Cyclotheonamide A (CtA, 1), a cyclic pentapeptide isolated from the marine sponge Theonella sp., is an inhibitor of serine proteases such as alpha-thrombin and trypsin. We describe, in detail; our total synthesis of CtA by a convergent [3 + 2] fragment-condensation route. The requisite protected amino acid starting materials-were processed and converted into two segments, A (13) and B (21), which were coupled with BOP reagent in 75% yield (65% With BOP-CL) to give pentapeptide intermediate 23. After selective removal of the terminal protecting groups On 23, the critical macrocyclization was effected with BOP-Cl in 65% yield under high-dilution conditions to provide 25 (25% yield for 23 --> 25). Macrocycle 25 was then processed in four steps to CtA (1), which was isolated and purified by, HPLC (trifluoroacetate salt). The synthetic CtA was identical to the natural product by 500-MHz H-1 NMR, 100-MHz C-13 NMR, HPLC, TLC, FAB-MS, optical rotation, and bioassay. The C-13 NMR spectrum of CtA in D2O. shows virtually exclusive population by the hydrated form of the alpha-keto amide (gem-diol. structure). We also synthesized cyclotheonamide B (CtB, 2) by utilizing key amine intermediate 28, through an analogous three-step "end game". Our chemical protocol offers a useful vehicle for the systematic preparation of cyclotheonamide analogues, and because of the late-stage primary amine intermediate, analogues with a modified N-acyl or N-alkyl substituent should be conveniently accessible. This could be important for satisfying the hydrophobic S-3 binding pocket of thrombin (viz; Figure 3), which is vacant for the CtA-thrombin complex but effectively utilized by-the standard D-Phe-Pro-Arg tripeptide inhibitors. Other chemical highlights of the synthesis include (1) homologation of a protected arginal via a cyanohydrin to obtain the h-Arg subunit,(2) use throughout of a monoprotected guanidine, and (3) macrocyclic lactam formation with an unprotected hydroxyl substituent. The characteristics of CtA as a thrombin inhibitor were also examined. Either competitive, Michaelis-Menten kinetics or slow,;tight-binding kinetics were, observed, depending on the substrate, the thrombin concentration, and the order of addition of components. Given sufficient time for equilibration of CtA and thrombin, slow-binding inhibition is generally displayed.