A Fluorescence-Lifetime-Based Binding Assay for Class IIa Histone Deacetylases

Article abstract of DOI:10.1002/chem.201605140

Class IIa histone deacetylases (HDACs) show extremely low enzymatic activity and no commonly accepted endogenous substrate is known today. Increasing evidence suggests that these enzymes exert their effect rather through molecular recognition of acetylated proteins and recruiting other proteins like HDAC3 to the desired target location. Accordingly, class IIa HDACs like bromodomains have been suggested to act as “Readers” of acetyl marks, whereas enzymatically active HDACs of class I or IIb are called “Erasers” to highlight their capability to remove acetyl groups from acetylated histones or other proteins. Small-molecule ligands of class IIa histone deacetylases (HDACs) have gained tremendous attention during the last decade and have been suggested as pharmaceutical targets in several indication areas such as cancer, Huntington's disease and muscular atrophy. Up to now, only enzyme activity assays with artificial chemically activated trifluoroacetylated substrates are in use for the identification and characterization of new active compounds against class IIa HDACs. Here, we describe the first binding assay for this class of HDAC enzymes that involves a simple mix-and-measure procedure and an extraordinarily robust fluorescence lifetime readout based on [1,3]dioxolo[4,5-f]benzodioxole-based ligand probes. The principle of the assay is generic and can also be transferred to class I HDAC8.

Full text of DOI:10.1002/chem.201605140

A Journal of
Accepted Article
Title: A fluorescence lifetime based binding assay for class IIa histone
deacetylases
Authors: Christian Meyners, Monique Mertens, Pablo Wessig, and
Franz-Josef Meyer-Almes
This manuscript has been accepted after peer review and appears as an
Accepted Article online prior to editing, proofing, and formal publication
of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: Chem. Eur. J. 10.1002/chem.201605140
Link to VoR: http://dx.doi.org/10.1002/chem.201605140
Supported by

Chemistry - A European Journal
10.1002/chem.201605140
FULL PAPER
A fluorescence lifetime based binding assay for class IIa histone
deacetylases
[
a]
[b]
[b]
[a]
Christian Meyners , Monique Mertens , Pablo Wessig* and Franz-Josef Meyer-Almes*
[
6]
Abstract: Class IIa HDACs show extremely low enzymatic activity
and no commonly accepted endogenous substrate is known today.
Increasing evidence suggests that these enzymes exert their effect
rather through molecular recognition of acetylated proteins and
recruiting other proteins like HDAC3 to the desired target location.
Accordingly, class IIa HDACs like bromodomains have been
suggested to act as “Readers” of acetyl marks, whereas
enzymatically active HDACs from class I or IIb are called “Erasers”
to highlight their capability to remove acetyl groups from acetylated
histones or other proteins. Small molecule ligands of class IIa
histone deacetylases (HDACs) gained tremendous attention during
the last decade and were suggested as pharmaceutical target in
several indication areas such as cancer, Huntington’s disease or
muscular atrophy. Up to now only enzyme activity assays with
artificial chemically activated trifluoroacetylated substrates are in use
for the identification and characterization of new active compounds
against class IIa HDACs. Here, we describe the first binding assay
for this class of HDAC enzymes with simple mix-and-measure
procedure and extraordinary robust fluorescence lifetime readout
based on [1,3]dioxolo[4,5-f]benzodioxole-based ligand probes. The
assay principle is generic and can also be transferred to class I
HDAC8.
clinical trials. Most of these compounds are pan inhibitors,
which target mainly class I and class IIb HDACs and cause
severe
side
effects
like
fatigue,
nausea
and
[
7,8]
thrombocytopenia.
An approach to overcome these unwanted
side effects is the development of more specific inhibitors. Class
IIa HDACs are different from other HDAC isoforms since they
show very week catalytic activity against conventional acetylated
substrates and have a large N-terminal domain through which
they interact with several transcription factors. Furthermore,
they are able to recruit class I HDACs via protein-protein
interactions resulting in large corepressor complexes like NcoR
and CoREST. There has been an extensive scientific dispute
whether class IIa HDACs possess endogeneous catalytic activity.
In fact, the catalytic deacetylation activity is about 1000-fold
weaker than in class I HDACs and there is no commonly
accepted specific substrate identified for class IIa enzymes
today.
immunoprecipitated,
measured which is most likely due to co-purified class I
[9]
[
10]
[
9,11,12]
However,
if
class
IIa
HDACs
are
a
lysine deacetylase activity can be
[
13-15]
HDACs.
The vanishing activity of class IIa HDACs can be
H967Y mutation, whereas
dramatically increased, by
a
a
mutation of the corresponding Y into H in class I HDACs leads to
inactivation confirming that class IIa HDACs are very inefficient
deacetylases due to this difference in a pivotal amino acid
position. Furthermore, transcriptional repression of MEF2 by
HDAC4 was shown to be mediated only by protein-protein
[
9]
Introduction
[
9,16]
interaction and not to depend on deacetylase activity.
Accordingly, class IIa HDACs have also been suggested as
epigenetic readers of acetyl marks with dissociation constants
within the range of binding affinities reported for bromodomains
Members of the histone deacetylase (HDAC) family have
emerged as promising protein target for the treatment of
different diseases like cancer, neurodegenerative diseases and
[
17-19]
(
K
d
= 10-100µM).
These findings have culminated in the
[
1-3]
parasitic infections.
The 18 human members of the HDAC
currently broadly accepted view of a mostly non-catalytic role of
[11,17,20-22]
family are divided into four classes: Class I consists of HDAC1,
class IIa HDACs.
Although their function is as yet little
2
5
, 3 and 8, class II HDACs are subdivided into class IIa (HDAC4,
, 7 and 9) and IIb (HDAC6 and 10), HDAC11 is the only
understood huge effort has been directed towards the
[
20,23]
development of specific HDAC class IIa inhibitors.
The
member of class IV and the sirtuins which are class III HDACs
therapeutic indication area for HDAC class IIa inhibitors range
from cancer to neurodegenerative diseases like Chorea
+
are not zinc dependent but require NAD for their catalytic
[4]
[
14,22,24]
activity. Class I HDACs are mostly recognised for their ability to
control the expression of important genes via the deacetylation
Huntington.
The ongoing efforts in the development of
HDAC inhibitors and the large pharmacological interest clearly
indicate an urgent need for devoted assays suitable for high
throughput screening (HTS) campaigns. Most commercially
available assay systems for the evaluation of HDAC inhibitors
rely on the deacetylation of a substrate and subsequent
[
5]
of lysine residues of histone proteins. They have therefore
emerged to be the number one target of all HDAC proteins.
Currently, there are four HDAC inhibitors approved by the FDA
for the treatment of cancer and several more are tested in
[
25-27]
enzymatic conversion into a quantifiable fluorophore.
As
class IIa HDACs are believed to act mainly as readers that
recognise acetylated lysines, chemically activated substrates
[
[
a]
b]
C.Meyners, Prof. Dr. F.-J. Meyer-Almes
Fachbereich Chemie- und Biotechnologie
Hochschule Darmstadt
Haardtring 100, 64295 Darmstadt (Germany)
E-mail: franz-josef.meyer-almes@h-da.de
M. Mertens, Prof. Dr. P. Wessig
Institut für Chemie
[
28]
were developed exploiting a trifluoroacetyled lysine.
Direct
HDAC binding assays rely on the displacement of usually
[29-32]
hydroxamic acid based probes.
Recently, such an assay
was published for HDAC like prokaryotic enzymes. The authors
used the environment sensitivity of [1,3]dioxolo[4,5-
Universität Potsdam
Karl-Liebknecht-Str. 24-25, 14476 Potsdam (Germany)
E-mail: wessig@uni-potsdam.de
f]benzodioxole (DBD) dyes to monitor the binding of
a
hydroxamic acid DBD conjugate via a large change of its
fluorescence lifetime upon binding. Thus a stable signal was
produced, which was insensitive to autofluorescence in the UV
region. The presented assay was further exploited to determine
Supporting information for this article is given via a link at the end of
the document.
This article is protected by copyright. All rights reserved.

