A panel of subunit-selective activity-based proteasome probes

Article abstract of DOI:10.1039/c001036g

Mammals express seven different catalytically active proteasome subunits. In order to determine the roles of the different proteolytically active subunits in antigen presentation and other cellular processes, highly specific inhibitors and activity-based probes that selectively target specific active sites are needed. In this work we present the development of fluorescent activity-based probes that selectively target the β1 and β5 sites of the constitutive proteasome.

Full text of DOI:10.1039/c001036g

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
A panel of subunit-selective activity-based proteasome probes†‡
Martijn Verdoes,§a Lianne I. Willems,a Wouter A. van der Linden,a Boudewijn A. Duivenvoorden,a
Gijsbert A. van der Marel,a Bogdan I. Florea,a Alexei F. Kisselev*b and Herman S. Overkleeft*c
Received 15th January 2010, Accepted 9th April 2010
First published as an Advance Article on the web 6th May 2010
DOI: 10.1039/c001036g
Mammals express seven different catalytically active proteasome subunits. In order to determine the
roles of the different proteolytically active subunits in antigen presentation and other cellular processes,
highly specific inhibitors and activity-based probes that selectively target specific active sites are needed.
In this work we present the development of fluorescent activity-based probes that selectively target the
b1 and b5 sites of the constitutive proteasome.
b1 sites are apparently redundant. Thus, it is not clear why b1 sites
are present in all eukaryotes from yeast to humans.
Introduction
The proteasome inhibitor bortezomib (Velcade, PS-341)4 is
being used for the treatment of multiple myeloma5 and mantle cell
lymphoma,6 and at least three other inhibitors are tested clinically.
The role of individual active sites as drug targets in cancer has
not been fully elucidated. The b5 and b5i sites are the primary
targets of all inhibitors used in the clinic, but the majority of them
also target other sites. For example, bortezomib also inhibits b1
sites of the constitutive proteasome and b1i and b2i sites of the
immunoproteasome.7,8 Whether or not the co-inhibition of b1/b1i
and b2i sites contributes to anti-neoplastic activity of bortezomib
is not clear. Using newly developed specific inhibitors of the b1 and
b5 sites (Fig. 1A, NC-001 (1) and NC-005 (2), respectively), we
have recently demonstrated that inhibition of the b1 sites sensitizes
myeloma cells to b5 selective inhibitors.9 This suggests that the b1
sites, despite their apparent redundancy, have to be considered
co-targets for anti-neoplastic drugs. Furthermore, proteasomes
are involved in the generation of antigenic peptides loaded in
MHC class I complexes, but the contribution of each separate
active proteasome subunit to the epitope repertoire has not yet
been defined, largely due to the lack of cell permeable site-specific
inhibitors.
Proteasomes degrade the majority of proteins in mammalian cells
and are established targets for the development of anticancer
drugs. All eukaryotic proteasomes have three types of active sites.
The site located on the b1 subunit is referred to as caspase-
like, the site on subunit b2 as trypsin-like and the site on the
b5 subunit as chymotryptisin-like, although the subunits are
rather more promiscuous in their substrate preference than is
suggested by this designation. Four additional proteolytically
active sites are present on proteasomes in specific cell types in
vertebrates. In cells of the immune system, the b1i, b2i and the
b5i subunits replace the corresponding constitutive subunits in
newly formed proteasome particles called immunoproteasomes.1
Cortical thymic epithelial cells express an additional proteolytic
subunit, the b5t, which replaces b5i in immunoproteasomes to
form the thymoproteasome.2
This diversity of active sites in eukaryotic proteasomes is in a
stark contrast to the much simpler proteasomes in bacteria and
archaea, which possess only one or two types of active sites. In
prokaryotic proteasomes there are proteolytic active sites on all
14 b-subunits. The total number of active sites on eukaryotic
proteasomes is 6 (two of each type). The reasons for such dramatic
reduction in total number of active sites (14 to 6) in favor of more
diverse specificity are not clear. Site-directed mutagenesis in yeast
revealed that the different active sites play specific roles in protein
degradation, and that their contribution to growth, viability and
stress resistance of yeast strains are strikingly different.3 According
to these studies, the b5 sites are the most important, whereas the
aLeiden Institute of Chemistry, Gorlaeus Laboratories, Leiden University,
PO Box 9502, 2300 RA Leiden, The Netherlands
bDepartment of Pharmacology and Toxicology, Norris Cotton Cancer
Center, Dartmouth Medical School, 1 Medical Center Drive, HB7936,
Lebanon, NH 03756, USA. E-mail: Alexei.F.Kisselev@Dartmouth.edu
Fig. 1 (A) Schematic representation of the eukaryotic 20S proteasome
and previously described b1 and b5 specific proteasome inhibitors. The
proteolytically active b subunits are marked in red. (B) Recognition
elements I and II and warheads a–c resulting in 6 hybrid structures, Ia–c
and IIa–c.
cLeiden Institute of Chemistry and Netherlands Proteomics Centre, Gorlaeus
Laboratories, Einsteinweg 55, 2333 CC Leiden, The Netherlands. E-mail:
h.s.overkleeft@chem.leidenuniv.nl
† This paper is part of an Organic & Biomolecular Chemistry web theme
issue on protein labelling and chemical proteomics. Guest editors: Ed Tate
and Matthew Bogyo.
In order to determine the roles of the different proteolytically ac-
tive subunits in antigen presentation and other cellular processes,
highly specific inhibitors and activity-based probes that selectively
target specific active sites are needed. In this study we use the
inhibitors we previously reported as being selective for the b1 and
‡ Electronic supplementary information (ESI) available: 1H NMR spectra.
See DOI: 10.1039/c001036g
§ Present address: Department of Pathology, Stanford University School
of Medicine, Stanford, CA 94305, USA
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b5 sites,9 Ac-Ala-Pro-Nle-LeuEK (1) and Napht-Tyr(OMe)-Phe-
LeuEK (2), respectively, as the basis for the development of a panel
of fluorescent site-specific activity-based probes.
with TMS–diazomethane to give methyl ester 5. Refluxing in
methanol in the presence of an excess of hydrazine resulted in
the b1 recognition element peptide hydrazide 6. The b5 recognition
peptide hydrazide was prepared by condensation of Boc-protected
tyrosine methyl ether 7 with phenylalanine methyl ester to give
dipeptide 8. After acidic cleavage of the Boc protective group
and capping with 2-(naphthalen-2-yl)-acetic acid, methyl ester 9
was reacted with hydrazine in refluxing methanol to give peptide
hydrazide 10. The recognition element peptide hydrazides were
treated with tert-butyl nitrite under acidic conditions to generate
an acyl azide in situ. After addition of base, the warhead amines
(synthesized according to literature procedures12–14) were reacted
with the activated peptides to give the hybrid structures Ia–c and
IIa–c.
The proteasome inhibition profile of the panel of 6 modified
oligopeptides was determined in competition experiments versus
the fluorescent broad-spectrum activity-based proteasome probe
MV151.15 Human Embryonic Kidney (HEK293T) cell lysates
expressing the constitutive proteasome were exposed to increasing
concentrations of the inhibitors for one hour. Residual proteasome
activity was labeled with MV151, after which the proteins were
denatured, separated on SDS-PAGE and visualized using a fluo-
rescence scanner (Fig. 2A). Apparent IC50 values were determined
by quantification of the fluorescent gel bands (Fig. 2A and
Table 1). As reported, inhibitors Ia16 and Ic (1)9 selectively block
the b1 subunit. The selectivity and potency of the methyl vinyl
sulfone-equipped b1 recognition peptide Ib is in the same order of
magnitude as the phenol vinyl sulfone Ia, but both are one order
of magnitude less potent than the b1 selective epoxyketone Ic.
