Diselenolane-mediated cellular uptake

Article abstract of DOI:10.1039/c7sc05151d

The emerging power of thiol-mediated uptake with strained disulfides called for a move from sulfur to selenium. We report that according to results with fluorescent model substrates, cellular uptake with 1,2-diselenolanes exceeds uptake with 1,2-dithiolanes and epidithiodiketopiperazines with regard to efficiency as well as intracellular localization. The diselenide analog of lipoic acid performs best. This 1,2-diselenolane delivers fluorophores efficiently to the cytosol of HeLa Kyoto cells, without detectable endosomal capture as with 1,2-dithiolanes or dominant escape into the nucleus as with epidithiodiketopiperazines. Diselenolane-mediated cytosolic delivery is non-toxic (MTT assay), sensitive to temperature but insensitive to inhibitors of endocytosis (chlorpromazine, methyl-β-cyclodextrin, wortmannin, cytochalasin B) and conventional thiol-mediated uptake (Ellman's reagent), and to serum. Selenophilicity, the extreme CSeSeC dihedral angle of 0° and the high but different acidity of primary and secondary selenols might all contribute to uptake. Thiol-exchange affinity chromatography is introduced as operational mimic of thiol-mediated uptake that provides, in combination with rate enhancement of DTT oxidation, direct experimental evidence for existence and nature of the involved selenosulfides.

Full text of DOI:10.1039/c7sc05151d

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Diselenolane-mediated cellular uptake†
Nicolas Chuard,a Amalia I. Poblador-Bahamonde,a Lili Zong,a Eline Bartolami,a
Cite this: DOI: 10.1039/c7sc05151d
b
a
Jana Hildebrandt,b Wolfgang Weigand,
Naomi Sakaia and Stefan Matile
*
The emerging power of thiol-mediated uptake with strained disulfides called for a move from sulfur to
selenium. We report that according to results with fluorescent model substrates, cellular uptake with
1,2-diselenolanes exceeds uptake with 1,2-dithiolanes and epidithiodiketopiperazines with regard to
efficiency as well as intracellular localization. The diselenide analog of lipoic acid performs best. This
1,2-diselenolane delivers fluorophores efficiently to the cytosol of HeLa Kyoto cells, without detectable
endosomal capture as with 1,2-dithiolanes or dominant escape into the nucleus as with
epidithiodiketopiperazines. Diselenolane-mediated cytosolic delivery is non-toxic (MTT assay), sensitive
to temperature but insensitive to inhibitors of endocytosis (chlorpromazine, methyl-b-cyclodextrin,
wortmannin, cytochalasin B) and conventional thiol-mediated uptake (Ellman's reagent), and to serum.
Selenophilicity, the extreme CSeSeC dihedral angle of 0ꢀ and the high but different acidity of primary
and secondary selenols might all contribute to uptake. Thiol-exchange affinity chromatography is
introduced as operational mimic of thiol-mediated uptake that provides, in combination with rate
enhancement of DTT oxidation, direct experimental evidence for existence and nature of the involved
selenosulfides.
Received 4th December 2017
Accepted 2nd January 2018
DOI: 10.1039/c7sc05151d
rsc.li/chemical-science
Unlike simple alkanes or peroxides, the most favorable CXXC
dihedral angle q in acyclic diselenides 1 and disuldes 2 is ꢁ90ꢀ
(X ¼ S, Se, Fig. 1).1 Decreasing q in cyclic disuldes causes the
increase of ring tension in 1,2-dithianes with 62ꢀ over
1,2-dithiolanes 3–4 with 27–35ꢀ to approach the maximum
epidithiodiketopiperazine (ETP) 5 with ꢁ0ꢀ.2 Earlier, we found
the dependence of the ability of cyclic disuldes as delivery
vehicles of otherwise cell-impermeable cargos to the ring
tension.1,2 Mechanistic studies support that the ring tension
promotes dynamic covalent thiol-disulde exchange on the cell
surface1–6 to initiate uptake.1,2 Asparagusic acid (SA) derivative 4
efficiently delivers functional peptides and objects as large as
intact liposomes.6
This emerging power of strain-promoted thiol-mediated
uptake with cyclic disuldes called for cyclic diselenides.
