Levuglandins (LG)D2 and LGE2 are γ-ketoaldehyde levulinaldehyde derivatives with prostanoid side chains produced by spontaneous rearrangement of the endoperoxide intermediate PGH2 in the biosynthesis of prostaglandins. Covalent adduction of LGs with the amyloid peptide Aβ1–42 promotes formation of the type of oligomers that have been associated with neurotoxicity and are a pathologic hallmark of Alzheimer’s disease. Within 1 min of their generation during the production of PGH2 by cyclooxygenation of arachidonic acid, LGs are sequestered by covalent adduction to proteins. In view of this high proclivity for covalent adduction, it is understandable that free LGs have never been detected in vivo. Recently a catabolite, believed to be an oxidized derivative of LGD2 (ox-LGD2), a levulinic acid hydroxylactone with prostanoid side chains, was isolated from the red alga Gracilaria edulis and detected in mouse tissues and in the lysate of phorbol-12-myristate-13-acetate-treated THP-1 cells incubated with arachidonic acid. Such oxidative catabolism of LGD2 is remarkable because it must be outstandingly efficient to prevail over adduction with proteins and because it requires a unique dehydrogenation. We now report a concise total synthesis that confirms the molecular structure proposed for ox-LGD2. The synthesis also produces ox-LGE2, which readily undergoes allylic rearrangement to Δ6-ox-LGE2.

Oxidative stress and angiogenesis have been implicated not only in normal phenomena such as tissue healing and remodeling but also in many pathological processes. However, the relationships between oxidative stress and angiogenesis still remain unclear, although oxidative stress has been convincingly demonstrated to influence the progression of angiogenesis under physiological and pathological conditions. The retina is particularly susceptible to oxidative stress because of its intensive oxygenation and high abundance of polyunsaturated fatty acyls. In particular, it has high levels of docosahexanoates, whose oxidative fragmentation produces 4-hydroxy-7-oxo-5-heptenoic acid lactone (HOHA-lactone). Previously, we found that HOHA-lactone is a major precursor of 2-(ω-carboxyethyl)pyrrole (CEP) derivatives, which are tightly linked to age-related macular degeneration (AMD). CEPs promote the pathological angiogenesis of late-stage AMD. We now report additional mechanisms by which HOHA-lactone promotes angiogenesis. Using cultured ARPE-19 cells, we observed that HOHA-lactone induces secretion of vascular endothelial growth factor (VEGF), which is correlated to increases in reactive oxygen species and decreases in intracellular glutathione (GSH). Wound healing and tube formation assays provided, for the first time, in vitro evidence that HOHA-lactone induces the release of VEGF from ARPE-19 cells, which promotes angiogenesis by human umbilical vein endothelial cells (HUVEC) in culture. Thus, HOHA-lactone can stimulate vascular growth through a VEGF-dependent pathway. In addition, results from MTT and wound healing assays as well as tube formation experiments showed that GSH-conjugated metabolites of HOHA-lactone stimulate HUVEC proliferation and promote angiogenesis in vitro. Previous studies demonstrated that HOHA-lactone, through its CEP derivatives, promotes angiogenesis in a novel Toll-like receptor 2-dependent manner that is independent of the VEGF receptor or VEGF expression. The new studies show that HOHA-lactone also participates in other angiogenic signaling pathways that include promoting the secretion of VEGF from retinal pigmented epithelial cells.

Our research on the roles of lipid oxidation in human disease is guided by chemical intuition. For example, we postulated that 2-(ω-carboxyethyl)pyrrole (CEP) derivatives of primary amines would be produced through covalent adduction of a γ-hydroxyalkenal generated, in turn, through oxidative fragmentation of docosahexaenoates. Our studies confirmed the natural occurrence of this chemistry, and the biological activities of these natural products and their extensive involvements in human physiology (wound healing) and pathology (age-related macular degeneration, autism, atherosclerosis, sickle cell disease, and tumor growth) continue to emerge. This perspective recounts these discoveries and proposes new frontiers where further developments are likely. Perhaps more significantly, it depicts an effective chemistry-based approach to the discovery of novel biochemistry.

