Short Total Synthesis of [15N5]-Cylindrospermopsins from 15NH4Cl Enables Precise Quantification of Freshwater Cyanobacterial Contamination

Article abstract of DOI:10.1021/jacs.8b03071

Fresh water cyanobacterial algal blooms represent a major health risk because these organisms produce cylindrospermopsin, a toxic, structurally complex, zwitterionic uracil-guanidine alkaloid recognized by the EPA as a dangerous drinking water contaminant. At present, the ability to detect and quantify the presence of cylindrospermospin in water samples is severely hampered by the lack of an isotopically labeled standard for analytical mass spectrometry. Herein, we present a concise, scaled total synthesis of ¹⁵N cylindrospermosin from ¹⁵N ammonium chloride, which leverages a unique stereoselective intramolecular double conjugate addition step to assemble the tricyclic guanidine core. In addition to providing the first pure isotopically labeled probe for precise quantification of this potent biotoxin in fresh water sources, our results demonstrate how unique constraints associated with isotope incorporation compel novel solutions to synthesis design.

Full text of DOI:10.1021/jacs.8b03071

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Short Total Synthesis of [15N5]-Cylindrospermopsins from 15NH4Cl
Enables Precise Quantification of Freshwater Cyanobacterial Contamination
Artur K. Mailyan, Joanna L. Chen, Weiwei Li, Arturo A.
Keller, Shawn Sternisha, Brian G. Miller, and Armen Zakarian
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.8b03071 • Publication Date (Web): 19 Apr 2018
Downloaded from http://pubs.acs.org on April 19, 2018
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Short Total Synthesis of [15N5]-Cylindrospermopsins from
15NH4Cl Enables Precise Quantification of Freshwater Cya-
nobacterial Contamination
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Artur K. Mailyan,† Joanna L. Chen,† Weiwei Li,‡ Arturo A. Keller,‡ Shawn M. Sternisha,§ Brian G. Miller,§ Armen
Zakarian*,†
† Department of Chemistry and Biochemistry, University of California, Santa Barbara, California 93110, United States
‡Bren School of Environmental Science and Management, University of California, Santa Barbara, California 93106, United States
§ Department of Chemistry and Biochemistry, Florida State University, Florida 32310, United States.
ABSTRACT: Fresh water cyanobacterial algal blooms represent a major health risk because these organisms produce cylindrospermopsin, a
toxic, structurally complex, zwitterionic uracil-guanidine alkaloid recognized by the EPA as a dangerous drinking water contaminant. At pre-
sent, the ability to detect and quantify the presence of cylindrospermospin in water samples is severely hampered by the lack of an isotopical-
15
15
ly-labeled standard for analytical mass spectrometry. Herein, we present a concise, scaled total synthesis of N cylindrospermosin from N
ammonium chloride, which leverages a unique stereoselective intramolecular double conjugate addition step to assemble the tricyclic guani-
dine core. In addition to providing the first pure isotopically labeled probe for precise quantification of this potent biotoxin in fresh water
sources, our results demonstrate how unique constraints associated with isotope incorporation compel novel solutions to synthesis design.
