Modulation of the inhibitor properties of dipeptidyl (acyloxy)methyl ketones toward the CaaX proteases

Article abstract of DOI:10.1016/j.bmc.2010.07.041

Dipeptidyl (acyloxy)methyl ketones (AOMKs) have been identified as mechanism-based inhibitors of certain cysteine proteases. These compounds are also inhibitors of the integral membrane proteins Rce1p and Ste24p, which are proteases that independently mediate a cleavage step associated with the maturation of certain isoprenylated proteins. The enzymatic mechanism of Rce1p is ill-defined, whereas Ste24p is a zinc metalloprotease. Rce1p is required for the proper processing of the oncoprotein Ras and is viewed as a potential target for cancer therapy. In this study, we synthesized a small library of dipeptidyl AOMKs to investigate the structural elements that contribute to the inhibitor properties of this class of molecules toward Rce1p and Ste24p. The compounds were evaluated using a fluorescence-based in vitro proteolysis assay. The most potent dipeptidyl AOMKs contained an arginine residue and the identity of the benzoate group strongly influenced potency. A 'warhead' free AOMK inhibited Rce1p and Ste24p. The data suggest that the dipeptidyl AOMKs are not mechanism-based inhibitors of Rce1p and Ste24p and corroborate the hypothesis that Rce1p is not a cysteine protease.

Full text of DOI:10.1016/j.bmc.2010.07.041

Bioorganic & Medicinal Chemistry 18 (2010) 6230–6237
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Modulation of the inhibitor properties of dipeptidyl (acyloxy)methyl
ketones toward the CaaX proteases
Anne-Marie R. Dechert a, James P. MacNamara a, Sarah R. Breevoort b, Emily R. Hildebrandt b,
Ned W. Hembree a, Adam C. Rea a, Duncan E. McLain a, Stephen B. Porter b, Walter K. Schmidt b,
,
*
Timothy M. Dore a,
*
a Department of Chemistry, University of Georgia, Athens, GA 30602-2556, United States
b Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA 30602-7229, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Dipeptidyl (acyloxy)methyl ketones (AOMKs) have been identified as mechanism-based inhibitors of cer-
tain cysteine proteases. These compounds are also inhibitors of the integral membrane proteins Rce1p
and Ste24p, which are proteases that independently mediate a cleavage step associated with the matu-
ration of certain isoprenylated proteins. The enzymatic mechanism of Rce1p is ill-defined, whereas
Ste24p is a zinc metalloprotease. Rce1p is required for the proper processing of the oncoprotein Ras
and is viewed as a potential target for cancer therapy. In this study, we synthesized a small library of
dipeptidyl AOMKs to investigate the structural elements that contribute to the inhibitor properties of this
class of molecules toward Rce1p and Ste24p. The compounds were evaluated using a fluorescence-based
in vitro proteolysis assay. The most potent dipeptidyl AOMKs contained an arginine residue and the iden-
tity of the benzoate group strongly influenced potency. A ‘warhead’ free AOMK inhibited Rce1p and
Ste24p. The data suggest that the dipeptidyl AOMKs are not mechanism-based inhibitors of Rce1p and
Ste24p and corroborate the hypothesis that Rce1p is not a cysteine protease.
Received 25 April 2010
Revised 12 July 2010
Accepted 16 July 2010
Available online 21 July 2010
Keywords:
Ras converting enzyme (Rce1p)
Sterile mutant 24 (Ste24p)
(Acyloxy)methyl ketone
Protease
Ras
CaaX protein
Post-translational modification
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
turnover of the unprocessed intermediate.5,9,10 The Ras subfamily
of small GTP-binding proteins11 are CaaX proteins having a prom-
Many eukaryotic proteins bear a C-terminal CaaX tetrapeptide
motif, where C is cysteine, a is typically an aliphatic amino acid,
and X is one of several amino acids, that directs an ordered series
of post-translational modifications (Fig. 1).1–3 These include the
covalent addition of an isoprenoid lipid to the cysteine by either
farnesyl or geranylgeranyl transferase (FTase or GGTase),4 a prote-
olytic step that trims away the aaX portion,5,6 and methyl esterifi-
cation of the resultant new carboxyl terminus by isoprenylcysteine
methyltransferase (ICMT).7,8 These modifications are critical to the
activity of many CaaX proteins; defects in the processing pathway
can result in non-functional or mislocalized protein, or enhanced
inent role in carcinogenesis.2,3 Hence, Ras proteins and Ras-regula-
tory proteins are considered targets for anticancer therapeutics.2,12
The CaaX endoproteases Ras converting enzyme 1 (Rce1p) and
sterile mutant 24 (Ste24p), both first identified in Saccharomyces
cerevisiae, mediate the carboxyl terminal proteolysis step of CaaX
protein maturation.5 Despite functional similarity, Rce1p and
Ste24p lack primary sequence similarity.5 Orthologs of both prote-
ases are present in humans,13,14 mice,15 Caenorhabditis elegans,16
plants, and other eukaryotic organisms.17,18 Both are integral
membrane proteins with multiple predicted transmembrane
spans19,20 that localize to the endoplasmic reticulum (ER) with
the catalytic sites presumably facing the cytosol where their sub-
strates are located.19,21 Ste24p is a zinc-dependent metallopro-
tease,5,19,22 possessing a consensus HEXXH motif (H is histidine,
E is glutamate, and X is any amino acid) that is required for prote-
olytic activity. The zinc chelator 1,10-phenanthroline inhibits the
proteolytic activity, but the addition of zinc reactivates it.19,22
SeveralreportshaveimplicatedRce1pasacysteineprotease,23–25
but mutation20 and bioinformatic26 studies provide a stronger case
that it is a metalloprotease. The only conserved cysteine among
eukaryotic Rce1p orthologs (Cys251 in yeast Rce1p) is not required
Abbreviations: ABz, ortho-aminobenzoate; AOMK, (acyloxy)methyl ketone; DCC,
N,N0-dicyclohexylcarbodiimide; DIPEA, N,N-diisopropylethylamine; HEPES, 4-(2-
hydroxyethyl)piperazine-1-ethanesulfonic acid; HOBt, 1-hydroxybenzotriazole;
HBTU, O-(benzotriazol-1-yl)-N,N,N0,N0-tetramethyluronium hexafluorophosphate;
ICMT, isoprenylcysteine methyltransferase; Pbf, pentamethyl-2,3-dihydrobenzofu-
ran-5-sulfonyl; QL,
acid; TPCK, N-tosyl-
e
L
-dinitrophenyl lysine; TEA, triethylamine; TFA, trifluoroacetic
-phenylalanine chloromethyl ketone.
* Corresponding authors. Tel.: +1 706 583 0423; fax: +1 706 542 9454 (T.M.D.);
tel.: +1 706 583 8241; fax: +1 706 542 1738 (W.K.S.).
