Development of a fluorescent chalcone library and its application in the discovery of a mouse embryonic stem cell probe

Article abstract of DOI:10.1039/c2cc31662e

We report the first fluorescent diamino-chalcone library and its application in the discovery of a mouse embryonic stem cell (mESC) probe. CDg4, a novel green fluorescent mESC probe was discovered through a high-content image based screening of 160 member

Full text of DOI:10.1039/c2cc31662e

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Development of a fluorescent chalcone library and its application in the
discovery of a mouse embryonic stem cell probew
a
a
Sung-Chan Lee,z Nam-Young Kang,z Sung-Jin Park,a Seong-Wook Yun,a
Yogeswari Chandrana and Young-Tae Chang*ab
Received 6th March 2012, Accepted 11th May 2012
DOI: 10.1039/c2cc31662e
We report the first fluorescent diamino-chalcone library and its
application in the discovery of a mouse embryonic stem cell
(mESC) probe. CDg4, a novel green fluorescent mESC probe
was discovered through a high-content image based screening of
160 members of the chalcone library. Interestingly, the molecular
binding target of CDg4 was identified as the glycogen of the stem
cell colony surface, rather than a conventional protein target from
an intracellular source.
Fig. 1 Structural design of Chalcone Library (CLA).
The diversity directed probe development approach by fluorescent
library synthesis and high throughput screening has been
demonstrated as a powerful tool for novel sensor/probe
discovery.1 Considering the limited number of currently available
fluorescent scaffolds, elucidation of novel chemical structures with
desirable fluorescence properties is one of the most important
components in expanding the diversity coverage of the tool boxes
in novel sensor/probe discovery. Herein, we report the first
diamino-chalcone based fluorescent dye library and the discovery
of a novel green fluorescent probe for mouse embryonic stem cells
(mESC).
emission intensity compared to other known mono oxygen or
mono amino chalcone fluorescent dyes.3
The synthesis of the CLA library is outlined in Scheme 1. Nitro
chalcone 1 was prepared by microwave assisted condensation of
4-nitroacetophenone and N-alkyl-4-aminobenzaldehyde using
pyrrolidine as a base. Intermediate 1 was loaded to 2-chlorotrityl
chloride resin and the nitro group was converted to an amino
group via SnCl2 reduction. The reduced amino group was then
coupled with 160 commercially available acid chlorides to
generate structural diversity within the chalcone scaffold. Mild
acidic cleavage with 2% trifluoroacetic acid in dichloromethane
was followed by simple filtration through a silica flock to afford
160 library compounds. Without further purification, LC–MS
Chalcones are considered to be precursors of flavonoids and
isoflavonoids in nature, and have been known to possess
various biological activities.2 Besides their medicinal applications,
the photophysical and photochemical properties of chalcones
have been neglected for a long time, whilst some chalcone
derivatives with proper electron push–pull pairs are known to
be fluorescent (Fig. 1).3
By analyzing the structures of known fluorescent chalcone
compounds, we designed a novel fluorescent ChaLcone Amide
library (CLA) as shown in Fig. 1. CLA compounds are
designed to have one amide and one amine on each side of
the scaffold as electron donors, to enhance the fluorescence
a Laboratory of Bioimaging Probe Development,
Singapore Bioimaging Consortium, Agency for Science,
Technology and Research (A*STAR), Biopolis, Singapore 138667
b Department of Chemistry, National University of Singapore,
MedChem Program of Life Sciences Institute,
National University of Singapore, 3 Science Drive 3,
Singapore 117543. E-mail: chmcyt@nus.edu.sg
w Electronic supplementary information (ESI) available: Experimental
procedures, spectroscopic data, cell image data. See DOI: 10.1039/
c2cc31662e
Scheme 1 Synthetic scheme: (a) 2-ClTr chloride resin, pyridine,
CH2Cl2, 5 h, RT; (b) SnCl2, 6% H2O in DMF, RT, overnight;
(c) acid chlorides, pyridine, CH2Cl2, 30 min.; (d) 2% TFA in CH2Cl2;
DMF = dimethylformamide, TFA = trifluoroacetic acid, RT = room
temperature, 2-ClTr = 2-chlorotrityl, *R is from 160 commercial acid
chlorides (Table S1w).
z These authors contributed equally.
c
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Chem. Commun., 2012, 48, 6681–6683 6681

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analysis of the library showed high purities (average purity
was 85% at 430 nm) and their spectroscopic properties were
measured individually and fully documented in Table S2w.
