Precursor directed biosynthesis of an orthogonally functional erythromycin analogue: Selectivity in the ribosome macrolide binding pocket

Article abstract of DOI:10.1021/ja304682q

The macrolide antibiotic erythromycin A and its semisynthetic analogues have been among the most useful antibacterial agents for the treatment of infectious diseases. Using a recently developed chemical genetic strategy for precursor-directed biosynthesis and colony bioassay of 6-deoxyerythromycin D analogues, we identified a new class of alkynyl- and alkenyl-substituted macrolides with activities comparable to that of the natural product. Further analysis revealed a marked and unexpected dependence of antibiotic activity on the size and degree of unsaturation of the precursor. Based on these leads, we also report the precursor-directed biosynthesis of 15-propargyl erythromycin A, a novel antibiotic that not only is as potent as erythromycin A with respect to its ability to inhibit bacterial growth and cell-free ribosomal protein biosynthesis but also harbors an orthogonal functional group that is capable of facile chemical modification.

Full text of DOI:10.1021/ja304682q

Article
pubs.acs.org/JACS
Precursor Directed Biosynthesis of an Orthogonally Functional
Erythromycin Analogue: Selectivity in the Ribosome Macrolide
Binding Pocket
Colin J. B. Harvey,†,∞ Joseph D. Puglisi,⊥ Vijay S. Pande,†,∥ David E. Cane,# and Chaitan Khosla*,†,‡,§
‡
§
∥
†Departments of Chemistry, Chemical Engineering, Biochemistry, and the Biophysics Program, Stanford University, Stanford,
California 94305, United States
⊥Department of Structural Biology, Stanford University School of Medicine, Stanford, California 94035, United States
#Department of Chemistry, Brown University, Providence, Rhode Island 02912, United States
S
* Supporting Information
ABSTRACT: The macrolide antibiotic erythromycin A and
its semisynthetic analogues have been among the most useful
antibacterial agents for the treatment of infectious diseases.
Using a recently developed chemical genetic strategy for
precursor-directed biosynthesis and colony bioassay of 6-
deoxyerythromycin D analogues, we identified a new class of
alkynyl- and alkenyl-substituted macrolides with activities
comparable to that of the natural product. Further analysis
revealed a marked and unexpected dependence of antibiotic
activity on the size and degree of unsaturation of the precursor.
Based on these leads, we also report the precursor-directed biosynthesis of 15-propargyl erythromycin A, a novel antibiotic that
not only is as potent as erythromycin A with respect to its ability to inhibit bacterial growth and cell-free ribosomal protein
biosynthesis but also harbors an orthogonal functional group that is capable of facile chemical modification.
reported total synthesis of 1a5−9 and all of the clinically used
analogues of the natural product are semisynthetically derived.
INTRODUCTION
The polyketides are a large class of natural products with
■
Precursor-directed biosynthesis represents a promising
clinically important activities ranging from anticancer (doxor-
alternative to semisynthesis or total synthesis approaches as a
ubicin) to immunosuppression (FK-506) and, perhaps most
means to access analogues of clinically important polyketide
prominently, antibacterial. A prototypical polyketide, eryth-
natural products.10,11 This strategy exploits this simplicity of
romycin A (1a), is produced by the soil bacterium
precursors and early intermediates in polyketide biosynthesis by
incorporating variants thereof into the corresponding natural
Saccharopolyspora erythraea and has been in clinical use as a
macrolide antibiotic for more than half a century.1,2 Its
product analogue. This approach has been used to synthesize a
medicinal chemistry has been extensively explored with an
number of bioactive erythromycin analogues.12−18
eye toward improving its therapeutic potential.3,4 While these
efforts have yielded significant advances, such as the discovery
of the second-generation macrolides, clarithromycin and
A major limitation of earlier approaches for polyketide
discovery via precursor directed biosynthesis was the scale on
which such experiments needed to be conducted. The low
azithromycin, as well as the third-generation ketolide,
telithromycin, the intrinsic structural complexity of 1a has
greatly hindered more fundamental variations in the core
structure to obtain agents of improved potency and
antibacterial spectrum.
