Atropurpuran (1), isolated from Aconitum hemsleyanum, is a unique
non-alkaloidal diterpenoid, which possesses an unprecedented cage-like
pentacyclic skeleton. Intriguingly, the B-E rings of 1 constitute the
highly-symmetrical tetracyclo[5.3.3.04,9.04,12]tridecane skeleton, which includes
two bicyclo[2.2.2]octane units. This unusual cage-like structural motif is only
found in nature, and there had been no synthetic report of this skeleton before our
synthetic study reported in 2011. Herein, we report the total synthesis of 1 based on
an intramolecular Diels-Alder reaction of masked ortho-benzoquinone (MOB).
The preparation of MOB was commenced from commercially available
tetralone 10. The synthetic elaboration including Buchwald-Hartwig reaction,
Mukaiyama aldol reaction, and Luche reduction afforded ketone 17. Installation of
two alkenes to ketone 17 was successful in a diastereoselective manner. Oxidative
dearomatization of phenol gave MOB 20. One-pot intramolecular Diels-Alder/
ring-closing metathesis reaction of MOB 20 afforded pentacyclic compound 22 in
68% yield. Construction of the remaining quaternary carbon center at C4 position
was achieved by using aldehyde 28 with t-BuOK and MeI. According to Qin’s total
synthesis, 1,2-reduction of ketone 30 completed the total synthesis of 1 in 27 steps
from tetralone 10.
The stemona alkaloids, isolated from Stemonaceae plants, consist of over 100 natural
products. Among these alkaloids, ca. 40 natural products belong to stemoamide-type alkaloids,
and contain the tricyclic core structure of stemoamide (1) comprised of a γ-lactone, a γ-lactam, and an azepane ring. The stemoamide-type alkaloids such as saxorumamide (2), isosaxorumamide (3), and stemonine (4) are tetracyclic compounds, which have additional γ-lactone to 1. We envisioned that stemoamide (1) could serve as a common precursor to tetracyclic natural products, and considered employing two successive coupling reactions of
the five-membered building blocks (Scheme 1). The first coupling reaction would be the
vinylogous Michael addition of 2-oxypyrrole 8 to chiral α,β-unsaturated lactone 7. The
second would be the chemoselective nucleophilic addition of the γ-lactone equivalent 10 to
stemoamide (1). The key to success of our strategy was the differentiation between the γ-
lactone and the γ-lactam of 1.
We commenced our unified synthesis of stemoamide-type alkaloids 1 ~ 4 by synthesis
of stemoamide (1) (Scheme 2). The vinylogous Michael reaction and subsequent reduction of
enamide 13 smoothly proceeded to afford bicyclic compound 9. After three-step conversion
from 9, we accomplished the total synthesis of 1 in 7 steps with 20.6% overall yield (Scheme
3). With stemoamide (1) in hand, we attempted the chemoselective nucleophilic addition to 1.
The lactone-selective nucleophilic addition with lithiated furan 17 provided both
saxorumamide (2) and isosaxorumamide (3) (Scheme 4). On the other hand, the lactamselective
nucleophilic addition was achieved through chemoselective reduction with
[IrCl(CO)(PPh3)2] and (Me2HSi)2O5), followed by acid-mediated vinylogous Mannich
reaction with siloxyfuran 27 to afford stemonine (4) (Scheme 6).
Avenaol (1), which was isolated from the allelopathic plant black oat (Avena
strigosa Schreb.) by Yoneyama and coworkers, is the first reported natural C20
germination stimulant structurally related to strigolactones (SLs). The structure of
avenaol is characterized by a bicyclo[4.1.0]heptanone skeleton with an all-cis
substituted cyclopropane on which the three main substituents are positioned in the
same direction. Avenaol shows potent germination-stimulating activity for
Phelipanche ramose seeds, but low activities for Striga hermonthica and
Orobanche minor. To date, no total synthesis of avenaol (1) has been reported.
Because of its unique structure, important biological activity, and limited
availability from the natural source, we started a synthetic project of avenaol.
In this presentation, we would like to present the first total synthesis of avenaol
(1), which features the following reactions: (i) Rh-catalyzed intramolecular
cyclopropanation of an allene (6a → 11a), (ii) Ir-catalyzed stereoselective
double-bond isomerization (5d → 4), (iii) differentiation of two hydroxymethyl
groups by intramolecular SN1 reaction (3 → 16), and (iv) regioselective C-H
oxidation of tetrahydropyran (17 → 19). This synthetic route through an
alkylidenecyclopropane effectively avoids undesired side reactions including
ring-opening of a cyclopropane. The proposed structure of avenaol, especially the
all-cis-substituted cyclopropane was confirmed to be correct by our total synthesis.
Calyciphylline F (1) was isolated from the leaves of Daphniphyllum calycinum as a novel class of
daphniphyllum alkaloids by Kobayashi and co-workers in 2007. The structural features of 1 include
unique fused-pentacyclic skeleton containing a 8-azatricyclo[184.108.40.206.]nonane system that consists of a tropane skeleton, three quaternary carbon centers, and seven contiguous stereogenic carbon centers. The bioactivities of 1 have not been elucidated yet because of its scarce supply. Thus we aim for the first total synthesis of 1 and establishing efficient processes for bioactivity study.
We started this project from development of concise [4+3] cycloaddition reaction of 2-oxyally
cations with pyrroles to furnish tropane skeleton, a platform for complex framework of 1 (Scheme 1). During the course of total synthesis of 1, we planed the late-stage bridgehead radical reaction for the introduction of side chain and efficient intramolecular [4+3] cycloaddition reaction for construction of tricyclic tropinone 7, a key intermediate of 1 (Scheme 2).
First, we obtained the desired tricyclic tropinone 7 as major product via intramolecular [4+3]
cycloaddition reaction from a precursor 8, containing pyrrole and 2-silyloxy-allylalcohol moieties
(Scheme 3). Next, 7 was converted to methylester 15' which was subjected to the cyclization reaction in the presence of KHMDS and TMSCl to afford the desired cycloadduct 16 as a single stereoisomer (Scheme 4). Then we successfully synthesized pentacyclic skeleton 22 through radical cyclization of iodoalkane 21 (Scheme 5). Finally we examined the bridgehead radical reaction with model xanthate 23. Protonation of tertiary amine was found to be crucial for this radical reaction (Scheme 6).Treatment of 23 with V-40, (TMS)3SiH and methyl acrylate in the presence of Tf2NH gave the desired product 24. The attempt to synthesize 1 by using this protocol is currently underway.
(+)-Gracilamine (1), isolated from Galanthus gracilis by Ünver and Kaya in 2005, is a
member of amaryllidaceae alkaloid, which possesses seven stereogenic centers involving all
carbon quaternary stereocenter at C3 on the unprecedented pentacyclic core structure (A to
D rings). Since the synthetically challenging structure of 1, many synthetic efforts have
been investigated, and so far total synthetic works (including formal synthesis) have been
reported from five research groups of Ma, Gao, Snyder, Yu and Banwell, respectively. But
no enantioselective synthesis of 1 has been reported, and the absolute configuration of 1 is
not known. Herein, we disclose an enantioselective synthetic (+)-gracilamine (1) based
upon a diastereoselective dearomative phenolic coupling reaction and regioselective
intramolecular aza-Michael reaction. In our synthetic process, organocatalytic
aza-Friedel-Crafts reaction developed by our group was efficiently applied for the
construction of stereocenter at C9a, whose stereochemistry allowed us to control the
remaining stereocenters in 1.
Asymmetric aza-Friedel-Crafts reaction with sesamol (4) and N-Boc aldimine 17 was
examined in the presence of catalyst (R,R)-2. Under the previously developed conditions,
aza-Friedel-Crafts reaction took place smoothly, and desired 18 was obtained in 94% yield
with 91% ee, which was increased to be 99% ee after recrystallization with hexane. The
construction of B and E rings included C3a stereocenter were achieved by diastereoselective
dearomative phenolic coupling reaction and subsequent regioselective intramolecular
aza-Michael reaction of phenol 22. Then, D ring was constructed by means of
intramolecular Mannich-type reaction of ketone 30 with α-keto ester 7. Finally,
(+)-gracilamine (1) was obtained by reduction of ketone, detosylation and methylation of
amino group. The optical rotation of our synthetic 1 [[α]25D = +16.4 (c 0.11)] showed good
agreement with previously report data [[α]25D = +21.8 (c 0.13)].
Hexahydropyrroloindole alkaloids possess structural diversity and a broad range of
biological activities. Among these compounds, (+)-gliocladin C (1), produced by a strain of
microorganism, Gliocladium roseum found in the sea hare Aplysia kurodai, is known to possess potent cytotoxicity against murine P388 lymphocytic leukemia cells (ED50 = 2.4 µg/mL). Moreover, the analogous compounds T988 A-C (2a-c), isolated from the New Zealand fungi Tilachlidium sp. (CANU-T988) by Munro and co-workers in 2004, are known to possess a potent cytotoxicity against cultured P388 leukemia cells. These compounds are composed of di- or triketopiperazine skeleton in conjunction with the pyrroloindole skeleton including a quaternary carbon center with indolyl group at the 10b position. These intriguing structural features have inspired a number of synthetic chemists to develop synthetic routes. Herein, we report our recently completed total syntheses of 1 and 2a-c via AgNTf2-mediated Friedel-Crafts alkylation and α-bromination on the diketopiperazine ring.
