Naphthyridine-based receptors for flurometric detection of urea and biotin

Article abstract of DOI:10.1007/s10847-009-9707-6

Naphthyridine-based receptors 1-4 have been designed and synthesized for the recognition of urea in CHCl₃ containing 1% CH₃CN. Receptor 1 also binds biotin and its methyl ester with moderate binding constant values. In comparison, re

Full text of DOI:10.1007/s10847-009-9707-6

J Incl Phenom Macrocycl Chem (2010) 67:271–280
DOI 10.1007/s10847-009-9707-6
ORIGINAL ARTICLE
Naphthyridine-based receptors for flurometric detection
of urea and biotin
•
Kumaresh Ghosh Tanushree Sen
Received: 15 September 2009 / Accepted: 9 November 2009 / Published online: 27 November 2009
Ó Springer Science+Business Media B.V. 2009
Abstract Naphthyridine-based receptors 1–4 have been
designed and synthesized for the recognition of urea in
CHCl3 containing 1% CH3CN. Receptor 1 also binds biotin
and its methyl ester with moderate binding constant values.
In comparison, receptor 2 is less efficient in recognising
biotin and its methyl ester analogue. Receptor 1 binds urea
and biotin with binding constant values of 1.02 9 104 and
1.08 9 104 M-1, respectively, in CHCl3 containing 1%
CH3CN and shows significant change in emission of
naphthyridine upon complexation. Characterization and
urea [3], biotin [4], carbohydrates [5], carboxylic acids [6],
amino acids [7] etc. Among these substrates of biological
significance, urea is important one because it is toxic,
pollutant and causes serious biological disorders [8, 9]. It
has a well-defined geometry, and it can form at least six
hydrogen bonds. The detection of this small molecule by
suitable synthetic receptor is therefore important. In this
regard, very few fluorescent receptors for urea recognition
are known in the literature. Goswami et al. reported a
pyridine-based macrocyclic receptor for the fluorometric
detection of urea [10]. Chetia and Iyer showed that a 2,6-
bis(2-benzimidazole)pyridine receptor is able to bind urea
and exhibits a concomitant change in fluorescence [11].
Our anthracene-based open and macrocyclic receptors for
fluorometric detection of urea are mentionable [12]. Non-
fluorescent receptors such as crown ether-based receptors,
developed by Pedersen [3] and carboxylic acid containing
crown ether receptor of Reinhoudt [13] are known to bind
urea. Reinhoudt et al. introduced the concept of using an
electrophilic center to bind urea in the cavity of crown
ether [14]. Bell et al. synthesized polyaza heterocycles
which were effective for urea recognition [15]. Recently,
we have shown that a pyridine amide-based macrocyclic
receptor can form a strong inclusion complex with urea
[16]. Goswami et al. reported the solubilization and rec-
ognition of urea using a simple dinaphthyridine receptor
[17].
1
sensing properties of the receptors were evaluated by H
NMR, UV–vis and fluorescence spectroscopic methods.
Keywords Biotin recognition ꢀ Urea recognition ꢀ
Fluorometric detection ꢀ Naphthyridine-based receptors
Introduction
Recognition of individual molecules is one of the most
fundamental processes both in chemistry and biology. The
study of synthetic receptors that mimic the biological
processes is important in the area of molecular recognition
[1, 2]. In designing such receptors judicious arrangement of
hydrogen-bonding sites has received a great deal of recent
attention. Considerable efforts have been made in last few
decades to design receptors for bioactive substrates like
Similarly, among the substrates biotin (referred to as
vitamin H in human) is an essential cofactor for a number
of enzymes that have diverse metabolic functions [18].
Structurally it consists of a pentanoic acid side chain and a
cis-fused bicyclic moiety with sulfur atom in the ring, as
evidenced from crystallographic studies [19]. The crystal
structure of biotin shows that the carboxyl group of one
biotin molecule is intermolecularly hydrogen bonded to the
Electronic supplementary material The online version of this
article (doi:10.1007/s10847-009-9707-6) contains supplementary
material, which is available to authorized users.
K. Ghosh (&) ꢀ T. Sen
Department of Chemistry, University of Kalyani, Kalyani,
Nadia 741235, India
e-mail: ghosh_k2003@yahoo.co.in
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J Incl Phenom Macrocycl Chem (2010) 67:271–280
urea linkage of the other biotin molecule. The valeryl chain
is severely twisted from the maximally extended all trans-
conformation. In an effort to bind this interesting biological
substrate, careful design of molecular receptor that can
bind both the urea and acid parts together is necessary. A
limited number of designed receptors are known in the
literature. In this aspect, the use of Troger’s base receptor
as reported by Wilcox and co-workers was noteworthy
[20]. Claramunt et al. reported the recognition of biotin
methyl ester [21]. Goswami et al. demonstrated that pyri-
dine amide-based simple receptor is able to bind biotin
itself involving both carboxylic acid and cyclic urea part of
biotin [4]. We have also recently reported the recognition
of biotin by a number of synthetic receptors, which are
benzthiazole [22] and naphthyridine [23] based.
