Hydrogen bonding and attenuation of the rate of enzymic catalysis

Article abstract of DOI:10.1021/ja983063e

Hydrogen bonds between a small molecule and an enzyme can potentially contribute significantly to the stability of the complex. Such electrostatic interactions can also lower energy barriers for reactions by solvation of high-energy species. A novel type

Full text of DOI:10.1021/ja983063e

VOLUME 120, NUMBER 50
DECEMBER 23, 1998
© Copyright 1998 by the
American Chemical Society
Hydrogen Bonding and Attenuation of the Rate of Enzymic Catalysis
Zhi-Hong Li, Alexey Bulychev, Lakshmi P. Kotra, Irina Massova, and
Shahriar Mobashery*
Contribution from the Department of Chemistry, Wayne State UniVersity, Detroit, Michigan 48202-3489
ReceiVed August 25, 1998
Abstract: Hydrogen bonds between a small molecule and an enzyme can potentially contribute significantly
to the stability of the complex. Such electrostatic interactions can also lower energy barriers for reactions by
solvation of high-energy species. A novel type of inhibitor is described in this report, which was designed to
take advantage of a hydrogen bond that it makes to the active-site histidine of chymotrypsin to attenuate its
basicity. Substrates of chymotrypsin acylate the active-site serine (of the catalytic triad), and the acyl-enzyme
intermediate undergoes deacylation in a second step of the catalytic turnover. The active-site histidine (of the
catalytic triad) serves as the general base in both steps of the turnover process. Such attenuation of basicity by
hydrogen bonding was expected to impair catalysis by the enzyme. Two molecules of this type were synthesized
that are based on the structure of the chymotrypsin substrate Ac-L-Ala-L-Ala-Gly-L-Phe methyl ester. These
were methyl (2R,3R)-5-(N-acetyl-L-alanyl-L-alanyl)amino-2-benzyl-3-hydroxylpentanoate (1) and methyl (2R,3S)-
5-(N-acetyl-L-alanyl-L-alanyl)amino-2-benzyl-3-hydroxylpentanoate (2). Compound 1 acylated chymotrypsin,
but the acyl-enzyme species resisted deacylation. On the other hand, compound 2 did not even have the ability
to acylate the active-site serine. Molecular modeling supported the assertion that compound 1 makes a critical
hydrogen bond to the active-site histidine at the acyl-enzyme stage, whereas compound 2 does so at the
preacylation complex. The concepts described herein are of general interest and should find applications for
inhibition of enzymes that employ general acid-base chemistry for their catalytic processes.
Hydrogen bonding is an electrostatic interaction that is
ubiquitous in biological molecules.1 It is one of the factors that
contributes to protein folding, to substrate recognition by
enzymes, and to the energetics of turnover in enzymic chemistry.
An enzyme may make hydrogen bonds to the ground-state or
transition-state species. If the strength of a given hydrogen bond
to the transition-state species is stronger than that to the ground
state, the hydrogen bond lowers the energy barrier for the
enzymic reaction by solvating the high-energy transition-state
species. The effects of these interactions could range from
modest to significant. A typical hydrogen bond may contribute
as much as 2-3 kcal/mol toward binding or lowering of energy
barriers, although effect as much as 11 kcal/mol have been
documented for enzymic systems.2 Furthermore, the possibility
for existence of low-barrier or short, strong hydrogen bonds,
which may have the potential to contribute to enzymic catalysis
even more significantly, has been the subject of much discussion
recently.3 Indeed, the manifestation of the effects of such an
interaction between an enzyme and substrate can be realized
even at sites relatively remote from the seat of the reaction.4
The contribution of hydrogen bonds has often been a factor
in design of molecules that mimic either the transition-state or
ground-state species in design of inhibitors.5 We disclose herein
a novel type of enzyme inhibitor that exploits the nature of a
(1) (a) Hibbert, F.; Emsley, J. AdV. Phys. Org. Chem. 1990, 26, 255.
Perrin, C. L.; Nielson, J. B. Annu. ReV. Phys. Chem. 1997, 48, 511. (b) A
partially covalent nature has also been attributed to strong hydrogen
bonds: Gilli, P.; Bertolasi, V.; Ferretti, V.; Gilli, G. J. Am. Chem. Soc.
1994, 116, 909.
(2) Roestamadji, J.; Grapsas, I.; Mobashery, S. J. Am. Chem. Soc. 1995,
117, 11060.