Diketopiperazine 18 was derived from L-tryptophan methyl ester 14 and D-serine
derivative 15 through condensation and formation of the diketopiperazine ring. Treatment of
18 with NBS in CH2Cl2 gave bromopyrroloindoline 12 high stereoselectively. We found that the judicious choice of solvent was crucial for the high stereoselectivity of this bromocyclization. Indole adduct 19 was then obtained in excellent yield by treatment of bromopyrroloindoline 12 with AgNTf2 in the presence of 5-bromoindole. After several conversions including oxidative cleavage of the terminal olefin, we achieved the total synthesis of (+)-gliocladin C (1). Furthermore, we developed a synthetic route to T988s via 21 as the synthetic intermediate. For chemoselective C-H oxidation at the C11a position, we found that the control of stereochemistry of the C3 position was crucial. Thus, we succeeded in α-bromination of 23 with NBS in the presence of AIBN under the microwave irradiation to obtain enamine 25 in quantitative yield. Finally, the total synthesis of (+)-T988 C (2a) and the first total synthesis of (+)-T988 A (2c) and (+)-T988 B (2b) were accomplished via formation of the disulfide bridge.
Juglorubin (1) was isolated from Streptomyces sp. 815 and GW4184 in 1993 (Figure 1).
This compound has an unusual 6/6/5/9/6-fused pentacyclic ring system containing a
cyclopentadienyl anion and a 9-membered lactone. Juglorubin might be biogenetically
synthesized from juglomycin C (2) via juglocombins A/B (3/4), because they were isolated
from the same Streptomyces strains (Scheme 1). The key steps in the proposed biosynthesis involve the dimerization reaction of juglomycin C (2) to establish the 6/6/5/6/6-fused pentacyclic flamework of juglocombins A/B (3/4), and the skeletal transformation from
juglocombins A/B to juglorubin through the C–C bond cleavage reaction. Herein, the first
total synthesis of juglorubin (1) by a strategy inspired by the proposed biosynthesis is
Juglocombins A/B (3/4), the hypothetical biosynthetic precursors, were successfully
synthesized using a bioinspired dimerization of juglomycin C derivative (7) and a
photoinduced reduction of epoxide in resultant dimer 6 as key reactions. The desired skeletal
transformation proceeded by treatment of 3/4 with MS3A and NaCl in MeCN under an
oxygen atmosphere to afford juglorubin in 26% yield. Furthermore, the reaction
intermediate 16 was obtained in 78% yield by the use of NaOAc as a weak base.
Juglorubin dimethyl ester (1') and 1-O-acetyljuglorubin dimethyl ester (17) were also
prepared from juglocombins A/B dimethyl ester (3'/4'). Broad absorption bands in the
region of 200-600 nm were observed in the UV-vis spectrum of 1'. Broad emission bands
spanning from the visible to the near-infrared region (550-900 nm) were detected in the
fluorescent spectrum of 1'. The maximum emission wavelength (λem. max) of 1' was 630 nm. The absolute quantum yield (φ) of 1' in acetonitrile was 5.3%.
Carthamin (1), a Japanese traditional red pigment, is a constituent of the flower petals of
safflower. After a long-standing structural investigation, the currently accepted planar
structure was independently proposed by Obara and Matsumoto. However, its
stereochemistry was deduced by comparison of the synthetic analogues by Sato, and the
total synthesis has remained unachieved. The structural features include 1) C-glycoside
composed of a D-glucose and a quinol, 2) the stereogenic center associated with the
tert-alcohol, 3) the dimer of the C-glycosyl quinochalcone units flanked by one carbon, and 4) a highly conjugated system that is the origin of the color. Charmed by such a complex and unusual structure, we became interested in the total synthesis. Herein, we will report the first total synthesis of 1 through the construction of the quinol C-glycoside structure by
means of oxidative dearomatization.
As a potential access to the C-glycosyl quinochalcone as the key intermediate for the
dimerization, we initially studied the oxidative dearomatization of C-glycosyl phenols by
using hypervalent iodine reagent. It turned out that choice of the 2-O-protecting group of the sugar moiety was crucial for the success of this conversion. By employing the 2-O-acyl
protection, the oxidative dearomatization reaction proceeded smoothly, giving the desired
dearomatization product in an excellent yield. Through this first-generation synthetic
venture, we noted a photo-lability of the geometry of the chalcone double bond. A good
news was that the impaired geometry could be corrected to the right (E)-geometry at the
stage of advanced intermediates with an internal hydrogen bonding, undergoing the
Toward the stereo-controlled generation of the quaternary center, we planned the
second-generation approach based on a group-selective functionalization of
pseudo-Cs-symmetric cyclic dienone derivative. Pleasingly, the group-selective
bromoacetoxylation enabled the projected desymmetrization, giving the corresponding
bromodienone with the requisite stereochemistry. Further transformation allowed
construction of the key C-glycosyl quinochalcone structure in a stereo-defined manner.
Assembly of two C-glycosyl quinochalcone units to the central one carbon followed by
global deprotection by using formic acid gave the targeted compound 1. The synthetic
material was fully identical in all respects with the authentic specimen.
Two new cyclopentadecane antibiotics, named mangromicins A and B, were
separated out from the culture broth of Lechevalieria aerocolonigenes K10-0216.
The chemical structures of the two novel compounds were elucidated by
instrumental analyses, including various NMR, MS and X-ray crystallography.
Mangromicins A and B consist of cyclopentadecane skeletons with a
tetrahydrofuran unit and a 5,6-dihydro-4-hydroxy-2-pyrone moiety.
Mangromicins A and B showed in vitro antitrypanosomal activity with IC50 values of 2.4 and 43.4 μg/ml, respectively. An enantioselective total synthesis of
(+)-mangromicin A has also been accomplished. The tetrahydrofuran ring of
mangromicin A, possessing a tetrasubstituted carbon center, was constructed by
Mukaiyama-type vinylogous alkylation via a cyclic oxocarbenium intermediate
derived from a γ-hydroxy ketone with ideal stereoselectivity, and the
4-hydroxydihydropyrone scaffold was generated via Dieckmann condensation at a
late stage of the total synthesis. The reliable asymmetric synthesis of
(+)-mangromicin A has revealed the absolute configuration of naturally occurring
Casuarinin and stachyurin are ellagitannins isolated from chestnut and oak. Castalagin
and vescalagin are known as analogues thereof. Structural features common to these
compounds are the presence of D-glucose in an open chain form and hexahydroxydiphenoyl
(HHDP) or nonahydroxytriphenoyl (NHTP) groups linked to the glucose moiety through a
C-glycosidic bond. The presence of the C-glycosidic bond has greatly influenced generation of their analogues and expression of biological activities. Here, we describe the synthetic study of these ellagitannins, including the development of efficient method for construction of the C-glycosidic bond and the total synthesis of casuarinin.
The following transformations completed the total synthesis of casuarinin. For the
comprehensive synthesis of C-glycoside ellagitannins, we embraced the synthetic strategy
based on the presumed biosynthesis of casuarinin, in which pedunculagin and liquidambin
are the key intermediates. Thus, we synthesized an equivalent of pedunculagin using the
oxidative coupling of 4-O-benzylated gallates as the key reaction. The opening of the
pyranose ring in the pedunculagin-equivalent as an oxime followed by introduction of the
galloyl group at O-5 provided an equivalent of liquidambin. Treatment of this compound
with acid constructed the C-glycosidic bound to furnish the skeleton of casuarinin as a single diastereomer. Finally, we achieved the first total synthesis of casuarinin by removal of the
A synthetic route toward leucinostatin A, a modulator of tumor-stroma interactions, was
established, in which catalytic asymmetric reactions developed in this laboratory were
employed: nitroaldol reaction for the synthesis of HyLeu, thioamide-aldol reaction,
Strecker-type reaction, and alcoholysis of 3-methylglutaric anhydride for the synthesis of
AHMOD. Careful analysis of the NMR data, HPLC profiles, and biologic activity suggested
that the correct structure of leucinostatin A is the epimeric form of the reported structure; the
secondary alcohol within the AHMOD residue has an R-configuration (epi-1).
Biologically active oligo peptides consisting of less than 20 amino acids are particularly
valuable for the development of drugs. In fact, they account for three-fourths of the existing
marketed peptide drugs. Feglymycin is a biologically active oligopeptide containing 13 amino
acids including highly racemizable 3,5-dihydroxyphenylglycines (Dpgs). Biological activity
of feglymycin involving strong anti-HIV activity and moderate antimicrobial activity and its
unique helical conformation make feglymycin an attractive lead compound for drug
development. We achieved a total synthesis of feglymycin based on a linear/convergent hybrid
synthetic approach. Our originally developed micro-flow amide bond formation enabled the
efficient preparation of hexapeptide and heptapeptide intermediates containing highly
racemizable Dpgs based on a linear synthetic approach that was previously considered
impossible. The developed synthetic approach will be useful for the rapid preparation of
feglymycin analogues in the future. Our micro-flow amide bond formation uses triphosgene,
and only emits CO2 and the HCl salt of DIEA. One of the advantages of using micro reactors is the ease of scaling up. Our developed process can be scaled by either continuous running or by the numbering up of the microreactors. This process will enable a practical preparation of biologically active oligopeptides containing highly racemizable amino acids.
