Lorenz and Diederichsen
CH2â), 32.6 (Cys-CH2â), 40.1 (Acm-CH2), 46.5 (Fmoc-CH), 48.1
(Ala-CHR), 49.7 (Ala-CHR), 52.0, 52.6, 52.8, 53.4 (Ser-CHR,
Cys-CHR), 56.8 (Val-CHR), 56.9 (Val-CHR), 63.2 (Ser-CH2â),
64.3 (Ser-CH2â), 65.9 (Fmoc-CH2, Z-CH2), 73.7 (Tce-CH2), 78.0
(tBu-quart C), 94.7 (Tce-CCl3), 120.0 (Fmoc-C5), 125.2 (Fmoc-
C2), 127.0, 127.6, 127.8, 128.3 (Fmoc-C3, Fmoc-C4, Z-aromat
CH), 136.6 (Z-quart C), 140.6 (Fmoc-C6), 143.7 (Fmoc-C1),
154.9, 155.9, 156.0 (Boc-CO, Fmoc-CO, Z-CO), 168.0, 168.1,
169.9, 170.1, 170.3, 170.4, 170.7, 170.8, 172.0, 172.8 (Ser-CO,
Val-CO, Cys-CO, Ala-CO, Acm-CO); HRMS (ESI) calcd for
dried (Na2SO4), filtered, and concentrated in vacuo. Flash
chromatography (SiO2, 2 × 15 cm, 15% methanol-ethyl
acetate, 0.3% HOAc) afforded 13b (52.7 mg, 44.2 µmol, 66%)
after coevaporation with toluene (3 × 5 mL) as an amorphous
white solid: TLC (20% methanol/ethyl acetate, 0.5% HOAc)
Rf 0.33; [R]25 -36 (c 0.05, MeOH); mp 165-168 °C; UV
D
(MeOH) λmax(ꢀ) 264 nm; IR (KBr) νmax 3420, 2926, 1684, 1654,
1560, 1541, 1458, 1399, 1249, 1168, 1062, 742 cm-1; 1H NMR
(DMSO-d6, 600 MHz) δ 0.82-0.88 (m, 12H, Val-CH3), 1.20 (d,
3H, 3J ) 7 Hz, Ala-CH3), 1.26 (d, 3H, 3J ) 7 Hz, Ala-CH3),
1.36 (s, 9H, tBu-CH3), 2.05-2.16 (m, 2H, Val-Hâ), 2.84 (dd,
C
64H85Cl3N10O19S2Na ([M + Na]+) 1489.4392, found 1489.4415.
(Z)-D-Ser {F m oc-D-Ser [Boc-L-Ala -L-Cys(Acm )-L-Va l]-L-
3
2
1H, J ) 11 Hz, J ) 13 Hz, Cys-Hâ), 2.90-3.05 (m, 3H, Cys-
Hâ), 3.42-3.54 (m, Ser-Hâ, revealed by two-dimensional
spectroscopy), 3.97-4.03 (m, 1H, Ser-HR), 4.09-4.15 (m, 1H,
Ala-HR), 4.21 (t, 1H, 3J ) 8 Hz, Fmoc-CH), 4.24-4.41 (m, 8H,
Fmoc-CH2, Val-HR, Ser-HR, Ser-Hâ), 4.42-4.47 (m, 1H, Ala-
HR), 4.91-4.97 (m, 1H, Cys-HR), 4.99-5.04 (m, 3H, Z-CH2,
Cys-HR), 6.86 (d, 1H, 3J ) 8 Hz, NH), 7.28-7.36 (m, 7H, Fmoc-
H3, Z-aromat CH), 7.38-7.43 (m, 2H, Fmoc-H4), 7.53-7.59
Ala -L-Cys(Acm )-L-Va l}-OH 12b. Compound 11b (230 mg,
0.16 mmol) was dissolved in 90% aqueous acetic acid (6 mL)
and cooled to 0 °C, and zinc powder (512 mg, 7.83 mmol, 50
equiv) was added. The resulting suspension was stirred for
2.5 h at 0 °C; the zinc was filtered off, and the filtrate was
concentrated in vacuo. The residue was treated with cold 1 N
aqueous HCl (15 mL) and extracted with ethyl acetate (3 ×
20 mL). The combined organic phases were washed with
saturated aqueous NaCl (10 mL), dried (MgSO4), filtered, and
concentrated in vacuo. Flash chromatography (SiO2, 2 × 18
