Mechanistic insight into the inactivation of carboxypeptidase A by α-benzyl-2-oxo-1,3-oxazolidine-4-acetic acid, a novel type of irreversible inhibitor for carboxypeptidase A with no stereospecificity

Article abstract of DOI:10.1021/jo010421e

On the basis of the active site topology and enzymic catalytic mechanism of carboxypeptidase A (CPA), a prototypical zinc-containing proteolytic enzyme, α-benzyl-2-oxo-1,3-oxazolidine-4-acetic acid (1), was designed as a novel type of mechanism-based inactivator of the enzyme. All four possible stereoisomers of the inhibitor were synthesized in an enantiomerically pure form starting with optically active aspartic acid, and their CPA inhibitory activities were evaluated to find that surprisingly all of the four stereoisomers inhibit CPA in a time dependent manner. The inhibited enzyme did not regain its enzymic activity upon dialysis. The inactivations were prevented by 2-benzylsuccinic acid, a competitive inhibitor that is known to bind the active site of the enzyme. These kinetic results strongly support that the inactivators attach covalently to the enzyme at the active site. The analysis of ESI mass spectral data of the inactivated CPA ascertained the conclusion from the kinetic results. The values of second-order inhibitory rate constants (k_obs/[I]_o) fall in the range of 1.7-3.6 M^-1 min^-1. The lack of stereospecificity shown in the inactivation led us to propose that the ring cleavage occurs by the nucleophilic attack at the 2-position rather than at the 5-position and the ring opening takes place in an addition-elimination mechanism. The tetrahedral transition state that would be generated in this pathway is thought to be stabilized by the active site zinc ion, which was supported by the PM3 semiemprical calculations. In addition, a-benzyl-2-oxo-1,3-oxazolidine-5-acetic acid (18), a structural isomer of i was also found to inactivate CPA in an irreversible manner, reinforcing the nucleophilic addition-elimination mechanism. The present study demonstrates that the transition state for the inactivation pathway plays a critical role in determining stereochemistry of the inactivation.

Full text of DOI:10.1021/jo010421e

6462
J . Org. Chem. 2001, 66, 6462-6471
Mech a n istic In sigh t in to th e In a ctiva tion of Ca r boxyp ep tid a se A
by r-Ben zyl-2-oxo-1,3-oxa zolid in e-4-a cetic Acid , a Novel Typ e of
Ir r ever sible In h ibitor for Ca r boxyp ep tid a se A w ith No
Ster eosp ecificity
Sang J . Chung, Suhman Chung, Hyun Soo Lee, Eun-J ung Kim, Kyung Seok Oh,
Hyuk Soon Choi, Kwang S. Kim, Yeoun J in Kim, J ong Hoon Hahn, and Dong H. Kim*
Center for Biofunctional Molecules and Division of Molecular and Life Sciences, Pohang University of
Science and Technology, San 31 Hyojadong, Pohang 790-784, Korea
dhkim@ postech.ac.kr
Received April 23, 2001
On the basis of the active site topology and enzymic catalytic mechanism of carboxypeptidase A
(CPA), a prototypical zinc-containing proteolytic enzyme, R-benzyl-2-oxo-1,3-oxazolidine-4-acetic
acid (1), was designed as a novel type of mechanism-based inactivator of the enzyme. All four possible
stereoisomers of the inhibitor were synthesized in an enantiomerically pure form starting with
optically active aspartic acid, and their CPA inhibitory activities were evaluated to find that
surprisingly all of the four stereoisomers inhibit CPA in a time dependent manner. The inhibited
enzyme did not regain its enzymic activity upon dialysis. The inactivations were prevented by
2-benzylsuccinic acid, a competitive inhibitor that is known to bind the active site of the enzyme.
These kinetic results strongly support that the inactivators attach covalently to the enzyme at the
active site. The analysis of ESI mass spectral data of the inactivated CPA ascertained the conclusion
from the kinetic results. The values of second-order inhibitory rate constants (kobs/[I]o) fall in the
range of 1.7-3.6 M-1 min-1. The lack of stereospecificity shown in the inactivation led us to propose
that the ring cleavage occurs by the nucleophilic attack at the 2-position rather than at the 5-position
and the ring opening takes place in an addition-elimination mechanism. The tetrahedral transition
state that would be generated in this pathway is thought to be stabilized by the active site zinc
ion, which was supported by the PM3 semiemprical calculations. In addition, R-benzyl-2-oxo-1,3-
oxazolidine-5-acetic acid (18), a structural isomer of 1 was also found to inactivate CPA in an
irreversible manner, reinforcing the nucleophilic addition-elimination mechanism. The present
study demonstrates that the transition state for the inactivation pathway plays a critical role in
determining stereochemistry of the inactivation.
In tr od u ction
of CPA. The enzyme inhibitor design principles developed
using CPA as a model enzyme6 are of importance because
the principles may be transferred to the zinc proteases
of medicinal interest, leading to generation of lead
compounds for new drug development.7 We wish to report
herein a novel inhibitor design concept that exploits the
Carboxypeptidase A (CPA, EC 3.4.17.1) is a prototypi-
cal zinc-containing proteolytic enzyme that cleaves off the
C-terminal amino acid residue having a hydrophobic side
chain.1 The enzyme also catalyzes the hydrolysis of esters
having structural feature similar to that of peptide
substrate.1 The X-ray crystal structure of the enzyme has
been known to the resolution of 1.54 Å.2 There are
numerous zinc proteases of phathological importance
such as angiotensin-converting enzyme,3 enkephalinase,4
and matrix metalloproteases5 whose active site structures
and catalytic mechanisms are thought to resemble those
(6) For example: (a) Ner, S. K.; Suckling, C. J .; Bell, A. R.;
Wriggleswoarth, R. J . Chem. Soc. Chem. Commun. 1987, 480-482.
(b) Kemp, A.; Tedford, M. C.; Suckling, C. J . Bioorg. Med. Chem. Lett.
1991, 1, 557-562. (c) Kemp, A.; Ner, S. K.; Rees, L.; Suckling, C. J .;
Tedford, M. C.; Bell, A. R.; Wrigglesworth, R. J . Chem. Soc., Perkin
Trans. 2 1993, 741-748. (d) Husbands, S.; Suckling, C. A.; Suckling,
C. J . Tetrahedron 1994, 50, 9729-9742. (e) Mobashery, S.; Ghosh, S.
S.; Tamura, Y.; Kaiser, E. T. Proc. Natl. Acad. Sci. U.S.A. 1990, 87,
1578-1582. (f) Tanaka, Y.; Grapsas, I.; Dakoji, S.; Cho, Y. J .;
Mobashery, S. J . Am. Chem. Soc. 1994, 116, 7475-7480. (g) Massova,
I.; Martin, P.; de Mel, S.; Tanaka, Y.; Edwards, B.; Mobashery, S. J .
Am. Chem. Soc. 1996, 118, 12479-12480. (h) Lee, K. J .; Kim, D. H.
Bioorg. Med. Chem. Lett. 1996, 6, 2431-2436. (i) Lee, K. J .; Kim, D.
H. Bioorg. Med. Chem. Lett. 1996, 6, 2431-2436. (j) Kim, D. H.; Lee,
K. J . Bioorg. Med. Chem. Lett. 1997, 7, 2607-2612. (k) Yun, M.; Park,
C.; Kim, S.; Nam, D.; Kim, S. C.; Kim, D. H. J . Am. Chem Soc. 1992,
114, 2281-2282. (l) Ryu, S.-E.; Choi, H.-J .; Kim, D. H. J . Am. Chem
Soc. 1997, 119, 38-41.
(1) (a) Hartsuck, J . A.; Lipscomb, W. N. In The Enzymes, 3rd ed.;
Boyer, P., Ed.; Academic: New York, 1971; Vol. 3, pp 1-56. (b)
Christianson, D. W.; Lipscomb, W. N. Acc. Chem. Res. 1989, 22, 62-
69.
(2) Rees, D. C.; Lewis, M.; Lipscomb, W. N. J . Mol. Biol. 1983, 168,
367-387.
(3) (a) Erdo¨s, E. G. Circul. Res. 1975, 36, 247-255. (b) Bu¨nning, P.;
Holmquist, B.; Riordan, J . F. In Biological Functions of Proteinases;
Hotzer, Tachesche Eds.; Springer-Verlag: Heidelberg, 1979; pp 269-
275. (c) Cushman, D. W.; Cheung, H. S.; Sabo, E. F.; Ondetti, M. A.
Biochemistry 1977, 16, 5484-5491.
(7) For example, (a) Ondetti, M. A.; Rubin, B.; Cushman, D. W.
Science 1977, 196, 441-444. (b) Chorev, M.; Shavitz, R.; Goodman,
M.; Minick, S.; Guillemen, R. Science 1979, 204, 1210-1212. (c) Kim,
D. H.; Guinosso, C. J .; Buzby, G. C., J r.; Herbst, D. R.; McCaully, R. J .
J . Med. Chem. 1983, 26, 394-403. (d) Gaffard, J . T.; Skidgel, R. A.;
Erdos, E. G.; Hersh, L. B. Biochemistry 1983, 22, 3265-3271.
(4) Grafford, J . T.; Skidgel, R. A.; Erdo¨s, E. G.; Hersh, L. B.
Biochemistry 1983, 22, 3265-3271.
(5) Whittaker, M.; Floyd, C. D.; Brown, P.; Gearing, A. J . H. Chem.
Rev. 1999, 99, 2735-2776.
