Peptidyl epoxides: Novel selective inactivators of cysteine proteases

Article abstract of DOI:10.1021/ja954261y

Peptidyl epoxides were designed as selective pseudo-mechanism-based inactivators of cysteine proteases. Both threo- and erythro-peptidyl epoxides were synthesized and tested as potential inactivators of serine proteases (chymotrypsin, subtilisin, and elastase) and of cysteine proteases (papain, cathepsin B, and clostripain). Four tripeptidyl epoxides (Cbz-Gly-Leu-Phe-epoxide, Cbz-Ala-Ala-Phe-epoxide, Cbz-Gly-Leu-Ala-epoxide, and Cbz-Ala-Ala-Ala-epoxide), bearing amino acid sequences similar to those of good substrates or known inhibitors of the serine proteases, were tested in this study. Neither the threo- nor the erythro-peptidyl epoxides exhibited any inhibitory activity toward the serine proteases, even at high concentration and long incubation time. Nor did the threo-peptidyl epoxides inhibit the cysteine proteases. On the other hand, the erythro-peptidyl epoxides were time- and concentration-dependent inactivators of the cysteine proteases. Furthermore, stereoselectivity toward the natural L-amino acid at the P₁ position was also exhibited upon inhibition of papain. In order to demonstrate selectivity within the cysteine protease family, two other erythro-peptidyl epoxides (Cbz-Phe-Ala-epoxide and Cbz-Phe-O-Bn-Thr-epoxide) were synthesized and tested as inhibitors of the three cysteine proteases. These new peptidyl epoxides exhibited selective inactivation of cysteine proteases, with second-order rate constants (k(i)/K(i)) ranging over 4 orders of magnitude (0.04-330 M^-1 s^-1). Thus, this new family of highly selective cysteine protease inhibitors offers mechanistic implications and may have useful applications.

Full text of DOI:10.1021/ja954261y

J. Am. Chem. Soc. 1996, 118, 3591-3596
3591
Peptidyl Epoxides: Novel Selective Inactivators of Cysteine
Proteases1
Amnon Albeck,* Shulamit Fluss, and Rachel Persky
Contribution from the Department of Chemistry, Bar Ilan UniVersity, Ramat Gan 52900, Israel
ReceiVed December 20, 1995X
Abstract: Peptidyl epoxides were designed as selective pseudo-mechanism-based inactivators of cysteine proteases.
Both threo- and erythro-peptidyl epoxides were synthesized and tested as potential inactivators of serine proteases
(chymotrypsin, subtilisin, and elastase) and of cysteine proteases (papain, cathepsin B, and clostripain). Four tripeptidyl
epoxides (Cbz-Gly-Leu-Phe-epoxide, Cbz-Ala-Ala-Phe-epoxide, Cbz-Gly-Leu-Ala-epoxide, and Cbz-Ala-Ala-Ala-
epoxide), bearing amino acid sequences similar to those of good substrates or known inhibitors of the serine proteases,
were tested in this study. Neither the threo- nor the erythro-peptidyl epoxides exhibited any inhibitory activity
toward the serine proteases, even at high concentration and long incubation time. Nor did the threo-peptidyl epoxides
inhibit the cysteine proteases. On the other hand, the erythro-peptidyl epoxides were time- and concentration-
dependent inactivators of the cysteine proteases. Furthermore, stereoselectivity toward the natural L-amino acid at
the P1 position was also exhibited upon inhibition of papain. In order to demonstrate selectivity within the cysteine
protease family, two other erythro-peptidyl epoxides (Cbz-Phe-Ala-epoxide and Cbz-Phe-O-Bn-Thr-epoxide) were
synthesized and tested as inhibitors of the three cysteine proteases. These new peptidyl epoxides exhibited selective
inactivation of cysteine proteases, with second-order rate constants (ki/Ki) ranging over 4 orders of magnitude (0.04-
330 M-1 s-1). Thus, this new family of highly selective cysteine protease inhibitors offers mechanistic implications
and may have useful applications.
Proteases play an essential role in all biological systems.
Their activity is subjected to tight regulation by endogenous
specific inhibitors.2 This delicate balance may be perturbed by
environmental factors or heredity, leading to excessive enzy-
matic activity associated with different disease states. High
blood pressure, cerebral and coronary infarction, inflammation,
emphysema, muscular dystrophy, rickets, and rheumatoid
arthritis are just a few to mention.3 Some bacterial and viral
diseases are also intimately associated with specific protease
activity, when these microorganisms utilize their own proteases
for protein procession. Thus, it is clear that selective inhibition
of specific proteolytic enzymes could be of high therapeutic
interest. Mechanistic studies have also stimulated development
of selective protease inhibitors. Peptidyl chloromethanes,4
peptidyl trifluoromethanes,5 and peptidyl phosphonates6 are just
a few such examples.
substrate’s amide scissile bond by an enzyme residue.7 On the
basis of theoretical calculations, it was recently suggested that
protonation of the substrate scissile bond (either on oxygen or
on nitrogen) takes place prior to or concerted with the nucleo-
philic attack of the thiolate of cysteine proteases.8 In serine
proteases, on the other hand, the nucleophilic attack by the serine
hydroxide precedes the protonation step, thus leading to a
negatively charged tetrahedral intermediate. This constitutes a
subtle mechanistic difference between the serine and cysteine
proteases. Such a mechanistic difference was earlier depicted
as a possible explanation for the selectivity of some inhibitors
toward cysteine proteases.9
Epoxides are weak electrophiles and are stable in neutral or
basic aqueous media.10 However, they become highly electro-
philic upon protonation. This character was exploited in the
development of a few protease inhibitors. Epoxides derived
from allyl amides and esters exhibited weak inhibition of
cathepsin B and papain.11 A few peptidyl epoxides were
demonstrated to inhibit aspartic proteases.12 There, too, pro-
tonation by the active site aspartic acid may activate the epoxide
moiety for nucleophilic attack by an enzyme residue. This
principle was also utilized for inhibition of carboxypeptidase
A, a metalloprotease in which Zn2+ can activate an epoxide.13
Serine and cysteine proteases share a similar catalytic
mechanism, which is based on a nucleophilic attack of the
X Abstract published in AdVance ACS Abstracts, April 1, 1996.
(1) Abbreviations: amino acids are written in their three-letter codes;
Bn, benzyl; Cbz, benzyloxycarbonyl; BAEE, NR-benzoyl-L-arginine ethyl
ester; BTEE, N-benzoyl-L-tyrosine ethyl ester; Succ-AAA-pNA, N-succinyl-
Ala-Ala-Ala p-nitroanilide; Succ-AAPF-pNA, N-succinyl-Ala-Ala-Pro-Phe
p-nitroanilide; ONp, o-nitrophenol; TFA, trifluoroacetic acid; DIBAL,
diisobutylaluminum hydride; mCPBA, 3-(chloroperoxy)benzoic acid; Tris,
tris(hydroxymethyl)aminomethane.
