Replacing proline at the apex of heptapeptide-based chloride ion transporters alters their properties and their ionophoretic efficacy

Article abstract of DOI:10.1039/b206570n

A membrane anchored heptapeptide, (C₁₈H₃₇)₂NHCOCH₂OCH ₂CO-NH-Gly-Gly-Gly-Pro-Gly-Gly-Gly-OCH₂Ph, has proved to be a selective chloride anion transporter that functions in phospholipid bilay

Full text of DOI:10.1039/b206570n

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Replacing proline at the apex of heptapeptide-based chloride ion
transporters alters their properties and their ionophoretic efficacy
Paul H. Schlesinger,*a Riccardo Ferdani,b Jolanta Pajewska,b Robert Pajewskib and
George W. Gokel*b
a
Cell Biology and Physiology, Washington University School of Medicine, 660 S. Euclid Ave.,
St. Louis MO 63110, USA
Program in Bioorganic Chemistry, Division of Bioorganic Chemistry, Departments of
b
Molecular Biology & Pharmacology, Washington University School of Medicine, 660 S.
Euclid Ave., St. Louis MO 63110, USA. E-mail: ggokel@molecool.wustl.edu;
Fax: 314/362-9298 or 7058; Tel: 314/362-9297
Received (in Montpellier, France) 5th July 2002, Accepted 28th August 2002
First published as an Advance Article on the web 12th December 2002
A membrane anchored heptapeptide, (C18H37)2NHCOCH2OCH2CO–NH–Gly–Gly–Gly–Pro–Gly–Gly–Gly–
OCH2Ph, has proved to be a selective chloride anion transporter that functions in phospholipid bilayer
membranes. When Pro was replaced by the natural amino acid Leu, the activity decreased dramatically. In the
present study, Pro is replaced by pipecolic acid (homoproline, Pip); the resulting anchored heptapeptide is a
membrane active, chloride selective transporter.
Chloride membrane transport is a ubiquitous process in living
systems. An array of chloride channels has been studied for
some time and understanding their function is clearly an
important goal.1,2 The only solid state structures of chloride
transporters were reported very recently.3 The X-ray structures
of the ClC Clꢀ channels from the prokaryotes Salmonella
enterica serovar typhimurium and Escherichia coli were reported
family of abiotic chloride channels.11,12 We now report related
compounds that have quite different activities depending on
what appear to be very subtle differences.
Results and discussion
Design and synthesis of SCMTR
˚
˚
at 3.0 A and 3.5 A resolutions, respectively. These structures
reveal that the chloride channels are quite complicated and dis-
tinct from those of voltage gated cation channels. Both ClC
channels incorporate two subunits of 18 a-helices that function
as independent channel pathways. Their association is coordi-
Our design schematic for a chloride ion transporter (s ynthetic
c hloride membrane t r ansporter, SCMTR or ‘‘scimitar’’)
2
˚
nated through a 2300 A contact surface. To date, four major
families of chloride channels have been described.4,5
The complexity of the chloride channel structures results
from many regulatory influences and a long evolutionary his-
tory as a fundamental cellular function. In early organisms,
chloride transport essential to volume and pH control must
have been achieved by significantly simpler structures. Cer-
tainly, chloride permeability in contemporary cells is essential
for volume, pH, and membrane potential regulation and these
same features must have been required, at least to some extent,
in early organisms.6,7 The work reported here results from an
effort to develop a simple, abiotic compound that would func-
tion as a chloride transporter in phospholipid bilayers.
An effort by Tomich and coworkers8–10 has been reported to
develop a chloride channel by using modified proteins. Specifi-
cally, C-K4-M2GlyR is a peptide prepared by modifying either
the N- or C-terminus of the M2 transmembrane sequence of
the brain glycine receptor by the addition of lysine residues.
The resulting peptide, C-K4-M2GlyR, was found to act as a
chloride channel when it was inserted into the apical mem-
brane of cultured renal epithelial monolayers. Our effort to
develop a chloride transporter differed. We used what is cur-
rently known about chloride channel proteins as a guide and
inspiration for the design of a synthetic, chloride selective
transporter. As a result, we recently reported the preparation
and limited characterization of the first example of this new
60
New J. Chem., 2003, 27, 60–67
DOI: 10.1039/b206570n
This journal is # The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2003

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Scheme 1
involved the development of three molecular sectors. These
included a hydrocarbon anchor, a peptide fragment expected
to serve as the channel portal and filter, and the connector.
Diglycolic acid, HOCOCH2OCH2COOH, was selected to be
the link between the peptide and the hydrocarbon anchors.
The latter were constructed from a dialkylamine, giving an
overall anchor of R2NCOCH2OCH2COOH. The peptide was
joined to the anchor’s C-terminus to give, schematically,
R2NCOCH2OCH2CO-peptide.
Fig. 1 Ac–GGGPGGG–Bzl shown in a structural figure (a) and ren-
dered in the CPK representation (b). Calculated structures of CH3NH–
Gly–Pro–Gly–OCH3 (c) and CH3NH–Gly–Pip–Gly–OCH3 (d).
The anchor unit was designed to correspond approximately
to the size and shape of a phospholipid membrane monomer.
The twin diamine chains mimicked the two fatty acid chains of
a natural phospholipid. Phospholipids contain a glycerol unit,
to which the fatty acids are attached by acyl links, resulting in
a region of intermediate polarity. The midpolar regime was
emulated by diglycolic acid, which contains carbonyl groups
and an ether oxygen. Diglycolic acid is especially attractive
for this application because its readily formed anhydride reacts
cleanly with diamines as shown in Scheme 1.
The peptide’s design was guided by structural patterns
found in natural chloride transporters. It was hoped that these
would lead to chloride selectivity in our abiotic compound.
Based upon the following lines of evidence, we have identified
a critical role for the peptide in the ion selectivity of naturally
occurring chloride transporters.6 (1) Proline is present in the
conserved motif GKxGPxxH in the anion pathway of all
ClC chloride protein channels.13,14 (2) The cation selectivity
of nicotinic acetylcholine receptor is altered to anion selectivity
when proline is inserted into the native channel’s selectivity fil-
ter.15,16 (3) The presence of a so-called ‘‘hinge-bend’’ regime in
channel-forming peptides is attributed to the proline in the
sequence GxxP.17 (4) Proline is reported to induce a surface
‘‘kink’’ in various proteins.18 (5) Studies with proinsulin’s
C-peptide show that the presence of a helix–loop–helix motif
at the apex of the peptide’s ‘‘loop’’ conformation is required
for ion channel activity.19 The combination of structural
observations and functional implications suggested to us the
importance of incorporating this particular element into our
ion portal design.
