New Acyloxymethyl Ketones: Useful Probes for Cysteine Protease Profiling

Article abstract of DOI:10.1055/s-0035-1562781

Peptidyl-acyloxymethyl ketones (AOMKs) belong to a class of selective, irreversible inhibitors (activity-based probes) widely used as chemical tools of investigating proteins, for example, in activity-based protein profiling. The synthesis of the AOMKs has always been challenging and current methodologies involve both solution and solid-phase synthesis. Herein, the synthesis of a new scaffold useful for the preparation of peptidyl-AOMKs is reported and it is demonstrated that the new synthetic probes bearing a 4-functionalized 2,6-dimethylbenzoate efficiently inhibit cysteine proteases like cathepsin B.

Full text of DOI:10.1055/s-0035-1562781

SYNTHESIS
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9
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© Georg Thieme Verlag Stuttgart · New York
2016, 48, A–G
paper
A
A. G. Coman et al.
Paper
Synthesis
New Acyloxymethyl Ketones: Useful Probes for Cysteine Protease
Profiling
Anca G. Comana §
Peptidyl-AOMKs
Codruta C. Paraschivescua §
Niculina D. Hadadeb
Andrei Juncuc
Ovidiu Vlaicuc
Costin-Ioan Popescuc
Mihaela Matache*a
O
CO2H
O
H
N
O
N
H
O
O
4-carboxymethoxy-
2,6-dimethylbenzoate scaffold
peptidyl chain
a University of Bucharest, Faculty of Chemistry, Department of Organic Chemistry,
Biochemistry and Catalysis, 90 Panduri Street, 050663 Bucharest, Romania
mihaela.matache@g.unibuc.ro
IC50 = 0.7 μM against cathepsin B
b Faculty of Chemistry and Chemical Engineering, ‘Babeș-Bolyai’ University, 11 Arany
Janos Str., 400028 Cluj-Napoca, Romania
c Institute of Biochemistry of the Romanian Academy, 296 Splaiul Independetei
Avenue, 060031 Bucharest, Romania
§ These authors contributed equally to this work
Received: 26.04.2016
Accepted after revision: 18.06.2016
Published online: 29.07.2016
studies established the selectivity of the dipeptidyl-AOMKs
for cathepsin B as well as dependency of the inhibition rates
on the pKa of the carboxylate leaving group. At first, the
synthesis of the peptidyl-AOMKs was accomplished in solu-
tion for short sequences of only two amino acids, by trans-
formation of the carboxyl group into the corresponding di-
azomethyl ketone, halomethyl ketone and, finally, the acyl-
oxymethyl ketone.4a Demand for longer and more complex
peptide sequences led to development of solid-phase strat-
egies (SPPS). First method was reported by Mujica and
Jung,5 who developed the synthesis of the aspartyl acyl-
oxymethyl ketone, on carboxy-functionalized resins. This
strategy is very convenient for amino acids bearing side-
chain functional groups. Later, Ellman et al.6 developed a
solid-phase methodology, which uses the ketone carbonyl
group of the halomethyl ketone for direct attachment,
through a hydrazone bond, onto a carbazate-linked solid
support and further growth of the peptide sequence. An-
other strategy that proved to be very useful for synthesizing
longer peptidyl-AOMKs was developed by Bogyo et al.3j
namely synthesis of the C-terminal target peptide by SPPS
and subsequent solution transformation into the peptidyl-
diazomethyl ketone, bromomethyl ketone and peptidyl-
AOMK, respectively. Thus, to the best of our knowledge, a
general synthetic method for peptidyl-AOMKs is not avail-
able. Fmoc-solid-phase synthesis of the peptidyl-AOMKs
has always been challenging, on one hand because the ben-
zoate moiety lacks a functional group for the attachment to
the solid support and, on the other hand, as a result of the
susceptibility to hydrolysis of the ester moiety to the basic
conditions required for Fmoc deprotection. However, this
latter difficulty has been solved by using diethylamine as
deprotecting reagent.3k
DOI: 10.1055/s-0035-1562781; Art ID: ss-2016-t0294-op
Abstract Peptidyl-acyloxymethyl ketones (AOMKs) belong to a class of
selective, irreversible inhibitors (activity-based probes) widely used as
chemical tools of investigating proteins, for example, in activity-based
protein profiling. The synthesis of the AOMKs has always been challeng-
ing and current methodologies involve both solution and solid-phase
synthesis. Herein, the synthesis of a new scaffold useful for the prepara-
tion of peptidyl-AOMKs is reported and it is demonstrated that the new
synthetic probes bearing a 4-functionalized 2,6-dimethylbenzoate effi-
ciently inhibit cysteine proteases like cathepsin B.
Key words acyloxymethyl ketones, activity-based probes, peptidyl-
AOMKs, 2,6-dimethylbenzoates, solid-phase peptide synthesis
Activity-based protein profiling (ABPP)1 is a powerful
tool for protein investigation that makes use of synthetic
small molecules (ABPs: activity-based probes) able to spe-
cifically and covalently bind target proteins in a complex bi-
ological mixture. The reactive group of an ABP is usually an
electrophilic moiety that is susceptible to the attack of ami-
no acids nucleophilic groups within the protein active site.2
The acyloxymethyl ketones (AOMKs), in particular pep-
tidyl-AOMKs, represent a class of compounds that have
found great applicability in ABPP, especially due to their se-
lectivity for cysteine proteases versus serine proteases.2,3
The inactivation mechanism of the cysteine-proteases by
peptidyl-AOMK follow a nucleophilic substitution (the thiol
group of the active site cysteine attacks the electrophilic
carbon of the ketone yielding a S–C covalent bond2c).
