Multivalent display of the antimicrobial peptides BP100 and BP143

Article abstract of DOI:10.3762/bjoc.8.237

Carbohydrates are considered as promising templates for the display of multiple copies of antimicrobial peptides. Herein, we describe the design and synthesis of chimeric structures containing two or four copies of the antimicrobial peptides KKLFKKILKYL-N

Full text of DOI:10.3762/bjoc.8.237

Multivalent display of the antimicrobial peptides
BP100 and BP143
Imma Güell1,§, Rafael Ferre1,§, Kasper K. Sørensen2,§, Esther Badosa3,
Iteng Ng-Choi1, Emilio Montesinos3, Eduard Bardají1, Lidia Feliu1,
Knud J. Jensen*2 and Marta Planas*1
Full Research Paper
Open Access
Address:
Beilstein J. Org. Chem. 2012, 8, 2106–2117.
1LIPPSO, Department of Chemistry, University of Girona, Campus
Montilivi, 17071 Girona, Spain, 2IGM, Faculty of Life Sciences
University of Copenhagen, DK-1871 Frederiksberg, Denmark and
3Laboratory of Plant Pathology, Institute of Food and Agricultural
Technology-CIDSAV-CeRTA, University of Girona, Campus Montilivi,
17071 Girona, Spain
doi:10.3762/bjoc.8.237
Received: 27 July 2012
Accepted: 07 November 2012
Published: 03 December 2012
Associate Editor: K. N. Ganesh
Email:
Knud J. Jensen* - kjj@life.ku.dk;
Marta Planas* - marta.planas@udg.edu
© 2012 Güell et al; licensee Beilstein-Institut.
License and terms: see end of document.
* Corresponding author
§ Equally contributing authors
Keywords:
antimicrobial activity; carbopeptides; multimeric structures; oxime
ligation; phytopathogenic bacteria
Abstract
Carbohydrates are considered as promising templates for the display of multiple copies of antimicrobial peptides. Herein, we
describe the design and synthesis of chimeric structures containing two or four copies of the antimicrobial peptides
KKLFKKILKYL-NH2 (BP100) and KKLfKKILKYL-NH2 (BP143) attached to the carbohydrate template cyclodithioerythritol
(cDTE) or α-D-galactopyranoside (Galp). The synthesis involved the preparation of the corresponding peptide aldehyde followed
by coupling to an aminooxy-functionalized carbohydrate template. After purification, the multivalent display systems were obtained
in high purities (90–98%) and in good yields (42–64%). These compounds were tested against plant and human pathogenic bacteria
and screened for their cytotoxicity on eukaryotic cells. They showed lower MIC values than the parent peptides against the bacteria
analyzed. In particular, the carbopeptides derived from cDTE and Galp, which contained two or four copies of BP100, respectively,
were 2- to 8-fold more active than the monomeric peptide against the phytopathogenic bacteria. These results suggest that
preassembling antimicrobial peptides to multimeric structures is not always associated with a significant improvement of the
activity. In contrast, the carbopeptides synthesized were active against human red blood cells pointing out that peptide preassembly
is critical for the hemolytic activity. Notably, peptide preassembly resulted in an enhanced bactericidal effect.
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Introduction
The emergence of antibiotic resistance is a major problem in chemical manipulation [23-25]. Jensen and co-workers reported
human health as well as in agronomy and there is a consider- the synthesis of carbopeptides and carboproteins bearing several
able current interest in developing novel antimicrobial com- copies of a peptide or a protein tethered in a carbohydrate
pounds [1,2]. In recent years, a growing number of studies have template. They developed an efficient strategy for the synthesis
shown that antimicrobial peptides constitute a potential alter- of these carboproteins, in which C-terminal peptide aldehydes
native to traditional antibiotics. To date, a very large number of are ligated by oxime bond formation to aminooxyacetyl-func-
antimicrobial peptides have been produced based on naturally tionalized monosaccharide templates, such as cyclodithioery-
occurring products or by de novo design, and most of them thritol (cDTE) or α-D-galactopyranoside (Galp) [23,26,27].
display a broad spectrum of antimicrobial activities [3,4].
Despite their remarkable structural diversity, these peptides Here we describe the use of carbohydrate templates for the
share several characteristics: the sequence length is typically display of multiple copies of antimicrobial peptides. Our aim in
between 12 to 59 amino acids, they bear a positive charge of +2 this study was to evaluate whether the assembly of cecropin
to +9, and they show an amphipathic character. The electro- A-melittin antimicrobial undecapeptide hybrids on a carbohy-
static interaction of antimicrobial peptides with membranes is a drate template could provide carbopeptides with improved
key factor in determining their biological activity [1,3,5-8]. For biological properties. In particular, KKLFKKILKYL-NH2
many of these peptides, membrane disruption is considered the (BP100) and KKLfKKILKYL-NH2 (BP143) were selected
primary mechanism of cell death. Although a general under- based on their high antibacterial activity against the plant patho-
standing has been achieved, the precise mechanism of genic bacteria Erwinia amylovora, Xanthomonas axonopodis
peptide–membrane interaction and cell killing has not been pv. vesicatoria and Pseudomonas syringae pv. syringae [28,29].
