Precursor-directed biosynthesis of aminofulvenes: New chalanilines from endophytic fungus chalara sp.

Article abstract of DOI:10.3390/molecules26154418

The plant endophyte Chalara sp. is able to biotransform the epigenetic modifier vorinostat to form unique, aniline-containing polyketides named chalanilines. Here, we sought to expand the chemical diversity of chalaniline A-type molecules by changing the aniline moiety in the precursor vorinostat. In total, twenty-three different vorinostat analogs were prepared via two-step synthesis, and nineteen were incorporated by the fungus into polyketides. The highest yielding substrates were selected for large-scale precursor-directed biosynthesis and five novel compounds, including two fluorinated chalanilines, were isolated, purified, and structurally characterized. Structure elucida-tion relied on 1D and 2D NMR techniques and was supported by low-and high-resolution mass spectrometry. All compounds were tested for their bioactivity but were not active in antimicrobial or cell viability assays. Aminofulvene-containing natural products are rare, and this high-yielding, precursor-directed process allows for the diversification of this class of compounds.

Full text of DOI:10.3390/molecules26154418

molecules
Article
Precursor-Directed Biosynthesis of Aminofulvenes: New
Chalanilines from Endophytic Fungus Chalara sp.
Mahsa Khoshbakht 1, Jason Srey 1, Donovon A. Adpressa 1, Annika Jagels 2 and Sandra Loesgen 1,2,
*
1
Department of Chemistry, Oregon State University, Corvallis, OR 97331, USA;
khoshbam@oregonstate.edu (M.K.); sreyj@oregonstate.edu (J.S.); donovon.adpressa@gmail.com (D.A.A.)
Whitney Laboratory for Marine Bioscience, Department of Chemistry, University of Florida, Gainesville,
FL 32080, USA; annika.jagels@whitney.ufl.edu
2
*
Correspondence: sandra.loesgen@whitney.ufl.edu; Tel.: +1-904-201-8437
Abstract: The plant endophyte Chalara sp. is able to biotransform the epigenetic modifier vorinostat
to form unique, aniline-containing polyketides named chalanilines. Here, we sought to expand the
chemical diversity of chalaniline A-type molecules by changing the aniline moiety in the precursor
vorinostat. In total, twenty-three different vorinostat analogs were prepared via two-step synthesis,
and nineteen were incorporated by the fungus into polyketides. The highest yielding substrates were
selected for large-scale precursor-directed biosynthesis and five novel compounds, including two
fluorinated chalanilines, were isolated, purified, and structurally characterized. Structure elucida-
tion relied on 1D and 2D NMR techniques and was supported by low- and high-resolution mass
spectrometry. All compounds were tested for their bioactivity but were not active in antimicrobial
or cell viability assays. Aminofulvene-containing natural products are rare, and this high-yielding,
precursor-directed process allows for the diversification of this class of compounds.
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Citation: Khoshbakht, M.; Srey, J.;
Adpressa, D.A.; Jagels, A.; Loesgen, S.
Precursor-Directed Biosynthesis of
Aminofulvenes: New Chalanilines
from Endophytic Fungus Chalara sp..
Molecules 2021, 26, 4418. https://
doi.org/10.3390/molecules26154418
Keywords: precursor-directed biosynthesis; fungal metabolite; aminofulvenes; biotransformation
1. Introduction
Fungal natural products keep surprising us with unprecedented chemical diversity
derived from complex biosynthetic machineries. A multitude of applications for fun-
gal metabolites have been found to aid humankind: from the antibiotic penicillin, the
cholesterol-lowering agent lovastatin, to the immunosuppressant cyclosporin, to name a
Academic Editors: Roman Dembinski
and Igor Alabugin
few [
and the number of biosynthetic gene clusters present in these fungi, which encode for spe-
cialized small molecules, is much larger than the chemical diversity found [ ]. Together
with the estimate that only a small fraction of fungi have been chemically explored thus far,
the potential of chemical discovery from fungi remains high [ ]. Additionally, fungi are
masters in biotransformation and have been shown to harbor unique enzymes enabling
them to conquer challenging environments, food sources, and lifestyles [ ]. This has been
exploited in biotechnological applications; approximately half of the commercially available
enzymes used in industry are of fungal origin [10]. Some fungi are known to biotransform
small molecules and/or biosynthesize highly chemoreactive species. One example is the
compound maximiscin from Tolypocladium sp. which results from highly reactive intermedi-
ates that can detoxify various synthetic and naturally derived antifungals via nucleophilic
substitutions [11]. Previously, we found that the endophyte Chalara sp. is able to biotrans-
form the epigenetic modifier vorinostat, also known as suberanilohydroxamic acid, to form
unique, aniline-containing polyketides named chalanilines A and B (Figure 1) [12]. In our
studies, chalaniline B, an unusual aminoxanthone, exhibited micromolar antimicrobial
activity against multidrug-resistant Staphylococcus aureus strain ATCC# BAA-44. Recently,
the total synthesis of chalaniline B was achieved, which allowed us to expand its antimi-
crobial assessment [13]. To our surprise, the synthetic intermediate deshydroxymethyl
1,2]. In recent years, an increasing number of fungal genomes have been sequenced
Received: 17 June 2021
Accepted: 19 July 2021
Published: 22 July 2021
3,4
5–7
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations.
