Investigating the Toxicity of the Aeruginosin Chlorosulfopeptides by Chemical Synthesis

Article abstract of DOI:10.1002/anie.201602755

Harmful algal blooms are becoming more prevalent all over the world, and identification and mechanism-of-action studies of the responsible toxins serve to protect ecosystems, livestock, and humans alike. In this study, the chlorosulfopeptide aeruginosin 8

Full text of DOI:10.1002/anie.201602755

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International Edition: DOI: 10.1002/anie.201602755
German Edition: DOI: 10.1002/ange.201602755
Algal Blooms
Investigating the Toxicity of the Aeruginosin Chlorosulfopeptides by
Chemical Synthesis
Manuel Scherer, Dominik Bezold, and Karl Gademann*
Abstract: Harmful algal blooms are becoming more prevalent
all over the world, and identification and mechanism-of-action
studies of the responsible toxins serve to protect ecosystems,
livestock, and humans alike. In this study, the chlorosulfopep-
tide aeruginosin 828A, which rivals the well-known toxin
microcystin LR in terms of crustacean toxicity, has been
synthesized for the first time. Furthermore, three congeners
with different permutations of the chloride and sulfate groups
were prepared, thereby enabling toxicity studies without the
risk of contamination by other natural toxins. Toxicity assays
with the sensitive crustacean Thamnocephalus platyurus dem-
onstrated that the introduction of a sulfate group leads to
pronounced toxicity, and NMR spectroscopic evidence sug-
gests that the chloride substituent modulates the conformation,
which in turn might influence protease inhibition.
H
armful algal blooms are increasingly prevalent in many
freshwater and marine ecosystems, and their massive occur-
rence poses a severe threat to drinking water supplies,
fisheries, and recreational areas alike.[1] Research on the
most prevalent cyanobacterial toxin microcystin provided
important insight into its toxicity and molecular mecha-
nisms,[2] which led the World Health Organization (WHO) to
set threshold values considered safe for drinking water.[2]
Over the last years, many additional cyanobacterial strains
that do not produce microcystin have been discovered, which
resulted in the characterization of new compounds considered
to be toxic to aquatic organisms.[3] However, their mecha-
nisms of action and the molecular basis of the processes
leading to death remain unclear for many structures.[3]
Figure 1. Aeruginosin 828A (1) and aeruginoside 126A (2) as well as
the synthetic analogues aeruginosin 748A (3) and 794A (4).
out for these isolated compounds, we sought to investigate
their toxicity by chemical synthesis followed by in vivo
toxicity studies. In particular, we were interested in 1) syn-
thesizing and characterizing peptides with all permutations
with regard to the sulfate and chloride groups present
(Figure 1), and 2) evaluating the effect of these functional
units on toxicity. Herein, we report the successful synthesis of
four natural products and putative congeners, which then
enabled toxicity studies with the crustacean Thamnocephalus
platyurus.
As the first two targets for the synthesis, we chose the
naturally occurring chlorosulfopeptide aeruginosin 828A (1),
which bears a sulfated xylose (Xyl) residue and a chloroleu-
cine (Cleu) unit, and aeruginoside 126A (2),[6] which is devoid
of sulfate and chloride groups. Interestingly, chlorosulfopep-
tide 1 was isolated from a toxic Planktothrix strain lacking
microcystin production, whereas peptide 2 originates from
a Planktothrix strain that is capable of producing micro-
cystins.[7] These observations support the hypothesis that
chlorosulfopeptide 1 restores the toxic phenotype of the
cyanobacterium, whereas compound 2 should be less toxic.
