Combining semi-synthesis with plant and microbial biocatalysis: New frontiers in producing a chemical arsenal against cancer

Article abstract of DOI:10.1039/c8ra02184h

Natural products (NPs) that exhibit anticancer activities are frequently not potent enough to be used clinically as therapeutics. Semi-synthesis and metabolic engineering are promising approaches for producing more efficacious derivatives of anticancer NPs (ACNPs), but each technique alone can be inefficient at obtaining specific ACNP derivatives that may be suspected to have enhanced anticancer activity. Here, we demonstrate that the methods of semi-synthesis and biocatalysis can be used as modules in succession and in different combinations to produce 6,8-dibromogenkwanin, a derivative of the ACNP apigenin. Further, we demonstrated that soybean seed coats can be used as a biocatalyst to convert brominated flavonoids into multiple derivatives. A strength of the combinatorial (bio)synthesis approach was that the order of the modules could be rearranged to increase the yield of the desired product. At lower treatment concentration (5 μM), 6,8-dibromogenkwanin exhibited enhanced antiproliferative activities against HT-29 colorectal adenocarcinoma cancer cells under normoxic and hypoxic conditions compared to its ACNP precursors, but not at higher concentrations. Dose-response analyses suggested that dibromogenkwanin had a distinct mode-of-action compared to apigenin. Thus, this proof-of-concept paper demonstrates combinatorial (bio)synthesis as an approach that can be used to produce novel chemistries for anticancer research.

Full text of DOI:10.1039/c8ra02184h

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PAPER
Combining semi-synthesis with plant and microbial
biocatalysis: new frontiers in producing a chemical
Cite this: RSC Adv., 2018, 8, 21332
arsenal against cancer†
c
Samuel Gary,a Janet Adegboye,‡b Brian Popp,
Jean-Christophe Cocuron,§b
d
Brooklyn Woodruma and Nik Kovinich
*
Natural products (NPs) that exhibit anticancer activities are frequently not potent enough to be used
clinically as therapeutics. Semi-synthesis and metabolic engineering are promising approaches for
producing more efficacious derivatives of anticancer NPs (ACNPs), but each technique alone can be
inefficient at obtaining specific ACNP derivatives that may be suspected to have enhanced anticancer
activity. Here, we demonstrate that the methods of semi-synthesis and biocatalysis can be used as
modules in succession and in different combinations to produce 6,8-dibromogenkwanin, a derivative of
the ACNP apigenin. Further, we demonstrated that soybean seed coats can be used as a biocatalyst to
convert brominated flavonoids into multiple derivatives. A strength of the combinatorial (bio)synthesis
approach was that the order of the modules could be rearranged to increase the yield of the desired
product. At lower treatment concentration (5 mM), 6,8-dibromogenkwanin exhibited enhanced
antiproliferative activities against HT-29 colorectal adenocarcinoma cancer cells under normoxic and
hypoxic conditions compared to its ACNP precursors, but not at higher concentrations. Dose–response
analyses suggested that dibromogenkwanin had a distinct mode-of-action compared to apigenin. Thus,
this proof-of-concept paper demonstrates combinatorial (bio)synthesis as an approach that can be used
to produce novel chemistries for anticancer research.
Received 12th March 2018
Accepted 4th June 2018
DOI: 10.1039/c8ra02184h
rsc.li/rsc-advances
cellular target, (ii) the chemical stability, or (iii) the transport.
Modication of the NP structure can also reduce the number of
Introduction
cellular targets and thus adverse side effects.
Bioactive natural products (NPs) commonly must be adminis-
tered at concentrations that are too high to permit clinical use.
High drug dosages generally increase the likelihood of off-target
molecular interactions that can disrupt cellular processes and
lead to adverse side effects. For this reason, drugs that bind
single cellular targets at low micromolar or lesser concentra-
tions are most suitable for use as drugs. The vast majority of
small molecules that were approved by the U.S. FDA as anti-
cancer drugs between 1981 and 2014 were derived from NPs and
were mostly semisynthetic derivatives.1 The modication of NPs
by semi-synthesis generally serves to improve drug efficacy by
enhancing: (i) the strength of molecular interaction with the
Semi-synthesis methods for chemical modication are
generally highly efficient and amenable to scale-up. However,
approaches can have limitations, particularly in maintaining
stereochemistry or in making certain regiospecic modica-
tions without the need for additional blocking agents that can
be costly or environmentally unfriendly. With the advent of
genetic engineering, biocatalysts such as engineered bacterial
or yeast have emerged as important alternatives for drug
modication. The benets of biocatalysis are that enzymes
generally maintain stereochemistry and are highly regiospecic.
