Synthesis of short cationic antimicrobial peptidomimetics containing arginine analogues

Article abstract of DOI:10.1002/psc.2435

Worldwide efforts are underway to develop new antimicrobial agents against bacterial resistance. To identify new compounds with a good antimicrobial profile, we designed and synthesized two series of small cationic antimicrobial peptidomimetics (1-8) containing unusual arginine mimetics (to introduce cationic charges) and several aromatic amino acids (bulky moieties to improve lipophilicity). Both series were screened for in vitro antibacterial activity against a representative panel of Gram-positive (Staphylococcus aureus and Staphylococcus epidermidis) and Gram-negative (Escherichia coli and Klebsiella pneumoniae) bacterial strains, and Candida albicans. The biological screening showed that peptidomimetics containing tryptophan residues are endowed with the best antimicrobial activity against S. aureus and S. epidermidis in respect to the other synthesized derivatives (MIC values range 7.5-50μg/ml). Moreover, small antimicrobial peptidomimetics derivatives 2 and 5 showed an appreciable activity against the tested Gram-negative bacteria and C. albicans. The most active compounds (1-2 and 5-6) have been tested against Gram-positive established biofilm, too. Results showed that the biofilm inhibitory concentration values of these compounds were never up to 200μg/ml. The replacement of tryptophan with phenylalanine or tyrosine resulted in considerable loss of the antibacterial action (compounds 3-4 and 7-8) against both Gram-positive and Gram-negative bacterial strains. Furthermore, by evaluating hemolytic activity, the synthesized compounds did not reveal cytotoxic activities, except for compound 5.

Full text of DOI:10.1002/psc.2435

Research Article
Received: 12 December 2011
Revised: 13 June 2012
Accepted: 20 June 2012
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI 10.1002/psc.2435
Synthesis of short cationic antimicrobial
peptidomimetics containing arginine analogues
Leonardo Baldassarre, Francesco Pinnen, Catia Cornacchia, Erika Fornasari,
Luigina Cellini, Marina Baffoni and Ivana Cacciatore*
Worldwide efforts are underway to develop new antimicrobial agents against bacterial resistance. To identify new compounds with
a good antimicrobial profile, we designed and synthesized two series of small cationic antimicrobial peptidomimetics (1–8)
containing unusual arginine mimetics (to introduce cationic charges) and several aromatic amino acids (bulky moieties to
improve lipophilicity). Both series were screened for in vitro antibacterial activity against a representative panel of Gram-positive
(Staphylococcus aureus and Staphylococcus epidermidis) and Gram-negative (Escherichia coli and Klebsiella pneumoniae) bacterial
strains, and Candida albicans. The biological screening showed that peptidomimetics containing tryptophan residues are
endowed with the best antimicrobial activity against S. aureus and S. epidermidis in respect to the other synthesized derivatives
(MIC values range 7.5–50mg/ml). Moreover, small antimicrobial peptidomimetics derivatives 2 and 5 showed an appreciable
activity against the tested Gram-negative bacteria and C. albicans. The most active compounds (1–2 and 5–6) have been tested
against Gram-positive established biofilm, too. Results showed that the biofilm inhibitory concentration values of these
compounds were never up to 200 mg/ml. The replacement of tryptophan with phenylalanine or tyrosine resulted in considerable
loss of the antibacterial action (compounds 3–4 and 7–8) against both Gram-positive and Gram-negative bacterial strains.
Furthermore, by evaluating hemolytic activity, the synthesized compounds did not reveal cytotoxic activities, except for
compound 5. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.
Keywords: antimicrobial peptidomimetics; arginine analogues; antibacterial activity; antifungal activity; microbial biofilm
bacteria to mammals. AMPs show antimicrobial activity against
several microorganisms such as bacteria, yeasts, fungi, and
Introduction
protozoa [10]. Currently, more than 850 AMPs have been charac-
The discovery of penicillin in 1928 by Alexander Fleming opened the
terized and most of them can be classified in alpha-helical
so-called antibiotic era, prior to which infections were life-threatening
peptides, peptides with three disulfide bonds, and proline or
diseases. Currently, conventional antibiotics possess growth-
tryptophan-rich peptides [11]. Despite this wide range of struc-
inhibitory or toxic activity on a broad range of pathogenic bacteria.
tures, most of the AMPs share common structural features. They
However, recently an increased frequency of bacterial mutations
are composed of 12–50 amino acids, 50% of which possess a
has resulted in a significantly augmented incidence of antibiotic
lipophilic chain and have a positive net charge [12]. Such struc-
resistance, and infections caused by antibiotic-resistant bacteria
tural arrangement confers them high affinity and selectivity for
have become a global health care problem of the 21st century [1].
bacterial membranes. The frequent mode of action of AMPs is
Indeed, if resistance was initially a problem only for hospitalized
partly related to cell membrane permeabilization and lysis. The
patients, gradually it extended into the community, causing severe
infections difficult to diagnose and treat [2].
Moreover, bacteria often choose a sessile biofilm lifestyle charac-
terized by single cells or micro colonies embedded in a hydrated
exact mechanism of membrane disruption by AMPs is not com-
pletely known, even though several models have been proposed
to explain the effect of these compounds. In this context – using
membrane potential sensitive dyes and fluorescently labeled
matrix – made of extracellular polymeric substances [3] – which
confers to the bacterial population a significant antimicrobial
tolerance when compared with the free-living counterparts [4,5].
*
Correspondence to: Ivana Cacciatore, Dipartimento di Scienze del Farmaco,
Università G. d’Annunzio, Via dei Vestini 31, 66100 Chieti, Italy. E-mail:
cacciatore@unich.it
The biofilm state represents a natural part of the life cycle of micro-
organisms, and the recognized presence in human infections poses
serious concerns in term of eradication. In fact, biofilm-related
infections can tolerate antimicrobial agents at concentrations of
10–1000 times higher than that needed to kill the equivalent
planktonic bacteria [6]. Thus, the development of novel antimicrobial
pharmaceuticals capable of overcoming these difficulties is a major
challenge of modern medicinal chemistry.
In the search for new and alternative sources of antibiotics, a
place of primary importance is occupied by AMPs [7–9]. AMPs are
ribosomally synthesized peptides that constitute a key component
of innate immune system of a wide number of organisms, from
Dipartimento di Scienze del Farmaco, Università G. d’Annunzio, Via dei Vestini
31, 66100 Chieti, Italy
Abbreviations used: AMP, antimicrobial peptides; ATCC, American tissue culture
collection; BEC, biofilm eradication concentration; BIC, biofilm inhibitory concentra-
tion; CLSI, clinical laboratory standards institute; DIPEA, N,N-diisopropylethylamine;
DMF, dimethylformamide; HBTU, O-benzotriazole-N,N,N0,N0-tetramethyl-
uronium-hexafluoro-phosphate; HOBt, hydroxybenzotriazole; MBC, minimum
bactericidal concentration; MIC, minimum inhibitory concentration; SAMPs,
small antimicrobial peptidomimetics; TEA, triethylamine; TFA, trifluoroacetic
acid; THF, tetrahydrofuran.
J. Pept. Sci. 2012
Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.

BALDASSARRE ET AL.
peptides – AMPs have been found to interact with membranes
and tend to divide peptides into two classes: (1) membrane disrup-
tive (barrel stave, toroidal, carpet, and micellar aggregate models)
and (2) non-membrane disruptive (intracellular targets) [13,14]. In
membrane disruptive models, AMPs have been proposed to disrupt
the membrane bilayer with consequent loss of bacterial membrane
functionality. Independently of the chosen membrane perturbation
model, it is important to consider the interaction that peptides
are able to establish in a complex environment of a cell
membrane. In fact, because AMPs commonly fold up into different
amphipathic structures – separating the hydrophobic and the
cationic parts – they are able to adhere to the surface of the
bacteria through electrostatic interactions, resulting in a rapid
lysis of a broad range of bacteria, which decreases the likelihood
of developing resistance [15].
acids (such as L-tryptophan, L-tyrosine, and L-phenylalanine),
respectively, in order to confer both lipophilic properties and
positive-charged portions. The second series of SAMPs deriva-
tives (5–8) contains the same set of three aromatic amino acids,
but the positive charges are conferred to the molecule by
another guanidine derivative such as 3,5-diguanidino benzoic acid.
Both series were screened for in vitro antibacterial activity against a
representative panel of Gram-positive (Staphylococcus aureus and
Staphylococcus epidermidis) and Gram-negative (Escherichia coli
and Klebsiella pneumoniae) bacterial strains, and Candida albicans.
Materials and Methods
Substances
L-Arginine-L-tryptophan benzyl ester, N,N0-bis(tert-butoxycarbonyl)-
S-methyl-isothiourea, Boc-L-tryptophyl-L-tryptophan, L-tryptophyl-
L-tryptophan methyl ester trifluoacetate salt, and 3,5-bis
((tert-butoxycarbonyl)amino)benzoic acid were prepared as
described in literature [17,28–30]. All other chemicals and all
solvents were purchased from Sigma-Aldrich (Sigma-Aldrich
Corp., Saint Louis, MO, USA). Mueller–Hinton broth and
microtiter plates were obtained from Becton Dickinson (Becton
Dickinson, Franklin Lakes, NJ, USA). Mueller–Hinton agar and
tryptic soy broth were from Oxoid (Oxoid, Dardilly, France), and
glucose was purchased from Sigma-Aldrich.
As a result, much attention has been given to AMPs, which are
rightfully thought to be a novel class for antibacterial treatment.
Nevertheless, therapeutic application of AMPs is hampered by
many unresolved problems, such as low bioavailability, limited
metabolic and proteolytic stability, high production cost, and
allergenic properties [16]. In order to increase peptides’ half-life,
many strategies involving several formulations, ways of administra-
tion, and different levels of chemical modification are possible.
Introduction of D-amino acids, amidation at the N-terminus, non-
natural amino acids, and peptide cyclization are the most common
strategies to increase peptide stability [15]. Because there are
few studies about the pharmacokinetics of AMPs and the effects
of all the aforementioned modifications, considerable additional
researches on these issues are urgently needed. Strøm et al.
[17] – before synthesizing small peptidomimetics with antimi-
crobial activity – focused their attention on the research of the
pharmacophore for antibacterial activity in cationic peptides.
