A new inhibitor design strategy for carboxypeptidase A as exemplified by N-(2-Chloroethyl)-N-methylphenylalanine

Article abstract of DOI:10.1016/S0968-0896(00)00239-X

N-(2-Chloroethyl)-N-methylphenylalanine was designed and synthesized in an optically active form as a novel class of mechanism-based inactivator for carboxypeptidase A (CPA). It was anticipated that the chloroethylamino moiety of the CPA bound inhibitor undergoes an intramolecular S_N2 reaction to generate a chemically reactive species (an aziridinium ion) which is expectedly subjected to a nucleophilic attack by the carboxylate of Glu-270, leading to covalent modification of the carboxylate. The irreversible nature of the inhibition of CPA by the inhibitor was supported by the kinetic data: the enzyme lost its enzymic activity in a time-dependent manner in the presence of the inhibitor and the inactivated CPA failed to regain the activity upon dialysis. Interestingly, the (R)-isomer that belongs to the D-series was more potent than its enantiomer.

Full text of DOI:10.1016/S0968-0896(00)00239-X

Bioorganic & Medicinal Chemistry 9 (2001) 237±243
A New Inhibitor Design Strategy for Carboxypeptidase A as
Exempli®ed by N-(2-Chloroethyl)-N-methylphenylalanine
Jung Dae Park, Kyung Joo 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 7 June 2000; accepted 25 August 2000
AbstractÐN-(2-Chloroethyl)-N-methylphenylalanine was designed and synthesized in an optically active form as a novel class of
mechanism-based inactivator for carboxypeptidase A(CPA). It was anticipated that the chloroethylamino moiety of the CPA
bound inhibitor undergoes an intramolecular SN2 reaction to generate a chemically reactive species (an aziridinium ion) which is
expectedly subjected to a nucleophilic attack by the carboxylate of Glu-270, leading to covalent modi®cation of the carboxylate.
The irreversible nature of the inhibition of CPAby the inhibitor was supported by the kinetic data: the enzyme lost its enzymic
activity in a time-dependent manner in the presence of the inhibitor and the inactivated CPAfailed to regain the activity upon
dialysis. Interestingly, the (R)-isomer that belongs to the d-series was more potent than its enantiomer. # 2001 Elsevier Science
Ltd. All rights reserved.
Introduction
hydrophobic side chain.10,11 Three binding and one
catalytic sites have been identi®ed at the active site of
CPA.10,11 The active site zinc ion that is coordinated
tightly to the backbone amino acid residues of His-69,
Glu-72 and His-196 is essential for the enzymic catalytic
hydrolysis reaction of substrate. Amolecule of water is
bound loosely to the zinc ion as the fourth ligand. The
zinc ion activates the water molecule in collaboration
with the carboxylate of Glu-270, generating a powerful
nucleophilic hydroxyl group that attacks at the carbonyl
carbon of the scissile peptide bond. The other important
role of the zinc ion is that it stabilizes the tetrahedral
transition state that is generated by the hydroxyl attack
The metal assisted substitution reactions that are
commonly found in solution chemistry may be of value
for designing inhibitors that are e€ective for
metalloproteases.1À4 Recently, Mobashery and co-
workers have successfully exploited the metal (Lewis
acid) mediated rate enhancement in the nucleophilic
substitution reaction of an unacitvated alkyl halide to
design an irreversible inhibitor for carboxypeptidase A
(CPA).5 2-Haloethylamines readily undergo an intra-
molecular substitution reaction in an aqueous medium
to form aziridinium ions that are known to be relatively
stable.6,7 Such a cyclization reaction may also be facili-
tated by the coordination of the halide leaving group to
a Lewis acid.8,9 We conceived a novel design strategy
for an inactivator of carboxypeptidase A, which takes
advantage of the Lewis acid assisted aziridinium forma-
tion reaction. This paper describes that N-(2-chloroethyl)-
N-methylphenylalanine is a new class of CPAirreversible
inhibitor that illustrates the novel design strategy.
0
in the catalytic process. The Arg-145 residue and the S1
subsite pocket are intimately involved in the binding of
substrate to CPA. The guanidinium moiety of Arg-145
forms bifurcated hydrogen bonds with the terminal
0
carboxylate of substrate, and the S1 subsite pocket
accommodates the aromatic ring present in the side
chain of the cleavable C-terminal amino acid residue,
thus serving as the primary substrate recognition pocket
(Fig. 1).
