Hippuryl-α-methylphenylalanine and hippuryl-α-methylphenyllactic acid as substrates for carboxypeptidase A. Syntheses, kinetic evaluation and mechanistic implication

Article abstract of DOI:10.1016/S0968-0896(00)00006-7

(R)- and (S)-Hippuryl-α-methylphenylalanine [(R)- and (S)-Hipp-α- MePhe] and (S)-hippuryl-α-methylphenyllactic acid [(S)-Hipp-α-MeOPhe] were synthesized and evaluated as substrates for carboxypeptidase A (CPA) in an effort to shed further light on the catalytic mechanism of the enzyme. The rate of CPA-catalyzed hydrolysis of (S)-Hipp-α-MePhe was reduced by 105-fold compared with that of (S)-Hipp-Phe, but the hydrolysis rate of (S)-Hipp-OPhe was lowered by only 6.8-fold by the introduction of a methyl group at the α- position. (R)-Hipp-α-MePhe failed to be hydrolyzed initially, then started to undergo hydrolysis in about 2 h at a much reduced rate. The results of present study may be envisioned on the basis of the proposition that while peptide substrate is hydrolyzed via a tetrahedral transition state formed by the attack of the zinc-bound water molecule at the peptide carbonyl carbon, ester hydrolysis takes the path that involves an anhydride intermediate generated by the attack of the carboxylate of Glu-270 at the ester carbonyl carbon. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0968-0896(00)00006-7

Bioorganic & Medicinal Chemistry 8 (2000) 815±823
Hippuryl-ꢀ-methylphenylalanine and Hippuryl-ꢀ-
methylphenyllactic Acid as Substrates for Carboxypeptidase A.
Syntheses, Kinetic Evaluation and Mechanistic Implication
Mijoon Lee and Dong H. Kim*
Center for Biofunctional Molecules and Department of Chemistry, Pohang University of Science and Technology,
San 31 Hyojadong, Pohang 790-784, South Korea
Received 6 October 1999; accepted 20 December 1999
AbstractÐ(R)- and (S)-Hippuryl-a-methylphenylalanine [(R)- and (S)-Hipp-a-MePhe] and (S)-hippuryl-a-methylphenyllactic acid
[(S)-Hipp-a-MeOPhe] were synthesized and evaluated as substrates for carboxypeptidase A (CPA) in an e€ort to shed further light
on the catalytic mechanism of the enzyme. The rate of CPA-catalyzed hydrolysis of (S)-Hipp-a-MePhe was reduced by 105-fold
compared with that of (S)-Hipp-Phe, but the hydrolysis rate of (S)-Hipp-OPhe was lowered by only 6.8-fold by the introduction of
a methyl group at the a-position. (R)-Hipp-a-MePhe failed to be hydrolyzed initially, then started to undergo hydrolysis in about
2 h at a much reduced rate. The results of present study may be envisioned on the basis of the proposition that while peptide
substrate is hydrolyzed via a tetrahedral transition state formed by the attack of the zinc-bound water molecule at the peptide
carbonyl carbon, ester hydrolysis takes the path that involves an anhydride intermediate generated by the attack of the carboxylate
of Glu-270 at the ester carbonyl carbon. # 2000 Elsevier Science Ltd. All rights reserved.
Introduction
substrate-like inhibitors of the enzyme.2 Accordingly, it
was thought to be of interest to know the e€ect that
might be brought about by the introduction of a methyl
group at the a-position of a substrate such as hippuryl-
phenylalanine,3 a substrate used widely for inhibition
kinetics. In this respect, hippuryl-a-methylphenyllactic
acid was thought to be of interest to study as well. This
paper describes syntheses and kinetic evaluations of
these compounds as substrates for CPA and their
implications to the catalytic mechanism of the enzyme.
