Synthesis of complex intermediates for the study of a dehydratase from borrelidin biosynthesis

Article abstract of DOI:10.3762/bjoc.10.55

Herein, we describe the syntheses of a complex biosynthesis-intermediate analogue of the potent antitumor polyketide borrelidin and of reference molecules to determine the stereoselectivity of the dehydratase of borrelidin polyketide synthase module 3. Th

Full text of DOI:10.3762/bjoc.10.55

Synthesis of complex intermediates for the study of a
dehydratase from borrelidin biosynthesis
Frank Hahn*,‡1,2, Nadine Kandziora‡1, Steffen Friedrich1
and Peter F. Leadlay2
Full Research Paper
Open Access
Address:
Beilstein J. Org. Chem. 2014, 10, 634–640.
1Institut für Organische Chemie und Biomolekulares
Wirkstoffzentrum, Leibniz Universität Hannover, Schneiderberg 1B,
30167 Hannover, Germany and 2Department of Biochemistry,
University of Cambridge, 80 Tennis Court Road, Cambridge CB2
1GA, United Kingdom
doi:10.3762/bjoc.10.55
Received: 30 August 2013
Accepted: 30 January 2014
Published: 11 March 2014
Email:
This article is part of the Thematic Series "Natural products in synthesis
and biosynthesis".
Frank Hahn* - frank.hahn@oci.uni-hannover.de
* Corresponding author ‡ Equal contributors
Guest Editor: J. S. Dickschat
Keywords:
© 2014 Hahn et al; licensee Beilstein-Institut.
License and terms: see end of document.
aldol reaction; coenzyme A; natural products; pig liver esterase;
polyketide biosynthesis; protection groups
Abstract
Herein, we describe the syntheses of a complex biosynthesis-intermediate analogue of the potent antitumor polyketide borrelidin
and of reference molecules to determine the stereoselectivity of the dehydratase of borrelidin polyketide synthase module 3. The
target molecules were obtained from a common precursor aldehyde in the form of N-acetylcysteamine (SNAc) thioesters and
methyl esters in 13 to 15 steps. Key steps for the assembly of the polyketide backbone of the dehydratase substrate analogue were a
Yamamoto asymmetric carbocyclisation and a Sakurai allylation as well as an anti-selective aldol reaction. Reference compounds
representing the E- and Z-configured double bond isomers as potential products of the dehydratase reaction were obtained from a
common precursor aldehyde by Wittig olefination and Still–Gennari olefination. The final deprotection of TBS ethers and methyl
esters was performed under mildly acidic conditions followed by pig liver esterase-mediated chemoselective hydrolysis. These
conditions are compatible with the presence of a coenzyme A or a SNAc thioester, suggesting that they are generally applicable to
the synthesis of complex polyketide-derived thioesters suited for biosynthesis studies.
Introduction
Borrelidin (1) is a macrolactone polyketide natural product with which are built-up by unconventional biosynthesis mechanisms
promising antibacterial, antimalarial, anticancer and anti-angio- [5,6]. The carbonitrile for example is probably formed by allylic
genesis activities, which are probably caused by the inhibition oxidation of the 12-methyl group in 12-desnitrile-12-methylbor-
of threonyl-tRNA synthetase and apoptosis induction by relidin to the corresponding aldehyde and transamination to the
caspase activation [1-4]. It bears several unusual structural amine followed by oxidation [6]. In the course of our studies on
elements like a cyclopentane ring and a carbonitrile (Figure 1a), borrelidin biosynthesis, we became interested in the formation
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Beilstein J. Org. Chem. 2014, 10, 634–640.
of the (12Z,14E)-diene [7]. Z-configured double bonds are Our aim was to assay the stereochemical course of the dehy-
much rarer in polyketides than their E-configured counterparts, dratase of polyketide synthase (PKS) module 3 (BorDH3) in
and their biosynthesis has not been thoroughly investigated yet vitro. Therefore, the surrogate 5a for BorDH3 as well as refer-
[7-9].
ence molecules such as 6a and 6b and the corresponding methyl
esters 7a and 7b, which resemble the potential assay products or
Gene cluster analysis suggested that the double bond at pos- easily accessible derivatives of it, are required (Figure 1c,
ition 12 in 1 is installed by the dehydratase of polyketide Scheme 1).
synthase (PKS) module 3 (BorDH3). Characteristic residues in
the active site of the preceding ketoreductase point towards a During chain elongation and reductive processing by PKSs,
3D configuration of the BorDH3 precursor 3 [10,11]. Further- their intermediates are bound to acetyl carrier proteins (ACPs)
more, we have shown in a previous study that BorDH3 prefer- via a 4'-phosphopantetheine arm (Figure 1b). It has been shown
entially accepts the 2D,3D-configured precursor, if all four for other PKS domains that the recognition of this prosthetic
potential stereoisomers of 3-hydroxy-2-methyl-SNAc- group is essential for proper substrate orientation in the active
pentanoate model substrates are presented [7].