Results and discussion
The majority of proteasome inhibitors are short peptides with
an electrophilic group at the C-terminus.10 This electrophilic
“warhead” reacts with the proteasome’s catalytic N-terminal
threonines. Previously, we demonstrated that swapping the peptide
portions and warheads of known proteasome inhibitors can
alter active site specificities and potencies.11 We decided to
test whether changing the warheads on compounds 1 and 2
improves their potency and specificity. The best characterized
and most commonly used proteasome warheads are aldehydes,
vinyl sulfones, boronates and epoxyketones. Of these, only vinyl
sulfones and epoxyketones are suitable for use in activity-based
probes, since only these form covalent irreversible bonds with
the proteasome active sites that are not destroyed during heating
under denaturing conditions, a mandatory step preceding analysis
of probe-treated samples by SDS-PAGE. Therefore, we have
synthesized derivatives of 1 and 2 armed with leucine phenol vinyl
sulfone (LeuVS-PhOH),12 leucine methyl vinyl sulfone (LeuVS)13
and leucine epoxyketone (LeuEK)14 to arrive at a panel of 6
potential subunit specific proteasome inhibitors (Fig. 1B). All
inhibitors used in the study were synthesized by coupling the
recognition fragments to the leucine-derived warheads. In order to
avoid epimerization at the P2 position, we used the azide coupling
(Scheme 1). Hence, the synthesis commenced with the preparation
of the hydrazides of the peptidic recognition elements. Fmoc-
based solid phase peptide synthesis using HMPB-functionalized
MBHA resin gave immobilized acetyl capped tripeptide 3. After
mild acidic cleavage from resin, the crude peptide was treated
Fig. 2 Proteasome profiling screen of the hybrids Ia–c and IIa–c using
MV151. (A) HEK293T lysates (10 mg total protein) were incubated with the
indicated concentrations of compounds Ia–c and IIa–c for 1 h at 37 ◦C. The
residual proteasome activity was fluorescently labeled by incubation with
1 mM MV151 for 1 h at 37 ◦C. (B–C) The fluorescent band intensity for
each subunit was quantified to give competition curves as shown in (B) for
IIb and in (C) for IIc.
Scheme 1 Synthesis of the hybrid structures. Reagents and conditions: (i)
1% TFA in DCM, 30 min, 3¥. (ii) TMS–diazomethane (2 equiv.), 15 min.,
85% from Fmoc-Nle-resin. (iii) Hydrazine monohydrate (60 equiv.),
MeOH, reflux, 1.5 h, 92%. (iv) (a) HCl (2.8 equiv.), tBuONO (1.1 equiv.),
DMF–EtOAc (1 : 1, v/v), -25 ◦C, 4 h. (b) TFA·H-Ra–c (1.1 equiv.), DiPEA
(3.8 equiv.), -25 ◦C to RT, 15 h, Ia 77%, Ib 34%, Ic 63%, IIa 77%, IIb
53%, IIc 66%. (v) HCl·H-Phe-OMe (1 equiv.), BOP (1 equiv.), DiPEA
(2.2 equiv.), DCM, 15 h, quant. (vi) TFA/DCM 1 : 1 (v/v), 15 min.
(vii) 2-(Naphthalen-2-yl)-acetic acid (1 equiv.), BOP (1 equiv.), DiPEA
(3.3 equiv.), DCM, 15 h, 68%. (viii) Hydrazine monohydrate (60 equiv.),
MeOH, reflux, 2.5 h, 95%.
In the panel of inhibitors containing the b5 recognition element,
phenol vinyl sulfone IIa is a more selective inhibitor of the b5
subunit than the parent epoxyketone IIc (2), but still targets
b1 and b2 at higher concentrations. However, vinyl sulfone IIb
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Table 1 Apparent IC50 values (mM) for Ia–c and IIa–ca
and subsequent capping with azido glycine afforded resin 17
(Scheme 3). Mild acidic cleavage, followed by reaction with TMS–
diazomethane resulted in azido containing peptide methyl ester 19,
which was converted to the corresponding hydrazide (20). An azide
coupling with the LeuEK warhead resulted in the b1 selective two-
step activity-based proteasome probe 21.9 Reaction with BODIPY
dye 22 by a copper catalysed Huisgen [2 + 3] cycloaddition then
afforded the fluorescently labeled probe 23 (Scheme 3).
Subunit
b1
b2
b5
Compound
Ia
4.0 0.67
7.8 0.86
0.66 0.12
32 16
>100
>100
>100
>100
>100
78 23
>100
>100
>100
373 91
0.07 0.01
0.11 0.02
0.013 0.001
Ib
Ic
IIa
IIb
IIc
0.28 0.04
a In the gels shown in Fig. 2, the fluorescent band intensity for each subunit
was quantified in each lane to give competition curves, which were then
used to calculate apparent IC50 values. IC50 values for irreversible inhibitors
do not refer to inhibition constants (IC50 π Ki). It is the observed 1/2 max
inhibition concentration for the specific experimental setup.
is a very potent and very selective inhibitor for the b5 subunit
(Fig. 2B). Although about 10 times more potent for b5, the parent
epoxyketone IIc (2) was much less selective, since it also blocks
the b2 subunit, leaving b1 as the sole active proteasome subunit
(Fig. 2C).
Three inhibitors were chosen as starting points for the develop-
ment of fluorescent activity-based probes: Ic that is selective for
b1, IIb as a selective and potent inhibitor for b5 and IIc inhibiting
b2 and b5. It was reasoned that replacing the aromatic naphthyl
acetic acid in inhibitors IIb and IIc (2) by azido-BODIPY17
would not affect the specificity of the resulting fluorescent probes.
The synthesis of the b5 targeted probes commenced with the
transformation of the Boc-protected dipeptide methyl ester 8 into
the corresponding hydrazide 11 (Scheme 2). Azide coupling with
LeuVS gave methyl vinyl sulfone 12. Acidic deprotection and
subsequent capping of the free amine with azido-BODIPY-OSu
(13) gave the fluorescent probe 14. Similarly, b2/b5 targeting probe
16 was constructed from Boc-protected tripeptide epoxyketone 15.
For the synthesis of a fluorescent b1 selective probe, we
took advantage of the previously described green fluorescent
acetylene functionalized BODIPY dye 22.18 The azide equipped
intermediate 21 is easily accessible by replacing the acetyl in Ic
with an azido glycine. Standard solid-phase peptide synthesis
Scheme 3 Synthesis of the fluorescent b1 selective proteasome probe
23. Reagents and conditions: (i) 1% TFA in DCM, 30 min, 3¥.
(ii) TMS–diazomethane (2 equiv.), 15 min, 90% from Fmoc-Nle-resin.
(iii) Hydrazine monohydrate (60 equiv.), MeOH, reflux, 1.5 h, quant.
(iv) (a) HCl (2.8 equiv.), tBuONO (1.1 equiv.), DMF–EtOAc (1 : 1, v/v),
-25 ◦C, 4 h. (b) TFA·H-Leu-epoxyketone (1.1 equiv.), DiPEA (3.8 equiv.),
-25 ◦C to RT, 15 h, 23%. (v) 22, CuSO4 (10 mol%), sodium ascorbate
(15 mol%), Tol.–H2O–tBuOH (1 : 1 : 1, v/v/v), 80 ◦C, 22 h, 65%.
Having synthesized the fluorescent derivatives of the subunit
selective inhibitors, their proteasome labeling profile and cell-
permeability were determined (Fig. 3). Fluorescently tagged
epoxomicin analogues 2418 and 2519 (Fig. 3D), which label
all proteolytically active proteasome subunits, were used as a
reference. Probe 23, designed to specifically target the b1 active
site, indeed shows a marked preference for b1 in HEK293T cell
lysates (Fig. 3A, left panel). At higher concentrations it starts
Scheme 2 Synthesis of azido-BODIPY probes 14 and 16. Reagents and conditions: (i) Hydrazine monohydrate (60 equiv.), MeOH, reflux, 2 h, 88%.
