Mechanism-based carbocyclic inhibitors of dehydroquinate synthase
Abstract
A mechanism-based approach to inhibitor design is applied to the study of dehydroquinate synthase. Carbocyclic inhibitors are identified that mimic either the substrate 3-deoxy- scD-arabino-heptulosonic acid 7-phosphate (DAHP), a reactive intermediate, or a transition state. The enzymology associated with these inhibitors uncovers important clues regarding the enzyme mechanism. A strategy for the synthesis of carbocyclic phosphonate inhibitors is described which is based on nucleophilic phosphonomethylation of epoxides. Epimeric carbaphosphonate (1S-($1\alpha,3\alpha, 4\beta, 5\alpha)$) -1,3,4-trihydroxy-5-(phosphonomethyl)cyclohexane-1-carboxylic acid, a diastereoisomer of the substrate analogue carbaphosphonate (1R-($1\alpha, 3\beta, 4\alpha, 5\beta$)) -1,3,4-trihydroxy-5-(phosphonomethyl)cyclohexane-1-carboxylic acid, is efficiently synthesized in ten steps from quinic acid. Epimeric carbaphosphonate is a nanomolar-level, slowly reversible inhibitor of dehydroquinate synthase with an association rate constant k$\sb{\rm on}$ = 1.5 $\times$ 10$\sp4$ M$\sp{-1}\rm s\sp{-1}$, a dissociation rate constant k$\sb{\rm off}$ = 1.1 $\times$ 10$\sp{-4}$ s$\sp{-1}$, and an inhibition constant K$\sb{\rm i}$ = 7.3 $\times$ 10$\sp{-9}$ M. In order to probe the origin of the slowly reversible inhibition displayed by the carbaphosphonates, C-4 deoxycarbaphosphonate is synthesized and tested in vitro. C-4 Deoxycarbaphosphonate is a surprisingly potent, slowly reversible inhibitor (k$\sb{\rm on}$ = 1.0 $\times$ 10$\sp3$ M$\sp{-1}\rm s\sp{-1}$, k$\sb{\rm off}$ = 5.4 $\times$ 10$\sp{-4}$ s$\sp{-1}$, K$\sb{\rm i}$ = 5.4 $\times$ 10$\sp{-7}$ M). This indicates that, contrary, to previous hypotheses, the enzyme-catalyzed oxidation of the C-4 hydroxyl group is not essential for slow-binding inhibition of dehydroquinate synthase. The first apparent irreversible inhibitor of dehydroquinate synthase is also described. Incubation of C4-ketocarbaphosphonate with the enzyme resulted in pseudo first order loss of enzyme activity which was not recovered even after dialysis of the inactive complex. The inhibition observed with ketocarbaphosphonate suggests a general strategy to inhibit other biologically important enzymes which also exploit nicotinamide dinucleotide (NAD$\sp{+}$) as a catalyst rather than a cosubstrate. Additional DHQ synthase inhibitors possessing a C-4 carbonyl are targeted for synthesis, using a new strategy based on intramolecular functionalization of quinic acid derivatives. Efforts using this strategy for the synthesis of two reactive intermediate analogues are described. The newly developed synthetic methodology also provides access to inhibitors which may function as transition state analogues.
Degree
Ph.D.
Advisors
Frost, Purdue University.
Subject Area
Organic chemistry
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