10.1021/jo010421e CCC: $20.00 © 2001 American Chemical Society
Published on Web 08/22/2001

Inactivation of Carboxypeptidase A
J . Org. Chem., Vol. 66, No. 19, 2001 6463
unique ring cleavage reaction of oxazolidinone hetero-
cycle, and synthesis of optically active R-benzyl-2-oxo-
1,3-oxazolidine-4-acetic acid as a CPA inactivator.8 Sur-
prisingly, all four possible stereoisomers of the inhibitor
were found to inactivate irreversiblly the enzyme with
comparable potency. The lack of stereospecificity shown
in the inactivation has been probed to envision that the
inactivation reaction occurs via the nucleophilic addi-
tion-elimination mechanism initiated by the attack of
the carboxylate of Glu-270 at the 2-position of the
oxazolidinone in the enzyme bound inhibitor.
Resu lts a n d Discu ssion
The active site of CPA has been well characterized.1
The most important residues at the active site are Glu-
270 and Arg-145. The former is directly involved in the
catalytic hydrolysis of substrate, and the latter forms
hydrogen bonds with the C-terminal carboxylate of
substrate. In addition, there is present a hydrophobic
pocket the primary function of which is to recognize
substrate by accommodating the aromatic side chain in
the P1′ residue of substrate. The catalytically essential
zinc ion is held by His-69, Glu-72, His-195, and a
molecule of water.
In the CPA-catalyzed hydrolysis of ester substrate, the
scissile bond of the substrate becomes activated by the
coordination of its carbonyl oxygen to the zinc ion at the
active site of the enzyme. The activated carbonyl carbon
is subjected to a nucleophilic attack by the carboxylate
of Glu-270 to form an anhydride intermediate that
hydrolyzes to products with regeneration of the enzyme.
This pathway is referred to as the anhydride mecha-
nism.9 There has also been proposed an alternative
pathway in which the carboxylate serves as a general
base, activating the zinc bound water molecule which,
in turn, attacks at the activated carbonyl carbon of the
substrate to generate a tetrahedral transition state.10 It
is generally believed that the latter mechanism is in
operation for the hydrolysis of peptide substrate.
F igu r e 1. Rationale for designing 1 as a mechanism-based
inactivator of CPA.
the cleavage of oxazolidinone also occurs by the treatment
of carboxylic acids, generating esters of 2-aminoethanol.13
These observations suggest that under the acidic condi-
tions the 5-position of the ring becomes an electrophilic
center presumably as a result of the protonation on the
carbamoyl moiety in the ring. We envisioned that a
metallic Lewis acid such as the zinc ion present at the
active site of CPA might also be able to activate oxazo-
lidinone for a nucleophile attack.
On the basis of the topology of the active site of CPA,
the proposed catalytic mechanism, and the unique prop-
erty of oxazolidinone, we have designed R-benzyl-2-oxo-
1,3-oxazolidine-4-acetic acid (1) as an enzyme-activated
irreversible inhibitor for CPA.
Oxazolidinones are relatively stable chemical entities
under ordinary conditions as evidenced by the fact that
the heterocycle constitutes the molecular framework of
most widely used chiral auxiliary in asymmetric synthe-
sis of chiral organic compounds.11 Nevertheless, the
heterocycle reportedly undergoes ring cleavage reac-
tion under certain conditions: As early as in 1885,
Nemirowsky reported that treatment of oxazolidinone
with hydrochloric acid affords 2-chloroethylamine as a
hydrochloride salt.12 Recently, Hsieh documented that
Due to its structural similarity to substrates of CPA,
1 is expected to bind the enzyme to form a Michaelis
complex. In the complex, the carboxylate group of the
inhibitor would form hydrogen bonds to the guanidinium
moiety of Arg-145 and the phenyl ring anchors in the
hydrophobic S1′ pocket of the enzyme (Figure 1).14 This
binding mode was thought to pose the oxazolidinone
carbonyl oxygen of the inhibitor in close proximity to the
active site zinc ion to form a coordinative bond with the
zinc ion, resulting in the activation of the 5-position for
a nucleophilic attack. An SN2-type nucleophilic attack by
the carboxylate of Glu-270 at the activated center may
ensue. The unstable carbamic acid moiety that is gener-
ated in the process is expected to lose carbon dioxide to
form an amino functionality which may undergo an
intramolecular rearrangement, converting the ester link-
age into a thermodynamically more stable amide bond.
In the end, the inhibitor becomes covalently attached to
the carboxylate and impairs the enzymic power of CPA
permanently (Figure 1). To examine stereospecificity
associated with the inhibition, we have synthesized all
(8) A preliminary account of the present work has been published:
Kim, D. H.; Chung, S. J .; Kim, E.-J .; Tian, G. R. Bioorg. Med. Chem.
Lett. 1998, 8, 859-864.
(9) (a) Makinen, M. W.; Kuo, L. C.; Dymowski, J . D.; J affer, S. J . J .
Biol. Chem. 1979, 254, 356-366. (b) Makinen, M. W.; Fukuyama, J .
M.; Kuo, L. C. J . Am. Chem. Soc. 1982, 104, 2667-2669. (c) Sander,
M. E.; Witzel, H. Biochem. Biophys. Res. Commun. 1985, 132, 681-
687. (d) Suh, J .; Park, T. H.; Hwang, B. K. J . Am. Chem. Soc. 1992,
114, 5141-5146. (e) Britt, B. M.; Peticolas, W. L. J . Am. Chem. Soc.
1992, 114, 5295-5303.
(10) (a) Breslow, R.; Schepartz, A. Chem. Lett. 1987, 1-4. (b)
Breslow, R.; Wernick, D. L. Proc. Natl. Acad. Sci. U.S.A. 1977, 107,
1303-1307. (c) Galdes, A.; Auld, D. S.; Vallee, B. L. Biochemistry 1986,
25, 646-651. (d) Kim, H.; Lipscomb, W. N. Biochemistry 1990, 29,
5546-5555.
(11) (a) Seyden-Penne, J . Chiral Auxiliaries and Ligands in Asym-
metric Synthesis, J ohn Wiley: New York, 1995; pp 72-74. (b) Ager,
D. J .; Prakash, I.; Schaad, D. R. Aldrichimica Acta 1997, 30, 3-12.
(12) Nemirowsky, J . J . J . Prakt. Chem. 1885, 31, 179.
(13) Hsieh, H. H. U.S. Patent 1984, 4,444,694.
(14) Kim, D. H.; Kim, K. S.; Park, J . K. Bull. Korean Chem. Soc.
1994, 15, 805-807.

6464 J . Org. Chem., Vol. 66, No. 19, 2001
Chung et al.
Sch em e 1a
Sch em e 2a
a
Reagents, conditions, and (yields): (a) LDA (2.2 equiv), HMPA,
and then BnBr, -78 °C, THF (73%); (b) tBuSAlMe2, CH2Cl2, 0 °C
to rt; (c) MsCl, Et3N, CH2Cl2, 0 to 5 °C (two-step yield: 88%); (d)
pyridine, 70 °C (64%); (e) Hg(OCOCF3)2, dioxane-water, 50 °C
(64%).
a
Reagents, conditions, and (yields): (a) LHMDS (2 equiv),
BnBr, -78 °C (46%); (b) 1 N NaOH, dioxane-water (5:1), rt, 24 h
(90%); (c) isobutyl chloroformate, 1-ethylmorpholine, DME, -10
°C, 5 min, and then NaBH4, -10 °C, 5 min; (d) TsCl, pyridine, rt,
7 d, (two-step yield: 33%); (e) H2/Pd-C (80%).
four possible stereoisomers of the inhibitor in an enan-
tiomerically pure form and evaluated each of them for
the CPA inactivating property.
Synthetic pathway for (RS,4R)-1 is shown in Scheme
1. D-Aspartic acid was converted to N-protected dibenzyl
ester (2a ) which was then treated with benzyl bromide
under Baldwin’s conditions15 to give 3a . Trans-benzylated
product was obtained exclusively. Regioselective hydroly-
sis to yield 4a was achieved by the treatment of the
dioxane solution of 3a with 1 N NaOH solution according
to the method reported by Berger and Katchalski.16 The
acid moiety was then reduced to the corresponding
alcohol using the method described by Rodriguez et al.,17
and the treatment of the N-protected amino alcohol (5a )
thus obtained with tosyl chloride in pyridine afforded
(RS,4R)-1 as a benzyl ester (6a ).18 Hydrogenolysis of the
latter in the presence of Pd/C afforded (RS,4R)-1. In an
analogous fashion, (RR,4S)-1 was synthesized starting
with L-aspartic acid.
Scheme 2 outlines synthetic path for the preparation
of (RS,4S)-1. Lactone 7 obtained from L-aspartaic acid
by the route reported by Yoda et al.19 was benzylated as
described by Takahashi et al.20 to afford exclusively trans-
benzylated product 8. Treatment of 8 with the Weinreb’s
reagent21 and subsequent methanesulfonylation of the
resulted alcohol moiety gave 9 having the erythro con-
figuration. Simple warming of 9 in pyridine produced 10
in 64% yield. Hydrolysis of the thioester moiety in 10 to
give (RS,4S)-1 was achieved by the method described by
Masamune et al.22
F igu r e 2. Plot of - ln(v/vo) vs incubation time, showing time-
dependent loss of CPA activity by (RS,4R)-1 ([, [I]0 ) 30 mM;
O, [I]0 ) 28 mM; 4, [I]0 ) 26 mM; ×, [I]0 ) 24 mM; *, [I]0 )
22mM; b, [I]0 ) 20mM; +, [I] 0 ) 18 mM; ], [I] 0 ) 16 mM; 9,
[I] ) 14 mM; 2, [I]0 ) 0).
