
complexation of the carboxylate end and the naphthyridine
sites are hardly bonded to the cyclic urea motif of biotin salt.
If the naphthyridine sites were really involved, the
excimer at 453 nm would be destroyed in principle. But
this has not happened. On the contrary, the gradual
disappearance of the excimer in the presence of biotin salt
in CH3CN containing 1.2% DMSO indicates that both the
isophthaloyl diamide core and the naphthyridine sites of 2
are simultaneously involved in complexation of biotin salt.
This is insignificant in the case of biotin methyl ester due to
high steric demand and electroneutrality of the ester group
(Figure 9(b)). The simultaneous involvement of both the
naphthyridine site and the diamide core of 2 in
complexation of biotin salt was proved by the titration
experiment of 2 in CH3CN containing 1.2% DMSO in the
presence of acetate where the excimer emission was
almost unperturbed (Figure 9(c)).
Based on these, we suggested the possible modes of
binding of biotin salt by receptors 1 and 2 (Figure 11). In the
suggested modes, the carboxylate ion is bonded in the cleft
under the influence of both hydrogen-bonding and charge–
charge interactions and the bicyclic urea motif is adhered to
the pendant naphthyridine sites via hydrogen bonds
(1A/2A). The other equilibrium forms 1B, 2B and 2C are
also possible in solution where only the carboxylate ion is
hydrogen bonded, leaving the cyclic urea motif uncom-
plexed. In our earlier publication, we reported that the
anthracene-based pyridinium salt 3 under an isophthaloyl
spacer is able to bind carboxylate, especially aliphatic
carboxylate anion, in the cleft involving both hydrogen-
bonding and charge–charge interactions (16). This finding,
additionally, enable us to suggest that the carboxylate part
of biotin carboxylate salt is particularly involved in binding
in the isophthaloyl diamide core according to the modes
suggested in Figure 11. Based on the results, it was thus

Supramolecular Chemistry
87
Figure 7. (a) Changes in fluorescence spectra for 2 ðc ¼ 6:54 £ 1025 MÞ in DMSO upon addition of tetrabutylammonium salt of biotin.
(b) Changes in fluorescence spectra for 2 ðc ¼ 6:54 £ 1025 MÞ in DMSO upon addition of biotin methyl ester. (c) Comparison in emission
of 2 in the presence of 10 equiv. biotin salt and biotin methyl ester. (d) Titration curves for biotin salt and biotin methyl ester with 2.
concluded that receptor 1 binds both the ends of biotin salt
in either the DMSO or CH3CN containing 1.2% DMSO
solvent. On the contrary, receptor 2 favours synergic
binding (Figure 11, 2A) only in CH3CN containing 1.2%
DMSO of low polarity over the case of DMSO itself, where
the binding of the cyclic urea motif at the naphthyridine site
is hardly possible either due to locking of the lower rim for
the formation of a strong excimer or due to disposition of
the naphthyridines at the distal positions.
1H NMR study
For further confirmation, 1H NMR of both receptors 1 and 2
in DMSO-d6 was recorded both in the presence and absence
of the tetrabutylammonium salt of biotin as well as biotin
methyl ester. In 1H NMR of 1, the amide protons Ha and Hc
moved less downfield ðDdNH ¼ þ0:18 ppm; DdNH
¼
a
c
þ0:03 ppmÞ in the presence of equivalent amount of
tetrabutylammonium salt of biotin carboxylic acid.
The same was true for symmetrical bis-amide receptor 2
Figure 8. (a) Changes in fluorescence spectra of 2 ðc ¼ 6:54 £ 1025 MÞ in different solvents, (b) changes in absorbance of 2
ðc ¼ 6:54 £ 1025 MÞ in different solvents and (c) change in emission of 2 at different concentrations in the solvent CH3CN containing
1.2% DMSO.

88
K. Ghosh et al.
Figure 9. Changes in fluorescence spectra for 2 ðc ¼ 6:54 £ 1025 MÞ in CH3CN containing 1.2% DMSO: (a) upon addition of
tetrabutylammonium salt of biotin, (b) biotin methyl ester and (c) tetrabutylammonium acetate.
