Synthesis of Marine Cyclodepsipeptide IB-01212
performed using 2,5-dihydroxybenzoic acid (DHB). 1H, 13C,
TOCSY, ROESY, GHMBC, and GHSQC spectra were recorded
on either 400 and 500 MHz. 1H and 13C NMR chemical shifts are
expressed in parts per million (ppm) downfield from tetramethylsilyl
chloride (TMS) as internal reference. The coupling constants are
expressed in Hertz and the multiplicities of the signals are indicated
as follows: s (singlet), d (doublet), t (triplet), q (quartet) and m
(multiplet).
equiv), and DIEA (978 µL, 5.8 mmol, 6 equiv) in DMF. After 1 h
of coupling, the reaction gave a negative result for the chroranil
test. The Fmoc group was removed as described above, and Fmoc-
Ser(tBu) (2.94 g, 7.7 mmol, 8 equiv) was coupled sequentially with
DIPCDI (588 µL, 3.8 mmol, 4 equiv) using the asymmetric
anhydride method. After 3 h of coupling, the reaction gave a
negative result for the chroranil test. The Fmoc group was removed
by treatment with piperidine/DMF (1 × 1 min, 2 × 10 min). Next,
the N,N-Me2Leu-OH (307 mg, 1.92 mmol, 2 equiv) was coupled
with PyBOP (999.2 mg, 1.92 mmol, 2 equiv), HOAt (261 mg, 1.92
mmol, 2 equiv), and DIEA (978 µL, 5.8 mmol, 6 equiv) for 2 h.
The reaction was verified by the ninhydrin test.30
Once the tetrapeptide was synthesized, it was cleaved from the
resin with TFA/DCM (1:99) (5 × 1 min). The combined filtrates
were evaporated to dryness under reduced pressure. The residue
was resuspended in TFA/H2O/TES (95:2.5:2.5) and stirred for 2 h
to eliminate the tBu group completely. The TFA was evaporated
under reduced pressure, and the residue was dissolved in CH3CN/
H2O (1:1) and then lyophilized to give the target compound (333
mg, 78% yield) with a purity of 71% as determined by HPLC (tR
7.8 min, condition A). MALDI-TOF calcd for C28H46N4O6 534.34,
found m/z 535.25 [M + H]+, 557.26 [M + Na]+, 573.24 [M +
K]+).
To a solution of the crude tetrapeptide (30 mg, 0.056 mmol) in
DCM/DMF/NMI (9:0.8:0.2, 10 mL) were added MSNT (60 mg,
0.152 mmol, 4 equiv) and DIEA (117 µL, 0.672 mmol, 12 equiv).
The mixture was stirred, and the cyclization was monitored by
HPLC-MS. Upon completion of the reaction, the solvent was
removed by evaporation under reduced pressure. The cyclic peptide
was dissolved in CH3CN/H2O (1:1) and purified by semipreparative
RP-HPLC using a linear gradient from 15% to 70% of CH3CN
(+0.05% TFA) in H2O (+0.1% TFA) for 30 min, 20 mL/min,
detection at 220 nm, to give the title product (0.7 mg, 0.7 µmol,
2.3% yield, 98% purity). HPLC analysis (tR ) 29.1 min, condition
B) and MALDI-TOF calcd for C56H88N8O10 1032.66, found m/z
1034.33 [M + H]+, 1056.28 [M + Na]+, 1072.24 [M + K]+.
Strategy 2. Linear Synthesis. Wang resin (1 g, 0.82 mmol/g)
was placed in a 20 mL polypropylene syringe fitted with a
polyethylene filter disk. After washing of the resin, a solution of
Fmoc-N-MePhe (658.3 mg, 1.64 mmol, 2 equiv), MSNT (486 mg,
1.64 mmol, 2 equiv), NMI (130 µL, 1.64 mmol, 2 equiv), and DIEA
(855 µL, 4.92 mmol, 6 equiv) was added. The mixture was stirred
for 3 h. The remaining hydroxyl functions were acetylated by
treatment with Ac2O/NMI/DIEA/DMF (2:1:1:6) for 30 min. The
Fmoc group was removed as described above. Next, Fmoc-N-
MeLeu (603 mg, 1.64 mmol, 2 equiv) was coupled using PyBOP
(853.5 mg, 1.64 mmol, 2 equiv), HOAt (223.2 mg, 1.64 mmol, 2
equiv) and DIEA (570 µL, 3.28 mmol, 4 equiv) in DMF. After
Fmoc removal and washing, Fmoc-Ser(tBu)-OH (2.515 g, 6.56
mmol, 8 equiv) was coupled with DIPCDI (508 µL, 3.28 mmol, 4
equiv) using the asymmetric anhydride method. After 3 h of
coupling, the Fmoc group was removed and N,N-Me2Leu-OH (262
mg, 1.64 mmol, 2 equiv) was incorporated with DIPCDI (254 µL,
1.64 mmol, 2 equiv) and HOAt (223 mg, 1.64 mmol, 2 equiv).
