Subclass-specific labeling of protein-reactive natural products with customized nucleophilic probes

Article abstract of DOI:10.1002/chem.201405009

Natural products represent a rich source of bioactive compounds that constitute a large fraction of approved drugs. Among those are molecules with electrophilic scaffolds, such as Michael acceptors, b-lactams, and epoxides that irreversibly inhibit essent

Full text of DOI:10.1002/chem.201405009

DOI: 10.1002/chem.201405009
Full Paper
&
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)
E-mail: stephan.sieber@tum.de
[+] 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.
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Figure 1. Selective detection of protein-reactive natural products. Metabolic extracts are incubated with a nucleophilic probe based on a naphthalene scaf-
fold. The tagging increases retention on HPLC, UV detection, and analysis by MS. Nu: nucleophile.
the complexity of the secretome samples. Moreover, the at-
tached tag increased the molecular weight and hydrophobicity
of the molecular adducts, which improved their separation by
HPLC and identification by mass spectrometry. The approach is
thus complementary to several excellent previous studies by
the Carlson, Cravatt, Li, and Sello
cleophilic centers. The unique UV-absorption characteristics
(local maximum at around l=303 nm), in combination with
the high hydrophobicity, enhance the identification of small,
hydrophilic natural products in reversed-phase LC separation
and UV detection. Additionally, this medium-sized molecular
groups, in which conjugation
methods were utilized for the li-
gation of reversible-binding nat-
ural products with characteristic
functionalities (for example, alco-
hols, ketones, amines, and free
carboxylic acids).[7]
Results and Discussion
Probe design and synthesis
Specific probes for electrophilic
natural products must fulfil the
Figure 2. A) Structures of probes with various nucleophilic and electron-donating properties. B) Electrophilic mole-
following criteria: They must
contain a representative set of
nucleophiles, such as thiols,
amines, or alcohols. They need
to be incorporated into a rigid
scaffold that increases the UV
absorption, the molecular mass,
and the retention of trapped
molecules during HPLC. More-
over, this scaffold must be ame-
nable to the incorporation of li-
gands that enhance the reactivi-
cules tested for reactivity with the nucleophilic probes. Bn: benzyl.
Scheme 1. Representative synthetic pathways for nucleophilic probes Cap3 and [Cap5]2. a) Me2SO4, K2CO3, ace-
ty of the corresponding nucleo- tone, reflux, 88%; b) NBS, acetonitrile, RT, 52%; c) 1) nBuLi (1.00 equiv), THF, ꢀ788C; 2) 5-(trimethylsilyl)pent-4-ynal,
THF, ꢀ788C to RT, 64%; d) TFA, Et3SiH, CH2Cl2, RT, 10 min, 78%; e) for Cap3: 1) nBuLi, THF, ꢀ788C; 2) DPPA, THF,
phile. Naphthalene scaffolds rep-
ꢀ788C to RT; 3) Red-Al, THF, 08C; for [Cap5]2: 1) nBuLi, THF, ꢀ788C, 30 min; 2) sulfur powder, THF, ꢀ788C to RT,
resent such a desired structural
18 h; 3) Red-Al, THF, ꢀ108C, 1 h; f) TBAF, THF, Cap3: 51% from 5, [Cap5]2: 13% from 5. DPPA:
backbone that can be equipped
diphenylphosphorylazide; NBS: N-bromosuccinimide; Red-Al: sodium bis(2-methoxyethoxy)aluminum hydride;
with the desired heteroatom nu- TBAF: tetrabutylammonium fluoride; TFA: trifluoroacetic acid; TMS: trimethylsilyl.
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entity can serve as a distinct mass label in mass spectrometry
through a predictable mass shift of the captured natural prod-
ucts, which represents an advantage in the case of low-molec-
ular-weight target compounds. In addition, fine tuning of the
nucleophilic reactivity is mediated through the appropriate at-
tachment of electron-donating groups with +I (alkyl) and +M
(methoxy) effects (Figure 2A). Finally, reactive sites can be
placed in close proximity to an adjacent hydrogen-bond ac-
ceptor in the peri position, which may further facilitate partial
deprotonation of the nucleophilic center and thus increase the
reactivity.[8]
probe Cap5 in quantitative yield (Figure 1 in the Supporting
Information).[9] TCEP can act as a nucleophile for Michael ac-
ceptor systems as well,[10] so traces had to be removed after
the reduction by filtration over silica.