They reported that three lipophilic bulky moieties and two
positively charged residues were necessary for the activity
against Gram-negative bacteria; whereas two positively charged
residues and two lipophilic bulky residues were required for
the activity toward Gram-positive bacteria. On the basis of this
pharmacophore motif, several molecules have been designed
and synthesized, thus originating a novel class of SAMPs
[18–20]. They retained the AMPs antimicrobial activity and were
active toward staphylococcal biofilms [21], also showing
improved pharmacokinetic properties [22]. In particular, high
antibacterial activity was obtained with the introduction of bulky
groups as aromatics but with a larger size than a phenyl group
[23], and of charged moieties as the side chains of arginine or
lysine [17,18,24]. Other research groups have lately reported
successful implementations of this pharmacophore. Knappe
et al. [25,26] found that replacing arginine residues with mimetic
analogues protected the AMPs from serum degradation without
affecting their antimicrobial activity, whereas Karstad et al. [27]
reported that the introduction of unnatural amino acids side
chain in the sequence of small cationic antimicrobial peptide
increased their stability toward chymotryptic degradation.
On the basis of these data, we designed and synthesized new
SAMPs derivatives (1–8). Starting from the most active com-
pound reported by Strom et al. [17] – the dipeptide L-arginine-L-
tryptophan benzyl ester – and in order to identify compounds
with a good antimicrobial profile, we designed and synthesized
two series of SAMPs containing arginine analogues and several
aromatic amino acids.
Synthesis and Purification
Microanalyses were performed on a 1106 Carlo Erba CHN analyzer
(Carlo Erba, Milan, Italy).1H and 13C NMR spectra were recorded on
a Varian VXR 300-MHz spectrometer (Varian Medical Systems, Inc.,
Palo Alto, CA, USA). Chemical shifts are reported in parts per
million (d) downfield from the internal standard Me4Si. The LC-
MS/MS system consisted of an LCQ (Thermo Finnigan, San Jose,
CA, USA) ion trap mass spectrometer equipped with an electro-
spray ionization (ESI) source. The capillary temperature was set at
300 ꢀC and the spray voltage at 4.25 kV. The fluid was nebulized
using nitrogen (N2) as both the sheath gas and the auxiliary gas.
The identity of new compounds was confirmed by 1H and 13C
NMR data and LC-MS/MS measurements; homogeneity was
confirmed by TLC on silica gel Merck 60 F254 (Merck & Co., Inc.,
Whitehouse Station, NJ, USA). Solutions were routinely dried over
anhydrous sodium sulfate prior to evaporation. Chromatographic
purifications were performed using Merck 60 70–230 mesh ASTM
silica gel column. Analytical HPLC was carried out on a Waters
600 HPLC (Waters Co., Milford, MA, USA) equipped with an X-
Bridge BEH130 C18, 5 mm, 4.6 ꢁ 250 mm column with Waters
2996 PDA detector, using a solvent system of H2O/CH3CN (0.1%
TFA) in the form of a linear gradient from 10 to 80% of CH3CN
in 30 min and a flow rate of 1 ml/min. All the final products (1–8)
were obtained as trifluoacetate salts with purity greater than
95%, determined by analytical HPLC at 254 and 280 nm.
Boc-L-tryptophyl-L-tryptophan-4-((Boc)aminomethyl)benzylamide (1a)
To a stirred solution of Boc-L-tryptophyl-L-tryptophan (1.69 g,
3.44 mmol) in dry DMF (15 ml), HOBt (567 mg, 4.20 mmol),
HBTU (1.56 g, 4.16 mmol), DIPEA (1.32 ml, 7.57 mmol), and 4-
(tert-butoxycarbonyl-aminomethyl)benzylamine (813 mg, 3.44 mmol)
were added at 0 ꢀC. After 10 min at 0 ꢀC and 2 h at room temper-
ature, the reaction mixture was evaporated under vacuum. The
residue was taken up in EtOAc and washed twice with 5% citric
acid, saturated aqueous NaHCO3, and brine. The organic layer
In the first series (peptidomimetics 1–4), we chose the 1-(4-
(aminomethyl)benzyl)guanidine and aromatic a-guanidino amino
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Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.
J. Pept. Sci. 2012

SYNTHESIS AND ANTIMICROBIAL ACTIVITY OF SHORT CATIONIC PEPTIDOMIMETICS
was dried over Na2SO4 and the solvent evaporated in vacuum.
The product was purified by column chromatography eluting with
CHCl3/MeOH (99 : 1 to 95 : 5) yielding 1a (2.09 g, 86%). Rf = 0.43,
CHCl3/MeOH (95 : 5); 1H NMR (DMSO-d6) d: 1.23 and 1.84
(18H, 2 ꢁ s, OBut), 2.90–3.30 (4H, m, 2 ꢁ Trp b-CH2), 4.06–5.57
(5H, m, 1H Trp a-CH, 4H Ar CH2), 4.57 (1H, m, Trp a-CH), 6.82
(1H, d, J = 8.1 Hz, Trp NHCOBut), 6.93–7.54 (15H, m, 5H Trp IndH,
5H Trp IndH, 4H ArH, and 1H NHCOBut), 7.91 (1H, d, J = 7.8 Hz,
Trp NHCO), 8.36 (1H, t, NHCO), 10.78 and 10.82 (1H, 2 ꢁ br s, Trp
IndNH). 13C NMR (DMSO-d6) d: 28.25 and 28.50 (2ꢁ Trp b-CH2),
28.75 and 29.93 (2 ꢁ OBut), 42.57 and 43.80 (2 ꢁ Ar CH2), 54.13 and
56.16 (2 ꢁ Trp a-CH), 78.42 and 79.86 (2ꢁ OBut), 110.39–139.26
(aromatics), 155.93 and 156.46 (2ꢁ OCONH), 171.84 and 172.31
(2ꢁ CO). Anal. Calcd for C40H48N6O6: C, 67.78; H, 6.83; N, 11.86.
Found: C, 67.80; H, 6.80; N, 11.87.
was performed as described for compound 1a. The crude
product obtained was purified by column chromatography
using CHCl3/MeOH (95 : 5) as eluant to give 2a (1.40 g, 81%).
Rf = 0.56, CHCl3/MeOH (95 : 5). 1H NMR (CDCl3) d: 1.42
and 1.46 (18H, 2 ꢁ s, OBut), 3.06 (1H, dd, J = 5.1, 15.0Hz, Trp
b-CHA), 3.29 (1H, dd, J = 4.8, 14.4 Hz, Trp b-CHB), 4.06 (1H, dd,
J = 6.9, 14.7Hz, ArCHA), 4.26 (2H, d, J = 5.7Hz, ArCH2), 4.30
(1H, J = 7.2, 15.5Hz, ArCHB), 4.44 (1H, m, Trp a-CH), 5.20–5.41
(2H, 2 ꢁ m, NHCOBut), 5.96 (1H, m, NHCO), 6.57–7.64 (9H, m, 5H
Trp IndH, and 4H ArH), 8.67 (1H, br s Trp IndNH). 13C NMR (CDCl3)
d: 28.55 and 28.67(2 ꢁ OBut), 29.00 (Trp b-CH2), 43.29 and
44.61 (2ꢁ Ar CH2), 55.33 (Trp a-CH), 80.08 and 80.27 (2ꢁ OBut),
109.73–138.37 (aromatics), 155.65 and 156.53 (2ꢁ OCONH),
171.76 (CO). Anal. Calcd for C29H38N4O5: C, 66.64; H, 7.33; N, 10.72.
Found: C, 66.68; H, 7.34; N, 10.73.
N,N0-bis(Boc)amidino-L-tryptophan-4-((N,N0-bis(Boc)amidino)
N-Amidino-L-tryptophyl-L-tryptophan-4-((N-amidino)aminomethyl)
benzylamide (1)
aminomethyl)benzylamide (2c)
To a solution of 1a (1.83 g, 2.58 mmol) in dry CH2Cl2 (28 ml), TFA
was added (2.8 ml). The mixture was stirred at room temperature
under N2. After 1.5 h, the solution was evaporated to dryness and
the residue was repeatedly evaporated with ethyl ether to give
1b in quantitative yield. The product was used without further
purification (1.86 g, 98%).
Deprotection of 2a (615 mg, 1.18 mmol) was performed as described
for 1a to give 2b (640 mg, 98%). The product was used without
further purification.
Guanylation of 2b (680 mg, 1.24 mmol) was carried out according
to the general procedure. The crude product was purified by column
chromatography eluting with CH2Cl2/EtOAc (9 : 1) yielding 2c
(479 mg, 48%). Rf = 0.52, CH2Cl2/EtOAc (9 : 1). 1H NMR (CDCl3) d:
1.43, 1.44, 1.49 and 1.50 (36H, 4 ꢁ s, OBut), 3.09 (1H, dd, J= 9.3,
14.1 Hz, Trp b-CHA), 3.38 (1H, dd, J= 5.1, 14.1 Hz, Trp b-CHB), 3.97
(1H, dd, J= 4.5, 14.4 Hz, Ar CHA), 4.49 (1H, J= 6.9, 14.7 Hz, Ar CHB),
4.52 (2H, d, J= 5.1 Hz, Ar CH2), 4.83 (1H, m, Trp a-CH), 6.03 (1H, t,
J= 5.4 Hz, NHCO), 6.70–7.97 (9H, m, 5H Trp IndH, and 4H ArH), 8.29
(1H, br s Trp IndNH), 8.62 (1H, t, J= 5.1 Hz, NHCO), 9.04 (1H, d,
J= 6.9 Hz, NHCO) 11.32 and 11.57 (2H, 2 ꢁ br s, NHCOBut).
13C NMR (CDCl3) d: 28.28, 28.31, 28.44, 28.51 (4 ꢁ OBut), 28.96
(Trp b-CH2), 43.25 and 44.88 (2 ꢁ Ar CH2), 55.10 (Trp a-CH),
79.28, 79.84, 83.46 and 83.71 (4 ꢁ OBut), 110.59–137.71
(aromatics), 152.90 and 153.59 (2 ꢁ OCON) 155.61 and 156.73
(2 ꢁ OCONH), 163.47 and 163.73 (2 ꢁ CN) 170.70 (CO). Anal.
Calcd for C41H58N8O9: C, 61.02; H, 7.24; N, 13.89. Found: C,
61.02; H, 7.23; N, 13.90.
