"Singapore green": A new fluorescent dye for microarray and bioimaging applications

Article abstract of DOI:10.1021/ol802700w

(Figure Presented) We have synthesized Singapore Green (SG), a structural hybrid of Tokyo Green and Rhodamine 110. This new dye is a green light-emitting substitute for 7-aminocoumarin, a blue fluorescent dye widely used in enzymatic assays. SG-conjugated

Full text of DOI:10.1021/ol802700w

ORGANIC
LETTERS
2009
Vol. 11, No. 2
405-408
“Singapore Green”: A New Fluorescent
Dye for Microarray and Bioimaging
Applications
Junqi Li and Shao Q. Yao*
Departments of Chemistry and Biological Sciences, NUS MedChem Program of the
Office of Life Sciences, National UniVersity of Singapore, Singapore 117543
chmyaosq@nus.edu.sg
Received November 22, 2008
ABSTRACT
We have synthesized Singapore Green (SG), a structural hybrid of Tokyo Green and Rhodamine 110. This new dye is a green light-emitting
substitute for 7-aminocoumarin, a blue fluorescent dye widely used in enzymatic assays. SG-conjugated peptide substrates were successfully
synthesized and used in microarray-based protease substrate profiling and live-cell imaging experiments.
Fluorogenic peptide substrates, including those employing
latent fluorophores, internally quenched and fluorescence
resonance energy transfer (FRET)-based substrates, have
emerged as indispensable tools in the profiling and visualiza-
tion of protease activities both in vitro and in vivo.1 They
are also widely used in high-throughput screening of protease
inhibitors. Among the various types of reagents used,
fluorogenic peptide substrates containing a C-terminally
capped coumarin derivative (i.e., 7-amino-4-methylcoumarin
(AMC) or 7-amino-4-carbamoylmethylcoumarin (ACC)) are
arguably the most useful for substrate specificity profiling
experiments, as only cleavage at the amide bond between
the peptide and the coumarin moiety will release the highly
fluorescent coumarin.2 Consequently, coumarin-based fluo-
rogenic peptide libraries have been employed to study the
substrate specificities of numerous therapeutically important
proteases, including caspases, thrombin, cathepsins, and
many others.2 In recent years, several attempts have been
made to introduce these substrate libraries to microarray-
based applications where further miniaturization and higher
throughput of enzymatic assays can be achieved.3,4 We and
others recently reported the immobilization of coumarin-
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10.1021/ol802700w CCC: $40.75
Published on Web 12/19/2008
2009 American Chemical Society

