Peptidyl cyclopropenones: Reversible inhibitors, irreversible inhibitors, or substrates of cysteine proteases?

Article abstract of DOI:10.1002/pro.2260

Peptidyl cyclopropenones were previously introduced as selective cysteine protease reversible inhibitors. In the present study we synthesized one such peptidyl cyclopropenone and investigated its interaction with papain, a prototype cysteine protease. A set of kinetics, biochemical, HPLC, MS, and ¹³C-NMR experiments revealed that the peptidyl cyclopropenone was an irreversible inhibitor of the enzyme, alkylating the catalytic cysteine. In parallel, this cyclopropenone also behaved as an alternative substrate of the enzyme, providing a product that was tentatively suggested to be either a spiroepoxy cyclopropanone or a gamma-lactone. Thus, a single family of compounds exhibits an unusual variety of activities, being reversible inhibitors, irreversible inhibitors and alternative substrates towards enzymes of the same family.

Full text of DOI:10.1002/pro.2260

Peptidyl cyclopropenones: Reversible
inhibitors, irreversible inhibitors, or
substrates of cysteine proteases?
Meital Cohen, Uriel Bretler and Amnon Albeck*
Department of Chemistry, The Julius Spokojny Bioorganic Chemistry Laboratory, Bar Ilan University, Ramat Gan, 52900, Israel
Received 11 December 2012; Accepted 25 March 2013
DOI: 10.1002/pro.2260
Published online 30 March 2013 proteinscience.org
Abstract: Peptidyl cyclopropenones were previously introduced as selective cysteine protease
reversible inhibitors. In the present study we synthesized one such peptidyl cyclopropenone and
investigated its interaction with papain, a prototype cysteine protease. A set of kinetics,
biochemical, HPLC, MS, and 13C-NMR experiments revealed that the peptidyl cyclopropenone was
an irreversible inhibitor of the enzyme, alkylating the catalytic cysteine. In parallel, this
cyclopropenone also behaved as an alternative substrate of the enzyme, providing a product that
was tentatively suggested to be either a spiroepoxy cyclopropanone or a gamma-lactone. Thus, a
single family of compounds exhibits an unusual variety of activities, being reversible inhibitors, irre-
versible inhibitors and alternative substrates towards enzymes of the same family.
Keywords: cyclopropenone; cysteine proteases; enzyme inhibition; irreversible inhibition; alternative
substrate
Cyclopropenones are intriguing chemical entities,
as they are both electrophilic and basic. They may
undergo nucleophilic attack at either of the olefinic
carbons or at the carbonyl carbon. On the other hand,
protonation of the carbonyl oxygen will yield a 2p aro-
matic hydroxycyclopropenyl cation [Fig. 1(b)].3–5
Introduction
Peptidyl cyclopropenones [Fig. 1(a)] were introduced
as selective cysteine protease inhibitors.1,2 They
exhibited amino acid sequence selectivity and stereo-
selectivity in the inhibition of a few enzymes of this
family. They did not inhibit serine- and aspartic pro-
teases. Detailed mechanistic analysis of the inhibi-
tion process of the cysteine protease m-calpain
revealed fully reversible competitive inhibition.1
These reactions may all be manifested in the
active site of cysteine proteases: the catalytic cyste-
ine can attack the cyclopropenone carbonyl to form a
thiohemiacetal or add across the C@C double bond.
Experimental6–8 and computational9–12 studies sug-
gest that protonation of a substrate in the active site
of cysteine proteases leads to the formation of a neu-
tral (protonated) tetrahedral intermediate. Thus, a
peptidyl cyclopropenone inhibitor may become proto-
nated in the enzyme active site, forming the aro-
matic hydroxycyclopropenyl cation. All of the above
reactions are reversible and therefore fit the
described inhibition mechanism.1 Alternatively, pep-
tidyl cyclopropenones may behave as classical com-
petitive inhibitors, occupying the enzyme active site
without undergoing any covalent modification.
Abbreviations: Boc, t-butoxycarbonyl; BSA, bovine serum
albumin; Cbz, benzyloxy carbonyl; COSY, correlated spec-
troscopy; DEPT, Distortionless Enhancement by Polarization
Transfer; DMSO, dimethyl sulfoxide; DTNB, 5,5’-dithiobis
(2-nitrobenzoic acid); DTT, 1,4-dithiothreitol; EDTA, ethylene-
diaminetetraacetic acid; HMBC, Heteronuclear Multiple Bond
Correlation; HMQC, Heteronuclear Multiple-Quantum Correla-
tion; Me, methyl; NOSEY, Nuclear Overhauser effect spec-
troscopy;
THF,
tetrahydrofuran;
TMEDA,
N,N,N’,N’-
tetramethylethylenediamine; TMS, tetramethylsilane.
Grant sponsor: The Marcus Center for Pharmaceutical and
Medicinal Chemistry at Bar Ilan University.
*Correspondence to: Amnon Albeck, Department of Chemistry,
Bar Ilan University, Ramat Gan 52900, Israel.
E-mail: amnon.albeck@biu.ac.il
In this article we describe our studies of the in-
hibition of the prototype cysteine protease papain by
C
V
788 PROTEIN SCIENCE 2013 VOL 22:788—799
Published by Wiley-Blackwell. 2013 The Protein Society