Yet, a shortcoming of using biocatalysis to modify NPs can be
that enzymes are very limited in the substrates that they accept.
For these reasons, novel methods are continuously being
developed in metabolic engineering and semi-synthesis to
circumvent the limitations to generate new drug molecules that
have enhanced capability and efficiency. Yet, there has been
little focus on combining the methods of biocatalysis and semi-
synthesis to (bio)synthesize derivatives of NPs that are sus-
pected to possess enhanced medicinal activities but cannot be
efficiently produced by either method alone.
aUndergraduate Intercollegiate Biochemistry Program, Division of Plant and Soil
Sciences, West Virginia University, 333 Evansdale Drive, 26506, Morgantown, USA.
E-mail: nikovinich@mail.wvu.edu
bDepartment of Molecular Genetics, Ohio State University, Columbus, Ohio, USA
cEugene Bennett Department of Chemistry, West Virginia University, Morgantown, USA
dDivision of Plant and Soil Sciences, West Virginia University, Morgantown, USA
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c8ra02184h
‡ Current address: Lerner College of Medicine, Cleveland Clinic, Cleveland, USA.
§ Current address: University of North Texas, 1504 W. Mulberry st., Denton, TX
76201.
Flavonoids are a family of polyphenol secondary metabolites
that have a 3-phenylchromen-4-one backbone. More than 6000
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avonoids are biosynthesized in plants. Epidemiological studies anticancer activities.8 Based on these studies, we hypothesized
have associated avonoids with diverse health benets including that methylating the 7-position in addition to brominating the
reduced incidence of the onset of certain types of cancers. This has 6- and 8-positions of apigenin would enhance its anticancer
stimulated the testing of various avonoids as antiproliferative and activity. Since 6,8-dibromogenkwanin could not be produced
antitumor agents. Despite the notable anticancer activities of using current semi-synthesis or biocatalysis protocols alone, we
a broad spectrum of avonoids, few molecules have exhibited proposed a novel combinatorial approach. Beginning with the
strong enough potency to warrant their assessment as therapeutics anticancer NP (ACNP) apigenin, we aimed to determine whether
in clinical trials. Exceptions include the soybean-derived isoavone the methods of semi-synthesis and microbial biocatalysis could
genistein, a highly specic inhibitor of protein tyrosine kinase be performed in succession in different combinations to
(PTK) that blocks the mitogenic effect mediated by EGF.2 Genistein produce 6,8-dibromogenkwanin. Further, we demonstrated
is currently the focus of Phase 2 clinical trials for the treatment of soybean seed coats as a plant biocatalyst for converting
colorectal, bladder, brain and other types of cancers. Since the vast brominated avonoids that did not exhibit enhanced anti-
majority of avonoids are not potent enough for clinical use, this cancer activity into novel derivatives. The combinatorial (bio)
has stimulated efforts to develop derivatives that have enhanced synthesis approach can be viewed as a novel approach that takes
anticancer activities. A successful example is avopiridol, a semi- advantage of the strengths of each method (i.e. the high yield of
synthetic derivative of rotukine that was approved by the Food semi-synthesis, the regiospecicity of recombinant enzymes
and Drug Administration (FDA) to treat B-cell chronic lymphocytic expressed in microbial systems, and the promiscuity of plant
leukemia (B-CLL) and acute myeloid leukemia (AML) and is in metabolism) to generate a plethora of structural variants with
Phase I – II clinical trials for the treatment of a number of other increasing structural complexity from an ACNP (Fig. 1).
cancers. Since so many avonoid molecules have moderate anti-
proliferative and/or antitumor activities, a logical step in devel-
oping novel anticancer drugs may be to modify such molecules in
ways that are suspected to enhance their bioactivities.
Materials and methods
Materials
The rice (Oryza sativa) OsNOMT gene codon-optimized for
expression in E. coli (GenBank accession MF991211) was
synthesized by Invitrogen (Carlsbad, CA). Apigenin and gen-
kwanin were from Carbosynth (San Diego, CA) and VWR (Rad-
nor, PA), respectively. NBS was from Sigma Aldrich (St. Louis,
MO). UPLC-MS solvents were Optima grade and ash chroma-
tography solvents were from Fisher Scientic (Hampton, NH).