Dipetidyl-AOMKs were first developed by the group of
Krantz4 as an alternative to the aldehyde- or the halomethyl
ketone-based inhibitors for cathepsin B and calpains. These
© Georg Thieme Verlag Stuttgart · New York — Synthesis 2016, 48, A–G

B
A. G. Coman et al.
Paper
Synthesis
I
I
CHO
CHO
OH
CHO
OR
9a: R = CH2COOtBu
9b: R = Bn
a)
b)
e)
f)
g)
OH
OH
OTBS
OTBS
2
3
4
7
8
h)
c)
CN
CN
COOH
d)
OTBS
OH
OR
5
6
1: R = CH2COOtBu
10: R = CH2COOH
i)
j)
11: R = Bn
12: R = H
Scheme 1 Synthesis of compound 1. Reagents and conditions: a) KI, KIO3, HCl, MeOH, 0 °C, 30 min, r.t., 20 h, 60%; b) tert-butyldimethylchlorosilane
(TBSCl, 1.1 equiv), imidazole (2.2 equiv), DMF, 0 °C, argon, 30 min, 96%; c) CuCN (1.12 equiv), DMF, reflux, 2 h, 85%; d) i) NaOH (25 equiv), H2O/EtOH,
1 h, reflux, 80% or ii) HCl, 3 h, reflux, 84%; e) n-BuLi (1.4 equiv), THF, –78 °C, DMF (2 equiv), argon, 3 h, 72%; f) KF (1.5 equiv), MeCN/H2O, 88%; g) tert-
butyl bromoacetate or benzyl bromide (1.1 equiv), KI (1.1 equiv), K2CO3 (1.1 equiv), acetone, r.t., overnight, 70% for 9a and 42% for 9b; h) KMnO4 (1.5
equiv), 1,4-dioxane/H2O, r.t., 4 h, 47% for 1 and 41% for 11; i) TFA 25% in CH2Cl2, quant; j) HBr (33 wt% in AcOH), 40%.
In this context, we report herein the synthesis of 4-sub-
stituted 2,6-dimethylbenzoic acids as new scaffolds useful
for the preparation of peptidyl-AOMKs and their assays as
inhibitors of cysteine proteases. These probes can be syn-
thesized using both solution and SPSS strategies since the
attachment to the solid support was designed to occur
through derivatization in position 4 of the benzoate moiety.
Enzyme inhibition experiments using cathepsin B7 were
performed in order to investigate whether this modification
of the benzoate moiety influence the interaction with the
active site of the protein.
Our design for a new AOMK scaffold that derives from 4-
hydroxy-2,6-dimethylbenzoic acid was based on the hy-
pothesis that modification of the 2,6-disubstituted benzo-
ate moiety will not affect the inhibition efficiency.4a There-
fore, we designed compound 1 (Scheme 1) to contain a tert-
butyl protected carboxyl group which, after grafting on any
amino acid and deprotection, enables covalent attachment
on the solid-support and the subsequent possibility to grow
any peptide sequence.
Synthesis of compound 1 was achieved through a six-
step strategy, starting from 3,5-dimethylphenol (Scheme
1). Iodination8 of the phenol 2 led to the corresponding iodo
derivative 3 in 60% yield that was further treated9 with tert-
butyldimethylchlorosilane to provide the protected phenol
4 in 96% yield. Initially, we attempted to introduce the car-
boxyl group through an ipso substitution10 of the iodine
with the cyano group (compound 5, 85%) and further hy-
drolysis of the nitrile. All efforts to hydrolyze the nitrile
group failed either under basic or acidic conditions. In all
cases, the reaction occurred only with the deprotection of
the TBS group and the 2,6-dimethyl-4-hydroxybenzonitrile
(6) was recovered (see the Supporting Information).
Therefore, our attention was turned towards introduc-
tion of the formyl group, taking into account that TBS acts
as an efficient protecting group for phenol group in both
lithiation and formylation.9 Unlike previous reported proce-
dures, which starts from the corresponding bromo deriva-
tive, in the presence of s-BuLi or MeLi-LiBr complex, the
preparation of the aldehyde 8 was optimized to successfully
use n-BuLi. Thus, use of the readily available iodo derivative
4 affords employment of more convenient n-BuLi as lithia-
tion reagent (1.4 equiv) to enable formation of the product
7 in very good yields, without detection of any by-prod-
ucts.9 Deprotection of the TBS group provided the desired
aldehyde 8 in 88% yield.
In order to avoid selectivity issues, aldehyde 8 was first
reacted with tert-butyl bromoacetate or benzyl bromide,
leading to the esters 9, which were further oxidized to the
carboxylic derivatives 1 and 11. Screening through the well-
known oxidizing agents for efficient oxidation of the alde-
hydes to carboxylic acids indicated as the optimum choice
the ‘classical’ potassium permanganate (1.5 equiv) in a wa-
ter/1,4-dioxane mixture. This procedure provided the tar-
get compounds in 47% (for 1) and 41% (for 11) and complete
recovery of the unreacted aldehydes. No oxidation of the
methyl groups was detected when the reaction was per-
formed at room temperature for 4 hours.
The attempt to perform the oxidation reaction of 9a us-
ing Oxone11 as the oxidizing agent led to the formate ester
(see the Supporting Information), while the same reaction
© Georg Thieme Verlag Stuttgart · New York — Synthesis 2016, 48, A–G

C
A. G. Coman et al.
Paper
Synthesis
performed with silver(I) oxide resulted in the deprotection
of the tert-butyl ester. Diacid 10 was obtained by stirring
compound 1 with a solution of 25% TFA in dichloromethane
in 83% yield, while the benzyl group in compound 11 was
removed using HBr 33 wt% solution in acetic acid.
TFA to enable the formation of 19 in 83% yield. Compounds
16, 17, and 19 were tested for their capacity to inhibit
cathepsin B activity at high concentration (1 mM) indicat-
ing a similar inhibitory activity (data not shown).
Further, in order to improve the efficiency of the
cathepsin B inhibition, the peptidyl-AOMK probes 23, 24,
and 26 were synthesized (Scheme 3). Thus, the probes were
obtained in solution by reaction between 2,6-dimethylben-
zoic acid, 2,4,6-trimethylbenzoic acid, or compound 1, re-
spectively, and the peptidyl bromomethyl ketone 22. Pep-
tide 22 was prepared from the corresponding diazomethyl
ketone 21, which was, in its turn, obtained from peptide 20.
The N-acetylated peptide 20 was synthesized by classical
SPPS protocol, using Wang resin, in 90% yield (Scheme 3).
