Total synthesis and stereochemical assignment of burkholdac b, a depsipeptide HDAC Inhibitor

Article abstract of DOI:10.1021/ol202197q

Three diastereomers of burkholdac B were prepared by total synthesis, enabling the full stereochemical assignment of the natural product. It is proposed that burkholdac B is identical to thailandepsin A independently isolated by Cheng from the same strain

Full text of DOI:10.1021/ol202197q

ORGANIC
LETTERS
2011
Vol. 13, No. 24
6334–6337
Total Synthesis and Stereochemical
Assignment of Burkholdac B, a
Depsipeptide HDAC Inhibitor
Hanae Benelkebir,† Alison M. Donlevy,‡ Graham Packham,‡ and A. Ganesan*,†
School of Pharmacy, University of East Anglia, Norwich Research Park, Norwich NR4
7TJ, United Kingdom, and Cancer Research UK Centre, University of Southampton,
Faculty of Medicine, Southampton General Hospital, Tremona Road, Southampton
SO16 6YD, United Kingdom
a.ganesan@uea.ac.uk
Received August 12, 2011; Revised Manuscript Received October 21, 2011
ABSTRACT
Three diastereomers of burkholdac B were prepared by total synthesis, enabling the full stereochemical assignment of the natural product. It is proposed
that burkholdac B is identical to thailandepsin A independently isolated by Cheng from the same strain of Burkholderia thailandensis. Burkholdac B is
the most potent among depsipeptide histone deacetylase inhibitors in growth inhibition of the MCF7 breast cancer cell line with an IC50 of 60 pM.
Lysine acetylation is a reversible protein post-translational
modification found in both prokaryotes and eukaryotes.1
Acetylation changes the side chain’s size and charge and
orchestrates proteinꢀprotein and proteinꢀDNA interac-
tions within the nucleus and other cellular compartments.
The conversion of N-acetyl lysine back to lysine in proteins
is catalyzed by histone deacetylases (HDACs). There are 18
HDACs in the human genome, 11 of which are zinc-
dependent while the 7 sirtuins are NADþ-dependent.2
Inhibitors of the zinc-dependent HDACs are potential
therapeutic agents for a variety of diseases.3 Natural
products are often a rich source of biologically active
compounds,4 and the HDACs are a case in point. Natural
product HDAC inhibitors such as trichostatin and apici-
din are widely used biological tools, while the depsipeptide
FK228 isolated5 from Chromobacterium violaceum No.
968 (Figure 1) is a clinically approved anticancer drug.
FK228 is a prodrug that undergoes intracellular dis-
ulfide bond reduction to release the free thiol that acts as a
zinc-binding warhead attheHDACactivesite. Subsequent
to the discovery of FK228, other depsipeptide HDAC
inhibitors with an identical warhead were identified. The
spiruchostatins isolated6 from Pseudomonas sp. have a
statine unit incorporated in the peptide backbone, while
largazole7 from the marine cyanobacterium Symploca
† University of East Anglia.
‡ University of Southampton.
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r
10.1021/ol202197q
Published on Web 11/17/2011
2011 American Chemical Society

