Total synthesis of marinostatin, a serine protease inhibitor isolated from the marine bacterium Pseudoallteromonas sagamiensis

Article abstract of DOI:10.1016/j.tetlet.2009.02.213

Marinostatin (MST) (1) isolated from a marine organism is a serine protease inhibitor consisting of 12 amino acids with two internal ester linkages that are formed between the β-hydroxyl and β-carboxyl groups, Thr³-Asp⁹ and Ser⁸

Full text of DOI:10.1016/j.tetlet.2009.02.213

Tetrahedron Letters 50 (2009) 2377–2380
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Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Total synthesis of marinostatin, a serine protease inhibitor isolated
from the marine bacterium Pseudoallteromonas sagamiensis
Misako Taichi a,b, Toshimasa Yamazaki c, Terutoshi Kimura a, Yuji Nishiuchi a,b,
*
a SAITO Research Center, Peptide Institute, Inc., Ibaraki, Osaka 567-0085, Japan
b Department of Chemistry, Graduate School of Science, Osaka University, Toyonaka, Osaka 560-0043, Japan
c Protein Research Unit, National Institute of Agrobiological Science, Tsukuba, Ibaraki 305-8602, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Marinostatin (MST) (1) isolated from a marine organism is a serine protease inhibitor consisting of 12
amino acids with two internal ester linkages that are formed between the b-hydroxyl and b-carboxyl
groups, Thr3-Asp9 and Ser8-Asp11. We synthesized MST by a regioselective esterification procedure
employing two sets of orthogonally removable protecting groups at the side-chains of Asp and Ser/Thr.
We optimized the esterification conditions to preferentially form the intramolecular ester linkages with-
out any significant aspartimide (Asi) formation at Asp9 and Asp11. The inhibitory potency of the synthetic
MST against subtilisin (Ki, 0.6 nM) was comparable with a reported value for native MST (1.5 nM).
Ó 2009 Elsevier Ltd. All rights reserved.
Received 3 February 2009
Revised 26 February 2009
Accepted 27 February 2009
Available online 4 March 2009
Keywords:
Ester linkage
Marinostatin (MST)
Protease inhibitor
Regioselective esterification
MST (1), a 12-residue peptide isolated from the marine bacte-
rium Pseudoalteromonas sagamiensis, contains two ester linkages
that are formed between the b-hydroxy and b-carboxyl groups,
Thr3-Asp9 and Ser8-Asp11 (Fig. 1).1 MST (1) is a serine protease
inhibitor with a strong potency against subtilisin, chymotrypsin
and elastase at an enzyme-inhibitor ratio of 1:1, but not against
trypsin.2 Its inhibitory potential is largely attributable to the
hydrogen bond linking the backbone NH proton of Arg5 and the
carbonyl oxygen atom of the ester linkage of Thr3-Asp9, which sta-
bilizes the scissile bond of Met4 (P1)-Arg5 (P10) for proteases.3 In
general, it is known that serine protease inhibitors have a rigid
hydrophobic core derived from cross-connecting disulfide bridges
in order to be able to directly bind to the catalytic sites of prote-
ases. In the case of MST, however, the characteristic ester linkages
may take the place of disulfide bridges to make it such a small-
sized protease inhibitor for the cardinal inhibitory activity. Due
to the interesting features of MST structure and biological activity,
we tried to develop a protocol, which would allow us to synthesize
not only the natural product to confirm its reported primary struc-
ture including ester linkages, but also its analogues possessing pro-
tease specificities that are different from those of the natural
product.