this solvent. Therefore, its spectrum could be taken
only in CDCl3 that was pre-treated with a base
(K2CO3) or in CD3CN. Indeed, removal of the acetal
protecting group from 6 was achieved by simply dis-
solving it in CHCl3. Condensation of the anion of 5
with Boc-valinal14 afforded compound 6 as a 3:1 dia-
stereomeric mixture (SS:SR, respectively). The rest
of the synthesis was carried out on the diastereomeric
mixture, and the two diastereomers of cyclopropenone
inhibitor 9 were separated by chromatography.
13C-labeled inhibitor
Inhibitor 9 was also prepared with selective 13C
labeling on its cyclopropenone carbonyl carbon,
starting from labeled phenyl acetone 1. We tried a
few approaches to prepare the latter, the best of
which was activation of 13C-labeled phenylacetic
acid as a Weinreb amide,15 followed by condensation
with MeLi (Scheme 2).
Enzyme inhibition
Time- and concentration-dependent inhibition was
observed upon incubation of the cysteine protease
papain with the S isomer of peptidyl cyclopropenone
9 (Fig. 2). The corresponding kinetic parameters are
Ki 5 46 lM and ki 5 0.021 min21. The R isomer
was about two fold less reactive (Ki 5 98 lM and ki
5 0.020 min21).
Figure 1. Peptidyl cyclopropenones. (a) General structure of
peptidyl cyclopropenones. (b) Possible electrophilic and basic
modes of reactivity of cyclopropenones.
In order to confirm the irreversibility of papain
inhibition by peptidyl cyclopropenone 9, the enzyme-
inhibitor complex (less than 30% residual enzymatic
activity) was dialyzed extensively (2 3 4 h) against
phosphate buffer solution containing DTT. No regen-
eration of enzymatic activity was observed. As a con-
trol, active papain that was similarly dialyzed
retained about 90% of its enzymatic activity.
Peptidyl cyclopropenone 9 (the S isomer) was
also tested as an inhibitor of cathepsin B, another
cysteine protease that was previously reported to be
inhibited reversibly by this family of inhibitors.1
Concentration-dependent and time-independent in-
a peptidyl cyclopropenone, in order to identify the
specific mode of reversible inhibition, and the many
surprises we encountered.
Results
Inhibitor synthesis
The synthesis of the peptidyl cyclopropenone inhibi-
tor 9 follows the synthesis developed by Ando et al.1
with some modifications (Scheme 1).
The reaction to form the cyclopropenone acetal
5 is very sensitive to the stoichiometric ratio of the
base and the dihalide substrate 4, and therefore
required a very clean substrate and a carefully
titrated base. A ratio < 2 afforded a mixture of the
product 5 and a chlorocyclopropanone acetal inter-
mediate, whereas a ratio > 2 led to the formation of
t-butoxycyclopropanone acetal, the product of addi-
tion of the base across the C5C double bond of the
required product. A recent publication describes the
elimination of t-butanol from this product, to regen-
erate the cyclopropenone acetal.13 The cycloprope-
none acetal was extremely acid labile: it was not
stable upon silica chromatography, and therefore it
was purified on a deactivated alumina column. Fur-
thermore, the acetal decomposed in CDCl3, used for
the NMR analysis, due to the usual slight acidity of
hibition indicated reversible inhibition, with IC50 5
85 lM under the specific experimental conditions.
The reversible nature of the inhibition of cathepsin
B was further validated by full recovery of enzy-
matic activity upon dialysis, as compared with active
enzyme control.
Alkylation site
The alkylation site on papain by the irreversible
peptidyl cyclopropenone inhibitor 9 was identified as
follows:
(a) Activated papain was separated from excess DTT
by gel filtration. Complete separation (3 frac-
tions) was achieved, as determined by measuring
the absorption (A280) of each fraction for enzyme
concentration and by interacting a sample of
Cohen et al.
PROTEIN SCIENCE VOL 22:788—799 789

Scheme 1. Synthesis of peptidyl cyclopropenone 9.
each fraction with DTNB (Ellman’s reagent)16
and measuring the absorption (A412) of the reac-
tion solution for free thiol concentration. The
active enzyme was then incubated with inhibitor
9. Samples from the incubation solution were
removed at time points along the inhibition pro-
cess, and measured for both residual enzymatic
activity and free thiol concentration. Very good
correlation between these two values of each
sample was demonstrated (Fig. 3).
correlated with the residual 30% enzymatic activ-
ity. The control samples of the free enzymes,
both the native and the denatured, exhibited
thiol concentration correlated with above 90% en-
zymatic activity.
The above two experiments confirm that pep-
tidyl cyclopropenone
active-site cysteine.
9 alkylates papain on its
(b) Activated papain was separated from excess DTT
by gel filtration, as above. The enzyme fraction
was split into two parts, one serving as a control
(active enzyme) and one incubated with inhibitor
9 till 70% inhibition was measured. Each of the
two samples, that of the inhibited enzyme and
the control active enzyme, was split again. One
part was treated with DTNB to determine the
concentration of free active-site thiol. The second
part was first denatured with 6N guanidinium
chloride and then treated with DTNB. The meas-
urements show that both samples of the inhib-
ited enzyme, the native and the denatured, had
practically identical concentration of thiols,
Fate of the 13C Labeling
Papain was inhibited with the specifically 13C la-
beled inhibitor 9, in an attempt to identify the struc-
ture of the inhibitor’s carbonyl carbon in the
inhibition complex. The process involved activation
of the enzyme, separation of the active enzyme from
various inactive forms by mercury affinity chroma-
tography,17 inhibition of the active enzyme by la-
beled inhibitor 9, and measuring of the 13C-NMR
spectrum of the inhibition complex [Fig. 4(c)]. The
same process was also carried out with an unlabeled
inhibitor 9 [Fig. 4(b)]. A difference spectrum of these
two spectra should present only species associated
with the labeled inhibitor and the products of its
Scheme 2. Synthesis of 13C-specifically labeled starting material phenyl acetone 1.
790 PROTEINSCIENCE.ORG
Peptidyl Cyclopropenones as Cysteine Protease Inhibitors

