Synthesis and biological characterization of arylomycin B antibiotics

Article abstract of DOI:10.1021/np200163g

Antibiotics are virtually always isolated as families of related compounds, but the evolutionary forces underlying the observed diversity are generally poorly understood, and it is not even clear whether they are all expected to be biologically active. Th

Full text of DOI:10.1021/np200163g

ARTICLE
pubs.acs.org/jnp
Synthesis and Biological Characterization of Arylomycin B Antibiotics
Tucker C. Roberts, Peter A. Smith, and Floyd E. Romesberg*
Department of Chemistry, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, United States
S Supporting Information
b
ABSTRACT: Antibiotics are virtually always isolated as families of related
compounds, but the evolutionary forces underlying the observed diversity are
generally poorly understood, and it is not even clear whether they are all
expected to be biologically active. The arylomycin class of antibiotics is
comprised of three related families that are differentiated by nitration,
glycosylation, and hydroxylation of a conserved core scaffold. Previously, we
reported the total synthesis of an A series member, arylomycin A2, as well as
the A series derivative arylomycin C16 and showed that both are active against a
broader spectrum of bacteria than previously appreciated. We now report the
total synthesis of a B series analogue, arylomycin B-C16, and its aromatic amine
derivative. While the aromatic amine loses activity against all bacteria tested,
the B series compound shows activities that are similar to the A series
compounds, except that it also gains activity against the important pathogen
Streptococcus agalactiae.
acteria produce a large assortment of compounds that kill
other bacteria, possibly to gain advantage over competing
2002 from a strain of Streptomyces23 and were shown to act via
the novel mechanism of inhibiting type I signal peptidase
(SPase).22,24,25 Nonetheless, the development of these com-
pounds as therapeutics was abandoned after it was concluded
that they have activity against only a few Gram-positive bac-
teria22,23 and, due to poor penetration, no activity against any
Gram-negative bacteria.22 However, after reporting the first
synthesis of an arylomycin, the A series member arylomycin A2
and its derivative arylomycin C16 (Figure 1),26 we recently
reported that these A series compounds actually have a remark-
ably broad spectrum of activity, including potent activity against
both Gram-positive and Gram-negative bacteria, but activity is
limited by the natural presence of a resistance-conferring Pro
residue in the signal peptidase of some bacteria (including most
bacteria originally tested).25 We also observed that several
bacteria lacking the resistance-conferring Pro, for example, Strepto-
coccus agalactiae, are resistant to the arylomycins, suggesting that in
some bacteria additional mechanisms of resistance exist.
Because it has been suggested that the arylomycin B series of
antibiotics may have a different spectrum of activity,23 we were
interested in the synthesis and evaluation of a B series compound,
as any differences in activity might shed more light on potential
resistance mechanisms and also further elucidate the potential of
the arylomycin scaffold as a therapeutic. Moreover, we envi-
sioned that the synthesis would provide access to the amino
derivative 1 (Figure 1), which on the basis of the known
biosynthetic pathways of other antibiotics14,18ꢀ20 could repre-
sent a biosynthetic precursor to the arylomycin B compounds.
B
microorganisms for limited resources.1ꢀ7 Most if not all of these
antibiotics are produced as families of related compounds;
however the biological relevance of this diversity is not well
understood.8 It has been suggested that many of the related
compounds might have important biological functions and thus
that their presence is a result of selection.9,10 Conversely, it has
also been argued that the diversity results from the action of
enzymes with broad substrate tolerance functioning in nonspe-
cific biosynthetic pathways or from selection for diversity
itself.8,11,12 Characterizing the biological activity of the related
compounds, as well as intermediates within their biosynthetic
pathways, is expected to provide insight into how and why
antibiotics evolved. From a chemical perspective, nitro substitu-
tion is a particularly interesting modification, especially at aro-
matic positions, due to the large effects expected on the
physicochemical properties of the compounds.13,14 While nitro
substituents are generally rare among natural products,15 they are
relatively more common among antibiotics,13,16,17 such as chlor-
amphenicol and pyrrolnitrin, which both have aromatic nitro
groups that are thought to be biosynthesized from the corres-
ponding aromatic amines.14,18ꢀ20
The arylomycin family of antibiotics is comprised of three
related series of compounds, each possessing a conserved core
lipohexapeptide containing a C-terminal tripeptide macrocycle
and a variable N-terminal fatty acid.21,22 The A series compounds
have an unmodified core, the B series compounds are nitrated
(Figure 1), and the lipoglycopeptides are differentiated by
glycosylation, and in some cases hydroxylation, of the core
hydroxyphenylglycine. The arylomycins were first isolated in
Received: October 11, 2010
Published: May 05, 2011
r
Copyright
2011 American Chemical Society and
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have elegantly circumvented the same problem by not protecting
the phenolic oxygen of the iodo-nitro-tyrosine. Under the
conditions of the reaction, the free phenol is deprotonated, and
perhaps this facilitates oxidative insertion by increasing electron
density at the arylꢀiodide bond or by chelating boron. However,
an advantage to our route is that the SuzukiꢀMiyaura coupling
may proceed with a protected phenol (the phenol groups cannot
be protected after installation of the boronic ester27), which
increases the yield of this critical step with the arylomycin A series
compounds and also eliminates a postcyclization protection step
that might be less attractive for the future synthesis of derivatives.
