Azapeptides as inhibitors and active site titrants for cysteine proteinases

Article abstract of DOI:10.1021/jm970802d

Ester and amide derivatives of α-azaglycine (carbazic acid, H₂NHCOOH), α-azaalanine, and α-azaphenylalanine (i.e., AC-L-Phe-NHN(R)CO-X, where X = H, CH₃, or CH₂Ph, respectively) were synthesized and evaluated as inhibitors

Full text of DOI:10.1021/jm970802d

1344
J . Med. Chem. 1998, 41, 1344-1351
Aza p ep tid es a s In h ibitor s a n d Active Site Titr a n ts for Cystein e P r otein a ses
Ruye Xing and Robert P. Hanzlik*
Department of Medicinal Chemistry, University of Kansas, Lawrence, Kansas 66045-2506
Received November 25, 1997
Ester and amide derivatives of R-azaglycine (carbazic acid, H2NNHCOOH), R-azaalanine, and
R-azaphenylalanine (i.e., Ac-L-Phe-NHN(R)CO-X, where X ) H, CH3, or CH2Ph, respectively)
were synthesized and evaluated as inhibitors of the cysteine proteinases papain and cathepsin
B. The ester derivatives inactivated papain and cathepsin B at rates which increased
dramatically with leaving group hydrophobicity and electronegativity. For example, with 8
(R ) H, X ) OPh) the apparent second-order rate constant for papain inactivation was 67 600
M-1 s-1. Amide and P1-thioamide derivatives do not inactivate papain, nor are they substrates;
instead they are weak competitive inhibitors (0.2 mM < Ki < 4 mM). Inactivation of papain
involves carbamoylation of the enzyme, as demonstrated by electrospray mass spectrometry.
Active site titration indicated a 1:1 stoichiometry for the inactivation of papain with 8, and
both inactivated papain and cathepsin B are highly resistant to reactivation by dialysis (t1/2
>
24 h at 4 °C). Azaalanine derivatives Ac-L-Phe-NHN(CH3)CO-X inactivate papain ca. 400-
900-fold more slowly than their azaglycine analogues, consistent with the planar configuration
at NR of the P1 residue and the very substantial stereoselectivity of papain for L- vs D- residues
at the P1 position of its substrates. Azaglycine derivative 9 (R ) H, X ) OC6H4NO2-p)
inactivates papain extremely rapidly (>70 000 M-1 s-1), but it also decomposes rapidly in buffer
with release of nitrophenol (kobs ) 0.13 min-1); under the same conditions 8 shows <7%
hydrolysis over 24 h. This nitrophenol release probably involves cyclization to an oxadiazolone
since 17 (R ) CH3, X ) OC6H4NO2-p), which cannot form an isocyanate, releases nitrophenol
almost as rapidly (kobs ) 0.028 min-1). Cathepsin C, another cysteine proteinase with a rather
different substrate specificity (i.e., aminopeptidase), was not inactivated by 8, indicating that
the inactivation of papain and cathepsin B by azapeptide esters is a specific process. Their
ease of synthesis coupled with good solution stability suggests that azapeptide esters may be
useful as active site titrants of cysteine proteinases and probes of their biological function in
vivo.
In tr od u ction
kinetic specificity for small substrates based on the Ac-
L-Phe-Gly-X motif, where X is an alcohol, amine, or
peptide leaving group.8,9 Cathepsin B is a mammalian
lysosomal proteinase which is secreted by some tumors
and has been implicated in facilitating tumor metasta-
sis. It has a similar protein folding pattern to that of
papain and a somewhat similar active site structure and
mechanism. However, because of a small insertion
(relative to papain) in its primary sequence that affects
the “prime side” of its substrate binding cleft, it has
significant dipeptidyl carboxypeptidase activity in ad-
dition to its endoproteinase activity.10,11 Like papain,
cathepsin B readily cleaves Ac-L-Phe-Gly-X type sub-
strates, and for both enzymes this kinetic specificity
carries over to substrate-like inhibitors as well.10,12,13
Although papain and cathepsin B are not closely related
to serine proteinases in terms of protein structure, they
are similar in utilizing an acyl enzyme mechanism of
catalysis and in having an active site histidine posi-
tioned to help deprotonate the active site nucleo-
phile.14,15
The number of reports on the involvement of cysteine
proteinases in a wide range of biological processes has
grown enormously in the past decade. Cysteine pro-
teinases are found in plants, bacteria, protozoa, and
fungi, as well as in mammals where both endogenous
and virus-induced forms are known. They play impor-
tant roles in a multitude of physiological as well as
pathological processes including lysosomal function,
bone resorption, hormone biosynthesis and inactivation,
certain viral and protozoal infections, and even apop-
tosis or “programmed cell death” (for reviews, see refs
1 and 2). With increased awareness of the widespread
and often pivotal involvement of cysteine proteinases
in disease processes has come increased interest in the
development of inhibitors for these enzymes. As an
extension of our own efforts in this direction,3-5 we wish
to report here on the inhibition of two related cysteine
proteinases, papain and cathepsin B, by a series of
azapeptide analogues of peptide substrates for these
enzymes.
Many inhibitors of cysteine and serine protein-
ases1,16-19 contain a reactive aldehyde or ketone group
that undergoes reversible covalent adduct formation
with the active site nucleophile group. Because such
adducts resemble transition states or reaction interme-
diates in substrate hydrolysis, they are very tightly
Papain is an endoproteinase from plants that cleaves
at peptide bonds having a large hydrophobic side chain
6,7
in their P2
position. It also displays considerable
* Address correspondence to this author. Tel.: 785-864-3750. Fax:
785-864-5326. E-mail: rhanzlik@rx.pharm.ukans.edu.
S0022-2623(97)00802-9 CCC: $15.00 © 1998 American Chemical Society
Published on Web 03/20/1998

Azapeptides as Inhibitors for Cysteine Proteinases
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 8 1345
Sch em e 1a
Ta ble 1. Apparent Second-Order Rate Constants for
Inactivation of Papain and Cathepsin B by Azapeptide Esters
and Amides
k2/Ki (M-1 s-1
)
compd
structure
papain
19
cathepsin B
1
2
AcPheNHNHCOOMe
AcPheNHNHCOOEt
47
3
AcPheNHNHCOOiBu
55
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
AcPheNHNHCOOCH2CH2Cl
AcPheNHNHCOOCH2CH2Br
AcPheNHNHCOOCH2CCl3
AcPheNHNHCOOCH2Ph
AcPheNHNHCOOPh
AcPheNHNHCOOC6H4NO2-p
AcPheNHNHCONH2
AcPheNHNHCONHC6H4NO2-p
AcPheNHNHCSNH2
AcPheNHN(CH3)COOMe
AcPheNHN(CH3)COOEt
AcPheNHN(CH3)COOCH2CCl3
AcPheNHN(CH3)COOPh
AcPheNHN(CH3)COOC6H4NO2-p
AcPheNHN(CH2Ph)COOPh
1150
1250
11800
30500
67600
>70000a
4
54
90
730
310
7730
0
0
1
0.03
0.05
0.05
12
110
a
0.3b
a
(a) H2N-NH2; (b) H2N-NHCH3; (c) ROCOCl; (d) Me3SiNCO;
0.34b
18c
(e) p-O2NC6H4NCO; (f) Me3SiNCS.
incubating it with valine methyl ester. As their report
appeared we were also involved in examining the
interaction of a series of azapeptides with papain and
cathepsin B. In this manuscript we present results with
azapeptides 1-18 which both confirm and extend the
work of Magrath and Abeles.