8,9
Copyright:
© 2021 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
Molecules 2021, 26, 4418. https://doi.org/10.3390/molecules26154418
https://www.mdpi.com/journal/molecules

Molecules 2021, 26, 4418
2 of 10
chalaniline B (1-anilino-2,8-dihydroxyxanthone) (Figure 1) was the most potent compound
tested, with MIC values of 8
µg/mL (25
µM) against both methicillin-resistant S. aureus
and B. subtilis. Aniline moieties are rare in natural products [14
,15], but have been used
extensively by synthetic chemists, for example, in dyes and early antibiotic development,
or in medicinal chemistry to optimize a drug lead [16]. In one example, the introduction of
aniline substituents enhanced the antifungal activity of aminoquinolones [17,18] and in
another, methoxy substituents on the aniline moiety increased brain permeability in the
development of a new drug to treat Alzheimer’s disease [19]. Here, we sought to expand
the chemical diversity of chalaniline A-type molecules by changing the aniline moiety in
the precursor vorinostat. Precursor-directed biosynthesis yielded five novel compounds
and their bioactivity in antimicrobial and cell viability assays was tested.
Figure 1. Chalanilines A and B isolated from Chalara sp. and deshydroxymethyl chalaniline B made
by chemical synthesis.
2. Results
Vorinostat analogs were prepared in a two-step synthesis following previous reports
(Figure 2A) [20]. Briefly, commercially available suberic acid monomethyl ester and differ-
ent anilines were coupled using 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC)
and hydroxybenzotriazole (HOBt), followed by hydroxyl amination under basic condi-
tions as the second step. After adjusting to pH 7 with hydrochloric acid, the vorinostat
analogs were purified using silica column chromatography and analyzed by mass spec-
trometry (MS) and nuclear magnetic resonance (NMR) and the data compared to published
reports [21–23]. Overall yields of the derivatives were 60–75% over two steps.
Next, the vorinostat analogs were supplemented to Chalara cultures (Figure 2D).
Previously, we have found that the full vorinostat structure is required for high yields in the
biotransformation; aniline or acetanilide were not incorporated or were only incorporated
in trace amounts [12]. One can hypothesize that the suberoyl chain aids in cell permeability,
and once inside the fungal cells, the aniline moiety is released by catabolic processes similar
to the ones found in human serum [24]. Isofusidienols, as well as chalanilines from Chalara
sp., can be envisioned biosynthetically from a xanthone arene epoxidation, forming a highly
reactive aldehyde on the xanthofulvene, which is able to react with free aniline to form
chalaniline A (SI Figure S33) [12]. Here, we optimized the feeding experiment and found
that the addition of vorinostat precursors (in DMSO, final concentration 1 mM) to fungal
cultures, followed by cultivation for 12–19 days, provided the highest yields of chalanilines
in the Chalara cultures (SI Figures S34 and S35). In total, twenty-three different vorinostat
analogs were prepared and nineteen were incorporated into the polyketide backbone by
the fungus as shown by low-resolution mass spectrometry (SI Figure S36). We selected
five vorinostat analogs with the most promising yields for large-scale precursor-directed
biosynthesis, using 2 L cultures of fungus, and were able to isolate unoptimized yields of
1–4 mg/L of compounds 1–5.

Molecules 2021, 26, 4418
3 of 10
Figure 2.
(
A
) Synthesis of twenty-three vorinostat analogs following published procedures (R1 and
R2 for different amine substituents) [20]. (
B
) Nineteen amines were biotransformed into chalaniline A
) Overview of precursor-
structures. ( ) Four amine moieties were not incorporated by the fungus. (
C
D
directed biosynthesis of chalaniline A-type molecules by supplementing Chalara sp. cultures with
vorinostat analogs.
2.1. Structure Elucidation
3-Fluoro chalaniline A (
1
) was isolated as a yellow amorphous solid in a yield of
3.2 mg/L. The HRESIMS gave an m/z value of 394.1088 [M + H]+ (calcd for C22H17FNO5,
394.1085;
416.0905;
∆
ppm = 0.8) and an m/z value of 416.0904 [M + Na]+ (calcd for C22H16FNO5Na,
∆
ppm = 0.2), resulting in a molecular formula of C22H16FNO5. The UV spectrum
showed maxima at 380, 308, and 244 nm, representing the chalaniline A-type backbone.
The 1H NMR spectrum exhibited a broad hydroxyl peak (δH 13.75), an N-H resonance
(
δH 11.84) with a large coupling constant (14.5 Hz) to one methine (δH 8.74), seven aro-
matic/olefinic hydrogens, one methoxy, and one methyl group (Table 1). The 13C NMR
spectrum displayed two carbonyls, two methyl carbons, and eighteen olefinic carbons.
The 3-fluoro benzene moiety exhibited 1J (244.9 Hz), 2J (26.2 and 21.4 Hz), and 3J (9.4 Hz)
C-F coupling constants in the carbon spectrum, and the proton spectrum revealed two
3J (10.7 and 8.0 Hz) H-F coupling constants (Table 1). The structure of
1 was determined
by conventional 2D NMR experiments. COSY and HSQC correlations helped to establish
aromatic rings A to C. HMBC correlations between H-6 of the fulvene and0C-11 of the
0
bridging methine, connections between N-H and the aniline ring (C-2 and C-6 ), as well as
0
the correlation between H-11 and C-2 supported the structural assignment. The placement
of the fluorine at C-30 was confirmed by C-F coupling constant analysis (Figure 3).
As previously established by quantum mechanical calculations [12], chalanilines exist
as zwitterions in solution (Figure 1). The NMR data found for 1–3 with the large trans
coupling constant between N-H and H-11 confirm the iminium with delocalized double
bond character.