firmly established for many peptides. Several models have been BP100 displayed minimum inhibitory concentration (MIC)
proposed to account for the morphologic changes in the values in the range 2.5–7.5 μM and also showed low hemolysis
membrane induced by antimicrobial peptides, such as pore for- (22% at 150 μM). BP143, which contains one D-amino acid,
mation, lysis or peptide translocation into the cytoplasm. Either was as active, significantly less hemolytic (2% at 150 μM), and
model relies on relatively high local density and synergy of more stable to protease degradation than BP100. Thus,
monomeric peptides.
carbopeptides 1–3 were designed and synthesized by linking
two or four copies of BP100 and BP143 on a cDTE or a Galp
It has occasionally been reported that an improved binding to template (Figure 1). The antibacterial and hemolytic activities
microbial membranes and thus an improved antimicrobial and the bactericidal effect of the carbopeptides 1–3 were
activity can be achieved if several copies of an antimicrobial analyzed and will be discussed.
peptide are linked together to form multimeric species [9-15].
Results
The hypothesis of the assembly model is based on prenucle-
ating a number of appropriately spaced and oriented antimicro- Design and synthesis of the carbopeptides
bial peptide monomers in order to reduce the threshold concen- Two-stranded and four-stranded carbopeptides 1–3 were
tration required for their activity. The interaction of these multi- designed by coupling of two or four copies of a C-terminal
valent species should a priori make the interaction with the undecapeptide aldehyde to an aminooxy-functionalized
microbial membrane strongly and entropically more favorable. carbohydrate template (Figure 1). The previously reported
Representative studies include multimeric derivatives of alame- cyclodithioerythritol (cDTE) was used as a template for the
thicin [16] and the dimeric antimicrobial peptide A3-APO [17]. two-stranded carbopeptides 1 and 2 [23,26,27]. The α-D-
Moreover, studies of multivalent antimicrobial peptides are of galactopyranoside (Galp) template was required to obtain the
growing importance because they could represent an interesting four-stranded carbopeptide 3. The sequence of the peptide alde-
tool to provide insights into the modes of action of antimicro- hydes was based on the peptides KKLFKKILKYL-NH2
bial peptides, which are generally poorly understood.
(BP100) and KKLfKKILKYL-NH2 (BP143), which have been
shown to display high antibacterial activity [28,29].
Several scaffolds, such as linear and cyclic peptides, alkyne-
functionalized dendrimers, a branched lysine core, and also a Templates cDTE and Galp functionalized with aminooxyacetic
polymaleic polymer, have been exploited as templates for the acid were prepared as before [23,30]. Peptide aldehydes
synthesis of multivalent antimicrobial peptides [18-22]. Carbo- KKLFKKILKYLG-H (4) and KKLfKKILKYLG-H (5), derived
hydrates are promising candidates as templates for the display from BP100 and BP143, respectively, were prepared by using a
of functional groups due to their inherent multifunctionality, the standard backbone amide linker (BAL) strategy (Scheme 1).
relative rigidity of their structure, and the ease of regioselective PALdehyde was coupled onto an amino-functionalized
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Figure 1: Structure of the carbopeptides 1–3.
TentaGel (TG) resin followed by reductive amination with isopropylcarbodiimide (DIPCDI) with microwave heating at
aminoacetaldehyde dimethyl acetal, which served as a precursor 60 °C. Peptide chain elongation followed standard 9-fluorenyl-
for the C-terminal glycinal residue. Next, acylation of the sec- methoxycarbonyl (Fmoc) solid-phase chemistry. After chain
ondary nitrogen with Fmoc-Leu-OH was mediated by N,N’-di- assembly was completed, peptides were released from the
Scheme 1: Solid-phase synthesis of the peptide aldehydes 4 and 5.
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support by treatment with trifluoroacetic acid (TFA), which With templates and peptide aldehydes in hand, the four-
removed the tert-butyl (t-Bu)/tert-butyloxycarbonyl (Boc) stranded structure 3 was prepared from Galp and peptide alde-
groups and also hydrolyzed the acetal to the required aldehyde. hyde 4 (Scheme 2). First, the reaction was conducted in a 1:1
When the cleavage was performed by using TFA/triisopropylsi- solution of CH3CN and acetate buffer (0.1 M, pH 4.76) at room
lane (TIS)/H2O (95:2.5:2.5), the corresponding peptide alcohol temperature for 2 h, but the expected carbopeptide 3 was not
was obtained. It has been previously reported that silanes under obtained. Since the use of aniline as nucleophilic catalyst for
acidic conditions can promote the reduction of aldehydes [27]. oxime ligation has been described to enhance reaction rates by
Cleavage of the peptidyl resins with TFA/H2O (95:5) followed up to three orders of magnitude [31-33], we assayed the forma-
by purification by reversed-phase high-performance liquid chro- tion of 3 using the previous CH3CN/acetate buffer solution but
matography (RP-HPLC) afforded peptide aldehydes 4 and 5 in containing aniline (100 mM). Under these conditions we could
99% purity, and their structure was confirmed by electrospray- not detect the carbopeptide 3 even when increasing the reaction
ionization mass spectrometry (ESIMS).
time up to 48 h. Results were improved by lyophilizing the
Scheme 2: Synthesis of the carbopeptides 1–3.
