Synthesis of Trifluoromethyl Ketone Containing Amino Acid Building Blocks for the Preparation of Peptide-Based Histone Deacetylase (HDAC) Inhibitors

Article abstract of DOI:10.1055/s-0037-1609945

Trifluoromethyl ketones (TFMKs) are electrophilic moieties which hydrate readily in aqueous media to give geminal diols. This ability has been exploited for the development of histone deacetylase (HDAC) inhibitors, because HDAC enzymes contain a Zn ²⁺ ion which may be chelated by this functionality. Interestingly, TFMKs are exceptional Zn ²⁺-binding groups for targeting the intriguing class IIa HDAC isozymes, involved in transcription factor recruitment and gene regulation. Here, we have developed a scalable and inexpensive synthetic procedure for preparation of the enantiomerically pure TFMK-containing amino acid building block (S)-2-amino-9,9,9-trifluoro-8-oxononanoic acid (Atona). In addition, we propose a protecting group strategy applicable to automated solid-phase peptide synthesis and demonstrate the ability of Atona-containing peptides to inhibit the enzymatic activity of class IIa HDACs with nanomolar potency. We envision that this synthesis will motivate the further development of peptide-based probes for the study of class IIa HDACs.

Full text of DOI:10.1055/s-0037-1609945

SYNTHESIS
0
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9
-
7
8
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© Georg Thieme Verlag Stuttgart · New York
2018, 50, 4037–4046
feature
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en
C. Moreno-Yruela et al.
Feature
Synthesis
Synthesis of Trifluoromethyl Ketone Containing Amino Acid
Building Blocks for the Preparation of Peptide-Based Histone
Deacetylase (HDAC) Inhibitors
Carlos Moreno-Yruela
0
0
0
0
0-
0
0
3
2-
4
5
0
6-
5
1
9
O
O
COOEt
COOEt
O
Christian A. Olsen*
0
0
0
0
0-
0
0
2
2-
9
5
3
8-
9
4
2
+
+
Br
F3C
O
CF3
AcHN
OH
Center for Biopharmaceuticals & Department of Drug Design
and Pharmacology, Faculty of Health and Medical Sciences,
University of Copenhagen, 2100 Copenhagen, Denmark
cao@sund.ku.dk
O
5–6 steps
CF3
1,3-dioxane
protection
COOH
O
O
O
NH2
Ac
PGHN
>99% ee
CF3
Class IIa HDAC inhibitor
Nanomolar potency
SPPS
Received: 28.06.2018
Accepted after revision: 09.08.2018
Published online: 29.08.2018
of epigenetic effectors through protein–protein interac-
tions.7,8 However, these HDAC isotypes have not been char-
acterized as extensively as class I and class IIb HDACs, and
their biological roles are still not completely understood.7,9
The development of organic molecules targeting HDACs
often takes advantage of the presence of the Zn2+ ion in the
active site. Common probes and inhibitors contain a Zn2+-
binding moiety such as hydroxamic acid, o-aminoanilide, or
thiol, attached to a hydrophobic linker that allows it to
reach into the pocket.5 In the case of class IIa HDACs, trifluo-
romethyl ketones (TFMKs) have proven to be one of the
most effective Zn2+-binding groups (Figure 1, a).10–12
DOI: 10.1055/s-0037-1609945; Art ID: ss-2018-z0437-fa
Abstract Trifluoromethyl ketones (TFMKs) are electrophilic moieties
which hydrate readily in aqueous media to give geminal diols. This abil-
ity has been exploited for the development of histone deacetylase
(HDAC) inhibitors, because HDAC enzymes contain a Zn2+ ion which
may be chelated by this functionality. Interestingly, TFMKs are excep-
tional Zn2+-binding groups for targeting the intriguing class IIa HDAC
isozymes, involved in transcription factor recruitment and gene regula-
tion. Here, we have developed a scalable and inexpensive synthetic pro-
cedure for preparation of the enantiomerically pure TFMK-containing
amino acid building block (S)-2-amino-9,9,9-trifluoro-8-oxononanoic
acid (Atona). In addition, we propose a protecting group strategy appli-
cable to automated solid-phase peptide synthesis and demonstrate the
ability of Atona-containing peptides to inhibit the enzymatic activity of
class IIa HDACs with nanomolar potency. We envision that this synthesis
will motivate the further development of peptide-based probes for the
study of class IIa HDACs.
Key words trifluoromethyl ketones, histone deacetylase, histone
deacetylase inhibitors, epigenetic, amino acids, peptides, protecting
groups
The Zn2+-dependent histone deacetylases (HDACs) are a
family of enzymes that hydrolyze various acylamide groups
on the side chains of lysine residues and polyamines.1,2 De-
regulation of HDACs has been associated with neurological
and autoimmune diseases,3,4 and a handful of HDAC inhibi-
tors have reached the clinic for the treatment of cancer.5,6
Interestingly, a subset of the 11 human enzyme isotypes
(HDACs 4, 5, 7, and 9, grouped into class IIa HDACs) does
not exhibit enzymatic activity against any physiologically
relevant acyl modifications tested in vitro thus far, which
correlates with a catalytic tyrosine being replaced by histi-
dine.7 Instead of being hydrolases in vivo, class IIa HDACs
have been speculated to be responsible for the recruitment
Figure 1 (a) Chemical structure of two class IIa HDAC inhibitors bear-
ing trifluoromethyl ketone Zn2+-binding groups (compounds 1 and
2).10,11 Peptide macrocycle 2 contains the (S)-2-amino-9,9,9-trifluoro-
8-oxononanoic acid (Atona) amino acid residue (cyan shading).
(b) Binding mode of hydrated inhibitor 1 to the Zn2+ ion in the active
site of HDAC4 (PDB: 2VQJ). (c) Proposed binding mode of the side chain
of Atona to Zn2+ in the active site of class IIa HDACs.
© Georg Thieme Verlag Stuttgart · New York — Synthesis 2018, 50, 4037–4046

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C. Moreno-Yruela et al.
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Synthesis
These highly electrophilic moieties, also employed for
the inhibition of serine proteases13–16 and other hydrolytic
enzymes,17–22 are readily hydrated in aqueous media, which
generates a geminal diol that chelates the Zn2+ ion (Figure 1,
b and c).2,11,19,23 This feature, together with the ability of the
trifluoromethyl group to fill space left by the tyrosine to
histidine mutation, has been exploited for developing in-
hibitors of class IIa HDACs.10–12,23–38 Similarly, nonphysiolog-
ically relevant ε-N-trifluoroacetyllysine-based substrates
have become important for the assaying of class IIa HDAC
inhibitors.7,8,11
Peptide macrocycles constitute an important type of
HDAC inhibitors,39 but just two TFMK-containing macrocy-
cles have been reported and both exhibited remarkable
HDAC inhibitory potencies.10,31 Here, we present a straight-
forward, scalable, and cost-effective synthetic strategy for
the preparation of building blocks based on the Atona ami-
no acid (Figure 1, a: cyan shading in compound 2). Our
strategy employs modified Dakin–West conditions for the
generation of the TFMK warhead and enzymatic resolution
for the derivatization enabling subsequent separation of the
desired enantiomer. In addition, protection of the amino
acid side chain was achieved via cyclic ketal formation,
which opens the opportunity for its application in standard
Fmoc/t-Bu solid-phase peptide synthesis (SPPS). This build-
ing block will simplify future efforts toward class IIa HDAC-
targeting peptides for unraveling the function and biologi-
cal role of these enzyme isotypes.