(2) Proteinase Inhibitors; Research Monographs in Cell and Tissue
Physiology; Barrett, A. J., Salvesen, G. Eds.; Elsevier: Amsterdam, 1986;
Vol. 12, Chapters 7-20, pp 301-612.
(3) (a) Rich, D. H. In ComprehensiVe Medicinal Chemistry; Hansch, C.,
Sammes, P. C., Taylor, J. B., Eds.; Pergamon Press: Oxford, 1990; Vol. 2,
pp 391-441. (b) Fischer, G. Nat. Prod. Rep. 1988, 465-495. (c) Powers,
J. C.; Harper, J. W. In Proteinase Inhibitors; Research Monographs in Cell
and Tissue Physiology; Barrett, A. J., Salvesen, G., Eds.; Elsevier:
Amsterdam, 1986; Vol. 12, pp 55-152.
(6) (a) Kaplan, A. P.; Bartlett, P. A. Biochemistry 1991, 30, 8165-8170.
(b) Bartlett, P. A.; Hanson, J. E.; Giannousis, P. P. J. Org. Chem. 1990,
55, 6268-6274. (c) Hanson, J. E.; Kaplan, A. P.; Bartlett, P. A.
Biochemistry 1989, 28, 6294-6305.
(7) (a) Fersht, A. Enzyme Structure and Mechanism, 2nd ed.; W. H.
Freeman and Co.: New York, 1985; pp 405-426. (b) Walsh, C. Enzymatic
Reaction Mechanisms; W. H. Freeman and Co.: New York, 1979; pp 53-
107.
(8) Arad, D.; Langride, R.; Kollman, P. A. J. Am. Chem. Soc. 1990,
112, 491-502.
(9) Rich, D. H. In Proteinase Inhibitors; Research Monographs in Cell
and Tissue Physiology; Barrett, A. J., Salvesen, G., Eds.; Elsevier:
Amsterdam, 1986; Vol. 12, pp 153-178.
(4) (a) Markland, F. S.; Shaw, E.; Smith, E. L. Proc. Natl. Acad. Sci.
U.S.A. 1968, 61, 1440-1447. (b) Schoellmann, G.; Shaw, E. Biochemistry
1963, 2, 252-255.
(5) (a) Brady, K.; Liang, T.-C.; Abeles, R. H. Biochemistry 1989, 28,
9066-9070. (b) Liang, T.-C.; Abeles, R. H. Biochemistry 1987, 26, 7603-
7608.
(10) Pocker, Y.; Ronald, B. P.; Anderson, K. W. J. Am. Chem. Soc. 1988,
110, 6492-6497.
(11) Giordano, C.; Gallina, C.; Consalvi, V.; Scandurra, R. Eur. J. Med.
Chem. 1990, 25, 479-487.
0002-7863/96/1518-3591$12.00/0 © 1996 American Chemical Society

3592 J. Am. Chem. Soc., Vol. 118, No. 15, 1996
Figure 1. Erythro (left) and threo (right) peptidyl epoxides.
Albeck et al.
Table 1. erythro:threo Ratio of Synthetic Peptidyl Epoxidesa
peptidyl epoxide
erythro:threo ratio
Cbz-Ala-Ala-Ala
Cbz-Ala-Ala-Phe
Cbz-Gly-Leu-Ala
Cbz-Gly-Leu-Phe
Cbz-Phe-Ala
2.5
13.3
6.7b
4.6
Scheme 1. Synthetic Approach toward (A) threo-Peptidyl
Epoxides and (B) erythro-Peptidyl Epoxides
3.6b
6.1
Cbz-Phe-O-Bn-Thr
a Synthesized according to the “erythro” procedure and crystallized.
This product mixture was used for the inhibition studies. b Ratio after
chromatography.
and Persky (see Scheme 1B).18 They were utilized in the
inhibition studies without being separated from the residual threo
isomer (see Table 1).
Interaction with Serine Proteases. The serine proteases
chymotrypsin and subtilisin were preincubated with each of the
threo-peptidyl epoxides Cbz-Gly-Leu-Ala-epoxide, Cbz-Gly-
Leu-Phe-epoxide, Cbz-Ala-Ala-Phe-epoxide, and Cbz-Ala-Ala-
Ala-epoxide. Inhibitor concentrations were almost at saturation
in the 10% (v/v) acetonitrile-buffer preincubation solution (1.0,
0.5, 1.0, 5.0 mM, respectively). No loss of enzymatic activity
was detected after 1 h of incubation.
Chymotrypsin and subtilisin were also preincubated with each
of the erythro-peptidyl epoxides Cbz-Gly-Leu-Ala-epoxide,
Cbz-Gly-Leu-Phe-epoxide, and Cbz-Ala-Ala-Phe-epoxide, while
the serine protease elastase was preincubated with Cbz-Ala-
Ala-Ala-epoxide and Cbz-Ala-Ala-Phe-epoxide. Inhibitor con-
centrations were almost at saturation in the 10% (v/v) aceto-
nitrile-buffer preincubation solution (ranging from 0.5 to 5.0
mM, as above). Here, too, no loss of enzymatic activity was
detected even after long incubation time (up to 2.5 h, except
for Cbz-Gly-Leu-Phe-epoxide, which started precipitating after
about 1 h).
Interaction with Cysteine Proteases. The activated cysteine
proteases papain and cathepsin B were preincubated with each
of the threo-peptidyl epoxides Cbz-Gly-Leu-Ala-epoxide, Cbz-
Gly-Leu-Phe-epoxide, Cbz-Ala-Ala-Phe-epoxide, and Cbz-Ala-
Ala-Ala-epoxide. Once again, inhibitor concentrations were
almost at saturation. No loss of enzymatic activity was detected
after incubation for 1 h.
On the other hand, time- and concentration-dependent inhibi-
tion was observed upon incubation of the cysteine proteases
papain, cathepsin B, and clostripain with the erythro isomer of
peptidyl epoxides. One typical graphical presentation of the
inactivation process is shown in Figure 2. The corresponding
kinetic parameters are summarized in Tables 2-4. It should
be noted that various erythro/threo mixtures were used for this
part of the study (see Table 1), and therefore, the actual second-
order rate constants for inactivation are somewhat larger then
those measured and presented in the tables.
As part of our efforts to design selective protease inhibitors,
and in order to validate the above-suggested mechanistic
difference between serine and cysteine proteases, (2S,3S)-3-[N-
(benzyloxycarbonyl)amino]-1,2-epoxy-4-phenylbutane (Cbz-
Phe-epoxide) was designed and synthesized as a potential
selective inactivator of cysteine proteases. We postulated that
it would be activated by protonation in the active site of cysteine
proteases, followed by irreversible alkylation of the active site
cysteine, but would be unreactive toward the serine proteases.
Indeed, it exhibited time- and concentration-dependent inactiva-
tion of cysteine proteases, while it did not inhibit serine
proteases.14
Here, we extended this approach to longer peptidyl epoxides
(see Figure 1), in order to generalize the selectivity between
the two protease families, to demonstrate selectivity within the
family of cysteine proteases, and to gain some insight into the
stereoselectivity of the inhibition reaction. We suggest that
peptidyl epoxides can be considered as mechanism-based
inactivators of cysteine proteases.
