Peptaibolin analogues by incorporation of α,α-dialkylglycines: Synthesis and study of their membrane permeating ability

Article abstract of DOI:10.1016/j.tet.2015.12.079

Analogues of Peptaibolin, a peptaibol with antibiotic activity, incorporating α,α-dialkylglycines (Deg, Dpg, and Ac₆c) at selected positions were synthesised by MW-SPPS and fully characterized. A control analogue incorporating l-alanine was also prepared. The native peptide and the analogues were studied by fluorescence spectroscopy for their membrane permeating activity. Small unilamellar vesicles (SUVs) of egg phosphatidylcholine/cholesterol (70:30) containing an encapsulated fluorescence probe (6-carboxyfluorescein) were used as membrane models. The assays of carboxyfluorescein release from SUVs upon peptide addition showed that Peptaibolin-Dpg and Peptaibolin-Ac₆c are the most active peptides. These results indicate that the structure of the α,α-dialkylglycines is crucial for the membrane permeating ability of these Peptaibolin analogues.

Full text of DOI:10.1016/j.tet.2015.12.079

Tetrahedron 72 (2016) 1024e1030
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Peptaibolin analogues by incorporation of
a,a-dialkylglycines:
synthesis and study of their membrane permeating ability
a
a
a
V a^ nia I.B. Castro , Carina M. Carvalho , Rui D.V. Fernandes ,
a
b
a,
*
Sílvia M.M.A. Pereira-Lima , Elisabete M.S. Castanheira , Susana P.G. Costa
Centre of Chemistry (CQUM), University of Minho, Campus de Gualtar, 4710-057 Braga, Portugal
a
b
Centre of Physics (CFUM), University of Minho, Campus de Gualtar, 4710-057 Braga, Portugal
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 31 July 2015
Received in revised form 28 December 2015
Accepted 29 December 2015
Available online 31 December 2015
Analogues of Peptaibolin, a peptaibol with antibiotic activity, incorporating
and Ac c) at selected positions were synthesised by MW-SPPS and fully characterized. A control analogue
incorporating -alanine was also prepared. The native peptide and the analogues were studied by fluo-
rescence spectroscopy for their membrane permeating activity. Small unilamellar vesicles (SUVs) of egg
phosphatidylcholine/cholesterol (70:30) containing an encapsulated fluorescence probe (6-
carboxyfluorescein) were used as membrane models. The assays of carboxyfluorescein release from
a,a-dialkylglycines (Deg, Dpg,
6
L
Keywords:
a,a-Dialkylglycines
Peptaibolin
Membrane permeating peptides
Model membranes
6
SUVs upon peptide addition showed that Peptaibolin-Dpg and Peptaibolin-Ac c are the most active
peptides. These results indicate that the structure of the
permeating ability of these Peptaibolin analogues.
a,
a-dialkylglycines is crucial for the membrane
Ó 2016 Elsevier Ltd. All rights reserved.
Fluorescence spectroscopy
1
. Introduction
occurring AMPs isolated from soil fungi has interesting structural
features: they all bear a C-terminal amino alcohol and a variable
17,18
Microbial resistance to classical antibiotics is a serious world-
wide problem and as such there is a fast-growing interest in the
design and synthesis of new therapeutic agents as alternatives to
the established drugs. To address this issue the scientific commu-
nity has been focussing on natural antimicrobial peptides (AMPs),
which are ubiquitous components of the innate defence mecha-
nisms of animals, plants and microorganisms, known to be active
against bacteria, fungi and protozoans. AMPs present a large va-
riety of sizes, amino acid composition and secondary structures,
and can interfere with several intercellular processes.
theless, most AMPs present a cationic and hydrophobic nature that
allows them to adopt amphipathic conformations. This enables
their interaction with microbial membranes and leads to the dis-
ruption of membrane integrity either by pore formation that allows
the leakage of cell contents, or by acting in a detergent like man-
ner.
resist AMPs action,
are considered lead compounds for the development of a new class
number of
-dialkylglycine present is
valine (Iva) and -diethylglycine (Deg) have also been detec-
This class of amino acids is frequently used in the
a,
a-dialkylglycines in their composition.
The major
a,
a
a-aminoisobutyric acid (Aib) but iso-
a,a
19,20
ted.
construction of peptides with pharmacological interest since they
are not recognized by hydrolytic enzymes, thus rendering peptides
more resistant to biodegradation and increasing their bio-
1
,2
21,22
availability.
Another remarkable feature about
a,a-dia-
3
,4
lkylglycines is the tetrasubstitution at the central carbon atom that
results in the restriction of the conformational space available
around peptide bonds, thus yielding peptides with more defined
2
,5
Never-
2
3e28
conformations.
Albeit interesting this is also responsible for
the fact that the synthesis of peptide analogues bearing these
amino acids is usually a major synthetic challenge that can only be
overcome by taking advantage of synthetic methodologies more
6
e11
29e32
Despite the fact that some microorganisms are known to
recently used in peptide chemistry.
12,13
due to their broad spectrum of action AMPs
Peptaibolin (Ac-Leu-Aib-Leu-Aib-Phol) is the smallest member
of the peptaibols family. This five-residue peptide was first isolated
by H u€ lsmann et al. from Sepedonium sp. and Sepedonium ampullo-
14e16
of antibiotic pharmaceuticals.
3
3
34
Among the various classes of peptides with recognized antibi-
sporum and later synthesized using solution or solid phase
3
0
otic activity one can find peptaibols. This family of naturally
strategies. Although initial findings suggested that Peptaibolin
and its Iva analogue (with Iva at the position of Aib) possessed no
3
4
affinity towards membranes, recent in silico studies suggest that
membrane affinity might be increased by the substitution of the
*
Corresponding author. E-mail address: spc@quimica.uminho.pt (S.P.G. Costa).
http://dx.doi.org/10.1016/j.tet.2015.12.079
040-4020/Ó 2016 Elsevier Ltd. All rights reserved.
0

V.I.B. Castro et al. / Tetrahedron 72 (2016) 1024e1030
1025
Aib residues by more structurally constrained and more hydro-
Ac-Leu-Aaa-Leu-Aaa-Phol
27
phobic
a,a-dialkylglycines.
3
Bearing the above facts in mind and our interest in the synthesis
3
5e37
and application of
first time the synthesis of several Peptaibolin analogues bearing
different -dialkylglycines at the positions of the Aib residues and
a
,
a
-dialkylglycines,
herein we report for the
R
R'
a) R = R' = CH3
(Aib)
(Ala)
(Deg)
(Dpg)
(Ac6c)
a,
a
b) R = H, R' = CH
3
C
c) R = R' = CH CH
3
the evaluation of their membrane permeating ability by carboxy-
fluorescein leakage assays from model membranes (small uni-
lamellar vesicles of egg phosphatidylcholine/cholesterol, 70:30).
2
Aaa =
N
C
2 2 3
d) R = R' = CH CH CH
H
e) R = R' = -(CH2)5-
O
Fig. 1. Structure of Peptaibolin 3a and its analogues 3bee.
2
2
. Results and discussion
HOBt), before they were added to the Wang resin (a 5 equiv excess
.1. Synthesis of native Peptaibolin and Peptaibolin
was used), and coupled using a standard MW protocol (5 min,
ꢀ
analogues
28 W, 75 C). To ensure complete activation of the
a
,a
-dia-
lkylglycines, considering their stereochemical constraints, the ac-
tivation mixture was allowed to react for 1.5e2 h, before being
added to the resin and subjected to microwave irradiation. This
ensures that the active esters are transformed into the corre-
The synthesis of Peptaibolin analogues 3cee by substitution of
the native Aib residues by other -dialkylglycines was envisaged
a,a
in order to obtain information about the influence of the different
side chains in terms of length and bulk on the membrane perme-
ating activity of the resulting peptides. The native Peptaibolin, Ac-
Leu-Aib-Leu-Aib-Phol, 3a was synthesized to act as a positive
control for the subsequent membrane permeation studies and the
Peptaibolin analogue bearing an alanine instead of Aib, Ac-Leu-Ala-
Leu-Ala-Phol 3b was thought as the negative control (Fig. 1). The
synthesis of the peptides was carried out by a microwave-assisted
solid phase peptide synthesis (MW-SPPS) protocol.
41,42
sponding oxazolinones before coupling.
After removal of the Fmoc group from the last amino acid in the
sequence, the N-terminal was acetylated by treatment with acetic
anhydride in the presence of a base (N,N-diisopropylethylamine,
DIPEA). Finally, the peptide was cleaved from the resin by a reductive
cleavage protocol using in situ generated lithium borohydride to
yield the required C-terminal amino alcohol (phenylalaninol, Phol)
(Scheme 2).