of hydrogen, carbon, nitrogen and oxygen for this purpose. To
ensure structural integrity of the analytical standard, isotope inser-
tion at non-exchangeable positions throughout the synthesis is
essential. Based on these two key requirements, we chose to pursue
Introduction
In recent years, cyanobacterial algal blooms have occurred with
increasing frequency near population centers worldwide, causing
significant socioeconomic impact and risk to human health.1 Cylin-
drospermopsin (CYN)2 and its naturally occurring 7-epi-3 and 7-
deoxy-4 congeners are potent biotoxins released into fresh water
sources during such blooms5 (Figure 1a,b). In June 2015, the US
Environmental Protection Agency released a drinking water health
advisory on cylindrospermopsins detailing this emerging environ-
mental threat.6 In addition to the acute toxicity risks, exposure to
cylindrospermopsin and its derivatives is linked to hepatotoxicity,
gastrointestinal inflammation, hemorrhages, anorexia and liver
failure.7
the
total
synthesis
of
[15N1,15N3,15N16,15N18,15N19]-
cylindrospermopsin ([15N5]-CYN, [15N5]-1, Figure 1c). The deci-
sion to incorporate 15N in the cylindrospermopsin scaffold required
us to rethink past synthetic strategies applied to the total synthesis
of CYN and structurally related alkaloids, as these approaches are
incompatible with efficient label installation at non-exchangeable
atomic positions. In addition, these prior strategies, while elegant in
design and execution, are laborious, requiring 20 or more steps, and
present uncertainties regarding scalability.15,16,17,18,19,20,21,22 Similarly,
preparation of labeled cylindrospermospins via fermentation using
enriched nutrients such as Na15NO3 has proven untenable.23 These
key concerns prompted the development of a de novo approach,
whereby we explored whether isotope incorporation at the design
stage could lead to an improved synthetic strategy that is more
effective than biosynthetic or synthetic strategies reported previ-
ously.
High-performance liquid chromatography coupled with mass
spectrometry is the preferred method for cylindrospermopsin de-
tection in environmental samples.8,9,10,11,12,13 In particular, isotope-
dilution mass spectrometry (IDMS)14 provides an attractive quanti-
fication method, as it uses an isotope of the analyte as an internal
reference standard to ensure high sensitivity and precision in the
measurement. The application of IDMS to cylindrospermopsin
analysis, however, is hampered by the lack of isotopically labeled
cynlidrospermopsins. To solve this important problem, we coupled
recent advances in synthetic chemistry and enzymatic bio-
oxidation to prepare 7-epi- and 7-deoxy-cylindrospermopsin from
[15N]-ammonium chloride, achieving >99.5% isotope incorpora-
tion into all five nitrogen atoms of these complex alkaloid natural
products.
Several principal considerations factored into the synthesis plan-
ning. Clearly, the effective introduction of the 15N isotope becomes
a major factor. Compared to a typical total synthesis effort, the
payoff of a convergent versus linear, atom-by-atom assembly is now
substantially enhanced because all but the most elemental sources
15
15
of the N isotope (15NH4Cl or aqueous NH3) become impracti-
24
cal. Thus, a convergent design with preassembled intermediates
containing several N isotopes is essential. The common metric of
15
To facilitate mass spectrometry data interpretation, we sought an
isotope of CYN with mass difference of at least 5 atomic units rela-
tive to the most abundant natural isotope. We considered isotopes
synthetic efficiency, namely, minimizing the number of individual
steps based on robust, reproducible chemical transformations be-
comes especially critical given the costs of the starting iso-
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topes.25,26,27 Finally, all intended transformation must ensure reten-
tion of the isotopic label.
strate for a hydroxyl-directed stereoselective delivery of free [15N3]-
guanidine that we previously developed for this application.28
1
2
3
4
5
6
7
8
a
The success of this plan rests on identifying a suitable substituent
Z, which must first activate the diene for addition of guanidine,
then facilitate C7 hydroxylation, and eventually be converted to
uracil. Although several options can be considered, heterocyclic
substituents such as 2,4-dimethoxypyrimid-6-yl appeared to be
especially enticing as precursors of uracil. Various electron-
deficient heterocylic groups are known to stabilize carbanions, and
activation of double bonds by substitution with heterocycles for
nucleophilic addition has been reported.29,30,31,32 What remained
uncertain was whether diene activation for double addition of guan-
idine, a centerpiece of our strategy, was feasible. After a careful
analysis supported by experimentation, we identified 2,4-
dimethoxypyrimid-6-yl as group Z because of its potential to meet
these criteria most effectively.
