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Technische Universität München

Lehrstuhl für organische Chemie II




Oliver Alexander Battenberg

Vollständiger Abdruck der von der Fakultät für Chemie

der Technischen Universität München zur Erlangung des akademischen Grades eines

Doktors der Naturwissenschaften genehmigten Dissertation.

Vorsitzender: Univ.-Prof. Dr. Johannes Buchner Prüfer der Dissertation: 1. Univ.-Prof. Dr. Stephan A. Sieber

2. TUM Junior Fellow Dr. Sabine Schneider Die Dissertation wurde am 6. Februar 2013 bei der Technischen Universität München eingereicht und durch die Fakultät für Chemie am 5. März 2013 angenommen.

Diese Arbeit wurde an den Fachbereichen Chemie der Ludwig Maximilian Universität und der Technische Universität München unter Leitung von Herrn Prof. Dr. Stephan A. Sieber in der Zeit von Oktober 2009 bis Februar 2013 durchgeführt.

Danksagung Herrn Prof. Dr. Stephan A. Sieber danke ich für die Aufnahme in seinen Arbeitskreis, die ansprechende und herausfordernde Themenstellung sowie die hervorragende Förderung dieser Arbeit.

Bei den Mitgliedern der Prüfungskommission bedanke ich mich für die Bemühungen bei der Bewertung dieser Arbeit.

An Frau Dr. Sabine Schneider richtet sich mein Dank für die Bereitschaft sich für die Erstellung des Zweitgutachtens zur Verfügung zu stellen.

Mona Wolf und Katja Bäuml danke ich für ihre Hilfsbereitschaft und ihr unermüdliches Engagement für einen organisierten Laboralltag.

Für die zahlreichen wissenschaftlichen und persönlichen Unterredungen sowie die hauptanteilige Übernahme der angefallenen Korrekturarbeiten gilt mein außerordentlicher Dank Dr. Matthew Nodwell, der durch seine stete, motivierende Hilfestellung einen wichtigen Beitrag zu dieser Arbeit geleistet hat.

Besonderer Dank richtet sich auch an meine Laborkollegen Dr. Maximilian Pitscheider, Tanja Wirth, Dr. Thomas Menzel, Georg Rudolf und Franziska Mandl für die hervorragende Unterstützung, das aufgeschlossene Entgegenkommen und die anregenden Gespräche.

Abschließend gilt mein größter Dank Daniela Falkner, meiner Familie und meinem Freundeskreis für die besondere Unterstützung während der gesamten Promotionszeit.



Activity-based protein profiling (ABPP)

Protein-reactive natural products

Ring-strained scaffolds

Michael acceptor systems

Other reactive moieties


Aryl azides






Evaluation of -pyrones and pyrimidones as photo affinity probes for activity based protein profiling.



Results and Discussion



Synthesis of a benzophenone-alkyne tag for reversible inhibitor-target profiling......... 53  Introduction

–  –  –

Experimental Section

Target profiling of 4-hydroxyderricin in S. aureus reveals seryl-tRNA synthetase binding and inhibition by covalent modification



Results and Discussion




α-Pyrones and pyrimidones as photoaffinity probes

Synthesis of a benzophenone-alkyne tag for affinity based protein profiling............ 86  Target profiling of 4-hydroxyderricin in S. aureus


α-Pyrone und Pyrimidone als Photoaffinitätssonden

Synthese eines Benzophenon-Alkin-Tags für das affinity based protein profiling..... 91  Target-Identifizierung von 4-Hydroxyderricin in S. aureus






–  –  –

Natural products are a unique class of organic molecules with distinct biological activities. The increasing knowledge about the molecular mechanism of biological processes identified this group of compounds as an almost unlimited source of bioactive molecules with a high therapeutic potential. In contrast to synthetic compound libraries natural products have coevolved with biological systems and therefore show a remarkably selectivity for the interaction with biological structures.[1-2] Since about 47% of the drugs that were approved from 1981 to 2006 are derived from natural products[3] additional screening programs might be a starting point to face the increasing demand for novel therapeutic strategies.[4] The investigation and understanding of the complex interactions of a natural product in a biological system are challenging but a number of valuable methods are available to unravel the mechanism of natural product -protein interactions.[5] Since the active cellular processes in living organisms are dominated by protein functions the proteome analysis of all natural product caused cellular changes is an important approach[1]. Progress in the application of mass spectrometry within the last decade has paved the way for activity-based protein profiling (ABPP) experiments by protein directed small molecule probes capably of covalent protein modification.[6] The design and application of these usually natural product derived probes with an appropriate reporter group enables a comprehensive proteome analysis and provides useful information about protein targets in complex biological systems.[7] The concept of ABPP is based on the design of a reactive probe that covalently modifies protein targets and subsequently offers a suitable readout mechanism for the identification of labeled

enzymes. A typical ABPP probe therefore combines at least three functionalities (Figure 1):

1.) A binding group for affinity mediated target binding. These structure motives are responsible for the selective non-covalent interactions with a complementary protein structures. Because natural products are fine-tuned for the interaction with proteins these compounds are an ideal source for a binding group.

2.) A reactive group for the covalent modification of the target. These reactive groups might be part of the binding groups’ structure (for example intrinsic reactivity of protein-reactive natural products) or have to be introduced by the addition of electrophilic or photo-reactive chemotypes for the covalent target binding.[7] 3.) A reporter group for visualization or purification protocols usually fluorescence dyes and biotin-tags.

