Chemoenzymatic synthesis of spinosyn A

Article abstract of DOI:10.1002/anie.201407806

Following the biosynthesis of polyketide backbones by polyketide synthases (PKSs), post-PKS modifications result in a significantly elevated level of structural complexity that renders the chemical synthesis of these natural products challenging. We report herein a total synthesis of the widely used polyketide insecticide spinosyn A by exploiting the prowess of both chemical and enzymatic methods. As more polyketide biosynthetic pathways are characterized, this chemoenzymatic approach is expected to become readily adaptable to streamlining the synthesis of other complex polyketides with more elaborate post-PKS modifications.

Full text of DOI:10.1002/anie.201407806

Angewandte
Chemie
DOI: 10.1002/anie.201407806
Macrolide Total Synthesis
Hot Paper
Chemoenzymatic Synthesis of Spinosyn A**
Hak Joong Kim, Sei-hyun Choi, Byung-sun Jeon, Namho Kim, Rongson Pongdee,
Qingquan Wu, and Hung-wen Liu*
Abstract: Following the biosynthesis of polyketide backbones
by polyketide synthases (PKSs), post-PKS modifications result
in a significantly elevated level of structural complexity that
renders the chemical synthesis of these natural products
challenging. We report herein a total synthesis of the widely
used polyketide insecticide spinosyn A by exploiting the
prowess of both chemical and enzymatic methods. As more
polyketide biosynthetic pathways are characterized, this che-
moenzymatic approach is expected to become readily adapt-
able to streamlining the synthesis of other complex polyketides
with more elaborate post-PKS modifications.
Spinosyn A (1), a commercially important, polyketide-
derived insecticide isolated from Saccharopolyspora spi-
nosa,[6] consists of four fused rings, seventeen stereogenic
centers, and multiple functional groups, including two unusual
carbohydrate moieties, all of which are built through post-
PKS modifications. Total syntheses of this complex frame-
work have been reported by the Evans, Paquette, and Roush
groups.[7–10] A Diels–Alder reaction (Evans, Roush) or an oxy-
Cope rearrangement (Paquette) was employed to construct
the octahydro-as-indacene core in these syntheses. Since, in
all cases, the stereochemical control was directed by the
conformation of the reactant, a major challenge of these early
syntheses was the development of effective routes to assem-
ble the stereocontrolling template motifs. To streamline the
synthesis of more complex polyketides, we envisioned that the
challenge of devising strategies for the stereo- and regio-
chemical control may better be met by using the correspond-
ing biosynthetic enzymes.[11,12]
Natural products are rich sources of lead compounds for the
development of new pharmaceuticals. Yet, their structural
complexity renders their chemical synthesis challenging.
There is a pressing need to develop efficient strategies for
the synthesis of natural-product-based or -like libraries for the
drug discovery program.[1,2]
Macrolide polyketides are an important class of natural
products having widespread clinical applications. Due to their
complex structures, this class of secondary metabolites have
often been targets for synthetic chemists to showcase newly
developed methods. Over the years, chemical approaches to
prepare macrolides have improved significantly to the point
where Krische et al. reported a 14-step synthesis of deoxy-
erythronolide B in 2013.[3] Such remarkable innovations have
greatly advanced the synthesis of linear polyketides and
macrolactone backbones, which are biosynthetically assem-
bled through the catalysis of polyketide synthases (PKSs).[4]
However, many polyketides possess additional structural
sophistication due to various post-PKS modifications of the
initially formed linear and monocyclic structures.[5] Syntheses
of polyketides whose structures are derived from a series of
complicated post-PKS modifications are much more demand-
ing.
