N-Terminal peptidic boronic acids selectively inhibit human ClpXP

Article abstract of DOI:10.1039/c004247a

The synthesis and development of N-terminal peptidic boronic acids as protease inhibitors is reported. N-Terminal peptidic boronic acids interrogate the S′ sites of the target protein for selectivity and provide a new strategy that complements the currently known peptidic α-amino boronic acids (C-terminal boronic acids). After screening a series of N-terminal peptidic boronic acids, the first selective inhibitor of human ClpXP, an ATP-dependent serine protease present in the mitochondrial matrix, was discovered. This should facilitate the understanding of the physiological function of this protease The Royal Society of Chemistry 2010.

Full text of DOI:10.1039/c004247a

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
N-Terminal peptidic boronic acids selectively inhibit human ClpXP†‡
Kenneth Knott,a Jennifer Fishovitz,b Steven B. Thorpe,a Irene Leeb and Webster L. Santos*a
Received 16th March 2010, Accepted 13th May 2010
First published as an Advance Article on the web 4th June 2010
DOI: 10.1039/c004247a
The synthesis and development of N-terminal peptidic boronic acids as protease inhibitors is reported.
N-Terminal peptidic boronic acids interrogate the S¢ sites of the target protein for selectivity and provide
a new strategy that complements the currently known peptidic a-amino boronic acids (C-terminal
boronic acids). After screening a series of N-terminal peptidic boronic acids, the first selective inhibitor
of human ClpXP, an ATP-dependent serine protease present in the mitochondrial matrix, was
discovered. This should facilitate the understanding of the physiological function of this protease
Introduction
An increasing number of boron-containing small molecules and
peptides are being investigated for their medical potential in
the treatment of various diseases.1 For example, benzoxaborole
AN2690, a compound with a novel mode of action of targeting
leucyl-transfer RNA synthetase, is currently in phase III clinical
trial for the treatment of onychomycosis.2–4 Others incorporate a-
aminoboronic acids into peptides to generate mechanism-based
inhibitors of therapeutically relevant serine proteases including
chymotrypsin,5,6 thrombin,7 dipeptidyl peptidase,8 and HCV NS3
protease.9
Because peptidic boronic acids inhibit serine proteases, a series
of dipeptidyl boronic acids were hypothesized to be potent
and selective inhibitors of the proteasome, which contains a
threonine residue in the active site.10 This was confirmed when
a reversible covalent bond with the N-terminal threonine residue
was established.11 Gratifyingly, the first boron-containing drug,
Bortezomib (Velcade), was approved by the FDA for the treatment
Fig. 1 Complementary peptidic boronic acid strategies.
of multiple myeloma and mantle cell lymphoma.12
Peptidic a-amino boronic acids are excellent inhibitors of
we report the synthesis and development of a complementary
approach, using N-terminal peptidic boronic acids, to harvest the
potential of residues on the opposite side (P¢-sites). We reason
that the S¢-sites may provide unique selectivity for a number of
biologically important proteases. In particular, the S¢-sites are
attractive when the S-sites do not achieve the desired selectivity and
inhibitory activity. In the design of N-terminal peptidic boronic
acids, the carboxyl group is replaced with a boronic acid moiety
and the nitrogen derived from the scissile amide bond is changed to
carbon because the B–N bond is labile. The terminal boronic acid
residue can be conveniently modified to incorporate both natural
and unnatural amino acid functional groups.
proteases because of the ability of Lewis basic residues located
in the enzyme active site to bind to the empty p-orbital of boron
forming a stable “ate” complex, which mimics the tetrahedral
intermediate formed during amide bond hydrolysis.5,6,13–15 When
the protease substrate is known, this strategy becomes attractive
because the P-site residues can readily be incorporated to provide
a good starting point for inhibitor development. It is well-known,
however, that substrate selectivity within the enzyme active site
relies heavily on both the P/S- and P¢/S¢-site complementarity
(Fig. 1). However, current methods incorporating the boronic
acid moiety only take advantage of the binding pocket on P/S
side of the scissile amide bond (C-terminal boronic acid). Herein,
To evaluate the utility of our approach, we compared the
inhibition profiles of a series of N-terminal peptidic boronic acids
toward proteases Lon and ClpXP, which are found in the matrix of
human mitochondria. Both Lon and ClpXP are serine proteases
but they only degrade proteins in the presence of ATP. Lon is a
homo-oligomeric complex whereas ClpXP is a hetero-oligomeric
complex.16–18 Recently, it has been shown that ATP-dependent
proteolytic activity can be up-regulated by cellular oxidative
stress, especially during cardiac ischemia/reperfusion.19,20 As Lon
and ClpXP are both present in the mitochondrial matrix,21 the
aDepartment of Chemistry, Virginia Tech, Blacksburg, Virginia 24061, USA.