Covalent conjugates of nutrients including sugars, amino acids, fatty acids, and vitamins
represent a recently emerging class of medicines and supplements. Such conjugates are also
present endogenously in our body, exerting a myriad of biological activities. These “nutrient
conjugates” tend to have two interesting properties: (i) potential to exert biological activity
that individual nutrients are unable to generate; (ii) higher safety profiles as they are
composed of endogenous nutrients. In this study, we constructed a conceptually new
chemical library of “nutrient conjugates,” a systematic collection of covalently coupled
multiple nutrients. The library was subjected to a number of cell-based screenings, and
subsequently, we isolated the nutrient conjugates that inhibited SREBP (sterol regulatory
element-binding protein), a transcription factor that orchestrates lipid synthesis and
metabolism. Mechanistic analysis of one of the careening hits, referred to E2, indicated that
E2 inhibits SREBP by inhibiting glucose transport and subsequent activation of AMPK
(AMP-activated protein kinase). Moreover, E2 inhibited the increase in blood glucose level
after glucose load in mice. Nutrient conjugate libraries might prove to be a rich source of
medicines, supplements, and chemical tools for biology with a number of biological
activities including energy metabolism.
The microenvironment in tumor is suffered from limited supply of oxygen and
nutrients. Some cancer cells such as pancreatic cancer are known to adapt these
severe conditions and grow, together with acquiring resistance to cancer
chemotherapy and irradiation. Therefore, selective growth inhibitors against
cancer cells under nutrient-starved conditions have the potential to be
anticancer drugs with novel mode of action, and to be chemical tools for
elucidating the adaptation mechanism to the conditions. Recently, we isolated
novel polyketides named biakamides A-D (1-4) as selective growth inhibitors
against human pancreatic cancer PANC-1 cells under glucose-deprived
conditions. In this work, total synthesis, structure-activity relationship (SAR)
study, and mechanistic analysis of biakamides were executed.
The planer structures of biakamides were elucidated by the detailed analyses of
NMR spectra, and the stereostructure at C-23 of biakamide A, B was determined
by modified Mosher method. Then, total syntheses of all stereoisomers at C-4
and C-6 of biakamides were executed, to determine the stereostructures of the
natural product. Compare analyses of NMR/CD spectra and specific rotations of
the synthetic isomers with those of natural products showed that the absolute
stereochemistries of all biakamides are (4R, 6S).
Next we explored the SAR of biakamides to develop easily accessible analogs,
and to know the participation of the substructure to their activity against
PANC-1 cells under glucose-deprived conditions. We found that dedimethyl
biakamide C analog (9), which is easily accessible, possessed the comparable
growth inhibitory activity under glucose-deprived condition to natural
biakamide C. Then, we investigated the detailed SAR study using various
analogs based on compound 9 as a scaffold. It revealed that the terminal acyl
chain is important for interacting with target molecule, and structural
modification of the amide part including thiazole is acceptable. On the contrary,
whole structure of biakamide is necessary for exhibiting its activity.
Finally, we tried to analyze the action mechanism of biakamides. Biakamide C
reduced expression level of GRP78 and phosphorylation level of Akt (473Ser),
known marker proteins of cancer cells adapted to nutrient-starved conditions.
Besides, biakamide C selectively inhibited mitochondrial respiratory complex I.
So, we designed and synthesized a probe 13, having a diazirine moiety and an
alkyne moiety, to confirm the intracellular localization of biakamides.
Fluorescence imaging study using photoaffinity labeling and click chemistry
revealed that the probe 13 was selectively accumulated at mitochondria in
PANC-1 cells. So, we concluded that the growth inhibitory activity of biakamides
was caused by the inhibition of mitochondrial respiratory complex I.
Oligomers of the 42-mer amyloid-β protein (Aβ42), rather than fibrils, cause memory
loss and synaptic dysfunction in the pathogenesis of Alzheimer’s disease (AD). The
aggregation mechanism of Aβ42 is explained by the nucleation-dependent polymerization
model that consists of nucleation and elongation phases. During the nucleation phase,
Aβ42 monomer gradually forms low-molecular-weight intermediates, called “nuclei”, which
are closely related to the formation of toxic oligomers of Aβ42 (12 ~ 24-mer: 2 or 3 × n).
Uncaria rhynchophylla is one ingredient of “Yokukansan”, a Kampo medicine, which is widely available for treating AD symptoms. Previously, extracts of U. rhynchophylla were found to delay the aggregation of Aβ42, but the active principles have yet to be identified. Here we identified uncarinic acid A ~ D as inhibitors of Aβ42 aggregation in the acetone extracts of the dried branches of U. rhynchophylla. Moreover, they acted as specific inhibitors of the nucleation phase of Aβ42 aggregation.1) Uncarinic acid C (3) was semi-synthesized from saponin A (10), an abundant by-product of rutin purified from Uncaria elliptica. Subsequent structure-activity studies on 3 indicated that both the C-27 ferulate and C-28 carboxylic acid group were necessary to its preventive activity. Ion mobility-mass spectrometry suggested that 3 could target the dimer ~ tetramer by its direct interaction with Aβ42 through the salt bridge between C-28 carboxylic acid group and Lys16,28 side chain, resulting in the suppression of further oligomerization. The 1H–15N SOFAST-HMQC NMR of Aβ42 in the presence of 3 revealed the significant perturbation of chemical shifts of His13,14, Lys16,28, and Arg5, which were close to the intermolecular β-sheet region (Gln15~Ala21). Given the potent inhibition of Aβ42-induced neurotoxicity by 3, the present findings may aid the development of toxic oligomer-specific inhibitors for AD therapy.
1) Yoshioka, T. et al., J. Nat. Prod.2016, 79, 2521-2529.
“Fairy rings” is the phenomenon in which turfgrass grows or dies in a circle. In
previous reports, we discovered that fairy rings were induced by 2-azahypoxanthine (AHX)
and imidazole-4-carboxamide (ICA) produced by one of the fairy rings forming fungi,
Lepista sordida. AHX is metabolized to 2-aza-8-oxohypoxanthine (AOH) in plants. AHX,
ICA and AOH are named fairy chemicals (FCs). Furthermore, we proved the existence of
endogenous FCs and succeed in isolation and structural determination of some metabolites
of FCs. Thus, we thought that the pathway producing them in plants was a novel purine
pathway. However, the biosynthetic pathway of FCs in plants has not been disclosed
The precursor of FCs in chemically synthesis is 5-aminoimidazole-4-carboxamide
(AICA) and we have found AHX is biosynthesized from AICA in plants. In feeding
experiments with stable isotope-labeled AICA, it was revealed that ICA was also
biosynthesized from AICA in plants. Furthermore, we developed a new detection and
quantification method of FCs and succeed in quantification the endogenous content of that.
In addition, by using this method, we found that FCs metabolites (two kinds of
AOH-β-D-glucosides, ICA-riboside, and ICA-ribotide) endogenously exist in rice.
AHX-ribotide, presumptive precursor of AHX, is chemically synthesized from
AICA-ribotide and NO donor (NOC5). We found that AHX-ribotide was converted from
AHX by hypoxanthine-guanine phosphoribosyltransferase obtained from rice, and a novel
compound, AOH-ribotide, was also converted from AOH by the enzyme.
Fosfomycin is a clinically approved broad-spectrum antibiotic produced by
Streptomyces and Pseudomonas. The structural feature of fosfomycin is the
characteristic phosphonate group that is attached to the C1 of methyl oxirane.
Although five genes were identified to be involved in the fosfomycin biosynthesis
in Streptomyces by genetic analysis and in vitro enzymatic analysis with
recombinant enzymes, the biosynthetic mechanism that links between the
2-hydroxyethylphosphonate (HEP) and 2-hydroxypropylphosphonate (HPP)
intermediates was elusive.
In this study, functional analysis of a cobalamin-dependent radical
S-adenosyl-L-methionine C-methyltransferase Fom3 that is coded in the
fosfomycin biosynthetic gene cluster was carried out. When cytidylylated
2-hydroxyethylphosphonate (HEP-CMP) was incubated in the presence of SAM,
methylcobalamin (MeCbl), methylviologen, NADH and dithiothreitol (DTT) with
reconstituted Fom3, the production of cytidylylated 2-hydroxypropylphosphonate
(HPP-CMP) was clearly observed. The production of 5'-deoxyadenosine (5'-dA)
was also observed to indicate that the 5'-deoxyadenosyl radical was generated to
abstract the hydrogen atom of the substrate HEP-CMP. In the presence of “HEP”
instead of HEP-CMP, the production of 5'-dA was not detected, confirming that
HEP is not an appropriate substrate of Fom3. This result suggests that the CMP
moiety of HEP-CMP is critical for substrate recognition by Fom3. Therefore,
HEP-CMP is likely an intermediate in the fosfomycin biosynthetic pathway.
The Fom3 reaction product was also isolated to confirm the chemical structure by
NMR. In the 1H-NMR spectrum of the product, two sets of doublet methyl signals
were observed around 1.15 ppm to indicate that Fom3 produced a mixture of two
diastereomers. The ratio of the (S)- and (R)-HPP-CMP in the Fom3 reaction
product was almost equal, thereby revealing that there is no stereoselectivity during
The production of S-adenosyl-L-homocysteine (SAH) was also observed,
indicating that MeCbl is regenerated by SAM for the catalytic cycle. The
production ratio of HPP-CMP, 5'-dA and SAH was almost equal. Based on these
results, we propose the Fom3 reaction mechanism as follows. At first,
5'-deoxyadenosyl radical generated from the reductive cleavage of SAM abstracts
one of the hydrogen atoms at C2 of HEP-CMP to generate the substrate radical
intermediate, which reacts with the methyl group on MeCbl at the opposite side
from SAM and the [4Fe-4S] cluster to give (S)- and (R)-HPP-CMP. The generated
cob(II)alamin is reduced by DTT to cob(I)alamin, which is methylated in the
presence of SAM to regenerate MeCbl.
