Gracia et al.
Procedures, and DMF (5 × 0.5 min). The loading was 0.50 mmol/
g, as calculated by Fmoc determination.
MALDI-TOF-MS calcd for C64H89N9O13 1,191.66; found m/z
1,092.17 [M + H]+, 1,214.14 [M + Na]+, 1,230.10 [M + K]+.
H-Orn(Boc)-D-allo-Ile-D-allo-Thr(&)-D-allo-Ile-D-Val-Phe-
ZDhb-Val& (N-Component 1). The protected peptide (6) (50 mg,
42 µmol) was dissolved in DMF (5 mL), then DEA (130 µL, 30
equiv) was added and the mixture was stirred for 90 min. The
solvent was removed by evaporation under reduced pressure. The
crude product was purified by HPLC (Symmetry C8 5 µm, 30 ×
100 mm), linear gradient of MeCN (30% to 75% in 15 min) MeCN
(+0.05% TFA) in water (+0.05% TFA), 20 mL/h, detection at 220
nm. The product was characterized by HPLC (Rt 8.7 min, Condition
A) and MALDI-TOF-MS calcd for C49H79N9O11 969.59; found m/z
970.87 [M + H]+, 870.78 [M - Boc]+.
N-Component 1: H-Orn(Boc)-D-allo-Ile-D-allo-Thr(&)-D-allo-
Ile-D-Val-Phe-ZDhb-Val& from pNZ-Orn(Boc)-OH.
pNZ-Orn(Boc)-D-allo-Ile-D-allo-Thr(&)-D-allo-Ile-D-Val-Phe-
ZDhb-Val& (7). The synthesis was performed in the same way as
for compound 6, but Fmoc-Orn(Boc)-OH was replaced by pNZ-
Orn(Boc)-OH.
[Fmoc-D-allo-Ile-D-allo-Thr(&)-D-allo-Ile-D-Val-O-TrtCl-re-
sin][Alloc-Val&] (2). Fmoc-D-allo-Ile-OH (707 mg, 2 mmol, 4
equiv), Fmoc-D-allo-Thr-OH (free hydroxy group) (683 mg, 2
mmol, 4 equiv), and Fmoc-D-allo-Ile-OH (707 mg, 2 mmol, 4 equiv)
were added sequentially to the above obtained H-D-Val-O-TrtCl-
resin (1) using DIPCDI (310 µL, 2 mmol, 4 equiv) and HOBt (307
mg, 2 mmol, 4 equiv) in DMF (2.5 mL). In all cases, after 90 min
of coupling, the ninhydrin test was negative. Removal of Fmoc
group and washings were performed as described in General
Procedures. Alloc-Val-OH (502 mg, 2.5 mmol, 5 equiv) was
coupled with DIPCDI (387 mg, 2.5 mmol, 5 equiv) in the presence
of DMAP (30.6 mg, 0.25 mmol, 0.5 equiv) and DIPEA (88 µL,
0.5 mmol, 1 equiv) for 45 min. This coupling was repeated twice
in the same conditions. An aliquot of the peptidyl-resin was treated
with TFA and the HPLC (Rt 14.2 min, column A) of the crude
obtained after evaporation showed a purity of >98%. ESMS calcd
for C45H63N5O11 849.45; found m/z 850.1 [M + H]+, 871.9 [M +
Na]+.
[Fmoc-Orn(Boc)-D-allo-Ile-D-allo-Thr(&)-D-allo-Ile-D-Val-O-
TrtCl-resin][Alloc-Val&] (3). The Fmoc group of the peptide resin
(2) was removed, and Fmoc-Orn(Boc)-OH (912 mg, 2 mmol, 4
equiv) was added using DIPCDI (310 µL, for 2.0 mmol and 4 equiv;
and 388 µL, for 2.5 mmol and 5 equiv) and HOBt (307 mg, for
2.0 mmol and 4 equiv; and 395 mg, for 2.5 mmol and 5 equiv) for
90 min. Ninhydrin test after the incorporation was negative. An
aliquot of the peptidyl-resin was treated with TFA and the HPLC
(Rt 12.8 min, column A) of the crude obtained after evaporation
showed a purity of 90%. ESMS calcd for C56H81N7O14 1,063.58;
found m/z 1,086.77 [M + Na+]+.
