8960 J. Am. Chem. Soc., Vol. 120, No. 35, 1998
Clark et al.
dipeptide 6-w as its free acid in 51% yield (ESI-MS, [M - H]- calcd
) 512.3, found ) 513).
m; m2 2.71, 3.34, m; d 2.97, 3.47, m; d* 2.87, 3.56, m), N-CH2CH2-
CH2CHCH2 (m1 1.77, 1.92, m; m2 1.10, 1.29, m; d 1.61, 1.62, m; d*
1.64, 1.77, m), N-CH2CH2CH2CHCH2 (m1 2.02, 2.21, m; m2 1.93,
m; d 2.01, 2.08, m; d* 1.88, 2.13, m), N-CH2CH2CH2CHCH2 (m1,
m2, d, d* 4.92-5.87, m), CRH (m1 5.08, ovl; m2 3.63, ovl; d 5.77,
ovl; d* 5.87, ovl), CâH3 (m1 1.36, ovl, d, J ) 7.4 Hz; m2 1.58, d, J )
6.8 Hz; d 0.95, d, J ) 6.8 Hz; d* 1.05, d, J ) 6.8 Hz); D-Phe3, N-H
(m1 7.92, d, J ) 6.1 Hz; m2 6.95, d, J ) 6.8 Hz; d 8.74, d, J ) 8.8 Hz;
d* 8.93, d, J ) 9.4 Hz), CRH (m1 5.02, m; m2 5.06, m; d 5.48, m; d*
5.44, m), CâH2 (m1 2.98, 3.01, m; m2 2.97, 3.01, m; d 3.03, 3.06, m;
d* 3.05, 3.10), Cδ,ꢀ,êH (m1, m2, d, d* 7.03-7.46, m); L-MeN-Hag4,
N-CH3 (m1 2.99, s; m2 2.67, s; d 2.98, s; d* 2.85, s), CRH (m1 5.10,
m; m2 4.54, m; d 5.68, m; d* 5.62, m), CâH2 (m1 1.58, 1.91, m; m2
1.49, 1.77, m; d 1.30, 1.62, m; d* 1.12, 1.66, m), CγH2 (m1, m2 1.49-
2.21, m; d 1.81, m; d* 1.75, m), CδH and CꢀH2 (m1, m2, d, d* 4.92-
5.87, m). ESI-MS: [M + H]+ calcd ) 1117.6, found ) 1118.
cyclo[(-L-Phe1-D-MeN-Ala2-L-Leu3-D-MeN-Ala4)2-] (9). Linear oc-
tapeptide (-L-Leu-D-MeN-Ala-L-Phe-D-MeN-Ala)2 (9-x) was synthesized
on N-Boc-D-MeN-Ala-(OCH2)-Pam resin, cleaved with HF, and isolated
as its TFA salt after purification by RP-HPLC (ESI-MS, [M + H]+
calcd ) 879.5, found ) 880). Cyclization of 45 mg of 9-x (46 µmol)
and purification as described above afforded 26 mg of 9 (30 µmol,
65%). 1H NMR (500 MHz, 293 K, 3.0 mM, CDCl3): L-Phe1, N-H
(m 7.20, ovl; d 8.65, d, J ) 8.2 Hz; d* 8.83, d, J ) 8.2 Hz), CRH (m
4.83, m; d 5.26, m; d* 5.27, m), CâH2 (m 3.03, 3.14, m; d 2.81, 2.96,
m; d* 2.91, 3.05, m), Cδ,ꢀ,êH (m, d, d* 7.16-7.27, m); D-MeN-Ala2,
N-CH3 (m 2.62, s; d 2.79, s; d* 2.80, s), CRH (m 5.25, ovl; d 5.92,
q, J ) 6.8 Hz; d* 5.86), CâH3 (m 1.19, ovl; d 1.11, d, J ) 6.8 Hz; d*
1.03, d, J ) 6.8 Hz); L-Leu3, N-H (m 6.92, d, J ) 8.2 Hz; d 8.80, d,
J ) 9.5 Hz; d* 8.48, d, J ) 8.8 Hz), CRH (m 4.60, m; d 5.14,; d*
5.09, m), CâH2 (m 1.37, 1.47, m; d 1.32, 1.56, m; d* 1.29, 1.43, m),
CγH (m 1.66, m; d 1.61, m; d* 1.51, m), Cδ1,δ2H3 (m 0.91, 0.92, ovl;
d 0.82, d, J ) 6.1 Hz, 0.95, ovl; d* 0.87, 0.93, ovl), D-MeN-Ala4,
N-CH3 (m 2.71, s; d 3.25, s; d* 3.27, s), CRH (m 5.26, ovl; d 5.79,
q, J ) 6.8 Hz; d* 5.86, ovl), CâH3 (m 1.26, ovl; d 1.15, d, J ) 6.8 Hz;
d* 1.23, ovl). ESI-MS: [M + H]+ calcd ) 861.5, found ) 862.
