Target-selective fluorescent "switch-on" protein labeling by 6π-azaelectrocyclization

Article abstract of DOI:10.1039/c1ob05320e

Application of azaelectrocyclization and FRET techniques to lysine groups enabled the selective and sensitive detection of a target protein from a mixture, with high fluorescence contrast.

Full text of DOI:10.1039/c1ob05320e

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Target-selective fluorescent “switch-on” protein labeling by
6p-azaelectrocyclization†
Katsunori Tanaka,* Masataka Kitadani and Koichi Fukase*
Received 28th February 2011, Accepted 3rd June 2011
DOI: 10.1039/c1ob05320e
Application of azaelectrocyclization and FRET techniques
to lysine groups enabled the selective and sensitive detection
of a target protein from a mixture, with high fluorescence
contrast.
groups onto lysine residues by reaction with unsaturated aldehyde
probes (such as probe 1 shown in Scheme 1) at very low
concentrations (~10-8 M) within a short period of time (10–30
min) at room temperature.9 Under such mild reaction conditions,
the labels could be introduced onto the more “exposed” surface
lysines at a level of one or two molecules per protein. The
native activity of the biomolecules and/or cell surfaces, therefore,
was retained. Using the electrocyclization protocol, the receptor-
mediated accumulation, circulatory residence, and trafficking of
glycoproteins and/or lymphocytes were successfully visualized by
non-invasive PET and fluorescence imaging. Site-selective non-
destructive modification of the target proteins was also achieved by
directing reactive groups to a specific site using a small-molecule
ligand of the protein (see Scheme 1).10 Labeling took place via
Schiff base formation and subsequent electrocyclization. The
ligand was then automatically cleaved from the conjugated protein
via autooxidation of the 1,2-dihydropyridine and hydrolysis of the
ester linkage connected to the ligand.
The ligand-directed approach described above may be applied
to the selective labeling of target proteins in cell lysates, on cell
surfaces, or even in living animals. Usually, selective labeling of a
target protein in a mixture of biomolecules, e.g., by fluorescence
reporter groups, yields poor fluorescence contrast between the
labeled protein and the unreacted probes. Sensitive detection of
the targeted protein is quite difficult unless the labeled proteins are
purified, the label-treated cells are washed, or the unreacted probes
are preferentially excreted from the serum during labeling in living
animals. The development of labeling probes that are fluorescently
silent by default (“caged” fluorescence) but are “switched on”
when they selectively react with the target proteins would go a long
way to removing the unlabeled background fluorescence signal.
Kikuchi and co-workers recently reported an elegant “switch-
on” method using a designed fluorescence quenching substrate
in which the quencher on the label was specifically released by a
b-lactamase, E166NTEM, fused to the proteins and/or expressed
on the cell surfaces.11 Hamachi and co-workers also developed
a “switch-on” probe that utilized their LDT strategy.7b In this
communication, we describe a unique strategy based on the
electrocyclization technique, in which fluorescence quenching
systems may be applied to an unsaturated aldehyde probe 1
(Scheme 1), which is only unlocked when an azaelectrocyclization-
induced cascade reaction proceeds on a target lysine. This new
strategy enabled sensitive detection of a target protein in an
equimolar mixture containing seven non-targeted proteins and
Chemical methods for labeling proteins have garnered much atten-
tion in the fields of molecular imaging due to their straightforward
procedures, general applicability, and the small size of chemical
labels, at least relative to the genetically-encoded protein-based la-
bels, such as Green Fluorescent Protein (GFP).1 Recently, a variety
of new chemical methods2 that can be combined with biological
techniques have been actively investigated.3 For example, the
Cu(I)-mediated Sharpless–Meldal click reaction employs an azide
moiety that may be genetically introduced at desired positions
within a protein. Bio-friendly (Cu(I)-free) versions of the click re-
action using strained acetylenes4 or the Staudinger reaction5 have
been developed by Bertozzi and co-workers. These reactions have
been successfully applied to fluorescence imaging on cell surfaces.
