Fluorogenic Strain-Promoted Alkyne-Diazo Cycloadditions

Article abstract of DOI:10.1002/chem.201502242

Fluorogenic reactions, in which non- or weakly fluorescent reagents produce highly fluorescent products, are attractive for detecting a broad range of compounds in the fields of bioconjugation and material sciences. Herein, we report that a dibenzocyclooctyne derivative modified with a cyclopropenone moiety (Fl-DIBO) can undergo fast strain-promoted cycloaddition reactions under catalyst-free conditions with azides, nitrones, nitrile oxides, as well as mono- and disubstituted diazo-derivatives. Although the reaction with nitrile oxides, nitrones, and disubstituted diazo compounds gave cycloadducts with low quantum yield, monosubstituted diazo reagents produced 1H-pyrazole derivatives that exhibited an approximately 160-fold fluorescence enhancement over Fl-DIBO combined with a greater than 10 000-fold increase in brightness. Concluding from quantum chemical calculations, fluorescence quenching of 3H-pyrazoles, which are formed by reaction with disubstituted diazo-derivatives, is likely due to the presence of energetically low-lying (n,π?) states. The fluorogenic probe Fl-DIBO was successfully employed for the labeling of diazo-tagged proteins without detectable background signal. Diazo-derivatives are emerging as attractive reporters for the labeling of biomolecules, and the studies presented herein demonstrate that Fl-DIBO can be employed for visualizing such biomolecules without the need for probe washout.

Full text of DOI:10.1002/chem.201502242

DOI: 10.1002/chem.201502242
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Fluorogenic Strain-Promoted Alkyne–Diazo Cycloadditions
FrØdØric Friscourt,[a, b] Christoph J. Fahrni,[c] and Geert-Jan Boons*[a]
Abstract: Fluorogenic reactions, in which non- or weakly flu-
orescent reagents produce highly fluorescent products, are
attractive for detecting a broad range of compounds in the
fields of bioconjugation and material sciences. Herein, we
report that a dibenzocyclooctyne derivative modified with
a cyclopropenone moiety (Fl-DIBO) can undergo fast strain-
promoted cycloaddition reactions under catalyst-free condi-
tions with azides, nitrones, nitrile oxides, as well as mono-
and disubstituted diazo-derivatives. Although the reaction
with nitrile oxides, nitrones, and disubstituted diazo com-
pounds gave cycloadducts with low quantum yield, mono-
substituted diazo reagents produced 1H-pyrazole derivatives
that exhibited an approximately 160-fold fluorescence en-
hancement over Fl-DIBO combined with a greater than
10000-fold increase in brightness. Concluding from quantum
chemical calculations, fluorescence quenching of 3H-pyra-
zoles, which are formed by reaction with disubstituted
diazo-derivatives, is likely due to the presence of energetical-
ly low-lying (n,p*) states. The fluorogenic probe Fl-DIBO was
successfully employed for the labeling of diazo-tagged pro-
teins without detectable background signal. Diazo-deriva-
tives are emerging as attractive reporters for the labeling of
biomolecules, and the studies presented herein demonstrate
that Fl-DIBO can be employed for visualizing such biomole-
cules without the need for probe washout.
