FRET-based imaging of transbilayer movement of pepducin in living cells by novel intracellular bioreductively activatable fluorescent probes

Article abstract of DOI:10.1039/c3ob27445d

To elucidate the mechanisms of direct transmembrane penetration of pepducins, which are artificial lipopeptide G protein-coupled receptor (GPCR) modulators, we developed two types of FRET-based probes, Pep13-FL-SS-Dab (13) targeting the inner leaflet of the lipid bilayer and Pep13-Dab-SS-FL (14) targeting the cytosol, respectively. They are composed of a pepducin moiety and a fluorescent switch component consisting of 5(6)-carboxyfluorescein (FAM) as a fluorophore and dabcyl as a quencher connected through disulfide bond linkage. When they are internalized into the cytosol, intracellular glutathione can cleave the disulfide bond to release the quencher, which results in a turn-on fluorescence signal. Using these probes, we performed live cell imaging of transbilayer movements of pepducins on MCF-7 cells for the first time. The results suggested that the lipid moiety of the probes facilitated pepducin flipping across and tethering to the membrane. The present study raises the possibility of applying the probe architecture for direct intracellular drug delivery. The Royal Society of Chemistry 2013.

Full text of DOI:10.1039/c3ob27445d

Organic &
Biomolecular Chemistry
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PAPER
FRET-based imaging of transbilayer movement of
pepducin in living cells by novel intracellular
Cite this: Org. Biomol. Chem., 2013, 11,
3030
bioreductively activatable fluorescent probes†
Mieko Tsuji, Satoshi Ueda, Tasuku Hirayama, Kensuke Okuda, Yoshiaki Sakaguchi,
Aoi Isono and Hideko Nagasawa*
To elucidate the mechanisms of direct transmembrane penetration of pepducins, which are artificial lipo-
peptide G protein-coupled receptor (GPCR) modulators, we developed two types of FRET-based probes,
Pep13-FL-SS-Dab (13) targeting the inner leaflet of the lipid bilayer and Pep13-Dab-SS-FL (14) targeting
the cytosol, respectively. They are composed of a pepducin moiety and a fluorescent switch component
consisting of 5(6)-carboxyfluorescein (FAM) as a fluorophore and dabcyl as a quencher connected
through disulfide bond linkage. When they are internalized into the cytosol, intracellular glutathione can
cleave the disulfide bond to release the quencher, which results in a turn-on fluorescence signal. Using
these probes, we performed live cell imaging of transbilayer movements of pepducins on MCF-7 cells for
the first time. The results suggested that the lipid moiety of the probes facilitated pepducin flipping
across and tethering to the membrane. The present study raises the possibility of applying the probe
architecture for direct intracellular drug delivery.
Received 18th December 2012,
Accepted 13th March 2013
DOI: 10.1039/c3ob27445d
www.rsc.org/obc
ligand-binding site of receptors without crossing the plasma
membrane barrier, pepducins are supposed to interact with
Introduction
One of the most important issues in drug development is the the intracellular portion of target receptors on the inner leaflet
innovation of versatile systems to deliver drugs across the cell of the lipid bilayer.12 Pepducins consist of a synthetic peptide
membrane. Endocytosis is a major mechanism of cellular derived from the appropriate intracellular loop domains of
entry for the currently used drug delivery systems, e.g., nano- GPCRs, typically 10–20 amino acids in length, and a hydro-
carriers and cell-penetrating peptides (CPPs), for membrane- phobic moiety, e.g., palmitate. The general versatility of the pep-
impermeable molecules.1,2 In the process of penetration, ducin technology in targeting diverse GPCRs is considered to be
endosomal escape pathways for promoting endosomal release due to the ability of their hydrophobic appendant, which
and cytoplasmic delivery are considered a limiting step of anchors in the plasma membrane and sequentially flips across
effective intracellular drug delivery to the cytosol without the lipid bilayer, to transfer the attached peptide onto the intra-
entrapment and degradation in endosomes and lysosomes.3,4 cellular surface of the membrane, where the peptide is able to
On the other hand, we anticipate that a direct penetration interact with the target inner loop domain of GPCRs.12,13
pathway not involving the endocytosis would achieve a more
To explore the remarkable ability of pepducins to trans-
straightforward and versatile drug delivery into the cytosol.5–7 locate through the cell membrane, some studies demonstrated,
We focus on the unique cell-penetrating feature of lipidated by using photochemistry-based approaches, that pepducins
peptides called pepducins discovered as allosteric modulators are localized at the inner leaflet of the plasma membrane and
of GPCRs in the early 2000s.8,9 To date, pepducins and related interact with their target GPCR directly.9,13,14 By using flow
lipopeptides have been developed to specifically modulate the cytometric analysis, Covic et al. were the first to show that the
activity of diverse GPCRs and other membrane proteins.10,11 fluorescein-conjugated pepducin for protease-activated recep-
Unlike classical GPCR modulators that act at the extracellular tor 1 (PAR1), which is a highly expressed GPCR in platelets,
could be delivered to circulating mouse platelets and penetrate
into the cells.9 Wielders et al. also demonstrated that pepdu-
Laboratory of Pharmaceutical and Medicinal Chemistry, Gifu Pharmaceutical
cins can flip across the lipid bilayer. They performed a fluore-
scence resonance energy transfer (FRET)-based assay by flow
University, 1-25-4 Daigaku-nishi, Gifu, 501-1196, Japan.
