Exploring dual electrophiles in peptide-based proteasome inhibitors: Carbonyls and epoxides

Article abstract of DOI:10.1039/c4ob00893f

Peptide epoxyketones are potent and selective proteasome inhibitors. Selectivity is governed by the epoxyketone dual electrophilic warhead, which reacts with the N-terminal threonine 1,2-amino alcohol uniquely present in proteasome active sites. We studied a series of C-terminally modified oligopeptides featuring adjacent electrophiles based on the epoxyketone warhead. We found that the carbonyl moiety in the natural warhead is essential, but that the adjacent epoxide can be replaced by a carbonyl, though with considerable loss of activity. the Partner Organisations 2014.

Full text of DOI:10.1039/c4ob00893f

Organic &
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Exploring dual electrophiles in peptide-based
proteasome inhibitors: carbonyls and epoxides†
Cite this: Org. Biomol. Chem., 2014,
12, 5710
Bo-Tao Xin,a Gerjan de Bruin,a Martijn Verdoes,b Dmitri V. Filippov,a
Gijs A. van der Marela and Herman S. Overkleeft*a
Peptide epoxyketones are potent and selective proteasome inhibitors. Selectivity is governed by the
epoxyketone dual electrophilic warhead, which reacts with the N-terminal threonine 1,2-amino alcohol
uniquely present in proteasome active sites. We studied a series of C-terminally modified oligopeptides
featuring adjacent electrophiles based on the epoxyketone warhead. We found that the carbonyl moiety
in the natural warhead is essential, but that the adjacent epoxide can be replaced by a carbonyl, though
with considerable loss of activity.
Received 1st May 2014,
Accepted 18th June 2014
DOI: 10.1039/c4ob00893f
www.rsc.org/obc
Introduction
The ubiquitin-proteasome system (UPS) is the major cytosolic
and nuclear protein degradation pathway in many organisms.1
The 26S proteasome, composed of a catalytic 20S core particle
and a 19S regulator particle, is responsible for the turnover of
proteins tagged for degradation through poly-ubiquitin chains.
In higher vertebrates, the constitutive proteasome core 20S
particle contains three distinct catalytic activities, namely β1
(cleaving after acidic amino acids), β2 (cleaving after basic
amino acids) and β5 (cleaving after hydrophobic amino
acids).2 In immunoproteasomes, these catalytic subunits are
replaced by β1i, β2i and β5i, respectively.3 Cortical thymic epi-
thelial cells uniquely express β5t subunits, which may replace
β5i to form thymoproteasomes.4 To date numerous protea-
some inhibitors have been reported. These include both
natural products and synthetic compounds.5 Of note, protea-
somes are validated drug targets in oncology and the peptide
boronic acid, bortezomib6 as well as the peptide epoxyketone,
carfilzomib,7 are used in the clinic for the treatment of
multiple myeloma and mantle cell lymphoma.
Fig. 1 Structures of epoxomicin (1) and peptide α-keto-aldehyde (2)
that are at the basis of the here presented studies.
Fig. 2 Mechanism of peptide epoxyketone (A) and peptide α-keto-
aldehyde (B) mediated proteasome inactivation.
lytic activities are characterized by an N-terminal threonine in
which the hydroxyl acts as the nucleophile with the amine as
catalytic base. Such catalytic sites are rare in nature and
peptide epoxyketones appear ideally suited to react with the
1,2-amino-alcohol moiety in these N-terminal threonine resi-
dues.9 In the inhibition mechanism, the carbonyl of the epoxy-
ketone warhead is first attacked by the N-terminal threonine
γ-hydroxyl (Fig. 2) after which the α-amine attacks the epoxide
ring, resulting in morpholine ring formation.
A large number of epoxyketone warhead containing inhibi-
tors of proteasome have been reported10 including the afore-
mentioned clinical drug, carfilzomib. A related class of
proteasome inhibitors more recently investigated in detail and
that also capitalize on the reactive 1,2-amino alcohol present
Carfilzomib is a member of the family of peptide epoxy-
ketone proteasome inhibitors. The first compound identified
in this class is the natural product; epoxomicin (Fig. 1, 1).8
Epoxomicin features an epoxyketone electrophilic trap, also
referred to as the warhead, which renders this compound highly
selective towards the proteasome catalytic sites. Proteasome cata-
aLeiden Institute of Chemistry, Leiden University, Einsteinweg 55, 2300 RA Leiden,
The Netherlands. E-mail: h.s.overkleeft@chem.leidenuniv.nl
bDepartment of Tumor Immunology, Nijmegen Centre for Molecular Life Sciences,
Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands
†Electronic supplementary information (ESI) available. See DOI:
10.1039/c4ob00893f
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Fig. 3 Target structures 3–8 that are at the basis of the here-presented
studies.
in proteasome active sites comprises the peptide α-keto-alde-
hydes (Fig. 1, 2).11 In a subsequent study, Groll and co-workers
reported a series of N-benzyloxycarbonyl-trileucinyl-1,2-dicar-
bonyl derivatives based on this motif and identified the
α-ketoamide electrophile as the most effective inhibitor of this
series.12 In α-keto-aldehyde 2, the carbonyl occupying the posi-
tion normally occupied by the amide carbonyl of the scissile
peptide bond is attacked first by the N-terminal threonine
γ-hydroxyl to reversibly form the hemi-ketal. This then further
condenses to form through a cyclic carbinolamine intermedi-
ate the corresponding 5,6-dihydro-2H-1,4-oxazine. In contrast
to epoxyketone-mediated inhibition, this process is reversible,
but the analogy is remarkable: two adjacent electrophilic
carbons are brought near to the proteasome N-terminal threo-
nine 1,2-amino alcohol to form after double nucleophilic
attack a six-membered adduct. This analogy invites the ques-
tion whether hybrid structures varying in the number and
position of carbonyl moieties (featuring an sp2 hybridized
electrophilic carbon) and epoxides (with an sp3 electrophilic
carbon) would be effective proteasome inhibitors. We decided
to put this question to the test by the design, synthesis and
evaluation as proteasome inhibitors of compounds 3–8
(Fig. 3).
N-Benzyloxycarbonyl-trileucinyl epoxyketone (ZL3-epoxy-
ketone) 3 is a known10a and easily accessible broad-spectrum
proteasome inhibitor and a close analogue of the natural
product, epoxomicin. The compound features an α-keto-
epoxide as the electrophilic trap with the two electrophilic
carbons hybridized as sp2–sp3. In compound 4 the direction of
the epoxyketone is inverted and the two electrophilic carbons
are now sp3–sp2-hybridized. Compound 5 features two adja-
cent epoxides (sp3–sp3) and compound 7, as close analogue to
lead structure 2, features two adjacent carbonyls (sp2–sp2).