We have previously demonstrated that 2-benzyl-3,4-
epoxybutanoic acid (1) inactivates CPAvery rapidly
with extremely high eciency.12À14 Analysis of the X-
ray crystal structures of the inactivated CPArevealed
that, like substrates, the terminal carboxylate in 1 forms
hydrogen bonds with the guanidinium moiety of Arg-
Results and Discussion
CPAis a prototypic zinc-containing exomonopeptidase
that cleaves the C-terminal amino acid residue having a
*Corresponding author. Tel.:+82-562-279-2101; fax: +82-562-279-
5877. E-mail: dhkim@vision.postech.ac.kr
0
145 and the hydrophobic phenyl ring anchors in the S1
0968-0896/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(00)00239-X

238
J. D. Park et al. / Bioorg. Med. Chem. 9 (2001) 237±243
subsite pocket of CPA. The oxirane ring of the CPA
bound 1 undergoes a ring cleavage reaction initiated by
the nucleophilic attack of the carboxylate of the cataly-
tic residue (Glu-270), leading to covalent modi®cation
of the carboxylate with formation of an ester linkage. It
occurred to us that the oxirane ring in 1 might be
replaced by an aziridinium moiety with retention of the
CPAinactivating property. The aziridinium moiety in
the potential inhibitor, 2, is expected to undergo an SN2
type nucleophilic ring cleavage reaction at the active site
of CPAby the attack of the carboxylate of Glu-270 to
result in a covalent bond formation, tethering the inhi-
bitor to the Glu-270 carboxylate (Fig. 2). The CPA
whose catalytic carboxylate is covalently modi®ed can
no longer perform its catalytic function and becomes
inactivated.
It has been known for a long time that aziridinium ions
15À17
undergo alkylation reactions with DNAbases.
Nitrogen mustard anticancer agents such as mechlor-
ethamine (bis-dichloroethylmethylamine) cross link
DNAvia aziridinium intermediates that are generated
under physiological conditions. Thus, inhibitor 2 was
thought to be generated at the active site of CPAfrom
N-(2-chloroethyl)-N-methylphenylalanine (3). Being a
derivative of Phe having the terminal carboxylate and a
hydrophobic benzyl group at the position a to the car-
boxylate, 3 would satisfy the structural requirements for
it to bind CPAand forms a Michaelis complex. We
envisioned that the chloro group of the CPAbound 3
may rest within the coordination sphere of the active
site zinc ion, and its leaving ability in the SN2 type
cyclization reaction would be enhanced, facilitating the
formation of the aziridinium ion.5,8,9 Thus the potential
inhibitor (2) would be generated at the active site of
CPA.
Figure 1. Schematic representation of the active site of CPA.
The designed inhibitor, 3, was prepared in an optically
pure form following the route outlined in Scheme 1
starting with (S)-phenylalanine methyl ester. Reductive
amination of the starting material with benzaldehyde
a€orded the corresponding N-benzyl derivative, (S)-4,
which was allowed to react with iodomethane to give
(S)-5. Hydrogenolysis of (S)-5 in the presence of Pd/C
furnished (S)-6. Treatment of (S)-6 with ethylene oxide
in acetonitrile by the method reported by Chini et al.
gave (S)-7 in 85% yield.18 The hydroxyl group in (S)-7
was converted into chloro group by allowing (S)-7 to
react with thionyl chloride, giving (S)-8, which was then
treated with concentrated hydrochloric acid to a€ord
the desired compound, (S)-3, as a hydrochloride salt.
(R)-3 was synthesized in a parallel route starting with
(R)-phenylalanine methyl ester.
Figure 2. (a) The oxirane ring in the CPAbound 2-benzyl-3,4-epoxy-
butanoic acid (1, BEBA) undergoes a ring cleavage reaction initiated
by the nucleophilic attack of the carboxylate of Glu-270, resulting in
the covalent modi®cation of the carboxylate. (b) Compound 2 is
expected to covalently modify the Glu-270 carboxylate in a similar
fashion.
Scheme 1. (a) Benzaldehyde, sodium cyanoborohydride (0.7 equiv), Et3N, MeOH, rt, 2 h, 83%; (b) iodomethane, K2CO3, DMF, rt, 7 h, 81%;
(c) H2, Pd/C, MeOH, rt, 5 h, 95%; (d) ethylene oxide, sodium perchlorate, MeCN, 35 ꢀC, 8 h, 85%; (e) SOCl2, CHCl3, rt, 12 h, 95%; (f) concd HCl,
re¯ux, 90%.