The entire biosynthetic pathway of spinosyn A has been
elucidated as shown in Scheme 1.[13–19] Post-PKS modifica-
tions of the aglycone core commence following release of
macrolactone 11 from the PKS acyl carrier protein. The
monocyclic intermediate 11 is first primed by the SpnJ-
catalyzed dehydrogenation at C-15 (11!12),[15] which facil-
itates the subsequent 1,4-dehydration catalyzed by SpnM.[19]
The resulting intermediate 13 is susceptible to a transannular
[4+2]-cycloaddition reaction (13!14) that can be accelerated
by SpnF.[19] This cyclization step to form the tricyclic
hexahydro-1H-indene intermediate 14 has attracted much
attention, because SpnF may operate as a Diels–Alder-
ase.[19–21]
ꢀ
Formation of the C C bond between C-2 and C-14, which
is catalyzed by SpnL, occurs after the rhamnosylation of 14
via the action of SpnG.[17,18,22] This affords the tetracyclic core
in 16, and the attached rhamnose moiety is subsequently
permethylated in the presence of the SAM-dependent
methyltransferases SpnH, SpnI, and SpnK to produce pseu-
doaglycone 17.[18] SpnP completes the biosynthesis of spino-
syn A by catalyzing the final coupling of 17 with foros-
amine.[13,23] The two sugar appendages (rhamnose and foros-
amine) are both biosynthesized from TDP-d-glucose (2)
through pathways that have been fully established (see
Scheme 1A).[14,16,24]
[*] Prof. Dr. H. J. Kim, Dr. S.-h. Choi, B.-s. Jeon, Dr. N. Kim,
Prof. Dr. R. Pongdee, Dr. Q. Wu, Prof. Dr. H.-w. Liu
Division of Medicinal Chemistry, College of Pharmacy
and Department of Chemistry, University of Texas at Austin
Austin, TX 78712 (USA)
E-mail: h.w.liu@mail.utexas.edu
Prof. Dr. H. J. Kim
Department of Chemistry, Korea University (Republic of Korea)
Considering the wealth of information available in the
chemical literature concerning the biosynthesis of poly-
ketides, we envisioned that a chemoenzymatic synthesis,
which exploits advantages offered by both chemical and
biological approaches, would provide a practical alternative
to conventional chemical synthesis for the assembly of
complex polyketide structures. Herein, we report the first
chemoenzymatic synthesis of spinosyn A encompassing the
Prof. Dr. R. Pongdee
Current address: Department of Chemistry
Sewanee: The University of the South (USA)
[**] This work was supported by grants from the National Institutes of
Health (GM035906 and GM040541) and the Welch Foundation (F-
1511).
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201407806.
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reaction with propionate 25, carrying
a Crimminsꢀ chiral auxiliary.[28] The
anticipated syn-aldol adduct 26 was
obtained in good yield with high dia-
stereoselectivity (> 19:1). The auxili-
ary group of 26 was reductively cleaved
after O-silyl protection and the result-
ing hydroxyl group was oxidized using
TPAP to afford aldehyde 27. Incorpo-
ration of a homoallyl extension was
achieved by Brownꢀs asymmetric ally-
lation methodology.[29,30] The desired
homoallylic alcohol 28 was produced
as a single isomer at C-15.[31,32] The
terminal olefin of 28 was transformed
to a primary alcohol by Lemieux–
Johnson oxidation under buffered con-
ditions (pH 7) followed by NaBH4
reduction. Introduction of a 1H-phe-
nyltetrazolyl-5-thioether moiety onto
29 to give 30 was accomplished under
Mitsunobu conditions. Conversion of
30 to sulfone 31 was found problematic
as common oxidants gave either a par-
tially oxidized sulfoxide or no product
at all. The desired sulfone was even-
tually obtained in 91% yield after
overnight incubation at 48C with
Scheme 1. Established biosynthetic pathway for spinosyn A (1).
chemical preparation of the spinosyn polyketide backbone
and the subsequent application of post-PKS modification
enzymes to complete the final construction. Our results
demonstrate that a “one-pot, multistep” chemoenzymatic
synthesis is a viable and effective approach to prepare
complex polyketide natural products.
The first phase of our synthesis is the preparation of
macrolactone 11, which is biosynthesized through the action
of five polyketide synthases SpnA–E. Although 11 has never
been isolated, its structure was deduced according to the
established principles of linear polyketide chain assembly by
type I PKSs[4] as well as the predicted hydroxyl group
stereochemistry which was based on sequence analysis of
the ketoreductase domains.[25] The retrosynthetic analysis for
the preparation of 11 is depicted in Scheme 2. The linear
polyketide chain 18 is assembled from three fragments via two
Scheme 2. Retrosynthetic analysis. TES=triethylsilyl, TBS=tert-butyl-
dimethylsilyl.
ꢀ
C C bond coupling reactions: a Julia–Kocienski olefination
between sulfone 19 (fragment A) and aldehyde 20 (frag-
ment B) to form the D12-(E) olefin, and a palladium-catalyzed
Stille coupling using stannyl dienoate 21 (fragment C) to
generate the C-5/C-6 linkage. Subsequent cyclization of 18 by
Yamaguchi macrolactonization completes the synthesis of 11
following global deprotection.
