Synthesis and Biochemical Evaluation of Biotinylated Conjugates of Largazole Analogues: Selective Class I Histone Deacetylase Inhibitors

Article abstract of DOI:10.1002/ijch.201600130

The synthesis of biotinylated conjugates of synthetic analogues of the potent and selective histone deacetylase (HDAC) inhibitor largazole is reported. The thiazole moiety of the parent compound's cap group was derivatized to allow the chemical conjugation to biotin. The derivatized largazole analogues were assayed across a panel of HDACs 1–9 and retained potent and selective inhibitory activity towards the class I HDAC isoforms. The biotinylated conjugate was further shown to pull down HDACs 1, 2, and 3.

Full text of DOI:10.1002/ijch.201600130

Full Paper
DOI: 10.1002/ijch.201600130
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Synthesis and Biochemical Evaluation of Biotinylated
Conjugates of Largazole Analogues: Selective Class I Histone
Deacetylase Inhibitors
Le Zhao,[a] Christine E. Dunne,[a] Dane J. Clausen,[a] Justin M. Roberts,[b] Joshiawa Paulk,[b] Haining Liu,[c]
Olaf G. Wiest,[c] James E. Bradner,[b] and Robert M. Williams*[a, d]
This paper is dedicated to Prof. Stuart L. Schreiber on the occasion of his 60th birthday. We are also very pleased to dedicate this
manuscript to Profs. Stuart L. Schreiber and K. C. Nicolaou on being awarded the Wolf Prize.
Abstract: The synthesis of biotinylated conjugates of syn- derivatized largazole analogues were assayed across a panel
thetic analogues of the potent and selective histone of HDACs 1–9 and retained potent and selective inhibitory
deacetylase (HDAC) inhibitor largazole is reported. The activity towards the class I HDAC isoforms. The biotinylated
thiazole moiety of the parent compound’s cap group was conjugate was further shown to pull down HDACs 1, 2, and
derivatized to allow the chemical conjugation to biotin. The 3.
Keywords: biotin · cancer · inhibitors · largazole · peptidomimetics
25 1. Introduction
been the most challenging aspect in the development of
HDAC inhibitors to date. Eighteen HDACs have been isolated,
the Schreiber laboratory spearheading this effort.[10] The
HDACs of general therapeutic interest are within classes I and
II; specifically, class I includes four HDACs (1, 2, 3, and 8)
that are much smaller and primarily located in the nucleus,
compared to class II HDACs (4, 5, 6, 7, and 9), which each
contain ~1000 amino acid residues and are expressed in both
the cytoplasm and nucleus.[11] Adverse side effects have been
observed in currently marketed pan-HDAC inhibitors, which
26
27 The marine natural product largazole was isolated from marine
28 cyanobacteria Symploca sp. off of the Florida coastline by
29 Luesch and co-workers.[1] Largazole’s sixteen-membered
30 cyclodepsipeptide structure (Figure 1), containing both a
31 thioester tail and (S)-3-hydroxy-7-mercaptohept-4-enoic acid
32 moieties, constitutes the structural basis of a promising
33 antitumor prodrug.[2] Research efforts have explored the
34 therapeutic use of largazole towards not only cancer therapy
35 but also corneal angiogenesis, rheumatoid arthritis, neuro-
36 degenerative diseases, osteoporosis, and numerous other
37 disease areas.[3] The cyclodepsipeptide natural product and
38 histone deacetylase inhibitor (HDACi) romidepsin (FK228) is
39 currently an FDA-approved therapeutic for the treatment of
40 cutaneous T-cell lymphoma (CTCL).[4] Vorinostat (SAHA), a
41 simple hydroxamate, was the first HDACi approved by the
42 FDA for human clinical use and is a non-selective “pan”
43 inhibitor of all the HDAC isoforms.[5] Panobinostat, the pan-
44 HDACi LBH-589 developed by Novartis, has been approved
45 for multiple myeloma combination therapy as well.[6] Larga-
46 zole, FK228, and the spiruchostatins,[7] which all contain the
47 same (S)-3-hydroxy-7-mercaptohept-4-enoic acid moiety, are
48 all potent biochemical inhibitors of the class I HDACs and
49 exert their HDAC inhibitory activity through binding of the
50 sulfhydryl residue to the active site zinc ion of the class I
51 HDACs. Our laboratory and others have incorporated the (S)-
52 3-hydroxy-7-mercaptohept-4-enoic acid moiety into numerous
53 synthetic analogues, which in most cases provides selective
54 and potent class I HDAC inhibitors.[8]
[a] L. Zhao, C. E. Dunne, D. J. Clausen, R. M. Williams
Department of Chemistry
Colorado State University
Fort Collins
Colorado 80523 (USA)
Tel.: (+1) 970-491-6747
E-mail: robert.williams@colostate.edu
[b] J. M. Roberts, J. Paulk, J. E. Bradner
Department of Medical Oncology
Dana-Farber Cancer Institute
Boston
Massachusetts 02115 (USA)
[c] H. Liu, O. G. Wiest
Department of Chemistry and Biochemistry
University of Notre Dame
Notre Dame
Indiana 46556-5670 (USA)
[d] R. M. Williams
University of Colorado Cancer Center
Aurora
Colorado 80045 (USA)
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Selective HDAC isoform inhibition is essential for the
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/ijch.201600130.
56 development of a safe and efficient therapeutic.[9] This has also
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fragment), and (3) the depsipeptide versus the corresponding
peptide isosteres (Figure 1).[13a]
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Herein we describe synthetic routes to access both
depsipeptide and peptide isostere analogues of the parent
natural product largazole with a functional handle projecting
from the thiazole moiety of the cap group that permits the
chemical conjugation of other molecular entities that might
prove valuable for cell-specific targeting and as probes for
biochemical and cellular function (Figure 2). Our synthetic
route utilizes our original strategic four-component convergent
disconnection deployed in the efficient total synthesis of
largazole reported by this laboratory in 2008.[8a]
2. Results and Discussion
2.1 Design
Prior molecular modeling studies of largazole, performed in
collaboration with the Wiest laboratory, revealed that the cap
group of largazole has the unique ability to interact with the
protein surface of the HDAC enzyme and projects away from
the active site in unencumbered space, providing an oppor-
tunity for chemical functionalization.[13a,14] We reasoned that
thiazole derivatization would be chemically tractable and
would allow for the installation of functionality that would not
hinder the Zn2+ binding arm of this scaffold from interaction
with the Zn2+ atom ensconced at the active site of the enzyme.
With the dual objective of not abrogating the Zn2+ binding
role of the sulfhydryl-containing side chain[15] and incorporat-
ing a chemically flexible handle for conjugating cancer-cell-
specific targeting motifs and other reporter molecules, we
Figure 1. Largazole (1), the parent natural product, and largazole
peptide isostere (2).