Phe-Ala-Thr-Met-Arg-Tyr-Pro-Ser-Asp-Ser-Asp-Glu
1
Figure 1. Structure of MST (1).
tection reaction4 of the resulting diester 2. To perform regioselec-
tive esterification, two sets of orthogonally removable side-chain
protecting groups were required for Ser/Thr and Asp on 3. The
tBu group was introduced to the side-chain functional groups of
Thr3 and Asp9, and the t-butyldimethylsilyl (TBS) and 2-chlorotrityl
[Trt(2-Cl)] groups were introduced to those of Ser8 and Asp11
,
respectively. The other side-chain-protecting groups and the N/C-
terminal ones must remain unchanged in peptide synthesis using
both Boc and Fmoc chemistry techniques and must be removed
by HF treatment. Considering this, the 3-pentyl (Pen) group5 was
introduced to the phenolic hydroxyl group of Tyr6, since it is com-
patible with Boc and Fmoc chemistry but is readily cleavable by the
standard HF procedure without the formation of any significant
amount of alkyltyrosine rearrangement product. The linear MST
molecule was assembled by solid-phase peptide synthesis (SPPS)
with Fmoc chemistry onto the C-terminal dipeptide 4, which was
attached to a Trt(2-Cl) resin through the b-carboxyl group of
Asp11 posterior to remove the tBu group (Scheme 2). Peptide elon-
gation was carried out with an ABI 433A peptide synthesizer using
FastMocÒ protocols of coupling with Fmoc-amino acid/HCTU/
The synthetic plan followed the retrosynthetic analysis shown
in Scheme 1. MST (1) can be obtained by sequential intramolecular
esterification of the linear peptide 3 followed by a final HF depro-
* Corresponding author. Tel.: +81 72 643 4411; fax: +81 72 643 4422.
E-mail address: yuji@peptide.co.jp (Y. Nishiuchi).
0040-4039/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2009.02.213

2378
M. Taichi et al. / Tetrahedron Letters 50 (2009) 2377–2380
Table 2
1
Effects of solvents and peptide concentrations on the esterification reaction between
Ser8 and Asp11 mediated by the MNBA method
Entry
Solvent
Peptide concentration
(10ꢁ3M)
Ratio of products
9:10:dimera
Z-Phe-Ala-Thr-Met-Arg-Tyr-Pro-Ser-Asp-Ser-Asp-Glu-OBzl
5
6
7
8
9
CH2Cl2
CH2Cl2
CH2Cl2/DMF (3:1)
DMF
CH2Cl2
4.6
1.0
4.6
4.6
4.6b
66:1:33
69:0:31
84:4:12
74:22:4
94:3:3
Tos Pen
OcHx
Bzl
2
a
Dimer refers to cyclic and linear dimerized peptides.
Pseudo-high dilution procedure involving progressive addition of the linear
Z-Phe-Ala-Thr-Met-Arg-Tyr-Pro-Ser-Asp-Ser-Asp-Glu-OBzl
b
peptide 8 to the reaction mixture for 3.5 h.
X1
Tos Pen
Y1 X2 Bzl Y2 OcHx
3
X = X = Bu, Y = TBS, Y = Trt(2-Cl) resin
t
1
2
1
2
Scheme 1. Retrosynthetic analysis of 1.
1)TFA
Fmoc-Asp(O Bu)-Glu(OcHx)-OBzl
t
Fmoc-Asp-Glu(OcHx)-OBzl
2)Cl-Trt(2-Cl)-resin,
DIEA
Trt(2-Cl)-resin
4
5
Fmoc SPPS
HFIP/CHCl3
rt, 1 h
Z-Phe-Ala-Thr-Met-Arg-Tyr-Pro-Ser-X-Ser-Asp-Glu-OBzl
Bu TosPen TBS Bzl OcHx
3
t
68%
6
7
: X=Asp(O Bu)
t
: X=Asi
TBAF, AcOH, THF
Z-Phe-Ala-Thr-Met-Arg-Tyr-Pro-Ser-Asp-Ser-Asp-Glu-OBzl
Bu
rt, 2 h
94%
8
t
Tos Pen
O Bu Bzl
OcHx
t
Scheme 2. Synthesis of the protected linear peptide 8.