Chemistry - A European Journal
10.1002/chem.201605140
FULL PAPER
the affinity of a set of HDAC inhibitors using a competitive
[
33]
binding assay. Unfortunately, these probes are not applicable
to class IIa HDACs as hydroxamic acids are known to be mostly
[34,35]
weak inhibitors of these enzymes.
In fact, until today there is
a lack of direct binding assays for HDAC class IIa enzymes
although molecular recognition of acetylated lysine residues is
supposed to be their primary mode of action. In this study, a
binding assay is developed to fill this gap and meet the urgent
need for a binding assay applicable to class IIa HDACs.
Results and Discussion
The assay development was inspired by a binding assay for
class I and IIb HDACs using a ligand probe containing a
hydroxamate group. In this study a series of DBD probes with a
trifluoromethylketone (TFMK) pharmacophoric group instead of
Scheme 2. Dynamic equilibrium between to tautomers of compound 13,14a.
a
hydroxamic acid were synthesised, since compounds
containing a TFMK group have been described as reasonable
[35,36]
ligands for class IIa HDACs.
For the synthesis of the DBD ligands a convergent route, shown
in scheme 1, was chosen. Starting with the synthesis of DBD
amines 6 and 7 by coupling of the known acyl and ester DBD
acids
1 and 2 to N-boc protected diethylamine 3, the
intermediates 4,5 were subsequently deprotected catalysed by
[
37]
trifluoroacetic acid (TFA).
For the TFMK building blocks the
synthesis of 12a,b starts with commercially available glutaric
acid diethyl ester (10a) and adipic acid monomethyl ester (10b).
The synthesis of 12c starts with compound 10c which was
synthesised by dehydration of pimelic acid
8 with acetic
anhydride as dehydration agent according to the known
procedure of Cisneros et al. and solvolysis of the resulting
[
38]
anhydride 9 with methanol. Although the diethyl ester 10a was
transferred to the corresponding TFMK 12a according to the
procedure of Okano et al., this procedure has not worked for the
[
39]
preparation of the TFMKs 12b,c. Therefore another route was
used, which starts with conversion of the carboxylic moieties of
1
0b,c to the TFMKs 11b,c with conditions described by Biovin et
[
40]
al.. Then, the monomethyl ester moieties were saponified with
lithium hydroxide to give the TFMKs 12b,c similar to the
procedure of Frey et al..
[
36]
Finally the DBD amines 6,7 are
coupled to the TFMKs 12a-c under standard peptide coupling
conditions.
Especially for compounds 13,14a a mixture of two tautomers
was observed, where the cyclic forms 13,14a’ exist
predominantly to the linear in CDCl
3
, shown in scheme 2.
Dissolving the samples in MeOH leads to a shift of equilibrium to
1
9
the linear forms 13,14a. This could be shown by F-NMR,
where the samples were concentrated from methanol and
subsequently spectra in CDCl were recorded (Fig. S1 and S2).
3
Specifically, for 13a a ratio of 1:1 between 13a and 13a’ could
be observed. Recording a second spectrum after a few hours
showed a ratio of 10:1 with 13a’ as the predominant form. A
similar behaviour was monitored for compound 14a. Thus we
assume a dynamic equilibrium between the different tautomers
depending on the solvent.
This article is protected by copyright. All rights reserved.

Chemistry - A European Journal
10.1002/chem.201605140
FULL PAPER
Scheme 1. Synthesis of DBD dyes 13a-c and 14a-c. Reagents and
conditions: i) 1. (COCl)
7 %, 5: 90 %); ii) TFA, DCM (6: 94 %, 7: 98 %); iii) Ac
reflux (10c: over 2 steps 51 %); v) NaOEt, CF COOEt, reflux; vi) 1.) (COCl)
DCM, DMF, 30 min 0°C, then rt; 2.) TFAA, Pyridine, 0°C; vii) 30 % H SO
reflux (12a: over 2 steps 17 %); viii) LiOH, THF, H
2
, DCM, DMF, 30 min 0°C, then rt; 2.) DIEA, DCM, 3 (4:
8
2
O, reflux; iv) MeOH,
3
2
,
,
2
4
2
O (over 2 steps: 12b: 41 %,
2c: 35 %); ix) TBTU, DIEA, DMF (13a: 89 %, 13b: 71 %, 13c: 90 %, 14a:
4 %, 14b: 89 %, 14c: 98 %).
1
8
The obtained ligands were first analysed for their potential to
inhibit human HDAC1-8. As expected the hydroxamic acid DBD
probe 15 was inactive towards class IIa HDACs and active
towards the other HDAC isoforms (Table. 1). In contrast, 13b
with TFMK moiety showed inhibitory activity against class IIa
HDACs in the micromolar range and preference for HDAC3 and
8
. While the reduction of the spacer to 3 CH
resulted in a mostly inactive compound, the increment by one
spacer unit from 4 to 5 CH units of 13c resulted in an increase
2
units of 13a
2
of the inhibitory activity. This effect was stronger pronounced on
class IIa HDACs with factors between 14 and 220, compared to
factors between
2 and 5 for the other HDACs. Similar
observations were made for the ester variants 14a-c of the DBD
probes, which showed in general IC50-values similar to the acyl
DBD probes 13a-c. Only the inhibition of HDAC8 by 14a was
about 15 times stronger than for its acyl counterpart.
This article is protected by copyright. All rights reserved.

Chemistry - A European Journal
10.1002/chem.201605140
FULL PAPER
Table 1. Inhibition of HDACs by DBD probes. IC50-values (µM) were determined using a fluorogenic enzyme activity assay as described in the experimental
section.
Probe
3a
3b
HDAC1
HDAC2
HDAC3
HDAC4
HDAC5
HDAC6
HDAC7
HDAC8
1
>50
1.6±0.1
0.82±0.03
>50
>50
7.6±0.4
3.2±0.1
>50
>50
10±1
2.2±0.3
0.010±0.002
14±1
4.0±1.0
1.4±0.2
0.10±0.01
5.3±0.5
2.4±0.1
0.21±0.03
>50
>50
1.9±0.2
1.6±0.1
4.8±0.3
0.091±0.008
0.021±0.001
0.31±0.02
0.14±0.01
0.058±0.003
3.2±0.3
1
0.11±0.01
0.042±0.003
>50
3.8±0.2
0.79±0.1
>50
1
3c
4a
0.036±0.006
3.6±0.2
1
14b
2.5±0.1
1.0±0.1
0.53±0.02
21±1
0.18±0.01
0.075±0.002
2.5±0.5
3.9±0.2
0.032±0.002
>50
2.9±0.5
1.0±0.1
5.2±0.9
2.5±0.1
14c
4.3±0.4
1.0±0.03
0.030±0.009
>50
15
Next, the change of FLT upon binding of the DBD ligands to
HDAC isoforms 4, 5, 7 and 8 was measured in titration
experiments. The unbound acyl probes exhibited a FLT in the
range of 1.6 ns. Upon addition of the respective HDAC isoform
the FLT of the DBD probes 13b and 13c increased in a
concentration dependent manner, while binding of 13a was not
detectable in the investigated concentration range (Fig. 1 and
Table 2). The largest change in FLT was determined for the
binding of 13b to HDAC4 with a difference of 6.1 ns. In general
the shorter probe 13b exhibited with at least 1.9 ns a larger
change in FLT than the prolonged probe 13c, which showed
differences between 0.89 and 1.9 ns. The concentration
dependency of the change in the FLT of the DBD probes was
further exploited to determine the K
d
-values for the binding. The
determined K -values were comparable to the determined IC50
d
-
values and were found to be between 1.2 µM and 0.01 µM. The
strong increase in affinity upon increment of the spacer by one
CH
2
unit towards class IIa HDACs was confirmed by the
-values. For binding to HDAC8 both probes
determined K
d
behaved quite equal and bound with a comparable affinity and
exhibited a similar change in their FLT.
The ester variants exhibited a long FLT of 25 ns in their unbound
state, which theoretically decreases upon binding. Unfortunately,
this was only observable for the binding of 14a to HDAC8. Here
the FLT decreased by 13 ns and it was possible to determine a
K -value of 0.69 µM (Table 2 and Fig. S3). The other ester
d
variants showed no difference in their FLT neither for the binding
to HDAC8 nor for the binding to class IIa HDACs.
This article is protected by copyright. All rights reserved.

Chemistry - A European Journal
10.1002/chem.201605140
FULL PAPER
Figure 1. Binding of DBD probe 13c to HDACs. Fluorescence decay curves of
0.025 nM 13c in the presence of increasing HDAC8 concentrations (a). The
binding of the DBD probe resulted in a concentration dependent increase in
the FLT of the probe, which was exploited to determine the binding degree as
described in the experimental section. The binding degree of 13c is plotted
against the concentration of HDAC4 (dots), HDAC5 (squares), HDAC7
(triangles) or HDAC8 (circles)(b). The continuous line represents the
calculated fit curve to equation 3. The determined binding constants are
summarised in Table 2. Inhibition of enzymatic HDAC activity by the DBD
probe 13c (c). HDAC activity of HDAC1 (dots), HDAC2 (squares), HDAC7
(triangles) and HDAC8 (circles) was determined as described in the
experimental section. The continuous line represents the calculated fit curve to
equation 1, which yields the IC50-values summarized in Table 1.
Table 2. Changes in FLT and binding constants, K
d
, of the binding of DBD probes to HDAC4, 5, 7 and 8.
HDAC5 HDAC7
/ µM ΔFLT / ns / µM ΔFLT / ns
>10 >10
Probe
HDAC4
/ µM ΔFLT / ns
>10
HDAC8
/ µM ΔFLT / ns
>10
K
d
K
d
K
d
K
d
13a
13b
13c
14a
-
6.1±0.2
0.89±0.02
-
-
-
3.9±0.1
1.23±0.03
-
-
1.2±0.2
0.40±0.03
0.078±0.008
-
1.92±0.05
1.28±0.03
-
0.38±0.04
0.046±0.008
-
0.051±0.006
0.024±0.003
0.69±0.06
2.43±0.06
1.87±0.05
−13.2±0.3
0.010±0.003
-
In order to determine the specificity of the binding and to
simulate the performance of the assay under screening
conditions a number of known HDAC inhibitors were used in
typical screening concentrations and incubated with HDAC4 or 8
and 50 nM of the DBD probe 13b. After an incubation of one
hour the fluorescence decay was measured and the FLT was
determined. Compared to the FLT of the HDAC probe complex a
decrease of the FLT was observable for all HDAC inhibitors (Fig.
specific reference inhibitors PCI-34051 and TMP269 were both
only active on their respective target HDAC isoform.
2). Thereby, the weak HDAC4 and HDAC8 inhibitors e.g. SAHA
and TSA showed a smaller decrease of the FLT than the
stronger competitors SATFMK and PDF306. Furthermore, the
This article is protected by copyright. All rights reserved.

