The main focus of this dissertation was set on the development of a general solid-phase approach to the natural product class of 3-amino-6-hydroxy-2-piperidone (Ahp) containing cyclodepsipeptides. This natural product class is, as described already, known for its potent and most intriguingly non-covalent inhibition of S1 serine proteases. The potent inhibition is achieved by adapting the so-called canonical conformation which has been recognized as a structural motif in proteins and peptides of natural and synthetic sources mediating a non- covalent inhibition of serine proteases. [17-20] At the beginning of the project, solution-phase
syntheses of two Ahp containing cyclodepsipeptides, micropeptin T-20 and somamide A, had been published.[100-102] Although these syntheses finally attained the synthesis of the
respective natural product, they are highly specialized and require multi-step solution phase syntheses. Nevertheless, Yokokawa et. al. could prove in their syntheses that the crucial Ahp hemiaminal residue can readily be generated from a precursor aldehyde macrolactam at a late stage of the peptide synthesis. In fact, the Ahp hemiaminal forms spontaneously from the precursor aldehyde.[100-102]
Figure 42: General structure of the Ahp containing depsipeptides. The Ahp hemiaminal is formed
spontaneously from a precursor aldehyde macrocycle, with the equilibrium being on the side of the hemiaminal structure.
Accordingly, the synthetic outline for a solid-phase approach to the class of Ahp containing cyclodepsipeptides envisioned the development a suitable precursor unit that can be coupled to solid support and, after the assembly of the peptide sequence, can be converted to an aldehyde residue (and thus ultimately to the Ahp unit). This approach was envisaged to pave the way to tailor-made Ahp cyclodepsipeptides because the unifying element of the substance class (i. e. the Ahp unit) was chosen as the anchoring point to the solid phase. As a proof of principle for this approach, a solid phase synthesis of the natural product Ahp
cyclodepsipeptide symplocamide A was chosen. Symplocamide A was only discovered recently[24] and no synthesis of this natural product had been reported before this PhD
project.
Figure 43: Symplocamide A, an Ahp cyclodepsipeptide isolated from a Symploca sp. from Papua New Guinea.[24]
The natural product was chosen as the synthetic target for the validation of the newly developed solid-phase strategy.
In a second step after its synthesis, a validation of its reported biological activity and investigation of further bioactivities were planned.
After the successful development of a solid phase-based synthesis of Ahp containing cyclodepsipeptides, the follow-up question of the significance of the Ahp moiety in Ahp cyclodepsipeptides was to be addressed. The contribution of the Ahp moiety to the induction of a canonical conformation in the cyclodepsipeptide is well-described in literature. As the synthesis of such Ahp-containing cyclodepsipeptides is however rather laborious, the development of peptidic analogs of the Ahp containing cyclodepsipeptides that could retain the canonical conformation and thereby the biological activity was an aim in this PhD thesis.
To this end, analogs in which the Ahp unit was replaced by other amino acids were investigated for their biological activity. The elucidation of suitable Ahp mimics would enable a quick and if necessary combinatorial access to canonical conformation inhibitors and therefore represent valuable tools for chemical biology investigations of serine proteases.
Figure 44: General structure of structure-activity analogs derived from symplocamide A. The amino acids
In a second project, the synthesis of the natural product symplostatin 4 and derivatives that would enable the biological investigation as well as the identification of the molecular target of symplostatin 4 by the means of an activity-based approach was planned.
Symplostatin 4 is a linear peptide isolated from a cyanobacterium of the Symploca genus and shows a structural similarity to the potent cytotoxic agents dolastatin 10 and 15 which are lead compounds in cancer therapy. As dolastatin 10 and 15 are potent disruptors of microtubule polymers, symplostatin was also tested on microtubule interaction, displaying an effect significantly lower than dolastatins 10 and 15, but leading to the same cellular phenotypes.[103]
Interestingly, the natural product gallinamide A isolated from a Schyzothryx sp., was published just shortly before symplostatin 4 was disclosed and later studies demonstrated that both natural products have the same structure.[103,104] However, gallinamide A emerged from a screen of compounds isolated from marine cyanobacteria and displayed a promising antimalarial activity.[104] Symplostatin 4 initially attracted interest as a target structure for this dissertation owing to the prominent Michael acceptor system that is accompanied by the rather unusual methyl-methoxypyrrolinone. In an activity-based approach, a probe generated on the basis of symplostatin 4 could be a valuable tool for the investigation of proteases that are known targets of α,β-unsaturated carbonyls. Moreover, the methyl- methoxypyrrolinone unit could be studied in terms of its influence on the activity and selectivity of the natural product once a target is identified.
Figure 45: Symplostatin 4 and derivatives for biological studies. A: The natural product symplostatin 4, a natural
product isolated from a Symploca sp. from Key Largo.[103] B: Derivatives of symplostatin 4 for biological studies. Differentially tagged derivatives should be accessible by the reaction of alkyne-modified analogs with differentially tagged azides using click chemistry.
In addition, some chemical biology studies with symplostatin 4 such as a target identification in different organisms were envisaged. To this end, two collaborations with groups from different research fields were established. First, in collaboration with the van der Hoorn group (MPI Cologne), the effect of symplostatin 4 in the model plant Arabidopsis thaliana was to be investigated and the targets of symplostatin 4 and its analogs were to be identified. In addition, the molecular basis of the antimalarial activity of Symplostatin 4 should be investigated. To this end, a collaboration with the Bogyo group (Stanford University) was initiated; this studies aimed at the determination of the potency of the antimalarial effect of symplostatin 4 as well as the target identification of this compound in