Fluorogenic azidofluoresceins for biological imaging

Article abstract of DOI:10.1021/ja308203h

Fluorogenic probes activated by bioorthogonal chemical reactions can enable biomolecule imaging in situations where it is not possible to wash away unbound probe. One challenge for the development of such probes is the a priori identification of structure

Full text of DOI:10.1021/ja308203h

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Communication
Fluorogenic Azidofluoresceins for Biological Imaging
Peyton Shieh, Matthew John Hangauer, and Carolyn R Bertozzi
J. Am. Chem. Soc., Just Accepted Manuscript • Publication Date (Web): 01 Oct 2012
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Fluorogenic Azidofluoresceins for Biological Imaging
Peyton Shieh,†,¥ Matthew J. Hangauer,†,¦,¥ and Carolyn R. Bertozzi*,†,‡,§
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Departments of Chemistry† and Molecular and Cell Biology‡ and Howard Hughes Medical Institute§, University of
California, Berkeley, California 94720, United States
KEYWORDS (Word Style “BG_Keywords”). If you are submitting your paper to a journal that requires keywords,
provide significant keywords to aid the reader in literature retrieval.
Supporting Information Placeholder
light.5,10,11 Such wavelengths are not ideal for biological
ABSTRACT: Fluorogenic probes activated by bioorthog-
imaging due to high levels of autofluorescence and poor
onal chemical reactions can enable biomolecule imaging
tissue penetrance.13
in situations where it is not possible to wash away un-
An obvious improvement upon these designs would be
the development of azido or alkynyl fluorogenic probes
with longer excitation and emission wavelengths. Some
attempts at achieving this goal have been made. Re-
cently, an azido BODIPY probe was reported, but the
compound was unstable and unable to specifically react
with alkynes on cellular biolmolecules.6 An alkynyl ben-
zothiazole analog that emits at ~500 nm has been re-
ported but its fluorescence enhancement upon reaction
with azides was a modest 3.5-fold in aqueous buffer.11 A
screen of 200 combinations of various alkynyl xan-
thenes and organic azides produced two reagent pairs
that reacted to give a fluorescence enhancement of 10-
fold in organic solvent.12 The utility of these alkyne/azide
pairs in biological settings remains unclear. Thus, fluo-
rogenic azido or alkynyl probes that perform well as cell
imaging reagents remain an important goal.
bound probe. One challenge for the development of
such probes is the a priori identification of structures that
will undergo a dramatic fluorescence enhancement by
virtue of the chemical transformation. With the aid of
density functional theory calculations reported previously
by Nagano and coworkers, we identified azidofluorescein
derivatives that were predicted to undergo an increase in
fluorescence quantum yield upon Cu-catalyzed or Cu-
free cycloaddition with linear or cyclic alkynes, respec-
tively. Four derivatives were experimentally verified in
model reactions, and one, a 4-azidonaphthyl fluorescein
analog, was further shown to label alkyne-functionalized
proteins in vitro and glycoproteins on cells with excellent
selectivity. The azidofluorescein derivative also enabled
cell imaging under no-wash conditions with good signal
above background. This work establishes a platform for
the rational design of fluorogenic azide probes with
spectral properties tailored for biological imaging.