(ii) (a) HCl (2.8 equiv.), tBuONO (1.1 equiv.), DMF–EtOAc (1 : 1, v/v), -25 ◦C, 4 h. (b) TFA·H-LeuVS (1.1 equiv.), DiPEA (3.8 equiv.), -25 ◦C to RT,
15 h, 75%. (iii) TFA/DCM 1 : 1 (v/v), 30 min. (iv) 13 (1 equiv.), DiPEA (1 equiv.), DCM, 15 h, 14 54%, 16 27%. (v) (a) HCl (2.8 equiv.), tBuONO
(1.1 equiv.), DMF–EtOAc (1 : 1, v/v), -25 ◦C, 4 h. (b) TFA·H-LeuEK (1.1 equiv.), DiPEA (3.8 equiv.), -25 ◦C to RT, 15 h, 71%.
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Fig. 3 Labeling profile of probe 14, 16 and 23. (A) HEK293T lysates (10 mg total protein) were incubated with the indicated concentrations of 23, 24
(left panel), 14 (middle panel) or 16 (right panel) for 1 h at 37 ◦C. (B) HEK293T cells (some 5 ¥ 105 cells) were exposed to the indicated concentrations of
23 or 24 (left panel), 14 or 25 (middle panel) or 16 (right panel) for 2 h at 37 ◦C. (C) Quantification of fluorescent labeling of living cells treated with 23
(left panel), 14 (middle panel) and 16 (right panel). Corresponding gels are shown in Fig. 3B. (D) Structures of reference probes 24 and 25.
to label b5, presumably because of the increase in steric bulk
and hydrophobicity due to BODIPY addition. To atest the cell
permeability, living HEK293T cells were exposed to increasing
concentrations of the probe (Fig. 3B, left panel). The fact that a
similar labeling profile is observed shows that the introduction of
the BODIPY dye has no dramatic effect on the cell permeability
of the probe. Plotting of the fluorescent band intensity gave an
SDS-PAGE. Using the probes labeled with different fluorophores
developed in this work should eliminate this problem. In order to
demonstrate that this is indeed the case, we have treated extracts
simultaneously with the b1-specific probe 23 and either of b5-
specific probes 14 and 16 (Fig. 4). Both subunits were detected
and clearly resolved with 2-color imaging.
apparent EC50 value of 62
5 nM for b1 labeling (Fig. 3C,
left panel). When using 1 mM probe, one can selectively saturate
the labeling of roughly all b1 sites, leaving most of the b5 sites
untouched.
HEK293T cell lysates incubated with azido-BODIPY methyl
vinyl sulfone probe 14 showed specific b5 labeling (Fig. 3A, middle
panel), with only minimal labeling of what is presumably b1 at high
concentrations. A similar labeling profile is observed when living
HEK293T cells are exposed to the fluorescent probe (Fig. 3B,
middle panel). Quantification of the fluorescent labeling revealed
an apparent EC50 value of 163 5 nM (Fig. 3C, middle panel).
Probe 16, designed to target b5 and, at higher concentrations,
the b2 site, indeed labeled both sites in lysates (Fig. 3A, right
panel). In living cells b5 labeling appears to reach a maximum
around 1 mM, after which the labeling intensity starts to decrease,
whereas the labeling of the b2 subunit keeps increasing (Fig. 3B
and C, right panel). The reason for this phenomenon remains to
be investigated.
Fig. 4 Labeling and, hence, chemical knock-down of specific proteasome
active sites. HEK293T lysates (10 mg total protein) were incubated with
the indicated concentrations of probes for 1 h at 37 ◦C. Merged image
of fluorescence scanned at lex 488, lem 520 nm in green and lex 532, lem
560 nm in red.
Conclusions
Building on our expertise in developing site-specific proteasome
inhibitors9,16 we describe here the development of fluorescent
subunit-specific activity-based proteasome probes 14, 16 and 23.
We have also made the intriguing observation that replacing the
epoxyketone warhead in compound 2 for a vinyl sulfone increases
Poor resolution of b5 and b1 subunits is often a problem
in the analysis of subunit-specific inhibition and labeling on
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the specificity of the b5-targeting inhibitors, with the methyl vinyl
sulfone inhibitor IIb being the most b5 specific. An advantage of
the fluorescent selective proteasome probes is the better resolution
of the b1 and b5 subunits on the SDS-PAGE gels using 2 color
imaging. The ability to chemically knock-down specific active sites
and being able to validate and monitor this using the fluorescent
readout will facilitate the elucidation of the roles of the individual
proteasome active sites as drug targets in cancer and in other
biological processes such as MHC class I antigen presentation.
5 mol%) in DCM for 2 h. The resin was filtered and washed
with DCM (2¥), followed by a second condensation cycle. The
loading of the resin was determined to be 0.46 mmol g-1 (4.28 g,
1.97 mmol, 98%) by spectrophotometric analysis. The obtained
resin was submitted to two cycles of Fmoc solid-phase synthesis
with Fmoc-Pro-OH and Fmoc-Ala-OH, respectively, as follows:
(a) deprotection with piperidine–NMP (1 : 4, v/v, 20 min.), (b)
wash with NMP (3¥), (c) coupling of Fmoc amino acid (5 mmol,
2.5 equiv.) in the presence of BOP (2.2 g, 5 mmol, 2.5 equiv.) and
DiPEA (0.99 ml, 6 mmol, 3 equiv.) in NMP and shaken for at least
2 h, (d) wash with NMP (3¥) and DCM (3¥), yielding resin-bound
Fmoc-Ala-Pro-Nle. Couplings were monitored for completion
by the Kaiser test. After Fmoc deprotection of 1.2 mmol, the
resin-bound tripeptide was capped with acetic anhydride (0.57 ml,
6 mmol, 5 equiv.) and DiPEA (1.98 ml, 12 mmol, 10 equiv.) for
15 min. Mild acidic cleavage of resin 3 with 1% TFA in DCM (3 ¥
10 min.) resulted in Ac-Ala-Pro-Nle-OH 4 which was used without
purification. The crude peptide 4 was dissolved in MeOH–Tol.
(1 : 1) and treated with TMS–diazomethane (1.2 ml 2 M in hexanes,
2.4 mmol, 2 equiv.) for 15 min before being coevaporated with
Tol. (3¥). Purification by flash column chromatography (DCM →
3% MeOH in DCM) yielded the title compound as a white solid
Experimental
General
All reagents were commercial grade and were used as received
unless indicated otherwise. Toluene (Tol.) (purum), ethyl acetate
(EtOAc) (puriss.), diethyl ether (Et2O) and light petroleum
ether (PetEt) (puriss.) were obtained from Riedel-de Hae¨n.
Dichloroethane (DCE), dichloromethane (DCM), dimethyl form-
˚
amide (DMF) and dioxane (Biosolve) were stored on 4 A
molecular sieves. Methanol (MeOH) and N-methylpyrrolidone
(NMP) were obtained from Biosolve. Reactions were monitored by
TLC-analysis using DC-alufolien (Merck, Kieselgel60, F254) with
detection by UV-absorption (254 nm), spraying with 20% H2SO4 in
ethanol followed by charring at ~150 ◦C, by spraying with a solu-
tion of (NH4)6Mo7O24·4H2O (25 g L-1) and (NH4)4Ce(SO4)4·2H2O
(10 g L-1) in 10% sulfuric acid followed by charring at ~150 ◦C, or
spraying with an aqueous solution of KMnO4 (20%) and K2CO3
(10%). Column chromatography was performed on Screening Di-
vices Silica gel (0.040–0.063 nm). LC/MS analysis was performed
on a LCQ Adventage Max (Thermo Finnigan) equipped with a
Gemini C18 column (Phenomenex). The applied buffers were A:
H2O, B: MeCN and C: 1.0% aq. TFA. HRMS were recorded
on a LTQ Orbitrap (Thermo Finnigan). 1H- and 13C-APT-NMR
spectra were recorded on a Jeol JNM-FX-200 (200/50 MHHz),
Bruker DPX-300 (300/75 MHz), Bruker AV-400 (400/100 MHz)
equipped with a pulsed field gradient accessory, a Bruker AV-
500 (500/125 MHz) or a Bruker DMX-600 (600/150 MHz)
with cryoprobe. Chemical shifts are given in ppm (d) relative
to tetramethylsilane as internal standard. Coupling constants are
given in Hz. All presented 13C-APT spectra are proton decou-
pled. Optical rotations were measured on a Propol automatic
polarimeter (sodium D line, l = 589 nm). Boc-LeuVS-PhOH,12
Boc-LeuVS,13 Boc-LeuEK14 and were synthesized as described in
literature.
