Syntheses and spectroscopic properties of energy transfer systems based on squaraines

Article abstract of DOI:10.1016/S0040-4020(03)00383-1

The purpose of this project was to prepare fluorescent dyes that could absorb energy at relatively short wavelengths, and fluoresce in the near-IR region. To achieve this, copper- and palladium-mediated C-N couplings were used to prepare the 'cassettes', i.e the carbazole derivative 3b and the carbazole-, phenothiazine-, and phenoazine-squaraines 4b-d. These compounds have carbazole, phenothiazine, and phenoazine donor-components that absorb around about 300-320nm, and squaraine acceptor-parts that fluoresce in the range 650-700nm. The efficiencies of energy transfer from the donor to the acceptor, and the overall quantum yields of the cassettes were determined.

Full text of DOI:10.1016/S0040-4020(03)00383-1

Tetrahedron 59 (2003) 3109–3116
Syntheses and spectroscopic properties of energy transfer systems
based on squaraines
Guan-Sheng Jiao,a Aurore Loudet,a Hong Boon Lee,a Stanislav Kalinin,b
b
Lennart B.-A. Johansson and Kevin Burgess
a
,
˚
*
aDepartment of Chemistry, Texas A&M University, P.O. Box 30012, College Station, TX 77841-3012, USA
˚ ˚
Department of Chemistry: Biophysical Chemistry, Umea University, S-90 187 Umea, Sweden
b
Received 17 January 2003; revised 5 March 2003; accepted 5 March 2003
Abstract—The purpose of this project was to prepare fluorescent dyes that could absorb energy at relatively short wavelengths, and fluoresce
in the near-IR region. To achieve this, copper- and palladium-mediated C–N couplings were used to prepare the ‘cassettes’, i.e the carbazole
derivative 3b and the carbazole-, phenothiazine-, and phenoazine-squaraines 4b–d. These compounds have carbazole, phenothiazine, and
phenoazine donor-components that absorb around about 300–320 nm, and squaraine acceptor-parts that fluoresce in the range 650–700 nm.
The efficiencies of energy transfer from the donor to the acceptor, and the overall quantum yields of the cassettes were determined. q 2003
Elsevier Science Ltd. All rights reserved.
1. Introduction
they emit in the near IR, and this region is far removed
from the fluorescence of any biomolecules.14 We were
particularly interested in carbazole and similar heterocycles
as donors since these absorb in the UV region at around
300 nm.
‘Donor acceptor cassettes’ (compounds with a moiety to
absorb at low wavelength and another to emit at a higher
wavelength) based on through-space fluorescence energy
transfer are common in biotechnology.1–8 In these, a
molecular fragment, the ‘donor’, harvests energy from an
excitation source and transmits it through space, via dipole–
dipole coupling, to an ‘acceptor’ that then fluoresces. The
donor and acceptor fragments in these cassettes are
connected via a non-conjugated linker (Fig. 1(a)). We are
investigating another type of cassette in which the donor and
acceptor fragments are conjugated.9,10 This change is
apparently subtle, but it can have profound effects on the
rates of energy transfer (ET) from the donor to the acceptor,
and the ‘apparent Stokes’ shifts’ observed (Fig. 1(b)).11
Conjugated ET systems of this kind have been investigated
extensively in material science.12,13 Cassettes of this kind
could absorb at short wavelength and emit efficiently at
much longer wavelengths, and might therefore be useful in
applications that involve several multiplexed dyes, excited
at one wavelength and observed at well dispersed longer
wavelengths. The work described here was undertaken as
part of our project to put these ideas to practice. Specifically,
this manuscript describes syntheses and spectral investi-
gations of novel through-bond ET cassettes in which donors
are directly conjugated to a squaraine acceptor (Fig. 1(c)).