The biological activity of arylomycin B-C16 and its derivative 1
was characterized by determining the minimal inhibitory con-
centration (MIC) required to inhibit the growth of wild-type
Staphylococcus epidermidis, Staphylococcus aureus, Escherichia coli,
and Pseudomonas aeruginosa (Table 1). In addition, for compar-
ison with the previously reported activity of arylomycin C16,
MICs were also determined against isogenic strains of S. aureus,
E. coli, and P. aeruginosa that were rendered sensitive to the
arylomycins by mutation of the resistance-conferring Pro to a
residue that does not confer resistance (P29S in the S. aureus
protein and P84L in the E. coli and P. aeruginosa proteins).25 We
also examined activity against a mutant strain of S. epidermidis
that was evolved to be resistant to arylomycin C16.34
As with the A series derivative arylomycin C16, arylomycin
B-C16 has potent activity against wild-type S. epidermidis (Table 1)
and no activity against wild-type S. aureus, E. coli, or P. aeruginosa
(MIC >128 mg/mL). Also like the A series compound, arylo-
mycin B-C16 has activity against the mutant strains of S. aureus,
E. coli, and P. aeruginosa and significantly less activity against the
mutant strain of S. epidermidis. In fact, the level of arylomycin
B-C16 activity against virtually all strains tested is indistinguish-
able from that of arylomycin C16 (Table 1). Thus, we conclude
that the activity of the B series compound is limited via the same
mechanism that limits the A series compounds, the presence of a
resistance-conferring Pro in SPase.25
The similar activities observed for the arylomycin A and B
series compounds were somewhat surprising given a previous
report that the B series derivatives have greater activity against
several species of Gram-positive soil bacteria.23 To generate a
better assessment of the relative activities of the A and B series
arylomycins, we examined a broad range of bacteria that have
been reported to be sensitive to the arylomycins, including B.
brevis, which was previously reported to be significantly more
sensitive to the B series compounds.23,25 However, we found that
the A and B series arylomycins displayed nearly identical
activities against almost all of these bacteria as well, suggesting
that, in contrast to previous reports, nitration does not generally
increase the activity of the arylomycins. Interestingly, under the
conditions we employed, neither the A nor B series arylomycins
demonstrated appreciable activity against B. brevis (MIC >64 μg/
mL). To determine whether the disagreement with the literature
is a result of a difference in growth conditions, we replicated the
conditions of the previous report (0.8% nutrient broth þ 0.5%
NaCl, grown with aeration by shaking).23 Although, we found
that the arylomycins do display some activity under these condi-
tions (MIC ∼2 μg/mL), the growth observed in the absence of
drug was very poor, and importantly, the activities of the A and B
series arylomycins were again indistinguishable. Removing the
0.5% NaCl restored robust growth but also eliminated the
arylomycin sensitivities, suggesting that poor growth conditions
can predispose some bacteria to arylomycin sensitivity.
Figure 1. Structure of the arylomycin derivatives characterized in this study.
’ RESULTS AND DISCUSSION
The arylomycins are naturally lipidated with fatty acids of
varying alkyl chain length (ranging in length from 12 to 16), but
because we found previously that the C16 tail of arylomycin C16
optimizes activity and because the majority of data available for
comparison are with this compound, our initial efforts to
synthesize a B series derivative targeted the analogous derivative,
denoted arylomycin B-C16 (Figure 1). Our synthesis (Scheme 1)
drew in part from our previously reported synthesis of arylomy-
cin A2,26 as well as the synthesis of Dufour and colleagues,27 and
commenced with construction of the nitrated tyrosine building
block 5. Boc protection of 3-nitro-tyrosine followed by iodination
of the phenol using benzyltrimethylammonium dichloroiodate28
gave compound 3, which was transformed to compound 4 in 82%
yield over three steps. After attempts to transform 4 into the
corresponding boronic ester using Miyaura’s boration conditions
failed, we found that the desired tripeptide 10 was readily
prepared by coupling 5 to dipeptide 9, which was synthesized
from the iodinated N-Me-hydroxyphenylglycine 6 that was
prepared as described by Dufour.27 The tripeptide was then
subjected to SuzukiꢀMiyaura coupling conditions (PdCl2(dppf)/
NaHCO3 in DMF) and Boc deprotected to give compound 11 in
42% yield over two steps. Compound 11 was then coupled to the
lipotripeptide tail 12 using DEPBT,29,30 yielding the protected
arylomycin analogue in 44% yield.
As reported with arylomycin A2, treatment of the protected B
series analogue with EtSH and 1.0 M AlBr3 in CH2Br2 under
inert atmosphere at 50 °C resulted in global deprotection.
