Biocatalytic synthesis of pikromycin, methymycin, neomethymycin, novamethymycin, and ketomethymycin

Article abstract of DOI:10.1021/ja404134f

A biocatalytic platform that employs the final two monomodular type I polyketide synthases of the pikromycin pathway in vitro followed by direct appendage of d-desosamine and final C-H oxidation(s) in vivo was developed and applied toward the synthesis of a suite of 12- and 14-membered ring macrolide natural products. This methodology delivered both compound classes in 13 steps (longest linear sequence) from commercially available (R)-Roche ester in >10% overall yields.

Full text of DOI:10.1021/ja404134f

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Article
Biocatalytic Synthesis of Pikromycin, Methymycin,
Neomethymycin, Novamethymycin, and Ketomethymycin
Douglas Andrew Hansen, Christopher M Rath, Eli Eisman, Alison Narayan, Jeffrey
D. Kittendorf, Jonathan David Mortison, Yeo Joon Yoon, and David H. Sherman
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/ja404134f • Publication Date (Web): 18 Jun 2013
Downloaded from http://pubs.acs.org on June 19, 2013
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Biocatalytic Synthesis of Pikromycin, Methymycin,
Neomethymycin, Novamethymycin, and Ketomethymycin
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Douglas A. Hansen,α† Christopher M. Rath,† Eli B. Eisman,† Alison R. H. Narayan,† Jeffrey D.
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Kittendorf,†β Jonathan D. Mortison,π† Yeo Joon Yoon,° and David H. Sherman*α†π‡
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α
Department of Medicinal Chemistry, University of Michigan, Ann Arbor, MI 48109
π
Department of Chemistry, University of Michigan, Ann Arbor, MI 48109
‡
Department of Microbiology & Immunology, University of Michigan, Ann Arbor, MI 48109
βAlluvium Biosciences, Ann Arbor, MI 48108
†
Life Sciences Institute, University of Michigan, Ann Arbor, MI 48109
°Division of Nano Sciences, Ewha Womans University, Seoul 120ꢀ750, South Korea
AUTHOR INFORMATION
*Corresponding Author
davidhs@umich.edu
Present Addresses
C.M.R. – Novartis Institute for Biomedical Research, Emeryville, CA 94608
J.D.M. ꢀ Harvard University, Cambridge, MA 02138
Keywords: Chemoenzymatic Synthesis, Biocatalysis, Pikromycin, Macrolides, Polyketide Synthase
Graphical Abstract:
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Abstract
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A biocatalytic platform that employs the final two monomodular type I polyketide synthases
(PKS) of the pikromycin pathway in vitro followed by direct appendage of Dꢀdesosamine and
final CꢀH oxidation(s) in vivo was developed and applied toward the synthesis of a suite of 12ꢀ
and 14ꢀmembered ring macrolide natural products. This methodology delivered both compound
classes in thirteen steps (longest linear sequence) from commercially available (R)ꢀRoche ester in
>10% overall yields.
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Introduction
Biocatalysis offers tremendous potential to complement conventional methods in synthetic
chemistry.1 Traditionally used for discrete chemical reactions, recent chemoenzymatic advances
have enabled cascades that streamline synthesis by reducing step counts, increasing yields, and
minimizing waste generation.2ꢀ3 While such processes are becoming increasingly prevalent, they
are typically employed in the production of relatively simple synthons for starting material or
later incorporation into mature molecules. Conversely, the biosynthetic machinery involved in
the generation of complex secondary metabolites could theoretically be leveraged at late stages
of a synthesis to achieve similar improvement in step counts and overall yields. Specifically,
modular type I polyketide synthases (PKSs) are of interest as a single polypeptide chain is
capable of performing multiple regioꢀ and stereospecific reactions while stabilizing otherwise
labile oligoketide substrates.
