Stereochemistry in enzyme inhibition: Synthesis and evaluation of enantiomerically pure 2-benzyl-3-formylpropanoic acids as inhibitors of carboxypeptidase A

Article abstract of DOI:10.1016/S0957-4166(99)00421-8

Both enantiomers of 2-benzyl-3-formylpropanoic acid were synthesized in five steps starting with hydrocinnamic acid and each enantiomer assayed for inhibitory activity against carboxypeptidase A to find that the (R)-form is 674-fold more potent than its e

Full text of DOI:10.1016/S0957-4166(99)00421-8

Pergamon
Tetrahedron: Asymmetry 10 (1999) 3769–3776
Stereochemistry in enzyme inhibition: synthesis and evaluation of
enantiomerically pure 2-benzyl-3-formylpropanoic acids as
inhibitors of carboxypeptidase A
Dong H. Kim ∗ and Suhman Chung
Center for Biofunctional Molecules and Department of Chemistry, Pohang University of Science and Technology,
San 31 Hyojadong, Pohang 790-784, South Korea
Received 26 July 1999; accepted 13 September 1999
Abstract
Both enantiomers of 2-benzyl-3-formylpropanoic acid were synthesized in five steps starting with hydrocinna-
mic acid and each enantiomer assayed for inhibitory activity against carboxypeptidase A to find that the (R)-form
is 674-fold more potent than its enantiomer. The finding that the (R)-form which belongs to the L-series is mostly
responsible for the inhibitory activity accords with the explanation that the present inhibitor is a transition state
analog inhibitor because, as such, its stereochemistry should belong to the same series as that of the substrate, i.e.,
the L-series. The gem-diol form of the inhibitor generated in situ mimics the transition state in the catalytic process.
© 1999 Published by Elsevier Science Ltd. All rights reserved.
1. Introduction
Enzyme inhibitors are compounds that bind enzymes at the active site but fail to undergo the chemical
reaction that substrates would. Design of enzyme inhibitors has been the subject of intensive research
especially in reference to the discovery of lead compounds as new therapeutic agents.1 Galardy and
Kortylewicz2 reported that 2-benzyl-3-formylpropanoic acid in the racemic form is a potent competitive
inhibitor for CPA,3 a much studied prototypic zinc-containing protease which catalyzes the cleavage of
the C-terminal amide bond of peptide substrate. The enzyme shows specificity for peptides having the C-
terminal amino acid residue with a hydrophobic side chain such as Phe. In connection with our ongoing
efforts directed to define the stereochemistry associated with inhibition of enzymes,4 it was thought to be
of interest to find which enantiomeric form of the inhibitor is responsible for its CPA inhibitory activity.
This paper describes efficient syntheses of both enantiomers of 2-benzyl-3-formylpropanoic acid in an
enantiomerically pure form and their evaluation as inhibitors for CPA.
∗
Corresponding author. Tel: (82) 562-279-2101; fax: (82) 562-279-5877; e-mail: dhkim@vision.postech.ac.kr
0957-4166/99/$ - see front matter © 1999 Published by Elsevier Science Ltd. All rights reserved.
PII: S0957-4166(99)00421-8

