Role of spacer in single- or two-step FRET: Studies in the presence of two connected cryptands with properly chosen fluorophores

Article abstract of DOI:10.1039/c000681e

Two cryptand molecules are connected viap-xyloyl, benzene-1,4-dicarbolyol and 9,10-dimethylene anthracene. Each cryptand is further derivatized with fluorophores such that electronic absorption of one fluorophore overlaps emission of the other. This way, three different systems L₁, L ₂ and L₃ have been synthesized to get a better view of the effect on the distance in single- and two-step fluorescence resonance energy transfer (FRET) process. These molecules are probed for FRET in the presence of a metal ion as input. L₁ and L₂ exhibit poor performance in single step FRET while in the case of L₃, a large two-step FRET process is operational with Cu(ii) or Hg(ii) as input.

Full text of DOI:10.1039/c000681e

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Role of spacer in single- or two-step FRET: studies in the presence of two
connected cryptands with properly chosen fluorophores†
Kalyan K. Sadhu,a Soumit Chatterjee,b Susan Sena and Parimal K. Bharadwaj*a
Received 12th January 2010, Accepted 18th February 2010
First published as an Advance Article on the web 22nd March 2010
DOI: 10.1039/c000681e
Two cryptand molecules are connected via p-xyloyl, benzene-1,4-dicarbolyol and 9,10-dimethylene
anthracene. Each cryptand is further derivatized with fluorophores such that electronic absorption of
one fluorophore overlaps emission of the other. This way, three different systems L1, L2 and L3 have
been synthesized to get a better view of the effect on the distance in single- and two-step fluorescence
resonance energy transfer (FRET) process. These molecules are probed for FRET in the presence of a
metal ion as input. L1 and L2 exhibit poor performance in single step FRET while in the case of L3, a
large two-step FRET process is operational with Cu(II) or Hg(II) as input.
such cryptands are used to design the overall system, the nature
of the spacer between the two plays a crucial role in single-
and two-step FRET. To check the role of the spacer in single-
and two-step FRET, we present here three different systems L1–
Introduction
The long range fluorescence resonance energy transfer (FRET)
takes place between chromophores of overlapping emission and
absorption bands as described by the Fo¨rster theory.1 It is an
L3 (Scheme 1). This study is also important from the perspec-
important tool capable of providing information on structure and
tive of getting valuable clues for photoinduced charge transfer
dynamics in macromolecules2 as well as self-assembled structures.3
Different configurational designs as well as techniques for single-
and two-step FRET with/without metal ions are covered in some
recent reviews.4 We have been using aza-oxa cryptands5 to study
processes.
In L1 and L2, two cryptand molecules are attached to an-
thracene and 7-nitrobenz-2-oxa-1,3-diazole (hereafter, diazole)
with different spacers. On the other hand, in L3 the two cryptands
are derivatized with bis-quinoline (donor) and bis-diazoles (ac-
FRET in the presence of transition/heavy metal ions. When two
ceptor) and connected through 9,10-dimethylanthracene spacer.
aChemistry Department, Indian Institute of Technology Kanpur, Kanpur,
208016, India. E-mail: pkb@iitk.ac.in
The excitation and emission peaks of quinoline, anthracene and
diazole are 316 and 330–400, 350 and 390–460, 475 nm and 480–
540 nm respectively. This light partitioning of the fluorophores
are the reason for choosing these fluorophores for the systems.
This phenomenon is better explained from the schematic energy
diagram (Fig. 1).
bChemistry Department, Indian Institute of Technology Bombay, Powai,
Mumbai, 400076, India
† Electronic supplementary information (ESI) available: Experimental sec-
tion involves characterization of the ligands L1–L3: Mass, 13C- NMR, 1H-
NMR, UV-vis and fluorescence spectral data. See DOI: 10.1039/c000681e
Scheme 1 Chemical structures of the compounds.
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Fig. 1 An energetic diagram showing the photoinduced processes occurring in L1, L2 and L3 (both in the presence and absence of metal ions).
1 ¥ Ar–H), 6.97 (d, 2H, 2 ¥ Ar–H), 7.07 (m, 4H, 4 ¥ Ar–H), 7.19
(m, 1H, 1 ¥ Ar–H), 8.05 (d, 2H, 2 ¥ Ar–H); 13C-NMR (125 MHz,
CDCl3, 25 ◦C, TMS, d): 157.08, 155.99, 155.53, 144.70, 144.43,
144.13, 144.01, 135.12, 134.71, 131.10, 129.20, 124.46, 122.94,
122.49, 121.40, 121.09, 112.09, 111.99, 67.65, 64.71, 54.48, 48.76,
33.92, 32.02, 31.52, 30.37, 29.32, 25.99, 22.79; ESI-MS (m/z): 884
(75%) [Ld–1]+. Anal. calcd. for C45H47N11O9: C, 61.01; H, 5.35; N,
17.39%. Found: C, 61.10; H, 5.49; N, 17.25%.
