Detailed Mechanistic Study of the Non-enzymatic Formation of the Discoipyrrole Family of Natural Products

Article abstract of DOI:10.1021/jacs.5b13320

Discoipyrroles A-D (DPA-DPD) are recently discovered natural products produced by the marine bacterium Bacillus hunanensis that exhibit anticancer properties in vitro. Initial biosynthetic studies demonstrated that DPA is formed in the liquid fermentation medium of B. hunanensis from three secreted metabolites through an unknown but protein-independent mechanism. The increased identification of natural products that depend on non-enzymatic steps creates a significant need to understand how these different reactions can occur. In this work, we utilized ¹⁵N-labeled starting materials and continuous high-sensitivity ¹H-¹⁵N HMBC NMR spectroscopy to resolve scarce reaction intermediates of the non-enzymatic discoipyrrole reaction as they formed in real time. This information guided supplemental experiments using ¹³C- and ¹⁸O-labeled materials to elucidate the details of DPA's non-enzymatic biosynthesis, which features a highly concerted pyrrole formation and necessary O₂-mediated oxidation. We have illustrated a novel way of using isotopically enhanced two-dimensional NMR spectroscopy to interrogate reaction mechanisms as they occur. In addition, these findings add to our growing knowledge of how multicomponent non-enzymatic reactions can occur through inherently reactive bacterial metabolites.

Full text of DOI:10.1021/jacs.5b13320

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Article
Detailed Mechanistic Study of the Non-enzymatic
Formation of the Discoipyrrole Family of Natural Products
Dominic A. Colosimo, and John B. MacMillan
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.5b13320 • Publication Date (Web): 29 Jan 2016
Downloaded from http://pubs.acs.org on February 4, 2016
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Detailed Mechanistic Study of the Non-enzymatic Formation
of the Discoipyrrole Family of Natural Products
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Dominic A. Colosimo and John B. MacMillan*
Department of Biochemistry, University of Texas Southwestern Medical Center. 5323 Harry Hines Blvd, Dallas, TX USA
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5390
ABSTRACT: The discoipyrroles AꢀD (DPAꢀDPD) are recently discovered natural products produced by the marine bacteria, Ba-
cillus hunanensis, which exhibit antiꢀcancer properties in-vitro. Initial biosynthetic studies demonstrated that DPA was formed in
the liquid fermentation media of B. hunanensis from three secreted metabolites through an unknown, but protein independent mechꢀ
anism. The increased identification of natural products who depend on nonꢀenzymatic steps create a significant need to understand
15
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how these different reactions can occur. Here, we utilized N labeled starting materials and continuous highꢀsensitivity Hꢀ N
HMBC NMR to resolve scarce reaction intermediates of the nonꢀenzymatic discoipyrrole reaction as they formed in real time. This
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information guided supplemental experiments, using C and O labeled materials, to elucidate the details of discoipyrrole A’s nonꢀ
enzymatic biosynthesis which features a highly concerted pyrrole formation and necessary O mediated oxidation. We have illusꢀ
2
trated a novel way of using isotopically enhanced twoꢀdimensional NMR to interrogate reaction mechanisms as they occur. In addiꢀ
tion, these findings added to our growing knowledge of how multiꢀcomponent nonꢀenzymatic reactions can occur through inherentꢀ
ly reactive bacterial metabolites.
INTRODUCTION
The discoipyrroles (DPs) are a family of natural products
that were isolated from the bacterial fermentation of Bacillus
hunanensis strain SNAꢀ048. The DPs were shown to be inhibiꢀ
tors of discoidin domain receptorꢀ2 (DDR2) dependent cell
migration of BR5 human foreskin fibroblasts and potent cytoꢀ
1
toxins to DDR2 mutant nonꢀsmall cell lung cancer cell lines .