To confirm our hypothesis that the modification in posi-
tion 4 of the benzoate moiety does not have negative im-
pact on the inhibition efficiency, the IC50 values were de-
termined for the probes 20, 23, 24, and 26 against cathepsin
B. The biochemical assays were performed as described in
the experimental section by fluorescence measurements,
using the fluorogenic substrate Nα-CBZ-Arg-Arg-7-amido-
4-methylcoumarin. As expected, probe 20 had no inhibito-
ry activity at the maximum concentration tested (20 μM),
while the IC50 of probe 23 was found to be 7 μM (Table 1).
The inhibitory efficiency significantly improved for probes
24 and 26 down to 0.7 μM. Thus, we have demonstrated
that substitution in position 4 of the 2,4-dimethylbenzoate
scaffold with a carboxymethyloxy residue does not affect
the biological activity of the probe.
O
O
a)
N2
FmocHN
FmocHN
OH
b)
13
14
R
O
O
c)
FmocHN
FmocHN
Br
O
O
16: R = H 40%
15
17: R = Me 50%
18: R = OCH2COOtBu, 40%
19: R = OCH2COOH, 83%
d)
Scheme 2 Synthesis of compounds 16–19. Reagents and conditions: a)
i) isobutyl chloroformate, N-methylmorpholine, –10 °C, 30 min; ii)
CH2N2/Et2O (in situ), 0 °C, 3 h, 70%; b) HBr (33 wt% in AcOH), THF, 0 °C,
15 min, 88%; c) KF, MeCN, r.t., 12 h and i) for compound 16: 2,6-di-
methylbenzoic acid, 40%, ii) for compound 17: 2,4,6-trimethylbenzoic
acid, 50%, iii) for compound 18: acid 1, 40%; d) TFA (25% in CH2Cl2),
83%.
Next, Fmoc-Leu-OH (13) (Scheme 2) was chosen as
model amino acid to perform the transformation of the car-
boxyl group into the AOMK moieties through a previously
described three-step strategy.4a Preparation of the diazo-
methyl ketone 14 was followed by conversion into the bro-
mo derivative 15 and subsequent synthesis of the acyl-
oxymethyl ketones 16–18. Compound 18 was treated with
We recently reported12 a solid-phase strategy for the
synthesis of fluoromethyl ketones (FMKs) based on the
functionalization of the FMK amino acid, with a bifunction-
al linker, through a ketalization reaction of the ketone car-
bonyl that enables attachment to the solid support and sub-
O
O
O
HN
HN
O
HN
O
O
O
O
O
H
H
H
SPPS
a)
b)
N2
N
N
Br
N
OH
HO
N
N
N
H
H
H
Wang resin
O
O
O
c)
20
21
22
R
O
O
H
H
N
O
N
N
H
O
O
O
23: R = H
24: R = Me
25: R = OCH2COOtBu
26: R = OCH2COOH
d)
Scheme 3 Synthesis of acyloxymethyl ketones 23–26; yield of precursor 20 by SPPS: 90%. Reagents and conditions: a) i) isobutyl chloroformate,
N-methylmorpholine, –10 °C, 30 min; ii) CH2N2/Et2O (in situ), 0 °C, 3 h, 47%; b) HBr (33 wt% in AcOH), THF, 0 °C, 15 min, 30%; c) 2,6-dimethylbenzoic
acid or 2,4,6-trimethylbenzoic acid or compound 1, respectively, KF, MeCN, r.t., 12 h, 91% for 23, 85% for 24, and 50% for 25; d) TFA 25% in CH2Cl2, 57%.
© Georg Thieme Verlag Stuttgart · New York — Synthesis 2016, 48, A–G

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A. G. Coman et al.
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Synthesis
Table 1 Inhibition of Cathepsin B by AOMKs 20, 23, 24, 26
coated aluminum F254 plates. All plates were visualized by UV irradi-
ation at 254 nm. Preparative column chromatography was performed
on Merck silica gel 60 (0.040–0.063 mm).
Compound
IC50
>20 μM
For the preparation of aldehyde 8, see the Supporting Information.
20
23
24
26
7 μM
0.7 μM
0.7 μM
tert-Butyl 2-(4-Formyl-3,5-dimethylphenoxy)acetate (9a)
Compound 8 (1.5 g, 10 mmol) was dissolved in acetone (30 mL), and
K2CO3 (1.52 g, 11 mmol), KI (1.83 g, 11 mmol), and tert-butyl bromoa-
cetate (1.63 mL, 11 mmol) were added. The reaction mixture was al-
lowed to stir at r.t. overnight and the solvent was removed in vacuo.
The residue was partitioned between H2O and EtOAc. The organic lay-
er was washed with H2O, brine and dried (anhyd MgSO4). Column
chromatography yielded 1.8 g (70%) of the pure product 9a as a color-
less oil; Rf = 0.53 (silica gel, EtOAc/hexane = 1:4).
1H NMR (300 MHz, CDCl3): δ = 10.47 (s, 1 H, CHO), 6.57 (s, 2 H, HAr),
4.55 (s, 2 H, CH2), 2.59 (s, 6 H, CH3), 1.48 (s, 9 H, t-C4H9).
13C NMR (75 MHz, CDCl3): δ = 191.8, 167.5, 161.1, 144.7, 126.8, 115.4,
sequent growth of the required peptide sequence. Hence,
we approached this strategy for the preparation of the pep-
tidyl-AOMKs and attempted to synthesize a similar deriva-
tive of 16 through use of the carbonyl ketone and a bifunc-
tional linker (see Scheme S1 in the Supporting Informa-
tion). We used 2,2-bis(hydroxymethyl)propanoic acid as
the starting material, transformed the hydroxyl groups into
silyl ethers (compound 28 in the Supporting Information)
and attempted to perform the ketalization reaction, in the
presence of TMSOTf, under anhydrous and inert conditions.