E-mail addresses: wschmidt@bmb.uga.edu (W.K. Schmidt), tdore@chem.uga.edu
(T.M. Dore).
for enzyme function,20 seriously weakening the case for
a
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.07.041

Anne-Marie R. Dechert et al. / Bioorg. Med. Chem. 18 (2010) 6230–6237
6231
hydrolyzable isoprenylated peptides and isoprenoids),23,24,37,38
natural products (barangcadoic and rhopaloic acids39 and scalar-
anes40), and small-molecules discovered in biochemical screens
of chemical libraries.41,42
We previously reported that peptidyl (acyloxy)methyl ketones
(AOMKs) inhibit both Rce1p and Ste24p.43 This compound class is
best known for potently and irreversibly modifying certain cysteine
proteases, presumably through covalent modification of the active
site cysteine.44,45 Curiously though, only one cysteine residue is con-
served among Rce1p orthologs and none is conserved among Ste24p
enzymes. Furthermore, the conserved cysteine in the Rce1p family
(Cys251 in yeast Rce1p sequence) is not required for activity, and
AOMKs inhibit the C251A mutant.20,43 The inhibition of the CaaX
proteases by AOMKs indicates that this compound class represents
an important new tool for the study of the CaaX proteases. By com-
parison, AOMKs perform more consistently than TPCK, a widely de-
scribed chloromethyl ketone Rce1p inhibitor.16,37,43,46 Moreover,
AOMKs are the first agents described that inhibit both Rce1p and
Ste24p. Thus, these compounds have potential for leading to a better
understanding of CaaX protease enzymology.
In this study, we investigated the structural elements of AOMKs
(Figs. 2 and 3) as they contribute to the inhibitory properties of this
compound class against yeast Rce1p and Ste24p in a fluorescence-
based in vitro proteolysis assay. In particular, we have determined
Figure 1. Post-translational modifications associated with CaaX proteins.
cysteine-based mechanism. Seven residues appear important for
optimum enzymatic activity of yeast Rce1p: Glu156, His194, His248
,
Glu157, Tyr160, Asn252 20
activity of yeast Rce1p and probably define the catalytic site, while
the latter four optimize activity and substrate specificity. Glu156
.
The first three are critical for the proteolytic
,
His194, and His248 are not in close proximity in the primary sequence.
These presumed catalytic residues are conserved among Rce1p
orthologs, and in the case of trypanosomal Rce1p, are also required
for activity.27 These residues must be in close three-dimensional
proximity if they indeed are part of the active site and are hypothe-
sized to reside at or within the membrane.20 Such a membrane-
embeddedactive siteis atypical forproteases, buthasbeen proposed
for presenilins, site-2 protease, rhomboid, and the signal peptide
peptidase.28
Rce1p and Ste24p have distinct, but partially overlapping, sub-
strate specificities, which suggests that they have different cellular
functions. Rce1p cleaves Ras and Ras-related proteins, whereas
Ste24p does not cleave yeast Ras2p5 or human farnesylated
K-Ras,21 which have CaaX motifs consisting of CIIS and CVIM
residues, respectively. Mammalian Ste24p cleaves pre-lamin A
(CSIM).29,30 Both proteases act on the precursor to the yeast a-factor
mating pheromone (CVIA).31 Knockout studies in mice have shown
that Rce1p is required for embryonic and cardiac development,15,32
and Ste24p is required for proper skeletal and muscular develop-
ment.29,30 Because of its involvement in pre-lamin A processing, hu-
man Ste24p deficiency is also connected to human progeroid
disorders.33–35
Figure 2. Dipeptidyl AOMK compounds synthesized for this study.
Inhibition of Rce1p is an attractive anticancer strategy because
it would be expected to impede Ras-induced oncogenic transfor-
mation, while not affecting the maturation of Ste24p-dependent
substrates.2,3 Furthermore, mouse embryonic fibroblasts deficient
in Rce1p are more sensitive to an FTase inhibitor than wild type
cells,36 indicating the potential for combination therapies. Inhibi-
tors of Rce1p fall into four categories: non-specific protease inhib-
itors (N-tosyl-
L
-phenylalanine chloromethyl ketone (TPCK),24,25,37
organomercurials,25 Zn2+, and Cu2+),25 substrate mimetics (non-
Figure 3. Other AOMKs.

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Anne-Marie R. Dechert et al. / Bioorg. Med. Chem. 18 (2010) 6230–6237
how the structural profile of the benzoate moiety and amino acid
substitutions of the peptidyl group modulate the inhibitory prop-
erties of AOMKs.
2. Results
2.1. Dipeptidyl AOMK synthesis
A library of three series of dipeptidyl AOMKs, each with differ-
ent benzoyloxy groups (Fig. 2), were synthesized using the method
described by Krantz44 and outlined in Schemes 1–3. Benzyloxycar-
bonyl-protected phenylalanine (1, Scheme 1) was reacted with
N-hydroxysuccinimide and DCC to generate the succinimide ester
Scheme 3. See Figure
(a) N-hydroxysuccinimide, DCC, THF, 0 °C, 15 h, 93%; (b)
2
for definitions of R0. Reagents and conditions:
-arginine(Pbf), TEA, CO2,
L
dioxane, H2O, pH 8, rt, 20 h, 48%; (c) isobutyl chloroformate, N-methylmorpholine,
THF, ꢁ20 °C, 2 h; (d) CH2N2, Et2O, 0 °C; (e) HCl, AcOH, 0 °C, 1 min, 22% for three steps;
(f) benzoic acids a–g, KF, DMF, rt, 4 h, 22–45%; (g) TFA/H2O (95:5), 0 °C, 4 h.
Scheme 1. See Figure 2 for definitions of R0. Reagents and conditions: (a) N-
hydroxysuccinimide, DCC, THF, 0 °C, 18 h, 95%; (b) L-alanine, TEA, CO2, dioxane,
1b, which was coupled to alanine to provide dipeptide 2. The iso-
butyl formate of 2 was treated with diazomethane to generate
the a-diazo ketone, which was converted to the bromomethyl ke-
H2O, pH 8, rt, 20 h, 51%; (c) isobutyl chloroformate, N-methylmorpholine, THF,
ꢁ20 °C, 2 h; (d) CH2N2, Et2O, 0 °C; (e) HBr, AcOH, CH2Cl2, 0 °C, 1 min, 15% for three
steps; (f) benzoic acids a–g, KF, DMF, rt, 4 h, 8–75% yield.
tone 3 by brief exposure to HBr in acetic acid. Displacement of the
bromide by the benzoates a–g resulted in the AOMKs 4a–g in
yields of 8–75%. Similarly, DCC coupling of e-Boc-protected lysine
to the succinimide 1b provided dipeptide 5 (Scheme 2), which
was converted to the bromomethyl ketone 6 using the Krantz pro-
cedure in 73% yield. The bromomethyl ketone 6 served as the start-
ing point for the syntheses of the benzoyl esters 7a–g, which
proceeded smoothly, albeit in low yields (ꢀ35%). Deprotection of
7a–g with trifluoroacetic acid (TFA) provided AOMKs 8a–g after
purification by HPLC. Removal of the Boc group from 5 with TFA
in dichloromethane gave dipeptide 9 for control experiments.
