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Fluorescence antibunching is a well-known technique for determining the number of independent emitters per molecule or molecular complex. It was rarely applied to autofluorescent proteins due to the necessity of collecting large numbers of fluorescence photons from a single molecule, which is usually impossible to achieve with rather photolabile autofluorescent proteins. Here, we measure fluorescence antibunching on molecules in solution, allowing us to accumulate data over a large number of molecules. We use that method for determining an average stoichiometry of molecular complexes. The proposed method is absolute in the sense that it does not need any calibration or referencing. We develop the necessary theoretical background and check the method on pure dye solutions and on molecular complexes with known stoichiometry.

Selective and potent P2Y(11) receptor antagonists have yet to be developed, thus impeding an evaluation of this G protein-coupled receptor mainly expressed on immune cells. Taking suramin with moderate inhibitory potency as a template, 18 ureas with variations of the methyl groups of suramin and their precursors were functionally tested at P2Y(11), P2Y(1), and P2Y(2) receptors. Fluorine substitution of the methyl groups of suramin led to the first nanomolar P2Y(11) antagonist (8f, NF157, pK(i): 7.35). For selectivity, 8f was also tested at various P2X receptors. 8f displayed selectivity for P2Y(11) over P2Y(1) (>650-fold), P2Y(2) (>650-fold), P2X(2) (3-fold), P2X(3) (8-fold), P2X(4) (>22-fold), and P2X(7) (>67-fold) but no selectivity over P2X(1). QSAR studies confirm that residues with favored resonance and size parameters in the aromatic linker region can indeed lead to an increased potency as is the case for 8f. A symmetric structure linking two anionic clusters seems to be required for bioactivity. 8f may be helpful for studies evaluating the physiological role of P2Y(11) receptors.