We develop a fluorescent ruthenium metalloglycocluster for use as a powerful molecular probe in evaluating the binding between carbohydrates and lectins by fluorescence emission (FE) and fluorescence polarization (FP) analyses. Changes in the FE and FP of these metalloglycoclusters are measured following the addition of lectin [peanut agglutinin (PNA), Ricinus communis agglutinin 120, Concanavalin A (ConA), or wheat germ agglutinin] or tetanus toxin c-fragment (TCF). After the addition of PNA, the FE spectrum of [Ru(bpy-2Gal)3] shows a new emission peak and the FP value of [Ru(bpy-2Gal)3] increases. Similarly, the FE spectrum of [Ru(bpy-2Glc)3] shows a new emission peak and the FP value increases on addition of ConA. Because other combinations of metalloglycoclusters and lectins show little change, specific binding of galactose to PNA and that of glucose to ConA are confirmed by the FE and FP measurements. Resulting dissociation constants (Kd) prove that the metalloglycoclusters with highly clustered carbohydrates show higher affinity for the respective lectins than those with less clustered carbohydrates. Furthermore, specific binding of [Ru(bpy-2Gal)3] to TCF was confirmed by the FP measurement.
We have developed a fluorescent ruthenium metalloglycocluster as a powerful molecular probe for evaluating a binding
event between carbohydrates and lectins by fluorescence emission (FE) and fluorescence polarization (FP) analysis. The
fluorescent ruthenium metalloglycoclusters, [Ru(bpy-2Gal)3] and [Ru(bpy-2Glc)3], possess clustered galactose and
glucose surrounding the ruthenium center. Changes in FE and FP of these metalloglycoclusters were measured by adding
each lectin (Peanut agglutinin (PNA), Ricinus communis agglutinin 120 (RCA), Concanavalin A (ConA), or Wheat germ
agglutinin (WGA)) or tetanus toxin c-fragment (TCF). Following the addition of PNA, the FE spectrum of [Ru(bpy-
2Gal)3] showed new emission peak and the FP value of [Ru(bpy-2Gal)3] increased. Similarly, the FE spectrum of
[Ru(bpy-2Glc)3] showed new emission peak and the FP value increased following the addition of ConA. Since other
combinations of the metalloglycoclusters and lectin caused little change, specific bindings of galactose to PNA and
glucose to ConA were proved by the FE and FP measurement. From nonlinear least-squares fitting, dissociation
constants (Kd) of [Ru(bpy-2Gal)3] to PNA was 6.1 μM, while the Kd values of [Ru(bpy)2(bpy-2Gal)] to PNA was ca. 10-4
M. Therefore, the clustered carbohydrates were proved to increase affinity to lectins. Furthermore, the FP measurements
proved specific binding of [Ru(bpy-2Gal)3] to TCF.
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