2
a
.1.1. Synthesis of Fmoc-
-dialkylglycines 2cef, necessary for the Fmoc-based MW-SPPS
-aminoisobutyric acid 2a
-alanine 2b (Fmoc-Ala-OH), used as
-Dialkylglycines 1cef, obtained pre-
viously through a methodology based on an Ugi multicomponent
a
,
a
-dialkylglycines 2cef. Fmoc protected
Fmoc
Phe
piperidine 20% DMF
,a
protocol, were prepared except for Fmoc-
Fmoc-Aib-OH) and Fmoc-
commercially acquired.
a
MW (30 s + 3 min, 28 W, 75 °C)
(
L
a,a
H2N
Phe
reaction,2
9,35,37
were
reacted
with
N-(9-
fluorenylmethoxycarbonyloxy) succinimide (Fmoc-OSu). This re-
action was carried out in two different solvent mixtures, namely 5%
sodium carbonate aqueous solution with 1,4-dioxane or acetone,
Fmoc-Aaa-OH
DIC, 6-Cl-HOBt
dry DMF
MW (5 min, 28 W, 75 °C)
depending on the solubility properties of the initial
a,a-dia-
piperidine 20% DMF
MW (30 s + 3 min, 28 W, 75 °C)
lkylglycines 1cef. N-protection was achieved in good to excellent
yields (66e98%) (Scheme 1) and the prepared amino acid de-
rivatives were characterized by NMR spectroscopy and were in
3
8e40
accordance with previously reported data.
Fmoc Leu Aaa Leu Aaa Phe
piperidine 20% DMF
O
O
MW (30 s + 3 min, 28 W, 75 °C)
N
O
O
O
R
R
C
O
O
C
OH
Ac O, DIPEA
2
base
O
N
H
DMF
+
R
R
Ac Leu Aaa Leu Aaa Phe
C
OH
2
c) R= CH
d) R= (CH
e) R= -(CH
f) R= CH CH(CH
2
CH
3
HCl.H
2
N
C
O
2
)
2
CH
3
2 5
) -
1
2
3
)
2
NaBH4/LiBr
Aaa = Aib
Ala
Scheme 1. Preparation of Fmoc-
a
,a
-dialkylglycines 2cef.
Deg
Dpg
Ac6c
Ac Leu Aaa Leu Aaa Phol
Scheme 2. MW-SPPS of Peptaibolin 3a and its analogues 3bee.
2
.1.2. MW-SPP synthesis of peptides 3aee. Peptides 3aee were
synthesized on a manual microwave-assisted peptide synthesizer
CEM Discover). The amino acid coupling reactions were based on
the Fmoc protocol and a preloaded Wang resin was used, namely
Fmoc-Phe-Wang (with functionalization degree of about
.5e0.6 mmol/g). The preloaded and all other Fmoc protecting
(
The purity of the peptides was checked by analytical HPLC and
samples of each peptide were purified by semi-preparative HPLC
using a Europa Peptide C18 column and ACN/water mixtures (1:1 or
2:1) as eluent. The peptides were obtained in fair to excellent yields
a
0
1
13
groups were removed with 20% piperidine in DMF solution using
and characterized by H and C NMR spectroscopy and high-
resolution mass spectrometry (Table 1). In general, as -dia-
ꢀ
a standard MW protocol (30 sþ3 min, 28 W, 75 C). All Fmoc amino
a,a
0
acids were dissolved in dry DMF containing N,N -diisopro-
lkylglycines side chain length increases, a decrease in the yield of
pylcarbodiimide (DIC) and 6-chloro-N-hydroxybenzotriazole (6-Cl-
peptide obtained can be observed, except for Peptaibolin-Deg 3c

1026
V.I.B. Castro et al. / Tetrahedron 72 (2016) 1024e1030
Table 1
Synthesis and characterization data of Peptaibolin 3a and analogues 3bee
Peptide
Yield (%)a
1H and 13C NMR
-C Aaab (ppm)
a
a
-CH Phol (ppm)
2
CH OH (ppm)
3
3
3
3
3
a
b
c
d
e
Ac-Leu-Aib-Leu-Aib-Phol
Ac-Leu-Ala-Leu-Ala-Phol
Ac-Leu-Deg-Leu-Deg-Phol
Ac-Leu-Dpg-Leu-Dpg-Phol
89
87
22
60
32
56.42, 56.88
48.40
63.85
62.76, 62.87
59.00, 59.36
4.23e4.34
4.13e4.26
4.01e4.12
4.72
3.60e3.72
3.25e3.32
3.25e3.33
3.20e3.30
3.21e3.33
Ac-Leu-Ac
6
c-Leu-Ac
6
c-Phol
4.12e4.18
a
Relative to the resin initial loading and after preparative HPLC purification.
Aaa refers to Ala or a,a-dialkylglycines at the native Aib positions.
b
whose lower yield is due to poor solubility properties. An attempt
to use Fmoc-Dibg-OH in the synthesis of the corresponding
Peptaibolin-Dibg derivative was made but only the dipeptide Ac-
Dibg-Phol 3f was isolated.
At [peptide]/[lipid]z80 (Fig. 2), where near full membrane
permeation is achieved for the most active peptides, the perme-
ating ability follows the sequence Peptabolin-Dpg 3d>Peptabolin-
Ac
b, pointing to a correlation between the length and bulk of the
side chain of the unnatural -dialkylglycines and the ability of the
6
c 3e>Peptabolin-Deg 3c>Peptabolin-Aib 3a>Peptabolin-Ala
3
a,a
2
.2. Permeation of model membranes by peptides 3aee
corresponding peptide to permeate the model membranes.
At low [peptide]/[lipid] ratios (below 30), the permeation ability
Peptaibols are known to interact with phospholipid bilayers, by
follows
d>Peptabolin-Aib 3a>Peptabolin-Deg 3c>Peptabolin-Ala 3b. It
should be noted that the peptides containing Dpg and Ac c have
the
order
Peptaibolin-Ac
6
c
3eꢁPeptabolin-Dpg
permeating its structure and allowing the leakage of the contents of
the vesicle. Therefore, the permeating properties of Peptaibolin and
its analogues were studied through the release of an entrapped
fluorophore, carboxyfluorescein (CF), in small unilamellar vesicles
3
6
a similar behaviour throughout the range of [peptide]/[lipid] ratios
used, with several intersections between the corresponding curves
(Fig. 2). Although very active at high [peptide]/[lipid] ratios,
Peptaibolin-Deg 3c presents a low permeating ability at [peptide]/
(
SUVs) of egg phosphatidylcholine/cholesterol 70:30, used as
43,44
membrane models.
The percentage of CF release with in-
creasing [peptide]/[lipid] ratio (until ca. 80), for the synthesized
peptides 3aee, is shown in Fig. 2.
It can be observed that the analogues bearing 1-amino-1-
6
cyclohexane carboxylic acid (Peptaibolin-Ac c) 3e and a,a-dipro-
pylglycine (Peptaibolin-Dpg) 3d are the ones with larger
permeation ability, that is always higher than for the native
Peptaibolin 3a, and is significant even at low [peptide] to [lipid]
ratio. At high [peptide] to [lipid] ratio (above 80), the peptide
[lipid]<60.
The influence of the composition of model membranes in the
permeability cannot be discarded and may justify the negligible
permeating ability in 1-palmitoyl-2-oleoyl-sn-glycero-3-
phosphocholine (POPC) membranes predicted for the native Pep-
2
7
taibolin by molecular dynamics simulations, contrarily to what is
observed in Fig. 2. It is also expected that the packing of the hy-
drophobic phospholipid chains in membrane, determining mem-
brane fluidity, play a major role in the permeation ability.
Phosphatidylcholine from egg yolk is a natural mixture of different
phosphatidylcholines (PC), varying in chain length and degree of
unsaturation. The main components are PC 16:0, PC 18:0 (both
saturated and rigid at room temperature) and PC 18:1 (unsaturated
containing
very active. As expected, a very low membrane permeating ability
was detected for the analogue 3b with -alanine.
a,a-diethylglycine (Peptaibolin-Deg) 3c also becomes
L
27
Recent molecular dynamics simulations showed that mem-
brane affinity might be increased by the substitution of the Aib
residues by more structurally constrained and more hydrophobic
4
5
and fluid). Variations in composition can affect the membrane
a
,a
-dialkylglycines. In particular, it was shown that Peptaibolin-
c and also a peptide bearing -dihexylglycine, its related
acyclic side chain counterpart, were able to induce -helix-type
4
6
fluidity and the size of phosphatidylcholine SUVs.
