Chemiluminescent energy-transfer cassettes based on fluorescein and nile red

Article abstract of DOI:10.1002/anie.200603307

The long and the short of it: Luminol chemiluminesces with a beautiful blue color; however, to be useful for biotechnological applications, the emission must be shifted to much longer wavelengths. Energy-transfer cassettes like that shown in the picture p

Full text of DOI:10.1002/anie.200603307

Communications
DOI: 10.1002/anie.200603307
Chemiluminescence
Chemiluminescent Energy-Transfer Cassettes Based on Fluorescein
and Nile Red**
Junyan Han, Jiney Jose, Erwen Mei, and Kevin Burgess*
The two most common ways to induce chemiluminescence in
purely organic, nonbiological systems are to treat either
oxalate esters or luminol derivatives with basic hydrogen
peroxide.[1,2] Both these types of mixtures emit light at
relatively short wavelengths, which are not ideal for applica-
tions in biotechnology. Luminol, for instance, emits in the
range 420–450 nm, depending on the solvent media.[3] Inti-
mate mixtures of oxalate esters or luminol,[4] an oxidant, and
an acceptor dye give longer wavelength emissions through
intermolecular energy transfer. This results in the mesmeriz-
ing, long-lived emissions seen in “light-stick devices”. How-
ever, the options for forming discrete probes for biotech-
nology that emit at longer, and generally more useful,
wavelengths are limited.[5–10]
gave organic-soluble, easily chromatographed intermediates,
and the PMB group is removed in the closing stages of the
synthesis through treatment with trifluoroacetic acid (TFA).
Thus, Scheme 1 shows the syntheses that evolved to form
compounds 1 and 2. In both routes, the cyclic hydrazide 3 was
bis-N-protected, then elaborated through Sonogashira reac-
tions[12] featuring derivatives of 5-bromofluorescein[13] and 2-
hydroxy nile red.[14] The route to the cassettes would have
been more convergent if an alkyne derivative of luminol
could have been coupled with halogenated/triflated acceptors,
but that approach was ineffective.
It is hard to describe in words the spectacular chemilu-
minescence of these compounds without films of the experi-
An ongoing project in our group features twisted, but
otherwise conjugated, donor and acceptor cassettes for
labeling biomolecules.[10,11] The motivation for this is that
energy transfer can occur through bonds as well as through
space, hence it can be relatively fast and efficient. All our
published research to date features cassettes based on UV-
absorbing donors, like compound A. We thought it would be
intriguing to make cassettes where the donor might be
activated chemically instead. Oxalate esters are not useful
donors for through-bond energy-transfer cassettes because it
is impossible to conjugate an acceptor to the oxalate frag-
ment. Consequently, luminol-based systems were selected.
Described herein are the syntheses and spectroscopic proper-
ties of the fluorescein- and nile red based, chemically
activated cassettes 1 and 2.
Nearly all luminol derivatives are almost insoluble in most
organic media, and this makes them extremely difficult to
manipulate. After considerable experimentation, one solution
to this problem emerged: bis(N-protection) of compounds
like 3 with 4-methoxybenzyl (PMB) groups. This approach
[*] J. Han, J. Jose, Prof. K. Burgess
Department of Chemistry
Texas A&M University
Box 30012, College Station, TX 77841-3012 (USA)
Fax: (+1)979-845-8839
E-mail: burgess@tamu.edu
Dr. E. Mei
Department of Chemistry
University of Pennsylvania
Philadelphia, PA 19104 (USA)
[**] We thank Prof. Robin M. Hochstrasser (University of Pennsylvania)
for helpful discussions and the National Institutes of Health
(GM72041) and The Robert A. Welch Foundation for providing
financial support.
Supporting information for this article is available on the WWW
under http://www.angewandte.org or from the author.
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Scheme 1. Preparation of a) the fluorescein cassette 1 and b) the nile red based cassette 2. MW=microwave treatment, Tf=trifluoromethane-
sulfonyl, TBAF=tetrabutylammonium fluoride, TMS=trimethylsilyl.
ments (Figure 1). For cassette 1, 100-mLaliquots of the
compound (10À5 m, in pH 10 aqueous Na2CO3/NaHCO3 buffer
solution) were added with stirring to a sample cell containing
CuSO4 (1.5 ” 10À3 m) and H2O2 (2.0 ” 10À3 m). Cassette 2 is not
very soluble in aqueous media and, in any case, the quantum
yield for nile red emission is less than 0.1 in water.
Consequently, for 2, potassium tert-butoxide in THF
(10À2 m) was added to the compound dissolved in anhydrous
N,N-dimethylformamide (DMF; 10À5 m). This experiment was
done open to the air, and oxygen is presumed to be the
oxidant. Luminol, under the conditions used for cassette 1,
gives a bright blue emission. However, if efficient energy
transfer occurs for compounds 1 and 2, then it is expected that
yellow/green and red chemiluminescence are emitted, which