10.1021/ja983063e CCC: $15.00 © 1998 American Chemical Society
Published on Web 12/03/1998

13004 J. Am. Chem. Soc., Vol. 120, No. 50, 1998
Li et al.
specific hydrogen bond between the inhibitor and the enzyme
to shut down the catalytic machinery.
Scheme 1
We decided to apply this method of inhibition to chymo-
trypsin, a prototypic serine protease for which high-resolution
crystal structures are known. The catalytic machinery of
chymotrypsin, as in other serine proteases, is dominated by the
catalytic triad of Asp-His-Ser, of which serine undergoes
acylation and deacylation in the course of substrate turnover.
The histidine of the triad activates the serine hydroxyl for the
acylation process and a water molecule for the deacylation step.
Hence, it serves as a general base in both steps. We reasoned
that the donation of a hydrogen bond from a substrate analogue
to the histidine imidazole group would attenuate its basicity.
One could envision this in two ways: the hydrogen bond would
tie up the lone pair of electrons on the imidazole ring, so it
would not be available for activation of the active-site serine,
and the additional hydrogen bond solvates the general base
effectively. Each principle would lower the basicity of the
general base and would impair the catalytic machinery of
chymotrypsin in either the preacylation complex or the acyl-
enzyme intermediate stage.
Our design of the inhibitor(s) commenced with the crystal
structure of chymotrypsin modified by Pro-Gly-Ala-Tyr.6 Mo-
lecular modeling indicated that a hydroxyl moiety â to the ester
carbonyl would be poised for interaction with the His-57
imidazole. Furthermore, both R and S stereoisomers at that site
could allow for such an interaction, although the hydrogen bond
by the R stereoisomer to the imidazole ring appeared to be
stronger. Therefore, we envisioned two potential inhibitors,
compounds 1 and 2. These compounds are peptidomimetic
analogues of the chymotrypsin substrate 3. Since evaluation of
the effect of the hydroxyl group in 1 and 2 was central to our
concept for inhibition, we also decided to study compound 4,
which lacks that functional group.
optical purity of the product was analyzed by NMR. The
conversion of 6 to 7 was effected in 78% yield by the general
procedure for regioselective reduction with the borane-dimethyl
sulfide complex in the presence of a catalytic amount of sodium
borohydride in THF.8 Treatment of 7 with p-toluenesulfonyl
chloride gave selective tosylation of the primary hydroxyl group.
Subsequent treatment of the tosylate with potassium carbonate
in methanol afforded 8. Nucleophic opening of the epoxide ring
with potassium cyanide gave 9 in 85% yield. Medium-pressure
catalytic hydrogenation of 9 in the presence of palladium-on-
charcoal yielded 10a, which was subsequently coupled with
N-Cbz-L-Ala-L-Ala. The Cbz protecting group was replaced by
the acetyl group to afford methyl (2R,3R)-5-(N-acetyl-L-alanyl-
L-alanyl)amino-2-benzyl-3-hydroxypentanoate (1). The (2R,3S)
analogue was synthesized starting from the N-acylated oxazo-
(4) Fersht, A. R.; Shi, J. P.; Wilkinson, A. J.; Blow, D. M.; Carter, P.;
Waye, M. M. Y.; Winter; G. P. Angew. Chem., Int. Ed. Engl. 1984, 23,
467. Wells, T. N. C.; Fersht, A. R. Nature 1985, 316, 656.
Compounds 1, 2 and 4 were synthesized according to Scheme
1. Dimethyl L-malate (5) was stereoselectively benzylated by
following the procedure reported by Seebach and Wasmuch7
to give 6. No diastereomeric impurity was detected when the
(5) Abraham, M. H.; Duce, P. P.; Prior, D. V.; Barratt, D. G.; Morris, J.
J.; Taylor, P. J. J. Chem. Soc., Perkin Trans 2 1989, 1355. Biller, S. A.;
Sofia, M. J.; DeLange, B.; Forster, C.; Gordon, E. M.; Harrity, T.; Rich, L.
C.; Ciosek, C. P. J. Am. Chem. Soc. 1991, 113, 8522. Liu, S.; Hanzlik, R.
P. Biochim. Biophys. Acta 1993, 1158, 264. Maveyraud, L.; Massova, I.;
Birck, C.; Miyashita, K.; Samama, J. P.; Mobashery, S. J. Am. Chem. Soc.
