Biosynthesis of Fluorinated Peptaibols Using a Site-Directed Building Block Incorporation Approach

Article abstract of DOI:10.1021/acs.jnatprod.7b00189

Synthetic biological approaches, such as site-directed biosynthesis, have contributed to the expansion of the chemical space of natural products, making possible the biosynthesis of unnatural metabolites that otherwise would be difficult to access. Such m

Full text of DOI:10.1021/acs.jnatprod.7b00189

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Article
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Biosynthesis of Fluorinated Peptaibols Using a Site-Directed Building
Block Incorporation Approach
Jose Rivera-Chavez,† Huzefa A. Raja,† Tyler N. Graf,† Joanna E. Burdette,‡ Cedric J. Pearce,§
́
́
and Nicholas H. Oberlies*,†
†Department of Chemistry and Biochemistry, University of North Carolina at Greensboro, P.O. Box 26170, Greensboro, North
Carolina 27412, United States
‡Department of Medicinal Chemistry and Pharmacognosy, University of Illinois at Chicago, Chicago, Illinois 60612, United States
§Mycosynthetix, Inc., 505 Meadowlands Drive, Suite 103, Hillsborough, North Carolina 27278, United States
S
* Supporting Information
ABSTRACT: Synthetic biological approaches, such as site-
directed biosynthesis, have contributed to the expansion of the
chemical space of natural products, making possible the
biosynthesis of unnatural metabolites that otherwise would be
difficult to access. Such methods may allow the incorporation of
fluorine, an atom rarely found in nature, into complex secondary
metabolites. Organofluorine compounds and secondary metab-
olites have both played pivotal roles in the development of
drugs; however, their discovery and development are often via
nonintersecting tracks. In this context, we used the biosynthetic
machinery of Trichoderma arundinaceum (strain MSX70741) to
incorporate a fluorine atom into peptaibol-type molecules in a
site-selective manner. Thus, fermentation of strain MSX70741 in
media containing ortho- and meta-F-phenylalanine resulted in the biosynthesis of two new fluorine-containing alamethicin F50
derivatives. The fluorinated products were characterized using spectroscopic (1D and 2D NMR, including 19F) and spectrometric
(HRESIMS/MSn) methods, and their absolute configurations were established by Marfey’s analysis. Fluorine-containing
alamethicin F50 derivatives exhibited potency analogous to the nonfluorinated parent when evaluated against a panel of human
cancer cell lines. Importantly, the biosynthesis of fluorinated alamethicin F50 derivatives by strain MSX70741 was monitored in
situ using a droplet−liquid microjunction−surface sampling probe coupled to a hyphenated system.
ased on the literature and Dictionary of Natural Products,
approximately 250 000 secondary metabolites have been
ration in lead molecules or drugs could impact drastically on
physicochemical properties, resulting in changes in absorption,
distribution, metabolism, and molecular interactions in vivo21
and in vitro.22 Due to the potential of fluorine-containing
molecules in drug discovery, there has been a focus on the
development of new synthetic and semisynthetic strategies to
incorporate this atom into organic molecules, particularly in a
site selective manner.13,23,24 However, the incorporation of a
fluorine atom into structurally complex natural products
remains a challenge, likely because of the perception that
most of the fluorination reagents could degrade the parent
molecule provided by nature.25
An alternative approach to modify natural product scaffolds
is to employ precursor-directed biosynthesis,26 using the
biosynthetic machinery of microorganisms to incorporate
fluorinated building blocks into natural products.27−30 This
technique has been widely used in the past, generating a vast
number of microbial natural product analogues, with cyclo-
B
isolated from plants, microorganisms, and other sources
(particularly marine life).1,2 Of these, about 4700 are
halogenated,3 and just 5 contain a fluorine atom,4−7 with no
reports of natural fluorometabolites derived from fungi. Nature
has been a fertile source for drug leads, particularly in the
realms of anticancer and antimicrobial agents.8−11 Alternatively,
drugs that include at least one fluorine atom (∼274 up to
200912) represent approximately 25−30% of pharmaceuticals,13
including some of the top-selling drugs, such as sofosbuvir
(Sovaldi; annual revenue of $9.4 billion in the U.S.),
rosuvastatin (Crestor; annual revenue of $8.5 billion), and
sitagliptin (Januvia; annual revenue of $5.0 billion).13−15
However, these two classes of molecules do not often intersect,
perhaps limiting the combination of privileged scaffolds in
natural products16−18 with the beneficial properties of fluorine
atoms in medicinal chemistry.13
The high electronegativity, small atomic radius, and low
polarizability of the C−F bond are some of the unique features
that make fluorine so attractive from the point of view of
medicinal chemistry,19,20 such that, the benefits of incorpo-
Received: March 5, 2017
© XXXX American Chemical Society and
American Society of Pharmacognosy
A
DOI: 10.1021/acs.jnatprod.7b00189
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Figure 1. Structures of alamethicin F50 (1), ortho-F-Pheol alamethicin F50 (2), meta-F-Pheol alamethicin F50 (3), trichokonin VI (4), ortho-F-Pheol
trichokonin VI (5), and meta-F-Pheol trichokonin VI (6). The amino acid residue targeted for modification is highlighted in blue, whereas the red
residues highlight the difference between alamethicin F50 (1) and trichokonin VI (4).