Modified Dakin–West conditions have been employed
for the synthesis of various TFMK-containing HDAC inhibi-
tors before, including small molecules and peptides.10,24,31–
33,40
However, the success of the reaction is highly variable
and most efficient for rather simple molecules,24,32,40 thus
posing a major limitation for late-stage functionalization of
amino acids and peptides.10,31 Other methods rely on the
reaction of the Ruppert–Prakash reagent (CF3TMS) with es-
ters or Weinreb amides,35,38,41–43 on Claisen condensation
with trifluoroacetates followed by decarboxylation under
forcing conditions,44,45 or on recently developed radical re-
actions with vinyl sulfones.46 Nevertheless, a synthetic pro-
cedure compatible with peptide chemistry is yet to be es-
tablished and the preparation of a TFMK-containing amino
acid building block remains a more robust strategy. Previ-
ous synthetic efforts in our group highlighted the limita-
tions behind current TFMK preparation methods.32 There-
fore, our initial strategy was based on the introduction of
this functionality on 6-bromohexanoyl chloride as a first
step, as reported by Marsilje et al.,40 because a simple mole-
cule would increase the chances of success for the modified
Dakin–West transformation. Even though the reaction yield
was acceptable (>70% in some cases), the 7-bromo-1,1,1-
trifluoroheptan-2-one product was volatile, which caused
problems during workup. Furthermore, the reaction of this
intermediate with diethyl acetamidomalonate provided a
mixture of products, which could only be avoided by pro-
tecting the TFMK group. Marsilje et al. had already masked
this moiety in the form of a 1,3-dioxolane, which could be
removed with BBr3 in CH2Cl2.40 However, as these condi-
Biographical Sketches
Carlos Moreno-Yruela stud-
ied chemistry at the University
of Zaragoza (Spain) where he
worked under the supervision
of Dr. Raquel Andreu on the de-
velopment of push–pull organic
dyes with second-order nonlin-
ear optical properties. He then
obtained his M.Sc. in Drug Dis-
covery with distinction from the
University of Surrey (UK), focus-
ing for his thesis on the synthe-
sis of magnetically-tagged
molecularly imprinted polymers
with potential application in
protein biosensors. After a short
period working on a JAE scholar-
ship from the Spanish Research
Council, he joined the laborato-
ry of Prof. Christian A. Olsen in
2016 to perform his Ph.D. stud-
ies on the development of pep-
tide-based chemical tools for
the study of histone deacetylase
(HDAC) function.
Christian A. Olsen received
his M.Sc. from the Technical
University of Denmark in 2000
and his Ph.D. from the Danish
University of Pharmaceutical
Sciences in 2004. After inde-
pendently working on the
development of novel peptido-
mimetics, he undertook a post-
doctoral fellowship with Prof.
Ghadiri at The Scripps Research
Institute. In 2010 he returned to
a faculty position at the Techni-
cal University of Denmark and
in 2014 he accepted his current
position as professor at the Uni-
versity of Copenhagen. He is a
recipient of the Lundbeck Foun-
dation Fellowship (2010), the
EFMC Award for a Young Medici-
nal Chemist in Academia
(2014), and an ERC Consolidator
Grant in 2016. His research in-
terests include foldamers, HDAC
inhibitors, and quorum sensing
modulators, as well as the inves-
tigation of protein lysine acyla-
tion.
© Georg Thieme Verlag Stuttgart · New York — Synthesis 2018, 50, 4037–4046

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C. Moreno-Yruela et al.
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Synthesis
O
COOEt
O
COOEt
O
d
b, c
Br
R
AcHN
Ot-Bu
AcHN
Ot-Bu
COOEt
3: R = OH
5
6
a
4: R = Ot-Bu
COOEt
O
COOH
O
COOH
O
e, f, g
h, i, j
+
AcHN
CF3
AcHN
CF3
RHN
CF3
7
8
9a: R = Fmoc
9b: R = Cbz
Scheme 1 Synthesis of Atona building blocks 9a and 9b. Reagents and conditions: (a) t-BuOH, DCC, DMAP, CH2Cl2, r.t., overnight, 75%; (b) NaI, acetone,
reflux, overnight, 96%; (c) NaH, diethyl acetamidomalonate, DMF, r.t., overnight, 91%; (d) LiCl, H2O, DMSO, 150 °C, overnight, 81%; (e) TFA, CH2Cl2, r.t.,
1.5 h; (f) SOCl2, CH2Cl2, 50 °C, 2 h; (g) TFAA, pyridine, CH2Cl2, r.t., 1.5 h; then H2O, r.t., 30 min, 28–48% (from 6); (h) NaOH, EtOH/H2O (1:1), r.t., 1 h; (i)
Acylase I from Aspergillus melleus, CoCl2·6H2O, phosphate buffer, 40 °C, overnight; (j) Fmoc or Cbz N-hydroxysuccinimide ester, 1,4-dioxane, r.t., over-
night, 41–48% from (S)-7.
tions would not be compatible with peptide chemistry,
combined with the problematic volatility of 7-bromo-1,1,1-
trifluoroheptan-2-one and its derivatives, we proceeded
with the strategy presented in Scheme 1 instead.
compound 7 was selected as the starting point for introduc-
ing a ketal protecting group (Scheme 2), thus avoiding the
volatility issues mentioned above for 7-bromo-1,1,1-trifluo-
roheptan-2-one.
The procedure reported herein was inspired by the syn-
thesis of an L-α-aminosuberic acid building block reported
by Kahnberg et al.47 First, 6-bromohexanoic acid (3) was
protected as a tert-butyl ester, converted into the corre-
sponding iodide through the Finkelstein reaction, and re-
acted with diethyl acetamidomalonate in the presence of
NaH to afford compound 5. Then, Krapcho decarboxylation
yielded compound 6, which is where the present synthesis
deviates from the previously mentioned route. Here, the
tert-butyl group was removed under acidic conditions, fol-
lowed by treatment with first SOCl2 and then trifluoroace-
tic anhydride and pyridine, before water was finally added
to effect the modified Dakin–West reaction to give 7.48 The
yields for this transformation were lower and varied more
between experiments (28–48% based on three individual
reactions) compared to the previous attempt on 6-bromo-
hexanoyl chloride. However, the reaction was scalable and
still considered to be early in the route, so we decided to
continue with this strategy. The ethyl ester of 7 was hydro-
lyzed and Acylase I from Aspergillus melleus (>0.5 U/mg)49
was utilized to selectively deacetylate the S-enantiomer (L-
amino acid) from the racemic mixture.47,50 Finally, the re-
sulting free amine was protected as common carbamates
(Fmoc or Cbz) to give Atona building blocks 9a and 9b in 6–
13% overall yield and over 99% ee (calculated using Marfey’s
method, see the Supporting Information Figure S1).51,52
Compounds 9a and 9b represent protected versions of
Atona ready to be introduced into potential peptide-based
probes. However, the electrophilic character of the TFMK
group makes it prone to formation of unwanted species
during peptide synthesis. Thus, Atona derivatives 9a and 9b
are best suited for introduction in the last step of a peptide
synthesis to avoid the formation of truncated species. Find-
ing a suitable TFMK protecting group was therefore consid-
ered to broaden the scope of this building block. This time,
n
COOEt
a
O
O
7
AcHN
CF3
10a: n = 0
10b: n = 1
10c: n = 2
COOH
O
O
O
AcHN
CF3
11
b, c, d
10b
+
COOH
O
FmocHN
CF3
12
Scheme 2 Synthesis of side-chain-protected Atona building block 12.
Reagents and conditions: (a) 2-chloroethanol, 3-chloro-1-propanol or
4-chloro-1-butanol, K2CO3, DMF, r.t. or 100 °C, 5–23%; (b) NaOH,
EtOH/H2O (1:1), r.t., 1 h; (c) Acylase I from Aspergillus melleus, CoCl2·6H2O,
phosphate buffer, 40 °C, overnight; (d) Fmoc N-hydroxysuccinimide es-
ter, 1,4-dioxane, r.t., overnight, 60% from (S)-10b.
First, 1,3-dioxolane protection was attempted, which
had been reported to be readily introduced under mild ba-
sic conditions and to be cleaved using strong Lewis acids.40
Thus, it was not surprising that compound 10a, bearing this
moiety, remained protected after overnight incubation in
4% CF3SO3H in TFA (v/v), rendering it undesirable for SPPS.
We then aimed to synthesize more labile 1,3-dioxane and
1,3-dioxepane protecting moieties (compounds 10b and
10c, respectively), for which we had to increase the tem-
perature of the reaction to 100 °C. This provided the desired
compounds, but it also led to the formation of byproducts.