Carboxypeptidase A (CPA) is a prototypic zinc-con-
taining proteolytic enzyme, representing a large family
of physiologically important zinc proteases such as
angiotensin converting enzyme and matrix metallopro-
teases.1 The enzyme catalyzes the hydrolysis of the C-
terminal amide bond of peptide substrate and shows
speci®city for oligopeptide having the C-terminal resi-
due with a hydrophobic side chain.1 CPA also catalyzes
the hydrolysis of esters having a structural feature simi-
lar to the peptide substrate.1 Thus, the enzyme cleaves
esters of a-hydroxy acid such as b-phenyllactic acid. As
a representative zinc protease, the enzyme has received
much attention as a model enzyme for devising design
protocols of inhibitors that are e€ective against zinc
proteases of medicinal interest. Recently, Asante-
Appiah et al. reported that at the active site of CPA
there exists a small hydrophobic cavity that can accom-
modate a methyl group present at the a-position of
*Corresponding author. Tel.: +82-562-279-2101; fax: +82-562-279-
5877; e-mail: dhkim@vision.postech.ac.kr
0968-0896/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(00)00006-7

816
M. Lee, D. H. Kim / Bioorg. Med. Chem. 8 (2000) 815±823
Scheme 1. (a) NMP, 70 ꢀC, 24 h, (S,S)-7, 39%; (R,S)-8, 38%; (b) CF3SO3H, MeOH, 80 ꢀC, 20 h, 70%; (c) HCl/dioxane, 100 ꢀC, 20 h; propylene
oxide, EtOH, re¯ux, 0.5 h, 90%; (d) H+, MeOH, re¯ux, 12 h, 90%; (e) hippuric acid, NMM, isobutyl chloroformate, THF, 10 ꢀC to rt, 12 h, 52%;
.
(f) LiOH H2O (3 equiv), MeOH/H2O/THF, rt, 24 h, 90%.
Results and Discussion
Compound (R)-2 was prepared from (R,S)-8 with
[a]d=+58.0ꢀ (EtOH) following the same sequences of
reactions as used for the preparation of (S)-2 (Scheme 2).
Synthesis of (S)-Hipp-a-MePhe (2) is outlined in
Scheme 1. The key intermediate, (S)-a-MePhe (10) was
prepared following the route described by Obrecht et
al.4 Compound 6 was obtained by benzylation of 4-
methyl-2-phenyl-1,3-oxazol-5(4H)-one according to the
literature method.5 Treatment of racemic 5 with (S)-
phenylalanine cyclohexylamide in N-methylpyrolidin-2-
one gave a mixture of diastereomers in the ratio of 1:1,
which was readily separated by column chromatography.
Diastereomer, (S,S)-7 having [a]d=+1.8ꢀ (EtOH) was
treated with tri¯uoromethanesulfonic acid in methanol
to yield (S)-9. The stereochemistry of the diastereomer
has been previously established by X-ray crystallography
to be the (S,S)-con®guration.4 Acidic hydrolysis of (S)-9
and subsequent esteri®cation gave (S)-a-MePhe methyl
ester (11) which was coupled with hippuric acid by
the mixed anhydride method to give (S)-12. The
desired compound was obtained by alkaline hydrolysis
of (S)-12 with lithium hydroxide in an overall yield of
10% from 5.
Scheme 3 depicts the synthetic route for the preparation
of 5. (S)-a-MeOPhe (17) was prepared by the general
route described by Seebach et al.6 with a minor mod-
i®cation. Thus, the conversion of 15 into 16 was carried
out under the conditions of Greiner and Ortholand.7
The coupling of 18 with N-t-Boc-glycine to give 19 was
achieved in a 62% yield by the method of Zhao et al.,8
which is known to be highly e€ective for esteri®cation of
sterically hindered tert-alcohols. Removal of the pro-
tection group in 19 followed by the N-benzoylation and
then hydrogenolysis produced 5 in an overall yield of
12% from 13.
The unnatural substrates thus synthesized were assayed
as substrates of CPA. The rate of the hydrolysis of (S)-2
was found to be lower considerably compared with that
of 1. The kinetic parameters for the enzymic hydrolysis
reaction were obtained from the Lineweaver±Burk plot9

M. Lee, D. H. Kim / Bioorg. Med. Chem. 8 (2000) 815±823
817
Scheme 2. (a) CF3SO3H, MeOH, 80 ꢀC, 20 h, 70%; (b) HCl/dioxane, 100 ꢀC, ꢀ20 h; propylene oxide, EtOH, re¯ux, 0.5 h, 90%; (c) H+, MeOH,
24 h, 90%.
.
re¯ux, 12 h, 90%; (d) hippuric acid, NMM, isobutyl chloroformate, THF, 10 C to rt, 12 h, 52%; (e) LiOH H2O (3 equiv), MeOH/H2O/THF, rt,
(Fig. 1) and are collected in Table 1. The kcat value was
reduced by 245-fold by the replacement of the hydrogen
at the a-position in 1 with a methyl group, although the
binding anity is improved. The apparent second-order
rate constant (kcat=Km) was reduced by 105-fold. On the
other hand, the enantiomer of (S)-2 failed to be hydro-
lyzed by the enzyme initially, then started to undergo
hydrolysis in about 2 h at a much reduced rate (Fig. 2).
