First artificial acidic fluorescent receptors for caffeine and other xanthine alkaloids

Article abstract of DOI:10.1007/s10847-009-9680-0

Recognition of three xanthine alkaloids caffeine, theobromine and theophylline with acidic fluorescent receptors has been achieved for the first time through the host-guest interaction. The designed receptors are based on the choice of different fluorophores as spacers which can accommodate the bicyclic xanthine guests in between the binding phenolic hydroxyl or carboxyl groups. Among all the xanthine alkaloids, caffeine being the strongest base, its binding constants were maximum though all the nitrogens of caffeine are methylated. Photochemically the trans isomers were converted to the cis isomers which generated cavities for better binding of the xanthine alkaloids. Receptor 4-{4-[4′-(3-Carboxypropoxy)phynylazo]phenoxy}butyric acid (5) has been shown to be most effective in enhancement of fluorescence intensity with caffeine complexation. The binding properties have been studied by a combination of experimental techniques such as UV-visible and fluorescence spectroscopy. Also solid state binding performance between receptor 4,4′-Diazenyldiphenol (4) and caffeine has been described.

Full text of DOI:10.1007/s10847-009-9680-0

J Incl Phenom Macrocycl Chem (2010) 67:99–108
DOI 10.1007/s10847-009-9680-0
ORIGINAL ARTICLE
First artificial acidic fluorescent receptors for caffeine and other
xanthine alkaloids
•
Ajit Kumar Mahapatra Prithidipa Sahoo
•
•
Shyamaprosad Goswami Hoong-Kun Fun
•
Chin Sing Yeap
Received: 5 May 2009 / Accepted: 9 September 2009 / Published online: 2 October 2009
Ó Springer Science+Business Media B.V. 2009
Abstract Recognition of three xanthine alkaloids caffeine,
theobromine and theophylline with acidic fluorescent
receptors has been achieved for the first time through the
host–guest interaction. The designed receptors are based on
the choice of different fluorophores as spacers which can
accommodate the bicyclic xanthine guests in between the
binding phenolic hydroxyl or carboxyl groups. Among all
the xanthine alkaloids, caffeine being the strongest base, its
binding constants were maximum though all the nitrogens of
caffeine are methylated. Photochemically the trans isomers
were converted to the cis isomers which generated cavities
for better binding of the xanthine alkaloids. Receptor 4-{4-
[40-(3-Carboxypropoxy)phynylazo]phenoxy}butyric acid (5)
has been shown to be most effective in enhancement of fluo-
rescence intensity with caffeine complexation. The binding
properties have been studied by a combination of experi-
mental techniques such as UV-visible and fluorescence
spectroscopy. Also solid state binding performance between
receptor 4,40-Diazenyldiphenol (4) and caffeine has been
described.
Introduction
Xanthine alkaloids are a class of alkylated oxopurines such
as caffeine (1), theobromine (2) and theophylline (3). These
are frequently consumed alkaloids [1]. Among these caffeine
is one of the most consumed alkaloidal drug [2, 3]. These are
natural constituent of tea, coffee, guarana paste, cola nuts and
cocoa beans etc. These are also added to many popular soft
drinks and these alkylated oxopurines are also widely used in
the counter medications including analgesic, bronchodilator,
diet aids, cold/flu remedies etc. Natural compounds like
catechin [4], theaflavin [5], cyclodextrin [6, 7], potassium
chlorogenate [8, 9] etc. are capable of binding caffeine or
theobromine through hydrophobic interactions. Caffeine
shows heterogeneous association with different dicarboxylic
acids [10–13], hydroxy acid [14] and simple heterogeneous
stacks with methyl gallate [15, 16], in their solid state.
Recently we [17, 18] and several other research groups
[19, 20], accomplished the synthesis of artificial alkylated
xanthine receptors. It has been recently described a novel
simple fluorescence receptor for caffeine by Waldvogel and
coworkers [21]. They used scaffolds of triphenyleneketals
based on amidic type H-bonding receptors for caffeine. It is
also known that the interaction of caffeine has been studied
with polyphenols which are present in the preparation of
black tea, coffee [22, 23] and tea creaming. In aqueous
solutions, the interaction of caffeine has also been studied
with zinc based porphyrin peptide type receptor [24].