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2. Results and discussion
Scheme 1 outlines the synthetic route for the preparation of (R)-2-benzyl-3-formylpropanoic acid.
2-Allyl-3-phenylpropanoic acid obtained by allylation of hydrocinnamic acid was tethered to (R)-4-
benzyl-2-oxazolidinone, an Evans chiral auxiliary, to give 3 as a diastereoisomeric mixture (Scheme 1).
The mixture was readily separated by column chromatography to give a crystalline product and an
oil in the ratio of 1:1. The crystalline product, the stereochemistry of which was assigned to be
(20S,4R) (see Scheme 1), was converted into the benzyl ester of 2-allyl-3-phenylpropanoic acid, (S)-
4, by treatment with BnOLi. Ozonolysis of (S)-4 followed by treatment with thiourea according to
the method reported by Gupta et al.5 transformed the olefinic moiety to a formyl group, giving (R)-
5. Hydrogenolysis of the methanolic solution of (R)-5 in the presence of Pd/C removed the benzyl
group to give (R)-1 as a hydroxylactone. Treatment of the latter with aqueous KOH afforded the target
compound of R-configuration as a potassium salt (R)-1a (Scheme 2). Treatment of the hydroxylactone
with dicyclohexylamine afforded a crystalline salt (R)-1b.
Scheme 1. Reagents, conditions, and yields: (a) LDA (2.1 equiv.), allyl bromide (1.1 equiv.), 0°C, THF (84%); (b) i. (COCl)2,
CH2Cl2; ii. n-BuLi (1.0 equiv.), (R)-(+)-4-benzyl-2-oxazolidinone, −78°C, THF (86%)
Scheme 2. Reagents, conditions, and yields: (c) n-BuLi (1.5 equiv.), BnOH, 0°C, THF (84%); (d) i. O3, MeOH; ii. thiourea
(60%); (e) i. H2, Pd/C, MeOH; ii. for a, aq. KOH (1.0 equiv.) (92%), for b, dicyclohexylamine, Et2O (96%)
The assigned stereochemistry was ascertained by oxidizing (R)-1b to give known (R)-2-benzylsuccinic
acid (Scheme 3). Thus, the [α]D value of 2-benzylsuccinic acid obtained from oxidizing (R)-1b by the
method of Kraus and Taschner6 was +26.5 which is in good agreement with the literature value7 of +27.2

D. H. Kim, S. Chung / Tetrahedron: Asymmetry 10 (1999) 3769–3776
3771
for (R)-2-benzylsuccinic acid. In a similar sequence of reactions, we obtained (S)-1 from (20R,4R)-3 as
an oil after chromatographic separation of the diastereoisomeric mixture of 3.
Scheme 3. Reagents, conditions, and yields: (f) NaClO2, 2-methyl-2-butene, NaH2PO4, t-BuOH–H2O (72%)
Each enantiomer of 1 thus synthesized was assayed as the potassium salt for its CPA inhibitory activity
according to the method reported by Galardy and Kortylewicz2 to find that the inhibitory activity resides
mostly with (R)-1 having the Ki value of 0.38ꢀ0.03 µM for the potassium salt (Fig. 1). The Ki value of
(S)-1a was shown to be 256ꢀ19 µM. Racemic 1 was reported to have the Ki value of 0.48ꢀ0.1 µM.2
Figure 1. The Dixon plot for the inhibition of CPA with potassium (R)-2-benzyl-3-formylpropanoate (R)-1a (substrate, Cl-CPL;
Tris buffer of pH 7.5; 25°C)
When the inhibitors were assayed as dicyclohexylamine salts, the inhibitory potencies were consider-
ably reduced as can be seen from Table 1, which is probably due to lowered solubility of these organic
salts in the assay medium.
Previously, Galardy and Kortylewicz proposed that the high binding affinity of 2-benzyl-3-
formylpropanoic acid is due to the facile tendency of its formyl moiety, in an aqueous solution, to form a
gem-diol that mimics the tetrahedral transition state generated in the CPA-catalyzed proteolytic process
(Fig. 2).2 In the CPA-catalyzed peptide cleavage, the active site water molecule that is activated dually
by the active site zinc ion and the carboxylate of Glu-270 attacks the carbonyl carbon of the scissile
peptide bond to form a tetrahedral transition state (Fig. 2a).8 Transition state analogs in the enzymic
reaction have been known to bind the active site with high affinity.9 The present observation that (R)-1,
which belongs to the L-series, is mostly responsible for the observed inhibitory activity of 1 accords
with the proposition, since the stereochemistry of the substrate is retained at the transition state.
3. Conclusion
Although the role of the chirality of the inhibitor in enzyme inhibitions is of utmost importance
especially in the design of therapeutically useful inhibitors, the factors controlling and determining the

