Total Synthesis of (6R,10R,13R,14R,16R,17R,19S,20R,21R,24S, 25S,28S,30S,32R,33R,34R,36S,37S,39R)-Azaspiracid-3 Reveals Non-Identity with the Natural Product

Article abstract of DOI:10.1002/anie.201711006

A convergent and stereoselective total synthesis of the previously assigned structure of azaspiracid-3 has been achieved by a late-stage Nozaki–Hiyama–Kishi coupling to form the C21?C22 bond with the C20 configuration unambiguously established from l-(+)-tartaric acid. Postcoupling steps involved oxidation to an ynone, modified Stryker reduction of the alkyne, global deprotection, and oxidation of the resulting C1 primary alcohol to the carboxylic acid. The synthetic product matched naturally occurring azaspiracid-3 by mass spectrometry, but differed both chromatographically and spectroscopically.

Full text of DOI:10.1002/anie.201711006

A Journal of the Gesellschaft Deutscher Chemiker
Angewandte
Chemie
International Edition
www.angewandte.org
Accepted Article
Title: Total Synthesis of (6R,10R,13R,14R,16R,17R,19S,20R,21R,24S,
25S,28S,30S,32R,33R,34R,36S,37S,39R)-Azaspiracid-3 Reveals
Non-Identity with the Natural Product
Authors: Craig J. Forsyth, Nathaniel T. Kenton, Daniel Adu-
Ampratwum, Antony A. Okumu, Zhigao Zhang, Yong Chen,
Son Nguyen, Jianyan Xu, Yue Ding, Pearse McCarron, Jane
Kilcoyne, Frode Rise, Alistair L. Wilkins, and Christopher O.
Miles
This manuscript has been accepted after peer review and appears as an
Accepted Article online prior to editing, proofing, and formal publication
of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201711006
Angew. Chem. 10.1002/ange.201711006
Link to VoR: http://dx.doi.org/10.1002/anie.201711006
http://dx.doi.org/10.1002/ange.201711006

10.1002/anie.201711006
Angewandte Chemie International Edition
COMMUNICATION
Total Synthesis of (6R,10R,13R,14R,16R,17R,19S,20R,21R,24S,
25S,28S,30S,32R,33R,34R,36S,37S,39R)-Azaspiracid-3 Reveals
Non-Identity with the Natural Product
Nathaniel T. Kenton,τ Daniel Adu-Ampratwum,τ Antony A. Okumu, Zhigao Zhang, Yong Chen, Son
Nguyen, Jianyan Xu, Yue Ding, Pearse McCarron, Jane Kilcoyne, Frode Rise, Alistair L. Wilkins,
Christopher O. Miles, and Craig J. Forsyth *
This work is dedicated to Professor Yoshito Kishi on the occasion of his 80th birthday.
Abstract: A convergent and stereoselective total synthesis of the
previously assigned structure of azaspiracid-3 has been achieved
via a late stage NHK coupling to form the C21‒C22 bond with the
C20 configuration unambiguously established from L-(+)-tartaric acid.
Post-coupling steps involved oxidation to an ynone, modified Stryker
reduction of the alkyne, global deprotection, and oxidation of the
primary alcohol to the carboxylic acid. The synthetic product
matched naturally occurring azaspiracid-3 by mass spectrometry,
but differed both chromatographically and spectroscopically.
in potential human food sources, and toxicological studies.[4]
The structures of the AZAs are diverse due to primary
biosynthesis and subsequent bivalve metabolism. Mussels
concentrate and convert primary AZAs into toxic AZA
metabolites.[2,5] There are some 59 AZA structures recognized,
that warrant monitoring to reduce human poisonings. The
provision of certified AZA reference standards of known
structures is an important need.[6] Also, probing the toxicology of
the AZAs benefits from synthetic inputs and requires an
accurate understanding of the relationship between chemical
structure and function.
The azaspiracids (AZAs) are lipophilic toxins produced by
some marine dinoflagellates of the family Amphidomataceae
(figure 1).[1] Widespread AZA occurrence and concentration by
filter-feeding bivalves serves as a conduit into human food
chains.[2] Incidental human consumption of AZAs raises health
concerns ranging from acute diarrheic shellfish poisoning to
chronic cardiomyopathy and neurotoxicity.[3] This has spurred
extensive surveillance efforts to detect and quantify AZA content
R1
8
H
1
O
O
H
O
H
OH
O
19
OH
R2
O
HO
20
H
τ
[*]
Co-authors contributed equally to the research described.