Experimental
Materials
All solvents and thionyl chloride were purified prior to use.
The purified solvents were found to be free from impurities,
moisture and were transparent in the region of interest. All other
reagent grade chemicals including metal salts were acquired from
Aldrich that were used as received. The metal salts were hydrated
as mentioned in the Aldrich catalogue. For chromatographic
separation, 100–200 mesh silica gel (Acme Synthetic Chemicals)
was used. The reactions were carried out under a N2 atmosphere
unless otherwise mentioned.
Synthesis of L1a. To a solution of Ld (0.45 g; 0.5 mmol)
in dry acetonitrile (20 ml), anhydrous K2CO3 (1.5 g, excess)
was added and stirred for 15 min. Freshly recrystallized 1,4-bis-
bromomethylbenzene (0.13 g, 0.5 mmol) was added to it along
with a crystal of KI and the reaction mixture was allowed to reflux
for 24 h. After cooling to RT, K2CO3 was removed by filtration.
The red filtrate was evaporated to dryness under reduced pressure,
washed several times with water and then extracted with CHCl3.
The organic layer, after drying over anhydrous Na2SO4, evaporated
to dryness to obtain a dark red solid as L1a.
Synthesis
Synthesis of L1. The synthetic route for the cryptand deriva-
tives is illustrated in Scheme 2.
Synthesis of Lo. Lo has been synthesized in good yield follow-
ing a procedure6 as reported earlier from our laboratory. The crude
product was recrystallized from acetonitrile as colorless crystals.
This sample was used for further derivatization.
Yield: 85(%); mp: 120 ◦C; 1H-NMR (400 MHz, CDCl3, 25 ◦C,
TMS, d): 3.03 (m, 12H, 6 ¥ CH2), 3.12 (m, 12H, 6 ¥ CH2), 4.25 (s,
6H, 3 ¥ CH2), 5.29 (s, 2H, CH2), 5.96 (s, 2H, CH2), 6.65 (m, 6H, 6 ¥
Ar–H), 6.80 (d, 2H, Ar–H), 6.98 (m, 3H, 3 ¥ Ar–H), 7.08 (m, 3H,
3 ¥ Ar–H), 7.24 (m, 2H, 2 ¥ Ar–H), 7.25 (m, 2H, 2 ¥ Ar–H), 7.45
(d, 2H, 2 ¥ Ar–H); 13C-NMR (100 MHz, CDCl3, 25 ◦C, TMS,
d): 149.38, 137.66, 131.2, 129.3, 127.15, 127.76, 125.43, 124.38,
123.14, 119.60, 57.12, 49.77, 47.09, 45.08; ESI-MS (m/z): 1069
(35%) [L1a]+. Anal. calcd. for C53H54BrN11O9: C, 59.55; H, 5.09; N,
14.41%. Found: C, 59.60; H, 5.04; N, 14.44%.
Synthesis of Ld. To a solution of Lo (0.56 g; 1 mmol) in dry ace-
tonitrile (20 ml), anhydrous K2CO3 (1.5 g, excess) was added and
stirred for 15 min. Freshly recrystallized 4-chloro-7-nitrobenz-2-
oxa-1,3-diazole (0.30 g, 1.5 mmol) was added to it and was allowed
to reflux for 72 h. After cooling to RT, K2CO3 was removed by
filtration. The red filtrate was evaporated to dryness under reduced
pressure, washed several times with water and then extracted with
CHCl3. The organic layer, after drying over anhydrous Na2SO4,
evaporated to dryness to obtain a red solid that was a mixture of
mono-, bis- and tris-derivatives. Compound Ld was subsequently
eluted out with chloroform : methanol (98 : 2 (v/v)) as the eluent.
The dark red solid obtained was recrystallized from acetone.
Yield: 45(%); mp: 130 ◦C; 1H-NMR (500 MHz, CDCl3, 25 ◦C,
TMS, d): 2.60-2.93 (m, 16H, 8 ¥ CH2), 2.98–3.11 (m, 8H, 4 ¥ CH2),
4.07 (s, 2H, 1 ¥ CH2), 4.24 (s, 4H, 2 ¥ CH2), 4.99 (br, 1H, NH),
6.64 (m, 4H, 4 ¥ Ar–H), 6.75 (m, 2H, 2 ¥ Ar–H), 6.91 (m, 1H,
Synthesis of D2. D2 has been synthesized following
a
procedure7 reported earlier from our laboratory.
