Design of mechanism-based carboxypeptidase A inactivators on the basis of the X-ray crystal structure and catalytic reaction pathway

Article abstract of DOI:10.1016/S0968-0896(98)00082-0

The X-ray crystal structure of the complex of carboxypeptidase A (CPA) and Gly-Tyr, has been documented. The crystal structure reveals that both the amide carbonyl oxygen and the terminal amino nitrogen of Gly-Tyr coordinate to the active site zinc ion of CPA in a bidentate fashion, whereby the zinc-bound water molecule is displaced by the amino group. As to the catalytic mechanism of CPA, it is generally believed that while in the cases of ester substrates the carboxylate of Glu-270 functions as the nucleophile which attacks the scissile carbonyl carbon (anhydride pathway), in the case of peptide substrates the zinc-bound water molecule attacks the scissile peptide bond (general base pathway). In light of the X-ray crystal structure and the proposed catalytic mechanism for the enzyme, it is envisioned that the ester bond of O-(hydroxyacetyl)-l-β-phenyllactic acid (l-1) would be hydrolyzed by the attack of the carboxylate of Glu-270 to generate an anhydride intermediate. The latter intermediate would then undergo an intramolecular rearrangement initiated by the attack of the hydroxyl to result in to form an ester bond with the Glu-270 carboxylate. This ester formation impairs the catalytic activity of CPA. We have demonstrated using kinetic analysis that l-1 is indeed an inactivator for the enzyme having the k(inact)/K(I) value of 0.057M^-1s^-1. We have also demonstrated that N-(hydroxyacetyl)-l-phenylalanine (l-2) inactivates the enzyme with the k(inact)/K(I) value of 0.071M^-1s^-1, suggesting that the carboxylate becomes to attack the peptide carbonyl carbon to generate the same anhydride intermediate as that formed in the inactivation of CPA by l-1. The formation of the anhydride intermediate rather than a tetrahedral transition state that is expected for peptide type substrates was envisioned to occur on the ground that the zinc-bound water molecule is displaced by the hydroxyl of l-2 upon binding to the enzyme. Copyright (C) 1998 Elsevier Science Ltd.

Full text of DOI:10.1016/S0968-0896(98)00082-0

BIOORGANIC &
MEDICINAL
CHEMISTRY
Bioorganic & Medicinal Chemistry 6 (1998) 1613±1622
Design of Mechanism-Based Carboxypeptidase A Inactivators
on the Basis of the X-ray Crystal Structure and Catalytic
Reaction Pathway1
Kyung Joo Lee and Dong H. Kim*
Department of Chemistry and Center for Biofunctional Molecules, Pohang University of Science and Technology,
San 31 Hyojadong, Pohang 790-784, Korea
Received 2 March 1998; accepted 10 April 1998
AbstractÐThe X-ray crystal structure of the complex of carboxypeptidase A (CPA) and Gly-Tyr, has been docu-
mented. The crystal structure reveals that both the amide carbonyl oxygen and the terminal amino nitrogen of Gly-Tyr
coordinate to the active site zinc ion of CPA in a bidentate fashion, whereby the zinc-bound water molecule is displaced
by the amino group. As to the catalytic mechanism of CPA, it is generally believed that while in the cases of ester
substrates the carboxylate of Glu-270 functions as the nucleophile which attacks the scissile carbonyl carbon (anhy-
dride pathway), in the case of peptide substrates the zinc-bound water molecule attacks the scissile peptide bond
(general base pathway). In light of the X-ray crystal structure and the proposed catalytic mechanism for the enzyme, it
is envisioned that the ester bond of O-(hydroxyacetyl)-l-b-phenyllactic acid (l-1) would be hydrolyzed by the attack of
the carboxylate of Glu-270 to generate an anhydride intermediate. The latter intermediate would then undergo an
intramolecular rearrangement initiated by the attack of the hydroxyl to result in to form an ester bond with the Glu-
270 carboxylate. This ester formation impairs the catalytic activity of CPA. We have demonstrated using kinetic ana-
lysis that l-1 is indeed an inactivator for the enzyme having the kinact/KI value of 0.057 M 1 s 1. We have also
demonstrated that N-(hydroxyacetyl)-l-phenylalanine (l-2) inactivates the enzyme with the kinact/KI value of
0.071 M 1 s 1, suggesting that the carboxylate becomes to attack the peptide carbonyl carbon to generate the same
anhydride intermediate as that formed in the inactivation of CPA by l-1. The formation of the anhydride intermediate
rather than a tetrahedral transition state that is expected for peptide type substrates was envisioned to occur on the
ground that the zinc-bound water molecule is displaced by the hydroxyl of l-2 upon binding to the enzyme. # 1998
Elsevier Science Ltd. All rights reserved.
Introduction
development of design methodologies of inhibitors for
proteases have received much attention.3 Of these pro-
teases, carboxypeptidase A (CPA)4 bears a special
importance. As a prototypical zinc containing proteo-
lytic enzyme, it has served as a target enzyme in the
development of new inhibitor design strategies5 which
can be useful for zinc proteases of medicinal interest.6
In this report we wish to describe a new design strategy
which we have developed for CPA on the bases of
the X-ray crystallographic structure of the enzyme
complexed with a slowly hydrolyzing substrate and
Proteases are an important class of enzymes, playing
key roles in a wide variety of biological processes such
as the digestion of ingested food, clotting of blood,
control of blood pressure and the production of hor-
mones.2 Their aberrant function, usually in the direction
of excessive activity, leads to the development of physi-
cal and mental ailment. Accordingly, inhibitors of
proteases are important as therapeutic agents, and
a catalytic mechanism proposed for the enzymic
reaction.
