BUER, LEVIN AND MARSH
1
charged residues in the AMP and the negatively charged lipid
head groups that predominate in bacterial membranes initiate
AMP binding. Once bound, AMPs may disrupt the lipid bilayer
by various mechanisms including the formation of toroidal pores,
as is the case for MSI-78. MSI-78 undergoes a conformational
change from unstructured to a-helical upon lipid binding. We
have previously used MSI-78 labeled with trifluoroethylglycine
H NMR (400 MHz, CD OD): d 4.30–4.23 (dd, J= 10.0, 4.4Hz, 1H),
3
4.20–4.12 (2H) 3.71 (s, 3H), 2.27–1.90 (dm, J= 107.6 Hz, 2H), 1.41 (s,
9H). F NMR (376 MHz, CD
19
3
OD): d 71.69 (s). See Figure S3 for spectra.
(S)-2-amino-4-((1,1,1,3,3,3-hexafluoro-2-(trifluoromethyl)propan-2-yl)
oxy)butanoic acid (pFtBSer, 4)
LiOH (250 mg, 10.5 mmol) was slowly added to 3 (1.83 g,
4
stirred for 12 h at room temperature. Upon completion, the
mixture was cooled to 0 C and acidified to pH 2 with 1 M HCl.
THF was removed by rotary evaporation, and the resultant
mixture was extracted with ethyl acetate (4 Â 25 ml). The ethyl
acetate layers were combined and washed with sat. aq. NaCl
(tFeG) [4,16] to study this interaction. These earlier studies
2
.2 mmol) dissolved in 40 ml THF and 20 ml H O. The reaction
provide a benchmark by which we can compare the signal
intensity and lipid binding induced chemical shift changes of
the pFtBSer-containing MSI-78 analogs.
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Material and Methods
2 4
solution (40 ml), dried over Na SO , filtered, and concentrated.
The concentrate of Boc-L-perfluoro-t-butyl-homoserine was
Synthesis of pFtBSer
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dissolved in 20 ml of CH
2
Cl
2
and cooled to 0 C before the addi-
(S)-2-((tert-butoxycarbonyl)amino)-4-hydroxybutanoic acid (1)
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tion of 2.8 ml TFA. Reaction proceeded at 0 C for 4 h, and solvent
was removed by rotary evaporation followed by three additions of
10 ml toluene and rotary evaporation to remove toluene and
L-homoserine (5.0 g, 42 mmol) was dissolved in 50 ml THF and
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7
1
5 ml H
2
O then cooled to 0 C. Sodium bicarbonate (10.58 g,
residual TFA. No further purification was needed to give 4 as
26 mmol) was added slowly to the stirring mixture followed
1
an off-white solid (1.07 g, 76% yield). H NMR (400 MHz, D
2
O):
by addition of di-t-butyl dicarbonate (13.75 g, 63 mmol). The reac-
tion was allowed to warm to room temperature, and progress
followed via TLC. Upon completion, the mixture was cooled
d 4.23–4.12 (dm, J = 7.2 Hz, 2H), 3.76–3.70 (t, J = 6.4 Hz, 1H),
19
2
2
.25–2.04 (dm, J = 30.8 Hz, 2H). F NMR (376MHz, D O): d 70.61
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(s). See Figure S4 for the spectra.
to 0 C and acidified to pH 2 with 1 M HCl. THF was removed by
rotary evaporation, and the resultant mixture was extracted with
ethyl acetate (4 Â 80 ml). The ethyl acetate layers were combined
and washed with sat. aq. NaCl solution (100 ml), dried over
Peptide Synthesis
Fmoc-protected SPPS on Rink Amide MBHA resin was used to
produce all three peptides as described previously. Resin cleavage
with TFA/triisopropylsilane/water (95 : 2.5: 2.5) yielded the desired
product with no cleavage of the perfluoro-t-butyl group observed.