Deprotection of 6 under similar conditions provided bromide 8h
in 54% yield.
The synthesis of the arginine series of AOMKs proved more diffi-
cult (Scheme 3). Coupling of Pbf-protected L-arginine to the benzyl-
oxycarbonyl-protected phenylalanine produced dipeptide 10 in 93%
yield, but the subsequent reactions to convert 10 to a halomethyl ke-
tone behaved inconsistently, despite the report of a related Pbf-pro-
tected arginine substrate undergoing the transformation in 65%
yield.47 Quenching the diazo intermediate with HBr gave unsatisfac-
tory results; using HCl generated the chloro ketone 11a. Regardless
the acid, reactions typically yielded hydroxymethyl ketone 11b, and
attempts to convert it to the desired AOMK with the appropriate
benzoyl chloride failed. When the reaction sequence was successful,
we obtained 11a in 22% yield. Displacement of the chloride with
benzoic acids a–g provided Pbf-protected AOMKs 12a–g in 22–
45% yield. Pbf-protected dipeptides 12a–g were successfully depro-
tected to give the arginine series of AOMKs 13a–g after purification
by HPLC. The deprotection step to convert bis(trifluoromethyl)ben-
zoate 12e to 13e often resulted in decomposition, but we were able
to obtain a sufficient quantity of 13e for biological studies.
Scheme 2. See Figure
(a) N-hydroxysuccinimide, DCC, THF, 0 °C, 15 h, 93%; (b)
dioxane, H2O, pH 8, rt, 20 h, 55%; (c) isobutyl chloroformate, N-methylmorpholine,
THF, ꢁ20 °C, 2 h; (d) CH2N2, Et2O, 0 °C; (e) HBr, AcOH, CH2Cl2, 0 °C, 1 min, 73% for
three steps; (f) benzoic acids a–g, KF, DMF, rt, 4 h, ꢀ35%; (g) TFA, CH2Cl2, 0 °C,
53–93%.
2
for definitions of R0. Reagents and conditions:
-lysine( -Boc), TEA, CO2,
To explore the role of the benzoate group, we prepared a dipep-
tidyl AOMK possessing an N-(b-phenethylamide) (17, Scheme 4) in
place of the benzoyloxymethyl group. This ‘warhead-free’ dipep-
tide was synthesized by coupling b-phenethylamine to protected
dipeptide 10, followed by removal of the Pbf group with TFA.
L
e

Anne-Marie R. Dechert et al. / Bioorg. Med. Chem. 18 (2010) 6230–6237
6233
ABz group is quenched by QL until CaaX protease-mediated prote-
olysis cleaves the peptide to liberate the quenching group. The as-
say was carried out in 96-well plates with fluorescence output
measured using a fluorescence microplate reader. Decreased fluo-
rescence output compared to a DMSO control indicated inhibition
of proteolysis.
The proteolytic activities of Rce1p and Ste24p relative to a
DMSO control in the presence of 100 lM AOMK-based inhibitors
are summarized in Figure 5. Control peptides lacking the benzoyl
ester group (5, 6, 9) and tripeptidyl AOMKs (14, 15) were not sig-
nificant inhibitors of either Rce1p or Ste24p. Overall, AOMKs con-
taining alanine (4a–g) were the least inhibitory, while those with
arginine (13a–g) were the most potent. The most inhibitory com-
pound in the assay was Z-FR-CH2OBz(2,4,6-trimethyl) (13d). The
dipeptidyl AOMKs demonstrated similar inhibitory strength
against Ste24p and Rce1p, though certain compounds demon-
strated slightly preferential inhibition of Ste24p over Rce1p (4b,
4g, 13a, 13c–f, 17), with ‘warhead-free’ dipeptide 17 having the
most dramatic preference.
Scheme 4. Reagents and conditions: (a) HOBt, HBTU, DIPEA, CH2Cl2, 0 °C, then rt
overnight, 39%; (b) TFA/H2O (95:5), 0 °C, 2 h, 33%.
2.2. Fluorescence-based proteolysis assay
The inhibition by the dipeptidyl AOMK compounds was evalu-
ated using a fluorescence-based in vitro CaaX proteolysis assay
(Fig. 4).42,43,48 ER membranes enriched for either yeast Rce1p or
Ste24p were used as the source of enzyme activity. Two different
fluorogenic substrates based on K-Ras4b were used to monitor
the proteolytic activity. For Rce1p, ABz-KSKTKC(farnesyl)QLIM
3. Discussion
was used, where ABz is ortho-aminobenzoate and QL is e-dinitro-
Peptidyl AOMKs are mechanism-based inhibitors of cysteine
proteases.44 They covalently and irreversibly attach to the active
site cysteine. The potency and reactivity of the AOMK can be tuned
phenyl lysine. For Ste24p, ABz-KSKTKC(farnesyl)VIQL was used
since the Rce1p substrate is not effectively recognized by Ste24p.43
In both substrates, fluorescence resulting from excitation of the
Figure 4. Fluorescence-based assay for Rce1p endoprotease activity. Assay for Ste24p is identical except substrate is ABz-KSKTKC(farnesyl)VIK-DNP. ABz is
ortho-aminobenzoate and DNP is -dinitrophenyl.
e
Figure 5. Effect of dipeptidyl AOMK derivatives (100 lM) on the endoprotease activity of Ste24p and Rce1p relative to a DMSO control in the fluorescence-based assay.
Values are the average of at least four replicates. The error bars represent the standard deviation of the measurement. Statistically significant (p <0.05) differences between
Ste24p and Rce1p inhibition are marked with an asterisk.

6234
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by modifying the benzoate leaving group moiety. The better the
leaving group capability of the benzoate, the more potent the
inhibitor when there is rapid, reversible binding of the inhibitor.
Since Ste24p is a zinc-dependent metalloprotease, it is surprising
that the AOMKs inhibit its activity. Earlier studies suggest that
Rce1p is a cysteine protease, in part because it responds to classic
cysteine protease inhibitors, such as chloromethyl ketones and
organomercurials, but not to classic serine, metallo-, and aspartyl
protease inhibitors.23–25 A mechanistic-based inhibition of Rce1p
by AOMKs is thus plausible if it were indeed a cysteine protease.
This conclusion, however, should be viewed cautiously. One con-
cern is that mechanism-based protease inhibitors often have
cross-reactivity with side chains other than the intended targets,
which obscures the identification of the mechanism of the catalytic
pathway. Second, mutational analyses on the only cysteine con-
served among Rce1p eukaryotic orthologs, specifically Cys251 of
yeast Rce1p, have presented conflicting results. In one study, the
C251A mutant is reported to be inactive,25 but another found that
it retains wild type activity in assays for protease activity.20 Third,
bioinformatic studies across a broader range of potential orthologs
do not reveal a conserved cysteine.26 It is considerably more likely
that Rce1p is not a cysteine protease and that, as with Ste24p,
AOMKs act in a non-mechanistic fashion to inhibit Rce1p.
we propose that dipeptidyl AOMKs are likely reversible, non-com-
petitive inhibitors of the CaaX proteases.