site and catalysis with natural stereoselectivity [13]. To mimic
the ACP-bound state of PKS intermediates, their analogues, free
A commonly accepted model suggests that DHs from PKS I SNAc thioesters, are used in enzyme assays.
systems catalyze the removal of water by syn-dehydration and
that a 2D,3D-configured precursor should lead to an E-config- Alternatively, ACP-bound substrates can be conveniently
ured BorDH3 product [10]. However, in the case of borrelidin obtained by loading coenzyme A (CoA) thioesters onto active
this is in contradiction to the structure of the natural product in site serine residues of recombinant ACPs by using 4'-phospho-
which a Z-configured double bond is present at position 12. In pantetheinyl transferases [12,14]. However, coenzyme A
the borrelidin gene cluster, there is no obvious gene coding for thioesters are synthetically hard to access, especially if the sub-
an isomerase, which might be able to catalyze the inversion of a strate structure is complex.
double bond configuration. Consequently, the Z-configured
Results and Discussion
double bond must be installed either directly by BorDH3 or by
E/Z-isomerisation of an initially formed E-configured double Retrosynthetic analysis of target molecules
bond via a not yet elucidated mechanism in downstream biosyn- One common feature of activated biosynthesis intermediate
thetic processes.
analogues such as 5a is their relative low stability. In the natural
Figure 1: a) Structure of borrelidin (1); b) PKS intermediates are attached to an acyl carrier protein domain (ACP) via a 4'-phosphopantetheine linker,
giving acyl-ACPs (2); c) BorDH3 catalyzes the dehydration of ACP-bound 3-hydroxyacylate 3 to one of the ACP-bound enoates 4a or 4b. The con-
figuration of the double bond in the dehydration product is presently unknown. In a previous study, we have shown that BorDH3 only accepts surro-
gates with the shown 2D,3D-configuration if incubated with simple 3-hydroxy-2-methyl-SNAc pentanoates [7,12].
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Scheme 1: Retrosynthetic analysis of surrogate substrates for BorDH3 and reference molecules for enzyme assays (5a, 6a, 6b, 7a and 7b); TBS =
tert-butyldimethylsilyl, PMB = p-methoxybenzyl, LG = leaving group.
context, PKS intermediates are quickly processed by down- groups should be cleavable under mildly acidic or esterase-
stream domains or tailoring enzymes. However, if analogues are catalyzed conditions. As the presence of a methyl ester would
synthesized chemically and isolated, it has to be taken into prevent the selective introduction of one thioester into 5a by
account that they often tend to undergo destructive side reac- saponification–thioesterification, we planned transesterification
tions. Therefore, mild reaction conditions are required, and the from a suitably activated carboxylic acid derivative 8. Alter-
synthetic routes to them should preferably be organized in a natively, direct introduction into 11 with appropriate SNAc
divergent fashion that allows flexible access to all target com- thioester building blocks was planned for the synthesis of 6a
pounds from a stable late-stage synthetic intermediate. For the and 6b.
synthesis of the target compounds presented in this study, a
strategy via the common precursor aldehyde 11 was envisaged Starting from aldehyde 11, a common precursor for
(Scheme 1).
all molecules required in this study, aldol reaction and
following transesterification should lead to thioester 8. The
The situation is additionally complicated by the fact that the bismethyl esters 7a and 7b as well as the SNAc thioesters 6a
polyketide part contains a cyclopentyl carboxylate, which and 6b should be accessible from aldehyde 11 through a
necessitates differentiation of the carboxyl groups at the termini Horner–Wadsworth–Emmons reaction and the Still–Gennari
to permit regioselective thioester formation. We decided to olefination as well as by a Wittig olefination with stabilised
avoid late redox transformations. Instead, we achieved differen- phosphoranes, respectively. The aldehyde 11 should be acces-
tiation by choice of a chemoselective protection group strategy sible from the known molecule 13 via 12 [15].
with removal conditions that are compatible for SNAc
Synthesis of the common precursor alde-
thioesters (Scheme 1).
hyde 11
We envisaged the usage of TBS ether for the protection of the The synthesis of the common precursor aldehyde 11 was
secondary hydroxy group and to protect the carboxylic acid as accomplished from di(menth-1-yl)succinate (14) through a
its methyl ester in the precursors 8, 9a, 9b, 10a and 10b. These known route described by Omura et al. (Scheme 2) [15-17]. The
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Beilstein J. Org. Chem. 2014, 10, 634–640.