Amino-group carrier protein (AmCP) is involved in the biosynthesis of
non-proteinogenic amino acid, (2S,6R)-diamino-(5R,7)-dihydroxy-heptanoic acid
(DADH), a key building block for a novel di-peptide compound, vazabitide A. We
screened 848 actinomycetes strains using polymerase chain reaction to
investigate the diversity of this newly identified biosynthetic machinery
mediated by AmCP. Successive draft genome sequencing of positive strains
revealed twelve putative gene clusters containing AmCP genes. Among them,
the heterologous expression of a putative gene cluster from Streptomyces
sp. SoC090715LN-17 led to the discovery of a novel natural product, s56-p1
that possesses a unique hydrazone unit attached to a DADH-derived amino acid.
This result shows the structural diversity of natural products biosynthesized via
AmCP-mediated machinery and paves the way for investigation of untapped
AmCP-containing gene clusters distributed among actinomycetes.
Further, in vivo and in vitro characterization of enzymes encoded in
the gene cluster for s56-p1 revealed the biosynthetic pathway for
hydrazineacetic acid (HAA), a putative precursor of the unique hydrazone unit of
s56-p1. This finding would give insight into the mechanism of a
nitrogen-nitrogen covalent bond formation in natural products biosynthesis.
Fungal polyketides, synthesized by iterative polyketide synthases (IPKSs), comprise a structurally diverse family of natural products, displaying a wide range of biological activities. Nature can increase the structural diversity of polyketides by collaboratively pairing IPKSs in the same biosynthetic pathway. Collaborative IPKSs are frequently colocalized in the same biosynthetic gene cluster, a genetic feature that can be utilized in the genome mining of new natural products.
One strategy to install nitrogen-containing functional groups in fungal polyketides is through the action of hybrid megasynthases known as PKS-NRPSs. The cis-acting NRPS module can amidate the completed polyketide acyl chain with a specific amino acid selected by the adenylation domain. The aminoacyl polyketide can then be cyclized into compounds that contain a nitrogen heterocycle, such as tetramate. Hence, one would expect pairing a PKS-NRPS with other IPKSs can lead to additional structural diversity.
Here we demonstrate a new mode of collaboration between an HRPKS and a PKS-NRPS, in which the HRPKS is responsible for the biosynthesis of free, unnatural amino acid that is subsequently incorporated by the PKS-NRPS to generate new succinimide and maleimide containing natural products.
Non-heme iron/α-ketoglutarate (αKG) dependent dioxygenases catalyse key
oxidation reactions to afford complex molecular structures in the fungal meroterpenoid
biosynthesis. AusE and PrhA are two dioxygenases that catalyse intriguing oxidative
rearrangement reactions in austinol and paraherquonin biosynthetic pathways, respectively.
AusE and PrhA have high sequence similarity (~78%), and both enzymes accept the same
preaustinoid A1 substrate but generate different products, preaustinoid A3 and berkeleydione.
Here we describe the X-ray crystal structures of apo AusE and PrhA, as well as PrhA
complexed to Fe(II), αKG, and preaustinoid A1 at under 2.1 Å resolution. Comparison of
the crystal structures revealed several key active site residues that are proximal to the
substrate and are different in AusE and PhrA. Mutation of the identified PhrA active site
residues to mimic those of AusE (A232S/V150L) resulted in a PrhA-A232S/V150L mutant
that catalyses an AusE-type reaction to produce preaustinoid A3. Subsequently, we solved
the X-ray crystal structure of PrhA-A232S/V150L complexed to Fe(II), αKG, and
preaustinoid A1 or preaustinoid A2. In the co-crystal structure with preaustinoid A1, the
distance between C2 of preaustinoid A1 and Fe(II) was reduced by 0.9 Å compared to the
wild type PrhA, providing evidence for the position of the initial hydrogen abstraction.
Furthermore, we observed that the C5 position of preaustinoid A2 is proximal to the
catalytic Fe center in the co-crystal structure of the PrhA mutant with preaustinoid A2,
indicating that the hydrogen abstraction at C5 triggers the sequential reaction to form the
spirolactone ring of preaustinoid A3. We also generated PrhA-A232S/V150L/M241V triple
mutant and found that in this mutant generates several novel compounds through four steps
oxidation from preaustinoid A1. This study presents the structural basis of a novel reaction
mechanism in multifunctional dioxygenases, in combination with mutagenesis studies that
successfully led to the engineering of a new function for these enzymes.
Previously, the seed coat pigment of small red beans (Vigna angularis) was
presumed to be anthocyanin with brown colored tannins. We isolated
anthocyanin pigment, cyanin (1), from small red beans in 1996, but the content
in the seed coat was very low, less than 0.01 mg/g dry seed coat. Therefore, the
seed-coat color was not due to anthocyanin.
We identified two purple pigment in the seed coat of small red beans, which was
responsible to the purple color of an-paste, and determined the structure. In this
study, we will report the structure and chemical properties of the purple pigments.
An aqueous acetonitrile solution containing trifluoroacetic acid was analyzed by
PDA detected ODS-HPLC. Two pigment peaks which exhibit purple color in
strong acidic condition were found. From 12 Kg of beans we extracted the
pigment and purified using repeated column chromatography to be obtained AZ-
1 (2) and AZ-2 (3), respectively. From the various NMR experiments and MS
analysis, the partial structure of AZ-1 (2) was determined. However, the
structure could not be determined. Then, we degraded AZ-1 (2) to AZ-1d (4) and
determined the structure, first. From the structure of AZ-1d (4) we could finally
determine the structure of AZ-1 (2) and named catechinopyranocyanidin A. It
has a condensed ring structure with catechin and cyanidin.
In the process of black tea production, various catechin dimers are produced from pyrogallol-type and catechol -type catechin by enzymatic oxidation. Dimerization of pyrogallol-type catechin affords dehydrotheasinensins stereoselectively. DFT optimization of dehydrotheasinensin C suggested that the selectivity was accounted for intramolecular π-π stacking between two A-rings in aqueous solution. It was supported by 1H NMR chemical shifts and NOE in D2O, as well as comparison of ECD spectra in water and CH3CN assisted by DFT calculations.
On the other hand, oxidative coupling between pyrogallol-type and catechol-type catechins affords theaflavins with a benzotropolone chromophore. Plane structure of the chromophore is produced via a bicyclo[3.2.1]octane-type intermediate, and formation of the intermediate was non-stereoselective coupling between catechol quinone and pyrogallol, which was shown by trapping a pair of epimers of intermediates. In this study, a newly developed facile method for theaflavin preparation was applied. Furthermore, structure of goupiolone B was revised by biomimetic synthesis related to theaflavin production.
Binding affinity to protein is one of the important property for biological activity.
Recently, we have developed “Target protein oriented natural products isolation
(TPO-NAPI)” method to isolation of bioactive natural products. Protein
immobilized sepharose beads were prepared and mixed with extract of natural
resources. After incubation and wash process, bound compounds were released from
protein beads by addition EtOH. Hairy and enhancer of split 1 (Hes1) is a repressor
type basic helix-loop-helix factor which inhibits neural stem cells (NSCs)
differentiation. Hes1 beads gave two new compounds 1, 2 which accelerated NSCs
differentiation. Notch intracellular domain (NICD) is a transcriptional factor in
Notch signal pathway. Aciculatin 5 was found as a Notch inhibitor by NICD beads
method. Compound 5 also enhanced the differentiation of NSCs. Hedgehog (Hh)
signal is one of the central signals in cell maintenance and differentiation. GLI1, a
transcriptional factor in Hh signal, was immobilized on beads, which gave five
natural products 9-13. This TPO-NAPI method would be useful to isolate target
protein binding natural products from extract of natural resources.
The genus Kopsia, belonging to the family Apocynaceae, is an abundant source
of monoterpenoid indole alkaloids possessing structural diversity and significant biological
activities. In our chemical studies on novel bioactive alkaloids, we have found several
unique monoterpenoid indole alkaloids, such as kopsiyunnanines A-M and andranginine
from Kopsia arborea native to Yunnan Province in China.
Kopsiyunnanine K (1) has an unprecedented azepane-fused tetrahydro-β-
carboline ring skeleton and the structure was confirmed by total synthesis utilizing
Ireland-Claisen rearrangement and diastereoselective Pictet-Spengler cyclization as a key
step. Kopsiyunnanine L (2) has a novel 2,3,4,5-tetrahydro-1H-benzazepin skeleton
rearranged from a Strychnos-type monoterpenoid indole alkaloid. Kopsiyunnanine M (3) is a
new type of bisindole alkaloid consisting of Aspidospermatan-type and Strychnos-type
Andranginine (4) was first isolated as a racemate from Craspidospermun
verticillatum. The first asymmetric total synthesis of 4 utilizing asymmetric Morita-Baylis-Hillman reaction and diastereoselective intramolecular Diels-Alder reaction has revealed
that 4 isolated from K. arborea existed as a scalemic mixture.
Resiniferatoxin (1) is a daphnane diterpenoid, isolated from the latex of Euphorbia
resinifera. Compound 1 was revealed to exhibit strong analgesic properties by acting as a
potent activator of transient receptor potential vanilloid 1 (TRPV1). The structure of 1
consists of a fused 5/7/6-membered carbocyclic framework and a unique orthoester moiety.
The promising therapeutic activities and the highly complex architecture have gained
intense interest in the chemical community. A number of synthetic efforts have been
reported, but the only total synthesis of a daphnane diterpenoid disclosed to date is that of 1
by Wender and co-workers. Herein, we describe the asymmetric synthesis of 1 by
employing a novel radical-based strategy.
