5426
H. Vilac¸ a et al. / Tetrahedron 70 (2014) 5420e5427
Pbf), 137.25 (C Pbf), 137.66 (C Phe), 156.10 (C]N Arg), 157.42 (C Pbf),
168.50 (C]O Gly), 169.07 ( C]O Asp), 169.92 (C]O Asp), 170.80
(C]O Phe), 172.00 (CH2C]O), 172.27 (C]O Arg), 173.07 (CO2H)
ppm. HRMS [MþH] (EI): calcd C49H71N8O12S 995.49067; found
995.48985.
ArH), 8.19 (1H, br s, NH Gly), 8.54e8.55 (1H, m, NHCH2) ppm. 13
NMR (100 MHz, DMSO):
¼12.24 (CH3 Pbf), 17.41 (CH2CH2), 17.57
(CH3 Pbf), 18.92 (CH3 Pbf), 18.02 (CH3 Val), 19.11 (CH3 Val), 24.07
(COCH2CH2), 25.48 (
CH2 Arg), 27.57 or 27.59 (3ꢂCH3), 28.27 (2-
CH3 Pbf), 29.05 ( CH2 Arg), 30.33 ( CH Val), 33.91 (COCH2CH2),
34.07 (NHCH2), 37.58 ( CH2 Asp), 37.93 ( CH2 Phe), 40.18 ( CH2
Arg), 41.93 (CH2 Gly), 42.44 (3-CH2 Pbf), 49.32 ( CH Asp), 52.26
CH Arg), 54.25 ( CH Phe), 57.75 ( CH Val), 80.09 (C(CH3)3), 86.27
(2-C Pbf),116.22 (C Pbf),120.39 (CH),121.16 (CH),121.16 (CH),124.29
(C Pbf), 126.22 (CH Phe), 127.94 (CH Phe), 129.21 (CH Phe), 131.41 (C
Pbf), 134.10 (C Pbf), 136.13 (C), 137.25 (C Pbf), 137.51 (C Phe), 145.93
(C), 147.66 (C), 156.02 (C]N), 157.42 (C Pbf), 168.54 (C]O), 169.03
(C]O), 170.12 (C]O), 170.87 (C]O), 170.89 (C]O), 172.06 (C]O),
C
b
d
g
b
b
b
b
d
4.2.7. Synthesis of peptide 6. Peptide 5 (0.10 mmol, 0.11 g) was
dissolved in DMF (5 mL) and the mixture cooled in an ice bath.
HOBt (0.10 mmol, 0.01 g), HBTU (0.10 mmol, 0.04 g), and prop-
argylamine (0.10 mmol, 0.01 mL) were added, waiting
5 min between each addition. DIPEA (0.21 mmol, 0.04 mL) was
added and the mixture was left stirring at room temperature for 2
days. The DMF was removed under reduced pressure. Peptide 6 was
precipitated from water (0.10 g, 92%) as a white solid. 1H NMR
a
(a
a
a
172.11 (C]O) ppm. HRMS [MþH] (EI): calcd
C58H78N15O11S
(400 MHz, DMSO):
3ꢂCH3), 1.39e1.41 (2H, m,
1.47e1.62 (2H, m, CH2 Arg), 1.64 (2H, m, CH2CH2CH2), 1.91 (1H, m,
CH Val), 1.99 (3H, s, CH3 Pbf), 2.12 (2H, dt, J¼2.4 and 7.2 Hz,
CH^CCH2), 2.21 (2H, t, J¼7.6 Hz, CH2C]O), 2.21e2.22 (1H, m, CH2
Asp), 2.37 (1H, s, CH2 Asp), 2.41 (3H, s, CH3 Pbf), 2.46 (3H, s, CH3
Pbf), 2.75 (1H, t, J¼2.4 Hz, CH^CCH2), 2.78 (1H, d, J¼9.6 Hz, CH
Phe), 2.95 (2H, s, 3-CH2 Pbf), 2.97 (1H, d, J¼8.8 Hz, CH Phe),
2.98e3.02 (2H, m,
CH2 Arg), 3.06 (1H, t, J¼2.4 Hz, CH^CCH2NH),
3.68 (2H, t, J¼6.8 Hz, CH2 Gly), 3.79 (2H, ddq, J¼2.4, 5.6, and 17.6 Hz,
CH^CCH2NH), 4.09 (1H, dd, J¼7.4 and 8.6 Hz, CH Val), 4.20 (1H, q,
J¼7.6 Hz, CH Arg), 4.53e4.57 (1H, m, CH Asp), 4.59e4.63 (1H, m,
CH Phe), 6.39 (1H, br s, NH), 6.66 (2H, br s, 2ꢂNH), 7.13e7.17 (1H,
m, ArH Phe), 7.19e7.23 (4H, m, ArH Phe), 8.02 (1H, d, J¼8.8 Hz, NH
d
¼0.75 (6H, d, J¼6.4 Hz, CH3 Val), 1.32 (9H, s,
1192.57205; found 1192.57320.
