Structure-Based Design of Inhibitors Selective for Human Proteasome β2c or β2i Subunits

Article abstract of DOI:10.1021/acs.jmedchem.8b01884

Subunit-selective proteasome inhibitors are valuable tools to assess the biological and medicinal relevance of individual proteasome active sites. Whereas the inhibitors for the β1c, β1i, β5c, and β5i subunits exploit the differences in the substrate-binding channels identified by X-ray crystallography, compounds selectively targeting β2c or β2i could not yet be rationally designed because of the high structural similarity of these two subunits. Here, we report the development, chemical synthesis, and biological screening of a compound library that led to the identification of the β2c- and β2i-selective compounds LU-002c (4; IC₅₀ β2c: 8 nM, IC₅₀ β2i/β2c: 40-fold) and LU-002i (5; IC₅₀ β2i: 220 nM, IC₅₀ β2c/β2i: 45-fold), respectively. Co-crystal structures with β2 humanized yeast proteasomes visualize protein-ligand interactions crucial for subunit specificity. Altogether, organic syntheses, activity-based protein profiling, yeast mutagenesis, and structural biology allowed us to decipher significant differences of β2 substrate-binding channels and to complete the set of subunit-selective proteasome inhibitors.

Full text of DOI:10.1021/acs.jmedchem.8b01884

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Article
Structure-based design of inhibitors selective
for human proteasome #2c or #2i subunits
Bo-Tao Xin, Eva Huber, Gerjan de Bruin, Wolfgang Heinemeyer, Elmer Maurits, Christopher
Espinal, Yimeng Du, Marissa Janssens, Emily S. Weyburne, Alexei Kisselev, Bogdan I. Florea,
Christoph Driessen, Gijsbert A. van der Marel, Michael Groll, and Herman S. Overkleeft
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b01884 • Publication Date (Web): 18 Jan 2019
Downloaded from http://pubs.acs.org on January 18, 2019
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Structure-based design of inhibitors selective for
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human proteasome β2c or β2i subunits
Bo-Tao Xin†,1, Eva M. Huber§,1, Gerjan de Bruin†, Wolfgang Heinemeyer§, Elmer Maurits†,
Christofer Espinal†, Yimeng Du†, Marissa Janssens†, Emily S. Weyburne#, Alexei F. Kisselev#&,
Bogdan I. Florea†, Christoph Driessen‡, Gijsbert A. van der Marel†, Michael Groll§,*and
Herman S. Overkleeft†,*
†Gorlaeus Laboratories, Leiden Institute of Chemistry and Netherlands Proteomics Centre,
Einsteinweg 55, 2333 CC Leiden, The Netherlands
§Center for Integrated Protein Science at the Department Chemie, Lehrstuhl für Biochemie,
Technische Universität München, 85748 Garching, Germany
‡Department of Hematology and Oncology, Kantonsspital St. Gallen, 9007 St. Gallen,
Switzerland
#Department of Molecular and Systems Biology and Norris Cotton Cancer Center, Geisel School
of Medicine at Dartmouth, 1 Medical Centre Drive HB7936, Lebanon, New Hampshire 03756,
United States of America.
*To whom correspondence should be addressed.
&Current address: Department of Drug Discovery and Development, Harrison School of
Pharmacy, Auburn University, Auburn Al 36849 USA
1These authors contributed equally.
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KEYWORDS Constitutive proteasome, immunoproteasome, epoxyketone, vinyl sulfone, subunit
selectivity, ligand binding
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ABSTRACT
Subunit-selective proteasome inhibitors are valuable tools to assess the biological and medicinal
relevance of individual proteasome active sites. While inhibitors for the β1c, β1i, β5c and β5i
subunits exploit differences in the substrate binding channels identified by X-ray crystallography,
compounds selectively targeting β2c or β2i could not yet be rationally designed due to the high
structural similarity of these two subunits. Here we report the development, chemical synthesis
and biological screening of a compound library that led to the identification of the β2c- and β2i-
selective compounds LU-002c (4; IC50 β2c: 8 nM, IC50 β2i/β2c: 20-fold) and LU-002i (5; IC50 β2i:
220 nM, IC50 β2c/β2i: 45-fold), respectively. Co-crystal structures with β2-humanized yeast
proteasomes visualize protein-ligand interactions crucial for subunit specificity. Altogether,
organic syntheses, activity-based protein profiling, yeast mutagenesis and structural biology
allowed us to decipher significant differences of β2 substrate binding channels and to complete
the set of subunit-selective proteasome inhibitors.
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Introduction
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Proteasomes are proteolytic machines responsible for the degradation of misfolded proteins
localized in the cytosol and nucleus of eukaryotic cells1. Their 20S core particles (CPs) are C2-
symmetrical barrel-shaped complexes assembled of 28 subunits that are arranged in four stacked
seven-membered rings2. The two outer rings are made of seven α-subunits (α1-7) and the two
inner rings consist of seven homologous yet distinct β-subunits (β1-7). In ubiquitously expressed
constitutive proteasomes (cCPs), the proteolytic activities reside within the subunits β1c
(caspase-like activity), β2c (trypsin-like activity) and β5c (chymotrypsin-like activity)3. In
lymphoid-tissues these subunits are replaced by their interferon-γ-inducible counterparts, β1i
(LMP2), β2i (MECL-1) and β5i (LMP7)4, yielding so-called immunoproteasome particles (iCPs)
that preferentially generate antigenic peptides with high affinity for major histocompatibility
complex (MHC) class I receptors5.
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Proteasomes are validated drug targets in oncology and numerous, structurally diverse inhibitors
of natural and non-natural origin have been reported so far6. Most synthetic compounds are N-
terminally capped peptides of 2-4 residues with a C-terminal electrophilic warhead that forms a
covalent linkage with the nucleophilic hydroxyl group and possibly the free N-terminus of
threonine-1 (Thr1) of catalytically active proteasomal β-subunits7. Subunit specificity of peptidic
ligands is largely determined by the sequence of the peptide fragment, although the nature of the
warhead can confer selectivity as well8. The first generation boronic acid bortezomib and the
second generation epoxyketone carfilzomib target more than one subunit at a time and therefore
are considered broad-spectrum proteasome inhibitors6a. Bortezomib and carfilzomib are now
approved drugs for the treatment of multiple myeloma9,10. Current industrial and academic drug
design efforts focus on the development of subunit-selective proteasome inhibitors and their
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potential therapeutic use in chronic inflammatory diseases. For instance, the first
immunoproteasome-selective compound KZR-61611, an analog of ONX 091412, has recently
entered phase 1b/2 clinical trials for the treatment of lupus erythematosus. Besides medical
issues, selective inhibition of individual proteasome subunits may aid investigations on the
involvement of these sites in different cellular pathways including MHC class I antigen
presentation and control of cytokine levels. Although there is an overlap in the substrate
preferences of cCP and iCP subunits, distinct structural features and amino acid linings of the
substrate binding channels β1c and β1i as well as β5c and β5i could be identified and
subsequently allowed for the development of specific inhibitors12-13. Design of inhibitors
targeting exclusively β2c or β2i however remained challenging due to the high structural
similarity between the trypsin-like active sites13d. In 2018, Liskamp and co-workers reported a
set of β2-selective inhibitors. However, these compounds, which are characterized by a sulfonyl
fluoride as the C-terminal electrophile, a basic P1 residue and a free N-terminus, display limited
preference for either β2c or β2i.14 As well, Kezar Life Sciences developed a epoxyketone
inhibitor with moderate selectivity for human β2i11.