E-mail: hnagasawa@gifu-pu.ac.jp; Fax: +81-58-230-8112; Tel: +81-58-230-8112
cytometry using two different types of 7-nitro-benzo[c][1,2,5]-
†Electronic supplementary information (ESI) available: Experimental data for
oxadiazol-4-yl phospholipids, which are supposed to be dis-
tributed to the inner or outer leaflet of the plasma membrane,
intermediate chemicals, HPLC data of the probes, and additional confocal
microscopy imaging data. See DOI: 10.1039/c3ob27445d
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respectively, and rhodamine-labeled pepducin.13 Although agonist, P1pal-13, developed by Covic et al.8,9 The probes are
these studies provided useful photoprobes of pepducin composed of pepducin and a FRET component consisting of
respectively to show their localization at the inner surface of 5(6)-FAM as a fluorophore and dabcyl as a quencher, con-
the plasma membrane appropriately, they made indirect obser- nected through a disulfide bond linkage (Fig. 1). The disulfide
vations using their probes by flow cytometry. On the other linker between the FRET donor and acceptor moieties was
hand, to obtain a better understanding of biological pheno- built from L-lysine, 3-mercaptopropionic acid and 2-mercap-
mena through the observation of cellular dynamics, live cell toethylamine linearly to be flexible and sterically unhindered.
imaging has taken on growing importance in life sciences.
The pepducin moiety is derived from the sequence of ten
Then, we focused on the large difference between the extra- C-terminal residues of the intracellular third loop and the sub-
and intra-cellular redox environments to visualize the trans- sequent three residues of the adjacent transmembrane helix 4
verse movement of pepducin through the cell membrane. For of PAR1.8,9 A palmitoyl group is bound to the intermediary
decades, the redox-sensitive disulfide linkers have been used L-lysine residue between pepducin and the FRET component.
for prodrug, caged compound chemistry, and the drug delivery Disulfide linkage has been shown to be applicable for drug
system.15,16 In particular, the disulfide bonds are widely delivery in vivo because of its stability in serum and outside of
applied for the FRET-based sensors in which the linkage of cells.20–22 In the present probes, the disulfide linkage between
the donor and the acceptor is sensitive to redox state in the quencher and fluorophore in the FRET system enables the
various live cell imaging systems.17–19 In this context, we con- rapid release of the dye and the enhancement of a specific
sider that it is possible to distinguish the cytoplasmic surface fluorescence signal through bioreductive cleavage inside the
of the cell membrane from the periplasmic surface by using cell. Because Pep13-FL-SS-Dab (13) has a membrane-anchorable
the disulfide-containing FRET system.
fluorophore while Pep13-Dab-SS-FL (14) possesses a cytosolic
Herein, we report the design and synthesis of novel bio- diffusible fluorophore, it would be possible to distinguish
reductively activatable FRET probes to analyze the transbilayer between endocytotic uptake and the direct penetration pathway,
movement of lipidated peptides in living cells by fluorescence and to reveal the mode of traversing the cell membrane of pep-
microscopy. We further demonstrate substantial functions of ducins by using the present probes in living cells.
the lipidated peptide moiety to flip across the membrane and
All the peptide parts of the probes shown in Scheme 1 were
to achieve rapid delivery of fluorescein as a cargo into the obtained by Fmoc solid-phase synthesis (SPPS). We also syn-
cytosol using pepducin-based probes.
thesized acetyl analogs of short pepducins 11 and 12 compris-
ing the seven C-terminal amino acid residues of 13 and 14 as
model compounds to examine the bioreductive response of
their fluorescence switch components by glutathione (GSH)
under physiological conditions. After construction of the
peptide sequences, the α-amino group of L-lysine was sub-
jected to acetylation or palmitoylation. Then, the ε-amino
Results and discussion
Design and synthesis of bioreductively activatable FRET based
probes
To visualize the flipping movement of pepducins across the group was conjugated with another L-lysine linker that was
membrane in live cells, we designed two types of FRET-based further coupled with S-trityl-3-mercaptopropionic acid at the
imaging agents, Pep13-FL-SS-Dab (13) and Pep13-Dab-SS-FL α-amino group and with dabcyl-OSu (2) or 5(6)-FAM-OSu (3)
(14), which were based on the structure of the PAR1 pepducin at the ε-amino group on the resin. The peptides 6–10 were
Fig. 1 (a) Structures of FRET probes. (b, c) Putative fluorescence responses of the probes. When the probes are in the extracellular milieu, fluorescence emission of
5(6)-FAM is quenched (OFF). The disulfide bond is cleaved by intracellular GSH to restore fluorescence signals (ON). Pep13-FL-SS-Dab (13) has a membrane-anchor-
able fluorophore (b), while Pep13-Dab-SS-FL (14) has a cytosolic releasable fluorophore (c).
