Cell-penetrating γ-peptide/antimicrobial undecapeptide conjugates with anticancer activity

Article abstract of DOI:10.1016/j.tet.2012.02.003

In this study, we combined a cell-penetrating γ-peptide, PEG-1, with antimicrobial undecapeptides in order to provide compounds with anticancer properties against MDA-MB-231 human breast cancer cells. We demonstrated that the conjugates were more cytotoxic than Ac-PEG-1 and the parent undecapeptides. We also evaluated the toxicity of the conjugates against non-malignant cells. The peptide conjugate with the best biological profile was BP77-PEG-1, which, at 10 μM, showed a 71% growth inhibition in MDA-MB-231 cells and only a 17% inhibition in non-malignant cells. Therefore, this study suggests that PEG-1 mediated the undecapeptide delivery into cancer cells and that these conjugates are the proof-of-concept of this strategy to generate improved anticancer drugs based on peptides.

Full text of DOI:10.1016/j.tet.2012.02.003

Tetrahedron 68 (2012) 4406e4412
Contents lists available at SciVerse ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Cell-penetrating
g
-peptide/antimicrobial undecapeptide conjugates with
anticancer activity
a
,
,
c
,
y
b
b
d
Cristina Roses y, Daniel Carbajo b , Gloria Sanclimens , Josep Farrera-Sinfreu , Adriana Blancafort ,
ꢀ
ꢁ
d
a
a
d
,
a
a,
*
*
ꢁ
Gloria Oliveras , Anna D. Cirac , Eduard Bardají , Teresa Puig , Marta Planas , Lidia Feliu
,
Fernando Albericio c f, Miriam Royo b
,
e
,
,c,
*
a
ꢀ
ꢁ
Laboratori d’Innovacio en Processos i Productes de Síntesi Organica (LIPPSO), Departament de Química, Universitat de Girona, Campus Montilivi, 17071 Girona, Spain
b Combinatorial Chemistry Unit, Barcelona Science Park, Baldiri Reixac 10, 08028 Barcelona, Spain
c CIBER-BBN, Networking Centre on Bioengineering, Biomaterials, and Nanomedicine, Spain
d Medicine School, Girona Biomedical Research Institute (IDIBGi), University of Girona, 17071 Girona, Spain
e Institute for Research in Biomedicine, Barcelona Science Park, Baldiri Reixac 10, 08028 Barcelona, Spain
f
ꢀ
Department of Organic Chemistry, University of Barcelona, Martí i Franques 1-11, 08028 Barcelona, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
In this study, we combined a cell-penetrating g-peptide, PEG-1, with antimicrobial undecapeptides in
Received 30 September 2011
Received in revised form 26 January 2012
Accepted 3 February 2012
order to provide compounds with anticancer properties against MDA-MB-231 human breast cancer cells.
We demonstrated that the conjugates were more cytotoxic than Ac-PEG-1 and the parent un-
decapeptides. We also evaluated the toxicity of the conjugates against non-malignant cells. The peptide
Available online 9 February 2012
conjugate with the best biological profile was BP77-PEG-1, which, at 10 mM, showed a 71% growth in-
hibition in MDA-MB-231 cells and only a 17% inhibition in non-malignant cells. Therefore, this study
suggests that PEG-1 mediated the undecapeptide delivery into cancer cells and that these conjugates are
the proof-of-concept of this strategy to generate improved anticancer drugs based on peptides.
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
g-Peptides
Anticancer
Antimicrobial peptides
Cell-penetrating peptides
Foldamers
1. Introduction
currently available conventional drugs, which typically interact
with a specific target protein. Upon binding, they disrupt cell
In spite of great advances in cancer therapy, there is consider-
able current interest in developing anticancer agents with a new
mode of action due to the development of resistance by cancer cells
toward current anticancer drugs.1 A growing number of studies
have shown that some antimicrobial peptides, which are toxic to
bacteria but not to normal mammalian cells, exhibit a broad
spectrum of cytotoxic activity against cancer cells.2
Although antimicrobial peptides display a wide structural di-
versity, many of them are short, cationically charged, and able to
form amphipathic secondary structures.3 Their exact mode of ac-
tion is not completely understood. However, there is a consensus
that antimicrobial peptides act by disrupting negatively charged
bacterial and cancer cell membranes to which they are electro-
statically attracted, leading to cell lysis and death,1b,3d unlike
membranes, possibly by transient pore formation or disruption of
lipid packing.1b,3d Based on this mode of action, these peptides are
unlikely to cause rapid emergence of resistance because it would
require significant alteration of membrane composition, which is
difficult to occur.3g,4 In addition, there is increasing evidence that
apart from membrane damage, other mechanisms may be involved
including intracellular targets.1b,2b,3b,d Some of these AMPs showed
remarkable selectivity to cancer cells versus untransformed pro-
liferating cells.1b,3a,b,d
Despite the excellent properties displayed by antimicrobial
peptides, the development of peptide-based drugs is hampered by
their limited access to the intracellular space. This obstacle has been
tried to overcome by using diverse methods to promote the cellular
uptake of exogenous molecules. Cell-penetrating peptides (CPPs)
have become one of the most efficient and explored transporters for
achieving intracellular access.5 CPPs are usually short cationic se-
quences and may be derived from natural sequences (i.e., TAT
peptide6) or be de novo designed peptides (polyarginine7 or trans-
portan8). The conjugation of biologically active peptides to CPPs
offers advantages, such as low toxicity and selective and controlled
* Corresponding authors. Tel.: þ34 972 419628; fax: þ34 972 419617 (T.P.);
tel.: þ34 972 418274; fax: þ34 972 418150 (L.F.); tel.: þ34 934037122; fax: þ34
934020496 (M.R.); e-mail addresses: teresa.puig@udg.edu (T. Puig), lidia.feliu@
udg.edu (L. Feliu), mroyo@pcb.ub.es (M. Royo).