Figure 2. Time- and concentration-dependent inhibition of papain by peptidyl cyclopropenone 9. Papain was activated in KPi
buffer (100 mM, pH 7.0) containing EDTA (1 mM) and DTT (0.5 mM). The active enzyme solution (200 lL) was incubated with
various concentrations of inhibitor (100 lL in DMSO) in KPi buffer solution (700 lL). At time points, aliquots from the inactivation
solution (100 lL) were treated with substrate solution (20 lL of Cbz-Gly-ONp 5 mM in CH3CN) in KPi buffer (880 lL). The hydro-
lysis rate was assayed spectrophotometrically at 404 nm. The insert is a replot of 1/kobs vs 1/[I], from which the inhibition pa-
rameters Ki and ki were determined.
interaction with the enzyme. The difference spec-
trum [Fig. 4(d)] has two signals: a resonance at 159
ppm, which corresponds to the free inhibitor 9 [Fig.
the NMR experiment solution was separated from
the soluble enzyme by centrifugation. HPLC analy-
sis of the precipitate revealed the two peaks at 33
4(a)], and
a new resonance at 176 ppm. Both
resonances are relatively narrow, about 19 Hz, indi-
cating that they are not associated with the protein.
Formation of the 176 ppm signal was enzyme-de-
pendent; it was not observed in the spectrum of 13C-
labeled inhibitor 9 incubated in either buffer alone
(with EDTA and DTT, but without the enzyme),
buffer with BSA, or buffer with denatured papain.
The enzyme-inhibitor incubation solution from
the NMR experiment, which includes the excess la-
beled inhibitor and the new compound represented
by the 176 ppm signal, was chromatographed by
HPLC. The chromatogram [Fig. 5(a)] is character-
ized by a peak of the free inhibitor, eluting at 33.7
min, and another peak at 38.2 min. The formation of
the new peak at 38.2 min was time-dependent,
growing in on the expense of the free inhibitor peak
at 33.7 min [Fig. 5(b)]. As controls, active enzyme in
buffer, which contained EDTA and DTT, [Fig. 5(c)]
and a solution of the inhibitor in buffer (including
EDTA and DTT) and BSA [Fig. 5(d)] were also chro-
matographed. The 38.2 min peak is clearly the prod-
uct of some enzyme – inhibitor 9 interaction, as it
does not appear in either the solution of the buffer
and the activating agents or a solution of the inhibi-
tor with a nonrelevant protein.
Figure 3. Linear correlation between residual enzymatic
activity and free active-site thiol upon partial inhibition of the
enzyme papain by peptidyl cyclopropenone 9. Activated papain
(16 mg) was purified from small molecules (DTT and EDTA) by
Sephadex G-15 chromatography, and was assayed for both
activity and free thiol concentration (by titration with DTNB).
The enzyme was then incubated with inhibitor 9. Aliquots from
the inactivation solution were removed at various time points,
and assayed for their residual enzymatic activity and for the
remaining free thiol concentration. An active enzyme solution
was similarly treated and assayed as a control.
Small organic molecules tend to precipitate from
aqueous solutions. Indeed, a precipitate formed in
Cohen et al.
PROTEIN SCIENCE VOL 22:788—799 791

Figure 4. 13C-NMR spectra of specifically labeled cyclopropenene 9 and papain at 150 MHz. (a) Peptidyl cyclopropenone 9 in
DMSO-d6 and KPi buffer with DTT and EDTA (0.5 mM and 1mM respectively). (b) Papain inhibited by unlabeled peptidyl cyclo-
propenone 9. (c) Papain inhibited by 13C-labeled peptidyl cyclopropenone 9. (d) Difference spectrum (c-b).
and 38 min whereas the corresponding 13C-NMR
spectrum in DMSO-d6 was characterized by two
resonances, at 156 and 173 ppm. (The 3 ppm shift
from the original spectrum stems from the change in
solvents, from 10% DMSO in water to DMSO alone).
No enzyme signals were observed. This experiment
confirms that the new 176 ppm resonance in the
13C-NMR spectrum belongs to a small molecule, sepa-
rated from the enzyme. Furthermore, it correlates
this resonance with the HPLC peak at 38 min.
Finally, the 38 min peak from the HPLC was collected
and submitted to mass spectroscopy analysis. HRMS
of the compound was characterized by a mass of
479.254, which corresponds to a molecular formula of
C28H35N2O5 (protonated compound, MH1, calculated
mass of 479.251). Interestingly, this mass is identical
to the mass of peptidyl cyclopropenone 9. A second
peak in the mass spectrum corresponds to a fragment
with a mass of 435.265 (MH1-CO2, calculated mass of
435.261). The loss of 44 Da in the MS analysis is typi-
cal to carbamate-protected peptides and amino acids.
cysteine proteases), whereas our detailed study was
carried out on papain. Furthermore, the two studies
also differ in the exact identity of the papain inhibi-
tor: both studies were carried out on the same pep-
tide sequence and cyclopropenone substituent, but
the previous study employed a cyclohexyl methoxy-
carbonyl protecting group whereas inhibitor 9 in
this study is Cbz-protected. This difference also
affected the inhibitor concentrations used.
It is also interesting to note that, in contrast to
its irreversible covalent inhibition of papain, pep-
tidyl cyclopropenone 9 was a reversible inhibitor of
cathepsin B (IC50 5 85 lM), another cysteine prote-
ase, as was also observed in the previous study.1
Thus, the same compound exhibits two very differ-
ent inhibitory activities towards two enzymes
belonging to the same family of proteases.
Time-dependent loss of enzymatic activity is
indeed reminiscent of irreversible covalent inhibi-
tion, but it could also represent reversible slow tight
binding inhibition.18 The fact that enzymatic activity
was not recovered upon dialysis of the enzyme-inhib-
itor complex indicates that indeed the inhibition is
irreversible. It may still be argued that a very low
off-rate (koff) would leave most of the enzyme in its
complex with the inhibitor even under a few hours
of dialysis. The DTNB titration experiments resolve
the reversibility issue. It clearly shows that the in-
hibitor alkylates the catalytic thiol (the only free
thiol in papain), and that this bond is stable even
under denaturation conditions.
Discussion
The irreversible mode of inhibition of papain by pep-
tidyl cyclopropenone 9, observed in the present
study, is markedly different from the reversible com-
petitive inhibition pattern previously observed for
this type of inhibitors.1,2 It should be noted that the
previous detailed mechanistic study was carried out
on m-calpain (though the same inhibition pattern
was also observed for papain and a few other
792 PROTEINSCIENCE.ORG
Peptidyl Cyclopropenones as Cysteine Protease Inhibitors