Mr. N. T. Kenton, Dr. D. Adu-Ampratwum, Dr. A. A. Okumu, Prof.
Dr. C. J. Forsyth, Department of Chemistry and Biochemistry, The
NH
O
21
O
40
H
22
H
Ohio State University, 151 W. Woodruff Ave, Columbus, Ohio,
43220, USA, E-mail: forsyth.14@osu.edu
Dr. Z. Zhang, Dr. J. Xu, Shanghai Hengrui Pharmaceutical Inc., No.
279 Wenjing Road, Shanghai, P.R. China, 200245
Dr. Y. Chen, Asymchem Life Science, No. 71 7th Ave., TEDA,
Tianjin, P.R.China, 300000
O
O
H
Dr. S. Nguyen, Johnson Matthey Pharma Services, 25 Patton Road
Devens, MA 01434
Dr. Y. Ding, Viva Biotech Ltd, 581 Shenkuo Rd., Pudong District,
Shanghai, China, 201203
Dr. P. McCarron, Measurement Science and Standards, National
Research Council Canada, 1411 Oxford St., Halifax, Nova Scotia,
B3H 3Z1 Canada
1: AZA1 R1 = H
2: AZA2 R1 = CH3 R2 = CH3
3: AZA3 R1 = H R2 = H
R2 = CH3
Dr. J. Kilcoyne, Marine Institute, Rinville, Oranmore, Co. Galway,
Ireland
Prof. Dr. F. Rise, Department of Chemistry, University of Oslo, N-
0315 Oslo, Norway
Figure 1. Published structures of AZAs 1‒3.[9]
Prof. Dr. A. L. Wilkins, Norwegian Veterinary Institute, P.O. Box 750
Sentrum, NO-0106 Oslo, Norway; and Chemistry Department,
University of Waikato, Private Bag 3105, 3240 Hamilton, New
Zealand
Dr. C. O. Miles, Norwegian Veterinary Institute, P.O. Box 750
Sentrum, NO-0106 Oslo, Norway; and Measurement Science and
Standards, National Research Council Canada, 1411 Oxford St.,
Halifax, Nova Scotia, B3H 3Z1, Canada
The structure of AZA1 was originally outlined by Yasumoto,
Satake, and co-workers in 1998[1a] and refined by subsequent
extensive efforts.[7] Nicolaou and Satake correlated oxidative
degradation fragments of AZA1 with synthetic products to
reduce the structural possibilities,[8] which resulted in total
syntheses of AZA1‒3 and seemed to complete the structural
assignments.[9] AZA3 is accepted to be the C22-desmethyl
variant of AZA1,[1b] with spectroscopic and synthetic data to
support that AZA1‒3 otherwise share identical stereochemistry.
Supporting information for this article is given via a link at the end of
the document.
This article is protected by copyright. All rights reserved.

10.1002/anie.201711006
Angewandte Chemie International Edition
COMMUNICATION
In mussels, oxidation of the C22 methyl group of AZA1 may
generate the C22 carboxylic acid AZA17, which, upon C22
decarboxylation, yields AZA3.[10] Reported herein is the total
synthesis of the previously assigned structure of AZA3[1b,8b] and
its direct comparison with naturally occuring AZA3.
oxidation of the secondary alcohol gave ketone 19, which was
converted to silyl enol ether 13. Partner aldehyde 12 was
derived from diol 20[16] via PMB ether 21. An efficient Mukaiyama
aldol reaction[14] between 12 and 13 generated β-hydroxyl ketone
22. Conversion to γ-hydroxyl ketone 11 allowed reductive
cyclization to stereoselectively generate trans-THF 24.[11,17]
Aldehyde 10 was derived simply from 24.
