SAR STUDY OF MARINOSTATIN
over anhydrous MgSO4. The filtrate was concentrated in vacuo.
The product was precipitated with ether, and the precipitates
were collected by filtration and dried in vacuo: yield 0.12 g (81%);
ESI-MS: 1067.1 ([M+2H]2+), theoretical value: 1067.0.
resin which was assembled onto a Fmoc-Asp[OTrt(2-Cl) resin]-
Glu(OcHx)-OBzl (0.39 g, 0.25 mmol) as described above for MST (1)
to yield 0.41 g (70%). The ester formation of Ser8-Asp11, followed
by that of Ser3-Asp9, was performed as reported previously [4] to
obtain 39 mg (60%). ESI-MS: 1368.5 ([M+H]+), theoretical value:
1368.6; amino acid analysis: Asp 1.98 (2), Ser 2.62 (3), Glu 1.00 (1),
Ala 1.02 (1), Met 0.89 (1), Tyr 0.96 (1), Phe 0.97 (1), Arg 0.97 (1), Pro
1.00 (1).
Z-Phe-Ala-Thr-Met-Arg(Tos)-Tyr(Pen)-Pro-Ser-Asp-Ser(Bzl)-Asp-
Glu(OcHx)-OBzl (with the ester linkages of Thr3-Asp9 and
Ser8-Asp11
)
[Ser8-Asp11]-MST(1–12) (3)
Z-Phe-Ala-Thr(tBu)-Met-Arg(Tos)-Tyr(Pen)-Pro-Ser-Asp(OtBu)-
Ser(Bzl)-Asp-Glu(OcHx)-OBzl with the ester linkage of Thr3-Asp9
(0.29 g, 0.14 mmol) was dissolved in TFA (100 ml) at 0 ◦C, and
then allowed to react for 1 h at room temperature. After removal
of excess TFA in vacuo, the product was precipitated with
diethylether, and collected by filtration, and dried to yield 0.27 g
(97%); ESI-MS: 1011.4 ([M+2H]2+), theoretical value: 1010.9. A
solution of this peptide (50 mg, 25 µmol) and TEA (3.4 µl, 25 µmol)
in anhydrous DCM/DMF (5/1, v/v, 2.4 ml) was added to a solution
of MNBA (10 mg, 30 µmol), DMAP (1.5 mg, 12 µmol), and TEA
(4.1 µl, 25 µmol) in anhydrous DCM/DMF (5/1, v/v, 3.6 ml) with a
mechanical driven syringe for 3.5 h. After being stirred for 19 h
at room temperature, the reaction mixture was quenched with
water at 0 ◦C, and then extracted with chloroform. The organic
layer was washed successively with 5% NaHCO3, water, 5% citric
acid, water, and brine, and then dried over anhydrous MgSO4. The
filtrate was concentrated in vacuo. The product was precipitated
with ether and the precipitates were collected by filtration, and
dried to yield 40 mg (81%). ESI-MS: 1002.5 ([M+2H]2+), theoretical
value: 1001.9.
This peptide was prepared as described above for [Ser3]-
MST(1–12) (2) to yield 39 mg (60%). ESI-MS: 1400.5 ([M+H]+),
theoretical value: 1400.6; amino acid analysis: Asp 2.02 (2), Thr 0.98
(1), Ser 1.82 (2), Glu 1.02 (1), Ala 1.01 (1), Met 0.94 (1), Tyr 1.00 (1),
Phe 1.01 (1), Arg 1.00 (1), Pro 1.03 (1).
Linear MST(1–12) (4)
The protected peptide was synthesized with an ABI 433A peptide
synthesizer on a Boc-Glu(OcHx)-PAM resin (0.59 g, 0.5 mmol)
using in situ neutralization protocols of coupling with Boc-amino
acid/HCTU/6-Cl-HOBt/DIEA (4/4/4/6 eq) in NMP (single coupling,
acetylation after each coupling step). The following side-chain-
protected Boc-amino acids were employed: Asp(OcHx), Arg(Tos),
Ser(Bzl),Thr(Bzl),andTyr(BrZ).Thepeptideresin(0.96 g,0.25 mmol)
was treated with HF (12 ml) in the presence of p-cresol (3.0 ml,
28 mmol) and 2-PySH (0.14 g, 1.3 mmol) at −2 to −5 ◦C for 1 h to
give a crude product, which was purified by RP-HPLC as described
above to obtain 0.21 g (60%). ESI-MS: 1418.2 ([M+H]+), theoretical
value: 1418.5; amino acid analysis: Asp 1.99 (2), Thr 0.97 (1), Ser
1.79 (2), Glu 1.00 (1), Ala 0.99 (1), Met 0.98 (1), Tyr 0.99 (1), Phe 0.99
(1), Arg 0.99 (1), Pro 1.04 (1).