All peptides were purified by reverse phase HPLC using a linear
gradient of 95% water, 4.9% acetonitrile and 0.1% TFA for solvent
A and 9.9% water, 90% acetonitrile and 0.1% TFA for solvent B, with
a flow rate of 10 ml/min on a Waters C18 preparatory column. After
lyophilization, peptides were dissolved in water to 20mg/ml, and
all residual TFA was removed by passing this solution through a
Stratosphere SPE column (Varian), which was conditioned with
Na
8
4
2
SO
4% yield). H NMR (400 MHz, CD
.8 Hz, 1H), 3.61–3.53 (m, 2H), 1.69–2.05 (dm, J = 96 Hz, 2H), 1.34
4
, filtered and concentrated to give 1 as a clear oil (7.6 g,
1
3
OD): d 4.02–3.92 (dd, J = 7.2,
(s, 9H). See Figure S1 for spectrum.
(S)-methyl 2-((tert-butoxycarbonyl)amino)-4-hydroxybutanoate (2)
Generation of diazomethane required the use of appropriate
glassware, free of scratches or ground glass. KOH (5.0 g) was
dissolved in 8 ml H O and 10 ml ethanol in a round bottom flask
2
equipped with a Teflon stir bar. To this, Diazald (5.86 g) dissolved
in 75 ml diethyl ether was added. This flask was slowly warmed
5
ml 50 : 50 water/MeOH. The column was then rinsed with 4 ml
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from room temperature to 50 C as ethereal diazomethane
methanol, and ten equivalents of formic acid were added to the
peptide solution before lyophilization. F NMR spectra confirmed
the complete removal of residual TFA; stock peptide concentrations
were determined using F NMR with a known concentration of
TFA as an internal reference. Peptide identities were confirmed
using MALDI-MS with a matrix of a-cyano-4-hydroxycinnamic acid.
was directly distilled into the reaction flask containing 1 (2.0 g,
19
9
.18 mmol) dissolved in 25 ml ethyl acetate. After distillation
of diazomethane was complete, the reaction flask was gently
19
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heated at 40 C followed by rotary evaporation to concentrate
1
giving 2 as a light yellow solid (2.0 g, 93% yield). H NMR
(
(
(
3
400 MHz, CD OD): d 4.26–4.20 (dd, J = 8.8, 4.4 Hz, 1H), 3.67
s, 3H), 3.62–3.52 (m, 2H), 2.00–1.72 (dm, J = 71.6 Hz, 2H), 1.40
s, 9H). See Figure S2 for spectrum.
Lipid Preparation
(
DMPC), 1,2-dimyristoyl-sn-glycero-3-phospho-(1’-rac-glycerol) (DMPG)
(
S)-methyl 2-((tert-butoxycarbonyl)amino)-4-((1,1,1,3,3,3-hexafluoro-2-
and 1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC) were
purchased from Avanti Polar Lipids (Avanti Polar Lipids Inc., Alabaster,
Alabama, USA). Isotropic bicelles were made in PBS buffer, pH 7.4 with
(trifluoromethyl)propan-2-yl)oxy)butanoate (3)
Ph P (3.38 g, 12.87 mmol) was added to 2 (2.0 g, 8.58 mmol)
3
dissolved in 60 ml dry THF and stirring in a three-neck round
10% D O by adding a solution of 3 : 1 DMPC/DMPG to a solution of DHPC
2
bottom flask fitted with a condenser. The solution was cooled
giving q= 0.5 resulting in a clear, non-viscous solution.
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to 0 C under N , and DIAD (2.6 g, 2.53 ml) was added dropwise
2
over 10 min. After stirring for an additional 10 min, perfluoro-t-
Circular Dichroism
butanol was added dropwise, and the reaction was heated at
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5
0 C for 20 h. Upon completion, solvent was removed by rotary
To examine the secondary structure, we recorded CD spectra of pep-
tides with an Aviv 62DS (Aviv Biomedical Inc., Lakewood, New Jersey,
USA) spectropolarimeter at 25 C. Samples typically contained 50 mM
peptide in buffered solution in either the presence or absence of
50 mM SDS micelles.
evaporation, and mixture was directly purified using silica column
chromatography with a gradient of 0% to 10% ethyl acetate in
hexanes; fractions were combined and solvent removed using
rotary evaporation giving 3 as a waxy solid (1.83 g, 49% yield).
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Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.
J. Pept. Sci. 2013