Squaraine acceptors are particularly attractive because
2. Results and discussion
2.1. Syntheses
Our preliminary studies led to the preparation of compounds
like 1 in which the squaraine fragment was formed from
resorcinol derivatives; however, the products had low
quantum yields (data not shown). In retrospect, this is
unsurprising because the 2,6-dihydroxyl substituents facili-
tate tautomerization processes, leading to radiationless
decay.15 Similar effects do not apply to the second class
of cassettes we prepared, i.e. 2 and others containing
3,3-dimethylindolenine fragments. The improvement in
quantum yields was not marked, however, because another
detrimental characteristic comes into play for unsym-
metrical squaraines; specifically, rotation about the bond
labeled ‘a’ is facile because resonance favors single bond
character here relative to bond ‘b’. This effect makes
electronic transmission through the squaraine system
more difficult than it would otherwise be, depressing the
quantum yield. Consequently, this paper focuses on the
symmetrical squaraines 3 and 4 incorporating 3,3-dimethyl-
indolenine and 2-methylbenzothiazole fragments, respec-
tively (Fig. 2).
Keywords: squaraines; fluorescence; energy-transfer; C–N couplings.
*
Corresponding author. Tel.: þ1-979-845-4345; fax: þ1-979-845-8839;
e-mail: asymm@tamu.edu
0040–4020/03/$ - see front matter q 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0040-4020(03)00383-1

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G.-S. Jiao et al. / Tetrahedron 59 (2003) 3109–3116
2.2. Spectroscopic studies
A series of spectroscopic measurements were undertaken to
test if compounds 3b and 4b–d behave as cassettes
(rather than as simple conjugated molecules) and, if so,
to quantitate their efficiencies. First, UV spectra of the
molecules were recorded. If the UV spectrum of a particular
system resembles that which would be obtained by super-
imposing UV spectra of the corresponding discrete donor
and acceptor components, then the molecule is behaving as
a cassette. Alternatively, if the UV is different, then the
compound is behaving as a conjugated system. Figure 3(a)
shows the UV spectra of compound 3b overlaid with the
spectrum of carbazole and 3a, i.e. the donor and acceptor
components, respectively. The UV spectrum of 3b is very
close to that which would be obtained by adding the donor
(carbazole) and acceptor (3a) components. Consequently,
this molecule behaves as a cassette with non-planar donor
and acceptor systems in the ground state. Similar data were
obtained for 4b–d indicating they all function as cassettes
(Fig. 3).
One of the criteria that define a good cassette is quenching
of the donor fluorescence. If this quenching is complete then
the ET efficiency is 100%, where:
ET efficiency ¼
{100 £ ½1 2 ðfluorescence intensity of donor in cassetteÞ=
ðfluorescence intensity of free donorÞꢀ%}
In our systems the fluorescence of the free carbazole donor
was so weak that accurate ET efficiency measurements
proved to be impossible. In practice, it was also difficult to
accurately measure the fluorescence quantum yield when
exciting the donor group of the cassettes studied here.
Thus, two important characteristics of the cassettes, ET
efficiencies and fluorescence quantum yields, could not be
measured directly.
ET efficiencies were estimated using another approach to
circumvent the experimental difficulties outlined above; this
involved the following line of reasoning. The efficiency of
ET in a cassette must be close to 100% if the fluorescence
quantum yield of the acceptor group is the same when
excited at the donor, or at the acceptor absorption. That the
ratios of the fluorescence excitation and the UV absorption
spectra of the cassettes were constant over the whole
spectral region provides evidence for ET efficiencies close
to unity. Fluorescence lifetimes of the squaraine acceptor
group in all the cassettes were also measured and shown to
be very similar irrespective of the excitation wavelength.
The results obtained are summarized in Table 1 (details are
given in Section 4).
Figure 1. (a) Through-space ET cassettes, (b) through-bond ET cassettes,
(c) the acceptor fragments used in this study.
Scheme 1(a) describes the syntheses of the cassettes 3b.
After N-alkylation of the iodoindolenine to give 5b, copper-
mediated couplings16 were used to introduce the carbazole
unit giving compounds 6b in moderate yield (53%).
Palladium-mediated couplings to achieve the same result
were investigated briefly, but with little success.17 Com-
pound 6b was condensed with squaric acid18 to give the
desired product as a blue metallic solid. The control
compound 3a was prepared via an abbreviated version of
this sequence. Preparation of the cassettes 4 was achieved
via a similar procedure (Scheme 1b) except that a
palladium-mediated C–N coupling was used. The most
notable difference between the two approaches is that the
metal-catalyzed reactions gave higher yields in the second
sequence (76–90%).17
The quantum yields were determined for irradiation of 3b
and 4b–d in the acceptor region (Table 1). The values
(0.23–0.37) show moderate fluorescence efficiencies. Rates
of ET between the donor and the acceptor are likely to be
fast relative to any thermal energy loss, so the quantum
yields of the cassettes when irradiated in the donor region

G.-S. Jiao et al. / Tetrahedron 59 (2003) 3109–3116
3111
Figure 2. Donor–acceptor cassettes in this research.
are likely to approximate to those of the acceptor
components within them.