*Corresponding author. E-mail: dhkim@vision.postech.ac.kr
0968-0896/98/$Ðsee front matter # 1998 Elsevier Science Ltd. All rights reserved
PII: S0968-0896(98)00082-0

1614
K. J. Lee, D. H. Kim/Bioorg. Med. Chem. 6 (1998) 1613±1622
Results and Discussion
Design rationale
Carboxypeptidase A is a zinc containing proteolytic
enzyme which selectively cleaves C-terminal amino acid
residue having a hydrophobic side chain.4 The active
site of CPA consists of a hydrophobic pocket (primary
substrate recognition site) which is primarily responsible
for the substrate speci®city, a guanidinium moiety of
Arg-145 which forms hydrogen bonds to the carboxy-
late of substrate, and Glu-270 whose carboxylate plays a
critical role functioning either as a nucleophile to attack
the scissile carboxamide carbonyl carbon of the sub-
strate or as a base to activate the zinc bound water
molecule, which in turn attacks the scissile peptide
bond. The active site zinc ion is coordinated to the
backbone amino acid residues of His-69, Glu-72, His-
196, and a molecule of water.7 Upon binding a substrate
to the enzyme, the scissile amide carbonyl oxygen is
coordinated to the zinc ion, whereby the carbonyl car-
bon becomes an electrophilic center.
the active site of the enzyme in light of their structural
similarity to Gly-Tyr: Expectedly, its terminal carboxyl-
ate forms hydrogen bonds with the guanidinium moiety
of backbone Arg-145 residue and the benzyl aromatic
ring ®ts in the primary substrate recognition pocket of
the enzyme. There then would follow the coordination
of both the carbonyl oxygen and the terminal hydroxyl
to the active site zinc ion of the enzyme. In this complex,
the catalytic carboxylate of Glu-270 would attack the
scissile ester carbonyl carbon of the ligand to generate
an anhydride intermediate with expulsion of the b-phenyl-
lactic acid. The anhydride intermediate thus formed
may then undergo an intramolecular rearrangement
initiated by the attack of the hydroxyl group at the
anhydride carbonyl carbon as depicted in Figure 1. The
rearrangement reaction would result in to modify cova-
lently the catalytically essential carboxylate of Glu-270
in the form of an ester, causing to an impairment of the
enzymic activity of CPA (Fig. 1).12
Gly-Tyr is a slowly hydrolyzing substrate for CPA with
kcat value of 0.9 min 1.8 The X-ray crystal structure of
the CPA complexed with Gly-Tyr reveals that both the
amide carbonyl oxygen and terminal amino nitrogen of
Gly-Tyr bind to the active site zinc ion of CPA in a
bidentate fashion.9 The X-ray crystal structure also
reveals that the terminal carboxylate is hydrogen bon-
ded to the guanidinium moiety of Arg-145 and the ben-
zyl group is anchored in the primary substrate
recognition pocket at the active site of the enzyme. The
zinc bound water molecule in the native CPA is dis-
placed by the terminal amino group.
In general, proteases catalyze the hydrolysis of esters as
well, often with a rate acceleration higher than that for
peptides. The detailed catalytic mechanism of CPA
remains to be established but it is generally believed that
the hydrolysis of ester substrates by CPA takes place
with an involvement of an anhydride intermediate which
is formed by the nucleophilic attack of the carboxylate
of Glu-270 on the ester carbonyl carbon (anhydride
pathway).10 On the other hand, in the hydrolysis of
peptide substrate the zinc bound water molecule which
is activated by the carboxylate of Glu-270 attacks on the
scissile carboxamide carbonyl carbon with the genera-
tion of a transient tetrahedral intermediate that col-
lapses to products (general base pathway).11
N-(Hydroxyacetyl)-l-phenylalanine (l-2) was thought to
be a ligand of interest. As a peptide, the CPA-catalyzed
hydrolysis of it may proceed by the general base
mechanism, but since in the CPA±l-2 complex the zinc-
bound water molecule is expected to be replaced by the
hydroxyl group of the ligand, there is present no longer
nucleophilic water molecule in this complex, and as a
consequence the carboxylate of Glu-270 may attack the
scissile amide carbonyl carbon. If this would take place,
then one can expect that the generation of the same anhy-
dride intermediate as that postulated for l-1, leading to
inactivation of CPA (Fig. 1). We have also evaluated the
analogues of these two potential inactivators, in which
the terminal hydroxyl is replaced with a benzyloxy
group as an e€ort to verify the proposed design rationale.
On the basis of the X-ray structural data of the
.
CPA Gly-Tyr complex and the catalytic mechanism
proposed for the hydrolysis of ester substrates,10 we
have envisioned that O-(hydroxyacetyl)-l-b-phenyllactic
acid (l-1) would be a potential mechanism-based inac-
tivator for CPA. The compound is expected to bind to

K. J. Lee, D. H. Kim/Bioorg. Med. Chem. 6 (1998) 1613±1622
1615
Synthesis
Coupling of glycolic acid with phenylalanine methyl
ester using DCC followed by alkaline hydrolysis a€or-
ded 2 (Scheme 2).
Compound 1 was synthesized by allowing benzyloxy-
acetyl chloride to react with b-phenyllactic acid benzyl
ester in the presence of pyridine in THF followed by
hydrogenolysis to remove the benzyl moieties (Scheme 1).
Benzyloxyacetyl chloride was allowed to react with
phenylalanine methyl ester in the presence of pyridine to
give 10 which was then treated with alkaline solution
to a€ord 5. Similarly, coupling of benzyloxyacetyl
chloride with l-N-methylphenylalanine13 gave the
Compound
4 was prepared by the treatment of
benzyloxyacetyl chloride with l-b-phenyllactic acid
(Scheme 1).
Figure 1. Schematic illustration of the rationale used for designing l-1 and l-2 as mechanism-based inactivators for CPA.