(1.45 g, 0.006 mol) was added followed by catalytic
amount of Cu2O. Reflux was continued for further 18 h.
The desired amine 6a was isolated in 60% yield after
extraction with chloroform followed by purification
through column chromatography using 35% ethyl acetate
in petroleum ether (60–80 °C). Mp was noted as 170 °C.
1H NMR (400 MHz, CDCl3) d 8.63 (d, 1H, J = 4.36 Hz),
7.80 (m, 2H), 7.69 (t, 1H), 7.48 (d, 1H, J = 7.8 Hz), 7.22 (m,
1H), 6.82 (d, 1H, J = 8.56 Hz), 6.61 (d, 1H, J = 8.48 Hz),
5.69 (s, 2H), 4.96 (s, 2H). FTIR (m cm-1, KBr): 3473, 3364,
3188, 2913, 1638, 1595, 1512, 1432, 1324, 1246, 1137.
7-(Benzyloxy)-1,8-naphthyridin-2-amine (6b)
A solution of benzyl alcohol (0.3 g, 0.003 mol) in dry THF
was stirred with NaH (0.07 g, 0.003 mol) for 2 h under
mild reflux. To it N-(7-chloro-1,8-naphthyridin-2-yl) bu-
tyramide 5b (0.76 g, 0.003 mol), dissolved in dry THF was
added followed by catalytic amount of Cu2O at RT.
Reaction was continued for further 16 h at refluxing con-
dition. Solvent was removed under reduced pressure; res-
idue was dissolved in CHCl3 (35 mL) and washed with
water. The CHCl3 layer was dried over anhydrous Na2SO4,
concentrated under vacuum and the crude mass was puri-
fied through column chromatography [eluent: 50% ethyl
acetate in petroleum ether (60–80 °C)]. The desired prod-
uct was obtained as semi solid (0.47 g, 62% yield).
1H NMR (500 MHz, CDCl3) d 7.79 (d, 1H, J = 8.5 Hz),
7.75 (d, 1H, J = 8.7 Hz), 7.49 (d, 2H, J = 7.4 Hz), 7.38
(t, 2H, J = 7.6 Hz), 7.32 (t, 1H, J = 7.4 Hz), 6.75 (d, 1H,
J = 8.5 Hz), 6.59 (d, 1H, J = 8.5 Hz), 5.58 (s, 2H), 5.08
(s, 2H). FTIR (m cm-1, KBr): 3336, 2926, 1671, 1627, 1601,
1513, 1382, 1323, 1270, 1139.
Thus the noteworthy progress made in this horizon
inspired us to work on the recognition of urea and biotin. In
this paper, we report the design and synthesis of a series of
naphthyridine-based receptors for recognition of urea,
biotin and their derivatives.
6
6
O
O
N
N
O
O
N
O
O
O
O
NH
HN
NH
HN
N
3
N
N
N
N
N
N
N
N
O
O
O
N
N
O
O
O
N
N
4
2
1
Materials and methods
Materials
5-octyloxy-N1,N3-bis(7-(pyridin-2-ylmethoxy)-1,8-
naphthyridin-2-yl)isophthalamide (1)
All the solvents were dried by usual procedures prior to
use. All the reactions were carried out under nitrogen. IR
and UV spectra were recorded on Perkin Elmer model
L120-00A and Lambda-25, respectively. Fluorescence was
In dry THF solution of 5-octyloxyisophthaloyldichloride
(0.2 g, 0.001 mol), a solution of 7-(pyridin-2-ylmethoxy)-
1,8-naphthyridin-2-amine 6a (0.46 g, 0.002 mol) and Et3N
(0.26 mL, 0.002 mol) in dry THF was added dropwise with
constant stirring. The reaction was continued for 18 h.
Solvent was removed under reduced pressure, washed with
20% NaHCO3 solution and extracted with CHCl3 con-
taining 3% CH3OH. The organic layers were collected,
washed with water and dried over anhydrous Na2SO4.
After evaporation of solvent it was passed through a silica
gel column (eluent: 3% CH3OH in CHCl3) and receptor 1
was obtained as off white solid (0.09 g, 20% yield). Mp
124 °C.
recorded by PerkinElmer LS55 instrument. For 1H and 13
C
NMR spectra Bruker 400 and 500 MHzs were used.
Melting points were recorded in open capillaries and are
uncorrected.
