N-linked peptidoresorc[4]arene-based receptors as noncompetitive inhibitors for α-chymotrypsin

Article abstract of DOI:10.1021/jo102592f

This paper deals with the design, synthesis, and evaluation of a new series of receptors for protein surface recognition. The design of these agents is based around the attachment of four constrained dipeptide chains onto a central resorc[4]arene scaffold. By varying the sequence, nature, and stereochemistry of the chains we prepared anionically functionalized N-linked peptidoresorc[4] arenes 12, 13, and 17 by Pd/C-catalyzed hydrogenation of the corresponding benzyl esters 10, 11, and 16. From this family of receptors we have identified noncompetitive inhibitors of α-chymotrypsin (ChT), which function by binding to the surface of the enzyme in the neighborhood of the active site cleft (K_i values ranging from 12.4 ± 5.1 μM for free carboxylic acid (+)-12b to 0.76 ± 0.14 μM for benzyl ester (-)-16a). For anionically functionalized receptors 12, 13, and 17 the ChT inhibition is based essentially on electrostatic interaction, and the bound enzyme can be released from the resorcarene surface by increasing the ionic strength, with its activity almost completely restored. For receptors with terminal benzyl ester groups (10 and 16) a hydrophobic network can be suggested.

Full text of DOI:10.1021/jo102592f

ARTICLE
pubs.acs.org/joc
N-Linked Peptidoresorc[4]arene-Based Receptors as Noncompetitive
Inhibitors for R-Chymotrypsin
Ilaria D’Acquarica,† Antonella Cerreto,† Giuliano Delle Monache,† Fabiana Subrizi,† Alberto Boffi,‡
||
Andrea Tafi,§ Stefano Forli,§, and Bruno Botta*,†
†Dipartimento di Chimica e Tecnologie del Farmaco and ‡Dipartimento di Scienze Biochimiche, Sapienza Universitꢀa di Roma,
P. le Aldo Moro 5, 00185 Roma, Italy
§Dipartimento Farmaco Chimico Tecnologico, Universitꢀa degli Studi di Siena, Via Aldo Moro, 53100 Siena, Italy
S Supporting Information
b
ABSTRACT: This paper deals with the design, synthesis, and
evaluation of a new series of receptors for protein surface
recognition. The design of these agents is based around the
attachment of four constrained dipeptide chains onto a central
resorc[4]arene scaffold. By varying the sequence, nature, and
stereochemistry of the chains we prepared anionically functio-
nalized N-linked peptidoresorc[4]arenes 12, 13, and 17 by Pd/
C-catalyzed hydrogenation of the corresponding benzyl esters
10, 11, and 16. From this family of receptors we have identified
noncompetitive inhibitors of R-chymotrypsin (ChT), which
function by binding to the surface of the enzyme in the
neighborhood of the active site cleft (Ki values ranging from
12.4 ( 5.1 μM for free carboxylic acid (þ)-12b to 0.76 (
0.14 μM for benzyl ester (ꢀ)-16a). For anionically functiona-
lized receptors 12, 13, and 17 the ChT inhibition is based
essentially on electrostatic interaction, and the bound enzyme
can be released from the resorcarene surface by increasing the ionic strength, with its activity almost completely restored. For
receptors with terminal benzyl ester groups (10 and 16) a hydrophobic network can be suggested.
’ INTRODUCTION
exposed surface of proteins.11 Moreover, a further approach to
recognition was predicted in the insertion of charged hydrophilic
carboxylate groups12 or lipophilic benzyl groups in the terminal
amino acid, which might contact complementary residues across
the surface of the protein, by electrostatic or hydrophobic interac-
tions, respectively. With the aim of proposing our N-linked
peptidoresorc[4]arenes as synthetic receptors for protein
surface binding, we therefore prepared anionically functionalized
macrocycles by the ex novo synthesis of N-linked peptidoresorc-
[4]arene benzyl esters 10, 11, and 16, which were in turn
hydrolyzed to the corresponding free carboxylic acids 12, 13,
and 17. In preliminary studies, we tested the effective inter-
action between 12 and human serum albumin (HSA), the most
common serum protein. Afterward, we explored the binding
and the concomitant inhibition of R-chymotrypsin (ChT),
a popular substrate for these experiments,7,12 which was
shown to be inhibited by a specific family of calix[4]arene
receptors.9b,d
Because of their key role in a number of biological processes,
proteinꢀprotein interactions (PPI)1 are attractive and important
targets for the design of novel therapeutics. Synthetic receptors
targeted at protein surfaces provide an alternative paradigm to
active site inhibition for the disruption of such interactions.2
Covalent modification of a protein with a polymer offers the
opportunity of irreversibly modifying its biological activity.3 On
the other hand, noncovalent interactions of proteomimetics, i.e.,
molecules that mimic the structure and function of extended
regions of protein surfaces, offer the possibility of reversible
binding and modulation of the protein function itself. Among
these synthetic receptors, small molecules,4 R-helix mimetics
based on terphenyl or terephthalamide scaffolds,5 multidentate
ligands,6 functionalized nanoparticle scaffolds,7 and macrocyclic
systems, such as porphyrins8 and calixarenes,9 have been success-
fully used as protein inhibitors. We have recently introduced a
new class of resorc[4]arenes based on the attachment of four
constrained dipeptide chains onto a central macrocyclic scaffold.10a,b
The flexible conformation and the occurrence of multiple con-
tacts by a variable peptide sequence offered by the system
appeared theoretically adapt for binding the convex and solvent
Received: January 7, 2011
Published: May 02, 2011
r
2011 American Chemical Society
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’ RESULTS AND DISCUSSION
described,13 was hydrolyzed to tetracarboxylic acid 2 and subse-
quently treated with thionyl chloride in dry benzene, affording
the corresponding acid chloride derivative 3 in quantitative yield.
For the preparation of L-valyl-L-leucine benzyl ester (6a,
Figure 2), commercial N-Boc-L-valine 4a was coupled with L-leucine
benzyl ester p-toluensulfonate 5a in the presence of HOBT and
triethylamine to afford N-Boc-L-valyl-L-leucine benzyl ester,
which after deprotection with TFA/DCM, gave the dipeptide
6a as TFA salt. Analogously, L-leucyl-L-valine benzyl ester 9a, as
TFA salt, was prepared starting from commercial N-Boc-L-leucine
7a and L-valine benzyl ester p-toluensulfonate 8a. In both cases, the
overall yield was about 85%. The same peptide coupling procedure
was applied to the DD-dipeptides series: D-valyl-D-leucine benzyl
ester 6b and D-leucyl-D-valine benzyl ester 9b, TFA salts, were
obtained with comparable yields, starting from the pairs 4b/5b and
7b/8b, respectively. Notably, because 5b and 8b were not easily
commercially available, they were ad hoc synthesized as TFA salts.
The overall yields of the dipeptides were about 80%.
The four 6a, 6b, 9a, and 9b dipeptides were conjugated in-
dividually with macrocycle 3 as follows: DIPEA was added under
nitrogen atmosphere to a solution of 3 in dry THF, and the re-
action mixture was stirred at room temperature; after the addition of
the proper dipeptide benzyl ester in THF, the mixture was re-
fluxed for 1 h. Standard purification gave, in 65ꢀ75% yields, the
four N-linked peptidoresorc[4]arenes benzyl esters 10a, 10b,
11a, and 11b (Figure 1), which in turn, by hydrogenation with
10% Pd/C, afforded the free carboxylic acids 12a, 12b, 13a, and
13b (Figure 1). After the positive experimental results of the
binding between 12 and HSA (vide infra), four more compounds
were synthesized, aiming at doubling the number of terminal
functional groups, either for a lipophilic or electrostatic interac-
tion with proteins. L-Valyl-L-aspartic acid dibenzyl ester (15a)
and D-valyl-D-aspartic acid dibenzyl ester (15b) were thus pre-
pared by the usual procedure, starting from the proper commer-
cial N-Boc-valine (4a or 4b) and the proper aspartic acid dibenzyl
ester (commercial 14a or ad hoc synthesized 14b). The diben-
zylated peptidoresorc[4]arenes 16a and 16b, obtained by the
coupling with 3 of 15a and 15b, respectively, finally gave the
corresponding free dicarboxylic acids 17a and 17b by Pd/C
hydrogenation in quantitative yields. All of the new compounds
were structurally confirmed by NMR spectroscopy (see Tables
1Sꢀ3S in the Supporting Information) and electrospray ioniza-
tion high-resolution mass spectrometry (ESI-HRMS, see the
Supporting Information for a collection of spectra), as well as by
the optical rotation values (see Table 1).
