Synthesis of a peptidocalix[4]arene library and identification of compounds with hydrolytic activity

Article abstract of DOI:10.1016/j.bioorg.2004.07.001

A 120 member library of peptidocalix[4]arenes was synthesized and screened for catalysis of the hydrolysis of p-nitrophenyl acetate. His-Ser-His-calix[4] arene was found to catalyze this reaction with v₀ = 3.24 × 10^-8 M/s, an increase of 1520% above background and 30% above the tripeptide (His-Ser-His) alone.

Full text of DOI:10.1016/j.bioorg.2004.07.001

Bioorganic Chemistry 33 (2005) 16–21
www.elsevier.com/locate/bioorg
Synthesis of a peptidocalix[4]arene library
and identification of compounds
with hydrolytic activity
R. Elizabeth Brewster, Brian G.A. Dalton,
Suzanne Beckham Shuker *
School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, GA 30329-0400, USA
Received 11 June 2004
Available online 23 September 2004
Abstract
A 120 member library of peptidocalix[4]arenes was synthesized and screened for catalysis of
the hydrolysis of p-nitrophenyl acetate. His-Ser-His-calix[4]arene was found to catalyze this
reaction with v0 = 3.24 · 10ꢀ8 M/s, an increase of 1520% above background and 30% above
the tripeptide (His-Ser-His) alone.
ꢀ 2004 Elsevier Inc. All rights reserved.
Keywords: Calixarenes; Catalysis; Solid-phase synthesis; Amino acids; Peptides
1. Introduction
Calix[4]arenes have been extensively studied for their ability to act as synthetic re-
ceptors and enzyme mimics. As hosts, calixarenes have been found to bind a wide
range of compounds, including amino acids [1], carboxylic acids [2], and other small,
organic molecules [3]. Calix[4]arenes substituted on the upper rim with ligands that
*
Corresponding author. Fax: +1 404 894 2295.
E-mail address: shuker@chemistry.gatech.edu. (S.B. Shuker).
0045-2068/$ - see front matter ꢀ 2004 Elsevier Inc. All rights reserved.
doi:10.1016/j.bioorg.2004.07.001

R.E. Brewster et al. / Bioorganic Chemistry 33 (2005) 16–21
17
chelate zinc [4] or copper [5] have been used to mimic hydrolytic enzymes and
catalyze the hydrolysis and transesterification of phosphonate esters. Ca-
lix[4]arene-4-sulfonates [6] have been shown to facilitate the regioselective cleavage
of cytidine 20,30-cyclic phosphate. Additionally, calix[4]arenes substituted with imi-
dazoles on the upper rim have been found to catalyze the hydrolysis of p-nitrophenyl
esters [7]. The success of these studies indicates that the functionality on the upper
rim of a calix[4]arene can be manipulated to design catalysts for a variety of reac-
tions. Peptides are an attractive choice for these substituents due to their wide range
of functional groups and inherent chirality. The binding pocket formed by amino ac-
ids incorporated at the upper rim of a calix[4]arene can take on the characteristics of
an enzyme-like active site, which is expected to confer selectivity and potency to the
catalyst.
We have reported the solid phase synthesis of a peptidocalix[4]arene [8] using
standard Fmoc peptide synthesis techniques, and we are currently investigating
the ability of peptidocalix[4]arenes to act as molecular receptors and enzyme mimics.
The development of these molecules is facilitated through the use of parallel, solid
phase synthesis, which allows rapid preparation of large numbers of compounds.
This paper details the synthesis and screening of a peptidocalix[4]arene library to
identify compounds that catalyze ester hydrolysis.
2. Materials and methods
2.1. General methods
The solvents and all reagents used in this study were purchased from commercial
suppliers and were used as received.
2.2. Tripropoxytetranitrocalix[4]arene anhydride 2
Tripropoxytetranitrocalixarene carboxylic acid 1 [8] (0.1g, 0.12mmol) and dic-
yclohexylcarbodiimide (0.012g, 0.060mmol) were stirred in 2mL of CH2Cl2. After
3h, the precipitate was removed by filtration and the filtrate was concentrated in vac-
uo to yield 2 (0.99g, 99%) as a yellow solid. This compound was used without further
purification.
2.3. Synthesis of library
Microtiter plates were amine functionalized [9] and the addition of tripropoxytet-
ranitrocalix[4]arene anhydride 2 to the glass was accomplished using DMAP as a
catalyst. The nitrocalix[4]arene 3 was treated with SnCl2 Æ 2H2O to provide the
corresponding tetraamino derivative 4. The appropriate amino acids were added
sequentially using Fmoc-protected amino acids, HBTU, and DIPEA. Treatment
with 20% piperidine removed the Fmoc protecting groups and with 95% TFA
removed the side chain protecting groups.