4-Hydroxy-7-oxo-5-heptenic acid (HOHA)-lactone is a biologically active oxidative truncation product released (t1/2 = 30 min at 37 °C) by nonenzymatic transesterification/deacylation from docosahexaenoate lipids. We now report that HOHA-lactone readily diffuses into retinal pigmented epithelial (RPE) cells where it is metabolized. A reduced glutathione (GSH) Michael adduct of HOHA-lactone is the most prominent metabolite detected by LC-MS in both the extracellular medium and cell lysates. This molecule appeared inside of ARPE-19 cells within seconds after exposure to HOHA-lactone. The intracellular level reached a maximum concentration at 30 min and then decreased with concomitant increases in its level in the extracellular medium, thus revealing a unidirectional export of the reduced GSH-HOHA-lactone adduct from the cytosol to extracellular medium. This metabolism is likely to modulate the involvement of HOHA-lactone in the pathogenesis of human diseases. HOHA-lactone is biologically active, e.g., low concentrations (0.1–1 μM) induce secretion of vascular endothelial growth factor (VEGF) from ARPE-19 cells. HOHA-lactone is also a precursor of 2-(ω-carboxyethyl)pyrrole (CEP) derivatives of primary amino groups in proteins and ethanolamine phospholipids that have significant pathological and physiological relevance to age-related macular degeneration (AMD), cancer, and wound healing. Both HOHA-lactone and the derived CEP can contribute to the angiogenesis that defines the neovascular “wet” form of AMD and that promotes the growth of tumors. While GSH depletion can increase the lethality of radiotherapy, because it will impair the metabolism of HOHA-lactone, the present study suggests that GSH depletion will also increase levels of HOHA-lactone and CEP that may promote recurrence of tumor growth.

γ-Hydroxy-α,β-unsaturated aldehydes, generated by oxidative damage of polyunsaturated phospholipids, form pyrrole derivatives that incorporate the ethanolamine phospholipid (EP) amino group such as 2-pentylpyrrole (PP)-EP and 2-(ω-carboxyalkyl)pyrrole (CAP)-EP derivatives: 2-(ω-carboxyethyl)pyrrole (CEP)-EP, 2-(ω-carboxypropyl)pyrrole (CPP)-EP, and 2-(ω-carboxyheptyl)pyrrole (CHP)-EP. Because EPs occur in vivo in various forms, a complex mixture of pyrrole-modified EPs with different molecular weights is expected to be generated. To provide a sensitive index of oxidative stress, all of the differences in mass related to the glycerophospholipid moieties were removed by releasing a single CAP-ethanolamine (ETN) or PP-ETN from each mixture by treatment with phospholipase D. Accurate quantization was achieved using the corresponding ethanolamine-d4 pyrroles as internal standards. The product mixture obtained by phospholipolysis of total blood phospholipids from sickle cell disease (SCD) patients was analyzed by LC-MS/MS. The method was applied to measure CAP-EP and PP-EP levels in blood plasma from clinical monitoring of SCD patients. We found uniformly elevated blood levels of CEP-EP (63.9 ± 9.7 nM) similar to mean levels in blood from age-related macular degeneration (AMD) patients (56.3 ± 37.1 nM), and 2-fold lower levels (27.6 ± 3.6 nM, n = 5) were detected in plasma from SCD patients hospitalized to treat a sickle cell crisis, although mean levels remain higher than those (12.1 ± 10.5 nM) detected in blood from healthy controls. Plasma levels of CPP-EPs from SCD clinic patients were 4-fold higher than those of SCD patients hospitalized to treat a sickle cell crisis (45.1 ± 10.9 nM, n = 5 versus 10.9 ± 3.4 nM, n = 6; p < 0.002). PP-EP concentration in plasma from SCD clinic patients is nearly 4.8-fold higher than its level in plasma samples from SCD patients hospitalized to treat a sickle cell crisis (7.06 ± 4.05 vs 1.48 ± 0.92 nM; p < 0.05). Because CAP-EPs promote angiogenesis and platelet activation, the elevated levels present in SCD blood can contribute to the hypercoaguability and vaso-occlusive events that are critical pathophysiologic features of SCD.