26 has been partially attributed to the lack of selectivity towards
27 HDAC isoforms.[12]
28
To date we have developed syntheses of more than sixty
29 analogues of largazole by modifying three main motifs of the
30 parent natural product.[8,13] We have interrogated structural
31 changes to (1) the zinc-binding domain, (2) the surface
32 recognition unit or so-called “cap group” (thiazoleÀthiazoline
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Figure 2. Thiazole-derivatized depsipeptides (3), thiazole-derivatized peptide isosteres (4), depsipeptideÀbiotin conjugate (5), and peptide
isostereÀbiotin conjugate (6).
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modified our convergent synthetic route, utilizing previously
established disconnections.[8b] This design was probed using
our previously established[16] molecular dynamics (MD)
protocol for the study of HDAC inhibitors. Figure 3 shows the
model of 6 bound to the crystal structure of HDAC8 (PDB
code: 4RN0).[14] As envisioned in the original design, the
biotin and the amide linker protrude away from the binding
site, leaving all contacts of the protein with the cap group and
the zinc binding domain intact. It is therefore expected that the
We chose to evaluate the installation of both hydroxyl
group handles and amino group handles for chemical
conjugation studies (Figure 2). The amine handle allows for
extensive conjugation across a series of cancer-relevant bio-
logical molecules via robust amide linkages, which might
prove resistant to proteolytic cleavage in vitro and in vivo,
whereas the hydroxyl residue allows for chemical conjugation
through more labile ester linkages.[15] Biotin conjugation has
been widely used to study and identify small-molecule drug
targeting to cellular proteins and has been used in the context
of numerous pull-down experiments.[16]
10 binding of 5 and 6 will mimic largazole and its amide isostere,
11 respectively. Although no crystal structure of largazole
12 derivatives to HDAC1À3 is currently available, these findings
13 should also apply to these HDACs due the previously
14 described similarities in binding modes.[13a,14]
2.2 Synthesis
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Scheme 1 shows the synthetic strategy we have successfully
used to access largazole derivatives and we have applied this
to the preparation of compounds 3 and 4.[8c] The two halves,
depsipeptide or peptide isostere fragments (9) and
thiazoleÀthiazoline moiety (10), are utilized to access the pre-
macrocyclization species. Macrocyclization to S-trityl-pro-
tected precursors is accessed through standard peptide
coupling techniques. The two halves can then be further
dissected into the b-amino acid or b-hydroxy acid fragments
(12, X=NH2, OH, respectively), protected l-valine (11), a-
methyl cysteine (13), and the substituted thiazole cyano
precursor (14).
As shown in Scheme 2, commercially available acrolein
(15) was reacted with trityl mercaptan followed by a Wittig
olefination to yield aldehyde 16. Alcohol stereochemistry was
established through the use of a Crimmins-type aldol con-
densation, the product of which was then cleaved using 2-
(trimethylsilyl)ethanol. Amino acid EDCI coupling between
the free alcohol of 18 and l-valine was successful in providing
compound 19.
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As illustrated in Scheme 3, the corresponding peptide
isostere precursor was accessed via Boc-protected amino acid
Figure 3. MD refined model of the complex of 6 bound to HDAC8.
Scheme 1. Retrosynthetic pathway for cap group derivatization of the thiazole fragment of parent compounds of largazole (X=O or NH).
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Scheme 4. Synthesis of fragment 30 for cap group assembly.
Reagents and conditions: (a) LDA, I2, THF, À788C, 74%; (b) tetrakis
(triphenylphosphine)palladium(0), sodium bicarbonate, DMEÀH2O
(3:1), reflux, 68%.
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Scheme 2. Synthesis of compound 19 for depsipeptide series.
Reagents and conditions: (a) TrtSH, Et3N, CH2Cl2; (b) (formyl-
methylene)triphenylphosphorane, PhH, 808C, 77% over two steps;
(c) (R)-1-(4-benzyl-2-thioxothiazolidin-3-yl)ethanone, TiCl4, DIPEA,
CH2Cl2, À788C, 76%; (d) 2-(trimethylsilyl)ethanol, imidazole, CH2
Cl2, 83%; (e) N-Fmoc-Val-OH, EDCI, DIPEA, DMAP, CH2Cl2, 77%.
nation followed by iodination provided cross-coupling pre-
cursor 28. Suzuki coupling with boronic acid 29 provided
compound 30. It is important to note that additional aromatic
boronic acids have been used to access derivatives successfully
and will be reported elsewhere.
Scheme 5 illustrates the depsipeptide macrocycle assem-
bly. The free alcohol of 30 could either be protected with
TBDPS for direct conjugation via ester linkages or subjected
to Mitsunobu reaction using tert-butyl ((4-nitrophenyl)
sulfonyl)carbamate, which provided compound 31. Cyclo-
condensation of 31 with a-methyl cysteine gave carboxylic
acids 32:33 as a 1:1 mixture that was carried forward as a
mixture.[19] Peptide coupling of fragment 19 provided 34+35
as a 1:1 mixture that was deprotected and cyclized to furnish
the desired species 36.
Scheme 3. Synthesis of fragment 26 for peptide isostere macrocycle.
Reagents and conditions: (a) 4-methylmorpholine, isobutyl chlor-
oformate, THF, À408C; NaBH4, MeOH, À208C, 66%; (b) oxalyl
chloride, DMSO, DIPEA, CH2Cl2, À658C; (c) methyltriphenylphos-
phonium bromide, KHMDS, THF, À788C, 80% over two steps; (d)
3-buten-1-ol, Grubbs catalyst, 2nd generation, CH2Cl2, 508C, 25%;
(e) TsCl, Et3N, DMAP, CH2Cl2, 82%; (f) TrtSH, KOtBu, THF, 87%;
(g) LiOH, THF, MeOH, 508C, 96%; (h) MeOH, EDCI, DIPEA,
DMAP, CH2Cl2, 77%; (i) TFA, CH2Cl2; (j) N-Boc-Val-OH, PyBOP,
DIPEA, CH2Cl2, 88% over two steps.
42 derivative 20.[8b] Reduction of the carboxylic acid (20 to 21),
43 Swern oxidation, and subsequent Wittig olefination provided
44 terminal alkene 22. Grubb’s cross-metathesis with 3-buten-1-
45 ol was employed to provide compound 23. The yield at this
46 stage was very modest and alternative routes were explored
47 but were either unsuccessful or resulted in epimerization of
48 the amino group.[7,17] Tosylation and reaction with trityl thiol
49 provided protected thiol 24, which then underwent methyl
50 ester formation to provide compound 25. N-t-Boc deprotection
51 using TFA exposed the free amine group, which was then
52 coupled to N-t-Boc protected l-valine through the use of
53 PyBOP, furnishing fragment 26.
Scheme 5. Synthesis of depsipeptide macrocycle compound 36.