Table 1
Effects of coupling reagents on the esterification reaction between Ser8 and Asp11 performed in CH2Cl2 at the peptide concentration of 4.6 ꢀ 10ꢁ3
M
Z-Phe-Ala-Thr-Met-Arg-Tyr-Pro-Ser-Asp-Ser-Asp-Glu-OBzl
Bu
Tos Pen
O Bu Bzl
t
OcHx
t
Table 1 and 2
4.6 mM
9
+
8
Z-Phe-Ala-Thr-Met-Arg-Tyr-Pro-Ser-Asp-Ser-Asi-Glu-OBzl
Bu Tos Pen O Bu Bzl OcHx
t
t
10
+
dimer
Entry
Coupling reagents
Additives
—
DMAP (0.5 equiv)
DMAP (0.5 equiv)
DMAP (0.5 equiv)
Base
Ratio of products
9:10:dimera
9: 91: 0
62: 4: 34
66: 1: 33
61: 5: 34
1
2
3
4
PyBOP (3.0 equiv)
TCB-Clb (1.2 equiv)
MNBA (1.2 equiv)
DICc (1.2 equiv)
DIEA (5.0 equiv)
TEA (2.2 equiv)
TEA (5.0 equiv)
—
a
Dimer refers to cyclic and linear dimerized peptides.
Trichlorobenzoyl chloride.
Diisopropylcarbodiimide.
b
c

M. Taichi et al. / Tetrahedron Letters 50 (2009) 2377–2380
2379
MNBA, DMAP, TEA,
CH2Cl2/DMF (5/1, v/v)
TFA, rt, 1 h
97%
Z-Phe-Ala-Thr-Met-Arg-Tyr-Pro-Ser-Asp-Ser-Asp-Glu-OBzl
9
rt, 4.6 mM,
slow addition (3.5 h),
21.5 h, 73%
TosPen
Bzl
OcHx
11
HF/ -cresol, -2 to -5oC, 1 h
p
Z-Phe-Ala-Thr-Met-Arg-Tyr-Pro-Ser-Asp-Ser-Asp-Glu-OBzl
1
44% after HPLC purification
Tos Pen
Bzl
OcHx
2
Scheme 3. Synthetic route for 1.
6-Cl-HOBt/DIEA (4:4:4:8) in NMP. After completion of the chain
assembly, the peptide resin 3 was treated with HFIP/chloroform
(1:4, v/v) for 1 h to afford 6. However, 6 was found to be accompa-
nied by the aspartimide (Asi)-peptide 7 between Asp9 and Ser10
(ꢂ10%) during chain elongation using repeated Fmoc deprotection
performed by 20% piperidine/NMP (2.5 min ꢀ 4). The Asi formation
could be significantly reduced (<2%) by substituting 20% morpho-
line/NMP for 20% piperidine/NMP in the Fmoc deprotection reac-
tion although prolonged deprotection steps (5 min ꢀ 4) were
required for complete removal of the Fmoc group. Removal of
the TBS group at Ser8 on 6 was achieved by treatment with
TBAF/AcOH in THF to give 8 having free hydroxyl and carboxyl
groups at Ser8 and Asp11, respectively.
To examine the conditions for preferentially forming the intra-
molecular ester linkage with Ser8-Asp11, the linear peptide 8 in
hand was subjected to esterification that was performed in CH2Cl2
at a peptide concentration of 4.6 ꢀ 10ꢁ3 M with the aid of various
coupling reagents (Table 1). This reaction more or less accompa-
nied Asi formation at Asp11, regardless of the coupling reagent
type, in addition to intra- and/or intermolecular esterification.
The PyBOP method (entry 1) almost quantitatively converted the
Asp11-peptide 8 to the Asi11-peptide 10, whereas the 2-methyl-6-
nitrobenzoic anhydride (MNBA)/DMAP method developed by Shi-
ina et al.6 was observed to be accompanied by a small amount of
Asi11 formation. However, the latter resulted in the ratio of the cyc-
lic monomer 9 and the cyclic/linear dimer to be 2 to 1 since intra-
molecular esterification did not occur predominantly (entry 3). We
therefore tried to optimize the conditions to preferentially form
the intramolecular ester linkage when using the MNBA/DMAP
method by changing the solvent and the peptide concentration
as shown in Table 2. Increasing the concentration of DMF in the
reaction mixture increased the extent of Asi11 formation (entries
7 and 8), while its formation remained at a minor level as long
as the reaction was performed in CH2Cl2, regardless of the peptide
concentration (entry 5, 6). In addition, no significant changes with
the ratio of 9 and the cyclic/linear dimer were observed even by
lowering the peptide concentration in CH2Cl2 to 1 ꢀ 10ꢁ3 M (entry
6). Therefore, a pseudo-high dilution procedure involving progres-
sive addition of the linear peptide 8 to the reaction mixture con-
taining MNBA/DMAP in CH2Cl2 was employed to accelerate the
intramolecular esterification. This could be successfully performed
to preferentially produce the cyclic monomer 9 without any signif-
icant amounts of side products such as the cyclic/linear dimer or
Asi11-peptide 10 (entry 9).