NMR solvents were from Cambridge Isotope Laboratories
(Tewksbury, MA). HT-29 and HCT 116 cells were generous gis
from Houjian Cai (University of Georgia).
Apigenin is a member of the avone subclass of avonoids
that has notable anticancer activity but must be administered at
relatively high concentrations (50–100 mM) to inhibit the
proliferation of cancer cells by more than 50%.3,4 Apigenin
induces apoptosis at these high treatment concentrations
possibly by inhibiting a pathway that results in NF-kB activa-
tion.3–5 Due to its insufficient potency, apigenin is a prime
candidate for the improvement of its anticancer activities by
derivatization. Halogenation of the 6- and 8- positions of the
phenylchromen ring is a modication that has been found to
enhance the antioxidant activity and lipophilicity of several
avonoids, and render them more potent in inhibiting the
proliferation of cancer cell lines compared to their precursor
NPs.6,7 Further, methylation of specically the 7-hydroxyl of
apigenin to produce genkwanin signicantly alters the
Biocatalysis with E. coli expressing synthetic OsNOMT
Synthetic OsNOMT was Gateway recombined into pDEST42
using the recombinase LR Clonase II (Thermosher Scientic,
Pittsburgh, PA). Fully sequenced constructs were transferred
Fig. 1 Schematic representation of the combinatorial (bio)synthesis approach for producing derivatives of anticancer natural products (ACNPs)
for bioactivity testing. The process begins with an ACNP; mainly a plant phenylpropanoid or (iso)flavonoid. The ACNP may be modified by semi-
synthesis prior to being fed to a bacteria, yeast, or plant cultures. Subsequent to a preliminary round of modification, the unnatural derivative may
be further altered by feeding to plant cultures prior to being testing in the activity panel. Each successive round of modification increases the
structural complexity of the compound. The activity panel consists of antiproliferation assays of human cancer cell lines.
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into E. coli BL21(DE3) pLysS (Novagen). For optimal biocatalysis
of apigenin, cells were cultured in 30 mL of LB broth in 250 mL
asks to an OD600 of 0.8, then induced with 1 mM of IPTG.
Aer 4 hours, apigenin or 6,8-dibromoapigenin was added to
a nal concentration of 60 mM from a 6 mM stock in DMSO.
Following overnight culture, the bacterial pellet was extracted
with 80% ethanol (v/v) by heating 80 ꢀC for 3 hours. Ethanol was
evaporated under a stream of nitrogen gas, water was added to 5
mL, and the aqueous phase was extracted three times with an
equal volume of ethyl acetate. The bacteria growth medium was
also extracted three times with an equal volume of ethyl acetate.
Residues from bacteria pellet and growth medium were resus-
pended in ethanol and pooled for analysis by LC-MS or DMSO-
d6 for analysis by NMR.
Chemical analyses
Compounds were structurally characterized by exact mass
UPLC-PDA-MSn and by 1H NMR spectroscopy. Reaction extracts
were evaporated under a stream of nitrogen gas and weighed.
For UPLC-PDA-MSn, residues were dissolved in ethanol and
analyzed by the method of Farrell et al. 2017 (ref. 12) following
the addition of an equal volume of water. For NMR, residues
were dissolved in DMSO-d6. For determination of percent yield
of reactions, MS parent ion or PDA peak areas at 337 nm were
compared among reactant NP and brominated products to
determine relative amounts.
For soybean biocatalysis reactions, extracts were analyzed
using an Agilent 1290-QTRAP 5500 LC-MSn (AB Sciex Framing-
ham, MA) in precursor ion scanning mode to identify molecules
that have lost a bromine during MSn fragmentation. Extract
(5 mL) was injected onto a symmetry 3.5 mM, 4.6 ꢂ 75 mm C18
Semi-synthesis
column incubated at 30 ꢀC with a ow rate of 1 mL minꢁ1
.
Bromination of avonoids was performed as indicated.9 Briey,
N-bromosuccinimide (0.036 mmol) in 0.5 mL acetone was
added dropwise to 0.018 mmol of avonoid in 0.5 mL acetone.
