Bioorthogonal Click and Release Reaction of Iminosydnones with Cycloalkynes

Article abstract of DOI:10.1002/anie.201708790

We report the discovery of a new bioorthogonal click-and-release reaction involving iminosydnones and strained alkynes. This transformation leads to two products resulting from the ligation and fragmentation of iminosydnones under physiological conditions. Optimized iminosydnones were successfully used to design innovative cleavable linkers for protein modification, thus opening up new areas in the fields of drug release and target-fishing applications. This click-and-release technology offers the possibility of exchanging tags on proteins for functionalized cyclooctynes under mild and bioorthogonal conditions.

Full text of DOI:10.1002/anie.201708790

A Journal of the Gesellschaft Deutscher Chemiker
Angewandte
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Accepted Article
Title: Discovery of New Click and Release Reactions by Screening of
Mesoionics and Cycloalkynes Combinations
Authors: Frédéric Taran, Sabrina Bernard, Davide Audisio, Riomet
margaux, Sarah Bregant, Antoine Sallustrau, lucie plougastel,
Elodie Decuypere, Sandra Gabillet, Ramar Arun Kumar, Jijy
Elyian, Minh Nguyet Trinh, Oleksandr Koniev, Alain Wagner,
and Sergii Kolodych
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using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201708790
Angew. Chem. 10.1002/ange.201708790
Link to VoR: http://dx.doi.org/10.1002/anie.201708790
http://dx.doi.org/10.1002/ange.201708790

10.1002/anie.201708790
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COMMUNICATION
Discovery of New Click and Release Reactions by Screening of
Mesoionics and Cycloalkynes Combinations
Sabrina Bernard,[a]∫ Davide Audisio,[a]∫ Margaux Riomet,[a] Sarah Bregant, [b] Antoine Sallustrau,[a] Lucie
Plougastel,[a] Elodie Decuypere,[a] Sandra Gabillet,[a] Ramar Arun Kumar,[a] Jijy Elyian,[a] Minh Nguyet
Trinh,[c] Oleksandr Koniev,[d] Alain Wagner,[c] Sergii Kolodych[d] and Frédéric Taran[a] *
Abstract: We report the discovery of a new bioorthogonal click and
release reaction involving imino-sydnones and strained alkynes. This
transformation leads to two products resulting from both ligation and
fragmentation of imino-sydnones under physiological conditions.
Optimized imino-sydnones were successfully used to design
innovative cleavable linkers for protein modifications opening new
areas in the fields of drug release and target fishing applications. This
click and release technology offers for the first time the possibility to
exchange tags on proteins with functionalized cyclooctynes under
mild and bioorthogonal conditions.
trigger (Scheme 1a).[10] More recently, M. S. Robillard et al.
exploited the powerful tetrazine/trans-cyclooctene click reaction in
a similar approach[11] (Scheme 1b) and proved its usefulness for
in vivo drug release.[12] These nice examples of controlled and
specific drug release opened a new way in designing prodrugs
but could also have a strong impact for antibody-drug-conjugate
development[13-16] in particular and innovative targeted
therapeutics in general.[17]
The ability to selectively form and break chemical bonds in
chemically complex and uncontrollable biological media is a long-
standing goal for chemists interested in modifying biological
materials. Bioorthogonal chemical reactions address precisely
this challenge and therefore are of major importance in the field
of chemical biology. Despite the high demanding specifications,
several efficient bioorthogonal ligation reactions have emerged
during the last 15 years and are now routinely used in the field of
chemical biology.[1-3] More recently, cleavage methods have also
received much attention leading to the development of cleavable
linkers based on acid, nucleophile, oxidant, reductant, metal-
induced or photo-induced cleavages.[4] Although these cleavable
linkers found nice applications,[5-9] they often lacked the level of
bioorthogonality generally observed for ligation reactions
therefore limiting their use in living systems. Significant advances
in this area were accomplished by using a bioorthogonal click
reaction to generate an unstable adduct decomposing in
fragments through bond breaking. This strategy was first
described by J-C. Florent et al. using the Staudinger ligation as a
Scheme 1. Conceptual blueprint for the development of click and release
strategies. a) Traditional approach based on the Staudinger reaction. b) Recent
approach based on the benchmark tetrazine-transcyclooctene reaction. c) Our
approach takes advantage of the peculiar reactivity of mesoionics towards
cycloaddition reactions.