Figure 5. HPLC analysis of the papain – inhibitor 9 inhibition process. HPLC was carried out on Nucleosil 12035 C18 column,
at a flow rate of 1 mL=min, applying linear gradients of 100% to 20% water-acetonitrile in 40 min. (a) The reaction mixture after
short incubation of papain with inhibitor 9. (b) The reaction mixture after 12 h incubation of papain with inhibitor 9. (c) Papain in
buffer (100 mM KPi) and EDTA (1 mM) and DTT (0.5 mM). (d) Ppetidyl cyclopeopenone 9 incubated in buffer (with EDTA and
DTT) and BSA for 12 h.
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PROTEIN SCIENCE VOL 22:788—799 793

Figure 6. HPLC analysis of the reaction product of inhibitor 9 with NaOH. HPLC was carried out on Nucleosil 12035 C18 col-
umn, at a flow rate of 1 mL=min, applying linear gradients of 100% to 20% water-acetonitrile in 40 min. (a) Purified product
from the interaction of papain with inhibitor 9. (b) The product mixture arising from treatment of 9 with one eq. of NaOH. (c) Co-
injection of the two products, from the enzymatic- and the base treatment reactions.
From the mechanistic point of view, the above
results rule out the simple reversible binding of the
cyclopropenone inhibitor 9 in the enzyme active-site
and the possibility of inhibitor protonation in the
active-site to form a 2p aromatic hydroxy cycloprope-
nium species. It probably also eliminates the option
of a nucleophilic attack of the catalytic thiol at the
inhibitors cyclopropenone carbonyl, to yield a thiohe-
miacetal. Such a covalent complex is expected to
undergo hydrolysis under the denaturation condi-
tions, which would be manifested in a titratable
thiol. A reasonable mechanism then is the addition
of the nucleophilic catalytic thiol across the cyclopro-
penone C@C double bond.
13C-NMR had previously been employed to
study covalent interactions between proteases and
various inhibitors.19–24 In this study we employed
this approach in order to identify the structure of
794 PROTEINSCIENCE.ORG
Peptidyl Cyclopropenones as Cysteine Protease Inhibitors

Scheme 3. Suggested mechanisms for the interaction of cyclopropenone 9 with papain. (a) Irreversible inhibition of papain by
peptidyl cyclopropenone 9. (b) Peptidyl cyclopropenone 9 as an alternative substrate of the enzyme. (c) An alternative product
resulting from the interaction of papain with cyclopropenone 9.
cyclopropenone inhibitor 9 in its complex with the
enzyme papain. Unfortunately, the 13C-NMR spec-
trum of the complex did not reveal any clear new
signal that could be assigned to the complex. This
may be attributed to the fact that the labeled carbon
does not carry any protons, and therefore it is
expected to be small (despite the 100% labeling).
Furthermore, it is expected to be a very wide peak
(in the order of 50 Hz) due to its association with a
protein bound carbon.
substrate of papain (Scheme 3). We suggest that the
catalytic cysteine can attack both carbons of the
C5C double bond of the cyclopropenone inhibitor. It
is not unusual for the catalytic cysteine of cysteine
proteases to attack the carbon adjacent to the
would-be scissile bond carbon. This was previously
observed with peptidyl chloromethyl ketones, pep-
tidyl diazomethyl ketones, peptidyl acyloxymethyl
ketones,25 and peptidyl epoxides.19 Thus, Michael
addition at the carbon more distant from the pep-
On the other hand, the 13C-NMR experiment
provided a surprise in the form of a new signal at
176 ppm. This signal belongs to a small molecule,
not attached to the enzyme. This small molecule is
only formed when both cyclopropenone 9 and active
papain are present, but not in the presence of other
proteins (BSA) or denatured papain. Thus, peptidyl
cyclopropenone 9 is not only an irreversible inhibitor
of the cysteine protease papain, but also a substrate
of the enzyme. HPLC and MS analyses provide some
insight into the structure of the product of this
interaction: this product elutes slower than cyclopro-
penone 9 from the reversed-phase HPLC column,
indicating that it is more hydrophobic. Its mass
spectrum (both HRMS molecular peak and fragmen-
tation pattern) is practically identical to that of
cyclopropenone 9. This new product could not be
fully characterized due to the small amount of its
production by an enzyme that is being irreversibly
inhibited at the same time.
tide provides a stable covalent enzyme-inhibitor
complex (Scheme 3a). On the other hand, addition
of the catalytic cysteine across the inhibitor’s C@C
double bond by attack of the thiol on the carbon
closer to the peptide and protonation of the other
C@C carbon would yield an a-hydroxy sulfide in-
termediate [Scheme 3(b)]. This intermediate could
then undergo intramolecular nucleophilic substitu-
tion reaction, in which the hydroxyl group displa-
ces the thiol to form a spiroepoxide product that
can leave the enzyme’s active site. This product is
more hydrophobic than the starting material, pep-
tidyl cyclopropenone 9, but they both have the
same molecular weight, as was observed in our
HPLC and MS experiments.
This product is formally the result of an intra-
molecular addition of the hydroxyl across the cyclo-
propenone double bond. Indeed, interaction of
cyclopropenone 9 with one equivalent of NaOH
gave
a mixture of products, one of which co-
On the basis of the above experimental data, we
can suggest possible mechanisms for the inhibition
of papain by peptidyl cyclopropenone 9 and for the
process in which cyclopropenone 9 is an alternative
migrated with the enzyme product on an HPLC
reversed-phase column in two different eluting
systems (water-acetonitrile and water-methanol)
(Fig. 6).
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PROTEIN SCIENCE VOL 22:788—799 795

