Sugar amino acid based peptide epoxyketones as potential proteasome inhibitors

Article abstract of DOI:10.1016/j.bioorg.2010.04.004

This paper describes the synthesis and biological evaluation of nine epoxomicin-derived sugar amino acid containing peptide epoxyketones. The title compounds are assembled from six sugar amino acid dipeptide isosteres and are synthesized using solution-phase peptide synthesis protocols. Although neither of the compounds displays inhibitory activity towards any of the proteasome active sites, our approach holds promise towards the development of structurally new proteasome inhibitors. It is likely that the central sugar amino acid dipeptide isoster needs to be designed such that it closely resemble dipeptides at position P2 and P3 in proteasome substrates inhibitors, such as the Thr-Ile dipeptide present in the lead compound, epoxomicin.

Full text of DOI:10.1016/j.bioorg.2010.04.004

Bioorganic Chemistry 38 (2010) 202–209
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Sugar amino acid based peptide epoxyketones as potential proteasome inhibitors
*
**
Martijn D.P. Risseeuw, Bogdan I. Florea, Gijsbert A. van der Marel, Herman S. Overkleeft , Mark Overhand
Gorlaeus Laboratories, Leiden Institute of Chemistry, Leiden University, Leiden, The Netherlands
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 1 April 2010
Available online 28 April 2010
This paper describes the synthesis and biological evaluation of nine epoxomicin-derived sugar amino acid
containing peptide epoxyketones. The title compounds are assembled from six sugar amino acid dipep-
tide isosteres and are synthesized using solution-phase peptide synthesis protocols. Although neither of
the compounds displays inhibitory activity towards any of the proteasome active sites, our approach
holds promise towards the development of structurally new proteasome inhibitors. It is likely that the
central sugar amino acid dipeptide isoster needs to be designed such that it closely resemble dipeptides
at position P2 and P3 in proteasome substrates inhibitors, such as the Thr-Ile dipeptide present in the lead
compound, epoxomicin.
Keywords:
Dipeptide isoster
Sugar amino acid
Epoxyketone
Proteasome
Activity-based probe
Ó 2010 Elsevier Inc. All rights reserved.
1. Introduction
that provide structural integrity to the 20S complex and that are
the interaction sites for specific proteins in the 19S caps. The 20S in-
The 26S proteasome is involved in the majority of cytosolic and
nuclear proteolysis events in mammalian cells [1]. Proteasomal pro-
teolysis takes place in an ATP- and ubiquitin-dependent fashion [2].
The proteasome products themselves, small oligopeptides ranging
from three to about twenty amino acid residues, are further pro-
cessed by a range of aminopeptidases to ultimately deliver amino
acids that can be reused in ribosomal protein synthesis. A small por-
tion of the oligopeptides produced by the proteasome is recruited by
the immune system and ends up on major histocompatibility com-
plex class I molecules onthe cellsurface for immunesurveillance [3].
Next to the constitutively expressed 26 proteasome two immune
tissue specific proteasome particles have been identified to date,
namely the immunoproteasome [4] and the thymoproteasome [5].
The biochemistry, biology and immunology of the proteasome, as
part of the ubiquitin–proteasome pathway [6] has been extensively
reviewed [1–7] in recent years. Of relevance for the here described
research is the nature and differences in activity of the catalytic enti-
ties that reside in the core proteasome particles [8]. The 26S protea-
some is assembled from two distinct multiprotein subunit species.
Of these, the 19S cap contains amongst others the ubiquitin-recogn-
ising domains and ATPase activities that together control proteaso-
mal processing. Either one or two 19S caps are associated with the
core 20S proteasome particle. The 20S proteasome has a C2-sym-
metrical barrel-like overall shape and consists of 28 protein subunits
stacked in four rings of seven subunits each. The two outer rings
ner rings contain seven distinct b-subunits and in eukaryotes three
of these possess proteolytic activity. With the aid of fluorogenic sub-
strates, oligopeptides of a specific sequential nature and equipped
with a fluorogenic leaving group situated at the C-terminus, the
specificity of these is determined as chymotryptic for the b5-subunit
(cleaving preferentially C-terminal of hydrophobic amino acid resi-
dues), tryptic for the b2-subunit (cleaving preferentially C-terminal
of basic amino acid residues) and caspase-like for the b1-subunit
(cleaving preferentially C-terminal of acidic amino acid residues)
[9]. The substrate specificity however is not as narrow as the desig-
nations indicate, and in fact determination of the actual substrate
specificity of the proteasome catalytic activities and their role in hu-
man health and disease is subject of extensive research [8–10]. The
same holds true for the catalytic subunits unique to the immunopro-
teasome (b5i, b5i and b5i) [11] and the thymoproteasome (b5t) [12].
The clinical relevance of these research activities is underscored by
the recent development of the peptide boronic acid Bortezomib as a
drug for the treatment of multiple myeloma [13] Bortezomib inhib-
its both b1 and b5 while leaving b2 largely intact and it is thought
that this specificity is at the basis of the mode of action of this clin-
ically approved drug [14].
The development of proteasome inhibitors is a widely pursued
research objective both for fundamental and practical (clinical)
reasons [15]. With a few notable exceptions [16] most effective
proteasome inhibitors are composed of a short oligopeptidic se-
quence equipped C-terminally with an electrophilic trap [8–10].
A prominent example is the natural product and proteasome inhib-
itor, epoxomicin [17] (Fig. 1, left structure) in which the C-terminal
epoxyketone features as the electrophilic trap. The N-terminal
threonine that resides in the proteasome catalytic sites reacts with
the epoxyketone moiety to form a morpholine ring and in this
consist of seven homologous but sequentially distinct
a-subunits
* Corresponding author.
** Corresponding author.
E-mail addresses: h.s.overkleeft@chem.leidenuniv.nl (H.S. Overkleeft), over-
hand@chem.leidenuniv.nl (M. Overhand).
0045-2068/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved.
doi:10.1016/j.bioorg.2010.04.004

M.D.P. Risseeuw et al. / Bioorganic Chemistry 38 (2010) 202–209
203
O
O
H
N
O
O
O
N
SAA
N
H
N
H
N
H
N
N3
O
O
O
O
HO
O
Fig. 1. Epoxomicin (left) and general design of the tagged sugar amino acid containing analogues (right).
fashion the active sites are covalently and irreversibly modified
[18]. Epoxomicin is a broad-spectrum proteasome inhibitor and
targets all catalytic subunits, with some preference for b2 and b5.