ðDdNH ¼ þ0:16 ppm; DdNH ¼ 0 ppmÞ. The small change
to perform experiments due to partial solubility of
receptors 1 and 2 in solvents such as CHCl3 and CH3CN.
Nevertheless, in both 1 and 2, the pyridyl Ho protons
underwent a significant downfield shift upon complexation
ðDdH ¼ þ0:07 ppm for 1; DdH ¼ þ0:09 ppm for 2Þ.
a
b
in chemical shift of the amide protons during complexation
was due to the interference of DMSO, which is known as a
competitive binding partner. The binding of DMSO in the
core of isophthaloyl diamide is well established and has
been shown by Kovalchuk et al. (17). DMSO was used
o
o
More importantly, the change in chemical shift of the key
Figure 10. Plot of IM=IEx versus guest concentration for receptor 2 with: (a) biotin salt, biotin methyl ester and acetate in CH3CN
containing 1.2% DMSO and (b) biotin salt, biotin methyl ester and acetate in DMSO.

Supramolecular Chemistry
89
Figure 11. Hydrogen-bonded complex of biotin carboxylate with receptors 1 and 2.
amide protons of both 1 and 2 in the presence of biotin
methyl ester was insignificant. Even the pyridyl Ho protons
in both 1 and 2 did not exhibit any measurable change in
chemical shift similar to that of 1 and 2 with biotin salt.
This was explained to be due to the steric nature of the
ester group for which the ester carbonyl was refused in the
binding process into the isophthaloyl diamide core. To
realise the effect of DMSO in the binding process, we
further studied the hydrogen-bonding interactions of both
1 and 2 with biotin salt and biotin methyl ester by 1H NMR
in CD3CN containing 1.2% DMSO-d6. Interestingly,
during titration of 1 with biotin salt, the amide protons
Ha, Hb and Hc of 1 underwent significant downfield
chemical shifts. In the presence of biotin methyl ester, the
changes in chemical shift values of Ha, Hb and Hc were
relatively less than the case with biotin salt. Figure 12, in
this aspect, shows the spectral changes during titration of 1
with biotin salt. In comparison to 1, receptor 2 under
similar condition showed weak interactions. During
titration with biotin salt, the amide proton Ha of 2
exhibited relatively greater change than the amide proton
Hb (Figure 13). In both 1 and 2, the amide proton Hb
appeared as a broad singlet, which upon successive
addition of biotin salt became too broad to detect. This is,
indeed, much pronounced in the case of receptor 2.
A closer look into the chemical shift values of urea protons
of biotin salt reveals that during complexation with 1,
significant downfield shifts of the urea signals take place.
In contrast to this, urea protons of biotin salt moves upfield
on complexation with 2, although the amide proton Hb
showed a downfield shift. This underlines the fact that the
bicyclic urea motif of biotin salt interacts with the pendant
naphthyridines of 2 not in a plane which contains
naphthyridine. This is possibly due to steric feature
exerted by the two naphthyridines. In the case of 1, the
cyclic urea motif interacts in a common plane so that urea
protons come in hydrogen bond distances with the
naphthyridine ring nitrogens. On the other hand, a
simultaneous downfield shift of Hp upon complexation
was appreciable. This may be either due to the
participation of Hp in the formation of CZH· · ·O hydrogen
bonds with carboxylate ion that stabilise the complex via
the mode B or a closer approach of the amide carbonyl
oxygen to Hp upon complexation via mode A (Figure 11).
Between A and B, the form A is more probable in solution
due to a greater number of hydrogen bonds in the complex.
