Protein surface recognition by synthetic receptors: A route to novel submicromolar inhibitors for α-chymotrypsin

Article abstract of DOI:10.1021/ja981504o

A family of synthetic receptors for protein surface recognition has been prepared. The receptors are based on a design in which four peptide loops are arrayed around a central calilx[4]arene core. By varying the sequence of the loop regions a range of differently functionalized receptor surfaces approximately 450 ?² in area can be prepared. From this family we have identified potent inhibitors of chymotrypsin that function by binding to the surface of the protein. The most potent of these (1) shows slow binding kinetics in an analogous manner to several of the natural protein proteinase inhibitors. Detailed kinetic analysis showed 1 to be a competitive inhibitor with K(i) and K(i)* values of 0.81 and 0.11 μM, respectively.

Full text of DOI:10.1021/ja981504o

8
J. Am. Chem. Soc. 1999, 121, 8-13
Protein Surface Recognition by Synthetic Receptors: A Route to
Novel Submicromolar Inhibitors for R-Chymotrypsin
Hyung Soon Park, Qing Lin, and Andrew D. Hamilton*
Contribution from the Department of Chemistry, Yale UniVersity, New HaVen, Connecticut 06511
ReceiVed May 1, 1998
Abstract: A family of synthetic receptors for protein surface recognition has been prepared. The receptors
are based on a design in which four peptide loops are arrayed around a central calilx[4]arene core. By varying
the sequence of the loop regions a range of differently functionalized receptor surfaces approximately 450 Å2
in area can be prepared. From this family we have identified potent inhibitors of chymotrypsin that function
by binding to the surface of the protein. The most potent of these (1) shows slow binding kinetics in an
analogous manner to several of the natural protein proteinase inhibitors. Detailed kinetic analysis showed 1 to
be a competitive inhibitor with Ki and Ki* values of 0.81 and 0.11 µM, respectively.
Introduction
inhibitors is known to bind to surface regions and/or the active
sites of different serine proteases. The crystal structure of
7
Every protein contains a unique distribution of functional
groups (charged, hydrogen bonding, and hydrophobic) on its
exterior surface. In many cases, these regions are involved in
important interactions to other proteins as part of multisubunit,
reactivity modulating, or signaling complexes. The design of
synthetic agents that recognize and bind to specific regions of
R-chymotrypsin (ChT) bound to Kunitz inhibitor shows exten-
sive interactions from negatively charged regions on the inhibitor
to several basic residues on the chymotrypsin surface close to
8
the active site. Figure 2 shows the disposition of lysines and
1
arginines on the surface of chymotrypsin and suggests that a
complementary synthetic receptor might bind to this region of
the protein and block the approach of a substrate to the active
site.
2
a protein surface offers a new approach to enzyme inhibitors
or to the disruption of key protein-protein interactions (as in
Figure 1). We are interested in developing a modular and
potentially general approach to synthetic receptors that bind to
protein surfaces. Our design takes its origin from the antigen
recognition sites of antibodies that use changes in the sequence
and conformation of six hypervariable loops to complex a wide
range of protein surfaces. We have recently introduced a new
class of protein surface receptors in which four peptide loops
are attached to a central calixarene scaffold (Figure 1). The
Results and Discussion
Inhibitor Design and Synthesis. Our approach to surface
binding inhibitors of chymotrypsin involved the synthesis by
9
solid-phase methods of a series of cyclic peptides containing
3
a 5-nitro-3-aminomethylbenzoic acid dipeptide mimetic (Figure
1
0
3
A). Reduction of the nitro group followed by reaction with
4
the tetra-acid chloride derivative of calilx[4]arene tetracarboxylic
acid and removal of any side chain protecting groups gave a
series of receptors containing four identical peptide loops (Figure
result is a concave molecular surface approximately 450-500
2
Å in area whose recognition characteristics can be readily
changed by varying the sequence of the cyclic peptide. In the
present paper we report the preparation of a family of tetra-
loop receptors and from them identify potent surface binding
inhibitors of R-chymotrypsin.5
The serine proteases represent interesting targets for protein
surface recognition due to their central role in biological and
3
B).
In targeting the predominantly cationic active site region of
R-chymotrypsin, our initial approach was to construct receptors
in which each peptide loop contained two anionic residues
(
(
GDGD, receptor 1) or one anionic and one hydrophobic residue
GDGY, 2), such that they might stretch across the surface of
6
medicinal chemistry. Also, a large family of natural protein
the protein and contact multiple basic residues. For comparison
purposes we also prepared the corresponding receptor containing
four bis-cationic (GKGK, 3) peptide loop derivatives, a simpli-
fied octa-carboxylic acid derivative of calixarene 4 and the nitro-
substituted single loop analogue 5.
(
1) Stites, W. E. Chem. ReV. 1997, 97, 1233-1250.
(2) Zutshi, R.; Brickner, M.; Chmielewski, J. Curr. Opin. Chem. Biol.
1
998, 62-66
3) Davies, D. R.; Padlan, E. A.; Sheriff, S. Annu. ReV. Biochem. 1990,
9, 439-473.
