The Bioorthogonal Isonitrile-Chlorooxime Ligation

Article abstract of DOI:10.1021/jacs.9b07632

Bioorthogonal reactions are valuable tools for the selective labeling and imaging of natural products and proteins. Here, we present the reaction between isonitriles and chlorooximes as a ligation that proceeds quickly (k ≈ 1 M^-1 s^-1

Full text of DOI:10.1021/jacs.9b07632

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The Bioorthogonal Isonitrile–Chlorooxime Ligation
Rebecca Schaefer, Mattia Riccardo Monaco, Mao Li,
Alina Tirla, Pablo Rivera-Fuentes, and Helma Wennemers
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.9b07632 • Publication Date (Web): 11 Nov 2019
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The Bioorthogonal Isonitrile–Chlorooxime Ligation
Rebecca J. B. Schäfer,‡ Mattia R. Monaco,‡ Mao Li, Alina Tirla, Pablo Rivera-Fuentes, and
Helma Wennemers*
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Laboratory of Organic Chemistry, ETH Zurich, D-CHAB, Vladimir-Prelog-Weg 3, Zurich 8093, Switzerland
HO
HO
ABSTRACT: Bioorthogonal reactions are valuable tools
for the selective labeling and imaging of natural products
and proteins. Here, we present the reaction between
isonitriles and chlorooximes as a ligation that proceeds
quickly (k ~ 1 M–1s–1) and with high chemoselectivity in an
aqueous environment. Imaging of metabolically labeled
cell surface glycans underlined the tolerance of the ligation
to common functional groups in cellular systems. Live-cell
dual labeling experiments revealed that the isonitrile–
chlorooxime ligation is orthogonal to the strain-promoted
azide–alkyne cycloaddition (SPAAC).
Bio-
Bio-
H2O
HCl
N
N
H
C
molecule
+
molecule
N
N
R
R
Cl
O
= e.g. fluorophore or affinity tag
R
1.17 Å
unobtrusive size
unique reactivity
R
N
C
R
N
C
zwitterion
carbene
Figure 1. Isonitrile-chlorooxime ligation.
Furthermore, isonitrile moieties are stable in the pH
range of 4–9.11 They are not known in higher eukaryotes
but occur in several natural products extracted, e.g., from
marine sponges, suggesting their compatibility with living
systems and orthogonality to mammalian systems.11 Prior
studies had shown that they can be applied for
bioorthogonal chemistry with tetrazines, but only tertiary
isonitriles provided stable products.12
In recent years, biocompatible chemical transformations
have had a profound impact on bioimaging and our
understanding of cellular processes.1-3 Biomolecules that
are endowed with non-natural chemical moieties
(“reporters”) can be selectively reacted with
a
complementary functional group bound, for example, to a
detectable probe. Such bioorthogonal reactions have
allowed for the localization, visualization, and/or isolation
of cellular components such as glycans,3,4 lipids,5 nucleic
acids,6 and proteins.7 A reaction amenable to such a
ligation must not only be fast and chemoselective at low
concentrations, but also tolerate a plethora of electrophiles
and nucleophiles that are present in biomolecules.8
Moreover, the reaction partners and the ligation product
must be stable under physiological conditions. The product
should also be small and chemically inert to avoid
interference with the activity of the target biomolecule.
Ideally, a new ligation reaction should also be orthogonal
to established ligation methods to allow for dual labeling.9
We envisioned chlorooximes as chemoselective
reaction partners. They are known to react in organic
solvents with isonitriles to form -hydroximino amides, a
functionality of small molecular size.13 In organic solvents,
this reaction requires a base to favor the elimination of
hydrochloric acid, and thus the unleashing of a reactive
nitrile oxide species.13,14 We hypothesized that this process
could also occur under physiological conditions, initiated
by water-assisted generation of nitrile oxide (I) from the
chlorooxime (eq 1).15 This intermediate would react with
the isonitrile and form a reactive nitrilium ion (II), which
would be readily intercepted by water to yield the desired
product (eq 1).
Herein, we introduce the coupling between isonitriles
and chlorooximes as a bioorthogonal reaction (Figure 1).
