A FRET-based fluorogenic phosphine for live-cell imaging with the Staudinger ligation

Article abstract of DOI:10.1002/anie.200704847

Azide imaging: A fluorogenic phosphine based on a FRET-quenching mechanism allows for live-cell imaging of azido sugars by the Staudinger ligation. This design strategy can accommodate numerous fluorophores and complementary quenchers, enabling extension to multi-color imaging. In the image shown, the nuclei are blue while the cell surfaces and Golgi are green. (Figure Presented).

Full text of DOI:10.1002/anie.200704847

Communications
DOI: 10.1002/anie.200704847
Live-Cell Imaging
A FRET-Based Fluorogenic Phosphine for Live-Cell Imaging with the
Staudinger Ligation**
Matthew J. Hangauer and Carolyn R. Bertozzi*
Fluorescent labeling is a central tool for studying localization,
trafficking, and expression levels of biomolecules in live cells.
Biologists routinely rely on this information to assist in the
study of cellular processes. While fluorescent fusion proteins
and other genetically encoded tags have been used to image
specific proteins in live cells,[1] analogous labeling techniques
have not been available for imaging biomolecules not directly
encoded by the genome, including glycans, lipids, and other
metabolites. As a result, a lag in cell-based studies of these
molecules has occurred despite their documented importance
in many essential biological processes.[2]
The metabolic labeling technique can be used to introduce
bioorthogonal chemical reporters into cellular biomolecules
without genetic manipulation.[3] Subsequent treatment with
an exogenously delivered probe allows direct tagging of the
target biomolecule. The most versatile bioorthogonal chem-
ical reporter is the azide. Metabolic labeling of biopolymers
with azido amino acids,[4] sugars,[5] lipids,[6] and cofactors[7]
have all been realized in live cells. Once in place, the azides
can be reacted with triaryl phosphines by the Staudinger
ligation[8] and alkynes by either a copper-catalyzed [3+2]
cycloaddition (i.e., click chemistry)[9] or a strain-promoted
[3+2] cycloaddition.[10] While the copper catalyst required for
click chemistry has been reported to be toxic to cells in some
cases,[4a,10c] the Staudinger ligation reagents have no apparent
toxicity,[11] rendering it attractive for studies with live cells or
organisms.
negatively charged fluorophore; other fluorophores suffered
from nonspecific cell binding and, accordingly, high back-
ground labeling and low sensitivity. This finding underscores
the major challenge posed by direct fluorescence imaging
approaches: how to minimize background fluorescence to
increase the signal-to-noise ratio.
An ideal labeling reagent would remain nonfluorescent
until bound to its target. This “fluorogenic” principle has been
widely employed for nucleic acid detection[14] and enzyme
activity assays.[15] We previously explored phosphine–cou-
marin analogues in which the lone pair of electrons on
phosphorus quenched the fluorophore to which it was directly
attached.[16] During the Staudinger ligation, oxidation to the
phosphine oxide enhanced fluorescence by 60-fold. However,
phosphines are prone to nonspecific air oxidation as well, a
side reaction that produced high background fluorescence in
cell-imaging experiments. More recently, fluorogenic naph-
thalimide and coumarin dyes have been designed to label
azide- or alkyne-modified biopolymers using click chemis-
try.[5c,17] While suitable for fixed cells, the toxicity of the
Fluorescent phosphine probes have been used for direct
imaging of various azide-bearing biomolecules with the
Staudinger ligation in cell-free environments.[12] Recently,
we applied phosphine-based dyes to image azides on the
surface of live cells.[13] Notably, significant labeling above
background levels could only be achieved using a highly
[*] M. J. Hangauer, Prof. C. R. Bertozzi
Departments of Chemistry and Molecular and Cell Biology
and Howard Hughes Medical Institute
University of California and The Molecular Foundry
Lawrence Berkeley National Laboratory
Berkeley, CA 94720 (USA)
Fax: (+1)510-643-2628
E-mail: crb@berkeley.edu
[**] The authors thank Dr. Nicholas Agard and Prof. Isaac Carrico for the
mDHFR samples, Dr. Jennifer Prescher for Ac4ManNAz, Prof.
Christopher Chang for the use of the fluorimeter, and Dr. Jennifer
Czlapinski, Pamela Chang, Jeremy Baskin, and Dr. Christopher de
Graffenried for helpful advice and discussion. This work was
supported by a NDSEG Fellowship (to M.J.H.) and NIH grant
GM058867.