of an ‘‘arch’’ flanked with glycine residues. We propose that
having the heptapeptide reside at the top of the mid-polar
regime of the membrane bilayer is critical. In this position it
can form an uncharged, chloride-selective portal. The resulting
formation of an aqueous pathway for ions to the interior of the
bilayer was anticipated. If this pathway was maintained long
enough for ion diffusion through the bilayer to occur, channel
activity should result. We anticipated that by adding a hydro-
phobic membrane anchor of appropriate length, the peptide
would be positioned to generate a functional ‘‘hinge-bend’’
portal at the membrane surface.19
The membrane anchor
As noted above, the membrane anchor was designed to mimic
a phospholipid monomer in size, polarity, and functional
group position. The anchor is a dialkylamino derivative of
diglycolic acid: R2NCOCH2OCH2COOH.20 A related anchor
system was reported several years ago21,22 but no experimental
details were included and no yield or characterization data
appear in the Beilstein database.23 We were able to construct
R2NCOCH2OCH2COOH conveniently by the reaction of
one equivalent each of diglycolic anhydride and dialkylamine
(e.g., dioctadecylamine) in refluxing toluene. After heating
for 48 hours, the solvent was removed and the nearly pure
R2NCOCH2OCH2COOH was crystallized from CHCl3 to
afford the analytically pure product in nearly 90% yield. It is
interesting to note that the reaction of bis(2-methoxyethyl)-
amine with diglycolic anhydride is reported to be much faster
than the rates we observed for the anhydride with simple di-
alkylamines.21 We confirmed this rate difference, although
the experimental procedure in the literature does not work as
reported for the methoxyethylamine.
We surmised that an anchored peptide would insert deeply
into the outer leaflet of a phospholipid bilayer. This, we felt,
would lead to a controlled membrane disruption of the second
leaflet resulting in a transmembrane pore. The advantage of
this approach was thought to be that the kinetics of insertion
into the outer leaflet of the bilayer would be more favorable
than any system that required transverse relaxation (flip-flop).
Further, by limiting the length of our anchor to the thickness
of a single bilayer, we anticipated that the detergent activity of
this compound would be minimized, limiting the formation of
membrane disks at high concentrations.24
We settled on a heptapeptide sequence having proline as its
central point for the reasons described above. The simplest
heptapeptide containing a ‘‘central’’ proline is Gly–Gly–
Gly–Pro–Gly–Gly–Gly (GGGPGGG). Analyses using mole-
cular models and simple computational methods suggested
that GGGPGGG would have a v-shaped conformation.
Further, distance measurements across a computer-generated
model of the ‘‘v’’ show that the C- and N-termini have a max-
˚
imum center-to-center separation of ꢁ8 A and the apex of the
‘‘v’’ makes an angle of about 75ꢂ. Space-filling (CPK) molecu-
lar models show that the available space within the ‘‘v’’ is 6–8
˚
A, depending on how compressed are the two GGG chains. A
computational model of CH3CH2CONH–GGGPGGG–
OCH2Ph is shown in Fig. 1. Measurement of the model shows
˚
that the two remotest carbonyl groups are 8.8 A apart in the
illustrated conformation.
In 2, [CH3(CH2)17]2NCOCH2OCH2CO–GGGPGGG–
OCH2Ph, the peptide sequence places proline at the pinnacle
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Fig. 2 Carboxyfluorescein release by 2 from liposomes. Concentra-
tions of 2: (bottom to top): 12.6 mM, 25.3 mM, 63.3 mM, 127 mM,
190 mM, and 253 mM. The two highest concentrations are nearly super-
imposed in this graph.
vesicle, CF self-quenches. Under appropriate conditions, its
exit from vesicles may readily be detected. We prepared phos-
pholipid liposomes in the presence of 20 mM carboxyfluores-
cein (CF). Dequenching of intravesicular CF was followed
spectrofluorometrically in a thermostated cuvette (see Experi-
mental Section). Data obtained for 2 over a range of concen-
trations are shown in Fig. 2. On the timescale shown, the
data for 2 at 190 mM and 253 mM are nearly superimposable.
The CF transport data show that 2 forms well-behaved
pores that release the dye in a controlled fashion. The critical
test, however, of a putative chloride transporter is whether it
fosters chloride release from liposomes. We used unilamellar
liposomes (150 ꢄ 16 nm) prepared from 30% (w/w) 1,2-di-
oleoyl-sn-glycero-3-phosphate and 70% 1,2-dioleoyl-sn-gly-
cero-3-phosphocholine.
Compound 2 mediated chloride release that was rapid, com-
plete, and concentration dependent.11 Both 1 and 2 release
carboxyfluorescein, a process related to chloride transport.
We have recently noted that CF release by 1 (10Pro), which
has shorter anchor chains, is about 10-fold greater25 than for
2 (18Pro).11 Thus CF release was complete in less than 100 s
when either 1 or 2 was present at a concentration of ꢁ15
mM or ꢁ250 mM respectively. We infer from this that the as
yet untested chloride transport rates will show a comparable
relationship.
Scheme 2
Synthesis of the compounds studied
Four target compounds were prepared for the studies reported
here. Each procedure began with the reaction of either didecyl-
amine or dioctadecylamine with diglycolic anhydride. The
product was (C10H21)2NCOCH2OCH2COOH or (C18H37)2-
NCOCH2OCH2COOH, respectively. We use shorthand to des-
ignate these diglycolic amide (DGA) derivatives: 102DGA–OH
and 182DGA–OH. The C-terminal tripeptide was prepared as
a benzyl ester. It was coupled (1-(3-dimethylaminopropyl)-3-
ethylcarbodiimide hydrochloride) with TsOHꢃH2N–GGG–
OCH2Ph to give R2DGA–Gly–Gly–Gly–OCH2Ph. The
C-terminal benzyl group was removed by hydrogenolysis in
EtOH.
Replacement of proline by leucine in
2 (182DGA–
GGGPGGG–Bzl ! 182DGA–GGGLGGG–Bzl, 5) led to
reduced CF release. Thus, equal CF release rates could be
achieved when [5] ¼ 154 mM and [2] ¼ 24.1 mM.11 This is at
least a 6-fold reduction in activity, apparently resulting from
the loss of the rigid ‘‘hinge-bend’’ enforced by proline’s cyclic
structure.
The tetrapeptide required to complete the heptapeptide
sequence was prepared by coupling TsOHꢃH2N–GGG–
OCH2Ph with N–Boc–Pro–OH to give Boc–Pro–Gly–Gly–
Gly–OCH2Ph (Z–PGGG–Bzl). The Boc group was removed
(HCl, dioxane) and the final coupling between R2DGA–
Gly–Gly–Gly–OH and H2N–PGGG–OCH2Ph gave either
102DGA–GGGPGGG–Bzl (1) or 182DGA–GGGPGGG–Bzl
(2). Compounds 3 and 4 were prepared analogously except
that pipecolic acid (homoproline) replaced proline in the hep-
tapeptide. The sequence is shown below in Scheme 2 for the
preparation of either 1 or 2 and details for the synthesis of
compounds 1–4 are recorded in the Experimental Section.