The mixture was stirred at 22 ꢀC for 43 min, then extracted three
times with an equal volume of ethyl acetate to remove succini-
mide. Reaction with apigenin yielded a bright yellow powder
whereas genkwanin yielded a brown-orange amorphous solid.
6,8-Dibromo-genkwanin was puried from mono- and tri-
brominated products by ash chromatography on silica gel 60
(particle size 0.040–0.063 mm, 230–400 mesh ASTM) from Sili-
Solvent A and B were acetonitrile and water in 0.1% formic acid,
respectively. The composition of A was 20% from 0–2 min, 20–
95% from 2–18 min, 95% 18–20 min, then held at 20% for 4 min
to stabilize pressure prior to the following injection. MSn
settings are listed in ESI, Table S3.†
Human cell culturing and in vitro proliferation assays
HT-29 cells were cultured in DMEM (Gibco, Gaithersburg, MD)
with 10% FBS (Fisher Scientic, Hampton, NH). HCT-116 cells
were in 45% DMEM, 45% F12, and 10% FBS medium. Cells were
cultured in a GALAXY CO48R HT CO2 and N2-equipped tri-gas
incubator (Eppendorf, Hauppauge, NY) at 37 ꢀC, 5% CO2,
95% humidity and 19% or 0.5% O2 for normoxia and hypoxia,
respectively. All compounds were dissolved in DMSO to 10 mM
´
cycle (Ville de Quebec, QC) using a gradient 10% / 40% ethyl
acetate/hexanes with constant 5% methanol. Fractions were
evaluated by TLC and like samples combined for analysis by 1H
NMR spectroscopy.
ꢀ
and stored at ꢁ80 C. Treatments were prepared fresh by seri-
Biocatalysis with soybean seed coats
ally diluting stock solutions to 90, 60, 30, 10, and 5 mM in
culture medium. For treatments, trypsinized cells were diluted
with culture medium. 5000 cells in 100 mL medium were loaded
into a 96-well plate. Aer 1 hour at 37 ꢀC to allow for cell
adhesion, the medium was replaced with 100 mL of medium
containing compounds. Aer 48 hours, 10 mL of MTT reagent
(5 mg mLꢁ1 in PBS) was added (ThermoFisher, Waltham, MA).
Aer 4 hours, 100 mL of isopropanol/0.04 N HCl was added to
dissolve formazan and Abs570 nm was measured. For hypoxia
treatments, cells cultured in normoxia were transferred to 0.5%
O2 for 20 hours then treated with compounds for 24 hours.
Results represent an average of the means of two independent
experiments, each included three biological replicates, and
ꢃthe standard error (S.E.). Data were analyzed using single
factor analysis of variance (ANOVA) followed by Tukey's post hoc
analysis.
Glycine max (L.) Merr. line 107 was obtained from the Agriculture
and Agri-Food Canada Soybean EMS-mutagenesis collection 2005
(M. Morrison, Eastern 140 Ontario Oilseed Research Centre,
Ottawa). This line was selected due to its early maturation and its
IiRT genotype. Plants were grown in an incubator as indicated.10
On ice, growing seeds (25–75 mg fresh weight) were isolated from
pods and the seed coats were dissected from the embryos.11 Seed
coats (8–10), hydrogen peroxide (1 mM nal) (Sigma-Aldrich St.
Louis, MO), and dibromoapigenin (1 mM nal, from 100 mM
stock dissolved in DMSO, 0.05% Tween20, 5% MeOH) were
added to 970 mL of Milli-Q®-puried water in a 24-well microtiter
plate. Plates were incubated in the dark for 24 hours at 22 ꢀC on
a rotary shaker (100 rpm). Seeds and reaction medium were then
extracted for analysis of reaction products. Seeds were lyophilized
to dryness then extracted with 80% ethanol (20 mL mgꢁ1 dry
weight) for 24 hours in the dark at 22 ꢀC on a rotary shaker (100
rpm). Milli-Q®-puried water used to rinse the microtiter plates
was pooled with the reaction medium. The mixture was extracted
three times with equal volumes of ethyl acetate. Following
evaporation to dryness under a stream of nitrogen gas, the
residue was resuspended with seed extract. The combined seed
Results
Biocatalysis and semi-synthesis can be combined to (bio)
synthesize 6,8-dibromogenkwanin with different efficiencies
depending on their order
and reaction medium extract was then ltered through 0.2 mm for To directly test our hypothesis of whether promiscuous NP
analysis by LC-MSn.