1996, 118, 7435. Lee, S. L.; Alexander, R. S.; Smallwood, A.; Trievel, R.;
Mersinger, L.; Weber, P. C.; Kettner, C. Biochemistry 1997, 36, 13180.
(6) Dixon, M. M.; Matthews, B. W. Biochemistry 1989, 28, 7033. Dixon,
M. M.; Brennan, R. G.; Matthews, B. W. Int. J. Biol. Macromol. 1991, 13,
89.
(3) Frey, P. A.; Whitt, S. A.; Tobin, J. B. Science 1994, 264, 1927.
Cleland, W. W.; Kreevoy, M. M. Science 1994, 264, 1887. Perrin, C. L.
Science 1994, 266, 1665. Scheiner, S.; Kar, T. J. Am. Chem. Soc. 1995,
117, 6970. Tobin, J. B.; Whitt, S. A.; Cassidy, C. S.; Frey, P. A.
Biochemistry 1995, 34, 6919. Schwartz, B.; Drueckhammer, D. G. J. Am.
Chem. Soc. 1995, 117, 11902. Shan, S.; Herschlag, D. J. Am. Chem. Soc.
1996, 118, 5515. Shan, S.; Loh, S.; Herschlag, D. Science 1996, 272, 97.
Ash, E. L.; Sudmeier, J. L.; De Fabo, E. C.; Bachovchin, W. W. Science
1997, 278, 1128. Cassidy, C. S.; Lin, J.; Frey, P. A. Biochemistry 1997,
36, 4576.
(7) Seebach, D.; Wasmuch, D. HelV. Chim. Acta. 1980, 63, 197.
(8) Saito, S.; Hasegawa, T.; Inaba, M.; Nishida, R.; Fuji, T.; Nomizu,
S.; Moriwake, T. Chem. Lett. 1984, 1389.

H-Bonding and the Rate of Enzymic Catalysis
J. Am. Chem. Soc., Vol. 120, No. 50, 1998 13005
recovery of full activity. Furthermore, analysis of the inhibition
mixture for over 20 h did not reveal the formation of any
products of turnover of this compound in the presence of the
enzyme. These results collectively indicate that 2 formed the
preacylation complex for its competitive inhibition of the
enzyme, but resisted serine acylation. However, 1 behaved as
a mechanism-based inactivator for chymotrypsin, as evidenced
by a time-dependence for loss of activity associated with enzyme
acylation. Once the acyl-enzyme intermediate formed, it resisted
deacylation. The enzyme inactivation proceeded with the
following kinetic parameters: kinact ) (1.4 ( 0.2) × 10-3 s-1
,
Figure 1. (A) Dixon plot for reversible competitive inhibition of
chymotrypsin by compound 2. (B) The double-reciprocal plot of the
first-order rate constant for inactivation of chymotrypsin vs concentra-
tions of compound 1.
KI ) 2.8 ( 0.7 mM, and kinact/KI ) 0.6 ( 0.1 M-1 s-1 (Figure
1B). The inhibited complex underwent deacylation with the
attendant recovery of activity in a very slow process with krec
) (4.2 ( 0.5) × 10-6 s-1 (i.e., t1/2 for recovery of activity was
1.9 days). The dissociation constant for the formation of the
preacylation complex with this enzyme inactivator (Ki) was
evaluated at 620 ( 150 µM.13 Therefore, as per design
principles, we believe that this compound interacted with the
histidine imidazole in the second step, such that once the acyl-
enzyme species formed, it could not undergo ready deacylation.
However, comparison of the values for kinact/KI for compound
1 with that for kcat/Km for 4 indicates that acylation of the active-
site serine is not a problem for compound 1 (the two values are
comparable). One discerns this from the following argument.
Serine proteases operate by the following kinetic scheme:
lidinone 119 in the presence of 2 equiv of diisopropylethylamine
with N-Cbz-3-aminopropanal to afford the erythro-isomer 12.10
Removal of the Cbz group from compound 13a facilitated a
spontaneous cyclization of the product to give the corresponding
six-membered lactam. The absolute stereochemistry at positions
2 and 3 were established by determination of the X-ray structure
of this lactam. The erythro-adduct 12 was readily hydrolyzed
without racemization of either center to the corresponding acid
upon treatment with 1.5 equiv of lithium hydroxide and 4 equiv
of hydrogen peroxide in THF-water (0 °C). Following the
esterification, 13a was transformed into the corresponding deoxy
methyl ester 13b by treatment with phenyl chlorothionoformate
and DMAP, followed by tributyltin hydride and di-tert-
butylperoxide in good yield. As described above, when 13a was
subjected to deprotection in methanol, almost a quantitative yield
of the corresponding lactam was obtained, which proved helpful
in the establishment of the absolute chemistry. However, when
the deprotection was performed in methanol containing 2 equiv
of HCl, hydrochloric acid salts of 10b and 10c were obtained.
