Synthesis of fluorescent-labeled aeruginosin derivatives for high-throughput fluorescence correlation spectroscopy assays

Article abstract of DOI:10.1016/j.bmcl.2007.02.045

The design and solid-phase synthesis of effective fluorescent-labeled aeruginosin derivatives and their application to the fluorescence correlation spectroscopy (FCS)-based competitive binding assay of an aeruginosin library are described. The phenolic hy

Full text of DOI:10.1016/j.bmcl.2007.02.045

Bioorganic & Medicinal Chemistry Letters 17 (2007) 2904–2907
Synthesis of fluorescent-labeled aeruginosin derivatives for
high-throughput fluorescence correlation spectroscopy assays
Yoichiro Hoshina,a Yoshifumi Yamada,b Hiroshi Tanaka,a
Takayuki Doia and Takashi Takahashia,*
aDepartment of Applied Chemistry, Graduate School of Science and Engineering, Tokyo Institute of Technology,
2-12-1 Ookayama, Meguro, Tokyo 152-8552, Japan
bLife Science Group, Olympus Corporation, 2-3 Kuboyama-cho, Hachioji-shi, Tokyo 192-8512, Japan
Received 23 January 2007; revised 15 February 2007; accepted 19 February 2007
Available online 22 February 2007
Abstract—The design and solid-phase synthesis of effective fluorescent-labeled aeruginosin derivatives and their application to the
fluorescence correlation spectroscopy (FCS)-based competitive binding assay of an aeruginosin library are described. The phenolic
hydroxyl group on the (R)-3-(4-hydroxyphenyl)lactic acid (D-Hpla) residue was observed to be suitable for connecting Rhodamine
green derivative with minimum loss of biological activity. In addition, the FCS-based binding assay of the library using fluorescent-
labeled chemical probes was also achieved.
Ó 2007 Elsevier Ltd. All rights reserved.
Biologically active natural products are useful as bio-
chemical probes for the discovery of new drug targets
or biomarkers.1 Fluorescent-labeled chemical probes
would be useful not only in high-throughput screening
(HTS) assays of libraries of compounds2 but also in pre-
venting the distribution of the target proteins in living
cells and organisms.3 However, attaching a fluorescent
unit to such biologically active natural products, which
frequently contain various functional groups, can be
problematic, resulting in significant loss of biological
activity and non-specific interactions of the chemical
probes with various proteins.
numbers of compounds or biomolecules present in small
quantities.
Aeruginosin 298-A (1) isolated from Microcystis
aeruginosa (NIES-298) is composed of (R)-3-(4-hydroxy-
phenyl)lactic acid (D-Hpla), D-Leu, 2-carboxy-6-hydrox-
yoctahydroindole (L-Choi), and argininol (L-Argol), and
exhibits inhibitory activity for serine proteases (Fig. 1).5
A variety of related compounds have been isolated as
protease inhibitors.6 A single crystal X-ray analysis of
thrombin–hirugen complexed with aeruginosin 298-A,7
and that of the trypsin–aeruginosin 98-B complex8 sug-
gest that the tetrapeptide structure could serve as an
effective scaffold for development of a variety of serine
protease inhibitors, which would require the develop-
ment of effective approaches for the synthesis of aeru-
ginosin-related compounds.9 We recently reported on
the solid-phase synthesis of a small combinatorial
library based on the structure of aeruginosins.10 The
inhibitory activity of tetrapeptide 2 composed of
D-Hpla-D-Leu-L-Choi-agmatine (Agma) was found to
be 300 times more potent than that of 1. We thus con-
cluded that fluorescent-labeled aeruginosin derivatives
would be effective biochemical probes for competitive
binding assays based on FCS. Herein, we describe the
synthesis of biologically active fluorescent-labeled
Fluorescence polarization or fluorescence correlation
spectroscopy (FCS) is a single-molecule detection tech-
nique using fluorescent-labeled chemical probes and
enables the binding affinity of the chemical probes to
proteins in solution to be estimated by simple manipula-
tion.4 FCS has enormous potential for miniaturized
HTS, because the fluorescent readout signal is insensi-
tive to the assay volume well below 1 lL. Hence, the
FCS assay is considered to be useful for testing large
Keywords: Chemical biology; Solid-phase synthesis; Combinatorial
chemistry; Fluorescence correlation spectroscopy.
*
Corresponding author. Tel.: +81 3 5734 2120; fax: +81 3 5734
2884; e-mail: ttak@apc.titech.ac.jp
aeruginosin derivatives and its application to
a
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.02.045