Figure 1. A FRET-based fluorogenicphosphine for live-cell imaging.
a) The design of a quenched phosphine–fluorophore that is activated
upon Staudinger ligation with azides. b) Compound 1, possessing
fluorescein (green) and disperse red 1 (dark red) moieties.
Supporting information for this article is available on the WWW
under http://www.angewandte.org or from the author.
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Chemie
copper reagent precludes the use of such dyes for live-cell
imaging.
Here, we report the design of a fluorogenic phosphine
reagent that can image azides on live cells with minimal
background. The reagent, compound 1 (Figure 1), comprises
a phosphine-tethered fluorophore moiety that is quenched
intramolecularly by an ester-linked fluorescence resonance
energy transfer (FRET) quencher, disperse red 1.[18] Stau-
dinger ligation of compound 1 with azides results in cleavage
of the ester and concomitant unquenching.
Nonspecific phosphine oxidation should not interfere with
the FRET quenching efficiency; hence, this design overcomes
the significant shortcoming of our previously described
fluorogenic phosphine. As a fluorescein analogue, compound
1 also benefits from spectral properties that are better suited
for live-cell imaging than earlier coumarin and naphthalimide
dyes.
The synthesis of compound 1 is described in detail in the
Supporting Information and is outlined in Scheme 1. Briefly,
carboxylic acid 2[19] was protected to yield tert-butyl ester 3.
Subsequent mild saponification of the methyl ester provided
compound 4, which was converted to triaryl phosphine 5 by
palladium cross-coupling with diphenylphosphine. Esterifica-
tion with commercially available disperse red 1 gave 6, which
was then deprotected to afford acid 7. Coupling of fluorescein
derivative 8[13] with 7 yielded compound 1.
A model reaction of 1 and benzyl azide was performed in
aqueous KH2PO4 (10 mm)/acetonitrile (1:1) (Scheme 2). The
Staudinger ligation to form 9 occurred with an apparent
second-order rate constant of 0.0038 Æ 0.0008mÀ1 sÀ1. As
expected based on previous kinetic and mechanistic studies,[20]
replacing the methyl ester of earlier Staudinger ligation
reagents with the disperse red 1 ester did not affect the
reaction rate.
Scheme 1. Synthesis of phosphine 1. Reagents and conditions:
a) tBuOH, DMAP (0.5 equiv), EDAC, CH2Cl2, 84%; b) LiOH
(1.5 equiv), MeOH/H2O (3:1), 94%; c) HPPh2, K2CO3, Pd(OAc)2
(0.3 mol%), CH3CN, reflux, 72%; d) disperse red 1, DMAP (0.1 equiv),
DCC, CH3CN (83%); e) TFA (26 equiv), TES (5 equiv), CH2Cl2 (87%);
f) 8, HATU, DIPEA, DMF (65%). DCC=N,N’-dicyclohexylcarbodiimide,
DIPEA=N,N-diisopropylethylamine, DMAP=4-dimethylaminopyridine,
EDAC=3-(3-dimethylaminopropyl)-1-ethylcarbodiimide, HATU=O-
(7-azabenzotriazol-1-yl)-N,N,N’,N’-tetramethyluronium hexafluorophos-
phate, TES=triethylsilane, TFA=trifluoroacetic acid.
We next measured the photophysical parameters of 1 and
its ligation product 9 (Table 1). Also, the phosphine oxide
derived from 1 (referred to as 1-oxide, see the Supporting
Information) was synthesized and analyzed. Importantly, 1
and 1-oxide were found to be essentially nonfluorescent
(quantum yields for both were < 0.01). Therefore, this FRET-
based fluorogenic phosphine will not suffer from background
fluorescence in the event of nonspecific phosphine oxidation.
In contrast to 1 and 1-oxide, Staudinger ligation product 9 was
strongly fluorescent, with a quantum yield of 0.64 Æ 0.02,
reflecting an increase in fluorescence quantum yield relative
to 1 of at least 170-fold. From these data, it is clear that 1
exhibits very efficient intramolecular FRET quenching and is
unquenched upon Staudinger ligation with an azide.