[Fmoc-Orn(Boc)-D-allo-Ile-D-allo-Thr(&)-D-allo-Ile-D-Val-O-
TrtCl-resin][Alloc-Phe-ZDhb-Val&] (4). The Alloc group of the
peptide resin (3) was removed with Pd(PPh3)4 (58 mg, 0.05 mmol,
0.1 equiv) in the presence of PhSiH3 (617 µL, 5 mmol, 10 equiv),
followed by washings with diethyldithiocarbamate 0.02 M (3 ×
15 min). Alloc-Phe-ZDhb-OH (666 mg, 2 mmol, 4 equiv) and HOAt
(273 mg, 2 mmol, 4 equiv) were dissolved in DMF (1.25 mL) and
added to peptidyl-resin. DIPCDI (310 µL, 2 mmol, 4 equiv) was
then added, and the mixture was stirred for 5 h. The ninhydrin test
was negative. After washings with DMF and CH2Cl2, an aliquot
of the peptidyl-resin was treated with TFA-H2O (1:99) for 1 min,
and the product was characterized by MALDI-TOF-MS: calcd for
C68H95N9O16 1,293.69; found m/z 1,294.35 [M + H]+, 1,316.39
[M + Na]+, 1,333.34 [M + K]+.
[Fmoc-Orn(Boc)-D-allo-Ile-D-allo-Thr(&)-D-allo-Ile-D-Val-
OH][H-Phe-ZDhb-Val&] (5). The Alloc group of the peptide resin
(4) was removed with Pd(PPh3)4 (58 mg, 0.05 mmol, 0.1 equiv) in
the presence of PhSiH3 (617 µL, 5 mmol, 10 equiv), the resin was
washed with sodium diethyldithiocarbamate in DMF 0.02 M (3 ×
15 min), and the protected peptide was cleaved from the resin by
TFA-CH2Cl2 (1:99) (5 × 30 s). The filtrate was collected on H2O
(4 mL) and the H2O was partially removed under reduced pressure.
MeCN was then added to dissolve solid that formed during the
removal of H2O, and the solution was lyophilized to give 700 mg
of title compound (578 µmol, 99% yield) of the title compound
with a purity of > 91% as checked by HPLC (Column A, Rt 8.59
min)], which was used without further purification. MALDI-TOF-
MS calcd for C64H91N9O14 1,209.67; found m/z 1,210.45 [M + H]+,
1,232.51 [M + Na]+, 1,248.45 [M + K]+.
H-Orn(Boc)-D-allo-Ile-D-allo-Thr(&)-D-allo-Ile-D-Val-Phe-
ZDhb-Val& (N-Component 1). The protected peptide (14.7 mg,
12.8 µmol) was dissolved in 1.6 mM HCl in DMF (10 mL), SnCl2
(3.8 g, 20 mmol) was then added, and the mixture was stirred until
HPLC (Column A) showed the completion of the reaction (1 h).
The solvent was removed by evaporation under reduced pressure.
The crude product was purified by HPLC (Symmetry C8 5 µm, 30
mm × 100 mm), gradient of MeCN (30% to 75% in 15 min) MeCN
(+0.05% TFA) in water (+0.05% TFA), 20 mL/h, detection at 220
nm, to give the title product (4.8 mg, 4.9 µmol, 40% yield). The
product was characterized by HPLC (Rt 8.2 min, Column A) and
by MALDI-TOF-MS: calcd for C49H79N9O11 969.59; found m/z
992.35 [M + Na]+, 870.34 [M - Boc]+, 892.34 [M + Na - Boc]+.
