Initial Analysis of the Arylomycin D Antibiotics

Article abstract of DOI:10.1021/acs.jnatprod.9b01174

The arylomycins are a class of natural product antibiotics that inhibit bacterial type I signal peptidase and are under development as therapeutics. Four classes of arylomycins are known, arylomycins A-D. Previously, we reported the synthesis and analysis of representatives of the A, B, and C classes and showed that their spectrum of activity has the potential to be much broader than originally assumed. Along with a comparison of the mechanism of acquired and innate resistance, this led us to suggest that the arylomycins are latent antibiotics, antibiotics that once possessed broad-spectrum activity, but which upon examination today, have only narrow spectrum activity due to prior selection for resistance in the course of the competition with other microorganisms that drove their evolution in the first place. Interestingly, actinocarbasin, the only identified member of the arylomycin D class, has been reported to have activity against MRSA. To confirm and understand this activity, several actinocarbasin derivatives were synthesized. We demonstrate that the previously reported structure of actinocarbasin is incorrect, identify what is likely the correct scaffold, confirm that scaffold has activity against MRSA, and determine the origin of this activity.

Full text of DOI:10.1021/acs.jnatprod.9b01174

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Initial Analysis of the Arylomycin D Antibiotics
Yun Xuan Tan, David S. Peters, Shawn I. Walsh, Matthew Holcomb, Diogo Santos-Martins,
Stefano Forli, and Floyd E. Romesberg*
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ABSTRACT: The arylomycins are a class of natural product antibiotics
that inhibit bacterial type I signal peptidase and are under development
as therapeutics. Four classes of arylomycins are known, arylomycins A−
D. Previously, we reported the synthesis and analysis of representatives
of the A, B, and C classes and showed that their spectrum of activity has
the potential to be much broader than originally assumed. Along with a
comparison of the mechanism of acquired and innate resistance, this led
us to suggest that the arylomycins are latent antibiotics, antibiotics that
once possessed broad-spectrum activity, but which upon examination
today, have only narrow spectrum activity due to prior selection for
resistance in the course of the competition with other microorganisms
that drove their evolution in the first place. Interestingly, actinocarbasin, the only identified member of the arylomycin D class, has
been reported to have activity against MRSA. To confirm and understand this activity, several actinocarbasin derivatives were
synthesized. We demonstrate that the previously reported structure of actinocarbasin is incorrect, identify what is likely the correct
scaffold, confirm that scaffold has activity against MRSA, and determine the origin of this activity.
interest due to an apparently narrow spectrum of activity that
y curing common infections that were once often fatal,
Bantibiotics revolutionized healthcare. However, their use included only a few Gram-positive bacteria.23,27,28 However,
following our report of the first total synthesis of an
arylomycin,19 our subsequent work to synthesize19,29−32 and
characterize the activity33 of the arylomycins suggested that they
have the potential for a much broader spectrum of activity,
including against Gram-negative pathogens.32,34 In addition to
facilitating our work to understand the effects of SPase
inhibition35−38 and the mechanisms of resistance,33,39−41 these
efforts led to pharmaceutical interest in the development of an
arylomycin therapeutic.42−44
Characterization of the mechanisms of innate and acquired
resistance to the arylomycins led us to suggest that they were
“latent antibiotics”,33,45 antibiotics that were once potent and
broad spectrum but whose activity is limited not by poor
properties but rather by their prior selection of resistance. We
speculate that all antibiotics developed as part of a competition
between microorganisms to select for resistance in susceptible
competitors, in fact in proportion to their potency, which in turn
creates selection pressure for the evolution of producer strains
that synthesize a variant of the antibiotic that regains activity.
The latent antibiotics hypothesis is based on the suggestion that
has applied an unprecedented selection pressure for the
evolution of resistance. Antibiotic resistance in the United
States alone is estimated to account for more than two million
illnesses and over 23 000 deaths each year.1 Worldwide
monitoring has revealed a loss of efficacy of standard treatments
for hospital acquired infections and urinary tract infections and,
in some cases, treatment failure.2 New antibiotics are
increasingly difficult to discover,3 at least in part because the
pharmaceutical industry typically requires broad-spectrum
activity in order to recoup investment.4,5 Thus, it is crucial to
identify new antibiotics, particularly broad-spectrum agents
acting by novel mechanisms of action.
Protein secretion is an essential aspect of bacterial physiology,
and the inhibition of secretion has long been suggested to be a
promising approach to antibiotic development.6 In particular,
bacterial type I signal peptidase (SPase), an endopeptidase
required to release mature proteins from their N-terminal signal
peptides that target them for translocation across the
cytoplasmic membrane,7−16 has long been appreciated as an
attractive target for antibiotic development6,13,16−23 due to its
conservation, essentiality, and relative accessibility on the outer
leaflet of the plasma membrane.24,25 Moreover, its proteolytic
mechanism (i.e., Ser-Lys dyad and si-face attack) is unique
relative to the more common Ser-His-Asp triad proteases,26
suggesting that selective inhibition should be possible.
Received: December 5, 2019
The arylomycins are a class of natural product antibiotics that
inhibit SPase, but which initially attracted little development
© XXXX American Chemical Society and
American Society of Pharmacognosy
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many natural product antibiotics may cycle through phases of
broad and narrow spectrum activity as mediated by the
competitive coevolution of producer and susceptible micro-
organisms. While cyclical, periods of broad-spectrum activity are
likely to be shorter than periods of narrow-spectrum activity.
This is because the cyclic processes for a given antibiotic and
different prey microorganisms are likely to be entrained by the
much faster rate at which resistance evolves compared to the rate
at which new active variants of the antibiotic are evolved
(selection pressure on prey is much greater than that on
predators), thereby reducing the spectrum of the antibiotic
across a broad range of competitors. Indeed, we have presented
evidence that arylomycin’s narrow spectrum results from having
selected for resistance in the past.33 By analogy to the success of
medicinal chemistry in overcoming specific resistance mecha-
nisms acquired by selection during clinical use of an antibiotic,
the likelihood of success in optimizing latent antibiotics to regain
broad-spectrum activity is much higher than that of de novo
scaffold discovery.
different lipopeptide tail that both is sulfonated and contains a
unique β-amino acid. Moreover, actinocarbasin is reported to
have activity against S. aureus, which the other natural products
do not. However, it is not clear which of the unique aspects of
the D class arylomycin impart it with activity against S. aureus.