Reagents and conditions: (a) tert-butyl ((4-nitrophenyl)sulfonyl)
carbamate, diethyl azodicarboxylate, triphenylphosphine, THF, 90%;
(b) a-methyl cysteine, sodium carbonate, triethylamine, MeOH,
reflux, 59%; (c) 19, MeCNÀdiethylamine (10:1); (d) PyBOP, DIPEA,
DCM, 58% over two steps; (e) DCMÀTFA (5:1); (f) HOBt, HATU,
DIPEA, MeCN, 20% over two steps.
For the assembly of the corresponding peptide isostere, 37
was reacted with LiOHÀH2O to provide a free carboxylic acid
that underwent intramolecular HOBt and HATU coupling with
the deprotected Boc amino group after subjection to TFA to
provide compound 39 (Scheme 6).
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Functionalization of the thiazole ring is depicted in
55 Scheme 4.[18] Compound 30 with the hydroxymethyl handle
56 was accessed via cyano-thiazole compound 27. LDA deproto-
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Scheme 6. Synthesis of peptide isostere macrocycle compound 39. Reagents and conditions: (a) 26, DCMÀTFA (10:1); (b) PyBOP, DIPEA,
DCM; (c) lithium hydroxideÀH2O, THFÀMeOHÀH2O (2:2:1); (d) DCMÀTFA (10:1); (e) HOBt, HATU, DIPEA, MeCN, 12% over five steps.
Table 1. IC50 values (nM) of compounds 1, 2, 5, and 43 across
HDAC isoforms 1–3, 6, and 8.
2.3 Biochemical Evaluation
HDAC isoform IC50 (nM)
Compounds 1, 2, 5, and 43 were tested across an HDAC
Compound
1
2
3
6
8
isoform panel of HDACs 1–9 to determine the biochemical
inhibitory potency of these species relative to the parent
natural product. The IC50 values, reported in nM in Table 1,
represent a summary of HDAC inhibitory activity. The peptide
isostere thiol of parent largazole, 2, displayed inhibitory
activity below 4 nM for HDACs 1–3, proving more potent
than the parent natural product 1. The newly derivatized
compound at the thiazole cap group of parent largazole, 43
(Figure 4), displayed sub-nanomolar inhibitory activity for
HDACs 1 and 3 and below 3 nM for HDAC 2. A decrease in
potency was seen across all derivatives for HDACs 6 and 8, as
expected.
Largazole (1)
2
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5
10.09
1.95
0.166
0.2388
18.65
3.38
2.61
2.84
9.09
2.59
0.734
1.064
165.6
102
14.9
1068
255.3
94
36.51
129.3
BiotinÀlargazole conjugate 5 exhibited very little change
in IC50 activity when compared to compound 43. IC50 values
below 3 nM were maintained across HDACs 1–3 for 5 and 43.
A full representation of the full HDAC panel for 43 (Fig-
ure 5A) and 5 (Figure 5B) is depicted in Figure 5.
Figure 4. Structure of depsipeptide analogue with a hydroxymethyl
handle off of the thiazole cap group.
Affinity chromatography assays were performed with the
largazoleÀbiotin derivative 5 to detect HDACs 1–3 binding in
cell lysate. Streptavidin beads were added after a 16 h
incubation with compound 5 or vehicle in 200 mg of HeLa
nuclear extract. Figure 6 shows immunoblot analysis of the
resulting eluates, confirming the enrichment of HDACs 1–3
from streptavidin beads conjugated to compound 5 over mock
pull down (vehicle only) or input samples.
Compounds 36 and 39 were each subjected to treatment
41 with thiophenol and 2 M KOH in acetonitrile to remove the
42 nosyl protecting group and expose the free amine for coupling
43 to biotin.[20] NaHCO3 was used to promote the amide coupling
44 of biotin for both the depsipeptide and peptide largazole
45 derivatives. Biotin linker 40 was used due to the stability of
46 the amide bond, reliable synthesis for highly functionalized
47 molecules, and the increased length allowed for solubility.
48 Trityl deprotection using DCMÀTFA and iPr3SiH provided
49 largazoleÀbiotin derivatives 5 and 6 in 41% and 36% yield,
50 respectively, as shown in Scheme 7. This convergent route can
51 be utilized to access additional amide-linked biological
52 conjugates.
3. Conclusion
We have developed a versatile, successful synthesis of new
cap group derivatives of the potent natural product largazole
with functional handles for conjugating probe molecules,
reporters, and potential cancer-cell-specific targeting moieties.
The potent biochemical activity observed for the thiazole-
derivatized analogues reported herein demonstrates that the
unencumbered cap group chemical space is a fertile area for
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Scheme 7. Synthesis of depsipeptideÀbiotin conjugate (5) and peptide isostereÀbiotin conjugate (6). Reagents and conditions: (a) thiophenol,
2 M potassium hydroxide, MeCN; (b) sodium bicarbonate, MeCN; (c) DCMÀTFA (20:1), triisopropylsilane, 41% (depsipeptide derivative)
over three steps, 36% (peptide isostere) over three steps.
Figure 5. Inhibition of HDAC activity by largazole thiol and biotinylated largazole thiol. (A,B) Inhibition of HDAC 1–9 activity by (A) 43 and (B)
5 is presented in doseÀresponse format. Data represent mean values of quadruplicate data Æ s.d. IC50 curves were fit by logistic regression.
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Figure 6. 1 mM largazoleÀbiotin (5) in 200 mL of HeLa nuclear extract (~200 mg); 1 mg of streptavidin beads added after 16 h incubation with
5.
29 future investigations into increasing the selectivity and
imeter Autopol III. FT-IR spectra were collected on a Thermo
Scientific Nicolet iS50.
30 therapeutic potential of largazole analogues. We are inten-
31 sively investigating the chemical conjugation of other mole-
32 cules to the largazole scaffold via the approach described
33 herein and will report on this in due course.
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4.2 tert-Butyl ((4-Cyano-5-iodothiazol-2-yl)methyl)carbamate
(28)
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36 4. Experimental Section
To a solution of thiazole 27 (85 mg, 0.36 mmol) in 7.2 mL of
THF at À788C, 1.1 mL of LDA (0.88 mmol, 0.8 M in THF)
was added dropwise (it should be noted that the addition of
more than three equivalents of LDA would have resulted in
decomposition of SM). The mixture was stirred at À788C for
20 min, then a THF solution (1.44 mL) of I2 (365 mg,
1.44 mmol) was added. After stirring at the same temperature
for 5 min, the solution was treated with saturated aq. NH4Cl,
diluted with ethyl acetate, separated, washed with brine, dried
over Na2SO4, filtered, and concentrated under reduced
pressure. The residue was purified by flash column chroma-
tography on silica gel (0 to 9% ethyl acetate in DCM) to
afford 97.4 mg (74% yield) of thiazole iodide 28 as a brown
solid.