H2O2 in the presence of an ammonium molybdate catalyst.[33]
Although cleavage of the TES group at C-21 was observed, it
could be re-introduced quantitatively to complete the syn-
thesis of fragment A (19).
Synthesis of fragment A (19) was initiated by applying
Soaiꢀs asymmetric ethylation to Weinreb amide 22[26] using
(ꢀ)-DBNP as the chiral catalyst (Scheme 3).[27] Mosher ester
analysis of product 23 confirmed the S configuration at C-21.
Aldehyde 24, prepared by O-silylation of 23 followed by
DIBAL-H reduction, was utilized in an asymmetric aldol
Preparation of fragment B (20) started with regioselective
epoxide opening of PMB-protected (S)-glycidol (32) using
lithiated 1,3-dithiane to give 33. After O-silyl protection
followed by hydrolysis of the 1,3-dithiane moiety, another
application of Brownꢀs allylation methodology afforded 35
with the desired stereochemistry at C-9 (d.r. > 20:1). Incor-
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Scheme 4. A) Synthesis of fragment C (21) and B) completion of
preparation of 11. Reagents and conditions: a) Bu3SnH, AIBN, ben-
zene, reflux, 50%; b) MnO2, acetone, RT, 85%; c) EtO2CCH2P(O)-
(OEt)2, NaH, THF, 08C to RT, 74%; d) KHMDS, THF, ꢀ788C, 82%;
e) 21, [Pd2(dba)3], Ph3As, DMF, RT, 70%; f) PPTS, EtOH, 08C;
g) LiOH, THF, CH3OH, H2O, reflux, 2 steps 62%; h) 2,4,6-
Cl3C6H3COCl, Et3N, THF; DMAP, toluene, RT, 75%; o) HF·pyr, EtOH,
48C, 4 days, 64%. AIBN=azobisisobutyronitrile, KHMDS=potassium
bis(trimethylsilyl)amide, dba=dibenzylideneacetone, PPTS=pyridi-
nium p-toluenesulfonate, DMAP=4-dimethylaminopyridine.
Scheme 3. Synthesis of A) fragment A (19) and B) fragment B (20).
Reagents and conditions: a) (ꢀ)-DBNP, Et2Zn, hexanes, 08C, 66%,
92% ee; b) TESOTf, 2,6-lutidine, CH2Cl2, ꢀ788C; c) iBu2AlH, toluene,
ꢀ788C, 2 steps 78%; d) TiCl4, (ꢀ)-sparteine, CH2Cl2, 08C, 84%;
e) TBSOTf, 2,6-lutidine, CH2Cl2, ꢀ788C; f) LiBH4, Et2O, CH3OH, 08C, 2
steps 75%; g) TPAP, NMO, 4 ꢀ M.S., CH2Cl2, RT; h) (+)-Ipc2B(allyl),
Et2O, ꢀ788C, 2 steps 60%; i) TBSOTf, 2,6-lutidine, CH2Cl2, ꢀ788C,
87%; j) OsO4, NMO, THF, acetone, pH 7 buffer, RT; k) NaIO4, THF,
pH 7 buffer, RT; l) NaBH4, EtOH, RT, 3 steps 67%; m) PTSH, PPh3,
DIAD, THF, RT, 86%; n) (NH4)6Mo7O24, H2O2, EtOH, H2O, 48C;
o) TESOTf, 2,6-lutidine, CH2Cl2, ꢀ788C, 2 steps 91%; p) 1,3-dithiane,
nBuLi, THF, 08C, 94%; q) TBSOTf, 2,6-lutidine, CH2Cl2, ꢀ788C, 95%;
r) MeI, CaCO3, CH3CN, H2O, reflux, quantitative; s) (+)-Ipc2B(allyl),
THF, ꢀ788C; t) TBSOTf, 2,6-lutidine, CH2Cl2, ꢀ788C, 2 steps 67%;
u) OsO4, NaIO4, 2,6-lutidine, dioxane, H2O, RT, 83%; v) CrCl2, HCI3,
dioxane, THF, RT, 87%; w) DDQ, aq. CH2Cl2, RT; x) Dess–Martin
periodinane, CH2Cl2, RT, 2 steps 83%. OTf=trifluoromethane-
sulfonate, TPAP=tetrapropylammonium perruthenate, NMO=N-
methylmorpholine-N-oxide, Ipc=isopinocampheyl, PTSH=1-phenyl-
tetrazole-5-thiol, DIAD=diisopropyl azodicarboxylate, DDQ=2,3-
dichloro-5,6-dicyano-1,4-benzoquinone.