In the initial report of DPA (1) we demonstrated its producꢀ
tion in the aqueous fermentation media of SNA-048 via a proꢀ
tein independent multiꢀcomponent reaction from three starting
materials; 4ꢀhydroxysattabacin (4), anthranilic acid (5), 4ꢀ
hydroxybenzaldehyde (6). The nonꢀenzymatic nature of this
reaction was demonstrated through a series of feeding studies
in fermentation media depleted of bacteria and proteins via
filtration and heating, respectively. We were further able to
recapitulate the chemistry in organic solvent via a threeꢀstep
procedure that was later utilized towards the total synthesis of
discoipyrrole D (3) reported by the May laboratory .
Figure 1. Metabolites found in the fermentation media of B.
hunanensis strain SNAꢀ048.
2
Previously described microbial natural products that utilize
2
It is well established that bacteria most often use compliꢀ
mentary enzymes to sequentially catalyze bond formation in
natural product biosynthesis, and that these proteins are exꢀ
a nonꢀenzymatic step feature attack of an electrophilic sp
carbon of a lateꢀstage enzymatic intermediate by a nucleoꢀ
5ꢀ6
phile . In the case of 1 however, the selfꢀassembly of 4, 5,
and 6 requires the formation of four covalent bonds as well as
two oxidations. Deciphering the mechanism by which the disꢀ
coipyrroles are formed will expand our general knowledge 0f
the types of nonꢀenzymatic reactions to be expected in the
milieu of microbial fermentation.
pressed from highly conserved and organized clusters of genes
3
.
However, there have been increasing number of cases where
isolated secondary metabolites differ from their predicted
structure due to nonꢀenzymatic reactions stemming from serꢀ
4
endipitous proximity of complimentary reactive metabolites .
The knowledge gained from interrogating these mechanisms
has fueled inventive and effective techniques to generate anaꢀ
5
logs with improved biological activities .
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Figure 2. Initial studies of formation of DPA in the fermentation flask were unsuccessful due to low reagent yields, interfering
media components, and complicated analytical sample preparation. To combat these issues, precursors were combined in deuterated
organic solvent and observed constantly by NMR. The base 4ꢀ(dimethylamino)pyridine (DMAP) was used to simulate the basic
environment of the fermentation flask (pH = ~9). The oxidation of the acyloin of 4ꢀhydroxysattabacin to form a diketone was
known to be a rate limiting step in DPA formation, thus the diketone was prepared with DessꢀMartin periodinane (DMP)
beforehand
to
increase
the
efficiency
of
the
model
system.
This understanding will improve our ability to predict natuꢀ
ral products that may undergo these reactions. Specific to the
discoipyrroles, further understanding of the mechanism of
formation can aid in our medicinal chemistry efforts.
sources of 4, 5, and 6. These experiments are described in
detail in the supplemental information (SI Text and SI Figures
1ꢀ5).
To investigate the nonꢀenzymatic multiꢀcomponent forꢀ
mation of the discoipyrrole scaffold we took advantage of our
previously established synthetic model reaction system in orꢀ
ganic solvent. This model takes advantage of starting with the
oxidized 4ꢀmethoxysattabacin (7) (Figure 2), the formation of
which we have previously found to be the rateꢀlimiting step in
both the fermentation media and organic solvent. Additionally,
the model system removes analytical barriers associated with
the complex fermentation media. Past successes using NMR
active isotope labels for studying reactions through use of
RESULTS AND DISCUSSION
Herein we report the details of the nonꢀenzymatic discoipyrꢀ
role formation obtained by a combination of NMR and MS
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approaches utilizing isotopically labeled substrates ( C, N,
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and O). In particular, N labeled substrates were used for
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highly sensitive NMR experiments to identify Hꢀ N heteroꢀ
nuclear correlations of key intermediates. The increased sensiꢀ
tivity of the isotope label allowed for short (~30 minute) exꢀ
periments capable of detecting low abundance intermediates in
complicated mixtures. This approach could be applied to the
study of mechanisms of heteronuclear bond forming reactions,
most notably applied to multiꢀcomponent reactions.
