Perfluoroaryl Azide Staudinger Reaction: A Fast and Bioorthogonal Reaction

Article abstract of DOI:10.1002/anie.201705346

We report a fast Staudinger reaction between perfluoroaryl azides (PFAAs) and aryl phosphines, which occurs readily under ambient conditions. A rate constant as high as 18 m^?1 s^?1 was obtained between methyl 4-azido-2,3,5,6-tetrafluorobenzoate and methyl 2-(diphenylphosphanyl)benzoate in CD₃CN/D₂O. Furthermore, the iminophosphorane product was stable toward hydrolysis and aza-phosphonium ylide reactions. This PFAA Staudinger reaction proved to be an excellent bioothorgonal reaction. PFAA-derivatized mannosamine and galactosamine were successfully transformed into cell-surface glycans and efficiently labeled with phosphine-derivatized fluorophore-conjugated bovine serum albumin.

Full text of DOI:10.1002/anie.201705346

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Accepted Article
Title: Perfluoroaryl Azide-Staudinger Reaction: A Fast and
Bioorthogonal Reaction
Authors: Madanodaya Sundhoro, Seaho Jeon, JaeHyeung Park, Olof
Ramström, and Mingdi Yan
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201705346
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10.1002/anie.201705346
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Perfluoroaryl Azide–Staudinger Reaction: A Fast and
Bioorthogonal Reaction
Madanodaya Sundhoro,[a] Seaho Jeon,[a] Jaehyeung Park,[a] Olof Ramström,*[a][b] Mingdi Yan*[a][b]
Abstract: We report a fast Staudinger reaction between perfluoroaryl
azides (PFAAs) and aryl phosphines, occurring readily under ambient
conditions. A rate constant as high as 18 M-1 s-1 was obtained
between methyl 4-azido-2,3,5,6-tetrafluorobenzoate and methyl 2-
(diphenylphosphanyl)benzoate in CD3CN/D2O. In addition, the
iminophosphorane product was stable toward hydrolysis and aza-
phosphonium ylide reactions. The PFAA-Staudinger reaction proved
to be an excellent bioothorgonal reaction. PFAA-derivatized mannose
and galactose were successfully transformed into cell surface glycans
and efficiently labeled with phosphine-derivatized fluorescent bovine
serum albumin.
The Staudinger reaction, the formation of an iminophosphorane
Figure 1. A) Staudinger ligation. B) PFAA-Staudinger reaction leading to stable
iminophosphorane.
from an azide and a phosphine,1 proceeds through nucleophilic
attack of the phosphine on the distal nitrogen of the azide to form
the phosphazide intermediate, which decomposes to the
iminophosphorane upon release of N2.2,3 When exposed to water,
Mechanistic studies on the Staudinger reaction between phenyl
the iminophosphorane is readily hydrolyzed to the corresponding
azides and triphenylphosphines revealed that the reaction rate
amine and phosphine oxide. The reaction has recently gained
can be enhanced by introducing electron-donating groups on the
renewed interest, owing to the search for highly efficient
phosphine or electron-withdrawing groups on the phenyl azide.2,6
bioorthogonal reactions.4
Since electron-rich phosphines are more susceptible to oxidation
The pioneering work applying the Staudinger reaction as a
in the biological environment,8 the alternative of using electron-
bioorthogonal reaction involves the introduction of an electrophilic
deficient azides would be a more logical solution. It was reported
ester trap on the phenyl phosphine (Fig. 1A).5 In the resulting
that iminophosphoranes formed from electron-deficient azides
Staudinger ligation reaction, the nucleophilic iminophosphorane
were more stable, and had lower hydrolysis rates.6,9-11 The P=N
attacks the ester intramolecularly to form a cyclic intermediate,
bond can be stabilized by steric hindrance.12,13 Encouraged by
which, upon hydrolysis, gives the conjugated amide product.6 The
these findings, we envisaged efficient Staudinger reactions using
reaction has been successfully applied to metabolic engineering,
perfluoroaryl azides (PFAAs). The F atoms lower the LUMO of the
where azide-derivatized N-acetylmannosamine was converted to
aryl azides, giving PFAAs unique reactivities towards, for example,
sialic acid derivatives, allowing the expression of azido groups on
enamines, thioacids and aldehydes.14-19 Here, we report that
the cell surface. Subsequent Staudinger ligation with biotin-
PFAAs undergo fast Staudinger reactions with aryl phosphines
derivatized phosphine followed by fluorescent avidin allowed the
under ambient conditions to give stable iminophosphoranes (Fig.
