End group engineering of artificial ion channels

Article abstract of DOI:10.1039/b509668e

Toward developing nanostructures for single molecule detection, we report the synthesis of new derivatives of artificial devices that possess ion channel activity. The membrane stability and the ion transport ability of these multiple crown α-helical peptides have been optimized by modulating the polarity of N- and C-terminal groups with incorporation of hydrophilic non-ionic units. Circular dichroism and ATR studies confirmed the α-helical conformation and membrane incorporation of new nanostructures while ²³Na NMR assays shown a significant increase of their ion transport ability. the Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2006.

Full text of DOI:10.1039/b509668e

View Article Online / Journal Homepage / Table of Contents for this issue
PAPER
www.rsc.org/njc | New Journal of Chemistry
End group engineering of artificial ion channels
Franc¸
ois Otis,a Normand Voyer,*a Ange Polidorib and Bernard Puccib
Received 12th July 2005, Accepted 11th August 2005
First published as an Advance Article on the web 1st September 2005
DOI: 10.1039/b509668e
Toward developing nanostructures for single molecule detection, we report the synthesis of new
derivatives of artificial devices that possess ion channel activity. The membrane stability and the
ion transport ability of these multiple crown a-helical peptides have been optimized by
modulating the polarity of N- and C-terminal groups with incorporation of hydrophilic non-ionic
units. Circular dichroism and ATR studies confirmed the a-helical conformation and membrane
incorporation of new nanostructures while 23Na NMR assays shown a significant increase of their
ion transport ability.
If we consider that such an adsorption could be due to a
Introduction
hydrophobic interaction between the leucine side chains of
crown peptides and the lipid chains, it can be assumed that
increasing the hydrophilicity of the terminal head groups by
means of non-ionic moieties should stabilize the transmem-
brane active form. Herein, we report the synthesis of a series of
crown modified peptides 2, 3, 4, analogs of 1, having variable
non-ionic polyhydroxylated groups derived from tris(hydrox-
ymethyl)aminomethane (Fig. 2). By using CD, FTIR and
ATR techniques the incorporation and orientation of these
different amphiphilic peptides in model membranes have been
studied.
The potential miscellaneous applications of artificial com-
pounds mimicking the conductivity properties of ion channel
proteins have stirred considerable efforts towards developing
such compounds. Many successful approaches have been
devised leading to a variety of artificial ion channels with their
own advantages and disadvantages.1 Our approach exploits
nanoscale peptidic frameworks that orient multiple crown
ethers to form a polar transmembrane channel for ions.
Herein, we report the molecular engineering of the end groups
of a prototype crown peptide channel in order to improve their
apolar membrane incorporation and active orientation.
We have reported the synthesis of various peptides endowed
with six aligned crown ethers2 and demonstrated their single-
channel activity in planar lipid bilayers and ion transport
ability in vesicle experiments.3,4 It is noteworthy that the
chosen synthetic pathway provides several advantages: rapid
synthesis by using solid phase technique, easy preparation of a
collection of analogs and, above all, facile purification of the
targeted compounds. For example, a series of synthetic helical
peptides that are oligomers of a repeating unit with five leucine
residues and two 21-crown-7 phenylalanines appropriately
positioned, exhibit an a-helical structure with the crown ethers
aligned on one side of the helix axis. Under an a-helical
conformation, the 21 residue peptide (n ¼ 3) can span a
bilayer membrane and act, under certain conditions, as artifi-
cial ion channel.5
Results and discussion
Synthesis
The synthesis of polar groups starts with commercially avail-
able tris(hydroxymethyl)methylamine (TRIS), which was first
protected with tert-butoxydimethylsilyl groups (Scheme 1) to
give 5.6,7 Reaction of 5 with succinic anhydride leads to the
N-terminal end group 6, ready to be coupled. On the other
hand, coupling N-benzyloxycarbonyl aminohexanoic acid8,9
with 5 provided 8, which after hydrogenation yielded the
C-terminal end group 9 (Scheme 2).
Preparation of 21mer peptide from N-BOC leucine and
(21-C-7)-L-Phe on oxime resin was described previously.5
Introduction of polar end groups is illustrated in Scheme 3.