The reaction failed most probably due to the sterical hin-
drance brought by the 2,6-dimethylbenzoate scaffold or the
amino acid bulky side-chain moiety. To clarify this point,
the corresponding Gly-AOMK was synthesized (compound
27 in the Supporting Information) and subjected to the ke-
talization reaction with the 2,2-bis(hydroxymethyl)propa-
noic acid silyl ether in the same conditions. Failure of this
reaction indicated that the bulky AOMK residue prevents
closure of the 1,3-dioxane ring, unlike the much smaller
fluorine atom in the case the fluoromethyl ketone.
82.9, 65.4, 28.2, 21.2.
HRMS (APCI+): m/z [M + H]+ calcd for C15H21O4: 265.1434; found:
265.1433.
4-(Benzyloxy)-2,6-dimethylbenzaldehyde (9b)
Compound 9b was synthesized starting from aldehyde 8 (240 mg, 1.6
mmol) using benzyl bromide (2 mL, 1.68 mmol) following the same
experimental procedure as described for 9a, and purified by column
chromatography; yield: 160 mg (42%); white solid; mp 68–70 °C; Rf =
0.57 (silica gel, EtOAc/PE = 1:4).
1H NMR (300 MHz, CDCl3): δ = 10.48 (s, 1 H, CHO), 7.40–7.42 (over-
lapped peaks, 3 H, HAr), 7.35–7.38 (m, 2 H, HAr), 6.68 (s, 2 H, HAr), 5.11
(s, 2 H, CH2), 2.61 (s, 6 H, CH3).
13C NMR (75 MHz, CDCl3): δ = 191.8, 162.0, 144.6, 136.4, 128.84,
In conclusion, we have described synthesis of a new
functionalized 2,6-dimethylbenzoate scaffold useful to pre-
pare peptidyl-AOMKs. The synthetic strategy starts from
simple and commercially available reagents and reactants
and furnishes the tert-butyl ester of the 4-substituted 2,6-
dimethylbenzoate moiety that is acid sensitive and can be
easily deprotected. Graft of this scaffold on amino acids or
peptidyl sequences was further performed and the probes
were assayed against cathepsin B to evaluate the inhibitory
efficiency. The IC50 value of the new probe is similar to the
well encountered C-terminal peptidyl derived 2,6-dimeth-
ylbenzoate or 2,4,6-trimethylbenzoate, providing, thus, evi-
dence that our new 4-functionalized 2,6-dimethylbenzoate
moiety does not interfere in the reaction.
128.4, 127.6, 126.3, 115.8, 70.0, 21.3.
HRMS (APCI+): m/z [M + H]+ calcd for C16H17O2: 241.1223; found:
241.1239.
4-[2-(tert-Butoxy)-2-oxoethoxy)-2,6-dimethylbenzoic Acid (1)
Aldehyde 9a (500 mg, 1.9 mmol) was dissolved in 1,4-dioxane (1 mL),
and KMnO4 (360 mg, 2.27 mmol) in H2O (10 mL) was added. The reac-
tion mixture was allowed to stir at r.t. for 3 h while monitoring the
reaction by TLC. The mixture was filtered, the brown solid thoroughly
washed with H2O, and acidified. The aqueous phase was extracted
with EtOAc, the organic layer was washed with brine, and dried (an-
hyd MgSO4). Evaporation of the solvent in vacuo afforded 200 mg
(47%) of pure white solid; mp 102–104 °C; Rf = 0.51 (silica gel,
EtOAc/PE = 1:1).
1H NMR (300 MHz, CDCl3): δ = 6.58 (s, 2 H, HAr), 4.51 (s, 2 H, CH2), 2.42
(s, 6 H, CH3), 1.48 (s, 9 H, t-C4H9).
13C NMR (75 MHz, CDCl3): δ = 173.8, 167.9, 159.0, 139.3, 125.1, 114.3,
82.7, 65.6, 28.1, 21.2.
HRMS (ESI–): m/z [M – H]– calcd for C15H19O5: 279.1227; found:
279.1234.
The air- and water-sensitive reactions were performed using anhyd
solvents and inert atmosphere. Anhyd THF was distilled over Na and
benzophenone. All other commercial solvents and reagents were
used without further purification. NMR spectra were recorded on
Bruker NMR spectrometers operating at 300 MHz or 500 MHz for 1H
and 75 MHz or 125 MHz for 13C. Chemical shifts (δ) are reported in
parts per million (ppm) using residual solvent peak as internal refer-
ence. High-resolution mass spectra were recorded on a ThermoScien-
tific (LTQ XL Orbitrap) spectrometer using APCI or ESI technique and
Orbital Ion Trap mass analyzer. TLC was performed on silica gel 60
4-(Carboxymethoxy)-2,6-dimethylbenzoic Acid (10)
Compound 1 (0.48 g, 1.71 mmol) was dissolved in 25% TFA in CH2Cl2
(1 mL) and the solution was stirred at r.t. for 5 h. The resulting white
precipitate was filtered, washed with CH2Cl2, and dried; yield: 375 mg
(quant) as a white solid; mp 203–205 °C; Rf = 0.2 (silica gel,
EtOAc/PE = 1:1).
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A. G. Coman et al.
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1H NMR (500 MHz, CDCl3): δ = 6.62 (s, 2 H, HAr), 4.67 (s, 2 H, CH2), 2.24
(S)-(9H-Fluoren-9-yl)methyl 1-(2-Bromoacetyl)-3-methylbutyl-
(s, 6 H, CH3).
carbamate (15)
13C NMR (125 MHz, CDCl3): δ = 170.5, 170.1, 157.6, 136.0, 128.2,
113.3, 64.2, 19.8.
HRMS (ESI–): m/z [M – H]– calcd for C11H11O5: 223.0601; found:
223.0613.
Diazomethyl ketone 14 (3.2 g, 8.5 mmol) was dissolved in THF (25.5
mL) and the solution was cooled to 0 °C. A mixture of HBr (33 wt% in
AcOH and H2O in a 1:2 volumetric ratio) was added dropwise. The re-
action mixture was allowed to stir until no evolution of gas was ob-
served (10–15 min) and diluted with EtOAc (20 mL). The organic layer
was washed with H2O, brine, and sat. aq NaHCO3. The organic layer
was dried (anhyd MgSO4) and the solvent removed in vacuo to yield
the product as a pure white solid in 88% yield (3.2 g); mp 112–113 °C;
Rf = 0.65 (silica gel, EtOAc/pentane = 1:3).