<0.2b
a
b
c
See text. kobs/[I], with [I] ) 0.95 mM. kobs/[I], with [I] ) 0.20
mM.
bound; consequently they are also potent competitive
inhibitors. The greater nucleophilicity of cysteine (thi-
olate) vs serine probably accounts for the selective
inhibition or inactivation of cysteine proteinases by
peptidyl nitriles, Michael acceptors, epoxides, diaz-
omethanes, acyloxymethyl ketones, and O-acylhydrox-
amates. Peptidyl halomethanes irreversibly inactivate
both cysteine and serine proteinases but by different
processes, i.e., alkylation of the active site thiolate20 vs
the active site histidine,21,22 respectively.
Ch em istr y
Azapeptides 1 and 2 were prepared by mixed anhy-
dride coupling of Ac-L-Phe with commercially available
methyl and ethyl carbazates, respectively. For the
synthesis of compounds 3-9 (Scheme 1), Ac-L-Phe-OMe
was condensed with hydrazine and the resulting acetyl-
L-phenylalaninehydrazide (19) was acylated with the
requisite chloroformate esters. Compounds 10-12 were
prepared by condensation of 19 with trimethylsilyl
isocyanate, p-nitrophenyl isocyanate, or trimethylsilyl
isothiocyanate, respectively; the silyl groups were evi-
dently lost during workup or chromatography. For
preparation of 13-17, the requisite hydrazide (20) was
prepared by condensation of Ac-L-Phe-OMe with meth-
ylhydrazine, followed by chromatographic separation of
the two regioisomeric hydrazide products, which are
In h ibitor Design
Replacing the R carbon of the P1 amino acid of a
peptide substrate with
a nitrogen generates an
azapeptide23-26 in which the P1 amino acid is formally
a derivative of carbazic acid (H2NNHCOOH). This
modification decreases the electrophilicity of the P1
carbonyl group and changes the geometry of the R
position from tetrahedral to trigonal (i.e., planar, achiral)
so that the P1 residue is neither D nor L but “in between”.
For these reasons some azapeptides are more resistant
to enzymatic hydrolysis than their normal peptide
analogues. On the other hand, azapeptide derivatives
with suitably reactive leaving groups acylate serine
proteases forming carbamoyl enzymes which undergo
deacylation very slowly. As a result, enzyme accumu-
lates in a catalytically inactive from. This approach has
been widely studied in regard to serine proteinases24,27-31
and other serine hydrolases such as acetyl cholinest-
erase for which carbamoylating inhibitors are important
therapeutic agents.
1
easily distinguishable by H NMR.30 In this case the
major product (59% yield) was the desired isomer Ac-
L-Phe-NHNHCH3. Acylation of the latter with the
appropriate chloroformate esters afforded 13-17.
Resu lts a n d Discu ssion
The catalytic mechanism of serine and cysteine pro-
teinases involves three principle steps, namely, sub-
strate recognition and noncovalent binding, acylation
of the active site nucleophile with release of the leaving
group, and deacylation or acyl transfer to water (eq 1).
(1)
A few azapeptide-based inhibitors have been designed
for cysteine proteinases, but most have been thiol-
alkylating halomethyl ketone analogues rather than
carbamoylating agents.32-35 An exception to this is the
work of Magrath and Abeles36 who reported that aza-
peptide 1 (structure in Table 1) and several of its
analogues were inactivators of papain. The interme-
diacy of a stable acyl enzyme was inferred on the basis
of an increasing rate of inactivation from 1 to 3 to 8
and on the reactivation of the inhibited enzyme by
With serine proteinases, azapeptide substrates with
poor leaving groups fail to acylate the enzyme and hence
are at best competitive inhibitors. Azapeptides with
more reactive leaving groups acylate the active site
serine, but the resulting carbamoyl enzymes are less
reactive than the normal acyl enzymes. Thus, depend-

1346 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 8
Xing and Hanzlik
Ta ble 2. Specificity Constants for Substrates and Inactivators
of Papain and Cathepsin B
compd
papain
cathepsin B
k
cat/Km (M-1 s-1
)
Cbz-Gly-ONp
203 000
800 000
120 000
853 000
123 000
450 000
80 000
Cbz-L-Ala-ONp
Cbz-L-Phe-ONp
Cbz-L-Lys-ONp
776 000
k2/Ki (M-1 s-1
)
Ac-L-Phe-AGly-OPh
Ac-L-Phe-AAla-OPh
Ac-L-Phe-APhe-OPh
67 600
110
0.3
7 730
18
<<0.2
competitive inhibitors (Ki ) 0.21, 3.3, and 3.9 mM,
respectively).
Compounds 4-8 also inactivate cathepsin B, and
although there are fewer data (Table 1) and the rates
are lower than with papain, the same dependence of
inactivation rate on leaving group electronegativity and
hydrophobicity is clearly evident suggesting that this
pattern of behavior may be a general one. Azapeptide
inactivators are still subject to the substrate specificity
of the particular enzyme, however. For example, cathe-
psin C, another member of the papain clan,15 has
considerable sequence similarity to papain and cathe-
psin B but has a very different substrate specificity. It
is an aminopeptidase with broad specificity that also
hydrolyzes small dipeptide substrates such as glycyl-L-
phenylalanine p-nitroanilide.41,42 Lacking a free amino
terminus, neither 6 nor 8 show any tendency to inac-
tivate cathepsin C, suggesting that their inactivation
of papain and cathepsin B is a specific process.
F igu r e 1. Effect of Ac-L-Phe-AGly-OCH2CCl3 (6) on progress
curves for papain-catalyzed hydrolysis of Cbz-Gly-ONp. The
assay substrate (50 µM) and inhibitor (concentrations indicated
on figure) were combined in a cuvette, and the reaction was
initiated by addition of papain (0.0125 µM).
ing on the particular enzyme and azapeptide, the
deacylation rate may be anywhere from somewhat
reduced to effectively zero. In the latter case the
compounds behave like irreversible affinity-labeling
agents for the enzyme.