Traditionally, alteration of natural product scaffolds has been
achieved via semisynthesis or total synthesis. The former
approach depends heavily upon modification of the naturally
installed functionality. This can be particularly restrictive in the
case of 1a, where regioselectivity is a major challenge. On the
other hand, total synthesis of complex natural product
analogues is often hampered by practical scalability of a
challenging production process. For example, despite extensive
efforts by synthetic chemists, there exists to date a single
conversion efficiency of precursor into product, coupled with
challenging fermentation procedures associated with actino-
mycete bacteria, necessitated the evaluation of alternative
synthetic precursors on a relatively large scale in a low-
throughput fashion. To circumvent these limitations, we
recently engineered an Escherichia coli strain that combines
the power of precursor directed biosynthesis of erythromycin
analogues with the possibilities of a single colony bioassay19,20
(Figure 1). For example, when a synthetic precursor (2) that
mimics the natural diketide intermediate in the biosynthesis of
6-deoxyerythronolide B (3a, 6-dEB) is fed to a culture of E. coli
Received: May 22, 2012
Published: June 28, 2012
© 2012 American Chemical Society
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Figure 1. A chemical genetic strategy for rapid discovery of new macrolide antibiotics via precursor directed biosynthesis in E. coli. The modular
organization of the DEBS assembly line is shown, with each circle representing a distinct active site. The KR domain is shown as a dimer because it
consists of an N-terminal structural subdomain and a C-terminal catalytic subdomain. Plasmids pBP175#8 and pBP130 express a truncated derivative
of the 6-deoxyerythronolide B synthase (DEBS) lacking the loading didomain and module 1. Plasmids pHL74 and pHL80* express proteins
responsible for sugar biosynthesis and glycosyl transfer. Plasmid pBP175#8 also expresses the propionyl-CoA carboxylase genes from S. coelicolor.
When synthetic diketide 2 is fed to this engineered E. coli strain, it is converted into the corresponding 6-deoxyerythromycin D product 4 via the
combined action of all of these enzymes.
Table 1. Plasmids and Bacterial Strains Used Throughout This Work
Strain
Description
Plasmid
pBP130
Gene Products
DEBS2-DEBS3
Strain
Description
Escherichia coli
HYL3
A derivative of E. coli BAP1 (BL21 ΔprpRBCD
(sfp)) evolved for improved polyketide
production.
Aglycone biosynthesis
pET21
(carb)
pBP175#8 PccAB-DEBS Module2
Aglycone biosynthesis
pET28
(kan)
pHL74
EryCIII-EryCII-TylAI-DesIV-
DesI-DesII-DesV-DesVI
TDP-desosamine
biosynthesis and transfer
pGZ119
(cm)
pHL80*
GroES-GroEL-TylCVII-
EryBIV-EryBVI-EryBV-
TylCIII-EryBII
TDP-mycarose
biosynthesis and
transfer, Chaperone
pCDF-duet
(strep)
Saccharopolyspora
erythraea A34
A mutant of S. erythraea with a greatly diminished
None
None
capacity for 6-DEB production
Bacillus subtilis
A representative, macrolide susceptible, gram
positive bacterium.
HYL3/pBP130/pBP175#8/pHL74/pHL80* (Table 1), the
precursor is converted into the corresponding glycosylated
macrolide, 6-deoxyerythromycin D (4). The bioactivity of this
glycosylated macrolide can be conveniently assayed in a colony
assay on a petri dish containing appropriate concentrations of
the desired synthetic precursor.
Here, we have used this system to biosynthesize and screen a
series of novel erythromycin analogues. Our findings have not
only yielded equipotent, orthogonally functionalized analogues
of erythromycin A, but also shed light on an unexpected steric
relationship in the macrolide binding pocket of the bacterial
ribosome.
ability of their ultimate biosynthetic product to produce a halo
of growth inhibition around single colonies of E. coli HYL3/
pBP130/pBP175#8/pHL74/pHL80* (Figure 2a,b). As sum-
marized in Table 1, this engineered host-vector system has
several features that improve the efficiency of precursor
directed biosynthesis. A key advantage of this method for
investigating macrolide structure−activity relationships is that it
requires only small quantities of a synthetic precursor, thereby
enabling analysis of a larger precursor library than possible
previously. While this assay is inherently qualitative in nature,
owing to its extremely low detection limit it provides a useful
tool for the efficient screening and identification of active
compounds (Figure 2c).