4. Conclusion
Based upon the discovery that peptidyl (acyloxy)methyl
ketones are inhibitors of Rce1p and Ste24p,43 we investigated the
structural elements of AOMKs that contribute to the inhibitory
properties of this compound class. In particular, we explored
how the structural profile of the benzoate moiety and amino acid
substitutions of the second amino acid modify the inhibitory prop-
erties of AOMKs. We generated a 21-member library of AOMKs by
coupling three benzyloxycarbonyl-protected dipeptides to seven
benzoic acids, and evaluated their ability to inhibit Rce1p and
Ste24p in a fluorescence-based proteolysis assay. The data reveal
that the inhibitory activity of the peptidyl AOMKs is strongly
influenced by the identity of the amino acid in the second position
(Z-FA < Z-FK < Z-FR) and the structure of the benzoate. Activity by a
‘warhead-free’ AOMK suggests that the inhibition of both Rce1p
and Ste24p is not mechanism-based, as it is with cysteine prote-
ases, but rather is a result of non-covalent binding of the AOMK
to the active site of the protease. This corroborates other work that
suggests that Rce1p is not a cysteine protease.20,26
In a previous study,43 we investigated the inhibition of Rce1p and
Ste24p by compounds 4d, 8d, 13b, 14, and 15 and found that dipep-
tidyl AOMKs 8d and 13b inhibited yeast Rce1p and Ste24p. In the
current study, the in vitro activity of these five compounds was reas-
sessed, and we found good agreement with the previous data, except
for the inhibition of Rce1p by 13b and of Ste24p by 8d. In the current
work, we found that these AOMKs were weaker inhibitors than pre-
viously reported. Possible explanations for the difference includethe
following: (1) the membranes containing Rce1p and Ste24p had dif-
ferent specific activities (i.e., the amount of active enzyme per milli-
gram of membrane was different), so the AOMKs could have
partitioned into the membrane differently, altering the concentra-
tion of free AOMK present,43 and (2) the AOMKs and substrate pep-
tides used in theprevious study were from differentsources, so there
might be batch to batch variations in the samples.
5. Material and methods
5.1. Reagents and general methods
All reagents were purchased from commercial sources and used
without further purification with the following exceptions: THF
was dried on an activated alumina column immediately prior to
use and DMF was distilled under vacuum and stored over molecu-
lar sieves. Thin layer and column chromatography were performed
on precoated Silica Gel 60 F254 plates and 230–400 mesh Silica Gel
60, respectively. Flash chromatography was performed on a Flash+
system (Biotage, Charlottesville, VA). HPLC analysis (analytical and
preparative) was performed on a Varian ProStar HPLC system using
Microsorb C-18 reverse phase columns and a diode array UV detec-
tor. 1H NMR spectra were recorded on Varian MercuryPlus
400 MHz spectrometer. Mass spectrometry was performed on a
Sciex API-1 Plus quadruple mass spectrometer with an electro-
spray ionization source or a Bruker Autoflex MALDI-TOF. ESI-HRMS
was performed on a Bruker 4.7 T FT-MS. CaaX protease substrates
ABz-KSKTKC(farnesyl)QLIM and ABz-KSKTKC(farnesyl)VIQL were
commercially synthesized by Anaspec.
Our previous work43 showed that inhibition of Rce1p by 8d was
not readily reversible through washing with salt or urea buffers,
but 8d did not inhibit Rce1p in a time-dependent manner, suggest-
ing that binding was reversible. Compound 8d reduced the Vmax of
Rce1p in a dose-dependent manner without significantly changing
the Km value from that observed in the absence of inhibitor. Anal-
ysis of the kinetic data suggested that 8d was a tightly bound,
reversible, non-competitive inhibitor of Rce1p.
Our current studies indicate that the leaving group ability of the
benzoyl ester does not correlate well with the inhibitory properties
of the dipeptidyl AOMKs. For example, the conjugate acids of the
benzoates in AOMKs 13b, d, and g have similar pKa values (3.21,
3.43, and 3.44, respectively), but 13b and g are far less potent than
13d in affecting the activity of Rce1p and Ste24p. This finding is
inconsistent with a cysteine protease mechanism, because a better
leaving group would be expected to better inhibit the enzyme,44
assuming that the AOMKs undergo rapid and reversible binding
and have similar Km values. The kinetic data previously reported
for 8d43 suggest that this is a reasonable assumption. The observa-
tion that the bromide 8h, which possesses an excellent leaving
group, is not much better at inhibiting Rce1p than other AOMKs
also does not support mechanism-based inhibition. Furthermore,
if the dipeptidyl AOMKs were acting as mechanism-based inhibi-
5.2. Synthesis of dipeptidyl (acyloxy)methyl ketones
Compounds 1b, 2, 3, 4a–g, 7a, 7d–f, 8a, 8d–f,44 5, 6,49 9,50 and
13b45 are known in the literature. N-Hydroxysuccinimide ester
1b is also available commercially. All of the dipeptidyl (acyl-
oxy)methyl ketones were synthesized by the method described
by Krantz et al.44 except compounds 13b, 14, and 15, which were
obtained from Matthew Bogyo.45 All compounds were purified by
HPLC prior to the protease assay, then stored at ꢁ20 °C in neat
form or at ꢁ80 °C as a stock solution in DMSO. HPLC analysis of
several dipeptidyl AOMKs (8c and g and 13a, c, d, and f) did not re-
veal decomposition of the compounds during prolonged storage
(>6 months) under these conditions. 1H NMR analysis of 8h after
prolonged storage (>3 years) as a solid at ꢁ20 °C did not indicate
that it had degraded.
tors, then dipeptide 17, where the
a-acyloxy group has been re-
placed with an amide, should not inhibit the proteases. Dipeptide
17 is characterized as ‘warhead-free’ because it lacks a benzoate
leaving group, which renders it incapable of covalently attaching
to a putative sulfur nucleophile in the active site. As an alternative,
5.2.1. Z-Phe-Lys(Boc)–CH2–O-(2,6-dimethyl)benzoate (7b)
2,6-Dimethylbenzoic acid (50.7 mg, 0.338 mmol) was added to
a solution of bromide 6 (100.5 mg, 0.166 mmol) in dry DMF

Anne-Marie R. Dechert et al. / Bioorg. Med. Chem. 18 (2010) 6230–6237
6235
(3 mL) and KF (70.1 mg, 1.21 mmol). After the reaction was stirred
at room temperature overnight, it was diluted with ether (20 mL)
and washed with water (3 ꢂ 5 mL). With each wash, EtOAc was
added to help separate the layers. The organic portion was washed
with saturated NaHCO3 (2 ꢂ 5 mL) and brine (2 ꢂ 5 mL), dried over
MgSO4, and concentrated under vacuum to a white solid (98.0 mg).