4) Hamuro, Y.; Calama, M. C.; Park, H. S.; Hamilton, A. D. Angew.
Chem., Int. Ed. Engl. 1997, 36, 2680-2683.
5) For other examples of surface binding synthetic inhibitors see: Regan,
(
5
(
(6) See, for example: Ripka, W. C.; Vlasuk, G. P. Ann. Rep. Med. Chem.
1997, 32, 71-89.
(
(7) Laskowski, M.; Sealock, R. W. In The Enzymes; Boyer, P. D., Ed.;
Academic Press: New York, 1971; pp 375-473.
J.; McGarry, D.; Bruno, J.; Green, D.; Newman, J.; Hsu, C. Y.; Kline, J.;
Barton, J.; Travis, J.; Choi, Y. M.; Volz, F.; Pauls, H.; Harrison, R.;
Zilberstein, A.; Ben-Sasson, S. A.; Chang, M. J. Med. Chem. 1997, 40,
(8) Capasso, C.; Rizzi, M.; Menegatti. E.; Ascenzi, P.; Bolognesi, M. J.
Mol. Recognit. 1997, 10, 26-35.
3
408-3422. Tilley, J. W.; Chen, L.; Fry, D. C.; Emerson, S. D.; Powers,
(9) Bach, A. C.; Eyermann, C. J.; Gross, J. D.; Bower, M. J.; Harlow,
R. L.; Weber, P. C.; DeGrado, W. F. J. Am. Chem. Soc. 1994, 116, 3207-
3219. Smythe, M. L.; Von Itzstein, M. J. Am. Chem. Soc. 1994, 116, 2725-
2733.
G. D.; Biondi, D.; Varnell, T.; Trilles, R.; Guthrie, R.; Mennona, F.; Kaplan,
G.; LeMahieu, R. A.; Carson, M.; Han, R. J.; Liu, C. M.; Palermo, R.; Ju,
G. J. Am. Chem. Soc. 1997, 119, 7589-7590. Li, S.; Gao, J.; Satoh, T.;
Friedman, T. M.; Edling, A. E.; Koch, U.; Choksi, S.; Han, X.; Korngold,
R.; Huang, Z. W. Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 73-78.
(10) Nigam, M.; Seong, C. M.; Qian, Y.; Blaskovich, M. A.; Hamilton,
A. D.; Sebti, S. M. J. Biol. Chem. 1993, 268, 20695-20698.
1
0.1021/ja981504o CCC: $18.00 © 1999 American Chemical Society
Published on Web 12/28/1998

Protein Surface Recognition by Synthetic Receptors
J. Am. Chem. Soc., Vol. 121, No. 1, 1999 9
Figure 1. Schematic representation of enzyme inhibition by protein surface recognition.
Figure 2. Distribution of basic residues on the surface of chymotrypsin (A, side view; B, top view).
length of time that 1 was incubated with the enzyme before
addition of the substrate. A plot of initial velocity vs incubation
-
5
-6
time at [1] ) 3.09 × 10 M and [ChT] ) 4.29 × 10
M
(
Figure 4) shows that slow binding is occurring with a half-life
of 135 ( 18 min. This property of slow binding inhibition is
similar to that seen with certain protein inhibitors of the serine
7
proteases (e.g., R1-trypsin inhibitor with kallikrein or modified
1
1
pancreatic trypsin inhibitor with trypsin ) whose half-lives are
also measured in hours. In this initial screening no detectable
inhibition was seen with the monomeric cyclic peptide 5 (with
GDGD sequence) clearly pointing to the importance of multiple
peptide loops in enzyme binding.
Initial Screening for Inhibition Activity. This family of
receptors was screened for its ability to inhibit bovine pancreatic
That the inhibition of R-chymotrypsin by 1 was due to
specific complex formation was confirmed by nondenaturing
gel electrophoresis on agarose gel (Figure 5). Comparison of
lanes 1 and 2 shows that 1 and R-chymotrypsin (1 equiv) form
a new species with opposite polarity to the uncomplexed
enzyme. No binding is seen to soybean trypsin inhibitor (TI;
lanes 3 and 4) and the ChT-TI complex migrates toward the
anode in a similar but distinct manner (lane 5).
-
7
R-chymotrypsin by treating the enzyme (3.03 × 10 M) with
-
6
solutions of 1, 2, or 3 (9.2 × 10 M) in phosphate buffer for
a 24 h induction period followed by addition of the chromogenic
substrate, N-benzoyltyrosine-p-nitroanilide (BTNA). Receptor
1
(GDGD) was found to decrease hydrolysis to 10% of that of
the untreated enzyme compared to little or no inhibition by 2
and 3. In contrast, another serine protease, elastase, showed no
inhibition with 1, 2, or 3, underlining the selectivity of 1 for
the surface of R-chymotrypsin. The inhibition depended on the
(11) Vincent, J. P.; Lazdunski, M. Biochemistry 1972, 11, 2967-2977.

10
J. Am. Chem. Soc., Vol. 121, No. 1, 1999
Park et al.
Figure 3. Designs of artificial receptors for chymotrypsin surface recognition.
