Optically active 2-benzyl-3-methanesulfinylpropanoic acid: Synthesis and evaluation as inhibitors for carboxypeptidase A

Article abstract of DOI:10.1016/S0968-0896(03)00481-4

All four possible stereomers of 2-benzyl-3-methanesulfinylpropanoic acid were synthesized and evaluated as inhibitors for carboxypeptidase A to find that the isomer having the (2S,4S)-configuration is most potent followed by isomers of (2R,4S)- and (2S,4R)-configurations. The stereochemical preferences shown by the isomers of the inhibitor in binding to the enzyme suggest that the sulfoxide oxygen in the inhibitor fails to ligate the active site zinc ion but may form a hydrogen bond with the guanidinium moiety of Arg-127 like the carbonyl oxygen of scissile peptide bond of oligopeptide substrate of the enzyme does. It may thus be inferred that a sulfoxide moiety may serve as an isosterer of a carboxamide moiety.

Full text of DOI:10.1016/S0968-0896(03)00481-4

Bioorganic & Medicinal Chemistry 11 (2003) 4377–4381
Optically Active 2-Benzyl-3-methanesulfinylpropanoic Acid:
Synthesis and Evaluation as Inhibitors for Carboxypeptidase A
Jing-Yi Jin,a Guan Rong Tiana and Dong H. Kimb,*
aDepartment of Chemistry, Yanbian University, 105 Gongyuan Road, YanJi, Jilin Province, 133002, PR China
bCenter for Integrated Molecular Systems, Division of Molecular and Life Sciences, Pohang University of Science and Technology,
San 31Hyoja-dong, Namku, Pohang 790-784, South Korea
Received 12 May 2003; accepted 14 July 2003
Abstract—All four possible stereomers of 2-benzyl-3-methanesulfinylpropanoic acid were synthesized and evaluated as inhibitors
for carboxypeptidase A to find that the isomer having the (2S,4S)-configuration is most potent followed by isomers of (2R,4S)- and
(2S,4R)-configurations. The stereochemical preferences shown by the isomers of the inhibitor in binding to the enzyme suggest that
the sulfoxide oxygen in the inhibitor fails to ligate the active site zinc ion but may form a hydrogen bond with the guanidinium
moiety of Arg-127 like the carbonyl oxygen of scissile peptide bond of oligopeptide substrate of the enzyme does. It may thus be
inferred that a sulfoxide moiety may serve as an isosterer of a carboxamide moiety.
# 2003 Elsevier Ltd. All rights reserved.
We have been involved in developing design strategies
of enzyme inhibitors that are effective against zinc pro-
teases. These proteolytic enzymes are family of enzymes
having a catalytically essential zinc ion at the active site
and play important roles in the intracellular processing
and degradation of proteins.1 Many of these enzymes
contain a conserved consensus sequence, HEXXH that
ligates the zinc ion at the enzyme active site. In the
endeavor to developenzyme inhibitor design strategies,
we have used carboxypeptidase A (CPA) as a model
target enzyme.2 CPA is a prototypical zinc protease
whose active site structure and catalytic mechanism
have been well established.3 The enzyme catalyzes the
hydrolysis of the terminal amide bond of peptide sub-
strate and shows specificity for oligopeptide having the
C-terminal residue with a hydrophobic side chain, such
as Phe and Tyr. The enzyme also hydrolyzes ester bond
in a compound, structure of which closely resemble the
peptide substrate. At the active site of CPA there is
present, in addition to the zinc ion, the S10 pocket (pri-
mary substrate recognition site) which accommodates
an aromatic ring such as phenyl and hydroxyphenyl
moieties present in Phe and Tyr, respectively. The roles
played by the zinc ion are 2-fold: (i) It takes a water
molecule as the fourth ligand and activates it by low-
ering its pKa, thus it to serve as an effective nucleophile
in the scission of peptide bond, and (ii) stabilizes toge-
ther with the guanidinium moiety of Arg-127 the gem-
diolate tetrahedral transition state that is generated by
the nucleophilic attack of the water molecule on the
sessile peptide bond.