1H NMR (400 MHz, CDCl3): δ = 7.77 (d, 3J = 8.0 Hz, 2 H, HAr-Fmoc), 7.59
(d, 3J = 8.0 Hz, 2 H, HAr-Fmoc), 7.41 (t, 3J = 8.0 Hz, 2 H, HAr-Fmoc), 7.32
(t, 3J = 8.0 Hz, 2 H, HAr-Fmoc), 5.13 (s, 1 H, CONH), 4.59–4.64 (m, 1 H,
NHCH), 4.42–4.53 (m, 2 H, CH2-Fmoc), 4.21 (t, 3J = 8.0 Hz, 1 H, CH-
Fmoc), 4.02 (d, 2J = 12.0 Hz, 1 H, CHHBr), 3.96 (d, 2J = 12.0 Hz, 1 H,
CHHBr), 1.60–1.66 [overlapped peaks, 2 H, CHHCH(CH3)2], 1.40–1.48
[m, 1 H, CH(CH3)], 0.97 [d, 3J = 8.0 Hz, 3 H, (CH3)2CH], 0.95 [d, 3J = 8.0
Hz, 3 H, (CH3)2CH].
4-(Benzyloxy)-2,6-dimethylbenzoic Acid (11)
Compound 11 was synthesized from 9b (140 mg, 0.58 mmol) using
the same procedure as described for acid 1 and was obtained in 41%
yield (50 mg); white solid; mp 109–110 °C; Rf = 0.13 (silica gel,
EtOAc/PE = 1:2).
1H NMR (300 MHz, CDCl3): δ = 7.34–7.44 (overlapped peaks, 5 H, HAr),
6.69 (s, 2 H, HAr), 5.07 (s, 2 H, CH2), 2.45 (s, 6 H, CH3).
13C NMR (75 MHz, CDCl3): δ = 174.6, 159.9, 139.4, 136.7, 128.8, 128.2,
127.6, 124.6, 114.6, 69.9, 21.3.
HRMS (ESI–) m/z [M – H]– calcd for C16H15O3: 255.1016; found:
255.1023.
13C NMR (100 MHz, CDCl3): δ = 201.4, 158.5, 143.6, 141,4, 127.8,
127.1, 124.9, 120.1, 66.8, 56.4, 47.3, 40.7, 30.9, 24.9, 23.2.
HRMS (ESI+): m/z [M + H]+ calcd for C22H25BrNO3: 430.1012; found:
430.1002.
4-Hydroxy-2,6-dimethylbenzoic Acid (12)
Compound 11 (180 mg, 1.08 mmol) was dissolved in HBr (33 wt% in
AcOH, 3.5 mL) and left to react at r.t. for 1 h. H2O was added and the
aqueous phase was extracted with EtOAc (3 × 10 mL). The combined
organic phases were washed with brine, dried (anhyd MgSO4), and
evaporated in vacuo. The residue was chromatographed (using gradi-
ent of 25% of PE in EtOAc until 100% EtOAc to yield 70 mg (40%) of 12;
white solid; mp 168–170 °C; Rf = 0.41 (silica gel, EtOAc/PE = 1:2).
(S)-3-({[(9H-Fluoren-9-yl)methoxy]carbonyl}amino)-5-methyl-2-
oxohexyl 2,6-Dimethylbenzoate (16)
Bromomethyl ketone 15 (617 mg, 1.4 mmol) was dissolved in MeCN
(10 mL) and the solution was cooled in an ice bath. KF (244 mg, 4.2
mmol) and 2,6-dimethylbenzoic acid (258 mg, 1.72 mmol) were add-
ed and the mixture was stirred overnight at r.t. The solvent was re-
moved and the residue was dissolved in EtOAc. The organic layer was
washed with H2O, brine, and sat. aq NaHCO3, dried (anhyd MgSO4),
and the solvent removed in vacuo. Column chromatography furnished
the pure product as a white solid in 40% yield (292 mg); mp 104–105
°C; Rf = 0.43 (silica gel, EtOAc/pentane = 1:5).
1H NMR (500 MHz, CD3OD): δ = 6.45 (s, 2 H, HAr), 2.28 (s, 6 H, CH3).
13C NMR (125 MHz, CD3OD): δ = 174.1, 159.2, 138.2, 127.2, 115.4,
20.3.
HRMS (ESI–): m/z [M – H]– calcd for C9H9O3: 165.0546; found:
165.0559.
(S)-(9H-Fluoren-9-yl)methyl (1-Diazo-5-methyl-2-oxohexan-3-
yl)carbamate (14)
1H NMR (300 MHz, CDCl3): δ = 0.96 [d, 3J = 3.6 Hz, 6 H, (CH3)2CH],
1.47–1.54 [m, 1 H, CH(CH3)], 1.66–1.72 [overlapped peaks, 2 H, CH-
HCH(CH3)2], 2.40 (s, 6 H, CH3-AOMK), 4.23 (t, 3J = 6.4 Hz, 1 H, CH-
Fmoc), 4.41–4.52 (overlapped peaks, 2 H, CHH-Fmoc), 4.56–4.70 (m, 1
H, NHCH), 4.96 (d, 2J = 17.0 Hz, 1 H, CHHOCO), 5.05 (d, 2J = 17.0 Hz, 1
H, CHHOCO), 5.15 (d, 3J = 8.1 Hz, CONH), 7.05 (d, 3J = 7.6 Hz, 2 H, HAr-
AOMK), 7.21 (t, 3J = 7.6 Hz, 1 H, HAr-AOMK), 7,32 (t, 3J = 7.6 Hz, 2 H,
HAr-Fmoc), 7,40 (t, 3J = 7.6 Hz, 2 H, HAr-Fmoc), 7.60 (d, 3J = 7.6 Hz, 2 H,
HAr-Fmoc), 7.77 (d, 3J = 7.6 Hz, 2 H, HAr-Fmoc).
