Epoxides related to dioncoquinone B: Synthesis, activity against multiple myeloma cells, and search for the target protein

Article abstract of DOI:10.1016/j.tet.2018.04.056

Epoxide 2b is an analog of the synthetic intermediate 2a en route to the polyketide-derived antitumoral naphthoquinone dioncoquinone B (1), isolated from cell cultures of the tropical liana Triphyophyllum peltatum (Dioncophyllaceae). Compound 2b was found to induce strong apoptosis in multiple myeloma cells at a concentration (EC₅₀ = 3.5 μM), distinctly lower than that of 1 and any related analog, without exerting significant toxicity against normal blood cells. Preliminary studies showed that 2b follows different SAR rules as compared to the naphthoquinones. Among the series of synthesized epoxides, 2b was the most active one and was thus, after biotinylation, subjected to mass spectrometry-based affinity capture experiments aiming at the identification of target proteins. The MS data revealed 2b to address proteins that are associated with stress regulation processes which are critical for multiple myeloma cell survival.

Full text of DOI:10.1016/j.tet.2018.04.056

Accepted Manuscript
Epoxides related to dioncoquinone B: Synthesis, activity against multiple myeloma
cells, and search for the target protein
Xia Cheng, Guoliang Zhang, Raina Seupel, Doris Feineis, Daniela Brünnert, Manik
Chatterjee, Andreas Schlosser, Gerhard Bringmann
PII:
S0040-4020(18)30445-9
10.1016/j.tet.2018.04.056
TET 29471
DOI:
Reference:
To appear in: Tetrahedron
Received Date: 8 March 2018
Revised Date: 13 April 2018
Accepted Date: 17 April 2018
Please cite this article as: Cheng X, Zhang G, Seupel R, Feineis D, Brünnert D, Chatterjee M, Schlosser
A, Bringmann G, Epoxides related to dioncoquinone B: Synthesis, activity against multiple myeloma
cells, and search for the target protein, Tetrahedron (2018), doi: 10.1016/j.tet.2018.04.056.
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ACCEPTED MANUSCRIPT
Graphical Abstract
Epoxides related to dioncoquinone B: Synthesis, activity against multiple myeloma
cells, and search for the target protein
Xia Chenga, Guoliang Zhanga, Raina Seupela, Doris Feineisa, Daniela Brünnertb, Manik Chatterjeeb,
Andreas Schlosserc, Gerhard Bringmanna,*
a Institute of Organic Chemistry, University of Würzburg, Am Hubland, D-97074 Würzburg
b Translational Oncology, Comprehensive Cancer Center Mainfranken, University Hospital of Würzburg, Versbacher Straße 5, D-97078 Würzburg
c Rudolf Virchow Center for Experimental Biomedicine, University of Würzburg, Josef-Schneider-Straße 2, D-97080 Würzburg
O
O
O
O
O
100
S
MeO
Biotinylation
( )
O
n
( )
3
N
H
N
H
O
O
H
H
N
N
75
50
Me
Me
HN
NH
4
2b
O
Biotin
Linker
Anti-MM active
epoxide
25
0
Mass spectrometry-based
capture experiments
0
1
2
3
4
5
6
7
8
9
10
Proteins associated with
stress regulation processes
2b (mM)
———
*
Corresponding author. Tel.: +49-931-318-5323; fax: +49-931-318-4755; e-mail: bringman@chemie.uni-wuerzburg.de (G. Bringmann)

ACCEPTED MANUSCRIPT
Tetrahedron
journal homepage: www.elsevier.com
Epoxides related to dioncoquinone B: Synthesis, activity against multiple myeloma
cells, and search for the target protein
Xia Chenga, Guoliang Zhanga, Raina Seupela, Doris Feineisa, Daniela Brünnertb, Manik Chatterjeeb,
Andreas Schlosserc, Gerhard Bringmanna,*
a Institute of Organic Chemistry, University of Würzburg, Am Hubland, D-97074 Würzburg
b Translational Oncology, Comprehensive Cancer Center Mainfranken, University Hospital of Würzburg, Versbacher Straße 5, D-97078 Würzburg
c Rudolf Virchow Center for Experimental Biomedicine, University of Würzburg, Josef-Schneider-Straße 2, D-97080 Würzburg
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
Epoxide 2b is an analog of the synthetic intermediate 2a en route to the polyketide-derived
antitumoral naphthoquinone dioncoquinone B (1), isolated from cell cultures of the tropical liana
Triphyophyllum peltatum (Dioncophyllaceae). Compound 2b was found to induce strong
apoptosis in multiple myeloma cells at a concentration (EC50 = 3.5 µM), distinctly lower than
that of 1 and any related analog, without exerting significant toxicity against normal blood cells.
Preliminary studies showed that 2b follows different SAR rules as compared to the
naphthoquinones. Among the series of synthesized epoxides, 2b was the most active one and
was thus, after biotinylation, subjected to mass spectrometry-based affinity capture experiments
aiming at the identification of target proteins. The MS data revealed 2b to address proteins that
are associated with stress regulation processes which are critical for multiple myeloma cell
survival.
Available online
Keywords:
Epoxides
Naphthoquinones
Multiple myeloma
Target identification
Biotinylation
2018 Elsevier Ltd. All rights reserved
.
INA-610-14 have been used during that work, which showed that
dioncoquinone B (1) exerts a pronounced cytotoxicity towards
MM cells (EC50 = 11 µM), without any noticeable toxic effects
against normal peripheral mononuclear blood cells (PBMCs).10
For more in-depth investigations on the anti-MM potential of
those naphthoquinones, a synthetic pathway (Scheme 1),
affording 1 in larger quantities and simultaneously providing a
small library of related compounds, has been developed.11
1. Introduction
Multiple myeloma (MM), the second most prevalent
hematological malignancy, is caused by an excess of abnormal
plasma cells forming tumors in multiple locations within the
bone marrow, thus leading to osteolytic bone destruction,
impaired hematopoiesis and renal failure.1-4 Several novel
therapies have substantially improved patient outcomes over the
past decade. However, with a five-year survival of only 49%,5
cure still remains elusive. In particular, once MM patients
become refractory to the major classes of anti-MM agents
(proteasome inhibitors, immunomodulatory drugs, and antibodies
against SLAMF7 and CD38), their median overall survival is less
than twelve months.6-9 Therefore, therapy-refractory patients
have the most urgent unmet clinical need for novel therapeutic
approaches.
OH
O
HO
OH
Me
Isolation
O
1
1. conc. H2SO4,
THF
2. BBr3, CH Cl2
2
from callus cultures
of T. peltatum
Solid callus cultures of the tropical liana Triphyophyllum
peltatum (Dioncophyllaceae) have previously been found to
2
O
OMe
O
O
MeO
MeO
produce
structurally
unique
polyketide-derived
O
Me
O
4
4
naphthylisoquinoline alkaloids along with a series of highly
oxygenated, previously unknown naphthoquinones, among them
dioncoquinone B (1) (Scheme 1).10,11 This naphthoquinone is
structurally related to the naphthalene part of the alkaloids. To
study more closely the tumor-specific activities of 1 and related
naphthoquinones, malignant cells of the human MM cell line
———
by SAR
Me
O
O
2a
2b
Total synthesis
Scheme 1. Preparation of dioncoquinone B (1) by isolation and by total
synthesis via the epoxide 2a, and structure of its optimized analog 2b.
*
Corresponding author. Tel.: +49-931-318-5323; fax: +49-931-318-4755; e-mail: bringman@chemie.uni-wuerzburg.de (G. Bringmann)