To simplify the retrosynthetic dissection of 1, we have developed radical-based approach
to assemble a highly oxygenated carbon skeleton. The caged orthoester structure of 1
motivated us to utilize a three-component radical coupling. Accordingly, 1 was
retrosynthetically disassembled into A-ring 6, allyl stannane 7 and C-ring 5, which would be
derived from D-ribose (10).
First, 5 was synthesized from D-ribose (10). The six-membered C-ring structure of 1
was assembled by a ring-closing metathesis of diene 12, which was derived from 10 via
installation of two carbon chains. Stereoselective functionalization of the C-ring provided
α-alkoxy selenide 5 as a radical precursor. The key three-component radical coupling
reaction between 5-7 enabled us to elongate the nine-carbon unit and introduce the three
stereocenters in a single step. Then, the following seven-step transformation, including
stereoselective C4-hydroxylation, afforded bisxanthate 34. B-ring formation by a 7-endo
radical cyclization built the C8-stereocenter, and produced ABC-ring framework 2. Finally,
the total synthesis of 1 was accomplished from 2 through the orthophenylacetate formation,
the C7-allylic oxidation, and the installation of homovanilloyl group by SN2 displacement as the key transformations.
In conclusion, we have developed a novel radical-based synthetic route to 1. The
present approach demonstrated the power and versatility of the radical reactions to realize
the construction of highly oxygenated structures.
The structurally complicated and interesting terpenens pallambins A-D were isolated from the liverwort Pallavicinia ambigua. These natural products have 4-6 rings, 7-10 contiguous stereogenic centers which can be considered as challenging architectures in organic synthetic chemistry. Two elegant total syntheses of pallambins have been reported, one from the Wong group and the other from Carreira group. The former route furnished pallambins C and D in 38 steps from the Wieland-Miescher ketone, and the latter gave pallambins A and B in 23 steps from a fluvene. We have achieved an efficient and concise total synthesis of pallambins C and D in only 11 pots, wherein protecting group manipulations is absent.
Our total synthesis of pallambins C and D commences with inexpensive furfuryl alcohol 10. Eschenmoser-Claisen rearrangement of 10 followed by reduction gave aldehyde 9 in a single vessel with decent yield. Robinson annulation of 9 and ethyl vinyl ketone afforded the corresponding cyclohexenone derivative 11 in moderate yield. TMS-enol ether 12 was obtained via Michael addition of a vinyl cuprate species followed by trapping the resulted enolate by TMSCl. Chemoselective oxidative cleavage of furan moiety was carried out in the presence of methylene blue with oxygen to give keto-aldehyde 13. The intramoleculer Mukaiyama aldol reaction was successfully accomplished in good yield and with excellent diastereoselectivity. Surprisingly, conversion of 6 to acetal 14 was achieved by treatment with CH(OMe)3 and SnCl4, and the intermediate 14 could be transformed into alkyl bromide 15 by successive addition of AcBr in the same reaction vessel. A reduction of the alkyl bromide followed by desaturation and elimination of methanol proceeded smoothly delivering enol ether 15 in good yield. Fortunately, we established the new transformation of enol ethers into the corresponding difunctionalized adducts by using SnCl4 and I2. The desired iodo-diester 18 was obtained in excellent yield and with perfect diastereoselectivity under this condition. Finally, the total synthesis of pallambins C and D was accomplished by engineering four transformations in one-pot.
Halichondrins are polyether macrolides, originally isolated from the marine sponge Halichondria okadai by Uemura, Hirata, and coworkers.2) Due to their intriguing structural architecture and extraordinary antitumor activity, halichondrins have received much attention from the scientific community. Although our group have previously reported efficient syntheses of halichondrins and norhalichondrins, an attempt of synthesizing homohalichondrins via the same route gave only a modest success.3)
In this presentation, we report a convergent, efficient, and scalable synthesis of all the members, including homohalichondrins A~C, in the halichondrin class of natural products. We have designed two new fragments, which were prepared from same intermediate, by disconnecting between C37-C38. During the course of this study, we have developed new coupling reactions. NHK coupling for aldehyde 6 and vinyl iodide 7 proceeded without protection on C30 hydroxy group by adding excess of Cp2ZrCl2 and DTBMP. Final coupling between 2 and 3 was accomplished with newly developed Zr/Ni-mediated one-pot ketone synthesis. By developing these assembly chemistry, halichondrins A and B were synthesized in 28 steps (29 steps for halichondrins C) from commercially available D-galactal.4)
1) This work was performed in the Kishi laboratory at Department of Chemistry, Harvard
University, Cambridge, Massachusetts, U.S.A.
2) (a) D. Uemura, Y. Hirata, et al., J. Am. Chem. Soc.1985, 107, 4796. (b) Y. Hirata, D. Uemura, Pure Appl. Chem.1986, 58, 701.
3) A. Ueda, A. Yamamoto, D. Kato, and Y. Kishi, J. Am. Chem. Soc.2014, 136, 5171.
4) (a) K. Yahata, N. Ye, Y. Ai, K. Iso, and Y. Kishi, submitted for publication. (b) Y. Ai, N.
Ye, Q. Wang, K. Yahata, and Y. Kishi, submitted for publication.
Asymmetric total synthesis of (–)-morphine (1) has been accomplished in 18 steps from commercially available 7-methoxy-2-tetralone (7). Our synthesis features a simple transformation from the readily available chiral intermediate (3), reported earlier by d’Angelo and co-workers. Introduction of the requisite oxygen functionality to the 8-position of the tetralone could be achieved by Friedel-Crafts acylation and ensuing Baeyer-Villiger oxidation. Upon treatment of dienol 15 with acid at 0 °C, a facile cyclization proceeded to form the E-ring (16). The diene thus formed was later converted to γ-hydroxy-α,β-unsaturated ketone 20 by means of photooxygenation and triethylamine. Dehydration of 20 was effected by triflic anhydride and subsequent removal of 2,4-dinitrobenzenesulfonyl group generated a mixture of neopinone (21) and codeinone (22). Conversion of the mixture to (–)-morphine was performed by following the known procedures.
A multi-step protocol for calculating 13C chemical shifts for moderate-size flexible organic molecules was developpled. The protocol features a series of an initial molecular mechanics step to identify all reasonable low-energy conformers, a series of quantum chemical steps of increasing complexity to estimate Boltzmann weights and density functional calculations to obtain 13C spectra for all contributing conformers.
This protocol was first applied to approximately 700 diverse natural products (MW: 140-930, average 360) established based on the X-ray crystallographic analyses to give small the RMS errors (average 2.0 ppm, max 4 ppm), demonstrating its high reliability. It was then applied to the other 1000 natural products of which structures were determined without X-ray crystallographic analyses (MW: 140-620, average 320). The obtained 13C shifts for the majority of these compounds were within agreement (RMS ＜4.0 ppm), as expected. However, the RMS errors for 〜10% of the natural products were substantially larger than those to allow. These were reinvestigated to propose the alternate structures in some cases. The suggested new structures afford agreeable RMS errors.
The protocol has been fully automated inside the Spartan molecular modeling program, and can be executed by general desktop computers. So, it would support or challenge NMR structure assignments for many natural product chemists.
Structure elucidation of natural products is a still challenging field because of their scarcity that prevents chemists from fully accessing to analytical experiments especially to X-ray diffraction analysis. In 2013, we reported an X-ray technique, the crystalline sponge method, that does not require the crystallization of samples.
Here, we introduce our recent results on the absolute configuration determination of noncrystalline
natural products using the NMR-coupled crystalline sponge method. Combining ‘de novo’ information of XRD study with that of NMR study resulted in elucidating the complete structure of an unknown natural product much faster and more reliably than conventional tandem studies. We also show the structure determination of a major component in crude extracts from natural source. These results are expected to accelerate a structural elucidation of natural products and provide novel scaffolds, which are useful for the natural product-derived drug discovery research.
Marine sponges are known to be rich sources of biologically active and structurally
unique compounds. A large number of marine natural products have been discovered
from the order of Dictyoceratida, which is well known as a rich source of structurally
highly diverse terpenes including sesterterpenes, norsesterterpenes, sesquiterpene
quinones, and furan-containing terpenes. In our search for the novel metabolites from
marine sponges, four new sesquiterpenes (1–4) together with a known sesquiterpene,
O-methyl nakafuran-8 lactone (5), were obtained from the marine sponge, Lamellodysidea herbacea, collected in Indonesia. Their planar structures were elucidated by analyses of spectroscopic data. Compounds 1 and 2 were structurally unique bridged polycyclic furanosesquiterpenes. Although several polycyclic furanosesquiterpenes were isolated from the marine sponges to date, the skeletons of 1 and 2 were the first compounds containing the unprecedented frameworks. Compounds 3 and 4 were obtained as an inseparable mixture in the ratio of 1:1 and shown to be 11-epimers. We determined the absolute configurations of 1–4 by interpretation of the calculated ECD spectra using the additivity relations of chromophores. The biosynthesis of 1–5 from farnesyl pyrophosphate were proposed.