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kinetic isotopes , to elucidate biosynthetic pathways , and in
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the identification of natural products led us to utilize C and
N labeled substrates to probe the multiꢀcomponent reaction
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by NMR and MS.
We began our studies of the discoipyrrole formation by exꢀ
amining the origins of the precursor molecules 4, 5, and 6. 4
resembles members of a growing family of acyloinꢀcontaining
molecules that are products of condensation between two amiꢀ
no acids. 5 and 6 are known degradation products of tryptoꢀ
phan and tyrosine, respectively (KEGG Compounds C00108
To track the formation of reaction intermediates, the chemiꢀ
cal shifts and heteronuclear correlations of key carbon and
nitrogen atoms around the pyrrole core were observed in real
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time. Utilizing [1ꢀ C]ꢀpꢀhydroxybenzaldehyde and
Nꢀ
anthranilic acid allowed for observation of heteronuclear
NMR correlations from these isotope labels during each of the
key bond forming steps. All of the following experiments,
unless otherwise noted, were run using 1.0 equivalent of 7, 2.0
equivalents of 5 and 6 along with 3 equivalents of DMAP. The
reactions were carried out in DMSO. For the reactions run in
an NMR tube, they were run in a total volume of 700 µL in
DMSOꢀd6.
7
and C00633) .
To confirm the amino acid origins of 4, we utilized seꢀ
quences of known TPPꢀdependent acyloin synthases to disꢀ
cover homologous candidate genes in the annotated genome of
8
Bacillus hunanensis strain SNA-048 (SI Figure 3) . Particularꢀ
ly of use were the recent findings by Park et al. describing the
sattabacin producing acyloin synthase known as Thzk1050
8a
from Thermosporothrix hazakensis . Based on the presence
of these putative biosynthetic genes in SNA-048, we perꢀ
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formed feeding studies with C isotopeꢀlabeled [2ꢀ C]ꢀLꢀ
13
tyrosine and [2ꢀ C]ꢀLꢀleucine and confirmed their incorporaꢀ
tion into 4 (SI Figure 4). Additional feeding studies and/or
genetic analyses were performed to conclude the amino acid
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c)
O
HO C
HO
HO
2
C
HO
O
HO C
2
HO
O
2
HO
O
4
O
O
15N
15N
fast
15N
15N
O
OH
OH
CO H
O
2
H
15NH
O
O
OH
O
2
O
6
5
O
I
II
III
IV
1
15
1
15
Figure 3. Reaction time course monitored by Hꢀ N NMR a) Representative time points from Hꢀ N HMBC experiments. Major
1
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correlations have been highlighted using colored boxes on the spectra. b) Construction of reaction timeline from Hꢀ N HMBC
experiments allows for design of additional NMR experiments to elucidate structures of intermediates. c) Structures of
intermediates determined through use of 1 and 2D NMR data.
Experimentally we began by carrying out the multiꢀ
component reaction with Nꢀanthranilic acid and conducted
continuous reaction monitoring in 30 minute intervals for 36
hours using Hꢀ N HMBC. This methodology provided eviꢀ
dence of CꢀN bond formation through developing Hꢀ N corꢀ
relations, specifically to downfield H signals of the isobutyl
chain of 7. Due to the spectral range of N NMR, the
chemical shift of anthranilateꢀderived intermediates provides
key structural information on the nature of the CꢀN bond of an
intermediate (SI Figure 6).
coipyrroles. The key COSY correlations were δ 2.17 and 2.38
ppm to a septet at δ 1.28 ppm that was further coupled to meꢀ
thyl doublets at δ 0.62 and 0.70 ppm. The slightly downfield
chemical shift of the diastereotopic methylene and the
chemical shift indicated an electron withdrawing environment,
potentially suggesting a pyrrole ring. This was confirmed by
1
5
1
15
1
5
N
1
15
1
1
5
15
1
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N
Hꢀ C HMBC correlations from methylene protons to carbons
at δ 127.0 and 138.5 ppm consistent with structure of I. The
1
13
absence of Hꢀ C HMBC correlations between these carbons
1
and exchangeable NH H signals further suggested the forꢀ
The first experiment performed provided a time frame for
mation of the pyrrole ring.