Oxidative stress causes lipid-derived oxidative modification of biomolecules that has been implicated in many pathological states. Phospholipids containing polyunsaturated fatty acids are major targets of free radical-initiated oxidation. Phospholipids that incorporate docosahexaenoate (DHA) are highly enriched in important neural structures including the brain and retina, where DHA comprises 40% and 60% of total fatty acids, respectively. Oxidative fragmentation of 2-docosahexaenoyl-1-palmityl-sn-glycerophosphocholine generates esters of 4-hydroxy-7-oxohept-5-enoic acid (HOHA) and 4-keto-7-oxohept-5-enoic acid (KOHA) with 2-lysophosphatidylcholine, HOHA-PC, and KOHA-PC. Covalent HOHA adducts that incorporate the primary amino groups of proteins and ethanolamine phospholipids in carboxyethylpyrrole (CEP) derivatives were detected immunologically with anti-CEP antibodies in human tumors, retina, and blood. Now, we generated an anti-OHdiA antibody to test the hypothesis that KOHA adducts, which incorporate the primary amino groups of proteins or ethanolamine phospholipids in 4-oxo-heptanedioic (OHdiA) monoamide derivatives, are present in vivo. However, whereas the anti-CEP antibody is highly specific and does not cross-react with the OHdiA monoamide epitope, the anti-OHdiA monoamide antibody cross-reacted with CEP epitopes making it of little value as an analytical tool for OHdiA monoamides but suggesting the possibility that OHdiA monoamides would exhibit receptor-mediated biological activity similar to that of CEP. An LC-MS/MS method was developed that allows quantification of OHdiA derivatives in biological samples. We now find that KOHA-PC forms OHdiA monoamide adducts of proteins and ethanolamine phospholipids and that OHdiA-protein levels are significantly higher than OHdiA-ethanloamine phospholipid levels in blood from healthy human subjects, 0.45 μM and 0.18 μM, respectively (n = 3, and p = 0.027). OHdiA monoamide epitopes are angiogenic, causing TLR2-dependent adhesion and tube formation by human umbilical vein endothelial cells. OHdiA monoamide epitopes are only slightly less potent than CEP epitopes that contribute to the pathological angiogenesis of age-related macular degeneration and tumor growth.

Isolevuglandins (isoLGs) are stereo and structurally isomeric γ-ketoaldehydes produced through free radical-induced oxidation of arachidonates. Some isoLG isomers are also generated through enzymatic cyclooxygenation. Post-translational modification of proteins by isoLGs is associated with loss-of-function, cross-linking and aggregation. We now report that a low level of modification by one or two molecules of isoLG has a profound effect on the activity of a multi subunit protease, calpain-1. Modification of one or two key lysyl residues apparently suffices to abolish catalytic activity. Covalent modification of calpain-1 led to intersubunit cross-linking. Hetero- and homo-oligomers of the catalytic and regulatory subunits of calpain-1 were detected by SDS–PAGE with Western blotting. N-Acetyl-glycyl-lysine methyl ester and β-amyloid(11–17) peptide EVHHQKL were used as models for characterizing the cross-linking of protein lysyl residues resulting from adduction of iso[4]LGE2. Aminal, bispyrrole, and trispyrrole cross-links of these two peptides were identified and fully characterized by mass spectrometry. Aminal and bispyrrole dimers were both detected. Furthermore, a complex mixture of derivatives of the bispyrrole cross-link containing one or more additional atoms of oxygen was found. Interesting differences are evident in the predominant cross-link type generated in the reaction of iso[4]LGE2 with these peptides. More aminal cross-links versus bispyrrole are formed during the reaction of the dipeptide with iso[4]LGE2. In contrast, more bispyrrole versus aminal cross-links are formed during the reaction of EVHHQKL with iso[4]LGE2. It is tempting to speculate that the EVHHQKL peptide–pyrrole modification forms noncovalent aggregates that favor the production of covalent bispyrrole cross-links because β-amyloid(11–17) tends to spontaneously oligomerize.

