Total synthesis of Herbarin A and B, determination of their antioxidant properties and toxicity in zebrafish embryo model

Article abstract of DOI:10.1016/j.bmcl.2015.01.065

Herbarin A and B were isolated from the fungal strains of Cladosporium herbarum found in marine sponges Aplysina aerophoba and Callyspongia aerizusa. Total synthesis of Herbarin A and B was achieved by carrying out a multi-step synthesis approach, and the antioxidant properties were evaluated using FRAP assay. Toxicity of these compounds was determined using a zebrafish embryo model.

Full text of DOI:10.1016/j.bmcl.2015.01.065

Bioorganic & Medicinal Chemistry Letters 25 (2015) 1192–1195
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Total synthesis of Herbarin A and B, determination of their
antioxidant properties and toxicity in zebrafish embryo model
Julia Heimberger a, Hannah C. Cade a, Jihan Padgett b, Vinoth Sittaramane b, Abid Shaikh a,
⇑
a Department of Chemistry, Georgia Southern University, 521 College of Education Drive, Statesboro, GA 30460-8064, USA
b Department of Biology, Georgia Southern University, 1332 Southern Drive, Statesboro, GA 30460-8042, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Herbarin A and B were isolated from the fungal strains of Cladosporium herbarum found in marine
sponges Aplysina aerophoba and Callyspongia aerizusa. Total synthesis of Herbarin A and B was achieved
by carrying out a multi-step synthesis approach, and the antioxidant properties were evaluated using
FRAP assay. Toxicity of these compounds was determined using a zebrafish embryo model.
Published by Elsevier Ltd.
Received 6 December 2014
Revised 27 January 2015
Accepted 29 January 2015
Available online 4 February 2015
Keywords:
Total synthesis
Antioxidant properties
Natural products
Toxicity
Zebrafish
Herbarin A and B were recently isolated by Jadulco and co-
workers1 from two strains of the fungi Cladosporium herbarum
found in sponges Aplysina aerophoba and Callyspongia aerizusa,
respectively. Spectroscopic analysis of ethyl acetate extracts from
ing blocks and using a modified approach described by Wilcox
and group.3 3-Methyl-2,4-pentanedione (3) was treated with
NaNH2 and then reacted with dry ice to provide diketo-acid (4)
in 77% crude yield. Diketo-acid (4) without any purification was
then treated with HF to afford the 4-hydroxypyrone (5) in about
39% yield. Additionally, etherification provided 4-methoxypyrone
(6) and upon oxidation with SeO2 selectively converted the methyl
group at position C-6 to aldehyde (7) in quantitative yield. After
successful execution of the synthetic scheme, aldehyde (7) was
synthesized in multi-gram quantities to be utilized in further
reactions.
Aldehyde (7) was then reacted with phosphonate ester (8) using
Horner–Wadsworth–Emmons coupling strategy to obtain an
exclusively trans-alkene (9) in about 90% yield.4 Hydrolysis of the
ester group with 3M HCl yielded Herbarin B (2) as a white solid.
All products were purified using column chromatography and
structure elucidation was carried out using various spectroscopic
techniques including NMR and mass-spectrometry. Lastly, the
spectroscopic data for Herbarin B was compared with literature
data to confirm the product formation. Herbarin B was deliberately
chosen as the first synthetic target considering it’s lesser complex-
ity compared to Herbarin A.
these fungi revealed two new a-pyrone derived structures, Herba-
rin A and B. Various spectroscopic techniques such as, NMR and
mass-spectrometry were used for structural elucidation. To deter-
mine the initial biological activity, a feeding assay was performed
on polyphagous pest insect larvae and brine shrimp larvae.1,2 As a
consequence of their potent toxicity towards insect and brine
shrimp larvae and unique chemical structure, we decided to pur-
sue the laboratory syntheses of Herbarin A and B. A multistep syn-
thetic approach provided the target compounds in multi-gram
scale quantity, which is not possible from the natural source. Hav-
ing the larger quantities allowed for further investigation of the
potential toxicity and antioxidant properties of these natural prod-
ucts (Fig. 1).
Herein, we describe the synthesis of target compounds using a
multi-step reaction sequence as described in Scheme 1. We identi-
fied an aldehyde (7) as the crucial intermediate that can afford
both the target compounds. As a first step, our research efforts
were directed towards the synthesis of 4-hydroxypyrone (5) by
utilizing commercially available starting materials as basic build-
After successfully accomplishing the synthesis of Herbarin B,
Herbarin A was targeted by using a similar synthetic scheme. The
aldehyde intermediate (7) was treated with commercially avail-
able predominantly trans-phosphonate ester (10) to form an exclu-
⇑
Corresponding author. Tel.: +1 912 478 0973; fax: +1 912 478 0699.
E-mail address: malnu@georgiasouthern.edu (A. Shaikh).
http://dx.doi.org/10.1016/j.bmcl.2015.01.065
0960-894X/Published by Elsevier Ltd.