2
Tetrahedron
ACCEPTED MANUSCRIPT
Table 1. Synthesis of epoxides 2a-h from their respective naphthoquinone precursors 3a-h under basic conditions.
X1
X1
O
4
O
4
5
X2
X2
X3
5
6
6
H2O2, base
solvent
O
O
4
7
2
1
X3
7
2
Me
Me
1
O
O
3a-h
2a-h
Starting
material
Entry
Product
X1
X2
X3
Reaction conditions
Yield (%)
1
2
3a11
3b
2a11
2b11
OMe
H
OMe
OMe
H
H
K2CO3, THF
K2CO3, THF
97
99
3
4
5
6
3c25
3d11
3e
2c24
2d11
2e
H
H
H
H
Me
OH
H
H
H
NaOH, MeOH/H2O
K2CO3, THF
93
92
72
87
CO2Me
OMe
NaOH, MeOH/H2O
K2CO3, THF
3f11
2f11
OMe
7
8
3g11
3h24
2g11
2h
OMe
OMe
OMe
Cl
OMe
H
NaOH, MeOH/H2O
NaOH, MeOH/H2O
99
71
In the course of these investigations, we also tested the anti-
standard therapy of MM.26,27 Epoxide 2e possessing a carboxylate
function at C-6, was found to be the most potent compound (EC50
= 0.6 µM) – yet combined with an even stronger cytotoxicity
against the non-malignant PMBCs (EC50 = 0.4 µM). The overall
best anti-MM activity was displayed by 2b, with an EC50 value of
3.5 µM, while lacking any cytotoxic effects in normal PMBCs.
In total, the structure-activity relationship (SAR) studies
showed that for a good anti-MM activity (and low cytotoxicity),
C-5 and C-7 should be unsubstituted, and C-6 should bear an
alkoxy group, and there should be no free hydroxy functions.
This SAR profile clearly distinguished the epoxide of the general
structure 2 from naphthoquinones of type 1, where we had
previously found that there should be free OH groups at C-3, C-
5, and/or C-6, evidencing that such epoxides 2 follow a clearly
different mode of action as compared to the previously described
naphthoquinones.
MM potential of selected synthetic precursors, among them the
intermediate epoxide 2a, which can easily be prepared from the
respective naphthoquinone precursor and can then be converted
into the target molecule 1 under acidic conditions (Scheme 1).
These investigations revealed that 2a and some related epoxides
of the general structure 2 showed a distinctly better activity than
the naphthoquinone 1, combined with a significantly reduced
cytotoxicity.
In this paper, we report on the synthesis of 2a and on the
SAR-guided preparation of a whole series of related epoxides of
type 2. Among them, the oxygen-poorer analog 2b was found to
induce strong apoptosis in MM cell lines at a concentration (3.5
µM), lower than that of any of the tested dioncoquinones and
their related synthetic analogs.10,11
We further describe the labeling of 2b by biotinylation for the
identification of the target protein and ensuing mass
spectrometry-based capture experiments aiming at the
identification of the target proteins.
Table 2. EC50 values (µM) of INA-6 multiple myeloma cells and
peripheral mononuclear blood cells (PMBCs) treated with epoxides 2a-h.
EC50 (µM)
2. Results and discussion
Entry
Compound
INA-6
PBMC
2.1. Synthesis and biotesting of the epoxides
1
2
2a
2b
2.8
3.5
7.4
The synthesis of the required naphthoquinones 3a, 3c, 3d, 3f-
3h was done according to known experimental procedures11,24,25
or, in the case of 3b and 3e, to analogous protocols, following in
principle three different approaches, either by a Stobbe strategy,
via a Grignard route, or by a Diels-Alder pathway.
NR
3
4
5
6
7
8
2c
2d
2e
2f
2.0
13.0
0.6
2.0
37.0
0.4
Likewise in analogy to previous protocols,11,24 epoxidation of
the naphthoquinones 3a-h succeeded by using hydrogen peroxide
under different optimized conditions, nearly all of the compounds
were obtained in very good yields and purity (Table 1).
5.0
11.0
3.8
2g
2h
2.2
3.0
4.5
The epoxides were tested for their growth-retarding activity
against MM cells according to a protocol established earlier,10-14
by determination of the viability of INA-6 cells 3 d after
treatment. The results (Table 2) showed that all of the epoxides
(except for compound 2d, which was the only one with an EC50
value of > 10 µM) exhibited significant antitumoral activities
against INA-6 cells in a low micromolar range, similar to that of
the DNA-alkylating agent melphalan, which is utilized in the
9
1
11.0
2
NR
3
10
melphalan
NR: not reached.
a Activity against the multiple myeloma cell line INA-6.
b Cytotoxicity against normal peripheral mononuclear blood cells (PBMC).
2.2. Biotinylation of the epoxide 2b

2b
The high anti-MM activity of the epoxides, in particular of 2b,
made it rewarding to launch first investigations on the mode of
action.
ACCEPTED MANUSCRIPT
Biotinylation
O
O
O
Despite the growing number of successful examples,
identification of molecular targets still remains a complex
challenge.28-30 One of the most powerful and frequently used
methods is the derivatization of a bioactive molecule to a
pulldown probe to determine the cellular binding partners by a
protein affinity isolation combined with mass-spectrometric
analysis of the captured proteins. Biotin as such an efficient
affinity tag, which enables a target enrichment utilizing the
strong non-covalent interaction with streptavidin,29,31 was chosen
for the labeling of the anti-MM active epoxide 2b.
S
6
O
( )
n
( )
3
N
H
N
H
O
H
H
N
N
Me
HN
NH
Biotin
Linker
AntiꢀMM active
epoxide
O
4a: n = 1
4b: n = 3
Scheme 2. General approach to a biotinylated derivative of the epoxide
2d.
The design of a functionalized derivative of 2b (Scheme 2)
was planned to be based on the attachment of biotin to the
known11 epoxide 2d through a triazol linker, by applying the
Cu(I)-catalyzed Huisgen reaction (CuAAC, 'click reaction').32-35
This mild method seemed to be well suited in order to avoid any
ring opening reaction of the epoxide function of 2d, although
epoxides of type 2 had previously been found to be relatively
stable towards nucleophiles.36
As the site for the attachment of a linker-tethered biotin
molecule, the oxygen function at C-6 was addressed because the
above-described structure-activity studies had shown that this
position should bear an alkoxy function, as given in the tethered
compound 4. This seemed better than any derivatization at C-5 or
C-7. A molecule of type 4 should also permit easy variation of
the chain length within the central linker part.
to the monoether 10 in acceptable yields (Scheme 3), but the
following substitution of the remaining bromine by inorganic
azide unfortunately resulted in a ring cleavage of the epoxide
function. For this reason, the dibromide 8 was first substituted
with azide to give a mixture of the desired 1-azido-6-bromo-
hexane along with unreacted 8. This mixture was directly
submitted to the etherification of 2d, which succeeded without
ring cleavage reaction, leading to the mixture of the desired
azido-functionalized epoxide 9 and the undesired bromide 10.
Despite the unsatisfying yield thus overall obtained, this
sequence was acceptable due to the small quantity of labeled
compound 4 finally needed. The copper(I)-catalyzed [3+2]
cycloaddition of 9 with the different biotin alkynes 7a and 7b
gave the functionalized epoxides 4a and 4b, respectively
(Scheme 3).
As starting material for the click reaction, the biotin residue
was chosen to contain the alkyne part, while the bioactive
epoxide was planned to constitute the azide part. The preparation
of the known alkyne 7a (Scheme 3) was achieved according to a
established procedure from literature.37 The synthesis of the new
analog 7b, bearing a longer chain, followed the same protocol.
Desymmetrization of the commercially available 1,6-
dibromohexane (8) by reaction with the phenol function of 2d led
2.3. Synthesis of shorter, less functionalized analogs of 4
In addition to the biotinylated analogs 4a and 4b, we also
prepared biotin-free, less functionalized derivatives, to make sure
that the linker had no influence on the anti-MM activity. Because
of the increased chain length, with a simultaneously larger
number of functional groups in 4 as compared to the epoxide 2b,
analogs of types 12 and 14 were synthesized (Scheme 4). The
O
Br
S
Br
( )
n
O
N
H2N
H
H
8
H
6a: n = 1
6b: n = 3
1. NaN3, DMF
2. Cs2CO3, 2d,
acetone
Cs2CO3, 2d,
acetone
HN
NH
5
37
O
lit.
71%
21%
71%
(
O
O
O
O
S
( )
O
O
(
n
(
(
2
N
Br
N3
2
H
H
O
O
H
H
Me
Me
7a: n = 1
7b: n = 3
HN
NH
9
10
O
O
NaN3, DMF
CuSO4ꢁ5H2O,
NaAsc,
CH2Cl2/H2O
n = 1 27 %
n = 3 31 %
O
O
O
S
( )
O
n
N
H
N
H
H
O
H
N
N
Me
HN
NH
4a: n = 1
4b: n = 3
O
Scheme 3. Synthesis of the azido-functionalized epoxide 9 and its click reaction with the biotin alkynes 7a and 7b to give the target molecules 4a and
4b, respectively.

4
Tetrahedron
ACCEPTED MANUSCRIPT
CuSO4ꢁ5H2O,
O
O
O
( )
O
( )
O
n
Me
n
NaAsc, CH2Cl2/H2O
Me
N3
N
O
O
N
N
n = 1 24 %
n = 2 17 %
11a: n = 1
11b: n = 2
Me
12a: n = 1
12b: n = 2
Me
9
O
O
O
O
O
CuSO4ꢁ5H2O
NaAsc, CH2Cl2/H2O
O
N3
( )
( )n
O
n
N
H
Me
N
H
N
O
Me
O
n = 1 33 %
n = 3 22 %
N
N
Me
13a: n = 1
13b: n = 3
9
Me
14a: n = 1
14b: n = 3
O
O
Scheme 4. Analogous synthesis of biotin-free derivatives of 2b.
Table 3. EC50 values (µM) of INA-6 multiple myeloma cells and
peripheral mononuclear blood cells (PMBCs) treated with the
biotinylated derivatives 4a/b, or with their biotin-free derivatives 9, 10,
12a/b, and 14a/b.
commercially available alkynes 11a and 11b and the likewise
known38 alkynyl acetamides 13a and 13b were submitted to click
reactions with 10 applying the same conditions as for the
preparation of the target molecules 4, leading to similar yields.
EC50 (µM)
Entry
Compound
2.4. Antitumoral activities and drug target identification
INA-6
PBMC
In contrast to the highly active epoxide 2b, its biotinylated
analogs 4a and 4b showed no anti-MM activity anymore (Table
3), probably as a consequence of decreased cell permeability due
to the large lipophilicity or the increased molecular size.29,31 This
assumption was confirmed by the already decreased activity of
the synthesized smaller, biotin-free analogs 12 and 14 (Figure 1).
The azido and bromo derivatives 9 and 10 showed good anti-MM
activity, with EC50 values of 3.3 µM and 7.2 µM respectively,
similar to that of the epoxide 2b, yet accompanied by an
increased cytotoxicity towards PMBCs. The more lipophilic
derivatives 12a and 12b, by contrast, exerted their anti-MM
activities at higher concentrations only. The closer analogs 14a
and 14b, with only biotin missing, and the labeled compounds 4a
and 4b themselves did not even reach measurable EC50 values in
the whole-cell assay.
1
2
2b
9
3.5
3.3
NR
5.5
3
4
5
6
7
10
7.2
16.2
25.9
NR
6.8
NR
NR
NR
NR
12a
12b
14a
14b
NR
8
9
4a
4b
NR
NR
NR
NR
NR: not reached.
a Activity against the multiple myeloma cell line INA-6.
b Cytotoxicity against normal peripheral mononuclear blood cells (PBMC).
100
100
100
75
50
75
50
75
50
25
0
25
0
25
0
0
1
2
0
1
2
3
4
5
6
0
1
2
3
5
6
8
9
3
4
5
6
7
8
9
10
4
7
10 12
11
9 (mM)
2b (mM)
10 (mM)
100
100
100
75
50
75
50
75
50
25
0
25
0
25
0
0
10
0
1
20
30
40
50
2
3
4
5
6
7
8
9
10
0
5
15
4b (mM)
20
10
25
12b (mM)
14b (mM)
Figure 1. Anti-MM effect of the highly active epoxide 2b, the derivatives 9, 10, 12b and 14b, and the biotinylated epoxide 4b (data of 12a and 14a are
not shown). INA-6 multiple myeloma cells were treated either with DMSO as the solvent control or with increasing concentrations of 2b, 9, 10, 12b,
14b, and 4b for 3 d. The viable fractions of the treated cells were determined by annexin V-FITC/PI staining using flow cytometry. Shown are mean
values and standard deviations of three independent experiments.