2-(ω-Carboxyethyl)pyrrole (CEP) derivatives of proteins were previously shown to have significant pathological and physiological relevance to age-related macular degeneration, cancer and wound healing. Previously, we showed that CEPs are generated in the reaction of ε-amino groups of protein lysyl residues with 1-palmityl-2-(4-hydroxy-7-oxo-5-heptenoyl)-sn-glycero-3-phosphatidylcholine (HOHA-PC), a lipid oxidation product uniquely generated by oxidative truncation of docosahexanenate-containing phosphatidylcholine. More recently, we found that HOHA-PC rapidly releases HOHA-lactone and 2-lyso-PC (t1/2 = 30 min at 37 °C) by nonenzymatic transesterification/deacylation. Now we report that HOHA-lactone reacts with Ac-Gly-Lys-OMe or human serum albumin to form CEP derivatives in vitro. Incubation of human red blood cell ghosts with HOHA-lactone generates CEP derivatives of membrane proteins and ethanolamine phospholipids. Quantitative analysis of the products generated in the reaction HOHA-PC with Ac-Gly-Lys-OMe showed that HOHA-PC mainly forms CEP-dipeptide that is not esterified to 2-lysophosphatidycholine. Thus, the HOHA-lactone pathway predominates over the direct reaction of HOHA-PC to produce the CEP-PC-dipeptide derivative. Myleoperoxidase/H2O2/NO2– promoted in vitro oxidation of either 1-palmityl-2-docosahexaneoyl-sn-glycero-3-phosphatidylcholine (DHA-PC) or docosahexaenoic acid (DHA) generates HOHA-lactone in yields of 0.45% and 0.78%, respectively. Lipid oxidation in human red blood cell ghosts also releases HOHA-lactone. Oxidative injury of ARPE-19 human retinal pigmented epithelial cells by exposure to H2O2 generated CEP derivatives. Treatment of ARPE-19 cells with HOHA-lactone generated CEP-modified proteins. Low (submicromolar), but not high, concentrations of HOHA-lactone promote increased vascular endothelial growth factor (VEGF) secretion by ARPE-19 cells. Therefore, HOHA-lactone not only serves as an intermediate for the generation of CEPs but also is a biologically active oxidative truncation product from docosahexaenoate lipids.

Oxidation of docosahexaenoate phospholipids produces 4-hydroxy-7-oxo-hept-5-eonyl phospholipids (HOHA-PLs) that react with protein lysyl ε-amino residues to generate 2-ω-carboxyethylpyrrole (CEP) derivatives, endogenous factors that induce angiogenesis in the retina and tumors. It seemed likely, but remained unproven, that HOHA-PLs react with ethanolamine phospholipids (EPs) in vivo to generate CEP-EPs. We now show that CEP-EPs are present in human blood at 4.6-fold higher levels in age-related macular degeneration plasma than in normal plasma. We also show that CEP-EPs are pro-angiogenic, inducing tube formation by human umbilical vein endothelial cells by activating Toll-like receptor 2. CEP-EP levels may be a useful biomarker for clinical assessment of AMD risk and CEP-associated tumor progression and a tool for monitoring the efficacy of therapeutic interventions.

A β-hydroxy hydroperoxide was obtained through base-catalyzed disproportionation of a hydroperoxy endoperoxide available by singlet oxygenation of cyclohepta-1,4-diene. Vitamins E and C induce fragmentation of this β-hydroxy hydroperoxide generating aldehydes, especially in the presence of redox active metal ions such as those present in vivo, e.g., under conditions of “iron overload”. This chemistry may contribute to the oxidative cleavage of polyunsaturated fatty acyls that produces similar aldehydes, which damage proteins and DNA through covalent adduction resulting in “oxidative injury”.