y
These two authors contributed equally to this work.
0040-4020/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2012.02.003

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C. Roses et al. / Tetrahedron 68 (2012) 4406e4412
4407
cell delivery compared with other administration vectors,5 being
considered a promising strategy for the design of anticancer agents.
A drawback of this strategy is the low stability of these CPPs to
proteases that it can prevent the drug to reach its target, forcing an
increment of the doses to keep the activity and consequently gen-
erating an increase in the toxicity. Some alternatives with good
protease stability have been described keeping internalizing prop-
explore its anticancer activity. Moreover, taking profit of the cell-
uptake properties offered by -peptide 1 (see Fig. 1), we decided to
design new -peptide 1/CECMEL11 undecapeptide conjugates. The
conjugation of these antimicrobial peptides to 1 can promote its in-
ternalization, which can interact with intracellular targets increasing
the cytotoxic effect, i.e., by disruption of mitochondrial membranes.15
g
g
As a linker between the CECMEL11 undecapeptides and g-pep-
erties, like diverse oligomers with foldamer properties, such as
tide 1, it was decided to use a short monodisperse bifunctional
polyethylene glycol (PEG) unit (8-amino-3,6-dioxaoctanoic acid),
generating the peptide PEG-1. Polyethylene glycol has many prop-
erties that make it an ideal carrier for peptides, such as high water
solubility, high mobility in solution and low immunogenicity. PEG is
often attached to the N or C termini of peptides.16 Besides increasing
overall size, this modification also protects peptides from exo-
peptidases and therefore increases their overall stability in vivo.
In this report, we describe the design and synthesis of PEG-1/
antimicrobial undecapeptide conjugates. The influence of the
11
peptoids,9 arylamide oligomers,10
b
-
and g
-peptides.12 Some of
these foldamers have not only been explored as potential CPPs, but
also as antimicrobial compounds with highly remarkable results.13
Generally both applications derived in similar sequence pattern
that combine hydrophobic/aromatic residues with cationic groups.
In 2004, Farrera-Sinfreu and co-workers described a new family of
g
-peptides hexamers based on the trifunctional amino acid cis 4-
aminoproline that adopt a C9 ribbon in H2O.12a This adopted sec-
ondary structure gives to these peptides a tridimensional pattern
with high amphipaticity character that contribute to their in-
g-peptide on the anticancer activity is also discussed.
ternalization properties. Series of these g-peptide diversely func-
tionalized on the Na of cis 4-aminoproline were synthesized and
have proven capacity for cellular uptake.12b These peptides also
showed lowcytotoxicityand high resistance to proteases, properties
that convert them in a promising class of CPPs, specially peptide 1
that showed a 40% of internalization capacity compared to TAT
peptide (Fig. 1) considered a gold standard in the field.
2. Results and discussion
2.1. Design and synthesis
The undecapeptides were representative examples of the 125-
member CECMEL11 library.14 The selected peptides showed
Fig. 1. Structures of the peptide conjugates.
Recently, Badosa and co-workers designed linear undecapeptides
(CECMEL11) to be used in plant protection.14 The general structure of
this library was R-X1KLFKKILKX10L-NH2, where X1 and X10 corre-
sponded to amino acids with various degrees of hydrophobicity and
hydrophilicity (Leu, Lys, Phe, Trp, Tyr, Val) and R included different
N-terminal derivatizations (H, Ac, Ts, Bz, Bn). The antimicrobial
evaluation of the CECMEL11 library led to the identification of pep-
a good balance between antimicrobial and hemolytic activities.
Their sequences differ from the N-terminal derivatization (H, Ac, Ts
or Bz) and the residues at positions 1 (Lys or Leu) and 10 (Val, Phe,
Tyr or Lys). Peptide conjugates were designed by linking the cor-
responding antimicrobial undecapeptide to the cell-penetrating
peptide PEG-1 (Fig. 1).