Scheme 1. Reagents and conditions: (a) l-b-Phenyllactic acid, pyridine, cat. DMAP, THF, 80%; (b) l-b-Phenyllactic acid benzyl
ester, pyridine, THF, 81%; (c) H2, Pd/C (1 atm), MeOH, quant.

1616
K. J. Lee, D. H. Kim/Bioorg. Med. Chem. 6 (1998) 1613±1622
N-methylphenylalanine 8 which was debenzylated and
hydrolyzed to give 3 as a sodium salt (Scheme 3).
p-chlorocinnamoyl)-l-b-phenyllactic acid (Cl-CPL)14 as
the substrate. Semilogarithmic plot of the percent activ-
ity remaining versus time at di€erent concentrations of
inhibitor gave straight lines in the cases of l-1 and l-2
(Fig. 2), suggesting that the inhibition of CPA by these
compounds occurs in an irreversible fashion. Their
enantiomers failed to show the time-dependent loss of
the enzymic activity. The rate of each inhibition was
reduced in the presence of 2-benzylsuccinic acid,15 a well
known potent competitive inhibitor for CPA, suggesting
that these inhibitors bind to the active site of CPA as 2-
benzlylsuccinic acid does (Fig. 3). Kinetic parameters
for the irreversible inhibition, KI and kinact were esti-
mated according to the method of Kitz and Wilson16 as
reported17 previously (Fig. 4) and are listed in Table 1.
The ®rst order rate constant can be expressed as
kobs=k0[inhibitor]n, where n is the order of inhibitor in
its reaction with enzyme.18 Thus, the slope in the plot of
log kobs versus log [inhibitor] corresponds to n. There
were obtained slopes of 0.81 and 0.85 for l-1 and l-2,
respectively, to suggest that the inhibitions occur in a 1:1
Kinetics
Enzymic assay and inhibition kinetics were carried out
at 25 ꢀC in Tris bu€er (0.05 M) of pH 7.5 using O-(trans-
Scheme 2. Reagents and conditions: (a) l-Phe-OCH3, HCl,
DCC, HOBt, NEM, THF, 45%; (b) 1N LiOH, MeOH, 90%.
Scheme 3. Reagents and conditions: (a) N-Methyl-l-Phe-OCH3, Et3N, THF, 84%; (b) H2, Pd/C, MeOH, 90%; (c) 1N NaOH,
MeOH, 90%; (d) l-Phe-OCH3-HCl, pyridine, THF, 75%; (e) 1N LiOH, MeOH, 95%.
Figure 2. Loss of enzyme activity of CPA as a function of incubation time.

K. J. Lee, D. H. Kim/Bioorg. Med. Chem. 6 (1998) 1613±1622
1617
stoichiometry (Fig. 5). The involvement of the terminal
hydroxyl group of the inhibitors in the irreversible inhi-
bition of the enzyme was supported by the observation
that O-(benzyloxyacetyl)-l-b-phenyllactic acid (4) and
O-(benzyloxyacetyl)-l-Phe (5) failed to inactivate the
enzyme. The inactivated enzyme failed to regain its
enzymic activity upon dialysis for 2 days, con®rming the
irreversible nature of the inhibitions. The partition ratio
of the inhibition, which is a measure of the eciency of
an inactivator was determined by the titration method19
(Fig. 6) and included in Table 1. All the substrate ana-
logues including 1 and 2 synthesized for the present
study were evaluated as competitive inhibitors for CPA,
and their reversible inhibitory constants (Ki) which re¯ect
the anity of the inhibitor for the enzyme were calculated
from the respective Dixon plot20 and listed in Table 1.
Discussion
As described above, substrate analogue l-1 was
designed as a mechanism-based inactivator for CPA on
the basis of the X-ray crystal structure of the CPA
complexed with Gly-Tyr, a slowly hydrolyzing sub-
strate9 and a proposed mechanism10 of the catalytic
action. The kinetic analyses (Table 1) together with
experimental observations presented above suggest
Figure 3. The rate of inactiviation by l-1 was reduced by the
presence of benzylsuccinic acid.
Figure 5. Determination of the reaction order in the inactiva-
tion of CPA by l-1 and l-2.
Figure 4. Double reciprocal plot of kobs versus [inactivator]o.
Values of kinact and KI were calculated from y intercept and the
slope, respectively.
Table 1. Kinetic parameters in the inhibition of CPA
Inhibitor
Ki
kinact
KI
(mM) (M 1 s
kinact/KI Partition
1
1
(mM)
(min
)
)
ratio
l-1
d-1
l-2
d-2
l-3
l-4
l-5
400
2,000a
1,320
1,800
7,000a
0.012
Ð
3.51
Ð
0.057
Ð
1246
Ð
0.044 10.3
0.071
Ð
Ð
1234
Ð
Ð
Ð
Ð
Ð
Ð
Ð
Ð
Ð
Ð
Ð
4.7
140
Ð
Ð
Ð
Figure 6. Determination of partition ratio by the titration
method.
aApproximate values due to poor binding ability.

1618
K. J. Lee, D. H. Kim/Bioorg. Med. Chem. 6 (1998) 1613±1622
strongly that l-1 is indeed a mechanism-based inacti-
vator for CPA, suggesting that the design rationale
employed is valid (Fig. 1). There may be a question of
stability of the newly generated ester linkage which
modi®es the catalytic carboxylate of Glu-270 towards
hydrolysis by bulk water. In this regard, it should be
noted that our previous works17,21 as well as a recent
report of Massova et al.22 which demonstrate that the
covalently modi®ed CPA at the carboxylate of Glu-270
in the form of an ester is highly stable and resists to be
hydrolyzed at pH even at 10.
CPA activity up to the concentration of 24 mM when
incubated with CPA. The result supports the propo-
sition that the hydrolytic cleavage of the amide or ester
is the crucial step in the inactivation chemistry.