Compounds 6 and 8 finally feature an enone system, thus
Michael acceptors with the two electrophilic carbons now sep-
arated by one carbon. We thought to include these latter com-
pounds because their ease of synthesis and because conjugate
addition has been shown to be another valid strategy in the
design of peptide-based proteasome inhibitors.13
Scheme 1 Synthesis of 4ab. Reagents and conditions: (a) (i) 12 (cat.),
toluene–DCM, aq. 50% K3PO4, −20 °C, 60 h; (ii) aq. 37% HCHO, aq. 50%
K3PO4, RT, 60 h; (b) CeCl3·7H2O, NaBH4, MeOH; (c) (i) tBuOOH, VO
(acac)2, DCM; (ii) Dess–Martin periodinane, DCM; (d) 50% TFA–DCM;
(e) (i) tBuONO, HCl, DMF–DCM, −30 °C; (ii) 15a or 15b, DiPEA–DMF.
11 via an enantioselective Mannich reaction catalyzed by
quinine-based catalyst 12, followed by Horner olefination.
According to chiral HPLC analysis, compound 11 was obtained
as a 1 : 3 mixture of enantiomers and we continued the syn-
thesis with this mixture. After reduction of 11 with CeCl3·7H2O
and NaBH4, compounds 13ab were obtained in equal amounts
and were separated by silica gel column chromatography. It
should be noted that both compounds exist as enantiomeric
mixture, in a 1 : 3 ratio. We could not determine the absolute
stereochemistry of the individual compounds and therefore
decided to continue the synthesis with both.
After diastereoselective epoxidation (tBuOOH and
VO(acac)2) and Dess–Martin oxidation of the hydroxyl group,
diastereomeric compounds 14a and 14b were obtained. Treat-
ment with TFA yielded the leucine keto-epoxides 15a and 15b.
Compound 16 was prepared according to literature methods.15
Finally, compounds 4a and 4b were prepared by standard
azide peptide coupling of hydrazide 16 with either of the Leu-
keto-epoxides 15a and 15b. During reverse HPLC purification
of 4a and 4b, there was a small peak next to the main peak,
which had the same mass as the product according to the
LC/MS analysis. We argued that this most probably is the enantio-
meric impurity which was introduced in compound 11. This
impurity was removed HPLC purification, and NMR-analysis
showed clearly that only one diastereomer was isolated for
both compounds 4a and 4b. Therefore, we assume that the
sole difference between 4a and 4b lies in the orientation of the
Results and discussion
The synthesis of inverted epoxyketone 4 commenced with an epoxide ring. Again, we could not establish the absolute
enantioselective Mannich reaction (Scheme 1). Phosphonate 9 stereochemistry.
and sulfonate 10 were prepared following literature pro-
The synthesis of the di-epoxides commenced with Wittig
cedures,14 and used in a one-pot procedure to form compound olefination of the Cbz-Leu-epoxyketone 21, prepared following
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Scheme 3 Synthesis of
6
and 7. Reagents and conditions: (a) (i)
tBuONO, HCl, DMF–DCM, −30 °C; (ii) 25 or 26, DiPEA–DMF. (b) O3,
PPh3, DCM, −78 °C to RT.
ketone part led to a dramatic drop in proteasome inhibitory
potency (4a and 4b compared with 3).
Compound 4b proved almost completely inactive whereas
diastereoisomer 4a inhibited β1 and β2 at high concentration
(>300 µM). In case of di-epoxides 5a and 5b, inhibition of β1
and β2 subunits was observed at concentration above 100 µM,
while β5 is not even completely blocked at 1 mM. Dicarbonyl
derivative 7 displayed some β5-selectivity in a manner similar
to ZL3-epoxyketone 3. Enones 6 and 8 showed similar inhibi-
tory potential. At high concentration (100 µM), β1 was comple-
tely inhibited whereas β2 and β5 were only partially inhibited.
Although being slightly less potent than 3, diketo compound 7
Scheme 2 Synthesis of 5ab. Reagents and conditions: (a) N,O-
dimethylhydroxylamine hydrochloride, HCTU, DiPEA, DCM; (b) (i) 2-bro-
mopropene, tBuLi, Et2O; (ii) NaBH4, CeCl3·7H2O, MeOH; (c) (i) VO(acac)2,
tBuOOH, DCM; (ii) Dess Martin periodinane, DCM; (d) (i) 50% TFA–DCM;
(ii) CbzCl, DiPEA, DCM; (e) [Ph3PCH3]+Br−, KHMDS, THF, RT to −78 °C;
(f) m-CPBA, DCM; (g) Pd/C, 1,4-cyclohexadiene, THF–MeOH; (h) (i)
tBuONO, HCl, DMF–DCM, −30 °C; (ii) 24a or 24b, DiPEA–DMF.
the route of synthesis as published in the patent literature.16 showed complete β5 inhibition already at 10 µM, whereas
Briefly, Boc-leucine 17 was transformed into Weinreb amide 18 β1 and β2 were inhibited at much higher concentrations.
to which was added 2-vinyllithium, prepared for this purpose
Our results underscore the fact that the epoxyketone geo-
through transmetallation of 2-bromopropene with tert-butyl- metry as present in the natural product epoxomicin and syn-
lithium. The intermediate enone was stereoselectively reduced thetic compounds featuring the same adjacent sp3–sp2
under Lüche conditions to provide after separation of the electrophilic carbons is the most effective design where it
formed diastereomers allylic alcohol 19 in good yield and comes to proteasome inhibition. The sp2-carbonyl in peptide
enantiomeric purity. Sharpless allylic epoxidation followed by epoxyketones situated at the position resembling the amide
Dess Martin oxidation gave leucine epoxyketone 20. Removal carbonyl in a scissile amide bond of a proteasome substrate
of the Boc group and installment of the N-Cbz protective group appears highly important for effective inhibition. Interestingly,
gave key intermediate 21 in good overall yield (Scheme 2). peptide vinyl sulfones – another major class of proteasome
Wittig olefination of Cbz-Leu-epoxyketone 21 gave alkene 22, inhibitors – position an sp2-hybridized electrophilic carbon for
and ensuing epoxidation with m-CPBA yielded epoxides 23a nucleophilic 1,4-addition at the same location whereas argu-
and 24b in equal amounts. The absolute stereochemistry of ably the covalent but reversible inhibition effected by peptide
these two compounds, which were obtained as enantiomeri- boronic acids18 proceeds starting from an sp2-hybridized
cally pure di-epoxides, again could not be determined.