Introduction
acid containing a reporter can be introduced into proteins by
using genetic-code-expansion technology.[5] Bioorthogonal re-
actions have also found application in many other fields of re-
search, and, for example, are commonly employed for the
modification of polymers and nanoparticles for the develop-
ment of smart materials.[6]
Over the past decade, bioorthogonal reactions have opened
new avenues for the study of complex biomolecules in cells
and organisms.[1] Proteins, lipids, and glycans have been suc-
cessfully visualized by the selective incorporation of an abiotic
chemical functionality (reporter) that can be reacted with
a complementary bioorthogonal functional group linked to
Organic azides are the most versatile reporters due to their
small size and virtual absence in biological systems.[7] They can
be conjugated by Staudinger ligation using modified phosphi-
nes,[3b,8] CuI-catalyzed cycloaddition with terminal alkynes
(CuAAC),[9] or by strain-promoted alkyne–azide cycloaddition
(SPAAC).[10] The latter type of reaction, which employs strained
alkynes, such as difluorinated cyclooctyne (DIFO),[11] dibenzylcy-
clooctynol (DIBO),[12] and bicyclononyne (BCN),[13] is attractive,
because it is fast and does not require a potentially toxic metal
catalyst. Other commonly employed bioorthogonal reactions
include the inverse-electron-demand Diels–Alder cycloaddition
between alkenes and tetrazines.[14]
a diverse set of probes, such as biotin and fluorescent tags.[2]
A
commonly employed approach for incorporating a chemical
reporter into biomolecules is by feeding a metabolic precursor
functionalized with the reporter.[3] Alternatively, an exogenous-
ly administered enzyme and a corresponding activated biosyn-
thetic substrate modified by the chemical reporter can be em-
ployed.[4] It has also been demonstrated that an artificial amino
[a] Dr. F. Friscourt, Prof. Dr. G.-J. Boons
Complex Carbohydrate Research Center
University of Georgia, 315 Riverbend Road
Athens, GA 30602 (USA)
Bioorthogonal reactions, in which non- or weakly fluorescent
reagents produce highly fluorescent products, offer the possi-
bility to greatly expand the detection of a broad range of com-
pounds and are particularly advantageous for applications, in
which probe washout is not possible or desirable. A number of
such fluorogenic probes have been developed based on
CuAAC,[15] Staudinger ligation,[16] and inverse-electron-demand
Diels–Alder reactions.[17] All these probes were designed based
on the concept of quenching established fluorophores, such
as anthracene,[18] 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene
(BODIPY),[19] or coumarins[20] with a bioorthogonal moiety, such
as an azide or tetrazine. Upon reaction, the quenching func-
tionality is converted to the corresponding cycloadduct and
Fax: (+1)706-542-4412
E-mail: gjboons@ccrc.uga.edu
[b] Dr. F. Friscourt
Present address:
Institut EuropØen de Chimie et Biologie
UniversitØ de Bordeaux, INCIA, CNRS UMR 5287
2 rue Robert Escarpit, 33607 Pessac (France)
[c] Prof. Dr. C. J. Fahrni
School of Chemistry and Biochemistry and Petit Institute for
Bioengineering and Bioscience, Georgia Institute of Technology
901 Atlantic Drive, Atlanta, GA 30332 (USA)
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/chem.201502242.
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fluorophore emission is restored. Although in principle the
same approach could be used for strain-promoted cycloaddi-
tions, the corresponding tags would inevitably be much larger
compared to the rather inconspicuous propargyl groups used
in CuAAC, and the increased molecular size might adversely
affect the properties of target molecules, especially with
regard to their biodistribution and biological activity. For this
mately half the size of an azido group, thus providing unique
opportunities for orthogonal labeling of cellular components.
The favorable properties of nitrones, nitrile oxides, and diazo
reagents prompted us to explore whether they could also be
employed for fluorescence turn-on reactions with Fl-DIBO (1).
reason, the preferred approach for developing a catalyst-free Results and Discussion
fluorogenic click reagent is to utilize azide as the tagging
Reaction of Fl-DIBO (1) with various 1,3-dipolar compounds
moiety and to integrate the cyclooctyne group into the fluoro-
phore structure. A first attempt towards this goal was reported
by Bertozzi and co-workers, who designed the fluorogenic re-
agent CoumBARAC by fusing the cyclooctyne ring with a cou-
marin fluorophore.[21] Although the reaction product with 2-azi-
doethanol exhibited a tenfold increased emission compared to
unreacted CoumBARAC, it exhib-
The previously reported Fl-DIBO probe 1 has the advantage of
combining ease of preparation (five-step synthesis with 35%
overall yield) with strong fluorescence enhancement upon re-
action with azides, such as benzyl azide 2 (Scheme 1A).[22] In-
trigued by the Fl-DIBO turn-on properties, we hypothesized
ited a low fluorescence quantum
yield and required excitation
around 300 nm, a wavelength
regime that is incompatible with
many biological applications. Re-
cently, we reported a dibenzocy-
clooctyne derivative (1, Figure 1,
Fl-DIBO), which upon reaction
with an azide produces a cyclo-
addition product that is more
than 1000-fold brighter com-
pared to the unreacted reagent,
shows a large Stokes shift, and
can be excited above 350 nm,
the typical cut-off wavelength of
standard fluorescence micro-
scopes.[22] Quantum chemical
calculations indicated that fluo-
rescent activation was due to an
enhancement
in
oscillator
strength of the S0–S1 transition
upon triazole formation.