Figure 2. (A) Full-scan MS data of MSX70741 grown in PDA medium (control). (B) Full-scan MS data of MSX70741 grown in PDA supplemented
with a racemic mixture of ortho-F-DL-Phe. (C) Full-scan MS data of MSX70741 grown in PDA supplemented with a racemic mixture of meta-F-DL-
Phe. (D) Full-scan MS data of MSX70741 grown in PDA supplemented with a racemic mixture of para-F-DL-Phe. All cultures were sampled in situ
using a droplet-liquid microjunction-surface sampling probe (droplet−LMJ−SSP) coupled to a hyphenated system (UPLC−PDA−HRMS−MS/
+
MS). In panels B and C, the peaks corresponding to the fragment b7 (m/z 792.4411 and 792.4409 for 2 and 3, respectively), indicating the
incorporation of fluorine (19F), are boxed in red. Note, the peak at m/z 906.5656 in panel A is not associated with the targeted molecule.
sporins likely being one of the most explored examples.26,31,32
There are challenges with this approach, because the building
block selected for fluorine incorporation may not be able to
compete with the natural moiety, or simply not be compatible
with the enzymes involved in the biosynthesis of the desired
product. Another challenge is that, in many of the cases, the
organisms must be genetically modified in order to disrupt the
biosynthetic pathway, so as to permit the incorporation of the
fluorinated building blocks.25,28−30,33−35
To probe the applicability of precursor-directed biosynthesis
with fungal cultures, and in order to contribute to the
expansion of the chemical space of natural products,8,36,37
a
site-directed building block incorporation approach was applied
to a wild type ascomycete using fluorinated amino acids as the
source of fluorine. Previously, we reported the isolation of
B
DOI: 10.1021/acs.jnatprod.7b00189
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a
Table 1. NMR Data for Alamethicin F50 (1), ortho-F-Pheol Alamethicin F50 (2), and meta-F-Pheol Alamethicin F50 (3)
2
3
residue
Ac
position
δC
type
δH, m (J in Hz)
2.05, s
δC
type
δH, m (J in Hz)
2.05, s
1
2
172.5
22.4
175.5
57.4
23.8
26.6
C
172.5
22.4
175.5
57.4
23.8
26.6
C
CH3
C
CH3
C
Aib1
1
2
C
C
3
CH3
CH3
1.46, s
1.53, s
8.63, s
CH3
CH3
1.46, s
1.54, s
8.64, s
4
NH
1
Pro2
175.6
65.7
29.7
C
175.6
65.7
29.7
C
2
CH
CH2
4.25, t (8.4)
1.80, m
CH
CH2
4.25, t (8.4)
1.80, m
3
2.34, m
2.34, m
b
b
4
5
27.1
49.9
CH2
CH2
1.97, m
27.1
49.9
CH2
CH2
1.97, m
2.08, m
2.08, m
3.48, td, (10.5, 6.3)
3.95, m
3.49, td, (10.5, 6.3,)
3.95, m
Aib3
1
2
178.5
57.4
23.1
27.4
C
178.5
57.4
23.1
27.4
C
C
C
3
CH3
CH3
1.54, s
1.56, s
7.62, s
CH3
CH3
1.54, s
1.56, s
7.62, s
4
NH
1
Ala4
Aib5
177.2
54.1
17.1
C
177.2
54.1
17.0
C
2
CH
CH3
4.09, m
CH
CH3
4.09, m
3
1.48, d, (7.7)
7.56, d, (5.6)
1.48, d, (7.7)
7.56, d, (5.6)
NH
1
177.8
57.3
23.1
27.1
C
177.8
57.3
23.1
27.1
C
2
C
C
3
CH3
CH3
1.54, s
1.56, s
7.93, s
CH3
CH3
1.54, s
1.56, s
7.93, s
4
NH
1
Ala6
178.1
53.9
16.9
C
178.1
53.8
16.9
C
2
CH
CH3
4.02, m
1.53, d, overlapped
7.92, brs
CH
CH3
4.01, m
1.53, d, overlapped
7.91, brs
3
NH
1
Gln7
175.8
58.1
27.1
C
175.8
58.1
C
2
CH
CH2
3.94, m
2.15, m
CH
CH2
3.94, m
2.15, m
3
27.3,
2.30, m
2.30, m
4
32.6
CH2
C
2.34, m
32.5
CH2
C
2.34, m
2.54, ddd, (15.4, 9.8, 5.6)
2.54, ddd, (15.4, 9.8, 6.3)
5
177.3
177.1
NH
8.00, d, (5.6)
6.77, brs
7.99, d, (4.9)
6.77, brs
5-NH2
7.44, brs
7.45, brs
Aib8
Val9
1
2
178.2
57.6
23.3
27.4
C
178.2
57.6
23.3
27.4
C
C
C
3
CH3
CH3
1.52, s
1.55, s
8.09, s
CH3
CH3
1.52, s
1.55, s
8.08, s
4
NH
1
175.3
65.7
30.4
19.6
20.8
C
175.3
65.7
30.6
19.6
20.8
C
2
CH
CH
CH3
CH3
3.58, dd, (9.8, 3.5)
2.25, m
CH
CH
CH3
CH3
3.58, dd, (9.3, 3.2)
2.25, m
3
4
1.00, d, (6.3)
1.13, d, (6.3)
7.49, d, (4.9)
1.00, d, (6.4)
1.13, d, (6.3)
7.49, d, (4.9)
5
NH
1
Aib10
179.0
57.6
26.8
27.1
C
179.0
57.6
26.8
27.1
C
2
C
C
3
CH3
CH3
1.54, s
1.56, s
8.22, s
CH3
CH3
1.54, s
1.56, s
8.22, s
4
NH
C
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Table 1. continued
2
3
residue
Gly11
position
δC
type
δH, m (J in Hz)
δC
type
δH, m (J in Hz)
1
2
173.0
45.1
C
173.0
45.0
C
CH2
3.67, m
3.94, m
CH2
3.67, dd (16.8, 5.6)
3.95, m
NH
1