Figure 1. Structures of depsipeptide HDAC inhibitors with
amino acid side chains indicated in red.
Figure 2. Cheng’s original (1, 2) and revised (3, 4) structures of
thailandepsins and Brady’s burkholdacs (5, 6).
sp. is an ester prodrug rather than a disulfide. The struc-
tural complexity and potent biological activity of these
depsipeptides has led to intensive efforts directed at total
and analogue synthesis by ourselves8 and others.9ꢀ12
Recently, the Cheng group characterized the FK228 gene
cluster.13 The biosynthesis involves a modular “assembly
line” hybrid of nonribosomal peptide synthase and polyke-
tide synthase. By genome mining for homologous open read-
ing frames, Cheng predicted Burkholderia thailandensis E264
to be a depsipeptide producer and discovered thailandepsins
A (1) and B (2) as disclosed in a patent (Figure 2).14
Intriguingly, one amide bond is replaced by a hemiam-
inal;a rare but not unprecedented motif in cyclic peptides.15
We explored a thailandepsin A synthesis via cyclization of an
aldehyde amine with the disulfide bridge in place but found
this to be a complex reaction yielding multiple products.
Since the patent provided no characterization data apart
from low-resolution MS that did not match the proposed
structures, we set aside further work on these compounds.
Cheng later revised the thailandepsin structures to more
plausible spiruchostatin congeners 3 and 4 (University of
Wisconsin;Madison Research Foundation presentation,
2010). While no stereochemical assignment was made, we
postulated that the statine was the syn diastereomer and all
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amino acids in the D-series by analogy to the spiruchosta-
tins (Figure 1). A remaining ambiguity is the isoleucine
stereochemistry. Epimerization of the R-chiral center of
L-Ile gives D-allo-Ile, whereas epimerization of R- and
β-chiral centers gives D-Ile. As both are found in natural
products,16 either diastereomer is possible and this can be
resolved only by synthesis.
E264 by overexpression of transcription factors to drive
secondary metabolite production.20 The burkholdacs are
isomeric to Cheng’s revised thailandepsins, and it is possible
that they are unique natural products. However, the isola-
tion of two distinct sets of depsipeptides from the same
bacterial strain seems unlikely. We believe Cheng’s thailan-
depsins are identical to Brady’s burkholdacs and the latter
became our new goal for synthesis.
The connectivity in the Brady structures was secured by
2D NMR. Although Brady did not assign stereochemistry,
he suggested the amino acids present in burkholdac B to be
L-Ile, D-Cys, and L-Met as the biosynthesis gene cluster
contains only one epimerase domain. Our own hypothesis
based on spiruchostatin homologywould predict all amino
acids to be of D-stereochemistry. We targeted three stereo-
isomers of burkholdac B 6 for total synthesis: (1) the Brady
proposal 6a with L-Ile, D-Cys, and L-Met; (2) the diaster-
eomer 6b with D-Ile, D-Cys, and D-Met; (3) the diastereo-
mer 6c with D-allo-Ile, D-Cys, and D-Met.
Scheme 1. Total Synthesis of Thailandepsin A 3a
Scheme 2. Total Synthesis of the Three Diastereomers of
Brady’s Burkholdac B
We first prepared the thailandepsin A diastereomer 3a
containing D-Ile (Scheme 1). The statine 7 was obtained by
Claisen condensation of the pentafluorophenyl ester of
Boc-D-Met and allyl acetate followed by ketone reduction
and Boc deprotection.17 Successive PyBOP-mediated cou-
plings with Fmoc-D-Cys and Fmoc-D-Ile gave the tripep-
tide 8. Amide bond formation with β-hydroxy acid 9,
obtained by our previously described asymmetric aldol
reaction8a with the FujitaꢀNagao auxiliary,18 provided
linear seco-hydroxy ester 10. Since thailandepsins are
sterically unencumbered next to the ester bond (unlike
FK228), macrolactonization8c with the Shiina reagent19
was successful and disulfide bond formation completed the
synthesis of 3a. As we were unable to obtain a sample of
thailandepsin A or spectral data, the identity of the natural
product remained unresolved.
Meanwhile, the Brady group reported the isolation of
burkholdacs A (5) and B (6) from Burkholderia thailandensis
Following the route described for statine 7, the statines
11aꢀc containing L-Ile, D-Ile, and D-allo-Ile side chains
were individually prepared from the corresponding isoleu-
cine diastereomer. These were then carried forward to the
linear hydroxy esters 12aꢀc, which upon macrolactoniza-
tion and disulfide bridging furnished depsipeptides 6aꢀc
(Scheme 2). Although the efficiency of macrocyclization
(16) Bevan, K.; Davies, J. S.; Hassall, C. H.; Phillips, D. A. S. J.
Chem. Soc. D: Chem. Commun. 1969, 1246.
(17) Preciado, A.; Williams, P. G. J. Org. Chem. 2008, 73, 9228–9234.
(18) Nagao, Y.; Yamada, S.; Kumagai, T.; Ochiai, M.; Fujita, E. J.
Chem. Soc., Chem. Commun. 1985, 1418–1419.
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7535–7539.
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1536–1539.
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Org. Lett., Vol. 13, No. 24, 2011