detection of cell surface glycans by flow cytometry. Although high
1B). In addition, the reaction showed excellent bioothorgonality
background noise was often observed,7 the Staudinger ligation is
and was successfully applied for cell surface labeling.
considered superior for in vivo labelling compared to the strain-
When an equimolar amount of PFAA 1a and phosphine 2a
promoted alkyne-azide cycloaddition (SPAAC) reaction, where,
(Fig. 2A) were mixed in acetonitrile (100 mM) at room
for example, the alkynes could bind strongly to murine serum
temperature, the solution immediately turned yellow. The color
albumin and were sequestered from azide-labelled tissues.8
remained for 4 min and was followed by evolution of nitrogen gas.
The product, iminophosphorane 3aa, was obtained in quantitative
yield (>99%), and the structure was confirmed by single crystal x-
[a]
[b]
Dr. M. Sundhoro, Dr. S. Jeon, Dr. J. Park, Prof. Dr. M. Yan, Prof. Dr.
O. Ramström
Department of Chemistry, University of Massachusetts Lowell, 1
University Ave., Lowell, MA 01854, United States
E-mail: mingdi_yan@uml.edu; olof_ramstrom@uml.edu
Prof. Dr. M. Yan, Prof. Dr. O. Ramström
Department of Chemistry, KTH – Royal Institute of Technology,
Teknikringen 30, S-10044 Stockholm, Sweden
E-mail: mingdi@kth.se; ramstrom@kth.se
ray crystallography (Fig. 2B).
The Staudinger reaction can proceed through first- or
second-order kinetics depending on whether the rate-limiting step
is unimolecular decomposition or bimolecular formation of the
intermediate phosphazide.20 In the present case, the reaction
between 1a and 2a followed second order as can be seen from
the kinetic analysis (Fig. 2C). The observed rate constant, 3.68 ±
0.03 M-1s-1, is six times higher than that between 1-azido-4-
nitrobenzene and triphenylphosphine (0.611 M-1s-1),2 the fastest
Supporting information for this article is given via a link at the end of
the document.
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Staudinger reaction reported, and 1940 times higher than that of
the classic Staudinger ligation reaction (kobs= 1.9 × 10-3 M-1s-1).6
The effect of solvent on the reaction rate was next investigated
(Table 1). In general, the observed rate constant increased with
the polarity of the solvent (Entries 1-4). Consistent with this
observation is the rate acceleration with the addition of D2O
(Entries 5-7), where an observed rate constant of 18.3 M-1s-1 was
obtained with 50% D2O (v/v) (Entry 7). These results are in
agreement with a polar transition state that is more stabilized in
polar solvents.2
(R2 = 0.99) with a small, positive ρ value (0.43). These results
suggest a buildup of negative charge and a small influence of the
PFAA’s para-substituent on the reaction rate. The reaction was
repeated with 1-azido-4-nitrobenzene (1b), the non-fluorinated
analog of compound 1d. This reaction was not completed after 30
min (Entry 2, Table 2), and displayed a 32 times lower rate in
comparison to the reaction with its perfluorinated analog 1d
(Entry 4).
Table 2. Scope of PFAAs.
Entry
Azide
1a
kobs (M-1s-1)a
3.68 ± 0.03
0.153 ± 0.001
3.92 ± 0.07
4.97 ± 0.08
2.73 ± 0.06
0.91 ± 0.01
Yield (%)b
1
2
3
4
5
6
91
41
95
95
93
82
1b
1c
1d
Figure 2. A) Reaction between 1a and 2a. B) X-ray single crystal structure of
3aa. C) Kinetic analysis; conditions: [1a]0 = 2.5 mM, [2a]0 = 2.5 mM, CD3CN,
25 °C; concentrations monitored by 1H NMR with spectra recorded every 60 s;
1e
kobs (2nd order): 3.68 ± 0.03 M-1 s-1
.
1f
[a] 2nd order; reactions were followed with 1H NMR over the course of 30 min.
[b] NMR yield at 30 min.
Table 1. Effect of solvent on reaction rate between PFAA 1a and phosphine
2a.
The scope of the phosphine was next investigated (Table 3).
Introducing an electron-withdrawing methyl ester on the phenyl
group increased the rate slightly (Entry 1 vs. Entry 2).