Molecules 2021, 26, 4418
4 of 10
a
b
c
Table 1. 1H and 13C NMR spectroscopic data for compounds
1–
5
(all recorded in DMSO-d6), JHH in Hz, JCF
,
JHF,
* partially assigned from 1D data.
1
2
3
4
5
Pos.
δC Type
δH
δC Type
δH
δC Type
δH
δC Type
δH
δC Type
δH
1
2
161.3, C
111.2, CH
145.0, C
106.9, CH
155.0, C
105.3 C
120.6, CH
119.6, CH
111.1, C
176.2, C
107.7, C
161.8, C
111.5, CH
145.3, C
107.3, CH
155.5, C
105.0, C
121.0, CH
119.2, C
111.4, C
176.6, C
108.1, C
161.3, C
111.0, CH
144.9, C
106.9, CH
155.0, C
104.8, C
120.7, CH
118.9, C
111.1, C
176.2, C
107.6, C
161.9, C
109.3, CH
142.5, C
106.0, CH
154.9, C
107.8, C
122.0, CH
124.7, C
110.7, C
174.7, C
107.3, C
161.7, C
109.8, CH
143.3, C
106.3, CH
155.1, C
107.1, C
122.7, CH
122.3, C
110.9, C
175.7, C
107.4, C
6.57, s
6.88, s
6.56, s
6.87, s
6.56, s
6.89, s
6.37, s
6.70, s
6.41, s
6.71, s
3
4
4a
5
6
7
7a
8
8a
9
7.69, s
7.69, s
7.73, s
7.31, s
9a
10
160.6, C
21.7, CH3
160.7, C
21.7, CH3
160.3, C
21.7, CH3
154.2, C
21.5, CH3
155.0, C
21.6, CH3
2.38, s
8.74, d (14.5) a
2.38, s
8.68, d,
(14.5) a
2.38, s
8.73, d
(14.7) a
2.32, s
8.50, s
2.32, s
8.61, s
11
145.1, CH
146.1, C
145.2, CH
147.0, CH
152.1, CH
12
13
14
164.2, C
51.2, CH3
164.7, C
51.6, CH3
164.2, C
51.1, CH3
55.5, CH3
164.9, C
50.0, CH3
55.1, CH3
165.0, C
50.3, CH3
3.77, s
3.77, s
3.76, s
3.84, s
3.65, s
3.75, s
3.76, s
140.7, C
(10.3) b
135.5, C
(2.6) b
10
20
30
140.1, C
104.0, CH
160.4, C
133.1, C
121.4, CH
114.2, CH
156.1, C
133.7, C *
112.7, CH *
125.5, CH *
105.28, CH
120.5, CH,
7.10, d
(8.7) a
6.91, d
(8.8) a
7.61, d (10.7) c
7.12, t (8.0) c
7.68, m
7.23, t (2.0) a
7.10, brd
7.49, m *
(26.2) b
(8.3) b
162.8, C
(244.9) b
112.6, CH
(21.4) b
116.5, CH,
(23.0) b
7.36, t
(8.7) a
160.6, C
6.87, dd (8.0,
2.0) a
7.65, d
(7.3) a
40
4a0
50
111.9, CH
123.3, CH
134.2, C
(244.8) b
131.5, CH
(9.4) b
7.88, d
(7.0) a
7.53, q (8.0) a
7.44, d (8.0) a
130.7, CH
110.7, CH
7.40, t (8.1) a
127.8, CH
114.8, CH
7.21, dd (8.1,
2.0) a
60
70
80
123.6, CH *
126.7, CH *
124.0, CH
129.0, C
7.49, m *
(2.8) b
7.49, m *
8.39, d
(7.0) a
8a0
13.84,
brd
OH
13.75, brd
13.83, brd
14.97, brd
14.91, brd
11.89, d
11.83, d
(14.7) a
NH
11.84, d (14.5) a
(14.5) a
Figure 3. Chemical structures of new chalanilines 1–5 with selected 2D NMR correlations.
4-Fluoro chalaniline A (
2
) was isolated as a yellow amorphous solid and the HRES-
+
IMS provided an m/z value of 394.1088 [M + H] (calcd for C22H17FNO5, 394.1085;
∆
ppm = 0.8) and m/z value of 416.0909 [M + Na]+ for the sodium adduct (calcd for

Molecules 2021, 26, 4418
5 of 10
C22H16FNO5Na, 416.0905;
the spectrum of
∆
ppm = 1.0). The 1H NMR spectrum of
2
was very similar to
1
, the only differences can be found in the number of aromatic signals
and the coupling and integration pattern for the fluoro-benzene moiety. Two sets of aro-
matic signals, with each an integration of ~2H, supported the symmetric, para-substituted
fluoro-aniline incorporation (Table 1). The 13C spectrum showed twenty signals: one
methyl, one methoxy, two carbonyl, one phenolic, and fifteen olefinic carbons. Only two
aromatic protons and carbons could be found due to the symmetry in the 40-fluoro aniline
1
moiety. J C-F coupling could be found 0for0C-40 (244.8 Hz), 2J C-F coupling of 23.0 Hz for
0
0
0
C-3 /5 , 3J C-F coupling of 8.3 Hz for C 2 /6 , and 4J C-F coupling (2.6 Hz) for C-1 (Table 1).
Two-dimensional NMR experiments were analyzed to complete the structure suggestion
for
2, with key HMBC correlations from N-H to both the fulvene (C-5) and the 4-fluoro
anilino ring C-20/60, and also from the H-11 to C-20 (Figure 3).
