Affinity precipitation using thermo-responsive, water-soluble polymers as matrix; synthetic studies toward the ageliferins

Isolated from marine sponges, many bromopyrrole alkaloids have become natural products of intense scientific interest. The oroidin-derived class of dimeric bromopyrrole alkaloids that include ageliferin, bromoageliferin, and dibromoageliferin exhibit interesting biological properties, including actomycin ATPase, antiviral and antibacterial activities. As a prelude to the total synthesis of ageliferin, an intramolecular Diels-Alder (IMDA) reaction was explored as a means to introduce the three contiguous stereocenters. Toward this end, various IMDA precursors were synthesized, and several strategies for the synthesis of IMDA substrates were devised and explored. Affinity chromatography is a powerful technique that enables the purification of a specific protein from a complex mixture. However, traditional affinity chromatography techniques are somewhat limited due to the solid state of the matrix. To overcome these limitations, the utility of a soluble polymer as an alternative affinity matrix was explored for the isolation of natural product receptors. This polymer displays physical properties that make it an ideal matrix for protein isolation and purification. The parent polymer is soluble in aqueous solution at 4 ?C and precipitates once the mixture reaches 32 ?C. Furthermore, employing this polymer for affinity chromatography may limit the nonspecific binding of proteins. It is also possible to determine the ligand loading using standard analytical techniques such as 1H and 13C NMR. For proof of concept, a dexamethasone-containing macroligand was synthesized to isolate the known glucocorticoid receptor. In addition, a cyclosporin A-containing macroligand was synthesized and employed to isolate the well known and more robust cyclophilins. In this work, it was demonstrated that indeed the ligand loading can be determined by 1H NMR technique. In addition, it was established, as expected, that the water solubility of the macroligands varies based on the hydrophilicity and hydrophobicity of the ligand and degree of ligand loading.