Here we report the rational design and experimental
validation of azide-functionalized fluorogenic probes
based on the widely-used blue-excitation green-emission
fluorescein scaffold. Our design capitalized on a theoret-
ical model developed by Nagano and coworkers that
enables prediction of fluorescence quantum yield based
on density functional theory calculations.14 Nagano
demonstrated that the pendant aryl ring of fluorescein
can quench fluorescence via photoinduced electron
transfer.15 This quenching can be increased or de-
creased in a predictable manner by altering the electron
density of the aryl ring using various substituents. More
importantly, Nagano and coworkers also demonstrated a
direct relationship between experimentally determined
fluorescence quantum yield and the calculated HOMO
energy (EHOMO) of the pendant aryl ring at the B3LYP/6-
31G(d) level of theory. Thus, in principle, by calculating
the EHOMO values of the aryl ring, one can predict the
fluorescence quantum yield of the corresponding fluo-
rescein and identify suitable targets for experimental
evaluation. This insight has guided the design of other
activatable fluorescein-based probes.16,17
Bioorthogonal chemistry has enabled the visualization
and study of biomolecules that cannot be tagged with
genetically encoded fluorescent proteins.1 Much work
has been devoted to expanding the toolbox of
bioorthogonal reactions, and these efforts can be com-
plemented by the development of fluorogenic probes.2
Such probes are typically endowed with a functionality
that suppresses fluorescence. Its transformation during
the reaction creates a new functionality that no longer
quenches fluorescence of the underlying system, result-
ing in a fluorescence enhancement. Such probes offer
significant advantages for imaging studies where it is not
possible to wash away unreacted probe, such as real-
time imaging of dynamic processes in cells or visualiza-
tion of molecules in live organisms.
One of the most widely used bioorthogonal reactions
is the azide-alkyne [3+2] cycloaddition to form a triazole.
This reaction has enabled the selective visualization of
azide- or alkyne-labeled proteins, glycans, nucleic acids,
Applying this concept to the development of a fluoro-
genic azidofluorescein, we performed computational
analyses of a panel of fluorescein analogs in which the
azide group was directly appended to a variety of aryl
substituents (Table S1). We anticipated that triazole for-
and lipids.3 Several
azide-4-7
and alkyne7-11
-
functionalized fluorogenic probes have been reported,
largely based on coumarins4,8, naphthalimides7 and oth-
er systems that require UV excitation and emit blue
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mation would lower the aryl EHOMO value relative to that
(Scheme 2). In one pot, we treated 10 with two equiva-
1
2
3
4
of the azide-functionalized substrate, reducing photoin-
duced electron transfer and resulting in fluorescence
turn-on (Figure 1). We focused our analysis on targets
that could be readily synthesized from commercially
available
lents of sodium hydride, then with tert-butyllithium to ef-
fect lithium halogen exchange; coupling with 5, afforded,
after acid workup, compound 11. Deacetylation using
BF3-OEt2 in MeOH24, diazotization, and azide displace-
ment afforded the desired azidofluorescein 4.
5
6
7
8
9
To synthesize the triazole products for photophysical
measurements, azidofluoresceins 1-4 were further re-
acted with model alkynes 12-14 (Figure 2) under Cu-
catalyzed (for compound 12) or Cu-free (13 [also abbre-
viated DIFO] and 14 [DIMAC]) conditions.25,26 The fluo-
rescence quantum yields of all compounds were then
measured in pH 7.4 phosphate-buffered saline (PBS)
(Table 1).27 Each triazolylfluorescein showed an increase
in fluorescence quantum yield compared to the parent
azidofluorescein. The relationship between fluorescence
quantum yield and calculated EHOMO followed a trend
similar to that observed by Nagano (Figure 2). As a point
of comparison, we analyzed known 5-azidofluorescein
(N3-fluor, Table 1), which is currently used as an alkyne-
specific probe but is not reported to be fluorogenic.28,29
The calculated EHOMO values of 5-azidofluorescein and
its corresponding triazole product with 12 were -0.242
and -0.246 Hartrees, respectively, suggesting that both
compounds should be highly fluorescent. Consistent with
prediction, we found the two compounds to have fluo-
rescent quantum yields of 0.75 and 0.59, respectively.
a)
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b)
Figure 1: Computation-aided design of fluorogenic az-
idofluorescein analogs. a. Relationship between pen-
dant aryl EHOMO and fluorescence quantum yield. The
ideal EHOMO values of the azido and triazolylfluorescein
analogs (left and right, respectively) are indicated with
brackets.14 b. Structures of target azidofluoresceins 1-4.
Interestingly, the fluorescence enhancement of 1-4
upon triazole formation was dependent on the structure
of the alkyne substrate, but not in a systematic way.