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microphotograph of Cylindrospermopsis raciborskii
Preparation of [15N]-labeled Precursors. While the initial valida-
tion of the entire total synthesis was carried out with the natural
isotope, we performed the scaled synthesis starting with 15NH4Cl as
depicted in Scheme 1. [15N]-Ammonium chloride proved to be the
most economical source of dry liquid [15N]-ammonia on the labor-
atory scale, affording >95% yields of the reagent reproducibly upon
treatment with NaOH in solid phase as long as the scale exceeds 0.5
g. [15N2]-2,4-Dimethoxy-6-bromopyrimidine [15N2]-5 was ob-
b
O
H
N
O
O
O
O
O
O
S
S
5
NH
HO
O
O
O
O
12
3 NH
O
10
7
7
X
1
N
H
N19
N
NH
NH
H
H
18
15
N
H
N16
H
cylindrospermopsin, CYN
X = OH, 7-epi-cylindrospermopsin
X = H, 7-deoxycylindrospermopsin
c
15
tained by the initial conversion of NH3 to urea,33 its condensation
with diethyl malonate to [15N2]-barbituric acid [15N2]-4, treatment
double alkene addition
of guanidine
CH oxidation
at C7
O
O
O
with POBr3, and methanolysis with 2 equiv. of NaOMe. In contrast
OR
OR
S
15NH
O
O
10
8
15
HO
7
to the synthesis of urea, the reaction of NH3 with diphenyl thio-
Z
Z
15N
H
15N
H
15N
15N
15NH
15N
15N
O
carbonate gave ammonium thiocyanate, pyrolysis of which was
required to access [15N2]-thiourea.34 The preparation of [15N3]-
guanidine hydrochloride was accomplished in four steps by S-
methylation, treatment with Boc2O, displacement of MeS group
15NH
H
15N
15N
Mbs
Mbs
H
i, Mbs = 4-methoxybenzenesulfonyl
ii
[
15N5]-CYN
Z = electron-withdrawing group
alkyne-diene
rearrangement
15
isotope availability
is a key constraint
with NH3 in chloroform, and complete hydrolysis with 12 M HCl
at reflux. The resulting [15N3]-guanidine hydrochloride and [15N2]-
5 constitute the source of all 15N isotopes in the target alkaloid.
H215N 15NH2
15NH
H215N 15NH2
Z
O
O
Total synthesis of [15N5]-Cylindrospermopsins. Advancing the
synthesis of [15N5]-cylindrospermopsin, bromopyrimidine [15N2]-5
was subjected to Sonogashira coupling with 5-hexyn-1-ol (Scheme
2). After Swern oxidation of [15N2]-7 to [15N2]-8, efficient
enantioselective crotylation was achieved using Leighton’s reagent
935 (99% yield, dr 10:1, er 97:3). To shorten the synthesis, we
explored direct conversion of [15N2]-7 to [15N2]-10 using a
powerful method developed by Krische and co-workers.36 Although
the enantiocontrol was excellent (er 98:2), lower
diastereoselectivity (4:1) and yield (21%) thwarted its application
for the scaled synthesis of [15N2]-10 (see the supporting
information for additional details). The next objective was the
appendage of guanidine unit to the unactivated terminal alkene in
[15N2]-10. This was achieved by a two-stage approach described
O
15N 15
NH
Z
OH
15 N
H
directed cyclo-
guanidylation
Mbs
15NH4Cl
iv
iii
Figure 1. (a) A confocal optical microscopy image of Cylindrospermop-
sis raciborskii, (b) the structures of cylindrospermopsins, and (c) the
outline of the synthetic design for [15N5]-CYN.