8.34, brt, (5.6)
8.34, brt, (5.7)
Leu12
175.8
54.1
41.5
C
175.8
54.0
41.5
C
2
CH
CH2
4.46, m
1.59, overlapped
1.96, m
CH
CH2
4.45, m
1.59, overlapped
1.96, m
3
4
5
25.6
21.3
23.4
CH
1.91, m
25.6
21.3
23.4
CH
1.91, m
CH3
CH3
0.92, d, (6.3)
0.94, d, (6.3)
8.11, d, (8.4)
CH3
CH3
0.92, d, (6.3)
0.94, d, (6.3)
8.11, d, (7.8)
6
NH
1
Aib13
174.9
58.1
23.7
26.7
C
174.9
58.1
23.7
26.6
C
2
C
C
3
CH3
CH3
1.61, s
1.54, s
8.40, s
CH3
CH3
1.61, s
1.54, s
8.40, s
4
NH
1
Pro14
176.4
64.6
30.0
C
176.4
64.6
30.0
C
2
CH2
CH2
4.38, dd, (9.1, 6.3)
1.80, m
CH2
CH2
4.39, dd, (8.9, 6.5)
1.80, m
3
2.35, m
2.35, m
b
b
4
5
26.9
50.6
CH2
CH2
1.99, m
26.9
50.5
CH2
CH2
1.99, m
2.08, m
2.08, m
3.73, m
3.73, m
3.88, dt, (11.2, 6.3)
3.88, dt, (11.9, 6.2)
Val15
1
2
175.3
64.3
30.5
19.4
20.2
C
175.3
64.3
30.5
19.4
20.2
C
CH
CH
CH3
CH3
3.73, m
2.34, m
CH
CH
CH3
CH3
3.73, m
3
2.34, m
4
0.97, d, (6.3)
1.07, d, (6.3)
7.63, d, overlapped
0.98, d, (6.5)
1.07, d, (6.4)
7.63, d (8.0)
5
NH
1
Aib16
Aib17
Gln18
177.6
57.6
23.4
27.4
C
177.6
57.6
23.4
27.4
C
2
C
C
3
CH3
CH3
1.54, s
1.54, s
7.58, s
CH3
CH3
1.54, s
1.54, s
7.59, s
4
NH
1
178.8
57.7
23.4
27.4
C
178.8
57.7
23.4
27.4
C
2
C
C
3
CH3
CH3
1.53, s
1.55, s
7.81, s
CH3
CH3
1.53, s
1.55, s
7.81, s
4
NH
1
175.6
57.0
28.0
33.2
C
175.5
57.0
28.0
33.1
C
2
CH
CH2
CH2
4.01, m
CH
CH2
CH2
4.01, m
3
2.25, m
2.25, m
4
2.43, dt, (15.4, 8.4)
2.62, dt, (15.4, 7.7)
2.43, dt, (15.5, 8.6)
2.62, dt, (15.2, 8.0)
5
177.4
C
177.4
C
NH
7.78, d, (5.6)
6.78, brs
7.79, d, (5.4)
6.79, brs
5-NH2
7.44, brs
7.45, brs
Gln19
1
2
3
4
174.1
55.7
27.9
32.9
C
174.0
55.6
27.9
32.9
C
CH
CH2
CH2
4.15, m
1.99, m
2.19, m
2.34, m
CH
CH2
CH2
4.16, m
2.01−2.05, m
2.23, m
2.34, m
5
177.3
C
177.3
C
NH
7.86, d, (7.7)
6.62, brs
7.87, d, (7.5)
6.63, brs
5-NH2
7.35, brs
7.35, brs
D
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Table 1. continued
2
3
residue
position
δC
type
δH, m (J in Hz)
δC
type
δH, m (J in Hz)
Pheol20/F-Pheol20
1
2
3
65.1
52.9
31.1
CH2
CH
3.65, brt
4.25, m
64.9
54.0
37.7
CH2
CH
3.63, brt
4.16, m
CH2
2.70, dd, (14.0, 9.1)
3.07, dd, (14.0, 4.9)
CH2
2.72, dd, (14.2, 9.7,)
2.98, dd, (13.9, 4.8)
4
126.5, d, (15.4)
162.6, d, (242.7)
115.5, d, (22.1)
129.1, d, (8.4)
124.9, d, (3.7)
132.9, d, (4.4)
C
142.6, d, (7.4)
117.1, d, (21.0)
160.0, d, (242.4)
113.8, d, (21.0)
130.7, d, (8.2)
126.3, d, (2.6)
C
5
CF
CH
CH
CH
CH
CH
CF
CH
CH
CH
7.06, brd, (10.2)
6
7
6.99, t, (9.1)
7.18, dd, (7.1, 7.0)
7.04, t, (7.7)
6.88, td, (8.5, 2.3)
7.24, t, (7.6)
8
9
7.37, t, (7.7)
7.10, d, (7.6)
NH
1-OH
7.32, d, (9.1)
5.23, t, (6.6)
7.22, d, overlapped
5.27, t, (6.6)
c
c
F
−119.7 , m
−115.8 , m
a
b
c
1
Data recorded in CD3OH. H (700 MHz), 13C (175 MHz), and 19F (470 MHz). Signals may be exchangeable. Recorded at 470 MHz.
Figure 3. Key HMBC, TOCSY, and NOESY correlations for compounds 1−3.
alamethicin F50 (1; Figure 1) as the main constituent in an
extract of fungal strain MSX70741 (Figure S1, Supporting
Information), a Trichoderma isolate from the Mycosynthetix
library.38 Compound 1 is a long-chain peptaibol (20 amino acid
residues), which contains a high proportion of α-amino-
isobutyric acid (Aib, 8 residues), includes an acyl substituted N-
terminus, and has a C-terminal phenylalaninol (Pheol)
moiety.39−41 Taking into account this structural information,
we selected the Pheol20 building block as an attractive target for
the introduction of a fluorine atom into the alamethicin F50
molecule (1) in a site-selective manner. We hypothesized that
doing so would not drastically impact the biosynthesis of 1, and
the resulting analogue should retain the α-helical conformation,
which is a key feature for the biological activity of peptaibols as
membrane modifiers and pore-forming antibiotics.42−44 There-
fore, in this communication we present the in vivo synthesis, as
well as the isolation, structure elucidation, and biological
evaluation of two new fluorinated-alamethicin F50 analogues,
which were biosynthesized in wild type fungal species using the
noncanonical amino acids ortho and meta F-substituted
phenylalanine (Phe).