Next to potency, subunit specificity is considered a major research
theme in the research community elaborating proteasome inhibi-
tors for both fundamental reasons (which subunit is responsible
for the generation of a given peptide, for instance in the context
of antigen presentation) and applied reasons (consider the subunit
specificity of the therapeutic agent Bortezomib, is this the optimal
pharmacological profile?). For these reasons considerable efforts
are aimed at the generation and evaluation of synthetic peptide
based proteasome inhibitors. These efforts can be roughly divided
in two distinct research themes, with the first characterised as re-
search aimed at variation of the C-terminal electrophilic trap and
the second as research aimed at variation of the oligopeptidic pro-
teasome recognition segment. With respect to the first strategy,
next to peptide epoxyketones [17,19] and peptide boronic acids
[13,20] peptide aldehydes [21], peptide vinyl sulphones [22] and
other peptide-based Michael acceptors [22] have been studied.
With respect to the latter, both peptide fragments composed of
by N-acylation with bromoacetyl bromide and subsequent bro-
mide displacement by azide to give the protected, azide tagged
N-terminal residue 3. Treatment with lithium hydroxide in a
water/dioxane mixture provided the free carboxylic acid 4 in 72%
overall yield.
With the suitably protected sugar amino acids and N-terminal
N-methylated isoleucine in hand the synthesis of the peptide
epoxyketones was undertaken. The strategy followed is depicted
in Scheme 2. Leucine epoxyketone 5, prepared according to the lit-
erature procedure [19a], was condensed with sugar amino acid a–f
under the agency of HCTU and DIPEA. Staudinger reduction of the
azide in the resulting dipeptides 6 was followed by condensation
with isoleucine derivative 4, and for this transformation the re-
agent combination consisting of EDC, HOAt and lutidine in a mix-
ture of DMF and dichloromethane proved most efficient. These
conditions have been succesfully applied previously in peptide
couplings of N-methyl-N-acyl amino acids with minimal racemiza-
tion. Epimerization of the
a-carbon of the N-terminal isoleucine
residue could however not be avoided. This provided stereomeric
mixtures of peptide epoxyketones 7a–f. Sugar amino acids b, c
and d gave rise to separable pairs of diastereomers (7bI/7bII, 7cI/
7cII and 7dI/7dII), although we were not able to identify the nature
of the chiral carbon (marked ꢀ in the scheme) of the individual dia-
stereomers. Sugar amino acids a, e and f produced intractable mix-
tures of diastereoisomers, at least after HPLC purification.
Epimerisation such as described above is a known hindrance in
peptide chemistry, especially during block couplings of (oligo)pep-
tidic fragments such as executed here. An attractive strategy to
avoid or suppress epimerisation of the carboxylate partner in pep-
tide block couplings is to make use of acyl azides as the activated
species [32]. This strategy was investigated here as well, and to this
end, the required Boc-protected isoleucine hydrazone 10 was read-
ily prepared starting from N-methyl-Fmoc-isoleucine 8 (Scheme
3). Ensuing in situ formation of the acyl azide by treatment with
HCl and tert-butyl nitrite followed by addition of amines 7a–f, ob-
tained after Staudinger reduction of 6a–f (see Scheme 2) and DIPEA
proved however abortive, and no tripeptides were isolated from
the resulting reaction mixtures.
proteinogenic [23] and non-proteinogenic [13]
been generated, also in a combinatorial approach [24] Pharmaco-
phores composed of molecular entities different from the -amino
a-amino acids have
a
acid configuration are remarkably scarce in the proteasome inhib-
itor literature [25]. Given the fact that sugar amino acids, subject of
this work, have met with some success in research on the design of
conformationally constrained and physiologically stable inhibitors
of other enzymes (RNAse [26], protein:farnesyl transferase [27])
we set out to investigate whether the assembly of sugar amino acid
based peptide epoxyketones would lead to effective and possibly
subunit-selective proteasome inhibitors.
The general structure of the sugar amino acid containing pep-
tide epoxyketones envisaged is depicted in Fig. 1 (right structure).
The C-terminal leucine epoxyketone moiety present in epoxomicin
is recognised by the three proteolytic activities of the 26S protea-
some and it is known [28] that some subunit specificity can be
achieved by variation of the threonine–isoleucine–isoleucine se-
quence at the positions P2–P4. We decided to keep the N-terminal,
N-methylated isoleucine residue intact and replace the threonine–
isoleucine dipeptide sequence with the panel of sugar amino acids
a–f depicted in Fig. 2. Of the panel of sugar amino acids used deriv-
atives a–e were subject of studies described elsewhere by us [29]
and others [30]. The N-terminal acetyl cap as present in epoxomi-
cin finally is replaced by azidoacetyl, enabling activity-based pro-
filing [31] as an alternative strategy compared to competition
assays to establish potential proteasome reactivity. We here report
on the synthesis and evaluation as potential proteasome inhibitors
of a panel of eight peptide epoxyketones encompassing sugar ami-
no acids a–f.
In the next experiment, peptide epoxyketones 7a–f were
screened on their proteasome inhibitor potential in a competition
experiment using the BODIPY–peptide–vinyl sulphone, MV151
(Fig. 3), as a read-out [22a–c]. In this experiment, lysates of the hu-
man hybridoma kidney cell line, HEK 293T, were treated with final
concentrations of 0, 1, 10 and 100 lM and incubated for one hour
prior to treatment with MV151. The proteins were denatured, re-
solved by SDS PAGE and monitored by a Typhoon fluorescence
scanner to identify fluorescent protein bands. As can be seen
(Fig. 4 lane 0) tissue cell lysate treated with MV151 without pre-
incubation with one of the putative inhibitors reveal three fluores-
cent bands corresponding to the proteasome catalytic actives
(from top down) b2, b1, and b5. Essentially the same picture
emerges irrespective of the nature and end concentrations of the
peptide epoxyketones added to the tissue lysate, indicating that
neither of the produced compounds holds any inhibitory potential
towards the proteasome. In some cases, there seems to be a slight
2. Results
The synthesis of the panel of peptide epoxyketones commences
with N-methylation of Fmoc-Ile–OH 1 according to the literature
procedure [32] to give Boc-(N-Me)-isoleucine
2 (Scheme 1).
