Combinatorial discovery of novel fluorescent dyes based on Dapoxyl

Article abstract of DOI:10.1016/S0040-4039(02)00999-1

We have developed a combinatorial method for the fast and effective synthesis of a library based on a known fluorescent dye, Dapoxyl using parallel solution-phase chemistry. By screening the 140 new compounds using fluorescence-based assays, we identified three new fluorescent dyes with novel properties.

Full text of DOI:10.1016/S0040-4039(02)00999-1

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 43 (2002) 5083–5086
Combinatorial discovery of novel fluorescent dyes based
on Dapoxyl™
Qing Zhu,a Hai-Shin Yoon,b Puja B. Parikh,b Young-Tae Changb and Shao Q. Yaoa,c,
*
aDepartment of Chemistry, National University of Singapore, 3 Science Drive 3, Singapore 117543, Singapore
bDepartment of Chemistry, New York University, New York, NY 10003, USA
cDepartment of Biological Sciences, National University of Singapore, 3 Science Drive 3, Singapore 117543, Singapore
Received 2 April 2002; revised 17 May 2002; accepted 24 May 2002
Abstract—We have developed a combinatorial method for the fast and effective synthesis of a library based on a known
fluorescent dye, Dapoxyl™ using parallel solution-phase chemistry. By screening the 140 new compounds using fluorescence-based
assays, we identified three new fluorescent dyes with novel properties. © 2002 Elsevier Science Ltd. All rights reserved.
Fluorescent dyes are widely used in the detection, quan-
titation, identification and characterization of inorganic
and organic compounds, and of biological structures
and processes.1 Recently, the use of fluorescent dyes to
label biologically important molecules such as DNA,
proteins, drugs etc. has grown markedly, particularly as
a result of advances in biotechnology.2 Little, however,
is known regarding how subtle structural changes in a
dye give rise to profound effects in its fluorescence
properties, and in many cases these effects are less than
trivial to predict. Until now,3 most research directed
towards the discovery of new fluorescent dyes has been
limited to the synthesis of individual compounds and
studies of their fluorescence properties, one compound
at a time.4 This, as a result, has greatly hindered the
rapid development of novel fluorescent dyes.
Dapoxyl™ dyes (2-pyridyl-5-aryl-oxazoles) and ana-
logues are commercially available and are used widely
as pH indicators and molecular probes.4 They have
attracted our attention not only because of their envi-
ronment-dependent sensitivity, but also their unique
properties which allows for their selective accumulation
in acidic organs such as lysosomes.5
be readily adaptable to automation, representing a cost-
effective tool in the field of preparative chemistry,
which increases throughput and reagent diversity.7 In
this paper, we describe the development of a fast,
parallel synthesis of fluorescent dyes based on the core
structure of Dapoxyl™. By combining the ability of
combinatorial chemistry to manipulate substituted
groups on the dye efficiently, together with available
instrumentation for high-throughput, fluorescence-
based screening, we hoped to identify new dyes that
possess novel fluorescence properties such as a large
Stokes shift, high quantum yields and favorable pH and
solvent profiles.
The synthesis of the dye library is shown in Fig. 1.
First, individual analogues of nicotinic acid A (27 in
total) were reacted with individual analogues of 2-
aminoacetophenone B (nine in total; Fig. 1) in the
presence of 1,3-diisopropylcarbodiimide (DIC), using
parallel solution-phase chemistry, to generate amides C,
which, without further purification, were first evapo-
rated to dryness, then concentrated sulfuric acid was
added.8 The mixture was stirred overnight to afford D.
Purification of D was achieved by addition of ice water
and 25% ammonia solution to the crude product, and
the resulting aqueous solution was extracted with ethyl
acetate. The organic layer was washed with sat. NaCl
and evaporated to give the final product D in 40–80%
purity for subsequent fluorescence screenings. In all,
with this solution-phased parallel approach, a total of
140 new compounds of type D were successfully synthe-
sized (as judged by TLC of the products and their
fluorescence spectra), of which more than 20% were
Over the last 15 years, combinatorial chemistry has
grown to become one of the most powerful tools used
in the areas of drug discovery, solid-state materials,
homogenous and heterogeneous catalysts, polymeric
materials and so on.6 Combinatorial approaches, based
on both solid-phase and solution-phase chemistry may
* Corresponding author. Tel.: +65-68741683; fax: +65-67791691;
e-mail: chmyaosq@nus.edu.sg
0040-4039/02/$ - see front matter © 2002 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(02)00999-1

