Synthesis of a TMC-95A Ketomethylene Analogue by Cyclization via Intramolecular Suzuki Coupling

Article abstract of DOI:10.1021/ol035178f

(Equation presented) A TMC-95A analogue extended at the C-terminus with NleΨ[COCH₂]Gly-Ala-Ala-NH₂ was synthesized via side-chain cyclization of the linear precursor by a Suzuki cross-coupling reaction in solution to analyze the effe

Full text of DOI:10.1021/ol035178f

ORGANIC
LETTERS
2003
Vol. 5, No. 19
3435-3437
Synthesis of a TMC-95A Ketomethylene
Analogue by Cyclization via
Intramolecular Suzuki Coupling
Markus Kaiser,† Carlo Siciliano,† Irmgard Assfalg-Machleidt,‡ Michael Groll,†
,†
Alexander G. Milbradt,† and Luis Moroder*
Max-Planck-Institut fu¨r Biochemie, AG Bioorganische Chemie,
82152 Martinsried, Germany, and Adolf-Butenandt-Institut der
Ludwig-Maximilians-UniVersita¨t, 80336 Mu¨nchen, Germany
moroder@biochem.mpg.de
Received June 25, 2003
ABSTRACT
A TMC-95A analogue extended at the C-terminus with NleΨ[COCH ]Gly-Ala-Ala-NH was synthesized via side-chain cyclization of the linear
2
2
precursor by a Suzuki cross-coupling reaction in solution to analyze the effect of additional P′ residues on the inhibitory potency against
yeast proteasome.
Degradation of cytosolic proteins is mainly regulated by the
ubiquitin-proteasome pathway1 where upon ATP-dependent
ubiquitinylation, enzymatic digestion of the unfolded polypep-
tide chains occurs within a multienzymatic machinery called
26S proteasome.2 Its proteolytic core chamber is constituted
by the 20S proteasome,3 and the molecular basis of the
enzymatic mechanism was derived mainly from X-ray
analysis of the 20S proteasome from T. acidophilum or yeast
complexed with synthetic4 or natural inhibitors such as
lactacystin5 and epoxomicin.6 More recently, a new natural
product from Apiospora montagnei Sacc. TC1093, i.e., TMC-
95A, was recognized as a potent and competitive proteasome
inhibitor.7 Crystallographic analysis of the TMC-95A/pro-
teasome complex revealed binding of the inhibitor in a â-type
extended conformation to the S-subsite of the protein and,
thus, in a substrate-like mode.8
From this structural analysis, a simplified TMC-95A
analogue was derived that was found to retain most of the
inhibitory potency.9 Since no information is available so far
about the substrate-binding mode to the S′-subsite, it was
compelling to investigate the use of the TMC-95A analogue
† Max-Planck-Institut fu¨r Biochemie, AG Bioorganische Chemie.
‡ Adolf-Butenandt-Institut der Ludwig-Maximilians-Universita¨t.
(1) Hershko, A.; Ciechanover, A. Annu. ReV. Biochem. 1998, 67, 425-
479.
(2) Voges, D.; Zwickl, P.; Baumeister, W. Annu. ReV. Biochem. 1999,
68, 1015-1068.
(3) (a) Bochtler, M.; Ditzel, L.; Groll, M.; Hartmann, C.; Huber, R. Annu.
ReV. Biophys. Biomol. Struct. 1999, 28, 295-317. (b) Coux, O.; Tanaka,
K.; Goldberg, A. L. Annu. ReV. Biochem. 1996, 65, 801-847.
(4) Lo¨we, J.; Stock, D.; Jap, B.; Zwickl, P.; Baumeister, W.; Huber, R.
Science 1995, 268, 533-539.
(5) Groll, M.; Ditzel, L.; Lo¨we, J.; Stock, D.; Bochtler, M.; Bartunik,
H. D.; Huber, R. Nature 1997, 386, 463-471.
(6) Groll, M.; Kim, K. B.; Kairies, N.; Huber, R.; Crews, C. M. J. Am.
Chem. Soc. 2000, 122, 1237-1238.
(7) (a) Koguchi, Y.; Kohno, J.; Nishio, M.; Takahashi, K.; Okuda, T.;
Ohnuki, T.; Komatsubara, S. J. Antibiot. 2000, 53, 105-109. (b) Kohno,
J.; Koguchi, Y.; Nishio, M.; Nakao, K.; Kuroda, M.; Shimizu, R.; Ohnuki,
T.; Komatsubara, S. J. Org. Chem. 2000, 65, 990-995.
(8) Groll, M.; Koguchi, Y.; Huber, R.; Kohno, J. J. Mol. Biol. 2001,
311, 543-548.
(9) Kaiser, M.; Groll, M.; Renner, C.; Huber, R.; Moroder, L. Angew.
Chem. 2002, 114, 817-820. Kaiser, M.; Groll, M.; Renner, C.; Huber, R.;
Moroder, L. Angew. Chem., Int. Ed. 2002, 41, 780-783.
10.1021/ol035178f CCC: $25.00 © 2003 American Chemical Society
Published on Web 08/27/2003