Secondly, the molecules have poor water solubilities and
this is likely to make their conjugation to biomolecules
difficult. Our current research on similar ET cassettes is
focused on solving these problems.
3. Conclusions
In conclusion, several cassettes based on squaraine
fragments acting as acceptors (3b and 4b–d) were prepared.
These cassettes transfer energy very efficiently to their
acceptor parts when irradiated in the donor absorption
region (ca. 300 nm). With respect to fluorescence emission,
the cassettes behave like dyes with a huge Stokes’ shift,
absorbing around 300 nm and fluorescing in the near IR
region (637–693 nm). Emission at such long wavelengths is
good for probing biological systems which do not fluoresce
in this region, i.e. DNA, RNA, the majority of proteins, and
many other biomolecular types. The quantum yields of the
acceptors in the cassettes are high, and we infer that they are
also high for the cassettes when irradiated in the donor
region. Despite these attributes, cassettes 3b and 4b–d have
some disadvantages with respect to tagging biological
molecules. First, the donor fragments do not absorb very
strongly, hence they cannot supply the acceptor well.
4. Experimental
4.1. General
4.1.1. Preparation of compound 5b. 5-Iodo-2,3,3-tri-
methyl-3H-indole19 (1.00 g, 3.50 mmol) and ethyl iodo-
acetate (0.901 g, 4.21 mmol) were weighed into a sealed
tube. The mixture was heated at 858C for 12 h. After cooling
to room temperature, 1N NaOH solution (20 mL) was added
and stirred for 1 h. The reaction mixture was extracted
with dichloromethane three times. The organic layers were
combined and evaporated to a residue which was purified by
flash column chromatography in 10% EtOAc/hexane to give
the product as a brown oil (0.988 g, 76%). 1H NMR
(300 MHz, CDCl3) d 7.40 (dd, J¼9.0, 2.0 Hz, 1H), 7.36
(d, J¼2.0 Hz, 1H), 6.30 (dd, J¼9.0, 2.0 Hz, 1H), 4.19 (q,
J¼8.0 Hz, 2H), 4.19 (s, 2H), 3.94 (d, J¼3.0 Hz, 1H), 3.87

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G.-S. Jiao et al. / Tetrahedron 59 (2003) 3109–3116
Scheme 1. Syntheses of (a) cassette 3b; and (b) cassettes 4b–d.
(d, J¼3.0 Hz, 1H),1.35 (s, 6H), 1.24 (t, J¼8.0 Hz, 3H); 13
C
flash column chromatography using 20% EtOAc/hexane
1
to give the product as a white solid (134 mg, 53%). H
NMR (75.4 MHz, CDCl3) d 168.6, 160.8, 145.35, 140.27,
136.30, 131.07, 107.51, 80.71, 75.53, 61.47, 44.41, 44.25,
30.04, 14.34; HR-MS (MALDI) for C15H19INO2 (MþHþ)
calc’d 372.0461, found 372.0445.
NMR (300 MHz, CDCl3) d 8.16 (d, J¼8.0 Hz, 2H), 7.43–
7.26 (m, 8H), 6.68 (d, J¼8.0 Hz, 1H), 4.33 (s, 2H), 4.28
(q, J¼8.0 Hz, 2H), 4.02 (d, J¼3.0 Hz, 1H), 3.96 (d,
J¼3.0 Hz, 1H), 1.45 (s, 6H), 1.32 (t, J¼8.0 Hz, 3H); 13C
NMR (300 MHz, CDCl3) d 168.94, 161.48, 144.99,
141.71, 139.22, 136.35, 131.12, 129.38, 126.95, 125.96,
123.21, 121.67, 120.47, 120.41, 119.67, 119.54, 110.76,
110.00, 107.53, 105.79, 75.71, 61.59, 44.63, 44.52,
30.20,14.43.