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Scheme 1 Reagents and conditions: (a) MeOH–AcOH, Ar, r.t., 24 h; (b) HOSu, EDC·HCl, DMF, 0 °C, 30 min to r.t., overnight; (c) 1, Et3N, DMF, 0 °C, 30 min to r.t., over-
night; (d) (i) Fmoc-AA-OH, HOBt·H2O, DIPCI, DMF, r.t., 1.5 h; (ii) 20% piperidine, DMF, r.t., 20 min; (i), (ii) repeat; (e) Fmoc-L-Lys (Mmt)-OH, HBTU, DIPEA, DMF, r.t., 5 h;
(f) 20% piperidine, DMF, r.t., 20 min; (g) Ac2O, pyridine, DMF, r.t., 1.5 h; or C15H31COOH, HBTU, DIPEA, DMF, r.t., 5 h; (h) AcOH–TFE–CH2Cl2, r.t., 4 × 15 min; (i) Fmoc-
L-Lys (Mmt)-OH, HBTU, DIPEA, DMF, r.t., 5 h; ( j) 20% piperidine, DMF, r.t., 20 min; (k) Trt-S(CH2)2COOH, HBTU, DIPEA, DMF, r.t., 5 h; (l) AcOH–TFE–CH2Cl2, r.t.,
4 × 15 min; (m) 2 or 3, DIPEA, r.t., overnight; (n) thioanisole–m-cresol–EDT–H2O–TFA, r.t., 3 h; (o) 4 or 5, DMF and 100 mM phosphate buffer (pH 6.0–8.0), r.t.,
20 min–24 h; (p) HOSu, EDC·HCl, DMF, 0 °C, 30 min to r.t., overnight; (q) (i) TFA–CH2Cl2 (1 : 1), 0 °C, 10 min; (ii) 3, Et3N, DMF, 0 °C, 15 min to r.t., overnight; (r) 20%
piperidine, DMF, r.t., 30 min; (s) (i) 16, DIPEA, DMF, 0 °C, 30 min to r.t., 34 h; (ii) CH2Cl2–TFA–TIPS, r.t., 1 h; (t) 4, DMF and 100 mM phosphate buffer (pH 8.0), r.t., 2 h.
cleaved from the resins, purified by reversed-phase (RP) HPLC, according to the physiological range of intracellular GSH con-
and identified by electrospray ionization mass spectrometry. centration, which was reported to be between 1 and 15 mM,23
Activated disulfide derivatives 4 and 5 synthesized from dabcyl while it is in the micromolar range (1–10 μM) in blood.24 As
acid and 5(6)-FAM, respectively, were conjugated with peptides shown in Fig. 2, the fluorescence derived from 11 and 12 rose
6–10 to afford the corresponding disulfide derivatives 11–15 rapidly to be 25-fold greater even after 1 min and then
(Scheme 1). As controls, we also synthesized compound 15, an increased time-dependently until reaching a plateau at about
acetyl analog of Pep13-FL-SS-Dab (13), and compound 20 con- 50-fold within 15 min. These data indicate that the disulfide
sisting of only the FRET component without the pepducin linker could be cleaved fast enough to detect the transverse
moiety to evaluate the functions of the palmitoyl group and movement of pepducin in plasma membrane in the presence
peptide moiety in their membrane penetration.
of physiological concentration of GSH. We also measured the
fluorescence intensity of the model compound 11 treated with
various concentrations of GSH for 15 min (Fig. S7†). The
fluorescence intensity increased dose-dependently in the range
of 1 to 6 mM GSH, while a nominal increase in fluorescence
was observed in the range of 1 to 10 μM GSH. Therefore, the
present FRET system using a disulfide linker could function in
the range of physiological intracellular concentrations of GSH,
but extracellular levels of GSH may not be detectable.
Evaluation of FRET probes for the response to reductive
activation
To examine the probe’s response to reductive activation, we
used the acetyl short peptide analogs 11 and 12 as model
probes with releasable dabcyl or fluorescein, respectively. The
model probes (0.1 μM) were treated with GSH (5 mM,
1 : 50 000 molar ratio) at 37 °C in HEPES buffer (pH 7.5) and
their fluorescence emissions (λex = 488 nm, λem = 520 nm) were
monitored over time. The GSH concentration was set to 5 mM
Next, we analyzed the products of the GSH cleavage reac-
tions of compounds 11 and 12 by LC-MS. For this study,
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Fig. 2 (a) The reaction of model peptides 11 and 12 with GSH. (b, d) Time courses of the fluorescence spectrum of 11 (b) and 12 (d) at a dose of 0.1 μM treated
with GSH (5 mM), before (0 min) and from 1 to 15 min after GSH addition. (c, e) HPLC analyses of the reactions of 11 (c) and 12 (e) at a dose of 1 mM with GSH
(5 mM), before (0 min) and 30 min after GSH addition at 37 °C. Column: a Waters Symmetry C18 analytical column (Waters, 4.6 × 75 mm), Buffer A: 0.05% formic
acid in ultra-pure water, B: 0.05% formic acid MeCN. Linear gradient (B): 10% for 5 min, 10–95% for 15 min and then 95% for 5 min, flow rate: 0.5 mL min−1
.
compound 11 or 12 (1 mM) was incubated with 5 mM GSH the concentration of 1 μM for 15 min, and then observed with
(1 : 5 molar ratio) in HEPES buffer (pH 7.5) for 30 min at the confocal laser-scanning microscope after changing the
37 °C. After 30 min, the peak of starting material 11 or 12 medium and washing. Nuclear co-staining with Hoechst 33342
decreased and three new peaks appeared in each experiment. (1.0 μg mL−1) was also demonstrated before the treatment with
The mass spectra of the new peaks revealed that the disulfide the probe. Pep13-FL-SS-Dab (13)-treated cells showed a distinct
bonds of compounds 11 and 12 were reductively cleaved to membrane-localized fluorescence pattern (Fig. 3a), whereas
generate the corresponding thiol components, 6 and 21 or 7 Pep13-Dab-SS-FL (14)-treated cells showed diffuse cytosolic
and 22, respectively. The disulfide dimers 23 and 24 were fluorescence (Fig. 3b). When the cells were pre-incubated with
obtained by air oxidation of 21 and 22, respectively. These N-ethylmaleimide (NEM) before the treatment with Pep13-
results indicated that the FRET probes are susceptible to FL-SS-Dab (13) in order to deplete reduced thiols both in the
reductive cleavage of their disulfide bonds by GSH to restore medium and in the cells, fluorescence signals were signifi-
the fluorescence rapidly under physiological conditions.
cantly low (Fig. 3c), indicating that the fluorescence enhance-
ment is definitely attributed to free thiols.