Cleavage of the 21mer peptide from resin using the amine 9 as
nucleophile provided the desired C-terminal polar head pep-
tide 3, after deprotection of alcohols with TBAF. Deprotec-
tion of N-BOC group from 21mer peptide using 50%
trifluoroacetic acid (TFA) in CH2Cl2 followed by the coupling
of acid 6 using DIC/HOBt as reagent in DMF/CH2Cl2 (1 : 1),
lead to the formation of the intermediate 10, still attached to
the resin. A portion of the resin was used to obtain N-terminal
polar head peptide 2 by cleavage with DBU in MeOH/THF
(1 : 9), followed by deprotection of hydroxyl groups with
acetic acid.10 The remaining of resin 10 was cleaved with
amine 9 and deprotected with TBAF to produce peptide 4
with polar heads at both extremities. Purification of peptide 1
was achieved by reversed-phase HPLC, while peptides 2–4
In order to provide even more efficient functional ion
channels, the active orientation of peptides must be enhanced,
i.e. the helical structure should reside most often in the
transmembrane orientation and the adsorption at the surface
of the membrane must be disfavoured (Fig. 1).
a
´
Departement de Chimie and Centre de Recherche sur la fonction, la
structure et l’inge´nierie des prote´ines, Faculte´ des sciences et de
´
´
´
genie, Universite Laval, Quebec, Canada G1K7P4. E-mail:
normand.voyer@chm.ulaval.ca; Fax: +1-418-656-7916;
Tel: 1-418-656-3613
b
`
´
Laboratoire de Chimie Bioorganique et des Systemes Moleculaires
Vectoriels, Faculte des sciences, Universite d’Avignon, 33, rue Louis
´
´
Pasteur, Avignon, France
ꢀc
This journal is the Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2006
New J. Chem., 2006, 30, 185–190 | 185

View Article Online
Fig. 1 Proposed mode of action for the channel activity of peptide 1.
were purified on Sephadex LH-20. All peptides were success-
1
fully characterized by H NMR and mass spectroscopy and
purity was checked by reversed-phase HPLC.
Conformational studies
Conformational studies performed by circular dichroism (CD)
and FT-IR provided strong evidence that peptide nanostruc-
tures such as 1 exist mainly under a helical conformation and
in a monomeric, unaggregated state in several solvents
(MeOH, TFE and 1,2-dichloroethane), as well as in bilayer
membranes prepared with egg yolk lecithin.4,11 To verify the
influence of the polar head groups on the required helical
conformation, CD experiments have been performed with 2, 3,
and 4. Results, illustrated in Fig. 3, reveal an increase of
peptide helicity in the cases of the engineered peptide 2–4 as
compared to 1. This indicates that increasing polarity at one or
both ends of the 21mer peptidic framework does not interfere
with the correct conformation of the chain and, indirectly,
with the alignment of crown rings.
Fig. 2 21Mer crown peptide derivatives with polar head groups
subject to investigation.
shown that peptide 1 is partially incorporated in DMPC
membranes.11 On the basis of previous biophysical studies,
we proposed that the channel peptides are in a two-state
incorporation equilibrium, one active at 301 parallel to the
lipid hydrocarbon chains and the other inactive at 901 (see
Fig. 1).11 In order to verify the effect of increasing end group
polarity and hydrophilicity on the active orientation of hex-
acrown peptide in membranes, we performed ATR experi-
ments on compounds 2, 3, and 4. For each sample, orientation
of lipid chains and peptide were calculated from the symmetric
CH2 stretching band of the lipid hydrocarbon tails and CQO
stretching amide I band of the peptide.12–14 Table 1 compares
dichroic ratios (R) and mean angles of incorporation y
Membrane incorporation
Activity of channel compounds such as 1 depends greatly on
their orientation in the bilayer membrane. Using polarized
infrared ATR spectroscopy, measurement of the orientation of
peptidic framework within bilayer membrane has previously
Scheme 1 Synthesis of N-terminal polar head group: a) TBDMS-Cl, imidazole, DMF; b) succinic anhydride, DMAP, CH2Cl2.