Receptors Design and Synthesis. An important class of
protein surface receptors based on the attachment of four cyclic
peptides to a calix[4]arene scaffold was developed by Hamilton
and targeted to the serine protease ChT.9b Moving from Hamilton’s
results, we performed preliminary molecular modeling studies
suggesting the design of tetra-anionically functionalized recep-
tors that might target the predominantly cationic region of ChT
surrounding the active site. We have previously functionalized
resorc[4]arene octamethyl ethers at the feet with valine units10c
and later exploited their capability of enantiodiscriminating amino
acidic guests in the gas phase by mass spectrometry. Afterward, we
moved to design valyl-leucine and leucyl-valine peptidoresorc-
[4]arenes10a,b inspired by the evidence that N- or C-linked peptides
endow calixarenes with novel properties, such as recognition of
protein surfaces and carbohydrates, formation of stable inclusion
complexes, and self-assembly phenomena. Resorc[4]arene core
in the cone conformation was chosen for its high flexibility and for
the presence of four all-cis substituents that could preorganize the
peptide chain on the same side of the scaffold.
Following the scheme of our previous work,10a resorc[4]arene
octamethyl ether tetraester 1 (Figure 1), obtained as previously
NMR Analysis of N-Linked Peptidoresorc[4]arenes. As
noticed in our previous paper,10a for the presence of chiral centers
in the side chains, the four symmetry planes (C4v) of the starting
Figure 1. N-Linked peptidoresorc[4]arene benzyl esters (10, 11,
and 16) and free acids (12, 13, and 17).
Figure 2. Building blocks of the dipeptide chains.
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Table 1. Physical Properties of Chiral N-Linked Peptidoresorc[4]arenes
compd
peptide sequence
ESI-HRMS (m/z)
mp (°C)
[R]20D (c, solvent)
(ꢀ)-10a
(þ)-10b
L-Val-L-Leu-OBz
D-Val-D-Leu-OBz
L-Leu-L-Val-OBz
D-Leu-D-Val-OBz
L-Val-L-Leu-OH
D-Val-D-Leu-OH
L-Leu-L-Val-OH
D-Leu-D-Val-OH
L-Val-L-Asp-OBz
D-Val-D-Asp-OBz
L-Val-L-Asp-OH
D-Val-D-Asp-OH
1043.53604 ([M þ 2Na]2þ
)
220ꢀ221
221ꢀ222
248ꢀ249
251ꢀ252
212ꢀ213
209ꢀ211
135ꢀ136
135ꢀ136
218ꢀ219
218ꢀ219
201ꢀ202a
203ꢀ204a
ꢀ80.5 (1.0, CHCl3)
þ79.4 (1.0, CHCl3)
ꢀ76.7 (0.3, CHCl3)
þ75.9 (1.0, CHCl3)
ꢀ55.5 (1.0, MeOH)
þ52.5 (1.0, MeOH)
ꢀ49.2 (0.6, MeOH)
þ46.3 (0.7, MeOH)
ꢀ35.7 (0.5, MeOH)
þ35.9 (0.5, MeOH)
ꢀ18.3 (0.1, MeOH)
þ17.7 (0.1, MeOH)
1043.53635 ([M þ 2Na]2þ
1043.53549 ([M þ 2Na]2þ
1043.53656 ([M þ 2Na]2þ
)
)
)
(ꢀ)-11a
(þ)-11b
(ꢀ)-12a
566.62079 ([M ꢀ 4H þ Na]3ꢀ
566.62022 ([M ꢀ 4H þ Na]3ꢀ
566.62026 ([M ꢀ 4H þ Na]3ꢀ
566.61975 ([M ꢀ 4H þ Na]3ꢀ
)
)
)
)
(þ)-12b
(ꢀ)-13a
(þ)-13b
(ꢀ)-16a
1228.01773 ([M þ 2Na]2þ
)
(þ)-16b
1228.01416 ([M þ 2Na]2þ
)
(ꢀ)-17a
843.33097 ([M ꢀ 2H]2ꢀ
)
(þ)-17b
843.32839 ([M ꢀ 2H]2ꢀ
)
a With decomposition.
to the fully deprotonated molecule as sodium adduct ([M ꢀ
4H þ Na]3ꢀ), at m/z 566.61975ꢀ566.62079, in agreement with
their molecular formula. For free dicarboxylic acids 17a,b, a
diagnostic peak at [M ꢀ 2H]2ꢀ was found in the mass spectrum
(negative mode), at m/z 843.32839ꢀ843.33097. Melting points
were normally restricted in a 1.0 °C interval, but the free
dicarboxylic acids (ꢀ)-17a and (þ)-17b decomposed in-
stead of melting. Finally, optical rotations for the two members
of each enantiomeric pair were comparable in most cases
(see Table 1), excluding any significant racemization. However,
for the enantiomeric pairs 12a,b and 13a,b quite different values
were obtained (vide infra). Notably, the signs of optical rotation
are correlated with the stereochemistry of peptidoresorc[4]arenes:
the L,L-series always show negative values.
Circular Dichroism (CD) and Enantioselective HPLC Anal-
ysis. The discrepancy between the optical rotations of the DD-
and LL-forms of compounds 12 and 13 prompted us to examine
their enantiomeric purity by circular dichroism (CD) measure-
ments and enantioselective HPLC analysis.
The UV spectra in TRIS buffer 0.1 M at pH 7.4 of both 12 and
13 couples of enantiomers (Figure 4a,b, top traces) exhibit
absorption bands at the same wavelength maxima (210 and
280 nm) attributed to the expected 1La and 1Lb electronic tran-
sitions.14 On the contrary, the CD spectra of each enantiomeric
(ꢀ)-12a/(þ)-12b and (ꢀ)-13a/(þ)-13b pair are mirror images
(Figure 4a,b, bottom traces) and display the bisignate Cotton
effect centered at the above-mentioned wavelengths.
The first approach by enantioselective HPLC with a teicopla-
nin-based column (CHIROBIOTIC T)15a led to the enantiose-
lective discrimination of the 12a/12b pair, but not 13a/13b. By
contrast, the enantiomeric pairs of both free acids were success-
fully separated using a CHIRALPAK QN-AX (8S,9R) column,
which has been proved to perform highly enantioselective
separations of a very broad spectrum of chiral acids, such as
N-derivatized amino acids, R-aryloxyalkyl and R-arylalkyl
carboxylic acids like profens.15bꢀd In fact, such a column, based
on a quinine (QN)ꢀcarbamate derivative (Figure 1S, Support-
ing Information), is a weak anionic exchanger and is able to
perform electrostatic interactions between oppositely charged
functionalities of the analyte and the chiral selector bonded.
Enantiomeric excesses (ee, %) of peptidoresorc[4]arenes 12a,b
and 13a,b, checked by enantioselective HPLC on the above-
mentioned column, were in the 95ꢀ97% range.