Synthesis of L1. To a solution of L1a (0.54 g; 0.5 mmol) in dry
acetonitrile (20 ml), anhydrous K2CO3 (1.5 g, excess) was added
and stirred for 15 min. Freshly prepared D2 (0.47 g, 0.5 mmol) was
added to it along with a crystal of KI and the reaction mixture
was allowed to reflux for 24 h. After cooling to RT, K2CO3 was
removed by filtration. The red filtrate was evaporated to dryness
under reduced pressure, washed several times with water and
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Scheme 2 Synthetic route to L1. Reagent and conditions: (i) 4-Chloro-7-nitrobenz-2-oxa-1,3-diazole, Toluene, reflux (ii) 1,4-bisbromomethylbenzene,
MeCN, KI, reflux, 24 h, (iii) 9-bromomethylanthracene, MeCN, KI, reflux, 24 h and (iv) K2CO3, MeCN, KI, reflux,72 h.
then extracted with CHCl3. The organic layer, after drying over
anhydrous Na2SO4, was evaporated to dryness to obtain a dark
red solid as L1.
was added and the reaction mixture was allowed to stir at
room temperature for 6 h. The orange solid was filtered and
washed thoroughly with DCM. Excess methanol was added for
esterification of the acid chloride derivative. It was filtered and
purified by column chromatography. The ester derivative was
finally hydrolyzed to obtain a dark red acid derivative as L2a.
Yield: 84(%); mp: 214 ◦C; 1H-NMR (400 MHz, d6-DMSO,
25 ◦C, TMS, d): 1.91 (m, 12H, 6 ¥ CH2), 2.74 (m, 4H, 2 ¥ CH2),
2.85 (m, 4H, 2 ¥ CH2), 3.25 (s, 2H, CH2), 3.95 (m, 4H, 2 ¥ CH2),
4.15 (m, 4H, 2 ¥ CH2), 6.63 (m, 2H, 2 ¥ Ar–H), 6.84 (m, 3H, 3 ¥
Ar–H), 6.96 (m, 3H, 3 ¥ Ar–H), 7.16 (m, 3H, 3 ¥ Ar–H), 7.24 (m,
2H, 2 ¥ Ar–H), 7.35 (m, 3H, 3 ¥ Ar–H), 7.89 (m, 2H, 2 ¥ Ar–H),
8.04 (m, 2H, 2 ¥ Ar–H); ESI-MS (m/z): 1034 (40%) [L2a]+. Anal.
calcd. for C53H51N11O12: C, 61.56; H, 4.97; N, 14.90%. Found: C,
61.62; H, 5.01; N, 14.84%.
Yield: 52(%); mp: 136 ◦C; 1H-NMR (400 MHz, CDCl3, 25 ◦C,
TMS, d): 1.79 (m, 48H, 24 ¥ CH2), 2.01 (m, 4H, 2 ¥ CH2), 3.32
(m, 4H, 2 ¥ CH2), 5.92 (s, 12H, 6 ¥ CH2), 6.70 (m, 4H, 4 ¥ Ar–H),
7.46-7.53 (m, 16H, 16 ¥ Ar–H), 8.03 (m, 6H, 6 ¥ Ar–H), 8.19 (m,
6H, 6 ¥ Ar–H), 8.28 (m, 6H, 6 ¥ Ar–H), 8.50 (m, 6H, 6 ¥ Ar–H),
9.24 (m, 6H, 6 ¥ Ar–H); 13C-NMR (100 MHz, CDCl3, 25 ◦C,
TMS, d): 157.07, 155.99, 155.53, 144.69, 144.42, 144.13, 144.00,
135.12, 134.71, 131.10, 129.19, 124.45, 122.93, 122.49, 121.40,
121.08, 112.08, 111.98, 67.64, 66.19, 65.05, 64.71, 55.21, 54.48,
53.55, 48.76, 33.93, 32.02, 31.59, 30.38, 30.20, 29.60, 29.04, 28.66,
25.10, 22.79, 14.23; ESI-MS (m/z): 1928 (40% scan range 1400–
2000) [L1]+. Anal. calcd. for C116H118N16O12: C, 72.25; H, 6.17; N,
11.62%. Found: C, 72.22; H, 6.18; N, 11.60%.
Synthesis of L2. To a solution of L2a (0.52 g; 0.5 mmol) in
dry DCM (20 ml) thionyl chloride (0.06 g, 0.5 mmol) was added
and the reaction mixture was allowed to stir at room temperature
for 6 h. The solvent and excess thionyl chloride were removed
by evaporation under reduced pressure. The orange solid was
Synthesis of L2. The synthetic route for this compound is
illustrated in Scheme 3.