To a stirred solution of 1b (770 mg, 1.04 mmol) in dry THF
(10 ml), 3,4-dimethyl-1H-pyrazole-1-carboximidamidenitrate salt
(2.1 g, 10.45 mmol) and TEA (1.75 ml, 12 mmol) were added. After
16 h at reflux, the solvent was evaporated in vacuum and the
crude product purified by a Waters 600 HPLC system equipped
with an X-BridgeW Prep BEH130 C18, 5 mm (10 ꢁ 250 mm) column.
The purification was carried out using a solvent system of
H2O/CH3CN (0.1% TFA) in the form of a 30 min gradient: 10–90%
CH3CN in 20 min, 90% CH3CN for 5 min, and 90–10% CH3CN in
5 min, yielding 248 mg (40%) of 1. 1H NMR (DMSO-d6) d: 2.95–3.19
(4H, m, 2 ꢁ Trp b-CH2), 4.22 (2H, dd, J= 5.7, 13.2 Hz Ar CH2), 4.30
(2H, d, J= 6.0 Hz, Ar CH2), 4.45 (1H, m, Trp a-CH), 4.63 (1H, m, Trp
a-CH), 6.93–7.73 (22H, m, 5H Trp IndH, 5H Trp IndH, 4H ArH, and
8H NH), 7.72 (1H, d, J= 8.1 Hz, NHCN) 8.09 (1H, t, J= 5.7 Hz, NHCO),
8.42 (1H, d, J= 8.1 Hz,Trp NHCO), 8.51 (1H, t, J= 5.4 Hz, NHCN),
10.82 and 10.92 (1H, 2 ꢁ br s, Trp IndNH). 13C NMR (DMSO-d6) d:
28.61 and 28.90 (2 ꢁ Trp b-CH2), 42.53 and 44.39 (2 ꢁ Ar CH2),
54.49 and 55.40 (2 ꢁ Trp a-CH), 109.30–139.03 (aromatics), 157.02,
and 157.61 (2 ꢁ CN), 170.10 and 171.61 (2 ꢁ CO). Anal. Calcd for
C36H38F6N10O6: C, 52.68; H, 4.67; N, 17.07. Found: C, 52.65; H, 4.68;
N-amidino-L-tryptophan-4-((N-amidino)aminomethyl)benzylamide (2)
Deprotection of 2c (417 mg, 0.52 mmol) was carried out as
1
described for 1a, yielding 2 (320 mg, 98%). H NMR (DMSO-d6)
N, 17.10. MS (ESI): m/z 297.4 [M + 2H]++
.
d: 3.02 (1H, dd, J = 7.8, 15.0 Hz, Trp b-CHA), 3.23 (1H, dd, J = 4.8,
14.4 Hz, Trp b-CHB), 4.25 (2H, d, J = 5.4 Hz, Ar CH2), 4.30
(2H, d, J = 6.0 Hz, Ar CH2), 4.45 (1H, m, Trp a-CH), 6.92–7.67
(17H, m, 5H Trp IndH, 4H ArH, and 8H NH), 7.78 (1H, d,
J = 8.70 Hz, NHCN), 8.12 (1H, t, J = 6.3 Hz, NHCN), 8.66 (1H, t,
J= 5.6 Hz, NHCO), 10.94 (1H, br s Trp IndNH). 13C NMR (DMSO-d6)
d: 28.77 (Trp b-CH2), 42.77 and 44.32 (2 ꢁ Ar CH2), 55.69
(Trp a-CH), 109.13–138.82 (aromatics), 157.20, 157.58 (2 ꢁ CN),
170.70 (CO). Anal. Calcd for C25H28F6N8O5: C, 47.32; H, 4.45; N,
17.66. Found: C, 47.33; H, 4.46; N, 17.69. MS (ESI): m/z 204.2
General procedure for guanylation reactions of compounds 2b–8b
Guanylation reactions of compounds 2b–8b were carried out
according to the following general procedure. To a stirred solution
of peptidomimetic trifluoroacetate (1 mmol) in dry DMF (6 ml),
TEA (0.93 ml, 6.6 mmol), N,N0-bis(tert-butoxycarbonyl)-S-methyl-
isothiourea (310 mg, 2.1 mmol), and HgCl2 (598 mg, 2.2 mmol)
were added under N2 at 0 ꢀC. After 3.5 h, the mixture was diluted
with EtOAc, filtered through a pad of Celite, and washed with
water. The aqueous layer was extracted twice with EtOAc. The
combined organic layers were washed with brine, dried over
Na2SO4, and evaporated.
[M + 2H]++
.
Boc-L-phenylalanine-4-((Boc)aminomethyl)benzylamide (3a)
Coupling of 4-(tert-butoxycarbonyl-aminomethyl)benzylamine
(891 mg, 3.77 mmol) with Boc-L-phenylalanine (1.00 g, 3.77 mmol)
was performed as described for compound 1a. The crude
product obtained was purified by column chromatography using
Boc-L-tryptophane-4-((Boc)aminomethyl)benzylamide (2a)
Coupling of 4-(tert-butoxycarbonyl-aminomethyl)benzylamine
(777 mg, 3.29 mmol) with Boc-L-tryptophan (1.00 g, 3.29 mmol)
J. Pept. Sci. 2012
Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.
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BALDASSARRE ET AL.
CHCl3/MeOH (98 : 2) as eluant to give 3a (1.68 g, 98%). Rf = 0.69,
CHCl3/MeOH (98 : 2). 1H NMR (CDCl3) d: 1.32 and 1.43
(18H, 2 ꢁ s, OBut), 3.04 (1H, d, J = 7.7 Hz, Phe b-CH2), 4.23
(1H, d, J = 6.9 Hz, ArCH2), 4.29 (2H, d, J = 6.0 Hz, ArCH2), 4.34
(1H, m, Phe a-CH), 4.94 (1H, t, J = 6.9 Hz, NHCOBut), 5.19
(1H, d, J = 7.9 Hz, NHCOBut), 6.33 (1H, t, J = 6.0 Hz, NHCO),
7.00–7.24 (5H, m, 5H Phe Ar, and 4H ArH). 13C NMR (CDCl3)
d: 28.48 and 28.64 (2 ꢁ OBut), 38.53 (Phe b-CH2), 43.29
and 44.50 (2 ꢁ Ar CH2), 56.20 (Phe a-CH), 79.71 and 80.37
(2 ꢁ OBut), 127.11–138.45 (aromatics), 155.65 and 156.13
(2 ꢁ OCONH), 171.40 (CO). Anal. Calcd for C27H37N3O5: C,
67.06; H, 7.71; N, 8.69. Found: C, 67.10; H, 7.69; N, 10.75.
Tyr ArH), 6.89 (1H, d, J= 8.9 Hz, NHCOBut), 7.01 (2H, d, J= 9.5 Hz,
Tyr ArH), 7.06–7.18 (4H, m, ArH), 7.34 (1H, t, J= 5.9 Hz, NHCOBut),
8.33 (1H, t, J=5.5 Hz, NHCO) 9.17 (1H, s, Tyr OH). 13C NMR (CDCl3)
d: 28.84 and 28.92 (2 ꢁ OBut), 37.36 (Tyr b-CH2), 42.41 and
43.77 (2 ꢁ Ar CH2), 56.99 (Tyr a-CH), 79.40 and 78.61 (2 ꢁ OBut),
115.73–139.27 (aromatics), 155.96 and 156.42 (2 ꢁ OCONH),
172.51 (CO). Anal. Calcd for C27H37N3O6: C, 64.91; H, 7.46; N, 8.41.
Found: C, 64.91; H, 7.47; N, 8.43.
N,N0-bis(Boc)amidino-L-tyrosine-4-((N,N0-bis(Boc)amidino)aminomethyl)
benzylamide (4c)
Deprotection of 4a (950 mg, 1.84 mmol) was performed as described
for 1a to give 4b (968 mg, quantitative). The product was used
without further purification.
N,N0-bis(Boc)amidino-L-phenylalanine-4-((N,N0-bis(Boc)amidino)
aminomethyl)benzylamide (3c)
Guanylation of 4b (900 mg, 1.71 mmol) was carried out according
to the general procedure. The crude product was purified by column
chromatography eluting with CH2Cl2/EtOAc (9 : 1) yielding 4c
(964 mg, 72%). Rf = 0.71, CH2Cl2/EtOAc (3 : 1). 1H NMR (CDCl3) d:
1.42, 1.45, 1.49, and 1.50 (36H, 4 ꢁ s, OBut), 2.82 (1H, dd, J= 6.2,
13.2 Hz, Tyr b-CHA), 3.10 (1H, dd, J= 5.7, 13.8 Hz, Tyr b-CHB), 3.94
(1H, dd, J= 4.2, 14.1 Hz, Ar CHA), 4.47–4.70 (4H, m, 1H Ar CHB, 1H
Tyr a-CH, 2H Ar CH2), 6.10 (1H, t, J= 5.9 Hz, NHCO), 6.44 (2H, d,
J= 8.7 Hz, Tyr ArH), 6.87–6.92 (4H, m, ArH), 7.11 (2H, d, J= 8.4 Hz,
Tyr ArH), 8.76 (1H, t, J= 7.4 Hz, NHCO), 8.92 (1H, d, J= 7.8 Hz, NHCO)
11.35 and 11.61 (2H, 2 ꢁ br s, NHCOBut).13C NMR (CDCl3) d: 28.27,
28.32, 28.34, 28.46 (4 ꢁ OBut), 37.79 (Tyr b-CH2), 43.06 and 44.40
(2 ꢁ Ar CH2), 56.34 (Tyr a-CH), 79.65, 80.16, 83.63 and 83.92
(4 ꢁ OBut), 115.54–137.07 (aromatics), 152.94 and 153.58
(2 ꢁ OCON) 155.78 and 156.72 (2 ꢁ OCONH), 163.35 and
163.46 (2 ꢁ CN) 169.89 (CO). Anal. Calcd for C39H57N7O10: C,
59.75; H, 7.33; N, 12.51. Found: C,59.74; H, 7.28; N, 12.52.
Deprotection of 3a (843 mg, 1.65 mmol) was performed as described
for 1a to give 3b (843mg, quantitative). The product was used
without further purification.