Live-cell imaging of transmembrane movement of pepducin
lipopeptides using Pep13-FL-SS-Dab (13) and Pep13-Dab-SS-FL (14)
On the other hand, it is known that the disulfide bond can
be cleaved on the exofacial surface of the cells by protein di-
sulfide isomerase (PDI).25 Therefore, we also carried out pre-
treatments of the cells with 5,5′-dithiobis(2-nitrobenzoic acid)
(DTNB), a membrane-impermeable SH blocker, or bacitracin, a
We performed live cell imaging experiments using MCF-7 cells
with Pep13-FL-SS-Dab (13) and Pep13-Dab-SS-FL (14) and con-
focal microscopy. MCF-7 cells were treated with the probes at
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Fig. 3 Confocal microscopic images and bright field (BF) images of live MCF-7 cells. (a) 1 μM of Pep13-FL-SS-Dab (13) for 15 min. (b) 1 μM of Pep13-Dab-SS-FL (14)
for 15 min. (c) Cells were incubated with 50 μM of NEM prior to treatment with 1 μM of 13 for 15 min. (d) Cells were incubated with 100 μM of DTNB prior to treat-
ment with 1 μM of 13 for 15 min. BF images were overlaid by nuclear staining (Hoechst 33342). Excitation was provided with a 488 nm laser.
PDI inhibitor, to exclude the possibility of fluorescence emis- charged fluorescein as a cargo to be delivered to the intracellu-
sion of probe 13 caused by cleavage of the disulfide bond with lar juxtamembrane region or the cytosol. Furthermore, the
the exofacial thiol-dependent enzyme.26 As shown in Fig. 3d, absence of the palmitoyl group or lipopeptide pepducin
DTNB was unable to impede the disulfide bond reduction of moiety in the control compounds 15 and 20 caused the loss of
probe 13. Bacitracin also did not affect the fluorescent localiz- the ability to penetrate the cell membrane (Fig. S11†). Thus,
ation of 13 at the plasma membrane (Fig. S8b†). These data the FRET component does not have the ability for membrane
show that intracellular GSH made the dominant contribution penetration in itself. Previous studies have demonstrated that
to the reductive cleavage of the disulfide linker.
conjugation of drugs to lipids enhances cellular uptake to
Furthermore, the confocal image of cells without washing increase their intracellular concentration and assist in proper
after treatment with probe 14 showed intense fluorescence subcellular targeting.27–30 Our data support a role for a lipid
within the cells and undetectable fluorescence in the media moiety in the direct penetrating ability of pepducins.
(Fig. S10a†). To study the membrane permeability of the thiol
fluorescein 22, which may be released from probe 14 by intra-
cellular reduction, disulfide dimer 24 and fluorescein diacetate
25 (see the ESI†) were synthesized as the precursor of 22. Com-
Conclusion
pound 24 was treated with an excess amount of DTT (20 eq. × 2)
at room temperature for 120 min to convert into 22 in 82%
calculated from HPLC peak area (Fig. S9†). When the cells were
incubated with the reaction mixture of 0.5 μM of 24 with DTT
for 15 min, no significant fluorescence was detected (Fig. S10b†).
In the treatment with compound 25, which can easily permeate
into the cytoplasm and be converted into 22 by intracellular
esterases and GSH, diffuse cytosolic fluorescence was observed
remarkably (Fig. S10c†). These results indicate that 22 is
impermeable to the plasma membrane and that the reductive
activation of probe 14 specifically occurs in the cytoplasm.
Taken together, the probes localize in the inner leaflets of
the plasma membrane, exposing their bioreduction-responsive
disulfide component to the cytoplasmic surface to be subjected
to reductive activation. Pep13-FL-SS-Dab (13) released the dabcyl
moiety into the cytosol and the remaining FAM-labeled pepdu-
cin moiety remained tethered to the inner leaflet of the mem-
brane for more than 30 min, while Pep13-Dab-SS-FL (14)
released fluorescein rapidly into the cytosol by intracellular GSH
reduction as shown in our concept of probe design (Fig. 1).
These results raise the possibility that our probe architec-
ture enables cell-impermeable compounds such as negatively
We designed and synthesized novel cellular bioreductively acti-
vatable FRET probes based on pepducins, Pep13-FL-SS-Dab
(13) targeting the intracellular juxtamembrane region and
Pep13-Dab-SS-FL (14) targeting the cytosol. These probes
enabled for the first time live cell imaging of the unique trans-
bilayer movement of pepducins, which revealed that the lipid
moiety of pepducin plays a pivotal role in flipping across the
plasma membrane and in tethering its peptide moiety to the
inner leaflet of the bilayer. Furthermore, these studies demon-
strated the great potential of applying the present probe archi-
tecture to simple and straightforward intracellular drug delivery
systems. Currently, we are optimizing the peptide sequence and
lipid structure for intracellular targeting of different cells for the
development of pepducin-based drug delivery systems.