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template prior to the oxime ligation. In this case, the reaction (MIC of 5 to 10 μM). The cDTE-containing carbopeptide 1 was
afforded 3 with or without the presence of aniline in the solvent two- to eight-fold (MIC of 0.6 to 5 μM) more active than
mixture. Best conditions involved the treatment of lyophilized BP100. In contrast, carbopeptide 2 derived from cDTE and
Galp with peptide aldehyde 4 in (1:1) CH3CN/acetate buffer for BP143 showed similar activity to the parent peptide (MIC of
4 h, followed by the addition of an extra amount of 4 dissolved 2.5 to 5 μM). Against the human pathogenic bacteria, the three
in (1:1) CH3CN/acetate buffer containing aniline (100 mM). carbopeptides 1–3 showed higher activity than the corres-
The reaction was completed after 2.5 h affording 3, which was ponding parent peptide, with E. coli and S. enterica being the
purified by RP-HPLC (90% purity, 64% yield) and character- most sensitive pathogens (MIC of 1.2 to 5 μM).
ized by mass spectrometry.
The toxicity of carbopeptides 1–3 to eukaryotic cells was also
The synthesis of carbopeptide 1 was achieved by oxime liga- evaluated and it was determined as the ability to lyse erythro-
tion of cDTE, previously lyophilized, with peptide aldehyde 4 cytes in comparison to melittin. Percent hemolysis at 50 and
in a 1:1 solution of CH3CN and acetate buffer (0.1 M, pH 4.76) 150 μM is included in Table 1. Results showed that the parent
containing aniline (100 mM) at room temperature for 3 h. The peptides BP100 and BP143 were not hemolytic at these concen-
crude mixture was purified yielding 1 in 98% purity and 42% trations. In contrast, carbopeptides 1–3 were able to lyse
yield. This methodology was extended to the preparation of erythrocytes at both concentrations suggesting that peptide
carbopeptide 2 derived from peptide aldehyde 5 with one preassembly is critical for the hemolytic activity.
D-amino acid in the sequence. The reaction was followed by
HPLC being completed after 3 h. After purification, carbopep- Moreover, we analyzed the bactericidal effect of carbopeptides
tide 2 was obtained in 98% purity and 62% yield. The structure 1–3 against E. amylovora and S. enterica by comparing the time
of carbopeptides 1 and 2 was confirmed by mass spectrometry.
course to kill suspensions of mid-logarithmic-phase cultures of
these bacterial strains. As shown in Figure 2, the monomeric
peptides BP100 and BP143 showed a bactericidal effect against
Biological activity
Carbopeptides 1–3 were tested for in vitro growth inhibition of both strains, but it was slower against S. enterica. Both peptides
phytopathogenic bacteria Erwinia amylovora, Xanthomonas exhibited an initial fast killing stage of 30–60 min, followed by
axonopodis pv. vesicatoria, and Pseudomonas syringae pv. a slower rate against E. amylovora. Notably, carbopeptides 1–3
syringae, and of human pathogenic bacteria Escherichia coli, were significantly more active against both pathogens and were
Staphylococcus aureus, Lysteria monocytogenes, and Salmo- able to kill 99.99% of all cells within 30–90 min at 2.5 μM.
nella enterica at 0.6, 1.2, 2.5, 5, 7.5, 10 and 20 μM (Table 1).
The antibacterial activity of BP100 and BP143 was also evalu- Circular dichroism
ated for comparison purposes.
We investigated the secondary structures of carbopeptides 1 and
3 by analyzing their CD spectra at 100 μM in (i) 10 mM sodium
Against phytopathogenic bacteria, carbopeptides 1 and 3 phosphate buffer at pH 7.4; (ii) 50% (v/v) trifluoroethanol
displayed higher activity than the parent peptide BP100. (TFE) in 10 mM sodium phosphate buffer at pH 7.4; and (iii)
Carbopeptide 3, derived from Galp, was the most active with 10 mM SDS in 10 mM sodium phosphate buffer at pH 7.4
minimum inhibitory concentration (MIC) values from four- to (Figure 3). Carbopeptides 1 and 3 had similar CD spectra. In
eight-fold lower (MIC of 0.6 to 2.5 μM) than those of BP100 phosphate buffer they displayed a disordered structure while in
Table 1: Antibacterial activity (MIC) and cytotoxicity of parent peptides and carbopeptides.
MIC (μM)
Ecb
Hemolysisa (%)
Compound
Eab
Xavb
Pssb
Sab
Lmb
Seb
50 μM
150 μM
KKLFKKILKYL-NH2 (BP100)
KKLfKKILKYL-NH2 (BP143)
7.5–10 5–7.5
2.5–5 5–7.5
5–7.5
2.5–5
2.5–5
>20
>10
>20
5–10
5–7.5
2.5–5
3 ± 0.1
2 ± 2.8
22 ± 2.8
22 ± 2.6
99 ± 5.2
92 ± 7.2
100 ± 1.1
10–20 >20
(KKLFKKILKYL-C2H4N)2-cDTE (1) 2.5–5
0.6–1.2 1.2–2.5 2.5–5
2.5–5 2.5–5
1.2–2.5 0.6–1.2 0.6–1.2 1.2–2.5 >20
10–20 5–7.5
100 ± 4.1
96 ± 0.9
100 ± 18.8
(KKLfKKILKYL-C2H4N)2-cDTE (2)
(KKLFKKILKYL-C2H4N)4-Galp (3)
2.5–5
1.2–2.5 10–20 7.5–10 2.5–5
5–7.5 2.5–5
aPercentage hemolysis plus confidence interval. bEa, Erwinia amylovora; Xav, Xanthomonas axonopodis pv. vesicatoria; Pss, Pseudomonas syringae
pv. syringae; Ec, Escherichia coli; Sa, Staphylococcus aureus; Lm, Lysteria monocytogenes; Se, Salmonella enterica.