E-mail: santosw@vt.edu; Fax: +1 540 231 3255; Tel: +1 540 231 5041
bDepartment of Chemistry, Case Western Reserve University, Cleveland,
Ohio 44106, USA
† This paper is part of an Organic & Biomolecular Chemistry web theme
issue on chemical biology.
‡ Electronic supplementary information (ESI) available: Detailed synthesis
for compounds 6g1/6g2, 1H and 13C NMR spectra for all compounds
synthesized, LC/MS of peptides used in this study and IC50 determination
of WLS6a. See DOI: 10.1039/c004247a
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observed up-regulation in ATP-dependent proteolytic activity
could be attributed to one or both enzymes. The contribution of
the respective protease in protecting mitochondria from oxidative
stress is not known. Therefore, the development of selective
inhibitors against the respective protease will provide valuable
chemical tools to interrogate the physiological functions of the
two mitochondrial proteases. Furthermore, several physiological
protein substrates have been identified for human Lon (hLon)
but not for human ClpXP (hClpXP). The availability of selec-
tive inhibitors should benefit the identification of physiological
substrates of hClpXP when used in conjunction with proteomic
studies.
b-boronic acids 4a–4f were smoothly attached using HCTU as the
coupling agent generating peptides 5a–5g (Scheme 2). Subsequent
cleavage from the resin also achieved a concomitant removal
of the pinacol ester protecting group affording 6a–6g. Peptides
6g1 and 6g2 were synthesized as in Scheme 1 but enantiopure
bis(pinanediolato)diboron was used.28 HPLC purification using
C18 column provided analytically pure material for subsequent
assays.
Using a FRET assay previously developed for hLon,29 we
adapted the same protocol to determine inhibitors for hClpXP
using FRETN 89–98 as the substrate. FRETN 89–98 is an 11
amino acid peptide that contains an anthranilamide/nitrotyrosine
fluorescence donor/acceptor pair. In the intact peptide, the
fluorescence is quenched, but upon cleavage, the fluorophore and
quencher separate and an increase in fluorescence emission can be
measured. Under the assay conditions used here, hClpXP shows
the expected ATP-dependent activity as illustrated by an increase
in fluorescence emission, which can be monitored in the absence
or presence of modulators (data not shown). Initial screening of
N-terminal peptidic boronic acids WLS6a–6g at 20 mM indicated
that diasteromers 6g1 and 6g2 inhibited both hLon and hClpXP
but 6b showed no inhibition (Fig. 2A). In contrast, WLS6a, 6e
and 6f inhibited hClpXP but not hLon. Encouraged by these
results, we then tested these compounds at a higher concentration
(1 mM) and discovered that after preincubation of the enzyme
with the inhibitor for 30 min at room temperature, the proteolytic
activity of hClpXP was almost completely inhibited (>90%) by
N-terminal boronic peptide WLS6a while hLon was unaffected
(Fig. 2B). 6e and 6f also inhibited hClpXP but to a lesser
Results and discussion
Motivated by these goals, we set out to evaluate the extent to which
N-terminal peptidic boronic acids can be exploited as inhibitors
of hLon and hClpXP. Synthesis of N-terminal boronic acid
building blocks commenced with the installation of the b-boronic
ester moiety (Scheme 1). Treatment of a,b-unsaturated esters 1a–
1d with bis(pinacolato)diboron, CuCl, DPEphos, NaOtBu and
MeOH22–24 or a mixed diboron developed in our laboratory25
readily provided b-boronic esters 2a–2d. Functional groups on
the a-position were introduced by enolization of 2c/2d with
LDA followed by alkylation with tert-butyl bromoacetate or
benzyl bromide to yield 3e and 3f, respectively. Saponification
with LiOH in THF–H2O or catalytic hydrogenation provided the
requisite b-boronic acids 4a–4f. Using solid phase peptide syn-
thesis, the sequence GRQAY26,27 was attached to Wang resin and
Scheme 1 Synthesis of N-terminal boronic acid monomers.