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2.4. Screening of library
The library was screened for the hydrolysis of p-nitrophenyl acetate (NPA) as fol-
lows. Into each well was added 74lM NPA in 100mM sodium acetate buffer, pH
5.0. Negative controls contained no calix[4]arene catalyst, while positive controls
contained 0.2mM KOH. The production of p-nitrophenol was monitored by the
change in UV-Vis absorbance at 324nm over 24h.
2.5. Synthesis of peptidcalixarenes on beads
The peptidocalix[4]arenes were synthesized according to literature procedures [8]
with exception that the anhydride derivative 2 of tripropoxytetranitrocalixarene car-
boxylic acid 1 was used to facilitate addition of the calixarenes to the solid support.
The loading was determined by measuring Fmoc release from the terminal amino
acids [10].
3. Results and discussion
A calix[4]arene library with four identical tripeptides on the upper rim was synthe-
sized on the surface of wells of 96-well glass microtiter plates. Each tripeptide con-
tained serine and histidine, two of the amino acids found in the catalytic triad of
serine proteases and esterases. The third amino acid was varied and all possible se-
quences—HSX, SHX, HXS, SXH, XHS, and XSH, where X, any of the 20 common
amino acids—were prepared, giving a library of 120 compounds. The preparation of
the calix[4]arene scaffold and the synthesis of the peptidocalix[4]arenes on solid sup-
port are shown in Scheme 1.
This screen revealed six compounds that catalyzed the hydrolysis: SerGluHis-ca-
lix[4]arene (SerGluHisCX), HisSerHisCX, GluSerHisCX, AsnSerHisCX, GluHisS-
erCX, and HisSerGluCX. These six compounds were synthesized on a larger scale
on Argopore-NH2 beads and were studied for their ability to catalyze the hydrolysis
of p-nitrophenyl acetate (shown in Scheme 1). The beads were screened by incubat-
ing them with 61.8mM NPA in 100mM sodium acetate buffer (pH 5.0)/acetonitrile
50/50 to give approximately equimolar amounts of catalyst and substrate. Controls
included either unmodified beads or beads with AlaAlaAlaCX. Samples were taken
every 30min for 8h. These results are shown in Fig. 1. UV–Vis spectroscopy indicat-
ed that these peptidocalix[4]arenes were capable of facilitating the hydrolysis with
SerGluHisCX, HisSerHisCX, and GluSerHisCX being the most efficient. AlaAla-
AlaCX showed no hydrolytic activity, as did the unmodified beads.
The initial rates for the hydrolysis of NPA by SerGluHisCX, HisSerHisCX or
GluSerHisCX were determined and compared to the initial rates of reaction for
the analogous tripeptides directly attached to the Argopore-NH2 beads (SerGluHis,
HisSerHis, and GluSerHis). The tripeptides were prepared directly attached to the
beads using standard Fmoc-protected peptide coupling techniques without the ca-
lix[4]arene scaffold. This comparison was meant to determine if the preorganization

R.E. Brewster et al. / Bioorganic Chemistry 33 (2005) 16–21
19
Scheme 1. Synthesis of peptidocalix[4]arenes on solid support. aAmine functionalized well of a microtiter
plate.
Fig. 1. Initial release of p-nitrophenol from PNA catalyzed by peptidocalix[4]arenes, monitored by UV–
Vis spectroscopy.

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Table 1
Initial rates for the hydrolysis of NPA in 50/50 acetonitrile/buffer (pH = 5.0)
Catalyst
v0 (10ꢀ8 M/s)
v0(cat)/v0(bkgd)
v0(pep-CX)/v0(pep)
—
0.2 0.1
1.7 0.2
1.7 0.4
2.4 0.3
1.2 0.4
0.8 0.3
3.2 0.1
—
—
—
GluSerHis
SerGluHis
HisSerHis
GluSerHisCX
SerGluHisCX
HisSerHisCX
8.5 0.5
8.4 0.6
12.2 0.5
6.2 0.6
4.1 0.6
16.2 0.5
—
—
0.7 0.3
0.5 0.4
1.3 0.1
[catalyst]/tripeptide = 0.48mM. [NPA]/[cat] = 500. Each point is the average of three runs.
of tripeptides on the upper rim of the calix[4]arene scaffold enhanced the catalytic
ability of these compounds over the tripeptides alone. Therefore, the amount of cat-
alyst used for the determination of the initial rates was based on the concentration of
the tripeptides and not on the concentration of the whole calixarene compound,
which allowed the comparison of the tripeptideꢀs ability to catalyze the hydrolysis
with and without calixarene scaffold. The initial rates for all six compounds were de-
termined using 0.48mM ([catalyst]/tripeptide) of SerGluHisCX, HisSerHisCX or
GluSerHisCX or 0.48mM SerGluHis, HisSerHis, or GluSerHis and 240mM NPA
([NPA]/[catalyst] = 500). These data are shown in Table 1. HisSerHisCX proved to
be the most efficient catalyst with v0 = 3.2 · 10ꢀ8 M/s at 240mM NPA. Under these
conditions, the presence of the calix[4]arene scaffold results in a 30% increase in the
initial rate of reaction over that of HisSerHis alone and a 1520% increase over that of
background hydrolysis (without catalyst). Interestingly, the tripeptides attached di-
rectly to the beads also proved to be quite effective at catalyzing this reaction in
all cases, and even better than the calix[4]arene catalysts for the tripeptides GluSer-
His and SerGluHis. Therefore, in only one case (HisSerHisCX) the calix[4]arene
scaffold enhanced the catalytic ability of the active tripeptide. Most importantly,
all six compounds showed a dramatic increase in reaction rate (from 310 to
1520%) over that of the background.
4. Conclusions
We have synthesized a peptidocalix[4]arene library and screened it for the hydro-
lysis of NPA. This revealed three compounds (SerGluHisCX, HisSerHisCX, and
GluSerHisCX) capable of hydrolyzing NPA. Further analysis indicated HisS-
erHisCX is the most efficient catalyst, giving an initial rate that is 1520% higher than
the background and 30% greater than that of HisSerHis in the absence of ca-
lix[4]arene scaffold. This is the first step in the development of these compounds as
novel, recyclable catalysts. We are currently preparing calix[4]arenes that contain
two or more different peptides at the upper rim and we expect these more diverse
peptidocalix[4]arenes to have improved catalytic ability. Additionally, we are contin-
uing to study the ability of the calix[4]arene scaffold to improve the catalytic efficien-
cy of the tripeptides.

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21
Acknowledgments
Acknowledgment is made to the donors of The Petroleum Research Fund,
administered by the American Chemical Society, and to the Georgia Institute of
Technology for support of this research. Suzanne B. Shuker is the Blanchard
Professor of Chemistry.
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