An antibody raised against acridinium hapten 1 is shown to catalyze the retro Diels−Alder reaction of the anthracene−HNO cycloadduct 2 to release anthracene 4 and nitroxyl (HNO). Nitroxyl is oxidized to nitric oxide (NO) in the presence of superoxide dismutase. Since the enzyme superoxide dismutase is ubiquitous in vivo, this catalytic antibody system may be equivalent to NO-synthase. Antibody catalysis is triggered by recognition of the phenyl rings in hapten 1 at an angle near that of the transition state of the retro Diels−Alder reaction of 2. Acridinium hapten 1 is in chemical equilibrium with its conjugate Lewis base 9-hydroxyacridane 1-OH (pK ∼ 8.2). Catalytic antibody 9D9 (K M(2) = 100 μM, k cat = 0.07 min-1, k uncat = 3 × 10-4 min-1) is the result of an heterologous immunization and binds both 1 (K i = 16.6 μM at pH 6.1) and 1-OH (K i = 0.9 μM at pH 9.0). The more tightly bound neutral conjugate base 1-OH probably represents a more accurate mimic of the transition state of the retro Diels−Alder process.

A general assay for monitoring catalysis by fluorescence in real time has been developed by use of an antibody sensor. The sensor consists of a product-specific antibody tightly bound to a product analogue covalently labeled with the fluorescent tag acridone. Acridone fluorescence is quenched in the bound state. The reaction is monitored by following the fluorescence increase caused by displacement of the acridonelabeled product from the antibody combining site by the released product. Fluorescence detection of enzymatic hydrolysis of a b-galactoside and butyrate by b-galactosidase and esterase, respectively, are demonstrated. The assay operates by modulation of fluorescence intensity at 445 nm, a signal compatible with currently available instruments measuring in 96-well or 384-well plastic microtiter plates. The method is potentially general and only limited by binding selectivities of the product versus the substrate that can be encountered in antibodies.

No abstract

No abstract

Synthetic details are presented for a series of more or less rigid, “parallel‐in‐plane” preoriented bis(diazenes), with N=N/N=N distances (d) of 3.3−2.9 Å and interorbital angles (ψ) of 142−164° (X‐ray crystal structures). DFT calculations (B3LYP/6−31G*) and one‐/two‐electron reduction experiments with the two least preoriented, most “distant” bis(diazenes) (dN=N/N=N ca. 3.3 Å; ψ 142−146°) provide more insight into the structural prerequisites for bis(homoconjugative) in‐plane electron delocalization in 4N/5e radical anions and 4N/6e dianions. (© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2003)