Combinatorial discovery of full-color-tunable emissive fluorescent probes using a single core skeleton, 1,2-dihydropyrrolo[3,4-β]indolizin-3-one

Article abstract of DOI:10.1021/ja8020268

We developed a novel fluorescent core skeleton, 1,2-dihydropyrrolo[3,4-β]indolizin-3-one, by complexity-generating one-pot reactions through 1,3-dipolar cyclization followed by oxidative aromatization. This fluorescent core skeleton can accommodate various wavelengths of emission maxima by changing the electronic properties of substituents, which was postulated by computational studies. The full-color-tunable emission maxima were achieved with a single core skeleton by changing the substituents using the combinatorial approach. These novel fluorophores have excellent photophysical and photochemical properties: moderate to excellent quantum yields, resistance to the photobleaching, pH-independent fluorescence, large Stokes shifts, druglike lipophilicity for membrane permeability, etc. Further, we successfully demonstrated the bioapplication of fluorophores B1 and B5 in the immunofluorescence for visualizing cellular compartments of HeLa cells. Copyright

Full text of DOI:10.1021/ja8020268

Published on Web 08/23/2008
Combinatorial Discovery of Full-Color-Tunable Emissive Fluorescent Probes
Using a Single Core Skeleton, 1,2-Dihydropyrrolo[3,4-ꢀ]indolizin-3-one
Eunha Kim, Minseob Koh, Jihoon Ryu, and Seung Bum Park*
Department of Chemistry, Seoul National UniVersity, Seoul 151-747, Korea
Received March 19, 2008; E-mail: sbpark@snu.ac.kr
Scheme 1. Synthetic Scheme of Fluorescent Core Skeletona
Fluorescent probes have been used extensively as research tools
in biological science, clinical diagnosis, and drug discovery because
of their high sensitivity and ease of handling.1 In addition to their
applications in biomedical research, the study of fluorescent
materials has become a popular research area because of their
industrial application as organic light-emitting diodes (OLEDs).2
Despite the high demand of fluorophores, there exists only a limited
number of fluorescent core skeletons with flexibility in their
synthetic strategies for tunable emission wavelengths.3 Many
strategies for the discovery of fluorescence probes have been
empirically pursued, one at a time lacking flexibility. In addition,
the rational design of fluorescent probes with specific emission
wavelengths and high quantum yields is still difficult owing to the
a Reagents and conditions: (a) R,ꢀ-unsaturated aldehydes, AcOH,
Na2SO4, CH2Cl2, room temp; then NaBH4 in MeOH, 0 °C; (b) bromoacetyl
bromide, TEA, CH2Cl2, -78 °C; (c) pyridines; (d) DBU in toluene; then
DDQ; (e) HF/pyridine/THF (v:v:v ) 5:5:90), then TMSOMe.
complexity of the underlying photophysical phenomena.4 In this
paper, we report the de novo design and discovery of novel
fluorescent core skeletons with full-color-tunable emissive properties
using the combinatorial approach.5
The discovery of this novel core skeleton was based on our original
attempt for synthetic pathway development of a novel core skeleton
using diversity-oriented synthesis,6 and we identified a novel molecular
framework 6 which was partially formed via spontaneous aromatization
from cycloadducts 5. This fluorescent core skeleton 6 was designed
to be synthesized with excellent substituent-pending potentials for the
construction of diverse fluorescent compounds. Prior to the construction
of a fluorescent-compound library, we pursued a rational design
through theoretical calculation for the identification of electronic
perturbation positions on our core skeleton. The orbital shapes of the
highest occupied molecular orbital (HOMO) and the lowest unoccupied
molecular orbital (LUMO) of our fluorophore were examined;10 the