Synthesis of L2a. To a solution of Ld (0.44 g; 0.5 mmol) in
dry DCM (20 ml) terephthaloyl dichloride (0.11 g; 0.55 mmol)
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Scheme 3 Synthetic route to L2. Reagent and conditions: (i) terephthaloyl dichloride, DCM, RT, 24 h, water (ii) 9-bromomethylanthracene, MeCN, KI,
reflux, 24 h and (iii) thionyl chloride, DCM, RT, 72 h.
dissolved in dry DCM and to this solution one equivalent of D2
was added. The final mixture was stirred for 12 h and then filtered
and washed thoroughly with hot water. The product was obtained
as a dark orange solid as L2.
washed several times with water and then extracted with CHCl3.
The organic layer, after drying over anhydrous Na2SO4, evaporated
to dryness to obtain a dark red solid as L3a.
Yield: 85(%); mp: 118 ◦C; 1H-NMR (400 MHz, CDCl3, 25 ◦C,
TMS, d): 2.44 (s, 2H, CH2), 2.45 (s, 2H, CH2), 3.02 (m, 6H, 3 ¥
CH2), 3.31 (m, 12H, 6 ¥ CH2), 3.52 (m, 6H, 3 ¥ CH2), 4.63 (s,
6H, 3 ¥ CH2), 7.11 (m, 2H, 2 ¥ Ar–H), 7.41-7.48 (m, 12H, 12 ¥
Ar–H), 7.97-8.01 (m, 2H, 2 ¥ Ar–H), 8.09-8.13 (m, 2H, 2 ¥ Ar–
H), 8.36-8.45 (m, 4H, 4 ¥ Ar–H), 8.75-8.93 (m, 2H, 2 ¥ Ar–H);
13C-NMR (100 MHz, CDCl3, 25 ◦C, TMS, d): 207.00, 157.15,
156.77, 139.24, 132.17, 130.98, 130.08, 129.88, 129.00,128.43,
127.18, 125.35, 125.08, 120.87, 114.04, 111.19, 53.39, 33.78, 31.88,
31.60, 30.90, 29.65, 29.47, 29.33, 29.12, 28.91, 22.65, 14.09; FAB-
MS (m/z): 1124 (50%) [L3a]+. Anal. calcd. for C61H58ClN11O9: C,
65.15; H, 5.20; N, 13.70%. Found: C, 65.08; H, 5.22; N, 13.74%.
Yield: 35(%); mp: 207 ◦C; 1H-NMR (400 MHz, d6-DMSO,
25 ◦C, TMS, d): 1.67 (m, 48H, 24 ¥ CH2), 2.01 (m, 4H, 2 ¥ CH2),
3.11 (m, 4H, 2 ¥ CH2), 5.28–5.36 (m, 8H, 4 ¥ CH2), 6.92 (m, 3H,
3 ¥ Ar–H), 6.97 (m, 3H, 3 ¥ Ar–H), 7.47–7.54 (m, 24H, 24 ¥ Ar–
H), 7.99–8.05 (m, 10H, 6 ¥ Ar–H), 8.20–8.31 (m, 6H, 6 ¥ Ar–H),
8.52 (m, 4H, 4 ¥ Ar–H); FAB-MS (m/z): 1957 (50%) [L2]+. Anal.
calcd. for C116H114N16O14: C, 71.22; H, 5.87; N, 11.46%. Found: C,
71.26; H, 5.93; N, 11.39%.
Synthesis of L3. The synthetic route for this compound is
illustrated in Scheme 4.
Synthesis of L3a. To a solution of Ld (0.45 g; 0.5 mmol) in
dry acetonitrile (20 ml), anhydrous K2CO3 (1.5 g, excess) was
added and stirred for 15 min. Freshly recrystallized 9,10-bis-
chloromethylanthracene (0.14 g, 0.5 mmol) was added to it along
with a crystal of KI and the reaction mixture was allowed to reflux
for 24 h. After cooling to RT, K2CO3 was removed by filtration.
The red filtrate was evaporated to dryness under reduced pressure,
Synthesis of L3b. To a solution of Lo (0.28 g; 0.5 mmol)
in dry acetonitrile (20 ml), anhydrous K2CO3 (1.5 g, excess)
was added and stirred for 15 min. Freshly recrystallized 2-bis-
bromomethylquinoline (0.17 g, 0.75 mmol) was added to it along
with a crystal of KI and the reaction mixture was allowed to reflux
for 48 h. After cooling to RT, K2CO3 was removed by filtration.
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Scheme 4 Synthetic route to L3. Reagent and conditions: (i) 9,10-bischloromethylanthracene, MeCN, KI, reflux, 48 h, (ii) 2-bromomethylquinoline,
MeCN, KI, reflux, 24 h and (iii) K2CO3, MeCN, KI, reflux, 72 h.