Results
Synthesis. threo-Peptidyl epoxides were stereoselectively
synthesized according to a procedure originally introduced by
Luly and co-workers for R-amino epoxides,15 adapted for
peptidyl epoxides by Rich and co-workers,16 and recently
improved by Albeck and Persky.17 This procedure is described
in Scheme 1A. The threo-peptidyl epoxides were purified by
HPLC, and their isomeric purity was analyzed by HPLC prior
to the inhibition studies.17
erythro-Peptidyl epoxides were stereoselectively synthesized
via a short and efficient route recently introduced by Albeck
(12) (a) Moore, M. L.; Fakhoury, S. A.; Bryan, W. M.; Bryan, H. G.;
Tomaszek, T. A., Jr.; Grant, S. K.; Meek, T. D.; Huffman, W. F. In
Peptides: Chemistry and Biology; Proceedings of the American Peptide
Symposium, 12th; Smith, J. A.; Rivier, J. E. Eds.; ESCOM: Leiden, 1992;
pp 781-782. (b) Grant, S. K.; Moore, M. L.; Fakhoury, S. A.; Tomaszek,
T. A., Jr.; Meek, T. D. Bioorg. Med. Chem. Lett. 1992, 2, 1441-1445. (c)
Johnson, R. L. In Peptides: Structure and Function; Proceedings of the
American Peptide Symposium, 8th; Hruby, V. J., Rich, D. H. Eds.; Pierce
Chem. Co.: Rockford, IL, 1983; pp 587-590.
(13) Kim, D. H.; & Kim, K. B. J. Am. Chem. Soc. 1991, 113, 3200-
3202.
(14) Albeck, A.; Persky, R.; Kliper, S. Bioorg. Med. Chem. Lett. 1995,
5, 1767-1772.
(15) Luly, J. R.; Dellaria, J. F.; Plattner, J. J.; Soderquist, J. L.; Yi, N.
J. Org. Chem. 1987, 52, 1487-1492.
Discussion
Selectivity between Serine and Cysteine Proteases. Both
isomers (erythro and threo) of tripeptidyl epoxides were
stereoselectively synthesized and tested as potential inactivators
of serine and cysteine proteases. The peptide sequences were
chosen to match those of good substrates or known inhibitors
of the serine proteases chymotrypsin, subtilisin, and elastase.19
Therefore, we expected that the enzymes and these specific
peptidyl epoxides would form Michaelis-type complexes, plac-
ing the epoxide moiety near the active site serine. Nevertheless,
(16) (a) Romeo, S.; Rich, D. H. Tetrahedron Lett. 1994, 35, 4939-4942.
(b) Romeo, S.; Rich, D. H. Tetrahedron Lett. 1993, 34, 7187-7190. (c)
Rich, D. H.; Sun, C.-Q.; Vara Prasad, J. V. N.; Pathiasseril, A.; Toth, M.
V.; Marshall, G. R.; Clare, M.; Mueller, R. A.; Hauseman, K. J. Med. Chem.
1991, 34, 1222-1225.
(18) Albeck, A.; Persky, R. Tetrahedron 1994, 50, 6333-6346.
(19) (a) Powers, J. C.; Lively, M. O.; Tippet, J. T. Biochim. Biophys.
Acta 1977, 480, 246-261. (b) Powers, J. C. In Chemistry and Biology of
Amino Acids, Peptides and Proteins; Weinstein, B., Ed.; Marcel Dekker:
New York, 1977; Vol. 4, pp 66-178.
(17) Albeck, A.; Persky, R. J. Org. Chem. 1994, 59, 653-657.

Peptidyl Epoxides as InactiVators of Cysteine Proteases
J. Am. Chem. Soc., Vol. 118, No. 15, 1996 3593
Table 4. Kinetic Parameters for the Inactivation of Clostripain by
erythro-Peptidyl Epoxides
inhibitor sequence
t1/2 (min)
[I] (mM)
kobs/[I] (M-1 s-1
)
Cbz-Phe-Ala
Cbz-Phe-O-Bn-Thr
270
250
1.0
0.1
0.04
0.46
fluoromethanes,21 peptidyl halomethanes,19b and peptidyl boronic
acids.22
The erythro isomers of the same peptidyl epoxides were found
to be moderate inhibitors of the cysteine proteases papain and
cathepsin B. They exhibited time- and concentration-dependent
inhibition, in agreement with covalent inactivation. Processing
of the data led to the kinetic parameters of the inhibition process,
according to the minimal inhibition scheme
ki
E + I y
and according to eq 1.23
1/kobs ) (Ki/ki)(1/[I]) + 1/ki
\
Kzi E‚I
9
8 E-I
(1)
These results clearly demonstrate the selectivity of erythro-
peptidyl epoxides as inhibitors of cysteine proteases.
Stereoselectivity. The inactivation of cysteine proteases by
peptidyl epoxides exhibited high stereoselectivity with respect
to two chiral centers.
The erythro isomer of the four tripeptidyl epoxides (Cbz-
Ala-Ala-Ala-epoxide, Cbz-Ala-Ala-Phe-epoxide, Cbz-Gly-Leu-
Ala-epoxide, and Cbz-Gly-Leu-Phe-epoxide) inhibited the cys-
teine proteases papain and cathepsin B, while the corresponding
threo isomer did not, even upon long incubation time at
saturation concentrations.
Figure 2. (A) Time course of inactivation of cathepsin B by Cbz-
Phe-O-Bn-Thr-epoxide, in 80 mM phosphate buffer, containing 10%
(v/v) acetonitrile, 0.5 mM DTT, and 2 mM EDTA, at 25 °C. Inhibitor
concentration: (O) 0 µM; (b) 1 µM; (]) 3 µM; ([) 5 µM; (4) 10
µM; (2) 30 µM; (0) 100 µM. (B) Replot of 1/kobs vs 1/[I].
A second aspect of the stereoselectivity expressed in the
inactivation process relates to the preference for the natural L
(S) isomer of the amino epoxide analog at the P1 position.24 In
comparing the two peptidyl epoxides erythro-Cbz-Gly-Leu-Phe-
epoxide and erythro-Cbz-Gly-Leu-D-Phe-epoxide, the latter
exhibited an apparent second-order rate constant of inactivation
only 3% that of the corresponding rate constant of the former
L-peptidyl epoxide. It has been previously demonstrated that
the synthetic procedure for preparation of erythro-peptidyl
epoxides involves about 3% epimerization at the CR of the P1
amino acid.18 Therefore, we attribute most of the 3% residual
inhibitory activity of the D isomer to contamination by the
corresponding L isomer. Thus, we conclude that the inactivation
process exhibits >99:1 stereoselectivity, preferring the natural
L isomer at the P1 position of the inhibitor.
