Role of the Backbone Conformation at Position 7 in the Structure and Activity of Marinostatin, an Ester-Linked Serine Protease Inhibitor

Article abstract of DOI:10.1002/cbic.201200315

Rational design of inhibitors: The cis-amide backbone at position 7 in the serine protease inhibitor marinostatin was replaced with an E or Z olefin. The E olefin analogue was not active, but the Z analogue was. The cis conformation might play a critical role in organizing a canonical structure for binding to proteases.

Full text of DOI:10.1002/cbic.201200315

DOI: 10.1002/cbic.201200315
Role of the Backbone Conformation at Position 7 in the Structure and
Activity of Marinostatin, an Ester-Linked Serine Protease Inhibitor
Misako Taichi,[a] Toshimasa Yamazaki,[c] and Yuji Nishiuchi*[a, b]
Marinostatin (MST, Scheme 1), isolated from the marine organ-
ism Pseudoaltermonus sagamiensis, is a serine protease inhibi-
tor that consists of 12 amino acids with two internal ester link-
ages formed between the b-hydroxyl and b-carboxyl groups,
Thr3–Asp9 and Ser8–Asp11.[1–3] MST strongly inhibits subtilisin
sential for inhibitory activity. Of particular importance is that
the cis conformation at Pro7 contributes to a rigid turn struc-
ture in Tyr6–Pro7 that promotes the internal hydrogen bond
between Arg5 and the Thr3–Asp9 ester linkage, which not
only protects the scissile bond but also maintains structural ri-
gidity. Thus, it was expected that replacing the cis amide bond
with a trans one would result in a loss of the potency. Howev-
er, [Ala7]-MST was found to retain significant inhibitory activity,
albeit two orders of magnitude less potent than the original
level, even though it had the trans conformation at Ala7 in the
enzyme-free solution structure. This conflicted with the strict
substrate/inhibitor recognition of serine proteases, and so im-
plied that the trans conformation at Ala7 might isomerize to
the cis one that is responsible for expressing, even if only to
a small extent, the canonical structure bound to proteases. To
exclude the possibility of isomerization at Ala7, we designed
the MST olefin analogue 1, in which an E olefin is substituted
for the amide of Tyr6–Ala7 so as to keep the trans backbone
structure. In addition, Z-olefin analogue 2 was synthesized to
confirm regeneration of the original inhibitory activity when
the MST molecule adopts the cis conformation at position 7.
The olefin frameworks of 1 and 2 were substituted for MST(6–
7) as olefin dipeptide isosteres TyrY[(E)-CH=CH]Gly (3) and
Scheme 1. Primary structure of MST.
(Ki =1.5 nm), chymotrypsin, and elastase at an enzyme/inhibitor
ratio of 1:1, but is not active against trypsin.[2] Its strong inhibi-
tory potential is attributed to the internal hydrogen bond link-
ing the backbone NH proton of Arg5 to the carbonyl oxygen
atom of the Thr3–Asp9 ester linkage to protect the scissile
Met4–Arg5 peptide bond.[3] Most serine protease inhibitors are
composed of 14 to 190 amino acids, and bind to their cognate
enzymes in a substrate-like manner. They have an exposed
binding loop that possesses a characteristic canonical confor-
mation stabilized mostly by crosslinked disulfide bridges, al-
though global three-dimensional structures are not com-
mon.[4,5] MST is the smallest serine protease inhibitor from a
natural source, and the structure of its reactive site, which is
prescribed by the ester linkages, is considered to adopt the
same canonical conformation as those of the typical serine
proteases.
TyrY[(Z)-CH=CH]Gly (4), which were prepared by employing a
[8]
´
modified Julia–Kocienski olefination and a Wittig olefination,
respectively. The synthesis of 3 commenced with the Mitsuno-
bu reaction of the mono-TBS-protected (TBS=tert-butyldime-
thylsilyl) alcohol 5 and the thiol 6 followed by molybdenum-
mediated oxidation[9] of the resulting sulfide to give sulfone 7
In a previous study,[6] we synthesized MST by regioselective
esterification, employing two sets of orthogonally removable
side-chain-protecting groups for Asp and Thr/Ser. The solution
structure of MST revealed that the Ramachandran angles of
the reactive site, MST(1–6), coincide with those of the binding
loop of the turkey ovomucoid third domain (OMTKY3), a repre-
sentative serine protease inhibitor.[7] In addition, a SAR study of
MST indicated that the Thr3–Asp9 ester linkage, the conforma-
tion of cis-Pro7, and the N-terminal Phe1–Ala2 residues are es-
(Scheme 2A). The modified Julia-Kocienski olefination of Boc-
´
Tyr(Dcb)-H (8) and sulfone 7 proceeded smoothly to afford
exclusively the desired E olefin unit 9 when KHMDS/DMF was
used as solvent. The Fmoc derivative 3 was prepared by re-
moval of the TBS group from 9 followed by a two-step oxida-
tion and conversion of the Na protecting group. To construct
the Z olefin framework of 4, a Wittig reaction was performed
on the phosphonium salt 10 and the tyrosine aldehyde 8
(Scheme 2B). The Z olefin unit 11 was obtained with a high
selectivity (E/Z=3:97) in 69% yield by using NaHMDS in THF.