Synthesis of 1–4
7-(Pyridin-2-ylmethoxy)-1,8-naphthyridin-2-amine (6a)
To a stirred solution of 2-hydroxymethylpyridine (0.6 g,
0.005 mol) in dry THF, NaH (0.14 g, 0.006 mol) was
added and refluxed for 3 h under nitrogen atmosphere.
After cooling, 2-acetamido-7-chloro-1, 8-naphthyridine 5a
1H NMR (500 MHz, CDCl3) d : 9.17 (2H, s, –NHCO–),
8.64 (2H, d, J = 5 Hz), 8.58 (2H, d, J = 10 Hz), 8.19
(3H, d, J = 10 Hz), 8.04 (2H, d, J = 10 Hz), 7.72 (2H, t,
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J Incl Phenom Macrocycl Chem (2010) 67:271–280
273
J = 10 Hz), 7.68 (2H, s), 7.52 (2H, d, J = 10 Hz), 7.25–
7.23 (2H, m), 7.07 (2H, d, J = 10 Hz), 5.74 (4H, s), 4.11
(2H, t, J = 5 Hz), 1.89–1.82 (m, 2H), 1.53–1.50 (m, 2H),
1.40–1.30 (m, 8H), 0.90 (t, 3H, J = 5 Hz). 13C (125 MHz,
CDCl3) 165.1, 160.4, 156.8, 154.4, 149.8, 139.5, 139.3,
137.1, 136.2, 123.2, 122.6, 118.7, 118.2, 117.8, 117.6,
113.4, 113.3, 112.9, 69.3, 69.0, 32.2, 29.7, 29.6, 29.5, 26.3,
23.1, 14.5. FTIR (m cm-1, KBr): 3363, 3062, 2922,
2851, 1682, 1612, 1458, 1187, 1133. Mass (ESI): 763.2
(M ? H)?, 556.3, 425.2, 382.3, 362.2.
1H NMR (500 MHz, CDCl3) d : 8.66 (d, 2H, J = 4.8 Hz),
7.96 (d, 2H, J = 8.6 Hz), 7.72 (t, 2H, J = 7.7 Hz), 7.54 (d,
2H, J = 7.8 Hz), 7.24 (t, 2H, J = 6.7 Hz), 6.98 (d, 2H,
J = 8.6 Hz), 5.75 (s, 4H).13C (125 MHz, CDCl3) d: 164.9,
157.2, 154.7, 149.8, 139.4, 137.1, 123.1, 122.7, 115.9, 111.5,
69.2. FT-IR m cm-1 (KBr): 3417, 2925, 2850, 1611, 1572,
1502, 1433, 1327. UV (1% CH3CN in CHCl3, c =
2.44 9 10-5 M) kmax (nm) 286, 292, 298, 304, 311, 318,
326, 344. Mass (ESI): 345.2 (M ? H)?, 367.2 (M ? Na)?.
2,7-Bis(benzyloxy)-1, 8-naphthyridine (4)
N1,N3-Bis(7-(benzyloxy)-1,8-naphthyridin-2-yl)-5-
propoxyisophthalamide (2)
In dry THF, benzyl alcohol (136 mg, 1.26 mmol) was
stirred with NaH (36 mg, 1.5 mmol) for 1 h under mild
refluxing condition. A solution of 2,7-dichloro-1,8-naph-
thyridine (100 mg, 0.5 mmol) in dry THF was added to it
at RT and reflux was continued for further 8 h. The solvent
was evaporated, washed with water and extracted with
CHCl3 (3 9 20 mL). The organic layers were taken toge-
ther, left over anhydrous Na2SO4 and concentrated. After
purification of the crude mass through column chroma-
tography using 20% ethyl acetate in petroleum ether (60–
80 °C) as eluent, compound 4 was isolated as white solid
(110 mg, 64% yield). Mp 100 °C.
A solution of 7-(benzyloxy)-1,8-naphthyridin-2-amine 6b
(0.45 g, 0.002 mol) was added to a dry THF solution of 5-
octyloxyisophthaloyldichloride (0.2 g, 0.001 mol) along
with Et3N (0.26 mL, 0.002 mol). The reaction mixture was
stirred at RT for 14 h. Solvent was evaporated under vac-
uum, neutralized with saturated NaHCO3 solution and
extracted with CHCl3. Organic layers were separated,
washed with water and dried over anhydrous Na2SO4. The
layer was concentrated and purified through column chro-
matography using 2% CH3OH in CHCl3. Receptor 2 was
obtained as yellow solid (0.41 g, 40% yield). Mp 104 °C.