Entirely chemical methods that use protein-specific ligands to
direct labeling using probe molecules, involving post-affinity label-
ing modifications (P-ALM), have been thoroughly investigated by
Hamachi and co-workers.6 After cleavage of the ligands from the
labeled proteins, the recovered activity was found to provide a sen-
sitive reporter for fluorescent imaging. Recently, a ligand-directed
tosyl (LDT) strategy, which selectively labeled target proteins with-
out removing the ligand, was reported by the same group. The la-
beling reaction simultaneously released the protein ligand from the
labeled protein.7 Successful application of this protocol involves
the fluorescence labeling of carbonic anhydrase in human blood
cells, lactose-binding congerin in mucus tissues of conger eel skin,
and the biotin-labeling of endogenous FKBP12 in Jurkat cells.7a
In vivo fluorescence labeling of the carbonic anhydrase was also
achieved by intravenous injection of the probe into living mice.7a
We have independently been investigating a lysine-based pro-
tocol for labeling peptides, proteins, and living cell surfaces,
based on rapid 6p-azaelectrocyclization.8,9 This method was used
to efficiently and selectively introduce both DOTA (1,4,7,10-
tetraazacyclodecane-1,4,7,10-tetraacetic acid) and fluorescent
Address, Department of Chemistry, Graduate School of Science, Osaka Uni-
versity, 1-1 Machikaneyama-cho, Toyonaka-shi, Osaka, 560-0043, Japan.
E-mail: ktzenori@chem.sci.osaka-u.ac.jp, koichi@chem.sci.osaka-u.ac.jp;
Fax: +81-6-6850-5419; Tel: +81-6-6850-5391
† Electronic supplementary information (ESI) available: Experimental
details, characterization data, and copies of 1H NMR, HPLC and
fluorescence spectra. See DOI: 10.1039/c1ob05320e
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Scheme 1 Target-selective and fluorescence “switch-on” mechanism of protein labeling by 6p-azaelectrocyclization.
peptides by achieving high fluorescence contrast from the labeled
proteins without the need for isolation from the mixture.
Our “switch-on” labeling mechanism was hinted at by our
earlier non-destructive labeling of human serum albumin (HSA,
MW = 66 000)10 and was investigated here using this model protein
(Scheme 1). We have designed an unsaturated aldehyde 1 as the
ligand-directed label-activated “switch-on” fluorescence probe.
7-Diethylaminocoumarin was introduced at the C9 position of
the probe and the 4-[4-(dimethylamino)phenylazo]benzoic acid
derivative (DABCYL) fluorescence quencher was attached at the
C3 position via an ester linkage. The structural arrangement,
shown in 1, achieved efficient quenching of the coumarin flu-
orescence by DABCYL so that the probe 1 itself was non-
fluorescent. 7-Diethylaminocoumarin is not only a “caged”
fluorescence reporter group, but it also functions as a ligand of
HSA:12 it strongly binds HSA through the subdomain IB (amino
acid residues 107–196).13 We showed that loading of this ligand
on the unsaturated aldehyde successfully directed the selective
labeling of Lys137, among the 59 HSA lysines present, through
azaelectrocyclization.10
Thus, incubation of HSA with probe 1 was hypothesized to favor
the selective labeling of lysines situated close to the ligand-binding
site, i.e., Lys137, through Schiff base formation followed by rapid
azaelectrocyclization. We envisioned that the 1,2-dihydropyridine
derivative would produce zwitterions through autooxidation and
hydrolysis. The cascade process would then release the DABCYL
fluorescence quencher, thereby selectively recovering the coumarin
fluorescence within the target protein.