Compound 5a.20 62%. 1H NMR (300 MHz, CDCl3) d 7.21–
7.16 (m, 2H), 6.88 (t, J¼7.8 Hz, 1H), 6.57 (d, J¼7.8 Hz,
1H), 4.29 (s, 2H), 4.26 (q, J¼7.2 Hz, 2H), 3.99 (d, J¼
2.4 Hz, 1H), 3.91 (d, J¼2.4 Hz, 1H), 1.45 (s, 6H), 1.30 (t,
J¼7.2 Hz, 3H); 13C NMR (75.4 MHz, CDCl3) d 169.2,
161.8, 145.7, 137.6, 127.8, 122.3, 119.5, 105.4. 74.7, 61.5,
44.6, 44.5, 30.3, 14.5.
4.1.3. Preparation of squaraine 3b. Compound 6b (24 mg,
0.06 mmol) and squaric acid (3 mg, 0.03 mmol) were
placed in a round-bottom flask. A mixture of n-BuOH/
toluene (15 mL, 1:1 v/v) was added. The mixture was
refluxed for 16 h. Water was azeotropically removed by
using a Dean–Stark trap. After cooling to room tempera-
ture, the reaction mixture was filtered to give a dark-blue
solid, which was further washed with ether three times
(15 mg, 66%). A sample for analysis was obtained by
further purification through flash column chromatography.
1H NMR (300 MHz, CDCl3) d 8.16 (d, J¼8.0 Hz, 4H),
4.1.2. Preparation of compound 6b. Compound 5b
(200 mg, 0.54 mmol), carbazole (90 mg, 0.54 mmol),
1,2-trans-diaminocyclohexane (12 mg, 0.11 mmol), CuI
(5 mg, 0.03 mmol), and Cs2CO3 (369 mg, 1.13 mmol)
were weighed into a round-bottom flask, then dioxane
(2 mL) was added. The mixture was heated at 1008C for
17 h. After cooling to room temperature, the reaction
mixture was diluted with ether and filtered through a pad of
celite. The residue after removal of solvent was purified by

G.-S. Jiao et al. / Tetrahedron 59 (2003) 3109–3116
3113
7.56–7.27 (m, 16H), 7.10 (d, J¼8.0 Hz, 2H), 6.02 (s, 2H),
4.33 (q, J¼7.2 Hz, 4H), 1.83 (br s, 12H), 1.36 (t, J¼7.2 Hz,
6 H); HR-MS (MALDI) for C58H51N4O6 (MþHþ) calcd
899.3809, found 899.3825.
121.42, 115.68, 110.30, 63.27, 50.62, 17.91, 14.59; HR-MS
(MALDI) for C24H21N2O2S (MþHþ) calcd 401.1324,
found 401.1264.
1
Compound 8a.21 86%. H NMR (300 MHz, DMSO-d6) d
Compound 3a. 91%. 1H NMR (300 MHz, CDCl3) d 7.37 (t,
J¼7.2 Hz, 2H), 7.31 (d, J¼7.2 Hz, 2H), 7.19 (t, J¼7.2 Hz,
2H), 6.91 (d, J¼7.2 Hz, 2H), 5.87 (s, 2H), 4.76 (br s, 4H),
4.26 (q, J¼7.2 Hz, 4H), 1.81 (br s, 12H), 1.29 (t, J¼7.2 Hz,
6H); HR-MS (MALDI) for C34H37N2O6 (MþHþ) calcd
569.2652, found 569.2627.
8.52 (d, J¼7.8 Hz, 1H), 8.30 (d, J¼7.8 Hz, 1H), 7.90 (t,
J¼7.8 Hz, 1H), 7.82 (t, J¼7.8 Hz, 1H), 5.91 (s, 2H), 4.24 (q,
J¼7.2 Hz, 2H), 3.23 (s, 3H), 1.25 (t, J¼7.2 Hz, 3H); 13C
NMR (75.4 MHz, DMSO-d6) d 180.31, 166.00, 141.78,
130.38, 129.29, 129.04, 125.62, 117.34, 63.30, 50.45, 17.84,
14.60.
