Synthesis and Purification of OT-B12 Conjugate

B₁₂‐DBCO was prepared by combining B₁₂ (25.0 mg, 0.018 mmol) with 1,1′‐carbonyl‐di‐(1,2,4‐trizole) (10.0 mg, 0.061 mmol) in 3 ml of n‐methyl‐pyrrolidone and stirring for 1 h under argon, at which time sulpho‐DBCO‐amine (25.0 mg, 0.059 mmol) and TEA (50 μl) were added to the solution. After an additional hour, a second equivalent of sulpho‐DBCO‐amine and TEA were added, and the reaction stirred overnight. B₁₂‐DBCO was purified using RP‐HPLC (H₂O + 0.1% TFA and MeOH from 1% MeOH/H₂O + 0.1% TFA to 70% MeOH/H₂O + 0.1% TFA in 15 min) to produce B₁₂‐DBCO at 92% purity in 80% yield. MALDI‐TOF‐MS expected m/z = 1808, observed m/z = [M‐CN⁻]⁺ 1782. The B₁₂‐DBCO (12.0 mg, 0.011 mmol) was then reacted with OT (10.0 mg, 0.010 mmol) (Figure 3) by dissolving both compounds in 4:1 DMF/H₂O and allowing the red‐coloured solution to stir gently at room temperature overnight. OT‐B₁₂ was purified using RP‐HPLC (H₂O + 0.1% TFA and MeCN from 1% MeCN/H₂O + 0.1% TFA to 70% MeCN/H₂O + 0.1% TFA in 15 min) to produce OT‐B₁₂ to 95% purity in stochiometric yields. MALDI‐TOF‐MS expected m/z = 2856, observed m/z = [M + H₂O⁺]⁺ 2873.