ACCEPTED MANUSCRIPT
Figure 2. Identification of potential protein targets from the affinity capture experiment with 4b and INA-6 cell lysate (for 4a not shown). Proteins
significantly enriched over the control are represented by closed circles. Outliers are shown in green, extreme outliers in red. Protein ratios and
intensities are median values from three biological replicates (n replicates >1). The size of the circles correlates with the number of identified razor and
unique peptides. Carboxylases were enriched specifically with the streptavidin control beads as they have biotin as the prosthetic group.
These results were not discouraging, because they only
showed that the biotinylated molecules 4a and 4b were not active
in the whole-cell test system, certainly due to the biotin residue,
which should prevent the permeation through the cell
membrane.29,31 Thus, it seemed obvious that the mechanistic
investigations should inevitably be performed in cell lysate.
For the target identification, the biotinylated compounds 4a
and 4b were subjected to mass spectrometry-based affinity
capture experiments. The magnetic bead preparation, cell lysis,
drug target enrichment and in-solution digestion followed
previously published experimental procedures.14 Therefore, the
biotinylated inhibitors 4a and 4b loaded on streptavidin magnetic
beads and empty beads as the control were incubated with INA-6
cell lysate under the same condition (3 h at 4 °C). After washing
the precipitate, the captured proteins were eluted with lithium
dodecyl sulfate (LDS) sample buffer, precipitated with acetone,
and digested in solution with trypsin. The proteins were analyzed
by nanoLC-MS/MS and label-free quantification (LFQ). To
achieve reliable results, three biological replicates of each biotin-
labeled compound were investigated.
The data set showed a clear enrichment of several proteins
(Figure 2). Of note, the proteins targeted by the biotinylated
epoxides 4a and 4b were similar to each other. One of the most
prominent hits was peroxiredoxin 1 (PRDX1), a redox‐regulating
protein that has been reported to eliminate reactive oxygen
species (ROS) and regulate cell growth, differentiation, and
apoptosis.38 PRDX1 has been found to be overexpressed in
human cancer including MM, and to functionally play a tumor-
promoting role in progression and metastasis.39-41 Thus, PRDX1
mediates anti‐apoptotic effects through interactions with
several ROS‐dependent effectors such as ASK1 and JNK. In
addition, PRDX1 can associate to key cancer transcription factors
such as p53, c-Myc and NF‐κB and can modulate their anti-
apoptotic activity.38 Another significantly enriched protein,
thioredoxin domain-containing 5 (TXNDC5), is a recently
discovered endoplasmic reticulum (ER) resident disulfide
isomerase, which has been implicated in oxidative-stress
associated diseases including cancer. Thus, TXNDC5 acts as a
chaperon-mediating protein folding and correct formation of
disulfide bonds through its thioredoxin domains.42 Recently,
TXNDC5 has been shown to act as a super-enhancer to
dysregulate c-Myc expression, and thus contributing to tumor
progression in MM.43 Like TXNDC5, the protein disulfide
isomerase A6 (PDIA6) acts as an ER-resident chaperon, co-
regulates UPR signaling, and thus contributes to protein
homeostasis.44,45 A recent study has identified a group of protein
homeostasis-relevant genes including PDIA6 which are
significantly regulated in therapy-resistant MM cells
underscoring the critical role of ER-stress associated processes in
MM.46 Accordingly, further ER-associated proteins were found
to be enriched: i) thioredoxin-related transmembrane protein 1
(TMX1), another ER-resident PDI family member, which
catalyzes protein folding and thiol-disulfide interchange reactions
and which is known to be involved in ER stress regulation, and
ii) Sec23B and SEC24C (Sec23 homolog B, coat complex II
component and SEC24 homolog C, COPII coat complex
component), both are components of the coat protein complex II
(COPII) and promote formation of transport vesicles from the
ER.47 Although all these enriched proteins indicate a functional
link of compound 2b to ER stress response mechanisms, which
are considered to be critical for tumor cell survival in MM,
further investigation on the potential target is needed and will be
reported in due course.48