The pale yellow filtrate was evaporated to dryness under reduced
pressure, washed several times with water and then extracted with
CHCl3. The organic layer, after drying over anhydrous Na2SO4,
evaporated to dryness to obtain a pale yellow solid as a mixture
of mono-, bis- and tris-derivatives. The product L3b was isolated
by column chromatography as reported earlier.7
Yield: 25(%); mp: 86 C; H-NMR (400 MHz, CDCl3, 25 C,
TMS, d): 2.16 (m, 24H, 12 ¥ CH2), 4.24 (s, 6H, 3 ¥ CH2), 4.81 (m,
4H, 2 ¥ CH2), 6.62 (m, 3H, 3 ¥ Ar–H), 6.83 (m, 4H, 4 ¥ Ar–H),
7.19 (m, 3H, 3 ¥ Ar–H), 7.35 (m, 3H, 3 ¥ Ar–H), 7.53 (m, 4H, 4 ¥
Ar–H), 7.86 (m, 3H, 3 ¥ Ar–H), 8.05 (m, 4H, 4 ¥ Ar–H); 13C-NMR
(100 MHz, CDCl3, 25 ◦C, TMS, d): 155.47, 144.42, 134.61, 123.09,
121.47, 112.09, 64.58, 32.02, 29.79, 29.45, 22.78, 14.22; ESI-MS
(m/z): 842 (20%) [L3b]+. Anal. calcd. for C53H59N7O3: C, 75.59; H,
7.06; N, 11.64%. Found: C, 75.50; H, 7.01; N, 11.75%.
under reduced pressure, washed several times with water and
then extracted with CHCl3. The organic layer, after drying over
anhydrous Na2SO4, evaporated to dryness to obtain a dark red
solid as L3.
Yield: 22(%); mp: 160 ◦C; 1H-NMR (400 MHz, CDCl3, 25 ◦C,
TMS, d): 1.92 (m, 48H, 24 ¥ CH2), 2.25 (m, 4H, 2 ¥ CH2), 3.11
(m, 4H, 2 ¥ CH2), 5.09 (m, 12H, 6 ¥ CH2), 7.27 (m, 6H, 6 ¥ Ar–
H), 7.44 (m, 12H, 12 ¥ Ar–H), 7.48 (m, 6H, 6 ¥ Ar–H), 7.91 (m,
6H, 6 ¥ Ar–H), 8.00 (m, 6H, 6 ¥ Ar–H), 8.24 (m, 3H, 3 ¥ Ar–
H), 8.45 (m, 3H, 3 ¥ Ar–H), 8.86 (m, 6H, 6 ¥ Ar–H); 13C-NMR
(100 MHz, CDCl3, 25 ◦C, TMS, d): 206.99, 139.23, 134.55, 134.08,
129.92, 127.52, 127.16, 114.02, 53.40, 33.77, 31.87, 30.89, 30.13,
29.64, 29.45, 29.31, 29.11, 28.89, 22.64, 14.09; FAB-MS (m/z):
1930 (50%) [L3]+. Anal. calcd. for C114H116N18O12: C, 70.93; H,
6.06; N, 13.06%. Found: C, 70.82; H, 6.12; N, 13.11%.
◦
◦
1
Synthesis of L3. To a solution of L3a (0.56 g; 0.5 mmol) in dry
acetonitrile (20 ml), anhydrous K2CO3 (1.5 g, excess) was added
and stirred for 15 min. To this mixture, L3b (0.42 g, 0.5 mmol)
was added along with a crystal of KI and the reaction mixture
was allowed to reflux for 72 h. After cooling to RT, K2CO3 was
removed by filtration. The red filtrate was evaporated to dryness
Physical measurements
Compounds L1, L2 and L3 were characterized by elemental anal-
1
yses, H-NMR, 13C-NMR and mass (positive ion) spectroscopy.
1H-NMR and 13C-NMR spectra were recorded on a JEOL JNM-
LA400 FT (400 MHz and 100 MHz respectively) instrument in
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CDCl3. ESI mass spectra were recorded on a WATERS Q-TOF
Premier mass spectrometer. The ESI capillary was set at 2.8 kV
and the cone voltage was 30V. Melting points were determined
with an electrical melting point apparatus by PERFIT, India
and were uncorrected. UV-visible spectra were recorded on a
JASCO V-570 spectrophotometer at 293 K and the average of
three measurements was taken. The deviations in molar absorption
coefficients were in the last digit only. Steady-state fluorescence
spectra were obtained using a Perkin-Elmer LS 50B luminescence
spectrometer at 293 K with excitation and emission band-pass of
5 nm. Steady-state fluorescence anisotropy data were obtained
using a Perkin-Elmer LS 55 Fluorimeter at 293 K with a slit
width of 5 nm and an integration time of 40 s. A time correlated
single photon counting experiment was performed using an IBH
Fluorocube spectrometer. The excitation wavelength was 295 nm.
