Concise Chemoenzymatic Synthesis of Fasamycin A

Article abstract of DOI:10.1021/acs.joc.1c00526

We report the development of a chemoenzymatic approach toward fasamycin A, a halogenated naphthacenoid that exhibits activities against methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus faecalis. The synthesis was accomplished in a convergent manner: two fragments were combined together in a Sammes annulation to afford a dimethylnaphthacenone system. Finally, an enzymatic halogenation was employed to introduce the requisite chlorine substituent of the natural product at a late stage.

Full text of DOI:10.1021/acs.joc.1c00526

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Concise Chemoenzymatic Synthesis of Fasamycin A
Jian Li and Hans Renata*
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sı Supporting Information
ABSTRACT: We report the development of a chemoenzymatic approach toward fasamycin A, a halogenated naphthacenoid that
exhibits activities against methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus faecalis. The synthesis was
accomplished in a convergent manner: two fragments were combined together in a Sammes annulation to afford a
dimethylnaphthacenone system. Finally, an enzymatic halogenation was employed to introduce the requisite chlorine substituent
of the natural product at a late stage.
INTRODUCTION
■
Antibiotic resistance is becoming one of the most pressing
threats to human health, especially with the emergence of
multidrug resistant (MDR) pathogens that prove recalcitrant
1
to existing treatments. Highlighting the urgency of the matter,
the World Health Organization recently released a list of 12
“priority pathogens” that exhibit resistance to a number of
antibiotics, including carbapenem-resistant Acinetobacter bau-
manii and Pseudomonas aeruginosa, methicillin-resistant Staph-
ylococcus aureus (MRSA), and vancomycin-resistant Enter-
ococcus faecalis (VRE). Given their involvement in “biological
warfare” among bacteria, many bacterial secondary metabolites
are known to be effective antimicrobials, and these natural
products make up the majority of antibiotics that are used in
2
the clinic today. Despite this success, pathogenic bacteria
constantly evolve new modes of resistance, and new antibiotic
candidates need to be constantly advanced to address this
challenge.
In 2011, Brady and co-workers discovered two chlorinated
polyaromatic natural products, namely fasamycins A (1) and B
Figure 1. Structures of fasamycins A and B and related natural
products.
(
2), through heterologous expression of an environmental
3
DNA gene cluster (Figure 1). Fasamycin A was noted to
exhibit potent antibacterial activities against MRSA (MIC =
Special Issue: Natural Products: An Era of Discovery
in Organic Chemistry
3
.1 μg/mL) and VRE (MIC = 0.8 μg/mL) by inhibiting FabF,
a key enzyme in the biosynthesis of type II fatty acid in
bacteria. FabF has been shown to be indispensable for
bacterial cell viability and currently represents an under-
developed target in antibacterial drug discovery. To date, only
4
Received: March 4, 2021
5
a few small molecules, mostly natural products, have been
©
XXXX American Chemical Society
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A

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Article
reported to act as inhibitors of this enzyme. By analyzing the
organization of the gene cluster, Brady and co-workers also
proposed a late stage enzymatic C−H chlorination reaction
catalyzed by a flavin-dependent halogenase (FDH) on
to forge the key quaternary carbon of 5. Gratifyingly, union of
alkene 10 and boronic acid 11 under Sigman’s conditions
proceeded uneventfully (Scheme 2) to provide aldehyde 12 in
6
naphthacemycin B1 to furnish the fasamycins. Based on its
Scheme 2. Synthesis of Enone 5
unique biogenesis and its potential to be a lead compound for
antibiotic development, we set out to develop a chemo-
enzymatic synthesis of fasamycin A.
The medicinal potential and the unique framework of this
natural product family have stimulated two elegant chemical
7
,8
syntheses by the groups of Shia and Kraus. Additionally, Shia
has also reported the first synthesis of a related natural product,
9
BE-24566B, also known as ABX. Inspired by these chemical
syntheses and the biosynthetic proposal, our retrosynthesis for
1
starts from disconnection of its C−Cl bond via a late stage
enzymatic chlorination to reveal naphthacemycin B1 (3).