Presumably, structure-dependent pathways for nonradia-
tive decay contribute to the observed fluorescence of the
triazole products. Nonetheless, half of the azidofluores-
cein-alkyne pairs gave a fluorescence turn-on of >10-
fold, indicating that rational design principles can more
efficiently identify fluorogenic reagent pairs than undi-
rected screening approaches.
bromo- or iodoaryl starting materials with a substituent
ortho to the halogen.18 Table S1 shows the 15 analogs
chosen for computational studies as well as the EHOMO
values of the aryl azides and corresponding triazoles
calculated at the B3LYP 6-31G(d) level of theory.
Calculations indicated that all compounds, with one
exception, undergo the expected decrease in aryl EHOMO
value upon triazole formation. From these compounds,
four (1-4, Fig. 1b) were chosen as targets for synthesis
and further evaluation. These candidates possessed
azide EHOMO values of -0.196 to -0.210 Hartrees and tria-
zole EHOMO values of -0.203 to -0.220 Hartrees. Given
the relationship between fluorescence quantum yield
and EHOMO value, we anticipated that compounds 1-4
would undergo an increase in fluorescence quantum
yield at pH 7.4 upon triazole formation.19
Scheme 1: Synthesis of azidofluorescein 1.
Br
tBuLi,
then 5,
Br
HCl, NaNO2
then KOH, pyrrolidine
then HCl
N
N
N
H2O
64%
THF
64%
NH2
TBSO
O
OTBS
6
7
Compounds 1, 2 and 3 were prepared by addition of
the corresponding aryl organolithium compound to a
protected xanthone derivative, an approach reported
previously by Tsien and coworkers (Scheme 1).22 To
generate compound 1, we subjected commercially avail-
able bromoaniline 6 to diazotization and then protected
the diazonium group by reaction with pyrrolidine,20,21 af-
fording aryl triazene 7. The aryl triazene underwent lithi-
um halogen exchange and addition to bis-TBS-protected
xanthone 522 to afford fluorescein derivative 8. Treatment
of this compound with acid unmasked the diazonium
group, which was displaced by azide anion to afford az-
idofluorescein 1. Using this same strategy, azidofluores-
ceins 2 and 3 were prepared (see Supporting Infor-
mation). Attempts to convert 4-bromo-6-aminoindole, the
starting material for compound 4, to the corresponding
triazene were unsuccessful.23 Instead, we opted to start
from acetylated indole 10, prepared from known com-
pound 9 by iron reduction and acetylation in acetic acid
HO
O
N
O
HO
O
O
O
5
HCl, NaN3
H2
O
53%
N3
N
N
1
8
Scheme 2: Synthesis of azidofluorescein 4.
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Br
Br
OMe
Fe, ∆
O
1
2
3
4
5
6
7
8
OMe
NO2
NaH, then tBuLi,
AcOH
51%
N
O2N
Me
N
5
then
H
H
then HCl
THF
9
10
58%
O
HO
Me
O
HO
O
O
1) BF3-OEt2, MeOH
2) NaNO2, NaN3, 3:1 AcOH/H2O
30% over 2 steps
O
N
H
N
N
N3
H
H
4
11
9
MeO
MeO
F
F
10
11
12
13
14
O
OH
N
N
H
CO2H
O
O
HO
O
Figure 3: Relationship between calculated pendant aryl
HOMO and fluorescence quantum yield of azidofluoresce-
12
13 (DIFO)
14 (DIMAC)
E
Figure 2: Alkynes used for triazolylfluorescein synthesis.
15
16
17
18
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20
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ins and triazolylfluoresceins. The grey line denotes the
relationship between aryl EHOMO and fluorescence quan-
tum yield determined in Ref. 14. Points represent exper-
imental data from individual azidofluoresceins (triangles)
and triazolylfluoresceins (circles). The points corre-
sponding to compound 1 and its triazole products are
highlighted in red.
