Syntheses and kinetic evaluation of racemic and optically active 2-benzyl-2-methyl-3,4-epoxybutanoic acids as irreversible inactivators for carboxypeptidase A

Article abstract of DOI:10.1016/S0968-0896(01)00340-6

Racemic and optically active 2-benzyl-2-methyl-3,4-epoxybutanoic acids were synthesized and evaluated as inactivators for carboxypeptidase A, a representative zinc-containing proteolytic enzyme. Only the threo-form of the inactivator is effective and its potency in terms of k_inact/K_I value is lower by 42-fold compared with 2-benzyl-3,4-epoxybutanoic acid, indicating that the α-methyl group affects adversely in the inactivation contrary to the expectation that it would enhance the inactivation activity of the inhibitor through additional interactions of the methyl group with a small cavity (α-methyl hole) present next to the S₁′ hydrophobic pocket. Of the enantiomeric pair, the inactivator having the (2S,3R)-configuration is more potent than its enantiomer by 44-fold. The observed kinetic results may be rationalized on the basis that the methyl group in the inactivator having the (2R,3S)-configuration experiences the van der Waals repulsive interactions with the bottom of the active site crevice in binding to CPA, casting a doubt on the presence of the so-called α-methyl hole at the active site of carboxypeptidase A. Copyright

Full text of DOI:10.1016/S0968-0896(01)00340-6

Bioorganic & MedicinalChemistry 10 (2002) 913–922
Syntheses and Kinetic Evaluation of Racemic and Optically Active
2-Benzyl-2-methyl-3,4-epoxybutanoic Acids as Irreversible
Inactivators for Carboxypeptidase A
Mijoon Lee and Dong H. Kim*
Division of Molecular and Life Sciences and Center for Integrated Molecular Systems, Pohang University of Science and Technology,
San 31 Hyojadong, Pohang 790–784, Republic of Korea
Received 1 August 2001; accepted 21 September 2001
Abstract—Racemic and optically active 2-benzyl-2-methyl-3,4-epoxybutanoic acids were synthesized and evaluated as inactivators
for carboxypeptidase A, a representative zinc-containing proteolytic enzyme. Only the threo-form of the inactivator is effective and
its potency in terms of kinact/KI value is lower by 42-fold compared with 2-benzyl-3,4-epoxybutanoic acid, indicating that the a-
methyl group affects adversely in the inactivation contrary to the expectation that it would enhance the inactivation activity of the
0
inhibitor through additionalinteractions of the methylgroup with a smallcavity (
a-methylhoel) present next to the S
hydro-
1
phobic pocket. Of the enantiomeric pair, the inactivator having the (2S,3R)-configuration is more potent than its enantiomer by 44-
fold. The observed kinetic results may be rationalized on the basis that the methyl group in the inactivator having the (2R,3S)-
configuration experiences the van der Waals repulsive interactions with the bottom of the active site crevice in binding to CPA,
casting a doubt on the presence of the so-called a-methylhole at the active site of carboxypeptidase A. # 2002 Elsevier Science Ltd.
All rights reserved.
Introduction
0
145, a large hydrophobic pocket at the S1 subsite, and
Arg-127. The Arg-145 forms bifurcated hydrogen bonds
0
Zinc metalloproteases constitute an important class of
proteolytic enzymes and have received much attention.1
These proteases play vital roles in numerous biological
processes.1 Of these enzymes, carboxypeptidase A
(CPA) is the most extensively studied and well char-
acterized protease which cleaves the C-terminal amino
acid residue having a hydrophobic side chain from pro-
tein substrate.2 The zinc ion that is essentialfor the
catalytic activity is coordinated to His-69 His-196 and
Glu-72 and a molecule of water that attacks the scissile
peptide bond to generate a tetrahedraltransition state.
The carboxylate of Glu-270 is known to function as a
generalbase that activates the zinc bound water moel-
cule by abstracting a proton to form a hydroxide ion
that attacks the scissile peptide bond.2 However, in the
case of ester hydrolysis, the carboxylate was shown to
attack directly the carbonyl carbon of the substrate,
which is activated by the zinc ion.3,4 At the active site of
CPA, there are present three binding sites, that is Arg-
with the terminalcarboxyal te of substrates, and the S
1
subsite pocket accommodates the side chain of the C-
terminalamino acid residue thus to serve as the primary
substrate recognition pocket.5 The guanidinium moiety
of Arg-127 forms a hydrogen bond to the carbonyl
oxygen of the scissile peptide bond.6
Recently, Asante-Appiah et al. reported that 2-ethyl-2-
methylsuccinic acid (1) is a potent inhibitor for CPA.7
.
The X-ray crystalstructure of CPA 1 complex led them
to propose that the high inhibitory potency of 1 is due
to additionalinteractions that are in effect between the
methylgroup of 1 and a small hole next to the hydro-
phobic pocket at the active site of CPA. These authors
suggested that improvement of potency of a CPA inhi-
bitor may be brought about by the incorporation of a
methylgroup at its a-position. 2-Benzyl-3,4-epoxy-
butanoic acid (2) is one of the most potent irreversible
inhibitor reported for CPA.8 The epoxide ring in the
enzyme-bound 2 is believed to be activated by the zinc
ion at the active site and undergoes a SN2 type ring
cleavage by the attack of the carboxylate of Gly-270 to
result in covalent modification of the carboxylate.9,10
*Corresponding author. Tel.: +82-54-279-2101; fax: +82-54-279-
5877; e-mail: dhkim@postech.ac.kr
0968-0896/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(01)00340-6

914
M. Lee, D. H. Kim / Bioorg. Med. Chem. 10 (2002) 913–922
Scheme 2. (a) BH3ꢀ SMe2, DMF, 0 ꢁC, 97%; (b) Cs2CO3, MeOH;
BnBr, DMF, rt, 60%; (c) DMSO, (COCl)2; Et3N, À78 ꢁC to rt, 89%;
(d) Ph3PMeI, KHMDS, THF, À78 ꢁC–40 ꢁC, 45%; (e) mCPBA,
K2HPO4, CH2Cl2, 0 ꢁC to rt, 55%; (f) H2, Pd/C, MeOH, rt, 95%.
Scheme 1. (a) MeI, K2CO3, MeOH, reflux, 87.5%; (b) BnBr, K2CO3,
MeOH, reflux, 88.5%; (c) NaBH4, MeOH/H2O, 0 ꢁC to rt, 85.5%; (d)
SOCl2, pyridine, 0–50 ꢁC, 68.5%; (e) LiOHꢀ H2O, MeOH/H2O/THF,
rt, 85.9%; (f) (i) Cs2CO3, MeOH, rt; (ii) BnBr, DMF, rt, 88.2%; (g)
mCPBA, K2HPO4, CH2Cl2, 0 ꢁC to rt, threo, 55%; erythro, 30%; (h)
H2, Pd/C, MeOH, rt, 95%.
It was thought therefore that introduction of a methyl
group at the a-position of 2 would enhance the binding
affinity for CPA leading to improvement of the inacti-
vation potency. This paper describes syntheses of 2-
benzyl-2-methyl-3,4-epoxybutanoic acid (3) as racemic
and optically active forms and their kinetic evaluation
as irreversible inhibitors for CPA.
Scheme 3. (a) LiOHꢀ H2O, MeOH/THF/H2O, rt, 95%; (b) (i) Cs2CO3,
MeOH; ii) BnBr, DMF, rt, 60%; (c) DMSO, (COCl)2; Et3N, À78 ꢁC
to rt, 85%; (d) Ph3PMeI, KHMDS, THF, À78–40 ꢁC, 45%; (e)
mCPBA, K2HPO4, CH2Cl2, 0 ꢁC to rt, 50%; (f) H2, Pd/C, MeOH, rt,
95%.