The synthesis of 1 was carried out from the Boc-protected
diamine 2, which was previously prepared by Stille coupling as
a key step (Scheme 2 and Supplementary Information †).9a,10
Two Boc protecting groups in 2 were removed by treatment
with 20% TFA in CH2Cl2 to give the unstable diamine, which
was immediately reacted with the succinimidyl ester of 4-[4-
(dimethylamino)phenylazo]benzoic acid to selectively introduce
the fluorescence quencher at the C3 position of 2 in 62% yield
over two steps. The remaining anilino nitrogen of 3 was treated
with 7-diethylaminocoumarin-3-carboxylic acid in the presence of
HATU and triethylamine in DMF to provide a coupling product in
76% yield. TBDPS deprotection was then achieved in 87% yield by
Scheme 2 Synthesis of the “switch-on” labeling probe: a) 20% TFA,
CH2Cl2,
◦C; b) 4-[4-(dimethylamino)phenylazo]benzoic acid-OSu,
CH2Cl2, rt, 62% (2 steps); c) 7-diethylamino-3-carboxylic acid, HATU,
Et3N, DMF, rt, 76%; d) TBAF, AcOH, THF,
IBX-polystyrene, CH2Cl2, rt, quant.
0
0
◦C, 87%; e)
reaction with TBAF buffered with acetic acid in THF. Finally, the
allylic alcohol was quantitatively oxidized by 2-iodoxybenzoic acid
(IBX) immobilized on polystyrene in CH2Cl2 for 2 h to afford the
desired aldehyde probe 1. The unstable aldehyde 1, the structure of
1
which was rapidly confirmed by the characteristic H NMR and
MS, was immediately used for fluorescence and/or labeling ex-
periments. As expected, fluorescence analysis of 1 (concentration
of 1: 1.0 ¥ 10-7 M) detected no coumarin fluorescence signal at
480 nm (excitation at 420 nm) due to efficient FRET (quenching)
between the two dyes proximal to 1 (spectra similar to that shown
in Fig. 1a). The labeling-induced “switch-on” strategy was thereby
demonstrated using the HSA protein.
The probe◦1 was first incubated with HSA in 0.1 M phosphate
buffer at 25 C (incubation concentrations, HSA: 1.0 ¥ 10-8 M,
probe: 1.0 ¥ 10-7 M), and the resulting solution was directly
analyzed by fluorescence spectroscopy. The coumarin fluorescence
rapidly increased from 20 min to 2 days, and it reached the
maximum in 5 days. The results indicate the release of the DAB-
CYL quencher from the coumarin-labeled protein through the
electrocyclization-initiated reaction on the HSA lysines (Fig. 1b).
The formation of the zwitterion product was also confirmed by
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Fig. 2 Fluorescence spectra of a solution containing several proteins
and peptides after treatment with 1. Incubation and fluorescence analysis
were performed under conditions identical to those used for Fig. 1. (a)
Protein and peptide mixtures consisted of phospholipase A2, TGF-b3,
orosomucoid, anti-Tau antibody, somatostatin, neurotensin, and ACTH.
(b) Protein and peptide mixtures containing HSA. Blue: 1 h; red: 1 d;
green; 2 d after incubation.
Fig. 1 Fluorescence spectra of HSA solutions after treatment with
(a) alcohol 4 (aldehyde precursor) as a control and (b) probe 1. Both
incubation and fluorescence analysis were performed at 1.0 ¥ 10-8 M for
HSA, and at 1.0 ¥ 10-7 M for probes in PBS at room temperature (excitation
at 420 nm).
the MALDI-TOF-MS analysis (Supplementary Information†);
since the 1,2-dihydropyridine can be very rapidly oxidized and
hydrolyzed during the ionization by FAB, CI, ESI, and MALDI,
the time-course analysis of the 1,2-dyhydropyridine formation,
oxidation, and hydrolysis process was not possible by MS, as we
have also experienced previously.8b In clear contrast, fluorescence
was not enhanced for the alcohol precursor 4 upon incubation with
HSA under identical conditions (Fig. 1a). All concentrations, even
below 10-9 M HSA, were sensitively detected by the “switch-on”
probe 1 (see the Supplementary Information †).
