Design, synthesis and antimicrobial properties of non-hemolytic cationic α-cyclopeptoids

Article abstract of DOI:10.1016/j.bmc.2010.01.026

The synthesis and screening of neutral and cationic, linear and cyclic peptoids (N-alkylglycine peptidomimetics) is described. Structure-activity relationship studies show that the in vitro activities of the tested peptoids depend on both cyclization and

Full text of DOI:10.1016/j.bmc.2010.01.026

Bioorganic & Medicinal Chemistry 18 (2010) 2010–2018
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis and antimicrobial properties of non-hemolytic cationic
a-cyclopeptoids
a,
Daniela Comegna a, Monica Benincasa b, Renato Gennaro b, Irene Izzo a, , Francesco De Riccardis
*
*
a Deparment of Chemistry, University of Salerno, Via Ponte Don Melillo, 84084 Fisciano (SA), Italy
b Department of Life Sciences, University of Trieste, 34127 Trieste, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis and screening of neutral and cationic, linear and cyclic peptoids (N-alkylglycine peptidom-
imetics) is described. Structure–activity relationship studies show that the in vitro activities of the tested
peptoids depend on both cyclization and decoration with cationic groups. The most powerful N-lysine
cyclopeptoid derivatives showed good antifungal activity against Candida albicans (ATCC90029 and
L21) and Candida famata (SA550, Amph B-resistant) and low hemolytic activity. The effects of the cyclic
peptoids on membrane permeabilization were evaluated by the propidium iodide exclusion assay.
Ó 2010 Elsevier Ltd. All rights reserved.
Received 3 December 2009
Revised 8 January 2010
Accepted 9 January 2010
Available online 15 January 2010
Keywords:
Cyclic peptoids
Antibiotics
Solid-phase synthesis
1. Introduction
toids,10 showing reduced conformational freedom10,11 and
excellent membrane-permeabilizing activity.12
The development of functionalised artificial scaffolds capable of
mimicking pharmacologically relevant natural compounds is an
area of vibrant interest both for synthetic and medicinal chemists.1
One of the fields that poses more challenges and opportunities,
from the molecular and therapeutic point of view, is that of innate
immunity, which raises an effective, nonspecific, broad-spectrum
antimicrobial response based on different cellular and molecular
systems, including cationic antimicrobial peptides.2 The antibiotic
activities of these structurally diverse sets of peptides derive from
their action on microbial cytoplasmic membranes. The model pro-
posed by Shai–Matsuzaki–Huan3 (SMH) presumes alteration and
permeabilization of the phospholipid bilayer with irreversible
damage of the critical membrane functions. The fact that peptides
operating by the SMH mechanism kill microbes at micromolar con-
centrations4 and the recent reports demonstrating that efficient
membrane-active antibiotics can be prepared from monomers
In the present paper, we investigate the antimicrobial activities
of five new cyclic cationic hexameric -peptoids (5, 9, 15, 19, 23),
a
correlating their efficacy with the linear cationic (3, 7, 13, 17, 21)
and the cyclic neutral (1, 11) counterparts (Fig. 1). The synthesized
peptoids have been assayed against clinically relevant bacteria and
fungi, including Escherichia coli, Staphylococcus aureus, amphoteri-
cin B-resistant Candida albicans, and Cryptococcus neoformans.13
The purpose of this study is to explore the biological effects of
the cyclization on positively charged oligomeric N-alkylglycines,
with the idea to mimic the natural amphiphilic peptide antibiotics.
The long-term aim of the effort is to find a key for the rational
design of novel antimicrobial compounds using the finely tunable
peptoid backbone.
2. Results and discussion
other than
a
-amino acids,5 prompted the design and synthesis of
facially amphiphilic peptides2a and peptidomimetics.6 The confor-
mational constraints of the proposed backbones, have been mainly
The exploration for possible biological activities of linear and
cyclic a-peptoids, started with the assessment of the antimicrobial
activity of the known10a N-benzyloxyethyl cyclohomohexamer 1
(Fig. 1, Block I). This neutral cyclic peptoid was considered a prom-
ising candidate in the antimicrobial assays for its high affinity to
the first group alkali metals (Ka ꢀ 106 for Na+, Li+ and K+)10a and
its ability to promote Na+/H+ transmembrane exchange through
ion-carrier mechanism,14 a behavior similar to that observed for
valinomycin, a well known K+-carrier with powerful antibiotic
activity.15 However, determination of the MIC values showed that
1 did not exert any antimicrobial activity against a group of
achieved through periodic incorporation of bulky,
a-chiral, side
chains,7 or use of rigid scaffolds.5 Cyclization of linear peptide/pep-
toid precursors (as a mean to obtain conformational order), has
been often neglected,8 notwithstanding the fact that nature offers
a vast assortment of powerful cyclic antimicrobial peptides.9 How-
ever, macrocyclization of N-substituted glycines gives circular pep-
* Corresponding authors. Tel.: +39 089 969552; fax: +39 089 969603.
E-mail addresses: iizzo@unisa.it (I. Izzo), dericca@unisa.it (F. De Riccardis).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.01.026

D. Comegna et al. / Bioorg. Med. Chem. 18 (2010) 2010–2018
2011
OBn
Block I Compounds
Block II Compounds
O
N
O
N
BnO
N
O
O
N
N
O
O
N
N
N
O
O
OBn
N
O
N
O
O
BnO
O
O
N
N
11
1
+H2
N
OH
6
OBn
10
OBn
R
R
R
O
R
OBn
O
OBn
O
OBn
O
OBn
O
O
O
O
O
O
O
O
N
N
N
N
N
N
+H2
N
N
N
OH
+H2
N
N
N
OH
2
2
2
2
2, R = NHBoc
12, R = NHBoc
4, R = NHBoc
14, R = NHBoc
+
+
+
+
3, R = NH3
13, R = NH3
5, R = NH3
15, R = NH3
R
R
O
R
R
R
O
R
OBn
OBn
O
O
O
O
O
O
O
O
N
N
N
N
N
+H2
N
N
OH
+H2
N
N
N
OH
3
2
3
2
16, R = NHBoc
18, R = NHBoc
6, R = NHBoc
8, R = NHBoc
+
+
+
+
17, R = NH3
19, R = NH3
7, R = NH3
9, R = NH3
R
R
O
N
R
N
O
N
O
R
N
O
N
O
+H2
N
OH
R
O
22, R = NHBoc
N
6
+
23, R = NH3
O
20, R = NHBoc
+
21, R = NH3
R
R
Figure 1. Structures of synthesized linear and cyclic peptoids. Bn = benzyl group; Boc = t-butoxycarbonyl group.
selected pathogenic fungi, and of Gram-negative and Gram-posi-
tive bacterial strains even at concentrations up to 1 mM (Table 1).
Detailed structure–activity relationship (SAR) studies7,16 have
revealed that the amphiphilicity of the peptides/peptidomimetics
and the total number of positively charged residues, impact signif-
icantly on the antimicrobial activity. We, thus, reasoned that
resin, using a 1:1 solution of hexafluoroisopropanol (HFIP)/dichlo-
romethane (the purity of the oligomers was >95%, RP-HPLC analy-
sis). The slight acidity of the fluorinated secondary alcohol
detached the hexamers from the 2-chlorotrityl groups, preserving
the side chain t-butoxycarbonyl protection. Head-to-tail macrocyc-
lizations of the linear N-substituted oligoglycines 2 and 6 (Block I
compounds, Fig. 1), proceeded smoothly giving, under high dilu-
tion conditions (2.0 ꢁ 10ꢂ3 M) and in the presence of the efficient
coupling agent benzotriazole-1-yl-oxy-tris-pyrrolidino-phospho-
nium hexafluorophosphate (PyBOP), the cyclic peptoids 4 and 8.
The Boc-protected linear and cyclic precursors (2, 4, 6, and 8) were
deprotected with trifluoroacetic acid (TFA), to afford the couples 3/
5 and 7/9.
cationic versions of the cyclic
esting mimics of antimicrobial peptides and planned the synthesis
of mixed N-(4-aminobutyl)/N-benzyloxyethyl hexameric -pep-
a-peptoids 1 could represent inter-
a
toids 5, and 9 (Fig. 1, Block I compounds). In our investigation,
we also included the linear cationic precursors 3 and 7, in order
to evaluate the effect of macrocyclization on the antimicrobial
activity.