Guanylation of 3b (800 g, 1.56 mmol) was carried out according to
the general procedure. The crude product was purified by column
chromatography eluting with CH2Cl2/EtOAc (9 : 1) yielding 3c
(831 mg, 69%). Rf = 0.91, CH2Cl2/EtOAc (3 : 1). 1H NMR (CDCl3) d:
1.41, 1.47, 1.49 and 1.51 (36H, 4 ꢁ s, OBut), 3.14 (1H, dd, J= 7.5,
13.8 Hz, Phe b-CHA), 3.19 (1H, dd, J= 7.5, 13.8 Hz, Phe b-CHB), 4.27
(1H, dd, J= 5.4, 15.3 Hz, Ar CHA), 4.41 (1H, dd, J= 6.0, 15.3 Hz, Ar
CHB), 4.58 (2H, d, J= 5.1 Hz, Ar CH2), 4.74 (1H, m, Phe a-CH), 6.85
(1H, t, J=5.5 Hz, NHCO), 7.08–7.24 (9H, m, 5H Phe ArH, and 4H
ArH), 8.57 (1H, br s, NHCO), 8.80 (1H, d, J= 7.2 Hz, NHCO) 11.28 and
11.53 (2H, 2 ꢁ br s, NHCOBut).13C NMR (CDCl3) d: 28.22, 28.26,
28.40, 28.52 (4 ꢁ OBut), 37.67 (Phe b-CH2), 43.40 and 44.89 (2 ꢁ Ar
CH2), 56.17 (Phe a-CH), 79.59, 79.65, 83.43, and 83.72 (4 ꢁ OBut),
127.13–137.48 (aromatics), 152.90 and 153.39 (2 ꢁ OCON), 156.10
and 156.31 (2 ꢁ OCONH), 163.15 and 163.80 (2 ꢁ CN) 170.46 (CO).
Anal. Calcd for C39H57N7O9: C, 61.00; H, 7.48; N, 12.77. Found: C,
61.02; H, 7.51; N, 12.71.
N-amidino-L-tyrosine-4-((N-amidino)aminomethyl)benzylamide (4)
Deprotection of 4c (500 mg, 0.64 mmol) was carried out as
1
described for 1a, yielding 4 (566 mg, 97%). H NMR (DMSO-d6)
d: 2.74 (1H, dd, J = 7.2, 12.1 Hz, Tyr b-CHA), 2.95(1H, dd, J = 4.8,
12.5 Hz, Tyr b-CHB), 4.18–4.67(9H, m, 1H Tyr a-CH, 4H Ar CH2, 4H
NH),6.63 (2H, d, J=8.7Hz, Tyr ArH), 6.98 (2H, d, J= 8.9 Hz, Tyr ArH),
7.09–7.62(8H, m, 4H ArH, and 4H NH), 7.81(1H, d, J= 8.3 Hz, NHCN),
8.18 (1H, t, J=5.4 Hz, NHCN), 8.64 (1H, t, J= 6.0 Hz, NHCO). 13C NMR
(DMSO-d6) d: 38.23 (Tyr b-CH2),42.86 and 43.90 (2 ꢁ Ar CH2),56.57
(Tyr a-CH), 115.18–138.7 (aromatics),157.63, and 157.98 (2 ꢁ CN),
169.24 (CO). Anal. Calcd for C23H27F6N7O6: C, 45.18; H, 4.45; N,
16.03. Found: C, 45.21; H, 4.46; N, 16.02. MS (ESI): m/z 192.4
N-amidino-L-phenylalanine-4-((N-amidino)aminomethyl)
benzylamide (3)
Deprotection of 3c (500 mg, 0.65 mmol) was carried out as
1
described for 1a, yielding 3 (572 mg, 98%). H NMR (DMSO-d6)
d: 2.84 (1H, dd, J=5.5, 13.7 Hz, Phe b-CHA), 3.09 (1H, dd, J=5.4,
13.5 Hz, Phe b-CHB), 4.19 (1H, dd, J=5.6, 13.9 Hz, Ar CHA), 4.26–4.32
(3H, m, 1H Ar CHB, and 2H Ar CH2), 4.43 (1H, m, Phe a-CH), 4.81
(4H, br s, NH), 7.09–7.42 (13H, m, 5H Phe ArH, 4H ArH, and 4H NH),
7.88 (1H, d, J=9.0 Hz, NHCN), 8.16 (1H, t, J=6.0 Hz, NHCN), 8.69
(1H, t, J=5.7 Hz, NHCO). 13C NMR (DMSO-d6) d: 38.53 (Phe b-CH2),
42.67 and 44.27 (2 ꢁ Ar CH2), 56.25 (Phe a-CH), 127.85–138.74
(aromatics), 157.21 and 157.61 (2 ꢁ CN), 169.85 (CO). Anal. Calcd
for C23H27F6N7O5: C, 46.39; H, 4.57; N, 16.46. Found: C, 46.35;
[M + 2H]++
.
3,5-bis((Boc)amino)benzoyl-L-tryptophyl-L-tryptophan methyl ester (5a)
To a stirred solution of 3,5-bis((Boc)amino)benzoic acid (544 mg,
1.54 mmol) in dry DMF (15 ml), HOBt (254 mg, 1.88 mmol), HBTU
(647 mg, 1.70 mmol), DIPEA (0.59 ml, 3.39 mmol), L-tryptophyl-L-
tryptophan methyl ester TFA salt (800 mg, 1.54 mmol), and DIPEA
(0.27 ml, 1.54 mmol) were added at 0 ꢀC under N2. After 10 min at
0 ꢀC and 2.5 h at room temperature, the reaction mixture was
diluted with EtOAc and washed twice with 5% citric acid,
saturated aqueous NaHCO3, and brine. The organic layer was
dried over Na2SO4 and evaporated under vacuum. The crude
product was purified by column chromatography eluting with
CH2Cl2/EtOAc (5 : 1 to 1 : 1) to give 5a (610 mg, 54%). Rf = 0.73,
H, 4.49; N, 16.45. MS (ESI): m/z 184.7 [M + 2H]++
.
Boc-L-tyrosine-4-((Boc)aminomethyl)benzylamide (4a)
Coupling of 4-(tert-butoxycarbonyl-aminomethyl)benzylamine
(891 mg, 3.77 mmol) with Boc-L-phenylalanine (1.06 g, 3.77 mmol)
was performed as described for compound 1a. The crude
product obtained was purified by column chromatography
using CHCl3/MeOH (95 : 5) as eluant to give 4a (1.84 g, 99%).
1
Rf = 0.57, CHCl3/MeOH (95 : 5). H NMR (CDCl3) d: 1.31 and 1.35
1
(18H, 2 ꢁ s, OBut), 2.48 (1H, dd, J = 5.4, 13.8 Hz, Tyr b-CHA), 2.83
(1H, dd, J = 5.5, 14.0 Hz, Tyr b-CHB), 4.05 (3H, m, 2H ArCH2, 1H
Tyr a-CH), 4.23 (2H, d, J = 7.6 Hz, ArCH2), 6.64 (2H, d, J = 8.8 Hz,
CH2Cl2/EtOAc (1 : 1). H NMR (CDCl3) d: 1.53 (18H, s, OBut), 3.15–3.23
(3H, m, Trp b-CH2 and Trp b-CHA), 3.36 (1H, dd, J = 6.6, 15.0Hz,
Trp b-CHB), 3.62 (3H, s, OMe), 4.83 (1H, m, Trp a-CH), 4.97 (1H, m,
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Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.
J. Pept. Sci. 2012

SYNTHESIS AND ANTIMICROBIAL ACTIVITY OF SHORT CATIONIC PEPTIDOMIMETICS
Trp a-CH), 6.66 (1H, d, J = 7.8Hz, NHCO), 6.74–7.65 (14H, m, 5H Trp
IndH, 5H Trp IndH, and 3H ArH, 1H NHCO), 8.29 (1H, br s Trp
IndNH), 9.12 (2H, br s NHCOBut). 13C NMR (CDCl3) d: 26.55 and
27.75 (2 ꢁ Trp b-CH2), 28.57 (2ꢁ OBut), 52.66 and 52.84 (2ꢁ Trp
a-CH), 54.16 (OCH3), 81.14 (2ꢁ OBut), 108.81–139.46 (aromatics),
153.05 (2 ꢁ OCONH), 167.56, 171.67, and 172.46 (3 ꢁ CO). Anal.
Calcd for C40H46N6O8: C, 65.03; H, 6.28; N, 11.37. Found: C, 65.02;
H, 6.27; N, 11.35.
172.45 (2ꢁ CO). Anal. Calcd for C29H36N4O7: C, 63.03; H, 6.57; N,
10.14. Found: C, 63.00; H, 6.58; N, 10.13.
3,5-Bis((N,N0-bis(Boc)amidino)amino)benzoyl-L-tryptophan methyl
ester (6c)
Deprotection of 6a (1.00 g, 1.81 mmol) was carried out as described
for 1a, yielding 6b (1.03 g, 97%). The product was used without
further purification.
Guanylation of 6b (500 mg, 0.86 mmol) was carried out according
to the general procedure. The crude product was purified by
column chromatography eluting with CH2Cl2/EtOAc (9 : 1)
yielding 6c (385 mg, 53%). Rf = 0.57, CH2Cl2/EtOAc (9 : 1). 1H
NMR (CDCl3) d:1.46 and 1.51 (36H, 2 ꢁ s, OBut), 3.42 (2H, m, Trp
b-CH2), 3.68 (3H, s, OMe), 5.04 (1H, m, Trp a-CH), 6.80 (1H, d,
J = 7.80 Hz, NHCO), 7.06–8.14 (8H, m, 5H Trp IndH and 3H ArH),
8.21 (1H, br s, Trp IndNH), 10.47 (2H, br s, NHCN), 11.54 (2H, br
s, NHC OBut). 13C NMR (CDCl3) d: 28.21 (Trp b-CH2), 28.26 and
28.35 (2 ꢁ OBut), 52.64 (OMe), 53.39 (Trp a-CH), 80.29 and
84.25 (2 ꢁ OBut), 110.18-137.81 (Aromatics), 153.33 (2 ꢁ OCON),
153.87 (2 ꢁ OCONH), 163.35 (2 ꢁ CN), 166.45 and 172.45 (2 ꢁ CO).
Anal. Calcd for C41H56N8O11: C, 58.84; H, 6.74; N, 13.39. Found:
C, 58.85; H, 6.76; N, 13.37.
3,5-bis((N,N0-bis(Boc)amidino)amino)benzoyl-L-tryptophyl-L-tryptophan
methyl ester (5c)
Deprotection of 5a (210 mg, 0.28 mmol) was carried out as
described for 1a, yielding 5b (218 mg, 90%). The product was
used without further purification.