Here, via synthesis of the reported core scaffold and several
derivatives, we show that the reported structure of actino-
carbasin is incorrect, and with the synthesis of additional
derivatives, we identify what is likely the correct structure.
Moreover, we show that the unique dipeptide of the arylomycin
D lipopeptide tail is sufficient for activity against S. aureus, and
modeling studies identify the likely basis for the increased
activity. These data contribute to the structure−activity
relationships that are needed to develop the arylomycins as
therapeutics.
RESULTS AND DISCUSSION
■
As the absolute configuration of the cysteic acid in the natural
product was unknown, both diastereomers were targeted. It was
hypothesized that, as with other arylomycins, the lipid moiety of
actinocarbasin inserts into the lipid bilayer of the bacterial
membrane and, thus, does not strongly contribute to its
spectrum of activity.30 This allowed the simplified derivatives
1a and 1b to be targeted under the assumption that, as with the
similar class A−C arylomycin derivatives, activity would not be
significantly affected (Scheme 1). Installation of the aryl sulfates
at a late stage was desired due to their high polarity and relative
instability. Thus, derivatives 1a and 1b were retrosynthetically
simplified to the protected macrocycles 2a and 2b. In previous
syntheses, the key macrocyclization reaction was accomplished
with either an intramolecular Suzuki−Miyaura reaction or a
copper-mediated oxidative coupling.19,32 Here, the intra-
molecular Suzuki−Miyaura coupling was chosen to construct
the macrocycle due to the challenges associated with the
oxidative coupling of phenols with differing electronic proper-
ties.47,48 The resulting retrosynthetic precursor, linear tripeptide
4, can be further disconnected into three amino acids, of which
3-iodotyrosine (5) and Boc-Ala-OH are commercially available,
leaving only arylglycine 6 to be synthesized (Scheme 1).
The synthesis of arylglycine 6 began with 5-iodovanillin (7),
which was converted to styrene 8 in 70% yield over three steps.
Next, a Sharpless asymmetric aminohydroxylation reaction
provided amino alcohol 9. This was then converted to the amino
acid 10 via two sequential oxidations. The N-methylation of 10
was achieved through a standard two-step sequence that utilizes
the intermediacy of oxazolidinone ring 11 and a subsequent
reductive opening. Finally, Boc protection yielded the requisite
N-methylated arylglycine 6 to be used in the synthesis of linear
tripeptide 4 (Scheme 2A).
To date, four classes of arylomycins have been identified,
arylomycins A−D (Figure 1). The latent antibiotics hypothesis
The protected Ala-Tyr dipeptide 13 was synthesized from 3-
iodotyrosine in 76% yield, over four steps. Briefly, 3-
iodotyrosine (5) was protected as a methyl ester and coupled
to Boc-Ala-OH. The free phenol was then protected as a benzyl
ether, and the boronic ester installed using standard Miyaura
borylation conditions to yield dipeptide 13. This dipeptide was
deprotected to the free amine and coupled to arylglycine 6 to
yield the linear tripeptide 4 in 74% yield (Scheme 2B).
In beginning to explore conditions to effect the desired
macrocyclization, those analogous to optimized conditions from
previous arylomycin syntheses were tried first (Scheme 3).
Treatment of 4 under these conditions {[PdCl2(dppf)] and
K2CO3 in DMSO at 90 °C} returned an encouraging yield of
21% (Scheme 3B, table entry 1). This was increased to only 29%
Figure 1. Arylomycin family of natural product antibiotics.
suggests that these different classes may represent variations
produced as part of a competition between microorganisms. As
part of our work to test this possibility, we have reported the
synthesis of members of each of the A−C classes and indeed
demonstrated that, relative to the A series, the B series gains
activity against Streptococcus agalactiae.19,29,31 Interestingly,
relative to the other arylomycins, the only class D arylomycin
identified to date, actinocarbasin, discovered at Merck,46 has
several significant structural differences. These include a unique
pattern of sulfation of the macrocyclic core and a significantly
B
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Scheme 1. Retrosynthetic Analysis of Derivatives 1a and 1b
lenges presented by the presence of a second substituent on the
arylglycine. Common additives used to facilitate Suzuki−
Miyaura reactions (Scheme 3B, table entries 3−5) were
explored next. Gratifyingly, it was found that the addition of
CuBr increased the yield of the macrocyclization to 36%
(Scheme 3B, table entry 5). The yield was further increased to
45% when a higher loading of both [PdCl2(dppf)] and CuBr was
used (Scheme 3B, table entry 6). The reaction was typically
complete within 1.5 h and was amenable to scale-up, producing a
35−40% yield of macrocycle 14 even at larger scales. No
reaction was observed when CuBr was added alone with no
palladium catalyst, suggesting that CuBr acts only as a promoter
of the palladium-catalyzed Suzuki−Miyaura reaction (Scheme
3B, table entry 7). It has been demonstrated in previous
literature reports that the addition of copper(I) salts promotes
the transmetalation of the aryl boronic ester to the palladium
center.49 A similar increase in yield was observed when CuBr
was used as an additive with Pd(0)[(P(tBu)3]2 (Scheme 3B,
table entry 11), although the overall yield was still lower than the
that obtained with [PdCl2(dppf)] and CuBr.
Scheme 2. Synthesis of Arylglycine 6 and Linear Peptide 4
With the desired macrocycle 14 in hand, attention next turned
to the glycosylation of the hindered free phenol (Scheme 3).
Employing previously optimized conditions for the glycosyla-
tion of arylomycin C−C16 (4 equiv of trichloroacetimidate 3, 10
equiv of BF3·Et2O, CH2Cl2, rt), only small amounts of the
glycosylated product were observed, with the deprotected amine
being the major side product, demonstrating the challenging
nature of this substrate for modification. Indeed, there are few
literature reports of the glycosylation of ortho,ortho-disubsti-
tuted phenols of a similar nature. After optimization, it was
found that heating the reaction to reflux in acetonitrile improved
conversion of the starting material 14 and that increasing the
equivalents of trichloroacetimidate 3 was necessary to achieve
when 40 mol % of the catalyst was used (Scheme 3B, table entry
2). However, no further improvement could be achieved by
varying the catalyst, base, or solvent (see Supporting
Information for details), demonstrating the additional chal-
Scheme 3. Suzuki−Miyaura Macrocyclization and Glycosylation
C
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Scheme 4. Synthesis of (A) derivatives 1a and 1b; (B) derivative 23; (C) derivative 24; and (D) derivative 27
full conversion due to decomposition of the trichloroacetimidate
at higher temperatures. The glycosylation was ultimately
achieved with BF3·Et2O and 15 equiv of trichloroacetimidate
3 in refluxing acetonitrile, providing 15 in 75% yield (Scheme 3).
