Concise Synthesis of (+)-[13C4]-Anatoxin-a by Dynamic Kinetic Resolution of a Cyclic Iminium Ion

Article abstract of DOI:10.1002/anie.202004464

An asymmetric total synthesis of [¹³C₄]-anatoxin-a ([¹³C₄]-1) has been developed from commercially available ethyl [¹³C₄]-acetoacetate ([¹³C₄]-15). The unique requirements associated with isotope incorporation inspired a new, robust, and highly scalable route, providing access to 0.110 g of this internal standard for use in the detection and precise quantification of anatoxin-a in freshwater. A highlight of the synthesis is a method that leverages a cyclic iminium ion racemization to achieve dynamic kinetic resolution in an enantioselective Morita–Baylis–Hillman (MBH) cyclization.

Full text of DOI:10.1002/anie.202004464

A Journal of the Gesellschaft Deutscher Chemiker
Angewandte
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International Edition
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Accepted Article
Title: Concise Synthesis of (+)-[13C4]-Anatoxin-a by a Dynamic Kinetic
Resolution of a Cyclic Iminium Ion
Authors: Armen Zakarian, Jacob J. Lacharity, Artur K. Mailyan, and
Karen Y. Chen
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.202004464
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10.1002/anie.202004464
Angewandte Chemie International Edition
COMMUNICATION
Concise Synthesis of (+)-[13C4]-Anatoxin-a by a Dynamic Kinetic
Resolution of a Cyclic Iminium Ion
Jacob J. Lacharity†, Artur K. Mailyan†, Karen Y. Chen, and Armen Zakarian*[a]
[a]
[†]
J. J. Lacharity, Dr. A.K. Mailyan, K. Y. Chen, Prof. Dr. A. Zakarian
Department of Chemistry and Biochemistry
University of California, Santa Barbara, CA 93106-9510 (USA)
E-mail: zakarian@chem.ucsb.edu
J.J.L. and A.K.M. contributed equally.
Supporting information for this article is given via a link at the end of the document.
Abstract: An asymmetric total synthesis of [13C4]-anatoxin-a ([13C4]-
1) has been developed from commercially available ethyl [13C4]-
acetoacetate ([13C4]-15). The unique requirements associated with
isotope incorporation inspired a new, robust, and highly scalable
route, providing access to 0.110 g of this internal standard for use in
the detection and precise quantification of anatoxin-a in freshwater.
A highlight of the synthesis is a method that leverages a cyclic
iminium ion racemization to achieve dynamic kinetic resolution in an
enantioselective Morita-Baylis-Hillman (MBH) cyclization.
MeO2CO
MeO2C
[
13C4]-3 available, however:
• 2 equivalents required
lost through RCM
Trost
1999
Br
3
•
NHTs
(±)-2
O
Aggarwal
1999
HO
[
13C4]-5
Me
H
O
not available
OH
N
NHBn
Me
4
5
MeO2C
Seitz
2000
•HCl
Me
N
N
O
CO2Me
[
13C4]-7
not available
(+)-anatoxin-a (1)
OBz
6
7
3
Anatoxin-a (1) is a cyanobacterial toxin produced by several
Br
genera
of
blue-green
algae,
including
Anabaena,
[
13C4]-9
not available
N
1
Martin
2004
Me
Aphanizomenon, and Oscillatoria. This potent agonist of
nicotinic acetylcholine receptors (nAChRs) causes prolonged
excitation at the neuromuscular junction and a blockage of
further electrical transmission, which can lead to muscular
paralysis and death by asphyxiation.2 It has been associated
with the poisoning and deaths of livestock, domestic animals,
and waterfowl after exposure to cyanobacteria-contaminated
water.3
8
Cbz
O
CO2Me
N
lost through oxidative cleavage
H
9
Figure 1. Previous synthetic approaches to (+)-anatoxin-a and consideration
of their ability to produce an isotopically labelled internal standard.