Chemistry
The synthesis of (Æ)-3 was started with methylaceto-
acetate (4) which was subjected to methylation followed
by benzylation11 to afford methyl2-benzy-l2-methy-l3-
oxobutanoate (6). The latter was then reduced with
sodium borohydride in MeOH to give 7. The dehydra-
tion of 7 was carried out in the presence of thionyl
chloride and pyridine to yield a 1:1 mixture of 8 and
methyl 2-benzyl-2-methyl-3-cholorobutanoate.12 Alka-
line hydrolysis of this mixture with lithium hydroxide
gave 2-benzyl-2-methylbut-3-enoic acid (9). Methyl2-
benzyl-2-methyl-3-cholorobutanoate that resisted to be
hydrolyzed could be separated from the product. 2-
Benzyl-2- methylbut-3-enoic acid (9) was protected as a
benzylester to give 10, which was then subjected to
expoxidation using m-chloroperbenzoic acid (mCPBA)
under the condition reported by Albeck and Persky.13
The diastereoisomeric mixture thus obtained was sepa-
rated by column chromatography on silica gel to afford
threo-(Æ)- and erythro-(Æ)-11, respectively in the ratio
of 2:1. Catalytic hydrogenolysis of threo-(Æ)- and ery-
thro-(Æ)-11 gave the target compounds, threo-(Æ)- and
erythro-(Æ)-3, respectively (Scheme 1).
The synthetic route for the preparation of (2S,3R)-3 is
depicted in Scheme 2. Enzymatic hydrolysis of racemic
benzylmethylmalonic dimethylester using a-chymo-
trypsin according to the literature method14 afforded the
corresponding monoester, 12 having the (R)-configura-
tion. The methylester moiety in 12 was selectively
ꢀ
reduced with BH3 SMe2 to give hydroxymethylacid, 13
of which the carboxylate was converted into benzyl
ester to yield 14.15 Oxidation of the primary alcohol
moiety in 15 to the corresponding aldehyde (15) was
accomplished by the method of Swern16 and the sub-
sequent reaction with methyltriphenylphosphine bro-
mide under the Wittig conditions gave 16.17 The
conversion of 16 to (2S,3R)-3 was accomplished by the
same sequence of reactions as those used for the pre-
paration of (Æ)-3.
The synthesis of (2R,3S)-3 was commenced with the
alkaline hydrolysis of 18 that was prepared by the
method of Lee et al.18 to give hydroxymethylester ( 19).

M. Lee, D. H. Kim / Bioorg. Med. Chem. 10 (2002) 913–922
915
(2R,3S)-24 was prepared by the method reported by
Renaud et al.19 The hydrolysis of 24 in aqueous HBr
gave hydroxysuccinic acid (25) which was converted to
benzylester in the presence of n-Bu4NOH and BnBr.20
The subsequent four functionalgroup interconversions
to obtain epoxybutanoate (23) were accomplished
according to the method described by Lee et al.21 The
conversion of 26 into 27 was effected in moderate yield
by the regioselective reduction by means of borane–
dimethyl sulfide complex in the presence of a catalytic
amount of sodium borohydride in tetrahydrofuran.22
When 27 was treated with p-toluenesulfonyl chloride in
the presence of pyridine, the primary hydroxylgroup
was selectively tosylated. The subsequent treatment of
the tosylate (29) thus obtained with potassium carbon-
ate in methanolafforded (2 R,3S)-23 whose spectraldata
were identicalwith those of 23 that was obtained from
the epoxidation of 22, establishing the stereochemistry
of 23 and its hydrogenolysis product to be (2R,3S) con-
figuration.
Scheme 4. (a) HBr (aq), n-Bu4NBr (cat), reflux, 74%; (b) (i) n-
Bu4NOH, MeOH; (ii) BnBr, DMF/acetonitrile, rt, 79%; (c)
BH3ꢀ SMe2, NaBH4 (cat), 0 ꢁC to rt, 67%; (d) TsCl, pyridine, CH2Cl2,
rt, 68%; (e) K2CO3, MeOH, rt, 60%.
The conversion of 19 to (2R,3S)-3 was accomplished
following the same sequences of reactions as those used
for the preparation of (2S,3R)-3, as illustrated in
Scheme 3.
Biological Evaluation
The compounds thus synthesized were assayed for CPA
inhibitory activity at 25 ꢁC in Tris buffer (0.05 M) of pH
7.5 using hippuryl-l-phenylalanine (Hip-l-Phe) as the
substrate. Threo-(Æ)-3, (2S,3R)-3, and (2R,3S)-3 inhib-
ited CPA in a time-dependent fashion as shown in Fig-
ure 1 to suggest that they inhibit the enzyme in an
irreversible manner. The inactivated CPA by threo-3
failed to gain its enzymic activity upon dialysis for 2
days, supporting the irreversible nature of the inhibi-
tions. Kinetic parameters for the irreversible inhibitions,
The stereochemistry of the epoxidation product (23)
was confirmed by an alternative synthesis that starts
with 2419 as depicted in Scheme 4.
Table 1. Kinetic parameters for the inactivation of carboxypeptidase
A
Compds
kinact (minÀ1) KI (mM) kinact/KI (MÀ1minÀ1) Ki (mM)
(2S,3R)-2a
(2R,3S)-2 a
1.6
1.1
0.19
0.34
8400
3300
Threo-(Æ)-3 0.85Æ0.3
5.9Æ2
140Æ7
Erythro-(Æ)-3
NIb
(2S,3R)-3
(2R,3S)-3
0.71Æ0.2
2.2Æ0.6
320Æ28
7.2Æ0.3
1.1Æ0.07
5.7Æ0.4
0.03Æ0.003 5.4Æ0.5
Figure 1. Time dependent loss of CPA activity by threo-(Æ)-3 (sub-
strate, Hip-l-Phe, [S]=250 mM; [E]=1.3 mM; Tris buffer of pH 7.5;
temperature, 25 ꢁC).
aRef. 21.
bNI means no irreversible inhibition.
Figure 3. Time dependent loss of CPA activity by threo-(Æ)-3 in the
absence and presence of (Æ)-2-benzylsuccinic acid (substrate, Hip-l-
Phe, [S]=250 mM; [E]=1.3 mM; Tris buffer of pH 7.5; temperature,
25 ꢁC).
Figure 2. The double reciprocal plot of kobs versus [I]0 for the inacti-
vation of CPA by threo-(Æ)-3.

916
M. Lee, D. H. Kim / Bioorg. Med. Chem. 10 (2002) 913–922
KI and kinact were estimated from the respective double
reciprocalpolt according to the method of Kitz and
Wilson23 as exemplified by Figure 2, and are listed in
Table 1. A protection of the CPA inhibition by threo-3
was observed when CPA was preincubated with (Æ)-2-
benzylsuccinic acid, a known active site directed com-
petitive inhibitor of CPA,24 indicating that the inactiva-
tion by threo-3 takes place at the active site (Fig. 3).
(2S,3R)-3 and (2R,3S)-3 were assayed as competitive
inhibitors for CPA as well and the inhibition constant
Ki values were estimated from the Dixon plot25 (Fig. 4).
Erythro-(Æ)-3 failed to show the time-dependent loss of
the enzymic activity.
group on the CPA inactivation is stereospecific, that is,
the methylgroup in (2 R,3S)-3 brings about much more
profound effect than that in (2S,3R)-3. In the case of 2,
the stereoisomer having the (2S,3R)-configuration is
more active than its enantiomer by 2.6-fold.21 The (2S)-
configuration in these inhibitors corresponds to the d-
series. The replacement of the a-H in (2S,3R)-2 with a
methylgroup renders increase of the KI value by 12-fold
and decrease of the kinact by 2.3-fold with resultant
decrease of the kinact/KI value by 26-fold. In compar-
ison, the methylsubstitution at the
a-position of
(2R,3S)-2 caused increase of the KI value by 16-fold but
there was a profound 28-fold decrease of the kinact
value, indicating that the covalent modification reaction
is retarded markedly by the substitution.
Results and Discussion
Covalent modification reaction leading to inactivation
of an enzyme takes place in the complex that is formed
by binding the inhibitor to the enzyme at the active site.