3-Methoxy chalaniline A (3) was isolated as an amorphous yellow solid. The HRES-
IMS gave an m/z value of 406.1284 [M + H] + (calcd for C23H20NO6, 406.1285) and m/z
value of 428.1102 [M + Na]+ for the sodium adduct (calcd for C23H19NO6Na, 428.1105;
∆
ppm = 0.7). The 1H NMR spectrum of
3 showed a similar pattern of signals as previously
reported for chalaniline A [12], with the addition of a second methoxy singlet (δH 3.84,
δC 55.5) and fewer aromatic resonances. The 13C NMR spectrum exhibited twenty-three
signals: three methyl peaks, two carbonyl shifts, a phenol and seventeen olefinic signals
(Table 1). The methoxy group was placed meta to the aniline based on the observed cou-
pling constant (Table 1). HMBC correlations between the fulvene (H-6) and the bridging
methine (H-11), H-11 and the aniline ring (C-20), as well as correlations from the N-H into
the aromatic ring (C-20; C-60) established connect0ivity of the spin systems. The methoxy
group exhibited a strong HMBC correlation to C-3 , confirming its meta position (Figure 3).
4-Methoxy chalaniline A (
The HRESIMS showed an m/z of 406.1286 [M + H] (calcd for C23H20NO6, 406.1285;
4
) was isolated as a yellow amorphous solid with low yields.
+
∆
ppm = 0.2), indicative of a regioisomer of 3. The para-substitution in the 4-methoxy aniline
moiety became evident in the proton NMR with two doublets each with an integration of
~2H (Table 1). The 13C signals were mainly derived from 2D spectra and were similar to
the spectra of 2 and 3
. HMBC correlations from both the H-20 and H-30 of the aniline ring
to the bridging methine (C-11), as well as the correlation between bridging methine (H-11)
to the fulvene (C-6, C-9a) assisted in the structure assembly (Figure 3).
Notably, there are differences in the NMR data between compounds 1–3 and 4, 5 in
the 1H NMR spectrum due to different isolation procedures. When acid was used, the
compounds lacked the N-H signal as well as the large coupling to H-11. We believe this is
due to protonation effects by water found in the NMR solvent.
Naphthyl chalaniline A (
IMS gave an m/z value of 426.1341 [M + H]+ (calcd for C26H20NO5, 426.1336;
and an m/z value of 448.1160 [M + Na]+ for the sodium adduct (calcd for C26H19NO5Na,
448.1155;
ppm = 1.1) supporting a molecular formula of C26H19NO5. The 1H and 13
NMR spectra were similar to chalaniline A with additional signals for the naphthyl moiety
instead of the phenyl in the aromatic region (Table 1). Due to the low amount of , only a
5) was isolated as a yellow amorphous solid and the HRES-
∆
ppm = 1.2),
∆
C
5
partial assignment of the naphthyl moiety based on 1D NMR and chemical shift predictions,
but not 2D correlations, could be made (Table 1). HMBC correlations from the bridging
methine (H-11) to the fulvene (C-6, C-9a) and C-20 of the naphthyl ring were utilized to
connect the spin systems (Figure 3).
2.2. Bioactivity
Inhibition of cell viability of pure compounds 1-5 was evaluated against colon (HCT-
116) and melanoma (SK-MEL-5) cancer cell models by measuring the reduction of the
tetrazolium salt MTT (3-(4, 5 dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide) by
metabolically active cells [25]. No inhibition of cell viability was observed at a single dose of
10
µM against these cell lines. All isolated compounds displayed no antimicrobial activity
at a dose of 128
µg/mL against a panel of four Gram-positive bacteria (S. aureus ATCC

Molecules 2021, 26, 4418
6 of 10
25923, methicillin-resistant S. aureus ATCC BAA-41, multidrug-resistant S. aureus ATCC
BAA-44, and E. faecium ATCC 49032), two Gram-negative bacteria (P. aeruginosa ATCC
15422, E. coli ATCC 8739) as well as the yeast Candida albicans (ATCC 90027) following CLSI
guidelines (details in the experimental section) [26].
3. Discussion
Precursor-directed biosynthesis has emerged as a powerful tool to diversify complex
natural products [27]. It combines the best of two worlds; the flexibility and ease of organic
synthesis to access small molecule precursors with the selectivity and often high yields
found in biosynthetic processes. This strategy allows natural products to be made with
“unnatural” functional groups, for example fluorine atoms or alkyne sidechains, which
enable bio-orthogonal approaches for mode-of-action studies. Anilino-fulvenes are rarely
found in chemical databases and chalaniline A was the first example found in fungi to our
knowledge. The ability to utilize the fungal biosynthesis to incorporate different anilino
moieties enabled us to study this new compound class in more detail. Chalara sp. was
able to biotransform nineteen different vorinostat analogs; in particular, various anilines
were well tolerated. The incorporation of alkynes or halogens like fluorine and bromine
into a small molecule backbone can assist in the pull down of a potential molecular target
via click-chemistry, fluorine-19 NMR to enable binding studies to macromolecules, or
bromination, which might aid in crystallization attempts or ease in mass spectrometric
detection [28]. These modifications also provide reactive handles for further chemical
diversification efforts. We will continue our exploration of the activity of the chalanilines
in an in vivo zebrafish behavior assay [29] as well as in silico molecular docking to find
potential receptors and intracellular targets for these aminofulvenes.