As expected, (R)-2 inhibited the enzymic hydrolysis of
(S)-Hipp-Phe with the Ki value of 405 mM when CPA
was added to the incubation mixture of the substrate
and (R)-2 (Fig. 3).
(S)-2 may be envisioned on the ground of the hypothesis
proposed by Asante-Appiah et al. Thus, in the case of
(S)-2, the space required for the proton transfer is
occupied by the a-methyl group upon forming the
complex and as a result the proton delivery to the
departing amino group becomes prohibitive, causing
impedence of the catalytic activity (Fig. 5(b)) as mani-
fested by the lowering of the kcat=Km value by 105-fold.
Details of the CPA catalytic mechanism will be dis-
cussed below. The observation that (R)-2 commences to
undergo CPA-catalyzed hydrolysis in about 2 h may be
a re¯ection of the inherent propensity of an enzyme to
exert its catalytic action on the bound ligand. The CPA
molecule may possibly undergo an induced-®t con-
formational change upon forming a complex with (R)-2,
and begins to catalyze the hydrolysis reaction.
The hydrolysis rate of 4 that carries a methyl group at
the a-position of the ester (3) was also reduced com-
pared with that for 3 but much less profoundly: it was
reduced by 6.8-fold. The kinetic parameters were
obtained from the Lineweaver±Burk plot9 (Fig. 4) and
are listed in Table 2.
The catalytic mechanism of CPA remains to be the
subject of much debate in spite of intensive research
over the last several decades. The promoted-water
pathway has been proposed by Christianson and Lips-
comb mostly based on the X-ray structural studies of
CPA complexes formed with slowly hydrolyzing sub-
strates and transition state analogue inhibitors.13,14
According to them, the carboxylate of Glu-270 functions
as a base, abstracting a proton from the zinc-bound
water molecule to generate a nucleophilic hydroxide
ion, which, in turn, attacks at the carbonyl carbon of
the scissile peptide bond, generating a tetrahedral tran-
sition state (Fig. 5(a)). The scissile peptide bond of the
enzyme bound substrate is thought to be activated for
the nucleophilic attack by forming its carbonyl oxygen
a bifurcated hydrogen bond with the guanidinium of
Asante-Appiah et al. reported that 2-ethyl-2-methylsuc-
cinic acid is a potent competitive inhibitor for CPA.2
The X-ray crystal structure of the complex of CPA
formed with the R-form of the inhibitor revealed that
the a-methyl group occupies a small hydrophobic cav-
ity. The small cavity (methyl hole) located next to the
primary recognition pocket of the enzyme is, however,
not ®lled when substrates bind the enzyme, and thus it
was proposed that its function is to provide a space
needed for the protonated carboxylate of Glu-270 to
transfer a proton to the amino group of the tetrahedral
transition state in the catalytic process (Fig. 5(a)).2,12 The
decrease in the enzymic hydrolysis rate observed with

818
M. Lee, D. H. Kim / Bioorg. Med. Chem. 8 (2000) 815±823
ꢀ
Schemeꢀ3. (a) Pivalaldehyde (1.1 equiv), BF3 Et2O (3 equiv), ether, 0 C, 4 h, 94%; (b) LDA (1.05 equiv), MeI (1.5 equiv), THF:hexane (9:1), 78 ꢀC
.
.
to 20 C, 3 h, 47%; (c) NaOEt (cat.), EtOH, re¯ux, 10 min, 66%; (d) LiOH H2O (3 equiv), THF:MeOH:H2O (3:1:1), rt, 12 h, 96%; (e) Cs2CO3 (1
equiv), MeOH, rt, 30 min; BnBr (1 equiv), DMF, rt, 6 h, 86%; (f) tBoc-Gly-OH (3 equiv), Sc(OTf)3 (0.6 equiv), DMAP (3 equiv), rt, 10 ꢀC, 30 min;
EDCI (3 equiv), CH2Cl2, 10 to 40 ꢀC, 48 h, 62%; (g) CF3CO2H, rt, 1 h, 90%; (h) BzCl (1 equiv), Et3N (1 equiv), CH2Cl2, 0 ꢀC to rt, 1 h, 91%; (i) Pd/
C, H2, MeOH, rt, 1 h, 95%.
Figure 1. The progress curve (a) and the Lineweaver±Burk plot (b) for CPA catalyzed hydrolysis of (S)-Hipp-a-MePhe (Tris bu€er of pH 7.5 at
25 ꢀC).