MST (1)
Z-Phe-Ala-Thr-Met-Arg(Tos)-Tyr(Pen)-Pro-Ser-Asp-Ser(Bzl)-Asp-
Glu(OcHx)-OBzl with the ester linkages of Thr3-Asp9 and
Ser8-Asp11 (70 mg, 35 µmol) was treated with HF (2 ml) in the
presence of p-cresol (0.50 ml, 4.8 mmol) and 2-PySH (20 mg,
0.18 mmol) at −2 to −5 ◦C for 1 h to give a crude product,
which was purified by RP-HPLC using a YMC-Pak ODS column
(30 × 250 mm) at a flow rate of 20 ml/min. The RP-HPLC run was
eluted with a linear gradient of MeCN in 0.1% TFA (10–30%,
80 min) to obtain 21 mg (44%). MALDI-TOF MS: 1383.38 ([M+H]+),
theoretical value: 1383.46 ([M+H]+); amino acid analysis: Asp 1.98
(2), Thr 0.98 (1), Ser 1.99 (2), Glu 1.00 (1), Ala 1.04 (1), Met 0.94
[Thr3-Asp9]-MST(1–12) (5)
This peptide was prepared as described above for MST (1) to yield
36 mg (45%). ESI-MS: 1400.5 ([M+H]+), theoretical value: 1400.6;
amino acid analysis: Asp 2.00 (2), Thr 0.96 (1), Ser 1.80 (2), Glu 1.01
(1), Ala 1.00 (1), Met 0.94 (1), Tyr 0.98 (1), Phe 1.00 (1), Arg 1.00 (1),
Pro 1.02 (1).
[Thr3-Asp9]-MST(1–9) (6)
Protected peptide was synthesized with an ABI 433A peptide
synthesizer using Fmoc chemistry on an Fmoc-Asp(Wang resin)-
OBzl(0.40 g, 0.25 mmol). Thefollowing side-chain-protectedFmoc
amino acids were employed: Arg(Tos), Ser(Bzl), Thr(tBu), and
Tyr(Cl2Bzl). Z-Phe was used for N-terminal incorporation. The
chain assembly was performed by employing the same conditions
as those for MST (1) except for the Fmoc deprotection which was
carried out using 20% piperidine/NMP (2.5 min ×4). This peptide
was prepared as described above for [Ser3]-MST(1–12) (2) to yield
26 mg (52%). ESI-MS: 1069.4 ([M+H]+), theoretical value: 1069.5;
amino acid analysis: Asp 1.00 (2), Thr 0.97 (1), Ser 0.93 (1), Ala 1.01
(1), Met 0.90 (1), Tyr 0.99 (1), Phe 0.99 (1), Arg 1.00 (1), Pro 1.00 (1).
1
(1), Tyr 1.00 (1), Phe 0.99 (1), Arg 0.97 (1), Pro 1.00 (1); H-NMR
[H2O/D2O (v/v, 9/1)]: Phe1 δ 4.24 (Hα), 3.24, 3.09 (Hβ), 7.26 (2, 6H),
7.35 (3, 5H), 7.32 (4H). Ala2 8.64 (NH), 4.47 (Hα), 1.35 (Hβ). Thr3
8.72 (NH), 4.58 (Hα), 5.52 (Hβ), 1.35 (Hγ ). Met4 8.95 (NH), 4.66 (Hα),
2.32, 1.81 (Hβ), 2.58, 2.44 (Hγ ). Arg5 7.72 (NH), 4.10 (Hα), 1.79, 1.70
(Hβ) 1.70, 1.53 (Hγ ), 3.26, 3.18 (Hδ), 7.36 (NH), 6.94, 6.48 (NH2).
Tyr6 8.52 (NH), 4.52 (Hα), 2.96, 2.86 (Hβ), 7.11 (2, 6H), 6.83 (3, 5H).
Pro7 3.49 (Hα), 1.76, 1.63 (Hβ), 1.65, 1.56 (Hγ ), 3.62, 3.34 (Hδ). Ser8
7.19 (NH), 4.66 (Hα), 4.92, 4.25 (Hβ). Asp9 9.29 (NH), 4.47 (Hα), 2.97,
2.97 (Hβ). Ser10 8.59 (NH), 4.55 (Hα), 3.84, 3.79 (Hβ). Asp11 7.83
(NH), 4.78 (Hα), 2.72, 2.63 (Hβ). Glu12 8.36 (NH), 4.23 (Hα), 2.14,
1.93 (Hβ), 2.41, 2.41 (Hγ ).
[Thr3-Asp9]-MST(3–9) (7)
This peptide was prepared as described above for [Thr3-Asp9]-
MST(1–9) (6) to yield 26 mg (52%). ESI-MS: 851.4 ([M+H]+),
theoretical value: 851.4; amino acid analysis: Asp 1.00 (1), Thr
0.95 (1), Ser 0.88 (1), Met 0.92 (1), Tyr 0.97 (1), Arg 0.99 (1), Pro
1.00 (1).
Synthesis of MST Analogs
[Ser3]-MST(1–12) (2)
Z-Phe-Ala-Ser(tBu)-Met-Arg(Tos)-Tyr(Pen)-Ser(TBS)-Asp(OtBu)-
Ser(Bzl)-Asp-Glu(OcHx)-OBzl was detached from the peptide
c