Scheme 2 Solid-phase peptide synthesis of N-terminal peptidic boronic acids.
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Fig. 2 Screening of N-terminal peptidic boronic acids using a FRET
assay. 20 mM (A) or 1 mM (B) putative inhibitor was incubated with
300 nM hLon (black) or hClpXP (gray) at rt for 30 min prior to addition
of ATP and FRETN 89–98 substrate. All values are the average of three
experiments.
Fig. 3 Proteolytic cleavage of casein. Reactions containing 50 mM
HEPES (pH 8.1), 15 mM Mg(OAc)2, 5 mM DTT, and 1 mM hLon (A) or
hClpXP (B) were incubated for 1 min at 37 ◦C in the absence and presence
of 1 mM WLS6a. 5 mM ATP was added and the reactions were further
incubated for 1 min at 37 ◦C. Reactions were initiated with 10 mM casein
and aliquots were quenched at specific time points and run on a 12.5%
SDS-PAGE.
extent. After identifying compound WLS6a as the most effective
inhibitor of the series, we performed experiments under steady-
state conditions to estimate its potency. An apparent IC50 value of
29 mM 9 was obtained.28 Detailed kinetic assays to elucidate the
mechanism of inhibition as well as the true inhibition constants
for WLS6a are currently underway using an optimized peptidase
assay.
To confirm that WLS6a is a selective inhibitor of hClpXP, we
performed a secondary assay and investigated whether compound
WLS6a inhibited hClpXP and hLon mediated protein degrada-
tion using SDS-PAGE analysis. We used casein as the substrate
since it is the best known protein substrate for both hLon and
hClpXP.30,31 As expected, casein was completely degraded by hLon
in the absence of inhibitor while minimal inhibition is observed
in the presence of inhibitor as a small amount of casein persisted
after 15 min of incubation (Fig. 3A). It is clear, however, that
casein is completely hydrolyzed after 30 min. In the presence of
hClpXP, proteolytic cleavage of casein was complete within 2 h in
the absence of inhibitor but almost fully intact during the same
time course of the experiment upon incubation with inhibitor
WLS6a (Fig. 3B). These results are in complete agreement with
the FRET assay and suggest that WLS6a is a selective inhibitor
for hClpXP over hLon, even at a concentration of 1 mM. To the
best of our knowledge, compound WLS6a is the first reported
selective inhibitor for hClpXP. Although the exact mechanism of
inhibition is currently unknown, this discovery should facilitate
the understanding of the roles of hClpXP and hLon in the
maintenance of mitochondrial integrity.
Conclusions
In conclusion, we demonstrated the potential broad utility of N-
terminal peptidic boronic acids as protease inhibitors. For the
first time, boron-containing peptidic probes that interrogate the
S¢-site of proteases are available. We have applied this strategy in
the discovery of a selective inhibitor for hClpXP and provided a
useful chemical biology tool to investigate its function. Current
efforts along these lines as well as understanding the mechanism
of action are underway.
Experimental
NMR spectra were recorded on a JEOL EclipsePlus 500 or a
Varian INOVA 400. The peptides were purified on an Agilent
1200 semi-prep system using a Thermo Scientific Hyper Sil Gold
C-18 column. Purity of the final peptides was verified by a
Thermo TSQ ESI LC/MS. High resolution mass spectra were
taken on an Agilent 6220 Accurate Mass TOF LC/MS. 1H NMR
chemical shifts are reported in ppm with the solvent resonance
as the internal standard (CDCl3: 7.26 ppm). Peak multiplicity is
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described as follows: s (singlet), br s (broad singlet), d (doublet),
dd (doublet of doublets), m (multiplet) and coupling constant (J)
values are given in Hz. 13C NMR chemical shifts are reported in
ppm with the solvent resonance as the internal standard (CDCl3:
77.16 ppm). In all cases, except as otherwise noted, the signal
7.2 Hz, 2H), 1.37 (h, J = 7.5 Hz, 1H), 1.23 (s, 2H), 1.23 (s, 2H),
0.99 (d, J = 7.5 Hz, 3H).13C NMR (126 MHz, CDCl3) d 174.17,
83.08, 51.24, 37.40, 24.68, 24.59, 15.03, (B–C) 13.54. HRMS-ESI
(m/z): [M + Na]+ calcd for C11H21[11B]NaO4, 251.14305; found,
251.14333.