Sulfoxide is a polarized molecule having a partial nega-
tive charge on the oxygen atom, and thus it is expected
that the molecule might be utilized as a zinc coordinat-
ing groupin the design of CPA inhibitors. In this study,
we evaluate 2-benzyl-3-methanesulfinylpropanoic acid
(4) as a novel CPA inhibitor, in which a sulfoxide moi-
ety is incorporated into a molecular scaffold that can be
recognized by CPA and forms a complex. We have
synthesized all four possible stereoisomers of 4 and
investigated their binding mode to CPA. 2-Benzyl-3-
methylthiopropanoic acid (1), an intermediate in the
synthesis of 4 is also evaluated as a CPA inhibitor to
find that it binds the enzyme more strongly than 4.
Results and Discussion
Scheme 1 outlines the synthesis of CPA inhibitor 4 in an
optically active form. 2-Benzyl-3-methylthiopropanoic
acid (1) prepared by a literature method4 was converted
*Corresponding author. Tel.: +82-54-279-2101; fax: +82-54-279-
5877 or +82-54-279-8142; e-mail: dhkim@postech.ac.kr
0968-0896/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/S0968-0896(03)00481-4

4378
J.-Y. Jin et al. / Bioorg. Med. Chem. 11 (2003) 4377–4381
into the corresponding methyl ester (2), and the latter
was then subjected to enzymatic resolution using a-
chymotrypsin, whereby the enantiomer having the (S)-
configuration was selectively hydrolyzed.5 The assigned
stereochemistry for the acid thus obtained was ascer-
tained by the X-ray crystallographic determination of a
final product (4) obtained from (S)-1. The unreacted
(R)-2 was treated once more with a-chymotrypsin to
improve the optical purity. Since attempts at the oxida-
tion of (S)-1 to obtain directly 4 met with a difficulty in
the separation of products, a diastereomeric mixture,
(S)-1 was converted into its methyl ester, (S)-2 and the
latter was oxidized with sodium periodate to give a
mixture of (2S,4R)- and (2S,4S)-3, which was readily
separated on a silica gel column. The hydrolysis of the
ester moiety in (2S,4R)-3 was effected by following the
method of Fisher and Trinkle,6 which involves treat-
ment of the ester with lithium iodide in refluxing anhy-
drous ethyl acetate. The method is known to be highly
efficacious for ester dealkylation of compounds having
an amide carbonyl at the g-position to the ester moiety.
Under the reaction conditions, (2S,4R)-3 was converted
into (2S,4R)-4 without bringing about racemization.
Hydrolysis under basic conditions using an aqueous
lithium hydroxide solution gave an inseparable mixture,
and hydrolysis under acidic conditions (10% HCl)
caused partial racemization at the 4-positon to occur.7
In a similar fashion, remaining three stereoisomers of 4
were synthesized (Scheme 1). The stereochemical
assignments for the final products as well as inter-
Scheme 1.

J.-Y. Jin et al. / Bioorg. Med. Chem. 11 (2003) 4377–4381
4379
mediates in the syntheses are made on the basis of the
[a] values and the crystal structure determined for 4
having [a]=À7.5ꢀ. The latter compound was shown to
have the (2S,4R)-configuration by the analysis of X-ray
diffraction pattern.8
noted that the sulfoxide-bearing inhibitors, (2S,4S)-4
in particular, is a substrate analogue inhibitor for
CPA, binding CPA with its sulfoxide oxygen to form
a hydrogen bond with the guanidinium of Arg-127
like peptide substrate does. In this respect, it may be
stated that sulfoxide moiety serves as an isosterer of a
carboxamide.