In the case of 10c, an estimated 5% yield of the desired com-
pound was obtained and this protecting group was aban-
© Georg Thieme Verlag Stuttgart · New York — Synthesis 2018, 50, 4037–4046

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C. Moreno-Yruela et al.
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Synthesis
doned. Fortunately, compound 10b bearing a 1,3-dioxane
group was isolated in 23% yield and successfully converted
back to the ketone under CF3SO3H/TFA conditions. Hydroly-
sis, enzymatic resolution, and in situ protection of this in-
termediate generated compound 12 (2–4% overall yield
from 3), a side-chain-protected Atona analogue suitable for
Fmoc/t-Bu SPPS.
The incorporation of nonprotected and protected build-
ing blocks 9a and 12 into peptides was subsequently tested.
For this purpose, a linear 9-mer peptide inspired by the se-
quence of human histone 3 (H3) was synthesized via stan-
dard automated SPPS on Rink amide resin.53 The chosen se-
quence (-TARKSTGG-) features a lysine residue (H3K9)
known to be acetylated in human cells and thus recognized
by several HDACs among other epigenetic proteins.6,54 This
residue was substituted by Atona in the attempt to design a
substrate-based inhibitor. In addition, a C-terminal trypto-
phan residue was added due to its properties as a chromo-
phore for UV detection (Figure 2). Following SPPS synthesis,
cleavage from the resin and concomitant deprotection were
achieved by treatment with a standard TFA-containing
cocktail for 2 hours. When building block 9a was used, the
obtained peptide crude purity was only 23% (peptide 13,
Figure 2, a).55 Since fluoro ketones are stable toward imine
formation,56 the reason for this was considered to be the
formation of an eight-membered cyclic hemiaminal be-
tween the free N-terminal and the Atona TFMK group
during the coupling of the n – 1 amino acid. The reversible
character of this transformation would also explain the ab-
sence of a major truncated peptide impurity, but rather a
mixture of byproducts. We attempted to isolate the major
byproduct, appearing at tR ≈13.5 minutes; however, we
were not able to unambiguously determine its structure.
When employing building block 12, on the other hand, the
crude purity was significantly improved (45% for peptide
13a, Figure 2, b). Interestingly, this ketal protecting group
remained stable during TFA incubation, which allowed us
to purify and characterize peptide 13a. Also, in order to un-
mask the TFMK group of Atona, a crude peptide sample of
13a was exposed to a stronger acidic cocktail containing
TFA/CF3SO3H/H2O/i-Pr3SiH [80.5:12:5:2.5 (v/v)] for 2 hours.
Under these conditions, deprotected peptide 13 was ob-
tained in a similar purity as the protected material (43%,
Figure 2, b; see also CF3SO3H concentration screen per-
formed on purified peptide 13a in the Supporting Informa-
tion Figure S2).
Lastly, we tested the ability of the TFMK-functionalized
peptide 13 to inhibit class IIa HDACs by applying a fluoro-
genic, activity-based in vitro enzyme assay. We screened 13
against a panel of recombinant Zn2+-dependent HDACs, uti-
lizing an ε-N-acetyllysine-containing substrate (Ac-LGKac-
AMC) for HDACs 1–3 and 6,57 an ε-N-trifluoroacetyllysine
substrate (Ac-LGKtfa-AMC) for class IIa HDACs (HDACs 4, 5,
7, and 9) and HDAC8,8 and a recently discovered ε-N-myris-
toyllysine-containing substrate (Ac-ETDKmyr-AMC) for
HDAC11.58 Peptide 13 exhibited selectivity for inhibition of
class IIa HDACs and the class I HDAC8, for which it reduced
the deacylase activity below 50% at 0.1 μM concentration.
The remaining HDAC isotypes retained over 75% activity
under similar conditions and HDAC11 was not inhibited
(Figure 3, a). Furthermore, dose–response experiments re-
vealed inhibition constants (Ki values) in the nanomolar
range for peptide 13 against class IIa HDACs and HDAC8
(Figure 3, b; see full curves in the Supporting Information
Figure S3). The control compound, common HDAC inhibitor
trichostatin A, exhibited Ki values >20-fold higher in the
case of class IIa HDACs, which is in good agreement with
values previously reported by Bradner, Mazitschek, and co-
workers.8
In conclusion, we have developed a scalable synthesis of
protected building blocks of the unnatural amino acid Ato-
na, which does not require expensive starting materials or
laborious experimental conditions. In addition, we report a
side-chain protecting group which allows for the synthesis
of Atona-containing peptides using Fmoc/t-Bu SPPS. The
electrophilic trifluoromethyl ketone warhead of this build-
ing block is hydrated in aqueous media, and the resulting
geminal diol is able to chelate the Zn2+ ion present in the ac-
Figure 2 (a) Crude HPLC trace for the synthesis of peptide 13 using
building block 9a. (b) Crude HPLC trace for the synthesis of peptide 13a
containing dioxane-protected building block 12 (cyan trace), and for
the resulting crude peptide 13 after deprotection of the dioxane group
(black trace). HPLC traces were recorded at 280 nm (linear gradient
0–95% of solvent B in solvent A during t = 5–35 min).
© Georg Thieme Verlag Stuttgart · New York — Synthesis 2018, 50, 4037–4046

4041
C. Moreno-Yruela et al.
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Synthesis
setup using granular silica gel (60 Å pore size, 15–40 μm) as station-
ary phase and EtOAc/heptane as eluent. 1H NMR and 13C NMR spectra
were recorded on a Bruker Avance III HD instrument equipped with a
cryogenically cooled probe, at 600 MHz and 151 MHz, respectively.
19F NMR spectra were recorded on a Bruker Advance III instrument
equipped with a 5 mm BBFO probe, at 376 MHz. Chemical shifts are
reported in ppm relative to deuterated solvent as internal standard.
Assignment of NMR spectra was based on correlation spectroscopy
(COSY, HSQC, and HMBC spectra). UPLC-MS analyses were performed
on a Waters Acquity ultra high-performance liquid chromatography
system equipped with a C18 Phenomenex Kinetex column (50 × 2.1
mm, 1.7 μm, 100 Å) and PDA and SQ detectors, using a 0–95% linear
gradient of eluent II (0.1% HCOOH in MeCN) in eluent I (0.1% HCOOH
in MilliQ H2O) during t = 0.20–4.80 min at 0.6 mL/min flow rate. MAL-
DI-TOF mass spectra were recorded on a Bruker Microflex bench-top
MALDI system using α-cyano-4-hydroxycinnamic acid (CHCA) in
H2O/MeCN/TFA [50:50:0.1 (v/v)] as matrix. Analytical reversed-phase
HPLC was performed on an Agilent 1100 LC system equipped with a
C18 Phenomenex Kinetex column (150 × 4.60 mm, 2.6 μm, 100 Å)
and a diode array UV detector, using a 0–95% linear gradient of eluent
B (0.1% TFA in MeCN) in eluent A [MilliQ H2O/MeCN/TFA, 95:5:0.1
(v/v)] during t = 5–35 min, with a flow rate of 1 mL/min. High-resolu-
tion mass spectrometry was performed on a Bruker SolariX ESI in-
strument.
tert-Butyl 6-Bromohexanoate (4)
Figure 3 (a) Inhibitory potency of peptide 13 at 1.0 μM and 0.1 μM
concentration against the deacylase activity of HDACs 1–9 and 11. Bars
are based on two individual experiments performed in duplicate and
represent mean ± SD. *HDAC3 assayed with the deacetylase activating
domain of nuclear co-repressor 2 (NCoR2) for activity purposes. (b) Ki
inhibition constants of peptide 13 and reference compound trichostatin
A (TSA) against class IIa HDACs and HDAC8. Values are based on two
individual experiments performed in duplicate and represent mean
± SD.
