Flavin adenine dinucleotide (FAD), an essential cofactor in cellular metabolism, catalyses a wide range of redox reactions. The organic synthesis of FAD is typically conducted by coupling flavin mononucleotide (FMN) and adenosine monophosphate. The reported synthesis routes have certain limitations such as multiple reaction steps, low yields, and/or difficult-to-obtain starting materials. In this study, we report the synthesis of FAD nucleobase analogues using chemical and enzymatic methods with readily available starting materials achieved in 1-3 steps with moderate yields (10-51%). Further, we demonstrate that Escherichia coli glutathione reductase can use these analogues to catalyse the reduction of glutathione. Finally, we show that FAD nucleobase analogues can also be synthesized inside a cell from cellular substrates FMN and nucleoside triphosphates. This lays the foundation for their use in studying the molecular role of FAD in cellular metabolism and as biorthogonal reagents in biotechnology and synthetic biology applications.
Flavin adenine dinucleotide (FAD) is an essential redox cofactor in cellular metabolism. The organic synthesis of FAD typically involves coupling flavin mononucleotide (FMN) with adenosine monophosphate, however, existing synthesis routes present limitations such as multiple steps, low yields, and/or difficult‐to‐obtain starting materials. In this study, we report the synthesis of FAD nucleobase analogues with guanine/cytosine/uracil in place of adenine and deoxyadenosine in place of adenosine using chemical and enzymatic approaches with readily available starting materials, achieved in 1–3 steps with moderate yields (10–57 %). We find that the enzymatic route using Methanocaldococcus jannaschii FMN adenylyltransferase (MjFMNAT) is versatile and can produce these FAD analogues in high yields. Further, we demonstrate that Escherichia coli glutathione reductase is capable of binding and using these analogues as cofactors. Finally, we show that FAD nucleobase analogues can be synthesized inside a cell from cellular substrates FMN and nucleoside triphosphates by the heterologous expression of MjFMNAT. This lays the foundation for their use in studying the molecular role of FAD in cellular metabolism and as biorthogonal reagents in biotechnology and synthetic biology.
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