The glycosylated macrocycle 15 was sequentially coupled to
isobutyl-protected L-cysteic acid 16a and acid 17 with 3-
(diethoxyphosphoryloxy)-1,2,3-benzotriazin-4(3H)-one
(DEPBT) and NaHCO3 to provide 18a with a fully elaborated
lipopeptide backbone. Next, sequential removal of the acyl and
benzyl protecting groups provided 19a in 88% yield. The
fluorosulfates were to be installed utilizing the SuFEx chemistry
developed by the Sharpless group.50 Conditions originally
reported by Sharpless call for CH2Cl2 as solvent; however the
reaction was found to be sluggish under these conditions, again
likely due to the steric bulk around the fluorosulfation site.
Model studies were conducted with a simplified substrate and
D
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Figure 2. Chemical shifts of the (A) aromatic protons in the natural product, 1a, and 24 and (B) the sugar residues of the natural product and 24 in
DMSO-d6 consistent with the revised structure of actinocarbasin (arylomycin D).
identified DMF as a more ideal solvent. However, when 19a was
fluorosulfated using these optimized conditions, major side
products were obtained in which the free alcohols on the sugar
subunit were also sulfated. This side reaction could be
suppressed by adding water as a cosolvent. These final
conditions produced the desired difluorosulfated product 20a
in 61% yield (Scheme 4A).
The methyl ester and isobutyl ester protecting groups were
removed in two steps via Me3SnOH-mediated hydrolysis and
treatment with NaI, respectively. When conversion of the
fluorosulfates to the arylsulfates was carried out with Cs2CO3 in
ethylene glycol at room temperature, only the conversion of a
single fluorosulfate to the arylsulfate was observed. Fortunately,
complete conversion of both fluorosulfates was achieved at a
higher temperature of 60 °C to give arylomycin derivative 1a in
21% yield over three steps (Scheme 4A). Derivative 1b was
synthesized from glycosylated macrocycle 15 and protected D-
cysteic acid 16b in an analogous manner (Scheme 4A).
With derivatives 1a and 1b in hand, the NMR data of the two
derivatives could be compared with that of the authentic natural
product (kindly provided by Merck). From this comparison, the
cysteic acid subunit in actinocarbasin is likely the L-isomer. The
chemical shift (in MeOD) of the cysteic acid α-proton in the
natural product (5.35 ppm) is more similar to 1a (5.36 ppm)
than 1b (5.45 ppm). Additionally, this is consistent with results
from previously synthesized arylomycin A derivatives, in which
derivatives with L-amino acids in the position directly adjacent to
the macrocycle exhibited stronger antibacterial activity than
derivatives with D-amino acids in the same position.46 However,
further comparison of the NMR spectra revealed several
inconsistencies in the chemical shifts associated with the
macrocycle that could not be explained by the different
hydrophobic side chains in the synthesized derivatives. Notably,
the chemical shifts of the aromatic protons in 1a (and 1b) were
shifted downfield relative to the authentic natural product, with
some protons differing by as much as 0.6−0.9 ppm (see Figure
2A). Conversely, the chemical shifts of the aromatic protons in
unsulfated intermediate 19a were much closer to those of the
authentic product. This suggested that the sulfates of actino-
carbasin are not attached to the aromatic ring.
The antibacterial activity of 1a and 1b were evaluated by
determining the minimum inhibitory concentration (MIC)
required to inhibit the growth of Yersinia pestis, Staphylococcus
epidermidis, methicillin-resistant Staphylococcus aureus (MRSA),
and a hyperpermeable mutant strain of Escherichia coli, using a
standard microdilution susceptibility assay51 (Table 1). Neither
1a nor 1b exhibited activity against any of the bacteria that were
tested, including MRSA. This lack of recapitulated activity
further supported the hypothesis that the reported structure
does not represent that of actinocarbasin.
To begin to explore what the correct structure of actino-
carbasin might be, and to elucidate the origins of its increased
Table 1. MICs (μg mL−1) of Synthesized Derivatives against
Pathogens
a
pathogen
Y. pestis
S. epidermidis
A-C16
1a
1b
23
24
27
4
>64
>64
N.D.
>64
>64
>64
>64
N.D.
>64
>64
>64
>64
N.D.
>64
>64
4
8
>64
64
32
2
4
>64
>64
16
0.25
>64
16
c
E. coli
E. coli IMP
b
MRSA (USA300)
>64
a
b
MIC values of arylomycin A-C16 are included as a reference.
A
c
hyperpermeable strain of E. coli. N.D.: not determined.
E
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spectrum, three additional derivatives were synthesized. To
investigate the effect that the arylsulfates alone have on activity, a
derivative that differs from arylomycin C-C16 by the addition of
the arylsulfates was synthesized. Briefly, glycosylated macrocycle
15 was coupled to the original A-C16 tail 21 to yield the coupled
product, which was then subjected to a deprotection and
sulfation sequence similar to the one described above, ultimately
providing derivative 23 (Scheme 4B). Similar to 1a and 1b,
derivative 23 shows no activity against any bacteria tested
(Table 1), confirming that the arylsulfates alone can ablate
activity.
The unsulfated derivatives 24 and 27 were also synthesized,
since comparison of their antibacterial activities with those of
arylomycin A-C16 and C-C16, respectively, was expected to aid in
understanding the potential contribution to antibacterial activity
of the β-alanine-cysteic acid dipeptide. 24 was synthesized from
intermediate 18a in a four-step sequence. The deprotection
sequence had to be modified so that the Pd/C-catalyzed
hydrogenolysis was the final step in order to suppress base-
mediated acyl-group transfer from the sugar unit to the phenol
on the arylglycine subunit (Scheme 4C). Derivative 27 was
synthesized by the sequential coupling of 16a and 17 to
macrocycle 25, providing 26 in four steps. Finally, deprotection
of the C-terminal carboxylic acid and side-chain cysteic acid
yielded derivative 27 (Scheme 4D).