for the carbon directly attached to boron was not observed in 13
C
NMR because of quadrupolar relaxation. Bis(pinacolato)diboron
was obtained from both Boron Molecular and Frontier Scientific.
Sodium tert-butoxide and methyl methacrylate were obtained
from Fluka. Methyl acrylate, benzyl methacrylate, and benzyl
acrylate were obtained from Alfa Aesar. All amino acids and Wang
resin were obtained from Novabiochem. HCTU was obtained
from Peptides International. Anhydrous THF, DCM, and DMF
were used from 18 L Pure-PakTM tanks from Sigma Aldrich. All
other chemicals were obtained from Sigma Aldrich. All chemicals
were used without further purification.
Methyl 2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)cyclo-
pentanecarboxylate (2b)
1,3-Dicyclohexylimidazolium chloride, (ICy)Cl, was prepared
according to published literature procedure.33 To a suspension of
(ICy)Cl (5 mol%), CuCl (5 mol%) and NaOtBu (5 mol%) in THF
(3 mL) stirring for 1.5 h was added an additional equiv of NaOtBu.
After 1 h, bis(pinacolato)diboron in THF (1 mL) was added and
the reaction mixture was stirred for another 10 min. Methyl 1-
cyclopentene-1-carboxylate (194 mg), followed by methanol, was
added and stirred at rt overnight. The product was filtered through
Celite, dried, and purified by flash silica gel chromatography (0–
20% ethyl acetate in hexanes) to provide the product in 96%
yield (375 mg) as light yellow oil. This compound was isolated
General procedure A: b-boration of a,b-unsaturated esters
The following method was adapted from Yun et al.32 Copper
chloride (0.03 equiv), sodium tert-butoxide (0.09 equiv), and
DPEphos (0.03 equiv) were weighed into a 2-neck flask and
alternate cycles of nitrogen purge and vacuum were performed.
Anhydrous THF was added to the reaction flask by needle
and stirred for 45 min (the solution gradually darkened to a
greenish suspension). Bis(pinacolato)diboron (1.1 equiv) dissolved
in THF (1 ml) was added via syringe and stirred for 30 min
(suspension rapidly darkened to a deep arterial red). The reaction
flask was then cooled to 0 ◦C and the a,b-unsaturated ester (1
equiv) was added by syringe followed immediately by methanol
(4 equiv). After the solution was allowed to warm to rt overnight,
the suspension was filtered through Celite and dried under
reduced pressure. A white suspension appeared after addition
of hexanes, which was removed by filtration and the filtrate was
purified by flash silica gel chromatography (0–20% ethyl acetate in
hexanes). TLC analysis with 10% ethyl acetate/hexanes allowed
visualization of the product by CAM or aqueous KMnO4 stain.
1
as an unresolved mixture of cis/trans diastereomers. H NMR
(500 MHz, CDCl3) d 3.64 (s, 0.63 H), 3.63 (s, 2.31 H), 3.04-2.96
(m, 0.68 H), 2.84-2.75 (m, 0.09 H), 1.95-1.35 (m, 7H), 1.23-1.20 (m,
12H). 13C NMR (126 MHz, CDCl3) d 177.38, 83.11, 51.54, 46.83,
46.19, 30.97, 30.38, 28.87, 27.78, 26.50, 25.91, 25.00, 24.83, 24.72.
HRMS-ESI (m/z): [M + H]+ calcd for C13H23[11B]O4, 255.1762;
found, 255.1763.
