Synthesis of calix[4]arene library substituted with peptides at the upper rim

Article abstract of DOI:10.1016/j.tetlet.2003.10.215

A fluorescence-labeled calix[4]arene library substituted with peptides at the upper rim was synthesized. Screening of the library for binding a dye-labeled oligopeptide indicated that some peptidocalix[4]arenes selectively bind the oligopeptide. The chemo

Full text of DOI:10.1016/j.tetlet.2003.10.215

Tetrahedron
Letters
Tetrahedron Letters 45 (2004) 561–564
Synthesis of calix[4]arene library substituted with peptides
at the upper rim
Hideaki Hioki,* Yumiko Ohnishi, Miwa Kubo, Emi Nashimoto, Yukinori Kinoshita,
Miho Samejima and Mitsuaki Kodama
Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Yamashiro-cho, Tokushima 770-8514, Japan
Received 14 July 2003; revised 8 October 2003; accepted 31 October 2003
Abstract—A fluorescence-labeled calix[4]arene library substituted with peptides at the upper rim was synthesized. Screening of the
library for binding a dye-labeled oligopeptide indicated that some peptidocalix[4]arenes selectively bind the oligopeptide. The
chemosensitivity of the library members for a target peptide was also investigated.
ꢀ 2003 Elsevier Ltd. All rights reserved.
NHR1
AA3
AA2
AA1
NH
Calixarenes are widely used as a platform for artificial
receptors.1 Their potential to function as receptors
depends on the modification of the core calixarene.
Recently, we synthesized a fluorescence-labeled peptido-
calix[4]arene library2;3 substituted with peptides at the
lower rim (phenolic oxygen side of calixarene). In the
library, there were the host molecules that only selec-
tively bind the target peptide but also act as chemo-
sensors4 for the target. In that study, we demonstrated
that the split synthesis of libraries based on calixarenes
is an attractive approach for discovering the new host
molecules.2;5 Although most organic molecules bind
calixarene-based receptors at the upper rim (aromatic
nuclei side of calixarene), split or parallel synthesis of
the upper rim-modified calixarene libraries have not yet
been reported.6 In this communication, we report the
split synthesis of a fluorescence-labeled calix[4]arene
library substituted with peptides7 at the upper rim, and a
binding assay for a pentapeptide. The chemosensitivity
for the pentapeptide is also reported.
R1HN
AA3
AA2
AA1
HN
building blocks (AA1-AA3)
L-Ala, D-Ala, L-Leu, D-Leu,
L-Pro, D-Pro, L-Phe, D-Phe,
L-Ser, L-Tyr, L-Lys, L-Asp,
L-Gln, D-Gln, β-Ala
OR2
O
R 1=
O
R2O
R2O
R 2= n-decyl
8
O
HN
1
Figure 1. Upper rim-modified peptidocalix[4]arene library.
conformational change of the peptidocalix[4]arene
induced by binding the substrate was expected to change
the fluorescence spectra in the same manner as previ-
ously reported.2
The fluorescence-labeled peptide library is shown in
Figure 1. The substrate binding site, consisting of two-
armed tripeptides was directly attached to the amino-
methylated calix[4]arene at the upper rim. Two pyrenyl
groups were attached at the peptide N-terminal. The
Core-compound 6 was prepared according to Scheme 1.
Starting from dibenzyloxycalix[4]arene 28, n-decylation
and deprotection of benzyl groups at the lower rim
followed by selective bromination at upper rim afforded
3. Compound 3 was subjected to a third n-decylation at
the lower rim. After the remaining phenolic OH was
alkylated with MPM-protected 6-bromo-n-decanol, the
MPM group was removed by DDQ (2,3-dichloro-5,
Keywords: Calixarenes; Combinatorial library; Binding assay; Chemo-
sensors.
* Corresponding author. Tel: +81-88-622-9611; fax: +81-88-655-3051;
e-mail: hioki@ph.bunri-u.ac.jp
0040-4039/$ - see front matter ꢀ 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2003.10.215

