Design and synthesis of a solid-supported FR225659 derivative for its receptor screening

Article abstract of DOI:10.1021/ol049100q

(Equation Presented) We describe the design and synthesis of latex particles attached to an FR225659 derivative to identify its receptor proteins. Two key building blocks were prepared by two-step degradation of FR225659 under basic conditions. The design

Full text of DOI:10.1021/ol049100q

ORGANIC
LETTERS
2004
Vol. 6, No. 14
2477-2480
Design and Synthesis of a
Solid-Supported FR225659 Derivative for
Its Receptor Screening
Tatsuya Zenkoh,†,‡ Hidetaka Hatori,†,‡ Hiroshi Tanaka,§ Makoto Hasegawa,†,
Mamoru Hatakeyama,† Yasuaki Kabe,† Hiroyuki Setoi,†,| Haruma Kawaguchi,#
,†,∇
,§
Hiroshi Handa,* and Takashi Takahashi*
Department of Applied Chemistry, Tokyo Institute of Technology, Meguro,
Tokyo 152-8552, Japan, Frontier CollaboratiVe Research Center and Faculty of
Bioscience and Biotechnology, Tokyo Institute of Technology, Yokohama,
Kanagawa 226-8501, Japan, and Faculty of Science and Technology, Keio UniVersity,
Yokohama, Kanagawa 223-8552, Japan
ttak@apc.titech.ac.jp
Received May 17, 2004
ABSTRACT
We describe the design and synthesis of latex particles attached to an FR225659 derivative to identify its receptor proteins. Two key building
blocks were prepared by two-step degradation of FR225659 under basic conditions. The designed ligand showed an acceptable level of
biological activity to make it of potential value for use in affinity-supported receptor identification. Affinity purification of FR225659-binding
proteins using the latex nanoparticles provided three candidate receptor peptides for the biological activity.
Affinity chromatography technology1 using solid-supported
biologically active small molecules is a powerful methodol-
ogy for identifying target receptors and proteins of small
molecules from cell lysates. We have already reported
effective latex nanoparticles composed of a styrene-glycidyl
methacrylate copolymer for affinity chromatography.2,3 The
polyglycidyl surface shows relatively little nonspecific
adsorption of proteins and can be reacted with amino groups
to load ligands. Furthermore, the large total surface area is
especially effective for purifying receptors from a small
amount of cell lysate. As a result, latex nanoparticles attached
to biologically active natural products are highly effective
tools for chemical biology. Unfortunately, the immobilization
reactions that attach the biologically active natural products
† Frontier Collaborative Research Center, Tokyo Institute of Technology.
‡ Present address: Exploratory Research Laboratories, Fujisawa Phar-
maceutical Co., Ltd., 5-2-3 Tokodai, Tsukuba, Ibaraki 300-2698, Japan.
§ Department of Applied Chemistry, Tokyo Institute of Technology.
Present address: Faculty of Bioscience, Nagahama Institute of Bio-
Science and Technology.
| Present address: Medicinal Chemistry Research Laboratories, Fujisawa
Pharmaceutical Co., Ltd., 2-1-6 Kashima, Yodogawa-ku, Osaka 532-8514,
Japan.
# Keio University.
∇ Faculty of Bioscience and Biotechnology, Tokyo Institute of Technol-
ogy.
(2) (a) Wada, T.; Watanabe, H.; Kawaguchi, H.; Handa, H. Method
Enzymol. 1995, 254, 595-604. (b) Shimizu, N.; Sugimoto, K.; Tang, J.;
Nishi, T.; Sato, I.; Hiramoto, M.; Aizawa, S.; Hatakeyama, M.; Ohba, R.;
Hatori, H.; Yoshikawa, T.; Suzuki, F.; Oomori, A.; Tanaka, H.; Kawaguchi,
H.; Watanabe, H.; Handa, H. Nat. Biotechnol. 2000, 18, 877-881.
(3) For a review of latex nanoparticles, see: Kawaguchi, H. Prog. Polym.
Sci. 2000, 25, 1171-1210.
(1) (a) Cuatrecasas, P.; Wilchek, M.; Anfinsen, C. B. Proc. Natl. Acad.
Sci. U.S.A. 1968, 61, 636-643. (b) Cuatrecasas, P. Annu. ReV. Biochem.
1971, 40, 259-278. (c) Schreiber, S. L. Bioorg. Med. Chem. 1988, 6, 1127-
1152. (d) Harding, M. W.; Galat, A.; Uehling, D. E.; Schreiber, S. L. Nature
1989, 341, 758-760. (e) Stockwell, B. R.; Hardwick, J. S.; Tong, J. K.;
Schreiber, S. L. J. Am. Chem. Soc. 1999, 121, 10662-10663.
10.1021/ol049100q CCC: $27.50 © 2004 American Chemical Society
Published on Web 06/18/2004