This ligation benefits from the small size of the reaction
partners, a fast reaction rate, as well as the stability and
unobtrusive nature of the hydroxyimino amide ligation
products. We show the value of the isonitrile–chlorooxime
ligation for metabolic glycan labeling of cellular
membranes and its orthogonality to the strain-promoted
azide–alkyne cycloaddition (SPAAC).
We started to evaluate the reactivity of isonitriles with
chlorooximes by reacting lysine-derived isonitrile 1 with N-
hydroxybenzimidoyl chloride 2. No reaction was observed
in THF, but upon addition of water (phosphate buffered
saline (PBS) at pH 7.4) complete conversion to the -
hydroxyimino amide occurred within 2 h at a concentration
of 4.8 mM (Table 1, entries 1 and 2). This finding
underlined the importance of the aqueous media for the
The isonitrile functional group is attractive for
bioorthogonal ligations since it combines the unique
reactivity of a formally divalent carbon atom with a small
size (~1.17 Å bond length),10 and thus exhibits appealing
attributes for a “chemical reporter” (Figure 1).
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reaction. The transformation still proceeded quickly to
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labeling.19 The second-order kinetic law implies that both
the isonitrile and the chlorooxime are involved in the rate-
determining step, indicating that the C–C bond formation is
the rate-limiting event (eq 1). Presumably, the nitrilium ion
II (eq 1) is a highly reactive intermediate and its hydration
occurs fast. The observed high reactivity is in agreement
with the beneficial effect of water as a solvent in reactions
proceeding via polar transition states.20
1
2
3
4
5
6
7
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completion upon lowering the concentration of the
reactants to micromolar concentrations (Table 1, entries 3
and 4).16 At physiological temperature (37 °C), the reaction
proceeded faster (Table 1, entry 5). We next probed the
chemoselectivity by performing the reaction in the presence
of functional groups common in biological systems. These
competition experiments with 1 equivalent of each additive
showed that carboxylic acids, amines, amides, imidazole,
indole, alcohols, and alkenes do not compromise product
formation (Table 1, entries 6–12). Only thiols such as
glutathione (GSH) interfered with the reaction, but
excellent chemoselectivity was restored by performing the
ligation at pH 5 (Table 1, entries 13 and 14).17
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Table 1. Probing the reactivity and chemoselectivity of reactions
between isonitrile 1 and chlorooxime 2.
Figure 2. Reaction progress kinetic analysis for the reaction
between 1 and 2 in THF:PBS (1:2) at pH 7.4. a) Conversion to 3
measured by HPLC at 25°C and 37°C with 0.53 mM of 1 and
1.2 equiv. of 2. Data points (dots) were fitted with a 9th order
polynomial (line). b) Concentration of isonitrile 1 plotted against
the ratio of the reaction rate to the concentration of chlorooxime 2
(rate/[2]). Linear regression of values (grey) at 25 °C (blue, R2 =
0.987) and 37 °C (red, R2 = 0.991). Standard error of k ±0.005.
entry [1]
(mM)
THF:PBS
additivea
time
(h)
conv.b
(%)
1
2
4.8
1:0
2:3
2:3
1:2
1:2
2:3
2:3
2:3
2:3
2:3
2:3
2:3
2:3
–
–
2
n.o.c
>95
>95
>95
>95
>95
>95
>95
>95
>95
>95
>95
38
To probe the value of the isonitrile–chlorooxime
reaction in a more complex cellular environment we chose
metabolic labeling of cell-surface glycans with N-
acetylmannosamine derivatives as a testing ground, a
process that has been established by Reutter, Bertozzi, and
others.3,4 Peracetylation enhances the cellular uptake of N-
acetylmannosamines, which are subsequently converted
into tagged neuraminic acid through the action of
intracellular esterases and aldolases, and integrated into
glycoconjugates.21 We therefore prepared peracetylated N-
isocyanopropanoylmannosamine 4 (Ac4ManNC) that bears
4.8
2
3
1.06
0.53
0.53
0.53
0.53