cm, 20% methanol-ethyl acetate, 0.5% HOAc) furnished 12b
(198 mg, 0.15 mmol, 94%) after coevaporation with toluene (3
× 5 mL) as an amorphous white solid: TLC (20% methanol/
3
3
(m, br, 1H, NH), 7.62 (d, 1H, J ) 6 Hz, NH), 7.67 (d, 2H, J
) 7 Hz, Fmoc-H2), 7.77 (d, 1H, 3J ) 6 Hz, NH), 7.87 (d, 1H, 3J
) 8 Hz, Fmoc-H5), 7.88 (d, 1H, J ) 8 Hz, Fmoc-H5), 8.00 (d,
3
1H, 3J ) 5 Hz, NH), 8.10-8.21 (m, 2H, NH), 8.54 (d, 1H, 3J )
9 Hz, Cys-NH); 13C NMR (DMSO-d6, 150 MHz) δ 17.7, 17.9,
18.3, 18.8 (Val-CH3, Ala-CH3), 28.1 (tBu-CH3), 28.9 (Val-CHâ),
30.6 (Val-CHâ), 43.4 (Cys-CH2â), 47.7 (Fmoc-CH), 48.1 (Ala-
CHR), 48.5 (Ala-CHR), 51.8 (Cys-CHR), 52.3 (Cys-CHR), 54.3
(Ser-CHR), 55.0 (Ser-CHR), 57.4 (Val-CHR), 57.7 (Val-CHR),
65.1 (Fmoc-CH2/Z-CH2), 65.9 (Ser-CH2â), 66.7 (Fmoc-CH2/Z-
CH2), 78.3 (tBu-quart C), 119.9 (Fmoc-C5), 121.3 (Fmoc-C2),
127.2, 127.6, 128.2, 128.9 (Fmoc-C3, Fmoc-C4, Z-aromat CH),
137.4 (Z-quart C), 139.4 (Fmoc-C6), 142.5 (Fmoc-C1), 154.8,
155.4 (Boc-CO, Fmoc-CO, Z-CO), 169.2, 169.2, 170.0, 170.1,
171.0, 171.2 (Ser-CO, Val-CO, Cys-CO, Ala-CO); HRMS (ESI)
calcd for C56H72N8O17S2 ([M + H]+) 1193.4530, found 1193.4529.
ethyl acetate, 0.5% HOAc) Rf 0.14; [R]25 -45 (c 0.06, MeOH);
D
mp 124-128 °C; UV (MeOH) λmax(ꢀ) 265 nm; IR (KBr) νmax
3420, 2967, 1654, 1541, 1449, 1420, 1254, 1157, 742, 620 cm-1
;
1H NMR (DMSO-d6, 600 MHz) δ 0.78-0.85 (m, 12H, Val-CH3),
3
3
1.17 (d, 3H, J ) 7 Hz, Ala-CH3), 1.18 (d, 3H, J ) 7 Hz, Ala-
CH3), 1.36 (s, 9H, tBu-CH3), 1.76 (s, acetate-CH3), 1.83 (s, 3H,
Acm-CH3), 1.85 (s, 3H, Acm-CH3), 1.99-2.08 (m, 2H, Val-Hâ),
2.57-2.70 (m, 2H, Cys-Hâ), 2.87-2.94 (m, 1H, Cys-Hâ), 3.05-
3.10 (m, 1H, Cys-Hâ), 3.44-3.52 (m, 1H, Ser-Hâ, revealed by
two-dimensional spectroscopy), 3.85-3.90 (m, 1H, Ser-HR),
3.96-4.03 (m, 1H, Ala-HR), 4.07-4.12 (m, 1H, Val-HR), 4.13-
4.39 (m, 11H, Fmoc-CH, Fmoc-CH2, Acm-CH2, Ser-Hâ, Ala-
HR), 4.40-4.45 (m, 1H, Ser-HR), 4.48-4.54 (m, 1H, Cys-HR),
((Z)-D-Ser -L-Ala -L-Cys-L-Va l-F m oc-D-Ser -L-Ala -L-Cys-L-
Va l) (Ser in e Hyd r oxyl) Dila cton e Disu lfid e 14b. A sample
of 13b (23.8 mg, 19.9 µmol) was treated with TFA (1 mL), and
the mixture was stirred at 25 °C for 30 min. The TFA was
removed by evaporation in vacuo; the residue was treated with
1 N aqueous HCl (10 mL), extracted with ethyl acetate (3 ×
10 mL), dried (MgSO4), filtered, and evaporated in vacuo. The
resulting white solid was dissolved in 15 mL of CH2Cl2-DMF
(9:1) and directly used in the next step.