The decays were recorded at emission wavelengths of 420 nm
and 526 nm at a spectral resolution of 28 ps per channel. They
were fitted by the iterative de-convolution method using DAS
6.2 software. All the decays were found to be bi-exponential
indicating the presence of two components, one fast and one slow,
respectively.
Fluorescence quantum yields of all the compounds were
determined by comparing the corrected spectrum with that of
anthracene (f = 0.297) in ethanol8 taking the area under the total
emission. On the other hand, emission quantum yields due to
diazole fluorophore were checked by comparing the corrected
spectrum with that of quinine sulfate8 in 1 N H2SO4. The
complex stability constants Ks were determined9 from the change
in absorbance or fluorescence intensity resulting from the titration
of dilute solutions (~10-5–10-6 M) of the fluorophoric systems
against metal ion concentration. The reported values gave good
correlation coefficients (≥0.98).
due to diazole is observed at 478 nm. In the case of compound
L2, the characteristic bands of anthracene appear at 341, 354
and 381 nm and the diazole band appears at 481 nm. The slight
positional changes in the anthracene and diazole bands may be
due to different spacers in L1 and L2. The intramolecular charge
transfer (ICT) band position of diazole as well as the anthryl
and quinoline band positions in L3 are almost similar to our
previous observation.5b In L3, the quinoline moiety shows a peak
at 317 nm (Fig. 2b). On the other hand, the anthryl groups give
a broad band centering at 377 nm with a shoulder at 392 nm
without its characteristic vibrational structure. This is due to the
movement and interactions between the chromophores of the side
arms attached to the cryptand core.5a,10
In MeCN, L1 upon excitation at 350 nm, exhibits the
characteristic9 emission band of anthracene around 420 nm and
a broad emission centered at ~520 nm. Low intensity of emission
in either case is due to photoinduced electron transfer (PET)
from the tertiary nitrogen atom of the receptor to the excited
fluorophore. Very similar emission behaviour is observed in L2.
On the other hand, L3 shows triple emissions, the exact nature of
which depends on the wavelength of light used for excitation. Upon
excitation at 317 nm (where quinoline absorbs predominantly)
in MeCN, emission due to quinoline appears at ~345 nm along
with anthracene (~420 nm) and diazole (~520 nm) emissions.
However, all the emissions are of low intensity due to PET being
operational.
When a transition metal ion is added to L1 in MeCN, the
metal occupies11 the tren-end of the cavity engaging N lone-pairs
that block PET. Upon excitation at 350 nm, the quantum yield
of anthracene emission increases significantly (Fig. 3a) in the
presence of a transition metal or a heavy metal ion input. The
extent of enhancement depends upon the nature of the metal ion
(Table 1). Among the metal ions studied, Zn(II) shows the highest
enhancement of anthracene fluorescence. There is also a small
enhancement (within 5 times) of the diazole emission. Thus a very
small amount of FRET is operating between the anthracene and
diazole fluorophore, which does not change significantly in the
presence of metal ion input.
Results and discussion
In dry MeCN, the absorption bands characteristic8 of the 9-
substituted anthracene moiety of L1 (Fig. 2a) in MeCN solution
are observed at 349, 368 and 386 nm and the lower energy band
Fig. 2 (a) UV-vis spectra of L1, L1 + Cu(II), L1 + Zn(II), L1+ Hg(II), L1+H+ (conc. of L1 = 1.7 ¥ 10-5 M) (b) UV-vis spectra of L3, L3+ Cu(II), L3 + Zn(II),
L3 + Hg(II), L3 + H+ (conc. of L3 = 1.5 ¥ 10-5 M) in MeCN.
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Fig. 3 Emission spectrum of L1, L1 + Hg(II), L1 + Zn(II), L1 + Cu(II) and L1 + H+ (conc. of L1 = 1.7 ¥ 10-6 M) excitation at (a) 350 nm and (b) 478 nm
in MeCN.
Table 1 Fluorescence output of L1 and L2 with different ionic
inputsaa Notes: Experimental conditions: medium, dry MeCN; concentra-
tion of ligands, ~10-6 M; concentration of ionic input, 10-4 M; Excitation
at 317 nm, 350 nm and 476 nm with excitation band-pass of 5 nm,
emission band-pass, 5nm; temperature, 298 K; f calculated by comparison
of corrected spectrum with that of anthracene (f = 0.297) or quinine sulfate
(f = 0.95) taking the area under the total emission. The error in f is within
10% in each case, except free ligands, where the error in f is within 15%.