4. Materials and Methods
General experimental procedures. UV spectra were recorded on a BioRad Smart-
Spec3000. IR spectra were recorded on a Thermo Scientific Nicolet 6700 FT-IR spectrometer
(Thermo Fisher Scientific, Waltham, MA, USA). NMR spectra were acquired on a Bruker
Avance III 500 MHz or Bruker Avance III 700 MHz spectrometer equipped with a 5 mm
TXI probe or 5 mm BBO probe (Bruker, Billerica, MA, USA 500 MHz and 700 MHz). For
compound 4, we used AMRIS’ Agilent VNMRS-600 spectrometer with a unique 1.5 mm
High Temperature Superconducting (HTS) Cold Probe and AMRIS’ Bruker Neo-600 spec-
trometer equipped with a 1.7 mm TCI Cryoprobe, all with the residual solvent used as
an internal standard (DMSO: 2.50/39.50 ppm). Low-resolution ESI-MS and HRTOFMS
mass spectra were recorded in positive ionization mode on an Agilent 1100 series LC with
MSD 1946 or Agilent 1260 Infinity II LC with 6545 QTOF MS, respectively. Analytical
high-pressure liquid chromatography (HPLC, Agilent, Santa Clara, CA, USA) was per-
formed using an Agilent 1100 HPLC system equipped with a photodiode array detector.
The mobile phase consisted of ultra-pure water (A) and acetonitrile (MeCN) (B), both with
0.05% formic acid. A gradient method from 10% A to 100% B in 35 min at a flow rate of
0.8 mL/min was used. The column (Phenomenex Kinetex
®C18, 5 µm particle size, 150 mm
×
4.6 mm, Phenomenex, Torrance, CA, USA) was re-equilibrated before each injection and
◦
the column compartment was maintained at 30 C throughout each run. Semi-preparative
HPLC (Phenomenex Kinetex® C 18, 5
m particle size, 150 mm 10 mm) utilized isocratic
µ
×
elution conditions or a gradient system with a flow rate of 4 mL/min on an Agilent 1100
HPLC system operating at room temperature equipped with a photodiode array detector.
Preparative HPLC (Phenomenex Luna C18, 5
conducted at room temperature, using isocratic elution conditions or a gradient system
with a flow rate of 20 mL/min utilizing an Agilent 1260 Infinity series HPLC equipped with
µ
m particle size, 250 mm
×
21 mm) was
a DAD detector. All samples were filtered through a 0.45 µm nylon filter or centrifuged
at 14,000 rpm for five minutes before LCMS and HPLC analysis. Analytical thin layer
chromatography (TLC) was performed on pre-coated silica gel 60 F254 plates (Eppendorf,
Hamburg, Germany). TLC plates were visualized by UV (254 and 360 nm), and by spray-

Molecules 2021, 26, 4418
7 of 10
ing with anisaldehyde solution followed by heating at 80 ◦C. General reagents were from
Sigma-Aldrich Corp. and VWR International.
Preparation of vorinostat analogs. A solution of suberic acid monomethyl ester
(1.0 g, 5.3 mmol) in anhydrous dimethyl formamide (DMF) (20.0 mL) was treated with
EDC (1.2 g, 7.7 mmol), HOBt (1.0 g, 7.3 mmol), triethylamine (2.2 mL, 15.9 mmol), and
stirred at room temperature (rt) for 30 minutes. Commercially available amine derivatives
were first dissolved in DMF (10.0 mL) and then added to the reaction mixture. The
resulting mixtures were stirred overnight (15–20 h) at rt and then diluted with ethyl acetate
(EtOAc) (30.0 mL) and washed with concentrated hydrochloric acid (2 M, 8.0 mL), water
(12.0 mL), and saturated sodium chloride solution (brine, 5 mL). The organic layer was
dried with sodium sulfate (Na2SO4), filtered and evaporated to afford the ester derivatives.
The crude products were pure enough to directly move to the second step. The ester
derivatives in methanol (MeOH) (~20.0 mL) were added to freshly prepared hydroxylamine
(~20.0 mL) and potassium hydroxide (KOH) and stirred for 2 h. The solvent evaporated
and the reaction mixtures were extracted with EtOAc, dried with Na2SO4 and in vacuo.
The crude products containing vorinostat derivatives were purified with silica column
chromatography and structures proved via NMR and LCMS methods.
Precursors directed biosynthesis. Cultures of Chalara sp. 6661 were grown for seven
days in YPD medium (yeast extract 20 g/L, bacto peptone 20 g/L; dextrose 40 g/L; pH 6.0)
agar at 25 ◦C with a day/night cycle [12 30]. Then, 1 cm2 sections of agar/hyphae were
,
excised and used to inoculate 50 mL cultures of YPD. After 48 h of growth at ambient tem-
peratures with 200 rpm shaking, 1 M solutions of synthesized vorinostat derivatives [21 23
–
]
in dimethyl sulfoxide (DMSO) were used to bring duplicate cultures to a final treatment
concentration of 1 mM. Cultures were maintained at ambient light and temperature with
200 rpm shaking for 30 days before extraction. Two-liter cultures treated with vorinostat
derivatives were prepared in a similar fashion, with 1 cm2 hyphae/100 mL used for inocu-
lation and vorinostat application occurring at 48 h thereafter. Biosynthesis of chalaniline A
was monitored by LCMS from the day of inoculation until 26 days of cultivation; chalaniline
A production was observed after 12 days (see SI Figures S33 and S34). Precursor-directed
biosynthesis cultures were allowed to grow for ~20 days before extraction, and isolation of
compounds of interest.