The product was purified by recrystallization from EtOAc and hex-
anes to give a white powder (7b, 39.1 mg, 0.0580 mmol, 35%), mp
160–161 °C: 1H NMR (400 MHz, CDCl3) d 7.40–7.15 (m, 11H), 7.05
(d, J = 7.8 Hz, 2H), 6.48 (m, 1H), 5.35 (m, 1H), 5.09 (s, 2H), 4.92 (d,
J = 17 Hz, 1H), 4.79 (d, J = 16.6 Hz, 1H), 4.68 (m, 1H), 4.62 (m, 1H),
4.45 (m, 1H), 3.08 (m, 4H), 2.39 (s, 6H), 1.91 (m, 1H), 1.63 (m, 1H),
1.58 (m, 2H), 1.56 (s, 9H), 1.26 (m, 2H); MS (ESI) m/z calcd for
(C38H47N3O8) 673.3, found 674.3 [M+H]+, 696.3 [M+Na]+; HRMS
(ESI) m/z calcd for [M+H]+ 674.3441, found 674.3451.
J = 13.7, 8.3 Hz, 1H), 2.86 (t, J = 7.3 Hz, 2H), 1.87 (m, 1H), 1.60 (m,
3H), 1.36 (m, 2H); MS (ESI) m/z calcd for (C24H30BrN3O4) 503.1
(
79Br), 505.1 (81Br), found 504, 506 [M+H]+; HRMS (ESI) m/z calcd
for [M+H]+ 504.1498 (79Br), 506.1477 (81Br), found 504.1519,
506.1497.
5.2.8. Z-Phe-Arg(Pbf)-OH (10)
L-Arginine(Pbf) (250 mg, 0.586 mmol) was dissolved in a mix-
ture of dioxane (10 mL) and water (10 mL). Et3N (0.163 mL) was
added followed by solid carbon dioxide pieces until pH 8 was
achieved. Z-L-Phe-N-hydroxysuccinimide ester (1b, 230 mg,
0.586 mmol) was added and the solution was stirred for 20 h.
The reaction was quenched with 1 N HCl (50 mL) and the product
was extracted with EtOAc. The EtOAc extracts were washed suc-
cessively with 1 N HCl (50 mL), water (50 mL), and brine (25 mL);
dried over MgSO4; and concentrated under vacuum. The crude
product was reprecipitated from EtOAc and hexanes to produce
Z-Phe-Arg(Pbf)-OH (10) as a white solid (230 mg, 0.325 mmol,
55%), mp 95–100 °C: 1H NMR (400 MHz, CDCl3) d 7.60 (br, 1H),
7.35–7.10 (m, 10H), 5.90 (m, 1H), 5.10–4.80 (m, 3H), 4.65–4.38
(m, 3H), 3.11 (m, 3H), 2.95 (m, 1H), 2.90 (s, 2H), 2.52 (d,
J = 6.8 Hz, 3H), 2.45 (d, J = 5.9 Hz, 3H), 2.05 (s, 3H), 1.95–1.20 (m,
6H), 1.43 (s, 6H); MS (MALDI) m/z calcd for (C36H45N5O8S) 707.3,
found 708.3 [M+H]+, 730.3 [M+Na]+; HRMS (ESI) m/z calcd for
[M+H]+ 708.3067, found 708.3046.
5.2.2. Z-Phe-Lys(Boc)–CH2–O-(4-methyl)benzoate (7c)
Mp 149–151 °C; 1H NMR (400 MHz, CDCl3) d 7.97 (d, J = 8.3 Hz,
2H) 7.40–7.18 (m, 12H), 6.52 (m, 1H), 5.35 (m, 1H), 5.08 (s, 2H),
4.92 (d, J = 17.1 Hz, 1H), 4.81 (d, J = 17.1 Hz, 1H), 4.79 (m, 1H),
4.67 (m, 1H), 4.45 (m, 1H), 3.08 (m, 4H), 2.43 (s, 3H), 1.88 (m,
1H), 1.60, (m, 1H), 1.46 (m, 2H), 1.43 (s, 9H), 1.24 (m, 2H); MS
(ESI) m/z calcd for (C37H45N3O8) 659.3, found 660.3 [M+H]+, 682.3
[M+Na]+; HRMS (ESI) m/z calcd for [M+H]+ 660.3285, found
660.3322.
5.2.3. Z-Phe-Lys(Boc)–CH2–O-(4-nitro)benzoate (7g)
5.2.9. Z-Phe-Arg(Pbf)-CH2-Cl (11a)
Mp 159–160 °C; 1H NMR (400 MHz, CDCl3) d 8.33 (d, J = 8.8 Hz,
2H), 8.25 (d, J = 8.8 Hz, 2H) 7.40–7.18 (m, 10H), 6.48 (m, 1H), 5.32
(m, 1H), 5.09 (s, 2H), 5.00 (d, J = 17.1 Hz, 1H), 4.91 (d, J = 16.6 Hz,
1H), 4.72 (m, 1H), 4.60 (m, 1H), 4.46 (m, 1H), 3.10 (m, 4H), 1.88
(m, 1H), 1.60, (m, 1H), 1.48 (m, 2H), 1.43 (s, 9H), 1.25 (m, 2H);
MS (ESI) m/z calcd for (C36H42N4O10) 690.3, found 691.3 [M+H]+;
HRMS (ESI) m/z calcd for [M+H]+ 691.2979, found 691.2998.
Under
a
nitrogen atmosphere, isobutyl chloroformate
(0.109 mL, 0.830 mmol) was added dropwise to a solution of
Z-Phe-Arg(Pbf)-OH (10, 509.6 mg, 0.722 mmol) and N-methylmor-
pholine (0.099 mL, 0.902 mmol) in anhydrous THF (5 mL) at
ꢁ20 °C. The solution was allowed to stir for 15 min. Diazomethane
in ether (prepared from DiazaldÒ) was added to the reaction flask
dropwise. The solution was allowed to warm to room temperature
over 3 h, then cooled to 0 °C with an ice bath. A 1:1 mixture of HCl
and acetic acid (5 mL) was added dropwise. Once the evolution of
N2 subsided, the reaction was diluted with EtOAc and washed with
water (2 ꢂ 25 mL), brine (2 ꢂ 25 mL), and saturated NaHCO3
(2 ꢂ 25 mL). The solvent was evaporated to yield a yellow solid,
which upon purification by flash chromatography with 80% EtOAc
in hexanes provided Z-Phe-Arg(Pbf)-CH2-Cl (11a, 117.7 mg,
0.1590 mmol, 22%): 1H NMR (400 MHz, CDCl3) d 7.76 (m, 1H),
7.27–7.13 (m, 10H), 6.36 (s, 2H), 6.10 (br, 1H), 5.93 (m, 1H), 4.93
(m, 1H), 4.94 (d, J = 12.2 Hz, 1H), 4.86 (d, J = 12.2 Hz, 1H), 4.58
(m, 1H), 4.51 (m, 1H), 4.09 (m, 1H), 4.00 (m, 1H), 3.08 (m, 2H),
2.95 (m, 1H), 2.91 (s, 2H), 2.55 (s, 3H), 2.47 (s, 3H), 2.07 (s, 3H),
1.81 (m, 2H), 1.61, (m, 2H), 1.44 (s, 6H); MS (ESI) m/z calcd for
(C37H46ClN5O7S) 739.3, found 740.2 [M+H]+, 762.2 [M+Na]+, 778.2
[M+K]+; HRMS (ESI) m/z calcd for [M+H]+ 740.2884 (35Cl) and
742.2854 (37Cl), found 740.2907, 742.2895.
