L 6200

Unless otherwise stated, all reagents and solvents were obtained from Sigma‐Aldrich (Sweden) and used without further purification. No‐carrier‐added [¹¹C]CO₂ production was performed using a GEMS PETtrace cyclotron (GE, Uppsala, Sweden). The ¹⁴N(p, α)¹¹C reaction was employed in a pressurized gas target containing nitrogen (nitrogen 6.0) and 1% oxygen (oxygen 4.8) by bombardment with a proton beam (16.5 MeV). HPLC was performed using a Hitachi L‐6200 gradient pump and a Hitachi L‐4000 variable wavelength UV‐detector in a series with a Bioscan β⁺‐flow detector. Analytical HPLC was performed using a Hitachi L‐6200 gradient pump and a Hitachi L‐4000 variable wavelength UV‐detector in series with a Bioscan β⁺‐flow detector. The reverse phase column (Agilent Eclipse XDB‐C18, 5 μm, 4.6 × 150 mm) was eluted with a gradient between acetonitrile (A) and 100mM HCO₂NH₄ (B). The gradient was linear between 10% and 90% (B) over 5 min at a flow rate of 3 ml/min. Identification of all radioactive products was confirmed by co‐elution with the corresponding nonradioactive compound.

Liquid chromatography-mass spectrometry (LC-MS) (Mariner Biospectrometry workstation [McKinley Scientific, Sparta, NJ], Hitachi L-6200 [Hitachi, Tokyo, Japan]) was performed using a Supelco reversed-phase C-18 column (250 mm × 2 mm, 5 μm; Sigma-Aldrich) with an electrospray ionization (ESI) system (positive ion mode). The ESI mass spectrum was presented at 287 mass-to-charge ratio, corresponding to the [M+H]⁺ of brazilin (molecular weight, 286 g/mol).

The purified CGL1 was reduced with tri-n-butylphosphine in 7M guanidine-HCl, 10 mM EDTA, 0.5 M Tris-HCl pH 8.5. The reduced CGL1 was pyridylethylated with 4-vinyl pyridine at 25 °C for 4 h in the dark. The reduced and pyridylethylated CGL1 was chemically cleaved at the methionyl bonds with 1% CNBr in 70% (v/v) formic acid at 25 °C for 24 h by the method of Gross28 (link)35 (link)36 (link). The peptides generated by the CNBr cleavage were separated by reverse-phase high-performance liquid chromatography (RP-HPLC) using HITACHI model L-6200 and L-4200 liquid chromatographs on a Wakosil-II 5C18 AR column (4.6 × 100 mm, Wako Pure Chemical Industries Ltd., Osaka, Japan). The column was equilibrated with solvent A (0.1% trifluoroacetic acid; TFA), and the peptides were eluted at the flow rate of 1 mL/min using a linear gradient of 0–100% solvent B (acetonitrile/water/TFA, 80:20:0.1 [v/v/v]) at room temperature. The separated fractions were collected for sequencing. Automated Edman degradation37 (link)38 39 (link)40 (link) was performed using a gas phase protein sequencer (Shimadzu model PPSQ-21).