Elevated levels of lysophosphatidylcholine (lysoPC), present in oxidatively damaged low-density lipoprotein (oxLDL), are implicated in cardiovascular complications. LysoPC is generated by free radical-catalyzed oxidation of polyunsaturated PCs to oxidatively truncated phosphophatidylcholines (oxPCs). It is known that oxPCs are especially susceptible to hydrolysis by platelet-activating factor acetylhydrolase, a phospholipase (PL) A2 that exists in plasma largely in association with LDL. Drugs that aim to prevent the generation of lysoPC by inhibiting this PLA2-catalyzed hydrolysis are in advanced clinical trials. We now report that spontaneous deacylation oxPCs, such as 1-palmityl-2-(4-hydroxy-7-oxo-5-heptenoyl)-sn-glycero-3-phosphocholine, occurs readily under physiological conditions of temperature and pH (t1/2 = 30 min at 37 °C and pH 7.4). We also show that this reaction proceeds through an intramolecular transesterification mechanism. Because antiphospholipase drugs cannot block this nonenzymatic pathway to lysoPC, additional therapeutic measures may be needed to avoid the pathological consequences of the newly discovered biomolecular chemistry of oxPCs.

Biologically active phospholipids that incorporate an oxidatively truncated acyl chain terminated by a γ-hydroxyalkenal are generated in vivo. The γ-hydroxyalkenal moiety protrudes from lipid bilayers like whiskers that serve as ligands for the scavenger receptor CD36, fostering endocytosis, e.g., of oxidatively damaged photoreceptor cell outer segments by retinal pigmented endothelial cells. They also covalently modify proteins generating carboxyalkyl pyrroles incorporating the ε-amino group of protein lysyl residues. We postulated that γ-hydroxyalkenals could be generated, e.g., in the eye, through fragmentation of hydroperoxy endoperoxides produced in the retina through reactions of singlet molecular oxygen with polyunsaturated phospholipids. Since phospholipid esters are far more abundant in the retina than free fatty acids, we examined the influence of a membrane environment on the fate of hydroperoxy endoperoxides. We now report that linoleate hydroperoxy endoperoxides in thin films and their phospholipid esters in biomimetic membranes fragment to γ-hydroxyalkenals, and fragmentation is stoichiometrically induced by vitamin E. The product distribution from fragmentation of the free acid in the homogeneous environment of a thin film is remarkably different from that from the corresponding phospholipid in a membrane. In the membrane, further oxidation of the initially formed γ-hydroxyalkenal to a butenolide is disfavored. A conformational preference for the γ-hydroxyalkenal, to protrude from the membrane into the aqueous phase, may protect it from oxidation induced by lipid hydroperoxides that remain buried in the lipophilic membrane core.

Basic research, exploring the hypothesis that 2-(ω-carboxyethyl)pyrrole (CEP) modifications of proteins are generated nonenzymatically in vivo is delivering a bonanza of molecular mechanistic insights into age-related macular degeneration, autism, cancer, and wound healing. CEPs are produced through covalent modification of protein lysyl ε-amino groups by γ-hydroxyalkenal phospholipids that are formed by oxidative cleavage of docosahexaenate-containing phospholipids. Chemical synthesis of CEP-modified proteins and the production of highly specific antibodies that recognize them preceded and facilitated their detection in vivo and enabled exploration of their biological occurrence and activities. This investigational approach, from the chemistry of biomolecules to disease phenotype, is proving to be remarkably productive.

Basic research, exploring the hypothesis that γ-hydroxyalkenal phospholipids are generated in vivo through oxidative cleavage of polyunsaturated phospholipids, is delivering a bonanza of molecular mechanistic insights into cardiovascular disease. Rather than targeting a specific pathology, these studies were predicated on the presumption that a fundamental understanding of lipid oxidation is likely to provide critical insights into disease processes. This investigational approach, from the chemistry of biomolecules to disease phenotype, that complements the more common opposite paradigm, is proving remarkably productive.