The parent CECMEL11 undecapeptides BP15, BP33, BP76, BP77,
BP101, BP125, and BP126 were prepared using a Fmoc-Rink-MBHA
resin following a standard Fmoc/t-Bu strategy.14 After the synthesis,
peptides were cleaved from the resin by treatment with TFA/
tides with high antibacterial activity (MIC<7.5
hemolysis (2e6% at 50 M and 8e40% at 150
properties, these peptides can be considered as good candidates to
mM) and with low
m
mM). Based on these

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C. Roses et al. / Tetrahedron 68 (2012) 4406e4412
4408
triisopropylsilane (TIS)/H2O. HPLC and ESI-MS analysis of the crude
reaction mixtures showed the formation of the expected peptides
in good purities (>75%).
assembly was completed, the N-terminal Fmoc group was re-
moved by exposure to piperidine-NMP. BP15-PEG-1, BP33-PEG-
1, and BP76-PEG-1 were cleaved from the resin by treatment
with HF. In the case of N-terminal derivatized conjugates, after
removal of the Fmoc group, the resulting peptidyl resins were
treated with acetic anhydride (Ac2O)epyridineeCH2Cl2 (BP77-
PEG-1 and BP101-PEG-1), with p-toluenesulfonyl chloride
(p-TsCl), and N,N-diisopropylethylamine (DIEA) (BP125-PEG-1)
or with benzoyl chloride (BzCl) and DIEA (BP126-PEG-1), and
then cleaved with HF. The crude peptides were purified by pre-
parative reversed-phase HPLC. The purified peptide conjugates
were analyzed by HPLC (ꢀ95% purity) and their structures were
confirmed by MALDI-TOF (Table 1).
The cell-penetrating peptide PEG-1 was synthesized on
a 4-methylbenzhydrylamine (MBHA) resin following an Fmoc/
Alloc/Boc combined solid-phase strategy, a modified version of
the previously described (Scheme 1).12 Instead of the sequential
introduction of the alkylation moiety after each monomer cou-
pling, this new strategy allows the simultaneous introduction of
the same type of alkylation moiety once the backbone synthesis
is finished. This results in a protocol with fewer synthetic steps
and cleaner final peptide crudes. The seven conjugates were
obtained by stepwise coupling of the corresponding Fmoc-amino
acids to the growing peptide chain on the PEG-1-MBHA
resin (Scheme 2). The couplings were mediated by ethyl cyano-
glyoxylate-2-oxime (Oxyma) and N,N0-diisopropylcarbodiimide
(DIPCDI) in N-methylpyrrolidinone (NMP).17 Once the chain
For comparison purposes, Ac-PEG-1 was prepared by acetyla-
tion of the PEG-1-MBHA resin with Ac2OepyridineeCH2Cl2 fol-
lowed by acidolytic cleavage. RP-HPLC purification afforded the
pure compound.
Scheme 1. Solid-phase synthesis of PEG-1-MBHA.

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C. Roses et al. / Tetrahedron 68 (2012) 4406e4412
4409
Scheme 2. Solid-phase synthesis of peptide conjugates.
Table 1
general, N-terminal derivatization seems to be important for the
anticancer activity of these peptides.
The PEG-1/undecapeptide conjugates were much more effective
in inhibiting the growth of MDA-MB-231 adenocarcinoma cells
Characterization of peptide conjugates
Peptide conjugate
tRa (min)
Purityb (%)
93
ESI
BP15-PEG-1
5.35
747.95 [Mþ4H]4þ
996.51 [Mþ3H]3þ
744.11 [Mþ4H]4þ
991.84 [Mþ3H]3þ
759.92 [Mþ4H]4þ
1012.82 [Mþ3H]3þ
770.40 [Mþ4H]4þ
1026.96 [Mþ3H]3þ
774.29 [Mþ4H]4þ
1032.29 [Mþ3H]3þ
793.70 [Mþ4H]4þ
1057.99 [Mþ3H]3þ
781.22 [Mþ4H]4þ
1041.24 [Mþ3H]3þ
than the CECMEL11 peptides. At 30
conjugates did not differ from that of Ac-PEG-1 (>90% inhibition).
In contrast, at 10 M, whereas Ac-PEG-1 only caused 3% inhibition,
mM, the activity of the seven
BP33-PEG-1
BP76-PEG-1
BP77-PEG-1
BP101-PEG-1
BP125-PEG-1
BP126-PEG-1
7.43
7.05
7.20
7.62
7.60
7.73
95
96
99
95
98
98
m
peptide conjugates were 10e20 times more active, with inhibition
percentages between 34 and 71%. This result indicates that the
peptide moiety is responsible for the anticancer activity. The pep-
tide conjugate with the highest cytotoxic activity was BP77-PEG-1.
As compared with the CECMEL11 parent peptides at 10 mM, con-
jugation to PEG-1 increased the activity from 1.2 up to 10-fold. We
speculate that the superior cytotoxicity of the peptide conjugates
could be attributed to the cell-internalization efficiency of PEG-1,
which enables the transport of the antimicrobial peptides over the
cell membrane, enhancing their activity.
a
RP-HPLC retention time.