The elaboration of the t-butoxycarbonyl protected Block I pep-
toids, was accomplished via an expeditious mixed ‘monomer’,17
and ‘submonomer’,18 approach, consisting in an alternate attach-
ment of the N-fluorenylmethoxycarbonyl,N0-benzyloxyethyl gly-
cine,19 (25, Scheme 1), and a two step construction of the N-(4-
aminobutyl) glycine remnant. The oligomerization of the units
was performed on a 2-chlorotrityl resin (24). Once the construction
of the linear oligomers was completed, they were cleaved from the
Cationic peptoids 3, 5, 7, and 9 were tested against four patho-
genic fungi and three clinically relevant bacterial strains. The anti-
microbial activities are summarized in Table 1. The tests showed a
marked increase of the antibacterial and antifungal activities with
cyclization. The presence of charged amino groups also influenced
the antimicrobial efficacy, as shown by the activity of the bi- and
tricationic compounds 5 and 9, when compared with the ineffec-
tive 1.

2012
D. Comegna et al. / Bioorg. Med. Chem. 18 (2010) 2010–2018
OBn
OBn
O
25
a)
O
HO
O
N
Fmoc , DIPEA
O
NH
Cl
b) 20% Piperidine
in DMF
26
24
NHBoc
OBn
Br
, DIC
OH
c)
d)
27
O
O
O
N
NH
NHBoc
29
H2N
NHBoc
28
OBn
OBn
O
O
O
O
e)
25, PyBOP
N
N
NH
2, 6
f) 20% Piperidine
in DMF
30
Scheme 1. Mixed monomer/submonomer approach for the synthesis of oligomers
2
and 6. DIPEA = diisopropylethylamine; DIC = N,N0-diisopropyl carbodiimide;
PyBOP = benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate.
Encouraged by the promising results displayed by the N-Lys/N-
benzyloxyethyl cyclopeptoids, we decided to move towards ana-
logues produced with a fully sub-monomeric approach and, with
the aim to have a better defined SAR, presenting a pairwise in-
crease of cationic side-chains, interrupted by N-Phe residues (Block
II compounds, Fig. 1). The solid-phase synthesis of these new pep-
toids proceeded uneventfully, giving, after detachment from the
resin in the same condition reported for the congeners 2 and 6,
the linear oligomers 10, 12, and 16 (the purity of the oligomers
was >95%, RP-HPLC analysis). High dilution (2.0 ꢁ 10ꢂ3 M) head-
to-tail PyBOP-mediated macrocyclizations, gave the cyclic 11, 14
and 18. The four Boc-protected linear (12 and 16) and cyclic (14
and 18) precursors were deprotected with TFA, to yield the couples
13/15, and 17/19, respectively.
hemolytic activity, at variance with melittin, a well known hemo-
lytic peptide used as a control, which lysed over 90% of the cells at
20 lM concentration. Although preliminary, these results are
encouraging for the development of the cyclic peptoids under
investigation, as they indicate that these compounds display a sig-
nificant selectivity against fungal versus mammalian cells.
To understand the mechanism by which the peptoids act
against bacterial and fungal cells, their effect on membrane integ-
rity was evaluated by flow cytometry using the fluorescent probe
propidium iodide (PI), which only enters cells with damaged mem-
branes and stains their nucleic acids.20
The results obtained after treatment of C. albicans with different
concentrations of the most active peptoids of the two series, 9 and
19, are reported in Figure 2. Compounds 19, at 100 lM concentra-
The synthesis of fully N-lysinated peptoids was also planned.
The linear hexamer 20 was oligomerized on-resin and, once de-
tached from the solid support, it was cyclized using the same con-
ditions reported for the N-benzylated congeners, to give the fully
protected homo-oligomer 22. Both 20 and 22 were deprotected
in the presence of TFA, to afford peptoids 21 and 23.
Compounds 11, 13, 15, 17, 19, 21 and 23 were tested against
five pathogenic fungi and three bacterial strains. The MIC values
are summarized in Table 2. The results of this second array of anti-
microbial evaluations again demonstrated an increase of the anti-
fungal activities when the cyclic arrangement was present (for
instance, compare the results of linear 17 vs cyclic 19) and weak
antibacterial activity. It is interesting to note that the number of
the amino groups inserted on the cyclic peptoids strongly influ-
ences the MIC value for the antifungal activity. In particular, com-
pound 19 seems to have an optimal N-Lys/N-benzyloxyethyl
residue ratio and, subsequently, a higher potency.
tion, permeabilized a significant percentage of the yeast cells after
120 min of incubation. The percentage of PI-positive cells in-
creased up to 50% by prolonging the incubation time (Fig. 2A).
The permeabilizing effect of compound 9 on C. albicans cells is less
evident also at the maximal concentration used (Fig. 2B), and these
results correlate with the different potency showed by the pep-
toids (Tables 1 and 2).
The effect of 9 on bacterial membrane integrity is shown in
Figure 2C. No significant effect is evident on the E. coli membrane
even after incubation for 180 min at a concentration of 250 lM. In
contrast, the same dose of 9 caused an appreciable effect (>40% PI-
positive cells) on S. aureus cells, already after 60 min incubation,
indicating that this compounds interacts with a faster kinetics with
the membrane of Gram-positive microorganisms.
3. Conclusions
To be useful therapeutically, the active compounds should not
be toxic to host cells. As a preliminary evaluation of the toxicity
of 19 and 23, which show the most potent antifungal activity
among the peptoids analyzed in this work, their hemolytic activity
was tested on 0.5% suspension of human red blood cells. Both com-
Cyclization of amino acid-based molecules is a strategy that has
been used by many organisms to produce compounds for defense,
signaling and microbial competition. Compounds such as cyclo-
sporin A and congeners are assembled by peptide synthetases; oth-
ers are encoded by genes and ribosomally translated, such as
members of the microcins and bacteriocins from bacteria, cyclo-
tides from plants and theta-defensins from monkeys.21 All these
pounds, tested at 100
lM concentration (approximately 10-fold
higher than the best MIC values, Table 2), showed virtually no

D. Comegna et al. / Bioorg. Med. Chem. 18 (2010) 2010–2018
2013
9, 19 and 23, Tables 1 and 2), in spite of a similar net positive
charge. Finally, in the cyclic compounds, the antifungal potency
appears to directly correlate with the net positive charge, as
shown by compounds 19 and 23 that have the highest net
positive charge (+3 and +5, respectively) and are also the most
effective ones.
60
50
40
30
20
10
0
A
In conclusion, the synthesis, structural features, and antimicro-
bial activities of linear and cyclic homo- and heteroligomeric
a-
peptoids have been described. Two cyclic compounds in particular,
9 and 19, showed good antifungal activity and no toxicity toward
red blood cells at the concentrations tested. These results are the
first indication that cyclic peptoids can represent new motifs on
which to base artificial antibiotics. Investigations on the mecha-
nism of action of structurally diverse cationic cyclopeptoids are
currently underway.
0
30
60
90
120 150 180 210 240
Incubation time (min)
50 µM
100 µM
250 µM
30
25
20
15
10
5
B
4. Experimental section
4.1. Synthesis
4.1.1. General methods
All reactions involving air or moisture sensitive reagents were
carried out under a dry argon or nitrogen atmosphere using freshly
distilled solvents. Toluene and dichloromethane were distilled from
calciumhydrideunder argon. Glassware was flame-dried (0.05 Torr)
prior to use. When necessary, compounds were dried in vacuo over
P2O5, by azeotropic removal of water with toluene under reduced
pressure or by freeze-drying. Starting materials and reagents pur-
chased from commercial suppliers were generally used without
purification unless otherwise mentioned. Reactions were monitored
by TLC on silica gel plates (0.25 mm) and visualized by UV light or by
spraying with phosphomolybdic acid or ninhydrin solutions and
drying. Flash chromatography was performed on Silica Gel 60
(particle size: 0.040–0.063 mm) and the solvents employed were
of analytical grade. HPLC analysis were performed on a C18
reversed-phase analytical and preparative columns (Waters,
0
0
30
60
90
120 150 180 210 240
Incubation time (min)
25 µM
100 µM
250 µM
60
C
50
40
30
20
10
0
lBondapak, 10
lm, 125 Å 3.9 mm ꢁ 300 mm and 7.8 ꢁ 300 mm,
respectively) using a Modular HPLC System JASCO LC-NET II/ADC
equipped with a JASCO Model PU-2089 Plus Pump and a JASCO
MD-2010 Plus UV–vis multiple wavelength detector set at 220 nm.
