Communication
lenide-mediated uptake nevertheless exceeds disulfide-mediat-
ed uptake because the higher polarizability increases exchange
rates
and
selectivity,
2,13]
resulting
in
pronounced
[
selenophilicity.
Uptake with acyclic diselenides 6 (and dithiolanes 3 and 4)
suffers from endosomal capture probably because fast ex-
change with cell surface thiols results in stable selenosul-
[
11]
fides. With DiSeL 7 (and ETPs 5), after reacting with cellular
thiols, the proximal selenolates (and thiolates) remain depro-
tonated and thus reactive, assuring that the transporters can
move on and hop along disulfides and thiols to avoid endoso-
[
11]
mal capture and enter into cytosol and nucleus. DiSeL-medi-
ated uptake has been shown to be insensitive to common en-
[11]
docytosis inhibitors and non-toxic up to at least 100 mm.
These results on COC-mediated cellular uptake have been ob-
tained with fluorescein-labeled dichalcogenides 1–7 (Fig-
ure 1A). Preliminary results on biologically more relevant sub-
strates are available only for AspA 4, which is weakly active
[
14]
and suffers from endosomal capture. Based on the unique
[
11]
characteristics found for diselenolanes, the objective of this
study was to explore the compatibility of DiSeL-mediated
uptake with larger substrates of biological relevance. Using
mostly topics that have been developed recently to character-
[
5,6]
ize the large CPD polymers,
we here report that cellular
uptake of the tiny diselenolanes is compatible with the cyto-
solic delivery of a broad variety of functional substrates with
diameters up to 15 nm. The combination of this broad sub-
strate tolerance with insights from model studies call for a
Figure 2. Cytosolic protein delivery exemplified by Sav 9, with non-covalent
DiSeL tag 8, fluorescent probes 11, F-actin ligands 13 and controls. (A) In-
scale molecular models of Sav and DiSeL tag 8. (B-D) CLSM images of HeLa
Kyoto (B, C) and mouse myoblast C2C12 cells (D) incubated with (B) 12,
(C) 10 and (D) 17 (all 2.5 mm). Scale bar: 10 mm.
[6]
[15]
mode of action that envisions DiSeL as molecular walkers
that move along disulfide tracks in transmembrane proteins,
[
16]
causing temporary local denaturation and the appearance of
then loaded with the DiSeL tag 8 and phalloidin 13 in a 3:1
ratio. Added to mouse myoblast C2Cl2 cells, the resulting com-
plex 17 efficiently tracked actin fibers, thus demonstrating
DiSeL-driven delivery of functional proteins to the cytosol with
high efficiency (Figures 2D and S6).
[
17]
adaptive micellar membrane defects.
[
18]
The biotinylated DiSeL tag 8 was synthesized for general
non-covalent connection to the substrate through the tetrava-
[
5,6,19]
lent streptavidin (Sav) 9, a ꢀ60 kDa protein (Figure 2).
Complexes 10 were prepared by mixing Sav 9 with biotinylat-
ed fluorophores 11 and biotinylated DiSeL tag 8 in a 3:1 ratio.
All complex stoichiometries given in the following refer to
mixing ratios, real existing product mixtures are naturally more
The catalysis of bioorthogonal transformations by artificial
metalloenzymes for metabolic engineering in designer cells is
a topic of current concern that relies on efficient cytosolic de-
[6]
livery. We considered next the uncaging of fluorescent rhoda-
[
5,6,19]
[22]
complex.
The complexes 10 were efficiently taken up into
mine 110 (18) by the “deallocase” 19, i.e., a Sav equipped
HeLa Kyoto cells (Figure 2C), while control complexes 12 with-
out DiSeL tag 8 were inactive (Figure 2B). Analogous com-
plexes prepared with biotinylated AspA and ETP tags gave
clearly weaker uptake efficiency (Figures S1, S2). Cellular
uptake of complex 10 was observed in all tested, overall four
other cell lines (Figures 2 and S3).
with ruthenium complex 20 to catalyze the cleavage of Alloc
groups in caged 21, to further elaborate on scope and limita-
tions of DiSeL-mediated uptake (Figure 3).
Although the hydrophobic and non-fluorescent substrate 21
is known to spontaneously diffuse into cells, its addition after
deallocase 22 did not result in the appearance of fluorescence
in HeLa Kyoto cells from the product 18 or the probe in 22
(Figures 3A and S8). These results suggest that the proteins
failed to penetrate the cells. In clear contrast, the addition of
the doubly DiSeLated but non-fluorescent deallocase 23 gen-
erated increasing emission from HeLa Kyoto cells with increas-
ing concentration, demonstrating the bioorthogonal uncaging
of 21 by the cell-penetrating metalloenzyme 23 (Figures 3B
and S9). The deallocase showed efficient catalytic activity in all
tested cell lines (Figure S10). Confirming the central impor-
tance of delivery for bioorthogonal catalysis within cells, the
metalloenzyme 24 with higher stoichiometry of catalytic unit
Biotinylated phalloidin 13 was selected next as a functional
substrate to be delivered in the cytosol. Phalloidin is a toxic bi-
cyclic heptapeptide from death cap mushrooms that specifical-
ly binds to and prevents the depolymerization of actin
[
20]
fibers. To facilitate the formation of trifunctional Sav conju-
gates with the targeting unit 13, transport unit 8 and the fluo-
rescent reporter, Sav 9 was covalently modified by tetramethyl
rhodamine (TAMRA) fluorophores. The biotin binding sites
[21]
were protected first with the weakly bound 2-iminobiotin 14
to give complex 15, which in turn was reacted with the NHS-
[
18]
activated TAMRA 16. The resulting red-fluorescent Sav was
&
&
Chem. Eur. J. 2019, 25, 1 – 6
2
ꢁ 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ÝÝ These are not the final page numbers!
Bartolami, Eline