biosynthesis enzymes can use semi-synthetic NP derivatives as
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substrates, we rst brominated apigenin at 6- and 8-positions semi-synthesis and biocatalysis can be used in tandem and in
using a semi-synthesis protocol that was developed for dihy- different combinations to manipulate the yield of desired
droquercetin.9 We chose to brominate these positions since chemical products.
several avonoid derivatives that were halogenated at those
sites exhibited greater antioxidant activity, increased lip-
ophilicity, and were more effective against human gastric and
colorectal adenocarcinoma cell lines compared to their NP
precursors.6,7
Apigenin was converted to 6,8-dibromoapigenin with 97.5%
efficiency by semi-synthesis using the bromine donor N-bro-
Dibromogenkwanin has greater antiproliferative activity
against colorectal adenocarcinoma cells in vitro compared to
its natural precursors at low micromolar treatment
concentrations
mosuccinimide (Fig. 2A). Reaction products were identied by
exact mass LC-PDA-MSn and 1H NMR spectroscopy (ESI, Tables
S1 and S2†). Puried 6,8-dibromoapigenin was supplied to E.
coli that was engineered to express a codon-optimized version of
the rice (Oryza sativa) gene naringenin 7-O-methyltransferase
from (OsNOMT).13 E. coli converted 6,8-dibromoapigenin to 6,8-
dibromogenkwanin with 2.3% efficiency (mol molꢁ1), yielding
0.6 mg Lꢁ1 culture. Thus, the overall efficiency of the tandem
(bio)synthesis approach ‘A’ was 2.2% (mol molꢁ1) (Fig. 2A).
We hypothesized that an advantage of conducting semi-
synthesis and biocatalysis as separate modules was that the
order of the modules could be changed to manipulate reaction
efficiency. To test, we did the experiment in the reverse order
starting with biocatalysis. OsNOMT-expressing E. coli
produced genkwanin from apigenin with 64.7% efficiency
(mol molꢁ1) yielding 11.0 mg Lꢁ1 culture. Subsequently, gen-
kwanin was converted to 6,8-dibromogenkwanin with 37.7%
Colorectal cancer is a major cause of morbidity and mortality
throughout the world.14 It is the third most common cancer and
the fourth most common cause of death.15 Of the cancers that
begin in the colorectal region, the vast majority (over 95%) are
classied as adenocarcinomas.16
To determine whether 6,8-dibromogenkwanin had
enhanced antiproliferative activity compared to its natural
precursors genkwanin and apigenin, we compared their anti-
proliferation activities against colon cancer cells in vitro. We
chose gland-derived adenocarcinoma HT-29 and lining-derived
carcinoma HCT-116 cells because apigenin and genkwanin
have well characterized antiproliferative activities against these
cell types.8,17,18 Bioactivities were tested under normal atmo-
spheric oxygen conditions (normoxia, 19% O2) at which apige-
nin and genkwanin were previously assayed,8,17,18 and under
hypoxia (0.5% O2), a condition that is more physiologically
relevant due to its prevalence during tumorigenesis.19,20
At the lowest treatment concentration compared under
normoxia (5 mM), 6,8-dibromogenkwanin inhibited the prolif-
eration of HT-29 adenocarcinoma cells by 56.5%, whereas its NP
precursors and 6,8-dibromoapigenin only inhibited prolifera-
tion by 6–8% (Fig. 3A). Roughly similar levels of inhibition were
observed for the compounds under hypoxia (Fig. 3B). It took 30–
60 mM apigenin to cause a similar levels of inhibition. Inter-
estingly, at higher doses, 6,8-dibromogenkwanin did not result
in signicantly greater levels of inhibition, whereas the levels of
inhibition increased for apigenin with increasing
efficiency (mol molꢁ1
) by semi-synthesis. 6,8-Dibromo-
genkwanin was the major product, but we also observed lesser
amounts of monobrominated and tribrominated derivatives.