However, in this case, deprotection proceeded concomitantly
with reduction of the aromatic nitro group,31ꢀ33 yielding the
deprotected, aminated arylomycin 1 as the major product (19%
yield). Pleasingly, fully deprotected arylomycin B-C16 was obtai-
ned in 67% yield after 1.0 M AlBr3/CH2Br2 was added to a
stirring solution of the protected natural product under air
atmosphere at ambient temperature in 10% EtSH/CHCl3.
During the preparation of this article, Dufour and colleagues
reported the synthesis of arylomycin B2,27 which differs from our
approach mainly in the method used for macrocyclization. The
resistance of 5 to Miyaura’s boration conditions is possibly due to
reduced electron density within the aryl iodide bond. As des-
cribed above, we circumvented this problem by installing the
boronic ester at the MeHpg center, while Dufour et al. appear to
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Scheme 1. Arylomycin B-C16 Synthesis
Table 1. MICs of Arylomycin Derivatives (μg/mL)a
Although the nitration of the arylomycin core observed in the
B series arylomycin does not generally appear to increase activity,
we did find that arylomycin B-C16 shows significant activity
against S. agalactiae, which stands in sharp contrast to arylomycin
C16, which has no activity against S. agalactiae. Unlike the activity
observed against B. brevis, the activity of arylomycin B-C16
against S. agalactiae was independent of the media employed
(cation-adjusted Mueller Hinton II broth or Todd Hewitt
broth). From the perspective of the potential development of
the arylomycins as antibiotics, this is significant, as S. agalactiae,
also known as group B streptococcus, is a leading cause of
morbidity and mortality among newborns35,36 and costs the
United States alone an estimated $60 million annually.37 The
activity against S. agalactiae is also particularly noteworthy
because this pathogen is predicted to be sensitive to the
arylomycins25 (the sequenced strain of this species encodes
two SPases, neither of which possesses the resistance-conferring
Pro), but grows in the presence of >128 μg/mL arylomycin C16.
Interestingly, the sensitivity of S. agalactiae to arylomycin B-C16
is very similar to the sensitivity of the related species S.
pneumoniae and S. pyogenes to both the A and B series variants.
Thus, within the context of these related organisms, it does not
strain
arylomycin C16 arylomycin B-C16
1
S. epidermidis RP62A
S. aureus P29S PAS8001
E. coli P84L PAS0260
P. aeruginosa P84L PAS2008
S. epidermidis PAS9002b
B. brevis ATCC 8246
R. equi ATCC 6939
0.25
0.25
8
4
2
4
8
4
2
4
8
64
16
32
nd
>64
nd
nd
nd
nd
nd
nd
nd
>64
>64
16
32
4
R. opacus DSM 1069
S. agalactiae COH-1
1
>128
8
8
S. pyogenes M1-5448
S. pneumoniae R800
4
8
16
2
C. glutamicum DSM 44475
L. lactis ATCC 19257
2
16
32
a nd, not determined. b Evolved from S. epidermidis RP62A to be resistant
to arylomycin C16.25
appear that the B series compound gained activity against
S. agalactiae, but rather that the A series compound lost activity
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against S. agalactiae. An explanation consistent with this data is
that S. agalactiae possesses other resistance mechanisms that are
effective against the A series derivative but not the B series
derivative, such as a modifying enzyme, although other factors
such as differences in affinity that are specific to S. agalactiae or
differences in cell wall penetration cannot be ruled out. Regard-
less, the increased activity of the B series compound against
S. agalactiae suggests that the arylomycin nitration may have
evolved as an adaptation.
In contrast, relative to arylomycin C16 and arylomycin B-C16,
we found that the amino derivative 1 is significantly less active
against all bacteria tested (Table 1), suggesting either that it is not
an intermediate during arylomycin B synthesis or that potent
activity was not required for the evolution of the biosynthetic
pathway. The loss in activity is slightly larger against the Gram-
positive bacteria (32-fold) than against the Gram-negative
pathogens (8-fold). This loss in activity is perhaps surprising
considering that when bound to SPase, the amino group is
expected to be oriented into the solvent. A variety of possible
causes may underlie this loss in activity. Perhaps the ortho amino
group induces changes in solvation, either directly by interacting
with water molecules or indirectly by hydrogen-bonding with the
adjacent hydroxyl group, which disfavors binding. Alternatively,
the amino group may stabilize interactions within a different
environment where the arylomycin is not active, such as the
plasma membrane or micelles.
In conclusion, the synthesis of arylomycin B-C16 was achieved
via a modification of published protocols26,27,29 in nine steps
from 3-nitro-tyrosine and in 8% overall yield. Generally, the
spectrum of antibiotic activity of arylomycin B-C16 is limited by
the same specific mechanism of resistance as the A series
compounds, which is widespread in nature and reduces the
practical utility of these compounds as therapeutics. However,
just as many clinically used therapeutics have been reoptimized
to overcome specific mechanisms of resistance that evolved
during their clinical use, it is possible that derivatization of the
arylomycin scaffold may be able to reoptimize it for broad-
spectrum activity. Indeed, at least regarding S. agalactiae, the B
series compounds have a broader spectrum of activity than the A
series compounds and may represent the natural manifestation of
this reoptimization. Whatever the evolutionary history of the
arylomycins, the results support the possibility that the spectrum
of the arylomycins can be optimized by derivatization. The
current and previously reported syntheses of the A and B series
arylomycins should provide for ready access to different deriva-
tives, which should allow us to begin testing this hypothesis.