Azapeptide 1 has been reported36 to inactivate papain
with an apparent second-order rate constant (k2/Ki) of
13 M-1 s-1
. Under slightly different experimental
S1-P1 Interactions. Papain is known to have broad
tolerance for different amino acids at the P1 position of
its substrates, as long as they have the L-configuration,43
and cathepsin B has somewhat similar preferences.44
This is illustrated by the rate constants for hydrolysis
of the Cbz-amino acid derivatives in Table 2. Crystal-
lographic studies on papain20,45 and cathepsin B10,11
indicate that the side chains of L-amino acids in the P1
position either lie along the surface of the protein or
project out into solution and suggest that the side chains
of D-residues in the P1 position would give rise to
strongly unfavorable steric interactions with the enzyme
itself. P1-Azapeptides have planar geometry at their
R-position and hence their side chains would project “in
between” those of D- vs L-amino acids at this position.
It was therefore of interest to explore this aspect of
azapeptides as inactivators of papain. Magrath and
Abeles36 reported that Ac-L-Phe-AAla-OiBu does not
inactivate papain, but this ester, like compounds 13 and
14, has an unreactive leaving group. Thus these
compounds are poor inactivating agents and less than
ideal as probes for exploring the influence of configu-
ration at the R position.
Upon introducing more reactive leaving groups, as in
15 and 16, inactivation rates with the azaalanine
derivatives become measurable, although they are
greatly reduced by comparison to the azaglycine ana-
logues (Table 1). Comparing the ratios of inactivation
rate constants for P1-azaglycine vs P1-azaalanine de-
rivatives with different leaving groups (-OMe, 380;
-OEt, 940; -OCH2CCl3, 980; -OPh, 615) reveals only
little more than a 2-fold variation in this ratio across a
350-fold range of inactivation rate constants. On the
conditions with 1, we observed the same type of behavior
and a second-order rate constant of 19 M-1 s-1 (Table
1). As will be shown below, this inactivation is specific,
covalent, stoichiometric, and essentially irreversible on
the time scale of our experiments. The time course of
this type of enzyme inhibition is illustrated in Figure 1
using compound 6, which is conveniently faster-acting
than 1 but produces the same (inactive) carbamoyl
enzyme product. As can be seen, incubation of even low-
micromolar concentrations of 6 with papain leads to
complete inactivation of the enzyme in a matter of
minutes.
Specificity of Inactivation. Papain shows a marked
preference for hydrophobic as well as for reactive (i.e.,
acidic, electronegative) leaving groups.3,37-40 Across the
series of azapeptides 1-9 the interplay of these two
factors results in a gradual increase of over 3500-fold
in the rate of papain inactivation without changing the
peptide portion of the molecule (Table 1). The electro-
philicity of carbonyl carbons decreases in the order ester
> amide > urea. Azapeptide amide 10, having a urea-
like carbonyl, is thus a very poor inactivator and not a
substrate for papain. Even adding an electron-with-
drawing p-nitrophenyl substituent to the potential
leaving group nitrogen fails to make the carbonyl carbon
in 11 attractive to the cysteine nucleophile of papain.
Although papain hydrolyzes simple peptide amides
quite well, it does not hydrolyze peptide thioamides
unless they have an electron-withdrawing nitrogen
substituent such as p-nitrophenyl;3 thus 12 is virtually
inert to papain (Table 1). Nevertheless, compounds 10-
12 still interact noncovalently with papain reasonably
well, as shown by the fact that they are reasonable

Azapeptides as Inhibitors for Cysteine Proteinases
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 8 1347
inactivating azapeptide 22, does not inactivate trypsin.49
Sch em e 2
other hand, as shown in Table 2, for normal peptide
substrates with good leaving groups the P1 side chain
has little influence on hydrolysis rate, whereas for
azapeptides with good leaving groups there is a huge
decrease in inactivation rate constant (225 000-fold) as
the size of the side chain increases in the order H <
CH3 < CH2Ph. Once again, cathepsin B shows parallel
behavior to that of papain in both of these respects, and
the effect appears to be almost entirely steric in origin,
meaning that P1 substituents larger than hydrogen
greatly hinder productive binding to these enzymes so
that acylation rates are drastically reduced.
Oxadiazolones are relatively unreactive toward basic
hydrolysis25,50 and thus do not acylate the enzyme.
Replacing the acyl moiety on an azapeptide nitrophenyl
ester with an alkoxycarbonyl27 or dialkylcarbamoyl49
group prevents oxadiazolone formation and nitrophenol
release under mild conditions in buffer.
Azaalanine derivative 17 cannot undergo E1cb elimi-
nation to form an isocyanate, yet it still releases
nitrophenol quite readily in buffer solution (kobs ) 0.028
min-1). This suggests that the E1cb route is unlikely
to be a significant contributor to either the decompostion
of 9 in buffer or its inactivation of papain (unless the
enzyme both catalyzes isocyanate formation and traps
it very efficiently). Compound 17 also inactivates
papain, but much less rapidly than 9. We did not
measure the rate constant for this process, but incuba-
tion of 17 (128 µM) and papain (2.5 µM) for 30 min
followed by 10-fold dilution into assay mixture gave an
initial rate 55% of control. Thus the relative reactivities
of 9 and 17 are probably in accord with those of other
AGly/AAla ester pairs as discussed above. On the basis
of the above considerations, we conclude that 9 and 17
inactivate papain by the same mechanism as their other
less reactive analogues (i.e., direct acylation), that
formation of isocyanates in solution is not involved in
enzyme inactivation, and that release of nitrophenol is
due to intramolecular cyclization to a noninhibitory
oxadiazolone derivative.
Observations on Carbamoylated Papain. Papain in-
activated by 3 was reported36 to be resistant to reactiva-
tion by dialysis, although it could be reactivated by
incubation with a high concentration of valine methyl
ester (90 mM). This lipophilic substrate-like nucleophile
presumably binds to the S1′ region of the active site and
attacks the carbamoyl enzyme in a reversal of the k2
step of eq 1. We also observed that papain inactivated
by 6, which should give the same carbamoyl enzyme as
3, was extremely slow to regenerate under dialysis
conditions (t1/2 g 24 h at 4-5 °C). To demonstrate that
inactivation was indeed due to carbamoyl enzyme
formation, papain inactivated with excess 6 was dia-
lyzed briefly to remove noncovalently bound inhibitor
and lyophilized. Electrospray mass spectrometry showed
the resultant protein to have a molecular mass of
23 667.6 ( 3.6 Da, which is in excellent agreement with
the calculated mass of the anticipated S-carbamoyl
enzyme (23 665.3 Da).