Since its isolation, a plethora of synthetic approaches to the
natural isotope of anatoxin-a have been developed and
extensively reviewed.7 Its popularity as a synthetic target has
inspired several innovative routes (Figure 1). Elegant work has
been described by the Trost,8 Aggarwal,9 Seitz,10 and Martin11
groups relying on a variety of creative strategies including
asymmetric allylic amination, asymmetric deprotonation,
tropanone ring expansion, and enyne metathesis. These
impressive syntheses proceeded in as few as 8 steps (Seitz)
and up to 27% overall yield (Martin), truly setting a high bar for
future approaches to this important target.
Our goal was to provide practical synthetic access to an
isotopically labelled anatoxin-a that could be used as an internal
standard for highly sensitive and precise freshwater
quantification. In the initial stages of synthesis planning, there
were several important criteria that had to be taken into
consideration.12 First, isotope incorporation at non-exchangeable
positions throughout the synthesis is essential to preventing
isotope loss and maintaining a uniform molecular mass in the
final product. Second, to prevent any signal overlap during mass
spectral analysis, we sought to prepare a standard with a mass
difference of 4 atomic units compared to the natural anatoxin-a.
Finally, given the high cost of isotopically labelled starting
materials, the synthesis itself must be as concise as possible,
Cyanobacterial algal blooms have occurred with increasing
frequency in recent years, likely caused by factors such as rising
water temperatures and fertilizer runoff.4 These blooms pose a
significant threat to public safety due to the production of
cyanobacterial toxins, such as anatoxin-a. As such, methods for
the detection and quantification of anatoxin-a in fresh water are
critically important to human health.1b The analytical method
currently adopted by the Environmental Protection Agency for
anatoxin-a quantification in finished drinking water involves a
combination of liquid chromatography, electrospray ionization,
and tandem mass spectrometry (LC/ESI-MS/MS) using
deuterated phenylalanine (L-phenylalanine-d5) as an internal
standard.5 While this method is useful, its accuracy hinges on
the assumption that L-phenylalanine-d5 and anatoxin-a will have
identical behavior during sample preparation and analysis.6 A
more ideal standard, which would account for potential sample
loss and ion suppression from other components present in the
sample matrix, would be an isotopically labelled variant of
anatoxin-a; however, no such standard is available.
1
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10.1002/anie.202004464
Angewandte Chemie International Edition
COMMUNICATION
highly scalable, and rely on robust chemical transformations to
ensure reproducibility.
enantioselectivity for this one-step transformation were lower
compared to the route outlined in Scheme 2 (see Supporting
Information for more details).
MeO
OMe
H
N
MBH
C-N bond
formation
Ireland-Claisen
rearrangement
Coupling of alcohol [13C4]-14 with 13 in the presence of DCC
gave ester [13C4]-12 in 97% yield. Deprotonation with LDA
followed by addition of TBSCl and Ireland-Claisen
rearrangement of the resulting silyl ketene acetal delivered
O
*
OMe
reaction
N
Me
*
*
*
Ts
CO2H
*
*
*
Me
*
*
13C4]-10
*
*
Me
[
13C4]-1
[
13C4]-11
[
*
O
OTBS
[13C4]-11 in 95% yield and 81% ee.
Acid [13C4]-11 was
MeO
MeO
O
transformed into the corresponding acyl azide with (PhO)2PON3,
which then underwent Curtius rearrangement upon heating.