We have demonstrated that in
addition to azides, a number of
other 1,3-dipoles, such as nitro-
nes, nitrile oxides, and diazo de-
rivatives react readily with strain-
ed alkynes, such as DIBO.[23] Vari-
ous methods have been devel-
oped to incorporate these func-
tional groups into biomolecules,
and, for example, it was recently
shown that a diazo derivative of
N-acetylmannosamine endured
cellular metabolism and could
be incorporated into mammalian
cell-surface glycoproteins.[24] The
chemoselectivity of diazo and al-
kynyl groups enabled dual label-
ing of cells, and it was noted
that the diazo group is approxi- Scheme 1. Reaction of Fl-DIBO with various 1,3-dipoles.
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that strong fluorescence enhancements might be also ach-
ieved upon reaction with other 1,3-dipolar compounds, such
as nitrones, nitrile oxides, and diazo derivatives. Reaction of Fl-
DIBO 1 with nitrone 3 and nitrile oxide 4 generated the corre-
sponding cycloaddition products 6 and 7 in near quantitative
yields (Scheme 1A). Both reactions were performed at room
temperature in a mixture of dichloromethane and methanol
(4:1, v/v) and were completed within two hours.
termined the respective fluorescence quantum yields with qui-
nine sulfate as fluorescence standard (FF =0.54 in 1.0 n aque-
ous sulfuric acid).[26] All pertinent photophysical data are com-
piled in Table 1. Although the lowest-energy absorption bands
of all cycloadducts fall within a narrow range between l=
340–360 nm, the molar extinction coefficients differ by more
than one order of magnitude, with isoxazole 7 and pyrazoles
11 a and b being the strongest absorbers. Compared to Fl-
DIBO (1), the fluorescence emission maxima of the cycloaddi-
tion products are significantly blueshifted, and the observed
quantum yields vary over a large range. For example, fluores-
cence emission of 6 containing the non-aromatic 2,3-dihydroi-
soxazole ring was effectively quenched, whereas isoxazole de-
rivative 7 showed an eightfold fluorescence enhancement over
Fl-DIBO (1) and a redshifted emission maximum compared to
triazole 5 (514 vs. 489 nm). Although replacing the ester group
in 1H-pyrazole 11 a with a phenyl substituent in 11 b gave simi-
lar excitation and emission maxima along with a slightly im-
proved quantum yield, the fluorescence emission of derivatives
10b–d containing a disubstituted 3H-pyrazole ring was consis-
tently quenched. It is also noteworthy that all cycloadducts 3,
5, 7, and 9 exhibit large Stokes shifts (>6000 cmÀ1), making
them less prone to self-absorption.
Although many diazoalkanes are too reactive to be isolated
or used in a biological context, it is possible to stabilize these
derivatives to attain compounds that can be employed for bio-
orthogonal reactions. To identify a diazo derivative that exhib-
its optimal properties, we explored the influence of substitu-
ents on the rate of reaction with 1 and on the photophysical
properties of the corresponding cycloaddition products
(Scheme 1B). Diazo derivatives 8a–c and 9b were prepared in
good yield by MnO2-mediated oxidation of the corresponding
hydrazones (see the Supporting Information), 9-diazo-fluorene
8d was synthesized from the corresponding azido-derivative
following a reported protocol,[25] and diazoacetate 9a was
commercially available. Although reaction of Fl-DIBO (1) with
disubstituted diazo reagents 8a–d produced the respective
[3+2] cycloaddition products 10a–d, the monosubstituted
diazo reagents 9a and b gave the thermodynamically more
stable tautomers 11 a and b, respectively (Scheme 1B). The
structural assignments were based on chemical shift differen-
ces of the C3 ring carbon atom in the 13C NMR spectra. Consis-
tent with formation of a 3H-pyrazole ring, all disubstituted
diazo containing compounds produced cycloadducts with
chemical shifts around dꢀ110 ppm (105.8 ppm for 10b,
112.6 ppm for 10c, and 113.9 ppm for 10d). In contrast, the cy-
cloaddition products of the monosubstituted diazo reagents
produced substantially lower field resonances at 141.7 ppm
(11 a) and 144.3 ppm (11 b), indicating the presence of a 1H-
pyrazole ring.