cyclo[(-L-Phe1-D-MeN-Ala2-L-Hag3-D-MeN-Ala4)2-] (10). Linear oc-
tapeptide (-D-MeN-Ala-L-Hag-D-MeN-Ala-L-Phe)2 (10-x) was synthesized
on N-Boc-L-Phe-(OCH2)-Pam resin (0.54 mmol scale) and cleaved with
Bu4NF‚xH2O to give 230 mg of the desired product as its TFA salt
after purification by RP-HPLC (233 µmol, 43%; ESI-MS, [M + H]+
calcd ) 875.5, found ) 875). Cyclization of 112 mg of 10-x (113
µmol) and purification as described above afforded 68 mg of 10 (79
µMol, 70%). 1H NMR (500 MHz, 278 K, 7.5 mM, CDCl3): L-Phe1,
N-H (m 7.21, ovl; d 8.60, d, J ) 8.2 Hz; d* 8.82, d, J ) 8.2 Hz),
CRH (m 4.84, m; d 5.22, m; d* 5.25, m), CâH2 (m 2.98, 3.07, m; d
2.77, 2.95, m; d* 2.90, 3.02, m), Cδ,ꢀ,êH (m, d, d* 7.18-7.30, m);
D-MeN-Ala2, N-CH3 (m 2.63, s; d 2.76, s; d* 2.74, s), CRH (m 5.27,
ovl; d 5.96, ovl; d* 5.82, ovl), CâH3 (m 1.17, ovl; d 1.11, ovl; d*
1.01, d, J ) 6.1 Hz); L-Hag3, N-H (m 7.04, br; d 8.82, d, J ) 8.2 Hz;
d* 8.48, d, J ) 8.1 Hz), CRH (m 4.54, m; d 5.07, m; d* 5.03, m),
CâH2 (m 1.60, 1.73, m; d 1.59, 1.71, m; d* 1.50, 1.62, m), CγH2 (m
2.01, m; d 2.07, m; d* 1.98, m), CδH (m 5.65, m; d 5.74, m; d* 5.71,
m), CꢀH2 (m 4.85 (E), 4.90 (Z), m; d 5.00 (E), 5.04 (Z), m; d* 4.89
(E), 4.94 (Z), m); D-MeN-Ala4, N-CH3 (m 2.69, s; d 3.19, s; d* 3.22,
s), CRH (m 5.20, ovl; d 5.76, ovl; d* 5.93, ovl), CâH3 (m 1.23, ovl; d
1.12, ovl; d* 1.20, ovl). IR (293 K, 1 mM, CHCl3): 3310 (amide A);
1631, 1675 (amide I ); 1527 (amide II|). ESI-MS: [M + H]+ calcd
) 857.5, found ) 858.
Peptide cyclo[(-D-Phe-L-N-(3-(4-MeBn)thiopropyl)-Ala-D-Phe-L-
Ala)2-] (6-y) was prepared as follows: Linear octapeptide (-L-Ala-D-
Phe-L-N-(3-(4-MeBn)thiopropyl)-Ala-D-Phe)2 (6-x) was synthesized on
N-Boc-D-Phe-(OCH2)-Pam resin (0.56 mmol scale) using N-Boc-
protected D-Phe and L-Ala as well as protected dipeptide N-Boc-D-
Phe-L-N-(3-(4-MeBn)thiopropyl)-Ala (6-w) and cleaved with Bu4NF‚
xH2O (300 mg of crude; ESI-MS, [M + H]+ calcd ) 1246, found )
1248). Cyclization of 300 mg of the crude material as described above
afforded 150 mg of 6-y (122 µmol, 22%) after purification by RP-
HPLC. ESI-MS: [M + H]+ calcd ) 1228, found ) 1230.