Yields refer to chromatographically and spectroscopically (1H and
13C NMR) pure materials. The NMR spectra were recorded on Bruker
DRX 400 (1H at 400.13 MHz, 13C at 100.03 MHz), Bruker DRX 300 (1H
at 300.10 MHz, 13C at 75.48 MHz), and Bruker DRX 250 (1H at
0
30
60
90
120
150
180
Incubation time (min)
250 µM
100 µM
Figure 2. Flow cytometric evaluation of membrane integrity of fungal and bacterial
cells after treatment with peptoids 9 and 19. The percentage of fluorescent cells (%
PI-positive cells) after incubation of C. albicans ATCC 90029 with 19 (A) and 9 (B), or
after incubation of S. aureus (filled symbols) and E. coli (empty symbols) cells with 9
(C) is shown. Background values in the absence of peptoids (<3% of permeabilized
cells) were subtracted to each peptoid-treated sample. Results are mean values of at
least three independent determinations with similar results.
Table 1
MIC values of peptoids 1, 3, 5, 7, and 9, against pathogenic fungi and Gram-positive/
Gram-negative bacteria
Fungia
1
3
5
7
9
C. albicans ATCC 90029
C. albicans L21
>103
>103
>103
>103
360
360
180
90
>103
196
196
196
98
184
184
184
92
compounds exert a wide array of biological activities that range
from regulation of microbial competitions to a potent microbicidal
activity against bacteria, fungi and some viruses.22,23
Recent studies have suggested both a structural and functional
role for the cyclic backbone. In fact, cyclization of the above men-
tioned peptides improves their stability, for instance by removing
possible cleavage sites for exoproteases, as well as biological activ-
ity, as shown by the decreased biological activity of linear ana-
logues of RTD-1 and cyclotides relative to the native peptides.24
681
C. neoformans ATCC 90112
C. neoformans L1
>103
349
Bacteriaa
E. coli ATCC 25922
>103
>103
>103
360
>103
>103
>103
392
383
Salmonella typhimurium ATCC 14028
Staphylococcus aureus ATCC 25923
720
>792
392
>766
383
>720
a
The MIC value is defined as the lowest compound concentration resulting in a
In general the
a
-peptoids described in this study, which have a
complete inhibition of visible growth after 48 h incubation at 30 °C for fungi and
after 18 h incubation at 37 °C for bacteria. MIC values are in micromolar concen-
tration to allow comparison of the same number of molecules per assay. To convert
data to micrograms/milliliter, multiply the MIC value with the molecular weight/
1000. Molecular weights of the compounds are as follows (as trifluoroacetate salts):
1, 1147; 3, 1381; 5, 1249; 7, 1432; 9, 1300. The activity data of the cyclic cationic
peptoids (5 and 9) are underlined.
backbone of 18 atoms, are more active against fungi than bacte-
ria. In addition, as observed for natural cyclic peptides, we found
that the linear forms are less effective than their cyclic counter-
parts (compare for instance the antifungal activity of the linear
compounds 7, 17 and 21 with that of the respective cyclic forms

2014
D. Comegna et al. / Bioorg. Med. Chem. 18 (2010) 2010–2018
Table 2
DMF (3 ꢁ 3 mL), DCM (3 ꢁ 3 mL) and DMF (3 ꢁ 3 mL). The yield of
MIC values of peptoids 11, 13, 15, 17, 19, 21 and 23, against pathogenic fungi and
Gram-positive/Gram-negative bacteria
the loading step was determined on the absorption of dibenzoful-
vene–piperidine adduct (kmax = 301,
e
= 7800 Mꢂ1 cmꢂ1). The
Fungia
11
13
15
17
19
21
23
incorporation of the N-(N-tert-butoxycarbonylbutyl)glycine (26)
was performed by a sub-monomeric approach18 (see below) while
the N-(benzyloxyethyl)glycine was inserted by Fmoc solid-phase
peptide synthesis method until the desired oligomer was obtained.
According to Fmoc solid-phase synthesis, the coupling step was
accomplished by incubating the resin with a solution of N-Fmoc
N-alkylated glycine (0.68 mmol), PyBOP (0.66 mmol) and DIPEA
(1.36 mmol) in dry DMF (2 mL) on a shaker platform for 1 h, fol-
lowed by extensive washes with DMF (3 ꢁ 3 mL), DCM
(3 ꢁ 3 mL) and DMF (3 ꢁ 3 mL). Once the coupling was complete
the Fmoc group was deprotected with piperidine as already
described and the resin washed again to prepare it for the next
coupling. The cleavage was performed with 20% hexafluoro
isopropanol (HFIP) solution in DCM (v/v). The final products were
dissolved in 50% acetonitrile in HPLC grade water and analysed
by RP-HPLC (purity >95% for all the oligomers, conditions:
5% ? 100% B in 30 min [A: 0.1% TFA in water, B: 0.1% TFA in aceto-
nitrile], flow: 1.0 mL/min, 220 nm. C18 reversed-phase analytical
C. albicans ATCC 90029
C. albicans L21
C. neoformans ATCC 90112 >103 >103
>103 >103
>103 >103
>103
>103
>103
>103
>103
465
232
232
116
12
12
47
99
12
10
500
5
>103
>103
>103
>103
>103
5
C. neoformans L1
C. famata SA550
>103 788
n. t. n. t.
n. t. >103
Bacteriaa
E. coli ATCC 25922
>103 >103
>103 >103
>103
>103
>103
>103
465
465
786
>103
>103
>103
Salmonella typhimurium
ATCC 14028
Staphylococcus aureus
ATCC 25923
>103 >103
>103
>103
465
>103
>103
a
The MIC value is defined as the lowest compound concentration resulting in a
complete inhibition of visible growth after 48 h incubation at 30 °C for fungi and
after 18 h incubation at 37 °C for bacteria. The MIC values are in micromolar con-
centration to allow comparison of the same number of molecules per assay. To
convert data to micrograms/milliliter, multiply the MIC value with the molecular
weight/1000. Molecular weights of the compounds are as follows (as trifluoroace-
tate salts): 11, 883; 13, 1205; 15, 1073; 17, 1395; 19, 1263; 21, 1585; 23, 1453. The
activities of the cyclic cationic peptoids (15, 19 and 23) are underlined. n.t.: not
tested
column [Waters,
l
Bondapak, 10
lm, 125 Å, 3.9 mm ꢁ 300 mm])
and ESI mass spectrometry. The linear oligomers were subjected
to the cyclization reaction without further purification.
250.13 MHz, 13C at 62.50 MHz) spectrometers. Chemical shifts (d)
are reported in ppm relatively to the residual solvent peak (CHCl3,
d = 7.26, 13CDCl3, d = 77.0; CD2HOD, d = 3.34, 13CD3OD, d = 49.0;
CD2HCN: d = 1.94; 13CD3CN: d = 1.39 HDO, d = 4.79, 1,4-dioxane
[external standard for D2O], d = 67.19); and the multiplicity of each
signal is designated by the following abbreviations: s, singlet; d,
doublet; t, triplet; q, quartet; quint, quintuplet; m, multiplet; br,
broad. Coupling constants (J) are quoted in hertz. Homonuclear
decoupling, for the complexed peptoid 14, 2D NMR experiments
such as COSY-45, ROESY, HSQC and HMBC were also performed for
the full assignment of each signal. High resolution ESI-MS spectra
were performed on a Q-Star Applied Biosystem mass spectrometer.
ESI-MS analysis in positive ion mode was performed using a Finni-
gan LCQ Deca ion trap mass spectrometer (ThermoFinnigan, San
Josè, CA, USA) and the mass spectra were acquired and processed
using the Xcalibur software provided by Thermo Finnigan. Samples
were dissolved in 1:1 CH3OH/H2O, 0.1 % formic acid, and infused in
4.2.1. Compound 2
White foam; 1H NMR (400 MHz, CDCl3, 25 °C, mixture of rota-
mers): d = 7.34–7.28 (m, 20H, Ar-H), 4.58 (d, Jgem = 11.8 Hz, 1H,
–OCHHPh), 4.50 (d, Jgem = 11.2 Hz, 1H, –OCHHPh), 4.48 (d, Jgem
=
12.1 Hz, 1H, –OCHHPh), 4.54–4.37 (m, 5H, –OCH2Ph), 4.41–2.98
(m, 36H, –COCH2N–, –CH2CH2OBn, –CH2CH2OBn, –NCH2 (CH2)3-
NHBoc, –CH2NHBoc, overlapped), 1.76–1.27 (m, 26H, –NCH2
(CH2)2CH2NHBoc, –C(CH3)3); 13C NMR (100 MHz, CDCl3, 25 °C, mix-
ture of rotamers): d = 173.5 (br s), 172.9 (br s), 171.0 (br s), 169.7,
169.4, 169.2, 168.7, 168.6, 168.4, 168.2, 168.0, 167.8, 167.5 (br s),
156.0 (br s) (CO), 138.0 (br s), 137.6 (br s), 137.5 (br s) (Cipso-Bn),
128.6–127.4 (C-Ar), 78.7 (br s) (–C(CH3)3), 73.4, 73.2, 73.0, 72.9,
72.8 (–OCH2Ph), 69.7 (br s), 69.5, 69.0, 68.7, 68.5, 68.3, 68.1, 67.9,
67.8 (br s), 67.3 (br s), 65.7, 65.6, 65.4, 64.8, 64.5 (br s)
(–CH2CH2OBn), 53.4, 51.9 (br s), 51.7, 50.7, 50.5, 50.4, 49.9 (br s),
49.2, 48.9, 48.7, 48.5 (br s), 48.0 (br s), 47.7, 47. 5, 47.1, 46.1, 45.7,
(–COCH2N–, –CH2CH2OBn, –NCH2(CH2)3NHBoc), 41.7 (br s), 40.0
(br s), 39.9, 39.8 (–CH2NHBoc), 28.3 (–C(CH3)3), 26.9, 26.4, 26.2
(br s), 24.9, 24.6, 24.4 (br s), 24.3, 23.8 (br s), 23.0 (-NCH2-
(CH2)2CH2NHBoc); ESI MS: 1239.9 m/z [M+H+].