A solution of 6-bromohexanoic acid (3, 14.34 g, 73.5 mmol, 1.0 equiv)
in CH2Cl2 (120 mL) was cooled to 0 °C under N2 atmosphere. Thereaf-
ter, t-BuOH (20.7 mL, 221 mmol, 3.0 equiv), DMAP (0.90 g, 7.35 mmol,
0.1 equiv), and DCC (16.68 g, 80.9 mmol, 1.1 equiv) were added and
the mixture was allowed to reach r.t. and stir overnight. Then, the
white suspension was filtered and the solid was repeatedly washed
with CH2Cl2. The combined organic layers were concentrated in vacuo
and the crude product was absorbed on silica gel and purified by col-
umn chromatography (0–10% EtOAc in heptane) to afford 4 as a color-
less oil (13.82 g, 75%).
1H NMR (600 MHz, CDCl3): δ = 3.41 (t, J = 6.8 Hz, 2 H, BrCH2), 2.23 (t,
J = 7.4 Hz, 2 H, CH2CO), 1.87 (tt, J = 7.4, 6.8 Hz, 2 H, BrCH2CH2), 1.61 (p,
J = 7.4 Hz, 2 H, CH2CH2CO), 1.46 (p, J = 7.4 Hz, 2 H, BrCH2CH2CH2), 1.44
(s, 9 H, t-Bu).
13C NMR (151 MHz, CDCl3): δ = 173.0 (C=O), 80.3 (C(CH3)3), 35.5
(CH2CO), 33.7 (BrCH2), 32.6 (BrCH2CH2), 28.3 (C(CH3)3), 27.8
tive site of HDACs to provide nanomolar potency against
class IIa HDAC isotypes. Therefore, it will be a useful build-
ing block for the synthesis and development of peptide-
based HDAC inhibitors and probe compounds. We envision
that the Atona building block can be incorporated into im-
mobilized peptide probes or nucleosomes for pull-down of
class IIa HDACs from biological samples. This will be power-
ful for the study of the localization and protein–protein in-
teraction network of class IIa HDACs, similar to previous re-
search carried out on class I HDACs applying alternatively
functionalized peptides.59–61 This is currently under investi-
gation in our laboratories and will be reported in due
course.
(BrCH2CH2CH2), 24.4 (CH2CH2CO).
+
HRMS: m/z [M + H]+ calcd for C10H20BrO2
: 251.06412; found:
251.06425.
6-tert-Butyl 1,1-Diethyl 1-Acetamidohexane-1,1,6-tricarboxylate
(5)
Step 1. Compound 4 (13.61 g, 54.2 mmol, 1.0 equiv) and NaI (9.75 g,
65.0 mmol, 1.2 equiv) were suspended in acetone (150 mL) and the
reaction mixture was stirred at reflux under N2 atmosphere over-
night. The resulting yellow dispersion was concentrated in vacuo, dis-
solved in Et2O (300 mL), and washed with H2O (200 mL + 100 mL), sat.
aqueous Na2S2O3 (200 mL), and brine (100 mL). The organic layer was
dried over Na2SO4, filtered, and concentrated in vacuo to afford a clear
oil (tert-butyl 6-iodohexanoate, 15.55 g, 96%).
1H NMR (600 MHz, CDCl3): δ = 3.19 (t, J = 7.0 Hz, 2 H, ICH2), 2.22 (t,
J = 7.4 Hz, 2 H, CH2CO), 1.84 (tt, J = 7.4, 7.0 Hz, 2 H, ICH2CH2), 1.60 (p,
J = 7.4 Hz, 2 H, CH2CH2CO), 1.44 (p, J = 7.4 Hz, 2 H, ICH2CH2CH2), 1.44
(s, 9 H, t-Bu).
All reagents and solvents were of analytical grade and used, without
further purification, as obtained from commercial suppliers. H2O re-
fers to deionized water, unless otherwise stated. Anhydrous CH2Cl2
was obtained from a PureSolv system. Reactions were monitored by
TLC using silica gel coated plates (analytical silica gel 60, F-254) visu-
alized under UV light or by staining with a standard KMnO4 solution
or, alternatively, by UPLC-MS analysis. Column chromatography puri-
fication was performed on a dry column vacuum chromatography
© Georg Thieme Verlag Stuttgart · New York — Synthesis 2018, 50, 4037–4046

4042
C. Moreno-Yruela et al.
Feature
Synthesis
13C NMR (151 MHz, CDCl3): δ = 173.0 (C=O), 80.3 (C(CH3)3), 35.5
(CH2CO), 33.3 (ICH2CH2), 30.1 (ICH2CH2CH2), 28.3 (C(CH3)3), 24.2
(CH2CH2CO), 6.8 (ICH2).
ture was concentrated in vacuo and co-concentrated three more
times with a heptane/toluene (50:50) mixture to afford 7-acetamido-
8-ethoxy-8-oxooctanoic acid as an orange oil.
Step 2. A mixture of NaH (60% dispersion in mineral oil, 2.07 g, 51.7
mmol, 1.1 equiv) in DMF (10 mL) was cooled to 0 °C under N2 atmo-
sphere, and a solution of diethyl acetamidomalonate (10.22 g, 47.0
mmol, 1.0 equiv) in DMF (50 mL) was added dropwise during 30 min,
observing gas development. Then, the mixture was allowed to reach
r.t. and stir for 30 min, after which tert-butyl 6-iodohexanoate (15.55
g) dissolved in DMF (15 mL) was added portionwise. The reaction
mixture was stirred overnight at r.t. and a color change from yellow to
orange was observed. Then, the mixture was concentrated in vacuo
and dissolved in EtOAc (300 mL). The organic phase was washed with
H2O (3 × 100 mL) and brine (100 mL), dried over Na2SO4, filtered, and
concentrated in vacuo. The crude yellow oil was purified by column
chromatography (0–40% EtOAc in heptane) to afford 5 as a yellow oil
(16.64 g, 91%).
1H NMR (600 MHz, CDCl3): δ = 6.75 (s, 1 H, NH), 4.24 (q, J = 7.1 Hz, 4
H, CH2CH3), 2.36–2.25 (m, 2 H, CCH2), 2.17 (t, J = 7.5 Hz, 2 H, CH2CO),
2.03 (s, 3 H, CH3CO), 1.55 (p, J = 7.5 Hz, 2 H, CH2CH2CO), 1.43 (s, 9 H,
t-Bu), 1.31 (tt, J = 7.6, 7.5 Hz, 2 H, CH2CH2CH2CO), 1.25 (t, J = 7.1 Hz, 6
H, CH2CH3), 1.14–1.08 (m, 2 H, CCH2CH2).
13C NMR (151 MHz, CDCl3): δ = 173.1 (COOt-Bu), 169.1 (CONH), 168.3
(COOEt), 80.2 (C(CH3)3), 66.7 (CCH2), 62.6 (CH2CH3), 35.5 (CH2CO),
32.2 (CCH2), 28.9 (CH2CH2CH2CO), 28.3 (C(CH3)3), 25.0 (CH2CH2CO),
23.6 (CCH2CH2), 23.2 (CH3CO), 14.1 (CH2CH3).
1H NMR (600 MHz, DMSO-d6): δ = 11.92 (s, 1 H, COOH), 8.17 (d, J = 7.4
Hz, 1 H, NH), 4.15 (ddd, J = 8.8, 7.4, 5.3 Hz, 1 H, CH), 4.12–4.00 (m, 2 H,
CH2CH3), 2.18 (t, J = 7.3 Hz, 2 H, CH2CO), 1.84 (s, 3 H, CH3CO), 1.68–
1.60 (m, 1 H, CHCH2,a), 1.60–1.51 (m, 1 H, CHCH2,b), 1.47 (q, J = 7.3 Hz,
2 H, CH2CH2CO), 1.35–1.21 (m, 4 H, CHCH2CH2, CH2CH2CH2CO), 1.17 (t,
J = 7.1 Hz, 3 H, CH2CH3).