15
key N containing intermediates (Figure 3, Supplemental
Video 1). For example, from the timeꢀlapse video it can be
determined that the first major intermediate (red box in Figure
15
A weaker N signal of δ ꢀ254.9 ppm appeared with correlaꢀ
tions to an aromatic proton at δ 6.95 ppm and aliphatic signals
at δ 4.40 and 1.34 ppm. Using similar strategies as described
for I, correlations between these signals and to the isobutyl
chain led to structure II, the ketone tautomer of I.
1
5
3
a) with correlations from the N to protons on 7 begin apꢀ
pearing at two hours and disappear by 12 hours, whereas the
second major intermediate (blue box in Figure 3a) appears at 4
hours and disappears at 14 hours. From this timeline, we carꢀ
ried out additional 2D NMR experiments, such as H – H
COSY, H – C HSQC, and Hꢀ C HMBC to elucidate the
structural identity of the time sensitive intermediates (Figure
1
15
After 16 hours, the Hꢀ N HMBC correlations of I and II
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1
1
decrease, while a third N signal at δ ꢀ248.3 ppm appears with
correlations to protons at δ 7.24 and 1.69 ppm, representing
compound III. Up to 24 hours into the reaction, this signal
existed independently and continued to build in intensity. Due
to the apparent stability and abundance of the intermediate an
aliquot of the reaction was removed and subjected to LC/MS
analysis. The retention time and mass indicated that compound
III was the open form of discoipyrrole that exists in pH deꢀ
pendent equilibrium with 1. This reaction was repeated at a
later time to yield larger quantities of III, which was isolated
and characterized in full (SI Table I).
1
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1
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b).
The starting material Nꢀanthranilic acid has a N shift of δ
311.1 ppm and a strong correlation to aromatic protons at
δ 6.71 and 7.67 ppm. Immediately after the three reagents
15
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ꢀ
1
15
were added to the NMR in DMSOꢀd we saw Hꢀ N HMBC
6
1
5
correlations from a N shift of δ ꢀ225.0 ppm to protons at
δ 6.96, 2.17, and 2.38 ppm appear, suggesting bond formation
between 7 and 5. This was validated by further COSY and
HSQC correlations that established the H’s at δ 2.17 and 2.38
ppm to be the methylene of the isobutyl sideꢀchain of the disꢀ
1
After 24 hours, 1% TFA was added to the NMR tube and
1
15
monitoring via Hꢀ N HMBC was continued. In the first
15
NMR scan after acid addition, two N species at δ ꢀ251.3
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ppm, with H correlations to δ 7.29 and 1.67 ppm, and δ ꢀ
46.4 ppm, with proton correlations to δ 7.61, 1.75, and 1.44
unaffected by substitution of either the substitutents on the
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benzaldehyde or anthranilic acid moeities.
2
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5
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ppm, appeared. Within minutes, a major N species of δ ꢀ
63.8 ppm with correlations to H of δ 6.29, 2.06, and 1.91
ppm appeared. This was assigned as the final product, IV.
Isolation of the Oꢀlabeled analog of III allowed us to exꢀ
1
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plore the acidꢀinduced ring closure. Surprisingly, exposure to
+
TFA led to an isolated product with m/z [M + H] of 457.1774
+
O
(
expected [M + H] m/z = 457.1776) that indicated the loss of
1
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15NH
O label upon ring closure (SI Figure 9). This suggests under
H
2
HO
O
DMAP
DMSO-d
these anhydrous, acidic conditions that the elimination of H O
2
HO
O
CO H
15N 18OH
2
6
COOH
leads to imine formation, followed by rapid cyclization.