0.53
0.53
0.53
0.53
0.53
0.53
0.53
–
1.5
2
4
–
5d
6
–
1.2
≤ 3
≤ 3
≤ 3
≤ 3
≤ 3
≤ 3
≤ 3
≤ 3
≤ 3
Ac-Gln-OH
H-Trp-OH
H-Ser-OH
Boc-Hyp-OMe
H-Lys-OH
H-His-OH
palmitoleic acid
GSH
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8
9
an isonitrile moiety as
biotinsulfone-functionalized chlorooxime
a
chemical reporter and
as
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11
12
13
14e
5
a
complementary detectable probe (Figure 3a).22,23 We then
added mannosamine 4 at different concentrations to
medium containing live CHO K1 cells. After three days,
the chlorooxime biotinsulfone probe 5 was added (10 M,
5 min) and visualized by avidin-Alexa Fluor 488, which
emits green fluorescence. Confocal microscopy revealed
intense green fluorescence of the cellular membrane
(Figure 3b, right). In contrast, control cells that had not
been incubated with 4 showed only minimal background
fluorescence (Figure 3b, left). Quantification of the
fluorescence intensity by flow cytometry revealed a
concentration dependence of labeled cells over background
with the highest selectivity observed at 300 M of 4
(Figure 3c). Viability assays showed hardly any toxicity of
reporter 4 at 300 M and of probe 5 at 10 M
concentrations (Figures 3d and S13). These results
highlight the value of the isonitrile–chlorooxime ligation
for live-cell imaging. Detection of the isonitrile-labeled
sugar in lysates of cells that had been treated with 4 proved,
however, difficult and will require further optimization.22
2:3
GSH
>90
a
b
1 equiv. of additive. Conversion to 3 determined by HPLC
c
d
analysis of the reaction mixture. not observed. Reaction at
37 °C. e Reaction at pH 5 (citrate buffer, 55 mM).
Next, we determined the rate constant of the reaction by
monitoring the formation of hydroximino amide 3 over
time by HPLC (Figure 2). Reaction progress kinetic
analysis18 revealed a linear relationship between the
concentration of isonitrile 1 and the ratio of the reaction
rate to the concentration of chlorooxime 2 (rate/[2]), and
thus a second order kinetic law (Figure 2b). The second-
order kinetic constant is k = 0.74 M–1s–1 at 25 °C and is
even higher (k = 1.04 M–1s–1) at 37 °C. These reaction rates
are higher or comparable to those of the Staudinger ligation
(k = 10–4 – 10–3 M–1s–1) and the strain-promoted azide–
alkyne cycloaddition (SPAAC, k = 10–2 – 101 M–1s–1)
ligations that are commonly used for bioorthogonal
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O
O
S
a)
illustrate the value of the isonitrile–chlorooxime ligation
for dual labeling.
OH
Cl
X
N
AcO
AcO
1
2
3
4
HN
HN
NH
O
OAc
AcO
AF 488
H
H
O
N
N
CHO cells
4: X = CH2NC
4a: X = N3
OH
N
NC
NH
3
3
O
O
O
O
5
N
N
N
1. DIBO-AF 647
2. Chlorooxime 5
N3
4a and 4
5
6
7
8
O
3 days
3. Avidin-AF 488
OH
CHOcell
OH
1. CHO K1 cells,
3 d
-
AF 647
CO2
OH
HO
N
H
H
4
O
O
N
N
2. 5 (10 M)
5 min
biotin
sulfone
O
O
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b)
Figure 4. Dual labeling of cells by the isonitrile–chlorooxime
(green) and SPAAC ligation (red). Bottom: Confocal microscopy
images of CHO K1 cells grown in medium containing
4 (300 M)/4a (50 M) for 3 days, labeled with first DIBO-
AlexaFluor647 (red, left) and then probe 5/avidin AlexaFluro488
(green, middle) and overlay (yellow, right). All cell images are
overlaid with the blue channel (Hoechst nuclei stain).25
20 m
20 m
c)
d)
0
400
0
100
0
300
0
200
0
50
In conclusion, the coupling between isonitriles and
chlorooximes is an effective bioorthogonal ligation. The
transformation is fast (k ~ 1 M–1s–1), chemoselective, and
yields a moiety that is small and stable under physiological
conditions. The study puts forth the value of the
transformation for the imaging of metabolically labeled
glycans on cell membranes. The work also highlighted the
orthogonality to other ligations and its utility for dual
labeling experiments. Together, these features of the
isonitrile–chlorooxime ligation open exciting opportunities
for the use of isonitriles in chemical biology.