Ac
6
a,a
Another important factor is the amount of cholesterol, the latter
acting as modulator of the bilayer fluidity. Significant differences in
permeation of phosphatidylcholine/cholesterol 70:30 and 80:20
model membranes were found for short-sequence peptaibols
a
secondary structures of Peptaibolin in water, which are not present
in the native structure, despite no apparent correlation between
27
increased helicity and membrane permeating ability being found.
4
4
(
Harzianins HC). The role of these factors in the permeating
ability of the Peptaibolin analogues is being investigated and the
results will be presented in the near future.
3. Conclusions
The microwave-assisted solid phase synthesis of Peptaibolin
and a series of analogues by substitution of the native Aib residue
by -alanine or different -dialkylglycines was reported for the
L
a,a
first time, in fair to excellent yields. The native peptide, the negative
control analogue and the dialkylglycine-bearing analogues were
studied for their ability to interact with model membranes (phos-
phatidylcholine/cholesterol 70:30 vesicles) containing encapsu-
lated carboxyfluorescein. By addition of peptides at different
peptide to lipid molar ratios, it was possible to follow the release of
the entrapped fluorescent probe, monitoring the increase in its
fluorescence intensity over time, due to the decrease of the self-
quenching effect. The obtained results revealed that the Peptaibo-
6
lin analogues bearing Ac c and Deg are the peptides with higher
Fig. 2. Peptide-induced CF leakage at 20 min for different [peptide]/[lipid] ratios from
egg phosphatidylcholine/cholesterol (70:30) vesicles.
permeating ability, evidencing a correlation between the length

V.I.B. Castro et al. / Tetrahedron 72 (2016) 1024e1030
1027
and bulk of the
a
,
a
-dialkylglycines side chain and the ability of the
(5 equiv) in water (50 mL). Fmoc-OSu (1.1e2.0 equiv), dissolved in
acetone (10 mL), was added drop wise and the mixture was stirred
at room temperature overnight. The acetone was evaporated and
the aqueous phase washed with ethyl acetate (3ꢂ40 mL). The
aqueous phase was acidified to pH 3 with aqueous 10% citric acid
solution and kept in the cold to form a precipitate. The solid was
filtered, washed with cold water and dissolved in ethyl acetate,
corresponding peptides to permeate the membranes.
The successful synthesis of a series of Peptaibolin analogues
with enhanced membrane permeating capability may represent
a step forward in the improvement of the antimicrobial properties
of this type of AMPs.
4
4
. Experimental Section
followed by drying with anhydrous MgSO
solvent to dryness in a rotary evaporator.
4
and evaporation of the
.1. General
4
.2.3. N-(9-Fluorenylmethyloxycarbonyl)-
a,a-diethylglycine, Fmoc-
All melting points were measured on a Stuart SMP3 melting
Deg-OH (2c). By reaction of -diethylglycine hydrochloride 1c
a,a
point apparatus. Chromatography on silica gel was carried out on
Merck Kieselgel (230e240 mesh). UV/visible absorption spectra
(1.023 g, 5.95 mmol) and Fmoc-OSu (2.412 g, 7.15 mmol, 1.2 equiv)
using method A, compound 2c was obtained as colourless oil
(1.023 g, 72%) and the characterization in accordance to previously
(
200e700 nm) were obtained using a Shimadzu UV-2501PC spec-
3
8
ꢀ
1
trophotometer. NMR spectra were obtained on a Bruker Avance III
reported data. Mp¼98.2e102.2 C. H NMR (CDCl
¼0.74 (t, J¼6.8 Hz, 6H, 2ꢂCH Deg), 1.74e1.80 (m, 2H, CH
2.22e2.28 (m, 2H, CH
Deg), 4.14 (t, J¼6.8 Hz, 1H, H-9 Fmoc), 4.32
(d, J¼6.4 Hz, 2H, CH Fmoc), 5.56 (s, 1H, NH), 7.23 (dt, J¼1.1 and
3
, 400 MHz):
1
4
00 at an operating frequency of 400 MHz for H and 100.6 MHz for
d
H
3
2
Deg),
1
3
ꢀ
C using the solvent peak as internal reference at 25 C. All
2
chemical shifts are given in ppm using Me
4
Si as reference and J
2
values are given in Hz. Assignments were supported by spin
decoupling-double resonance and bidimensional heteronuclear
correlation techniques. Mass spectrometry analyses were per-
formed at the ‘C.A.C.T.I.dUnidad de Espectrometria de Masas’, at
University of Vigo, Spain. Microwave-assisted solid phase peptide
synthesis was carried out on a CEM Discover SPS equipment. Pep-
tide analysis and purification was carried out with analytical HPLC
6.4 Hz, 2H, H-2 and H-7 Fmoc), 7.32 (t, J¼7.4 Hz, 2H, H-3 and H-6
Fmoc), 7.52 (d, J¼7.2 Hz, 2H, H-4 and H-5 Fmoc), 7.68 (d, J¼7.2 Hz,
1
3
2H, H-1 and H-8 Fmoc). C NMR (CDCl
3
, 100.6 MHz):
d
C
¼8.29
-C Deg),
Fmoc), 119.96 (C-1 and C-8 Fmoc), 124.98 (C-4 and C-5
(2ꢂCH
3
Deg), 28.16 (2ꢂCH
2
Deg), 47.26 (C-9 Fmoc), 65.03 (a
66.28 (CH
2
Fmoc),127.03 (C-2 and C-7 Fmoc),127.66 (C-3 and C-6 Fmoc),141.31
(C-4a and C-4b Fmoc), 143.83 (C-1a and C-8a Fmoc), 154.41 (C]O
Fmoc), 178.27 (C]O acid).
using a Licrospher 100 RP18 (5 mm) column in a JASCO HPLC system
composed by a PU-2080 pump and a UV-2070 detector with
ChromNav software, and semipreparative HPLC with a Shimadzu
LC-8A, UV/Vis JASCO 875-UV detector and a Shimadzu C-RGA
4.2.4. N-(9-Fluorenylmethyloxycarbonyl)-
a,a-dipropylglycine, Fmoc-
Dpg-OH (2d). By reaction of -dipropylglycine hydrochloride 1d
a,a
Chromatopac register on a Europa Peptide 120 C18 (5
using ACN/water mixtures (1:1 or 2:1) with 0.1% TFA (
m
m) column
(0.420 g, 2.1 mmol) and Fmoc-OSu (0.708 g, 1.1 equiv, 2.31 mmol)
using method A, after recrystallization from dichloromethane/pe-
troleum ether, compound 2d was obtained as a colourless solid
(0.532 g, 66%) and the characterization in accordance to previously
l
det¼215 nm).
Fluorescence spectra were collected using a FluoroMax-4 spectro-
fluorometer (HoribadJobin-Yvon), equipped with double mono-
chromators in both excitation and emission and a temperature-
controlled cuvette holder. Fluorescence spectra were corrected for
the instrumental response of the system.
3
8
ꢀ
1
reported data. Mp¼143.3e145.5 C. H NMR (CDCl
¼0.91 (t, J¼6.4 and 7.2 Hz, 6H, 2ꢂCH Dpg), 1.10e1.13 (m, 2H,
CH Dpg), 1.28e1.34 (m, 2H, -CH Dpg), 1.74e1.81 (m, 2H, -CH
Dpg), 2.27e2.35 (m, 2H, -CH
Dpg), 4.23 (t, J¼6.6 Hz, 1H, H-9
Fmoc), 5.75 (s, 1H, NH), 7.33 (dt,
3
, 400 MHz):
d
H
3
g-
2
g
2
b
2
Fmoc-Aib-OH 2a, Fmoc-l-Ala-OH 2b, Fmoc-
L-Leu-OH, N-(9-
b
2
fluorenylmethoxy-carbonyloxy)succinimide (Fmoc-OSu), Fmoc-
Fmoc), 4.42 (d, J¼6.4 Hz, 2H, CH
2
0
Phe-Wang resin, N,N -diisopropylcarbodiimide (DIC), 6-chloro-N-
J¼1.0 and 7.4 Hz, 2H, H-2 and H-7 Fmoc), 7.41 (t, J¼7.6 Hz, 2H, H-3
and H-6 Fmoc), 7.58e7.64 (m, 2H, H-4 and H-5 Fmoc), 7.78 (d,
hydroxybenzotriazole (6-Cl-HOBt) and N,N-diisopropylethylamine
1
3
(
DIPEA) were purchased from AAPPTec and ACROS Organics and
J¼7.6 Hz, 2H, H-1 and H-8 Fmoc). C NMR (CDCl
¼13.93 (4ꢂCH Dpg), 17.32 (2ꢂ -CH Dpg), 37.63 (2ꢂ
47.33 (C-9 Fmoc), 63.94 ( -C Dpg), 66.12 (CH Fmoc), 120.20 (C-1
3
, 100.6 MHz):
used as received. -Phosphatidylcholine from egg yolk, choles-
L-
a
d
C
3
g
2
b
-CH Dpg),
2
Ò
Ò
terol, 6-carboxyfluorescein, Triton X-100 and Sephadex G-50
were purchased from SigmaeAldrich and used as received.
a
2
and C-8 Fmoc), 124.98 (C-4 and C-5 Fmoc), 127.60 (C-2 and C-7
Fmoc), 127.65 (C-3 and C-6 Fmoc), 141.33 (C-4a and C-4b Fmoc),
143.85 (C-1a and C-8a Fmoc), 154.00 (C]O Fmoc), 178.84 (C]O
acid).