Titration curves in Figures 12(b) and 13(b) indicate the
change in chemical shifts of the interacting protons and

90
K. Ghosh et al.
Figure 12. (a) Partial 1H NMR spectra (CD3CN containing 1.2% DMSO-d6, 400 MHz) of 1 upon addition of tetrabutylammonium salt
of biotin and (b) titration curves for 1 ðc ¼ 3:64 £ 1023 MÞ with tetrabutylammonium salt of biotin.
also present the stoichiometries attained by both 1 and 2 in
the binding process. The breaks of the titration curves at
½Gꢀ=½Hꢀ ¼ 1 indicated 1:1 stoichiometries of the com-
plexes. These notable findings thus prove that biotin salt is
comfortably complexed by 1 in the mode 1A. In contrast,
symmetrical diamide 2 is less efficient in complexation of
biotin salt and follows a binding structure 2A where the
naphthyridine amide hydrogens are involved in weak
hydrogen bonding, leaving urea protons uncomplexed.
In both the cases, the carboxylate end of biotin salt
is strongly complexed in the diamide cores involving
hydrogen-bonding and charge–charge interactions.
In the case of biotin methyl ester, all the amide and
Ho protons of 1 and 2 underwent a negligible downfield
shift, suggesting poor interactions. These detailed
experiments in 1H NMR thus fully support our
proposition on the binding structures (Figure 11).
Receptor 1 efficiently interacts with biotin salt and
follows the hydrogen-bonding structure 1A as shown in
Figure 11. Similarly, receptor 2 binds the same via the
Figure 13. (a) Partial 1H NMR spectra (CD3CN containing 1.2% DMSO-d6, 400 MHz) of 2 upon addition of tetrabutylammonium salt
of biotin and (b) titration curves for 2 ðc ¼ 3:64 £ 1023 MÞ with tetrabutylammonium salt of biotin.

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91
Table 1. Binding constants (Ka) of receptor 1.
Ka in M21 by the fluorescence method
Ka in M21 by the UV method
1
Ka in M21 by H NMR
CD3CN containing 1.2%
DMSO-d6
CH3CN containing 1.2%
DMSO
CH3CN containing 1.2%
DMSO
Guests
DMSO
DMSO
Biotin salt
Biotin methyl ester 6.33 £ 103
1.89 £ 104
1.19 £ 104
5.12 £ 104
5.60 £ 103
5.14 £ 104
2.57 £ 104
3.69 £ 103
1.44 £ 104
1.89 £ 104
8.89 £ 103
1.74 £ 104
2.72 £ 104
a
Acetate
a Not determined due to negligible change in H NMR.
1
Table 2. Binding constants (Ka) of receptor 2.
Ka in M21 by the fluorescence method
Ka in M21 by the UV method
1
Ka in M21 by H NMR
CD3CN containing 1.2%
DMSO-d6
CH3CN containing 1.2%
DMSO
CH3CN containing 1.2%
DMSO
Guests
DMSO
DMSO
Biotin salt
Biotin methyl ester 4.17 £ 103
9.70 £ 103
1.95 £ 104
4.22 £ 104
7.05 £ 102
1.91 £ 104
2.85 £ 104
2.59 £ 103
6.94 £ 103
1.11 £ 104
7.54 £ 103
1.14 £ 104
6.16 £ 102
a
Acetate
a Not determined due to negligible change in H NMR.
1
mode 2A, although the involvement of naphthyridines is,
indeed, less due to their closeness.
in solvents of different polarities, cannot be ignored during
interaction with receptors 1 and 2. Due to the more polar
character of the excited state, the binding constant values
determined by the fluorescence method are presumably
marginally different from the values obtained by the UV
method. The binding constant value for 1 with biotin salt
by the NMR method (19) is in accordance with the values
obtained by the fluorescence and UV methods. In the case
of 2, the binding constant value obtained for biotin salt by
the NMR method is found to be significantly less than the
values determined by the fluorescence and UV methods.
This is due to less involvement of the naphthyridine for
effective hydrogen bonding of the urea motif of biotin salt
like 1. Additionally, we suggest that there is an effective
self-association of the closely spaced pendant naphthyr-
idines in 2 in the NMR concentration range used
ðc ¼ 3:64 £ 1023 MÞ. This is evidenced from the emis-
sion spectra of 2 in different concentrations [Figure 8(c)].