0
100
0
0
0
0
)
(
0
0
0
0
0
0
5
0
0
0
0
0
0
0
1
0
0
)
–
0
0
1
2
5
0
0
0
0
0
+
(
(
1
2
3
1
2
3
4
5
5
5
,
,
)
)
–
–
(
conc. 4 [M]
conc. 4 [M]
4
4
Figure 3. a) Chemical reporters 4 and 4a and probe 5; Cell-
surface labeling of CHO K1 cells with isonitrile and
4
chlorooxime 5. b) Confocal microscopy images of CHO K1 cells.
Right: grown in medium containing 4 (300 uM) for 3 days,
labeled with 5 (10 M) for 5 min and stained with avidin
AlexaFluro488 (right); left: control cells grown in the absence of
4; all cell images are overlaid with the blue channel (Hoechst
33342 nuclei stain). c) Flow cytometry results at various
concentrations of 4 after labeling with probe 5. Error bars
represent standard deviation of three repeats, 10000 events per
repeat. d) Viability of CHO K1 cells (MTT assays) of various
concentrations of 4.
ASSOCIATED CONTENT
Supporting Information
Details on the syntheses and analyses of the presented
compounds and the cellular experiments. This material is
available free of charge via the Internet at http://pubs.acs.org.
Finally, we evaluated the compatibility of the isonitrile–
chlorooxime reaction with other chemoselective
ligations.4,9 We reasoned that the unique reactivity of
isonitriles should allow for compatibility with ligations
between azides and alkynes and tested the complementarity
of our method with the SPAAC.24 We therefore prepared
AUTHOR INFORMATION
Corresponding Author
* Helma.Wennemers@org.chem.ethz.ch
ORCID: 0000-0002-3075-5741
Author Contribution
the
azide-functionalized
N-acetylmannosamine
4a
‡These authors contributed equally.
(Ac4ManN3) and incubated CHO K1 cells with both the
isonitrile 4 and azide 4a. After three days, we added
strained cyclooctyne (DIBO) bearing the red fluorophore
AlexaFluor 647, as well as the biotinsulfone probe 5 and
avidin bearing the green fluorophore AlexaFluor 488 to test
for the incorporation of 4a and 4 into the cellular
membrane. Confocal microscopy revealed labeling of the
cellular membrane with both the green and the red
fluorophore (Figure 4). In the absence of the mannosamine
derivatives only minimal background labeling was
observed for both ligation reactions.22 These results
Notes
The authors declare no competing financial interests.
ACKNOWLEDGMENT
This project has received funding from the European Union’s
Horizon 2020 research and innovation programme under the
Marie Sklodowska-Curie grant agreement No 746087. We are
grateful to Dr. Matthew Aronoff for intellectual contributions,
thank Leyla Hernandez for assistance in cell culture and
Raphael Hofmann as well as Jakob Farnung from the Bode
group (ETH Zurich) for help with the Western Blot. We thank
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Grammel, M.; Hang, H. C. Chemical reporters for biological
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the Scientific Center for Optical and Electron Microscopy
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Meneghetti, F.; Novellino, E.; Tron, G. C. Multicomponent Reaction
of Z-Chlorooximes, Isocyanides, and Hydroxylamines as
Hypernucleophilic Traps. A One Pot Route to Aminodioximes and
Their Transformation into 5- Amino-1,2,4-oxadiazoles by Mitsunobu-
Beckmann Rearrangement. J. Org. Chem. 2015, 80, 9652. (b) Soeta,
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2016, 14, 694. For a review, see: Giustiniano, M.; Novellino, E.;
Tron, G. C. Nitrile N-Oxides and Nitrile Imines as New Fuels for the
Discovery of Novel Isocyanide-Based Multicomponent reactions.
Synthesis 2016, 48, 2721.
(7) For examples, see: (a) Griffin, B. A.; Adams, S. R.; Tsien, R.
