An artificial nickel chlorinase based on the biotin–streptavidin technology

Herein, we report on an artificial nickel chlorinase (ANCase) resulting from anchoring a biotinylated nickel-based cofactor within streptavidin (Sav). The resulting ANCase was efficient for the chlorination of diverse C(sp3)–H bonds. Guided by the X-ray analysis of the ANCase, the activity of the artificial chlorinase could be significantly improved. This approach opens interesting perspectives for late-stage functionalization of organic intermediates as it complements biocatalytic chlorination strategies.


Materials and methods
All commercially-available chemicals were purchased from Sigma-Aldrich, Acros Organics, TCI Europe, Fluorochem and used without further purification.All substrates and reference products were purchased from Sigma-Aldrich.Dry solvents were purchased from Acros Organics and used directly without further purification.The water used for all biological and catalytic experiments was purified with a Milli-Q Advantage system.

Instrumentation
1 H and 13 C spectra were recorded on a Bruker 500 MHz at room temperature.Chemical shifts are reported in ppm (parts per million) relative to TMS ( = 0.00 ppm for 1 H and 13 C).Signals are quoted as s (singlet), d (doublet), t (triplet), bs (broad singlet) and m (multiplet).Spectra were analyzed on MestReNova and calibrated relative to the residual solvent peak.Electron-Spray Ionization Mass Spectra (ESI-MS) were recorded on a Bruker FTMS 4.7T bioAPEX II.
High-resolution mass spectra (HRMS) were measured on a Bruker maXis 4G QTOF ESI mass spectrometer.GC-MS analysis of catalysis was performed on a Shimadzu GC-2010 Plus system coupled with a GC-MS-QP2020 detector (column: Agilent HP-5 (30 m × 0.25 mm × 0.25 μm)) using He as carrier gas and naphthalene as internal standard.GC analysis of catalysis was performed on a GC-FID equipped with a Astec ® CHIRALDEX TM G-TA Capillary GC Column (50 m 0.25 mm 0.12 μm) using He as carrier gas and naphthalene as internal standard.
Circular Dichroism (CD) spectra were recorded on a Chirascan from Applied Photophysics at 25 o C using a quartz cell (1 cm path length).Flash chromatography was performed on a Biotage Isolera or a Buchi Pure chromatography system.Preparative HPLC purifications were carried out with a Water Prep LC 4000 System with an Agilent XDB-C18 column (21.2 × 150 mm, 5 μm).No unexpected or unusually high safety hazards were encountered.
The residue was used for next step directly.Step 2: The residue from step 1 was dissolved in dry DCM (10 mL) and then was added dropwise at 0 º C within 1 h to a solution of 8aminoquinoline (929 mg, 6.45 mmol) and triethylamine (1.30 g, 19.3 mmol).The reaction mixture was stirred at room temperature overnight.The solution was filtered through a celite pad and concentrated in vacuo.The residue was purified by silica gel flash chromatography (DCM/EA = 98/2 -80/20) to afford the crude bis-amidoquinoline S3.A more thorough purification was carried out after the biotinylation step.

Synthesis of the cofactor [(Biot C4 -bAQ)Ni] 1: 4
Triethylamine (19 mg, 0.19 mmol, 4.0 equiv) was added to a solution of ligand S4 (0.047 mmol, 1.0 equiv) in dry DMF (3 mL) under a flow of nitrogen.The mixture was stirred for 15 min at room temperature, and then NiCl2• 6H2O (10 mg, 0.043 mmol, 1.1 equiv) was added.The mixture was stirred at room temperature overnight.The solvent was removed in vacuo.The resulting residue was purified by washing with DCM to afford the corresponding Ni-cofactor Green solid.[(Biot C4 -bAQ)Ni] 1 was synthesized following the general procedure in 82% yield. 1

HABA displacement titration for determining the binding constant of [(Biot C4 -bAQ)Ni] 1 for Sav WT
6][7] In a quartz cuvette, a solution of streptavidin WT (Sav WT, tetrameric, initial concentration 8 µM, 2.4 mL, 0.0192 µmol, 1.0 equiv) in PBS buffer (20 mM, pH 7) was added.A solution of 2-(4'hydroxyazobenzene)benzoic acid (HABA, 9.6 mM, 300 µL, 2.88 µmol, 150 equiv) in PBS buffer (20 mM, pH 7) was added and the mixture was incubated for 5 min to ensure full saturation of the biotin-binding sites.A blank (PBS buffer only) was measured at 506 nm and the absorbance of the HABA• Sav solution was determined.Aliquots of [(Biot C4 -bAQ)Ni] 1 (0.96 mM in DMSO) or biotin (0.96 mM in DMSO) were added to the HABA• Sav solution in 0.50 equiv.step (10 µL per step, up to 5.0 equiv).The CD spectrum (at 506 nm) was recorded 2 minutes after each addition and the molar ellipticity was plotted against the equivalents of [(Biot C4 -bAQ)Ni] 1 or biotin added.The decrease of the CD signal ceased once all the HABA was displaced by [(Biot C4 -bAQ)Ni] 1 or biotin.The measured data were fitted according to a published procedure. 8, 9 )) Abbreviations: The data collection was carried out at the Swiss Light Source beam line PSI at a wavelength of 1.0000 Å. XDS 10 was used for crystal indexing, integration and AIMLESS 11 for scaling, within the graphical interface CCP4i2 12 of the CCP4 suite.The structures were solved by molecular replacement using PHASER-MR 13 and the streptavidin structure PDB:7ZOF as search model.
Refinement was carried out by REFMAC5 14 and for structure modeling and electron-density visualization COOT 15 was used.Ligand restraints were generated using eLBOW 16 .Figures
were generated with PyMOL (the PyMOL Molecular Graphics System, Version 2.5.0,Schrödinger, LLC).Data collection and refinement statistics are listed in TableS1.The data have been deposited under PDB ID 8QQ3.Four monomers are present in the asymmetric unit (corresponding to the homotetrameric Sav WT structure, space group C121).Residual electron density in the Fo-Fc map was observed in the biotin-binding site of streptavidin for all four subunits, in molecule A-C the electron density around the cofactor was strong whereas in molecule D the electron density was weaker.Furthermore, anomalous dispersion density was observed.Modeling of the cofactor [(Biot C4 -bAQ)Ni] 1 into the electron density projected the nickel in the position of the anomalous density peak.Finally, in three of the four Sav subunits the cofactor was modelled.

Figure S4 .] 1 •Figure S5 .Figure S6 .
Figure S3.(a) Calibration curve used to determine TON for chlorocyclohexane, using naphthalene as internal standard.(b) GC-MS analysis of a mixture containing commercially available chlorocyclohexane, cyclohexanon and naphthalene, used as a sample for creating the calibration curve.(c) GC-MS analysis resulting from a reaction giving highest TON catalyzed