1
(0.36 g, 1.0 mmol, 85%). H NMR (300 MHz, CDCl3): d ppm
7.42 (d, J = 7.8 Hz, 1H), 7.14 (d, J = 7.5 Hz, 1H), 4.78 (m, 1H),
4.64 (m, 1H), 4.50 (m, 1H), 3.78 (m, 1H), 3.74 (s, 3H), 3.59 (m,
1H), 2.29 (m, 1H), 2.12 (m, 1H), 2.06–1.93 (m, 6H), 1.80 (m, 1H),
1.66 (m, 1H), 1.36 (d, J = 6.9 Hz, 3H), 1.28 (m, 4H), 0.87 (t, J =
6.8 Hz, 3H).
Ac-Ala-Pro-Nle-hydrazide (6)
Ac-Ala-Pro-Nle-OMe (5, 0.36 g, 1.0 mmol) was dissolved in
MeOH. Hydrazine monohydrate (2.9 ml, 60 mmol, 60 equiv.)
was added and the reaction mixture was refluxed for 1.5 h. Tol.
was added and the resulting white solid was filtered to give the
1
title compound (0.33 g, 0.92 mmol, 92%). H NMR (400 MHz,
CDCl3): d ppm 4.60 (q, J = 7.0 Hz, 1H), 4.46 (dd, J1 = 8.2, J2 =
4.6 Hz, 1H), 4.25 (dd, J1 = 8.4, J2 = 6.0 Hz, 1H), 3.85–3.77 (m,
1H), 3.69–3.60 (m, 1H), 2.26–2.13 (m, 1H), 2.11–2.01 (m, 1H),
2.00–1.91 (m, 5H), 1.82–1.71 (m, 1H), 1.71–1.60 (m, 1H), 1.42–
1.24 (m, 5H), 0.91 (t, J = 6.8 Hz, 3H). 13C NMR (100 MHz,
CDCl3): d ppm 174.12, 173.77, 173.45, 172.80, 61.49, 53.48, 48.50,
48.46, 32.88, 30.31, 28.97, 26.00, 23.36, 22.26, 16.88, 14.28.
Boc-Tyr(Me)-Phe-OMe (8)
HCl·H-Phe-OMe (2.16 g, 10 mmol) and Boc-Tyr(Me)-OH (7,
2.95 g, 10 mmol, 1 equiv.) were dissolved in DCM. BOP (4.42 g,
10 mmol, 1 equiv.) and DiPEA (3.64 ml, 16.5 mmol, 2.2 equiv.)
were added and the reaction mixture was stirred for 15 h before
being washed with 0.5 M HCl (aq.) and sat. aq. NaHCO3,
separated and dried over MgSO4. Purification by flash column
chromatography (20% EtOAc in PetEt → 40% EtOAc in PetEt)
yielded the title compound as a white solid (4.6 g, 10 mmol,
quant.). 1H NMR (200 MHz, CDCl3): d ppm 7.32–7.17 (m, 4H),
7.15–7.05 (m, 3H), 7.04–6.94 (m, 2H), 6.81 (d, J = 8.7 Hz, 2H),
6.38 (d, J = 7.8 Hz, 1H), 4.78 (q, J = 5.9, 5.9, 5.9 Hz, 1H), 4.38–
4.19 (m, 1H), 3.78 (s, 3H), 3.67 (s, 3H), 3.05 (dd, J = 5.9, 1.6 Hz,
2H), 2.96 (d, J = 6.7 Hz, 2H), 1.40 (s, 9H). 13C NMR (50 MHz,
Ac-Ala-Pro-Nle-OMe (5)
4-Methylbenzhydrylamine (MBHA)-functionalized polystyrene
resin (7.14 g, 0.7 mmol g-1, 5 mmol) was washed with NMP
(3¥) followed by addition of a preactivated mixture of 4-(4-
hydroxymethyl-3-methoxyphenoxy)-butyric acid (HMPB) linker
(3.6 g, 15.0 mmol, 3 equiv.), BOP (6.6 g, 15 mmol, 3 equiv.) and
DiPEA (5 mL, 30 mmol, 6 equiv.) in NMP. After 2 h of shaking,
the resin was washed with NMP (3¥) and DCM (3¥), dried and
used as such. Part of the resin (2 mmol) was transferred to a
flask, coevaporated with DCE (2¥), and condensed with Fmoc-
Nle-OH (2.12 g, 6 mmol, 3 equiv.) under the influence of DIC
(1.0 mL, 6.6 mmol, 3.3 equiv.) and DMAP (6.6 mg, 0.3 mmol,
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CDCl3): d ppm 171.40, 158.15, 155.30, 135.62, 129.98, 128.86,
128.31, 128.10, 126.64, 113.54, 78.93, 55.41, 54.77, 53.13, 51.80,
37.46, 37.15, 27.78.
added and the organic layer was separated, dried over MgSO4 and
concentrated in vacuo. The crude product was purified by flash
column chromatography.
(Tyr(Me)-Phe-OMe)-2-(naphthalen-2-yl)-acetamide (9)
Ac-Ala-Pro-Nle-Leu-4-hydroxyphenyl-vinylsulfone (Ia)
Boc-Tyr(Me)-Phe-OMe (8, 4.6 g, 10 mmol) was dissolved in TFA–
DCM 1 : 1 (v/v). The reaction mixture was stirred for 15 min
before being co-evaporated with Tol. (3¥). The crude TFA salt
was dissolved in DCM and 2-(naphthalen-2-yl)-acetic acid (1.86 g,
10 mmol, 1 equiv.), BOP (4.42 g, 10 mmol, 1 equiv.) and DiPEA
(5.46 ml, 33 mmol, 3.3 equiv.) were added. After being stirred for
15 h the reaction mixture was washed with 0.5 M HCl (aq.) and
sat. aq. NaHCO3, separated and dried over MgSO4. Purification
by flash column chromatography (PetEt → EtOAc, followed by
a second column: DCM → 30% EtOAc in DCM), washing with
H2O (3¥) and drying over MgSO4 gave the pure title compound
Following the general procedure for azide coupling, the title com-
pound was obtained from Boc-LeuVS-PhOH (61 mg, 0.17 mmol,
1.1 equiv.) and Ac-Ala-Pro-Nle-hydrazide (6, 53.3 mg, 0.15 mmol).
Purification by flash column chromatography (DCM → 6%
MeOH in DCM) gave Ia as colorless oil (68.4 mg, 0.12 mmol,
77%). 1H NMR (500 MHz, DMSO, T = 353 K): d ppm 7.68 (d,
J = 8.8 Hz, 2H), 6.92 (d, J = 8.8 Hz, 2H), 6.79 (dd, J1 = 15.0,
J2 = 5.1 Hz, 1H), 6.54 (dd, J1 = 15.1, J2 = 1.4 Hz, 1H), 4.69–4.59
(m, 1H), 4.56 (q, J = 7.0 Hz, 1H), 4.38 (dd, J1 = 8.2, J2 = 5.1 Hz,
1H), 4.17 (dd, J1 = 8.2, J2 = 6.0 Hz, 1H), 3.84–3.73 (m, 1H),
3.68–3.56 (m, 1H), 2.26–2.09 (m, 1H), 2.07–1.84 (m, 6H), 1.81–
1.59 (m, 3H), 1.58–1.49 (m, 1H), 1.47–1.38 (m, 1H), 1.36–1.24
(m, 7H), 0.97–0.83 (m, 9H). HRMS: calcd. for [C29H44N4O7SH]+
593.30035, found 593.30046.