the ESI source by using a syringe pump; the flow rate was 5 lL/min.
The capillary voltage was set at 4.0 V, the spray voltage at 5 kV, and
the tube lens offset at ꢂ40 V. The capillary temperature was 220 °C.
Data were acquired in MS1and MSn scanning modes. Zoom scan was
used in these experiments.
4.2.2. Compound 6
4.2. Mixed monomer/submonomer solid-phase synthesis of
linear peptoids 2 and 6
White foam; 1H NMR (400 MHz, CDCl3, 25 °C, mixture of rota-
mers): d = 7.32–7.29 (m, 15H, Ar-H), 4.52 (d, Jgem=12.8 Hz, 1H,
–OCHHPh), 4.51 (d, Jgem = 12.6 Hz, 1H, –OCHHPh), 4.49 (d, Jgem
=
Linear peptoids 2 and 6 were synthesized by alternating sub-
monomer solid-phase method18 with standard manual Fmoc so-
lid-phase peptide synthesis protocol.
12.8 Hz, 1H, –OCHHPh), 4.46–2.89 (m, 39H, –OCH2Ph, –COCH2N–,
–NCH2CH2OBn, –NCH2CH2OBn, –NCH2(CH2)3NHBoc, –CH2NHBoc
overlapped), 1.44–1.28 (m, 39H, –C(CH3)3, –NCH2(CH2)2
CH2NHBoc); 13C NMR (100 MHz, CDCl3, 25 °C, mixture of rota-
mers): d = 173.6, 173.4, 171.2, 170.8, 169.8, 169.5, 169.4, 169.2
(br s), 168.9 (br s), 168.4, 168.0 (br s), 167.4, 167.2, 166.7, 166.1,
156.4 (br s) (CO), 137.9 (br s), 137.5 (br s), 137.4 (br s) (Cipso-Bn),
128.4–127.5 (C-Ar), 79.3 (br s) (–C(CH3)3), 73.6 (br s), 73.5, 72.9,
(–OCH2Ph), 67.5 (br s) (–CH2CH2OBn), 50.6, 50.4, 50.2, 49.8, 49.5,
49.2, 48.8 (br s), 48.6 (br s), 48.3 (br s), 48.0, 47.8, 47.6, 47.5
(–COCH2N–, –CH2CH2OBn, –NCH2(CH2)3NHBoc), 40.0, 39.8 (br s),
39.6 (br s) (–CH2NHBoc), 29.6, 29.0, 27.0, 26.9, 26.7, 26.4, 25.1,
24.6, 23.3, 23.1 (–NCH2(CH2)2CH2NHBoc), 28.3 (-C(CH3)3); ESI
MS: 1276.8 m/z [M+H+].
Typically, 2-chlorotrityl chloride resin (2,a-dichlorobenzhydryl-
polystyrene crosslinked with 1% DVB; 100–200 mesh; 1.55 mmol/
g, 0.30 g, 0.46 mmol) was swelled in dry DMF (3 mL) for 45 min
and washed twice with dry DCM (3 mL). A solution of N-Fmoc N-
benzyloxyethyl glycine10a (25, 0.17 mmol in 3 mL of dry DCM)
and DIPEA (0.68 mmol) was added on a shaker platform for 1.5 h
at room temperature. The loaded resin was washed with dry
DCM (3 mL), then with a mixture of DCM/MeOH/DIPEA (17:2:1)
(2 ꢁ 3 mL) and finally with DMF (3 ꢁ 3 mL). Fmoc deprotection
was performed twice with 20% piperidine in DMF (3 mL) for
3 min and 7 min, respectively, followed by extensive washes with

D. Comegna et al. / Bioorg. Med. Chem. 18 (2010) 2010–2018
2015
4.3. Sub-monomer solid-phase synthesis of linear peptoids 10,
12, 16 and 20
4.3.3. Compound 16
White foam; 1H NMR (250 MHz, CDCl3, 25 °C, mixture of rota-
mers): d = 7.75–7.16 (m, 10H, Ar-H), 5.07 (br s, 4H), 4.30–3.41
(m, 16H), 3.25 (br s, 6H), 2.99 (br s, 10H), 1.34 (br s, 52H); 13C
NMR (62.5 MHz, CDCl3, 25 °C, mixture of rotamers): d = 173.0 (br
Linear peptoid oligomers 10, 12, 16 and 20 were synthesized
using a sub-monomer solid-phase approach18 In a typical synthesis
2-chlorotrityl chloride resin (2,
a-dichlorobenzhydryl-polystyrene
s), 170.8 (br s), 168.0 (br s), 156.3 (br s) (CO), 131.0–130.2 (Cipso-
crosslinked with 1% DVB; 100–200 mesh; 1.1 mmol/g, 0.30 g,
0.33 mmol) was swelled in dry DMF (3 mL) for 45 min and washed
twice with dry DCM (3 mL). The first sub-monomer was attached
onto the resin by adding 56 mg (0.40 mmol) of bromoacetic acid
Bn), 127.6 (br s) (C-Ar), 79.1 (br s) (–C(CH3)3), 50.2 (br s), 47.7
(br s), 39.8 (br s), 37.6 (br s), 35.3 (br s), 31.1 (br s), 30.2 (br s),
29.1 (br s), 27.9 (br s), 27.1 (br s), 25.1 (br s) (–NCH2Ph,
–COCH2N–, –N(CH2)4NHBoc, –C(CH3)3); ESI MS: 1225.5 m/z
[M+H+].
in dry DCM (3 mL) and 280 lL of DIPEA (1.6 mmol) on a shaker
platform for 30 min at room temperature, followed by washing
with dry DCM (3 mL) and then with DMF (3 ꢁ 3 mL). To the bromo-
acetylated resin was added a DMF solution (1 M, 3 mL) of the de-
sired amine (benzylamine or the commercially available [Aldrich]
N-tert-butoxycarbonyl-1,4-diaminobutane for the assembly of the
N-(phenylmethyl)glycine and N-(N-tert-butoxycarbonylbutyl)gly-
cine, respectively) the mixture was left on a shaker platform for
30 min at room temperature, then the resin was washed with
DMF (3 ꢁ 3 mL). Subsequent bromoacetylation reactions were
accomplished by reacting the aminated oligomer with a solution
4.3.4. Compound 20
White foam; 1H NMR (400 MHz, CDCl3, 25 °C, mixture of rota-
mers): d = 4.40–3.78 (m, 12H), 3.52–3.20 (m, 10H), 3.15–2.90 (m,
14H), 1.69–1.23 (m, 78H); 13C NMR (100 MHz, CDCl3, 25 °C, mix-
ture of rotamers): d = 172.7 (br s), 168.0 (br s), 156.4 (br s) (CO),
79.2 (br s) (–C(CH3)3), 48.1 (br s), 40.0 (br s), 29.6 (br s), 28.3 (br
s), 27.2 (br s), 25.4 (br s), 24.6 (br s), 22.6 (br s) (–COCH2N–,
–N(CH2)4NHBoc, –C(CH3)3); ESI MS: 1387.9 m/z [M+H+].
of bromoacetic acid in DMF (1.2 M, 2.5 mL) and 600
l
L of DIC for
4.4. General cyclization reaction (synthesis of 4, 8, 11, 14, 18,
22)
20 min at room temperature. The filtered resin was washed with
DMF (3 ꢁ 3 mL) and treated again with the amine in the same con-
ditions reported above. This cycle of reactions was iterated until
the target oligomer was obtained.