5.2.4. Z-Phe-Lys–CH2–O-(2,6-dimethyl)benzoate (8b)
TFA (1 mL) was added dropwise over 5 min to a solution of 7b
(53.0 mg, 0.0787 mmol) in CH2Cl2 (3 mL) at 0 °C. The solution
was warmed to room temperature over 2.5 h. The solvent was
evaporated, and the resulting sticky residue was reprecipitated
with EtOAc and ether, filtered, and dried under vacuum to give a
brownish solid, which was purified by HPLC, using a gradient of
25% CH3CN in water/TFA (0.1%) to 100% CH3CN, to provide a white
solid (8b, 24.1 mg, 0.0420 mmol, 53%), mp 136–139 °C: MS (ESI)
m/z calcd for (C33H39N3O6) 573.3, found 574.2 [M+H]+; HRMS
(ESI) m/z calcd for [M+H]+ 574.2917, found 574.2909.
5.2.5. Z-Phe-Lys–CH2–O-(4-methyl)benzoate (8c)
Mp 112–114 °C; MS (ESI) m/z calcd for (C32H37N3O6) 559.3,
found 560.3 [M+H]+; HRMS (ESI) m/z calcd for [M+H]+ 560.2760,
found 560.2781.
5.2.10. Z-Phe-Arg(Pbf)–CH2–O-benzoate (12a)
5.2.6. Z-Phe-Lys–CH2–O-(4-nitro)benzoate (8g)
Mp 134–145 °C; MS (ESI) m/z calcd for (C31H34N4O8) 590.2,
found 591.2 [M+H]+; HRMS (ESI) m/z calcd for [M+H]+ 591.2455,
found 591.2449.
Under a nitrogen atmosphere, Z-Phe-Arg(Pbf)-CH2-Cl (11a,
50 mg, 0.068 mmol) was dissolved in dry DMF (2 mL). Benzoic acid
(25 mg, 0.20 mmol) and KF (40 mg, 0.69 mmol) were added, and
the reaction was stirred at room temperature overnight. The reac-
tion was diluted with EtOAc, then successively washed with water
(5 mL), saturated NaHCO3 (5 mL), and brine (5 mL). The solvent
was dried over MgSO4 and evaporated to yield a residue, which
upon purification by flash chromatography with 70% EtOAc in hex-
anes provided Z-Phe-Arg(Pbf)–CH2–O-benzoate (12a, 24.8 mg,
0.0300 mmol, 44%.), mp 94–96 °C: 1H NMR (400 MHz, CDCl3) d
8.02 (m, 2H), 7.72 (br, 1H), 7.55 (m, 1H), 7.41 (m, 2H), 7.28–7.13
(m, 10H), 6.31 (br, 2H), 6.10 (br, 1H), 5.86 (br, 1H), 4.92 (m, 4H),
4.56 (m, 2H), 3.12 (m, 3H), 2.95 (m, 1H), 2.91 (s, 2H), 2.57 (s,
3H), 2.50 (s, 3H), 2.07 (s, 3H), 1.89 (m, 2H), 1.70 (m, 2H), 1.44 (s,
5.2.7. Z-Phe-Lys-CH2-Br (8h)
TFA (1.5 mL) was added dropwise over 5 min to a solution of 6
(50 mg, 0.083 mmol) in CH2Cl2 at 0 °C. The solution was warmed to
room temperature over 4 h. The solvent was evaporated, and the
resulting sticky residue was purified by HPLC with a gradient of
20% CH3CN in water/TFA (0.1%) to 100% CH3CN, providing a white
solid (8h, 22.7 mg, 0.0450 mmol, 54%), mp 141–143 °C: 1H NMR
(400 MHz, CD3OD) d 7.28 (m, 10H), 5.05 (s, 2H), 4.53 (m, 1H),
4.38 (m, 1H), 3.9 (s, 2H), 3.07 (dd, J = 13.7, 6.8 Hz,1H), 2.94 (dd,

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Anne-Marie R. Dechert et al. / Bioorg. Med. Chem. 18 (2010) 6230–6237
6H); MS (ESI) m/z calcd for (C44H51N5O9S) 825.3, found 826.2
[M+H]+, 848.4 [M+Na]+; HRMS (ESI) m/z calcd for [M+H]+
740.2884, found 740.2907.
remaining residue purified by HPLC, using 40% CH3CN in water/
TFA (0.1%) as the eluant, to provide Z-Phe-Arg–CH2–O-benzoate
(13a, 5.6 mg, 0.00975 mmol, 33%), mp 104–106 °C: MS (ESI)
m/z calcd for (C31H35N5O6) 573.3, found 574.3 [M+H]+; HRMS
(ESI) m/z calcd for [M+H]+ 574.2665, found 574.2648.
5.2.11. Z-Phe-Arg(Pbf)–CH2–O-(2,6-dimethyl)benzoate (12b)
Mp 99–102 °C; 1H NMR (400 MHz, CDCl3) d 7.35–7.10 (m, 11H),
7.04 (d, J = 7.3 Hz, 2H), 6.08 (br, 2 H), 5.55 (br, 1H), 4.98 (s, 2H), 4.80
(m, 2H), 4.61 (m, 1H), 4.51 (m, 1H), 3.11 (m, 3H), 3.03 (m, 1H), 2.92
(s, 2H), 2.57 (s, 3H), 2.51 (s, 3H), 2.38 (s, 6H), 2.07 (s, 3H), 1.96 (m,
2H), 1.68 (m, 2H), 1.44 (s, 6H); MS (MALDI) m/z calcd for
(C46H55N5O9S) 853.37, found 854.44 [M+H]+, 876.51 [M+Na]+;
HRMS (ESI) m/z calcd for [M+H]+ 854.3798, found 854.3775.
5.2.18. Z-Phe-Arg–CH2–O-(4-methyl)benzoate (13c)
MS (MALDI) m/z calcd for (C32H37N5O6) 587.3, found 588.2
[M+H]+; HRMS (ESI) m/z calcd for [M+H]+ 588.2822, found
588.2806.
