A R T I C L E S
Kaul et al.
The solution was filtered, and the solvent was removed under reduced
pressure, affording 10 as a yellow residue (2.76 g, 100% yield). HRMS
(MALDI-FTMS): calcd for MNa+ 507.1993, found 507.1994. 1H NMR
(500 MHz, CDCl3): δ 0.35 (s, 18H), δ 1.23 (t, J ) 6.0 Hz, 3H), δ
2.84 (t, J ) 6.6 Hz, 2H), δ 2.89 (t, J ) 6.6 Hz, 2H), δ 3.33 (t, J ) 6.4
Hz, 2H), δ 3.35 (t, J ) 6.4 Hz, 2H), δ 4.15 (q, J ) 6.0 Hz, 2H), δ 7.40
(s, 1H), δ 7.41 (s, 1H), δ 7.98 (s, 1H), δ 7.98 (s, 1H). 13C NMR (500
MHz, CDCl3): δ -0.7, 14.2, 25.6, 25.9, 34.1, 34.5, 60.7, 123.5, 123.7,
123.8, 123.9, 124.1, 132.1, 132.2, 134.4, 155.1, 173.6, 178.0.
119.1, 119.3, 119.8, 120.0, 124.7, 125.4, 127.7, 128.2, 128.3, 128.7,
130.5, 130.6, 136.7, 143.4, 143.5, 156.1, 156.3, 156.5, 166.5, 166.5,
172.8.
3-{2,8-Bis-(2-tert-butoxycarbonyl-ethyl)-6-[2-(9H-fluoren-9-yl-
methoxycarbonylamino)-ethyl]-dibenzofuran-4-yl}-propionic Acid
(4). Hydrolysis, hydrogenation, and Fmoc protection of the amino group
were carried out consecutively using the crude product from the
previous step. Hydrolysis was performed by dissolving 0.32 g (0.47
mmol) of 13 in 10 mL of 3:1 tetrahydrofuran:ethanol to which was
added 2.5 mL of 1 M LiOH in water. The reaction was complete after
12 h. The mixture was neutralized utilizing dilute HCl and diluted with
50 mL of dichloromethane. The aqueous layer was separated, and the
organic layer was dried (MgSO4). The solvent was removed under
reduced pressure. The crude residue was subjected to hydrogenation
as described previously for the preparation of 10 (10 mL of methanol
with 5 mg of 10% palladium on activated carbon). The crude product
from hydrogenation was dissolved in 10 mL of dichloromethane, and
Fmoc-OSu (0.23 g, 0.7 mmol) was added. The mixture was stirred at
room temperature for 12 h, and the solvent was removed under reduced
pressure. The desired product 4 was purified by flash column chro-
matography (7:3 hexane:ethyl acetate), affording 4 as a white foam
(0.34 g, 95% yield for these three steps combined). HRMS (MALDI-
3-(6-Iodo-2,8-bis-trimethylsilanyl-dibenzofuran-4-yl)-acrylic Acid
Ethyl Ester (11). A dry 50 mL round-bottom flask was charged with
2.6 g (5.4 mmol) of 10, 40 mL of benzyl alcohol, 0.56 g (5.4 mmol)
of triethylamine, and 1.78 g (6.4 mmol) of dipheynylphosphonic azide.
The solution was heated at reflux for 24 h and cooled to room
temperature. The solvent was removed under reduced pressure, and
the resulting oil was dissolved in ether (40 mL). This solution was
washed with 2 M citric acid (3 × 30 mL), 5% sodium bicarbonate (3
× 30 mL), and water (2 × 40 mL). The organic layer was dried (sodium
sulfate) and concentrated to afford 11 (2.22 g, 70% yield) as a pale
yellow oil. HRMS (MALDI-FTMS): calcd for MNa+ 612.2572, found
1
612.2566. H NMR (500 MHz, CDCl3): δ 0.35 (s, 18H), δ 1.21 (t, J
) 5.9 Hz, 3H), δ 2.81 (t, J ) 6.5 Hz, 2H), δ 3.22 (t, J ) 6.5 Hz, 2H),
δ 3.31 (t, J ) 6.5 Hz, 2H), δ 3.67 (t, J ) 6.5 Hz, 2H), δ 4.12 (q, J )
5.9 Hz, 2H), δ 5.09 (s, 2H), δ 7.29 (m, 5H), δ 7.39 (s, 1H), δ 7.40 (s,
1H), δ 7.98 (s, 1H), δ 7.99 (s, 1H). 13C NMR (500 MHz, CDCl3): δ
-0.5, 14.4, 25.7, 30.9, 34.5, 41.2, 60.7, 66.8, 122.3, 123.9, 124.0, 124.1,
124.2, 124.4, 128.2, 128.6, 132.3, 133.0, 134.7, 134.8, 136.8, 155.4,
155.6, 156.5, 173.2.