Pore conductance and rectification in planar bilayers
Planar lipid bilayers were prepared from asolectin and studied
under voltage clamp conditions (see Experimental Section and
ref. 26). The addition and mixing of 89.7 mM 182DGA–
GGGPGGG–Bzl (2, 18Pro, SCMTR) on the trans side of
the planar bilayer resulted in a current that was mildly rectify-
ing at negative potentials. The I–V relationship (ꢀ60 mV to
+60 mV) had a limiting slope of 349 pS. The 102DGA–
GGGPGGG–Bzl (1, 10Pro, SCMTR) at 5.2 mM was also
mildly rectifying and had a limiting slope of 573 pS. We have
previously noted that the 18Pro is selective for chloride over
potassium by > 10:1. The larger conductance of 10Pro with
an identical pore size as determined in Fig. 1 indicates that this
pore is relatively non-selective for chloride versus potassium.
Carboxyfluorescein dequenching and pore characterization
Carboxyfluorescein (CF) is a fluorescent dye that may readily
be incorporated into liposomes. When concentrated within the
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Fig. 4 Carboxyfluorescein release from phospholipid liposomes
mediated by compounds 1–4. The top (red, 10Pip, 3, 5 mM) and bot-
tom (blue, 18Pip, 4, 29 mM) traces are for 3 (10Pip) pipecholic acid
derivatives. The overlapping central traces (black above, 1, 10Pro,
0.167 mM; orange, 2, 18Pro, 63.3 mM) are for the proline-containing
heptapeptide.
Fig. 3 Models of proline (top) and pipecolic acid in tube and space
filling representations.
Substitution of proline in the heptapeptide sequence by leucine
or by pipecolic acid
be 5 mM/0.167 mM or 29.9. The didecyl-pipecolic (10Pip) acid
derivative (3) is somewhat more active at 5 mM than is the pro-
line analog at 0.167 mM, so we estimate that 10Pip (3) is about
20-fold less active than the proline counterpart (10Pro, 1). This
is a significant reactivity difference that may result from varia-
tions in bond angles, hydrophobicity, and/or other factors.
The more striking observation is that at a concentration of
29 mM, 18Pip (4) showed essentially no transport activity.
A direct comparison of 2 with 4 at identical concentrations
would have been more satisfactory. Unfortunately, when
[18Pip] > 29 mM, precipitation of the transporter occurred
and the experiment was compromised. We therefore under-
took the fluorescence dequenching experiment with 18Pro (2)
at a concentration of 25.3 mM. Within the error of these experi-
ments, this is an identical concentration. The results are shown
in Fig. 4. At identical concentrations, the activity of 4 is
unmeasurable compared to that of 2.
A comparison of the data presented in the graphs of Figs. 2
and 4 raises several interesting questions. First, do we expect
any activity from a compound in which pipecolic acid replaces
proline? As noted above, previous work showed that the
anchored heptapeptide lost nearly all of its activity when Leu
replaced Pro.11 This result correlated to known C-peptide
activity (see above).27 Pipecolic acid is far closer to proline29
than is leucine, although Pro and Leu have the same number
of carbon atoms. Proline and pipecolic acids are similar in size
and shape and the latter should function to form a conforma-
tional kink in the heptapeptide chain as does proline. Indeed,
in the C10-anchored system, pore formation for 10Pro (1)
was ꢁ20-fold greater than for 10Pip (3). Significant activity
for 10Pip was observed only at or above 5 mM concentrations,
compared to 167 nM for 10Pro.
The known peptide sequence of Clꢀ-conducting C-peptide27
contains proline at the loop apex in humans and in rats.28 In
contrast, the pig sequence has leucine in this position and this
peptide is not a chloride ion transporter.27 As noted above,
we found in previous studies that a Pro ! Leu replacement
led to diminished (ꢁ6-fold) activity. A more subtle variation
is the change from proline to pipecolic acid.29 This alteration
formally involves a 1-carbon ring expansion of the essential
amino acid. The structures of the bis(octadecyl) derivatives
are shown below as 2 and 4.
These two amino acids differ in several respects (Fig. 3).29
The difference in ring size changes the N–C–CO angles from
ꢁ112.5ꢂ to ꢁ110.5ꢂ. The presence of the additional carbon
atom in the ring also increases hydrophobicity but, as with
the bond angles, the change is expected to be small. The mole-
cular weights of these two compounds are 1168 Da and 1182
Da, respectively. This is a difference of only ꢁ1%. We also con-
sider the differences in bond angles and hydrophobicity to be
modest.
We have assessed the efficacy of the proline (1, 2) and pipe-
colic acid (3, 4) derivatives by CF dequenching (see above).
Selected data are presented in Fig. 4. The graph shows four
traces corresponding to various concentrations of 1–4. The
two nearly superimposed traces seen in the middle of the
graph are for 102DGA–GGGPGGGOBzl (1) and 182DGA–
GGGPGGG–OBzl (2). The CF release is essentially identical
but the concentration difference is dramatic: their activities dif-
fer by nearly 400-fold ([1] ¼ 0.167 mM, [2] ¼ 63.3 mM).
We may compare the activity of 102DGA–GGGPGGG–
OBzl (10Pro, 1) with 102DGA–GGGPipGGG–OBzl (10Pip,
3). If the traces were superimposed, the ratio of activity would
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A second question is why no pore formation is observed for
4 when 2 shows reasonable kinetics at an identical concentra-
tion. Note that 18Pro (2) is significantly less active than is
10Pro (1). A more compelling question, given the design and
conceptualization of these compounds, is why more effective
transport is observed generally with shorter anchors (1, 3 are
more active than 2 or 4). We have experimentally confirmed
this relationship in separate work but the explanation remains
obscure.
into important factors in anion transport. The results reported
here support this hypothesis. The ‘‘SCMTR’’ family of mole-
cules has solubility behavior with dramatic structural sen-
sitivity and aggregation properties that are complex and
structurally dependent. The pore activation step clearly
depends upon the anchor chain length; pore size is altered dra-
matically by the subtle change from proline to pipecolate. We
now anticipate that this novel family of compounds will be
useful to explore in detail the chemistry and biophysics of pore
formation and anion transport in lipid bilayers.