boron as well. Apparently, a situation in which the reactive
substrate/inhibitor is
After Cbz-removal (to give 24a/b) and condensation of these carbon/carbon-replacing atom in
a
warheads with dipeptide 16, 5a and 5b were obtained. (S)-4- offered to the active site in an sp2 geometry to yield after
Amino-2,6-dimethylhept-1-en-3-one 25, which we obtained in nucleophilic addition of the threonine–OH an sp3-adduct is
route to leucine epoxyketone 21, was condensation with 16 to ideal. The secondary electrophilic trap – the tertiary epoxide
obtain 6 (Scheme 3). Ozonolysis of 6 gave diketone 7. Com- carbon in epoxomicin – is amenable for modification to
pound 16 was converted in a similar sequence of events to give provide sp2 analogues, as is evidenced by peptide diketone 7
8 as an inseparable mixture of diastereomers.
in a result that complements the literature findings11,12 on
The inhibition properties of all compounds were deter- related compounds. It is however also obvious that inhibitory
mined in a competitive activity-based protein profiling assay potency is partially compromised in such sp2–sp2 hybridized
using broad-spectrum proteasome activity-based probe compounds.
BODIPY-epoxomicin 2717 as the read-out. The results (Fig. 4)
Our set of compounds completes a series of peptide epoxy-
reveal that interchanging the position of the epoxide and the ketone analogues as potential proteasome inhibitors that has
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Experimental
General
All reagents were of commercial grade and used as received
unless indicated otherwise. Methylene chloride (DCM), di-
methylformamide (DMF) and tetrahydrofuran (THF) were
stored over 4 Å molecular sieves. Reactions were conducted
under an argon atmosphere. Reactions were monitored by TLC
analysis by using DC-fertigfolien (Schleicher & Schuell, F1500,
LS254) with detection by UV absorption (254 nm), spraying
with an aqueous solution of KMnO4 (7%) and KOH (2%).
Column chromatography was performed on silica gel from
Screening devices (0.040–0.063 mm). 1H-NMR and
13C-APT-NMR spectra were recorded on Bruker AV-400
(400 MHz) or Bruker AV-600 (600 MHz) machines. Chemical
shifts are given in ppm (δ) relative to tetramethylsilane as an
internal standard. Coupling constants are given in Hz. Peak
assignments are based on 2D 1H-COSY and 13C-HSQC NMR
experiments. All presented 13C-APT spectra are proton
decoupled. LC/MS analysis was performed on a LCQ Advan-
tage Max (Thermo Finnigan) equipped with a Gemini C18
column (Phenomenex). HRMS was recorded on a LTQ Orbitrap
(Thermo Finnigan). For reverse phase HPLC purification of the
final compounds, an automated Gilson HPLC system equipped
with a C18 semiprep column (Gemini C18, 250 × 10 mm,
5μ particle size, Phenomenex) was used.
General procedure for Boc deprotection
The appropriate Boc-protected C-terminally modified leucine
derivative was dissolved in TFA–DCM (1 : 1, v/v) and stirred for
20 min. Co-evaporation with toluene (3×) afforded the TFA-
salt, which was used without further purification.
General procedure for azide peptide coupling
The hydrazide (16) was dissolved in 1 : 1 DMF–DCM (v/v) and
cooled to −30 °C. tBuONO (1.1 eq.) and HCl (4 M solution in
1,4-dioxane, 2.8 eq.) were added, and the mixture was stirred
for 3 h at −30 °C after which TLC analysis (10% MeOH–DCM,
v/v) showed complete consumption of the starting material.
The warhead-TFA salt was added to the reaction mixture as a
solution in DMF with 5.0 eq. of DiPEA and this mixture was
allowed to warm up to room temperature slowly overnight. The
mixture was diluted with EA and extracted with H2O (3×) and
brine. The organic layer was dried over MgSO4 and purified by
Fig. 4 Inhibition profile of compounds 3–8 and the structure of ABP (27).
been growing in the literature in recent years. We could not reverse phase HPLC.
determine the absolute stereochemistry of the various epox-
tert-Butyl (S)-(6-methyl-3-methylene-2-oxoheptan-4-yl)carba-
ides we studied. However, in all cases we obtained the respect- mate (11). Diphenylphosphorylketone 9 (798 mg, 1.54 mmol,
ive diastereoisomeric pairs as enantiomerically pure 1.1 eq.), compound 10 (917 mg, 2.80 mmol), and catalyst 12
compounds, neither of which proved potent proteasome (134 mg, 0.28 mmol, 0.1 eq.) were added to the flask and the
inhibitors. This result, coupled with our previous19 finding solids were suspended in a toluene–DCM (7 : 3) mixture
that a diastereomeric epoxyketone with respect to the chirality (14.0 mL), and the mixture was cooled to −20 °C. Aqueous
of the epoxide carbon in epoxomicin derivatives also results in K3PO4 (50% w/w, 3.8 mL, 14.0 mmol, 5.0 eq.) was then added
a drastic drop in activity, leads to the conclusion that the geo- in one portion. After the resulting biphasic mixture had been
metry and absolute stereochemistry of the epoxomicin vigorously stirred at the same temperature for 60 h, aq. form-
warhead is indeed ideally suited for optimal proteasome aldehyde (37% w/w, 1.0 mL, 14.0 mmol, 5.0 eq.) was added,
inactivation.