1H NMR (400 MHz, CDCl3) d : 9.06 (s, 2H), 8.58 (d,
2H, J = 8.5 Hz), 8.19 (d, 2H, J = 8.8 Hz), 8.17 (s, 1H),
8.02 (d, 2H, J = 8.6 Hz), 7.67 (s, 2H), 7.51 (d, 4H,
J = 7.2 Hz), 7.42–7.33 (m, 6H), 6.99 (d, 2H, J = 8.6 Hz),
5.61 (s, 4H), 4.11 (t, 2H, J = 6.4 Hz), 1.88–1.85 (m, 2H),
1.37-1.19 (m, 10H), 0.88 (t, 3H, J = 6.3 Hz). 13C
(100 MHz, CDCl3) 165.1, 164.6, 160.0, 154.1, 152.9,
139.2, 138.7, 136.5, 135.9, 128.5, 128.2, 128.1, 118.2,
117.1, 117.06, 113.04, 112.2, 68.9, 68.5, 31.8, 29.3, 29.2,
29.01, 25.9, 22.6, 14.1. FTIR (m cm-1, KBr): 3364, 3063,
2923, 2853, 1682, 1614, 1531, 1505, 1439, 1326, 1274,
1131. Mass (ESI): 760.9 (M ? H)?, 597.4, 583.6, 542.5.
1H NMR (400 MHz, CDCl3) d: 7.94 (d, 2H, J = 8.6 Hz),
7.51 (d, 4H, J = 7.2 Hz), 7.41 (t, 4H, J = 8.0 Hz), 7.33
(t, 2H, J = 8.0 Hz), 6.91 (d, 2H, J = 8.6 Hz), 5.62
(s, 4H).13C (100 MHz, CDCl3) d 164.9, 154.6, 138.8, 136.9,
128.5, 128.2, 127.9, 115.3, 111.1, 68.4. FT-IR m cm-1 (KBr):
3418, 3063, 3031, 2925, 1607, 1560, 1502, 1435, 1326, 1259.
UV (1% CH3CN in CHCl3, c = 2.44 9 10-5 M) kmax (nm)
293, 299, 305, 312, 319, 326. Mass (ESI): 343.4 (M ? H)?,
365.4 (M ? Na)?.
Results and discussion
Synthesis
2,7-Bis(pyridin-2-ylmethoxy)-1, 8-naphthyridine (3)
Receptors 1 [24] and 2 were synthesized according to the
Scheme 1. Coupling of 2-acetylamino-7-chloro-1,8-naph-
thyridine 5a, obtained by known method [25], with 2-
hydroxymethylpyridine in dry THF in the presence of NaH
and a catalytic amount of Cu2O gave compound 6a.
Reaction of amine 6a with 5-octyloxyisophthaloyl diacid
chloride gave the receptor 1 in 20% yield. The same
reaction conditions were applied to the synthesis of
receptor 2 (Scheme 1). Here, 2-butrylamino-7-chloro-1,8-
naphthyridine 5b was coupled with benzyl alcohol, under
the same reaction condition to give amine 6b as the sole
product. The synthesis of compounds 3 and 4 was started
with 2,7-dichloro-1,8-naphthyridine which was obtained by
known method [25]. Coupling of 2-hydroxymethylpyridine
A dry THF solution of 2-hydroxymethylpyridine (166 mg,
1.51 mmol) was stirred with NaH (45 mg, 1.87 mmol)
under mild refluxing condition for 1.5 h. To this solution
2,7-dichloro-1,8-naphthyridine (100 mg, 0.5 mmol) was
added along with catalytic amount of Cu2O. The reaction
was further continued for 10 h. Solvent was removed under
vacuum and the solid mass was redissolved in CHCl3
(30 mL). The organic layer was collected, washed with
water (3 9 20 mL), dried over anhydrous Na2SO4 and
finally concentrated under reduced pressure. The residue
was purified by column chromatography (eluent: 40% ethyl
acetate in petroleum ether (60 – 80 °C)) to give compound
3 as brown solid (95 mg, 55% yield). Mp 72 °C.
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Complexation properties
RCH2OH, NaH, dry THF
reflux, cat. Cu2O
O
O
N
N
NH2
Cl
N
N
N
H
R'
R
1H-NMR study
5a: R' = CH3
5b: R' = C3H7
6a: R = pyridyl
6b: R = phenyl
However, to understand the binding properties of the
receptors in solution, 1H NMR spectra were recorded in dry
CDCl3. In each case, powder urea was added to CDCl3
solutions of the receptors, followed by thorough sonication
for about 10 min. The excess urea was removed by filtra-
tion, and the filtrate of each receptor solution was used to
5-octyloxyisophthaloyl dichloride
Et3N, dry THF, rt
1: R = pyridyl
2: R = phenyl
Scheme 1 The syntheses of receptors 1 and 2
RCH2OH, NaH, dry THF
reflux, cat. Cu2O.