Hydrolysis of the intermediate isocyanate with NaOSiMe3 gave a
primary amine, which was tosylated to give [13C4]-19 in 83%
yield over 2 steps.18 Treatment with CSA resulted in pyrrolidine
formation and simultaneous removal of the TBS group. In the
same pot, addition of TEMPO and PhI(OAc)2 gave ketone [13C4]-
20, and subsequent treatment with DBU led to migration of the
double bond into the α,β-position, delivering [13C4]-10 in 83%
yield over 2 steps. Enone [13C4]-10 served as a precursor for the
penultimate MBH cyclization. After extensive experimentation, it
was found that the use of tetrahydrothiophene as the
nucleophile in the presence of Me3SiOTf in MeCN at –30 °C
gave [13C4]-21 in 78% yield and 80% ee, which could be
enriched to 98% ee following recrystallization.19 Heating [13C4]-
21 with ethylene diol and PPTS generated an acetal, tosyl group
removal was successfully accomplished through reduction with
magnesium, and in situ hydrolysis of the acetal gave [13C4]-
anatoxin-a in 72% yield as its hydrochloride salt. High resolution
mass spectral analysis revealed the final product has 99.1% 13C
incorporation at all four positions.
During optimization of the iminium MBH cyclization, we noticed
an unexpected relationship between reaction temperature and
enantiopurity of the cyclized product. At temperatures above –
30 °C, significant racemization was observed. This caught us by
surprise, given the apparent lack of an epimerizable carbon in
the molecule. Tanner and co-workers observed the same
phenomenon in their synthesis of anatoxin-a, in which they used
an MBH cyclization on a similar precursor.11 The authors
propose that racemization occurs through a hydride shift of the
initially formed cyclic iminium ion. However, this mechanism
seems improbable given that [1,3]-hydride shifts are symmetry-
forbidden.20 It is conceivable that this racemization involves a
simple trace acid-catalyzed isomerization of the initially-formed
C-monosubstituted imine to the more stable C-disubstituted
imine (i to ii, Scheme 3a). However, at present the exact origin
of epimerization remains unknown.
OH
MeO
MeO
O
esterification
O
*
O
*
13
O
*
OTBS
EtO
Me
*
OH OTBS
*
Me
*
*
*
Me
*
[
13C4]-15
*
*
*
[
13C4]-12
[
13C4]-14
*Denotes a 13C atom
Scheme 1. Synthesis plan for [13C4]-anatoxin-a.
Inspired by the past approaches described above, we attempted
to identity a suitable route to isotopically labelled anatoxin-a that
would satisfy these criteria. Previous syntheses, while succinct
and high yielding, appeared to be unfeasible in this regard due
to a lack of commercially available isotopically labelled starting
materials or synthetic transformations that would result in
isotope loss (Figure 1). We therefore decided to develop a de
novo synthesis of [13C4]-anatoxin-a ([13C4]-1) from a suitable
source of heavy isotopes. We envisioned a synthetic route in
which the final C-C bond of anatoxin-a would be constructed
from an iminium Morita-Baylis-Hillman (MBH) cyclization of
enone [13C4]-10 (Scheme 1).13,14 The cyclization precursor [13C4]-
10 would be derived from acid [13C4]-11, which in turn would
arise from an Ireland-Claisen rearrangement of allylic ester
[13C4]-12.15 Disconnection of the ester C-O bond identified chiral
alcohol 14 and carboxylic acid 15 as potential materials for the
introduction of isotopes into the synthesis. After a thorough
survey of commercially available isotopically labelled
compounds, ethyl [13C4]-acetoacetate ([13C4]-15) was determined
to be an excellent precursor to alcohol [13C4]-14. This material
serves as the sole source of all heavy isotopes in the synthesis.
More importantly, these isotopes are placed at non-
exchangeable positions throughout the designed route. Herein,
we describe our successful approach to [13C2,13C3,13C10,13C11]-
anatoxin-a ([13C4]-1) from [13C4]-15 and the development of a
dynamic kinetic resolution process through enantioselective
MBH cyclization.