Alternatively, the enzymatic product could be
the result of some rearrangement=ring expansion to
yield a g-lactone product [Scheme 3(c)].
was washed with 0.5N HCl (30 mL), saturated
NaHCO3 solution (50 mL) and brine (50 mL). Drying
over MgSO4, filtration and evaporation gave the clean
phenyl acetone 1 (0.71 g, 84% yield). 1H-NMR: d 7.3-7.2
(m, 5H); 3.69 (d, J56.6 Hz, 2H); 2.15 (d, J55.7 Hz, 3H).
13C-NMR: d 206.5, 134.4, 129.5, 128.9, 127.2, 51.2 (d,
J538 Hz), 29.4 (d, J5 41 Hz). MS: m=z 136 (MH1, 7),
123 (16), 91(40). HRMS: m=z for 12C813CH11O (MH1):
calcd. 136.0843, found 136.0812.
Materials and Methods
Synthesis
General. Anhydrous THF and TMEDA were dried
and freshly distilled from sodium=benzophenone.
CH2Cl2 was dried on molecular sieves and used
without distillation. Chromatography refers to flash
column chromatography,26 carried out on silica gel
60 (230–400 mesh ASTM, E. Merck), or aluminum
oxide (Brockmann activity 3, Fluka) when indicated.
TLC was performed on E. Merck 0.2 mm percolated
silica gel F-254 plates. Compounds were detected by
UV light (254 nm) and=or by staining with vanillin,
Cl2=KI-tolidine.27 1H- and 13C-NMR spectra were
recorded at 600, 300, or 200 MHz and 150 or 75
MHz, respectively, in CDCl3. Chemical shifts are
reported relative to internal TMS. Some 1H-NMR
assignments were supported by 2D homonuclear
COSY and NOSEY experiments. 13C-NMR assign-
ments were supported by DEPT or 2D HMQC and
HMBC experiments. Mass spectra were recorded in
DCI mode with methane as the reagent gas.
a-Chlorophenyl acetone (2). Phenyl acetone 1 (5
mL, 36.7 mmol) was dissolved in CH2Cl2 (50 mL),
and sulfuryl chloride (3.6 mL, 44.8 mmol) was added
slowly at 0ꢀC. After stirring at room temperature for
5 h, water (50 mL) was added, and the separated
aqueous layer was extracted with CH2Cl2 (2 3 50
mL). The combined organic layer was washed with
brine and dried over anhydrous MgSO4. Filtration
and evaporation afforded the a-chloroketone 2 as
slight yellow oil, which was used without purifica-
tion (6.04 g, 97% yield). 1H-NMR: d 7.43-7.36 (m,
5H); 5.35 (s, 1H); 2.21 (s, 3H). 13C-NMR: d 200.2,
135.2, 129.3, 129.2, 127.9, 66.7, 25.9. MS: m=z 170
(M1, 4), 168 (M1, 14), 118 (17), 127 (60), 125 (100),
90 (31). HRMS: m=z for C9H9O37Cl (M1): calcd.
170.0312, found 170.0328, for C9H9O35Cl (M1):
calcd. 168.0342, found 168.0345, for 12C813CH9O35Cl
(M1): calcd. 169.0375, found 169.0374.
Phenylacetic acid N,O-dimethylhydroxamate.
To
a suspension of carbamoylimidazolium salt
2-Chlorobenzyl-2,5,5-trimethyl-1,3-dioxane
(1-f[methoxy(methyl)amino]-carbonylg-3-methyl-1H-
imidazol-3-ium iodide)15 (2.38 g, 8.07 mmol) in aceto-
nitrile (48 mL) were added phenyl acetic acid (1.09
g, 8.07 mmol) and Et3N (1.1 mL, 8.07 mmol). The
reaction was stirred at room temperature overnight.
The solvent was removed in vacuo and the residue
was dissolved in CH2Cl2 (50 mL) and 0.2 N HCl (50
mL) was added. The aqueous layer was extracted
with CH2Cl2 (3 3 50 mL). The combined organic
layers were washed with 0.2 N HCl (100 mL), 0.5 M
K2CO3 (100 mL), and brine, dried over anhydrous
MgSO4, filtered and evaporated to give clean
hydroxamate as colorless oil (1.38 g, 95% yield). 1H-
NMR: d 7.6-7.3 (m, 5H); 3.77 (s, 2H); 3.59 (s, 3H);
3.19 (s, 3H). 13C-NMR: d 135.0, 129.4, 128.6, 126.8,
61.4, 39.5, 36.2. MS: m=z 180 (MH1, 60), 118 (67),
(3). To a solution of a-chloroketone 2 (6.04 g, 35.8
mmol) in dry toluene (50 mL) were added neopentyl
glycol (6.71 g, 64.4 mmol) and p-toluensulfonic acid
(136 mg, 0.72 mmol). Then the mixture was refluxed
over night in a Dean-Stark apparatus. After cooling to
room temperature, water (50 mL) was added. The
layers were separated and the aqueous layer was re-
extracted with hexane (2 3 50 mL). Then the com-
bined organic layers were washed with saturated
NaHCO3 solution and brine successively and dried
over anhydrous MgSO4. Filtration and evaporation
afforded crude acetal 3, which was used without puri-
fication (8.67 g, 95% yield). 1H-NMR: d 7.6-7.5 (m,
2H); 7.37-7.25 (m, 3H); 4.96 (s, 1H); 3.61 (d, J511.7
Hz, 1H); 3.57 (d, J511.7 Hz, 1H); 3.49 (dd, J511.7, 2.1
Hz, 1H); 3.47 (dd, J511.7, 2.1 Hz, 1H); 1.40 (s, 3H);
0.98 (s, 3H); 0.79 (s, 3H). 13C-NMR: 137.7, 128.4,
128.2, 127.8, 99.3, 71.0, 70.1, 66.8, 30.1, 22.7, 22.3,
16.2. MS: m=z 257 (MH1, 3) 255 (MH1, 7), 129 (100).
91 (100). HRMS: m=z
C
10H14NO2 (MH1) calcd.
180.1025 found 180.0989.
Phenyl acetone (1). To dry THF (20 mL) under ar-
gon atmosphere at –78ꢀC, was added a solution of
MeLi-LiBr (1.5M in ether, 16.5 mL) and 13C-labeled
phenylacetic acid N,O-dimethylhydroxamate (1.14 g,
6.36 mmol) dissolved in dry THF (10 mL). After 3 h the
solution was warmed to 0ꢀC in an ice bath. After addi-
tional 3 h, the resulting mixture was quenched by add-
ing water (30 mL). The aqueous layer was extracted
with ether (2 3 50 mL) and the combined organic layer
HRMS: m=z for C14H20O235Cl (MH1): calcd. 255.1109,
found 255.1152; for
C
13CH20O237Cl (MH1): calcd.
12
13
12
258.1156, found 258.1202; for
(MH1): calcd. 256.1185, found 256.1167.
C
13CH20O235Cl
13
2-Bromomethyl-2-(chlorobenzyl)-5,5-dimethyl-
1,3-dioxane (4). Acetal 3 (0.54 g, 2.17 mmol) was
dissolved in dried chloroform (30 mL), and pyridin-
ium hydrobromide perbromide (0.66 g, 2.07 mmol)
796 PROTEINSCIENCE.ORG
Peptidyl Cyclopropenones as Cysteine Protease Inhibitors