Here, we envisioned an opportunity to functionally character-
ize the FDH (hereby termed FasV) from fasamycin biosyn-
thesis for the first time and perform initial investigations into
7
its biocatalytic utility (Scheme 1). Similar to Shia’s approach,
Scheme 1. Retrosynthetic Analysis of Fasamycin A
1
1
5
0% yield. A SnCl -induced intramolecular Friedel−Crafts
4
cyclization gave benzylic alcohol 13 in 61% yield and this
alcohol was directly oxidized to the corresponding enone in
12
one pot by using IBX as an oxidant to complete the synthesis
of enone 5.
Toward lactone 4, we noted that a related structure had
been prepared by Shia through the use of ring substitution/
functionalization of a resorcinol derivative. We envisioned that
a ring annulation strategy would allow for a more expedient
13
14
access. Toward this goal, Brassard’s diene 6 and alkyne 7
were utilized to construct lactone 14 through a sequential
Diels−Alder reaction and acid-induced lactonization (Scheme
3
). Triflation of the phenol group and conversion of the triflate
Scheme 3. Synthesis of Lactone 16 and Completion of the
Synthesis of Naphthacemycin B1
we deemed the use of an annulation strategy to construct the
central ring of 3 to be the most convergent and strategic. To
this end, a convergent assembly of 3 was envisioned through a
Sammes annulation featuring lactone 4 and enone 5 and
installation of the pendant arene by a Suzuki coupling. Lactone
4
could be traced back to Brassard’s diene 6 and ynoate 7 via a
Diels−Alder and lactonization sequence. Finally, enone 5
could be synthesized through a formal annulation involving
resorcinol derivative 8 and an appropriate 6C coupling partner
(
e.g., 9).
RESULTS AND DISCUSSION
■
We first focused our efforts toward identifying an efficient
route to enone 5. The same fragment has been synthesized
previously by Shia through the use of hydrogen-atom-transfer
group into arene side chain via standard Suzuki coupling
conditions provided lactone 16 in 82% overall yield. LDA-
induced Sammes annulation between 5 and 16, followed by
treatment with HCl furnished the desired dimethylnaphthace-
none ring system. Finally, global deprotection with BBr3
(
HAT)-based Giese coupling, followed by Friedel−Crafts
acylation and oxidation state adjustment. Seeking to establish a
complementary disconnection with reduced overall step-count,
we envisioned the use of Sigman’s redox-relay Heck reaction
10
B
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proceeded in 40% yield (over two steps) to complete our
synthesis of naphthacemycin B1.
always the major chlorinated product. Among the combina-
tions tested, the FasV/CtcQ pair gave the highest conversion
though the overall chlorination yield was still unsatisfactory.
While the presence of bischlorinated products could be
detected through HPLC extracted ion chromatogram analysis
of the crude reaction, the amounts detected were too low to
allow further isolation and structural determination. At this
stage, we also tested a range of different pHs and temperatures
for the reaction (Table 1 and Supplementary Table S1) and
found that a pH range of 7.4−8.0 was optimal for the reaction
and that temperature had minimal effect on the outcome of the
With naphthacemycin B1 in hand, we set out to functionally
characterize FasV and investigate its use in the final C−H
chlorination step. FasV shares 59.8% and 55.4% sequence
identity with ForV, a FDH from formicamycin biosynthesis,
and AbxH, a FDH from BE-24566B biosynthesis. While no
biosynthetic studies have been performed on the fasamycins,
some of the late-stage tailoring steps in formicamycin and BE-
15,16
2
4566B have begun to be elucidated.
Notably, in frame
deletion of forV resulted in the production of nonchlorinated
precursors to the formicamycins and in trans complementation
rescued the production of the formicamycins. Additionally, in
vitro assays had been performed to verify the role of AbxH in
BE-24566B biosynthesis.