Pikromycin (1) is a PKS derived macrolide antibiotic belonging to the ketolide subclass,
originally isolated in 1950.4ꢀ5 The subsequent discovery of erythromycin A (2) and its success as
a broad spectrum antibiotic initiated drug discovery efforts through isolation and semiꢀsynthetic
modification,6 with 3rd generation macrolides treating front line infections almost sixty years
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later. Through onꢀgoing strain improvement programs, fermentationꢀderived erythromycin
provides the starting material for a number of clinically relevant antibiotics including 2nd
generation clarithromycin, azithromycin and the 3rd generation ketolides telithromycin (4),
cethromycin, and solithromycin. Despite the availability of erythromycin as a commodity fineꢀ
chemical, rigorous structure activity relationship (SAR) studies of the ketolide class are
constrained by the inherent limitations of semiꢀsynthesis using this complex natural product. For
example, methods to directly remove, add, or modify alkyl groups in the macrolactone ring
remain elusive with current synthetic technology. In contrast, de novo production of ketolides
though total synthesis offers the opportunity to explore previously inaccessible chemical space
for discovery of new antiꢀinfective agents.7ꢀ10 At present, this remains a formidable goal as
conventional synthetic approaches understandably suffer from practical limitations, even in the
case of natural macrolides or their simplified macrolactone11ꢀ12 skeletons. Total syntheses have
been previously reported for erythromycin13ꢀ15 and pikromycin,16 however, the innate complexity
of these compounds requires high step counts, resulting in low overall yields. An underexplored
route to macrolactone scaffolds employs PKS enzymes in a controlled in vitro environment.
Development of biocatalytic approaches using modular PKSs has the potential to provide ready
access to natural macrolactones, and increase our functional understanding of these complex
proteins toward creating libraries of novel macrolides in an efficient manner.
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Bacterial PKS pathways mediate the extension and processing of polyketide chains in a
stepwise stereospecific manner, typically adding two (acetate) to three (propionate) carbon atoms
per module.17 In the case of propionate extension, methylmalonylꢀcoenzyme A (MMꢀCoA, 5)
serves as a masked enolate of the extender unit. The acyl transferase (AT) domain selects for
methylmalonate and transfers this subunit to the phosphopantetheine (Ppant) arm of the acyl
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carrier protein (ACP), where the ketosynthase (KS) domain accepts a growing chain elongation
intermediate from the previous module and catalyzes a decarboxylative Claisen condensation
placing the extended chain onto the ACP for reductive tailoring. The ketoreductase (KR),
dehydratase (DH), and enoyl reductase (ER) domains modify the chain at the β position,
(depending on the module and the presence of processing domains), while the terminal
thioesterase domain (TE) catalyzes cyclization of the macrolactone product.18ꢀ21
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Streptomyces venezuelae ATCC 15439 produces a suite of 12ꢀ and 14ꢀmembered ring
macrolide antibiotics with lateꢀstage PKS modules (e.g. PikAIII and PikAIV) in the biosynthetic
pathway demonstrating substrate flexibility.22ꢀ23 The unnatural PikAIIIꢀTE fusion protein
produces the 12ꢀmembered ring macrolactone 10ꢀdeoxymethyonolide (10ꢀdml, 7) from the Nꢀ
acetylcysteamine thioester (NAC) (21) of the natural Pik pentaketide (6). Incubation with both
PikAIII and PikAIV monomodules results in two propionate extensions followed by
macrocyclization to yield the 14ꢀmembered ring aglycone narbonolide (nbl, 8).24 Although
considerable progress has been made in microꢀscale analysis of modular PKS systems using
advanced polyketide chain elongation intermediates,19,23ꢀ24 we were motivated to explore the
possibility of a more efficient and scalable biocatalytic system.
From a practical perspective, preparative scale (>1 mmol) biocatalysis by type I modular
PKSs has been impeded by a number of technical challenges including (i) slow loading of
substrate onto the KS (ii) stoichiometric and costꢀprohibitive cofactors (iii) the large, fragile
nature of PKS proteins. Accordingly, we sought to address these challenges and develop
multifunctional PKS enzymes as options in the toolbox of synthetic chemistry. Finally, tailoring
of the macrolactones to macrolides was envisioned through whole cell biotransformation to
append Dꢀdesosamine and perform CꢀH oxidation(s) by the PikC monooxygenase (Scheme 2).