1
FTMS): calcd for MNa+ 784.3456, found 784.3449. H NMR (500
MHz, CDCl3): δ 1.40 (s, 18H), δ 2.53 (t, J ) 7.5 Hz), δ 2.61 (m,
3H), δ 2.65 (m, 1H), δ 2.83 (m, 1H), δ 2.95 (t, J ) 6.4 Hz, 1H), δ
3.02 (m, 3H), δ 3.14 (m, 2H), δ 3.30 (m, 2H), δ 3.62 (m, 2H), δ 4.03
(m, 0.5H), δ 4.07 (m, 1H), δ 4.18 (m, 0.5H), δ 4.40 (m, 1H), δ 5.17
(m, 0.5H), δ 6.78 (m, 0.5H), δ 7.11 (m, 2H), δ 7.26 (t, J ) 6.2 Hz,
2H), δ 7.37 (t, J ) 6.2 Hz, 2H), δ 7.42 (m, 1H), δ 7.53 (m, 1H), δ
7.59 (t, J ) 5.2 Hz, 2H), δ 7.73 (t, J ) 5.2 Hz, 2H). 13C NMR (500
MHz, CDCl3): δ 26.5, 27.0, 28.1, 29.5, 31.0, 36.4, 37.7, 41.2, 43.3,
47.0, 47.1, 67.0, 67.5, 80.4, 118.5, 119.9, 125.0, 127.0, 127.7, 128.0,
128.4, 135.5, 135.6, 135.7, 141.2, 141.3, 143.6, 143.8, 153.6, 153.7,
153.8, 154.3, 156.4, 158.5, 172.3.
3-[6-(2-Benzyloxycarbonylamino-ethyl)-2,8-diiodo-dibenzofuran-
4-yl]-propionic Acid Ethyl Ester (12). An oven-dried 50 mL round-
bottom flask was charged with 1.27 g (2.0 mmol) of 11 and 1.10 g
(8.0 mmol) of K2CO3 and purged with nitrogen. After 11 was dissolved
using 20 mL of carbon tetrachloride, the suspension was cooled to 0
°C. To the suspension was added 1.30 g (8.0 mmol) of iodine
monochloride in 10 mL of carbon tetrachloride via syringe. The mixture
was stirred at 0 °C for 1 h and was poured into a solution of 3 g of
NaHSO3 in 50 mL of water. The mixture was stirred at room
temperature for 1 h, and the CCl4 was collected. Removal of CCl4
afforded a white precipitate, which was collected by filtration. Recrys-
tallization from benzene-hexane gave 1.41 g (95%) of 12 as colorless
crystals (mp 199-201 °C). HRMS (MALDI-FTMS): calcd for MNa+
General Method for Solid-Phase Peptide Synthesis (SPPS).
Automated solid-phase peptide synthesis was performed on an Applied
Biosystems 433A peptide synthesizer. All syntheses were performed
on a 0.1 mmol scale using the standard Fmoc-based FastMoc coupling
chemistry provided by the system’s software. Briefly, the coupling
reactions were carried out in N-methylpyrrolidone (NMP) using 10
equiv of amino acid and the activating agents 2-(1H-benzotriazol-1-
yl)-1,1,3,3 tetramethyluronium hexafluorophosphate (10 equiv) and
1-hydroxybenzotriazole (10 equiv) in the presence of diisopropylethy-
lamine (10 equiv). The N-terminal Fmoc deprotection was achieved
using 20% piperidine in DMF for 20 min. The templates 1a and 1b
(1.2 equiv) were incorporated manually using 1.2 equiv of the activating
agents HBTU/HOBt in DMF (2 mL) in the presence of 2.2 equiv of
diisopropylethylamine (2 M in NMP). The coupling was carried out
for 24 h before loading the resin back on the synthesizer for further
extension of the peptide chain. Peptide cleavage and side-chain
deprotection were carried out by gently agitating the crude peptide-
resin in Reagent K (1 mL per 0.025 mmol of crude peptide) (Reagent
K: 82.5% TFA, 5% m-cresol, 5% thioanisole, 5% water, and 2.5%
EDT) for 2 h at room temperature. Crude peptide was precipitated in
tert-butyl methyl ether, centrifuged, redissolved in DMF, and purified
by HPLC using a 10-50% linear gradient in solvent B over 60 min.