Pore size for 18Pro, 2, vs. 10Pip, 3
Planar bilayer conductance studies permit us to assess the
effective size of the pore that is formed. This is typically given
in terms of conductance rather than diameter because ion slec-
tivity as well as pore size can determine the current. As shown
in the graph of Fig. 5, ‘‘C18’’ represents 182DGA–GGG-
PGGG–OBzl, 2, and ‘‘C10’’ corresponds to 102DGA–
GGGPipGGG–OBzl, 3. The size of the resulting pores was
determined to be the same when using CF (Fig. 2) so that
the increase in conductance by 10Pro probably results from
lowered ion selectivity. This pore passes both chloride and
potassium; since they are traveling in opposite directions in
response to the applied voltage, the resulting current is
summed. The graph shows identical chloride selectivity
(x-intercept), which indicates that the striking difference in
slope results from a change in pore size. Admittedly, these
compounds display large stability and activity differences in
pore activation. However, these are single pore experiments
in which thermodynamic and kinetic characteristics are only
indirectly reflected. We have previously observed that pore
sizes for 1 and 2 (C10Pro vs. C18Pro chains, proline derivative)
do not differ significantly.25 Thus, we did not expect ring size
per se to lead to a difference in pore size. The data shown in
Fig. 5 show that the pore sizes for 2 and 3 differ by nearly
4-fold. This is a remarkable difference considering that the pep-
tide chains of 2 and 3 differ in such a subtle way.
Experimental
General
All reaction solvents were freshly distilled and reactions were
conducted under N2 unless otherwise specified. Et3N was dis-
tilled from KOH and stored over KOH. CH2Cl2 was distilled
from CaH2 . Column chromatography was performed on silica
gel 60 (230–400 mesh). Thin layer chromatography was per-
formed with silica gel 60 F254 plates with visualization by
UV light (254 nm) and/or by phosphomolybdic acid (PMA)
spray. Starting materials were purchased from Aldrich Chemi-
1
cal Co., and used as received unless otherwise indicated. H-
NMR spectra were recorded at 300 MHz and are reported
in the following manner: chemical shifts are reported in ppm
downfield from internal tetramethylsilane (integrated intensity,
multiplicity (b ¼ broad; s ¼ singlet; d ¼ doublet; t ¼ triplet;
m ¼ multiplet, bs ¼ broad singlet, etc.), coupling constants
in Hertz, assignment). 13C-NMR spectra were obtained at 75
MHz and referenced to CDCl3 (d 77.0). Infrared spectra were
recorded in KBr unless otherwise noted and were calibrated
against the 1601 cmꢀ1 band of polystyrene. Melting points
were determined on a Thomas Hoover apparatus in open
capillaries and were uncorrected. Optical rotations were
recorded on a Perkin-Elmer Model 214 polarimeter.
N,N-Didecyl-3-oxaglutaramide (102DGA)
Conclusion
A solution of didecylamine (5.0 g, 16.8 mmol) and diglycolic
anhydride (2.2 g, 18.5 mmol) in THF (40 mL) was refluxed
for 48 h. The solvent was evaporated and the crude product
was dissolved in Et2O (150 mL) and washed with 10% aqueous
HCl (2 ꢅ 20 mL), dried (MgSO4) and crystallized from Et2O
to give a white solid (5.61 g, 88%), mp 51–52 ꢂC. 1H-NMR
CDCl3 : 0.87 (6H, t, J ¼ 6.9 Hz, –CH2CH3), 1.25 (28H,
pseudo-s, CH3(CH2)7CH2CH2N), 1.54 (4H, bs, CH3(CH2)7-
CH2CH2N), 3.07 (2H, t, J ¼ 7.8 Hz, CH3(CH2)7CH2CH2N),
3.34 (2H, t, J ¼ 7.8 Hz, CH3(CH2)7CH2CH2N), 4.21 (2H, s,
COCH2O), 4.38 (2H, s, COCH2O). 13C-NMR: 14.1, 22.7,
26.8, 26.9, 27.4, 28.6, 29.2, 29.3, 29.4, 29.5, 31.8, 46.8, 71.3,
73.2, 170.6, 171.7. IR (CHCl3): 3401, 3269, 2957, 2923, 2853,
Our study of these anchored heptapeptides was based upon the
premise that a synthetic, chemically simple, chloride transpor-
ter would be instructive for the study of anion transport across
bilayer lipid membranes. We hoped that defined changes in
transporter structure would alter activity and provide insight
1713, 1627, 1467, 1431, 1377, 1255, 1150, 1137, 1062, 923 cmꢀ1
.
.
TsOH GGG–OCH2Ph
H2N–GGG–OH (3.0 g, 15.9 mmol) and p-toluenesulfonic acid
(monohydrate, 3.6 g, 18.9 mmol) were added to a mixture of
benzyl alcohol (20 mL) and toluene (30 mL). The mixture
was heated to reflux and water was removed by using a
Dean–Stark trap. When no more water appeared in the distil-
late, heating was stopped. The mixture was cooled to room
temperature, diluted with ether (50 mL) and cooled in an ice
water bath for 2 h. Crystalline TsOHꢃH2N–GGG–Bzl was
collected on a filter, washed with ether (50 mL) and dried.
After recrystallization from methanol–ether (1:1), the salt (5.5 g,
77%) melted at 176–177 ꢂC. 1H-NMR CDCl3 : 2.34 (3H, s,
CH3Ph), 3.74 (2H, s, Gly NCH2), 3.97 (4H, s, Gly NCH2),
5.14 (2H, s, PHCH2O), 7.21 (2H, d, J ¼ 8.4 Hz, tosyl HAr),
7.30–7.35 (5H, m, Ph HAr), 7.69 (2H, d, J ¼ 8.4 Hz, tosyl
Fig. 5 Planar lipid bilayers were generated on a 0.25 mm orifice in a
plastic cuvette. When a bilayer was present, C18Pro or C10Pip was
added to the cis chamber at 40 or 9 mM respectively in a 450:150
mM KCl gradient (cis to trans). Currents were then determined as
the voltage across the bilayer was varied. The slope of the current–vol-
tage plot is the conductance as determined from the inverse of Ohm’s
law (1/R ¼ I/E). The x intercept is the reversal potential that is calcu-
lated by the Nernst equation to be ꢁ29 mV for a 450:150 KCl gradient
with a highly selective chloride channel. The slopes for the lines are
shown in picoSiemens (pS) and millivolts (mV).
64
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HAr). 13C-NMR: 21.4, 41.7, 42.1, 43.2, 68.1, 127.2, 129.5,
129.6, 129.9, 130.2, 137.5, 142.1, 143.7, 168.4, 171.4, 172.2.
IR (KBr): 3331, 3083, 1747, 1670, 1545, 1456, 1406, 1362,
Calcd for C23H32N4O7 : C, 57.97; H, 6.77; N, 11.76%. Found:
C, 57.87; H, 6.76; N, 11.39%.
1202, 1125, 1035, 1011, 913, 817, 736, 685 cmꢀ1
.