Y. Hoshina et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2904–2907
2905
HO
R3
H
SiEt2
SiEt2
O
H
O
NH
CO2H
FmocHN
N
H
*
O
N
8
N
N
NH2
H
H
H
O
a, b
H
O
R1
OH
R2O
H
H
N
N
Boc
D-Hpla
D-Leu L-Choi L-Argol or Agma
CO2Allyl
FmocHN
CO2Allyl
O
1: R1 = CH2OH (L-Argol), R2 = H, R3 = 2-propyl (* = R)
2: R1 = H (Agma), R2 = H, R3 = 2-propyl (* = R)
7
(0.79 mmol/g)
9
3: R1 = CH2OH, R2 = -(CH2)3NH-Rhodamine green, R3 = 2-propyl (* = R)
NBoc
H2N
4: R1 = CH2OH, R2 = H, R3 = -(CH2)3-Rhodamine green (* = S)
SiEt2
N
NHBoc
3
H
OTBS
c, d
5: R1 = C(=O)NH(CH2)3NH-Rhodamine green, R2 = H, R3 = 2-propyl (* = R)
6: R1 = H, R2 = -(CH2)3NH-Rhodamine green, R3 = 2-propyl (* = R)
O
10
H
H
O
NBoc
N
H
H2N
N
FmocHN
N
NHBoc
3
O
H
O
OTBS
O
Rhodamine green
11
CO2H
CO2H
HN
HO
OTBS
12
BocHN
O
3
H
Figure 1. Aeruginosin 298-A 1, its derivative 2, and fluorescent-labeled
aeruginosin derivatives 3–6.
e, f, g
H
O
O
NH
NH2
N
H
N
N
N
H
3
H
O
R2HN
O
OH
OH
3
13: R2 = H
3: R2 = Rhodamine green
h
high-throughput binding assay of an aeruginosin library
based on FCS.
Scheme 1. Reagents and conditions: (a) TMSOTf, 2,6-lutidine, CH2Cl2;
(b) Fmoc-D-Leu-OH, 8, DIC, HOBt, 20% DMF–CH2Cl2; (c) Pd(PPh3)4,
dimedone, THF; (d) 10, DIC, HOBt, 20% DMF–CH2Cl2; (e) 20%
piperidine–DMF; (f) 12, DIC, HOBt, 20% DMF–CH2Cl2; (g) TFA–
H2O–CH2Cl2 (10:1:9); then PS-NMM, 50% MeOH–CH2Cl2; (h) Rho-
damine green-OSu, MP-carbonate, DMF–H2O.
We designed four Rhodamine-labeled aeruginosin deriv-
atives 3–6 as fluorescent-labeled chemical probes
(Fig. 1). Rhodamine green is a favorable fluorophore
for FCS. The chemical probes 3–5 are based on the
structure of 1, in which the positions where the Rhod-
amine is attached are different. The position of the fluo-
rescent label influences their protease inhibitory activity
and binding affinity to the receptor proteins. Based on
the crystal structures of the aeruginosin derivatives,7,8
the terminal Hpla residue would be a suitable location
to attach Rhodamine. The Rhodamine conjugate 6
was based on the structure of the more potent
compound 2.
procedure (details are provided in the supplementary
material).
We initially conducted binding assays of chemical
probes 3–6 for trypsin based on FCS and their inhibi-
tory activities against trypsin (Table 1). Aeruginosin
298-A derivatives 3 and 6 in which the Rhodamine green
was linked to the Hpla moiety showed moderate to tight
binding affinities. The binding affinity of the Agma
derivative 6 was 30 times higher than 3. On the other
hand, the aeruginosin 298-A derivatives 4 and 5 showed
reduced binding affinity to trypsin. These results indicate
that the position of the attachment of the fluorescent
group is a major factor in the design of such chemical
probes.
The preparation of the Rhodamine-labeled derivative 3
was based on the previously reported solid-supported
synthesis10 as shown in Scheme 1. The solid-supported
L-Choi 7 attached through the silyl ether was used as
the starting material. Removal of the N-Boc protecting
group without cleavage of the silyl linker followed by
coupling with leucine 8 provided the dipeptide 9. Cleav-
age of the allyl ester using a palladium catalyst followed
by amidation of the resulting carboxylic acid with argol
10 afforded tripeptide 11. The N-Fmoc group was
removed from the solid-supported tripeptide 11 by treat-
ment with 20% piperidine in DMF. Coupling of the
resulting amine with carboxylic acid 12, followed by
release from the resin and cleavage of all the protecting
groups under acidic conditions, provided the amino
derivative 13. The resulting crude 13 was reacted with
Rhodamine green carboxylic acid succinimidyl ester in
the presence of macroporous triethylammonium methyl-
polystyrene-carbonate (MP-carbonate),11 and pure 3
was obtained by reverse phase HPLC. The preparation
of Rhodamine derivatives 4–6 followed the same
Table 1. Binding affinities (Kd) to trypsin by FCS and inhibitory
activities against trypsin
Compound
Binding affinity
Kd (lM)
Inhibition of trypsin
IC50 (lM)
1
2
3
4
5
6
–
19
0.063
1.5
–
0.18
>100
>100
0.0059
>100
3.7
0.15