The cleavage was performed by treating twice the resin, previ-
ously washed with DCM (3 ꢁ 3 mL), with 3 mL of 20% HFIP in DCM
(v/v) on a shaker platform at room temperature for 30 min and
5 min, respectively. The resin was then filtered away and the
combined filtrates were concentrated in vacuo. The final products
were dissolved in 50% acetonitrile in HPLC grade water and ana-
lysed by RP-HPLC (purity >95% for all the oligomers, conditions:
5% ? 100% B in 30 min [A: 0.1% TFA in water, B: 0.1% TFA in aceto-
nitrile], flow: 1.0 mL/min, 220 nm. C18 reversed-phase analytical
A
solution of the linear peptoid (2, 6, 10, 12, 16, 20,
0.222 mmol), previously co-evaporated three times with toluene,
was prepared under nitrogen in dry DMF (20 mL). The mixture
was added drop-wise by syringe pump in 2 h to a stirred solution
of PyBop (462 mg, 0.890 mmol) and DIPEA (230 lL, 1.320 mmol) in
dry DMF (40 mL) at room temperature in anhydrous atmosphere.
After 12 h the resulting mixture was concentrated in vacuo, diluted
with CH2Cl2 (20 mL) and a solution of HCl (0.5 N, 20 mL). The mix-
ture was extracted with CH2Cl2 (2 ꢁ 20 mL) and the combined
organic phases were washed with water (12 mL), dried over anhy-
drous Na2SO4, filtered and concentrated in vacuo. The cyclic prod-
ucts were dissolved in 50% acetonitrile in HPLC grade water and
analysed by RP-HPLC (purity >85% for all the cyclic oligomers, con-
ditions: 5% ? 100% B in 30 min [A: 0.1% TFA in water, B: 0.1% TFA
in acetonitrile], flow: 1.0 mL/min, 220 nm. C18 reversed-phase
column [Waters,
and ESI mass spectrometry. The linear oligomers were subjected
l
Bondapak, 10
l
m, 125 Å, 3.9 mm ꢁ 300 mm])
to the cyclization reaction without further purification.
analytical column [Waters,
lBondapak, 10 lm, 125 Å, 3.9 mm
4.3.1. Compound 10
ꢁ 300 mm]) and ESI mass spectrometry (zoom scan technique).
The crude residues were purified by HPLC on a C18 reversed-phase
preparative column, conditions: 20% ? 100% B in 40 min [A: 0.1%
TFA in water, B: 0.1% TFA in acetonitrile], flow: 2.0 mL/min,
220 nm. The samples were dried in a falcon tube under low
pressure.
White foam; 1H NMR (400 MHz, CDCl3, 25 °C, mixture of rota-
mers): d = 7.61–7.02 (m, 30H, Ar-H), 4.80–3.54 (m, 24H,
–NCH2Ph, –COCH2N–, overlapped); 13C NMR (100 MHz, CDCl3,
25 °C, mixture of rotamers): d = 173.7, 173.5, 173.0, 172.6,
172.2 (br s), 171.3, 170.8, 169.3, 169.0 (br s), 168.4 (br s),
168.3 (br s), 167.9 (br s), 167.3 (br s) (CO), 137.0–135.6 (Cipso
-
Bn), 130.3–125.2 (C-Ar) 53.4 (br s), 52.2, 51.8 (br s), 51.7, 51.5,
51.4, 51.3, 51.2, 51.1, 50.9, 50.8, 50.6, 50.4, 50.2, 50.0, 49.8 (br
s), 49.6, 49.1 (br s), 48.6 (br s), 48.5 (br s), 47.9, 47.8, 47.5,
47.4, 47.3, 47.2, 47.0, 48.8, 46.7, 46.6, 46.5 (–NCH2Ph,
–COCH2N–); ESI MS: 901.7 m/z [M+H+].
4.4.1. Compound 4
White amorphous solid (154 mg, yield 57%). 1H NMR (250 MHz,
CDCl3, 25 °C, mixture of rotamers): d = 7.36–7.18 (m, 20H, Ar-H),
4.85 (br s, 2H, –NHBoc), 4.58–4.44 (m, 4H, –OCH2Ph), 4.38
(d, Jgem = 11.4 Hz, 1H, –OCHHPh), 4.36 (d, Jgem = 11.8 Hz, 1H, –
OCHHPh), 4.30 (d, Jgem = 12.1 Hz, 1H, –OCHHPh), 4.29
(d, Jgem = 11.4 Hz, 1H, –OCHHPh), 3.80–2.89 (m, 36H, –COCH2N–,
–NCH2CH2OBn, –NCH2CH2OBn, –NCH2(CH2)2CH2NHBoc over-
lapped), 1.76–1.27 (m, 26H, –NCH2(CH2)2CH2NHBoc, –C(CH3)3);
13C NMR (62.5 MHz, CDCl3, 25 °C, mixture of rotamers): d = 170.2
(br s), 169.9 (br s), 169.4 (br s), 156.0 (br s) (CO), 137.8 (br s) (Cip-
so-Bn), 129.3–126.3 (C-Ar), 78.7 (br s) (–C(CH3)3), 75.5 (br s), 73.0
(br s), 70.8 (br s), 70.2 (br s), 68.7 (br s), 67.1 (br s), 66.4 (br s),
65.9 (br s) (–OCH2Ph, –CH2CH2OBn), 52.3, 52.1, 51.2, 51.0, 50.9,
50.2, 50.0, 49.2 (br s), 48.3, 47.9, 46.9, 46.1 (br s), 45.5, 43.8 (br
s), 42.0, 39.9, 37.5 (–COCH2N–, –CH2CH2OBn, –NCH2(CH2)3NHBoc,
–CH2NHBoc), 31.2, 29.2 (br s), 28.6, 28.3, 27.2 (br s), 27.0, 26.1
(br s), 25.2, 24.8, 24.0 (br s) (–NCH2(CH2)2CH2NHBoc, –C(CH3)3);
ESI MS: 1243.7 m/z [M+Na+].
4.3.2. Compound 12
White foam; 1H NMR (400 MHz, CDCl3, 25 °C, mixture of rota-
mers): d = 7.33–7.20 (m, 20H, Ar-H), 4.63–2.92 (m, 28H, –NCH2Ph,
–COCH2N–, –NCH2(CH2)3NHBoc, –CH2NHBoc overlapped), 1.42–
1.22 (m, 26H, –C(CH3)3, –NCH2(CH2)2CH2NHBoc); 13C NMR
(100 MHz, CDCl3, 25 °C, mixture of rotamers): d = 172.3 (br s),
171.9 (br s), 169.3, 169.5, 169.0, 168.8 (br s), 168.5 (br s), 156.3
(br s) (CO), 135.3–133.2 (Cipso-Bn), 129.4–126.1 (C-Ar), 79.5, 77.4
(-C(CH3)3), 52.2, 51.9 (br s), 51.6, 51.3, 51.1 (br s), 49.1, 48.8,
48.6, 48.3 (br s), 48.0 (br s), 47.5, 44.4, 44.0, 43.8, 41.2, 40.1, 39.8
(br s), 38.6, 27.0 (br s), 26.9, 26.6, 25.2 (br s), 24.9, 24.6, 23.3
(–NCH2Ph, –COCH2N–, –N(CH2)4NHBoc), 28.3 (–C(CH3)3); ESI MS:
1063.8 m/z [M+H+].

2016
D. Comegna et al. / Bioorg. Med. Chem. 18 (2010) 2010–2018
4.4.2. Compound 8
4.5. Global deprotection of linear and cyclic peptoids (synthesis
of 3, 5, 7, 9, 13, 15, 17, 19, 21, 23)
White amorphous solid (134 mg, 48% yield). 1H NMR (400 MHz,
CDCl3, 25 °C, mixture of rotamers): d = 7.34–7.26 (m, 15H, Ar-H),
4.69 (br s, 3H, –NHBoc), 4.53–3.02 (m, 42H, –OCH2Ph, –OCCH2N–,
–NCH2CH2OBn, –NCH2CH2OBn, –NCH2(CH2)2CH2NHBoc, over-
lapped), 1.51.1.25 (m, 39H, –C(CH3)3, –NCH2(CH2)2CH2NHBoc);
13C NMR (100 MHz, CDCl3, 25 °C, mixture of rotamers): d = 171.9
(br s), 171.6 (br s), 169.7 (br s), 169.2, 168.8, 167.7, 156.2 (br s),
Typically, the N-tert-butoxycarbonyl protected peptoid (2, 4, 6,
8, 12, 14, 16, 18, 20, 22, 0.030 mmol) was dissolved in a mixture
of CH2Cl2/TFA 1:1 (1 mL) and stirred for 2 h at room temperature.