Chemistry - A European Journal
10.1002/chem.201605140
FULL PAPER
HDAC8 the binding of the probe 13b was more affine, which
allowed a reliable determination of the K
compounds.
I
-values of the tested
Figure 3. Determination of binding constants of the binding of unlabeled
HDAC inhibitors to HDAC8 (a) and HDAC4 (b). A serial dilution of the
unlabeled HDAC inhibitors PDF306 (dots), SATFMK (squares), TMP269
Figure 2. Displacement of the DBD 13b by competitive HDAC inhibitors. The
FLT of 0.125 µM of the DBD probe 13b was measured alone, in presence of
(triangles) or PCI34051 (circles) was incubated with either 0.45 µM HDAC8
and 0.125 µM of the DBD probe 13b or 0.8 µM HDAC4 and 0.125 µM of the
DBD probe 13c. After equilibration the FLT of the probes was measured and
translated into binding degree as described in the experimental section. The
continuous line represents the calculated fit to equation 4, which yields the
binding constants summarised in Table 3.
0.4 µM HDAC8 (a) or 0.75 µM HDAC4 (b) and with addition of 10 µM of the
indicated inhibitor.
For the strong competitors the concentration dependency of the
displacement of the probe was exploited to determine the
respective K
and the probe 13b were quite steep and produced equal K
values around 0.08 µM for all three inhibitors (Fig. S4). This and
the high error in the determined K -values indicated that these
I
-values. The resulting curves for binding to HDAC4
I
-
I
Table 3. Comparison of inhibitor binding constants, K determined by the
fluorescence life time binding assay with K
d
-values determined by ITC and
I
IC50-values of inhibitors of HDAC4 and HDAC8.
values could not be determined reliably, which originates
presumably from the high difference in affinity between the
probe and the inhibitors. To overcome these limitations the more
affine binding probe 13c was used. The resulting curves allowed
HDAC4
HDAC8
Compd
K
µM
I
K
d
IC50
/ µM
K
/ µM
I
K
d
IC50
/ µM
/
/ µM
/ µM
a more exact determination of the K
agreement with K -values determined by ITC and IC50-values
from an enzyme activity assay (Fig. 3, Fig. S5 and Table 2). For
I
-values, which were in good
d
SATFMK
0.012
±0.003
0.047
±0.01
0.10
±0.01
0.023
±0.005
0.08
±0.01
0.035
±0.002
This article is protected by copyright. All rights reserved.

Chemistry - A European Journal
10.1002/chem.201605140
FULL PAPER
1
Some compounds with a DBD backbone show in the H-NMR spectra
PDF306
TMP269
PCI34051
0.23
0.22
±0.04
0.40
±0.02
0.12
±0.01
0.058
±0.01
0.18
±0.06
signals of an AB-system caused by the methylene group belonging to the
DBD. This will be marked as “AB” and the given value in Hz is the
distance between the inner signals of such a spin system. The outer
signals of higher order are usually too small to be able to perform
analysis.
±
0.02
0.14
-
-
0.26
±0.03
-
-
-
-
±
0.01
-
-
0.028
0.024
±
0.006
±0.002
tert-Butyl
(2-(2-(4,8-dibutyrylbenzo[1,2-d:4,5-d']bis([1,3]dioxole)-2-
yl)acetamido)ethyl)-carbamate (4): To a cooled solution (0°C) of the
carboxylic acid 1 (102.4 mg, 281.05 µmol) in anhydrous DCM (25 mL)
was added oxalyl chloride (0.2 mL, 2.33 mmol, 8.3 eq.) and one drop of
anhydrous DMF. After 4 h the solution was concentrated under reduced
pressure. The resulting solid was dissolved in anhydrous DCM (20 mL)
and slowly dropped to a solution of DIEA (100 µL, 574.09 µmol, 2.0 eq.)
and tert-butyl (2-aminoethyl)carbamate 3 (50 µL, 324.56 µmol, 1.1 eq.) in
anhydrous DCM (20 mL). The solution was stirred over night for 18 h and
Conclusions
3
then put into a saturated aqueous solution of NaHCO (50 mL). The
aqueous phase was extracted with DCM (4x50 mL), washed with
aqueous HCl (0.1 N, 1x50 mL) and with saturated aqueous NaCl (1x50
mL). The combined organic phases were dried (MgSO ), filtered and
4
concentrated under reduced pressure. The residue was purified by silica
The presented assay is the first reported direct binding assay for
class IIa HDACs. The assay relies on DBD probes which show a
large change in FLT up to 6.1 ns upon binding to the enzymes.
The general procedure is extremely simple (mix and measure),
without any additional incubation step for the reactions of target
and/or detection enzymes and exploits the proven robust FLT
readout resulting in a high performance assay suitable for HTS
applications. The assay is capable to identify strong as well as
weak ligands under typical screening conditions and the binding
constants of strong binders can be determined reliably showing
good correlation with IC50-values determined by enzyme activity
assays. The inhibitory activity of the DBD probes against other
HDAC isoforms also suggest that they may be used as generic
tools in binding assays for class I and IIb HDACs.
gel chromatography (DCM/MeOH 99:1→ 92:8) to afford 4 as an orange
solid (124.4 mg, 87 %). R = 0.50 (DCM/MeOH 25:1); m. p.: 164-165°C;
f
1
3
H-NMR (300 MHz, CDCl
Hz, 1H, CH); 6.09 (s, 2H, CH
CH ); 3.33-3.19 (m, 2H, CH ); 2.89 (d, J = 5.0 Hz, 2H, CH
.2 Hz, 4H, CH ); 1.76-1.62 (m, 4H, CH ); 1.41 (s, 9H, CH
); C-NMR (75 MHz, CDCl , 27°C): δ = 196.4 (2C,
C=O); 166.8 (1C, C=O); 156.8 (1C, C=O); 141.1 (2C, Cquart.); 140.4 (2C,
3
, 27°C): δ = 6.75 (bs, 1H, NH); 6.62 (t, J = 5.2
2
); 5.30 (bs, 1H, NH); 3.46-3.34 (m, 2H,
3
3
2
2
2
); 2.87 (t, J =
3
); 0.96 ppm (t,
7
2
2
3
13
J = 7.4 Hz, 6H, CH
3
3
C
C
quart.); 110.5 (1C, CH); 110.0 (2C, Cquart.); 102.6 (1C, CH
quart.); 45.6 (2C, CH ); 42.0 (1C, CH ); 40.8 (1C, CH ); 40.3 (1C, CH
2
2
); 79.6 (1C,
2
2
2
);
28.3 (3C, CH ); 17.1 (2C, CH ); 13.7 ppm (2C, CH ); IR (ATR): v = 3293,
3
2
3
̅
−
1
2
966, 1682, 1654, 1436, 1279, 1236, 1175, 1087, 1036 cm ; HRMS: (EI)
m/z = 506.2272 [M ], calc.: 506.2264.
+
Diethyl 2-(2-((2-((tert butoxycarbonyl)amino)-
oxoethyl)benzo[1,2-d:4,5-d']bis- ([1,3]dioxole)-4,8-dicarboxylate (5):
ethyl)amino)-2-
Experimental Section
Compound 5 was synthesised from the carboxylic acid 2 (98.7 mg,
All reagents and solvents were purchased from commercial suppliers and
used with further purification only if necessary. SAHA was bought from
Cayman chemicals, TSA and PCI-34051 were bought from Selleckchem.
HDAC isoforms 1, 2, 3 and 6 were ordered from BPS Bioscience. The
compounds SATFMK, PDF306 and 15 were made as described
2
4
=
2
5
3
1
1
1
7
67.99 µmol) by following the procedure described above for compound
. The desired product was obtained as yellow solid (123.3 mg, 90 %). R
f
1
0.34 (DCM/MeOH 25:1); m. p.: 165-167°C; H-NMR (300 MHz, CDCl
3
,
3
7°C): δ = 6.61 (t, J = 5.3 Hz, 1H, CH); 6.10 (AB, J = 0.46 Hz, 2H, CH
2
);
);
);
, 27°C): δ =
66.8 (1C, C=O); 161.8 (2C, C=O); 156.6 (1C, C=O); 141.9 (2C, Cquart.);
41.2 (2C, Cquart.); 110.7 (1C, CH); 103.2 (2C, Cquart.); 102.9 (1C, CH );
9.5 (1C, Cquart.); 61.7 (2C, CH ); 42.1 (1C, CH ); 40.5 (1C, CH ); 40.3
1C, CH ); 28.3 (3C, CH ); 14.2 ppm (2C, CH ); IR (ATR): v = 3275, 2977,
3
.26 (bs, 1H, NH); 4.38 (q, J = 7.1 Hz, 4H, CH
2
); 3.44-3.33 (m, 2H, CH
); 2.90 (d, J = 5.3 Hz, 2H, CH ); 1.41 (s, 9H, CH
); C-NMR (75 MHz, CDCl
2
[
33,41,42]
elsewhere.
Malakhov et al.. Reactions were performed under an inert atmosphere
nitrogen or argon) whenever anhydrous solvents were used and were
monitored by thin layer chromatography (TLC) using aluminium sheets
coated with silica gel (SiO -60, F254). Flash chromatography was carried
SUMO protease 1 (Ulp 1) was made as described by
3
.33-3.20 (2H, CH
2
2
3
[
43]
3
13
.36 ppm (t, J = 7.1 Hz, 6H, CH
3
3
(
2
2
2
2
2
out on silica gel 60 (40-63 µm). NMR spectra were recorded on a Bruker
(
2
3
3
̅
Avance 300. Chemical shifts are reported as δ in parts per million (ppm)
−1
1710, 1656, 1450, 1294, 1261, 1155, 1080 cm ; HRMS: (EI) m/z =
1
relative to the solvent signal ( H-NMR: CDCl
3
7.26 ppm, MeOD-d
4
3.31
49.15 ppm). For F-NMR
+
510.1840 [M ], calc.: 510.1850.
13
19
ppm, C-NMR: CDCl
3
77.0 ppm, MeOD-d
4
trifluoroacetic acid (TFA) (-76.55 ppm) was used as external standard.
Assignments of NMR spectra are based on correlation spectroscopy
2
-(2-(4,8-Dibutyrylbenzo[1,2-d:4,5-d']bis([1,3]dioxole)-2-
yl)acetamido)ethan-1-aminium 2,2,2-trifluoroacetate (6): To a solution
of DCM/TFA (3:1, 12 mL) was added compound 4 (96.0 mg, 189.52
µmol). After 30 min all volatile compounds are removed and the residue
was suspended in toluene. The solvent was removed under reduced
pressure and this procedure was repeated once again. Then the crude
product was dissolved in hot MeOH, cooled to RT and precipitated with
PE. Filtration and drying under reduced pressure yield to the title
(COSY, HSQC). High resolution mass spectra (HRMS) were measured
on a quadrupole time-of-flight (TOF) mass spectrometer. Melting points
are uncorrected and were determined in an Elektrothermal 9100 melting
point instrument. IR spectra were recorded on a Perkin Elmer FTIR
spectrometer Spectrum 2.
Synthesis of DBD-Probes
Notice:
1
compound 6 as an orange solid (92.4 mg, 94 %). m. p.: 149-151°C; H-
NMR (300 MHz, [D
3
4
]MeOH, 25°C): δ = 6.67 (t, J = 5.3 Hz, 1H, CH),
3
3
2 2
6.14 (AB, J = 1.2 Hz, 2H, CH ), 3.54 (t, J = 5.9 Hz, 2H, CH ), 3.14 (t, J
3
3
=
2 2
5.9 Hz, 2H, CH ), 3.00 (d, J = 5.3 Hz, 2H, CH ), 2.96 (t, J = 7.2 Hz,
This article is protected by copyright. All rights reserved.