Keywords Fluorescence sensors ꢀ Caffeine ꢀ
Theobromine ꢀ Theophylline ꢀ UV–vis titration ꢀ
Co-crystal
For the determination of caffeine, standard chromato-
graphic methods are available such as (HP)TLC, HPLC-UV
and CE2. Howeverall the above methods require a longperiod
of time, so methods requiring shorter time for analysis is
needed. A very few spectroscopic [25], approaches and the
applications of molecular imprinted polymers (MIPS) occur
in combination with quartz microbalance [26]. However these
A. K. Mahapatra (&) ꢀ P. Sahoo ꢀ S. Goswami
Department of Chemistry, Bengal Engineering and Science
University, Shibpur 711103, India
e-mail: mahapatra574@gmail.com
H.-K. Fun ꢀ C. S. Yeap
X-ray Crystallography Unit, School of Physics, Universiti Sains
Malaysia, 11800 Penang, Malaysia
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J Incl Phenom Macrocycl Chem (2010) 67:99–108
types of sensors also required a long time and large concen-
tration of analyte (caffeine). As such a promising approach is
to use chemo-sensors, which represent a vivid field in supra-
molecular chemistry. This problem has been tackled by
supramolecular chemists by devising several tailor-made
chemo-receptors [27] which allow a design based interaction
to occur in solution at very low concentration in the order of
10-5 (M). Various synthetic receptors have recently been
discovered for caffeine [17–20], theobromine [18, 28, 29] and
theophylline [30]. The most of the chemo-sensors are ana-
lytically useful in response to the binding event [31].The field
of chemical sensor is therefore very attractive due to its easier
applications and measurements by spectroscopical means.
Fluorometric methods are extremely sensitive to appropri-
ately designed systems. Most of the fluorescent chemo-sen-
sors are based on hydrogen bonding phenomenon [21]. We
have recently described a fluorescence sensing property of
theobromine by simply using 2,6-diaminopyridine [18].
However, there is no such systematic study yet on detection of
very minute quantity of caffeine, theobromine and theophyl-
line in beverages and foods. It is, therefore, important and of
major interest in sensor and/or separation technology to apply
the concept of directmolecular interactionsbetween alkylated
xanthines and synthetic receptors.
Remarkably the binding sensitivity of these receptors
towards caffeine is more than towards theobromine and
theophylline using our previous work amide based receptors
[17]. Our designed receptors for xanthine alkaloidal moiety
combines the acidic part of the receptors which favours the
H-bonding, with the ring oxygen and most basic imidazole
‘N’ of the guests. Due to the presence of azo sensing systems
(4 and 5), receptors form complexes with xanthine deriva-
tives through cis–trans isomerisation process.
Experimental section
General comments
All the chemicals were reagent grade and were used as
1
supplied. The H NMR spectra were recorded on Bruker-
AM-200 and AM-500 spectrometers. The 1H NMR
chemical shift values are expressed in ppm (d) relative to
CHCl3 (d = 7.26 ppm). UV–visible and fluorescence
spectra measurements were performed on a JASCO V530
and a PerkinElmer LS-55 spectrofluorimeter respectively.
The receptors and guests were dissolved in dry UV
grade acetonitrile and only for the guest theobromine
stopper volumetric flask was sonicated for half an hour.
The corresponding absorbance values during titration were
noted and used for the determination of binding constant
values. Binding constant were determined by the using
expression A0/A - A0 = [eM/(eM - eC)](Ka-1 Cg-1 ?1),
where eM and eC are molar extinction co-efficients for
receptor and the hydrogen bonding complex respectively at
selected wavelengths, A0 denotes the absorbance of the
free receptors at the specific wavelength and Cg is the
concentration of methyl xanthine guests. The measured
absorbance A0/A - A0 as a function of the inverse of the
methyl xanthine guest concentration fits a linear relation-
ship, indicating a 1:1 complexation of the receptor and
methyl xanthine. The ratio of the intercepts to the slope
was used to determine the binding constant Ka.
We now report a series of novel azo, naphthyridine and
naphthalene based receptors 4–7 (Scheme 1) which easily
bind to caffeine, theobromine and theophylline (Scheme 2).
N
N
N N
O
O
HO
OH
O
O
HO
OH
Receptor 4
Receptor 5
O
O
O
N
N
O
O
O
O
O
OH
OH
HO
OH
Receptor 6
Receptor 7
Synthesis of receptors 4–7
Scheme 1 Fluorescence receptors 4–7
Our designs are based on the structural features of xanthine
alkaloids. Caffeine, theobromine and theophylline are dif-
ficult recognition targets as their nitrogen’s are fully or
partly methylated causing N-CH3 moiety a poor recogni-
tion site for binding. Theobromine is also a difficult rec-
ognition substrate for its poor solubility in chloroform as
well as in methanol.