Figure 3. Symmetry in the achiral molecule precursor (left) and the
final chiral product (right).
cone conformer 1 are no longer present in the N-linked peptido
derivatives 10ꢀ13, 16, and 17 (Figure 3). The planes R1/R3 and
R2/R4 contain the chiral centers, whereas the two planes A/C
and B/D separate two moieties that are no longer mirror images
(the DD-configuration of a chain should correspond the LL-con-
figuration of the opposite chain). Therefore, C-1 and C-3 carbons,
for instance, are no longer equivalent and give two distinct signals.
However, a C4 symmetry axis is still present, since the chains,
including the chiral center, are superimposable after a 90° rotation.
The C-1 carbon will thus be equivalent to C-7, C-13, and C-19
and C-3 will coincide with C-9, C-15, and C-21. Carbons (and
protons) equidistant from the chiral centers, such as C-e/C-i
(or H-e/H-i), show a single signal as also observed in the
achiral resorc[4]arene 1 precursor, whereas all of the other nuclei
cumulatively produce two signals. The distribution pattern of
carbons and protons in Tables 1Sꢀ3S (Supporting Information)
is in agreement with these considerations.
Physical Characterization of N-Linked Peptidoresorc-
[4]arenes. Physical properties of N-linked peptidoresorc[4]arenes
are summarized in Table 1. In the ESI-HRMS characterization of
peptidoresorc[4]arene benzyl esters 10, 11, and 16 (positive
mode), a strong tendency was observed to form complexes with
sodium (Na) and potassium (K) ion impurities, as already
reported.10a The most intense peak in the spectrum was that
for the double charged sodium adduct ion [M þ 2Na]2þ, at m/z
1043.53604ꢀ1043.53656 for 10 and 11 (which are indeed
regioisomers), and at m/z 1228.01773 for 16. The corresponding
monocharged species, [M þ Na]þ, were also observed. The free
acids 12 and 13, which are indeed regioisomers, displayed in the
mass spectra (negative mode) the most abundant ion corresponding
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Figure 4. UV (top traces) and CD (bottom traces) spectra in TRIS buffer 0.1 M at pH 7.4 of enantiomeric resorc[4]arenes (a) (ꢀ)-12a/(þ)-12b and
(b) (ꢀ)-13a/(þ)-13b (4.0 ꢁ 10ꢀ5 M).
Preliminary Binding Studies with HSA. The ability of N-
linked peptidoresorc[4]arenes to establish interactions with pro-
teins was preliminary tested on HSA. To demonstrate the effective
binding of receptor 12 to HSA, a CD experiment was carried out
with a double sector quartz cuvette. Solutions of HSA (5 ꢁ 10ꢀ6 M)
and of resorcarene derivatives (ꢀ)-12a or (þ)-12b (5 ꢁ
10ꢀ6 M) in 0.005 M sodium phosphate buffer (pH = 7.4) were
introduced separately into each sectors of the cuvette. A series of
CD spectra were recorded before mixing the two solutions (Figure 5,
top traces a,b, solid lines) and a second one after mixing (dotted
lines). The CD spectra before mixing showed intense negative
bands at ∼208 and 222 nm, which are typical of proteins with
high R-helical content. The corresponding spectra obtained after
the mixing with receptors (ꢀ)-12a and (þ)-12b were similar in
shape, indicating that the structure of HSA after mixing is still
predominantly R-helical. The difference between the spectra of
resorcarene derivatives collected after and before the mixing with
HSA provided CD spectra (Figure 5, bottom traces a,b, dotted
lines), which, as compared with the respectively normalized spectra
(solid lines), showed a consistent change in the absorptions. The
differences between the spectral profiles and peak positions of
the resorcarene CD bands before and after mixing with HSA
indicate that the molecule is located in a dissymmetric environ-
ment after exposure to the protein. These observations indicate
the formation of a complex between HSA and the resorcarene
derivative, not affecting the structural integrity of HSA.
Initial Screening for Inhibition Activity. The inhibition activ-
ity of the synthesized N-linked peptidoresorc[4]arenes was eval-
uated by in vitro assays with a chromogenic substrate toward
bovine pancreatic R-chymotrypsin (R-ChT), a serine protease
which was already shown to be inhibited by a specific family of
calix[4]arene receptors.9b ChT hydrolyzes proteins selectively
attacking the protein chain next to aromatic residues such as
tyrosine (Tyr) and phenylalanine (Phe), but it has also affinity for
other amino acids bearing apolar side chains (e.g., methionine,
leucine).16 The protein, which is characterized by the pre-
sence of positive charges on its surface, as suggested by the is-
oelectric point of 8.8, has been considered a suitable target for
studying the interaction with receptors containing terminal car-
boxylic acids, in anionic form at the physiological pH.7,12 At the
same pH, some basic amino acids surrounding the active site are
protonated: electrostatic interactions with the positively
charged patches of ChT are thus expected to lead to a
consequent inhibition of the enzyme. Inhibition assays
should thus provide a useful handle for studying the proteinꢀ
receptor complex.
Inhibition of ChT by peptidoresorc[4]arenes free acids 12
and 13 was investigated by monitoring at 405 nm the rate of
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Figure 5. Top traces: CD spectra obtained in a double sector cuvette of HSA (5 ꢁ 10ꢀ6 M) in 0.005 M sodium phosphate buffer (pH = 7.4) before
(solid lines) and after (dotted lines) mixing with (a) (ꢀ)-12a (5 ꢁ 10ꢀ6 M) or (b) (þ)-12b (5 ꢁ 10ꢀ6 M) solutions. Bottom traces: CD spectra of
(a) (ꢀ)-12a or (b) (þ)-12b obtained by the difference between the spectra after and before the mixing with HSA (dotted lines), compared with the
respectively normalized spectra (solid lines).
enzymatic hydrolysis of the chromogenic substrate N-succinyl-
Ala-Ala-Pro-Phe-p-nitroanilide (Suc-AAPF-pNA) to p-nitroaniline,
after the mixing of protein and receptors. Control experi-
ments were carried out under identical conditions without the
addition of resorc[4]arenes. The inhibitory effect, determined by
treating the enzyme (4.0 ꢁ 10ꢀ8 M) with solutions of 12a, 12b,
13a, or 13b (4.0 ꢁ 10ꢀ5 M) in 0.005 M sodium phosphate buffer
for various preincubation periods followed by addition of Suc-
AAPF-pNA in 2-methoxyethanol (cosolvent), was shown to be
time-independent (see Table 2). Receptors (ꢀ)-12a and (þ)
-12b were found to decrease the Suc-AAPF-pNA hydrolysis to 13
and 11%, respectively, as compared with those of the untreated
enzyme, whereas (ꢀ)-13a and (þ)-13b led to a smaller inhibi-
tion. The major inhibition by the pair 12a/12b (with the valyl-
leucine instead of leucyl-valine sequence) can be attributed to a
preference of the enzyme for the cleavage of dipeptide chains
containing leucine next to hydrophobic residues. The dipeptide
configuration seems to be not so important, even though the DD-
derivatives were slightly more active than the LL-enantiomers,
which are the natural ligands of the enzyme.
(þ)-12b, which was treated at concentrations ranging from 4.0 ꢁ
10ꢀ6 to 1.0 ꢁ 10ꢀ4 M with ChT maintained at a fixed con-
centration (4.0 ꢁ 10ꢀ8 M) for a 90 min preincubation period.
