General chemoenzymatic route to two-stereocenter triketides employing assembly line ketoreductases

Article abstract of DOI:10.1039/c9cc07966a

Modular polyketide synthases (PKSs) are enzymatic assembly lines that fuse carbon fragments into complex chiral products. Here, their synthetic logic is employed to chemoenzymatically generate two-stereocenter triketides. Each of the four stereoisomers was constructed in a stereocontrolled manner using C-acylation and two PKS ketoreductases possessing opposite stereoselectivities.

Full text of DOI:10.1039/c9cc07966a

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General chemoenzymatic route
to two-stereocenter triketides employing
assembly line ketoreductases†
Cite this: Chem. Commun., 2020,
56, 157
Received 11th October 2019,
Accepted 26th November 2019
a
a
Zhicheng Zhang,a Alexis J. Cepeda,a Mireya L. Robles,
Kaan Kumru,
Melissa Hirsch,
Jina A. Zhoua and Adrian T. Keatinge-Clay
*
b
b
DOI: 10.1039/c9cc07966a
rsc.li/chemcomm
Modular polyketide synthases (PKSs) are enzymatic assembly lines Claisen-like condensation of malonyl thioesters in the active sites of
that fuse carbon fragments into complex chiral products. Here, KSs. While KRs have been utilized as robust biocatalysts,13 KSs
their synthetic logic is employed to chemoenzymatically generate have not.14 However, since a directly analogous C-acylation reaction
two-stereocenter triketides. Each of the four stereoisomers was is known,15,16 a chemoenzymatic synthesis of polyketides through
constructed in a stereocontrolled manner using C-acylation and cycles of C-acylation and KR biocatalysis should be achievable.17,18
two PKS ketoreductases possessing opposite stereoselectivities.
Aiming at a general synthesis of polyketides, we sought to
demonstrate the chemoenzymatic construction of chiral trike-
Natural products are often lead compounds in the discovery and tides and set our goal as the stereocontrolled synthesis of a
development of new medicines, with polyketides more than any library of four stereoisomers containing chiral centers at their
other class of natural product giving rise to clinically-valuable b- and d-carbons. Two KRs with opposite stereoselectivities
pharmaceuticals (e.g., the antibacterial erythromycin, the immu- were employed – the KR from the sixth module of the myco-
nosuppressant rapamycin, and the anticancer agent epothilone).1 lactone PKS (MycKR6)19 and the KR from the second module of
Unfortunately, the low natural abundance of polyketides that the tylosin PKS (TylKR2)20 (updated module definition used21,22).
possess desired bioactivities coupled with their complex structures Both MycKR6, an A-type KR that generates L-b-hydroxy sub-
hamper their development as medicines. Advances have been stituents, and TylKR2, a B-type KR that generates D-b-hydroxy
made in obtaining complex polyketides such as discodermolide2 substituents, have been shown to be stereoselectively-robust
and bryostatin3 through total synthesis, yet a general strategy to biocatalysts when excised from their assembly lines.23–26
precisely set stereocenters during the union of carbon fragments b-Hydroxyl groups generated by KRs need to be protected before
has not yet been realized. While automated technologies have been C-acylation due to the sensitivity of this reaction to labile protons
utilized to rapidly synthesize biopolymers such as polypeptides4,5 (another consideration is the spontaneous cyclization of the
and DNA6,7 for almost half a century, no similar process has been d-hydroxy thioester product).
developed for polyketides.
Our group is inspired by polyketide synthase (PKS) assembly
lines and how they construct complex polyketides from simple
building blocks (Fig. 1).8–11 During polyketide biosynthesis,
intermediates are covalently attached to acyl carrier protein
(ACP) domains that dock with assembly line enzymes such as
ketosynthases (KSs) that catalyze carbon–carbon bond formation
and ketoreductases (KRs) that mediate stereocontrolled reduc-
tions that forge either one or two stereocenters.12 The extension
of intermediates is accomplished through the decarboxylative
a Department of Chemistry, The University of Texas at Austin,
Fig. 1 Polyketide assembly lines provide inspiration for the chemoenzy-
matic synthesis of polyketide fragments. If the power of the ketosynthase
(KS) domains that make carbon–carbon bonds and ketoreductase (KR)
domains that set stereocenters were accessed, the general synthesis of
diverse, chiral fragments would be possible.
100 E. 24th St., Austin, TX 78712, USA
b Department of Molecular Biosciences, The University of Texas at Austin, 100 E.