based enzyme substrates onto microarray platforms and used
them to profile substrate specificities of proteases.4 Since
coumarin dyes are excited in the UV region (maximum λex
∼350 nm), these strategies have not been effective due to
high fluorescence backgrounds and the lack of microarray
scanners with UV light sources. For similar reasons, cou-
marin-based peptide substrates are rarely used in live-cell
imaging experiments.1b
quinone and the nonfluorescent spirolactone forms of R110
reduces fluorescence output.7b,c Our new fluorophore, Sin-
gapore Green (SG), is a hybrid of R110 and the fluorescein
analogue 2-Me TokyoGreen (Figure 1),8 with a phenolic
group on one end providing a chemical handle (for solid-
phase peptide synthesis, microarray immobilization, and
potentially other applications in cell-based experiments) and
an amino group on the other end serving as the point of
conjugation to a peptide sequence. We reasoned that ami-
dation at the amino group of SG by a peptide should suppress
the fluorescence of the dye. Similar fluorescence-quenching
effects have been observed in other coumarin- and rhodamine-
based peptide conjugates.5 Cleavage of the amide bond by
a protease should release the highly fluorescent SG, thus
reporting protease activity accordingly. Herein, we report the
synthesis and characterizations of SG, the solid-phase
synthesis of SG-conjugated peptides, as well as their
applications in microarray-based and live-cell imaging ex-
periments.
Figure 1. Structures of common fluorophores used in fluorogenic
peptide substrates (ACC and Rhodamine 110) and fluorophores
from which Singapore Green was derived (Rhodamine 110 and
Tokyo Green).
We sought to replace coumarin with a new fluorophore
having excitation and emission wavelengths in the visible
range, so that it is suitable for both microarray and live-cell
imaging applications. We turned to other fluorescein and
rhodamine fluorophores that have been used for labeling
reagents and enzymatic assays.5 Of these, Rhodamine 110
(R110)-based substrates are well-established peptide probes
for serine and cysteine proteases.6,7 Despite the desirable
fluorescence properties of R110, several drawbacks hinder
the direct use of these substrates: (1) R110-conjugated
peptides require both peptide groups to be cleaved in order
to generate maximum fluorescence and thus are not suitable
for quantitative studies of linear enzyme kinetics;5,6a (2)
“asymmetric” versions of these dyes containing a single
peptide cleavage site lack an immobilization handle which
is essential for both solid-phase peptide synthesis and
microarray applications;7b-d (3) equilibrium between the
Figure 2. (A) Fluorescence spectra of SG1. Fluorescence increase
from cleavage of Ac-DEVD-SG1 by caspase-3 (blue line) and
caspase-7 (red line) over the background fluorescence (orange line).
As shown in Scheme 1, the synthesis of SG started with
the formation of the asymmetric xanthone 1, which was
generated by Ullman-type coupling between 3-acetami-
dophenol and 2-chloro-4-nitrobenzoic acid based on a
published procedure.9 Diazotization of the amino group and
hydrolysis of the diazonium salt yields the phenol 2 which
underwent methylation or alkylation with a linker unit to
give 3. The nitro group was subsequently reduced to give
4a and 4b. Subsequent trityl protection and Grignard addition
of o-tolylmagnesium bromide followed by deprotection of
the trityl group afforded the fluorophores SG1 and SG2,
respectively. SG1 was found to have excitation maxima at
469 and 493 nm and emission maxima at 517 nm in ethanol
(Figure 2a), similar to fluorescein (494 and 521 nm respec-
tively, in water), and is thus compatible with the 488 nm
argon-ion laser used in most microarray scanners and
fluorescence microscopes. SG1 has a quantum yield of 0.50
and an extinction coefficient of 28500 M-1cm-1, which is
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Scheme 1. Synthesis of Peptide Conjugates of SG1 and SG2
reasonably bright for most applications. The photostability
of SG was tested by irradiating the dye with a fluorimeter;
results indicated that SG is at least as photostable as
fluorescein. To test if peptide conjugates of SG1 can be used
as fluorogenic probes to detect enzymatic activity, Ac-
DEVD-SG1 was synthesized as an analogue of Ac-DEVD-
AMC (a coumarin-based substrate for caspase-3 and -7 from
commercial sources). Ac-DEVD-SG1 was weakly fluores-
cent. Upon incubation with caspase-3 and -7, however, there
was a time-dependent increase in fluorescence, which follows
typical Michaelis-Menten curves (Figure 2b), indicating that
SG-based peptide conjugates are indeed suitable green light-
emitting substitutes of their coumarin counterparts.
single major peak in LCMS profiles; see the Supporting
Information), thus demonstrating the compatibility of SG2
with standard Fmoc chemistry.
We next sought to test the feasibility of using peptide
conjugates of SG2 for reporting protease activities on the
microarray. We picked 10 peptide sequences from comm-
ericially available p-nitroanilide or coumarin-based substrates
targetting various proteases and replaced the p-nitroaniline
chromophore or coumarin fluorophore with SG2. To render
the fluorophore compatible with Fmoc-based chemistry used
in solid-phase peptide synthesis, the amino end of SG2 was
protected with Fmoc, giving 8, and the hydroxyl-containing
linker located on the other end was oxidized to an aldehyde
with Dess-Martin periodinane to give 9. The compound was
subsequently loaded onto a threonine-functionalized ami-
nomethyl polystyrene resin by acid-catalyzed oxazolidine
formation (Supporting Information). Peptide synthesis then
proceeded using standard Fmoc chemistry on solid phase.
Final cleavage of the peptides from the solid support gave
the desired aldehyde-functionalized peptides in good or
excellent purity (most crude products except P9 showed a
Figure 3. (A) Strategy for detecting protease activity on microarray.
(B) Enzyme “fingerprints” obtained (clockwise from top left:
caspase-3, caspase-7, R-chymotrypsin, subtilisin). Peptides were
spotted in triplicate (vertically). (C) Time-dependent kinetic profiles
obtained from the peptide microarray. Peptides were spotted in
duplicate (vertically). See the Supporting Information for details
of spotting patterns.
We next spotted the peptides onto alkoxyamine-function-
alized glass slides to generate the corresponding peptide
Org. Lett., Vol. 11, No. 2, 2009
407