In the inactivation of CPA by (2R,3S)- and (2S,3R)-2
the carboxylate of Glu-270 is known to attack at the 3-
position of the oxirane heterocycle of the enzyme-bound
inhibitor.9,10 In this SN2 type ring cleavage of the oxi-
rane ring, the carboxylate is known to attack the elec-
trophilic center from the direction of 180ꢁ to the
cleavable C–O bond of the ring. Therefore, in order for
the covalent bond formation reaction to take place at
the active site of CPA, the carboxylate nucleophile
should be disposed spatially on the same line of the
cleavable C3–O bond of the oxirane ring in the enzyme-
bound inhibitor. Apparently, in the inactivation of CPA
by (2R,3S)- and (2S,3R)-2, the requisite spatial
arrangement for the nucleophilic cleavage of the oxirane
ring is well satisfied. The kinetic results obtained with
(2R,3S)- and (2S,3R)-3 may be rationalized by the pro-
position that the methylgroup of (2 R,3S)-3 may not fit
in the so-called a-methylhoel when the inhibitor binds
CPA and as a result the half of the molecule including
the oxirane ring would be pushed outward compared
with that of the enzyme-bound (2R,3S)-2. As a con-
sequence, the oxirane ring in (2R,3S)-3 and the carbox-
ylate of Glu-270 are no longer optimally aligned for the
SN2 type ring cleavage reaction. Accordingly, the rate of
the covalent modification reaction of the catalytic car-
boxylate, that is, the kinact, value would be diminished.
Figure 5A depicts schematically the binding mode of
(2R,3S)-3 to CPA and the ensuing covalent modifica-
tion reaction of the carboxylate. On the other hand, in
binding of (2S,3R)-3 to CPA the a-methylgroup woudl
The threo form of 2 are rationally designed fast acting
and highly potent pseudomechanism-based inactivators
of CPA,8 which inhibits the enzyme irreversibly by
covalent modification of the carboxylate of Glu-270.9,10
In the present study, we have evaluated an analogue of
2 in which the a-H is replaced with a methyl group,
expecting that the new inhibitor would have improved
inactivating property for CPA. This expectation was
based on the proposition set forth by Asante-Appiah et
al.7 that there is present a small cavity at the active site
of CPA. The cavity referred by them to as the a-methyl
hole is situated next to the substrate recognition pocket
at the S1 subsite of CPA and thus can accommodate a
methylgroup at the a-position of C-terminalresidue of
substrate and inhibitor. The unexpectedly high CPA
inhibitory potency (Ki=0.11 mM) shown by 1 has been
attributed to the additionalinteractions of the methyl
group with the cavity.7
Like inactivator 2, only the threo form of 3 showed CPA
inactivating activity. As can be seen from Table 1, threo-
(Æ)-3 is less active than the parent inhibitor, that is
threo-(Æ)-2 by 42-fold26 in terms of the second order
inactivation rate constant (kinact/KI), which reflects
effectiveness of an enzyme inctivator. Erythro-(Æ)-3 did
not exhibit the CPA inactivation activity, which is in
line with the observation made with inhibitor 2. It was
thought to be of interest to examine inhibitory potency
of each enantiomer of threo-3 and the effect of the a-
methylgroup on the inactivation of CPA. We have
found that (2S,3R)-3 is more potent than its enantiomer
by 44-fold, indicating that the effect of the a-methyl
Figure 4. The Dixon plot for the inhibition of CPA with (2S,3S)-3 (a) and (2R,3S)-3 (b). (substrate, Cl-CPL; Tris buffer of pH 7.5; temperature,
25 ꢁC).

M. Lee, D. H. Kim / Bioorg. Med. Chem. 10 (2002) 913–922
917
Figure 5. Proposed binding modes of (2R,3S)- and (2S,3R)-3 to CPA and ensuing nucleophilic attack of the Glu-270 carboxylate on the oxirane of
the enzyme-bound inhibitors, leading to covalent modification of the enzyme.
not experience the van der Waals repulsive interactions
since the methylgroup is expected to rest in the open
space filled with water. Thus, the spatial alignment
required for the nucleophilic ring cleavage reaction is
the proposition that the two reaction centers for the
covalent bond formation reaction, that is, the carbox-
ylate of Glu-270 and the oxirane of the inhibitor are
disarranged from the optimal alignment for the nucleo-
philic ring cleavage reaction due to the van der Waals
repulsive interactions between the methyl group in the
inhibitor and the bottom of the active site crevice of CPA.
The present study highlights that the proper alignment of
the two reaction centers is crucialin order for a chemical
reaction to take place at the active site of an enzyme.
Furthermore, this study casts a doubt on the validity for
the presence of the so-called a-methylhoel at the active
site of CPA and efforts to improve the potency of existing
inhibitors for CPA by introduction of a small alkyl group
at the a-position to its carboxylate may not be fruitful.
.
maintained in this CPA (2S,3R)-3 complex. The ring
opening reaction for the covalent modification of CPA
would proceed at the rate comparable to that for the
inactivation of CPA by (2S,3R)-2 (Fig. 5B). As a con-
sequence, (2S,3R)-3 is 44-fold more potent than
(2R,3S)-3 in terms of the kinact/KI value. The lower Ki
value of 1.1 mM obtained with (2S,3R)-3 compared
with that (5.7 mM) for (2R,3S)-3 tends to support the
proposition that the reduced binding affinity of (2R,3S)-
3 is due to the methylgroup projected toward the bot-
tom of the active site cleft preventing the inhibitor from
binding to the enzyme active site. The Ki value repre-
sents reversible inhibitory constant that reflects the affi-
nity of an inhibitor for enzyme. Recently, we have
reported about the effect of the a-methylgroup on the
CPA-catalyzed hydrolysis of ester substrate: Hippuryl-
l-phenyllactic acid and hippuryl-l-a-methylphenyllactic
acid were found to hydrolyze essentially at the same
rate, suggesting that the attack of the carboxylate of
Glu-270 of CPA is not deterred by the a-methyl
group.27 This observation suggests that the diminution
of the CPA inactivation rate caused by the introduction
of a methylgroup at the a-position of (2R,3S)-2 may
not be due to the methylgroup blocking the carboxylate
to attack the electrophilic center of the oxirane ring in
the inhibitor but likely due to disarranging the spatial
alignment of the two reaction centers for the covalent
modification of the Glu-270 carboxylate.
Experimental
All chemicals were of reagent grade obtained from
Aldrich Chemical Co. and solvents were purified before
use. Flash chromatography was performed on silica gel
60 (230–400 mesh) and thin layer chromatography
(TLC) was carried out on silica coated glass sheets
(Merck silica gel 60 F-254). All melting points were
determined on a Thomas-Hoover capillary melting
1
point apparatus and were not corrected. H NMR and
13C NMR spectra were measured on a Bruker AM 300
(300 MHz) instrument using tetramethylsilane as the
internalstandard. IR spectra were recorded on a Bruker
Equinox 55 FT-IR spectrometer. Opticalrotations were
measured on a Rudolp Research Autopol III digital
polarimeter. Mass spectra and elemental analyses were
performed at CentralMachine and Facitlies Shop,
Pohang University of Science and Technology, Pohang,
Korea. High resolution mass spectra were performed by
Mass Spectrometry Analysis Team, Korea Basic Science
Institute, Taegu, Korea.