After incubation of all of the probes with the electrophiles
for 2 h at equimolar concentrations in organic solvent (DMSO/
acetonitrile), the reaction products were analyzed by HPLC.
The alcohol- and amine-based nucleophilic probes yielded no
significant substrate conversion with any electrophile (data not
shown). Satisfyingly, the thiol-based probes quantitatively
modified the maleimide scaffold in organic solvent (Figure 2 in
the Supporting Information), as well as under physiological
conditions in aqueous phosphate-buffered saline (PBS; 18 h,
10–20-fold excess, pH 7.4; Table 2 in the Supporting Informa-
tion). In addition, thiol probe Cap6 without the peri substitu-
tion showed a significantly higher conversion (50%) of the ep-
oxide than the sterically constrained probe Cap5. The results
of this study furthermore suggest that the current probe
design does not provide a sufficient reactivity of alcohols and
amines towards the electrophilic centers. Only thiols, as the
strongest nucleophile in this series, were able to react inde-
pendently of the presence of the acceptor. Cap6 revealed the
best results for capturing maleimide and epoxide scaffolds, so
we utilized this probe for all subsequent studies.
A divergent synthetic strategy was chosen to obtain the de-
sired amino- and thiol-substituted naphthalene probes Cap3–
Cap6. The representative synthetic pathways for Cap3 and
Cap5 are displayed in Scheme 1. Bismethoxynaphthalene 2
was halogen functionalized and then selectively alkylated in
the peri position to introduce the alkyne to afford general
building block 5. Final installation of the nucleophilic center
was accomplished by lithiation and amination for Cap3 or thio-
lation for Cap5 with the respective electrophiles. Cap5 was
found to be prone to oxidative dimerization during synthesis;
therefore, the stable dimer [Cap5]2 served as the synthetic
target. Additionally, analogues lacking the acceptor moiety in
the peri position to the nucleophilic group were prepared to
compare the influence of the ac-
ceptor moiety on the nucleophil-
ic reactivity (Figure 2A). The syn-
theses of hydroxy-based probes
Cap1 and Cap2 were accom-
plished through a natural-prod-
uct-based approach by starting
from juglone. Synthetic details
are provided in the Supporting
Information.
Probe reaction with prominent
electrophilic entities
All probes were tested for reac-
tivity towards
a collection of
common electrophilic groups
(Figure 2B). Thiol-based probes
had to be reduced before incu-
bation with electrophiles be-
cause the electron-enriched
naphthalene
p system
was
prone to oxidation to form thiol
dimers. Several reduction meth-
ods were screened to identify
a
high-yielding
procedure
(Table 1 in the Supporting Infor-
mation). Remarkably, only the
tris(2-carboxyethyl)phosphine
(TCEP) promoted conversion in
a
slightly acidified aqueous
Figure 3. Reactivity of thiol probe Cap6 with the natural product showdomycin (Sh). HPLC analysis of the reaction
of showdomycin with Cap6 in aqueous buffered medium (PBS/DMSO, 9:1; pH 7.4, 308C, 18 h). HRMS of *Sh–Cap6
medium developed by White-
sides et al. provided the reduced adducts calcd for C20H21NO7S [M]ꢀ: 418.0966; found: 418.09664; diff.: 2.73 ppm.
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Figure 4. Profiling of probe specificity towards a panel of electrophiles. A) Structures of natural products or natural-product-inspired compounds. B) Conver-
sion of compounds with Cap6. Calculated p values for the different electrophiles: p=1.58ꢁ10ꢀ2 for Pp, p=1.49ꢁ10ꢀ4 for FM209, and p=5.02ꢁ10ꢀ5 for Sh.
For FM209 and Sh, the p values are statistically highly significant (***p<0.005); for phosphomycin, the results are statistically significant (*p<0.05). C) Re-
versed-phased HPLC-ESI-HRMS traces of showdomycin, phosphomycin, and FM209 before and after reaction with Cap6.