followed by another portion of aq. K3PO4 (50% w/w, 15.4 mL,
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56.0 mmol, 20.0 eq.). The mixture was stirred at room temp- The organic layer was dried over anhydrous MgSO4 and
erature for 60 h. The organic layer was then charged directly concentrated in vacuo. The crude product was used in the next
on a silica gel chromatographic column, the aqueous phase step without any purification. Dess–Martin periodinane (496 mg,
was extracted with toluene (2 × 7.0 mL), and the extracts were 1.17 mmol, 3.0 eq.) was suspended in anhydrous DCM, put
charged as well. Purification by column chromatography (1% under argon atmosphere and cooled to 0 °C. The crude
EtOAc–pentane→5% EtOAc–pentane) gave 11 (469.7 mg, product was co-evaporated with toluene, dissolved in an-
1.8 mmol, 66% yield). The enantiomeric excess (ee) of the hydrous DCM and added to the first solution. The reaction
product was determined by HPLC (Chiralcel OD (250 × mixture was stirred overnight and allowed to warm up to room
4.6 mm), n-hexane–i-PrOH 99 : 1, 1.0 mL min−1, λ = 254 nm: temperature. Sat. aq. NaHCO3 was added and the layers were
τmax = 5.612 min, τmin = 6.526 min, 50.8% ee). 1H NMR separated. The aqueous layer was extracted with EtOAc and the
(400 MHz, CDCl3): δ 6.04 (s, 1H), 5.94 (s, 1H), 5.18 (s, 1H), 4.44 combined organic layers were washed with H2O, brine, dried
(s, 1H) 2.33 (s, 3H), 1.54–1.44 (m, 3H), 1.42 (s, 9H), 2.04 over anhydrous MgSO4 and concentrated in vacuo. The crude
(s, 3H), 0.93–0.89 (m, 6H). 13C NMR (100 MHz, CDCl3): δ 199.91, product was purified by column chromatography (1% EtOAc–
155.32, 148.78, 126.62, 79.23, 52.68, 43.96, 28.50, 25.32, 23.57, pentane→10% EtOAc–pentane) to yield the title compound
22.65, 22.29. LC-MS (linear gradient 10→90% MeCN–H2O, (14a, 84.0 mg, 0.31 mmol, 79%). 14b was prepared in the same
0.1% TFA, 12.5 min): Rt (min): 7.68 (ESI-MS (m/z): 255.80 method and on the same scale, yielding the title compound
(M+)). HRMS calculated for C14H26NO3 256.19072 [M + H]+; (14b, 42.1 mg, 0.15 mmol, 40%). 14a: 1H NMR (400 MHz,
found 256.19085. [α]2D1 +9.8 (C = 1, CHCl3).
CDCl3): δ 4.48–4.43 (m, 2H), 2.92–2.88 (m, 2H), 2.05 (s, 3H),
tert-Butyl ((2R/S,4R)-2-hydroxy-6-methyl-3-methyleneheptan- 1.68–1.63 (m, 1H), 1.43 (s, 10H), 1.29–1.21 (m, 1H), 0.96–0.90
4-yl)carbamate (13a and 13b). A solution of compound 11 (m, 6H). 13C NMR (100 MHz, CDCl3): δ 206.60, 155.48, 79.57,
(400 mg, 1.57 mmol) in methanol was put under argon atmos- 64.92, 49.30, 47.00, 40.92, 28.33, 24.90, 24.59, 23.55, 21.25.
phere and CeCl3·7H2O (882 mg, 2.36 mmol, 1.5 eq.) was LC-MS (linear gradient 10→90% MeCN–H2O, 0.1% TFA,
added. The solution was cooled to 0 °C, before NaBH4 12.5 min): Rt (min): 7.65 (ESI-MS (m/z): 271.80 (M+)). 14b:
(71.2 mg, 1.88 mmol, 1.2 eq.) was added in 3 portions over 1H NMR (400 MHz, CDCl3): δ 5.33 (d, J = 10 Hz, 1H), 3.50–3.44
10 min. After stirring for 30 min., the reaction was quenched (m, 1H), 3.15 (d, J = 4.8 Hz, 1H), 2.97 (d, J = 5.2 Hz, 1H), 2.01
with glacial acetic acid and toluene was added after an (s, 3H), 1.67–1.51 (m, 2H), 1.44 (s, 10H), 1.29–1.21 (m, 1H),
additional 20 min at 0 °C. The solvents were removed in vacuo 0.93–0.89 (m, 6H). 13C NMR (100 MHz, CDCl3): δ 208.64,
and the oily residue was dissolved in an EtOAc–H2O mixture. 155.48, 79.41, 52.59, 51.26, 40.73, 28.43, 24.92, 24.48, 23.17,
The organic layer was washed with H2O and brine, dried over 22.04. LC-MS (linear gradient 10→90% MeCN–H2O, 0.1% TFA,
anhydrous MgSO4 and concentrated in vacuo. Purification by 12.5 min): Rt (min): 7.87 (ESI-MS (m/z): 271.80 (M+)).
column chromatography (2% EtOAc–pentane→10% EtOAc– HRMS calculated for C14H26NO4 272.18563 [M + H]+; found
pentane) gave 13a (153.4 mg, 0.6 mmol, 38% yield) and 13b 272.18592.
(105.0 mg, 0.4 mmol, 26% yield). 13a: 1H NMR (400 MHz,
Benzyl ((S)-1-(((S)-1-(((S)-1-((R/S)-2-acetyloxiran-2-yl)-3-methyl-
CDCl3): δ 5.11 (s, 1H), 5.02 (s, 1H), 4.75 (s, 1H), 4.34 (q, J = butyl)amino)-4-methyl-1-oxopentan-2-yl)amino)-4-methyl-1-
6.8 Hz, 1H), 4.21(s, 1H), 1.69–1.66 (m, 1H), 1.41 (s, 11H), 1.35 oxopentan-2-yl)carbamate (4a and 4b). Compound 15a was
(d, J = 8 Hz, 3H), 0.95 (d, J = 6.8 Hz, 6H). 13C NMR (100 MHz, prepared by the general procedure for Boc deprotection (14a,
CDCl3): δ 156.04, 155.07, 110.34, 79.88, 69.45, 48.96, 44.71, 14.9 mg. 55 µmol, 1.1 eq.), followed by the general procedure
28.52, 25.25, 23.35, 22.39, 22.00. 13b: 1H NMR (100 MHz, for azide coupling using 16 (19.6 mg, 50 µmol, 1 eq.). Purifi-
CDCl3): δ 5.18 (s, 1H), 5.05 (s, 1H), 4.69 (s, 1H), 4.31 (q, J = cation by preparative HPLC (60%→90% MeCN–H2O) yielded
6.4 Hz, 1H), 4.20 (q, J = 7.6 Hz, 1H), 1.70–1.65 (m, 1H), 1.45 the title compound (4a, 4.9 mg, 9.2 µmol, 18%). Compound 4b
(s, 11H), 1.38 (d, J = 6.4 Hz, 3H), 0.99 (d, J = 6.8 Hz, 1H). was prepared in the same method and same scale, yielding the
13C NMR (100 MHz, CDCl3):
δ
155.85, 155.54, 109.21, title compound (4b, 6.9 mg, 13.0 µmol, 26%). 4a: 1H NMR
79.82, 68.79, 50.48, 44.76, 28.54, 25.15, 23.22, 22.84, 22.24. (600 MHz, CDCl3): δ 7.26–7.15 (m, 5H), 5.03 (s, 2H), 4.75 (t, J =
HRMS calculated for C14H28NO3 258.20637 [M + H]+; found 2.4 Hz, J = 3.2 Hz, 1H), 4.20 (m, 1H), 4.03 (t, J = 7.2 Hz, J =
258.20654.