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Figure 2: Kinetics of bactericidal activity on E. amylovora and S. enterica in the presence of peptides and carbopeptides. Viable cells were deter-
mined at different time intervals.
SDS and in 50% TFE they became moderately structured, with should expect at least a two-fold decrease in the MIC values,
the degree of secondary structure formation being higher in the and for carbopeptide 3, incorporating four peptide sequences,
latter solvent. In both cases, two local minima near 205 and the activity should decrease at least four-fold. Against the
220 nm, and a positive band near 195 nm were observed. In phytopathogenic bacteria, carbopeptides 1 and 3, derived from
50% TFE, we estimated an α-helical content of 41% for BP100, displayed a multimeric effect that was lower than
carbopeptide 1 and of 19% for carbopeptide 3. Monomeric expected. In contrast, carbopeptide 2 derived from BP143
undecapeptide BP100 also became moderately structured in containing one D-amino acid did not improve the activity of the
50% TFE with an α-helical content of 21%.
parent peptide. Against the human pathogens, we did not
observe a significant decrease of the MIC values for any of the
three carbopeptides. According to Kallenbach and co-workers,
Discussion
Several studies have demonstrated that covalently tethering a multivalent cationic antimicrobial peptides may display a lower
number of peptides to a template can improve overall avidity activity than expected due to the potential immobilization of the
for targets such as cell membranes [9-15]. In this study we used cationic sequences onto the negatively charged bacterial
carbohydrate templates for the design of multivalent antimicro- surfaces, which may impede them to reach the bacterial
bial peptides targeted to control plant and human pathogenic membrane [15]. Similar observations were described by Shai
bacteria and explored whether multivalency enhances the and co-workers for covalently linked pentameric bundles of
antimicrobial activity. These multivalent constructs were cationic antimicrobial peptides [14]. These studies demon-
obtained by preassembling the antimicrobial peptides BP100 strated that the large volume and the charge of the bundles
and BP143 to a cDTE or a Galp carbohydrate template through made their passage through the peptidoglycan or the
oxime formation. They were tested against E. amylovora, lipopolysaccharide layers difficult, therefore preventing them
X. axonopodis pv. vesicatoria, P. syringae pv. syringae, E. coli, from accessing the inner membrane. In contrast, despite the fact
S. aureus, S. enterica, and L. monocytogenes.
that a significant decrease of the MIC values was not observed,
the preassembly of multiple copies of BP100 and BP143 did
In agreement with previous studies, we observed that the effec- have an important effect on the bactericidal activity. The results
tiveness of the designed carbopeptides depended on the showed that the carbopeptides 1–3 were significantly more
bacteria, the peptide, and the structure of the template [14,15]. potent than the corresponding monomers, which is in agree-
For carbopeptides 1 and 2 containing two monomeric units we ment with previous studies [34,35]. This ability to kill the
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Figure 3: CD spectra of (a) carbopeptide 1 and of (b) carbopeptide 3 in: (i) 10 mM sodium phosphate buffer at pH 7.4 (broken line, - - -); (ii) 10 mM
SDS in 10 mM sodium phosphate buffer at pH 7.4 (solid line, ___), and (iii) 50% (v/v) trifluoroethanol in 10 mM sodium phosphate buffer at pH 7.4
(broken dotted line, ··−··−).
bacteria rapidly confers them a greater capacity to control bacte- troscopy, the preassembly of BP100 did not result in a more
rial cell growth, which may be important for their future poten- organized structure, especially for carbopeptide 3, which
tial use as bactericidal agents.
suggests that the high hemolysis observed for carbopeptides
cannot be attributed to a higher degree of secondary-structure
Unlike the parent peptides BP100 and BP143, carbopeptides formation. A likely explanation for the higher hemolytic
1–3 exhibited high activity towards erythrocytes. It has been activity displayed by carbopeptides could be ascribed to their
shown that peptide preassembly may favor the formation of a different interaction with zwitterionic membranes compared to
more organized amphipathic peptide structure compared to the peptide monomers, as has been described in previous reports
monomers, which has been correlated with a higher degree of [14,36].