Table 2. Kinetic Parameters for the Inactivation of Papain by
erythro-Peptidyl Epoxides
inhibitor sequence
ki (min-1
)
Ki (mM)
ki/Ki (M-1 s-1
)
Cbz-Ala-Ala-Ala
Cbz-Ala-Ala-Phe
Cbz-Gly-Leu-Ala
Cbz-Gly-Leu-Phe
Cbz-Gly-Leu-D-Phe
Cbz-Phec
0.07a
1.75
0.31
0.20
0.02
0.06
1.91
1.08
0.64
1.56
0.04a,b
6.14
0.21
0.04
0.03
0.57
0.32
0.03
Cbz-Phe-Ala
Cbz-Phe-O-Bn-Thr
2.08
16.66
a Since inactivation was measured in a single inhibitor concentration
(1 mM), this value represents kobs/[I]. b See Discussion. c Taken from
Albeck et al.14
These stereoselectivities support an active-site-directed in-
activation, in contrast to a nonspecific bimolecular reaction.
Selectivity within the Family of Cysteine Proteases. For
most foreseen practical applications of protease inhibition, high
selectivity is essential. Therefore, a second set of dipeptidyl
Table 3. Kinetic Parameters for the Inactivation of Cathepsin B
by erythro-Peptidyl Epoxides
inhibitor sequence
ki (min-1
)
Ki (mM)
ki/Ki (M-1 s-1
)
Cbz-Ala-Ala-Ala
Cbz-Gly-Leu-Phe
Cbz-Phec
Cbz-Phe-Ala
Cbz-Phe-O-Bn-Thr
0.15a
1.14b
4.05
1.94
333.33
(20) (a) Delbaere, T. J.; Brayer, G. D. J. Mol. Biol. 1987, 183, 89-103.
(b) Hassall, C. H.; Johnson, W. H.; Kennedy, A. J.; Roberts, N. A. FEBS
Lett. 1985, 183, 201-205. (c) Galpin, I. J.; Wilby, A. H.; Place, G. A.;
Baynon, R. J. Int. J. Peptide Protein Res. 1984, 23, 477-486. (d)
Thompson, R. C. Biochemistry 1973, 12, 47-51.
(21) (a) Stein, R. L.; Strimpler, A. M.; Edwards, P. D.; Lewis, J. J.;
Mauger, R. S.; Schwartz, J. A.; Stein, M. M.; Trainor, D. A.; Wildonger,
R. A.; Zottola, M. A. Biochemistry 1987, 26, 2682-2689. (b) Dunlap, R.
P.; Stone, P. J.; Abeles, R. H. Biochem. Biophys. Res. Commun. 1987, 145,
509-513. (c) Imperiali, B.; Abeles, R. H. Biochemistry 1986, 25, 3760-
3767.
0.09
0.07
0.20
0.37
0.60
0.01
a Since inactivation was measured in a single inhibitor concentration
(1 mM), this value represents kobs/[I]. b Measurements were limited to
the linear range, where [I] e Ki, due to solubility problems. Therefore,
only the second-order rate constant was obtained. c Taken from Albeck
et al.14
no irreversible inactivation was observed with either of the
erythro- or threo-peptidyl epoxides studied. This is in sharp
contrast to the formation of covalent adducts between serine
proteases and a variety of peptide-based reversible and irre-
versible inhibitors such as peptidyl aldehydes,20 peptidyl tri-
(22) Kettner, C. A.; Shenvi, A. B. J. Biol. Chem. 1984, 259, 15106-
15114.
(23) Kitz, R.; Wilson, I. B. J. Biol. Chem. 1962, 237, 3245-3249.
(24) The P and P′ for substrates and S and S′ for enzyme subsites
terminology is used. See: Schechter, I.; Berger, A. Biochem. Biophys. Res.
Commun. 1967, 27, 157-162.

3594 J. Am. Chem. Soc., Vol. 118, No. 15, 1996
Albeck et al.
Scheme 2. Proposed Mechanism for the Inactivation of Cysteine Proteases by erythro-Peptidyl Epoxidesa
a In this scheme protonation precedes alkylation, but the two chemical steps may be concerted (see text).
epoxides, especially designed to exhibit high selectivity within
the family of cysteine proteases, was synthesized. The specific
amino acid sequences chosen for these inhibitors were based
on reviewing the selectivity of three cysteine proteasesspapain,
cathepsin B, and clostripainstoward some peptide substrates25
and other peptide inhibitors.26
In general, the second-order rate constants for inhibition of
cysteine proteases by peptidyl epoxides are significantly lower
than those exhibited by corresponding peptidyl diazomethanes
and peptidyl chloromethanes. This is due, at least in part, to
lower Ki values of the latter inhibitors. Such Ki values are
significantly lower than the actual dissociation constants from
the Michaelis-type complex as a result of a fast reversible
chemical step (a reversible nucleophilic attack of the active-
site thiolate on the inhibitor carbonyl) that precedes the
irreversible alkylation step. Such a reversible step is not feasible
in the case of peptidyl epoxides, and therefore their Ki values
represent the actual dissociation constants.
The results presented in Table 2 show an about 240-fold
increase in the second-order rate constant of inactivation of
papain upon going from the peptidyl epoxide with a peptide
sequence Cbz-Ala-Ala-Ala to the sequence Cbz-Phe-O-Bn-Thr.
This increase arises mainly from a decrease of the Ki values,
which in the simplest model represent the dissociation constants
of the enzyme-inhibitor Michaelis-like complexes. This is in
good agreement with the general preference of papain to
hydrolyze amide bonds of substrates having a large hydrophobic
residue at the P2 position.25 The selectivity is much more
pronounced in the inactivation of cathepsin B, which exhibits
a similar preference for P2 hydrophobic substrates.26c,27 Cbz-
Phe-O-Bn-Thr-epoxide inhibited the enzyme with a second-order
rate constant 170-fold higher than that displayed by Cbz-Phe-
Ala-epoxide and 2200-fold higher than that displayed by Cbz-
Ala-Ala-Ala-epoxide. Once again, most of the selectivity is
manifested in the kinetic parameter Ki.
Comparison of the effectiveness of a given inhibitor toward
various cysteine proteases also reveals a high degree of
selectivity. The inhibitor Cbz-Phe-O-Bn-Thr-epoxide inacti-
vates cathepsin B 20 times faster than it inactivates papain and
725 times faster than clostripain, while the inhibitor Cbz-Phe-
Ala-epoxide inhibits papain and cathepsin B 50 times faster than
it inhibits clostripain. The two most extreme cases observed
in this study reveal an almost 4 orders of magnitude difference
in the second-order rate constant.
Mechanistic Implications. Kollman and co-workers sug-
gested that, in serine proteases, the nucleophilic attack of the
active site serine on the substrate amide bond precedes proto-
nation. In cysteine proteases, on the other hand, protonation
of the scissile amide bond occurs prior to or concomitant with
the nucleophilic attack.8 Such initial protonation is feasible in
cysteine proteases due to the existence of a thiolate-imidazo-
lium ion pair in the Michaelis complex and in the free enzyme.28
On the other hand, in serine proteases the active site configu-
ration does not allow initial protonation since the serine and
histidine residues are neutral at the corresponding stages.
