Grotenbreg et al.
2.42 (s, 3H, CH3 Tos). 13C NMR (100 MHz, CDCl3): δ 168.1
(COOMe, CdO Phth), 144.9 (Cq Tos), 134.2 (CH Phth), 132.4
(Cq Tos), 131.3 (Cq Phth), 129.8, 128.1 (CH Phth), 77.2 (C6),
73.4 (C2), 71.6 (C4), 70.6 (C5), 68.9 (C7), 53.5 (C3), 52.5 (OMe),
21.5 (CH3 Tos). ATR-IR (thin film): 3456.5, 2923.9, 1774.4,
1708.8, 1386.7, 1359.7, 1190.0, 1174.6, 1118.6, 1095.5, 966.3,
in 20 min; m/z 1070.8 [M + H]+, 536.1 [M + H]2+) and purified
by RP-HPLC (linear gradient of 3.0 CV; 40f50% B; Rt 1.9 CV).
1H NMR (600 MHz, CD3OH): δ 8.90 (d, 1H, NH DPhe5, JNH,HR
) 3.5 Hz), 8.68 (d, 1H, NHR Orn3, JNH,HR ) 8.1 Hz), 8.62 (d,
1H, NH Leu4, JNH,HR ) 9.4 Hz), 8.61 (d, 1H, NHR Orn8, JNH,HR
) 8.9 Hz), 8.56 (d, 1H, NH Leu9, JNH,HR ) 8.9 Hz), 8.07 (t, 1H,
NH SAA1, JNH,7 ) 6.1 Hz), 7.86 (br s, 2H, NHδ Orn3,8), 7.74 (d,
813.9, 719.4 cm-1. [R]23 +20.4 (c 1.0, CHCl3). MS (ESI): m/z
D
506.0 [M + H]+, 528.3 [M + Na]+. HRMS: calcd for C23H23-
1H, NH Val7, JNH,HR ) 8.6 Hz), 7.55 (d, 1H, NH Val2, JNH,HR
)
NO10SNH4 523.1386, found 523.1396.
8.5 Hz), 7.38-7.21 (m, 5H, Har), 4.98 (m, 1H, HR Orn3), 4.71
(m, 1H, HR Orn8), 4.65 (m, 1H, HR Leu4), 4.56 (m, 1H, HR Leu9),
4.51 (m, 1H, HR DPhe5), 4.34 (m, 1H, HR Pro6), 4.24 (m, 1H, HR
Val2), 4.06 (m, 1H, HR Val7), 3.95 (m, 2H, H3,6 SAA1), 3.86 (dd,
1H, H5 SAA1, J5,4 ) 5.2 Hz, J5,6 ) 3.0 Hz), 3.78 (dd, 1H, H4
SAA1, J4,5 ) 5.2 Hz, J4,3 ) 6.5 Hz), 3.72 (m, 1H, Hδd Pro6),
3.36 (m, 1H, H7d SAA1), 3.31 (m, 1H, H7u SAA1), 3.07 (dd, 1H,
Hâd DPhe5, Jâd,âu ) 12.6 Hz, Jâd,R ) 5.0 Hz), 3.02 (m, 1H, Hδd
Orn3), 2.98 (m, 1H, Hδd Orn8), 2.96 (m, 3H, Hδu Orn3, Hδu Orn8,
Hâu DPhe5), 2.50 (m, 3H, H2 SAA1, Hδu Pro6), 2.28 (m, 1H, Hâ
Val7), 1.99 (m, 3H, Hâd Pro6, Hâd Orn3, Hâ Val2), 1.83 (m, 1H,
Hâd Orn8), 1.74 (m, 2H, H Orn3), 1.71 (m, 2H, Hâu, Pro6),
The tosylate (3.42 g, 6.76 mmol) was then dissolved in DMF
(35 mL) and NaN3 (4.4 g, 67.6 mmol) was added. The reaction
mixture was stirred at 80 °C for 48 h and subsequently
concentrated. The residue was diluted with water and ex-
tracted twice with EtOAc. The combined organic layers were
successively washed with saturated aqueous NaHCO3 and
brine, dried (MgSO4), and concentrated. The crude product was
applied to a silica gel column (60f80% EtOAc in light PE) to
1
yield azide 12 (2.15 g, 5.72 mmol, 85%) as a white foam. H
NMR (400 MHz, CDCl3): δ 7.80-7.72 (m, 4H, Phth), 4.72 (d,
1H, H2, J2,3 ) 10.4 Hz), 4.39 (dd, 1H, H4, J4,3 ) 10.4 Hz, J4,5
)
γ
γd
1.67 (m, 1H, Hâu Orn3), 1.66 (m, 2H, Hγ Orn8), 1.64 (m, 3H,
Hâ, Leu9), 1.59 (m, 1H, Hγu Pro6), 1.56 (m, 2H, Hâd, Leu4),
9.2 Hz), 4.25 (dd, 1H, H3, J3,4 ) 10.4 Hz, J3,2 ) 10.4 Hz), 3.59-
3.41 (m, 4H, H5, H6, H7), 3.55 (s, 3H, OCH3). 13C NMR (100
MHz, CDCl3): δ 168.3, 168.2 (COOMe, CdO Phth), 134.3 (CH
Phth), 131.4 (Cq Phth), 123.5 (CH Phth), 78.5 (C6), 73.4 (C2),
71.8 (C4), 71.8 (C5), 53.6 (C3), 52.6 (OMe), 51.2 (C7). ATR-IR
(thin film): 3455.0, 2100.3, 1774.4, 1741.0, 1705.0, 1436.9,
1384.8, 1286.4, 1114.8, 1066.6, 1010.6, 964.3, 873.7, 719.4
cm-1. [R]23D +54.4 (c 1.00, CHCl3). MS (ESI): m/z 377.2 [M +
H]+, 398.9 [M + Na]+. HRMS: calcd for C16H16N4O7NH4
394.1363, found 394.1340.