The final compounds as well as an intermediate in the
synthesis were assayed for their inhibitory activity
against CPA as described in the literature9 using O-
(trans-p-chlorocinnamoyl)-l-b-phenyllactic acid (Cl-
CPL) as the substrate. The Ki values were estimated
from the respective Dixon plot and are collected in
Table 1. The inhibitory activities exhibited by them are
disappointingly weak but nevertheless enough for
probing the stereospecificity in binding of these inhibi-
tors to CPA. Inhibitor 4 having the (2S,4S)-configur-
ation was found to be most potent among the four
stereoisomers, but its enantiomer having the (2R,4R)-
configuration failed to show a CPA inhibitory activity
upto the concentration of 10 mM. The other two ste-
reoisomers in a mirror image relationship, that is,
(2S,4R)- and (2R,4S)-4 bind CPA poorly but interest-
ingly with nearly equal potency. Previously, Mock and
coworkers investigated CPA inhibitory activity of 5
having a sulfoximine moiety as the zinc ligating group
to reveal that (2S,4R)-5 is more potent than (2S,4S)-5
by 9-fold.10 They have attributed the higher potency of
(2S,4R)-5 over (2S,4S)-5 to the ability of its imino
groupto coordinate the active site zinc ion, suggesting
that the S!O groupin sulfoximine moiety fails to
coordinate the metal ion. This explanation is consistent
with the observation that the inhibitory potency of 2-
benzyl-3-methanesulfonylpropanoic acid (Ki=910 mM)
is much reduced compared with Ki=0.26 mM for
(2S,4R)-5.11 Thus, our observation that there operates a
stereochemical preference in the CPA inhibition with 4
suggests that although the sulfoxide oxygen fails to
interact with the zinc ion, it may form a hydrogen bond
with the guanidinium moiety of Arg-127, and the weak
binding affinity shown by 4 may be surmised on the
ground that a hydrogen bond is much weak compared
with the interactions between a metal ion and ligand.12
Christianson and Lipscomb argued on the basis of X-ray
crystallographic analysis of the binding modes of transi-
tion state analogue inhibitors of CPA13 that not the zinc
ion but the guanidinium of Arg-127 of CPA interacts,
albeit weakly, with the carbonyl oxygen of the scissile
peptide bond, and stabilizes the oxyanion of the gem-diol
tetrahedral transition state generated by the attack of a
water molecule on the scissile bond. The arguement has
been substantiated by Rutter and coworkers by the site
specific mutagenesis study.14 It may then be arguably
It is worth noting that 1 shows a significant CPA inhi-
bitory activity (Table 1). In fact, (S)-1, the stereo-
chemistry of which belongs to the that of the P10 residue
of substrate, i.e., the l-series, was shown to be the most
potent CPA inhibitor having the Ki value of 100 mM
among the compounds evaluated in the present study. 2-
Benzyl-3-mercaptopropanoic acid in which the methyl-
thio moiety in 1 is replaced with a mercapto group is
one of the most potent inhibitors of CPA, and it has
been suggested that its sulfhydryl groupcoordinates the
active site zinc ion in a deprotonated form.15 The dras-
tically reduced binding affinity of 1 in comparison with
2-benzyl-3-mercaptopropanoic acid supports the pro-
position that the thiolate rather than thiol group of
mercapto-bearing CPA inhibitors ligates the zinc ion: if
the mercapto group ligates the zinc ion with its proton
being attached to the sulfur atom, the replacement of
the hydrogen with a methyl groupwould not diminish
the binding affinity in such a drastic extent. Replace-
ment of the sulfur atom in 1 with a carbon to obtain 2-
benzylpentanoic acid16 attenuated the inhibitory
potency by 3-fold, suggesting that the sulfide moiety is
not an effective zinc ligating group.
Conclusion
All four possible stereoisomers of 4 were synthesized and
evaluated as competitive inhibitors of CPA to find that
(2S,4S)-4 binds the enzyme most tightly followed by
(2R,4S)- and (2S,4R)-4. The enantiomer of (2S,4S)-4 fails
to bind the enzyme. The stereochemical preference shown
by these inhibitors suggests that the sulfoxide oxygen of
(2S,4S)-4 may form a hydrogen bond with the guanidi-
nium of Arg-127 like the carbonyl oxygen of scissile pep-
tide bond of substrate when it forms a Michaelis complex
with CPA. It may thus be inferred that a sulfoxide may
serve as an isosterer of carboxamide moiety.