Percentage determined by RP-HPLC at 210 nm of pure compounds.
b
The activity on non-malignant human cells (fibroblasts N1) was
also assessed for all the compounds at 10 and 30
peptides were non toxic, and only BP77 caused significant growth
inhibition at 30 M (73%). These values correlate with the low
hemolytic activity found for these peptides in previous studies
(0e6% at 50 M; 8e40% at 150
M).14 Modification of the peptides
with PEG-1 resulted in an increase of the cytotoxicity against N1
cells. The percentage of growth inhibition at 30 M was of 79% for
mM. CECMEL11
2.2. Cytotoxicity
m
The anticancer activity of the CECMEL11 undecapeptides and
that of the undecapeptides/PEG-1 conjugates was screened in
MDA-MB-231 human breast adenocarcinoma cells by determining
m
m
the growth inhibition percentage at 10 and 30
mM peptide con-
m
centration using the standard 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT) assay.18 MTT is a fast, sensi-
tive, and economical assay that allows to evaluate the anti-
proliferation properties of different compounds. Growth inhibition
values are summarized in Table 2.
The CECMEL11 undecapeptides showed low activity against
breast carcinoma cells at 10 mM. They produced an inhibition per-
centage of cell growth between 5 and 39%, being the most active
compounds BP76 and the N-terminal derivatized peptides BP77,
BP125, and BP126 (24e39%). The inhibition percentage of these
Ac-PEG-1 and of >90% for all peptide conjugates, pointing out that
the toxicity could be mainly attributed to PEG-1. Interestingly, at
10
mM the peptide conjugates were not significantly cytotoxic
against non-malignant cells. A growth inhibition <10% was ob-
served for four sequences and only two conjugates displayed an
inhibition percentage >20%. Notably, BP77-PEG-1 only produced
a 17% of growth inhibition. This low toxicity together with the
marked anticancer activity displayed by this peptide conjugate,
suggest that BP77-PEG-1 can be considered a proof-of-concept for
future studies that will explore the use of these strategy to produce
new anticancer conjugates that combine other anticancer peptides
four peptides increased up to 51e81% at 30
mM. The highest activity
was observed for BP77 (81%). These results pointed out that, in
with peptide 1 or related g-peptides with CPPs properties.

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C. Roses et al. / Tetrahedron 68 (2012) 4406e4412
4410
Table 2
Cytotoxicity against MDA-MB-231 cells and non-malignant fibroblasts (N1)
Peptide
MDA-MB-231 growth
inhibitiona (%)
N1 growth inhibitiona (%)
Peptide
conjugate
MDA-MB-231 growth
inhibitiona (%)
N1 growth inhibitiona (%)
10
m
M
30
m
M
10
m
M
30
m
M
10
m
M
30
m
M
10
m
M
30 mM
Ac-PEG-1
3ꢂ0.06
34ꢂ0.06
49ꢂ0.06
49ꢂ0.04
71ꢂ0.02
49ꢂ0.09
59ꢂ0.06
47ꢂ0.13
93ꢂ0.01
93ꢂ0.01
92ꢂ0.01
92ꢂ0.01
92ꢂ0.01
92ꢂ0.01
92ꢂ0.01
92ꢂ0.01
nac
79ꢂ0.08
92ꢂ0.01
93ꢂ0.01
93ꢂ0.01
93ꢂ0.01
93ꢂ0.01
93ꢂ0.01
93ꢂ0.01
BP15
BP33
BP76
BP77
BP101
BP125
BP126
5ꢂ0.04
5ꢂ0.02
39ꢂ0.09
32ꢂ0.03
db
12ꢂ0.03
16ꢂ0.01
51ꢂ0.03
81ꢂ0.02
d
nac
nac
nac
nac
d
2ꢂ0.14
nac
BP15-PEG-1
BP33-PEG-1
BP76-PEG-1
BP77-PEG-1
BP101-PEG-1
BP125-PEG-1
BP126-PEG-1
16ꢂ0.01
9ꢂ0.05
9ꢂ0.09
17ꢂ0.14
28ꢂ0.10
25ꢂ0.07
nac
nac
73ꢂ0.07
d
28ꢂ0.02
24ꢂ0.05
62ꢂ0.02
61ꢂ0.05
nac
5ꢂ0.16
16ꢂ0.10
nac
a
Percentage of cell growth inhibition plus standard deviation. Values were obtained from duplicate experiments performed in triplicate.
Not determined.
na: non active.
b
c
3. Conclusions
Dihydroxybenzoic acid (DHB) was used as a matrix and was pur-
chased from Aldrich.
In summary, we have showed that by combining an antimicro-
bial peptide and a cell-penetrating -peptide effective anticancer
g
4.2. General method for solid-phase synthesis of CECMEL11
undecapeptides
agents are obtained. The efficacy of CECMEL11 undecapeptides to-
ward breast cancers cells was several folds higher when conjugated
to PEG-1 as compared to treatment with antimicrobial peptides
alone. This result suggests that the covalent linking to PEG-1 was
able to successfully deliver peptides into the cancer cells, enhancing
their efficiency. Moreover, the most active peptide conjugate BP77-
PEG-1 also showed low toxicity against normal cells. These results
constitute a proof of the usefulness of these g-peptides as cellular
transporters and open the possibility to conjugate them to other
therapeutic peptides with the aim to improve their action.