was added. After refluxing for 2 h, it was cooled to
room temperature, and then water (50 mL) was
added. The aqueous layer was extracted with chloro-
form, and the combined organic layer was washed
with water, saturated NaHCO3 solution, and brine
successively and dried over anhydrous MgSO4. Fil-
tration and evaporation afforded crude dihalide 4,
which was used without purification. The crude can
be recrystallized from hexane to give the desired
The aqueous solution was extracted with ether
(3330 mL). The combined organic layer was washed
with brine and dried over anhydrous MgSO4. Filtra-
tion and evaporation gave a crude product, which
was purified by aluminum oxide (brockmann 3) col-
umn chromatography (hexane:EtOAc 6:1) to give a
mixture of the diastereomers 6 and recovered 5. The
two diastereomers 6 were dissolved in CHCl3 to
afford the desired diastereomers 7 (0.36 g, 53%
yield) and the diol, which was separated by chroma-
1
dihalide 4 (0.62 g, 88% yield). H-NMR: d 7.6-7.5 (m,
1
2H); 7.3-7.3 (m, 3H); 5.29 (s, 1H); 3.94 (d, J511.7
Hz, 1H); 3.62 (d, J511.4 Hz, 1H); 3.57 (d, J511.7
Hz, 1H); 3.56 (d, J511.1 Hz, 1H); 3.52 (dd, J511.7,
2.4 Hz, 1H); 3.43 (dd, J511.7, 2.4 Hz, 1H); 0.80 (s,
3H); 0.76 (s, 3H). 13C-NMR: d 136.4, 129.9, 128.6,
127.8, 97.9, 71.2, 71.0, 63.6, 29.7, 27.8, 22.3. MS:
tography (hexane:EtOAc 3:1). H-NMR: d 8.0-7.9 (m,
2H); 7.6-7.4 (m, 3H); 5.22 (d, J57. 2 Hz, 1H mi);
5.11 (d, J52.4 1H ma); 5.10 (s, 1H mi); 4.78 (d,
J56.6 Hz, 1H ma); 3.75 (ddd, J58.1, 7.2, 2.4 Hz, 1H
ma); 3.41 (m, 1H mi); 2.39 (m, 1H mi); 2.03 (dsept,
J5 8.1, 6.6 Hz, 1H ma); 1.31 (s, 9H ma); 1.26 (s, 9H
mi); 1.15 (d, J56.6 Hz, 3H); 1.09 (d, J56.6 Hz, 3H);
1.087 (d, J56.6 Hz, 3H); 1.03 (d, J56.6 Hz, 3H). 13C-
NMR: d 158.5, 157.6, 156.5, 156.0, 155.7, 154.7,
132.9, 132.8, 132.6, 122.9, 81.1 (mi), 80.8 (ma), 73.2
(mi), 71.4 (ma), 62.4 (mi), 61.3 (ma), 29.8 (ma, mi),
28.1, 28.0 (ma, mi), 20.1 (ma), 20.0 (ma), 19.7 (mi),
19.1 (mi). MS: m=z 332 (3), 276 (100), 232 (14).
HRMS: m=z for (MH1) C19H26NO4: calcd. 332.1862,
found 332.1834; for (MH1-C4H8) C15H18NO4: calcd.
m=z (M-Cl) 299 (5) 297 (5), 209 (90), 207 (90), 69
(100). HRMS: m=z for
calcd. 334.0290
C
13CH19O2BrCl (MH1):
12
13
found
334.0291;
for
12
C
13CH18O281Br (M-Cl): calcd. 300.0503 found
13
12
300.0408; for
C
13CH18O279Br (M-Cl): calcd.
13
298.0524 found 298.0442; for C14H18O279Br (M-Cl):
calcd. 297.0490 found 297.0468.
Phenyl cyclopropenone Acetal (5). To solution of
potassium tert-butoxide (0.59 g, 5.23 mmol) in THF
(5 mL) and HMPA (1.37 mL, 7.83 mmol) was added
via cannula dihalide 4 (0.87g, 2.61 mmol) in THF (5
mL). The reaction was carried under argon atmos-
phere. After stirring for 4 h at 0ꢀC the reaction was
allowed to warm up to room temperature and stirred
overnight. Then water (20 mL) was added, and the
aqueous solution was extracted with hexane (3 3 20
mL). The combined organic layer was washed with
brine and dried over anhydrous MgSO4. Filtration
and evaporation gave a crude product, which was
purified by deactivated (brockmann 3) aluminum ox-
ide chromatography (hexane: EtOAc 30:1) to afford
compound 5. (0.39 g, 60% yield). The CDCl3 for
NMR was passed through K2CO3 (Alternatively
276.1236,
found
276.1175;
for
(MH1)
12
C
13CH26NO4: calcd. 333.1895, found 333.1926;
18
for (MH1-C4H8)
C
13CH18NO4: calcd. 277.1269,
12
14
found 277.1219.
2-f(2S)-2-amino-1-hydroxy-3-methylbutylg-3-phe-
nylcyclopropenone hydrochloride (8). To solu-
tion of 7 (87 mg, 0.26 mmol) in 1,4-dioxane (0.25
mL) and water (5.6 mL) 4N HCl in 1,4-dioxane (0.8
mL) was added at room temperature. After stirring
for 30 min, the solution was evaporated in warm
bath to afford compound 8 as highly hygroscopic
salt. (54 mg, 90% yield). 1H-NMR: d 8.1-8.0 (m, 2H);
7.7-7.5 (m, 3H); 5.41 (d, J54.5 Hz, 1H mi); 5.19 (d,
J56.6 Hz, 1H ma); 3.42 (dd, J56.3, 5.4 Hz, 1H ma);
3.33 (dd, J59.6, 4.5 Hz, 1H mi); 2.28 (septd, J56.9,
5.4 Hz, 1H ma); 2.08 (dsept, J59.6, 6.9 Hz, 1H mi);
1.21 (d, J56.9 Hz, 1H mi); 1.19 (d, J56.9, 1H ma);
1.16 (d, J56.9 Hz, 1H mi); 1.158 (d, J56.9 Hz, 1H
ma). 13C-NMR: d 157.5, 157.1, 154.4, 134.8, 133.9,
130.5, 123.7, 68.6 (mi), 68.0 (ma); 62.6 (mi), 60.8
(ma), 30.1 (mi), 29.3 (ma), 19.9 (mi), 19.7 (ma), 17.4
(ma, mi). MS: m=z 232 (100), 214 (50).
1
CD3CN was used as solvent). H-NMR: d 7.68 (s, 1H);
7.64-7.61 (m, 2H); 7.50-7.35 (m, 3H); 3.74 (s, 4H); 1.14
(s, 3H); 1.06 (s, 3H). 13C-NMR: d 135.6, 130.1, 129.7,
128.9, 125.9, 114.7, 83.0, 77.8, 30.5, 22.6, 22.4. MS:
m=z 217 (M1, 100). HRMS: m=z for
(MH1): calcd. 218.1262 found 218.1251.
C
13
13CH17O2
12
2-f(2S)-2-Bocamine-1-hydroxy-3-methyl-butylg-3-
phenylcyclopeopenone (7). To solution of
a
5
(0.82 g, 3.8 mmol) and dry N,N,N’,N’-tetramethyle-
thylenediamine (1.14 mL, 7.8 mmol) in dry THF (8
mL), was added n-BuLi (1.2 M in THF, 3.16 mL) at -
78ꢀC over 5 min. After the mixture was stirred for
20 min, a solution of N-Boc-valinal14 (0.42 g, 2.11
mmol) in dry THF (4 mL) was added via cannula.
The mixture was stirred for 2 h at -78ꢀC. After addi-
tion of 1:4 water:THF (4 mL) the mixture was
diluted with ether and extracted with water (33).
2-f(2S)-2-(Cbz-Leucyl-amino)-1-hydroxyl-3-meth-
ylbutylg-3-phenyl-cyclopropenone (9). Cbz-Leu-
OH (77 mg, 0.29 mmol), PyBOP (154 mg, 0.29
mmol) and the hydrochloride salt 8 (60 mg, 0.26
mmol) were dissolved in CH2Cl2 (2 mL) and Et3N
(0.13 mL, 9.5 mmol) was added. After 15 min of stir-
ring at room temperature, the pH checked for basic
condition and the mixture was stirred for additional
2.5 h. It was then quenched with an aq KHSO4
Cohen et al.
PROTEIN SCIENCE VOL 22:788—799 797