increment of Asi9 formation associated with the use of DMF could
be kept to a minimum by reducing the DMF concentration as much
as possible. Thus, the pseudo-high dilution procedure performed in
CH2Cl2/DMF (v/v, 5/1) predominantly led to intramolecular esteri-
fication with Thr3-Asp9 to afford the diester-peptide 2 in a 73%
yield with the proportions of the products being 90/2/8 for 2/
Asi9-peptide/cyclic or linear dimer. The diester-peptide 2 was trea-
ted with anhydrous HF in the presence of p-cresol (v/v, 8/2) at
ꢁ2 °C to ꢁ5 °C for 1 h to remove all the protecting groups and
the product 1 was obtained in a 44% yield after purification by
RP–HPLC.7 Spectral and analytical data of synthetic MST were in
good agreement with those of the literature data,1,8although we
had no opportunity to directly compare the synthetic peptide with
the natural one by an analytical procedure using RP-, ion ex-
change-HPLC or capillary zone electrophoresis. As for inhibitory
activity against subtilisin, the Ki value was measured using Suc-
Ala-Ala-Pro-Phe-MCA as a substrate and its potency (Ki, 0.6 nM)
was comparable with the reported value for native MST
(1.5 nM).1,9,10
In conclusion, we have achieved the first total synthesis of MST
(1) by regioselective formation of the intramolecular ester linkages,
Thr3-Asp9 and Ser8-Asp11, with the aid of MNBA-mediated esterifi-
cation as a key step. By applying the structural motif of MST, the
preparation of analogues is in progress for rationally designing pro-
tease inhibitory specificities that are different from those of the
natural product.
Acknowledgement
This work was supported partly by the Program for Promotion
of Fundamental Studies in Health Science of National Institute of
Biomedical Innovation (NIBL).
References and notes
1. (a) Kanaori, K.; Kamei, K.; Taniguchi, M.; Koyama, T.; Yasui, T.; Takano, R.;
Imada, C.; Tajima, K.; Hara, S. Biochemistry 2005, 44, 2462–2468; (b) Taniguchi,
M.; Kamei, K.; Kanaori, K.; Koyama, T.; Yasui, T.; Takano, R.; Harada, S.; Tajima,
K.; Imada, C.; Hara, S. J. Pept. Res. 2005, 66, 49–58.
2. Imada, C.; Maeda, M.; Hara, S.; Taga, N.; Simidu, U. J. Appl. Bacteriol. 1986, 60,
469–476.
3. Takano, R.; Imada, C.; Kamei, K.; Hara, S. J. Biochem. 1991, 110, 856–858.
4. (a) Sakakibara, S.; Shimonishi, Y.; Kishida, Y.; Okada, M.; Sugihara, H. Bull.
Chem. Soc. Jpn. 1967, 40, 2164–2167; (b) Sakakibara, S.; Kishida, Y.; Nishizawa,
R.; Shimonishi, Y. Bull. Chem. Soc. Jpn. 1968, 41, 438–441.
5. Bódi, J.; Nishiuchi, Y.; Nishio, H.; Inui, T.; Kimura, T. Terahedron Lett. 1998, 39,
7117–7120.