Heterologous expressions of FasV and ForV as N-His6-
tagged proteins resulted in satisfactory yields of soluble
21
reaction. The use of Opt13 to regenerate the NADH cofactor
did not lead to any improvement in the reaction conversion.
22
23
Chitnumsub and Chaiyen and Lewis had discussed the
possibility of hypohalous acid leakage as an alternative
mechanism for FDH-catalyzed halogenation. In light of the
low efficiency of our reaction, we sought to determine if this
pathway was operative with FasV. To this end, we tested the
halogenation reaction in the absence of FasV and in the
presence of a variant of FasV containing mutation at its active-
site lysine residue (K75A). In both cases, no formation of 1
could be observed, suggesting that 1 was not formed from the
reaction of 3 with free HOCl in our system.
proteins, particularly in the presence of cochaperones
GroES/EL, allowing us to start screening for an optimal set
of C−H chlorination conditions. The two purified FDHs were
17
tested in combination with five FAD reductases, namely Fre,
SfFre (a Fre homologue from S. formicae, the native producer
18
19
20
of the formicamycins), SsuE, CtcQ, and HpaC (Table 1).
The enzymatic reaction was next performed on preparative
scale, and purification of the reaction mixture by preparative
TLC afforded fasamycin A in pure form. To benchmark the
enzymatic chlorination strategy, naphthacemycin B1 was
submitted to electrophilic chlorination with several small-
molecule reagents. However, intractable mixtures were
consistently observed across all conditions tested. This
observation suggests that, despite its low efficiency, the
enzymatic chlorination proceeds under much milder con-
ditions than conventional chemical halogenations. While the
low conversion was disappointing, it is worth noting that
during the characterization of the halogenase from BE-24566B
biosynthesis, Lei, Qu, and co-workers also observed only low
conversion to the halogenated products despite the use of high
a
Table 1. Enzymatic Halogenation Screening
halogenase
reductase
conversion
isolated yield of 1 (%)
FasV
FasV
FasV
FasV
Fre
0%
0%
ND
ND
ND
5
SfFre
SsuE
CtcQ
HpaC
Fre
SfFre
SsuE
CtcQ
HpaC
CtcQ
CtcQ
CtcQ
CtcQ
16
enzyme loading. Furthermore, this issue could potentially be
3% as a mixture
7% as a mixture
3−5% as a mixture
0%
addressed with future enzyme engineering efforts or genome
mining for enzyme homologues. In light of the challenges
associated with evolving inefficient enzymes for improved
activities, we anticipate that the latter strategy would facilitate
the identification of a better starting point for an evolution
FasV
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ForV
ForV
ForV
ForV
ForV
FasV
FasV
N.A.
FasV K75A
0%
3% as a mixture
5% as a mixture
0%
4% as a mixture
4% as a mixture
24
campaign. A recent report from Lewis and co-workers on the
utility of family-wide activity profiling of FDHs to uncover new
enzymes with unique substrate preferences bodes well for the
realization of this idea.
b
c
0%
0%
CONCLUSION
■
a
Standard conditions for screening: naphthacemycin B1 (0.5 mM, 1
equiv), NADH/NADPH (1 mM, 2 equiv), FAD (100 μM, 20 mol
), halogenase (5 μM, 1 mol %), Reductase (5 μM, 1 mol %), buffer
20 mM phosphate pH 7.4, 100 mM NaCl, 5 mL total volume), 12 h.
Reaction was performed in the presence of Opt13 (5.0 μM) and
Na HPO ·5H O (10 mM). Reaction was performed at 30 °C.