Most of the library compounds showed similar optical properties
with a maximum absorbance at 430 nm, and emission at 560 nm.
Quantum yields were distributed between 0.1 and 0.2 and the
cLogP (average 4.5) were in a reasonable range for biological
applications according to the Lipinski rule (Fig. S1w).4 In rare
cases, lower quantum yield (o0.1) was observed when strong
electron withdrawing groups were introduced on the amide
group. This may be attributed to the fact that the strong electron
withdrawing groups attenuate the electron donor’s pushing effect
to the chalcone scaffold. The structure and quantum yield
relationships are summarized in Table S3 and Fig. S2w.
For novel biological applications, the synthesized CLA
library was screened for mESC selective probes. Using whole
cell image based screening, our group has previously reported
one yellow (CDy1) and one blue (CDb8) mESC probe.5 We
envisioned the green color of CLA would allow the chance of
complementary multi-color imaging probe development in the
green range. All the CLA compounds were screened in mESC
and mouse embryonic fibroblast (MEF) as a negative control
in 384 well plates. The cell images were recorded by the
automated imaging microscope system, ImageXpress Microt,
and the fluorescent images were analyzed by cellular fluorescence
intensity using MetaXpresss image processing software. From
this initial screening, we selected 3 compounds that displayed
selective staining for mESC. Subsequently, we validated the
3 candidate compounds by flow cytometry to identify CDg4
(Compound of Designation green 4), as the only compound
which works both for imaging and flow cytometry (Fig. 2).
For further characterization of CDg4 in biological applications,
we induced differentiation of CDg4-stained mESC by removing
LIF from the culture media and allowing them to form embryoid
Fig. 3 Confocal images for a mESC colony stained by CDg4. Merged
fluorescent images of CDg4 and Hoechst 33 342 (left), CDg4
fluorescent image (middle: 2 mM of CDg4 is incubated for 1 h) and
nucleus staining image with Hoechst 33 342 (right). Scale bar: 10 mm.
Images of stained cell colonies were taken by ꢁ40 objective lenses.
bodies and spontaneously differentiate. The 3 types of germ layer
cells, which were confirmed by immunostaining of lineage specific
markers, were not stained by CDg4 (Fig. S3w). Then we split
the mESC and stained them with CDg4 at each passage to
investigate the effect of CDg4 on mESC proliferation. The
numbers of mESC colonies counted from the CDg4-stained
groups during 3 serial passages were 56 ꢀ 6, 56 ꢀ 6 and
57 ꢀ 2 per 4.05 mm2, which were not statistically different
from 50 ꢀ 5, 53 ꢀ 8 and 51 ꢀ 7 obtained from a non-treated
group (Fig. S4w). These results suggest that CDg4 does not
affect the normal growth, colony formation and differentiation
of mESC. For the subcellular localization analysis of CDg4 by
multi-colour imaging, we stained mESC using CDy1, CDb8, CDg4
and Hoechst 33 342. The multi-colour images (Fig. S5, S6w) and
confocal microscopy (Fig. 3) showed that CDg4 stains only the
outside of a mESC colony.
To validate the generality of the mESC staining property of
CDg4, we attempted to stain the E14 mESC cell line and a
mouse induced pluripotent stem cell (iPSC) which was generated
from the MEF of Oct4-GFP transgenic mice by ectopic expression
of Oct-4, Sox2, Klf4 and c-Myc. E14 was stained by CDg4 with a
similar pattern as shown in our mESC cell line (Fig. S7w). For
GFP expressing iPSC colony staining, we took the images before
and after CDg4 staining. The image taken after CDg4 staining
obviously showed larger area, which implies that the outsides of
the colonies were stained by CDg4 (Fig. S8w).
The stem cell colony surface is composed of numerous
polysaccharides, lipids, lipoproteins, and glycolipids which play
roles both in cell–cell interaction and embryo development.6
To investigate the possible binding target of CDg4, we performed
a systematic fluorescence response assay for CDg4 against
82 biological analytes in vitro (Fig. S9w).7 To our surprise,
CDg4 selectively responded to only glycogen among 82 analytes,
increasing its fluorescent intensity.