The dose-responsive inhibition of ChT by (þ)-12b at pH 7.4 in
0.005 M sodium phosphate buffer showed that, in the presence of
(þ)-12b at a 4.0 ꢁ 10ꢀ6 M concentration, the activity of ChT
decreased to 43% (this value represents an average of three in-
dependent sets of experiments, SD < 3%). Further increase in the
concentration of (þ)-12b (4.0 ꢁ 10ꢀ5 M) resulted in a decrease
of the initial velocity to 11% (dose-responsive inhibition plots are
reported in Figure 2S, Supporting Information). Nonlinear
regression analysis to fit the MichaelisꢀMenten data (Figure 6,
top) yielded the apparent maximum enzymatic reaction rate
(Vmax = 7.3 ꢁ 10ꢀ3 abs secꢀ1) and the MichaelisꢀMenten
constant (Km = 96.9 ( 7.2 μM), whereas Dixon plots17a
(Figure 6, bottom right) were used to measure the inhibition
constant (Ki = 12.4 ( 5.1 μM). The kinetic studies pointed
out that receptor (þ)-12b behaves as a noncompetitive
inhibitor of ChT because it decreases the Vmax values, with
minor effects on the Km constant.17b On these bases, it can be
envisaged that (þ)-12b does not compete directly with the
substrate molecules, though it may affect the size and shape of
the catalytic cleft.
Peptidoresorc[4]arene (þ)-12b. To get more information
on the inhibition mechanism of peptidoresorc[4]arenes toward
ChT, we focused our attention on the most effective inhibitor
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Table 2. Residual Activity (%) of ChT (4.0 ꢁ 10ꢀ8 M) after Mixing with Peptidoresorc[4]arenes 12, 13, 16, and 17 in Comparison
with the Activity of the Native Enzyme
To determine some possible binding areas of (þ)-12b on the
surface of ChT, a computational docking experiment was carried
out using the program AutoDock 4.0.18 The calculations point to
a complex of the type shown in Figure 7, where the carboxylate
groups in (þ)-12b make electrostatic interactions with several of
the basic residues (Lys-36, Lys-90, Lys-175, and Arg-145) surround-
ing the active site cleft of ChT. Some of these amino acids were
already found to be involved in ChT complexes with calix-
[4]arene receptors.9b,d
The proposed ChT inhibition by peptidoresorc[4]arene
(þ)-12b through a specific complex formation was confirmed
by nondenaturing gel electrophoresis on 1% agarose gel at pH 7.4
(Figure 8).9b,g In the absence of receptor (lane 1), the protein
(ChT, 2.0 ꢁ 10ꢀ5 M) migrated almost completely toward the
cathode, in agree with its positively charged surface. At a (þ)
-12b/ChT molar ratio of 1:1 (lane 2), two bands were observed
and attributed to unbound ChT (near to the cathode) and to a
complex between ChT and (þ)-12b, which migrated toward the
anode. When the concentration of receptor (þ)-12b was in-
creased to 1.0 ꢁ 10ꢀ4 M (lane 3) and to 2.0 ꢁ 10ꢀ4 M (lane 4),
the majority of ChT molecules appeared to be bound to receptor
(þ)-12b, as shown by the lower intensity of the bands in the
cathode region. At a (þ)-12b/ChT molar ratio of 50:1 (lane 5),
only the band corresponding to the bound ChT was observed.
To sum up, comparison of the lanes shows that ChT and receptor
(þ)-12b form a new species with different polarity to uncom-
plexed enzyme. Since no free enzyme appeared on the gel starting
from lane 4, the binding ratio of (þ)-12b/ChT should be
between 10:1 and 50:1.
To shed light on some other possible interactions between pep-
tidoresorc[4]arenes and ChT, the investigation was extended to
the available benzyl ester (þ)-10b, which was the synthetic
precursor of the analyzed free acid. The inhibition experiments,
carried out under the same conditions as for (þ)-12b (see
Table 2), resulted in only 5% residual activity of the enzyme
after incubation with (þ)-10b at a concentration of 4.0 ꢁ
10ꢀ5 M, increasing to 68% with the macrocycle at 4.0 ꢁ
10ꢀ6 M (time-independent inhibition). A different type of
interaction, actually hydrophobic, by peptidoresorc[4]arenes
was thus shown to be possible for the ChT inhibition (vide
infra), since free acid (þ)-12b was a much weaker inhibitor of
ChT than its benzyl ester homochiral precursor (þ)-10b.
Peptidoresorc[4]arenes 17a,b. The receptors pair 17a/17b
features a change in the nature of the dipeptide chain (valyl-
aspartic acid instead of valyl-leucine, as compared with 12a/12b),
but mainly a 2-fold number of ionizable carboxylic groups
and, as expected, a major amount of electrostatic interactions.
However, only the LL-derivative (ꢀ)-17a exhibited a larger inhibi-
tion activity than the homochiral (ꢀ)-12a (see Table 2) at the
same concentration: numerically, only 5% of residual activity of
ChT was found at a concentration of 4.0 ꢁ 10ꢀ5 M. The
inhibition resulted to be time-independent and was still signifi-
cant (28% of residual ChT activity) at a 4 ꢁ 10ꢀ6 M concentra-
tion of receptor. Surprisingly, in the presence of the DD-derivative
(þ)-17b, ChT exhibited a 71% of residual activity after 90 min.
The inhibitory activity of (þ)-17b was shown to be time-
dependent (100% of residual ChT activity was found after
180 min). As in the case of (þ)-12b, for comparative purposes
we tested the inhibition activity of benzyl esters (ꢀ)-16a and
(þ)-16b, which were the synthetic precursors of receptors 17a,b:
in these cases, because the benzyl esters were slightly soluble in
phosphate buffer, higher concentrations than 4 ꢁ 10ꢀ6 M were
not allowed, and the cosolvent (2-methoxyethanol) was replaced
by dimethyl sulfoxide (DMSO) for the preparation of stock sol-
utions of receptors. The final concentration of DMSO (2.4 ꢁ
10ꢀ2 M) was indeed much lower than those needed for influencing
The change in mobility of ChT after addition of receptor
(þ)-12b may depend on both an increased size and an attenua-
tion of the enzyme surface charge due to binding and complex
formation: several cationic residues are indeed present near the
active site cleft of ChT. These findings suggested that complexa-
tion between ChT and (þ)-12b resulted in the formation of
neutral particles but gave no information on the type of binding,
which was assumed to be primarily, but not purely, electrostatic.
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Figure 6. Noncompetitive inhibition of ChT (4.0 ꢁ 10ꢀ8 M) by receptor (þ)-12b. Top: nonlinear regression analysis to fit the MichaelisꢀMenten
data in the absence of inhibitor (Km = 96.9 ( 7.2 μM). Left: LineweaverꢀBurk plot at three fixed Suc-AAPF-pNA concentrations in the absence (b) and
presence of 4 μM (1), 20 μM ((), and 40 μM (9) of (þ)-12b. Right: Dixon plot at three fixed Suc-AAPF-pNA concentrations, namely (2) 10, (b) 100,
and (9) 1000 μM. Ki = 12.4 ( 5.1 μM.
Figure 7. Calculated structure (crossed stereoview by AutoDock 4.0)
for the interaction of (þ)-12b with ChT. The protein structure is
displayed as a Connolly surface, while the 3D structure of the docked
Figure 8. Nondenaturing gel electrophoresis of ChT and receptor
ligand is explicitly shown colored by atom type. Uncharged amino acids
are colored in yellow, negatively charged in red and positively charged in
blue. Few relevant basic residues are tagged. The active site cleft entrance
is marked by a black arrow.
