DOI: 10.1002/chem.201405009
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Metabolome Analysis
Subclass-Specific Labeling of Protein-Reactive Natural Products
with Customized Nucleophilic Probes
Georg C. Rudolf, Maximilian F. Koch, Franziska A. M. Mandl, and Stephan A. Sieber*[a]
Abstract: Natural products represent a rich source of bioac-
tive compounds that constitute a large fraction of approved
drugs. Among those are molecules with electrophilic scaf-
folds, such as Michael acceptors, b-lactams, and epoxides
that irreversibly inhibit essential enzymes based on their cat-
alytic mechanism. In the search for novel bioactive mole-
cules, current methods are challenged by the frequent redis-
covery of known chemical entities. Herein small nucleophilic
probes that attack electrophilic natural products and en-
hance their detection by HPLC-UV and HPLC-MS are intro-
duced. A screen of diverse probe designs revealed one com-
pound with a desired selectivity for epoxide- and maleimide-
based antibiotics. Correspondingly, the natural products
showdomycin and phosphomycin could be selectively tar-
geted in extracts of their natural producing organism, in
which the probe-modified molecules exhibited superior re-
tention and MS detection relative to their unmodified coun-
terparts. This method may thus help to discover small, elec-
trophilic molecules that might otherwise easily elude detec-
tion in complex samples.
Introduction
ed the targets of these specialized scaffolds, such as showdo-
mycin, a potent maleimide antibiotic with activity against
Staphylococcus aureus and multiple-drug-resistant derivatives.[6]
The molecule inhibits pathogen growth by targeting two es-
sential cell-wall-biosynthesis enzymes through covalent modifi-
cation of a cysteine residue. Intrigued by this mode of action,
we became interested to explore the subclass of reactive elec-
trophiles in greater detail by establishing a directed isolation
procedure.
Nature provides a rich source of bioactive compounds with
a huge diversity of pharmacological activities. In medicine,
these compounds constitute up to 50% of all currently applied
drugs and serve as a great source for privileged structures in
biomimetic-compound screening programs.[1] These structures
have been optimized during evolution to bind and inhibit es-
sential cellular proteins and enzymes. Whereas a large fraction
of compounds facilitate this inhibition by reversible interaction,
a certain subclass of natural products is designed to attach co-
valently with the active site.[2] This subclass of so-called pro-
tein-reactive natural products adopts a precise reactivity and
specificity profile that ensures target selectivity. One advantage
of covalent inhibition is, for example, an increase in potency
due to a prolonged duration of the corresponding biological
effect.[3] In fact, many natural-product-derived drugs, such as b-
lactam antibiotics and aspirin, rely on this covalent-inhibition
principle.[4] Scaffolds that exhibit protein reactivity are diverse
and include several prominent electrophilic moieties, such as
Michael acceptors, b-lactams, b-lactones, or epoxides. Some
microorganisms produce an additional set of electrophilic scaf-
folds, such as maleimides.[5] In previous studies, we investigat-
Although technological advancements over the last decades
have enabled the discovery and structure elucidation of many
compounds by NMR spectroscopy and mass spectrometry, the
field is confronted with several fundamental challenges. First,
the extraction, fractionation, and isolation of natural products
requires very sensitive analytical methods in order to detect
low-abundance compounds. Second, the physical properties of
some natural products, such as showdomycin (hydrophilic,
poorly UV active), are not compatible with established screen-
ing procedures and may evade detection. Third, a large
number of compounds are usually detected, but only a fraction
can be analyzed by detailed follow-up studies. Selection crite-
ria have to be established, for example, based on desired activ-
ities, in order to choose promising compounds.
Herein, we introduce nucleophilic tags that selectively bind
the electrophilic moieties of natural-product antibiotics
(Figure 1). We synthesized several thiol, hydroxy, and amine
nucleophiles and screened these molecules against various
protein-reactive compounds and natural products. We found
a suitable reactivity and selectivity of electron-rich, thiol-substi-
tuted naphthalenes with showdomycin and phosphomycin.
Application of the probe in the corresponding producer-strain
extracts facilitated UV detection of tagged natural products at
a unique wavelength of 303 nm, which significantly reduced
[a] Dr. G. C. Rudolf,+ M. Sc. M. F. Koch,+ M. Sc. F. A. M. Mandl,
Prof. Dr. S. A. Sieber
Center for Integrated Protein Science CIPSM
Institute of Advanced Studies IAS, Department Chemie
Lehrstuhl fꢀr Organische Chemie II, Technische Universitꢁt Mꢀnchen
Lichtenbergstrasse 4, 85747 Garching (Germany)
[+] These authors contributed equally to this work.
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/chem.201405009.
Chem. Eur. J. 2015, 21, 1 – 8
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ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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