Here, we report evidence for the production of ozone in human disease. Signature products unique to cholesterol ozonolysis are present within atherosclerotic tissue at the time of carotid endarterectomy, suggesting that ozone production occurred during lesion development. Furthermore, advanced atherosclerotic plaques generate ozone when the leukocytes within the diseased arteries are activated in vitro. The steroids produced by cholesterol ozonolysis cause effects that are thought to be critical to the pathogenesis of atherosclerosis, including cytotoxicity, lipid-loading in macrophages, and deformation of the apolipoprotein B-100 secondary structure. We propose the trivial designation "atheronals" for this previously unrecognized class of steroids.

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.

Recently, we showed that antibodies catalyze the generation of hydrogen peroxide (H2O2) from singlet molecular oxygen (1O2*) and water. Here, we show that this process can lead to efficient killing of bacteria, regardless of the antigen specificity of the antibody. H2O2 production by antibodies alone was found to be not sufficient for bacterial killing. Our studies suggested that the antibody-catalyzed water-oxidation pathway produced an additional molecular species with a chemical signature similar to that of ozone. This species is also generated during the oxidative burst of activated human neutrophils and during inflammation. These observations suggest that alternative pathways may exist for biological killing of bacteria that are mediated by potent oxidants previously unknown to biology.

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.

Recent work in our laboratory showed that products formed by the antibody-catalyzed water-oxidation pathway can kill bacteria. Dihydrogen peroxide, the end product of this pathway, was found to be necessary, but not sufficient, for the observed efficiency of bacterial killing. The search for further bactericidal agents that might be formed along the pathway led to the recognition of an oxidant that, in its interaction with chemical probes, showed the chemical signature of ozone. Here we report that the antibodycatalyzed water-oxidation process is capable of regioselectively converting antibody-bound benzoic acid into para-hydroxy benzoic acid as well as regioselectively hydroxylating the 4-position of the phenyl ring of a single tryptophan residue located in the antibody molecule. We view the occurrence of these highly selective chemical reactions as evidence for the formation of a shortlived hydroxylating radical species within the antibody molecule. In line with our previously presented hypothesis according to which the singlet-oxygen ( 1 O* 2) induced antibody-catalyzed wateroxidation pathways proceeds via the formation of dihydrogen trioxide (H 2O3), we now consider the possibility that the hydroxylating species might be the hydrotrioxy radical HO 3• , and we point to the remarkable potential of this either H 2O3-or O3-derivable species to act as a masked hydroxyl radical (HO • ) in a biological environment.

Continuing studies into the utility of poly(ethylene glycol) (PEG)-supported triarylphosphines as functional polymer reagents in liquid-phase organic synthesis (LPOS) are being pursued. This report describes the synthesis and NMR characterization of an aryl−alkyl ether-linked PEG-triarylphosphine derivative (2) and its subsequent application in LPOS. The utility of 2 as a mild stoichiometric reagent for ozonide reduction has been demonstrated, and a direct comparison between 2, a Merrifield resin-bound triarylphosphine derivative, and a solution-phase triphenylphosphine reagent revealed that the highest observed yields occur under liquid-phase conditions. Transformation of phosphine 2 into a phosphonium salt (3) then allowed the inherent aqueous solubility of PEG-functionalized moieties to be exploited by enabling a Wittig reaction, between a range of aldehydes and 3, to occur under mildly basic aqueous conditions. This led to the generation of substituted stilbenes in good to excellent yields. Finally, regeneration of 2 was achieved by reduction of the PEG-supported triphenylphosphine oxide byproduct 4 with alane (100% conversion, 75% yield). This combination of reaction, recovery, and regeneration expands the utility of PEG-supported triarylphosphine reagents across the spectra of both organic chemistry and solution-phase combinatorial strategies.