Mechanistic Considerations. Compound 9 contains
the most reactive leaving group in the series, and it
inactivates papain too rapidly for us to determine k2/Ki
as we did for its congeners. In buffer, 9 also releases
p-nitrophenol quite rapidly (kobs ) 0.13 min-1). Because
release of p-nitrophenol from 9 can generate potentially
inhibitory byproducts (see below), we performed several
experiments to characterize the inactivation of papain
by 9. In the first experiment compound 9 (5 µM) was
added to papain (0.125 µM), and as rapidly as the
solution could be mixed an aliquot was removed and
diluted 10-fold into assay buffer containing Cbz-G-
ONp46 (50 µM); the observed activity was <2% that of
a control reaction in which 9 was omitted, indicating
that extremely rapid inactivation had occurred. Next,
9 (5 µM) and Cbz-G-ONp (50 µM) were added simulta-
neously to prediluted papain (0.0125 µM); the initial
rate observed was reduced compared to the control with
9 omitted, and it continued to decline steadily reaching
zero within ca. 9-10 min as shown for 6 in Figure 1. In
the third experiment 9 (5 µM) was allowed to stand in
assay buffer for 40 min (ca. 7.5 times its half-life) before
addition of Cbz-G-ONp (50 µM) and papain (0.0125 µM);
the initial rate was ca. 90% that of a control reaction in
which 9 was omitted altogether, and it declined only
slightly over the next 5 min, indicating that nothing
with significant inhibitory activity remained in solution.
In contrast, HPLC studies showed that 6 and 7 were
unchanged after standing in buffer for 24 h at room
temperature, while the more reactive 8 had hydrolyzed
only to the extent of 4-7%. Thus, while 9 is quite
unstable in buffer its decomposition products are not
papain inactivators; the other azapeptides are stable in
buffer for extended periods of time.
In addition to direct hydrolysis, other possible routes
for nitrophenol release from 9 include an E1cb mecha-
nism giving rise to a peptidyl isocyanate (Scheme 2,
path a) and an intramolecular cyclization giving rise to
an oxadiazolone (Scheme 2, path b). Alkyl and peptidyl
isocyanates inactivate serine proteases by carbamoy-
lating the active site serine47 or, in special circum-
stances, the active site histidine.48 On the other hand,
oxadiazolone 21, formed by cyclization of the trypsin-
Stoichiometry of Inactivation and Active Site Titration
of Papain. A common problem in dealing with cysteine
proteinases is that of determining the operational
normality of a given enzyme preparation or solution.
One accepted approach to this is active site titration
with [L-trans-(epoxysuccinyl)leucinamido](4-guanidino)-
butane, also known as E-64.51 This agent alkylates the
active site cysteine of papain and many other cysteine

1348 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 8
Xing and Hanzlik
and the filtrate evaporated to give a white paste which was
partitioned into chloroform (20 mL), washed with NaHCO3
solution and water, and then dried. Recrystallization from
ethyl acetate and ether afforded the desired product as a white
solid (0.51 g, 46%). Mp: 167-169 °C. 1H NMR (CDCl3): δ
1.95 (s, 3H), 3.05 (dd, 1H), 3.14 (dd, 1H), 3.72 (s, 3 H), 4.74 (q,
1H), 6.25 (d, 1H), 6.70 (s, 1H), 7.25 (m, 5H), 8.27 (s, 1H). MS
(CI, NH3): m/z 297 (M‚NH4+, 19), 280 (MH+, 100), 190 (58),
120 (26). Anal. (C13H17N3O4) C, H, N.
N-(Acetyl-L-p h en yla la n yl)a za glycin e Eth yl Ester (2).
2 was synthesized from ethyl carbazate as described above for
1. The crude product was chromatographed over silica gel
eluted with methanol/chloroform (10:90, v/v); recrystallization
from ether/hexane gave a white solid (0.56 g, 64%). Mp: 157-
159 °C. 1H NMR (CDCl3): δ 0.92 (t, 3H), 1.60 (s, 3H), 2.57
(dd, 1H), 2.85 (dd, 1H), 3.81 (q, 2H), 4.96 (m, 1H), 6.34 (d, 1H),
6.72 (s, 1H), 6.90 (m, 5H), 8.50 (s, 1H). MS (CI, NH3): m/z
311 (M‚NH4+, 40), 294 (MH+, 100), 190 (53), 120 (40). Anal.
(C14H19N3O4) C, H, N.
N-Acetyl-L-p h en yla la n in e h yd r a zid e (19). N-Ac-L-Phe-
nylalanine (10.4 g, 80 mmol) was dissolved in 80 mL of
methanol with 0.5 mL of concentrated H2SO4 and refluxed at
80 °C for 6 h. The methanol was removed by rotary evapora-
tion and the residue dissolved in CHCl3 which was then
extracted with 5 mL of saturated NaHCO3 and 5 mL of H2O,
dried, and concentrated by rotary evaporation. Recrystalli-
zation from CHCl3/hexane yielded Ac-L-Phe-OCH3 (9.98 g,
90%). Mp: 87-88 °C. 1H NMR (CDCl3): δ 1.95 (s, 3H), 3.10
(dq, 2H), 3.70 (s, 3H), 4.86 (q, 1H), 6.00 (s, 1H), 7.05-7.30 (m,
5H). The latter (2.21 g, 10 mmol) and hydrazine hydrate (5.01
g, 100 mmol) were dissolved in 20 mL of MeOH and kept at
room temperature for 2 days. After removal of solvent the
residue was washed successively with EtOH, ether, and
hexane which yielded a white solid (2.02 g, 92%). Mp: 178-
179 °C. 1H NMR (CDCl3): δ 1.92 (s, 3H), 2.97 (m, 2H), 3.74
(s, 2H), 4.53 (q, 1H), 6.04 (d, 1H), 7.04 (s, 1H), 7.20 (m, 6H).
MS (CI, NH3): m/z 222 (MH+, 100), 207 (45), 191 (27), 163
(24), 120 (28). Anal. (C11H15N3O2) C, H, N.
N1-(Acetyl-L-p h en yla la n in yl)-N2-m eth ylh yd r a zin e (20).
Ac-L-PheOMe (1.58 g, 7.0 mmol) was dissolved in methylhy-
drazine (1.38 g, 30.0 mmol) and kept at room temperature for
24 h. After removal of the excess methylhydrazine by rotary
evaporation, the residue was chromatographed on silica gel
eluted with 10% MeOH in CHCl3. The major product (0.98 g,
59% yield, compound 19) was a white solid. Mp: 201-204
°C. Rf 0.22 (silica gel, MeOH/CHCl3, 10:90, v/v). 1H NMR
(CDCl3): δ 1.96 (s, 3H), 2.46 (s, 3H, -NHNHCH3), 2.98 (q,
1H), 3.05 (q, 1H), 4.50 (q, 1H), 6.10 (d, 1H), 7.05 (s, 1H), 7.20
(m, 6H). The minor product (0.26 g, 12% yield, compound 20)
had Rf 0.30 and showed an N-methyl peak (-N(CH3)NH2) at
3.05 ppm in the 1H NMR.
Gen er a l P r oced u r e for Syn th esis of Aza glycin e P ep -
tid es 3-9. The general procedure was to dissolve Ac-L-
PheNHNH2 and 1 equiv of N-methylmorpholine in CHCl3 at
-10 °C followed by slow dropwise addition of 1 equiv of a
chloroformate ester dissolved in 5 mL of CHCl3. The reaction
mixture was stirred at -10 °C for 30 min and at room
temperature for an additional 6 h. After addition of more
CHCl3, the organic phase was extracted with saturated
NaHCO3 and then H2O and dried. Evaporation of the CHCl3
left a white paste as crude product, from which pure products
were isolated by chromatography and/or recrystallization.