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Results
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Synthesis of Pik Pentaketide. Retrosynthetically, we envisioned disconnection at the enone
double bond, simplifying the necessary building blocks to α,β unsaturated ketone 18, a type II
olefin,25 matched to undergo cross metathesis with type I fragment 19. Synthesis of enone 18
began with TBS protection of (R)-Roche ester (15),26ꢀ27 which was subsequently reduced and
iodinated to yield 16. Alkylation with (S,S)ꢀpseudoephedrine propionamide,28 displacement of
the auxiliary with nꢀBuLi and RuO4 oxidation29 furnished saturated acid 17. Conversion of 17 to
enone 18 was envisioned through oxidation of the corresponding silyl enol ether.30 Trapping the
kinetic enolate of 17 with LDA/TMSCl at ꢀ78 °C proved problematic as epimerization was
observed through a presumed silyl ketene intermediate. Employment of LHMDS/TMSCl at ꢀ78
°C facilitated smooth trapping of the enolate31 and subsequent IBX oxidation of the crude silyl
enol ether yielded compound 18.30 This twoꢀstep sequence initially demonstrated poor
reproducibility but was improved through quenching excess LHMDS/TMSCl with a drop wise
addition of acetone prior to an aqueous quench to provide 18 in good yield.32 Common
metathesis conditions were evaluated with 1.5 equivalents of silyl ether 19 and 3 mol% of HGꢀ
II,33 where the highest yields were achieved when the reaction was run neat at 50 °C for 12
hours. Cleavage of the silyl ether with hydrogen fluoride in acetonitrile afforded the unactivated
pentaketide 5. With the desired substrate 20 in hand, we considered alternatives to NAC
thioesters in an effort to increase efficiency of loading target KS domains to initiate PKS chain
extension.
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Synthesis and Evaluation of Pentaketide Thioesters. We envisioned four Pik pentaketide
substrates of varying reactivity and aqueous solubility for direct PKS loading. NAC 21 was
synthesized as described previously,24 while 22-24 were generated from corresponding
disulfides.34 Activated pentaketides 21-24 were incubated with purified PikAIIIꢀTE, extender
unit, and hydride donor under nonꢀoptimized conditions (Figure 2). QTOFꢀLC/MS analysis of
50 ꢁL reaction mixtures after one hour revealed significant differences in conversion between
aryl thioesters and traditionally employed NAC thioesters. Thiophenol activated 22 showed a 4ꢀ
fold increase in conversion to 10ꢀdml while 23 and 24 demonstrated 2ꢀfold increases.
Furthermore, 22 showed no detectable hydrolysis in the absence of enzyme or in the presence of
denatured enzyme, while both 23 and 24 underwent detectable hydrolysis. Compounds 21 and
22 were further analyzed via HPLC calibrated against a 10ꢀdml standard curve providing
absolute quantification along the reaction time course. Having demonstrated the superiority of
thiophenolꢀactivated pentaketide 22 to the previously employed 21, attention was turned toward
exploring alternatives to superstoichiometric quantities of MMꢀCoA (5) and NADPH as essential
coꢀfactors.
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Evaluation of Superstoichiometric Co-Factors. Replacement of MMꢀCoA is requisite to attain
a practical chemoenzymatic platform due to its cost (>$1000/25 mg)35 and poor atom economy.
Khosla and coworkers reported methylmalonyl Nꢀacetylcysteamine (MMꢀNAC, 25) as a
surrogate for MMꢀCoA in the erythromycin pathway (DEBS) albeit at higher concentrations.36
In our hands, gram scale synthesis of MMꢀNAC was hindered by difficulties in separation from
sideꢀproducts due to the extreme hydrophilicity of the compound. An alternative twoꢀstep route
proceeded through compound 27, a crystalline solid available in a single step from methyl
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Meldrum’s acid (26).37 Transthioesterification38 in aqueous sodium bicarbonate followed by
acidification with cation exchange resin, washing of the aqueous phase with CH2Cl2 and
subsequent lyophilization provided rapid access to decagrams of 25. The stoichiometric
dependence of MMꢀNAC was examined in a stop point assay after 4 hours with both PikAIIIꢀTE
and PikAIII/PikAIV (Figure 3). While enzymatic NADPH recycling was potentially problematic
in the presence of electrophilic thioesters, the glucoseꢀ6ꢀphosphate/glucoseꢀ6ꢀphosphate
dehydrogenase system39 proved sufficiently robust to be employed without modification.
Ultimately, NADP+ could be recycled effectively at 10 mol% relative to pentaketide substrate.
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Semi-Preparative and Preparative PKS catalysis. Following coꢀfactor optimization, we
examined the scalability of the in vitro macrolactoneꢀforming PKS reactions. Scaleꢀup of
analytical 50 ꢁL reactions to semiꢀpreparative 200 mL reactions (0.2 mmol 22) while
maintaining identical concentrations and stoichiometry provided 10ꢀdml (7) in 53% yield and
acetylꢀnarbonolide (28) in 47% yield, respectively after silica gel chromatography (Figure 4).