The Trt protecting group was removed completely by treatment
with TFA/TES/DCM (1.5:5:93.5) for 2 h. The TFA salt was
neutralized by washing with DIEA/DCM (1:19). Fmoc-N-MePhe
(1.316 g, 3.28 mmol, 4 equiv) was then esterified with MSNT (972
mg, 3.28 mmol, 4 equiv), NMI (260 µL, 3.28 mmol, 4 equiv) and
DIEA (1.140 mL, 6.56 mmol, 8 equiv) for 3 h. A second coupling
using similar conditions was carried out. Fmoc-N-MeLeu (1.205
g, 3.28 mmol, 4 equiv) was coupled with DIPCDI (254 µL, 1.64
mmol, 2 equiv) overnight. Similarly, Fmoc-Ser(tBu)-OH (2.515 g,
6.56 mmol, 8 equiv) was coupled with DIPCDI (508 µL, 3.28
mmol, 4 equiv) using the asymmetric anhydride method. Finally,
N,N-Me2Leu (262 mg, 1.64 mmol, 2 equiv) was incorporated with
Solid-phase syntheses were carried out in polypropylene syringes
(10-20 mL) fitted with a polyethylene porous disk. Solvents and
soluble reagents were removed by vacuum. Removal of the Fmoc
group was carried out with piperidine/DMF (1 × 1 min, 2 × 10
min) and DBU/piperidine/DMF/toluene (5:5:70:20) (1 × 1 min, 2
× 5 min). Washings between deprotection, coupling, and again,
deprotection steps were carried out with DMF (5 × 1 min) and
DCM (5 × 1 min) using 10 mL solvent/g resin per treatment.
Peptide syntheses and washes were performed at 25 °C. Solid-phase
syntheses were monitored by RP-HPLC of the intermediates
obtained from cleavage of small aliquots of peptidyl resin.
Fmoc-Ser(Trt)-OPfp. Fmoc-Ser(Trt)-OPfp was prepared as
described by Packman et al.29 To a solution of Fmoc-Ser(Trt)-OH
(2.63 mmol, 1.5 g) in DCM/DMF (9:1) (10 mL) was added
pentafluorophenol (2.63 mmol, 0.48 g) and DCC (3 mmol, 0.62 g)
at 0 °C. Upon reaction completion (2 h), the dicyclohexylurea was
filtered off. The filtrate was evaporated, and the residue was
resuspended in EtOAc. The organic phase was washed with 10%
aqueous Na2CO3 and H2O. The organic phase was dried over
MgSO4 and evaporated to yield the desired Fmoc-Ser(Trt)-OPfp
(1.7 g, 89% yield) with a purity of 97.5% as determined by HPLC
(tR ) 17.59 min, condition A). TLC of the crude product indicated
only one major component, which was UV positive. MALDI-TOF
calcd for C43H30F5NO5 735.20, found m/z 757.96 [M + Na]+,
773.86 [M + K]+.
Synthesis of Fmoc-Ser(Trt)-MeLeu-OH. To a solution of
Fmoc-Ser(Trt)-OPfp (0.95 mmol, 0.7 g) in DCM (5 mL) was added
N-MeLeu (1.5 mmol, 0.22 g) dissolved in DCM/DMF (1:1). The
mixture was treated with DIEA (0.95 mmol, 162 µL) and a catalytic
amount of HOBt. The mixture was left to stir at room temperature
and the reaction was monitored by TLC with UV-visualization
and RP-HPLC. Upon reaction completion, the solvent was removed
by evaporation under reduced pressure, and the resulting solid was
dissolved in EtOAc. The solution was washed with 10% citric acid,
saturated brine, and H2O. The organic phase was dried over Na2-
SO4, filtered, and evaporated to dryness to give Fmoc-Ser(Trt)-N-
MeLeu-OH (0.5 g, 76%) with a purity of 18% as determined by
HPLC (tR ) 16.18 min, condition A). The product was purified by
semipreparative RP-HPLC using a gradient from 50% to 100% of
CH3CN (+1% TFA) to H2O (+0.5% TFA). The pure product was
obtained with a 99.3% purity and a 14% global yield. MALDI-
TOF calcd for C44H44N2O6 696.32, found m/z 719.24 [M + Na]+,
735.21 [M + K]+.
Strategy 1. Dimerization of Heterodetic Fragments. ClTrt-Cl
resin (0.6 g, 1.6 mmol/g) was placed in a 10 mL polypropylene
syringe fitted with a polyethylene filter disk and then resin washed
with DCM (5 × 2 min), DMF (5 × 2 min), and DCM (5 × 2
min). A solution of Fmoc-N-MePhe-OH (240 mg, 0.6 mmol) and
DIEA (162 µL, 1 equiv) in DCM (2 mL) was added, and the
mixture was stirred for 5 min. DIEA (326 µL, 1.92 mmol, 2 equiv)
was added, and the mixture was stirred for 1 h. The reaction
quenched by the addition of MeOH (400 µL) with stirring for 15
min. The Fmoc-N-MePhe-O-Trt-Cl-resin was then washed with
DCM (3 × 1 min) and DMF (3 × 1 min) and treated with
piperidine/DMF (1 × 1 min, 2 × 10 min) and DBU/piperidine/
toluene/DMF (5:5:20:70, 1 × 1 min, 2 × 5 min). The loading
calculated by Fmoc determination was 0.7 mmol/g. The Fmoc-N-
MeLeu (705 mg, 1.92 mmol, 2 equiv) was added using PyBOP
(999.2 mg, 1.6 mmol, 2 equiv), HOAt (261.4 mg, 1.92 mmol, 2
(30) Kaiser, E.; Colescott, R. L.; Bossinger, C. D.; Cook, P. I. Anal.
Biochem. 1970, 34, 595.
(29) Packman, L. C.; Quibell, M.; Johnson, T. Pept. Res. 1994, 7, 125.
J. Org. Chem, Vol. 71, No. 9, 2006 3343