fluorescence increased significantly in a time-dependent manner
in the presence of the target protein (Fig. 2b). The degree of
fluorescence enhancement was similar to that observed in the
HSA/probe spectra shown in Fig. 1b, indicating selective reaction
with HSA. To demonstrate that the fluorescence enhancement
arose from the preferential labeling of HSA lysines, the mixed so-
lution was directly analyzed by reverse-phase and size-partitioning
gel-filtration HPLC with fluorescence detection. To confirm this
analysis, MALDI-TOF-MS was also used for a few proteins (Sup-
plementary Information †). Coumarin fluorescence was detected
only at the elution time of the HSA, indicating ligand-directed
selective labeling of this protein. Overall, the ligand-directed
“switch-on” probe 1 labeled and sensitively detected a target
protein in a solution containing seven non-target protein/peptides,
all present at 10-8 M.
In summary, we developed a label-initiated “switch-on” probe
for detecting target proteins using a fluorescence quenching
system. This strategy selectively and sensitively detected a target
protein by making use of the unique reactivity of an unsaturated
aldehyde probe 1 on the target lysine. The target lysine was
selectively labeled with a fluorescently “caged” probe 1 that could
be loaded onto a high-affinity ligand of the target protein. The
electrocyclization products were susceptible to autooxidation, so
the fluorescence quencher present on the labeled protein was
automatically released under both neutral and physiological con-
ditions. Although many “fluorogenic” protocols in combination
with various biological techniques directed to sensitive fluores-
cence labeling have been reported,11 purely chemical methods that
employ novel reactivity are quite rare.7b The results described here
are applicable to the efficient labeling of target proteins in cell
The promising fluorescence spectra drove examination of the
labeling selectivity for the HSA protein of the ligand-directed
probe 1 in the context of other proteins and/or peptides. Bovine
pancreatic phospholipase A2 (14 kDa), TGF-b3 (25 kDa), oroso-
mucoid (44 kDa), and anti-Tau antibody (150 kDa) were tested as
non-target proteins, and somatostatin (1.6 kDa), neurotensin (1.7
kDa), and ACTH (2.9 kDa) were tested as non-target peptides
(Supplementary Information †). A 1.0 ¥ 10-8 M concentration (in
PBS) of each protein or peptide was incubated in the presence of a
10 ¥ concentration of the probe 1. Gratifyingly, no detectable
coumarin fluorescence was observed at 480 nm over 2 days.
Fluorescence enhancement was not observed, even at 1.0 ¥ 10-6
M
peptide or protein concentrations (Supplementary Information †).
The data clearly showed preferential labeling of HSA by the probe
1 under mild reaction conditions. Selective labeling was efficiently
achieved via the strong coumarin-protein interactions.
Last, a mixture of these seven proteins and peptides (each 1.0
¥ 10-8 M in PBS) was treated with the probe 1 (1.0 ¥ 10-7 M)
in the absence or presence of HSA at the same concentration as
the other biomolecules (Fig. 2). Coumarin fluorescence at 480 nm
was not observed in the absence of HSA (Fig. 2a), whereas the
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lysates as well as on cell surfaces without the need for isolation
and/or washing procedures. Alternatively, direct application to
living animals permits the use of the labeled proteins and/or
cells for molecular imaging immediately following injection of the
probe. These studies are currently in progress in our laboratory.
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Parts of this work were financially supported by a grant from
Grants-in-Aid for Scientific Research No. 19681024, 19651095 and
23681047 from the Japan Society for the Promotion of Science,
Collaborative Development of Innovative Seeds from Japan
Science and Technology Agency (JST), the New Energy and Indus-
trial Technology Development Organization (NEDO, project ID:
07A01014a), Research Grants from the Yamada Science Founda-
tion, as well as the Molecular Imaging Research Program, Grants-
in-Aid for Scientific Research from the Ministry of Education,
Culture, Sports, Science and Technology (MEXT) of Japan.
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