However, oxidation of alcohol 12, which is formed by removal
of the TBS group, led to a mixture of by-products, probably
due to interaction between the NH proton of the Tyr isostere
and the resulting aldehyde, which are spatially localized in the
Z configuration. To avoid this side reaction, another Boc group
had to be introduced into the secondary amine of the Tyr iso-
stere in order to perform oxidation of the alcohol 13 to the
carboxylic acid 14 (83% yield for two steps), which was con-
verted to the Fmoc derivative 4. The olefin dipeptide isosteres
3 and 4 were clearly separated and no contamination by the
other isomer in either product was observed by RP-HPLC; this
[a] Dr. M. Taichi, Prof. Dr. Y. Nishiuchi
SAITO Research Center, Peptide Institute, Inc.
7-2-9 Saito-Asagi, Ibaraki, Osaka 567-0085 (Japan)
E-mail: yuji@peptide.co.jp
[b] Prof. Dr. Y. Nishiuchi
Graduate School of Science, Osaka University
1-1 Machikaneyama, Toyonaka, Osaka 560-0043 (Japan)
[c] Dr. T. Yamazaki
Biomolecular Research Unit, National Institute of Agrobiological Sciences
2-1-2 Kannondai, Tsukuba, Ibaraki 305-8602 (Japan)
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cbic.201200315.
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indicates that both products possessed a high degree of iso-
meric purity. The starting linear protected peptide (15) for the
E olefin analogue was assembled by solid-phase peptide syn-
thesis with Fmoc chemistry onto the C-terminal dipeptide,
which was attached to a Trt(2-Cl) resin through the b-carboxyl
group of Asp11 as reported previously (Scheme 3).[6] Peptide
Scheme 3. Synthesis of E olefin analogue 1. a) HFIP/CHCl3 (4:1, v/v), RT, 1 h,
79%; b) TASF, DMF, RT, 1 h, 95%; c) MNBA, DMAP, TEA, CH2Cl2/DMF (5:1, v/v),
RT, 2.5 h, 89%; d) TFA, 08C to RT, 1 h, 96%; e) MNBA, DMAP, TEA, CH2Cl2/
DMF (5:1, v/v), RT, 3 h, 88%; f) HF/p-cresol (4:1, v/v), À5 to À28C, 1 h, 18%.
cHx=cyclohexyl, HFIP=1,1,1,3,3,3-hexafluoropropan-2-ol, MNBA=2-methyl-
6-nitrobenzoic anhydride, TEA=triethylamine, TASF=tris(dimethylamino)sul-
fonium difluorotrimethylsilicate.
chain elongation was carried out by using 1 min preactivation
of coupling with Fmoc derivative/2-(6-chloro-1H-benzotriazole-
1-yl)-1,1,3,3-tetramethylaminium hexafluorophosphate (HCTU)/
6-chloro-1-hydroxy-1H-benzotriazole (6-Cl-HOBt)/DIEA (4:4:4:8
equiv)—except for coupling of the E olefin dipeptide iso-
stere 3, which was incorporated by the N,N’-diisopropyl carbo-
diimide (DIC)/HOBt method because reaction mediated by
HCTU/6-Cl-HOBt or PyBOP was accompanied by isomerization
from the b,g-unsaturated structure to the a,b-unsaturated one.