1
record the H NMR spectra. In case of receptor 1 signifi-
O
N
N
O
Cl
N
N
Cl
cant downfield shifts of the amide protons (Dd = 2.05) and
isophthaloyl peri proton (Dd = 0.52) were observed in the
presence of urea. In addition, a new peak at d 6.37 for urea-
protons was observed (Fig. 2). The proton integration ratio
indicated the formation of 1:1 complex and importantly,
dilution of this 1:1 complex in CDCl3 did not show any
R
R
3: R = pyridyl
4: R = phenyl
Scheme 2 The syntheses of receptors 3 and 4
1
change in H NMR spectra thereby suggesting a strong
and benzyl alcohol with 2,7-dichloro-1,8-naphthyridine in
dry THF in the presence of NaH and catalytic amount of
Cu2O afforded the receptors 3 and 4 in 55% and 64% yield,
respectively (Scheme 2).
complexation. In comparison to 1, receptor 2 showed weak
interaction as evidenced from the appearance of a weak
signal at d 5.55 for urea (Fig. 3) followed by small
downfield shifts of amide protons and the isophthaloyl peri
proton (Hb) (Dd for amide NH 1.36 and Dd for isophthaloyl
peri proton 0.34; Fig. 3). The weak interaction is attributed
due to the presence of phenyl ring, which does not have
any hydrogen bond acceptor for a significant contribution
to the interaction process. From the integration values it
was found that receptor 2 was bound with *63% urea in
CDCl3. Thiourea, N, N0-dimethyl urea etc., were also
individually added to the CDCl3 solutions of 1 and 2. After
sonication and filtration, clear solutions were used to record
the 1H NMR spectra. Careful analysis of the results showed
that thiourea, N, N0-dimethylurea interacted weakly with
Molecular modeling
In the designed receptors naphthyridine has been taken as
hydrogen bonding site as well as fluorescent probe for
monitoring the recognition event.
The optimization of the geometries of 1 and 2 was
performed by MMX calculation.1 It is evident from Fig. 1
that the open cleft of 1 is able to make a strong complex
with both urea and biotin utilizing hydrogen bond donors
and acceptors together. Similarly, receptor 2, which does
not have pyridine rings at the lower rim, also interacts with
urea in the cleft involving same number of hydrogen bonds
like receptor 1. Only the difference in between the com-
plexes of urea with receptors 1 and 2 is lying with the
marginal change in hydrogen bond distances (see Fig. 1)
which influence the receptor 1 to from a stable urea com-
plex. Due to absence of hydrogen bonding site at the lower
rim of 2, biotin is complexed at the diamide core involving
only the cyclic urea part leaving carboxylic acid uncom-
plexed (Fig. 1d). The energy values of the complexes of 1
and 2 and the intermolecular hydrogen bond distances are
displayed in Fig. 1. MMX calculations of the complexes of
1 and 2 with N,N0-dimethylurea and thiourea were also
carried out in gas phase (see Supplementary Material) and
they were found to be less stable due to formation of less
number of hydrogen bonds.
1
both receptors 1 and 2. In H NMR, weak signals for urea
protons appeared at d 4.55 and 4.45 during interactions
with 3 and 4, respectively. This suggested that receptors 3
and 4 are poor binder of urea.
A similar study was performed with biotin and its
methyl ester. Figures 4 and 5 highlight the changes
occurring during hydrogen bonding interactions of recep-
tors 1 and 2 with biotin and biotin methyl ester. It is evident
from Figs. 4 and 5 that the amide protons of both 1 and 2
are shifted more downfield in the presence of biotin methyl
ester. In presence of biotin acid, the amide protons were
shifted to the lesser extent. This is due to the existence of
two possible modes of complexation of biotin with the
receptors 1 and 2 in solution (Fig. 6). Receptor 1 may
complex biotin via the modes A and B, which may remain
in equilibrium in solution. Similarly, for receptor 2, forms
C and D are possible. In case of biotin methyl ester the
possibility of hydrogen bonding of the ester group into
the diamide core is negligible due to the bulky nature of the
ester group. Therefore, while biotin has the chance to be
1
Energy minimization was carried out using MMX (PC Model
Serena Software (1993). Molecular modeling was performed using
standard constants, and the dielectric constant was maintained at 1.5.