RESULTS
Several factors were considered when designing the diketides
to be screened. Previous work has shown that the stereo-
chemistry of the α-methyl and β-hydroxyl groups, particularly
their syn orientation, is important for diketide incorporation by
module 2.21 With this in mind, 2b, the enantiomer of the
■
Precursor Screening. In earlier studies, we described the
application of a colony bioassay for the directed evolution of
erythromycin biosynthesis in E. coli.19,20 Here, we adapted this
assay to evaluate a set of diketide precursors based on the
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Figure 2. Screening of diketide analogues in a single colony assay. (a) Synthetic route for diketide production (see Supplementary Methods for
details). (b) Diketide library. Compounds 2a−2m, 5a−b were used in an initial screen, while compounds 2n−2r were subsequently assayed. None of
these diketides themselves had detectable bioactivity. Table S2 illustrates the potential macrolide products of feeding each diketide. (c)
Representative petri dish from the initial screening of compound 2i. Dark halos of growth inhibition can be seen in the B. subtilis overlaid lawn,
where E. coli colonies capable of producing a bioactive compound from the provided precursor had been grown. For the purposes of this work, this
was deemed a strong halo. The table summarizes the halo strength from each diketide, classified as a strong, weak, or nonexistent halo. (d) Results
from the secondary screen of C7-, C8-, and C9-diketides. The readout of this assay is the minimum amount (% v/v) of a 24-h fermentation culture
medium that causes growth inhibition of B. subtilis.
natural diketide 2a, and 2c, its chain-extended analogue, were
the only compounds in which this portion of the precursor was
altered. The lack of a growth inhibition halo upon feeding of
these latter two diketides is consistent with their expected
reduced level of incorporation into the biosynthetic pathway14
and the fact that the macrolide ring conformation is highly
conserved in the crystal structures of most ribosome-bound
macrolides.22,23
Compounds 5a and 5b were also examined, because previous
studies showed that these α,β-unsaturated thioesters are
processed in unusual ways by DEBS (Scheme S2).24 Whereas
5a is recognized as a triketide, presumably due to the syn-
relationship between the methyl and hydroxyl groups, 5b is
processed as a diketide that is transformed into a 16-membered
ring. Perhaps not surprisingly, neither compound gave a
detectable halo, presumably because both aglycones are poorly
recognized by the necessary glycosyl transferases.
The tolerance of this antibiotic screen for steric bulk was
examined by feeding diketides 2d−2h. The benzylated diketide
2d is known to be transformed by an engineered variant of
DEBS into the corresponding erythromycin D analogue,
although the bioactivity of this compound is unknown.13
Therefore, the purpose of feeding 2d−2h was twofold: to test
whether or not benzylated 4 was produced at appreciable levels
and was sufficiently bioactive to yield a signal, and if so, to
determine whether altered electron density (2e, 2g), steric
features (2h), or flexibility (2f) could improve upon this signal.
Our data revealed that aromatic functionality at this position of
the macrolide is not sufficiently well tolerated to effect
detectable growth inhibition (Figure 2c). Of the 14 unnatural
diketides tested in this initial screen, only 3 resulted in visible
halos: 2i, 2j, and 2k. Of particular interest was the signal
produced by 2i, which was indistinguishable from that
produced by 2a, the natural diketide (Figure 2c). As it is
rather unlikely that 2i would be a better substrate for DEBS
KS2 than 2a, we hypothesized that the corresponding
macrolide analogue had bioactivity at least equivalent to that
of 4a. Also notably, both 2j and 2k, which were shorter than 2i
by a single methylene and possessed zero or one degree of
unsaturation, respectively, produced only weak halos. While
these shorter diketides gave diminished signals, 2l and 2m, the
two initially tested linear precursors longer than 2i, produced
no signal. These results prompted us to examine systematically
whether the strong signal seen for 2i was due to the length
and/or the degree of unsaturation of the pendant arm.