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Recently, we published a set of activity-based protein profiling (ABPP) probes and inhibitors
selective for each of the six catalytic activities of human cCP and iCP, including compounds LU-
002c (β2c) and LU-002i (β2i; Figure 1)15. Here, we describe the design, synthesis and screening
of focused compound libraries that allowed us to identify these β2c and β2i inhibitors,
respectively. Crystallographic data on humanized yeast proteasomes in complex with selective
ligands provide insights into their mode of binding and reveal so far unnoticed differences in
substrate and inhibitor specificity for the trypsin-like active sites of cCP and iCP.
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LU-102 (1)
N3
O
O
O
H
N
H
Apparent IC50 values (M)
2c 0.007
N
S
N
H
2i 0.011
O
O
5c 0.77
5i 0.71
NH2
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1c/1i > 1.0
LU-112 (2)
N3
O
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H
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Apparent IC50 values (M)
2c 0.004
2i 0.050
5c 21.0
1c/1i /5i > 50.0
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O
NH2
NH2
ONX-0914 (3)
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Apparent IC50 values (M)
2c 1.1
2i 0.59
5c 0.054
5i 0.0057
1c 0.46
1i > 1.0
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LU-002c (4)
N3
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S
Apparent IC50 values (M)
2c 0.008
2i 0.32
5c/5i/1c/1i > 10.0
N
H
O
O
NH2
O
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O
LU-002i (5)
O
H
N
N
N
H
N
H
Apparent IC50 values (M)
2i 0.22
2i/5c/5i/1c/1i > 10.0
O
O
O
Figure 1. Chemical structures and IC50 values for the lead structures LU-102 (1)16, LU-112 (2)16
and ONX 0914 (3)12 that guided the development of the β2c- and β2i-selective compounds LU-
002c (4) and LU-002i (5), respectively. IC50 values were measured by competitive ABPP.
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Results
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Development of selective inhibitors for subunit β2c
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The previously identified vinyl sulfone inhibitor LU-102 (Figure 1), which inhibits β2c and β2i
with similar potency16, was used as a starting point for creating selective β2c ligands. We
generated a compound library based on the vinyl sulfone warhead and the 4-aminomethylphenyl
side chain on P1 of LU-102, as these moieties proved to be crucial for β2-selectivity in general16.
In a first step, we replaced the N-cap of LU-102 by a set of groups often found in peptide-based
proteasome inhibitors (6-12). Next, we synthesized compounds with relatively small amino acid
side chains in the P2 position (4, 13-20) and finally incorporated bulky aliphatic side chains at P2
and P3 (21-36). In total, 32 compounds were prepared using established protocols for the
chemical synthesis of the 4-aminomethylphenylalanine vinyl sulfone warhead and solution-phase
coupling of the peptide vinyl sulfones to the corresponding alpha-amino acids (see Supporting
Information)27.
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All compounds were evaluated for β2c/β2i inhibition by our competitive ABPP assay at final
concentrations of 0.01, 0.1, 1.0 as well as 10.0 M and apparent IC50 values were determined
(Table 1). Among the N-cap series 6-12, compound 7 (pyrazine N-cap) showed the highest
selectivity for β2c over β2i (40-fold), but also decreased potency for β2c compared to LU-102
(23-fold). Screening of small P2 residues (compounds 4, 13-20) identified several ligands with
both good selectivity and potency for β2c: 4 (P2 alanine; 10 nM, 32-fold selectivity over β2i), 13
(P2 serine; 11 nM, 47-fold), 15 (P2 methoxyserine; 8 nM, 25-fold), 16 (P2 threonine; 8 nM, 41-
fold) and especially 18 (P2 glycine; 26 nM, 224-fold). Combining 2-methylthiazole N-caps (20)
with bulky P2 or P3 residues (21-36) revealed several potent and selective β2c compounds, as
well: see for instance, compounds 20 (P2 methoxyserine, P3 leucine; 72 nM, 14-fold), 22 (P2
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leucine, P3 cyclohexyl; 18 nM, 30-fold), 30 (P2 cyclohexyl-homoalanine, P3 leucine; 11 nM, 25-
fold) and 36 (P2 and P3 cyclohexyl 40 nM, 10.5-fold). Altogether, based on the data shown in
Table 1 we conclude that 1) subunit β2c accepts small as well as bulky P2 residues but disfavors
oversized P3 side chains; and 2) that β2i disfavors small P2 side chains and large P3 groups.
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To establish apparent IC50 values more accurately and to obtain insights into co-inhibition of β1c,
β1i, β5c and β5i activities, we selected the compounds 4, 7, 13, 16, 18, 20, 22 and 25 for further
analysis. In our competitive ABPP assay using Raji cell extracts (containing both cCPs and iCPs)
a wider range of final concentrations were tested. All compounds inhibited β2c at low nanomolar
concentrations (Table 2). Inhibitors 4, 13, 18 and 20, featuring small side chains on P2, displayed
considerably enhanced selectivity for β2c over β2i (≥27-fold) compared to LU-102 (1.6-fold;
Table 2) with 18 being the most selective (54-fold).
Next, we assessed the inhibitory effects in living RPMI-8226 cells (Table 3). Initial screenings
identified compound 4 as the most active, and we included this compound as LU-002c in our
suite of subunit-selective proteasome inhibitors15. In subsequent studies we identified compound
16 to be even more potent and selective and we dubbed this compound LU-012c.