N-(Acetyl-L-p h en yla la n in yl)a za glycin e Isobu tyl Ester
(3). Chromatography of the crude product on silica gel gave
pure 3 as a white solid. Yield: 84%. Mp: 159-160 °C (lit.
mp 157-159 °C).36 1H NMR (CDCl3): δ 0.90 (d, 6H), 1.90 (s,
3H), 3.00 (q, 1H), 3.15 (q, 1H), 3.86 (d, 2H), 4.78 (q, 1H), 6.46
(d, 1H), 6.86 (s, 1H), 7.22 (m, 5H), 8.63 (s, 1H). MS (CI, NH3):
m/z 339 (M‚NH4+, 2), 322 (MH+, 35), 248 (54), 190 (100), 162
(30), 120 (50). Anal. (C16H23N3O4) C, H, N.
F igu r e 2. Active site titration of papain with E-64 and
azapeptides 6 and 8. Papain (0.125 µM) was preincubated with
6, 8, or E-64 at room temperature for 60 min. Remaining
activity was determined by adding 50 µM Cbz-Gly-ONp as
substrate. See text for details.
proteinases extremely rapidly and irreversibly. To
establish the stoichiometry of the reactions of 6 and 8
with papain, we compared them to E-64 in an active
site titration experiment. Variable aliquots of com-
pound (0-125 pmol) were combined with fixed aliquots
of papain (125 pmol in 1.0 mL of buffer); substrate was
added after 60 min at room temperature, and active
enzyme was measured. The results, shown in Figure
2, indicate that 8, like E-64, inactivates papain with 1:1
stoichiometry. Compound 6 appears to be less efficient,
but this is probably a consequence of incomplete reaction
with papain during the 60-min reaction period rather
than competing hydrolysis of 6 in buffer. Since aza-
paptides such as 8 are easily synthesized and can be
tailored to the specificity requirements of individual
cysteine proteinases, they may offer advantages over
E-64 as active site titrants for this family of enzymes.
Exp er im en ta l Section
Mercuripapain, cathepsin B from bovine spleen, cathepsin
C from bovine spleen, and [L-trans-(epoxysuccinyl)leucinamido]-
(4-guanidino)butane (E-64) were purchased from Sigma Chemi-
cal Co. (St. Louis, MO). Solvents and other chemicals were
obtained from standard commercial sources and used without
further purification. All reactions were carried out at room
temperature unless otherwise stated. Organic extracts were
dried over anhydrous Na2SO4 or MgSO4 prior to concentration
by rotary evaporation. HPLC analyses were carried out using
a C-18 reverse-phase column (Alltech Econosil, 10 µm, 4.6 ×
250 mm) eluted with 40% CH3CN in H2O at a flow rate of 1
mL/min and monitored at 220 nm. Thin-layer chromatogra-
phy was performed on glass plates (2.5 × 10 cm) precoated
with silica gel GHLF (250 µm; Analtech) eluted with 10%
methanol in chloroform. NMR spectra were recorded on a GE
QE Plus instrument. Melting points were determined in open
capillary tubes using a Thomas-Hoover apparatus and are
uncorrected.
N-(Acetyl-L-p h en yla la n yl)a za glycin e Meth yl Ester (1).
N-Ac-L-phenylalanine (0.83 g, 4.0 mmol) was activated with
N-methylmorpholine (1.05 equiv) and isobutyl chloroformate
(1.05 equiv) in THF (15 mL) at -20 °C as described.4 After
stirring for 10 min at -20 °C, methyl carbazate (0.36 g, 4.0
mmol) in 3 mL of THF was added, and the reaction was stirred
at room temperature for 3 h. The reaction mixture was filtered
N-(Acetyl-L-p h en yla la n in yl)a za glycin e 2-Ch lor oeth yl
Ester (4). 4 was obtained by chromatography on silica gel
followed by recrystallization from CHCl3/Et2O. Yield: 64%.
Mp: 158-159 °C. 1H NMR (acetone-d6): δ 1.80 (s, 3H), 2.82

Azapeptides as Inhibitors for Cysteine Proteinases
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 8 1349
(q, 1H), 3.15 (q, 1H), 3.72 (t, 2H), 4.26 (t, 2H), 4.70 (m, 1H),
7.22 (m, 5H), 8.32 (s, 1H), 9.20 (s, 1H). MS (CI, NH3): m/z
328 (MH+, 1), 292 (5), 248 (3), 232 (2), 190 (39), 162 (41), 120
(100). Anal. (C14H18N3O4Cl) C, H, N.
386 (MH+, 0.4), 248 (4), 222 (100), 190 (77), 164 (59), 120 (97).
Anal. (C18H19N5O5) C, H, N.
N-(Acetyl-L-ph en ylalan in yl)th iosem icar bazide (12). Ac-
L-PheNHNH2 (0.44 g, 2.0 mmol) in 15 mL of EtOH/THF (1:1)
was combined with trimethylsilyl isothiocyanate (0.52 g, 4.0
mmol) as described for compound 10. After removal of solvent
by rotary evaporation, the residue was chromatographed
yielding a white solid which was recrystallized from methanol/
ether. Yield: 33%. Mp: 197-199 °C. 1H NMR (DMSO): δ
1.74 (s, 3H), 2.75 (q, 1H), 2.96 (q, 1H), 4.28 (m, 1H), 6.88 (s,
1H), 7.22 (m, 5H), 7.92 (s, 1H), 8.24 (d, 1H), 9.32 (s, 1H). MS
(CI, NH3): m/z 281 (MH+, 1), 280 (M+, 1), 204 (1), 190 (11),
162 (16), 120 (72), 43 (100). Anal. (C12H16N4O2S) C, H, N.
N-(Acetyl-L-p h en yla la n in yl)a za a la n in e Meth yl Ester
(13). 13 was obtained as a white solid following chromatog-
raphy followed by recrystallization from EtOH/hexane.
Yield: 69%. Mp: 141-142 °C. 1H NMR (CDCl3): δ 1.90 (s,
3H), 2.97 (s, 3H), 3.08 (m, 2H), 3.62 (s, 3H), 4.73 (q, 1H), 6.72
(s, 1H), 7.22 (m, 5H), 8.80 (s, 1H). MS (CI, NH3): m/z 294
(MH+, 3), 190 (6), 162 (1), 105 (2), 84 (21), 47 (100). Anal.
(C14H19N3O4) C, H, N.
N-(Acetyl-L-ph en ylalan in yl)azaalan in e Eth yl Ester (14).
14 was obtained as a white solid following chromatography
followed by recrystallization from EtOH/hexane. Yield: 65%.