Conclusion
Contrary to the expectation that the substitution of the
a-H of 2, a pseudomechanism-based inactivator for
CPA, with a methylgroup woudl augment the inhibi-
tory potency through the additionalinteractions of the
methylgroup with the so-caled a-methylhole at the active
site of CPA, the inactivation potency of the a-methylated
inhibitor was lowered. For example, the a-methylation of
(2R,3S)-2 results in the decrease of the second order rate
constant by 453-fold. This diminution of the second
order inactivation rate constant may be rationalized by
Methyl 2 - methyl - 3 - oxobutanoate (5). A mixture of
methyliodide (9.4 mL, 0.15 mo)l, methylacetoacetate
(16.2 mL, 0.15 mol) and anhydrous powdered K2CO3
(20.7 g, 0.15 mol) in MeOH (100 mL) was refluxed. As
the refluxing started, the evolution of carbon dioxide
was observed. When the gas evolution ceased (ꢂ3 h),
the reaction mixture was poured on to cold water (30

918
M. Lee, D. H. Kim / Bioorg. Med. Chem. 10 (2002) 913–922
mL) and the resulting mixture was concentrated to
about 50 mL under reduced pressure. The residue was
extracted with ethylacetate (3 Â50 mL). The combined
extracts were washed with 0.5 N HCl(50 mL), dried
over MgSO4, and evaporated. The residue thus
obtained was purified by column chromatography to
give a colorless oil (14.6 g, 87.5%). IR (neat) 1738, 1720
20.5, 21.7, 43.5, 45.1, 45.9, 50.4, 52.2, 54.0, 54.4, 63.3,
63.4, 114.4, 127.0, 127.4, 128.4, 128.7, 130.4, 130.7,
137.0, 137.2, 137.6, 141.8, 174.6, 176.0.
2-Benzyl-2-methylbut-3-enoic acid (9). Lithium hydrox-
ide (960 mg, 33 mmol) solution in water (10 mL) was
added to a solution of 1:1 mixture of 8 and methyl2-
benzyl-2-methyl-3-chlorobutanoate (2.4 g, 11 mmol)
dissolved in THF/MeOH (3:1, 30 mL). The reaction
mixture was stirred at room temperature for 24 h. After
evaporation of the solvent under reduced pressure, the
residue was extracted with ethylacetate (3 Â30 mL). The
combined extracts were washed with brine, dried over
MgSO4, and evaporated to afford methyl2-benzy-l2-
methyl-3-chlorobutanoate (1.1 g, 80%). The basic aqu-
eous solution was then acidified with 2 N HCl (40 mL)
and extracted with ethylacetate (3 Â30 mL). The com-
bined extracts were washed with brine and dried over
MgSO4. Evaporation of the solvent gave the desired
product (0.9 g, 85.9%). 9: IR (neat) 1703 cmÀ1; FAB
HRMS calcd for C12H15O2 (MH+) 191.1072, found
1
cmÀ1; H NMR 300 MHz (CDCl3) d 1.24 (d, J=1.61
Hz, 3H), 2.12 (s, 3H), 3.42 (q, J=1.61 Hz, 1H), 3.62 (s,
3H).
Methyl 2-benzyl-2-methyl-3-oxobutanoate (6). The ben-
zylation was carried out in the same manner as that
used for the synthesis of 5 using benzylbromide instead
of iodomethane. The crude product was recrystallized
from hexane to give colorless crystal in 88.5% yield.
Mp=58–59 ꢁC; IR (KBr) 1743, 1715 cmÀ1; FAB MS m/
z 221 (MH+); FAB HRMS calcd for C13H17O3 (MH+)
1
221.1178, found 221.1171; H NMR 300 MHz (CDCl3)
d 1.26 (s, 3H), 2.12 (s, 3H), 3.01, 3.25 (2d, J=13.7 Hz,
2H), 3.67 (s, 3H), 7.03–7.25 (m, 5H); 13C NMR
300 MHz (CDCl3) d 19.3, 26.8, 40.9, 52.7, 61.2, 127.2,
128.6, 130.5, 136.8, 173.2, 205.5. Anal. calcd for
C13H16O3: C, 70.89; H, 7.32. Found: C, 71.17; H, 7.11.
1
191.1069; H NMR 300 MHz (CDCl3) d 1.28 (s, 3H),
2.89, 3.12 (2d, AB, J=13.2 Hz, 2H), 5.10 (d, J=17.5
Hz, 1H), 5.16 (d, J=10.8 Hz, 1H), 6.13 (dd, J=10.8,
17.8 Hz, 1H), 7.11–7.15 (m, 5H); 13C NMR 300 MHz
(CDCl3) d 20.2, 45.7, 50.3, 115.2, 127.2, 128.5, 130.9,
137.3, 141.2, 182.8. methyl 2-benzyl-2-methyl-3-chloro-
butanoate: 1H NMR 300 MHz (CDCl3) d 1.24, 1.25 (2s,
3H), 1.49, 1.58 (2d, AB, J=6.6, 6.8 Hz, 3H), 2.68–3.20
(m, 2H), 3.62, 3.64 (2s, 3H), 4.46, 4.55 (2q, J=6.8, 6.6
Hz, 1H), 7.11–7.31 (m, 5H); 13C NMR 300 MHz
(CDCl3) d 15.3, 19.0, 21.7, 43.5, 45.1, 52.2, 54.0, 54.4,
63.3, 63.4, 127.4, 128.7, 130.4, 137.0, 137.2, 174.6.
Methyl 2-benzyl-2-methyl-3-hydroxybutanoate (7). To a
solution of 6 (2.2 g, 10 mmol) in MeOH (10 mL) was
added NaBH4 (380 mg, 10 mmol) in water (0.5 mL) at
0 ꢁC. The reaction mixture was stirred at room tem-
perature for 1 h and quenched with 2 N HCl(10 mL)
followed by extraction with ethyl acetate (3Â20 mL).
The combined extracts were washed with brine (30 mL),
dried over MgSO4, and evaporated under reduced pres-
sure. The residue was purified by column chromato-
graphy to give a colorless oil (1.9 g, 85.5%). IR (neat)
3418, 1725 cmÀ1; FAB MS m/z 239 (MH+), 221
Benzyl 2-benzyl-2-methylbut-3-enoate (10). To a stirred
solution obtained by dissolving 9 (0.50 g, 2.65 mmol) in
10 mL of MeOH was added Cs2CO3 (0.86 g, 2.65 mmol)
and the stirring continued for 30 min. The mixture was
concentrated in vacuo and the resulting white solid was
suspended in 10 mL of DMF. To this solution, benzyl
bromide (330 mL, 2.78 mmol) was added and the
resulting mixture was stirred overnight at room tem-
perature. The reaction mixture was diluted with water
(20 mL) and extracted with ethylacetate (3 Â10 mL).
The combined extracts were washed with water, 0.5 N
HCl, and brine, dried over MgSO4, and evaporated
under reduced pressure to give a crude product which
was purified by column chromatography to afford 10
(0.65 g, 88.2%). IR (neat) 1722 cmÀ1; FAB HRMS
calcd for C18H21O3 (MH+) 285.1491, found 285.1485;
1H NMR 300 MHz (CDCl3) d 1.29 (s, 3H), 2.89, 3.12
(2d, AB, J=13.2 Hz, 2H), 5.10 (d, J=17.4 Hz, 1H),
5.14 (s, 2H), 5.15 (d, J=10.7 Hz, 1H), 6.14 (dd, J=10.7,
17.4 Hz, 1H), 7.07–7.38 (m, 5H); 13C NMR 300 MHz
(CDCl3) d 17.2, 45.9, 50.4, 66.9, 114.6, 127.0, 128.4,
128.5, 128.9, 130.7, 136.4, 137.5, 141.7, 175.4.
1
(MH+ÀH2O); H NMR 300 MHz (CDCl3) d 1.06, 1.08
(2s, 3H), 1.19 (d, J=6.3 Hz, 3H), 2.76, 2.83 (2d, AB,
J=13.2 Hz, 2H), 3.06, 3.19 (2d, AB, J=13.2 Hz, 1H),
3.60, 3.66 (2s, 3H), 3.75–3.81, 3.98–4.02 (m, 1H), 7.07–
7.27 (m, 5H); 13C NMR 300 MHz (CDCl3) d 16.2, 17.6,
41.8, 42.9, 52.0, 52.9, 72.0, 126.9, 128.5, 130.4, 137.4,
138.8, 176.9, 177.4.