hemolysis [14,36]. BP100 was originally designed based on an
ideal α-helical Edmunson wheel and showed a moderate degree In summary, this study provides an example of carbopeptides
of secondary-structure formation by CD. As shown by CD spec- with high antimicrobial activity and highlights the importance
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of peptide preassembly for the hemolysis and for the bacteri- Gemini 110 Å C18 (4.6 × 50 mm, 3 µm particle size) with a
cidal activity. The structure of these carbopeptides includes two gradient of 5–80% B over 5 min and 80–100% B over 5 min
or four copies of the antimicrobial peptides BP100 or BP143 (buffer A: 0.1% formic acid in H2O; buffer B: 0.1% formic acid
linked to a cDTE or a Galp carbohydrate template. Even though in CH3CN) at a flow rate of 1 mL/min. Carbopeptides were
these multivalent compounds exhibited low MIC values against analyzed on a C4 Phenomenex Jupiter 300 Å (4.6 × 150 mm,
plant and human pathogenic bacteria, a significant multimeric 5 µm particle size) with a gradient of 5–20% B over 2 min,
effect was not observed. Our results support the notion that 20–40% B over 8 min and 40–100% B over 2 min at a flow rate
preassembly of antimicrobial peptides to multimeric structures of 1 mL/min.
prior to contact with the microbial surface does not necessarily
lead to a significant improvement of the antimicrobial activity. Preparative RP-HPLC was performed on a similar Dionex Ulti-
Mate 3000 system, equipped with a standard thermostatted
Experimental
column compartment (TCC) and with UV detection at 220 nm.
General methods
Solvents used for RP-HPLC were (A) H2O with 0.1% TFA, (B)
The synthesis of Boc2-Aoa-OH was described in an earlier CH3CN with 0.1% TFA, (C) H2O and (D) CH3CN. A
publication from our laboratories [23]. It is now available from Phenomenex Gemini-NX C18 110 Å column (100 × 21.20 mm,
Polypeptide Laboratories (NeoMPS, Strassbourg, France). 5 µm particle size) was used, running at a flow rate of
Automated peptide synthesis was carried out on a fully auto- 10.0 mL/min at room temperature with gradients from 5–55% B
mated Syro II peptide synthesizer (MultiSynTech). Manual over 24 min and 55–100% B over 11 min (method A) and at
peptide synthesis was performed in polypropylene syringes 42 °C with gradients running from 5–50% B over 24 min,
fitted with a polyethylene porous disk. Solvents and soluble 50–95% B over 11 min and 95–100% B over 2 min (method B).
reagents were removed by suction. Chemicals were purchased A FeF Chemicals C4 250 Å column (20 × 250 mm, 5 μm
from commercial suppliers Sigma–Aldrich, Fluka, particle size) was used running at a flow rate of 15 mL/min
NovaBiochem (Schwalbach, Germany) or Iris Biotech GmbH at 42 °C with a linear gradient of 5–95% B over 28 min
(Marktredwitz, Germany) and used without further purification. (method C).
Microwave heating was performed in a Biotage Initiator instru-
ment (Biotage, Uppsala, Sweden).
ESIMS analyses were performed on a MSQ Plus Mass Spec-
trometer (Thermo) or by using an Esquire 6000 ESI ion Trap
An acetate buffer (0.1 M, pH 4.76) was prepared by dissolving LC–MS (Bruker Daltonics). High-resolution mass spectro-
an equal amount of NaOAc·3H2O (10 mmol) and AcOH metry (HRMS) was recorded on a Bruker MicroToF-Q MALDI
(10 mmol) in water (200 mL) and then adjusting to pH 4.76 instrument by using a hybrid quadrupole time-of-flight mass
with NaOH (1 M aq). An acetate buffer (0.1 M, pH 4.76) spectrometer (University of Zaragoza).
containing aniline (100 mM) was prepared by dissolving aniline
(10 mmol) in the previous acetate buffer (100 mL). A UV spec- Circular dichroism (CD) measurements were obtained using a
trophotometer was used to quantify the amount of Fmoc Jasco spectropolarimeter (J-810, Easton, MD, USA) at 25 °C.
cleaved.
Spectra were obtained in a fused quartz cell with 1 mm path
length over a wavelength range 190–260 nm at 0.1 nm intervals,
Peptides were analyzed under standard analytical HPLC condi- 50 nm/min speed, 0.5 s response time, and 1 nm bandwidth.
tions with a Dionex instrument composed of an UV–vis Dionex
Solid-phase synthesis of peptides BP100
UVD170U detector, a P680 Dionex pump, an ASI-100 Dionex
automatic injector, and Chromeleon 6.60 software. Detection and BP143
was performed at 220 nm. Analysis was carried out with a Peptides were synthesized manually by the solid-phase method
Kromasil 100 C18 (40 mm × 4.6 mm, 3.5 μm) column with a using standard Fmoc chemistry. Fmoc-Rink-MBHA resin
2–100% B linear gradient over 7 min at a flow rate of (0.56 mmol/g) was used as a solid support. Couplings of the
1 mL/min. Solvent A was 0.1% aq TFA, and solvent B was corresponding amino acids Fmoc-Leu-OH, Fmoc-Phe-OH,
0.1% TFA in CH3CN.