After cleavage of the Trt(2-Cl) resin and the TBS group on
Asp11 and Ser8, respectively, the resultant peptide, 16, was
subjected to intramolecular esterification by using Shiina re-
agent, 2-methyl-6-nitrobenzoic anhydride (MNBA),[10] to prefer-
entially produce the cyclic monomer 17 in a 89% yield, with
a small amount of by-products such as aspartimide (Asi) pep-
tide formed at Asp11, and cyclic/linear dimers. The second in-
tramolecular esterification with Thr3–Asp9 was performed by
employing the same procedure with Ser8–Asp11. The diester
Scheme 2. Synthesis of A) E olefin dipeptide isostere 3 and B) Z olefin dipep-
tide isostere 4. a) PPh3, DIAD, 1-phenyl-1H-tetrazol-5-thiol (6), THF, 08C, 93%;
b) H2O2 aq., (NH4)6Mo7O14·4H2O, EtOH, 08C, 10 h, 72%; c) Boc-Tyr(Dcb)-H (8),
KHMDS, DMF, À78 to 08C, 2 h, 40%; d) TBAF, THF, RT, 50 min, 83%; e) i: DMP,
NaHCO3, CH2Cl2, 08C to RT, 35 min; ii: NaClO2, NaH2PO4, DMSO/H2O (4:1, v/v),
08C to RT, 40 min, 69% for two steps; f) i: TFA, 08C to RT, 40 min; ii: Fmoc-
OSu, DIEA, DMF/H2O (4:1, v/v), 08C to RT, 2 h, 89% for two steps; g) Boc-Tyr-
(Dcb)-H (8), NaHMDS, THF, À788C to 08C, 2.5 h, 69%; h) TBAF, THF, RT,
50 min, quant; i) i: Boc2O, LiHMDS, THF, 08C, 40 min; ii: TBAF, THF, RT, 96%
for two steps; j) i: DMP, NaHCO3, CH2Cl2, 08C to RT, 1.5 h; ii: NaClO2,
NaH2PO4, DMSO/H2O (4:1, v/v), 08C to RT, 35 min, 83% for two steps; k) i:
TFA, 08C to RT, 40 min; ii: Fmoc-OSu, DIEA, DMF/H2O (4:1, v/v), 08C to RT,
1.5 h. Boc2O=di-tert-butyl dicarbonate, Dcb=2,6-dichlorobenzyl, DIAD=di-
isopropyl azadicarboxylate, DIEA=N,N-diisopropylethylamide, DMP=Dess–
Martin periodinate, Fmoc-OSu=N-(9-fluorenylmethoxycarbonyloxy)succini-
mide, KHMDS=potassium bis(trimethylsilyl)amide, LiHMDS=lithium bis(tri-
methylsilyl)amide, NaHMDS=sodium bis(trimethylsilyl)amide, TBAF=tetra-
butylammonium fluoride, TFA=trifluoroacetic acid.
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peptide was treated with HF in the presence of p-
cresol at À5 to À28C for 1 h to remove all the pro-
tecting groups with no loss of the ester linkages, and
the product 1 was obtained after purification by RP-
HPLC. The Z olefin analogue 2 was synthesized by
employing the same procedure as for 1. Upon cycli-
zation of the olefin analogues 1 and 2, no significant
differences were observed between the E and Z
olefin dipeptide isosteres in terms of the efficiency of
yielding individual ester linkages.
The solution structures of 1 and 2 as determined
by NMR are shown in Figure 1. As we had expected,
the f and y backbone dihedral angles of P2–P2’ in 2
were comparable to those in MST, whereas those in
1 were like those in [Ala7]-MST (Table 1). Comparison
of the solution structure of 2 with the crystal struc-
ture of OMTKY3 bound to subtilisin Carlsberg (CARL;
PDB ID: 1YU6)[11] revealed that the structures of the
P4–P2’ regions of these two inhibitors superimposed
well, with a root-mean-square deviation (RMSD) of
0.70 ꢁ for the main-chain N, Ca and C atoms (Fig-
ure 2A). Furthermore, the side chain of Tyr6 in 2
adopted the same conformation as the correspond-
ing Tyr20 in OMTKY3, which forms a hydrophobic
stacking interaction with Phe189 in CARL. These
structural similarities demonstrate that even in the
free form, the Z olefin analogue 2 has the P4–P2’
structure preferentially organized to bind to CARL. In
contrast, the P4–P2’ region of the E olefin ana-
logue 1 has a significantly different structure, partic-
ularly in the Arg5(P1’)–Tyr6(P2’) region that assumes
a tight bI-turn-like structure and might disrupt favor-
able interactions between the inhibitor and enzyme
(Figure 2B). These observations clearly indicated that
the cis backbone conformation at position 7 could
play a critical role in prescribing the canonical struc-
ture for the reactive site of MST(P2–P2’). Thus, the E
olefin analogue 1 completely lost inhibitory activity
against subtilisin, whereas the Z olefin analogue 2
retained its potency to a substantial level (Table 2). It
should be noted that subtilisin cannot interact with
a MST analogue that possesses a trans conformation
at position 7. These results demonstrated that iso-
merization at Ala7 of [Ala7]-MST can lead to binding
to proteases in a canonical manner. The structure of
the larger ring in MST, that is, MST(3–9), which is
governed by the ester linkage with Thr3–Asp9 and
cis-Pro7, is considered to be strengthened by the
smaller ring MST(8–11). When the smaller ring of
MST opened, isomerization took place at Pro7 to de-
crease the amount of the cis isomer to 85%; this
lowered the potency (Ki =14 nm). By the same
Figure 1. NMR solution structures of A) E olefin analogue 1 and B) Z olefin analogue 2.