Additional experimental support corroborating this hypoth-
esis was provided by Blom and co-workers, who reported 1 to
be highly toxic.[4] Surprisingly, the toxicity of peptide 2 had
not been investigated, which provided an additional stimulus
for the work reported herein. To further evaluate this
hypothesis and to study the influence of the structure on
toxicity, we chose the synthetic analogues aeruginosin 748A
Chlorosulfopeptides such as aeruginosin 828A have
recently emerged as harmful compounds restoring the toxic
phenotype of microcystin-deficient bacteria (Figure 1).[4]
Whereas chlorosulfolipids such as danicalipin have been
intensively studied by chemical synthesis with regard to the
structural requirements for toxicity,[5] research on chlorosul-
fopeptides has to date been restricted to compounds obtained
by isolation from natural sources.[4] As contamination with
other toxins or biologically active compounds cannot be ruled
[*] M. Scherer, D. Bezold, Prof. Dr. K. Gademann
Department Chemie, Universitꢀt Basel
St. Johanns-Ring 19, 4056 Basel (Switzerland)
M. Scherer, Prof. Dr. K. Gademann
Department Chemie, Universitꢀt Zꢁrich
Winterthurerstrasse 190, 8057 Zurich (Switzerland)
E-mail: karl.gademann@uzh.ch
Supporting information and the ORCID identification numbers for
the authors of this article can be found under http://dx.doi.org/10.
1002/anie.201602755.
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(3) and 794A (4) as targets, as 3 contains the chloroleucine
moiety, but no sulfate group, and 4 features the sulfate group,
but is devoid of chloride substitution.
product formation while remarkably leaving the dihydropyr-
role moiety untouched.
The l-2-carboxy-6-octahydroindole (l-Choi) subunit was
synthesized following the aza-Prins route developed for the
total synthesis of oscillarin,[13,14] which gave the l-Choi
building block 9 in 6.4% yield over eleven synthetic steps.
The chloroleucine derivative 10 was synthesized over ten
steps and in an overall yield of 7% from isobutyraldehyde
according to a method that was used in the total synthesis of
chlorodysinosin A.[15] Peptide coupling of chloroleucine
derivative 10 with the MOM-protected phenyllactic acid
(Pla) derivative 11[16] followed by oxidation of the alcohol to
the acid gave the Cleu/Pla building block 12. For the
derivatives of aeruginosin 828A lacking the chloride substitu-
ent, a building block without the chlorine atom was synthe-
sized through the coupling of phenyllactic acid derivative 11
with leucine (Leu) methyl ester (13).[17] Cleavage of the
methyl ester gave the final intermediate 14 in excellent yield.
With all subunits in hand, we proceeded with the assembly
of the different building blocks (Scheme 2). Our strategy
started with the challenging a-xylosylation of the l-Choi core
unit 9. Starting from building block 6, several donors
containing a sulfate group protected as the trichloroethyl
ester at the O4 position were prepared.[18] However, all
attempts to use these donors with protected sulfate groups
for the glycosylation were not successful, as the electron-
withdrawing nature of the sulfate group reduced the reactivity
(“disarming”) of the different donors,[19] leading to either
little conversion or the preferred formation of the b-anomer.
We therefore decided to introduce the sulfate group at a later
We started with the preparation of the required building
blocks for the various chlorosulfopeptides (Scheme 1). The
selective installation of the sulfate group at the O4 position of
the xylosyl moiety required orthogonal protection of the O4
position with respect to positions O2 and O3. Therefore, we
used a base-labile protecting group for O4 and an acid-labile
group for O2 and O3. The xylose moiety was synthesized
starting from the known xyloside 5, which was obtained from
commercially available d-xylose in three steps.[8] Regioselec-
tive protection of the O4 hydroxy group with a benzoyl (Bz)
protecting group was achieved in 81% yield using benzoyl
chloride and Me2SnCl2 as catalyst.[9] The O2 and O3 hydroxy
groups were protected as the butane diacetal (BDA) using
2,3-butanedione, trimethyl orthoformate, and camphorsul-
fonic acid.[10] Initial attempts of the BDA protection had
always led to mixtures of isomers; however, after optimiza-
tion of the reaction conditions, xylose derivative 6 could be
obtained as a single diastereoisomer.
The 1-(N-amidino-D3-pyrrolino)ethyl (Adc) side chain 8
was synthesized following a slightly modified procedure by
Hanessian and co-workers,[11] which led to the intermediate
dihydropyrrole 7 in ten synthetic steps and 8% overall yield
from commercially available a-methylene-g-butyrolactone.
