Solid phase combinatorial synthesis of a xanthone library using click chemistry and its application to an embryonic stem cell probe

Article abstract of DOI:10.1039/c1cc11962a

We report the first solid phase synthesis of a xanthone library CX and its application to embryonic stem cell probe development. The CX library was further derivatised with an activated ester resin to provide an acetylated CX (CXAC) library. Screening of these libraries led to the discovery of a novel fluorescent mESC probe, CDb8.

Full text of DOI:10.1039/c1cc11962a

View Article Online / Journal Homepage / Table of Contents for this issue
Dynamic Article Links
ChemComm
Cite this: Chem. Commun., 2011, 47, 7488–7490
www.rsc.org/chemcomm
COMMUNICATION
Solid phase combinatorial synthesis of a xanthone library using click
chemistry and its application to an embryonic stem cell probew
Krishna Kanta Ghosh,a Hyung-Ho Ha,b Nam-Young Kang,c Yogeswari Chandranc and
Young-Tae Chang*ac
Received 7th April 2011, Accepted 17th May 2011
DOI: 10.1039/c1cc11962a
We report the first solid phase synthesis of a xanthone library CX
and its application to embryonic stem cell probe development. The
CX library was further derivatised with an activated ester resin to
provide an acetylated CX (CXAC) library. Screening of these libraries
led to the discovery of a novel fluorescent mESC probe, CDb8.
The structural similarity of the xanthone to rosamine library,
which CDy1 belongs to, was also considered for the library
design. Herein, we report the first solid phase synthesis of
a click xanthone library (CX), and its acetyl derivatives, a
CXAC library and the identification of a novel blue fluorescent
compound, CDb8, which selectively stains mESC compared to
MEF and other differentiated cells.
Fluorescent probe molecules have been widely used in bioimaging
and medicinal applications for several decades due to their
high sensitivity and easy visibility. As an alternative to a
traditional knowledge-based Target Oriented Approach
(TOA),1 recently several research groups including our group
Compared to the relatively well studied fluorescent scaffolds,
such as BODIPY, rosamine, coumarin, cyanine and styryl,5 the
xanthone scaffold is still in its infancy for library synthesis and
sensor application. Copper catalysed Huisgen’s 1,3-dipolar
cycloaddition reaction between an azide and a terminal alkyne
has become one of the most popular click reactions because
of its superior regioselectivity, simple reaction conditions,
biocompatibility, and excellent yield.6 The simplicity of this
reaction has made it an attractive choice for construction of
various chemical libraries. Although a simple synthesis of a
xanthone library has been reported using solution phase
click chemistry,7 the biological application of the library
compounds may require careful purification of the product
from the potentially toxic copper catalyst or other impurities.
In order to overcome these problems, we adopted a solid
phase chemistry for the combinatorial diversification of the
xanthone core.
introduced
a Diversity Oriented Fluorescence Library
Approach (DOFLA) for rapid discovery of novel fluorescent
probes and sensors. Several DOFL scaffolds including
coumarin,2a,b dapoxyl,2c styryl,2d hemicyanine,2e rosamine,3a
and BODIPY3b have been developed and their potential
application to imaging probe development demonstrated.
DOFLA has a unique advantage over TOA especially when
information about the target analyte is not available.
Recently, we reported the discovery of the first embryonic
stem cell (ESC) probe CDy1 (Compound of Designation
yellow 1, lex/lem
=
535/570 nm)4 through DOFLA,
incorporating a high-throughput screening of the rosamine
library in ESC and mouse embryonic fibroblast (MEF). While
powerful, the yellow region of the fluorescence by CDy1 has a
significant color overlapping problem when multi-color staining
was tried together with standard YFP (yellow fluorescent
protein). To overcome this color limitation, we expanded
our library to blue/green or red/near IR light emitting scaffolds.
The xanthone scaffold was chosen for the blue library due to its
superior photophysical properties such as high photostability,
large stock shift (120 nm) and moderate quantum yield.
a Department of Chemistry & MedChem Program of Life Sciences
Institute, National University of Singapore, 117543 Singapore,
Singapore. E-mail: chmcyt@nus.edu.sg; Fax: +65-6779-1691;
Tel: +65-6516-7794
b College of Pharmacy, Sunchon National University, 540-742,
South Korea
Scheme 1 Synthesis of CX derivatives. Reagents and conditions:
(a) K2CO3, Cu, DMF, 130 1C, 12 h; (b) H2SO4, 90 1C, 1 h;
(c) 1-Boc piperazine, DMSO, 90 1C, 12 h; (d) NH2–NH2ÁH2O, Pd/C,
EtOH, 90 1C, 4 h; (e) H2O/AcOH (1 : 1), NaNO2, 0 1C, 1 h; (f) NaN3,
0 1C to r.t., 1 h; (g) 10% TFA in DCM, r.t., 2 h; (h) DMF/DCM,
DIEA, Trityl-Cl, r.t., 12 h; (i) DMF/piperidine (4 : 1), CuI, ascorbic
acid, RCCH, r.t., 16 h; (j) 2% TFA in DCM, r.t., 10 min.
c Laboratory of Bioimaging Probe Development,
Singapore Bioimaging Consortium, Agency for Science,
Technology and Research (A*STAR), 11 Biopolis Way,
#02-02 Helios Building, 138667 Singapore, Singapore
w Electronic supplementary information (ESI) available. See DOI:
10.1039/c1cc11962a
c
7488 Chem. Commun., 2011, 47, 7488–7490
This journal is The Royal Society of Chemistry 2011