To a 10-mL round-bottom flask equipped with a magnetic stir bar
was added 110 mg of cyclo[(-D-Phe-L-N-(3-(4-MeBn)thiopropyl)-Ala-
D-Phe-L-Ala)2-] (6-y) (90 µmol, 1 equiv) followed by 5 mL of TFA
and 19.5 µL of anisole. The solution was cooled to 4 °C, treated with
53.5 mg of Tl(CF3CO2)3, and stirred at 4 °C for 18 h. After evaporation
of the solvent under a stream of nitrogen, the resulting residue was
subjected to purification by RP-HPLC to give 20 mg of the desired
bicyclic product 6 (20 µMol, 22%). 1H NMR (500 MHz, 293 K, 10.0
mM, CDCl3): D-Phe1, N-H (m 7.20, br; d 8.19, br), CRH (m 4.91, m;
d 5.38, m), CâH2 (m 3.07, m; d 2.76, 2.95, m), Cδ,ꢀ,êH (m, d 7.05-
7.31, m); L-N-(dithiopropyl)-Ala2, N-CH2CH2CH2S- (m 2.83, 3.19,
m; d 3.17, 3.59, m), N-CH2CH2CH2S- (m 1.43, 1.56, m; d 1.57,
1.69, m), N-CH2CH2CH2S- (m 1.94, 2.11, m; d 2.50, 3.29, m), CRH
(m 5.15, br; d 4.73, br); CâH3 (m 1.11, br s; d 0.83, br s); D-Phe3,
N-H (m 7.73, br; d 8.01, br), CRH (m 4.61, m; d 4.68, m), CâH2 (m
2.85, 2.99, m; d 3.00, m), Cδ,ꢀ,êH (m, d 7.05-7.31, m); L-Ala4, N-H
(m 6.30, br; d 6.81, br), CRH (m 4.51, br; d 5.00, br), CâH3 (m 1.12,
br s; d 1.08, br s). ESI-MS: [M + H]+ calcd ) 1019.5, found )
1020.
cyclo[(-D-Phe1-L-PenN-Ala2-D-Phe3-L-Hag4)2-] (7). Linear octapep-
tide (-D-Phe-L-PenN-Ala-D-Phe-L-Hag)2 (7-x) was synthesized on N-Boc-
L-Hag-(OCH2)-Pam resin (0.4 mmol scale) and cleaved with Bu4NF‚
xH2O to give 143 mg of the desired product as its TFA salt after
purification by RP-HPLC (117 µmol, 29%). Cyclization of 143 mg
of 7-x (117 µmol) and purification as described above afforded 67 mg
of 7 (62 µmol, 53%). 1H NMR (500 MHz, 293 K, CDCl3): D-Phe1,
N-H (m 7.56, br; d* 8.69, d, J ) 9.5 Hz; d 9.04, d, J ) 9.5 Hz), CRH
(m 4.87, m; d* 5.34, m; d 5.33, m), CâH2 (m 3.00, 3.25, m; d 2.87,
2.99, m; d * 2.85, 3.27, m), Cδ,ꢀ,êH (m, d, d* 7.02-7.46, m); L-PenN-
Ala2, N-CH2CH2CH2CHCH2 (m 2.93, 2.94, m; d 3.02, 3.57, m; d*
3.06, 3.66, m), N-CH2CH2CH2CHCH2 (m 1.07, 1.19, m; d 1.70, 1.77,
m; d*1.86, 1.95, m), N-CH2CH2CH2CHCH2 (m 1.84, m; d 2.02, 2.17,
m; d* 2.08, 2.24, m), N-CH2CH2CH2CHCH2 (m 5.61, m; d 5.90, m;
d* 5.95, m), N-CH2CH2CH2CHCH2 (m 4.89 (E), 4.93 (Z), m; d 5.08
(E and Z), m; d* 5.09 (Z), 5.13 (E), m), CRH (m 5.12, ovl; d 5.79, ovl;
d* 5.48, ovl), CâH3 (m 1.16, ovl; d 1.13, d, J ) 6.8 Hz; d* 1.06, d, J