Compound 1 was next tested with an azide-modified
protein (Figure 2). Recombinant murine dihydrofolate reduc-
tase (mDHFR) containing azidohomoalanine in place of
native methionine residues,[19] as well as native mDHFR as a
control, were incubated with 12.5 mm 1 for 20 h at room
temperature. The crude reaction mixtures were analyzed by
SDS-PAGE, and the gel was imaged by fluorescence, reveal-
ing azide-specific labeling with no detectable background
fluorescence.
incubated with peracetylated N-a-azidoacetylmannosamine
(Ac4ManNAz) for three days in order to introduce N-a-
azidoacetyl sialic acid (SiaNAz) into their cell surface,
secreted, and Golgi-resident glycans.[8,11a] The Ac4ManNAz-
treated CHO cells were incubated with 25 mm 1 for 8 h at
378C and subsequently analyzed by flow cytometry. Robust
fluorescent labeling was observed for cells treated with both
Ac4ManNAz and 1 (Figure 3). By contrast, control cells
lacking azides but treated with 1 displayed minimal fluores-
cence. Importantly, we did not observe any nonspecific ester
hydrolysis by cellular esterases that would liberate the
quencher prematurely and create unwanted background
fluorescence.
Finally, we evaluated compound 1 for live-cell imaging by
fluorescence microscopy. HeLa cells were treated with
Ac4ManNAz for 40 h, rinsed, and then incubated with 50 mm
Compound 1 was then employed to label azides displayed
on live cells. Chinese hamster ovary (CHO) cells were
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Figure 3. Flow cytometry analysis of live Ac4ManNAz-treated CHO
cells labeled by phosphine 1. Cells were treated with Ac4ManNAz for
three days and then phosphine 1 for 8 h. Control cells were either
untreated or treated with phosphine 1 alone. Error bars represent the
standard deviation of the mean for triplicate measurements.
Scheme 2. Model Staudinger ligation.
Table 1: Photophysical parameters of phosphine probes.
e [mÀ1 cmÀ1 [a]
]
labs [nm][a] lem [nm][a,b] FF
[a–c]
Probe
1 for 8 h at 378C. Bright cell-surface labeling was observed for
cells displaying azides (Figure 4a), with essentially no back-
ground labeling observed for cells lacking azides (Figure 4b).
HeLa cells bearing SiaNAz residues also demonstrated
intracellular labeling that colocalized with a live-cell Golgi
marker (Figure 4a, top row), as well as a Golgi-protein-
specific antibody (Figure 4a, bottom row). Because 1 was
shown to be live-cell impermeant in other assays (data not
shown), the Golgi labeling observed in this experiment likely
reflects the internalization of labeled cell-surface glycans
rather than direct labeling of Golgi-resident azides. In fact, we
recently observed this phenomenon with difluorinated cyclo-
octyne imaging reagents as well.[10d] Also, 1 was shown to be
nontoxic to the cells by exclusion of propidium iodide, a
reagent that selectively stains the nuclei of dead cells
(Figure 4). Additionally, lack of increased staining relative
to untreated cells with early apoptosis marker Annexin V
confirmed that 1 is not cytotoxic (see Figure S2 in the
1
6600Æ200
505
505
515
520
520
0.00372Æ0.00005
0.00521Æ0.00004
0.64Æ0.02
1-oxide 18800Æ300
9
44000Æ1000 501
[a] Measured in phosphate-buffered saline (PBS), pH 7.0. [b] lex =
470 nm. [c] Relative quantum yields of fluorescence (FF) measured
using fluorescein as the standard.
Figure 2. Labeling of azido-mDHFR with compound 1. Purified azido-
mDHFR (left lane) and native mDHFR (right lane) were labeled with
12.5 mm 1 for 20 h at RT in PBS under denaturing conditions. The
crude reaction mixtures were separated by SDS-PAGE and the gel was
analyzed by fluorescence imaging (top row) and by Zn stain to reveal
total protein content (bottom row).
Figure 4. Fluorescence microscopic images of Ac4ManNAz-treated HeLa cells labeled with phosphine 1. a) Top row: Live HeLa cells treated with
Ac4ManNAz, 1 (FITC channel), and the live-cell Golgi marker BODIPY TR C5-ceramide (Cy3 channel); bottom row: HeLa cells treated with
Ac4ManNAz, 1, then fixed, permeabilized, and treated with anti-Golgin 97 mouse mAb and goat anti-mouse IgG-AlexaFluor647 (Cy5 channel).
b) Live HeLa cells treated with 1 only. All cells were treated while alive with nuclear stain Hoechst 33342 (DAPI channel) and viability stain
propidium iodide (Cy3 and Cy5 channels). The lack of nuclear fluorescence in Cy3 and Cy5 channels indicates propidium iodide exclusion from
cells. The scale bars represent 10 mm.
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Received: October 18, 2007
Published online: February 27, 2008
Keywords: azides · fluorescent probes · glycosylation ·
.
phosphines · Staudinger ligation
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