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38 4.1 General Experimental Methods
39
40 All reactions were run under argon in oven- or flame-dried
41 glassware. Thin-layer silica gel chromatography (TLC) was
42 used to monitor reactions on 0.25 mm silica gel 60F plated
43 with fluorescent indicator from Merck. Various stains were
44 used to visualize TLC plates, including but not limited to
45 anisaldehyde and Seebach’s with gentle heating. Column
46 chromatography and preparatory TLC were used to purify all
47 compounds. The solvent systems described below were run on
48 silica gel 60 (230–400 mesh) purchased from Sorbent Tech-
49 nologies. Reagents were purchased from commercial sources
50 and used either directly or after drying. HNMR and 13CNMR
1HNMR (400 MHz, CDCl3) d 5.29 (s, 1H), 4.57 (d, J=
6.4 Hz, 2H), 1.46 (s, 9H); 13CNMR (100 MHz, CDCl3) d
176.5, 155.6, 134.6, 113.8, 85.0, 81.0, 42.6, 28.3; IR (neat)
3364, 2927, 1678, 1516; HRMS (ESI): m/z calcd. for C10H12
IN3NaO2S+ (M+Na)+ 387.9587, found 387.9579.
1
51 were recorded using Varian 300, 400, or 500 MHz NMR
52 spectrometers. Chemical shifts are reported in ppm relative to
53 CHCl3 at d=7.27 (1HNMR) and d=77.23 (13CNMR) or
54 tetramethylsilane (TMS) at d=0.00. Mass spectra were
55 obtained on a Fisions VG Autospec. Optical rotations were
56 collected at 589 nm on a Rudolph Research Automatic Polar-
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4.3 tert-Butyl ((4-Cyano-5-(4-(hydroxymethyl)phenyl)
thiazol-2-yl)methyl)carbamate (30)
4.5 (R)-2’-(((tert-Butoxycarbonyl)amino)methyl)-4-me-
thyl-5’-(4-(((4-nitrophenyl)sulfonamido)methyl)phenyl)-4,5-di-
hydro-[2,4’-bithiazole]-4-carboxylic Acid (33)
Thiazole iodide 28 (550 mg, 1.5 mmol), 4-(hydroxymethyl)
phenylboronic acid 29, (343 mg, 2.3 mmol), NaHCO3
(380 mg, 4.5 mmol), and Pd(PPh3)4 were combined, and
16 mL of solvent (DME/H2O=3/1, v/v) was added. The
mixture was heated to 1208C and refluxed at that temperature
for 14 h, then cooled to room temperature and diluted with
Thiazole nitrile 31 (377 mg, 0.6 mmol), a-methyl-cysteine-
HCl (124 mg, 0.72 mmol), and NaHCO3 (76 mg, 0.9 mmol)
were combined and 10 mL of methanol was added. To the
mixture was added Et3N (0.16 mL, 1.2 mmol). After refluxing
at 708C for 36 h, the solvent was removed under reduced
pressure. The residue was purified by flash column chroma-
tography on silica gel (9% to 30% ethyl acetate in DCM) to
afford 230 mg (32:33=1:1, 59%) of 32 and 33 as a yellow
foam. 32 and 33 were used as a mixture for the next reaction
without further purification. An analytically pure sample could
be obtained by esterification, separation, then hydrolysis.
10 water. DME was evaporated, and the aqueous layer was
11 extracted with ethyl acetate and separated. The organic layer
12 was washed with brine, dried over Na2SO4, filtered, and
13 concentrated under reduced pressure. The residue was purified
14 by flash column chromatography on silica gel (0 to 11% ethyl
15 acetate in DCM) to afford 67 mg (19% yield) of protonated
16 compound 27 and 354 mg (68% yield) of desired coupling
17 product 30 as a white solid.
1
(R=H, 33), [a]D28 =À88 (c=1.126 in CH3OH); HNMR
(400 MHz, CD3OD) d 7.81 (dd, J=7.8, 1.2 Hz, 1H), 7.74 (dd,
J=7.6, 1.4 Hz, 1H), 7.66 (ddd, J=7.8, 7.6, 1.4 Hz, 1H), 7.59
(ddd, J=7.8, 7.6, 1.2 Hz, 1H), 7.33 (d, J=8.1 Hz, 2H), 7.25
(d, J=8.1 Hz, 2H), 4.46 (s, 2H), 4.29 (s, 2H), 3.76 (d, J=
11.3 Hz, 1H), 3.22 (d, J=11.3 Hz, 1H), 1.45 (s, 3H), 1.45 (s,
9H); 13CNMR (100 MHz, CDCl3) d 176.0, 169.5, 162.6,
156.8, 147.7, 141.6, 140.0, 138.2, 133.8, 133.2, 132.1, 130.2,
129.7, 128.9, 127.5, 124.3, 84.8, 79.6, 46.3, 41.7, 41.5, 27.3,
22.6; IR (neat) 3120, 2974, 2938, 1693, 1665, 1392, 1365,
1347, 1172, 1154, 1079, 863; HRMS (ESI): m/z calcd. for C27
18
1HNMR (400 MHz, CDCl3) d 7.61 (d, J=8.2 Hz, 2H),
19 7.41 (d, J=8.2 Hz, 2H), 5.64 (br s, 1H), 4.70 (s, 2H), 4.52 (d,
20 J=4.6 Hz, 2H), 2.81 (br s, 1H), 1.45 (s, 9H); 13CNMR (100
21 MHz, CDCl3) d 169.1, 143.7, 128.7, 128.1, 127.5, 127.0,
22 120.4, 115.3, 114.6, 80.8, 64.2, 42.3, 28.3 (3 3); IR (neat)
23 3332, 2978, 2227, 1689, 1513, 1279, 1248, 910, 728; HRMS
24 (ESI): m/z calcd. for C17H19N3O2S+ (M+Na)+ 368.1039,
25 found 368.1030.
26
27
+
H30N5O8S3 (M+H)+ 648.1251, found 648.1231.
28
29
4.4 tert-Butyl ((4-Bromophenyl)sulfonyl)(4-(2-(((tert-
butoxycarbonyl)amino)methyl)-4-cyanothiazol-5-yl)benzyl)-
30 carbamate (31)
4.6 2-(Trimethylsilyl)ethyl (S,E)-3-((((R)-2’-(((tert-
31
Butoxycarbonyl)amino)methyl)-4-methyl-5’-(4-(((4-nitrophe-
nyl)sulfonamido)methyl)phenyl)-4,5-dihydro-[2,4’-bithiazo-
le]-4-carbonyl)-L-valyl)oxy)-7-(tritylthio)hept-4-enoate (35)
32 Benzyl alcohol 30 (35 mg, 0.1 mmol), NsNHBoc (45 mg,
33 0.15 mmol), and PPh3 (39 mg, 0.15 mmol) were combined and
34 1 mL of THF was added. To the mixture was added DEAD
35 (40% in toluene, w/w, 75 mL, 0.15 mmol) at 08C. After
36 stirring at 08C for 30 min, the solution was washed with 1 N
37 HCl, dried over Na2SO4, filtered, and concentrated under
38 reduced pressure. The residue was purified by flash column
39 chromatography on silica gel (0% to 4% ethyl acetate in
40 DCM) to afford 57 mg (90% yield) of 31 as a yellow solid.
19 (336 mg, 0.4 mmol) was dissolved in 40 mL of CH3CN.