) 7.4 Hz); D-Phe3, N-H (m 7.30, br; d 8.50, d, J ) 7.5 Hz; d* 8.69,
d, J ) 8.1 Hz), CRH (m 4.73, m; d 4.53, m; d* 4.63, m), CâH2 (m
2.97, 3.02, m; d 2.77, 3.27, m; d* 2.88, 3.09, m), Cδ,ꢀ,êH (m, d, d*
7.02-7.46, m); L-Hag4, N-H (m 6.56, d, J ) 8.1 Hz; d 6.51, d, J )
8.2 Hz; d* 6.45, br), CRH (m 4.61, m; d 4.94, m; d* 5.16, m), CâH2
(m 1.69, 1.78, m; d 1.06, 1.20, m; d* 1.20, 1.28, m), CγH2 (m 1.89,
m; d 1.14, m; d* 1.52, m), CδH (m 5.75, m; d 5.45, m; d* 5.55, m),
CꢀH2 (m 5.01 (E and Z), m; d 4.77 (E), 4.90 (Z), m; d* 4.83 (E), 4.93
(Z), m). IR (293 K, 1 mM, CHCl3): 3304 (amide A); 1627, 1654,
1687 (amide I ); 1523 (amide II|). ESI-MS: [M + H]+ calcd ) 1089.6,
found ) 1090.
cyclo[(-D-Phe1-L-PenN-Ala2-D-Phe3-L-MeN-Hag4)2-] (8). Linear oc-
tapeptide (-D-Phe-L-PenN-Ala-D-Phe-L-MeN-Hag)2 (8-x) was synthesized
on N-Boc-L-MeN-Hag-(OCH2)-Pam resin (0.4 mmol scale) and cleaved
cyclo[(-L-Phe1-D-MeN-Ala2-L-Ile3-D-MeN-Ala4)2-] (11). Linear oc-
tapeptide (-L-Phe-D-MeN-Ala-L-Ile-D-MeN-Ala)2 (11-x) was synthesized
on N-Boc-D-MeN-Ala-(OCH2)-Pam resin (0.4 mmol scale) and cleaved
with HF to give 100 mg of the desired product as its TFA salt after
purification by RP-HPLC (101 µmol, 25%; ESI-MS, [M + H]+ calcd
) 879.5, found ) 880). Cyclization of 100 mg of 11-x (101 µmol)
and purification as described above afforded 50 mg of 11 (58 µmol,
57%). 1H NMR (500 MHz, 293 K, 22.1 mM, CDCl3): L-Phe1, N-H
(m 7.05, br; d 8.71, d, J ) 7.0 Hz; d* 8.87, d, J ) 6.5 Hz), CRH (m
4.85, m; d 5.28, m; d* 5.28, m), CâH2 (m 3.04, m; d 2.93, 3.13, m; d*
2.91, 3.17, m), Cδ,ꢀ,êH (m, d, d* 7.16-7.26, m); D-MeN-Ala2, N-CH3
(m 2.55, s; d 2.81, s; d* 2.72, s), CRH (m 5.17, ovl; d 5.80, q, J ) 6.8
50c
with Bu4NHSO4-K2CO3 to give 281 mg of the desired product as
its TFA salt after purification by RP-HPLC (225 µmol, 56%).
Cyclization of 281 mg of 8-x (225 µmol) and purification as described
above afforded 35 mg of 8 (35 µmol, 16%). 1H NMR (500 MHz, 293
K, CDCl3): D-Phe1, N-H (m1 8.50, d, J ) 6.8 Hz; m2 7.00, d, J )
10.2 Hz; d 8.87, d, J ) 8.9 Hz; d* 8.77, d, J ) 9.5 Hz), CRH (m1 4.63,
m; m2 4.91, m; d 5.41, m; d* 5.49, m), CâH2 (m1 2.56, 2.69, m; m2
2.99, 3.12, m; d 3.05, 3.10, m; d* 3.03, 3.06, m), Cδ,ꢀ,êH (m1, m2, d,
d* 7.03-7.46, m); L-PenN-Ala2, N-CH2CH2CH2CHCH2 (m1 3.31, 3.49,