In summary, we have accomplished the chemoenzymatic
synthesis of fasamycin A. The western and eastern segments
were concisely prepared and convergently assembled to furnish
the naphthacemycin core skeleton. By relying on the native
FDH from fasamycin biosynthesis, a late-stage enzymatic
chlorination reaction was performed to install the chlorine
substituent of the natural product. This work also provided the
first direct biochemical confirmation of the halogenation
activity of FasV and ForV and presented our initial foray
toward evaluating their performance and viability as late-stage
halogenation biocatalysts. Finally, the route described herein
lays down the blueprint for future chemoenzymatic preparation
of other halogenated naphthacenone natural products and will
%
(
b
c
2
3
2
Despite extensive screening, only low conversions to the
chlorinated products could be observed and several reductases
Fre and SfFre) failed to give any desired products at all.
Additionally, a mixture of monochlorination regioisomers was
consistently detected, though LC/MS analyses and comparison
with authentic product standard showed that fasamycin A was
(
C
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facilitate the discovery of new antibiotics and their SAR
studies.
dried over anhydrous Na
solvent under vacuum, the residue was subjected to flash column
chromatography for purification using EtOAc/petroleum ether (1:10
2
SO
4
, and filtered. After removal of the
→
1:4) as eluent to give aldehyde 12 (971 mg, 50%) as a pale yellow
EXPERIMENTAL SECTION
1
■
oil: H NMR (400 MHz, acetone-d
6
) δ 9.59 (s, 1 H), 7.51−7.23 (m,
1
0 H), 6.64−6.58 (m, 2 H), 6.54 (s, 1 H), 5.10 (s, 4 H), 2.25−2.11
General Materials and Methods. Unless otherwise noted, all
chemicals and reagents for chemical reactions were purchased at the
highest commercial quality and used without further purification.
Reactions were monitored by thin-layer chromatography (TLC) and
liquid chromatography/mass spectrometry (LC/MS). TLC was
performed with 0.25 mm E. Merck silica plates (60F-254) using
short-wave UV light as the visualizing agent and ninhydrin, KMnO4,
or phosphomolybdic acid and heat as developing agents. LC/MS was
performed with Agilent 1260 Infinity System equipped with Poroshell
1
3
1
(m, 2 H), 1.93−1.83 (m, 2 H); C{ H} NMR (101 MHz, acetone-
d
6
) δ 202.5, 160.7, 151.5, 138.3, 129.1, 128.4, 128.3, 106.3, 99.9, 70.3,
+
40.4, 37.9, 36.2, 32.1; HRMS (ESI-TOF) m/z [M + Na] calcd for
Na 411.1931, found 411.1933.
6,8-Bis(benzyloxy)-4,4-dimethyl-1,2,3,4-tetrahydronaphthalen-
-ol (13). To a stirred solution of aldehyde 12 (971 mg, 2.50 mmol)
in CH Cl (10 mL) was added SnCl (3 mL, 1.0 M in CH Cl ) at
C H O
26 28 3
1
2
2
4
2
2
−
4
78 °C. The reaction mixture was stirred at the same temperature for
h and then quenched with saturated aq NaHCO (20 mL). The
1
20 EC-C18 column (3.0 × 50 mm, 2.7 μm). NMR spectra were
3
resulting mixture was extracted with EtOAc (3 × 20 mL), and the
combined organic phases were washed with brine (150 mL) and dried
over anhydrous Na SO . After filtration and evaporation of the
solvent under vacuum, the residue was subjected to flash column
chromatography for purification using EtOAc/petroleum ether (1:3
recorded on a Bruker spetrometer and calibrated using residual
undeuterated solvent. Optical rotations were measured on Autopol IV
polarimeter (Rudolph Research Analytical). Electrocompetent E. coli
BL21(DE3) were purchased from Lucigen.
2
4
Plasmid Generation for Enzyme Expression. With the
exception of HpaC, expression vectors for all enzymes used in this
work were obtained via DNA synthesis from Twist Bioscience. Briefly,
the codon-optimized DNA sequences of the respective enzymes were
inserted between the NdeI and XhoI restriction sites within the
commercial pET28a(+) vector. The resulting vectors were used
directly to transform electrocompetent E. coli BL21(DE3).