TFA-mediated removal of the Boc protective group was followed
reduction of fluorescence at 100
lM
final concentrations.

204
M.D.P. Risseeuw et al. / Bioorganic Chemistry 38 (2010) 202–209
O
O
O
H
H
O
O
O
HO
N3
OBn
HO
BnO
N3
OBn
HO
BnO
N3
OBn
BnO
OBn
OBn
OBn
a
c
b
O
H
O
H
H
O
O
O
HO
HO
BnO
N3
OBn
N3
OBn
O
HO
N3
OBn
OBn
OBn
e
d
f
Fig. 2. Structure of the sugar amino acid scaffolds used in the here described study.
O
O
ref 33
i, ii
iii
OH
O
N3
O
N3
OH
BocHN
BocN
N
N
O
O
O
O
Fmoc-L-Ile-OH 1
2
3
4
Scheme 1. Reagents and conditions: (i) TFA, CH2Cl2. (ii) First bromoacetyl bromide, DBU, DMF, then NaN3 (76% over the two steps). (iii) LiOH, H2O/dioxane 1:1 (95%).
O
O
O
O
i
ii
SAA
N
H
N
NH3.TFA
SAA
N
H
N3
N3
O
O
O
O
5
6a-f
iii
O
O
O
O
O
O
O
H
O
H
O
H
O
N
O
N
*
O
N
*
N
H
N
N3
N
H
N
H
OBn
N3
*
N
N
H
OBn
N3
H
H
O
O
O
O
O
O
BnO
OBn
BnO
BnO
OBn
OBn
OBn
7bI and 7bII
7cI and 7cII
7dI and 7dII
O
O
O
O
O
O
H
H
O
H
N
O
O
O
O
N
N
N3
O
N
N
H
N
H
OBn
N3
N3
N
H
N
H
OBn
N
H
H
O
O
O
O
O
O
OBn
BnO
OBn
OBn
7a
7e
7f
Scheme 2. Reagents and conditions: (i) sugar amino acid a–f, HCTU, DIPEA, CH2Cl2 (79–93%). (ii) a. Me3P, H2O/THF, b. 10, TFA, rt, c. tBuONO, HCl, ꢁ30 °C, then DiPEA, DMF,
EtOAC, rt. (iii) First Me3P, H2O/THF, then 4, EDC, HOAt, lutidine, DMF, CH2Cl2 followed by HPLC purification (11–55% two steps).
However, incubation of tissue cell lysate with a 1 mM final concen-
tration of these specific examples led to no significant increase in
labelling inhibition (data not shown). In contrast with the SAA-
based compound library, epoxomicin shows a very potent protea-
some inhibition, competing out MV151 at concentrations as low as
1 lM.

M.D.P. Risseeuw et al. / Bioorganic Chemistry 38 (2010) 202–209
205
O
i
ii
H
N
H
N
OH
N3
FmocN
FmocN
NHBoc
N
NHBoc
O
O
O
8
9
10
Scheme 3. Reagents and conditions: (i) BocNHNH2, HCTU, DIPEA, CH2Cl2 (85%). (ii) First DBU, DMF, then bromoacetyl bromide, then sodium azide (72%).
Fig. 3. Structure of the fluorescent proteasome inhibitor MV151.
protective groups, on compounds featuring sugar amino acids of
a different structural and stereochemical nature, and on com-
pounds featuring sugar amino acids structurally closely related to
the Thr-Ile dipeptide are needed to form a definite conclusion
whether sugar amino acids are useful building blocks in the con-
struction of effective, selective and possibly physiologically inert
proteasome inhibitors.
7bI
7a
10 100
7bII
1
β2
β1
β5
7f
7cII
7cII
1
10 100
1
1
10 100
0
0
1
1
10 100
β2
4. Experimental
β1
β5
4.1. Synthesis
7e
10 100
7dI
10 100
7dII
10 100
1
All reactions described were performed under an argon atmo-
sphere and at ambient temperature unless stated otherwise. All
solvents, except water, were purchased from Biosolve and used
as received. Amino acids were purchased from Novabiochem. All
other reagents were purchased from Sigma–Aldrich or Acros and
were used as received. Reactions were monitored by TLC analysis
using TLC aluminium sheets (Merck, Silica gel 60, F254) with detec-
tion by spraying with a solution of (NH4)6Mo7O24ꢂ4H2O (25 g/L)
and (NH4)4Ce(SO4)4ꢂ2H2O (10 g/L) in H2SO4 (10%) followed by char-
ring. Column chromatography was performed on 60 Å silica gel
β2
β1
β5
epoxomicin
neg
M
1
10 100
Fig. 4. Compounds 7a–f do not inhibit the active proteasome subunits.
(40–63
gan LTQ Orbitrap Mass spectrometer. 1H- and 13C-APT-NMR spec-
tra were recorded with Bruker AV-400 (400/100 MHz)
spectrometer. Chemical shifts are given in ppm (d) relative to tet-
ramethylsilane as an internal standard (1H NMR) or CDCl3 13C
lm). High resolution spectra were recorded with a Finni-
a
3. Conclusion
(
NMR). Coupling constants are given in Hz. Optical rotations were
measured with a Propol automatic polarimeter (k = 589 nm) and
IR (ATR-IR) spectra were recorded with a Perkin Elmer Paragon
1000 FT-IR Spectrometer. Preparative HPLC purifications were car-
ried out using a Gilson preparative HPLC system equipped with a
This paper details the design and synthesis of nine sugar amino
acid containing peptide epoxyketones. Evaluation of these in a pro-
teasome inhibitor competition assay revealed that none of these
compounds possess affinity of one of the proteasome catalytic ac-
tives. Thus, replacement of the central isoleucine–threonine dipep-
tide as present in the parent compound, epoxomicin, with either of
the six sugar amino acids applied appears detrimental to the inhib-
itory activity. Lack of activity may be caused by the secondary
structure inherent to the sugar amino acids used and that imposes
secondary structural features that are at odds with proteasomal
recognition. Alternatively, either the substitution pattern or the
presence of benzyl ethers, or a combination thereof, prohibits
proteasomal inhibition. Future research on compounds free of
Phenomenex Gemini C18 column (150 ꢃ 21.20 mm, 5
lm) using
a water/acetonitrile/TFA gradient system.
