b-Hairpin Peptides
FULL PAPER
produced a residue which was washed with several volumes of ether, and
was then carefully decanted.The remaining residue was taken up into
ethyl acetate (50 mL) and washed with water (210 mL).The resulting
organic solution was dried with MgSO4 and evaporated to dryness, result-
ing in a clear oil.Compound 4 was purified by means of silica-gel chro-
matography (column flushed with 100 mL CH2Cl2; product eluted with
CH2Cl2/MeOH 10:1) to give a white solid (0.200 g, 0.362 mmol, 72%
yield for two steps). 1H NMR (600 MHz, CDCl3): d = 7.954 (d, 1H),
7.748 (d, 2H), 7.652 (m, 2H), 7.58 (m, 3H), 7.384 (dd, 2H), 7.299 (dd,
2H), 5.325 (d, 1H), 4.405 (s, 2H), 4.208 (t, 1H), 3.320 (m, 1H), 3.238 (m,
1H), 2.862 (s, 3H), 1.96 (m, 1H), 1.70 ppm (m, 3H); ESIMS: m/z: calcd
for: 553.15; found: 553.2.
(t, 1H), 3.160 (m, 1H), 3.045 (m, 1H), 2.739 (s, 6H), 2.16–2.28 ppm (m,
2H); ESIMS: m/z: calcd for: 368.17; found: 368.2.
Trimethylated lysine, ornithine, and diaminobutyric acid: Trimethylated
amino acids were synthesized by following the procedure of Kretsinger
and Schneider.[11] The peptides were synthesized by using Fmoc-
Lys(Me)2-OH purchased from either Anaspec, Bachem, Fmoc-Orn(Me)2-
OH or Fmoc-Dab(Me)2-OH synthesized by methods described above.
The dimethylated amino acid-containing peptides (0.100 mmol scale)
were reacted prior to cleavage from the resin with MTBD (18 mL,
0.125 mmol) and methyl iodide (62 mL, 1 mmol) in DMF (5 mL) for 4 h
with bubbling N2 in a peptide synthesis flask stoppered with a vented
septum.After washing the resin with DMF (3), CH 2Cl2 (3), and
drying, the peptide was cleaved with a cocktail of 90% TFA/5% Triiso-
propylsilane/5% H2O for 3 h.The peptide was then purified by standard
HPLC methods.
An identical procedure was followed for the synthesis of Fmoc-monome-
thyldiaminobutyric acid (oNBS), by using Boc-diaminobutyric acid
(0.358 g, 1.64 mmol). The product was isolated as a clear oil (0.180 g,
0.334 mmol). 1H NMR (600 MHz, CDCl3): d = 7.928 (d, 1H), 7.739 (d,
2H), 7.655 (m, 2H), 7.57 (m, 3H), 7.373 (dd, 2H), 7.30 (dd, 2H), 5.478
(d, 1H), 4.463 (s, 2H), 4.234 (t, 1H), 3.334 (m, 1H), 3.213 (m, 1H), 2.885
(s, 3H), 2.195 (m, 1H), 1.855 ppm (m, 1H); ESIMS: m/z: calcd for:
539.14; found: 539.2.
Peptide synthesis and purification: Peptide synthesis was performed on
an Applied Biosystems Pioneer peptide synthesizer by using standard
FMOC solid-phase peptide synthesis methodology.Non-commercially
available amino acids were in some cases coupled by hand.Peptides
were purified with reverse-phase HPLC, lyophilized, and characterized
by MALDI or ESI-TOF mass and NMR spectroscopy.
Fmoc-dimethyl ornithine and Fmoc-dimethyl diaminobutyric acid (6a):
Boc-ornithine 1a (0.175 g; 0.75 mmol) was taken up in MeOH (10 mL) in
a 100 mL round-bottomed flask with stirring.Formaldehyde (340 mL,
37%, 11 mmol) was added to the solution, which was then allowed to stir
for 5 min.The flask was flushed with N 2, followed by the addition of
0.33 g of 10% Pd/C. Next, the flask was plugged with a rubber septum
NMR spectroscopy: NMR samples were made in concentrations of ap-
proximately 1 mm and analyzed on a Varian Inova 600 MHz spectrome-
ter.Samples were dissolved in D 2O buffered to pD 4.0 (uncorrected)
with 50 mm [D3]NaOAc, unless otherwise noted.Amine and amide reso-
nances were assigned in 60% H2O solutions. 1D NMR spectra were col-
lected by using 32 K data points and between 8 and 64 scans by using a
1–3 s presaturation.All 2D NMR experiments used pulse sequences from
the Chempack software including TOCSY, DQCOSY, gCOSY, and
NOESY. 2D NMR scans were taken with 16–64 scans in the first dimen-
sion and 64–256 scans in the 2nd dimension.All spectra were analyzed
by using standard window functions (Sinebell and Gaussian).Mixing
times of 0.5 or 0.6 s were used in the NOESY spectra. Assignments were
made by using standard methods as described by Wüthrich.[27] Tempera-
ture calibrations were made by using MeOH and ethylene glycol stand-
ards.