Chemistry - A European Journal
10.1002/chem.201605140
FULL PAPER
3
4
H, CH
2
), 1.82-1.60 (m, 4H, CH
C-NMR (75 MHz, [D ]MeOH, 25°C): δ = 199.0 (2C, C=O), 171.6 (1C,
C=O), 143.4 (2C, Cquart.), 142.6 (2C, Cquart.), 113.1 (1C, CH), 112.0 (2C,
quart.), 104.9 (1C, CH ), 47.4 (2C, CH ), 43.0 (1C, CH ), 41.7 (1C, CH ),
9.0 (1C, CH ), 19.0 (2C, CH ), 14.9 ppm (2C, CH ); IR (ATR): v = 3275,
099, 2964, 1660, 1567, 1439, 1282, 1187, 1119, 1039, 837, 723 cm
2
), 1.00 ppm (t, J = 7.4 Hz, 6H, CH
3
);
CDCl
3
, 25°C): δ = -80.0 ppm (s, 3F, CF
3
); IR (ATR): v
̅
= 2952, 2869,
.
1
3
−1
4
1763, 1735, 1438, 1404, 1368, 1291, 1203, 1152, 1023 cm
C
3
3
2
2
2
2
[36]
7,7,7-Trifluoro-6-oxoheptanoic acid (12b):
The carboxylic acid 12b
[36]
2
2
3
̅
was synthesised similar to the procedure described by Frey et al.. The
trifluoromethyl ketone 11b (1.02 g, 4.81 mmol) was dissolved in THF (20
mL) at RT and treated with an aqueous solution of LiOH (2 M, 24 mL) for
−
1
;
+
HRMS: (ESI) m/z = 407.1811 [MH ], calc.: 407.1818.
8
h. Then the reaction mixture was washed with MTBE (2x10 mL) and
2
-(2-(4,8-Bis(ethoxycarbonyl)benzo[1,2-d:4,5-d']bis([1,3]dioxole)-2-
acidified with aqueous HCl (1 N) to pH 2. The inorganic phase was
extracted with EtOAc (4x 20mL) and the combined organic phases were
dried (MgSO ), filtered and concentrated under reduced pressure. The
4
desired trifluoromethyl ketone 12b was obtained as a white solid (0.67 g,
70 %) and was used without further purification. The analytical data are
in agreement with those reported.
yl)acetamido)ethan-1-aminium 2,2,2-trifluoroacetate (7): Compound 7
was synthesised from 5 (60.2 mg, 117.92 µmol) by following the
procedure described above for compound 6. The desired product was
obtained as yellow solid (60.8 mg, 98 %). m. p.: decomp. at 176°C; H-
NMR (300 MHz, [D
1
3
6
]DMSO, 25°C): δ = 8.33 (t, J = 5.4 Hz, 1H, NH);
3
2
7.87 (bs, 3H, NH); 6.62 (t, J = 5.2 Hz, 1H, CH); 6.12 (s, 2H, CH ); 4.28
3
3
[
36]
(
q, J = 7.1 Hz, 4H, CH
CH ); 2.88-2.83 (2H, CH ); 1.26 ppm (t, J = 7.1 Hz, 6H, CH
75 MHz, [D ]DMSO, 25°C): δ = 168.1 (1C, C=O); 161.8 (2C, C=O);
41.9 (2C, Cquart.); 141.8 (2C, Cquart.); 112.1 (1C, CH); 103.6 (2C, Cquart.);
03.3 (1C, CH ); 62.0 (2C, CH ); 41.5 (1C, CH ); 39.4 (1C, CH ); 37.2
); 15.0 ppm (2C, CH ); IR (ATR): v = 3270, 3091, 2984, 1719,
661, 1451, 1298, 1268, 1177, 1091, 1022, 955, 723 cm ; HRMS: (ESI)
2 2
); 3.43-3.28 (2H, CH ); 2.88 (d, 2H, J = 5.3,
8
1
,8,8-Trifluoro-7-oxooctanoic acid (12c): The trifluoromethyl ketone
2c was synthesised from the methylester 11c (737.5 mg, 3.26 mmol) by
3
13
2
2
3
); C-NMR
(
1
1
6
following the procedure described above for compound 12b. The crude
product was purified by silica gel chromatography (DCM/MeOH 30:1) to
afford the trifluoromethyl ketone 12c as highly viscous oil (425.1 mg,
2
2
2
2
(1C, CH
2
3
̅
61 %). The analytical data are in agreement with those reported.
−
1
1
+
m/z = 410.1310 [MH ], calc.: 410.1325
N-(2-(2-(4,8-Dibutyrylbenzo[1,2-d:4,5-d']bis([1,3]dioxole)-2-
yl)acetamido)ethyl)-6,6,6-trifluoro-5-oxohexanamide (13a): To
a
[
38]
7
-Methoxy-7-oxoheptanoic acid (10c):
Compound
9
was
mixture of the carboxylic acid 12a (20.0 mg, 108.63 µmol, 1.5 eq.), DIEA
26.0 µL, 149.26 µmol, 2.0 eq.) and TBTU (35.0 mg, 109.00 µmol, 1.5
synthesised according to the procedure of Cisneros et al. from pimelic
acid (3.02 g, 18.84 mmol) and then refluxed in anhydrous methanol (10
mL) for 5 h. After removal of the solvent under reduced pressure the
residue was purified by distillation (100°C, 1.2 · 10 mbar) to yield 10c
3
as a colorless oil (1.67 g, 51 % over 2 steps). H-NMR (300 MHz, CDCl ,
(
eq.) in anhydrous DMF (1.5 mL) was added the amine 6 (38.7 mg, 74.36
µmol, 1.0 eq.). After 16 h the solvent was removed under reduced
pressure and the residue was dissolved in DCM and worked up with
aqueous HCl (1 N) and saturated aqueous NaCl. The organic phase was
−
2
1
3
2
CH
5°C): δ = 10.82 (s, 1H, OH); 3.63 (s, 3H, CH
3
); 2.32 (t, J =7.5 Hz, 2H,
); 1.70-1.54 (m, 4H, CH ); 1.41-125
); C-NMR (75 MHz, CDCl , 25°C): δ = 179.8 (1C,
C=O); 174.1 (1C, C=O); 51.4 (1C, CH ); 33.7 (2C, CH ); 28.4 (1C, CH );
); IR (ATR): v = 2950, 2866, 1733,
dried (MgSO
crude product was purified by silica gel chromatography
DCM/MeOH/TEA 100:1:0.1). After washing with saturated aqueous
NaCl and drying over MgSO the trifluoromethyl ketone 13a was obtained
4
), filtered and concentrated under reduced pressure and the
3
2
); 2.29 (t, J =7.6 Hz, 2H, CH
2
2
13
ppm (m, 2H, CH
2
3
(
3
2
2
4
2
1
1
4.4 (1C, CH
2
); 24.2 ppm (1C, CH
2
̅
as an orange solid (37.7 mg, 89 %). NMR spectra have shown that the
title compound exists as a mixture of two tautomers. Which tautomer is
predominant depends on the solvent. The present data shows a 1:1
−
1
+
708, 1437, 1198, 1171 cm ; HRMS: (EI) m/z = 175.0964 [MH ], calc.:
75.0965.
1
19
mixture (judged by H- und F-NMR). R
f
= 0.49 (DCM/MeOH 10:1); m.
1
Methyl 8,8,8-trifluoro-7-oxooctanoate (11c): Compound 11c was
p.: 124-127°C; H-NMR (300 MHz, CDCl
3
, 27°C): δ = 6.90 (bt, 0.5H, NH);
[
40]
synthesised similar to the procedure described by Biovin. To a stirred
solution of pimelic acid monomethyl ester 10c (1.36 g, 7.81 mmol) in
anhydrous DCM (20 mL) was added one drop of anhydrous DMF. After
cooling to 0°C oxalyl chloride (1.2 mL, 15.62 mmol, 2.0 eq.) was added
slowly and the solution was stirred for 18 h at RT. After removing of all
volatile compounds under reduced pressure the corresponding acid
chloride of 10c was dissolved in anhydrous DCM (50 mL) and cooled to
6.74 (bt, 0.5H, NH); 6.68-6.49 (1.5H, NH, CH); 6.18-6.05 (2H, CH
2
);
4.26-4.12 (m, 0.5H, CH); 3.87-3.73 (m, 0.5H, CH); 3.56 - 3.24 (3H,
3
CH
Hz, 1H, CH
ppm (t, J = 7.4 Hz, 6H, CH
2
); 3.00 - 2.78 (6H, CH
); 2.12-1.93 (1.5H, CH, CH
2
); 2.57-2.35 (1.5H, CH, CH
); 1.91-1.62 (6H, CH
); C-NMR (75 MHz, CDCl , 27°C): δ =
2
); 2.29 (t, J = 7.1
2
2
2
); 0.97
3
13
3
3
196.7 (2C, C=O); 196.6 (2C, C=O); 172.7 (1C, C=O); 172.3 (1C, C=O);
168.2 (1C, C=O); 167.0 (1C, C=O); 141.2 (2C, Cquart.); 140.4 (2C, Cquart.);
0
°C. Treated with trifluoroacetic anhydride (4.4 mL, 31.25 mmol, 4.0 eq.)
110.6 (2C, Cquart.); 110.5 (2C, Cquart.); 110.1 (1C, CH); 102.6 (1C, CH
102.5 (1C, CH ); 45.7 (2C, CH ); 42.2 (1C, CH ); 39.9 (1C, CH ); 39.8
(1C, CH ); 35.5 (1C, CH ); 34.2 (1C, CH ); 31.6 (1C, CH ); 18.2 (1C,
CH ); 17.1 (2C, CH ); 17.0 (2C, CH
MHz, CDCl
2
);
and pyridine (3.8 mL, 46.87 mmol, 6.0 eq.) the solution was stirred for 30
min at 0°C. Ice water (20 mL) was added slowly and the solution was
then stirred additionally for 60 min. After the addition of distilled water
2
2
2
2
2
2
2
2
9
1
2
2
2
3
); 13.7 ppm (2C, CH ); F-NMR (282
(100 mL) to the solution and the separation of the organic phase, the
3
, 25°C): δ = -77.6 (s, 3F), -79.7 ppm (s, 3F); IR (ATR): v
̅
=
−
1
inorganic phase was extracted with DCM (4x50 mL). The combined
organic phases were washed with a saturated aqueous solution of NaCl,
3283, 2964, 2877, 1685, 1660, 1438, 1283, 1204, 1120, 1093 cm
;
+
HRMS: (EI) m/z = 572.1986 [M ], calc.: 572.1982.
dried over MgSO
residue was purified by silica gel chromatography (DCM/PE 1:1) to obtain
1c as a pale yellow oil (1.58 g, 58 %), which was contaminated with
small amounts of starting material which cannot separated from the
4
and concentrated under reduced pressure. The
Diethyl
2-(2-oxo-2-((2-(6,6,6-trifluoro-5-oxohexanamido)ethyl)ami-
1
no)ethyl)benzo[1,2-d:4,5-d']bis([1,3]dioxole)-4,8-dicarboxylate (14a):
Following the procedure described above for 13a, the trifluoromethyl
ketone 14a was obtained from the carboxylic acid 12a (18.0 mg, 97.77
µmol, 1.3 eq.), DIEA (26 µL, 149.26 µmol, 2.0 eq.), TBTU (33.59 mg,
1
product. H-NMR (300 MHz, CDCl
3
, 25°C): δ = 3.65 (s, 3H, CH
); 2.31 (t, J = 7.4 Hz, 2H, CH ); 1.75-1.53 (4H, CH
); C-NMR (75 MHz, CDCl , 25°C): δ = 191.4
q, J(C,F) = 34.8 Hz, 1C, C=O); 176.0 (1C, C=O); 115.5 (q, J(C,F) =
92.0 Hz, 1C, CF ); 52.3 (1C, CH ); 36.0 (1C, CH ); 33.7 (1C, CH ); 28.0
1C, CH ); 24.4 (1C, CH ); 21.9 ppm (1C, CH ); F-NMR (282 MHz,
3
); 2.71 (t,
3
3
J = 7.2 Hz, 2H, CH
.43-1.28 ppm (m, 2H, CH
2
2
2
);
13
1
(
2
3
104.61 µmol, 1.4 eq.) and the amine 7 (38.7 mg, 73.80 µmol) in
2
1
anhydrous DMF (1.5 mL) as a yellow solid (35.7 mg, 84 %). NMR spectra
have shown that the title compound exists as a mixture of two tautomers.
Which tautomer is predominant depends on the solvent. The present
2
(
3
3
2
2
19
2
2
2
1
19
f
data shows a 1:1 mixture (judged by H- und F-NMR). R = 0.47
This article is protected by copyright. All rights reserved.