The resulting solution was concentrated to 100 lL, under a nitro-
gen stream, and poured in cold diethyl ether (2 mL). The white pre-
cipitate was washed three times with cold diethyl ether (2 mL) and
dried under high vacuum. The presence of the CF3COOH reso-
nances in the 13C NMR spectrum depends on the concentration
of the sample in the NMR experiment. Not all the samples showed
evident 13CF313COOH NMR resonances, even though all the pep-
toids were isolated as trifluoroacetate salts.
156.0 (br s) (CO), 138.4, 138.3, 137.9 (br s), 137.6, 137.5 (Cipso
-
Bn), 128.4–127.7 (C-Ar), 79.1 (br s) (–C(CH3)3), 74.0, 73.8, 73.5
(br s), 73.3, 73.2 (–OCH2Ph), 69.9, 69.4, 68.8, 66.6 (–CH2CH2OBn),
51.2, 50.5, 49.6 (br s), 49.4, 49.0 (br s), 48.8, 48.5 (br s), 48.2,
48.0, 47.6 (br s), 46.6, 45.5, 45.3 (–COCH2N–, –CH2CH2OBn,
–NCH2(CH2)3NHBoc), 40.3 (br s), 40.0 (br s) (–CH2NHBoc), 34.4,
29.7, 27.0 (br s), 26.2 (br s), 26.0, 25.2 (br s), 24.0
(–NCH2(CH2)2CH2NHBoc), 28.4 (-C(CH3)3); ESI MS: 1280.9 m/z
[M+Na+].
4.5.1. Compound 3
White foam (41.4 mg, quantitative yield); 1H NMR (300 MHz,
D2O, 25 °C, mixture of rotamers): d = 7.40–7.32 (m, 20H, Ar-H),
4.59–2.90 (m, 44H, –OCCH2N–, –NCH2CH2OBn, –NCH2CH2OBn,
4.4.3. Compound 11
White amorphous solid (90 mg, yield 46%). 1H NMR (400 MHz,
CDCl3, 25 °C, mixture of rotamers): d = 7.40–7.06 (m, 30H, Ar-H),
4.86–3.25 (m, 24H, –NCH2Ph, –COCH2N–, overlapped); 13C NMR
(100 MHz, CDCl3, 25 °C, mixture of rotamers): d = 171.9, 171.3,
169.6, 168.9, 168.6, 167.9 (CO), 137.2–135.4 (Cipso-Bn), 129.3–
125.4 (C-Ar), 53.0 (br s), 51.6, 50.2 (br s), 48.9 (br s), 48.6, 48.4,
47.7, 47.5 (br s) (–NCH2Ph, –COCH2N–); ESI MS: 883.9 m/z
[M+H+]; HR-ESI MS: m/z 883.4183 [M+H]+ (calcd for C54H55N6O6
883.4178).
—NCH2ðCH2Þ3NH þ, —NðCH2Þ3CH2NH3 overlapped), 1.48 (br s,
þ
8H, —NCH2ðCH2Þ3 CH2NH þ); 13C NMR (75 MHz, D2O, 25 °C, mix-
2
3
ture of rotamers): d = 172.7, 171.5, 170.2, 169.9 (br s), 169.7 (br
s), 167.2, 167.1, 166.7, 165.8 (br s), 165.5 (br s), 163.3 (br s),
2
162.4 (br s), 162.2, 161.8 (CO), 162.6 (q, JC,F = 35 Hz, –COCF3)
1
116.0 (q, JC,F = 289 Hz, –CF3), 137.2 (br s), 136.8 (Cipso-Bn),
128.4–128.0 (C-Ar), 72.8 (br s), 72.6 (br s), 72.2 (br s) (–OCH2Ph),
67.1 (br s), 66.9 (br s), 66.6, 65.8, 65.6, 64.3 (br s), 64.0 (br s)
(–CH2CH2OBn), 48.7, 48.4, 48.0, 47.4 (br s), 47.0 (br s), 46.9 (br s),
46.7 (–COCH2N–, –CH2CH2OBn, –NCH2(CH2)3NH2), 38.7 (br s)
(—CH2NH3þ), 23.8, 23.7 (br s), 23.1 (br s) (–NCH2(CH2)2CH2NH3+);
ESI MS: 1039.4 m/z [M+H+]; HR-ESI MS: m/z 1039.5873 [M+H]+
(calcd for C56H79N8O11 1039.5863).
4.4.4. Compound 14
White amorphous solid (118 mg, yield 51%). 1H NMR (400 MHz,
CDCl3, 25 °C, mixture of rotamers) d: 7.49–7.01 (m, 20H, Ar-H),
5.39 (br s, 2H, –NHBoc), 4.86–2.93 (m, 28H, –NCH2Ph, –COCH2N–,
–NCH2(CH2)3NHBoc, –CH2NHBoc, overlapped), 1.42–1.22 (m, 26H,
–C(CH3)3, –NCH2(CH2)2CH2NHBoc); 13C NMR (100 MHz, CDCl3,
25 °C, mixture of rotamers): d = 169.6, 169.4, 169.1, 168.7, 168.5,
168.4, 168.3, 168.1, 156.3 (br s) (CO), 135.3–133.2 (Cipso-Bn),
129.4–126.1 (C-Ar), 79.5, 77.4 (–C(CH3)3), 53.0, 51.5, 51.4, 51.3,
51.1, 50.9, 50.8, 50.5, 50.4, 50.2 (br s), 49.8 (br s), 49.4 (br s),
49.1, 49.0, 48.8 (br s), 48.6, 48.0 (br s), 47.6, 47.4 (br s) (–NCH2Ph,
–COCH2N–), 40.3 (br s), 40.0 (br s), 30.2 (br s), 30.0 (br s), 29.7 (br
s), 29.4, 29.3, 27.3, 27.1, 26.9, 25.9, 25.7, 25.3 (br sbr s), 25.2, 25.1,
24.8 (br s), 22.6 (br s) (–N(CH2)4NHBoc), 28.3 (–C(CH3)3); ESI MS:
1045.9 m/z [M+H+].
4.5.2. Compound 5
White amorphous solid (37.5 mg, quantitative yield); 1H NMR
(250 MHz, CD3OD, 25 °C, mixture of rotamers): d = 7.58–7.29
(m, 20H, Ar-H), 4.82–2.78 (m, 44H, –COCH2N, –NCH2CH2OBn,
þ
–NCH2CH2OBn, —NCH2ðCH2Þ3NH3þ, —CH2NH3 overlapped), 1.90–
1.41 (m, 8H, —NCH2ðCH2Þ2CH2NH3þ); 13C NMR (62.5 MHz, D2O/
CD3CN 1:1, 25 °C, mixture of rotamers): d = 169.5 (br s), 169.2 (br
s), 168.5 (br s) (CO), 137.3 (br s) (Cipso-Bn), 127.9–127.3 (C-Ar),
72.2 (br s), 71.8 (br s) (–OCH2Ph), 67.6, 67.3, 67.2, 67.1, 66.1, 65.0
(br s) 63.8, 62.9 (–CH2CH2OBn), 50.2 (br s), 49.3(br s), 48.9 (br s),
48.3 (br s), 47.5 (br s), 47.2 (br s), 47.1 (br s), 44.8 (br s), 38.4 (br
s), 23.34 (br s), 23.0 (br s) (–COCH2N–, –CH2CH2OBn,
—NðCH2Þ4NH3þ); ESI MS: 1021.4 m/z [M+H+]; HR-ESI MS: m/z
1021.5751 [M+H]+ (calcd for C56H77N8O10 1021.5757).
4.4.5. Compound 18
White amorphous solid (142 mg, 53 % yield). 1H NMR (400 MHz,
CDCl3, 25 °C, mixture of rotamers): d = 7.30–7.18 (m, 10H, Ar-H),
5.24–3.41 (m, 20H), 3.39–3.12 (m, 6H), 3.10–2.82 (m, 10H), 1.54–
1.20 (m, 52H); 13C NMR (100 MHz, CDCl3, 25 °C, mixture of
rotamers): d = 170.1 (br s), 168.8 (br s), 167.6 (br s), 165.6 (br s),
156.7 (br s), 156.3 (br s) (CO), 129.1, 128.2, 127.1, 126.2, 125.8
(C-Ar), 79.1 (br s) (–C(CH3)3), 53.4, 49.2 (br s), 46.2 (br s),
39.8 (br s), 29.6 (br s), 28.3 (br s), 27.2 (br s), 26.3 (br s), 26.2 (br
s) (–NCH2Ph, –COCH2N–, –N(CH2)4NHBoc, –C(CH3)3); ESI MS:
1229.8 m/z [M+Na+].