Compounds 10b and 10c were transformed into the correspond-
ing tetrapeptide analogues 2 and 4 according to the procedure
described above.
For this scheme, the following kinetic parameters are
derived: kcat ) (k2k3)/(k2 + k3), Km ) (k3Ks)/(k2 + k3), and kcat/
Km ) k2/Ks, assuming that k-1 > k2. The ratios of kcat/Km and,
by analogy, of kinact/KI, are directly proportional to the rate of
enzyme acylation.
Modeling was attempted to rationalize these findings. Whereas
the confirmation of these observations should await crystal-
lographic findings, preliminary modeling indicated that the
hydroxyl group of compound 1 interactes with the active-site
histidine at the acyl-enzyme intermediate and that of 2 does so
at the preacylation complex. Hence, 1 acylates the active-site
serine and resists deacylation, whereas 2 is incapable of enzyme
acylation.
Compounds 1 and 2 serve as prototypes for this novel type
of enzyme inhibitor, and they demonstrate clearly the proof of
the concept disclosed herein. The strategy described in this report
should in principle find applications for many enzymes that
employ general acid-base chemistry for their catalytic pro-
cesses.14
Compound 4 is simply a substrate for chymotrypsin (kcat
)
0.054 ( 0.003 min-1 and Km ) 420 ( 50 µM, kcat/Km ) 2.1 (
0.3 M-1 s-1), which means that it acylated the enzyme and the
complex underwent deacylation. The following two substrates
have similar structures to our compounds and they have been
studied with chymotrypsin: Ac-L-Ala-L-Ala-L-Phe-OMe (Km )
30 µM, kcat ) 60 s-1, kcat/Km ) 2 × 106 M-1 s-1 11a
)
and Suc-
L-Ala-L-Ala-L-Pro-L-Phe-pNA (Km ) 43 µM, kcat ) 45 s-1, kcat/
Km ) 1 × 106 M-1 s-1).11b Compound 4 is a poorer substrate
for chymotrypsin by a few orders of magnitude. The reason for
this difference in tunover property may have to do with the
fact that the amine moiety of the C-terminal amino acid of the
substrate makes a critical hydrogen bond to the main-chain
carbonyl of Ser-214 of chymotrypsin.6,12 This interaction is
obviously absent for the binding of 4 to the active site.
Material and Methods
1H and 13C NMR spectra were recorded on either a Varian Gemini-
300 or a Varian Unity-500 spectrometer. Chemical shifts are reported
in ppm from tetramethylsilane on the δ scale. Optical rotations were
determined with a Jasco DIP-370 digital polarimeter. Infrared and mass
spectra were recorded on Nicolet 680 DSP and Kratos MS 80RFT
On the other hand, both 1 and 2 inhibited the enzyme, and
each did so in a different manner. Compound 2 served as a
competitive inhibitor of the enzyme (Ki ) 2.2 ( 0.8 mM; Figure
1A). Dilution of the inhibited chymotrypsin mixture into a
solution of substrate for the enzyme allowed for the immediate
(13) Ki is a dissociation constant, whereas KI is a more complex parameter
for inhibition, such as Km is for turnover.
(9) Evans, D. A.; Mathre, D. J.; Scott, W. L. J. Org. Chem. 1985, 50,
1830.
(14) The crystal structure of chymotrypsin modified by the mechanism-
based inactivator 3-benzyl-6-chloro-2-pyrone indicates that a carboxylate
from the inhibitor, which is generated on active-site modification of the
enzyme, interacts weakly (3.4 Å) with the active-site histidine (Ringe, D.;
Mottonen, J. M.; Gelb, M. H.; Abeles, R. H. Biochemistry 1986, 25, 5633).
The complex undergoes slow deacylation, which may be attributable to
this weak interaction and the fact that the acyl-enzyme species is an R-â-
unsaturated ester.
(10) Interestingly, the aldol condensation in the presence of 1.0 equiv
of the base led to the selective formation of the anti-aldol; the experimental
details for this observation will be described elswhere.