→
1:1) as eluent to give alcohol 13 (592 mg, 61%) as a pale yellow
1
oil: H NMR (400 MHz, acetone-d ) δ 7.54−7.29 (m, 10 H), 6.65 (d,
6
J = 2.3 Hz, 1 H), 6.58 (d, J = 2.4 Hz, 1 H), 5.20−5.07 (m, 4 H),
5
.05−4.94 (m, 1 H), 1.93−1.76 (m, 2 H), 1.53−1.38 (m, 1 H), 1.29
13 1
(s, 3 H), 1.17 (s, 3H); C{ H} NMR (101 MHz, acetone-d ) δ
6
159.7, 158.7, 148.7, 138.4, 138.3, 129.1, 129.1, 128.4, 128.4, 128.3,
128.1, 120.7, 104.7, 98.5, 70.5, 70.2, 62.1, 34.7, 33.7, 31.5, 30.8, 27.9;
HRMS (ESI-TOF) m/z [M + Na] calcd for C26
found 411.1937.
6,8-Bis(benzyloxy)-4,4-dimethylnaphthalen-1(4H)-one (5). To a
stirred solution of alcohol 13 (300 mg, 0.772 mmol) in DMSO (3
mL) was added IBX (476 mg, 1.70 mmol) at 22 °C. The resulting
mixture was heated to 100 °C in an oil bath and stirred at that
temperature for 48 h before it was cooled to 22 °C and diluted with
EtOAc (20 mL). The resultant mixture was sequentially washed with
saturated aq Na
organic phases were dried over anhydrous Na
solvent was evaporated under vacuum, and the residue was rapidly
purified by flash column chromatography with EtOAc/petroleum
ether (1:10 → 1:2) to give enone 5 (249 mg, 84%) as a colorless oil.
Characterization data for this compound are in agreement with
Generation of FasV K75A Mutant. Site-directed mutagenesis to
generate FasV K75A mutant was performed by using standard
Quikchange PCR method with primers containing the desired K75A
mutation and wild-type FasV as the PCR template. The resulting PCR
product was digested with DpnI, gel purified, repaired using
NEBuilder HiFi DNA Assembly kit (NEB product no.: 2621) and
used directly to transform electrocompetent E. coli BL21(DE3).
Enzyme Expression and Purification. Expression vectors were
used directly to transform electrocompetent E. coli strain BL21(DE3).
Sonication was performed using a Qsonica Q500 sonicator. Purified
enzymes were accessed via immobilized metal ion affinity
chromatography with HisTrap HP column. Recombinant E. coli
BL21(DE3) cells harboring plasmids that encode for the appropriate
enzyme variants were cultured under standard conditions with IPTG
induction. Optionally, soluble expression of FasV and ForV could be
improved through coexpression of the chaperones GroES/EL under
+
H
28
O
3
Na 411.1931,
S O
2 2 3
(10 mL) and brine (10 mL). The combined
SO and filtered. The
2
4
7
previous literature report.
25
previously reported conditions. Cell were harvested by centrifuga-
tion (4 °C, 15 min, 3000g), and the cell pellet was stored at −20 °C
or below for at least 2 h. Purification was performed with an AKTA
pure FPLC system (GE Healthcare). The thawed cell pellet was
resuspended in Ni−NTA buffer A (25 mM Tris·HCl, 200 mM NaCl,
7-Hydroxy-5-methoxyisobenzofuran-1(3H)-one (14). To a stirred
solution of alkyne 7 (1.72 g, 10.0 mmol) in toluene (10 mL) was
added Brassard’s diene 6 (2.43 g, 12.0 mmol) at 22 °C. The resulting
mixture was heated to 110 °C in an oil bath and stirred at the same
temperature for 12 h before it was cooled to 22 °C. HCl was added
(10 mL, 1.0 M in ethyl acetate), and the resulting mixture was stirred
for 3 h. The organic phase was diluted with 100 mL ethyl acetate,
washed with brine (100 mL), dried over anhydrous Na SO , and
2
5 mM imidazole, pH 9.0, 4 mL/g of cell wet weight) and lysed by
sonication (3 × 1 min, 50% duty cycle). The lysate was centrifuged at
5000g for 30 min at 4 °C to remove cell debris. The collected
1
2
4
supernatant was subjected to a Ni-NTA chromatography step using a
Ni sepharose column (HisTrap-HP, GE Healthcare, Piscataway, NJ).