are characteristic of fluorescein and nile red, respectively.
This is exactly what we saw. Cassette 1 gave a bright yellow/
green emission, whereas 2 glowed with a less-intense red
color. No trace of blue in the emission was seen in either case.
Figure 1. Pictures of a) luminol, b) cassette 1, and c) cassette 2 when
activated with an oxidant.
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Communications
Compounds 9 and 10 were prepared as controls (see the
chemiluminescence output of the phthalate derived from
luminol and the UV absorption of the acceptor part of
cassettes 9 and 10 is shown in Figure 2b. Normalized
chemiluminescence emissions for 1 and 2 are shown in
Figure 2c. The emissions of 1 and 2 are sharp and character-
istic of the acceptors only; no chemiluminescence from the
donor was detected.
Supporting Information) as it is thought that the excited
species from luminol derivatives involves the corresponding
phthalate dianions. Indeed, ESIMS analysis of cassettes 1 and
2 after the oxidative activation revealed the presence of 9 and
10, respectively. This was confirmed through HPLC analyses
in the case of 1.
Figure 2a shows normalized UV absorption and fluores-
cence spectra for 9 and 10. The extent of overlap between the
Sometimes, the eyes can play tricks on the brain and that
is partially true in this case. The chemiluminescence from
cassette 2 appears to be weaker than that for 1, but the
quantitative data collected in Table 1 indicates that this is not
Table 1: Selected spectroscopic properties of luminol, 1, 9, 2, 10, and 11.
Cmpd.
UV
Fluorescence
Chemiluminesence
labs max [nm] lfl max [nm]
lchemi max [nm] Rel. Fchemi
luminol[a]
1[a]
–
–
442
524
–
634
–
100
61
–
>100
–
494
493
558
558
–
518
519
628
628
–
9[a]
2[b]
10[b]
11
412
0.02[c]
[a] In carbonate/bicarbonate buffer solution. [b] In anhydrous DMF.
[c] From reference [6].
the case. Chemiluminescent quantum yields measured rela-
tive to luminol indicate that 2 actually emits more strongly.
Probably, chemiluminescence from 2 appears to be weaker
than that from 1 because the human eye is about five times
more sensitive to light near the emission maximum of
fluorescein than it is to light emitted from the nile red
acceptors.[15]
The relative quantum yields for chemiluminescence that
are presented in Table 1 use luminol as a standard. However,
the donor fragments of cassettes 1 and 2 do not
have the amino substituent of luminol. Small
changes to the luminol structure tend to reduce
its chemiluminescence dramatically.[3] If the
emissions from cassettes 1 and 2 are compared
with the hydrazide 11 (which has a much lower
absolute quantum yield for chemilumines-
cence), then the data for cassettes 1 and 2
would appear to be even more impressive.
Experimentally, it is extremely challenging to determine
the extent of energy transfer through bonds and through
space in twisted but otherwise conjugated cassettes. For the
UV-activated system A, we asserted that through-bond
energy transfer must be fast and efficient by considering
rates of energy transfer.[16] However, direct observation of
rates is hard in chemically activated systems in which
excitation of the donor occurs continuously. Further, we
have so far been unable to prepare the logical control
compounds for comparison: those in which the alkyne linker
of cassettes 1 or 2 are replaced by an ethylene fragment. In
any case, through-space energy transfer for those controls
might differ considerably from that occurring in 1 or 2
because the orientation of the donor and acceptor fragments
would be dynamic in the reduced compounds. However, the
Figure 2. a) Normalized UV/Vis (UV) and fluorescence (Fl) spectra of
9 in pH 10 aqueous sodium carbonate/bicarbonate buffer solution,
and of 10 in anhydrous DMF; b) normalized chemiluminescence (CL)
spectrum of luminol (blue), UV/Vis absorption bands of compound 9
(green) and compound 10 (red); c) normalized chemiluminescence
spectra of luminol (blue), cassette 1 (green), and cassette 2 (red).
In =normalized intensity.
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through-space energy-transfer cassette B, based on luminol,
was prepared approximately four decades ago and does
provide an interesting comparison.[17–19] The reported relative
chemiluminescence quantum yield for this compound (lumi-
nol standard) is significantly less than that measured here for
cassettes 1 and 2. It may be that, just as in our UV-activated
cassettes like A, rapid and efficient energy transfer can occur
for the systems that facilitate the possibility of through-bond
energy transfer.
chemically activated energy transfer can be applied in
biotechnology.
Received: August 13, 2006
Revised: December 6, 2006
Published online: January 19, 2007
Keywords: UV/Vis spectroscopy · fluorescent probes ·
.
FRET (fluorescence resonant energy transfer) · imaging agents ·
luminescence
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