Chemistry - A European Journal
10.1002/chem.201605140
FULL PAPER
1
(
DCM/MeOH 10:1); m. p.: 155-158°C; H-NMR (300 MHz, CDCl
3
, 25°C):
); 4.37
); 4.25-4.11 (m, 0.5H, CH); 3.86-3.70 (m, 0.5H,
DIEA (35 µL, 200.93 µmol, 2.7 eq.), TBTU (27.3 mg, 85.02 µmol, 1.1 eq.)
and the amine 6 (39.2 mg, 75.32 µmol, 1.0 eq.) in anhydrous DMF (4.0
δ = 6.78 (bs, 0.5H, NH); 6.72-6.36 (2H, NH, CH); 6.11 (s, 2H, CH
2
3
(
q, J = 7.1 Hz, 4H, CH
CH); 3.61-3.20 (3H, CH
CH ); 2.52-2.32 (1.5H, CH, CH
.75 (m, 2.5H, CH ); 1.38 ppm (t, J= 7.1 Hz, 6H. CH
MHz, CDCl , 25°C): δ = 172.6 (1C, C=O); 172.2 (1C, C=O); 168.3 (1C,
C=O); 167.3 (1C, C=O); 161.9 (2C, C=O); 141.9 (2C, Cquart.); 141.2 (2C,
quart.); 110.9 (1C, CH); 110.7 (1C, CH); 103.2 (2C, Cquart.); 102.9 (1C,
CH ); 61.7 (2C, CH ); 42.2 (1C, CH ); 41.7 (1C, CH ); 40.3 (2C, CH );
9.6 (2C, CH ); 35.5 (1C, CH ); 34.1 (1C, CH ); 32.3 (1C, CH ); 31.5 (1C,
CH ); 18.1 (1C, CH ); 15.4 (1C, CH
MHz, CDCl
405, 2976, 1720, 1654, 1449, 1296, 1176, 1078, 1023 cm ; HRMS: (EI)
2
mL).
Purifying by silica gel chromatography (DCM/MeOH/TEA
3
2
); 3.02-2.88 (2H, CH
2
); 2.82 (t, J = 6.8 Hz, 1H,
); 2.27 (t, J = 7.0 Hz, 1H, CH ); 2.10-
); C-NMR (75
10:1:0.0→10:1:0.1) give the title compound as an orange solid (40.3 mg,
3
1
2
2
2
90 %). R
MHz, CDCl
5.3 Hz, 1H, CH); 6.11 (s, 2H, CH
f
= 0.53 (DCM/MeOH 10:1); m. p.: 185-186°C; H-NMR (300
3
13
3
1
2
3
3
, 25°C): δ = 6.72 (bt, 1H, NH); 6.60 (bt, 1H, NH); 6.59 (t, J =
); 3.47-3.36 (4H, CH ); 2.93-2.86 (6H,
); 2.22 (t, J = 7.4 Hz, 2H, CH ); 1.77-
); 0.97 ppm (t, J = 7.4 Hz, 6H,
, 25°C): δ = 196.6 (2C, C=O); 173.8 (1C,
C=O); 167.0 (1C, C=O); 141.2 (2C, Cquart.); 140.4 (2C, Cquart.); 110.6 (1C,
CH); 110.1 (2C, Cquart.); 102.6 (1C, CH ); 45.7 (2C, CH ); 42.2 (1C, CH );
40.2 (1C, CH ); 39.9 (1C, CH ); 36.1 (1C, CH ); 35.9 (1C, CH ); 28.2 (1C,
CH ); 25.0 (1C, CH ); 22.0 (1C, CH ); 17.1 (2C, CH
F-NMR (282 MHz, CDCl
275, 2933, 1710, 1658, 1557, 1451, 1293, 1262, 1155, 1079, 1051 cm
3
2
2
3
3
CH
2
); 2.71 (t, J = 7.1 Hz, 2H, CH
); 1.41-1.24 (m, 2H, CH
); C-NMR (75 MHz, CDCl
2
2
3
C
1.58 (8H, CH
2
2
13
2
2
2
2
2
CH
3
3
3
2
2
2
2
19
2
2
2
); 14.2 ppm (2C, CH
3
); F-NMR (282
2
2
2
3
, 25°C): δ = -77.9 (s, 3F); -79.9 ppm (s, 3F); IR (ATR): v
̅
=
2
2
2
2
-
1
3
2
2
2
2
3
); 13.7 (2C, CH );
+
19
m/z = 576.1548 [M ], calc.: 576.1567.
3
̅
, 25°C): δ = -79.8 ppm (s, 3F); IR (ATR): v =
-
3
;
1
+
HRMS: (EI) m/z = 600.2304 [M ], calc.: 600.2295.
N-(2-(2-(4,8-Dibutyrylbenzo[1,2-d:4,5-d']bis([1,3]dioxole)-2-
yl)acetamido)ethyl)-7,7,7-trifluoro-6-oxoheptanamide
(13b):
Following the procedure described above for 13a, the trifluoromethyl
ketone 13b was obtained from the carboxylic acid 12b (20.0 mg, 108.63
µmol, 1.5 eq.), DIEA (26 µL, 149.26 µmol, 2.0 eq.), TBTU (35.0 mg,
Diethyl 2-(2-oxo-2-((2-(8,8,8-trifluoro-7-oxooctanamido)ethyl)ami-
no)ethyl)benzo[1,2-d:4,5-d']bis([1,3]dioxole)-4,8-dicarboxylate (14c):
Following the procedure described above for 13a, the trifluoromethyl
ketone 14c was obtained from the carboxylic acid 12c (17.9 mg, 84.37
µmol, 1.1 eq.), DIEA (35 µL, 200.93 µmol, 2.7 eq.), TBTU (27.2 mg,
84.71 µmol, 1.1 eq.) and the amine 7 (39.2 mg, 74.75 µmol, 1.0 eq.) in
anhydrous DMF (4.0 mL). Purifying by silica gel chromatography
(DCM/MeOH/TEA 20:1:0.1) give the title compound as a yellow solid
109.00 µmol, 1.5 eq.) and the amine 6 (38.7 mg, 74.36 µmol) in
anhydrous DMF (1.5 mL). Purifying by silica gel chromatography
(
DCM/MeOH/TEA 100:1:0.1) give the title compound as an orange solid
26.8 mg, 71 %). R = 0.47 (DCM/MeOH 10:1); m. p.: decomp. at 175°C;
H-NMR (300 MHz, CDCl , 25°C): δ = 6.82 (bs, 1H, NH); 6.65 (bs, 1H,-
NH); 6.59 (t, J = 4.8 Hz, 1H, CH); 6.11 (s, 2H, CH ); 3.54-3.33 (4H,
CH ); 2.97-2.83 (6H, CH ); 2.73 (bt, 2H, CH ); 2.26 (bt, 2H, CH ); 1.88-
.56 (8H, CH ); 0.97 ppm (t, J = 7.3 Hz, 6H, CH ); C-NMR (75 MHz,
CDCl , 25°C): δ = 196.7 (2C, C=O); 173.3 (1C, C=O); 167.2 (1C, C=O);
41.2 (2C, Cquart.); 140.4 (2C, Cquart.); 110.6 (2C, Cquart.); 110.0 (1C, CH);
02.6 (1C, CH ); 45.7 (2C, CH ); 42.2 (1C, CH ); 40.0 (1C, CH ); 39.9
); 36.1 (1C, CH ); 35.7 (1C, CH ); 24.5 (1C, CH ); 21.8 (1C,
); 17.1 (2C, CH ); 13.7 ppm (2C, CH ); F-NMR (282 MHz, CDCl
= 3284, 2962, 2936, 2877,
678, 1642, 1560, 1436, 1281, 1089 cm ; HRMS: (EI) m/z = 586.2128
(
f
1
3
3
2
(44.4 mg, 98 %). R
f
= 0.48 (DCM/MeOH 10:1); m. p.: decomp. at 195°C;
