Mixed bioengineering-chemical synthesis approach for the efficient preparation of δ7-dafachronic acid

Article abstract of DOI:10.1021/ol5006642

Combining bioengineering with chemical synthesis has enabled an efficient method for producing δ7-dafachronic acid, a steroidal hormone associated with nematode germline longevity. Saccharomyces cerevisiae was engineered to produce 7,24-cholestadienol, a convenient starting material for a four-step synthesis of δ7-dafachronic acid.

Full text of DOI:10.1021/ol5006642

Letter
pubs.acs.org/OrgLett
Mixed Bioengineering−Chemical Synthesis Approach for the
Efficient Preparation of Δ7-Dafachronic Acid
Matias I. Kinzurik, Lachezar V. Hristov, Seiichi P. T. Matsuda,* and Zachary T. Ball*,†
†
†
,†,‡
†
‡
Department of Chemistry and Department of Biochemistry and Cell Biology, Rice University, Houston, Texas 77005, United States
S Supporting Information
*
ABSTRACT: Combining bioengineering with chemical syn-
thesis has enabled an efficient method for producing Δ7-
dafachronic acid, a steroidal hormone associated with
nematode germline longevity. Saccharomyces cerevisiae was
engineered to produce 7,24-cholestadienol, a convenient
starting material for a four-step synthesis of Δ7-dafachronic
acid.
arasitic nematodes cause serious problems for human
health and agriculture. For example, the human parasites
Scheme 1. Selected Examples of Starting Material Selection
for Synthesis of Δ7-Dafachronic Acid
a
P
Ancylostoma and Necator spp affect one in every 10 people on
the planet and are a leading cause of iron-deficient anemia
1
worldwide, while plant parasitic nematodes are an important
2
and unsolved threat to agriculture. The natural product Δ7-
3
dafachronic acid (1) disrupts nematode development by
halting the lethargic dauer developmental stage though
4
interactions with the nuclear receptor DAF-12. DAF-12
signaling pathways are conserved across numerous parasitic
5
−7
nematodes,
and so the novel mechanism of action of Δ7-
dafachronic acid has inspired synthetic and biological study of
the compound for its anti-nematode potential.
8
−14
In this
paper, we report a semisynthetic route, developing engineered
mutant yeast S. cerevisiae to produce the precursor sterol 2 that
allows efficient and high-yielding access to Δ7-dafachronic acid
(1).
The complexity of steroid structure has long created
synthetic challenges, requiring control over stereochemistry,
degree of unsaturation, and oxygenation site. To date, routes to
Δ7-dafachronic acid have started from commercially available
a
Producing the engineered sterol 2 minimizes the need for oxidation-
8
12
11
steroidal building blocks including β-stigmasterol, diosgenin,
state alterations.
9
,15
3β-hydroxychol-5-en-24-oic acid,
and hyodeoxycholic acid
(
a selection of starting materials is shown in Scheme 1).
shown to prevent yeast growth, indicating that the late stages
Despite the similarities to Δ7-dafachronic acid, these
compounds still require routes of more than 10 steps to
forge the target compound (1), often using reagents that are
challenging for efficient synthesis on scale. A central challenge
in these syntheses is that readily available sterols and steroids
contain functionality that is not trivial to manipulate toward
Δ7-dafachronic acid.
of Δ7-dafachronic acid biosynthesis would likely be incompat-
21
ible with efficient bioproduction. We have developed a
semisynthesis of Δ7-dafachronic acid by manipulating yeast
sterol biosynthetic pathways to bioengineer a mutant strain that
could efficiently produce a compound with sufficient structural
similarity and functional handles to be converted into the target
structure through chemical synthesis.
Engineering mutant organisms to produce a target structure
16−18
The ergosterol biosynthetic pathway accesses intermedi-
directly is a compelling alternative,
made to elucidate the precise steps in the biosynthetic
pathway.
and efforts have been
2
2,23
ates
that display structural features that are desirable in a
1
9,20
But there are limits to the reactions that can be
carried out efficiently in a producing organism. For example,
accumulation of carboxylate-containing steroids has been
Received: March 3, 2014
Published: April 9, 2014
©
2014 American Chemical Society
2188
dx.doi.org/10.1021/ol5006642 | Org. Lett. 2014, 16, 2188−2191

