Synthesis and Stereochemical Revision of the C31–C67 Fragment of Amphidinol 3

Article abstract of DOI:10.1002/anie.201712167

Amphidinol 3 (AM3) is a marine natural product produced by the dinoflagellate Amphidinium klebsii. Although the absolute configuration of AM3 was determined in 1999 by extensive NMR analysis and degradation of the natural product, it was a daunting task because of the presence of numerous stereogenic centers on the acyclic carbon chain and the limited availability from natural sources. Thereafter, revisions of the absolute configurations at C2 and C51 were reported in 2008 and 2013, respectively. Reported herein is the revised absolute configuration of AM3: 32S, 33R, 34S, 35S, 36S, and 38S based on the chemical synthesis of partial structures corresponding to the C31–C67 fragment of AM3 in combination with degradation of the natural product. The revised structure is unique in that both antipodal tetrahydropyran counterparts exist on a single carbon chain. The structural revision of AM3 may affect proposed structures of congeners related to the amphidinols.

Full text of DOI:10.1002/anie.201712167

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Title: Synthesis and Stereochemical Revision of the C31-C67 Section
of Amphidinol 3
Authors: Yuma Wakamiya, Makoto Ebine, Mariko Murayama, Hiroyuki
Omizu, Nobuaki Matsumori, Michio Murata, and Tohru Oishi
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10.1002/anie.201712167
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Synthesis and Stereochemical Revision of the C31–C67 Section of
Amphidinol 3
Yuma Wakamiya, Makoto Ebine, Mariko Murayama, Hiroyuki Omizu, Nobuaki Matsumori, Michio Murata, and
Tohru Oishi*
Abstract: Amphidinol 3 (AM3) is a marine natural product produced by the
dinoflagellate Amphidinium klebsii. Although the absolute configuration of
AM3 was determined in 1999 by extensive NMR analysis and degradation
of the natural product, it was a daunting task due to the presence of
numerous stereogenic centers on the acyclic carbon chain and the limited
availability from natural sources. Thereafter, revisions of the absolute
configuration at C38–C39 was determined by the JBCA method, the
observed key J value for C38–C39 was in the medium range (i.e.,
3JH38,H39 = 5.1 Hz). If the assignment of the absolute configuration at
C38 of R determined by the JBCA method is wrong, the absolute
configurations at C32, C33, C34, C35, and C36 should be also
incorrect. Therefore, it is important to confirm the absolute
configurations at C2 and C51 were reported in 2008 and 2013, respectively. configuration at C38. The similar situation was observed for C36–C37
Herein, we revised the absolute configuration of AM3 to be 32S, 33R, 34S,
35S, 36S, and 38S based on the chemical synthesis of partial structures
corresponding to the C31–C67 section of AM3 in combination with
degradation of the natural product. The revised structure is unique in that
both antipodal tetrahydropyran counterparts exist on a single carbon chain.
The structural revision of AM3 may affect proposed structures of congeners
related to the amphidinols.
of KmTx2, and the proposed structure was supported by DP4 chemical
shift analysis.4g Herein, we report the chemical synthesis of the partial
structures of AM3, degradation of the natural product and synthetic
intermediates, and chemical correlation of these compounds to confirm
the absolute configuration of AM3.
Amphidinol 3 (AM3) is a marine natural product produced by the
dinoflagellate Amphidinium klebsii.1 AM3 elicits potent antifungal and
hemolytic activities, while its mode of action is not fully understood.2
The planar structure of AM3 was reported in 1991,1a however,
determination of the absolute configuration was a daunting task due to
the limited availability of the natural product and the presence of a
number of stereogenic centers located on the acyclic carbon chain. It
was finally achieved in 1999 with the aid of extensive NMR analysis
including the J-based configuration analysis (JBCA) method3 (Figure
1a).1b To date, a number of congeners of amphidinols and related
compounds,4 have been identified. Among these congeners, the
structure of karlotoxin-2 (KmTx2), reported in 20104f and revised in
2015,4g is interesting in that the THP rings are reported to be antipodal
to those of AM3 (Figure 1c).
Because the absolute configuration of the B-ring of KmTx2 is
antipodal to that of AM3, we have confirmed the absolute
configuration of the B-ring of AM3 to be correct by degradation of the
natural product and chemical correlation.5c On the other hand, the
absolute configurations at C2 and C51 were revised to be R and S,
respectively, by chemical syntheses of partial structures and chemical
correlation (Figure 1b).5a,d The reason for the misassignment at C51
was the difficulty in applying the JBCA method because the key J
Figure 1. (a) Originally proposed structure of amphidinol 3
(AM3) in 1999. (b) Structure of AM3 revised in 2013. (c)
Structure of karlotoxin 2 (KmTx2) revised in 2015.
