Director, Chemical Biology
Modulation of biological processes by small molecules raises the fundamental question which compound classes are likely endowed with biological activity. Natural products and their structural scaffolds represent the biologically relevant and prevalidated regions of chemical structure space explored by nature. The underlying 3D-structures of evolutionary selected natural products (NPs) define structural prerequisites for binding to proteins because NPs are a) biosyn-thesized by enzymes (proteins) and b) they exert their biological functions by binding to them. As a consequence, the structural scaffolds of natural product classes are endowed with relevance to nature and provide evolutionarily selected starting points in chemical structure space for compound collection design and development. Biology oriented synthesis (BIOS) builds on these arguments and employs core structures primarily, but not exclusively delineated from natural products as scaffolds of compound collections. In the course of our BIOS program we have synthesized various focused NP-inspired compound collections frequently in collaboration with Andrey Antonchick and Kamal Kumar.
Novel strategies for the synthesis of natural product-inspired compounds
We developed the first enantioselective inverse demand imino-Diels-Alder (IEDIDA) reaction between electron-poor chromone dienes and cyclic imines by using zinc/Binol complexes as catalyst to access the centrocountin scaffold [1, 2] [Eschenbrenner-Lux et al. 2014, Figure 1A]. In addition, a compound library of 530 tetrahydroindolo[2,3-a]quinolizines was generated .
Gold complexes are powerful catalysts for addition and cyclization reactions involving diverse nucleophiles and alkynes. A novel and efficient one-step procedure for the synthesis of chiral CAAC–AuI complexes through a mechanistically unique cyclization–rearrangement sequence was discovered by us . A two-step reaction sequence including a catalytic Pictet–Spengler cyclization followed by Au(I) catalyzed intramolecular hydroamination of acetylenes gave access to a range of complex heterocyclic frameworks based on biologically relevant indole/oxindole scaffolds. A related cascade polycyclization of a designed β-carboline embodying a 1,5-enyne group yielded analogues of the alkaloid harmicine . A gold (I) catalyzed cycloisomerization of indolyl-1,6-enynes via 5-exo-dig cyclization was achieved which passes through an intermediate whose fate can be steered to yield different indole polycyclic scaffolds through various intra- and intermolecular cyclization reactions. One of the key transformations of the indolyl-1,6-enynes was a formal [2+2+2] cycloaddition reaction with various aldehydes to yield natural product-like tetracyclic indoles . We also reported on a divergent skeletal rearrangement of 1,7-enynes to exocyclic allenes and tricyclic hexahydro-anthracenes by catalysis with a cationic gold(I) complex .
Within the context of our compound library development program enantioselective dipolar cycloadditions have proven to be powerful synthesis methods with wide scope. Thus, we developed one of the first highly enantioselective intramolecular 1,3-dipolar cycloadditions which yielded pyrrolidino-piperidines and, in tandem with a stereoselective intermolecular 1,3-dipolar cycloaddition, complex piperidino-pyrrolizidines in a one-pot reaction sequence  [Vidadala et al. 2015, Figure 1B]. A catalytic enantioselective intermolecular cycloaddition reaction between carbonyl ylides derived from diazodiketoesters and tropone as the dipolarophile yielded a diverse range of 5-alkoxylactone derivatives . We demonstrated that azomethine ylides such as isoquinolinium methylides can act as efficient dipoles to participate in [3+2] cycloaddition reactions with allenoates and allenones catalysed by phosphines. By means of this methodology, pyrrolo-isoquinolines can be accessed regioselectively . An asymmetric multicomponent cascade reaction consisting of the aerobic copper-catalyzed oxidation of cyclopentadiene to cyclopentadienone followed by subsequent enantioselective catalytic double 1,3-dipolar cycloaddition of azomethine ylides yielded complex natural product-inspired products with eight stereocenters. In this transformation a chiral copper complex catalyzes two mechanistically different cycles in one multicomponent cascade reaction . Moreover, we developed a formal 1,3-dipolar cycloaddition of coumarin with α-iminoesters for the preparation of 6,6,5-tricyclic compounds , and obtained a pyrrolocoumarine library with fully substituted pyrrole moiety by means of a sequential one-pot synthesis which involves N-terminal deprotection of a N-substituted Boc-glycine O-aryl ester embodying an ortho-alkyne substituent, azomethine ylide formation with an aldehyde, intramolecular [3+2] cycloaddition and subsequent aromatization . An efficient strategy for enantiodivergent synthesis of NP-inspired hybrid compounds was established embodying both tropane and pyrrolidinone NP fragments  [ Xu et al. 2016, Figure 1C]. For the asymmetric synthesis of structurally complex hexahydro-aza-pentalenones embodying four consecutive stereogenic centers including two quaternary centers, one of which is an all-carbon-quaternary center, an enantioselective [3+2] cycloaddition reaction of azomethine ylides and substituted cyclopentenones was developed .