Reactions were magnetically stirred and monitored by thin-layer
chromatography (TLC) with 0.25 mm Whatman precoated silica gel
(with fluorescence indicator) plates. Flash chromatography was per-
formed with silica gel (particle size 40ꢀ63 μm, EMD chemicals). 1H and
13C NMR spectra were recorded on Bruker DRX 500 or Bruker DRX
600 spectrometers. Chemical shifts are reported relative to either chloro-
form (δ 7.26) or methanol (δ 3.31) for 1H NMR and either chloroform
(δ 77.16) or methanol (δ 49.00) for 13C NMR. High-resolution time-of-
flight mass spectra were measured at the Scripps Center for Mass
Spectrometry. ESI mass spectra were measured on either an HP Series
1100 MSD or a PESCIEX API/Plus. For all compounds exhibiting
atropisomerism or isolated as semipure mixtures, NMR spectra are
provided in the Supporting Information. Yields refer to chromatogra-
phically and spectroscopically pure compounds unless otherwise stated.
All preparative reversed-phase chromatography was performed using
Dynamax SD-200 pumps connected to a Dynamax UV-D II detector
(monitoring at 220 nm) on a Phenomenex Jupiter C18 column (10 μm,
2.12 ꢁ 25 cm, 300 Å pore size). All solvents contained 0.1% TFA;
solvent A, H2O; solvent B, 90% acetonitrile/10% H2O. All samples were
loaded onto the column at 0% B, and the column was allowed to
equilibrate ∼10 min before a linear gradient was started. Retention times
are reported according to the linear gradient used and the % B at the time
the sample eluted.
Synthesis of Compound 4. To a solution of 3-nitro-tyrosine
(1 g, 4.4 mmol, 1 equiv) in acetone/H2O (1:1, 10 mL and treated
with NaHCO3 (554 mg, 1.5 equiv) and Boc2O (946 μL, 1 equiv),
and allowed to stir overnight. The reaction was acidified with 5% citric
acid (pH 3) and extracted 3ꢁ with EtOAc; then the combined organic
fractions were washed with brine, dried over sodium sulfate, and
concentrated. The crude material was then iodinated by a modification
of a procedure by Kajigaeshi et al.28 The crude material (1.37 g,
4.2 mmol, 1 equiv) was taken up in a 5:2 mixture of DCM/MeOH
(56 mL), treated with BTMA-ICl2 (1.6 g, 1.1 equiv) and NaHCO3
(2.47g, 7equiv), andallowedtostirovernight. ThesolidNaHCO3 was then
filtered, and the filtrate was concentrated and acidified with 5% citric acid
(pH 3). The aqueous layer was extracted 3ꢁ with EtOAc, and the
combined organic layers were dried over sodium sulfate and concen-
trated. The crude material (1.89 g, 4.19 mmol, 1 equiv) was dissolved in
acetone, treated with K2CO3 (2.9 g, 5 equiv) and MeI (1.3 mL, 5 equiv),
and heated to reflux over two days. The reaction mixture was then
allowed to cool to room temperature and quenched with a small amount
of water, and the volatiles were evaporated. Citric acid (5%, pH 3) and
EtOAc were added, then separated, and the aqueous layer was extracted
2ꢁ with EtOAc. The combined organic layers were washed with brine,
dried over sodium sulfate, and concentrated. The crude material was
purified via column chromatography (0ꢀ0.5% MeOH in DCM) to yield
compound 4 (1.67 g, 82% yield over three steps). 1H NMR (CDCl3, 500
MHz) (ppm): 7.80 (d, J = 1.5 Hz, 1H), 7.56 (d, J = 1.5 Hz, 1H), 5.12
(d, J = 6.5 Hz, 1H), 4.54ꢀ4.53 (m, 1H), 3.94 (s, 3H), 3.76 (s, 3H), 3.18
(dd, J = 5.0 Hz, J = 14.0 Hz, 1H) 2.98 (dd, J = 6.5 Hz, J = 14.0 Hz, 1H)
1.41 (s, 9H). 13C NMR (CDCl3, 500 MHz) (ppm): 171.5, 155.0, 152.1,
144.9, 143.8, 135.1, 126.4, 94.3, 80.5, 62.8, 54.2, 52.8, 37.0, 28.4. MS (ESI):
m/z (M þ Naþ) 503.0. Compound 4 (127 mg, 0.27 mmol, 1 equiv) was
then dissolved in DCM (2.5 mL) and treated with TFA (0.5 mL). When
TLC analysis indicated the complete consumption of starting material, the
volatiles were evaporated and the residue was dried under vacuum. The
residue was then taken up in EtOAc and saturated NaHCO3, the aqueous
layer was extracted 3ꢁ with EtOAc, and the combined organic layers were
dried over sodium sulfate and concentrated. The resulting compound
5 (101 mg) was used without characterization or further purification.