Methyl 2-benzyl-2-methylbut-3-enoate (8). To an ice-
chilled solution obtained by dissolving 7 (1.8 g, 8.1
mmol) in pyridine (2 mL) was added SOCl2 (3.5 mL,
24.3 mmol) and then the reaction mixture was stirred at
50 ꢁC for 24 h. The reaction was quenched by pouring
into 30 mL of ice water and extracted with ethylacetate
(3Â10 mL). The organic layer was dried over MgSO4
and evaporated to afford yellowish oil. The crude pro-
duct was purified by column chromatography to give 1:1
mixture of 8 and methyl 2-benzyl-2-methyl-3-chloro-
butanoate (1.2 g, 68.5%). 8: IR (neat) 1722 cmÀ1; FAB
HRMS calcd for C13H17O2 (MH+) 205.1229, found
1
205.1219; H NMR 300 MHz (CDCl3) d 1.24, 1.25 (2s,
3H), 1.28 (s, 3H), 1.49, 1.58 (2d, AB, J=6.6, 6.8 Hz,
3H), 2.68–3.20 (m, 2H), 2.89, 3.12 (2d, AB, J=13.2 Hz,
2H), 3.62, 3.64 (2s, 3H), 3.68 (s, 3H), 4.46, 4.55 (2q,
J=6.8, 6.6 Hz, 1H), 5.11 (d, J=17.6 Hz, 1H), 5.16 (d,
J=10.8, 1H), 6.13 (dd, J=17.6, 10.8 Hz, 1H), 7.11–7.31
(m, 10H); 13C NMR 300 MHz (CDCl3) d 15.3, 19.0,
Benzyl 2-benzyl-2-methyl-3,4-epoxybutanoate (threo-(Æ)
-11) and erythro-(Æ)-11). To a mixture of 10 (560 mg,
2.0 mmol) in CH2Cl2 (40 mL) and K2HPO4 (480 mg,
12.0 mmol) in water (0.5 mL) was added mCPBA (3.76
g, 12.0 mmol, 50–60%) at 0 ꢁC. The solution was stirred
vigorously at rt for 48 h. The white precipitate was

M. Lee, D. H. Kim / Bioorg. Med. Chem. 10 (2002) 913–922
919
1
filtered off and washed with CH2Cl2. The filtrate was
washed with 5% aqueous NaHCO3 solution, water and
brine, then dried over MgSO4 and evaporated under
reduced pressure. The residue (a mixture of four dia-
stereomers) was separated into threo and erythro iso-
mers by column chromatography on silica gel to give
180 mg (30%) of erythro-(Æ)-11, and 330 mg (55%) of
195.1021, found 195.1019; H NMR 300 MHz (CDCl3)
d 1.16 (s, 3H), 2.89–3.01 (dd, 2H), 3.58–3.59 (d, 2H),
7.18–7.32 (m, 5H); 13C NMR 300 MHz (CDCl3) d 19.5,
41.1, 49.0, 67.3, 127.1, 128.6, 130.8, 136.7, 181.8. Anal.
calcd for C11H14O3: C, 68.02; H, 7.27. Found: C, 68.28;
H, 7.11.
threo-(Æ)-11. threo-(Æ)-11: IR (neat) 3028, 1722 cmÀ1
;
Benzyl (S)-2-benzyl-2-methyl-3-hydroxypropanate (14).
To a MeOH (30 mL) solution of 13 (1.7 g, 8.75 mmol)
was added Cs2CO3 (2.85 g, 8.75 mmol) and the stirring
was continued for 30 min. The mixture was con-
centrated in vacuo and the resulting white solid was
suspended in 30 mL of DMF. To this solution, benzyl
bromide (1.0 mL, 8.75 mmol) was added and the
resulting mixture was stirred overnight at rt. The reac-
tion mixture was diluted with water (50 mL) and
extracted with ethylacetate (3 Â30 mL). The combined
extracts were washed with water, 0.5 N HCl, and brine,
then dried over MgSO4, and evaporated under reduced
pressure to give a crude product which was purified by
column chromatography to afford 14 (1.50 g, 60%) as
FAB MS m/z 297 (MH+); FAB HRMS calcd for
1
C19H21O3 (MH+) 297.1491, found 297.1491; H NMR
300 MHz (CDCl3) d 1.02 (s, 3H), 2.56 (m, 1H), 2.72 (t,
J=4.2 Hz, 1H), 2.96, 3.04 (2d, AB, J=13.4 Hz, 2H),
3.34 (m, 1H), 7.16–7.31 (m, 5H); 13C NMR 300 MHz
(CDCl3) d 15.3, 42.4, 43.9, 47.7, 55.9, 65.9, 126.9, 128.3,
128.7, 130.3, 136.0, 136.5, 174.5. erythro-(Æ)-11: IR
(neat) 3028, 1722 cmÀ1; FAB MS m/z 297 (MH+); FAB
HRMS calcd for C19H21O3 (MH+) 297.1491, found
1
297.1489; H NMR 300 MHz (CDCl3) d 1.04 (s, 3H),
2.71–2.76 (m, 2H), 2.97, 3.08 (2d, AB, J=13.3 Hz, 2H),
3.19 (t, J=3.4 Hz, 1H), 5.11 (s, 2H), 7.09–7.35 (m, 10
H), 9.90 (br s, 1H); 13C NMR 300 MHz (CDCl3) d 17.1,
44.0, 45.0, 48.0, 55.2, 67.0, 127.1, 128.6, 128.7, 129.0,
130.5, 136.0, 137.0, 174.5.
25
an oil. ½aŠD À3:0ꢁ(c 1.0, CHCl3); IR (neat) 3450, 1735
cmÀ1; FAB MS m/z 285 (MH+), 267 (MH+ÀH2O); H
1
NMR 300 MHz (CDCl3) d 1.24 (s, 3H), 3.01 (s, 2H),
3.63, 3.72 (2d, AB, J=11.13 Hz, 2H), 5.20 (s, 2H), 7.17–
7.44 (m, 5H); 13C NMR 300 MHz (CDCl3) d 19.8, 41.5,
49.5, 67.0, 127.1, 128.6, 129.1, 130.8, 136.2, 176.9.
threo-(Æ)-2-Benzyl-2-methyl-3,4-epoxybutanoic acid
(threo-(Æ)-3). A methanol(3 mL) soul tion containing
threo-(Æ)-11 (300 mg, 1.0 mmol) and a catalytic
amount of Pd/C was stirred under an atmosphere of H2
gas at rt. After 2 h, the reaction mixture was filtered
through Celite to remove the catalyst and concentrated
in vacuo to give threo-(Æ)-3 (200 mg, 95%) as an color-
less oil. IR (neat) 3028, 1706 cmÀ1; FAB HRMS calcd for
Benzyl (S)-2-benzyl-2-methyl-2-formylacetate (15). To a
solution of oxalyl chloride (360 mL, 4.2 mmol) in anhy-
drous CH2Cl2 (3 mL) was added dropwise a solution of
DMSO (390 mL, 5.6 mmol) in CH2Cl2 (2 mL) at À63 ꢁC
(CHCl3/dry ice) over a period of 15 min and the mixture
was further stirred for 10 min at À63 ꢁC. To this solu-
tion, the solution of 14 (0.79 g, 2.8 mmol) in CH2Cl2 (20
mL) was then added dropwise over 10 min at À63 ꢁC.
