I am working on a reductive amination conjugation reaction between a tag I've synthesized and a glycan (I am using lactose to test out the protocol). Total molecular weight of the conjugate is 689 g/mol. The tag includes a TEMPO group. I've tried varying concentrations of glacial acetic acid and a water negative control to decompose the sodium cyanoborohydride. The solvent was then evaporated under vacuum and the insoluble salt was removed before analysis. After analysis via mass spec, I've noticed that the TEMPO was removed from the conjugate in all samples (including the negative control). I've determined that the TEMPO was undesirably removed from the tag to form 1-hydroxy-2,2,6,6-tetramethylpiperidine via reaction with BH2CN. The rest of the tag reacted to form tag+BHCN.

Also, I should mention that I did indeed notice hydrogen gas bubbling once the acetic acid solutions were added to the samples: BH3CN- + H+ --> BH2CN + H2 .

I am wondering if anyone can figure out what could possibly work in preventing this destruction of the conjugate. I am thinking of repeating this same experiment but at lower temperatures (please note that I ran the original experiment at RT) to see if the TEMPO-based conjugate can be preserved. What do you think about this?