It is worthy noting that only 1 and 2 having the l-con-
®guration are able to inactivate the enzyme and their
mirror image forms in the d-series just competitive
inhibitors. It appears that in the case of 2, the stereo-
chemistry is re¯ected in the ®rst step of the inactivation,
i.e. the cleavage of the amide bond because the Ki values
of both enantiomers are comparable. On the other
hand, in the case of 1, one can not draw any conclusion
because d-1 is shown to bind the enzyme very poorly.
We notice in Table 1 that unexpectedly the kinact
(0.044 min 1) for l-2 is signi®cantly larger than that
(0.012 min 1) for l-1, but no explanation can be o€ered
presently for the observation.
We found that peptide l-2 is also an e€ective inactivator
for CPA, suggesting that the carboxylate of Glu-270
functions as the nucleophile which attacks the peptide
bond in the absence of the zinc-bound water molecule as
discussed in the design rationale. This explanation is
consistent with the conclusion derived from molecular
dynamic calculations performed by Banci et al.23 on a
complex of CPA and Val-Leu-Phe-Phe. They reported
that when the zinc-bound water molecule is displaced, the
Glu-270 carboxylate becomes to attack the peptide car-
bonyl carbon to generate an anhydride intermediate.23
Conclusion
l-1 and l-2 are a new type of mechanism-based inacti-
vators for CPA, which have been designed rationally on
the basis of the X-ray crystal structure of the enzyme
which is complexed with a slowly hydrolyzing substrate,
Gly-Tyr and a catalytic mechanism proposed for the
enzyme. In designing them we have exploited the subtle
di€erence in physical and chemical properties between
an amino and a hydroxyl group. When Gly-Tyr is bound
to the enzyme, the nucleophilic carboxylate of Glu-270
becomes involved in an ionic interaction with the terminal
amino group of Gly-Tyr, and as a result the carboxylate
is no longer to function as a nucleophile. On the other
hand, in the cases of 1 and 2, the carboxylate is free to
attack the electrophilic center of the ester or amide car-
bonyl carbon of the inactivators, leading to irreversible
inhibition of the enzyme with a covalent modi®cation.
The design strategy demonstrated in this work may be
of considerable interest to those who are engaged in the
rational design of inhibitors for zinc-containing proteases.
The high partition ratio exhibited by these inactivators
suggests that the turn over process (path b) competes
favorably with the inactivation, i.e. there appears to
occur extensive hydrolytic cleavage of the anhydride
intermediate by water with regeneration of the enzyme
(Fig. 1). While it is generally desirable for mechanism-
based inactivators to have a small partition ratio, there
are present some important exceptions: the partition
ratio24 for sulbactam, a mechanism-based inactivator
for b-lactamase is 1000±3000, yet it is currently being
used clinically in conjunction with penicillins in treat-
ment of bacterial infections. The order of inactivation
reaction calculated from the plot of log kobs versus log
[I]o is 0.81 for l-1 and 0.85 for l-2, indicating that CPA
interacts with both inhibitors in a 1:1 stoichiometry in
the inactivation. To con®rm that the hydroxyl group in
those inactivators is required for them to exhibit the
inactivation property, compounds l-4 and l-5 in which
the hydroxyl in l-1 and l-2 are replaced with a benzyl-
oxy group were evaluated. Although their Ki values are
almost 100 times lower than those for l-1 and l-2, pos-
sibly due to anchoring of the second benzyl moiety in
the hydrophobic S2 pocket of CPA , they did not exhibit
a time-dependent loss of enzymic activity up to the con-
centration of 15 mM. These observations are consistent
with the proposition that the hydroxyl group in these
inactivators is directly involved in the inactivation of
CPA.25 It has been known that CPA does not hydrolyze
N-methylated peptides.26 To gain an additional suppor-
tive evidence for the design rationale discussed above,
compound 3, a N-methylated analogue of 2 was pre-
pared. Expectedly, 3 showed no time-dependent loss of
Experimental
Melting points were taken on a Thomas-Hoover capil-
1
lary melting point apparatus and were uncorrected. H
NMR and 13C NMR spectra were recorded on a Bruker
AM 300 (300 MHz) instrument using tetramethylsilane
as the internal standard. IR spectra were recorded on a
BOMEM FT IR M100-C15 spectrometer. Mass spectra
were obtained with KRATOS MS 25 RFA spectro-
meter. High resolution mass spectra were recorded on a
JEOL JMX-HX110/110A by the Korea Basic Science
Center, Taejon, Korea. Flash chromatography was per-
formed on silica gel 60 (230±400 mesh) and thin layer

K. J. Lee, D. H. Kim/Bioorg. Med. Chem. 6 (1998) 1613±1622
1619
chromatography (TLC) was carried out on silica coated
glass sheets (Merck silica gel 60 F-254). Elemental ana-
lyses were performed at the Basic Science Center,
Kyungbook National University, Taegu, Korea.
duct as a yellow oil (2.79 g, 45%). IR (neat) 3200±3400,
1
1738, 1650, 1520 cm
;
1H NMR (CDCl3) d 3.10±3.21
(m, 2H, benzylic), 3.73 (s, 3H), 4.08 (s, 2H, CH2O),
4.89±4.95 (m, 1H, a-H), 6.81 (d, J=9 Hz, amide NH),
7.10±7.32 (m, 5H).