With a decrease in concentration of 2 in CH3CN
containing 1.2% DMSO, the monomer emission of
naphthyridine was found to increase followed by a change
in emission for excimer or exciplex at higher wavelength.
At higher concentration, the monomer emission of 2 was
almost level of and, in turn, the emission was observed
only in the excimer region. This simple experiment
suggests that at higher concentration, receptor 2 exhibits
self-association for which the hetero-association with
biotin salt is less in magnitude. The hetero-association is
significant at lower concentrations, used for recording the
UV and fluorescence spectra. The biotin salt is
comfortably complexed in the open cleft of 1 with a
higher value than that of receptor 2.
Binding study
Complexation-induced change in chemical shift values of
the key amide protons of both 1 and 2 in CD3CN
containing 1.2% DMSO-d6 was used to determine the
binding constant values (Tables 1 and 2). To realise the
binding potencies of both 1 and 2, the binding constants
were also determined from the UV–vis and fluorescence
titrations and the stoichiometries of the complexes were
confirmed from the break of the titration curves [see the
inserts of Figures 6(d), 7(d), 12(b) and 13(b)] as well as
from the UV Job plots (Figures 3 and 4). In all the cases,
receptors 1 and 2 were found to maintain a 1:1
stoichiometry with biotin salt and biotin methyl ester.
For a complex of 1:1 stoichiometry, the binding constant
value was determined by using the data obtained from
both the fluorescence and UV titrations (Tables 1 and 2)
(18). As can be seen from Tables 1 and 2, receptors 1 and 2
are efficient binder of tetrabutylammonium salt of biotin
rather than biotin methyl ester and especially in the
CH3CN containing 1.2% DMSO solvent. DMSO being a
competitive hydrogen bond acceptor interacts with the
isophthaloyl diamide cores of 1 and 2 and reduces the
binding constant values (17). The presence of CH3CN
containing 1.2% DMSO has also a marked effect on the
binding event for which the binding constant values
reported in Tables 1 and 2 are close in magnitude for each
guest. The degree of self-association of the biotin salt as
well as biotin ester, which may vary to different extents

92
K. Ghosh et al.
After completion of the reaction, the solvent was
Conclusions
evaporated and the residual mass was extracted with
CHCl3–CH3OH mixture (3£ 20 ml). The organic extract
was next washed with NaHCO3 solution (3£ 15 ml) and
dried over anhydrous Na2SO4. The solvent was removed
under vacuum and the residual mass was purified by silica
gel column chromatography using ethyl acetate–petroleum
ether (4:1) as the eluent to afford compound 4 (300 mg,
21% yield, mp 1528C).
1H NMR (DMSO-d6, 400 MHz): d 10.6 (s, 1H, amide
NH), 8.95 (s, 1H), 8.64 (brt, 1H), 8.43 (s, 1H), 8.32
(d, J ¼ 4 Hz, 1H), 8.20 (d, J ¼ 8 Hz, 1H), 8.09 (d,
J ¼ 7.6 Hz, 1H), 8.05 (d, J ¼ 7.6 Hz, 1H), 7.64 (t,
J ¼ 7.6 Hz, 1H), 7.42–7.39 (t, J ¼ 8 Hz, 1H), 3.25 (q,
J ¼ 8 Hz, 2H), 1.58–1.53 (m, 2H), 0.91 (t, J ¼ 8 Hz, 3H)
ppm. 13C NMR (DMSO-d6, 125 MHz): d 166.4 (two amide
carbonyl carbons overlapped), 145.5, 142.8, 136.6, 135.9,
135.4, 131.2, 131.0, 129.3, 128.2, 127.5, 124.4, 41.9, 23.2,
12.3ppm. FT-IR (KBr) n: 3324, 3314, 2967, 2926, 1670,
1636, 1544, 1419cm21. Mass (ESI): 284.2 [MþH]þ, 225.2.
In summary, naphthyridine-based symmetrical and unsym-
metrical pyridinium amides 1 and 2 bearing multiple
hydrogen-bonding sites were synthesised. All the receptors
form 1:1 complexeswith tetrabutylammonium salt of biotin
as well as biotin methyl ester and are found to be effective
in sensing biotin salt rather than biotin methyl ester.