Experimental procedures
General
Protected amino acids and NovaSyn TGR resin were purchased
from Novabiochem (Merck) and other chemicals were pur-
chased from Sigma-Aldrich, Wako or TCI. RP-HPLC analyses
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were performed on 20-AD series (Shimadzu) equipped with a column (Waters, 4.6 × 75 mm), flow rate 0.5 mL min−1, linear
Waters Symmetry C18 analytical column (Waters, 4.6 × 75 mm, gradient of solvent B into A, 10 to 50% B over 15 min. r.t. =
flow rate 0.5 mL min−1) or an Inertsil C4 analytical column 5.6, 5.8 min, HRMS (ESI) m/z: calcd for C77H111N17O18S2+
(GL Sciences, 3 × 150 mm, flow rate 0.5 mL min−1). For ana- [M + 2H]2+ 796.9001, found 796.8983.
lysis, a solvent system consisting of 0.05% formic acid in ultra-
Compound 7. The title compound was prepared by the
pure water (v/v, solvent A) and 0.05% formic acid in MeCN (v/v, above method to give 47.7 mg (26% from resin). Analytical
solvent B) was used and the eluting products were detected by HPLC conditions: column, a Waters Symmetry C18 analytical
light absorption at 220 nm and 496 nm. For preparative HPLC, column (Waters, 4.6 × 75 mm), flow rate 0.5 mL min−1, linear
a Cosmosil 5C18-ARII preparative column (Nacalai Tesque, gradient of solvent B into A, 10 to 90% B over 15 min. r.t. =
20 × 250 mm, flow rate 8 or 5 mL min−1), a Cosmosil 5C18-AR 5.0 min, HRMS (ESI) m/z: calcd for C71H116N20O13S2+
II (Nacalai Tesque, 10 × 250 mm, flow rate 3 or 5 mL min−1), [M + 2H]2+ 743.4292, found 743.4282.
a Bensil 5-C18 preparative column (Bentech, 20 × 250 mm,
Compound 8. The title compound was prepared by the
flow rate 8 or 5 mL min−1), or an Inertsil WP300 C4 preparative above method to give 32.2 mg (15% from resin). Analytical
column (GL sciences, 20 × 150 mm, flow rate 8 or 5 mL min−1
)
HPLC conditions: column, a Waters Symmetry C18 analytical
was used. For preparative HPLC, a solvent system consisting of column (Waters, 4.6 × 75 mm), flow rate 0.5 mL min−1, linear
0.1% TFA in ultra-pure water (v/v) and 0.1% TFA in MeCN (v/v) gradient of solvent B into A, 10 to 90% B over 15 min. r.t. =
was used and the eluting products were detected by UV light 6.8 min, HRMS (ESI) m/z: calcd for
C
115H183N27O26S2+
absorption at 220 nm. MPLC separation was performed on [M + 2H]2+ 1194.1690, found 1194.1642.
C18 Ultrapack column or C4 Hi-Flash column (YAMAZEN)
Compound 9. The title compound was prepared by the
with YFLC W-Prep 2XY (YAMAZEN) using a solvent system con- above method and purified by MPLC (C4 and C18) and then
sisting of 0.1% TFA in ultra-pure water (v/v) and 0.1% TFA in by RP–HPLC to give 10.6 mg (3.7% from resin). Analytical
MeCN (v/v), and the eluting products were detected by UV light HPLC conditions: column, a Waters Symmetry C18 analytical
absorption at 220 nm. High-resolution mass spectra using column (Waters, 4.6 × 75 mm), flow rate 0.5 mL min−1, linear
electron spray ionization (ESI) were obtained on LCMS-IT-TOF gradient of solvent B into A, 30 to 60% B over 15 min. r.t. =
(Shimadzu). Fluorescence spectra were measured by FP-6600 6.2 min, HRMS (ESI) m/z: calcd for
C
109H188N30O21S3+
(JASCO). The purities of all isolated peptides were ≥95% as [M + 3H]3+ 760.8012, found 760.8008.
determined by analytical reversed-phase HPLC.
Compound 10. The title compound was prepared by the
above method to give 13.8 mg (5.2% from resin). Analytical
HPLC conditions: column, a Waters Symmetry C18 analytical
Solid phase peptide synthesis
Peptides were synthesized manually by Fmoc-based solid column (Waters, 4.6 × 75 mm), flow rate 0.5 mL min−1, linear
phase peptide synthesis (SPPS) on NovaSyn TGR resin. Trt for gradient of solvent B into A, 10 to 50% B over 15 min. r.t. =
L-Asn, Pbf for L-Arg, t-Bu for L-Ser and Boc for L-Lys were used 4.8 min, HRMS (ESI) m/z: calcd for
for side-chain protection. The Fmoc group was deprotected by [M + 4H]4+ 548.5334, found 548.5322.
20% piperidine in DMF for 20 min. The protected peptide
C
101H159N27O26S4+
Preparation of disulfide peptide31
resin was manually constructed in 0.10 mmol scale on
NovaSyn TGR resin (0.20–0.25 mmol g−1). Fmoc-amino acids The solution of thiol peptide (6–10) and compound 4 or
were coupled with five equivalents Fmoc-amino acid using 5 (2 eq.) in DMF and 100 mM sodium phosphate buffer
DIPCI (5 eq.) and HOBt·H2O (5 eq.) in DMF at room temp- (pH 6.0–8.0) was stirred at room temperature for an appropri-
erature for 1.5 h. For the coupling of Fmoc-L-Lys(Mmt)-OH, ate length of reaction time. The mixture was purified by HPLC
palmitic acid or Trt-S(CH2)2COOH, the 3 eq. of each compound to give disulfide peptide as a freeze-dried powder.