13C NMR (151 MHz, DMSO-d6): δ = 174.4 (COOH), 172.3 (COOEt),
169.4 (CONH), 60.3 (CH2CH3), 51.9 (CH), 33.5 (CH2CO), 30.8 (CHCH2),
28.0 (CH2CH2CH2CO), 25.0 (CHCH2CH2), 24.2 (CH2CH2CO), 22.2
(CH3CO), 14.1 (CH2CH3).
Step 2. 7-Acetamido-8-ethoxy-8-oxooctanoic acid (≤11.7 mmol, 1.0
equiv) was suspended in anhyd CH2Cl2 (45 mL) at 0 °C under N2 atmo-
sphere, and SOCl2 (1.71 mL, 23.4 mmol, 2.0 equiv) was added drop-
wise. Then, the temperature was increased slowly to 50 °C and the
mixture was stirred for 2 h. The reaction was judged complete by
UPLC-MS analysis of an aliquot quenched with MeOH. The dark mix-
ture was then cooled to r.t., followed by addition of TFAA (4.88 mL,
35.1 mmol, 3.0 equiv), and dropwise addition of pyridine (4.73 mL,
58.5 mmol, 5.0 equiv) with a water bath to control the temperature.
This mixture was allowed to stir for 1.5 h before addition of excess
H2O (10 mL), and then agitated for an additional 30 min. The reaction
crude mixture was diluted with CH2Cl2 (100 mL), washed with H2O
(2 × 150 mL), dried over Na2SO4, filtered, and concentrated in vacuo.
Column chromatography (25–85% EtOAc in heptane) afforded 7 as a
dark red oil (1.02–1.75 g, 28–48%, based on three individual reac-
tions).
+
HRMS: m/z [M + H]+ calcd for C19H34NO7
: 388.23298; found:
388.23372.
1H NMR (600 MHz, CDCl3): δ = 6.21 (d, J = 8.0 Hz, 1 H, NH), 4.57 (td,
J = 7.6, 5.3 Hz, 1 H, CH), 4.19 (q, J = 7.1 Hz, 2 H, CH2CH3), 2.69 (t, J = 7.1
Hz, 2 H, CH2CO), 2.02 (s, 3 H, CH3CO), 1.88–1.75 (m, 1 H, CHCH2,a),
1.71–1.57 (m, 3 H, CHCH2,b, CH2CH2CO), 1.34 (d, J = 5.8 Hz, 4 H,
CHCH2CH2, CH2CH2CH2CO), 1.27 (t, J = 7.1 Hz, 3 H, CH2CH3).
13C NMR (151 MHz, CDCl3): δ = 191.5 (q, J = 35.1 Hz, COCF3), 172.6
(COOEt), 170.5 (CONH), 115.6 (q, J = 292.2 Hz, CF3), 61.7 (CH2CH3),
52.2 (CH), 36.2 (CH2CO), 32.4 (CHCH2), 28.3 (CH2CH2CH2CO), 24.9
(CHCH2CH2), 23.2 (CH2CH2CO), 22.2 (CH3CO), 14.2 (CH2CH3).
8-tert-Butyl 1-Ethyl 2-Acetamidooctanedioate (6)
Compound 5 (10.12 g, 26.1 mmol, 1.0 equiv), LiCl (1.65 g, 39.0 mmol,
1.5 equiv), and H2O (0.94 mL, 52.2 mmol, 2.0 equiv) were suspended
in DMSO (50 mL), and the yellow mixture was stirred at 150 °C under
N2 atmosphere overnight. The resulting maroon solution was repeat-
edly extracted with EtOAc (100 mL + 100 mL + 50 mL), and the com-
bined organic layer was washed with H2O (2 × 100 mL) and brine
(100 mL), dried over Na2SO4, filtered, and concentrated in vacuo. The
crude material was purified by column chromatography (30–75%
EtOAc in heptane) to afford 6 as an orange oil (6.67 g, 81%).
19F NMR (376 MHz, CDCl3): δ = –79.4 (CF3).
1H NMR (600 MHz, CDCl3): δ = 5.97 (d, J = 8.0 Hz, 1 H, NH), 4.58 (ddd,
J = 8.0, 7.2, 5.3 Hz, 1 H, CH), 4.20 (q, J = 7.1 Hz, 2 H, CH2CH3), 2.19 (t,
J = 7.4 Hz, 2 H, CH2CO), 2.02 (s, 3 H, CH3CO), 1.88–1.78 (m, 1 H,
CHCH2,a), 1.71–1.61 (m, 1 H, CHCH2,b), 1.61–1.51 (m, 2 H, CH2CH2CO),
1.43 (s, 9 H, t-Bu), 1.40–1.24 (m, 7 H, CHCH2CH2, CH2CH2CH2CO,
CH2CH3).
13C NMR (151 MHz, CDCl3): δ = 173.2 (COOt-Bu), 172.8 (COOEt), 169.8
(CONH), 80.2 (C(CH3)3), 61.6 (CH2CH3), 52.3 (CH), 35.5 (CH2CO), 32.6
(CHCH2), 28.8 (CH2CH2CH2CO), 28.3 (C(CH3)3), 25.0 (CHCH2CH2), 24.9
(CH2CH2CO), 23.4 (CH3CO), 14.3 (CH2CH3).
HRMS: m/z [M + H]+ calcd for C13H21F3NO4
312.14209.
: 312.14172; found:
+
(R)-2-Acetamido-9,9,9-trifluoro-8-oxononanoic Acid (8)
Compound 8 was obtained in the synthesis of compounds 9a and 9b,
purified by column chromatography [Rf (8) = 0.14, Rf (9a) = 0.68, Rf
(9b) = 0.70, 0.25% AcOH in EtOAc], and isolated as a white solid [154
mg (63%) and 115 mg (47%), respectively].
1H NMR (600 MHz, CDCl3): δ = 6.69 (d, J = 7.8 Hz, 1 H, NH), 4.58–4.46
(m, 1 H, CH), 2.79–2.66 (m, 2 H, CH2CO), 2.05 (s, 3 H, CH3), 1.91–1.79
(m, 1 H, CHCH2,a), 1.78–1.59 (m, 3 H, CHCH2,b, CH2CH2CO), 1.45–1.26
(m, 4 H, CHCH2CH2, CH2CH2CH2CO).
+
HRMS: m/z [M + H]+ calcd for C16H30NO5
: 316.21185; found:
316.21200.
13C NMR (151 MHz, CDCl3): δ = 191.7 (q, J = 34.9 Hz, COCF3), 175.3
(COOH), 172.2 (CONH), 115.6 (q, J = 292.1 Hz, CF3), 52.5 (CH), 36.3
CH2CO), 31.7 (CHCH2), 28.3 (CH2CH2CH2CO), 25.0 (CHCH2CH2), 22.8
(CH3), 22.1 (CH2CH2CO).
Ethyl 2-Acetamido-9,9,9-trifluoro-8-oxononanoate (7)
Step 1. Compound 6 (3.70 g, 11.7 mmol, 1.0 equiv) was suspended in
CH2Cl2 (30 mL), and TFA (15 mL) was added. The reaction was com-
plete after 1.5 h, as judged by UPLC-MS, after which the reaction mix-
19F NMR (376 MHz, CDCl3): δ = –79.4 (CF3).
+
HRMS: m/z [M + H]+ calcd for C11H17F3NO4
: 284.11042; found:
284.11076.