13C NMR (75 MHz, CDCl3): δ = 202.8, 169.1, 156.2, 143.8, 141.5, 135.8,
132.6, 129.8, 127.9, 127.8, 127.2, 125.2, 125.1, 120.2, 67.1, 66.7, 55.9,
47.4, 40.4, 24.7, 23.2, 20.1.
HRMS (ESI+): m/z [M + Na]+ calcd for C31H33NO5Na: 522.2251; found:
522.2235.
To a solution of Fmoc-Leu-OH (13; 5.3 g, 15 mmol) in anhyd THF (50
mL) at –10 °C were added N-methylmorpholine (2.1 mL, 18.75 mmol)
and isobutyl chloroformate (2.25 mL, 17.25 mmol). The mixture was
stirred at –10 °C for 30 min, then a solution of diazomethane in Et2O
(15–20 mmol, generated in situ from ethereal α-nitrozomethylurea13
and dried over solid KOH) was added at 0 °C. The mixture was further
stirred for 3 h at r.t. and quenched with H2O (15 mL) and AcOH (1.5
mL). The mixture was diluted with EtOAc (50 mL), washed with H2O,
brine, and sat. aq NaHCO3. The organic layer was dried (anhyd MgSO4)
and the solvent removed in vacuo. Column chromatography on silica
gel furnished pure product 14 as a light-yellow oil in 70% yield (2.8 g);
Rf = 0.26 (EtOAc/pentane = 1:3).
1H NMR (300 MHz, CDCl3): δ = 7.77 (d, 3J = 8.0 Hz, 2 H, HAr-Fmoc), 7.59
(dd, 3J1 = 8.0 Hz, 2 H, HAr-Fmoc), 7.40 (t, 3J = 8.0 Hz, 2 H, HAr-Fmoc),
7.31 (t, 3J = 8.0 Hz, 2 H, HAr-Fmoc), 5.31 (s, 1 H, CH=N2), 4.40–4.52 (m,
2 H, CH2-Fmoc), 4.17–4.25 (overlapped peaks, 2 H, CH-Fmoc, NHCH),
1.41–1.74 [overlapped peaks, 3 H, CH(CH3), CH2CH(CH3)2], 0.94 [d, 3J =
8.0 Hz, 6 H, (CH3)2CH].
(S)-3-({[(9H-Fluoren-9-yl)methoxy]carbonyl}amino)-5-methyl-2-
oxohexyl 2,4,6-Trimethylbenzoate (17)
Compound 17 was synthesized following the same experimental pro-
cedure as described for 16, using bromomethyl ketone 15 (30 mg, 70
μmol) and 2,4,6-trimethylbenzoic acid (14 mg, 84 μmol) to yield 17
mg (50%) of 17 as a white solid; Rf = 0.34 (silica gel, EtOAc/PE = 1:4).
1H NMR (500 MHz, CDCl3): δ = 7.76 (d, 3J = 7.2 Hz, 2 H, HAr-Fmoc), 7.60
(d, 3J = 7.2 Hz, 2 H, HAr-Fmoc), 7.40 (t, 3J = 7.2 Hz, 2 H, HAr-Fmoc), 7.32
(t, 3J = 7.2 Hz, 2 H, HAr-Fmoc), 6.87 (s, 2 H, HAr-AOMK), 5.15 (d, 3J = 7.7
Hz, CONH), 5.03 (d, 2J = 17.0 Hz, 1 H, CHHOCO), 4.94 (d, 2J = 17.0 Hz, 1
13C NMR (75 MHz, CDCl3): δ = 194.4, 156.1, 143.8, 141.4, 127.8, 127.2,
125.2, 125.1, 120.1, 66.8, 54.5, 47.4, 41.4, 24.8, 23.2.
HRMS (ESI+): m/z [M + H – N2]+ calcd for C22H24N3O3: 350.1751;
found: 350.1740.
© Georg Thieme Verlag Stuttgart · New York — Synthesis 2016, 48, A–G

F
A. G. Coman et al.
Paper
Synthesis
H, CHHOCO), 4.43–4.51 (m, 3 H, CH2-Fmoc, NHCH), 4.22 (t, 3J = 6.5 Hz,
1 H, CH-Fmoc), 2.40 (s, 6 H, CH3-AOMK), 2.29 (s, 3 H, CH3-AOMK),
1.67–1.72 [m, 2 H, CH2CH(CH3)2], 1.49–1.54 [m, 1 H, CH(CH3)], 0.96 [d,
3J = 2.5 Hz, 6 H, (CH3)2CH].
13C NMR (125 MHz, CDCl3): δ = 202.9, 169.2, 156.2, 143.9, 141.5,
139.9, 136.0, 129.6, 128.6, 127.9, 127.3, 127.2, 125.2, 125.1, 120.1,
67.0, 66.7, 55.9, 47.4, 40.5, 24.8, 23.4, 21.3, 20.1.
(5.4 mmol, 4 equiv) using PyBOP (5.4 mmol, 4 equiv) and HOBt (5.4
mmol, 4 equiv) as coupling activating reagents and DIPEA (12 equiv)
as base dissolved in DMF. Deprotection of Fmoc group was achieved
using piperidine and the coupling efficiency was checked by Kaiser
test. Cleavage of the peptide was performed using a standard cleavage
cocktail (95% TFA, 2.5% H2O, and 2.5% TIS). Obtained as a white solid;
overall yield: 1.43 g (90%).
HRMS (APCI+): m/z [M + H]+ calcd for C20H38N3O5: 400.2806; found:
400.2822.
HRMS (ESI+): m/z [M + Na]+ calcd for C32H35NO5Na:536.2407; found:
536.2418.
Peptidyl-Diazomethyl Ketone 21
(S)-3-({[(9H-Fluoren-9-yl)methoxy]carbonyl}amino)-5-methyl-2-
oxohexyl 4-[2-(tert-Butoxy)-2-oxoethoxy]-2,6-dimethylbenzoate
(18)
Peptidyl-diazomethyl ketone 21 was synthesized following the same
experimental procedure as described for 14 from peptide 20 (200 mg,
0.5 mmol) to yield 100 mg (47%) of the pure product as a yellow oil
after purification by column chromatography (silica gel, starting with
EtOAc/MeOH = 99:1 and increasing the gradient up to 100% MeOH).