24th St., Austin, TX 78712, USA. E-mail: adriankc@utexas.edu
† Electronic supplementary information (ESI) available: Methods and character-
ization. See DOI: 10.1039/c9cc07966a
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The first two steps of this route are based on our previous
work but were performed using a modified procedure to obtain
higher purity.13 Propionyl chloride was added to Meldrum’s acid
in the presence of excess pyridine to afford propionyl-Meldrum’s
acid 1 without separation (70% crude yield).27 1 was refluxed
at 115 1C with NAC to provide b-keto-diketide 2 (61% yield).
Unreacted NAC could be removed with copper sulfate impreg-
nated silica during flash chromatography. The NAC handle,
compared with shorter handles such as ethanethiol, affords
greater stereocontrol during KR biocatalysis.28 b-Keto-diketide
2 (soluble to B100 mM in water) was incubated at the gram
scale overnight with either MycKR6 to provide 3a (65% yield)
or TylKR2 to provide 3b (58% yield) as measured by chiral HPLC in
comparison with standards.13 Through heating overnight in 5 M
NaOH, crude 3a and 3b were hydrolyzed to L-b-hydroxypentanoic
acid 4a and D-b-hydroxypentanoic acid 4b (51% and 59% yields,
respectively); this enabled the removal of NAC and its dimer
generated during the reduction reaction.
Several groups were considered to protect the b-hydroxyl
substituent, including acetyl, methyl, methoxymethyl, and silyl.
The acetyl group was attempted; however, as elimination reac-
tions predominated, the more stable methyl protecting group was
adopted. In the biosynthesis of polyketides, O-methyltransferase
domains frequently methylate b-hydroxyl substituents, therefore
the methyl group will not likely prevent entry into the active sites
of downstream enzymes such as KRs, which is a concern with the
larger silyl groups.29 The hydroxyl substituents of 4a and 4b were
methylated using a solution of n-butyllithium (n-BuLi) and DMSO
through treatment with MeI, generating b-methoxy acids 5a and 5b
(73% and 78% yield, respectively).30 The reaction was monitored
through the color change from the 4a/4b dianion (dark brown) to
the monoanion (light orange); although the reaction had been
developed to generate dimethylated alkoxy esters from hydroxy
acids, it mainly provided the desired monomethylated alkoxy
esters. The protected diketide acids 5a and 5b were C-acylated
using the magnesium salt of malonyl ethanethiol thioester to give
b-keto-triketides 6a and 6b (35% and 34% yields, respectively).15
This key, carbon–carbon bond-forming step mimicks the
decarboxylative condensation catalyzed by KSs.
Scheme 1 Chemoenzymatic route. (a) Pyridine (2.0 eq.), propionyl chloride
(1.0 eq.), DCM, overnight, 0 to 22 1C (76% yield); NAC (0.95 eq.), toluene,
115 1C, 5 h (61%), (b) either MycKR6 (A-type KR) or TylKR2 (B-type KR), GDH,
NADP+, pH 7.7, overnight, 22 1C (3a, 65%; 3b, 58%), (c) 5 M NaOH aq., 80 1C,
overnight (4a, 51%; 4b, 59%), (d) 2.5 M n-BuLi (3.0 eq.), DMSO, MeI (2.4 eq.),
under argon, 22 1C, overnight (5a, 73%; 5b, 78%), (e) 1,10-carbonyldiimidazole
(1.1 eq.), Mg(OEt)2 (0.55 eq.), malonyl ethanethiol thioester (1.1 eq.), anhydrous
THF, 22 1C, overnight (6a, 35%; 6b, 34%), (f–h) NAC, pH 8.5, 22 1C, 2 h, then
either MycKR6 or TylKR2, GDH, NADP+, pH 7.7, overnight, then 5 M NaOH,
70 1C, overnight (9aa, 57%; 9ab, 36%; 9ba, 44%; 9bb, 24%).