2906
Y. Hoshina et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2904–2907
Table 2. Competitive binding assays by FCS and inhibitory activities of aeruginosin derivatives against trypsin
Compound
FCS assay with 3 IC50 (lM)
FCS assay with 6 IC50 (lM)
Inhibition of trypsin IC50 (lM)
1
2.1
16
200
0.19
2.0
19
2
0.063
0.65
8.8
D-Hpla-D-Leu-L-Choi-L-Arg-OH
D-Hpla-D-Tyr-L-Choi-L-Argol
D-Hpla- D-Tyr-L-Choi-L-Arg-OH
D-Hpla-D-Tyr-L-Choi-Agma
D-Hpla-D-Tyr-L-Choi-Agma
D-Hpla-L-Phe-L-Choi-L-Arg-OH
D-Hpla-L-Phe-L-Choi-L-Arg-OH
D-Hpla-L-Phe-L-Choi-L-Arg-OH
20
78
130
9.3
23
17
1.0
0.51
>200
>200
190
59
0.15
>200
>200
12
>200
>200
78
>100
15
To demonstrate the utility of the chemical probes 3
and 6, we used them in an FCS-based competitive
binding assay of an aeruginosin library (Table 2).
The trypsin inhibitory activity of the library has been
reported previously.10 When the more active ligand 6
was applied to the FCS-based competitive binding
assay, the most active compound 2 in the binding assay
was identical with that explained by the inhibitory
activity. In addition, the order of the relative binding
affinity of the library was comparable to its inhibitory
activity. However, the binding assay using the less
active ligand 3 provided the incorrect compound 1 as
the most potent compound. These results indicated
that the additional hydroxymethyl group of 3 on the
Argol could promote specific or non-specific binding
of 3 to trypsin that did not lead to the inhibitory
activity.
References and notes
1. (a) Campbell, D. A.; Szardenings, A. K. Curr. Opin.
Chem. Biol. 2003, 7, 296; (b) Tietze, L. F.; Bell, H. P.;
Chandrasekhar, S. Angew. Chem., Int. Ed. 2003, 42, 3996;
(c) Ortholand, J.-Y.; Ganesan, A. Curr. Opin. Chem. Biol.
2004, 8, 271; (d) Boldi, A. M. Curr. Opin. Chem. Biol.
2004, 8, 281; (e) Ganesan, A. Curr. Opin. Biotech. 2004, 15,
584.
2. (a) Ruhl, T.; Daghish, M.; Buchynskyy, A.; Barche, K.;
¨
Volke, D.; Stembera, K.; Kempin, U.; Knoll, D.; Hennig,
L.; Findeisen, M.; Oehme, R.; Giesa, S.; Ayala, J.; Welzel,
P. Bioorg. Med. Chem. 2003, 11, 2965; (b) Li, J.; Kim, I.
H.; Roche, E. D.; Beeman, D.; Lynch, A. S.; Ding, C. Z.;
Ma, Z. Bioorg. Med. Chem. Lett. 2006, 16, 794.
3. Recent results of application of fluorescent-labeled bio-
logically active small molecules to biological evaluation.
(a) Berque-Bestel, I.; Soulier, J.-L.; Giner, M.; Rivail, L.;
Langlois, M.; Sicsic, S. J. Med. Chem. 2003, 46, 2606; (b)
Ho¨dl, C.; Strauss, W. S. L.; Sailer, R.; Seger, C.; Steiner,
R.; Haslinger, E.; Schramm, H. W. Bioconjugate Chem.
2004, 15, 359; (c) Cha, J. H.; Zou, M.-F.; Adkins, E. M.;
Rasmussen, S. G. F.; Loland, C. J.; Schoenenber, B.;
Gether, U.; Newman, A. H. J. Med. Chem. 2005, 48, 7513;
(d) Vukojevicacute, V.; Pramanik, A.; Yakovleva, T.;
Rigler, R.; Terenius, L.; Bakalkin, G. Cell Mol. Life Sci.
In conclusion, the design and solid-phase synthesis of
effective fluorescent-labeled aeruginosin derivatives and