Compound 18 was synthesized following the same experimental pro-
cedure as described for 16, using bromomethyl ketone 15 (100 mg,
0.23 mmol) and acid 1 (82 mg, 0.28 mmol) to yield 60 mg (40%) of a
white solid; mp 179–180 °C; Rf = 0.5 (silica gel, EtOAc/PE = 1:4).
1H NMR (300 MHz, CDCl3): δ = 7.76 (d, 3J = 7.4 Hz, 2 H, HAr-Fmoc), 7.58
(d, 3J = 7.4 Hz, 2 H, HAr-Fmoc), 7.40 (t, 3J = 7.4 Hz, 2 H, HAr-Fmoc), 7.31
(t, 3J = 7.4 Hz, 2 H, HAr-Fmoc), 6.57 (s, 2 H, HAr-AOMK), 5.17 (d, 2J = 8.2
Hz, 1 H, NH), 5.03 (d, 2J = 17.0 Hz, 1 H, CHHCOO), 4.93 (d, 2J = 17.0 Hz,
1 H, CHHCOO), 4.49 (s, 2 H, COCH2OCO), 4.44–4.65 [overlapped peaks,
3 H, CH2-Fmoc, CHCH2CH(CH3)2], 4.21 (t, 2J = 6.4 Hz, 1 H, CH-Fmoc),
2.39 (s, 6 H, CH3-AOMK), 1.65–1.71 (m, 2 H, CH2CH(CH3)2), 1.49–1.60
[m, 1 H, CH(CH3)2], 1.49 (s, 9 H, t-C4H9), 0.95 [d, 3J = 5.3 Hz, 6 H,
(CH3)2CH].
HRMS (ESI+): m/z [M + Na]+ calcd for C21H37N5O4Na: 446.2738; found
[M + Na]+ 446.2767, [M – N2]+ 396.2857.
Peptidyl-Bromethyl Ketone 22
Peptidyl-bromomethyl ketone 22 was synthesized following the same
experimental procedure as described for 15 from peptide 21 (100 mg,
0.23 mmol) to yield 32 mg (30%) of the pure product as a yellow oil.
HRMS (ESI+): m/z [M + H]+ calcd for C21H39BrN3O4: 476.2118; found:
476.2120.
13C NMR (75 MHz, CDCl3): δ = 202.9, 168.7, 167.8, 158.7, 156.2, 143.8,
141.5, 138.8, 127.8, 127.2, 125.7, 125.2, 125.1, 114.0, 82.6, 67.0, 66.7,
65.6, 55.7, 47.4, 40.5, 24.8, 23.4, 21.5, 20.7.
HRMS (ESI–): m/z [M – H – t-Bu]– calcd for C37H34NO8: 572.2279;
found: 572.2278.
Peptidyl-Acyloxymethyl Ketone 23
Peptidyl-acyloxymethyl ketone 23 was synthesized starting from
peptidyl-bromomethyl ketone 22 (5 mg, 10.5 μmol) using 2,6-di-
methylbenzoic acid (2 mg, 12.6 mmol) following the same experi-
mental procedure as described for 16 to yield 5.2 mg (91%) of the
pure product as a colorless oil.
(S)-2-[4-({[3-({[(9H-Fluoren-9-yl)methoxy]carbonyl}amino)-5-
methyl-2-oxohexyl]oxy}carbonyl)-3,5-dimethylphenoxy]acetic
Acid (19)
HRMS (ESI+): m/z [M + H]+ calcd for C30H48N3O6: 546.3535; found:
546.3538.
Compound 18 (50 mg, 0.08 mmol) was dissolved in 25% TFA in CH2Cl2
(3 mL). The reaction mixture was allowed to stir at r.t. until complete
conversion of the substrate. The solvent was removed in vacuo and
the product was passed through a short pad of silica gel (CH2Cl2/PE =
1:6) to yield the pure product as a white solid in 83% yield (34 mg).
1H NMR (500 MHz, DMSO-d6): δ = 7.89 (d, 3J = 8.0 Hz, 2 H, HAr-Fmoc),
7.79 (d, 3J = 8.0 Hz, 1 H, NH), 7.70 (d, 3J = 8.0 Hz, 2 H, HAr-Fmoc), 7.41
(t, 3J = 8.0 Hz, 2 H, HAr-Fmoc), 7.32 (t, 3J = 8.0 Hz, 2 H, HAr-Fmoc), 6.53
(s, 2 H, HAr-AOMK), 5.01 (d, 2J = 17.0 Hz, 1 H, CHHOCO), 4.93 d, 2J =
17.0 Hz, 1 H, CHHOCO), 4.38–4.46 (overlapped peaks, 2 H, CHH-
Fmoc), 4.24 (t, 3J = 6.6 Hz, 1 H, CH-Fmoc), 4.13–4.17 (overlapped
peaks, 3 H, CHNH, CH2CO2H), 2.26 (s, 6 H, CH3-AOMK), 1.58–1.63 [m, 1
H, CH(CH3)], 1.49–1.52 [overlapped peaks, 2 H, CHHCH(CH3)2], 0.88
(d, 3J = 6.5 Hz, 3 H, (CH3)2CH], 0.84 [d, 3J = 6.5 Hz, 3 H, (CH3)2CH].
Peptidyl-Acyloxymethyl Ketone 24
Peptidyl-acyloxymethyl ketone 24 was synthesized starting from
peptidyl-bromomethyl ketone 22 (5 mg, 10.5 μmol) using 2,4,6-
trimethylbenzoic acid (2 mg, 12.6 mmol) following the same experi-
mental procedure as described for 16 to yield 5 mg (85%) of the pure
product as a colorless oil.
HRMS (ESI+): m/z [M + H]+ calcd for C31H50N3O6: 560.3694; found:
560.3709.