Guanylation of 5b (197 mg, 0.26 mmol) was carried out according
to the general procedure. The crude product was purified by column
chromatography eluting with CH2Cl2/EtOAc (5 : 1 to 3 : 1), yielding
5c (113 mg, 42.32%). Rf = 0.63, CH2Cl2/EtOAc (3 : 1). 1H NMR (CDCl3)
d: 1.43 and 1.56 (36H, 2 ꢁ s, OBut), 3.10 (1H, dd, J= 7.2, 15.3 Hz, Trp
b-CHA), 3.28 (3H, m, Trp b-CH2 and Trp b-CHB), 3.71 (3H, s, OMe),
4.75–4.86 (2H, m, 2 ꢁ Trp a-CH), 6.46 (1H, d, J= 6.6 Hz, NHCO),
6.73–7.87 (14H, m, 1H NHCO, 5H Trp IndH, 5H Trp IndH, and 3H
ArH), 8.13 and 9.05 (2H, 2 ꢁ br s, Trp IndNH), 10.36 and 11.65
(4H, 2 ꢁ br s, NHCOBut). 13C NMR (CDCl3) d: 26.34 and 27.46
(2 ꢁ Trp b-CH2), 28.21 and 28.31 (2 ꢁ OBut), 52.47 and 52.64
(2 ꢁ Trp a-CH), 53.56 (OMe), 80.51 and 84.42 (2 ꢁ OBut),
108.46–136.36 (aromatics), 153.45 (2 ꢁ OCON), 154.31 (2 ꢁ OCONH),
163.37 (CN), 166.33, 171.11, and 172.52 (3 ꢁ CO). Anal. Calcd
for C52H66N10O12: C, 61.04; H, 6.50; N, 13.69. Found: C, 61.02; H,
6.51; N, 13.70.
3,5-Bis(N-amidino)aminobenzoyl-L-tryptophan methyl ester (6)
Deprotection of 6c (300 mg, 0.36 mmol) was performed as
described for 5c to give 6 (234 mg, 98%). 1H NMR (DMSO-d6)
d: 3.24 (2H, m, Trp b-CH2), 3.61 (3H, s, OMe), 4.72 (1H, m, Trp
a-CH), 6.97–7.71 (16H, m, 5H Trp IndH and 3H ArH, 8 NH), 8.97
(1H, d, J = 6.90 Hz, NHCO), 10.16 (2H, br s, NHCN), 10.85 (1H, br
s, Trp IndNH). 13C NMR (DMSO-d6) d: 29.31 (Trp b-CH2), 53.65
(OMe), 55.08 (Trp a-CH), 111.68–138.81 (aromatics), 157.03 (2 ꢁ CN),
165.35 and 172.92 (2 ꢁ CO). Anal. Calcd for C25H26F6N8O7: C, 45.19;
H, 3.94; N, 16.86. Found: C, 45.20; H, 3.93; N, 16.84. MS (ESI):
3,5-Bis(N-amidino)aminobenzoyl-L-tryptophyl-L-tryptophan methyl
ester (5)
m/z 219.2 [M + 2H]++
.
To a stirred solution of 5c (108 mg, 0.10 mmol) in dry CH2Cl2
(4 ml), TFA (4 ml) was added under N2. After 1.5 h, the solution
was evaporated to dryness and the residue was repeatedly
evaporated with ether to give 5 (85 mg, 96%). The product
was used without further purification. 1H NMR (DMSO-d6) d:
3.04–3.22 (4H, m, 2 ꢁ Trp b-CH2), 3.54 (3H, s, OMe), 4.56 and
4.85 (2H, m, 2 ꢁ Trp a-CH), 6.94–7.71 (21H, m, 5H Trp IndH, 5H
Trp IndH, 3H ArH, 8H NH2), 8.64–8.71 (2H, m, 2 ꢁ NHCO), 10.14
(2H, s, 2 ꢁ NHCN) 10.79 and 10.90 (2H, 2 ꢁ br s, Trp IndNH). 13C
NMR (DMSO-d6) d: 27.62 and 28.36 (2 ꢁ Trp b-CH2), 52.54 (OMe),
54.02 and 54.53 (2 ꢁ Trp a-CH), 109.97–137.43 (aromatics),
156.78 (2 ꢁ CN), 165.24, 172.57, and 172.90 (3 ꢁ CO). Anal. Calcd
for C36H36F6N10O8: C, 50.83; H, 4.27; N, 16.46. Found: C, 50.83;
3,5-Bis((Boc)amino)benzoyl-L-phenylalanine methyl ester (7a)
Coupling of 3,5-bis((Boc)amino)benzoic acid (700 mg, 1.99 mmol)
with L-phenylalanine methyl ester hydrochloride (428 mg, 1.99 mmol)
was performed as described for compound 5a. The crude
product obtained was purified by column chromatography
using CHCl3/MeOH (99 : 1) as eluant to give 7a (850 mg, 83%).
Rf = 0.65, CHCl3/MeOH (98 : 2). 1H NMR (CDCl3) d: 1.50 (18H,
s, OBut), 3.21 (2H, d, J = 5.7 Hz, Phe b-CH2), 3.72 (3H, s, OMe),
5.03 (1H, m, Phe a-CH), 6.63 (1H, d, J = 7.8 Hz, NHCO), 6.65
(2H, s, NHCOBut), 7.14–7.33 (5H, m, 5H Phe ArH), 7.38 (2H, d,
J = 1.8 Hz, ArH), 7.74 (1H, t, J = 1.8 Hz, ArH). 13C NMR (CDCl3)
d: 28.49 (OBut), 37.29 (Phe b-CH2), 52.68 (OMe), 54.24 (Phe a-CH),
81.11 (2ꢁ OBut), 112.24–139.65 (aromatics), 153.18 (2ꢁ OCONH),
167.45 and 172.48 (2 ꢁ CO). Anal. Calcd for C27H35N3O7: C, 63.14;
H, 6.87; N, 8.18. Found: C, 63.15; H, 6.89; N, 8.15.
H, 4.25; N, 16.48. MS (ESI): m/z 312.2 [M + 2H]++
.
3,5-Bis((Boc)amino)benzoyl-L-tryptophan methyl ester (6a)
Coupling of 3,5-bis((Boc)amino)benzoic acid (806 mg, 2.29 mmol)
with L-tryptophan methyl ester hydrochloride (583 mg, 2.29 mmol)
was performed as described for compound 5a. The crude
product obtained was purified by column chromatography
using CHCl3/MeOH (99 : 1) as eluant to give 6a (1.14 g, 90%).
Rf = 0.63, CHCl3/MeOH (95 : 5). 1H NMR (CDCl3) d: 1.50 (18H, s,
OBut), 3.41 (2H, d, J = 5.4 Hz, Trp b-CH2), 3.68 (3H, s, OMe), 5.06
(1H, m, Trp a-CH), 6.75 (1H, d, J = 8.1 Hz, NHCO), 7.07–7.56
(8H, m, 5H Trp IndH, and 3H ArH), 7.77 (1H, NHCOBut), 8.31
(1H, br s, Trp IndNH). 13C NMR (CDCl3) d: 27.82 (Trp b-CH2),
28.54 (OBut), 52.71 (OMe), 53.29 (Trp a-CH), 81.00 (2 ꢁ OBut),
109.82–139.73 (aromatics), 152.93 (2 ꢁ OCONH), 166.35, and
3,5-Bis((N,N0-bis(Boc)amidino)amino)benzoyl-L-phenylalanine methyl
ester (7c)
Deprotection of 7a (700 mg, 1.36 mmol) was carried out as
described for 1a, yielding 7b (722 mg, 98%). The product was
used without further purification.
Guanylation of 7b (500 mg, 0.92 mmol) was carried out according
to the general procedure. The crude product was purified by
column chromatography eluting with CH2Cl2/EtOAc (98 : 2)
yielding 7c (551 mg, 75%). Rf = 0.61, CH2Cl2/EtOAc (98 : 2). 1H
NMR (CDCl3) d:1.49 and 1.53 (36H, 2 ꢁ s, OBut), 3.15 (2H, dd,
J. Pept. Sci. 2012
Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.
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BALDASSARRE ET AL.
J = 6.3, 13.8 Hz, Phe b-CHA), 3.25 (2H, dd, J = 5.7, 13.5 Hz, Phe
b-CHB), 3.72 (3H, s, OMe), 5.00 (1H, m, Phe a-CH), 6.80
(1H, d, J = 7.8 Hz, NHCO), 7.21–7.31 (5H, m, Phe ArH), 7.88
(2H, d, J = 1.8 Hz, ArH), 8.18 (1H, t, J = 1.8, ArH), 10.52 (2H, br
s, NHCN), 11.56 (2H, br s, NHC OBut). 13C NMR (CDCl3) d:
28.18 and 28.26 (2 ꢁ OBut), 36.86 (Phe b-CH2), 52.54 (OMe),
54.07 (Phe a-CH), 80.25 and 84.36 (2 ꢁ OBut), 116.69–138.61
(aromatics), 153.34 (2 ꢁ OCON), 153.68 (2 ꢁ OCONH), 163.21
(2 ꢁ CN), 166.49 and 172.10 (2 ꢁ CO). Anal. Calcd for
(2 ꢁ CO). Anal. Calcd for C39H55N7O12: C, 57.55; H, 6.81; N,
12.05. Found: C, 57.58; H, 6.80; N, 12.07.
3,5-Bis(N-amidino)aminobenzoyl-L-tyrosine methyl ester (8)
Deprotection of 8c (300 mg, 0.37 mmol) was performed as
1
described for 5c to give 8 (232 mg, 98%). H NMR (DMSO-d6)
d: 2.95 (2H, dd, J = 5.8, 13.2 Hz Tyr b-CHA), 3.03 (2H, dd, J = 5.4,
14.1 Hz Tyr b-CHB), 3.60 (3H, s, OMe), 4.58 (1H, m, Tyr a-CH),
6.63 (2H, d, J = 8.4 Hz, Tyr ArH), 7.04 (2H, d, J = 8.7 Hz, Tyr
ArH), 7.34 (1H, t, J = 1.8 Hz, ArH), 7.56 (1H, t, J = 1.8 Hz, ArH),
7.74 (9H, br s, NH), 8.94 (1H, d, J = 7.8 Hz, NHCO), 10.22
(2H, br s, NHCN). 13C NMR (DMSO-d6) d: 36.19 (Tyr b-CH2),
52.68 (OMe), 55.36 (Tyr a-CH), 115.20–137.48 (aromatics),
156.64 (2 ꢁ CN), 165.41 and 172.79 (2 ꢁ CO). Anal. Calcd for
C
39H55N7O11: C, 58.71; H, 6.95; N, 12.29. Found: C, 58.68; H,
6.91; N, 12.30.