7.2 Hz, 1H), 2.80 (d, J = 4.4 Hz, 1H), 2.74 (d, J = 3.2 Hz, 1H),
tert-Butyl ((S)-1-((R/S)-2-acetyloxiran-2-yl)-3-methylbutyl)- 1.91 (s, 3H), 1.61–1.38 (m, 8H), 1.08–1.01 (m, 1H), 0.85–0.78
carbamate (14a and 14b). Compound 13a (100 mg, 0.39 mmol) (m, 18H). 13C NMR (150 MHz, CDCl3): δ 208.80, 175.62,
was dissolved in anhydrous DCM and cooled to 0 °C. Next, 174.88, 158.58, 129.48, 129.01, 128.85, 67.76, 65.82, 55.09,
VO(acac)2 (11 mg, 0.04 mmol, 0.1 eq.) and tBuOOH (0.21 ml 53.31, 49.64, 45.94, 41.94, 41.52, 40.21, 26.04, 25.95, 25.87,
5.5 M in decane, 1.17 mmol, 3.0 eq.) were added and the 24.52, 23.94, 23.39, 23.36, 21.94, 21.24. LC-MS (linear gradient
reaction mixture was stirred for 2 h, while allowing the temp- 10→90% MeCN–H2O, 0.1% TFA, 12.5 min): Rt (min): 8.39
erature to rise slowly to room temperature. The resulting (ESI-MS (m/z): 532.20, (M + H+)). 4b: 1H NMR (600 MHz,
purple solution was concentrated in vacuo and the residue was CDCl3): δ 7.26–7.16 (m, 5H), 5.00 (s, 2H), 4.26 (t, J = 5.2 Hz, J =
dissolved in EtOAc and washed with half saturated NaHCO3 4.8 Hz, 1H), 4.04 (m, 1H), 3.95 (m, 1H), 2.90 (m, 2H), 1.88
solution. The aqueous layer was extracted with EtOAc and (s, 3H), 1.61–1.27 (m, 9H), 0.86–0.74 (m. 18H). 13C NMR
the combined organic layer was washed with H2O and brine. (150 MHz, CDCl3): δ 208.95, 175.57, 174.32, 158.58, 138.21,
5714 | Org. Biomol. Chem., 2014, 12, 5710–5718
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129.47, 129.02, 128.89, 67.89, 63.26, 55.14, 53.29, 50.59, 49.57,
tert-Butyl ((S)-4-methyl-1-((R)-2-methyloxiran-2-yl)-1-oxopen-
42.05, 41.50, 40.64, 25.92, 25.90, 24.45, 23.74, 23.41, 21.96, tan-2-yl)carbamate (20). Compound 19 (0.786 g, 3.1 mmol)
21.86, 21.78. LC-MS (linear gradient 10→90% MeCN–H2O, was coevaporated with toluene, dissolved in DCM (30 mL) and
0.1% TFA, 12.5 min): Rt (min): 8.39 (ESI-MS (m/z): 532.20, cooled down to 0 °C. After addition of VO(acac)2 (0.041 g,
(M
[M + H]+; found 532.33796.
+
H+)). HRMS calculated for C29H46N3O6 532.33811 0.15 mmol, 0.05 eq.) the solution turned light blue-green and
subsequent addition of tBuOOH (1.7 mL 5.5 M solution,
tert-Butyl (S)-(1-(methoxy(methyl)amino)-4-methyl-1-oxopenta- 9.4 mmol, 3.0 eq.) resulted in a dark brown-purple reaction
n-2-yl)carbamate (18). Boc-Leu-OH (4.63 g, 20 mmol) was dis- mixture. After stirring for 1 h the reaction mixture was
solved in DCM (200 mL), followed by addition of HBTU removed from the ice bath. After 15 min, the reaction mixture
(9.10 g, 24 mmol, 1.2 eq.). After stirring for 5 min DiPEA was concentrated in vacuo, dissolved in EtOAc and washed
(10.5 mL, 60 mmol, 3.0 eq.) and N,O-dimethylhydroxyl- with a 1 : 1 mixture of sat. aq. NaHCO3 and H2O. The aqueous
amine·HCl (2.35 g, 24 mmol, 1.2 eq.) was added. TLC analysis indi- layer was extracted with EtOAc (3×) and the combined organics
cated completion after 2 h. The reaction mixture was diluted were washed with H2O and brine, dried over MgSO4 and con-
with EtOAc, washed with 1 M aqueous HCl solution, sat. aq. centrated in vacuo to give the crude intermediate as a yellow
NaHCO3 and brine, dried over MgSO4 and concentrated oil. Dess–Martin periodinane (1.974 g, 4.7 mmol, 1.5 eq.) was
in vacuo. Purification by column chromatography (10% EtOAc– dissolved in DCM (30 mL) and cooled down to 0 °C. The inter-
pentane→40% EtOAc–pentane) yielded the title compound as mediate was coevaporated with toluene, dissolved in DCM
1
a clear oil (4.6 g, 17 mmol, 83%). H NMR (400 MHz, CDCl3): (30 mL) and added to the Dess–Martin periodinane solution.
δ 5.07 (d, J = 9.0 Hz, 1H), 4.73 (s, 1H), 3.79 (s, 3H), 3.20 (s, 3H), After 1.5 h the reaction mixture was removed from the ice
1.78–1.66 (m, 1H), 1.44 (s, 11H), 0.97 (d, J = 6.5 Hz, 3H), 0.93 bath. After another 1.5 h TLC analysis indicated full conver-
(d, J = 6.7 Hz, 3H). 13C NMR (100 MHz, CDCl3): δ 155.63, 79.43, sion of the intermediate and the reaction was quenched with
61.57, 48.97, 42.11, 32.08, 28.35, 24.73, 23.34, 21.58. LC-MS sat. aq. NaHCO3 (30 mL). The aqueous layer was extracted with
(linear gradient 10→90% MeCN–H2O, 0.1% TFA, 15 min): DCM (2 × 15 mL). The combined organics were washed
Rt (min): 7.81 (ESI-MS (m/z): 274.93, (M + H+)).