Substituting the phenyl moieties with pentafluorophenyl groups
(2c) strongly impeded the reaction as no product was observed
after 30 min (Entry 3). This is consistent with the classic
Staudinger reaction in which the rate decreases when using an
electron-poor phosphine.2 When electron-donating groups were
introduced (2d, 2e, 2f), the phosphine was immediately converted
into the phosphazide (Fig. S16A-S18A) upon mixing with PFAA
1a. The decomposition of phosphazide was in this case the rate-
limiting step, following first order kinetics (Fig. S16). The rate of
phosphazide decomposition decreased with the electron donating
ability of the aryl substituent (Entries 4-6). For the reaction with
2d, the isolated yield was lower in comparison to the triphenyl
counterpart (63%) due to the production of oxidized phosphine.
Entry
Solvent
kobs (M-1s-1)a
0.83 ± 0.03
3.68 ± 0.03
2.37 ± 0.03
5.7 ± 0.1
Yield (%)b
1
2
3
4
5
6
7
CDCl3
79
91
91
95
97
96
97
CD3CN
(CD3)2CO
CD3OD
CD3CN/D2O (5/1)
CD3OD/D2O (5/1)
CD3CN/D2O (1/1)
6.5 ± 0.1
11.3 ± 0.3
18.3 ± 0.7
[a] 2nd order; reactions followed with 1H NMR by monitoring the methyl
protons in PFAA 1a over 30-45 min. [b] NMR yield at 30 min.
The iminophosphorane products proved stable over time.
When solutions of 3aa or 3ab in CD3CN were stored under
ambient conditions (Fig. S6-S7), or when 10% D2O was added
(Fig. S8-S9), the 1H NMR spectra did not change during a course
of 35 days. This demonstrates the high stability of the
iminophosphorane towards hydrolysis. Compounds 3aa or 3ab
were then mixed with an excess of CS2 or p-anisaldehyde in
The scope of the PFAA structures was studied, and high
reaction rates were observed for all PFAAs tested (Table 2).
Furthermore, the more electron-withdrawing the substituent on
the PFAA core, the higher the reaction rate is consistent with the
kinetics of the Staudinger reaction.2 In addition, the reactions
proved efficient, and high conversions (> 90%) were achieved
under ambient conditions in all cases. Hammett analysis
performed on the PFAAs (Graph S1) showed a linear correlation
1
CD3CN. After 35 days, no new peaks were detected in the H
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NMR spectra (Fig. S10-S13), demonstrating the inability of these
iminophosphoranes to undergo aza-Wittig reactions in
consequence to the low nucleophilicity of the fluorinated
iminophosphoranes.
as 4a, whereas 4c was much less efficient. Similar low labeling
efficiency of GlcNAc derivatives was also observed by others,
speculated as the restriction of GlcNAc into the sialic acid
pathway, and the ability of GlcNAc analogs to produce multiple
metabolic products.21-23
Table 3. Scope of phosphines
N
R2
R2
F
F
N
N
F
F
P
R1
R2
R2
N
F
F
N3
F
F
R2
k1
k2
F
P
R1
P
- N2
R1 R2
MeO2C
MeO2C
MeO2C
F
F
F
1a
2a-f
3aa-3af
F
F
F
R2
=
2b R1,R2
=
2c R1,R2
=
2a R1
=
F
MeO2C
F
O
2e R1,R2
=
2d R1
=
2f R1,R2
=
OMe
R2
=
NH
Entry
Phosphine
k1,obs (M-1s-1)a
k2,obs × 103 (s-1)a
-
Yield (%)b
1
2
3
4
5
6
2a
2b
2c
2d
2e
2f
3.68 ± 0.03
91
6.1 ± 0.7
4.1 ± 0.1
-
93
Figure 3. Cell surface labelling experiment using PFAA-Staudinger reaction.
-
-
-
-
NRc
96
1.56 ± 0.02
1.31 ± 0.01
0.56 ± 0.01
89
62
[a] Monitored by 1H NMR. [b] NMR yield at 30 min. [c] No reaction during the
course of 30 min.
The PFAA-Staudinger reaction was next tested for its
bioorthogonality (Fig. 3), employing the widely-studied N-
acetylneuraminic acid (Neu5Ac) metabolic pathway that also
accommodates other monosaccharides.5,21 PFAA-derivatized
acetylated Man (4a), Gal (4b), and Glc (4c) (Fig. 3) were
synthesized by
a straightforward protocol (cf. Supporting
Information). The labelling reagents, P2g-BSA-FITC or P2h-BSA-
FITC, were synthesized by treating BSA with fluorescein
isothiocyanate (FITC) followed by phosphines 2g or 2h, and
purified by dialysis. BSA was chosen for its ability to reduce non-
specific protein adsorption. A549 cells were incubated with 4a, 4b,
or 4c for 3 days,5 washed, and treated with P2g-BSA-FITC for 30
min. The extent of cell labelling was analysed using flow
cytometry. Results showed that cells treated with 0.2 mM of 4a
had ~4 times higher fluorescence intensity than the control (0 mM,
Fig. 4A), indicating the successful expression of PFAA on the cell
surface.