4 mL of diethylamine was added at 08C. The bath was
removed and the resulting solution was stirred at room
temperature for 2 h, then evaporated, azeotroped with toluene
(23 2 mL), and dried under vacuum. In another round flask,
acid 32/33 (230 mg, 0.35 mmol, mixture of 32 and 33, 1:1)
was dissolved in 55 mL of DCM. PyBOP (416 mg, 0.8 mmol)
and DIPEA (210 mL, 1.2 mmol) were added and the mixture
was allowed to stir at room temperature for 20 minutes. To the
resulting solution was added a DCM solution (total 25 mL) of
crude amine. After 3 h, the reaction was concentrated and
submitted immediately to column chromatography (0% to
25% ethyl acetate in DCM) to afford the macrocyclization
precursor (236 mg, 58%, 34:35=1:1) as a yellow foam. 34
and 35 were used as a mixture for the next reaction without
further purification.
41
1HNMR (400 MHz, CDCl3) d 8.32 (dd, J=8.3, 1.7 Hz,
42 1H), 7.79–7.69 (m, 5H), 7.54 (d, J=8.2 Hz, 1H), 5.54 (br s,
43 1H), 4.99 (s, 2H), 4.57 (d, J=5.9 Hz, 2H), 1.46 (s, 9H), 1.28
44 (s, 9H); 13CNMR (100 MHz, CDCl3) d 176.2, 169.2, 150.2,
45 147.6, 140.0, 134.5, 133.8, 133.1, 132.4, 132.0, 128.5, 128.4,
46 127.5, 124.6, 120.7, 114.7, 85.7, 80.8, 50.6, 42.5, 28.3 (3 3),
47 27.8 (3 3); IR (neat) 3353, 2980, 1730, 1543, 1367, 1280,
48 1249, 1151, 1123, 1062, 918, 852, 760; HRMS (ESI): m/z
49 calcd. for C28H31N5NaO8S2 (M+Na)+ 652.1506, found
50 652.1498.
+
51
52
53
54
55
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4.7 N-(4-((12Z,22Z,24R,5S,8S)-5-Isopropyl-24-methyl-3,6,10-tri-
oxo-8-((E)-4-(tritylthio)but-1-en-1-yl)-24,25-dihydro-7-oxa-4,11-
diaza-1(4,2),2(2,4)-dithiazolacyclododecaphane-15-yl)
benzyl)-4-nitrobenzenesulfonamide (36)
Free amine (6.0 mg, 9 mmol), biotin derivative 40 (8.1 mg,
18 mmol), and NaHCO3 (1.5 mg, 18 mmol) were combined.
DMF (0.3 mL) was added and the reaction was allowed to stir
for 14 h then concentrated under reduced pressure. The residue
was purified by flash column chromatography on silica gel
(100% AcOEt then 5% to 11% MeOH in DCM) then by
preparative TLC (9% MeOH in DCM three times) to afford
the pure coupling product 41 as a yellow oil.
Coupling product 41 was dissolved in 0.6 mL of DCM and
cooled to 08C. TFA (30 mL) and iPr3SiH (1.0 mL, 6 mmol)
were added to the solution at 08C. The bath was removed and
the reaction was allowed to stir at room temperature for 1 h.
The solvent was removed by argon flow and the residue was
purified by flash column chromatography on silica gel (0 to
9% MeOH in CHCl3) to afford 4.0 mg of free thiol 5 (53% for
two steps). This was further purified by preparative TLC (9%
MeOH in DCM two times) to afford the pure sample for
biological evaluation.
Acyclic precursor (236 mg, 0.2 mmol, mixture of 34 and 35,
1:1) was dissolved in 30 mL of DCM, and 6 mL of TFA was
added to the solution at 08C. The reaction was allowed to
warm to room temperature and stirred for 16 h. Solvents were
10 evaporated and the crude amino acid was azeotroped with
11 toluene (103 3 mL) to remove residual TFA. The crude amino
12 acid was then dissolved in 300 mL of CH3CN (to ~0.001 M)
13 and DIPEA (0.32 mL, 1.8 mmol) was added. The resulting
14 moderately opaque solution was allowed to stir for 10 min,
15 before an MeCN (10 mL) solution of HATU (230 mg,
16 0.6 mmol) and HOBt (81 mg, 0.6 mmol) was added dropwise.
17 The reaction was allowed to stir for 26 h, then concentrated
18 under reduced pressure. The residue was purified by flash
19 column chromatography on silica gel (0% to 11% AcOEt in
20 DCM for the first column, 25% to 75% ethyl acetate in
21 hexane for the second column, and 0% to 9% AcOEt in DCM
22 for the third column) to afford 40 mg (20% yield for two
[a]D25 = +52.08 (c=0.050 in CH3OH); 1HNMR (600
MHz, CD3OD,) 7.48 (d, J=8.0 Hz, 2H), 7.40 (d, J=8.0 Hz,
2H), 5.92–5.82 (m, 2H), 5.72–5.63 (m, 2H), 5.13 (d, J=
8.0 Hz, 1H), 4.59–4.55 (m, 2H), 4.49 (dd, J=7.7, 4.7 Hz, 1H),
4.44–4.40 (m, 3H), 4.30 (dd, J=7.7, 4.7 Hz, 1H), 3.77 (d, J=
11.6 Hz, 1H), 3.60 (s, 1H), 3.27 (d, J=11.6 Hz, 1H), 3.21–
3.15 (m, 5H), 2.98–2.91 (m, 2H), 2.76–2.69 (m, 3H), 2.56 (dd,
J=6.9, 6.9 Hz, 1H), 2.45 (dd, J=13.5, 6.5 Hz, 1H), 2.36 (m,
1H), 2.28 (dd, J=7.4, 7.4 Hz, 2H), 2.23–2.09 (m, 6H), 1.81
(s, 3H), 1.76–1.29 (m, 23H), 0.74 (d, J=6.9 Hz, 3H), 0.59 (d,
J=6.9 Hz, 3H); 13CNMR (125 MHz, CD3OD) 176.1, 176.0,
175.9, 175.8, 171.8, 170.4, 168,3, 167.2, 166.0, 144.7, 143.0,
142.5, 133.7, 131.4, 129.7, 129.1, 128.8, 84.0, 73.8, 64.3,
63.3, 61.6, 58.9, 57.0, 44.4, 43.6, 41.2, 41.0, 40.2, 38.7, 37.0,
36.9, 36.8, 35.2, 32.7, 30.1, 29.8, 29.5, 27.5, 26.9, 26.7, 26.6,
24.5, 19.8, 17.3; IR (neat) 3308, 2930, 2859, 1656, 1551,
23 steps) of 36 as a yellow foam.