Mp: 158-160 °C. 1H NMR (CDCl3): δ 1.21 (t, 3H), 1.95 (s,
3H), 3.03 (s, 3H), 3.05 (m, 2H), 4.10 (q, 2H), 4.68 (q, 1H), 6.33
(s, 1H), 7.25 (m, 6H), 8.23 (s, 1H). MS (CI, NH3): m/z 325
(M‚NH4+, 19), 308 (MH+, 68), 279 (100), 262 (82), 190 (32),
120 (23). Anal. (C15H12N3O4) C, H, N.
N-(Acetyl-L-p h en yla la n in yl)a za glycin e 2-Br om oeth yl
Ester (5). 5 was purified by chromatography and recrystal-
lized from CHCl3/Et2O. Yield: 94%. Mp: 151-153 °C. NMR
1H NMR (acetone-d6): δ 1.80 (s, 3H), 3.25 (q, 1H), 3.15 (q, 1H),
3.60 (s, 2H), 4.35 (m, 2H), 4.68 (m, 1H), 7.22 (m, 5H), 8.35 (s,
1H). MS (CI, NH3): m/z 372 (MH+, 2), 292 (9), 248 (2), 190
(61), 162 (51), 120 (100). Anal. (C14H18N3O4Br) C, H, N.
N-(Acetyl-L-p h en yla la n in yl)a za glyin e Tr ich lor oeth yl
Ester (6). 6 was isolated by chromatography on silica gel
using 10% MeOH/CHCl3. Recrystallization from CHCl3/Et2O
gave 6 as a white solid in 76% yield. Mp: 188-189 °C. 1H
NMR (CDCl3): δ 1.94 (s, 3H), 3.04 (q, 1H), 3.14 (q, 1H), 4.72
(s, 2H), 4.80 (m, 1H), 6.35 (d, 1H), 7.25 (m, 6H), 8.75 (s, 1H).
MS (CI, NH3): m/z 396 (MH+, 14), 265 (16), 248 (100), 190
(91), 188 (56), 162 (38), 131 (23), 120 (91). Anal. (C14 H16N3O3
Cl3) C, H, N.
N-(Acetyl-L-p h en yla la n in yl)a za glycin e Ben zyl Ester
(7). 7 was obtained in 54% yield as a white solid. Mp: 145-
146 °C. 1H NMR (CDCl3): δ 1.90 (s, 3H), 3.05 (q, 1H), 3.15 (q,
1H), 4.70 (d, 1H), 5.13 (s, 2H), 6.12 (s, 1H), 6.75 (m, 1H), 6.7-
7.4 (m, 10H), 8.24 (s, 1H). MS (CI, NH3): m/z 356 (MH+, 18),
190 (68), 162 (18), 120 (43). Anal. (C19H21N3O4) H, N; C:
calcd, 64.21; found, 63.30.
N-(Acetyl-L-p h en yla la n in yl)a za glycin e P h en yl Ester
(8). 8 was isolated by chromatography using 10% MeOH/
CHCl3. Recrystallization from CHCl3/Et2O gave a white solid
in 57% yield. Mp: 138-141 °C (lit. mp 137-140 °C).36 1H
NMR (CDCl3): δ 1.95 (s, 3H), 3.04 (q, 1H), 3.16 (q, 1H), 4.76
(q, 1H), 6.15 (d, 1H), 7.10 (s, 1H), 7.25 (m, 10H), 8.35 (s, 1H).
MS (CI): m/z 342 (MH+, 1), 265 (2), 248 (37), 188 (42), 114
(43), 94 (100), 66 (32), 43 (44).
N-(Acetyl-L-p h en yla la n in yl)a za a la n in e Tr ich lor oeth yl
Ester (15). 15 was obtained as a white solid following
chromatography and recrystallization from ethyl acetate and
ether. Yield: 69%. Mp: 132-134 °C. 1H NMR (CDCl3): δ
1.90 (s, 3H), 3.04-3.08 (m, 5H), 4.68 (m, 3H), 6.08-6.22 (d,
1H), 7.21 (m, 5 H), 8.55 (s, 1H). MS (CI, NH3): m/z 410 (MH+,
9), 279 (28), 262 (100), 202 (27), 190 (22), 128 (45), 120 (18).
Anal. (C15H18N3O4Cl3) C, H, N.
N-(Acetyl-L-ph en ylalan yl)azaglycin e p-Nitr oph en yl Es-
ter (9). After acylation of hydrazide 19 with p-nitrophenyl
chloroformate as described above, the reaction mixture was
diluted with ethyl acetate, extracted with 1 N HCl, saturated
NaHCO3, and H2O, and dried. Evaporation of the solvent gave
a solid residue, but upon attempted chromatography on silica
gel the column turned deep-yellow suggesting extensive hy-
drolysis. Therefore the product was isolated by crystallization
from ether/hexane in 26% yield. This compound is highly
unstable in water and was used without further purification.
Mp: 144-145 °C. 1H NMR (DMSO): δ 1.72 (s, 3H), 2.72 (q,
1H), 2.96 (q, 1H), 4.54 (m, 1H), 7.22 (m, 5H), 7.40 (d, 2H), 8.20
(d, 1H), 8.25 (d, 2H), 10.02 (s, 1H), 10.22 (s, 1H). MS (CI,
NH3): m/z 265 (23), 248 (100), 188 (40), 157 (59), 139 (31),
109 (22), 91 (25), 65 (95). Anal. (C18H18N4O6) H, N; C: calcd,
55.96; found, 54.60.
N-(Acetyl-L-p h en yla la n in yl)a za a la n in e P h en yl Ester
(16). 16 was obtained as a white solid following chromatog-
raphy and recrystallization from methanol and ether. Yield:
45%. Mp: 158-160 °C. 1H NMR (DMSO): δ 1.72 (s, 3H),
2.75 (m, 1H), 2.95 (s, 3H), 3.10 (s, 1H), 4.50 (m, 1H), 7.0-7.4
(m, 10H), 8.30 (d, 1H). MS (CI, NH3): m/z 262 (4), 202 (17),
170 (10), 128 (45), 94 (100). Anal. (C19H21N3O4) C, H, N.
N-(Acetyl-L-p h en yla la n in yl)a za a la in e p-Nitr op h en yl
Ester (17). 17 was obtained as a white solid following
recrystallization from ether and hexane. Yield: 41%. Mp:
168.5-170 °C. 1H NMR (DMSO): δ 1.72 (s, 3H), 2.75 (q, 1H),
2.95 (q, 1H), 2.96 (s, 3H), 4.50 (m, 1H), 7.22 (m, 5H), 7.26 (d,
2H), 7.52 (d, 1H), 8.24 (d, 2H), 8.30 (s, 1H). MS (CI, NH3):
m/z 262 (37), 220 (5), 202 (69), 170 (28), 128 (100), 109 (15),
91 (44), 65 (60), 43 (72). Anal. (C19H20N4O6) C, H, N.