H-NMR (300 MHz, CDCl , 25°C): δ = 6.75 (bt, 1H, NH); 6.64-6.56 (1H,
); 4.38
); 2.91 (d, J = 5.3 Hz, 2H,
1
2
2
2
2
3
3
13
3
1
2
3
NH); 6.60 (t, J = 5.3 Hz, 1H, CH); 6.11 (d, AB, J =1.6 Hz, 2H, CH
(q, J = 7.1 Hz, 4H, CH
2
3
3
3
2
); 3.49-3.34 (4H, CH
2
3
3
1
1
CH
2
); 2.71 (t, J = 7.0 Hz, 2H, CH
2
); 2.23 (t, J = 7.4 Hz, 2H, CH
2
); 1.73-
); 1.37 ppm (t, J = 7.1 Hz, 6H,
, 25°C): δ = 167.3 (1C, C=O); 161.8 (2C,
C=O); 141.9 (2C, Cquart.); 141.2 (2C, Cquart.); 110.8 (1C, CH); 103.2 (2C,
quart.); 102.9 (1C, CH ); 61.7 (2C, CH ); 42.1 (1C, CH ); 40.1 (1C, CH );
39.8 (1C, CH ); 36.1 (1C, CH ); 35.6 (1C, CH ); 28.2 (1C, CH ); 25.1 (1C,
CH ); 22.0 (1C, CH ); F-NMR (282 MHz, CDCl
= 3278, 2931, 1710, 1657,
3
2
2
2
2
1.58 (m, 4H, CH
2
); 1.41-1.28 (m, 2H, CH
2
13
(1C, CH
2
2
2
2
CH
3
); C-NMR (75 MHz, CDCl
3
1
9
CH
2
1
2
2
3
3
,
5°C): δ = -79.8 ppm (s, 3F); IR (ATR): v
̅
C
2
2
2
2
−
1
2
2
2
2
+
19
[M ], calc.: 586.2138.
2
2
); 14.2 ppm (2C, CH
3
3
,
2
1
6
5°C): δ = -79.5 ppm (s, 3F); IR (ATR): v
̅
-
1
557, 1450, 1293, 1261, 1155, 1079, 1051 cm ; HRMS: (EI) m/z =
Diethyl 2-(2-oxo-2-((2-(7,7,7-trifluoro-6-oxoheptanamido)ethyl)ami-
no)ethyl)benzo[1,2-d:4,5-d']bis([1,3]dioxole)-4,8-dicarboxylate (14b):
Following the procedure described above for 13a, the trifluoromethyl
ketone 14b was obtained from the carboxylic acid 12b (15.5 mg, 78.23
µmol, 1.1 eq.), DIEA (30 µL, 172.23 µmol, 2.5 eq.), TBTU (25.5 mg,
+
04.1869 [M ], calc.: 604.1880.
Protein expression and purification of HDACs
7
9.42 µmol, 1.1 eq.) and the amine 7 (36.6 mg, 69.79 µmol, 1.0 eq.) in
anhydrous DMF (3.0 mL). Purifying by silica gel chromatography
DCM/MeOH/TEA 10:1:0.0→10:1:0.1) give the title compound as a
yellow solid (36.5 mg, 89 %). R = 0.45 (DCM/MeOH 10:1); m. p.:
decomp. at 182°C; H-NMR (300 MHz, CDCl , 25°C): δ = 6.72 (bs, 1H,
NH); 6.67-6.46 (2H, NH, CH); 6.11 (s, 2H, CH
The genes encoding the catalytical domains of HDAC4 (amino acids 648-
1057), HDAC5 (amino acids 655-1122), HDAC7 (amino acids 518-952)
(
and full length HDAC8 were codon optimised and ordered from
GenScribt. The genes were delivered in a pUC57 plasmid and cloned
into a pET14b-SUMO (HDAC8) or a pET14b-SUMO-CPD (HDAC4, 5
and 7) vector. All proteins were recombinantly expressed in E.coli
f
1
3
3
2
); 4.38 (q, J = 7.1 Hz, 4H,
); 3.02-2.83 (m, 2H, CH ); 2.82-2.65 (m, 2H,
); 2.36-2.12 (m, 2H, CH ); 1.77-1.58 (m, 4H, CH
); C-NMR (126 MHz, CDCl , 25°C): δ = 161.9 (2C,
C=O); 141.9 (2C, Cquart.); 141.2 (2C, Cquart.); 110.8 (1C, CH); 103.2 (2C,
quart.); 102.9 (1C, CH ); 61.7 (2C, CH ); 42.2 (1C, CH ); 40.0 (1C, CH );
); 36.1 (1C, CH ); 35.7 (1C, CH ); 24.5 (1C, CH ); 21.8 (1C,
); F-NMR (282 MHz, CDCl , 25°C): δ = -79.8
= 3276, 2943, 1710, 1659, 1557, 1451, 1294,
CH
CH
2
); 3.57-3.30 (4H, CH
2
2
BL21(DE3) as fusion proteins, whereby they were N-terminally fused to a
3
); 1.37 ppm (t, J =
[43]
2
2
2
6xHis-SUMO tag.
HDACs 4, 5 and 7 were additionally fused to a C-
13
7.2 Hz, 6H, CH
3
3
terminal cysteine protease domain (CPD), which possess an
autocatalytic activity and cuts itself from the HDAC upon induction by
C
2
2
2
2
[44]
phytic acid. Bacterial cultures were grown at 37 °C in LB media to an
39.9 (1C, CH
2
2
2
2
optical density at 600 nm of 0.6 - 0.8. The protein expression was
induced by addition of 1 mM isopropyl-β-D-thiogalactopyranoside and the
cells were further cultured overnight at 25 °C. Cells were harvested by
centrifugation (15 min, 15.000xg, 4 °C) and the pellet was resuspended
in lysis buffer (50 mM HEPES, pH 8.0, 300 mM NaCl). Cells were lysed
by sonication and cell fragments were removed by centrifugation (30 min,
25000xg, 4 °C). The supernatant was loaded onto an immobilised metal
affinity chromatography using a cOmplete His-Tag purification resin
(Roche) which was equilibrated with lysis buffer. The resin was washed
with 20 volumes of lysis buffer. For the CPD fusion constructs the resin
19
CH
2
); 14.2 ppm (2C, CH
3
3
ppm (s, 3F); IR (ATR): v
̅
-
1
+
1
5
262, 1156, 1080, 1025 cm ; HRMS: (EI) m/z = 590.1739 [M ], calc.:
90.1723.
N-(2-(2-(4,8-Dibutyrylbenzo[1,2-d:4,5-d']bis([1,3]dioxole)-2-
yl)acetamido)ethyl)-8,8,8-trifluoro-7-oxooctanamide (13c): Following
the procedure described above for 13a, the trifluoromethyl ketone 13c
was obtained from the carboxylic acid 12c (18.2 mg, 85.78 µmol, 1.1 eq.),
This article is protected by copyright. All rights reserved.