(11) (a) Morihara, K.; Oka, T. Arch. Biochem. Biophys. 1977, 178, 188.
(b) DelMar, E. G.; Largman, C.; Brodrich, J. W.; Geokas, M. C. Anal.
Biochem. 1979, 99, 316.
(12) Kim, D. H. Bioorg. Med. Chem. Lett. 1993, 3, 1333.

13006 J. Am. Chem. Soc., Vol. 120, No. 50, 1998
Li et al.
spectrometers, respectively. Melting points were taken on an Electro-
thermal melting point apparatus and are uncorrected. Analytical high-
performance liquid chromatography was carried out on a Perkin-Elmer
Series 410 LC, using a Vydac C-18 column. Thin-layer chromatography
was performed with Whatman reagents 0.25 mm silica gel 60-F plates.
All other reagents were purchased from the Aldrich Chemical Co.
R-Chymotrypsin and N-succinyl-L-Ala-L-Ala-L-Pro-L-Phe p-nitroanilide
were purchased from Sigma Co. Spectrophotometric studies were
performed on Hewlett-Packard 8453 diode array instrument. Nonlinear
regression analysis was performed by the use of the SigmaPlot program
(Jandel Scientific). Other calculations were performed by the MS Excel
program. The PDB codes for the chymotrypsin that were used for the
design of the compounds are 1gct.pdb and 2gct.pdb (both at 1.8 Å
resolution).6 The numbering of the residues is according to the PDB
nomenclature, as described earlier.6 The optical rotation, melting point,
IR, NMR, and MS for each compound are provided in the Supporting
Information.
was concentrated in vacuo to give the title compound as a white solid
(234 mg, 100%).
Methyl (2R,3R)-5-(N-Cbz-L-alanyl-L-alanyl)amino-2-benzyl-3-hy-
droxypentanoate (14a). To a solution of N-Cbz-L-alanyl-L-alanine (294
mg, 1 mmol) in CH2Cl2 (10 mL) was added 1-hydroxybenzo-
triazole (150 mg, 0.9 mmol) and dicyclohexylcarbodiimide (185 mg,
0.9 mmol), and the mixture was stirred for 1 h. Subsequently, 10a (220
mg, 0.8 mmol) and triethylamine (81 mg, 0.8 mmol) were added to
the reaction mixture, which was stirred for an additional 24 h at room
temperature. After filtration, the filtrate was concentrated in vacuo, and
the residue was taken up in ethyl acetate, washed with 1 N HCl, 5%
NaHCO3, and saturated NaCl, and dried over MgSO4. The solvent was
evaporated, and the residue was purified by column chromatography
(silica gel, CHCl3:MeOH, 20:1) to give the title compound as a white
solid (375 mg, 73%).
Methyl (2R,3R)-5-(N-Acetyl-L-alanyl-L-alanyl)amino-2-benzyl-3-
hydroxypentanoate (1). A solution of 14a (126 mg, 0.2 mmol) in
methanol (10 mL) containing HCl (0.4 mmol) was hydrogenolyzed at
room temperature in the presence of 10% palladium on carbon for 30
min. Removal of the catalyst and concentration of the filtrate under
reduced pressure gave the corresponding deprotected compound (83
mg, 100%). This product was dissolved in 10% triethylamine methanol,
to which was added acetic anhydride (24.5 mg, 0.24 mmol, 22 µL)
while stirring vigorously. After acetylation was completed, the solvent
was evaporated under reduced pressure, and the residue was purified
by column chromatography (silica gel, CHCl3:MeOH, 20:1) to give
the title compound as a white solid (76 mg, 90%).