The synthesis commenced with a sodium borohydride reduction
of [13C4]-15 (Scheme 2). Silylation of the nascent alcohol with
TBSCl (96% yield over 2 steps) followed by partial reduction of
the ethyl ester gave aldehyde [13C4]-16. Homologation with
triethyl phosphonoacetate followed by reduction of the
intermediate α,β-unsaturated ester furnished allylic alcohol
[13C4]-17 in 84% yield over 2 steps. Sharpless asymmetric
epoxidation of [13C4]-17 gave epoxide [13C4]-18, which was then
immediately subjected to iodo-dehydroxylation conditions. 16
Reduction with zinc occurred with concomitant epoxide opening
to deliver chiral alcohol [13C4]-14 in 88% yield and 86% ee over 3
steps. It is worth mentioning that we did attempt to access [13C4]-
We became intrigued by the practical implications of the cyclic
imine undergoing facile racemization. At temperatures above –
30 °C, the rate of imine isomerization begins to outcompete the
rate of cyclization. We hypothesized that this phenomenon could
be leveraged to achieve dynamic kinetic resolution through the
use of
a chiral nucleophile to induce enantioselectivity.
Treatment of racemic cyclization precursor (±)-10 with Me3SiOTf
produces a series of rapidly equilibrating cyclic imines (i, ii, and
iii, Scheme 3a). If reaction parameters are adjusted such that
equilibration between iminium ions i and iii occurs rapidly
relative to cyclization, the product distribution should reflect the
difference in energy between the two diastereomeric transition
14 directly through enantioselective addition of divinyl zinc to
17
aldehyde [13C4]-16.
However, both the yield and
2
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10.1002/anie.202004464
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COMMUNICATION
Ti(Oi-Pr)4, (-)-DIPT
t-BuOOH, 4 Å MS
1. NaBH4
1. EtO2CCH2PO(OEt)2, NaH
2. TBSCl, ImH (96% yield)
1. I2, PPh3, ImH
O
*
O
*
O
*
OTBS
OTBS
OTBS
2. i-Bu2AlH
CH2Cl2, -15 °C, 12 h
3. i-Bu2AlH
2. Zn, NaI
O
EtO
Me
*
H
Me
HO
Me
HO
Me
*
*
*
*
*
*
84% yield (3 steps)
*
*
88% yield
(3 steps)
86% ee
*
*
*
*
13C4]-18
13C4]-16
[
13C4]-17
[
13C4]-15
[
[
MeO
MeO
O
CSA, MeOH
MeO
OMe
MeO
OMe
MeO
OMe
LDA, HMPA, TBSCl;
then MeOH
23 °C, 12 h;
1. (PhO)2PON3, i-Pr2NEt;
OH
DCC, DMAP 13
TEMPO, PhI(OAc)2
CH2Cl2, 23 °C, 8 h
NaOSiMe3
OMe
N
OH OTBS
THF, -78 to 0 °C, 3 h
2. TsCl, i-Pr2NEt
CH2Cl2, 23 °C, 12 h
O
Ts
*
NHTs
CO2H
*
*
Me
*
*
*
O
91% yield
83% (2 steps)
95% yield
97% yield
*
Me
*
*
*
*
*
*
*
*
*
[
13C4]-14
81% ee
*
*
13C4]-20
*
Me
Me
Me
*
[
O
[
13C4]-19
[
13C4]-11
[
13C4]-12
OTBS
OTBS
OTBS
DBU
PhH, 23 °C, 12 h
90% yield
Direct synthesis of (±)-[13C4]-14 to access racemic [13C4]-10:
S
(HOCH2)2, PPTS
PhH, 80 °C, 24 h;
•HCl
Ts
MgBr
Mg, MeOH, 23 °C, 1 h;
3 M HCl, 23 °C, 1 h
H
Me3SiOTf
OMe
O
O
*
N
N
O
*
OTBS
OH OTBS
Me
N
THF, 0 °C, 45 min.