After removal of the tBu groups at Thr3 and Asp9 by treating 9
with TFA, the second ester linkage with Thr3-Asp9 was built up
to obtain the fully protected MST (2) by employing the procedure
same as that for the first one with Ser8-Asp11, except for the reac-
tion solvent (Scheme 3). A mixture of CH2Cl2 and DMF (v/v, 5:1)
was needed to dissolve 11 due to its low solubility in CH2Cl2. The
6. Shiina, I.; Ibuka, R.; Kubota, M. Chem. Lett. 2002, 286–287.
7. The protected MST (2) (70 mg, 35
presence of p-cresol (0.50 ml) at ꢁ2 °C to ꢁ5 °C for 1 h to give a crude product
of 1, which was purified by RP-HPLC using YMC-Pak ODS column
lmol) was treated with HF (2.0 ml) in the
a
(30 ꢀ 250 mm) at a flow rate 20 ml/min. The RP–HPLC run was eluted with
an increasing gradient of CH3CN in 0.1% TFA (10–30%, 80 min) to obtain 1
(21 mg, 44%).

2380
M. Taichi et al. / Tetrahedron Letters 50 (2009) 2377–2380
8. Analytical and spectral data of 1: Amino acid analysis (after hydrolysis with 6 N
HCl at 110 °Cfor 22 h): Asp 1.98 (2), Thr 0.98 (1), Ser 1.99 (2), Glu 1.00 (1), Ala 1.04
(1), Met 0.94 (1), Tyr 1.00 (1), Phe 0.99 (1), Arg 0.97 (1), Pro 1.00 (1); MALDI-TOF
MS: 1383.38 ([M+H]+) theoretical value, 1383.46.
9. Green, N. M.; Work, E. Biochem. J. 1953, 54, 347–352.
10. The inhibitory activity of the synthetic MST (1) against subtilisin was measured
according to a literature procedure.1a Briefly, the substrate (Suc-Ala-Ala-Pro-
Phe-MCA) obtained from Peptide Institute, Inc. (Osaka, Japan) was dissolved in
1H and 13C NMR chemical shifts (ppm) [750 MHz, H2O/D2O (v/v, 9:1), 10 °C]
DMSO at concentration of 25 lM. Subtilisin Carlsberg (Calbiochem, Germany,
110 nM) was incubated with an appropriate amount of 1 in 2.0 ml of 25 mM
phosphate buffer (pH 7.0) containing 1.0 mM CaCl2 for 1 min at 30 °C. The
reaction was started by addition of 0.1 ml of the substrate solution. The release of
7-amino-4-methylcoumarin (AMC) was monitored by measuring fluorescence
intensity at 440 nm with excitation at 350 nm. The residual enzyme activity was
plotted against the concentration of 1 to calculate the Ki value.9
Residue
Phe1
NH
H
a
(C
a
)
Hb (Cb)
Others (C)
4.24 (56.22)
3.24, 3.09 (39.3)
2, 6H 7.26 (131.64)
3, 5H 7.35 (131.33)
4H 7.32 (130.24)
Ala2
Thr3
Met4
Arg5
8.64
8.72
8.95
7.72
4.47 (51.45)
4.58 (58.73)
4.66 (54.28)
4.10 (56.55)
1.35 (19.28)
5.52 (73.98)
2.32, 1.81 (31.57)
1.79, 1.70 (30.43)
Hc
Hc
Hc
1.35 (19.28)
2.58, 2.44 (31.68)
1.70, 1.53 (27.49)
Hd 3.26, 3.18 (42.92)
NH 7.36
NH2 6.94, 6.48
2, 6H 7.11 (132.83)
3, 5H 6.83 (117.94)
Tyr6
Pro7
8.52
4.52 (54.70)
3.49 (63.52)
2.96, 2.86 (39.98)
1.76, 1.63 (32.82)
Hc
1.65, 1.56 (24.28)
Hd 3.62, 3.34 (49.48)
Ser8
7.19
9.29
8.59
7.83
8.36
4.66 (54.78)
4.47 (54.65)
4.55 (56.40)
4.78 (51.60)
4.23 (55.59)
4.92, 4.25 (66.24)
2.97, 2.97 (36.20)
3.84, 3.79 (62.13)
2.72, 2.63 (38.26)
2.14, 1.93 (28.35)
Asp9
Ser10
Asp11
Glu12
Hc 2.41, 2.41 (32.70)