© Georg Thieme Verlag Stuttgart · New York — Synthesis 2018, 50, 4037–4046

4043
C. Moreno-Yruela et al.
Feature
Synthesis
(S)-2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-9,9,9-trifluo-
19F NMR (376 MHz, CDCl3): δ = –79.3 (CF3).
ro-8-oxononanoic Acid (9a)
+
HRMS: m/z [M + H]+ calcd for C17H21F3NO5
:
376.13663; found:
Compound 7 (485 mg, 1.7 mmol, 1.0 equiv) was suspended in EtOH (5
mL), and aqueous NaOH (1 M, 5 mL) was added under open-flask con-
ditions. After 1 h of stirring, the EtOH was removed in vacuo, the mix-
ture was diluted in phosphate buffer (0.1 M, pH 7.2, 50 mL), and the
pH was adjusted to 7.2 with aqueous HCl (2 M). A control NMR sam-
ple was taken by mixing a reaction aliquot (50 μL) with MeOD (450
μL). Then, the reaction mixture was warmed to 40 °C, and Acylase I
from Aspergillus melleus (41.2 mg, 24 mg/mmol)49 and CoCl2·6H2O
(11.9 mg, to reach 1 mM concentration) were added. The enzymatic
resolution was allowed to proceed overnight, and it was followed by
1H NMR spectroscopy with water suppression on samples prepared as
the control, by measuring the ratio between the NHCH and the CH2CO
signals (the reaction was judged complete when the ratio lowered to
50% relative to the initial control). The mixture was then basified to
pH 10 with aqueous NaOH (2 M), followed by addition of Fmoc N-hy-
droxysuccinimide ester (435 mg, 1.29 mmol, 0.75 equiv) in 1,4-diox-
ane (5 mL). The mixture was stirred at r.t. overnight, concentrated in
vacuo to remove the 1,4-dioxane, acidified to pH 2 with aqueous HCl
(2 M), and extracted with EtOAc (3 × 75 mL). The organic layer was
dried over Na2SO4, filtered, and concentrated in vacuo. Column chro-
matography (10–100% EtOAc in heptane, with 0.25% AcOH) afforded
9a as an off-white solid (189 mg, 48% based on (S)-7).62
376.13867.
Ethyl 2-Acetamido-7-(2-(trifluoromethyl)-1,3-dioxolan-2-yl)hep-
tanoate (10a)
Compound 7 (19.0 mg, 0.061 mmol, 1.0 equiv) and K2CO3 (24.9 mg,
0.18 mmol, 3.0 equiv) were suspended in anhydrous DMF (2 mL) un-
der N2 atmosphere. Then, 2-chloroethanol (12.1 μL, 0.18 mmol, 3.0
equiv) was added, and the mixture was stirred at r.t. for 2 h. The re-
sulting orange solution was diluted with H2O (20 mL) and extracted
with Et2O (3 × 8 mL). The combined organic layer was washed with
H2O (10 mL), dried over Na2SO4, filtered, and concentrated in vacuo.
Column chromatography (40–80% EtOAc in heptane) afforded 10a as
a clear oil (3.0 mg, 14%, reaction and isolation conditions were not op-
timized).
1H NMR (600 MHz, CDCl3): δ = 5.98 (d, J = 8.1 Hz, 1 H, NH), 4.59 (td,
J = 7.6, 5.3 Hz, 1 H, CH), 4.20 (q, J = 7.2 Hz, 2 H, CH2CH3), 4.17–4.12 (m,
2 H, CH2CH2,aO), 4.12–4.02 (m, 2 H, CH2CH2,bO), 2.02 (s, 3 H, CH3CO),
1.87–1.76 (m, 3 H, CHCH2,a, CH2CCF3), 1.69–1.61 (m, 1 H, CHCH2,b),
1.46–1.29 (m, 6 H, CH2CH2CCF3, CHCH2CH2, CHCH2CH2CH2), 1.28 (t,
J = 7.1 Hz, 3 H, CH2CH3).
13C NMR (151 MHz, CDCl3): δ = 172.8 (COO), 169.8 (CON), 123.8 (q,
J = 290.6 Hz, CF3), 106.4 (q, J = 30.7 Hz, CCF3), 67.1 (CH2CH2O), 61.6
(CH2CH3), 52.3 (CH), 32.6 (CHCH2), 31.7 (CH2CCF3), 29.2
(CHCH2CH2CH2), 25.1 (CHCH2CH2), 23.4 (CH3CO), 21.2 (CH2CH2CCF3),
14.3 (CH2CH3).
19F NMR (376 MHz, CDCl3): δ = –82.4 (CF3).
HRMS: m/z [M + H]+ calcd for C15H25F3NO5
1H NMR (600 MHz, CDCl3): δ = 7.76 (d, J = 7.5 Hz, 2 H, C4H, C5H),
7.65–7.50 (m, 2 H, C1H, C8H), 7.45–7.35 (m, 2 H, C3H, C6H), 7.31 (t,
J = 7.2 Hz, 2 H, C2H, C7H), 5.26 (d, J = 8.3 Hz, 1 H, NH), 4.62–4.32 (m, 3
H, NHCH, CH2O), 4.23 (t, J = 7.0 Hz, 1 H, C9H), 2.71 (t, J = 7.2 Hz, 2 H,
CH2CO), 1.98–1.84 (m,
NHCHCH2,b CH2CH2CO), 1.39 (d, J = 7.7 Hz,
CH2CH2CH2CO).
1
H, NHCHCH2,a), 1.76–1.58 (m,
3 H,
,
4
H, CHCH2CH2,
+
: 356.16793; found:
356.16812.
13C NMR (151 MHz, CDCl3): δ = 191.5 (q, J = 34.9 Hz, COCF3), 176.8
(COOH), 156.2 (OCON), 143.9 (C8a or C9a), 143.8 (C8a or C9a), 141.5
(C4a, C4b), 127.9 (C3, C6), 127.2 (C2, C7), 125.2 (C1, C8), 120.2 (C4,
C5), 115.7 (q, J = 292.2 Hz, CF3), 67.3 (CH2O), 53.7 (NHCH), 47.3 (C9),
36.3 (CH2CO), 32.2 (NHCHCH2), 28.3 (CH2CH2CH2CO), 25.0
(NHCHCH2CH2), 22.2 (CH2CH2CO).
Ethyl 2-Acetamido-7-(2-(trifluoromethyl)-1,3-dioxan-2-yl)hepta-
noate (10b)
Compound 7 (44.6 mg, 0.14 mmol, 1.0 equiv) and K2CO3 (59.4 mg,
0.43 mmol, 3.0 equiv) were mixed in anhydrous DMF (5 mL) under N2
atmosphere, followed by addition of 3-chloro-1-propanol (36.0 μL,
0.43 mmol, 3.0 equiv). The mixture was stirred at 100 °C for 2 h, after
which it was cooled, diluted with H2O (30 mL), and extracted with
Et2O (3 × 20 mL). The combined organic layer was washed with H2O
(30 mL), dried over Na2SO4, filtered, and concentrated in vacuo. Col-
umn chromatography (40–80% EtOAc in heptane) afforded 10b as a
clear oil (12.9 mg, 23%).
19F NMR (376 MHz, CDCl3): δ = –79.3 (CF3).
+
HRMS: m/z [M + H]+ calcd for C24H25F3NO5
: 464.16793; found:
464.16854.
(S)-2-(((Benzyloxy)carbonyl)amino)-9,9,9-trifluoro-8-oxononano-
ic Acid (9b)
1H NMR (600 MHz, CDCl3): δ = 5.97 (d, J = 8.1 Hz, 1 H, NH), 4.66–4.53
(m, 1 H, CH), 4.20 (q, J = 7.1 Hz, 2 H, CH2CH3), 4.16–4.06 (m, 2 H,
CH2CH2,aO), 4.00–3.90 (m, 2 H, CH2CH2,bO), 2.02 (s, 3 H, CH3CO), 1.99–
1.89 (m, 1 H, CH2,aCH2O), 1.89–1.78 (m, 1 H, CHCH2,a), 1.72–1.60 (m, 4
H, CH2,bCH2O, CHCH2,b, CH2CCF3), 1.51–1.41 (m, 2 H, CH2CH2CCF3),
1.39–1.24 (m, 4 H, CHCH2CH2, CHCH2CH2CH2), 1.28 (t, J = 7.1 Hz, 3 H,
CH2CH3).
13C NMR (151 MHz, CDCl3): δ = 172.8 (COO), 169.8 (CON), 124.5 (q,
J = 295.6 Hz, CF3), 95.75 (q, J = 29.1 Hz, CCF3), 61.7 (CH2CH2O), 61.6
(CH2CH3), 52.3 (CH), 33.7 (CH2CCF3), 32.6 (CHCH2), 29.3
(CHCH2CH2CH2), 25.1 (CHCH2CH2), 24.0 (CH2CH2O), 23.4 (CH3CO),
21.6 (CH2CH2CCF3), 14.3 (CH2CH3).
