Sodium borodeuteride

Desulfurization
of StsA was adapted from previous work on subtilosin A.^{38 (link)} NiCl₂ hexahydrate (500 μg,
Acros Organics) was added to a 2 mL screw-capped vial. GluC-digested
StsA (125 μg) was dissolved in 150 μL of 60% methanol:
Milli-Q water and transferred to the vial followed by 500 μg
of sodium borohydride (Fluka). The addition of sodium borohydride
causes rapid formation of the nickel boride catalyst as a fine black
particulate and gas evolution. The vial was quickly sealed and heated
for 5 min at 50 °C. Two further portions of 500 μg sodium
borohydride were added followed by 5 min of heating at 50 °C
after each portion. The reaction was quenched with 30 μL of
trifluoroacetic acid (TCI) and the nickel boride particulates were
removed by centrifugation. The supernatant was transferred to a 1.5
mL Eppendorf tube and dried using a Speedvac concentrator (Savant
ISS110). The white residue was reconstituted in 100 μL of 1%
methanol in Milli-Q water, desalted using a C₁₈ ZipTip,
and eluted into 80% methanol Milli-Q water with 1% acetic acid. For
desulfurization under deuterated conditions, the same method was used
with the following modifications: sodium borodeuteride was used in
place of sodium borohydride, anhydrous nickel chloride was used in
place of the hexahydrate salt, and 60% D₄ methanol–D₂O was used as the reaction solvent (Cambridge Isotope Laboratories).