The simultaneous binding of carboxylate ion and urea motif
is possibleinto the open cleftsof receptors 1 and 2. Receptor
1 binds both the carboxylate end and the bicyclic urea motif
of biotin salt involving both the isophthaloyl hetero-bis-
amide core and naphthyridine site together. On the contrary,
receptor 2 follows this mode preferably in CH3CN
containing 1.2% DMSO than pure DMSO where the
involvement of the naphthyridine site in complexation is
less. The carboxylate end of biotin salt is strongly
complexed in the isophthaloyl diamide core by
NZH· · ·O, CZH· · ·O hydrogen bonds and charge–charge
interactions and the binding of biotin salt is well reported by
a concomitant change in fluorescence of the appended
naphthyridine. Thus, in spite of not using additional
fluorophore in 1 and 2, both of them act as promising
fluorescent chemosensors for biotin salt rather than biotin
ester. To our knowledge, such fluorometric detection of
biotin salt by exploiting fluorescence property of
naphthyridine is a first-time approach in the literature.
N1-(7-[3-(3-[(Propylamino)carbonyl]benzoylamino)-1
pyridiniumyl]methyl[1,8]naphthyridin-2-yl)acetamide
hexafluorophosphate (1)
Compound 4 (0.07 g, 0.25 mmol) was treated with
2-N-acetyl-7-bromomethyl-1,8-naphthyridine (0.1 g,
0.38 mmol) in dry CH3CN and the reaction mixture was
refluxed for 48 h to afford the bromide salt 5 (60 mg,
43.2% yield). Compound 5 (0.04 g, 0.07 mmol) in MeOH
was subsequently treated with aqueous NH4PF6 solution to
carry out the anion exchange reaction. After heating with
stirring of the solution for 20 min, a precipitate appeared.
Filtration of the precipitate followed by thorough washing
with ether afforded receptor 1 in 67% yield (31 mg, mp
1748C).
Experimental section
General
Solvents were dried and distilled prior to use. Dichlor-
omethane was distilled from anhydrous CaCl2. Triethyla-
mine was distilled from calcium hydride. Acetonitrile was
dried over CaH2. All other commercially available
reagents were used without further purification. Melting
points were recorded in open capillaries and are
uncorrected. FT-IR spectra were recorded on a Perkin-
1H NMR (DMSO-d6, 400 MHz): d 11.37 (s, 1H, amide
NH), 11.09 (s, 1H, amide NH), 9.63 (s, 1H), 8.91 (brs, 1H),
8.79 (d, J ¼ 8 Hz, 1H), 8.64 (brt, 1H), 8.52–8.45 (m, 3H),
8.39 (d, J ¼ 8 Hz, 1H), 8.24 (brt, 1H, amide NH),
8.12–8.08 (m, 2H), 7.72–7.67 (m, 2H), 6.34 (s, 2H), 3.27
(m, 2H), 2.10 (s, 3H), 1.57–1.52 (m, 2H), 0.89 (t,
J ¼ 4 Hz, 3H) ppm. 13C NMR (DMSO-d6, 100 MHz): d
170.1, 166.0, 165.3, 157.1, 154.6, 153.8, 141.0, 139.5,
138.3, 137.1, 135.9, 135.1, 133.2, 130.7, 130.3, 128.7,
127.8, 126.9, 119.4, 119.0, 115.0, 64.4, 41.0, 23.9, 22.2,
11.3 ppm. FT-IR (KBr) n: 3428, 3309, 3067, 2967, 2876,
Elmer model L120-00A. H and 13C NMR spectra were
1
recorded on Bruker Avance 400 and 500 MHz. UV–vis
spectra were taken with a Lambda-25 spectrophotometer.
Fluorescence spectra were obtained with Perkin-Elmer
LS-50B and LS-55 spectrofluorimeters.