The chemical shifts of the aromatic protons in the unsulfated
derivative 24 are a near exact match to the authentic natural
product (see Figure 2A), further supporting the hypothesis that
the sulfates are not directly attached to the aromatic ring as
reported. Indeed, a closer examination of the NMR spectra of
derivative 24 reveals that the only significant differences were in
the chemical shifts of the sugar subunit. Most significantly, the
protons on residues 3 and 4 of the sugar of derivative 24 were
shifted upfield 0.8−0.9 ppm relative to the natural product, while
the other glycosyl protons were shifted only 0.15−0.5 ppm. A
similar upfield shift was observed for the carbons corresponding
to those labeled 3 and 4 (see Figure 2B). During mass analysis of
the authentic natural product a major fragmentation observed
was the loss of the sugar unit together with two neutral SO3 units
(see Supporting Information). This fragmentation, together
with the large upfield shift of the signals corresponding to the
protons at carbons 3 and 4 of the sugar on derivative 24 relative
to the natural product, suggests that the sulfates are not directly
attached to the phenols, but rather attached to positions 3 and 4
of the sugar (Figure 2).
activity of the arylomycin D series compared to that of the
arylomycin A series.
To explore how the β-alanine-cysteic acid dipeptide imparts
activity against MRSA, we generated a model of 27 bound to S.
aureus SPase using as reference a reported structure of an E. coli−
arylomycin complex.29 Given the structural similarity between
the E. coli and S. aureus SPases and the fact that both the E. coli
protein and macrocycle adopt virtually identical conformations
in complexes with different arylomycins,29,52 we assumed that
27 would bind the S. aureus protein with its macrocycle adopting
the same conformation observed in the E. coli complexes. This
argument is even stronger with 27, as it shares an identical
macrocycle with one of the arylomycins whose complex with E.
coli SPase has been solved.52 Thus, using a superposition of the
free S. aureus protein with the arylomycin-bound E. coli protein
that we generated with the MatchMaker application in
Chimera,53 we identified residues in the S. aureus protein that
were different from their E. coli counterparts and predicted to be
proximal to the arylomycin. The superposition identified four
such residues: Thr28, Tyr30, Thr31, and Lys33. The positions of
Lys33 and Tyr30 in the free protein were predicted to clash with
the arylomycin macrocycle, and thus to model an induced fit
binding, we first performed a docking simulation of the
macrocyclic core in which these two side chains were allowed
to sample different conformations. This identified a pocket
conformation and mode of binding that closely reproduced that
of the E. coli complex. This conformation was then used in a
subsequent docking of the macrocycle core decorated with
dipeptide and truncated tail, this time fixing the side chains of
Lys33 and Tyr30 in the optimal conformation, while allowing
the side chains of Thr28 and Thr31 to sample different
conformations. By visual inspection, the best scored pose was
discarded, as the projected orientation of the aliphatic tail was
not consistent with burial in the membrane (absent in the
model), which is observed in all arylomycin-bound SPase
structures and thought to be essential for binding. The second
best scored pose was scored only slightly lower but projected the
aliphatic tail toward the membrane and in a manner consistent
with the other structural data, and so was selected as the final
model (Figure 3). As expected, the model shows the OH groups
of the macrocycle to be oriented into the solvent. More
interestingly, it predicts packing interactions between the
methylenes of the β-alanine and the side chain of Pro29 and
between the cysteic acid and Tyr30, as well as the formation of a
bifurcated H-bond involving the cysteic acid side chain, the
The antibiotic activity of both 24 and 27 was explored to
elucidate the role of the modified β-alanine-cysteic acid
dipeptide. Both derivatives exhibited activity similar to that of
arylomycin A-C16 against Y. pestis, but less activity against S.
epidermidis and the hyperpermeable strain of E. coli. In contrast,
both derivatives gained activity against MRSA (Table 1), with
24 showing a level of activity against MRSA similar to that
reported for actinocarbasin. These MIC results, particularly that
of derivative 27, which differs from arylomycin A-C16 only in the
linear dipeptide portion, demonstrate that the β-alanine-cysteic
acid dipeptide imparts activity against MRSA and supports the
reassignment of the actinocarbasin core structure as that shown
in Figure 2. They also suggest that the presumed sugar sulfation
of actinocarbasin is not essential for its activity, at least against
the MRSA strain tested. Although the unsulfated derivatives
show a slight loss in activity against S. epidermidis, activity is not
completely ablated. This, together with the gain in activity
against MRSA, represents an overall gain in the spectrum of
Figure 3. Model of 27 bound to S. aureus SPase
F
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residual undeuterated solvent as an internal reference (chloroform-d:
amide O of the lipid tail, and the side chain of Thr28. Thus the
model suggests that the Thr-Pro sequence is important for
recognition.
1
1H NMR δ = 7.26 ppm, 13C NMR δ = 77.16 ppm; methanol-d4: H
NMR δ = 3.31 ppm, 13C NMR δ = 49.00 ppm; DMSO-d6: 1H NMR δ =
2.50 ppm, 13C NMR δ = 39.52 ppm). The following abbreviations were
used to explain NMR peak multiplicities: s = singlet, d = doublet, t =
triplet, q = quartet, m = multiplet, br = broad. High-resolution mass
spectra (HRMS) were recorded on an Agilent LC/MSD TOF mass
spectrometer by electrospray ionization time-of-flight (ESI-TOF)
reflectron experiments.
In conclusion, five derivatives were synthesized in our efforts
to explore the arylomycin D series and to elucidate the origins of
actinocarbasin’s activity against S. aureus. The synthesis of these
derivatives utilized a similar strategy to the majority of
previously reported syntheses of other arylomycin family
members. However, the presence of a second substituent on
the arylglycine and the increased steric bulk around the
macrocycle led to several challenges that were addressed along
the route. Comparison of the NMR spectra of the synthesized
derivatives with that of the authentic natural product, along with
mass fragmentation analysis, led us to suggest that actino-
carbasin is in fact sulfated at positions 3 and 4 of the sugar
subunit. However, MIC analysis of the synthesized derivatives
ultimately suggests that sulfation is not required for activity, but
rather that the β-alanine-cysteic acid dipeptide contributes
significantly to the antibacterial activity of the arylomycin D
series against MRSA. The model of 27 bound to S. aureus
provides a clear explanation for why the β-alanine-cysteic acid
dipeptide imparts activity against this pathogen (Figure 3). In
particular, the β-alanine is predicted to contribute to recognition
by packing with the side chain of Pro29. Interestingly, most
SPases do not contain a Pro at this site, and any other residue will
present a backbone H-bond donor at this position, which would
disrupt this interaction. Moreover, when a backbone NH is
present at this position in SPase, it is predicted to form a
hydrogen bond with the backbone O of the canonical amino
acids present in the other classes of arylomycins. Finally, we
previously showed that S. epidermidis evolves resistance to the A
family arylomycins by installation of a Pro at this position and
that other bacteria that already possess a homologous Pro may
be rendered sensitive by mutating it to any other natural amino
acid.33 When viewed from the latent antibiotics perspective, it is
interesting to speculate that the residue at position 29 (S. aureus
numbering) of SPase and the peptidic side chain of the
arylomycin scaffold are coevolving.