6
Tetrahedron
ACCEPTED MANUSCRIPT
3. Experimental section
portion of KMnO4 (840 mg, 5.32 mmol, 2.0 equiv) was added.
After 0.5 h the reaction mixture was cooled to room temperature
and 1N aqueous NaOH was added. The solution was filtered over
Celite and the solvent was concentrated in vacuo. The crude
product was purified by column chromatography (n-
hexane/EtOAc 10:1) to give the naphthoic acid 15 (403 mg, 2.16
mmol, 81%) as a white solid; mp 225−227 °C; IR (ATR) vmax
3400 (m), 2980 (w), 2916 (w), 2830 (w), 1670 (s), 1574 (m),
1475 (m), 1415 (w), 1383 (m), 1290 (m), 1244 (m), 1194 (m),
1099 (m), 958 (w), 906 (w), 877 (w), 800 (m), 764 (m), 712 (m),
3.1. General experimental procedures
All melting points were measured on a Reichert Kofler hot-
stage microscope and are uncorrected. IR spectra were recorded
on a Jasco FT/IR-410 spectrometer by the ATR technique. The
signal intensity is assigned using the following abbreviations: s
(strong), m (medium), w (weak). 1H and 13C spectra were
recorded at room temperature on a Bruker Avance-400
1
instrument (400 MHz for H and 100 MHz for 13C). They are
1
637 (m) cm-1; H NMR (400 MHz, CDCl3) δ 8.66 (s, 1H, Har),
internally referenced to residual solvent signals and are expressed
in parts per million (ppm). All coupling constants (J) are reported
in Hertz (Hz) and the following notations indicate the
multiplicity of the signals: b (broad), s (singlet), d (doublet), dd
(doublet of doublet), t (triplet), q (quartet), m (multiplet). MS and
HRMS measurements were performed on a Finnigan MAT-8200
instrument (EI) and a Bruker Daltonics micrOTOF-mass
spectrometer (ESI). Thin-layer chromatography was performed
on Merck precoated silica gel aluminum plates (60 F254) and
visualized by exposure to ultraviolet light at 254 nm and 365 nm
or by staining with a potassium permanganate solution. Column
chromatography was performed using silica gel (0.063 mm,
Machery-Nagel). All solvents were either purchased from
commercial suppliers or purified by standard techniques.
Reactions sensitive to air or moisture were carried out in flame-
dried glassware under nitrogen using standard Schlenk
techniques. 1-Pentyne and 1-hexyne were purchased from
commercial suppliers. The syntheses of the biotin alkynes and
acetamides were carried out according to published
procedures.37,38 The naphthoquinones en route to the epoxides 3a,
3c, 3d, 3f-h and the epoxides 2a, 2c, 2d, 2f, 2g were prepared as
reported in earlier papers.11,24,25
8.08 (dd, J = 8.7, 1.2 Hz, 1H, Har), 7.88 (d, J = 8.5 Hz, 1H, Har),
7.82 (d, J = 8.7 Hz, 1H, Har), 7.67 (s, 1H, Har), 7.40 (dd, J = 8.5,
1.2 Hz, 1H, Har), 2.56 (s, 3H, 2-CH3) ppm; 13C NMR (100 MHz,
CDCl3) δ 171.4 (CO2H); 139.0 (Car), 136.4 (Car), 132.0 (Car),
130.8 (Car), 129.5 (Car), 129.2 (Car), 127.8 (Car), 127.0 (Car),
125.7 (Car), 125.7 (Car), 22.1 (2-CH3) ppm; MS (EI, 70 eV) m/z
(rel int) 186 [M]+ (100), 169 [M-OH]+ (30), 141 [M-CO2H]+ (57);
HRMS (ESI) calcd for C12H10O2Na [M+Na]+ 209.0573, found
209.0572.
3.2.2.2. Methyl-6-methyl-2-naphthoate (16)
K2CO3 (162 mg, 1.18 mmol, 3.0 equiv) was added to a
solution of 6-methyl-2-naphthoic acid (15) (73.0 mg, 392 ꢀmol,
1.0 equiv) in acetone (10 mL) at 0 °C. After stirring the mixture
for 30 min, dimethyl sulfate (83.8 mg, 1.22 mmol, 63 ꢀL, 3.1
equiv) was added and the solution was refluxed for 1 h. The
reaction mixture was cooled down to room temperature and H2O
was added. After evaporation of acetone, the remaining aqueous
mixture was extracted with CH2Cl2. The combined organic layers
were dried over Na2SO4, concentrated in vacuo and the residue
was resolved by column chromatography (n-hexane/EtOAc 10:1)
to give the ester 16 (66.0 mg, 330 ꢀmol, 84%) as a colorless
solid; mp 119-120 °C; IR (ATR) vmax 2954 (w), 2926 (w), 2851
(w), 1707 (s), 1628 (w), 1473 (w), 1435 (m), 1336 (w), 1286 (s),
1190 (s), 1124 (m), 1093 (s), 972 (m), 897 (m), 843 (w), 810 (s),
3.2. Synthesis of the naphthoquinones for epoxides 3b and 3e
3.2.1. 6-Methoxy-2-methyl-1,4-naphthoquinone (3b)
1
777 (m), 750 (m), 723 (w) cm-1; H NMR (400 MHz, CDCl3) δ
K2CO3 (20.6 mg, 149 ꢀmol, 5.0 equiv) was added to a
solution of 6-hydroxy-2-methyl-1,4-naphthoquinone (5.6 mg,
29.8 ꢀmol, 1.0 equiv) in acetone (2 mL) at 0 ºC. After stirring the
mixture for 10 min, dimethyl sulfate (18.8 mg, 149 ꢀmol, 14.1
ꢀL, 5.0 equiv) was added and the reaction solution was stirred for
3 h. Then water was added and the mixture was neutralized with
1N aqueous HCl and extracted with CH2Cl2. The combined
organic layers were dried over Na2SO4, concentrated in vacuo
and the residue was resolved by column chromatography (n-
hexane/EtOAc 10:1) to give the naphthoquinone 3b (5.9 mg, 29.2
µmol, 98%) as a yellow solid; mp 142 °C; IR (ATR) vmax 2924
(w), 2851 (w), 1661 (m), 1592 (s), 1438 (w), 1361 (m), 1305 (s),
1243 (m), 1076 (m), 1027 (m), 946 (w), 896 (w), 850 (w) cm-1;
1H NMR (400 MHz, CDCl3) δ 8.04 (d, J = 8.6 Hz, 1H, Har), 7.49
(d, J = 2.7 Hz, 1H, Har), 7.18 (dd, J = 8.6, 2.7 Hz, 1H, Har), 6.80
(q, J = 1.5 Hz, 1H, CH), 3.94 (s, 3H, OCH3), 2.18 (d, J = 1.5 Hz,
3H, 2-CH3) ppm; 13C NMR (100 MHz, CDCl3) δ 185.3 (C=O),
184.8 (C=O), 164.1 (C-6), 148.7 (C-2), 135.4 (C-3), 134.5 (Car),
129.2 (Car), 125.9 (Car), 120.4 (Car), 109.4 (Car), 56.1 (OCH3),
16.7 (2-CH3) ppm; MS (EI, 70 eV) m/z (rel int.) 202 [M]+ (100),
174 [M-CO]+ (22), 134 (26); HRMS (ESI) calcd for C12H11O3
[M+H]+ 203.0703, found 203.0702.
8.57 (s, 1H, Har), 8.02 (dd, J = 8.6, 1.7 Hz, 1H, Har), 7.85 (d, J =
8.4 Hz, 1H, Har), 7.79 (d, J = 8.6 Hz, 1H, Har), 7.65 (s, 1H, Har),
7.38 (dd, J = 8.4, 1.7 Hz, 1H, Har), 3.97 (s, 3H, OCH3), 2.54 (s,
3H, 2-CH3) ppm; 13C NMR (100 MHz, CDCl3) δ 167.6 (CO2Me),
138.5 (Car), 135.9 (Car), 131.0 (Car), 130.9 (Car), 129.3 (Car),
129.1 (Car), 127.6 (Car), 126.9 (Car), 126.7 (Car), 125.5 (Car), 52.3
(OCH3), 22.1 (2-CH3) ppm; MS (EI, 70 eV): m/z (rel int) 200
[M]+ (78), 169 [M-OCH3]+ (100), 141 (32); HRMS (ESI) calcd
for C13H12O2Na [M+Na]+ 223.0730, found 223.0730.
3.2.2.3. Methyl -2-methyl-1,4-naphthoquinone-6-carboxylate (3e)
To a mixture of H5IO6 (299 mg, 1.31 mmol, 4.0 equiv) and
CrO3 (3.3 mg, 33.0 ꢀmol, 0.1 equiv) in MeCN (7 mL), 16 (66.0
mg, 330 ꢀmol, 1.0 equiv) in MeCN (7 mL) was added dropwise.
After stirring at 0 ºC for 4 h, the reaction mixture was filtered
over a short pad of Celite and washed with CH2Cl2. The solvent
was concentrated in vacuo and the residue was resolved by
column chromatography (n-hexane/EtOAc 97:3) to give the
naphthoquinone 3e (30.0 mg, 130 ꢀmol, 40%) as a yellow solid;
mp149-152 °C; IR (ATR) vmax 2950 (w), 2929 (w), 2852 (w),
1716 (s), 1660 (s), 1603 (m), 1439 (m), 1356 (w), 1259 (s), 1120
(m), 972 (m), 939 (m), 912 (m), 874 (m), 766 (m), 688 (m) cm-1;
1H NMR (400 MHz, CDCl3) δ 8.70 (dd, J = 1.7, 0.5 Hz, 1H, Har),
8.37 (dd, J = 8.1, 1.7 Hz, 1H, Har), 8.18 (dd, J = 8.1 Hz, 0.5 Hz,
1H, Har), 6.91 (q, J = 1.6 Hz, 1H, Har), 3.98 (s, 3H, OCH3), 2.22
(d, J = 1.6 Hz, 3H, 2-CH3) ppm; 13C NMR (100 MHz, CDCl3) δ
185.1 (C=O), 184.2 (C=O), 165.6 (CO2Me), 148.6 (Car), 136.2
3.2.2. Methyl-6-carboxylate-2-methyl-1,4-naphthoquinone (3e)
3.2.2.1. 6-Methyl-2-naphthoic acid (15)
A mixture of 2,6-dimethylnaphthalene (416 mg, 2.66 mmol,
1.0 equiv) and KMnO4 (2.10 g, 13.3 mmol, 5.0 equiv) in pyridine
(35 mL) and water (12 mL) was refluxed for 2 h. Then another