Figure 2. Comparison of P4–P2’ structures. A) The P4–P2’ region, Phe1–Tyr6, of the NMR
solution structure of the Z olefin analogue 2 is superimposed on the corresponding
region (Ala15–Tyr20) of OMTKY3 bound to CARL; PDB ID: 1YU6 by using the main-chain
atoms. The analogue 2 and OMTKY3 are shown as stick models in green and magenta,
respectively. The OMTKY3-interacting residues of CARL are shown as ball-and-stick mo-
dels. Hydrogen bonds and a stacking interaction observed for the OMTKY3–CARL com-
plex are indicated by blue and brown dotted lines, respectively. Nitrogen and oxygen
atoms are shown in blue and red, respectively. B) The P4–P2’ region, Phe1–Tyr6, of the
NMR solution structure of the E olefin analogue 1 is overlaid with the corresponding
region (Ala15–Tyr20) of OMTKY3 bound to CARL; PDB ID: 1YU6. The analogue 1 and
OMTKY3 are shown as stick models in blue and magenta, respectively. Nitrogen and
oxygen atoms, the OMTKY3-interacting residues of CARL, hydrogen bonds and a stacking
interaction observed for the OMTKY3-CARL complex are shown as in (A).
token, the smaller ring could help [Ala7]-MST isomerize its
trans conformation at Ala7 into the cis one, even if only to
a small extent. This might cause [Ala7]-MST to display inhibito-
ry activity against subtilisin, even if at two orders of magnitude
less potently than the original.
In summary, we have successfully synthesized the MST olefin
analogues 1 and 2 by employing stereoselectively constructed
E and Z olefin dipeptide isosteres, respectively. Analogue
1 completely lost inhibitory activity, but analogue 2 retained it.
This strongly suggested that the cis conformation at position 7
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tion 7 to express a canonical structure, analysis of their crystal
structures in complex with subtilisin is now in progress.
Table 1. f and y angles of the P2–P2’ regions in MST, [Ala7]-MST, E olefin
analogue 1, and Z olefin analogue 2.
P2 (Thr)
P1(Met)
P1’(Arg)
P2’(Tyr)
Keywords: enzyme inhibitors
spectroscopy · olefin dipeptide isosteres · structure–activity
relationships
·
marinostatin
·
NMR
[Ala7]-MST
f
y
f
y
f
y
f
y
À141.9
133.5
À138.8
156.9
À70.9
153.9
À59.5
152.8
À159.6
9.8
À159.5
17.2
À103.2
23.6
À101.5
26.1
À65.1
À81.3
À33.5
À61.4
À43.3
170.2
À47.2
162.0
À49.5
À55
1
À105.7
À70.2
À107.0
121.2
MST
2
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À110.9
112.8
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2377–2380.
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T. Teshima, T. Ohkubo, Y. Kobayashi, Y. Nishiuchi, J. Pept. Sci. 2010, 16,
329–336.
Table 2. Ki values of MST analogues against CARL.
MST analogues
Ki [nm][a]
Conformation of AA7
MST
2
[Ala7]-MST
1
0.59Æ0.05[b]
cis
cis
trans
trans
6.6Æ1.1
84.3Æ1.2[b]
>10000
[8] a) J. B. Baudin, G. Hareau, S. A. Julia, O. Ruel, Tetrahedron Lett. 1991, 32,
1175–1178; b) P. R. Blakemore, W. J. Cole, P. J. Kocienski, A. Morley, Syn-
lett 1998, 26–28.
[a] Means of 4–6 determinationsÆSD. [b] Data from ref. [7].
[9] P. J. Kocienski, A. Bell, P. R. Blakemore, Synlett 2000, 365–366.
[10] I. Shiina, M. Kubota, H. Oshiumi, M. Hashizume, J. Org. Chem. 2004, 69,
1822–1830.
[11] J. T. Maynes, M. M. Cherney, M. A. Qasim, M. Laskowski, Jr., M. N. G.
James, Acta Crystallogr. Sect. D Biol. Crystallogr. 2005, 61, 580–588.
plays a critical role in the MST molecule for prescribing the
canonical structure bound to proteases, and that [Ala7]-MST
should isomerize its trans-Ala7 into the cis one when it binds
to proteases. To verify that both enzyme-bound MST and
enzyme-bound [Ala7]-MST take the cis conformation at posi-
Received: May 15, 2012
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COMMUNICATIONS
Rational design of inhibitors: The cis-
amide backbone at position 7 in the
serine protease inhibitor marinostatin
was replaced with an E or Z olefin. The
E olefin analogue was not active, but
the Z analogue was. The cis conforma-
tion might play a critical role in organiz-
ing a canonical structure for binding to
proteases.
M. Taichi, T. Yamazaki, Y. Nishiuchi*
&& – &&
Role of the Backbone Conformation at
Position 7 in the Structure and
Activity of Marinostatin, an Ester-
Linked Serine Protease Inhibitor
ChemBioChem 0000, 00, 1 – 4
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chembiochem.org
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These are not the final page numbers!