(þ)-12b on 1% agarose gel at pH 6.0. Samples were loaded in the
middle of the gel: lane 1, ChT (2.0 ꢁ 10ꢀ5 M); lanes 2ꢀ5, (þ)-12b þ
ChT at increasing receptor-to-ChT molar ratios; lane 6, (þ)-12b
(2.0 ꢁ 10ꢀ5 M).
the enzyme activity.19 In the presence of receptor (þ)-16b, the
of weak hydrophobic interactions. Moreover, the LL-enantiomer
residual activity of ChT decreased by the time to 13% after
90 min to reach a minimum (6%) after 120 min. The time-
dependent inhibition supports the slow formation of a network
(ꢀ)-16a was shown to be the most potent inhibitor of ChT of all
the family of peptidoresorc[4]arenes: 5% of residual activity of
the enzyme was found in the presence of (ꢀ)-16a at a concentration
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Figure 9. Noncompetitive inhibition of ChT (4.0 ꢁ 10ꢀ8 M) by receptor (ꢀ)-16a. Left: LineweaverꢀBurk plot at three fixed Suc-AAPF-pNA
concentrations in the absence (() and presence of 0.4 μM (9), 1 μM (2), and 2 μM (b) of (ꢀ)-16a. Right: Dixon plot at three fixed Suc-AAPF-pNA
concentrations, (2) 10 μM, (9) 100 μM, and (() 1000 μM. Ki = 0.76 ( 0.14 μM.
of 4.0 ꢁ 10ꢀ6 M, and 60% at 4.0 ꢁ 10ꢀ7 M (dose-responsive
inhibition plots of ChT by (ꢀ)-16a are reported in Figure 3S,
Supporting Information). Notably, the inhibition was immediate
and time-independent. The fact that LL-conformation of dipep-
tides is coincident with the stereochemistry of the natural sub-
strates of the enzyme is not enough to explain the unexpected
results for interactions based exclusively on hydrophobicity.
Peptidoresorc[4]arene (ꢀ)-16a. To get more information on
the unexpected strong inhibition of benzyl ester (ꢀ)-16a toward
ChT, we performed the same set of experiments as for free acid
(þ)-12b (vide supra). Receptor (ꢀ)-16a was thus treated at
concentrations ranging from 4.0 ꢁ 10ꢀ7 to 4.0 ꢁ 10ꢀ6 M with
ChT maintained at a fixed concentration (4.0 ꢁ 10ꢀ8 M) for a 90
min preincubation period. Kinetic analysis, based on Lineweaverꢀ
Burk (Figure 9, left) and Dixon (Figure 9, right) plots,
pointed out that receptor (ꢀ)-16a is a noncompetitive inhibitor
of ChT (Ki = 0.76 ( 0.14 μM) because it decreases the Vmax
values without affecting the Km constant.17 However, receptor
(þ)-12b, featuring more complementary recognition surfaces to
ChT than receptor (ꢀ)-16a, showed a significantly lower in-
hibition potency (Ki = 12.4 ( 5.1 μM). To explain such strong
interaction between benzyl ester (ꢀ)-16a and ChT we may
invoke the chemical similarity between the terminal benzyl groups
and the benzyl side chain of Phe; in fact, examination of the
specificity requirements for ChT reveals that a critical interaction
for efficient binding of a substrate to this enzyme includes a
van der Waals interaction of the benzyl side chain of Phe with a
hydrophobic pocket adjacent to the active site of the enzyme.7a
Effect of Medium Ionic Strength on the ProteinꢀReceptor
Complexation. The noncovalent binding experiment performed
by nondenaturing gel electrophoresis (Figure 8, lanes 2ꢀ5)
showed that the binding process between inhibitor (þ)-12b
and ChT must be reversible. Therefore, we investigated the
possible occurrence, for the bound enzyme, to be released from
the macrocycle surface when the ionic strength of the medium is
increased. ChT (4.0 ꢁ 10ꢀ8 M) was thus incubated with in-
hibitor (þ)-12b (4.0 ꢁ 10ꢀ5 M) in the presence of sodium
chloride (NaCl) at five different concentrations ranging from 1.0 ꢁ
10ꢀ4 to 1.0 ꢁ 10ꢀ1 M, as already reported.6e,7b The samples were
incubated at room temperature for 90 min, a time sufficient for an
almost complete inhibition (∼ 90%) in the absence of NaCl. The
enzyme activity was monitored as above-described and com-
pared with the activity of a control solution containing only ChT
and NaCl under the same conditions (system named before
mixing, BM). The normalized residual activities of each sample
and the corresponding NaCl concentration are summarized in
Table 3.
In a second system (named after mixing, AM), NaCl at the
above-mentioned concentrations was added to the complex
ChT/(þ)-12b, as well as to a solution of pure ChT, after 90
min incubation period. Both mixtures were analyzed after further
90 min of incubation. This method was used to evaluate the effect
of ionic strength on the stability of the complex ChT/(þ)-12b,
once allowed to form; the results, i.e., the residual activities of the
enzyme and the corresponding NaCl concentrations, are also
collected in Table 3.
As expected, the residual activity of ChT increases with the
increase of NaCl concentration (BM mode): at NaCl concentra-
tions between 1.0 ꢁ 10ꢀ4 and 1.0 ꢁ 10ꢀ2 M (see Table 3), the
ionic strength is not strong enough to break completely
the electrostatic attraction between enzyme and inhibitor, and
the residual activity undergoes a small increase (23% and 36% of
the control). By contrast, the ChT residual activity increases
sharply in the presence of 5.0 ꢁ 10ꢀ2 M NaCl (95% of the
control) and is almost completely restored (∼ 98%) for NaCl
1.0 ꢁ 10ꢀ1 M. These findings confirm that the binding of
inhibitor (þ)-12b to the enzyme is mostly based on electrostatic
complementarities and, as such, is reversible and does not
denature the native conformation of ChT.
When we performed the same set of experiments on receptor
(ꢀ)-17a, which features a 2-fold number of ionizable carboxylic
groups for the replacement of leucine with an aspartate residue,
we observed that the formation of the complex ChT-receptor
seemed to be mitigated only partially by the increase of the ionic
strength: actually, the residual activity of the enzyme is still 50%
in the presence of NaCl 1.0 ꢁ 10ꢀ1 M (Table 3, system BM).
This result may be attributed to the higher number of ionizable
functional groups, and thus to a major amount of electrostatic
interactions to be broken.
The AM mode experiments, aimed at investigating the stability
of the complex ChT-receptor under the influence of the medium
ionic strength, substantially confirmed the mainly electrostatic
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Table 3. Residual Activity (%) of ChT (4.0 ꢁ 10ꢀ8 M) in the Presence of NaCl before Mixing (System BM) and after Mixing
(System AM) with Peptidoresorc[4]arenes (þ)-12b, (ꢀ)-17a, (þ)-10b, (þ)-16b, and (ꢀ)-16a, in Comparison with the Activity of
the Native Enzyme
NaCl
compda
system
0 M
1.0 ꢁ 10ꢀ4 M
1.0 ꢁ 10ꢀ3 M
1.0 ꢁ 10ꢀ2 M
5.0 ꢁ 10ꢀ2 M
1.0 ꢁ 10ꢀ1 M
(þ)-12b
BM
AM
BM
AM
BM
AM
BM
AM
BM
AM
11
8
23
10
3
36
10
5
36
20
5
95
62
17
64
9
98
92
50
100
51
15
32
40
2
(ꢀ)-17a
(þ)-10b
(þ)-16b
(ꢀ)-16a
5
3
4
3
10
5
6
11
17
22
2
13
13
4
13
16
5
13
13
4
12
14
3
1
1
1
2
2
2
a Receptors concentration: 4.0 ꢁ 10ꢀ5 M, except for receptors (ꢀ)-16a and (þ)-16b (4.0 ꢁ 10ꢀ6 M).
nature of the interaction between ChT and receptors (þ)-12b
and (ꢀ)-17a (the first complex is slightly more stable). Also in
this case, the investigation on (þ)-12b was extended to the
available benzyl ester (þ)-10b, which was its synthetic precursor:
the enzyme restored only partially its activity, which was 51% in the
presence of NaCl 1.0 ꢁ 10ꢀ1 M (Table 3, system BM), denoting
a low importance of ionic strength on the quality of interaction
between ChT and benzyl ester (þ)-10b. As a confirmation, the
stability of the complex was shown to be slightly influenced by
the ionic strength as well, since, at the same NaCl concentration,
ChT showed only 15% residual activity (AM mode).