J. D. Park et al. / Bioorg. Med. Chem. 9 (2001) 237±243
239
Both enantiomeric forms of 3 inhibited CPAin a time-
dependent fashion as shown in Figure 3 to suggest that
they inhibit the enzyme in an irreversible manner. A
protection of the CPAinhibition by 3 was observed
when CPAwas preincubated with benzylsuccinate, a
known active site directed competitive inhibitor of
CPA,19 indicating that the inactivation chemistry takes
place at the active site (Fig. 4). The irreversibility of the
CPAinhibition by 3 was ascertained by the dialysis
experiment: extensive dialysis over 2 days of the inacti-
vated CPAagainst the kinetic medium failed to regen-
erate the enzymic activity. It can be concluded from the
above kinetic and dialysis studies that the inhibitors
bind the enzyme at the active site to form a covalent
bond with the catalytic carboxylate of Glu-270 as
expected from the design rationale.
inhibitor and E-I0 is covalently modi®ed CPAby the
inhibitor. In the inactivation assay, if the inhibitor con-
centration is suciently greater than the total con-
centration of the enzyme, the observed ®rst-order rate
constant (kobs) is related to the total inhibitor con-
centration ([I]o) by Eq. (2), in which KI represents the
.
dissociation constant of the E I complex.
1
1
KI 1
ˆ
‡
ꢀ2†
kobs kinact kinact ‰IŠ
o
Eq. (2) may be reduced to Eq. (3) provided the KI value
is much greater than the inhibitor concentration to
show that the second-order inhibitory rate constant can
be expressed by kobs/[I]o.
The inactivation reaction of CPAby 3 may be repre-
.
sented by Eq. (1), in which E I represents the Michaelis
complex of the enzyme formed with the
kinact kobs
ˆ
ꢀ3†
KI
‰IŠ
o
E ‡ I ˆ EÁI ! E-I0
ꢀ1†
The reversible binding anity of 3 to CPAwas esti-
mated by the method of Dixon (Fig. 5) and values are
listed in Table 1 together with the second-order inhibi-
tory rate constants calculated using Eq. (3).
In contrast to nitrogen mustards, which undergo rapid
cyclization in aqueous medium,20 compound 3 was
shown to be fairly stable in the enzyme kinetic medium
when examined by NMR spectroscopy. The chemical
shift of the methyl group shown at 3.03 ppm remained
unchanged for 24 h. The inertness of 3 in the aqueous
Table 1. Kinetic constants for inhibition of carboxypeptidase A
Inhibitor
Ki (mM)
kobs/[I]o (MÀ1 sÀ1
)
(S)-3
(R)-3
rac-11
2.5
0.36
1.6
0.864
1.26
±
Figure 3. The plot of remaining CPAactivity versus time in the
presence of (S)-3 or (R)-3 to show that the inhibition occurs in a
time-dependent manner.
Figure 4. The inhibition of CPAby compound 3 is impeded by 2-
benzylsuccinic acid (BSA), an active site directed inhibitor for CPA.
Figure 5. The Dixon plot for the reversible inhibition of CPAby
compound 3.

240
J. D. Park et al. / Bioorg. Med. Chem. 9 (2001) 237±243
solution appears to be due to 3 existing mostly in the
zwitterionic form with the carboxylate. Nonetheless, a
minute amount of the nonzwitterionic form present in
equilibrium with the zwitterionic form would bind CPA
at the active site and undergoes a covalent bond forma-
tion reaction. The alkylation of DNAbases by azir-
idinium intermediates that are generated in situ from
nitrogen mustards is thought to proceed through an SN1
type reaction.21 However, it is highly unlikely that the
nucleophilic aziridinium ring cleavage at the active site
of CPAoccurs by the same mode as the alkylaton of
DNAbases because the formation of primary carboca-
tion from the aziridinium ion in a nonpolar environ-
ment such as at the active site of an enzyme is thought
to be a highly energetic process.
active site zinc ion that is liganded by Glu-72, His-69,
and His-196 is rested near the core of the enzyme.22
Inhibitor (R)-3 binds the active site of CPAwith its
chloroethylamino moiety being projected towards the
inside of the enzyme. Thus, the chloro group may pos-
sibly be positioned within the coordination sphere of the
zinc ion, and as a result the aziridinium ion formation
reaction is accelerated. On the other hand, the corre-
sponding chloro group in (S)-3 may not be rested in the
region where the chloro group can interact with the
active site zinc ion, thus aziridinium ion formation is
relatively slow. The overall result is that the inactivation
of CPAby ( S)-3 is slower compared with that by (R)-3.
