Multivalent design of apoptosis-inducing Bid-BH3 peptide-oligosaccharides boosts the intracellular activity at identical overall peptide concentrations

Article abstract of DOI:10.1002/chem.201202276

Multivalent peptide-oligosaccharide conjugates were prepared and used to investigate the multivalency effect concerning the activity of Bid-BH3 peptides in live cells. Dextran oligosaccharides were carboxyethylated selectively in the 2-position of the carbohydrate units and activated for the ligation of N-terminally cysteinylated peptides. Ligation through maleimide coupling was found to be superior to the native chemical ligation protocol. Monomeric Bid-BH3 peptides were virtually inactive, whereas pentameric peptide conjugates induced apoptosis up to 20-fold stronger at identical peptide concentrations. Comparison of lowly multivalent and highly multivalent peptide dextrans proved a multivalency effect in life cells which was specific for the BH3 peptide sequence. A potent blend of sugar and peptides: The intracellular activity of BH3 peptides is boosted upon their multivalent presentation on the oligosaccharide dextran. Induction of apoptosis by the pentavalent BH3 peptide dextran was visualized in cells of the human cell line Jurkat E6.1. by using a protein stain Annexin-V (purple; see figure). This flexible access to peptide dextrans should pave the way to further biological applications of these new materials. Copyright

Full text of DOI:10.1002/chem.201202276

DOI: 10.1002/chem.201202276
Multivalent Design of Apoptosis-Inducing Bid-BH3 Peptide–
Oligosaccharides Boosts the Intracellular Activity at Identical Overall
Peptide Concentrations
Martin Richter,[b, c] Alokta Chakrabarti,[d] Ivo R. Ruttekolk,[d] Burkhard Wiesner,[b]
Michael Beyermann,[b] Roland Brock,[d] and Jçrg Rademann*[a, b]
Abstract: Multivalent peptide–oligo-
saccharide conjugates were prepared
and used to investigate the multivalen-
cy effect concerning the activity of Bid-
BH3 peptides in live cells. Dextran oli-
gosaccharides were carboxyethylated
selectively in the 2-position of the car-
bohydrate units and activated for the
ligation of N-terminally cysteinylated
peptides. Ligation through maleimide
coupling was found to be superior to
the native chemical ligation protocol.
Monomeric Bid-BH3 peptides were
virtually inactive, whereas pentameric
peptide conjugates induced apoptosis
up to 20-fold stronger at identical pep-
tide concentrations. Comparison of
lowly multivalent and highly multiva-
lent peptide dextrans proved a multiva-
lency effect in life cells which was spe-
cific for the BH3 peptide sequence.
Keywords: apoptosis · cancer · oli-
gosaccharides · peptides · polymers ·
protein–protein interactions
Introduction
mains can effectively modulate PPIs, in other cases, they fail
to do so due to a lack of activity. Multivalency is a biologi-
cally relevant mechanism for converting low-affinity binding
into high-affinity interactions found ubiquitously in nature.[3]
Consequently, multivalent arrangements of peptides should
be able to potentiate the biological activity by increasing the
local ligand concentration and by targeting proteins that
present multivalent binding sites, for example, by the incor-
poration into membranes.[4–6] Therefore, the development of
a flexible synthetic access to multivalent and structurally de-
fined peptide-oligosaccharide conjugates (“peptide-dex-
trans”) was investigated.[7] Ideally, such a material was ex-
pected to enhance the biological potency of a multivalent
peptide conjugate relative to a monovalent peptide at iden-
tical peptide concentrations, if the targeted receptor protein
indeed was accessible to multivalent binding.
To test this hypothesis, the activity of multivalent Bid-
BH3-dextrans was investigated. Bid-BH3 is a pro-apoptotic
peptide, derived from the apoptosis-activating Bid pro-
tein.[8,9] Expression of the Bid-protein is stimulated by bind-
ing of the tetrameric oncoprotein p53 to damaged DNA and
leads to the induction of apoptosis through the interaction
of the BH3 helix with anti-apoptotic Bcl-proteins.[10,11] Muta-
tions of the p53 gene, which are found in>95% of all
tumors, are responsible for the shut-down of apoptosis in
cancer cells, constituting a crucial switch towards the surviv-
al of cancer cells.[12–14] Therefore, the re-stimulation of the
pro-apoptotic pathway downstream of p53 leads to the effi-
cient reversion of cancer cell survival and might have the
potential to kill cancer cells with mutated p53 selectively.
The Bid-BH3 peptide has been found to interact with the
anti-apoptotic Bcl-proteins Bcl-xl, Bcl-2, and Bcl-w, but also
the pro-apoptotic Bax protein in mitochondrial membranes,
Intracellular protein–protein interactions (PPIs) are essen-
tial for the transmission of information within and between
living cells.[1] Whereas numerous protein–protein interac-
tions have been predicted by proteome-wide interaction
screens such as yeast two-hybrid systems,[2] such methods
are prone to produce high numbers of false positives and
the functional relevance of many of the postulated protein–
protein interactions remains unclear. Therefore, chemical
tools to specifically disrupt and modulate protein–protein in-
teractions are in high demand. Whereas in some cases, pep-
tides corresponding to parts of the native interaction do-
[a] Prof. Dr. J. Rademann
Pharmaceutical/Medicinal Chemistry
Institute of Pharmacy, University of Leipzig
Brꢀderstrasse 34, 04103 Leipzig (Germany)
Fax : (+49) 341-97-36709
E-mail: rademann@uni-leipzig.de
[b] Dr. M. Richter,+ Dr. B. Wiesner, Dr. M. Beyermann,
Prof. Dr. J. Rademann
Leibniz Institut fꢀr Molekulare Pharmakologie (FMP)
Robert-Rçssle-Strasse 10, 13125 Berlin (Germany)
[c] Dr. M. Richter+
Freie Universitꢁt Berlin
Institute of Chemistry and Biochemistry
Takustrasse 3, 14195 Berlin (Germany)
[d] A. Chakrabarti,+ Dr. I. R. Ruttekolk, Prof. Dr. R. Brock
Nijmegen Centre for Molecular Life Sciences
Radboud University Nijmegen Medical Centre
PO Box 9101, 6500 HB Nijmegen (The Netherlands)
[+] These authors contributed equally to this work.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201202276.
