DESIGN AND PREPARATION OF SERINE-THREONINE PROTEIN PHOSPHATASE INHIBITORS BASED UPON THE NODULARIN AND MICROCYSTIN TOXIN STRUCTURES .1. EVALUATION OF KEY INHIBITORY FEATURES AND SYNTHESIS OF A RATIONALLY STRIPPED-DOWN NODULARIN MACROCYCLE

The natural nodularin and microcystin toxins are powerful but non-sele ctive inhibitors of the ubiquitous and structurally related eukaryotic Ser-Thr protein phosphatases, PP1 and PP2A, enzymes that are intimate ly involved in controlling cellular metabolism Both families of toxin are cyclic tri-isopeptides typified by the presence of two free carbox ylic acid groups, a dehydroamino acid moiety, and a large, rigid exocy clic lipophilic side-chain, To learn how to design specific inhibitors for each enzyme, the nature of specific interactions with potential i nhibitor-conferring moieties in the toxin was considered, Borohydride reduction of the dehydroalanine residue present in microcystin-LR, a p otential Michael acceptor, gave two diastereoisomeric dihydromicrocyst in products, Each of these displayed subnanomolar activities as inhibi tors for PP2A, as for the parent compound, indicating that the dehydro amino acid residue in microcystin and, probably, in nodularin, is not essential for activity, Other conserved features appeared to be requir ed to confer activity, hence strategies towards the synthesis of simpl ified non-dehydroamino acid-containing analogues of each macrocycle ty pe were considered, In each case it was planned to elaborate the lipop hilic side-chain functionality after the formation of the macrocyclic ring, In order to synthesize the precursor nodularin-type macrolactam, two peptide-bond disconnections of the ring were investigated using a model system, ha-OMe-gamma-Sar-(R)-Asp-alpha-OMe-beta-(S)-Phe-], one bond disconnecting between the sarcosine carboxy group and the (2R)-As p N-atom and the other disconnecting between the (2R)-Asp beta-carboxy group and the (2S)-Phe N-atom, preparation of the linear precursors w as achieved using solution-phase chemistry without incident, Macrolact amisation via the displacement of the beta-pentafluorophenyl ester of the (2R)-Asp alpha-methyl ester residue by the free amino group of the (2S)-phenylalanine residue proceeded in excellent yield (89%), but th e alternative strategy failed, Application of the successful macrolact amisation strategy to other nodularin macrocycles and to the construct ion of the microcystin-type macrocycle is described in the following a rticle.