Besides many similarities, disuldes and -selenides have also
important differences. In six-membered rings, a very similar q of
ꢁ60ꢀ (56ꢀ) has been conrmed.7 In the crystal structure of
1,2-diselenolanes like SeA 6, the CSeSeC dihedral angle is 0.2ꢀ,
contributing to an almost ideal, twist-free envelope structure of
the ve-membered ring.8,9 At the same time, the Se–Se bond
Fig.
1 (a) Selected CXXC dihedral angles, XX bond length and
absorption maxima of disulfides and diselenides, and the intrinsic pKa
of one thiol or selenol in their reduced form (X ¼ S, Se; R, R0, R00, see
Fig. 2). (b) B3LYP/6-311++G** energies of MeXXMe conformers. (c)
Minimized structure of the parent 1,2-dioxo- (O ¼ red), dithio- (S ¼
yellow) and diselenolane (Se ¼ orange) with angles (CXXC, CXX) and
bond length (XX), arrows: endo position.
aDepartment of Organic Chemistry, University of Geneva, Geneva, Switzerland. E-mail:
stefan.matile@unige.ch; Web: http://www.unige.ch/sciences/chiorg/matile/; Tel: +41
22 379 6523
bInstitute of Inorganic and Analytical Chemistry, Friedrich-Schiller University Jena,
Germany
† Electronic supplementary information (ESI) available: Detailed procedures and
results for all reported experiments. See DOI: 10.1039/c7sc05151d
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length increases from ꢁ2.3 A in relaxed diselenides 1 to 2.38 A
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˚
˚
To elaborate on diselenolane-mediated uptake, green-
in 1,2-diselenolane 6, and the absorption maximum shis uorescent uorescein isothiocyanate (FITC) conjugates were
320 nm to 430 nm.8–10 The CSSC dihedrals of 1,2-dithiolanes synthesized. SeL 7 was prepared from dichloride 8 (Fig. 2
maximize at 27ꢀ in SAs such as 4, and absorptions shi and Scheme S1†). The reaction with Na2Se2 afforded the
from 250 nm to maximal 340 nm.1 The crystal structures of 1,2-di-selenolane scaffold as in the previously reported diseleno
1,2-di-selenolanes show layers of selenium with chalcogen lipoic acid 9.19 Activation with N-hydroxysuccinimide (NHS)
bonds11 of down to 3.55 A length between the polarizable sele- afforded the “SeL tag” 10, an activated ester that reacted
˚
nium atoms of different molecules.8,9 In the solid state, the readily with amines such as FITC-amine 11 to afford SeL 7. The
strained di-selenolanes easily polymerize into a gum.9 In green-uorescent diselenolane SeA
6 was prepared from
solution, relaxed diselenides exchange up to 7 orders of diseleno asparagusic acid7,8 following the same route
magnitude faster than disuldes.12,13 The high acidity of sele- (Scheme S2†), and the acyclic diselenide 1 was obtained by
nols (pKa ꢁ 5.2) compared to thiols (pKa ꢁ 8.2) contributes to reaction of methylated diseleno cystine (SeC)20 with FITC
this increased reactivity by activating both nucleophile and (Scheme S3†). The control compounds SA 4 and ETP 5 were
leaving group for selenol–diselenide exchange in neutral and also prepared following previously reported procedures.1,2
slightly acidic water.12–17 However, although exchange is possible
In computational studies, the minima at 90ꢀ and maxima at
with both thiols and selenols,12 the formation of Se–Se bonds is 0ꢀ in the conformational proles of MeSeSeMe and MeSSMe
favored over mixed S–Se bonds.13 This selenophilicity has been were conrmed, whereas MeOOMe naturally minimized in anti
used elegantly to catalyze protein folding15 and protein denatur- conformation (Fig. 1b). The decreasing energy of the maxima
ation with di-selenides in the presence of immobilized thiols.16
from O to S and Se at 0ꢀ supported that ring tension in cyclic
Some of the characteristics of 1,2-diselenolanes are remi- diselenides is lower than in disuldes. For unsubstituted
niscent of ETPs 5 (i.e., pKa < 7, CXXC dihedral ꢁ0ꢀ, Fig. 1) and 1,2-dioxo-, dithio- and diselenolanes, this trend resulted in
could thus promise similarly outstanding uptake efficiencies envelope conformers with the chalcogen atom, the a carbon,
with dual activation at highest tension.2 Worthwhile to note in and the b carbon in endo position and CXX angles decreasing
this context is also the presence of eight out of the 25 human from 102ꢀ to 95ꢀ and 91ꢀ, respectively (arrows, Fig. 1).