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FULL PAPER
Figure 1. Design principles for bioactive BH3 peptides. Helical BH3 pep-
tides (red) bind to Bcl-xl proteins (blue) in the outer mitochondrial mem-
brane replacing the Bax protein (green). Oligomerization of Bax leads to
the formation of membrane pores and the release of cytochrome c into
the cytosol triggering the activation of caspases and resulting in apopto-
sis.
interactions that have been characterized functionally as
well as structurally (Figure 1).[15–17]
Apoptosis is triggered by induction of Bax oligomers in
the mitochondrial membrane. Whereas Bid-BH3 peptides
bind to Bcl-xl in homogeneous, cell-free in-vitro systems
with affinities at high nm to mm concentrations, no stimula-
tion of apoptosis has been found for the free peptide in
cells.[26] Only upon peptide lipidation or conformational fixa-
tion of the BH3 helix by hydrocarbon staples the induction
of apoptosis was observed.[10,19] In addition, small molecules
have been developed as BH3 analogues and are in clinical
trials to date.[20] Since activation of Bax involves formation
of a multimeric complex, we ask here whether multivalency
may be useful to increase the activity of Bid-BH3 peptides.
Results and Discussion
Scheme 1. Synthesis of peptide dextrans 8a–i: a) acryl amide, 1n NaOH,
30–508C, 0.1n HCl; b) N-(2-aminoethyl)-maleimide 5, EDC, pH 6.5;
c) cysteinyl peptide 7, TCEP, pH 6.5.
For the synthesis of peptide–oligosaccharide conjugates dex-
tran (a-(1!6)-linked oligo-d-glucose) was selected as a well
characterized, biocompatible polymer, which we have dem-
onstrated recently as an efficient carrier in intracellular ap-
plications.[21] Dextran 1 displayed an average molecular
sults were confirmed by colorimetric titration of polymer 2.
All subsequent experiments were conducted employing 2-
CED 2 with a loading of 1.2 mmolgꢀ1 polymer (13 CE
groups per dextran molecule). For the ligation with unpro-
tected peptides, the carboxy groups of polymers were con-
densed either with ethyl glycine thioester 3 and N-ethyl-N’-
3-dimethylamino-propyl carbodiimide (EDC) furnishing
polymer 4 for native chemical ligation or with EDC and 2-
(aminoethyl)-maleimide 5 yielding polymer 6 for malei-
mide-thiol couplings (Scheme 1).
In both cases the complete amidation of the carboxyethyl
groups could be confirmed by NMR spectroscopy of the
polymer (see the Supporting Information, Figures S4 and
S5). Activated dextrans 4 and 6 (each at 1 mm with 13 mm
activated carboxylates) were tested using both ligation pro-
tocols with N-cysteinylated R9-peptide 7 at concentrations
weight of 10 kDa and
a polydispersity index of 1.58
(Scheme 1). Thus, an average dextran molecule 1 was con-
structed of 62 glucose monomers, spanning a distance of
25 nm in its extended conformation, which corresponds to
the size of 8 globular proteins of 24 kDa. Dextran 1 was al-
kylated with acrylamide and the amides were hydrolyzed
yielding 2-carboxyethyl dextran (2-CED) 2, which was iso-
lated by precipitation followed by dialysis.[21,22] Using the re-
ported, optimized reaction conditions, carboxyethylation
was found to be entirely selective for the 2-position of glu-
cose as revealed by HMQC-HMBC NMR spectroscopic
analysis of 2 (see the Supporting Information, Figures S2
and S3). Loading of 2-CED was varied in a range between 6
and 25 carboxyethyl (CE) groups per dextran molecule and
1
was quantified by H NMR spectroscopy (see Figure 2). Re-
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J. Rademann et al.
analysis. Fluorescence correla-
tion spectroscopy was em-
ployed to verify the polymer
attachment of the fluorescein
dye. No traces of free, unat-
tached fluoresceinyl peptides
were detected in the polymer
product. Coupling efficiencies
correlated directly with peptide
concentration and provided
oligosaccharides with 3 to 7
peptide copies; relative stoi-
chiometries of peptides on the
polymer reflected the stoichi-
ometry in the reaction mixture
with no significant effect of
peptide sequences and lengths.
To investigate the intracellu-
lar multivalency effect of the
22-mer Bid-BH3 peptide and
to substantiate the findings
with all necessary control ex-
periments, nine peptide dex-
trans 8a–i and four free pepti-
des 9–12 were designed and
Figure 2. Overlay of HMBC (red) and HMQC (black) spectra showed that signals of CE-methylene groups 1’
correlated only with the signals of the glucose units in position 2, but not in position 3 or 4.
of 5 and 7 mm. The efficiency of the maleimide couplings
was found to be superior compared with native chemical li-
gation (max. yield of 75 vs. 30%, see the Supporting Infor-
mation, Tables S4 and S5). Thus, maleimide couplings at
pH 6.5 were preferred for all subsequent ligation reactions.