Table 1. Inhibition constants (Ki) for CPA
Compd
Ki (mM)
(RS)-1
(R)-1
(S)-1
(2S, 4R)-4
(2S, 4S)-4
(2R, 4S)-4
(2R, 4R)-4
0.74
1.46
0.1
3.86
0.56
2.79
NI
Experimental
Melting points were taken on a Thomas-Hoover capil-
lary melting point apparatus and were uncorrected. IR
spectra were recorded on a Bruker Equinox 55 FT-IR

4380
J.-Y. Jin et al. / Bioorg. Med. Chem. 11 (2003) 4377–4381
spectrometer. 1H NMR and 13C NMR spectra were
obtained with a Bruker AM 300 (300 MHz) NMR
spectrometer using tetramethylsilane as the internal
standard. High-resolution mass spectra were obtained
at Korea Basic Science Institute, Daejeon, Korea. Silica
gel 60 (230–400 mesh) was used for flash chromato-
graphy and thin layer chromatography (TLC) was car-
ried out on silica coated glass sheets (Merck silica gel 60
F-254). Elemental analyses were performed at Pohang
University of Science and Technology, Pohang, Korea.
CPA was purchased from Sigma Chemical Co. (Allan
form, twice crystallized from bovine pancrease, aqueous
suspension in toluene) and used without further pur-
ification. Cl-CPL, the substrate used in the kinetic
study, was synthesized as described in the literature.9 All
solutions for kinetic study were prepared by dissolving
in doubly distilled and deionized water. CPA stock
solutions were prepared by dissolving CPA in 0.05 M
Tris/0.5 M NaCl, pH 7.5 buffer solution and their con-
centrations were estimated from the absorbance at 278
nm (e278=64,200). The stock assay solutions were fil-
tered (GHP Acrodic syringe filter, pore size 0.2 mm)
before use. Perkin-Elmer HP 8453 UV–vis spectrometer
was used for UV absorbance measurements.
rated with sodium chloride, then extracted with ethyl
acetate (25 mLꢁ4). The extract was dried over MgSO4
and evaporated under reduced pressure. The residue
was purified by column chromatography (silica gel,
EtOAc/n-hexane=1:6) to give (S)-1. Yield, 45%. The
spectral data of (S)-1 are identical with those reported
in the literature4 for 1. Mp104–105 ꢀC. [a]2D0=À8.96 (c
0.05, CHCl3). Anal. calcd for C11H14O2S: C, 62.83; H,
6.71. Found: C, 62.66; H, 6.74.
(R)-2-Benzyl-3-methylthiopropanoic acid [(R)-1]
The crude (R)-2 from the enzymatic resolution of 2 was
treated with a-chymotrypsin in 0.01 M phosphate buffer
(25 mL) in order to improve the optical purity. When a
few mL of NaOH solution (0.2 N) was consumed, the
reaction mixture was extracted with ethyl acetate (30
mLꢁ4), and the combined extract was washed with
sodium bicarbonate solution (5%), dried over MgSO4,
and evaporated under reduced pressure to afford (R)-2
as oil. A portion of the residue (0.8 g) was dissolved in a
mixture of dioxane (8 mL) and LiOH solution (1.0 N,
4.0 mL), and stirred at room temperature for 12 h. The
solution was then acidified to pH 1 with HCl (1.0 N),
and extracted with ether (10 mLꢁ3) and the combined
extract was washed with brine, dried over MgSO4, and
evaporated in vacuo to give a solid residue which was
recrystallized from chloroform and n-hexane to give
(R)-1. Yield, 45%. Mp104–105 ꢀC. [a]D20=+8.70 (c
0.05, CHCl3). Anal. calcd for C11H14O2S: C, 62.83; H,
6.71, found: C, 62.71; H, 6.74.
2-Benzyl-3-methylthiopropanoic acid, methyl ester (2).