Results and Discussion
Synthesis Design. Following the logic outlined above, we formu-
lated a synthesis plan depicted in Fig 1c. [15N3]-Guanidine and
15
[15N2]-urea, each derived from NH4Cl, serve as precursors of the
tricyclic guanidine and uracil subunits of cylindrospermopsin, re-
spectively. The synthesis plan requires a late stage conversion of the
electron-withdrawing group Z in intermediate i to uracil and C–H
oxidation at C7. The electron-withdrawing group Z enables a key
simplification to diene ii by disconnection of two C–N bonds in
tricyclic guanidine i at C8 and C10. Stereoselective intramolecular
addition of guanidine to diene, although lacking precedent, is ex-
pected to ensure rapid assembly of the tricyclic guanidine subunit
of cylindrospermopsins. Alkyne iii was intended as a precursor to
diene ii via a transition metal-mediated rearrangement. As n essen-
tial element of the plan, homoallylic alcohol iv serves as the sub-
15
28
previously. First, free [ N3]-guanidine was attached to the hydroxy
group by a carbonyl linker in good yield using 1.2 equiv. of 15N-
labeled reagent. Second, electrophilic cycloguanidylation was
performed in the presence of N-iodosuccinimide, delivering [15N5]-
12 after treatment with 4-methoxybenzenesulfonyl chloride
(MbsCl) in 71% yield and 2.5:1 dr favoring the requisite
diastereomer.
Alkyne [15N5]-12 served as an effective precursor of the 1,3-diene
required for the key double addition of guanidine.37,38,39,40
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Scheme1. The preparation of [15N2]-2,4-dimethoxy-6-bromopyrimidine and [15N3]-guanidine hydrochloride from NH4Cl, the key precur-
sors constituting the source of all 15N isotopes in cylindrospermopsins.
1. POBr3, reflux, 110 oC, 3 h
CH2(CO2Et)2 NaOEt
(PhO)2CO, H2O, reflux, 6 h
94% yield from 15NH4Cl
2. NaOMe, MeOH, Et2O, 23 oC, 20 h
EtOH, reflux, 48 h
Br
MeO
15N 15N
O
O
H215N 15NH2
O
H15
N
15NH
90% yield
72% yield
7
8
9
OMe
O
15NH4Cl
NaOH
15N2]-4
[
15N2]-5
[
15NH3
4.15 g
10
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(PhO)2CS, H2O
sealed tube,
110 oC (oil bath
1. 15NH3, CHCl3, 23 oC, 96 h
2. 12 M aq. HCl, 100 oC, 1.5 h
1. CH3I, MeOH
160 oC, 2 h
N
H15
N
15NH2
2. Boc2O, NaHCO3
Boc15
N
15NHBoc
H215N 15NH2
S
temperature), 6 h
15NH4SC15
HCl
15NH2
2.3 g
SMe
15N2]-6
95% yield
from 15NH4Cl
57% yield
(6 cycles, 29%
per cycle)
99% yield
71% yield
[
Scheme 2. Incorporation of all five 15N isotopes into advanced intermediate [15N5]-16 from [15N2]-2,4-dimethoxy-6-bromopyrimidine
[15N2]-5 and [15N3]-guanidine hydrochloride.
4-BrC6H4
N
Si
9
Cl
N
4-BrC6H4
(COCl)2, Me2SO
Et3N, CH2Cl2
2 mol % Pd(PPh3)2Cl2, 4 mol % CuI
5-hexyn-1-ol, Et3N, 23 oC, 2 h
5 mol % Sc(OTf)3, CH2Cl2, ⎯20 o
C
MeO
15N 15N
OMe
Br
MeO
15N 15N
MeO
15N 15N
OMe
OH
O
98% yield
99% yield from 7, dr 10:1, er 97:3
OMe
[
15N2]-7
[
15N2]-5
[
15N2]-8
Im2CO, THF, 23 o
then THF, DMF, 23 o
C
C
N-iodosuccinimide
CH3CN, 0 oC, then
H15
N
15NH2
MeO
O
O
MeO
MeO
15N 15N
OMe
MbsCl, iPr2NEt, CH2Cl2
OH
O
O
15NH2
15N 15N
OMe
