Manipulating Diastereomeric Bicyclononynes to Sensitively Determine Enzyme Activity and Facilitate Macromolecule Conjugations.

Bicyclo[6.1.0]nonyne (BCN) is one of the most commonly used cycloalkynes in strain-promoted azide-alkyne cycloaddition (SPAAC). The synthesis of BCN produces two diastereomers, -BCN and -BCN. The potential significance of the different steric structures of the tricyclic fused rings in SPAAC products synthesized from the BCN diastereomers has not been previously studied. We first demonstrated that only -BCN could reduce the level of fluorescence quenching in SPAAC reaction products. The reduction was likely due to the presence of extended tricyclic fused ring systems. This hypothesis was supported by the synthesis of a fluorescence always-on construct by substituting -BCN for -BCN in a previously reported chemical probe that was characterized with good contact fluorescence quenching. We also synthesized -BCN derivatives to enhance the steric structural differences in the corresponding SPAAC products. A constitutional isomer of the azido-derivatized 5(6)-carboxyfluorescein [5(6)-FAM] was reacted with both --BCN and --BCN compounds. However, one form of the --BCN-based product did not augment contact fluorescence quenching, while a second --BCN product could not further reduce contact fluorescence quenching. Nevertheless, a new fluorescence turn-on chemical probe was employed to determine the activities of two serum biomarkers, butyrylcholinesterase and paraoxonase 1. Moreover, --BCN was exploited to successfully conjugate BSA with a 5-FAM derivative compound.