Neomacrophorin X (1) was isolated from Trichoderma sp. 1212-03. The HMBC spectral analysis indicated a unique [4.4.3]propellane framework, which was verified by the 1H and 13C chemical shift calculations based on DFT and subsequent comparison with experimental data obtained in CDCl3. The DFT-based ECD calculations were effective not only in determining the absolute configuration, but also confirming the relative structure. The predominant conformation of 1 was found to be solvent-dependent, with different conformations presenting different NMR and ECD profiles. Introduction of J-based analysis with a J-resolved HMBC aided in this investigation. This conformational alternation was reproduced by considering the solvation with the SM5.4 model in the calculation, although it was not sufficiently quantitative. Although the calculations without solvent effects suggested a conformer which satisfies the spectral profiles in CDCl3, post calculations with SM5.4 solvation protocol stabilized the second major conformer which reproduces the NMR and ECD profiles in CD3OD (CH3OH). Neomacrophorin X (1) is assumed to be biosynthesized by a coupling between the reduced form of anthraquinone and a neomacrophorin derivative. This hypothesis was supported experimentally by the isolation of pachybasin and chrysophanol, as well as acyclic premacrophorin (2) from the same fungus.
In the past, through enantioselective total synthesis, our laboratory has found that isodehydrothyrsiferol (5), a marine squalene-derived triterpene polyether isolated from the red alga Laurencia viridis, shows partial enantiodivergency in that six asymmetric centers in the ABC ring system (a dioxabicyclo[4.4.0]decane ring system with an attached bromine-containing tetrahydropyranyl ring that forms the core structure of the triterpene polyethers produced from the genus Laurencia) are enantiomeric to those of other members of the thyrsiferol family.1) In this presentation, our laboratory performed the total syntheis of aplysiol B and 22-hydroxy-15(28)-dehydrovenustatriol whose absolute configurations have never been determined to research the partial enantiodivergency in the thyrsiferol family in depth.
Aplysiol B, a member of the thyrsiferol family isolated from the sea hare Aplysia dactylomela, possesses feeding-deterrent and ichthyotoxic properties. However, the proposed structures 6a2) and 6b3) were in contradiction to the biogenetic hypothesis. Therefore, we reconsidered the biogenetic pathway of aplysiol B and synthesized the reasonable structure 6c through a key Shi epoxidation12) followed by a 5-exo cyclization and a subsequent 6-endo bromoetherification using BDSB.13) The spectral data and the optical rotation of synthetic 6c were in agreement with those reported for the natural sample.2) As a result, the first total synthesis of aplysiol B was accomplished, and the reported structures 6a and 6b were revised to 6c.
The planar structure of 22-hydroxy-15(28)-dehydrovenuatatriol was determined by NMR analysis.6) The stereostructure of the ABC ring system was elucidated by comparing the NMR data with those of dehydrothyrsiferol (4), whose absolute structure was known. However, the stereochemical relationship between the ABC ring system and D ring due to the intervening methylene chain and the absolute configuration has not been determined to date. Our laboratory synthesized the proposed structure 8a via a key Suzuki-Miyaura cross-coupling between the BC ring system 19 and D ring 20. However, the NMR spectra of synthetic 8a did not match with those of the reported data.6) We also synthesized 8b, a possible diastereomer of 8a, and the proposed structure 8a was revised to 8b. Moreover, we observed that the ABC ring system of 8b has the same absolute configuration as that of isodehydrothyrsiferol (5).
Considering an enantiodivergent phenomenon in the common skeleton of the thyrsiferol family, based on the biogenesis of the squalene-derived thyrsiferol family suggested by the Fernández group,4) we propose the biogenesis of 6c and 8b via the bromocation-initiated epoxide-opening cascade reaction of squalene pentaepoxide 37.
Maitotoxin (MTX) is a ladder-shaped polycyclic ether produced by the epiphytic dinoflagellate Gambierdiscus toxicus. MTX elicits potent Ca2+ influx activity. During the course of our structure-activity relationship studies of MTX by using chemically-synthesized model compounds corresponding to its partial structures, the C’D’E’F’ and WXYZA’B’C’ ring system have inhibited the MTX-induced Ca2+ influx (IC50 59 and 30 μM, respectively). Herein, we report synthesis and bioactivity other partial structures of MTX.
We synthesized the hydrophobic part corresponding to the QRS ring system, and found that
it elicited inhibitory activity (IC50 44 μM) hydrophobic comparable to above two partial structures.
In order to explore the importance of the hydrophilic region, not only the LMNO ring but also its enantiomer were synthesized. They showed only weak inhibitory activity and there was no significant difference between the enantiomers.
The amphiphilicity is one of the characteristic properties of MTX. Therefore, we synthesized the NOPQRS ring system corresponding to the border part of hydrophobic and hydrophilic region of MTX. It is interesting to note that the compound elicited no inhibitory activity, suggesting that presence of both hydrophobic and hydrophilic parts influenced the biological activity in contradictory manners
Maitotoxin (MTX, 1) is the largest (MW 3422) and most toxic natural product so far discovered; it was isolated from the dinoflagellate Gambierdiscus toxicus.1 It is involved in ciguatera fish poisoning, and induces cell death by activating a variety of Ca2+ channel in various cell types. The structure of MTX consists of 98 chiral centers, 32 cyclic ether rings, 21 methyl groups, and 2 sulfate groups (only the hydrophobic part of MTX are shown in Figure 1). The characteristic structure and bioactivity of MTX are interested by synthetic organic chemists, and so far several fragments of MTX have been synthesized by Nicolaou’s,2a-e Oishi’s,2f-h and our groups.2i-k
We have already reported stereoselective syntheses of the WXYZA’-ring2k and the C’D’E’F’-ring2j having a side chain. In this paper, we accomplished 1) a convergent synthesis of the RSTUV-ring system 4 via coupling of RS-ring carboxylic acid 6 and V-ring alcohol 5, an intramolecular Barbier reaction3 of the resulting iodo ester 7 with n-BuLi, and insertion of Me group to hemithioacetal 12, 2) a linear synthesis of the UVWXYZ-ring system 3 through 6-endo cyclization of vinyl epoxide,7 and SmI2-mediated reductive cyclization of aldehyde and β-alkoxy acrylate4 or β-alkoxy sulfoxide,5 and unique methyl insertion reaction,8 and 3) a convergent synthesis of the ent-ZA’B’C’D’-ring system 2 via the Suzuki-Miyaura cross coupling reaction7 of (Z)-vinyl iodide 25 with alkylborane devived from 26, hemithioacetal B’-ring formation, and reduction of the resulting hemithioacetal.9
Aflastatin A was isolated from the culture broth of Streptomyces sp. MRI142 by Sakuda and co-workers in 1996 (Figure 1). This natural product exhibits an inhibitory activity against the biosynthesis of carcinogen aflatoxin produced by a part of Aspergillus sp. The highly oxygenated structure of aflastatin A contains various typical polyketide structures including polyacetate, polypropionate, deoxypropionate and contiguous polyol motifs. We selected aflastatin A as a synthetic target in order to develop the efficient synthesis of these various polyketide motifs.
In the synthesis of the C22-C48 polyol segment, the contiguous polyol motif such as C27-C31 moiety would be efficiently synthesized by the aldol reaction. We examined the aldol reaction between aldehyde 7 derived from D-mannose and ketone 18 derived from L-tartric acid (Figure 4). In consequence, addition of SnCl2 gave the desired aldol adduct 19 in high stereoselectivity and we achieved the efficient synthesis of C22-C48 segment 21.
In the synthesis of the C3-C21 segment including deoxypropionate and propionate structures, we constructed these compounds by transformation of olefins of products 22, 29 and 33 synthesized from vinylketene silyl N,O-acetal 22 and ent-22 according to our previous studies on the remote asymmetric introduction-type alkylation and the aldol reaction (Figure 6 to 8). Streoselective
Birch reduction with a compound 23 was achieved by using 2-methyl -benzimidazole as a bulky proton to give the C4-C7 deoxypropionate 24 in only 2 steps. Appropriate oxidations of olefins of compound 29 such as regioselective epoxidation following hydroboration gave propionate 32, the C9-C15 moiety of aflastatin A. Coupling reactions including the Julia-Kocienski reaction between
C3-C8 and C9-C15 segment and the aldol reaction between C3-C15 and C16-C21 segment gave C3-C21 segment 40 (Figure 9).
In 2009, we isolated biselyngbyaside (BLS, 1), an 18-membered macrolide glycoside, from marine cyanobacterium Lyngbya sp. collected in Okinawa. BLS and its aglycone biselyngbyolide B (BLLB, 2) show growth-inhibitory activity against HeLa and HL60 cells. In addition, 1 inhibited RANKL-induced osteoclastgenesis and induced apoptosis of mature osteoclasts at a low concentration. In a recent study, we clarified that 1 and 2 strongly inhibited the ATPase activites of SERCA1a and 2a, and determined the X-ray crystal structure of 1 and 2 with SERCA1a. The synthetic studies of biselyngbyasides were reported by several groups. And we reported the total synthesis of biselyngbyolide A. In this conference, we will report about the first total synthesis of biselyngbyolide B and biselyngbyaside.
At first, we achieved the total synthesis of BLLB. We prepared the vinyl iodide 5 from 1,3-propanediol and (R)-stannene 6 from glycidol derivartive. The two segments were connected by esterification reaction using Shiina reagent and the 18-membered ring structure was constructed by intramolecular Stille coupling reaction. Finally, TBS group was removed to give BLLB.
To synthesize biselyngbyaside, we tried the direct glycosylation reaction from BLLB. However, even with the use of various conditions for the glycosylation reaction the glycoside bond could not been constructed. So, we introduced the sugar moiety before the connection of vinyl iodide 5 and stannane 6. TBS group of vinyl iodide 5 was removed followed by glycosylation reaction to give glycoside 27. Glycoside 27 was converted to carboxylic acid 30 in 5 steps. Carboxylic acid 30 and stannane 6 were connected using Mitsunobu reaction, though Shiina esterification reaction gave no desired compound. Finally, intramolecular Stille coupling reaction and removal of TES groups afforded BLS.