solution (0.5 g, 5 mL). The aqueous phase was
extracted with CH2Cl2 (3 3 20 mL), the combined
organic layers were washed with brine, dried over
MgSO4 and evaporated to give a crude product,
which was purified by chromatography (hexane:E-
tOAc 1:4) to afford the compound 9 as two separated
diastereomers. (61 mg, 49% yield, 9a:9b 5:1).
solution was purified from small molecules (DTT
and EDTA) by Sephadex G-15 chromatography (15
3 1 cm2 column, degassed 100 mM KPi buffer pH
7.0). Fractions of 1.5 mL were collected and their
A280 was measured. The enzyme-containing fractions
were pooled and the enzyme activity assayed. Con-
centration of free thiol in the enzyme fraction, as
well as the other fractions was determined by addi-
tion of aq. DTNB solution (30 lL, 10 mM) and meas-
2-f(1S,2S)-2-(Cbz-Leucyl-amino)-1-hydroxyl-3-
methylbutylg-3-phenyl-cyclopropenone
(9a).
1H-
NMR: d 8.027.9 (m, 2H); 7.62 (d, J56.9, 1H);
7.627.4 (m, 3H); 7.327.2 (m, 5H); 5.74 (d, J57.5 Hz,
1H); 5.07 (d, J52.9 Hz, 1H); 5.02 (d, J512.3 Hz,
1H); 4.91 (d, J512.3 Hz, 1H); 4.17 (brq, J58 Hz,
1H); 3.69 (br.dt, J58.1, 7.2, 2.4 Hz, 1H); 2.33 (d.sept,
J59.6, 6.6 Hz, 1H); 1.50 (nonet, J56.6 Hz, 1H); 1.31
(m, 1H); 1.06 (d, J56.6, 3H); 0.98 (d, J56.6 Hz, 3H);
0.7820.71 (m, 6H). 13C-NMR: d 174.9, 156.4, 156.2,
156.0, 154.9, 136.1, 133.0, 132.6, 129.2, 128.5, 128.2,
127.9, 122.8, 70.5, 67.2, 61.5, 53.9, 41.1, 28.0, 24.7,
22.8, 21.6, 19.7, 19.5. MS: m=z 479 (MH1, 87), 451
(97), 433 (100), 319 (35), 248 (52), 91 (76). HRMS:
uring A412 (e 5 13,600 M21 cm21 16
incubation.
)
after 5 min of
To correlate residual enzyme activity with free
thiol concentration, aliquots (1.5 mL) from the inac-
tivation solution were removed at various time
points. Totally, 10 lL of the aliquot was used for en-
zymatic activity assay (immediate dilution into the
assay buffer solution practically stopped the inacti-
vation reaction), and the rest was treated with
DTNB as above to determine the remaining free
thiol concentration. An active enzyme solution was
similarly treated and assayed as a control.
m=z for (MH1) C28H35N2O5: calcd. 479.2546, found
12
479.2560; for
C
13CH35N2O5: calcd. 480.2580,
Denaturation
27
found 480.2567.
Heat denaturation was carried out by boiling an
enzyme solution for 3 min. Chemical denaturation
was achieved by addition of guanidinium chloride
(at 6M final concentration). Residual enzyme activ-
ity and free thiol concentration were determined in
both cases.
2-f(1R,2S)-2-(Cbz-Leucyl-amino)-1-hydroxyl-3-
methylbutylg-3-phenyl-cyclopropenone (9b). 1H-NMR:
d 8.128.0 (m, 2H); 7.627.5 (m, 3H); 7.427.3 (m, 5H);
6.95 (d, J57.2, 1H); 5.34 (d, J57.2 Hz, 1H); 5.16 (bd,
J52.9 Hz, 1H); 5.04 (d, J512 Hz, 1H); 4.94 (d, J512
Hz, 1H); 4.17 (dt, J57.8, 6.6 Hz, 1H); 4.08 (ddd,
J59.6, 7.2, 2.4 Hz, 1H); 2.16 (d.sept, J59, 6.6 Hz,
1H); 1.5521.34 (m, 3H); 1.16 (d, J56.6, 3H); 1.09 (d,
J56.6Hz, 3H); 0.72 (d, J56.3, 6H). 13C-NMR: d 175.5,
156.4, 155.7, 154.4, 136.2, 133.1, 132.8, 129.3, 128.6,
128.3, 128.2, 123.0, 72.7, 67.3, 61.5, 53.9, 41.1, 29.4,
24.8, 22.6, 22.1, 20.2, 19.8. MS: m=z 479 (MH1, 100),
433 (60), 319 (37), 176 (53), 91 (87). HRMS: m=z for
(MH1) C28H35N2O5: calcd. 479.2546, found 479.2570.
Mercury affinity chromatography
Activated papain was separated from inactive pro-
tein by mercury affinity chromatography as previ-
ously described.17
13C-NMR analysis
Papain (100 mg) was activated and the active
enzyme was purified by mercury affinity chromatog-
raphy. The active enzyme solution was concentrated
to 10 mL through a 10,000 cutoff filter (Amicon),
diluted in potassium phosphate buffer (90 mL, 100
mM, pH 7.0), and reconcentrated to 2 mL by Amicon
and further concentrated into 0.5 mL by a stream of
nitrogen. To this solution was added a solution of in-
hibitor 9 in DMSO-d6 (0.1 mL, 12 mM), or DMSO-d6
alone (0.1 mL) in the control.
Biochemistry
Enzyme kinetics. Papain (EC 3.4.22.2) and ca-
thepsin B (EC 3.4.22.1) were activated, assayed, and
inhibited as previously described.7
Dialysis. A 1.2 mL solution of inhibited enzyme
(whose residual activity determined) was dialyzed
in a 3,500 Da cut-off dialysis bag against 400 mL of
phosphate buffer (100 mM KPi, pH 7.0, containing
0.5 mM DTT). The buffer solution was replaced
after 4 h, and dialysis continued for additional 4 h,
after which the enzyme activity was measured again.
As a control, active enzyme underwent the same
protocol.
HPLC analysis
Nucleosil 12035 C18 column (Macherey-Nagel) was
used at a flow rate of 1 mL=min, applying linear gra-
dients of either 100% to 20% water-acetonitrile in 40
min or 100% to 20% water-methanol in 50 min.
Conclusions
Peptidyl cyclopropenones were previously introduced
as competitive reversible inhibitors of cysteine pro-
teases. Their specific mechanism of interaction with
the enzyme active site was not clarified. In this
Free thiol determination. Activated papain (16
mg in 100 mL) was concentrated to about 3 mL in a
10,000 cutoff Amicon tube. The concentrated enzyme
798 PROTEINSCIENCE.ORG
Peptidyl Cyclopropenones as Cysteine Protease Inhibitors