mixture of either ortho-F-DL-Phe, meta-F-DL-Phe or para-F-DL-
Phe (500 ppm; see Figure S1 for photographs of the cultures)
were monitored in situ using a droplet−liquid microjunction−
surface sampling probe (droplet−LMJ−SSP) coupled to a
UPLC−PDA−HRMS−MS/MS system.45 All of these cultures
showed characteristic in-source ion peaks for alamethicin F50
(1), such as m/z 1963.1313 ([M + H]+; monoisotopic
+
precursor ion), 1189.6942 (b13 fragment), 982.0722 ([M +
2H]2+), 774.4505 (y7 fragment), and 655.0505 ([M + 3H]3+)
+
(Figure 2).38 Moreover, in the culture supplemented with meta-
F-DL-Phe, the mass spectrum also showed a set of peaks shifted
by 17.99 amu ([M + H]+= 1981.1241, and y7 = 792.4409), 9.00
+
amu ([M + 2H]2+ = 991.0670), and 6.00 amu ([M + 3H]3+
=
661.0471), indicating the incorporation of a fluorine atom (F =
18.9984 amu, exact mass) into alamethicin F50 (1) (Figures 1
and 2). Similar results were observed in the MS spectrum of
cultures supplemented with ortho-F-DL-Phe (Figure 2).
Importantly, incubating the microorganism with para-F-DL-
Phe did not result in the biosynthesis of the para-F-Pheol
alamethicin F50 analogue (Figure 2), as previously reported for
beauvericin,46 pseurotin and synerazol analogues,47 giving
insights into the specificity in building block recognition by
nonribosomal peptide synthetases (NRPS).48
To obtain enough material for structural characterization of
the putative fluorinated alamethicin F50 analogues, T.
arundinaceum strain MSX70741 was grown on rice and rice
supplemented with each of the fluorinated building blocks
RESULTS AND DISCUSSION
■
Strain MSX70741 was identified as Trichoderma arundinaceum
based on morphological and molecular characterization
(Figures S2−S4). Cultures of this strain grown in potato
dextrose agar (PDA) or PDA supplemented with a racemic
E
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Figure 4. Comparison of the 1H NMR spectra in the aromatic region (6.60−7.65 ppm) for alamethicin F50 (1; maroon), ortho-F-Pheol alamethicin
F50 (2; green), and meta-F-Pheol alamethicin F50 (3; navy). All spectra were recorded in CD3OH at 700 MHz. The spectra were identical from 0.75
to 6.50 ppm. For clarity, the signals belonging to the aromatic ring in each compound have been labeled and correspond to the data for the Pheol20
residues shown in Tables S3, S4, and S5 for compounds 1, 2, and 3, respectively.
(ortho/meta/para-F-DL-Phe), separately, following established
procedures (Scheme S1).49−52 HRESIMS analysis of the
extracts obtained after 21 days of fermentation confirmed the
presence of alamethicin F50 (1), as well as its ortho and meta-F-
Pheol analogues (Figure S11), and support the hypothesis of
the inability of the microorganism’s biosynthetic machinery to
incorporate fluorine by assimilation of para-F-Phe. Purification
of the organic extract (1:1 MeCN-MeOH) of these cultures
using a set of chromatographic procedures, and guided by MS
analysis for the fluorinated signals, led to the isolation of two
fluorinated peptaibols, namely, ortho-F-Pheol alamethicin F50
(2) and meta-F-Pheol alamethicin F50 (3), in ratios of 1:20 and
1:10 compared with the nonfluorinated parent (1).
analogues of alamethicin F50 (1), respectively (Figure 3). In
1
general, the H and 13C NMR spectra for compounds 2 and 3
were almost identical to those recorded for 1 (Table 1; Figure
4), the main differences being in the chemical shifts and
splitting of signals attributed to an ortho and meta F-substituted
phenyl (2 and 3, respectively), equivalent to the phenylalaninol
(Pheol20) moiety in 1 (Figure 4, Table 1). Thus, in the 13C
NMR spectrum for 2 and 3, a set of six doublets were displayed
in the aromatic region (δC 115−163 ppm), instead of four
singlet peaks observed for 1 (δC 127−140 ppm; Table 1). The
observation of splitting in a proton-decoupled 13C NMR
experiment confirmed the incorporation of the fluorinated
building blocks into the products, as noted by the prominent
JFC values (Table 1). Importantly, the presence of a fluorine
atom in 2 and 3 was verified by 19F NMR spectroscopy with δF
values of −119.7 and −115.8 ppm compared to δF values of
−119.7 and −114.8 ppm for the racemic mixtures of amino
acids ortho-F-DL-Phe and meta-F-DL-Phe, respectively (Tables 1
and S2). The 19F-NMR signals for products 2 and 3 had similar
19F−1H coupling patterns to those observed for their
corresponding building blocks, confirming their incorporation
into alamethicin F50 (1) (Table S2).