Aplyronine A (1) is a 24-membered macrolide isolated from sea hare by our group. Aplyronine A (1) shows cell growth inhibition, anticancer, and actin depolymerizing activities. Our previous works disclosed the novel action mechanism that the side chain part of aplyronine A binds actin, and then, the trimethylserine group interacts with tubulin α,β-heterodimer to form a ternary complex. Now, we investigate structure-activity relationship of aplyronine A for development of new anticancer drugs. We have already succeeded in total synthesis of aplyronine A, however, it required 86 steps. Thus, we designed a simpler analog, aplyronine A - swinholide A hybrid compound 3, which consists of the macrolactone part of aplyronine A and the side chain part of swinholide A (2), an actin depolymerizing dimeric macrolide with a structurally simpler side chain.
Our synthetic strategy contains esterification between C1-C19 segment 5 and C20-C34 segment 6, followed by intramolecular NHK coupling for construction of macrocycle. Finally, we synthesized hybrid compound 3 in 70 steps (vs 86 steps for aplyronine A). Hybrid compound 3 was found to show a potent cell growth inhibitory activity, revealing the importance of combination of macrocycle and side chain parts for potent bioactivity.
Lipopolysaccharides (LPS) are the major glycoconjugates in outer membrane of Gram-negative bacteria and act as potent stimulators of innate immunity. The active principle of LPS is lipid A, the terminal moiety of LPS.
Alcaligenes sp. have been known as opportunistic pathogens. Kiyono et al. showed that Alcaligenes faecalis inhabit human Peyer’s patches. Because Peyer’s patches play the important role to regulate the gut immunity, it is suggested that A. faecalis be associated with the regulation of the gut immunity.
In this study, we isolated LPS from A. faecalis and found that its LPS is lipooligosaccharide (LOS) with short oligosaccharide compared to general LPS. We also identified the chemical structure of LOS and revealed that this LOS is a novel compound consisting of nona-saccharide and multiple fatty acids. Furthermore, we synthesized A. faecalis lipid A from D-glucosamine hydrochloride (2) via key intermediate disaccharide 3. After the acylation of disaccharide intermediate, two phosphate groups were simultaneously introduced into 14, thus 15 was constructed efficiently. All protective groups were removed to accomplish the synthesis of A. faecalis lipid A 1a.
Introduction Phenolic glycolipids 1 and 2 (PGLs) are abundant in the cell envelope of Mycobacteria fungi, and are composed of a partially O-methylated oligosaccharide and a polyketide-derived diacylated lipid1) (Figure 1). In a previous investigation, the sugar part of PGLs appeared to possess immunomodulatory effect via inhibition of proinflammatory cytokine release2). However, mechanism of their biological effects and precise structure-activity relationships are not still clear. Therefore, we aimed to develop an effective method for the synthesis of partially O-methylated glycosides varying at the lipid part and evaluate their biological activity for understanding the mode of action.
Results Scheme 1 shows our strategy for synthesis of the chemical probes 3. The chemical probe 3 was synthesized in one-pot from the sugar part 4, the cyclic borane 5 and the functional part 6 by a sequential Suzuki-Miyaura coupling.3) The naphthyl group 6a would be a mimic of the lipid part. The photoaffinity labeling unit 6b and the fluorescent dye 6c would assist to identify the target receptor. Formation of 2-O-methyl-α-rhamnosides and fucosides would be a key step in the synthesis of PGL sugar parts. O-methylation of the C2 hydroxy group makes neighboring group participation promoting 1,2-trans glycosidic linakge not to be available. We found that glycosidation of glycosyl imidate 7b with I2 and Bu4N·OTf provided disaccharide 9 in good yield with excellent α-selectivity. The glycosidation method allowed one to synthesize the PGL-1 sugar part (4a) and the PGL-tb1 sugar part (4b) in a straight forward manner. We next developed the aforesaid Suzuki-Miyaura coupling (Scheme 5). The glycosides 4a were converted to the chemical probes 3a-c via a sequential Suzuki-Miyaura coupling using the cyclic boranes. The generated borinic acid 28 was inert at 60 °C and underwent next coupling reaction at higher reaction temperature to provide the asymmetrical functionalized alkanes in one-pot. Finally, immunomodulatory effect of PGL analogues on bone-marrow-derived macrophages stimulated with trehalose-6,6’-dimycolate (TDM) was evaluated (Figure 2). The PGL-tb1 disaccharide sugar part-derived glycolipid Ea possessed a strongest activity among the PGL-tb1 related glycosides. Fluorescent chemical probes Ac-Cc also showed comparable activity with glycolipid Aa-Ac.
Conclusion In this study, we developed direct and stereoselective glycosidation and sequential Suzuki-Miyaura coupling using cyclic boranes. We successfully synthesized the PGL sugar parts 4 and the chemical probes 3 by a sequential Suzuki-Miyaura coupling. We expected that the synthesized chemical probes would strongly asset to elucidate precise biological information of the PGL-promoting biological activity.
Under metal-free conditions, propargyl esters are stable functional groups that typically lack the electron-withdrawing inductive effects needed to participate in nucleophilic acyl substitution reactions. Herein we report an unusual observation where glycine propargyl ester derivatives were found to selectively form amide bonds with a series of linear alkylamines under mild, aqueous conditions in a base-independent manner. Through global reaction route mapping (GRRM) modeling calculations, it is speculated that reactivity may be primarily driven via hydrogen-bonding and intermolecular interactions, rather than base catalysis.
As a proof-of-concept, a site-specific C-terminal glycine peptide bioconjugation technique was designed and developed. The design of this approach relies on the selective reactivity of C-terminal glycine propargyl esters over that of aspartate and glutamate side chain-linked propargyl esters. Several small, unprotected peptides were successfully conjugated, which shows the potential for adaptation towards protein/peptide bioconjugation.
In another application, glycine propargyl ester probes were investigated for their ability to selectively react with polyamines over other biological amines (ex/ amino acids/proteins and monoamine neurotransmitters). Due to higher intracellular concentrations of polyamines within rapidly-growing cancer cells, in some cases by as much as twofold compared to nomal tissues, this work could provide the potential framework in cancer targeting therapeutic applications. Kinetic and cell-based experiments shown in this presentation hope to highlight the viability of htis approach.
Antibody-drug conjugates (ADCs) are used to deliver cytotoxic agents to tumor sites.
These conjugates, which leak out from neovascular vessels via the enhanced permeability
and retention (EPR) effect, bind to cell surface antigens and are internalized, thereby
releasing their cytotoxic agents site-specifically. Conjugation sites and drug-to-antibody
ratios influence the efficacy of ADCs; therefore, methods that allow homogeneous ADC
preparation are required. While conjugation site and drug-to-antibody ratios affect the
efficacy of ADCs, non-human type N-glycan structures are known to be immunogenic. We
will solve these two problems by conjugating drugs via a conserved N-glycan at Asn297.
The scheme is as follows: 1) Heterogeneous N-glycan on antibody was cleaved by endoS;
2) Human-type N-glycan from egg yolk was modified by azide addition and oxazoline
formation: 3) The modified N-glycan was added to the antibody carrying enzymatically
truncated N-glycan by endoS D233Q: 4) Drug carrying linker was conjugated via Huisgen
Reaction progress was monitored by HILIC column chromatography without enzymatic
digestion of the antibody. Using the above scheme, a homogeneous ADC was prepared.
Homogeneity was quantitated by mass spectroscopy after proteolytic digestion of the
The anti-tumor tetrahydroisoquinoline (THIQ) alkaloids, represented by
saframycin A (1), jorunnamycin A (2), renieramycin M (3) and ecteinascidin 743
(4), share a common pentacyclic scaffold that is biosynthesized by non-ribosomal
peptide synthetases (NRPS). We previously revealed unique biosynthetic
conversions catalyzed by SfmC, a module of NRPS, to forge the pentacyclic
scaffold. Two molecules of tyrosine derivative and a peptidyl aldehyde bearing a
cryptic fatty acyl chain are assembled by SfmC via iterative Pictet-Spengler
reactions followed by reduction of thioesters to liberate aldehyde intermediates.
Herein we report the chemo-enzymatic total synthesis of 2 by merging precise
chemical synthesis with in vitro engineered biotransformation. By optimizing
designer substrates compatible with SfmC through chemical synthesis, we
succeeded in efficient assembly of the appropriately functionalized pentacyclic
skeleton by streamlining the linkage between SfmC-catalyzed multi-step enzymatic
conversions and chemical manipulations of the intermediates to install amino
nitrile and N-methyl groups. This approach allowed very rapid access to the
elaborated pentacyclic skeleton in a single day starting from two simple synthetic
substrates (7, 15) without isolation of the intermediates. Further functional group
manipulations involving hydrolysis and oxidation allowed operationally simple and
expeditious synthesis of jorunnamycin A (2) that could be a versatile and common
precursor for the artificial production of other anti-tumor THIQ alkaloids and their
Sesame (Sesamum indicum) is a major oil crop rich in (+)-sesamin, (+)-sesamolin,
(+)-sesaminol and other lignans that are phenylpropanoid-derived specialized metabolites.