Development of β2i-selective inhibitors
For the development of β2i-selective compounds, we used ONX 0914 (3)12 as starting point
(Figure 1). Though ONX 0914 is a β5i selective inhibitor, it also targets other proteasome
subunits12, 13b (Figure 1) and shows slight selectivity for β2i over β2c (IC50 (β2i) 0.59 µM; IC50
(β2c) 1.1 µM, 1.9-fold)13b. During our efforts to create β5i-selective compounds, we noted that
substitution of the P1 phenylalanine in ONX 0914 for cyclohexylalanine enhances selectivity for
both β5i and β2i over the respective constitutive subunits (ratio β2c/β2i = 6) and that any
additional modifications of the P2 and P3 positions as well as the N-cap led to the loss of
activities for the trypsin-like sites13b. Based on these observations, we reasoned that large
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aliphatic amino acid residues at P1 might lead to β2i selective inhibitors. To probe this
hypothesis, a set of epoxyketone inhibitors with large hydrophobic P1 residues (compounds 5,
37-53, Table 4) was synthesized (for details see Supporting Information).
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The compounds were tested at final concentrations of 0.01, 0.1, 1.0 and 10.0 M by our
competitive ABPP assay and apparent IC50 values for inhibition of β2c and β2i were determined
(Table 4). In this first evaluation step, compounds 5 (P1 1-decalanine; 319 nM, >31-fold), 39 (P1
cyclohexyl-homoalanine; 215 nM, >46-fold), 41b (methylcyclohexylalanine, 264 nM, 24-fold)
and 44b (bicyclohexylalanine, 97 nM, 9-fold) showed the highest selectivity for β2i over β2c.
Next, inhibition of all six sites by compounds 5 and 39 were tested at a wider range of final
concentrations (Table 5). In this set-up, compound 5 proved to be the most selective β2i ligand
(ratio β2c/β2i: 67) as it did not inhibit any of the β1 and β5 proteasome subunits. By contrast,
epoxyketone 39 proved to be a dual inhibitor of both β2i and β5i with high selectivity over the
corresponding constitutive subunits (ratio β2c/β2i: 44; ratio β5c/β5i: 109).
Epoxyketone 5, the most selective β2i-inhibitor of the series, was termed LU-002i and published
as part of a set of compounds and ABPP probes to visualize all six catalytic activities of human
constitutive and immunoproteasomes15. However, the decalin moiety of 5 was synthesized as a
mixture of stereoisomers that could not be separated. To address the question whether one or
both of the possible stereomers are active, the following attempts were undertaken to synthesize
a stereomerically pure analogue of 5 (LU-002i). First, compounds with partially reduced
naphthyl rings containing only one chiral carbon center within the bicyclic system were
synthesized: 68 (R) and 71 (S) (Scheme 1; Supporting Information). In the competitive ABPP
assay in Raji cell lysates (Table 6) 68 was inactive, while 71 selectively targeted β2i though with
dramatic loss of potency (IC50 2.54 µM) compared to 5 (IC50 0.18 µM).
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Scheme 1. Synthesis of compounds 68 and 71.
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N
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O
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54 (R = CO2H)
55 (R = CN)
S
a
S
N
R
H2N
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H
60 (R = CN)
61 (R = CO2H)
62 (R = C(=O)NMe(OMe)
f
g
h
j
i
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63
65 (R = Boc)
O
k
66 (R = H)
BocHN
RHN
OH
O
BocHN
CO2H
O
O
O
l
O
H
N
N
NH2
N
N
H
H
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O
OMe
OH
O
O
O
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BocHN
O
H
N
N
O
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N
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H
H
O
O
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OMe
O
O
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O
H
N
N
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H
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H
O
O
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OMe
Reagents and conditions: (a) i) LiAlH4/Et2O, 99%; ii) TsCl/TEA/DCM, 97%; iii) NaCN/DMF,
95%; (b) i) KOH/ethylene glycol; ii) N,O-dimethylhydroxylamine hydrochloride,
HCTU/DiPEA/DCM, 49% over two steps; (c) LiAlH4/Et2O; (d) 58/CuSO4/DCM, 84% two-step
yield; (e) Et2AlCN/i-PrOH/THF, 58%; (f) i) 6 M HCl, reflux; ii) Boc2O/TEA/THF/H2O, 58%
over two steps; (g) N,O-dimethylhydroxylamine hydrochloride, HCTU/DiPEA/DCM, 77%; (h)
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tBuLi/2-bromopropene/Et2O, -78 °C, 78%; (i) NaBH4/CeCl3 7H2O/MeOH, 59%; (j) 1)
VO(acac)2/tBuOOH /DCM; 2) Dess-Martin periodinane/DCM, 33% over two steps; (k) TFA,
quantitative yield; (l) 1) 67, tBuONO/HCl (4N in dioxane) DCM/DMF, -30 °C; 2) 66, DiPEA,
DMF, 40% over two steps.
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In a second approach to unravel the active stereomer of 5, fully reduced decaline systems were
produced, yielding the peptide epoxyketones 74 and 77, respectively (Scheme 2, Supporting
Information). Competitive ABPP revealed that 74 inhibits β2i with an IC50 of 12.0 µM without
touching the other five active sites of cCP and iCP particles (Table 6). Compound 77 in turn
proved to be a potent β2i inhibitor (IC50 0.384 µM) with some cross reactivity against β2c (IC50
27.64 µM). Notably, the absolute stereochemistry of the P1 side chain in 77 matches that of the
corresponding carbon center in ligand 71, but it appears that a decaline at P1 (77) is more
effective for β2i inhibition than the corresponding partially oxidized bicyclic system (71).
Scheme 2. Synthesis of compounds 74 and 77.
O
OH
O
H
OH
O
O
O
H
N
O
N
a
N
N
H
H
O
O
O
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H
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72
OH
BocHN
73
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8
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4a
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O
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8a
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R
O
OH
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H
OH
O
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H
N
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N
a
N
N
H
H
O
O
O
77
H
69
75
OH
BocHN
76
O
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Reagents and conditions: (a) H2, PtO2, AcOH, 99%.
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With this information in hand, an enantiomerically pure diastereomeric set of peptide
epoxyketones 86 and 87 was synthesized (Scheme 3; Supporting Information). Compound 86
appeared to be a weak (IC50 33.71 µM) but selective β2i inhibitor, whereas epoxyketone 87
strongly inhibits β2i (IC50 0.189 µM) with β2c, β1c and β5i as off targets at high micromolar
concentrations (Table 6). Based on the assumption that carbon 1 in the decaline system of
compound 87 has the (S) configuration as in 71 and 77, and assuming that the catalytic
hydrogenation proceeded to deliver the decaline with cis,cis stereochemistry, the observed
results strongly suggest that the stereochemistry of the most active and selective β2i-inhibitor is
as shown in structure 87 (Scheme 3).
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Scheme 3. Synthesis of compounds 86 and 87.