Polymer 6 was loaded with mixtures of two different N-cys-
teinyl peptides yielding nine different peptide dextrans 8a–i
in one step (Table 1). Cysteinyl peptides were applied in
1.5–2 fold excess to the desired degree of substitution. The
remaining maleimide groups were deactivated by using 2-
mercaptoethanol for 16 h. Obtained peptide dextrans 8a–i
were analyzed after precipitation and dialysis by gel perme-
ation chromatography, NMR, and quantitative amino acid
synthesized (Table 1). Multivalent peptide dextrans 8c,d,e,h,
and 8i were produced as low valency (divalent) and dex-
trans 8a,b,f, and 8g as high valency (pentavalent) derivatives
to dissect the multivalency effect from the effect of oligosac-
charide attachment alone. In addition, two routes of admin-
istration were investigated; delivery by an oscillating electric
field, denominated as electroporation (or nucleofection),
and delivery using a cell-penetrating peptide. Peptide dex-
trans 8a–d were prepared without attachment of a cell-pen-
etrating peptide and were delivered by nucleofection, there-
by challenging the possibility that cell-penetrating peptide
(CPP) delivery was required for multivalent enhancement.
Dextrans 8e–i were synthesized with two copies of the N-
cysteinylated CPP nonaarginine 7 to enable delivery through
endosomal uptake. For this group of polymers, peptide dex-
tran 8e, carrying only two copies of the CPP, served as a
control to exclude that the CPP alone was sufficient to
induce apoptosis.
Table 1. Composition of tested peptide dextrans 8a–i and peptides (9–
12).
Structure
Name
7
CRRRRRRRRR-NH2
CR9
Peptide dextrans 8a,c,f,h were obtained from the malei-
mide-dextran 6 by coupling with the N-cysteinyl derivative
of the native 22-mer BH3 peptide (C-BH3), whereas
8b,d,g,i were ligated with the N-cysteinyl derivative of the
mutated, inactive control peptide mBH3 (C-mBH3) to
prove that the observed effect was specific to the BH3 se-
quence. The mBH3 peptide was rendered inactive by three
alanine mutations in positions 12, 17, and 19, which have
been described as essential for the interaction with Bcl-xl
protein.[15] For fluorescencelabeling, either N-cysteinyl-lysin-
yl-5(6)-carboxyfluorescein was used in the coupling mixture,
or fluoresceinyl-lysine was added C-terminally to both the
BH3 and mBH3 peptides.[23] Finally, non-conjugated, “free”
peptides were prepared for comparison, namely the 23-mer
8a
8b
8c
8d
8e
8 f
8g
8h
8i
9
Dextran 6 with 5 C-BH3[a] and 1 C-Fluo[b]
Dextran 6 with 5 C-mBH3[c] and 1 C-Fluo
Dextran 6 with 2 C-BH3 and 1 C-Fluo
Dextran 6 with 2 C-mBH3 and 1 C-Fluo
Dextran 6 with 2 CR9[d] and 1 C-Fluo
Dextran 6 with 5 C-BH3-Fluo and 2 CR9
Dextran 6 with 5 C-mBH3-Fluo and 2 CR9
Dextran 6 with 2 C-BH3-Fluo and 2 C-R9
Dextran 6 with 2 C-mBH3-Fluo and 2 C-R9
Fluo-BH3
Dex-5B
Dex-5mB
Dex-2B
Dex-2mB
Dex-2R
Dex-5B-2R
Dex-5mB-2R
Dex-2B-2R
Dex-2mB-2R
BH3
mBH3
BH3-R9
mBH3-R9
10
11
12
Fluo-mBH3
Fluo-BH3-R9
Fluo-mBH3-R9
[a] BH3: EDIIRNIARHLAQVGDSMDRSI-NH2. [b] C-Fluo: CK(N-flu-
oresceinyl-carboxy). [c] mBH3: EDIIRNIARHAAQVGASADRSI-NH2.
[d] R9: RRRRRRRRR-NH2.
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Apoptosis-Inducing Bid-BH3 Peptide-Oligosaccharides
FULL PAPER
Fluo-BH3 9 with an N-terminal lysinyl-N-e-fluoresceinyl-
carboxy residue, the mutated derivative Fluo-mBH3 10, and
the two C-terminal nonaarginine conjugates, 11 and 12, both
being 32 mer peptides.
8a–d were imported into Jurkat E6.1 cells by nucleofection,
a gentle way of electroporation, in which the plasma mem-
brane is temporarily permeabilized by an externally applied
electric field. Peptide dextrans 8e–i were imported into cells
by covalently attached nonaarginine, an established cell-pen-
etrating peptide.[24] After loading of cells with peptide dex-
trans at various concentrations the cellular distribution of
peptide dextrans was evaluated by confocal laser scanning
microscopy (Figure 3) and uptake was quantified employing
flow cytometry. First, apoptotic cells were visualized by ad-
dition of annexin-V labeled with the fluorescent dye Alexa
Fluor 647 to cells after washing. Annexin-V is a protein that
binds specifically to the membrane phospholipid phosphati-
dylserine (PS), which is externalized to the outer leaflet of
the plasma membrane in early apoptosis. Integrity of the
plasma membrane was validated by addition of trypan blue
(Figure 3) and propidium iodide (Figure 4), dyes, which do
Preparation of all free peptides (9–12) and the ligated
peptides (C-BH3 and C-mBH3) required the careful optimi-
zation of the synthesis protocols to obtain products of suffi-
cient quality and without deletion sequences. Efficient syn-
thesis of the BH3 sequence required the employment of the
pseudo-dipeptide building blocks Asp-Ser(YMe,MePro) and
Ala-Ser(YMe,MePro) in position 17 and 18 of the 23 mers
C-BH3 C-mBH3, FluoBH3 (9), FluomBH3 (10), as well as
in the 32 mers Fluo-BH3-R9 and Fluo-mBH3-R9 (11 and
12, respectively).[27]
The apoptosis-inducing activity of peptide dextrans 8a–i
(Table 1) was investigated at Jurkat E6.1 cells, an establish-
ed lymphoma cell line (Figures 3 and 5). Peptide dextrans
Figure 4. Cellular uptake of peptide dextrans 8 f,g (10 mm total BH3-pep-
tide concentration) after 30 min in Jurkat E6.1 cells. The green fluores-
cent dextran was homogeneously distributed inside the cells. The absence
of propidium iodide staining in the cells showed their good viability.