Thus, the overall efficiency of the tandem (bio)synthesis
approach ‘B’ was 24.4% (mol molꢁ1) (Fig. 2B), roughly 11-fold
greater than approach ‘A’. The specic activities for apigenin
and 6,8-dibromoapigenin substrates were 254.6 and 6.0 at
mgꢁ1 total E. coli protein, respectively. However, our metabo-
lite extraction protocol resulted in complete loss of enzyme
activity. Overall, our results demonstrated that modules of
Fig. 2 Module order affects the efficiency of (bio)synthesizing 6,8-dibromogenkwanin from apigenin. (A) In reaction scheme 1 apigenin was first
converted to 6,8-dibromo-apigenin by semi-synthesis module using the N-bromo-succinimide method. 6,8-dibromo-apigenin was then
converted to DiBrGenk by the biocatalysis module using bacteria engineered to express the rice enzyme OsNOMT. (B) In reaction scheme 2 the
modules were conducted in the reverse order. The percent yields of the individual steps and the cumulative final yield are shown in red and blue
fonts, respectively.
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concentrations. These results suggested a distinct mechanism genes encoding chalcone synthase (CHS) in those tissues.22
of action for 6,8-dibromogenkwanin and apigenin.
Thus IiRT genotypes have a yellow seed coat devoid of avonoids
In contrast to the effects observed for adenocarcinoma cells, but with all the enzymatic capacity to biosynthesize, modify,
the proliferation of HCT-116 carcinoma cells under normoxia and polymerize avonoid intermediates.
was inhibited equivalently by apigenin and 6,8-dibromo-
Dibromoapigenin supplied through the culture medium was
genkwanin (Fig. 3C). The effectiveness of both compounds (and converted to more than six brominated derivatives (Fig. 4A and
genkwanin) was reduced under hypoxia (Fig. 3D). 6,8-Dibro- B). The parent masses of the brominated metabolites were
moapigenin was least effective in both cell types under hypoxia identied by an LC-MSn method operating in precursor ion
and normoxia.
mode that specically identies molecules that have undergone
the loss of bromine groups during MSn fragmentation of parent
molecules (see Methods). To ensure that the detected metabo-
lites were specically derivatives of 6,8-dibromoapigenin, we
supplied seed coats apigenin as a comparator. No brominated
molecules were detected from seed coats that were supplied
apigenin (Fig. 4C).
Soybeans do not biosynthesize apigenin or its derivatives.11
To determine whether seed coats could metabolize a bromi-
nated version of a native avonoid intermediate, we fed seed
coats 6,8-dibromodihydroquercetin. Dihydroquercetin is an
intermediate in the biosynthesis of anthocyanins and proan-
thocyanidin (PA, a.k.a. condensed tannin) avonoids. The vast
majority was converted to a mixture of highly polar brominated
molecules, yet distinct brominated metabolites with greater and
lower molecular masses than 6,8-dibromodihydroquercetin
were also detected (Fig. 4D and E). Again, no brominated
metabolites were measured from seed coats that were supplied
the non-brominated avonoid precursor (Fig. 4F).
Plants for the diversication of NP derivatives: soybean seed
coats convert brominated avonoids into novel derivatives
Various avonoid biosynthesis enzymes have exhibited
promiscuous substrate specicities.13,21 As a component of the
proposed combinatorial (bio)synthesis approach for generating
derivatives of anticancer NPs (Fig. 1), we hypothesized that
plant tissues expressing avonoid enzymes could convert semi-
synthetic avonoids supplied through the culture medium into
novel derivatives. Since the semi-synthetic molecule 6,8-dibro-
moapigenin showed relatively low antiproliferative activities in
vitro, we decided to test whether soybean seed coats could
convert 6,8-dibromoapigenin into novel derivatives. Soybean
seed coats from an IiRT genotype11 were selected since they
express genes for avonoid biosynthesis throughout the seed
coat, but lack avonoids outside the hilum region due to
a naturally occurring RNA interference (RNAi) silencing mech-
anism that silences the expression of the avonoid biosynthesis
Fig. 3 Anti-proliferative effects of natural flavonoids and their 6,8-dibrominated derivatives on HT-29 adenocarcinoma and HCT-116 carcinoma
colorectal cells in vitro. (A) HT-29 cultured with various concentrations of compound for 48 hours under 19% O2. (B) HT-29 pre-cultured for 20
hours under 0.5% O2 then cultured with compound under the same condition for 24 hours. (C) HCT-116 cultured for 48 hours under 19% O2. (D)
HT-29 pre-cultured for 20 hours under 0.5% O2 then cultured with compound under the same condition for 24 hours. Different letters denote
significant differences, P < 0.05, ANOVA, Tukey post hoc.
