A hydrocarbon anchored peptide that forms a chloride-selective channel in liposomes.

Article abstract of DOI:10.1039/b200126h

The heptapeptide sequence Gly-Gly-Gly-Pro-Gly-Gly-Gly, when anchored to diglycolic acid derived (C18H37)2NCOCH2OCH2COOH, forms chloride-selective ion channels in phospholipid liposomes but the related heptapeptide Gly-Gly-Gly-Leu-Gly-Gly-Gly, and tripepti

Full text of DOI:10.1039/b200126h

A hydrocarbon anchored peptide that forms a chloride-selective channel
in liposomes†
b
a
a
a
Paul H. Schlesinger,* Riccardo Ferdani, Robert Pajewski, Jolanta Pajewska and George W.
Gokel*
a
a
Division of Bioorganic Chemistry, Bioorganic Chemistry Program and Department of Molecular Biology
&
Pharmacology, Washington University School of Medicine, 660 South Euclid Ave, Campus Box 8103, St.
Louis, MO 63110, USA
b
Department of Cell Biology, Washington University School of Medicine, 660 South Euclid Ave, Campus
Box 8103, St. Louis, MO 63110, USA. E-mail: ggokel@molecool.wustl.edu; Fax: 314/362-9298 or 7058;
Tel: 314/362-9297
Received (in Columbia, MO, USA) 17th December 2001, Accepted 13th February 2002
First published as an Advance Article on the web 19th March 2002
The heptapeptide sequence Gly-Gly-Gly-Pro-Gly-Gly-Gly,
when anchored to diglycolic acid derived (C18 NCO-
CH OCH COOH, forms chloride-selective ion channels in
phospholipid liposomes but the related heptapeptide Gly-
Gly-Gly-Leu-Gly-Gly-Gly, and tripeptide Gly-Gly-Gly do
not.
motif in C-peptide and this arrangement is required for ion
H
37
)
2
channel activity.22
2
2
37 2 2 2
We therefore set as our target (C18H ) NCOCH OCH CO-
G-G-G-P-G-G-G-OCH Ph, 1. Diglycolic anhydride was heated
2
at reflux with dioctadecylamine in toluene for 48 h. The
monoamide (C18 NCOCH OCH COOH ([18] DGA-OH)
was obtained in 87% yield after crystallization from CHCl (mp
81–82 °C). The acid, [18] DGA-OH, was coupled to
TsOH·H N-Gly-Gly-Gly-OCH Ph (Me N(CH NNCNNEt
(EDCI), Et N, CH Cl , 0–25 °C, 30 h) to afford 3. Hydro-
genolysis of 3 (H , Pd/C, 95% EtOH) afforded [18] DGA-G-G-
G-OH (96%, mp 163–164 °C). Coupling (EDCI, Et N, CH Cl
N-L-G-
Ph gave 2 (83%, mp
H
37
)
2
2
2
2
3
The transport of ions through phospholipid bilayers is mediated
by a variety of channels. Recent solid state studies of
2
2
2
2
2 3
)
1
,2
3
4
potassium, sodium, mechano-sensitive, and water chan-
3
2
2
5
6
nels have greatly advanced our understanding. Until this year,
no structural evidence had appeared that would correspondingly
aid our comprehension of transmembrane chloride channels
ClC). Even the remarkable solid state structure of the ClC
2
2
3
2
2
,
0–25 °C, 30 h) of [18]
G-G-OCH Ph or H
2
DGA-G-G-G-OH with either H
2
7
(
2
2
N-P-G-G-G-OCH
2
2
3
channel raises nearly as many questions as it resolves because it
is so inherently complex. Naturally occurring peptides such as
164–165 °C) or 1 (82%, mp 116–118 °C) respectively.
8
9
10
alamethicin, melittin, gramicidin and a number of synthetic
organic models have been developed to mimic cation channel
function.11 No organic chemical model of chloride channel
function has yet appeared although an anion-selective analogue
of the channel-forming peptide alamethicin12 has been reported.
Tomich and coworkers have developed a chloride-selective
peptide by modifying a known glycine-gated Cl-channel
peptide.13
The challenge to develop a functional, synthetic chloride
channel is great, especially considering the dearth of structural
information available on which to base a model. One possibility
was to modify our hydraphile cation channel model14 com-
pounds in accord with the electrostatic analyses of Dawson and
coworkers.15 Instead, we chose to devise a novel model system
consisting of three parts. A twin-tailed amine would serve as the
equivalent of the phospholipidAs fatty acyl chains. Diglycolic
2 2
acid, HOCOCH OCH COOH would connect the hydrophobic
residues to the headgroup and approximate the phospholipidAs
midpolar (acyl glycerol) regime.16 The overall length of the
For the reasons noted above, we hypothesize that proline is
critical to the channel forming activity of 1. Assessing the
release of chloride from liposomes mediated by 1, 2, and 3
tested this hypothesis. Phospholipid liposomes were prepared in
‘
anchor’ or phospholipid mimic would be determined by the
alkyl chains attached to the acid, i.e., R in R NCOCH O-
CH COOH.
2
2
2
We considered using such previously incorporated ‘portal
elements’ as crown ethers and cyclodextrins but ultimately
chose a different approach. It is known that proline plays a
24
2
00 mM KCl. A chloride selective resin electrode was used to
2
measure Cl concentration after extravesicular chloride had
been chromatographically exchanged for non-interfering ni-
trate.25 The data are shown in the two graphs of Fig. 1. The top
critical role in the chloride selectivity of naturally occurring
chloride transporters.17 We further noted that all members of the
2 2
panel presents data for [18] DGA-GGGPGGG-OCH Ph (1,
ClC family of chloride protein channels contain the conserved
motif GKxGPxxH in the putative anion pathway.18 It is known
that substitution of a proline into the intrinsic channel selectivity
filter of nicotinic acetylcholine receptors reverses the ion
147 mM) and [18] DGA-GGGLGGG-OCH Ph (2, 154 mM).
2
2
The slight concentration difference results from experimental
conditions and is not significant. The bottom panel presents the
selectivity.19 Proline may form a ‘hinge-bend’ regime (GxxP)
20
same data for [18] DGA-GGGPGGG-OCH Ph (1, 147 mM)
2
2
2 2
and compares it with [18] DGA-GGG-OCH Ph (3, 154 mM).
or it may induce a surface ‘kink’ in membrane transport
proteins.21 Finally, proline is at the apex of the helix–loop–helix
Compound 3, in which the peptide chain is truncated compared
to 1, showed substantially reduced chloride release. When
proline in 1 was replaced by leucine (2), chloride release was
again greatly reduced (Fig. 1B). We infer from these results that
†
Electronic supplementary information (ESI) available: analytical data for
1
, 2 and 3. See http://www.rsc.org/suppdata/cc/b2/b200126h/
8
40
CHEM. COMMUN., 2002, 840–841
This journal is © The Royal Society of Chemistry 2002

We thank the NIH and NSF for grants that supported this
work.
Notes and references
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+ 27
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2
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22
SO
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27
mine the anion selectivity of 1. In Fig. 2 and in previous work,
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22
2
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22
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3
2
3 1: mp 116–118 °C. 2: mp 164–165 °C. Additional details may be found
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Fig. 2 Fraction of Cl2 released with respect to time by 1 in the presence of
2
22
NO
3
, valinomycin, and SO
4
.
CHEM. COMMUN., 2002, 840–841
841