1
as a white solid (3.55 g, 6.8 mmol, 68%). H NMR (200 MHz,
CDCl3): d ppm 8.53 (d, J = 7.5 Hz, 1H), 8.27 (d, J = 8.7 Hz, 1H),
7.91–7.70 (m, 3H), 7.59 (s, 1H), 7.54–7.40 (m, 2H), 7.32–7.16 (m,
6H), 7.11 (d, J = 8.7 Hz, 2H), 6.71 (d, J = 8.7 Hz, 2H), 4.62–4.41
(m, 2H), 3.65 (s, 3H), 3.62 (d, J = 13.7 Hz, 1H), 3.58 (s, 3H), 3.49
(d, J = 14.0 Hz, 1H), 3.13–2.83 (m, 3H), 2.67 (dd, J1 = 13.7, J2 =
9.9 Hz, 1H).
Ac-Ala-Pro-NLe-Leu-methyl-vinylsulfone (Ib)
Following the general procedure for azide coupling, the ti-
tle compound was obtained from Boc-LeuVS (50.7 mg,
0.17 mmol, 1.1 equiv.) and Ac-Ala-Pro-NLe-hydrazide (6, 53.3 mg,
0.15 mmol). Purification by flash column chromatography
(DCM → 4% MeOH in DCM) gave Ib as white solid (26.1 mg,
51 mmol, 34%). 1H NMR (500 MHz, DMSO, T = 353 K): d ppm
7.84 (s, 1H), 7.63–7.53 (m, 2H), 6.74–6.58 (m, 2H), 4.63–4.48 (m,
2H), 4.39–4.31 (m, 1H), 4.16 (dd, J1 = 13.5, J2 = 7.7 Hz, 1H),
3.69–3.61 (m, 1H), 3.60–3.52 (m, 1H), 2.95 (s, 3H), 2.10–2.00 (m,
1H), 1.98–1.86 (m, 3H), 1.84 (s, 3H), 1.77–1.67 (m, 1H), 1.67–1.55
(m, 2H), 1.54–1.46 (m, 1H), 1.45–1.35 (m, 1H), 1.33–1.24 (m, 4H),
1.22 (d, J = 6.8 Hz, 3H), 0.93–0.84 (m, 9H). HRMS: calcd. for
[C24H42N4O6SH]+ 515.28978, found 515.28961.
(Tyr(Me)-Phe-hydrazinyl)-2-(naphthalen-2-yl)-acetamide (10)
To
a solution of (Tyr(Me)-Phe-OMe)-2-(naphthalen-2-yl)-
acetamide (9, 0.52 g, 1 mmol) in MeOH was added hydrazine
monohydrate (2.91 ml, 60 mmol, 60 equiv.). The reaction mixture
was refluxed for 2.5 h. The title compound precipitated as a
white solid and was filtered off and washed with MeOH (0.5 g,
1
0.95 mmol, 95%). H NMR (600 MHz, CDCl3): d ppm 9.22 (s,
1H), 8.25 (t, J = 7.9, 7.9 Hz, 2H), 7.86 (d, J = 7.8 Hz, 1H),
7.79 (d, J = 7.9 Hz, 1H), 7.77 (d, J = 8.5 Hz, 1H), 7.62 (s, 1H),
7.50–7.44 (m, 2H), 7.29–7.21 (m, 5H), 7.20–7.15 (m, 1H), 7.10 (d,
J = 8.6 Hz, 2H), 6.71 (d, J = 8.6 Hz, 2H), 4.59–4.51 (m, 2H),
4.29 (s, 2H), 3.65 (s, 3H), 3.64 (d, J = 12.5 Hz, 1H), 3.53 (d, J =
14.0 Hz, 1H), 2.99 (dd, J1 = 13.7, J2 = 5.6 Hz, 1H), 2.94 (dd,
J1 = 13.7, J2 = 3.9 Hz, 1H), 2.86 (dd, J1 = 13.7, J2 = 8.7 Hz,
1H), 2.70 (dd, J1 = 13.6, J2 = 10.1 Hz, 1H). 13C NMR (50 MHz,
CDCl3): d ppm 170.97, 170.00, 169.77, 157.66, 137.51, 133.91,
132.91, 131.71, 130.22, 129.50, 129.16, 128.07, 127.65, 127.41,
127.35, 127.21, 126.28, 125.93, 125.43, 113.30, 54.76, 53.98, 52.67,
42.34, 38.05, 36.70.
Ac-Ala-Pro-NLe-Leu-epoxyketone (Ic)
Following the general procedure for azide coupling, the title
compound was obtained from Boc-LeuEK (47.4 mg, 0.17 mmol,
1.1 equiv.) and Ac-Ala-Pro-NLe-hydrazide (6, 53.3 mg,
0.15 mmol). Purification by flash column chromatography
(DCM → 4% MeOH in DCM) gave Ib as colorless oil (47.1 mg,
95 mmol, 63%). 1H NMR (500 MHz, DMSO, T = 353 K): d ppm
7.80 (s, 1H), 7.73 (d, J = 7.3 Hz, 1H), 7.50 (d, J = 5.6 Hz, 1H),
4.60–4.48 (m, 1H), 4.47–4.41 (m, 1H), 4.41–4.36 (m, 1H), 4.22
(dd, J1 = 13.6, J2 = 7.9 Hz, 1H), 3.72–3.59 (m, 1H), 3.57–3.47
(m, 1H), 3.18 (d, J = 5.2 Hz, 1H), 2.96 (d, J = 5.2 Hz, 1H), 2.08–
1.95 (m, 1H), 1.95–1.86 (m, 2H), 1.83 (s, 3H), 1.73–1.61 (m, 2H),
1.56–1.46 (m, 1H), 1.42 (s, 3H), 1.41–1.31 (m, 2H), 1.30–1.23 (m,
4H), 1.20 (d, J = 6.7 Hz, 3H), 0.91 (d, J = 6.64 Hz, 1H), 0.88–
0.83 (m, 6H). HRMS: calcd. for [C25H42N4O6H]+ 495.31771, found
495.31755.
Synthesis of Ia–c and IIa–c; general procedure azide coupling
The peptide hydrazide was dissolved in DMF–EtOAc (1 : 1, v/v)
and cooled to -25 ◦C, before HCl (2.8 equiv., 4 M in 1,4-dioxane)
and tBuONO (1.1 equiv.) were added. The reaction mixture was
stirred for 4 h at -25 ◦C to form the corresponding acyl azide. Boc-
protected Leucine derived warhead Boc-LeuVS-PhOH,12 Boc-
LeuVS13 or Boc-LeuEK14 was dissolved in DCM–TFA (1 : 1,
v/v) and stirred for 30 min, before being coevaporated with Tol.
(3¥). The resulting warhead TFA-salt was dissolved in DMF and
DiPEA (3.8 equiv.) was added, before the mixture was combined
with the acyl azide mixture at -25 ◦C (NOTE: make sure the pH is
8–9. If not, add more DiPEA). The reaction mixture was allowed
to warm up to room temperature overnight. EtOAc and water were
(Tyr(Me)-Phe-Leu-4-hydroxyphenyl-vinylsulfone)-2-(naphthalen-
2-yl)-acetamide (IIa)
Following the general procedure for azide coupling, the title com-
pound was obtained from Boc-LeuVS-PhOH (61 mg, 0.17 mmol,
2724 | Org. Biomol. Chem., 2010, 8, 2719–2727
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1.1 equiv.) and (Tyr(Me)-Phe-hydrazinyl)-2-(naphthalen-2-yl)-
acetamide (10, 78.7 mg, 0.15 mmol). Crystallization from EtOAc
with PetEt gave IIa as a white solid (88.1 mg, 0.12 mmol, 77%).