The formation of the cyclic aziridinium ion is thought to
be the slowest step in the inactivation pathway. Figure 6
depicts schematically the di€erence in binding mode
between (S)- and (R)-3 to CPAand the suggested
inactivation pathways.
It is surprising to note that (R)-3, which belongs to the
d-series, is more e€ective as a CPAinactiator than its
enantiomer, i.e. (S)-3. This observation may possibly be
rationalized as follows: the X-ray crystal structures of
native and inhibitor bound CPAs revealed that the
2-Benzyl-5-chloropentanoic acid (11) was prepared in
order to verify that 3 is ®rst converted by the enzyme to
the chemically reactive aziridinium derivative, 2, that
interacts with a nucleophile present at the active site.
That is, 3 is a mechanism-based inactivator of CPA.
Compound 11 was synthesized starting with 5-chloro-
valeronitrile, which was benzylated in the presence of
LDAto give 10. Treatment of 10 with concentrated
hydrochloric acid under re¯ux conditions a€orded 11 as
a racemate (Scheme 2). We found that 11 does not
function as an irreversible inhibitor for CPAbut serves
merely as a poor competitive inhibitor for the enzyme
with a Ki value of 1.6 mM, which is in accord with the
design rationale. Apparently, 11 does not inactivate
CPAbecause it cannot generate the aziridinium ring
that is thought to play a critical role in the inactivation
of CPAby 3.
Conclusion
We have designed N-(2-chloroethyl)-N-methylphenyl-
alanine (3) as a novel class of mechanism-based inacti-
vator for CPA. The inhibitor is designed to generate a
chemically reactive intermediate 2 at the active site of
CPAupon binding to the enzyme. The nucleophilic ring
cleavage by the attack of the Glu-270 carboxylate on the
aziridinium moiety in the CPAbound 2 would result in
tethering of the inhibitor to the catalytic site of the
enzyme, which deprives the catalytic activity of the
enzyme. Interestingly, (R)-3 that belongs to the D-series
is more potent than its enantiomer.
Figure 6. (a) The CPAbound ( R)-3 undergoes the aziridinium ion
formation readily because the chloro group is coordinated to the
active site zinc ion. The aziridinium moiety thus formed reacts with the
carboxylate of Glu-270 to result in the covalent modi®cation of the
carboxylate. (b) The same inactivation reaction may also occur at the
active site of CPAwhen CPAis treated with ( S)-3 but at reduced rate
because the aziridinium ion formation is not facilitated by the active
site zinc ion.
Experimental
Melting points were taken on a Thomas-Hoover capil-
1
lary melting point apparatus and are 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
Bruker Equinox 55 FT-IR spectrometer. Mass spectra
were obtained with a Micro Mass Platform II 8410E
spectrometer. Silica gel 60 (230±400 mesh) was used for
Scheme 2. (a) LDA(1 equiv), benzyl bromide (1 equiv), THF, 0 ꢀC,
6 h, 54%; (b) concd HCl, re¯ux, 12 h, 46%.

J. D. Park et al. / Bioorg. Med. Chem. 9 (2001) 237±243
241
¯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 Center for Biofunctional Molecules,
Pohang University of Science and Technology, Pohang,
Korea.
standing. Mp 75±76 ꢀC; [a]D=+24.9ꢀ (c 0.3, CHCl3);
IR (neat) 1744 cmÀ1; 1H NMR 300 MHz (CDCl3) d 2.35
(s, 3H), 2.93±2.96 (d, 2H), 3.42±3.47 (t, 1H), 3.66 (s, 3H),
7.15±7.31 (m, 5H); 13C NMR 300 MHz (CDCl3) d 34.8,
39.6, 52.1, 64.7, 127.2, 128.9, 129.5, 137.3, 174.8.
(R)-N-Methylphenylalanine methyl ester ((R)-6). This
compound was synthesized from (R)-6 in a manner
analogous to that used for its enantiomer. [a]D=À25.1ꢀ
(c 1.0, CHCl3).