as an anchor at the S-subsite of the proteasome. Compound
1 would bridge the TMC-95A analogue from the S-subsite
with a nonscissible ketomethylene group for presentation of
a C-terminal peptide extension to the S′-subsite as outlined
in Figure 1.
decided to use a similar side-chain cyclization to synthesize
TMC-95A analogues.
Thus, in a second approach to the synthesis of compound
1, the linear precursor 5 was prepared following the
procedure illustrated in Scheme 1.
Scheme 1. Synthesis of the TMC-95A Analogue 1 Containing
a Ketomethylene Moiety in the Peptide Backbonea
Figure 1. Structure of the target compound 1 and the previously
synthesized TMC-95A analogue 29 and the proposed mode of
binding to the active sites of the 20S proteasome.
Our previous synthesis of the TMC-95A analogue was
based on the macrolactamization of a linear precursor as the
cyclization step. Similarly, all other syntheses of TMC-95A/B
reported so far rely on cyclization of the biaryl-containing
precursor via amide bond formation.10 Thus, in the first
instance, the synthesis of compound 1 was again attempted
via the macrolactamization approach. Yields of the cyclic
compound were poor and nonreproducible because of the
presence of the ketomethylene moiety and purification proved
to be exceedingly difficult.
The two natural products complestatin and chloropeptin
contain a biaryl system very similar to that of TMC-95A/
B.11 The synthesis of related cyclic structures of complestatin
and chloropeptin was achieved both by macrolactamization
and side-chain cyclization via Suzuki cross-coupling.12 We
a Reaction conditions: (a) (i) TFA/DCM (1:1); (ii) Boc-
NleΨ[COCH2]Gly-OBt, DIEA, NMP. (b) (i) TFA/DCM (1:1); (ii)
Boc-Trp(7-Br)-OH, PyBOP/HOBt, DIEA, NMP. (c) (i) TFA/DCM
(1:1); (ii) Boc-Leu-OH, PyBOP/HOBt, DIEA, NMP. (d) (i) TFA/
DCM (1:1); (ii) Boc-Tyr(3-BO2C6H12,4-Me)-OH, PyBOP/HOBt,
DIEA, NMP. (e) DMSO (20 equiv), glacial AcOH/concentrated
HCl (4:1), rt, 4 h. (f) K2CO3 (3 equiv), Pd(dppf)Cl2 (0.05 equiv),
DME/H2O (7:1), 80 °C, 5 h.
(10) (a) Lin, S.; Danishefsky, S. J. Angew. Chem. 2001, 113, 2020-
2024; Lin, S.; Danishefsky, S. J. Angew. Chem., Int. Ed. 2001, 40, 1967-
1970. (b) Lin, S.; Danishefsky, S. J. Angew. Chem. 2002, 114, 530-533.
Lin, S.; Danishefsky, S. J. Angew. Chem., Int. Ed. 2002, 41, 512-515. (c)
Albrecht, B. K.; Williams, R. M. Org. Lett. 2003, 5, 197-200. (d) Yang,
Z.-Q.; Kwok, B. H. B.; Lin, S.; Koldobskiy, M. A.; Crews, C. M.;
Danishefsky, S. J. ChemBioChem 2003, 4, 508-513. (e) Inoue, M.;
Sakazaki, H.; Furuyama, H.; Hirama, M. Angew. Chem. 2003, 115, 2758-
2761. Inoue, M.; Sakazaki, H.; Furuyama, H.; Hirama, M. Angew. Chem.,
Int. Ed. 2003, 42, 2654-2657.
The peptide chain was built-up in a stepwise manner from
H-Ala-Ala-NH2 in solution making use of intermediate NR-
(11) (a) Kaneko, I.; Kamoshida, K.; Takahashi, S. J. Antibiot. 1989, 42,
236-241. (b) Matsuzaki, K.; Ikeda, H.; Ogino, T.; Matsumato, A.;
Woodruff, H. B.; Tanaka, H.; Omura, S. J. Antibiot. 1994, 47, 1173-1174.
(c) Matsuzaki, K.; Ogino, T.; Sunazuka, T.; Tanaka, H.; Omura, S. J.
Antibiot. 1997, 50, 66-69.
3436
Org. Lett., Vol. 5, No. 19, 2003