0
(Hipp-L-Phe) as substrate. CPA was incubated with an
excess of the inhibitor and the loss of enzymic activity
was monitored at different time intervals. Figure 2 is an
exemplary plot for the inhibition of CPA with (RS,4R)-1.
All four diastereoisomers of 1 inhibited CPA in a time-
dependent manner, suggesting that the inhibitions occur
in an irreversible fashion.23 The irreversible nature of the
inhibitions was ascertained by dialysis experiment: The
inactivated enzyme failed to regain the enzymic activity
after dialysis against the buffered solution for 2 days.
Furthermore, the analysis of the electrospray ionization
(ESI) mass spectrum of the inactivated CPA in compari-
son with that of the native CPA confirmed the conclusion
derived from the kinetic analysis. The native CPA showed
peaks at 34070, 34103, 34127, 34154, 34181 and 34244
Da arising from allotypes of CPA24 and the inactivated
CPA by (RS,4R)-1 at 34268, 34301, 34322, 34346, and
34436 Da. The molecular weight differences (192 Da)
between the two spectra correspond to (CPA + 1) - CPA
- CO2.25
The compounds thus synthesized were assayed for
inhibitory activity against CPA using (S)-hippurylPhe
(15) Baldwin, J . E.; Moloney, M. G.; North, M. Tetrahedron 1989,
45, 6309-6318.
(16) Berger, A.; Katchalski, E. J . Am. Chem. Soc. 1951, 73, 4084-
4088.
(17) Rodriguez, M.; Llinares, M.; Doulut, S.; Heitz, A.; Martinez, J .
Tetrahedron Lett. 1991, 32, 923-926.
(18) Curran, T. P.; Pollastri, M. P.; Abelleira, S. M.; Messier, R. J .;
McCollum, T. A.; Rowe, C. G. Tetrahedron Lett. 1994, 35, 5409-5412.
(19) Yoda, H.; Nakagami, Y.; Tanabe, K. Tetrahedron: Asymmetry
1994, 5, 169-172.
The inactivation reaction of CPA by 1 may be repre-
sented by eq 1, in which E•I represents noncovalently
bound complex of the enzyme with the inhibitor
(20) (a) McGarvey, G. J .; Williams, J . M.; Hiner, R. N.; Matsubara,
Y.; Oh, T. J . Am. Chem. Soc. 1986, 108, 4943-4952. (b) Takahashi,
Y.; Hasegawa, S.; Izawa, T.; Kobayashi, S.; Ohno, M. Chem. Pharm.
Bull. 1986, 34, 3020-3024.
(23) Silvermann, R. B. Mechanism-Based Enzyme Inactivation:
Chemistry and Enzymology; CRC: Boca Raton, 1988; Vol. I, Chapter
1.
(24) (a) Bradshaw, R. A.; Ericsson, L. H.; Walsh, K. A.; Neurath, H.
Proc. Natl. Acad. Sci. U.S.A. 1969, 63, 1389-1394. (b) Pe´tra, P. H.;
Neurath, H. Biochemistry 1969, 8, 2466-2475. (c) Burkrinsky, J . T.;
Bjerrum, J .; MKadziola, A. Biochemistry 1899, 37, 16555-16564.
(21) Hatch, R. P.; Weinreb, S. M. J . Org. Chem. 1977, 42, 3960-
3961.
(22) Masamune, S.; Kamata, S.; Schilling, W. J . Am. Chem. Soc.
1975, 97, 3515-3516.

Inactivation of Carboxypeptidase A
J . Org. Chem., Vol. 66, No. 19, 2001 6465
Ta ble 1. Kin etic P a r a m eter s for CP A In a ctiva tion
E + I ) E•I f E-I′
(1)
a
inhibitor
kobs (min-1
)
kobs/[I]0 (M-1 min-1
)
partition ratio
and E-I′ is covalently modified CPA by the inhibitor. In
the inhibition assay, if the inhibitor concentration is
sufficiently greater than the total concentration of the
enzyme, kobs is related to the total inhibitor concentration
([I]o) by eq 2, in which KI represents the dissociation
constant of the E•I complex, and kinact represents inacti-
vation rate constant.26
(RS,4R)-1
(RR,4S)-1
(RR,4R)-1
(RS,4S)-1
0.0333
0.0453
0.0607
0.0723
1.665
2.265
3.035
3.615
94
120
133
121
a
Apparent first-order rate constant at inhibitor concentration
of 20 mM.
KI
1
1
1
[I]o
)
+
(2)
kobs kinact kinact
Equation 2 may be further reduced to give eq 3, if the
KI value is much greater than the [I]o value,27 showing
that the second-order inhibitory rate constant can be
expressed by kobs/[I]o. The irreversible inhibitory activities
of all four diastereoisomers of 1 toward CPA were
evaluated by the incubation method and their second-
order rate constants expressed in kobs/[I]o are listed in
Table 1. The values of kobs were calculated from plots of
ln v/vo vs incubation time.
F igu r e 3. Active site protection of (RS,4R)-1 caused CPA
inactivation by (R)-2-benzylsuccinic acid (BSA): (×, no inhibi-
tor; 2, (RS,4R)-1 (20 mM) and BSA (20 µM); 9, (RS,4R)-1 (20
µM) and BSA (10 µM); b, (RS,4R)-1 (20 mM).
kinact kobs
)
(3)
2-benzyl-3,4-epoxybutanoic acid30 whose oxirane ring is
cleaved by the SN2 type nucleophilic attack at the
3-position of the ring by the Glu-270 carboxylate. Out of
four stereoisomers of 2-benzyl-3,4-epoxybutanoic acid
only two diastereoisomers having the (2S,3R)- and (2R,3S)-
configurations that meet the stereochemical requirement
for the SN2 type ring cleavage reaction were active.30
We became to realize that the proposed binding mode
of the oxazolidinone moiety in the complex of 1 with CPA
is incongruous with the topology of the CPA active site
crevice. The distance between the zinc ion and the
carboxylate of Glu-270 is estimated to be 4.1-4.6 Å from
the X-ray crystal structure of CPA.2 On the other hand,
the distance between the carbonyl oxygen and a hydrogen
atom at the 5-position of oxazolidinone ring was learned
to be 4.09 Å from the X-ray crystal structure,31 indicating
that the heterocycle is too big to fit in the space between
the carboxylate and the zinc ion with its carbonyl oxygen
forming a coordinative bond to the metal ion. Accordingly,
a likely mode of resting of the heterocycle in the complex
involves that the oxazolidinone ring resides horizontally
between the carboxylate and the zinc ion. In this binding
mode, the nucleophilic attack at the 5-position of the ring
would be improbable. Instead, the carboxylate nucleo-
phile would attack at the 2-position of the heterocycle. A
recent report of Najer et al. is noteworthy in this regard.
They reported that treatment of oxazolidinone with
alkylamine affords N,N′-disubstituted urea,32 indicating
that in the absence of Lewis acid the nucleophilic attack
takes place not at the 5-position but at the 2-position of
the heterocycle and there occurs a ring cleavage by the
addition-elimination mechanism. On the basis of the
foregoing consideration, an alternative CPA inactivation
pathway that can account for the observed lack of
stereospecificity is proposed as depicted in Figure 4. The
KI
[I]o
Poor solubility of these compounds in the kinetic
medium prohibited the determination of the Ki values.
The protection from the inactivation by 2-benzylsuccinic
acid, a potent active site directed reversible inhibitor for
CPA28 was observed for all cases, indicating that the
inactivations take place at the active site (Figure 3). The
partition ratio (kcat/kinact) that reflects the efficiency of an
inactivator was determined for all four inhibitors by the
titration method,29 and these kinetic parameters are
included in Table 1.
Enzyme catalyzes chemical transformations stereospe-
cifically, and the stereospecificity has its origin in the
chiral nature of the enzyme active site where the binding
and chemical transformations of substrate take place. It
was surprising to learn that all four diastereoisomers of
1 inactivate CPA. The lack of stereospecificity shown in
the inactivation of CPA by 1 can hardly be accounted for
in terms of the design rationale. The SN2 type ring
cleavage reaction caused by the attacking at the 5-posi-
tion of the oxazolidinone requires the carboxylate nu-
cleophile to approach the electrophilic center in the
180°direction to the leaving group, and only two out of
the four diastereomers of 1 are thought to satisfy the
requirement, thus leading to inactivation of CPA. This
expectation is inferred from the inactivation of CPA by
(25) Suh and Kaiser reported that (+)-(R)-(trans-cinnamoyl)-R-
mercapto-â-phenylpropinate acylates the phenolic hydroxyly of Tyr-
248 and proposed that the inhibitor binds CPA in an inversed fashion
with its carboxylate binding to the active site zinc ion and the hydroxyl
of Tyr-248 attacks on the thioester moiety (Suh, J .; Kaiser, E. T.
Biochem. Biphys. Res. Commun. 1975, 64, 863-869). An analogous
binding of 1 to CPA may be feasible, but the possibility that the
cleavage of the unactivated oxazolidinone ring by the Tyr hydroxyl is
ruled out on the ground of the mass spectral data: if the hydroxyl
group is modified, it would be in the form of carbamate, and the
molecular weight difference would be 236 Da rather than 192 Da.
(26) Kitz, R.; Wilson, I. B. J . Biol. Chem. 1962, 237, 3245- 3249.
(27) The KI value of 576 mM was obtained for (RS,4R)-1 from the
Kitz-Wilson plot at high concentration (>20 mM) of the inactivator.
(28) Byers, L. D.; Wolfenden, R. Biochemistry 1973, 12, 2070-2078.