not enter healthy and early apoptotic cells. After this initial
demonstration of the activity of the conjugate, we investigat-
ed the impact of multivalency on the induction of apoptosis
using conjugates with different numbers of BH3 peptides
per polymers. For a reliable quantification of apoptosis in-
duction, the activity of the protease caspase-3 was meas-
ured.[25] This specific enzyme was selected as a biomarker, as
in every apoptotic process, caspase-3 is the central effector
protease, which is activated by a pro-apoptotic signal (such
as the presence of BH3 peptides) and then leads to the pro-
teolytic degradation of the cell. Notably, caspase-3 is activat-
ed only during apoptosis, but not in other processes leading
to cell death such as necrosis. Caspase-3 activation by pep-
tide-dextrans without nonaarginine was determined follow-
ing nucleofection of cells with peptide dextrans or with free
peptides. After washing with buffer, cells were incubated for
6 h at 378C and lyzed. The protein content of the cell lysate
was determined by the Bradford assay and equal amounts
of protein (30 mg) of each sample were incubated with a flu-
orogenic substrate of caspase-3 (Ac-DEVD-AMC = N-(N-
acetyl-aspartyl-glutamyl-valinyl-aspartyl)-7-amino-5-methyl
Figure 3. Induction of apoptosis by the pentavalent BH3 peptide dextran
8a (10 mm total peptide concentration) after 8 h in cells of the human cell
line Jurkat E6.1. Compound 8a was transferred into the cells by nucleo-
fection, a mild form of electroporation. Compounds 8a and the mutated
pentavalent control peptide dextran 8b were homogeneously distributed
in the cells as indicated by staining with the fluorescein label (left
column). Only 8a led to the membrane staining of cells with Annexin-V
(purple), characteristic for cells in early apoptosis. At the same time,
trypan-blue staining (red) was detectable only outside of living cells for
both 8a and 8b, indicative for intact, not yet permeabilized outer cell
membranes.
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J. Rademann et al.
coumarin)[25] in a 96-well plate and analyzed in a fluores-
cence spectrometer. Fluorescence intensity was recorded
after 3 h and the activity of caspase-3 was compared after
subtraction of the initial fluorescence (Figure 5A).
strating a clear multivalency effect for the pentavalent pep-
tide polymer architecture. In the next step, the same set of
experiments was conducted with peptide dextrans 8 f–i de-
livered by the attachment of two copies of nonaarginine
through endosomal uptake without nucleofection. Dextran
8e labeled with N-cysteinyl-nonaarginine amide and N-cys-
teinylfluorescein amide as well as dextrans 8 f–i functional-
ized with N-cysteinyl-nonaarginine amide and fluorescently
labeled Cys-BH3ACTHNUTRGNEUNG(Fluo) showed good uptake into cells with
two copies of the cell-penetrating peptide and no signs of
toxicity within the tested concentration range. After wash-
ing, the integrity of the cell membranes was indicated by the
complete exclusion of propidium iodide from the interior of
cells. Confocal laser scanning microscopy revealed a homo-
geneous distribution of fluorescein fluorescence in the cells
(the Supporting Information, Figure S3). No induction of
apoptosis was observed at up to 10 mm total peptide concen-
tration by the monomeric, R9-conjugated peptides 11 and
12 or by the dextran-conjugated R9 with mutated BH3-dex-
trans (8g,i, Figure 3B).
In contrast, apoptosis was induced efficiently by multiva-
lent BH3-carrying peptide dextrans 8 f,h. At 10 mm overall
BH3-peptide concentration the bivalent BH3-peptide dex-
tran Dex-2B-2R 8h induced caspase-3 to a sevenfold level
of activity. For comparison, the pentavalent BH3-peptide
dextran Dex-5B-2R 8 f increased the caspase-3 activity 17-
fold at the same overall BH3-peptide concentration corre-
sponding to a dextran concentration of 2 mm, demonstrating
again that the degree of multivalency had a strong positive
impact on activity. Whereas the sevenfold activity enhance-
ment for the divalent peptide polymer, 8h, can be attributed
partially to the protection from proteolytic degration exert-
ed by the polymer,[26] the additional amplification observed
for the pentavalent architecture (compound 8 f) can only
result from the multivalency of the construct. Therefore,
comparison of the biological activities of differentially
loaded peptide dextrans 8a, 8c, 8 f, and 8h proved conclu-
sively a strong multivalency effect for the intracellular inter-
actions of BH3 peptides. Furthermore, it could be confirmed
from the conducted control experiments that the observed
effect can be contributed to specific interactions of the BH3
peptides, because neither the nonaarginine nor the mutated
BH3 peptides had an effect on the biological activity.
Figure 5. Induction of apoptosis by Bid-BH3-containing peptide dextrans
in Jurkat E6.1 cells. A) Activation of caspase-3 in nucleofected cells and
B) with CPP-mediated delivery of peptide dextrans. Error bars reflect
the standard deviation of values in three independent sets of experi-
ments.
Conclusion
Whereas significant induction of apoptosis was observed
neither for the monovalent BH3 peptide 9 nor for the
mono-valent mutated BH3 (mBH3) peptide 10, and the mu-
tated BH3-dextran conjugates 8b,d, a considerable, concen-
tration-dependent induction of apoptosis was recorded for
the bi- and pentavalent BH3 peptide dextrans 8a,c at identi-
cal overall peptide concentrations of 10 mm. Remarkably, the
bivalent Dex-2B 8c increased the caspase activity by a
factor of 12, whereas caspase-3 activity in cells treated with
the pentavalent Dex-5B 8a increased by 21-fold, demon-
The presented results demonstrate the efficient and modular
synthesis of multivalent and multifunctional peptide dex-
trans. For this purpose, a protocol for selective carboxyethy-
lation of dextran in the 2-hydroxy position has been devel-
oped that was employed for the diverse derivatization of
dextran over a broad concentration range between 6 to 25
carboxyethyl groups per 10 kDa dextran. The biocompatible
CE-dextran carriers were activated either as maleimides or
as thioesters and were ligated with mixtures of N-terminally
cysteinylated peptides in a single step. By using this method,
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Apoptosis-Inducing Bid-BH3 Peptide-Oligosaccharides
FULL PAPER
groups per polymer, respectively. CE groups per dextran were quantified
by titration with 0.1n NaOH and phenolphthalein as indicator (the Sup-
porting Information, Table S2).
a small collection of differentially loaded, multivalent pep-
tide dextrans was designed and synthesized to investigate
the multivalency effect for the BH3 peptide in living cells.