Boc protection. For this purpose, Boc-NleΨ[COCH2]Gly-
OH, Boc-Trp(7-Br)-OH, and the 3-boronic ester derivative
of Z-Tyr(4-Me)-OH were prepared by well-established
procedures (see Supporting Information). Coupling of the
ketomethylene isostere to H-Ala-Ala-NH2 was carried out
with a preformed HOBt ester since standard in situ activation
procedures, e.g., PyBOP/HOBt, led to side reactions and thus
to low yields upon chromatographic purification. Acidic
cleavage of the NR-Boc group with TFA/DCM (1:1) was
followed by coupling of Boc-Trp(7-Br)-OH, Boc-Leu-OH,
and finally the Z-protected tyrosine-O-methyl 3-boronic ester
derivative with PyBOP/HOBt. Our previous studies have
clearly shown that cyclization to the constrained macrocyclic
ring system of TMC-95A is only possible if the C3-carbon
of the indole ring is sp3-hybridized.9,13 Therefore, oxidation
of the tryptophan side-chain to the 2-oxindole ring was
performed prior to cyclization. Because of the oxidatively
labile boronic ester, optimal conditions were assayed on
model compounds and a mixture of glacial acetic acid/
concentrated hydrochloric acid (4:1) containing 20 equiv of
DMSO14 was found to be the most suitable for conversion
of 4 to 5 in high yields (76%), resulting in a 1:1 mixture of
C3 diastereomers. The subsequent cyclization was performed
by an intramolecular Suzuki cross-coupling under standard
conditions,15 i.e., 3 equiv of K2CO3 as the base and Pd(dppf)-
Cl2 as the catalyst in DME/H2O (7:1). The base-catalyzed
enolization of the oxindole system provides the (S)-
configured precursor at the C3 atom as required for the
stereoselective cyclization.9 Upon RP-HPLC of the crude
product, the desired compound 1 was isolated in a 57%
yield.16
precursor represents a significant progress when compared
to the macrolactamization approach, as it tolerates a wider
spectrum of functional groups. Furthermore, it may well be
suited for the solid-phase synthesis of TMC-95A related
analogues.
The inhibitory potencies of compound 1 for all three
proteasome activities, i.e., chymotrypsin-like (CL), trypsin-
like (TL), and peptidyl-glutamyl-peptide-hydrolase (PGPH),
were determined by newly optimized enzyme assays for yeast
proteasome and compared to those of Ac-Leu-Leu-Nle-H and
compound 2 (Table 1).
Table 1. Inhibition of Yeast Proteasome (Ki [µM]) by
Compounds 1 and 2 and Ac-Leu-Leu-Nle-H
inhibitor
CL activity TL activity PGPH activity
compound 1
compound 2
Ac-Leu-Leu-Nle-H
9.1
2.4
1.4
60
55
364
g2000
g2000
g2000
According to the X-ray analysis of the TMC-95A/
proteasome complex,8 the C-terminal (Z)-propenylamide
group of the natural product acts as a P1 residue and Asn as
a P3 residue. From modeling experiments, a replacement of
these two residues by Nle and Leu, respectively, and the
extension of the peptide chain via a nonscissible ketometh-
ylene moiety with P′ residues for interaction with the S′-
subsite of the enzyme were expected to improve the binding
affinities, particularly for the CL active site. Therefore, rather
surprising was the lower inhibitory potency of the new TMC-
95A analogue 1. A comparative X-ray analysis of the
complexes of yeast proteasome with the previous TMC-95A
analogue 2 and compound 1 is expected to yield the structural
basis for the discrepancy with modeling experiments.
Similarly to what has been reported for the synthesis of
complestatin- and chloropeptin-related compounds,12 the
intramolecular cyclization via Suzuki coupling was found
to proceed at higher rates than the bimolecular coupling
required for the synthesis of the macrolactamization precur-
sor.
Acknowledgment. The study was supported by the SFB
469 (grants A2 and A6) of the Ludwig-Maximilians-
Universita¨t Mu¨nchen. Dr. R. D. Su¨ssmuth of the University
of Tu¨bingen is gratefully acknowledged for providing the
HRMS analysis.
The first synthesis of a TMC-95A related cyclic structure
by an intramolecular Suzuki cross-coupling of the linear
(12) Elder, A. M.; Rich, D. H. Org. Lett. 1999, 1, 1443-1446.
(13) Kaiser, M.; Milbradt, A. G.; Moroder, L. Lett. Pept. Sci. 2002, 9,
65-70.
(14) Savige, W. E.; Fontana, A. Int. J. Pept. Protein Res. 1980, 15, 285-
297.
(15) (a) Miyaura, N.; Suzuki, A. Chem. ReV. 1995, 95, 2457-2483. (b)
Suzuki, A. J. Organomet. Chem. 1999, 576, 147-168.
(16) All compounds prepared in this study exhibited spectroscopic
properties consistent with their proposed structures. Full experimental details
are included in Supporting Information.
Supporting Information Available: Synthetic procedures
and spectroscopic characterization of precursors and com-
pounds 1 and 5 and protocols for enzyme assays. This
material is available free of charge via the Internet at
http://pubs.acs.org.
OL035178F
Org. Lett., Vol. 5, No. 19, 2003
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