g
CH2 Arg), 1.40 (6H, s, 2-CH3 Pbf),
b
4.2.9. Synthesis of peptide 8. To a solution of peptide 6 (0.10 mmol,
0.10 g) in DMF (1 mL), 2,7-diazido-9,9-dioctyl-9H-fluorene
(0.15 mmol, 68.70 mg), copper(I) iodide (4.00 mmol, 0.80 mg), and
triethylamine (0.29 mmol, 0.04 mL) were added. The mixture was
left stirring at room temperature. The reaction was followed by 1H
NMR. After 72 h the solvent was removed and a mixture of peptide
8 and copper was precipitated from water. The solid was washed
with an aqueous solution of ethylenediamine tetraacetic acid
(EDTA) 0.1 M, giving peptide 8 (0.14 g, 97%) as a light brown solid.
b
b
b
b
b
d
a
a
a
1H NMR (400 MHz, DMSO, 70 ꢁC):
d
¼0.60 (4H, br s, 2ꢂCH2),
a
0.78e0.81 (12H, m, 2ꢂCH3 Val, 2ꢂCH3), 1.01e1.24 (18H, m, 9ꢂCH2),
1.32 (9H, s, 3ꢂCH3), 1.38e1.50 (4H, m, CH2 Arg, CH2 Arg), 1.60 (3H,
Asp), 8.06 (3H, d, J¼9.2 Hz,
a
NH Arg, NH Phe, NH Val), 8.17 (1H, t,
br s, 2ꢂCH3 Pbf), 1.95e1.99 (2H, m, CH2), 2.08 (8H, br s,
bCH Val, CH3
J¼5.2 Hz, NH Gly), 8.45 (1H, t, J¼5.2 Hz, CH^CCH2NH) ppm. 13C
Pbf, 2ꢂCH2), 2.25e2.42 (4H, m, CH2 Asp, CH2), 2.43 (3H, s, CH3 Pbf),
2.49 (3H, br s, CH3 Pbf), 2.74 (4H, br s, 2ꢂCH2), 2.89e2.92 (6H, m,
CH2 Phe, CH2 Arg, CH2 Pbf), 3.74 (2H, br s, CH2 Gly), 4e17 (1H, br s,
NMR (100 MHz, DMSO):
d
¼12.25 (CH3 Pbf), 17.40 (CH^CCH2), 17.56
(CH3 Pbf), 18.09 (CH3 Val), 18.91 (CH3 Pbf), 18.99 (CH3 Val), 24.07
(CH2CH2CH2), 25.45
CH2 Arg), 27.60 (3ꢂCH3), 27.76
(CH^CCH2NH), 28.28 (2-CH3 Pbf), 29.06 ( CH2 Arg), 30.58 ( CH
Val), 33.90 (CH2C]O), 37.62 ( CH2 Asp), 38.02 ( CH2 Phe), 40.29
CH2 Arg), 41.90 (CH2 Gly), 42.45 (3-CH2 Pbf), 49.24 ( CH Asp),
52.53 CH Arg), 54.13 CH Phe), 57.50 CH Val), 71.42
(g
aCH Val), 4.28 (1H, br s,
aCH Arg), 4.45 (2H, br s, CH2), 4.57(2H, br s,
b
b
a
CH Asp, CH Phe), 6.43 (1H, br s, NH), 6.62 (1H, br s, NH),
a
b
b
7.06e7.08 (2H, m, ArH), 7.14e7.20 (7H, m, ArH), 7.75e7.95 (8H, m,
2ꢂArH, 4ꢂNH), 8.29 (1H, br s, NH), 8.49e8.57 (2H, m, 2ꢂNH) ppm.
(d
a
(a
(
a
(a
13C NMR (100 MHz, DMSO, 70 ꢁC):
d¼11.71 (CH3), 13.27 (2ꢂCH3),
(CH^CCH2), 72.85 (CH^CCH2NH), 80.06 (C(CH3)3), 80.88
(CH^CCH2NH), 84.03 (CH^CCH2), 86.27 (2-C Pbf), 116.23 (C Pbf),
124.29 (C Pbf), 126.22 (CH Phe), 127.92 (CH Phe), 129.22 (CH Phe),
131.40 (C Pbf), 134.16 (C Pbf), 137.24 (C Pbf), 137.52 (C Phe), 156.01
17.01 (CH3), 18.32 (CH3), 18.69 (2ꢂCH3), 21.45 (CH2), 22.81 (CH2),
24.43 (CH2), 24.54 (CH2), 25.16 (CH2), 27.32 (3ꢂCH3), 27.84
(2ꢂCH3), 28.58 (CH2), 28.69 (CH2), 28.78 (CH2), 30.15 (CH), 30.63
(CH2), 33.93 (CH2), 34.36 (CH2), 37.12 (CH2), 37.38 (CH2), 38.57
(CH2), 39.85 (CH2), 41.90 (CH2), 42.27 (CH2), 45.97 (CH2), 49.37 (CH),
52.35 (CH), 54.19 (CH), 54.94 (C), 57.72 (CH), 79.79 (C(CH3)3), 85.80
(C), 113.52 (CH), 116.0 (C), 117.71 (CH), 119.79 (CH), 120.59 (CH),
120.84 (CH), 124.0 (C), 125.80 (CH), 127.57 (CH), 128.74 (CH), 131.17
(C), 134.16 (C), 136.82 (C), 137.00 (C), 137.95 (C), 139.28 (C), 145.46
(2ꢂC), 152.02 (C), 155.86 (C), 157.22 (C), 168.30 (C), 168.78 (C),
169.80 (C), 170.36 (C), 170.48 (C), 171.85 (C), 171.97 (C) ppm. HRMS
[MþH] (EI): calcd C81H114N15O11S 1504.85430; found 1504.85375.