MeCN, -30 °C, 72 h
Me
*
Ts
Me
*
*
*
*
*
*
H
Me
*
*
*
*
*
*
*
78% yield
72% yield
82% yield
*
*
*
*
Me
[
13C4]-21
13C4]-10
[
13C4]-16
(±)-[13C4]-14
[
O
80% ee
98% ee
recrystallization
[
13C4]-1•HCl
Scheme 2. Total synthesis of [13C4]-anatoxin-a.
states TS1 and TS2, in accordance with the Curtin-Hammett
principle.21 The ability to use racemic (±)-10 would obviate the
need to introduce chirality into alcohol 14. Racemic (±)-[13C4]-14
can be accessed directly through addition of vinylmagnesium
bromide to (±)-[13C4]-16 in 82% yield (Scheme 2 inset), reducing
the synthesis by 4 steps.
Our success using tetrahydrothiophene as a nucleophile in our
original route inspired us to explore a variety of chiral sulfur-
containing nucleophiles for enantioselective iminium MBH
cyclization. We began our experiments by employing
commercially available chiral sulfide 22 (Scheme 3b, entry 1).22
In this case, product 21 was isolated in high yield, but virtually
no enantioselectivity was achieved. We then screened the
camphor-derived sulfide 23 developed by Aggarwal and co-
workers.23 We were excited by the potential of this sulfide to
enantioselectivity was observed, with the cyclized products
being isolated in 40% yield, 62% ee (entry 7) and 29% yield,
a)
H
SiMe3
Me3Si
H
TfO
Ts
OTf Ts
N
O
O
H
N
N
N
N
H
SR2*
H
SR2*
Me
Me
TfO
ii
TfO
Me
TfO
Ts
Ts
Ts
Me
Me
SR2*
*R2S
i
iii
TfO
OTf
O
O
O
TS1
slow
TS2
Me3SiOTf, SR2*
T > -30 °C
fast
H
O
H
N
OMe
N
O
N
Me
Ts
Me
Me
racemic
O
(+)-1
O
(±)-10
(-)-1
b)
induce selectivity given its previous application in
Ts
chiral sufide
OMe
Me
O
N
24
N
S
Me3SiOTf
enantioselective MBH reactions.
Unfortunately, while the
Ts
Me
Me
Me
cyclized product was isolated in excellent yield, no
enantioselectivity was observed (entry 2). Our attention was
S
Me
Me
Me
22
23
O
then turned toward
a series of C2-symmetric trans-2,5-
(±)-10
21
sulfide temp. time yield ee
Ph
Ph
S
disubstituted thiolanes. The bis-primary alkyl substituted thiolane
24 gave the product in 68% yield with an unimpressive ee of –
6% (entry 3). Diphenyl thiolane 25 gave 21 in 22% ee with a
drop in yield to 53%.25 In this case, the recovered sulfide had
entry solvent
24
MeCN
1
23
24
25
26
27
28
29
30
30
29
30
0 °C
2 h 85%
-4%
S
MeCN
2
0 °C
3 h 85% 0%
2 h 68% -6%
22 h 53% -21%
25
MeCN
3
0 °C
partially epimerized to
a mixture of cis- and trans-2,5-
4a
5b
6
MeCN
-20 °C
-15 °C
0 °C
S
diphenylthiolane (dr 14:86; 89% ee for the recovered trans
isomer), likely due to formation of a benzylic carbocation under
the reaction conditions. To prevent epimerization, we sought to
install suitable electron withdrawing groups on the phenyl rings.
To this end, sulfide 26 was synthesized and applied in our
optimization. The para-CF3 groups did indeed prevent
racemization; however, the yield and purity of the product
declined drastically compared to 26 (entry 5). The use of
dicyclopentyl sulfide 27 gave the product in reasonable yield
with an improved ee of 44%. We then tested dicyclohexyl and
ditetrahydropyranyl thiolanes 28 and 29, postulating that
increasing the steric bulk of the thiolane substituents could
potentially lead to greater selectivity. In both cases, improved
F3C
CF3
26
MeCN-CH2Cl2
MeCN-CH2Cl2
MeCN-PhMe
MeCN-CH2Cl2
MeCN-CH2Cl2
MeCN-PhMe
MeCN-CH2Cl2
24 h 19%
-
3 h 59% 44%
3 h 40% 62%
3 h 29% 82%
2 h 50% 78%
24 h 42% 70%
4 h 28% 93%
S
27
7
0 °C
8
0 °C
9c
10
11
0 °C
S
28
-40 °C
-78 °C
S
aEpimerization of the sulfide was observed. bTBSOTf was
used instead of Me3SiOTf. cMe3SiOTf was added at -78 °C,
stirred for 1 h, then warmed to 0 °C and stirred 1 h.