5.2.19. Z-Phe-Arg–CH2–O-(2,4,6-trimethyl)benzoate (13d)
MS (MALDI) m/z calcd for (C34H41N5O6) 615.3, found 616.2
[M+H]+; HRMS (ESI) m/z calcd for [M+H]+ 616.3135, found
616.3161.
5.2.12. Z-Phe-Arg(Pbf)–CH2–O-(4-methyl)benzoate (12c)
Mp 88–91 °C; 1H NMR (400 MHz, CDCl3) d 7.92 (t, J = 8 Hz, 2H),
7.30–7.18 (m, 12H), 6.08 (br, 2H), 5.58 (br, 0.67H), 5.40 (br, 0.33H),
5.00 (d, J = 16.6 Hz, 2H), 4.98 (m, 1H), 4.83 (m, 1H), 4.62 (m, 1H),
4.45 (m, 1H), 3.11 (m, 3H), 3.00 (m, 1H), 2.93 (s, 2H), 2.58 (s,
3H), 2.51 (s, 3H), 2.41 (s, 3H), 2.08 (s, 3H), 1.90 (m, 2H), 1.67 (m,
2H), 1.45 (s, 6H); MS (ESI) m/z calcd for (C45H53N5O9S) 839.4, found
840.4 [M+H]+, 862.30 [M+Na]+; HRMS (ESI) m/z calcd for [M+H]+
840.3642, found 840.3637.
5.2.20. Z-Phe-Arg–CH2–O-(bis-2,6-trifluoromethyl)benzoate
(13e)
MS (ESI) m/z calcd for (C33H33F6N5O6) 709.2, found 710.0
[M+H]+; HRMS (ESI) m/z calcd for [M+H]+ 710.2413, found
710.2395.
5.2.21. Z-Phe-Arg–CH2–O-(4-methoxy)benzoate (13f)
MS (MALDI) m/z calcd for (C32H37N5O7) 603.3, found 604.3
[M+H]+; HRMS (ESI) m/z calcd for [M+H]+ 604.2771, found
604.2768.
5.2.13. Z-Phe-Arg(Pbf)–CH2–O-(2,4,6-trimethyl)benzoate (12d)
Mp 82–84 °C; 1H NMR (400 MHz, CDCl3) d 7.28–7.08 (m, 10H),
6.86 (s, 2H), 6.13 (br, 2H), 5.60 (br, 0.67H), 5.46 (br, 0.33H), 4.93
(m, 3H), 4.78 (m, 1H), 4.52 (m, 2H), 3.11 (m, 2H), 3.01 (m, 1H),
2.92 (s, 2H), 2.57 (s, 3H), 2.50 (s, 3H), 2.35 (s, 6H), 2.29 (s, 3H),
2.07 (s, 3H), 1.96 (m, 2H), 2.63 (m, 2H), 1.45 (s, 6H); MS (ESI) m/z
calcd for (C47H57N5O9S) 867.4, found 868.2 [M+H]+, 890.4
[M+Na]+; HRMS (ESI) m/z calcd for [M+H]+ 868.3955, found
868.3994.
5.2.22. Z-Phe-Arg–CH2–O-(4-nitro)benzoate (13g)
Mp 101–103 °C; MS (ESI) m/z calcd for (C31H34N6O8) 618.2,
found 619.0 [M+H]+; HRMS (ESI) m/z calcd for [M+H]+ 619.2516,
found 619.2499.
5.2.23. Z-Phe-Arg(Pbf)-(b-phenethylamine) (16)
5.2.14. Z-Phe-Arg(Pbf)–CH2–O-(bis-2,6-
trifluoromethyl)benzoate (12e)
Z-Phe-Arg(Pbf)-OH (10, 90.2 mg, 0.127 mmol) was dissolved in
CH2Cl2 (4.5 mL) and cooled to 0 °C. HOBt (19.5 mg, 0.147 mmol)
was added and the solution was stirred for 10 min at 0 °C. HBTU
(48.3 mg, 0.127 mmol) and DIPEA (0.024 mL, 0.127 mmol) were
added sequentially, and the solution was allowed to warm to room
temperature over 0.5 h. b-Phenethylamine (0.018 mL, 0.127 mmol)
was added and the solution was stirred overnight. The reaction
was diluted with CH2Cl2 (50 mL) and washed successively with
water (25 mL) and brine (25 mL). The solvent was dried over
Na2SO3 and evaporated to yield a residue, which upon purification
by flash chromatography with 1:1 THF/CHCl3 provided Z-Phe-
Arg(Pbf)-(b-phenethylamine) (16, 40.2 mg, 0.0496 mmol, 39%):
1H NMR (400 MHz, CDCl3) d 7.40–7.10 (m, 15H), 6.18 (br, 2H),
5.50 (br, 0.67H), 5.44 (br, 0.33H) 4.99 (m, 2H), 4.38 (m, 2H), 3.44
(m, 3H), 3.17 (m, 3H), 2.94 (s, 2H), 2.78 (m, 2H), 2.59 (s, 3H),
2.51 (s, 3H), 2.09 (s, 3H), 1.93 (m, 2H), 1.67 (m, 2H), 1.45 (s, 6H);
MS (ESI) m/z calcd for (C44H54N6O7S) 810.4, found 811.4 [M+H]+;
HRMS (ESI) m/z calcd for [M+H]+ 811.3853, found 811.3858.
1H NMR (400 MHz, CDCl3) d 7.94 (d, J = 8.3 Hz, 2H), 7.74 (t,
J = 7.8 Hz, 1H), 7.44 (br, 1H), 7.17 (m, 10H), 6.17 (br, 2H), 6.00
(br, 1H), 5.63 (d, J = 6.8 Hz, 1H), 4.94 (m, 4H), 4.62 (m, 1H), 4.55
(m, 1H), 3.12 (m, 3H), 2.97 (m, 1H), 2.92 (s, 2H), 2.55 (s, 3H),
2.49 (s, 3H), 2.07 (s, 3H), 1.90 (m, 2H), 1.63 (m, 2H), 1.44 (s, 6H);
MS (ESI) m/z calcd for (C46H49F6N5O9S) 961.3, found 962.4
[M+H]+, 984.2 [M+Na]+; HRMS (ESI) m/z calcd for [M+H]+
962.3233, found 962.3281.
5.2.15. Z-Phe-Arg(Pbf)–CH2–O-(4-methoxy)benzoate (12f)
Mp 83–85 °C; 1HNMR(400 MHz, CDCl3) d 8.00 (m, 2H), 7.30–7.15
(m, 10H), 6.91(m, 2H), 6.11 (br, 2H), 5.60(br, 0.67H), 5.43(br, 0.33H),
4.99 (m, 3H), 4.83 (m, 1H), 4.64 (m, 1H), 4.48 (m, 1H), 3.86 (s, 3H),
3.11 (m, 3H), 2.98 (m, 1H), 2.94 (s, 2H), 2.58 (s, 3H), 2.52 (s, 3H),
2.09(s, 3H), 1.93(m, 2H), 1.67(m, 2H), 1.45(s, 6H);MS(ESI) m/zcalcd
for (C45H53N5O10S) 855.4, found 856.4 [M+H]+; HRMS (ESI) m/z calcd
for [M+H]+ 856.3591, found 856.3574.