Peptides were synthesized manually by the solid-phase method
using standard Fmoc chemistry. Fmoc-Rink-MBHA resin
(0.56 mmol/g) was used as solid support. Couplings of the corre-
sponding amino acids Fmoc-Leu-OH, Fmoc-Phe-OH, Fmoc-
Lys(Boc)-OH, Fmoc-Ile-OH, Fmoc-Val-OH, and Fmoc-Tyr(t-Bu)-OH
(4 equiv) were performed using ethyl cyanoglyoxylate-2-oxime
(4 equiv), N,N0-diisopropylcarbodiimide (DIPCDI) (4 equiv) in DMF
at room temperature for 1 h. The completion of the reactions was
monitored by the Kaiser test.19 Fmoc group removal was achieved
with piperidine/DMF (3:7, 1ꢁ2 minþ1ꢁ10 min). After each cou-
pling and deprotection step, the resin was washed with DMF
(6ꢁ1 min) and CH2Cl2 (3ꢁ1 min) and air-dried. Once the peptide
sequence was completed, the Fmoc group was removed and, when
required, the resulting peptidyl resin was derivatized. Acidolytic
cleavage was performed by treatment of the resin with TFA/H2O/
triisopropylsilane (TIS) (95:2.5:2.5) for 2 h at room temperature.
Following TFA evaporation and Et2O extraction, the crude peptide
was dissolved in H2O/CH3CN, lyophilized, analyzed by HPLC, and
characterized by MS.
4. Experimental section
4.1. General methods
Commercially available reagents were used throughout without
purification. PEG-1, Ac-PEG-1, and peptide conjugates were ana-
lyzed under standard analytical RP-HPLC conditions with a Waters
liquid chromatography instrument equipped with a UV/vis detector.
Analysis was carried out with a Symmetry C18 (150 mmꢁ4.6 mm,
5 mm) column using a 0e100% B linear gradient over 15 min at a flow
rate of 1 mL/min. Solvent A was 0.1% aq TFA, and solvent B was 0.1%
TFA in CH3CN. Detection was performed at 210 nm.
4.2.1. BP15 (KKLFKKILKVL-NH2). Starting from resin Fmoc-Rink-
MBHA (50 mg), sequential couplings of the corresponding Fmoc-
protected amino acids and removal of the Fmoc group, followed
by acidolytic cleavage afforded BP15 (24 mg, 89% purity).
tR¼4.39 min. MS (ESI): m/z¼1356.83 [MþH]þ.
Undecapeptides were analyzed under standard analytical HPLC
conditions with a Dionex liquid chromatography instrument com-
posed of a UV/vis Dionex UVD170U detector, a P680 Dionex bomb,
an ASI-100 Dionex automatic injector, and CHROMELEON 6.60
software. Detection was performed at 220 nm. Analysis was carried
out with a Kromasil 100 C18 (40 mmꢁ4.6 mm, 3.5
m
m) column with
4.2.2. BP33 (LKLFKKILKVL-NH2). Starting from resin Fmoc-Rink-
MBHA (50 mg), sequential couplings of the corresponding Fmoc-
protected amino acids and removal of the Fmoc group, followed
by acidolytic cleavage afforded BP33 (26 mg, 92% purity).
tR¼4.46 min. MS (ESI): m/z¼1341.81 [MþH]þ.
a 2e100% B linear gradient over 7 min at a flow rate of 1 mL/min.
Solvent A was 0.1% aq TFA, and solvent B was 0.1% TFA in CH3CN.
UndecapeptidesESI-MSanalyses were performed with anEsquire
6000 ESI ion Trap LC/MS (Bruker Daltonics) instrument equipped
with an electrospray ion source. The instrument was operated in the
positive ESI(þ) ion mode. Samples (5
mass spectrometer ion source directly through an HPLC autosampler.
The mobile phase (80:20 CH3CN/H2O at a flow rate of 100 L/min)
was delivered by a 1100 Series HPLC pump (Agilent). Nitrogen was
employed as both the drying and nebulizing gas.
m
L) were introduced into the
4.2.3. BP76 (KKLFKKILKFL-NH2). Starting from resin Fmoc-Rink-
MBHA (50 mg), sequential couplings of the corresponding Fmoc-
protected amino acids and removal of the Fmoc group, followed
by acidolytic cleavage afforded BP76 (20 mg, 82% purity).
tR¼4.20 min. MS (ESI): m/z¼1404.87 [MþH]þ.
m
PEG-1 and the conjugates HPLC-ES analyses were performed on
Waters Alliance 2695 chromatography system equipped with Wa-
ters 995 photodiode array detector and ESI-MS Waters Micromass
ZQ on a Symmetry column with linear gradients of B: MeCN (with
0.1% FA) in A: H2O (with 0.1% FA) at a flow rate of 1 mL/min.
Mass spectra were recorded on a MALDI Voyager DE RP time-of-
flight (TOF) spectrometer (Applied Biosystems, Framingham). 2,5-
4.2.4. BP77 (Ac-KKLFKKILKFL-NH2). Synthesis started from resin
Fmoc-Rink-MBHA (50 mg) and after sequential couplings of the
corresponding Fmoc-protected amino acids and removal of the
Fmoc group, the resulting resin was treated with acetic anhy-
drideepyridineeCH2Cl2 (1:1:1, 2ꢁ30 min) and then washed with
CH2Cl2 (3ꢁ2 min), NMP (3ꢁ2 min) and CH2Cl2 (6ꢁ1 min).