After stirring the resulting slightly cloudy solution for
10 min at À63 ꢁC, Et3N (1.5 mL, 11.2 mmol) in CH2Cl2
(3 mL) was added dropwise over 15 min. The reaction
mixture was stirred overnight at rt and the reaction was
quenched by adding water (4.2 mL) to the rapidly stir-
red reaction solution at À63 ꢁC. The resulting slurry was
immediately poured into hexane (30 mL) and washed
with 100 mL of 20% saturated aqueous KHSO4. The
organic layers was collected and the aqueous layer was
back extracted with ether (2Â30 mL). The combined
organic layers were washed with saturated aqueous
NaHCO3, water, and brine, then dried over MgSO4,
and concentrated in vacuo to provide the crude product
which was purified by column chromatography to give
1
C12H15O3 (MH+) 207.1021, found 207.1011; H NMR
300 MHz (CDCl3) d 1.02 (s, 3H), 2.55–2.57 (m, 1H),
2.72 (t, J=4.2 Hz, 1H), 2.96, 3.04 (2d, AB, J=13.4 Hz,
2H), 3.33–3.36 (m, 1H), 7.16–7.31 (m, 5H); 13C NMR
300 MHz (CDCl3) d 15.9, 42.4, 43.9, 47.7, 55.9, 66.8,
127.4, 128.7, 130.7, 136.4, 181.0.
erythro-(Æ)-2-Benzyl-2-methyl-3,4-epoxybutanoic acid
(erythro-(Æ)-3). The hydrogenolysis was carried out as
described for the preparation of threo-(Æ)-3. IR (neat)
3028, 1706 cmÀ1; FAB HRMS calcd for C12H15O3
1
(MH+) 207.1021, found 207.1010; H NMR 300 MHz
(CDCl3) d 1.11 (s, 3H), 2.80–2.85 (m, 2H), 3.05, 3.14
(2d, AB, J=13.3 Hz, 2H), 3.24 (t, J=3.2 Hz, 1H), 7.22–
7.36 (m, 5H); 13C NMR 300 MHz (CDCl3) d 15.9, 42.5,
44.5, 47.7, 56.2, 127.4, 128.7, 130.7, 136.4, 181.0.
(S)-2-Benzyl-2-methyl-3-hydroxypropananoic acid (13).
To a solution of 1214 (4.0 g, 18.0 mmol) in dry THF (45
mL) was added dropwise borane dimethylsulfide (12.6
mL of 2 M solution in THF, 21.6 mmol) at 20 ꢁC. The
mixture was stirred for 6 h, then chilled at 0 ꢁC, quen-
ched with water (30 mL), and extracted with ethylace-
tate (3Â10 mL). The combined organic extracts were
washed with brine, dried over MgSO4, and con-
centrated. The resulting product was purified by column
chromatography on silica gel followed by recrystalliza-
tion from ether and hexane (3.4 g, 97%). mp=73.5–
25
15 (0.70 g, 89%) as an oil. ½aŠD À18:9ꢁ(c 1.0, CHCl3); IR
(neat) 1740, 1735 cmÀ1; FAB MS m/z 283 (MH+), 254
(MH+ÀH2O); FAB HRMS calcd for C18H19O3 (MH+)
1
283.1334, found 283.1358; H NMR 300 MHz (CDCl3)
d 1.28 (s, 3H), 3.07, 3.24 (2d, AB, J=13.6 Hz, 2H), 5.20
(s, 2H), 7.09–7.30 (m, 5H), 9.76 (s, 1H); 13C NMR
300 MHz (CDCl3) d 17.3, 40.7, 52.9, 59.3, 49.5, 69.6,
127.4, 128.7, 130.4, 135.8, 172.5, 199.7.
Benzyl (S)-2-benzyl-2-methylbut-3-enoate (16). KHMDS
(11.8 mL of 0.5 M solution in toluene, 5.22 mmol) was
added using a cannula to an ice-cooled solution of
methyltriphenylphosphonium bromide (1.86 g, 5.25
25
ꢁ
74.5 ꢁC; C½aŠD À12:6ꢁ(c 0.54, MeOH); IR (KBr) 3440,
3030, 1735 cmÀ1; FAB MS m/z 195 (MH+), 177
(MH+ÀH2O); FAB HRMS calcd for C11H15O3 (MH+)

920
M. Lee, D. H. Kim / Bioorg. Med. Chem. 10 (2002) 913–922
mmol) in THF (10 mL). The mixture was stirred vigor-
ously for 1 h, at which time it was cooled to À78 ꢁC.
Compound, 15 (0.70 g, 2.5 mmol) dissolved in THF (5
mL) was then added to the mixture using a cannula.
After being stirred at À78 ꢁC for 15 min, the mixture
was allowed to slowly warm to rt and then heated at
40 ꢁC for 12 h. The mixture was then cooled to rt and
quenched with methanol(1.0 mL) fool wed by addition
of aqueous solution of 20% Rochelle salts (5 mL). The
mixture was extracted with ethylacetate (3 Â20 mL).
The combined extracts were washed with water, brine,
and dried over MgSO4, and concentrated. The resulting
crude alkene was purified by chromatography on silica
gelto give 16 (310 mg, 45%) as a colorless oil.
Benzyl (R)-2-benzyl-2-methylbut-3-enoate (22). Com-
pound 22 was obtained from 21 following the procedure
25
used for the preparation of 16 in 45% yield. ½aŠD þ4:1ꢁ
(c 1.0, CHCl3).
Benzyl (2R,3S)-2-benzyl-2-methyl-3,4-epoxybutanoate
(23). Compound 23 was obtained from 22 following
the procedure used for the preparation of 17 in 50%
25
yield. ½aŠD þ20:4ꢁ (c 1.1, CHCl3).
(2R,3S) - 2 - Benzyl - 2 - methyl - 3,4 - epoxybutanoic acid
((2R,3S) -3). Compound (2R,3S)-3 was obtained from
19 following the procedure used for the preparation of
25
(2S,3R)-3 in 95% yield. ½aŠD þ18:8ꢁ (c 0.1, MeOH).
25
½aŠD À4:0ꢁ(c 1.0, CHCl3); IR (neat) 3027, 1722 cmÀ1; 1H
NMR 300 MHz (CDCl3) d 1.28 (s, 3H), 3.07, 3.24 (2d,
AB, J=13.6 Hz, 2H), 5.20 (s, 2H), 7.09–7.30 (m, 5H),
9.76 (s, 1H); 13C NMR 300 MHz (CDCl3) d 17.2, 45.9,
50.3, 66.9, 114.6, 127.0, 128.4, 128.5, 128.9, 130.7, 136.4,
137.5, 141.7, 175.4.
(2R,3S)-2-Benzyl-2-methyl-3-hydroxysuccinic acid (25).
Compound
(2R,3S)-2-benzyl-2-methyl-3-hydroxy-
succinic acid 1-ethylester ( 24,19 2.50 g, 8.5 mmol) was
dissolved in 48% aqueous solution of HBr (10 mL) in
the presence of n-Bu4NBr (500 mg, 1.5 mmol), and the
resulting mixture was refluxed for 4 h, then extracted
with ethylacetate (3 Â10 mL). The combined organic
layers were washed with water and brine, dried over
MgSO4, and evaporated under reduced pressure to give
the crude product which was recrystallized from
methanoland ethylacetate (1.5 g, 74%). Mp=203–
Benzyl (2S,3R)-2-benzyl-2-methyl-3,4-epoxybutanoate
(17). The synthesis was carried out as described for the
25
preparation of threo-(Æ)-11. ½aŠD À20:3ꢁ(c 1.2, CHCl3);
IR (neat) 3028, 1722 cmÀ1; 1H NMR 300 MHz (CDCl3)
d 1.02 (s, 3H), 2.55–3.57 (m, 1H), 2.72 (t, J=4.2 Hz,
1H), 2.96, 3.04 (2d, AB, J=13.4 Hz, 2H), 3.33–3.36 (m,
1H), 5.18 (s, 2H), 7.16–7.31 (m, 5H); 13C NMR
300 MHz (CDCl3) d 16.2, 42.7, 44.3, 47.9, 56.2, 67.1,
126.9, 128.3, 128.7, 130.3, 136.0, 136.5, 174.9.
25
205 ꢁC; ½aŠD À34:6ꢁ (c 0.95, MeOH); IR (KBr) 3028,
1706 cm?1; FAB HRMS calcd for C12H15O5 (MH+)
1
239.0920, found 239.0925; H NMR 300 MHz (CDCl3)
d 1.12 (s, 3H), 3.03, 3.17 (2d, AB, J=13.3 Hz, 2H), 4.32
(s, 1H), 4.61 (br s, 2H), 7.21–7.27 (m, 5H); 13C NMR
300 MHz (CDCl3) d 16.8, 41.5, 51.5, 75.3, 126.9, 128.3,
130.7, 137.1, 174.8, 177.3. Anal. calcd for C12H14O5: C,
60.50; H, 5.92. Found: C, 60.68; H, 5.71.