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COOH
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H
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Reagents and conditions: (a) H2, PtO2, AcOH, quantitative yield; (b) i) LiAlH4/Et2O, 92%; ii)
TsCl/TEA/DCM, 95%; 3) NaCN/DMF, 83%; (c) i) KOH/ethylene glycol; ii) N,O-
dimethylhydroxylamine hydrochloride, HCTU/DiPEA/DCM, 88% over two steps; (d)
LiAlH4/Et2O; (e) 58/CuSO4/DCM, 85% over two steps; (f) Et2AlCN/i-PrOH/THF, 75%.
To test whether compound 87 is the major active component of the stereomeric mixture that
makes up compound 5 (the previously described β2i-selective inhibitor, LU-002i15) both were
assessed in a competitive ABPP assay in Raji cell extracts at final inhibitor concentrations
ranging from 0 to 3 µM (Figure 2). Since both preparations are about equally active and selective,
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diastereomer 87 appears to be indeed the main active component in the stereomeric mixture that
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has previously been reported as LU-002i15.
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Figure 2. Comparative activity-based protein profiling assay of compounds 5 (LU-002i) and 87,
determined in Raji cell lysates.
Next, compound 87 was tested in intact RPMI-8226 cell lines, in comparison with the dual
β2i/β5i inhibitor 39. Cells were first treated with inhibitor at various concentrations, then lysed,
incubated with the ABPP mixture, denatured and resolved on SDS PAGE as described before.
Like in Raji cell lysates, compound 87 selectively targeted only β2i (IC50 0.159 µM) without
affecting the remaining proteolytically active proteasome subunits, whereas epoxyketone 39
inhibited both β2i (IC50 0.124 µM) and β5i (IC50 0.183 µM) (Figure 3). Thus, inhibitor 39
represents a co-inhibitor of β2i and β5i with potential medicinal relevance, especially because
targeting of β2 has previously been shown to sensitize cells to β5 inhibitors17 and dual subunit
inhibition is required for suppressing autoinflammatory reactions11.
Figure 3. Inhibition profiles of compounds 39 and 87, determined in intact RPMI-8226 cell lines.
As the next research objective we decided to investigate whether a β2i-selective activity-based
probe could be derived from LU-002i (5). Since attachment of a fluorescent tag at the N-
terminus of subunit-selective inhibitors may be detrimental to selectivity, we decided to graft the
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reporter group onto the tyrosine residue at P2 by substituting the methyl group for an
appropriately functionalized alkyl group (Scheme 4). The resulting activity-based probe 97 was
tested in Raji cell lysate to profile the proteasome activities. At a final concentration of 3 µM,
β2i-labeling was selective and could be easily distinguished (Figure 4A). In a competitive ABPP
assay with probe 97 labeling of β2i could be completely abolished by pre-incubation with LU-
002i (5, β2i) at 3 µM. The β2i signal was partially reduced after treatment with LU-002c (4, β2c)
at high concentrations, and completely abolished after pre-incubation with LU-102 (1, β2c/β2i)
(Figure 4B). Finally, a competitive ABPP assay with probe 97 side-by-side with the three-probe
mixture used previously in competitive ABPP experiments was carried out. This time treatment
with LU-002i (5, β2i) selectively blocked β2i-labeling by the three probes at 3 µM, whereas LU-
002c (4, β2c) completely prevented β2c identification (0.3 µM final concentration) and partially
inhibited β2i labeling. Furthermore, LU-102 (1, β2c/β2i) blocked both β2c- and β2i- labeling at 1
µM (Figure 4B). These results match those published earlier on these compounds against the
same set of probes15. Altogether these data demonstrates that ABP 97 is a potent and highly
selective ABP for visualizing β2i activities of human immunoproteasomes.
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Scheme 4. Synthesis of probe 97.
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O
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N3
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Reagents and conditions: (a) 89, K2CO3/DMF, 80%; (b) i) TFA, 99%; ii) Boc-Ala-OH, HCTU/
DiPEA/DCM, 93%; (c) i) TFA, 99%; ii) 2-morpholino acetic acid, HCTU/DiPEA/DCM, 32%;
(d) N2H4·H2O, MeOH, 99%; (e) tBuONO/HCl (4N in dioxane), DCM/DMF (1/1, v/v), -30 °C,
56%; (f) CuSO4, sodium ascorbate, DMF, 18%.
Figure 4. (A) Activity-based proteasome profiling using probe 97 at different concentrations.
Cocktail ABPs were added as control. (B) Left: Competitive activity-based protein profiling
assay using ABP 97 and the inhibitors 1 (LU-102, 0.1 µM), 4 (LU-002c, 0.03 µM) and 5 (LU-
002i, 0.1 µM). Right: Competitive ABPP assay with probe 97 side-by-side with the three-probe
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mixture used previously in competitive ABPP experiments and the inhibitors 1 (LU-102, 0.1
µM), 4 (LU-002c, 0.03 µM) and 5 (LU-002i, 0.1 µM).
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X-ray structures of selected inhibitors in complex with yeast and humanized CPs
In order to obtain more insights into the structural features that drive either β2c- or β2i-
selectivity of ligands, we aimed at determining X-ray structures of selected compounds in
complex with CPs. Since structural data on the human apo iCP are not available, we recently
developed chimeric yeast proteasomes, which feature key elements of human β5 subunits, as
structural tools18. Based on this work, we here created β2 humanized yeast proteasomes.
While yeast proteasome α subunits can be easily exchanged by human counterparts, replacement
of most β entities i.e. β1, β2, β5, β6 and β7 is lethal to yeast13d, 18-19. Strikingly however, the
single point mutation S171G suffices to rescue the lethal phenotype that is caused by substitution
of the endogenous yeast (y) β2 subunit with the human (h) β2c counterpart19. We created the
respective β2c chimeric yeast strain (Figures 5A, S8), purified and crystallized its mutant
proteasome. The X-ray structure (Table S13) revealed that the β2 propeptide was released from
the active site Thr1 and that the overall fold of the subunit was intact (Figure 6A). Although the
S171G mutation had no obvious impact on the structure of the matured mutant proteasome, it
likely supports subunit folding and proteasome assembly. Any pronounced effects of Gly171 on
β2 activity are excluded, as yeast viability does not depend on peptide bond hydrolysis by β220.
Since no rescuing mutation for the hβ2i subunit is known to date, we created various chimeric
hβ2i-yβ2 constructs and tested whether they can substitute wild-type (WT) yβ2. Surprisingly,
only a construct featuring the β2i amino acids 1-53 was viable (Figure 5B). As this sequence
covers the entire β2 substrate binding channel, we used this construct for structural analyses
(Table S13).
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Figure 5. Schematic representation of yeast (y) and human (h) β2 subunits and their propeptides.