24
1
[a]D26 = +198 (c=0.147 in CHCl3); HNMR (500 MHz,
25 CD3OD) d 7.91 (dd, J=7.8, 1.3 Hz, 1H), 7.80 (dd, J=7.8,
26 1.3 Hz, 1H), 7.71 (ddd, J=7.9, 7.5, 1.4 Hz, 1H), 7.64 (dddd,
27 J=7.7, 7.6, 5.1, 1.2 Hz, 1H), 7.57–7.18 (m, 15H), 5.71 (ddd,
28 J=16.1, 8.4, 6.7 Hz, 1H), 5.63 (m, 1H), 5.48 (dd, J=15.4,
29 6.3 Hz, 1H), 5.11 (d, J=17.5 Hz, 1H), 4.55 (d, J=3.8 Hz,
30 1H), 4.37 (s, 1H), 4.34 (d, J=17.5 Hz, 1H), 3.77 (d, J=
31 11.6 Hz, 1H), 3.24 (d, J=11.6 Hz, 1H), 3.20 (m, 1H), 2.89
32 (dd, J=15.9, 10.6 Hz, 1H), 2.67 (dd, J=15.9, 3.1 Hz, 1H),
33 2.20 (dd, J=17.3, 17.0 Hz, 1H), 2.12–2.02 (m, 2H), 1.92–1.88
34 (m, 2H), 1.77 (s, 3H), 1.59 (m, 1H), 0.73 (d, J=6.9 Hz, 3H),
35 0.57 (d, J=6.9 Hz, 3H); 13CNMR (125 MHz, CD3OD) d
36 175.8, 171.8, 170.3, 168.3, 167.2, 149.3, 146.3, 144.6, 142.9,
37 140.8, 135.4, 134.8, 133.8, 133.5, 133.0, 131.6, 131.3, 130.8,
38 129.3, 129.2, 128.9, 127.7, 125.8, 83.9, 73.6, 58.9, 47.6, 47.4,
39 44.4, 41.3, 41.0, 35.2, 32.5, 32.4, 30.8, 30.5, 27.4, 24.5, 23.8,
40 19.8, 17.3, 14.4; IR (neat) 3362, 2960, 2926, 2853, 1734,
41 1668, 1540, 1443, 1344, 1259, 1165, 1071, 972, 853; HRMS
+
1462, 1262, 667; HRMS (ESI): m/z calcd. for C50H72N9O8S4
(M+H)+ 1054.4381, found 1054.4346.
4.9 Methyl (S,E)-3-((S)-2-((R)-2’-(((tert-Butoxycarbonyl)
amino)methyl)-4-methyl-5’-(4-(((4-nitrophenyl)sulfonamido)-
methyl)phenyl)-4,5-dihydro-[2,4’-bithiazole]-4-carboxami-
do)-3-methylbutanamido)-7-(tritylthio)hept-4-enoate (38)
42 (ESI): m/z calcd. for C53H53N6O8S4 (M+H)+ 1029.2802,
+
43 found 1029.2778.
44
45
Boc-amine 26 (420 mg, 0.66 mmol) was dissolved in 10 mL
of DCM and 1 mL of TFA was added to the solution at 08C.
The reaction was allowed to warm to room temperature and
stirred for 2 h. Solvents were evaporated and the crude amino
acid was azeotroped with toluene (23 2 mL) to remove
residual TFA. Acid 32/33 (470 mg, ca. 0.72 mmol, 32:33=
1:1) was dissolved in 50 mL of DCM. PyBOP (730 mg,
1.4 mmol) and DIPEA (630 mL, 3.6 mmol) were added and
the mixture was allowed to stir at room temperature for 20
minutes. To the resulting solution was added a DCM solution
(total 10 mL) of crude amine. After 4 h, the reaction was
concentrated and submitted immediately to column chroma-
46 4.8 LargazoleÀBiotin Conjugate (5)
47
48 Thiophenol (10.4 mL, 0.1 mmol), KOH (3.6 mg, 64 mmol),
49 water (30 mL), and acetonitrile (1.2 mL) were combined to
50 give a colorless solution. To 8.2 mg (9.1 mmol) of Ns amine 36
51 was added 0.4 mL of the above solution at room temperature.
52 The reaction was allowed to stir for 2 h then diluted with
53 4 mL of hexane and submitted directly to column chromatog-
54 raphy (100% AcOEt then 25% MeOH in DCM) to afford
55 6.0 mg (78% yield) of free amine as a yellow oil.
56
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tography (16% to 33% ethyl acetate in DCM) to afford
macrocyclization precursor 37:38 (1:1) as a yellow foam.
For R=H 38, [a]D26 =À55.88 (c=0.163 in CHCl3);
1HNMR (400 MHz, CDCl3) d 8.04 (m, 1H), 7.67 (m, 1H),
7.61–7.58 (m, 2H), 7.36–7.34 (m, 10H), 7.27–7.14 (m, 13H),
6.63 (d, J=8.9 Hz, 1H), 6.46 (d, J=9.4 Hz, 1H), 5.87 (br s,
1H), 5.46 (ddd, J=15.4, 6.8, 6.3 Hz, 1H), 5.37–5.31 (m, 2H),
4.76 (m, 1H), 4.57 (d, J=6.0 Hz, 2H), 4.31 (m, 2H), 3.93 (dd,
J=8.9, 8.6 Hz, 2H), 3.67 (m, 2H), 3.56 (m, 1H), 3.53 (s, 3H),
dissolve in pure CHCl3, AcOEt, MeCN, or MeOH. Use 66%
AcOEt in DCM for developing TLC.)
[a]D30 = +17.58 (c=0.257 in CH2Cl2); 1HNMR (400
MHz, CD2Cl2) d 8.03 (dd, J=7.5, 2.2 Hz, 1H), 7.82 (dd, J=
7.8, 1.5 Hz, 1H), 7.72–7.68 (m, 2H), 7.31–7.18 (m, 23H), 6.92
(d, J=8.1 Hz, 1H), 6.72 (d, J=9.5 Hz, 1H), 6.63 (d, J=
10.5 Hz, 1H), 6.21 (dd, J=6.4, 6.4 Hz, 1H), 5.52–5.40 (m,
2H), 5.07 (dd, J=17.5, 8.4 Hz, 1H), 4.86 (m, 2H), 4.47 (dd,
J=10.4, 3.3 Hz, 1H), 4.35 (dd, J=6.4, 1.8 Hz, 1H), 4.24 (dd,
J=17.5, 3.1 Hz, 1H), 3.71 (d, J=11.7 Hz, 1H), 3.24 (d, J=
11.7 Hz, 1H), 2.67 (dd, J=14.6, 4.2 Hz, 1H), 2.52 (dd, J=
14.8, 9.1 Hz, 1H), 2.37 (m, 1H), 2.20 (dd, J=7.7, 7.1 Hz, 2H),
2.02–1.98 (m, 2H), 1.77 (s, 3H), 0.87 (dd, J=6.9, 6.6 Hz,
1H), 0.71 (d, J=6.8 Hz, 3H), 0.40 (d, J=6.8 Hz, 3H);
13CNMR (100 MHz, CD2Cl2) d 173.7, 170.1, 170.0, 166.0,
147.7, 144.8, 142.1, 141.9, 138.4, 133.7, 133.5, 132.9, 130.8,
130.5, 130.2, 129.6, 129.5, 128.3, 127.9, 127.8, 126.6, 126.5,
125.2, 83.8, 66.9, 58.5, 48.2, 47.4, 44.6, 41.3, 40.5, 38.7, 32.3,
31.9, 31.7, 31.6, 30.0, 24.0, 19.7, 16.0, 14.3; IR (neat) 3351,
2960, 2927, 1661, 1539, 1499, 1364, 1265, 1163, 1070, 844,
733, 700, 583; HRMS (ESI): m/z calcd. for C53H53N7NaO7S4
(M+Na)+ 1050.2781, found 1050.2801.