Benzyl 3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)propanoate
(2c)
Using general procedure A, beginning with benzyl acrylate, the
title product was isolated in 79% (1.151 g) yield as a light yellow
oil. 1H NMR (500 MHz, CDCl3) d 7.35-7.28 (m, 5H), 5.10 (s, 2H),
2.49 (t, J = 7.5 Hz, 2H), 1.21 (s, 12H), 1.04 (t, J = 7.5 Hz, 2H).
13C NMR (126 MHz, CDCl3) d 174.55, 136.33, 128.56, 128.16,
128.13, 83.32, 66.15, 28.92, 24.83. HRMS-ESI (m/z): [M + H]+
calcd for C16H23[11B]O4, 291.17012; found, 291.17712.
General procedure B: a-alkylation of borylation products
Methyl 3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)propanoate
(2d)
LDA was prepared in situ by adding n-butyl lithium (1.1 equiv)
drop-wise to diisopropylamine (1 equiv) dissolved in THF at
-78 ◦C for 45 min. Th◦e solution was warmed to 0 ◦C for 10 min and
again chilled to -78 C before cannulating into a THF solution
Using general procedure A, beginning with methyl acrylate, the
title product was isolated in 84% (1.789 g) yield as a colorless oil.
1H NMR (500 MHz, CDCl3) d 3.64 (s, 3H), 2.43 (t, J = 7.5 Hz,
2H), 1.23 (s, 12H), 1.01 (t, J = 7.5 Hz, 2H). 13C NMR (126 MHz,
CDCl3) d 175.15, 100.00, 83.31, 51.58, 28.68, 24.83. HRMS-ESI
(m/z): [M + Na]+ calcd for C10H19[11B]NaO4, 237.1274; found,
237.12754
◦
of the borylated compound at -78 C. After stirring for 30 min,
the alkyl halide was added and the temperature was maintained
at -78 ◦C for 15 min before warming to 0 ◦C. The reaction
was quenched with saturated aqueous ammonium chloride. The
aqueous layer was extracted three times with EtOAc and the
combined organic layers were washed with brine and dried over
sodium sulfate. TLC analysis was performed with 10% ethyl
acetate/hexanes and CAM stain. Flash silica gel chromatography
(0–20% ethyl acetate in hexanes) provided the purified product.
1-Benzyl 4-tert-butyl 2-((4,4,5,5-tetramethyl-1,3,2-dioxaborolan-
2-yl)methyl)succinate (3e)
Using general procedure B, beginning with 2c and alkylating with
tert-butyl bromoacetate, the product was isolated in 24% yield
(50 mg) as colorless oil. 1H NMR (500 MHz, CDCl3) d 7.36-7.27
(m, 5H), 5.11 (q, J = 12.5 Hz, 2H), 3.08-2.99 (m, 1H), 2.64 (dd,
J = 16.2, 8.1 Hz, 1H), 2.48 (dd, J = 16.2, 5.7 Hz, 1H), 1.39 (s,
9H), 1.19 (s, 6H), 1.18 (s, 6H), 1.17-1.13 (m, 1H), 0.99 (dd, J =
16.0, 7.0 Hz, 1H). 13C NMR (126 MHz, CDCl3) d 175.37, 171.18,
Methyl 3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butanoate
(2a)
Using general procedure A, beginning with methyl crotonate, the
title product was isolated in 81% (1.856 g) yield as a colorless oil.
1H NMR (500 MHz, CDCl3) d 3.64 (s, 3H), 2.40 (dd, J = 16.4,
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136.25, 128.51, 128.12, 128.08, 100.00, 83.41, 80.65, 66.36, 39.19,
37.44, 28.10, 24.85, 24.78. HRMS-ESI (m/z): [M + H]+ calcd for
C22H33[11B]O6, 405.2443; found, 405.2445.
The product was isolated in 92% yield (7 mg) as slightly yellow oil.
1H NMR (500 MHz, CDCl3) d 7.29-7.26 (m, 2H), 7.21-7.16 (m,
3H), 3.07 (dd, J = 13.4, 6.5 Hz, 1H), 2.95-2.87 (m, J = 8.7 Hz,
1H), 2.76 (dd, J = 13.4, 7.7 Hz, 1H), 1.21 (s, 6H), 1.20 (s, 6H),
1.04 (dd, J = 16.0, 8.8 Hz, 1H), 0.93 (dd, J = 16.0, 6.1 Hz, 1H).