Secondary structure elements, helices (H) and sheets (S) are numbered. (A) The full-length hβ2c
(green) and hβ2i (pink) subunits cannot substitute the endogenous yβ2 subunit (gray), neither
with their natural propeptides (pp; colored) nor with the yβ2 one (gray) (for details see
experimental procedures). Strikingly, the human β2c subunit can replace the yeast counterpart
when featuring the single point mutation S171G19. (B) Schematic illustration of human-yeast
chimeric β2i constructs according to panel (A). Sequences highlighted in pink were taken from
human β2i, whereas gray ones originate from the yeast β2 entity. All tested variants, except for
the construct encoding the residues 1-53 from human β2i, caused lethality when expressed in a
pup1Δ yeast strain.
Superposition of ligand-free β2c/i chimeric structures with the natural mouse counterpart13d
proved their structural similarity (Figure 6A, B). Subsequent crystal soakings with ONX 0914 as
a reference compound confirmed that the β2 proteolytic centers were reactive (Figure S9) and
visualized a similar binding mode for the inhibitor as in the respective mouse crystal structures13d
(Figure 6C, D). β2 subunits can accommodate bulky P1 residues without any pronounced
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conformational changes of the protein backbone (Figure S10A, B). The corresponding spacious
P1 binding site is created by Gly45 at the bottom of the S1 pocket13d. While the chemical nature
and the orientation of amino acid 45 differs between most proteasome subunits, Gly45 has been
preserved in β2 subunits throughout evolution13d. Though mutation of Gly45 to Ala does neither
impair yeast growth nor affect subunit folding and ligand binding, any additional increase of
residue 45 is predicted to sterically interfere with the surrounding protein side chains (Figures S8,
S11, S12, Table S13).
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Based on the structural similarity of human-yeast chimeric and mouse β2 active sites, a set of 29
ligand complex structures was determined with WT and β2 chimeric yeast proteasomes (Table
S13).
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Figure 6. Structural superpositions of the natural mouse β2c (A, C) and β2i (B, D) subunits with
their human-yeast chimeric counterparts in the ligand-free (A, B) and ONX 0914 bound (C, D)
states. Amino acids are labelled by the one-letter code. Hydrogen bonds are depicted as black
dashed lines. Hydrophobic interactions are highlighted by double arrows. Color coding is
according to Figure 5. Note that ONX 0914 has been previously modelled into the mouse β2
subunits as a morpholine adduct with Thr113d, while in the chimeric subunits it was built as a
seven-membered ring structure according to the revised reaction mechanism of epoxyketones
with Thr1.21 PDB IDs: 3UNE (mouse cCP), 3UNH (mouse iCP), 3UNB (mouse cCP:ONX 0914),
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3UNF (mouse iCP:ONX 0914), 6HTB (hβ2c chimera), 6HV3 (hβ2i chimera), 6HTC (hβ2c
chimera:ONX 0914), 6HV4 (hβ2i chimera:ONX 0914).
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The β2c-selective compound 4 (LU-002c) was found to be well stabilized in the β2c and β2i
active sites. Interactions of the 4-aminomethylphenyl group at P1 with the carboxylic amino acid
side chains in position 53 are supposed to be the driving force for the general β2-selectivity of 4
(LU-002c) as well as the related compounds LU-102 (1) and LU-112 (2) (Figure 1)16. Selectivity
for subunit β2c might be gained by dual anchoring of the 4-aminomethylphenyl group to Asp53
in β2c versus a single interaction with Glu53 in β2i (Figure 7A, C). In addition, the shorter P2
Ala side chain of 4 (LU-002c) compared to Leu in LU-102 increases β2c-selectivity by reducing
the potency for β2i (Figure 1). Most likely small P2 residues like Ala fail to undergo favorable
van der Waals interactions with Val48 in β2i (Figure 7C) and thereby lead to the observed β2c-
selectivity of 4 (LU-002c).
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Figure 7. Human-yeast chimeric proteasomes in complex with β2c- (4; green) and β2i- (39;
purple) selective inhibitors. (A, C, E, G) 2FO-FC electron density maps for the compounds bound
to the β2c (green) and β2i (purple) chimeric subunits, respectively, are shown as blue meshes
contoured to 1σ. (B, D, F, H) Structural superposition of ligand-free and ligand-bound chimeric
β2c and β2i subunits respectively. Polar and hydrophobic interactions are depicted according to
Figure 6. PDB IDs: 6HTB (hβ2c chimera), 6HTD (hβ2c chimera:4), 6HUV (hβ2c chimera:39),
6HV3 (hβ2i chimera), 6HV5 (hβ2c chimera:4), 6HVV (hβ2i chimera:39).
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For the most selective β2i-inhibitor, compound 5 (LU-002i), crystallographic data could only be
obtained with WT yCP (Figure S13, Table S14). We assume that the ligand could not be trapped
at the mutant β2 active site, since the reactivity of chimeric subunits is impaired18, and because
compound 5 is poorly soluble in aqueous solutions due to its apolar decaline moiety. Chimeric
proteasome structures in complex with 39 however could be achieved. 5 (LU-002i) and 39 are
derived from the epoxyketone inhibitor ONX 0914. Epoxyketones have recently been shown to
form seven-membered21 instead of six-membered22 ring structures with the nucleophilic Thr1
residue of proteasomal β subunits. Although the 1,4-oxazepane (seven-membered) ring structure
fits our experimental electron densities in most cases, we also have structural data which matches
better the six-membered 1,4-morpholine system (for example see Figure S13A, B). However, the
kind of irreversible covalent structure inhibitors form with Thr1 has no further implications for
drug development, as subunit selectivity of epoxyketone inhibitors is mostly gained by
interactions of the ligands’ side chains with the protein surroundings.
ONX 0914 slightly favors β2i over β2c12, which may be supported by an advantageous
hydrophobic interaction of its P2-methoxy group with Val48 of β2i, a contact that is not
provided in subunit β2c (Figure 6C, D). Furthermore, Asn22 hydrogen bonds to the amide
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oxygen atom of the morpholine cap of ONX 0914, while Glu22 in subunit β2c fails to provide
this additional stabilization (Figure 6C, D). The interaction with Asn22 in β2i is also observed
with other tripeptide ligands like 39 (Figure 7G, H), implying that peptide substrates in general
might be better stabilized in the β2i substrate binding channel than in the β2c one. Notably, a
similar observation has previously been reported for Thr22 in subunit yβ1/β1c13a.
The co-crystal structure of the β2i chimera with compound 39 shows a well-defined 2FO-FC
electron density map for the ligand (Figure 7G). Comparison of ligand-free and ligand-bound
states of the β2i chimera indicates a movement of His35 upon inhibitor binding (Figure 7H).
