Exposing Small-Molecule Nanoentities by a Nuclear Magnetic Resonance Relaxation Assay

Small molecules can self-assemble
in aqueous solution into a wide
range of nanoentity types and sizes (dimers, n-mers,
micelles, colloids, etc.), each having their own unique properties.
This has important consequences in the context of drug discovery including
issues related to nonspecific binding, off-target effects, and false
positives and negatives. Here, we demonstrate the use of the spin–spin
relaxation Carr–Purcell–Meiboom–Gill NMR experiment,
which is sensitive to molecular tumbling rates and can expose larger
aggregate species that have slower rotational correlations. The strategy
easily distinguishes lone-tumbling molecules versus nanoentities of
various sizes. The technique is highly sensitive to chemical exchange
between single-molecule and aggregate states and can therefore be
used as a reporter when direct measurement of aggregates is not possible
by NMR. Interestingly, we found differences in solution behavior for
compounds within structurally related series, demonstrating structure–nanoentity
relationships. This practical experiment is a valuable tool to support
drug discovery efforts.