Compounds 2 and 3 were isolated as white amorphous
powders. Their molecular formulas were established as
C92H150FN23O24 on the basis of the protonated molecule
peaks [M + H]+ at m/z 1981.1290 and 1981.1294 for 2 and 3,
respectively, in the HRESIMS spectrum (29 degrees of
unsaturation). The incorporation of the fluorine atom into
alamethicin F50 (1) was detected on the basis of the
characteristic mass shifts of [M + H]+, [M + 2H]2+, [M +
3H]3+, and y7+ ions (Figures S13, S21, and S31). The structures
of 2 and 3 were confirmed by exhaustive interpretation of 1D
and 2D NMR data, including 19F-NMR spectra (Figures S30
and S40). The 1H NMR (Table 1) spectra for 2 and 3 showed
resonances for 18 NH groups (δH 6.50−8.70 ppm), three NH2
(Gln7, Gln18, and Gln19), four aromatic protons among δH
6.80−7.40 ppm, 13 αH ranging δH 3.30−4.50 ppm, one acetyl
group at δH 2.05 ppm, and several signals in the shielded region
(δH 0.70−2.50 ppm), including 16 methyl singlets (Aib) and
eight methyl doublets, confirming 2 and 3 to be peptaibol
derivatives.38,53,54 Analysis of the 2D NMR data, in particular
COSY, TOCSY, and HMBC experiments (Figure 3), permitted
the assignment of the side chain for each amino acid (eight Aib,
three Gln, two Ala, two Val, two Pro, one Leu, one Gly, and
The amino acid sequences in 2 and 3 were also examined by
HRESIMS/MS (Figures S22 and S32). In the case of
compound 3, the full scan HRESIMS spectra exhibited several
common in-source ions, specifically [M + H]+, [M + 2H]2+, [M
+ 3H]3+, b13 and y7 fragments, with the latter two generated
from the cleavage between Aib13 and Pro14 (Figure S31).38 In
compound 3, for example, fragmentation of the ion b13+ at m/z
1189.69 gave peaks at m/z 934.5372, 849.4816, 750.4141,
665.3605, 537.3030, 466.2657, 381.2129, 310.1757, and
225.1231 indicating the successive losses of Aib13-Leu12-Gly11,
Aib10, Val9, Aib8, Gln7, Ala6, Aib5, Ala4, and Aib3 and supporting
the sequence AcAib1-Pro2-Aib3-Ala4-Aib5-Ala6-Gln7-Aib8-Val9-
Aib10-Gly11-Leu12-Aib13. Similar fragmentation of the [M +
+
+
3
2H]2+ ion, in particular the fragment y7 permitted the
+
one F-substituted Pheol moiety). On the other hand, JCH and
2JCH HMBC correlations between NH protons and the αC and
CO signals, as well as the NOESY correlations among αH
and NH protons of the neighboring amino acids, supported the
structures of 2 and 3 as the ortho-F-Pheol and meta-F-Pheol
assignment of the C-terminal fragment as Pro14-Val15-Aib16-
Aib17-Gln18-Gln19-(meta-F)Pheol20. Analogous mass spectro-
metric experiments were used to assign the amino acid
sequence in 2 (Figures S21 and S22).
F
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Journal of Natural Products
Article
The absolute configuration of each amino acid in 2 and 3 was
confirmed by acid hydrolysis, Marfey’s derivatization under
alkaline conditions, and analysis of the derivatives using a 10
min UPLC protocol.38 For this, the appropriate standards for
the D and L enantiomers of the ortho and meta F-Pheol building
blocks were prepared (Supporting Information).55 As expected,
the absolute configuration of all amino acids in 2 and 3 was L, as
previously reported for alamethicin F50 (1) by Ayers et al.38
Importantly, these results indicated that T. arundinaceum strain
MSX70741 incorporated only the L-enantiomer of the
fluorinated building blocks into the alamethicin F50 analogues
(2,3) (Figures S9 and S10). In previous studies, the absolute
configurations of the fluorinated analogues of beauvericin,
pseurotin, and synerazol were presumed to be the same as in
the parent compounds, based on the detection of one
diastereomer and specific rotation data.46,47
To assess the bioactivities of compounds 2 and 3, their IC50
values were determined against a panel of cancer cell lines
[MDA-MB-435 (melanoma), MDA-MB-231 (adenocarcino-
ma), and OVCAR3 (ovarian cancer)],51,52,56−58 In these
cytotoxicity assays, compounds 2 and 3 were equipotent to
their nonfluorinated parent, with IC50 values ranging from 4.8
to 6.4 μM (Table 2). These data suggested that the
incorporation of fluorine into the alamethicin F50 (1) molecule
did not drastically impact the cytotoxicity of the compounds.
(2) the optimum way to obtain the ortho fluorinated analogues
of alamethicin F50 was supplementing the media with 500 ppm
of ortho-F-DL-Phe, yielding 11.4 mg of a 2:5 ratio of 2/1.
Finally, to validate the protocol used for biosynthesis of
peptaibols fluorinated in the Pheol moiety at the C-terminal, we
selected Trichoderma albolutescens strain MSX57715 (details
about the strain identification are provided in the Supporting
Information, Figure S5), which biosynthesizes the peptaibol
trichokonin VI (4).38 Cultivation of this strain under the same
conditions used for strain MSX70741 led to the isolation of the
ortho and meta-F-Pheol analogues of trichokonin VI (4−6,
Figure 1). As observed with MSX70741, MSX57715 incorpo-
rated only the ortho and meta-F-L-Phe building blocks into
trichokonin VI (4), and not the para substituted analogue.
From the point of view of bioactivity, compounds 5 and 6
displayed potency similar to the nonfluorinated parent when
evaluated against the same panel of cancer cell lines, with IC50
values in the lower μM range (Table 2).
As demonstrated in this study, a site-directed building block
incorporation approach can be a powerful tool for studying, and
perhaps expanding upon, the chemical diversity available
through nature. Primarily, this approach facilitates the
incorporation of fragments that are rarely found in nature
into complex secondary metabolites. Second, these unnatural
metabolites, which may be otherwise difficult to obtain,
contribute to the expansion of chemical space around privileged
scaffolds.59 Moreover, these new biosynthetic products may
address some perceived challenges to the screening of natural
products,60 such as legal access to biodiversity, identification of
biological activity, and most recently, intellectual property
associated with composition of matter patents,60,61 which many
would consider the most desirable of “Orange Book” patents.62
In short, this approach imparts another way to translate natural
products discoveries into further development. In particular,
this methodology opens up new avenues for targeting the
biosynthesis of bioactive compounds (i.e., privileged scaffolds)
with potentially improved physicochemical and pharmacolog-
ical properties. This technique, in combination with appropriate
genomic approaches,8 may lead to the generation of valuable
compounds.
