Synthesis of Neomycin-Biotin Conjugate

Neomycin trisulfate (contains 6 amino groups per 1 molecule) was taken in a quantity of 0.6 mg (0.66 µM), and biotinamidohexanoyl-6-aminohexanoic acid N-hydroxysuccinimide ester was taken in a quantity of 3 mg (5 µM). Then both preparations were added to 50 µL of C₂H₅OH and incubated for 1 h. The activated biotin derivative was added in excess to minimize the number of unreacted neomycin amino groups. Next, 200 µL of the 10% BSA solution (0.3 µM) was added to the reaction mixture and incubated for 30 min. One BSA molecule contains more than 30 surface amino groups [23 (link)]. That is, 0.3 µM BSA is able to bind more than 9 µM of activated biotin. The resulting conjugate was purified using Amicon Ultracel 10 K filters (Millipore, USA), centrifuging the reaction mixture at 10,000× g for 15 min. The solution passed through the filter containing the target neomycin–biotin conjugate, whereas BSA, which has bound excess biotin, remained on the filter. The same methodology was used to synthesize streptomycin-biotin conjugate.