HPLC purifications were carried out using a Vydac C18 column (10
μm, 10 × 250 mm, 300 Å pore size) and Shimadzu LC-20AP pumps
connected to a Shimadzu CBM-20A detector (monitoring at 220 and
280 nm). Samples were loaded onto the column at 0% B and eluted
either in acidic conditions (A: 0.1% TFA in H2O, B: 0.1% TFA in
CH3CN, linear gradient 0 to 90% B) or in NH4OAc buffered conditions
(A: 20 mM NH4OAc in H2O, B: CH3CN, linear gradient 0 to 80% B).
Synthesis of Amino Alcohol 9. A solution of tert-butyl carbamate
(5.6 g, 47.7 mmol, 2.1 equiv) in n-PrOH (60 mL) was sequentially
treated with NaOH (1.8 g, 45.4 mmol, 2.0 equiv, dissolved in 110 mL of
water) and t-BuOCl (5.06 g, 46.5 mmol, 2.05 equiv). After 15 min of
stirring, the solution was cooled to 0 °C and a solution of
(DHQ)2PHAL (708 mg, 0.91 mmol, 4 mol % dissolved in 60 mL of
n-PrOH) was added. A solution of styrene 8 (9.0 g, 22.7 mmol dissolved
in 90 mL of n-PrOH) was then added followed by K2OsO2(OH)4 (220
mg, 0.59 mmol, 2.6 mol % dissolved in 0.6 mL of 0.4 M NaOH). The
reaction mixture was stirred for 1 h at 0 °C. n-PrOH was removed by
rotary evaporation, and the remainder was diluted with water and
extracted three times with EtOAc. The combined organic layers were
washed with water and brine, dried over Na2SO4, and concentrated
under reduced pressure. The crude material was purified by flash
column chromatography (SiO2, 40% EtOAc in hexanes) to give amino
alcohol 9 as a white solid (10.0 g, 83% yield, > 98% ee). Rf = 0.26 (30%
EtOAc in hexanes). Absolute configuration and enantiomeric excess
were determined by Mosher’s amide analysis with the free amine. 1H
NMR (600 MHz, chloroform-d) δ 7.42−7.36 (m, 4H), 7.34−7.33 (m,
1H), 7.32 (d, J = 2.0 Hz, 1H), 6.88 (d, J = 2.0 Hz, 1H), 5.21 (br s, 1H),
5.17 (s, 2H), 5.06 (d, J = 1.8 Hz, 2H), 4.64 (s, 1H), 3.79 (dd, J = 11.3,
4.2 Hz, 1H), 3.76−3.70 (m, 1H), 3.59 (s, 3H), 1.96 (br s, 1H), 1.44 (s,
9H); 13C NMR (151 MHz, CDCl3) δ 155.95, 151.36, 145.89, 137.81,
136.20, 129.23, 128.76, 128.39, 127.79, 113.37, 98.92, 93.20, 80.32,
71.29, 66.45, 58.54, 55.92, 28.47; ESI HRMS calcd for C22H28INO6 [M
+ Na]+ 552.0853, found 552.0868.
Synthesis of Compound 11. Amino alcohol 9 (10.0 g, 18.9 mmol)
was dissolved in CH2Cl2 (250 mL) and cooled to 0 °C. Dess-Martin
periodinane (8.8 g, 20.8 mmol, 1.1 equiv) was added, and the reaction
was allowed to warm to room temperature and stirred for 2 h. The
reaction mixture was quenched with the addition of a 1:1 mixture of
saturated aqueous NaHCO3 and saturated aqueous Na2S2O3 and
stirred for 30 min. The mixture was extracted with CH2Cl2 (3×). The
combined organic layers were dried over Na2SO4, and solvents were
removed under reduced pressure. The residue was dissolved in a
solution of tert-butanol/2-methyl-2-butene/THF (80 mL:40 mL:100
mL) and cooled to 0 °C. A solution of NaClO2 (3.2 g, 35.4 mmol, 1.9
equiv) in aqueous NaH2PO4 (10.8 g in 60 mL of H2O) was added, and
the mixture was allowed to stir at 0 °C for 1.5 h. The reaction mixture
was diluted with EtOAc/H2O and extracted with EtOAc (3×). The
combined organic layers were washed with brine and dried over
Na2SO4, and solvents were removed under reduced pressure. The crude
acid was dissolved in toluene (200 mL), and to it were added
paraformaldehyde (3.46 g, 115 mmol, 6.1 equiv) and p-toluenesulfonic
acid (360 mg, 1.89 mmol, 0.1 equiv). The mixture was refluxed in a
Dean−Stark apparatus for 2 h. The solvent was removed under reduced
pressure, and the residue was partitioned between EtOAc and saturated
NaHCO3 and extracted twice with EtOAc. The combined organic
layers were washed with brine, dried over Na2SO4, and concentrated
under reduced pressure. The crude material was purified by flash
column chromatography (SiO2, 20% EtOAc in hexanes) to give
oxazolidinone 11 as a yellow oil (5.26 g, 50% yield). Rf = 0.6 (30%
EtOAc in hexanes); 1H NMR (600 MHz, chloroform-d) δ 7.43−7.31
(m, 6H), 6.97 (s, 1H), 5.61 (s, 1H), 5.31 (d, J = 4.7 Hz, 1H), 5.18 (s,
The possible coevolution of the arylomycins and SPase is
fascinating and, if supported by future studies, would provide
strong support for the latent antibiotics hypothesis in general
and that the arylomycins in particular are latent antibiotics.