View Article Online
The general synthetic strategy of xanthone azide (Scheme 1)
involves the reaction between 2-chloro-4-nitro benzoic acid
and 3-fluorophenol followed by a cyclization in the presence of
concentrated sulfuric acid to give 3-fluoro-6-nitro xanthone, 1.
The 3-fluoro group was then replaced by tertiary-butoxycarbonyl
(Boc) protected piperazine and the nitro group was reduced
to aniline with hydrazine and Pd/C in ethanol to provide
intermediate 3. Treatment of 3 with sodium nitrite in water–
acetic acid (1 : 1) at 0 1C followed by reaction with sodium
azide afforded 6-azido-3-piperazine substituted xanthone, 4.
The reaction proceeded quantitatively, and subsequent
deprotection of the Boc group with trifluoroacetic acid
afforded the desired xanthone azide, 5. The xanthone azide
showed no fluorescence, maybe due to the quenching effect of
electron rich a-nitrogen. Once the click chemistry is performed
on the xanthone azide, the fluorescence was recovered. Thus,
the intermediate 5 was loaded onto the solid support and a
broad chemical diversity was introduced by 90 aliphatic and
aromatic terminal alkynes (Chart 1) in the presence of Cu(I).
The formation of the heterocyclic triazole moiety following
Huisgen’s 1,3-dipolar cycloaddition was highly efficient and
for most of the cases, the reaction was completed within
12 hours at room temperature. After acidic cleavage, the
entire library compounds were characterized by HPLC-MS
without further purification and 80 relatively pure compounds
(CX library, average purity is 93%, measured at 254 nm
(Table S1, ESIw)) were collected for further study. Most of
the library compounds showed a blue range of fluorescence as
Scheme 2 Synthesis of CXAC derivatives. Reagents and conditions:
1ÁDCM/ACN (7 : 1), NaHCO3, r.t., 2 h.
expected (excitation ranges from 360 to 370 nm and emission
ranges from 480 to 495 nm). The quantum yield of each molecule
varied from 0.02 to 0.16 reflecting diverse structural and electronic
characteristics.
The piperazine tag of the CX compounds can be easily used
for further diversification. To fully enjoy this possibility, we
synthesized an acetyl version of CX (CXAC) compounds
using solid phase activated ester chemistry. Firstly, active ester
resins were prepared by treating the nitrophenol resin with
acetyl chloride. The reaction of the resulting resin with the
highly reactive piperazine moiety of CX compounds in the
presence of mild base NaHCO3 afforded the corresponding
CXAC compounds (Scheme 2). The reaction completed within
2 hours and the products were obtained after a simple filtration
with an average purity of above 90% without further purification.
This simple but powerful chemistry can be applied to a broad
range of acyl group derivatization leaving the potential of
further diversification of CX.8 The resulting CXAC compounds
have an average excitation at 365 nm and emission at around
495 nm (Table S2, ESIw).
For the discovery of a stem cell selective blue probe, the
160 compounds of the CX and CXAC libraries were screened
against mESC, MEF and co-culture of the two cells using high
throughput cell imaging screening. For the primary screening,
the dye (1 mM) was incubated with cells and many of the
Fig. 1 Selective staining of mESC by CDb8. (a) Chemical structure of
CDb8; (b) mESC was selectively stained by CDb8 at 1 mM for 1 h.
Upper panel: mouse embryonic fibroblasts (MEF), middle: mouse
embryonic stem cells (mESC), lower panel: mESC on MEF feeder.
(c) Flow cytometry analysis of DMSO control cells. (d) Flow
cytometry analysis of CDb8 stained cells. The cells are loaded after
1 h incubation at 1 mM. B.F: bright field, scale bar: 100 mm.
Chart 1 Alkyne building blocks for a CX library.
c
This journal is The Royal Society of Chemistry 2011
Chem. Commun., 2011, 47, 7488–7490 7489