CD Studies. CD spectra were recorded using an Aviv model 202SF
circular dichroism spectrometer equipped with a Peltier temperature-
controlled cell holder using a 0.2 cm path length Suprasil quartz cell
(Hellma, Forest Hills, New York). Far-UV CD spectra were recorded
from 200 to 250 nm at 2 and 25 °C. The wavelength step size was 0.5
nm, and the averaging time used was 2 s per scan at each wavelength
step. The peptide sample was dissolved in 20 µM sodium phosphate
(pH 7.0). Thermal denaturation was monitored at 227 nm. The
temperature range utilized was from 2 to 98 °C with a 2 °C step size
with a 90 s equilibration time. Data for each point were averaged for
1
719.9714, found 719.9718. H NMR (500 MHz, CDCl3): δ 1.21 (t, J
) 5.9 Hz, 3H), δ 2.75 (t, J ) 6.4 Hz, 2H), δ 3.14 (t, J ) 6.4 Hz, 2H),
δ 3.20 (t, J ) 6.4 Hz, 2H), δ 3.63 (t, J ) 6.4 Hz, 2H), δ 4.12 (q, J )
5.9 Hz, 2H), δ 4.96 (br s, 1H), δ 5.07 (s, 2H), δ 7.29 (m, 5H), δ 7.59
(s, 1H), δ 7.59 (s, 1H), δ 8.05 (s, 1H), δ 8.07 (s, 1H). 13C NMR (500
MHz, CDCl3): δ 14.2, 24.8, 30.3, 33.8, 40.6, 60.6, 66.7, 86.3, 125.2,
125.3, 127.0, 128.0, 128.0, 128.1, 128.2, 128.5, 136.2, 136.7, 154.0,
154.2, 156.2, 172.4.
3-[6-(2-Benzyloxycarbonylamino-ethyl)-8-(2-tert-butoxycarbonyl-
vinyl)-4-(2-ethoxycarbonyl-ethyl)-dibenzofuran-2-yl]-acrylic Acid
tert-Butyl Ester (13). The synthesis of 13 was similar to that of 8.
tert-Butyl acrylate (0.44 g, 3.44 mmol) was allowed to react with 12
(1.2 g, 1.72 mmol), tri-o-tolylphosphine (0.16 g, 0.53 mmol), and
palladium(II)acetate (20 mg, 0.17 mmol). After flash column chroma-
tography on silica gel using 3:7 hexane:ethyl acetate solution as eluent,
the desired product (13) was obtained (0.96 g, 80% yield) as a colorless
oil. HRMS (MALDI-FTMS): calcd for MNa+ 720.3143, found
720.3156. 1H NMR (500 MHz, CDCl3): δ 1.21 (t, J ) 5.9 Hz, 3H), δ
1.56 (s, 18H), δ 2.79 (t, J ) 6.4 Hz, 2H), δ 3.20 (t, J ) 6.4 Hz, 2H),
δ 3.28 (t, J ) 6.4 Hz, 2H), δ 3.67 (t, J ) 6.4 Hz, 2H), δ 4.12 (q, J )
5.9 Hz, 2H), δ 5.07 (s, 2H), δ 6.42 (d, J ) 3.8 Hz, 1H), δ 6.45 (d, J
) 3.8 Hz, 1H), δ 7.29 (m, 5H), δ 7.46 (s, 1H), δ 7.47 (s, 1H), δ 7.68
(s, 1H), δ 7.71 (s, 1H), δ 7.93 (s, 1H), δ 7.94 (s, 1H). 13C NMR (500
MHz, CDCl3): δ 14.4, 25.3, 28.4, 30.8, 34.1, 40.9, 60.8, 66.8, 80.7,
9
11906 J. AM. CHEM. SOC. VOL. 124, NO. 40, 2002