3,5-Bis(N-amidino)aminobenzoyl-L-phenylalanine methyl ester (7)
Deprotection of 7c (300 mg, 0.48 mmol) was performed as
described for 5c to give 7 (297 mg, 99%). 1H NMR (DMSO-d6)
d: 3.07 (2H, dd, J = 3.6, 10.2 Hz Phe b-CHA), 3.17 (2H, dd, J = 4.8,
13.8 Hz Phe b-CHB), 3.63 (3H, s, OMe), 4.68 (1H, m, Phe
a-CH), 7.17–7.29 (5H, m, Phe ArH), 7.35 (1H, t, J = 2.0 Hz, ArH),
7.55 (2H, d, J = 2.1 Hz, ArH), 7.75 (8H, br s, NH), 9.00 (1H, d,
J = 7.8 Hz, NHCO), 10.16 (2H, br s, NHCN). 13C NMR (DMSO-d6) d:
36.28 (Phe b-CH2), 52.54 (OMe), 55.18 (Phe a-CH), 117.6–138.61
(aromatics), 156.13 (2 ꢁ CN), 165.21 and 172.80 (2 ꢁ CO). Anal.
Calcd for C23H25F6N7O7: C, 44.17; H, 4.03; N, 15.68. Found: C,
C
23H25F6N7O8: C, 43.06; H, 3.93; N, 15.28. Found: C, 43.08; H,
3.91; N, 15.27. MS (ESI): m/z 207.5 [M + 2H]++
.
Hydrophobicity
All SAMPs derivatives 1–8 were analyzed by RP-HPLC using a
solvent system of H2O/CH3CN (0.1% TFA) in the form of a linear
gradient from 10 to 80% of CH3CN in 30 min and a flow rate of
1 ml/min.
44.15; H, 4.05; N, 15.70. MS (ESI): m/z 199.6 [M + 2H]++
.
Antimicrobial Activity
3,5-Bis((Boc)amino)benzoyl-L-tyrosine methyl ester (8a)
Two Gram-positive (S. aureus ATCC 29213 and S. epidermidis
ATCC 35984) and two Gram-negative (E. coli ATCC 8739 and K.
pneumoniae 5F) strains, and one fungal strain (C. albicans ATCC
10231) – all biofilm producers – were used for the detection of
antibacterial activity of the SAMPs derivatives 1–8.
Coupling of 3,5-bis((Boc)amino)benzoic acid (700 mg, 1.99 mmol)
with L-tyrosine methyl ester (388 mg, 1.99 mmol) was performed
as described for compound 5a. The crude product obtained
was purified by column chromatography using CH2Cl2/EtOAc
(3 : 1) as eluant to give 8a (894 mg, 85%). Rf = 0.47, CH2Cl2/EtOAc
The peptidomimetic effect on planktonic cells was evaluated
by MIC and MBC using the broth microdilution method
according to CLSI guidelines [31]. Bacterial suspensions, grown
in Mueller–Hinton broth at logarithmic phase, were incubated
on microtiter plates at a concentration of 5 ꢁ 105 CFU/ml, with
several peptide dilutions – 1, 2.5, 5, 7.5, 10, 15.6, 25, 31.3, 50,
62.5, 75, 100, 125, 150, 200, 250, and 300 mg/ml – for 24 h at
37 ꢀC. The MIC was defined as the lowest concentration of
peptide giving a complete inhibition of visible growth in
comparison with a peptide-free control well, and the MBC
was determined as the lowest concentration at which no
bacterial growth occurred on Mueller–Hinton agar plates.
The MIC detection of C. albicans ATCC 10231 was performed
in Mueller–Hinton broth for an incubation of 48 h at 25 ꢀC. Data
were obtained from at least three independent experiments
performed in duplicate. The susceptibility of the bacterial strains
against ciprofloxacin was used as an internal standard during
MIC determinations. Another complete experiment was conducted
by using buffered peptone water – in the same experimental
conditions – to compare the MIC and MBC results obtained with
Mueller–Hinton broth.
The efficacy on established biofilm was evaluated by determining
BIC and BEC according to the method described by Johnson et al.
[32] with some modifications. Bacterial suspensions, grown in tryptic
soy broth supplemented with 0.5% (v/v) glucose at logarithmic
phase, were incubated on flat-bottomed microtiter plates at a
concentration of 5 ꢁ 105 CFU/ml. After 24 h of incubation at 37ꢀC,
the planktonic cells were gently removed and the wells were
washed with sterile phosphate-buffered saline (PBS) pH 7.3 and
filled with different peptide dilutions ranging from the MIC values
to a maximum concentration of 1200 mg/ml. The OD600 was
1
(3 : 1). H NMR (CDCl3) d: 1.45 (18H, s, OBut), 3.01 (2H, dd, J = 6.3,
13.8 Hz, Tyr b-CHA), 3.06 (2H, dd, J = 5.4, 14.0 Hz, Tyr b-CHB), 3.65
(3H, s, OMe), 4.94 (1H, m, Tyr a-CH), 6.64 (2H, d, J = 8.1 Hz,
Tyr ArH), 6.92 (3H, m, 2H Tyr ArH, and 1H NHCO), 7.08 (2H, s,
NHCOBut), 7.32 (2H, d, J = 2.0 Hz, ArH), 7.76 (1H, t, J = 2.0 Hz,
ArH). 13C NMR (CDCl3) d: 28.51 (OBut), 37.41 (Tyr b-CH2), 52.68
(OMe), 54.98 (Tyr a-CH), 81.00 (2 ꢁ OBut), 115.12–138.84
(aromatics), 153.45 (2 ꢁ OCONH), 168.25 and 172.32 (2 ꢁ CO).
Anal. Calcd for C27H35N3O8: C, 61.23; H, 6.66; N, 7.93. Found: C,
61.23; H, 6.67; N, 7.93.
3,5-Bis((N,N0-bis(Boc)amidino)amino)benzoyl-L-tyrosine methyl
ester (8c)
Deprotection of 8a (700 mg, 1.32 mmol) was carried out as
described for 1a, yielding 8b (714 mg, 97%). The product was
used without further purification.
Guanylation of 8 (500 mg, 0.90 mmol) was carried out according
to the general procedure. The crude product was purified by
column chromatography eluting with CH2Cl2/EtOAc (98 : 2)
yielding 8c (601 mg, 82%). Rf = 0.41, CH2Cl2/EtOAc (98 : 2). 1H
NMR (CDCl3) d:1.50 and 1.52 (36H, 2 ꢁ s, OBut), 3.03 (2H, dd,
J = 5.5, 13.8 Hz, Tyr b-CHA), 3.15 (2H, dd, J = 5.3, 13.5 Hz, Tyr
b-CHB), 3.70 (3H, s, OMe), 4.96 (1H, m, Tyr a-CH), 6.68
(2H, d, J = 8.4 Hz, Tyr ArH), 6.86 (1H, d, J = 7.9 Hz, NHCO), 7.04
(2H, d, J = 8.4 Hz, Tyr ArH), 7.83 (2H, d, J = 1.8 Hz, ArH), 8.14
(1H, t, J = 1.8 Hz, ArH), 10.49 (2H, br s, NHCN), 11.52 (2H, br s,
NHC OBut). 13C NMR (CDCl3) d: 28.20 and 28.27 (2 ꢁ OBut),
36.86 (Phe b-CH2), 52.61 (OMe), 54.56 (Phe a-CH), 80.15 and
84.21 (2 ꢁ OBut), 115.08–138.61 (aromatics), 154.04 (2 ꢁ OCON),
154.37 (2 ꢁ OCONH), 164.72 (2 ꢁ CN), 166.21 and 172.35
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SYNTHESIS AND ANTIMICROBIAL ACTIVITY OF SHORT CATIONIC PEPTIDOMIMETICS
measured at time 0 and after incubation for 24 h at 37 ꢀC. The BIC
was determined as the lowest concentration of the peptide inhibit-
ing growth in the supernatant fluid, confirmed by no increase in
optical density compared with the initial reading. The BEC was
determined as the lowest concentration at which no bacterial
growth occurred on tryptic soy agar plates. Data were obtained from
at least three independent experiments performed in duplicate.
Hemolytic Activity
Hemolytic activity of the reference compound was not tested
because it is known in literature [17]. Hemolytic activity of the
SAMPs derivatives 1–8 was tested against human red blood
cells (h-RBCs). Fresh human blood, collected with EDTA, was
centrifuged at 3000 rpm for 5 min then washed three times with
PBS pH 7.3. Red blood cells were diluted to 4% in PBS and incu-
bated with different peptide dilutions ranging from 1.25 to
200 mg/ml for SAMPs derivatives 1–2 and 5–6 and from 1.25 to
800 mg/ml for 3–4 and 7–8. After 1 h of incubation at 37 ꢀC, the
suspensions were sedimented by centrifugation and the release
of hemoglobin was determined by absorbance measurement at
405 nm and compared with a 0% hemolysis control (PBS) and a
100% hemolysis control (PBS with 1% v/v Triton X-100). The
percentage of hemolysis was calculated using the following
equation:
Scheme 1. Reagents and condition: (i) Boc-L-tryptophan-L-tryptophan,
HBTU, HOBt, DIPEA, DMF, 0 ꢀC, 10 min, and room temperature, 2.5 h; (ii)
TFA/CH2Cl2 (1 : 10), room temperature, 1.5 h; (iii) 3,4-dimethyl-1H-pyra-
zole-1-carboximidamide nitrate salt, TEA, THF, reflux, 16 h.
reactions were performed using the HBTU/HOBt method to
give the Boc-protected peptidomimetics 1a–8a. After the
removal of Boc-protecting groups, the introduction of guanidine
moiety in compound 1b to obtain 1, was carried out using 3,4-
dimethyl-1H-pyrazole-1-carboximidamidenitrate salt as guanyla-
tion reagent [33]. Unfortunately, the use of this strategy in the
synthesis of the SAMPs derivatives 2–8 resulted disappointing,
giving low yields and difficulties in the isolation and purification
of the products. Thus, for the peptidomimetics 2–8 an alternative
guanylation strategy was employed. The guanidine moiety was
added in the compounds 2b-8b as Boc-protected functionality
by reaction with N,N0-bis(Boc)-S-methyl-isothiourea in the
presence of mercury chloride [34]. After the cleavage of the
Boc-protecting group using TFA/CH2Cl2, the final products were
obtained in good yields as trifluoacetate salts.