[a]
[b]
S. Bernard, Dr. D. Audisio, M. Riomet, Dr. A. Sallustrau, L.
Plougastel, E. Decuypere, S. Gabillet, Dr. R. A. Kumar, Dr. E. Jijy,
Dr. F. Taran
Service de Chimie Bio-organique et Marquage DRF-JOLIOT-SCBM,
CEA, Université Paris-Saclay, F-91191, Gif-sur-Yvette, France
E-mail: frederic.taran@cea.fr
We recently identified sydnones as versatile dipole for the Cu-
catalyzed cycloaddition with terminal alkynes.[18] These
compounds react smoothly with cyclooctynes via initial
suprafacial [3+2] cycloaddition followed by spontaneous
cycloreversion through retro-Diels Alder (rDA) to afford a
cyclooctane-fused pyrazole with concomitant extrusion of
CO2.[19,20] Here, we explore the reactivity of mesoionics towards
cycloalkynes to devise new bioorthogonal reactions allowing
ligation and release of complex molecules (Scheme 1c). We
synthesized a library of 25 mesoionics (1-25, Figure 1c) and
combined them with 4 cycloalkynes (A-D, Figure 1d) of increased
strain. The corresponding 100 reactions were carried out
overnight at room temperature in a H2O/DMSO mixture and
analyzed by LC/MS (Figure 1e).
Dr. S. Bregant
Service d’Ingénierie Moléculaire des Protéines DRF-JOLIOT-
SIMOPRO, CEA, Université Paris-Saclay, F-91191, Gif-sur-Yvette,
France
Dr. A. Wagner, Dr. M. N. Trinh
Laboratory of Functional Chemo-Systems UMR 7199 CNRS-UdS,
67401, Illkirch, France
Dr. O. Koniev, Dr. S. Kolodych
Syndivia SAS, 650 Boulevard Gonthier d’Andernach 67400 Illkirch,
France
[c]
[d]
∫ These authors contributed equally to this work
Supporting information for this article is given via a link at the end of
the document.
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10.1002/anie.201708790
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COMMUNICATION
a)
b)
Unidentified
product(s)
Incomplete
reaction
Complete
reaction
No reaction
c)
1
2
3
4
5
6
7
8
9
10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
d)
e)
A
B
C
D
Figure 1. Screening of mesoionic/cycloalkynes combinations for click and release reactions. 100 experiments were carried out at 25 °C in water containing 50%
DMSO for 16 h, [mesoionic] = 8 mM, [cycloalkyne] = 24 mM. a) Reaction under investigation. b) Color code. c) Structures of the mesoionics used in this study. d)
Structures of the cycloalkynes used in this study. e) Screening results.
As expected, sydnones 1-3 reacted smoothly with
cycloalkynes B-D to afford the corresponding pyrazole products
quantitatively; cycloalkyne A did not display sufficient strain to
allow efficient reaction. Interestingly, imino-sydnones 4-6 display
exquisite reactivity towards cycloalkynes. The reaction of imino-
sydnone 4 and cyclooctyne B was further studied by NMR
indicating very clean reaction (Figure S1). The expected pyrazole
product was isolated in good yield together with benzyl urea
coming from the fragmentation of the exocyclic part of 4 (Scheme
2a). This finding suggested the formation of an acyl-isocyanate
resulting from the rDA step, evolving spontaneously to the
corresponding urea through hydrolysis and decarboxylation.
Dithiolium compounds 15, 18 and 19 also underwent clean
reactions with cyclooctyne B but no reaction occurred with the
more strained alkynes C and D probably for steric reasons. This
reactivity profile was confirmed in a series of reactions conducted
in flask with 15 (Scheme 2b and Figure S2). This new click
reaction liberating COS as release product is currently under
Scheme 2. Confirmation of hits. Reactions were carried out in flask at 50 mM
with 1.5 equiv. of cycloalkynes. NMR yields, (isolated yields).
investigation in our group. Similar reactivity was found with
dithiolium 18 (Scheme 2c) but reaction was found to be very slow
and the starting mesoionic compound moderately stable in pure
water therefore limiting its usefulness for bioorthogonal
applications. Although the biological properties of imino-sydnones
are well documented,[21-24] their reactivity with alkynes has been
To our delight, we found that electron withdrawing
substituents on N-aryl imino-sydnones and urea moieties
connected to the exocyclic nitrogen atom of the imino-sydnone
overlooked: only one paper in 1970 reported their reaction with
core had a strong beneficial impact on reaction kinetics (Table 1).