γ
γ
1.39 (m, 1H, Hâu Leu4), 0.95 (m, 3H, Hγd Val7), 0.94 (m, 3H,
Hγd Val2), 0.92 (m, 3H, Hγu Val2), 0.90 (m, 6H, Hδ Leu4), 0.88
(m, 3H, Hγu Val7), 0.86 (m, 6H, Hδ Leu9). ATR-IR (thin film):
3278.1, 3071.9, 2959.2, 2935.6, 2873.4, 1669.8, 1636.5, 1539.2,
1464.7, 1456.7, 1437.0, 1203.7, 1182.7, 1135.0, 1033.3, 1020.8,
837.1, 800.1, 722.6, 702.5 cm-1. HRMS: calcd for C53H87N11O12H
1079.6608, found 1070.6521.
cyclo-[SAA6-Val-Orn-Leu-DPhe-Pro-Val-Orn-Leu] (15c).
Analyzed by LC/MS (Rt 15.79 min; linear gradient 10f90% B
in 20 min; m/z 1054.8 [M + H]+, 528.2 [M + H]2+) and purified
by RP-HPLC (linear gradient of 3.0 CV; 35f55% B; Rt 2.7 CV).
1H NMR (600 MHz, CD3OH): δ 8.99 (d, 1H, NH DPhe5, JNH,HR
) 3.1 Hz), 8.76 (d, 1H, NHR Orn3, JNH,HR ) 6.7 Hz), 8.74 (d,
1H, NH Leu4, JNH,HR ) 7.6 Hz), 8.56 (d, 1H, NH Leu9, JNH,HR
) 9.2 Hz), 8.51 (d, 1H, NHR Orn8, JNH,HR ) 9.4 Hz), 8.11 (t,
1H, NH SAA1, JNH,7 ) 6.3 Hz), 7.87 (br s, 2H, NHδ Orn3,8),
7.69 (d, 1H, NH Val2, JNH,HR ) 8.5 Hz), 7.60 (d, 1H, NH Val7,
JNH,HR ) 9.3 Hz), 7.32-7.23 (m, 5H, Har), 4.90 (m, 1H, HR Orn8),
4.73 (m, 1H, HR Orn3), 4.64 (m, 1H, HR Leu4), 4.50 (m, 2H, HR
DPhe5, HR Leu9), 4.39 (m, 2H, HR Pro6, HR Val7), 4.13 (m, 1H,
HR Val2), 3.71 (m, 1H, Hδd Pro6), 3.52 (m, 1H, H7d SAA1), 3.44
(2, 2H, H2,3 SAA1), 3.41 (m, 1H, H6 SAA1), 3.08 (dd, 1H, Hâd
DPhe5, Jâd,âu ) 12.9 Hz, Jâd,R ) 4.9 Hz), 3.07 (m, 1H, H7u SAA1),
3.01 (m, 1H, Hδd Orn3), 2.93 (dd, 1H, Hâu DPhe5, Jâd,âu ) Jâd,R
) 12.9 Hz), 2.87 (m, 3H, Hδ Orn8, Hδu Orn3), 2.46 (m, 1H, Hδu
Pro6), 2.23 (m, 1H, Hâ Val7), 2.14 (m, 1H, Hâ Val2), 2.09 (m,
1H, H4d SAA1), 2.01 (m, 2H, Hâd Pro6, Hâd Orn3), 1.77 (m, 2H,
Hâ Orn3), 1.71 (m, 1H, H5d SAA1), 1.62 (m, 5H, Hâd Leu9, Hâd
Leu6, Hâd Orn8, Hâu Pro6, Hγu Orn3), 1.52 (m, 7H, Hâu, γd Orn8,
Hγ Leu4, Hγ Leu9, Hγ Pro6, H4u SAA1), 1.42 (m, 4H, Hâu, Leu9,
Hâu, Leu6, Hγu Orn8, H4u SAA1), 1.42 (m, 1H, Hâu Leu4), 1.02
(d, 3H, Hγd Val2 Jγ,â ) 6.7 Hz), 0.97 (d, 3H, Hγu Val2 Jγ,â ) 6.8
Hz), 0.92 (d, 6H, Hγ Val7), 0.93-0.87 (m, 12H, Hδ Leu4, Hδ
Leu9). ATR-IR (thin film): 3267.7, 3061.3, 2957.6, 2933.1,
2870.1, 1675.2, 1639.5, 1538.9, 1456.8, 1203.4, 1182.1, 1133.3,
1060.1, 1033.4, 838.3, 800.0, 749.1, 722.8, 701.7 cm-1. HRMS:
calcd for C53H87N11O11H 1054.6659, found 1054.6622.