The unique structural features of AM3, represented by the
presence of a long hydrophilic polyol chain, highly substituted
tetrahydropyran ring systems, and a hydrophobic polyene unit, have
attracted considerable attention in the synthetic community.7,8
Although pioneering synthetic studies of AM3 including the
tetrahydropyran ring and the polyene unit, the C31–C67 section by
Rychnovsky8f and the C43–C67 section by Roush8d,e and Paquette8i
have been reported, all of them were based on the originally proposed
structure reported in 1999. Therefore, there was no argument regarding
the stereochemistry of AM3 except for the synthetic study of the C1–
C31 polyol part by Evans8l based on the revised structure in 2013.
We planned to confirm the absolute configuration at C38 of
AM3 by comparing the NMR data between natural product and the
synthetic model compounds 1a or 1b (Figure 2). The C31–C67 section
1a corresponds to the revised structure of AM3 in 2013, whereas 1b
has an opposite stereochemistry at C32–C38. Both 1a and 1b would be
synthesized via alkenyllithium–aldehyde coupling8f,j between the B-
3
value was in the medium range (i.e., JH50,H51 = 3.4 Hz). A similar
situation was also observed for the assignment of the absolute
configuration at C38, that is, although the absolute configuration at
C39 was determined by the modified Mosher method6 and the relative
[*]
Y. Wakamiya, Dr. M. Ebine, M. Murayama, H. Omizu, Prof. Dr. N.
Matsumori, Prof. Dr. T. Oishi,
Department of Chemistry, Faculty and Graduate School of Science,
Kyushu University
744 Motooka, Nishi-ku, Fukuoka 819-0395 (Japan)
E-mail: oishi@chem.kyushu-univ.jp
Prof. Dr. M. Murata
Department of Chemistry, Graduate School of Science,
Osaka University
1-1 Machikeneyama, Toyonaka, Osaka 560-0043 (Japan)
Supporting information and the ORCID identification number(s) for
the author(s) of this article can be found under:
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10.1002/anie.201712167
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ring and the A-ring or its enantiomer with inversion of C39, followed
by Julia–Kocienski olefination,9 respectively.
Figure 2. Structures and synthetic plan of the model compounds
1a and 1b.
The synthesis of the C31–C67 part (1a) was achieved as shown
in Scheme 1. Protecting group manipulation of the known compound
25d (86%, 3 steps) afforded primary alcohol 3. Dess–Martin oxidation
followed by Horner–Wadsworth–Emmons olefination under the
Masamune–Roush protocol10 using phosphonate 4 afforded an α,β-
unsaturated ketone, which was hydrogenated to furnish saturated
methyl ketone 5 (81%, 3 steps). The ketone 5 was converted to
alkenyliodide 7 (70%, 3 steps) via enol triflation8f with Comins reagent
6,11 Stille coupling of the triflate with Me3SnSnMe3, and subsequent
tin-iodine exchange reaction. Iodide 7 was treated with t-BuLi to form
alkenyllithium 8, which was then coupled with aldehyde 912 to afford
secondary alcohol 10 with concomitant formation of the C43-epimer
1113 in a 2.2:1 ratio (61%, based on 7). After separation and protecting
group manipulation, the resulting primary alcohol 13 was oxidized to
an aldehyde and coupled with sulfone 145f by Julia–Kocienski
olefination.8f,i Global deprotection with HF·pyridine afforded the C31–
C67 part 1a as a single E-isomer (62%, 3 steps).
Next, we turned our attention to the synthesis of the diastereomer
1b as shown in Scheme 2. The secondary alcohol 1512 was converted to
mesylate (96%). Removal of the Bn group (88%) followed by
intramolecular SN2 reaction by treatment with K2CO3 afforded epoxide
16 (78%) with inversion of stereochemistry at C39. Nucleophilic ring-
opening of 16 by dilithium reagent 1714 furnished alcohol 18 (85%).8e,j
After protection of 18 as a TBS ether 19 (91%), the resulting terminal
alkyne was converted to iodoolefin via Ni-catalyzed regioselective
hydroalumination/iodination (76%).15 Removal of the PMB group
(99%) followed by protection as TES ether furnished iodoolefin 20
(84%). In an analogous sequence as shown in Scheme 1, coupling of
iodide 20 and aldehyde 9 was carried out to afford secondary alcohol
22 and C43-epimer 2313 (71% based on 20, 22:23 = 1.7:1). Further
transformation similar to Scheme 1 afforded the C31–C67 part 1b as a
mixture of the geometrical isomers at C52–C53 (E:Z = 3:1). The
isomers were separated by HPLC (33%).
Scheme 1. Synthesis of 1a.