O
O
N
O
N
CH3
CH3
H
H3C
N
H3C
O
N
N
N
N
HN
N
N
O
N
N
O
CH3
CH3
CH3
1
2
3
The most important task to design artificial receptors for
recognition of this type of bigger substrates is to place the
hydrogen bonding groups (donors and acceptors) in an
Scheme 2 Xanthine alkaloidal guests: caffeine (1), theobromine (2)
and theophylline (3)
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J Incl Phenom Macrocycl Chem (2010) 67:99–108
101
appropriate big cavity where the particular binuclear guest
can make maximum number of hydrogen bonds with the
host leading to a stronger complexation.
extracted by chloroform. The organic layer was separated
and dried over Na2SO4. The crude products were purified by
doing column chromatography in chloroform solvent. The
orange yellow powder was obtained (yield 80%); Mp.
102 °C; 1H NMR (CDCl3, 200 MHz): d (ppm): 7.87 (d, 4H,
J = 6 Hz), 6.98 (d, 4H, J = 6 Hz), 4.16 (t, 4H, J = 5 Hz),
4.09 (qt, 4H, J = 2.0 Hz), 2.53 (t, 4H, J = 5 Hz), 2.14
(qt, 4H, J = 4 Hz), 1.26 (t, 6H, J = 5 Hz). C24O6H30N2
(442.504), Anal. Calcd. C 65.142, H 6.833, O 21.694,
N 6.331; found C 65.132, H 6.833, O 21.689, N 6.337.
The receptors 4, 5, 6 and 7 were synthesized according
to the Schemes 3, 4, 5. Azo-diol 4 was prepared following
the known literature procedures [32]. O-alkylation of 4 and
14 were synthesized from ethyl-4-bromobutanoate in dry
acetone and K2CO3 mixture and subsequent hydrolysis of
the resultant product afforded the corresponding acidic
receptors 5 and 7 [33, 34]. Receptor 6 was prepared by the
reaction between 2,7-dihydroxy naphthyridine [35, 36] and
ethyl-4-bromobutyrate in the presence of K2CO3-dry ace-
tone at refluxing condition and the resulting product was
hydrolysed accordingly to give receptor 6. All the syn-
thesized compounds (4–7) were characterized structurally
4-{4-[4-(3-Carboxypropoxy)phynylazo]phenoxy}
butyric acid (5)
The diester was dissolved in a mixture of 10% NaOH
solution and EtOH (1:1) under reflux for 24 h. Then it is
acidified with conc. HCl in ice. A bright yellow ppt which
was filtered and recrystallised from acetone to give the final
product. (yield 60%); Mp. [ 250 °C; 1H NMR (DMSO-d6,
500 MHz): d (ppm): 11.07 (s, 2H), 7.69 (d, 4H, J = 6 Hz),
6.96 (d, 4H, J = 10 Hz), 4.04 (t, 4H, J = 7.5 Hz), 2.39
(qt, 4H, J = 9.0 Hz), 2.01 (t, 4H, J = 8.5 Hz). C20O6H22N2
(386.399), Anal. Calcd. C 62.167, H 5.738, O 24.844,
N 7.249; found C 62.159, H 5.441, O 28.721, N 8.366. TOF
MS: m/z: [M-2CO2]?: 301.2697.
1
using H NMR, mass, analysis, IR and UV-spectroscopic
method (supporting information).
4,40-Diazenyldiphenol (4)
A mixture of KOH (50 g, 760 mmol), p-nitrophenol (10 g,
72 mmol) and water (10 mL) was heated 120 °C for 1 h.
Then temperature is allowed to rise to 195–200 °C for
vigorous reaction. The large bubbles are produced. Then it
is dissolved into water. A dark red solution is produced. It
is acidified with pH 3 by conc. HCl and extracted with
ether. Then it is dried by Na2SO4 and recrystallised by 50%
EtOH (v/v) water. A brown crystalline product was
obtained. A co-crystal of X-ray quality of the receptor 4
with caffeine was obtained by slow evaporation from a 1:1
saturated solution of the compounds in 2:1 chloro-
form:methanol mixture solvent.