With regard to receptor (þ)-16b, which was supposed to
slowly form a network of hydrophobic interactions with ChT
(see Table 2), the experiments in the presence of increasing
concentrations of NaCl confirmed such hypothesis (see Table 3). In
fact, the reduced influence of the ionic strength on the formation
(system BM) and the stability (system AM) of the ChT-(þ)-16b
complex is evidenced by the values of 32% and 40%, respectively,
recovered at NaCl 1.0 ꢁ 10ꢀ1 M for the residual ChT activity.
Notably, the LL-enantiomer (ꢀ)-16a was confirmed to be the
most effective inhibitor of ChT activity, since the inhibition was
not significantly affected by the ionic strength (Table 3), both
before and after incubation of the inhibitor with enzyme.
(Lys-36, Lys-90, Lys-175, and Arg-145) surrounding the active
site cleft of ChT. Within the same framework, the benzyl ester
derivatives seem to be more effective: for example, free acid
(þ)-12b is a much weaker inhibitor of ChT than its benzyl ester
homochiral precursor (þ)-10b, as judged by the residual enzy-
matic activity after a 90 min incubation period. In the cases of
receptors 12 and 13, compounds with D,D-configuration were
more active than the L,L-derivatives. In particular, receptor
(þ)-12b was shown to be a noncompetitive inhibitor of ChT
(Ki = 12.4 ( 5.1 μM) because it decreases the Vmax values
without affecting the Km. However, for all of the other cases, the
most active compounds featured the L,L-configuration, which is
the sterical arrangement of the natural ligand. This is mainly
true for the 17a/17b pair: the arrangement of the eight carbox-
ylate groups, responsible for electrostatic interaction with the
enzyme seems to be significantly affected by the D,D-configura-
tion of the dipeptide chains (71% of residual enzymatic activity vs
5%). This trend was observed on the benzyl ester homochiral
precursor as well, where the LL-enantiomer (ꢀ)-16a was found to
be the most effective noncompetitive inhibitor of ChT activity,
with a Ki = 0.76 ( 0.14 μM. In this case, inhibition was not
significantly affected by the ionic strength, both before and after
mixing of the inhibitor with enzyme.
Focusing on the nature of the dipeptide chains, we noticed
that the terminal amino acids of peptidoresor[4]arenes 12, 13
and 17 could be placed in the following scale of inhibition
activity: aspartic acid (receptor 17) > leucine (receptor 12) >
valine (receptor 13), somehow corresponding to the 2-fold
number of interaction sites (for aspartic acid) and to the affinity
of ChT for the amino acids (for leucine), as a substrate.16 Anal-
ogously, to explain the strong hydrophobic interactions between
ChT and peptidoresor[4]arenes benzyl esters 10 or 16, we may
invoke the chemical similarity between the terminal benzyl
groups and the benzyl side chain of phenylalanine, one of the
main substrates of ChT.16 In fact, a van der Waals interaction of
the Phe benzyl side chain with a hydrophobic pocket adjacent to
the active site is one of the specificity requirements for ChT.
’ CONCLUSIONS
Our efforts into the design of novel N-linked peptidoresorc-
[4]arenes with appropriately modified functional groups proved
that they are able to interact with proteins such as HSA and acquire
inhibition properties vs enzymes such as ChT. For compounds
with terminal carboxylate functions (12, 13, and 17), the ChT
inhibition is based essentially on electrostatic interaction, and the
bound enzyme can be released from the resorcarene surface by
increasing the ionic strength, with its activity almost completely
restored. The inhibitors were not modified by ChT, as checked
by TLC analysis (CHCl3/MeOH mixtures) of mixtures of
peptidoresorc[4]arenes and enzyme incubated for 3 h, after
complex denaturation. For receptors with terminal benzyl ester
groups (10 and 16) a hydrophobic network can be instead
suggested. A computational docking experiment carried out on
(þ)-12b pointed to a complex where the carboxylate groups
make electrostatic interactions with several of the basic residues
’ EXPERIMENTAL SECTION
Materials. R-Chymotrypsin (Type II) from bovine pancreas
(ChT), N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide (Suc-AAPF-pNA),
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1
HPLC-grade acetonitrile, water, and glacial acetic acid were purchased
from Sigma Aldrich. HPLC-grade ammonium acetate was purchased
from Baker. Dimethyl sulfoxide (DMSO) and 2-methoxyethanol (analytical
grade) were from Carlo Erba. Reagents for electrophoresis were purchased
from commercial sources.
6b. Pale yellow powder. 65% overall yield. H NMR, 13C NMR, and
ESI-HRMS data are coincident with those reported for (ꢀ)-10a.
[R]20D: þ79.4 (c 1.0, CHCl3).
2,8,14,20-Tetrakis(L-leucyl-L-valinamido)resorc[4]arene
Benzyl Ester (11a). (ꢀ)-11a was obtained as described for (ꢀ)-10a,
starting from tetracarboxylic acid chloride 3 and dipeptide benzyl
ester 9a. Pale yellow powder. 75% overall yield. Mp: 248ꢀ249 °C. 1H
and 13C NMR signals are reported in the Supporting Information
(Tables 1S and 2S). ESI-HRMS (pos): m/z found 1043.53549 ([M þ
2Na]2þ), C116H152O24N8Na2 requires 1043.53520 (monoisotopic mass).
[R]20D: ꢀ76.7 (c 0.3, CHCl3).
Synthesis of Resorc[4]arene Octamethyl Ethers 1ꢀ3. Com-
pounds 1ꢀ3 were synthesized as previously described.10a
Preparation of N-Boc-D-aspartic Acid. To an ice-cooled solu-
tion of D-aspartic acid in 1,4-dioxane (1.5 g, 11.3 mmol in 20 mL) were
added NaOH 1 N (45 mL) and di-tert-butyl dicarbonate (2.5 g,
11.5 mmol). The mixture was left at room temperature for 3 h, acidified
to pH 1ꢀ2 with HCl 1 N, and extracted with ethyl acetate (5 ꢁ 80 mL).
The pooled organic extract, after drying over Na2SO4, gave N-Boc-D-
aspartic acid with a 63% yield.
2,8,14,20-Tetrakis(D-leucyl-D-valinamido)resorc[4]arene
Benzyl Ester (11b). (þ)-11b was obtained as described for (ꢀ)-10a,
starting from tetracarboxylic acid chloride 3 and dipeptide benzyl ester
9b. Pale yellow. 65% overall yield. 1H NMR, 13C NMR, and ESI-
HRMS data are coincident with those reported for (ꢀ)-11a.
[R]20D: þ75.9 (c 1.0, CHCl3).
General Procedure for the Preparation of Benzyl Esters
5b, 8b, and 14b as TFA Salts. Cs2CO3 (32.6 g, 100 mmol) was
added to an ice-cooled solution of the proper N-Boc-protected D-amino
acid (100 mmol) in DMF (200 mL). The mixture was stirred at 0 °C for
45ꢀ60 min. Benzyl bromide (11.9 mL, 17.1 g, 100 mmol) was added to
the reaction mixture, which was maintained under magnetic stirring at
0 °C for 30 min and allowed to reach room temperature overnight. The
reaction mixture was poured into H2O (800 mL) and extracted with
n-hexane (4 ꢁ 250 mL). The combined extracts were washed with H2O
(200 mL) and saturated NaCl, and then dried over Na2SO4. Filtration,
concentration, and drying under vacuum gave N-Boc-D-leucine benzyl
ester (95%) N-Boc-D-valine benzyl ester (95%) and N-Boc-D-aspartic
acid dibenzyl ester (86%) as colorless oils. Benzyl esters (1 mmol) were
deprotected by treatment with TFAꢀDCM (1:1, 25 mL). The reaction
mixture was stirred at 25 °C for 45 min and then evaporated to dryness;
the addition of a suitable hexane/Et2O mixture (50 mL) under vigorous
stirring yielded white solids that were filtered, washed several times with
hexane/Et2O, and dried under vacuum to afford the pure TFA salts 5b,
8b, and 14b in quantitative yields. For the characterization of the above
compounds, see the Supporting Information.
