Synthesis of Novel Analogs of Acetyl Coenzyme A: Mimics of Enzyme Reaction Intermediates

Abstract

An improved method for the synthesis of analogs of coenzyme A (CoA) and its thioesters, which are modified in the thiol or thioester moiety, has been developed using a combination of chemical and enzymatic reactions. The enzymes catalyzing the last two steps of CoA biosynthesis were used to prepare a CoA analog (lc) in which an amide bond is replaced by a thioester bond and the thiol group is replaced by a methyl group. Reaction of lc with a primary amine in aqueous solution results in aminolysis of the thioester linkage to form the desired CoA analog. Reaction with different amines permits the introduction of a variety of functional groups in place of the normal thiol or thioester group. This methodology has been used in the synthesis of five new analogs of acetyl-CoA in which the thioester sulfur is replaced by a methylene group and the acetyl group is replaced by carboxylate (14a), nitro (14b), carboxamide (14c), methyl sulfoxide (14d), and methyl sulfone (14e) groups. 14a-c were designed to mimic the possible enolate or enol intermediate in the reaction of citrate synthase and related enzymes. 14a and 14c are potent inhibitors of citrate synthase, with K-, values 1000-and 570-fold lower than the Km for acetyl-CoA, respectively. CD titrations indicate that 14a and 14c have low affinity for citrate synthase in the absence of oxaloacetate, consistent with their recognition as enol or enolate analogs. 14b is a poor inhibitor of citrate synthase, with affinity slightly lower than that for acetyl-CoA. These results are consistent with generation of the enol form of acetyl-CoA as the nucleophilic intermediate in the reaction of citrate synthase. 14d and 14e were designed to mimic the tetrahedral intermediate or transition state in the reaction of chloramphenicol acetyltransferase and related acetyl-CoA-dependent acetyltransferases. Both compounds are poor inhibitors of chloramphenicol acetyltransferase, with affinities slightly lower than that of acetyl-CoA, indicating that these compounds are not good mimics of the enzyme-bound tetrahedral intermediate or transition state.

Identification of yacE ( coaE ) as the Structural Gene for Dephosphocoenzyme A Kinase in Escherichia coli K-12

“…The K m determined for dephospho-CoA (0.74 mM) is much higher than the value reported for the dephospho-CoA kinase activity of the partially purified bifunctional enzyme from rat liver (0.01 mM) (19) and somewhat higher than the value previously measured for the C. ammoniagenes dephospho-CoA kinase (0.12 mM) (13). Like the rat liver enzyme, maximum activity is observed at a high pH.…”

A Specialized Citric Acid Cycle Requiring Succinyl-Coenzyme A (CoA):Acetate CoA-Transferase (AarC) Confers Acetic Acid Resistance on the Acidophile Acetobacter aceti

“…AarCH6 fluorescence emission showed a maximum at 343 nm, which was quenched 8% by the addition of dethiaacetylCoA, a nonhydrolyzable analogue of acetyl-CoA that has a methylene group replacing the sulfur atom (30). Fluorescence titrations, performed as described in the supplemental material, were used to determine an apparent K d (dissociation constant) (Table 3).…”

One-pot chemoenzymatic preparation of coenzyme A analogues

“…Previous chemoenzymatic approaches to the synthesis of CoA derivatives employed crude enzyme extracts and used pantothenic acid derivatives chemically phosphorylated at position 4 0 [10]. We reasoned that overexpression of the genes encoding E. coli PanK [17], PPAT [18], and DPCK [19] would provide a rapid means of generating novel CoA derivatives directly form readily accessible chemically prepared panthotenic acids.…”

“…Dimethylethylthioaluminum was generated by the slow addition of ethanethiol (5.66 g, 0.09 mol) to a cooled ()10°C) solution of trimethylaluminum (2 M sol. in hexanes, 47.2 ml) in dry CH 2 Cl 2 (146 ml) [10]. The resulting solution was stirred at 0°C for 5 min and then at room temperature for an additional 15 min.…”

“…This approach is limited by the nonspecific reactivity of these activated esters and possible unwanted side reaction at alternate functional groups of CoA. Methods to generate novel CoA analogues by means of either total chemical synthetic strategies [8] or by the use of crude enzyme preparations to adenylate and phosphorylate synthetically generated 4 0 -phosphopantothenate derivatives [9][10][11][12][13] have been reported. This latter approach has been very successfully used to prepare a number of CoA derivatives.…”