The observed precipitation of modular PKS proteins during the reaction spurred a final
evaluation of conditions, and transitioning from purified protein to crude cell lysate while
maintaining identical concentrations and stoichiometry demonstrated modest improvement
generating 10ꢀdml in 62% yield or acetylꢀnarbonolide in 55% yield. Increased enzymatic
stability enabled subsequent reactions to be conducted at a fourꢀfold greater concentration while
decreasing the stoichiometry of MMꢀNAC by a factor of two, affording 10ꢀdml in 66% yield or
acetylꢀnarbonolide in 55% yield. With final reaction conditions in hand, preparativeꢀscale
catalysis with 0.5 g of Pik pentaketide 22 (1.43 mmol) generated 10ꢀdml in 60% yield, and
acetylꢀnarbonolide in 49% yield.
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Biotransformation of Macrolactones to Macrolides. As the final step toward targeted
macrolides, we evaluated whole cell biocatalysis for introduction of Dꢀdesosamine and the final
p450ꢀmediated CꢀH oxidation(s). Engineered variants of Streptomyces venezuelae ATCC 15439
designated strains DHS200140 and YJ11241 were assayed for their ability to transform
macrolactones to macrolides (Figure 5). Observed variation in levels of bioconversion from
culture to culture (normalized by time and based on initial culture inoculation to addition of
macrolactone) motivated studies to isolate and evaluate growth phase dependence. Addition of
the macrolactone substrate to high OD600 DHS2001 cultures resulted in fast initial rates, but
incomplete conversions. By contrast, addition of macrolactone to preꢀlog phase cultures of
resulted in highest overall conversion to macrolide products. While conversion of 10ꢀdml to
methymycins (11-14) was undetectable with either strain, this was overcome by addition of
acetylꢀnarbonolide (28)42 to afford >99% conversion to target macrolides over 48 hours.
Addition of acetylꢀnarbonolide to narbonolide biotransformations increased rates and overall
conversion (>99%) in a similar manner. Bioconversion of 10ꢀdml with DHS2001 predominately
afforded neomethymycin and methymycin, with low levels of novamethymycin isolated as well.
S. venezuelae strain YJ112 mediated conversion of 10ꢀdml with superior access to higher
oxidation states including increased levels of novamethymycin, and enabled discovery of the
new macrolide ketomethymycin arising from a presumed dihydroxylation at the Cꢀ12 position.
Addition of narbonolide to S. venezuelae strain YJ112 provided pikromycin in 72% yield.
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Discussion
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Modern synthesis of polyketide natural products is routinely enabled by chiral auxiliary
methodology that predictably forms CꢀC bonds in a stereoselective, biomimetic manner.
Prominent examples, including Myers alkylation28,43 and Evans aldol44 protocols and variants
thereof, are commonly employed in constructing these complex secondary metabolites. Indeed,
a recent synthesis of narbonolide [>19 steps (longest linear sequence) and <3% overall yield]12
and related macrolactone 6ꢀdEB [22 steps (longest linear sequence) and 7.8% overall yield]45
relied heavily on these “practical laboratory emulations of the series of acylation/reduction
reactions performed by polyketide synthases.”46 For example, an aldol reaction with Nꢀ
propionylꢀ2ꢀoxazolidone and propionaldehyde mimics one round of KS catalyzed propionate
extension and KR reduction. In this approach, each new CꢀC bond formed requires further steps
to protect installed functionality, displace the auxiliary, and oxidize or otherwise activate the
chain for the next CꢀC bond construction. The thiophenol activated Pik pentaketide 22 was
synthesized in this manner [11 linear steps and 38% yield], demonstrating the typical strengths
(>90% yield per operation) and shortcomings (numerous redox and protecting group
manipulations) of this approach. PKS catalysis offers an attractive alternative to synthetic
emulators, as post CꢀC bond redox steps are performed on the protein with effective stabilization
of labile polyketide chains. Covalent attachment of the chain elongation intermediate to the ACP
eliminates the need for protective groups prior to subsequent elongation or cyclization.