Altogether, the pyrazoles 11 a and b displayed the strongest
fluorescence increase with a quantum yield of around 30%.
Combined with their large absorption cross-section at an exci-
tation wavelength of 370 nm, these cycloadducts are by more
than 10000-fold brighter compared to unreacted Fl-DIBO (1;
Table 1, last column), thus making monosubstituted diazo re-
agents potentially exciting chemical reporters as the photo-
physical properties of 1H-pyrazoles 11 a and b are more favora-
ble than those of cycloadducts of azides (5; Scheme 1).
Computational studies
The second-order rate constant for the cycloaddition reac-
tion of Fl-DIBO (1) and commercially available diazo ester 9a
was determined by monitoring product formation by H NMR
Intrigued by the strong fluorescence quenching of 3H-pyra-
zoles 10b–d, we were interested in exploring potential differ-
ences in their excited state manifolds compared to the highly
emissive 1H-pyrazoles 11 a and b. Of particular interest was the
1
spectroscopy in a mixture of CDCl3 and CD3OD (Figure S9 in
the Supporting Information). The measured second-order rate
constant of 2.4”10À3 mÀ1 sÀ1 was somewhat lower compared
s
to the cycloaddition with benzyl azide 2 (1.9”10À2 mÀ1 À1).[22]
3
question to what extent energetically low-lying triplet (n,p*)
states might contribute to fluorescence quenching in the 3H-
pyrazole derivatives.[27] For this purpose, we utilized com-
pounds 10b and 11 b as representative model compounds
and determined their vertical excitation energies based on
time-dependent density functional theory (TD-DFT) quantum
chemical calculations. Previous studies on Fl-DIBO (1) and tria-
zole 5 showed that the long-range corrected hybrid functional
CAM-B3LYP[28] with a polarized continuum model (PCM)[29] re-
produced absorption and emission data quite well for this
compound class. Following the same approach, we calculated
the energies of the first four excited singlet states at the
B3LYP/6-31+G(d)//B3LYP/6-31G(d) level of theory (Table 2) and
analyzed the nature of each transition based on electron densi-
ty difference plots (Figure 2). Consistent with the large molar
extinction coefficient of 10600mÀ1 cmÀ1 for the lowest-energy
absorption band, the first excited singlet state S1 of 11 b is do-
minated by a HOMO–LUMO transition with substantial oscilla-
The presence of the electron-withdrawing ethyl ester on the
diazo reagent significantly stabilizes the HOMO of the 1,3-
dipole, leading to a lower reactivity. Notably, diazo malonate
8a showed no reactivity with Fl-DIBO (1). Nevertheless, the ob-
served rate constant for the coupling with diazo ester 9a still
compares favorably with the reaction kinetics of the Stauding-
er ligation (k=1.5”10À4 mÀ1 sÀ1) or the cycloaddition reaction
of the first-generation cyclooctyne ALO with benzyl azide (k=
1.3”10À3 mÀ1 À1).[10a]
s
Photophysical characterization
To evaluate the change in fluorescence brightness of the cyclo-
addition products compared to Fl-DIBO (1), we acquired ab-
sorption and emission spectra in methanol (Figure 1) and de-
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Table 1. Photophysical properties of Fl-DIBO 1, as well as cycloadducts
5–7, 10b–d, and 11 a and b in MeOH at 298 K.
[b]
labs
eabs
[mÀ1 cmÀ1
lem
FF
Brightness[c] EF[d]
[a]
[nm]
]
[nm]
Fl-DIBO
(1)[e]
5[e]
6
7
10b
10c
10d
11 a
11 b
420
150
574
0.002
0.3 1
364
345
363
350
361
370
360
370
4600
1330
19250
1300
3000
2100
13400
10600
489
–
0.12
<0.001
0.016 300
0.001
0.002
0.002
0.30 4000
0.32 3400
550
1
1800
3
514
n.d.
n.d.
n.d.