(2S,3R) - 2 - Benzyl - 2 - methyl - 3,4 - epoxybutanoic acid
((2S,3R)-3). The synthesis was carried out as described
for the preparation of threo-(Æ)-3 and obtained as an
25
oil. ½aŠD À19:2ꢁ (c 0.5, CHCl3); IR (neat) 3028, 1706
1
cmÀ1; H NMR 300 MHz (CDCl3) d 1.02 (s, 3H), 2.55–
Dibenzyl (2R,3S)-2-benzyl-2-methyl-3-hydroxysuccinate
(26). A solution of n-Bu4NOH (11.0 mL of 1.0 M solu-
tion in MeOH, 11.0 mmol) was added to a solution of
25 (1.3 g, 5.5 mmol) in methanol (20 mL). The mixture
was concentrated in vacuo and the white residue was
suspended in 30 mL of dry DMF/acetonitrile (3:1).
Benzylbromide (1.3 mL, 11.0 mmo)l was added and the
mixture was stirred for 21 h at rt. The reaction mix-
ture was diluted with ether (100 mL), washed with
5% Na2S2O3 solution, 10% citric acid, 5% NaHCO3,
and brine. The organic solution was dried over MgSO4,
and evaporated under reduced pressure to give a crude
product which was purified by column chromatography
followed by recrystallization from petroleum ether
and hexane to afford 26 (1.8 g, 79% yield). Mp=57–
2.57 (m, 1H), 2.72 (t, J=4.2 Hz, 1H), 2.96, 3.04 (2d,
AB, J=13.4 Hz, 2H), 3.33–3.36 (m, 1H), 7.16–7.31 (m,
5H); 13C NMR 300 MHz (CDCl3) d 15.9, 42.4, 43.9,
47.7, 55.9, 127.4, 128.7, 130.7, 136.4, 181.0.
(R)-2-Benzyl-2-methyl-3-hydroxypropananoic acid (19).
An aqueous lithium hydroxide solution (0.324 g of
LiOH in 3 mL of water, 7.7 mmol) was added to a
solution of 1818 (1.33 g, 6.4 mmol) dissolved in THF/
MeOH (3:1, 12 mL). The reaction mixture was stirred at
rt for 12 h. After evaporation of the solvent under
reduced pressure, the residue was acidified with 2 N HCl
(10 mL) and extracted with ethylacetate (3 Â5 mL). The
combined extracts were washed with brine and dried
over MgSO4. Evaporation of the solvent gave 19 (1.18
g, 95%) which was recrystalized from diethyl ether and
25
58 ꢁC; ½aŠD þ11:4ꢁ (c 0.95, CHCl3); IR (KBr) 1725,
3450 cmÀ1; FAB HRMS calcd for C26H27O5 (MH+)
25
hexane. Mp=76–77 ꢁC; ½aŠD þ12:5ꢁ(c 0.5, MeOH).
419.1859, found 419.1847; H NMR 300 MHz (CDCl3)
1
d 1.10 (s, 3H), 2.97, 3.12 (2d, AB, J=13.3 Hz, 2H),
4.24–4.29 (m, 1H), 4.98–5.20 (m, 4H), 7.09–7.34 (m,
5H); 13C NMR 300 MHz (CDCl3) d 17.9, 41.6, 51.7,
67.3, 68.0, 75.8, 127.2, 128.6, 129.1, 129.2, 130.9, 135.3,
135.9, 136.6, 173.0, 174.7. Anal. calcd for C26H26O5: C,
74.62; H, 6.26. Found: C, 74.55; H, 6.05.
Benzyl (R)-2-benzyl-2-methyl-3-hydroxypropanate (20).
Compound 20 was obtained from 19 following the pro-
cedure used for the preparation of 14 in 60% yield.
25
½aŠD þ2:95ꢁ(c 1.0, CHCl3).
Benzyl (R) - 2 - benzyl - 2 - methyl - 3 - formylacetate (21).
Compound 21 was obtained from 20 following the pro-
cedure used for the preparation of 15 in 85% yield.
Benzyl (2R,3S)-2-benzyl-2-methyl-3,4-dihydroxybutano-
ate (27). Borane–dimethyl sulfide complex (1.6 mL of
2.0 M solution in THF, 3.2 mmol) was added dropwise
25
½aŠD þ19:0ꢁ(c 1.0, CHCl3).

M. Lee, D. H. Kim / Bioorg. Med. Chem. 10 (2002) 913–922
921
to a solution of 26 (1.4 g, 3.3 mmol) in dried THF (15
mL) at 0 ꢁC under nitrogen atmosphere, and the mix-
ture was stirred for 1 h. Sodium borohydride (0.05 g)
was slowly added to the mixture and stirring was con-
tinued for 30 min at rt and for 1 h at 30 ꢁC. The reaction
was quenched by slow addition of methanol (1.5 mL).
The mixture was stirred for a further 30 min and then
evaporated under reduced pressure. The residue was
purified by flash column chromatography on silica gel
and recrystallized from petroleum ether and diethyl
ether to give 27 as a white solid (0.7 g, 67% yield).
CPL) was synthesized according to the method reported
by Suh et al.28 A Perkin-Elmer HP 8453 UV–vis spec-
trometer was used for UV absorbance measurement in
kinetic experiments.
Determination of kinact and KI
A series of inactivation experiments were carried out
with inactivation concentrations within the range of 0.5
to 2.0 mM for (Æ)-threo-3, 0.5–1.4 mM for (2S,3R)-3,
and 2.0–5.0 mM for (2R,3S)-3. A solution of the inacti-
vator in 1:1 solution of DMSO and 0.05 M Tris/0.5 M
NaCl, pH 7.5 buffer was added to an enzyme solution in
0.05 M Tris/0.5 M NaCl, pH 7.5 buffer to afford a final
concentration of 1.3 mM enzyme in 15% DMSO for any
given inactivator concentration. The solutions were
incubated at 25 ꢁC. At 2.5 min intervals for (Æ)-threo-3
and (2S,3R)-3, and at 30 min intervals for (2R,3S)-3, 50
mL aliquots of the inactivation mixture were taken and
added to a 950 mL assay mixture containing 50 mL of 5
mM solution of Hip-l-Phe. The increase in the absor-
bance at 254 nm was monitored for the first 40 s of the
reaction immediately. A plot of the natural log of the
residualenzymic activity versus inactivation time gave a
straight line with a slope of Àkobs (Fig. 1). The values of
KI and kinact were calculated from the double reciprocal
plot of the kobs versus concentration of inhibitors
according to the method of Kitz–Wilson (Fig. 2).23
25
Mp=55.5–56.5 ꢁC; ½aŠD þ28:3ꢁ (c 1.0, CHCl3); IR (KBr)
3350, 1720 cmÀ1; EI MS m/z 314 (M+); FAB HRMS
calcd for C19H22O4 (MH+) 315.1596, found 315.1586;
1H NMR 300 MHz (CDCl3) d 1.11 (s, 3H), 2.93, 3.12
(2d, AB, J=13.2 Hz, 2H), 3.49–3.76 (m, 3H), 5.09 (s,
2H), 7.11–7.35 (m, 10H); 13C NMR 300 MHz (CDCl3)
d?18.1, 43.0, 50.0, 63.6, 67.3, 76.5, 126.3, 127.2, 128.6,
128.78, 129.0, 130.7, 135.7, 136.6, 176.9. Anal. calcd for
C19H22O4: C, 72.59; H, 7.05. Found: C, 72.79; H, 6.90.
Benzyl (2R,3S)-2-benzyl-2-methyl-3,4-epoxybutanoate
(23). To a solution of 27 (0.6 g, 1.9 mmol) and freshly
distilled pyridine (290 mL, 3.8 mmol) in CH2Cl2 (5 mL)
was added p-toluene sulfonylchloride (362 mg, 1.9
mmol) at 0 C under nitrogen atmosphere and the mix-
ture was stirred for 48 h at rt. Ethylacetate (10 mL) was
added to the reaction mixture, and the organic phase
was washed with 3 N aqueous hydrochloric acid, water,
and brine. The organic phase was then dried over
MgSO4 and evaporated under reduced pressure. The
residue (28) was used directly in the next reaction.