Table 2 Fluorescence output of L3 with different ionic inputsa
Quantum Yield f (Enhancement Factor)
L3 (lex = 317 nm)
Input
None
Mn(II)
Fe(II)
Co(II)
Ni(II)
Cu(II)
Zn(II)
Cd(II)
Ag(I)
Hg(II)
Pb(II)
H+
fQuin
fAnthr
fDiaz
0.0004
0.0014
0.0015
0.0005 (1)
0.0011 (3)
0.0011 (3)
0.0005 (1)
0.0015 (4)
0.0011 (3)
0.0014 (4)
0.0011 (3)
0.0022 (6)
0.0011 (3)
0.0025 (6)
0.0143 (10)
0.0369 (26)
0.0449 (32)
0.0165 (12)
0.1214 (87)
0.0682 (49)
0.0441 (32)
0.0323 (23)
0.2448 (175)
0.0466 (33)
0.0771 (55)
0.0251 (17)
0.0425 (28)
0.0319 (21)
0.0218 (15)
0.1225 (82)
0.0451 (30)
0.0368 (25)
0.0198 (13)
0.1231 (82)
0.0363 (24)
0.0282 (19)
Quantum Yield f (Enhancement Factor)
L1
L2
Inputs fAnthr (350 nm) fDiaz (478 nm) fAnthr (350 nm) fDiaz (478 nm)
None
Mn(II) 0.100 (100)
Fe(II) 0.090 (90)
Co(II) 0.076 (76)
Ni(II) 0.098 (98)
Cu(II) 0.142 (142)
Zn(II) 0.148 (148)
Cd(II) 0.043 (43)
Ag(I) 0.048 (48)
Hg(II) 0.052 (52)
0.001
0.001
0.001
0.001
0.048 (48)
0.071 (71)
0.079 (79)
0.045 (45)
0.083 (83)
0.094 (94)
0.091 (91)
0.072 (72)
0.168 (168)
0.101 (101)
0.064 (64)
0.074 (74)
0.095 (95)
0.073 (73)
0.076 (76)
0.101 (101)
0.088 (88)
0.033 (33)
0.030 (30)
0.048 (48)
0.066 (66)
0.025 (25)
0.056 (56)
0.060 (60)
0.076 (76)
0.040 (40)
0.072 (72)
0.112 (112)
0.107 (107)
0.095 (95)
0.113 (113)
0.102 (102)
0.040 (40)
observed with Hg(II). Among the 3d-transition metal ions, Cu(II)
shows a large enhancement of the anthracene monomer emission
with respect to the metal free L3. The data are collected in Table 2.
Hg(II) and Cu(II) show the highest two-step FRET in compound
L3. Other metal ions used as input show less enhancement
(Table 2). In order to verify that the fluorescence enhancement is
due to the metal ion only and not because of protonation, several
controlled experiments5 were carried out.
Pb(II)
0.094 (94)
0.044 (44)
H+
Upon excitation of diazole fluorophore at 476 nm, there is
significant fluorescence enhancement of the diazole emission in
the presence of a metal ion (Fig. 3b). When a transition metal ion
is added to L2 in MeCN, quite a similar behavior as in the case of
L1 is observed although the extent of the enhancement is different
(Table 1).
When L3 is excited at 317 nm, no substantial enhancement of
quinoline emission is observed. The enhancement factor remains
within a limit of ~6 (Table 2) which is quite ineffective. Instead, two
large emission bands at 420 nm and 520 nm are observed assignable
to the anthracene and diazole moieties (Fig. 4) due to a significant
amount of FRET from the excited quinoline to the diazole via
anthracene where the metal ion acts as a conduit. Metal ions on
coordination with the N atoms connect the different fluorophores.
The highest enhancement of anthracene emission due to single-
step FRET from the quinoline to the anthracene moiety is
Since Cu(II) and Hg(II) show high quantum yields among the
metal ions studied with the systems, Cu(II) is titrated with L1
and L2 while Hg(II) is titrated with L3. Both the emission bands
due to the anthracene and diazole moieties are monitored for
L1. The intensities of the anthracene emission gradually increase
attaining the maximum when one equivalent of Cu(II) is added.
Thereafter, no change in intensity is observed indicating a 1 : 1
formation of Cu(II) with the cryptand attached to the anthracene.