4-[8-(3-Carboethoxypropyl)-7-oxo-7,8-dihydro-
[1, 8]naphthyridine-2-yloxy]butyrate (14)
Solid K2CO3 was ground smoothly. 2,7-dihydroxy-1,8-
naphthyridine (2.0 g, 12.3 mmol), K2CO3 (3 g, 21 mmol)
ethyl 4-bromo butanoate (4.0 mL, 25 mmol) in acetone
(100 mL) was refluxed overnight under an inert atmosphere.
The reaction was evaporated to dryness, dissolved in water
and extracted with dichloromethane. The organic layer was
separated and dried over Na2SO4. The diester was isolated.
(yield 80%); Mp. 98 °C; 1H NMR (CDCl3, 200 MHz):
d (ppm): 7.71 (d, 1H, J = 8 Hz), 7.55 (d, 1H, J = 9 Hz),
6.59 (d, 1H, J = 8 Hz), 6.55 (d, 1H, J = 9 Hz), 4.51 (q, 4H,
J = 5 Hz), 4.48 (t, 2H, J = 4 Hz), 4.14 (t, 2H, J = 3 Hz),
4-{4-[4-(3-Carboethoxypropoxy)phynylazo]
phenoxy}butyrate (10)
The azodiol (4) (2 g, 9.34 mmol), K2CO3 (3 g, 21 mmol)
and ethyl 4-bromo butanoate (4.0 mL, 25 mmol) in acetone
was refluxed overnight under an inert atmosphere. The
reaction was evaporated to dryness, dissolved in water and
Scheme 3 Synthesis of
receptors 4 and 5
N N
OH
ethyl-4-bromo-
butanoate,
N N
O
O
solid NaOH
acetone,
K2CO3, reflux
150°C, 2 h
HO
OH
O
O
NO2
EtO
OEt
Receptor 4
8
9
NaOH(10%)
& EtOH 1:1
Receptor 5
60°C, 24h,
H3O+
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Scheme 4 Synthesis of
receptor 6
conc. H2SO4
conc. H2SO4
Malic acid
110°C, 2-3 h
H2N
N
NH2
ice, NaNO2
HO
N
N
OH
H2N
N
N
OH
11
10
12
NaOH(10%)
& EtOH 1:1
O
N
N
O
ethyl-4-bromobutanoate,
acetone,
Receptor 6
O
O
60°C, 24 h,
H3O+
K CO ,
DMF
3
2
OEt
OEt
reflux
13
Scheme 5 Synthesis of
receptor 7
ethyl-4-bromo-
butanoate,
acetone,
OH
O
NaOH(10%)
O
& EtOH 1:1
Receptor 7
HO
K CO ,
60°C, 24h,
H3O+
DMF
3
2
O
O
reflux
EtO
OEt
14
15
2.48 (t, 2H, J = 3 Hz), 2.42 (t, 2H, J = 7 Hz), 2.18 (qt, 4H,
J = 7 Hz), 1.24 (t, 6H, J = 4 Hz). C20O6H26N2 (390.430),
Anal. Calcd. C 61.525, H 6.712, O 24.587, N 7.175; found
C 61.532, H 6.721, O 24.581, N 7.181.
4.06 (t, 4H, J = 5.5 Hz), 2.39 (t, 4H, J = 7 Hz), 1.86–1.83
(m, 4H), 1.53 (t, 6H, J = 7 Hz), C22O6H28 (388.45), Anal.
Calcd. C 68.022, H 7.265, O 24.712; found C 68.013,
H 7.254, O 24.742.
4-[8-(3-Carboxypropyl)-7-oxo-7,8-dihydro-
2,7-Bis[[4-(carboxyl)propyl]oxo]naphthalene (7)
[1, 8]naphthyridine-2-yloxy]butyric acid (6)
The diester was dissolved in a mixture of 10% aqueous
NaOH solution and EtOH under reflux for 24 h. The
reaction mixture was evaporated and it was acidified with
conc. HCl into ice. The light yellow ppt was collected.
(yield 90%); Mp. 182 °C; 1H NMR (DMSO-d6, 400 MHz):
d (ppm): 12.48 (s, 2H), 7.63 (d, 2H, J = 8 Hz), 7.02–6.89
(m, 4H), 4.06 (t, 4H, J = 5.5 Hz), 2.39 (t, 4H, J = 7 Hz),
1.86–1.83 (m, 4H), C18O6H20 (332.347), Anal. Calcd.
C 65.050, H 6.065, O 28.884; found C 65.048, H 6.071,
O 28.877. TOF MS: m/z: [M ? Na]?: 355.1156.