.
HCl H2N–PGGG–OCH2Ph
Boc–PGGG–OCH2Ph (0.2 g, 0.42 mmol) was dissolved in 4 M
HCl in dioxane (10 mL) at 5 ꢂC and the reaction mixture was
stirred for 1 h. The solvent was evaporated in vacuo and the
residue was crystallized from 1:1 MeOH/Et2O (0.18 g, 100%)
to give the title compound as a colorless solid (mp 145–
146 ꢂC). 1H-NMR (CD3OD): 2.00–2.25 (4H, m, Pro
NCH2CH2CH2), 3.35–3.45 (2H, m, Pro NCH2CH2CH2),
3.90–4.05 (6H, m, Gly NCH2), 4.30–4.40 (1H, m, Pro NCH),
5.18 (2H, s, PHCH2O), 7.30–7.40 (5H, m, HAr). 13C-NMR:
25.2, 30.9, 42.1, 43.3, 43.7, 47.6, 61.4, 68.1, 129.5, 129.6,
129.9, 137.5, 170.9, 171.4, 171.8, 172.4.
102DGA–GGG–OCH2Ph
To 102DGA (2 g, 4.8 mmol) dissolved in CH2Cl2 (30 mL), 1-
(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride
(1.0 g, 5.3 mmol) was added and the mixture was stirred at
room temperature. After 0.5 h, TsOHꢃGGG–OCH2Ph (2.2 g,
4.8 mmol) and Et3N (2.0 mL) were added and reaction was
stirred at ambient temperature for two days. The reaction mix-
ture was evaporated in vacuo, the residue successively washed
with 5% citric acid (20 mL), water (20 mL), 5% NaHCO3 (20
mL), and brine (20 mL), dried (MgSO4), and evaporated.
The crude, oily product was purified by chromatography
(SiO2 , 2% MeOH–CH2Cl2) and afforded a colorless oil (2.42
g, 76%). 1H-NMR CDCl3 : 0.87 (6H, t, J ¼ 6.9 Hz,
–CH2CH3), 1.25 (28H, pseudo-s, CH3(CH2)7CH2CH2N), 1.50
(4H, bs, CH3(CH2)8CH2CH2N), 3.05 (2H, t, J ¼ 7.5 Hz,
CH3(CH2)7CH2CH2N), 3.25 (2H, t, J ¼ 7.5 Hz, CH3(CH2)7-
CH2CH2N), 3.95–4.05 (6H, m, Gly NCH2), 4.10 (2H, s,
COCH2O), 4.30 (2H, s, COCH2O), 5.13 (2H, s, PHCH2O),
7.30–7.35 (5H, m, HAr), 7.96 (1H, t, J ¼ 5.7 Hz, Gly CONH),
8.27 (1H, t, J ¼ 5.7 Hz, Gly CONH). 13C-NMR: 13.9, 22.5,
26.7, 26.9, 27.4, 28.6, 29.2, 29.3, 31.8, 41.0, 42.9, 46.3, 46.7,
66.9, 69.6, 71.7, 128.2, 128.4, 128.6, 135.3, 168.6, 169.7,
169.8, 170.0, 171.5. IR (film): 3292, 2915, 2848, 1750, 1650,
102DGA–GGGPGGG–OCH2Ph, 1
To 102DGA–GGG–OH (0.18 g, 0.31 mmol) suspended in
CH2Cl2 (20 mL), 1-(3-dimethylaminopropyl)-3-ethylcarbodii-
mide hydrochloride (0.07 g, 0.34 mmol) was added and the
reaction was stirred for 0.5 h. Then HClꢃPGGG–OCH2Ph
(0.13 g, 0.31 mmol) in CH2Cl2 (10 mL) containing Et3N
(0.10 mL) was added and reaction mixture was stirred for 48
h at room temperature. Then solvent was evaporated and the
crude, oily product was purified by chromatography (SiO2 ,
10% MeOH–CH2Cl2) and afforded 1 as colorless crystals
(0.17 g, 60%), mp 127–128 ꢂC. [a]20 ꢀ11.4ꢂ (c 1.265, CHCl3).
D
1H-NMR CDCl3 : 0.87 (6H, t, J ¼ 6.9 Hz, –CH2CH3), 1.25
(28H, pseudo-s, CH3(CH2)7 CH2CH2N), 1.47 (4H, bs,
CH3(CH2)7CH2CH2N), 1.80–2.20 (4H, m, Pro NCH2-
CH2CH2), 3.03 (2H, t, J ¼ 7.5 Hz, CH3(CH2)16CH2N), 3.23
(2H, t, J ¼ 7.5 Hz, CH3(CH2)8CH2N), 3.40–3.45 (1H, m,
Pro NCH2CH2CH2), 3.50–3.55 (1H, m, Pro NCH2CH2CH2),
3.90–4.05 (12H, m, Gly NCH2), 4.12 (2H, s, COCH2O), 4.26
(2H, s, COCH2O), 4.35 (1H, bs, Pro NCH), 5.12 (2H, s,
PHCH2O), 7.33 (5H, s, HAr), 7.66 (1H, bs, Gly CONH),
7.76 (1H, br, Gly CONH), 7.85 (1H, br, Gly CONH), 8.06
(1H, bs, Gly CONH), 8.31 (1H, bs, Gly CONH), 8.47 (1H,
bs, Gly CONH). 13C-NMR: 13.9, 22.5, 24.9, 26.8, 26.9, 27.5,
28.6, 29.2, 29.5, 29.6, 31.8, 41.1, 41.8, 42.5, 42.6, 42.8, 43.1,
46.1, 46.7, 61.1, 67.0, 69.1, 70.9, 128.2, 128.4, 128.6, 135.4,
168.5, 169.0, 170.3, 170.5, 170.6, 170.9, 171.2, 173.7. IR
(KBr): 3300, 2921, 2852, 1741, 1652, 1541, 1457, 1241, 1131,
1028, 720 cmꢀ1. Anal. Calcd for C48H78N8O11 : C, 61.12; H,
8.34; N, 11.88%. Found: C, 60.87; H, 8.26; N, 11.65%.
1538, 1465, 1195, 1130, 1031, 721, 697 cmꢀ1
.