O-(Benzyloxyacetyl)-L-ꢀ-phenyllactic acid benzyl ester
(6). To a solution of l-b-phenyllactic acid benzyl ester
(0.5 g, 1.95 mmol) and pyridine (0.46 g, 6 mmol) in THF
(10 mL) was added benzyloxyacetyl chloride (0.36 g,
2 mmol) dropwise at 0 ꢀC and the mixture was stirred at
room temperature for 8 h. The reaction mixture was
diluted with ethyl acetate (30 mL), washed with 3 N
HCl, saturated NaHCO3 solution, and brine, then dried
(MgSO4) and evaporated in vacuo. The residue was
puri®ed by column chromatography to give the product
N-(Hydroxyacetyl)-L-phenylalanine (L-2). A mixture of 7
(0.25 g, 1 mmol) and 1 N LiOH (2 mL) in methanol
(10 mL) was heated at 40 ꢀC for 1 h, then acidi®ed with
3 N HCl to pH of 1. It was extracted with ethyl acetate
(15 mLÂ3), washed with brine (10 mLÂ3), dried
(MgSO4), then concentrated in vacuo. The crude pro-
duct was recrystallized from ethyl acetate (10% n-hex-
ane) to give the product as a white solid (0.1 g, 45%).
mp 116±118 ꢀC; [a]d= +31.2ꢀ (c 1.0, DMSO); 1H
NMR (DMSO-d6) d 2.96±3.11 (m, 2H, benzylic), 3.76 (s,
2H, CH2O), 4.49±4.56 (m, 1H, a-H), 7.17±7.29 (m, 5H),
7.61 (d, J=8.1 Hz, CONH); 13C NMR (DMSO-d6) d
37.5, 53.0, 112, 127.9, 129.1, 130.0, 137, 172, 173; Anal.
calcd for C11H13NO4: C, 59.20; H, 5.87; N, 6.28. found:
C, 59.63; H, 5.93; N, 6.74.
as a colorless oil (0.64 g, 81%). IR (neat) 1755,
1
1725 cm
;
1H NMR (CDCl3) d 3.10±3.27 (m, 2H,
benzylic), 4.12 (d, J=7.2 Hz, 2H), 4.54 (s, 2H), 5.17 (s,
2H), 5.40±5.43 (m, 1H, a-H), 7.18±7.36 (m, 15H); 13C
NMR (CDCl3) d 38.3, 52.9, 67.7, 69.6, 73.8, 127.6±
135.9, 170.0, 172.0.
O-(Hydroxyacetyl)-L-ꢀ-phenyllactic acid (L-1). Com-
pound 6 (0.6 g) was dissolved in MeOH (5 mL) and was
hydrogenated (1 atm) in the presence of 10% Pd-C
(100 mg) at room temperature for 6 h. The mixture was
®ltered through a celite pad and washed with methanol.
The ®ltrate was concentrated in vacuo to give l-1 as a
colorless oil in a quantitative yield. [a]d= 23.0ꢀ (c 1,
N-(Hydroxyacetyl)-D-phenylalanine (D-2). Compound
d-2 was prepared following the same procedure as for l-
2 starting with d-phenylalanine. [a]d= 34.0ꢀ (c 1,
DMSO).
N-Benzyloxyacetyl-N-methyl-L-phenylalanine methyl ester
(8). To a solution of N-methyl-l-phenylalanine methyl
ester (0.5 g, 2.6 mmol) and triethylamine (0.44 mL,
3.12 mmol) in THF (15 mL) was added benzyloxyacetyl
chloride (0.41 mL, 2.6 mmol) dropwise at 0 ꢀC and the
mixture was stirred at room temperature for 5 h. The
reaction mixture was diluted with ethyl acetate (30 mL),
washed with 3 N HCl, saturated NaHCO3 solution, and
brine, then dried (MgSO4) and evaporated in vacuo.
The residue was puri®ed by column chromatography
(ethyl acetate/n-hexane=1/2) to give the product as a
colorless oil (0.6 g, 84%). This compound is a mixture of
1
CHCl3); IR (neat) 3000-3500 (br), 1750, 1720 cm 1; H
NMR (CDCl3) d 3.10±3.32 (m, 2H), 4.16 (d, J=18 Hz,
2H), 5.38 (m, 1H, a-H), 7.21±7.34 (m, 5H); 13C NMR
(CDCl3) d 37.4, 60.8, 73.5, 127.8, 129.1, 129.6, 135.6,
167.3, 173.3; EI HRMS calcd for C11H12O5: 224.0685,
found: 224.0685.
O-(Hydroxyacetyl)-D-ꢀ-phenyllactic acid (D-1). Com-
pound d-1 was prepared following the same procedure
as for l-1 starting with d-phenyllactic acid. [a]d=
+20.5ꢀ (c 1, CHCl3)
1
1
cis and trans amide (3:7 from the H NMR). H NMR
(CDCl3) d 2.86 and 2.93 (s, 3H), 3.08 (m, 1H, benzylic),
3.39 (m, 1H, benzylic), 3.69±3.77 (s, 3H), 4.10
(d, J=3.0 Hz, 2H), 4.44 (m, 2H), 4.90 (dd, J=11.2,
5.3 Hz, a-H, 0.3H), 5.38 (dd, J=11.2, 5.3 Hz, a-H,
0.7H).
N-(Hydroxyacetyl)-L-phenylalanine methyl ester (7). To
a stirred suspension of glycolic acid (2 g, 26.2 mmol),
dicyclohexylcarbodiimide (5.4 g, 26.2 mmol), and
hydroxybenzotriazole-hydrate (3.56 g, 26.2 mmol) in
THF (30 mL) were added l-phenylalanine methylester
hydrochloride (5.64 g, 26.2 mmol) and N-ethylmorpho-
line (3.4 mL, 26.2 mmol). The mixture was stirred at
room temperature for 28 h, diluted with ethyl acetate
(40 mL) and the precipitated dicyclohexylurea was
removed by ®ltration. The ®ltrate was washed with 10%
citric acid (20 mLÂ2), 10% sodium bicarbonate
(20 mLÂ2) solution and ®nally with brine (20 mLÂ3).
