Expanding the scope of copper artificial metalloenzymes: A potential fluorinase?

• Published in: Journal of inorganic biochemistry 2025-02, Vol.263, p.112777, Article 112777
• Main Authors: Lüddecke, Isabeau, Jarvis, Amanda G.
• Format: Article
• Language: English
• Subjects: Artificial metalloenzymes; Bacterial Proteins; Bioconjugation; Catalysis; Copper - chemistry; Copper-catalysed electrophilic fluorination; Halogenation; Metalloproteins - chemistry; Metalloproteins - metabolism; Oxidoreductases - chemistry; Oxidoreductases - metabolism
• ISSN: 0162-0134; 1873-3344; 1873-3344
• DOI: 10.1016/j.jinorgbio.2024.112777

Biocatalysts for fluorination are rare, and thus of great interest for artificial enzyme design. Biohybrid catalysts including Cu-based DNAzymes and dinucleotide catalysts can catalyse enantioselective electrophilic fluorination of β-ketoesters. Here we report the investigation of Cu-based artificial metalloenzymes as catalysts for electrophilic fluorination reactions. A library of artificial copper proteins was prepared by bioconjugation of bidentate and tridentate nitrogen ligands to cysteine variants of the Sterol Carrier Protein 2 L (SCP-2 L) and subsequent addition of Cu(II) salts. The resulting copper proteins were screened for activity for the fluorination of β-ketoesters using Selectfluor. Under aqueous acidic conditions it was observed that the designed catalysts did not outcompete the uncatalysed background reaction. This work highlights that careful consideration of substrate reactivity and background reactions is needed when considering potential reactions for artificial metalloenzyme catalysis. Artificial SCP-2 L variants were engineered with nitrogen-based copper-binding ligands via cysteine bioconjugation to serve as Lewis acid catalysts for the fluorination of β-ketoesters. The artificial metalloenzymes showed no increase in yield over the background reaction which is promoted in mildly acidic buffers compared to other solvents. [Display omitted] •Twenty-one modified SCP-2 L proteins containing nitrogen-ligands were prepared.•Successful Cu-binding was observed.•Cu-SCP-2 L ArMs do not outcompete the background reaction for the fluorination of β-ketoesters under aqueous conditions.