N-(Acetyl-L-p h en yla la n in yl)a za p h en yla la n in e P h en yl
Ester (18). A mixture of Ac-L-PheHNH2 (221 mg, 1.0 mmol)
and benzaldehyde (106 mg, 1.25 mmol) in 30 mL of MeOH
was refluxed for 8 h under N2. Concentration and recrystal-
lization from MeOH/ether give a white solid (255 mg, 83%
yield). 1H NMR (CDCl3): δ 2.0 (s, 3H), 3.0-3.3 (m, 4H), 4.7
(d, 1H), 7.1-7.4 (m, 10H). MS (CI, NH3): m/z 310 (MH+, 100),
163 (14), 162 (22), 120 (92), 106 (38).
The above material (Ac-L-PheNHNdCHPh, 255 mg, 0.83
mmol) was dissolved in dry THF, LiAlH4 (76 mg, 2.0 mmol)
was added, and the reaction mixture was refluxed for 4 h. After
cooling to room temperature, water (1.0 mL) was added
cautiously. The white precipitate which formed was removed
by filtration and the filtrate evaporated to give a yellow oil.
Recrystallization from ether/hexane gave Ac-L-PheNHNH-CH2-
Ph as a soft-yellow solid in 31% yield. MS (CI, NH3): m/z 312
(MH+, 100).
The latter product (80 mg, 0.26 mmol) was acylated with
phenyl chloroformate as described for compound 8. After silica
gel chromatography and recrystallization from ether and
hexane, compound 18 was obtained as a white solid in 37%
yield. Mp: 141-143 °C. 1H NMR (CDCl3): δ 1.90 (s, 3H),
N-(Acetyl-L-p h en yla la n yl)a za glycin e Am id e (10). Ac-
L-PheNHNH2 (0.44 g, 2.0 mmol) in 15 mL of THF/EtOH (1:1)
was combined with trimethylsilyl isocyanate (0.54 g, 4.0
mmol), and the mixture was heated to reflux for 20 h, after
which water was added and the mixture was stirred for 1 h.
After removal of solvent by rotary evaporation, the residue was
chromatographed yielding a white solid which was recrystal-
lized from methanol/ether. Yield: 56%. Mp: 123-126 °C. 1H
NMR (DMSO): δ 1.72 (s, 3H), 2.72 (q, 1H), 2.95 (q, 1H), 4.38
(m, 1H), 5.78 (s, 2H), 7.22 (m, 5H), 7.74 (s, 1H), 8.16 (d, 1H),
9.74 (s, 1H). MS (CI, NH3): m/z 265 (MH+, 13), 248 (7), 222
(54), 190 (83), 162 (100), 120 (84). Anal. (C12H16N4O3‚0.5H2O)
C, H, N.
N-(Acetyl-L-p h en yla la n in yl)a za glycin e p-Nitr oa n ilid e
(11). Ac-L-PheNHNH2 (0.22 g, 1.0 mmol) in 10 mL of THF
was combined with p-nitrophenyl isocyanate (0.16 g, 1.0 mmol)
in 5 mL of THF for 20 h. Removal of solvent and chromatog-
raphy on silica gel gave a faintly-yellow solid which was
recrystallized from THF and hexane. Yield: 48%. Mp: 193-
195 °C. 1H NMR (DMSO): δ 1.78 (s, 3H), 2.78 (m, 1H), 2.97
(m, 1H), 4.40 (s, 1H), 7.22 (m, 6H), 7.72 (d, 2H), 8.12 (d, 2H),
8.32 (s, 1H), 8.53 (s, 1H), 10.07 (s, 1H). MS (CI, NH3): m/z

1350 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 8
Xing and Hanzlik
(3) Foje, K. L.; Hanzlik, R. P. Peptidyl Thioamides as Substrates
and Inhibitors of Papain, and as Probes of the Kinetic Signifi-
cance of the Oxyanion Hole. Biochim. Biophys. Acta 1994, 1201,
447-453.
(4) Liu, S.; Hanzlik, R. P. Structure-Activity Relationships for
Inhibition of Papain by Peptide Michael Acceptors. J . Med.
Chem. 1992, 35, 1067-1075.
(5) Hanzlik, R. P.; Zygmunt, J .; Moon, J . B. Reversible Covalent
Binding of Peptide Nitriles to Papain. Biochim. Biophys. Acta
1990, 1035, 62-70.
(6) The nomenclature used to designate the individual residues in
a peptide (P1, P2, etc.) and individual enzyme subsites (S1, S2,
etc.) is that of Schechter and Burger, see ref 7.
3.00 (q, 1H), 3.10 (q, 1H), 4.65 (m, 3H), 6.00 (s, 1H), 7.3 (m,
15H), 8.40 (s, 1H). MS (CI, NH3): m/z 449 (M‚NH4+, 4), 432
(MH+, 16), 355 (40), 338 (100), 278 (24), 204 (40), 120 (13).
Anal. (C25H25N3O4) C, H, N.
En zym e Assa ys. Papain was purified and assayed as
described previously.4,5,52 Briefly, Hg-Sepharose 4B was
prepared according to Sluyterman and Wijdenes53,54 and found
to have a capacity of 2.24 µmol of 5,5′-dithiobis(2-nitrobenzoic
acid) (DTNB)/mL of resin bed. Commercial mercuripapain
(200 mg of crystallized suspension in 11.6 mL of 70% EtOH)
was added to 40 mL of column buffer (50 mM NaOAc, 100 mM
KCl, pH 5.0) containing 10 mM Na2SO3 and gently stirred at
room temperature for 45 min to allow the protein to dissolve.
(The original procedure53 calls for adding DMSO (10%, v/v) to
prevent protein aggregation, but we find this is not necessary;
when organic cosolvents are needed, for example, to improve
solubility of test compounds, up to 20% (v/v) acetonitrile may
be used with very little adverse effect on enzyme activity.) The
mixture was centrifuged at 18000g for 10 min, and the
supernatant was diluted to 78 mL with standard buffer and
loaded onto a Hg-Sepharose-4B column (bed volume 40 mL)
at a flow rate of 20 mL/h. The column was washed with 150
mL of column buffer and then with 200 mL of column buffer
containing 0.5 mM HgCl2. Active fractions were pooled (ca.
54 mL) and dialyzed against distilled water (4 °C, 10-kDa
cutoff membrane, 3 × 4000 mL). Lyophilization afforded
mercuripapain as a white solid power which could be stored
at -20 °C for long periods of time.
Solutions of active papain were prepared by dissolving 3 mg
of Hg-papain into 50 mL of standard buffer (50 mM K2HPO4,
1 mM EDTA, pH 6.3) and activating it by addition of
L-cysteine‚HCl (50 µL of 0.1 M solution/mL of papain solution)
at room temperature for 15 min. Papain assays contained 50
µM Cbz-Gly-ONp as substrate and 20% CH3CN in buffer. The
rate of substrate hydrolysis was followed at 340 nm with ∆ꢀ
of 6000 M-1 cm-1 and corrected for spontaneous chemical
hydrolysis of substrate.