To a mixture of 2-benzyl-3-methylthiopropanoic acid
(2.10 g, 10 mmol) and anhydrous methanol (10 mL) was
added thionyl chloride (0.84 mL, 11.5 mmol) and the
resulting solution was stirred at room temperature
overnight. After removing the solvent, the residue was
dissolved in ethyl acetate and the solution was washed
with saturated sodium bicarbonate solution, then with
brine, dried over MgSO4, and evaporated under reduced
pressure to give an oil which was purified by column
chromatography (silica gel, EtOAc/n-hexane=1:3) to
General procedure for oxidation of 2 to obtain
2-benzyl-3-methanesulfinylpropanoic acid,
methyl ester (3)
afford a colorless oil (2.17 g, 97%). IR (film, cmÀ1
)
Typically, (S)-2 (1.38 g, 6.15 mmol) was dissolved in a
50% aqueous methanol solution (124 mL), and to the
resulting solution was added sodium periodate (1.32 g,
6.15 mmol) under stirring at 0 ꢀC. The reaction mixture
was stirred vigorously for 30 min at 0 ꢀC, and most of
the organic solvent was removed under reduced pressure
at 35–40 ꢀC, then was added to aqueous NaS2O3 solu-
tion (5%, 40 mL), and extracted with ethyl acetate (30
mLꢁ3). The combined extracts was washed with brine,
dried over MgSO4, and evaporated under reduced pres-
sure to give a mixture of diastereomers, which was
separated by column chromatography (silica gel,
CHCl3/MeOH=200:3). (2S,4R)-3 (560 mg, 38%). Mp
1736. 1H NMR (300 MHz, CDCl3) d 7.30–7.14 (m, 5H),
3.62 (s, 3H), 3.00–2.89 (m, 3H), 2.77 (dd, J=7.8, 13.0
Hz, 1H). 2.61 (dd, J=5.2, 13.0 Hz, 1H). 2.07 (s, 3H).
13C NMR (75 MHz, CDCl3) d 174.79, 138.93, 129.27,
128.88, 126.95, 52.10, 47.94, 38.18, 36.03, 16.26. HRMS
calcd for C12H16O2S; 224.0871, found: 224.0873.
(S)-2-Benzyl-3-methylthiopropanoic acid, methyl ester
[(S)-2]. This was prepared in a similar fashion. (Yield,
95%). [a]2D0=À4.45 (c 0.23, CHCl3), HRMS calcd for
C12H16O2S; 224.0871, found: 224.0871.
66–68 ꢀC; IR (KBr, cmÀ1): 1730, 1032. 1H NMR H
1
(R)-2-Benzyl-3-methylthiopropanoic acid, methyl ester
[(R)-2]. (Yield, 95%). [a]2D0 À4.45 (c 0.16, CHCl3),
HRMS calcd for C12H16O2S; 224.0871, found:
224.0874.
NMR (300 MHz, CDCl3) d 7.33–7.22 (m, 3H), 7.17–
7.14 (m, 2H), 3.70 (s, 3H), 3.39–3.33 (m, 1H), 3.12 (dd,
J=6.8, 13.6 Hz, 1H), 3.05–2.92 (m, 2H), 2.70 (dd,
J=2.6, 12.8 Hz, 1H). 2.56 (s, 3H). 13C NMR (75 MHz,
CDCl3) d 174.13, 137.44, 129.38, 129.12, 127.46, 56.36,
52.55, 41.86, 39.90, 38.85. [a]2D0 À8.13 (c 0.015, CHCl3).
Anal. calcd for C12H16O3S: C, 59.97; H, 6.71, found: C,
59.79; H, 6.74. (2S,4S)-3: (oil) yield, 42%. IR (KBr,
cmÀ1): 1735, 1044. 1H NMR (300 MHz, CDCl3) d 7.30–
7.25 (m, 3H), 7.23–7.15 (m, 2H), 3.68 (s, 3H), 3.29–3.20
(m, 1H), 3.15–2.96 (m, 3H), 2.75 (dd, J=7.0, 13.0 Hz,
1H), 2.55 (s, 1H). 13C NMR (75 MHz, CDCl3) d 174.01,
137.62, 129.49, 129.07, 127.39, 55.33, 52.58, 41.77,
(S)-2-Benzyl-3-methylthiopropanoic acid [(S)-1]. Race-
mic 2 (2.68g, 11.96 mmol) was suspended in 0.01 M
phosphate buffer (35 mL) to which was added a-chy-
motrypsin (200 mg) and the resulting mixture was stir-
red slowly at room temperature. The pH of the mixture
was maintained at 7.5 by addition of NaOH solution
(0.2 N) using a pH-stat potentiometer. When 12 mL of
the alkaline solution was consumed (11 h), the reaction
mixture was made acidic with HCl (1.0 N), and satu-

J.-Y. Jin et al. / Bioorg. Med. Chem. 11 (2003) 4377–4381
4381
39.78, 37.71. [a]2D0 À7.06 (c, 0.11, CHCl3). HRMS calcd
for C12H16O3S: 240.0820. Found: 240.0820. (2R,4S)-3:
Mp66–68 C. [a]D =+8.39 (c 0.02, CHCl3). Anal. calcd
for C12H16O3S: C, 59.97; H, 6.71; found: C, 60.03; H,
6.77. (2R,4R)-3: (oil) yield, 41%. [a]2D0=+7.34 (c
0.15%, CHCl3). HRMS, calcd for C12H16O3S:
240.0820, found: 240.0820.