Scheme 1. Mechanisms of Time-Dependent Enzyme
Inhibition
initial EI complex (as in eq 1), and the second (Ki*) corresponds
to the dissociation constant of the second enzyme inhibitor
complex E*I, as defined by eq 2.
Figure 4. Time course for the inhibition of chymotrypsin by 1.
Kik6
[E][I]
k5 + k6 [EI] + [E*I]
K * )
)
(2)
i
These different slow binding inhibition mechanisms can be
investigated by preincubation of enzyme with inhibitor followed
by the measurement of initial velocities for substrate hydrolysis
as a function of preincubation time. In these cases, the inhibition
can be described by eq 3,
Figure 5. Nondenaturing gel electrophoresis on 1% agarose at pH
V /V ) exp(-kobst)
(3)
i
0
7
.4, sample loaded in the center of the gel (lane 1, ChT; lane 2, ChT+1;
lane 3, TI; lane 4, TI+1; lane 5, ChT+TI; lane 6, ChT+TI+1).
where V0 and Vi are the initial velocities of uninhibited and
inhibited reaction, respectively, and kobs is the first-order rate
constant for the interconversion between the initial and the
steady state. Figure 6 shows the fractional velocities for
N-benzoyltyrosine-p-nitroanilide hydrolysis by R-chymotrypsin
for different concentrations of inhibitor 1. Correlation of kobs
obtained for each curve in Figure 6) against [1] gave a nonlinear
plot (Figure 7) that is consistent with slow binding inhibition
through a two-step, enzyme-isomerization mechanism (Scheme
Slow Binding Inhibition. Slow binding inhibition is encoun-
tered more rarely than classical or tight binding inhibiton and
12
can result from three essential mechanisms (Scheme 1): simple
slow binding (mechanism B), in which the inhibitor binds to
the enzyme like a classical inhibitor but with a slow binding
rate; enzyme isomerization (mechanism C), in which a rapid
inhibitor binding step is followed by a slower conformational
change or isomerization; and irreversible inhibition (mechanism
(
1
2
1
C). Nonlinear curve fitting of these data by using eq 4 gave
values for Ki , k5, and k6 (Table 1).
1
3
D). In the case of mechanism B, the inhibition constant (Ki)
app
is defined simply as the equilibrium dissociation constant.
k5[I]
app
k4 [E][I]
kobs ) k +
(4)
K )
)
(1)
6
i
k3
[EI]
K
i
+ [I]
However, in mechanism C two inhibition constants must be
considered. The first (Ki) is the dissociation constant for the
A similar analysis was applied to the much weaker inhibitor
2. A plot of [2] vs kobs gave a linear relationship (Figure 8) that
is characteristic of a single step slow binding mechanism
(
12) Copeland R. A. In Enzymes: A Practical Introduction to Structure,
Mechanism, and Data Analysis; Wiley-VCH: New York, 1996; p 237.
13) Morrison, J. F.; Walsh, C. T. AdV. Enzymol. 1988, 61, 201-301.
1
2
(
Scheme 1B), as described by eq 5. However, in this case we
app
(
cannot exclude the possibility of mechanism C, since if Ki

Protein Surface Recognition by Synthetic Receptors
J. Am. Chem. Soc., Vol. 121, No. 1, 1999 11
a
Table 1. Summary of Rate and Equilibrium Constants for the Inhibition of Chymotrypsin by Receptors
(M- min )c
5
1
-1
4
k )
(min-1
c
k
or k
3
receptorsb
(min )d
-1
or k
(min )d
-1
K
(10-6 M)
K
i
* (10-6 M)
0.107 ( 0.007
6
i
1
(GDGD)
(GDGY)
(GKGK)
0.00652 ( 0.00005
79.8 ( 9.6
0.000986 ( 0.000048
0.00121 ( 0.00003
0.813 ( 0.033
15.2 ( 1.5
2
3
4
5
35.7 ( 3.2
26.4 ( 0.2
>50
a
-7
-7
Concentration of chymotrypsin was fixed as 3.5 × 10 M for 1, 2, and 4 and 7.0 × 10 M for 3 and 5 in 5% DMSO, pH 7.4, 5 mM phosphate
buffer at 25 °C. Substrate was BTNA and its concentration was fixed as 1.4 × 10 M. b [1] ) (0.555-7.40) × 10 M, [2] ) (5.83-11.7) × 10
-
4
-6
-6
M, [3] ) (1.00-5.00) × 10 M, [4] ) (1.05-4.20) × 10 M, [5] ) (1.00-5.00) × 10 M. c For 2. d For 1.
-
5
-5
-5
Figure 8. Plot of kobs vs [2].
Figure 6. Plot of fractional velocity vs preincubation time for several
concentrations of 1.
Figure 9. Plot of kobs vs [BTNA] at fixed concentrations of chymo-
trypsin and 1.
and 5, time dependent effects could not be observed due to the
very weak inhibition (less than 20% inhibition after 5 h of
incubation). In these cases, Lineweaver-Burk analysis was used
and the inhibition constants measured by this method are
collected in Table 1. Receptor 1 binds tightly to chymotrypsin
in both its initial (Ki ) 0.81 µM) and final (Ki* ) 0.11 µM)
enzyme-inhibitor complexes. In contrast, receptors 2 and 3 with
less complementary recognition surfaces show significantly
lower inhibition potency (Ki ) 15 and 36 µM, respectively).