Scheme 2 Synthesis of C-terminal polar head group: c) NaOH, H2O, Benzyloxycarbonyl chloride; d) 5, DCC, CH2Cl2; e) H2, Pd/C, MeOH.
ꢀc
This journal is the Royal Society of Chemistry the Centre National de la Recherche Scientifique 2006
186 | New J. Chem., 2006, 30, 185–190

View Article Online
Scheme 3 Synthesis of crown peptide derivatives with hydrophilic
head groups: a) 50% TFA/CH2Cl2; b) 6, DIC, HOBt, DIEA, CH2Cl2;
c)THF/MeOH (9 : 1), DBU, LiBr; d) AcOH; e) 9, CH2Cl2, AcOH
(cat); f) 1M TBAF/THF.
Fig. 3 CD spectra in TFE of peptide derivatives 1–4 at a concentra-
tion of 0.2 mM.
calculated for each crown peptides. On the basis of a two-state
incorporation equilibrium, it is possible to evaluate the parti-
cipation of each state to the resulting incorporation angle and
obtain a percentage of incorporation of peptides in the mem-
brane.11 First, the value obtained for the orientation angle of
pure DMPC (25.01) is in good agreement with those reported
in the literature.12
water solubility of 4, but also impedes slightly membrane
incorporation.
Ion transport studies
In order to obtain information on the ability of these new
channels to conduct ions, membrane transport studies were
done using the 23Na NMR method.15 This method permits a
good assessment of channel transport activity. Using Dy31 as
shift reagent, this technique provides the exchange rate of
sodium ions between the two sides of model membranes in the
presence of a functional channel and has been successfully
applied to Gramicidin D16 and artificial ion channels.5,17
Previous studies have demonstrated that transport activity of
peptide 1 represents 3% of the activity of Gramicidin D.5
Moreover, they have shown the importance of crown ether size
for sodium ion transport while supporting the monomolecular
channel mechanism of hexacrown peptides such as 1. To verify
the impact of polar terminal groups on transport abilities,
compounds 1, 2, 3 and 4 were tested. The results are reported
in Table 2 and are expressed as relative rates compared to
peptide 1 (100%).
As shown in Table 1, all peptides bearing a polar end group
demonstrate higher membrane incorporation. However, addi-
tion of a polar group at the N-terminal position has the most
pronounced effect on the stability of incorporated peptide.
Indeed, compound 2 demonstrates the best improvement in
incorporation, increasing from 28% for the fully protected
peptide 1 to 54% with 2 having polar end group at the N-
terminal position. Orientation of lipid chains varies in the
presence of 2 (27.11) compared with pure DMPC (25.01),
confirming that an important amount of the peptidic nanos-
tructure is incorporated into the membrane. Insertion of a
hydrophilic group at the C-terminal position also leads to a
significant enhancement of incorporation, 38% for peptide 3,
but to a lower extent as compared with 2. For crown peptide 4
bearing the polar head group at both termini, a 48% of
membrane incorporation is observed. This result points out
the lack of additivity of two polar head groups in the mem-
brane incorporation/stabilization processes. It is also possible
that the grafting of a second polar head, not only increases the
The main conclusion from theses studies is the increase of
transport activity for compound 2, the one with polar head at
the N-terminal position, which has an activity corresponding
to 271% of the one of fully protected peptide 1. This result is
in agreement with ATR results that demonstrated better
membrane incorporation for 2. Moreover, the enhancement
of transport activity for 4, even if more modest than the one
Table 1 ATR orientation studies on peptide nanostructures 1, 2, 3,
and 4 in planar DMPC bilayers
nC–H sym
namide 1
Incorporation
(%)a
Sample
R
y
R
y
Table 2 Rate constant (k) and relative transport rates for Na1 across
DMPC
1.08
1.13
1.12
1.15
1.14
25.01
28.01
27.11
28.91
28.31
a DMPC/DMPG bilayer membrane by 23Na NMR
1
2
3
4
a
2.03
2.71
2.28
2.54
54.21
41.81
49.01
44.41
28
54
38
48
Sample
k/sꢁ1 mMꢁ1
Relative rate (%)
1
2
3
4
2.4
6.5
1.9
2.8
100
271
80
Relates to the amount of peptide nanostructures parallel to the lipid
chain (Fig. 1)
117
ꢀc
This journal is the Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2006
New J. Chem., 2006, 30, 185–190 | 187

View Article Online
extremities. The ion transport efficiency and enhanced mem-
brane incorporation observed of N-monosubstituted peptide 2
allow the possibility of further engineering of the C-terminal
position of such peptide nanostructures in order to incorpo-
rate various recognition elements to target important biologi-
cal analytes. The results reported also open the door to the use
of more sophisticated head groups such as carbohydrate
derivatives for increased interaction with specific cell mem-
branes. Work is in progress along these lines.
