Abstract
Coenzyme A (CoA) is a ubiquitous cofactor present in every known organism. The thioesters of CoA are core intermediates in many metabolic processes, such as the citric acid cycle, fatty acid biosynthesis and secondary metabolism, including polyketide biosynthesis. Synthesis of CoA-thioesters is vital for the study of CoA-dependent enzymes and pathways, but also as standards for metabolomics studies. In this work we systematically tested five chemo-enzymatic methods for the synthesis of the three most abundant acyl-CoA thioester classes in biology; saturated acyl-CoAs, α,β-unsaturated acyl-CoAs (i.e., enoyl-CoA derivatives), and α-carboxylated acyl-CoAs (i.e., malonyl-CoA derivatives). Additionally we report on the substrate promiscuity of three newly described acyl-CoA dehydrogenases that allow the simple conversion of acyl-CoAs into enoyl-CoAs. With these five methods, we synthesized 26 different CoA-thioesters with a yield of 40% or higher. The CoA esters produced range from short- to long-chain, include branched and α,β-unsaturated representatives as well as other functional groups. Based on our results we provide a general guideline to the optimal synthesis method of a given CoA-thioester in respect to its functional group(s) and the commercial availability of the precursor molecule. The proposed synthetic routes can be performed in small scale and do not require special chemical equipment, making them convenient also for biological laboratories.
Highlights
Coenzyme A (CoA) plays a vital role in all three domains of life
This includes important enzymes in primary metabolism, as well as enzymes involved in natural product biosynthesis
We analyzed all reactions by analytical methylmalonyl-CoA (28) and determined the stereochemical outcome of the latter [7]
Summary
Coenzyme A (CoA) plays a vital role in all three domains of life. Its thioesters are key intermediates in many metabolic and biosynthetic pathways. 5% of all enzymes listed on the BRENDA database use CoA as a substrate or produce it [1]. This includes important enzymes in primary metabolism (e.g., acetyl-CoA carboxylase, fatty acid synthase, pyruvate dehydrogenase, etc.), as well as enzymes involved in natural product biosynthesis (e.g., polyketide synthases). Commercial availability of CoA esters, is limited to only a few compounds, such as acetyl-CoA or malonyl-CoA. This fact often requires researchers to synthesize their own CoA-thioesters starting from CoA or the even more basic building block pantothenic acid [3]. The main difficulties in working with CoA are its insolubility in organic solvents due to the three phosphate groups in the
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