The protein was eluted from the Ni sepharose column using 25 mM
Tris·HCl, 200 mM NaCl, 300 mM imidazole, pH 8.0. Ni-purified
protein was buffer exchanged into 0.05 M phosphate buffer (pH =
filtered. The solvent was evaporated under vacuum, and the residue
was purified by flash column chromatography with EtOAc/petroleum
ether (1:10 → 1:1) to give lactone 14 (810 mg, 45%) as a white
powder. Characterization data for this compound are in agreement
7
with previous literature report.
7
.0) using a 30 kDa MW cutoff centrifugal filter. Protein
7-(2,4-Bis(benzyloxy)-6-methylphenyl)-5-methoxyisobenzofur-
concentrations were determined by A280 with calculated extinction
coefficients as obtained at https://web.expasy.org/protparam. For
storage, proteins were portioned into 100 μL aliquots, flash frozen on
an-1(3H)-one (16). To a stirred solution of lactone 14 (901 mg, 5.0
mmol) in CH
10 mmol) and Tf
mixture was stirred at the same temperature for 2 h before it was
quenched with saturated aq NaHCO (20 mL). The resulting mixture
Cl
2
(15 mL) were sequentially added pyridine (804 μL,
2
O (1.01 mL, 6.0 mmol) at −20 °C. The reaction
2
liquid N , and stored at −80 °C.
2
4
-(3,5-Bis(benzyloxy)phenyl)-4-methylpentanal (12). A stirred
3
solution of Pd(CH CN) (OTs) (159 mg, 0.30 mmol), Cu(OTf)
was extracted with EtOAc (3 × 20 mL), and the combined organic
phases were washed with brine (150 mL) and dried over anhydrous
3
2
2
2
(
54 mg, 0.15 mmol), PyrOx (123 mg, 0.045 mmol), and 3 Å
molecular sieves (750 mg) in DMF (400 mL) was stirred under O2
for 10 min at 22 °C. To the resulting mixture were sequentially added
alcohol 10 (500 mg, 4.99 mmol) and boronic acid 11 (1.67 g, 5.00
mmol) at rt. The reaction mixture was stirred at room temperature for
Na SO . After filtration and evaporation of the solvent under vacuum,
2 4
the triflated lactone 4 was obtained as a pale yellow solid (1.56 g,
1
99%): H NMR (400 MHz, CDCl ) δ 6.96 (dd, J = 1.8, 0.9 Hz, 1 H),
3
1
3
1
6.92−6.85 (m, 1 H), 5.33−5.16 (m, 2H), 3.94 (s, 3H); C{ H}
NMR (151 MHz, CDCl ) δ 166.3, 166.1, 151.2, 147.0, 118.9 (q, J =
2
4 h before it was quenched with saturated aq NaHCO (400 mL).
3
3
The mixture so obtained was extracted with EtOAc (3 × 200 mL).
The combined organic phases were washed with brine (400 mL),
320.8 Hz), 111.0, 110.2, 106.3, 68.9, 56.7; HRMS (ESI-TOF) m/z
+
[M + Na] calcd for C H F O SNa 334.9808, found 334.9812.