Y. Specific covalent labeling of recombinant protein molecules inside
live cells. Science 1998, 281, 269. (b) Blackman, M. L.; Royzen, M.;
Fox, J. M. Tetrazine Ligation: Fast Bioconjugation Based on Inverse-
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Different Reactivity. J. Am. Chem. Soc. 2011, 133, 949; (c)
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Burley, G. A.; Ravoo, B. J. Orthogonal, metal-free surface
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alkene/alkyne cycloadditions. Chem. Sci. 2012, 3, 2479 (d)
Gutsmiedl, K.; Wirges C. T.; Ehmke, V.; Carell, T. Copper-Free
“Click” Modification of DNA via Nitrile Oxide-Norbornene 1,3-
Dipolar Cycloaddition. Org. Lett. 2009, 11, 2405; (e) Bode, J. W.;
Hachisu, Y.; Matsuura, T.; Suzuki, K. Amine-promoted
cyclocondensation of highly substituted aromatic nitrile oxides with
diketones. Tetrahedron Lett. 2003, 44, 3555.
1
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(21) Warren, L.; Felsenfeld, H. The Biosynthesis of Sialic Acids. J.
Biol. Chem., 1962, 237, 1421.
(22) For details, see the supporting information.
(23) The binding affinity between biotin sulfone and avidin is
partially reduced with respect to biotin (Kd ≈ 10–14 M): (a) Zenpleni.
J.; McCormick, D. B.; Mock, D. M. Identification of biotin sulfone,
bisnorbiotin methyl ketone, and tetranorbiotion-I-sulfoxide in human
urine. Am. J. Clin. Nutr. 1997, 65, 508; (b) Collot, M.; Sendid, B.;
Fievez, A.; Savaux, C.; Standaert-Vitse, A.; Tabouret, M. drucbert, A.
S.; Danzé, P. M.; Poulain, D.; Mallet, J.-M. Biotin Sulfone as a New
Tool for Synthetic Oligosaccharide Immobilization: Application to
Multiple Analysis Profiling and Surface Plasmonic Analysis of Anti-
Candida albicans Antibody Reactivity against  and  (1→2)
Oligomannoside. J. Med. Chem. 2008, 51, 6201.
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(15) (a) Kesornpun, C; Aree, T.; Mahidol, C.; Ruchiriwat, S.;
Kittakoop, P. Water-assisted Nitrile Oxide Cycloadditions: Synthesis
of Isoxazoles and Stereoselective Syntheses of Isoxazolines and 1,2,4-
Oxadiazoles. Angew. Chem. Int. Ed. 2016, 55, 3997. (b) Dignam, K.
J.; Hegarty, A. F. , Quain, P. L. Reactivity of 1,3-Dipoles in Aqueous
Solution. Part 1: Stereospecific Formation of Z-Amidooximes in the
Reaction of Benzonitrile Oxides with Amines. J. Chem. Soc., Perkin
Trans. 2, 1977, 11, 1457.
(16) Note, concentrations below 0.53 mM did not allow for reliable
product quantification by HPLC with UV detection.
(24) Ning, X.; Guo, J.; Wolfert, M. A.; Boons, G.-J. Visualizing
Metabolically Labeled Glycoconjugates of Living Cells by Copper-
Free and Fast Huisgen Cycloadditions. Angew. Chem. Int. Ed. 2008,
47, 2253.
(17) At acidic pH the concentration of thiolates is significantly
lower compared to pH 7.4. This result therefore implies that thiolates
are the interfering species.
(25) Note, the higher background fluorescence of the SPAAC
ligation is most likely due to the higher cellular uptake of DIBO-AF
647 compared to Avidine-AF 488, see ref. 4d for similar observations.
(18) Blackmond, D. G. Reaction Progress Kinetic Analysis: A
Powerful Methodology for Mechanistic Studies of Complex Catalytic
Reactions. Angew. Chem.Int. Ed. 2005, 44, 4302.
(19) For a review, see: Lang, K; Chin, J. W. Bioorthogonal
Reactions for Labeling Proteins. ACS Chem. Biol. 2014, 9, 16.
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Suggested Table of Content Graphic:
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HO
HO
H
Bio-
molecule
Bio-
molecule
H2O
N
N
C
+
N
N
R
O
pH 7.4
Cl
R
7
8
fast kinetics
9
small & stable ligation product
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42
43
44
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good chemoselectivity
compatible with living cells
20 µm
6
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