Angewandte
Chemie
Table 1: Reaction 10C-Cu.[a]
equivalent of nucleophiles (amine, thiol, acid, etc.) or electro-
philes (Michael acceptor, bromoalkane, aldehyde, etc.) and
second in the presence of concentrated solutions of proteins,
DNA, or sugars, in cell media as well as in blood plasma. The
yields of the crudes were then determined by sandwich
immunoassay. The yields of about half of the 42 hits were
significantly increased, but only those of 4 reactions (3F-Ir,
8D-Rh, 10B-Pd, and 10C-Cu) were optimized to more than
50% (Figure 2D).
Three of these reactions were only of limited interest in
terms of chemoselectivity and bioorthogonality, but were
nevertheless further explored on the mmol scale using
nontagged reactants and catalytic amounts of metals
(Scheme 1). We first focused our efforts on reaction 8D-Rh,
which apparently displayed the fastest kinetics in well plates
according to the screening results. The reaction was repro-
duced in a flask and was indeed complete in only 15 minutes
at room temperature using cationic rhodium (20 mol%) to
generate imidazolopyridine compounds through a 1,3-dipolar
cycloaddition of pyridinium salts with aromatic nitriles. Only
traces of product were detected in the absence of rhodium.
However, yields were moderate due to the fast degradation of
the pyridinium substrates under our reaction conditions.
Reactions of azides with bromoalkynes in the presence of the
iridium dimer [Ir(cod)Cl]2 generated a mixture of 1,4 and 1,5
regioisomers of bromotriazoles and dehalogenated 1,4-tria-
zole in various ratios with poor yields, depending of the
electronic enrichment of the alkyne. Exploration of reaction
10B-Pd revealed a new dehydrogenative Heck coupling of
Entry Cu/L
Conditions
Yield [%] 10C/10C’
1
2
3
4
5
–
DMF, Ar, 1538C
DMF, Ar, 608C
15
92
25/75
100/0
100/0
100/0
100/0
CuI/L1
CuSO4/AS[b]/L1 tBuOH/H2O, air, 608C 99
CuSO4/AS[b]/L2 H2O, air, 608C[c]
CuSO4/AS[b]/L2 tBuOH/H2O, air, 258C 67
99
[a] Experiments were carried at a concentration of 0.1m with 1 equiv of
reactants and 1 equiv of TEA for 14 h. [b] 2 equiv of AS. [c] 1 equiv of
N(CH2CH2OH)3 in place of TEA. AS=sodium ascorbate, TEA=trie-
thylamine.
combinations we tested. We first examined this reaction using
phenylsydnone 10a and alkyne C1 as model substrates
(Table 1). In accordance with the optimization data obtained
during the screening procedure, the reaction proceeded
efficiently under CuI–phenanthroline catalysis at room tem-
perature or with gentle heating to produce pure 1,4-pyrazole
10C. The thermal 1,3-dipolar cycloaddition of sydnones with
alkynes, known since Huisgenꢀs work in 1962,[11] has been of
limited success over the years because of harsh conditions and
low regioselectivity, which generates a mixture of pyra-
zoles.[12] Addition of CuI–phenanthroline complexes allows
the exclusive formation of 1,4-pyrazoles under milder con-
ditions (compare entry 1 with entries 2–5 in Table 1).
Convinced by its synthetic impact, we investigated the
substrate scope of this new copper-catalyzed reaction
(Scheme 2). The reaction was remarkably tolerant to all
tested functional groups that were present either in the
sydnone or the alkyne substrate.
For example, dansylated peptide 10C-20 was synthesized
quantitatively in water using this reaction. Although this Cu-
catalyzed sydnone–alkyne cycloaddition (CuSAC) reaction
has some limitations (reaction was unsuccessful with N-alkyl
sydnones), it has many advantageous features. The reaction
proceeded smoothly in many solvents, including biological
media, giving clean reactions with no trace of by-product. For
example, 10C-13 was synthesized with 88% yield at 378C in
pure human blood plasma.
À
À
sydnones with alkenes, allowing a C H/C H cross-coupling
at only 508C, but with moderate yields and low level of
regioselectivity.
The improvement of these reactions would merit further
investigation, but at this point our interest was entirely
focused on the reaction involving dipole 10, dipolarophile C,
and copper(I) salts. Indeed, according to the screening results
in Figure 2D, this particular combination appeared to be the
most efficient, chemoselective, and bioorthogonal of the 2816
With the reaction scope established, we next sought to
demonstrate the usefulness of the CuSAC reaction for
bioconjugation applications. BSA–sydnone conjugate,
obtained through standard peptide coupling using an excess
of sydnone 10b, was reacted with 1.5 equivalents (per
sydnone) of dansylated alkyne C2 under CuSAC conditions.
According to MALDI-TOF analysis, 75% of the sydnones on
BSA were transformed into pyrazoles, and sodium dodecyl
sulfate/polyacrylamide gel electrophoresis (SDS-PAGE)
analysis confirmed the effective dansylation of the protein
(Figure 3).
The CuSAC reaction might be considered a new example
of a highly effective and selective transformation that results
from the reactivity of in situ generated copper(I) acetylides. A
possible mechanism may involve coordination of N2 of the
Scheme 1. Interesting reactions identified by high-throughput screen-
ing (yields of isolated products). cod=cycloocta-l,5-diene, DIEA=dii-
sopropylethylamine, DMF=N,N-dimethylformamide, Tf=trifluorome-
thanesulfonyl.
Angew. Chem. Int. Ed. 2013, 52, 1 – 6
ꢀ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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