selenoproteins in cellular membranes, which could potentially
With tris(2-carboxyethyl)phosphine (TCEP), 1,2-diselenolane
participate in selenol–diselenide exchange reactions during the 9 was readily reduced to diselenol SeLR 120 (Fig. 2). However,
uptake of diselenides.17 Moreover, cellular uptake, particularly neither SeA nor SeL appeared to react with various thiols 13
gene transfection, with tension-free poly(diselenide)s and pol- (Fig. 3). These results implied that either diselenides do not
y(ditelluride)s has been reported to occur and differ sometimes undergo exchange reactions with thiolates or, unlike S–S
favorably from uptake with poly(disulde)s.18 In the following, homologs 14, the selenosulde intermediate products 15–17
we report uptake of 1,2-diselenolanes SeL 7 and SeA 6 to the ring close easily by intramolecular selenol–selenosulde
cytosol without endosomal capture as with the corresponding exchange. Their existence and nature was thus explored by
1,2-dithiolanes SL 3 and SA 4 or dominant escape into the thiol-exchange affinity chromatography (Fig. 3). Compared to
nucleus as with ETP 5.
a non-reactive carboxyuorescein (CF) standard (Fig. 3a), all
disuldes and diselenides showed delayed elution as expected
Fig. 2 Complete structure of all transporters used (neutral form), with representative synthesis and reduction for SeL 7. (a) (1) Several steps,
ref. 17, (2) NHS, DCC, THF, rt, 18 h, 53%; (b) several steps, ref. 1; (c) DMF, rt, 3 h, 14%; (d) TCEP, <5 min; (e) air.
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the secondary selenol in S–Se isomer 17 with a higher pKa is
a weaker nucleophile. The reduced reactivity of this S–Se isomer
17 with a secondary selenol could explain the detectability of the
second, DTT-dependent peak in the affinity chromatogram of
SeL 7 but not for SA 6 with primary selenols only (Fig. 3e, *). The
isomerization from more reactive 16 to less reactive 17 could
also account for the split of the rst-eluting peak into two,
a behavior that was unique for SeL 7 (Fig. 3e). An intense, very
broad, DTT-dependent second peak was also observed with the
acyclic diseleno cystine SeC 1 (Fig. 1 and 3f, *). The appearance
of this peak was as expected because of the absence of a nearby
selenol for rapid release by intramolecular exchange.
In the permanent presence of 50 mM DTT in the mobile
phase, the fast eluting double peak of SeL 7 at Rt ¼ 36.1 min was
followed by smaller but signicant additional peak (Fig. 3e, **).
A similar peak occurred also with ETP 5 (Fig. 3b, **). A possible
explanation of this observation was that DTT reduces SeL 7 to
the diselenol 12 (Fig. 2), which then offers the activated primary
selenolate to exchange with “hidden” disuldes 18 on the solid
phase to give the more stable selenosulde 17, which undergoes
ring closure or exchanges with DTT slowly enough to appear as
separate peak. The same arguments were applicable to ETP 5.
This plausible explanation implied the existence of less reactive
disuldes 18 on the solid phase that resist DTT but not the more
reactive SeLR 12 and reduced ETP 5. Although speculative, this
conclusion caught our full attention because it would be
consistent with unique properties of SeL 7 and ETP 5 with
regard to cellular uptake (see below).