Assembly of GS Analogues 14a-h. Resin-anchored pep-
tides 13a-d (100 µmol) were constructed from 1 according to
the general SPPS procedure. Reduction of the N-terminal azide
was accomplished by washing the solid support with 1,4-
dioxane (5 mL, 3 × 3 min) and dispersing it in 1,4-dioxane
(10 mL), to which trimethylphosphine (16 equiv, 1.6 mL, 1.6
mmol, 1 M in toluene) was added. The resin was shaken for 2
h, water (1 mL) was added, and shaking was continued
another 4 h. The resin was washed with 1,4-dioxane (5 mL, 3
× 3 min) and DCM (5 mL, 3 × 3 min). The peptides were
released from the resin, cyclized, and purified as described
above to yield the protected 14a (96%), 14b (63%), 14c (85%),
and 14d (78%), respectively, as amorphous white solids. The
assembly of peptides 13e-h was performed in a similar
manner, to furnish 14e (36%), 14f (43%), 14g (72%), and 14h
(78%), respectively.
Deprotection of 14a-h. The pivaloyl protection groups in
14a (32 mg, 24 µmol) and 14e (12 mg, 10 µmol) were removed
by dissolving the peptides in MeOH (5 mL), followed by
addition of NaOMe (16 equiv, 20 mg, 370 mmol) and stirring
overnight. The mixtures were neutralized with Amberlite IR-
120 (H+), filtered, and concentrated and the crudes were used
directly in the following Boc-deprotection step. For peptides
14d (17 mg, 13 µmol) and 14h (17 mg, 11.4 µmol), the
phthaloyl protection groups were removed by dissolving the
peptides in MeOH (5 mL), followed by addition of hydrazine-
monohydrate (50 equiv, 28 µL, 0.57 mmol). After refluxing for
16 h, the solvents were evaporated and the crude compounds
were used without further purification in the following Boc-
deprotection. Removal of the Boc protection groups in the
aforementioned peptides, as well as 14b (14 mg, 11.0 µmol),
14c (14 mg, 11 µmol), 14f (6 mg, 5.0 µmol), and 14g (12 mg,
10.3 µmol), was performed according to the general procedure
to give 15a (22.0 mg, 20.8 µmol, 87%), 15b (8.1 mg, 7.6 µmol,
69%), 15c (9.8 mg, 9.3 µmol, 85%), 15d (12.5 mg, 11.5 µmol,
88%), 15e (9.0 mg, 9.3 µmol, 93%), 15f (4.2 mg, 4.2 µmol, 84%),
15g (9.6 mg, 9.9 µmol, 96%), and 15h (6.9 mg, 6.7 µmol, 59%),
respectively, as white amorphous powders.
cyclo-[SAA7-Val-Orn-Leu-DPhe-Pro-Val-Orn-Leu] (15d).
Analyzed by LC/MS (Rt 12.92 min; linear gradient 10f90% B
in 20 min; m/z 1086.0 [M + H]+, 543.6 [M + H]2+) and purified
by RP-HPLC (linear gradient of 3.0 CV; 30f50% B; Rt 2.8 CV).
1H NMR (600 MHz, CD3OH): δ 8.92 (d, 1H, NH DPhe5, JNH,HR
) 3.3 Hz), 8.69 (d, 1H, NH Leu4, JNH,HR ) 9.2 Hz), 8.61 (d, 1H,
NHR Orn3, JNH,HR ) 8.9 Hz), 8.58 (d, 1H, NHR Orn8, JNH,HR
)
9.3 Hz), 8.47 (d, 1H, NH Leu9, JNH,HR ) 9.1 Hz), 8.09 (t, 1H,
NH SAA1, JNH,7 ) 6.1 Hz), 7.72 (d, 1H, NH Val7, JNH,HR ) 9.0
Hz), 7.70 (d, 1H, NH Val2, JNH,HR ) 9.1 Hz), 7.38-7.21 (m,
5H, Har), 4.97 (m, 1H, HR Orn3), 4.80 (m, 1H, HR Orn8), 4.64
(m, 1H, HR Leu4), 4.57 (m, 1H, HR Leu9), 4.49 (m, 1H, HR
DPhe5), 4.34 (m, 1H, HR Pro6), 4.32 (m, 1H, HR Val2), 4.07 (m,
cyclo-[SAA4-Val-Orn-Leu-DPhe-Pro-Val-Orn-Leu] (15a).
Prepared as described previously.11
cyclo-[SAA5-Val-Orn-Leu-DPhe-Pro-Val-Orn-Leu] (15b).
Analyzed by LC/MS (Rt 14.71 min; linear gradient 10f90% B
7858 J. Org. Chem., Vol. 69, No. 23, 2004