Synthetic and Biocatalytic Methods for the Chemoenzymatic Production of Novel Cryptophycin Anticancer Agents.

Abstract

The cryptophycin family of cyanobacterial peptolides contains exceptionally potent antimitotic anticancer agents. Active at levels significantly lower than currently approved cancer therapies, synthetic cryptophycin 52 was also effective against multi-drug resistant cancers. Phase II clinical trials revealed minor peripheral neurotoxicity, however, making synthetic derivatization a priority for the development of safe, effective cryptophycins for the treatment of cancer. Specifically, incorporation of heterocycles on unit A of cryptophycin was proposed to increase the solubility and stability, as well as reduce toxicity of the parent drugs. To this end, an efficient and divergent synthetic route to unit A analogues was developed and optimized for the production of a diverse library of heterocyclic functionality. Incorporation with units B, C, and D yielded fully elaborated, SNAc-thioester bound seco-cryptophycins as substrates for macrocyclization. Cryptophycin thioesterase (CrpTE) activity was reconstituted in vitro and used to demonstrate impressive inherent flexibility for a suite of heterocyclic substrates. CrpTE was then optimized for activity and displayed little preference for reaction temperature, buffer pH, or DMSO concentration. Incredibly, CrpTE was active at up to 50% DMSO and in a variety of organic solvents. In fact, a novel cosolvent system of 20% diglyme with 1% MCD more than doubled CrpTE conversion with a natural substrate mimic and proved to be an effective strategy for the chemoenzymatic cyclization of the 2-pyridyl derivatized cryptophycin 500. Joined with the complementary heterocyclic substrate flexibility of cryptophycin epoxidase (CrpE), a powerful method now exists to produce unique cryptophycins in a campaign to access better anticancer agents. This chemoenzymatic method should also provide a means to construct affinity probes for mechanism of action studies and interrogation of CrpTE and CrpE active site architecture.