463
469
1000
3
20
1
6
4
13
13000
11 000
[a] Determined at 370 nm. [b] Fluorescence quantum yield, quinine sul-
fate in 1.0 n H2SO4 as standard. [c] Product of the extinction coefficient
(at 370 nm) and the fluorescence quantum yield (eabsFF). [d] Enhance-
ment factor of the fluorophore brightness relative to Fl-DIBO (1). [e] Data
from Ref. [22].
Table 2. Computational data for cycloaddition products 10b and 11 b.[a]
State
Energy
TD-DFT
[eV][b]
Oscillator
strength
Exptl
absorption
[eV][c]
Exptl
eabs
[mÀ1 cmÀ1
[c]
]
10b
11 b
S1 (n,p*)
S2 (n,p*)
S3 (p,p*)
S4 (p,p*)
S1 (p,p*)
S2 (n,p*)
S3 (n,p*)
S4 (p,p*)
3.34
3.63
4.23
4.34
3.66
4.40
4.42
4.65
0.013
0.091
0.016
0.581
0.205
0.086
0.068
0.025
3.26
3.57
3.99
4.16
3.42
n.d.
n.d.
n.d.
800
1700
540
8300
10600
n.d.
n.d.
n.d.
[a] Calculated at the TD-CAM-B3LYP/6-31+G(d)//B3LYP/6-31G(d) level of
theory. [b] Vertical excitation energy. [c] Determined based on Gaussian fit-
ting as illustrated in Figure 3.
Figure 2. Excited state manifold for (A) pyrazole derivative 11 b and (B) 3H-
pyrazole derivative 10b. The dual color plots illustrate the total electron
density differences between each excited state and the corresponding
ground state (decreasing intensity shown in blue, increasing density red;
GS=ground state, S1, S2, S3, and S4 correspond to the four lowest-energy ex-
cited singlet states.
Figure 1. Absorption (solid traces; 10 mm solutions) and fluorescence emis-
sion (dashed traces; lexc =370 nm; OD370 =0.75) spectra of cycloadducts 5, 6,
7, and 11 a in MeOH. Inset: Visual comparison of the fluorescence intensity
of cycloadducts in MeOH with excitation at 365 nm.
tor strength. The corresponding electron density difference
plot resembles the pattern previously reported for the lowest
excited state of triazole 5[22] indicating a symmetry-allowed p–
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p* transition (Figure 2A). Although the closest higher-energy
excited singlet state S2 exhibits n,p* character involving the
carbonyl oxygen lone pairs, there is a large energy separation
of 0.74 eV between the two states such that the corresponding
3
triplet (n,p*) state should remain above S1 without interfering
with radiative deactivation. The proposed energy ordering is
supported by the inherently small singlet–triplet splitting of
(n,p*) states with DEST ꢀ0.1 eV.[30] In contrast, the lowest two
excited states of 3H-pyrazole 10b exhibit significant n,p* char-
acter with involvement of the nitrogen lone pairs on the 3H-
pyrazole ring (Figure 2B). Hence, the low fluorescence quan-
tum yield of 10b is likely caused by non-radiative deactivation
through intersystem crossing to the corresponding triplet
3(n,p*) states. The presence of an energetically low-lying (n,p*)
state is further supported by the experimental absorption
spectrum, which upon Gaussian fitting revealed a weak low-
energy band with a small molar extinction coefficient around
800mÀ1 cmÀ1 (Figure 3). Furthermore, the intensity and position
Figure 4. Visualizing diazo-labeled proteins with Fl-DIBO (1) as fluorogenic
reagent. A) Diazo-labeling of BSA (20 mgmLÀ1 in PBS) using NHS-activated
ester 12 (25 mm in DMSO) and subsequent reaction with Fl-DIBO (1)
(250 mm) overnight at 378C. B) In-gel visualization of labeled BSA with either
Fl-DIBO or DIBO-FITC by fluorescence imaging (top row; lexc =365 nm;
ldetec =480 nm) and by Coomassie Blue stain to reveal total protein content
(bottom row).