the activated alkyne dimethylacetylene dicarboxylate leading to
The combination of the two effects, as illustrated by compound
pyrazoles in organic solvents and at elevated temperatures.[25]
Im7, allows straightforward click and release reaction with a rate
constant of 0.18 M-1.sec-1 when mixed with alkyne B. This value
We thus focused our attention on the reaction of imino-sydnones
with cyclic alkynes and investigated the influence of the structure
of the mesoionic on the efficiency and speed of the reaction. A
series of imino-sydnones were synthesized and tested as new
dipoles for the reaction with bicyclo-[6.1.0]-nonyne B used as
model cyclooctyne reagent.
is comparable to the well-known strained promoted azide-alkyne
cycloaddition (SPAAC) click reaction.[26,27] The reaction rate can
be significantly increased to 3.15 and 29.2 M-1.sec-1 by the use of
the more reactive alkynes C and D.
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Table 1. Comparative kinetic studies of iminosydnones Im1-7.[a]
incubated in PBS with 1 mM solution of cyclooctyne B. The
resulting solution was analyzed by SDS-PAGE, confirming the
complete deconjugation of the fluorophore (compare lanes 1 and
2 in Figure 2b). The specificity of the reaction was tested by
incubating T1 with 1 mM of B in blood plasma. Complete release
of TAMRA was achieved in this complex biological mixture after
16 h of incubation (Figure 2b, lane 4). In a control experiment,
without addition of B, the conjugate remained stable in blood
plasma for more than 24 h (Figure 2b, lane 3). Finally, we
performed a series of release reactions in PBS, gradually
decreasing the concentration of B. Efficient release of TAMRA
was observed even at as low concentration of B as 63 µM,
corresponding to less than 2 eq. of B per imino-sydnone linker
(Figures S4 and S5).
Im
R
R’
Alkyne
K (M-1.sec-1) [b]
no reaction
H
B
B
B
B
B
B
B
C
C
D
Im1
Im2
Im3
Im4
Im5
Im6
Im7
Im7
Im7
Im7
C6H5
pTol-CO-
pTol-SO2-
tBuOCO-
pTol-NHCO-
pTol-NHCO-
pTol-NHCO-
pTol-NHCO-
pTol-NHCO-
pTol-NHCO-
0.006 ± 0.001
0.032 ± 0.005
0.019 ± 0.005
0.046 ± 0.002
0.008 ± 0.001
0.179 ± 0.002
1.47 ± 0.11
C6H5
C6H5
C6H5
C6H5
We next investigated the usefulness of imino-sydnones as
versatile cleavable linkers for protein fishing applications. To
overcome the harsh conditions required for the recovery of
biotinylated proteins from immobilized Streptavidin, several
approaches using cleavable linkers have already been described
in literature.[9,28-31] However, in addition to a low level of
bioorthogonality, none of these strategies offers the possibility of
attaching a label onto the protein while releasing it from the
support. The ability of imino-sydnones to click and cleave upon
mild and selective reaction with cyclooctynes offers the unique
opportunity of bioorthogonal trans-tagging release.
pHOC6H4
pEtOOCC6H4
pEtOOCC6H4
pEtOOCC6H4
pEtOOCC6H4
3.15 ± 0.11[c]
29.2 ± 2.9
[a] reactions were conducted with 1 equiv. of imino-sydnones and 1.5 equiv. of
alkyne in DMSO containing 20% PBS 0.1 M (pH 7.4) [b] determined under initial
rate conditions. [c] conducted in PBS 0.1 M containing 20% DMSO.
For this purpose, we designed Im11 bearing a biotin
separated from the imino-sydnone moiety by a Peg spacer and
an azide function for CuAAC ligation with alkynylated proteins
(Figure 2a). Very interestingly, we found that the CuAAC is
completely orthogonal towards the imino-sydnone under the
tested conditions. CuAAC conditions applied to alkynylated
Myoglobin (MyoAlk) used as model protein in presence of Im11
provided biotinylated Myoglobin (MyoBiot) that was easily
captured by Streptavidin beads (Figure 2c). Following capture
and supernatant removal, MyoBiot was smoothly released from
Streptavidin beads through addition of the commercially available
fluorescent cyclooctyne 26 (500 µM, 25 °C, 1 h). The results
indicated quantitative capture and conversion of MyoBiot into
fluorescent labeled Myoglobin (MyoTAMRA, compare lanes Ref
and 2 in Figure 2c). We next envisaged the capture and release
of Myoglobin from a complex proteome. The same trans-tagging
procedure was thus applied to MyoBiot mixed in liver extract.