1H NMR (400 MHz, DMSO): d ppm 10.64 (s, 1H), 8.29 (d, J =
8.0 Hz, 1H), 8.25 (d, J = 8.4 Hz, 1H), 8.06 (d, J = 8.4 Hz, 1H),
7.85 (d, J = 7.0 Hz, 1H), 7.80–7.72 (m, 2H), 7.62 (d, J = 8.7 Hz,
2H), 7.58 (s, 1H), 7.50–7.42 (m, 2H), 7.25–7.20 (m, 1H), 7.18–7.13
(m, 5H), 7.07 (d, J = 8.6 Hz, 2H), 6.95 (d, J = 8.7 Hz, 2H),
6.71–6.61 (m, 3H), 6.22 (dd, J1 = 15.1, J2 = 1.3 Hz, 1H), 4.58–
4.42 (m, 3H), 3.64 (s, 3H), 3.59 (d, J = 13.9 Hz, 1H), 3.48 (d, J =
13.9 Hz, 1H), 2.98–2.75 (m, 3H), 2.64 (dd, J1 = 13.7, J2 = 10.0 Hz,
1H), 1.62–1.50 (m, 1H), 1.39–1.25 (m, 2H), 0.81 (dd, J1 = 12.6,
J2 = 6.6 Hz, 6H). 13C NMR (100 MHz, DMSO): d ppm 170.99,
170.17, 169.71, 161.95, 157.58, 145.37, 137.14, 133.85, 132.81,
131.61, 130.10, 129.96, 129.85, 129.61, 129.39, 128.96, 127.99,
127.55, 127.32, 127.31, 127.24, 127.10, 126.35, 125.85, 125.35,
115.86, 113.21, 54.72, 54.02, 53.82, 47.03, 42.17, 41.96, 37.51,
36.59, 23.99, 22.88, 21.31. HRMS: calcd. for [C44H47N3O7SH]+
762.32075, found 762.32139.
126.98, 126.52, 126.19, 113.97, 79.36, 59.00, 55.11, 54.43, 54.01,
52.33, 50.11, 43.60, 39.94, 37.78, 36.31, 25.15, 23.30, 21.40, 16.71.
HRMS: calcd. for [C40H45N3O6H]+ 664.33811, found 664.33838.
Boc-Tyr(Me)-Phe-hydrazide (11)
Boc-Tyr(Me)-Phe-methyl ester (8, 1.17 g, 2.56 mmol) was dis-
solved in MeOH and hydrazine monohydrate (7.47 ml, 154 mmol,
60 equiv.) was added. The reaction mixture was refluxed for 2 h,
before being concentrated until white precipitate is formed. The
resulting suspension was cooled and the product was filtered off
(1.03 g, 2.25 mmol, 88%). 1H NMR (400 MHz, MeOD–CDCl3):
d ppm 7.26–7.08 (m, 5H), 7.03 (d, J = 8.6 Hz, 2H), 6.79 (d, J =
8.6 Hz, 2H), 4.55–4.50 (m, 1H), 4.18 (t, J = 6.6 Hz, 1H), 3.01 (dd,
J1 = 13.7, J2 = 7.0 Hz, 1H), 2.96–2.85 (m, 2H), 2.73 (dd, J1 =
13.5, J2 = 8.1 Hz, 1H), 1.34 (s, 9H). NMR (100 MHz, MeOD–
CDCl3): d ppm 171.77, 170.37, 158.08, 135.87, 129.70, 128.62,
128.08, 127.96, 126.37, 113.40, 79.72, 55.67, 54.53, 52.52, 37.25,
36.74, 27.46.
Boc-Tyr(Me)-Phe-Leu-methyl-vinylsulfone (12)
(Tyr(Me)-Phe-Leu-methyl-vinylsulfone)-2-(naphthalen-2-yl)-
acetamide (IIb)
Following the general procedure for azide coupling the
title compound was obtained from Boc-LeuVS (0.24 g,
0.83 mmol, 1.1 equiv.) and Boc-Tyr(Me)-Phe-hydrazide (11,
0.39 g, 0.75 mmol). Purification by flash column chromatography
(DCM → 1.5% MeOH in DCM) gave 12 (0.35 g, 0.56 mmol, 75%).
1H NMR (400 MHz, CDCl3): d ppm 7.29–7.22 (m, 3H), 7.09 (d,
J = 8.5 Hz, 2H), 7.03 (d, J = 6.1 Hz, 2H), 6.87 (d, J = 8.5 Hz,
2H), 6.82–6.65 (m, 2H), 6.47–6.37 (m, 2H), 4.84 (d, J = 3.0 Hz,
1H), 4.77–4.69 (m, 1H), 4.66 (dd, J1 = 13.0, J2 = 6.3 Hz, 1H), 4.16
(td, J1 = 7.8, J2 = 4.8 Hz, 1H), 3.80 (s, 3H), 3.32–3.23 (m, 1H),
3.03 (dd, J1 = 14.3, J2 = 5.0 Hz, 1H), 2.91 (s, 3H), 2.91–2.83 (m,
2H), 1.53–1.41 (m, 1H), 1.37–1.30 (m, 2H), 1.27 (s, 9H), 0.90 (d,
J = 6.4 Hz, 3H), 0.86 (d, J = 6.5 Hz, 3H).
Following the general procedure for azide coupling the title
compound was obtained from Boc-LeuVS (32 mg, 0.11 mmol,
1.1 equiv.) and (Tyr(Me)-Phe-hydrazinyl)-2-(naphthalen-2-yl)-
acetamide (10, 52 mg, 0.1 mmol). Column chromatography (DCM
→ 1.5% MeOH in DCM) gave the title compound as a white solid
1
(36.2 mg, 53 mmol, 53%). H NMR (400 MHz, CDCl3): d ppm
8.30 (d, J = 8.0 Hz, 1H), 8.24 (d, J = 8.3 Hz, 1H), 8.12 (d, J =
8.3 Hz, 1H), 7.85 (d, J = 7.3 Hz, 1H), 7.81–7.73 (m, 2H), 7.60 (s,
1H), 7.52–7.42 (m, 2H), 7.31–7.15 (m, 6H), 7.07 (d, J = 8.1 Hz,
2H), 6.68 (d, J = 8.3 Hz, 2H), 6.60 (dd, J1 = 15.2, J2 = 4.9 Hz,
1H), 6.32 (d, J = 15.3 Hz, 1H), 4.61–4.43 (m, 3H), 3.65 (s, 3H),
3.61 (d, J = 13.9 Hz, 1H), 3.49 (d, J = 14.0 Hz, 1H), 3.01 (dd,
J1 = 13.5, J2 = 6.6 Hz, 1H), 2.95 (s, 3H), 2.91–2.82 (m, 2H), 2.66
(dd, J1 = 14.6, J2 = 11.1 Hz, 1H), 1.68–1.55 (m, 1H), 1.47–1.29
(m, 2H), 0.85 (dd, J1 = 15.5, J2 = 6.5 Hz, 6H). HRMS: calcd. for
[C39H45N3O6SH]+ 684.31018, found 684.31060.
Azido-BODIPY-Tyr(Me)-Phe-Leu-methyl-vinylsulfone (14)
Boc-Tyr(Me)-Phe-LeuVS (12, 22 mg, 35 mmol) was dissolved in
TFA–DCM 1 : 1 (v/v). The reaction mixture was stirred for 30 min,
before being co-evaporated with Tol. (3¥). The crude TFA salt was
dissolved in DCM and N3-BODIPY-OSu17 (13, 20 mg, 35 mmol,
1 equiv.) and DiPEA (6 ml, 35 mmol, 1 equiv.) were added. The
reaction mixture was stirred for 5 h, before being concentrated.
