4-Halogeno-sydnones for fast strain promoted cycloaddition with bicyclo-[6.1.0]-nonyne

Article abstract of DOI:10.1039/c4cc03816a

New sydnone derivatives have been synthesized and screened for their capacity to undergo fast copper-free cycloaddition reaction with bicyclo-[6.1.0]-nonyne. The influences of substitution in positions N-3 and C-4 of sydnones have been particularly studied leading to the identification of highly reactive partners for bio-orthogonal ligation reactions.

Full text of DOI:10.1039/c4cc03816a

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4-Halogeno-sydnones for fast strain promoted
cycloaddition with bicyclo-[6.1.0]-nonyne†
Cite this: Chem. Commun., 2014,
50, 9376
Lucie Plougastel,a Oleksandr Koniev,b Simon Specklin,a Elodie Decuypere,a
Christophe Creminon,c David-Alexandre Buisson,a Alain Wagner,b Sergii Kolodych*b
´
Received 19th May 2014,
Accepted 13th June 2014
and Frederic Taran*a
´ ´
DOI: 10.1039/c4cc03816a
www.rsc.org/chemcomm
New sydnone derivatives have been synthesized and screened for
their capacity to undergo fast copper-free cycloaddition reaction
with bicyclo-[6.1.0]-nonyne. The influences of substitution in positions
N-3 and C-4 of sydnones have been particularly studied leading to the
identification of highly reactive partners for bio-orthogonal ligation
reactions.
Scheme 1 Strain promoted 1,3-dipolar cycloaddition of benzyl azide versus
phenyl sydnone with BCN. Reactions conditions: [dipoles] = 100 mM, [BCN] =
150 mM, 0.1 M (pH 7.4) PBS containing 10% DMSO. Kinetic constants were
calculated from initial velocity values (yields o 10%).
Among the described bio-orthogonal reactions, the strain promoted
azide–alkyne cycloaddition (SPAAC) reaction remains one of the
most popular click tools for in vitro as well as in vivo ligation
applications.1 To improve the kinetics of SPAAC, efforts have been
focused more on the development of new strained cycloalkynes2 sydnone–alkyne cycloaddition (SPSAC) by exploring the influence
than on the discovery of new dipoles. Nitrones,3 tetrazines4 and of the structure of sydnones on the kinetics of the reaction.
nitrile oxides5 are today the main reaction partners for cycloalkynes
Preliminary experiments indicated that, under our reaction
which can successfully replace azide compounds. Recently, our conditions (1.5 equiv. of BCN, pH 7.4 in PBS buffer), phenyl-
group has identified sydnones as new interesting dipole partners sydnone 4e is more than 4 times less reactive than regular
for the Cu-catalyzed cycloaddition reaction with terminal alkynes.6 azides such as benzyl azide 1a (Scheme 1).
Following this work, we investigated the copper-free version of this
We therefore synthesized a series of sydnones bearing
reaction. During the progress of our work, J. W. Chin et al. various substituents in positions 3 and 4 according to known
published the strain promoted cycloaddition reaction of phenyl- procedures.8 The SPSAC reactions of sydnones with BCN were
sydnones with bicyclo-[6.1.0]-nonyne (BCN, 2a).7 The determined carried out at room temperature in PBS containing 10% DMSO
rate constant was 0.054 MÀ1 sÀ1 which is in the same range as (w.w) and kinetics were observed for 2 hours by HPLC using an
SPAAC therefore stimulating the pursuit of our investigations.
internal standard (Table S1, ESI†). Rate constants were determined
An interesting structural feature of sydnones is the possibility under initial rate conditions.
of varying the reactivity of the dipole by adding appropriate
The results indicated in Table 1 clearly showed a strong
substitutions in positions 3 and 4 of the mesoionic ring. Here influence of the substituent nature both in positions 3 and 4 on
we described our attempts to improve the strain promoted the kinetics of the cycloaddition. Structural motifs increasing
the partial positive charge at the N-3 atom of the sydnone ring
were found to be beneficial to the reaction: whereas N-alkyl
sydnones were found to be inactive under our reaction condi-
tions (Table 1, entries 1 and 2), N-aryl sydnones bearing strong
a CEA, iBiTecS, Service de Chimie Bioorganique et de Marquage, Gif sur Yvette,
F-91191, France. E-mail: frederic.taran@cea.fr; Fax: +33 169087991;
electron withdrawing groups reacted up to 10 times faster than
N-phenyl sydnone 4e (compared entries 6–8 with entry 5 in
Table 1). A good correlation between SPSAC rate constants and
the Hammet s values was obtained (r = 1.35, Fig. S5, ESI†). The
presence of a halogen in position 4 induced the strongest
positive impact on the kinetics of the reaction. The increase
of the sydnone reactivity toward BCN appeared to be correlated
Tel: +33 169082685
b Laboratory of Functional Chemo-Systems UMR 7199 CNRS-UdS, LabEx MEDALIS,
74 route du Rhin, 67401, Illkirch, France