4.1.1. Compound 3
Boc-N,O-dimethyl-isoleucine [32] (2.6 g, 10 mmol) was taken
up in TFA (50 mL) allowed to stand for 10 min and concentrated
in vacuo. The residue was coevaporated twice with toluene
(50 mL) and taken up in DMF (100 mL). Bromoacetylbromide

206
M.D.P. Risseeuw et al. / Bioorganic Chemistry 38 (2010) 202–209
(1.8 mL, 20 mmol) and DBU (3.0 mL, 20 mmol) were added and
stirring is continued for 4 h during which the reaction mixture be-
came deep red in colour. Sodium azide (6.5 g, 100 mmol) was
added and stirring is continued over night. The reaction mixture
is diluted with ethyl acetate (500 mL) and washed with HCl (1 M,
200 mL, twice) and NaCl (satd. aq., 200 mL, twice). The organic
fraction was dried (Na2SO4) and taken to dryness. Silica gel chro-
matography (0 ? 25% EtOAc in toluene) yielded the product
(1.90 g, 7.29 mmol, 73%) as a pale yellow oil. (major rotamer) 1H
NMR (400 MHz, CDCl3) d = 5.00 (d, J = 10.8 Hz, 1H), 4.09–3.91 (m,
2H), 3.72 (s, 3H), 2.96 (s, 3H), 2.07–1.97 (m, 1H), 1.40–1.34 (m,
1H), 1.13–1.05 (m, 1H), 1.00–0.88 (m, 6H),13C NMR (100 MHz,
CDCl3) d = 171.25, 168.05, 60.20, 51.77, 50.55, 33.36, 30.42, 24.99,
15.47, 10.52; IR neat (cmꢁ1): 2968.9, 2361.6, 2105.4, 1735.9,
J = 7.6 Hz, 1H)3.32 (d, J = 5.0 Hz, 1H) 3.23, (dd, J = 9.6 Hz,
J = 7.2 Hz, 1H), 2.89 (d, J = 5.0 Hz, 1H), 1.55 (s, 3H), 1.52–1.45 (m,
2H), 1.18–1.13 (m, 1H), 0.86 (d, J = 6.4 Hz, 3H), 0.83 (d, J = 6.4 Hz,
3H); 13C NMR (100 MHz, CDCl3) d = 208.25, 168.81, 137.73,
137.57, 134.45, 128.92, 128.60, 128.50, 128.41, 128.33, 128.12,
127.88, 127.78, 127.68, 127.62, 84.09, 79.65, 78.91, 77.72, 76.97,
74.50, 74.20, 73.20, 64.66, 58.94, 52.29, 49.26, 40.31, 24.96,
23.24, 21.14, 16.63; IR neat (cmꢁ1): 3065.6, 2100.7, 1718.9,
1638.8, 1451.2, 1092.5, 734.5, 698.0, 535.6; ½a D20
ꢄ
+18 (c 1.0,
MeOH); HRMS: calcd for C43H49O7N4 ([M+H]) 733.35958 found
733.36023.
4.1.6. Compound 6d
1H NMR (400 MHz, CDCl3) d = 7.30–7.16 (m, 15 H), 6.63 (d,
J = 8.4 Hz, 1H), 4.87–4.53 (m, 7H), 3.96 (d, J = 7.6 Hz, 1H), 3.80–
3.75 (m, 2H), 3.73–3.64 (m, 3H), 3.32 (d, J = 5.0 Hz, 1H), 2.87 (d,
J = 5.0 Hz, 1H), 1.80–1.62 (m, 3H), 1.55–1.48 (m, 3H), 1.29–1.20
(m, 2H), 0.96–0.85 (m, 12 H); 13C NMR (100 MHz, CDCl3)
d = 208.07, 168.69, 138.018, 137.78, 137.52, 128.42, 128.30,
128.08, 127.85, 127.78, 127.73, 127.60, 84.21, 80.48, 79.60, 78.19,
77.34, 74.78, 74.31, 74.01, 61.31, 59.05, 52.35, 49.76, 39.99,
37.44, 25.14, 25.09, 23.29, 23.22, 21.28, 21.24, 16.69;; IR neat
(cmꢁ1): 2960.2, 2103.4, 1718.1, 1638.5, 1512.0, 1092.6, 734.6,
1660.1, 1452.0, 1265.6, 1138.6, 996.8, 740.6; ½a D20
ꢁ99.1 (c 2.0,
ꢄ
CHCl3); ESI-MS: [M+H]: 243.3.
4.1.2. General procedure I
Condensing the epoxyketone warhead to the azido acids.
Boc–Leucine–epoxyketone (68 mg, 0.25 mmol) is taken up in
CH2Cl2 (2 mL) and trifluoroacetic acid (2 mL) is added. The mixture
is allowed to stand for 30 min after which it is concentrated in va-
cuo. The residue is coevaporated twice with toluene (3 mL) and is
taken up in DMF (4 mL). To the solution the appropriate amino acid
is added (0.20 mmol) along with HCTU (0.25 mmol, 104 mg) and
695.4, 457.2;
½
a 2D0
+48 (c 1.0, CHCl3); HRMS: calcd for
ꢄ
C41H53O7N4 ([M+H]) 713.39088 found 713.39151.
DIPEA (1 mmol, 174 ll). The reaction mixture was stirred over-
night and diluted with 50 mL ethyl acetate. This solution was ex-
tracted with citric acid (10% aq., 30 mL, twice) and NaHCO3 (satd.
aq., 30 mL, twice). The organic fraction was dried on Na2SO4, and
concentrated. The residue was purified on column chromatography
(0 ? 50% EtOAc in toluene) yielding the product as a viscous oil
(79–93%).