–  –  –

A drawback of this approach is that the incorporation of these structure elements into the parent structure could change the natural products’ biological profile as well as negatively affect the probes solubility and cell permeability. This is why an alternative strategy has been developed that avoids large tags in the natural product structure. A useful circumvention is the modification of bioactive molecules and reporter groups with click chemistry ligation handles. The CuIcatalyzed Huisgen [3+2] cycloaddition of azides and terminal alkynes forms 1,2,3-triazoles under physiological conditions. It is the most prominent example of a bioorthogonal ligation reaction and facilitates the probes connection with a tag after the initial labeling event (Scheme 1)[8-10]

–  –  –

Live cells or cell lysates are therefore incubated with a suitable alkyne-containing ABPP probe to covalently label the protein targets. The ligation reaction is then carried out by the addition of the complementary azide-tag e.g. a fluorescent dye, a CuII salt, a reducing agent such as sodium ascorbate or tris(2-carboxyethyl)phosphine (TCEP) for the in situ Cu-reduction and a suitable ligand such as tris-(5-benzyl-1H-triazol-4-yl)-methanamine (TBTA) to form the active CuI complex.[11] Following the ligation reaction, separation of the proteins by SDS-PAGE and in-gel readout of the reporter-tag (fluorescence scanning) finally unravels probe modified proteins (Figure 2).[12]

Figure 2. General workflow of a click chemistry based ABPP experiment (Rh:

–  –  –

In order to identify the protein bands by a gel based approach the labeled proteins have to be selectively enriched for subsequent analysis to reduce the background of non-target proteins. An approach as outlined in Figure 3 is an avidin/biotin affinity purification protocol. Selective enzyme labeling by the protein reactive probe is followed by the click reaction with a tag that combines fluorescence readout and biotin enrichment capabilities for the visualization and purification.

Affinity-enrichment on immobilized avidin yields potential protein targets that are subsequently separated by SDS-PAGE and analyzed by in-gel fluorescence scanning. Fluorescent bands are cut, tryptically digested and applied to LC-MS/MS analysis to unravel protein identities (Figure 3).

Figure 3. Identification of labeled proteins by avidin/biotin affinity purification

–  –  –

These gel based ABPP-platforms feature fast, simple and simultaneous analysis of different natural product proteome labeling experiments with an easy comparison of the obtained results.

Although low resolution limits the universal application of this technique compared to gel-free [13-14] methods it is still is a very useful and frequently used analytical platform for protein target identification and therefore was exclusively applied in this work.

Protein-reactive natural products Electrophilic natural products with a covalent target-binding mechanism are ideal compounds for mode of action studies by ABPP. The general reaction mechanism of these compounds is based on a covalent attack of nucleophilic protein-residues (e.g. lysine-, threonine-, serine- or cysteineside-chains) on an electrophilic natural product center. This common electrophilicity facilitates the arrangement in subgroups due to similar chemical reactive moieties.[2, 12, 15] Ring-strained scaffolds Because of its historical significance and its importance in antibacterial therapies the β-lactams probably are the most famous electrophilic natural products. The most prominent example are penicillins originally discovered by Alexander Fleming. These compounds exert their biological effects via covalent binding of enzymes involved in bacterial cell wall biosynthesis. A nucleophilic attack of an activated serine residue at the β-lactam functional group causes an irreversible enzyme inhibition (Scheme 2).[16-17] The activities of these by definition called penicillin-binding proteins are essential for the cell-wall biosynthesis and therefore one of the most important antibacterial protein targets.[18]

–  –  –

In addition to β-lactams β-lactones, aziridines and epoxides are important electrophilic centers whose reactivity is caused by ring strain. Attacks of suitable nucleophiles at natural products containing these reactive moieties are often the basis for the biological activity of these compounds.[2, 15] A selection of representative molecules is show in Figure 4.

Figure 4. Selective representative natural products with ring strained reactive

–  –  –

Michael acceptor systems Michael acceptor scaffolds are next to ring strained systems another important subgroup of electrophilic natural products. A variety of different α,β unsaturated systems induce their interesting bioactivities by a molecular mechanism based on the attack of nucleophilic amino acid side chains on the Michal acceptor scaffold.

A prominent example is the metabolite wortmannin isolated from the fungus Penicillium wortmanii. The antifungal and mainly antiproliferative activity of this natural product is based on a covalent modification of the lipid kinase phosphatidylinositol 3-kinase (PI3K). A nucleophilic PI3K-lysine attacks the furan of wortmannin and causes an irreversible inhibition of this important anti-cancer target (Scheme 3).[12]

–  –  –

Chalcones are another interesting group of Michael acceptor containing natural products. The chalcones have a general structure as shown in Figure 5. The substitution pattern of the two aromatic rings determines different bioactivities including anticancer, anti-inflammatory and antifungal activities. Particularly interesting are the antibacterial data for 4-hydroxyderricin (4HD, [20-21] Figure7). Although no studies so far have investigated the molecular mechanisms for the

–  –  –

proposed protein reactivity therefore makes this structure an ideal starting compound for the design of an APBB probe to unravel the antibacterial mode of action.

–  –  –

Other reactive moieties Next to these two major groups of electrophilic natural products there is a variety of other electrophilic moieties for the covalent interaction with suitable biological nucleophiles.

Carbamates, chlorodihydroisoxazoles, 4-hydroxycoumarins, isothiocyanates, disulfides and αketoamides are other interesting reactive scaffolds with reported protein reactivity as well as the divers group of natural products with intracellular activation of the reactive electrophilic group. [15] Photocrosslinking ABPP using protein reactive probes is limited to investigate natural products that exert their biological effects via covalent inhibition. But many natural products exert their biological effect by reversible binding. The concept of proteome analysis by ABPP therefore needed to be expanded by the introduction of photo affinity labeling.[24-25] The design of a molecular photo-probe involves the addition of a photo-reactive group for the light induced covalent labeling of target structures (Figure 6). This concept is a very powerful approach to investigate the mechanism of natural products without intrinsic protein reactivity.

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