Extraction and isolation. Fungal cultures were treated with XAD-7 resin (10% w/v)
and left overnight after separation of fungal mycelia from culture broth by filtration. XAD-7
resin was collected by filtration, washed with 2 L of deionized water, and then extracted
with 2 L of 1:1 acetone:methanol mixture. Extract was concentrated before partitioning
between EtOAc and water. The aqueous layer was washed three times with EtOAc and
organic layers were combined to be concentrated to dryness in vacuo. Crude organic
extracts were first separated into fractions by normal phase chromatographic separation on
an ISCO flash chromatography system (3-fluoro vorinostat-treated culture: four fractions
eluted with gradient of dichloromethane (DCM):MeOH, 4-fluoro vorinostat-treated culture:
five fractions eluted with a gradient of DCM:MeOH, 3-methoxy vorinostat-treated culture:
six fractions eluted first with a gradient of hexane:EtOAc and then with EtOAc:MeOH,
4-methoxy vorinostat-treated culture: five fractions eluted with DCM:MeOH gradient,
naphthyl vorinostat-treated culture: seven fractions eluted with first 3:1 to 0:1 gradient of
hexane:EtOAc and then EtOAc:MeOH gradient) and were further isolated directly from
these fractions using preparative HPLC with isocratic mobile phases (3.2 mg of
fraction two using a 60%:40% MeCN:H2O isocratic run, 2.9 mg of from fraction two by
preparative HPLC using 60%:40% MeCN:H2O isocratic run, 3.5 mg of from fraction three
by using 55%:45% MeCN:H2O isocratic run, 1.0 mg of from fraction two by using 55%:45%
MeCN:H2O isocratic run, 1.1 mg of from fraction six using 65%:35% MeCN:H2O isocratic
1 from
2
3
4
5
run). Precursor incorporation studies were checked with LCMS analysis by preparing
10 mg/mL samples in MeCN.
3-Fluoro chalaniline A (
1
): yellow amorphous solid; IR (ATR):
ν
= 3370, 2925, 2854,
max: 380, 306, 244 nm;
max
1712, 1651, 1591, 1470, 1297, 1208, 1147, 827, 732 cm−1; UV (MeCN)
λ

Molecules 2021, 26, 4418
8 of 10
13C NMR (176 MHz, DMSO-d6) and 1H NMR (500 MHz, DMSO-d6) see Table 1; HRESIMS:
m/z 394.1088 [M + H]+ (calcd for C22H17FNO5 394.1085;
Na]+ (calcd for C22H16FNO5Na 416.0905; ∆ppm 0.2)
∆
ppm 0.8), m/z 416.0904 [M +
2): yellow amorphous solid; IR (ATR): ν = 3430, 2925, 2853,
max
4-Fluoro chalaniline A (
1712, 1651, 1589, 1508, 1466, 1365, 1209, 1098 cm−1; UV (MeCN)
λmax: 380, 308, 244 nm;
13C NMR (176 MHz, DMSO-d6) and 1H NMR (700 MHz, DMSO-d6) see Table 1; HRESIMS:
m/z 394.1088 [M + H]+ (calcd for C22H17FNO5 394.1085;
Na]+, (calcd for C22H16FNO5Na 416.0905; ∆ppm = 1.0)
∆ppm = 0.8), m/z 416.0909 [M +
3-Methoxy chalaniline A (
3
): yellow amorphous solid; IR (ATR):
ν
= 3290, 2924,
max
2850, 1703, 1647, 1601, 1510, 1470, 1252, 1190, 1050, 840, 735 cm−1; UV (MeCN)
λ
max = 383, 310, 246 nm; 13C NMR (176 MHz, DMSO-d6) and 1H NMR (700 MHz, DMSO-d6)
see Table 1; HRESIMS: m/z 406.1284 [M + H]+ (calcd for C23H20NO6 406.1285;
∆
ppm 0.2),
m/z 428.1102 [M + Na]+ (calcd for C23H19NO6Na 428.1005; ∆ppm 0.7)
4-Methoxy chalaniline A (4): yellow amorphous solid; IR (ATR): ν
max
= 3290, 2924,
1703, 1647, 1510, 1490, 1252, 1050, 820, 745 cm−1; UV (MeCN) max: 382, 308, 244 nm; 13
λ C
NMR (150 MHz, DMSO-d6) and 1H NMR (600 MHz, DMSO-d6) see Table 1; HRESIMS:
m/z 406.1286 [M + H]+, (calcd for C23H20NO6 406.1285; ∆ppm 0.2)
Naphthyl chalaniline A (
5
): yellow amorphous solid; IR (ATR):
ν
= 3410, 2926,
max
2852, 1737, 1647, 1614, 1465,1372, 1307, 1207, 1098, 830, 767 cm−1; UV (MeCN)
λmax: 374,
305, 242, 214 nm; 13C NMR (176 MHz, DMSO-d6) and 1H NMR (500 MHz, DMSO-d6) see
Table 1; HRESIMS: m/z 426.1341 [M + H]+ (calcd for C26H20NO5 426.1336;
∆ppm 1.2), m/z
448.1160 [M + Na]+ (calcd for C26H19NO5Na 448.1155; ∆ppm 1.1)
Antimicrobial Assays. Extracts and fractions were tested for inhibitory activity against
Staphylococcus aureus (ATCC 25923), methicillin-resistant Staphylococcus aureus (ATCC BAA-
41), multidrug-resistant Staphylococcus aureus (ATCC BAA-44), Pseudomonas aeruginosa
(ATCC 15442), Candida albicans (ATCC 90027), Candida krusei (ATCC 34135), and My-
cobacterium smegmatis (ATCC 14468) in microbroth assays performed following an estab-
lished protocol [26
,31]. Fractions and pure compounds were tested at a concentration of
128 g/mL. All human pathogens used in the study were acquired from the American
µ
Type Culture Collection (ATCC, Manassas, VA, USA).