O
18O
2
1
15
While monitoring Hꢀ N HMBC, we could detect no inꢀ
termediates prior to pyrrole formation, i.e. no combination of 5
with either 7 or with 6 alone. This indicated to us that forꢀ
mation of intermediate I was irreversible and the multiꢀ
component reaction occurred too rapidly to be detected by
isotope enhanced NMR. We hypothesized that there were
three potential pathways to form I (Figure 5, pathways 1 – 3).
To test these individually, we performed three independent
reactions, each with one of the substrates omitted. These reacꢀ
tions were performed using the described model conditions,
except when noted.
0
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5
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0
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9
0
O
O
8
Pathway 1 relies on initial formation of an aldimine between
5
and 6, setting up for a Mannich reaction. We monitored for
1 15
imine formation using H– N HMBC with an expected imine
15
N shift in the 0 – 100 ppm range. Under model conditions,
after 24 hours there was no formation of 9 (Figure 6). This
was further confirmed by LCꢀMS analysis. As a control reacꢀ
15
tion we were readily able to form aldimine 10 between
N
aniline and 6 under the model conditions to give a product
15
with a N δ at ꢀ65.0 ppm (Figure 6). This suggests that the
mechanism of discoipyrrole formation does not go through a
Mannich reaction. To further test this, we took commercially
available 9, 7 and DMAP in our model conditions and moniꢀ
tored for formation of III by LCꢀMS. Product was not detectꢀ
ed after 48 hours, well within the timeframe expected for
product formation to occur.
Figure 4. Formation of discoipyrrole analog 8 under an
18
–
atmosphere of O . [M – H] data confirming incorporation of
2
1
1
8
6
O with representative control experiment carried out with
O2.
13
To test pathway 2, [1ꢀ C]ꢀ6 and 7 were treated with DMAP
and monitored by NMR and LCꢀMS looking for the Claisenꢀ
Schmidt product. After 48 hours we only observed the presꢀ
These experiments demonstrated that the formation of the
pyrrole core of the discoipyrroles occurs rapidly to produce
the first major intermediate, I, which persists in equilibrium
with the tautomer II. The loss of oxygen indicated that forꢀ
mation of the CꢀN bond in I induces an elimination of water.
Subsequent oxidation of the pyrrolone ring to yield III, which
under exposure to acid undergoes cyclization to IV. It should
be noted that it is also possible that I is directly oxidized to III
and that we only observe II as part of the equilibrium with I.
To confirm this mechanism, we carried out the formation of
13
ence of [1ꢀ C]ꢀ6 and 7. Increasing reaction time, temperature,
or strength of the base did not facilitate any condensation. It is
possible that anthranilic acid could act as a catalyst for the
ClaisenꢀSchmidt reaction, however the complete lack of prodꢀ
uct formation under all of the conditions attempted is strong
evidence against pathway 2.
Pathway 3, in which 7 undergoes imine formation was testꢀ
ed by omitting 6 in the reaction. However to our surprise, after
24 hours the main product was a dimethoxy discoipyrrole anaꢀ
log. Utilizing NMR and LC/MS we observed 4ꢀ
methoxybenzaldehyde in the reaction, demonstrating that 7
could be degraded to form 4ꢀmethoxybenzaldehyde. Thereꢀ
fore, aldehyde contamination of reactions involving oxidized
sattabacin analogs prevented further analysis due to the shutꢀ
tling of intermediates to pyrrole formation.
