Trans cyclooctene nhs ester

The CS bioadhesives were synthesized using previously published methods. 17, 23 Two precursors, CS-Tz and CS-TCO, were produced through carbodiimide crosslinking between primary amine groups on the CS polymer backbone (low MW, Sigma-Aldrich, St Louis, MO) and N-hydroxysuccinimide (NHS) groups in tetrazine-NHS (Tz; Click Chemistry Tools, Scottdale, AZ) and trans-cyclooctene-NHS ester (TCO; Click Chemistry Tools). The Tz/TCO-NHS ester was dissolved in 2 mL dimethyl sulfoxide (62 mM) and mixed with 2 mL CS solution (10 mg/mL in 50 mM MES buffer at pH 6.0). The obtained solution was continuously stirred overnight. The conjugated CS with Tz and TCO solution was purified first via precipitation in excess acetone, washed in dimethylformamide, and then exhaustively dialyzed (molecular cutoff: 2 kDa) against 1% acetic acid and deionized water. After dialysis, CS-Tz/TCO solutions were lyophilized and stored at 4°C until further use. Freeze-dried CS-Tz/TCO was reconstituted in 50 mM MES buffer at 10 mg/mL. To form the bioadhesive, CS-Tz, CS-TCO, and 90 mg/mL co-crosslinker 4-arm PEG-propionaldehyde (10 kDa MW, Laysan Bio, Arab, AL) were mixed in a 5:5:1 ratio.