with H2O (3×) and brine, dried over MgSO4 and concentrated
tert-Butyl ((3R,4S)-3-hydroxy-2,6-dimethylhept-1-en-4-yl)- in vacuo. Purification by column chromatography (1% EtOAc–
carbamate (19). A solution of 2-bromopropene (3.3 mL, pentane→5% EtOAc–pentane) yielded the title compound
1
33.1 mmol, 3 eq.) in dry Et2O (40 mL) was cooled down to (391 mg, 1.4 mmol, 46%). H NMR (400 MHz, CDCl3): δ 4.89
−78 °C under argon atmosphere and stirred for 15 min before (d, J = 8.4 Hz, 1H), 4.38–4.27 (m, 1H), 3.29 (d, J = 4.9 Hz, 1H),
adding tBuLi (31.0 mL 1.6 M in pentane, 49.7 mmol, 4.5 eq.). 2.89 (d, J = 5.0 Hz, 1H), 1.78–1.66 (m, 1H), 1.52 (s, 3H), 1.48
The reaction mixture was stirred for 15 min. Weinreb amide 18 (dd, J = 9.8, 3.2 Hz, 1H), 1.41 (s, 9H), 1.22–1.12 (m, 1H), 0.95
(3.0 g, 11.1 mmol) was coevaporated with toluene and dis- (dd, J = 12.8, 6.6 Hz, 6H). 13C NMR (101 MHz, CDCl3):
solved in 10 mL Et2O. This solution was added dropwise to the δ 209.54, 155.63, 79.61, 58.98, 52.29, 51.37, 40.28, 28.31, 25.06,
reaction mixture during 30 min. The resulting reaction 23.41, 21.26, 16.75.
mixture was allowed to warm up to rt and quenched after 2 h
Benzyl ((S)-4-methyl-1-((R)-2-methyloxiran-2-yl)-1-oxopentan-
with sat. aq. NH4Cl (40 ml). The water layer was extracted with 2-yl)carbamate (21). tert-Butyl ((S)-4-methyl-1-((R)-2-methyl-
EtOAc (3×) and the combined organics were washed with oxiran-2-yl)-1-oxopentan-2-yl)-carbamate (20) (312 mg, 1.15 mmol)
brine, dried over MgSO4 and concentrated in vacuo. Purifi- was dissolved in 30% TFA in DCM and the reaction was stirred
cation by flash column chromatography (1% EtOAc– at room temperature for 30 min. All solvent was removed
pentane→10% EtOAc–pentane) yielded the product as clear oil in vacuo and co-evaporated with toluene for three times. The
(1.60 g, 6.3 mmol, 57%). The product obtained from last step deprotected compound was dissolved in 15 mL DCM and
(1.60 g, 6.3 mmol) was dissolved in methanol (60 mL), DiPEA (0.60 mL, 3.45 mmol, 3 eq.) was added. Then, CbzCl
followed by addition of CeCl3·7H2O (3.52 g, 9.5 mmol, 1.5 eq.). (0.25 mL, 1.73 mmol, 1.5 eq.) was added and the reaction was
After the solution turned clear, it was cooled down to 0 °C and stirred at room temperature overnight. All solvent was removed
NaBH4 (0.34 g, 8.8 mmol, 1.4 eq.) was added portion-wise. in vacuo and the residue was dissolved in EtOAc. The organic
After 5 min TLC analysis indicated complete conversion and layer was washed by H2O, brine, dried over anhydrous MgSO4
the reaction mixture was quenched with glacial AcOH (6 mL). and concentrated in vacuo. Purification by column chromato-
The mixture was concentrated, coevaporated with toluene, dis- graphy (1% EtOAc–pentane→5% EtOAc–pentane) yielded the
solved in EtOAc, washed with H2O and brine, dried over title compound (290.0 mg, 0.95 mmol, 83%). 1H NMR
MgSO4 and concentrated in vacuo. Purification by column (400 MHz, CDCl3): δ 7.37–7.23 (m, 5H), 6.90 (d, J = 8.4 Hz, 1H),
chromatography (10% EtOAc–pentane→50% EtOAc–pentane) 5.42 (d, J = 8.4 Hz, 1H) 5.07–4.99 (m, 2H), 4.42–4.36 (m, 1H),
1
yielded the title compound (1.39 g, 5.4 mmol, 86%). H NMR 3.27 (d, J = 5.2 Hz, 1H), 2.96 (d, J = 4.8 Hz, 1H), 1.74–1.65 (m,
(400 MHz, CDCl3): δ 5.03 (s, 1H), 4.93 (s, 1H), 4.14 (s, 1H), 3.83 1H), 1.53–1.34 (m, 4H), 1.28–1.20 (m, 1H), 0.97–0.88 (m. 6H).
(s, 1H), 1.75 (s, 3H), 1.63 (d, J = 5.9 Hz, 1H), 1.44 (s, 9H), 13C NMR (100 MHz, CDCl3): δ 209.27, 156.22, 136.21, 128.70,
1.31–1.15 (m, 2H), 0.97–0.82 (m, 6H). 13C NMR (100 MHz, 127.94, 127.41, 126.88, 66.84, 59.01, 52.20, 51.82, 40.21, 25.01,
CDCl3): δ 156.22, 144.96, 111.95, 111.34, 77.87, 51.07, 37.20, 23.37, 21.19, 16.67. LC-MS (linear gradient 10→90% MeCN–
28.48, 24.69, 23.87, 21.60, 19.52.
H2O, 0.1% TFA, 12.5 min): Rt (min): 7.88 (ESI-MS (m/z): 305.93,
This journal is © The Royal Society of Chemistry 2014
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(M
+
H+)). HRMS calculated for C17H24NO4 306.16998
Benzyl ((S)-4-methyl-1-(((S)-4-methyl-1-(((S)-3-methyl-1-((2S
or R, 2′R)-2′-methyl-[2,2′-bioxiran]-2-yl)butyl)amino)-1-oxo-
[M + H]+; found 306.17003.
Benzyl ((S)-5-methyl-2-((S)-2-methyloxiran-2-yl)hex-1-en-3-yl)- pentan-2-yl)amino)-1-oxopentan-2-yl)carbamate (5a and 5b).
carbamate (22). Methyltriphenylphosphonium bromide (543 mg, Compound 23a (39 mg, 0.12 mmol) was dissolved in 3 mL
1.52 mmol, 1.6 eq.) was suspended in 10 mL anhydrous THF anhydrous THF and 3 mL anhydrous MeOH. 1,4-Cyclohexa-
and then potassium bis(trimethylsilyl)amide (2.7 mL, 0.5 M in diene (0.4 mL, 5.52 mmol, 46 eq.) and 10% Pd–C (68 mg) were
toluene, 1.2 mmol, 1.4 eq.) was added drop by drop. The reac- added. After stirring at room temperature for 2 h, the reaction
tion was stirred at room temperature for 1 h and then cooled mixture was filtered through celite and solvents were removed
to −78 °C. Compound 18 (290.0 mg, 0.95 mmol) was dissolved in vacuo. The crude products 24a was directly used in next
in 5 mL anhydrous THF and added to the reaction mixture. step. 24b was prepared in the same method and the same
The reaction was stirred at −78 °C for 1 h and then stirred scale. Compound 5a and 5b were prepared by the general pro-
overnight while allowing to warm up to room temperature. The cedure for azide peptide coupling used 16 (43.1 mg, 0.11 mol,
reaction was quenched by saturated aqueous NH4Cl (30 mL)
1 eq.), 24a and 24b. Purification by preparative HPLC
and then extracted with EtOAc (3 × 30 mL). The combined (50%→100% MeCN–H2O) yielded the title compound
organic layer was washed by brine, dried over anhydrous (5a, 8.1 mg, 14.9 µmol, 14% and 5b, 9.4 mg, 17.2 µmol, 16%).