J. Heimberger et al. / Bioorg. Med. Chem. Lett. 25 (2015) 1192–1195
1193
O
O
O
OH
OH
O
O
O
O
O
Herbarin A
1
Herbarin B
2
Figure 1. Structures of Herbarin A and Herbarin B.
0
sively trans–trans-ester (11) in 81% yield, which on further hydro-
lysis with 3M HCl provided Herbarin A in 67% yield (Scheme 2).
Determination of antioxidant properties: Ferric Reducing Ability
of Plasma (FRAP) assay: A wide range of assays is known for mea-
suring the reducing capacity of antioxidants. The assays are carried
out at acidic (FRAP), neutral (TEAC), or basic (total phenols assay
FCR) conditions. The pH values have an important effect on the
reducing capacity of antioxidants.5 Herbarin A and B both have
carboxylic acid groups and to maintain the integrity of these mol-
ecules, we choose to use the acidic FRAP assay. Also FRAP assay has
several advantages such as high reproducibility, simple, rapidly
performed and showed the highest efficiency for acids and phe-
nols.6 Therefore, it would be an appropriate technique for deter-
mining antioxidant assay for Herbarin A and B. The FRAP assay,
is presented as a novel method for assessing ‘antioxidant power’.
Ferric to ferrous ion reduction at low pH causes a colored fer-
rous–tripyridyltriazine complex to form. FRAP values are obtained
by comparing the absorbance change at 593 nm in test reaction
mixtures with those containing ferrous ions in known concentra-
tion. Absorbance changes are linear over a wide concentration
range with antioxidant mixtures, including plasma, and with solu-
tions containing one antioxidant in purified form.7 The FRAP assay
is inexpensive, reagents are simple to prepare, results are highly
reproducible, and the procedure is straightforward and speedy.
The FRAP assay offers a putative index of antioxidant, or reducing
Figure 2. Antioxidant activity of Herbarin A and B.
potential of organic compounds within the technological reach of
every laboratory. The FRAP assay was employed as described in
the literature.8 The mechanism of this method is based on the
reduction of ferric 2,4,6-tripyridyl-s-triazine complex (Fe3+–TPTZ)
to its ferrous form (Fe2+–TPTZ) in the presence of antioxidants. A
solution of acetate buffer 25 mL, 2.5 mL of Fe3+–tripyridyl-triazine
(Fe3+–TPTZ) (10 mmol/L in 40 mmol/L of HCl, and 2.5 mL FeCl3
(20 mmol/L) was then prepared. This solution (300
mixed with Herbarin A and B (at 10, 20, 40, and 80
30 L of distilled H2O. The electron-donating capacity of the anti-
oxidant was measured by the change in absorbance at 593 nm;
the blue-colored Fe2+–tripyridyl-s-triazine (Fe2+–TPTZ) compound
was formed from colorless oxidized Fe3+–TPTZ. The absorbance of
the reaction mixture was measured spectrophotometrically at
593 nm after incubation at room temperature for 1 h. Calibration
curves were generated from aqueous solutions of FeSO4 at different
concentrations ranging from 0.1 to 1 mM. Both Herbarin A and B
showed activity towards antioxidant assay at 0.1–1 mM concentra-
tions (Fig. 2).
l
L) was then
lg/mL) and
l
OH
O
O
1. NaNH2
SeO2
O
O
O
O
O
ether/ -20 o
C
H+
dioxane
DMS
100 o
C
OH
K2CO3
O
2. CO2
O
O
5
O
O
6
O
O
80 o
C
4
3
H
7
O
O
O
O
EtO
EtO
O
P
O
3M HCl
70 oC
8
H
O
H
O
H
OH
OEt
KOt-Bu / toluene
O
O
O
O
O
THF / 0 oC-RT
H
O
H
9
O
7
Herbarin B
2
Scheme 1. Synthetic strategy for Herbarin B.
O
O
EtO
EtO
O
O
O
P
O
3M HCl
70 o
10
H
H
O
H
H
O
H
OH
C
OEt
KOt-Bu / toluene
O
O
O
O
O
THF / 0 oC-RT
H
H
O
H
H
O
7
Herbarin A
11
1
Scheme 2. Synthetic strategy for Herbarin A.