(4R,5S)-3-[(2R,3S)-5-Cbz-amino-2-benzyl-3-hydroxypentanoyl]-
4-methyl-5-phenyloxazolidin-2-one (12). Di-n-butylboryltriflate (1 M
in CH2Cl2, 12 mL, 12 mmol) was added dropwise to a stirred solution
of the N-acyloxazolidinone 11 (3.09 g, 10 mmol) in CH2Cl2 (50 mL)
at 0 °C. After the mixture was stirred at 0 °C for 10 min, diisopropyl-
ethylamine (DPEA; 1.55 g, 2.1 mL, 12 mmol) in CH2Cl2 (10 mL) was
added dropwise to the reaction mixture. The mixture was stirred at 0
°C for 30 min and was cooled to -78 °C. To the above enolate
solutionwas added a solution of N-Cbz-aminopropylaldehyde (2.47 g,
12 mmol) and DPEA (2.1 mL) in CH2Cl2 (15 mL). The reaction mixture
was stirred at -78 °C for 4 h, allowed to warm to 0 °C, and the reaction
was then quenched with a mixture of methanol (60 mL), aqueous
phosphate buffer (pH 7.0, 40 mL), and 30% H2O2 (40 mL). The aqueous
layer was extracted with CH2Cl2. The combined organic portion was
washed with saturated aqueous NH4Cl and saturated NaCl, dried over
MgSO4, and concentrated in vacuo. The residue was purified by column
chromatography (silica gel, EtOAc:hexane, 1:2) to give the title
compound as colorless oil (3.40 g, 66%). The unused starting
oxazolidinone was also recovered (1.05 g, 34%).
Methyl (2R,3S)-5-N-Cbz-amino-2-benzyl-3-hydroxypentanoate
(13a). A solution of 12 (1.03 g, 2.0 mmol) in a mixture of THF and
water (4:1, 20 mL) was cooled to 0 °C. The sequential addition of
30% H2O2 (0.9 mL, 8.0 mmol) and a solution of LiOH (126 mg, 3.0
mmol) in water (3 mL) to the mixture was followed by 1 h of stirring
at 0 °C. A solution of Na2SO3 (1.01 g, 8.0 mmol) in water (6 mL) was
added, and the resulting solution was stirred for 15 min. The organic
solvent was removed under reduced pressure, and the resulting aqueous
phase (pH 12) was washed with CH2Cl2 (3 × 20 mL). The aqueous
phase was cooled to 0 °C, acidified to pH ≈ 1 with 1 N HCl, and
extracted with EtOAc (5 × 20 mL). The organic phase was dried over
MgSO4 and concentrated under reduced pressure to give the corres-
ponding acid as a colorless oil (578 mg). The CH2Cl2 extract was dried
over MgSO4 and evaporated under reduced pressure. This procedure
allowed the recovery of the chiral oxazolidinone for reuse (335 mg,
95%). The above acid was dissolved in methanol (20 mL) containing
catalytic amount of HCl and was stirred at room temperature overnight.
The solvent was removed in vacuo, and the residue was taken up in
ethyl acetate (20 mL) and then washed with 5% NaHCO3, water, and
saturated NaCl, followed by drying over MgSO4. The solvent was
removed in vacuo, and the residue was purified by column chroma-
tography (silica gel, EtOAc:hexane, 1:2) to give the title compound as
a colorless oil (601 mg, 81%).
Dimethyl (2R,3S)-2-Benzyl-3-hydroxysuccinate (6). Butyllithium
(2.5 M solution in hexane; 28 mL, 70 mmol) was added dropwise to
a solution of diisopropylamine (9.9 mL, 70 mmol) in anhydrous THF
(50 mL) at 0 °C under a nitrogen atmosphere, and the mixture was
stirred for 30 min. Dimethyl (S)-2-hydroxysuccinate (5.3 g, 33 mmol)
in anhydrous THF (10 mL) was added dropwise to the solution of
lithium diisopropylamine, described above, at -78 °C, and the mixture
was stirred for 1 h. Benzyl bromide (6.0 g, 35 mmol) was added and
stirring was continued at -78 °C for 1 h and subsequently at -50 °C
for 5 h. Saturated aqueous ammonium chloride (20 mL) was added to
the mixture, and the solution was then washed with ethyl acetate. The
organic layer was washed with 5% NaHCO3 and saturated NaCl, was
dried over MgSO4, and was concentrated in vacuo. The residue was
purified by column chromatography (silica gel, EtOAc:hexane, 1:4) to
afford the title compound as a colorless oil (5.16 g, 62%).
Methyl (2R,3S)-2-Benzyl-3,4-dihydroxybutanoate (7). A borane-
methyl sulfide solution (2.0 M solution in THF; 2.32 mL, 4.64 mmol)
was added dropwise to a solution of 6 (1.17 g, 4.64 mmol) in anhydrous
THF (10 mL) at 0 °C under a nitrogen atmosphere, and the mixture
was stirred for 1 h. Sodium borohydride (8.8 mg, 0.23 mmol) was added
to the mixture, and stirring was continued for 30 min at room
temperature. The mixture was then quenched by the addition of dry
methanol (3 mL), followed by an additional 30 min of stirring. The
solvent was removed by evaporation in vacuo to give a colorless oil
that was purified by column chromatography (silica gel, EtOAc:hexane,
1:1) to give the title compound as a colorless oil (810 mg, 78%).