(29) Silvermann, R. B. Mechanism-Based Enzyme Inactivation:
Chemistry and Enzymology; CRC: Boca Raton, 1988; Vol. I, pp 22-
23.
(30) Lee, S. S.; Li, Z.-H.; Lee, D. H.; Kim, D. H. J . Chem. Soc., Perkin
Trans. 1 1995, 2877-2882
(31) Wouters, J .; Ooms, F.; Durant, F. Acta Crystallogr. 1997, C57,
895-897.
(32) (a) Najer, H.; Chabrier, P.; Giudicelli, R.; Mabille, P. Bull. Soc.
Chim. Fr. 1957, 1069-1072. (b) Najer, H.; Chabrier, P.; Giudicelli, R.;
Menin, J .; Duchemin, J . Bull. Soc. Chim. Fr. 1959, 1841-1844.

6466 J . Org. Chem., Vol. 66, No. 19, 2001
Chung et al.
F igu r e 5. Transition states that are generated by the PM3
semiemprical calculations for the inactivation of CPA by 1.
Each of the putative tetrahedral transition states formed by
the attack of Glu-270 carboxylate at the C-2 of oxazolidinone
of the CPA bound 1 is stabilized by the active site zinc ion.
Thin line represents key amino acid residues involved in the
transition state formation and thick line denotes inhibitor 1.
A: Transition state formed with (RR,4R)-1. B: Transition state
formed with (RR,4S)-1. C: Transition state formed with
(RS,4R)-1. D: Transition state formed with (RS,4S)-1.
F igu r e 4. Proposed reaction pathway for the inactivation of
CPA by 1.
was examined by the PM3 semiempirical calculations.36
Each of the diastereoisomers of 1 was docked to the X-ray
crystal structure of the CPA active site that consists of
Zn2+, His-69, Glu-72, His-196, Arg-127, Tyr-248 Arg-145,
and Glu-270, and the putative tetrahedral transition
states were optimized (Figure 5).37
Glu-270 carboxylate attacks at the 2-position of the
oxazolidinone of the CPA-bound 1 and opens the ring via
a tetrahedral transition state. Such a reaction does not
require the stringent directionality of the attacking
nucleophile. The approach of a nucleophile on an ester
carbonyl carbon is known to take place within a cone of
30° with the axis being 15° off of the normal to the
carbonyl plane.33
Being different from chemical reactions taken place in
solution, the chemical transformations that occur at the
active site of an enzyme can proceed only when the two
interacting species are poised within a critical distance
with proper stereochemical orientation.34 The lack of
stereospecificity observed in the inhibition of CPA by 1
may now be envisioned: Since the oxazolidinone ring is
not coordinated to the active site zinc ion, the heterocycle
in the CPA-bound 1 may be poised in close proximity to
the nucleophilic carboxylate and the tetrahedral transi-
tion states that are generated by the nucleophilic attack
are conceivably stabilized by the active site zinc ion
(Figure 4).
The transition state formed with (RS,4S)-1 is found to
be most stable. The calculated energies for transition
states in the reaction with (RR,4R)-, (RR,4S)-, and
(RR,4S)-1 are higher compared with that of the most
stable complex38 by 1.6, 4.0, and 5.3 kcal mol-1, respec-
tively.39 The plot of the relative energy against -ln(kobs
/
40
kobs[(aS,4S)-1]
)
for the four inactivation reactions yielded
a straight line, suggesting that all four diastereoisomers
inactivate CPA along the similar pathway that involves
the tetrahedral transition state which is stabilized by the
active site zinc ion and by hydrogen bondings with Arg-
127, Arg-147, and Tyr-248.
Compound 18 is an isomer of 1, in which oxazolidinone
ring is linked at its 5-position to the R-carbon of 3-phe-
nylpropionic acid. It was thought to be worthwhile to
evaluate 18 as an inactivator of CPA because if indeed
the inactivation of CPA by 1 takes place with the attack
Wipff et al. suggested that the stereospecificity in
enzymic reactions is determined in the transition states
rather than in the initial Michaelis complex formation.35
Transition state that is thought to be involved in the
oxazolidinone ring cleavage by the nucleophilic attack
(36) (a) Stewart, J . J . P. J . Comput. Chem. 1989, 10, 209-220. (b)
Stewart, J . J . P. J . Comput. Chem. 1989, 10, 221-264.
(37) In the optimization by the PM3 method, the active site structure
of CPA was fixed to the geometry of the X-ray crystal structure of CPA.
(38) The energy of the most stable transition state was set to zero.
(39) The difference in the energy state shown for the four transition
state may be much reduced in the real inactivation pathway because
most of the strains imposed for the calculations by using the rigid X-ray
structure would be expected to disappear in the real enzyme system,
i.e., most of the strain energies would be absorbed in conformational
changes of the enzyme.
(33) (a) Storm, D. R.; Koshland, D. E., J r. J . Am. Chem. Soc. 1972,
94, 5815-5825. (b) Bu¨rgi, H. B.; Dunitz, J . D.; Shefter, E. Acta
Crystallogr 1974, B30, 1517-1527. (c) Lightstone, F. C.; Bruice, T. C.
J . Am. Chem. Soc. 1996, 118, 2595-2605.
(34) Menger, F. M. Acc. Chem. Res. 1985, 18, 128-134.
(35) Wipff, G.; Dearing, A.; Weiner, P. K.; Blaney, J . M.; Kollman,
P. A. J . Am. Chem. Soc. 1983, 105, 997-1005.
(40) (kobs/kobs[(RS,4S)-1]) denotes the ratio of the kcat value for
diastereoisomer of 1 vs that for (RS,4S)-1.
a

Inactivation of Carboxypeptidase A
J . Org. Chem., Vol. 66, No. 19, 2001 6467
Sch em e 3a
Sch em e 4a
a
Reagents, conditions, and (yields): (a) t-BuOH, H2SO4, MgSO4,
CH2Cl2 (70%); (b) mCPBA, K2HPO4, CH2Cl2 (81%); (c) TFA,
CH2Cl2.
a
Reagents, conditions, and (yields): (a) NaN3, NH4Cl, EtOH-
H2O (96%); (b) H2, Pd/C, AcOH, MeOH-H2O (60%); (c) carbonyl-
diimidazole, CH2Cl2 (74%); (d) TFA, CH2Cl2 (73%)
of the carboxylate at the 2-posistion of the oxazolidinone,
18 should also be equally effective as a CPA inactivator.
Due to the ring nitrogen being a poor leaving group, it is
highly unlikely that the oxazolidinone undergoes ring
cleavage by the nucleophilic attack at the 4-positon of
the ring.
Ta ble 2. Kin etic P a r a m eter s for CP A In a ctiva tion
a
inhibitor
kobs (min-1
)
kobs/[I]0 (M-1 min-1
)
threo-11
erythro-11
0.0351
0.0773
1.755
3.685
a
Apparent first-order rate constant at inhibitor concentration
of 20 mM.
to find that both forms inhibit CPA in a time-dependent
and competitive manner. The inactivated enzyme did not
regenerate the catalytic activity upon dialysis against the
kinetic buffer, suggesting strongly that they are effective
as inactivators for CPA. Kinetic parameters for the
inactivation of CPA by 18 are listed in Table 2. It can be
seen from Table 2 that they inactivate CPA with potency
comparable to those of 1. These results provide an
additional supportive evidence for the proposed inactiva-
tion pathway that involves the attack of the Glu-270
carboxylate at the C-2 site of oxazolidinone of the CPA
bound 1 as depicted in Figure 4.
Synthesis of 18 commenced with vinylacetic acid.
2-Benzylvinylacetic acid that was obtained by benzylation
on vinylacetic acid was esterified following the method
reported by Wright et al.41 to give 12. Epoxidation of 12
with m-CPBA gave the diastereoisomeric mixture of 13a
and 13b in the ratio of 2:1 (Scheme 3). The mixture was
readily separated by column chromatography. Stero-
chemistry of 13a and its conversion products including
threo-18 was ascertained by the 2D 1H NMR NOE
spectrum obtained with 14a 42 that was prepared from
13a by the treatment with TFA: A positive NOE was
observed between the C3-H at 2.84 (ddd, J ) 11.5, 4.0,
4.0 Hz) and C4-H at 4.41 (dd, J ) 4.0, 3.0 Hz), indicating
that the benzyl and hydroxyl groups in 14a are on the
same side of the lactone ring, from which it was inferred
that the oxirane ring in 13a is oriented trans to the
benzyl moiety.
Treatment of 13a with sodium azide in the presence
of ammonium chloride in 60% ethanol solution afforded
15a . Hydrogenation of 15a in the presence of Pd/C
converted the azido into amine to give 16a which was
treated with carbonyldiimidazole in methylene chloride
to give 17a . The ester moiety in 17a was hydrolyzed by
treatment with TFA to yield threo-18 as a racemic
mixture (Scheme 4). The erythro form of 18 was synthe-
sized in a similar fashion from 13b.
Con clu sion
Inhibitor 1 represents a new type of mechanism-based
inactivator for CPA designed rationally on the basis of
the established active site topology of the enzyme and a
proposed catalytic mechanism for the enzymic reaction.
In the designing, the unique property of the oxazolidinone
heterocycle was exploited as a latent inactivating species.
Surprisingly, all four diastereoisomers of 1 inactivated
CPA. The unexpected lack of stereospecificity shown by
the inhibitor may be envisioned on the ground that the
Glu-270 carboxylate attacks at the carbamate carbonyl
carbon rather than the 5-position of the oxazolidinone of
CPA bound 1, and each of tetrahedral transition state
thus generated is stabilized by the active site zinc ion.