The diester was dissolved in a mixture of 10% aqueous
NaOH solution and EtOH under reflux for 24 h. The reaction
mixture was evaporated and it was acidified with conc. HCl
into ice. The light yellow ppt was collected. (yield 90%);
1
Mp. [ 250 °C; H NMR (DMSO-d6, 400 MHz): d (ppm):
12.08 (s, 2H), 8.04(d, 1H, J = 8 Hz), 7.85 (d, 1H, J = 9 Hz),
6.70 (d, 1H, J = 8 Hz), 6.48 (d, 1H, J = 9 Hz), 4.39 (t, 2H,
J = 6 Hz), 4.37 (t, 2H, J = 7 Hz), 2.39 (q, 2H, J = 7 Hz), 2.27
(q, 2H, J = 7 Hz), 1.99 (t, 2H, J = 6.8 Hz), 1.89 (t, 2H,
J = 7.1 Hz). C16O6H18N2 (334.324), Anal. Calcd. C 57.481,
H 5.427, O 28.714, N 8.379; found C 57.488, H 5.421, O
28.717, N 8.372. . TOF MS: m/z: [M ? Na]?: 358.1112.
Results and discussion
2,7-Bis[[4-(ethoxycarbonyl)propyl]oxo]naphthalene (16)
Interaction studies
A solution of 2,7-naphthalenediol (2.0 g, 12.5 mmol), solid
K2CO3 (3.6 g, 26 mmol), ethyl 4-bromo butanoate
(4.0 mL, 25 mmol) in acetone (100 mL) was refluxed
overnight under an inert atmosphere. The reaction was
evaporated to dryness, dissolved in water and extracted
with DCM. The organic layer was separated and dried over
Na2SO4. The diester was isolated. (yield 80%); Mp. 80 °C;
1H NMR (CDCl3, 200 MHz): d (ppm): 7.63 (d, 2H,
J = 8 Hz), 7.02–6.89 (m, 4H), 4.52 (q, 4H, J = 7 Hz),
The photophysical properties of 4, 5, 6 and 7 were
evaluated in acetonitrile solution (for theobromine 0.8%
DMSO containing acetonitrile was used). Here our designed
receptors are all acidic in nature and the guest xanthine
alkaloids are basic components due to the presence of
N-CH3 and imidazole nitrogen. Moreover it is reported that
caffeine extraction from tea leaves can be done by using
benzoic acid type acidic substrate. All three compounds
(except 4) reveal the same arrangement of hydrogen
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J Incl Phenom Macrocycl Chem (2010) 67:99–108
103
bonding acceptors that is complementary to that of the
binding site.
solution of receptor 4 results in a bathochromic shift of the
emission bands Fig. 1a by 6 nm and an increased signal
intensity, from 205 to 211 nm. A clear isobestic point was
observed at 297 nm.
To investigate the sensitivity of photo-physical property
of these receptors, we carried out both the UV and fluo-
rescence titrations at very low concentration to effectively
sense caffeine, theobromine and theophylline. All methyl
xanthines do not absorb light at wavelength greater than
310 nm, whereas azo, naphthyridine and naphthalene based
receptors show significant absorption in the range up to
347–400 nm respectively.
This result demonstrates that a complex formation of 4
with caffeine is taking place via hydrogen bonding. The
same phenomenon is observed in the case of cis isomer of
receptor 4 by similar experiment where only difference is
noted that no shoulder like peak occurs and sharp peak
appear at 273 nm (Fig. 1c) with the formation of clear
isobestic point at 300 nm. The formation of hydrogen
bonded weak interactions with different fluoro-acidic
groups, results stronger binding with basic xanthines.
Similar type of experiments were done with other guests 2
and 3 by UV–vis method with receptors 4 and 5 and their
results are shown in the supporting information.
UV-visible spectroscopic studies
The colorimetric sensing ability of the receptors 4–7 with
methyl xanthine alkaloids such as caffeine (1), theobro-
mine (2) and theophylline (3) in acetonitrile was monitored
by UV–vis absorption [37]. The solutions of guests (1–3)
(4.639 9 10-4 M) were respectively added to the solutions
of the receptors 4–7 (3 9 10-5 M). Figure 1a shows that
UV–vis absorption spectra of a mixture of receptor 4 (trans
form) with different concentrations of caffeine in acetoni-
trile. Addition of caffeine to a solution of receptor 4 results
a new absorption band at 272 nm which is gradually
enhanced, while the intensity of absorption at 211 nm
slowly gets decreased by the formation of shoulder like
peak which appear at 230 nm due to formation of new
complex at higher concentration. Addition of caffeine to a
Parallel investigations were carried out with a series
same methyl xanthine alkaloids (1–3) with receptors 6 and
7. A similar phenomenon of UV–vis absorptions was
observed in Figs. 2a and 3a. In all the cases increasing
concentration of guest, develops a new absorption band
which gradually gets enhanced and exhibits sharp upward
perturbation Fig. 4a. By contrast, most of the cases sig-
nificant changes are observed in the presence of caffeine,
so it is apparent that caffeine has more selectivity than
other xanthine guests. The selectivity of caffeine can be
rationalized on the basis of association constants (Table 1).