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Table 1
Inhibition of CPA-catalyzed hydrolysis of O-(trans-p-chlorocinnamoyl)-L-β-phenyllactate
Figure 2. (a) Schematic representation that shows the general-base pathway of CPA-catalyzed hydrolysis of peptide substrate.
A tetrahedral transition state that is generated by the attack of the activated water molecule at the active site on the scissile
carbonyl carbon of the substrate collapses to products; (b) 2-benzyl-3,3-dihydroxypropanoic acid that is generated from
2-benzyl-3-formylpropanoic acid in aqueous solution resembles structurally the tetrahedral transition state in the enzymic
reaction and binds the active site of CPA with high affinity
inhibitor stereospecificity are still poorly understood. We hope that the present observation will be of
value for designing transition state analog inhibitors of pharmacologically important zinc-containing
proteases such as angiotensin converting enzyme and matrix metalloproteases. Inhibitors of these
enzymes are valuable as potential therapeutic agents.

D. H. Kim, S. Chung / Tetrahedron: Asymmetry 10 (1999) 3769–3776
3773
4. Experimental
Melting points were taken on a Thomas–Hoover capillary melting point apparatus and are uncorrected.
1H NMR and 13C NMR spectra were recorded on a Bruker AM 300 (300 MHz) instrument using
tetramethylsilane as the internal standard. IR spectra were recorded on a Bruker Equinox 55 FT-
IR spectrometer. Flash chromatography was performed on silica gel (230–400 mesh) and thin layer
chromatography (TLC) was carried out on silica coated glass sheets (Merck silica gel 60 F-254). Optical
rotations were measured on a Rudolph Research Autopol III digital polarimeter. Elemental analyses were
performed at the Center of Biofunctional Molecules, Pohang University of Science and Technology,
Pohang, Korea.
4.1. 2-Allyl-3-phenylpropanoic acid 2
To an ice-chilled solution of diisopropylamine (9.2 mL, 70.2 mmol) in dry THF (50 mL) was slowly
added n-butyllithium (28.1 mL of 2.5 M solution in n-hexane, 70.2 mmol) under a nitrogen atmosphere.
After the mixture was stirred for 30 min, hydrocinnamic acid (5.0 g, 33.3 mmol) in dry THF (30 mL)
was added dropwise over a period of 20 min. The resulting yellowish solution was stirred for 40 min,
and then allyl bromide (3.2 mL, 37.0 mmol) was added dropwise. The reaction mixture was stirred for
3 h to give a colorless solution. The pH of the solution was adjusted to 2.5 with 3N HCl solution. The
organic layer was separated and extracted with saturated sodium bicarbonate solution. The aqueous layer
was acidified with 3N HCl solution to pH 2, saturated with sodium chloride, and extracted with ethyl
acetate (50 mL×3). The combined extracts were dried over anhydrous MgSO4, filtered, and evaporated
under reduced pressure. The residue was purified by column chromatography, eluting with a solution of
ethyl acetate and n-hexane (1:3) to give 2 (5.3 g, 84%) as a colorless oil. IR (neat) 1712 cm−1; 1H NMR
(CDCl3): δ 2.29–2.46 (m, 2H), 2.75–2.85 (m, 2H), 2.97–3.05 (m, 1H), 5.08–5.15 (m, 2H), 5.74–5.88 (m,
1H), 7.19–7.34 (m, 5H); 13C NMR (CDCl3): δ 36.0, 37.7, 47.4, 117.9, 126.9, 128.9, 129.3, 135.1, 139.2,
181.2.
4.2. (20R,4R)- and (20S,4R)-N-(20-Allyl-30-phenylpropanoyl)-4-benzyl-1,3-oxazolidin-2-one (20R,4R)-3
and (20S,4R)-3
To an ice-chilled solution of 2 (5.2 g, 27 mmol) in dry CH2Cl2 was added dropwise oxalyl chloride
(2.4 mL, 27 mmol). The reaction mixture was refluxed for 2 h, and then concentrated in vacuo to give
acyl chloride as a colorless oil. A solution of (R)-(+)-4-benzyl-2-oxazolidinone (4.8 g, 27 mmol) in
dry THF, stirred at −78°C under a nitrogen atmosphere, was treated dropwise with n-butyllithium (11
mL of 2.5 M solution in n-hexane, 27 mmol). After the mixture was stirred for 30 min, the above acyl
chloride dissolved in dry THF was added dropwise. The resulting mixture was stirred at −78°C for 30
min, then quenched with saturated ammonium chloride solution and partitioned between ether and water.
The combined ether extracts were washed with 5% sodium bicarbonate solution and brine, then dried
over anhydrous MgSO4, filtered, and evaporated under reduced pressure. The resulting diastereomeric
mixture (1:1) was separated by column chromatography, eluting with ethyl acetate and n-hexane (1:30)
to give (20S,4R)-3 (4.1 g, 43%) as a pale yellow oil and (20R,4R)-3 (4.1 g, 43%) as a white crystalline
solid.
1
Compound (20R,4R)-3: [α]D=−114.5 (c 0.7, CHCl3); IR (neat) 3062, 1779, 1697 cm−1; H NMR
(CDCl3): δ 2.36 (m, 1H), 2.54–2.58 (m, 1H), 2.60–2.68 (dd, 1H), 2.80–2.99 (m, 2H), 3.20–3.26 (dd,
1H), 3.82 (t, 1H), 3.99–4.03 (dd, 1H), 4.33 (m, 1H), 4.45 (m, 1H), 5.06–5.16 (m, 2H), 5.82–5.87 (m,