Organic Letters
Letter
a
Scheme 2. Anticipated Control of Ergosterol Pathways in a Mutant Yeast Strain Accumulating 7,24-Cholestadienol (2)
a
A representative sample of plausible enzyme pathways from lanosterol. Arrows blocked with an “ × ” indicate plausible natural pathways that have
been blocked by gene deletion in the triple mutant (ERG6/ERG5/ERG3) strain.
starting material for Δ7-dafachronic acid synthesis. Lanosterol
to the desired 7,24-cholestadienol (2) as the primary sterol
product (77%), along with minor amounts of zymosterol
(20%). These two sterols comprise ≥97% of the non-
saponifiable lipids isolated from the strain (Figure 1), as judged
by GC−MS and NMR analyses.
is the initial carbocyclic intermediate en route to sterols
(
Scheme 2). Lanosterol displays the tetracyclic ring system and
oxidation at C3 that are also present in Δ7-dafachronic acid,
and the C24−C25 olefin is a valuable handle for side-chain
modification. Some native yeast enzymes modify lanosterol to
more closely resemble Δ7-dafachronic acid: demethylation at 4,
4′, and 14 and isomerization of the C8−C9 olefin to C7−C8.
The plausible intermediate 7,24 cholestadienol (2) would be an
ideal starting material for chemical synthesis and could be
obtained if enzymatic steps that diverge from Δ7-dafachronic
acid are eliminated through mutation. However, because yeast
sterol biosynthesis is not rigidly linearthe order of the
transformations is variablehalting the action of a single
enzyme typically is insufficient to allow accumulation of a single
desired intermediate, such as 7,24 cholestadienol (2).
The enzyme ERG6 promotes the methylation of the side
chain. Deletion of that enzyme is desirable to retain the C24−
2
5 olefin, which is an excellent handle for side-chain
24
modification. An ERG6 knockout strain generated sterol
products with the C24−C25 olefin rather than the terminal
methylene, but the dominant products retained C5−C6 and
C22−C23 olefins (3, 4, and 8, see Table 1), which are absent in
Figure 1. GC analysis of crude nonsaponifiable-lipid products isolated
from the triple mutant (ERG6/ERG3/ERG5) strain, before (left, 77%
purity) and after (right, 91% purity) a single recrystallization from
Table 1. Sterol Composition of Mutant Yeast Strains
methanol. The desired 7,24-cholestadienol (2, t 10.6 min) was the
R
primary lipid produced, along with small amounts of zymosterol (3, tR
gene deletions
1
0.3 min).
ERG6/
ERG5
ERG6/ERG3/
ERG5
sterol
ERG6
7
,24-cholestadienol (2)
3
27
30
29
11
3
29
60
0
77
20
0
The clean conversion of lanosterol to these two sterols
zymosterol (3)
indicates that among sterol biosynthetic enzymes not
deliberately disrupted, only the sterol isomerase ERG2 leaves
meaningful amounts of its starting material unreacted. Delta-8
sterols are frequently recovered in modest amounts from
5
5
,7,24-cholestatrienol (4)
,7, 22, 24-cholestatetraenol (8)
0
all other sterols
8
≤3
27
yeast. Efforts to engineer yeast to redirect the ergosterol
pathway in favor of other desired structures may result in sickly
strains that require supplementation of the media with sterols
Δ7-dafachronic acid. ERG5 was then deleted by PCR-mediated
homologous recombination. The resultant ERG6/ERG5
double-knockout strain lost the ability to form the side-
chain diene and primarily accumulated sterol 4.
25
27
and even then exhibit decreased productivity. In contrast, the
triple mutant strain reported here is remarkably robust without
supplementing the growth media with sterols, and sterol 2 was
produced in concentrations of 28 mg/L of culture, performed
on 24-L scale. Pure material could be obtained by HPLC
Removing an additional downstream desaturase (ERG3)
2
6
resulted in the triple-knockout mutant (ERG6/ERG5/ERG3)
that successfully funneled the majority of lanosterol metabolites
2
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dx.doi.org/10.1021/ol5006642 | Org. Lett. 2014, 16, 2188−2191