Fig. 1 a UV spectral change of
trans-form of receptor 4 in
CH3CN (c = 3.271 9 10-5 M)
upon gradual addition of
caffeine (c = 4.639 9 10-4 M)
dissolved in CH3CN. b Binding
constant calculation curves for
receptor 4 versus guests. c UV
spectral change of cis-form of
receptor 4
2.5
2.0
1.5
1.0
0.5
0.0
380
360
Receptor 4
with
(a)
340
(b)
320
Caffeine
300
Theobromine
280
Theophylline
260
240
220
200
180
160
140
120
100
80
60
40
20
0
200
250
300
350
400
0
50 100 150 200 250 300 350 400 450
1/[G] x 103
Wave length, nm
2.0
1.6
1.2
0.8
0.4
(c)
0.0
200
250
300
350
400
450
wave length, nm
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0.50
0.45
0.40
0.35
0.30
0.25
0.20
0.15
0.10
0.05
0.00
(a)
(b)
25
20
15
10
5
Receptor 5
with
Caffeine
Theobromine
Theophylline
0
260
265
270
275
280
285
0
50
100
150
200
250
3
300
350
400
Wave length, nm
1/[G] x 10
Fig. 2 a UV spectral change of receptor 5 in CH3CN (c = 3.6269 9 10-5 M) upon gradual addition of caffeine (c = 4.639 9 10-4 M)
dissolved in CH3CN. b Binding constant calculation curves for receptor 5 versus guests
4500
4000
3500
3000
2500
2000
1500
1000
500
1.2
1.0
0.8
0.6
0.4
0.2
0.0
(b)
(a)
Receptor 6
with
Caffeine
Theobromine
Theophylline
0
20
220
240
260
280
300
0
1000 2000 3000 4000 5000 6000 7000
1/[G] x 103
Wave length, nm
Fig. 3 a UV spectral change of receptor 6 in CH3CN (c = 3.5928 9 10-5 M) upon gradual addition of caffeine (c = 4.639 9 10-4 M)
dissolved in CH3CN. b Binding constant calculation curves for receptor 6 versus guests
1300
1200
1100
1000
900
0.14
0.12
0.10
0.08
0.06
0.04
0.02
0.00
Receptor 7
vs
Caffeine
Theobromine
Theophylline
(b)
(a)
800
700
600
500
400
300
200
100
20 40
60
80 100 120 140 160 180 200 220
1/[G] x 103
300
310
320
330
340
Wavelength, nm
Fig. 4 a UV spectral change of receptor 7 in CH3CN (c = 3.4638 9 10-5 M) upon gradual addition of caffeine (c = 4.639 9 10-4 M)
dissolved in CH3CN. b Binding constant calculation curves for receptor 7 versus guests
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J Incl Phenom Macrocycl Chem (2010) 67:99–108
105
Table 1 Association constants of receptors 4, 5, 6 and 7 with xanthine alkaloids 1, 2 and 3 in CH3CN by UV–vis and fluorescence methods
Guests UV–vis method Fluorescence method
R4 (Ka M-1
)
R5 (Ka M-1
)
R6 (Ka M-1
)
R7 (Ka M-1
)
R4 (Ka M-1
)
R5 (Ka M-1
)
R6 (Ka M-1
)
R7 (Ka M-1
)
1
2
3
1.877 9 104
1.084 9 103
1.133 9 103
2.076 9 104
1.330 9 104
3.268 9 103
6.586 9 105
1.419 9 105
1.120 9 105
1.184 9 104
6.97 9 103
1.818 9 103
1.451 9 105
2.513 9 104
1.026 9 104
1.024 9 107
1.004 9 106
5.008 9 106
3.236 9 107
1.471 9 106
1.056 9 106
4.192 9 105
6.250 9 104
5.340 9 104
All the errors are 8%
For guests 1 and 3 dry CH3CN were used for all experiments
For guest 2 dry CH3CN containing 0.8% DMSO was used for experiments
Fluorescence study
slow increase of fluorescence intensity at different kmax is
observed according to their fluorophores (see supplemen-
tary data). The stronger fluorescence response was
observed for caffeine for all the receptors, especially for
the receptors 5 and 6. In case of receptor 5, large increase
of fluorescent intensity was observed. On the basis of
the change in fluorescent intensity, the association con-
stants (Ka) were determined and are shown in Table 1. The
values demonstrate that the association constants for caf-
feine are more among all of the studied guests. As caffeine
is more basic than other two methyl xanthine alkaloids
(2 and 3), it forms stronger complexes with the designed
receptors (Fig. 8).