Despite this structural flexibility and plasticity of the S1 pocket, the hydrogen bond between
His35 and Glu53 remains intact. Compared to β2i, the β2c active site appears to be more rigid, as
binding of 39 does not trigger any structural changes of His35 (Figure 7F). Presumably, the P1
side chain of 39 is less well defined in the β2c active site due to the tight anchoring of and the
resulting steric hindrance with His35 (Figure 7E, F). Thus, although β2 subunits in general
accept large P1 side chains, it appears that the plasticity of the β2i active site tolerates bulky
residues even more readily than β2c.
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Discussion
Here we describe the development and evaluation of a set of potent and selective inhibitors of
human β2c and β2i proteasome activities. Due to the structural similarities of mammalian β2c
and β2i subunits, no key guidelines for compound design strategies could be derived from crystal
structures so far13d. Thus, we used the previously described inhibitors LU-102 (1)16, LU-112 (2)16
and ONX 0914 (3)12 as starting points, which have no or only moderate preference for one of the
two human β2 subunits over the other. By changing the P sites of the ligands we disfavored the
most closely related subunit, either β2i or β2c, and gained selectivity.
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Substantial organic synthesis efforts and thorough empiric screening of compound libraries
derived from these lead structures finally led to the identification of selective compounds and to
the development of suitable probes for ABPP assays. Furthermore, previously unaddressed
stereochemistry issues on LU-002i (5) have now been resolved and the exact configuration of the
bioactive compound has been determined.
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Selected β2c and β2i inhibitors were analyzed by X-ray crystallography in complex with the WT
yeast CP and with chimeric human-yeast proteasomes, incorporating key elements of the human
β2c and β2i substrate binding channel, respectively. Despite the artificial character of chimeras
they were previously shown to serve as excellent structural tools18 and now again prove valuable
for explaining the selectivity patterns observed for the here described β2 compound libraries.
Both β2c and β2i can incorporate large P1 residues in their spacious S1 pocket. Due to favorable
hydrogen bond interactions with Asp/Glu53, LU-102 derivatives with their 4-
aminomethylphenyl side chain at P1 are in general more potent β2 inhibitors than ONX 0914-
based compounds, featuring apolar P1 residues16. Selectivity for β2c was gained by installing
small P2 residues on LU-102. Epoxyketones with bulky hydrophobic P1 residues and small P3
side chains were found to show β2i-selectivity. Because of the plasticity of the S1 pocket and the
flexibility of His35 in subunit β2i, large apolar P1 side chains can be better accommodated in β2i
than in β2c.
Taken together, we here present the most selective β2c and β2i ligands reported so far. As part of
a set of inhibitors and ABPs that is capable of disabling and visualizing the individual activities
of human constitutive and immunoproteasomes15, these compounds might become valuable tools
for fundamental as well as applied biochemical and biomedical research on proteasomes and
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hopefully elucidate more details on the biological role and impact of the trypsin-like active sites
of human proteasomes.
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Experimental Section
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General procedures
All reagents were of commercial grade and used as received unless indicate otherwise. The
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purity of all tested compounds is >95% on the basis of LC-MS and NMR. H- and C NMR
spectra were recorded on a Bruker AV-400 (400 MHz), AV-600 (600 MHz) or AV-850 (850
MHz) spectrometer. Chemical shifts are given in ppm (δ) relative to CD3OD or CDCl3 as
internal standard. Coupling constants are given in Hz and peak assignments are based on 2D 1H
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COSY and C HSQC NMR experiments. All C APT spectra are proton decoupled. LC-MS
analysis was performed on a Finnigan Surveyor HPLC system with a Gemini C18 50 × 4.60 mm
column (detection at 200−600 nm) coupled to a Finnigan LCQ Advantage Max mass
spectrometer with ESI. Methods used are: 15 min (0-0.5 min: 10% MeCN; 0.5-10.5 min: 10% to
90% MeCN; 10.5-12.5 min: 90% MeCN; 12.5-15 min: 90% to 10% MeCN) or 12.5 min (0-0.5
min: 10% MeCN; 0.5-8.5 min: 10% to 90% MeCN; 8.5-10.5 min: 90% MeCN; 10.5-12.5 min:
90% to 10% MeCN). HRMS was recorded on a LTQ Orbitrap (ThermoFinnigan). For reverse
phase HPLC purification, an automated Gilson HPLC system equipped with a C18 semiprep
column (Phenomenex Gemini C18, 5μm 250×10 mm) and a GX281 fraction collector was used.
General procedure for Boc removal
The appropriate Boc-protected C-terminally modified leucine derivative was dissolved in TFA
and stirred for 20 min. Co-evaporation with toluene (3x) afforded the TFA-salt, which was used
without further purification.
General procedure for azide couplings
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Compounds 6-53, 68, 71, 74, 77, 86, 87 and 97 were prepared via azide coupling of the
appropriate protected tripeptide hydrazide and either an epoxyketone amine or a vinyl sulfone
amines. Peptide hydrazides were prepared by hydrazinolysis of peptide methyl esters synthesized
as described in the Supporting Information. The hydrazide was dissolved in 1:1 DMF:DCM (v/v)
and cooled to -30 °C. tBuONO (1.1 eq.) and HCl (4N 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 epoxyketone or vinyl sulfone
amine was added as a free amine to the reaction mixture as a solution in DMF with 5.0 eq. of
DiPEA. The mixture was allowed to warm to r.t. overnight. The mixture was diluted with EtOAc
and extracted with H2O (3x) and brine. The organic layer was dried over MgSO4 and purified by
reverse phase HPLC. For compounds featuring Boc protecting groups, TFA was added and the
reaction mixture was stirred for 30 min. The crude was purified by reverse phase HPLC.
N3Phe-Leu-Ser-Phe(4-CH2NH2)VS TFA salt (13). The synthesis of tripeptide hydrazide N3Phe-
Leu-Ser(tBu)-NHNH2 is described in the Supporting Information. The title compound was
prepared according to the general procedure for azide coupling on a 50 μmol scale and purified
by HPLC (30%-40% MeCN-H2O) to yield 2.8 mg (3.8 μmol, 8%). 1H NMR (600 MHz, MeOD)
δ 7.46-7.23 (m, 9H), 6.85-6.81 (m, 1H), 6.76-6.73 (m, 1H), 4.39-4.27 (m, 2H), 4.20-4.17 (m,
1H), 4.11 (s, 2H), 3.84-3.81 (m, 1H), 3.76-3.73 (m, 1H), 3.25-3.22 (m, 1H), 3.07-2.98 (m, 3H),
2.95 (s, 3H), 1.69-1.52 (m, 3H), 1.02-0.88 (m, 6H). 13C NMR (150 MHz, MeOD) δ 174.33,
172.23, 171.81, 146.65, 139.69, 137.84, 133.00, 131.84, 131.26, 130.42, 130.24, 129.65, 128.10,
65.38, 62.75, 56.70, 53.80, 52.56, 44.09, 42.77, 41.38, 40.25, 38.72, 25.82, 23.46, 21.84. LC-MS
(linear gradient 10 → 90% MeCN/H2O, 0.1% TFA, 15.0 min): Rt (min): 6.27 (ESI-MS (m/z):
628.20, (M+H+)). HRMS calculated for C30H41N7O6S 628.29118 [M+H]+; found 628.29123.