Fine-tuned naphthalene thiols for maleimide and epoxide
capture
pound lacking the methoxy group in the peri position
(Figure 3). Importantly, the addition of the probe dramatically
altered the physical properties of showdomycin and led to
a significantly improved retention during reversed-phase C18
HPLC (there is no retention with unmodified showdomycin on
reversed-phase LC systems), as well as a specific detection at
l=303 nm. There is no stereo- or regiocontrol, so the nucleo-
The synthesis of the nucleoside antibiotic showdomycin was
adapted from a published procedure with slight modifica-
tions.[6,11] Incubation of the probes for 3 h with the natural
product revealed quantitative conversion with Cap6, the com-
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philic attack onto the showdomycin maleimide scaffold results
in the formation of isomer peaks, which slightly reduce the
overall signal intensity (Figure 3). Showdomycin is sterically
more constrained than the model substance N-ethylmaleimide,
so the trajectory of the attacking thiol group requires more
space in order to reach the shielded electrophilic center. Thus,
compound Cap5 with an adjacent methoxy hydrogen acceptor
did not show any reactivity, likely due to the additional steric
bulk (Figure 2 in the Supporting Information).
Labeling of showdomycin and phosphomycin in metabolic
extracts of natural producer strains
Due to the insufficient retention of showdomycin and phos-
phomycin on reversed-phase C18 columns (Figure 4C), direct
HPLC-MS analysis of the natural products in extracts of the
producing organisms is challenging. With a selective probe in
hand, we tested the modification of showdomycin and phos-
phomycin antibiotics in their producing strains: Streptomyces
showdoensis and S. fradiae, respectively.[12] HPLC analysis of an
S. showdoensis extract in the absence of probe Cap6 revealed
only a few peaks at the characteristic naphthalene absorption
wavelength. Importantly, addition of Cap6 resulted in the for-
mation of intense peaks with the expected retention time for
the showdomycin–probe isomers (Figure 5A).
In order to dissect the reactivity of probe Cap6, we com-
pared the reaction with that of naphthalene-1-thiol without
the electron-donating para-methoxy group. Incubation of
Cap6 and naphthalene-1-thiol with showdomycin revealed the
addition product only with the electron-rich Cap6 probe
(Figure 3 in the Supporting Information), which demonstrated
that a donor is mandatory for elevated nucleophilicity.
In addition, the methoxythionaphthalene mass tag led to
a characteristic shift in the MS spectrum, which facilitated easy
showdomycin–probe detection in both the MS base peaks as
function of elution time (Figure 6A), as well as in the sum of
all mass spectra (Figure 6B). Similarly, addition of Cap6 to the
extract of S. fradiae resulted in the selective identification of
the phosphomycin adducts by HPLC separation and detection
through UV and MS (Figure 5B and Figure 6C and D). Impor-
tantly, neither showdomycin nor phosphomycin could be de-
tected in the absence of Cap6, because the poor retention of
both compounds resulted in coelution with metabolites that
suppressed their ionization.
We next investigated the selectivity of capture probe Cap6
for maleimides and epoxides in a mixture of eight electrophilic
natural products (Figure 4A and B). Importantly, the probe
only converted showdomycin (95%), phosphomycin (36%),
and FM209 (83%), which indicated the desired specificity for
maleimide- and epoxide-containing molecules (Figure 4 in the
Supporting Information). Again, the tag significantly improved
the HPLC retention (Figure 5 in the Supporting Information) on
reversed-phase C18 columns, and the ionization of modified
showdomycin, phosphomycin, and FM209 could be increased
two–threefold under mild ESI conditions (Figure 4C; extended
gradient shown in Figure 6 in the Supporting Information). The
MS detection limit for Sh–Cap6 and Pp–Cap6 is in the range
of 100–150 pmol (Figure 7 in the Supporting Information).
Figure 5. Showdomycin and phosphomycin identification in Streptomyces producer strains. A) HPLC traces of S. showdoensis extracts before and after treat-
ment with Cap6. B) HPLC traces of S. fradiae extracts before and after treatment with Cap6.
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Figure 6. Showdomycin and phosphomycin identification in Streptomyces producer strains. A) Base peaks of all mass spectra as a function of elution time
(50–600 Da) for S. showdoensis extracts before and after treatment with Cap6. B) Sum of all mass spectra from 1–16 min/over the whole gradient of S. show-
doensis extracts before and after treatment with Cap6. C) Base peaks of all mass spectra as a function of elution time (50–600 Da) of S. fradiae extracts before
and after treatment with Cap6. D) Sum of all mass spectra from 1–16 min/over the whole gradient of S. fradiae extracts before and after treatment with
Cap6.