1194
J. Heimberger et al. / Bioorg. Med. Chem. Lett. 25 (2015) 1192–1195
Figure 3. Quantitative summary of lethality and teratogenicity in zebrafish embryos.
Figure 4. Zebrafish development in Herbarin A and Herbarin B treatment.
Toxicity assay: To assess the biological value of Herbarin A and B,
observed toxicity and teratogenicity at similar concentrations
(Fig. 4C and D). We performed at least 3 replicates of the zebrafish
assay. Statistical analyses using student t-test revealed significant
we used a zebrafish embryo screening assay. Herbarin A and B
were tested for toxicity and teratogenicity in zebrafish
embryos.9–13 These assays were performed using an established
literature procedure.9–13 Briefly, wild-type zebrafish embryos were
collected from spawn tanks and allowed to grow until 6 hpf (hours
post fertilization) at standard conditions.14 At 6 hpf, embryos were
observed under a microscope for embryonic development and only
normally developing embryos were transferred to wells in 24-well
plate and allowed to grow in 1 mL of treatment solution for up to
2 dpf (days post fertilization). Observations were made at 24 hpf
and solutions were replaced as necessary. Herbarin A and B were
dissolved in 100% DMSO after purification, further they were resus-
pended in E3 (embryonic medium) at various concentrations
(Figs. 3 and 4) for zebrafish assay. Therefore our controls were
WT (wild-type) embryos in E3 with same concentrations of DMSO
(Fig. 4A and B) as in Herbarin A and B solutions. This study had
identified that Herbarin B is more biologically active than Herbarin
A (Figs. 3 and 4). Lethality assay was performed at concentrations
from 9 nM to 475 nM and these studies revealed that Herbarin B
has a LD50 (50% lethal dose) of 190 nM (Fig. 3). Herbarin B dis-
played dynamically increasing lethality from 9 nM to 190 nM con-
centrations beyond which it is completely lethal (Fig. 3). Herbarin
B was also 100% teratogenic at concentrations of 9–190 nM (Fig. 4).
Teratogenic phenotypes were specific showing a shortened body
axis resembling gastrulation defects during early development,
dorsal curvature of the trunk, tail malformations and pericardial
edema (Fig. 4E and F; black arrows denote dorsal curvature of
trunk and tail malformations). While Herbarin B was lethal and
teratogenic, Herbarin A appears to be a docile compound with no
⁄
difference (Fig. 3; p <0.001) between DMSO control and Herbarin
B observations and no significant differences (Fig. 3; ⁄⁄p <0.001)
between DMSO control and Herbarin A treatment. In summary,
the zebrafish assays have identified that Herbarin B is more biolog-
ically active compared to Herbarin A.
In conclusion, we have successfully achieved the synthesis of
Herbarin A and Herbarin B utilizing commercially available start-
ing materials and following known literature synthetic protocols.
All the products were characterized using analytical techniques.
The antioxidant activity of target compounds was investigated.
Furthermore, the toxicity assay using zebrafish embryo was also
investigated. The bioassay results indicated the potent toxicity of
Herbarin B.
Acknowledgments
Financial support provided by Georgia Southern University, Col-
lege Office of Under-graduate Research (COUR-GSU) to HCC and
National Science Foundation, USA (DMR 1229292) is gratefully
acknowledged.
Supplementary data
Supplementary data (experimental procedure and NMR charac-
terization data of all the products) associated with this article can
be found, in the online version, at http://dx.doi.org/10.1016/j.bmcl.
2015.01.065.

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Nesnas, N. J. Nat. Prod. 2011, 74, 455.
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