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J Incl Phenom Macrocycl Chem (2010) 67:271–280
275
˚
˚
˚
˚
˚
Fig. 1 Energy optimized geometries of (a) 1. urea complex [E =
˚
53.01 kcal/mol, a = 2.17 A, b = 2.50 A, c = 2.02 A, d = 1.84 A,
a = 2.21 A, b = 2.41 A, c = 2.28 A, d = 2.69 A, e = 3.36 A,
˚
˚
˚
˚ ˚ ˚
f = 1.85 A, g = 2.94 A, h = 2.99 A]; (d) 2. biotin [E = 68.50 kcal/
˚ ˚ ˚ ˚ ˚
mol, a = 2.31 A, b = 2.57 A, c = 1.96 A, d = 1.89 A, e = 2.72 A,
˚
˚
e = 1.80 A, f = 2.92 A, g = 3.09 A]; (b) 2. urea [E = 56.08 kcal/mol,
˚
˚
˚
˚
˚
˚
a = 2.18 A, b = 2.45 A, c = 2.02 A, d = 1.82 A, e = 1.81 A,
˚
˚
f = 1.88 A, g = 2.55 A]
˚
f = 3.07 A, g = 3.2 A]; (c). 1. biotin [E = 80.81 kcal/mol,
˚
1
Fig. 2 (a) Partial H NMR (400 MHz, CDCl3) spectra of receptor 1
Fig. 5 (a) Partial 1H NMR (400 MHz) spectra of receptor 2
(c = 2.82 9 10-3 M) and its 1:1 complex with (b) biotin acid and
(c) biotin methyl ester in CDCl3
(c = 2.81 9 10-3 M). (b) 1:1 complex of receptor 1 with urea
complexed into the cleft of receptor 1 via the modes A and
B, biotin methyl ester will follow only one binding mode
(A0) giving the preference of complexation of cyclic urea
part into the diamide core of 1 (Fig. 7). This is also true
for receptor 2. The pendant pyridyl groups in 1 play key
role in the binding event. It is obvious from Fig. 6 that
the dimaide core and the pendant pyridine rings are
simultaneously involved in the binding of both acid and
urea parts of biotin. In contrast, receptor 2 binds only one
part (either acid or cyclic urea) of biotin and thus exhibits
weak binding. However, amongst the different modes,
modes B and D are only favourable due to compact
binding of the cyclic urea motif with more number of
hydrogen bonds.
1
Fig. 3 (a) Partial H NMR (400 MHz, CDCl3) spectra of receptor 2
(c = 2.82 9 10-3 M). (b) 1:1 complex of receptor 2 with urea
After having this information from 1H NMR, we carried
out fluorescence and UV–vis titrations to understand
the binding potencies, selectivities and sensitivities of the
receptors with the guests mentioned in the study. All the
guests were dissolved in CHCl3 containing 1% CH3CN
and direct titration method was followed to evaluate the
binding constant values.
Fig. 4 (a) Partial 1H NMR (400 MHz) spectra of receptor 1
(c = 2.89 9 10-3 M) and its 1:1 complex with (b) biotin and (c)
biotin methyl ester in CDCl3
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Fig. 6 Possible modes of
complexation of biotin with 1
(A and B) and 2 (C and D)
O
O
O
O
O
O
6
6
6
6
O
O
O
O
O
O
O
O
N
N
N
N
H
N
N
N
N
H
H
H
H
H
H
H
O
O
O
S
N
N
N
N
N
O
S
H
H
N
N
N
N
N
N
HN
H
NH
HN
NH
H
H
H
N
N
O
N
N
N
O
S
S
O
O
H
H
H
O
H
H
O
O
O
N
H
N
N
N
H
O
H
H
O
H
N
O
O
N
O
O
N
N
(C)
(D)
(A)
(B)
Fig. 7 Possible mode of
complexation of biotin methyl
ester with 1 and 2
O
O
N,N'-Dimethyl urea
Thiourea
6
O
O
N
N
H
H
Urea
N
N
HN
H
NH
H
N
O
N
Biotin Methyl Ester
Biotin Acid
S
O
O
Me
X
X
O
0.0
0.1
0.2
0.3
0.4
A'
Δ
I/I0
X = N; 1, X = CH; 2
Fig. 8 Fluorescence ratio (I-I0/I0) of receptor 1 (c = 1.49 9 10-5 M)
at 360 nm upon addition of 7 equiv of a particular guest in CHCl3
containing 1% CH3CN
Fluorescence and UV studies
The effect on absorption and emission spectra of naph-
thyridine-based receptors 1 and 2 was recorded in presence
of urea, biotin and their derivatives in CHCl3 containing
1% CH3CN. Receptor 1 (c = 1.49 9 10-5 M) showed an
emission at 360 nm when excited at 320 nm in CHCl3
containing 1% CH3CN. Upon gradual addition of biotin,
biotin methyl ester, urea, thiourea and N, N0-dimethyl urea
individually to the solution of receptor 1 in CHCl3 con-
taining 1% CH3CN, the emission of naphthyridine was
increased to the different extents (Fig. 8).
perturbation of the excited np* state in a destabilizing
manner for which the pp* state becomes the lowest energy
singlet excited state [26].
Simultaneous UV–vis titrations of 1 with the same
guests were carried out in CHCl3 containing 1% CH3CN.