Synthesis of Compound 7. To a solution of compound 627 (300
mg, 0.74 mmol, 1 equiv) in DMF (7.4 mL) was added sequentially
H-Ala-OBn HCl (160 mg, 1 equiv), EDC (170 mg, 1.2 equiv), HOBt
(100 mg, 1 equiv), and NaHCO3 (71 mg, 1.15 equiv), and the reaction
’ EXPERIMENTAL SECTION
General Experimental Procedures. Dry solvents were pur-
chased from Acros. Commercially available amino acids were purchased
from Bachem (Torrence, CA), Chem-Impex (Wood Dale, IL), or
Novabiochem (EMD Chemicals, Gibbstown, NJ). Celite 545 filter aid
(not acid washed) was purchased from Fisher. Anhydrous 1-hydroxy-
benzotriazole (HOBt) was purchased from Chem-Impex. All other
chemicals were purchased from Fisher/Acros or Aldrich. Abbreviations:
THF, tetrahydrofuran; EtOH, ethanol; MeOH, methanol; AcOH, acetic
acid; DCM, dichloromethane; DMF, N,N-dimethylformamide; EDC,
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride; EtOAc,
ethyl acetate; Hex, hexanes; Ar, argon; TFA, trifluoroacetic acid; BTMA
ICl2, benzyltrimethylammonium dichloroiodate; MeI, iodomethane;
DEPBT, 3-(diethoxyphosphoryloxy)-1,2,3-benzotriazin-4(3H)-one.
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was allowed to stir overnight. Dilute NaHCO3 was added, and the
aqueous phase was extracted 3ꢁ with EtOAc. The combined organic
layers were washed with 5% citric acid (pH 3), water, and brine, then
dried over sodium sulfate, and concentrated. The crude material
(353 mg, 0.62 mmol, 1 equiv) was taken up in acetone (6.2 mL), and
to this solution were added K2CO3 (428 mg, 5 equiv) and MeI (386 μL,
10 equiv). The mixture was allowed to stir overnight at reflux in a sealed
vial; then the solvent was evaporated, water was added, and the aqueous
phase was extracted 3ꢁ with EtOAc. The combined organic layers were
washed with brine, dried over sodium sulfate, and concentrated. The
crude material was purified via column chromatography (0.75% MeOH
in DCM) to give the product (189 mg, 44% yield over two steps). 1H
NMR (CDCl3, 600 MHz) (ppm): 7.80 (s, 1H), 7.37ꢀ7.29 (m, 6H),
6.74 (d, J = 8.4 Hz, 1H), 6.30 (d, J = 7.2 Hz, 1H), 5.74 (br s, 1H),
5.22ꢀ5.15 (m, 2H), 4.70ꢀ4.66 (m, 1H), 3.87 (s, 3H), 2.70 (s, 3H), 1.48
(s, 9H), 1.44 (d, J = 7.2 Hz, 3H). 13C NMR (CDCl3, 600 MHz) (ppm):
172.6, 169.3, 158.2, 140.4, 135.4, 130.6, 129.3, 128.8, 128.6, 128.4, 114.2,
110.7, 86.2, 80.9, 67.4, 56.6, 55.4, 53.6, 48.5, 31.7, 28.5, 18.3. MS (ESI):
m/z (M þ Naþ) 605.1.
up in DCM (4.0 mL) and treated with TFA (0.8 mL). The reaction was
monitored via TLC, and when starting material was no longer present,
the volatiles were evaporated under a stream of nitrogen. DCM was
added and evaporated under nitrogen twice more, and the crude residue
was dissolved in EtOAc. The organic layer was washed with saturated
NaHCO3, driedoversodium sulfate, and concentrated. The crude material
was purified via pipet column chromatography (9% MeOH in DCM) to
give the product (29.7 mg, 42% yield). Multiple species were observed by
NMR due to atropisomerism. MS (ESI): m/z (M þ Hþ) 501.1.
Synthesis of Compound 12. Compound 12 was synthesized via
standard Fmoc/piperidine solid-phase peptide synthesis. Fmoc-Gly-OH
was loaded onto chlorotrityl chloride resin with DIEA at a loading
density of 0.61 mmol/g; then the constituent amino acids, Fmoc-D-Ala-
OH and Fmoc-N-Me-D-Ser(OBn)-OH, were coupled to the resin using
HCTU/DIEA (3 equiv:6 equiv) in DMF followed by palmitic acid
coupling with HCTU/DIEA (3 equiv:6 equiv) in DMF and enough
DCM to completely dissolve the acid. Cleavage from the resin was
achieved using 1% TFA in DCM using protocols supplied by Novabio-
chem. The product was purified via HPLC (linear gradient, 0.66% B per
minute, product eluted at 97% B) to give compound 12 (173 mg, 30%
yield after loading of Gly).