BH3 peptide dextrans were delivered to the interior of cells
either by nucleofection or through the attachment of two
copies of N-cysteinyl nonaarginine as cell-penetrating pepti-
des. The obtained multivalent peptide dextrans displayed
specific biological activity in the induction of apoptosis. In-
dependent of the mode of cellular import and of the protec-
tion against proteolytic degradation exerted by the polymer,
this activity was significantly potentiated by multivalent pre-
sentation of the peptides. To our knowledge this is the first
example of a multivalency effect proven with synthetic pep-
tide constructs for an intracellular protein target. The dem-
onstrated methodology should enable the investigation of
homo- and hetero-multivalency effects for other PPIs in
cells as well. The flexible access to peptide dextrans should
pave the way to further biological applications of these new
materials.
Glycine thioethyl ester hydrochloride 3: Compound 15 (23.1 g,
105.3 mmol) was stirred in 4n HCl in dioxane (10 mL) for 90 min. The
mixture was concentrated in vacuo and the product precipitated in cold
Et2O. The precipitate was filtered, washed with Et2O and dried to pro-
vide the product as white solid (15.34 g, 96%). 1H NMR (D2O,
300 MHz): d=1.22 (t, 3H, J=7.4 Hz, SCH2CH3), 2.98 (q, 2H, J=7.4 Hz,
SCH2CH3), 4.11 ppm (s, 2H, CH2COSEt); 13C NMR (D2O, 75 MHz): d=
13.3 (SCH2CH3), 23.08 (SCH2CH3), 46.63 (CH2COSEt), 194.92 ppm
(COSEt); HRMS (ESI-TOF): m/z: calcd for C4H9NOS: 120.0478 [M+
H]+; found: 120.0476.
N-(2-Aminoethyl)maleimide (5): Compound 14 (4.6 g, 19.2 mmol) was
dissolved in CH2Cl2 (35 mL) at 08C. To this solution, TFA (27 mL) was
added and stirred for 1 h, during which time the solution was allowed to
reach room temperature. The mixture was concentrated and precipitated
with cold Et2O (50 mL). Trituration with cold Et2O (3ꢃ50 mL) followed
by evaporation of remaining solvent provided a white solid as product
(2.5 g, 93%). 1H NMR (CDCl3, 300 MHz): d=3.18 (t, 2H, J=5.8 Hz,
NH3+CH2CH2), 3.79 (t, 2H, J=5.8 Hz, NH3+CH2CH2N), 6.86 ppm (s,
+
2H, COCH2CH2CON); 13C NMR (CDCl3, 75 MHz): d=34.92 (NH3
CH2CH2), 38.28 (NH3+CH2CH2), 134.6 (COCH2CH2CO), 172.53 ppm
(COCH2CH2CO); HRMS (ESI-TOF): m/z: calcd for C6H8N2O2: 141.0659
[M+H]+; found: 141.0662.
S-Ethyl-thiocarbonyl-ethyl amidoethyl dextran (thioester dextran) 4:
Compound 2 (0.2 g, 0.02 mmol) and 3 (0.23 g, 1.49 mmol) were dissolved
in water (1 mL). After adjusting the pH value of 6.5 with sodium bicar-
bonate, EDC (0.14 g, 0.74 mmol) was added. After 1 h (0.14 g,
0.74 mmol) EDC was added again and the solution was stirred overnight.
The product was precipitated with MeOH and washed several times with
MeOH. The precipitate was dissolved in water (2.5 mL) and purified on
a PD-10 column. After lyophilization a white solid was obtained as prod-
uct (0.19 g, 77%). 1H NMR (D2O, 300 MHz): d=1.17 (t, 3H, J=7.4,
SCH2CH3), 2.59 (t, 2H, J=5.4, CH2COSEt), 2.86 (q, 2H, J=7.4,
Experimental Section
Materials: All chemicals for synthesis were purchased from Sigma Al-
drich (Germany) and used without further purification. Annexin-V Assay
Kit was obtained from Invitrogen (Karlsruhe, Germany), resins for SPPS
from Rapp Polymere (Tꢀbingen, Germany), PD-10 columns from Sigma
Aldrich (Germany) and cell culture medium from PAN biotech (Aiden-
bach, Germany). Automated peptide synthesis was performed at an
Activo P11 (Cambridge, England) synthesizer. Cell Line Nucleofector
Kit was purchased from AMAXA (Cologne, Germany). 1H and
13C NMR measurements were performed on a Bruker AVANCETM
300 MHz spectrometer. 2D NMR measurements were performed on a
Bruker AVANCETM 600 MHz spectrometer. UV spectra were obtained
with a JASCO V-550 UV/Vis spectrometer. Amino acid analysis was per-
formed by Genaxxon Bioscience GmbH (Ulm, Germany).