13C NMR (126 MHz, CDCl3) d 181.23, 139.32, 129.29, 128.42,
126.42, 83.43, 42.74, 39.80, 24.88, 24.72. HRMS-ESI (m/z): [M +
H]+ calcd for C16H23[11B]O4, 289.1617; found, 289.1617.
Methyl 2-benzyl-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-
yl)propanoate (3f)
Using general procedure B, beginning with 2d and alkylating with
benzyl bromide, the title product was isolated in 44% yield (62 mg)
as colorless oil. 1H NMR (400 MHz, CDCl3) d 7.28-7.12 (m, 5H),
3.60 (s, J = 0.6 Hz, 3H), 3.01 (dd, J = 13.1, 6.8 Hz, 1H), 2.91-2.81
(m, 1H), 2.73 (dd, J = 13.1, 7.5 Hz, 1H), 1.21 (s, 6H), 1.20 (s, 6H),
1.03 (dd, J = 15.8, 8.8 Hz, 1H), 0.92 (dd, J = 15.9, 6.2 Hz, 1H).
13C NMR (126 MHz, CD2Cl2) d 176.30, 139.81, 129.14, 128.26,
126.23, 83.20, 51.32, 43.08, 40.14, 24.69, 24.51, 13.72. HRMS-
ESI (m/z): [M + H]+ calcd for C17H25[11B] O4, 305.1922; found,
305.1910.
Solid-phase peptide synthesis
To a cooled solution of Fmoc-Tyr(tBu)-OH (1241 mg, 2.70 mmol)
in CH2Cl2 (15 ml) and minimum volume of DMF (1.5 ml) at 0 ◦C
was added DIC in CH2Cl2 (1 ml). After 30 min, a gelatinous
precipitate developed, which was redissolved in DMF (3 ml).
After an additional 1 h, CH2Cl2 was removed under rotary
evaporator, and the product was dissolved in minimum volume
of DMF and saved for coupling onto Wang resin. In a Poly-
Prep column, Wang resin (800 mg, 0.3 mmol g-1 loading) was
swollen in CH2Cl2 (2 mL, 2 ¥ 15 min) then suspended in DMF
(2 mL, 15 min). To a suspension of the resin bubbling with argon,
tyrosine anhydride (from above) and one small crystal of DMAP
were added. After 45 min, the resin was filtered, washed with
DMF (3¥), CH2Cl2 (3¥) and deprotected with 20% piperidine
in DMF. Further couplings were performed through standard
solid phase peptide synthesis using N-a-Fmoc protected L-amino
acids, HCTU and DIEA in DMF. Solid phase synthesis was
done on a vacuum manifold (Qiagen) outfitted with 3-way Luer
lock stopcocks (Sigma) in either Poly-Prep columns or Econo-
Pac polypropylene columns (Bio-Rad). The resin was mixed in
solution by bubbling argon during all coupling and washing
steps. Fmoc-Ala-OH, Fmoc-Gln(Trt)-OH, Fmoc-Arg(Pbf)-OH,
and Fmoc-Gly-OH were coupled sequentially to provide Fmoc-
Gly-Arg-Gln-Ala-Tyr-Wang resin. Beads were washed extensively
with DMF between reactions, and the couplings were tested for
completeness via Kaiser test.
3-(4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-yl)butanoic acid (4a)
Compound 2a was dissolved in 2 ml THF and hydrolyzed with 4
equiv of LiOH dissolved in 2 ml H2O at room temperature for 1 h.
The product was isolated in 89% (48 mg) yield as colorless oil. 1H
NMR (500 MHz, CDCl3) d 2.49-2.35 (m, 2H), 1.32 (s, 1H), 1.19 (s,
6H), 1.19 (s, 6H), 0.98 (d, J = 7.5 Hz, 3H). 13C NMR (126 MHz,
CDCl3) d 180.40, 83.45, 37.61, 24.85, 24.76, 15.16, (B–C) 13.44.
HRMS-ESI (m/z): [M - H]- calcd for C10H19[11B]O4, 213.1304;
found, 213.1309.