4.1.4. Preparation of compound 7b. Carbazole (184 mg,
1.1 mmol), 5-bromo-2-methylbenzothiazole (228 mg, 1.00
mmol), and sodium tert-butoxide (134 mg, 1.40 mmol)
were placed in a Schlenk tube. Tris(dibenzylideneacetone)-
dipalladium (23 mg, 0.02 mmol) and 2-(di-t-butylphosphino)-
biphenyl (12 mg, 0.04 mmol) were weighed in glove box,
the tube was removed from the box, then toluene (2 mL)
was added. The mixture was heated at 1008C for 2 h. After
cooling to room temperature, the reaction mixture was
diluted with ether and filtered through a pad of celite. The
residue after removal of solvent was purified by flash
column chromatography using 20% EtOAc/hexane to give
the product as a white solid (256 mg, 82%). 1H NMR
(300 MHz, CDCl3) d 8.23–8.19 (m, 3H), 8.02 (d, J¼8.7 Hz,
1H), 7.58 (dd, J¼8.1, 1.8 Hz, 1H), 7.51–7.42 (m, 4H), 7.35
(td, J¼8.1, 1.8 Hz, 2H), 2.93 (s, 3H); 13C NMR (75.4 MHz,
CDCl3) d 169.23, 154.83, 141.29, 136.23, 126.36, 126.08,
124.18, 123.74, 122.80, 121.19, 120.67, 120.41, 110.02,
20.62; HR-MS (ESI) for C20H15N2S (MþHþ) calcd
315.0956, found 315.0926.
Compound 8c. 68%. 1H NMR (300 MHz, DMSO-d6) d 8.66
(dd, J¼8.7, 1.8 Hz, 1H), 8.36 (d, J¼1.8 Hz, 1H), 7.82 (dd,
J¼8.7, 1.8 Hz, 1H), 7.24 (dd, J¼7.2, 1.8 Hz, 2H), 7.07–
6.97 (m, 4H), 6.39 (dd, J¼7.2, 1.8 Hz, 2H), 5.84 (s, 2H),
4.17 (q, J¼7.2 Hz, 2H), 3.25 (s, 3H), 1.18 (t, J¼7.2 Hz, 3H);
13C NMR (75.4 MHz, DMSO-d6) d 180.89, 165.19, 143.14,
142.76, 142.59, 128.63, 127.53, 127.35, 127.12, 124.01,
123.80, 122.27, 118.30, 115.98, 62.53, 49.87, 17.16, 13.84;
HR-MS (MALDI) for C24H21N2O2S2 (MþHþ) calcd
433.1045, found 433.1066.
Compound 8d. 70%. 1H NMR (300 MHz, DMSO-d6) d 8.75
(d, J¼8.7 Hz, 1H), 8.62 (d, J¼1.8 Hz, 1H), 7.90 (dd, J¼8.7,
1.8 Hz, 1H), 6.84–6.64 (m, 6H), 5.87 (dd, J¼7.5, 1.5 Hz,
2H), 5.86 (s, 2H), 4.20 (q, J¼7.2 Hz, 2H), 3.26 (s, 3H), 1.19
(t, J¼7.2 Hz, 3H); 13C NMR (75.4 MHz, DMSO-d6) d
181.09, 164.87, 143.22, 143.12, 139.69, 133.25, 130.60,
128.64, 127.79, 123.55, 122.07, 119.62, 115.47, 113.33,
62.36, 49.82, 17.11, 13.67; HR-MS (MALDI) for
C24H21N2O3S (MþHþ) calcd 417.1273, found 417.1276.
Compound 7c. 76%. 1H NMR (300 MHz, CDCl3) d 8.31 (s,
1H), 8.02 (d, J¼5.7 Hz, 1H), 7.37 (dd, J¼8.4, 1.8 Hz, 1H),
7.07–7.04 (m, 2H), 6.86–6.82 (m, 4H), 6.29–6.25 (m, 2H),
2.90 (s, 3H); 13C NMR (75.4 MHz, CDCl3) d 169.09, 155.52,
144.51, 139.44, 135.71, 127.42, 127.14, 127.08, 124.97,
123.80, 122.94, 120.76, 116.60, 20.59; HR-MS (ESI) for
C20H15N2S2 (MþHþ) calcd 347.0671, found 347.0653.