Compound collections for chemical biology research
A withanolide compound collection with the characteristic trans-hydrindane dehydro-δ-lactone scaffold was stereoselectively synthesized and biological investigation of the collection revealed novel and potent inhibitors of the Hedgehog signaling pathway, which bind to the Smoothened protein  [Švenda et al. 2015, Figure 2].
In addition, we prepared a militarinone-inspired 4-hydroxy-2-pyridone collec-tion and analyzed the compounds for neuritogenic properties, which led to the discovery of the stress pathway serine/threonine kinase MAP4K4 as a target of the neuritogenic 4-hydroxy-2-pyridones  [Schröder et al. 2015].
Modulators of signaling B4 lipid-modified proteins
Lipid-modified proteins play important roles in biology. Rab (Ras-related in brain) GTPases are master regulators of intracellular vesicular transport and their malfunctions have been implicated in an increasing number of inherited and acquired pathologies. Chemical modulation of Rab function has remained elusive since the Rab proteins interact with their effectors through extensive protein-protein interactions (PPIs) currently not amenable to small molecule inhibition. Derived from α-helical binding motifs of Rab-interacting proteins we designed stabilized peptides by means of hydrocarbon–peptide stapling to inhibit protein-protein interactions involving Rab8 as a representative GTPase, and thereby developed the first chemical modulator of a Rab PPI  [Spiegel et al. 2014, Figure 3A]. Chemically orthogonal ring-closing olefin and alkyne metathesis was developed and successfully employed to evolve a monocyclic peptide inhibitor of the small GTPase Rab8 into a bicyclic ligand . Ras GTPases are lipidated proteins that regulate important cellular processes such as growth, differentiation, and apoptosis. 20-30% of all human tumors harbor Ras mutations. However, despite numerous efforts there are no Ras-targeting drugs in the clinic so far. In a close collaboration with the Bastiaens and the Wittinghofer group, we developed small molecules that bind to the prenyl-binding pocket of the Ras chaperone PDEδ and inhibit the interaction of PDEδ with KRas and the Rheb GTPase. Inhibition impaired K-Ras signaling and proliferation of human pancreatic ductal adenocarcinoma cells. Structure-based compound design guided the identification of pyrazolopyridazinones as a 2nd generation chemotype with improved pharmacological characteristics  [Papke et al. 2016, Figure 3B]. Furthermore, lipid-modified peptides, representative of different lipoprotein classes were synthesized and analyzed for binding to the relevant chaperones PDEδ, UNC119a, UNC119b, and galectins-1 and -3. PDEδ was found to recognize S-isoprenylated C-terminal peptidic structures, whereas UNC119 proteins bound only mono-N-myristoylated peptides .
Numerous bioactive natural products are macrocyclic and macrocycles are advantageous synthesis targets for research programs in drug discovery and chemical biology. We employed an efficient solid-phase/cyclorelease method for the synthesis of macrocyclic depsipeptides inspired by the very potent F-actin stabilizing depsipeptides of the jasplakinolide/geodiamolide class and validated this approach in the first total synthesis of the actin-stabilizing cyclodepsipeptide seragamide . A fluorescent-tagged jasplakinolide analogue was applied as powerful tool to study actin-relate processes by means of STED microscopy .
Target identification and assay development
In order to harness the potential of the synthesized compounds for chemical biology research, focussed compound collections or the entire COMAS library were subjected to several biochemical (inhibition of the kinesin HSET, Unc119 interactions) and cell-based screening (e. g. Wnt and Hedgehog signaling) assays. Establishment of structure-activity correlations enabled hit improvement. For the best compounds their cellular targets were identified and validated employing a combination of quantitative proteomics, computational approaches, biochemical, biophysical, genetic, molecular and cell-biological techniques. The natural product podoverine A and di/tripeptoids were identified as inhibitors of microtubule dynamics and/or to target karyopherins [24, 25]. Withanolide-inspired small molecules proved to potently inhibit Hedgehog signaling by targeting the Smoothened protein . In collaboration with MPI Münster, a phenotypic screening for moni-toring small molecule-induced reprogramming of epiblast stem cells to embryonic stem cells (ESCs) yielded Triamteren (TR) as hit which was developed into the more potent Epiblastin A. This compound efficiently induces reprogramming of epiblast stem cells to pluripotent ESCs by engaging casein kinase Iα [26, 27] [Ursu et al. 2016, Figure 4A]. A phenotypic assay was employed to identify neuritogenic 4-hydroxy-2-pyridones. Target deconvolution uncovered the stress pathway serine/threonine kinase MAP4K4 as the target .