The experiment was then repeated using P2h-BSA-FITC
following the same protocol as above, and the labeled cells were
analyzed by flow cytometry. Similar to the results of P2g-BSA-
FITC, cells treated with 0.2 mM of 4a displayed the highest
fluorescence intensity (Fig. 4B). Furthermore, P2h-BSA-FITC
showed higher labeling efficiency, displayed ~11 times higher
intensity than the control (0 mM) compared to P2g-BSA-FITC (~4).
P2h-BSA-FITC was thus chosen as the labeling agent to test
PFAA 4b and 4c. As can be seen from Fig. 4B, 4b was as efficient
Figure 4. Fluorescence intensity of labelled cells measured by flow cytometry.
(A) A549 cells were incubated with 4a at 0, 0.04, 0.09, 0.2 mM for 3 days
followed by P2g-BSA-FITC (1 µM) for 30 min. (B) A549 cells were incubated with
4a at 0, 0.09, 0.2 mM, 4b at 0.2 mM or 4c at 0.2 mM for 3 days followed by P2h
BSA-FITC (1 µM) for 30 min.
-
Fluorescence microscopy was furthermore used to image the
labeled cells. For this, A549 cells were first incubated with 4a at
0.29 mM for 3 days and then treated with P2h-BSA-FITC for 1, 5,
10, 15, or 30 min. The green fluorescence that resulted from P2h-
BSA-FITC was clearly seen on the cells (Fig. 5). Successful
labeling was already visible at 5 min. High fluorescence intensities
were observed at 15 min, and remained unchanged after 30 min.
A549 cells were then incubated with 4a, 4b or 4c for 3 days,
followed by P2h-BSA-FITC for 30 min, and the cell nuclei were
stained with Hoechst 33342 which fluoresces blue. Green
fluorescence was clearly seen on the cells (Fig. 6, I), where the
signals were strong in the case of 4a and 4b (Fig. 6A, 6B), and
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much weaker in the case of 4c (Fig. 6C). This is consistent with
the flow cytometry results (Fig. 4B) showing the lower labelling
efficiency for 4c. Control samples prepared without treating the
cells with 4 showed no visible green fluorescence (Fig. 6D).
the labelling agent (1 µM) tagged with a conventional dye (FITC),
affording clear images with low background noises.
Acknowledgements
We thank the National Institutes of Health (R01GM080295 and
R21AI109896, to MY) for the financial support of this work. We
acknowledge Dr. Andreas Fischer for his contribution in
performing X-ray crystallography, and Dr. Nanjing Hao for his help
in fluorescence microscopy.
Conflict of interest
The authors declare no conflict of interest.
Figure 5. Fluorescence images of labelled cells. Cells were incubated with 0.3
mM 4a for 3 days, followed by P2h-BSA-FITC for 1, 5, 10, 15, or 30 min.
Keywords: Staudinger reaction • perfluoroaryl azide •
bioorthogonal reaction • metabolic labelling
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Figure 6. Fluorescence images of A549 cells treated for 3 days with 0.3 mM of
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In summary, we have developed a fast PFAA-Staudinger
reaction, with rate constants of up to 18 M-1s-1 in CD3CN/D2O (1/1).
The iminophosphorane product was found stable towards
hydrolysis thus eliminating the need of an ester trap in the
Staudinger ligation. The iminophosphorane was also inert
towards aza-Wittig reagents. Furthermore, PFAA-derivatized
ManNAc and GalNAc were taken up by A549 cells and
successfully probed by the PFAA-Staudinger reaction. The
labelling reaction was fast, carried out using low concentration of
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Entry for the Table of Contents (Please choose one layout)
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[a]
Madanodaya Sundhoro, Seaho
[a]
[a]
Jeon, Jaehyeung Park, Olof
Ramström,*[b] and Mingdi Yan*[a][b]
Page No. – Page No.
Fast, Bioorthogonal Staudinger
Reaction
Perfluoroaryl azides (PFAAs) react with aryl phosphines in a fast reaction under
ambient conditions, yielding kinetically stable iminophosphorane products. The
PFAA-Staudinger reaction proved to be an excellent bioorthogonal reaction and
was successfully applied to cell surface labeling.
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