A second-generation set of diketides, 2n−2r, were
synthesized and screened alongside 2a as well as 2i−2k in a
slightly more sensitive (albeit also qualitative) liquid culture
assay (Figure 2d). Two unexpected trends were noted. First,
based on the finding that 2n produced a stronger signal than 2j
or 2k and 2i > 2p > 2o, the bioactivity resultant from feeding
these diketides appeared to be related to their degree of
terminal unsaturation. More importantly, the C8 diketides (2i,
2o, 2p) gave clearly more active products than either the C7 or
C9 analogues. We therefore undertook preparative production
and isolation of selected macrolide analogues in order to
explore their antibacterial properties more rigorously.
Macrolide Analogue Bioactivity. The macrolide 6-
deoxyerythromycin D (4a) and its analogues (4b−4f), were
isolated from a 5 to 20 L culture of E. coli HYL3/pBP130/
pBP175#8/pHL74/pHL80*, as described in the Materials and
Methods. Their bioactivities were examined against a
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representative Gram-positive bacterium Bacillus subtilis (Table
2). These results were, for the most part, consistent with the
occluding passage of the nascent peptide. This causes the
translating ribosome to stall and eventually dissociate from the
mRNA. We therefore compared the efficacy of 1a versus 1b as
inhibitors of translation, using a chloramphenicol acetyltransfer-
ase cell-free translation system.29,30 As shown in Figure 3, the
inhibitory activities of the two macrolides are equivalent.
Table 2. MIC Values for Selected Macrolide Antibiotics in B.
subtilis Culture
Compound
MIC in B. subtilis culture (nM)
1a
1b
4a
4b
4c
4d
4e
4f
25
25
250
500
500
250
125
2,000
>50,000
>50,000
3a
3b
trend observed in the halo assay. Remarkably, compound 4e
was able to inhibit the growth of B. subtilis at a 2-fold lower
concentration than the natural product 4a. Analogue 4d, where
the terminal acetylene was replaced by an olefin, had
comparable bioactivity to that of 4a, whereas the fully saturated
analogue 4c had a 2-fold lower activity. Changing precursor
length also had a marked effect on bioactivity, as evidenced by
the differences in the antibacterial activities of 4b, 4d, and 4f.
Taken together, our findings suggest that substitution at the C-
13 position can have an unexpectedly strong influence on
macrolide antibiotic activity.
Whereas the identification of an orthogonally functionalized
analogue of 4 with improved bioactive properties was
encouraging, due to the absence of the 6-OH and 12-OH
groups, the macrolides considered to this point were an order
of magnitude less active than erythromycin A (1a vs 4a in
Table 2). We therefore produced macrolide 1b, the
erythromycin A counterpart of 4e.
Figure 3. Inhibition of translation by 1a and 1b in a cell-free system.
While the macrolide inhibition mechanism described above
has been well accepted for some time, the structural details of
the macrolide−ribosome interaction have only recently come
to light. With the solution of the crystal structures of many
prokaryotic ribosomes in the past decade, the ability to probe
specific antibiotic interactions through cocrystallization has led
to tremendous improvement in the understanding of the mode
of action of macrolide antibiotics.22,23,28,31,32 Using the available
crystal structure of 1a bound to the E. coli ribosome22 we have
now developed a structural model to rationalize our structure−
activity data by modifying the C-13 substituent of erythromycin
A so as to correspond to 1b. Subsequent minimization of the
bound structure using the MMF94 force field resulted in the
binding model shown in Figure 4.