In collaboration with TU Dortmund/FU Berlin and the University of Leeds we discovered the transient receptor potential channels TRPC4/5 as targets of the natural product Englerin A  [Akbulut et al. 2015, Figure 4B].
Covalent probes are at the heart of “reactive proteomics”. We introduced isoxazolium salts (Woodward’s reagent K) as novel chemotype to this field, which may covalently label proteins at nucleophilic amino acids. Surprisingly WRK-based probes were selective for a few proteins in the human proteome  [Qian et al. 2016].
1. Eschenbrenner-Lux, V., Kuchler, P., Ziegler, S., Kumar, K., and Waldmann, H. (2014). An Enantioselective Inverse-Electron-Demand Imino Diels-Alder Reaction. Angew Chem Int Edit 53, 2134-2137.
2. Duckert, H., Pries, V., Khedkar, V., Menninger, S., Bruss, H., Bird, A.W., Maliga, Z., Brockmeyer, A., Janning, P., Hyman, A., et al. (2012). Natural product-inspired cascade synthesis yields modulators of centrosome integrity. Nat Chem Biol 8, 179-184.
3. Sankar, M.G., Mantilli, L., Bull, J., Giordanetto, F., Bauer, J.O., Strohmann, C., Waldmann, H., and Kumar, K. (2015). Stereoselective synthesis of a natural product inspired tetrahydroindolo[2,3-a]-quinolizine compound library. Bioorgan Med Chem 23, 2614-2620.
4. Kolundzic, F., Murali, A., Perez-Galan, P., Bauer, J.O., Strohmann, C., Kumar, K., and Waldmann, H. (2014). A Cyclization-Rearrangement Cascade for the Synthesis of Structurally Complex Chiral Gold(I)-Aminocarbene Complexes. Angew Chem Int Edit 53, 8122-8126.
5. Danda, A., Kumar, K., and Waldmann, H. (2015). A general catalytic reaction sequence to access alkaloid-inspired indole polycycles. Chem Commun 51, 7536-7539.
6. Pérez-Galán, P., Waldmann, H., and Kumar, K. (2016). Building polycyclic indole scaffolds via gold(I)-catalyzed intra- and inter-molecular cyclization reactions of 1,6-enynes. Tetrahedron 72, 3647-3652.
7. Meiss, R., Kumar, K., and Waldmann, H. (2015). Divergent Gold(I)-Catalyzed Skeletal Rearrangements of 1,7-Enynes. Chem-Eur J 21, 13526-13530.
8. Vidadala, S.R., Golz, C., Strohmann, C., Daniliuc, C.G., and Waldmann, H. (2015). Highly enantioselective intramolecular 1,3-dipolar cycloaddition: a route to piperidino-pyrrolizidines. Angewandte Chemie 54, 651-655.
9. Murarka, S., Jia, Z.J., Merten, C., Daniliuc, C.G., Antonchick, A.P., and Waldmann, H. (2015). Rhodium(II)-Catalyzed Enantioselective Synthesis of Troponoids. Angew Chem Int Edit 54, 7653-7656.
10. Jia, Z.J., Daniliuc, C.G., Antonchick, A.P., and Waldmann, H. (2015). Phosphine-catalyzed dearomatizing [3+2] annulations of isoquinolinium methylides with allenes. Chem Commun 51, 1054-1057.
11. Potowski, M., Merten, C., Antonchick, A.P., and Waldmann, H. (2015). Catalytic Aerobic Oxidation and Tandem Enantioselective Cycloaddition in Cascade Multicomponent Synthesis. Chem-Eur J 21, 4913-4917.
12. Potowski, M., Golz, C., Strohmann, C., Antonchick, A.P., and Waldmann, H. (2015). Biology-oriented synthesis of benzopyrano[3,4-c]pyrrolidines. Bioorgan Med Chem 23, 2895-2903.
13. Vidadala, S.R., and Waldmann, H. (2015). One-pot synthesis of a natural product inspired pyrrolocoumarine compound collection by means of an intramolecular 1,3-dipolar cycloaddition as key step. Tetrahedron Lett 56, 3358-3360.
14. Xu, H., Golz, C., C, S., Antonchick, A., and Waldmann, H. (2016). Enantiodivergent Combination of Natural Product Scaffolds Enabled by Catalytic Enantioselective Cycloaddition. Angewandte Chemie doi: 10.1002/anie.201602084.
15. Dakas, P.Y., Waldmann, H., and Kumar, K. (2015). Natural Product Inspired Enantioselective Synthesis of Hexahydro-Aza-Pentalenones. Heterocycles.
