A combined experimental and theoretical study of the pH‐dependent binding mode of NAD+ by water‐soluble molecular clips

The highly selective recognition process of NAD+ and NADH (as important cofactors of many redox enzymes) by molecular clips in aqueous solution is studied systematically by a combined experimental and quantum‐chemical approach. The strongly pH‐dependent complexation‐induced 1H NMR shifts of the guest molecule indicate that in buffered aqueous solution at pH = 7.2 the nicotinamide ring, the active site of NAD+, is preferentially bound inside the cavity of the molecular clip, whereas in pure water under slightly acidic conditions both units (the nicotinamide ring as well as the adenine moiety) are located outside the cavity. The latter finding is explained by a competing self‐aggregation of NAD+ which prohibits the recognition process. In addition, the investigation of the NAD+ fragments NMNA, NMN, and AMP as well as the comparison of measured and computed chemical shieldings provides information on possible binding modes. Under equal conditions the binding of NADH to the molecular clip is significantly weaker than that of NAD+, so that the oxidation states (NAD+/NADH) can be distinguished by the molecular clips. The nucleotides NMN and AMP are bound less strongly by the molecular clips than NAD+. The weaker binding indicates that multiple aromatic π−π and cation−π host–guest interactions only possible in NAD+ have a synergetic effect on the complex stability. In addition to the inhibition of the cofactor NAD+, a further implication is the development of sensors since a quenching of fluorescence is observed for specific molecular clips by the addition of NAD+. Copyright © 2009 John Wiley & Sons, Ltd. The highly selective recognition process of NADR and NADH (as important cofactors of many redox enzymes) by molecular clips in aqueous solution is studied systematically by a combined experimental and quantum‐chemical approach.