Moreover, it would provide a fascinating glimpse into the
molecular details underlying the competition between arylomy-
cin producers and their competitors. Regardless, actinocarbasin
appears to use its β-alanine-cysteic acid dipeptide to recognize
the Thr28-Pro29-Tyr30 sequence of S. aureus SPase, and this
understanding should prove useful for efforts both in our
laboratory and in the pharmaceutical industry to develop the
arylomycins as broad-spectrum therapeutics.
EXPERIMENTAL SECTION
■
General Experimental Procedures. All reactions were carried out
under an argon atmosphere with dry solvents using anhydrous
conditions unless otherwise stated. Reagents were purchased at the
highest quality commercially available and used without further
purification, unless otherwise stated. Yields refer to chromato-
graphically and spectroscopically (1H NMR) homogeneous materials,
unless otherwise stated. Reactions were monitored by thin-layer
chromatography (TLC) carried out on 0.25 mm E. Merck silica gel
plates (60F-254) using UV light as the visualizing agent and/or KMnO4
and heat as a developing agent. Flash silica gel chromatography was
performed using E. Merck silica gel (60, particle size 0.043−0.063 mm).
NMR spectra were recorded on Bruker DRX-600 and calibrated using
G
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2H), 5.14−5.04 (m, 3H), 3.58 (s, 3H), 1.43 (br s, 9H); 13C NMR (151
MHz, CDCl3) δ 170.39, 152.05, 151.55, 146.86, 135.98, 128.95,
128.79, 128.45, 128.11, 127.78, 112.74, 98.94, 93.06, 82.82, 79.92,
78.32, 71.38, 58.53, 28.27; ESI HRMS calcd for C23H26INO7 [M − H]−
554.0681, found 554.0680.
(1.05 g, 2.42 mmol, 15 equiv dissolved in 1 mL of CH3CN) was added
in one portion. The reaction mixture was refluxed for an additional 3 h;
then the reaction was cooled to room temperature and quenched with a
saturated aqueous NaHCO3 solution. The mixture was extracted with
EtOAc (3×), and the combined organic layers were washed with brine,
dried over Na2SO4, and concentrated under reduced pressure. The
crude material was purified by flash column chromatography (SiO2,
80% EtOAc in hexanes then 10% MeOH in CH2Cl2) to give 15 as a
yellow solid (109 mg, 75% yield). Rf = 0.36 (10% MeOH in CH2Cl2).
1H NMR (600 MHz, chloroform-d) multiple isomers, see spectrum in
Supporting Information; 13C NMR (151 MHz, CDCl3) multiple
isomers, see spectrum in Supporting Information.; ESI HRMS calcd for
C48H53N3O14 [M + H]+ 896.3600, found 896.3603.
Synthesis of Arylglycine 6. To a stirred solution of 11 (5.0 g, 9.0
mmol) in CH2Cl2 (20 mL) at 0 °C was added triethylsilane (5.8 mL,
36.0 mmol, 4 equiv) followed by TFA (20 mL). The reaction was
allowed to warm to room temperature and stirred for 18 h. The mixture
was concentrated under reduced pressure, dissolved in water (42 mL),
and treated with solid NaHCO3 at 0 °C until pH 7. To the resulting
solution was added 1 N aqueous NaOH (18 mL, 18.0 mmol, 2 equiv) at
0 °C, followed by Boc2O (2.9 g, 13.5 mmol, 1.5 equiv) in THF (60 mL).
The mixture was allowed to warm to room temperature and stirred for 8
h. THF was removed under reduced pressure, and the aqueous layer
was washed twice with hexane, then treated with 6 N aqueous HCl until
pH 2−3. The aqueous layer was extracted with EtOAc (3×), and the
combined organic layers were washed with brine, dried over Na2SO4,
and concentrated to give arylglycine 6 as a pale yellow foam (4.6 g, 64%
Synthesis of Compound 2a. A solution of 16a (163 mg, 0.502
mmol, 3 equiv) in THF (8 mL) was treated sequentially with NaHCO3
(141 mg, 1.67 mmol, 10 equiv) and DEPBT (250 mg, 0.837 mmol, 5
equiv) and stirred at room temperature for 30 min. Glycosylated
macrocycle 15 (150 mg, 0.167 mmol, dissolved in 2 mL of THF) was
added, and the reaction stirred at room temperature for a further 16 h.
The reaction was partitioned between EtOAc and saturated aqueous
NaHCO3 solution and extracted twice with EtOAc. The organic layers
were combined and washed with brine, dried over Na2SO4, and
concentrated under reduced pressure. The crude material was purified
by flash column chromatography (SiO2, 60% EtOAc in hexanes) to give
2a as a white solid (100 mg, 50% yield). Rf = 0.48 (67% EtOAc in
1
yield), which was used without further purification. H NMR (600
MHz, chloroform-d) δ 7.42−7.34 (m, 6H), 7.25 (s, 1H), 6.87 (s, 1H),
5.83 (br s, 1H), 5.09 (s, 2H), 2.65 (s, 3H), 1.49 (s, 9H); 13C NMR (151
MHz, CDCl3) δ 174.73, 156.72, 146.39, 145.13, 135.50, 131.43,
128.90, 128.82, 128.75, 128.07, 122.69, 113.47, 85.34, 81.42, 71.64,
31.58, 28.46; ESI HRMS calcd for C21H24INO6 [M − H]− 512.0576,
found 512.0583.
1
hexanes). H NMR (600 MHz, chloroform-d) multiple isomers, see
spectrum in Supporting Information; 13C NMR (151 MHz, CDCl3)
multiple isomers, see spectrum in Supporting Information; ESI HRMS
calcd for C60H74N4O20S [M + H]+ 1203.4690, found 1203.4689.
Synthesis of Compound 18a. To a solution of 2a (80 mg, 0.066
mmol) in CH2Cl2 (3.6 mL) was slowly added TFA (0.4 mL) at 0 °C.
The solution was allowed to warm to room temperature and stirred for
1.5 h. The solvents were removed under reduced pressure to yield the
free amine used in the next step without purification. A solution of 17
(87 mg, 0.266 mmol, 4 equiv) in THF (4 mL) was treated sequentially
with NaHCO3 (56 mg, 0.66 mmol, 10 equiv) and DEPBT (99 mg,
0.332 mmol, 5 equiv) and stirred at room temperature for 30 min. The
above free amine (dissolved in 2 mL of THF) was added, and the
reaction stirred at room temperature for a further 16 h. The reaction
was partitioned between EtOAc and saturated aqueous NaHCO3
solution and extracted twice with EtOAc. The organic layers were
combined and washed with brine, dried over Na2SO4, and concentrated
under reduced pressure. The crude material was purified by flash
column chromatography (SiO2, 5% MeOH in CH2Cl2) to give 18a as a
white solid (58 mg, 62% yield). Rf = 0.24 (80% EtOAc in hexanes). 1H
NMR (600 MHz, chloroform-d) multiple isomers, see spectrum in
Supporting Information; 13C NMR (151 MHz, CDCl3) multiple
isomers, see spectrum in Supporting Information; ESI HRMS calcd for
C74H101N5O20S [M + H]+ 1412.6833, found 1412.6843.