1
0
7
3
6
D
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Characterization data for this compound are in agreement with the
previous literature report.
were added NADH (1 mM final concentration), FAD (100 μM final
concentration), 0.25 mL of naphthacemycin B1 stock solution (10
mM in methanol, ca. 0.5 mM final concentration), FasV (5.0 μM final
concentration), and CtcQ (5.0 μM final concentration). The vial was
sealed and shaken for 12 h at 22 °C, 200 rpm. The mixture was
extracted with ethyl acetate (5 mL). To provide sufficient material for
isolation, 20 reactions were performed in parallel and combined at the
end. The combined organic layers were concentrated under vacuum
and purified by preparative thin-layer chromatography (EtOAc/
7
The above triflated lactone 4 was taken to the next step without
further purification. To a stirred solution of the triflate and boronic
acid 15 (2.44 g, 7.0 mmol) in toluene (20 mL) were sequentially
added Pd(PPh ) (289 mg, 0.25 mmol) and K PO (10 mL, 1.0 M in
3
4
3
4
water). The resultant mixture was heated to 110 °C in an oil bath and
stirred at the same temperature for 8 h before it was cooled to 22 °C.
The reaction mixture was diluted with 50 mL ethyl acetate, washed
with brine (100 mL), dried over anhydrous Na SO , and filtered. The
petroleum ether 1:2) to give fasamycin A (1.3 mg, 5.2%) as a yellow
2
4
1
solvent was evaporated under vacuum, and the residue was purified by
flash column chromatography with EtOAc/petroleum ether (1:50 →
solid: H NMR (600 MHz, methanol-d
4
) δ 7.34 (s, 1 H), 7.05 (d, J =
2.5 Hz, 1 H), 6.72 (d, J = 2.5 Hz, 1 H), 6.45 (d, J = 1.0 Hz, 1 H), 6.26
(dd, J = 2.4, 0.8 Hz, 1 H), 6.23 (d, J = 2.3 Hz, 1 H), 2.06 (s, 3 H),
1
:10) to give lactone 16 (1.91 g, 82%) as a colorless oil.
1
3
1
Characterization data for this compound are in agreement with
previous literature report.
2.04 (s, 3 H), 1.88 (s, 3 H); C{ H} NMR (151 MHz, methanol-d )
4
7
δ 191.6, 166.6, 165.5, 160.3, 157.5, 155.5, 150.0, 149.2, 143.5, 140.9,
Naphthacemycin B1 (3). To a stirred solution of lactone 16 (446
mg, 1.00 mmol) and enone 5 (384 mg, 1.00 mmol) in THF (4 mL)
was added LDA (0.6 mL, 2.0 M in THF/heptane/ethylbenzene, 1.2
mmol) at −78 °C. The reaction mixture was warmed to 22 °C and
stirred at that temperature for 48 h before it was quenched with HCl
138.2, 124.4, 122.6, 117.8, 117.0, 109.9, 108.8, 106.3, 103.6, 100.8,
+
40.7, 30.3, 30.2, 20.7; HRMS (ESI-TOF) m/z [M + Na] calcd for
2
4
C H21ClO Na 515.0868, found 515.0877; [α] = +5.7 (c = 0.20 in
2
7
7
D
MeOH).
(
3.0 mL, 1.0 M in ethyl acetate). The resulting mixture was extracted
ASSOCIATED CONTENT
sı Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.joc.1c00526.