R1 position at the LUMO has a significantly smaller lobe than that at
the HOMO, whereas their difference at the R2 position is less
pronounced. On the basis of this calculation, we reasoned that the
introduction of an electron-donating group (EDG) on the R1 position
could trigger a bathochromic shift in our fluorescent compounds, while
it was difficult to systematize the electronic perturbation of the
substituents on R2 position onto the influence on emissive properties.7
Through the deliberation of computational studies, the construction
of various fluorophores in a single molecular framework was carried
out using the combinatorial approach with five R,ꢀ-unsaturated
aldehydes (R1, A-E) and five pyridine derivatives (R2, 1-5) of
different electronic properties: A series of several straightforward
reactions yield azomethine ylide precursor 4. The crude products of 4
were then subjected to 1,3-dipolar cycloaddition8a in toluene in the
presence of DBU. The resulting cycloaddition products 5 were
successfully transformed into our desired products 6, 1,2-dihydro-
pyrrolo[3,4-ꢀ]indolizin-3-one, via the subsequent oxidative aromati-
zation with DDQ in a one-pot fashion with reasonable yields (63% in
three-step yields, Scheme 1).8b Overall, a total of 24 novel fluorescent
compounds with a single core skeleton were successfully synthesized
by solution-phase parallel synthesis.
With the compilation of the fluorescent-compound library, we
evaluated the photochemical properties of each individual fluoro-
phore in our library. As shown in Table 1, the overlaid fluorescent
emission spectra highlight the development of full-color-tunable
emissive fluorophores.10 It is unprecedented to achieve a dramatic
change in the fluorescence properties within a single molecular
framework simply by changing the substituents at just two variation
points of the fluorophore. The direct comparison of the electronic
properties of the R1 substituents clearly demonstrates the tunablity
of this core skeleton. The electron-donating potentials are increased
in the following order: methyl (A5), phenyl (B5), 2-methoxyphenyl
(C5), 2-thiophenyl (D5), and 4-dimethylamino phenyl (E5) on a
single skeleton with fixed R2 substituent such as acetyl group. The
corresponding emission wavelengths of A5, B5, C5, D5, and E5
are 471, 507, 508, 540, and 613 nm, respectively. The bathochromic
shift of emission wavelengths is consistent with our initial prediction
based on our quantum mechanics calculation. Changes of the
substituents on R2 position also trigger the notable bathochromic
shift, which required further investigations for the rationalization.
Among our novel fluorophores, we chose B5 for direct comparison
with the well-known fluorescent molecule fluorescein since B5 has a
comparable quantum yield [ΦF: 0.74] and considerably larger Stokes
shift that enables intense fluorescent signals with the minimal back-
ground.11 In addition, B5 is more resistant to photobleaching than
fluorescein. Fluorescein is well-known to have multiple ionization
equilibria, which leads to pH-dependent absorption and emission,
whereas, because of its novel molecular framework, the fluorescence
intensity and maximum emission wavelength of B5 are not influenced
by pH value in the range 4-9 (physiological range of in vivo system),
that is an important element in bioimaging and bioapplication.12 To
confirm the bioapplicability of our fluorophores, we employed them
for protein modification. Because our novel fluorescent molecules were
designed with hydroxy functionality, the functional-group inter-
9
12206 J. AM. CHEM. SOC. 2008, 130, 12206–12207
10.1021/ja8020268 CCC: $40.75
2008 American Chemical Society