This stabilization of the transition state may also explain
the facile cleavage of the relatively stable oxazolidinone
by the enzyme under the ambient conditions, leading to
covalently modify the nucleophilic carboxylate. The new
design concept may hold the promise of a viable approach
for designing selective inactivators of metalloproteases.
Compound 18 thus prepared in racemic threo and
erythro forms were assayed for CPA inactivating activity
(41) Wright, S. W.; Hageman, D. L.; Wright, A, S.; Mcclure, L. D.
Tetrahedron Lett. 1997, 38, 7345-7348.
Exp er im en ta l Section
(42) Compound 14a has been reported previously but the assigned
stereochemistry was not established (Maruoka, O.; Tanabe, G.; Sano,
K.; Minematsu, T.; Momese, T. J . Chem. Soc., Perkin Trans 1 1994,
1883-1845.).
Gen er a l Meth od s. Melting points were measured on a
capillary melting point apparatus and are uncorrected. 1H
and 13C NMR spectra were recorded on a 300 MHz NMR

6468 J . Org. Chem., Vol. 66, No. 19, 2001
Chung et al.
spectrometer using tetramethylsilane as an internal standard.
ESI mass spectra were recorded on CPA (native and inacti-
vated by (RS,4R)-1)) that was dissolved in formic acid and then
diluted with acetonitrile. Flash chromatography was per-
formed on silica gel 60 (230-400 mesh). Elemental analyses
were performed at the Center for Biofunctional Molecules,
Pohang University of Science and Technology, Pohang, Korea,
and the Korea Basic Science Center, Taegu, Korea. All
chemicals were of reagent grade and obtained from Aldrich
Chemical Co. The solvents were purified before use.
(rR,4S)-r-Ben zyl-2-oxo-1,3-oxa zolid in e-4-a cet ic Acid
Ben zyl Ester (6a ). To a cooled solution of 4a (4.7 g, 11.38
mmol) in 1,2-dimethoxyethane (30 mL) were successively
added 1-ethylmorpholine (1.6 mL, 1.44 g, 12.5 mmol) and
isobutyl chloroformate (1.61 mL, 1.7 g, 12.5 mmol) at -15 °C.
After the mixture was stirred for 3 min, a solution of sodium
borohydride (0.82 g, 34.16 mmol) in water (9 mL) was added
in one portion. After a few minutes, the reaction mixture was
quenched by the addition of water (500 mL). The resulting
solution was extracted with ethyl acetate (200 mL × 3), and
the combined organic layer was dried over anhydrous mag-
nesium sulfate. Evaporation of the organic solvent under
reduced pressure afforded a reduction product 5a , which was
added in a dried pyridine solution (20 mL) containing an excess
amount of p-toluenesulfonyl chloride (7 g). The reaction
mixture was stirred at room temperature for 7 days and
diluted with ether (200 mL). The ether solution was washed
with 10% sulfuric acid (50 mL × 2), water, and brine and dried
over anhydrous magnesium sulfate. The oily residue thus
obtained from evaporation of the solvent was purified via flash
chromatography (10% methanol in chloroform). The product
which was slowly crystallized on evaporation of the solvent
was recrystallized from ethanol to afford white crystalline 6a
(1.2 g, 33%): mp 105-106 °C; [R]D +19.2° (c 0.5, CHCl3); IR
(KBr) 3296 (NH), 1749 (CdO), 1722 (CdO) cm-1; 1H NMR (300
MHz; CDCl3) δ 2.88 (m, 3H), 4.01 (m, 2H), 5.04 (s, 2H), 5.72
(s, 1H), 6.90-7.30 (m, 10H); EIMS m/z 325 (M+). Anal. Calcd
for C19H19NO4: C, 70.14; H, 5.88; N, 4.30. Found: C, 70.05;
H, 5.81; N, 4.54.
(2R,3S)-N-ter t-Bu tyloxycar bon yl-3-ben zylaspar tic Acid
Diben zyl Ester (3a ). To a solution of hexamethyldisilazane
(58.6 mL, 50 g, 278 mmol) in THF (400 mL) was added
n-butyllithium in hexane (10 M, 24.5 mL, 245 mmol) at 0 °C
under nitrogen atmosphere and the mixture was stirred for
10 min, then cooled to -78 °C. A solution of N-tert-butyloxy-
carbonyl-L-aspartic acid dibenzyl ester (2a ) (46 g, 111.2 mmol)
in THF (200 mL) was added slowly to the mixture and the
reaction mixture was allowed to stir for 3 h at the same
temperature. Benzyl bromide (26.4 mL, 38 g, 222 mmol) was
added and the reaction mixture was stirred for an additional
4 h, and quenched by addition of 3 N hydrochloric acid to pH
∼3. The aqueous layer was saturated with sodium chloride,
and then extracted with ethyl acetate (200 mL × 2). The
combined organic layer was dried over anhydrous magnesium
sulfate and evaporated under reduced pressure to give an oily
residue which crystallized on standing. The product was
recrystallized from methanol to afford white crystalline 3a (26
g, 46%): mp 82.5-84 °C; [R]D -0.9° (c 1.0, CHCl3); IR (KBr)
(rR,4S)-r-Ben zyl-2-oxo-1,3-oxa zolid in e-4-a cet ic a cid
ben zyl ester (6b) was prepared from 4b in 30% yield
following the procedure used for the preparation of 6a : mp
105-106 °C; [R]D -19.2° (c 0.5, CHCl3).
3396 (NH), 1733 (CdO), 1717 (CdO), 1503 (CdO) cm-1 1H
;
NMR (300 MHz, CDCl3) δ 1.45 (s, 9H), 2.84 (dd, 1H), 3.08 (dd,
1H), 3.42 (sextet, 1H), 4.50 (d, 1H), 4.92-5.10 (m, 4H), 5.56
(d, 1H) and 7.10-7.32 (m, 15H); MS-FAB m/z 504 (MH+) and
526 (M + Na+). Anal. Calcd for C30H33NO6: C, 71.55; H, 6.60;
N, 2.78. Found: C, 71.44; H, 6.72; N, 2.83.
(rR,4S)-r-Ben zyl-2-oxo-1,3-oxa zolid in e-4-a cetic Acid
((rR,4S)-1). A solution of 6a (1 g, 3 mmol) and 10% palladium
on charcoal (0.2 g) in methanol (10 mL) was stirred under
hydrogen atmosphere at room temperature for 2 h and filtered.
Concentration of the filtrate under reduced pressure gave the
solid residue which was recrystallized from water to afford
white crystalline product (560 mg, 80%): mp 178-179 °C; [R]D
-2.4° (c 0.5, EtOH); IR (KBr) 3277 (NH), 3500-2500 (acid OH)
1716 (CdO) and 1700 (CdO) cm-1; 1H NMR (300 MHz; DMSO-
d6) δ 2.75 (m, 2H), 2.85 (dd, 1H), 3.97 (quintet, 1H), 4.24 (dd,
1H), 4.37 (t, 1H), 7.16-7.30 (m, 5H) 7.93 (s, 1H) and 12.45 (s,
1H); EIMS m/z 191 (M+ - CO2), 235 (M+). Anal. Calcd for
C12H13NO4: C, 61.29; H, 5.53; N, 5.96. Found: C, 61.05; H,
5.54; N, 5.91.
(rS,4R)-r-Ben zyl-2-oxo-1,3-oxa zolid in e-4-a cet ic a cid
((RS,4R)-1) was prepared from 6b in 85% yield following the
procedure used for the preparation of (RR,4S)-1: mp 178-179
°C; [R]D +2.4° (c 0.5, EtOH). Anal. Calcd for C12H13NO4‚1/
5H2O: C, 60.37; H, 5.66; N, 5.87. Found: C, 60.66; H, 5.50; N,
5.50.
(3S,4S)-3-Ben zyl-4-(ter t-bu tyloxyca r bon yla m in o)-γ-bu -
tyr ola cton e (8a ). To a stirred solution of lithium diisopro-
pylamide (50 mmol) in THF (40 mL)-HMPA (40 mL) at -78
°C was added dropwise a solution of compound (S)-4-(tert-
butyloxycarbonylamino)-γ-butyrolactone (7a) (4.1 g, 20.4 mmol)
in THF (50 mL). After 2 h benzyl bromide (5.1 g, 30 mol) was
added slowly at the same temperature and the resulting
mixture was stirred for 3 h, acidified with 10% aqueous citric
acid to pH 3 and the organic layer was separated. The aqueous
layer was extracted with ethyl acetate (100 mL × 3). The
combined organic layer was washed with 10% aqueous citric
acid (100 mL × 3), saturated aqueous NaHCO3 (100 mL × 2),
brine (100 mL), dried over anhydrous sodium sulfate, and
concentrated under reduced pressure. Purification via flash
chromatography (10∼25% ethyl acetate in hexane) gave 8a
(4.33 g, 73%) as white crystals: mp 89-90 °C (recrystallized
from ether-hexane); [R]D -20.3° (c 0.62, CHCl3); IR (CHCl3)
3300 (NH), 1776 (CdO), 1712 (CdO) cm-1; 1H NMR (300 MHz,
CDCl3) δ 7.32-7.19 (m, 5H), 4.55, (br s, 1H), 4.29-4.15 (m,
2H), 3.88 (dd, J ) 8.72, 6.41 Hz, 1H), 3.19 (dd, J ) 14.04, 4.23
Hz, 1H), 2.95 (dd, J ) 13.40, 7.09 Hz, 1H), 2.78 (dd, J ) 7.38,
(2S,3R)-N-ter t-Bu tyloxycar bon yl-3-ben zylaspar tic Acid
Diben zyl Ester (3b) was obtained in 53% yield from N-tert-
butyloxycarbonyl-D-aspartic acid dibenzyl ester (2b) following
the procedure used for the preparation of 3a : mp 82.5-84 °C;
[R]D +1.0° (c 1.0, CHCl3).