Cell proliferation assay. Cytotoxic activities of extracts and pure compounds were eval-
uated against colon (HCT-116) and melanoma (SK-MEL-5) cancer models by measuring the
reduction of the tetrazolium salt MTT (3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium
bromide) by metabolically active cells following standard procedures [32,33].
Supplementary Materials: The following are available online. NMR spectra of compounds
(S1–S32), HPLC chromatograms (S33 and S34), vorinostats made in this study and scope of
biotransformation (S35 and S36)
1–
5
Author Contributions: M.K., J.S. performed experiments, data analysis, and prepared manuscript,
D.A.A. developed synthesis, feeding protocols, and supervised data analysis, A.J. assisted with
compound isolation and NMR acquisition and analysis. D.A.A. and S.L. conceived and designed the
study. All authors have read and agreed to the published version of the manuscript.
Funding: This research was funded by OSU and UF start-up funds and by the National Science
Foundation under grant CH 1808717, CH 2020110.
Data Availability Statement: NMR and MS raw files can be requested from the authors.
Acknowledgments: We thank Steve Huhn (OSU), Ion Ghiviriga (UF), and James Rocca (UF, AMRIS)
for excellent NMR support. We acknowledge the support of the Oregon State University’s NMR
Facility, funded in part by the National Institutes of Health, HEI grant 1S10OD018518, and by the
M. J. Murdock Charitable Trust grant no. 2014162. We acknowledge the University of Florida’s
McKnight Brain Institute at the National High Magnetic Field Laboratory’s Advanced Magnetic
Resonance Imaging and Spectroscopy (AMRIS) Facility, which is supported by the US NSF Coopera-
tive Agreement No. DMR-1644779 and the State of Florida. A portion of this work was carried out
using a 1.5 mm High-Temperature Superconducting Cryogenic Probe developed with support from
NIH award R01 EB009772. We thank E. N. Kaweesa, P. E. Mandelare, and C. Zhu for performing

Molecules 2021, 26, 4418
9 of 10
the cytotoxicity and antimicrobial assays, respectively. We would like to thank Paul Blakemore for
continued support.
Conflicts of Interest: The authors declare no conflict of interest.
Sample Availability: Samples of the compounds, if available in enough quantity, are available from
the authors.
References
1.
2.
3.
Alberti, F.; Foster, G.D.; Bailey, A.M. Natural products from filamentous fungi and production by heterologous expression. Appl.
Microbiol. Biotechnol. 2017, 101, 493–500. [CrossRef]
Keller, N.P. Fungal secondary metabolism: Regulation, function and drug discovery. Nat. Rev. Microbiol. 2019, 17, 167–180.
[CrossRef]
Okada, B.K.; Seyedsayamdost, M.R. Antibiotic dialogues: Induction of silent biosynthetic gene clusters by exogenous small
molecules. FEMS Microbiol. Rev. 2017, 41, 19–33. [CrossRef]
4.
5.
Li, G.; Lou, H.X. Strategies to diversify natural products for drug discovery. Med. Res. Rev. 2018, 38, 1255–1294. [CrossRef]
Greco, C.; Keller, N.P.; Rokas, A. Unearthing fungal chemodiversity and prospects for drug discovery. Curr. Opin. Microbiol. 2019
51, 22–29. [CrossRef]
,
6.
7.
Aly, A.H.; Debbab, A.; Proksch, P. Fungal endophytes: Unique plant inhabitants with great promises. Appl. Microbiol. Biotechnol.
2011, 90, 1829–1845. [CrossRef]
Hyde, K.D.; Xu, J.; Rapior, S.; Jeewon, R.; Lumyong, S.; Niego, A.G.T.; Abeywickrama, P.D.; Aluthmuhandiram, J.V.S.; Brahaman-
age, R.S.; Brooks, S.; et al. The amazing potential of fungi: 50 ways we can exploit fungi industrially. Fungal Divers. 2019, 97,
1–136. [CrossRef]
8.
9.
Vylkova, S. Environmental pH modulation by pathogenic fungi as a strategy to conquer the host. PLoS Pathog. 2017, 13, e1006149.
[CrossRef]
Wang, Y.; Dai, C.-C. Endophytes: A potential resource for biosynthesis, biotransformation, and biodegradation. Ann. Microbiol.
2011, 61, 207–215. [CrossRef]