The organic extract was dried (MgSO4) and evaporated
in vacuo. The residue was puri®ed by column chroma-
tography (ethyl acetate/n-hexane=1/1) to give the pro-
N-Hydroxyacetyl-N-methyl-L-phenylalanine sodium salt
(3). A solution of 8 (0.5 g, 1.46 mmol) in methanol
(10 mL) was hydrogenated (1 atm) in the presence of
10% Pd-C (100 mg) at room temperature for 6 h. The
mixture was ®ltered through a Celite pad and washed
with methanol. The ®ltrate was concentrated in vacuo to
give N-hydroxyacetyl-N-methyl-l-phenylalanine methyl
ester (9) in a quantitative yield. This product (9) was
dissolved in methanol (10 mL) and 1 N NaOH
(1.4 mmol) was added and stirred at room temperature

1620
K. J. Lee, D. H. Kim/Bioorg. Med. Chem. 6 (1998) 1613±1622
for 1 h. The mixture was concentrated in vacuo to give
the product as a hygroscopic solid in a quantitative
yield. This compound is also a mixture of cis and trans
amide (4:6 from 1H NMR). This product was isolated as
General remarks for kinetic experiments
All solutions were prepared by dissolving in doubly dis-
tilled and deionized water. Stock assay solutions were
®ltered before use. Carboxypeptidase A was purchased
from Sigma Chemical Co. (Allan form, twice crystal-
lized from bovine pancreas, aqueous suspension in
toluene) and used without further puri®cation. CPA
stock solutions were prepared by dissolving the enzyme
in 0.05 M Tris/0.5 M NaCl, pH 7.5 bu€er solution and
the concentrations were determined from the absor-
bance at 278 nm (e=6.42Â104 M 1 cm 1). O-(trans-p-
chlorocinnamoyl)-l-b-phenyllactate (Cl-CPL) was syn-
thesized according to the method reported by Suh et al.14
and the decrease in the absorbance at 320 nm was followed
at 25 ꢀC. A Perkin-Elmer HP 8452 or HP8453 UV/VIS
spectrometer was used in enzyme inhibition studies.
1
a sodium salt. H NMR (D2O) d 2.63 and 2.76 (s, 3H),
2.83±2.90 (m, 1H, benzylic), 3.08±3.25 (m, 1H, benzylic),
3.89 (t, J=16 Hz, 1H), 4.09 (m, 1H), 4.65 (dd, J=12.0,
4.5 Hz, a-H, 0.6 H), 4.65 (dd, J=12.0, 4.5 Hz, a-H, 0.4
H); 13C NMR (D2O) d 30.5 and 30.8, 35.1 and 35.6, 59.5
and 59.9, 61.6 and 63.8, 126.9, 127.3, 128.9, 129.2, 129.4,
138.7 and 138.9, 173.5 and 174.0, 176.5 and 177.7.
O-(Benzyloxyacetyl)-L-ꢀ-phenyllactic acid (4). To
a
solution of l-b-phenyllactic acid (0.5 g, 3 mmol), N,N-
dimethylaminopyridine (70 mg, 0.6 mmol) and pyridine
(0.7 g, 9 mmol) in THF (10 mL) was added benzylox-
yacetyl chloride (0.47 mL, 3 mmol) dropwise at 0 ꢀC and
the mixture was stirred at room temperature for 12 h.
The reaction mixture was diluted with ethyl acetate
(30 mL), washed with 3 N HCl, saturated NaHCO3
solution, and brine, then dried (MgSO4) and evaporated
in vacuo. The residue was puri®ed by column chroma-
tography to give the product as a colorless oil (0.75 g,
Determination of Ki
Initial velocities were calculated from the linear initial
slopes of the change in absorbance where the amount of
substrate consumed was less than 10%. The Ki values
were then estimated from the semireciprocal plot of the
initial velocity versus the concentration of the inhibitors
according to the method of Dixon.20 Two concentra-
tions of the substrate (50 and 100 mM) of Cl-CPL were
used. Typically, enzyme stock solution was added to
various concentrations of inhibitors in 0.05 M Tris/
0.5 M NaCl, pH 7.5 bu€er (1 mL cuvette) , and the
initial rates were measured immediately.
80%). [a]d= 18.2ꢀ (c 1.2, CHCl3); IR (neat) 1755,
1
1722 cm
;
1H NMR (CDCl3) d 3.13±3.34 (m, 2H,
benzylic), 4.17 (d, J=6.3 Hz, 2H), 4.55 (s, 2H), 5.41 (dd,
J=8.9, 4.0 Hz, 1H, a-H), 7.24±7.40 (m, 10H), 8.8 (br,
1H, CO2H); 13C NMR (CDCl3) d 37.5, 67.1, 73.1, 73.7,
127.7±137.2, 170.4, 174.9; EI MS 314 (M+), 296, 208,
91.
N-(Benzyloxyacetyl)-L-phenylalanine (5). To a solution
of l-phenylalanine methyl ester (0.5 g, 2.3 mmol) and
pyridine (0.55 g, 6.9 mmol) in THF (15 mL) was added
benzyloxyacetyl chloride (0.44 g, 2.3 mmol) dropwise at
0 ꢀC and the mixture was stirred at room temperature
for 6 h. The reaction mixture was diluted with ethyl
acetate (30 mL), washed with 3 N HCl, saturated
NaHCO3 solution, and brine, then dried (MgSO4) and
evaporated in vacuo to give N-(benzyloxyacetyl)-l-phe-
nylalanine methyl ester (10) as a colorless oil. This pro-
duct (10) was dissolved in MeOH (20 mL) and 1 N
NaOH (5 mL) was added and the resulting mixture was
stirred at room temperature for 2.5 h. The mixture was
acidi®ed with 3 N HCl, extracted with ethyl acetate
(10 mLÂ2), washed with brine, dried (MgSO4), then
concentrated in vacuo. The crude product was recrys-
tallized from ethyl acetate (30% n-hexane) to give the
Determination of KI and kinact
Into the cuvette (1 mL) containing 595±635 mL of 0.05 M
Tris/0.5 M NaCl, pH 7.5 bu€er solution, 300 mL of
1 mM solution of Cl-CPL in the same bu€er (10%
dimethyl sulfoxide, ®nal concentration is 300 mM), 15±
55 mL of 136 mM stock solution of inactivator in dime-
thyl sulfoxide (®nal concentrations are 2.0±7.5 mM for
l-1 and 5.4±10.8 mM for l-2) and 5±45 mL of dimethyl
sulfoxide to make a total concentration of dimethyl
sulfoxide to 10%, was added 50 mL of 8 nM solution of
CPA in the Tris bu€er (®nal concentration is 0.4 nM),
and the change in absorbance at 320 nm was recorded
over a time interval of 0±200 s. The values of kobs were
calculated from the progress curves using the computer
assisted spectrophotometer and values of KI and kinact
were calculated, respectively, from the slope and the y
intercept of straight lines in Figure 4. In the calculation,
the Km values of 113 mM was used.