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D. H. Kim, S. Chung / Tetrahedron: Asymmetry 10 (1999) 3769–3776
1H), 7.18–7.34 (m, 10H); 13C NMR (CDCl3): δ 36.7, 38.4, 38.7, 44.4, 55.9, 66.3, 117.8, 126.8, 127.7,
128.8, 129.3, 129.5, 129.8, 135.5, 135.8, 139.3, 153.4, 175.7.
Compound (20S,4R)-3: mp 86–88°C; [α]D=−30.7 (c 0.5, CHCl3); IR (neat) 3027, 1777, 1696 cm−1;
1H NMR (CDCl3): δ 2.30 (m, 1H), 2.40–2.49 (m, 2H), 2.79–2.86 (dd, 1H), 2.97–3.10 (m, 2H), 4.03–4.13
(m, 2H), 4.33 (m, 1H), 4.64 (m, 1H), 5.01–5.10 (m, 2H), 5.75–5.81 (m, 1H), 6.99–7.29 (m, 10H); 13C
NMR (CDCl3): δ 36.7, 38.0, 38.5, 44.7, 55.5, 66.2, 117.7, 126.9, 127.6, 128.8, 129.3, 129.8, 135.6,
139.3, 153.5, 175.7. Anal. calcd for C22H23NO3: C, 75.62; H, 6.63; N, 4.01. Found: C, 75.87; H, 6.52;
N, 3.61.
4.3. Benzyl (S)-2-allyl-3-phenylpropanoate (S)-4
To an ice-chilled solution of benzyl alcohol (2.1 g, 19.4 mmol) in dry THF (50 mL) was slowly
added n-butyllithium (5.7 mL of 2.5 M solution in n-hexane, 14.3 mmol) under a nitrogen atmosphere.
After the mixture was stirred for 1 h, a solution of (20S,4R)-3 (3.3 g, 9.4 mmol) in dry THF (20 mL)
was added dropwise over a period of 30 min. The resulting mixture was stirred at −78°C for 30 min,
then quenched with saturated ammonium chloride solution and partitioned between ether and water. The
combined ether extracts were washed with 5% sodium bicarbonate solution and brine, then dried over
anhydrous MgSO4, filtered, and evaporated under reduced pressure. The residue was purified by column
chromatography, eluting with a solution of ethyl acetate and n-hexane (1:10) to give (S)-4 (2.2 g, 84%)
1
as a colorless oil. [α]D=+25.8 (c 0.5, CHCl3); IR (neat) 3029, 1731, 1496 cm−1; H NMR (CDCl3): δ
2.32–2.46 (m, 2H), 2.80–2.91 (m, 2H), 2.94–3.03 (m, 1H); 13C NMR (CDCl3): δ 36.5, 38.2, 47.7, 66.5,
117.5, 126.7, 128.4, 128.6, 128.8, 129.3, 135.5, 136.4, 139.5, 175.0.
4.4. Benzyl (R)-2-allyl-3-phenylpropanoate (R)-4
This compound was synthesized from (20R,4R)-3 in a manner analogous to that used for its enantiomer.
[α]D=−24.2 (c 0.6, CHCl3).
4.5. Benzyl (R)-2-benzyl-3-formylpropanoate (R)-5
Ozonized oxygen was bubbled through a solution of (S)-4 (0.5 g, 1.8 mmol) in anhydrous MeOH (10
mL) at −10°C for 30 min. Nitrogen was then bubbled through the solution to displace any remaining
ozone and the solution was added to a solution of thiourea (70 mg, 0.9 mmol) in anhydrous MeOH at
0°C under stirring. Thiourea S-dioxide started depositing as white crystals within 15 min. After stirring
for another 40 min, the reaction mixture was filtered and evaporated under reduced pressure. The residue
was diluted with ether and washed with 5% sodium bicarbonate and water. The resulting solution was
evaporated under reduced pressure and purified by flash column chromatography to give (R)-5 as a
1
colorless oil (0.3 g, 60%). [α]D=+16.1 (c 0.6, CHCl3); IR (neat) 3030, 1778, 1731 cm−1; H NMR
(CDCl3): δ 2.52–2.60 (dd, 1H), 2.77–2.93 (m, 2H), 3.06–3.13 (dd, 1H), 3.25–3.31 (m, 1H), 5.13 (s, 2H),
7.13–7.39 (m, 5H), 9.72 (s, 1H); 13C NMR (CDCl3): δ 38.0, 41.3, 44.8, 67.1, 127.2, 128.7, 129.0, 129.1,
129.4, 136.1, 138.4, 174.4, 200.3.
4.6. Benzyl (S)-2-benzyl-3-formylpropanoate (S)-5
This compound was synthesized from (R)-4 in a manner analogous to that used for its enantiomer.
[α]D=−15.3 (c 0.5, CHCl3).