MgSO4 and concentrated in vacuo. Purification by column 5a: 1H NMR (400 MHz, MeOD): δ 7.26–7.17 (m, 5H), 4.98 (s, 2H),
chromatography (1% EtOAc–pentane→10% EtOAc–pentane) 4.39–4.35 (m, 1H), 4.28–4.25 (m, 1H), 4.06–4.02 (m, 1H), 2.67
yielded the title compound (240 mg, 0.79 mmol, 83%). (d, J = 5.2 Hz, 1H), 2.54–2.52 (m, 2H), 2.45 (d, J = 5.2 Hz, 1H),
1H NMR (400 MHz, CDCl3): δ 7.35–7.26 (m, 5H), 5.20–5.03 (m, 1.61–1.37 (m, 9H), 1.29 (s, 3H), 0.88–0.79 (m, 18H). 13C NMR
5H), 4.16–4.10 (m, 1H), 2.85 (d, J = 4.8 Hz, 1H), 2.73 (d, J = (100 MHz, MeOD): δ 175.37, 174.62, 158.52, 138.17, 129.47,
5.2 Hz, 1H), 1.70–1.60 (m, 1H), 1.49–1.25 (m, 5H), 0.96–0.89 129.02, 128.82, 67.68, 61.59, 55.74, 54.87, 53.23, 52.59, 46.71,
(m, 6H). 13C NMR (100 MHz, CDCl3): δ 155.68, 151.38, 136.53, 41.94, 41.80, 40.10, 25.91, 25.85, 25.64, 24.18, 23.41, 23.37,
129.40, 128.47, 128.40, 128.05, 110.73, 66.83, 56.85, 54.25, 21.97, 21.86, 21.38, 18.93. LC-MS (linear gradient 10→90%
49.78, 44.98, 25.11, 23.28, 21.56. LC-MS (linear gradient MeCN–H2O, 0.1% TFA, 12.5 min): Rt (min): 8.22 (ESI-MS (m/z):
1
10→90% MeCN–H2O, 0.1% TFA, 12.5 min): Rt (min): 6.81 546.27, (M + H+)). 5b: H NMR (600 MHz, MeOD): δ 7.25–7.16
(ESI-MS (m/z): 304.07, (M
C18H26NO3 304.19072 [M + H]+; found 304.19086.
+
H+)). HRMS calculated for (m, 5H), 5.00–4.94 (m, 2H), 4.29–4.25 (m, 2H), 4.06–4.01
(m, 1H), 2.59–2.49 (m, 4H), 1.58–1.20 (m, 12H), 0.83–0.73
Benzyl ((S)-3-methyl-1-((2R/S, 2′R)-2′-methyl-[2,2′-bioxiran]- (m, 18H). 13C NMR (150 MHz, MeOD): δ 175.17, 174.55,
2-yl)butyl)-carbamate (23a and 23b). Compound 22 (240 mg, 158.53, 138.14, 129.48, 129.02, 128.82, 67.69, 63.11, 56.90,
0.79 mmol) was dissolved in 15 mL anhydrous DCM and 54.97, 53.39, 52.61, 47.91, 47.68, 41.93, 41.76, 40.49, 25.84,
m-CPBA (585.0 mg. 2.37 mmol, 3 eq.) was added. The reaction 25.74, 25.67, 24.10, 23.39, 23.35, 22.18, 22.02, 21.53, 18.75.
was stirred at room temperature overnight. DCM was removed LC-MS (linear gradient 10→90% MeCN–H2O, 0.1% TFA,
in vacuo and the residue was dissolved EtOAc and washed by 12.5 min): Rt (min): 8.22 (ESI-MS (m/z): 546.27, (M + H+)). HRMS
saturated NaS2O3, saturated NaHCO3, brine, dried over an- calculated for C30H48N3O6 546.35376 [M + H]+; found 546.35375.
hydrous MgSO4 and concentrated in vacuo. Purification by
Benzyl
((S)-1-(((S)-1-(((S)-2,6-dimethyl-3-oxohept-1-en-4-yl)
column chromatography (1% EtOAc–pentane→10% EtOAc– amino)-4-methyl-1-oxopentan-2-yl)amino)-4-methyl-1-oxopen-
pentane) yielded the title compounds (23a, 99 mg, 0.31 mmol, tan-2-yl)carbamate (6). Compound 25 was prepared according
39% and 23b, 65.0 mg, 0.20 mmol, 26%). 23a: 1H NMR to the literature procedures, followed by the general procedure
(400 MHz, CDCl3): δ 7.41–7.29 (m, 5H), 5.17–5.08 (m, 2H), for azide peptide coupling using 16 (121.7 mg, 0.31 mol, 1 eq.)