product as a white solid (0.36 g, 48%). mp 114±115 ꢀC;
[a]d= +25.4ꢀ (c 1, CHCl3); IR 1730, 1652 cm
;
1H
1
NMR (CDCl3) d 3.11±3.27 (m, 2H, benzylic), 3.98 (s,
2H), 4.49 (d, J=5.8 Hz, 2H), 4.88 (m, 1H, a-H), 7.04 (d,
J=7.8 Hz, CONH), 7.15±7.38 (m, 10H); 13C NMR
(CDCl3) d 37.7, 52.9, 69.4, 73.8, 127.7±137.0, 170.8,
175.2. Anal. Calcd for C18H19NO4: C, 69.00; H, 6.11; N,
4.47. Found: C, 69.48; H, 6.02; N, 4.66.
Active site protection test
To a preincubated solution of 1±10 mM benzylsuccinate
and 1 mM of CPA in 0.05 M Tris/0.5 M NaCl, pH 7.5

K. J. Lee, D. H. Kim/Bioorg. Med. Chem. 6 (1998) 1613±1622
1621
bu€er for 10 min was added an inactivator (®nal con-
centration is 15.6 mM for l-1, and 13.4 mM for l-2,
dimethyl sulfoxide content: 10%) and the whole mixture
was incubated at room temperature. At speci®ed inter-
vals, 50 mL samples of the incubation mixture were
removed and added to a 950 mL assay mixture and the
activity was monitored at 320 nm (Figure 3).
J.; Lohr, N. S.; Ho€sommer, R. D.; Joshua, H.; Ruyle, W. V.;
Rothrock, J. W.; Aster, S. D.; Maycock, A. L.; Robinson, F.
M.; Hirschman, R.; Sweet, C. S.; Ulm, E. H.; Gross, D. M.;
Vassil, T. C.; Stone, C. A. Nature (London) 1980, 288, 280±283.
(d) Ga€ord, J. T.; Skidgel, R. A.; Erdos, E. G.; Hersh, L. B.
Biochemistry 1983, 22, 3265±3271. (e) Kim, D. H.; Guinosso,
C. J.; Buzby, Jr., G. C.; Herbst, D. R.; McCaully, R. J.; Wicks,
T. C. J. Med. Chem. 1983, 26, 394±403.
6. (a) Redshaw, S. In Medicinal Chemistry, 2nd edn, Roberts,
S. M; Ganellin, C. R. Eds; Academic Press, San Diego, CA,
1993, pp 163±186. (b) Whal, R. C.; Dunlap, R. P. Ann. Rep.
Med. Chem. 1989, 25, 177±184. (c) Hagman, W. K.; Lark, M.
W.; Becker, J. Ann. Rep. Med. Chem. 1996, 31, 231±240.
Determination of the partition ratios
A series of solutions (500 mL each) containing 14.5 mM of
CPA and various molar equivalents of inactivators were
prepared to give [I]o/[E]o ratios of 0 to 1200 in 0.05 M
Tris/0.5 M NaCl, pH 7.5 bu€er, supplemented with
15% dimethyl sulfoxide and were stirred gently at 4 ꢀC
for 16 h. The incubation mixtures were then dialyzed in
the same bu€er solution at 4 ꢀC for 28 h. Subsequently
50 mL samples of each solution were diluted into 450 mL
of the same bu€er. Then a 30 mL portion of each dilu-
tion was added to 970 mL of the assay mixture and the
activity was measured immediately. Partition ratio was
calculated from the plot of fraction of activity remaining
versus [I]o/[E]o.
7. Kim, D. H.; Kim, K. S.; Park, J. K. Bull. Korean Chem. Soc.
1994, 15, 805±807.
8. Izmiya, N.; Uchio, H. J. Biochem. 1959, 46, 235±245.
9. (a) Rees, D. C.; Lewis, M.; Honzatko, R. B.; Lipscomb, W.
N.; Hardman, K. D. Proc. Natl. Acad. Sci. U.S.A. 1981, 78,
3408±3412. (b) Mackenzie, N. E.; Fagerness, P. E.; Scott, A. I.
J. Chem. Soc., Chem. Commun. 1985, 635±637. (c) Chris-
tianson, D. W.; Lipscomb, W. N. Proc. Natl. Acad. Sci. U.S.A.
1986, 83, 7568±7572.
10. (a) Makinen, M. W.; Yamamura, K.; Kaiser, E. T. Proc.
Natl. Acad. Sci. U.S.A. 1976, 73, 3882±3886. (b) Makinen, M.
W.; Kuo, L. C.; Dymowski, J. J. J. Biol. Chem. 1979, 254, 356±
366. (c) Kuo, L. C.; Makinen, M. W. J. Biol. Chem. 1982, 257,
24±27. (d) Suh, J.; Hong, S.-B.; Chung, S. J. Biol. Chem. 1986,
261, 7112±7114. (e) Suh, J.; Cho, W.; Chung, S. J. Am. Chem.