Table 2. Bioactivity Data of Compounds 1−6
a
IC50 (μM)
compd
MDA-MB-435
MDA-MB-231
OVCAR3
1
4.9
6.4
5.6
5.9
1.5
4.8
2
3
4.8
5.9
6.3
4
4.8
5.1
5.1
5
4.7
5.0
5.0
6
2.8
4.3
4.4
Paclitaxel (taxol)
0.0005
0.009
0.002
a
IC50 values were determined as the concentration required to reduce
cellular proliferation by 50% relative to the untreated controls
following 72 h of continuous exposure.
In summary, we report the biosynthesis of fluorine
containing analogues of the peptaibols alamethicin F50 (2,3)
and trichokonin VI (5,6) using a site-directed building block
incorporation approach. Importantly, the biosynthesis of these
products was carried out using wild type Trichoderma strains.
Biosynthesis of products 2, 3, 5, and 6 represent the first report
of the application of a site-directed building block incorporation
approach targeting the incorporation of a fluorine atom into
peptaibol type molecules. Notably, examination of the ability of
Trichoderma species to incorporate the fluorinated building
blocks was monitored in situ, facilitating the identification of the
products in an early stage of the study, before the scaling up of
the cultures.
In an attempt to stimulate the fungus to increase the
biosynthesis of the fluorinated analogues of alamethicin F50, an
experiment was designed as follows: T. arundinaceum was
cultured on PDA for 3 days. Subsequently, an agar plug with
mycelium from the leading edge of the colony was used to
inoculate 10 mL of liquid medium containing 2% of soy
peptone, 2% dextrose, and 1% yeast extract (YESD). After 3
days of growth, the liquid media was used to inoculate either
autoclaved rice (10 g of rice and 20 mL of H2O; control), or
autoclaved rice containing either: (1) 130 mg (powder) of
ortho-F-DL-Phe; (2) 37.5 mg of ortho-F-DL-Phe in 5.0 mL of
H2O (final concentration: 1250 ppm); (3) 15 mg of ortho-F-DL-
Phe in 2.0 mL of H2O (final concentration: 500 ppm); or (4)
15 mg of ortho-F-L-Phe in 2.0 mL of H2O (final concentration:
500 ppm) (Scheme S2). All four cultures were incubated for 21
days and then extracted and analyzed by UPLC-PDA-MS. The
fluorinated analogues were detected in all cultures. Subsequent
isolation of compound 2 by HPLC-PDA-MS following the
protocol described in the experimental section indicated that
(1) supplementing the microorganism with 130 mg of ortho-F-
DL-Phe increased the ratio of compounds 2/1 to 1:1, although
the overall yield of product declined significantly (1 mg), and
EXPERIMENTAL SECTION
■
General Experimental Procedures. NMR experiments were
conducted in CD3OH with presaturation of the OH peak at δH 4.92
ppm (wet experiment). NMR instrumentation was a JEOL ECA-500
NMR spectrometer operating at 500 MHz for H, 470 MHz for 19F,
1
and 125 MHz for 13C, or an Agilent 700 MHz NMR spectrometer
1
equipped with a cryoprobe, operating at 700 MHz for H and 175
MHz for 13C. All chemical shifts were referenced to the residual
solvent peaks (δH 3.31 and δC 49.0). HRESIMS data were obtained
using a Thermo QExactive Plus mass spectrometer (ThermoFisher
G
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Journal of Natural Products
Article
Scientific) paired with an electrospray ionization source. Monitoring
the biosynthesis of secondary metabolites in fungal cultures in situ was
performed using the droplet-LMJ-SSP coupled with a Waters Acquity
ultraperformance liquid chromatography (UPLC) system (Waters
Corp.) to a Thermo QExactive Plus via procedures described
previously by Sica et al.45 Briefly, extractions were performed using
Fisher Optima LC/MS grade solvents consisting of 50:50 MeOH-
H2O. An initial 5 μL of solvent was drawn into the syringe. Droplets of
4 μL were dispensed onto the surface of the sample at a rate of 2 μL/s,
held on the surface for 2 s, and withdrawn back into the syringe at the
same rate. This extraction process was repeated in triplicate for a single
spot prior to injection into the UPLC−MS system. The higher-energy
collisional dissociation (HCD) used a normalized energy of 35 for all
the compounds to obtain MS/MS data. The UPLC separations were
performed using an Acquity BEH C18 column (50 mm × 2.1 mm,
internal diameter, 1.7 μm) equilibrated at 40 °C and a flow rate set at
0.3 mL/min. The mobile phase consisted of a linear MeCN-H2O
(acidified with 0.1% formic acid) gradient starting at 15% MeCN to
100% MeCN over 8 min. The mobile phase was held for another 1.5
min at 100% MeCN before returning to the starting conditions. The
HPLC separations were performed using a Varian ProStar HPLC
system connected to a ProStar 335 photodiode array detector (PDA)
with UV detection set at 195 and 210 nm. Preparative HPLC
purifications of isolated compounds were performed on a Phenomenex
Synergi 4 μm particle size C12 column (21 × 250 mm) at a flow rate of
20.0 mL/min. Flash column chromatography was carried out with a
Teledyne ISCO Combiflash Rf connected to ELSD and PDA
detectors, with the latter having UV detection set at 200−400 nm,
all according to established protocols.51,58,63,64 All solvents were
obtained from Fisher Scientific and used without further purification.