However, electrostatic effects are not the sole contributor to
strong binding since derivative 4, with a similar number of
carboxylic acid groups, is a much weaker inhibitor of R-chy-
motrypsin than 1.
Figure 7. Plot of kobs vs [1].
.
Ki*app, the concentration of the EI complex would be
negligible and a linear relationship between [I] and kobs would
be seen.12
[
I]
app
kobs ) k 1 +
(5)
4
(
)
Ki
To determine the type of slow binding inhibition (e.g.,
competitive, noncompetitive, etc.) and hence to obtain appropri-
ate values for Ki or Ki*, we measured the dependence of kobs
on the concentration of substrate. With receptor 1, kobs was seen
to decrease as the substrate concentration increased, which is
(14) In the case of slow binding competitive inhibition the appropriate
relationships are:
k
app
[S]
kobs
)
and Ki ) K 1 +
i
(
)
1
+ [S]/Km
Km
behavior unique to the competitive mechanism of slow binding
See: Tian, W. X.; Tsou, C. L. Biochemistry 1981, 34, 808.
inhibition (Figure 9).1
4,15
Full data for inhibition of R-chymo-
(
15) It should be noted that sufficiently high concentrations of substrate
trypsin by each receptor are summarized in Table 1. For 3, 4,
could not be reached to observe saturation effects.

12
J. Am. Chem. Soc., Vol. 121, No. 1, 1999
Park et al.
The observation of competitive inhibiton of R-chymotrypsin
put in the refrigerator for 6 h. After filtration the resin was washed
extensively by using the same procedure as above. The resin was
coupled with 2.5 mmol of a preformed symmetric anhydride of Fmoc-
Gly-OH and DMAP (0.306 g, 2.5 mmol) and the vessel was shaken at
room temperature for 4 h. The following steps were then performed:
by 1 lends support to the proposal that the receptor is binding
close to the active site of the enzyme and blocking the approach
16
of the substrate. In addition, a competitive binding experiment
showed, by nondenaturing gel electrophoresis (Figure 2, lane
(a) The resin was washed with DMF (2×), MeOH (2×), CH
2
Cl
2
(2×),
6
), that 1 (3.5 equiv) is able to displace trypsin inhibitor from
MeOH (2×), and DMF (2×); (b) the Fmoc group was removed by
using 20% piperidine in DMF for 45 min; (c) the resin was washed
using the same procedure as in step a; (d) Fmoc-Amnb-OH (0.420 g,
1.0 mmol, 2 equiv), BOP (0.443 g, 1.0 mmol, 2 equiv), HOBt (0.135
g, 1.0 mmol, 2 equiv), and DIEA (0.18 mL, 1.0 mmol, 2 equiv) in 10
mL of DMF were added and the reaction vessel was shaken for 2 h;
and (e) the ninhydrin test was performed to check the completion of
the coupling, and recoupling was performed if necessary. Steps afe
were repeated for the other amino acid residues using the same
equivalents until the desired linear peptide was assembled.
its 1:1 complex with R-chymotrypsin. This suggests that 1 is
binding to a comparable region of R-chymotrypsin, possibly
interacting with several cationic residues (including lysines 36,
9
0, 175, 177 and arginine 145) near the active site cleft. This
interaction may either lead to a disruptive conformational change
in the protein or interfere with the approach of the substrate to
the active site. However, receptor 1 is also not modified by the
enzyme, suggesting no direct contact with the active site.
Incubation of R-chymotrypsin and 1 for 5 h followed by
denaturation of the complex and reverse phase HPLC analysis
The resin was thoroughly washed with DMF (2×), MeOH (2×),
CH
2
Cl
2
(3×), and MeOH (2×) and dried in vacuo overnight. The resin
2 2
(
elution solvent, 7-90% acetonitrile:water containing 0.1%
was treated with 1% TFA/CH
were drained into 1-2 mL of ice cold DMF. The CH
by evaporation in vacuo and the peptide was precipitated by dropping
the DMF solution into 100 mL of H O. The precipitate was collected
Cl
(5×, 2 min each), and the filtrates
TFA) showed that unreacted receptor was recovered.
In summary, we have developed a novel series of inhibitors
of R-chymotrypsin based on the specific interaction of a
synthetic receptor with the exterior of the protein. We are
currently extending this strategy to less symmetrical receptors
and to different classes of proteins.
