Synthesis of nodularin analogues as potential protein phosphatases inhibitors

Abstract

Reversible phosphorylation of proteins on serine, threonine and tyrosine residues, is now widely accepted to be the principal mechanism for the control of intracellular events in eukaryotic and prokaryotic cells. The nodularins are known to be potent inhibitors of serine/threonine protein phosphatases, PPlc and PP2Ac, with sub-nanomolar inhibition constants. They have been shown to form covalent adducts with the enzymes and are known to be potent hepatotoxins and liver promoters. Nodularin has the general structure: cyclo [(R)-eryphro-beta-methyl-iso-Asp-(S)-X-Adda-(R)-iso- Glu-N-methyldehydrobutyric acid)], where (S)-X is a variable S-amino acid and Adda is the unique beta-amino acid, (2S,3S,8S,9S)-3-amiao-9-methoxy-2,6,8-trimethyl-10-phenyldeca-4,6- dienoic acid. In order to investigate the mode of inhibition and also to probe the active-site binding interactions, we decided to synthesis new analogues of nodularin. Specific inhibitors for either PP1 or PP2A are not presently available, but would be useful biochemical tools in delineating the individual physiological roles of these enzymes. We decided to syntliesise, the potential inhibitor cyclo [betaAla-(2R)-Glu-alpha-OMe-gamma-Pro-(2R)-Asp-alpha-OMe-gamma-(25)-Phe], a stripped-down nodularin macrocycle, and also an analogue which is suitable for synthetic elaboration at the "Adda position". Using solution phase peptide synthesis (LPPS), four such nodularin analogues (both (25)- and (2R)-proline) were synthesised in seventeen steps. The cyclisation between the Phe and Asp residues were carried out using DIPEA under conditions of high dilution. NMR studies (TOCSY, ROESY) have elucidated the three dimensional structures which have been shown to be similar to the natural product, nodularin. A shorter synthesis of these nodularin analogues was developed using solid phase peptide synthesis (SPPS). Two solid phase synthesise of the nodularin macrocycles, cyclo-[betaAla-(2R)- Glu-alpha-OMe-gamma-Pro-(2R)-Asp-beta-(25)-Phe]; one in which Fmoc-(2S)-Phe-betaAla-(2R)-Glu-alpha-OMe-gamma-Pro-(2R)-Asp(alphaO-Wang Resin)-beta-OAllyl is deprotected and then cyclised on the resin prior to cyclisation were found to be successful. Even though the resin-bound synthesis gave low yields for the cyclisation step, compared to the situation in solution, it offered advantages in the construction of the linear isopentapeptide precursor. Initial studies have shown that the nodualrin analogues 130 and 131 are moderate inhibitors (IC50 2.8 mmol) of PP1 when tested using the malachite green system. Studies towards the synthesis of incorporating more suitable Adda functionalities, and the development of a radiolabelled protein phosphatase assay are currently being investigated within the group.