15N 15
NH
15N 15N
OMe
H15
N
15NH2
86% yield
15 N
H
71% yield, dr 2.5 :1
15NH
Mbs
15N2]-10
[
15N5]-11
[
15N5]-12
[
5 grams
OMe
15N
CO2Me
MeOH, iPr2NEt, CH3CN
82 oC, 24 h
25 mol % (PPh3)4Pd, 0.5 equiv PhCO2H
1,4-dioxiane, 101 oC, 1 h
O
O
O
MeO
15N 15
NH
15N 15N
OMe
15N
OMe
H
CH3
15N
15NH
15 N
75% yield
H
15N
Mbs
15N5]-14
[
15N5]-13, E:Z 6:1
Mbs
[
3 grams
MeO
MeO
15N
15N
15N
15N
CO2Me
O
OH
NaOMe, MeOH, 23 oC, 0.5 h
8
OMe
OMe
CH3
15N
15N
15N
CH3
15N
15N
15N
>93% yield from 13
dr 3:2 (at C8)
Mbs
15N5]-16
Mbs
[
[
15N5]-15
We identified (Ph3P)4Pd and benzoic acid at reflux in dioxane as
the optimal reaction system for the challenging substrate
containing two heterocyclic substituents. Diene [15N5]-13,
obtained in 75% yield as a 6:1 mixture of E and Z isomers, was now
poised to implement the key cyclization to the tricyclic guanidine
ring system of CYNs. Simple heating [15N5]-13 in 14% methanol in
rapid methanolysis to [15N5]-14. Remarkably, the key addition of
guanidine to the diene took place concurrently with methanolysis
under these mild reaction conditions, and subsequent removal of
methyl carbonate in [15N5]-14 was accomplished with NaOMe in
situ, affording [15N5]-16. The success of this process was essential
to ensure the brevity of the total synthesis, and demonstrated the
i
acetonitrile (v/v) in the presence of Pr2NEt at reflux resulted in
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Scheme 3. Total synthesis of [15N5]-CYN, [15N5]-7-epi-CYN, and 7-deoxy-CYN by a) C7 bio-hydroxylation with Cyrl and b) stereoselective
chemical oxidation via lithiation at C7.
a. Chemoenzymatic oxidation [most direct]
5
6
7
8
9
O
O
O
O
Cyrl (2.5 mol %)
O
O
7
H
1. 12 M aq. HCl,
100 oC, 3 h
15N
H
S
15N
S
FeSO4, ascorbic acid
2-ketoglutarate, 28 oC, 2 h
O
O
H
H
O
O
10
11
12
13
14
15
16
17
18
19
20
21
22
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25
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HO
H
O
15N
2. SO3•Py, DMF
O
15N
Me
[15N5]-16
15N
15N
15NH
Me
15N
15N
H
15NH
H
95% yield
95% yield, dr 25:1
H
8.6 mg scale
H
15N5]-3, [15N5]-7-deoxy-CYN
[
15N5]-1, [15N5]-CYN
[
O
b. Chemical oxidation [amenable gram to scale]
H
15N
OR
HO
12
O
15N
Me
15N
15N
15NH
H
H
H
[15N5]-16
SO3∙Py, DMF
77% yield from
[
15N5]-21, 75 mg
[
15N5]-22, (R = H)
MeO
MeO
15N
LDA, ⎯78 oC, 5 min
-
15N
15N
[
15N5]-1, (R = SO3
)
12 M aq. HCl
100 oC, 3 h
Et3SiCl, ImH
CH2Cl2
then oxidant
2 min
OSiEt3
OSiEt3
HO
7
8
OMe
OMe
15N
+
Me
Me
15N
15N
15N
15N
15N
15N
Mbs
15N5]-21
O
99% yield
dr 10:1
51% yield
H
93% yield
from 13
H
15N
(84% brsm)
Mbs
OR
H
dr 3:2 (at C8)
O
15N
15N5]-17
1.5 grams
[
[
Me
oxidant
15N
15NH
H
OH
O
O
15N
N
N
PhO2S
Ph
H
S
O2
[
15N5]-23, (R = H)
SO3∙Py, DMF
7% yield from
15N5]-21, 7 mg
(+)-20: dr 20:1
(⎯)-20: dr 25:1
19: dr 6:1
[
-
[
15N5]-2, ((R = SO3
15N5]-7-epi-CYN
)
[
activating properties of the 2,6-dimethoxy-6-pyrimidinyl substitu-
ent for addition of nitrogen nucleophiles to alkenes.
demonstrating the advantages of combining traditional organic
chemistry with enzymatic transformations.