Experimental
General
The oxime resin was prepared according to a reported proce-
dure using polystyrene beads (100–200 mesh 1% DVB, Ad-
vanced ChemTech, Louisville, KY).19 Resins with substitution
levels of around 0.5 mmol per gram of oxime group were used.
Boc-protected amino acids were purchased from Advanced
ChemTech. All solvents were Reagent, Spectro, or HPLC
grade quality purchased commercially and used without any
further purification except for DMF (degassed with N2),
dichloromethane (distilled), diethyl ether and THF (distilled
from sodium and benzophenone). Water used throughout the
studies was distilled and deionized using a Barnstead NANO-
purII system (Boston, MA) with four purification columns.
Phosphatidylglycerol (20 mg mLꢁ1 solution in CHCl3), phos-
phatidylcholine (20 mg mLꢁ1 solution in CHCl3) were pur-
chased from Avanti Polar-Lipids and used without further
purification. D2O was purchased from CDN Isotopes (Pointe-
Claire, QC, Canada) and used without further purification. All
other reagents were purchased from Sigma Aldrich Co. (Mil-
wauke, WI). Solid phase peptide synthesis was performed
manually using solid-phase reaction vessels equipped with a
coarse glass frit (ChemGlass, Vineland, NJ). 1H and 13C NMR
spectra were recorded on a Varian 400 MHz spectrometer.
Sonication was done using a Branson water bath model 3510.
Analyses by reverse phase HPLC were done with a Vydac C-4
column (0.45 cm ꢂ 25 cm). All solvents were degassed and
gradients of A (H2O/0.1% TFA) and B (49.95% CH3CN/
49.95% isopropanol/0.1% TFA) were used. CD measure-
ments, infrared ATR analyses, 23Na NMR measurements
and fluorescence vesicle lysis experiments have been carried
out as described previously.5,11
Fig. 4 Calcein leakage induced by addition of peptide nanostructures
1–4 and Boc-14mer-OMe. Complete leakage (100%) was obtained by
addition of a solution of Triton X-100 at 400 s.
for 2, indicates that hydrophilic non-ionic end groups can
penetrate the vesicle membrane. Indeed, in these experiments,
vesicles were formed before addition of crown peptides,
whereas, in ATR studies, phospholipid membranes were pre-
pared in the presence of peptides. Introduction of polar end
group only at the C-terminal position does not result in
increase transport ability and the rate is even lower for 3 than
for 1. Difficulty of incorporation and lower stability in mem-
brane may explain this lower activity, although more work is
necessary to understand the molecular details involved in this
case.
To verify that the activity observed by the 23Na NMR assay
is not due to lysis of vesicles, a control assay was performed.
The vesicle lysis assay is based on the increase of self quench-
ing fluorescence of calcein upon lysis.18 Fig. 4 reports calcein
release profiles induced by addition of 1, 2, 3, 4, and Boc-
14mer-OMe, precursor of 1, known to have an important lytic
activity.5,18 It appears that all 21mer analogs induced very
little lysis compared to the control, Boc-14mer-OMe. The
lower effect on the membrane induced by 3 reflects its apparent
feeble interactions with membranes and is in agreement with
ATR and NMR results. On the other hand, the very low level
of lysis observed confirmed that results obtained in the 23Na
NMR assay reflect the transport ability of artificial channels 2,
3, and 4.