4.72–4.69 (m, 1H), 4.34–4.27 (m, 1H), 2.74 (d, J = 5.2 Hz 1H), and compound 25 (92.0 mg. 0.34 mmol, 1.1 eq.). Purifica-
2.68 (d, J = 7.6 Hz, 1H), 2.65–2.63 (m, 2H), 1.72–1.66 (m, 1H), tion by column chromatography (10% EtOAc–pentane→30%
1.47(s, 3H), 1.44–1.35 (m, 2H), 0.99–0.85 (m, 6H). 13C NMR EtOAc–pentane) yielded the title compound (130.0 mg,
(100 MHz, CDCl3): δ 156.37, 128.74, 128.62, 128.42, 128.27, 0.25 mmol, 81%). 1H NMR (400 MHz, CDCl3): δ 7.35–7.29
66.90, 60.85, 54.49, 51.89, 48.42, 47.56, 40.13, 23.79, 22.52, (m, 5H), 6.90 (d, J = 8.4 Hz, 1H), 6.65 (d, J = 7.6 Hz, 1H),
21.20, 18.73. LC-MS (linear gradient 10→90% MeCN–H2O, 6.11 (s, 1H), 5.90 (s, 1H), 5.51 (d, J = 8.4 Hz, 1H) 5.40–5.35
0.1% TFA, 12.5 min): Rt (min): 7.56 (ESI-MS (m/z): 319.93, (m, 1H), 5.13–5.06 (m, 2H), 4.55 (m, 1H), 4.27 (m, 1H), 1.89
(M + H+)). 23b: 1H NMR (400 MHz, CDCl3): δ 7.41–7.27 (s, 3H), 1.68–1.34 (m, 9H), 0.96–0.86 (m. 18H). 13C NMR
(m, 5H), 5.12–5.08 (m, 2H), 4.89 (d, J = 9.2 Hz, 1H), 4.13–4.07 (100 MHz, CDCl3): δ 200.71, 172.19, 171.41, 156.34, 142.26,
(m, 1H), 2.80–2.73 (m, 3H), 2.53 (d, J = 5.2 Hz, 1H), 1.68–1.61 136.34, 128.64, 128.28, 128.12, 126.65, 67.12, 53.52, 51.82,
(m, 1H), 1.47(s, 3H), 1.47–1.33 (m, 2H), 0.99–0.88 (m, 6H). 51.26, 42.73, 41.59, 41.29, 25.05, 24.79, 23.47, 22.96, 22.80,
13C NMR (100 MHz, CDCl3): δ 156.26, 128.70, 128.63, 128.56, 22.37, 22.24, 21.86, 17.92. LC-MS (linear gradient 10→90%
128.28, 66.84, 61.48, 57.03, 51.17, 49.76, 49.70, 40.60, 23.91, MeCN–H2O, 0.1% TFA, 12.5 min): Rt (min): 8.69 (ESI-MS (m/z):
22.77, 21.65, 19.04. LC-MS (linear gradient 10→90% MeCN– 516.20, (M + H+)). HRMS calculated for C29H46N3O5 516.34320
H2O, 0.1% TFA, 12.5 min): Rt (min): 7.49 (ESI-MS (m/z): 319.93, [M + H]+; found 516.34302.
(M
+
H+)). HRMS calculated for C18H26NO4 320.18563
Benzyl ((S)-4-methyl-1-(((S)-4-methyl-1-(((S)-6-methyl-2,3-
dioxoheptan-4-yl)amino)-1-oxopentan-2-yl)amino)-1-oxopentan-
[M + H]+; found 320.18573.
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2-yl)-carbamate (7). Ozone was bubbled into a stirred solution
of 6 (130 mg, 0.25 mmol) in DCM at −78 °C for 15 min until
Acknowledgements
the blue colour of the saturated ozone solution persisted. The The Chinese Scholarship Council (CSC, to B-TX), the
solution was then purged with oxygen until the solution Netherlands Genomics Initiative (NGI, to HSO) and the
became colourless. Triphenylphosphine (131.1 mg, 0.50 mmol, Netherlands Organization for Scientific Research (NWO-CW, to
2 eq.) was added after purging with argon for 5 min and the HSO) are acknowledged for the financial support.
reaction was stirred overnight while warming up to room temp-
erature. Purification by preparative HPLC (50%→100% MeCN–
H2O) yielded the title compound (18.5 mg, 35.8 µmol, 14%).
1H NMR (400 MHz, CDCl3): δ 7.38–7.31 (m, 5H), 6.93 (d, J =
5.6 Hz, 1H), 6.54 (d, J = 8.0 Hz, 1H), 5.32 (d, J = 7.2 Hz, 1H),
References
5.14–5.11 (m, 2H), 4.93–4.90 (m, 1H), 4.47–4.42 (m, 1H), 4.18
(m, 1H), 2.35 (s, 3H), 1.68–1.43 (m, 9H), 0.93–0.87 (m. 18H)
13C NMR (100 MHz, CDCl3): δ 196.86, 172.50, 171.91, 156.53,
136.08, 128.74, 128.48, 128.23, 67.42, 53.82, 52.37, 51.43,
41.21, 40.33, 39.67, 25.13, 24.84, 24.20, 23.26, 23.04,
22.91, 22.12, 22.05, 21.61. LC-MS (linear gradient 10→90%
MeCN–H2O, 0.1% TFA, 12.5 min): Rt (min): 8.52 (ESI-MS (m/z):
518.13, (M + H+)). HRMS calculated for C28H44N3O6 518.32246
[M + H]+; found 518.32245.
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Benzyl ((S)-4-methyl-1-(((S)-4-methyl-1-(((S)-6-methyl-3-methy-
lene-2-oxoheptan-4-yl)amino)-1-oxopentan-2-yl)amino)-1-oxo-
pentan-2-yl)carbamate (8). Compound 26 was prepared by the
general procedure for Boc deprotection (compound 11, 52 mg.
0.19 mmol, 1.1 eq.), followed by the general procedure for
azide peptide coupling used 16 (67.8 mg, 0.17 mol, 1 eq.).
Purification by column chromatography (10% EtOAc–
pentane→30% EtOAc–pentane) yielded the title compound
(68.2 mg, 0.13 mmol, 78%).), 1H NMR (400 MHz, CDCl3):
δ 7.33–7.27 (m, 5H), 6.72 (d, J = 8.4 Hz, 1H), 6.42 (d, J = 7.6 Hz,
1H), 6.00 (s, 1H), 5.92 (s, 1H), 5.13–5.05 (m, 2H), 4.79–4.73
(m, 1H), 4.22–4.39 (m, 1H), 4.25–4.24 (m, 1H), 2.30 (s, 3H),
1.68–1.29 (m, 9H), 0.92–0.85 (m. 18H). 13C NMR (100 MHz,
CDCl3): δ 199.71, 172.34, 170.85, 156.35, 148.23, 136.26,
128.63, 128.28, 128.10, 126.87, 67.13, 53.63, 52.01, 50.34,
43.57, 41.56, 41.27, 25.35, 24.90, 23.10, 22.90, 22.81, 22.27,
22.06, 22.00. LC-MS (linear gradient 10→90% MeCN–H2O,
0.1% TFA, 12.5 min): Rt (min): 8.43 (ESI-MS (m/z): 516.20,
(M
+
H+)). HRMS calculated for C29H46N3O5 516.34320
[M + H]+; found 516.34316.
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Competition assay in cell lysate
Lysates of HEK-293 T cells were prepared by sonication in
3 volumes of lysis buffer containing 50 mM Tris pH 7.5, 1 mM
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19 In our work on β5-specific peptide epoxyketones we noted
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5718 | Org. Biomol. Chem., 2014, 12, 5710–5718
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