D. H. Kim, S. Chung / Tetrahedron: Asymmetry 10 (1999) 3769–3776
3775
4.7. Potassium (R)-2-benzyl-3-formylpropanoate (R)-1a
To a solution of (R)-5 (200 mg, 0.7 mmol) in anhydrous methanol (3 mL) was added a catalytic
amount of 10% Pd/C and the resulting suspension was stirred under hydrogen gas for 1 h. The catalyst
was removed by filtration and the filtrate was evaporated under reduced pressure to give hydroxylactone.
To 130 mg of hydroxylactone was added 0.68 mL of 1N KOH solution, and this mixture was stirred at
ambient temperature until the organic layer had disappeared. Lyophilization gave (R)-1a (150 mg, 92%)
1
as a white solid: mp 155–158°C; [α]D=−8.1 (c 0.5, CHCl3); H NMR (DMSO-d6): δ 1.87–1.94 (dd,
1H), 2.08–2.22 (m, 1H), 2.46–2.65 (m, 2H), 3.05–3.11 (dd, 1H), 7.08–7.26 (m, 5H), 9.49 (s, 1H); 13C
NMR (DMSO-d6): δ 39.4, 47.4, 50.2, 126.3, 128.9, 129.8, 142.8, 196.5.
4.8. Potassium (S)-2-benzyl-3-formylpropanoate (S)-1a
This compound was synthesized from (S)-5 in a manner analogous to that used for its enantiomer.
[α]D=+7.5 (c 0.5, CHCl3).
4.9. (R)-2-Benzyl-3-formylpropanoic acid, dicyclohexylamine salt (R)-1b
To a soluton of the above hydroxylactone (100 mg, 0.5 mmol) in ether was added dicyclohexylamine
(0.2 mL). The resulting salt was filtered and recrystallized from CH2Cl2–hexane to give (R)-1b (190 mg,
96% for two steps) as white crystals: mp 129–130°C; [α]D=−11.3 (c 0.4, CHCl3); IR (neat) 3500, 2939,
1
1715, 1561 cm−1; H NMR (CDCl3): δ 1.18–1.36 (m, 10H), 1.62–1.97 (m, 10H), 2.29–2.31 (dd, 1H),
2.45–2.58 (ddd, 1H), 2.66–2.74 (dd, 1H), 2.85 (m, 2H), 3.00 (m, 1H), 3.17–3.24 (dd, 1H), 7.15–7.29 (m,
5H), 9.16 (s, 1H); 13C NMR (CDCl3): δ 25.3, 25.7, 30.2, 39.0, 44.8, 44.9, 52.9, 126.5, 128.7, 129.5,
140.7, 179.1. Anal. calcd for C22H23NO3: C, 73.95; H, 9.44; N, 3.75. Found: C, 73.77; H, 9.29; N, 3.69.
4.10. (S)-2-Benzyl-3-formylpropanoic acid, dicyclohexylamine salt (S)-1b
This compound was obtained from (S)-5 in a manner analogous to that for its enantiomer. [α]D=+12.4
(c 0.5, CHCl3).
4.11. (R)-2-Benzylsuccinic acid (R)-6
To a solution of (R)-1b (50 mg, 0.13 mmol) and 2-methyl-2-butene (60 µL, 0.57 mmol) in t-BuOH
(2 mL) was added dropwise a solution containing sodium chlorite (73 mg, 0.80 mmol) and sodium