View Article Online
compounds smoothly stained cells to different extent. The
compounds were minimally toxic to the cells over several days
of incubation (Fig. S1, ESIw), demonstrating the suitability of the
library compounds for biological application, free from the toxic
copper catalyst. Dyes staining mESC stronger than MEF were
selected as primary hits (Fig. 1b) and further tested by flow
cytometry to confirm the selective staining of mESC. CXAC-59
was selected as the best dye out of total 160 compounds in terms
of selective staining of mESC and separation of the two cell
populations from flow cytometry (Fig. 1d). As the first blue
fluorescent compound which selectively stains mESC, we dubbed
the compound CDb8 (Compound of Designation blue 8) (Fig. 1a).
We also investigated the selectivity of CDb8 in differentiated
mESC and observed that the differentiated cells were not stained
by CDb8 (Fig. 1b; Fig. S2, ESIw).
In conclusion, we have successfully synthesized the first solid
phase combinatorial xanthone library (CX) using click chemistry.
The secondary derivatization of CX afforded a further library,
CXAC with high yield and purity, demonstrating the robust
acylation platform for new library generation. These xanthone
library compounds have demonstrated their biocompatibility in
terms of easy cell penetration and low toxicity. One mESC
selective compound CDb8 was identified as a blue color imaging
and flow cytometry probe for embryonic stem cells. Detailed
biological applications of CDb8 will be reported in due course.
This work was supported by an intramural funding from
A*STAR (Agency for Science, Technology and Research,
Singapore) Biomedical Research Council and A*STAR SSCC
Grant (SSCC10/024).
Notes and references
1 (a) R. Martinez-Manez and F. Sancenon, Chem. Rev., 2003, 103,
4419–4476; (b) C. Suksai and T. Tuntulani, Chem. Soc. Rev., 2003,
32, 192–202.
2 (a) M. S. Schiedel, C. A. Briehn and P. Bauerle, Angew. Chem., Int.
Ed., 2001, 40, 4677–4680; (b) K. Sivakumar, F. Xie, B. M. Cash,
S. Long, H. N. Barnhill and Q. Wang, Org. Lett., 2004, 6,
4603–4606; (c) J. Min, J. W. Lee, Y. H. Ahn and Y. T. Chang,
J. Comb. Chem., 2007, 9, 1079–1083; (d) Q. Li, J. S. Lee, C. Ha,
C. B. Park, G. Yang, W. B. Gan and Y. T. Chang, Angew. Chem.,
Int. Ed., 2004, 43, 6331–6335; (e) S. Wang and Y. T. Chang, J. Am.
Chem. Soc., 2006, 128, 10380–10381.
3 (a) Y. H. Ahn, J. S. Lee and Y. T. Chang, J. Am. Chem. Soc., 2007,
129, 4510–4511; (b) J. S. Lee, N. Y. Kang, Y. K. Kim, A. Samanta,
S. Feng, H. K. Kim, M. Vendrell, J. H. Park and Y. T. Chang,
J. Am. Chem. Soc., 2009, 131, 10077–10082.
4 C. N. Im, N. Y. Kang, H. H. Ha, X. 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, 7497–7500.
5 (a) A. Loudet and K. Burgess, Chem. Rev., 2007, 107, 4891–4932;
(b) L. Wu and K. Burgess, J. Org. Chem., 2008, 73, 8711–8718;
(c) T. Ueno, Y. Urano, K. Setsukinai, H. Takakusa, H. Kojima,
K. Kikuchi, K. Ohkubo, S. Fukuzumi and T. Nagano, J. Am.
Chem. Soc., 2004, 126, 14079–14085; (d) M. Ali, P. Dutta and
S. Pandey, J. Phys. Chem. B, 2010, 114, 15042–15051; (e) Z. Zhou
and C. J. Fahrni, J. Am. Chem. Soc., 2004, 126, 8862–8863;
(f) A. Samanta, M. Vendrell, R. Das and Y. T. Chang, Chem.
Commun., 2010, 46, 7406–7408.
6 (a) M. Meldal and C. W. Tornoe, Chem. Rev., 2008, 108, 2952–3015;
(b) M. G. Finn, H. C. Kolb, V. V. Fokin and K. B. Sharpless, Prog.
Chem., 2008, 20, 1–4.
7 J. Li, M. Hu and S. Q. Yao, Org. Lett., 2009, 11, 3008–3011.
8 Y. T. Chang, J. Choi, S. Ding, E. E. Prieschl, T. Baumruker,
J. M. Lee, S. K. Chung and P. G. Schultz, J. Am. Chem. Soc.,
2002, 124, 1856–1857.
c
7490 Chem. Commun., 2011, 47, 7488–7490
This journal is The Royal Society of Chemistry 2011