Selective and efficient release and trans-tagging of supported
Myoglobin was observed allowing quantitative recovery and
labelling of the protein (Figure 2c, lane 4 and Figures S6-10). This
result confirmed the high selectivity and bioorthogonality of the
imino-sydnone motif and highlighted its stability in complex
biological media such as liver extracts.
These results encouraged us to appraise the suitability of this
new click and release reaction for bioorthogonal protein
modification. To this purpose, we synthesized in gram scale
imino-sydnone Im9 as a cleavable bifunctional building block
(Figure 2a). NMR and LC/MS analyses confirmed that the
reaction of Im9 and cyclooctynes B or C leads to the formation of
the expected pyrazole bicyclic products with similar rate constants
as Im7 (Table S1). Stability of imino-sydnones in plasma were
also studied and showed half-life greater than 40 h at 37 °C. We
then designed and synthesized compound Im10 containing : i) a
maleimide function, suitable for conjugation with free cysteine
residues, ii) the imino-sydnone linker, susceptible to
bioorthogonal cleavage by cyclooctynes and iii)
a
carboxytetramethylrhodamine (TAMRA) as a fluorescent tag. A
humanized monoclonal antibody, trastuzumab T, was used as a
model protein to test our approach. Having the molecular weight
of over 145 000 Da, trastuzumab is composed of two heavy and
two light chains bound together by
4 disulfide bridges.
Trastuzumab was incubated with the phosphine TCEP at 37 °C
allowing for the partial reduction of interchain disulfide bonds,
followed by incubation with Im10 at 4 °C resulting in conjugate T1
with an average of 5.4 TAMRA derivatives per antibody (Figure
2b). In order to induce the release of TAMRA, conjugate T1 was
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Ref MM
1
2
MM
3
4
Figure 2. Applications of imino-sydnones as cleavable linkers. a) Synthesis of iminosydnones Im10 and Im11. b) Preparation of the trastuzumab-TAMRA conjugate
T1 and bioorthogonal cleavage induced by cyclooctyne B. Non-reducing SDS-PAGE analysis of the conjugate T1. Lane 1: conjugate T1 in PBS. Lane 2: conjugate
T1 after incubation with 1mM of B in PBS at 25 °C for 16 h. Lane 3: conjugate T1 after incubation in blood plasma at 25 °C for 24 h. Lane 4: conjugate T1 after
incubation with 1 mM of B in blood plasma at 25 °C for 16 h. c) Capture and release of Myoglobin. SDS-PAGE analysis of trans-tagging experiments on beads
using 26. MyoBiot in PBS was captured by Streptavidin beads and treated by PBS (lane 1) or 500 µM of 26 (lane 2). MyoBiot in liver extract was captured by
Streptavidin beads and treated by PBS (lane 3) or 500 µM of 26 (lane 4). Top panel: fluorescence scan; lower panel: coomassie blue staining. Ref = MyoTAMRA
reference prepared in vitro. MM = Molecular Markers.
In conclusion, we described a bioorthogonal click and release
reaction involving imino-sydnones and cyclooctynes as triggers.
The reaction allows complete release of a fluorophore from an
antibody and the full concomitant recovery and labelling of an
immobilized protein under mild, physiological conditions. Imino-
sydnones are tunable, stable, easy to prepare and orthogonal to
the CuAAC reaction. This unprecedented click and release
technology allows for the first time the trans-tagging of proteins
under physiological conditions with commercially available
functionalized cycloalkynes. We believe this transformation will
trigger new perspectives in research fields where trans-tagging
plays a major role for chemical modifications of biological
materials, such as target fishing and controlled drug release.
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Acknowledgements
This work was supported by the French Research National
Agency (ANR-14-CE06-0004). The authors thank David-
Alexandre Buisson, Céline Puente and Elodie Marcon for
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Keywords: Mesoionics • Click chemistry • Cleavage • Chemical
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COMMUNICATION
Sabrina Bernard, Davide Audisio,
Margaux Riomet,Sarah Bregant, Antoine
Sallustrau, Lucie Plougastel, Elodie
Decuypere, Sandra Gabillet Ramar Arun
Kumar, Jijy Elyian, Minh Nguyet Trinh,
Oleksandr Koniev, Alain Wagner, Sergii
Kolodych and Frédéric Taran
Click and Cleave: Imino-sydnones react smoothly with cycloalkynes under
physiological, metal-free, reaction conditions to form a click pyrazole product together
with a release urea product. Possible applications of this new reaction for drug
release and protein trans-tagging have been explored in proof-of-principle
experiments.
Page No. – Page No.
Discovery of New Click and Release
Reactions by Screening of
Mesoionics and Cycloalkynes
Combinations
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