Synthesis
Tris(tert-butyldimethylsilyloxymethyl)aminomethane 5. tert-
Butyldimethylsilyl chloride (6.7 g, 44.6 mmol) and imidazole
(6.3 g, 92.9 mmol) were dissolved in 5 mL DMF. Tris(hydroxy-
methyl)methyl amine (1.5 g, 12.4 mmol) was added and the
mixture was stirred at room temperature for 4 h. The product
was washed with H2O, extracted with CH2Cl2, dried over
MgSO4 and filtered. Solvent was removed in vacuo to give a
white powder (5.4 g, 95%): mp 32–34 1C. 1H NMR (400 MHz,
CDCl3) d: 3.55 (s, 6H, CH2–O), 0.89 (s, 27H, t-Bu), 0.05
(s, 18H, Si–CH3). 13C NMR (100 MHz, CDCl3) d: 64.3, 57.6,
26.1, 18.4, ꢁ5.3. ESMS m/z (M þ H1) 465.
Conclusion
We have described a simple and efficient approach to stabilize
in phospholipid membranes artificial ion channels based on
crown peptide nanostructures. Utilization of non-ionic hydro-
philic groups derived from TRIS represents an efficient alter-
native to increase the polarity at transmembrane peptide
[Tris(tert-butyldimethylsilyloxymethyl)methyl]amidosuccinic
acid 6. Succinic anhydride (0.71 g, 7.1 mmol) and 5 (3.0 g, 6.5
ꢀc
This journal is the Royal Society of Chemistry the Centre National de la Recherche Scientifique 2006
188 | New J. Chem., 2006, 30, 185–190

View Article Online
mmol) were dissolved in CH2Cl2. DMAP (60 mg, 0.49 mmol)
was added and the mixture was stirred at room temperature
for 2 h. The product was extracted with 5% NaHCO3 solu-
tion, acidified and extracted with CH2Cl2. The organic phase
was dried over MgSO4, filtered, and solvent was removed
added. The mixture was shaken mechanically for 2 h at room
temperature. The resin was filtered and washed thoroughly
with DMF (3 ꢂ 50 mL), MeOH (3 ꢂ 50 mL), DMF (3 ꢂ 50
mL), MeOH (3 ꢂ 50 mL) and then dried in vacuo. The N-BOC
group was deprotected by a 30 min treatment with a 50%
CF3COOH solution in DCM. The completion of coupling
reactions was monitored by the ninhydrin test.
1
in vacuo to give a white powder (2.0 g, 55%). H NMR (400
MHz, CDCl3) d: 6.75 (s, 1H, NH), 3.78 (s, 6H, CH2–O), 2.60
(t, 2H, CH2–COOH), 2.44 (t, 2H, CH2–CONH), 0.84 (s, 27H,
t-Bu), 0.00 (s, 18H, Si–CH3). 13C NMR (100 MHz, CDCl3) d:
175.9, 172.3, 62.4, 60.7, 31.6, 30.6, 26.0, 18.4, ꢁ5.4. ESMS m/z
(M þ H1) 564.
N-tert-BOC-(L-(21-C-7)-L-L-L-(21-C-7)-L)3-OMe 1. The
peptide on oxime resin N-tert-BOC-(L-(21-C-7)-L-L-L-(21-
C-7)-L)3-resin was synthesized in the same manner as de-
scribed previously.5 Cleavage of the 21mer peptide from 500
mg of this resin was performed in MeOH using 2 equiv. of
DBU.20 Purification was done by reverse phase HPLC (0–
100% B in 45 min). Solvents were removed in vacuo to give a
colorless oil that was dissolved in acetic acid, and lyophilized
to yield 31 mg of a fluffy white solid. 1H NMR (300 MHz,
CDCl3) d: 8.30–7.40 (m, 21H, NH), 6.85–6.60 (m, 18H, H Ar),
4.35–4.25 (m, 6H, aCH 21-C-7), 4.10–3.85 (m, 24H, 12 CH2–
OAr), 3.75–3.60 (m, 24H, 12 CH2–CH2–OAr), 3.60–3.40 (m,
114H, 48 CH2–O þ 15 Leu aCH þ CH3–O), 3.10–2.90 (m,
12H, 6 bCH2 21-C-7), 1.65–1.35 (m, 54H, 15 Leu bCH2 þ 15
Leu gCH þ 9 H t-Bu), 0.95–0.65 (m, 90H, Leu CH3). MALDI
TOF-MS m/z (M þ Na)1 4406.