16. Svenda, J., Sheremet, M., Kremer, L., Maier, L., Bauer, J.O., Strohmann, C., Ziegler, S., Kumar, K., and Waldmann, H. (2015). Biology-Oriented Synthesis of a Withanolide-Inspired Compound Collection Reveals Novel Modulators of Hedgehog Signaling. Angew Chem Int Edit 54, 5596-5602.
17. Schroder, P., Forster, T., Kleine, S., Becker, C., Richters, A., Ziegler, S., Rauh, D., Kumar, K., and Waldmann, H. (2015). Neuritogenic Militarinone-Inspired 4-Hydroxypyridones Target the Stress Pathway Kinase MAP4K4. Angew Chem Int Edit 54, 12398-12403.
18. Spiegel, J., Cromm, P.M., Itzen, A., Goody, R.S., Grossmann, T.N., and Waldmann, H. (2014). Direct Targeting of Rab-GTPase-Effector Interactions**. Angewandte Chemie-International Edition 53, 2498-2503.
19. Cromm, P.M., Spiegel, J., Grossmann, T.N., and Waldmann, H. (2015). Direct Modulation of Small GTPase Activity and Function. Angew Chem Int Edit 54, 13516-13537. 20. Papke, B., Murarka, S., Vogel, H.A., Martin-Gago, P., Kovacevic, M., Truxius, D.C., Fansa, E.K., Ismail, S., Zimmermann, G., Heinelt, K., et al. (2016). Identification of pyrazolopyridazinones as PDE[delta] inhibitors. Nat Commun 7.
21. Mejuch, T., van Hattum, H., Triola, G., Jaiswal, M., and Waldmann, H. (2015). Specificity of Lipoprotein Chaperones for the Characteristic Lipidated Structural Motifs of their Cognate Lipoproteins. Chembiochem 16, 2460-2465.
22. Arndt, H.D., Rizzo, S., Nocker, C., Wakchaure, V.N., Milroy, L.G., Bieker, V., Calderon, A., Tran, T.T.N., Brand, S., Dehmelt, L., et al. (2015). Divergent Solid-Phase Synthesis of Natural Product-Inspired Bipartite Cyclodepsipeptides: Total Synthesis of Seragamide A. Chem-Eur J 21, 5311-5316.
23. Lukinavicius, G., Reymond, L., D'Este, E., Masharina, A., Gottfert, F., Ta, H., Guther, A., Fournier, M., Rizzo, S., Waldmann, H., et al. (2014). Fluorogenic probes for live-cell imaging of the cytoskeleton. Nat Methods 11, 731-733.
24. Tran, T.T.N., Gerding-Reimers, C., Scholermann, B., Stanitzki, B., Henkel, T., Waldmann, H., and Ziegler, S. (2014). Podoverine A-a novel microtubule destabilizing natural product from the Podophyllum species. Bioorgan Med Chem 22, 5110-5116.
25. Vendrell-Navarro, G., Rua, F., Bujons, J., Brockmeyer, A., Janning, P., Ziegler, S., Messeguer, A., and Waldmann, H. (2015). Positional Scanning Synthesis of a Peptoid Library Yields New Inducers of Apoptosis that Target Karyopherins and Tubulin. Chembiochem 16, 1580-1587.
26. Ursu, A., Illich, D.J., Takemoto, Y., Porfetye, A.T., Zhang, M., Brockmeyer, A., Janning, P., Watanabe, N., Osada, H., Vetter, I.R., et al. (2016). Epiblastin A Induces Reprogramming of Epiblast Stem Cells Into Embryonic Stem Cells by Inhibition of Casein Kinase 1. Cell chemical biology 23, 494-507.
27. Illich, D.J., Zhang, M., Ursu, A., Osorno, R., Kim, K.P., Yoon, J., Arauzo-Bravo, M.J., Wu, G., Esch, D., Sabour, D., et al. (2016). Distinct Signaling Requirements for the Establishment of ESC Pluripotency in Late-Stage EpiSCs. Cell Rep 15, 787-800.
28. Akbulut, Y., Gaunt, H.J., Muraki, K., Ludlow, M.J., Amer, M.S., Bruns, A., Vasudev, N.S., Radtke, L., Willot, M., Hahn, S., et al. (2015). (-)-Englerin A is a Potent and Selective Activator of TRPC4 and TRPC5 Calcium Channels. Angew Chem Int Edit 54, 3787-3791.
29. Qian, Y., Schurmann, M., Janning, P., Hedberg, C., and Waldmann, H. (2016). Activity-Based Proteome Profiling Probes Based on Woodward's Reagent K with Distinct Target Selectivity. Angewandte Chemie.