SCH2CH3), 3.3–4.1 (m, CACTHUNTRGNEG(UN 2–6) H-dextran, OCH2CH2CO), 4.92 (s, anome-
ric H (unsubstituted)), 4.9–5.3 ppm (m, anomeric H); 13C NMR (D2O,
75 MHz): d=13.32 (SCH2CH3), 22.50 (SCH2CH3), 35.49 (CH2CONH),
48.70 (NHCH2CO), 59.95 (C(6)), 64.97 (OCH2CH2CO), 68.98 (C(5)),
69.64 (C(3)), 70.87 (C(2)), 72.87 (C(4)), 97.16 (C(1)), 174.14
(CONHCH2), 200.97 ppm (COSEt).
2-Carboxyethyl dextran (2-CED) 2: Dextran (5 g, 0.5 mmol, Mw 10 kDa,
PDI=1.58) was dissolved in a 1 n NaOH solution (50 mL). Acryl amide
(0.9 g, 12.5 mmol) was added and stirred at 308C. After 24 h the reaction
temperature was increased to 508C and stirred for additional 24 h. The
solution was neutralized the oligosaccharide products were precipitated
with methanol, solvents were removed after centrifugation (4000ꢃrpm,
5 min). After washing of precipitate with methanol the precipitate was
dried. The white solid was dissolved in water (25 mL) and dialyzed (ben-
zoylated dialysis tubing, with a pore size (nominal molecular weight cut-
off (NMWCO)) of 2000, Sigma Aldrich) twice with 0.1n HCl (10 L) and
water (2ꢃ10 L) over 24 h. The loading with carboxyethyl groups was
quantified by 1H NMR spectroscopy and confirmed by titration against
0.1n NaOH as described below. Positions of the CE-substitution were de-
termined by using HMBC-HMQC NMR spectroscopy as described
below. Substitution was found selectively in the 2-position under both
conditions described (see also Figure 2 and the Supporting Information,
S2). After lyophilization a white powder was obtained in a yield of 4 g
(72%, 13 carboxyethyl groups per dextran). 1H NMR (D2O, 300 MHz):
Maleimidoethyl amidoethyl dextran (maleimide dextran) 6: Compound 2
(0.2 g, 0.02 mmol) with 13 carboxy groups (0.26 mmol) and 5 (0.69 g,
2.69 mmol) were dissolved in water (1 mL). A pH of 6.5–7 was adjusted
and EDC (0.26 g, 1.35 mmol) added. After 1 h EDC (0.26 g, 1.35 mmol)
was added again and the solution was allowed to stir for 4 h. The mixture
was precipitated with methanol. The precipitate was washed with metha-
nol and DMF. The solid was dissolved in water (2.5 mL) and purified
over Sephadex G-25. Lyophilization led to product as a white solid
(0.19 g, 82%). Quantification of maleimide groups per dextran was per-
formed using 1H NMR spectroscopy (the Supporting Information, Fig-
ure S4). It could be proven that under the conditions used, all the carbox-
yl groups on the dextran reacted to the corresponding maleimidoethyl
amide groups. 1H NMR (D2O, 300 MHz): d=2.48 (t, 2H, J=4.7,
CH2CH2CO), 3.3–4.1 (m, CH2CH2CO, H (glucose units)), 4.98, ꢀ5.3 (m,
anomeric H), 6.89 ppm (s, 2H, COCHCHCO); 13C NMR (D2O, 75 MHz):
d=36.38 (CH2CONH), 37.12 (NHCH2CH2NH2), 39.97 (NHCH2CH2N),
65.35 (C(6)H2), 65.58 (C(OCH2CH2CONH), 67.94 (C(4) Glucose), 71.41
(C(5) Glucose), 72.57 (C(3)H Glucose), 74.68 (C(2) Glucose), 98.71
(C(1) Glucose), 179.29 (CONH), 181.82 ppm (CH2CONH).
d=2.65 (t, 2H, CH2COOH), 3.25–4.1 (m, CH2CH2COOH, CACTHNUTRGNEUG(N 2–6)H (glu-
cose units)), 4.93–5.30 ppm (m, 1H, C(1)H); 13C NMR (D2O, 75 MHz):
d=34.20 (CH2COOH), 65.03 (OCH2CH2COOH), 69.03 (C(4) glucose
unit), 69.67 (C(2) glucose unit), 70.89 (C(4) glucose unit), 72.89(C(3) glu-
cose unit), 97.18 (C(1) glucose unit, 175.39 ppm (CH2COOH). CE substi-
tution of dextran was varied by using different concentrations of acryl
amide in the reaction mixture. Under the reported conditions about 50%
of the employed acrylamide was attached to the polymer (the Supporting
Information, Table S1). With 5 g (0.5 mmol) dextran 25, 12.5, and
6.25 mmol of acrylamide yielded a loading of 25, 13, and 6 carboxyethyl
Peptide synthesis (7, 9, 10, 11, and 12): Peptides were synthesized man-
ually or by automated Fmoc-SPPS on TentaGel
S Ram amide
(0.2 mmolgꢀ1) resins. Fluorescein-labeled peptides were synthesized by
using preloaded Na-carboxyfluorescein-lysinyl amide TentaGel R Ram
resin. To avoid aspartimide formation, Fmoc-groups were cleaved using
20% piperidine and 0.1 n acetic acid solution in DMF. To decrease im-
purities by sequence termination pseudo-prolines were used. For the syn-
thesis of BH3-peptides, Asp17-Ser18 were substituted by Asp-Ser(YMe,-
Chem. Eur. J. 2012, 18, 16708 – 16715
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16713

J. Rademann et al.
MePro) and Ala17-Ser18 were substituted by Ala-Ser(YMe,MePro)[27]
Purification was performed by preparative RP-HPLC. Peptides were
identified by LCMS, HRMS and MALDI TOF.
.
Synthesis of nonaarginine dextran conjugates using maleimide thiol cou-
pling 16, 17: Compound 6 (10 mg, 1 mmol) loaded with 12 maleimide
groups (12 mmol) was dissolved in MES buffer (1 mL, pH 6.5). After de-
gassing, cysteinyl-nonaarginine amide 7 (6 mg, 5 mmol) was added under
nitrogen atmosphere. The pH value was checked and the mixture stirred
for 16 h. The solution was concentrated and the product precipitated
with MeOH. After several washing steps with MeOH and DMF the solid
was dissolved in water (2.5 mL) and purified twice using PD-10 columns.