6-(N-Benzyloxycarbonylamino)hexanoic acid 7. 6-Amino-
hexanoic acid (5.0 g, 38.1 mmol) was dissolved in H2O and
cooled to 0 1C. Alternatively, benzylchloroformate (8.1 g, 56.7
mmol) and 5N NaOH (60 mL) was added in portions, keeping
the reaction mixture at pH 10. The mixture was stirred at
room temperature for 1 h. The alkaline solution was washed
with diethyl ether and acidified to pH 2–3 with 1 N HCl. The
product was extracted with CH2Cl2, and dried over MgSO4.
After filtration, solvent was removed in vacuo to give a white
powder (8.6 g, 85%): mp 48–49 1C. 1H NMR (400 MHz,
CDCl3) d: 12.01 (s, 1H, COOH), 7.47–7.30 (m, 5H, Ar), 7.25
(t, 1H, NH), 5.02 (s, 2H, CH2-Ar), 2.99 (q, 2H, CH2–NH),
2.20 (t, 2H, CH2–COOH), 1.50 (m, 2H, CH2–CH2–NH), 1.41
(m, 2H, CH2–CH2–COOH), 1.26 (m, 2H, CH2–CH2–CH2–
NH). 13C NMR (100 MHz, CDCl3) d: 179.3, 156.7, 134.8–
128.3, 66.9, 41.0, 34.1, 29.8, 26.3, 24.4. ESMS m/z (M þ H1)
266.
6-(L-(21-C-7)-L-L-L-(21-C-7)-L)3-resin 10. N-tert-BOC-(L-
(21-C-7)-L-L-L-(21-C-7)-L)3-resin was deprotected by a 30
min treatment with a 50% CF3COOH solution in DCM.
The acid 6 (0.25 g, 0.45 mmol) was activated with DIC/HOBt
(5 equiv.) during 30 min at 0 1C in CH2Cl2/DMF (1 : 1), and
then added, with 1.5 equiv. of DIEA, to the deprotected 21mer
on resin (0.21 g, 0.089 mmol) swollen with CH2Cl2. The
mixture was shaken mechanically for 2 h at room temperature.
The resin was drained and washed thoroughly with DMF (3 ꢂ
50 mL), MeOH (3 ꢂ 50 mL), DMF (3 ꢂ 50 mL), MeOH (3 ꢂ
50 mL) and then dried in vacuo. The completion of coupling
reaction was monitored by the ninhydrin test.
[Tris(tert-butyldimethylsilyloxymethyl)methyl]6-(N-benzylox-
ycarbonylamino) hexanamide 8. At 0 1C, 7 (1.2 g, 4.5 mmol)
and DCC (0.93 g, 4.5 mmol) were dissolved in CH2Cl2. The
amine 5 (2.1 g, 4.5 mmol) dissolved in CH2Cl2 was added and
the mixture was stirred overnight at room temperature. Urea
was eliminated by filtration and the product was washed with
NaHCO3 5%, H2O, 1N HCl, H2O, and dried over MgSO4.
After filtration, solvent was removed in vacuo and purification
on silica gel (25% AcOEt/hexanes) gave a white powder (1.5 g,
47%): mp 88–89 1C. 1H NMR (400 MHz, CDCl3) d: 7.43–7.30
(m, 5H, Ar), 7.22 (t, 1H, NH(Z)), 6.73 (s, 1H, NH(Tris)), 5.00
(s, 2H, CH2–Ar), 3.67 (s, 6H, CH2–OSi), 2.96 (q, 2H, CH2–
NH), 2.10 (t, 2H, CH2–CO), 1.45 (m, 2H, CH2–CH2–NH),
1.38 (m, 2H, CH2–CH2–CO), 1.24 (m, 2H, CH2–CH2–CH2–
NH), 0.86 (s, 27H, t-Bu), 0.00 (s, 18H, Si–CH3). 13C NMR
(100 MHz, CDCl3) d: 172.4, 156.6, 136.8–128.3, 66.8, 61.8,
60.7, 41.1, 37.5, 29.9, 26.6, 26.1, 25.5, 18.4, ꢁ5.3. ESMS m/z
(M þ H1) 711.