A few families of selective cysteine protease inhibitors have
been described. However, this selectivity does not have
mechanistic implications, as it can be explained on different
grounds. E-64 probably places its reactive epoxide away from
the active site hydroxyl of serine proteases due to preferential
binding of one of its side chains in the enzyme’s S1 site
(hydrophobic in chymotrypsin and subtilisin and basic in
trypsin). In addition, a “reversed” (retro-amide) mode of
binding to serine proteases, analogous to its binding in the active
site of papain,29 may lead to a very high dissociation constant.
Similar arguments can explain why the synthetic inhibitor Cbz-
Phe-Gly-epoxide inhibited papain and cathepsin B but not
chymotrypsin.11 In fact, we believe that this compound is
actually a substrate of chymotrypsin and related serine proteases.
Peptidyl diazomethanes are also selective inhibitors of
cysteine proteases. The suggested mechanism of inactivation
involves a cyclic sulfonium intermediate.9 Such a mechanism
is possible only in the binding site of cysteine proteases, due to
the easy alkylation of sulfides to form sulfonium cations, while
the corresponding reaction involving oxonium ion in the binding
site of serine proteases would be extremely energetic, and
therefore not feasible.
Thus, in principle, the selectivity exhibited by the above two
families of inhibitors can be explained in terms of alkylation
ability and is unrelated to the timing of the protonation step.
Peptidyl epoxides were designed as inhibitors which utilize
the initial (or simultaneous) protonation step in cysteine
proteases. The inhibition of papain by Cbz-Phe-epoxide was
previously shown to be irreversible, and a good linear correlation
was found between the remaining enzymatic activity and the
concentration of free thiol in the enzyme active site.
A
significant rate acceleration in the inactivation process relative
to a model reaction in solution was observed.14 These results
as well as the high stereoselectivity and the amino acid sequence
selectivity exhibited by the peptidyl epoxide inhibitors toward
the cysteine proteases strongly support an active-site-directed,
enzyme-catalyzed mechanism of inactivation (see Scheme 2).
Taken together with the selectivity toward cysteine proteases
as opposed to serine proteases, our results support the suggested
Such selectivity and its correlation with the natural selectivity
of the enzymes toward synthetic peptides or natural proteins
(and with the respective selectivity of other peptide-based
inhibitors) further support the suggested active-site-directed
inactivation, and lay the grounds for further mechanistic studies.
(25) Berger, A.; Schechter, I. Philos. Trans. R. Soc. London 1970, B257,
249-264.
(26) (a) Rauber, P.; Angliker, H.; Walker, B.; Shaw, E. Biochem. J. 1986,
239, 633-640. (b) Shaw, E.; Wikstrom, P.; Ruscica, J. Arch. Biochem.
Biophys. 1983, 222, 424-429. (c) Green, G. D. J.; Shaw, E. J. Biol. Chem.
1981, 256, 1923-1928. (d) Leary, R.; Larsen, D.; Watanabe, H.; Shaw,
E. Biochemistry 1977, 16, 5857-5861. (e) Roffman, L. S. Ph.D. Thesis,
New York University, 1974, Order no. 74-30,038.
(28) (a) Polgar, L.; Halasz, P. Biochem. J. 1982, 207, 1-10. (b) Johnson,
F. A.; Lewis, S. D.; Shafer, J. A. Biochemistry 1981, 20, 44-48. (c) Lewis,
S. D.; Johnson, F. A.; Shafer, J. A. Biochemistry 1981, 20, 48-51.
(29) (a) Yamamoto, D.; Matsumoto, K.; Ohishi, H.; Ishida, T.; Inoue,
M.; Kitamura, K.; Mizuno, H. J. Biol. Chem. 1991, 266, 14771-14777.
(b) Varughese, K. I.; Ahmed, F. R.; Carey, P. R.; Hasnain, S.; Huber, C.
P.; Storer, A. C. Biochemistry 1989, 28, 1330-1332.
(27) Barrett, A. J.; Kirschke, H. Methods Enzymol. 1981, 80, 535-561.

Peptidyl Epoxides as InactiVators of Cysteine Proteases
J. Am. Chem. Soc., Vol. 118, No. 15, 1996 3595
Cbz-Ala-Ala-Ala-epoxide (86% yield after ethyl acetate chroma-
tography, erythro:threo ) 2.3:1) was crystallized from acetone/
hexane: 1H NMR (erythro) δ 1.141 (d, J ) 6.9 Hz, 3H, CH3), 1.346
(assignment may be inverted) (d, J ) 7 Hz, 3H, CH3), 1.384 (d, J )
7.1 Hz, 3H, CH3), 2.706 (dd, J ) 4.7, 2.7 Hz, 1H, CH2O), 2.751 (dd,
J ) 4.7, 3.6 Hz, 1H, CH2O), 2.937 (ddd, J ) 5.3, 3.6, 2.7 Hz, 1H,
CHO), 3.987 (sext, J ) 7.0 Hz, 1H, CHR), 4.15-4.30 (m, 1H, CHR),
4.488 (quint, J ) 7 Hz, 1H, CHR), 5.117 (s, 2H, CH2Ph), 5.464 (d, J
) 6.4 Hz, 1H, NH), 6.64 (d, J ) 8 Hz, 1H, NH), 6.71 (d, J ) 6.9 Hz,
1H, NH), 7.383 (br s, 5H, Ph); (threo) δ 1.257 (d, J ) 6.9 Hz, 3H,
CH3), 1.365 (assignment may be inverted) (d, J ) 7 Hz, 3H, CH3),
1.384 (d, J ) 7.1 Hz, 3H, CH3), 2.551 (dd, J ) 4.6, 2.5 Hz, 1H, CH2O),
2.72 (dd, J ) 4.6, 4 Hz, 1H, CH2O), 3.010 (dt, J ) 4, 2.5 Hz, 1H,
CHO), 4.15-4.30 (m, 2H, CHR), 4.438 (quint, J ) 7 Hz, 1H, CHR),
5.117 (s, 2H, CH2Ph), 5.46 (obscured, 1H, NH), 6.294 (d, J ) 8.5 Hz,
1H, NH), 6.71 (obscured, 1H, NH), 7.383 (br s, 5H, Ph); 13C NMR
(erythro) δ 15.87 (CH3), 18.21 (CH3), 18.45 (CH3), 45.86 (CH2O), 46.08
(CR), 48.99 (CR), 50.99 (CR), 54.08 (CHO), 67.22 (CH2Ph), 128.05,
128.55, 136.00 (Ph), 156.07 (OCON), 171.51, 172.19 (CON); (threo)
δ 18-19 (3C, CH3), 43.81 (CR), 44.47 (CH2O), 49.12 (CR), 50.77
(CR), 54.17 (CHO), 67.16 (CH2Ph), 128.25, 128.30, 136.07 (Ph);
HRMS (EI) calcd for C18H25N3O5 (M+) 363.1794, found 363.1799;
MS (EI) m/z (relative intensity) 277 (7), 210 (24), 185 (40), 143 (73),
134 (15), 108 (21), 91 (100).
difference in the catalytic mechanism of these two families of
proteases. Thus, peptidyl epoxides can be considered as
mechanism-based inhibitors of cysteine proteases.