10 3.45 (d, J=11.6 Hz, 1H), 3.19 (d, J=11.6 Hz, 1H), 2.52 (m,
11 2H), 2.13–1.98 (m, 2H), 1.73 (m, 1H), 1.44 (s, 12H), 1.26–
12 1.20 (m, 4H), 0.77 (d, J=6.7 Hz, 3H), 0.62 (d, J=6.7 Hz,
13 3H); 13CNMR (100 MHz, CDCl3) d 174.4, 171.7,169.8,
14 167.4, 162.3, 148.0, 144.8, 141.6, 137.8, 134.2, 132.3, 130.6,
15 130.5, 130.3, 129.6, 129.5, 129.4, 127.8, 127.6, 126.5, 124.5,
16 85.5, 80.4, 66.5, 58.6, 51.7, 47.2, 47.1, 43.7, 42.3, 40.6, 38.6,
17 31.4, 31.3, 30.5, 29.6, 24.1, 19.2, 18.4, 18.1, 17.0, 14.1, 12.6;
18 IR (neat) 3367, 2964, 2927, 1717, 1652, 1541, 1511, 1366,
19 1164, 972, 752; HRMS (ESI): m/z calcd. for C59H65N7NaO10
20 S4 (M+Na)+ 1182.3568, found 1182.3583.
21
22
23
24
+
4.10 N-(4-((12Z,22Z,24R,5S,8S)-5-Isopropyl-24-methyl-3,6,10-t-
rioxo-8-((E)-4-(tritylthio)but-1-en-1-yl)-24,25-dihydro-4,7,11-
triaza-1(4,2),2(2,4)-dithiazolacyclododecaphane-15-yl)benzyl)-
25
4.11 Largazole Peptide IsostereÀBiotin Conjugate (6)
26 4-nitrobenzenesulfonamide (39)
27
Thiophenol (10.4 mL, 0.1 mmol), KOH (3.6 mg, 64 mmol),
water (30 mL), and DMF (1.2 mL; acetonitrile was used as
solvent to deprotect the Ns group of the depsipeptide macro-
cyclic substrate 36 mentioned above, however this peptide
macrocycle 39 is not soluble in acetonitrile therefore DMF
was used here) were combined to give a colorless solution. To
8.5 mg (8.3 mmol) of Ns amine 39 was added 1.0 mL of above
solution at room temperature. The reaction was allowed to stir
for 3 h then evaporated and purified by column chromatog-
raphy (50% AcOEt in DCM then 50% MeOH in DCM) to
afford 6.1 mg (87% yield) of free amine as a yellow solid.
Free amine (6.1 mg, 7.2 mmol), biotin derivative 40
(8.2 mg, 14 mmol), and NaHCO3 (1.2 mg, 14 mmol) were
combined. DMF (0.5 mL) was added and the reaction was
allowed to stir for 15 h then concentrated under reduced
pressure. The residue was purified by flash column chroma-
tography on silica gel (100% AcOEt then 5% to 50% MeOH
in DCM) then by preparative TLC (11% MeOH in DCM two
times) to afford the pure coupling product 42 as a yellow
solid.
Coupling product 42 was combined with 0.6 mL of DCM
to give a suspension. TFA (30 mL) was added at 08C and the
suspension soon became a yellow solution. iPr3SiH (1.0 mL, 6
mmol) was added to the solution at 08C. The bath was
removed and the reaction was allowed to stir at room
temperature for 1 h. Solvent was removed by argon flow and
the residue was purified by flash column chromatography on
silica gel (4 to 50% MeOH in DCM) to afford 3.1 mg of free
thiol 6 (41% for two steps). This was further purified by
28 Acyclic precursor 37/38 obtained above was azeotroped with
29 toluene (23 2 mL) to remove residual AcOEt, then combined
30 with
LiOH·H2O
(139 mg,
3.3 mmol).
Solvent
31 (THFÀMeOHÀH2O, 2+2+1 mL) was added and the reaction
32 mixture was allowed to stir for 2.5 h. The reaction mixture
33 was diluted with 10 mL of water, then the pH was adjusted to
34 2 by using 1 N HCl. Organic solvents were evaporated and the
35 remaining aqueous layer was extracted with DCM (33 10 mL)
36 then dried over Na2SO4, filtered, and evaporated. The crude
37 acid was dissolved in 15 mL of DCM and 3 mL of TFA was
38 added to the solution at 08C. The reaction was allowed to
39 warm to room temperature and stirred for 3 h. Solvents were
40 evaporated and the crude amino acid was azeotroped with
41 toluene (23 2 mL) to remove residual TFA. The crude amino
42 acid was then taken up in 700 mL MeCN and DIPEA (1 mL,
43 5.7 mmol) was added. The resulting moderately opaque
44 solution was allowed to stir for 10 min, before HATU
45 (500 mg, 1.32 mmol) and HOBt (178 mg, 1.32 mmol) were
46 added. The reaction was allowed to stir for 14 h, then
47 concentrated and redissolved in AcOEt. The solution was
48 washed with saturated aqueous NH4Cl, NaHCO3, and brine,
49 dried over Na2SO4, filtered, and concentrated under reduced
50 pressure. The residue was purified by flash column chroma-
51 tography on silica gel (0% to 33% AcOEt in DCM for the
52 first column, 16% to 33% AcOEt in DCM for the second
53 column, then 33% to 66% AcOEt in DCM for PTLC) to
54 afford 80 mg (12% yield for five steps) of 39 as white foam.