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Fig. 4 LC-MSn chromatograms in precursor ion measuring derivatives of brominated flavonoids that were produced by soybean seed coats. (A)
6,8-Dibromoapigenin. (B) Brominated molecules that arose from culturing soybean seed coats with 6,8-dibromoapigenin. (C) No brominated
molecules were detected after culturing soybean seed coats with apigenin. (D) 6,8-Dibromodihydroquercetin. (E) Brominated molecules that
arose from culturing soybean seed coats with 6,8-dibromodihydroquercetin. (F) No brominated molecules were detected after culturing
soybean seed coats with dihydroquercetin.
apigenin as a substrate, genkwanin could be biosynthesized
with 64.7% efficiency. Genkwanin could then be dibrominated
Discussion
by semi-synthesis using NBS to yield 6,8-dibromogenkwanin
with up to 49.7% efficiency. Random mutagenesis screens could
potentially be used to improve the catalytic efficiency of
OsNOMT towards apigenin or 6,8-dibromoapigenin. For
example, this approach generated variants of human theta class
1-1 glutathione transferase enzyme (hGSTT1-1) that exhibited
up to a 20 000-fold increased kcat/KM for a chlorinated derivative
of coumarin.26 Random mutagenesis can also be used to expand
the promiscuity of enzymes,27 thus further enhancing their
usefulness as tools in the combinatorial (bio)synthesis toolbox.
Combinatorial (bio)synthesis relies heavily on the promis-
cuity of enzymes. Here, we demonstrated that plant tissues
should be considered for use as modules since soybean seed
coats could readily convert brominated avonoids fed through
the culture medium into novel brominated derivatives. While
the identities of the novel derivatives and their activities against
cancer would require much more extensive characterization, in
this work we have proven the concept that plant tissues can be
used as biocatalyst, an area of research that deserves more
investigation in the future. Many, or most plant secondary
metabolism enzymes are thought to be promiscuous, accepting
multiple substrates. This is thought to have been a major
contributor to the evolution of chemodiversity in plants.28,29 Yet,
the promiscuity of enzymes remains poorly understood, and
thus cannot be readily predicted based on amino acid sequence
Here, we demonstrated a novel approach for making a desired
derivative of an ACNP that could not be produced using either
semi-synthesis or biocatalysis protocols alone. By combining
modules of semi-synthesis and biocatalysis in tandem and in
different combinations, we were able to use a novel combined
method, named the combinatorial (bio)synthesis approach, to
make 6,8-dibromogenkwanin with varying efficiency depending
on the module order. Brominated and methylated derivatives of
apigenin were previously semi-synthesized to test for enhanced
antiproliferative activity against HepG2 hepatoma cells.23
However, the approach could not semi-synthesize 6,8-dibro-
mogenkwanin since the methylation of the 7-hydroxyl of api-
genin was not a favorable reaction. Biocatalysis is typically an
option for producing compounds that are difficult to semi-
synthesize. Enzymes that catalyze the halogenation of NPs
have been identied mainly from bacteria, but also from
plants.24 Chloroperoxidase enzymes from Caldariomyces fumago
were capable of halogenating the avanones, naringenin and
hesperetin, at C-6 and C-8 in the presence of either Clꢁ or Brꢁ.
However, they did not accept avones as substrates.25 Here, we
demonstrated that the enzyme naringenin 7-O-methyltransfer-
ase from rice (OsNOMT) expressed in E. coli could methylate the
7-hydroxyl of 6,8-dibromoapigenin. Yet, bioconversion to 6,8-
dibromogenkwanin occurred with only 2.3% efficiency. Yet, if
the module order was reversed and OsNOMT was provided
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or even protein modeling.26–28 This can limit our ability to
predict whether the desired products will be biosynthesized
from an exogenous input molecule. For instance, transgenic
roots of the medicinal plant Catharanthus roseus (Madagascar
periwinkle) engineered to express tryptophan halogenating
enzymes from a soil bacterium were found to biosynthesize
chlorinated alkaloid intermediates but not the anticancer nal
biosynthetic products vincristine and vinblastine.30 The accu-
mulation of intermediates could be helpful in identifying gene
targets for expanding promiscuity by random mutagenesis,27
suggesting a focus for future research.
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