The selective response of CDg4 on glycogen brought us to a
further focused screening using various structurally related
carbohydrate polymers. Interestingly, CDg4 showed almost no
response to dextran, amylose, dermatan sulphate, hyaluronic
acid and heparin, which are structurally similar carbohydrate
polymers to glycogen (Fig. 4a), but uniquely responded to
glycogen in a dose dependent manner reaching up to a 66 fold
fluorescence increase. This was a striking observation, especially
for dextran and amylose, which have exactly the same building
blocks, i.e. glucose, with glycogen. Glycogen is a highly
branched glucose polymer with a(1 - 4) linkage in the main
chain and a(1 - 6) branch points. Amylose is a linear polymer
Fig. 2 (a) The mESCs were co-cultured on MEF. The bright field
(left) and fluorescent image (right) of CDg4. Scale bar: 200 mm. Images
of stained cell colonies were taken by ꢁ4 objective lenses. (b) Flow
cytometry dot plot images of mESC and MEF stained with DMSO as
a control and CDg4 (lab: 430 nm, lem: 560 nm, e: 23 600, QY: 0.2 in
DMSO).
c
6682 Chem. Commun., 2012, 48, 6681–6683
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suggest that glycogen is the binding target of CDg4 in mESC.
This is an intriguing example of a fluorescent small molecule
targeting a carbohydrate, considering the fact that most of the
conventional small molecule targets in chemical genetics are
proteins.11
The glycogen specific probe CDg4 may enable the detection
of mESC and the quantification of embryonic surface glyco-
gen levels.
This study was supported by intramural funding and grant
10/1/21/19/656 from the A*STAR (Agency for Science, Tech-
nology and Research, Singapore) Biomedical Research
Council.
Notes and references
1 (a) M. Vendrell, J. S. Lee and Y. T. Chang, Curr. Opin. Chem.
Biol., 2010, 14, 383; (b) J. S. Lee, N. Y. Kang, Y. K. Kim,
A. Samanta, S. H. Feng, H. K. Kim, M. Vendrell, J. H. Park
and Y. T. Chang, J. Am. Chem. Soc., 2009, 131, 10077;
(c) N. Y. Kang, H. H. Ha, S. W. Yun, Y. H. Yu and
Y. T. Chang, Chem. Soc. Rev., 2011, 40, 3613; (d) R. K. Das,
A. Samanta, H. H. Ha and Y. T. Chang, RSC Adv., 2011, 1, 573.
2 (a) S. S. Mokle, M. A. Sayyed, Kothawar and Chopde, Int. J.
Chem. Sci., 2004, 2, 96; (b) H. K. Hsieh, L. T. Tsao, J. P. Wang and
C. N. Lin, J. Pharm. Pharmacol., 2000, 52, 163; (c) G. S. Viana,
M. A. Bandeira and F. Matos, Phytomedicine, 2003, 10, 189.
3 (a) M. Gaber, T. A. Fayed, S. A. El-Daly and Y. S. El-Sayed,
Photochem. Photobiol. Sci., 2008, 7, 257; (b) K. Rurack,
J. L. Bricks, G. Reck, R. Radeglia and U. Resch-Genger,
J. Phys. Chem. A, 2000, 104, 3087; (c) C. G. Niu, A. L. Guan,
G. M. Zeng, Y. G. Liu and Z. W. Li, Anal. Chim. Acta, 2006,
577, 264; (d) V. Marchi-Artzner, B. Lorz, C. Gosse, L. Jullien,
R. Merkel, H. Kessler and E. Sackmann, Langmuir, 2003, 19, 835;
(e) M. Ono, R. Ikeoka, H. Watanabe, H. Kimura, T. Fuchigami,
M. Haratake, H. Saji and M. Nakayama, ACS Chem. Neurosci.,
2010, 1, 598; (f) P. F. Wang and S. K. Wu, J. Photochem.
Photobiol., A, 1995, 86, 109; (g) D. G. Powers, D. S. Casebier,
D. Fokas, W. J. Ryan, J. R. Troth and D. L. Coffen, Tetrahedron,
1998, 54, 4085; (h) A. Ullah, F. L. Ansari, I. Haq, S. Nazir and
B. Mirzab, Chem. Biodiversity, 2007, 4, 203; (i) A. R. Jagtap,
V. S. Satam, R. N. Rajule and V. R. Kanetkar, Dyes Pigm., 2011,
91, 20.
Fig. 4 (a) Fluorescence increase of CDg4 spectra (10 mM) upon
interaction with serial concentrations of glycogen (ex: 430 nm).