Arg-127.15 The abstracted proton is then delivered to
the amino nitrogen of the leaving amino acid residue at
the transition state, making the collapse of the transi-
tion state to products facile (Fig. 5(a)). On the other
hand, Makinen and others were able to detect spectro-
scopically anhydride-type acyl-enzyme intermediates in
the CPA-catalyzed hydrolysis of ester type substrates
under low temperature conditions.16±21 On the basis of
these observations Makinen proposed that the catalytic
mechanism of CPA is initiated by the nucleophilic
attack of the carboxylate of Glu-270 on the carbonyl
carbon of the bound substrate to form an anhydride

M. Lee, D. H. Kim / Bioorg. Med. Chem. 8 (2000) 815±823
Table 1. Kinetic parameters for carboxypeptidase A catalyzed hydrolysis reactions
(S)-Hipp-Phe
819
(S)-Hipp-a-MePhe
((S)-2)
(R)-Hipp-a-MePhe
((R)-2)
(1)
3
5
7
Initial velocitya
1.39Â10
1.95Â10
9.23Â10
1
kcat (s
)
81 (67)b
0.33
313
NDc
ND
ND
405d
Km (mM)
kcat=Km (M
Ki (mM)
700 (690)b
11.6Â104 (9.7Â104)b
1
1
s
)
0.11Â104
aInitial velocity is de®ned as the increase of UV absorbance at 254 nm per second at the initial stage of the enzymic reaction ([E]=600 nM, [S]=250
mM).
bref 11.
cND denotes that the kinetic parameter was not determined.
dThe Ki value was obtained for the inhibition of the hydrolysis of (S)-Hipp-Phe by CPA.
Figure 2. Graphs showing the product formation against time for CPA catalyzed hydrolysis of (S)-Hipp-Phe (a) and (R)-Hipp-a-MePhe (b) at
[S]=250 mM and [CPA]=600 nM (Tris bu€er of pH 7.5 at 25 ꢀC).
intermediate (Fig. 6).16,17,20 The anhydride intermediate
is then hydrolyzed by the active site zinc bound water
molecule to yield products with regeneration of CPA.
This reaction path is generally referred to as the anhy-
dride mechanism. The accumulated experimental
data13±21 pertaining to the enzymic mechanism of the
CPA catalyzed hydrolysis reactions may be reconciled
by proposing that there may operate two di€erent cata-
lytic reaction paths depending on the nature of sub-
strate: natural substrates of peptide are hydrolyzed by
the promoted-water pathway, but in the hydrolysis of
esters the anhydride intermediates are involved.
zinc ion, whereas binding of a peptide does not, sup-
ports the proposition that the ester coordinates to the
active site zinc ion in binding to CPA.24 The altered
binding mode is thought to be responsible for the esters
taking the anhydride pathway. As an ester, 4 would also
bind CPA with its carbonyl oxygen being coordinated
to the zinc ion, and is hydrolyzed by the anhydride
pathway that does not require the proton transfer process
It is now well established that the carbonyl oxygen of
peptide substrate forms a hydrogen bond with the gua-
nidinium moiety of Arg-127 as demonstrated by the
X-ray crystal structure study15 and subsequently con-
®rmed by the site speci®c mutagenesis experiment.22
However, no X-ray structural date that can establish the
binding mode for ester substrates is available. It is not
unreasonable to expect that esters may bind di€erently
from peptides in the complex formation in light of their
molecular conformational di€erences. While a peptide
bears a planar conformation, ester moiety is known to
have a bent molecular shape23 with the dihedral angle of
54.5ꢀ. Thus, in forming the Michaelis complex with
CPA, the carbonyl oxygen of ester substrates may not
hydrogen bond to guanidinium of Arg-127, but instead
ligates to the zinc ion. The ®nding of Auld and Holm-
quist that binding of esters requires the presence of the
Figure 3. The Dixon plot10 for the inhibition of the CPA catalyzed
hydrolysis of (S)-Hipp-Phe by (R)-Hipp-a-MePhe (Tris bu€er of pH
7.5 at 25 ꢀC).

820
M. Lee, D. H. Kim / Bioorg. Med. Chem. 8 (2000) 815±823
Figure 4. The progress curve (a) and the Lineweaver±Burk plot (b) for CPA catalyzed hydrolysis of (S)-Hipp-a-MeOPhe (Tris bu€er of pH 7.5 at
25 ꢀC).
Table 2. Kinetic parameters for carboxypeptidase
hydrolysis reactions
A catalyzed
(S)-Hipp-OPhe (3)
(S)-Hipp-a-MeOPhe (4)
1
kcat (s
)
682 (424)a
72 (53)a
9.5Â106 (8.0Â106)a
477
344
1.4Â106
Km (mM)
kcat=Km (M
1
1
s
)
aref 3.
which is critical for the peptide substrate. Therefore, the
reaction path of the CPA-catalyzed hydrolysis of 4 is
not perturbed by the a-methyl group, and the hydrolysis
proceeds rapidly (Table 2) albeit not at the optimal rate
as observed with 3 (Fig. 6).