Scheme 1. Strategy for the Synthesis of Solid-Supported
FR225659 for Receptor Screening
Figure 1. Structure of FR225659 (1) and FR227366 (2).
to nanoparticles are often problematic, particularly when the
supporting molecule contains several unstable functional
groups.
FR225659 (1) and FR227366 (2) were isolated from the
cultured broth of fungal strain no. 19353, which originated
from a decayed leaf.4 Both molecules share a unique structure
that consists of a carboxylic acid and three unusual amino
acids (Figure 1). They also potently inhibit glucagon-induced
gluconeogenesis in vitro. Although their tripeptide structures
might cause them to be easily metabolized in vivo, they could
nevertheless serve as useful ligands for identifying new
antidiabetic drug targets. Therefore, we planned to synthesize
latex nanoparticles coupled to an FR225659 derivative for
receptor screening. Herein we describe the synthesis of an
FR225659 derivative bound via an aminoalkyl group onto
the solid-phase of latex nanoparticles and its use for
identifying candidate receptors.
The design of the solid-supported FR225659 derivative 3
is shown in Scheme 1. The supported tripeptide 3 was
selected for affinity chromatography because the biological
activity of the methyl ether 2 was comparable to that of the
phenol 1. It was envisaged that the tripeptide amine 5 would
undergo chemoselective acylation with the solid-supported
activated ester 4 to provide the solid-supported FR225659
derivative 3. An arginine moiety was selected instead of the
3-chloro-4-hydroxyl arginine to improve the chemical stabil-
ity of the resulting ligand.
with solid-supported amines often results in low yields due
to generation of the cyclic product. Selective deprotection
of the δ-amino group, followed by coupling of amidine 9
would provide the desired arginine derivatives.
The solid-phase strategy employed for the synthesis of
amine 5 was based upon the sequential coupling of four
building blocks 6-9; 2-chlorotrityl polystyrene 10 would be
used as the solid-support for the synthesis. The unique
carboxylic acid 6 and dipeptide 8 would both be obtained
by degradation of naturally occurring FR225659 (1). R-N-
Fmoc and δ-N-[1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylide-
ne)ethyl] (Dde)5 ornithine (7) were selected instead of the
arginine moiety, as the direct coupling of arginine derivatives
The preparation of building blocks 6 and 8 is shown in
Scheme 2. Hydrolysis of FR225659 (1) under mildly basic
conditions effected amide bond cleavage between the argi-
nine and proline moieties. Purification of the crude mixture
was accomplished by reverse-phase column chromatography
to afford dehydroamino acid derivative 12 in 81% yield and
a mixture containing the aromatic acyl derivative 11. The
dipeptide 12 was converted to the Fmoc derivative 8 by
acylation with Fmoc-Cl in quantitative yield. Further deg-
radation of the mixture containing 11 was achieved by
heating it under basic conditions; this provided the acid 13
in 58% yield from 1. Treatment of 13 with bromide 14 in
the presence of K2CO3 afforded the dialkylated product 15
in 91% yield, whose ester could be hydrolyzed to provide
acid 6 in 98% yield.
(4) (a) Ohtsu, Y.; Sasamura, H.; Tsurumi, Y.; Yoshimura, S.; Takase,
S.; Hashimoto, M.; Shibata, T.; Hino, M.; Fujii, T. J. Antibiot. 2003, 56,
682-688. (b) Ohtsu, Y.; Sasamura, H.; Shibata, T.; Nakajima, H.; Hino,
M.; Fujii, T. J. Antibiot. 2003, 56, 689-693. (c) Zenkoh, T.; Ohtsu, Y.;
Yoshimura, S.; Shigematsu, N.; Takase, S.; Hino, M. J. Antibiot. 2003, 56,
694-699.
(5) (a) Bycroft, B. W.; Chan, W. C.; Chhabra, S. R.; Hone, N. D. J.
Chem. Soc., Chem. Commun. 1993, 778-779. (b) Chhabra, S. R.; Hothi,
B.; Evans, D. J.; White, P. D.; Bycroft, B. W.; Chan, W. C. Tetrahedron
Lett. 1998, 39, 1603-1606.
The solid-phase synthesis of amine 5 is disclosed in
Scheme 3. Loading of the dipeptide 8 onto 2-chlorotrityl
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Org. Lett., Vol. 6, No. 14, 2004