The o/m/p-F-DL-Phe, o/m-F-D-Phe, and o/m-F-L-Phe were purchased
from Acros Organics. The standards of o/m-F-D-Pheol and o/m-F-L-
Pheol were prepared as detailed in the Supporting Information.
Fungal Strain Isolation and Identification. Mycosynthetix
fungal strain MSX70741 was isolated from wood collected in a
humid mountain forest (April 1993), whereas strain MSX57715 was
isolated from leaf litter in a predominately oak forest (October 1991)
both by Dr. Barry Katz.38 Both strains were used previously for the
isolation of peptaibols.38 A description of the procedures used to
identify these strains was outlined recently,65 and the specific details
are also provided in the Supporting Information (Table S1, Figures
S2−S5). MSX70741 was identified as Trichoderma arundinaceum,
whereas strain MSX57715 was identified as T. albolutescens. The
sequence data for both strains were deposited in GenBank (accession
numbers: ITS: KY630171, tef1: KY630169, KY630170, RPB2:
KY630166 for strain MSX70741 and accession numbers: tef1:
KY630167, KY630168, RPB2: KY630164, KY630165 for strain
MSX57715).
Fermentation, Extraction, and Isolation. Fungal strains
MSX70741 and MSX57715 were each grown on a malt extract agar,
and subsequently, a small piece from the leading edge of the colony
was transferred into YESD media (followed by incubation for 7 days at
22 °C with agitation at 125 rpm). The seed cultures were transferred
into 250 mL Erlenmeyer flasks containing 50 mL of rice, which was
prepared by adding a vitamin solution and twice the volume of rice
with H2O. These flasks were incubated at 22 °C until the culture
showed good growth. In the case of media supplemented with a
racemic mixture of ortho-F-DL-Phe (E2), meta-F-DL-Phe (E3), or para-
F-DL-Phe (E4), 100 mg of the amino acid were added to the culture
after a week of growth (Scheme S1).
To each solid fermentation culture of MSX70741 (EC, E2-E4; see
Scheme S1), 60 mL of 1:1 MeOH−CHCl3 were added, and the
resulting slurry was shaken for 16 h on an orbital shaker. These
mixtures were filtered under vacuum. To each filtrate 90 mL of CHCl3
and 150 mL of H2O were added, and the mixtures were stirred for 30
min and then transferred into a separatory funnel. The organic layer
was drawn off and dried in vacuo. This dried organic extract was
defatted by reconstituting in a mixture of 100 mL of 1:1 MeOH−
MeCN and 100 mL of hexane, and then partitioned in a separatory
funnel. The MeOH−MeCN layer was collected and concentrated in
vacuo. The resulting MeOH−MeCN extracts were then adsorbed on
Celite 545 (Acros Organics) and fractionated via flash chromatography
on a 4 g RediSep Rf Gold Si-gel column using a gradient solvent
system of hexane-CHCl3- MeOH at a flow rate of 18 mL/min over 90
column volumes (CV) for a duration of 24.0 min. Fractions were
collected every 9.0 mL and pooled according to the UV and ELSD
profiles, which resulted in four combined fractions in total (FI-FIV).
MS-directed resolution of fraction FIV from E2 and E3 (eluted with
100% MeOH, 92.4 and 136.0 mg, respectively) via reversed-phase
HPLC (Synergi column), using a linear gradient from 40% to 100%
MeCN in H2O (0.1% formic acid) at a flow rate of 20.0 mL/min over
30 min afforded seven subfractions (FIV‑1−FIV‑7). Fractions FIV‑1 (tR
18.5−19.5 min) from E2 and E3 were further characterized as
alamethicin F50 (1, 28.5 and 42.9 mg, respectively). Purification of
fractions FIV‑2 from E2 and E3 by MS-directed semipreparative HPLC
using the above-mentioned conditions at 4.60 mL/min led to the
isolation of compounds 2 (1.4 mg) and 3 (4.5 mg). Analogous
procedures were used to isolate compounds 4 (24.0 mg), 5 (3.4 mg),
and 6 (6.2 mg).
26
Alamethicin F50 (1). White powder; [α]D −6.5 (c 0.2,MeOH);
1
UV (MeOH) λmax (log ε) 204 (4.52) nm; H NMR (CD3OH, 700
MHz) and 13C NMR (CD3OH, 175 MHz), see Table S3; HRESIMS
m/z 1963.1388 [M + H]+ (calcd for C92H152N23O24, m/z 1963.1375).
o-F-Pheol-Alamethicin F50 (2). White powder; [α]D26 −7.0 (c 0.2,
MeOH); UV (MeOH) λmax (log ε) 204 (4.54) nm; 1H NMR
(CD3OH, 700 MHz) and 13C NMR (CD3OH, 175 MHz), see Table
1; HRESIMS m/z 1981.1290 [M + H]+ (calcd for C92H151FN23O24,
1981.1280).
27
m-F-Pheol-Alamethicin F50 (3). White powder; [α]D −1.0 (c
1
0.03, MeOH); UV (MeOH) λmax (log ε) 204 (4.52) nm; H NMR
(CD3OH, 700 MHz) and 13C NMR (CD3OH, 175 MHz), see Table
1; HRESIMS m/z 1981.1294 [M + H]+ (calcd for C92H151FN23O24,
1981.1280).
24
Trichokonin VI (4). White powder; [α]D −10.0 (c 0.2, MeOH);
UV (MeOH) λmax (log ε) 204 (4.57) nm; HRESIMS m/z 1937.1232
[M + H]+ (calcd for C90H150N23O24, 1937.1218).
25
o-F-Pheol-Trichokonin VI (5). White powder; [α]D −8.0 (c 0.3,
MeOH) ; UV (MeOH) λmax (log ε) 204 (4.54) nm; HRESIMS m/z
1955.1147 [M + H]+ (calcd for C90H149FN23O24, 1955.1124).