O
O
29
Scheme 3. a) Proposed dynamic kinetic resolution process through an
enantioselective aza-MBH reaction. b) Optimization of the chiral sulfide used
for enantioselective cyclization.
3
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10.1002/anie.202004464
Angewandte Chemie International Edition
COMMUNICATION
82% ee (entry 8), respectively. For sulfide 29, adding Me3SiOTf
at –78 °C, stirring for 1 h, and then allowing the mixture to stir at
0 °C for 1 h led to a substantial increase in the yield (50%) with a
similar ee (78%, entry 9). Finally, we also explored conditions in
which the reaction temperature was kept low enough to prevent
imine rearrangement (below –30 °C) in an attempt to achieve
simple kinetic resolution. Performing the cyclization at –40 °C
using sulfide 28 gave the cyclized product in a slightly improved
yield of 42% and 70% ee (entry 10). Using sulfide 29 and
performing the reaction at –78 °C, the cyclized product was
Keywords: anatoxin-a
•
isotopic total synthesis
•
•
enantioselective aza-Morita-Baylis-Hillman
iminium rearrangement
• chiral sulfide
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06/documents/anatoxin-a-report-2015.pdf
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a
remarkable
enantioselectivity of 93% ee. These observations indicate that
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https://www.epa.gov/sites/production/files/2017-
10/documents/epa_815-r-15-009_method_545.pdf
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Our approach to [13C4]-anatoxin-a was framed by a need to
develop a route which commenced from a commercially feasible
isotopically-labeled starting material and utilized transformations
that would retain all heavy isotopes. These constraints inspired
the first synthesis of [13C4]-1, relying on a Sharpless asymmetric
epoxidation as the enantiodetermining step, and an MBH
cyclization to forge the final bicyclic scaffold of the anatoxin-a
skeleton. The synthesis was completed in 12 steps from [13C4]-
15 and 14% overall yield through the kinetic resolution strategy.
As a testament to the scalability of this route, 0.110 g of [13C4]-1
have been prepared to date. The ability to access substantial
quantities of [13C4]-1 will enable the development of a new
analytical method for its precise and highly accurate
quantification in samples of fresh water. Finally, an observation
made during the penultimate MBH cyclization in our original
route prompted an investigation into a unique dynamic kinetic
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Zakarian, J. Am. Chem. Soc. 2018, 140, 6027.
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Acknowledgements
This work was supported by the NIH (NIGMS, R01-077379 and
NIEHS, R03 ES025345-01). J.J.L. thanks the Natural Sciences
and Engineering Research Council of Canada (NSERC) for a
postgraduate scholarship (PGS-D) and UCSB for a Chancellor’s
Fellowship. Dr. Hongjun Zhou is acknowledged for assistance
with NMR spectroscopy. Dr. Dmitriy Uchenik and the UCSB
mass spectroscopy facility are thanked for assistance with mass
spectral analysis.
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10.1002/anie.202004464
Angewandte Chemie International Edition
COMMUNICATION
Entry for the Table of Contents
We report an asymmetric total synthesis of the isotopically labelled cyanotoxin [13C4]-anatoxin-a. A critical feature of the synthesis
was the development of a unique enantioselective Morita-Baylis-Hillman cyclization that leverages a cyclic iminium ion racemization
to achieve dynamic kinetic resolution. The synthesis produced substantial quantities of this internal standard with 99.1% isotope
incorporation to enable the development of an analytical method for the precise quantification of anatoxin-a in freshwater.
5
This article is protected by copyright. All rights reserved.