(S)-N-Benzylphenylalanine methyl ester ((S)-4). Amix-
ture of (S)-phenylalanine methyl ester (10 g, 46.4 mmol),
benzaldehyde (5.4 mL, 51.4 mmol), triethylamine
(6.5 mL, 46.4 mmol) and sodium cyanoborohydride
(2.04 g, 32.5 mmol) in methanol (140 mL) was stirred at
room temperature for 2 h. Methanol was removed and
the residue was diluted with ethyl acetate, washed with
brine (50 mLÂ3), and dried over anhydrous MgSO4.
The solution was concentrated, and the crude product
was puri®ed by column chromatography (EA/n-Hex=
1/1) to give the product as a colorless oil (10.4 g,
83.2%). [a]D=À7.0ꢀ (c 0.6, CHCl3); IR (neat)
(S)-N-(2-Hydroxyethyl)-N-methylphenylalanine methyl
ester ((S)-7). Amixture of ( S)-6 (2.0 g, 10.3 mmol) and
sodium perchlorate (1.27 g, 10.3 mmol) in dried aceto-
nitrile (5 mL) was stirred until a solution was obtained.
To the solution was introduced an excess of epoxide
within 2 h at room temperature using a gas dispersing
tube. The mixture was stirred for 3 h at 35±40 ꢀC, then
diluted with water, and extracted with ethyl acetate. The
combined extracts were dried over anhydrous MgSO4.
Concentration of the organic phase gave the product as
a pale yellow oil (2.2 g, 90.0%). [a]D=À41.6ꢀ (c 0.5,
1
1733 cmÀ1; H NMR 300 MHz (CDCl3) d 2.99±3.02 (d,
2H), 3.56±3.60 (t, 1H), 3.68 (s, 3H), 3.74±3.87 (dd, 2H),
7.19±7.39 (m, 10H); 13C NMR 300 MHz (CDCl3) d
40.2, 52.0, 52.4, 62.5, 127.1, 127.4, 127.9, 128.8, 128.9,
129.4, 137.7, 140.0, 175.4.
CHCl3); IR (neat) 1732 cmÀ1, 3441 cmÀ1 1H NMR
;
300 MHz (CDCl3) d 2.36 (s, 3H), 2.71±2.80 (m, 2H),
2.91±3.13 (m, 2H), 3.43±3.48 (m, 2H), 3.53±3.56 (t, 1H),
3.68 (s, 3H), 7.19±7.33 (m, 5H); 13C NMR 300 MHz
(CDCl3) d 35.9, 37.1, 51.7, 57.0, 58.6, 68.4, 127.0, 128.9,
129.3, 138.7, 172.7.
(R)-N-Benzylphenylalanine methyl ester ((R)-4). This
compound was synthesized from (R)-phenylalanine
methyl ester in a manner analogous to that used for its
enantiomer. [a]D=+8.1ꢀ (c 1.0, CHCl3).
(R)-N-(2-Hydroxyethyl)-N-methylphenylalanine methyl
ester ((R)-7). This compound was synthesized from (R)-
6 in a manner analogous to that used for its enantiomer.
[a]D=À40.5ꢀ (c 1.0, CHCl3).
(S)-N-Benzyl-N-methylphenylalanine methyl ester ((S)-
5). Amixture of ( S)-4 (8.8 g, 32.7 mmol), potassium
carbonate (9.0 g, 65.2 mmol), and methyl iodide
(4.06 mL, 65.2 mmol) in dimethylformamide (100 mL)
was stirred at room temperature for 7 h. The residue
was diluted with ethyl acetate, washed successively with
water, 5% sodium thiosulfate (50 mLÂ3) and brine
(50 mLÂ3), and dried over anhydrous MgSO4. The
solution was concentrated, and the crude product was
puri®ed by column chromatography (EA/n-Hex=1/4)
to give the product as a colorless oil (7.4 g, 80.3%).
