Marinostatin (MST) isolated from a marine organism is a serine protease inhibitor consisting of 12 amino acids with two internal ester linkages formed between the β-hydroxyl and (β-carboxyl groups, ...Thr^3-Asp^9 and Ser^8-Asp^ . MST was synthesized by regioselective intramolecular esterification employing two sets of orthogonally removable side chain protecting groups for Ser/Thr and Asp. SAR study revealed that the ester linkage with Thr^3-Asp^9, the cis-conformation at Pro^7 and the N-terminal Phe^1-Ala^2 are the structural requirements for expression of the inhibitory activity. These findings were also supported by analyzing the solution and enzyme-bound structures of MST. Of particular note is that cis-Pro^7 may promote the internal hydrogen bond between the NH proton of Are and the carbonyl oxygen atom of the ester linkage with Thr^3-Asp^9 to protect its scissile bond of Met^4-Arg^5. This could be responsible for enhancing the potency. To elucidate the importance of backbone conformation at position 7, 16 and cis/trans-olefin analogs 17/18, in which cis/trans-olefins are substituted for the amide bond of Tyr^6-Ala^7, were synthesized. Although Ala^7 in 16 takes a trans-conformation in the solution structure, it takes a cis-conformation in the enzyme-bound structure. This implies that Ala^7 would isomerize from a trans to cis conformation when it binds to an enzyme, resulting in a certain inhibitory potency. However, the trans-olefin analog 18 lost the potency while the cis-olefin analog 17 displayed almost the same potency as that of MST. These results clearly indicated that the cis-conformation at position 7 is indispensable for binding to an enzyme in a canonical manner. By applying the structural motif of MST, we were able to rationally design protease inhibitory specificities that differed from those of the natural product.
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