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Q. Zhu et al. / Tetrahedron Letters 43 (2002) 5083–5086
Figure 1. Synthesis of the Dapoxyl™ library with building blocks A and B.
microplate reader made it simple to carry out the
randomly chosen and further characterized with LC-
MS to confirm unambiguously their purity and identity
(Table 1).
screening in a 96-well format in a rapid yet automated
fashion. Both excitation and emission spectra of the
compounds in different solvents under different pH
conditions (e.g. DMF, chloroform, methanol:water
(1:1), methanol and pH 4, 7, 10 buffer solutions9) were
recorded. The determined excitation maxima (uex=270–
380 nm) and the emission maxima (uem=380–480 nm)
covered a wide range of values. To our surprise,
replacement of an aromatic ring in the dye structure
Synthesis of some of the compounds (listed in Table 1)
was either not attempted or attempted unsuccessfully,
probably because most of them contain competing
functional groups (-COOH or -NH2) that interfere with
the regiospecific coupling between A and B. Other
groups, such as -OH in A24, were more tolerable and
thus in some cases required no protection in order to
carry out the condensation and the subsequent cycliza-
tion step to generate the desired products (e.g. A24B4).
Since our approach was to develop a simple and
efficient synthetic route for rapid access to a large
number of new compounds for subsequent fluorescence
screening, no attempt was made to introduce appropri-
ate protecting groups in some of the building blocks or
their corresponding intermediates.
with fused rings (i.e. A8,9,13B1–9) did not lead to drastic
red/blue shifts of excitation/emission wavelengths,
which have often been observed in other dyes, further
indicating the unpredictable effect of different sub-
stituents on the dye structure on its fluorescence prop-
erties. Out of all compounds screened, we identified
three compounds (A2B1, A2B7, A18B7; Fig. 2(a)) which
have unique fluorescence properties. They all have large
Stokes shifts and show strong fluorescence in aqueous
buffers under different pH conditions, and in the pres-
ence of organic solvents where most other compounds
have little or no fluorescence. In order to confirm this
All 140 compounds were screened for desired fluores-
cence properties. The use of a fluorescence-based

Q. Zhu et al. / Tetrahedron Letters 43 (2002) 5083–5086
Table 1. Analogues of Dapoxyl™ synthesized
5085
A1
A2
A3
A4
A5
A6
A7
A8
A9
A10
A11
A12
A13
A14
B1
B2
B3
B4
B5
B6
B7
B8
B9
ã
ã
ã
ã
ã
ã
ã
x
ã
ã
x
ã
x
ã
ã
ã
ã
ã
ã
ã
x
ã
ã
ã
ã
ã
ã
ã
x
ã
ã
ã
ã
ã
ã
ã
x
ã
ã
ã
ã
ã
ã
ã
x
ã
ã
ã
ã
ã
ã
ã
x
ã
ã
ã
ã
ã
ã
ã
x
ã
ã
ã
ã
ã
ã
ã
x
ã
ã
ã
ã
ã
ã
ã
x
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ã
ã
ã
ã
ã
ã
x
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ã
ã
ã
ã
ã
ã
x
ã
ã
ã
ã
ã
ã
ã
x
ã
ã
ã
ã
ã
ã
ã
x
x
ã
ã
ã
x
x
x
x
x
x
x
x
x
x
x
x
x
A15
A16
A17
A18
A19
A20
A21
A22
A23
A24
A25
A26
A27
B1
B2
B3
B4
B5
B6
B7
B8
B9
ã
ã
ã
ã
ã
ã
ã
x
ã
ã
ã
ã
ã
ã
ã
x
ã
ã
ã
ã
ã
ã
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ã
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x
x
x
ã: Synthesized successfully (as judged by TLC and fluorescence); x: not synthesized or attempted unsuccessfully.
Figure 2. (a) Structures of selected dyes; (b) spectra of the dyes. Each graph represents spectra of a dye under different conditions
(shown in different colors), where both excitation (left spectrum) and emission (right spectrum) under the same conditions, are
represented with the same color.
unambiguously, we subsequently synthesized these three
compounds individually, purified them to homogeneity
(by HPLC) and further characterized their fluorescence
properties with a conventional fluorimeter. For compari-
son, compound A18B2, which does not show the same
pH- and solvent-independent fluorescence profiles from
our screenings, was also synthesized.
tions in aqueous solutions and in the presence of organic
solvents such as 50% methanol, in good agreement with
results obtained from the high-throughput, 96-well-for-
mat screening, further validating both the synthetic and
the 96-well screening strategies we have adopted. It is
worth noting that all three compounds contain halogen
groups on their aromatic rings. It had previously been
demonstrated that halogen substitution affects the pH
profile of some known fluorescent dyes.1 The reference
compound, A18B2 on the other hand, shows strong pH-
As shown in Fig. 2(b) and Table 2, A2B1, A2B7 and A18B7
all show strong fluorescence under different pH condi-