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Despite the clear potential for synthetic utility and more than two decades of investigation,47ꢀ
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complexity of PKS enzymes. Initial development was complicated by codependent factors that
precluded optimizing individual reaction parameters in a stepwise manner. For example, while
sensitive radioꢀassays have been necessary to compensate for the inefficiency of most in vitro
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PKS reactions and the microꢀscale at which they are conducted, their qualitative readout and
lowꢀthroughput nature make them unsuitable for screening reaction conditions. Fortunately,
increased accessibility to high resolution LC/MS enabled us to avoid use of radioactive
substrates and establish basic parameters (Figure 2) from which to proceed.
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While evaluation of thioester leaving groups by QTOFꢀLC/MS illuminated striking
improvement in substrate conversion and ultimately enabled the development of simple HPLC
assays, initial experiments appeared less than promising. The NAC activated Pik pentaketide 21
is sparingly water soluble despite the polarity imparted by NAC, and substitution to more
hydrophobic thiophenol 22 led to a visible dispersion at 1mM concentrations. Fortunately, loss
of turbidity could be used to visually track the progress of the reaction, with consumption of
starting material rendering a clear solution. The superior conversion of aryl thioesters compared
to conventional NAC thioesters suggests that increased reactivity trumps possible KSꢀPpant
interactions. Of particular note is the apparent preference for the Pik pentaketide substrate to
load the KS active site cysteine over the ACP Ppant arm, as concomitant ACP acylation would
effectively remove the module from participating in catalysis. In retrospect, observed fidelity in
loading the PikAIII KS domain active site cysteine residue is surprising given the demonstrated
efficacy of thiophenol thioesters in loading Ppant arms of PCP domains.49ꢀ52 Ultimately, PKS
mediated conversion of Pik pentaketide proceeded with modest yield to 10ꢀdml [60% yield, 23%
overall from 15] or acetylꢀnarbonolide [49% yield, 18% overall from 15] at preparative scale,
generating ~250 mg of either macrolactone.
Chemical glycosylation of both macrolactones with Dꢀdesosamine53ꢀ54 and subsequent CꢀH
oxidation through PikC monooxygenase have been described,55 though complications obtaining
and attaching Dꢀdesosamine significantly impede this approach. This amino sugar can be
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chemically synthesized in nine steps,54,56 though it is more commonly harvested from
degradation of erythromycin A.57ꢀ58 Utilizing Dꢀdesosamine acquired from degradation requires
a multiꢀstep sequence of protection, activation, glycosylation, and deprotection. Further,
glycosylation of hindered secondary alcohols in either macrolactone has been challenging and
proceeds with moderate yields.16,54
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Alternatively, the direct biotransformation of macrolactones to macrolides presents an
attractive option in converting 10ꢀdml to methymycins (11–14) and narbonolide to pikromycin
(1).59 In this approach, the 10ꢀdml and narbonolide macrolactones are natural substrates for the
Dꢀdesosamine biosynthetic pathway and subsequent PikC oxidation within engineered variants
of S. venezuelae ATCC 15439. Indeed, strain YJ122 efficiently converted crude narbonolide to
pikromycin [72% yield, 13% overall from 15]. Bioconversion of 10ꢀdml to methymycins proved
to be tunable depending on the strain employed, with DHS2001 biotransformation affording
primarily monohydroxylated products [combined 66% yield, 15% overall from 15]. S.
venezuelae strain YJ112 mediated conversion of 10ꢀdml with increased accessibility to
dihydroxylated novamethymycin, and provided access to previously unknown ketomethymycin
[combined 71% yield, 16% overall from 15]. Careful analysis of wild type S. venezuelae ATCC
15439 culture revealed production of a compound having an identical mass and retention time to
synthetic ketomethymycin, suggesting that this compound is indeed a secondary metabolite made
in trace quantities under normal laboratory culture conditions.
Conclusion
Advances in polyketide chain elongation intermediate activation and optimization of in vitro
reaction parameters yielded practical type I PKS biocatalysis for scalable de novo macrolide
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production. The flexibility of the Pik pathway enabled divergent generation of either 10ꢀdml or
narbonolide and optimized whole cell biotransformations effectively tailored macrolactones to
macrolides, with S. venezuelae strain YJ112 enabling access to ketomethymycin bearing a
previously unobserved oxidation state at the Cꢀ12 position. This platform offers practical entry
to natural 12ꢀ and 14 membered ring macrolides from the Pik pathway, and the potential of this
system to produce unnatural macrolides will be reported in due course.
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Acknowledgments
This research was supported by Rackham Merit and American Foundation for Pharmaceutical
Education preꢀdoctoral fellowships (D.A.H.), the Life Sciences Research Foundation (A.R.H.N),
and NIH grant GM076477 and the Hans W. Vahlteich Professorship (to D.H.S.).