2
Cl was removed
2
2
on a glass filter, washed with H O, and dried in vacuo (0.35 g). The
2
Fmoc group was removed subsequently by treating the peptide with
10% Et
vacuo and the dibenzofulvene was extracted from the solid residue with
100 mL of Et O. The peptide was cyclized by treatment with BOP
0.443 g, 1.0 mmol), HOBt (0.135 g, 1.0 mmol), and DIEA (0.18 mL,
2
NH/ACN (20 mL) for 2 h. The solvents were then removed in
Experimental Section
2
(
Materials and Instruments. Buffer reagents were purchased from
Bio-Rad, and R-chymotrypsin (ChT), porcine pancreatic elastase and
N-benzoyltyrosine-p-nitroanilide(BTNA) were purchased from Sigma
and used without purification. Spectra Max 250 (Molecular Devices)
was used for simultaneous 96-well microplate assays. HPLC data were
obtained from DYNAMAX with Microsorb-MB (C18-reverse phase
column) column (Rainin). All equipment and reagents for electrophore-
1
.0 mmol) in 200 mL of DMF at room temperature overnight. The
DMF was evaporated in vacuo and the crude product was taken up in
00 mL of EtOAc and washed with 1 N NaOH (50 mL × 2), 1 N HCl
50 mL × 2), and brine (50 mL) and dried over Na SO . The solvent
was then evaporated and the residue was applied to a silica gel column
with 5% MeOH/CH Cl as the eluent to produce the desired cyclic
peptide (0.111 g, 32% overall yield from resin): mp >350 °C; H NMR
300 MHz, DMSO-d ) δ 9.58 (t, J ) 5.0 Hz, 1H), 8.81 (t, J ) 6.0 Hz,
H), 8.53 (s, 1H), 8.37 (s, 1H), 8.32 (d, J ) 9.4 Hz, 1H), 8.05 (t, J )
1
(
2
4
2
2
1
1
13
sis were purchased from Bio-Rad. H and C NMR data were collected
on a Bruker AM 500 NMR.
(
6
1
4
4
General Conditions for Enzyme Assay. For kinetic studies, a 5
mM sodium phosphate buffer of pH 7.4 was used. All solutions were
kept at 25 °C for the kinetic analyses and also for the incubation periods.
R-Chymotrypsin and different concentrations of the receptors were
mixed and kept at 25 °C for a fixed incubation period. An aliquot (190
µL) of the incubated solution was taken and to it was added 10 µL of
a stock solution of BTNA. Hydrolysis was followed by monitoring
product formation at 410 nm.
Electrophoresis. 1% of melted agarose in 5 mM of sodium
phosphate was poured on the glass plate to a thickness of 1 mm. Small
wells (5 µL volume) were made in the middle of the gel with a small
comb. Individual samples (4 µL) in buffer (plus 0.1% of tracking dye
and glycerol) were loaded on to the gel and a constant voltage (100 V)
was applied for aproximately 1 h to run the electrophoresis. At the
end of this time the gel was fixed in 25% acetic acid solution for 20
min and acetone for 10 min. The gel was dried by hot air and then
immersed in a 0.5% Coomasie Blue staining solution (10% acetic acid,
.0 Hz, 1H), 7.85 (t, J ) 5.6 Hz, 1H), 4.82 (m, 1H), 4.78 (m, 1H),
.73 (m, 1H), 4.14 (dd, J ) 16.8, 4.6 Hz, 1H), 3.96 (dd, J ) 13.0, 6.1
Hz, 1H), 3.90 (dd, J ) 13.3, 5.5 Hz, 1H), 3.78 (dd, J ) 11.0, 6.0 Hz,
1
2
H), 3.73 (dd, J ) 8.4, 4.3 Hz, 1H), 2.83 (dd, J ) 15.5, 5.3 Hz, 1H),
.62 (dd, J ) 15.9, 8.4 Hz, 1H), 2.40 (dd, J ) 15.5, 9.2 Hz, 1H), 2.34
(dd, J ) 15.9, 6.6 Hz, 1H), 1.40 (s, 9H), 1.38 (s, 9H); LR FAB-MS
+
m/e calcd for C28
H
39
N
6
O
11 [M + H] 635.3, found 635.5.
t
t
To a solution of cyclo(Gly-Asp(O Bu)-Gly-Asp(O Bu)Amnb) (0.217
g, 0.32 mmol) in 15 mL of MeOH was added 10% Pd/C (100 mg,
0
at room temperature for 7 h. The catalyst was removed by filtration
through Celite. The filtrate was concentrated and the residue was applied
to a chromatography column (SiO
CH Cl ) to give the title compound (0.060 g, 31%): mp 184-185 °C
dec; H NMR (300 MHz, DMSO-d
8
5
.094 mmol) carefully. The suspension was stirred under 1 atm of H
2
2 2 2
, 5% MeOH/CH Cl , 10% MeOH/
2
2
1
6
) δ 8.78 (dd, J ) 6.4, 3.9 Hz, 1H),
.37 (dd, J ) 7.0, 5.2 Hz, 1H), 8.23 (d, J ) 9.5 Hz, 1H), 8.08 (t, J )
.9 Hz, 1H), 7.96 (d, J ) 8.9 Hz, 1H), 6.81 (s, 2H), 6.54 (s, 1H), 5.24
4
0% methanol aqueous solution). The gel background was destained
(
1
s, 2H), 4.83 (m, 2H), 4.46 (dd, J ) 16.3, 7.4 Hz, 1H), 4.01 (dd, J )
with 10% acetic acid, 40% methanol, and 50% water solution until the
protein bands could be seen.