gas-phase and solvation energetics. J Am Chem Soc
110:7195–7200.
study we show that
a peptidyl cyclopropenone
behaves as an irreversible covalent inhibitor of a
cysteine protease, alkylating the catalytic cysteine of
papain. In parallel, it also behaves as an alternative
substrate of the enzyme. A detailed mechanism for
these two reactions was suggested. Thus, this study
draws attention to the reality that a family of com-
pounds may exhibit various modes of interaction
with different enzymes sharing the same catalytic
mechanism, and even a dual mode of interaction
with a single enzyme.
13. Sakaki T, Ando R (2007) 2-tert-Butoxy-3-phenylcyclo-
propanone acetal, a stable precursor of lithiated 2-phe-
nylcyclopropenone acetal. Tetrahedron Lett 48:
7011–7014.
14. Mirilashvili S, Chasid-Rubinstein N, Albeck A (2010)
Optically Active N- and C-terminal building blocks for
the synthesis of peptidyl olefin peptidomimetics. Eur J
Org Chem 4671–4686.
15. Grzyb JA, Shen M, Yoshina-Ishii C, Chi W, Brown RS,
Batey RA (2005) Carbamoylimidazolium and thiocarba-
moylimidazolium salts: Novel reagents for the synthe-
sis of ureas, thioureas, carbamates, thiocarbamates
and amides. Tetrahedron 61:7153–7175.
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