4.1.7. Compound 6e
1H NMR (400 MHz, CDCl3) d = 7.36–7.24 (m, 10H), 6.08 (d,
J = 8.4 Hz, 1H), 4.75–4.50 (m, 5H), 3.80 (d, J = 5.2 Hz, 2H), 3.69–
3.65 (m, 1H), 3.61–3.55 (m, 1H), 3.44 (t, J = 5.2 Hz, 1H), 3.29 (d,
J = 5.0 Hz, 1H), 3.28–3.23 (m, 1H), 2.86 (d, J = 5.0 Hz, 1H), 1.92–
1.88 (m, 1H), 1.83–1.80 (m, 1H), 1.81 (m, 1H), 1.57–1.50 (m, 4H),
1.30–1.24 (m, 1H), 0.99–0.95 (m, 6H); 13C NMR (100 MHz, CDCl3)
d = 208.65, 170.15, 137.59, 137.47, 128.42, 127.91, 127.87,
127.81, 127.65, 75.26, 74.26, 73.34, 72.92, 71.15, 58.94, 56.01,
52.30, 51.12, 50.13, 40.41, 25.49, 25.10, 23.30, 22.50, 21.37,
16.59; IR neat (cmꢁ1): 3293.9, 2959.4, 2096.0, 1718.0, 1635.8,
4.1.3. Compound 6a
1H NMR (400 MHz, CDCl3) d = 7.33–7.22 (m, 15 H), 6.65 (d,
J = 8.4 Hz, 1H), 4.90–5.55 (m, 7H), 3.90 (d, J = 8.4 Hz, 1H), 3.73–
3.67 (m, 2H), 3.65–3.58 (m, 3H), 3.34 (d, J = 4.8 Hz, 1H), 3.30 (dd,
J = 13.6 Hz, J = 3.2 Hz, 1H), 2.87 (d, J = 4.8 Hz. 1H), 1.64 (m, 1H),
1.55–1.47 (m, 4H), 1.26 (m, 1H), 0.95–0.85 (m, 6H); 13C NMR
(100 MHz, CDCl3) d = 208.22, 168.52, 138.03, 137.63, 137.51,
128.47, 128.42, 128.32, 128.18, 137.99, 127.92, 127.80, 127.73,
127.69, 85.14, 80.32, 77.93, 77.56, 77.49, 75.27, 74.83, 74.79,
59.07, 52.37, 50.80, 49.66, 39.81, 25.12, 23.23, 21.11, 16.66; IR neat
(cmꢁ1): 2871.6, 2363.9, 2100.8, 17.18.2, 1667.9, 1454.6, 1095.4,
1533.7, 1072.0, 735.1, 696.6; ½a D20
ꢄ
+79 (c 2.0, CHCl3); HRMS: calcd
for C31H39O6N4 ([M+H]) 563.28641 found 563.28614.
4.1.8. Compound 6f
1H NMR (400 MHz, CDCl3) d = 7.45 (m, 1H), 7.37–7.29 (m, 5H),
4.68–4.62 (m, 1H), 4.61–4.52 (m, 2H), 4.14–4.02 (m, 2H), 3.72–
3.69 (m, 1H), 3.54–3.47 (m, 3H), 3.43–3.37 (m, 1H), 3.32 (d,
J = 5.0 Hz, 1H), 2.87 (d, J = 5.0 Hz, 1H), 1.68 (m, 1H), 1.54–1.46
(m, 4H), 1.26–1.20 (m, 1H), 0.96–0.88 (m, 6H); 13C NMR
(100 MHz, CDCl3) d = 208.01, 169.54, 137.07, 128.33, 127.78,
127.65, 78.46, 73.39, 69.26, 68.75, 58.87, 52.11, 51.81, 49.43,
39.47, 24.94, 23.15, 21.01, 16.50; IR neat (cmꢁ1): 2959.7, 2102.7,
734.9, 697.6, 460.5; ½a D20
ꢁ12.8 (c 2.0, CHCl3); HRMS: calcd for
ꢄ
C
37H45O7N4 ([M+H]) 657.32828 found 657.32864.
4.1.4. Compound 6b
1H NMR (400 MHz, CDCl3) d = 7.31–7.21 (m, 15H), 6.62 (d,
J = 8.4 Hz, 1H), 4.89–4.55 (m, 7H), 3.92 (m, 1H), 3.77–3.71 (m,
2H), 3.69–3.59 (m, 2H), 3.52–3.47 (m, 1H), 3.28 (d, J = 4.8 Hz,
1H), 2.86 (d, J = 4.8 Hz, 1H), 1.66–1.60 (m, 1H), 1.55–1.48 (m,
4H), 1.27–1.20 (m, 1H), 1.17 (d, J = 6.8 Hz, 3H), 1.00–0.85 (m,
6H); 13C NMR (100 MHz, CDCl3) d = 207.74, 168.214, 137.67,
137.44, 137.13, 128.14, 128.10, 127.98, 127.73, 127.68, 127.65,
127.41, 127.33, 84.61, 79.83, 79.48, 77.71, 76.67, 74.69, 74.16,
73.66, 58.63, 56.58, 51.98, 49.39, 39.88, 24.80, 22.92, 20.95,
16.33, 12.68; IR neat (cmꢁ1): 3397.1, 2960.6, 2360.2, 2103.1,
1719.0, 1675.8, 1522.1, 1454.0, 1096.2, 532.2; ½a D20
ꢄ
+60.4 (c 2.0,
CHCl3); HRMS: calcd for C21H31O7N4 ([M+H]) 419.22890 found
419.22877.
4.1.9. General procedure II
Condensing the N-methyl-leucine azidoacetamide to the AA
epoxyketones.
Compound 3 (61 mg, 0.25 mmol) is taken up in 2 mL THF. To
this solution are added methanol (0.5 mL) and LiOH (250 lL,
4.0 M) and the reaction is stirred at 0 °C for 2 h. The reaction mix-
ture is acidified with HCl (1 M) to pH 2 and extracted with CH2Cl2
(2 ꢃ 2 mL). The organic layers are pooled dried (Na2SO4) and con-
centrated. The residue was used without further purification. The
1718.2, 1689.2, 1519.8, 1077.2, 733.3, 695.4; ½a D20
ꢄ
+9.6 (c 1.0,
CHCl3); HRMS: calcd for C38H46O7N4 ([M+H]) 671.34393 found
671.34434.