Prior syntheses of AZA1‒3[9] and ent-AZA1[11] relied upon late-
stage formation of the C20‒C21 bond that installed the C20
OTBDPS
stereogenic center via ketone reductions or
a
poorly
AcO
10
O
OTBS
H
1
diastereoselective[11b,c] coupling reaction. In the present
approach to AZA3 the key coupling is between C21 and C22 (6
+ 7 = 5, scheme 1). This was to minimize post-coupling
transformations and enhance mass throughput. It also provides
the C20 carbinol configuration at the outset knowingly and
unperturbed from L-(+)-tartaric acid. Thus, AZA3 was to arise
from C21 ketone 4 via global desilylation and C1 oxidation
(scheme 1). Ketone 4 was to derive from propargylic alcohol 5,
that would result from the convergent union of a C1‒C21
aldehyde (6) and a C22‒C40 alkyne (7) via an NHK reaction.[12]
O
O
13
H
H
20
H
21
13
O
10
12
OTBS
H
OPiv
O
O
H
OMe
1
H
O
OTBDPS
H
6
8
O
21
+
OTBDPS
1
AcO
OAc
O
OTBS
18
17
20
21
TBSO
OPiv
H
13
O
13
20 21
OPiv
I
O
O
H
12
13
OH
OTBS
H
H
OMe
11
10
9
TMS
O
OPiv
OH
O
O
20
HO 18
O
18
20
+
H
21
OH
21
TBSO
11
17
OH
OPMB
OTBS
L-(+)-tartaric acid
12
13
Scheme 2. Genesis of the C1‒21 domain. Ac = acetyl. Piv = pivaloyl. PMB =
4-methoxybenzyl.
H
1
OH
H
O
O
O
O
H
O
H
H
O
O
H
1
Ph
Ph
20
1. HCl, MeOH, 22 °C
2. PhCH(OMe)2
OH
O
HO
OTBS
21
TBDPSO
O
reference [15]
OH
O
H
OH
20
O
O
O
O
O
H
NH
O
O
O
O
40
H
+
21
HO 18
O
TsOH, CH2Cl2, 22 °C
(15a : 15b = 4 : 1)
67%, 2-steps
H
Teoc
20
18
21
O
N
21
OH
H
O
40
OH
O
OR
desilylation /
C1 oxidation
OH
15b
25
OTES
O
H
L-(+)-tartaric acid
TBSOTf
2,6-lutidine
CH2Cl2, 0 °C
87%
O
14
15a R = H
H
16 R = TBS
H2, Pd/C
90%
3
4
SO3.pyr.
DMSO
OPiv
(6R,10R,13R,14R,16R,17R,19S,20R,21R,24S,
25S,28S,30S,32R,33R,34R,36S,37S,39R)-AZA3
ynone formation /
conjugate reduction
TMSOTf
O
OH OR
20
TMSO
20
OPiv
21
i-Pr2NEt
Et3N, CH2Cl2
20
H
O
O
CH2Cl2
0 °C, 93%
-78 to 0 °C, 99%
18
H
O
H
OTBS
19
OTBS
17 R = H
18 R = Piv
OTBS
13
H
1
TBDPSO
O
O
H
OTBS
21
O
O
H
PivCl, pyr.
CH2Cl2
1
TBDPSO
H
OTBS
21
22
O
O
6
H
0
to 22 °C, 77%
H
HO
1. Anisaldehyde
dimethyl acetal,
PPTS, CH2Cl2, 22 °C
+
Teoc
NHK fragment
coupling
I
N
TBSO
OH
TBSO
OH
H
O
13
17
OPMB
40
Teoc
22
OH
O
N
40
H
O
O
2. DIBALH, CH2Cl2
-78 °C, 87% for 2 steps
20
21
OTES
H
O
O
SO3.pyr., DMSO,
i-Pr2NEt, CH2Cl2
0 °C, 97%
OTES
H
5
7
O
13, 2.7 equivalents
OR1
O
MgBr2.OEt2, CH2Cl2
TBSO
13
H
TBSO
OPiv
13
21
OTBS
Ac2O, DMAP
17
OPMB
Scheme 1. Retrosynthesis of 3. NHK = Nozaki-Hiyama-Kishi.[12] TBS = tert-
butyldimethylsilyl. TBDPS tert-butyldiphenylsilyl. Teoc 2-
trimethyl(ethyloxy)carbonyl. TES = triethylsilyl.