Figure 6. Schematic representation showing a proposed reaction path
for the CPA-catalyzed hydrolysis of ester-type substrates, for example,
3 and 4.
The Km value obtained for 4 is augmented compared
with that for 3 (Table 2), indicating that the a-methyl
group tends to deter the binding of 4 to the enzyme.
This observation suggests that the minor cavity next to
the primary substrate recognition pocket of CPA is not
optimally situated for accommodating the methyl group
at the a-position of the substrate as expected from the
proposition that its primary function is to provide a
space for the proton transfer in the enzymic hydro-
lysis of peptide substrates. The surprising ®nding of
Asante-Appiah et al.,2 that 2-ethyl-2-methylsuccinic
acid binds CPA with extremely high anity may be
explained. The ethyl group that is considerably smaller
0
than the aromatic side chain present in the P1 residue of
normal substrates can ®t freely in the primary recogni-
tion pocket. The inserted ethyl group may have su-
cient room for maneuvering inside the pocket and
enable the 2-methyl moiety to ®t fully in the small cavity
resulting in the strengthening of the binding of 2-ethyl-
2-methylsuccinic acid to CPA.
Figure 5. Schematic representations showing the tetrahedral transition state proposed for the CPA-catalyzed hydrolysis of normal peptide substrate
0
(1) (a) and unnatural substrate (2) whose P1 residue carries a methyl group at the a-position.

M. Lee, D. H. Kim / Bioorg. Med. Chem. 8 (2000) 815±823
821
Conclusion
3H), 1.96 (br. s, 1H), 3.19±3.40 (dd, 2H), 3.72 (s, 3H),
4.06±4.07 (d, 2H), 6.89±7.80 (m, 11H); 13C NMR 300
MHz (CDCl3) d 23.48, 41.99, 44.26, 53.13, 61.39,
127.59, 128.76, 128.98, 130.26, 132.24, 133.93, 136.21,
137.59, 168.07, 168.80, 174.35.
The substitution of the proton at the a-position of (S)-
Hipp-Phe with a methyl group causes the substrate to
be resistant to the enzymic hydrolysis, resulting in the
decrease of the second-order rate constant by 105-fold.
This is probably due to blocking of the approach of the
protonated carboxylate of Glu-270 to the departing
amino group in the transition state of the bond cleavage
process by the methyl group. In the case of the ester
substrate, the hydrolysis rate was lowered by only 6.8-
fold by the introduction of a methyl at the a-position.
The di€erent e€ects exhibited by the introduction of an
a-methyl group in the two di€erent classes of substrates
on the enzymic reaction may be explained on the basis
that they do not bind in the same mode at the enzyme
active site, and as a result take a di€erent reaction path.
The peptide bond is cleaved via a tetrahedral transition
state generated by the attack of the zinc-bound acti-
vated water molecule on the scissile amide bond, but
ester hydrolysis takes the path involving an anhydride
intermediate formed by the attack of the carboxylate of
the catalytic Glu-270 residue at the ester carbonyl car-
bon. The result of the present study is in accord with the
suggestion made by Breslow that there might exist two
di€erent pathways in the CPA catalyzed reaction.25
(S)-Hippuryl-ꢀ-methylphenylalanine ((S)-2). Lithium
hydroxide (46 mg, 1.1 mmol) solution in water (500 mL)
was added to a solution of 12 (130 mg, 0.37 mmol)
dissolved in THF:MeOH (3:1, 1.5 mL). The reaction
mixture was stirred at room temperature for 24 h. After
evaporation of the solvent under reduced pressure, the
residue was acidi®ed with 2 N HCl (2 mL) and extracted
with ethyl acetate (3Â5 mL). The combined extracts
were washed with brine and dried over MgSO4. Eva-
poration of the solvent gave 2 (110 mg, 90%) which was
recrystallized from ethyl acetate, diethyl ether and hex-
ane: mp=164.5±166 ꢀC; [a]d 49.0ꢀ (c 0.95, MeOH); IR
1
(KBr) 3412, 1715, 1677 cm
;
1H NMR 300 MHz
(D2O) d 1.60 (s, 3H), 3.26±3.37 (dd, 2H), 3.99±4.18 (m,
2H), 6.96±7.82 (m, 12H); 13C NMR 300 MHz (CDCl3) d
18.39, 41.57, 44.17, 61.14, 127.43, 127.77, 128.71,
130.46, 132.45, 133.47, 136.14, 168.77, 169.97, 176.71.
