Combinatorial rosamine library and application to in vivo glutathione probe

Article abstract of DOI:10.1021/ja068230m

Combinatorial approach on fluorescent rosamine library was developed using solid-phase chemistry. A highly selective library member (H22) toward reduced glutathione (GSH) was discovered, demonstrating the capability of in vivo GSH level sensing in live cells. Copyright

Full text of DOI:10.1021/ja068230m

Published on Web 03/23/2007
Combinatorial Rosamine Library and Application to in Vivo Glutathione Probe
Young-Hoon Ahn, Jun-Seok Lee, and Young-Tae Chang*
Department of Chemistry, New York UniVersity, New York, New York 10003
Received November 17, 2006; E-mail: yt.chang@nyu.edu
Scheme 1 . Synthesis Generating Rosamine Library and Building
Fluorescent compounds have been excellent tools for the sensitive
and specific detection of a variety of analytes.1 As an alternative
approach to conventional target-oriented rational design, we have
previously introduced combinatorial fluorescent styryl libraries and
successfully demonstrated the power of the diversity-oriented
approach by generating specific sensors for various bioanalytes
(DNA, RNA, â-amyloid protein, and organelles).2 Encouraged by
these results, we are expanding the general approach to different
fluorophores for better properties.
Blocksa
Rhodamine is a highly favorable fluorescence scaffold because
of its excellent photophysical properties such as high extinction
coefficient, high quantum yield (often near 1), high photostability,
pH-insensitivity (unlike fluorescein), and relatively long emission
wavelength (>500 nm).3 These superior and constant optical
properties of rhodamine partially originate from its rigid-core
structure. In addition to the fused xanthene structure, rhodamine
carries 2′-carboxylic acid, which constrains the rotation of the
9-phenyl ring.4 Therefore, we envisioned that removal of the 2′-
carboxylic acid group from rhodamine (called rosamine) will
introduce flexibility onto the rhodamine scaffold and thus would
generate a possible sensor candidate whose fluorescence properties
can be changed by an analyte environment. Here we report the
first combinatorial synthesis of a rosamine library and an in vivo
fluorescent probe for monitoring cellular glutathione (GSH) levels.
Most rhodamine derivatives including rosamine have been
prepared by condensation reactions under strong acidic conditions,5
requiring difficult or tedious purification. We have sought to
incorporate solid-phase chemistry to generate the final product
rosamine, circumventing acidic reflux conditions and time-consum-
ing purification steps (Scheme 1). Initially three different 3-amino-
6-nitro-9H-xanthone derivatives (S3, Y ) O, NH, S) were
synthesized. The first diversity was introduced by modifying the
3-amino group utilizing the 6-nitro group as a linker after reduction
to an amino group. Twelve different unsymmetrical xanthone
derivatives (S5, R1 building blocks, A-L) containing oxygen,
sulfur, and nitrogen bridges were synthesized (synthetic details for
each intermediate are in Supporting Information (SI)). Each
intermediate was loaded on the 2-chlorotrityl chloride resin (S6),
and reacted with 33 different Grignard reagents (R2 building block)
for the second diversity.6 An acidic cleavage from the resin resulted
in the dehydration, giving the fully conjugated rosamine derivatives.
All the compounds in the library were characterized by HPLC-
MS and 240 relatively pure compounds (average purity is 93%,
measured at 250 nm (see SI)) were collected for further study. The
library compounds have a wide range of spectral diversities
(excitation ranges from 480 to 545 nm and emission ranges from
530 to 605 nm). The quantum yield of each molecule highly varies
from 0.00025 to 0.89 reflecting diverse structural and electronic
characteristics.
a (a) K2CO3, Cu, DMF, 130 °C; (b) H2SO4, 80 °C; (c) modification for
R1 (see Supporting Information for each reaction); (d) SnCl2, EtOH, 90 °C;
(e) 3-chloro-trityl chloride resin, Pyr, CH2Cl2/DMF; (f) R2 Grignard reagent,
THF, 62 °C; (g) 1%TFA, CH2Cl2.
reduced glutathione (GSH) compared to other analytes (SI). In
physiological condition (pH 7.4, 50 mM HEPES), H22 (3 µM) was
further evaluated and showed a marked fluorescence increase upon
addition of GSH (5 mM) by ca. 11-fold in 30 min (Figure 1). The
quantum yield of H22 was 0.033 and 0.28 before and after addition
of GSH. It is noteworthy that H22 did not show any fluorescence
response to GSSG (5 mM) while several thiol-containing analytes
such as DTT, â-mercaptoethanol, and cysteine showed modest
For primary screening, the library compounds were screened for
fluorescence intensity change toward a variety of biorelevant
analytes. Notably, H22 exhibited a highly selective response toward
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10.1021/ja068230m CCC: $37.00 © 2007 American Chemical Society

C O M M U N I C A T I O N S
sulfoximine; GSH synthesis inhibitor)8 for 60 min also showed a
similar intensity decrease (SI), while untreated cells did not show
such a decrease in the same experiment duration. Taken together,
these experiments clearly demonstrate that H22 is able to sense
changes in the cellular GSH level in living cells.
GSH is the most abundant cellular thiol and plays the central
role in maintaining redox homeostasis, existing in a redox equi-
librium between the reduced (GSH) and oxidized (GSSG) form.8
With a high concentration of GSH (∼5 mM) in a cell, the ratio of
GSH over GSSG (ranging from 1 to 100)7d is of critical importance
in regulating the redox potential in a living cell. Although many
thiol detecting fluorescent dyes have been utilized for measuring
GSH,3,9 there are few cellular applications of such small molecule
probes for monitoring changes of the cellular GSH concentration.
In summary, we have developed the first rosamine library using
solid-phase chemistry as a potential fluorescence sensor set, and
described the potential of H22 as a glutathione probe in living cells.
Figure 1. Fluorescence responses of H22 (3 µM) toward GSH in 0, 0.01,
0.05, 0.1, 0.25, 0.5, 1.0, 2.5, 5 mM. H22 was incubated with GSH at room
temperature for 30 min in 50 mM HEPES, pH 7.4. Spectra were obtained
with excitation at 500 nm.
Acknowledgment. We gratefully acknowledge the National
Institutes of Health for financial support (Grant P20GM072029)
and for equipment grants for the NMR (Grant MRI-0116222) and
the capillary LC-ion trap mass spectrometer (Grant CHE-0234863).
Components of this work were conducted in a Shared Instrumenta-
tion Facility constructed with support from Research Facilities
Improvement Grant C06 RR-16572 from the NCRR/NIH. J.S.L.
was supported by a Korea Research Foundation Grant funded by
the Korean government (MOEHRD, Basic Research Promotion
Fund: KRF-2005-C00088)
Supporting Information Available: Synthesis and experimental
procedures and characterization data of all library compounds. This
material is available free of charge via the Internet at http://pubs.acs.org.
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Figure 2. Fluorescence microscopic images of live 3T3 cells: (A) cells
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