(2R,3S)-N-ter t-Bu tyloxycar bon yl-3-ben zylaspar tic Acid
â-Ben zyl Ester (4a ). To an ice-cooled solution of 3a (21.36 g,
42.4 mmol) in 80% aqueous dioxane (530 mL) was added an
aqueous solution of 1 N sodium hydroxide (42 mL, 42 mmol)
and 80% aqueous dioxane (540 mL). The resultant solution
was stirred at room temperature for 24 h, acidified with 3 N
hydrochloric acid at 0 °C to pH ∼3, and extracted with ether
(200 mL × 3). The combined extract was dried over anhydrous
magnesium sulfate, and filtered. Dicyclohexylamine (44 mmol)
was added to the filtrate, and chilled in a refrigerator for 24 h
to afford a fine crystalline solid (20.1 g, 80%). The solid was
collected and treated with cold 1 N hydrochloric acid (60 mL)
and extracted with ether (100 mL × 3). The combined extract
was dried over anhydrous magnesium sulfate. Evaporation of
the organic solvent afforded an oily residue which crystallized
on standing. Recrystallization from benzene-petroleum ether
gave white crystalline 4a (12 g, 68.5%): mp 108-110 °C; [R]D
+24° (c 1, CHCl3); IR (KBr) 3263 (NH), 1740 (CdO), 1706
1
(CdO), 1647 (CdO) cm-1; H NMR (300 MHz, CDCl3) δ 1.45
(s, 9H), 2.88 (dd, 1H), 3.13 (dd, 1H), 3.42 (sextet, 1H), 4.44
(dd, 1H), 5.08 (dd, 2H), 5.70 (d, 1H) and 7.20-7.41 (m, 10 H);
MS-FAB m/z 414 (MH+). Dicyclohexylamine salt: mp 178-
180 °C; IR (KBr) 3422 (NH), 1734 (CdO), 1712 (CdO) and 1635
(CdO) cm-1; 1H NMR (300 MHz; DMSO-d6) δ 1-1.5 (m, 19H),
1.57 (d, 2H), 1.70 (d, 4H), 1.95 (d, 4H), 2.69 (dd, 1H), 2.73-3.0
(m, 3H), 3.20 (m, 1H), 4.08 (d, 1H), 4.99 (dd, 2H), 5.95 (d, 1H)
and 7.00-7.40 (10H, m); MS-FAB m/z 595 (MH+). Anal. Calcd
for C23H27NO6: C, 66.81; H, 6.58; N, 3.39. Found: C, 67.12;
H, 6.55; N, 3.39.
(2S,3R)-N-ter t-Bu tyloxycar bon yl-3-ben zylaspar tic acid
â-ben zyl ester (4b) was prepared from 3b in 75% yield
following the procedure used for the preparation of 4a : mp
108-110 °C; [R]D -26° (c 1, CHCl3).

Inactivation of Carboxypeptidase A
J . Org. Chem., Vol. 66, No. 19, 2001 6469
5.14 Hz, 1H), 1.37 (s, 9H); 13C NMR (300 MHz, CDCl3) δ
176.64, 155.29, 137.38, 129.55, 127.50, 80.75, 71.36, 52.41,
47.25, 34.9, 28.64; EIMS m/z 291 (M+), 235 (M+ - C4H8). Anal.
Calcd for C16H21NO4: C, 65.96; H, 7.27; 4.81. Found: C, 65.90;
H, 7.39; 4.57.
(3R,4R)-3-Ben zyl-4-(ter t-b u t yloxyca r b on yla m in o)-γ-
bu tyr ola cton e (8b) was prepared from (R)-4-(tert-butyloxy-
carbonylamino)-γ-butyrolactone (7b) by the same method as
the preparation of 8a : mp 91-92 °C; [R]D +18.4° (c 0.63,
CHCl3). Anal. Calcd for C16H21NO4: C, 65.96; H, 7.27; 4.81.
Found: C, 66.18; H, 7.35; 4.68.
(rS,4S)-r-Ben zyl-2-oxo-1,3-oxa zolid in e-4-a cet ic Acid
((rS,4S)-1). A solution of 10a (492 mg, 1.6 mmol) and mercury
(II) trifluoroacetate (1.36 g, 3.2 mmol) in 80% aqueous dioxane
(40 mL) was stirred for 3 h at 50 °C and then cooled to 0 °C.
Hydrogen sulfide freshly prepared from Na2S and sulfuric acid
was introduced into the reaction mixture for 15 min to give a
black precipitation of mercury sulfide which was filtered off
through Celite pad. The filtrate was concentrated and purified
by flesh column chromatography (silica gel, 50-100% ethyl
acetate in hexane) followed by recrystallization from ethyl
acetate-hexane to give analytically pure (RS,4S)-1 (240 mg,
64%): mp 152.5-153.5 °C; [R]D -19.6° (c 0.52, EtOH); IR (KBr)
3292 (NH), 1734, 1690 (CdO) cm-1; 1H NMR (300 MHz, CDCl3
+ DMSO-d6) δ 7.44 (s, 1H), 7.36-7.18 (m, 5H), 4.47 (t, J )
8.82 Hz, 1H), 4.24 (dd, J ) 8.85, 5.70, 1H, 4.03 (m, 1H), 3.00
(m, 2H), 2.83 (t, J ) 7.35 Hz, 1H); 13C NMR (300 MHz, CDCl3
+ DMSO-d6) δ 174.0, 159.3, 137.9, 129.0, 128.5, 126.7, 68.2,
53.1, 51.8, 34.5; MS-FAB m/z 236 (MH+). Anal. Calcd for
C12H13NO4: C, 61.27; H, 5.57; N, 5.95. Found: C, 61.31; H,
5.60; N, 5.88.
(2S,3S)-2-Ben zyl-3-(ter t-b u t yloxyca r b on yla m in o)-4-
(m eth a n esu lfon yloxy)bu ta n oic Acid ter t-Bu tyl Th ioester
(9a ). tert-Butylthiol (1.16 mL, 10.3 mmol) was added to a
solution of trimethylaluminum (10.3 mmol) in dichloromethane
(8.15 mL) at 0 °C, and the resultant mixture was allowed to
warm to room temperature over 20 min with stirring. A
solution of compound 8a (1 g, 3.43 mmol) in dichloromethane
(8 mL) was added and the mixture was stirred overnight. The
mixture was cooled to -78 °C and quenched with ether (20
mL) followed by careful addition of 1 N hydrochloric acid (30
mL). The aqueous layer was extracted with ether (30 mL ×
2). The combined organic layer was washed with 1 N cold
hydrochloric acid (30 mL), saturated aqueous sodium bicar-
bonate (50 mL), and brine (50 mL), dried over anhydrous
sodium sulfate, and concentrated under reduced pressure to
give an oily product (1.37 g). To a solution of the oil (1.37 g,
3.4 mmol) in dichloromethane (60 mL) at 0 °C were succes-
sively added triethylamine (0.98 mL, 7 mmol) and methane-
sulfonyl chloride (0.48 mL, 7 mmol). The mixture was stirred
for 20 min at room temperature, and then washed with water
(20 mL), 0.5 N cold hydrochloric acid (20 mL), 5% aqueous
sodium bicarbonate (20 mL), brine (20 mL), dried over
anhydrous sodium sulfate and concentrated. Purification of the
residue via flash chromatography (20% ethyl acetate in hex-
ane) gave 9a (1.4 g, two step yield: 88%) as white crystals:
mp 129.5-130.5 °C (recrystallized from ether-hexane); [R]D
- 9.6° (c 0.57, CHCl3); IR (CHCl3) 3370 (NH), 1720-1660
(CdO) cm-1; 1H NMR (300 MHz, CDCl3) δ 7.28-7.11 (m, 5H),
4.89 (d, 8.1 Hz, 1H), 4.30-4.25 (m, 3H), 3.02 (s, 3H), 3.02-
2.91 (m, 3H), 1.45 (s, 9H), 1.32 (s, 9H); 13C NMR (300 MHz,
CDCl3) δ 201.2, 155.6, 138.1, 129.6, 128.8, 127.0, 80.7, 70.0,
57.5, 51.9, 49.2, 37.8, 36.5, 29.8, 28.7; MS-FAB m/z 460 (MH+),
404 (MH+ - C4H8). Anal. Calcd for C21H33NO6S2: C, 54.88; H,
7.24; N, 3.05. Found: C, 54.88; H, 7.51; N, 3.42.
(4R,rR)-r-Ben zyl-2-oxo-1,3-oxa zolid in e-4-a cetic a cid
((4R,RR)-1) was prepared from 10b by the same method as
that used for the preparation of 1c: mp 149-150 °C; [R]D
+20.0° (c 0.63, EtOH). Anal. Calcd for C12H13NO4: C, 61.27;
H, 5.57; N, 5.95. Found: C, 61.37; H, 5.56; N, 5.93.
(()-2-Ben zyl-3-bu ten oic Acid ter t-Bu tyl Ester (12).