Table 1: Fluorescence properties of azido- and triazol-
ylfuoresceins in pH 7.4 PBS. “1-12” denotes the triazol-
ylfluorescein product from reaction of 1 and 12. Other
triazolylfluoresceins are named analogously.
Compound
λex
λem
Ф500
In-
crease
fluorescein
490
497
495
499
499
501
510
513
518
517
517
521
--
--
Of the reagents tested, azidofluorescein 1 gave the
highest and most alkyne-independent increase in fluo-
rescence quantum yield at pH 7.4. Thus, we chose to
further evaluate its use in biological labeling. First, we
performed a model reaction with alkyne 12 under stand-
ard biological labeling conditions (CuSO4, ligand,30,31 and
sodium ascorbate). The expected fluorescence increase
was observed and the reaction was complete within 1 h
(Figure S2). We next demonstrated that azidofluorescein
1 can selectively label alkyne-tagged proteins. Bovine
serum albumin (BSA) was functionalized with alkynes by
modification of its lysine side chains with 4-pentynoyl-
NHS-ester. An alkene-modified control sample was pro-
duced by reaction of BSA with 4-pentenoyl-NHS ester.
Alkyne- or alkene-modified BSA, as well as unmodified
BSA, were incubated with 1, CuSO4, TBTA, and sodium
ascorbate in 95:5 pH 7.4 PBS/tert-butanol, and the sam-
ples were analyzed by SDS-PAGE. Fluorescence scan-
ning showed that only alkyne-modified BSA was chemi-
cally labeled, and in a Cu-dependent manner (Figure
S3).
1
0.024
0.70
0.81
0.72
0.0005
--
1-12
29x
34x
30x
--
1-DIFO
1-DIMAC
2
6
6
2-12
506
507
507
517
522
521
0.0018
0.0018
0.0007
3.2x
3.2x
1.4x
2-DIFO
2-DIMAC
3
496
499
499
499
497
499
497
497
492
494
516
520
519
518
518
519
520
516
511
516
0.057
0.72
--
3-12
13x
8.6x
13x
--
3-DIFO
3-DIMAC
4
0.49
0.72
0.0052
0.13
4-12
25x
3.7x
3.8x
--
4-DIFO
4-DIMAC
N3-fluor
N3-fluor-12
0.019
0.020
0.75
To evaluate azidofluorescein 1 as a biological imaging
reagent, Chinese Hamster Ovary (CHO) cells were met-
abolically
labeled
with
peracetylated
N-(4-
pentynoyl)mannosamine (Ac4ManNAl) (50 µM for 3
days) as previously described.32 These cells convert
Ac4ManNAl to the corresponding alkynyl-sialic acid,
which is then incorporated into cell-surface glycopro-
teins. Control cells were incubated with peracetylated N-
acetylmannosamine (Ac4ManNAc). The cells were then
fixed with paraformaldehyde and incubated with com-
pound 1, CuSO4, sodium ascorbate, and TBTA for 1 h.
After washing, the cells showed robust alkyne-
dependent labeling with 1 (Figure S4). The experiment
was then repeated, but without the washing step after
reaction with 1. As shown in Figure 4, bright cell-surface
fluorescence was observed for alkyne-labeled cells (Fig.
4b) compared to the background observed with control
0.59
0.79x
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(Ac4ManNAc-labeled) cells (Fig. 4d). By contrast, general strategy will be useful for the development of
1
2
3
4
5
6
7
8
whereas non-fluorogenic 5-azidofluorescein also labels
cells in an alkyne-dependent manner (Figure S5), the
strong fluorescence signal of this azide dye gave high
background under no-wash conditions (Figure 4f), there-
fluorogenic alkynyl fluorescein analogs as well. Indeed,
calculations indicate that conversion of aryl alkynes to
the corresponding triazoles results in a significant in-
crease in ELUMO
. In principle, alkynyl fluoresceins that
by obscuring cell-surface labeling of alkynyl sugars.
are internally quenched by PET, in this case from the
xanthene to the aryl ring, should undergo fluorescence
enhancement upon triazole formation.35 This notion, as
well as extension of the design principle to red-shifted
fluorophores, are interesting future directions.