Scheme 2
Figure 2. Elucidation of FR225659-specific binding proteins in
rat hepatocyte cell lysates. Lane 1 showed binding of proteins to
control particles in buffer (10mM Tris-HCl pH 7.4, 100 mM NaCl,
1.0 mM MgCl2, 0.2 mM EDTA, 10% glycerol, 0.1% Nonidet P-40,
1.0 mM CaCl2 and 0.5 mM DTT) at 4 °C for 1 h. Lane 2 showed
binding of proteins to FR225659-bound particles 3. Lanes 3-6
showed binding of proteins to FR225659-bound particles 3 in the
presence of FR225659 (1). The concentrations of 1 were 0.1 µg/
mL (lane 3), 1 µg/mL (lane 4), 10 µg/mL (lane 5), and 100 µg/mL
(lane 6).
resin6 (1.25 mmol/mg) was achieved using diisopropylethy-
lamine (DIEA) in dichloromethane to provide the solid-
supported dipeptide 16. Loading was determined to be
quantitative by cleavage from the resin under acidic condi-
tions, and by measurement of the mass recovery of 8.
Deprotection of the Fmoc group, followed by coupling with
ornithine 7 in the presence of tetramethylfluoroformidinium
hexafluorophosphate (TFFH)7 and DIEA, provided the solid-
supported tripeptide 17. Removal of the Fmoc group,
followed by acylation with benzoic acid derivative 6 using
DIC-HOBt in N-methylpyrrolidinone, afforded the solid-
supported acyl tripeptide 18. Finally, conversion of the
ornithine residue into an arginine was examined. Selective
deprotection of the Dde protecting group was achieved with
hydrazine. This was followed by coupling of the resulting
amine with amidine 98 to provide the Boc-protected guani-
dine 19. Cleavage of the product from the resin with acetic
acid-trifluoroethanol-dichloromethane (1:1:8), followed by
purification by preparative TLC, provided the tri-Boc-
protected carboxylic acid 20 in 82% yield from 16. Removal
of the tri-N-Boc groups in 40% TFA-dichloromethane
solution at ambient temperature provided the amine deriva-
tive 5 as its TFA salt in 91% yield with 95% purity. Its purity
was estimated by HPLC on the basis of total UV absorption
from 190 to 600 nm (interval, 2.0 nm) using a diode array
detector.
assay, the IC50 value of the amine derivative 5 (IC50 ) 0.830
µM) was comparable to that of FR225659 (1) (IC50 ) 0.530
µM) as a positive control. These results indicated that the
amino derivative 5 could be used to prepare the affinity
particles for its receptor identification.
Treatment of the activated ester 49 on the latex nanopar-
ticles with a 0.2 M solution of 5 in 0.2 M acetic acid sodium
salt buffer (500 µL) for 1 h at room temperature, followed
by amidation of the remaining activated esters with 2-hy-
droxyethylamine, provided the affinity particles 3. Conver-
sion of any remaining activated esters to the 2-hydroxyeth-
ylamides was effective for reducing nonspecific interactions
of the particles with proteins. HPLC analysis of the acid
degradation product of 3 showed a loading amount of 38.9
nmol/mg. The results of the FR225659 affinity binding
experiments using a cytoplasmic fraction of rat hepatocytes
are shown in Figure 2. Control latex particles10 showed no
binding proteins (lane 1). In contrast, FR225659-bound
particles 3 showed binding to a number of proteins (lane 2).
FR225659 (1) specifically inhibited the binding of particle
3 to three peptides I-III in a concentration-dependent manner
Glucagon-induced gluconeogenesis inhibitory activity in
primary cultured rat hepatocytes was examined.4b In this
(6) 2-Chlorotrityl resin (UniSphere 200) was purchased from IRORI, San
Diego, CA.
(7) Carpino, L. A.; El-Faham, A. J. Am. Chem. Soc. 1995, 117, 5401-
(9) Activated ester 4 was prepared by acylation of the amino-bearing
latex particles (SGNEGDEN)2b with succinic anhydride, followed by
esterification of resulting acids with N-hydroxysuccinimide.
(10) Control resin was prepared by amidation of the activated ester 4
with 2-hydroxyethylamine.
5402.
(8) (a) Robinson, S.; Roskamp, E. J. Tetrahedron 1997, 53, 6697-6705.
(b) Yong, Y. F.; Kowalski, J. A.; Thoen, J. C.; Lipton, M. A. Tetrahedron
Lett. 1999, 40, 53-56.
Org. Lett., Vol. 6, No. 14, 2004
2479

Scheme 3
(lanes 3-6). They possess apparent molecular masses of 36,
Acknowledgment. This work was performed under the
management of the Research Association for Biotechnology
as a part of the Industrial Science and Technology Frontier
Program supported by NEDO (New Energy and Industrial
Technology Development Organization).
37, and 60 kDa, respectively. The three peptides, which
appear to specifically bind to FR225659 in a dose-dependent
manner (Figure 2), may serve as candidate receptors for the
inhibition of glucagon-induced gluconeogenesis. Current
studies include sequence determination for the peptides,
which may play an important role in diabetes, and elucidation
of the biological outcomes induced by the binding of 1.
Supporting Information Available: Experimental pro-
cedures for the degradation and the solid-phase synthesis and
full characterization for compounds 5, 6, 8, 12, 13, 15 and
20. This material is available free of charge via the Internet
at http://pubs.acs.org.
In conclusion, we have described the synthesis of latex
nanoparticles 3 coupled to the bioactive FR225659 derivative
(loading 38.9 nmol/mg). Affinity purification of FR225659-
binding proteins using the latex nanoparticles 3 provided
three candidate peptides for the biological activity. Elucida-
tion of the biological roles of these peptides is in progress.
OL049100Q
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Org. Lett., Vol. 6, No. 14, 2004