consumed was less than 10%. The Ki values were
then estimated from the semireciprocal plot of the initial
velocity versus the concentration of the inhibitor accord-
ing to the method of Dixon.17 The correlation coeffi-
cients for the Dixon plots were above 0.990.
20
ꢀ
Acknowledgements
General procedure for conversion of 3 into 2-benzyl-3-
methanesulfinylpropanoic acid (4)
The authors express their thanks to the National Nat-
ural Science Foundation of China and Korean Science
and Engineering Foundation for the financial support
of this work.
Typically, lithium iodide (2.77 g, 20.6 mmol) was added
to a anhydrous ethyl acetate (75 mL) solution of
(2S,4R)-3 (920 mg, 3.75 mmol), and the resulting mix-
ture was heated under reflux for 8 h under N2. After
cooling to room temperature, the reaction mixture was
treated with water and the water layer was acidified to
pH 1.0 with an aqueous citric acid solution (10%), then
extracted with ethyl acetate (50 mLꢁ5). The combined
extract was washed with saturated Na2S2O3 solution (20
mL), dried over MgSO4, and evaporated under reduced
pressure to give a pale yellow oil which crystallized on
standing to obtain crystalline (2S,4R)-4, (394 mg, 47%).
References and Notes
1. Lipscomb, N. W.; Strater, N. Chem. Rev. 1996, 96, 2375.
2. (a) Kim, D. H.; Kim, K. B. J. Am. Chem. Soc. 1991, 113,
3200. (b) Kim, D. H.; Chung, S. J. Bioorg. Med. Chem. Lett.
1995, 5, 1667. (c) Lee, K. J.; Kim, D. H. Bioorg. Med. Chem.
Lett. 1996, 6, 2431. (d) Kim, K. J.; Joo, K.-J.; Lee, M.; Kim,
D. H. Bioorg. Med. Chem. 1997, 5, 1989. (e) Kim, D. H.; Lee,
K. J. Bioorg. Med. Chem. Lett. 1997, 7, 2607. (f) Lee, K. J.;
Kim, D. H. Bull. Korean Chem. Soc. 1997, 18, 1100. (g) Kim,
D. H.; Chung, S. J.; Kim, E.-J.; Tian, G. R. Bioorg. Med.
Chem. Lett. 1998, 8, 859. (h) Kim, D. H.; Jin, Y. Bioorg. Med.
Chem. Lett. 1999, 9, 691. (i) Lee, M.; Jin, Y.; Kim, D. H.
Bioorg. Med. Chem. 1999, 7, 1755. (j) Chung, S. J.; Kim, D. H.
Bioorg. Med. Chem. 2001, 9, 185.
3. Christianson, D. W.; Lipscomb, W. N. Acc. Chem. Res.
1989, 22, 62.
4. Mock, W. L.; Tsay, J.-T. Synth. Commun. 1988, 18, 769.
5. Kim, D. H.; Kim, Y. J. Bioorg. Med. Chem. Lett. 1993, 3,
2681.
6. Fisher, J. W.; Trinkle, K. L. Tetrahedron Lett. 1994, 35,
2505.
7. Tillett, J. G. Chem. Rev. 1976, 76, 747.
8. Crystallographic data (excluding structure factors) for the
structure have been deposited with the Cambridge Crystal-
lographic Data Center as supplementary publication nos.
CCDC 209253. Copies of the data can be obtained, free of
charge, on application to CCDC, 12 Union Road, Cam-
bridge, CB2 1EZ, UK (fax: +44-1223-336033 or e-mail:
deposit@ccdc.cam.ac.uk).