25
m-F-Pheol-Trichokonin VI (6). White powder; [α]D −5.0 (c 0.2,
MeOH) ; UV (MeOH) λmax (log ε) 204 (4.48) nm; HRESIMS m/z
1955.1149 [M + H]+ (calcd for C90H149FN23O24, 1955.1124).
Cytotoxicity Assay. Human melanoma cancer cells MDA-MB-
435, human breast cancer cells MDA-MB-231, and human ovarian
cancer cells OVCAR3 were purchased from the American Type
Culture Collection (Manassas, VA, U.S.A.). The cell lines were
propagated at 37 °C in 5% CO2 in RPMI 1640 medium, supplemented
with fetal bovine serum (10%), penicillin (100 units/mL), and
streptomycin (100 μg/mL). Cells in log phase of growth were
harvested by trypsinization followed by two washes to remove all
traces of enzyme. A total of 5000 cells were seeded per well of a 96-
well clear, flat-bottom plate (Microtest 96, Falcon) and incubated
overnight (37 °C in 5% CO2). Samples dissolved in DMSO were then
diluted and added to the appropriate wells (several concentrations;
total volume: 100 μL; DMSO: 0.5%). The cells were incubated in the
presence of test substance for 72 h at 37 °C and evaluated for viability
with a commercial absorbance assay (CellTiter 96 AQueous One
Solution Cell Proliferation Assay, Promega Corp, Madison) that
measured viable cells. IC50 values were determined as the
concentration required to reduce cellular proliferation by 50% relative
to the untreated controls following 72 h of continuous exposure.
Paclitaxel (taxol) was used as a positive control.
Optimization of Biosynthesis of Fluorinated Analogues. For
each different condition (Scheme S2) a seed of the fungal strain
MSX70741 was grown on liquid YESD followed by incubation for 3
days at 22 °C with agitation at 125 rpm. The seed culture was
transferred into 250 mL Erlenmeyer flasks containing 30 mL of rice
medium, prepared using 10 g of rice and twice the volume of rice with
H2O. These flasks were supplemented with 130 mg of ortho-F-DL-Phe
powder (Condition 1), 2.0 mL of a stock solution 7500 ppm of ortho-
H
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Journal of Natural Products
Article
F-DL-Phe (500 ppm, Condition 2), 5.0 mL of a stock solution 7500
ppm of ortho-F-DL-Phe (1250 ppm, Condition 3), or 2.0 mL of a stock
solution 7500 ppm of ortho-F-L-Phe (500 ppm, Condition 4) (Scheme
S2). The flasks were incubated at 22 °C until they showed good
growth and then extracted according to the procedure mentioned in
the fermentation, extraction, and isolation section.
Marfey’s Analysis. Approximately 0.2 mg of each amino acid
standard was weighed into separate glass 2 mL reaction vials. To each
standard was added 50 μL of H2O, 20 μL of 1 M NaHCO3, and 100
μL 1% Marfey’s reagent (Nα-(2,4-dinitro-5-fluorophenyl)-L-alanina-
mide) in acetone. The reaction mixtures were agitated at 40 °C for 1 h.
The reactions were halted by the addition of 10 μL of 2 N HCl. The
product of the reactions was dried under a stream of nitrogen and
dissolved in ∼1.7 mL of MeOH. Each derivatized standard was
injected individually (0.7 μL) onto the UPLC. Also, aliquots of all of
the derivatized standards were combined to give a mixed standard,
which was injected too. UPLC conditions were 10−70% MeOH in
0.1% of formic acid in H2O over 10 min on a BEH column, and the
eluent was monitored at 340 nm.
To generate the digested and derivatized peptaibols, approximately
0.2−0.3 mg of compounds 1−3 were weighed separately into 2 mL
reaction vials, to which was added 0.5 mL of 6 N HCl. The
compounds were hydrolyzed at 90 °C for 24 h, at which time they
were evaporated under a stream of nitrogen. To each hydrolysis
product was then added 25 μL of H2O, 10 μL of 1 M NaHCO3, and
50 μL of 1% Marfey’s reagent in acetone. The reaction mixtures were
agitated at 40 °C for 1 h. The reactions were halted by the addition of
5 μL of 2 N HCl. The mixtures were dried under a stream of nitrogen
and brought up in ∼200 μL of MeOH and injected onto the UPLC
using the same conditions as for the standards (Figures S7−S10).
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ASSOCIATED CONTENT
■
S
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jnat-
prod.7b00189.
Molecular phylogenetic trees of T. arundinaceum and T.
albolutescens; culture conditions of strains MSX70741
and MSX57715; experimental procedures; 1D and 2D
NMR spectra and tabulated data of compounds 1−3;
HRESIMS spectra of compounds 1−3; and Marfey’s
analysis of standards and compounds 1−3 (PDF)
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N.; Kaminsky, W.; Charman, S. A.; Matthews, D.; Rathod, P. K.;
Phillips, M. A. J. Med. Chem. 2014, 57, 5381−5394.
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AUTHOR INFORMATION
■
Corresponding Author
*E-mail: Nicholas_Oberlies@uncg.edu. Tel: 336-334-5474.
ORCID
Nicholas H. Oberlies: 0000-0002-0354-8464
Notes
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The authors declare no competing financial interest.
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ACKNOWLEDGMENTS
■
This research was supported via grant P01 CA125066 from the
National Cancer Institute/National Institutes of Health,
Bethesda, MD, USA. The high-resolution mass spectrometry
data were acquired in the Triad Mass Spectrometry Laboratory
at the University of North Carolina at Greensboro, Greensboro,
NC, USA. We thank Drs. V. Kertesz and G. J. Van Berkel
(Mass Spectrometry and Laser Spectroscopy Group, Chemical
Sciences Division, Oak Ridge National Laboratory, Oak Ridge,
TN, USA) for inspiration and guidance with the droplet-LMJ-
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