1
8
III identical conditions under an atmosphere of O . Monitorꢀ
2
ing of the reaction by LCꢀMS led to observation of a peak with
–
an m/z [M – H] of 475.2, a +2 shift from canonical III as eviꢀ
16
dent by the control reaction run with O (Figure 4). Acid free
2
18
isolation and high resolution mass spectrometry of the Oꢀ
labeled product gave an experimental value of an m/z [M +
+
+
H] of 477.1923 (expected [M + H] m/z = 477.1924). The
1
6
isolated + O control product gave an experimental value m/z
[M + H] of 475.1881 (expected [M + H] m/z = 475.1881) (SI
+
Figure 7ꢀ8). This unusual oxidation of the pyrrole moiety is
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Pathway 1
OH
1
2
3
4
5
6
7
8
9
HO
O
HO
O
2
CO H
CO H
2
CO H
2
15NH
15N
15NH
-H O
2
O
O
H
I
O
O
O
OH
O
O
Pathway 2
HO
2
C
HO
H215N
HO
HO
O
-H O
CO H
CO H
2
2
2
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O
4
15NH
15NH
-H O
-H
2
O
6
O
O
O
I
CO H
2
H
15NH2
O
H
OH
O
HO
O
O
O
5
Pathway 3
2
CO H
O
HO
HO C
HO C
2
HO
2
15N
2
-H O
15N
15N
I
HO
OH
O
O
O
O
O
Figure 5. Possible pathways to pyrrole core intermediate I.
densation between the C1 position of sattabacin and 4ꢀ
hydroxybenzaldehyde. We believe that the amination increasꢀ
es the acidity of the C1 methylene to cause the “snapping”
together of the three substituents. The resulting conjugated
olefin can then be attacked by the nitrogen of anthranilic acid
to form the five membered core.
DMAP,
DMSO-d
O
R
R
6
15N
NH2
H
6
H
HO
5
R = CO H
aniline R = H
9 R = CO H
2
10 R = H
2
CONCLUSION
CO H
2
15NH2
15NH2
The discoipyrroles presented a unique biosynthetic case, in
that they can be produced by a specific and concerted mechaꢀ
nism in aqueous media from diverse excreted metabolites. To
study how anthranilic acid, 4ꢀhydroxybenzadehyde, and 4ꢀ
hydroxysattabacin were able to undergo such a reaction we
constructed a model system in organic solvent and utilized
various analytical techniques, including the novel application
1
15
of Hꢀ N HMBC as a tool for monitoring reactions in situ.
1
15
Building on the foundation made possible by this technique,
Figure 6. Using Hꢀ N HMBC to monitor formation of
aldimine between 6 and aniline and 6 and 5.
13
we performed various isotope labeling experiments with C,
15
18
N, and O to elucidate the mechanism of nonꢀenzymatic
To circumvent this issue, we utilized 4ꢀhydroxysattabacin in
lieu of 7, removing the possibility for condensation with aldeꢀ
hyde coming from 7. No discernable amount of the desired
product was detected, however slightly increased temperatures
induced minor product formation. We reasoned that an equiꢀ
librium between the acyloin group and any imine might favor
the former, compared to the diketone of 7. Therefore, we set
out to trap potential amination products using reductive condiꢀ
tions. Indeed, reacting 4ꢀhydroxysattabacin and anthranilate in
the presence of sodium triacetoxyborohydride in dichloroꢀ
ethane with acetic acid yielded a reductive amination product
discoipyrrole formation. This multiꢀcomponent reaction was
characterized by the initial, rapid steps to induce pyrrole forꢀ
mation and the following changes in oxidation state that dicꢀ
tate the final closure of the lactone ring. Particularly useful
18
was the O labeling experiments which allowed us to clearly
demonstrate the atmospheric oxidation of the pyrrolone using
careful isolation and high resolution mass spectrometry. Furꢀ
thermore, the subsequent loss of this isotope was integral in
establishing the dehydration dependent lactone ring closure.
Importantly, once the details of the mechanism were uncovꢀ
ered, we constructed fermentation media with deuterated waꢀ
(
31% yield) within 16 hours (SI Figure 10). Reduction of 4ꢀ
hydroxysattabacin to the diol side product was prevalent, poꢀ
tentially obscuring the calculated reaction efficiency.
1
15
ter and used our Hꢀ N HMBC monitoring method to validate
discoipyrrole formation in biologically equivalent conditions
without organic solvent or added DMAP (SI Figure 11). We
observe identical intermediates and a similar reaction time
course under both conditions.