2
4.57-4.64 (m, 1H, Cys-HR), 4.95 (d, 1H, J ) 12 Hz, Z-CH2),
2
3
5.01 (d, 1H, J ) 12 Hz, Z-CH2), 6.60 (d, 1H, J ) 6 Hz, Ser-
NH), 6.86 (d, 1H, 3J ) 8 Hz, Ala-NH), 7.26-7.36 (m, 7H, Fmoc-
H3, Z-aromat CH), 7.37-7.43 (m, 2H, Fmoc-H4), 7.70 (d, 1H,
3J ) 8 Hz, Fmoc-H2), 7.72 (d, 1H, J ) 8 Hz, Fmoc-H2), 7.79
3
(d, 1H, 3J ) 6 Hz, Ser-NH), 7.87 (d, 2H, 3J ) 8 Hz, Fmoc-H5),
3
7.91-7.97 (m, 2H, Cys-NH, Val-NH), 8.08 (d, 1H, J ) 8 Hz,
The solution was cooled to 0 °C and under vigorous stirring
were added sequentially HOAt (2.71 mg, 19.9 µmol, 1 equiv),
NMM (6.58 µL, 59.8 µmol, 3 equiv) and DIC (31.2 µL, 199 µmol,
10 equiv). The mixture stirred at 4 °C for 36 h before being
poured onto cold 1n aqueous HCl (10 mL). The phases were
separated and the aqueous phase extracted with ethyl acetate
(2 × 10 mL). The combined organic phases were washed with
5% aqueous NaHCO3 (10 mL) and saturated aqueous NaCl (5
mL), dried (MgSO4), filtered and concentrated in vacuo.
Purification by flash chromatography (SiO2, 1 × 14 cm, ethyl
acetate) provided 14b (19.1 mg, 17.4 µmol, 87%) as amorphous
pale yellow solid: TLC (20% hexane/ethyl acetate) Rf 0.43;
3
Val-NH), 8.16-8.21 (m, 1H, Cys-NH), 8.26 (d, 1H, J ) 7 Hz,
Ala-NH), 8.54 (t, 1H, 3J ) 8 Hz, Acm-NH), 9.94 (s, br, 1H,
COOH); 13C NMR (DMSO-d6, 150 MHz) δ 17.9, 18.2, 18.8, 19.1
(Val-CH3, Ala-CH3), 22.5 (Acm-CH3), 22.9 (acetate-CH3), 28.1
(tBu-CH3), 29.8 (Val-CHâ), 30.4 (Val-CHâ), 32.3 (Cys-CH2â),
32.6 (Cys-CH2â), 40.3 (Acm-CH2, revealed by two-dimensional
spectroscopy), 47.7 (Ala-CHR/Fmoc-CH), 48.2 (Ala-CHR/Fmoc-
CH), 49.7 (Ala-CHR), 52.2 (Cys-CHR), 53.5 (Cys-CHR), 54.7
(Ser-CHR), 55.2 (Ser-CHR), 57.2 (Val-CHR), 57.8 (Val-CHR),
61.8 (Ser-CH2â), 65.1 (Fmoc-CH2/Z-CH2), 65.4 (Fmoc-CH2/Z-
CH2), 78.0 (tBu-quart C), 119.9 (Fmoc-C5), 121.3 (Fmoc-C5),
124.1 (Fmoc-C2), 127.2, 127.6, 128.2, 128.8 (Fmoc-C3, Fmoc-
C4, Z-aromat CH), 137.4 (Z-quart C), 139.3 (Fmoc-C6), 142.5
(Fmoc-C1), 154.9, 155.2 (Boc-CO, Fmoc-CO, Z-CO), 169.4,
169.9, 170.4, 170.7, 172.0, 172.7 (Ser-CO, Val-CO, Cys-CO, Ala-
CO, Acm-CO, acetate-CO); HRMS (ESI) calcd for C62H85N10O19S2
([M + H]+) 1337.5428, found 1337.5422.