OR2
-20 to -10 °C, 93%
22 R1 = H, R2 =PMB
23 R1 = Ac, R2 =PMB
11 R1 = Ac, R2 =H
=
=
12
CH2Cl2, 0 °C, 92%
DDQ, pH 7 buffer
CH2Cl2, 0 °C
SnCl4, Et3SiH
CH2Cl2, -78 °C
The bis-spiroketal
6 would derive from ketone 8 upon
deacetylation and thermodynamically driven intramolecular
transketalizations (scheme 2). Ketone 8 derives from the known
C1‒C12 alkynyl iodide 9[13] and C13‒C21 aldehyde 10. The
latter derives from acyclic keto-alcohol 11. A chelation-controlled
Mukaiyama aldol reaction[14] between aldehyde 12 and L-(+)-
tartrate-derived silyl enol ether 13 would predictably generate 11.
AcO
AcO
SO3.pyr., DMSO
H
H
i-Pr2NEt, CH2Cl2
13
O
H
OTBS
OTBS
21
OPiv
O
O
H
H
OR
0 °C, 95%
OPiv
10
24 R = TBS
+
25 R = H
PPTS, MeOH, 22 °C
74% of 25 for 3 steps
Preparation of the C13‒C21 fragment 10 is outlined in scheme 3.
Synthon 14 provided the C20 stereogenic center derived from L-
(+)-tartaric acid.[15] Vicinal acetonide 14 was converted to
benzylidenes 15a/15b. Silylation of 15a yielded masked triol 16,
which was hydrogenated to diol 17. Acylation of the primary and
Scheme 3. C13‒C21 Fragment assembly. DDQ = 2,3-dichloro-5,6-dicyano-
1,4-benzoquinone. DIBALH diisobutylaluminum hydride. DMAP 4-
dimethylaminopyridine. DMSO = dimethylsulfoxide. PPTS = pyridinium 4-
toluenesulfonate. pyr. pyridine. TBSOTf tert-butyldimethylsilyl
trifluoromethanesulfonate. TMS trimethylsilyl. TMSOTf trimethylsilyl
trifluoromethanesulfonate. TsOH = 4-toluenesulfonic acid.
=
=
=
=
=
=
This article is protected by copyright. All rights reserved.

10.1002/anie.201711006
Angewandte Chemie International Edition
COMMUNICATION
Aldehyde 10 was joined via an NHK reaction[12] with C1‒C12
iodoalkyne 9[13] to generate epimeric propargylic alcohols 26 that
were oxidized to ynone 27 (scheme 4). Conjugate reduction[18]
afforded ketone 8. C17-O-Deacetylation allowed acid-induced
bis-spiroketalization[11,19] to consolidate the ABCD-ring system in
29. Selective deprotection and oxidation gave the C1‒C21
aldehyde 6.
Stryker reduction also benefited from careful optimization:
inclusion of 1,2-bis(diphenylphosphino)benzene (BDP) was
critical for success.[18] Global deprotection of 4 was achieved
with freshly prepared TBAF solution provided primary alcohol
35. Two-step oxidation to the carboxylic acid completed the total
synthesis of 3. As observed by Evans,[11b,c] the C20 alcohol was
largely inert towards oxidation using unbuffered Dess-Martin
periodinane[22] conditions.
AcO
OTBDPS
1
OTBDPS
1
H
OTBS
1
3
H
OTBS
AcO
O
21
H
H
21
O
O
O
O
OPiv
I
H
10
OPiv
X
13
H
O
H
X
O
H
O
12
12
H
OMe
H
OMe
9
CrCl2, NiCl2,THF
22 °C, 82%
OTBS
26 X = CHOH
27 X = C=O
O
MnO2, CH2Cl2, 0 °C, 84%
CrCl2, NiCl2
H
OTBDPS
Teoc
6 + 7
(CuHPPh3)6, PhSiH3, H2O, PhMe, 22 °C
1
85%
4-t-Bu-pyridine
THF, 60%
N
OTBDPS
H
40
OTBS
RO
10
H
PPTS, CH2Cl2
-40 → 0 °C
41% for 2 steps
1
O
O
17
H
21
O
H
13
O
17
10
13
O
O
H
O
H
OPiv
OTBS
O
H
O
1
H
H
OMe
R = Ac
28 R = H
OTBDPS
21
OTES
O
OR
8
K2CO3, MeOH, 22 °C
29 R = Piv
30 R = H
DIBAL-H, CH2Cl2
H
O
-95 °C, 85%
O
DMP, pyr., H2O
CH2Cl2, 0 °C, 94%
H
O
H
6
5 X = CHOH
34 X = CO
DMP, pyridine
CH2Cl2, 70%
(CuHPPh3)6
BDP, PhSiH
H2O, PhMe
22 °C, 64%
OH
OH
O
H
Y
Scheme 4. Convergent assembly of the C1‒C21 domain. DMP = Dess-Martin
periodinane.