and flushed with H2 gas.A balloon of H was attached, and the reaction
2
stirred for 24 h.The contents of the flask were then filtered to remove
the catalyst and the solvent removed in vacuo to give compound 5a.The
reaction was monitored by TLC (CH2Cl2/MeOH 5:1) with ninhydrin
staining.Reaction completion was confirmed with RP-HPLC (C18
column; gradient of 5–60% CH3CN in water over 30 min). 1H NMR
(600 MHz, CDCl3): d = 5.66 (s, 1H), 4.04 (s, 1H), 2.94 (m, 1H), 2.76 (m,
1H), 2.66 (s, 6H), 1.72–1.81 (m, 3H), 1.52–1.60 (m, 1H), 1.38 ppm (s,
9H); ESIMS: m/z: calcd for: 260.17; found: 260.2. Compound 5a was
taken up into a solution of trifluoroacetic acid (5 mL) and triisopropylsi-
lane (250 mL) in CH2Cl2 (15 mL) and stirred for 30 min to remove the
Boc group.After removing the solvent in vacuo, the remaining residue
was triturated with ether, resulting in an oily precipitate.The material
was extracted into water (10 mL), frozen, and lyophilized to dryness.The
resulting oil was then dissolved in water (10 mL) and added to a 100 mL
pH studies: Three buffer solutions in D2O were used to analyze the sensi-
tivity of peptide stability to changes in pH: pH 7.4 (10 mm sodium phos-
phate buffer), pH 4 (50 mm sodium acetate buffer), and pH 1.10 (phos-
phoric acid buffer).pH values uncorrected for deuterium isotope effects.
NMR structure calculation and MD simulation: NOEs were classified as
strong, medium, or weak by visual inspection.Accordingly, upper bounds
for distance restraints were set at 5.0, 3.5, or 2.5 .[28] NMR structures
were calculated by using a simulated annealing protocol within the pro-
gram CNS Solve.[25] Hydrogen bonds were enforced with upper limits of
2.0 and assigned based on backbone amide shifts (see the Supporting
Information).In the calculation of WKL, 23 nonsequential NOEs were
used and 34 nonsequential NOEs were used in the calculation of the
structure of WKMe3L.All available backbone amide, H a, and side-chain
1H chemical shifts were also employed in the calculations by a harmonic
potential with a primary chemical shift force value of 10 (61 observed
chemical shifts; random coil values taken from 7 mers).Two rounds of si-
mulated annealing were employed in each calculation.In the first, 200
structures were generated from an extended starting structure.In the
second, 50 structures were generated starting from an averaged folded
structure taken from the initial run.The best structures were selected
from the second run based on total energy and visual inspection and
averaged.The average structures were subjected to an additional uncon-
strained conjugate gradient minimization.
round-bottomed flask equipped with
a stirbar.Sodium carbonate
(2.3 mmol, 0.240 g) was added to the solution, followed by dioxane
(5 mL).The resulting solution was stirred for 10 min at 0 8C.Fmoc-OSu
(0.825 mmol; 0.278 g) was dissolved in dioxane (5 mL) and added drop-
wise to the ice-cold solution over 10 min.The resulting mixture was
stirred for 1 h at 08C and 16 h at room temperature.The reaction was
followed by TLC (CH2Cl2/MeOH 10:1, ninhydrin staining).The mixture
was then washed with ether (225 mL) and acidified with HCl (1n) to
pH 3.The acidic solution was extracted with ethyl acetate (450 mL),
dried with sodium sulfate, and the solvent was removed in vacuo to give
0.252 g (0.659 mmol, 88% yield for two steps) of compound 6a as an
amorphous white solid. 1H NMR (600 MHz, CDCl3): d = 7.710 (d, 2H),
7.559 (dd, 2H), 7.346 (dd, 2H), 7.252 (dd, 2H), 6.149 (d, 1H), 4.304 (m,
2H), 4.150 (t, 1H), 3.111 (m, 1H), 2.984 (m, 1H), 2.746 (s, 6H), 1.90–
1.96 (m, 1H), 1.77–1.84 ppm (m, 3H); ESIMS: m/z: calcd for: 382.19;
found: 382.3.
An identical procedure was followed for the synthesis of Fmoc-dimethyl-
diaminobutyric acid, by using Boc-diaminobutyric acid (0.164 g;
0.75 mmol), 30% formaldehyde (340 mL), Pd/C (0.033 g) in MeOH
(10 mL), and H2O (1 mL). 1H NMR (600 MHz, CDCl3): d = 5.85 (s,
1H), 3.95 (s, 1H), 3.11 (m, 1H), 2.96 (m, 1H), 2.68 (s, 6H), 2.05–2.19 (m,
2H), 1.40 ppm (s, 9H). ESIMS: calcd: 246.16; found: 246.2. Deprotection
with TFA and reprotection with Fmoc-OSu by an identical procedure
gave 0.173 g of compound 7b as a clear oil (0.47 mmol, 63% yield for
two steps). 1H NMR (600 MHz, CDCl3): d = 7.737 (d, 2H), 7.590 (m,
2H), 7.374 (dd, 2H), 7.289 (dd, 2H), 6.239 (s, 1H), 4.331 (m, 2H), 4.185
The resulting average structure for WKMe3L was used as the starting
structure for an MD run in explicit water in AMBER.[29] Non-natural res-
idues were constructed in xLeap and parameterized based on existing
residues with scaled RESP charges (included in the Supporting Informa-
tion).The starting structure was solvated in an octahedral box by using
TIP3P water and the system charge neutralized with two chloride ions.
The solvated system was minimized and then equilibrated by heating
from 0 to 300 K over 20 ps.Finally, production MD were run at 300 K by
using PME electrostatics and periodic boundary conditions within
Chem. Eur. J. 2007, 13, 5753 – 5764
ꢀ 2007 Wiley-VCH Verlag GmbH & Co.KGaA, Weinheim
5763