Synthesis of Arylomycin B-C16. To a solution of compound 11
(29.2 mg, 58.4 μmol) and compound 12 (50 mg, 1.5 equiv) in THF
(0.5 mL) at 0 °C were added DEPBT (28.0 mg, 1.6 equiv) and NaHCO3
(5.0 mg, 1 equiv). The reaction was then allowed to warm to room
temperature and stirred overnight. The THF was then evaporated under
a stream of nitrogen, and the reaction was dried under vacuum. The
crude reaction mixture was taken up in EtOAc, washed 2ꢁ with
saturated NaHCO3, then brine, dried over sodium sulfate, and concen-
trated. The crude was purified via column chromatography (3% MeOH
in DCM, then 4.5% MeOH in DCM) to give the protected arylomycin
(14.7 mg, 44%). The protected arylomycin (10.0 mg, 9.4 μmol, 1 equiv)
was dissolved in CHCl3 (2 mL), treated with ethanethiol (180 μL, 250
equiv) and 1.0 M AlBr3 in CH2Br2 (189 μL, 20 equiv), and stirred in a
vial open to air at room temperature for 6 h. The reaction was quenched
by the addition of MeOH, and the volatiles were evaporated under a
stream of nitrogen. The crude material was taken up in MeOH and dried
twice more to remove any remaining ethanethiol; then it was dissolved
in MeOH, centrifuged, and purified via HPLC (linear gradient, 1.0% B
per minute, product eluted at 82% B) to give the product (5.8 mg, 67%
yield). Multiple species were observed by NMR due to atropisomerism.
ESI HRMS: m/z [(M þ H)þ] 926.4873 (calcd for C47H70N6O11
926.4869).
Synthesis of Compound 1. The protected arylomycin (6.3 mg,
6.0 μmol, 1 equiv) was dissolved in ethanethiol (300 μL) and 1.0 M
AlBr3 in CH2Br2 (120 μL, 20 equiv) and stirred in a vial for 5 h under Ar
at 50 °C. The reaction was quenched by the addition of MeOH, and the
volatiles were evaporated under a stream of nitrogen. The crude material
was taken up in MeOH and dried twice more to remove any lingering
ethanethiol; then it was dissolved in MeOH, centrifuged, and purified via
HPLC (linear gradient, 1.0% B per minute, product eluted at 75% B) to
give the product (1.0 mg, 19% yield). Multiple species were observed by
NMR due to atropisomerism. ESI HRMS: m/z [(M þ H)þ] 896.5123
(calcd for C47H70N6O11, 896.5128).
Determination of Antimicrobial Activity. Antimicrobial activ-
ity was examined using 18 bacterial strains, Staphylococcus epidermidis
RP62A, Staphylococcus aureus NCTC 8325, Escherichia coli MG1655,
Pseudomonas aeruginosa PAO1, Staphylococcus epidermidis RP62A
SpsIB(S29P) (PAS9001), Staphylococcus epidermidis RP62A SpsIB-
(S31P) (PAS9002),25 Staphylococcus aureus NCTC 8325 SpsB(P29S)
(PAS8001),25 Escherichia coli MG1655 LepB(P84L) (PAS0260),25
Pseudomonas aeruginosa PAO1 LepB(P84L) (PAS2008),25 Brevibacillus
brevis ATCC 8246, Rhodococcus equi ATCC 6939, Rhodococcus opacus
DSM 1069, Streptococcus agalactiae COH-1, Streptococcus pyogenes
Synthesis of Compound 8. Toasolutionof compound7(185mg,
0.36 mmol, 1 equiv) in DMSO (7 mL) under Ar were added sequentially
bispinacolatodiboron (95 mg, 1.05 equiv), potassium acetate (353 mg,
10 equiv), and PdCl2(dppf) (15 mg, 0.05 equiv). The mixture was
allowed to stir for 2.5 h at 80 °C, then cooled to room temperature,
diluted with water, and extracted 3ꢁ with EtOAc. The combined organic
layers were washed with brine, dried over sodium sulfate, and concen-
trated. The crude material was purified by abbreviated column chroma-
tography (35% EtOAc in Hex) (to minimize the time of exposure to
silica), giving compound 8 as a mixture of boronic acid and ester (118
mg, 64% yield). NMR spectra showed two sets of overlapping signals in a
1
3:1 ratio. H NMR (CDCl3, 600 MHz) (ppm): 7.61ꢀ7.59 (m, 1H),
7.37ꢀ7.31 (m, 5H), 6.81ꢀ6.76 (m, 1H), 6.31ꢀ6.18 (m, 1H), 5.74 (br
s), 5.20ꢀ5.12 (m, 2H), 4.73ꢀ4.66 (m, 1H), 3.83ꢀ3.80 (m, 3H),
2.68ꢀ2.67 (m, 3H), 1.47ꢀ1.40 (m, 12H), 1.34ꢀ1.33 (m, 9H). MS
(ESI): m/z (M þ Naþ) 605.3.