Atona building block 9b was prepared following the procedure de-
scribed for 9a, with addition of Cbz N-hydroxysuccinimide ester (249
mg, 1.29 mmol, 0.75 equiv) instead of the corresponding Fmoc re-
agent. Compound 9b was obtained as a yellow oil (132 mg, 41% based
on (S)-7).
1H NMR (600 MHz, CDCl3): δ = 7.39–7.34 (m, 3 H, C3H, C4H, C5H),
7.34–7.29 (m, 2 H, C2H, C6H), 5.27 (d, J = 8.3 Hz, 1 H, NH), 5.21–5.01
(m, 2 H, CH2O), 4.44–4.37 (m, 1 H, NHCH), 2.70 (t, J = 7.0 Hz, 2 H, CH2-
CO), 1.95–1.86 (m, 1 H, NHCHCH2,a), 1.78–1.55 (m, 3 H, NHCHCH2,b
,
CH2CH2CO), 1.48–1.27 (m, 4 H, CHCH2CH2, CH2CH2CH2CO).
13C NMR (151 MHz, CDCl3): δ = 191.5 (q, J = 35.0 Hz, COCF3), 177.1
(COOH), 156.2 (OCON), 136.2 (C1), 128.7 (CHPh), 128.4 (CHPh), 128.3
(CHPh), 115.7 (q, J = 292.1 Hz, CF3), 67.4 (CH2O), 53.7 (NHCH), 36.3
(CH2CO), 32.3 (NHCHCH2), 28.3 (CH2CH2CH2CO), 25.0 (NHCHCH2CH2),
22.2 (CH2CH2CO).
19F NMR (376 MHz, CDCl3): δ = –76.1 (CF3).
+
HRMS: m/z [M + H]+ calcd for C16H27F3NO5
: 370.18358; found:
370.18383.
© Georg Thieme Verlag Stuttgart · New York — Synthesis 2018, 50, 4037–4046

4044
C. Moreno-Yruela et al.
Feature
Synthesis
Ethyl 2-Acetamido-7-(2-(trifluoromethyl)-1,3-dioxepan-2-yl)hep-
tanoate (10c)
Enantiomeric Purity Determination (Marfey’s Method)51,52
Compound 9a (1.0 mg, 2.16 μmol, 1.0 equiv) was suspended in DMF
(42.2 μL), and piperidine (0.21 μL, 2.16 μmol, 1.0 equiv), 1,8-diazabi-
cyclo[5.4.0]undec-7-ene (0.01 μL, 0.06 μmol, 0.03 equiv), and 1-octa-
nethiol (1.87 μL, 10.8 μmol, 5.0 equiv) were added. The mixture was
agitated in a plastic tube at r.t. for 2 h and, thereafter, the sample was
concentrated to dryness by centrifugation in vacuo. The pellet was re-
suspended in DMF (50 μL), followed by addition of Nα-(2,4-dinitro-5-
fluorophenyl)-L-alaninamide (FDAA, Marfey’s reagent, 1.0 mg, 3.67
μmol, 1.7 equiv) in acetone (100 μL) and aqueous NaHCO3 (1 M, 20 μL,
9.2 equiv). The mixture was agitated at 40 °C for 1 h, quenched with
aqueous HCl (2 M, 10 μL), concentrated by centrifugation in vacuo,
and analyzed by UPLC-MS and HPLC (see the Supporting Information
Figure S1).
Compound 10c was synthesized following the same procedure as for
10b, using 4-chloro-1-butanol. Column chromatography, however,
was not sufficient for obtaining a sufficiently pure sample (estimated
yield: 5%).
+
HRMS: m/z [M + H]+ calcd for C17H29F3NO5
: 384.19923; found:
384.19948.
(R)-2-Acetamido-7-(2-(trifluoromethyl)-1,3-dioxan-2-yl)heptano-
ic Acid (11)
Compound 11 was obtained in the synthesis of compound 12, puri-
fied by column chromatography [Rf (11) = 0.16, Rf (12) = 0.58, 0.25%
AcOH in heptane/EtOAc (1:1)], and isolated as a white powder (37
mg, 59%).
1H NMR (600 MHz, CDCl3): δ = 6.00 (d, J = 7.6 Hz, 1 H, NH), 4.58 (td,
J = 7.5, 5.4 Hz, 1 H, CH), 4.19–4.04 (m, 2 H, CH2CH2,aO), 4.04–3.88 (m, 2
Compound 8 (4.2 mg, 14.8 μmol, 1.0 equiv) was suspended in aque-
ous HCl (2 M, 70 μL, 9.5 equiv) and incubated at 100 °C for 2 h in a
plastic tube. Then, the pH was adjusted to pH 7 with aqueous NaOH
(2 M, ~70 μL), and the mixture was diluted with H2O (156 μL). An ali-
quot (50 μL) was transferred to a separate plastic tube, treated with
Marfey’s reagent, and analyzed as for 9a.
H, CH2CH2,bO), 2.06 (s, 3 H, CH3), 2.02–1.84 (m, 2 H, CHCH2,a
,
CH2,aCH2O), 1.76–1.67 (m, 3 H, CHCH2,b, CH2CCF3), 1.62 (dt, J = 13.6,
3.6 Hz, 1 H, CH2,bCH2O), 1.55–1.28 (m, 6 H, CHCH2CH2, CHCH2CH2CH2,
CH2CH2CCF3).
13C NMR (151 MHz, CDCl3): δ = 175.2 (COOH), 171.3 (CON), 124.5 (q,
J = 294.7 Hz, CF3), 95.8 (q, J = 29.1 Hz, CCF3), 61.7 (CH2CH2O), 52.6
(CH), 33.7 (CH2CCF3), 31.9 (CHCH2), 29.2 (CHCH2CH2CH2), 25.2
(CHCH2CH2), 24.0 (CH2CH2O), 23.2 (CH3), 21.5 (CH2CH2CCF3).
Synthesis of Peptides 13 and 13a
Peptides were prepared through standard automated Fmoc solid-
phase peptide synthesis on a Biotage Syro Wave™ synthesizer. The
employed amino acid building blocks were Fmoc-Ala-OH, Fmoc-
Arg(Pbf)-OH, Fmoc-Gly-OH, Fmoc-Ser(t-Bu)-OH, Fmoc-Thr(t-Bu)-OH,
and Fmoc-Trp(Boc)-OH, and the synthesis was performed on a 20
μmol scale on pre-loaded TentaGel S RAM resin (0.24 mmol/g). Fmoc
deprotection steps were performed with piperidine/DMF [first, 2:3
(v/v), 3 min; then, 1:4 (v/v), 12 min], followed by resin washing with
DMF (2 × 45 s), CH2Cl2 (45 s), and DMF (2 × 45 s). Automated coupling
reactions were performed twice with Fmoc-Xaa-OH (200 μL, 0.5 M in
DMF, 5.0 equiv to resin loading), HBTU (210 μL, 0.48 M in DMF, 5.0
equiv), and i-Pr2NEt (100 μL, 2 M in NMP, 10.0 equiv) for 2 × 40 min,
followed by washing with DMF (3 × 45 s). Final capping was per-
formed with AcOH in a similar manner. Building blocks 9a and 12
were coupled manually using Fmoc-Xaa-OH (2.0 equiv), HATU (1.9
equiv), and i-Pr2NEt (3.9 equiv) in DMF (for a 0.2 M amino acid con-
centration) for 40 min. Peptides were cleaved and deprotected by in-
cubation with the TFA-containing peptide cleavage cocktail
TFA/DODT/H2O/i-Pr3SiH [92.5:2.5:2.5:2.5 (v/v)] for 2 h at r.t., followed
by concentration under a N2 stream, trituration with ice-cold Et2O (8
mL), and centrifugation for 3 min at 1600 RCF. Supernatants were dis-
carded, and pellets were washed with ice-cold Et2O (2 × 4 mL), dis-
solved in eluent A, and lyophilized. Peptide crude 13a was further
19F NMR (376 MHz, CDCl3): δ = –76.0 (CF3).