Purification by flash column chromatography (DCM → 1.5%
MeOH in DCM) afforded the title compound as a purple solid
(18.2 mg, 18.9 mmol, 54%). 1H NMR (400 MHz, CDCl3): d ppm
7.87 (d, J = 8.8 Hz, 2H), 7.29–7.20 (m, 3H), 7.07 (s, 1H), 7.04
(dd, J1 = 7.4, J2 = 1.5 Hz, 2H), 7.00–6.93 (m, 5H), 6.79 (d, J =
8.5 Hz, 2H), 6.71 (dd, J1 = 15.1, J2 = 4.6 Hz, 1H), 6.54 (d, J =
4.1 Hz, 1H), 6.52–6.44 (m, 2H), 6.21 (dd, J1 = 15.1, J2 = 1.1 Hz,
1H), 6.10–6.04 (m, 1H), 4.72–4.57 (m, 2H), 4.45 (q, J = 6.4 Hz,
1H), 4.10 (t, J = 5.9 Hz, 2H), 3.70 (s, 3H), 3.53 (t, J = 6.6 Hz,
3H), 3.19 (dd, J1 = 13.8, J2 = 5.4 Hz, 1H), 2.95–2.89 (m, 3H), 2.89
(s, 3H), 2.65–2.50 (m, 2H), 2.48 (s, 3H), 2.19–2.13 (m, 3H), 2.13
(s, 3H), 2.11–2.03 (m, 2H), 1.52–1.38 (m, 1H), 1.38–1.26 (m, 2H),
0.87 (t, J = 6.7 Hz, 6H). 13C NMR (100 MHz, CDCl3): d ppm
172.52, 170.81, 169.95, 159.55, 158.86, 158.32, 156.04, 147.21,
139.32, 135.87, 135.13, 134.04, 134.02, 130.77, 130.73, 130.69,
130.08, 129.60, 129.25, 129.21, 128.85, 128.80, 128.39, 127.41,
(Tyr(Me)-Phe-Leu-epoxyketone)-2-(naphthalen-2-yl)-acetamide
(IIc)
Following the general procedure for azide coupling the title
compound was obtained from Boc-LeuEK (76 mg, 0.28 mmol,
1.1 equiv.) and (Tyr(Me)-Phe-hydrazinyl)-2-(naphthalen-2-yl)-
acetamide (10, 0.13 g, 0.25 mmol). Column chromatography
(DCM → 2% MeOH in DCM) gave the title compound as a white
1
solid (0.11 g, 0.17 mmol, 66%). H NMR (400 MHz, CDCl3):
d ppm 7.86–7.75 (m, 1H), 7.73–7.67 (m, 2H), 7.52 (s, 1H), 7.49–
7.44 (m, 2H), 7.20–7.09 (m, 3H), 7.01–6.91 (m, 3H), 6.68 (d, J =
8.6 Hz, 2H), 6.57–6.47 (m, 1H), 6.40 (d, J = 8.3 Hz, 2H), 4.79–4.66
(m, 2H), 4.58 (dt, J1 = 19.8, J2 = 3.0 Hz, 1H), 3.65–3.45 (m, 5H),
3.24 (d, J = 4.9 Hz, 1H), 3.01–2.85 (m, 1H), 2.84–2.77 (m, 4H),
1.59–1.51 (m, 1H), 1.49 (s, 3H), 1.47–1.37 (m, 1H), 1.33–1.19 (m,
1H), 0.88 (dd, J1 = 13.1, J2 = 6.4 Hz, 6H).13C NMR (100 MHz,
CDCl3): d ppm 207.99, 171.32, 170.57, 170.47, 158.50, 136.27,
133.56, 132.57, 131.70, 130.02, 129.36, 128.92, 128.87, 128.61,
128.56, 128.29, 128.25, 127.77, 127.72, 127.56, 127.12, 127.02,
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127.31, 125.53, 123.01, 118.64, 118.60, 118.58, 114.29, 114.20,
64.45, 55.19, 54.25, 48.20, 47.91, 42.74, 42.42, 37.17, 36.37, 35.82,
28.74, 24.61, 22.80, 21.74, 19.59, 13.09, 9.57. HRMS: calcd. for
[C50H59BF2N8O7SH]+ 965.43613, found 965.43837.
4.77 (p, J = 7.0 Hz, 1H), 4.62 (dd, J1 = 8.1, J2 = 2.6 Hz, 1H),
4.50 (dt, J1 = 7.7, J2 = 5.5 Hz, 1H), 3.98 (d, J = 3.6 Hz, 2H),
3.74 (s, 3H), 3.70–3.55 (m, 2H), 2.39–2.29 (m, 1H), 2.23–2.08 (m,
1H), 2.08–1.99 (m, 1H), 1.98–1.89 (m, 1H), 1.87–1.76 (m, 1H),
1.71–1.59 (m, 1H), 1.40 (d, J = 6.9 Hz, 3H), 1.35–1.21 (m, 2H),
0.88 (t, J = 7.0 Hz, 3H).
Boc-Tyr(Me)-Phe-Leu-epoxyketone (15)
Following the general procedure for azide coupling the ti-
tle compound was obtained from Boc-LeuEK (0.22 g,
0.83 mmol, 1.1 equiv.) and Boc-Tyr(Me)-Phe-hydrazide (11,
0.39 g, 0.75 mmol). Purification by flash column chromatography
(DCM → 1.5% MeOH in DCM) gave 15 (0.32 g, 0.54 mmol,
Az-Ala-Pro-Nle-hydrazide (20)
Az-Ala-Pro-NLe-OMe (19, 89.3 mg, 0.23 mmol) was dissolved in
MeOH. Hydrazine monohydrate (0.67 ml, 13.8 mmol, 60 equiv.)
was added and the reaction mixture was refluxed for 2 h. Tol.
was added and the resulting white solid was filtered to give the
title compound (90 mg, 0.23 mmol, quant.).1H NMR (400 MHz,
MeOD): d ppm 4.65 (q, J = 7.0 Hz, 1H), 4.46 (dd, J1 = 8.2, J2 =
4.6 Hz, 1H), 4.23 (dd, J1 = 8.4, J2 = 6.0 Hz, 1H), 3.89 (s, 2H),
3.86–3.77 (m, 1H), 3.70–3.60 (m, 1H), 2.30–2.12 (m, 1H), 2.12–
1.91 (m, 2H), 1.83–1.72 (m, 1H), 1.71–1.61 (m, 1H), 1.44–1.25 (m,
7H), 0.92 (t, J = 6.9 Hz, 3H).
1
71%). H NMR (400 MHz, CDCl3): d ppm 7.26–7.11 (m, 3H),
7.10–7.04 (m, 4H), 7.04–7.01 (m, 1H), 6.90 (s, 1H), 6.78 (d, J =
8.5 Hz, 2H), 5.31 (d, J = 7.7 Hz, 1H), 4.77 (q, J = 6.9 Hz, 1H),
4.56 (dt, J1 = 9.8, J2 = 3.2 Hz, 1H), 4.41 (s, 1H), 3.73 (s, 3H),
3.25 (d, J = 4.4 Hz, 1H), 3.02 (dd, J1 = 14.0, J2 = 6.8 Hz, 1H),
2.98–2.88 (m, 3H), 2.84 (d, J = 4.9 Hz, 1H), 1.58–1.51 (m, 1H),
1.49 (s, 3H), 1.48–1.38 (m, 1H), 1.36 (s, 9H), 1.27–1.18 (m, 1H),
0.88 (dd, J1 = 12.3, J2 = 6.4 Hz, 6H).
Az-Ala-Pro-NLe-Leu-epoxyketone (21)
Azido-BODIPY-Tyr(Me)-Phe-Leu-epoxyketone (16)
Following the general procedure for azide coupling the ti-
tle compound was obtained from Boc-LeuEK (38.8 mg,
0.14 mmol, 1.1 equiv.) and Az-Ala-Pro-Nle-hydrazide (19,
53.2 mg, 0.13 mmol). Purification by flash column chromatog-
raphy (DCM → 2% MeOH in DCM) gave 21 (15.9 mg, 30 mmol,
Boc-Tyr(Me)-Phe-LeuEK (15, 21 mg, 35 mmol) was dissolved
in TFA–DCM 1 : 1 (v/v). The reaction mixture was stirred for
30 min, before being co-evaporated with Tol. (3¥). The crude
TFA salt was dissolved in DCM and N3-BODIPY-OSu17 (13,
20 mg, 35 mmol, 1 equiv.) and DiPEA (6 ml, 35 mmol, 1 equiv.)
were added. The reaction mixture was stirred for 15 h, before
being concentrated. Purification by flash column chromatography
(DCM → 1% MeOH in DCM) afforded the title compound as a
purple solid (8.8 mg, 9.3 mmol, 27%). 1H NMR (400 MHz, CDCl3):
d ppm 7.87 (d, J = 8.6 Hz, 2H), 7.27–7.19 (m, 3H), 7.11–7.05 (m,
3H), 7.00–6.92 (m, 5H), 6.76 (d, J = 8.2 Hz, 2H), 6.54 (d, J =
3.9 Hz, 1H), 6.32 (d, J = 7.6 Hz, 1H), 6.03 (d, J = 7.6 Hz, 1H), 5.88
(d, J = 7.0 Hz, 1H), 4.58–4.47 (m, 3H), 4.10 (t, J = 5.8 Hz, 2H),
3.73–3.70 (m, 3H), 3.53 (t, J = 6.6 Hz, 2H), 3.23 (d, J = 4.8 Hz,
1H), 3.08–2.89 (m, 4H), 2.88 (d, J = 4.8 Hz, 1H), 2.76–2.55 (m,
2H), 2.51 (s, 3H), 2.33–2.20 (m, 2H), 2.18 (s, 3H), 2.08 (td, J1 =
12.1, J2 = 6.1 Hz, 2H), 1.50 (s, 3H), 1.49–1.38 (m, 2H), 1.20–1.13
(m, 1H), 0.89 (dd, J1 = 13.7, J2 = 5.5 Hz, 6H). HRMS: calcd. for
[C51H59BF2N8O7H]+ 945.46406, found 945.46639.