dihydrogen phosphate monohydrate (74 mg, 0.54 mmol) in water (3 mL). The resulting pale yellow
solution was stirred at room temperature for 30 min, concentrated in vacuo, and diluted with water (5
mL). The aqueous solution was acidified to pH 2 with 3N HCl and extracted with three 5 mL portions
of ethyl acetate. The ethyl acetate layers were combined, dried with MgSO4, concentrated under reduced
pressure, and the residue was recrystallized from ether–hexane to give (R)-6 (20 mg, 72%) as a white
crystalline solid: mp 165–166°C [lit.: mp 161–163°C]; [α]D=+26.5 (c 0.7, ethyl acetate) [lit.: [α]D=+27.2
1
(c 2.9, ethyl acetate)]; H NMR (DMSO-d6): δ 2.18–2.25 (dd, 1H), 2.35–2.44 (dd, 1H), 2.70–2.75 (q,
1H), 2.84–2.91 (m, 2H), 7.15–7.29 (m, 5H).

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4.12. General remarks for kinetic experiment
All solutions were prepared by dissolving in doubly distilled and deionized water. Stock assay
solutions were filtered before use. Carboxypeptidase A was purchased from Sigma Chemical Co. (Allan
form, twice crystallized from bovine pancrease, aqueous suspension in toluene) 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. In the kinetic study, O-(trans-p-chlorocinnamoyl)-L-β-phenyllactate (Cl-CPL)
purchased from Sigma Chemical Co. was used as substrate and the decrease in the absorbance at 320 nm
was followed using a Perkin–Elmer HP 8453 UV/vis spectrometer at 25°C.
4.13. Determination of Ki
Initial velocities were calculated from the linear initial slopes of the change in absorbance where
the amount of substrate consumed was less than 10%. The Ki values were then estimated from the
semireciprocal plot of the initial velocity versus the concentration of the inhibitors according to the
method of Dixon. Two concentrations of the substrate of Cl-CPL were used. Typically, enzyme stock
solution was added to various concentrations of inhibitors in 0.05 M Tris/0.5 M NaCl, pH 7.5 buffer (1
mL cuvette), and the initial rates were measured immediately.
Acknowledgements
The authors wish to express their appreciation to the Korea Science and Engineering Foundation for
the financial support of this work.
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