Chemoenzymatic Total Synthesis of Ambruticins and Jerangolids
Final Report Abstract
The aim of this project was gaining a detailed understanding of the unusual enzymology of the ambruticin and jerangolid biosynthesis. Both polyketide pathways were proposed to contain a plethora of unusual and novel enzymatic transformations that might be exploited for chemoenzymatic synthesis of these compounds and potentially also for biocatalysis in general. The proposed synthetic work was fully accomplished. Substrate surrogates for the study of all relevant late-stage enzymes have been prepared. Among these are for example multiple tetraketidic, pentaketidic and natural product-like surrogates of biosynthetic intermediates and their analogues. All enzymes of interest have been produced by heterologous expression in E. coli. Enzymatic in vitro assays led to insights into the biosynthetic mechanisms of the most sections of the biosynthetic pathways. Notable discoveries are the full biochemical and structural characterization of the unusual bifunctional PKS (polyketide synthase) domain AmbDH3 with dehydratase and cyclase activity. This enzyme catalyses a cascade of dehydration and subsequent oxa-Michael addition to a tetrahydropyran. Furthermore, it was shown that a double bond shift is effected by the complex interplay between the trifunctional PKS domain AmbDH4 (dehydratase, epimerase and enoylisomerase) and the C- methyltransferase AmbM. This and the pyran cyclisation set up the rigid vinylpyran motif, which is characteristic for both studied natural products. The in vitro studies on the formation of the ambruticin middle fragment led to the discovery of a novel type of fatty acyl adenylate ligase (FAAL) domain with relaxed substrate specificity that participates in processing of the cyclopropane product. The actual mechanism of the divinylcyclopropane formation occurring was refined based on the results of X-ray crystal structure analysis, molecular modelling and chemical logic. A reductive Horner Wadsworth Emmons Olefination approach was developed and provided fast and flexible synthetic access to the highly sensitive polyene-ketothioesters essential for in vitro studies with the putative cyclopropane-forming PKS module AmbF. The O-methyltransferase JerF is a key enzyme in the tailoring of the western jerangolid part. This enzyme was fully biochemically characterised. Relevant progress in the production and characterisation of the late-stage-tailoring Rieske oxygenases of both pathways was also achieved. Of the studied enzymes, the cyclase AmbDH3 and the O-methyltransferase JerF and showed particularly attractive features for a general application in chemoenzymatic synthesis. Both enzymes exhibit relaxed substrate specificity and high selectivity for the formation of two vicinal stereocentres or methylenolether in the presence of other potential methylation sites, respectively. AmbDH3 can be used for the preparative-scale synthesis of chiral tetrahydropyrans carrying up to four stereocentres. Both enzymes were used in a first generation chemoenzymatic synthesis of the jerangolids. This route provided access to novel derivatives, which are currently undergoing biological testing. The synthetic material is also valuable for comprehensive studies on the late-stage Rieske oxygenase tailoring steps.
Publications
- A Dehydratase Domain in Ambruticin Biosynthesis Displays Additional Activity as a Pyran- Forming Cyclase; Angew. Chem. 2014, 126, 14464-14468; Angew. Chem. Int. Ed. 2014, 53, 14240-14244
G. Berkhan, F. Hahn
(See online at https://doi.org/10.1002/anie.201407979) - Opportunities for Enzyme Catalysis in Natural Product Chemistry; Tetrahedron 2015, 71, 1473–1508
S. Friedrich, F. Hahn
(See online at https://doi.org/10.1016/j.tet.2014.12.026) - Biosynthesis of oxygen and nitrogen-containing heterocycles in polyketides; Beilstein J. Org. Chem. 2016, 12, 1512-1550
F. Hemmerling, F. Hahn
(See online at https://doi.org/10.3762/bjoc.12.148) - Characterisation of the Broadly-Specific O-Methyl-transferase JerF from the Late Stages of Jerangolid Biosynthesis; Molecules 2016, 21, 1443
S. Friedrich, F. Hemmerling, F. Lindner, A. Warnke, J. Wunderlich, G. Berkhan, F. Hahn
(See online at https://doi.org/10.3390/molecules21111443) - The Interplay between a Multifunctional Dehydratase Domain and a C-Methyltransferase Effects Olefin Shift in Ambruticin Biosynthesis; Angew. Chem. Int. Ed. 2016, 55, 13589-13592; Angew. Chem. 2016, 128, 13787–13790
G. Berkhan, C. Merten, C. Holec, F. Hahn
(See online at https://doi.org/10.1002/anie.201607827) - An Unusual FAAL‐ACP Didomain in Ambruticin Biosynthesis; ChemBioChem 2018, 19, 1006–1011
F. Hemmerling, K. Lebe, J. Wunderlich, F. Hahn
(See online at https://doi.org/10.1002/cbic.201800084) - Heterocycle biosynthesis by sp3 C–H activation; Nat. Catal., 2018, 1, 905
F. Hahn
(See online at https://doi.org/10.1038/s41929-018-0194-2) - Insights into the dual Activity of a Bifunctional Dehydratase-Cyclase Domain; Angew. Chem. Int. Ed. 2018, 57, 343-347; Angew. Chem. 2018, 130, 349-353
K. H. Sung, G. Berkhan, T. Hollmann, L. Wagner, W. Blankenfeldt, F. Hahn
(See online at https://doi.org/10.1002/anie.201707774) - Total Synthesis of Complex Biosynthetic Late-Stage Intermediates and Bioconversion by a Tailoring Enzyme from Jerangolid Biosynthesis; J. Org. Chem. 2018, 83, 14091
F. Lindner, S. Friedrich, F. Hahn
(See online at https://doi.org/10.1021/acs.joc.8b02047)