Concentrated sulfuric acid (1.9 mL, 34 mmol) was added to a
vigorously stirred suspension of anhydrous magnesium sulfate
(16.4 g, 136 mmol) in dichloromethane (70 mL). After the
mixture was stirred for 15 min 2-benzyl-3-butenoic acid (11)
(6.0 g, 34 mmol) and 2-methyl-2-propanol (16 mL, 167 mmol)
were added successively. The reaction flask was sealed tightly
and stirred for 18 h at 25 °C. The reaction mixture was then
quenched with saturated aqueous sodium bicarbonate (50 mL)
and stirred until all magnesium sulfate had dissolved. The
organic phase was separated, washed with brine, dried over
magnesium sulfate, and evaporated under reduced pressure
to give the crude tert-butyl ester which was purified by column
chromatography (10% ethyl acetate in n-hexane) to give 12
as a colorless oil (5.5 g, 70%): 1H NMR (300 MHz, CDCl3) δ
1.35 (s, 9H), 2.87-2.85 (dd, 1H), 3.01-3.08 (dd, 1H), 3.20-
3.25 (q, 1H), 5.06-5.13 (m, 2H), 5.80-5.92 (m, 1H), 7.17-7.30
(m, 5H); 13C NMR (CDCl3) δ 28.3, 38.9, 53.2, 81.0, 117.4, 126.7,
128.6, 129.5, 136.5, 139.3, 173.0.
(2S,3S)-2-Ben zyl-3-(ter t-b u t yloxyca r b on yla m in o)-4-
(m eth a n esu lfon yloxy)bu ta n oic a cid ter t-bu tyl th ioester
(9b) was prepared from 8b by the same method as that used
for the preparation of 9a : mp 125-126 °C; [R]D +47.1° (c 0.50,
CHCl3). Anal. Calcd for C21H33NO6S2: C, 54.88; H, 7.24; N,
3.05. Found: C, 54.54; H, 7.23; N, 2.95.
(()-2-Ben zyl-3,4-ep oxybu ta n oic Acid ter t-Bu tyl Ester
(13a a n d 13b). A solution of 12 (3.0 g, 13 mmol), m-
chloroperbenzoic acid (7.4 g, 26 mmol), and dibasic potassium
phosphate (4.5 g, 26 mmol) in dichloromethane was stirred at
room temperature for 4 days. The resulting mixture was
quenched with 5% aqueous sodium bisulfite, and partitioned
between dichloromethane and water. The combined organic
extracts were washed with 5% aqueous sodium bicarbonate,
brine, and then dried over magnesium sulfate. After evapora-
tion of the solvent the resulting diastereomeric mixture was
separated by column chromatography (5% ethyl acetate in
n-hexane) to give 13a (1.7 g, 53%) and 13b (0.9 g, 28%) as
colorless oil. 13a : IR (CHCl3) 2979, 1725, 1455 cm-1; 1H NMR
(300 MHz, CDCl3) δ 1.42 (s, 9H), 2.31-2.42 (m, 2H), 2.69-
2.72 (t, 1H), 2.85-2.92 (dd, 1H), 3.00-3.07 (dd, 1H), 3.16-
3.20 (m, 1H), 7.19-7.32 (m, 5H); 13C NMR (CDCl3) δ 28.4, 35.7,
46.7, 51.8, 52.9, 81.7, 127.0, 128.8, 129.4, 138.6, 172.4. 13b:
(rS,4S)-r-Ben zyl-2-oxo-1,3-oxa zolid in e-4-a cet ic Acid
ter t-Bu tyl Th ioester (10a ). A solution of 9a (1 g, 2.18 mmol)
in pyridine (50 mL) was stirred for 3 h at 90 °C, cooled to room
temperature, and concentrated under reduced pressure. The
resulting oily residue was dissolved in ethyl acetate (200 mL),
and the mixture was washed with 0.5 N cold hydrochloric acid
(50 mL), brine (50 mL), dried over anhydrous sodium sulfate,
and concentrated under reduced pressure. Purification of the
residue via flash chromatography (30% ethyl acetate in hex-
ane) gave 10a (430 mg, 64%) as white crystals: mp 144-145
°C (recrystallized from ethyl acetate-hexane); [R]D -88.6° (c
0.54, CHCl3); IR (CHCl3) 3300 (NH), 1752 (CdO) cm-1 1H
;
1
IR (CHCl3) 2979, 2931, 1725, 1497 cm-1; H NMR (300 MHz,
NMR (300 MHz, CDCl3) δ 7.25-7.06 (m, 5H), 6.21 (s, 1H), 4.39
(t, J ) 8.84 Hz, 1H), 4.20 (dd, J ) 8.99, 5.67 Hz, 1H), 4.03 (m,
1H), 2.94 (dd, J ) 14.49, 3.97 Hz, 1H), 2.87-2.81 (m, 2H), 1.30
(s, 9H); 13C NMR (300 MHz, CDCl3) δ 200.8, 159.6, 137.2,
129.3, 128.8, 127.1, 68.2, 60.1, 54.0, 49.3, 35.7, 29.7; EIMS m/z
307 (M+). Anal. Calcd for C16H21NO3S: C, 62.51; H, 6.89; N,
4.56. Found: C, 62.89; H, 7.17; N, 4.69.
(rR,4R)-r-Ben zyl-2-oxo-1,3-oxa zolid in e-4-a cetic a cid
ter t-bu tyl th ioester (10b) was prepared from 9b by the same
method as that used for the preparation of 10a : mp 147-148
°C; [R]D +84.2° (c 0.50, CHCl3). Anal. Calcd for C16H21NO3S:
C, 62.51; H, 6.89; N, 4.56. Found: C, 62.41; H, 6.85; N, 4.57.
CDCl3) δ 1.35 (s, 9H), 2.38-2.45 (q, 1H), 2.70-2.72 (dd, 1H),
2.82-2.85 (dd, 1H), 3.07-3.13 (m, 3H), 7.20-7.32 (m, 5H); 13
C
NMR (CDCl3) δ 28.3, 36.4, 47.0, 51.8, 53.0, 81.7, 126.9, 128.7,
128.8, 129.5, 138.7, 172.4.
(()-cis-3-Ben zyl-4-h ydr oxytetr ah ydr ofu r an -2-on e (14a).
TFA (1 mL) was added to stirred solution of 13a (80 mg, 0.32
mmol) dissolved in methylene chloride (5 mL) at 0 °C. After
the reaction mixture was being stirred for 90 min at 0 °C, it
was evaporated under reduced pressure and the residue was
purified by column chromatography (hexane/ethyl acetate )
3:1) to give 14a (10 mg, 16%). IR (CHCl3) 3448, 1761 cm-1; 1H

6470 J . Org. Chem., Vol. 66, No. 19, 2001
Chung et al.
NMR (CDCl3) δ 1.99 (br s, 1H), 2.84 (m, 1H), 2.99 (dd, 1H),
3.25 (dd,1H), 4.29 (d, 2H), 4.41 (m, 1H), 7.23-7.37 (m, 5H);
13C NMR δ 29.8, 47.7, 69.2, 74.8, 127.0, 129.0, 129.3, 139.0,
180.0.
(0.3 mL, 3 mmol) and dichloromethane was stirred at room
temperature for 5 h. The volatile materials were evaporated
under reduced pressure and the remaining residue was
recrystallized from methanol-ether to give threo-18 (60 mg,
1
73%) as white crystals: mp 197-199 °C; H NMR (30 MHz,
(()-4-Azid o-2-ben zyl-3-h yd r oxybu ta n oic Acid ter t-Bu -
tyl Ester (15a ). To a solution of 13a (0.50 g, 2 mmol) in 60%
ethanol were added sodium azide (0.65 g, 10 mmol) and
ammonium chloride (0.22 g, 4 mmol). The mixture was heated
at 60 °C for 2 h, and the solvent was removed under reduced
pressure. The residue was suspended in ether and washed with
water. The organic layer was dried over magnesium sulfate
and concentrated to dryness under reduced pressure. The
resulting crude product was recrystallized from ether-hexane
to give 14a (0.56 g, 96%) as white crystals: mp 80-81 °C; IR
MeOH-d4) δ 2.84-2,97 (m, 3H), 3.44-3.50 (dd, 1H), 3.64-3.70
(t, 1H), 4.77-4.85 (m, 1H), 7.17-7.30 (m, 5H); 13C NMR
(MeOH-d4) δ 33.8, 44.0, 52.2, 77.1, 126.7, 128.5, 129.0, 138.4,
160.7, 174.0. Anal. Calcd for C12H13NO4: C, 61.27; H, 5.57; N,
5.95. Found: C, 61.18; H, 5.47; N, 5.68.
(()-r-Ben zyl-2-oxo-1,3-oxazolidin e-5-acetic acid (erythro-
18) was prepared in 74% yield from 17b (0.25 g, 0.9 mmol)
following the same procedure as that used for the preparation
of threo-18: mp 90-91 °C; 1H NMR (30 MHz, MeOH-d4) δ
2.93-3.12 (m, 3H), 3.42-3.50 (dd, 1H), 3.58-3.71 (t, 1H),
4.68-4.75 (m, 1H), 7.21-7.31 (m, 5H); 13C NMR (MeOH-d4) δ
34.0, 44.4, 51.5, 76.1, 126.8, 128.5, 129.0, 138.1, 160.7, 173.7.
Anal. Calcd for C12H13NO4: C, 61.27; H, 5.57; N, 5.95. Found:
C, 61.22; H, 5.68; N, 5.97.
(CHCl3) 3445, 2103, 1715, 1645 cm-1 1H NMR (300 MHz,
;
CDCl3) δ 1.37 (s, 9H), 2.71-2.84 (m, 1H), 2.93-3.05 (m, 2H),
3.32-3.43 (m, 2H), 3.65 (br s, 1H), 3.79-3.82 (q, 1H), 7.21-
7.34 (m, 5H); 13C NMR (CDCl3) δ 28.3, 36.1, 50.4, 55.8, 71.5,
82.5, 127.1, 128.9, 129.6, 138.4, 174.3.