4
.2. General method for the N-protection of
a,a-dia-
lkylglycines 2cef
4
.2.1. Method A. The amino acid hydrochloride 1 (2 mmol) was
dissolved in a mixture of aqueous 5% Na CO solution/1,4-dioxane
5:2). The mixture was placed in an ice bath and Fmoc-OSu
1.1e2 equiv) dissolved in 1,4-dioxane (4 mL) was added drop
CO so-
4.2.5. 1-(9-Fluorenylmethyloxycarbonylamino)-cyclohexyl-1-
carboxylic acid, Fmoc-Ac c-OH (2e). By reaction of 1-amino-1-
6
2
3
(
(
cyclohexane carboxylic acid hydrochloride 1e (0.973 g,
6.42 mmol) and Fmoc-OSu (2.597 g, 1.2 equiv, 7.70 mmol) using
method B, after recrystallization from dichloromethane and pe-
troleum ether, compound 2e was obtained as a light orange solid
(1.638 g, 70%) and the characterization in accordance to previously
wise. The final pH was adjusted to 8 with aqueous 5% Na
2
3
lution, if necessary. The mixture was stirred at room temperature
overnight. Water (15 mL) was added and the mixture was extracted
with diethyl ether/petroleum ether (1:3) (3ꢂ15 mL). The aqueous
layer was acidified to pH 3 with aqueous 10% citric acid solution and
extracted with ethyl acetate (3ꢂ15 mL). The organic layer was
washed with aqueous 5% citric acid solution (2ꢂ10 mL), followed by
washing with water as many times necessary until neutral. The
3
9,40
ꢀ
1
reported data.
Mp¼172.9e173.8 C. H NMR (CDCl
3
, 400 MHz):
0
0
0
d
H
¼1.37e1.51 (m, 2H, H-3 or H-5 Ac
6
c), 1.55e1.71 (m, 4H, H-3 or
0
0
0
0
H-5 and H-4 Ac
6
c), 1.78e1.90 (m, 2H, H-2 or H-6 Ac
6
c), 2.0e2.2
0
0
(m, 2H, H-2 or H-6 Ac
6
c), 4.23 (t, J¼6.0 Hz, 1H, H-9 Fmoc), 4.43 (d,
J¼4.0 Hz, 2H, CH Fmoc), 5.04 (s, 1H, NH), 7.31 (t, J¼7.2 Hz, 2H, H-2
2
organic layer was dried with anhydrous MgSO
evaporated to dryness in a rotary evaporator.
4
, filtered and
and H-7 Fmoc), 7.39 (t, J¼7.2 Hz, 2H, H-3 and H-6 Fmoc), 7.59 (d,
J¼7.6 Hz, 2H, H-4 and H-5 Fmoc), 7.76 (d, J¼7.6 Hz, 2H, H-1 and H-8
13
0
0
Fmoc). C NMR (CDCl
3
, 100.6 MHz):
d
C
¼21.09 (C-3 and C-5 Ac
6
c),
0
0
0
4.2.2. Method B. The amino acid hydrochloride 1 (1 equiv) was
25.03 (C-4 Ac
6
c), 32.24 (C-2 and C-6 Ac
6
c), 47.20 (C-9 Fmoc), 58.97
0
dissolved in a mixture of acetone (40 mL) and a solution of Na CO
2
3
(C-1 Ac
6
c), 66.76 (CH
2
Fmoc), 119.90 (C-1 and C-8 Fmoc), 125.17 (C-

1028
V.I.B. Castro et al. / Tetrahedron 72 (2016) 1024e1030
4
and C-5 Fmoc), 127.04 (C-2 and C-7 Fmoc), 127.63 (C-3 and C-6
Fmoc), 141.29 (C-4a and C-4b Fmoc), 143.79 (C-1a and C-8a Fmoc),
55.58 (C]O Fmoc), 179.22 (C]O acid).
under a nitrogen atmosphere, in an ice bath. Then, lithium bromide
(5 equiv) suspended in dry ethanol (4 mL/g of resin) was added. The
mixture was stirred vigorously for 15 min on ice and 15 min at room
temperature. The dried resin was added to this mixture and
allowed to stir at room temperature for 24 h. The organic solvent
mixture was separated from the resin by vacuum filtration and the
resin was washed with THF (2ꢂ15 mL), ethanol (2ꢂ15 mL) and ethyl
acetate (2ꢂ15 mL). The solvents were combined and a small
amount of acetic acid was added, just enough to decompose bo-
rohydride in excess. The solution was concentrated almost to dry-
ness on a rotary evaporator without heating and water was added
(25 mL). The resulting aqueous solution was extracted with ethyl
acetate (3ꢂ25 mL), and the combined organic extracts were
washed with water (15 mL) and saturated NaCl solution (2ꢂ15 mL).
1
4
.2.6. N-(9-Fluorenylmethyloxycarbonyl)-
Fmoc-Dibg-OH (2f). By reaction of -diisobutylglycine hydro-
chloride 1f (1.718 g, 7.68 mmol) and Fmoc-OSu (5.194 g, 2.0 equiv,
5.4 mmol) using method A, after column chromatography on silica
gel with mixtures of dichloromethane and methanol, compound 2f
a,a-diisobutylglycine,
a,a
1
was obtained as
a
colourless solid (3.085 g, 98%).
Mp¼143.3e145.5 C. 1H NMR (CDCl
ꢀ
, 400 MHz):
d
¼0.83 (d,
3
J¼6.4 Hz, 6H, 2ꢂCH
3
Dibg), 0.89 (d, J¼6.4 Hz, 6H, 2ꢂCH
3
Dibg),
Dibg),
1
2
9
.53e1.63 (m, 2H, 2ꢂ
g
-CH Dibg), 1.66e1.71 (m, 2H,
.40 (dd, J¼5.8 and 8.4 Hz, 2H, -CH
Fmoc), 5.94 (s, 1H, NH), 7.32 (dt,
b-CH
2
b
2
Dibg), 4.21 (t, J¼7.0 Hz, 1H, H-
Fmoc), 4.40 (d, J¼6.8 Hz, 2H, CH
2
The organic layer was dried with anhydrous MgSO
evaporated to dryness without heating.
4
and the solvent
J¼1.1 and 6.4 Hz, 2H, H-2 and H-7 Fmoc), 7.41 (t, J¼7.4 Hz, 2H, H-3
and H-6 Fmoc), 7.61 (d, J¼7.2 Hz, 2H, H-4 and H-5 Fmoc), 7.6 (d,
1
3
J¼7.6 Hz, 2H, H-1 and H-8 Fmoc). C NMR (CDCl
3
, 100.6 MHz):
-CH Dibg),
-C Dibg), 66.15
2
Fmoc), 119.95 (C-1 and C-8 Fmoc), 125.01 (C-4 and C-5 Fmoc),
4.3.5. Purification of the peptides by HPLC. The purity of the pep-
tides was checked by analytical HPLC and a sample of each peptide
was purified by preparative HPLC.
d
¼22.86 (2ꢂCH
4.82 (2ꢂ -CH
CH
3
Dibg), 23.69 (2ꢂCH
3
Dibg), 24.61 (2ꢂ
g
4
(
b
2
Dibg), 47.37 (C-9 Fmoc), 62.87 (a
127.02 (C-2 and C-7 Fmoc), 127.64 (C-3 and C-6 Fmoc), 141.33 (C-4a
4.3.6. Peptaibolin, Ac-L-Leu-Aib-L-Leu-Aib-L-Phol (3a). Starting from
and C-4b Fmoc), 143.92 (C-1a and C-8a Fmoc), 153.83 (C]O Fmoc),
Fmoc-Phe-Wang resin (1.5 g, 0.51 mmol/g), following the above
general methods, by sequential coupling of Fmoc-Aib-OH 2a and
Fmoc-L-Leu-OH, after reductive cleavage from the resin, the crude
þ
179.85 (C]O acid). HRMS (ESI): calcd for C25
H32NO
4
[M þH]:
410.23326; found: 410.23321.
peptide was obtained as a colourless solid (0.5727 g). A sample of
the crude product (0.0502 g) was purified by preparative HPLC
yielding the pure peptide as a colourless solid (0.0347 g, purifica-
tion yield 70%, global yield 89%). Mp¼209e211 C (224e225 C ).