After lyophilization the resulting conjugate was obtained as a white
powder. Results of amino acid analysis showed how much peptide per
polymer was coupled (see the Supporting Information, Table S4: 8 mg,
68%, 3 R9 groups per dextran).
General procedure for synthesis of dextran-peptide conjugates (8a–g): 6
(5 mg, 0.4 mmol) with 13 maleimide groups was dissolved in an MES
buffer (1 mL, pH 6.5). After degassing, cysteinyl-BH3ACTHNUTRGNE(NUG Fluo)-peptide
(7 mg, 2 mmol for 8 f and 3 mg, 1 mmol for 8h) and cysteinyl-nonaarginine
amide 7 (2 mg, 1 mmol) were added under a nitrogen atmosphere. The
pH value was checked and the mixture stirred for 16 h. The solution was
concentrated and the product precipitated with MeOH. After several
washing steps with MeOH and DMF the solid was dissolved in water
(2.5 mL) and purified twice using PD-10 columns. The desired conjugate
was obtained after lyophilization. Purity of the conjugates was checked
using fluorescence correlation spectroscopy (FCS). Results of amino acid
analysis showed how much peptide per polymer was coupled (see the
Synthesis of nonaarginine dextran conjugates using native chemical liga-
tion 18, 19: Thioester dextran 4 (10 mg, 1 mmol) loaded with 12 thioester
groups (12 mmol) was dissolved in phosphate buffer (1 mL, 100 mm
KH2PO4/Na2PO4 pH 7.4). After degassing, cysteinyl-nonaarginine amide
7 (6 mg, 5 mmol), TCEP (9 mg, 50 equiv) and 4-mercaptophenyl acetic
acid (5 mg, 32 equiv) were added under a nitrogen atmosphere. The pH
value was checked and the mixture stirred for 16 h. The solution was con-
centrated and the product precipitated with MeOH. After several wash-
ing steps with MeOH and DMF the solid was dissolved in water (2.5 mL)
and purified twice using PD-10 columns. The resulting conjugate was ob-
tained after lyophilization. Results of amino acid analysis showed how
much peptide per polymer was coupled (see the Supporting Information,
Table S5: 8 mg, 73%, 1.2 R9 groups per dextran).
Supporting Information, Table S3 (8 f: 7 mg, 60%, 5 BH3ACTHNUTRGENUG(N Fluo)- and 2
R9 groups per dextran)). Cellular uptake was shown in Jurkat E6.1 cells.
Viability of cells was checked with propidium iodide (see the Supporting
Information, Figure S6).
N-(tert-Butoxycarbonyl)-ethyl-1,2-diamine 13: A solution of Boc-anhy-
dride (8.7 g, 40 mmol) in CHCl3 (200 mL) was added dropwise to an ice-
cold solution of 1,2-diaminoethane (27 mL, 400 mmol) in CHCl3
(400 mL) over 3 h. After complete addition, the mixture was stirred at
08C for 30 min and afterwards allowed to warm to room temperature
overnight. The solvent was removed under reduced pressure and the resi-
due dissolved in 3 n Na2CO3 (300 mL) solution. The mixture was extract-
ed with CHCl3 (3ꢃ200 mL), the organic phase dried over MgSO4 and
Comparison of both ligation methods: To compare NCL and maleimide
thiol coupling, compounds 4 and 6 were reacted with different amounts
of cysteinyl-nonaarginine amide. Polymers were worked up as previously
described and analyzed by using amino acid analysis. Under the opti-
mized conditions 25% of the available cysteinyl peptides were coupled
to (S)-ethyl-thiocarbonyl-ethyl dextran using NCL. In contrast to that
75% of the available cysteinyl peptides were coupled on 4 using malei-
mide thiol coupling. Advantages and disadvantages of both ligation
methods are summarized in the Supporting Information, Table S6.
evaporated to provide the product as
a colorless oil (6.4 g, 98%).
1H NMR (300 MHz, CDCl3): d=1.35 (s, 9H, CCH3), 2.69 (t, 2H,
CH2NHBoc), 3.07 (q, 2H, CH2CH2NHBoc), 5.11 ppm (brs, 1H,
NHBoc); 13C NMR (75 MHz, CDCl3): d=27.87 (CCH3), 41.36
(H2NCH2CH2), 42.93 (CH2NHBoc), 78.52 (CCH3), 155.73 ppm
(NHCOO); HRMS (ESI-TOF): m/z: calcd for C7H16N2O2: 161.1285 [M+
H]+; found: 161.1285.
Laser scanning microscopy: HeLa-S cells seeded on cover slips were in-
cubated with peptides or peptide dextrans, dissolved in HEPES buffer,
for 30 min at 378C under a LSM-510 or LSM 710 laser scanning micro-
scope (Carl Zeiss MicroImaging, Jena, Germany) equipped with a 63
water objective. Immediately after adding the substance, pictures were
recorded every 60 sec for 30 min. After removing the peptide solution
and washing the coverslips with HEPES buffer, trypan blue was added to
prove the viability of treated cells and their membrane integrity.
1-(N-tert-Butoxycarbonyl)-2-aminoethyl) maleimide 14: Mono-protected
N-Boc-ethylenediamine 13 (5.1 g, 31.9 mmol) and Et3N (6.6 mL,
47.79 mmol) were dissolved in Et2O (60 mL) at 08C. Maleic anhydride
(3.1 g, 31.9 mmol) in 60 mL Et2O was added dropwise and the reaction
mixture was stirred for 4 h, during which time the solution was allowed
to reach room temperature. After concentrating the solution the residue
was dissolved in acetone (150 mL). Et3N (8.8 mL, 63.7 mmol) was added
and the mixture was heated to reflux. Acetic anhydride (4.5 mL,
47.8 mmol) was added and the solution was heated to reflux for 20 h.