Fmoc-D-Phe-OH, Fmoc-Lys(Boc)-OH, Fmoc-Ile-OH, and
Fmoc-Tyr(t-Bu)-OH (4 equiv) were performed using ethyl
Peptide aldehydes, carbohydrate templates and carbopeptides cyanoglyoxylate-2-oxime (Oxyma) (4 equiv), N,N′-diisopropyl-
were analyzed under standard analytical HPLC conditions with carbodiimide (DIPCDI) (4 equiv) in DMF at room temperature
a Dionex UltiMate 3000 with Chromeleon 6.80SP3 software. for 1 h. The completion of the reactions was monitored by the
Detection was performed at 215 nm. Peptide aldehydes and Kaiser test. Fmoc group removal was achieved with piperidine/
carbohydrate templates were analyzed with a Phenomenex DMF (3:7, 1 × 2 min + 1 × 10 min). After each coupling and
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deprotection step, the resin was washed with DMF (6 × 1 min) room temperature. DMF was used as solvent for all the
and CH2Cl2 (3 × 1 min) and air-dried. Once the peptide couplings except for Fmoc-Phe-OH and Fmoc-D-Phe-OH,
sequence had been completed, the Fmoc group was removed. which were coupled in N-methyl-2-pyrrolidinone (NMP). Each
Acidolytic cleavage was performed by treatment of the resin coupling step was repeated twice. The Fmoc group was
with TFA/H2O/TIS (95:2.5:2.5) for 2 h at room temperature. removed by treating the resin with piperidine/DMF (2:3, 3 min)
Following concentration in vacuo by evaporation of TFA and followed by two treatments of piperidine/DMF (1:4, 12 +
trituration with Et2O, the crude peptide was dissolved in H2O/ 15 min). After coupling and deprotection steps the resin was
CH3CN, lyophilized, analyzed by HPLC, and characterized by washed with DMF (3 × 1 min), CH2Cl2 (1 × 1 min) and DMF
ESIMS.
(3 × 1 min). Once the second amino acid was coupled, a Fmoc
quantification was performed to estimate the overall loss in
loading.
Peptide KKLFKKILKYL-NH2 (BP100)
HPLC: tR = 4.07 min (90% purity); ESIMS (m/z): 1420.87 Once the synthesis was completed, peptides were individually
[M + H]+.
cleaved from the resin with TFA/H2O (95:5) for 2 h. Following
concentration in vacuo by evaporation of TFA and trituration
with Et2O, the crude peptides were dissolved in H2O, analyzed
by HPLC, purified by preparative RP-HPLC using Method A
Peptide KKLfKKILKYL-NH2 (BP143)
HPLC: tR = 6.28 min (84% purity); ESIMS (m/z): 1420.87 and characterized by LC–MS.
[M + H]+.
Peptide aldehyde KKLFKKILKYLG-H (4)
HPLC: tR = 2.08 min (99% purity); ESIMS (m/z): 366.7
Solid-phase synthesis of peptide aldehydes 4
and 5
TentaGel S NH2 (TG) resin (0.27 mmol/g) was placed in a [M + 4H]4+, 488.6 [M + 3H]3+.
syringe and swelled with CH2Cl2 (3 × 10 min) and DMF (3 ×
10 min). The ortho backbone amide linker (o-BAL) was incor- Peptide aldehyde KKLfKKILKYLG-H (5)
porated by treating the resin twice with 5-(2-formyl-3,5-
dimethoxyphenoxy)pentanoic acid (4.0 equiv), N,N,N′,N′- HPLC: tR = 2.20 min (99% purity); ESIMS (m/z): 366.6
tetramethyl-O-(1H-benzotriazol-1-yl)uronium hexafluorophos- [M + 4H]4+, 488.5 [M + 3H]3+.
phate (HBTU) (3.8 equiv), 1-hydroxybenzotriazole (HOBt)
Preparation of the aminooxy-functionalized
(4.0 equiv), and N,N-diisopropylethylamine (DIPEA)
(7.8 equiv) in DMF at room temperature for 12 h. Washings templates cDTE and Galp
were performed with DMF (3 × 1 min), CH2Cl2 (3 × 1 min) and The synthesis of the functionalized cyclo-dithioerythritol
DMF (3 × 1 min). Next a reductive amination was performed by (cDTE) and α-D-galactopyranoside (Galp) templates have been
using aminoacetaldehyde dimethyl acetal (10 equiv) and described in earlier publications [23,30].
NaBH3CN (10 equiv) suspended in AcOH/DMF (1:99). This
suspension was added to the o-BAL-TG resin and the mixture Synthesis of carbopeptide (KKLFKKILKYL-C2H4N)2-
was heated in a microwave instrument at 60 °C for 10 min. The cDTE (1)
resin was washed with DMF (3 × 1 min), CH2Cl2 (3 × 1 min) (4R,5S)-1,2-Dithiane-4,5-diyl bis(Boc)2-Aoa (cDTE) (12.7 mg,
and dichloroethane (DCE) (3 × 1 min) and the reductive amina- 18.2 μmol) was dissolved in TFA/CH2Cl2 (1:1, 2 mL) and
tion step was repeated. Then, Fmoc-Leu-OH (10 equiv) was stirred for 1 h. The solution was then concentrated in vacuo,
dissolved in DCE/DMF (10:1), DIPCDI (5 equiv) was added, redissolved in a small amount of water and lyophilized to afford
and after preactivation for 10 min, the slurry was transferred to (4R,5S)-1,2-dithiane-4,5-diyl bis(2-Aoa) as a white powder.
the resin. The reaction mixture was heated in a microwave This template and the peptide aldehyde 4 (65.7 mg, 45.0 μmol)
instrument to 60 °C for 2 × 10 min, and then the resin was were dissolved in 6.2 mL of a 1:1 solution of CH3CN and an
washed with DMF (3 × 1 min), CH2Cl2 (3 × 1 min) and DMF acetate buffer (0.1 M, pH 4.76) containing aniline (100 mM).