4.5.3. Compound 7
White amorphous solid (43.0 mg, quantitative yield); 1H NMR
(400 MHz, D2O, 25 °C, mixture of rotamers): d = 7.40–7.34 (m,
15H, Ar-H), 4.55–2.90 (m, 42H, –OCH2Ph, –COCH2N–, –NCH2-
þ
CH2OBn, –NCH2CH2OBn, —NCH2ðCH2Þ2CH2NH3þ, —CH2NH3 over-
lapped), 1.75–1.45 (m, 12H, —NCH2ðCH2Þ2CH2NH3þ); 13C NMR
(100 MHz, D2O, 25 °C, mixture of rotamers): d = 172.6 (br s),
170.1 (br s), 170.0 (br s), 169.9 (br s), 169.8 (br s), 169.7 (br s),
169.6 (br s) (CO), 137.2 (br s) (Cipso-Bn), 128.4–127.9 (C-Ar), 72.8
(br s), 72.7 (br s) (–OCH2Ph), 67.0 (br s), 66.9 (br s), 65.7 (br s)
(–CH2CH2OBn), 48.7 (br s), 48.1 (br s), 47.3 (br s), 47.0 (br s),
46.6 (br s) (–COCH2N–, –CH2CH2OBn, –NCH2(CH2)3NH2), 38.7 (br
s), 38.4 (br s) (—CH2NH3þ), 24.1, 23.7 (br s), 23.5 (br s), 23.3
(—NCH2ðCH2Þ2CH2NH3þ); ESI MS: 976.2 m/z [M+H+]; HR-ESI MS:
m/z 976.5875 [M+H]+ (calcd for C51H78N9O10 976.5866).
4.4.6. Compound 22
White amorphous solid (176 mg, 58% yield); 1H NMR (400 MHz,
CDCl3, 25 °C, mixture of rotamers): d = 5.06 (br s, 4H), 4.61 (br s,
2H), 4.48–3.58 (m, 12H), 3.50–3.18 (m, 10H), 3.15–2.28 (m, 14H),
1.72–1.31 (m, 78H); 13C NMR (100 MHz, CDCl3, 25 °C, mixture of
rotamers): d = 172.3 (br s), 168.8 (br s), 156.6 (br s) (CO), 79.3 (br
s) (-C(CH3)3), 49.6 (br s), 40.0 (br s), 29.7 (br s), 28.3 (br s), 27.2
(br s), 25.0 (br s) (–COCH2N–, –N(CH2)4NHBoc, –C(CH3)3); ESI MS:
1392.0 m/z [M+Na+].
4.5.4. Compound 9
White amorphous solid (39.0 mg, quantitative yield); 1H NMR
(300 MHz, D2O, 25 °C, mixture of rotamers): d = 7.40–7.35 (m,

D. Comegna et al. / Bioorg. Med. Chem. 18 (2010) 2010–2018
2017
15H, Ar-H), 4.62–2.78 (m, 42H, –OCH2Ph, –COCH2N–,
24.5 (br s), 23.9 (br s), 26.2 (br s) (–NCH2Ph, –COCH2N–,
—NðCH2Þ4NH3þ); ESI MS: 807.7 m/z [M+H+]; HR-ESI MS: m/z
807.5244 [M+H]+ (calcd for C42H67N10O6 807.5240).
þ
–NCH2CH2OBn, –NCH2CH2OBn, —NCH2ðCH2Þ2CH2NH3þ, —CH2NH3
overlapped), 1.71–129 (m, 12H, —NCH2ðCH2Þ2CH2NH3þ); 13C
NMR (75 MHz, D2O, 25 °C, mixture of rotamers): d = 169.5 (br s),
169.1, 168.9, 168.7, 168.2, 167.8 (CO), 137.2 (br s) (Cipso-Bn),
128.4–127.5 (C-Ar), 72.8 (br s), 72.6 (br s), 72.2 (br s), 71.8
(–OCH2Ph), 67.3 (br s), 67.0 (br s), 65.6 (br s) (–CH2CH2OBn),
51.2, 49.8, 49.6, 49.0 (br s), 48.5 (br s), 48.3 (br s), 48.0 (br s),
47.6 (br s), 47.3 (br s), 47.2 (br s), 46.8 (br s) (–COCH2N–,
4.5.9. Compound 21
White amorphous solid (47.5 mg, quantitative yield); 1H NMR
(250 MHz, D2O, 25 °C, mixture of rotamers): d = 4.32–3.94 (m,
12H), 3.46–3.12 (m, 10H), 3.08–2.78 (m, 14H) 1.63–1.45 (br s,
24H); 13C NMR (62.5 MHz, D2O, 25 °C, mixture of rotamers):
d = 172.0 (br s), 169.2 (br s), 168.2 (br s), 164.9 (br s) (CO), 161.7
+
–CH2CH2OBn, –NCH2(CH2)3NH3 2), 38.7 (br s) (–CH2NH3+), 24.1,
23.7 (br s), 23.0 (–NCH2(CH2)2CH2NH3+); ESI MS: 958.5 m/z
[M+H+]; HR-ESI MS: m/z 958.5769 [M+H]+ (calcd for C51H76N9O9
958.5761).
(q, JC,F = 36 Hz, -COCF3) 115.0 (q, JC,F = 290 Hz, -CF3), 48.9 (br s),
46.8 (br s), 40.0 (br s), 37.7 (br s), 35.5 (br s), 24.6 (br s), 22.8 (br
s), 20.8 (br s) (–COCH2N–, —NðCH2Þ4NH3þ); ESI MS: 787.8 m/z
[M+H+]; HR-ESI MS: m/z 787.5871 [M+H]+ (calcd for C36H75N12O7
787.5876).
2
1
4.5.5. Compound 13
White amorphous solid (36.1 mg, quantitative yield); 1H NMR
(400 MHz, CD3CN, 25 °C, mixture of rotamers): d = 7.33–7.20 (m,
20H, Ar-H), 4.60–2.92 (m, 28H, –NCH2Ph, –COCH2N–, —NCH2
4.5.10. Compound 23
White amorphous solid (35.3 mg, 81% yield); 1H NMR
(400 MHz, D2O, 25 °C, mixture of rotamers): d = 4.80–3.50 (m,
12H), 3.41–3.02 (m, 10H), 3.02–2.81 (m, 14H) 1.78–1.39 (m,
24H); 13C NMR (100 MHz, D2O, 25 °C, mixture of rotamers):
d = 171.5 (br s), 170.6 (br s), 169.8 (br s), 168.9 (br s) (CO), 162.8
þ
þ
ðCH2Þ3NH3
,
—CH2NH3
overlapped), 1.86–1.48 (m, 8H,
—NCH2ðCH2Þ2CH2NH3þ); 13C NMR (100 MHz, CD3CN, 25 °C, mix-
ture of rotamers): d = 172.9, 171.8 (br s), 171.6, 171.2 (br s),
170.9 (br s), 170.6, 170.5, 170.3, 169.6, 168.5, 168.3, 168.0, 162.1
2
1
(br s) (CO), 139.6, 139.5, 139.3, 137.1, 137.0, 136.8, 136.7 (Cipso
-
(q, JC,F = 35 Hz, -COCF3) 116.3 (q, JC,F = 290 Hz, –CF3), 50.9 (br s),
49.3 (br s), 48.1 (br s), 38.9 (br s), 25.3 (br s), 24.0 (br s)
(–COCH2N–, —NðCH2Þ4NH3þ); ESI MS: 769.7 m/z [M+H+]; HR-ESI
MS: m/z 769.5767 [M+H]+ (calcd for C36H73N12O6 769.5771).
Bn), 131.8–129.5 (C-Ar), 53.2 (br s), 52.8, 52.3, 52.0, 51.8, 50.4
(br s), 49.4, 48.3, 47.0, 45.3, (–NCH2Ph, –COCH2N–), 40.8 (br s)
26.9, 26.7, 26.5, 26.4, 26.0, 25.2, 25.0 (—NðCH2Þ4NH3þ); ESI MS:
864.0 m/z [M+H+]; HR-ESI MS: m/z 863.4809 [M+H]+ (calcd for
C48H63N8O7 863.4814).
