Introduction of a highly photodurable and common-laser excitable fluorescent amino acid into a peptide as a FRET acceptor for protease cleavage detection

Article abstract of DOI:10.1246/cl.2010.818

Synthesis and photochemical properties of a benzoacridonecontaining fluorescent amino acid (badAla) which is photodurable and excitable by widely applicable 488 nm lasers was described. The amino acid carries a relatively small fluorophore that shows abso

Full text of DOI:10.1246/cl.2010.818

doi:10.1246/cl.2010.818
818
Published on the web June 26, 2010
Introduction of a Highly Photodurable and Common-laser Excitable Fluorescent
Amino Acid into a Peptide as a FRET Acceptor for Protease Cleavage Detection
Masumi Taki,* Yoshito Yamazaki, Yuto Suzuki, and Masahiko Sisido
Department of Bioscience and Biotechnology, Faculty of Engineering, Okayama University,
3-1-1 Tsushimanaka, Okayama 700-8530
(Received May 6, 2010; CL-100431; E-mail: taki@biotech.okayama-u.ac.jp)
Synthesis and photochemical properties of a benzoacridone-
OH
OH
O
Cl
1
O
OH
containing fluorescent amino acid (badAla) which is photo-
durable and excitable by widely applicable 488 nm lasers was
described. The amino acid carries a relatively small fluorophore
that shows absorption around 450-500 nm, emission above
500 nm with a high fluorescence quantum yield (0.65), and
relatively long fluorescence lifetime (17 ns). BadAla can be used
in solid-phase peptide synthesis without any precaution. A
peptide-containing badAla was used to measure caspase activity
via fluorescence resonance energy transfer (FRET).
COOEt
COOH
a
Boc
N
H2N
H
c, d
b
COOEt
O
Boc
HO
HN
O
N
H
HN
4
3
2
NH2
Fluorescent probes are widely used in peptide chemistry.
In particular, introducing donor and acceptor fluorescence
resonance energy transfer (FRET) pair in a single peptide is a
versatile technique to evaluate specific protease activity. Several
intramolecular FRET peptide substrates have been designed and
reported.1 The design generally includes D-X-A structure, in
which D is the fluorescent donor group and A is the acceptor
group, separated by a peptide sequence (X) containing the
protease cleavage site. If the proteolysis occurs, separation of the
donor and the acceptor results in a loss of energy transfer and
subsequent enhancement of donor fluorescence.
Scheme 1. Synthesis of badAla (4): (a) PCl5, POCl3; (b) Cu/Cu2O,
K2CO3, 2-methoxyethanol; (c) phosphoric acid; and (d) LiOHaq.,
THF.
Usually, peptides are derivatized with fluorescent probes at
specific positions after solid-phase peptide synthesis (SPPS) and
purification. The reactive groups are typically free amine at the
N-terminus and thiol group at the side chain. However, the
presence of other reacting groups in the same peptide makes the
labeling process difficult in many cases. Direct introduction of
the chromophores into the peptide sequence by using fluorescent
nonnatural amino acids can avoid the problem. However, there
are few fluorescent amino acids as monomers which are
applicable for SPPS. Recently, Deleris et al. synthesized two
different fluorescence nonnatural amino acids as tools for
protease cleavage detection by using FRET.2 However, severe
fluorescence quenching between the two fluorophores took
place, probably because they are prone to aggregate in aqueous
conditions. Once they aggregated, the emission of the fluoro-
phore is highly quenched. For this reason, we tried to find an
appropriate FRET pair of two fluorescent nonnatural amino
acids.
An acridone-containing fluorescent nonnatural amino acid
(acrydonylalanine: acdAla), which was initially reported by
Szymanska et al.,3 is a promising candidate; its small size may
minimize steric disturbance in the substrate recognition by
proteases. Moreover, it shows many excellent photophysical
properties such as high quantum yield, long fluorescence
lifetime, high photostability, excitability by a violet laser
(405 nm), and moderate solubility in aqueous solutions.3,4 Here
we synthesized a novel benzoacridone-containing amino acid
Figure 1. Absorption (left) and emission (right) spectra of badAla
(4) in EtOH (solid line) and in 50% EtOH/PBS buffer (dotted line).
(badAla (4)) which is excitable by widely applicable 488 nm
lasers and act as an efficient acceptor from acdAla (Scheme 1).
First, we synthesized 3-chloro-2-naphthoic acid (1), and
then it was coupled with Boc-protected aminophenylalanine-
ethyl ester 2. Initially, the coupling reaction was attempted under
reported conditions for the Ullmann-Jourdan reaction with 2-
chlorobenzoic acid and 2.3 However, only a trace of the coupled
product 3 was obtained. We found the reaction yield was greatly
improved when 2-methoxyethanol was used as the solvent and
Cu2O cocatalyst was present in the reaction mixture. Finally,
badAla (4) was successfully synthesized in 38% yield from 2.
Absorption and fluorescence spectra of badAla (4) are
typical of benzoacridone derivative (Figure 1). As we expected,
4 can be excited at longer wavelengths than that of acdAla; the
absorption band spans the region of 450-500 nm. When 4 was
excited at 488 nm, fluorescence emission above 500 nm was
observed. The absorption and emission bands allow us to excite
and detect the benzoacridone chromophore selectively, without
any interference from other natural chromophores of peptides
and proteins, or those inherently existing in living cells. As
shown in the Supporting Information,5 the overlap of the
emission spectrum of acdAla and the absorption spectrum of
Chem. Lett. 2010, 39, 818-819
© 2010 The Chemical Society of Japan
www.csj.jp/journals/chem-lett/