Synthesis of Compound 19a. Compound 18a (58 mg, 0.041
mmol) was dissolved in anhydrous MeOH (2.5 mL), and to it was
added 30% NaOMe in MeOH (22 μL, 0.123 mmol, 3.0 equiv) at 0 °C.
The mixture was stirred for 2 h at the same temperature. Amberlyst-15
ion-exchange resin was added to the reaction, and the mixture was
stirred for a further 30 min. The resin was filtered off, and solvents were
removed under reduced pressure. The residue and 10% Pd/C (44 mg,
0.041 mmol, 1.0 equiv) were suspended in MeOH (5.0 mL) and stirred
for 3 h at room temperature under a hydrogen atmosphere. The Pd/C
was filtered off through Celite, and solvents were removed under
reduced pressure to yield 19a (40 mg, 88% yield) as a white solid, which
was used without further purification. 1H NMR (600 MHz, methanol-
d4) multiple isomers, see spectrum in Supporting Information; 13C
NMR (151 MHz, MeOD) multiple isomers, see spectrum in
Supporting Information; ESI HRMS calcd for C54H83N5O17S [M +
H]+ 1106.5577, found 1106.5575.
Synthesis of Linear Tripeptide 4. To a solution of dipeptide 13
(1.06 g, 1.82 mmol, 1.4 equiv) in CH2Cl2 (18 mL) was slowly added
TFA (2 mL) at 0 °C. The solution was allowed to warm to room
temperature and stirred for 2 h. The solvent was evaporated, and the
residue was partitioned between EtOAc and saturated aqueous
NaHCO3 solution and extracted twice with EtOAc. The organic layers
were combined and washed with brine, dried over Na2SO4, and
concentrated under reduced pressure to yield the free amine used in the
next step without purification. The above amine was dissolved in
anhydrous DMF (12 mL) and treated sequentially with HOBt (400 mg,
2.6 mmol, 2.0 equiv), arylglycine 6 (670 mg, 1.3 mmol), and
triethylamine (0.22 mL, 1.56 mmol, 1.2 equiv). The mixture was
cooled to 0 °C, and EDC (500 mg, 2.6 mmol, 2.0 equiv) was added in
one portion. The reaction was allowed to warm to room temperature
and stirred for 14 h. The mixture was partitioned between EtOAc and
water and extracted twice with EtOAc. The organic layers were
combined and washed with water and brine, dried over Na2SO4, and
concentrated under reduced pressure. The crude product was purified
by flash column chromatography (SiO2, 50% EtOAc in hexanes) to give
tripeptide 4 as a white solid (950 mg, 74% yield). Rf = 0.33 (50% EtOAc
in hexanes). 1H NMR (600 MHz, chloroform-d) multiple isomers, see
spectrum in Supporting Information; 13C NMR (151 MHz, CDCl3)
multiple isomers, see spectrum in Supporting Information; ESI HRMS
calcd for C47H57BIN3O11 [M + H]+ 978.3203, found 978.3217.
Synthesis of Macrocycle 14. Linear tripeptide 4 (700 mg, 0.716
mmol), K2CO3 (990 mg, 7.16 mmol, 10 equiv), CuBr (205 mg, 1.43
mmol, 2.0 equiv), and PdCl2(dppf) (234 mg, 0.286 mmol, 0.4 equiv)
were suspended in anhydrous DMSO (50 mL, degassed by sparging
with Ar) and heated to 90 °C for 1.5 h under Ar. The reaction mixture
was cooled to room temperature, partitioned between EtOAc and a
dilute aqueous NH4Cl solution, and extracted twice with EtOAc. The
combined organic layers were washed with water and brine, dried over
Na2SO4, and concentrated under reduced pressure. The crude product
was purified by flash column chromatography (SiO2, 60% EtOAc in
hexanes) to give macrocycle 14 as a pale yellow solid (217 mg, 42%
1
yield). Rf = 0.34 (60% EtOAc in hexanes). H NMR (600 MHz,
chloroform-d) multiple isomers, see spectrum in Supporting
Information; 13C NMR (151 MHz, CDCl3) multiple isomers, see
spectrum in Supporting Information; ESI HRMS calcd for C41H45N3O9
[M + H]+ 724.3228, found 724.3223.
Synthesis of Compound 15. To a mixture of the macrocycle 14
(117 mg, 0.162 mmol) and activated 3 Å molecular sieves in anhydrous
CH3CN (7 mL) was added BF3/Et2O (0.2 mL, 1.62 mmol, 10 equiv) at
0 °C. The mixture was heated to reflux, and trichloroacetimidate 3
Synthesis of Compound 20a. Compound 19a (40 mg, 0.036
mmol) and NEt3 (0.1 mL, 0.36 mmol, 20 equiv) were dissolved in a 2:1
mixture of DMF and H2O (3.6 mL) and stirred for 24 h at room
temperature under a SO2F2 atmosphere. The reaction mixture was
partitioned between EtOAc and 0.5 N HCl, and the aqueous layer was
H
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Article
extracted twice with EtOAc. The organic layers were combined and
washed sequentially with 5% aqueous LiCl solution and brine, dried
over Na2SO4, and concentrated under reduced pressure to give 20a as a
white solid (28 mg, 61% yield), which was used without further
purification. 1H NMR (600 MHz, methanol-d4) multiple isomers, see
spectrum in Supporting Information; 13C NMR (151 MHz, MeOD)
multiple isomers, see spectrum in Supporting Information; ESI HRMS
calcd for C54H81F2N5O21S3 [M + H]+ 1270.4627, found 1270.4629.