4
.3. General procedure of the synthesis of peptides 3aee by
MW-SPPS
ꢀ
ꢀ
34
1
4.3.1. Preparation for coupling of preloaded Fmoc-Phe-Wang res-
H NMR (CDCl
(s, 3H, CH Aib), 1.38 (s, 3H, CH
3H, CH Aib), 1.58e1.63 (m, 2H,
and
3
, 400 MHz):
d
H
¼0.89e1.01 (m, 12H, 4ꢂCH
3
Leu), 1.36
Aib), 1.54 (s,
Leu), 1.66e1.75 (m, 3H, -CH
-CH Leu), 2.13 (s, 3H, CH
Phol), 3.60e3.72 (m, 2H, CH
-CH Leu), 4.01e4.07 (m, 1H,
-CH Phol), 7.15e7.17 (m, 1H, NH), 7.21e7.25 (m,
5H, Ph-H Phol), 7.28e7.30 (m, 1H, NH Aib), 7.85 (br s, 1H, NH Leu),
in. After swelling of the resin in DMF for 15 min, the resin was
subjected to an initial deprotection to remove the Fmoc group using
a solution of 20% piperidine in DMF (7 mL/g resin) for 30 s, under
3
3
Aib), 1.47 (s, 3H, CH
-CH
3
3
b
2
b
2
g
-CH Leu), 1.76e1.90 (m, 1H,
g
3
Ac),
OH),
ꢀ
microwave irradiation (power¼28 W, temperature¼75 C). The
2.72e2.82 (m, 2H,
3.92e3.98 (m, 1H,
b
-CH
2
2
resin was washed with DMF (2ꢂ10 mL), MeOH (2ꢂ10 mL) and DMF.
A fresh solution of 20% piperidine in DMF was added and allowed to
react for 3 min under microwave irradiation (power¼28 W,
a
a-CH Leu),
4.23e4.34 (m, 1H,
a
ꢀ
13
temperature¼75 C). The resin was washed with DMF (2ꢂ10 mL)
8.11 (br s, 1H, NH) 8.20 (br s, 1H, NH). C NMR (CDCl
¼21.39 (CH Leu), 22.02 (CH Leu), 22.41 (CH Leu), 22.81 (CH
Ac), 22.84 (CH Aib), 22.89 (CH Leu), 23.13 (CH Aib), 24.79 ( -CH
Leu), 25.24 ( -CH Leu) 26.81 (CH Aib), 27.36 (CH Aib), 36.90 (
CH Phol), 39.34 ( -CH Leu), 39.61 ( -CH Leu), 53.23 ( -CH Phol),
55.10 ( -CH Leu), 56.03 ( -CH Leu), 56.42 ( -C Aib), 56.88 ( -C Aib),
65.00 (CH OH), 126.22 (C-4 Phol), 128.19 (C-3 and C-5 Phol), 129.10
3
, 100.6 MHz):
and MeOH (2ꢂ10 mL) and this washing cycle was repeated five
d
C
3
3
3
3
times.
3
3
3
g
g
3
3
b-
4.3.2. Coupling of Fmoc-amino acids. The Fmoc-amino acid 2
2
b
2
b
2
a
(
5 equiv on the degree of functionalization of the resin) was dis-
a
a
a
a
0
solved in dry DMF (6 mL/g resin) and N,N -diisopropylcarbodiimide
2
(
DIC) (5 equiv) and 6-chloro-N-hydroxybenzotriazole (6-Cl-HOBt)
(C-2 and C-6 Phol), 138.38 (C-1 Phol), 173.48 (C]O Ac), 174.44 (C]
(
5 equiv) dissolved in dry DMF (2 mL/g) were added. The reaction
O Leu), 174.71 (C]O Leu), 175.39 (C]O Aib), 176.72 (C]O Aib).
þ
mixture was allowed to stir at room temperature for 1.5e2 h, for
the -dialkylglycines, and 20 min for the remaining amino acids,
HRMS (ESI): calcd for C31
590.39114.
52
H N
5
O
6
[M þH]: 590.39121; found:
a,a
before being added to the resin and subjected to microwave irra-
diation. The suspension was then subjected to microwave irradia-
4.3.7. Peptaibolin-Ala, Ac-
from Fmoc-Phe-Wang resin (2.0 g, 0.51 mmol/g), following the above
general methods, by sequential coupling of Fmoc- -Ala-OH 2b and
Fmoc- -Leu-OH, after reductive cleavage from the resin, the crude
peptide was obtained as a colourless solid (0.9450 g). A sample of the
crude product (0.0403 g) was purified by preparative HPLC yielding
L-Leu-L-Ala-L-Leu-L-Ala-L-Phol (3b). Starting
ꢀ
tion for 5 min (power¼25 W, temperature¼75 C). The resin was
washed with DMF (2ꢂ10 mL) followed by MeOH (2ꢂ10 mL) and
this washing cycle was repeated three times. The N-terminal Fmoc
group was removed as described above for the preparation of the
resin.
L
L
the pure peptide as a white solid (0.0385 g), purification yield 90%,
1
4
.3.3. N-terminal acetylation of the peptides. The resin with the
peptide was suspended in dry DMF (7 mL/g of resin) and DIPEA
14 equiv) and acetic anhydride (7 equiv) were added. The mixture
global yield 87%). H NMR (DMSO-d
6
, 400 MHz):
Ala), 1.18 (d, J¼7.2 Hz, 3H,
Leu), 1.55e1.57 (m, 2H, 2ꢂ -CH
Ac), 2.55e2.83 (m, 2H, -CH Phol), 3.25e3.32
OH), 3.80e3.84 (m, 1H, -CH Phol), 4.13e4.26 (m, 1H,
OH), 7.13e7.26 (m, 5H, Ph-H Phol), 7.53 (d, J¼8.4 Hz, 1H, NH Phol),
7.78 (d, J¼8.0 Hz, 1H, NH Ala), 7.79 (d, J¼7.6 Hz, 1H, NH Leu), 8.01 (d,
d
H
¼0.79e0.86 (m,
12H, 4ꢂCH
CH
Ala), 1.37e1.44 (m, 4H, 2ꢂ
Leu), 1.82 (s, 3H, CH
(m, 2H, CH
CH
3
Leu), 1.13 (d, J¼7.2 Hz, 3H, CH
3
(
3
b-CH
2
g
was allowed to stir at room temperature for 1.5 h. The resin was
washed with DMF (2ꢂ10 mL), MeOH (2ꢂ10 mL), DCM (3ꢂ10 mL)
and diethyl ether (3ꢂ10 mL). The resin was dried in a vacuum oven
at room temperature.
3
b
2
2
a
2
1
3
J¼8.0 Hz, 1H, NH Leu), 8.10 (d, J¼6.8 Hz, 1H, NH Ala). C NMR (DMSO-
, 100.6 MHz): Ala), 18.37 (CH Ala), 21.55 (CH Leu),
¼17.58 (CH
21.68 (CH Leu), 22.54 (CH Ac), 23.10 (CH Leu), 23.19 (CH Leu), 24.18
4.3.4. Reductive separation of the peptides from Wang resin. Sodium
d
6
d
C
3
3
3
borohydride (5 equiv) was suspended in dry THF (16 mL/g of resin)
3
3
3
3

V.I.B. Castro et al. / Tetrahedron 72 (2016) 1024e1030
1029
(
4
g
-CH Leu), 24.25 (
0.79 ( -CH
Leu), 48.40 (2ꢂ
Leu), 52.37 ( -CH Phol), 62.31 (CH
and C-5 Phol), 129.22 (C-2 and C-6 Phol), 138.00 (C-1 Phol), 169.73
g
-CH Leu), 36.48 (
-CH Ala), 51.26 (
OH), 126.02 (C-4 Phol), 128.19 (C-3
b
-CH
2
Phol), 40.57 (
b
-CH
2
Leu),
172.03 (C]O Dpg) 173.17 (C]O Leu). HRMS (ESI): calcd for
þ
b
2
a
a
a-CH Leu), 52.39 (
a-CH
C
39
H
68
N
5
O
6
[M þH]: 702.51641; found: 702.51662.