5. Biological activities
4.5.6. Compound 15
5.1. Bacterial and fungal strains and growth conditions
White amorphous solid (32.2 mg, quantitative yield); 1H NMR
(400 MHz, CD3CN, 25 °C, mixture of rotamers): d = 7.52–7.12 (m,
20H, Ar-H), 4.81–2.96 (m, 28H, –NCH2Ph, –COCH2N–, —NCH2
ðCH2Þ NH3þ, —CH2NH3þ, overlapped), 1.77–1.45 (m, 8H, —NCH2
ðCH2Þ3CH2NH þ); 13C NMR (100 MHz, CDCl3, 25 °C, mixture of rota-
E. coli ATCC 25922, Salmonella enterica serovar Typhimurium
ATCC 14028, and S. aureus ATCC 25923 were used for determining
the antibacterial and the membrane permeabilizing activities of
the peptoids. All strains were grown at 37 °C in Mueller–Hinton
(MH) broth (DIFCO) and all tests were performed in the same med-
ium using bacteria in logarithmic phase growth.
The fungal strains used in this study are the reference strains C.
neoformans ATCC 90112 and C. albicans ATCC 90029, and 3 clinical
strains isolated from immunocompromised patients (C. albicans
L21, C. famata SA550 and C. neoformans L1). Fungi were grown on
Sabouraud agar plates at 30 °C for 48 h. Fungal suspensions were
then prepared by picking and suspending five colonies in 5 mL of
sterile PBS. The turbidity of the conidial spore suspensions was
measured at 600 nm and was adjusted in RPMI-1640 medium (Sig-
ma–Aldrich) to obtain the appropriate cell density.
2
3
mers): d = 174.3, 171.9 (br s), 171.3 (CO), 138.4–137.3 (Cipso-Bn),
130.3–127.2 (C-Ar), 53.9, 52.9 (br s), 52.2 (br s), 50.9 (br s), 50.6
(br s), 49.7 (br s), 49.5 (br s), (–NCH2Ph, –COCH2N–), 40.5 (br s),
40.3 (br s) (—NCH2ðCH2Þ NH3þ, —NðCH2Þ3CH2NH3þ), 25.8 (br s),
25.5 (br s) (—NCH2ðCH2Þ3 CH2NH þ); ESI MS: 846.0 m/z [M+H+];
2
3
HR-ESI MS: m/z 845.4712 [M+H]+ (calcd for C48H61N8O6 845.4709).
4.5.7. Compound 17
White amorphous solid (31.0 mg, 74% yield); 1H NMR
(250 MHz, D2O, 25 °C, mixture of rotamers): d = 7.34–7.13 (m,
10H, Ar-H), 4.56–3.94 (m, 16H), 3.19 (br s, 6H), 2.82 (br s, 10H),
1.52–1.44 (m, 16H); 13C NMR (62.5 MHz, D2O, 25 °C, mixture of
rotamers): d = 172.1 (br s), 169.1 (br s), 168.4 (br s), 164.8 (br s)
5.2. Antimicrobial assays
2
1
(CO), 161.6 (q, JC,F = 35 Hz, –COCF3) 115.1 (q, JC,F = 290 Hz, –CF3),
133.9 (br s),130.0 (br s), 129.4 (br s), 128.9 (br s), 127.4 (br s),
126.7 (br s), 125.6 (br s), 124.4 (br s) (C-Ar), 52.0 (br s), 49.0 (br
s), 46.8 (br s), 45.1 (br s), 40.1 (br s), 37.8 (br s), 35.4 (br s), 32.0
(br s), 30.0 (br s), 28.7 (br s), 27.9 (br s), 22.7 (br s), 20.7 (br s)
(–NCH2Ph, –COCH2N–, —NðCH2Þ4NH3þ); ESI MS: 825.8 m/z
[M+H+]; HR-ESI MS: m/z 825.5339 [M+H]+ (calcd for C42H69N10O7
825.5345).
The antibacterial activity was evaluated by the broth microdilu-
tion susceptibility test following the guidelines of the National
Committee for Clinical Laboratory Standards, to determine the
Minimum Inhibitory Concentration (MIC) values. The MIC of each
compound was determined as previously described.13,25
Briefly, twofold serial dilutions of each compound were prepared
in 96-well polypropylene microplates (Sarstedt) in MH broth or
RPMI-1640 medium to a final volume of 50
a well without compound as a control. A total of 50
l
L. Each series included
L of the ad-
l
4.5.8. Compound 19
justed inoculum diluted in MH broth for bacteria (ꢀ2.5 ꢁ 105 cells/
mL) or in RPMI-1640 medium for fungi (ꢀ5 ꢁ 104 cells/mL) was
added to each well. Samples were then incubated at 37 °C overnight
or 30 °C for 48 h, respectively, for bacteria o fungi. The MIC was taken
as the lowest concentration of antimicrobial compound resulting in
the complete inhibition of visible growth after incubation. Data are
the mean of 4–6 independent determinations with values differing
by one dilution at maximum.
White amorphous solid (20.8 mg, 55% yield); 1H NMR
(400 MHz, D2O, 25 °C, mixture of rotamers): d = 7.42–7.15 (m,
10H, Ar-H), 4.78–3.85 (m, 16H), 3.29–3.10 (m, 6H), 2.90–2.76 (m,
10H), 1.59–1.37 (m, 16H); 13C NMR (100 MHz, D2O, 25 °C, mixture
of rotamers): d = 173.4 (br s), 169.5 (br s), 166.4 (br s), 165.6 (br s)
2
1
(CO), 162.8 (q, JC,F = 35 Hz, –COCF3) 116 (q, JC,F = 290.0 Hz, -CF3),
130.2, 129.1, 128.8, 127.8, 127.3, 127.3, 126.7, 126.7, 126.2 (C-
Ar), 51.0 (br s), 47.9 (br s), 43.9 (br s), 38.9 (br s), 38.6 (br s),

2018
D. Comegna et al. / Bioorg. Med. Chem. 18 (2010) 2010–2018
5.3. Membrane permeabilization
References and notes
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For the analyses with fungal cells, C. albicans or C. neoformans
subcultured on Sabouraud agar were diluted in RPMI-1640 med-
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were then incubated with the compound at 30 °C for different
times.
A 0.2
to the treated bacterial or fungal cells at a final concentration of
10 g/mL, and samples were acquired after 4 min of incubation
lm-filtered solution of PI (Sigma–Aldrich) was then added
l
at 37 °C or 30 °C. All experiments were conducted in triplicate.
Data analysis was performed with the WinMDI software (Dr J. Trot-
ter, Scripps Research Institute, La Jolla, CA, USA). Data are
expressed as means SD.
5.4. Hemolytic activity
Erythrocytes were prepared from freshly collected human blood
anticoagulated with citrate-dextrose. The assays were performed
in 10 mM phosphate buffer, pH 7.4, containing 139 mM NaCl, by
incubating 0.5% (v/v) erythrocyte suspensions with 100 lM cyclo-
peptoids for 60 min at 37 °C. The reaction was stopped with cold
buffer, and the supernatant was carefully collected after centrifu-
gation at 10,000g for 1 min. The released hemoglobin was
measured at 415 nm, and the percentage of hemolysis was deter-
mined as (Asample ꢂ Ablank)/(Atot ꢂ Ablank) ꢁ 100, where Ablank and
Atot correspond, respectively, to the hemolysis in the absence of
the sample and to 100% hemolysis as obtained by addition of
0.2% Triton X-100.27 Melittin at 20
control.
lM was used as a positive
18. Zuckermann, R. N.; Kerr, J. N.; Kent, S. B. H.; Moos, W. H. J. Am. Chem. Soc. 1992,
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Acknowledgments
19. The N-fluorenylmethoxycarbonyl,N0-benzyloxyethyl glycine was synthesised
in three steps, with an overall yield of 49%. See supporting information of Ref.
10a.
Financial support from the University of Salerno. We thank Pro-
fessor G. Bifulco (University of Salerno) for discussions and Miss
Gerardina Galdi for valuable experimental work.
20. Pina-Vaz, C.; Sansonetty, F.; Rodrigues, A. G.; Costa-Oliveira, S.; Tavares, C.;
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Antimicrob. Agents Chemother. 2008, 52, 944.
Supplementary data
24. Selsted, M. E. Curr. Protein Pept. Sci. 2004, 5, 365.
25. Benincasa, M.; Scocchi, M.; Podda, E.; Skerlavaj, B.; Dolzani, L.; Gennaro, R.
Peptides 2004, 25, 2055.
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Scocchi, M. Biochim. Biophys. Acta 2006, 1760, 1732.
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463, 58.
Synthesis, spectroscopic features, computational details and
atom coordinates for the structure of compound 14 as a Na+
complex are available. Supplementary data associated with this
article can be found, in the online version, at doi:10.1016/
j.bmc.2010.01.026.