(Car), 134.9 (Car), 134.8 (Car), 134.3 (Car), 132.4 (Car), 127.7 (Car),
3.3.5.
epoxide (2h)
6-Chloro-5-methoxy-2-methyl-1,4-naphthoquinone-2,3-
ACCEPTED MANUSCRIPT
127.0 (Car), 52.9 (OCH3), 16.7 (2-CH3) ppm; MS (EI, 70 eV) m/z
(rel int) 230 [M]+ (100), 199 [M-OCH3]+ (95), 171 (47); HRMS
(ESI) calcd for C13H11O4 [M+H]+ 231.0652, found 231.0645.
According to the general procedure 3.3.2., compound 2h was
synthesized starting from the naphthoquinone 3h24 (41.0 mg, 173
µmol, 1.0 equiv). The residue was purified by column
chromatography (n-hexane/EtOAc 9:1) to give the epoxide 2h
(29.0 mg, 122 µmol, 71%) as a yellow solid; mp 85 °C; IR
(ATR) vmax 2923 (w), 2853 (w), 1699 (s), 1572 (m), 1467 (m),
1404 (m), 1330 (m), 1289 (m), 1240 (s), 1141 (m), 1075 (m),
1029 (m), 965 (s), 851 (m), 750 (m), 717 (s) cm-1; 1H NMR (400
MHz, CDCl3) 7.71 (s, 1H, Har), 7.70 (s, 1H, Har), 3.97 (s, 3H,
OCH3), 3.86 (s, 1H, CH), 1.72 (s, 3H, 2-CH3) ppm; 13C NMR
(100 MHz, CDCl3) 191.8 (C=O), 190.8 (C=O), 155.5 (C-5),
136.6 (Car), 135.5 (Car), 132.4 (Car), 127.0 (Car), 123.9 (Car), 62.8
(OCH3), 62.0 (C-2), 61.7 (C-3), 14.6 (2-CH3) ppm; MS (EI, 70
eV) m/z (rel int) 254 [M]+ (33), 252 [M]+ (91), 239 [M-CH3]+
(17), 237 [M-CH3]+ (52), 211 (36), 209 (100); HRMS (ESI) calcd
for C12H9ClNaO4 [M+Na]+ 275.0082, found 275.0081.
3.3. General procedure for the epoxidation of the
naphthoquinones
3.3.1. Epoxidation with H2O2 and K2CO3 in THF
Aqueous H2O2 (30%, 90 equiv) was added with stirring to a
mixture of the naphthoquinone (1.0 equiv) in THF (25 µL µmol-
1) at room temperature. After addition of 1N aqueous Na2CO3
until the mixture had turned colorless, the reaction solution was
stirred for 3 h at room temperature. Saturated aqueous NaCl was
added and the mixture was extracted with EtOAc. The combined
organic layers were dried over Na2SO4 and concentrated in
vacuo.
3.3.2. Epoxidation by H2O2 and NaOH in MeOH/H2O
To a mixture of naphthoquinone (1.0 equiv) in H2O/MeOH
(1:4, 50 µL/µmol) 1N aqueous NaOH solution (0.5 equiv) was
added at room temperature. After the mixture was stirred for 10
minutes, aqueous H2O2 solution (30%, 1.5 equiv) was added and
stirred for 3 h. The mixture was neutralized with 1N aqueous HCl
and extracted with Et2O. The combined organic layers were dried
over Na2SO4, and the solvent was removed in vacuo.
3.4. Synthesis of the biotin-labeled analogs 4a/b and biotin-free
compounds 12a/b and 14a/b by 'click reaction'
3.4.1. Biotinamido-4-pentyne (7b)
To a solution of 537 (100 mg, 320 µmol. 1.0 equiv.) in DMF (8
mL), amino alkyne 6b49 (53.2 mg, 640 µmol, 2.0 equiv.) and
NEt3 (160 mg, 219 µL, 1.6 mmol, 5.0 equiv.) was added. After
stirring for 14 h the reaction mixture was concentrated in vacuo.
Purification by column chromatography (CH2Cl2/MeOH 8:1)
3.3.3. 6-Methoxy-2-methyl-1,4-naphthoquinone-2,3-epoxide (2b)
According to the general procedure 3.3.1., compound 2b was
synthesized starting from the naphthoquinone 3b (6.0 mg, 29.7
µmol, 1.0 equiv). Purification by column chromatography (n-
hexane/EtOAc 10:1) gave the epoxide 2b (6.4 mg, 29.4 µmol,
99%) as yellow solid; mp 89 °C; IR (ATR) vmax 1684 (s), 1592
(s), 1494 (m), 1433 (m), 1294 (s), 1232 (s), 1192 (m), 1057 (m),
1026 (m), 948 (m), 856 (s), 780 (m), 740 (s) cm-1; 1H NMR (400
MHz, CDCl3) δ 7.99 (d, J = 8.7 Hz, 1H, Har), 7.38 (d, J = 2.7 Hz,
1H, Har), 7.22 (dd, J = 8.7, 2.7 Hz, 1H, Har), 3.92 (s, 3H, OCH3),
3.84 (s, 1H, CH), 1.72 (s, 3H, 2-CH3) ppm; 13C NMR (100 MHz,
CDCl3) δ 192.0 (C=O), 190.8 (C=O), 164.7 (C-6), 134.2 (Car),
130.1 (Car), 125.4 (Car), 121.7 (Car), 109.9 (Car), 61.5 (C-2), 61.4
(C-3), 56.1 (OCH3), 14.9 (2-CH3) ppm; MS (EI, 70 eV) m/z (rel
int) 218 [M]+ (100), 203 [M-CH3]+ (82), 119 (51); HRMS (ESI)
calcd for C12H10NaO4 [M+Na]+ 241.0471, found 241.0470.
gave the biotin alkyne 7b (82.5 mg, 270 µmol, 84%) as a
20
D
colorless solid; mp 124 °C (CH2Cl2/MeOH); [α]
+50.9
(MeOH; c 0.1); IR (ATR) vmax 3288 (w), 2927 (w), 2853 €, 1696
(s), 1637 (s), 1546 (m), 1460 (m), 1426 (m), 1323 (m), 1265 (m),
1210 (m), 1153 (w), 1074 (m), 1044 (w), 997 (w), 939 (w), 857
1
(w), 815 (w), 758 (m), 713 (m), 648 (s) cm-1; H NMR (400
MHz, CDCl3) δ 4.49 (dd, J = 3.9, 3.1 Hz, 1H, NHCH), 4.30 (dd,
J = 7.9, 4.4 Hz, 1H, NHCH), 3.27 (t, J = 6.9 Hz, 2H, NHCH2),
3.24 – 3.17 (m, 1H, SCH), 2.93 (dd, J = 12.8, 5.0 Hz, 1H, SCH2),
2.71 (d, J = 12.8 Hz, 1H, SCH2), 2.27 – 2.14 (m, 5H, C=OCH2,
CH, CH2), 1.80 – 1.55 (m, 6H, CH2), 1.49 – 1.39 (m, 2H, CH2)
ppm; 13C NMR (100 MHz, CDCl3) δ 176.2 (NHC=O), 166.1
(((NH)2C=O), 84.1 (Cq), 70.0 (CH), 63.4 (NHCH), 61.6 (NHCH),
57.0 (SCH), 41.0 (SCH2), 39.4 (NHCH2), 36.8 (CH2C=O), 29.8
(CH2), 29.5 (CH2), 29.4 (CH2), 26.9 (CH2), 26.3 (CH2) ppm;
HRMS (ESI) calcd for C15H23N3NaO2S [M+Na]+ 332.1403,
found 332.1405.
3.3.4. Methyl-2-methyl-1,4-naphthoquinone-2,3-epoxide-6-car-
boxylate (2e)
3.4.2. 1-Azido-6-bromohexane (17)
According to the general procedure 3.3.2., compound 2e was
synthesized starting from the naphthoquinone 3e (62.0 mg, 269
µmol, 1.0 equiv). The residue was purified by column
chromatography (n-hexane/EtOAc 9:1) to give the epoxide 2e
(48.0 mg, 193 µmol, 72%) as a yellow solid; mp 135 °C; IR
(ATR) vmax 2954 (w), 2929 (w), 1716 (s), 1691 (s), 1606 (m),
1444 (m), 1400 (w), 1338 (w), 1263 (s), 1184 (m), 1122 (m), 974
(w), 947 (m), 876 (w), 850 (w), 795 (w), 759 (m), 714 (s), 640
(w) cm-1; 1H NMR (400 MHz, CDCl3) δ 8.57 (d, J = 1.7 Hz, 1H,
Har), 8.36 (dd, J = 8.1, 1.7 Hz, 1H, Har), 8.07 (d, J = 8.1 Hz, 1H,
Har), 3.97 (s, 3H, OCH3), 3.90 (s, 1H, CH), 1.74 (s, 3H, 2-CH3)
ppm; 13C NMR (100 MHz, CDCl3) δ 191.6 (C=O), 191.1 (C=O),
165.2 (CO2Me), 135.6 (Car), 135.2 (Car), 135.0 (Car), 132.2 (Car),
128.3 (Car), 127.97 (Car), 61.9 (C-2), 61.6 (C-3), 53.0 (OCH3),
14.7 (2-CH3) ppm; MS (EI, 70 eV) m/z (rel int) 246 [M]+ (36),
231 [M-CH3]+ (100), 218 [M-CO]+ (87), 215 [M-OCH3]+ (35),
187 (27); HRMS (ESI) calcd for C13H11O5 [M+H]+ 247.0601,
found 247.0603.
To a solution of 1,6-dibromohexane (45.0 g, 185 mmol, 28.2
mL, 1.0 equiv) in DMF (51 mL) and H2O (12 mL), NaN3 (13.3 g,
204 mmol, 1.1 equiv) was added. After the reaction mixture was
heated at 60 °C for 28 h, the solution was cooled down and then
extracted with CH2Cl2. The combined organic layers were dried
over Na2SO4 and concentrated in vacuo to give the product 17
(37.5 g, 182 mmol, 98 %) as a colorless liquid; IR (ATR) vmax
2953 (m), 2860 (w), 2089 (s), 1724 (w), 1456 (m), 1348 (w),
1
1249 (s), 897 (w), 728 (m), 642 (m) cm-1; H NMR (400 MHz,
CDCl3) δ 3.40 (t, J = 6.8 Hz, 2H, CH2Br), 3.26 (t, J = 6.8 Hz, 2H,
CH2N3), 1.86 (m, 2H, CH2CH2Br), 1.62 (m, 2H, CH2CH2N3),
1.47 (m, 2H, CH2(CH2)2Br), 1.39 (m, 2H, CH2(CH2)2N3) ppm;
13C NMR (100 MHz, CDCl3) δ 51.4 (CH2N3), 33.8 (CH2Br), 32.6
(CH2CH2Br), 28.8 (CH2CH2N3), 27.4 (CH2(CH2)2Br), 26.4
(CH2(CH2)2N3) ppm; MS (EI, 70 eV) m/z (rel int) 163 [M-N3]+
(2), 83 [M-BrN3]+ (37), 55 (47), 41 (100).