Several notable features are apparent from this model. First,
despite the complete lack of constraints on the model prior to
minimization, very little alteration of the macrocycle con-
formation is observed (Figure S2). This lack of disruption also
extended to the hydrogen bonds formed by the 3″ hydroxyl and
tertiary amine of the desosamine moiety, both known to be
vital for macrolide function. While all of these key interactions
remain intact, the bound structure of 1b differs from that of 1a
in the orientation of the C-13 substituent. In the case of 1b, this
substituent makes hydrophobic contacts with the wall of the
ribosome exit tunnel. In particular, the terminal acetylene is
predicted to fit into a groove formed by U746 of the 23S RNA
and Lys90 of protein L22. This tight conformational fit might
account at least partially for the trend observed in Table 2 since
a shorter pendent arm, such as that in 4b, would not provide
such a contact. Conversely, an increase in the length of the
substituent such as that in 4f would likely result in a steric clash
with G745.
Because there is not yet an efficient E. coli-based system
capable of direct production of analogues of 1a, we converted
E. coli derived 6-dEB (3a) and its aglycone analogues into fully
decorated macrolide antibiotics using a two-step procedure that
has been described previously (Scheme 1).25
Scheme 1. Conversion of Diketide 2i into 1b via a two-step
procedure
As reported in Table 2, compound 1b had comparable
bioactivity to 1a with an MIC of 25 nM, a 5-fold improvement
on the bioactivity of 4e. As controls, we confirmed that the
aglycones themselves were inactive.
Ribosomal Inhibition. Macrolides act by ribosomal
binding and inhibition of protein biosynthesis.22,26−28 Specif-
ically, 1a and its semisynthetic analogues bind in the
polypeptide exit tunnel of the large ribosomal subunit, thereby
A comparison of the model for 1b bound to the ribosome
exit tunnel with the crystal structure of telithromycin bound to
the same site is shown in Figure 4b. Telithromycin, a recently
launched ketolide antibiotic, lacks the cladinose sugar character-
istic of all other derivatives of 1. Its cocrystal structure, in
concert with RNA footprinting experiments, has suggested that
much of the improved binding affinity could result from the
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position would lead to extension of the pendant arm, a property
shown here to diminish bioactivity. Previous reports, however,
demonstrate that while the steric constraint reported here holds
for linear, unsaturated extensions of the C-13 position, the use
of flexible alkyl linkers for appending aromatic heterocycles can
lead to greatly improved bioactivity. For example, RNA
footprinting studies of one such compound, K-1325 6 (Figure
5a) bound to the ribosome, have demonstrated that aromatic
Figure 4. Binding of 1b. (a) Model of macrolide 1b docked into the
ribosome binding site of 1a. (b) Modeled structure of 1b (orange)
compared with the experimental crystal structure of telithromycin
(blue). The ribosomal residues interacting with the pendant arms are
labeled.
Figure 5. Ketolide binding. (a) Structures of select ketolides. (b)
Structure of ribosome bound 1b compared to residues crystallo-
graphically shown to interact with telithromycin and those chemically
protected by K-1325.
strong π−π stacking interaction between the aryl arm appended
to the 11−12 cyclic carbamate and the bridge formed by U2609
and A752. Our model for the ribosomal binding of 1b suggests
that the affinity could be further improved through interaction
with the wall of the ribosome exit tunnel. The plethora of facile
modifications available via the orthogonally reactive alkyne
makes this an attractive and feasible goal.
heterocycles appended by alkyl linkers to the C-13 position of a
ketolide led to the strong protection of U790 and A789, two
residues within Domain II of the 23S rRNA (Figure 5b).36
These residues are further down the ribosome exit tunnel,
distinct from but proximal to the known interactions of
telithromycin 7, suggesting that modifications to the C-13
position lead to unique constructive interactions as compared
to existing macrolides and ketolides. Figure 5b also serves to
demonstrate the appreciable volume in the ribosome exit
tunnel accessible for further small molecule binding inter-
actions. Taken together, this suggests that while we observe a
steric constraint in the series of compounds examined here, this
constraint is not absolute and does not obviate the potential of
1b as a lead for further macrolide development.
