I. The synthesis of YC-1 analogues for a photoaffinity study of soluble guanylate cyclase. II. Synthesis of kifunensine and analogues as inhibitors of endoplasmic reticulum alpha mannosidase I.

Abstract

The research presented in this thesis comprises work from two separate projects. The first project involved the design, synthesis and enzymatic screening of photolabile analogues of the substituted benzylindazole compound known as YC-1, a known activator of the enzyme soluble guanylate cyclase (sGC), for use in a photoaffinity study to identify the YC-1 binding site within sGC. Several benzophenone and azide-containing YC-1 analogues were synthesized and a 6-azido-YC-1 analogue, found to activate the enzyme in manner similar to YC-1, was used in a photoaffinity study and found to bind to the alpha-subunit of sGC. The second project presented in this thesis involved the development of large scale preparation of the potent class I alpha-mannosidase inhibitor kifunensine from L-ascorbic acid. In addition to providing a means to synthesize gram quantities of kifunensine, this route provided access to a number of novel <italic>N</italic>-1 substituted kifunensine analogues that may be used as biochemical tools in the study of class I alpha-mannosidases. One of the main goals of this thesis research was to design and synthesize a kifunensine pseudodisaccharide to mimic the Man(alpha1,2)Man disaccharide portion of the natural oligosaccharide substrate of class I alpha-mannosidases. Such a compound could prove useful in mechanistic studies of these enzymes and may lead to the development of kifunensine derivatives that are specific for endoplasmic reticulum alpha-mannosidase I over other class I alpha-mannosidases. To this end, several tethered pseudodisaccharide kifunensine analogues were synthesized that were shown to cause significant inhibition of human endoplasmic reticulum alpha-mannosidase I (human ER Man I) when assayed at 5 mM concentrations. In addition, an affinity-bound kifunensine analogue was prepared for use in the purification of class I alpha-mannosidases. While several of the <italic> N</italic>-1 substituted kifunensine analogues were good inhibitors of human ER Man I at 5 mM concentrations, the affinity-bound kifunensine analogue exhibited poor inhibition of this enzyme. The results obtained from this study demonstrate that <italic>N</italic>(1)-substituted kifunensine analogues are capable of inhibiting class I alpha-mannosidases, although they are not nearly as potent of inhibitors as kifunensine itself.