6.5, 7.0 Hz, 1H), 3.95 (dd, J ) 17.3, 6.7 Hz, 1H), 3.86 (dd, J ) 16.2,
.5 Hz, 1H), 3.66 (dd, J ) 13.8, 5.6 Hz, 1H), 3.60 (dd, J ) 11.8, 3.6
4
General Procedure for Solid-Phase Synthesis of Cyclic Peptide
Hz, 1H), 2.82 (dd, J ) 15.3, 5.4 Hz, 1H), 2.64 (dd, J ) 15.8, 8.5 Hz,
1
1
t
t
Sequences. (a) cyclo(Gly-Asp(O Bu)-Gly-Asp(O Bu)-Amab). To a
H), 2.40 (dd, J ) 15.3, 9.0 Hz, 1H), 2.27 (dd, J ) 15.8, 6.4 Hz, 1H),
25 mL vessel was added MBHA resin (0.50 g, substitution level )
1.01 mmol/g), HMPB (0.360 g, 1.5 mmol), BOP (0.664 g, 1.5 mmol),
.37 (s, 9H), 1.34 (s, 9H); LR FABMS m/e calcd for C28
H
41
N
6
O
9
[M
+
+
H] 605.3, found 605.4.
HOBt (0.203 g, 1.5 mmol), DIEA (0.26 mL, 1.5 mmol), and 5 mL of
peptide synthesis grade DMF. The mixture was shaken at room
temperature overnight. After all the liquids were removed, the resin
was washed thoroughly (20 mL volumes) with DMF (2×), MeOH (2×),
17
(b) Butoxycalix[4]arene tetracyclo-(GDGD) (1). To a solution
17
of butoxycalix[4]arene tetraacid (86 mg, 0.10 mmol) and oxalyl
chloride (254 mg, 2.0 mmol) in dry CH Cl (10 mL) was added DMF
0.025 mL) through a silica gel filter and the mixture was stirred at
2
2
(
CH
2
Cl
2
(2×), MeOH (2×), and DMF (2×). Kaiser test showed there
room temperature for 8 h. The reaction mixture was evaporated in vacuo
to obtain the acid chloride (102 mg). A solution of cyclo-(Gly-
Asp(O Bu)-Gly-Asp(O Bu)-Amab) (266 mg, 0.44 mmol) and DIEA
was no free amine left on the resin. To the resin was added Fmoc-
Gly-OH (0.744 g, 2.5 mmol), DCC (0.516 g, 2.5 mmol), and DMAP
t
t
17
(0.306 g, 2.5 mmol) in DMF/CH
2 2
Cl (2 mL/8 mL) and the vessel was
(
16) Segel, I. H. In Enzyme Kinetics; Wiley-Interscience: New York,
(17) Lin, Q.; Park, H. S.; Hamuro, Y.; Lee, C. S.; Hamilton, A. D.
Biopolymers (Peptide Science). In press.
1
975; p 125.

Protein Surface Recognition by Synthetic Receptors
J. Am. Chem. Soc., Vol. 121, No. 1, 1999 13
(
80 mg, 0.60 mmol) in dry CH
residue. The mixture was stirred at room temperature for 14 h. The
reaction mixture was purified by preparative TLC (SiO , first 10%
MeOH/CH Cl , later 25% MeOH/CH Cl ) to obtain the desired
compound as a yellow powder (281 mg, 89%): mp >350 °C; H NMR
2
Cl
2
(10 mL) was added to the evaporated
protected product. Further treatment with 25% TFA/CH
2 2
Cl (3 mL) at
room temperature for 2 h afforded the final product as its TFA salt (18
1
2
mg, 57%): mp >290 °C dec; H NMR (500 MHz, DMSO-d ) δ 9.97
6
2
2
2
2
(s, 4H), 8.71 (s, 4H), 8.42 (s, 4H), 8.33 (d, J ) 9.2 Hz, 4H), 8.23 (d,
J ) 6.5 Hz, 4H), 7.95 (s, 4H), 7.85 (s, 8H), 7.67 (s, 4H), 7.49 (d, J )
13.8 Hz, 4H), 7.29 (s, 4H), 4.50 (m, 8H), 4.22 (m, 8H), 3.94 (m, 24H),
3.75 (m, 4H), 3.59 (m, 4H), 2.76 (s, 16H), 1.93 (m, 12H), 1.68 (m,
1
(
(
7
3
4
300 MHz, DMSO-d
6
) δ 10.10 (broad s, 4H), 8.95 (broad, 4H), 8.49
broad, 4H), 8.25 (d, J ) 8.7 Hz, 4H), 8.09 (broad, 4H), 7.99 (m, 8H),
.67 (s, 4H), 7.60 (s, 8H), 7.37 (s, 4H), 4.81 (m, 8H), 4.51 (m, 8H),
.99 (m, 20H), 3.63 (m, 8H), 3.46 (m, 4H), 2.79 (m, 4H), 2.62 (m,
H), 2.31 (m, 8H), 1.97 (m, 8H), 1.47 (m, 8H), 1.35 (m, 72H), 1.02 (t,
4H), 1.51 (m, 28H), 1.30 (m, 20H), 1.02 (t, J ) 7.3 Hz, 12H); FAB-
+
MS calcd for C144
H
201
N
32
O
28 2827.28 (M + H ), found 2827.4.