Fig. 3 Thiol-exchange affinity chromatograms of (a) CF, (b) ETP 5, (c)
SA 4, (d) SeA 6, (e) SeL 7 and (f) SeC 1 in 10 mM Tris, 0.1 M NaCl, 1 mM
EDTA, pH 7.5 with a 0–50 mM DTT gradient at t ¼ 60–70 min (solid)
and constant 50 mM DTT from t ¼ 0 (dashed). * ¼ peaks indicative for
inert binding of 1, 4 and 7 to thiols on the stationary phase by strain-
releasing thiol-disulfide/diselenide exchange in the absence of DTT. **
¼ peaks indicative for labile binding of reduced forms (e.g. SeLR 12) to
disulfides on the stationary phase by thiol/selenol–disulfide exchange
in the presence of DTT. Structures are shown in neutral form, acidic
protons are indicated in parenthesis.
Further support of the possible thiol-diselenide exchange
was obtained by measuring the oxidation kinetics of DTT 19 to
DTTO 20 catalyzed by diselenides.15 In the presence of one
equivalent seleno lipoic acid 9, the halife time of DTT 19
decreased from t50 z 16 h (Fig. 4, B) to t50 z 6.9 h (Fig. 4, -).
This rate enhancement conrmed the occurrence of
thiol/diselenide exchange, the existence of intermediate 21 with
mixed S–Se bonds and fast oxidation of SeLR 120 by molecular
oxygen. The catalysis of DTT oxidation by 1,2-diselenolane 9
also demonstrated that the reloading of ring tension in 9 is less
for temporal covalent bonding with the solid phase through
thiol exchange (Fig. 3b–f, solid). Although the retention time of
these peaks was shortened a little (ꢁ1 min) in the presence of
DTT in the mobile phase (Fig. 3b–f, dashed), these retentions
supported but did not prove transient dynamic covalent
binding to the solid phase.
With SA 4, a second broad peak eluted only upon addition of
DTT to the mobile phase (Fig. 3c, *). This observation conrmed
the formation and stability of the ring-opened, strain-released
exchange product 14 by covalent attachment to the stationary
phase. The absence of this second peak with SeA 6 indicated
that ring-opened S–Se product 15, if formed on the exchange
column, ring closes rapidly back to SeA 6 (Fig. 3d). This result
was consistent with expectations from the reduced ring tension
in 1,2-diselenolanes compared to 1,2-dithiolanes, selenophi-
licity and the low pKa of the selenol (Fig. 1). The importance of
this low pKa was conrmed by the absence of this second
DTT-dependent peak also for ETP 5 with thiols of comparable
acidity (Fig. 1 and 3b).
With SeL 7, a second broad peak could be observed upon
addition of DTT to the mobile phase (Fig. 3e, *). This nding
provided direct experimental evidence for the occurrence of
thiol-diselenide exchange with SeL 7, i.e., existence of the
ring-opened S–Se product 16. This S–Se product 16 and not the Fig. 4 Oxidation of 500 mM 19 to 20 as a function of time in the
absence (B) and the presence of 500 mM 9 (-) and 22 (C) in D2O, pD
constitutional isomer 17 was presumably formed rst because
¼ 8.0, with linear fits to determine the t50. Structures are shown in
a primary selenolate with a lower pKa is a better leaving group
neutral form, acidic protons are indicated in parenthesis. Competing
than the secondary selenolate with a higher pKa. However, S–Se
isomer 16 was expected to exchange intramolecularly because
ring closures in reactive intermediate 21 are indicated with solid
(preferred) and dashed red arrows.
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favored than ring closure of DTT into the much less strained
DTTO 20 (Fig. 4, arrows).
Even more signicant rate enhancements for DTT oxidation
were observed with seleno asparagusic acid (SeA 22, Fig. 2 and
4, C). Compared to 1,2-diselenolane 9, DTT lifetimes dropped
further from t50 z 6.9 h to t50 z 3.9 h in the presence of SeA,
and the nal second-order rate constant increased from k z 1.4 ꢂ
10ꢃ2 Mꢃ1 sꢃ1 to k z 8.9 ꢂ 10ꢃ2 Mꢃ1 sꢃ1. These results provided
the experimental evidence for thiol-diselenide exchange for SeA
that could not be secured unambiguously by thiol-exchange
affinity chromatography (Fig. 3d).