After the solvent was removed under reduced pressure chromatography
of the dark brown oily residue over silica gel (EtOAc/hexane 1:1) gave
Fluorescence correlation spectroscopy: FCS measurements were per-
formed at room temperature on an LSM 510-ConfoCor 2 system and on
an LSM 710-ConfoCor 3 system (Carl Zeiss MicroImaging, Jena). Fluo-
rescent BH3-peptides, BH3-dextrans 8 f–i, respective mixtures of unreac-
tive CED 2, and fluorescent BH3-peptides were dissolved in water at a
concentration of 10–8 molLꢀ1. Each solution on cover slips (50 mL per
sample) were fixed in a measurement chamber. An argon ion laser
(488 nm) was used for excitation of fluorescein. Calculations of the auto-
correlation function as well as fitting of the autocorrelation function
were performed using ZEN 2010 software. Correlation functions account-
ing for free diffusion in three dimensions were fitted for one component
and yielded diffusional autocorrelation times of particles through the
confocal volume (the Supporting Information, Figure S7). In two compo-
nent fittings, it could be shown that the investigated peptide polymers
(e.g., 8 f) contained no free peptides.
Electroporation: Jurkat E6.1 cells (106) were resuspended in Nucleofec-
tor Solution V (100 mL). Peptide or peptide polymer was added in the re-
spective concentration and the solution was pipetted into LONZA nucle-
ofection cuvettes. Nucleofection was performed in an AMAXA Nucleo-
fector using program X-001 in accordance with the recommendations of
the manufacturer. Following nucleofection the medium (900 mL) was
added carefully and the cells incubated for 5 min at RT. Subsequently
cells were washed and resuspended in fresh medium.
1
the product as a white solid (3.7 g, 48.1%). H NMR (CDCl3, 300 MHz):
d=1.4 (s, 9H, CCH3), 3.34 (q, 2H, J=4.9 Hz, CH2CH2NHBoc), 3.66 (t,
2H, J=5.1 Hz, CH2CH2NHBoc), 4.74 (brs, 1H, NHBoc), 6.71 ppm (s,
2H, CHCHCON); 13C NMR (CDCl3, 75 MHz): d=27.83 (CCH3), 37.5
(CH2CH2NHBoc), 38.91 (CH2CH2NHBoc), 79.07 (CCH3), 133.96
(CHCHCON), 155.46 (NHCOO), 170.34 ppm (CONCO); HRMS (ESI-
TOF): m/z: calcd for C11H16N2O4: 241.1183 [M+H]+; found: 241.1188.
N-tert-Butoxycarbonyl-glycine thioethyl ester 15: N-Boc-glycine (24.7 g,
141.5 mmol) and carbonyldiimidazole (25 g, 154.2 mmol) were stirred in
dry THF (125 mL) at 208C under a nitrogen atmosphere for 30 min. Mer-
captoethanol (31.39 mL, 424.3 mmol) was added and the mixture was stir-
red overnight. After addition of 4 n NaOH (250 mL), the solution was
rapidly extracted with CH2Cl2 (3ꢃ150 mL). The organic phase was dried
over MgSO4, evaporated and redissolved in ethyl acetate. The solution
was filtered over silica gel and the solvent removed under reduced pres-
sure to provide pure product as yellowish oil (23 g, 75%). 1H NMR
(D2O, 300 MHz): d=1.23 (t, 3H, J=7.2 Hz, SCH2CH3), 1.44 (s, 9H, OC-
ACHTUNGTRENNUNG
AHCTUNGTRENNUNG
AnnexinV assay: Electroporated Jurkat E6.1 cells were washed with
medium, transferred to a 24-well tissue culture plate and incubated for
14 h in RPMI medium. The cells were washed in cold PBS buffer and in-
ACHTUNGTRENNUNG
16714
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ꢂ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2012, 18, 16708 – 16715

Apoptosis-Inducing Bid-BH3 Peptide-Oligosaccharides
FULL PAPER
cubated for 15 min with 5 mL AnnexinV Alexa Fluor 647 in 95 mL Annex-
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sample. Analysis was performed by flow cytometry on a FACS Calibur
System (Becton Dickinson, Heidelberg).
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Caspase-3 assay: Jurkat E6.1 cells (106) were incubated with peptide or
peptide dextran conjugate at the respective concentration for 6 h. After
washing with ice-cold PBS buffer, the cells were spun down at 1900 rpm
for 5 min and pellets were resuspended in lysis buffer (20 mm Tris/HCl,
150 mm NaCl, 1 mm EDTA, 1% Triton X-100, 1 protease inhibitor cock-
tail tablet (Roche Diagnostics Mannheim, Germany) per 50 mL buffer,
pH 7.7). After 30 min cooling on ice, the lysate was separated from the
remaining solid by centrifugation and protein content estimated by com-
mercially available Bradford Protein Assay Kit (Bio-Rad Laboratories
Munich, Germany). 30 mg protein aliquots of each sample were diluted in
caspase activity buffer (20 mm HEPES, 100 mm NaCl, 10 mm dithiothrei-
tol, 10% glycerol, pH 7.5). Finally, fluorogenic caspase-3 substrate (Ac-
DEVD-AMC) was added to a concentration of 2 mm. Cleavage efficiency
of active caspase-3 in solution was measured immediately after addition
of the substrate and after 3 h at 378C by measuring fluorescence increase
using a luminescence spectrometer LS50B (Perkin–Elmer, Norwalk, CT,
USA).
Acknowledgements
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Received: June 27, 2012
Published online: November 4, 2012
Chem. Eur. J. 2012, 18, 16708 – 16715
ꢂ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
16715