819
Table 1. Fluorescence quantum yields of various amino acids and
Boc-derivatives containing acridone moieties in MeOHa
Xaa
Boc-Xaa
acdAla sacdAla badAla acdAla sacdAla badAla
Non-degassed 0.61
Degassed
0.743
0.31
0.40
0.51
0.65
0.61
0.54
0.49
aFor the structures of amino acids, see Supporting Information.5
badAla (4) shows that FRET can occur between these two probes
if they are attached onto the same peptide.
Fluorescence decay time of tert-butoxycarbonyl-badAla
(Boc-badAla) in PBS buffer was 17 ns (see Supporting
Information5), which is close to that of Boc-acdAla (16 ns)4 or
Boc-sacdAla (14 ns).6 The fluorescence decay time is long
enough for eliminating inherent fluorescence from living cells.
The photodurability of badAla (4) was compared with those
of other fluorescent groups frequently used in biochemical
experiments. BadAla (4) retained more than 85% of its original
fluorescence intensity after 1 h irradiation, whereas fluorescence
intensities of anthranylalanine, BODIPY, and fluorescein de-
creased more rapidly under the same irradiation conditions.5
These data indicate that the benzoacridonyl group is tough
enough for prolonged measurement on fluorescence imagers or
confocal microscopes equipped with common-lasers.
Absolute values of fluorescence quantum yield for the
acridone-containing amino acids and their Boc-derivatives were
measured by a C9920-02 Absolute PL Quantum Yield Measure-
ment System (Hamamatsu photonics K. K.), and are shown in
Table 1. In every case, the quantum yield of the fluorescence
was sufficiently high compared to conventional fluorophores,
even when the solvent (MeOH) was not degassed.
The fluorescent nonnatural amino acids, acdAla and badAla
(4) as a FRET pair, were introduced into a peptide by
conventional SPPS. Without doing any HPLC purification, we
could observe a single peak in the HPLC measurement, and the
corresponding m/z value was found in MALDI-TOF-MS. This
suggests that side-reaction seldom occurred and these amino
acids are applicable to conventional SPPS. The sequence of
synthesized peptide was Glu-badAla-Asp-Glu-Val-Asp-
acdAla-Glu, which contains the caspase-3 cleavage site
(bold) to detect the enzymatic activity. Caspase-3 is a key
enzyme secreted during apoptosis, and measurement of caspase-
3 activity would provide valuable information about the
apoptotic mechanism. The two Glu units increased the solubility
of the peptide and their electric repulsion prevented aggregation
of the fluorophores. Without adding Glu units at both ends, the
fluorophores easily aggregated. The synthesized peptide showed
yellow fluorescence of the acceptor (badAla) when the donor
(acdAla) was excited, because intramolecular FRET occurred
from acdAla to badAla. The FRET quenching efficiency was
80%. In contrast, a mixture that contained an equimolar amount
of the monomeric donor (acdAla) and the acceptor (badAla)
showed acdAla fluorescence when the former was excited.5
When the peptide was incubated with caspase-3, the