reagent and employed to modify bovine serum albumin (BSA)
as a model protein. The diazo ester 12 was attached to the
protein surface using standard NHS-ester-coupling conditions
(Figure 4 A). Briefly, a solution of BSA (20 mgmLÀ1) in phos-
phate buffered saline (PBS; pH 7.4) was incubated with a solu-
tion of NHS-activated diazo ester 12 (25 mm) in DMSO over-
night at room temperature. The excess of low molecular
weight NHS-activated diazo ester was removed by spin filtra-
tion. Diazo-labeled BSA was then reacted with Fl-DIBO (1) at
378C for 18 h and analyzed by in-gel fluorescence imaging. As
depicted in Figure 3B, a strong fluorescent band (lane 3) was
observed at 66 kDa, confirming the ligation reaction, whereas
no reaction was observed in the absence of either Fl-DIBO
(lane 2) or diazo ester label (lane 4). As an additional control,
diazo-conjugated BSA was exposed to DIBO-FITC[22] to yield
a fluorescent band at 66 kDa (lane 5); however, faint labeling
was also observed when DIBO-FITC was reacted with unmodi-
fied BSA (lane 6). Cyclooctynes can react with thiol residues of
proteins leading to background labeling.[32] Gratifyingly, Fl-
DIBO did not exhibit fluorescence turn-on in the presence of
unmodified BSA (lane 4).
Figure 3. Gaussian fitting (red trace) for the low-energy portion of the ab-
sorption spectrum (black trace) of 3H-pyrazole derivative 10b and compari-
son to computed vertical excitation energies (blue lines) based on TD-DFT
calculations (B3LYP/6-31+G(d)//B3LYP/6-31G(d) level of theory, PCM solva-
tion with methanol). The dashed traces correspond to the individual bands
extracted from a fit with four Gaussian functions. The experimental absorp-
tion spectrum is plotted on a molar absorptivity scale (left), whereas the in-
tensities of the TD-DFT transitions are represented as oscillator strengths
(right).
of the adjacent bands at shorter wavelengths fit qualitatively
well with the oscillator strengths and transition energies pre-
dicted based on the TD-DFT calculations (Figure 3 and Table 2).
Altogether, it can be concluded that the poor brightness of
10b compared with 11 b is due to a much smaller absorption
cross-section combined with efficient fluorescence quenching,
both of which are the result of energetically low-lying (n,p*)
states.
Conclusion
We have explored fluorescence turn-on cycloadditions of Fl-
DIBO 1 with a number of 1,3-dipoles including azides, nitrile
oxides, nitrones, and diazo-derivatives. It has been found that
reactions with nitrile oxides, nitrones, and disubstituted diazo
derivatives give adducts, in which the fluorescence emission is
low or quenched. On the other hand, cycloadditions of Fl-DIBO
(1) with monosubstituted diazo reagents give adducts that can
tautomerize to aromatic 1H-pyrazoles (11 a and b) and exhibit
Protein-labeling experiment
Next, we explored Fl-DIBO (1) as fluorogenic labeling agent for
diazo-functionalized biomolecules. Because diazo ester 9a was
found to be more stable than phenyl diazo methane 9b, NHS-
activated diazo ester 12[31] was synthesized as amino-reactive
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an approximately 160-fold enhancement in fluorescence quan-
tum yield combined with more than 10000-fold increase in
brightness. Photophysical properties of the pyrazoles, such as
quantum yield, brightness, and Stokes shift, are more favorable
than those of triazoles formed by a cycloaddition with azides.
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Acknowledgements
This research was supported by the National Cancer Institute
(R01CA88986 to G.J.B.) and the National Institute of General
Medical Sciences (R01GM067169 to C.J.F.) of the US National
Institutes of Health.
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Keywords: bioorthogonal · click chemistry · cycloaddition ·
cyclooctyne · fluorogenic probes
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Published online on August 18, 2015
Chem. Eur. J. 2015, 21, 13996 – 14001
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