Chemistry - A European Journal
10.1002/chem.201605140
FULL PAPER
was afterwards incubated for one hour with 200 µM phytic acid in lysis
buffer. The SUMO fusion proteins were eluted by addition of 100 mM
imidazole in lysis buffer. The SUMO tag was cleaved by incubation with
456 nm. For the binding of the ester DBD ligands 14a-c the experiments
were conducted under the same conditions, but the band-pass filter was
exchanged to 520 nm and the pulse excitation occurred at 405 nm.
Binding of the DBD ligands resulted in an increased FLT, which was
exploited to determine the binding degree (B.D.) according to
20 µg Ulp 1 at 4 °C over night. The proteins were purified further by
gelfiltration using a Sephadex 200 16/60 column equilibrated with HDAC
buffer (50 mM HEPES, pH 8.0, 20 mM NaCl, 10 mM KCl). HDAC
containing fractions were pooled and concentrated. The protein
concentration was determined by using the bicinchoninic acid assay.
Protein solutions were supplemented with 10% glycerol, flash frozen with
liquid nitrogen and stored at -20 °C.
ꢄ−ꢄ푚ꢅ푛
퐵. 퐷. =
(2)
ꢄ푚푎푥−ꢄ푚ꢅ푛
where
A
denotes the FLT in the presence of
a
certain enzyme
concentration of interest, Amin the FLT in the absence of enzyme and
max the FLT in the presence of saturating enzyme concentrations.
A
Enzyme activity assay
From the determined binding isotherms the equilibrium dissociation
constant, K , can be fitted by exploiting the following equation
d
[
26]
The HDAC activity assay was conducted as described elsewhere.
In
brief a serial dilution of the DBD probe or inhibitor in assay buffer (25 mM
Tris-HCl, pH 8.0, 75 mM KCl, 0.001% Pluronic F-127) was placed in a
black 96-well microtiter plate (Greiner) and incubated with 1 nM of the
respective HDAC isoform for 30 min at 30 °C. The reaction was then
initiated by addition of either 50 µM Boc-Lys(Ac)-AMC (for HDAC1, 2, 3
and 6) or 20 µM Boc-Lys(trifluoracetyl)-AMC (for HDAC4, 5, 7 and 8) as
substrate. After an incubation for 60 minutes at 30 °C the reaction was
stopped by addition of 20 µM SAHA or SATFMK and the deacetylated
substrate was converted into a fluorescent product by addition of
[ꢀ퐿]
1
2
퐵. 퐷. = [퐿]ꢁ
=
ꢆ[퐸] + [ꢇ] + 퐾 ꢈ √([퐸] + [ꢇ] + 퐾 ) ꢈ 4[ꢇ] [퐸] ꢉ (3)
0
0
푑
0
0
푑
0
0
2[퐿]ꢁ
where [EL] is the equilibrium concentration of the complex between the
enzyme, E, and the respective DBD-ligand, L. Initial concentrations are
marked by the subscript 0 and other concentrations refer to the chemical
equilibrium state.
I
For the determination of the inhibitor binding constant K of unlabeled
0.5 mg/ml trypsin. The release of AMC was followed in a micro plate
HDAC inhibitors a serial dilution of the respective inhibitor in assay buffer
supplemented with 0.125 µM of the respective DBD probe was placed in
the afore mentioned microtiter plate. 0.45 to 0.8 µM of HDAC8 or HDAC4
reader and correlated to enzyme activity. Dose-response curves were
generated using graph pad prism and fitted to the following four
parameter logistic model yielding the respective IC50-value
[
45]
were added and the plates were incubated for
temperature. Afterwards the FLT of the DBD probe was determined as
stated above. The binding reaction was analyzed with simple
1 hour at room
(
ꢀ푚푎푥−ꢀꢁ)
퐸퐴 = 퐸 +
(1)
0
[(log 퐼퐶 ꢃ푥)ℎ]
1
ꢂ10
5ꢁ
a
competition model (E+L= EL and E+I=EI) where the inhibitor, I, binds to
the enzyme, E, and forms the enzyme-inhibitor complex, EI, which is in
competition to the formation of the complex EL between the DBD probe,
L, and the enzyme
where EA denotes enzyme activity at a certain inhibitor concentration x
and Emax and E are the determined enzyme activity at no and complete
inhibition; IC50 denotes the inhibitor concentration, at which half of the
enzyme is inhibited and h is the hill slope.
0
ꢌ
ꢌ
2
ꢍ
ꢍ
ꢋ퐼ꢂ
(ꢋ퐼ꢂ[ꢏ]ꢁ−[ꢀ]ꢁ)
ꢌ
ꢋ퐼ꢂ
ꢒ + ꢓꢊ
(ꢋ퐼ꢂ[ꢏ]ꢁ−[ꢀ]ꢁ)
ꢌ
Isothermal titration calorimetry
1
퐵. 퐷. = 2 ꢊꢈ
[ꢀ]ꢁ
[ꢎ]ꢁ
[ꢎ]ꢁ
ꢔ[ꢀ]ꢁꢋ퐼[퐿]ꢁ
ꢍ ꢂ1ꢑ
ꢍ
ꢐ
[ꢎ]ꢁ
ꢒ + ꢋ
(4)
ꢌ
ꢌ
ꢌ
ꢍ ꢐ ꢍ ꢂ1ꢑ
ꢎ]ꢁ [ꢎ]ꢁ
ꢍ ꢐ ꢍ ꢂ1ꢑ
[ꢎ]ꢁ [ꢎ]ꢁ
[
I
For the validation of the K -values determined by the fluorescence life
time binding assay binding of SATFMK and PDF306 to HDAC4 and
HDAC8 was investigated by ITC. Therefore, 20 to 40 µM HDAC in HDAC
buffer was placed in the sample cell of a PEAQ-ITC instrument (Malvern
Instruments Ltd) 25 °C. The syringe was loaded with 100 to 200 µM of
the inhibitor suspended in HDAC buffer. To minimise the heat of dilution
the DMSO and glycerol concentrations were adjusted to be identical in
the sample cell and syringe. Nevertheless, the resulting final DMSO
concentrations of up to 2% resulted in a reduction of the binding active
HDAC concentration which resulted in a observed stoichiometry between
Acknowledgements
The work has been supported by the Deutsche
Forschungsgemeinschaft (DFG Grant ME 3122/2-1).
Keywords: Fluorescent probes • Hydrolases • High-throughput
0.13 and 0.4. The measurements were carried out at 25 °C and the
screening
inhibitor was titrated by injecting 19 times 2 µl into the sample cell with a
spacing of 150 to 300 s. The amount of released heat for each injection
was determined by integrating the area under the curve by using the
MicroCal PEAQ-ITC Analysis software. The resulting data points were
the fitted to a one site binding model supplied with the software.
[
1]
2]
I. Hoshino, H. Matsubara, Surg. Today. 2010, 40, 809–15.
[
D.-M. Chuang, Y. Leng, Z. Marinova, H.-J. Kim, C.-T. Chiu, Trends
Neurosci. 2009, 32, 591–601.
[
3]
K. T. Andrews, A. Haque, M. K. Jones, Immunol. Cell Biol. 2011, 90,
66–77.
Fluorescence life time binding assay
[
[
[
4]
5]
6]
X.-J. Yang, E. Seto, Nat. Rev. Mol. Cell Biol. 2008, 9, 206–18.
I. Gregoretti, Y.-M. Lee, H. V. Goodson, J. Mol. Biol. 2004, 338,17–31.
S. Yoon, G. H. Eom, Chonnam Med. J. 2016, 52,1–11.
In order to determine the equilibrium dissociation constant of the binding
of the acyl DBD ligands 13a-c to HDAC4, 5, 7 and 8 a serial dilution of
the respective HDAC was placed in the afore mentioned microtiter plate
and mixed with 25 to 125 nM of the DBD ligand. After incubation for 30
minutes at 25 °C the fluorescence decay was measured using a LF502
nanoscan instrument from Berthold Detection Systems GmbH (Pforzheim,
Germany) behind a 630 nm band-pass filter after pulse excitation at
[7]
8]
P. A. Marks, R. Breslow, Nat. Biotechnol. 2007, 25, 84–90.
P. A. Marks, W.-S. Xu, J. Cell. Biochem. 2009,107, 600–608.
[
This article is protected by copyright. All rights reserved.