Fluorescence spectroscopy studies were also carried out in
order to evaluate the sensitivity or ascertain their excited
state properties of receptors 4–7 to operate as fluorescent
sensors for methylxanthines (1–3) (Figs. 5, 6, 7). The
receptor 4 (3.271 9 10-5 M) when excited at 401 nm in
acetonitrile produces large enhancements in the monomer
emission bands at 398 nm on gradual addition of caffeine
and much smaller changes occur in the corresponding
excimer band at 417 nm (Fig. 5a).
The similar changes are observed in the fluorescence
spectra of the receptor 5 (3.271 9 10-5 M) in acetonitrile
on adding up caffeine which are depicted in Fig. 6a. The
large increase in fluorescent intensity was observed.
Spectra (a) in Fig. 6a was measured in the absence of
caffeine where receptor 5 shows fluorescence spectrum
with kmax = 543 nm. As shown in spectra (o)–(q), 5 exhibits
high perturbations upon addition of caffeine. Analogous
investigations were carried out with the receptors 6 and 7
by adding caffeine in the titration experiments in the
fluorescence spectroscopy (Fig. 7a, b).
Crystallographic study of receptor 4 with caffeine
The crystal structure analysis reveals that an asymmetric
unit of co-crystal 4 contains a minimum one molecule of
caffeine and one molecule of azo-diol. The whole azo-diol
molecule is disordered over two position with a site
occupancy ratio of 0.638(5):0.362(5). The complex is
neutral as determined from X-ray structure analysis.
Co-crystal of 4 with caffeine crystallizes in the mono-
clinic P21/c space group. The caffeine and azo-diol
For other guests 2 and 3, parallel investigations were
carried out with a series of receptor 4–7. In all the cases
(b)
500
Receptor 4
with
Caffeine
Theobromine
(a)
Receptor 4 + caffeine
Receptor 4
400
10
Theophylline
300
200
100
0
5
0
50 100 150 200 250 300 350 400 450
380
400
420
440
460
480
500
520
Wavelength, nm
1 / [G] x 103
Fig. 5 a Fluorescence change of receptor 4 (c = 3.271 9 10-5 M) upon gradual addition of caffeine dissolved in CH3CN (kmax = 398 nm).
b Binding constant calculation curves for receptor 4 versus guests
123

106
J Incl Phenom Macrocycl Chem (2010) 67:99–108
500
400
300
200
100
0
q
(b)
1.05
1.04
1.03
1.02
1.01
1.00
(a)
p
o
Receptor 5
with
Theophylline
Caffeine
Theobromine
m
k
i
g
e
c
a
520
540
560
580
0
50
100
150
1/[G] x 103
200
250 300
350
400
Wavelength, nm
Fig. 6 a Fluorescence change of receptor 5 (c = 3.271 9 10-5 M) upon gradual addition of caffeine dissolved in CH3CN (kmax = 548 nm).
b Binding constant calculation curves for receptor 5 versus guests
600
600
500
400
300
200
100
(a)
(b)
500
400
300
200
100
0
360
400
440
480
320
360
400
440
Wavelength, nm
Wavelength, nm
Fig. 7 a Fluorescence change of receptor 6 (c = 3.5928 9 10-5 M) and b receptor 7 (c = 3.4638 9 10-5 M) upon gradual addition of caffeine
dissolved in CH3CN (kmax = 335 and 345 nm) respectively
and another hydroxyl group is linked with the oxygen of
another molecule of caffeine. It is normal that the basic N
of imidazole part of caffeine makes hydrogen bonds with
acidic phenolic hydrogen. Selected hydrogen bond
parameters are shown in Table 2. The acid–base pairs
interact in parallel via weak van der Waals forces to form
stacks. Co-crystal of receptor 4 with caffeine forms a wave-
like polymeric chain when viewed along c axis. The
another presentation is shown in Fig. 9c, which resembles
an ivy climber plant curling up a pole.