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C. Roses et al. / Tetrahedron 68 (2012) 4406e4412
4411
Acidolytic cleavage afforded BP77 (19 mg, 75% purity). tR¼4.72 min.
MS (ESI): m/z¼1446.91 [MþH]þ.
Part of the PEG-1-MBHA resin (100 mg) was treated with acetic
anhydrideepyridineeCH2Cl2 (1:1:1, 2ꢁ30 min) and then washed
with CH2Cl2 (3ꢁ2 min), NMP (3ꢁ2 min), and CH2Cl2 (6ꢁ1 min).
Acidolytic cleavage of the resulting resin afforded Ac-PEG-1.
4.2.5. BP101 (Ac-KKLFKKILKYL-NH2). Synthesis started from resin
Fmoc-Rink-MBHA (50 mg) and after sequential couplings of the
corresponding Fmoc-protected amino acids and removal of the
Fmoc group, the resulting resin was treated with acetic anhy-
drideepyridineeCH2Cl2 (1:1:1, 2ꢁ30 min) and then washed with
CH2Cl2 (3ꢁ2 min), NMP (3ꢁ2 min), and CH2Cl2 (6ꢁ1 min). Acid-
olytic cleavage afforded BP101 (22 mg, 82% purity). tR¼4.56 min.
MS (ESI): m/z¼1462.91 [MþH]þ.
4.4. General method for solid-phase synthesis of peptide
conjugates
PEG-1-MBHA resin was divided on seven parts and on each one
was built one of the antimicrobial peptides selected following
a Fmoc/t-Bu strategy on a standard solid-phase peptide synthesis.
Couplings of the corresponding amino acids Fmoc-Leu-OH, Fmoc-
Phe-OH, Fmoc-Lys(Boc)-OH, Fmoc-Ile-OH, Fmoc-Val-OH, and
Fmoc-Tyr(t-Bu)-OH (4 equiv) were performed using ethyl cyano-
glyoxylate-2-oxime (4 equiv), DIPCDI (4 equiv) in NMP at room
temperature for 3 h. The completion of the reactions was moni-
tored by the Kaiser test.19 Fmoc group removal was achieved with
piperidine/NMP (3:7, 1ꢁ2þ2ꢁ10 min). After each coupling and
deprotection step, the resin was washed with NMP (6ꢁ1 min) and
CH2Cl2 (3ꢁ1 min) and air-dried. Once the peptide sequence was
completed, the N-terminal Fmoc group was removed and, when
required, the resulting peptidyl resin was derivatized.
Acidolytic cleavage was performed by treating the resin with HF
in the presence of 10% anisole for 1 h at 0 ꢃC. Peptides were pre-
cipitated with cold anhydrous TMBE, dissolved in HOAc, and then
lyophilized. Crude peptides were purified by preparative HPLC
using a linear gradient of CH3CN (containing 1% of TFA) and H2O
(containing 1% of TFA). The purity of each fraction was verified by
analytical HPLC, HPLC-MS, and MALDI-TOF.
4.2.6. BP125 (Ts-KKLFKKILKKL-NH2). Synthesis started from resin
Fmoc-Rink-MBHA (50 mg) and after sequential couplings of the
corresponding Fmoc-protected amino acids and removal of the
Fmoc group, the resulting resin was treated with p-toluenesulfonyl
chloride (40 equiv) and DIEA (80 equiv) in CH2Cl2eNMP (9:1) for
1 h and then washed with CH2Cl2 (3ꢁ2 min), NMP (3ꢁ2 min), and
CH2Cl2 (6ꢁ1 min). Acidolytic cleavage afforded BP125 (18 mg, 76%
purity). tR¼4.53 min. MS (ESI): m/z¼1540.06 [MþH]þ.
4.2.7. BP126 (Bz-KKLFKKILKKL-NH2). Synthesis started from resin
Fmoc-Rink-MBHA (50 mg) and after sequential couplings of the
corresponding Fmoc-protected amino acids and removal of the
Fmoc group, the resulting resin was treated with benzoyl chloride
(40 equiv) and DIEA (80 equiv) in CH2Cl2eNMP (9:1) for 1 h and
then washed with CH2Cl2 (3ꢁ2 min), NMP (3ꢁ2 min), and CH2Cl2
(6ꢁ1 min). Acidolytic cleavage afforded BP126 (19 mg, 77% purity).
tR¼4.63 min. MS (ESI): m/z¼1489.98 [MþH]þ.
4.3. PEG-1-MBHA resin synthesis
4.4.1. BP15-PEG-1 (KKLFKKILKVL-PEG-1). Starting from resin PEG-
1-MBHA (100 mg), sequential couplings of the corresponding
Fmoc-protected amino acids and removal of the Fmoc group, fol-
lowed by acidolytic cleavage afforded BP15-PEG-1, which was pu-
rified by preparative HPLC (8 mg, 93% purity). MS (MALDI-TOF):
PEG-1 peptidyl resin was synthesized manually by the solid-
phase method using MBHA resin (500 mg, 0.63 mmol/g) as solid
support following a combined Fmoc/Boc/Alloc strategy. Peptide
synthesis was performed manually in a polypropylene syringe fitted
with a polyethylene porous disc. Solvents and soluble reagents were
removed by suction. Washings between deprotection, coupling, and
subsequentdeprotection steps were carriedout withDMF(5ꢁ1 min)
and DCM (5ꢁ1 min) using 10 mL solvent/g resin for each wash.