final oxidation state at C10 in 12 was installed after protective [18]. In this way, the aldol product was conveniently obtained
group removal to primary alcohol 15 followed by Dess–Martin as an inseparable 1:1 mixture of both 2,3-anti diastereomers 17a
oxidation, Pinnick oxidation and methylation. Methyl ester 12 and 17b in 57% yield over two steps [22]. Such thiophenol
was obtained in 10 steps with a good overall yield of 20%. esters readily undergo thiol-exchange reactions and are there-
Olefin cross metathesis of alkene 12 with crotonaldehyde in the fore suitable precursors for SNAc, pantetheine and CoA
presence of a second generation Grubbs catalyst required only a thioesters [14]. Accordingly, when we treated the mixture of
short filter column to isolate α,β-unsaturated aldehyde 11 in a thiophenol esters 17a and 17b with HSNAc and triethylamine
pure form.
in DMF, they underwent clean transesterification furnishing
SNAc thioesters 18a and 18b in 70% combined yield [20]
(Scheme 3).
With the mixture of 18a and 18b in hand, we turned to the mild
removal of the protection groups. For TBS cleavage, we
focused on conditions previously described by us for the
removal of an acid-sensitive dioxolane protection group from a
CoA thioester [23]. We evaluated the exposure of the model
CoA ester 20, synthesized from 11 by Pinnick oxidation and
CoA thioesterification via an intermediate activation as
N-hydroxysuccinimide ester, to several acids in H2O/THF
mixed solvent systems. In 20 the acyl part is similarly function-
alized as in the protected SNAc thioesters 18a and 18b.
However, the CoA thioester can be regarded as more
demanding in terms of sensitivity.
Scheme 2: Synthesis of the common precursor aldehyde 11. Com-
pound 13 was prepared in six steps and with an overall yield of 40%
via a known route by Omura et al. [15]. a) DDQ, CH2Cl2, rt, 30 min
(85%); b) DMP, CH2Cl2, rt, 2 h; c) NaOCl, 2-methyl-2-butene, t-BuOH,
phosphate buffer, rt, 16 h; d) TMS-CHN2, toluene/MeOH 2:3, rt, 40 min
(58% over three steps); e) second generation Grubbs catalyst, croton-
aldehyde, CH2Cl2, 40 °C, 120 min (88%); DDQ = 2,3-dichloro-5,6-
dicyano-1,4-benzoquinone, Men = (l)-menthyl, DMP = Dess–Martin
periodinane, TMS = trimethylsilyl.
The progress of the reaction was followed by mass spectrom-
etry. While the use of strong acids like p-toluenesulfonic acid or
hydrochloric acid led to the decomposition of 20, weaker acids
like acetic acid did not lead to any detectable product formation,
even after reaction times of up to 20 h. Gratifyingly, treatment
with a mixture of THF/HCOOH/H2O (6:3:1), gave a slow
conversion into the desired product 21 (see Figure S1 and
Figure S2 in Supporting Information File 1). Complete depro-
tection was achieved after 2 days.
Synthesis of DH substrate surrogate
For the synthesis of the DH substrate surrogate 5a we aimed at
an anti-selective aldol reaction, which permits efficient access The mixture of SNAc esters 18a and 18b was exposed to the
to the desired 2D,3D-stereoisomer followed by a smooth trans- established acidic deprotection conditions and the progress of
formation into the SNAc thioester in the presence of a methyl the reaction was monitored by mass spectrometry. After two
ester function. Amongst others, we tested Paterson’s lactate- days, complete TBS removal was obtained. The crude product
derived benzoyl auxiliary, Evans’ magnesium-catalyzed direct was subjected to selective methyl ester cleavage by using pig
aldol reaction, and an Abiko–Masamune-like aldol reaction by liver esterase (PLE) [24]. The target molecule was obtained as a
using a thiodesoxy variant of the norephedrine-derived auxil- mixture of both anti-diastereomers 5a and 5b after 15 steps in
iary on simplified model aldehydes as well as on aldehyde 11 7% overall yield.
[18-21]. However, in all these cases either the aldol reaction
itself proved to be low yielding (<20%) or removal conditions Synthesis of reference compounds
of chiral auxiliaries were destructive to the molecule.
We exploited a Wittig reaction with a suitable stabilized phos-
phorane for the synthesis of (E,E)-diene 6a (Scheme 4). The
The best results were obtained for an anti-selective variant, in Wittig compound 24 was obtained in two steps from 2-bromo-
which aldehyde 11 was reacted with an (E)-boron enolate propionic acid following synthetic procedures described for its
formed by the reaction of thiophenol propionate with chlorodi- acetic acid analogue and the respective methyl ester ylide 26
cyclohexylborane and dimethylethylamine, similar to the condi- [25,26]. The reaction of phosphorane 24 with 11 under neutral
tions described by Paterson et al. for lactate-derived auxiliaries conditions at 50 °C gave the desired diene 9a in very good 88%
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Beilstein J. Org. Chem. 2014, 10, 634–640.