Compound 7d. 90%. 1H NMR (300 MHz, CDCl3) d 8.04 (d,
J¼8.4 Hz, 1H), 7.98 (d, J¼1.8 Hz, 1H), 7.34 (dd, J¼8.4,
1.8 Hz, 1H), 6.75–6.56 (m, 6H), 5.96 (dd, J¼8.4, 1.5 Hz,
1H), 2.90 (s, 3H); 13C NMR (75.4 MHz, CDCl3) d 168.95,
155.77, 144.25, 137.40, 136.09, 134.68, 127.26, 125.30,
124.11, 123.47, 121.70, 115.73, 113.63, 20.45; HR-MS
(ESI) for C20H15N2OS (MþHþ) calcd 331.0899, found
331.0867.
4.1.6. Preparation of squaraine 4b. Compound 8b
(100 mg, 0.21 mmol), quinoline (27 mg, 0.21 mmol), and
squaric acid (12 mg, 0.10 mmol) were placed in a round-
bottom flask. A mixture of n-BuOH/toluene (15 mL, 1:1
v/v) was added. The mixture was refluxed for 12 h while
the water formed was azeotropically removed by using a
Dean–Stark trap. After cooling to room temperature, the
reaction mixture was filtered to give a dark-green solid,
which was further washed with ether 3£(81 mg, 83%). A
sample for analysis was obtained by purification through
flash column chromatography using 40% EtOAc/CH2Cl2.
1H NMR (300 MHz, CDCl3) d 8.17 (d, J¼7.8 Hz, 4H), 7.75
(d, J¼8.1 Hz, 2H), 7.48–7.21 (m, 16H), 5.88 (s, 2H), 4.78
(s, 4H), 4.29 (q, J¼7.2 Hz, 4H), 1.32 (t, J¼7.2 Hz, 6H); HR-
MS (MALDI) for C52H39N4O6S2 (MþHþ) calcd 879.2311,
found 879.2334.
4.1.5. Preparation of compound 8b. Compound 7b
(250 mg, 0.800 mmol) and ethyl bromoacetate (160 mg,
0.960 mmol) were weighed into a sealed tube. The mixture
was heated at 808C for 18 h. After cooling to room
temperature, the reaction mixture was poured into ether
and filtered to give a pale green solid which was washed
with ether 2£(322 mg, 84%). 1H NMR (300 MHz, DMSO-
d6) d 8.76 (s, 1H), 8.74 (d, J¼6.9 Hz, 1H), 8.31 (d, J¼
7.5 Hz, 2H), 8.13 (d, J¼7.2 Hz, 1H), 7.48 (d, J¼3.9 Hz,
4H), 7.39–7.34 (m, 2H), 5.92 (s, 2H), 4.25 (q, J¼7.2 Hz,
2H), 3.29 (s, 3H), 1.25 (t, J¼7.2 Hz, 3H); 13C NMR
(75.4 MHz, DMSO-d6) d 181.90, 166.07, 143.18, 140.49,
138.86, 127.88, 127.58, 127.31, 127.22, 123.80, 121.58,
Compound 4a. 96%. 1H NMR (300 MHz, CDCl3) d 7.55 (d,
J¼7.5 Hz, 2H), 7.36 (t, J¼7.5 Hz, 2H), 7.23 (t, J¼7.5 Hz,
2H), 7.02 (d, J¼7.5 Hz, 2H), 5.81 (s, 2H), 4.80 (s, 4H), 4.29
(q, J¼7.2 Hz, 4H), 1.32 (t, J¼7.2 Hz, 6H); HR-MS (ESI) for
C28H25N2O6S2 (MþHþ) calcd 549.1154, found 549.1132.
Compound 4c. 80%. 1H NMR (300 MHz, CDCl3) d 7.72 (d,
J¼8.4 Hz, 2H), 7.26 (dd, J¼8.4, 1.8 Hz, 2H), 7.11–7.08 (m,
4H), 7.02 (d, J¼1.8 Hz, 2H), 6.96–6.86 (m, 8H), 6.32 (dd,
J¼8.1, 1.8 Hz, 4H), 5.85 (s, 2H), 4.75 (s, 4H), 4.27 (q,
J¼7.2 Hz, 4H), 1.29 (t, J¼7.2 Hz, 6H); HR-MS (MALDI)
for C52H39N4O6S4 (MþHþ) calcd 943.1753, found
943.1709.