Additionally, the installation of triazole-linked aromatic
groups is also promising, as this type of functionality on the
western portion of the ketolide scaffold is extremely beneficial
to bioactivity, as demonstrated by Solithromycin 8 (Figure 5a),
a ketolide currently entering phase II clinical development.37 In
the context of these results, 1b represents a promising lead,
which will facilitate future medicinal chemistry efforts by
allowing the exploration of a position on the macrolide scaffold
that has, to this point, been varied only to a very limited extent.
DISCUSSION
■
Since the discovery of multimodular polyketide synthases,33,34
biosynthetic engineers have sought to exploit the assembly line
logic of these systems for the production of new and improved
antibiotics. Indeed, promising new macrolide antibacterial
agents have been prepared through combinations of bio-
synthetic engineering and medicinal chemical approaches (for
example, see Krokidis et al., 201035). Here we have reported the
discovery of 1b, an orthogonally functionalized, equipotent
analogue of the widely used macrolide erythromycin A that is
directly derived from a fermentation source. In the process, we
have uncovered an unexpectedly stringent steric constraint in
the portion of the ribosome exit tunnel that accommodates the
C-13 ethyl group of 1a.
The initial motivation for producing bioactive macrolides
possessing terminal unsaturation by precursor-directed biosyn-
thesis was the development of a system to facilitate the use of
medicinal chemistry for the exploration of the C-13 position.
Taken alone, the steric constraint reported here would suggest
that 1b does not fulfill this goal, as any modifications of this
CONCLUSION
Reported here is the discovery of 1b, an equipotent and
orthogonally functional macrolide antibiotic accessible by
■
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followed by extraction with EtOAc (2 × 1 L per liter culture), drying
over Na2SO4, and solvent removal in vacuo. Partial purification was
performed on a silica gel column (10−60% EtOAc in pentane, product
Rf ≈ 0.5 in 50% EtOAc in pentane). This resulted in a semipure yellow
oil in which the presence of the 6-dEB analogue was confirmed by
NMR.
fermentative means as a promising lead compound for
antibiotic development. This discovery serves to reinforce the
utility of an engineered E. coli based system for rapid and
sensitive screening of new macrolide antibiotics, while also
demonstrating the power of engineered biosynthesis for the
production of new starting materials for anti-infective medicinal
chemistry.
Conversion of the aglycone into the final antibiotic was performed
by microbial transformation in S. erythraea A34. All incubations of this
strain were performed at 30 °C. Initially, this strain was streaked on a
plate of R5 media and left to grow for 72 h. Mycelia were collected and
used to inoculate cultures in liquid R5 which were incubated with
shaking at for a further 72 h. These cultures were used to inoculate
third stage cultures in V2 media (20% v/v), which were left to shake
for a further 96 h. V2 cultures were used to inoculate the production
culture in an F1 medium (15% v/v). Cultures were incubated for 94 h
prior to feeding of the desired 6-dEB analogue (∼25 mg L−1 fed as a
50 mg mL−1 solution in DMSO). The cultures were monitored daily.
When antibiotic production had ceased (5−7 days), the supernatant
was clarified by centrifugation (2 h at 4420 g), basified to pH 11−12
with 2 M NaOH, and extracted with EtOAc (3 × 500 mL per 500 mL
of culture).
Compound purification was performed by HPLC with an RX-C8
column (9.4 mm × 250 mm) from Agilent Technologies (CH3CN
gradient, 0.1% NH4OH, 5% for 5 min, 5−80% in 150 min, 80−95% in
10 min). The compound identity was verified by NMR (Table S4) and
high-resolution mass spectrometry (Table S3)
Measurement of MIC in B. subtilis Culture. MIC values were
determined through serial dilution in 96-well plates. Compounds are
dissolved as 20 μM solutions in DMSO and added to a dilute (0.1% v/
v overnight culture) B. subtilis culture (1 μM, 5% DMSO v/v). A 2-
fold serial dilution down a 96-well plate was performed, and the plate
was left to incubate at 30 °C for 20 h. The MIC was defined as the
lowest concentration of drug that prevented the OD490 of a well from
reaching 1.00 (at saturation OD490 ≈ 0.2).