was variable, the yields are reported for a single experiment
and unoptimized.
Nevertheless, the additional isomers would not have been
otherwise made and have shed useful insights into the SAR
of depsipeptide HDAC inhibitors.
The NMR spectra of 6aꢀc show clear and significant
differences particularly in the resonances for the isoleucine
residue (Supporting Information). Diastereomer 6c con-
taining D-allo-Ile matches Brady’s 1H and 13C NMR data
and can be definitively assigned as burkholdac B and is
likely to be identical to Cheng’s thailandepsin A.
For the burkholdacs, the presence of a single epimerase
domain in the gene cluster does not lead to a natural
product with a single D-amino acid. The same lack of
congruence is seen in the related FK228 gene cluster, where
the natural product has two D-amino acids. In these
depsipeptides, the single epimerase may be acting in trans
fashion to invert the other amino acids or the acylation
domains may be accepting D-amino acids.
From the activity point of view, our results with 6a show
that introducing multiple L-amino acids into the depsipep-
tide skeleton is disadvantageous. Cheng’s revised structure
3 for thailandepsin A is similar in activity to other natural
products in this class, while burkholdac B 6c is outstanding
as an inhibitor of cell growth. Burkholdac B has a lipo-
philic Met residue, while other depsipeptide HDAC in-
hibitors contain smaller Gly, Ala, or Val amino acids at the
corresponding position. This change appears to be impor-
tant for the significant increase in activity and isoform
selectivity and suggests that further unnatural analogues
can be designed to optimize these features.
Table 1. IC50 Values (nM) of Depsipeptides in the Fluor-de-Lys
HDAC Enzyme Assay with HeLa Cell Extracts and Growth
Inhibition of the MCF7 Breast Cancer Cell Line
compound
HDAC (nM)a
MCF7 (nM)a
FK228
24 ( 4
0.8 ( 0.2
5.7 ( 0.7
5 ( 1
10.5 ( 1.8
410 ( 82
0.25 ( 0.05
0.06 ( 0.04
spiruchostatin A
5.3 ( 3.3
0.04 ( 0.3
8.3 ( 2.4
3312 ( 2472
3.1
largazole
3a
6a
6b
6c (burkholdac B)
5.0 ( 3.0
a The free thiol was generated for HDAC assays, while cell assays
were performed with the disulfide prodrug.
Note Added in Proof. Cheng has published the isolation of
thailandepsins (J. Nat. Prod. 2011, 74, 2031ꢀ2038) and con-
firmed that thailandepsin A and burkholdac B are identical.
Klausmeyer and coworkers at the NCI have isolated the
methionine sulfoxide of burkholdac B as an additional
natural product (J. Nat. Prod. 2011, 74, 2039ꢀ2044).
The “thailandepsin A” depsipeptide 3a and burkholdac B
diastereomers 6aꢀc were evaluated in HDAC enzyme and
cell line growth inhibition assays (Table 1). In both assays,
the diastereomer 6a with L-amino acids is less potent than
the natural products. Cheng’s transposed burkholdac 3a
and the diastereomers 6b and 6c are all nanomolar HDAC
inhibitors with similar activity to FK228 and spiruchostatin
A. In the MCF7 growth inhibition assay, the subnanomolar
activity of 6b,c is particularly noteworthy. This may be a
reflection of improved class I HDAC isoform selectivity as
reported by Brady or increased bioavailability or involve
other factors. Whatever the reasons, burkholdac B 6c is by
far the most potent of the depsipeptide natural products in
this cell assay. With an IC50 of 60 pM, burkholdac B is an
exciting lead for further investigation.
Structure elucidation was initially a major driver for
natural product total synthesis. With the sophistication of
current characterization methods, the structures of natural
products are usually unambiguous when the synthetic
endeavor commences. The thailandepsin/burkholdac case
is an exception with issues in both the atom connectivity
and stereochemistry.
Acknowledgment. Dedicated to Professor Clayton H.
Heathcock on the occasion of his 75th birthday. We are
grateful to Cancer Research UK and the COST Action
TD0905 ‘Epigenetics: Bench to Bedside’ for financial
support and the EPSRC National Mass Spectrometry
Service Centre, Swansea for HRMS data.
Note Added after Print Publication. This paper was pub-
lished ASAP November 17, 2011. The Table of Contents/
Abstract graphic, Schemes 1 and 2, Figures 1 and 2, and
the Supporting Information contained stereochemical
errors; the files were replaced and the Web edition
reposted January 6, 2012.
Supporting Information Available. Detailed experimen-
tal procedures and NMR spectra for all novel com-
pounds. This material is available free of charge via the
Internet at http://pubs.acs.org.
Several rounds of total synthesis were necessary before
the absolute structure of burkholdac B was confirmed.
Org. Lett., Vol. 13, No. 24, 2011
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