5.2.24. Z-Phe-Arg-(b-phenethylamine) (17)
5.2.16. Z-Phe-Arg(Pbf)–CH2–O-(4-nitro)benzoate (12g)
A solution of Z-Phe-Arg(Pbf)-(b-phenethylamine) (16, 24 mg,
0.029 mmol) in 95:5 TFA/water (10 mL) was stirred for 2 h at
0 °C. The solvent was evaporated and the remaining residue was
purified by HPLC using a 20-min linear eluant gradient from 30%
acetonitrile CH3CN in water/TFA (0.1%) to 100% CH3CN to provide
Z-Phe-Arg-(b-phenethylamine) (17, 5.4 mg, 0.0097 mmol, 33%) as
Mp 108–111 °C; 1H NMR (400 MHz, CDCl3) d 8.22 (m, 4H), 7.45
(br, 1H), 7.30–7.14 (m, 10H), 6.18 (br, 2H), 5.63 (br, 0.67H), 5.48
(br, 0.33H), 4.99 (m, 4H), 4.55 (m, 2H), 3.12 (m, 2H), 3.05 (m,
1H), 2.93 (s, 2H), 2.58 (s, 3H), 2.51 (s, 3H), 2.08 (s, 3H), 1.93 (m,
2H), 1.67 (m, 2H), 1.45 (s, 6H); MS (ESI) m/z calcd for
(C44H50N6O11S) 870.3, found 871.4 [M+H]+, 893.2 [M+Na]+; HRMS
(ESI) m/z calcd for [M+H]+ 871.3336, found 871.3368.
a
clear yellow film: 1H NMR (400 MHz, CD3OD)
d 8.30 (d,
J = 7.8 Hz, 1H), 8.13 (d, J = 7.8, 1H), 7.99 (t, J = 4.9, 1H), 7.70 (br,
1H), 7.25 (m, 15H), 5.05 (m, 2H), 4.35 (m, 1H) 4.26 (m, 1H), 4.08
(br, 1H), 3.37 (m, 2H), 3.27 (m, 1H), 3.07 (m, 2H), 2.92 (m, 3H),
2.76 (m, 2H), 1.76 (m, 2H), 1.60 (m, 2H); MS (ESI) m/z calcd for
(C31H39N6O4) 558.3, found 559.3 [M+H]+; HRMS (ESI) m/z calcd
for [M+H]+ 559.3033, found 559.3021.
5.2.17. Z-Phe-Arg–CH2–O-benzoate (13a)
Z-Phe-Arg(Pbf)–CH2–O-benzoate (12a, 24.4 mg, 0.295 mmol)
was stirred at 0 °C in 95:5 TFA/water (10 mL) until TLC indicated
the reaction complete. The solvent was evaporated and the

Anne-Marie R. Dechert et al. / Bioorg. Med. Chem. 18 (2010) 6230–6237
6237
3. Konstantinopoulos, P. A.; Karamouzis, M. V.; Papavassiliou, A. G. Nat. Rev. Drug
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::TRP1).6,51,52 Plasmid-bearing
versions of the strain were generated according to published meth-
ods.53 Strains were routinely grown at 30 °C using synthetic com-
plete dropout (SC–ura) medium.54 The over-expression plasmids
encoding yeast Rce1p (pWS479) and yeast Ste24p (pSM1282) have
been reported.19,20,42,55
9. Backlund, P. S., Jr. J. Biol. Chem. 1997, 272, 33175.
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5.4. In vitro fluorescence-based CaaX proteolysis assay
An established fluorescence-based assay was used to monitor
CaaX protease activity in the presence of candidate inhibi-
tors.42,43,48 In brief, the assay involved the mixing of a quenched
fluorogenic substrate with yeast membranes enriched for the
appropriate CaaX protease. Membranes used as the source of activ-
ity were isolated according to our reported methods20,22 and stored
at ꢁ80 °C as 1 mg/mL stocks in lysis buffer (50 mM Tris, pH 7.5,
0.2 M sorbitol, 1 mM EDTA, 0.2% NaN3) containing a protease
inhibitor cocktail (chymostatin, leupeptin, pepstatin, aprotinin,
and phenylmethylsulphonyl fluoride) that does not inhibit either
of the CaaX proteases. The membranes were typically diluted with
assay buffer (100 mM HEPES, pH 7.5, 5 mM MgCl2) to 0.5 mg/mL
14. Otto, J. C.; Kim, E.; Young, S. G.; Casey, P. J. J. Biol. Chem. 1999, 274, 8379.
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immediately before use. The substrate was diluted to 40
a 1 mM stock prepared in 4% DMSO. Assay assembly involved the
dispensing of 50- L aliquots of diluted membranes into the wells
lM from
l
of a black, clear-bottom, 96-well microplate. This was followed
by the addition of each compound to duplicate wells and a 15-
min pretreatment incubation at 30 °C. After pretreatment, the as-
say was initiated by adding 50
in final concentrations of 0.25 mg/mL membrane, 20
and 100 M inhibitor. The sample fluorescence was measured
lL of diluted substrate, resulting
lM substrate,
l
every 30–60 s over a 60-min time course at 30 °C using a BioTek
Synergy™ HT microplate fluorometer equipped with a 320/420-
nm excitation/emission filter set. The collected data were exported
to Microsoft Excel and graphed as a function of fluorescence versus
time, and the initial velocities determined. These values were used
to calculate the percentage activities relative to a DMSO-treated
sample, which was included as a control in each reaction set.
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Acknowledgments
We are grateful to Dr. Margot Paulick for technical advice and
critical discussions and to Dr. Matthew Bogyo for donations of sev-
eral AOMKs. This work was supported in part through a Georgia
Cancer Coalition Distinguished Cancer Clinician/Scientist Scholar
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Award and National Institutes of Health
Grant (GM067092)
(W.K.S.), an NSF CAREER Award (T.M.D.), a University of Georgia
(UGA) BHSI Summer Research Fellowship (S.R.B.), a UGA SURO
Summer Research Fellowship (A.C.R.), and a UGA CURO Summer
Research Fellowships (N.W.H. and J.P.M.).
Supplementary data
45. Kato, D.; Boatright, K. M.; Berger, A. B.; Nazif, T.; Blum, G.; Ryan, C.; Chehade, K.
A. H.; Salvesen, G. S.; Bogyo, M. Nat. Chem. Biol. 2005, 1, 33.
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Supplementary data (1H NMR spectra and HPLC chromatograms
for new compounds) associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2010.07.041.
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