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H. Hioki et al. / Tetrahedron Letters 45 (2004) 561–564
Br
Br
ii
i
OH
OR1
OBn
OH
HO
BnO
Br
R1O
HO
NC
2
3
Br
CN
iii
iv
OR1
OR1
OR2
OR2
R1O
R1O
R1O
R1O
4
5
NHMoz
MozHN
OR1
H
O
N
R1O
R1O
9
O
6
R1 = n-decyl R2 = (CH2)10OH
Scheme 1. Synthesis of the core-compound 6 of library 1. Reagents and conditions: (i) 1. NaH, n-decylbromide, DMF, 86%; 2. H2, Pd(OH)2, EtOH/
benzene 2/1, 80%; 3. HPyBr3, CHCl3/MeOH, 10/1, 98%; (ii) 1. NaH, n-decylbromide, DMF, 72%; 2. NaH, MPMOC10H20Br, DMF, 82%; 3. DDQ,
aq NaHCO3/CH2Cl2 1/10, 100%; (iii) Zn(CN)2, DPPF, Pd2(dba)2, DMF, 140 ꢁC, 74%; (iv) 1. BH3ÆTHF, THF, reflux; 2. 6 M HC1, reflux, ; 3. Moz-S,
K2CO3, l,4-dioxane/H2O 4/1, 78% in three steps; 4. IBX, DMF, 98%; 5. NaClO2, NaHPO4Æ2H2O, 2-methyl-2-butene t-BuOH/H2O (15/4) 82%; 6.
aminomethylated polystyrene resin, DIC, HOBt, CH2C13. Abbreviations: DMF ¼ N,N0-dimethylformamide, MPM ¼ p-methoxyphenylmethyl,
DDQ ¼ 2,3-dichloro-5,6-dicyano-l,4-benzoquinone, DPPF ¼ 1,10-bis(diphenylphosphino)ferrocene, Moz-S ¼ S-p-methoxybenzyloxycarbonyl-4,6-
dimethyl-2-mercaptopyrimidine, Moz ¼ p-methoxybenzyloxycarbonyl, DIC ¼ 1,3-diisopropylcarbodiimide, HOBt ¼ 1-hydroxybenzotriazole.
6-dicyano-1,4-benzoquinone) to give 4. Compound 4
1
R-NH-L-Tyr(O-t-Bu)-Gly-Gly-L-Phe -L-Leu-OMe
has a cone conformation,9 which was determined by H
7a: R =
NMR analysis. Palladium-catalyzed cyanation of 4 was
achieved in good yield.10 The cyano group was reduced
by borane and the resulting two aminomethyl groups
were protected by Moz (p-methoxybenzyloxycarbonyl)
groups. Finally, the hydroxy group in R2 was oxidized
and the resulting carboxylic acid was condensed with
aminomethylated polystyrene resin to prepare the pep-
tide library on a solid support.
H
O
N
O2N
N
N
N
4
O
O
7b: R = CH3(CH)14CO
Figure 2. Structure of the analytes 7a and 7b.
The peptide library was produced using the previously
reported procedure.2;5 After removal of the N-Moz
groups in 6,11 a split synthesis was run on both sides of
the arms to append a tripeptide using 15 Fmoc–amino
acids as building blocks. Because 15 amino acids were
used as building blocks, this library consists of
153 ¼ 3375 peptidocalixarenes. After removal of the N-
terminal Fmoc group in the tripeptide, the library was
condensed using 2-pyreneacetic acid as a fluorophore.
Finally, 1 was obtained by treating with trifluoroacetic
acid to remove the protective groups on the side chain of
the peptides.12
fying that only a few of the library members bind pep-
tide 7a. All the colored beads were isolated and decoded
to identify their amino acid sequences (Table 1).
Remarkably, only 4 of 3375 library members were
identified and all the colored beads had an
AA2. -Phe frequently appeared in AA3.
L-Tyr at
D
The solid support free 8 in Figure 3, which possessed
most frequently appearing peptide sequences in the
screening shown in Table 1, was selected as the
chemosensor for analyte 7b.13 Compound 8 was pre-
pared in solution phase.
The Leu5 enkephalin derivative 7 in Figure 2 was chosen
as an analyte. For the binding study of the library 1,
approximately 3.0 · 10ꢀ5 mol dmꢀ3 dye-labeled peptide
7a was incubated with ca. 5 mg of the library on beads in
CHCl3 for 3 days. Only a few beads turned red, signi-
The fluorescence spectrum of 8 at 1.0 · 10ꢀ6 mol dmꢀ3 in
CHCl3 is shown in Figure 4. In the absence of 7b, the
fluorescence spectrum of 8 exhibited dual emission
resulting from the monomer (382 and 400 nm) and the
excimer (470 nm). The emission at 470 nm was assigned