5086
Q. Zhu et al. / Tetrahedron Letters 43 (2002) 5083–5086
Table 2. Fluorescence properties of selected dyes in different solutions9
Methanol:water (1:1)a
pH 4
(nm)
pH 7
(nm)
pH 10
(nm)
m0 (×10−3
)
uex
/
(nm)
€
uex
/
€
uex
/
uex
/
€
f
em
f
em
f
em
em
A2B1
A2B7
A18B7
A18B2
20.3
28.4
23.9
23.9
350/410
352/400
325/400
339/450
0.63
0.88
0.74
0.74
364/444
360/453
373/477
380/450
0.66
0.74
0.60
0.04
350/410
350/400
325/400
360/460
0.50
0.49
0.59
0.07
354/410
350/420
328/406
340/466
0.55
0.52
0.39
0.14
b
a The quantum yields were determined using 2-pyridyl-5-phenyloxazole in methanol: water (1:1) as the reference standard (€f=0.88).
b An example of a dye screened which does not have the desired fluorescence profile.
as well as solvent-dependent fluorescence profiles, cor-
relating well with those generated from microplate
screenings. Quantum yields, as well as m0 of the com-
pounds were also determined and showed similar values
to that of the original Dapoxyl™.
5. Diwu, Z.; Chen, C. S.; Zhang, C.; Klaubert, D.; Haugland,
R. P. Chem. Biol. 1999, 6, 411–418.
6. (a) Wang, J.; Yoo, Y.; Gao, C.; Takeuchi, I.; Sun, X.;
Chang, H.; Xiang, X. D.; Schultz, P. G. Science 1998, 279,
1712–1714; (b) Jandeleit, B.; Schaefer, D. J.; Powers, T. S.;
Tuner, W. H.; Weinberg, W. H. Angew. Chem., Int. Ed.
1999, 38, 2494–2532.
7. (a) Edwards, P. J.; Gardner, M.; Klute, W.; Smith, G. F.;
Terrett, N. K. Curr. Opin. Drug. Discov. Develop. 1999, 2,
321; (b) Floyd, C. D.; Leblanc, C.; Whitaker, M. Prog.
Med. Chem. 1999, 36, 91; (c) Balkenhohl, F.; Bussche-
Hu¨nnefeld, C.; Lansky, A.; Zechel, C. Angew. Chem., Int.
Ed. Engl. 1996, 35, 2288.
In conclusion, we have developed a new method for
fast and effective synthesis of a fluorescent dye library
based on the known dye Dapoxyl™, using parallel
solution-phase chemistry. By combining this method
with high-throughput fluorescence screening, we have
identified three new dyes that possess novel fluorescence
properties. We are currently assessing other dyes in this
library for novel fluorescence properties. This approach
may be general and applicable to the screening of other
fluorescent dye libraries.
8. Representative example of dye synthesis. Synthesis of 2-
pyridyl-5-(4-fluorophenyl)oxazole (A18B7): Isonicotinic
acid A18 (0.02 mmol) and 2-amino-4%-fluoroacetophenone
B7 (0.02 mmol) were dissolved in 0.5 mL DMF, and 50 mL
DIC was added to the solution. After 1 h, the reaction was
filtered and evaporated under vacuum. Concentrated sul-
furic acid (0.5 mL) was added and the reaction was stirred
overnight before quenching with 5 mL of ice water and 2
mL of 25% ammonia. The product was extracted into 30
mL of ethyl acetate, followed by washing with a sat. NaCl
solution, and dried with anhyd. MgSO4. Evaporation in
vacuo gave the crude product with purity typically ranging
from 40 to 80%. Further purification and characterization
were performed on an LCQ-MAT LC-MS (Finnigan,
USA). HPLC was performed using Phenomenex RP-18
(analytic: 5 mm, 4.6×250 mm; semi-preparative: 5 mm,
10×250 mm) columns with an acetonitrile–water gradient
(with 0.1% TFA). Fluorescence was recorded using a
SpectraMAX™ Gemini XS fluorescence plate reader
Acknowledgements
Funding support from the National University of Sin-
gapore is acknowledged.
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1
(Molecular Devices, USA). Mp 152–153°C, H NMR (300
MHz, CDCl3, 25°C): l=8.78 (broad s, 2H), 7.96 (broad s,
2H), 7.73 (m, 2H), 7.47 (s, 1H), 7.18 (m, 2H). ESI-MS:
m/z (%): 241.3 (100) [M++1].
9. Compounds were dissolved in an organic solvent (MeOH
or DMSO). A drop of the solution was added to 2 mL of
freshly prepared aqueous buffer solutions (Thomas Scien-
tific, USA). The amount of organic solvent was estimated
to be <0.5% of the total volume.