Methyl (2R,3S)-2-Benzyl-3,4-epoxybutanoate (8). Freshly distilled
pyridine (4 mL) and toluenesulfonyl chloride (7.63 g, 40 mmol) were
added to a solution of 7 (4.48 g, 20 mmol) in dry dichloromethane (48
mL) at 0 °C under a nitrogen atmosphere. The mixture was stirred for
12 h at 0 °C and was then acidified (pH 3-4) with 1N HCl. The organic
layer was washed with water and saturated NaCl, dried over MgSO4,
and concentrated in vacuo to give the monotosylate as a semisolid (5.67
g). This residue was dissolved in methanol (50 mL) and was treated
with potassium carbonate (2.07 g, 15 mmol) while being chilled in
ice-water temperature. After 1 h, the mixture was quenched by the
addition of water and was washed with ethyl acetate. The ethyl acetate
layer was washed with saturated NaCl and dried over Na2SO4, and the
solvent was evaporated. The residue was purified by column chroma-
tography (silica gel, EtOAc:hexane, 1:4) to give the title compound as
a colorless oil (2.75 g, 67%).
Methyl (2R,3S)-2-Benzyl-4-cyano-3-hydroxybutanoate (9). An-
hydrous potassium cyanide (146 mg, 2.32 mmol) was added to a
solution of 8 (240 mg, 1.16 mmol) in acetonitrile (5 mL), and the
mixture was stirred overnight at room temperature. The solvent was
evaporated in vacuo, and the residue was purified by column chroma-
tography (silica gel, EtOAc:hexane, 1:3) to give the title compound as
a colorless oil (230 mg, 85%).
Methyl (2R,3R)-5-Amino-2-benzyl-3-hydroxypentanoate Hydro-
chloride (10a). A solution of 9 (200 mg, 0.858 mmol) in anhydrous
methanol (10 mL) containing HCl (2.58 mmol) was hydrogenated under
40 psi at room temperature in the presence of 10% palladium on carbon
(200 mg) for 2 h. After removal of the catalyst by filtration, the filtrate
Methyl (2R,3S)-5-(N-Cbz-L-alanyl-L-alanyl)amino-2-benzyl-3-
hydroxypentanoate (14b). A solution of 13a (410 mg, 1.10 mmol) in

H-Bonding and the Rate of Enzymic Catalysis
J. Am. Chem. Soc., Vol. 120, No. 50, 1998 13007
5 mL of anhydrous methanol containing HCl (2.2 mmol) was subjected
to hydrogenolysis at room temperature in the presence of 10% palladium
on carbon (400 mg). After filtration, the solvent was evaporated in
vacuo to give a white solid (300 mg). To a solution of N-Cbz-L-Ala-
L-Ala (485 mg, 1.65 mmol) in CH2Cl2 (5 mL) were added HOBt (148
mg, 1.1 mmol) and DCC (227 mg, 1.1 mmol). The mixture was stirred
at room temperature for 1 h. To this mixture was added the above
amine salt (300 mg, 1.1 mmol), followed by stirring at room-
temperature overnight. After filtration, the filtrate was washed with
5% NaHCO3, 1 N HCl, water, and saturated NaCl and dried over
MgSO4. The solvent was removed in vacuo, and the residue was purified
by column chromatography (silica gel, CHCl3:MeOH, 40:1) to give
the title compound (485 mg, 86%).
The recovery of activity of inactivated enzyme was studied in the
following manner. After 20 h of incubation with 10 mM of compound
1 (residual enzyme activity was less than 5%), the solution was
subjected to ultrafiltration to remove the excess of inhibitor. The inactive
enzyme was reconstituted to the original volume with 100 mM
phosphate buffer, pH 7.8. Enzyme activity was monitored over a number
of hours.