The changes in absorbance of 1 upon complexation of
biotin and urea are represented in Figs. 9b and 10b,
respectively. Fluorescence titration data were used to
determine the binding constant values [27]. For the com-
plexes of 1 with biotin and urea, I0/DI as function of the
inverse of guest concentration fits a linear relationship,
indicating 1:1 stoichiometry of receptor/guest complex.
The ratio of the intercept versus slope gave the binding
constant (Ka) value (see Supplementary Material).
During titrations of receptor 1 with biotin, biotin methyl
ester and urea, strong fluorescence enhancement effect was
observed. For example, the spectral changes in fluores-
cence of 1 during titrations with biotin and urea are
represented in Figs. 9a and 10a, respectively. Upon com-
plexation of biotin and urea, the emission peak of 1 at
360 nm for naphthyridine underwent a red shift of 3 nm,
suggesting a strong hydrogen bonding interaction. During
titration no other spectral changes were observed in the
emission spectra. The gradual increase of intensity of the
monomer emission of naphthyridine motif during com-
plexation is explained by hydrogen bond mediated
As can be seen from Table 1, simple receptor 1 shows
selectivity for biotin and urea. Urea, being smaller in size,
is preferentially complexed than thiourea in the excited
state. Weak binding of thiourea is attributed to the bigger
size of the sulfur atom that poorly fits into the cavity than
urea. The polarisability of the sulfur atom and charge
transfer phenomena is also the responsible factors for the
weaker hydrogen bond interaction of sulfur atom [28].
123

J Incl Phenom Macrocycl Chem (2010) 67:271–280
277
Fig. 9 (a) Change in emission
of 1 (c = 1.49 9 10-5 M) and
(b) change in absorbance of 1
0.8
0.6
0.4
0.2
0.0
(a) 800
(b)
(c = 1.49 9 10-5 M) upon
600
gradual addition of biotin in
CHCl3 containing 1% CH3CN
400
200
0
350
400
450
500
550
600
300
320
340
360
380
Wavelength (nm)
Wavelength(nm)
Fig. 10 (a) Change in emission
0.8
0.6
0.4
0.2
0.0
750
600
450
300
150
0
(b)
(a)
of 1 (c = 1.49 9 10-5 M) and
(b) change in absorbance of 1
(c = 1.49 9 10-5 M) upon
gradual addition of urea in
CHCl3 containing 1% CH3CN
300
320
340
360
380
350
400
450
500
550
600
Wavelength (nm)
Wavelength (nm)
other hand, the absorbance at 343 nm gradually decreased
upon addition of biotin, urea and their derivatives (Sup-
plementary Material). The change in absorbance of the
naphthyridine motif at 343 nm was considered for deter-
mination of binding constant and we were unable to
determine satisfactorily. In such situation the dilution
effect during the course of titration cannot be ignored.
In order to evaluate the sensing and binding ability of 3
as a receptor for urea, fluorescence studies were carried out
in CHCl3 containing 1% CH3CN. Receptor 3 gave emis-
sion at 343 nm when excited at 310 nm. Addition of urea,
thiourea or N,N0-dimethylurea individually to the solution
of 3 caused marginal change in emission. Figure 11, for
example, represents the change in emission and absorption
spectra of 3 upon adding urea up to 15 equivalents.
Upon adding urea the absorptions of the bands at 310,
318 and 325 nm of 3 were decreased without showing any
other spectral change. The similar change was also
observed for thiourea and N,N0-dimethylurea. From the
fluorescence and UV titrations data, it was difficult to
determine the reliable association constant values for 3
with urea, thiourea or N,N0-dimethylurea.
Table 1 Binding constant values of 1 from fluorescence in CHCl3
containing 1% CH3CN
Guests
Ka (M-1 a
)
Biotin acid
Biotin methyl ester
Urea
1.08 9 104
8.2 9 103
1.02 9 104
Thiourea
N,N0-dimethyl urea
b
b
a
Binding constant values were determined at wavelength 360 nm
Binding constant values were not determined due to minor change
b
N,N0-dimethylurea is weakly complexed into the open cleft
due to its bulky nature.