Whereas Hanessian and co-workers had chosen a Staudinger
reduction for the conversion of the azide into the amine, we
opted for a catalytic hydrogenation using Lindlar catalyst.[12]
This procedure led to an increased yield of 96% with no side
Scheme 1. Synthesis of the different building blocks. Reagents and conditions: a) benzoyl chloride, Me2SnCl2, DIPEA, THF/H2O (9:1), 258C, 81%;
b) 2,3-butanedione, HC(OCH3)3, CSA, MeOH, 678C, 69%; c) Lindlar cat., H2, MeOH, 258C, 96%; d) PyBOP, 2,6-lutidine, CH2Cl2, 258C, 81%;
e) LiOH, THF/H2O (5:3), 258C, 98%. Boc=tert-butoxycarbonyl, Bz=benzoyl, Cbz=carboxybenzyl, CSA=camphorsulfonic acid, DIPEA=N,N-
diisopropylethylamine, MOM=methoxymethyl, PyBOP=(benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate, THF=tetrahydro-
furan.
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of the secondary amine of the l-Choi group, which is
responsible for long reaction times of several days for the
coupling. The elimination can be explained by the formation
of an oxazolone intermediate during the peptide coupling that
is readily deprotonated, leading to the elimination of HCl.[24]
To overcome the problem of elimination, different bases, such
as NMM, DIPEA, or NaHCO3, were tested in combination
with DEBPT as the coupling reagent, but without success.
Adjusting the temperature also had no critical influence on
the outcome of the reaction. We therefore started screening
different coupling reagents (e.g., PyBOP, bromotripyrrolidi-
nophosphonium hexafluorophosphate (PyBrOP), and 2-(7-
aza-1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexa-
fluorophosphate (HATU)), and found 4-(4,6-dimethoxy-
1,3,5-triazin-2-yl)-4-methylmorpholinium
chloride
(DMTMM) to be the best promoter for the coupling.[25]
With DMTMM as coupling reagent and NMM as base, the
coupling was completed after only two hours, resulting in an
excellent yield of 87% with no elimination of HCl. Hydrolysis
of methyl ester 17 to acid 18 was achieved in 0.1n aqueous
LiOH solution. The progress of the reaction was monitored
by ultra-performance liquid chromatography (UHPLC),
which allowed for timely quenching of the reaction, as
longer reaction times again led to the elimination of HCl.
Acid 18 was further coupled with the Adc unit 8 using PyBOP
as the coupling reagent and 2,6-lutidine as the base to give
tetrapeptide 19 in a moderate yield of 60%. Sulfatation of the
O4 hydroxy group of the xylose moiety was achieved using an
excess of SO3–pyridinium complex in pyridine. Global
deprotection of the Boc, MOM, and BDA groups was carried
out in TFA/CH2Cl2 solution to obtain aeruginosin 828A (1) in
72% yield over two steps. When intermediate 19 was directly
subjected to global deprotection under acidic conditions,
aeruginosin 748A (3), which lacks the SO3 group, was
obtained in 70% yield.
Following the reaction sequence developed for the syn-
thesis of aeruginosin 828A, we were also able to synthesize
the analogues aeruginoside 126A (2) and aeruginosin 794A
(4). Coupling of the Leu/Pla dipeptide 14 with l-Choi/Xyl 16
gave tripeptide 20 in moderate yield. Ester hydrolysis of 20
and further coupling with the Adc side chain 8 gave
tetrapeptide 21, which served as an intermediate for the
synthesis of aeruginoside 126A (2) and synthetic analogue 4 in
good yields. Comparison of the NMR spectra of synthetic and
previously isolated aeruginoside 126A (2) showed slight
differences in the chemical shifts. These differences might
result either from different amounts of water in the NMR
samples or from a residual counterion as a result of the HPLC
purification. Similar observations have already been reported
by our group for different natural products.[26] However,
definitive proof that the synthetic and isolated samples are
identical can only be obtained by recording NMR spectra of
equimolar mixtures of the two samples or by HPLC co-
injection. Furthermore, the absolute configuration of the
xylose moiety had not been assigned during the isolation
work, which, however, should result in larger differences in
the NMR spectra in the case of isomeric xyloses.