■
with EtOAc (3 × 50 mL). The combined organic phases were washed
with brine (150 mL) and dried over anhydrous Na SO . After
filtration and removal of the solvent under vacuum, the residue was
purified by flash column chromatography with EtOAc/petroleum
ether (1:60 → 1:10) to give 374 mg of prenaphthacemycin B1 (17) as
yellow oil: H NMR (400 MHz, CDCl ) δ 7.66−7.54 (m, 2 H),
7
(
6
5
*
2
4
Protein and DNA sequences, H and 13C NMR data,
and HPLC traces (PDF)
1
1
3
.52−7.29 (m, 15 H), 7.13−7.00 (m, 4 H), 6.99−6.90 (m, 2 H), 6.88
d, J = 2.3 Hz, 1 H), 6.83 (d, J = 2.5 Hz, 1 H), 6.64−6.56 (m, 2 H),
AUTHOR INFORMATION
Corresponding Author
.51 (d, J = 2.3 Hz, 1 H), 5.30 (s, 2 H), 5.18 (s, 2 H), 5.14 (s, 2 H),
.12−5.01 (m, 2 H), 4.93 (s, 2 H), 3.94 (s, 3 H), 2.08 (s, 3 H), 1.75
■
1
3
1
(
s, 3 H), 1.73 (s, 3 H); C{ H} NMR (101 MHz, CDCl ) δ 187.8,
Hans Renata − The Scripps Research Institute, Department of
Chemistry, Jupiter, Florida 33458, United States;
orcid.org/0000-0003-2468-2328; Email: hrenata@
scripps.edu
3
1
1
1
1
7
65.8, 163.4, 162.2, 160.0, 158.5, 156.8, 155.4, 145.0, 140.3, 138.9,
37.7, 137.4, 137.2, 136.8, 136.2, 128.9, 128.8, 128.7, 128.6, 128.2,
28.1, 127.9, 127.8, 127.8, 127.1, 126.9, 126.9, 126.7, 120.7, 118.2,
14.4, 113.5, 109.5, 107.4, 106.1, 104.9, 99.4, 98.5, 71.1, 70.4, 70.4,
+
0.2, 55.4, 39.2, 34.5, 33.8, 21.0; HRMS (ESI-TOF) m/z [M + Na]
Author
calcd for C H O Na 855.3293, found 855.3301.
56
48
7
Jian Li − The Scripps Research Institute, Department of
Chemistry, Jupiter, Florida 33458, United States
To a stirred solution of prenaphthacemycin B1 (17) in CH Cl (3
2
2
mL) was added BBr (2.0 mL, 1.0 M in CH Cl ) at −78 °C. The
3
2
2
resulting mixture was warmed to 22 °C and stirred at the same
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.joc.1c00526
temperature for 8 h before it was quenched with saturated aq
NaHCO (20 mL). The resultant mixture was extracted with EtOAc
3
(
3 × 20 mL), and the combined organic phases were washed with
Notes
brine (50 mL) and dried over anhydrous Na SO . After filtration and
evaporation of the solvent under vacuum, the residue was subjected to
2
4
The authors declare no competing financial interest.
flash column chromatography for purification using EtOAc/
ACKNOWLEDGMENTS
petroleum ether (1:3 → 1:1) as eluent to give naphthacemycin B1
■
1
3
(183 mg, 40%) as a yellow solid: H NMR (600 MHz, methanol-d )
Funding for this work is generously provided by the National
Institutes of Health grants GM128895 (H.R.) and S10
OD021550 (for 600 MHz NMR instrumentation). We thank
Prof. David H. Sherman and Dr. Amy E. Fraley for providing
plasmid pET11a-HpaC (encoding for HpaC flavin reductase).
We acknowledge the Roush, Bannister, and Shen laboratories
for generous access to their reagents and instrumentation.
4
δ 7.36 (s, 1 H), 7.05 (d, J = 2.5 Hz, 1 H), 6.72 (d, J = 2.5 Hz, 1 H),
6
2
1
.65 (d, J = 2.3 Hz, 1 H), 6.27 (dd, J = 2.4, 0.8 Hz, 1 H), 6.24 (dd, J =
.3, 0.6 Hz, 1 H), 6.21 (d, J = 2.2 Hz, 1 H), 1.88 (d, J = 0.7 Hz, 3 H),
1
3
1
.71 (s, 3 H), 1.69 (s, 3 H); C{ H} NMR (151 MHz, Methanol-d )
4
δ 210.5, 191.6, 166.9, 166.6, 166.3, 160.1, 157.5, 155.8, 155.4, 146.5,
1
1
42.9, 140.8, 138.3, 124.5, 122.5, 118.2, 116.1, 110.1, 108.8, 108.7,
07.6, 106.9, 102.0, 100.8, 39.7, 34.7, 34.5, 20.7; HRMS (ESI-TOF)
+
m/z [M + Na] calcd for C H O Na 481.1258, found 481.1264.
2
7
22
7
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