In summary, the passage through thiol-exchange affinity
columns complemented by DTT oxidation catalysis reected
pertinent aspects of cellular uptake well (see below).1,2 The results
demonstrated compatibility of 1,2-diselenolanes with strain-
promoted thiol-mediated uptake. Moreover, evidence became
available for dynamic covalent exchange with thiols on the cell
surface, possible hopping along thiols or disuldes, facile intra-
cellular release, possible participation of less reactive cellular
disuldes and thiols, and clear differences between diselenides
SeL 7, SeA 6 and SeC 1 as well as disuldes SA 4 and ETP 5.
The quantitative detection of cellular uptake was compli-
cated by the uorescence quenching with 1,2-diselenolanes.
Compared to already strongly quenched 1,2-dithiolane SA 4,
1,2-diselenolanes SeL 7 and SeA 6 displayed even weaker
emission of FITC in buffer by factors of 4.6 and 3.3, respectively
(Fig. 5b, solid, blue, red vs. green). Adding the 3.4-fold
quenching of free FITC by 1,2-dithiolane in 4,1 the total
quenching by 1,2-diselenolane calculated to a factor of up to
15.6. Fluorescence partially recovered upon reduction with
TCEP (Fig. 2, 5a and b). Compared to reduced SA 4, correction
factors of 1.9 and 1.5 were obtained for SeLR 12 and reduced
form of SeA 6 (Fig. 5b, dashed, blue, red vs. green). For the
quantication of uptake experiments, correction factors for
closed cycles were applied.
Fig. 6 CLSM images of HeLa Kyoto cells after 1 h of incubation with
50 mM (top) and 100 mM (bottom) SA 4 (a), SeA 6 (b) and SeL 7 (c) in
Leibovitz medium at 37 ꢀC, together with Hoechst 33342 to stain the
nuclei (blue). Scale bar ¼ 10 mm. LP ¼ 0.8% for CF and 6.5% for
Hoechst.
confocal laser scanning microscopy (CLSM). Flow cytometry
analysis with 10 mM of diselenides in Leibovitz medium at
37 ꢀC showed a gradually increasing uptake with time, with
1,2-diselenolanes being much more active than 1,2-dithiolane
SA 4, and with SeL 7 outperforming SeA 6 (Fig. 5c).
These impressions were conrmed in CLSM images, taken
aer one hour of incubation. Dithiolane SA 4 showed the known
punctate emission characteristic for substantial capture in
endosomes, together with signicant homogeneous emission
from the cytosol (Fig. 6a). SeA 6 showed stronger and more
homogeneous emission at 50 mM but was visibly cytotoxic at
100 mM (Fig. 6b). With SeL 7, intact cells were stained brightly
and homogeneously including nuclei, at 50 mM and also at
100 mM (Fig. 6c).
Cytotoxicity was determined with the MTT assay, an assay
that reports on the metabolic activity of the cells. Like SA 4 and
ETP 5, SeL 7 was not cytotoxic up to concentrations as high
as 100 mM (Fig. 7a). As indicated by CLSM images (Fig. 6b),
cytotoxicity started to emerge at 100 mM of SeA 6, although
The uptake of the green-uorescent SA 4, SeA 6 and SeL 7
into HeLa Kyoto cells was monitored by ow cytometry and
Fig. 5 (a) Absorption spectra of the isolated motifs FITC (black) and
SeA (22, blue) before (solid) and after treatment with TCEP (dashed). (b) Fig. 7 (a) Cell viability from MTT assay with 0 ((ꢃ) normalized to 1.0), 10
FITC emission from conjugates SA 4 (green), SeA 6 (blue) and SeL 7 (+) and 100 mM (++) of SeL 7, SeA 6 or polyarginine (pR) in HeLa cells.
(red, 100 mM, PBS) before (solid) and after (dashed) reduction with (b) Flow cytometry data for HeLa Kyoto cells that were incubated with
TCEP. (c) Flow cytometry results (mean ꢄ error of three independent 10 mM SeL 7 at 37 ꢀC (normalized to 1.0) and 4 ꢀC, and cells that were
measurements) for uptake of 10 mM SA 4, SeA 6 and SeL 7 and preincubated with inhibitors of endocytosis (CPZ, mbCD, wort, cytoB)
untreated (blank, b) HeLa Kyoto cells after incubation for 1 h (black), 2 h and thiol-mediated uptake (DTNB, 1.2 mM), followed by incubation
(hashed) and 4 h (grey), corrected for emission in closed form (b, solid). with 7 at 37 ꢀC, and incubation with 10% serum at 37 ꢀC. Shown are
*Not determined.
average values ꢄ error from three independent measurements.