4
O
NH
H
H
40
O
O
TBAF, THF
-78 °C
O
The C22‒C40 coupling partner 7 was obtained from alkyne 31[20]
(scheme 5) via iodination and allylic ether modification. Prior
installation of the C22 iodine atom was critical to consistently
obtain high yields for oxidative scission of the PMB ether, as
rapid decomposition resulted upon treatment of 31 with DDQ.
H
79%
35 Y = CH2OH
3 Y = CO2H
1. DMP, CH2Cl2, 0 °C
2. NaClO2, 2-methyl-2-butene
NaH2PO4 / H2O, t-BuOH, 22 °C
78% for 2 steps
R
I
Teoc
22
22
Teoc
DDQ
pH 7 buffer
N
Scheme 6. Completion of the synthesis of 3. BDP
= 1,2-bis(diphenyl-
N
40
H
O
40
H
O
phosphino)benzene. TBAF = tetra-n-butyl-ammonium fluoride.
O
O
O
O
CH2Cl2, 0 °C
99%
OPMB
H
OR
H
31 R = H
32 R = I
NIS, AgOTf, DMF
22 °C, 99%
33 R = H
7 R = TES
TESOTf, 2,6-lut.
CH2Cl2, -78 °C, 90%
Scheme 5. Elaboration of the C22‒C40 domain. AgOTf = silver trifluoro-
methanesulfonate. DMF = N,N-dimethylformamide. NIS = N-iodosuccinimide.
PMB
=
4-methoxybenzyl. TES
=
triethylsilyl. TESOTf
=
triethylsilyl
trifluoromethanesulfonate. Teoc = 2-trimethyl(ethyloxy)carbonyl. 2,6-lut. = 2,6-
dimethylpyridine.
The carbon skeleton of 3 was obtained from 6 and 7 via an
optimized NHK reaction (scheme 6).[12] Key to the rapid success
of this reaction was the use of 4-tert-butylpyridine as an
additive.[21] This seems to solublize and activate the metal salts
and buffer acidity, while also accelerating the reaction rate. In
the absence of 4-tert-butylpyridine, 7 underwent decomposition
under the NHK reaction conditions. The resultant epimeric
propargylic alcohols 5 were oxidized to ynone 34, which was
chemoselectively reduced to ketone 4. This modified Lipshutz-
This article is protected by copyright. All rights reserved.

10.1002/anie.201711006
Angewandte Chemie International Edition
COMMUNICATION
Biotechnology ERA-NET; project number 604814. (“Enhanced
Biorefining Methods for the Production of Marine Biotoxins and
Microalgae Fish Feed”), and The Research Council of Norway is
acknowledged for support through the Norwegian NMR Platform,
NNP (226244 / F50).
Keywords: azaspiracids • total synthesis • structure
determination • natural products • toxins
[1]
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Silke, T. Yasumoto, J. Am. Chem. Soc. 1998, 120, 9967–9968; b) K.
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J. Jeong, Toxicon 2012, 60, 830–839; e) U. Tillmann, D. Jaen, L.
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Miles, P. Hess, Toxicon, 2013, 65, 81–89; c) J. Kilcoyne, C. Nulty, T.
Jauffrais, P. McCarron, F. Herve, B. Foley, F. Rise, S. Crain, A. L.
Wilkins, M. J. Twiner, P. Hess, C. O. Miles, J. Nat. Prod. 2014, 77,
2465–2474; d) D. O'Driscoll, Z. Škrabáková, K. J. James, Toxicon 2014,
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Food Chem. 2015, 63, 5083–5091.