Hemolysisð%Þ ¼ ½ðOD405sample ꢂ OD4050% lysis controlÞ=
ðOD405100% lysis control ꢂ OD4050% lysis controlÞꢃ ꢁ 100:
Results and Discussion
Synthesis
The SAMPs derivatives 1–8 reported in Figure 1 were synthesized
according to the strategies shown in Schemes 1–4. The coupling
Figure 1. Structures of synthesized SAMPs derivatives 1–8.
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BALDASSARRE ET AL.
Scheme 2. Reagents and conditions: (i) Boc-L-tryptophan, HBTU, HOBt, DIPEA, DMF, 0 ꢀC, 10 min, and room temperature, 2.5 h; (ii) TFA/CH2Cl2 (1 : 10),
room temperature, 1.5 h; (iii) N,N0-bis(tert-butoxycarbonyl)-S-methyl-isothiourea, HgCl2, TEA, DMF, 0 ꢀC, 3 h; (iv) TFA/CH2Cl2 (1 : 10), room temperature,
1.5 h.
Hydrophobic Parameters
In order to investigate the correlation between hydrophobicity
on antimicrobial activity, the overall hydrophobicity was evalu-
ated by measuring the retention time (tR) of each individual
peptidomimetic using RP-HPLC. The tR values, determined
for all synthesized SAMPs derivatives 1–8, were in the range
11.87–14.56 min as shown in Table 1. The highest values (14.69
and 14.56 min) were observed for the reference compound and
peptidomimetic 5, respectively, showing a high affinity to the
HPLC hydrophobic phase. All other synthesized analogues showed
reduced retention times, except for SAMPs derivatives 1 and 7
(13.34 min) (Table 1). By evaluating these data, as we expected,
the replacement of the tryptophan residue with less lipophilic
amino acids, such as phenylalanine or tyrosine, reflected on the
total hydrophobicity of the synthesized peptidomimetics.
A clear correlation between retention time on the C18 column
(i.e. affinity for the hydrophobic column surface) and antibacterial
activity was not found, except for peptidomimetic 5.
Antimicrobial Activity
The antimicrobial activity of the SAMPs derivatives 1–8 was
evaluated in vitro against two reference Gram-positive strains
(S. aureus and S. epidermidis), two reference Gram-negative strains
(E. coli and K. pneumoniae), and one fungal strain (C. albicans)
(Table 2a).
We synthesized and tested the reference compound Arg-Trp-OBzl
and our SAMPs derivatives 1–8 on different panel of reference
bacteria respect to that used by Strom et al. [17]. The MIC values
Scheme 3. Reagents and conditions: (i) L-tryptophyl-L-tryptophan
methyl ester, HBTU, HOBt, DIPEA, DMF, 0 ꢀC, 10 min, and room tempera-
ture, 2.5 h; (ii) TFA/CH2Cl2 (1 : 10), room temperature, 1.5 h; (iii) N,N0-bis
(tertbutoxycarbonyl)-S-methyl-isothiourea, HgCl2, TEA, DMF, 0 ꢀC, 3 h; (iv)
TFA/CH2Cl2 (1 : 1), room temperature, 1.5 h.
Scheme 4. Reagents and conditions: (i) L-tryptophan methyl ester, HBTU, HOBt, DIPEA, DMF, 0 ꢀC, 10 min, and room temperature, 2.5 h; (ii) TFA/CH2Cl2
(1 : 10), room temperature, 1.5 h; (iii) N,N0-bis(tert-butoxycarbonyl)-S-methyl-isothiourea, HgCl2, TEA, DMF, 0 ꢀC, 3 h; (iv) TFA/CH2Cl2 (1 : 1), room temper-
ature, 1.5 h.
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SYNTHESIS AND ANTIMICROBIAL ACTIVITY OF SHORT CATIONIC PEPTIDOMIMETICS
Table 1. Hydrophobicity of SAMPs derivatives 1–8 measured as
retention time (tR) on C18 column with solvent system of
acetonitrile/water in the form of a linear gradient starting from 10
to 80% of CH3CN in 30 min
Den Hertog et al. [40], the candidacidal effect of SAMPs derivatives
2 and 5 could be related to the impairment of the functional and
structural integrity of cell membrane.
The difference of susceptibility between Gram-positive and
Gram-negative bacteria was not investigated. Considering the
lower activity against Gram-negative bacteria, with respect to
Gram-positive species, the effect of SAMPs derivatives on estab-
lished biofilms was evaluated only against S. aureus ATCC 29213
and S. epidermidis ATCC 35984. The most active SAMPs deriva-
tives (1–2 and 5–6), against the tested Gram-positive bacteria,
were chosen to evaluate their capability to disrupt the mature
biofilms (Table 2b). Results showed BIC values ranging from
fourfold to 20-fold higher than the corresponding MIC values,
whereas BECs were never under 800 mg/ml for all peptidomi-
metics tested. Thus, all synthesized SAMPs derivatives 1–2 and
5–6 showed an appreciable inhibiting activity on established
Gram-positive biofilms (BIC < 200 mg/ml), i.e. peptidomimetic 1
(100 mg/ml) but a lower capacity to eradicate the biofilms
(BEC > 800). The relatively low BIC values probably reflect the
ability of the compounds to act on slow-growing or non-growing
bacteria [9], whereas the high BEC values are likely due to the
difficulties of peptidomimetics to diffuse in the extracellular
biofilm polymers (EPS). In fact, the polysaccharide intercellular
adhesin, a positively charged homopolymer of beta-1,6 linked
to N-acetylglucosamine residues, is considered to play a critical
role in the development and maturation of Staphylococcus
biofilms [41]. This cell surface-associated EPS, being positively
charged, acts as an intercellular adhesin and binds the negatively
charged bacterial cells together via electrostatic interactions,
resulting in an increased cellular accumulation [42].
SAMPs derivatives
MW
tR (min)
Arg-Trp-OBzl
523.46
820.29
634.53
595.49
611.49
850.72
664.51
625.48
641.48
14.69
13.34
12.78
12.13
11.93
14.56
12.96
12.34
11.87
1
2
3
4
5
6
7
8
against Gram-positive species ranged from 7.5 to 200 mg/ml, except
for peptidomimetic 4 with MIC values >300. The SAMPs derivatives
5 and 6 revealed the most antibacterial activity – among the tested
peptidomimetics – both against S. aureus ATCC 29213 (10 mg/ml)
and S. epidermidis ATCC 35984 (7.5 mg/ml). SAMPs derivatives 1
and 2 were moderately active; in particular, peptidomimetic 2
showed MIC values of 25 mg/ml against both S. aureus ATCC 29213
and S. epidermidis ATCC 35984. However, our most active SAMPs
derivatives (1–2 and 5–6) showed better MIC values with respect
to the reference compound (75 mg/ml). Other tested peptidomi-
metics showed no valuable antimicrobial activity against
Gram-positive bacteria (Table 2a). The MIC values, recorded in
SAMPs derivatives 5, 1, and 3 against S. aureus ATCC 29213,
and 7 against S. epidermidis ATCC 35984 (Table 2a), corresponded
to the bactericidal values.
Hemolytic Activity
By evaluating the biological results, the order of activity against
Gram positive bacteria can be rationalized on the basis of the
different structural features between the SAMPs derivatives 1–4
with respect to 5–8, probably due to the spatial disposition of
the cationic groups in the two series of analogues. In the SAMPs
derivatives 1–4, the positively charged moieties are at the oppo-
site sides of the molecule, whereas in peptidomimetics 5–8, they
are closer in space. It could be interesting to note that the spatial
disposition in SAMPs derivatives 1–4 could affect the capacity of
the molecules to effectively assume the amphipathic conforma-
tion with separate hydrophobic and hydrophilic portions, as
required to exert antimicrobial activity [35,36]. The introduction
of arginine mimetics in all SAMPs derivatives 1–8 as cationic
residue in place of lysine was based on the hypothesis that the
guanidine group of arginine would interact more strongly with
the negatively charged phospholipids of the bacterial cell
membrane by forming both electrostatic and hydrogen-bonding
interactions [37]. It is important to note that the replacement of
the tryptophan residue by phenylalanine or tyrosine does signif-
icantly alter the antibacterial activity (SAMPs derivatives 3–4 and
7–8). These data were not surprising because tyrosine and
phenylalanine were less effective in providing bulk and lipophili-
city than tryptophan, thus lowering antibacterial activity [38,39].
The SAMPs derivatives 2, 5, and 6 revealed the same low
efficacy against the tested Gram-negative bacteria and C. albicans
(Table 2a). In particular, K. pneumoniae 5F was inhibited and killed
by using peptidomimetic 2 at 31.3 mg/ml, whereas peptidomi-
metic 5 was active against E. coli ATCC 8739 at 25 mg/ml. C.
albicans ATCC 10231 was inhibited and killed at 50 and 62.5 mg/ml
by using SAMPs derivatives 2 and 5, respectively. As explained by
All analyzed peptidomimetics did not produce human blood
hemolysis at their MIC values. In particular, all SAMPs derivatives
never expressed hemolytic activity higher than 33% at their
maximum concentration tested, except for peptidomimetic 5,
which displayed a cytotoxic effect showing hemolytic activity
over 50% at 100 mg/ml.
The dose–response curves for the hemolytic activity of the
SAMPs derivatives were shown in Figure 2. A weak linear trend
between hemolytic activity and lipophilicity was found, and
peptidomimetic 5 – the most hydrophobic one – being the most
hemolytic as well. On the other hand, there is not a clear correla-
tion between antimicrobial potency and hemolytic activity for
the SAMPs derivatives 1, 2, and 6.
Conclusion
In conclusion, novel antimicrobial peptidomimetics containing
the 1-(4-(aminomethyl)benzyl)guanidine, or the 3,5-diguanidino
benzoic acid, and aromatic a-guanidino amino acids have been
synthesized.
In the present study, all the synthesized peptidomimetics
fulfilled the pharmacophore model of short AMPs with antimicro-
bial activity by having a net charge of +2 and an amphipathic
structure. It was observed that the presence of tryptophan
moieties favored the antibacterial activity especially against
Gram-positive bacterial strains. On the contrary, compounds
bearing phenylalanine or tyrosine as bulky groups resulted in a
considerable loss of the antibacterial action (SAMPs derivatives
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SYNTHESIS AND ANTIMICROBIAL ACTIVITY OF SHORT CATIONIC PEPTIDOMIMETICS
of analogues with increased amphipathicity and elevated net
positive charge of the molecule. Russ. J. Bioorg. Chem. 2004; 30(5):
458–465.
