P maleimidophenyl isocyanate

Hydroxyapatite (20 µm, ⩾97%, synthetic), (3-mercaptopropyl) triethoxysilane (MPTES) (⩾95%), Propan-2-ol (puriss, p.a., ACS reagent, ⩾99.8% (GC)), HCl (ACS reagent, 37%), potassium hydroxide (reagent grade, 90%), mEthanol (CHROMASOLV^®, ⩾99.9%), p-maleimidophenyl isocyanate (PMPI) (purum, ⩾97%) and sodium borohydride (NaBH₄) (99.99% trace metals basis) were acquired from Sigma-Aldrich Ltd (UK). Ethanol (absolute, analytical reagent grade), dimethyl sulfoxide (DMSO) (analytical reagent grade) and Ellman’s reagent (5,5′-Dithio-bis-(2-nitrobenzoic acid)) were acquired from Fisher Scientific (UK). Acheson Silver DAG was acquired from Agar Scientific (UK). VICTREX^® PEEK™ 450PF (25 µm, easy fine flow) was acquired from Victrex plc (UK). Kapton^® polyimide film was acquired from DuPont™ (USA). Loctite^® Frekote^® 44-NC mould release agent was acquired from Henkel (Germany). Distilled water acquired from an arium^® advance EDI pure water system by Sartorius (Germany).

17.5 mg of 6-amino-2-cyanobenzothiazole (CBT, Sigma) (0.1 mmol) and 10.7 mg of p-maleimidophenyl isocyanate (Sigma) (0.05 mmol) were dissolved in 1 mL of anhydrous dimethyl sulfoxide and stirred for 30 min at room temperature. The product was purified by preparative high-performance liquid chromatography (HPLC) on an Agilent 1260 Infinity system equipped with a Supelcosil PLC-18 12 μm, 250 × 21.2 mm column. The mobile phase consisted of 0.1% formic acid in H₂O (buffer A), and 0.1% formic acid in acetonitrile (buffer B). A gradient from 5 to 95% of buffer B in buffer A was run over 30 min at a flow rate of 5 mL min⁻¹. The separation was monitored at 335 nm. The identity of the product was confirmed by mass spectrometry (MS) and nuclear magnetic resonance (NMR) (Supplementary Fig. 1a, b).