Thiol–thioester exchange in 0.33 M HEPES (pH 8.5) using 10 eq.
of NAC converted 6a to 7a and 6b to 7b. After this handle swap, the
pH was adjusted to 7.7 and the NADPH regeneration system as well
as either MycKR6 or TylKR2 was added to generate a product with
We designed the route to begin with N-acetylcysteamine (NAC) an L- or D-orientated b-hydroxyl group, respectively. Purifications of
opening propionyl-Meldrum’s acid 1 to generate b-keto-diketide 2 8aa, 8ba, 8ab, and 8bb were challenging due to byproducts such as
(Scheme 1). Next, incubation of 2 with MycKR6 or TylKR2 and a the NAC dimer. However, heating at 70 1C in aqueous 5 M NaOH
nicotinamide adenine dinucleotide phosphate (NADPH) regenera- converted the thioesters into carboxylic acids and enabled the
tion system [D-glucose and glucose dehydrogenase (GDH)] would removal of byproducts to facilitate the purification of triketide
yield either the L- or the D-b-hydroxy-diketide 3.13 The hydrolysis of products 9aa, 9ba, 9ab, and 9bb (57%, 44%, 36%, and 24% yield,
3 would afford the corresponding b-hydroxy acid 4, and protection respectively, through three steps).
would yield protected acid 5. C-Acylation of 5 with malonyl
Chemical standards for triketide thioesters 8aa, 8ab, 8ba, and
ethanethiol thioester would generate b-keto-triketide 6. In situ 8bb as well as the corresponding carboxylic acids 9aa, 9ab, 9ba,
thiol–thioester exchange of 6 with NAC would give NAC-bound and 9bb were prepared (Scheme 2). Commercially-available 10 was
b-keto-triketide 7, which should then be reduced by MycKR6 hydrogenated at 100 atm using (Sa)-Ir-spiroSAP or (Ra)-Ir-spiroSAP
or TylKR2 to yield a b-hydroxy triketide 8. Subsequent hydro- as the catalyst to give enantiomers 11a or 11b, respectively.31,32 11a
lysis would provide a two-stereocenter triketide acid 9.
and 11b were hydrolyzed by heating overnight in a solution of
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respectively, through two steps). Coupling with NAC yielded 8aa,
8ab, 8ba, and 8bb.
The syn-isomers 9aa and 9bb can be distinguished from the
anti-isomers 9ab and 9ba through 1H NMR most apparently
through the signal from the C5 hydrogen geminal to the methoxy
group (4.2–4.3 ppm); however, impurities from the enzymatic
reductions often gave rise to overlapping signals. The relative
configurations of the isomers could be assessed through 13C
NMR using Hoffmann’s method of distinguishing between
b-alkoxy alcohol diastereomers.33 The sum of the signals for
the hydroxy- and alkoxy-bearing carbons should be lower for
anti-isomers (in the chair made by the hydrogen bond between
the hydroxy and alkoxy groups either the substituent on the
hydroxy- or the alkoxy-bearing carbon is axial for the anti-
isomers, resulting in steric hindrance and relatively upfield
signals compared to the signals from the hydroxy- and alkoxy-
bearing carbons in the syn-isomers that possess equatorial
substituents) (Fig. 2A). For the chemoenzymatically- or
chemically-generated syn-isomers 9aa and 9bb, the signals
from the hydroxy-bearing C3 and the alkoxy-bearing C5 were
82.0 ppm and 67.9 ppm, respectively, and for the chemoenzy-
matically- or chemically-generated anti-isomers 9ab and 9ba,
Scheme 2 Synthesis of standards. (a) Either (Sa)-Ir-spiroSAP, 100 atm H2,
MeOH, 22 1C, 48 h for 11a or (Ra)-Ir-spiro-SAP, 100 atm H2, MeOH, 22 1C,
48 h for 11b (both yields 499%), (b) 5 M NaOH, 80 1C, overnight (4a, 51%
and 4b, 59%), (c) 2.5 M n-BuLi (3.0 eq.), DMSO, MeI (2.4 eq.), under argon,
22 1C, overnight (5a, 73%; 5b, 78%), (d) 1,10-carbonyldiimidazole (1.1 eq.),
Mg(OEt)2 (0.55 eq.), malonyl ethanethiol thioester (1.1 eq.), anhydrous THF,
22 1C, overnight (6a, 35%; 6b, 34%), (e) sodium methoxide (10.0 eq.),
anhydrous MeOH, 22 1C, 6–9 h, (12a, 27%; 12b, 16%), (f–g) either (Sa)-Ir-spiroSAP,
100 atm H2, MeOH, 22 1C, 48 h then 5 M NaOH, 60 1C, overnight for 9aa and
9ba or (Ra)-Ir-spiroSAP, 100 atm H2, MeOH, 22 1C, 48 h then 5 M NaOH, 60 1C,
overnight for 9ab and 9bb (9aa, 77%; 9ab, 97%; 9ba, 83%; 9bb, 86%). (h) THF,
4-dimethylaminopyridine (0.5 eq.), 1-ethyl-3-(3-dimethylaminopropyl)carbo-
diimide-HCl (2.3 eq.), NAC (5.0 eq.), 22 1C, overnight.