Chemistry - A European Journal
10.1002/chem.201605140
FULL PAPER
[
9]
A. Lahm, C. Paolini, M. Pallaoro, M. Nardi, P. Jones, P. Neddermann,
S. Sambucini, M. J. Bottomley, P. lo Surdo, A. Carfí, U. Koch, R. de
Francesco, C. Steinkühler, P. Gallinari, Proc. Natl. Acad. Sci. U. S. A.
[27]
F. Halley, J. Reinshagen, B. Ellinger, M. Wolf, A. L. Niles, N. J. Evans,
T. A. Kirkland, J. M. Wagner, M. Jung, P. Gribbon, S. Gul, J. Biomol.
Screening 2011,16,1227–1235.
2007, 104,17335–17340.
[
28]
29]
D. Riester, D. Wegener, C. Hildmann, A. Schwienhorst, Biochem.
Biophys. Res. Commun. 2004, 324, 1116–1123.
[
10]
11]
A. Clocchiatti, E. di Giorgio, F. Demarchi, C. Brancolini, Cell.
Signalling 2013, 25, 269–276.
[
R. K. Singh, T. Mandal, N. Balasubramanian, G. Cook, D. Srivastava,
[
M. Lobera, K. P. Madauss, D. T. Pohlhaus, G. Q. Wright, M. Trocha,
D. R. Schmidt, E. Baloglu, R. P. Trump, M. S. Head, G. A. Hofmann,
M. Murray-Thompson, B. Schwartz, S. Chakravorty, Z. Wu, P. K.
Mander, L. Kruidenier, R. A. Reid, W. Burkhart, B. J. Turunen, J. X.
Rong, C. Wagner, M. B. Moyer, C. Wells, X. Hong, J. T. Moore, J. D.
Williams, D. Soler, S. Gosh, M. A. Nolan, Nat. Chem. Biol. 2013, 9,
Anal. Biochem. 2011, 408, 309–315.
[30]
D, Riester, C. Hildmann, P- Haus, A. Galetovic, A. Schober, A.
Schwienhorst, F.-J. Meyer-Almes, Bioorg. Med. Chem. Lett. 2009,19,
3651–3656.
3
19–325
[31]
[32]
[33]
[34]
C. Meyners, M. G. Baud, M. J. Fuchter, F.-J. Meyer-Almes, J. Mol.
Recognit. 2014, 27, 160–72.
[12]
P. Jones, S. Altamura, R. de Francesco, P. Gallinari, A. Lahm, P.
Neddermann, M. Rowley, S. Serafini, C. Steinkühler, Bioorg. Med.
Chem. Lett. 2008, 18, 1814–1819
L. Neumann, K. von Kӧnig, D. Ullmann, Methods Enzymol. 2011, 493,
299–320.
[
[
[
13]
14]
15]
W. Fischle, F. Dequiedt, M. J. Hendzel, M. G. Guenther, M. A. Lazar,
W. Voelter, E. Verdin, Mol. Cell. 2002, 9, 45-57
C. Meyners, R. Wawrzinek, A. Krämer, S. Hinz, P. Wessig, F.-J.
Meyer-Almes, Anal. Bioanal. Chem. 2014, 406,4889–4897.
A: Clocchiatti, E. di Giorgio, S. Ingrao, F.-J. Meyer-Almes, C. Tripodo,
C. Micelli, G. Rastelli, Drug Discovery Today 2015, 20, 718–735
C. Brancolini, FASEB J. 2013, 27, 942–954.
[
35]
A. S. Madsen, H. M. Kristensen, G. Lanz, C. A. Olsen,
ChemMedChem 2014, 9, 614–626.
E. di Giorgio, A. Clocchiatti, S. Piccinin, A. Sgorbissa, G. Viviani, P.
Peruzzo, S. Romeo, S. Rossi, A. P. dei Tos, R. Maestro, C.
Brancolini, Mol. Cell. Biol. 2013, 33, 4473–4491
[36]
R. R. Frey, C. K. Wada, R. B. Garland, M. L. Curtin, M. R.
Michaelides, J. Li, L. J. Pease, K. B. Glaser, P. A. Marcotte, J. J.
Bouska, S. S. Murphy, S. K. Davidsen, Bioorg. Med. Chem. Lett.
2002, 12, 3443–3447.
[16]
D. B. Sparrow, E. A. Miska, E. Langley, S. Reynaud-Deonauth, S.
Kotecha, N. Towers, G. Spohr, T. Kouzarides, T. J. Mahun, EMBO J.
1999, 18, 5085–5098
[37]
R. Wawrzinek, J. Ziomkowska, J. Heuveling, M. Mertens, A.
Herrmann, E. Schneider, P. Wessig, Chem. – Eur. J. 2013,19, 17349–
17357.
[
17]
18]
C. H, Arrowsmith, C. Bounta, P. V. Fish, K. Lee, M. Schapira, Nat.
Rev. Drug Discov. 2012,11, 384–400
[
J. E. Bradner, N. West, M. L. Grachan, E. F. Greenberg, S. J.
[38]
J. A. Cisneros, E. Bjꢀrklund, I. Gonzꢁlez-Gil, Y. Hu, A. Canales, F. J.
Medrano, A. Romero, S. Ortega-Gutiérrez, C. J. Fowler, M. L. López-
Rodríguez, J. Med. Chem. 2012, 55, 824–836.
Haggarty, T. Warnow, R. Mazitschek, Nat. Chem. Biol. 2010, 6, 238–
243
[
19]
20]
S. Mujtaba, L. Zeng, M. M. Zhou, Oncogene 2007, 26, 5521–5527
[39]
[40]
T. Okano, T. Sakaida, S. Eguchi, J. Org. Chem. 1996, 61, 8826–8830.
J. Boivin, L. el Kaim, S. Z. Zard, Tetrahedron 1995, 51, 2573–2584.
[
E. di Giorgio, E. Gagliostro, C. Brancolini, Cell. Mol. Life Sci. 2015, 72,
73–86
[41]
T. K. Nielsen, C. Hildmann, D. Riester, D. Wegener, A: Schwienhorst,
[
21]
22]
H. L. Fitzsimons, Neurobiol. Learn Mem. 2015, 123, 149–158
R. Ficner, Acta Crystallogr., Sect. F: Struct. Biol. Cryst. Commun.
2007, 63, 270–273.
[
M. Mielcarek, D. Zielonka, A. Carnemolla,J. T. Marcinkowski, F.
Guidez, Front. Cell. Neurosci. 2015, 9, 42.
[42]
P. Jones, M. J. Bottomley, A. Carfí, O. Cecchetti, F. Ferrigno, P. L.
Surdo, J. M. Ontoria, M. Rowley, R. Scarpelli, C. Schultz-Fademrecht,
C. Steinkühler, Bioorg. Med. Chem. Lett. 2008, 18, 3456–3461.
[
23]
24]
C. A. Luckhurst, P. Breccia, A. J. Stott, O. Aziz, H. L. Birch, R. Bürli,
S. J. Hughes, R. E. Jarvis, M. Lamers, P. M. Leonard, K. L. Matthews,
G. McAllister, S. Pollack, E. Saville-Stones, G. Wishart, D. Yates, C.
Dominguez, ACS Med. Chem. Lett. 2015, 7, 34–39.
[43]
[44]
M. P. Malakhov, M. R. Mattern, O. A. Malakhova, M. Drinker, S. D.
Weeks, T. R. Butt, J. Struct. Funct. Genomics 2004, 5, 75–86.
[
M.-C. Choi, T. J. Cohen, T. Barrientos, B. Wang, M. Li, B. J.
A. Shen, P. J. Lupardus, M. Morell, E. L. Ponder, A. M. Sadaghiani, K.
Simmons, J. S. Yang, G. A. Cox, Y. Zhao, T. P. Yao, Mol. Cell. 2012,
C. Garcia, M. Bogyo, PloS one, 2009, 4, e8119.
47, 122–132.
[45]
A. Vølund, Biometrics 1978, 34, 357–365.
[
25]
26]
N. A. Wolfson, C. A. Pitcairn, E. D. Sullivan, C. G. Joseph, C. A.
Fierke, Anal. Biochem. 2014, 456, 61–69
[
D. Wegener, C. Hildmann, D. Riester, A. Schwienhorst, Anal.
Biochem. 2003, 321, 202–208.
This article is protected by copyright. All rights reserved.

Chemistry - A European Journal
10.1002/chem.201605140
FULL PAPER
Entry for the Table of Contents (Please choose one layout)
Layout 1:
FULL PAPER
Text for Table of Contents
Author(s), Corresponding Author(s)*
Page No. – Page No.
Title
((Insert TOC Graphic here: max.
width: 5.5 cm; max. height: 5.0 cm))
Layout 2:
FULL PAPER
Author(s), Corresponding Author(s)*
((Insert TOC Graphic here; max. width: 11.5 cm; max. height: 2.5 cm))
Page No. – Page No.
Title
Text for Table of Contents
This article is protected by copyright. All rights reserved.