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Morp-Ala-Tyr(Me)-HomoCha-EK TFA salt (39). The synthesis of Boc-HomoCha-EK is
described in the Supporting Information and the Boc protecting group was removed according to
the general procedure. The title compound was prepared according to the general procedure for
azide coupling on a 50 μmol scale and purified by HPLC (30%-45% MeCN-H2O) to yield 12.3
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mg (17.2 μmol, 34%). H NMR (600 MHz, MeOD) δ 7.25-7.01 (m, 2H), 6.91-6.67 (m, 2H),
4.60-4.57 (m, 1H), 4.48-4.28 (m, 2H), 3.77 (s, 3H), 3.71-3.70 (m, 4H), 3.21 (d, J = 4.9 Hz, 1H),
3.09-2.88 (m, 4H), 2.84-2.79 (m, 1H), 2.56-2.37 (m, 4H), 1.83-1.63 (m, 6H), 1.52-1.39 (m, 4H),
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1.38-1.16 (m, 9H), 0.97-0.83 (m, 2H). C NMR (150 MHz, MeOD) δ 209.19, 174.20, 173.30,
171.99, 159.94, 131.41, 130.02, 114.75, 67.85, 62.40, 60.01, 55.75, 55.60, 54.71, 53.06, 52.92,
49.65, 38.48, 38.15, 34.61, 34.02, 29.11, 27.72, 27.44, 27.38, 18.65, 16.84. LC-MS (linear
gradient 10 → 90% MeCN/H2O, 0.1% TFA, 12.5 min): Rt (min): 6.23 (ESI-MS (m/z): 601.33,
(M+H+)). HRMS calculated for C32H48N4O7 601.35958 [M+H]+; found 601.35945.
Morp-Ala-Tyr(Me)-1-(R)-TetraNal-EK TFA salt (68). The synthesis of Boc-1-TatraNal-EK is
described in the Supporting Information and the Boc protecting group was removed according to
the general procedure. The title compound was prepared according to the general procedure for
azide coupling on a 56 μmol scale and purified by HPLC (30%-45% MeCN-H2O) to yield 14.2
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mg (22.4 μmol, 40%). H NMR (400 MHz, MeOD) δ 7.22-7.13 (m, 2H), 7.12-7.01 (m, 4H),
6.88-6.77 (m, 2H), 4.63-4.59 (m, 2H), 4.41-4.36 (m, 1H), 4.06-3.84 (m, 6H), 3.78 (s, 3H), 3.17-
13
2.98 (m, 2H), 2.93-2.67 (m, 5H), 2.14-1.54 (m, 6H), 1.45 (s, 3H), 1.35 (d, J = 7.1 Hz, 3H). C
NMR (100 MHz, MeOD) δ 209.40, 174.19, 173.10, 165.04, 160.01, 140.66, 137.91, 131.40,
130.18, 129.98, 129.94, 127.07, 126.54, 114.84, 64.84, 59.89, 58.37, 55.83, 55.67, 53.93, 52.88,
51.64, 50.57, 39.22, 37.99, 36.17, 30.02, 29.64, 20.16, 18.11, 16.76. LC-MS (linear gradient 10
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→ 90% MeCN/H2O, 0.1% TFA, 12.5 min): Rt (min): 6.10 (ESI-MS (m/z): 635.00, (M+H+)).
HRMS calculated for C35H46N4O7 635.34393 [M+H]+; found 635.34371.
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Morp-Ala-Tyr(Me)-1-(S)-TetraNal-EK TFA salt (71). The synthesis of Boc-1-TatraNal-EK is
described in the Supporting Information and the Boc protecting group was removed according to
the general procedure. The title compound was prepared according to the general procedure for
azide coupling on a 50 μmol scale and purified by HPLC (30%-45% MeCN-H2O) to yield 16.5
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mg (26.0 μmol, 52%). H NMR (400 MHz, MeOD) δ 7.24-7.13 (m, 3H), 7.13-6.98 (m, 3H),
6.86-6.78 (m, 2H), 4.77-4.59 (m, 2H), 4.41-4.36 (m, 1H), 4.00-3.86 (m, 6H), 3.21 (d, J = 5.0 Hz,
1H), 3.10 (dd, J = 14.0, 6.0 Hz, 1H), 3.03-2.64 (m, 5H), 2.02-1.60 (m, 6H), 1.45 (s, 3H), 1.33 (d,
J = 7.2 Hz, 3H). 13C NMR (100 MHz, MeOD) δ 208.97, 174.18, 173.57, 164.98, 160.01, 141.21,
138.13, 131.41, 130.09, 130.01, 129.75, 126.83, 126.76, 114.84, 114.75, 64.82, 60.04, 58.35,
55.89, 55.66, 53.91, 53.05, 51.05, 50.54, 38.67, 38.02, 35.13, 30.57, 27.13, 20.23, 18.10, 16.86.
LC-MS (linear gradient 10 → 90% MeCN/H2O, 0.1% TFA, 12.5 min): Rt (min): 6.18 (ESI-MS
(m/z): 635.07, (M+H+)). HRMS calculated for C35H46N4O7 635.34393 [M+H]+; found
635.34370.