General Procedure for the Preparation of Dipeptide Ben-
zyl Esters 6a,b, 9a,b, and 15a,b as TFA Salts. The proper amino
acid benzyl ester as TFA or p-toluensulfonate salt (5.5 mmol), HOBT
(0.75 g, 5.5 mmol), Et3N (0.96 mL, 7.1 mmol), and the proper N-Boc-
protected amino acid (5.5 mmol) were dissolved in DCM (250 mL).
DCC (1.14 g, 5.5 mmol) was added to the solution cooled in an ice bath.
After 4 h at room temperature, the solvent was removed under vacuum,
and the residue was purified by silica gel column chromatography
(hexaneꢀethyl acetate, 7.5:2.5) to yield the N-Boc-protected dipeptide
benzyl esters as white powders (yields 80ꢀ85%). Dipeptides (0.9 mmol)
were deprotected by treatment with TFAꢀDCM (1:1, 25 mL) as de-
scribed previously to afford the pure TFA salts 6a,b, 9a,b, and 15a,b in
quantitative yields. For the characterization of the above compounds, see
the Supporting Information.
2,8,14,20-Tetrakis(L-valyl-L-leucinamido)resorc[4]arene
Benzyl Ester (10a). DIPEA (0.42 mL, 2.5 mmol) was added under
nitrogen to a dry THF solution of tetracarboxylic acid chloride 3 (150 mg,
0.15 mmol in 45 mL). The mixture was stirred for 40 min at room
temperature, and a dry THF solution of 6a (322 mg, 0.9 mmol in 30 mL)
was added dropwise in a 1 h period. The reaction mixture was held at
reflux under nitrogen for 1 h. Evaporation of the solvent and purification
by column chromatography (CHCl3/MeOH mixtures) gave peptido-
resorc[4]arene (ꢀ)-10a as a pale yellow powder (70% overall yield).
Mp: 220ꢀ221 °C. 1H and 13C NMR signals are reported in the
Supporting Information (Tables 1S and 2S). ESI-HRMS (pos): m/z
found 1043.53604 ([M þ 2Na]2þ), C116H152O24N8Na2 requires
1043.53520 (monoisotopic mass). [R]20D: ꢀ80.5 (c 1.0, CHCl3).
2,8,14,20-Tetrakis(D-valyl-D-leucinamido)resorc[4]arene
Benzyl Ester (10b). (þ)-10b was obtained as described for (ꢀ)-10a,
starting from tetracarboxylic acid chloride 3 and dipeptide benzyl ester
2,8,14,20-Tetrakis(L-valyl-L-aspartic acid amido)resorc-
[4]arene Benzyl Ester (16a). (ꢀ)-16a was obtained as described
for (ꢀ)-10a, starting from tetracarboxylic acid chloride 3 and dipeptide
benzyl ester 15a. Pale yellow powder. 54% overall yield. Mp: 218ꢀ219 °C.
1H and 13C NMR signals are reported in the Supporting Information
(Table 3S). ESI-HRMS (pos): m/z found 1228.01773 ([M þ 2Na]2þ),
C136H152O32N8Na2 requires 1228.01647 (monoisotopic mass).
[R]20D: ꢀ35.7 (c 0.5, MeOH).
2,8,14,20-Tetrakis(D-valyl-D-aspartic acid amido)resorc-
[4]arene Benzyl Ester (16b). (þ)-16b was obtained as described
for (ꢀ)-10a, starting from tetracarboxylic acid chloride 3 and dipeptide
benzyl ester 15b. Pale yellow powder. 56% overall yield. 1H NMR, 13
C
NMR, and ESI-HRMS data are coincident with those reported for
(ꢀ)-16a. [R]20D: þ35.9 (c 0.5, MeOH).
General Procedure for Pd/C Hydrogenation of Resorc-
[4]arene Benzyl Esters to Resorc[4]arene Free Acids 12a,b,
13a,b, and 17a,b. After two vacuum/nitrogen cycles to replace air
inside the reaction tube, a mixture of the proper resorc[4]arene benzyl
ester (0.05 mmol) and 10% Pd/C (70 mg) in absolute EtOH (4 mL)
and dry THF (4 mL) was vigorously stirred at room temperature under 1
atm of hydrogen for 24 h. The reaction mixture was filtered using a
membrane filter (Millipore, Millex-LH, 0.45 μm), and the filtrate was
concentrated to provide the product, which was dissolved twice in ethyl
acetate to remove residual EtOH. The ethyl acetate solution was finally
dried under high vacuum at 25 °C, to give resorc[4]arene free acids
12a,b, 13a,b, and 17a,b in quantitative yields.
2,8,14,20-Tetrakis(L-valyl-L-leucinamido)resorc[4]arenecar-
boxylic Acid (12a). (ꢀ)-12a was obtained by Pd/C hydrogenation of
(ꢀ)-10a. Mp: 212ꢀ213 °C. 1H and 13C NMR signals are reported in the
Supporting Information (Tables 1S and 2S). ESI-HRMS (neg): m/z
found 566.62079 ([M ꢀ 4H þ Na]3ꢀ), C88H124O24N8Na requires
566.62142 (monoisotopic mass). [R]20D: ꢀ55.5 (c 1.0, MeOH). En-
antiomeric excess (ee, %) of (ꢀ)-12a, checked by enantioselective
HPLC, was in the 95ꢀ97% range.
2,8,14,20-Tetrakis(D-valyl-D-leucinamido)resorc[4]arenecar-
boxylic Acid (12b). (þ)-12b was obtained by Pd/C hydrogenation of
(þ)-10b. 1H NMR, 13C NMR, and ESI-HRMS data are coincident with
those reported for (ꢀ)-12a. [R]20D: þ52.5 (c 1.0, MeOH).
2,8,14,20-Tetrakis(L-leucyl-L-valinamido)resorc[4]arene-
carboxylic Acid (13a). (ꢀ)-13a was obtained by Pd/C hydrogena-
tion of (ꢀ)-11a. Mp: 135ꢀ136 °C. 1H and 13C NMR signals are
reported in the Supporting Information (Tables 1S and 2S).
ESI-HRMS (neg): m/z found 566.62026 ([M ꢀ 4H þ Na]3ꢀ),
C88H124O24N8Na requires 566.62142 (monoisotopic mass). [R]20D: ꢀ49.2
(c 0.6, MeOH). Enantiomeric excess (ee, %) of (ꢀ)-13a, checked by
enantioselective HPLC, was in the 95ꢀ97% range.
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2,8,14,20-Tetrakis(D-leucyl-D-valinamido)resorc[4]areneca-
rboxylic Acid (13b). (þ)-13b was obtained by Pd/C hydrogenation
of (þ)-11b. 1H NMR, 13C NMR, and ESI-HRMS data are coincident
with those reported for (ꢀ)-13a. [R]20D: þ46.3 (c 0.7, MeOH).
2,8,14,20-Tetrakis(L-valyl-L-aspartic acid amido) resorc-
[4]arenecarboxylic Acid (17a). (ꢀ)-17a was obtained by Pd/C
preincubation period, and then 20 μL of a stock solution of Suc-AAPF-
pNA in 2-methoxyethanol were added to reach a final substrate con-
centration of 1 ꢁ 10ꢀ4 M. Enzymatic hydrolysis was followed by
monitoring p-nitroaniline formation at 405 nm for 1 min. The assays
were performed in triplicate, and the averages are reported in Table 2
(SD < 3%). The activity of the native ChT (control) was taken to be
100%, and from this value the residual enzymatic activity (%) was
calculated.
1
hydrogenation of (ꢀ)-16a. Mp: 201ꢀ202 °C dec. H and 13C NMR
signals are reported in the Supporting Information (Table 3S). ESI-
HRMS (neg): m/z found 843.33097 ([M ꢀ 2H]2ꢀ), C80H102O32N8
requires 843.33056 (monoisotopic mass). [R]20D: ꢀ18.3 (c 0.1, MeOH).