+
HRMS: m/z [M + H]+ calcd for C14H23F3NO5
: 342.15228; found:
342.15277.
(S)-2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-7-(2-(trifluo-
romethyl)-1,3-dioxan-2-yl)heptanoic Acid (12)
Compound 10b (138 mg, 0.37 mmol, 1.0 equiv) was treated similar to
7 for the synthesis of 9a. Column chromatography (10–50% EtOAc in
heptane, with 0.25% AcOH) afforded 12 as a clear oil (58 mg, 60%).
This amino acid building block was stable under aqueous acid condi-
tions and could therefore be purified by reversed-phase preparative
HPLC.
1H NMR (600 MHz, CDCl3): δ = 7.76 (d, J = 7.5 Hz, 2 H, C4H, C5H),
7.64–7.55 (m, 2 H, C1H, C8H), 7.40 (t, J = 7.5 Hz, 2 H, C3H, C6H), 7.32
(t, J = 7.5 Hz, 2 H, C2H, C7H), 5.21 (d, J = 8.3 Hz, 1 H, NH), 4.58–4.35
(m, 3 H, NHCH, CHCH2O), 4.23 (t, J = 7.0 Hz, 1 H, C9H), 4.17–4.07 (m, 2
H, CH2CH2,aO), 4.00–3.89 (m, 2 H, CH2CH2,bO), 2.01–1.85 (m, 2 H,
NHCHCH2,a, CH2,aCH2O), 1.77–1.66 (m, 3 H, NHCHCH2,b, CH2CCF3), 1.61
(dt, J = 13.5, 3.6 Hz, 1 H, CH2,bCH2O), 1.54–1.21 (m, 6 H, CHCH2CH2,
CHCH2CH2CH2, CH2CH2CCF3).
13C NMR (151 MHz, CDCl3): δ = 176.6 (COOH), 156.2 (OCON), 144.0
(C8a or C9a), 143.8 (C8a or C9a), 141.5 (C4a, C4b), 127.9 (C3, C6),
127.2 (C2, C7), 125.2 (C1, C8), 124.5 (q, J = 294.6 Hz, CF3), 120.2 (C4 or
C5), 120.1 (C4 or C5), 95.8 (q, J = 29.1 Hz, CCF3), 67.2 (CHCH2O), 61.7
(CH2CH2O), 53.8 (NHCH), 47.3 (C9), 33.8 (CH2CCF3), 32.3 (NHCHCH2),
29.3 (CHCH2CH2CH2), 25.3 (CHCH2CH2), 24.0 (CH2CH2O), 21.6
(CH2CH2CCF3).
treated with
a
cocktail containing TFA/CF3SO3H/H2O/i-Pr3SiH
[80.5:12:5:2.5 (v/v)] for 2 h, followed by concentration, trituration,
centrifugation, and washing as before. Peptides were purified by pre-
parative HPLC on an Agilent 1260 LC system equipped with a C18
Phenomenex Luna column (250 × 20 mm, 5 μm, 100 Å) and a diode
array UV detector at 20 mL/min flow rate. A linear gradient of eluent
B in eluent A (0–30% or 10–40%) over 30 min was applied, and frac-
tions containing the desired peptide were identified by UPLC-MS or
MALDI-TOF MS, lyophilized, and analyzed by HPLC.
HRMS: m/z (13) [M + H]+ calcd for C46H70F3N14O14+: 1099.51425;
found: 1099.51450.
HRMS: m/z (13a) [M + H]+ calcd for C49H76F3N14O15+: 1157.55612;
found: 1157.55492.
19F NMR (376 MHz, CDCl3): δ = –76.0 (CF3).
+
HRMS: m/z [M + H]+ calcd for C27H31F3NO6
: 522.20980; found:
522.20979.
© Georg Thieme Verlag Stuttgart · New York — Synthesis 2018, 50, 4037–4046

4045
C. Moreno-Yruela et al.
Feature
Synthesis
Histone Deacetylase (HDAC) Inhibition Assays
References
Peptide 13 was screened for inhibition of the deacylase activity of
HDACs 1–9 and 11 in a fluorogenic in vitro assay adapted to Corning
microtiter 96-well plates (half-area wells). Assays were performed
twice, with internal duplicates, control wells without enzyme, and
reference wells containing a set concentration of 7-amino-4-methyl-
coumarin (AMC). HDAC enzyme stock solutions (3–50 μM) were pur-
chased from BPS Bioscience, and from Millipore in the case of HDAC7.
Substrate DMSO stock solutions (20 mM) were prepared in-house,
following previously reported procedures for synthesis of the AMC-
containing peptide substrates Ac-LGKac-AMC,57,63 Ac-LGKtfa-AMC,8,63
and Ac-ETDKmyr-AMC.64 Substrate and inhibitor concentrations were
calculated based on absorbance measured on a Thermo Scientific
NanoDropC instrument.
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Inhibitor (0.1–10 μM), substrate, and enzyme were incubated in 4-(2-
hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES) buffer [50
mM HEPES, pH 7.4, 100 mM KCl, 0.001% Tween 20 (v/v), 0.5 mg/mL
bovine serum albumin; 25 μL total volume] for 30 min at 37 °C.
Thereafter, a trypsin solution in HEPES buffer (0.2 mg/mL, 25 μL) was
added to each well and the assay was allowed to develop for 15 min at
r.t. Fluorescence at 460 nm (with excitation at 360 nm) was recorded
for each well on a Tecan Safire II plate reader, and data were corrected
by subtraction of the background. Assay conditions: HDAC1 (0.5 nM;
with Ac-LGKac-AMC, 20 μM), HDAC2 (0.5 nM; with Ac-LGKac-AMC,
20 μM), HDAC3/NCoR2 (1.0 nM; with Ac-LGKac-AMC, 20 μM), HDAC4
(0.05 nM; with Ac-LGKtfa-AMC, 20 μM), HDAC5 (0.1 nM; with Ac-
LGKtfa-AMC, 120 μM), HDAC6 (0.5 nM; with Ac-LGKac-AMC, 20 μM),
HDAC7 (0.5 nM; with Ac-LGKtfa-AMC, 40 μM), HDAC8 (0.5 nM; with
Ac-LGKtfa-AMC, 200 μM), HDAC9 (0.5 nM; with Ac-LGKtfa-AMC, 80
μM), and HDAC11 (1.0 nM; with Ac-ETDKmyr-AMC, 50 μM).
Dose–response experiments were performed similarly for peptide 13
(1.6–5000 nM) and commercial trichostatin A (0.08–250 μM) against
HDAC4 (0.01 nM), HDAC5 (0.1 nM), HDAC7 (0.05 nM), HDAC8 (0.3
nM), and HDAC9 (0.1 nM), with substrates as before. The Cheng–Pru-
soff equation [Ki = IC50/(1 + [S]/KM)] was employed for converting IC50
values into Ki values, with the assumption of a standard fast-on–fast-
off mechanism of inhibition, and using KM values reported by Bradner,
Mazitschek, and co-workers.8
Funding Information
This work was supported by the Carlsberg Foundation (2013-01-
0333; C.A.O.) and the University of Copenhagen (Ph.D. fellowship for
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Acknowledgment
We thank Dr. Hans M. Maric for fruitful discussions, and Dr. Andreas
S. Madsen and Dr. Iacopo Galleano for providing enzyme substrates.
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Supporting Information
Supporting information for this article is available online at
https://doi.org/10.1055/s-0037-1609945.
S
u
p
p
ortioInfgrmoaitn
S
u
p
p
ortiInfogrmoaitn
© Georg Thieme Verlag Stuttgart · New York — Synthesis 2018, 50, 4037–4046

4046
C. Moreno-Yruela et al.
Feature
Synthesis
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© Georg Thieme Verlag Stuttgart · New York — Synthesis 2018, 50, 4037–4046