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G.-S. Jiao et al. / Tetrahedron 59 (2003) 3109–3116
Figure 3 (continued)

G.-S. Jiao et al. / Tetrahedron 59 (2003) 3109–3116
3115
Figure 3. (a), (c), (e) and (g) Overlaid absorption spectra of the donor fragments, the acceptor fragments, and the complete cassettes; all in CHCl3 solution.
(b), (d), (f) and (h) Fluorescence spectra of the cassettes; all excited at ,300 nm or 590 (630) nm in CHCl3 solution.
Table 1. Key spectroscopic data for the compounds 3a,b and 4b–d
Compound
Absorption lmax
below 350 nm (nm)
Fluorescence
emission lmax (nm)
Apparent Stokes’
shift (nm)
Energy transfer
efficiency, ET (%)a
Quantum yield
Fb
3a
3b
4a
4b
4c
4d
279, 344
293, 340
310, 350
292, 339
257, 315
319
637
659
679
693
691
688
6c
–
0.23
0.31
0.33
0.37
0.33
0.36
366, 319
10c
401, 354
434, 376
369
,100
–
,100
,100
,100
a
Determined indirectly, see Section 4.
Excited at 590 nm for 3a,b and 630 nm for 4a–d.
Actual Stokes’ shift for 3a and 4a.
b
c
Compound 4d. 62%. 1H NMR (300 MHz, CDCl3) d 7.75 (d,
J¼8.4 Hz, 2H), 7.23 (dd, J¼8.4, 1.8 Hz, 2H), 7.02 (d,
J¼1.8 Hz, 2H), 6.75–6.60 (m, 14H), 5.94 (dd, J¼8.1,
1.5 Hz, 4H), 5.86 (s, 2H), 4.77 (s, 4H), 4.27 (q, J¼7.2 Hz,
4H), 1.30 (t, J¼7.2 Hz, 6H); HR-MS (MALDI) for
C52H39N4O8S2 (MþHþ) calcd 911.2209, found 911.2254.
increased by increasing the concentration of the cassettes,
but then the fluorescence spectra become distorted due to
the re-absorption in the acceptor region. Therefore, we used
the following procedures to indirectly estimate the ET
efficiency of the cassettes 3b and 4b–d.
(1) The absorption spectrum {A(lex)} and the corrected
excitation spectrum {F(lex)} were compared. If the ET
efficiency is 1, then one expects to find a ratio
between these spectra which is constant at all
wavelengths. The ratios obtained are listed in Table 2.
These data suggest that the ET efficiency is close to 1
for cassettes 3b, 4b, and 4d. The deviation for 4c can
4.2. Indirect determination of ET efficiencies
In practice it is extremely difficult to directly measure the
ET efficiency of the cassettes 3b and 4b–d because of the
very low absorbance in the donor region as compared to
the acceptor region. However, the donor absorption can be
Table 2. The ratios of absorptions and fluorescence excitation spectra {A(lex)/F(lex)} and the average lifetimes of the acceptor group upon excitation of the
donor and the acceptors of cassettes 3b and 4b–d
Compound
Average A(lex)/F(lex), lex¼300–400 nma
Average lifetime (ns), lex¼300–350 nm
Average lifetime (ns), lex¼630 nm
4b
4c
4d
3b
1.03
1.33
0.88
0.99
2.78
3.04
2.95
1.56
2.60
2.83
2.79
1.46
a
The spectra are scaled so that the average ratio A(lex)/F(lex) at lex¼600–690 nm is 1.

3116
G.-S. Jiao et al. / Tetrahedron 59 (2003) 3109–3116
Rosenblum, B. B.; Menchen, S. M.; Graham, R. J.;
Constantinescu, A.; Upadhya, K. G.; Cassel, J. M. Nucleic
Acids Res. 1997, 25, 2816–2822.
be explained by a lower ET efficiency, or by presence
of pure donor.
(2) We further measured the fluorescence lifetimes of the
cassettes 3b and 4b–d upon excitation in the donor and
acceptor regions. If the timescale of ET is ,100 ps or
slower, we expect to observe negative components in
the fluorescence decay. No such component was found.