Bovine spleen cathepsin B was assayed in 25 mM sodium
acetate buffer, pH 5.3, containing 1 mM EDTA as described
by Bajkowski et al.55 Stock cathepsin B was activated by 10
mM dithiothreitol and assayed with 80 µM Cbz-Lys-ONp as
substrate in buffer containing 20% CH3CN (v/v). The rate of
substrate hydrolysis was monitored at 326 nm with ∆ꢀ of 9060
M-1 cm-1. The concentration of cathepsin B was determined
by titration with protease inhibitor E-64 according to Barrett
et al.56
(7) Schechter, I.; Berger, A. On the Size of the Active Site in
Proteases. Papain. Biochem. Biophys. Res. Commun. 1967, 27,
157-162.
(8) Brocklehurst, K. Acyl Group Transfer - Cysteine Proteinases.
In Enzyme Mechanisms; Page, M. I., Williams, A., Eds.; The
Royal Society of Chemsitry: London, 1987; pp 140-158.
(9) Storer, A. C.; Menard, R. Recent Insights into Cysteine Protease
Specificity: Lesons for Drug Design. In Protease: Evolution,
Function and Inhibitor Design; McKerrow, J . H., J ames, M. N.
G., Eds.; ESCOM Science Publishers: Leiden, 1996; pp 33-46.
(10) Musil, D.; Zucic, D.; Engh, R. A.; Mayr, I.; Huber, R.; Papovic,
T.; Turk, V.; Towatari, T.; Katunuma, N.; Bode, W. The Refined
2.15 A X-ray Crystal Structure of Human Liver Cathepsin B:
the Structural Basis for its Specificity. EMBO J . 1991, 10, 2321-
2330.
(11) Turk, D.; Podobnik, M.; Popovic, T.; Katunuma, N.; Bode, W.;
Huber, R.; Turk, V. Crystal Structure of Cathepsin B Inhibited
with CA030 at 2.0-Angstrom Resolution: A Basis for the Design
of Specific Epoxysuccinyl Inhibitors. Biochemistry 1995, 34,
4791-4797.
(12) Elliott, E.; Sloane, B. F. The Cysteine Protease Cathepsin B in
Cancer. In Cysteine Proteases: Evolution, Function and Inhibitor
Design; McKerrow, J . H., J ames, M. N. G., Eds.; ESCOM Science
Publishers: Leiden, 1996; 12-32.
(13) Hasnain, S.; Huber, C. P.; Rowan, A. D.; Mort, J . S. Investigation
of Structure Function Relationships in Cathepsin B. Biol. Chem.
Hoppe-Seyler 1992, 373, 413-418.
(14) Fink, A. L. Acyl Group Transfer - The Serine Proteinases. In
Enzyme Mechanisms; Page, M. I., Williams, A., Eds.; The Royal
Society of Chemsitry: London, 1987; pp 159-177.
(15) Barrett, A. J .; Rawlings, N. D. Families and Clans of Cysteine
Peptidases. In Cysteine Proteases: Evolution, Function and
Inhibitor Design; McKerrow, J . H., J ames, M. N. G., Eds.;
ESCOM Science Publishers: Leiden, 1996; pp 1-11.
(16) Shaw, E. Cysteinyl Proteinases and their Selective Inactivation.
Adv. Enzymol. 1990, 63, 271-347.
(17) Rasnick, D. Small Synthetic Inhibitors of Cysteine Proteases.
In Cysteine Proteases: Evolution, Function and Inhibitor Design;
McKerrow, J . H., J ames, M. N. G., Eds.; ESCOM Science
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Cathepsin C from bovine spleen (Sigma type X) was assayed
at 37 °C in 50 mM sodium citrate, pH 5.0, containing 0.5 mM
EDTA and 10 mM NaCl. It was activated by 5 mM dithio-
threitol at room temperature for 15 min. Enzyme activity was
monitored at 410 nm (∆ꢀ ) 9300 M-1 cm-1) at 37 °C with 4
mM Gly-Phe-pNA as substrate.42
Inhibitors were evaluated using the method of Kitz and
Wilson57 as described previously.4 Inactivation rate constants
(kobsd, s-1) were determined from the slopes of semilog plots of
enzyme activity vs time. At least five time points were used
for each plot, and r2 values were g0.94 in most cases.
Apparent second-order rate constants (k2/Ki, M-1 s-1) were
determined from replots of kobs vs [I] using at least five values
of [I] (except as indicated in Table 1); in these plots r2 values
were generally 0.90-0.98. The resulting uncertainty on the
rate constants (slope values) is probably in the order of 10%
or less for the more active compounds and 20% or more for
compounds showing only marginal activity.
(18) Baggio, R.; Shi, Y.-Q.; Wu, Y.-Q.; Abeles, R. H. From Poor
Substrates to Good Inhibitors: Design of Inhibitors for Serine
and Thiol Proteases. Biochemistry 1996, 35, 3351-3353.
(19) Mehdi, S. Synthetic and Naturally Occurring Protease Inhibitors
Containing an Electrophilic Carbonyl Group. Bioorg. Chem.
1993, 21, 249-259.
(20) Drenth, J .; Kalk, K. H.; Swen, H. M. Binding of Chloromethyl
Ketone Substrate Analogues to Crystalline Papain. Biochemistry
1976, 15, 3731-3738.
(21) Kreutter, K.; Steinmetz, A. C. U.; Liang, T.-C.; Porok, M.; Abeles,
R. H.; Ringe, D. Three-dimensional Structure of Chymotrypsin
Inactivated
with
(2S)-N-Acetyl-L-alanyl-L-phenylalanyl
R-chloroethane: Implications for the Mechanism of Inactivation
of Serine Proteases by Chloroketones. Biochemistry 1994, 33,
13792-13800.
(22) Navia, M. A.; McKeever, B. M.; Springer, J . P.; Lin, T.-Y.;
Williams, H. R.; Fluder, E. M.; Dorn, C. P.; Hoogsten, K.
Structure of Human Neutrophil Elastase in Comples with a
Peptide Chloromethyl Ketone Inhibitor at 1.84 Å Resolution.
Proc. Natl. Acad. Sci. U.S.A. 1989, 86, 7-11.
(23) Aza-amino acid residues in which the R-methine of an amino
acid residue is replaced by a nitrogen atom are designated by
placing an “A” before the standard three-letter abbreviation or
an “a” before the standard one letter abbreviation for the
particular amino acid, as described by Powers et al. (ref 24).
(24) Powers, J . C.; Bond, R.; Carroll, D. L.; Gupton, B. F.; Kam, C.-
M.; Nishino, N.; Sakamoto, M.; Tuhy, P. M. Reaction of Aza-
peptides with Human Leukocyte Elastase and Procine Pancre-
atic Elastase. J . Biol. Chem. 1984, 259, 4288-4294.
Ack n ow led gm en t. This work was supported by a
fellowship to R. Xing from the Marion Merrell Dow
Scientific Education Partnership Foundation.
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