To address one of the most challenging phases of the synthesis,
the stereospecific hydroxylation of the C7 methylene group of
[15N5]-7-deoxy-CYN ([15N5]-3), we explored enzymatic biooxida-
tion. This conversion represents the final step of CYN biosynthesis
As a comparison to the enzymatic approach, and to gain access
the 7-epi congener of cylindrospermospin, we also investigated
chemical oxidation of [15N5]-16 (Scheme 3b). Silylation of [15N5]-
16 with Et3SiCl (TESCl) afforded tricyclic guanidine [15N5]-17 in
93% overall yield from diene [15N5]-13 as a separable 3:2 mixture of
diastereomers at C8, favoring the required R configuration. Re-
placement of methyl carbonate with the TES group in [15N5]-17
facilitated two practical objectives 1) enabling a simple chromato-
graphic separation of C8 diastereomers, and 2) compatibility with
basic conditions required for the ensuing C7 metallation and hy-
droxylation. Although the substrate was sensitive to the nature and
stoichiometry of the base, we found that rapid lithiation with LDA
(5 min, –78 °C) followed by a quench with an oxaziridine afforded
the diastereomeric C7 hydroxylation products in good yields along
with recovered starting material. The diastereomer ratio is depend-
ent on the oxaziridine: rac-19 afforded a 6:1 dr, while camphor
based reagent 20 or ent-20 gave 25:1 and 20:1 dr in favor of [15N5]-
1, respectively. The optimized hydroxylation was scaled to 1.50 g of
and is catalyzed by CyrI, an α-ketoglutarate-dependent non-heme
iron oxygenase. We produced pure, recombinant CyrI (Oscillatoria
strain PCC7926) in E. coli and applied the enzyme in the direct
oxidation of ([15N5]-3), which was readily generated from [15N5]-
16 by acidic hydrolysis (12 M HCl, reflux) and O-sulfonation
(SO3•Py, DMF, 95% overall yield) (Scheme 3a).41 The enzyme
showed high selectivity and did not accept a substrate lacking the
O12 sulfate, suggesting that O-sulfonation must precede C7 hy-
droxylation in the biosynthesis of CYN. Importantly, CyrI enabled
a catalytic, protecting-group-free oxidation to afford the final natu-
ral product in 93% yield with >90:1 diastereoslectivity achievable
with high enzyme loading. The biocatalytic synthesis produced
15
uniformly N labeled CYN in 10 total steps with 15% overall yield,
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[15N5]-17, providing [15N5]-21 as a pure isomer in 46% yield along
se efforts provided a high-quality isotopically labeled probe for the
reliable detection and quantification of cyanotoxins in fresh water
sources with the highest limits of detection reported to date. Our
results demonstrate how atypical constraints associated with iso-
tope incorporation empower the development of novel approaches
to organic synthesis design.
1
2
3
4
5
6
7
8
with 41% of starting material. Complete hydrolysis to uracil along
with the removal of the Mbs group was achieved on 0.350 g scale of
[15N5]-21 by reflux in 12 M HCl for 3 h, resulting in a minor epi-
merization at C7 (10:1). The mixture of epimers [15N5]-22 and
[15N5]-23 was submitted to O-sulfonylation with SO3•Py complex
18
in DMF. After optimizing the reagent loading, we observed highly
ASSOCIATED CONTENT
selective monosulfation of the diols at the C12 OH group. The
resulting mixture was readily separated by reverse-phase HPLC,
affording 75 mg of [15N5]-CYN (77% yield) and 6.8 mg of [15N5]-7-
epi-CYN (7% yield) in a single attempt. By comparison with bioox-
idation using CyrI, the chemical oxidation shows improved scala-
bility, but introduces one additional step in the total synthesis with
slightly diminished overall yield and lower stereoselectivity.