Fig. 2 (A) The relative configurations of the triketide stereoisomers can be
readily assigned using Hoffmann’s method of summing the 13C NMR shifts
for the C3 and C5 signals: the anti-isomers (e.g., 9ba) give a smaller d(C3) + d(C5)
value than syn-isomers (e.g., 9aa) due to axial substituents on the chair
formed by the hydroxyl and the methoxy groups. (B) Chiral chromato-
graphy of chemoenzymatically-generated triketides 8aa, 8ab, 8ba, and
8bb using two systems: an OC-H column coupled with a UV detector
(left) and an IF3 column coupled with a time-of-flight mass spectrometer
(right). The OC-H column was able to resolve the syn-products, while the
IF3 column was able to resolve the anti-products. Retention times
matched those of standards (Tables S1 and S2, ESI†).
5 M NaOH to generate 4a and 4b. Protection and extension of 4a
and 4b into 6a and 6b were performed as in the chemoenzymatic
route. Alcoholysis converted thioesters 6a and 6b to oxoesters 12a
and 12b (27% and 16% yields, respectively; 30% of starting material
was unreacted and could be reused). 12a was reduced using (Sa)-Ir-
spiroSAP or (Ra)-Ir-spiroSAP and hydrolyzed to provide 9aa or 9ab,
(77% and 97% yields, respectively, through two steps).31,32 Like-
wise, 12b was reduced using (Sa)-Ir-spiroSAP or (Ra)-Ir-spiroSAP
and hydrolyzed to provide 9ba or 9bb (83% and 86% yields,
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the signals from the hydroxy-bearing C3 and the alkoxy-bearing Conflicts of interest
C5 were 79.7 ppm and 65.3 ppm, respectively. In agreement with
Hoffmann’s method, the sum of the signals for the syn-isomers
was greater than the anti-isomers, by 4.9 ppm.
There are no conflicts of interest to declare.
Notes and references
Chemoenzymatically- and chemically-generated triketide
stereoisomers 8aa, 8ab, 8ba, and 8bb were also compared using
complementary, chiral chromatography systems – an OC-H
column coupled with a UV detector and an IF3 column coupled
with a time-of-flight mass spectrometer (Fig. 2B and Fig. S1,
Table S1, ESI†). The anti-isomers, 8ab and 8ba, eluted before
the syn-isomers, 8aa and 8bb, in both chromatographies. While
the anti-isomers could not be resolved from one another on the
OC-H column, they could be on the IF3 column, and while the
syn-isomers could not be resolved from one another on the IF3
column, they could be on the OC-H column. In all four cases,
the targeted stereoisomer of the chemoenzymatic synthesis
matched the chemically-generated stereoisomer. The A-type
MycKR6 and the B-type TylKR2, known to be stereoselectively
robust towards b-ketodiketide NAC thioesters,13 proved to be
stereoselectively robust towards triketide NAC thioesters. KRs
thus have an advantage over most asymmetric catalysts in the
reduction of 1,3 polyol substrates in that they override the
influence of neighboring stereocenters which commonly affect
stereocontrol.32,34
The chemoenzymatic synthesis of a library of two-stereo-
center triketide fragments was accomplished with overall yields
of 2.0% for 9aa, 1.3% for 9ab, 1.7% for 9ba, and 0.9% for 9bb
(in each case the targeted stereoisomer comprises at least 90%
of all possible stereoisomers; stereoisomeric compositions are
reported in Table S1, ESI†). This marks an initial success for the
described general polyketide synthesis and a starting point for
optimization. The yields should be improved, and a protecting
group that can be easily removed at the end of the synthesis but
does not affect KR stereocontrol should be identified. As many
KRs are able to set two stereocenters when reducing a-substituted,
b-ketoacyl substrates,12 the described route may be utilized to
generate libraries of three- or four-stereocenter triketides, for
example through the use of methylmalonyl ethanethiol thioester
in C-acylation reactions. The biocatalytic employment of other
PKS enzymes such as dehydratases and enoylreductases could
further diversify the functionality of synthesized fragments. Reaction
optimization should allow another round of C-acylation and
reduction to generate tetraketides at milligram levels. If the
described, stereoselective synthesis could be generally employed
in the construction of stereocomplex fragments, it would be a
boon to synthetic methodology as well as the development of
new medicines.
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