Anal. calcd for C19H20N2O4: C, 67.05; H, 5.92; N, 8.23.
Found: C, 66.82; H, 5.89; N, 7.95.
(R)-Hippuryl-ꢀ-methylphenylalanine ((R)-2). The synthe-
sis was carried out as described for (S)-hippuryl-a-
methylphenylalanine starting from (R,S)-8: mp=165±
166 ꢀC; [a]d +48.5ꢀ (c 1.0, MeOH).
Experimental
Melting points (mp) were taken on a Thomas±Hoover
1
capillary mp apparatus and were uncorrected. H and
(S)-Ethyl ꢀ-methylphenyllactate (16). Ethanol solution
of 15 (3.3 g, 13.3 mmol) and a catalytic amount of
sodium ethoxide was re¯uxed for 10 min, then evapo-
rated under reduced pressure to a€ord 1.8 g (66%) of 16
as an oil. [a]d 20.9ꢀ (c 2.1, CHCl3); IR (neat) 3300,
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 Bruker
Equinox 55 FT±IR spectrometer. Silica gel 60 (230±400
mesh) was used for ¯ash chromatography and thin layer
chromatography (TLC) was carried out on silica coated
glass sheets (Merck silica gel 60 F-254). Elemental
analyses were performed at the Basic Science Center,
Kyungbook National University, Taegu, Korea. A
Perkin±Elmer HP 8453 UV±vis spectrometer was used
in enzyme inhibition studies.
1
1762 cm 1; H NMR 300 MHz (CDCl3) d 1.23±1.27 (t,
3H), 1.49 (s, 3H), 2.90±3.10 (dd, 2H), 3.23 (br. s, 3.23),
4.11±4.19 (q, 2H), 7.19±7.30 (m, 5H); 13C NMR 300
MHz (CDCl3) d 14.59, 26.27, 46.79, 62.18, 75.43,
127.30, 128.55, 130.55, 136.47, 176.53.
(S)-ꢀ-Methylphenyllactic acid (17). Lithium hydroxide
solution obtained by dissolving lithium hydroxide
(0.725, 17.3 mmol) in water (3 mL) was added to a
solution of 16 (1.8 g, 8.6 mmol) in THF:MeOH (3:1, 12
mL). The reaction mixture was stirred at room tem-
perature for 12 h. After evaporation of the solvent
under reduced pressure, the residue was acidi®ed with 2
N HCl (10 mL) and extracted with ethyl acetate (3Â5
mL). The combined extracts were washed with brine
and dried over MgSO4. Evaporation of the solvent gave
17 (1.5 g, 96%) which was recrystallized from diethyl
ether and hexane. Mp=114±116 ꢀC (lit.26 117.5±119 ꢀC);
[a]d 32.1ꢀ (c 1.0, CHCl3) (lit.26 16.4ꢀ (c 5.7, dioxane);
IR (KBr) 3446, 1652 cm 1; 1H NMR 300 MHz (CDCl3)
d 1.47 (s, 3H), 2.89±3.16 (dd, 2H), 7.25±7.28 (m, 6H);
13C NMR 300 MHz (CDCl3) d 26.28, 46.38, 75.00,
127.04, 128.35, 130.72, 136.90, 178.64.
(S)-Methyl hippuryl-ꢀ-methylphenylalaninate (12). To a
mixture of hippuric acid (192 mg, 1.1 mmol) and N-
methylmorpholine (NMM, 120 mL, 1.1 mmol) in dry
THF (5 mL) was added isobutyl chloroformate (140 mL,
1.1 mmol) at 15 ꢀC. (S)-a-Methylphenylalanine methyl
ester hydrochloride ((S)-11, 250 mg, 1.1 mmol) was
neutralized with NMM (120 mL, 1.1 mmol) in DMF (1
mL), and added slowly to the reaction mixture. The
resulting mixture was allowed to warm up to room
temperature and stirred for 12 h. The hydrochloride of
NMM was removed by ®ltration and washed with THF.
The combined ®ltrate and washings were concentrated
under reduced pressure and diluted with ethyl acetate.