11 Bucki R, Levental I, Janmey PA. Antibacterial peptides a bright
future or a false hope. Curr. Med. Chem. Anti-Infect. Agents. 2007;
6: 175–184.
12 Haug BE, Strøm MB, Svendsen JSM. The medicinal chemistry of short
lactoferricin-based antibacterial peptides. Curr. Med. Chem. 2007;
14(1): 1–18.
13 Giuliani A, Pirri G, Nicoletto SF. Antimicrobial peptides: an overview of
a promising class of therapeutics. Cent. Eur. J. Biol. 2007; 2: 1–33.
14 Melo MN, Ferre R, Castanho MARB. Antimicrobial peptides: linking
partition, activity and high membrane-bound concentrations. Nature
Rev. Microbiol. 2009; 7(3): 245–250.
15 Godballe T, Nilsson LL, Petersen PD, Jensse NH. Antimicrobial b-peptides
and a-peptoids. Chem. Biol. Drug Des. 2011; 77: 107–116.
16 Dathe M, Nikolenko H, Klose J, Bienert M. Cyclization increases the
antimicrobial activity and selectivity of arginine- and tryptophan-
containing hexapeptides. Biochemistry 2004; 43(28): 9140–9150.
17 Strøm MB, Haug BE, Skar ML, Stensen W, Stiberg T, Svendsen JS. The
pharmacophore of short cationic antibacterial peptides. J. Med. Chem.
2003; 46(9): 1567–1570.
18 Haug BE, Stensen W, Kalaaji M, Rekdal ꢀ, Svendsen JS. Synthetic
antimicrobial peptidomimetics with therapeutic potential. J. Med.
Chem. 2008; 51(14): 4306–4314.
19 Sharma RK, Reddy RP, Tegge W, Jain R. Discovery of Trp-His and
His-Arg analogues as new structural classes of short antimicrobial
peptides. J. Med. Chem. 2009; 52(23): 7421–7431.
Figure 2. Dose–response curve of hemolytic activity of SAMPs deriva-
tives 1–8 against h-RBCs. Peptidomimetics 1–2 refer to y-axis on the right
whereas peptidomimetics 3–8 refer to the y-axis on the left.
3–4 and 7–8) against both Gram-positive and Gram-negative
bacterial strains. In this study, an appreciable antifungal activity
was observed only for SAMPs derivatives 2 and 5.
In light of the results presented in this work and taking into
account that this is preliminary studies, we can conclude that
the synthesized SAMPs derivatives 1–2 and 6 showed a
promising antimicrobial activity both in planktonic and sessile
growth mode. Further studies are needed to well investigate
their potential antimicrobial activity against a wide panel of
Gram-positive strains.
20 Hansen T, Alst T, Havelkova M, Strøm MB. Antimicrobial activity of small
beta-peptidomimetics based on the pharmacophore model of short
cationic antimicrobial peptides. J. Med. Chem. 2010; 53(2): 595–606.
21 Flemming K, Klingenberg C, Cavanagh JP, Sletteng M, Stensen W,
Svendsen JS, Flaegstad T. High in vitro antimicrobial activity of
synthetic antimicrobial peptidomimetics against staphylococcal
biofilms. J. Antimicrob. Chemother. 2009; 63(1): 136–145.
22 Hansen T, Ausbacher D, Flaten GE, Havelkova M, Strøm MB. Synthesis
of cationic antimicrobial b(2,2)-amino acid derivatives with potential
for oral administration. J. Med. Chem. 2011; 54(3): 858–868.
23 Haug BE, Stensen W, Stiberg T, Svendsen JS. Bulky nonproteinogenic
amino acids permit the design of very small and effective cationic
antibacterial peptides. J. Med. Chem. 2004; 47(17): 4159–4162.
24 Svenson J, Karstad R, Flaten GE, Brandsdal B, Brandl M, Svendsen JS.
Altered activity and physicochemical properties of short cationic
antimicrobial peptides by incorporation of arginine analogues. Mol.
Pharm. 2009; 6(3): 996–1005.
Acknowledgements
Financial support from Ministero dell’ Istruzione, dell’Università e
della Ricerca (MIUR) is gratefully acknowledged.
25 Knappe D, Kabankov N, Hoffmann R. Bactericidal oncocin derivatives
with superior serum stabilities. Int. J. Antimicrob. Agents 2011; 37
(2011): 166–170.
Conflict of Interest
26 Knappe D, Henklein P, Hoffmann R, Hilpert K. Easy strategy to protect
antimicrobial peptides from fast degradation in serum. Antimicrob.
Agents Chemother. 2010; 54(9): 4003–4005.
The authors have declared no conflict of interest.
27 Karstad R, Isaksen G, Brandsdal B, Svendsen JS, Svenson J. Unnatural
amino acid side chains as S1, S10, and S20 probes yields cationic anti-
microbial peptides with stability toward chymotryptic degradation. J.
Med. Chem. 2010; 53(15): 5558–5566.
28 Gers T, Kunce D, Markowski P, Izdebski J. Reagents for efficient conversion
of amines to protected guanidines. Synthesis 2004; 2004(1): 37–42.
29 Appleton DR, Babcock RC, Copp BR. Novel tryptophan-derived dipep-
tides and bioactive metabolites from the sea hare Aplysia dactylomela.
Tetrahedron 2001; 57(51): 10181–10189.
References
1 Hentzer M, Givskov M. Pharmacological inhibition of quorum sensing
for the treatment of chronic bacterial infections. J. Clin. Invest. 2003;
112(9): 1300–1307.
2 Alanis AJ. Resistance to antibiotics: are we in the post-antibiotic era?
Arch. Med. Res. 2005; 36(6): 697–705.
3 Donlan RM, Costerton JW. Biofilms: survival mechanisms of clinically
relevant microorganisms. Clin. Microbiol. Rev. 2002; 15: 167–193.
4 Lewis K. Persister cells. Annu. Rev. Microbiol. 2010; 64: 357–372.
5 Lewis K. Riddle of biofilm resistance. Antimicrob. Agents Chemother.
2001; 45: 999–1007.
6 Jefferson KK. What drives bacteria to produce a biofilm? FEMS
Microbiol. Lett. 2004; 236(2): 163–173.
7 Hancock R, Chapple DS. Peptide antibiotics. Antimicrob. Agents
Chemother. 1999; 43(6): 1317–1323.
30 Kumar A, Meijer EW. Novel hyperbranched polymer based on urea lin-
kages. Chem. Commun. 1998; 1998(16): 1629–1630.
31 Clinical Laboratory Standards Institute. 2006. Performance Standards
for Antimicrobial Susceptibility. 16th Informational Supplement
M100-S16. Clinical Laboratory Standards Institute: Wayne, Pa, USA.
32 Johnson SA, Goddard PA, Iliffe C, Timmins B, Rickard AH, Robson G,
Handley PS. Comparative susceptibility of resident and transient hand
bacteria to para-chloro-meta-xylenol and triclosan. J. Appl. Microbiol.
2002; 93(2): 336–344.
8 Boman HG. Antibacterial peptides: basic facts and emerging
concepts. J. Intern. Med. 2003; 254(3): 197–215.
33 Bannard RAB, Casselman AA, Cockburn WF, Brown GM. Guanidine
compounds II. Preparation of mono- and N,N-di-alkylguanidines.
Can. J. Chem. 1958; 36: 1541–1549.
9 Liu Z, Brady Z, Young A, Rasimick B, Chen K, Zhou C, Kallenbach
NR. Length effects in antimicrobial peptides of the (RW)n series. Anti-
microb. Agents Chemother. 2007; 51(2): 597–603.
34 Zhao-Xia G, Cammidge AN, Horwell DC. A convenient and versatile
method for the synthesis of protected guanidines. Synth. Commun.
2000; 30(16): 2933–2934.
10 Smirnova MP, Afonin VG, Shpen’ VM, Tiagotin IV, Kolodkin NI.
Structure–function relationship between analogues of the anti-
bacterial peptide indolicidin. I. Synthesis and biological activity
J. Pept. Sci. 2012
Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/jpepsci

BALDASSARRE ET AL.
35 Sundriyal S, Sharma RK, Jain R, Bharatam PV. Minimum require-
ments of hydrophobic and hydrophilic features in cationic peptide
antibiotics (CPAs): pharmacophore generation and validation
with cationic steroid antibiotics (CSAs). J. Mol. Mod. 2008; 14(4):
265–278.
36 Wessolowski A, Bienert M, Dathe MA. Antimicrobial activity of argi-
nine- and tryptophane-rich hexapeptides: the effects of aromatics
clusters, D-amino acid substitution and cyclization. J. Peptide Res.
2004; 64(4): 159–169.
37 Thaker HD, Sgolastra F, Clements D, Scott RW, Tew GN. Synthetic
mimics of antimicrobial peptides from triaryl scaffolds. J. Med. Chem.
2011; 54(7): 2241–2254.
38 Dougherty DA. Cation-p interactions in chemistry and biology: a new
view of benzene, Phe, Tyr, and Trp. Science 1996; 271(5246): 163–168.
39 Chan DI, Prenner EJ, Vogel HJ. Tryptophan- and arginine-rich antimi-
crobial peptides: structures and mechanisms of action. Biochim. Bio-
phys. Acta 2006; 1758(9): 1184–1202.
40 Den Hertog AL, Van Marle J, Van Veen HA, Van’t Hof W, Bolscher JGM,
Veerman ECI, Nieuw Amerogan AV. Candidacidal effects of two anti-
microbial peptides: histatin 5 causes small membrane defects, but
LL-37 causes massive disruption of the cell membrane. Biochem. J.
2005; 388: 689–695.
41 O’Gara JP. Ica and beyond: biofilm mechanisms and regulation in
Staphylococcus epidermidis and Staphylococcus aureus. FEMS Microbiol.
Lett. 2007; 270(2): 179–188.
42 Singh R, Ray P, Das A, Sharma M. Enhanced production of exopolysac-
charide matrix and biofilm by a menadione-auxotrophic Staphylococcus
aureus small-colony variant. J. Med. Microbiol. 2010; 59(Pt 5): 521–527.
wileyonlinelibrary.com/journal/jpepsci
Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.
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