Morp-Ala-Tyr(Me)-1-DecAla-EK TFA salt (74). The synthesis of Boc-1-DecAla-EK is
described in the Supporting Information and the Boc protecting group was removed according to
the general procedure. The title compound was prepared according to the general procedure for
azide coupling on a 50 μmol scale and purified by HPLC (40%-50% MeCN-H2O) to yield 14.6
1
mg (22.8 μmol, 46%). H NMR (400 MHz, MeOD) δ 7.20-7.11 (m, 2H), 6.86-6.78 (m, 2H),
4.62-4.51 (m, 2H), 4.38-4.33 (m, 1H), 4.07-3.83 (m, 6H), 3.77 (d, J = 3.7 Hz, 3H), 3.22 (d, J =
12 Hz, 1H), 3.06-3.01 (m, 1H), 2.95 (d, J = 12 Hz, 1H), 2.85-2.79 (m, 1H), 1.84-1.13 (m, 25H).
13C NMR (100 MHz, MeOD) δ 209.81, 174.11, 173.35, 164.81, 159.95, 131.42, 130.00, 114.77,
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64.78, 60.08, 58.26, 55.64, 53.89, 52.81, 50.50, 50.29, 39.17, 38.74, 38.20, 33.84, 29.05, 27.91,
26.60, 22.38, 20.22, 18.11, 16.88. LC-MS (linear gradient 10 → 90% MeCN/H2O, 0.1% TFA,
12.5 min): Rt (min): 6.75 (ESI-MS (m/z): 641.13, (M+H+)). HRMS calculated for C35H52N4O7
641.39088 [M+H]+; found 641.39081.
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Morp-Ala-Tyr(Me)-1-DecAla-EK TFA salt (77). The synthesis of Boc-1-DecAla-EK is
described in the Supporting Information and the Boc protecting group was removed according to
the general procedure. The title compound was prepared according to the general procedure for
azide coupling on a 23 μmol scale and purified by HPLC (40%-50% MeCN-H2O) to yield 6.8
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mg (10.6 μmol, 46%s). H NMR (400 MHz, MeOD) δ 7.19-7.13 (m, 2H), 6.84-6.80 (m, 2H),
4.63-4.60 (m, 1H), 4.53-4.50 (m, 1H), 4.40-4.34 (m, 1H), 4.01-3.90 (m, 6H), 3.78 (d, J = 1.9 Hz,
3H), 3.17 (d, J = 5.1 Hz, 1H), 3.07-3.02 (m, 1H), 2.94 (d, J = 5.1 Hz, 1H), 2.88-2.80 (m, 1H),
1.85-1.07 (m, 25H). 13C NMR (100 MHz, MeOD) δ 209.46, 174.13, 173.41, 164.82, 159.97,
131.43, 129.96, 114.77, 64.78, 59.91, 58.27, 55.62, 53.90, 52.92, 50.97, 50.52, 43.08, 39.49,
39.11, 38.25, 36.48, 33.73, 27.99, 27.69, 26.76, 26.54, 22.32, 21.21, 18.10, 16.87. LC-MS (linear
gradient 10 → 90% MeCN/H2O, 0.1% TFA, 12.5 min): Rt (min): 6.79 (ESI-MS (m/z): 641.07,
(M+H+)). HRMS calculated for C35H52N4O7 641.39088 [M+H]+; found 641.39070.
Morp-Ala-Tyr(Me)-1-DecAla-EK TFA salt (86). The synthesis of Boc-1-DecAla-EK is
described in the Supporting Information and the Boc protecting group was removed according to
the general procedure. The title compound was prepared according to the general procedure for
azide coupling on a 25 μmol scale and purified by HPLC (30%-45% MeCN-H2O) to yield 8.6
1
mg (11.4 μmol, 46%). H NMR (500 MHz, MeOD) δ 7.15 (d, J = 8.7 Hz, 2H), 6.85-6.80 (m,
2H), 4.62-4.59 (m, 1H), 4.55-4.52 (m, 1H), 4.40-4.35 (m, 1H), 4.01-3.91 (m, 6H), 3.78 (s, 3H),
3.21 (d, J = 5.1 Hz, 1H), 3.07-3.03 (m, 1H), 2.95 (d, J = 5.1 Hz, 1H), 2.85-2.81 (m, 1H), 1.83-
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1.52 (m, 10H), 1.47-1.41 (m, 5H), 1.37-1.18 (m, 10H). C NMR (125 MHz, MeOD) δ 209.83,
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174.13, 173.36, 164.80, 159.96, 131.42, 129.99, 114.77, 64.78, 60.09, 58.26, 55.64, 53.90, 52.81,
50.49, 50.29, 39.21, 39.17, 38.75, 38.20, 34.85, 33.84, 29.05, 27.92, 27.91, 26.60, 22.38, 20.22,
18.12, 16.87. LC-MS (linear gradient 10 → 90% MeCN/H2O, 0.1% TFA, 12.5 min): Rt (min):
6.92 (ESI-MS (m/z): 641.13, (M+H+)). HRMS calculated for C35H52N4O7 641.39088 [M+H]+;
found 641.39065.
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Morp-Ala-Tyr(Me)-1-DecAla-EK TFA salt (87). The synthesis of Boc-1-DecAla-EK is
described in the Supporting Information and the Boc protecting group was removed according to
the general procedure. The title compound was prepared according to the general procedure for
azide coupling on a 44 μmol scale and purified by HPLC (30%-45% MeCN-H2O) to yield 12.7
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mg (16.8 μmol, 38%). H NMR (500 MHz, MeOD) δ 7.15 (d, J = 8.6 Hz, 2H), 6.84-6.79 (m,
2H), 4.66-4.58 (m, 1H), 4.53-4.50 (m, 1H), 4.39-4.35 (m, 1H), 4.02-3.92 (m, 6H), 3.17 (d, J =
5.1 Hz, 1H), 3.07-3.03 (m, 1H), 2.94 (d, J = 5.1 Hz, 1H), 2.86-2.82 (m, 1H), 1.84-1.18 (m, 25H).
13C NMR (125 MHz, MeOD) δ 209.47, 174.13, 173.41, 164.81, 159.96, 131.43, 129.96, 114.77,
64.76, 59.91, 58.24, 55.64, 53.89, 52.93, 50.96, 50.55, 43.06, 39.48, 39.10, 38.25, 36.48, 33.72,
27.98, 27.68, 26.75, 26.53, 22.31, 21.21, 18.08, 16.87. LC-MS (linear gradient 10 → 90%
MeCN/H2O, 0.1% TFA, 12.5 min): Rt (min): 6.88 (ESI-MS (m/z): 641.13, (M+H+)). HRMS
calculated for C35H52N4O7 641.39088 [M+H]+; found 641.39077.
Morp-Ala-Tyr(O-C2H4-BODIPY(FL))-1-DecAla-EK (97). The synthesis of compound 95 is
described in the Supporting Information. Compound 95 (23 mg, 33 μmol) and BODIPY-FL-
alkyne 9623 (13 mg, 40 μmol, 1.2 eq.) were dissolved in DMF. An aqueous solution of sodium
ascorbate (100 μl, 25 μmol, 0.75 eq.) and an aqueous solution of CuSO4 (100 μL, 17 μmol, 0.5
eq.) were added. The reaction mixture was stirred at r.t. overnight. The reaction mixture was
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