2
4.3.10. Peptaibolin-Ac
from Fmoc-Phe-Wang resin (1.5 g, 0.51 mmol/g), following the above
general methods, by sequential coupling of Fmoc-Ac c-OH 2e and
Fmoc- -Leu-OH, after reductive cleavage from the resin, the crude
6 6 6
c, Ac-L-Leu-Ac c-L-Leu-Ac c-L-Phol (3e). Starting
(C]O Ac), 171.56 (C]O Leu), 171.72 (C]O Leu), 172.36 (C]O Ala),
þ
1
5
72.43 (C]O Ala). HRMS (ESI): calcd for C29
62.36074; found: 562.35978.
H N O
48 5 6
[M þH]:
6
L
peptide was obtained as colourless oil (0.3840 g). A sample of the
crude product (0.0370 g) was purified by preparative HPLC yielding
the pure peptide as a colourless solid (0.0341 g, purification yield 43%,
4
.3.8. Peptaibolin-Deg, Ac-L-Leu-Deg-L-Leu-Deg-L-Phol (3c). Starting
from Fmoc-Phe-Wang resin (1.5 g, 0.60 mmol/g), following the
above general methods, by sequential coupling of Fmoc-Deg-OH
2
the crude peptide was obtained as a colourless solid (0.3593 g). A
sample of the crude product (0.1042 g) was purified by preparative
1
global yield 32%). H NMR (DMSO-d
6
, 400 MHz):
Ac
c, 2ꢂ
Ac), 1.96e2.08 (m, 4H, 2ꢂCH
d
H
¼0.81e0.94 (m,
-CH and 2ꢂ
Ac c),
-CH
-CH Leu),
-CH Phol), 6.94 (d, J¼9.2 Hz, 1H, NH Leu or Phol),
c), 7.17e7.20 (m, 4H, H-
, H-3, H-5 and H-6 Phol), 7.64 (d, J¼6.0 Hz, 1H, NH Leu or Phol), 7.96
1
CH
2H, 4ꢂCH
3
Leu), 1.08e1.74 (m, 22H, 8ꢂCH
2
6
g
b-
c and Fmoc-L-Leu-OH, after reductive cleavage from the resin,
2
Leu), 1.89 (s, 3H, CH
3
2
6
2
.53e2.59 (m, 1H,
Phol), 3.21e3.33 (m, 2H, CH
4.12e4.18 (m, 1H,
b
-CH
2
Phol), 2.84 (dd, J¼4.8 and 8.8 Hz, 1H,
b
2
2
OH), 3.81e3.89 (m, 2H, 2ꢂ
a
HPLC yielding the pure peptide as colourless solid (0.0318 g, pu-
rification yield 30%, global yield 22%).
1
a
6
H NMR (DMSO-d ,
7
2
.10e7.14 (m, 1H, H-4 Phol), 7.15 (s,1H, NH Ac
6
4
00 MHz):
ꢂCH Deg), 0.75e0.88 (m, 12H, 4ꢂCH
-CH Leu and -CH Deg), 1.51e1.66 (m, 3H,
Leu), 1.74e1.90 (m, 2H, -CH Deg), 1.87 (s, 3H, CH
m, 4H, 2ꢂ -CH Deg), 2.55e2.61 (m, 1H, -CH
J¼4.4 and 9.2 Hz, 1H, -CH Phol), 3.25e3.33 (m, 2H, CH
.01e4.12 (m, 3H, 2ꢂ -CH Leu and -CH Phol), 7.11e7.12 (m, 1H,
H-4 Phol), 7.16e7.19 (m, 4H, H-2, H-3, H-5 and H-6 Phol), 7.34 (s,
d
H
¼0.12 (t, J¼8.0 Hz, 3H, CH
3
Deg), 0.50e0.56 (m, 9H,
Leu), 1.37e1.49 (m, 5H, b-
3
CH
3
3
13
(
s, 1H, NH Ac
6
c) 8.31 (d, J¼5.6 Hz, 1H, NH Leu). C NMR (DMSO-d
Ac c), 21.33 (CH Leu), 22.01 (CH Leu),
Leu), 22.95 (CH Leu), 24.16 ( -CH Leu), 24
Ac c), 25.01 (CH Ac c), 30.78 (CH Ac c),
Ac c), 36.50 ( -CH -CH
Leu), 52.62 ( -CH Leu), 52.83 ( -CH Phol), 53.30 (
-C Ac c), 59.36 ( -C Ac OH), 125.70 (C-
4 Phol), 127.83 (C-3 and C-5 Phol), 129.16 (C-2 and C-6 Phol), 139.35
C-1 Phol), 171.13 (C]O Ac), 171.83 (C]O Ac c), 173.28 (C]O Ac c),
6
,
2
,
g
b
2
2
b-CH and g-CH
100.6 MHz):
d
C
¼21.04 (4ꢂCH
2
6
3
3
b
2
3
Ac), 2.08e2.20
2
2.35 (CH
1 ( -CH Leu), 24.87 (CH
1.14 (CH Ac
Leu), 39.08 (
CH Leu), 59.00 (
3
Ac), 22.46 (CH
3
3
g
(
b
2
b
2
Phol), 2.89 (dd,
OH),
3
g
2
6
2
6
2
6
b
2
2
3
2
6
c), 31.47 (2ꢂCH
2
6
b
2
Phol), 38.87 (
b
2
4
a
a
b-CH
2
a
a
a-
a
6
a
6 2
c), 63.02 (CH
1
H, NH Deg), 7.48 (d, J¼8.8 Hz, 1H, NH Leu), 7.55 (s, 1H, NH Deg),
13
7
.98 (d, J¼7.6 Hz, 1H, NH Leu) 8.21 (d, J¼6.8 Hz, 1H, NH Phol).
C
(
6
6
NMR (DMSO-d
6
, 100.6 MHz):
d
C
¼7.61 (CH
Ac), 20.90 (CH
Leu), 23.00 (CH Leu), 23.22 (CH
-CH Leu), 26.50 ( -CH Deg), 26.71 (
Deg), 27.11 ( -CH Deg), 36.30 ( -CH Phol), 39.91 (
Leu), 40.12 ( -CH Leu), 52.58 ( -CH Leu), 52.77 ( -CH Phol),
2.95 ( -CH Leu), 63.42 (CH -C Deg), 125.84 (C-4
3
Deg), 7.72 (CH
Leu), 21.11 (
Leu), 24.11 (
-CH Deg),
3
Deg),
1
C
73.66 (C]O Leu) 175.45 (C]O Leu). HRMS (ESI): calcd for
7
.86 (2ꢂCH
Leu), 21.37 (CH
CH Leu), 24.35 (
7.03 ( -CH
CH
3
Deg), 20.82 (CH
3
3
b
-CH
2
þ
H N O
37 60 5 6
[M þH]: 670.45381; found: 670.45324.
3
3
3
g-
g
b
2
b
2
4.3.11. Attempted synthesis of Peptaibolin-Dibg: isolation of Ac-Dibg-
2
b
2
b
2
b
2
b-
L-Phol (3f). Starting from Fmoc-Phe-Wang resin (1.5 g, 0.60 mmol/
g), following the above general methods, by sequential coupling of
2
b
2
a
a
5
a
2
OH), 63.85 (2ꢂ
a
Fmoc-Dibg-OH 2f and Fmoc-
L-Leu-OH, after reductive cleavage
Phol), 128.06 (C-3 and C-5 Phol), 128.96 (C-2 and C-6 Phol), 139.31
from the resin, only the dipeptide Ac-Dibg-Phol was isolated as off-
(
C-1 Phol), 169.95 (C]O Leu) 170.60 (C]O Ac), 171.03 (C]O Leu),
1
white solid (0.0123 g). H NMR (DMSO-d
J¼6.8 Hz, 3H, CH Dibg), 0.63 (d, J¼6.4 Hz, 3H, CH
Dibg), 1.04e1.11 (m, 1H, -CH Dibg), 1.35e1.45 (m,
Dibg), 1.55e1.61 (m, 1H, -CH Dibg), 1.81 (s, 3H, CH Ac),
.07e2.21 (m, 2H, -CH Dibg), 2.65e2.71 (m, 1H, -CH Phol),
-CH Phol), 3.20e3.25 (m, 1H, CH OH),
OH), 3.98e4.04 (m, 1H, -CH Phol), 4.75 (br s,
6
, 400 MHz):
d
H
¼0.37 (d,
171.80 (C]O Deg), 173.10 (C]O Deg). HRMS (ESI): calcd for
þ
3
3
Dibg), 0.73e0.78
C
H
35 60
N
5
O
6
[M þH]: 646.4538; found: 646.4533.