Conclusions
In the present study peptidyl epoxides were introduced as
novel selective cysteine protease inhibitors. High selectivity
is manifested in all aspects of this study, starting with stereo-
selective syntheses of both erythro and threo isomers of peptidyl
epoxides, through selectivity of the inhibition process between
serine and cysteine proteases, and finally stereoselectivity and
sequence selectivity of inhibition within the family of cysteine
proteases. Implications on the catalytic mechanism of cysteine
proteases were also considered.
Finally, we believe that besides possible future applications
of these cysteine protease inhibitors, peptidyl epoxides could
serve as a useful tool to identify cysteine protease activity and
a probe for the determination of the spatial arrangement of the
active site residues.
Experimental Section
Cbz-Phe-Ala-CH2Br (45% yield after 1:1 ether/hexane chroma-
1
tography) was crystallized from ether/hexane: mp 142-143 °C; H
General Procedure. Enzymes, amino acids, protected amino acids,
and protected peptides were from Sigma Chemical Co. They were
used without further purification. 1H and 13C NMR spectra were
recorded at 300 or 200 MHz and 75 or 50 MHz, respectively, in CDCl3,
unless otherwise specified. Chemical shifts are reported on the δ scale
with TMS resonance in CDCl3 or solvent resonance in other solvents
NMR δ 1.260 (d, J ) 7.1 Hz, 3H, CH3), 3.041 (d, J ) 7.0 Hz, 2H,
CH2â), 3.851 (s, 2H, CH2Br), 4.478 (q, J ) 7.3 Hz, 1H, CHR (Phe)),
4.669 (quint, J ) 7.1 Hz, 1H, CHR (Ala)), 5.034 (d, J ) 12.4 Hz, 1H,
CH2 (Cbz)), 5.055 (d, J ) 12.4 Hz, 1H, CH2 (Cbz)), 5.615 (d, J ) 7.8
Hz, 1H, NH (Phe)), 6.841 (d, J ) 5.8 Hz, 1H, NH (Ala)), 7.15-7.35
(m, 10H, Ph); 13C NMR δ 17.15 (CH3), 31.68 (CH2Br), 38.42 (Câ
(Phe)), 52.06 (CR (Phe)), 56.13 (CR (Ala)), 67.13 (CH2 (Cbz)), 127.14,
127.90, 128.17, 128.48, 128.67, 129.20, 135.98 (Ph), 155.96 (OCON),
170.92 (CON), 200.17 (CO); HRMS calcd for C21H24BrN2O4 (MH+)
447.0919, 449.0899, found 447.0802, 449.0777; MS m/z (relative
intensity) 447, 449 (MH+, 49, 50), 403 (33), 405 (25), 391 (26), 367
(100), 225 (60), 210 (31), 91 (72).
1
as an internal standard. All H NMR assignments were supported by
homonuclear decoupling experiments, while 13C NMR assignments were
supported by off-resonance heteronuclear decoupling or 2-D hetero-
COSY experiments. Mass spectra were recorded in CI mode with either
isobutane or ammonia as the reagent gas, unless otherwise indicated.
TLC was performed on E. Merck 0.2 mm precoated silica gel F-254
plates, and viewed by either UV light or Cl2/KI-tolidine.30 Flash column
chromatography31 was carried out on silica gel 60 (230-400 mesh
ASTM, E. Merck).
Cbz-Phe-Ala-epoxide (65% yield after 1:1 ether/hexane chroma-
tography, erythro:threo ) 3.6:1) was crystallized from ether/hexane:
mp 136-137 °C; 1H NMR (erythro) δ 1.028 (d, J ) 6.8 Hz, 3H, CH3),
2.600 (dd, J ) 4.1, 3.2 Hz, 1H, CH2O), 2.64 (obscured, 1H, CHO),
2.672 (t, J ) 4.1 Hz, 1H, CH2O), 2.995 (dd, J ) 13.6, 7.8 Hz, 1H,
CH2â), 3.121 (dd, J ) 13.6, 6.3 Hz, 1H, CH2â), 3.898 (m, 1H, CHR
(Ala)), 4.369 (q, J ) 6.7 Hz, 1H, CHR (Phe)), 5.083 (s, 2H, CH2 (Cbz)),
5.41 (d, J ) 6 Hz, 1H, NH (Phe)), 5.78 (d, J ) 5 Hz, 1H, NH (Ala)),
7.15-7.35 (m, 10H, Ph); (threo) δ 2.07 (dd, J ) 4, 3 Hz, 1H, CH2O),
2.501 (t, J ) 4 Hz, 1H, CH2O), 2.87 (dt, J ) 4, 2.5 Hz, 1H, CHO);
13C NMR (erythro) δ 15.90 (CH3), 38.86 (Câ (Phe)), 46.00 (CH2O),
46.40 (CR (Ala)), 53.89 (CHO), 56.60 (CR (Phe)), 67.19 (CH2 (Cbz)),
127.15, 128.05, 128.23, 128.55, 128.77, 129.30, 136.18, 136.38 (Ph),
170.22 (CON); HRMS calcd for C21H25N2O4 (MH+) 369.1814, found
369.1795; MS m/z (relative intensity) 369 (MH+, 100), 325 (10), 277
(14), 261 (10), 254 (12), 233 (10), 217 (14), 210 (19), 91 (94).
O-Benzyl-L-threonine trifluoroacetic acid salt (1 mmol) was
prepared in quantitative yield by stirring N-t-Boc-O-benzyl-L-threonine
in 4 mL of TFA/CH2Cl2 (1/1 by volume) at room temperature for 2 h,
followed by evaporation to dryness: 1H NMR δ 1.171 (d, J ) 6.4 Hz,
3H, CH3), 3.849 (br s, 1H, CHR), 4.027 (dq, J ) 3.5, 6.4 Hz, 1H,
CHâ), 4.241 (d, J ) 11.4 Hz, 1H, CH2O), 4.443 (d, J ) 11.4 Hz, 1H,
CH2O), 7.05-7.20 (m, 5H, Ph), 7.292 (br s, 3H, NH3), 10.928 (br s,
CO2H); 13C NMR δ 15.99 (CH3), 58.27 (CR), 71.09 (Câ), 71.72
(CH2O), 128.02, 128.18, 128.50, 136.62 (Ph), 170.67 (CO2H).
Cbz-Phe-O-benzyl-Thr was prepared by standard coupling (DCC,
NHS) of Cbz-Phe and O-benzyl-Thr. The dipeptide product was
isolated by ether/water extraction, acidification (HCl) of the aqueous
phase to pH 1.5, extraction of the aqueous phase with ether, drying of
the latter organic phase (MgSO4), filtration, and evaporation to dryness.