N1-Propyl-N3-(3-pyridyl)isophthalamide (4)
The unsymmetrical hetero-bis-amide 4 was obtained by
dropwise addition of n-propylamine (0.291 g, 4.92 mmol)
and 3-aminopyridine (0.463 g, 4.92 mmol) to isophthaloyl
diacid chloride (1 g, 4.92 mmol) in dry dichloromethane
(DCM) at high dilution condition under nitrogen atmos-
phere. Triethylamine (4.92 mmol) was added in each amine
solution during the reaction. After completion of addition
of the amines, the reaction mixture was stirred overnight.
The progress of the reaction was monitored by TLC.
1686, 1639, 1459 cm21
.
Mass (ESI): 483.3
[M2 PF6 þ1]þ, 441.4, 284.3.
N1,N3-Di(3-pyridyl)isophthalamide (6)
The symmetrical bis-amide 6 was obtained by coupling of
3-aminopyridine (1.2 g, 12.8 mmol) with isophthaloyl

Supramolecular Chemistry
93
diacid chloride (1.3 g, 6.4 mmol) in dry DCM followed by
addition of triethylamine (1.4 mmol) under nitrogen
atmosphere. The reaction mixture was allowed to stirring
for overnight. After completion of the reaction, the solvent
was removed under vacuum. The residual mass was
extracted with CHCl3 –CH3OH mixture (3£ 20 ml).
The organic layer was washed with NaHCO3 solution
(3£ 15 ml) and dried over anhydrous Na2SO4. The solvent
was removed and the residual mass was purified by column
chromatography over silica gel using chloroform–
methanol (4:1) as the eluent to afford the desired
compound 6 (730 mg, 36% yield, mp 2108C).
noted. Then, for the complexes of receptors 1 and 2 with
guests, [A0/(A 2 A0)] as a function of the inverse of guest
concentration was plotted. The plot fits a linear
relationship, indicating the 1:1 stoichiometry of the
receptor–guest complex. The ratio for the intercept versus
slope gives the association or binding constant (Ka) for
the receptor–guest complex shown in Tables 1 and 2.
A similar experiment was done with 1 and 2 in the
fluorescence and the emission intensities were recorded to
evaluate the binding constant values. In NMR, Dd/[G ] as a
function of Dd was plotted. The plot fits a linear
relationship and the slope of the curve gives the binding
constant value. Stoichiometries of the complexes were
determined from the break of the titration curves which
were obtained from the plot of Dd versus [G ]/[H ].
1H NMR (DMSO-d6, 400 MHz): d 10.69 (s, 2H, amide
NH), 8.96 (d, J ¼ 2 Hz, 2H), 8.59 (s, 1H), 8.33 (d,
J ¼ 4 Hz, 2H), 8.23–8.18 (m, 4H), 7.74 (t, J ¼ 8 Hz, 1H),
7.43 (d, J ¼ 8 Hz, 1H), 7.42 (d, J ¼ 8 Hz, 1H) ppm. 13C
NMR (DMSO-d6, 125 MHz): d 166.2, 145.6, 142.9, 136.5,
135.5, 131.8, 129.6, 128.2, 128.0, 124.4 ppm. FT-IR (KBr)
n: 3418, 3054, 1676, 1613, 1585, 1556, 1481, 1429 cm21
Mass (ESI): 319.1 [MþH]þ, 225.2, 130.2.
.
Acknowledgements
We thank CSIR, New Delhi, India for financial support. A.R.S.
thanks the University of Kalyani, West Bengal, India for a
University Research Fellowship. We acknowledge DST, New
Delhi, India for providing facilities in the department under DST
FIST Programme.
N1-[7-(3-[(3-[(1-[7-(Acetylamino)[1,8]naphthyridin-2-
yl]methyl-3-pyridiniumyl)amino]
carbonylbenzoyl)amino]-1-pyridiniumylmethyl)
[1,8]naphthyridin-2-yl]acetamide
dihexafluorophosphate (2)
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8.79 (brd, 2H), 8.63 (brt, 2H), 8.51 (d, J ¼ 8 Hz, 2H), 8.46
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