55 (The product does dissolve in pure DCM and mixtures of
56 DCM/AcOEt, DCM/MeOH and CHCl3/MeOH but does not
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preparative TLC (11% MeOH in DCM two times) to afford
the pure sample for biological evaluation.
extract (1 mg/mL total protein) at a final concentration of
1 mM and 0.1%, respectively. After 16 h incubation at 48C,
200 mg streptavidin Dynabeads (MyOne Streptavidin T1;
ThermoFisher Scientific), equilibrated in NER buffer at 1 mg/
mL, was added to each proteinÀcompound mixture and
incubated for 1 h at 48C with gentle rotation. Beads were then
collected, washed four times with wash buffer (10 mM HEPES
pH 8.0, 300 mM NaCl, 10 mM KCl, 1.5 mM MgCl2, and
0.2% Triton X-100), and finally resuspended and boiled in 90
mL of 1X NuPAGE LDS sample buffer (ThermoFisher
Scientific) to elute bound protein. Eluate and input samples
were run on 4–12% Bis-Tris NuPAGE gels (ThermoFisher
Scientific) and transferred to PVDF membranes. Membranes
were blocked for 1 h at RT in Odyssey blocking buffer (TBS;
LI–COR Biosciences) and then incubated overnight at 48C in
blocking buffer supplemented with 0.2% Tween-20 and a
primary antibody diluted 1:1000 (anti-HDAC1, Cell Signaling
#2062; anti-HDAC2, Cell Signaling #2540; anti-HDAC3, Cell
Signaling #2632). Next, membranes were washed three times
with TBST at RT (5 min each) and incubated for 1 h at RT in
blocking buffer supplemented with 0.2% Tween-20, 0.01%
SDS, and IRDye 800CW goat anti-rabbit IgG (LIÀCOR
Biosciences) diluted 1:7000. Following three 5 min washes in
TBST, membranes were rinsed in TBS and imaged on the
Odyssey CLx imaging system (auto intensity, 84 mm resolu-
tion, high quality; LI–COR Biosciences).
[a]D29 = +88.08 (c=0.10 in CH3OH); 1HNMR (600
MHz, CD3OD,) 7.49 (d, J=8.2 Hz, 2H), 7.41 (d, J=8.2 Hz,
2H), 5.70–5.56 (m, 3H), 5.17 (d, J=17.5 Hz, 1H), 4.93 (m,
1H), 4.91 (ddd, J=12.2, 5.9, 4.1 Hz, 1H), 4.52 (d, J=3.0 Hz,
1H), 4.48 (m, 1H), 4.44 (s, 2H), 4.37 (d, J=17.5, 1H), 4.29
(dd, J=7.9, 4.5 Hz, 1H), 3.71 (d, J=11.9 Hz, 1H), 3.65 (s,
1H), 3.60 (s, 1H), 3.40 (d, J=11.9 Hz, 1H), 3.21–3.14 (m,
10 4H), 2.92 (dd, J=12.8, 5.0 Hz, 1H), 2.77–2.69 (m, 2H), 2.64
11 (dd, J=14.9, 4.0 Hz, 1H), 2.55 (1H, dd, J=6.9, 6.9 Hz),
12 2.47–2.40 (m, 2H), 2.35 (dd, J=13.5, 6.8 Hz, 1H), 2.28 (dd,
13 J=7.4, 7.4 Hz, 1H), 2.18 (dd, J=13.5, 7.2 Hz, 2H), 1.88 (3H,
14 s), 1.75–1.36 (m, 23H), 0.73 (d, J=6.9 Hz, 3H), 0.41 (d, J=
15 6.9 Hz, 3H); 13CNMR (125 MHz, CD3OD) 176.2, 176.0,
16 175.9, 175.8, 172.2, 172.1, 169.6, 167.7, 160.0, 144.8, 142.8,
17 142.7, 132.5, 131.3, 129.8, 129.0, 128.7, 84.2, 64.3, 63.4,
18 61.6, 59.2, 57.0, 44.7, 43.6, 41.1, 41.0, 40.1, 39.1, 37.5, 37.0,
19 36.9, 36.8, 33.3, 30.7, 30.1, 29.7, 29.5, 27.5, 26.9, 26.7, 26.6,
20 24.6, 24.3, 20.0, 15.8; IR (neat) 3300, 2927, 2854, 1677,
21 1548, 1435, 1205, 1132, 801, 724; HRMS (ESI): m/z calcd.
22 for C50H72N10NaO7S4
23 1075.4379.
24
25
+
(M+Na)+ 1075.4360, found
26 4.12 HDAC Biochemical Assay
27
28 Compounds 1, 2, 5, and 43 were tested against HDACs 1–9
29 and the activity was determined with an optimized homoge-
30 nous assay performed in a 384-well plate. Reactions were
31 performed in assay buffer (50 mM HEPES, 100 mM KCl,
32 0.001% Tween-20, 0.05% BSA, pH 7.4; additional 200 mM
33 TCEP was added for HDAC6) and followed by fluorogenic
34 release of 7-amino-4-methylcoumarin from the substrate upon
35 deacetylase and trypsin enzymatic activity. Fluorescence
36 measurements were obtained every five minutes using a
37 multilabel plate reader and plate-stacker (Envision; Perkin-
38 Elmer). Each plate was analyzed by plate repeat, and the first
39 derivative within the linear range was imported into analytical
40 software (Spotfire DecisionSite). Replicate experimental data
41 from incubations with inhibitor were normalized to DMSO
42 controls ([DMSO] <0.5%). The IC50 was determined by
43 logistic regression with unconstrained maximum and mini-
44 mum values. The recombinant, full-length HDAC protein
45 (BPS Biosciences) was incubated with fluorophore conjugates
46 substrate, MAZ1600 and MAZ1675 at Km =[substrate].
47
Notes
The authors declare no competing financial interest. The
manuscript was written through contributions of all authors.
All authors have given approval to the final version of the
manuscript.
Acknowledgements
This work was supported by the National Institutes of Health
(Grant 1RO1 CA152314 to R. M. W. and J. E. B.). We also
acknowledge support from Dr. Christopher Rithner for NMR
support and Donald Dick for mass spectra experimental
assistance. Mass spectra were obtained on instruments
supported by the NIH Shared Instrument Grant GM49631.
J. E. B. acknowledges support by grants from the National
Cancer Institute (1K08CA128972) and the Burroughs-Well-
come Foundation (CAMS).
48
49 4.13 Biochemical Pull-Down Studies
50
51 HeLa cells (ATCC) were cultured in DMEM supplemented
52 with 10% FBS and Pen Strep (100 U/mL). Nuclear extracts
53 were collected from ~250 mL of HeLa cell pellet using NE-
54 PER extraction reagents (ThermoFisher Scientific) according
55 to the manufacturer’s protocol. For pull-down studies, com-
56 pound 5 or DMSO was added to 200 mL of HeLa nuclear
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Received: October 10, 2016
Accepted: && &&, 0000
Published online on && &&, 0000
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L. Zhao, C. E. Dunne, D. J. Clausen,
J. M. Roberts, J. Paulk, H. Liu, O. G.
Wiest, J. E. Bradner, R. M. Williams*
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Synthesis and Biochemical Evalua-
tion of Biotinylated Conjugates of
Largazole Analogues: Selective Class
I Histone Deacetylase Inhibitors
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