was reacted with DIPEA (10 eq.) and HBTU (2.9 eq.) in DMF at
Compound 11. The title compound was prepared by the
room temperature for 5 h. Acetylation was performed with above method from compounds 6 (10 mg, 0.005 mmol) and 4
acetic anhydride (10 eq.) and pyridine (10 eq.) in DMF at room (4.4 mg, 0.010 mmol) in DMF (0.9 mL) and phosphate buffer
temperature for 1.5 h. The Mmt protecting group was removed (pH 6.0, 0.1 mL) for 5 h to give 5.42 mg (48%). Analytical
by treatment with AcOH–TFE–CH2Cl2 (1 : 2 : 7) (4 × 15 min). HPLC conditions: column, a Waters Symmetry C18 analytical
Dabcyl-OSu (2) or 5(6)-FAM-OSu (3) (1.5 eq.) was coupled by column (Waters, 4.6 × 75 mm), flow rate 0.5 mL min−1, linear
treatment with DIPEA (5 eq.). The protected peptide resin gradient of solvent B into A, 10% B for 5 min then 10 to 90% B
(500 mg) was treated with thioanisole–m-cresol–EDT–H2O–TFA over 15 min. r.t. = 10.8 min, HRMS (ESI) m/z: calcd for
(5 : 5 : 5 : 5 : 80 (v/v), 5 mL) at room temperature for 3 h. Cold C94H133N21O19S2 [M + 3H]3+ 640.3092, found 640.3085. (See
3+
Et2O was added to a reaction mixture and the resulting precipi- the HPLC chart in Fig. S1†.)
tate was collected by centrifugation. The precipitate was
washed three times with Et2O and purified by RP-HPLC to give above method from compounds 7 (10 mg, 0.005 mmol) and 5
peptide as a freeze-dried powder to be analyzed by HPLC. (5.4 mg, 0.01 mmol) in DMF (0.9 mL) and phosphate buffer
Compound 12. The title compound was prepared by the
Compound 6. The title compound was prepared by the (pH 8.0, 0.1 mL) for 1.5 h to give 8.5 mg (74%). Analytical
above method to give 24.0 mg (11% from resin). Analytical HPLC conditions: column, a Waters Symmetry C18 analytical
HPLC conditions: column, a Waters Symmetry C18 analytical column (Waters, 4.6 × 75 mm), flow rate 0.5 mL min−1, linear
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Organic & Biomolecular Chemistry
gradient of solvent B into A, 10% B for 5 min, 10 to 95% B over of solvent B into A, 30 to 95% B over 60 min. r.t. = 13.6 min,
15 min and then 95% B for 5 min. r.t. = 10.7 min, HRMS (ESI) HRMS (ESI) m/z: calcd for C30H30N2O9S+ [M + H]+ 593.1588,
m/z: calcd for C94H133N21O19S2 [M + 3H]3+ 640.3092, found found 593.1560.
3+
640.3095. (See the HPLC chart in Fig. S2†.)
Compound 20. The solution of compound 19 (15 mg,
Pep13-FL-SS-Dab (13). The title compound was prepared by 0.026 mmol) and compound 4 (23 mg, 0.052 mmol) in DMF
the above method from compounds 8 (12.0 mg, 0.005 mmol) (0.9 mL) and 100 mM phosphate buffer (pH 8.0, 0.1 mL) was
and 4 (4.4 mg, 0.010 mmol) in DMF (1.9 mL) and phosphate stirred at room temperature for 2 h. The solvent was removed
buffer (pH 8.0, 0.1 mL) for 20 min to give 8.0 mg (50%). in vacuo and the residue was purified by HPLC to give 20
Analytical HPLC conditions: column, a Waters Symmetry C18 (4.0 mg, 1.7% yield from Fmoc-L-Lys(Boc)-OH) as a red freeze-
analytical column (Waters, 4.6 × 75 mm), flow rate 0.5 mL dried powder. Analytical HPLC conditions: column, a Waters
min−1, linear gradient of solvent B into A, 10 to 90% B over Symmetry C18 analytical column (Waters, 4.6 × 75 mm), flow
15 min. r.t.
132H207N31O27S2 [M + 5H]5+ 543.4960, found 543.4985. (See 90% B over 15 min. r.t. = 12.0 min. HRMS (ESI) m/z: calcd for
=
7.7 min, HRMS (ESI) m/z: calcd for rate 0.5 mL min−1, linear gradient of solvent B into A, 10 to
5+
C
C47H44N6O10S2 [M − H]− 917.2644, found 917.2660. (See the
−
the HPLC chart in Fig. S3†.)
Pep13-Dab-SS-FL (14). The title compound was prepared by HPLC chart in Fig. S6†.)
the above method from compounds 9 (23 mg, 0.008 mmol)
and 5 (8.7 mg, 0.016 mmol) in DMF (1.44 mL) and phosphate
Fluorescence spectroscopy
buffer (pH 6.0, 0.16 mL) for 24 h to give 1.3 mg (5.2%). Analyti-
cal HPLC conditions: an Inertsil C4 analytical column (GL
sciences, 3 × 150 mm), flow rate 0.5 mL min−1, linear gradient
of solvent B into A, 10 to 90% B over 15 min. r.t. = 7.8 min,
Fluorescence spectra were recorded at 37 °C using a JASCO
FP6600 with a slit width of 5 nm and 10 nm for excitation and
emission, respectively. The path length was 1 cm with a cell
volume of 3.0 mL.
HRMS (ESI) m/z: calcd for C132H201N31O27S2 [M + 2H]2+
2+
HPLC analysis of the disulfide bond cleavage
1357.2291, found 1357.2243. (See the HPLC chart in Fig. S4†.)