8
Tetrahedron
ACCEPTED MANUSCRIPT
3.4.3. 6-[(6′-Bromohexyl)oxy]-2-methyl-1,4-naphthoquinone-2,3-
epoxide (10)
gel (CH2Cl2/MeOH 50:1→10:1) to give product 4a (8.34 mg,
13.7 µmol, 17 %) as a yellow oil; IR (ATR) vmax 2925 (m), 2854
(m), 2158 (w), 2027 (m), 1978 (m), 1699 (s), 1653 (s), 1593 (m),
1560 (m), 1543 (m), 1523 (m), 1508 (m), 1459 (m), 1289 (s),
1045 (m), 731 (m), 650 (w) cm-1; 1H NMR (400 MHz, MeOD) δ
7.96 (d, J = 8.7 Hz, 1H, Har), 7.85 (s, 1H, NCH), 7.36 (d, J = 2.7
Hz, 1H, Har), 7.29 (dd, J = 8.7, 2.7 Hz, 1H, Har), 4.48 (dd, J = 7.9,
4.4 Hz, 1H, NHCH), 4.43-4.38 (m, 4H, NCH2, NHCH2), 4.28
(dd, J = 7.9, 4.4 Hz, 1H, NHCH), 4.11 (t, J = 6.4 Hz, 2H, OCH2),
3.88 (s, 1H, CH), 3.21-3.15 (m, 1H, SCH), 2.92 (dd, J = 12.7, 4.9
Hz, 1H, SCH2), 2.72-2.66 (m, 3H, SCH2, CH2), 2.23 (t, J = 7.3
Hz, 2H, C=OCH2), 1.98-1.89 (m, 2H, CH2), 1.85-1.77 (m, 2H,
CH2), 1.75-1.66 (m, H, CH2), 1.65 (s, 3H, CH3), 1.55-1.52 (m,
2H, CH2), 1.45-1.35 (m, 4H, CH2) ppm; 13C NMR (100 MHz,
MeOD) δ 193.0 (C=O), 192.1 (C=O), 176.0 (NHC=O), 166.1 (C-
6), 165.4 ((NH)2C=O), 146.3 (CqCH2), 135.5 (Car), 130.7 (Car),
126.4 (Car), 124.3 (NCH), 122.1 (Car), 111.6 (Car), 69.7 (OCH2),
62.7 (C-2), 62.4 (NHCH), 62.3 (CH), 61.6 (NHCH), 57.0 (SCH),
51.3 (NCH2), 41.1 (SCH2), 36.5 (NHCH2), 35.6 (C=OCH2), 31.2
(CH2), 29.8 (CH2), 29.7 (CH2), 29.4 (CH2), 27.1 (CH2), 26.7
(CH2), 26.4 (CH2), 14.9 (2-CH3) ppm; MS (EI, 70 eV) m/z (rel
int) 551 (5), 523 (5), 367 (5), 97 (17), 71 (19), 69 (23), 57 (33);
HRMS (ESI) m/z calcd for C30H38N6NaO6S [M+Na]+ 633.2466,
found 633.2474.
Cs2CO3 (79.8 mg, 245 µmol, 5.0 equiv) was added with
stirring to a mixture of epoxide 2d11 (10.0 mg, 49.0 µmol, 1.0
equiv) in acetone (2 mL) at room temperature. After 8 (1.20 g,
750 µL, 4.90 mmol, 100 equiv) was added, the mixture was
stirred for 30 min at room temperature. Then the solution was
filtered through a short pad of celite and washed with EtOAc.
The solvent was removed in vacuo and the residue was purified
by column chromatography (n-hexane/EtOAc 250:1) to give
product 10 (12.8 mg, 34.8 µmol, 71 %) as a colorless oil; IR
(ATR) vmax 2927 (m), 2858 (w), 1738 (s), 1693 (s), 1597 (s),
1439 (w), 1366 (m), 1328 (m), 1297 (m), 1230 (s), 1059 (w), 949
(w), 748 (m) cm-1; 1H NMR (400 MHz, CDCl3) δ 7.98 (d, J = 8.7
Hz, 1H, Har), 7.35 (d, J = 2.6 Hz, 1H, Har), 7.20 (dd, J = 8.7, 2.6
Hz, 1H, Har), 4.16-4.01 (m, 2H, OCH2), 3.83 (s, 1H, CH), 3.43 (t,
J = 6.7 Hz, 2H, BrCH2), 1.97-1.77 (m, 4H, CH2), 1.72 (s, 3H,
CH3), 1.56-1.44 (m, 4H, CH2) ppm; 13C NMR (100 MHz, CDCl3)
δ 192.1 (C=O), 190.8 (C=O), 164.2 (Car), 134.2 (Car), 130.1 (Car),
125.3 (Car), 122.0 (Car), 110.4 (Car), 68.7 (CH2O), 61.6 (CqCH3),
61.4 (CH), 33.9 (CH2Br), 32.8 (CH2), 29.0 (CH2), 28.0 (CH2),
25.3 (CH2), 15.0 (CH3) ppm; MS (EI, 70 eV) m/z (rel int) 367
[M]+ (4), 69 [C5H9]+ (17), 57 (24); HRMS (ESI) calcd for
C17H19BrNaO4 [M+Na]+ 389.0359, found 389.0352.
3.4.4. 6-[(6′-Azidohexyl)oxy]-2-methyl-1,4-naphthoquinone-2,3-
epoxide (9)
3.4.7. Biotinylated epoxide 4b
According to the general procedure 3.6.3., compound 4b was
synthesized from azide 9 (23.3 mg, 70.7 µmol, 1.0 equiv) and 6b
(26.2 mg, 84.8 µmol, 1.2 equiv). The residue was purified by
column chromatography on deactivated (NH3, 7.5% w/w) silica
gel (CH2Cl2/MeOH 50:1→30:1) to give product 4b (14.2 mg,
22.2 µmol, 31 %) as an orange oil; IR (ATR) vmax 2925 (s), 2858
(m), 1697 (m), 1596 (w), 1459 (m), 1290 (m), 1127 (s), 1059 (s),
Cs2CO3 (2.71 g, 8.20 mmol, 5.0 equiv) was added with
stirring to a mixture of epoxide 2d11 (335 mg, 1.64 mmol, 1.0
equiv) in acetone (500 mL) at room temperature. After 17 (33.8
g, 164 mmol, 100 equiv) was added, the mixture was stirred for 4
h at room temperature. Then the solution was filtered through a
short pad of celite and washed with EtOAc. The solvent was
removed in vacuo and the residue was purified by column
chromatography (n-hexane/EtOAc 500:1) to give product 9 (112
mg, 0.34 mmol, 21 %) as a yellow oil; IR (ATR) vmax 2937 (w),
2861 (w), 2094 (m), 1691 (s), 1596 (s), 1495 (w), 1467 (w), 1438
(w), 1327 (m), 1295 (s), 1236 (m), 1195 (w), 1090 (w), 1060 (w),
1
896 (w), 817 (w) cm-1; H NMR (400 MHz, MeOD) δ 1.40-1.48
(m, 4H, CH2), 1.52-1.61 (m, 4H, CH2), 1.65 (s, 3H, 2-CH3), 1.66-
1.73 (m, 2H, CH2), 1.78-1.88 (m, 4H, CH2), 1.90-1.99 (m, 2H,
CH2), 2.20 (t, J = 7.3 Hz, 2H, C=OCH2), 2.67-2.74 (m, 3H,
SCH2, CH2), 2.91 (dd, J = 12.7, 5.0 Hz, 1H, SCH2), 3.20 (t, J =
6.9 Hz, 3H, NHCH2, SCH), 3.89 (s, 1H, CH), 4.11 (t, J = 6.3 Hz,
2H, OCH2), 4.29 (dd, J = 7.8, 4.5 Hz, 1H, NHCH), 4.39 (t, J =
7.0 Hz, 2H, NCH2), 4.48 (dd, J = 7.9, 4.2 Hz, 1H, NHCH), 7.29
(dd, J = 8.7, 2.7 Hz, 1H, Har), 7.37 (d, J = 2.6 Hz, 1H, Har), 7.77
(s, 1H, NCH), 7.97 (d, J = 8.7 Hz, 1H, Har) ppm; 13C NMR (100
MHz, MeOD) δ 14.9 (2-CH3), 23.6 (CH2), 26.4 (CH2), 26.9
(CH2), 27.1 (CH2), 29.5 (CH2), 29.8 (CH2), 30.2 (CH2), 30.7
(CH2), 31.2 (CH2), 36.8 (C=OCH2), 39.6 (NHCH2), 41.1 (SCH2),
51.2 (NCH2), 57.0 (SCH), 61.6 (NHCH), 62.3 (CH), 62.4
(NHCH), 62.7 (C-2), 69.7 (OCH2), 111.5 (Car), 122.1 (Car), 123.4
(NCH), 126.4 (Car), 130.7 (Car), 135.5 (Car), 147.0 (CqCH2), 164.6
((NH)2C=O), 165.4 (C-6), 176.1 (NHC=O), 191.7 (C=O), 193.0
(C=O) ppm; MS (EI, 70 eV) m/z (rel int) 180 (4), 98 (12), 57
(13), 55 (22); HRMS (ESI) m/z calcd for C32H42N6NaO6S
[M+Na]+ 661.2779, found 661.2788.
1
1016 (w), 948 (m), 879 (w), 856 (w), 783 (w), 742 (m) cm-1; H
NMR (400 MHz, CDCl3) δ 7.97 (d, J = 8.7 Hz, 1H, Har), 7.35 (d,
J = 2.6 Hz, 1H, Har), 7.20 (dd, J = 8.7, 2.6 Hz, 1H, Har), 4.11-4.04
(m, 2H, OCH2), 3.83 (s, 1H, CH), 3.29 (t, J = 6.8 Hz, 2H,
N3CH2), 1.87-1.79 (m, 2H, CH2), 1.71 (s, 3H, CH3), 1.68-1.58
(m, 2H, CH2), 1.54-1.42 (m, 4H, CH2) ppm; 13C NMR (100 MHz,
CDCl3) δ 192.1 (C=O), 190.8 (C=O), 164.2 (Car), 134.2 (Car),
130.1 (Car), 125.3 (Car), 122.0 (Car), 110.4 (Car), 68.7 (CH2O),
61.6 (C-2), 61.4 (C-3), 51.5 (CH2N3), 29.0 (CH2), 28.9 (CH2),
26.6 (CH2), 25.7 (CH2), 15.0 (2-CH3) ppm; MS (EI, 70 eV) m/z
(rel int) 329 [M]+ (4), 258 [M-C5H11]+ (5), 204 (19), 189 (53), 98
(70); HRMS (ESI) calcd for C17H19N3NaO4 [M+Na]+ 352.1268,
found 352.1264.
3.4.5. General procedure for the 'click reaction'
The reaction was carried out under light exclusion. To a
solution of azide 9 (1.0 equiv) in CH2Cl2/H2O (1:1, 170
µL/µmol) CuSO4ꢁ5H2O (1.0 equiv), sodium ascorbate (1.0 equiv)
and the respective alkyne in excess were added. After stirring for
2 d at room temperature, the solvent was evaporated.
3.4.8. Biotin-free derivative 12a
According to the general procedure 3.6.3., compound 12a was
synthesized from azide 9 (10.0 mg, 30.4 µmol, 1.0 equiv) and 1-
pentyne (690 mg, 10.1 mmol, 1.0 mL, 332 equiv). The residue
was purified by column chromatography on deactivated (NH3,
7.5% w/w) silica gel (CH2Cl2/MeOH 500:1→50:1) to give
product 12a (2.88 mg, 7.25 µmol, 24 %) as a yellow oil; IR
(ATR) vmax 2917 (s), 2852 (m), 1694 (m), 1596 (m), 1465 (s),
1289 (s), 1087 (w), 1058 (w), 737 (m) cm-1; 1H NMR (400 MHz,
MeOD) δ 7.96 (d, J = 8.7 Hz, 1H, Har), 7.73 (s, 1H, NCH), 7.36
3.4.6. Biotinylated epoxide 4a
According to the general procedure 3.6.3., compound 4a was
synthesized from azide 9 (27.0 mg, 82.0 µmol, 1.0 equiv) and
6a37 (27.7 mg, 98.4 µmol, 1.2 equiv). The residue was purified by
column chromatography on deactivated (NH3, 7.5% w/w) silica