(C]N), 157.42 (C Pbf), 168.46 (C]O Gly), 169.01 (
(C]O Asp), 170.69 (C]O Val), 170.71 (C]O Phe), 172.01 (CH2C]O),
b
C¼O Asp), 170.01
172.14 (C]O Arg) ppm. HRMS [MþH] (EI): calcd C52H74N9O11
S
1032.52230; found 1032.52280.
4.2.8. Synthesis of peptide 7. To a solution of peptide 6 (0.05 mmol,
0.05 g) in DMF (2 mL), 1,4-diazidobenzene (0.05 mmol, 7.40 mg),
copper(I) iodide (0.90
mmol, 0.20 mg), and triethylamine
(0.10 mmol, 0.01 mL) were added and the mixture was left stirring
at room temperature. The reaction was followed by TLC (chloro-
form/methanol, 9:1). After 84 h the solvent was removed and
peptide 7 was precipitated from water (0.04 g; 82%). 1H NMR
4.2.10. Synthesis of peptide 9. To peptide 7 (0.02 mmol, 0.03 g)
trifluoroacetic acid (TFA) (0.70 mL) was added and the mixture was
left stirring at room temperature for 5 h. The solvent was removed
and the solid obtained was washed with an aqueous solution of
EDTA 0.1 M and water. Peptide 9 (9.40 mg, 44%) was obtained as
(400 MHz, DMSO):
1.33e1.40 (2H, m,
m, CH2 Arg), 1.58e1.65 (3H, m,
(1H, m, CH Val), 1.98 (3H, s, CH3 Pbf), 2.11e2.13 (2H, m, CH2CH2-
triazole), 2.18e2.26 (3H, m, CH2 Asp, COCH2CH2), 2.39e2.45 (1H,
m, CH2 Asp), 2.40 (3H, s, CH3 Pbf), 2.45 (3H, s, CH3 Pbf), 2.75e2.79
(1H, m, CH2 Phe), 2.93 (2H, s, 3-CH2 Pbf), 2.93e3.00 (1H, m, CH2
Phe), 2.98e3.02 (2H, m, CH2 Arg), 3.67e3.72 (2H, m, CH2 Gly), 4.13
(1H, dd, J¼6.0 and 14.4 Hz, CH Val), 4.19e4.21 (1H, m, CH Arg),
4.38e4.46 (2H, m, NHCH2), 4.50e4.58 (1H, m, CH Asp), 4.58e4.65
(1H, m,
d
¼0.74 (6H, br s, CH3 Val), 1.31 (9H, s, 3ꢂCH3),
CH2 Arg), 1.40 (6H, s, 2-CH3 Pbf), 1.40e1.52 (1H,
CH2 Arg, COCH2CH2), 1.90e1.96
g
b
b
a green solid. 1H NMR (400 MHz, DMSO):
1.60e1.64 (3H, m,
Arg, CH Val, CH2 Arg), 2.00e2.15 (2H, m, CH2CH2-triazole),
2.15e2.27 (2H, m, COCH2CH2), 2.27e2.42 (2H, m, CH2 Asp),
2.73e2.86 (1H, m, CH2 Phe), 2.94e3.06 (1H, m, CH2 Phe), 3.07
(2H, m, CH2 Arg), 3.68 (2H, br s, CH2 Gly), 4.10 (1H, br s, CH Val),
4.26 (1H, br s, CH Arg), 4.41e4.50 (3H, m, NHCH2, CH Asp), 4.56
(1H, br s,
d
¼0.74 (6H, br s, CH3 Val),
b
b
CH2 Arg, COCH2CH2), 1.80e2.00 (4H, m, CH2
b
b
b
g
b
b
b
b
b
b
d
d
a
a
a
a
a
a
a
CH Phe), 7.21e7.28 (7H, br s, ArH Phe, 2ꢂNH), 7.30 (1H, s,
a
CH Phe), 6.39 (1H, br s, NH), 6.67 (2H, br s, 2ꢂNH),
CH), 7.88 (1H, s, CH), 8.07 (8H, m, 4ꢂNH, ArH), 8.56 (1H, br s, NH),
7.13e7.17 (1H, m, ArH Phe), 7.21e7.22 (4H, m, ArH Phe), 7.29 (1H, s,
CH triazole), 7.88 (1H, s, CH triazole), 8.07e8.12 (8H, m, 4ꢂNH,
8.66 (1H, br s, NH), 12.15 (1H, br s, CO2H) ppm. 13C NMR (100 MHz,
DMSO):
d
¼17.34 (CH2CH2), 18.87 (CH3 Val), 24.14 (COCH2CH2),