[R]25 -64 (c 0.03, MeOH); mp 165-168 °C; UV (MeOH) λmax
-
D
(ꢀ) 265 nm; IR (KBr) νmax 3422, 2961, 2924, 2364, 1617, 1576,
1458, 1385, 1249, 1169 cm-1; 1H NMR (DMSO-d6, 600 MHz) δ
3
3
0.86 (d, 6H, J ) 7 Hz, Val-CH3), 0.88 (d, 6H, J ) 7 Hz, Val-
CH3), 1.22-1.27 (m, 6H, Ala-CH3), 2.17-2.28 (m, br, 2H, Val-
Hâ), 2.61-2.68 (m, br, 2H, Cys-Hâ), 2.81 (dd, 2H, 3J ) 11 Hz,
2J ) 14 Hz, Cys-Hâ), 4.08-4.43 (m, 13H, Fmoc-CH, Fmoc-
CH2, Ala-HR, Ser-HR, Ser-Hâ, Val-HR), 5.06-5.11 (m, br, 2H,
Z-CH2), 5.21-5.28 (m, br, 2H, Cys-HR), 7.28-7.34 (m, 2H,
Fmoc-H3), 7.34-7.38 (m, 5H, Z-aromat CH), 7.39-7.43 (m, 2H,
(Z)-D-Ser [F m oc-D-Ser (Boc-L-Ala -L-Cys-L-Va l)-L-Ala -L-
Cys-L-Va l]-OH Disu lfid e 13b. A solution of 12b (90.0 mg,
67.3 µmol) in 30 mL of CH2Cl2-MeOH (15:1) was added
dropwise to a solution of iodine (171 mg, 673 µmol, 10 equiv)
in 80 mL of CH2Cl2-MeOH (10:1), and the mixture was stirred
at 25 °C for 2.5 h. The mixture was cooled to 0 °C, and 5%
aqueous Na2S2O3 was added slowly until the excess iodine was
discharged and the color disappeared. The phases were
separated, and the organic phase was washed with 1 N
aqueous HCl (80 mL) and saturated aqueous NaCl (50 mL),
3
Fmoc-H4), 7.50-7.57 (m, br, 2H, NH), 7.66 (d, 2H, J ) 8 Hz,
3
3
Fmoc-H2), 7.74 (d, 2H, J ) 7 Hz, Ala-NH), 7.87 (d, 1H, J )
3
7 Hz, Fmoc-H5), 7.88 (d, 1H, J ) 7 Hz, Fmoc-H5), 8.12-8.20
(m, 2H, Val-NH), 8.65-8.71 (m, 2H, Cys-NH); 13C NMR
(DMSO-d6, 150 MHz) δ 18.1 (Ala-CH3), 18.2 (Val-CH3, Ala-
CH3), 18.89 (Val-CH3), 18.94 (Val-CH3), 30.0 (Val-CHâ), 30.2
3926 J . Org. Chem., Vol. 69, No. 11, 2004