Scheme 2. Synthesis of aeruginosin 828A (1) and aeruginoside 126A
(2) as well as derivatives 3 and 4. Reagents and conditions: a) 6, NIS,
AgOTf, Et2O, 258C, 50%; b) PdCl2, Et3SiH, Et3N, 258C, 88%; c) 12 or
14, DMTMM, NMM, CH2Cl2, 0!258C (17: 87% from 16; 20: 58%
from 16); d) 0.1n LiOH, THF/H2O (5:3), 258C (18: 77% from 17; 21:
95% from 20); e) 8, PyBOP, 2,6-lutidine, CH2Cl2, 258C (19: 60% from
18; 22: 64% from 21); f) SO3·pyridine, pyridine, 508C; g) CH2Cl2/TFA
(10:1), 258C (1: 72% from 19; 4: 75% from 22); h) CH2Cl2/TFA
(10:1), 258C (3: 70% from 19; 2: 73% from 22). DMTMM=4-(4,6-
dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride, NIS=
N-iodosuccinimide, NMM=4-methylmorpholine, TFA=trifluoroacetic
acid.
stage of the synthesis and focused on donors with the benzoyl
protecting group at O4 and different leaving groups at the
C1 position instead. Among the donors surveyed, xyloside 6,
with thiophenol as the leaving group, in combination with NIS
as activator and AgOTf as promoter, showed the most
promising selectivity and reactivity.[20] After optimization, the
xylosylation gave the desired product with an a/b ratio of 5:3
and a good yield of 81%. Attempts to remove the Cbz group
by catalytic hydrogenation (Pd/C, Pd(OH)2, and H2)[11]
resulted in little or no conversion. Pleasingly, when 15 was
treated with PdCl2 and Et3N dissolved in triethylsilane
according to a method developed by Birkofer and co-work-
ers,[21] the desired free amine 16 was formed in 88% yield.
Next, we investigated the coupling of amine 16 with the
Cleu side chain 12. Initial attempts with 3-(diethoxyphos-
phoryloxy)-1,2,3-benzotriazin-4(3H)-one (DEBPT) as cou-
pling reagent and 2,6-lutidine as base led to the elimination of
HCl.[22] Similar behavior or racemization had already been
observed in the syntheses of other aeruginosins.[15,23] These
undesired side reactions are likely due to the poor reactivity
It is interesting to note that the late-stage intermediates
containing the Cleu side chain, including 1 and 3, appeared as
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Figure 2. The amide bond rotamers of the l-Choi–Cleu and l-Choi–Leu
units are in equilibrium.
Figure 3. To study the acute toxicity of compounds 1–4 towards
T. platyurus, the mortalities were determined as a function of the
concentration of these compounds.
a single rotamer, or as a large excess of one rotamer (> 50:1),
in the H NMR spectra (Figure 2). Analysis of the ROESY
1
spectra of 1 and 3 indicates that the trans rotamer dominates,
as nuclear Overhauser effects (NOEs) were observed
between Cleu H2 and Choi (H7, H7’, and H7a). However,
the intermediates devoid of the chloride substituent, includ-
ing 2 and 4, appeared as rotameric mixtures (ca. 4:1) around
the l-Choi–Leu peptide bond. The presence of two rotamers
for 2 had already been reported by Dittmann and co-
workers[6] and was additionally confirmed by the exchange
cross-peaks between the methyl groups of both rotameric
leucine units in the ROESY spectra of 2 and 4. The trans
rotamers of 2 and 4 showed similar NOEs as described before,
whereas the cis rotamers gave NOEs between the Leu H2 and
l-Choi H2 hydrogen atoms. The halogen substituent there-
fore appears to have a critical effect on the conformation of
the different aeruginosins by restricting the rotation around
the l-Choi–Leu amide bond; a related phenomenon has
already been observed by Hanessian and co-workers for
unnatural aeruginosin hybrids. Furthermore, these authors
suggested that such conformational effects increase protease
binding.[27]
With the four derivatives in hand, we were interested in
evaluating the effect of the sulfate and chloride groups on
bioactivity. The environmental toxicity was studied in stan-
dard assays with the sensitive freshwater crustacean Tham-
nocephalus platyurus. For this assay, six concentrations
ranging from 0.41 mm to 100 mm for 1 and 1.2 mm to 150 mm
for 2, 3, and 4 were tested in an acute toxicity assay (24 h).
