Controllable stereoinversion in DNA-catalyzed olefin cyclopropanation via cofactor modification†

The assembly of DNA with metal-complex cofactors can form promising biocatalysts for asymmetric reactions, although catalytic performance is typically limited by low enantioselectivities and stereo-control remains a challenge. Here, we engineer G-quadruplex-based DNA biocatalysts for an asymmetric cyclopropanation reaction, achieving enantiomeric excess (eetrans) values of up to +91% with controllable stereoinversion, where the enantioselectivity switches to −72% eetrans through modification of the Fe-porphyrin cofactor. Complementary circular dichroism, nuclear magnetic resonance, and fluorescence titration experiments show that the porphyrin ligand of the cofactor participates in the regulation of the catalytic enantioselectivity via a synergetic effect with DNA residues at the active site. These findings underline the important role of cofactor modification in DNA catalysis and thus pave the way for the rational engineering of DNA-based biocatalysts.


Materials and Methods
Unless otherwise noted, all chemicals and reagents for chemical reactions were obtained from commercial suppliers (Sigma-Aldrich, Acros, TCI, Frontier scientific) and used without further purification. The DNA sequences were all purchased from Sangon (Shanghai, China). The DNA strand concentrations were determined by measuring the UV absorbance of sample at 260 nm by using the molar extinction coefficient values provided by the manufacturer. Water purified on a Milli-Q A10 water purification system (specific resistance of 18.2 MΩ at 25 o C) was used for all experiments.
High Performance Liquid Chromatography (HPLC). The enantioselectivity was determined by Agilent HPLC 1260 analysis using Daicel chiralcel OJH column and Daicel CHIRALPAK-IJ column with a UV-detector by using ethanol, isopropanol and n-hexane as eluents at 25 °C. Circular Dichroism (CD) Spectroscopy. All CD spectra were recorded on a dual beam DSM 1000 CD spectrophotometer (Olis, Bogart, GA) with a 10 mm or 1.5 mm path-length quartz cell. Each measurement was recorded from 220 to 400 nm at 20 ºC under N 2 purge. The scan rate was 0.5 nm per second. The average scan for each sample was subtracted by a background CD spectrum of corresponding buffer solution.
UV Melting Experiment. UV melting experiments were carried out on Shimadzu 2450 S4 spectrophotometer (Shimadzu, Japan) equipped with a Peltier temperature control accessory. A sealed quartz cell with a path length of 1.0 cm was used. The UV melting curves of the G-quadruplexes and G4-based biocatalysts were monitored by UV absorption at 295 nm with a heating rate of 0.5 °C/min. Data were analyzed by using Origin 8 software. The melting temperatures (T m ) can be obtained from the best sigmoidal curve fit of the melting profile.

UV-Vis Absorption Titration Experiments. Absorption spectra were measured on
Shimadzu 2600 spectrophotometer (Shimadzu, Japan) with a 1 cm path-length quarter cell. UV-vis absorption titrations were carried out by the stepwise addition of Gquadruplex solution to a cell containing FeTMPyPn (n = 4, 3, 2). Absorption spectra were recorded in the range of 300-550 nm at room temperature. The titration was terminated when the wavelength and intensity of the Soret band for FeTMPyPn did not change any more upon three successive additions of G-quadruplexes. Fluorescence quench titration assay. 2 Fluorescently labelled oligonucleotide was dissolved in assay buffer (potassium phosphate buffer 10 mM, pH 7.0), which is in agreement with the catalytic buffer. The resultant strand concentration of oligonucleotide was 100 nM. FeTMPyPn (n = 4, 3, 2) was prepared at a concentration of 5 mM in water and diluted to an appropriate concentration before titration. The fluorescence experiments were recorded on a FLS920 fluorescence spectrometer (Edinburgh) with a 1 cm path length quartz cuvette at 20 o C. Fluorescence intensity of FAM-labeled DNA was recorded after the addition of FeTMPyPn. Interval time S6 between two titration points was 10-15 minutes in order to reach the binding equilibrium. Each quench titration assay was conducted in triplicate. If not stated otherwise, the titration curve was fitting as one site specific binding by using software

S8
The reaction mixture was allowed to stirrer at room temperature overnight. The pure product was obtained after flash chromatography.

Synthesis of cofactor FeTMPyPn. 4
The pH of an aqueous solution of H 2 TMPyP (0.15 mmol) in 60 ml water was adjusted to 2 (with 1 M HCl), and a 40-fold molar excess FeCl 2 .4H 2 O was added and the solution was stirred and heated under reflux. The course of the metalation was followed by the decrease of the fluorescence of the metal-free porphyrin using UV light at 356 nm. The metalation was completed in 24 hours. The solution was filtered through a filter paper.
The Fe porphyrin was precipitated as the PF 6salt with a saturated aqueous solution of NH 4 PF 6 (2 ml). The precipitate was thoroughly washed with diethyl ether (5 × 5 mL).
The dried precipitate was then dissolved in acetone (the smallest possible amount) and precipitated as the chloride salt with saturated acetone solution of methyl-trinoctylammonium chloride (2 mL). The precipitate was washed with acetone and dissolved in the smallest possible amount of water. The whole precipitation procedure was repeated once again to ensure high purity.