The solution was washed with 0.5 N NaHCO3, 0.5 N
HCl, water, and brine, then dried (MgSO4), and evapo-
rated under reduced pressure. The residue was puri®ed
by column chromatography to give (S)-12 as an oil (150
mg, 52%). [a]d 72.1ꢀ (c 0.5, MeOH); IR (neat) 1723,
1652, 1647 cm 1; 1H NMR 300 MHz (CDCl3) d 1.60 (s,
(S)-Benzyl ꢀ-methylphenyllactate (18). To a solution of
17 (0.62 g, 3.4 mmol) dissolved in 10 mL of MeOH was
added Cs2CO3 (1.12 g, 3.4 mmol) and stirred for 30 min.

822
M. Lee, D. H. Kim / Bioorg. Med. Chem. 8 (2000) 815±823
After the evaporation of the solvent under the reduced
pressure, cesium salt of lactate was suspended in 10 mL
DMF. Benzyl bromide (430 mL, 3.6 mmol) was added
dropwise to the reaction mixture and stirred for 6 h at
room temperature. The reaction mixture was poured
onto ice (20 g) and extracted with ethyl acetate (3Â20
mL). The combined extracts were washed with water
(30 mL), 0.5 N HCl (30 mL), and brine (30 mL), dried
over MgSO4, and evaporated under the reduced pres-
sure to give a white solid. The crude product was
recrystallized from petroleum ether to give 0.80 g (86%)
of 18 (white crystal). Mp=59.5±61.0 ꢀC; [a]d 70.8ꢀ (c
0.54, CHCl3); IR (KBr) 3446, 1733 cm 1; 1H NMR 300
MHz (CDCl3) d 1.55 (s, 3H), 2.92±3.13 (dd, 2H), 5.16 (s,
2H), 7.10±7.42 (m, 10H); 13C NMR 300 MHz (CDCl3) d
26.27, 46.83, 67.94, 75.67, 126.29, 127.33, 128.62,
128.95, 129.08, 130.48, 135.51, 136.19, 176.36.
reduced pressure. The product was puri®ed by chroma-
tography on a silica gel column to give 21 (770 mg 91%)
as a colorless oil. [a]d 30.0ꢀ (c 0.40, CHCl3); IR (neat)
1
1739, 1652, 1539 cm 1; H NMR 300 MHz (CDCl3) d
1.64 (s, 3H), 3.10±3.36 (dd, 2H), 4.13±4.32 (m, 2H), 5.16
(s, 2H), 7.09±7.86 (m, 15H); 13C NMR 300 MHz
(CDCl3) d 21.68, 42.38, 44.20, 67.71, 82.67, 127.55,
127.66, 128.72, 128.81, 128.97, 129.01, 130.49, 130.92,
132.24, 134.03, 134.79, 135.62, 167.83, 169.59, 171.61.
(S)-Hippuryl-ꢀ-methylphenyllactic acid (4). A methanol
(3 mL) solution containing 21 (700 mg, 1.6 mmol) and a
catalytic amount of Pd/C was stirred for 1 h at room
temperature. The catalyst was removed by ®ltration,
and the solvent was evaporated under the reduced
pressure to give a resin (526 mg, 95%). [a]d 39.5ꢀ (c
1.3, CHCl3); IR (KBr) 1733, 1652, 1538 cm 1; 1H NMR
300 MHz (CDCl3) d 1.54 (s, 3H), 3.01±3.35 (dd, 2H),
4.21±4.23 (m, 2H), 7.14±7.38 (m, 10H); 13C NMR 300
MHz (CDCl3) d 18.24, 21.56, 42.46, 43.63, 82.19,
127.60, 127.74, 128.69, 129.01, 131.32, 132.39, 133.60,
134.93, 168.86, 169.86, 175.07. Anal. calcd for C19H19
NO5: C, 66.85; H, 5.61; N, 4.10. Found: C, 66.56; H,
5.93; N, 3.85.
(S)-Benzyl t-Boc-glycyl-ꢀ-methylphenyllactate (19). A
stirring mixture of 18 (0.90 g, 3.3 mmol), scandium tri-
¯ate (0.98 g, 2.0 mmol), t-Boc-Gly-OH (1.75 g, 10.0
mmol), and DMAP (1.22 g, 10.0 mmol) in anhydrous
CH2Cl2 (7 mL) was cooled to 10 ꢀC in an ice-salt bath
for 30 min. EDCI (1.98 g, 10.0 mmol) was added and
the stirring was continued for 30 min. The resulting
mixture was allowed to warm up to room temperature
over the period of 2 h and stirred at 40 ꢀC for 48 h. The
reaction mixture was diluted with 20 mL of CH2Cl2 and
washed with 2Â20 mL of 0.1 N HCl, 2Â20 mL of 0.1 N
NaHCO3, 20 mL of brine, then 20 mL of water. The
organic phase was dried over MgSO4 and the solvent
was removed under reduced pressure. The product was
puri®ed by chromatography on a silica gel column to
General remarks for kinetic experiments
All solutions were prepared by dissolving in doubly
distilled 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 pancrease, aqueous suspension in tol-