microarray via chemoselective oxime bond formation (Figure
3a). As a proof-of-concept experiment, the immobilized
peptides were treated with four different proteases (caspase-
3, caspase-7, R-chymotrypsin, and subtilisin). The microaray
was subsequently scanned with a microarray scanner equipped
with a green light source. As shown in Figure 3b, images
obtained immediately revealed discerning “substrate finger-
prints” of each enzyme. Of the proteases used, enzymes with
broader substrate specificity (subtilisin and R-chymotrypsin)
cleaved multiple substrates to different extents, while highly
specific proteases (caspase-3 and -7) cleaved only its optimal
substrate sequence, Ac-DEVD-SG2. The fluorescence read-
outs generally correlated well with the corresponding enzy-
matic reactions carried out in microplate-based assays (data
not shown). We next examined whether the SG-based peptide
microarray could be used to obtain quantitative enzyme
kinetic data by incubating four selected peptides with four
different enzymes in a time-dependent experiment (Figure
3c). Fluorescence intensities were quantified and fitted to
kinetic curves to obtain kobs values which reflected the
substrate preferences of the proteases (see the Supporting
Information). Taken together, these experiments indicate the
applicability of SG-based substrates in microarray-based
protease profiling experiments.
To demonstrate that our SG-based substrates can be used
for live-cell imaging, we tested the ability of Ac-DEVD-
SG1 to image apoptosis in live cells (Figure 4). Caspase-3
and -7 are key mediators of this important biological process
where improper regulation of caspase activity has detrimental
pathological and physiological effects. To image apoptosis,
numerous peptide- and protein-based probes (including R110
peptide conjugates) have been developed for the sensitive
detection of caspase activity.10,11 We thus evaluated Ac-
DEVD-SG1 as a fluorogenic probe for reporting caspase-3
and -7 activity in apoptotic HeLa cells. As shown in Figure
4, cells treated with Ac-DEVD-SG1 developed a strong green
fluorescence upon apoptosis induction with staurosporine
(bottom vs top left panels). In contrast, no significant increase
in green fluorescence was observed in nonapoptotic cells
even after extended incubation time (Supporting Informa-
tion). Nonapoptotic cells remained viable after treatment with
the probe for more than 3 h, indicating that the probe was
not cytotoxic (data not shown). These results suggest that
SG-based substrates could serve as useful fluorogenic probes
for live-cell imaging of protease activities. To accommodate
future bioimaging applications, the phenolic handle in SG-
based probes may be conveniently modified with cellular
localization sequences, protein tranduction domains, etc.
Figure 4. Detecting caspase activity in live Hela cells with Ac-
DEVD-SG1. Bottom panel shows fluorescence incease from
cleavage of Ac-DEVD-SG1 after apoptosis induction (with stau-
rosporine). Cells were injected with 50 µM of Ac-DEVD-SG1 (with
tetramethylrhodamine-dextran as marker to identify injected cells
in RFP channel) and imaged. Inset: DIC images of the cells. Scale
bar ) 15 µm.
In summary, we have developed a new fluorophore that
possesses desirable chemical and fluorescence properties
suitable for biomedical applications. In this report, we have
shown that peptide conjugates of this new fluorophore can
be readily synthesized using standard solid-phase peptide
chemistry, and conveniently immobilized on a microarray
for high-throughput substrate specificitiy profiling of pro-
teases. We further showed that these probes are equally
amenable for live-cell imaging of protease activities. We
anticipate that these probes will find wide applications in
the emerging field of catalomics.12
Acknowledgment. We thank Souvik Chattophadhaya for
technical advice on bioimaging and Candy Lu for microarray
experiments. Funding support was provided by the Ministry
of Education (R143-000-280-112) and the Agency for
Science, Technology, and Research (A*Star) of Singapore
(R154-000-274-305).
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Supporting Information Available: Experimental pro-
cedures, characterization of new compounds, and additional
images. This material is available free of charge via the
Internet at http://pubs.acs.org.
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