Peptidyl-Acyloxymethyl Ketone 25
Peptidyl-acyloxymethyl ketone 25 was synthesized starting from
peptidyl-bromomethyl ketone 22 (10 mg, 21 μmol) using acid 1 (7.4
mg, 25.2 μmol) following the same experimental procedure as de-
scribed for 16 to yield 7 mg (50%) of the pure product as a colorless
oil.
13C NMR (125 MHz, DMSO-d6): δ = 203.8, 170.8, 168.2, 159.8, 156.3,
143.8, 140.9, 137.2, 127.7, 127.4, 127.1, 125.3, 124.1, 120.2, 113.8,
67.5, 66.6, 65.4, 56.3, 46.9, 38.2, 24.1, 23.2, 21.1.
HRMS (ESI+): m/z [M + H]+ calcd for C36H58N3O9: 676.4168; found:
676.4139.
HRMS (ESI–): m/z [M – H]– calcd for C33H34NO8: 572.2279; found:
572.2279.
Peptidyl-Acyloxymethyl Ketone 26
Peptide 25 (7 mg, 10 μmol) was dissolved in a 25% solution of TFA in
CH2Cl2 (1 mL). The reaction mixture was allowed to stir at r.t. over-
night and the solvent was evaporated to yield 3.5 mg (57%) of the pure
product as a colorless oil.
Peptide 20
Peptide 20 was synthesized following the standard SPPS procedure
on Wang resin. 1.5 g Wang resin (loading 0.9 mmol/g, 1 equiv) swelled
in DMF was followed by coupling of each Fmoc-protected amino acid
© Georg Thieme Verlag Stuttgart · New York — Synthesis 2016, 48, A–G

G
A. G. Coman et al.
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Synthesis
HRMS (ESI+): m/z [M + H]+ calcd for C32H50N3O9: 620.3542; found:
620.3511.
(3) For recent examples of applications of peptidyl-AOMKs, see:
(a) Edgington, L. E.; Verdoes, M.; Ortega, A.; Withana, N. P.; Lee,
J.; Syed, S.; Bachmann, M. H.; Blum, G.; Bogyo, M. J. Am. Chem.
Soc. 2013, 135, 174. (b) Puri, A. W.; Lupardus, P. J.; Deu, E.;
Albrow, V. E.; Garcia, K. C.; Bogyo, M.; Shen, A. Chem. Biol. 2010,
17, 1201. (c) Edgington, L. E.; van Raam, B. J.; Verdoes, M.;
Wierschem, C.; Salvesen, G. S.; Bogyo, M. Chem. Biol. 2012, 19,
340. (d) Torkar, A.; Lenarci, B.; Lah, T.; Dive, V.; Devel, L. Bioorg.
Med. Chem. Lett. 2013, 23, 2968. (e) Paulick, M. G.; Bogyo, M.
ACS Chem. Biol. 2011, 6, 563. (f) Lee, J.; Bogyo, M. ACS Chem. Biol.
2010, 5, 233. (g) Chong, C.-M.; Gao, S.; Chiang, B.-Y.; Hsu, W.-H.;
Lin, T.-C.; Chen, T.-C.; Lin, C.-H. ChemBioChem 2011, 12, 2306.
(h) Dechert, A.-M. R.; MacNamara, J. P.; Breevoort, S. R.;
Hildebrandt, E. R.; Hembree, N. W.; Rea, A. C.; McLain, D. E.;
Porter, S. B.; Schmidt, W. K.; Dore, T. M. Bioorg. Med. Chem.
2010, 18, 6230. (i) Henzing, A. J.; Dodson, H.; Reid, J. M.;
Kaufmann, S. H.; Baxter, R. L.; Earnshaw, W. C. J. Med. Chem.
2006, 49, 7636. (j) Blum, G.; Mullins, S. R.; Keren, K.; Fonovic,
M.; Jedeszko, C.; Rice, M. J.; Sloane, B. F.; Bogyo, M. Nat. Chem.
Biol. 2005, 1, 203 . (k) 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. (l) Edem, P. E.; Czorny, S.;
Valliant, J. F. J. Med. Chem. 2014, 57, 9564.
Enzyme Inhibition Assays
Cathepsin B (0.1 U) from bovine spleen (Sigma) was incubated for 30
min at 37 °C with various concentrations of compounds 20, 23, 24,
and 26, respectively, in the reaction buffer (NaOAc 50 mM, pH = 5,
EDTA 2.5 mM, β-mercaptoethanol 10 mM, Tween 20 0.1%, DMSO 1%).
Following compound incubation, the fluorogenic substrate Nα-CBZ-
Arg-Arg-7-amido-4-methylcoumarin was added at a final concentra-
tion of 10 μM. The reaction was further incubated for 5 min at 37 °C.
Fluorescence measurements (λex = 360 nm, λem = 460 nm) were per-
formed and the data were fitted to a sigmoidal 4 parameters curve.
IC50 values (Table 1) were determined using the GraphPad Prism
software.
Acknowledgment
Financial support by the National Research Council (CNCS) of Roma-
nia, Project PN-II-RU-TE-2012-3-0471 is gratefully acknowledged.
(4) (a) Krantz, A.; Copp, L.; Coles, P.; Smith, R.; Heard, S. Biochemis-
try 1991, 30, 4678. (b) Pliura, D. H.; Bonaventura, B. J.; Smith, R.
A.; Coles, P. J.; Krantz, A. Biochem. J. 1992, 288, 759.
(5) Mujica, M. T.; Jung, G. Synlett 1999, 1933.
(6) Lee, A.; Huang, L.; Ellman, J. A. J. Am. Chem. Soc. 1999, 121, 9907.
(7) Turk, V.; Turk, B.; Turk, D. EMBO J. 2001, 20, 4629.
Supporting Information
Supporting information for this article is available online at
http://dx.doi.org/10.1055/s-0035-1562781.
S
u
p
p
ortioInfgrmoaitn
S
u
p
p
ortiInfogrmoaitn
(8) Getman, D. P.; Periana, R. A.; Riley, D. P. US Patent 4879398,
1989.
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© Georg Thieme Verlag Stuttgart · New York — Synthesis 2016, 48, A–G