1
23%). H NMR (400 MHz, CDCl3): d ppm 7.16 (d, J = 7.4 Hz,
1H), 7.08 (d, J = 7.7 Hz, 1H), 6.40 (d, J = 8.0 Hz, 1H), 4.77 (p,
J = 6.9 Hz, 1H), 4.62–4.54 (m, 2H), 4.30 (dt, J1 = 7.8, J2 = 5.7 Hz,
1H), 3.98 (d, J = 5.0 Hz, 2H), 3.71–3.62 (m, 1H), 3.61–3.55 (m,
1H), 3.31 (d, J = 5.0 Hz, 1H), 2.89 (d, J = 5.0 Hz, 1H), 2.35–2.27
(m, 1H), 2.20–2.09 (m, 1H), 2.07–1.99 (m, 1H), 1.98–1.90 (m, 1H),
1.85–1.75 (m, 1H), 1.69–1.53 (m, 2H), 1.51 (s, 3H), 1.39 (d, J =
6.8 Hz, 3H), 1.36–1.20 (m, 6H), 0.94 (dd, J1 = 6.5, J2 = 2.4 Hz,
6H), 0.88 (t, J = 7.1 Hz, 3H). HRMS: calcd. for [C25H41N7O6H]+
536.31911, found 536.31980.
Green fluorescent b1specific probe (23)
b1 selective probe 21 (7.7 mg, 0.014 mmol) was reacted with
acetylene-functionalised BODIPY 2218 (4.7 mg, 0.014 mmol,
1.0 equiv.) catalysed by CuSO4 (0.05 ml 28 mM in H2O, 1.4 mmol,
10 mol%) and sodium ascorbate (0.05 ml 44 mM in H2O, 2.2 mmol,
15 mol%) in a mixture of Tol.–H2O–tBuOH (final ratio 1 : 1 : 1,
v/v/v, 0.6 ml) at 80 ◦C for 22 h. The mixture was then allowed to
cool to room temperature and concentrated in vacuo. Purification
by column chromatography (DCM → 2% MeOH in DCM) gave
the fluorescent probe 23 (8.6 mg, 9.3 mmol, 65%) as an orange
solid. 1H NMR (400 MHz, DMSO): d ppm 8.66 (d, J = 6.8 Hz,
1H), 8.09 (d, J = 7.2 Hz, 1H), 7.80–7.77 (m, 2H), 6.23 (s, 2H),
5.07 (s, 2H), 4.56–4.53 (s, 1H), 4.38–4.33 (m, 2H), 4.24–4.17 (m,
1H), 3.58–3.55 (m, 2H), 3.19–3.17 (m, 1H), 3.02–2.96 (m, 3H),
2.72 (t, J = 7.2, 7.2 Hz), 2.41 (s, 6H), 2.40 (s, 6H), 1.98–1.94
(m, 2H), 1.89–1.81 (m, 4H), 1.64–1.62 (m, 4H), 1.47–1.44 (m,
1H), 1.41 (s, 3H), 1.36–1.31 (m, 2H), 1.24–1.20 (m, 7H), 0.91
(d, J = 6.8 Hz, 6H), 0.88–0.81 (m, 3H). 13C NMR (100 MHz,
DMSO): d ppm 208.28, 171.87, 170.92, 170.39, 165.03, 153.06,
146.68, 140.90, 130.72, 123.50, 121.69, 59.18, 58.93, 51.99, 51.64,
51.22, 49.60, 46.70, 46.41, 38.33, 31.90, 30.81, 29.43, 28.78, 27.61,
Az-Ala-Pro-Nle-OMe (19)
Resin-bound Fmoc-Ala-Pro-Nle (synthesis described above,
0.25 mmol) was deprotected with piperidine–NMP (1 : 4, v/v,
20 min), washed with NMP (3¥) and DCM (3¥), and capped with
azido acetic acid (63 mg, 0.63 mmol, 2.5 equiv.) under the influence
of BOP (0.28 g, 0.63 mmol, 2.5 equiv.) and DiPEA (0.12 ml,
0.75 mmol, 3 equiv.) for 15 h. Mild acidic cleavage of resin 17
with 1% TFA in DCM (3 ¥ 10 min.) and co-evaporation with Tol.
(3¥) resulted in the crude Az-Ala-Pro-NLe-OH 18 which was used
without purification. The crude peptide was dissolved in MeOH–
Tol. (1 : 1) and treated with TMS–diazomethane (0.25 ml 2 M in
hexanes, 0.5 mmol, 2 equiv.) for 15 min before being co-evaporated
with Tol. (3¥). Purification by flash column chromatography
(DCM → 2.5% MeOH in DCM) yielded the title compound as
1
a white solid (89.3 mg, 0.23 mmol, 90%). H NMR (400 MHz,
CDCl3): d ppm 7.25 (d, J = 7.4 Hz, 1H), 7.11 (d, J = 7.7 Hz, 1H),
2726 | Org. Biomol. Chem., 2010, 8, 2719–2727
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The Royal Society of Chemistry 2010
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27.20, 24.53, 24.40, 23.16, 21.92, 20.98, 17.07, 16.52, 15.83, 14.08,
13.92. HRMS: calcd. for [C44H64BF2N9O6H]+ 864.51134, found
864.51332.
Hans van den Elst and Nico Meeuwenoord for HPLC and LC-MS
assistance.
References and notes
Competition and labeling experiments in vitro
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indicated concentrations of the inhibitors or fluorescent probes
(100 ¥ solution in DMSO) for 2 h, before being washed with PBS
(2¥) and harvested. The cells were permeated in digitonin lysis
buffer (4 ¥ pellet volume, 50 mM Tris pH 7.5, 250 mM sucrose,
5 mM MgCl2, 5 mM DTT, 0.025% digitonin) for 5 min on ice
and centrifuged at 16.100 rcf for 20 min at 4 ◦C. The supernatant
containing the cytosolic fraction was collected and the protein
content was determined by Bradford assay. In case of a competi-
tion experiment, the lysates were exposed to MV151 (1 mM) for
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Laemli’s sample buffer containing beta-mercapto-ethanol and the
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Cy3/Tamra settings (lex 532, lem 560) for MV151 and the azido-
BODIPY functionalized probes 14, 16 and 25 or lex 488 nm, lem
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Acknowledgements
19 B. I. Florea, M. Verdoes, N. Li, W. A. Van der Linden, P. P. Geurink,
H. Van den Elst, T. Hofmann, A. de Ru, P. Van Veelen, K. Tanaka,
K. Sasaki, S. Murata, H. Den Dulk, J. Brouwer, F. Ossendorp, H. S.
Overkleeft, manuscript submitted for publication.
This work was supported by The Netherlands Organization for
Scientific Research (NWO), The Netherlands Genomics Centre
Initiative (NGI) and the NCI (grant RO1CA124634). We thank
This journal is
The Royal Society of Chemistry 2010
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©