4.1.5. Compound 6c
appropriate epoxyketone amide (150 lmol) was taken up in THF
1H NMR (400 MHz, CDCl3) d = 7.36–7.16 (m, 20 H), 6.58 (d,
J = 8.8 Hz, 1H), 4.87–4.40 (m, 8H), 4.03 (d, J = 7.6 Hz, 1H), 3.93
(dd, J = 9.6 Hz, J = 7.6 Hz, 1H), 3.74 (t, J = 7.2 Hz, 1H), 3.60 (t,
(2 mL) and water (0.1 mL). To this solution Me3P in THF (3 mL,
1.0 M) was added and the solution was stirred for 1 h. The reaction
mixture is concentrated in vacuo and coevaporated twice with

M.D.P. Risseeuw et al. / Bioorganic Chemistry 38 (2010) 202–209
207
toluene (3 mL). The crude amine was taken up in CH2Cl2 (2.5 mL)
and DMF (0.5 mL) and the freshly prepared N-methyl-leucine azi-
2H), 1.06–0.97 (m, 1H), 0.93–0.82 (m, 12H); 13C NMR (100 MHz,
CDCl3) d = 208.84, 174.96, 168.98, 168.45, 137.85, 137.65, 137.49,
137.29, 128.68, 128.47, 128.35, 128.32, 128.28, 128.05, 127.92,
127.75, 127.71, 127.62, 127.44, 84.52, 79.82, 79.01, 77.62, 77.10,
61.56, 59.06, 53.02, 52.49, 50.59, 49.66, 39.91, 31.60, 30.26,
24.99, 24.49, 23.19, 21.08, 16.76, 15.29, 10.27; IR neat (cmꢁ1):
2963.7, 2360.1, 2107.8, 1718.0, 1669.9, 1270.3, 1028.0, 530.6;
ESI-MS: [M+H]: 917.20.
doacetamide (ꢅ250
ꢁ30 °C and under stirring EDC (144 mg, 750
250 mol) and 2,6 lutidine (30 l, 250 mol) are added. Stirring
lmol) was added. The solution was cooled to
lmol), HOAt (35 mg,
l
l
l
is continued for 16 h after which the reaction mixture is diluted
with ethyl acetate (20 mL). The ethyl acetate fraction was ex-
tracted with citric acid (10% aq., 10 mL, twice) and NaHCO3 (satd.
aq., 10 mL, twice). The organic fraction was dried on Na2SO4, and
concentrated. The products were purified and where possible sep-
arated on reversed phase HPLC using a water/acetonitrile/TFA gra-
dient system. The yields were 35–55% for epimeric mixtures and
11–27% for diastereomerically pure compounds.
4.1.14. Compound 7cII
1H NMR (400 MHz, CDCl3) d = 7.39–7.22 (m, 20 H), 5.34 (dd,
J = 8.4, J = 2.8, 1H), 4.82–4.54 (m, 7H), 4.43–4.38 (m, 1H), 4.00–
3.92 (m, 3H), 3.76–3.70 (m, 2H), 3.66 (t, J = 8.3 Hz, 1H), 3.39 (d,
J = 5.0 Hz, 1H), 3.10 (dd, J = 9.8 Hz, J = 7.6 Hz, 1H), 2.96 (s, 3H),
2.91 (d, J = 5.0 Hz, 1H), 2.02 (m, 1H), 1.62 (m, 1H), 1.58–1.47 (m,
4H), 1.34–1.20 (m, H2), 0.94–0.75 (m, 12H); 13C NMR (100 MHz,
CDCl3) d = 208.49, 175.18, 168.84, 168.55, 137.94, 137.84, 137.82,
137.09, 128.86, 128.52, 128.41, 128.39, 128.31, 128.22, 128.02,
127.95, 128.87, 127.78, 127.73, 85.28, 79.67, 79.48, 78.06, 77.14,
74.76, 74.40, 73.52, 61.77, 59.09, 52.93, 52.44, 50.95, 49.84,
39.87, 32.24, 38.87, 32.24, 30.13, 25.50, 25.19, 23.33, 21.15,
16.73, 14.70, 10.81; IR neat (cmꢁ1): 2963.7, 2361.8, 2108.3,
1718.0, 1675.8, 1272.2, 1090.8, 531.6; ESI-MS: [M+H]: 917.20;
ESI-MS: [M+H]: 917.13.
4.1.10. Compound 7a
1H NMR (400 MHz, CDCl3) d = 7.28–7.25 (m, 15 H), 7.07 (d,
J = 8.0 Hz, 1H), 6.91 (m, 1H), 4.91–4.55 (m, 8H), 4.03–3.93 (m,
2H), 3.84 (d, J = 9.2 Hz, 1H), 3.73–3.60 (m, 3H), 3.55–3.49 (m,
1H), 3.45–3.40 (m, 1H), 3.35 (d, J = 5.0 Hz, 1H), 3.19 (t, J = 8.8 Hz,
1H), 2.97 (s, 3H), 2.89 (d, J = 5.0 Hz, 1H), 2.17–2.10 (m, 1H), 1.69–
1.63 (m, 1H), 1.54–1.46 (m, 4H), 1.38–1.22 (m, 2H), 1.08–0.98
(m, 1H), 0.94–0.85 (m, 12 H); 13C NMR (100 MHz, CDCl3)
d = 208.60, 169.79, 169.75, 168.76, 138.15, 137.63, 137.48,
128.58, 128.46, 128.36, 128.17, 127.97, 127.84, 127.75, 127.61,
85.69, 80.40, 77.98, 77.84, 76.95, 75.41, 75.04, 74.81, 61.94,
59.21, 52.53, 51.06, 50.33, 39.51, 39.36, 31.64, 30.53, 25.21,
24.68, 23.24, 21.10, 16.75, 15.32, 10.29; IR neat (cmꢁ1): 2963.7,
2361.7, 2107.9, 1718.1, 1651.9, 1270.5, 1096.2, 732.2; ESI-MS:
[M+H]: 841.7.
4.1.15. Compound 7dI
1H NMR (400 MHz, CDCl3) d = 7.50–7.20 (m, 15 H), 4.90–4.46
(m, 9 H), 4.05–3.36 (m, 10H) 2.96–2.89 (m, 4H), 2.25 (m, 1H),
1.70–1.20 (m, 10 H), 1.01–0.80 (m, 19H), 13C NMR (100 MHz,
CDCl3) d = 208.88, 170.20, 169.12, 168.80, 168.70, 168.31, 138.15,
137.82, 137.64, 137.55, 137.51, 128.65, 128.62, 128.56, 128.49,
128.47, 128.45, 128.42, 128.36, 128.24, 128.14, 128.11, 128.08,
128.02, 127.93, 127.80, 127.75, 127.72, 127.69, 127.57, 85.92,
85.83, 82.62, 81.12, 80.63, 80.35, 80.21, 79.16, 78.49, 77.81,
77.46, 77.26, 76.72, 75.39, 75.32, 75.16, 75.02, 74.89, 74.77,
74.35, 69.23, 68.89, 59.28, 54.44, 52.63, 51.11, 51.02, 50.17,
47.14, 46.91, 46.62, 39.75, 36.65, 31.93, 31.79, 31.70, 25.47,
25.33, 24.98, 24.66, 23.70, 23.31, 21.98, 21.61, 21.44, 16.79,
15.38, 10.51; IR neat (cmꢁ1): 2959.0, 2360.1, 2108.4, 1652.0,
1094.3, 735.1, 699.0, 529.9; ESI-MS: [M+H]: 898.0.