It was transferred to the next reaction without further purification: 1H
NMR δ 1.124 (d, J ) 6.1 Hz, 3H, CH3), 2.951 (dd, J ) 13.7, 8.3 Hz,
1H, CH2â (Phe)), 3.17 (dd, J ) 13, 5 Hz, 1H, CH2â (Phe)), 4.165 (dq,
J ) 1.9, 6.1 Hz, 1H, CHâ (Thr)), 4.359 (d, J ) 11.7 Hz, 1H, CH2O
Synthesis. The synthesis of threo- and erythro-peptidyl epoxides
was previously described.17,18 New erythro-peptidyl epoxides, Cbz-
Ala-Ala-Ala-epoxide, Cbz-Phe-Ala-epoxide, and Cbz-Phe-O-Bn-Thr-
epoxide, were synthesized according to the same procedure.18
Cbz-Ala-Ala-Ala (93% yield): 1H NMR (CD3OD) δ 1.332 (d, J )
7.2 Hz, 3H, CH3), 1.358 (d, J ) 8 Hz, 3H, CH3), 1.385 (d, J ) 7.3 Hz,
3H, CH3), 4.134 (q, J ) 7.2 Hz, 1H, CHR), 4.352 (m, 2H, CHR), 5.061
(d, J ) 12.8 Hz, 1H, CH2Ph), 5.088 (d, J ) 12.8 Hz, 1H, CH2Ph),
7.27-7.34 (m, 5H, Ph); 13C NMR (CD3OD) δ 17.66 (CH3), 18.06
(CH3), 18.22 (CH3), 49 (CR), 50.06 (CR), 51.99 (CR), 67.69 (CH2Ph),
128.80, 128.98, 129.44 (Ph), 174.44, 175.24, 175.72 (CO); HRMS calcd
for C17H24N3O6 (MH+) 366.1665, found 366.1654; MS m/z (relative
intensity) 348 (10), 277 (16), 258 (84), 240 (18), 212 (20), 187 (22),
169 (28), 141 (31), 116 (69), 91 (100).
Cbz-Ala-Ala-Ala-CH2Br (53% yield after 1:1 ethyl acetate/hexane
f ethyl acetate chromatography): 1H NMR δ 1.37 (d, J ) 7 Hz, 3H,
CH3), 1.381 (d, J ) 7.2 Hz, 6H, CH3), 4.05 (d, J ) 13 Hz, 1H, CH2-
Br), 4.07 (d, J ) 13 Hz, 1H, CH2Br), 4.242 (quint, J ) 6.6 Hz, 1H,
CHR), 4.489 (quint, J ) 7.2 Hz, CHR), 4.720 (quint, J ) 7.1 Hz, CHR),
5.13 (br s, 2H, CH2Ph), 5.52 (d, J ) 6 Hz, 1H, NH), 6.83 (d, J ) 6
Hz, 1H, NH), 7.13 (d, J ) 6 Hz, 1H, NH), 7.32-7.36 (m, 5H, Ph); 13
C
NMR δ 16.92 (CH3), 17.31 (CH3), 17.76 (CH3), 31.90 (CH2Br), 48.67
(CR), 51.07 (CR), 52.38 (CR), 67.28 (CH2Ph), 128.03, 128.29, 128.56
(Ph), 171.86, 172.25 (CO); HRMS calcd for C18H24N3O5Br (MH+)
442.0978, 444.0957, found 442.0747, 444.0714; MS m/z (relative
intensity) 364 (5), 362 (7), 320 (13), 277 (48), 249 (11), 206 (20), 134
(24), 91 (100).
(30) Krebs, K. G.; Heusser, D.; Wimmer, H. In Thin Layer Chroma-
tography, 2nd ed.; Stahl, E., Ed.; Springer-Verlag: New York, 1969; p
862.
(31) Still, W. C.; Kahn, M.; Mitra, A. J. Org. Chem. 1978, 43, 2923-
2925.

3596 J. Am. Chem. Soc., Vol. 118, No. 15, 1996
Albeck et al.
(Bn)), 4.510 (d, J ) 11.7 Hz, 1H, CH2O (Bn)), 4.60-4.70 (m, 2H,
CHR), 4.96 (d, J ) 13 Hz, 1H, CH2O (Cbz)), 5.01 (d, J ) 13 Hz, 1H,
CH2O (Cbz)), 5.536 (d, J ) 8.1 Hz, 1H, NH), 6.046 (d, J ) 7.6 Hz,
1H, NH), 7.10-7.30 (m, 15H, Ph), 10.080 (br s, CO2H); 13C NMR δ
16.08 (CH3), 38.21 (Câ (Phe)), 56.16, 56.70 (CR), 67.12 (CH2O (Cbz)),
71.11 (CH2O (Bn)), 74.03 (Câ (Thr)), 126.99, 127.71, 127.86, 128.08,
128.34, 128.40, 128.57, 129.26, 135.62, 135.85, 137.33 (Ph), 156.30
(OCON), 172.76 (CO2H), 173.62 (CON).
Cbz-Phe-O-benzyl-Thr-CH2Br (25% total yield from Cbz-Phe) was
crystallized from CH2Cl2/hexane: mp 143-144 °C; 1H NMR δ 1.028
(d, J ) 6.3 Hz, 3H, CH3), 3.060 (d, J ) 7.0 Hz, 2H, CH2â (Phe)),
3.706 (d, J ) 14.3 Hz, 1H, CH2Br), 3.808 (d, J ) 14.3 Hz, 1H, CH2-
Br), 4.030 (dq, J ) 2.5, 6.3 Hz, 1H, CHâ (Thr)), 4.364 (d, J ) 11.7
Hz, 1H, CH2O (Bn)), 4.509 (d, J ) 11.7 Hz, 1H, CH2O (Bn)), 4.550
(dt, J ) 8.0, 7.0 Hz, 1H, CHR (Phe)), 4.786 (dd, J ) 7.8, 2.5 Hz, 1H,
CHR (Thr)), 5.009 (d, J ) 12.3 Hz, 1H, CH2 (Cbz)), 5.063 (d, J )
12.3 Hz, 1H, CH2 (Cbz)), 5.556 (d, J ) 8.0 Hz, 1H, NH (Phe)), 6.877
(d, J ) 7.8 Hz, 1H, NH (Thr)), 7.15-7.40 (m, 15H, Ph); 13C NMR δ
15.53 (CH3), 33.57 (CH2Br), 38.28 (Câ (Phe)), 56.29 (CR (Phe)), 60.64
(CR (Thr)), 67.00 (CH2 (Cbz)), 70.96 (CH2O (Bn)), 73.54 (Câ (Thr)),
127.05, 127.73, 127.83, 128.01, 128.36, 128.60, 129.12, 135.97, 137.25
(Ph), 155.78 (OCON), 171.40 (CON), 198.56 (CO); HRMS calcd for
C29H31BrN2O5 (MH+) 567.1495, 569.1474, found 567.1478, 569.1282;
MS m/z (relative intensity) 567, 569 (MH+, 5, 5), 523 (4), 525 (4),
487 (11), 445 (5), 379 (5), 282 (7), 254 (6), 210 (9), 162 (4), 164 (4),
108 (4), 91 (100).
dissolved in 960 µL of buffer. The catalytic reaction was initiated by
addition of 20 µL of enzyme solution. The concentration of the
enzymes were set such that, under substrate saturation (Vmax) conditions,
the initial velocity of hydrolysis was about 10-3 OD/s.