Compound 15. The title compound was prepared by the
above method from compounds 10 (6.6 mg, 0.003 mmol) and 4
(2.6 mg, 0.006 mmol) in DMF (1.9 mL) and phosphate buffer
(pH 6.0, 0.1 mL) for 24 h to give 5.0 mg (46%). Analytical HPLC
conditions: column, a Waters Symmetry C18 analytical column
(Waters, 4.6 × 75 mm), flow rate 0.5 mL min−1, linear gradient
of solvent B into A, 10 to 90% B over 15 min. r.t. = 5.3 min,
Compound 11 or 12 was dissolved in 100 mM HEPES buffer
(pH 7.5) at a concentration of 1 mM, and reacted with 5 mM
GSH for 30 min at 37 °C. The reaction mixtures were diluted to
0.5 mM with 0.05% formic acid in MeCN, and then analysed
by LC-MS.
Cell culture
3+
HRMS (ESI) m/z: calcd for C118H175N31O27S2 [M + 3H]3+
MCF-7 cells were maintained in MEM supplemented with 10%
FBS and 50 U mL−1 penicillin and 0.05% streptomycin and
kanamycin under the conditions of 5% CO2 at 37 °C.
839.7488, found 839.7484. (See the HPLC chart in Fig. S5†.)
Compound 18. Compound 17 (120 mg, 0.17 mmol), which
was prepared as shown in the ESI,† was treated with 20%
piperidine in DMF at room temperature for 30 min. Cold Et2O
was added to the reaction mixture and the resulting precipitate
was collected by centrifugation to give compound 18 (32 mg,
35% crude yield) as a yellow solid. Analytical HPLC conditions:
column, a Waters Symmetry C18 analytical column (Waters,
4.6 × 75 mm), flow rate 0.5 mL min−1, linear gradient of
solvent B into A, 10 to 90% B over 15 min. r.t. = 5.4 min,
5.6 min, HRMS (ESI) m/z: calcd for C27H22N2O8 [M − H]−
503.1460, found 503.1456.
Compound 19. Compounds 18 (32 mg, 0.06 mmol) and 16
(38 mg, 0.11 mmol) were dissolved in DMF (5.0 mL) and
DIPEA (51 μL, 0.29 mmol). The solution was stirred at 0 °C for
30 min and then at room temperature for 34 h. The solvent
was removed in vacuo and the residue was purified by MPLC
(C18) to give a yellow freeze-dried powder. The yellow powder
was treated with CH2Cl2–TFA (1 : 1, 10 mL) and TIPS (18 μL,
0.088 mmol) at room temperature for 1 h. Et2O was added to
the reaction mixture and the resulting precipitate was collected
by centrifugation to give compound 19 (16 mg, 45% crude
Confocal microscopy
Confocal fluorescence images were acquired with a Zeiss LSM
700 laser-scanning microscope system. Experiments were per-
formed with a 20× objective lens. MCF-7 cells (7.5 × 104 cells
per well) were plated onto a 35 mm glass-bottomed dish
(treated TC, Greiner) and were allowed to grow to 60–70% con-
fluence. Cells were preincubated with Hoechst 33342 (1.0 μg
mL−1, from 0.1 mg mL−1 stock solution in water) for 10 min
for nuclear staining, and then treated with probes (1 μM, 1%
DMSO as a cosolvent) at 37 °C for 15 min in MEM (−) (without
FBS). Next, the cells were washed with MEM (−) and fresh
medium was added. For the experiments using N-ethylmale-
imide (NEM), the cells were preincubated with 50 μM NEM in
MEM (−) for 30 min prior to the treatment with the probe in
MEM (−) for 15 min.
Abbreviations
yield for two steps) as a yellow solid. Analytical HPLC con- EDC·HCl
ditions: column, a Waters Symmetry C18 analytical column
1-[3-(Dimethylamino)propyl]-3-ethylcarbodiimide
hydrochloride
N-Hydroxysuccinimide
(Waters, 4.6 × 75 mm), flow rate 0.5 mL min−1, linear gradient HOSu
3036 | Org. Biomol. Chem., 2013, 11, 3030–3037
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Organic & Biomolecular Chemistry
Paper
9 L. Covic, M. Misra, J. Badar, C. Singh and A. Kuliopulos,
Nat. Med., 2002, 8, 1161–1165.
DIPEA
N,N-Diisopropylethylamine
HOBt·H2O N-Hydroxybenzotriazole hydrate
10 K. O’Callaghan, A. Kuliopulos and L. Covic, J. Biol. Chem.,
2012, 287, 12787–12796.
DIPCI
TFE
1,3-Diisopropylcarbodiimide
Trifluoroethanol
11 K. E. Carlson, T. J. McMurry and S. W. Hunt 3rd, Drug Dis-
covery Today, 2012, 9, e33–e39.
12 J. Miller, A. Agarwal, L. A. Devi, K. Fontanini,
J. A. Hamilton, J. P. Pin, D. C. Shields, C. A. Spek,
T. P. Sakmar, A. Kuliopulos and S. W. Hunt 3rd, Ann. N. Y.
Acad. Sci., 2009, 1180(Suppl 1), E1–12.
HBTU
2-(1H-Benzotriazol-1-yl)-1,1,3,3-tetramethyl-
aminium hexafluorophosphate
1,2-Ethanedithiol
EDT
GSH
HEPES
Glutathione
2-[4-(2-Hydroxyethyl)piperazin-1-yl]ethanesulfonic
acid
13 S. J. H. Wielders, A. Bennaghmouch, C. P. M. Reutelingsperger,
E. M. Bevers and T. Lindhout, J. Thromb. Haemost., 2007, 5,
571–576.