4.1.11. Compound 7bI
1H NMR (400 MHz, CDCl3) d = 7.35–7.25 (m, 15 H), 4.92–4.59
(m, 7 H), 4.50 (d, J = 11.2 Hz, 1H), 4.40 (m, 1H), 4.00–3.91 (m,
2H), 3.80 (d, J = 9.0 Hz, 1 H), 3.76–3.70 (m, 1H), 3.65–3.58 (m,
1H), 3.47–3.37 (m, 2H), 3.35 (d, J = 4.9 Hz, 1H), 3.00 (s, 3H), 2.90
(d, J = 4.9 Hz, 1H), 2.11 (m, 1H), 1.62 (m, 1H) 1.52 (m, 4H), 1.36–
1.17 (m, 2H), 1.01–0.80 (16 H); 13C NMR (100 MHz, CDCl3)
d = 208.86, 168.93, 168.70, 168.37, 138.05, 137.74, 137.53,
128.49, 128.45, 128.34, 128.28, 128.18, 128.05, 127.94, 127.78,
127.75, 127.71, 85.96, 80.38, 79.93, 77.79, 77.11,75.36, 74.81,
74.16, 59.17, 52.57, 50.90, 50.07, 44.37, 44.05, 39.86, 32.27,
30.83, 25.28, 24.73, 23.26, 21.33, 16.74, 15.24, 13.26, 10.40; IR neat
(cmꢁ1): 2963.8, 2360.3, 2108.7, 1718.1, 1653.3, 1090.3, 699.9,
526.1; ESI-MS: [M+H]: 855.2.
4.1.16. Compound 7dII
1H NMR (400 MHz, CDCl3) d = 7.45–7.19 (m, 15 H), 4.90–4.60
(m, 7 H), 4.55–4.30 (m, 2H), 4.00 (d, J = 3.7 Hz, 2H), 3.88–3.65 (m,
3H) 3.51–3.42 (m, 2H) 3.36 (d, 5.0 Hz, 1H), 3.24 (dd, J = 9.9 Hz,
J = 1.8 Hz, 1H) 2.94 (s, 3H), 2.84 (d, J = 5.0 Hz, 1H), 2.11 (m 1H),
1.59–1.34 (m, 8H), 1.10 (m, 2H), 1.01–0.78 (m, 19 H); 13C NMR
(100 MHz, CDCl3) d = 208.03, 169.00, 168.54, 168.16, 138.32,
138.28, 137.62, 128.61, 128.49, 128.42, 128.37, 128.29, 128.15,
128.12, 127.98, 127.93, 127.74, 127.70, 127.64, 127.60, 127.51,
86.54, 81.84, 79.39, 78.70, 77.51, 75.36, 74.86, 74.69, 62.83,
59.14, 52.44, 51.31, 50.30, 46.51, 39.61, 36.50, 31.38, 30.22,
25.75, 25.41, 24.85, 23.93, 23.32, 21.47, 21.30, 16.82, 14.81,
10.86; IR neat (cmꢁ1): 2958.7, 2361.4, 2108.1, 1718.1, 1669.2,
1455.0, 1090.7, 910.7, 732.2, 698.2; ESI-MS: [M+H]: 898.0.
4.1.12. Compound 7bII
1H NMR (400 MHz, CDCl3) d = 7.33–7.25 (m, 15 H), 4.92–4.71
(m, 5H), 4.69–4.61 (m, 2H), 4.04–3.92 (m, 2H), 3.82–3.68 (m,
3H), 3.41–3.33 (m, 2H), 3.27 (dd, J = 9.9 Hz, J = 2.2 Hz, 1H), 2.94
(s, 3H), 2.87 (d, J = 5.0 Hz, 1H), 2.11 (m, 1H) 1.70 (m, 1H), 1.56–
1.46 (m, 5H), 1.30 (m, 1H), 1.06 (m, 1H), 1.00–0.78 (m, 15 H); 13C
NMR (100 MHz, CDCl3) d = 208.25, 168.81, 168.27, 168.20,
138.17, 138.09, 137.53, 128.53, 128.46, 128.29, 128.00, 127.95,
127.73, 127.70, 127.65, 86.36, 80.52, 79.67, 78.35, 77.07, 75.40,
74.76, 74.45, 62.21, 59.11, 51.08, 50.20, 44.11, 52.45, 51.08,
50.20, 44.11, 39.74, 31.91, 30.16, 25.87, 25.34, 23.32, 21.38,
16.77, 14.76, 13.46, 11.07; IR neat (cmꢁ1): 2970.2, 2361.9,
2108.8, 1664.0, 1454.5, 1049.9, 528.4; ESI-MS: [M+H]: 855.2.