Chymotrypsin (from bovine pancreas, EC 3.4.21.1) was assayed in
100 mM potassium phosphate buffer pH 7.0, by followng the hydrolysis
of BTEE (20 mM in DMSO) at 256 nm.32
Subtilisin (Sigma protease type XXVII) was assayed in 100 mM
potassium phosphate buffer, pH 7.0, by following the hydrolysis of
Succ-AAPF-pNA (5 mM in DMSO) at 404 nm.33
Elastase (from porcine pancreas, EC 3.4.21.36) was assayed in 100
mM Tris‚HCl buffer, pH 8.0, by following the hydrolysis of Succ-
AAA-pNA (4.4 mM in the same buffer) at 412 nm.34
Papain (EC 3.4.22.2) and cathepsin B (from bovine spleen, EC
3.4.22.1) were activated at 25 °C for 1 h in 80 mM potassium phosphate
buffer, pH 7.0, containing 0.5 mM DTT and 2 mM EDTA. They were
assayed in 100 mM potassium phosphate buffer, pH 7.0, by following
the hydrolysis of Cbz-Gly-ONp (1.25 mM in CH3CN) at 404 nm.11
Clostripain (EC 3.4.22.8) was activated at 25 °C for 2 h in 1 mM
Ca(OAc)2 aqueous solution, containing 0.5 mM DTT. It was assayed
in 25 mM potassium phosphate buffer, pH 7.6, containing 2.5 mM
DTT, by following the hydrolysis of BAEE (12 mM in water) at 254
nm.35
Inactivation Studies. The discontinuous method was applied.
Typically, the enzyme studied was incubated at 25 °C with the particular
inhibitor dissolved in CH3CN (volume of organic solvent not exceeding
10% of the total volume). Aliquots were removed periodically and
diluted into assay solution containing the substrate, and the residual
enzymatic activity was measured. A control preincubation solution,
containing all of the ingredients except for the inhibitor itself, was run
and assayed in parallel. Values of kobs (the apparent inactivation rate
constant) were calculated from semilog plots of percent residual
enzymatic activity vs time (as (ln 2)/t1/2 or by fitting the graphs to the
Cbz-Phe-O-benzyl-Thr-epoxide (95% yield, erythro:threo ) 5.7:
1
1) was crystallized from CH2Cl2/hexane: mp 110-112 °C; H NMR
(erythro) δ 1.075 (d, J ) 6.3 Hz, 3H, CH3), 2.680 (m, 2H, CH2O
(epoxide)), 2.715 (obscured, 1H, CHO (epoxide)), 2.948 (dd, J ) 13.5,
8.3 Hz, 1H, CH2â (Phe)), 3.138 (dd, J ) 13.5, 5.8 Hz, 1H, CH2â (Phe)),
3.376 (ddd, J ) 8.7, 7.4, 1.5 Hz, 1H, CHR (Thr)), 3.854 (dq, J ) 1.5,
6.3 Hz, 1H, CHâ (Thr)), 4.373 (d, J ) 11.5 Hz, 1H, CH2O (Bn)), 4.415
(ddd, J ) 5.8, 6.5, 8.3 Hz, 1H, CHR (Phe)), 4.568 (d, J ) 11.5 Hz,
1H, CH2O (Bn)), 5.062 (d, J ) 12.3 Hz, 1H, CH2 (Cbz)), 5.083 (d, J
) 12.3 Hz, 1H, CH2 (Cbz)), 5.452 (d, J ) 6.5 Hz, 1H, NH (Phe)),
6.015 (d, J ) 8.7 Hz, 1H, NH (Thr)), 7.15-7.40 (m, 15H, Ph); (threo)
δ 1.103 (d, J ) 6 Hz, 3H, CH3), 2.020 (br s, 1H, CH2O (epoxide)),
2.471 (t, J ) 4.1 Hz, 1H, CH2O (epoxide)), 3.058 (dd, J ) 13.4, 7.2
Hz, 1H, CH2â (Phe)), 3.088 (m, 1H, CHO (epoxide)), 3.14 (obscured,
1H, CH2â (Phe)), 3.688 (dq, J ) 3.1, 6.3 Hz, 1H, CHâ (Thr)), 4.210
(dt, J ) 9.0, 2.4 Hz, 1H, CHR (Thr)), 4.4 (obscured, 1H, CHR (Phe)),
4.480 (d, J ) 11.9 Hz, 1H, CH2O (Bn)), 4.600 (d, J ) 11.9 Hz, 1H,
CH2O (Bn)), 5.07 (obscured, 2H, CH2 (Cbz)), 5.323 (d, J ) 6.5 Hz,
1H, NH (Phe)), 6.01 (obscured, 1H, NH (Thr)), 7.15-7.40 (obscured,
15H, Ph); 13C NMR (erythro) δ 16.24 (CH3), 38.98 (Câ (Phe)), 47.37
(CH2O (epoxide)), 51.14 (CR (Thr)), 56.30 (CHO (epoxide)), 56.30
(CR (Phe)), 67.03 (CH2 (Cbz)), 71.12 (CH2O (Bn)), 73.32 (Câ (Thr)),
127.02, 127.62, 127.73, 127.97, 128.13, 128.34, 128.46, 128.66, 129.18,
136.14, 136.28, 137.92 (Ph), 155.70 (OCON), 170.85 (CON); (threo)
δ 15.88 (CH3), 38.56 (Câ (Phe)), 42.75 (CH2O (epoxide)), 50.44 (CR
(Thr)), 70.74 (CH2O (Bn)), 74.56 (Câ (Thr)); HRMS (EI) calcd for
C29H32N2O5 (M+) 488.2311, found 488.2290; MS (EI) m/z (relative
intensity) 488 (M+, 3), 382 (4), 323 (8), 243 (6), 210 (5), 169 (5), 142
(5), 119 (6), 91 (100).
t
exponential equation % activityt ) 100e-k ). A replot of 1/kobs vs
obs
1/[I] yielded the inactivation kinetic parameters ki and Ki.23
Acknowledgment. We thank Dr. S. Albeck for critical
reading of the paper. This research was supported by The Israel
Science Foundation administered by The Israel Academy of
Sciences and Humanities.
Supporting Information Available: 1H and 13C NMR
spectra of all new compounds (15 pages). This material is
contained in many libraries on microfiche, immediately follows
this article in the microfilm version of the journal, can be ordered
from the ACS, and can be downloaded from the Internet; see
any current masthead page for ordering information and Internet
access instructions.
JA954261Y
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Enzymatic Assays. All enzymatic assays were carried out at 25
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µL substrate solution (in the indicated concentration and solvent) was
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