cleavage site could be cleaved, resulting in the separation of
the donor-acceptor distances, leading to the recovery of the
donor fluorescence. The proteolysis-induced fluorescence recov-
ery is shown in Figure 2. With an increased incubation time, the
emission peak of the donor around 450 nm greatly recovered.
Figure 2. Continuous fluorometric assay of caspase-3 (0.3 ¯g,
Sigma) with Glu-badAla-Asp-Glu-Val-Asp-acdAla-Glu (0.2 ¯M)
in a buffer (50 mM HEPES (pH 7.4), 0.1% CHAPS, 10 mM DTT,
100 mM NaCl, and 1 mM EDTA) at 25 °C. Around 450 nm, from
bottom to top, fluorescence spectra were recorded at t = 0, 1, 3, 5, and
24 h after addition of the protease (-ex = 360 nm).
Along with this moment, we also observed slight enhancement
of the acceptor fluorescence around 530 nm. We assume that
quenching occurred to some extent in the double-labeled peptide
concomitantly with FRET prior to the caspase-3 cleavage.2,7
In conclusion, the new nonnatural amino acid (badAla (4))
was useful as a FRET acceptor for protease cleavage detection.
To the best of our knowledge, it is the smallest fluorescent
amino acid which can be excitable by widely applicable lasers
at 488 nm and applicable to SPPS without any protection/
deprotection. We also found that 4 shows moderately high
fluorescence quantum yield, long lifetime, and is durable to
prolonged photoirradiation. The acridone-containing amino
acids,8 when introduced into peptides and proteins as the FRET
pair, will be a useful probe for monitoring protease cleavage
under currently available laser equipments such as fluorescence
imagers or confocal microscopes.
The authors acknowledge Dr. K. Suzuki and Mr. M. Kamiya
for the measurement of the absolute fluorescence quantum
yields. The authors also thank Prof. H. Ishida and Dr. T. Sato for
kind discussions and assistance. This work was supported by the
Ministry of Education, Culture, Sports, Science and Technology,
Grant-in-Aid for Young Scientists.
References and Notes
1
2
H. Y. Sun, R. C. Panicker, S. Q. Yao, Biopolymers 2007, 88, 141.
T. Berthelot, J.-C. Talbot, G. Laïn, G. Déleris, L. Latxague,
J. Pept. Sci. 2005, 11, 153.
3
4
A. Szymańska, K. Wegner, L. Łankiewicz, Helv. Chim. Acta 2003,
86, 3326.
H. Hamada, N. Kameshima, A. Szymańska, K. Wegner, L.
Łankiewicz, H. Shinohara, M. Taki, M. Sisido, Bioorg. Med.
Chem. 2005, 13, 3379.
5
6
Supporting Information is available electronically on the CSJ-
Journal Web site, http://www.csj.jp/journals/chem-lett/index.html.
M. Taki, Y. Suzuki, Y. Yamazaki, M. Sisido, in Peptide Science
2006, ed. by H. Ishida, H. Mihara, The Japanese Peptide Society,
2006, p. 12.
7
8
S. Mizukami, K. Kikuchi, T. Higuchi, Y. Urano, T. Mashima, T.
Tsuruo, T. Nagano, FEBS Lett. 1999, 453, 356.
Boc- or Fmoc-protected fluorescent amino acids (acdAla and
badAla) are currently available from Watanabe Chemical Indus-
tries.
Chem. Lett. 2010, 39, 818-819
© 2010 The Chemical Society of Japan
www.csj.jp/journals/chem-lett/