Fmoc
Boc
Trt
9-Fluorenylmethoxycarbonyl
t-Butoxycarbonyl
Trityl
14 J. M. Janz, Y. Ren, R. Looby, M. A. Kazmi, P. Sachdev,
A. Grunbeck, L. Haggis, D. Chinnapen, A. Y. Lin, C. Seibert,
T. McMurry, K. E. Carlson, T. W. Muir, S. Hunt III and
T. P. Sakmar, J. Am. Chem. Soc., 2011, 133, 15878–15881.
15 K. R. West and S. Otto, Curr. Drug Discovery Technol., 2005,
2, 123–160.
Mmt
Pbf
4-Methoxytrityl
2,2,4,6,7-Pentamethyldihydrobenzofuran-5-
sulfonyl
TIPS
Triisopropylsilane
Acknowledgements
16 S. Santra, C. Kaittanis, O. J. Santiesteban and J. M. Perez,
J. Am. Chem. Soc., 2011, 133, 16680–16688.
This research was supported in part by a grant from the Sasa-
gawa Scientific Research Grant from the Japan Science Society
(24-340 to M.T.), the Saijiro Endo Memorial Foundation for
Science and Technology (to S.U.) and a Grant-in-Aid for Young
Scientists (Start-up) (19890179 to S.U.) from the Japan Society
for the Promotion of Science (JSPS). The authors thank
Dr H. Fukumitsu and Dr H. Soumiya (Gifu Pharmaceutical
University) for their helpful suggestions, Prof. M. Suzui
(Nagoya City University) for his gift of cells, and Ms Emi Inaba
for her technical assistance.
17 J. Razkin, V. Josserand, D. Boturyn, Z.-h. Jin, P. Dumy,
M. Favrot, J. L. Coll and I. Texier, ChemMedChem, 2006, 1,
1069–1072.
18 S. Foillard, L. Sancey, J.-L. Coll, D. Boturyn and P. Dumy,
Org. Biomol. Chem., 2009, 7, 221–224.
19 R. J. Christie, C. J. Tadiello, L. M. Chamberlain and
D. W. Grainger, Bioconjugate Chem., 2009, 20, 476–480.
20 L. R. Jones, E. A. Goun, R. Shinde, J. B. Rothbard,
C. H. Contag and P. A. Wender, J. Am. Chem. Soc., 2006,
128, 6526–6527.
21 E. A. Dubikovskaya, S. H. Thorne, T. H. Pillow,
C. H. Contag and P. A. Wender, Proc. Natl. Acad.
Sci. U. S. A., 2008, 105, 12128–12133.
22 A. H. Henkin, A. S. Cohen, E. A. Dubikovskaya, H. M. Park,
G. F. Nikitin, M. G. Auzias, M. Kazantzis, C. R. Bertozzi and
A. Stahl, ACS Chem. Biol., 2012, 7, 1884–1891.
References
1 L. M. Bareford and P. W. Swaan, Adv. Drug Delivery Rev.,
2007, 59, 748–758.
23 Y. Hou, Z. Guo, J. Li and P. G. Wang, Biochem. Biophys. Res.
Commun., 1996, 228, 88–93.
2 F. Madani, S. Lindberg, Ü. Langel, S. Futaki and
A. Gräslund, J. Biophys., 2011, 2011, 414729.
24 L. Ducry and B. Stump, Bioconjugate Chem., 2010, 21, 5–13.
25 J. Yang, H. Chen, I. R. Vlahov, J.-X. Cheng and P. S. Low,
Proc. Natl. Acad. Sci. U. S. A., 2006, 103, 13872–13877.
26 R. Mandel, H. J.-P. Ryser, F. Ghani, M. Wu and D. Peak,
Proc. Natl. Acad. Sci. U. S. A., 1993, 90, 4112–4116.
27 L. Rajendran, V. Udayar and Z. V. Goodger, Trends Pharma-
col. Sci., 2012, 33, 215–222.
28 L. Rajendran, H.-J. Knölker and K. Simons, Nat. Rev. Drug
Discovery, 2010, 9, 29–42.
29 P. Wikstrom, H. Kirschke, S. Stone and E. Shaw, Arch.
Biochem. Biophys., 1989, 270, 286–293.
3 A. K. Varkouhi, M. Scholte, G. Storm and H. J. Haisma,
J. Controlled Release, 2011, 151, 220–228.
4 V. Torchilin, Drug Discovery Today, 2008, 5, e95–e103.
5 S. Katayama, H. Hirose, K. Takayama, I. Nakase and
S. Futaki, J. Controlled Release, 2011, 149, 29–35.
6 T. Takeuchi, M. Kosuge, A. Tadokoro, Y. Sugiura, M. Nishi,
M. Kawata, N. Sakai, S. Matile and S. Futaki, ACS Chem.
Biol., 2006, 1, 299–303.
7 M. Futami, Y. Watanabe, T. Asama, H. Murata, H. Tada,
M. Kosaka, H. Yamada and J. Futami, Bioconjugate Chem.,
2012, 23, 2025–2031.
30 M. D. Resh, BBA–Mol. Cell Res., 1999, 1451, 1–16.
31 D. Bonnet, K. Thiam, E. Loing, O. Melnyk and H. Gras-
Masse, J. Med. Chem., 2001, 44, 468–471.
8 L. Covic, A. L. Gresser, J. Talavera, S. Swift and
A. Kuliopulos, Proc. Natl. Acad. Sci. U. S. A., 2002, 99,
643–648.
This journal is © The Royal Society of Chemistry 2013
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