c CEA, iBiTecS, Service de Pharmacologie et d’Immunoanalyse, Gif sur Yvette,
F-91191, France
† Electronic supplementary information (ESI) available: Synthetic protocols and
analytical data of sydnone synthesis, kinetic studies and protein modification.
See DOI: 10.1039/c4cc03816a
9376 | Chem. Commun., 2014, 50, 9376--9378
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Table 1 Influence of the sydnone structure on the SPSAC reactiona
b
c
Entry
4
R
X
KSyd
KSyd/KN3
1
2
3
4
5
6
7
8
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
4n
4o
4p
4q
CH3
H
H
H
H
H
H
H
H
CH3
C6H5
CF3
CN
Cl
Br
I
No reaction
No reaction
—
—
CH2CO2H
pOMeC6H4
pMeC6H4
C6H5
pCO2HC6H4
pCF3C6H4
pNO2C6H4
C6H5
C6H5
C6H5
C6H5
C6H5
0.006 Æ 0.001
0.032 Æ 0.001
0.027 Æ 0.002
0.059 Æ 0.001
0.199 Æ 0.002
0.289 Æ 0.012
0.018 Æ 0.002
0.027 Æ 0.001
0.008 Æ 0.001
0.003 Æ 0.001
0.872 Æ 0.034
0.592 Æ 0.021
0.306 Æ 0.008
0.798 Æ 0.065
1.593 Æ 0.034
0.05
0.27
0.22
0.49
1.64
2.29
0.14
0.22
0.07
0.02
7.21
4.89
2.53
6.60
13.16
Fig. 1 1H NMR spectroscopic analysis of the competitive reaction
between chlorosydnone 4q and azide 1b with BCN in CD3OD at 25 1C.
9
10
11
12
13
14
15
16
17
attached to BSA (used as model protein) by standard peptide
coupling procedures with an excess of reagents. Fluorescent labeling
of solutions of the corresponding BSA–sydnone conjugates, used at
50 mg mLÀ1, was then carried out with one equivalent per sydnone of
the BCN–TAMRA conjugate 2b (Fig. 2).
C6H5
C6H5
pCO2HC6H4
pCO2HC6H4
Br
Cl
The results obtained by SDS-PAGE and MALDI-TOF analysis
confirmed the superior ability of 4-chlorosydnones to react with
2b allowing an effective and fast labeling of the protein. Kinetic
data indicated that more than 85% of the 4-chlorosydnones on
BSA were transformed into pyrazoles in less than 5 min (60% in
30 min for non-chlorinated sydnones).
In summary, we reported new sydnone reactants which may
represent an interesting new tool for bioorthogonal cycloadditions
with strained alkynes. 4-Chloro-sydnones were found to react with
BCN more than 10 times faster than regular azides and more than
30 times faster than nonsubstituted sydnones. The high reactivity
of chlorosydnones was applied to the fast and efficient fluorescent
labeling of a model protein and found some utility in particular for
the labeling, or the sorting, of low-abundant proteins.
a
Reactions were conducted with 100 mM of sydnones and 150 mM of
BCN in 0.1 M (pH 7.4) PBS containing 10% DMSO. MÀ1 sÀ1
. Rate
b
c
constant ratio calculated from the value obtained with benzyl azide.
with the electro negativity of the halogen (Cl 4 Br4 I, entries 13–15,
Table 1). However, all other electron withdrawing groups tested
in this study were found to be prejudicial to the reaction
(entries 11 and 12, Table 1).
According to our results the simple presence of a Cl atom in
position 4 of the sydnone induced a 30-fold increase of the
SPSAC kinetics (compare entry 5 with 13 in Table 1). We then
tried to combine this halogen effect with appropriate substitutions
in the N-aryl group of sydnones. Unfortunately, chlorosydnones
bearing strong electron withdrawing groups like pNO2-phenyl in
position 3 were found to be difficult to isolate. We however
succeeded in synthesizing sydnone 4q bearing a carboxylic group
useful for further anchoring to proteins. Sydnone 4q is a stable
compound which reacts more than 10 times faster than azides
(entry 17, Table 1).
This work was supported by the French National Research
Agency (ANR, ClickScreen project). We thank Dr Sarah Bregant
To confirm this result we decided to run a competitive
cycloaddition reaction by mixing equimolar amounts of BCN
2a, azide 1b and chlorosydnone 4q in CD3OD, the reaction was
followed by NMR analysis. A complete reaction was observed in
a few minutes leading to the pyrazole cycloadduct 5b almost
quantitatively; only traces of triazole 3b were detected (Fig. 1
and Fig. S4, ESI†).
Sydnones are usually very stable mesoionic compounds,
even in vivo.9 Because the presence of a halogen in position 4
of the sydnone heterocycle might alter its reactivity towards
nucleophiles, we checked the stability of sydnones 4p and 4q. Our
results indicated perfect stability of these halogeno-sydnones and
clean SPSAC reaction with BCN in biological media such as serum
or in a 10 mM glutathione solution (Fig. S1–S3, ESI†). To
demonstrate the advantage of using 4-chlorosydnones for bio-
conjugation, we investigated the application of this reaction for
protein labeling. Sydnones 4f and 4q were first covalently
Fig. 2 Protein labelling by SPSAC. Structure of sydnones and BCN
reactants.
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9378 | Chem. Commun., 2014, 50, 9376--9378
This journal is ©The Royal Society of Chemistry 2014