102DGA–GGG–OH
102DGA–GGG–OCH2Ph (2.3 g, 3.4 mmol) was dissolved in
absolute ethanol (40 mL) and 10% Pd/C (0.15 g) was added
and this mixture was shaken under 60 psi H2 for 3 h. The reac-
tion mixture was heated to reflux and filtered through a celite
pad. The solvent was evaporated under reduced pressure to
1
afford a white solid (1.92 g, 96%), mp 120–122 ꢂC. H-NMR
CD3OD: 0.90 (6H, t, J ¼ 6.9 Hz, –CH2CH3), 1.29 (32H,
pseudo-s, CH3(CH2)8CH2N), 1.57 (4H, bs, CH3(CH2)7-
CH2CH2N), 3.21 (2H, t, J ¼ 7.8 Hz, CH3(CH2)8CH2N), 3.35
(2H, t, J ¼ 7.8 Hz, CH3(CH2)7CH2N), 3.93 (2H, s, Gly
NCH2), 3.94 (2H, s, Gly NCH2), 3.97 (2H, s, Gly NCH2),
4.12 (2H, s, COCH2O), 4.40 (2H, s, COCH2O).13C-NMR:
IR (KBr): 3319, 3269, 3084, 2922, 2852, 1727, 1651, 1604,
1557, 1467, 1428, 1413, 1377, 1283, 1248, 1210, 1129, 1031,
955, 905, 722, 684 cmꢀ1
.
N,N-Dioctadecyl 3-oxaglutaramide (182DGA)
A solution of dioctadecylamine (2.0 g, 3.8 mmol) and diglyco-
lic anhydride (0.44 g, 3.8 mmol) in toluene (50 mL) was
refluxed for 48 h. The solvent was evaporated and the crude
product crystallized from CHCl3 to give a white solid (2.12
Boc–PGGG–OCH2Ph
Boc-L-Proline (1.43 g, 6.7 mmol), TsOHꢃGGG–O–CH2Ph (3.0
g, 6.7 mmol), and Et3N (2.80 mL) were dissolved in CH2Cl2
(40 mL) and cooled to 5 ꢂC. EDCI (1.34 g, 7 mmol) was added
and reaction was stirred at ambient temperature for 3 days.
Solvent was evaporated and residue was dissolved in EtOAc
(50 mL) and washed with saturated aqueous NH4Cl (25 mL)
and brine (25 mL), dried over MgSO4 and evaporated. The
crude, oily product was purified by chromatography (SiO2 ,
5% MeOH–CH2Cl2) and afforded colorless crystals (2.25 g,
1
g, 87%), mp 80–81 ꢂC. H-NMR CDCl3 : 0.87 (6H, t, J ¼ 6.9
Hz, –CH2CH3), 1.25 (60H, pseudo-s, CH3(CH2)15CH2CH2N),
1.55 (4H, bs, CH3(CH2)15CH2CH2N), 3.07 (2H, t, J ¼ 7.8 Hz,
CH3(CH2)16CH2N), 3.34 (2H, t, J ¼ 7.8 Hz, CH3(CH2)16-
CH2N), 4.21 (2H, s, COCH2O), 4.38 (2H, s, COCH2O). 13C-
NMR: 14.2, 22.8, 26.9, 27.0, 27.5, 28.7, 29.4, 29.5, 29.6, 29.7,
29.8, 32.0, 47.0, 71.4, 73.2, 171.0, 172.2. IR (KBr): 2918,
2850, 1748, 1602, 1488, 1472, 1463, 1431, 1356, 1224, 1159,
1
71%, mp 54–55 ꢂC). H-NMR CDCl3 : 1.42 (9H, s, C(CH3)3),
1.80–2.20 (4H, m, Pro NCH2CH2CH2), 3.35–3.55 (2H, m,
Pro NCH2CH2CH2), 3.85–4.20 (7H, m, Gly NCH2 , Pro
NCH), 5.15 (2H, s, PHCH2O), 7.05 (2H, bs, Gly CONH),
7.30–7.35 (5H, m, HAr), 7.80 (1H, bs, Gly CONH). 13C-
NMR: 24.6, 28.3, 29.4, 41.1, 43.0, 43.3, 47.2, 60.7, 66.9, 80.9,
128.4, 128.5, 128.7, 135.4, 155.8, 169.6, 170.0, 173.9. IR
(KBr): 3310, 3066, 2976, 2933, 1753, 1667, 1540, 1455, 1408,
1366, 1245, 1174, 1129, 1031, 974, 912, 773, 739, 698. Anal.
1135, 1045, 1013, 990, 920, 885, 729, 720, 689, 643 cmꢀ1
.
182DGA–GGG–OCH2Ph
To 182DGA–OH (1g, 1.5 mmol) dissolved in CH2Cl2 (30 mL),
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochlor-
ide (0.31 g, 1.6 mmol) was added and the mixture was stirred
at room temperature. After 0.5 h, TsOHꢃH2N–GGG–OCH2Ph
New J. Chem., 2003, 27, 60–67
65

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(0.66 g, 1.5 mmol) and Et3N (0.6 mL) were added and the mix-
ture was stirred at ambient temperature overnight. The reac-
tion mixture was successively washed with water (20 mL),
0.5 M HCl (20 mL), water (20 mL), 10% Na2CO3 (20 mL),
and brine (20 mL), dried (MgSO4), evaporated and the residue
crystallized from MeOH to afford a white solid (1.26 g, 89%),
mp 41–42 ꢂC. 1H-NMR CDCl3 : 0.86 (6H, t, J ¼ 6.9 Hz,
–CH2CH3), 1.24 (60H, pseudo-s, CH3(CH2)15CH2CH2N),
1.49 (4H, bs, CH3(CH2)15CH2CH2N), 1.61 (1H, H2O), 3.04
(2H, t, J ¼ 7.5 Hz, CH3(CH2)16CH2N), 3.24 (2H, t, J ¼ 7.5
Hz, CH3(CH2)16CH2N), 3.95–4.05 (6H, m, Gly NCH2), 4.09
(2H, s, COCH2O), 4.29 (2H, s, COCH2O), 5.12 (2H, s,
PHCH2O), 7.23 (1H, t, J ¼ 6.0 Hz, Gly CONH), 7.30–7.35
(5H, m, HAr), 7.93 (1H, t, J ¼ 5.7 Hz, Gly CONH), 8.27 (1H,
t, J ¼ 5.7 Hz, Gly CONH). 13C-NMR: 13.9, 22.5, 26.7, 26.9,
27.4, 28.6, 29.2, 29.3, 29.6, 31.8, 41.0, 42.9, 46.3, 46.7, 66.9,
69.6, 71.7, 128.2, 128.4, 128.6, 135.3, 168.6, 169.7, 169.8,
170.0, 171.5. IR (KBr): 3293, 2916, 2849, 1749, 1651, 1544,
1467, 1196, 1128, 1031, 721, 697 cmꢀ1. Anal. Calcd for
C53H94N4O7 + 0.5 H2O: C, 70.11; H, 10.54; N, 6.17%. Found:
C, 70.18; H, 10.55; N, 6.18%.
C64H110N8O11 : C, 65.83; H, 9.50; N, 9.60%. Found: C,
65.38; H, 9.68; N, 9.16%.