4.1.17. Compound 7e
1H NMR (400 MHz, CDCl3) d = 7.36–7.24 (m, 10 H), 4.75–4.47
(m, 6H), 4.02–3.88 (m, 2H), 3.82–3.78 (m, 1H), 3.76–3.70 (m,
1H), 3.63–3.57 (m, 1H), 3.51–3.44 (m, 2H), 3.36–3.31 (m, 2H),
2.98–2.90 (m, 3H), 2.88 (d, J = 4.9 Hz, 1H), 2.09 (m, 1H), 1.94–
1.90 (m, 1H), 1.87–1.80 (m, 1H), 1.74 (m, 1H), 1.53–1.46 (m, 4H),
1.37–1.23 (m, 2H), 1.02–0.77 n(m, 13H); 13C NMR (100 MHz,
CDCl3) d = 209.08, 208.92, 170.56, 170.53, 169.86, 169.63, 168.75,
168.68, 138.63, 137.50, 137.45, 128.28, 127.90, 127.82, 127.73,
127.69, 127.62, 127.60, 127.47, 76.14, 76.10, 74.76, 74.38, 72.88,
4.1.13. Compound 7cI
1H NMR (400 MHz, CDCl3) d = 7.38–7.23 (m, 20H), 5.38 (dd,
J = 8.7 Hz, J = 2.6 Hz, 1H), 4.82–4.74 (m, 2H), 4.71–4.56 (m, 5H),
4.51–4.46 (m, 1H), 4.01 (d, 8.0 Hz, 1H), 3.96–3.82 (m, 3H), 3.74
(t, J = 7.5 Hz, 1H), 3.55 (t, J = 7.9 Hz, 1H), 3.37 (d, J = 4.9 Hz, 1H),
3.12 (dd, J = 9.7 Hz, J = 7.5 Hz, 1H), 2.93 (d, J = 4.9 Hz, 1H), 2.71 (s,
3H), 2.08 (m, 1H), 1.57 (s, 3H), 1.54–1.46 (m, 2H), 1.33–1.16 (m,

208
M.D.P. Risseeuw et al. / Bioorganic Chemistry 38 (2010) 202–209
72.74, 72.63, 72.06, 70.99, 70.95, 61.61, 61.16, 58.93, 56.13, 55.85,
52.30, 50.51, 50.42, 50.29, 42.40, 39.87, 39.70, 31.77, 31.55, 30.04,
29.86, 25.68, 24.97, 24.89, 24.70, 24.49, 23.19, 21.95, 21.14, 16.54,
15.23, 14.38, 10.96, 10.19; IR neat (cmꢁ1): 2963.9, 2366.2, 1718.0,
1654.0, 1268.7, 1025.8, 526.7; ESI-MS: [M+H]: 748.0.
buffer (50 mM Tris HCl pH 7.4, 250 mM sucrose, 5 mM MgCl2,
1 mM DTT, 2 mM ATP, 0.025% digitonin) for 15 min on ice. After
2 ꢃ 30 s sonication pulses, cell debris were removed by cold centri-
fugation, yielding a protein concentration of 5 lg/ll determined by
Bradford assay with a BSA calibration curve. For the competition
study, every compound was diluted at the indicated concentration
4.1.18. Compound 7f
in 10
37 °C for 1 h and the residual proteasome activity was captured
with 0.5 M MV151 for the next hour at 37 °C. Proteins were re-
solved by 12.5% PAA SDS–PAGE gels and the wet gel slabs were im-
aged with a Typhoon fluorescence scanner (GE Healthcare). As
ll reaction volume containing 10 lg protein incubated at
1H NMR (400 MHz, CDCl3) d = 7.40–7.25 (m, 5H), 4.72–4.64 (m,
1H), 4.62–4.50 (m, 3H), 4.18–3.82 (m, 4H), 3.70–3.40 (m, 4H),
3.37–3.21 (m, 3H), 3.00–2.85 (m, 4H), 2.08 (m, 1H), 1.68 (m, 1H),
1.54–1.23 (m, 6H), 1.07–0.76 (m, 13H); 13C NMR (100 MHz, CDCl3)
d = 208.73, 208.59, 170.34, 170.19, 170.19, 170.02, 169.83, 168.86,
168.54, 137.43, 137.31, 129.54, 128.42, 127.87, 127.79, 127.77,
127.71, 127.61, 78.85, 78.55, 73.39, 73.34, 70.28, 70.09, 69.31,
69.15, 61.46, 61.29, 58.99, 58.95, 52.37, 52.31, 50.59, 50.53,
49.75, 49.71, 39.81, 39.63, 39.47, 39.26, 31.75, 31.62, 30.06,
29.81, 25.73, 25.05, 25.02, 24.52, 23.19, 21.07, 21.05, 16.65,
16.63, 15.13, 14.35, 10.92, 10.18; IR neat (cmꢁ1): 2963.8, 2360.4,
2107.6, 1718.2, 1654.0, 1027.8, 523.6; ESI-MS: [M+H]: 747.6.
l
negative control, 10 lg protein was denatured by boiling with 1%
SDS prior to 1 h exposure to 0.5% MV151.
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Fmoc-N-methyl-isoleucine (3.80 g, 10.3 mmol) was taken up in
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(2.0 g, 15.1 mmol), HCTU (6.20 g, 15 mmol) and DIPEA (5.2 mL,
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ken to dryness. Silica gel chromatography (0 ? 25% EtOAc in tolu-
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86%). 1H NMR (400 MHz, DMSO, 352 K) d = 7.87 (d, J = 7.2 Hz, 2H),
7.65 (d, J = 7.2 Hz, 2H), 7.41 (t, J = 7.2 Hz, 2H), 7.33 (t, J = 7.2 Hz,
2H), 4.50–4.45 (m, 1H), 4.38–4.31 (m, 2H), 4.18 (m, 1H), 2.78 (s,
3H), 1.91 (m, 1H), 1.40 (s, 9H), 1.26–1.17 (m, 1H), 0.88–0.80 (m,
7H); 13C NMR (100 MHz, CDCl3) d = 168.00, 155.10, 154.60, 143.57,
140.39, 127.09, 126.59, 126.54, 124.51, 124.43, 119.49, 78.69,
66.40, 60.80, 46.53, 32.00, 29.12, 27.64, 23.93, 14.77, 9.82; IR neat
(cmꢁ1): 2969.8, 2360.1, 1651.7, 1668.1, 1451.9, 1311.7, 1147.3,
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758.1, 739.5;
½
a 2D0
ꢄ
ꢁ103.2 (c 0.5, CHCl3); HRMS: calcd for
C27H36O5N3 ([M+H]) 482.26495 found 482.26477.
4.1.20. Compound 10
Compound 9 (2.83 g, 5.88 mmol) is taken up in DMF (60 mL). To
this solution is added DBU (1.8 mL, 12.0 mmol) and the reaction
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the reaction mixture became deep red in colour. Sodium azide
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organic fraction was dried (Na2SO4) and taken to dryness. Silica
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0.89 (t, J = 7.6 Hz, 3H); 13C NMR (100 MHz, CDCl3) d = 169.61,
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15.32, 10.18; IR neat (cmꢁ1): 3289.4, 2964.7, 2103.7, 1725.1,
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Five 15 cm petri dishes of exponentially growing Hek293T were
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