The diverse biological activities of γ-hydroxyalkenal phospholipids and their involvement in disease are the subject of intense study. Phospholipid aldehydes, such as the 4-hydroxy-7-oxohept-5-enoic acid ester of 2-lyso-phosphatidylcholine (HOHA-PC), the 5-hydroxy-8-oxo-6-octenoic acid ester of 2-lyso-PC (HOOA-PC), and the 9-hydroxy-12-oxododec-10-enoic acid ester of 2-lyso-PC (HODA-PC), are generated by oxidative cleavage of polyunsaturated fatty acyl phospholipids. To facilitate investigations of their chemistry and biology, we now report efficient total synthesis of HOOA, HODA, and HOHA phospholipids. Because the target γ-hydroxyalkenals readily decompose through oxidation of the aldehyde group to a carboxylic acid or through cyclization to furans, these synthesis generate the sensitive functional array of the target phospholipids under mild conditions from acetal derivatives that are suitable for long-term storage.Herein, we report efficient total synthesis of HOOA-, HODA-, and HOHA-phosphatidylcholines.

Protein modifications in which the ε-amino group of lysyl residues is incorporated into a 2-(ω-carboxyethyl)pyrrole (CEP) are mediators of age-related macular degeneration (AMD). They promote both angiogenesis into the retina (‘wet AMD’) and geographic retinal atrophy (‘dry AMD’). Blood levels of CEPs are biomarkers for clinical prognosis of the disease. To enable mechanistic studies of their role in promoting AMD, for example, through the activation of B- and T-cells, interaction with receptors, or binding with complement proteins, we developed an efficient synthesis of CEP derivatives, that is especially effective for proteins. The structures of tryptic peptides derived from CEP-modified proteins were also determined. A key finding is that 4,7-dioxoheptanoic acid 9-fluorenylmethyl ester reacts with primary amines to provide 9-fluorenylmethyl esters of CEP-modified proteins that can be deprotected in situ with 1,8-diazabicyclo[5.4.0]undec-7-ene without causing protein denaturation. The introduction of multiple CEP-modifications with a wide variety of CEP:protein ratios is readily achieved using this strategy.Proteins in which the ε-amino groups of lysyl residues are incorporated into 2-(ω-carboxyethyl)-pyrroles are mediators of age-related macular degeneration. We report an efficient synthesis that accommodates a wide variety of CEP:protein ratios. Reaction of proteins with 4,7-dioxoheptanoic acid 9-fluorenylmethyl ester, and in situ deprotection with DBU provides CEP–proteins without causing denaturation. The structures of tryptic peptides derived from CEP–proteins were also determined.

(E)-4-Hydroxynon-2-enal (HNE), an electrophilic bifunctional cytotoxic lipid peroxidation product, forms covalent adducts with nucleophilic side chains of amino acid residues. HNE-derived adducts have been implicated in many pathophysiological processes including atherosclerosis, diabetes, and Alzheimer’s disease. Tritium- and deuterium-labeled HNE (d4-HNE) were used orthogonally to study adduction with proteins and individual nucleophilic groups of histidyl, lysyl, and cysteine residues. Using tritium-labeled HNE, we detected the binding of 486 molecules of HNE per low-density lipoprotein (LDL) particle, significantly more than the total number of all reactive nucleophiles in the LDL particle. This suggests the formation of adducts that incorporate multiple molecules of HNE with some nucleophilic amino acid side chains. We also found that the reaction of a 1:1 mixture of d4-HNE and d0-HNE with N-acetylhistidine, N-acetyl-Gly-Lys-OMe, or N-acetyl cysteine generates 1:1, 2:1, and 3:1 adducts, which exhibit unique mass spectral signatures that aid in structural characterization. A domino-like reaction of initial 1:1 HNE Michael adducts of histidyl or lysyl nucleophiles with multiple additional HNE molecules forms 2:1 and 3:1 adducts that were structurally characterized by tandem mass spectrometry.