10. Biotechnological Use of Fungal Enzymes. In Fungi; Wiley: Hoboken, NJ, USA, 2017; pp. 201–225.
11. Du, L.; You, J.; Nicholas, K.M.; Cichewicz, R.H. Chemoreactive Natural Products that Afford Resistance against Disparate
Antibiotics and Toxins. Angew. Chem. Int. Ed. Engl. 2016, 55, 4220–4225. [CrossRef]
12. Adpressa, D.A.; Stalheim, K.J.; Proteau, P.J.; Loesgen, S. Unexpected Biotransformation of the HDAC Inhibitor Vorinostat Yields
Aniline-Containing Fungal Metabolites. ACS Chem. Biol. 2017, 12, 1842–1847. [CrossRef]
13. Khoshbakht, M.; Thanaussavadate, B.; Zhu, C.; Cao, Y.; Zakharov, L.N.; Loesgen, S.; Blakemore, P.R. Total Synthesis of Chalaniline
B: An Antibiotic Aminoxanthone from Vorinostat-Treated Fungus Chalara sp. 6661. J. Org. Chem. 2021. [CrossRef]
14. Dunn, T.B.; Ellis, J.M.; Kofink, C.C.; Manning, J.R.; Overman, L.E. Asymmetric Construction of Rings A D of Daphnicyclidin-Type
−
Alkaloids. Org. Lett. 2009, 11, 5658–5661. [CrossRef]
15. Hong, B.-C.; Shr, Y.-J.; Wu, J.-L.; Gupta, A.K.; Lin, K.-J. Novel [6 + 2] Cycloaddition of Fulvenes with Alkenes: A Facile Synthesis
of the Anislactone and Hirsutane Framework. Org. Lett. 2002, 4, 2249–2252. [CrossRef]
16. Naksomboon, K.; Poater, J.; Bickelhaupt, F.M.; Fernández-Ibáñez, M.Á. para-Selective C–H Olefination of Aniline Derivatives via
Pd/S,O-Ligand Catalysis. J. Am. Chem. Soc. 2019, 141, 6719–6725. [CrossRef]
17. Wang, J.; Liu, J.; Xie, Z.; Li, J.; Li, J.; Hu, L. Design, synthesis and biological evaluation of mogrol derivatives as a novel class of
AMPKα2β1γ1 activators. Bioorg. Med. Chem. Lett. 2020, 30, 126790. [CrossRef]
18. Yang, R.; Du, W.; Yuan, H.; Qin, T.; He, R.; Ma, Y.; Du, H. Synthesis and biological evaluation of 2-phenyl-4-aminoquinolines as
potential antifungal agents. Mol. Divers. 2020, 24, 1065–1075. [CrossRef]
19. Srivastava, P.; Tripathi, P.N.; Sharma, P.; Shrivastava, S.K. Design, synthesis, and evaluation of novel N-(4-phenoxybenzyl)aniline
derivatives targeting acetylcholinesterase, β-amyloid aggregation and oxidative stress to treat Alzheimer’s disease. Bioorg. Med.
Chem. 2019, 27, 3650–3662. [CrossRef]
20. Soli, E.D.; Braun, M.P. Synthesis of [phenyl-U-14C]aryl and [8-14C]carboxy labeled tracers of vorinostat. J. Labelled Comp.
Radiopharm. 2006, 49, 437–443. [CrossRef]
21. Salmi-Smail, C.; Fabre, A.; Dequiedt, F.; Restouin, A.; Castellano, R.; Garbit, S.; Roche, P.; Morelli, X.; Brunel, J.M.; Collette,
Y. Modified cap group suberoylanilide hydroxamic acid histone deacetylase inhibitor derivatives reveal improved selective
antileukemic activity. J. Med. Chem. 2010, 53, 3038–3047. [CrossRef] [PubMed]
22. Rebelo, B.A.; Santos, R.B.; Ascenso, O.S.; Nogueira, A.C.; Lousa, D.; Abranches, R.; Ventura, M.R. Synthesis and biological effects
of small molecule enhancers for improved recombinant protein production in plant cell cultures. Bioorg. Chem. 2020, 94, 103452.
[CrossRef]
23. Remiszewski, S.W.; Sambucetti, L.C.; Atadja, P.; Bair, K.W.; Cornell, W.D.; Green, M.A.; Howell, K.L.; Jung, M.; Kwon, P.; Trogani,
N.; et al. Inhibitors of human histone deacetylase: Synthesis and enzyme and cellular activity of straight chain hydroxamates. J.
Med. Chem. 2002, 45, 753–757. [CrossRef]

Molecules 2021, 26, 4418
10 of 10
24. Thangavel, S.; Rajamanikandan, R.; Friedrich, H.B.; Ilanchelian, M.; Omondi, B. Binding interaction, conformational change, and
molecular docking study of N-(pyridin-2-ylmethylene)aniline derivatives and carbazole Ru(II) complexes with human serum
albumins. Polyhedron 2016, 107, 124–135. [CrossRef]
25. Vichai, V.; Kirtikara, K. Sulforhodamine B colorimetric assay for cytotoxicity screening. Nat. Protoc. 2006, 1, 1112–1116. [CrossRef]
26. CLSI. Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically, 7th ed.; National Committee for
Clinical Laboratory Standards: Wayne, PA, USA, 2018.
27. Kennedy, J. Mutasynthesis, chemobiosynthesis, and back to semi-synthesis: Combining synthetic chemistry and biosynthetic
engineering for diversifying natural products. Nat. Prod. Rep. 2008, 25, 25–34. [CrossRef]
28. Hughes, C.C. Chemical labeling strategies for small molecule natural product detection and isolation. Nat. Prod. Rep. 2021
[CrossRef]
.
29. Haney, W.A.; Moussaoui, B.; Strother, J.A. Prolonged exposure to stressors suppresses exploratory behavior in zebrafish larvae. J.
Exp. Biol. 2020, 223, jeb224964. [CrossRef]
30. Lösgen, S.; Magull, J.; Schulz, B.; Draeger, S.; Zeeck, A. Isofusidienols: Novel chromone-3-oxepines produced by the endophytic
fungus Chalara sp. Eur. JOC 2008, 4, 698–703.
31. CLSI. Performance Standards for Antimicrobial Susceptibility Testing M100, 30th ed.; Clinical and Laboratory Standards Institute:
Wayne, PA, USA, 2020.
32. Kmail, A.; Lyoussi, B.; Zaid, H.; Saad, B. In vitro assessments of cytotoxic and cytostatic effects of Asparagus aphyllus, Crataegus
aronia, and Ephedra alata in monocultures and co-cultures of Hepg2 and THP-1-derived macrophages. Pharmacogn. Commun. 2015
5, 165–172. [CrossRef]
,
33. Mosmann, T. Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. J.
Immunol. Methods 1983, 65, 55–63. [CrossRef]