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ported contributions from thiol-mediated dynamic covalent
oligochalcogenide exchange[9–14] to the uptake of BPS5 1
(Supporting Information, Figure S8). According to the MTT
assay, none of the tested COCs were cytotoxic under
experimental conditions (Leibowitz, 24 h, concentrations up
to 50 mm; Supporting Information, Figure S9).
Compared to other COCs, benzopolysulfanes offer differ-
ent reactivity, culminating in ring contraction and expansion
from trisulfides to nonasulfides, i.e., 1–3, 16–21,[3] reminiscent
of elemental sulfur Sn, with pentasulfides 1–3 being clearly
preferred, followed by trisulfides 16 (Figure 2a,e and Sup-
porting Information, Figure S38).[1–3] The mechanism of these
reversible interconversions remains under debate, with tran-
sient ring opening by traces of nucleophilic impurities the
most likely explanation.[1–3,6,21] BPS chemistry further
includes sulfur replacement, nucleophilic displacement and
oxidation, radicals, metal coordination, and photochemis-
try,[1–3] presumably much influenced, if not determined by the
strings of electrophilic s holes next to nucleophilic lone pairs
on the lined-up sulfur atoms.[22] The low pKa values of
thiophenols and persulfides facilitate ring opening to give
interconvertible reactive intermediates, like RI-1 and RI-2,
with preserved reactivity even in slightly acidic water. With
ETP2 5 and diselenolanes, such less basic thiolates and
selenolates were thought to account for mobility, i.e., their
hypothetical mode of action as molecular walkers, walking
along transmembrane disulfide tracks in membrane pro-
teins.[13] In a neutral, deuterated phosphate buffer, the
1H NMR spectrum of BPS5 3 remained unchanged at least
for two weeks (Figure 2b; BPSn have nearly identical
spectra). In the HPLC, equilibration with BPS3 16 was
detectable within hours (Figure 2e and Supporting Informa-
tion, Figure S22 and S38). In the presence of thiols (dithio-
threitol, DTT, and glutathione 22, GSH), BPS5 3 transformed
Scheme 1. a) 6 steps, Scheme S1;[3] b) S2Cl2, 08C to rt, 24 h, 20%;[3]
c) FL–NH2, DMF, rt, 12 h, 14%; d) 6 steps, Scheme S5;[11] e) 1. NH3,
MeOH, rt, 30 min, 2. S2Cl2, CH2Cl2, 08C to rt, 2 h, 58%; f) 3 steps;
g) 10 steps, Scheme S6. PNP=p-nitrophenyl.
Biotinylated and TEGylated BPS5 2 and 3 were prepared
correspondingly. ETP4 4 was prepared from ethylamine 12,
which was converted into heterocycle 13 as described.[11]
Methanolysis of thioesters followed by treatment with
S2Cl2,[18] removal of the tert-butyl ester in 14, and reaction
with FL–NH2 yielded ETP4 4. A close congener of ETP2 5,[11]
ETP2 6 was newly prepared from amine 15, also to explore the
advantages of a phenyl group during synthesis.
Cellular uptake of COCs into HeLa Kyoto cells was
monitored by both flow cytometry and confocal laser scan-
ning microscopy (CLSM). The flow cytometry data (Fig-
ure 1a) were evaluated considering the different degree of
fluorescence quenching by intact or reduced COCs (Support-
ing Information, Figures S1, S2, S6, and Table S1).[12] Inde-
pendent of any corrections applied, the uptake of BPS5 1
exceeded all other COCs. With correction, BPS5 1 was
approximately 10-times more active than the previous best
in the sulfur series, ETP2 5,[11] and approximately 140-times
more active than the best explored asparagusic acid (AspA)
derivative 7.[10] Tetrasulfide ETP4 4 showed less uptake than
disulfide ETP2 5. This result demonstrated that simple
oligomer effects in ring-expanded COCs fail to explain the
power of BPS5 1. The approximately 2.4-times higher activity
of new phenoxyethyl ETP2 6 compared to ethyl ETP2 5
suggested that the known contributions of aromatic rings to
cellular uptake[19] might also apply to COC-mediated uptake.
However, the inactivity of control 8 confirmed that contribu-
tions from such secondary ion–p interactions at the mem-
brane–water interface[19] to the activity of BPS5 1 are almost
negligible.
CLSM images confirmed that BPS5 1 is more active than
all other COCs (Figure 1b–g). Concentration and time
dependence analysis revealed binding to the plasma mem-
brane with efficient delivery to the cytosol and particularly
nucleus within one hour (Supporting Information, Figures S4
and S5). Many interpretations are possible for reduced
activity at 48C, including hindered endocytosis, decelerated
oligochalcogenide exchange kinetics, or membrane stiffening
(Supporting Information, Figure S6). Insensitivity toward
several inhibitors indicated the absence of uptake by cla-
thrin-mediated endocytosis (chlorpromazine), caveolae-
mediated endocytosis (methyl-b-cyclodextrin), and macro-
pinocytosis (wortmannin, cytochalasin B; Supporting Infor-
mation, Figure S7).[9–15,20] A drop in activity to 70% upon
preincubation with 2 mm Ellmanꢀs reagent (DTNB) sup-
1
rapidly into multicomponent mixtures, with H NMR signa-
tures changing with the substrate, time, and pH (Figure 2c–d
and Supporting Information, Figures S10–S17).
According to HPLC analysis combined with low- and
high-resolution mass spectrometry (MS), the reaction of
BPS5 1 with GSH affords a dynamic-covalent network that
includes unprecedented cyclic oligomers 23, from dimers 232
to heptamers 237, with up to nineteen sulfur atoms in
a macrocycle of thirty-three atoms, besides the expected di-
and mono-GSH-BPS2 conjugates 24 and 25, and reduced
BPS2 26 (Figure 2 and Supporting Information, Figures S24
and S39–S46). The identification of these large cyclic BPS
oligomers was interesting because oligomer effects have been
shown to account for thiol-mediated uptake with ordinary
disulfides.[14,23] In contrast, AspA 7 remained intact even with
large excess of GSH (Supporting Information, Figure S36).
This very important difference in reactivity was consistent
with the weaker uptake activity of AspA, thus supporting that
dynamic-covalent networks matter for the mode of action of
BPS. In agreement with the dynamic nature, the composition
of the product library from BPS5 1 and GSH was altered by
the subsequent addition of disulfides (GSSG, Figure 2g and
Supporting Information, Figure S27; lipoic acid, Supporting
Information, Figures S29 and S48). Although less efficiently,
BPS networks also formed with only disulfides (GSSG,
2
ꢀ 2019 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2019, 58, 1 – 6
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