Scheme 3: Synthesis of the BorDH3 substrates. a) Thiophenolpropionate, Cy2BCl, Me2EtN, Et2O, −78 °C to −20 °C, 16 h (57% over two steps from
12); b) HSNAc, Et3N, DMF, rt, 16 h (70%); c) THF/HCOOH/H2O (6:3:1), rt, 2 d; d) pig liver esterase, phosphate buffer, rt, 5 d (81% over two steps);
e) NaOCl, 2-methyl-2-butene, t-BuOH, phosphate buffer (pH 7.0), rt, 16 h; f) N-hydroxysuccinimide, N,N’-dicyclohexylcarbodiimide, THF, rt, 16 h (50%
over three steps from 12); g) HSCoA·3Li, THF/H2O (2:1) adjusted to pH 8.0 by phosphate buffer, 35 °C, 18 h; DMF = N,N-dimethylformamide.
Scheme 4: Synthesis of reference compounds for the BorDH3 assay. a) 24, CH2Cl2, 50 °C, 3 h (88% over two steps); b) THF/HCOOH/H2O (6:3:1),
rt, 2 d (62% for 7a, 57% for 7b); c) pig liver esterase, phosphate buffer, rt, 3 d (67% over two steps from 9a); d) 25, NaH, THF, 0 °C, 3 h (18% over
two steps from 12) or 26, CH2Cl2, 50 °C, 21 h (64% over two steps from 12); e) 27, 18-crown-6, K2CO3, THF, −20 °C then 0 °C for 5 h (47% over two
steps from 12); f) HSNAc, EDC, DMAP, CH2Cl2, rt, 16 h (78%); g) PPh3, H2O, 70 °C, 11 h, then NaOH, rt (64%).
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Beilstein J. Org. Chem. 2014, 10, 634–640.
yield and in nearly perfect E-selectivity. The protection groups a highly efficient and less common late stage ester hydrolysis
were removed from 9a under the previously established condi- by using pig liver esterase. Preliminary tests suggest the
tions to finally give target SNAc thioester 6a in a total yield of compatibility of these conditions with coenzyme A thioesters,
12% over 14 steps. Alternative attempts to synthesize 9a and 9b indicating that the chosen strategy has broad potential in the
by Horner–Wadsworth–Emmons olefination and Still–Gennari future synthesis of similarly (or even more) complex polyke-
olefination with the respective phosphonates gave no reaction tide-derived coenzyme A thioesters.
product at all (Horner–Wadsworth–Emmons olefination) or
only the (E,E)-diene 10a instead of the desired E,Z-configured Thus, the presented strategy enables for the first time the in
product 10b (Still–Gennari olefination).
vitro assaying of a potential Z-selective dehydratase domain
from a polyketide synthase with suitable precursor molecules.
In order to obtain alternative reference compounds for both These experiments are currently ongoing in our lab.
potential products of the BorDH3 enzyme assay, we synthe-
sized double bond isomers 7a and 7b. To be able to compare
Supporting Information
them to the products in the crude BorDH3 assay mixture, the
latter will be transformed into their corresponding methyl esters
The supporting information provides reaction details,
by saponification and following methylation with trimethylsilyl-
analytical data, and copies of 1H and 13C NMR spectra.
diazomethane.
Supporting Information File 1
Procedures.
The fully protected E-isomer 10a was obtained in 18% yield by
a Horner–Wadsworth–Emmons reaction with phosphonate 25
or, alternatively, in 64% yield by a Wittig reaction with stabi-
lized phosphorane 26. The Z-isomer 10b was synthesized by
Still–Gennari olefination using phosphonate 27 in 47% yield.
The yield for the deprotection step was satisfying (61% for 7a
and 57% for 7b) in both cases, indicating that both isomers are
configurationally stable under these conditions. The overall
yield over 13 steps was 8% for 7a and 5% for 7b.
[http://www.beilstein-journals.org/bjoc/content/
supplementary/1860-5397-10-55-S1.pdf]
Supporting Information File 2
NMR spectra.
[http://www.beilstein-journals.org/bjoc/content/
supplementary/1860-5397-10-55-S2.pdf]
(E,E)-diene 6a will find application in the comparative NMR Acknowledgements
evaluation of a large scale BorDH3 enzyme assay of substrates We thank the Marie Curie program of the European Union and
5a and 5b. Together with the successful synthesis of 5a and 5b the Emmy Noether program of the DFG for funding. We would
and the availability of the isomers 7a and 7b, this sets the stage also like to thank the NMR facility and the mass facility of the
to determine the stereoselectivity of BorDH3.
OCI Hanover as well as Prof. Andreas Kirschning and his group
for general support and helpful discussion.
Conclusion
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