According to the general procedure 3.6.3., compound 14b was
(d, J = 2.6 Hz, 1H, Har), 7.29 (dd, J = 8.7, 2.6 AHzC, 1CHE, HPaTr),E4.D38 MANUSCRIPT
(t, J = 7.0 Hz, 2H, NCH2), 4.11 (t, J = 6.3 Hz, 2H, OCH2), 3.88
(s, 1H, CH), 2.65 (t, J = 7.4 Hz, 2H, CqCH2), 1. 89-1.89 (m, 2H,
CH2), 1.86-1.77 (m, 2H, CH2), 1.72-1.65 (m, 2H, CH2), 1.65 (s,
3H, 2-CH3), 1.58-1.49 (m, 2H, CH2), 1.43-1.35 (m, 2H, CH2),
0.95 (t, J = 7.4 Hz, 3H, CH3) ppm; 13C NMR (100 MHz, MeOD)
δ 193.0 (C=O), 192.1 (C=O), 165.4 (C-6), 149.1 (CqCH2), 135.5
(Car), 130.7 (Car), 126.4 (Car), 123.2 (NCH), 122.1 (Car), 111.5
(Car), 69.7 (OCH2), 62.7 (CqCH3), 62.4 (CH), 51.1 (NCH2), 31.2
(CH2), 28.8 (CH2), 28.3 (CH2), 27.1 (CH2Cq), 26.4 (CH2), 23.8
(CH2), 14.9 (2-CH3), 14.0 (CH3) ppm; MS (EI, 70 eV) m/z (rel
int) 396 [M-H]+ (23), 381 [M-O]+ (14), 369 [M-CO]+ (21), 194
(19), 96 (26), 83 (42), 55 (84); HRMS (ESI) m/z calcd for
C22H27N3NaO4 M+Na]+ 420.1894, found 420.1888.
synthesized from azide 9 (23.3 mg, 70.7 µmol, 1.0 equiv) and
13b38 (10.6 mg, 84.8 µmol, 1.2 equiv). The residue was purified
by column chromatography on deactivated (NH3, 7.5% w/w)
silica gel (CH2Cl2/MeOH 500:1→100:1) to give product 14b
(7.19 mg, 15.8 µmol, 22 %) as a yellow oil; IR (ATR) vmax 2938
(w), 2862 (w), 1691 (s), 1592 (m), 1437 (w), 1371 (m), 1330 (m),
1230 (m), 1174 (m), 1058 (w), 856 (w), 742 (w), 613 (w) cm-1;
1H NMR (400 MHz, MeOD) δ 7.95 (d, J = 8.7 Hz, 1H, Har), 7.80
(s, 1H, NCH), 7.34 (d, J = 2.7 Hz, 1H, Har), 7.27 (dd, J = 8.7, 2.7
Hz, 1H, Har), 4.39 (t, J = 6.9 Hz, 2H, NCH2), 4.10 (t, J = 6.3 Hz,
2H, OCH2), 3.88 (s, 1H, CH), 3.73-3.66 (m, 2H, NHCH2), 2.72
(t, J = 7.4 Hz, 2H, CqCH2), 2.33 (s, 3H, CH3), 1.98-1.88 (m, 4H,
CH2), 1.85-1.77 (m, 2H, CH2), 1.64 (s, 3H, 2-CH3), 1.58-1.49 (m,
2H, CH2), 1.42-1.35 (m, 2H, CH2) ppm; 13C NMR (100 MHz,
MeOD) δ 193.0 (C=O), 192.1 (C=O), 175.2 (NHC=O), 165.4 (C-
6), 148.1 (CqCH2), 135.5 (Car), 130.7 (Car), 126.4 (Car), 123.4
(NCH), 122.1 (Car), 111.5 (Car), 69.6 (OCH2), 62.7 (CqCH3), 62.4
(CH), 51.2 (NCH2), 45.3 (NHCH2), 31.1 (CH2), 29.8 (CH2), 29.4
(CH2), 27.1 (CH2), 26.4 (CH3), 26.4 (CH2), 23.6 (CH2Cq), 14.9
(2-CH3) ppm; MS (EI, 70 eV) m/z (rel int) 83 (70), 55 (71), 41
(100); HRMS (ESI) m/z calcd for C24H31N4O5 [M+H]+ 455.2289,
found 455.2293.
3.4.9. Biotin-free derivative 12b
According to the general procedure 3.6.3., compound 12b was
synthesized from azide 9 (27.0 mg, 82.0 µmol, 1.0 equiv) and 1-
hexyne (720 mg, 1.0 mL, 8.77 mmol, 107 equiv). The residue
was purified by column chromatography on deactivated (NH3,
7.5% w/w) silica gel (CH2Cl2/MeOH 500:1→100:1) to give
product 12b (5.80 mg, 14.1 µmol, 17 %) as a yellow oil; IR
(ATR) vmax 2933 (m), 2860 (w), 1692 (s), 1596 (s), 1494 (w),
1439 (m), 1328 (s), 1295 (s), 1229 (s), 1137 (w), 1089 (m), 1059
1
(m), 948 (m), 856 (w), 743 (m) cm-1; H NMR (400 MHz,
3.5. Cell culture
MeOD) δ 7.96 (d, J = 8.7 Hz, 1H, Har), 7.72 (s, 1H, NCH), 7.36
(d, J = 2.6 Hz, 1H, Har), 7.28 (dd, J = 8.7, 2.6 Hz, 1H, Har), 4.37
(t, J = 7.0 Hz, 2H, NCH2), 4.10 (t, J = 6.3 Hz, 2H, OCH2), 3.88
(s, 1H, CH), 2.67 (t, J = 7.4 Hz, 2H, CqCH2), 1.98-1.88 (m, 2H,
CH2), 1.87-1.77 (m, 2H, CH2), 1.64 (s, 3H, 2-CH3), 1.68-1.59 (m,
2H, CH2), 1.58-1.49 (m, 2H, CH2), 1.44-1.32 (m, 4H, CH2), 0.93
(t, J = 7.4 Hz, 3H, CH3) ppm; 13C NMR (100 MHz, MeOD) δ
193.0 (C=O), 192.1 (C=O), 165.4 (C-6), 149.2 (CqCH2), 135.5
(Car), 130.7 (Car), 126.4 (Car), 123.1 (NCH), 122.1 (Car), 111.5
(Car), 69.7 (OCH2), 62.7 (C-2), 62.4 (CH), 51.1 (NCH2), 32.8
(CH2), 31.2 (CH2), 29.8 (CH2), 27.1 (CH2), 26.4 (CH2), 26.0
(CH2Cq), 23.2 (CH2), 14.9 (2-CH3), 14.2 (CH3) ppm; MS (EI, 70
eV) m/z (rel int) 98 (3), 73 (4), 57 (8); HRMS (ESI) m/z calcd for
C23H29N3NaO4 [M+Na]+ 434.2050, found 434.2053.
The human MM cell line INA-6 was a gift from Dr. Martin
Gramatzki (Kiel, Germany). Cells were cultured in a humidified
incubator at 37°C and 5% CO2, in RPMI-1640 medium (Sigma,
Deisenhofen, Germany) supplemented with 2 ng/mL interleukin
6, 10% FBS (PAA part of GE Healthcare, Pasching, Austria),
100 U/ml penicillin, 100 ꢀg/mL streptomycin (Sigma Aldrich
GmbH), 1 mM sodium pyruvate (Sigma Aldrich), and 2 mM L-
glutamine (Sigma).
3.6. Viability assessment
Viability was determined by staining with propidium iodide
(PI) and annexin V labelled with fluoresceinisothiocyanate
(FITC) as described before.50 Briefly, 8,000 INA-6 cells were
seeded and treated with the respective drug concentrations for 3
days INA-6 cells were washed with PBS buffer, incubated in
binding buffer (10 mM HEPES/NaOH, pH 7.4, 140 mM NaCl,
and 2.5 mM CaCl2) containing 2.5 mL annexin V-FITC and 1
3.4.10. Biotin-free derivative 14a
According to the general procedure 3.6.3., compound 14a was
synthesized from azide 9 (23.3 mg, 70.7 µmol, 1.0 equiv) 13a38
(8.24 mg, 84.8 µmol, 1.2 equiv). The residue was purified by
column chromatography on deactivated (NH3, 7.5% w/w) silica
gel (CH2Cl2/MeOH 500:1→50:1) to give product 14a (10.0 mg,
23.5 µmol, 33 %) as a yellow oil; IR (ATR) vmax 2939 (w), 2862
(w), 1691 (s), 1660 (m), 1596 (s), 1548 (w), 1438 (w), 1329 (m),
mg/mL
PI,
and
analyzed
by
flow
cytometry
(FACSCalibur/CELLQuest; Becton Dickinson, Heidelberg,
Germany). Thereby, Annexin V was prepared following a
known51 protocol and coupled to FITC (Sigma, Deisenhofen,
Germany; F725). Whereas the early apoptotic stage can only be
1
1294 (s), 1230 (s), 1058 (w), 948 (w), 856 (w), 742 (w) cm-1; H
NMR (400 MHz, MeOD) δ 7.95 (d, J = 8.7 Hz, 1H, Har), 7.86 (s,
1H, NCH), 7.35 (d, J = 2.6 Hz, 1H, Har), 7.28 (dd, J = 8.5, 2.5
Hz, 1H, Har), 4.40 (t, J = 3.5 Hz, 4H, NCH2, NHCH2), 4.10 (t, J =
6.3 Hz, 2H, OCH2), 3.88 (s, 1H, CH), 1.96 (s, 3H, 2-CH3), 1.95-
1.89 (m, 2H, CH2), 1.85-1.76 (m, 2H, CH2), 1.64 (s, 3H, CH3),
1.58-1.48 (m, 2H, CH2), 1.43-1.33 (m, 2H, CH2) ppm; 13C NMR
(100 MHz, MeOD) δ 193.0 (C=O), 192.1 (C=O), 173.2
(NHC=O), 165.4 (C-6), 146.2 (CqCH2), 135.5 (Car), 130.7 (Car),
126.4 (Car), 124.2 (NCH), 122.1 (Car), 111.5 (Car), 69.6 (OCH2),
62.7 (C-2), 62.4 (CH), 51.2 (NCH2), 35.7 (NHCH2), 31.2 (CH2),
29.8 (CH2), 27.1 (CH2), 26.4 (CH2), 22.4 (CH3), 14.9 (2-CH3)
ppm; MS (EI, 70 eV) m/z (rel int) 398 [M-CO]+ (5), 343 (2), 83
(17), 55 (29); HRMS (ESI) m/z calcd for C22H26N4NaO5 [M+Na]+
449.1795, found 449.1804.
detected by binding of annexin
V
to translocated
phosphatidylserine residues at the external cell membrane, later
apoptotic stages, in which cellular membrane integrity is lost, can
additionally be visualized by incorporation of the DNA-binding
agent PI. Thus, the cell fraction that is negative for both annexin
V-FITC and PI is considered viable. On the basis of the
respective INA-6 viable cell fractions, the means, standard
deviations, dose−response curves, and EC50 values for
compounds 2a-h, 4a, 4b, 9, 10, 12a, 12b, 14a and 14b were
calculated using GraphPad Prism software version 7 (GraphPad
Software Inc., La Jolla, USA).
3.7. Drug target identification
3.7.1. Magnetic bead preparation
3.4.11. Biotin-free derivative 14b