It is also true that the average lifetimes must be longer
when the donor is excited directly. We obtained the
average lifetimes shown in Table 2. The experimental
accuracy suggests that the average lifetimes are very
similar.
8. Rosenblum, B. B.; Lee, L. G.; Spurgeon, S. L.; Khan, S. H.;
Menchen, S. M.; Heiner, C. R.; Chen, S. M. Nucleic Acids Res.
1997, 25, 4500–4504.
9. Burgess, K.; Burghart, A.; Chen, J.; Wan, C.-W. New
Chemistry of BODIPY Dyes, and BODIPY Dye Cassettes
Featuring Through-Bond Energy Transfer; SPIE BiOS 2000.
The International Symposium on Biomedical Optics, San Jose,
CA, 2000.
10. Burghart, A.; Thoresen, L. H.; Chen, J.; Burgess, K.;
¨
Bergstrom, F.; Johansson, L. B.-A. Chem. Commun. 2000,
2203–2204.
Acknowledgements
11. Speiser, S. Chem. Rev. 1996, 96, 1953–1976.
12. Wagner, R. W.; Lindsey, J. S. J. Am. Chem. Soc. 1994, 116,
9759–9760.
We wish to thank Lars H. Thoresen for useful discussions.
Support for this work was provided by The National
Institutes of Health (HG/GM01745), and The Robert
A. Welch Foundation.
13. McQuade, D. T.; Pullen, A. E.; Swager, T. M. Chem. Rev.
2000, 100, 2537–2574.
14. Slavik, J. In Near-Infrared Dyes for High Technology
Applications; Daehne, S., Resch-Genger, U., Wolfbeis, O. S.,
Eds.; Application of Red Fluorescent Probes for the
Measurement of Individual Cell Cytoplasmic Ph Values.
Kluwer Academic: Dordrecht, 1998; Vol. 52, pp 115–124.
15. Law, K.-Y. In Organic Photochemistry, Ramamurthy, V., Ed.;
Absorption, Fluorescence Emission, and Photophysics of
Squaraines. Marcel Dekker: New York, 1997; Vol. 1, pp
519–584.
References
1. Hung, S.-C.; Ju, J.; Mathies, R. A.; Glazer, A. N. Anal.
Biochem. 1996, 243, 15–27.
2. Lee, L. G.; Spurgeoun, S. L.; Rosenblum, B. Energy Transfer
Dyes with Enhanced Fluorescence; Perkin–Elmer: US, 1997;
p 3.
16. Klapars, A.; Antilla, J. C.; Huang, X.; Buchwald, S. L. J. Am.
Chem. Soc. 2001, 123, 7727–7729.
3. Lee, L. G.; Rosenblum, B.; Spurgeoun, S. L. Fluorescent
Energy Transfer Dyes—Useful for Labelling Dideoxynucleo-
tides, Oligonucleotides; etc. Perkin–Elmer: US, 1998; p 2.
4. Berti, L.; Xie, J.; Medintz, I. L.; Glazer, A. N.; Mathies, R. A.
Anal. Biochem. 2001, 292, 188–197.
17. Old, D. W.; Harris, M. C.; Buchwald, S. L. Org. Lett. 2000, 2,
1403–1406.
18. Sprenger, H.-E.; Ziegenbein, W. Angew. Chem. Int. Ed. 1967,
6, 553–554.
19. Moreau, M. F.; Lapalus, F.; Meyniel, G. Eur. J. Med. Chem.-
Chim. Ther. 1974, 9, 274–280.
5. Berti, L.; Medintz, I. L.; Tom, J.; Mathies, R. A. Bioconjugate
Chem. 2001, 12, 493–500.
20. Tilford, C. H. J. Med. Chem. 1971, 14, 1020–1021.
21. Galera, C.; Vaquero, J. J.; Navio, J. L. G.; Alvarez-Builla, J.
J. Heterocycl. Chem. 1986, 23, 1889–1892.
6. Nampalli, S.; Zhang, W.; Rao, T. S.; Xiao, H.; Kotra, L. P.;
Kumar, S. Tetrahedron Lett. 2002, 43, 1999–2003.
7. Lee, L. G.; Spurgeon, S. L.; Heiner, C. R.; Benson, S. C.;