radical to propargyl alcohol (38) afforded (E)-vinylstannane
39. Oxidation of the hydroxyl group followed by Horner–
Wadsworth–Emmons olefination of the resulting aldehyde
furnished dienoate 21 in excellent overall yield. With all
fragments in hand, a Julia–Kocienski olefination connecting
19 and 20 was achieved using KHMDS at ꢀ788C to give the
desired product 40 with E selectivity in 82% yield (Scheme
4B). Then, a palladium-catalyzed Stille coupling between 40
and fragment C (21) effectively generated the linear ketide
18. The final steps to complete the synthesis of 11 involved
chemoselective deprotection of the TES group at C-21,
hydrolysis of the ethyl ester moiety to afford seco-acid 41,
and lactonization under Yamaguchi conditions to give macro-
lactone 42.[36] The finishing global deprotection of 42 proved
to be more challenging and partial deprotection or decom-
position was encountered under various conditions. We were
pleased to find that treatment of 42 with HF·pyridine in
ethanol at 48C for roughly 4 days eventually led to successful
production of 11 in satisfactory yield (64%).
In the second phase of our synthesis, we opted to take
advantage of our knowledge regarding spinosyn biosynthesis
and the availability of all post-PKS tailoring enzymes, and
apply a series of enzymatic transformations in one pot to
convert macrolactone 11 to 17-pseudoaglycone (17). As
shown in Figure 1A, the genes responsible for post-PKS
modifications in the spinosyn pathway are translationally
controlled by at least four operons (operons I–IV).[13] In our
poration of the terminal vinyl iodide group began with TBS
protection of 35 followed by oxidative cleavage of the
terminal olefin by Jinꢀs procedure.[34] The resulting aldehyde
36 was subjected to Takai iodoolefination[35] providing 37 with
> 9:1 E/Z stereoselectivity. Subsequent oxidative removal of
the PMB ether followed by Dess–Martin oxidation completed
the synthesis of fragment B (20).
Preparation of fragment C (21) was straightforward as
shown in Scheme 4A. Regioselective addition of tributytin
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profiles and yields of a series of incubations containing 12 and
TDP-l-rhamnose with SpnM, SpnG, SpnL, and varied con-
centrations of SpnF (0 to 20 mm) were analyzed. Our results
showed that addition of 20 mm of SpnF clearly suppressed the
formation of minor byproducts and elevated the yield of the
tetracyclic octahydro-as-indacene product (Figures S1 and
S2).[32] Hence, the concentration of SpnF in the in vitro
experiment was set at 20 mm.
Having the concentrations of all enzymes required for
post-PKS modifications adjusted, the one-pot reaction was
conducted by incubation of 1 mm 11, excess S-adenosyl
methionine (SAM), and TDP-l-rhamnose (5) with the
aforementioned enzymes in 50 mm Tris·HCl buffer (pH 8) at
308C. As shown in Figure 1B, transformation of 11 to product
17 was achieved with an overall conversion yield estimated to
be 19.6% (average yield per step = 81.6%) based on HPLC
analysis.[32]
To complete the synthesis of spionsyn A (1), the attach-
ment of forosamine at C-17 of 17 was attempted using SpnP,
which is the glycosyltransferase assigned for this transforma-
tion.[13] Specifically, SpnP was incubated with all enzymes
involved in TDP-d-forosamine biosynthesis (SpnO, SpnN,
SpnQ, SpnR, and SpnS),[14,16] TDP-4-keto-6-deoxyglucose (3),
and 17 in one pot. Unfortunately, production of 1 was not
observed. Further sequence alignment and crystal structural
analysis suggested that SpnP belongs to a group of glycosyl-
transferases requiring an auxiliary protein for activation.[23]
However, no putative auxiliary protein gene could be found
in the spinosyn biosynthetic gene cluster. Thus, the failure of
SpnP to forosamylate 17 might be due to the absence of the
cognate auxiliary protein to reconstitute its activity in vitro.