MATERIALS AND METHODS
■
Materials. All solvents used for extraction and purification were
ACS grade purchased from Fisher scientific. The synthetic procedures
used to prepare diketides 2a−2r have been described previously12,38
(Figure 2a and Scheme S1). These syntheses were performed using
chemicals of the highest available quality from Sigma-Aldrich.
General Methods. All cultures grown in LB broth were
supplemented with antibiotics as required at the following
concentrations: 100 mg L−1 carbenicillin (Teknova, Hollister, CA),
50 mg L−1 kanamycin (IBI Scientific, Peosta, IA), 34 mg L−1
chloramphenicol (Sigma-Aldrich, St Louis, MO), and 50 mg L−1
streptomycin (Santa Cruz Biotechnology, Santa Cruz, CA). Non-
standard media used in the growth of S. erythraea (V1, F1) are defined
in Table S1. Unless otherwise noted, NMR data were collected on a
Varian 600 MHz spectrometer (Agilent Technologies, Santa Clara,
CA) while HR-MS data were collected using ESI-MS in an Exactive
benchtop Orbitrap UPLC-MS (Thermo Scientific, Waltham, MA).
Screening of Diketide Library. Cultures of E. coli HYL3/
pBP130/pBP175#8/pHL74/pHL80* were grown in LB media
overnight to a stationary phase. These cultures were diluted 1000×
with LB broth, and 100 μL were plated on cellophane covered LB agar
containing 1 mg mL−1 sodium propionate and 10−100 μM of the
diketide to be tested. These plates were left to incubate at 30 °C for 60
h. The cellophane was then removed, and soft agar containing B.
subtilis was overlaid. After overnight incubation, the plates were
analyzed for halos of growth inhibition where individual colonies of E.
coli had been located (Figure 2c).
Production of 6-Deoxyerythromycin D Analogues in E. coli.
Cultures of E. coli HYL3/pBP130/pBP175#8/pHL74/pHL80* were
grown in LB broth at 37 °C overnight to a stationary phase. These
were then used to inoculate larger scale cultures in LB broth (1% v/v),
which were grown at 37 °C to OD600 = 0.5. The cell pellets were
collected by centrifugation (15 min @ 4420g) and resuspended at 4
°C in 5% of the original volume of LB media. Cultures were induced
by addition of 200 μM β-D-1-thiogalactopyranoside (IPTG) along with
the addition of 2.5 g L−1 sodium propionate and the desired diketide at
100 μM. This production culture was left to shake at 30 °C for 72 h.
Following this 30 °C incubation, the supernatant was clarified by
centrifugation (1 h @ 4420g), and 2 M NaOH was added to bring the
supernatant pH to 11−12. Extraction with EtOAc (3×), drying over
Na2SO4, and removal of solvent in vacuo resulted in a yellow oil.
Initial purification was performed with acid−base back extraction.
The yellow oil was suspended in 10 mL of H2O, acidified to pH = 2
with 2 M HCl, and extracted with Et2O (5 × 10 mL). The pH was
then adjusted to pH = 12 with 2 M NaOH and extracted with EtOAC
(5 × 10 mL). The EtOAc extract was then dried over Na2SO4 and the
solvent was removed in vacuo, yielding a semipure macrolide.
Final purification was performed by HPLC with an RX-C8 column
(9.4 mm × 250 mm) from Agilent Technologies (CH3CN gradient,
0.1% formic acid, 0−25% in 10 min, 25−40% in 80 min, 40−95% in 10
min). The compound identity was verified by NMR (Table S5) and
high-resolution mass spectrometry (Table S3).