Mp122–124 ꢀC. IR (KBr, cmÀ1) 3429, 1718, 1016. H
1
NMR (300 MHz, CDCl3) d 9.22 (br, 1H), 7.33–7.18 (m,
5H), 3.33–3.29 (m, 1H), 3.20 (dd, J=5.6, 13.8 Hz, 1H),
3.03 (t, J=12.9 Hz, 1H), 2.93 (dd, J=8.3, 13.8 Hz, 1H),
2.74 (dd, J=2.4, 12.9 Hz, 1H), 2.55 (s, 3H). 13C NMR
(75 MHz, CDCl3) d 175.06, 138.96, 129.90, 129.24,
127.41, 55.90, 42.18, 39.53, 38.41. [a]2D0=À7.5 (c 0.1%,
CHCl3). Anal. calcd for C11H14O3S: C, 58.38; H, 6.24;
found: C, 58.20; H, 6.23. (2S,4S)-4: (48%). Mp109–
110 ꢀC. IR (KBr, cmÀ1) 3440, 2912,1722, 1007. 1H
NMR (300 MHz, CDCl3) d 10.68 (br, 1H), 7.30–7.19
(m, 5H), 3.29 (dd, J=6.2, 12.6 Hz, 1H), 3.22–3.12 (m,
2H), 3.03–2.99 (m, 1H), 2.74 (dd, J=5.1, 12.6 Hz, 1H),
2.60 (s, 3H). 13C NMR (75 MHz, CDCl3) d 175.12,
139.19, 129.84, 129.21, 127.34, 55.83, 42.13, 39.30,
37.56. [a]2D0=À6.5 (c 0.1%, CHCl3). Anal. calcd for
C11H14O3S: C, 58.38; H, 6.24; found: C, 58.30; H, 6.29.
20
ꢀ
(2R,4S)-4: (43%). Mp122–124 C. [a]D =+7.2 (c 0.1%,
CHCl3). Anal. calcd for C11H14O3S: C, 58.38; H, 6.24.
Found: C, 58.23; H, 6.31. (2R,4R)-4: (41%). Mp109–
110 ꢀC. [a]D20=+6.7 (c 0.1%, CHCl3). Anal. calcd for
C11H14O3S: C, 58.38; H, 6.24. Found: C, 58.16; H, 6.33.
9. Suh, J.; Kaiser, E. T. J. Am. Chem. Soc. 1976, 98, 1940.
10. Mock, W. L.; Zhang, J. Z. J. Biol. Chem. 1991, 266, 6903.
11. Mock, W. L.; Tsay, J.-Y. J. Am. Chem. Soc. 1989, 111,
4467.
12. Fabbrizzi, L.; Licchelli, M.; Rabaioli, G.; Taglietti, A.
Coor. Chem. Rev. 2000, 205, 85.
Determination of Ki value
13. (a) Christianson, D. W.; Lipscomb, W. N. Proc. Natl.
Acad. Sci. U.S.A. 1987, 84, 1512. (b) Christianson, D. W.;
Lipscomb, W. N. J. Am. Chem. Soc. 1988, 110, 5560.
14. Phillips, M. A.; Fletterick, R.; Rutter, W. J. J. Biol. Chem.
1990, 265, 20692.
15. Ondetti, M. A.; Condon, M. E.; Reid, J.; Sato, E. F.;
Cheung, H. S.; Cushman, D. W. Biochemistry 1979, 18, 1427.
16. McElvain, S. M.; Kent, R. E.; Stevens, C. L. J. Am. Chem.
Soc. 1946, 68, 1922.
The enzyme stock solution was added to a solution
containing Cl-CPL (final concentrations: 50 and 100
mM) and inhibitor (five different final concentrations in
the range of 0.5–2 Ki) in 0.05 M Tris/0.5 M NaCl, pH
7.5 buffer (1-mL cuvette), and the change in absorbance
at 320 nm was measured immediately. The final con-
centration of CPA was 18 nM. Initial velocities were
then calculated from the linear initial slopes of the
change in absorbance where the amount of substrate
17. Dixon, M. Biochem. J. 1953, 55, 170.