(e) Iminodiacetic Acid Diethyl Ester. A solution of iminodiacetic
acid (2.66 g, 0.02 mol) and concentrated H SO (2.0 mL) in 80 mL of
EtOH was refluxed under N for 4 h. The solvent was then evaporated
and 100 mL of H O was added. The resulting solution was neutralized
with 1.0 N NaOH, and the solution was extracted with Et O (150 mL
× 2) and the organic layer was separated and dried over MgSO . The
ether was evaporated to give the title compound as an oil (2.54 g,
67%): 1H NMR (300 MHz, CDCl
) δ 4.18 (q, J ) 7.2 Hz, 4H), 3.44
1
3
J ) 8.4 Hz, 12H); C NMR (75 MHz, DMSO-d
8
2
6
) δ 120.1, 117.8,
0.0 (2C), 75.0, 49.1, 48.8, 44.2, 42.3, 41.4, 37.5, 36.9, 31.9, 30.5,
7.6, 18.9, 14.0. To the above compound (298 mg, 0.093 mmol) was
Cl (3 mL) and the mixture was stirred
2
4
2
2
added TFA (3 mL) and dry CH
2
2
2
at room temperature for 1 h and then evaporated under reduced pressure.
The crude product was passed through anion-exchange resin (Amberlite
IRA-400(OH), water) and cation-exchange resin (Amberlite IR 120
4
3
13
(
plus), water) to remove ions. Water was lyophilized to give the title
(s, 4H), 1.83 (s, 1H), 1.26 (t, J ) 7.2 Hz, 6H); C NMR (125 MHz,
1
compound (229 mg, 90%): mp >350 °C; H NMR (300 MHz, DMSO-
CDCl
3
8 4
) δ 172.0, 61.1, 50.4, 14.4; HR EI-MS m/e calcd for C H15NO
+
d
4
8
8
6
) δ 10.02 (s, 4H), 8.81 (s, 4H), 8.48 (s, 4H), 8.31 (d, J ) 8.7 Hz,
[M] 189.1001, found 189.1008.
H), 7.97 (m, 12H), 7.64 (s, 4H), 7.55 (s, 8H), 7.30 (s, 4H), 4.76 (m,
(f) Butoxycalix[4]arene Octaacid Ethyl Ester. To a suspension of
butoxycalix[4]arene tetraacid (84.2 mg, 0.102 mmol)1 in 5 mL of
7
H), 4.52 (m, 8H), 4.0-3.4 (m, 32H), 2.8-2.6 (m, 8H), 2.5-2.3 (m,
1
3
H), 1.97 (m, 8H), 1.50 (m, 8H), 1.01 (t, 8.1 Hz, 12 H); C NMR (75
2 2 2
CH Cl was added (COCl) (0.15 mL, 1.72 mmol) and a catalytic
MHz, DMSO-d
6
) δ 172.2, 171.7, 170.9, 170.4, 169.3, 168.5, 165.1,
amount of DMF, and the mixture was stirred at room temperature
1
7
59.0, 140.2, 138.8, 134.4, 134.2, 128.7, 128.3, 121.4, 119.9, 117.7,
overnight. The solvent and excess (COCl)
residue was redissolved in 2.0 mL of CH Cl
2
acid diethyl ester (1.3 M CH Cl /THF solution, 2.0 mL, 2.6 mmol)
and 40 µL of DIEA were added to above solution, and the mixture
was stirred at room temperature overnight. After evaporation, the residue
2
were evaporated and the
/THF (4/1). Iminodiacetic
5.0, 49.1, 44.3, 42.2, 41.3, 36.1, 31.9, 30.5, 18.9, 14.0; HR FAB-MS
2
2
+
m/e calcd for C128
H N
145 24
O
44 (M + H ) 2721.9767, found 2721.985.
2
(
c) Butoxycalix[4]arene tetracyclo-(GDGY) (2). To a solution of
1
7
butoxycalix[4]arene tetraacid (22.5 mg, 0.027 mmol) in 5 mL of dry
CH Cl was added oxalyl chloride (146 mg, 1.1 mmol) and a catalytic
amount of DMF (0.2 µL), and the mixture was stirred at room
temperature overnight. The reaction mixture was evaporated in vacuo
to give the acid chloride. A solution of cyclo-(Gly-Asp(O Bu)-Gly-
2
2
was taken up into 20 mL of CH
0.1 N HCl and saturated brine and then dried over Na
was removed to give the product as an oil (0.148 g, 96%): H NMR
(500 MHz, CDCl ) δ 6.95 (s, 8H), 4.45 (d, J ) 13.1 Hz, 4H), 4.20 (m,
2
Cl
2
and the solution was washed with
2
SO . The solvent
4
1
t
3
t
Tyr(O Bu)-Amab) (78.0 mg, 0.12 mmol) and DIEA (15 mg, 0.12 mmol)
in dry CH Cl (5 mL) was added to the acid chloride and the mixture
2 2
16H), 4.15 (s, 8H), 3.95 (m, 16H), 3.17 (d, J ) 13.1 Hz, 4H), 1.89 (p,
J ) 7.7 Hz, 8H), 1.42 (m, 8H), 1.27 (t, J ) 7.0 Hz, 24H), 1.00 (t, J )
was stirred at room temperature for 2 days. The solvent and excess
reagent were evaporated and the residue was applied to a Sephadex
LH-20 gel filtration column eluted with CH
portions were collected and evaporated to give the fully protected
product. Further treatment with 25% TFA/CH Cl (5 mL) at room
temperature for 2 h afforded the final product as a light-yellow powder
7.4 Hz, 12H); 13C NMR (125 MHz, CDCl
3
) δ 171.4, 169.4, 169.0,
134.5, 128.6, 128.1, 75.3, 61.6, 60.9, 52.1, 47.9, 32.0, 31.3, 19.3, 14.2,
+
2
Cl
2
. The appropriate
14.0; HR FAB-MS m/e calcd for C80
found 1509.7437.