6-(L-(21-C-7)-L-L-L-(21-C-7)-L)3-OMe 2. Cleavage of the
21mer peptide from 100 mg of 10 resin was performed in
MeOH (10%)/THF using 2 equiv. of DBU. Deprotection was
completed in acetic acid after 90 h. Purification was done on
Sephadex LH-20 in MeOH. Solvents were removed in vacuo to
give a colorless oil that was dissolved in acetic acid, and
lyophilized to obtain 2 as a fluffy white solid. Purity was
verified by reverse-phase HPLC (0–100% B in 45 min). 1H
NMR (400 MHz, CDCl3) d: 8.45–7.80 (m, 22H, NH), 6.85–
6.55 (m, 18H, H Ar), 4.30–3.80 (m, 54H, 6 aCH 21-C-7 þ 12
CH2–Oar þ 12 CH2–CH2–OAr), 3.80–3.40 (m, 120H, 51
CH2–O þ15 Leu aCH þ OCH3), 3.35–2.90 (m, 16H, 6 bCH2
21-C-7 þ CH2–CO), 1.90–1.20 (m, 45H, 15 Leu bCH2 þ15
Leu gCH), 0.95–0.60 (m, 90H, Leu CH3). MALDI TOF-MS
m/z (M þ Na)1 4504.
[Tris(tert-butyldimethylsilyloxymethyl)methyl]6-aminohexana-
mide 9. The protected amine 8 (66 mg, 0.093 mmol) was
deprotected in MeOH with H2/Pd on activated carbon for 2
h at room temperature. The mixture was filtered on Celite,
solvent was evaporated and the amine 9 was used in next step
without further purification.
N-tert-BOC-(L-(21-C-7)-L-L-L-(21-C-7)-L)3-9 3. Cleavage
of the peptide N-tert-BOC-(L-(21-C-7)-L-L-L-(21-C-7)-L)3-
resin from the oxime resin was performed in CH2Cl2 using 1
equiv. of 9 and acetic acid in catalytic amount. Deprotection
was effected with 2.5 equiv. of TBAF in THF. Purification was
done on Sephadex LH-20 in MeOH. Solvents were removed
Typical procedure for amino acids coupling on solid support.
The amino acid (5 equiv.) was activated with DIC/HOBt
during 30 min at 0 1C in CH2Cl2/DMF (1 : 1), then added
to the resin swollen in DMF and 1.5 equiv. of DIEA were
ꢀc
This journal is the Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2006
New J. Chem., 2006, 30, 185–190 | 189

View Article Online
in vacuo to give a colorless oil that was dissolved in acetic acid,
and lyophilized to obtain 3 as a fluffy white solid. Purity was
verified by reverse-phase HPLC (0–100% B in 45 min). 1H
NMR (400 MHz, CDCl3) d: 8.50–7.70 (m, 23H, NH), 6.85–
6.50 (m, 18H, H Ar), 4.30–3.80 (m, 54H, 6 aCH 21-C-7 þ 12
CH2–OAr þ 12 CH2–CH2–OAr), 3.80–3.40 (m, 117H, 51
CH2–O þ 15 Leu aCH), 3.35–2.85 (m, 16H, 6 bCH2 21-C-7 þ
CH2–NH þ CH2–CO), 2.00–1.30 (m, 60H, 15 Leu bCH2 þ
15 Leu gCH þ 3 CH2–(CH2)3–CH2 þ 9 H t-Bu), 1.00–0.65
(m, 90H, Leu CH3). MALDI TOF-MS m/z (M þ Na)1 4603.
60, 6405; (b) U. Koert, L. Al-Momani and J. R. Pfeifer, Synthesis,
2004, 8, 1129; (c) I. I. Stoikov, I. S. Antipin and A. I. Konovalov,
Russ. Chem. Rev., 2003, 72, 1055; (d) G. W. Gokel and A.
Mukhopadhyay, Chem. Soc. Rev., 2001, 30, 274 and references
cited therein.