Ongoing efforts to unravel the origins of the cholesterol 5,6-secosterols (1a and 1b) in biological systems have revealed that the two known chemical routes to these oxysterols; ozonolysis of cholesterol (3) and Hock-cleavage of 5-α-hydroperoxycholesterol (4a), are distinguishable based upon the ratio of the hydrazone derivatives (2a-b) formed in each case and this ratio offers an insight into the chemical origin of the secosterols in vivo.In a recent report, Pratt and co-workers 1 have shown that Hock-cleavage of 5α-hydroperoxycholesterol (4a), that can arise from the singlet oxygen ene reaction with cholesterol (3), occurs faciley under acidic conditions in organic solvents leading to the formation of primarily cholesterol 5,6-secosterol atheronal-B (1b). Atheronal-A (1a) is either not formed at all, or is a minor component in participating solvents such as ethanol (Fig. 1).We discovered the cholesterol 5,6-secosterols 1a and 1b within human atherosclerotic plaque material in vivo 2 and surmised that, based upon the wealth of literature in the field of chemical-, biological-and auto-oxidation of cholesterol at the time, 3, 4 that only ozone was capable of generating 1a from cholesterol (3). 2 In fact, we considered the presence of 1a [measured as 2a after extraction and derivatization with 2,4-dinitrophenyl (2,4-DNP) hydrazine] within inflammatory arteries as indirect evidence that an oxidant with the chemical signature of ozone may be being generated during atherosclerosis progression. We also showed that 1a undergoes an almost instantaneous aldolization process to form 1b in whole blood and therefore the cholesterol 5,6-secosterols 1a and 1b were both potential signature molecules for cholesterol ozonolysis in vivo. 2 The oxysterols 1a and 1b are proatherogenic 5 and induce protein misfolding and amyloidogenesis 6 in a number of biologically-relevant proteins. We find that the 2,4-DNP hydrazone of atheronal-B (2b) is observed at levels above untreated hLDL (~ 0.01 % of 3), after 2,4-DNP hydrazine derivization of hLDL that has been oxidized in aqueous buffer by superoxide anion ( Tables 1-3).The photosensitized and hPMN-mediated oxidation of cholesterol in hHDL in aqueous buffer (PBS, pH 7.4) parallel the observations with hLDL in that the 2,4-DNP hydrazone of cholesterol secosterol 2b is elevated above untreated levels and no 2a is observed (ESI Tables 4-5). Similarly, the photosensitized oxidation of 3 in liposomes (DOPC 80 mol %; 3 5 mol %; PIP-2 15 mol %) in PBS (pH 7.4) implicates a 1 O 2 pathway to 2b, because the measured levels of 2b are elevated in PBS containing D 2 O, relative to H 2 O (ESI Table 6).One of the clearest observations of these aqueous hLDL oxidation studies is that the 2,4-DNP hydrazone of atheronal-A (2a) is only observed after ozonolysis (Table 1). Thus, passage of an ozone/oxygen mixture over the surface of an hLDL solution containing catalase in aqueous buffer (pH 7.4), followed by 2,4-DNP hydrazine derivatization of the lipid extract yields 2a and 2b in a ratio of ~ 4:1...

SUMMARY Epidemiologic and clinical evidence points to an increased risk of cancer when coupled with chronic inflammation. However, the molecular mechanisms that underpin this interrelationship remain largely unresolved. Herein we show that the inflammation-derived cholesterol 5,6-secosterol aldehydes, atheronal-A (KA) and –B (ALD), but not the PUFA-derived aldehydes 4-hydroxynonenal (HNE) and 4-hydroxyhexenal (HHE), induce misfolding of wild-type p53 into an amyloidogenic form that binds thioflavin T and Congo Red dyes but cannot bind to a consensus DNA sequence. Treatment of lung carcinoma cells with KA and ALD leads to a loss of function of extracted p53, as determined by analysis of extracted nuclear protein and in activation of p21. Our results uncover a plausible chemical link between inflammation and cancer and expands the already pivotal role of p53 dysfunction and cancer risk.