(
m, 6H, 2ꢂCH
3
g
2
2
2
3
1
H,
b
-CH
2
g
3
4
.3.9. Peptaibolin-Dpg, Ac-L-Leu-Dpg-L-Leu-Dpg-L-Phol (3d). Starting
b
2
b
2
from Fmoc-Phe-Wang resin (1.5 g, 0.51 mmol/g), following the
above general methods, by sequential coupling of Fmoc-Dpg-OH 2d
and Fmoc-L-Leu-OH, after reductive cleavage from the resin, the
crude peptide was obtained as a colourless solid (0.4427 g). A
sample of the crude product (0.0370 g) was purified by preparative
HPLC yielding the pure peptide as colourless solid (0.0318 g, puri-
fication yield 76%, global yield 60%). H NMR (DMSO-d
.89e2.94 (m, 1H,
.39e3.45 (m, 1H, CH
b
2
2
2
a
2
H, CH OH), 7.13e7.16 (m,1H, H-4 Phol), 7.21e7.26 (m, 5H, H-2, H-3,
13
H-5 and H-6 Phol and NH Dibg), 7.59 (d, J¼8.0 Hz, 1H, NH Phol).
NMR (DMSO-d ,100.6 MHz): Dibg), 21.13 (CH Dibg),
¼23.03 (CH
3.50 (CH Ac), 23.46 ( -CH Dibg), 23.88 (CH Dibg), 23.86 ( -CH
Dibg), 23.88 (CH Dibg), 36.07 ( -CH Phol), 43.20 ( -CH Dibg),
3.45 ( -CH Dibg), 53.45 ( -CH Phol), 62.43 (CH OH), 125.85 (C-4
Phol), 128.10 (C-3 and C-5 Phol), 128.94 (C-2 and C-6 Phol), 139.32
C
6
d
C
3
3
2
3
g
3
g
1
6
, 400 MHz):
Leu
Dpg),
Dpg
-CH
Phol),
OH), 3.98e4.13 (m, 3H,
-CH Phol), 4.72 (br s, 1H, CH OH), 7.10e7.20 (m,
H, Ph-H Phol), 7.39 (s, 1H, NH Dpg), 7.47 (d, J¼8.4 Hz, 1H, NH), 7.61
b
b
3
2
2
d
H
¼0.58 (t, J¼7.2 Hz, 3H, CH
and CH
Dpg) 0.79e0.91 (m, 12H, 3ꢂCH
-CH
Dpg), 1.33e1.81 (m, 4H, 2ꢂ
-CH Leu), 1.84 (s, 3H, CH
-CH Phol), 2.89e2.94 (m,1H,
-CH Leu and CH
3
Leu), 0.64e0.78 (m, 12H, 3ꢂCH
3
4
b
2
a
2
3
3
Dpg and
g-CH
2
0
.92e1.20 (m, 6H, 3ꢂ
g
2
b
-CH
2
(
C-1 Phol), 167.94 (C]O Ac), 173.22 (C]O Dibg). HRMS (ESI): calcd
and 2ꢂ
g
3
Ac), 1.98e2.20 (m, 4H, 2ꢂ
b
2
þ
for C21
35
H N
2
O
3
[M þH]: 363.26485; found: 363.26512.
Dpg), 2.57e2.62 (m,1H,
b
2
b
-CH
2
3
2
5
.20e3.30 (m, 4H, 2ꢂ
g
2
4.4. Membrane permeation studies
ꢂa
-CH Leu and
a
2
The membrane permeation ability of Peptaibolin and its ana-
(
s, 1H, NH Dpg), 7.93 (d, J¼7.6 Hz, 1H, NH) 8.18 (d, J¼7.2 Hz, 1H, NH).
ꢀ
logues was monitored at 22 C using the carboxyfluorescein-
1
3
C NMR (DMSO-d
Leu), 14.13 (CH Leu), 14.22 (CH
CH -CH Dpg), 20.87 (CH
Ac), 22.87 (CH Dpg), 23.14 (CH
-CH Phol), 36.06 (
-CH Dpg), 39.92 (
-CH Leu), 52.71 ( -CH Leu), 53.04 (
-C Dpg), 62.87 ( -C Dpg), 63.30 (CH OH), 125.70 (C-4
Phol), 127.89 (C-3 and C-5 Phol), 128.80 (C-2 and C-6 Phol), 139.32
C-1 Phol), 168.78 (C]O Ac), 170.44 (C]O Dpg), 170.85 (C]O Leu),
6
, 100.6 MHz):
d
C
¼13.83 (CH
3
Leu), 13.95 (CH
3
-
43,44
entrapped vesicle technique.
3
3
Leu), 15.87 (
g
-CH
2
Dpg), 16.27 (
g
2
Dpg), 16.36 (2ꢂ
2.33 (CH
4.25 ( -CH Leu), 35.96 (
Dpg), 36.64 ( -CH
0.02 ( -CH Leu), 52.48 (
Phol), 62.76 (
g
2
3
Dpg), 21.46 (CH
Dpg), 23.96 (
-CH Dpg), 36.39 (
-CH Leu),
-CH
3
Dpg),
g-CH Leu),
4
.4.1. Vesicles preparation. The small unilamellar vesicles (SUVs)
were composed of egg phosphatidylcholine and cholesterol
70:30). The SUVs preparation was carried out in a concentrated
2
2
3
3
b
3
g
2
b
2
b-
(
CH
4
2
b
2
Dpg), 36.72 (
a
b
2
b
2
solution of 6-carboxyfluorescein (CF) in HEPES (4-(2-
hydroxyethyl)-1-piperazineethanesulfonic acid) buffer (pH¼7.4).
An ethanolic solution of the lipid mixture was injected in the CF
b
2
a
a
a
a
2
47
solution under vortexing (ethanolic injection method), followed
(
by sonication for 10 min (with pauses of 1 min) at 71 W and 5.5 J in

1030
V.I.B. Castro et al. / Tetrahedron 72 (2016) 1024e1030
a Q-Sonica Q125 sonicator, to obtain the SUVs. The vesicles with
9. Bechinger, B.; Salnikov, E. S. Chem. Phys. Lipids 2012, 165, 282e301.
0. Fjell, C. D.; Hiss, J. A.; Hancock, R. E. W.; Schneider, G. Nat. Rev. Drug Discov.
012, 11, 37e51.
1. Iftemi, S.; De Zotti, M.; Formaggio, F.; Toniolo, C.; Stella, L.; Luchian, T. J. Pept. Sci.
2014, 20, 517e525.
2. Bauer, M. E.; Shafer, W. M. Biochim. Biophys. Acta-Biomembr. 2015, 1848,
101e3111.
3. Maria-Neto, S.; de Almeida, K. C.; Macedo, M. L. R.; Franco, O. L. Biochim. Bio-
phys. Acta-Biomembr. 2015, 1848, 3078e3088.
4. Lohan, S.; Bisht, G. S. Mini-Rev. Med. Chem. 2013, 13, 1073e1088.
5. M eꢀ ndez-Samperio, P. Infect. Drug Resist. 2014, 7, 229e237.
1
entrapped fluorophore were separated from non-encapsulated CF
2
Ò
by size exclusion chromatography, using Sephadex G-50 as gel
1
filtration medium and HEPES buffer (pH¼7.4) as eluent. The frac-
1
tions containing the vesicles with entrapped CF were identified by
3
measuring the absorbance (
tensity (
to the ones with simultaneously higher absorption (higher CF
concentration) and lower emission (higher CF self-quenching).
l
abs¼461 nm) and fluorescence in-
1
l
exc¼488 nm; em¼520 nm), these fractions corresponding
l
1
1
6. Hol ꢀa skov ꢀa , E.; Galuszka, P.; Fr eꢀ bort, I.; Tufan Oz, M. Biotechnol. Adv. 2015, 33,
005e1023.