their application to the FCS-based competitive binding
assay of an aeruginosin library are described. The
phenolic hydroxy group on the Hpla residue was found
to be suitable for connecting Rhodamine green deriva-
tive with minimum loss of biological activity. An FCS-
based competitive binding assay using the appropriate
fluorescent-labeled derivative 6 gave results that were
comparable to those of the protease inhibition assay.
In addition, the FCS-based binding assay of the library
using fluorescent-labeled chemical probes would be an
effective method because of its small observed-volume
and high sensitivity.
´
2005, 62, 535; (e) Vazquez, M. E.; Blanco, J. B.; Salvadori,
S.; Trapella, C.; Argazzi, R.; Bryant, S. D.; Jinsmaa, Y.;
Lazarus, L. H.; Negri, L.; Ginnini, E.; Lattanzi, R.;
Colucci, M.; Balboni, G. J. Med. Chem. 2006, 49, 3653.
4. (a) Eigen, M.; Rigler, R. Proc. Natl. Acad. Sci. U.S.A.
1994, 91, 5740; (b) Wohland, T.; Friedrich, K.; Hovius, R.;
Vogel, H. Biochemsitry 1999, 38, 8671; (c) Moore, K. J.;
Turconi, S.; Ashman, S.; Ruediger, M.; Haupts, U.;
Emerick, V.; Pope, A. J. J. Biomol. Screen. 1999, 4, 335.
5. Murakami, M.; Okita, Y.; Matsuda, H.; Okino, T.;
Yamaguchi, K. Tetrahedron Lett. 1994, 35, 3132.
6. (a) Murakami, M.; Ishida, K.; Okino, T.; Okita, Y.;
Matsuda, H.; Yamaguchi, K. Tetrahedron Lett. 1995, 36,
2785; (b) Matsuda, H.; Okino, T.; Murakami, M.;
Yamaguchi, K. Tetrahedron 1996, 52, 14501; (c) Shin,
H.; Matsuda, H.; Murakami, M.; Yamaguchi, K. J. Org.
Chem. 1997, 62, 1810; (d) Kodani, S.; Ishida, K.; Mura-
kami, M. J. Nat. Prod. 1998, 61, 1046; (e) Ishida, K.;
Matsuda, H.; Okino, T.; Murakami, M. Tetrahedron 1999,
55, 10971; (f) Banker, R.; Carmeli, S. Tetrahedron 1999,
55, 10835.
Acknowledgment
This work was supported in part by NEDO (the New
Energy and Industrial Technology Development Orga-
nization) in Japan.
Supplementary data
7. Steiner, J. L. R.; Murakami, M.; Tulinsky, A. J. Am.
Chem. Soc. 1998, 120, 597.
8. Sandler, B.; Murakami, M.; Clardy, J. J. Am. Chem. Soc.
1998, 120, 595.
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.bmcl.
2007.02.045.

Y. Hoshina et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2904–2907
2907
´
9. (a) Valls, N.; Lopez-Canet, M.; Vallribera, M.; Bonjoch, J.
J. Am. Chem. Soc. 2000, 122, 11248; (b) Valls, N.; Lopez-
esikan, V.; Shibuguchi, T.; Nemoto, T.; Kisugi, T.; Okino,
T.; Shibasaki, M. Proc. Nat. Acad. Sci. U.S.A. 2004, 101,
5433.
´
Canet, M.; Vallribera, M.; Bonjoch, J. Chem. Eur. J. 2001,
7, 3446; (c) Wipf, P.; Methot, J.-L. Org. Lett. 2000, 2,
4213; (d) Ohshima, T.; Gnanadesikan, V.; Shibuguchi, T.;
Fukuta, Y.; Nemoto, T.; Shibasaki, M. J. Am. Chem. Soc.
2003, 125, 11206; (e) Fukuta, Y.; Ohshima, T.; Gnanad-
10. Doi, T.; Hoshina, Y.; Mogi, H.; Yamada, Y.; Takahashi,
T. J. Comb. Chem. 2006, 8, 571.
11. MP-carbonate was purchased from Argonaut Technolo-
gies (now Biotage).