En zym e Assa ys. Carboxypeptidase A (Sigma Chemical Co.
Type II, twice recrystallized) showed one band when analyzed
by 15% SDS polyacrylamide gel electrophoresis.43 Hippuryl-
L-phenylalanine and Tris acid/base were purchased from the
Sigma Chemical Co. Tris buffer of pH 7.5 (0.05 M Tris)
containing 0.5 M NaCl was used. The commercial CPA (50 µM)
was suspended in the buffer (950 µL) and its concentration
was determined by optical density measurements at 278 nm
(ꢀ278 ) 64 200 M-1 cm-1). The enzyme stock solution (1.6 µM)
was prepared by dilution of the enzyme solution in the buffer.
The inhibitor stock solutions (40 mM) were prepared by
dissolution of each inhibitor (9.4 mg, 40 µmol) in DMSO (260
µL) followed by addition of the buffer (740 µL). Enzyme activity
was measured by monitoring hydrolysis of hippuryl-L-pheny-
lalanine (substrate) at 254 nm using a computer-assisted UV
spectrophotometer (Hewlett-Packard 8453 diode array spec-
trophotometer).
Mea su r em en ts of th e Tim e-Dep en d en t Loss of th e
En zym ic Activity. A portion of the inhibitor stock solution
was added to the same volume of the enzyme stock solution
in an ice-water bath to give an incubation mixture of the
enzyme (1.6 µM). While the mixture was incubated at room-
temperature aliquots (20 µL) of the mixture were taken, at 5
min intervals, into the buffer solution (980 µL) of the substrate
to give 300 µM and 32 nM of final concentrations of the
substrate and the enzyme, respectively. The remaining enzyme
activity was measured by monitoring the hydrolysis of the
substrate at 254 nm for 60 s. Values of kobs (the apparent
inactivation rate constant) were calculated from semilogarithm
plots of residual enzymic activity vs time.
Deter m in a tion of P a r tition Ra tios. A series of solutions
(100 µL each) containing CPA (250 µM) and various molar
equivalent amount of the inhibitor were prepared to give [I]0/
[E]0 ratios of 0 to 200 in Tris buffer containing DMSO (13%)
and were incubated at 4 °C for 24 h. The incubated mixtures
were dialyzed in the same buffer at 4 °C for 48 h during which
the buffer solution was changed five times. A 30 µL of each
dialyzed mixture was added to 970 µL of assay mixture (final
[S]0 ) 500 µM) and the activity was measured immediately.
A solution of no inhibitor was used as the reference setting
100%. A plot of the percent enzyme activity remaining vs the
ratio of the molar equivalents of the inhibitors for CPA was
constructed, in which the extrapolation to the ratio gives
turnover number (The plots not shown). The turnover number
minus 1 gives partition ratio.
(()-4-Azid o-2-ben zyl-3-h yd r oxybu ta n oic a cid ter t-bu -
tyl ester (15b) was prepared in 97% yield from 13b (0.80 g,
3.2 mmol) following the same procedure as that described for
15a : colorless oil; IR (CHCl3) 3449, 2103, 1719 cm-1; 1H NMR
(CDCl3) δ 1.30 (s, 9H), 2.77-2.84 (m, 1H), 2.89-2.97 (dd, 1H),
3.04-3.10 (m, 2H), 3.36-3.46 (m, 1H), 3.97-4.03 (q, 1H),
7.20-7.32 (m, 5H); 13C NMR (CDCl3) δ 28.2, 34.5, 51.9, 55.0,
71.6, 82.1, 126.9, 128.8, 129.5, 139.0, 173.4.
(()-4-Am in o-2-ben zyl-3-h yd r oxybu ta n oic Acid ter t-Bu -
tyl Ester , Acetic Acid Sa lt (16a ). To a solution of 15a (0.52
g, 1.8 mmol) in 50% aqueous methanol was added a catalytic
amount of 10% Pd/C and acetic acid (0.16 mL). The resulting
mixture was stirred under hydrogen atmosphere for 5 h. The
catalyst was removed by filtration and the filtrate was
evaporated under reduced pressure. The crude product was
recrystallized from methanol-ether to give 16a (0.35 g, 60%)
as white crystals: mp 149-150 °C; IR (CHCl3) 3418, 1714,
1644 cm-1; 1H NMR (CDCl3) δ 1.30 (s, 9H), 1.93 (s, 3H), 2.71-
2.78 (m, 1H), 2.88-2.96 (m, 3H), 3.04-3.09 (dd, 1H), 3.95-
4.01 (m, 1H), 7.17-7.29 (m, 5H); 13C NMR (CDCl3) δ 24.1, 28.3,
35.2, 44.3, 52.5, 70.1, 81.8, 126.8, 128.7, 129.5, 138.9, 173.2,
178.9.
(()-4-Am in o-2-ben zyl-3-h yd r oxybu ta n oic a cid ter t-bu -
tyl ester , a cetic a cid sa lt (16b) was prepared in 60% yield
from 15b (0.82 g, 2.8 mmol) following the same procedure as
that used for 16a (0.55 g,): mp 89-90 °C; IR (CHCl3) 3418,
1
1722, 1631 1520 cm-1; H NMR (CDCl3) δ 1.27 (s, 9H), 1.99
(s, 3H), 2.68-2.76 (m, 1H), 2.83-3.03 (m, 3H), 3.16-3.21 (dd,
1H), 3.85-4.03 (m, 1H), 7.17-7.28 (m, 5H); 13C NMR (CDCl3)
δ 24.4, 28.2, 35.6, 43.9, 53.7, 69.7, 81.8, 126.7, 128.6, 129.5,
139.1, 173.1, 180.5
(()-r-Ben zyl-2-oxo-1,3-oxa zolid in e-5-a cetic Acid ter t-
Bu tyl Ester (17a ). Carbonyldiimidazole (0.10 g, 0.6 mmol)
was added to a suspension of 16a (0.20 g, 0.6 mmol) in
dichloromethane. After being stirred at room temperature for
5 h, the reaction mixture was washed with 5% sodium
bicarbonate and brine and dried over magnesium sulfate. The
residue resulted from evaporation of the solvent was recrystal-
lized from dichloromethane-hexane to give 17a (0.13 g, 74%)
as white crystals: mp 91-92 °C; IR (CHCl3) 2978, 1752, 1644,
1493 cm-1 1H NMR (30 MHz, CDCl3) δ 1.34 (s, 9H), 2.82-
;
2.99 (m, 3H), 3.45-3.51 (t, 1H), 3.59-3.65 (t, 1H), 4.75-4.82
(m, 1H), 5.80 (br, 1H), 7.18-7.31 (m, 5H); 13C NMR (CDCl3) δ
28.2, 34.0, 44.0, 52.6, 76.6, 82.3, 127.2, 129.0, 129.5, 137.9,
159.9, 170.8.
Ir r ever sibility of th e In h ibition . Incubation mixtures of
CPA and the inhibitors were prepared as described above. A
reference mixture was prepared analogously by mixing the
CPA and the buffer containing DMSO (26%). When the
mixtures were incubated for 3 days at 4 °C the mixtures
(()-r-Ben zyl-2-oxo-1,3-oxa zolid in e-5-a ceta te ter t-bu tyl
ester (17b) was prepared in 73% yield from 16b (0.50 g, 1.5
mmol) following the same procedure as that used for 17a : mp
155-156 °C; IR (CHCl3) 3271, 2981, 1755, 1720 cm-1; 1H NMR
(CDCl3) δ 1.33 (s, 9H), 2.94-3.05 (m, 2H), 3.14-3.22 (m, 1H),
3.41-3.46 (t, 1H), 3.69-3.75 (t, 1H), 4.67-4.75 (m, 1H), 6.10
(br s, 1H), 7.20-7.33 (m, 5H); 13C NMR (CDCl3) δ 28.3, 35.2,
44.9, 52.9, 76.3, 82.4, 127.1, 128.8, 129.7, 137.7, 159.8, 170.0.
(43) A comparative activity assay of the commercial enzyme with
homogeneous CPA obtained by overexpressing bovine CPA gene in E.
coli (Cho, J . H.; Kim, D.-H.; Lee, K. J .; Kim, D. H.; Choi, K. Y.
Biochemistry, in press) showed that both enzymes are essentially
equipotent.
(()-r-Ben zyl-2-oxo-1,3-oxazolidin e-5-acetic Acid (th r eo-
18). A solution of 17a (0.10 g, 0.3 mmol) in trifluoroacetic acid

Inactivation of Carboxypeptidase A
J . Org. Chem., Vol. 66, No. 19, 2001 6471
containing inhibitors showed less than 5% of remaining
activity against the reference. The mixtures were transferred
into dialysis bags and dialyzed in the buffer for 3 days. The
enzyme activity after the dialysis was measured.
Active Site P r otection by (R)-2-Ben zylsu ccin ic Acid .
A mixture of the inhibitor (40 mM) and (R)-2-benzylsuccinic
acid (40 µM) in the buffer containing DMSO (26%) was added
to the same volume of the enzyme stock solution. The mixture
was incubated at room temperature, and the remaining
activity at certain time intervals was measured as described
above.
Ack n ow led gm en t. We express our sincere thanks
to the Korea Science and Engineering Foundation for
the financial support of this work. S.C. is a recipient of
BK21 stipend.
J O010421E