Boc–PipGGG–Bzl
Boc-L-Pipecolic acid (0.50 g, 2.2 mmol), TsOHꢃGGG–O–
CH2Ph (0.98 g, 2.2 mmol), and Et3N (1.50 mL) were dissolved
in CH2Cl2 (20 mL) and cooled to 5 ꢂC. EDCI (0.46 g, 2.4
mmol) was added and the reaction was stirred at ambient tem-
perature for 3 days. Solvent was evaporated and the residue
was dissolved in EtOAc (50 mL), washed with citric acid
(5%, 25 mL), Na2CO3 (25 mL), brine (25 mL), dried over
MgSO4 , and evaporated. The crude, oily product was purified
by chromatography (SiO2 , 5% MeOH–CH2Cl2) to afford col-
orless crystals (0.97 g, 91%), mp 56–58 ꢂC. [a]20 ꢀ36.1ꢂ (c
D
1
1.045, CHCl3). H-NMR CDCl3 : 1.45 (9H, s, C(CH3)3), 1.59
(4H, bs, Pip CH2), 2.15 (2H, bs, Pip CH2), 3.00 (1H, bs, Pip
CH2), 3.85–4.15 (7H, overlapping signals due to Gly CH2
and Pip CH2), 4.69 (1H, s, Pip CH), 5.15 (2H, s, CH2Ph),
6.96 (1H, bs, NH), 7.10 (1H, bs, NH), 7.30–7.50 (5H, m,
HAr). 13C-NMR: 20.4, 24.7, 25.8, 28.3, 41.3, 42.6, 42.9, 43.2,
55.1, 67.2, 80.8, 128.3, 128.5, 128.6, 135.2, 156.1, 169.1,
169.6, 172.6. IR (KBr): 3325, 2938, 1752, 1664, 1530, 1457,
1409, 1366, 1253, 1189, 1164, 1032, 989, 870, 751, 699 cmꢀ1
.
182DGA–GGG–OH
.
H2N–PipGGG–OCH2Ph HCl
182DGA–GGG–OCH2Ph (1.0 g, 1.1 mmol) was dissolved in
absolute ethanol (100 mL) and 10% Pd/C (0.2 g) was added
and this mixture was shaken under 60 psi H2 for 3 h. The reac-
tion mixture was heated to reflux and filtered hot through a
celite pad. The solvent was evaporated under reduced pressure
to afford a white solid (0.86 g, 96%), mp 163–164 ꢂC. 1H-NMR
CD3OD: 0.90 (6H, t, J ¼ 6.9 Hz, –CH2CH3), 1.29 (60H,
pseudo-s, CH3(CH2)15CH2CH2N), 1.57 (4H, bs, CH3(CH2)15-
CH2CH2N), 3.21 (2H, t, J ¼ 7.8 Hz, CH3(CH2)16CH2N), 3.35
(2H, t, J ¼ 7.8 Hz, CH3(CH2)16CH2N), 3.93 (2H, s, Gly
NCH2), 3.94 (2H, s, Gly NCH2), 3.97 (2H, s, Gly NCH2),
4.12 (2H, s, COCH2O), 4.40 (2H, s, COCH2O). 13C-NMR:
IR (KBr): 3285, 3084, 2925, 2852, 1740, 1650, 1551, 1467,
1420, 1378, 1219, 1128, 1033, 1011, 721, 681 cmꢀ1. Anal. Calcd
for C46H88N4O7 : C, 68.28; H, 10.96; N, 6.92%. Found: C,
67.97; H, 10.92; N, 6.81%.
Boc–PipGGG–OCH2Ph (0.2 g, 0.25 mmol) was dissolved in 4
M HCl in dioxane (10 mL) at 5 ꢂC and the reaction mixture
was stirred for 1 h. The solvent was evaporated in vacuo and
the residue was used in the next reaction without further
purification.
102DGA–GGGPipGGG–OCH2Ph, 3
To 102DGA–GGG–OH (0.36 g, 0.61 mmol) suspended in
CH2Cl2 (20 mL), 1-(3-dimethylaminopropyl)-3-ethylcarbodi-
imide hydrochloride (0.12 g, 0.64 mmol) and HOBt (0.09 g,
0.64 mmol) were added and the reaction mixture was stirred
for 0.5 h. Then HClꢃPipGGG–OCH2Ph (0.29 g, 0.61 mmol)
in CH2Cl2 (20 mL) containing Et3N (0.09 mL, 0.64 mmol)
was added and the reaction mixture was stirred for 48 h at
room temperature. The reaction mixture was evaporated
in vacuo, the residue successively washed with 5% citric acid (20
mL), water (20 mL), 5% NaHCO3 (20 mL), and brine (20
mL), dried (MgSO4), and evaporated. The crude, oily product
was purified by chromatography (SiO2 , 5% MeOH–CH2Cl2)
182DGA–GGGPGGG–OCH2Ph, 2
182DGA–GGG–OH (0.31 g, 0.39 mmol) and 1-(3-dimethyla-
minopropyl)-3-ethylcarbodiimide hydrochloride (0.08 g, 0.42
mmol) were stirred for 0.5 h in CH2Cl2 (40 mL) and
HClꢃH2N–PGGG–OCH2Ph (0.16 g, 0.39 mmol) in CH2Cl2
(10 mL) containing Et3N (0.15 mL) was added. The mixture
was stirred for 48 h at room temperature, solvent was evapo-
rated, and the residue was crystallized from MeOH to give 2
and afforded 3 as a white solid (0.49 g, 84%), mp 78–80 ꢂC.
[a]20 ꢀ22.5 (c 1.065, CHCl3). H-NMR CDCl3 : 0.87 (6H, t,
1
D
J ¼ 6.9 Hz, –CH2CH3), 1.25 (28H, pseudo-s, CH3(CH2)7-
CH2CH2N), 1.50 (8H, bs, overlapping signals due to
CH3(CH2)7CH2CH2N and Pip CH2), 2.13 (2H, bs, Pip CH2),
2.85 (1H, bs, Pip CH2) 3.05 (2H, t, J ¼ 7.5 Hz, CH3-
(CH2)7CH2CH2N), 3.25 (2H, t, J ¼ 7.5 Hz, CH3(CH2)7-
CH2CH2N), 3.55–3.65 (1H, m, Pip CH2), 3.80–4.15 (12H, m,
Gly NCH2), 4.10 (2H, s, COCH2O), 4.27 (2H, s, COCH2O),
5.06 (1H, bs, Pip CH), 5.14 (2H, s, PHCH2O), 7.34 (5H,
pseudo s, HAr), 7.49 (1H, bs, Gly CONH), 7.55 (2H, bs, Gly
CONH), 7.85 (1H, bs, Gly CONH), 7.98 (1H, bs, Gly CONH),
8.29 (1H, bs, Gly CONH). 13C-NMR: 14.0, 20.2, 22.6, 24.9,
25.7, 26.8, 27.0, 27.6, 28.8, 29.2, 29.3, 29.4, 29.5, 31.8, 41.2,
41.4, 42.7, 43.0, 43.3, 46.2, 46.8, 53.4, 67.0, 69.2, 71.2, 128.1,
128.3, 128.5, 135.3, 168.3, 169.1, 170.0, 170.1, 170.2 170.4,
170.8, 171.9. IR (CHCl3): 3309, 3069, 2926, 2854, 1748,
1651, 1540, 1457, 1259, 1193, 1129, 1029 cmꢀ1. Anal. Calcd
for C49H80N8O11 : C, 61.48; H, 8.42; N, 11.71%. Found: C,
61.12; H, 8.35; N, 11.43%.