H. Hioki et al. / Tetrahedron Letters 45 (2004) 561–564
Table 1. Peptide sequences of colored beads in 1 for 7a
563
Compound
AA1
AA2
AA3
Frequencya
1a
1b
1c
1d
D
D
-Leu
-Leu
L
L
L
L
-Tyr
D
D
D
D
-Phe
14
4
-Tyr
-Tyr
-Tyr
-Leu
-Phe
-Phe
L
L
-Tyr
-Ala
2
1
a Number of beads isolated.
NHR1
L-Tyr
In conclusion, we synthesized a fluorescence-labeled
calix[4]arene library substituted with peptides at the
upper rim. The binding selectivity of 7a for library
members was higher than that of the lower rim-modified
calixarene library. Solid supported core compound 6
might serve as a useful platform for split synthesis of
libraries to produce artificial receptors using not only
peptides but also various types of building blocks.
R1HN
D-Phe
D-Phe
L-Tyr
D-Leu
NH
D-Leu
HN
O
R1 =
R2 = n-decyl
OR2
OR2
R2O
Acknowledgements
R2O
8
This work was supported in part by a SUNBOR
GRANT from the Suntry Institute for Bioorganic
Research and a Grant-in-Aid for Scientific Research
from the Ministry of Education, Science, and Culture of
Japan (No. 15608002).
Figure 3. Chemosensor for analyte 7b.
[7b] / 10-6 mol dm-3
0
95
190
380
760
References and Notes
1. For selected books on calixarene chemistry, see: Gutsche,
C. D. Calixarenes Revisited. In Monographs in Supramo-
lecular Chemistry; Stoddart, F. J., Ed.; The Royal Society
of Chemistry: Cambridge, 1998; Vol. 6; Mandolini, L.;
Ungaro, R. Calixarenes in Action; Imperial College Press:
London, 2000.
2. Hioki, H.; Kubo, M.; Yoshida, H.; Bando, M.; Ohnishi,
Y.; Kodama, M. Tetrahedron Lett. 2002, 43, 7949–7952.
3. For recent review for receptor libraries, see: Peczuh, M.
W.; Hamilton, A. D. Chem. Rev. 2000, 100, 2479–2494;
Lavigne, J. J.; Anslyn, E. V. Angew. Chem., Int. Ed. 2001,
40, 3118–3130.
4. For a recent review on calixarene-based chemosensors,
see: Diamond, D.; Nolan, K. Anal. Chem. 2001, 73, 22A–
29A.
400
450
500
λ / nm
550
600
Figure 4. Fluorescence emission spectroscopic change of 8 in CHCl3
upon addition of peptide 7b at 20 ꢁC. [8] ¼ 1.0 · 10ꢀ6 dmꢀ3. Excitation
wavelength: 344 nm.
5. Hioki, H.; Yamada, T.; Fujioka, C.; Kodama, M.
Tetrahedron Lett. 1999, 40, 6821–6825.
to the intramolecular excimer in this concentration.14
The monomer emission was dominant compared with
the excimer emission. The addition of analyte 7b to the
solution of 8 enhanced the fluorescence of excimer.
In contrast, the intensity of the monomer emission
decreased depending on the concentration of the ana-
lyte.15 This result indicated that the analyte 7b brings the
two pyrenyl groups of 8 closer together. The spectral
change was quite different from a previously reported
lower rim-modified peptidocalixarene sensor, which
enhances the fluorescence of both the monomer and
excimer emission, depending on the concentration of 7b.
Both results were different from the sodium ion-selective
fluorescence-labeled calixarene sensor reported by Jin
et al.16 Studies to clarify the difference in action, sensing
abilities of the other analyte are in progress.
6. A solid supported calix[4]arene substituted by trialanine
on the upper rim was reported, see: Shuker, S. B.;
Esterbrook, J.; Gonzalez, J. Synlett 2001, 23, 210–213.
7. As protein receptors, calix[4]arenes modified at the upper
rim by cyclic peptide were reported, see: Blaskovich, M.
A.; Lin, Q.; Delarue, F. L.; Sun, J.; Park, H. S.; Coppola,
D.; Hamilton, A. D.; Sebti, S. M. Nature Biotechnol. 2000,
18, 1065–1070, and references cited therein.
8. van Loon, J. D.; Arduini, A.; Coppi, L.; Verboom, W.;
Pochini, A.; Ungaro, R.; Harkema, S.; Reinhoudt, D. N.
J. Org. Chem. 1990, 55, 5639–5646.
9. Calixarenes, Monographs in Supramolecular Chemistry;
Gutsche, C. D., Stoddart, F. J., Eds.; The Royal Society of
Chemistry: Cambridge, 1989; Vol. 1.
10. Hioki, H.; Nakaoka, R.; Maruyama, A.; Kodama, M.
J. Chem. Soc., Perkin. Trans. 1 2001, 3265–3268.

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H. Hioki et al. / Tetrahedron Letters 45 (2004) 561–564
11. Protective groups on the side chains were cleaved by
treating twice with 5% iPr3SiH–1% TFA/CH2Cl2 (v/v) at
room temperature for 1 h.
12. N-Moz groups were cleaved by treating twice with 1%
TFA/CH2Cl2 (v/v) in the presence of PhSH (30 equiv) at
room temperature for 1 h.
13. Analyte 7b possesses a palmitoyl group at the N-terminal
(R) instead of the dye moiety in 7a. The N-terminal was
changed from the dye to the palmitoyl group because the
fluorescence emission of 8 overlapped with the absorption
of 7a.
14. The ratio (monomer/excimer) was not affected by the
concentration of 8 in the range of 10ꢀ6 to 10ꢀ8 mol dmꢀ3
.
The result indicates that the emission at 470 nm was
assigned to an intramolecular excimer.
15. The association constant (Kass) of 5 against peptide 2b was
estimated to be about 3.0 · 103 mol dmꢀ3 from the eximer
fluorescence intensity using the Benesi–Hildebrand plot17.
16. Jin, T.; Ichikawa, K.; Koyama, T. J. Chem. Soc., Chem.
Commun. 1992, 499–501.
17. Connors, K. A. Binding Constants; Wiley-Interscience,
1987; p 141.