Measurement of Translation Inhibition. The IC50 values
reported for translation inhibition were measured using a modified
version of the PURExpress Δ Ribosome in vitro protein synthesis kit
(New England BioLabs, Ipswich MA, #E3313). The provided stock
ribosomes were diluted to 1 μM with ribosome dilution buffer (RD
Buffer: 20 mM HEPES, pH 7.6, 10 mM Mg(OAc)2, 30 mM KCl, 7
mM β-mercaptoethanol). Each reaction was performed on a 10 μL
scale with the following components: 3.3 μL of Solution A, 1 μL of
factor mix, 0.2 μL of murine RNase inhibitor (NEB #M0314S), 1.5 μL
of RD Buffer, 1 μL of ribosome solution (final concentration of 100
nm), 1 μL of nuclease-free water, and 0.5 μL of an antibiotic solution
in DMSO (final concentration of 0.1 nM−25 μM). The reactions were
initiated by the addition of 80 ng of pUCCAT9L,39 a plasmid
containing the gene for chloramphenicol acetyltransferase (CAT)
under the control of a T7 promoter. The reactions were incubated at
37 °C for 2 h, and the production of CAT was assayed
spectrophotometrically as follows: 10 μL of reaction mixture were
added to 1 mL of assay buffer (100 mM Tris-HCl pH 7.6, 100 μM
acetyl CoA, and 0.4 mg mL−1 5,5′-dithiobis(2-nitrobenzoic acid)). The
addition of chloramphenicol to a concentration of 100 μM began the
reaction, and the change in absorbance at 412 nM was monitored for 5
min.30,40
Production of Analogues of Erythromycin A. These
compounds were produced using a two-step procedure (Scheme 1).
Cultures of E. coli HYL3/pBP130/pBP175#8 were grown in LB media
with antibiotics at 37 °C overnight to stationary phase. 1 L cultures in
LB media with antibiotics were then inoculated (1% v/v) and left to
grow to OD600 0.5 at 37 °C. These cultures were then induced with
200 μM IPTG concomitant with the addition of 200 μM diketide and
2.5 g L−1 sodium propionate and left to shake at 18 °C for 72 h.
Clarification of the supernatant by centrifugation (1 h at 4420 g) was
Modeling of Ribosomal Binding. The modeling was based upon
the recently solved crystal structure of erythromycin A bound to the
ribosome of E. coli22 (PDB ID code 3OFR). The structure of bound 1
was extracted and imported to VIDA (OpenEye scientific software,
Santa Fe, NM), where carbon-15 was modified to yield 1b. This
modified structure was then returned to its original biological context,
and the modified bound structure was minimized with the MMF94
force field using LigandScout (inte:ligand, Maria Enzersdorf, Austria).
12264
dx.doi.org/10.1021/ja304682q | J. Am. Chem. Soc. 2012, 134, 12259−12265

Journal of the American Chemical Society
Article
(11) Kirschning, A.; Taft, F.; Knobloch, T. Org. Biomol. Chem. 2007,
5, 3245−3259.
ASSOCIATED CONTENT
* Supporting Information
Spectroscopic and analytical data, further modeling, and
detailed methods. This material is available free of charge via
the Internet at http://pubs.acs.org.
■
S
(12) Ashley, G. W.; Burlingame, M.; Desai, R.; Fu, H.; Leaf, T.;
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AUTHOR INFORMATION
Corresponding Author
khosla@stanford.edu
■
(15) Goss, R. J. M.; Hong, H. Chem. Commun. (Camb) 2005, 3983−
3985.
(16) Desai, R. P.; Rodriguez, E.; Galazzo, J. L.; Licari, P. Biotechnol.
Prog. 2004, 20, 1660−1665.
Present Address
∞Stanford Genome Technology Center, Department of
Biochemistry, Stanford University School of Medicine,
Stanford, CA 94305.
(17) Frykman, S.; Leaf, T.; Carreras, C.; Licari, P. Biotechnol. Bioeng.
2001, 76, 303−310.
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(19) Lee, H. Y.; Harvey, C. J. B.; Cane, D. E.; Khosla, C. J. Antibiot.
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Notes
The authors declare no competing financial interest.
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(21) Schnarr, N.; Chen, A.; Cane, D.; Khosla, C. Biochemistry 2005,
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ACKNOWLEDGMENTS
■
This research was supported by grants from the National
Institutes of Health (GM 087934 to C.K., GM 022172 to
D.E.C., GM 51266 to J.D.P., and GM 062868 to V.S.P.).
C.J.B.H. is a recipient of a Predoctoral Fellowship from the
Center for Molecular Analysis and Design (CMAD) at Stanford
University.
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