Fluorescent intensity was measured at 560 nm. The same concentra-
tions of dextran, dermatan sulphate, hyaluronic acid, amylose and
heparin were used. (b) Histogram of flow cytometry analysis. 0.5%
amylase was treated to mESC after 2 mM of CDg4 staining. The CDg4
signal was measured by flow cytometry. (c) CDg4 fluorescent images
(left) and bright field (right) images of mESC and mouse neurosphere
(NSP). Scale bar: 100 mm. (d) Glycogen quantification assay for mESC
and neurosphere (NSP).
with only a(1 - 4) linkage, and dextran has a(1 - 6) main
chains with short a(1 - 3) branches. We speculate the selective
response of CDg4 to glycogen is due to the unique secondary
structure derived from the a(1 - 6) branched glucose
structure. Also, glycogen is a globular shaped polymer with
longer branch units and a larger exposed area, compared to
dextran and amylose, which are in relatively more packed
forms.8 Glycogen is known for energy storage and the
accumulation is dramatically increased during early embryonic
development.9 High glycogen accumulation in the inner cell
mass (ICM) and also inter cellular localization has been
demonstrated as depicted in Fig. S10w.9c To confirm if
glycogen on the mESC colony surface is indeed responsible
for the selective staining of CDg4 to mESC in comparison to
other cell types, we hydrolyzed the surface glycogen of mESC
using amylase.
4 C. A. Lipinski, F. Lombardo, B. W. Dominy and P. J. Feeney,
Adv. Drug Delivery Rev., 2001, 46, 3.
5 (a) C. N. Im, N. Y. Kang, H. H. Ha, X. Z. Bi, J. J. Lee, S. J. Park,
S. Y. Lee, M. Vendrell, Y. K. Kim, J. S. Lee, J. Li, Y. H. Ahn,
B. Feng, H. H. Ng, S. W. Yun and Y. T. Chang, Angew. Chem.,
Int. Ed., 2010, 49, 7497; (b) K. K. Ghosh, H. H. Ha, N. Y. Kang,
Y. Chandran and Y. T. Chang, Chem. Commun., 2011, 47, 7488.
6 (a) A. V. Nairn, A. Kinoshita-Toyoda, H. Toyoda, J. Xie,
K. Harris, S. Dalton, M. Kulik, J. M. Pierce, T. Toida,
K. W. Moremen and R. J. Linhardt, J. Proteome Res.,
2007, 6, 4374; (b) R. J. Linhardt and T. Toida, Acc. Chem. Res.,
2004, 37, 431; (c) B. Thisse and C. Thisse, Dev. Biol., 2005,
287, 390.
The amylase treatment dramatically reduced the fluores-
cence intensity of mESC stained by CDg4 (Fig. 4b) indicating
that the molecular binding target of CDg4 is the glycogen
located on the surface of the mESC colony. Next, we examined
the CDg4 staining selectivity in neurosphere (NSP) to rule out
the possibility that CDg4 simply stains clustering cells. It was
clearly demonstrated that mouse NSP was not stained by
CDg4, despite their similar physical morphology (Fig. 4c).
To confirm the connection of this selectivity to glycogen
content on the colony surface, we quantified10 the glycogen in
each type of stem cell and found that mESC has 9 times higher
glycogen content than NSP (Fig. 4d). Altogether, these data
7 J. S. Lee, H. K. Kim, S. H. Feng, M. Vendrell and Y. T. Chang,
Chem. Commun., 2011, 47, 2339.
8 (a) B. J. Gibbons, P. J. Roach and T. D. Hurley, J. Mol. Biol.,
2002, 319, 463; (b) J. Lomako, W. M. Lomako and W. J. Whelan,
Biochim. Biophys. Acta, Gen. Subj., 2004, 1673, 45;
(c) A. M. Myers, M. K. Morell, M. G. James and S. G. Ball, Plant
Physiol., 2000, 122, 989.
9 (a) C. B. Ozias and S. Estern, Biol. Reprod., 1973, 8, 467;
(b) W. Dormeyer, D. V. Hoof, S. R. Braam, A. J. R. Heck,
C. L. Mummery and J. Krijgsveld, J. Proteome Res., 2008,
7, 2936; (c) S. Kolle, M. Stojkovic, K. Prelle, M. Waters,
E. Wolf and F. Sinowatz, Biol. Reprod., 2001, 64, 1826.
10 F. Huijing, Clin. Chim. Acta, 1970, 30, 567.
11 D. P. Walsh and Y. T. Chang, Chem. Rev., 2006, 106, 2476.
c
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Chem. Commun., 2012, 48, 6681–6683 6683