Trichloro-s-triazine Activation and Coupling

The three reactive chlorines on TsT have dramatically different reactivities toward nucleophiles in aqueous solution. The first chlorine is reactive toward hydroxyls as well as primary and secondary amine groups at 4°C and a pH of 9.0 (Abuchowski, 1977a Mumtaz and Bachhawat, 1991). Once the first chlorine is coupled, the second one requires at least room temperature conditions at the same pH to react efficiently. If two chlorines are conjugated to nucleophilic groups, the third is even more difficult to couple, requiring at least 80°C at alkaline 

The following protocol for mPEG activation using TsT and its coupling to proteins is based on the protocols of Abuchowski et al. (1977b) and Gotoh et al. (1993). 

Note All operations should be done in a fume hood. Dispose of hazardous waste according to EPA guidelines. 

Dissolve 5.5 g of TsT in 400 ml of anhydrous benzene which contains 10 g of anhydrous sodium carbonate. 

Add to the TsT solution, 50 g of mPEG-5000 (monomethoxypolyethylene glycol having a molecular weight of 5000). Mix well to dissolve. 

---