Synthesis of Compound 10. Compound 8 (118 mg, 0.19 mmol,
1 equiv) was taken up in 95% EtOH (2 mL), 10% Pd/C (38 mg, 1/3 by
weight) was added, and the mixture was placed under an atmosphere of
H2. The reaction was allowed to proceed until TLC analysis indicated
the complete consumption of starting material. The mixture was then
filtered through Celite and concentrated. To a solution of this crude
compound 9 (94 mg, 0.19 mmol, 1 equiv) and compound 5 (101 mg,
0.27 mmol, 1.4 equiv) in AcCN/DMF (2.2:1, 2 mL) were added
sequentially HOBt (64 mg, 2.5 equiv) and EDC (80 mg, 2.2 equiv),
and the reaction was allowed to stir overnight. Dilute NaHCO3(aq) was
then added to the reaction, and the aqueous phase was extracted 3ꢁ with
EtOAc. The combined organic layers were washed with 5% citric acid,
water, and brine, then dried over sodium sulfate and concentrated. The
crude material was purified via abbreviated column chromatography
(3% MeOH in DCM) to provide 10 as a mixture of boronic acid and
ester (130 mg, 80%). MS (ESI): m/z (M þ Naþ) 877.2 (ester).
Synthesis of Compound 11. A solution of compound 10 (118
mg, 0.14 mol, 1 equiv) and NaHCO3 (118 mg, 10 equiv) in DMF
(4.2 mL) was purged several times via cycling with vacuum and Ar and
sealed with a crimped septum. To this solution was added, via syringe, a
solution of PdCl2(dppf) (23.0 mg, 0.2 equiv) in DMF (2.8 mL) that had
been sparged with Ar for ∼15 min. The resulting mixture was submitted
to several more cycles of vacuum and Ar, then heated to 80 °C. The
mixture was then cooled to room temperature, and water was added.
The aqueous phase was extracted with EtOAc 3ꢁ, then washed with
water and brine, dried over sodium sulfate, and concentrated. The crude
material was subjected to abbreviated column chromatography (4%
MeOH in DCM) to remove most of the Pd species, then used without
further purification. The resulting semipure material (83 mg) was taken
960
dx.doi.org/10.1021/np200163g |J. Nat. Prod. 2011, 74, 956–961

Journal of Natural Products
ARTICLE
M1-5448, Streptococcus pneumoniae R800, Corynebacterium glutamicum
ATCC 44475, and Lactococcus lactis ATCC 19257. Minimum inhibitory
concentrations were determined from at least three independent
experiments using the CLSI broth microdilution method. Briefly,
inocula were prepared by suspending bacteria growing on solid media
into the same type of broth used in the MIC experiment and diluting to a
final concentration of 1 ꢁ 107 colony forming units/mL. A 5 μL amount
of this suspension was added to the wells of a 96-well plate containing
100 μL of media with the appropriate concentrations of compound. The
MICs of E. coli, P. aeruginosa, S. aureus, S. epidermidis, R. equi, R. opacus,
C. glutamicum, and B. brevis were determined in cation-adjusted Mueller
Hinton II broth. MICs of S. pyogenes and S. pneumoniae were determined
in Todd Hewitt broth. The MICs of S. agalactiae were determined in
cation-adjusted Mueller Hinton II broth and in Todd Hewitt broth
(MIC values differed by at most 2-fold between these two media). The
MIC of L. lactis was determined in Trypticase soy yeast broth. To
replicate previous measurements of arylomycin A and B series com-
pounds against B. brevis, 106 cfu of B. brevis were inoculated into 14 mL
culture tubes containing 1 mL of 0.8% nutrient broth supplemented with
0.5% NaCl, the appropriate concentration of compounds, and DMSO at
a final concentration of 1%. Cultures were shaken at 28 °C for 24 h.
Identical experiments were also performed using cation-adjusted Mueller
Hinton II broth and using 0.8% nutrient broth without NaCl. In all cases
MICs were defined as the lowest concentration of compound to inhibit
visible growth.
(14) Ju, K. S.; Parales, R. E. Microbiol. Mol. Biol. Rev. 2010,
74, 250–272.
(15) He, J.; Hertweck, C. J. Am. Chem. Soc. 2004, 126, 3694–3695.
(16) Choi, Y. S.; Zhang, H.; Brunzelle, J. S.; Nair, S. K.; Zhao, H. Proc.
Natl. Acad. Sci. U. S. A. 2008, 105, 6858–6863.
(17) Herbert, R. B.; Knaggs, A. R. Tetrahedron Lett. 1990,
31, 7517–7520.
(18) Kirner, S.; Hammer, P. E.; Hill, D. S.; Altmann, A.; Fischer, I.;
Weislo, L. J.; Lanahan, M.; van Pee, K. H.; Ligon, J. M. J. Bacteriol. 1998,
180, 1939–1943.
(19) Kirner, S.; Vanpee, K. H. Angew. Chem., Int. Ed. 1994, 33,
352–352.
(20) He, J.; Magarvey, N.; Piraee, M.; Vining, L. C. Microbiology
2001, 147, 2817–2829.
(21) Holtzel, A.; Schmid, D. G.; Nicholson, G. J.; Stevanovic, S.;
Schimana, J.; Gebhardt, K.; Fiedler, H. P.; Jung, G. J. Antibiot. 2002,
55, 571–577.