7. Chugh, J. K.; Wallace, B. A. Biochem. Soc. Trans. 2001, 29, 565e570.
8. Duclohier, H. Chem. Biodivers. 2007, 4, 1023e1026.
9. Whitmore, L.; Wallace, B. A. Eur. Biophys. J. 2004, 33, 233e237.
€
1
1
1
1
1
4.4.2. Peptide-induced CF leakage monitored by fluorescence emis-
sion. In the CF leakage assays, the phospholipid concentration was
kept constant (0.6 mM). Increasing [peptide]/[lipid] molar ratios
were obtained by adding successive aliquots of methanolic solu-
tions of peptides, keeping the final methanol concentration below
20. Stoppacher, N.; Neumann, N. K. N.; Burgstaller, L.; Zeilinger, S.; Degenkolb, T.;
Br u€ ckner, H.; Schuhmacher, R. Chem. Biodivers. 2013, 10, 734e743.
21. Vlieghe, P.; Lisowski, V.; Martinez, J.; Khrestchatisky, M. Drug Discov. Today
2010, 15, 40e56.
5
% by volume. After rapid and vigorous stirring, the time course of
22. Liskamp, R. M. J.; Rijkers, D. T. S.; Kruijtzer, J. A. W.; Kemmink, J. Chembiochem
2011, 12, 1626e1653.
fluorescence change corresponding to CF release was recorded at
2
3. Toniolo, C.; Crisma, M.; Formaggio, F.; Peggion, C. Biopolym. Pept. Sci. 2001, 60,
96e419.
4. Tanaka, M. Chem. Pharm. Bull. 2007, 55, 349e358.
l
em¼520 nm (1.0 nm bandpass) with exc¼488 nm (1.0 nm band-
l
3
pass). The percentage of released CF (%CF) at time t due to peptide
2
addition was determined using Eq. 1:
25. Aravinda, S.; Shamala, N.; Balaram, P. Chem. Biodivers. 2008, 5, 1238e1262.
6. Maekawa, H.; Ballano, G.; Toniolo, C.; Ge, N.-H. J. Phys. Chem. B 2011, 115,
168e5182.
27. Castro, T. G.; Mica
28. Castro, T. G.; Mica e^ lo, N. M. J. Phys. Chem. B 2014, 118, 9861e9870.
2
5
ðF ꢃ F Þ
t
0
e^ lo, N. M. J. Phys. Chem. B 2014, 118, 649e658.
%CF ¼
ꢂ 100
0
(1)
is
ðF ꢃ F Þ
T
29. Pinto, F. C. S. C.; Pereira-Lima, S. M. M. A.; Maia, H. L. S. Tetrahedron 2009, 65,
9165e9179.
where F
the fluorescence intensity in the presence of peptide, and F
fluorescence intensity with total membrane rupture and CF release
obtained by addition of 50 L of a 10% Triton X-100 solution). The
0
is the fluorescence intensity in the absence of peptide, F
t
30. Hjørringgaard, C. U.; Pedersen, J. M.; Vosegaard, T.; Nielsen, N. C.; Skrydstrup, T.
J. Org. Chem. 2009, 74, 1329e1332.
T
is the
31. Pedersen, S. L.; Tofteng, A. P.; Malik, L.; Jensen, K. J. Chem. Soc. Rev. 2012, 41,
(
m
1826e1844.
fluorescence intensity increased over time, confirming the rupture
of the vesicles and consequent release of CF. The time course for
each peptide/membrane interaction assay was 20 min.
32. Ben Haj Salah, K.; Inguimbert, N. Org. Lett. 2014, 16, 1783e1785.
3
3
3
3
3. H u€ lsmann, H.; Heinze, S.; Ritzau, M.; Schlegel, B.; Gr €a fe, U. J. Antibiot. 1998, 51,
1055e1058.
4. Crisma, M.; Barazza, A.; Formaggio, F.; Kaptein, B.; Broxterman, Q. B.; Kam-
phuis, J.; Toniolo, C. Tetrahedron 2001, 57, 2813e2825.
5. Costa, S. P. G.; Maia, H. L. S.; Pereira-Lima, S. M. M. A. Org. Biomol. Chem. 2003, 1,
Acknowledgements
1475e1479.
6. Pinto, F. C. S. C.; Pereira-Lima, S. M. M. A.; Ventura, C.; Albuquerque, L.;
The authors acknowledge Funda c¸ a~ o para a Ci ^e ncia e a Tecnologia
Gon c¸ alves-Maia, R.; Maia, H. L. S. Tetrahedron 2006, 62, 8184e8198.
37. Aguiam, N. R.; Castro, V. I.; Ribeiro, A. I. F.; Fernandes, R. D. V.; Carvalho, C. M.;
(
Portugal) and FEDER-COMPETE-QREN-EU for financial support
Costa, S. P. G.; Pereira-Lima, S. M. M. A. Tetrahedron 2013, 69, 9161e9165.
through projects PTDC/QUI-BIQ/118389/2010 (FCOMP-01-0124-
3
3
4
8. Arduin, M.; Spagnolo, B.; Cal oꢁ , G.; Guerrini, R.; Carr aꢁ , G.; Fischetti, C.; Trapella,
C.; Marzola, E.; McDonald, J.; Lambert, D. G.; Regoli, D.; Salvadori, S. Bioorg.
Med. Chem. 2007, 15, 4434e4443.
9. Galoppini, C.; Meini, S.; Tancredi, M.; Di Fenza, A.; Triolo, A.; Quartara, L.;
Maggi, C. A.; Formaggio, F.; Toniolo, C.; Mazzucco, S.; Papini, A.; Rovero, P. J.
Med. Chem. 1999, 42, 409e414.
0. Lescrinier, T.; Hendrix, C.; Kerremans, L.; Rozenski, J.; Link, A.; Samyn, B.; van
Aerschot, A.; Lescrinier, E.; Eritja, R.; van Beeumen, J.; Herdewijn, P. Chem.dEur.
J. 1998, 4, 425e433.
FEDER-020906),
FEDER-037302),
PEst-C/QUI/UI0686/2013
PEst-C/FIS/UI0607/2013
(F-COMP-01-0124-
(F-COMP-01-0124-
FEDER-022711) and the Portuguese NMR network (PTNMR,
Bruker Avance III 400-Univ. Minho).
References and notes
4
1. Jiang, W.; Costa, S. P. G.; Maia, H. L. S. Org. Biomol. Chem. 2003, 1, 3804e3810.
1
. Hancock, R. E. W.; Chapple, D. S. Antimicrob. Agents Chemother. 1999, 43,
4
4
4
4
4
2. Jiang, W.-Q.; Ventura, C.; Costa, S. P. G.; Albuquerque, L.; Gon c¸ alves-Maia, R.;
1317e1323.
Maia, H. L. S. J. Pept. Sci. 2005, 11, 472e480.
2. Wimley, W. C. ACS Chem. Biol. 2008, 5, 905e917.
3. Toniolo, C.; Crisma, M.; Formaggio, F.; Peggion, C.; Monaco, V.; Goulard, C.;
Rebuffat, S.; Bodo, B. J. Am. Chem. Soc. 1996, 118, 4952e4958.
4. Lucaciu, M.; Rebuffat, S.; Goulard, C.; Duclohier, H.; Molle, G.; Bodo, B. Biochim.
Biophys. Acta 1997, 1323, 85e96.
3. Reddy, K. V. R.; Yedery, R. D.; Aranha, C. Int. J. Antimicrob. Agents 2004, 24,
536e547.
4
5
6
. Wang, G.; Li, X.; Wang, Z. Nucleic Acids Res. 2009, 37, D933eD937.
. Brogden, K. A. Nat. Rev. Microbiol. 2005, 3, 238e250.
. Bocchinfuso, G.; Palleschi, A.; Orioni, B.; Grande, G.; Formaggio, F.; Toniolo, C.;
Park, Y.; Hahm, K.-S.; Stella, L. J. Pept. Sci. 2009, 15, 550e558.
5. Papahadjopoulos, D.; Miller, N. Biochim. Biophys. Acta-Biomembr. 1967, 4,
6
24e638.
6. Cohen, D. E.; Angelico, M.; Carey, M. C. J. Lipid Res. 1990, 31, 55e70.
7. Kremer, J. M. H.; Esker, M. W. J. V. D.; Pathmamanoharan, C.; Wiersema, P. H.
Biochemistry 1977, 16, 3932e3935.
7
8
. Melo, M. N.; Castanho, M. A. R. B. Front. Immunol. 2012, 3, 1e4.
. Mihajlovic, M.; Lazaridis, T. Biochim. Biophys. Acta-Biomembr. 2012, 1818,
274e1283.
4
1