Under similar experimental conditions, titrations of
receptor 2 were conducted in CHCl3 containing 1%
CH3CN (see Supplementary Material). In contrast to
receptor 1, upon increasing amounts of addition of urea,
biotin and their derivatives individually, to the receptor
solution of 2, minor change in fluorescence intensity of
naphthyridine moiety was observed; suggesting weak
1
interactions (also see explanation in H NMR study). Due
Under similar experimental condition, 4 was also titra-
ted with urea, thiourea and N,N0-dimethylurea in CHCl3
containing 1% CH3CN (see Supplementary Material). Here
also we got similar findings as observed with the receptor
to the minor and irregular changes in emission of 2 upon
complexation, reliable binding constant values were diffi-
cult to determine from fluorescence titration results. On the
123

278
J Incl Phenom Macrocycl Chem (2010) 67:271–280
Fig. 11 (a) Emission spectra of
3 (c = 2.44 9 10-5 M) and
(b) absorption spectra
350
300
250
200
150
100
50
(a)
(b) 0.60
(c = 2.44 9 10-5 M) during
titration with urea in CHCl3
containing 1% CH3CN
0.45
0.30
0.15
0.00
0
280
300
320
340
360
380
350
400
450
500
Wavelength (nm)
Wavelength (nm)
3. There was no appreciable change in emission of 4 upon
adding urea or its derivatives. After the addition of 10
equivalent amounts of guests, the emission intensity at
343 nm (when excited at 310 nm) was gradually
decreased. This decrease in emission intensity may occur
due to dilution effect.
Fig. 13a, while emission at 363 nm in CHCl3 is markedly
increased or ‘switched on’ with a red shift of 4 nm in
presence of equivalent amount of urea to the solution of 1,
it is decreased in presence of thiourea. A similar observa-
tion was made with the receptor 2 in dry CHCl3 (Fig. 13b).
Figure 14a displays the change in emission spectra upon
dilution of the complex of 1.urea. The change in emission
of the naphthyridine moiety at 366 nm for the 1:1 complex
of 1 with urea is presented in Fig. 14b, showing an almost
linear response with complex concentration. The same
behavior was noticed with the urea complex of receptor 2
in CHCl3 (see Supplementary Material).
Thus the experimental results as corroborated above
represent that only the receptor 1 is a better binder of urea
and biotin due to the placement of the pyridyl ring at the
lower rim.
In order to realize the complexing abilities of 1 and 2
with urea in less polar solvent CHCl3, we further carried
out the fluorescence and UV titration experiments by
dilution of the 1:1 complexes of urea. Figure 12a, in this
regard, indicates the change in absorbance of naphthyridine
with 1-urea complex concentration. The linear change in
absorbance at 366 nm in 1 demonstrated 1:1 stoichiometry
of the complex of 1 with urea (Fig. 12b).
Binding constant values were determined by dilution
method [29] using UV titration results in dry CHCl3 and
receptor 1 shows about a 10 times higher binding constant
value (Ka = 8.73 9 105 M-1) in CHCl3 than the case in
CHCl3 containing 1% CH3CN (cf. Table 1). On the other
hand, receptor 2 exhibits a binding constant value of
1.15 9 105 M-1, which is less than 1. It is fact that
CH3CN, being more polar than CHCl3 reduces the hydro-
gen bonding interactions, for which guest induced change
in emission of 2 in comparison to 1, in CHCl3 containing
1% CH3CN was too less to consider for determination of
the binding constant values.
A similar UV study with 2 gave linear change in
absorbance at 343 nm, which indicated 1:1 stoichiometry
of the complex of 2 with urea (Supplementary Material).
To understand the sensing property of receptors 1 and 2,
fluorescence titrations by dilution method were performed
in the presence and absence of urea. It is evident from
Fig. 12 (a) UV spectra of the
complex 1. urea
0.8
(a)
0.4
(b)
(c = 8.66 9 10-6 M) and its
change of absorbance on
0.6
0.3
0.2
0.1
0.0
dilution; (b) plot of absorbance
versus concentration of the 1:1
complex of urea with 1
0.4
0.2
0.0
2x10-6
4x10-6
6x10-6
8x10-6
1x10-5
0
300
320
340
360
Wavelength (nm)
[Complex] in M
123

J Incl Phenom Macrocycl Chem (2010) 67:271–280
279
Fig. 13 (a) Emission spectra of
(a)
(b)
600
1 (c = 8.66 9 10-6 M) and its
1:1 complexes with urea and
thiourea; (b) Emission spectra
1:1 complex with urea
Receptor 2
160
500
of 2 (c = 1.26 9 10-5 M) and
its 1:1 complexes with urea and
thiourea
120
80
40
0
1:1 complex with thiourea
400
1:1 complex with urea
Receptor 1
300
200
100
1:1 complex with thiourea
0
350
400
450
500
550
600
350
400
450
500
550
600
Wavelength (nm)
Wavelength (nm)
Fig. 14 (a) Change in emission
of 1 (c = 8.66 9 10-6 M) in
CHCl3 in presence of
stoichiometric amounts of urea
and the dilution spectra of the
1:1 complex (kex = 320 nm);
(b) plot of fluorescence intensity
versus concentration of the 1:1
complex of urea with 1
600
500
400
300
200
100
0
(a)
(b)
600
500
400
300
200
100
0
2x10-6
4x10-6
6x10-6
8x10-6
1x10-5
350
400
450
500
550
600
0
[Complex]
Wavelength (nm)
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123