Photoenzymatic Synthesis of α-Tertiary Amines by Engineered Flavin-Dependent "Ene"-Reductases.

α-Tertiary amines are a common motif in pharmaceutically important molecules but are challenging to prepare using asymmetric catalysis. Here, we demonstrate engineered flavin-dependent 'ene'-reductases (EREDs) can catalyze radical additions into oximes to prepare this motif. Two different EREDs were evolved into competent catalysts for this transformation with high levels of stereoselectivity. Mechanistic studies indicate that the oxime contributes to the enzyme templated charge-transfer complex formed between the substrate and cofactor. These products can be further derivatized to prepare a variety of motifs, highlighting the versatility of ERED photoenzymatic catalysis for organic synthesis.

N-methylmesoporphyrin IX fluorescence as a reporter of strand orientation in guanine quadruplexes.

Guanine quadruplexes (GQ) are four-stranded DNA structures formed by guanine-rich DNA sequences. The formation of GQs inhibits cancer cell growth, although the detection of GQs in vivo has proven difficult, in part because of their structural diversity. The development of GQ-selective fluorescent reporters would enhance our ability to quantify the number and location of GQs, ultimately advancing biological studies of quadruplex relevance and function. N-methylmesoporphyrin IX (NMM) interacts selectively with parallel-stranded GQs; in addition, its fluorescence is sensitive to the presence of DNA, making this ligand a possible candidate for a quadruplex probe. In the present study, we investigated the effect of DNA secondary structure on NMM fluorescence. We found that NMM fluorescence increases by about 60-fold in the presence of parallel-stranded GQs and by about 40-fold in the presence of hybrid GQs. Antiparallel GQs lead to lower than 10-fold increases in NMM fluorescence. Single-stranded DNA, duplex, or i-motif, induce no change in NMM fluorescence. We conclude that NMM shows promise as a 'turn-on' fluorescent probe for detecting quadruplex structures, as well as for differentiating them on the basis of strand orientation.