uene) and used without further puri®cation. CPA stock
solutions were prepared by dissolving the enzyme in
0.05 M Tirs/0.5 M NaCl, pH 7.5 bu€er solution.
Enzyme concentrations were estimated from the absor-
bance at 278 nm (E278=64,200). (S)-Hippuryl-phenyl-
alanine ((S)-Hipp-Phe) was purchased from Sigma
Chemical Co. (S)-Hippuryl-phenyllactic acid ((S)-Hipp-
OPhe) was prepared according to the literature
method.27
give 19 (1.00 g, 62%) as a colorless oil. [a]d 20.8ꢀ (c
1
0.80, CHCl3); IR (neat) 1739, 1717, 1652 cm
;
1H
NMR 300 MHz (CDCl3) d 1.45 (s, 9H), 1.56 (s, 3H),
3.03±3.30 (dd, 2H), 3.89±3.92 (m, 2H), 4.93 (s, 1H), 5.11
(s, 2H), 7.10±7.36 (m, 10H); 13C NMR 300 MHz
(CDCl3) d 21.63, 28.73, 42.98, 44.18, 67.67, 80.40, 82.30,
126.30, 127.61, 128.67, 128.72, 128.95, 130.91, 134.88,
135.66, 155.943, 169.76, 171.65.
(S)-Benzyl glycyl-ꢀ-methylphenyllactate (20). To a solu-
tion of 19 (900 mg, 0.75 mmol) in CH2Cl2 (5 mL) was
added CF3CO2H (3 mL) and stirred at room tempera-
ture for 1 h. Evaporation of the excess tri¯uoroacetic
acid and solvent yielded 20 (680 mg 90%) as a yellow
oil. [a]d 14.7ꢀ (c 0.80, CHCl3); IR (neat) 1750, 1684
cm 1; 1H NMR 300 MHz (CDCl3) d 1.59 (s. 3H), 3.07±
3.22 (dd, 2H), 3.84±3.87 (m, 2H), 5.01±5.11 (m, 2H),
7.00±7.41 (m, 10H), 7.82±7.93 (br. s, 2H); 13C NMR 300
MHz (CDCl3) d 21.32, 41.05, 44.40, 68.28, 84.66,
127.93, 128.79, 128.82, 129.04, 129.35, 130.66, 133.95,
135.04, 166.65, 171.32.
Determination of kcat and Km
Data obtained by repeated measurements of the change
in absorbance upon complete hydrolysis of several
known concentrations of each substrate was converted
to initial velocities by using observed ÁE ((S)-Hipp-
OPhe)=332 M 1 cm 1, ÁE ((S)-Hipp-a-MeOPhe)=318
1
1
1
M
cm 1, ÁE ((S)-Hipp-a-MePhe)=210 M cm at
254 nm. The kcat and Km values were then estimated
from the double reciprocal plot of the initial velocity
versus the concentration of the substrate.9 Typically,
enzyme stock solution was added to various concentra-
tions of substrates in 0.05 M Tris/0.5 M NaCl, pH 7.5
bu€er (1 mL cuvette), and the initial rates were mea-
sured immediately.
(S)-Benzyl hippuryl-ꢀ-methylphenyllactate (21). To a
solution of 20 (640 mg, 1.95 mmol) dissolved in 5 mL
CH2Cl2 was added triethylamine (300 mL, 2.15 mmol)
and benzoyl chloride (200 mL, 2.15 mmol) alternatingly
at 0 ꢀC. The reaction mixture was stirred for 1 h at room
temperature and then washed with water, 0.1 N HCl,
0.1 N NaHCO3, and brine. The organic phase was dried
over MgSO4 and the solvent was removed under
Determination of Ki
The Ki values were estimated from the semireciprocal
plot of the initial velocity versus the concentration of
the inhibitors according to the method of Dixon.10 Two

M. Lee, D. H. Kim / Bioorg. Med. Chem. 8 (2000) 815±823
823
concentrations of the substrate were used. Typically,
enzyme stock solution was added to various concentra-
tions of inhibitors in 0.05 M Tris/0.5 M NaCl, pH 7.5
bu€er (1 mL cuvette), and the initial rates were mea-
sured immediately using a microcomputer-interfaced
UV spectrometer.
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Acknowledgements
The authors express their thanks to the Korea Science
and Engineering Foundation for ®nancial support of
this work.
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