(22) Kulanthaivel, P.; Kreuzman, A. J.; Strege, M. A.; Belvo, M. D.;
Smitka, T. A.; Clemens, M.; Swartling, J. R.; Minton, K. L.; Zheng, F.;
Angleton, E. L.; Mullen, D.; Jungheim, L. N.; Klimkowski, V. J.; Nicas,
T. I.; Thompson, R. C.; Peng, S. B. J. Biol. Chem. 2004,
279, 36250–36258.
(23) Schimana, J.; Gebhardt, K.; Holtzel, A.; Schmid, D. G.;
Sussmuth, R.; Muller, J.; Pukall, R.; Fiedler, H. P. J. Antibiot. 2002, 55,
565–570.
(24) Paetzel, M.; Goodall, J. J.; Kania, M.; Dalbey, R. E.; Page,
M. G. P. J. Biol. Chem. 2004, 279, 30781–30790.
(25) Smith, P. A.; Roberts, T. C.; Romesberg, F. E. Chem. Biol. 2010,
17, 1223–1231.
(26) Roberts, T. C.; Smith, P. A.; Cirz, R. T.; Romesberg, F. E. J. Am.
Chem. Soc. 2007, 129, 15830–15838.
’ ASSOCIATED CONTENT
Supporting Information. 1H and 13C NMR spectra of
S
b
(27) Dufour, J.; Neuville, L.; Zhu, J. Chem.—Eur. J. 2010, 16, 10523–
10534.
compounds 1, 8, 10, 11, 12, and arylomycin B-C16. This material
is available free of charge via the Internet at http://pubs.acs.org.
(28) Kajigaeshi, S.; Kakinami, T.; Yamasaki, H.; Fujisaki, S.; Kondo,
M.; Okamoto, T. Chem. Lett. 1987, 2109–2112.
(29) Dufour, J.; Neuville, L.; Zhu, J. P. Synlett 2008, 15, 2355–2359.
(30) Boger, D. L.; Kim, S. H.; Mori, Y.; Weng, J. H.; Rogel, O.;
Castle, S. L.; McAtee, J. J. J. Am. Chem. Soc. 2001, 123, 1862–1871.
(31) Paradisi, C.; Scorrano, G. Acc. Chem. Res. 1999, 32, 958–968.
(32) Montanari, S.; Paradisi, C.; Scorrano, G. J. Org. Chem. 1999,
64, 3422–3428.
’ AUTHOR INFORMATION
Corresponding Author
*E-mail: floyd@scripps.edu.
(33) Montanari, S.; Paradisi, C.; Scorrano, G. J. Org. Chem. 1991,
56, 4274–4279.
(34) Smith, P. A.; Roberts, T. C.; Romesberg, F. E. Chem. Biol. 2010,
’ ACKNOWLEDGMENT
This work was supported by the Office of Naval Research
(Awards N000140310126 and N000140810478) and the Na-
tional Institutes of Health (1R21AI081126).
17, 1223–1231.
(35) Apgar, B. S.; Greenberg, G.; Yen, G. Am. Fam. Physician 2005,
71, 903–910.
(36) Sherris Medical Microbiology: An Introduction to Infectious Dis-
eases; Ryan, K.; Ray, C.; Ryan, K. J., Eds.; McGraw Hill: New York, 2003.
(37) Centersfor DiseaseControl. InProvisionalRecommendations for
the Prevention of Perinatal Group B Streptococcal Disease, July 29, 2010.
’ REFERENCES
(1) Allen, H. K.; Donato, J.; Wang, H. H.; Cloud-Hansen, K. A.;
Davies, J.; Handelsman, J. Nat. Rev. Microbiol. 2010, 8, 251–259.
(2) Baltz, R. H. J. Ind. Microbiol. Biotechnol. 2006, 33, 507–513.
(3) Czaran, T. L.; Hoekstra, R. F.; Pagie, L. Proc. Natl. Acad. Sci.
U. S. A. 2002, 99, 786–790.
(4) D’Costa, V. M.; Griffiths, E.; Wright, G. D. Curr. Opin. Microbiol.
2007, 10, 481–489.
(5) Laskaris, P.; Tolba, S.; Calvo-Bado, L.; Wellington, L. Environ.
Microbiol. 2010, 12, 783–796.
(6) Lynch, M. Proc. Natl. Acad. Sci. U. S. A. 2007, 104 (Suppl. 1),
8597–8604.
(7) Martinez, J. L. Proc. Biol. Sci. 2009, 276, 2521–2530.
(8) Fischbach, M. A.; Clardy, J. Nat. Chem. Biol. 2007, 3, 353–355.
(9) Stone, M. J.; Williams, D. H. Mol. Microbiol. 1992, 6, 29–34.
(10) Williams, D. H.; Stone, M. J.; Hauck, P. R.; Rahman, S. K. J. Nat.
Prod. 1989, 52, 1189–1208.
(11) Firn, R. D.; Jones, C. G. Mol. Microbiol. 2000, 37, 989–994.
(12) Firn, R. D.; Jones, C. G. Nat. Prod. Rep. 2003, 20, 382–391.
(13) Winkler, R.; Hertweck, C. Chembiochem 2007, 8, 973–977.
961
dx.doi.org/10.1021/np200163g |J. Nat. Prod. 2011, 74, 956–961