Powdered potassium carbonate (190 mg, 1.4 mmol) was
added to a solution of 28 (0.7 g, 1.5 mmol) in methanol
(10 mL) while being chilled in an ice bath, and the
resulting mixture was stirred for 10 min at rt. The reac-
tion was quenched with water and extracted with ethyl
acetate (3Â10 mL). The combined extracts were washed
with brine, dried over MgSO4, and evaporated under
reduced pressure and the residue was purified by flash
column chromatography on silica gel to give 23 as a
Active site protection test24
CPA (1.3 mM) was incubated with (Æ)-2-benzylsuccinic
acid (2 and 5 mM) for 10 min at 25 C. Subsequently,
(2S,3R)-3 (or (2R,3S)-3) was added to the mixture to
give a finalinactivator concentration of 500 mM. At 2.5-
min intervals, 50 mL aliquots of the incubation mixture
were removed and added to a 950 mL assay mixture con-
taining 250 mM of Hip-l-Phe and the remaining enzymic
activity was monitored at 254 nm at 25 ꢁC (Fig. 3).
Determination of Ki
25
colorless oil (340 mg, 60%). ½aŠD þ19:9ꢁ (c 0.75, CHCl3);
IR (neat) 1725, 1270 cmÀ1; 1H NMR 300 MHz (CDCl3)
d 1.02 (s, 3H), 2.53–2.56 (m, 1H), 2.69 (t, J=4.2 Hz,
1H), 2.91, 3.02 (2d, AB, J=13.3 Hz, 2H), 3.31–3.34 (m,
1H), 5.16 (s, 2H), 7.05–7.34 (m, 10H); 13C NMR
300 MHz (CDCl3) d 16.2, 42.7, 44.3, 47.9, 56.3, 67.2,
127.2, 128.5, 128.6, 130.0, 136.1, 136.6, 174.9.
Two different concentrations of Cl-CPL (50 and 100
mM) were used. A series of assay mixtures containing
both the substrate (Cl-CPL) and various concentrations
of the inactivator [in the range 0.5–2.0 mM for (2S,3R)-
3 and 1.0–10.0 mM for (2R,3S)-3] were prepared in
0.05 M Tris/0.5 M NaCl, pH 7.5 buffer. Enzyme stock
solution was added to assay mixture to afford a final
enzyme concentration of 20 nM in a totalvoulme of
1000 mL. The initialrates of enzymic reaction were
measured immediately using a microcomputer-inter-
faced UV spectrometer. The Ki values were then esti-
mated from the semireciprocal plot of the initial velocity
versus the concentration of the inhibitors according to
the method of Dixon (Fig. 4).25
General remarks for kinetic experiments
All solutions were prepared by dissolving in double dis-
tilled and deionized water. Stock assay solutions were
filtered 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 purification. CPA stock
solutions were prepared by dissolving the enzyme in
0.05 M Tris/0.5 M NaCl, pH 7.5 buffer solution.
Enzyme concentrations were estimated from the absor-
bance at 278 nm (e278=64,200). Hippuryl-l-phenylala-
nine (Hip-l-Phe) was purchased from Sigma Chemical
Co. O-(trans-p-chlorocinnamyl)-l-b-phenyllactate (Cl-
Dialysis
Solutions of the inactivators (1–10 mM) and CPA (1
mM) in 0.05 M Tris/0.5 M NaCl, pH 7.5 buffer were
incubated at 4 ꢁC for 48 h. The mixture was then dia-
lyzed for 24 h at rt against 0.05 M Tris/0.5 M NaCl, pH

922
M. Lee, D. H. Kim / Bioorg. Med. Chem. 10 (2002) 913–922
7.5 buffer. The buffer was changed every 6 h. After dia-
lysis, 50 mL aliquots of inactivation mixture were
removed and added to 950 mL of assay mixture. The
remaining enzymic activity was monitored at 254 nm.
The enzyme failed to show the proteolytic activity.
8. Kim, D. H.; Kim, K. B. J. Am. Chem. Soc. 1991, 113, 3200.
9. Yun, M.; Park, C.; Kim, S.; Nam, D.; Kim, S. C.; Kim,
D. H. J. Am. Chem. Soc. 1992, 114, 2281.
10. Ryu, S.-E.; Choi, H.-J.; Kim, D. H. J. Am. Chem. Soc.
1997, 119, 38.
11. Kagabu, S.; Kojima, Y. J. Chem. Edu. 1992, 69, 420.
12. Schwartz, A.; Madan, P. J. Org. Chem. 1986, 51, 5463.
13. Albeck, A.; Persky, R. J. Org. Chem. 1994, 59, 653.
14. Bjorkling, F.; Boutelje, J.; Gatenbeck, S.; Hult, K.; Norin,
T.; Szmulik, P. Tetrahedron 1985, 41, 1347.
Acknowledgements
The authors express their thanks to the Korea Science
and Engineering Foundation for financialsupport of
this work and the Ministry of Education and Human
Resources for the BK21 fellowship provided to M.L.
15. Fadel, A.; Canet, J.-L.; Salaun, J. Tetrahedron Lett. 1989,
30, 6687.
16. Mancuso, A. J.; Huang, S.-L.; Swern, D. J. Org. Chem.
1978, 43, 2480.
17. Fadel, A.; Canet, J.-L.; Salaun, J. Tetrahedron:Asymmetry
1993, 4, 27.
18. Lee, M.; Jin, Y.; Kim, D. H. Bioorg. Med. Chem. 1999, 7,
1755.
References and Notes
19. Renaud, P.; Hurzeler, M.; Seebach, D. Helv. Chim. Acta
1987, 70, 292.
20. Scaman, C. H.; Palcic, M. M.; McPhalen, C.; Gore, M. P.;
Lam, L. K. P.; Vederas, J. C. J. Biol. Chem. 1991, 266, 5525.
21. Lee, S. S.; Li, Z.-H.; Lee, D. H.; Kim, D. H. J. Chem.
Soc., Perkin Trans. 1 1995, 2877.
1. Lipscomb, W. N.; Strater, N. Chem. Rev. 1996, 96,
2375.
2. (a) Christianson, D. W.; Lipscomb, W. N. Acc. Chem. Res.
1989, 22, 62. (b) Quiocho, F. A.; Lipscomb, W. N. Adv. Pro-
tein Chem. 1971, 25, 1.
3. Makinen, M. W.; Yamamura, K.; Kaiser, E. T. Proc. Natl.
Acad. Sci. U.S.A. 1976, 73, 3882.
4. Kuo, L. C.; Makinen, M. W. J. Am. Chem. Soc. 1985, 107, 5255.
5. Kim, D. H.; Kim, K. S.; Park, J. K. Bull. Korean Chem.
Soc. 1994, 15, 805.
22. Saito, S.; Hasegawa, T.; Inaba, M.; Nishida, R.; Fujii, T.;
Nomizu, S.; Moriwake, T. Chem. Lett. 1984, 1389.
23. Kitz, R.; Wilson, I. B. J. Biol. Chem. 1962, 237, 3245.
24. Byers, L. D.; Wolfenden, R. Biochemistry 1973, 12, 2070.
25. Dixon, M. Biochem. J. 1953, 55, 170.
26. The kinact/KI value of 5850 MÀ1 minÀ1 for threo-(Æ)-2 was
used for the calculation of the relative potency.
27. Lee, M.; Kim, D. H. Bioorg. Med. Chem. 2000, 8, 815.
28. Suh, J.; Kaiser, E. T. J. Am. Chem. Soc. 1976, 98, 1940.
6. Phillips, M. A.; Fletterick, R.; Rutter, W. J. J. Biol. Chem.
1990, 265, 20692.
7. Asante-Appiah, E.; Seetharaman, J.; Sicheri, F.; Yang,
D. S.-C.; Chan, W. W.-C. Biochemistry 1997, 36, 8710.