2,8,14,20-Tetrakis(D-valyl-D-aspartic acid amido)resorc-
[4]arenecarboxylic Acid (17b). (þ)-17b was obtained by Pd/C
hydrogenation of (þ)-16b. 1H NMR, 13C NMR, and ESI-HRMS data
are coincident with those reported for (ꢀ)-17a. [R]20D: þ17.7 (c 0.1,
MeOH).
Kinetic assay. ChT activity was measured using Suc-AAPF-pNA as
substrate in the absence or presence of (þ)-12b or (ꢀ)-16a inhibitors,
under identical conditions as activity assay. The reaction mixture contained
three fixed Suc-AAPF-pNA concentrations (10 μM, 100 μM, and 1000 μM)
with inhibitors concentrations equal to 0, 4, 20, and 40 μM for (þ)-12b
and 0, 0.4, 1, and 2 μM for (ꢀ)-16a.
Statistical Analysis. All assays were run in triplicate. Graphs were
plotted using Microcal Origin software, version 6.0. Values of the slopes
and intercepts were obtained by the linear regression analysis using the
same software. Ki is given as the mean ( SD of three values calculated by
using the Dixon plots at three different substrate concentrations. Values
of Vmax and Km were recovered by nonlinear regression analysis based on
the MichaelisꢀMenten equation (R2 = 0.99872).
Ultraviolet (UV) and Circular Dichroism (CD) Experiments.
UV and CD spectra of receptors 12 and 13 in TRIS buffer 0.1 M at pH
7.4 (4.0 ꢁ 10ꢀ5 M) were obtained using a quartz cuvette with a 1 cm
path length at a constant temperature of 25 °C. Eight scans were taken
for each sample at 0.2 nm intervals from 190 to 320 nm, at a rate of
20 nm/min. Binding experiments of receptors (ꢀ)-12a and (þ)-12b
with HSA were performed using a double sector quartz cuvette with a
0.9 cm path length, at a constant temperature of 25 °C. Three averaged
scans for each CD spectrum were taken at 0.2 nm intervals from 190 to
320 nm, at a rate of 20 nm/min. In these experiments, all samples were
dissolved in 0.005 M sodium phosphate buffer (pH = 7.4) at the same
concentration (5 ꢁ 10ꢀ6 M). The spectra of receptors (ꢀ)-12a and
(þ)-12b alone were subtracted from the spectra of receptors (ꢀ)-12a
and (þ)-12b and HSA, to give difference spectra of receptors (ꢀ)-12a
and (þ)-12b in the presence of HSA (Figure 5).
Docking Analysis. The input conformation for receptor (þ)-12b
was generated by a conformational search carried out with the Macro-
Model/Maestro software package (Schr€odinger)20 equipped with Am-
ber* force field.21 The search was performed on the rotatable bonds of
the four dipeptide side chains of the molecule, which were moved in 3D
space with the aim of locating a reliable local minimum energy geometry.22
In this work, the default settings of Monte Carlo Multiple Minimum
search protocol were left unchanged. Analysis of possible binding areas
of (þ)-12b on the surface of ChT was then carried out by using the
program AutoDock 4.0.18 Further details are given in the Supporting
Information.
Enantioselective HPLC of Peptidoresorc[4]arenes 12 and
13. Enantioselective HPLC separations were performed on a CHIR-
ALPAK QN-AX (8S,9R) stainless steel column (250 ꢁ 4.0 mm i.d.). A
mixture of acetonitrileꢀ18 mM ammonium acetate (90:10) was used as
the mobile phase. The pH of this mixture (apparent pH) was adjusted to
8.2 by adding HPLC-grade glacial acetic acid. Flow rate: 1.0 mL/min;
UV detection at 284 nm. Retention (k0) and enantioselectivity (R)
’ ASSOCIATED CONTENT
Supporting Information. 1H and 13C NMR signals of
S
b
peptidoresorc[4]arenes 10ꢀ13 and 16ꢀ17. Structure of the
CHIRALPAK QN-AX (8S,9R) chiral stationary phase. Dose-
responsive inhibition plots of ChT by (þ)-12b and by (ꢀ)-16a.
ESI-HRMS spectra of peptidoresorc[4]arenes (ꢀ)-10a,
factors obtained were the following: for peptidoresorc[4]arene 12, k0L
=
2.75 (corresponding to 12a); k0D = 3.27 (corresponding to 12b); R =
1.19. For peptidoresorc[4]arene 13, k0D = 3.13 (corresponding to 13a);
k0D = 3.98 (corresponding to 13b); R = 1.27. Enantiomeric excesses
(ee, %) were in the 95ꢀ97% range.
1
(ꢀ)-11a, (ꢀ)-12a, (ꢀ)-13a, (ꢀ)-16a, and (ꢀ)-17a. H and
13C NMR spectra of peptidoresorc[4]arenes (þ)-10b, (ꢀ)-11a,
(þ)-12b, (ꢀ)-13a, (ꢀ)-16a, and (þ)-17b. Docking analysis and
clustering of focused docking experiment. This material is
available free of charge via the Internet at http://pubs.acs.org.
Gel Electrophoresis. Agarose gels were prepared in 0.005 M
sodium phosphate buffer (pH = 6.0) at a 1% final agarose concentration,
the buffer being the mobile phase. Appropriately sized wells (40 μL
volume) were formed by placing a comb in the middle of the gel. A ChT
2 ꢁ 10ꢀ4 M stock solution in 0.005 M sodium phosphate buffer
(pH = 7.4) was used to prepare individual samples (30 μL) of the
appropriate ChT/receptor (þ)-12b ratio. After a 90 min incubation
period at room temperature, 8 μL of 33% glycerol containing tracking dye
(bromophenol blue) were added, and a constant voltage (70 V) was
applied for approximately 1 h to run the electrophoresis. At the end, the
gel was placed in a staining solution (0.5% Coomassie Blue, 40%
methanol, 10% acetic acid in water) for 1 h, followed by extensive
destaining (40% methanol, 10% acetic acid aqueous solution) until the
protein bands became evident.
’ AUTHOR INFORMATION
Corresponding Author
* Tel.: þ390649912781. Fax: þ390649912780. E-mail: bruno.
botta@uniroma1.it.
Present Addresses
Department of Molecular Biology, The Scripps Research
Institute, La Jolla, California 92037-1000
General Conditions for Enzymatic Activity Assay. All experi-
ments were performed in 0.005 M sodium phosphate buffer (pH = 7.4).
All solutions were kept at 25 °C during incubation periods and the
kinetic analysis. The desired concentrations of the different receptors
were achieved by adding a proper quantity of stock solutions in 2-methox-
yethanol (for 10, 12 and 13) or DMSO (for 16 and 17) to 1 mL of the
phosphate buffer solution of ChT (4.0 ꢁ 10ꢀ8 M). ChT and different
concentrations of the receptors were kept at 25 °C for a fixed
’ ACKNOWLEDGMENT
We acknowledge financial support from FIRB, grant no.
RBPR05NWWC_006, Sapienza Universitꢀa di Roma, Italy (Funds
for Selected Research Topics 2008ꢀ2010) and Fondazione Roma
(Roma, Italy). This work was partially supported by the “Istituto
PasteurꢀFondazione Cenci Bolognetti”, Sapienza Universitꢀa di
Roma, Italy. We gratefully acknowledge Professor F. Gasparrini
4406
dx.doi.org/10.1021/jo102592f |J. Org. Chem. 2011, 76, 4396–4407

The Journal of Organic Chemistry
ARTICLE
(Sapienza Universitꢀa di Roma, Italy) for assistance with enantio-
selective HPLC analysis of peptidoresorc[4]arenes 12 and 13
and for helpful discussions.
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