O
HN
AcO
AcO
AcO
O
fix;
OAc
N3
I
N3
N3
1
Cu , TBTA,
Ac4ManNAl
3 days
9
no wash
10
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ASSOCIATED CONTENT
Supporting Information. Experimental and synthetic pro-
cedures, materials, and supporting figures and tables. This
material is available free of charge via the Internet at
http://pubs.acs.org.
AUTHOR INFORMATION
¥ Equal contribution
Corresponding Author
* crb@berkeley.edu
Present Address
Figure 4: No-wash cell labeling with azidofluorescein
probes. a. Outline of the no-wash labeling experiment
using Ac4ManNAl and 1. CHO cells were metabolically
labeled with Ac4ManNAl or Ac4ManNAc for 3 d. The
cells were then then reacted with 25 µM probe as well as
the other reagents for Cu-catalyzed click chemistry for 1
h. b-g. Top row: fluorescence images; Bottom row: cor-
responding DIC images. b,c. Ac4ManNAl-treated cells
reacted with 1 (inset shows a 3-fold magnification to
highlight cell-surface labeling). d,e. Ac4ManNAc-treated
cells reacted with 1. f,g. Ac4ManNAl-treated cells react-
ed with 5-azidofluorescein. Scale bar = 50 µm. Exposure
time/fluorescence cutoffs were 150 ms/150-400 for b, d and
10 ms/500-900 for f.
¦ School of Medicine, University of California, San Fran-
cisco
ACKNOWLEDGMENT
This work was funded by NIH grant GM58867 to C.R.B. We
would like to thank Prof. C. Chang (UC Berkeley) for the
generous use of his fluorimeter. M.J.H. was supported by a
National Defense Science and Engineering Graduate Fel-
lowship.
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A potential problematic side reaction of aryl azides is
their reduction to the corresponding anilines by endoge-
nous thiols. This reaction has been demonstrated to
proceed under physiological conditions,33 and indeed it
undermined the use of azidonaphthalimides as biological
imaging reagents.7 Aryl azide reduction has also been
used to detect intracellular hydrogen sulfide.34 Calcula-
tions indicate that this reduction pathway would not be
an issue for 1, as the predicted aryl EHOMO value of the
aniline reduction product is higher than that of the start-
ing aryl azide (-0.189 Hartrees vs. -0.210 Hartrees). To
demonstrate this experimentally, we reduced 1 with di-
thiothreitol and measured the fluorescence quantum
yield of the arylamine product. As expected, reduction
resulted in a decrease in fluorescent quantum yield at pH
7.4 from 0.024 to 0.0067, indicating that, if this side reac-
tion occurs in vivo, it would not contribute significantly to
background fluorescence.
In summary, by taking advantage of the observed rela-
tionship between EHOMO of a pendant aryl substituent and
fluorescence quantum yield, we were able to identify
potential fluorogenic azidofluoresceins. One azidofluo-
rescein, 1, was suitable for no-wash imaging of cells.
This compound is cell permeable and may be suitable
for intracellular imaging of biomolecule-associated cy-
clooctynes as well (Figure S6). We anticipate that this
(10) Key, J. A.; Cairo, C. W. Dyes and Pigments 2010, 88,
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whether measurements at pH 13, as performed by
Nagano, would reconcile the observed differenced
but, for most part, the fluorescence quantum yields of
1-4 were unchanged at higher pH.
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(28) Rotman, A.; Heldman, J. Biochemistry 1981, 20,
5995-5999.
(18) This ortho substituent restricts rotation around the Car-
yl-Caryl bond and is necessary for maintaining predict-
able fluorescence quantum yields (See ref 14).
(19) Nagano and coworkers have shown that while the
general trend relating EHOMO to quantum yield is simi-
lar at different pH values, the exact EHOMO values at
which fluorescence dramatically increases are pH-
dependent (See ref 14). We performed our calcula-
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