A solution of R-chymotrypsin (final concentration 68 µM) in 50
mM Tris, 50 mM CaCl2, pH 7.8, was mixed with a solution of 4 in the
same buffer (final concentration 1.25 mM), and the mixture was
incubated at room temperature. Aliquots (50 µL) were removed from
the mixture at different time points. The reaction was stopped by
separation of the low molecular weight species from the protein by
ultrafiltration through a Microcon device, and 10-µL portions of the
filtrate were analyzed for determination of concentration of 4 by
HPLC: tR ) 22.8 min (Vydac C-18 4.6 mm × 25 cm, 25% acetonitrile
isocratic for 10 min, linear gradient 25-95% acetonitrile for 10 min,
95% isocratic acetonitrile for 10 min, 0.5 mL/min). The eluent was
monitored at 215 nm on a Perkin-Elmer UV detector, and the area
under the signals was integrated by Dynamax MacIntegrator. Concen-
trations of 4 in the course of the kinetic runs were calculated from
similar determinations for known concentrations of the compound used
to generate a calibration curve. Kinetic parameters (kcat and Km) for
enzymic turnover of the substrate were calculated from the complete
hydrolysis curves, using the integrated form of the Michaelis-Menten
equation.15
Methyl (2R,3S)-5-(N-Acetyl-L-alanyl-L-alanyl)amino-2-benzyl-3-
hydroxypentanoate (2). The title compound was prepared according
to the procedure described for 1 from 126 mg (0.2 mmol) of 14b to
afford 75 mg (90%) of 2 as a white solid.
Methyl (2R)-5-N-Cbz-amino-2-benzylpentanoate (13b). DMAP
(488 mg, 4 mmol) and phenyl chlorothionoformate (192 mg, 1.1 mmol)
were added to the solution of 13a (345 mg, 0.93 mmol) in CH2Cl2 (10
mL). The mixture was stirred at room temperature for 4 h. Ethyl acetate
(50 mL) was added to the reaction mixture, then washed with 1N HCl,
5% NaHCO3, water, and saturated NaCl, and was dried over MgSO4.
The solvent was removed in vacuo, and the residue was purified by
column chromatography (silica gel, EtOAc:hexane, 1:5) to give an
unstable phenyl thiocarbonate as a yellowish oil (333 mg, 71%).
Tributyltin hydride (2 mL) and di-tert-butyl peroxide (12 mg) were
added to the thiocarbonate (175 mg, 0.345 mmol), and the mixture
was stirred at 50 °C for 3 h. The reaction mixture was directly subjected
to column chromatography (silica gel, EtOAc:hexane, 1:3) to give the
title compound as a colorless oil (98 mg, 80%).
Methyl (2R)-5-(N-Cbz-L-alanyl-L-alanyl)amino-2-benzylpentanoate
(14c). The title compound was prepared according to the procedure
for 14b from 57 mg (0.16 mmol) of 13b to afford 75 mg (94%) of 14c
as a white solid.
Methyl (2R)-5-(N-Acetyl-L-alanyl-L-alanyl)amino-2-benzylpen-
tanoate (4). The title compound was prepared according to the
procedure for 1 from 373 mg (0.75 mmol) of 14c to afford 265 mg
(87%) of 4 as a white solid.
Kinetics. A typical inactivation experiment was performed as
described below. A solution of 1 in water was added to a solution of
R-chymotrypsin (0.44 µM final concentration) in 50 mM Tris, 50 mM
CaCl2 buffer, pH 7.8, in a total volume of 200 µL, to give final
concentrations of 1 in the range of 1-10 mM. Aliquots (10 µL) of the
inactivation mixture were diluted into the 1-mL assay mixture contain-
ing 250 µM N-succinyl-L-Ala-L-Ala-L-Pro-L-Phe p-nitroanilide in the
same buffer. The initial rate of hydrolysis of the substrate was monitored
at 400 nm.
The dissociation constants (Ki) for compounds 1 and 2 for R-chy-
motrypsin were calculated by the method of Dixon.16 A series of assay
mixtures containing both N-succinyl-L-Ala-L-Ala-L-Pro-L-Phe p-nitroa-
nilide as the substrate (either 50 or 250 µM), and various concentrations
of compounds as inhibitor (100-500 µM) were prepared in 50 mM
Tris, 50 mM CaCl2, pH 7.8. A portion of the enzyme was added to
give a final concentration of 44 nM in a total volume of 1.0 mL. The
enzyme activity was determined immediately.
Supporting Information Available: Detailed information
for optical rotation, melting point, IR, NMR and MS for each
compound is provided (5 pages, print/PDF). See any current
masthead page for ordering information and Web access
instructions.
JA983063E
(15) Cornish-Bowden, A. In Fundamentals of Enzyme Kinetics; Portland
Press: 1995; p 45.
(16) Dixon, M. Biochem. J. 1953, 55, 170.