Consequently, chemical glycosylation was adopted instead,
and treatment of 17 and d-forosamine with BF3·OEt2
successfully led to spinosyn A (1).[32]
In summary, a chemoenzymatic strategy was effectively
applied in our synthesis of spinosyn A. Construction of
monocyclic precursor 11 was achieved chemically by assem-
bling three synthesized fragments in a linear form followed by
a controlled macrolactonization. The more challenging post-
PKS modifications converting 11 to 17 were accomplished
using a total of eight enzymes all in one pot. While the overall
enzymatic transformations were highly effective, it was rather
unfortunate that direct transformation of 11 to spinosyn A (1)
was hampered by the lack of the putative auxiliary protein for
SpnP. This prevented the exploitation of the full capacity of
the spinosyn biosynthetic machinery. Nevertheless, the feasi-
bility of a chemoenzymatic synthesis of a complex polyketide-
derived natural product has been demonstrated. When
compared to the reported chemical syntheses of spinosyn A,
our chemoenzymatic approach is not without its shortcom-
ings. In particular, an in-depth understanding of the biosyn-
thetic pathway is an essential prerequisite with its own set of
challenges. Thus, it may be premature at the present time to
claim a general applicability of the chemoenzymatic approach
for the synthesis of complex natural products. However,
continuing innovation in the fields of synthetic chemistry and
natural product biosynthesis indicate that the current techni-
cal impediments to chemoenzymatic synthesis will soon be
overcome. This approach is thus expected to become more
Figure 1. A) The organization of putative operons encoding enzymes
responsible for the post-PKS modifications in the spinosyn biosyn-
thetic gene cluster. B) HPLC analysis of the enzymatic tandem
reactions to convert 11 to 17; 11 alone (a), enzyme reaction mixture
(b), and 17 reference (c).
previous study of O-methylation of the rhamnose moiety in
spinosyn A, it was found that prudent coordination of the
three rhamnose methyltransferases in vitro could generate
distinct methylation patterns of rhamnose.[18] In fact, accu-
mulation of mono- and dimethylated products could be
avoided when 10 mm SpnI, 5 mm SpnK, and 1 mm SpnH were
used to permethylate rhamnose. These results suggested that
the in vivo control of rhamnose methylation is likely achieved
via differential expression of these methyltrans-ferase genes,
spnH, spnI, and spnK, which are located on three different
operons (III, II, and I, respectively, see Figure 1A).[18]
Hence, proper adjustment of the relative concentrations
of enzymes catalyzing the tailoring reactions of spinosyn A
biosynthesis may also be crucial to minimizing the possible
accumulation of dead-end intermediates in a one-pot con-
version of 11 to 17. While the actual mechanism for in vivo
metabolic flux control is likely to be more complicated, we
proceeded on the assumption that the expression levels of the
genes (i.e., the concentration of the encoded enzymes) from
the same operon would be similar. Thus, guided by results
from the rhamnose permethylation work, we set the concen-
trations of enzymes from operon I (SpnM, SpnL, SpnK, and
SpnJ) at 5 mm, those from operon II (SpnI) at 10 mm, and those
from operon III (SpnH, SpnG) at 3 mm.[32]
SpnF catalyzes the [4+2] cycloaddition of 13 to yield 14
(Scheme 1).[19] The cyclization could also occur in the absence
of SpnF, albeit at a reduced rate. Since 13 is susceptible to
Michael addition by cellular nucleophiles and/or radicals, the
physiological function of SpnF may be to prevent the
formation of byproducts from such off-path reactions by
accelerating the cycloaddition step. Since SpnF is the sole
gene encoded in operon IV, the appropriate concentration of
SpnF used in the incubation must be determined separately.
Accordingly, a model system was devised in which the product
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Received: July 31, 2014
Published online: && &&, &&&&
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Communications
Macrolide Total Synthesis
H. J. Kim, S.-h. Choi, B.-s. Jeon, N. Kim,
R. Pongdee, Q. Wu,
H.-w. Liu*
&&&& — &&&&
Chemoenzymatic Synthesis of
Spinosyn A
First chemistry, then biology: For the total
synthesis of spinosyn A, the monocyclic
macrolide precursor was constructed by
conventional chemical methods. The
synthetically challenging structural ele-
ments were introduced by the use of eight
enzymes in a “one-pot” reaction system.
Final chemical forosamylation of 17-
pseudoglycone completed the total syn-
thesis.
6
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Angew. Chem. Int. Ed. 2014, 53, 1 – 6
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