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Captions
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Figure 1. Macrolide antibiotics including ketolide 3,60 an intermediate en route to telithromycin.
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from erythromycin A.
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Scheme 1. Retrosynthetic analysis. PikC: P450 catalyzing monoꢀ and dihydroxylation of YCꢀ17
(9) and narbomycin (10) macrolides. DesIꢀVIII: Dꢀdesosamine biosynthetic pathway catalyzing
glycosylation of 10ꢀdeoxymethynolide (7) and narbonolide (8) macrolactones. PikAIIIꢀTE or
PikAIII/AIV: PKS modules capable of converting Pik pentaketide (6) to macrolactones 10ꢀ
deoxymethynolide (7) or narbonolide (8).
Scheme 2. Second generation24 approach to Pik pentaketide.
Figure 2. Synthesis and evaluation of activated Pik pentaketides. Enzymatic reaction
conditions: 1mM Pik pentaketide, 10mM MMꢀNAC (vide infra), 0.5mM NADP+, 2.5mM
glucoseꢀ6ꢀphosphate, 0.5 unit/mL glucoseꢀ6ꢀphosphate dehydrogenase, 8mM 2ꢀvinylpyridine,
and 1ꢁM purified PikAIIIꢀTE, 1ꢀ24 hours. Conversion of 21 and 22 to 10ꢀdeoxymethyonolide
(7) was monitored (HPLC) with data represented as mean ± standard deviation. n=3
Figure 3. Synthesis and evaluation of methylmalonyl Nꢀacetylcysteamine (MMꢀNAC, 25)
Enzymatic reaction conditions: 1mM Pik pentaketide 22, 1ꢀ20mM MMꢀNAC, 0.5mM NADP+,
2.5mM glucoseꢀ6ꢀphosphate, 0.5 unit/mL glucoseꢀ6ꢀphosphate dehydrogenase, 8mM 2ꢀ
vinylpyridine, and 1ꢁM purified PikAIIIꢀTE or PikAIII/PikAIV, 4 hours. Conversion of
pentaketides 21 and 22 to 10ꢀdeoxymethyonolide (6) or narbonolide (7) was monitored (HPLC)
with data represented as mean ± standard deviation. n=3
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Figure 4. Semipreparative and preparative PKS cascade to macrolactones. Enzymatic reaction
conditions: 1mMꢀ4mM Pik pentaketide 22, 10ꢀ20 equiv. MMꢀNAC, 0.1mM NADP+, 2.5ꢀ10mM
glucoseꢀ6ꢀphosphate, 0.5ꢀ2 unit/mL glucoseꢀ6ꢀphosphate dehydrogenase, 1ꢀ4ꢁM purifieda or cell
freeb PikAIIIꢀTE or PikAIII/PikAIV, 4 hours. Narbonolide was acetylated to 28 prior to
chromatography to prevent degradation. Yields are calculated from isolated macrolactone after
chromatography.
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Figure 5. Biotansformation of macrolactones to macrolides. Biotransformation conditions:
macrolactone added to 100 mg/L with 2.5 mg/L acetylꢀnarbonolide (28) when culture reached
OD600 0.1, incubated for 48 hours. methymycin (12) : R1=OH, R2=H, neomethymycin (11) :
R1=H, R2=OH, novamethymycin (14) : R1=OH, R2=OH, ketomethymycin (13) : R1=H, R2=O.
Yields are calculated as the sum of differentially oxidized macrolides after HPLC purification.
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Figure 1:
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Scheme 1:
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Scheme 2.
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Figure 2.
Figure 3.
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Figure 4.
Figure 5.
O
O
O
O
O
HO
R1
NMe2
O
HO
NMe2
O
O
OH
O
O
HO
R2
O
O
7
OH
O
O
H
O
O
O
O
11-14
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1
combined
yield (%)
ketomethymycin
(13)
methymycin neomethymycin novamethymycin
strain
pikromycin
yield (%)
strain
YJ112
(12)
3.5
3.5
(11)
(14)
:
:
:
:
:
:
YJ112
71
66
5
3
1
72
DHS2001
7
1
trace
Associated Content
Supporting Information Available: Full experimental details and spectroscopic data. This
material is available free of charge via the Internet at http://pubs.acs.org/.
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