Soc. 1985, 107, 4530±4535. (f) Sander, M. E.; Witzel, H.
Biochem. Biophys. Res. Commun. 1985, 132, 681±687. (g) Britt,
B. M.; Peticolas, W. L. J. Am. Chem. Soc. 1992, 114, 5295±
5303.
Acknowledgements
The authors wish to express their sincere thanks to the
Pohang University of Science and Technology and the
Korea Science and Engineering Foundation for the
®nancial support of this work.
11. (a) Breslow, R.; Wernick, D. L. Proc. Natl. Acad. Sci.
U.S.A. 1977, 74, 1303±1307. (b) Breslow, R.; Wernick, D. L. J.
Am. Chem. Soc. 1976, 98, 259±261. (c) Nau, H.; Riordan, J. F.
Biochemistry 1975, 14, 5285±5294. (d) Galdes, A.; Auld, D. S.;
Vallee, B. L. Biochemistry 1983, 22, 1888±1893. (e) Galdes, A.;
Auld, D. S.; Vallee, B. L. Biochemistry 1986, 25, 646±651. (f)
Breslow, R.; Shepartz, A. Chem. Lett. 1987, 1±4.
References and Notes
1. A preliminary account of this work has been published:
Kim, D. H.; Lee, K. J. Bioorg. Med. Chem. Lett. 1997, 7, 2607±
2612.
2. (a) The Enzymes, Boyer, P. D., Academic Press, New York,
1971, Vol. III. (b) Fersht, A. Enzyme Structure and Mechanism,
2nd edn; W. H. Freeman Co.: Cambridge, England, 1984: pp
405±426.
12. The slow hydrolysis of Gly-Tyr was attributed to the for-
mation of a salt bridge between the carboxylate of Glu-270 and
the terminal amino group of the ligand.9
13. Hass, G. M.; Neurath, H. Biochemistry 1971, 10, 3535±
3540.
3. (a) Protease Inhibitors; Barrett, A. J.; Salvesen, G. Eds.;
Elsevier: Amsterdam, 1986. (b) Rich, D. H. In Comprehensive
Medicinal Chemistry; Hansch, C.; Sammes, P. C.; Taylor, J. B.
Eds.; Pergamon Press: Oxford, 1990; Vol. 2, pp 391±441. (c)
Fischer, G. Nat. Prod. Rep. 1988, 465±495. (d) Walpole, C. S.
J.; Wrigglesworth, R. Nat. Prod. Rep. 1989, 311±346.
14. Suh, J.; Kaiser, E. T. J. Am. Chem. Soc. 1976, 98, 1940±
1947.
15. (a) Beyers, L. D.; Wolfenden, R. Biochemistry 1973, 12,
2070±2078. (b) Beyers, L. D.; Wolfenden, R. J. Biol. Chem.
1972, 247, 606±608.
4. (a) Quiocho, F. A.; Lipscomb, W. N. Adv. Prot. Chem. 1971,
25, 1±78. (b) Christianson, D. W.; Lipscomb, W. N. Acc. Chem.
Res. 1989, 22, 62±69. (c) Lipscomb, W. N.; Strater, N. Chem.
Rev. 1996, 96, 2375±2433.
16. Kitz, R.; Wilson, I. B. J. Biol. Chem. 1962, 237, 3245±3249.
17. Lee, S. S.; Li, Z.-H.; Lee, D. H.; Kim, D. H. J. Chem. Soc.
Perkin Trans. 1, 1995, 2877±2882.
18. Levy, H. M.; Leber, P. D.; Ryan, E. M. J. Biol. Chem.
1963, 238, 3654±3659.
5. (a) Ondetti, M. A.; Rubin, R.; Cushman, D. W. Science,
1977, 196, 441±444. (b) Cushman, D. W.; Cheung, H. S.; Sabo,
E. F.; Ondetti, M. A. Biochemistry 1977, 16, 5484±5941. (c)
Patchett, A. A.; Harris, E.; Tristram, R. W.; Wyvratt, M. J.;
Wu, M. T.; Taub, D.; Peterson, E. R.; Ikeler, T. J.; ten Broeke,
J.; Payne, L. G.; Ondeyka, D. L.; Thorsett, E. D.; Greenlee, W.
19. Silverman, R. Mechanism-Based Enzyme Inactivation:
Chemistry and Enzymology; CRC: Boca Raton, FL, 1988; Vol.
I, pp 22±23.
20. Dixon, M. Biochem. J. 1953, 55, 170±171.

1622
K. J. Lee, D. H. Kim/Bioorg. Med. Chem. 6 (1998) 1613±1622
21. (a) Kim, D. H.; Kim, K. B. J. Am. Chem. Soc. 1991, 113,
3200±3202. (b) Yun, M.; Park, C.; Kim, S.; Nam, D.; Kim, S.
C.; Kim, D. H. J. Am. Chem. Soc. 1992, 114, 2281±2282. (c)
Ryu, S.-E.; Choi, H.-J.; Kim, D. H. J. Am. Chem. Soc. 1997,
119, 38±41.
24. Labia, R.; Lelievre, V.; Peduzzi, J. Biochim. Biophys. Acta
1980, 611, 351±357.
25. We observed that Gly-l-Phe and Gly-l-phenyllactic acid
undergo exclusive hydrolytic cleavage when incubated with
CPA, suggesting that they are not inhibitors but substrates for
CPA.
22. Massova, I.; Martin, P.; Mel, S.; Tanaka, Y.; Edwards, B.;
Mobashery, S. J. Am. Chem. Soc. 1996, 118, 12479±12480.
23. Banci. L.; Bertini, I.; La Penna, G. Proteins: Structure,
Funtions, Genetics 1994, 18, 186±197.
26. Stahmann, M.; Fruton, J. S.; Bergmann, M. J. Biol. Chem.
1946, 164, 753±760.