Having synthesized the model compounds 1a and 1b, their NMR
data were compared with those of the natural product. The differences
in the chemical shifts at C31–C51 portion of AM3 and 1a or 1b are
shown in Figure 3. For both diastereomers, chemical shifts at C52–C67
corresponding to the polyene moiety are identical to those of AM3,12
but those at the C31–C33 terminus deviate because the structures are
different from AM3. Large deviations for 1a were observed in both 1H
and 13C chemical shifts, which clearly indicates that the proposed
structure of AM3 is incorrect. As for 1b, deviations are almost within
error range, however, non-negligible deviations were observed for C38
and H40a. Considering the observed NOE between CH3 (C69) and CH2
(C70) groups, AM3 is likely to take tightly bending conformation with
C30–C31 olefin close to C38–C41.2a Conformational differences
between the model compounds and natural product may cause the
difference in NMR chemical shifts, but analysis of 1b revealed the
compound to have a conformation similar to that of AM3.12 Therefore,
the deviation of the chemical shifts at C38–C41 may be due to the
magnetic anisotropic effect caused by C30–C31 olefin which is lacking
3
in the structure of 1b. It is noteworthy that the estimated JH38,H39
values of 1a and 1b (4.6 Hz and 5.4 Hz, respectively) support the
difficulty in applying the JBCA method to this system.
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Therefore, as shown in Scheme 3, we decided to convert our
samples to MTPA esters 26a–c which lack the C30–C31 double bond
but retain the C38–C39 portion.1b The sample corresponding to 38R
diastereomer 26a was prepared from 10, the precursor of 1a, and the
38S diastereomer 26b from 22, the precursor of 1b. Although
preparation of 26c from natural product was not an easy task due to the
limited availability of AM3, we obtained an adequate amount of 26c
for 1H NMR analysis from no more than ca. 0.3 mg of AM3.
Scheme 2. Synthesis of 1b.
Scheme 3. Degradation of 10, 22, and AM3.
Then, 1H NMR data (600 MHz, CDCl3) of (S)-MTPA esters 26a
(38R, 39R) and 26b (38S, 39R) were compared with those of (S)-
MTPA ester 26c derived from the natural product. The differences in
the chemical shifts at C36–C47 between 26c and 26a or 26b are shown
in Figure 4. It is obvious that deviations between 26c and 26a are large
(red bars), but chemical shifts of 26c are identical to those of 26b (blue
bars). Therefore, the correct absolute configurations at C32–C36 and
C38 are opposite to those in the originally proposed structure, namely,
they should be revised to 32S, 33R, 34S, 35S, 36S, and 38S (Figure 5a).
Based on these results, it was revealed that AM3 is a unique natural
product having both antipodal tetrahydropyran counterparts on a single
carbon chain (C33–C38 and C45–C50 sections).
Figure 3. Differences in chemical shifts between AM3 and the
synthetic fragments 1a and 1b. (a) 1H NMR (600 MHz, 1:2
C5D5N/CD3OD), (b) 13C NMR (150 MHz, 1:2 C5D5N/CD3OD).
The x- and y-axes represent carbon number and Δδ in ppm,
respectively. Red and blue bars represent Δδ = δAM3 –
δsynthetic 1a or 1b, respectively.
Figure 4. Differences in 1H NMR (600 MHz, CDCl3) chemical
shifts between degradation products 26c derived from AM3 and
the synthetic fragments 26a and 26b. Red and blue bars
represent Δδ = δ26c – δ26a or 26b, respectively.
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Although biosynthetic pathways of the amphidinols are not fully
elucidated, a 13C metabolite labeling pattern was reported by the
feeding experiment of 13C-enriched acetate (Figure 5b).16 The labeling
patterns are not symmetrical between the A- and B-ring moieties, and
the arrangement of the A- and B-rings is anti-parallel, head-to-tail to
tail-to-head (Figure 5c). Therefore, it is plausible that the two antipodal
tetrahydropyran moieties were constructed coincidentally in the
biosynthetic pathway, and it is not considered to be unnatural
phenomenon. It has been reported that enantiomeric natural products
can arise from a single or different species, and that both diastereomers
possessing enantiomeric partial structures can arise from a single
species.17,18 It is interesting to note that both enantiomers of the partial
The authors declare no conflict of interest.
Keywords: Amphidinol 3 • Natural Product• Structure
Elucidation • Organic Synthesis • Stereochemistry
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In conclusion, syntheses of the C31–C67 part (1a) of AM3 and the
diastereomer at C32–C36 and C38 (1b) were achieved to confirm the
absolute configuration of the natural product. By comparison of the
NMR data of 1a and 1b with those of AM3 in combination with the
degradation of the natural product, the absolute configuration of AM3
was revised to be 32S, 33R, 34S, 35S, 36S, and 38S. The present results
suggest that structures of AM3 congeners should also be corrected, and
investigations toward this end are currently in progress.
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Acknowledgements
This work was supported in part by JST ERATO Lipid Active
Structure, and JSPS KAKENHI Grant Numbers JP24750092,
JP15K17857, JP15K13645, and JP16H01159 in Middle Molecular
Strategy.
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Yuma Wakamiya, Makoto Ebine, Mariko
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Synthesis and Stereochemical
Revision of the C31–C67 Section of
Amphidinol 3
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