(3 × 1 min). Peptide elongation was performed on the fully The reaction mixture was stirred for 3 h and purified by prepar-
automatic synthesizer MultiSynTech Syro II by repeated cycles ative RP-HPLC using Method B (24.3 mg, 42% yield).
of amino acid coupling, Fmoc group removal and washings.
The amino acid couplings were carried out by using the corres- HPLC: tR = 2.50 min (98% purity); ESIMS (m/z): 1594.5
ponding Fmoc-amino acid (6 equiv), HBTU (5.8 equiv), HOBt [M + 2H]2+, 1063.5 [M + 3H]3+, 797.8 [M + 4H]4+, 638.4
(6 equiv) and DIPEA (11.7 equiv) shaking the mixture for 2 h at [M + 5H]5+, 532.2 [M + 6H]6+; HRMS–MALDI (m/z):
2114

Beilstein J. Org. Chem. 2012, 8, 2106–2117.
[M + Na]+ calcd for C156H264N36NaO30S2, 3208.9753; found, (Institut de Tecnologia Agroalimentària, Universitat de Girona,
3208.9737.
Spain), and Xanthomonas axonopodis pv. vesicatoria 2133-2
(Instituto Valenciano de Investigaciones Agrarias, Valencia,
Spain). Moreover, the following bacterial strains were also
used: Escherichia coli ATCC NCTC 5934, Listeria monocyto-
Synthesis of carbopeptide (KKLfKKILKYL-C2H4N)2-
cDTE (2)
(4R,5S)-1,2-Dithiane-4,5-diyl bis(2-(Boc)2-Aoa) (cDTE) genes ATCC 15313, Staphylococcus aureus subsp. aureus
(4.8 mg, 6.9 μmol) was dissolved in TFA/CH2Cl2 (1:1, 1 mL) ATCC 9144 and Salmonella enterica subsp. enterica LT2
and stirred for 1 h. The solution was then concentrated in vacuo, ATCC 15277.
redissolved in a small amount of water and lyophilized to afford
(4R,5S)-1,2-dithiane-4,5-diyl bis(2-Aoa) as a white powder. All bacteria were stored in Luria Bertani (LB) broth except for
This template and the peptide aldehyde 5 (30.2 mg, 20.6 μmol) L. monocytogenes, which was stored in Brain Heart Infusion
were dissolved in 1:1 solution (2 mL) of CH3CN and an acetate (BHI) broth (Oxoid, Hampshire, United Kingdom) supple-
buffer (0.1 M, pH 4.76) containing aniline (100 mM). The reac- mented with glycerol (20%) and maintained at −80 °C.
tion mixture was stirred for 3 h and purified by preparative E. amylovora and P. syringae pv. syringae were scrapped from
RP-HPLC using Method B (13.6 mg, 62% yield).
LB agar after growing for 24 h at 25 °C; X. axonopodis pv.
vesicatoria was scrapped from LB agar after growing for 48 h
HPLC: tR = 2.51 min (98% purity); ESIMS (m/z): 1594.1 at 25 °C; S. aureus, E. coli and S. enterica were scrapped from
[M + 2H]2+, 1063.5 [M + 3H]3+, 797.9 [M + 4H]4+, 638.4 LB agar after growing for 24 h at 37 °C; L. monocytogenes was
[M + 5H]5+, 532.2 [M + 6H]6+; HRMS–MALDI (m/z): scrapped from BHI agar after growing for 24 h at 37 °C. The
[M + H]+ calcd for C156H265N36O30S2, 3186.9753; found, cell material was suspended in sterile water to obtain a suspen-
3186.9820.
sion of 108 CFU mL−1.
Synthesis of carbopeptide (KKLFKKILKYL-C2H4N)4- Antibacterial activity
Galp (3)
As described in [28], lyophilized compounds were solubilized
Methyl 2,3,4,6-tetra-O-((Boc)2-Aoa)-α-D-Galp (3.4 mg, in sterile Milli-Q water to a final concentration of 1000 μM and
2.6 μmol) was dissolved in TFA/CH2Cl2 (1:1, 0.5 mL) and filter sterilized through a 0.22 μm pore filter. For minimum
stirred for 30 min at room temperature. The solution was then inhibitory concentration (MIC) assessment, dilutions of the
concentrated to dryness, redissolved in a small amount of water compounds were made to obtain a final concentration of 200,
and lyophilized to afford methyl 2,3,4,6-tetra-O-Aoa-α-D-Galp 100, 75, 50, 25, 12.5 and 6 μM. Twenty microliters of each
as a white powder. This lyophilized template was dissolved in dilution were mixed in a microtiter plate well with 20 μL of the
1.2 mL of a 1:1 solution of CH3CN and an acetate buffer corresponding suspension of the bacterial indicator, 160 μL of
(0.1 M, pH 4.76). Peptide aldehyde 4 (19.3 mg, 13.2 μmol) was Trypticase Soy Broth (TSB) (BioMèrieux, France) for
added, and the mixture was stirred at room temperature. The E. amylovora, P. syringae pv. syringae, X. axonopodis pv.