(S)-N-(2-Chloroethyl)-N-methylphenylalanine methyl ester
((S)-8). Amixture of ( S)-7 (1.36 g, 5.73 mmol) and
thionyl chloride (0.84 mL, 11.5 mmol) in chloroform
(25 mL) was stirred at room temperature for 12 h. The
solution was evaporated and the residue was recrys-
tallized from ether and methanol to give a white powder
(1.57 g, 93.5%). Mp 109±110 ꢀC; [a]D=À46.9ꢀ (c 0.5,
MeOH); IR (neat) 1744 cmÀ1 1H NMR 300 MHz
;
(D2O) d 2.92 (s, 3H), 3.15±3.35 (m, 2H), 3.50±3.68 (m,
2H), 3.55 (s, 3H), 3.80±3.83 (m, 2H), 4.40±4.66 (m, 1H),
7.16±7.28 (m, 5H); 13C NMR 300 MHz (D2O) d 33.1,
37.8, 39.3, 54.1, 56.4, 68.3, 126.3, 128.4, 129.6, 134.2,
168.7.
[a]D=À81.6ꢀ (c 0.5, CHCl3); IR (neat) 1732 cmÀ1; H
1
NMR 300 MHz (CDCl3) d 2.31 (s, 3H), 2.98±3.10 (m,
2H), 3.57±3.59 (t, 2H), 3.67 (s, 3H), 3.77±3.82 (d, 1H),
7.15±7.27 (m, 10H); 13C NMR 300 MHz (CDCl3) d
36.2, 38.3, 51.5, 59.2, 67.7, 126.7, 128.7, 129.1, 129.7,
138.8, 139.6, 172.7.
(R)-N-(2-Chloroethyl)-N-methylphenylalanine
methyl
ester ((R)-8). This compound was synthesized from (R)-
7 in a manner analogous to that used for its enantiomer.
[a]D=+46.8ꢀ (c 0.4, MeOH).
(R)-N-Benzyl-N-methylphenylalanine methyl ester ((R)-
5). This compound was synthesized from (R)-4 in a
manner analogous to that used for its enantiomer.
[a]D=+90.8ꢀ (c 0.5, CHCl3).
(S)-N-(2-Chloroethyl)-N-methylphenylalanine ((S)-3). A
mixture of (S)-8 (0.22 g, 0.75 mmol) and concd HCl
(5 mL) was re¯uxed for 1 h, then evaporated under
reduced pressure. The residue was recrystallized from
ether and ethanol to give a white powder (quantitative).
Mp 142±143 ꢀC; [a]D=+23.4ꢀ (c 1.0, MeOH); IR (neat)
(S)-N-Methylphenylalanine methyl ester ((S)-6). Amix-
ture of (S)-5 (3.77 g, 13.3 mmol) and 20% acetic acid
(3 mL) in methanol (50 mL) was hydrogenated using
Pd/C at atmospheric pressure for 5 h. The mixture was
®ltered, and the ®ltrate was concentrated and partitioned
between ethyl acetate and sodium carbonate solution.
Concentration of organic phase gave the product as a
pale yellow oil (quantitative) which solidi®ed on
1
1731 cmÀ1; H NMR 300 MHz (D2O) d 2.90 (s, 3H),
3.16±3.25 (m, 2H), 3.47±3.61 (m, 2H), 3.80±3.84 (t, 2H),
4.15 (m, 1H), 7.21±7.29 (m, 5H); 13C NMR 300 MHz
(D2O) d 33.2, 37.9, 39.1, 56.1, 69.2, 128.2, 129.5, 129.6,

242
J. D. Park et al. / Bioorg. Med. Chem. 9 (2001) 237±243
.
134.8, 170.4. Anal. calcd for C12H17Cl2NO2 1/4H2O: C,
51.0; H, 6.24; N, 4.96. Found: C, 51.25; H, 6.26; N, 4.94.
pH 7.5 bu€er (1 mL cuvette), and the change in absor-
bance at 254 nm was measured immediately. Initial
velocities were then 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 (Fig. 5).
(R)-N-(2-Chloroethyl)-N-methylphenylalanine
((R)-3).
This compound was synthesized from (R)-8 in a man-
ner analogous to that used for its enantiomer.
[a]D=À21.7ꢀ (c 1.0, MeOH).
2-Benzyl-5-chlorovaleronitrile (10). To an ice chilled
solution of diisopropylamine (2.3 mL, 17.8 mmol) in
dried THF (100 mL) was added slowly n-butyllithium
(7.1 mL of 2.5 M solution in n-hexane, 17.8 mmol)
under nitrogen gas. After the mixture was stirred for
30 min, 9 (2.0 mL, 17.8 mmol) in dried THF (20 mL)
was added dropwise over a period of 20 min. The
resulting solution was stirred for 40 min, and then ben-
zyl bromide (2.1 mL, 17.8 mmol) was added dropwise.