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Fig. 8 CLSM images of HeLa Kyoto cells after incubation with 10 mM
SeL 7 for 1 h (a), 2 h (b) and 4 h (c) (arrows indicate selected nucleoli),
z-stacks ((e) 4 h, distances in z-axis) and 10 mM SeC 1 (4 h (d)); (a),
(b) and (d) together with Hoechst 33342, blue. Scale bar ¼ 10 mm.
LP ¼ 0.8% for CF and 6.5% for Hoechst.
Fig. 9 Comparative summary of intracellular location of SeC 1, SA 4,
ETP 5,2 and SeL 7, and a tentative uptake mechanism for SeL 7.
endocytosis and result in mostly endosomal capture, exactly as
with SA 4 (Fig. 3f vs. 3c and 9).6a It is understood that SeC 1 is not
a perfect acyclic mimic of SeL 7 because it contains an
additional uorophore. However, this difference was expected
to be irrelevant with regard to the uptake mechanism under
investigation, the second uorophore released at the cell
surface should be simply washed away before imaging. The
agreement between mechanistic expectations and observed
results in cells and on thiol-exchange columns supported that
these assumptions are correct and SeC 1 and SeL 7 properly reveal
the intrinsic differences between cellular uptake of acyclic and
strained cyclic diselenides. Given the demonstrated usefulness of
SA 4 to deliver peptides, liposomes and polymersomes also to the
cytosol along this route,6 this implied that delivery mediated by
acyclic SeC 1 could be of substantial practical interest.
The clear difference of SA 4 and SeC 1 compared to ETP 5 and
SeL 7 suggested that for dominant delivery to cytosol and
nucleus with little endosomal capture, the release from covalent
conjugates is essential (Fig. 9). The similar affinity chromato-
gram of the most active ETP 5 and SeL 7, particularly the
unique, DTT-dependent peak (Fig. 3, **), implied the transient
existence of the fully reduced form and its exchange with less
reactive cellular disuldes during uptake (12, Fig. 9). For
1,2-diselenolane-mediated uptake, this added up to a putative
mechanism that begins with exchange of poorly reactive thiols
13 on the cell surface with SeL 7 (insensitivity to DTNB). The
obtained selenosulde 16, covalently attached to the cell
surface, rearranges to isomer 17 that is stable enough for
detection by affinity chromatography (Fig. 3d*). Somewhere
during uptake, thiol exchange with selenosulde isomer 17
releases SeLR 12, which exchanges with less reactive cellular
disuldes 18 before it can back-oxidize to 7 (interpretation of
**peak in affinity chromatogram, Fig. 3). Further exchange with
other cellular thiols 13 and disuldes 18 is possible until SeL 7
or SeLR 12 are released into the cytosol (Fig. 4 and 9). The result
for diselenolane-mediated cytosolic delivery with SeL 7 is a quite
spectacular target hopping mechanism on different oxidation
levels that will be extremely difficult to clarify in detail (Fig. 9).
10 mM CPPs remained clearly more toxic (Fig. 7a). Studies on
SeA 6 were thus discontinued.
Diselenolane-mediated uptake of SeL 7 was less sensitive to
temperature than ETP-mediated uptake,2 for instance (Fig. 7b).
Insensitivity toward inhibitors supported that SeL does not
enter cells by clathrin-mediated endocytosis (chlorpromazine;
CPZ), caveolae-mediated endocytosis (methyl-b-cyclodextrin;
mbCD) and macropinocytosis (wortmannin; wort, cytochalasin
B; cytoB).5 The presence of 10% serum slightly decelerated
uptake without reducing nal efficiency signicantly. As for ETP
5 but neither for SA 4 nor SL 3, uptake of SeL 7 was insensitive to
DTNB, an inhibitor of conventional thiol-mediated uptake. In
agreement with results from thiol-exchange affinity columns
(Fig. 3), this might suggest that SeL 7 and ETP 5 can exchange
also with less reactive thiols on the cell surface that are beyond
reach of Ellman's reagent.