Two units of Fmoc-8-amine-3,6-dioxaoctanoic acid were coupled
consecutively to the resin using standard DIPCDI/HOBt coupling
system (3 equiv). Then, alternate incorporations of Fmoc-Amp(Boc)-
OH (3 equiv) and Fmoc-Amp(Alloc)-OH (3 equiv) were carried out
with DIPCDI (3 equiv) and ethyl cyanoglyoxyl-2-oxime (3 equiv) in
DMF for 2 h. The resin was washed with DMF (5ꢁ1 min) and DCM
(5ꢁ1 min) after each coupling. Couplings were monitored by the
m/z¼747.95 [Mþ4H]4þ, 996.51 [Mþ3H]3þ
.
4.4.2. BP33-PEG-1 (LKLFKKILKVL-PEG-1). Starting from resin PEG-
1-MBHA (100 mg), sequential couplings of the corresponding
Fmoc-protected amino acids and removal of the Fmoc group, fol-
lowed by acidolytic cleavage afforded BP33-PEG-1, which was
purified by preparative HPLC (16 mg, 95% purity). MS (MALDI-TOF):
m/z¼744.11 [Mþ4H]4þ, 991.84 [Mþ3H]3þ
.
4.4.3. BP76-PEG-1 (KKLFKKILKFL-PEG-1). Starting from resin PEG-
1-MBHA (100 mg), sequential couplings of the corresponding
Fmoc-protected amino acids and removal of the Fmoc group, fol-
lowed by acidolytic cleavage afforded BP76-PEG-1, which was pu-
rified by preparative HPLC (12 mg, 96% purity). MS (MALDI-TOF):
Kaiser test.19 Once the alternate Boc/Alloc
a-amino protected skele-
ton had been built, the Na-Boc protecting group was removed, al-
kylation of the amino group was performed by on-resin reductive
amination using phenylacetaldehyde (5 equiv for each amine) in 1%
HOAc in DMF for 30min and then treating the resin with NaBH3CN
(5 equiv for each amine) in MeOH for 2 h. After that, the resin was
washed with DMF (5ꢁ1 min) and DCM (5ꢁ1 min). Alkylation was
monitored by chloranil test.20 Then, Na-Alloc protecting group was
removed with Pd(PPh3)3/PhSiH (0.1:10) (2ꢁ10 min in DCM) and al-
kylation with isovaleraldehyde was performed as above. After re-
moval of last Fmoc protecting group (which provided 1-MBHA) an
aliquot of PEG-1-MBHA was cleaved to check the peptidyl resin
quality by acidolytic cleavage with HFand PEG-1 wasobtained, which
was dissolved in H2O/CH3CN, lyophilized and analyzed by HPLC (88%
purity). tR¼6.28 min. MS (MALDI-TOF): m/z¼MS (MALDI-TOF):
m/z¼1503.85 [MþH]þ, 1525.85 [MþNa]þ. ESI found: 752.8; 502.3.
Finally, an additional unit of Fmoc-8-amine-3,6-dioxaoctanoic
acid was ad added to the 1-MBHA resin as described above ren-
dering PEG-1-MBHA-resin.
m/z¼759.92 [Mþ4H]4þ, 1012.82 [Mþ3H]3þ
.
4.4.4. BP77-PEG-1 (Ac-KKLFKKILKFL-PEG-1). Synthesis started
from resin PEG-1-MBHA (100 mg) and after sequential couplings of
the corresponding Fmoc-protected amino acids and removal of the
Fmoc group, the resulting resin was treated with acetic anhy-
drideepyridineeCH2Cl2 (1:1:1, 2ꢁ30 min) and then washed with
CH2Cl2 (3ꢁ2 min), NMP (3ꢁ2 min) and CH2Cl2 (6ꢁ1 min). Acid-
olytic cleavage afforded BP77-PEG-1, which was purified by pre-
parative HPLC (18 mg, 99% purity). MS (MALDI-TOF): m/z¼770.40
[Mþ4H]4þ, 1026.96 [Mþ3H]3þ
.