Figure 2. LC-HRMS chromatograms of A: natural AZA3 (tR 20.23 min, m/z
828.4888), and B: synthetic 3 (tR 25.89 min, m/z 828.4883) (LC-MS method 1;
see SI).
Comparative LC-MS analysis of 3 and an authentic sample of
AZA3 showed nearly indistinguishable MS/MS spectra, but a
different retention time was observed for each (figure 2).
Comparison of the 1H and 13C NMR spectroscopic data of
synthetic 3 and AZA3 revealed significant differences in the
appearance of the C19-H multiplet and the chemical shifts of the
C20-H and C22-H axial multiplets.[23,24] The possibility that the
stereochemistry at C19 or C20 had been compromised en route
[3]
a) A. Furey, S. O’Doherty, K. O’Callaghan, M. Lehane, K. J. James,
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Vieira, L. M. Botana, Toxicol. Sci. 2016, 151, 104–114; d) M. J. Twiner,
G. J. Doucette, A. Rasky, X.-P. Huang, B. L. Roth, M. C. Sanguinetti,
Chem. Res. Toxicol. 2012, 25, 1975–1984.
to
3 was seriously considered; subsequent experiments
established with certainty, however, that the structure of
synthetic 3 is as represented in schemes 1 and 6.[25] It was thus
concluded that synthetic (6R,10R,13R,14R,16R,17R,19S,20R,
[4]
a) S. Leonardo, M. Rambla-Alegre, J. Diogene, I. A. Samdal, C. O.
Miles, J. Kilcoyne, C. K. O'Sullivan, M. Campas, Biosens.
21R,24S,25S,28S,30S,32R,33R,34R,36S,37S,39R)-3,
which
Bioelectron. 2017, 92, 200–206; b) R. Rossi, C. Dell'Aversano, B. Krock,
P. Ciminiello, I. Percopo, U. Tillmann, V. Soprano, A. Zingone, Anal.
Bioanal. Chem. 2016, 409, 1121–1134; c) P. McCarron, S. D. Giddings,
K. L. Reeves, P. Hess, M. A. Quilliam, Anal. Bioanal.
Chem. 2015, 407, 2985–2996; d) I. A. Samdal, K. E. Løvberg, L. R.
Briggs, J. Kilcoyne, J. Xu, C. J. Forsyth, and C. O. Miles, J. Agric.
Food Chem. 2015, 63, 7855–7861; e) R. Draisci, L. Palleschi, E.
Ferretti, A. Furey, K. J. James, M. Satake, T. Yasumoto, J. Chromatogr.
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Experimental Section
Please see the Supporting Information for comprehensive experimental
details.
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This work was sponsored by the NIH (R01-ES10615), The Ohio
State University (OSU), and the Forsyth Research Fund (CJF).
We thank Dr. L. Xiao (OSU) for administrative assistance, and
Prof. Dr. A. B. Dounay, and Drs. J. Aiguade-Bosch, J. Hao, L. K.
Geisler, Y. Li, and F. Zhao for foundational research
contributions. This work was supported by the First Call for
Transnational
Research
Projects
within
the
Marine
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2017, xxx.
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COMMUNICATION
COMMUNICATION
A convergent and
stereospecific total
synthesis of the
previously assigned
structure of the marine
neurotoxin azaspiracid-
3 reveals non-identity
and thus necessitates
structural revision of
Author(s), Corresponding Author(s)*
H
1
O
O
H
O
H
OH
O
OH O
19
O
Page No. – Page No.
OH
18
20
HO
20
19
21
OH
H
HO 21
O
OH
NH
O
22
O
40
H
L-(+)-tartaric acid
H
Total Synthesis of
O
(6R,10R,13R,14R,16R,17R,19S,20R,
21R,24S,25S,28S,30S,32R,33R,34R,
36S,37S,39R)-Azaspiracid-3 Reveals
Non-Identity with the Natural
Product
O
H
the primary
azaspiracids.
(6R,10R,13R,14R,16R,17R,19S,20R,21R,24S,
25S,28S,30S,32R,33R,34R,36S,37S,39R)-
3
Azaspiracid-3
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