Crystal data are as follows (CCDC No. 726709):
C20H20N6O4, Mr = 408.42, 100 K, Monoclinic, P21/c, a =
˚
˚
˚
Fig. 8 The molecular structure (ORTEP diagram) of receptor 4 and
caffeine
10.1958(2) A, b = 7.8938(2) A, c = 25.4275(5) A, a =
90.00°, b = 111.0550(10)°, c = 90.00°, V = 1909.86(7)
A , Z = 4, l(Mo Ka) = 0.103 cm-1, Dc = 1.420 g cm-3
,
3
˚
component form a two component assembly involving an
intermolecular O–HꢀꢀꢀO and O–HꢀꢀꢀN hydrogen bonds. In
azo-diol, one hydroxyl group binds with the 5-membered
imidazole nitrogen (most basic) of one caffeine molecule
F(000) = 856, range of indices = -14,14; -9,11; -36,27,
no. of reflections collected = 24211, Unique reflections =
5892, Rint = 0.033, Reflections with I [ 2r(I) = 4,013,
No. of parameters = 423, R(F2), F [ 2r(F2) = 0.0461,
123

J Incl Phenom Macrocycl Chem (2010) 67:99–108
107
Table 2 Selected hydrogen bond parameters of co-crystal of caffeine with receptor 4
˚
d(D-H)/A
˚
˚
D-HꢀꢀꢀA
d(HꢀꢀꢀA)/A
d(DꢀꢀꢀA)/A
h(D-HꢀꢀꢀA)/deg
O1A-H1AAꢀꢀꢀN5a
O2A-H2AAꢀꢀꢀO3b
C12A-H12AꢀꢀꢀO3c
C17-H17AꢀꢀꢀO2Ad
C18-H18BꢀꢀꢀO1Ae
C18-H18BꢀꢀꢀN5
0.8200
0.8200
0.9300
0.9300
0.9600
0.9600
0.9600
0.9600
1.9800
1.8900
2.4700
2.4900
2.4900
2.5500
2.6200
2.3300
2.772(5)
2.654(12)
3.289(4)
162.00
155.00
147.00
111.00
166.00
107.00
160.00
106.00
2.958(11)
3.427(5)
2.9772(15)
3.5400(15)
2.747(17)
C18-H18CꢀꢀꢀN5d
C19-H19CꢀꢀꢀO4
a
Symmetry codes: x - 1, y, z; x, -y ? 3/2, z - 1/2; –x ? 1, -y ? 1, -z ? 2; –x ? 1, y ? 1/2, -z ? 3/2; x ? 1, y, z
b
c
d
e
Fig. 9 Illustration of the
co-crystal structure of receptor
4 (red) with caffeine (blue)
a proposed H-bonding structure
b 1D polymeric wave-like chain
viewed down the
O
N
N
N
N
O
crystallographic c axis.
HO
c supramolecular network
which resembles an ivy climber
plant curling up a pole
O
N
N N
N
N
N
(a)
O
OH
(c)
(b)
wR(F2), F [ 2r(F2) = 0.1104, R(F2), all data = 0.0771,
wR(F2), all data = 0.1232.
medicinally and technically highly relevant analytes.
Together with the results from the X-ray crystallography, a
consistent picture of binding of the receptors with caffeine
is presented.
Conclusion
Acknowledgments This work is supported by the UGC [F. No. 34-
340/2008(SR)], New Delhi, India. S.G. thanks CSIR and DST,
Government for financial supports. We appreciate the help of IACS,
Jadavpur, Kolkata for NMR and mass spectra and also for elemental
analysis. We would also like to thank the Malaysian Government and
Universiti Sains Malaysia.
In summary, we have prepared the acidic receptors 4, 5, 6
and 7 based on different fluorescence probes. The simple
hydrogen bonding acidic fluoro-receptors formed stronger
complexes with methylxanthines. During the course of the
investigations, it was found that the binding of caffeine with
acidic receptors is stronger than that for theobromine and
theophylline. The more basic nature of the caffeine among
the other xanthines studied suggest stronger association
constants with the acidic receptors. Here in fact, caffeine
guest leads to a signal increase by a factor up to 10 and
produces a reliable and fast identification method of the
caffeine content in beverages. However, this system rep-
resents a powerful chemosensor method for the detection of
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