Organic Letters
Letter
purification; alternatively, a single recrystallization from
methanol afforded sterol 2 in 91% purity (69% mass recovery).
The subsequent chemical synthesis has been successfully
applied to both HPLC-purified sterol 2 and to sterol 2
contaminated with small amounts of isomer 3, in which case
the mixture of Δ7- (2) and Δ8-isomers (3) is carried
throughout the synthesis.
Scheme 3. Synthesis of Δ7-Dafachronic Acid (1)
With a biosynthetic route to a suitable precursor in hand, we
set about developing a synthesis of Δ7-dafachronic acid from
sterol 2 that would be efficient and compatible with larger scale
production. The ketone at C3 was first installed by palladium-
catalyzed oxidation in an O atmosphere, affording ketone
2
2
8,29
10.
This transformation was performed first because
subsequent side-chain functionalization chemistry was incom-
patible with the C3 hydroxyl. While approaches to side chain
allylic functionalization exist, such as selenium dioxide
3
0,31
32
oxidation
or circuitous multistep methods, we were
interested in finding a more environmentally friendly route with
improved yield. There is some precedent for palladium-
mediated side chain allylic functionalization with linear
3
3,34
polyisoprene substrates,
but we were unable to oxidize
the terminal methyl groups under a variety of conditions.
We turned instead to olefin metathesis. Despite the dearth of
examples in the literature of trisubstituted olefin synthesis from
3
5,36
trisubstituted starting materials,
we were pleased to find
that cross-metathesis with Grubbs’ second-generation catalyst
and methyl methacrylate provided the desired ester product,
which was then saponified to unsaturated acid 11. In initial
studies, we found that methacrolein was also a competent
substrate for this reaction, affording the corresponding enal
product in 82% yield.
sterol starting materials may be a valuable approach for the
preparation of related Δ7-dafachronic analogues, of interest for
40
medical and biological purposes, with subtle variation in
unsaturation and oxidation state.
3
7,38
ASSOCIATED CONTENT
Supporting Information
Finally, directed hydrogenation
with [RuCl(p-cymene)-
■
(
(S)-(−)-H -BINAP)]Cl yielded the desired product with 95:5
*
S
8
11
diastereoselectivity, consistent with literature precedent. For
the case at hand, the distal nature of the C24 stereocenter
meant that we were unable to assess diastereomeric purity by
Experimental procedures and characterization of all synthetic
products. This material is available free of charge via the
Internet at http://pubs.acs.org.
1
chromatographic means or by 1D H NMR. Instead, integrating
1
13
a H− C HSQC spectrum of the reaction product solved this
problem, allowing reliable diastereoselectivity determination
without resorting to circuitous derivatization methods. Action
of the enantiomeric (R)-catalyst provided the analogous (25R)
compound in 91% yield and 93:7 diastereoselectivity for
spectroscopic comparison and biological testing.
AUTHOR INFORMATION
Corresponding Authors
■
*
*
E-mail: matsuda@rice.edu.
E-mail: zb1@rice.edu.
Notes
The combination of engineered yeast and synthetic
chemistry offers remarkably efficient access to Δ7-dafachronic
acid (1) (Scheme 3). Four synthetic steps produce the target in
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
58% overall yield, featuring three transition-metal-catalyzed
We acknowledge financial support from the Robert A. Welch
Foundation Research Grant No. C-1680 (Z.T.B.) and Research
Grant No. C-1323 (S.P.T.M.).
reactions and a simple ester saponification. The only
stoichiometric reagents in the sequence are O , H , LiOH,
2
2
and methyl methacrylate. At the same time, thoughtful
manipulation of the S. cerevisiae ergosterol pathway allows
production of 7,24-cholestadienol (2), which replicates many of
the challenging features of Δ7-dafachronic acid, including
C7−C8 unsaturation. The synthetic precursor 7,24-cholesta-
dienol (2) comprises 77% of the nonsaponifiable lipids
extracted from cultured yeast and is produced in culture at
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