During this process the quantum yield of the diazole emission
increases slowly due to a small amount of FRET from anthracene
to diazole. Thereafter, with an increase in concentration of Cu(II)
the intensities of the diazole emission increases rapidly and it
is saturated after 1 : 1 complexation of Cu(II) with the cryptand
attached to the diazole. Since diazole is electron-withdrawing, the
metal ion enters the cavity of the cryptand attached to the anthryl
groups.12 When L3 is titrated with Hg(II), the emission bands due
4152 | Dalton Trans., 2010, 39, 4146–4154
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Table 3 Interchromophoric distances of L3 with different ionic inputs. (a) Distance between quinoline and anthracene and (b) distance between
anthracene and diazole
a
2
b
2
b
c
c,d
(a)
E (%)
fd
J/M-1cm3
<k >max
<k >min
R0,av
Rav
L3
3
0.0030
0.1586
0.1397
9.02 ¥ 10-14
8.89 ¥ 10-14
8.27 ¥ 10-14
J/M-1cm3
7.14 ¥ 10-15
8.23 ¥ 10-15
7.50 ¥ 10-15
1.47
1.53
1.73
0.61
0.60
0.53
10
20
19
R0,av
11
14
18
18
15
11
Rav
21
10
13
L3 + Cu(II)
L3 + Hg(II)
(b)
83
96
E (%)
2
85
86
a¢
2
b
2
b
c
c,d
fd
<k >max
<k >min
L3
0.0009
0.1341
0.0200
1.75
2.46
1.20
0.43
0.32
0.72
L3 + Cu(II)
L3 + Hg(II)
a Quantum yield of only bis-quinoline substituted cryptand in MeCN. a¢Quantum yield of mono-anthracene substituted cryptand in MeCN. b The limits on
2
k are obtained by the measurements of the fluorescence anisotropy of the donor and acceptor fluorophores. c All the distances are in A. d An uncertainty
˚
in the derived intramolecular separation of approximately 15% about the limit of R.
Fig. 4 (a) Emission spectrum of L3, L3 + H+, L3 + Zn(II) and L3 + Hg(II) (conc. of L3 = 1.7 ¥ 10-6 M) excitation at 317 nm in MeCN. (b) Emission
spectra of L3 in the presence of an increasing concentration of Hg(II) in MeCN.
to anthracene and diazole moieties (Fig. 4b) are monitored and
1 : 2 formation is observed.
Hg(II) and Cu(II) complexes do not differ much while the average
lifetime of the Hg(II) complex is actually found to be slightly
less than both the free L3 ligand and the Cu(II)-L3 complex,
and consequently substantiated the maximum intensity among
them as found in steady-state studies. The interchromophoric
distances are estimated by steady-state energy transfer experiments
following a protocol reported in the literature.13 The distances
between quinoline and anthracene, in the case of Hg(II) and Cu(II)
The binding constant (Ks) values for the cryptand moieties
attached to anthracene in L1 and L2 with Cu(II) are 1.6 ¥ 106 M-1
and 1.5 ¥ 106 M-1 respectively. The Ks values for the cryptand
moieties attached to diazole in L1 and L2 with Cu(II) are 2.4 ¥
105 M-1 and 2.2¥ 105 M-1 respectively. The binding constant
values suggest that at first Cu(II) binds in the cryptand attached to
anthracene and next to the other cryptand attached to the diazole
moieties. For L3 the overall binding constant value determined
from the fluorescence intensity data with Hg(II) is found to be
3.1 ¥ 1011 M-2.
˚
˚
complexes of L3, afforded values of 11 A and 15 A respectively.
On the other hand, the distances between anthracene and diazole,
in the case of Hg(II) and Cu(II) complexes of L3, afforded values
˚
˚
of 13 A and 10 A respectively (Table 3).
The photodynamics of L3 and its complexes were affected
mostly for 420 nm emission with the incorporation of the metal
ions. With the inclusion of Cu(II) and Hg(II), the average lifetime
of the complexes were enhanced. For the Cu(II)-L3 complex, the
lifetime almost doubled with an increase in the contribution of
the slow component, but with a concomitant decrease in the fast
component contribution. Whereas, the Hg(II)-L3 complex, at the
same wavelength, the average lifetime was enhanced 1.5 times
more than that of the free ligand, but with a large decrease of the
slow component contribution thereby justifying the large increase
in fluorescence intensity at 420 nm. At 526 nm emission, both
Conclusion
In conclusion, a two-step FRET in L3 is found to be quite
significant in the presence of Cu(II) and Hg(II) ions. However,
a single-step FRET in L1 and L2 is insignificant even in the
presence of transition/heavy metal ions. Thus, the present systems
provide a starting point to design single/multi-step FRET where a
suitable choice of spacer between the donor–acceptor fluorophores
is essential.
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15889; (d) C. G. dos Remedios and P. D. Moens, J. Struct. Biol., 1995,
115, 175; K. E. Sapsford, L. Berti and I. L. Medintz, Minerva Biol.,
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Acknowledgements
P. K. B. acknowledges the financial support from DRDO, New
Delhi, India. K. K. S. and S. S. thanks the CSIR, India and
S. C. thanks the UGC, India for fellowships. We thank Dr
A. Datta from Indian Institute of Technology Bombay for time-
resolved fluorescence and fluorescence anisotropy measurements
and helpful discussions.
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©