2-(4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-yl)cyclopenta-
necarboxylic acid (4b)
Compound 2b was dissolved in 2 ml THF and hydrolyzed with
4 equiv of LiOH dissolved in 2 ml H2O at room temperature for
1 h. The title product was isolated in 92% yield (112 mg) as a
1
white solid. H NMR (500 MHz, CDCl3) d 2.84 (q, J = 8.6 Hz,
1H), 1.99-1.81 (m, 3H), 1.73-1.62 (m, 2H), 1.61-1.47 (m, 2H), 1.25
(s, 12H). 13C NMR (126 MHz, CDCl3) d 181.56, 83.52, 46.62,
30.37, 28.90, 26.60, 24.74. HRMS-ESI (m/z): [M - H]- calcd for
C12H21[11B]O4, 239.146; found, 239.1458.
Coupling of boronic ester monomers
Following the same procedure for SPPS as above, boronic ester
monomers 4a–f were each coupled to the above sequence. The
couplings were tested for completeness via Kaiser test.
4-tert-Butoxycarbonyl-2-((4,4,5,5-tetramethyl-1,3,2-dioxaborolan-
2-yl)methyl)butanoic acid (4e)
Compound 3e was dissolved in 2 ml MeOH in the presence of
10% Pd/C for 30 min under a large balloon of hydrogen. The
crude mixture was filtered through Celite and purified by flash
silica gel chromatography to provide the product in 83% yield (55
Side-chain deprotection and peptide cleavage
The peptides were cleaved and deprotected by a 6 hour treatment
with 94 : 1 : 2.5 : 2.5 TFA/TIS/H2O/EDT (v/v). The protocol also
removed the pinacol protecting group. After peptide cleavage,
TFA was removed under reduced pressure. The resulting yellow
peptide was washed several times with cold diethyl ether, dried
under reduced pressure and was purified on an Agilent 1200
semi-prep HPLC using a Thermo Scientific Hyper Sil Gold C-18
column.
1
mg) as a colorless oil. H NMR (500 MHz, CDCl3) d 3.02-2.91
(m, J = 28.0 Hz, 1H), 2.59 (dd, J = 16.3, 7.8 Hz, 1H), 2.47 (dd,
J = 16.3, 5.5 Hz, 1H), 1.41 (s, 9H), 1.21 (s, 6H), 1.20 (s, 6H), 1.09
(dd, J = 15.8, 8.0 Hz, 1H), 0.94 (dd, J = 15.9, 7.1 Hz, 1H). 13C
NMR (126 MHz, CDCl3) d 181.40, 171.57, 83.25, 80.99, 39.02,
37.45, 28.09, 24.80, 24.76, (B–C) 14.19. HRMS-ESI (m/z): [M -
H]- calcd for C15H27[11B]O6, 313.1901; found, 313.1812.
Fluorescent peptidase assay
2-Benzyl-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-
yl)propanoic acid (4f)
ClpXP peptidase activity was monitored using a previously
designed assay34 which utilizes an 11-amino acid peptide
(FRETN 89–98) that contains an anthranilamide-nitrotyrosine
donor/quencher pair on opposite termini. Fluorescent peptidase
Compound 3f was dissolved in 1 ml THF and hydrolyzed with 4
equiv of LiOH dissolved in 1 ml H2O at room temperature for 1 h.
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assays were performed on a Fluoro-Max3 spectrophotometer
at 37 ◦C using excitation and emission wavelengths of 320 nm
and 420 nm, respectively. Reactions containing 50 mM HEPES
(pH 8.1), 5 mM Mg(OAc)2, 2 mM DTT, and 300 nM ClpXP were
incubated in the absence and presence of 1 mM ATP for 1 min at
37 ◦C. Reactions were initiated by the addition of 500 mM FRETN
89–98 and fluorescence emission was monitored for 1200 s. In
all assays, a mixed substrate was used (90% non-fluorescent 10%
fluorescent) to avoid the inner filter effect.35
Miller Jeffress Memorial Trust. We thank Jason Hudak for
assistance in the production of recombinant human ClpXP.
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Acknowledgements
We gratefully acknowledge financial support from Virginia Tech
Department of Chemistry and the Thomas F. Jeffress and Kate
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3456 | Org. Biomol. Chem., 2010, 8, 3451–3456
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The Royal Society of Chemistry 2010
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