Aer longer incubation times, SeL 7 was efficiently delivered
also at 10 mM (Fig. 8a–c). Z-stacks for SeL 7 conrmed that the
homogeneous emission originates from cytosol (Fig. 8e).
Corroborative support for delivery to cytosol and nucleus could
be seen by the poor emission from nucleoli (Fig. 8d, arrows).
Entry into the nucleus but not the nucleoli contrasted nicely to
CPDs and other polycationic carriers that can accumulate in
nucleoli, presumably due to interactions with polyanionic
DNA,5 and to ETP 5, which targets the nucleus including
nucleoli.2
The acyclic diseleno cystine SeC 1 showed weaker activity
and mostly produced the punctate emission characteristic for
endosomal capture (Fig. 8d and 9). This behavior was similar to
SA 4 (Fig. 9) and in excellent agreement with mechanistic
expectations from thiol exchange affinity columns (Fig. 3). More
specically, the selenosuldes obtained by thiol-diselenide
exchange of SeC 1 on the cell surface are much more stable
than with SeL 7 because the proximal covalent selenol for ring
closure and release is missing (Fig. 3f vs. Fig. 3e). Uptake of this
covalently attached selenosulde could then occur by
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The same conclusions apply to the similarly promising cytosolic
delivery with ETP 5.
Acknowledgements
Several possible additions and alternatives to this putative
uptake mechanism exist. For instance, the free selenol next to
the selenosulde in conjugates 16 or 17 could exchange with
another exofacial disulde to produce a second selenosulde
and afford a more stable conjugate, whereas further dimeriza-
tion of oligomerization are unlikely due to high dilution and
fast ring closure (Fig. 9). Glutathione at the cell surface could be
involved and possibly initiate uptake with partially or fully
reduced SeLR 12, exchanging rst with exofacial disuldes 18,
instead of oxidized SeL 7, exchanging rst with exofacial thiols
13 (Fig. 9).3p,3q Intracellular retention could contribute to uptake
efficiencies and should inuence intracellular location21
(uptake of underivatized CF was negligible under experimental
conditions1,21). These and other uncertainties concerning
a complex mode of action should not distract from the key facts
disclosed in this report: (a) Cytosolic delivery by SeL 7 is
outstanding (Fig. 9).22 (b) Clear differences to SeA 6 (cytotoxic,
Fig. 6b and 7a; insensitive to affinity columns, Fig. 3d and e)
indicate that a secondary selenol is essential (they very well
reect the distinct differences between SL and SA with regard to
cellular uptake1 and polymerization on surfaces23 and in solu-
tion24). (c) Clear differences to SeC 1 (endosomal capture, Fig. 9;
permanent retention on affinity columns, Fig. 3f and e) indicate
that a strained cyclic diselenide is essential. (d) All trends,
covering diselenides and disuldes from molecular modeling to
affinity chromatography and intracellular localization, are
consistent with conceptual expectations.
In summary, the move from sulfur to selenium affords the
best carriers identied in this series so far. Cellular uptake
mediated by 1,2-diselenolane 7, i.e., diseleno lipoic acid SeL,
exceeds the performance of the best 1,2-dithiolane, i.e. SA 4 as
well as the best disulde at maximal ring tension, i.e. ETP 5,
with regard to both efficiency and intracellular localization. No
trace of endosomal capture as with 1,2-dithiolane 4, no trace of
escape into the nucleus as with ETP 5: diselenolane 7 delivers
mostly to the cytosol (Fig. 9). The uorescence images are
particularly impressive with the strong quenching exerted by
1,2-diselenolanes taken into consideration (Fig. 5). The
homologous 1,2-dithiolane SL 3 is clearly less performing not
only than SeL 7 but also SA 4.1
We thank Xavier Martin-Benlloch for contributions to synthesis,
the Roux group for assistance with cell culture, the NMR, the MS
2.0 and the Bioimaging Platform for services, and the University
of Geneva, the Swiss National Centre of Competence in
Research (NCCR) Chemical Biology, the NCCR Molecular
Systems Engineering and the Swiss NSF for nancial support.
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Conflicts of interest
There are no conicts of interest to declare.
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