Supporting Information
9
Full experimental procedures and copies of 1H, 13C, 15N NMR spec-
tra and crystallographic information is provided in the Supporting
Information. The Supporting Information is available free of charge on
the ACS Publications website.
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With the supply of [15N5]-CYN secured by total synthesis, its de-
velopment as an analytical standard for the precise quantification of
CYN by LC/ESI-MS/MS was undertaken. Based on reported con-
centrations of the cyanotoxin in environmental samples, we devel-
oped an assay for the detection of cylindrospermopsin in a broad
range from 10 ng/L to 100 µg/L. While the EPA limit for cylin-
drospermopsin in drinking water has been proposed to be 1 µg/L,
AUTHOR INFORMATION
Corresponding Author
* zakarian@chem.ucsb.edu
Present Addresses
†If an author’s address is different than the one given in the affiliation
line, this information may be included here.
,42
7
concentrations below this level still represent an environmental
hazard due to bioaccumulation and uncertain health risks of chron-
ic sub-lethal exposure.43 The limit of detection (LOD, Signal/Noise
= 3) and the limit of quantification (LOQ, Signal/Noise = 10) for
the method were determined to be 2.5 ng/L and 8.25 ng/L respec-
tively, the highest sensitivity reported to date.8-13 The evaluation of
matrix effect for analyte quantification was performed using envi-
ronmental samples free of native CYN that were spiked with 100
µg/L. In all cases the observed recoveries were nearly 100%. This
method was then used to quantify cylindrospermopsin in environ-
mental water samples in fourteen locations in Southern California
where potential contamination or water resources was suspected.44
Notably, availability of [15N5]-CYN afforded the reliable and pre-
cise detection of cylindrospermopsin in Lake Casitas, a previously
undescribed location of cyanotoxin contamination, where we de-
tected the concentration of 157 ng/L of the toxin.
Author Contributions
The manuscript was written through contributions of all authors. / All
authors have given approval to the final version of the manuscript. /
‡These authors contributed equally. (match statement to author
names with a symbol)
Funding Sources
Any funds used to support the research of the manuscript should be
placed here (per journal style).
Notes
Any additional relevant notes should be placed here.
ACKNOWLEDGMENT
This work was supported by NIH (NIGMS, R01-077379, R01-115388
and NIEHS, R03 ES025345-01). 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. Dr. Guang Wu and the UCSB X-ray analytical facility
are acknowledged for assistance with and the X-ray crystallography. We
are especially grateful to Anastasiia S. Minakova for assistance with
enzymatic oxidation experiment.
Concluding Remarks
In summary, we developed a strategy for the concise total syn-
thesis of cylindrospermopsins that is directly applicable for 15N
isotope incorporation. We demonstrated the scalability of the syn-
thesis by preparing 0.125 g of [15N5]-CYN in 14 steps (longest
linear sequence) and 9% overall yield from [15N]-ammonium chlo-
ride, along with 7.5 mg of [15N5]-7-epi-CYN and 20 mg of [15N5]-7-
deoxy-CYN. In the natural isotope series, the total synthesis of
CYN was completed from commercially available 2,4-dimethoxy-6-
bromopyrimidine (5) in only 10 steps via the enzymatic oxidation
route (15% yield from 5), and 11 steps by chemical C7 hydroxyla-
tion (11% yield), representing a substantial improvement in brevity
and scalability compared to the previous efforts. Furthermore, the-
Dr. Ben Lopez and the NRI-MCDB Microscopy Facility at UCSB are
acknowledged for obtaining microphotographs of Cylindrospermopsis
raciborskii.
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TOC graphic:
O
O
O
H
S
O
O
HO
14 steps
O
15N
H
15NH4Cl
15N
15N
15NH
H
H
[
15N5]-cylindrospermopsin
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