N-Component 1: H-Orn(Boc)-D-allo-Ile-D-allo-Thr(&)-D-allo-
Ile-D-Val-Phe-ZDhb-Val& from Alloc-Orn(Boc)-OH
H-Phe-ZDhb-O-TrtCl-resin (8). Cl-TrtCl-resin (1 g, 1.64 mmol/
g) was placed in a 20 mL polypropylene syringe fitted with a
polyethylene filter disk. The resin was then washed with CH2Cl2
(5 × 0.5 min), and a solution of Alloc-Phe-ZDhb-OH (232 mg,
0.7 mmol, 0.42 equiv) and DIPEA (0.41 mL) in CH2Cl2 (2.5 mL)
was added. This mixture was then stirred for 15 min. Extra DIPEA
(0.81 mL, total 7 mmol) was added, and the mixture was stirred
for an additional 45 min. The reaction was arrested by adding
MeOH (800 µL), after stirring for 10 min. The Alloc-Phe-ZDhb-
O-TrtCl-resin was subjected to washings with CH2Cl2 (3 × 0.5
min) and DMF (3 × 0.5 min), and the Alloc group was removed
with Pd(PPh3)4 (58 mg, 0.05 mmol, 0.1 equiv) in the presence of
PhSiH3 (617 µL, 5 mmol, 10 equiv) in CH2Cl2. The resin was
washed as described in General Procedures. The loading was 0.68
mmol/g, as calculated by Fmoc determination.
[Alloc-Orn(Boc)-D-allo-Ile-D-allo-Thr(&)-D-allo-Ile-D-Val-Phe-
ZDhb-OH][H-Val&] (9). Fmoc-D-Val-OH (678 mg, 2 mmol, 4
equiv), Fmoc-D-allo-Ile-OH (707 mg, 2 mmol, 4 equiv), Fmoc-D-
allo-Thr-OH (free hydroxy group) (683 mg, 2 mmol, 4 equiv),
Fmoc-D-allo-Ile-OH (707 mg, 2 mmol, 4 equiv), and Alloc-Orn-
(Boc)-OH (630 mg, 2 mmol, 4 equiv) were added sequentially to
the above obtained H-Phe-ZDhb-O-TrtCl-resin (8) using DIPCDI
(310 µL, 2 mmol, 4 equiv) and HOBt (307 mg, 2 mmol, 4 equiv)
in DMF (2.5 mL). In all cases, after 90 min of coupling, the
ninhydrin test was negative. Removal of Fmoc group and washings
were performed as described in General Procedures. Fmoc-Val-
OH (848.2 mg, 2.5 mmol, 5 equiv) was coupled with DIPCDI (387
mg, 2.5 mmol, 5 equiv) in the presence of DMAP (30.6 mg, 0.25
mmol, 0.5 equiv) and DIPEA (88 µL, 0.5 mmol, 1 equiv) for 45
min. This coupling was repeated twice in the same conditions. After
removal of the Fmoc group as described in General Procedures,
the protected peptide was cleaved from the resin by TFA-CH2Cl2
(1:99) (5 × 30 s). Filtrate was collected on H2O (4 mL), and the
H2O was partially removed under reduced pressure. MeCN was
then added to dissolve the solid that formed during H2O removal,
Fmoc-Orn(Boc)-D-allo-Ile-D-allo-Thr(&)-D-allo-Ile-D-Val-Phe-
ZDhb-Val& (6). The protected peptide (5) was dissolved in CH2-
Cl2 (580 mL, 1 mM), and HOBt (137 mg, 2.3 mmol) dissolved in
the minimum volume of DMF to dissolve HOBt, DIPEA (302 µL,
1.73 mmol, 3 equiv), and DIPCDI (356 µL, 2.3 mmol, 4 equiv)
were added. The mixture was stirred for 1 h, and the course of the
cyclization step was then checked by HPLC (column A, Rt 12.4
min). The solvent was removed by evaporation under reduced
pressure and the product was used without further purification.
7202 J. Org. Chem., Vol. 71, No. 19, 2006