H
109
N
4
O
24 [M + H] 1509.7432,
2
2
(g) Butoxycalix[4]arene Octaacid (5). A solution of butoxycalix-
[4]arene octaacid ethyl ester (0.135 g, 0.089 mmol) and LiOH (0.112
1
(52.6 mg, 66%): mp >350 °C dec; H NMR (500 MHz, DMSO-d
6
) δ
.99 (s, 4H), 9.15 (s, b, 4H), 8.74 (s, 4H), 8.42 (d, J ) 8.4 Hz, 4H),
.41 (s, 4H), 8.19 (d, J ) 7.2 Hz, 4H), 7.96 (s, 4H), 7.84 (s, 8H), 7.63
g, 2.67 mmol) in 10 mL of THF/H
temperature overnight. After THF was evaporated, H
the aqueous layer was washed with Et
2
O (1/1) was stirred at room
9
8
2
O was added and
2
O (10 mL × 2). The aqueous
(
s, 4H), 7.55 (s, 4H), 7.31 (s, 4H), 6.99 (d, J ) 8.4 Hz, 8H), 6.64 (d,
J ) 8.3 Hz, 8H), 4.75 (m, 4H), 4.58 (m, 8H), 4.43 (m, 4H), 4.01 (s, b,
H), 3.94 (d, J ) 14.7 Hz, 4H), 3.74 (m, 12H), 3.57 (m, 8H), 2.90
dd, J ) 13.8, 5.3 Hz, 4H), 2.80 (dd, J ) 16.4, 5.9 Hz, 4H), 2.63 (dd,
solution was acidified to pH 2.0 with 1 N HCl. The precipitate was
filtered off and dried in vacuo to give a light yellow solid (90.7 mg,
1
8
(
79%): mp 186-188 °C; H NMR (500 MHz, DMSO-d
6
) δ 9.21 (s, b,
8H), 6.84 (s, b, 8H), 4.35 (d, J ) 13.0 Hz, 4H), 3.94 (s, b, 24H), 3.28
(d, J ) 13.1 Hz, 4H), 1.88 (m, 8H), 1.44 (s, b, 8H), 0.99 (t, J ) 7.3
J ) 13.6, 9.0 Hz, 4H), 2.48 (m, 4H), 1.96 (m, 8H), 1.49 (m, 8H), 1.03
(
t, J ) 7.3 Hz, 12H); ES-MS calcd for C148
H
161
N
24
O40 2915.97 (M +
Hz, 12H); 13C NMR (125 MHz, DMSO-d
6
) δ 170.3, 169.7, 157.7,
+
H ), found 2914.74 ( 0.38.
134.5, 127.9, 127.2, 74.7, 51.3, 48.2, 31.6, 30.3, 18.8, 13.8; LR FAB-
+
(
d) Butoxycalix[4]arene tetracyclo-(GKGK) (3). To a solution of
butoxycalix[4]arene tetraacid (9.2 mg, 0.011 mmol) in 2 mL of dry
CH Cl was added oxalyl chloride (29 mg, 0.23 mmol) and a catalytic
amount of DMF (0.1 µL), and the mixture was stirred at room
temperature overnight. The reaction mixture was evaporated in vacuo
to give the acid chloride. A solution of cyclo-(Gly-Lys(BOC)-Gly-
Lys(BOC)-Amab) (32.0 mg, 0.044 mmol) and DIEA (6 mg, 0.048
MS m/e calcd for C64H N
77 4
O
24 [M] 1285.3, found 1285.5.
Acknowledgment. We thank the National Institutes of
Health for financial support of this work and Dr. Yoshitomo
Hamuro for preliminary experiments on the preparation of
receptor 1.
2
2
2 2
mmol) in dry CH Cl (2 mL) was added to the acid chloride and the
mixture was stirred at room temperature for 2 days. The solvent and
excess reagent were evaporated and the residue was applied to a
Supporting Information Available: Experimental details
and results (6 pages, print/PDF). See any current masthead page
for ordering information and Web access instructions.
Sephadex LH-20 gel filtration column eluted with CH
2 2
Cl . The
appropriate portions were collected and evaporated to give the fully
JA981504O