Conclusion
lene core structure, because of its excellent UV absorption, dis-
tinct mass shift, hydrophobic properties, and the wealth of
possible modifications that allow fine tuning of the nucleophi-
licity. Whereas the introduction of H-bond acceptors in the peri
position lowered the steric accessibility and, thus, the applica-
bility, the incorporation of electron-donating groups led to
a significant increase in reactivity. Among the tested nucleo-
philes, one probe with a methoxy donor and a nucleophilic
thiol group proved to be optimal to react selectively with ep-
oxides and maleimides in the presence of other electrophiles,
such as b-lactams and Michael acceptors. The applicability of
the probe for capturing natural products was demonstrated in
complex extracts. Although showdomycin and phosphomycin
were difficult to detect in the absence of the probe, probe ad-
The discovery of novel natural products is a major challenge
that can only be advanced by improved detection methods.[13]
Methods to modify a conserved structural motif, such as an
amine or carboxylic acid group, have been introduced previ-
ously through, for example, chemical coupling procedures.[7a–c]
Due to the wealth of bioactive compounds containing an elec-
trophilic center, which is crucial for their mechanism of action,
a method to selectively capture these protein-reactive natural
products by a nucleophilic trap is desired. Recently, Mitchell
and co-workers utilized dithiothreitol as a small tag for the
analysis of natural products containing dehydrated amino
acids.[14] In our proof of principle study, we applied a naphtha-
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dition resulted in modified compounds with improved reten-
tion times, UV absorption, and MS ionization. The additional in-
corporation of a bioorthogonal tag, such as an azide or alkyne,
would further allow the selective enrichment of captured natu-
ral products. This could be useful, in particular, to obtain quan-
tities of low-abundance compounds suitable for structural
characterization by NMR spectroscopy. In fact, some of our
probes are already equipped with alkyne substituents through
robust synthetic operations. However, care must be taken in
the selection of the alkyne position on the naphthalene scaf-
fold because the Cap5 probe was unreactive, probably due to
steric restrictions. Moreover, in order to expand the coverage
of the probes to additional protein-reactive natural products,
such as b-lactams, b-lactones, and Michael acceptors, the
design of the nucleophilic naphthalene traps will need to in-
clude electronically enriched benzene scaffolds with less steric
hindrance, heteroaromatic systems with different electron-do-
nating groups to fine tune the reactivity, and positively
charged ligands to further increase MS detectability.
Syntheses and additional biochemical assays
For details see the Experimental Section in the Supporting Informa-
tion.
Acknowledgements
We thank Mona Wolff for excellent technical support and M.
Nodwell for scientific discussion. S.A.S. was supported by the
Deutsche Forschungsgemeinschaft (SFB749, SFB1035, and
FOR1406), an ERC starting grant (259024), and the Center for
Integrated Protein Science Munich CIPSM. G.C.R. and M.F.K.
thank the TUM Graduate School for project funding and the
ERC for financial support.
Keywords: chemical probes
· epoxides · maleimides ·
metabolome analysis · natural products
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Metabolite extract solution (100 mL) was treated with nucleophilic
probe stock solution (10 mL; 25 mm), which was filtered prior to
use over a small pad of silica and further eluted into the extract
with acetonitrile (500 mL). Samples were evaporated to dryness and
redissolved in 20% DMSO/PBS buffer (pH 7.4; 100 mL). Reaction
mixtures were filtered through a 0.45 mm centrifugal filter and then
analyzed by analytical reversed-phase HPLC (C18 column, 3.5 mm,
4.6ꢁ100 mm, gradient 2!100% 0.1% (v/v) formic acid in acetoni-
trile/water 90:10 (B) over 25 min) and reversed-phase HPLC-ESI-
HRMS (C18 column, 3.5 mm, 4.6ꢁ100 mm, gradient 2!100% B
over 30 min).
Received: August 26, 2014
Published online on && &&, 0000
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FULL PAPER
Playing tag with electrophilic natural
products: Nucleophilic probes facilitate
the directed identification of electrophil-
ic natural products in complex extracts
of producing microorganisms with im-
proved HPLC retention and MS detec-
tion (see figure).
&
Metabolome Analysis
G. C. Rudolf, M. F. Koch, F. A. M. Mandl,
S. A. Sieber*
&& – &&
Subclass-Specific Labeling of Protein-
Reactive Natural Products with
Customized Nucleophilic Probes
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