Antibody catalysis of the oxidation of water.

Recently we reported that antibodies can generate hydrogen peroxide (H2O2) from singlet molecular oxygen (1O2*). We now show that this process is catalytic, and we identify the electron source for a quasi-unlimited generation of H2O2. Antibodies produce up to 500 mole equivalents of H2O2 from 1O2*, without a reduction in rate, and we have excluded metals or Cl- as the electron source. On the basis of isotope incorporation experiments and kinetic data, we propose that antibodies use H2O as an electron source, facilitating its addition to 1O2* to form H2O3 as the first intermediate in a reaction cascade that eventually leads to H2O2. X-ray crystallographic studies with xenon point to putative conserved oxygen binding sites within the antibody fold where this chemistry could be initiated. Our findings suggest a protective function of immunoglobulins against 1O2* and raise the question of whether the need to detoxify 1O2* has played a decisive role in the evolution of the immunoglobulin fold.

Antibodies have the intrinsic capacity to destroy antigens.

Research throughout the last century has led to a consensus as to the strategy of the humoral component of the immune system. The essence is that, for killing, the antibody molecule activates additional systems that respond to antibody-antigen union. We now report that the immune system seems to have a previously unrecognized chemical potential intrinsic to the antibody molecule itself. All antibodies studied, regardless of source or antigenic specificity, can convert molecular oxygen into hydrogen peroxide, thereby potentially aligning recognition and killing within the same molecule. Aside from pointing to a new chemical arm for the immune system, these results may be important to the understanding of how antibodies evolved and what role they may play in human diseases.

A one-pot multistep approach to alpha-azido-phosphonate and phosphonothioate diesters: key intermediates in the synthesis of haptens for the generation of antibody ligases.

A four-step, one-pot synthesis of mixed alpha-azido-phosphonates and phosphonothioates 12a-d is described. This chemistry has provided a facile route to haptens 6a b and 7 that have been employed for the elicitation of antibody ligases. Five hapten-specific antibodies have been identified as modest catalysts of a model peptide ligation reaction between thioester 1b and thiol 2 to give the amide product 5.

A soluble polymer-supported triflating reagent: a high-throughput synthetic approach to aryl and enol triflates.

[reaction: see text] The high-yielding synthesis and application of the first example of a polymer-supported reagent for the preparation of trifluoromethanesulfonates (triflates) is described. This new reagent efficiently triflates aryl alcohols and lithium enolates in high yield (>90%). A simple precipitation and filtration to remove the excess reagent and byproduct facilitate purification of the triflate products. The PEG-supported approach is highly efficient, as the PEG-supported byproduct can be quantitatively recovered and recycled into reagent 1.

A bait and switch hapten strategy generates catalytic antibodies for phosphodiester hydrolysis.

General base catalysis supplied by the histidine-12 (H-12) residue of ribonuclease (RNase) A has long been appreciated as a major component of the catalytic power of the enzyme. In an attempt to harness the catalytic power of a general base into antibody catalysis of phosphodiester bond hydrolysis, the quaternary ammonium phosphate 1 was used as a bait and switch hapten. Based on precedence, it was rationalized that this positively charged hapten could induce a counter-charged residue in the antibody binding site at a locus suitable for it to deprotonate the 2'-hydroxyl group of the anhydroribitol phosphodiester substrate 2. After murine immunization with hapten 1, mAb production yielded a library of 35 antibodies that bound to a BSA-1 conjugate. From this panel, two were found to catalyze the cyclization-cleavage of phosphodiester 2. Kinetic studies at pH 7.49 (Hepes, 20 mM) and 25 degreesC showed that the most active antibody, MATT.F-1, obeyed classical Michaelis-Menten kinetics with a Km = 104 microM, a kcat = 0.44 min-1, and a kcat/kuncat = 1.7 x 10(3). Hapten 1 stoichiometrically inhibits the catalytic activity of the antibody. MATT.F-1 is the most proficient antibody-catalyst (1.6 x 10(7) M-1) yet generated for the function of phosphodiester hydrolysis and emphasizes the utility of the bait and switch hapten paradigm when generating antibody catalysts for processes for which general-base catalysis can be exploited.