Synthesis of Arylomycin Derivative 1a. A mixture of 20a (28
mg, 0.022 mmol) and trimethyltin hydroxide (20 mg, 0.11 mmol, 5
equiv) in anhydrous DCE (2 mL) was heated to 70 °C for 16 h, then
cooled to room temperature. The solvent was removed under reduced
pressure, the residue was partitioned between EtOAc and 0.5 N HCl,
and the aqueous layer was extracted twice with EtOAc. The organic
layers were combined and washed with brine, dried over Na2SO4, and
concentrated under reduced pressure. The residue and sodium iodide
(50 mg, 0.33 mmol, 15 equiv) were suspended in acetone (2 mL) and
heated to 50 °C for 4 h, then concentrated under reduced pressure. The
crude material was taken up in MeOH and purified by reverse-phase
preparatory HPLC (0.1% TFA conditions) to yield the product as a
rotatable bonds were assigned with AutoDockTools55 using default
parameters.
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jnatprod.9b01174.
■
sı
Procedures and characterization, Table S1, Figures S1−
S2, NMR spectra (PDF)
AUTHOR INFORMATION
Corresponding Author
■
Floyd E. Romesberg − Department of Chemistry, The Scripps
Research Institute, La Jolla, California 92037, United States;
orcid.org/0000-0001-6317-1315; Email: fromesberg@
synthorx.com
Authors
1
white solid (8.4 mg, 32% yield) after lyophilization. H NMR (600
Yun Xuan Tan − Department of Chemistry, The Scripps Research
Institute, La Jolla, California 92037, United States
David S. Peters − Department of Chemistry, The Scripps Research
Institute, La Jolla, California 92037, United States;
orcid.org/0000-0003-0942-4224
Shawn I. Walsh − Department of Chemistry, The Scripps Research
Institute, La Jolla, California 92037, United States
Matthew Holcomb − Department of Chemistry, The Scripps
Research Institute, La Jolla, California 92037, United States
Diogo Santos-Martins − Department of Integrative Structural
and Computational Biology, The Scripps Research Institute, La
Jolla, California 92037, United States
MHz, methanol-d4) multiple isomers, see spectrum in SI (compound
S6a); 13C NMR (151 MHz, MeOD) multiple isomers, see spectrum in
Supporting Information; ESI HRMS calcd for C49H71F2N5O21S3 [M +
H]+ 1200.3844, found 1200.3845. The above white solid (4.5 mg, 3.75
μmol), dried cesium carbonate (36 mg, 0.11 mmol, 30 equiv), and
activated 3 Å molecular sieves were suspended in anhydrous ethylene
glycol (0.4 mL) in a flame-dried flask and heated to 60 °C for 20 min
under Ar. The reaction was cooled to room temperature, then diluted
by CH3CN and purified by reverse-phase preparatory HPLC
(NH4OAc-buffered conditions) to yield derivative 1a as a white solid
(2.9 mg, 65% yield) after lyophilization. 1H NMR (600 MHz,
methanol-d4) δ 7.51 (d, J = 9.3 Hz, 1H), 7.48 (d, J = 2.2 Hz, 1H),
7.20 (d, J = 9.3 Hz, 1H), 6.94 (s, 1H), 6.86 (br s, 1H), 6.33−6.24 (m,
1H), 5.38−5.33 (m, 1H), 5.26 (s, 1H), 4.77 (q, J = 6.3, 5.8 Hz, 1H),
4.54 (br s, 1H), 4.34−4.29 (m, 1H), 3.85 (dd, J = 9.4, 3.1 Hz, 1H),
3.53−3.45 (m, 2H), 3.38−3.32 (m, 2H), 3.25−3.10 (m, 4H), 3.10−
3.01 (m, 3H), 2.46 (dt, J = 6.2 Hz, 2H), 2.26−2.16 (m, 2H), 1.65−1.55
(m, 2H), 1.37−1.22 (m, 26H), 0.90 (t, J = 7.0 Hz, 3H), 0.62 (d, J = 6.1
Hz, 3H); 13C NMR (151 MHz, MeOD) δ 180.09, 176.51, 173.64,
173.25, 170.29, 163.31, 149.28, 147.86, 146.38, 136.55, 135.79, 133.10,
132.46, 132.32, 130.60, 128.40, 124.46, 121.53, 105.40, 73.74, 71.94,
71.79, 71.41, 64.32, 56.52, 54.80, 52.76, 50.82, 49.85, 37.18, 36.71,
36.47, 35.93, 33.08, 30.81, 30.77, 30.70, 30.58, 30.48, 30.44, 27.07,
24.11, 23.74, 19.13, 17.56, 14.45; ESI HRMS calcd for C49H73N5O23S3
[M − 3H]3− 397.4541, found 397.4533.
Stefano Forli − Department of Integrative Structural and
Computational Biology, The Scripps Research Institute, La Jolla,
California 92037, United States; orcid.org/0000-0002-
5964-7111
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jnatprod.9b01174
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
1
All H and 13C NMR spectra, as well as complete procedures and
This work was supported in part by NSS (Ph.D.) A*STAR
(predoctoral fellowship to Y.X.T.) and a National Science
Foundation Graduate Research Fellowship (NSF/DGE-
1346837 to D.S.P.).
characterizations for all other synthetic intermediates and arylomycin
derivatives, have been supplied as Supporting Information.
Biological Methods. Minimum inhibitory concentrations were
determined using the Clinical and Laboratory Standards Institute broth
microdilution method51 and the following laboratory strains of
bacteria: Y. pestis KIM6+, S. epidermidis RP62A, E. coli K-12
MG1655, E. coli IMP BAS901, methicillin-resistant S. aureus USA300.
Briefly, antibiotics were prepared as 2-fold dilutions in 96-well plates
containing cation-adjusted Mueller-Hinton II broth containing 0.002%
v/v TWEEN-80. Stock solutions of antibiotics were made in DMSO.
Wells were inoculated from a fresh plate scrape diluted to a final
concentration of 5 × 105 CFU/mL and incubated at 37 °C. The MICs
were defined as the lowest concentrations of antibiotic to prevent visible
growth after 24 h of incubation as observed from a minimum of three
independent determinations.
Computational Methods. Molecular docking calculations were
performed to model the binding of compound 27 to S. aureus SPase
using AutoDock 4.254 and the structures of S. aureus and E. coli SPases
(PDB: 4WVG and PDB: 3S04, respectively). Because the lipid
membrane was not included in the model, the lipid tail of 27 was
truncated at the first carbon. Structures of both ligand and target were
prepared according to the standard AutoDock protocol,55 removing all
waters and nonstandard residues. Atom types, partial charges, and
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