C O M M U N I C A T I O N S
Table 1. Photophysical Data of All Fluorophores (A1-E5)
The representative fluorophores (B1 and B5) with amine moiety
were then charged with a maleimide group for the specific
conjugation with thiol moiety of biopolymers.10 As shown in Figure
1, our fluorophores were labeled on protein-of-interests and
successfully applied in immunocytochemistry via direct labeling
of 1°Ab (Erbitux) or labeling of 2°Ab to visualize nucleol and
epidermal growth factor receptor (EGFR) as model systems.
In summary, we have developed a full-color-tunable fluorescent
core skeleton, 1,2-dihydropyrrolo[3,4-ꢀ]indolizin-3-one, by com-
plexity-generating one-pot reactions. This core skeleton can ac-
commodate various emission maxima simply by changing substit-
uents at the R1 and R2 positions, having different electronic
properties. These novel fluorophores have excellent photophysical
and photochemical properties, resistance to photobleaching, moder-
ate to excellent quantum yields, pH-independent fluorescence, large
Stokes shifts, and druglike lipophilicity for membrane permeability.
Further, we have successfully demonstrated the bioapplication of
our fluorophore in the immunofluorescence.
d
Cpd
R1
R2
clogP λabs (nm)a λem (nm)b gap (eV)c ΦF
A1 methyl
A2 methyl
A3 methyl
A4 methyl
A5 methyl
B1 phenyl
B2 phenyl
B3 phenyl
B4 phenyl
B5 phenyl
C1 o-methoxy phenyl
C2 o-methoxy phenyl
C3 o-methoxy phenyl
C4 o-methoxy phenyl
C5 o-methoxy phenyl
D1 thiophenyl
D2 thiophenyl
D3 thiophenyl
D4 thiophenyl
D5 thiophenyl
E1 p-dimethylaminophenyl hydrogen 3.15
E2 p-dimethylaminophenyl methoxy 3.16
E3 p-dimethylaminophenyl phenyl
E4 p-dimethylaminophenyl nitrile
E5 p-dimethylaminophenyl acetyl
hydrogen 1.59
methoxy 1.61
326
-
434
-
2.92
2.71
2.55
2.53
2.27
2.80
2.64
2.47
2.41
2.17
2.82
2.64
2.46
2.39
2.15
2.55
2.46
2.30
2.13
1.92
2.53
2.44
2.17
2.03
1.78
0.41
-
phenyl
nitrile
acetyl
3.48
1.06
1.12
349
342
396
298
320
350
388
403
320
323
381
391
404
322
335
283
398
349
298
311
403
428
440
433
460
471
420
481
490
497
507
461
465
489
493
508
481
500
515
529
540
495
480
530
509
613
0.19
0.76
0.82
0.57
0.27
0.83
0.65
0.74
0.37
0.26
0.69
0.55
0.71
0.08
0.03
0.11
0.15
0.35
0.10
0.03
0.21
0.13
0.15
hydrogen 2.97
methoxy 2.99
phenyl
nitrile
acetyl
4.87
2.45
2.50
hydrogen 2.34
methoxy 2.36
Acknowledgment. This work was supported by the Korea
Science and Engineering Foundation (KOSEF), MarineBio Tech-
nology Program funded by Ministry of Land, Transport, and
Maritime Affairs (MLTM), Korea, and the Research Program for
New Drug Target Discovery grant from the Ministry of Education,
Science & Technology (MEST). E. Kim and M. Koh are grateful
for the award of the Seoul Science Fellowship award and for the
BK21 fellowship awards.
phenyl
nitrile
acetyl
4.23
1.82
1.87
hydrogen 2.84
methoxy 2.85
phenyl
nitrile
acetyl
4.72
2.31
2.36
5.04
2.63
2.67
Supporting Information Available: Experimental procedures,
complete spectroscopic characterization data of all new compounds,
and results of computational studies. This material is available free of
charge via the Internet at http://pubs.acs.org.
a Only the longest absorption maxima are shown. b Excited at the maximum
excitation wavelength. c Value of calculated energy gap between the HOMO and
LUMO. d Absolute fluorescence quantum yield. Absolute quantum yields of
known fluorescent dyes in various wavelengths were measured to confirm the
reliability of the system [anthracene: ΦF ) 0.27 (reported: 0.27);9a fluorescein:
ΦF ) 0.76 (reported: 0.79);9b cresyl violet: ΦF ) 0.48 (reported:
0.54)9c].
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Figure 1. Immunofluorescence image of HeLa cell. Nucleus stained by
Hoechst (A, J); nucleol targeted by antinucleol 1°Ab and visualized by B5-
labeled antimouse 2°Ab (B, G) or B1-labeled antimouse 2°Ab (D); Actin
visualized by TRITC-labeled phalloidin (E); EGFR targeted by Erbitux and
visualized by B1-labeled antihuman 2°Ab (H); EGFR visualized by B5-
labeled Erbitux (K). Panels C, F, I, and L are merged images of A and B,
D and E, G and H, and J and K, respectively.10
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