Despite the value of lignans for their health-promoting properties, the biosynthesis of
(+)-sesamolin and (+)-sesaminol remained elusive. A genome-based genetic approach
identified that the deletion of four C-terminal amino acids (Del4C) in a P450 enzyme
CYP92B14 resulted in low (+)-sesamolin content in sesame seeds. Enzyme assays
confirmed that recombinant CYP92B14, but not Del4C, biosynthesized (+)-sesamolin from
(+)-sesamin through oxidative rearrangement of α-oxy-substituted aryl groups. Unexpectedly,
CYP92B14 also converted (+)-sesamin to (+)-sesaminol in vitro. Furthermore, conversion of
(+)-sesamin into (+)-sesaminol and (+)-sesamolin by CYP92B14 was enhanced when
co-expressed with sesamin synthase CYP81Q1, implying functional coordination of
CYP81Q1 with CYP92B14. The discovery of CYP92B14 not only uncovers the last steps in
sesame lignan biosynthesis but highlights the remarkable catalytic plasticity of P450s that
contributes to metabolic diversity in nature.
Rice produces an array of labdane-related diterpenoids that function as phytoalexins or/and
allelochemicals. The biochemical function of the rice diterpene synthases has been
elucidated. The downstream oxygenation reactions have been proposed to be catalyzed by
cytochromes P450 (CYPs), with subsequent further oxidation catalyzed by short-chain
alcohol dehydrogenases/reductases (SDRs). In this study, we report the CYP and SDR
activities which propose the biosynthetic pathway of Oryzalexins and Momilactone A (13).
SDRs induced by the elicitor chitin were assayed with the rice diterpenoid biosynthetic
intermediates. Two of these SDRs clearly appear to act in oryzalexin biosynthesis, with
OsSDR110C-MI3 readily oxidizing the 3α-hydroxyl of Oryzalexin D (8), while
OsSDR110C-MS3 can also oxidize the accompanying 7β-hydroxyl.
Momilactone A (13) is a rice diterpenoid which is produced from syn-pimaradiene (14).
CYP76M8, CYP99A2, CYP99A3, and CYP701A8 have been reported to react with
syn-pimaradiene. In addition to these CYPs, an SDR catalyzing the final step in production
of Momilactone A (13) has been identified. However, the order in which the CYPs react has
been unclear. This is evaluated here using our Escherichia coli metabolic engineering
system and in vitro enzyme assays. Various pairs of CYPs were co-expressed with the
requisite reductase, in E. coli also engineered to produce syn-pimaradiene (14).
syn-Pimaradiene-19,6β-hemiacetal (20) was detected in E. coli culture co-expressing
CYP99A3 and CYP76M8, and syn-pimaradiene-3β,6β-diol (16) was detected in E. coli culture co-expressing CYP701A8 and CYP76M8. Moreover, syn-pimaradiene-19,6β-hemiacetal (20) could be further oxidized to the corresponding syn-pimaradien-19,6β-olide (21) by OsSDR110C-MS1. It also was found that CYP701A8 was able to oxidize syn-pimaradien-19,6β-olide (21) to Momilactone A (13).
Bifunctional terpene synthases (BFTSs) are attractive target for genome mining because
they yield di/sesterterpenes with unique polycyclic structures. Bioinformatics analysis of
N-terminal terpene cyclase domains of BFTSs allowed us to categorize them into 6 clades.
Interestingly, all functionally characterized BFTSs, such as PaFS, PaPS, and AcOS, were
classified into Clade B. Taken that those BFTSs catalyze cyclization via carbocation
intermediate with 5-11 bicyclic ring system, it was proposed that the amino acid sequence
reflects the initial cyclization mode, which is most likely related to the initial conformation
of a linear prenyl diphosphate. In this study, we examined genome mining focusing on
BFTSs classified into Clade A.
Heterologous expression of five BFTS genes into Aspergillus oryzae or Escherichia coli allowed us to isolate structurally related compounds, three sesterterpene hydrocarbons and two sesterterpene alcohols. Computational analysis of the cyclization mechanism of NfSS,
which produced a most elaborated sesterterpene alcohol, revealed two kinetically and
thermodynamically favorable pathways. The cyclization mechanism was supported by the
analysis of in vivo and in vitro enzymatic reactions with isotopically labeled precursors.
Interestingly, the structures of carbocation intermediates proposed in one of the computed
pathway were in good agreement with those of isolated compounds, suggesting that
functionally characterized BFTSs follow the cyclization mechanism. Additionally,
co-expression of those BFTSs with modification enzyme genes enabled us to isolate
Steroids, with a tetracyclic molecular framework, have versatile, strong and important
biological activities. Given their importance in medicine and biology, the efficient
synthesis of steroid is still an important subject in synthetic organic chemistry in spite of the
many previous synthetic studies.
Recently, we proposed the term “pot economy”, since one-pot operations constitute an
effective method both for carrying out several transformations and forming several bonds in
a single pot, while at the same time eliminating several purification steps, minimizing
chemical waste generation, and saving time. Based on this concept, we have reported a
one-pot synthesis of (-)-oseltamivir and a three-pots synthesis of PGE1 methyl ester.
We have accomplished a total synthesis of estradiol methyl ether (6) via five pots using
organocatalyst as follows: One of the key reactions is a domino reaction of diphenylprolinol
silyl ether 1 mediated asymmetric Michael reaction between nitroalkane 2 and enal 3, and
intramolecular aldol reaction, which afforded bicyclo[4.3.0]nonane derivative 5, possessing
A, C, and D rings of steroid with excellent diastereo- and enantioselectivities. The
strereochemistry of 5 was found to be identical to that of the steroid.
After formation of 5, cyanohydrine formation, protection by xanthate and dehydration
afforded compound 10 in the first pot. Compound 10 has been contained all carbons for
estradiol methyl ether. Then, nitro group and xanthate moiety was reduced in one-pot to
afford 12. The ketone and nitrile moiety of 12 was reduced and protected by silyl ether to
afford 18. Then, next six reactions can be conducted sequentially in a single vessel:
Pinnick-Kraus oxidation, diastereoselective hydrogenation, acid chloride formation,
Friedel-Crafts acylation, deprotection of silyl ether and hydrogenolysis of benzylketone
afforded estradiol methyl ether (6). This synthesis is fewest pot synthesis of estradiol
Frondosin A (1), isolated from the marine sponge Dysidea frondosa by Freyer and
co-workers,1 is a member of a family of five norsesquiterpenoid natural products. Frondosin A (1) was found to inhibit the binding of the interleukin-8 and exhibit anti-HIV activity.
Recently, we reported that Rh2(S-TCPTTL)4 (5) is a highly effective catalyst for enantioselective tandem carbonyl ylide formation-cyclization reactions of
α-diazo-β-ketoester with arylallene dipolarophiles, providing cycloadducts in good to high
yields and with enantioselectivities of up to 99% ee as well as with perfect exo
diastereoselectibity. In conjunction with our continuing interest in the carbonyl ylide
cycloaddition strategy for the synthesis of natural products, we herein report catalytic
asymmetric total synthesis of frondosin A (1) by exploiting Rh(II)-catalyzed intermolecular
1,3-dipolar cycloaddition as one of the key steps.
The 1,3-dipolar cycloaddition of carbonyl ylide derived derived from α-diazo-β-ketoester 2a with 2 equiv of 2,5-dimethoxyphenylallene (3a) using 1 mol % of Rh2(S-TCPTTL)4 (5) afforded the exo-cycloadduct 4a in 78% yield with 82% ee. Reductive cleavage of the ether-bridge in iodoalcohol followed by protection of the primary alcohol as its TIPS ether and treatment of tertiary alcohol with LiHMDS and methyl chloroformate furnished allylic carbonate 17.
Palladium-catalyzed regio- and stereoselective formate reduction of 17 provided 18 as a
major product, which was transformed to allyl alcohol 9. 1,3-Transposition of primary
allylic alcohol of 9 followed by oxidation gave enone 8, which was further converted to enol
triflate 7 by 1,4-addition of alkylcopper reagent and subsequent triflation. Reductive Heck
cyclization of 7 using Pd(OAc)2 and formic acid in the presence of diisopropylamine in N-methylpyrrolidone provided frondosin dimethyl ether (6) in good yield. Finally, oxidation
of 6 and subsequent reduction of the corresponding quinone with zinc in acetic acid
completed the total synthesis of frondosin A (1).
Callophycoic acid A (1) was isolated by Kubanek and co-workers in 2007 from
extracts of the red alga Callophycus serratus (Solieriaceae), which was collected near
Cakau-i-Ra Reef, Fiji. This natural product is the first example of diterpene–benzoic acid in
macroalgae. The structure is characterized by a brominated tricyclic skeleton containing an
all-carbon quaternary stereocenter. It showed antibacterial activity against
vancomycin-resistant Enterococcus faecium as well as antimalarial activity and cytotoxicity
against human cancer cell lines. Interested in these features, we have been involved in
synthetic studies of 1.
In order to develop a method for construction of the all-carbon quaternary stereocenter
in 1, we examined allylboration reaction of sugar-derived aldehydes. The reaction of
D-glyceraldehyde derivative 2 or D-erythrose derivative 5 with geranylboronate 3 proceeded
diastereoselectively, giving the γ-adduct possessing a quaternary stereocenter. This method
was applied to the stereoselective synthesis of homoallylic alcohol 9-D from D-xylose
derivative 7. By using boronate 8 instead of 3, higher diastereoselectivity was achieved. The
γ-adduct 9 was converted into disubstituted olefin 12, which possesses the benzoic acid
moiety. Then 12 was transformed to alkenyl bromides 19 and 20, and radical cyclizations
were attempted to construct the cyclohexane ring. When triacetate 20 was used as the
substrate, the desired cyclized product 21-a was preferentially obtained.