2 N. Voyer, J. Am. Chem. Soc., 1991, 113, 1818.
3 N. Voyer and M. Robitaille, J. Am. Chem. Soc., 1995, 117, 6599.
4 J.-C. Meillon and N. Voyer, Angew. Chem., Int. Ed. Engl., 1997,
36, 967.
5 E. Biron, F. Otis, J.-C. Meillon, M. Robitaille, J. Lamothe, P. Van
Hove, M.-E. Cormier and N. Voyer, Bioorg. Med. Chem., 2004, 12,
1279.
6 J. Kohda, K. Shinozuka and H. Sawai, Tetrahedron Lett., 1995, 36,
5575.
6-(L-(21-C-7)-L-L-L-(21-C-7)-L)3-9 4. Cleavage of the
21mer peptide from 500 mg of 10 resin was performed as
described for 3 using 1 equiv. of 9. Deprotection was done with
2.5 equiv. TBAF in THF. Purification was done on Sephadex
LH-20 in MeOH. Solvents were removed in vacuo to give a
colorless oil that was dissolved in acetic acid, and lyophilized
to obtain 4 as a fluffy white solid. Purity was checked by
reverse-phase HPLC (0–100% B in 45 min). 1H NMR (400
MHz, CDCl3) d: 8.40–7.60 (m, 24H, NH), 6.85–6.55 (m, 18H,
H Ar), 4.30–3.80 (m, 54H, 6 aCH 21-C-7 þ 12 CH2–OAr þ 12
CH2–CH2–OAr), 3.80–3.40 (m, 123H, 54 CH2–O þ 15 Leu
aCH), 3.35–2.80 (m, 20H, 6 bCH2 21-C-7 þ CH2–NH þ 3
CH2–CO), 1.90–1.20 (m, 51H, 15 Leu bCH2 þ 15 Leu gCH þ
3 CH2–(CH2)3–CH2), 0.95–0.65 (m, 90H, Leu CH3). MALDI
TOF-MS m/z (M þ Na)1 4707.
7 E. J. Corey and A. Venkateswarlu, J. Am. Chem. Soc., 1972, 94,
6190.
8 J. K. Lee, K. C. Ahn, D. W. Stoutamire, S. J. Gee and B. D.
Hammock, J. Agric. Food Chem., 2003, 51, 3695.
9 J. C. Sheehan and G. P. Hess, J. Am. Chem. Soc., 1955, 77, 1067.
10 A. Pichette, N. Voyer, R. Larouche and J.-C. Meillon, Tetrahedron
Lett., 1997, 38, 1279.
11 E. Biron, N. Voyer, J.-C. Meillon, M.-E. Cormier and M. Auger,
Biopolymers, 2000, 55, 364.
12 E. Okamura, J. Umemrua and T. Takenaka, Biochim. Biophys.
Acta, 1986, 856, 68.
13 W. Hubner and H. H. Mantsch, Biophys. J., 1991, 59, 1261.
¨
14 M. Bouchard and M. Auger, Biophys. J., 1993, 65, 2484.
15 F. G. Riddell and M. K. Hayer, Biochim. Biophys. Acta, 1985, 817,
313.
16 D. Buster, J. Hinton, F. Millett and D. Shungu, Biophys. J., 1988,
53, 145.
17 E. Abel, G. E. M. Maguire, O. Murillo, I. Suzuki, S. L. De Wall
and G. W. Gokel, J. Am. Chem. Soc., 1999, 121, 9043.
18 Y. R. Vandenburg, B. D. Smith, E. Biron and N. Voyer, Chem.
Commun., 2002, 17, 1694.
19 W. F. DeGrado and E. T. Kaiser, J. Org. Chem., 1980, 45, 1295.
20 N. Voyer, A. Lavoie, M. Pinette and J. Bernier, Tetrahedron Lett.,
1994, 35, 355.
References
1 Many reviews on artificial ion channel have been published. See for
example: (a) S. Matile, A. Som and N. Sorde, Tetrahedron, 2004,
´
ꢀc
This journal is the Royal Society of Chemistry the Centre National de la Recherche Scientifique 2006
190 | New J. Chem., 2006, 30, 185–190