reaction was followed by HPLC. After 4 h, an additional vesicatoria, S. aureus, E. coli and S. enterica or BHI for
amount of peptide aldehyde 4 (20 mg, 13.7 μmol) dissolved in L. monocytogenes to a total volume of 200 μL. Three replicates
1.1 mL of a 1:1 solution of CH3CN and an acetate buffer for each strain, compound and concentration were used. Posi-
(0.1 M, pH 4.76) containing aniline (100 mM) was added. After tive controls contained water instead of compound, and nega-
further 2.5 h, the reaction was completed. The reaction mixture tive controls contained compounds without bacterial suspen-
was purified by RP-HPLC using Method C (10.7 mg, 64% sion. Microbial growth was automatically determined by optical
yield).
density measurement at 600 nm (Bioscreen C, Labsystem,
Helsinki, Finland). Microplates were incubated at 25 °C for
HPLC: tR = 6.23 min (90% purity); ESIMS m/z: 1566.3 plant pathogenic bacteria and at 37 °C for foodborne bacterial
[M + 4H]4+, 1253.8 [M + 5H]5+, 1045.1 [M + 6H]6+, 895.7 strains, with 20 s shaking before hourly absorbance measure-
[M + 7H]7+, 784.2 [M + 8H]8+, 697.2 [M + 9H]9+, 627.5 ment, for 48 h. The experiment was repeated twice. The MIC
[M + 10H]10+ ([M]+ calcd for C311H526N72O62, 6262.022 Da). was taken as the lowest compound concentration with no
growth at the end of the experiment.
Bacterial strains and growth conditions
As described in [28], the following plant pathogenic and food- Bactericidal activity
borne bacterial strains were used: Erwinia amylovora The bactericidal activity was determined for peptides BP100
PMV6076 (Institut National de la Recherche Agronomique, and BP143 and for carbopeptides 1–3 by using E. amylovora
Angers, France), Pseudomonas syringae pv. syringae EPS94 and S. enterica as indicators of plant pathogenic and foodborne
2115

Beilstein J. Org. Chem. 2012, 8, 2106–2117.
bacteria. Ringer bacterial suspensions at 1–1.5 × 106 CFU mL−1 residue ellipticity at 222 nm. Values for
and
,
were incubated in a 2.5 µM concentration of the corresponding corresponding to 0 and 100% helix content at 222 nm,
compound, including also a negative control containing only were estimated to be −2000 and −30000 deg cm2 dmol−1,
Ringer solution. A total volume of 13 mL suspension was incu- respectively [37].
bated for 3 h at room temperature. Aliquots of 1.5 mL were
removed at 30 min intervals, tenfold-diluted if necessary, and
Supporting Information
spread in triplicate onto LB agar plates by using a spiral plater
(Eddy Jet; IUL Instruments, Spain). After 24 h of incubation at
Supporting Information File 1
25 °C for E. amylovora, and 37 °C for S. enterica, the concen-
trations of the suspensions were obtained with an automatic
counter (Flash and Go; IUL Instruments, Spain) by incorpor-
ating the software Counter Mat version 5.0. Values are
expressed as percentages of survival from the start of the
experiment.
HPLC, ESIMS of peptide aldehydes 4 and 5. HPLC,
ESIMS, and HRMS of carbopeptides 1–3.
[http://www.beilstein-journals.org/bjoc/content/
supplementary/1860-5397-8-237-S1.pdf]
Acknowledgements
Hemolytic activity
Imma Güell is recipient of a predoctoral fellowship from the
Generalitat de Catalunya. This work was supported by grants
AGL2006-13564/AGR and AGL2009-13255-C02-02/AGR
from MICINN of Spain.
As described in [28], the hemolytic activity of the compounds
was evaluated by determining hemoglobin release from erythro-
cyte suspensions of fresh human blood (5% vol/vol). Blood was
aseptically collected using a BD vacutainer K2E System with
EDTA (Belliver Industrial State, Plymouth, U.K.) and stored for
less than 2 h at 4 °C. Blood was centrifuged at 6000g for 5 min,
washed three times with tris(hydroxymethyl)aminomethane
(TRIS) buffer (10 mM TRIS, 150 mM NaCl, pH 7.2) and
diluted. Compounds were solubilized in TRIS buffer to a final
concentration of 500, 300 and 100 μM. Fifty microliters of
human red blood cells were mixed with 50 μL of the compound
solution and incubated under continuous shaking for 1 h at
37 °C. Then, the tubes were centrifuged at 3500g for 10 min.
Eighty microliter aliquots of the supernatant were transferred to
100-well microplates (Bioscreen) and diluted with 80 μL of
Milli-Q water. Hemolysis was measured as the absorbance at
540 nm with a Bioscreen plate reader. Complete hemolysis was
determined in TRIS buffer plus melittin at 100 µM
(Sigma–Aldrich, Madrid, Spain) as a positive control. The
percentage of hemolysis (H) was calculated by using the
following equation: H = 100 × [(Op−Ob)/(Om−Ob)], where Op
was the density for a given compound concentration, Ob for the
buffer, and Om for the melittin positive control.
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