The reaction mixture was stirred for 6 h. The reaction
was quenched by the addition of saturated solution of
ammonium chloride (50 mL). The organic layer was
separated and the aqueous layer was washed with ethyl
acetate (3Â20 mL). The combined organic layers were
washed with brine, dried over anhydrous MgSO4, and
evaporated to dryness in vacuo. The crude product was
puri®ed by chromatography (EA/n-Hex=1/8) to give
Determination of kobs
Into a 1 mL cuvette were added 850 mL of bu€er solu-
tion (0.05 M Tris/0.5 M NaCl, pH 7.5), 100 mL of 5 mM
solution of Hipp-l-Phe in the same bu€er, and 20 mL of
10 mM stock solution of inactivator in the same bu€er.
To this cuvette was added 30 mL of 13.7 mM solution of
CPAin the Tris bu€er, and the change in absorbance at
254 nm was recorded over a time interval of 0±240 min.
The values of kobs were calculated from the progress
curves using the computer assisted spectrophotometer.
Active site protection test
To demonstrate that the inactivation is active site
directed, a competitive experiment with benzylsuccinic
acid, a well known competitive CPAinhibitor, was car-
ried out. To a preincubated solution of 2 mM benzyl-
succinic acid and 1 mM enzyme in 0.05 M Tris/0.5 M
NaCl, pH 7.5 bu€er for 10 min was added an inacti-
vator and the whole mixture was incubated at room
temperature. At some intervals 50 mL samples of the
inactivation mixture were removed and added to a
950 mM assay mixture and the activity was monitored at
254 nm. From the data Figure 4 was obtained.
the product as an oil (2.0 g, 54%). IR (neat) 2239 cmÀ1
;
1H NMR 300 MHz (CDCl3) d 1.76 (m, 2H), 1.99 (m,
2H), 2.82 (m, 1H), 2.91 (m, 2H), 3.55 (t, 2H), 7.30 (m,
5H); 13C NMR 300 MHz (CDCl3) d 29.5, 30.3, 33.7,
38.8, 44.4, 121.7, 127.8, 129.2, 129.4, 137.0.
2-Benzyl-5-chloropentanoic acid (11). Amixture of 10
(1.0 g, 4.8 mmol) and concd HCl (15 mL) was re¯uxed for
12 h, then evaporated under reduced pressure. The crude
product was puri®ed by chromatography (EA/n-Hex=
1/2) to give the product as an oil (0.5 g, 46%). IR (neat)
Dialysis
1
1705 cmÀ1; H NMR 300 MHz (CDCl3) d 1.73 (m, 2H),
1.81 (m, 2H), 2.72 (m, 1H), 3.00 (dd, 1H), 3.50 (t, 2H),
7.22 (m, 5H); 13C NMR 300 MHz (CDCl3) d 29.3, 30.6,
38.5, 44.9, 47.1, 127.0, 128.9, 129.3, 139.0, 181.7;
HRMS (FAB+) calcd for (C12H15O2Cl, Na+):
249.0658; found: 249.0658.
Solutions of 5±20 mM inactivator and 1 mM CPAin
0.05 M Tris/0.5 M NaCl, pH 7.5 bu€er were incubated
at 4 ꢀC for 48 h. The mixture was then dialyzed for 24 h
at room temperature against 0.05 M Tris/0.5 M NaCl,
pH 7.5 bu€er. The bu€er was changed every 6 h. The
enzyme failed to show the proteolytic activity.
General remarks for kineticexperiments
All solutions were prepared by dissolving in doubly
distilled and deionized water. Stock assay solutions were
®ltered before use. Carboxypeptidase Awas purchased
from Sigma Chemical Co. (Allan form, twice crystal-
lized from bovine pancrease, aqueous suspension in tol-
uene) and used without further puri®cation. CPAstock
solutions were prepared by dissolving the enzyme in
0.05 M Tris/0.5 M NaCl, pH 7.5 bu€er solution. Hip-
puryl-l-phenylalanine (Hipp-l-Phe) purchased from
Sigma Chemical Co. was used as substrate for CPAand
the decrease in the absorbance at 254 nm was followed
at 25 ꢀC. APerkin±Elmer HP 8453 UV/VIS spectro-
meter was used in enzyme inhibition studies.
Acknowledgement
The authors thank the Korea Science and Engineering
Foundation for the generous support of this work.
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