as a white solid (0.37 g, 82%), mp 116–118 ꢂC. [a]20 ꢀ8.9 (c
D
1
1.055, CHCl3). H-NMR CDCl3 : 0.87 (6H, t, J ¼ 6.9 Hz, –
CH2CH3), 1.25 (60H, pseudo-s, CH3(CH2)15CH2CH2N), 1.47
(4H, bs, CH3(CH2)15CH2CH2N), 1.80–2.20 (4H, m, Pro
NCH2CH2CH2), 3.03 (2H, t, J ¼ 7.5 Hz, CH3(CH2)16CH2N),
3.23 (2H, t, J ¼ 7.5 Hz, CH3(CH2)16CH2N), 3.40–3.45 (1H, m,
Pro NCH2CH2CH2), 3.50–3.55 (1H, m, Pro NCH2CH2CH2),
3.90–4.05 (12H, m, Gly NCH2), 4.12 (2H, s, COCH2O), 4.26
(2H, s, COCH2O), 4.35 (1H, bs, Pro NCH), 5.12 (2H, s,
PHCH2O), 7.33 (5H, s, HAr), 7.66 (1H, bs, Gly CONH),
7.76 (1H, br, Gly CONH), 7.85 (1H, br, Gly CONH), 8.06
(1H, bs, Gly CONH), 8.31 (1H, bs, Gly CONH), 8.47 (1H,
bs, Gly CONH). 13C-NMR: 13.9, 22.5, 24.9, 26.8, 26.9, 27.5,
28.6, 29.2, 29.5, 29.6, 31.8, 41.1, 41.8, 42.5, 42.6, 42.8, 43.1,
46.1, 46.7, 61.1, 67.0, 69.1, 70.9, 128.2, 128.4, 128.6, 135.4,
168.5, 169.0, 170.3, 170.5, 170.6, 170.9, 171.2, 173.7. IR
(KBr): 3301, 2922, 2853, 1740, 1653, 1540, 1457, 1242, 1131,
1029, 720. MS (ESI): m/z [M + Na]+ calculated for
C64H110N8O11Na 1189, observed 1190. Anal. Cald for
182DGA–GGGPipGGG–OCH2Ph, 4
182DGA–18–GGG–OH (0.20 g, 0.25 mmol), 1-(3-dimethyl-
aminopropyl)-3-ethylcarbodiimide hydrochloride (0.05 g,
66
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8
9
J. M. Tomich, D. Wallace, K. Henderson, K. E. Mitchell, G.
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D. P. Wallace, J. M. Tomich, J. W. Eppler, T. Iwamoto, J. J.
Grantham and L. P. Sullivan, Biochim. Biophys. Acta, 2000,
1464, 69–82.
0.28 mmol) and HOBt (0.04 g, 0.28 mmol) were suspended in
CH2Cl2 (20 mL) and stirred for 0.5 h. HClꢃH2N–PipGGG–
OCH2Ph (0.12 g, 0.25 mmol) in CH2Cl2 (5 mL) containing
N-methylmorpholine (0.05 mL, 0.46 mmol) was added and
the mixture was stirred for 48 h at room temperature. The sol-
vent was evaporated off and the residue was crystallized from
10 J. R. Broughman, K. E. Mitchell, R. L. Sedlacek, T. Iwamoto,
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C451–C458.
11 P. H. Schlesinger, R. Ferdani, J. Liu, J. Pajewska, R. Pajewski,
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2002, 124, 1848–1849.
12 P. H. Schlesinger, R. Ferdani, R. Pajewski, J. Pajewska and G. W.
Gokel, Chem. Commun., 2002, 840–841.
13 C. Fahlke, H. T. Yu, C. L. Beck, T. H. Rhodes and A. L. George,
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MeOH to give 4 as a white solid (0.24 g, 82%), mp 164–165 ꢂC.
1
[a]20 ꢀ15.1ꢂ (c 1.10, CHCl3). H-NMR CDCl3 : 0.87 (6H, t,
D
J ¼ 6.9 Hz, –CH2CH3), 1.25 (60H, pseudo-s, CH3(CH2)15-
CH2CH2N), 1.50 (8H, bs, overlapping signals due to
CH3(CH2)15CH2CH2N and Pip CH2), 2.13 (2H, bs, Pip
CH2), 2.85 (1H, bs, Pip CH2) 3.05 (2H, t, J ¼ 7.5 Hz,
CH3(CH2)16CH2N), 3.25 (2H, t, J ¼ 7.5 Hz, CH3(CH2)16-
CH2N), 3.55–3.65 (1H, m, Pip CH2), 3.80–4.15 (12H, m, Gly
NCH2), 4.10 (2H, s, COCH2O), 4.27 (2H, s, COCH2O), 5.04
(1H, bs, Pip CH), 5.14 (2H, s, PHCH2O), 7.34 (5H, pseudo
s, HAr), 7.49 (1H, bs, Gly CONH), 7.55 (2H, bs, Gly CONH),
7.85 (1H, bs, Gly CONH), 7.98 (1H, bs, Gly CONH), 8.29
(1H, bs, Gly CONH). 13C-NMR: 14.1, 20.1, 22.7, 24.8, 25.8,
26.9, 27.1, 27.6, 29.3, 29.4, 29.6, 29.7, 31.9, 41.2, 41.4, 42.8,
43.0, 43.3, 46.3, 46.9, 50.7, 53.6, 67.0, 69.4, 71.3, 128.2,
128.4, 128.6, 135.4, 168.4, 169.4, 169.9, 170.0, 170.1, 170.3,
170.4, 170.9, 172.0. IR (KBr): 3315, 2921, 2851, 2359, 1749,
1656, 1541, 1468, 1262, 1130, 1029, 722, 698 cmꢀ1. Anal. Calcd
for C65H112N8O11 : C, 66.07; H, 9.55; N, 9.48%. Found: C,
66.34; H, 9.63; N, 9.32%.
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`
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