For every concentration, three to four experiments with 10 to
16 animals per experiment were conducted, and the mortality
after 24 h was determined by visual inspection of the animals
(Figure 3). Blom et al. had reported an LC50 value of 22.4 mm
against T. platyurus for 1,[4] which is only slightly higher than
the toxicity of the known biotoxin microcystin.[28] Our assay
with synthetic aeruginosin 828A showed a comparable tox-
icity of 34.5 mm. Analogue 3, which lacks the sulfate group,
showed a toxicity of 24.2 mm, which is in the same range as
that of 1. Interestingly, the dechloro derivative 4 showed an
increased and potent toxicity of 12.8 mm. As demonstrated
above by NMR spectroscopy, the chlorine substituent has
a strong impact on the conformation of the aeruginosins. It
has also been reported that the chlorine substituent in the
leucine moiety is important for the inhibition of enzymes such
as thrombin.[13b,27] For toxicity, however, this “chlorine effect”
appears to be detrimental, which is likely due to the restricted
rotation around the peptide bond between the l-Choi and the
leucine residues, which could result in an entropic penalty.
Most interestingly, derivative 2, which contains neither the
sulfate group nor a chlorine substituent, showed a significantly
lower toxicity of 57.7 mm. Overall, the bioassays support the
hypotheses that 1) the introduction of either chloride or
sulfate groups leads to increased toxicity, and 2) the chloride
group in combination with the sulfate moiety leads to an
attenuation of toxicity. Both hypotheses are supported by
ecological observations: Chlorosulfopeptides such as 1 are
produced in microcystin-deficient, but still toxic cyanobac-
teria,[4] which supports the hypothesis that these chlorosulfo-
peptides restore the toxic phenotype. In contrast, the much
less toxic congener 2, which lacks both groups, is found in
a microcystin-producing strain.[7] Therefore, a cyanobacterium
can restore its toxicity by the introduction of one sulfate
group upon loss of the gene for microcystin production
(switching from 2 to 4).
In conclusion, the first total synthesis of aeruginosin 828A
(longest linear sequence: 18 steps, overall yield: 13% from
known building block 9) has been reported. With the
developed synthetic route, aeruginoside 126A and the syn-
thetic analogues aeruginosin 794A and aeruginosin 748A
were readily synthesized. Key features of the synthesis
include the a-xylosylation of the l-Choi core unit 9 and the
peptide coupling of amine 16 with Cleu moiety 12. The
synthesized compounds were tested with regard to their
toxicity against T. platyurus, demonstrating that the sulfate
group has a critical effect on toxicity. This finding supports the
hypothesis that chlorosulfopeptides are important for restor-
ing the toxic phenotype in strains that are incapable of
microcystin production. This work therefore supports the
conclusion that the toxicity of cyanobacteria should not be
assessed in terms of microcystin production alone.
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We thank Priv.-Doz. Dr. D. Hꢀussinger and S. Jurt for
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for X-ray crystallographic analysis.
Keywords: algal blooms · medicinal chemistry ·
natural products · total synthesis · toxicology
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Received: March 18, 2016
Published online: && &&, &&&&
Angew. Chem. Int. Ed. 2016, 55, 1 – 6
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
5
These are not the final page numbers!

Angewandte
Communications
Chemie
Communications
Algal Blooms
Toxic brew: Four chlorosulfopeptide con-
geners that are thought to be responsible
for the adverse effects of algal blooms
were prepared by chemical synthesis. The
roles of the chloride and sulfate groups
were then established by toxicity assays
with the crustacean Thamnocephalus pla-
tyurus.
M. Scherer, D. Bezold,
K. Gademann*
&&&& — &&&&
Investigating the Toxicity of the
Aeruginosin Chlorosulfopeptides by
Chemical Synthesis
6
www.angewandte.org
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2016, 55, 1 – 6
These are not the final page numbers!