10
Tetrahedron
ACCEPTED MANUSCRIPT
Pierce streptavidin magnetic beads (catalog no. 88816,
linear gradients from 3% to 32% acetonitrile/0.1% formic acid.
In-solution digests were analyzed with 50 cm analytical
columns and a 180 min gradient (MS scan resolution of 60,000,
MS/MS scan resolution 7,500, HCD fragmentation, and top
speed method with a maximum 3 s cycle time).
Singly charged precursors were excluded from selection, and a
dynamic exclusion list was applied. EASY-IC was used for
internal calibration for all runs.
Thermo) and biotinylated inhibitor in DMSO were equilibrated to
room temperature. For each sample, 50 ꢀL of bead slurry was
washed with 500 ꢀL of a 1:1 mixture of DMSO and 50 mM
borate buffer at pH 8.5. To saturate the beads with the inhibitor,
we used 500 nmol of the biotinylated inhibitor in 400 ꢀL of a 1:1
mixture of DMSO and 50 mM borate buffer at pH 8.5.
Streptavidin beads and biotinylated inhibitor were incubated
(under rotation) overnight at room temperature. Control beads
were prepared without inhibitor accordingly. The supernatant
was discarded, and the beads were washed twice with 1 mL of
50% DMSO. After a second wash, the bead slurry was
transferred to a new reaction tube and was washed again twice
with 500 ꢀL of Pierce IP lysis buffer (catalog no. 87787,
Thermo).
3.9. MS data analysis
For MS raw data file processing, database searches, and
quantification, MaxQuant version 1.5.3.30 was used.[54] Searches
were performed against the H. sapiens reference proteome
database (UniProt) and additionally, a database containing
common contaminants. The search was performed with tryptic
cleavage specificity with three allowed miscleavages.
3.7.2. Cell lysis
INA-6 cells (5 × 107) were lysed with 2 mL of IP Pierce lysis
buffer together with 20 ꢀL of Halt protease inhibitor cocktail
(catalog no. 78430, Thermo Fisher Scientific, Massachusetts,
USA). Cells were incubated on ice for 10 min with periodic
mixing. Lysates were cleared by centrifugation at 16,000g at 4 °C
for 5 min, and protein concentration was determined (BCA
Protein Assay, Thermo Fisher Scientific).
Protein identification was under the control of the false-
discovery rate (<1% FDR on protein and peptide level). In
addition to MaxQuant default settings, the search was performed
against the following variable modifications: protein N-terminal
acetylation, Gln to pyro-Glu formation (N-term. Gln), and
oxidation (Met). For protein quantification, LFQ intensities were
used.55 Protein groups with less than two identified razor/unique
peptides were dismissed. Experiments for which in-solution and
in-gel digests were performed were combined as technical
replicates during analysis.
For further data analysis, in-house developed R scripts were
used. Missing LFQ intensities in the control samples were
imputed with values close to the baseline, i.e., with values from a
standard normal distribution with a mean of the 1% quantile of
the log10-transformed LFQ intensities (of inhibitor and
corresponding control sample) and a standard deviation of 0.05.
Significantly enriched proteins were identified by using robust
statistics based on intensity binned boxplots (at least 200 proteins
per bin), proteins outside the 1.5- or 3-fold interquartile range
(IQR) of the distribution were classified as significant (1) or
extreme significant (2) outliers, respectively.
3.7.3. Drug target enrichment
Inhibitor-loaded beads and control beads were incubated with
cleared INA-6 cell lysate (5 mg total protein) for 3 h at 4 °C and
overhead rotation. Beads were washed four times with 1 mL of
20 mM HEPES buffer at pH 7.5, 115 mM NaCl, 1.2 mM CaCl2,
1.2 mM MgCl2, 2.4 mM K2HPO4, and 0.5% NP-40. Proteins
were eluted with 360 ꢀL of 1× lithium dodecyl sulfate (LDS)
sample buffer (Invitrogen) and reduced by adding 40 ꢀL of 500
mM DTT and boiling the sample for 10 min at 70 °C. Samples
were alkylated with
a final concentration of 120 mM
iodoacetamide at room temperature in the dark. The eluates were
separated from the beads with a magnet. Proteins were
precipitated by adding the 4-fold sample volume of acetone.
Precipitation was performed overnight at −20 °C. Pellets were
washed three times with 1 mL of acetone.
Acknowledgments
3.7.4. In-solution digestion
This
work
was
supported
by
the
Deutsche
Precipitated proteins were dissolved in 0.5% sodium
deoxycholate (SDC, Sigma-Aldrich) in 100 mM ammonium
hydrogen carbonate. Digests were performed with trypsin
(trypsin-to-protein ratio 1:200) overnight at 37 °C. SDC was
removed by extraction with ethyl acetate.52 Peptides were dried in
a vacuum concentrator (Concentrator 5301, Eppendorf) to
remove remaining ethylaceate. Peptides were desalted using C18
stage tips.53 Each stage tip was prepared with three disks of C18
Empore SPE disks (3M) in a 200 ꢀL pipet tip. Peptides were
eluted with 80% acetonitrile/0.1% formic acid, dried in a vacuum
concentrator, and stored at −20 °C. Peptides were dissolved in
2% acetonitrile/0.1% formic acid prior to nanoLC-MS/MS
analysis.
Forschungsgemeinschaft (KFO 216 “Characterization of the
Oncogenic Signaling Network in Multiple Myeloma:
Development of Targeted Therapies”), and by the German
Excellence Initiative to the Graduate School of Life Sciences,
University of Würzburg (grant to GLZ). We thank Dr. M. Grüne
and P. Altenberger for the NMR experiments, Dr. M. Büchner, F.
Dadrich, A. Heckmann, and J. Adelmann for the mass spectra,
and Stefanie Kressmann and Matthias Bach for excellent
technical assistance.
Supplementary data
Supplementary data including NMR spectra (1H, 13C) and
detailed results from affinity capture experiments associated with
this article can be found, in the online version, at
http://dx.doi.org/10.1017/j.tet.2018.xx.xxx.
3.8. NanoLC-MS/MS analysis
NanoLC-MS/MS analyses were performed on an Orbitrap
Fusion (Thermo) equipped with an EASY-Spray ion source and
coupled to an EASY-nLC 1000 (Thermo). Peptides were loaded
on a trapping column (2 cm × 75 ꢀm ID, PepMap C18, 3 ꢀm
particles, 100 Å pore size) and separated on EASY-Spray
analytical columns (75 ꢀm ID, PepMap C18, 2 ꢀm particles, 100
Å pore size reverse phase material) with 200 nL/min flow and

24. Lieberherr, C.; Zhang, G.; Singethan, K.; Kendl, S.; Vogt, V.; Maier, J.;
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