The efficiency of these omission experiments compared to
the complete reaction containing all three reactants indicated
that the mechanism was enhanced by a concerted flow of reacꢀ
tivity. Our current hypothesis is that pyrrole formation can
proceed through amination of 7 by anthranilic acid to an imine
intermediate that can be trapped by a ClaisenꢀSchmidt conꢀ
Aryl amines are found to participate in many of the recent
examples of natural products amenable to nonꢀenzymatic inꢀ
corporation including the ammosamides, elansolids, and oxaꢀ
5
ꢀ6
zinin A . Through our mechanistic studies of the DPs, we
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have shown that anthranilic acid acts as the initiating factor
through imine formation with sattabacin and that the ortho
carboxylic acid substitution of anthranilate drives the selectiviꢀ
ty as seen in the aldimine formation experiments (Figure 6).
Our work and others suggest that aryl amines play a signifiꢀ
cant role in nonꢀenzymatic reactions and that aryl substitution
patterns, the nature of the amination sites, and the stability of
intermediates all contribute to the complexity of these reacꢀ
tions. Using these types of nucleophiles could be a potentially
cost effective and simple way to screen bacterial libraries for
natural products amenable to nonꢀenzymatic perturbations.
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Concordantly, detection methods using N labels such as the
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(5) Pan, E.; Oswald, N. W.; Legako, A. G.; Life, J. M.; Posner, B.
A.; Macmillan, J. B. Chem. Sci. 2013, 4, 482ꢀ488.
Hꢀ N HMBC demonstrated here could provide quick and
high sensitivity analysis.
(
6) (a) Lin, Z.; Koch, M.; Abdel Aziz, M. H.; GalindoꢀMurillo, R.;
Tianero, M. D.; Cheatham, T. E.; Barrows, L. R.; Reilly, C. A.;
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ASSOCIATED CONTENT
Supporting Information
(
c) Steinmetz, H.; Zander, W.; Shushni, M. A. M.; Jansen, R.; Gerth,
K.; Dehn, R.; Dräger, G.; Kirschning, A.; Müller, R. ChemBioChem
012, 13, 1813ꢀ1817.
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General procedures, data tables, NMR spectra, and additional data
are described. This material is available free of charge via the
Internet at http://pubs.acs.org.
2
(
Kanehisa, M.; Goto, S.; Sato, Y.; Kawashima, M.; Furumichi, M.;
Tanabe, M. Nuc. Acids Res. 2014, 42, D199ꢀ205.
(8) (a) Park, J. S.; Kagaya, N.; Hashimoto, J.; Izumikawa, M.;
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AUTHOR INFORMATION
Corresponding Author
*
john.macmillan@utsouthwestern.edu
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Notes
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The authors declare no competing financial interest.
ACKNOWLEDGMENT
9
63.
10) (a) Mahmud, T. J. Labelled Comp. Radiopharm. 2007, 50,
We acknowledge Jonathan R. Goodman and Dr. Wade C. Winꢀ
kler (University of Maryland) for their gracious contribution in
the sequencing and processing of the Bacillus hunanensis SNA-
(
1039ꢀ1051; (b) Esquenazi, E.; Jones, A. C.; Byrum, T.; Dorrestein, P.
C.; Gerwick, W. H. Proc. Natl. Acad. Sci. U S A 2011, 108, 5226ꢀ31;
(c) Rinkel, J.; Dickschat, J. S. Beilstein J. Org. Chem. 2015, 11, 2493ꢀ
2508.
0
48 genome. We acknowledge following grants for funding this
project: Welch Foundation Iꢀ1689 and NIH R01CA1499833, NIH
U01CA176284, the Chilton/Bell Foundation and the Martha Steiꢀ
ner family.
(11) (a) Vizcaino, M. I.; Crawford, J. M. Nat. Chem. 2015, 7, 411ꢀ
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Kegler, C.; Lorenzen, W.; Brachmann, A. O.; Grun, P. Chemistry
012, 18, 2342ꢀ8; (c) Kwon, Y.; Park, S.; Shin, J.; Oh, D. C. Arch.
Pharm. Res. 2014, 37, 967ꢀ71.
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