4.4.5. BP101-PEG-1 (Ac-KKLFKKILKYL-PEG-1). Synthesis started
from resin PEG-1-MBHA (100 mg) and after sequential couplings of
the corresponding Fmoc-protected amino acids and removal of the
Fmoc group, the resulting resin was treated with acetic anhy-
drideepyridineeCH2Cl2 (1:1:1, 2ꢁ30 min) and then washed with

ꢀ
C. Roses et al. / Tetrahedron 68 (2012) 4406e4412
4412
CH2Cl2 (3ꢁ2 min), NMP (3ꢁ2 min) and CH2Cl2 (6ꢁ1 min). Acid-
olytic cleavage afforded BP101-PEG-1, which was purified by pre-
parative HPLC (2 mg, 95% purity). MS (MALDI-TOF): m/z¼774.29
CTQ2009-07758 (LF), and CIT-090000-2008-10 (TP)), the ‘Gen-
eralitat de Catalunya’ (2009SGR 1024), the Institute for Research
in Biomedicine, and the Barcelona Science Park, and the CIBER-
BBN, Networking Centre on Bioengineering, Biomaterials, and
Nanomedicine. D.C. is a PCB fellow and C.R. and A.B. were re-
cipient of a predoctoral fellowship from the MICINN and Uni-
versity of Girona, respectively. Financial support was also
provided by grant from the University of Girona (Grant for RþD
Projects of Health Sciences).
[Mþ4H]4þ, 1032.29 [Mþ3H]3þ
.
4.4.6. BP125-PEG-1 (Ts-KKLFKKILKKL-PEG-1). Synthesis started
from resin PEG-1-MBHA (100 mg) and after sequential couplings of
the corresponding Fmoc-protected amino acids and removal of the
Fmoc group, the resulting resin was treated with p-toluenesulfonyl
chloride (40 equiv) and DIEA (80 equiv) in CH2Cl2eNMP (9:1) for
1 h and then washed with CH2Cl2 (3ꢁ2 min), NMP (3ꢁ2 min), and
CH2Cl2 (6ꢁ1 min). Acidolytic cleavage afforded BP125-PEG-1,
which was purified by preparative HPLC (17 mg, 98% purity). MS
References and notes
(MALDI-TOF): m/z¼793.70 [Mþ4H]4þ, 1057.99 [Mþ3H]3þ
.
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4.4.7. BP126-PEG-1 (Bz-KKLFKKILKKL-PEG-1). Synthesis started
from resin PEG-1-MBHA (100 mg) and after sequential couplings of
the corresponding Fmoc-protected amino acids and removal of the
Fmoc group, the resulting resin was treated with benzoyl chloride
(40 equiv) and DIEA (80 equiv) in CH2Cl2eNMP (9:1) for 1 h and
then washed with CH2Cl2 (3ꢁ2 min), NMP (3ꢁ2 min), and CH2Cl2
(6ꢁ1 min). Acidolytic cleavage afforded BP126-PEG-1, which was
purified by preparative HPLC (18 mg, 98% purity). MS (MALDI-TOF):
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.
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4.5. Cell lines and cell culture
MDA-MB-231 human breast Caucasian adenocarcinoma cells
were obtained from the ATCC (American Type Culture Collection
Rockville, MD, USA). Cells were routinely grown in Dulbecco’s
modified Eagle’s medium (DMEM, Gibco, Berlin, Germany) con-
taining 10% fetal bovine serum (FBS, Bio-Whittaker, Walkersville,
MD, USA), 1%
L-glutamine, 1% sodium pyruvate, 50 U/ml penicillin,
and 50 g/ml streptomycin (Gibco). The cells remained free of
m
Mycoplasma and were propagated in adherent culture according to
established protocols. Cells were maintained at 37 ꢃC in a humidi-
fied atmosphere of 95% air and 5% CO2.
10. (a) Iriondo-Alberdi, J.; Laxmi-Reddy, K.; Bouguerne, B.; Staedel, C.; Huc, I.
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4.6. Cell growth inhibition assay
Peptides were dissolved in sterile Phosphate Saline Buffer (PBS)
12. (a) Farrera-Sinfreu, J.; Zaccaro, L.; Vidal, D.; Salvatella, X.; Giralt, E.; Pons, M.;
Albericio, F.; Royo, M. J. Am. Chem. Soc. 2004, 126, 6048; (b) Farrera-Sinfreu, J.;
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to get a final stock solution of 3200 mM. Sensitivity was determined
using a standard colorimetric MTT assay. Briefly, MDA-MB-231 cells
were plated out at a density of 7ꢁ103 cells/100
ml/well in 96-well
microtiter plates and allowed an overnight period for attachment.
Then, the medium was removed and fresh medium along with the
corresponding peptide concentration (10 or 30 mM) were added to
the cultures. Agents were not renewed during the 48 h of cell ex-
posure, and control cells without agents were cultured under the
same conditions. Following treatment, cells were fed with drug-free
medium (100 ml/well) and MTT solution (10 mL, 5 mg/ml in PBS), and
incubation was prolonged for 3 h at 37 ꢃC. After carefully removing
the supernatants, the MTT-formazan crystals formed by metaboli-
cally viable cells were dissolved in dimethyl sulfoxide (DMSO,100 ml/
ꢀ
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plate reader (Model SPECTRAmax 340 PC (380) reader). Using con-
trol optical density (OD) valuesÓ, test OD values (T), and time zero OD
values (T0), the growth inhibition (IC value) at every concentration
was calculated from the equation, 100ꢁ[(TꢄT0)/(CꢄT0)]¼IC.
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Acknowledgements
This work was partially supported by the Spanish Ministry
of Science and Innovation (MICINN) (CTQ2008-00177 (MR),
20. Christensen, T. Acta Chem. Scand. 1979, 33, 760.