Free-radical reactions induced by OH-radical attack on cytosine-related compounds: a study by a method combining ESR, spin trapping and HPLC.

Free-radical reactions induced by OH-radical attack on cytosine-related compounds were investigated by a method combining ESR, spin trapping with 2-methyl-2-nitrosopropane and high-performance liquid chromatography (HPLC). Cytidine, 2'-deoxycytidine, cytidine 3'-monophosphate, cytidine 5'-monophosphate, 2'-deoxycytidine 5'-monophosphate and their derivatives, of which 5,6-protons at the base moiety were replaced by deuterons, and polycytidylic acid (poly(C] were employed as samples. OH radicals were generated by X-irradiating an N2O-saturated aqueous solution. Five spin adducts were separated by HPLC. Examination of them by ESR spectroscopy and UV photospectrometry showed that spin adducts assigned to C5 and C6 radicals due to OH addition to the 5,6 double-bond, a deaminated form of the spin adduct derived from a C5 radical due to the cyclization reaction between C5' of the sugar and C6 of the base, and a spin adduct assigned to the C4' radical due to H abstraction by OH radicals were produced. From these results the sites of OH-radical attack and the subsequent radical reactions in cytosine-related compounds were clarified.     

A micellar prodrug of paclitaxel conjugated to cyclotriphosphazene

A novel water soluble and biodegradable cyclotriphosphazene-paclitaxel conjugate was prepared by reacting 2'-succinyl paclitaxel with cyclotriphosphazenes bearing equimolar glycyl-L-lysine and methoxy poly(ethylene glycol) as side groups. The macromolecular conjugate was found to self-assemble in aqueous solution to form stable micelles with a mean hydrodynamic diameter of 24.7 nm and a low critical micelle concentration of 10 mg/L. The present conjugate exhibited lower than free paclitaxel but reasonably high in vitro cytotoxicity against selected human tumor cells due to their hydrolytic degradation in PBS solution.     

Asymmetric synthesis of functionalized carbocycles and heterocycles

Anions of 1-halo-4-hexenyl phosphonamides derived from chiral, enantiopure C2 symmetrical 1,2-diamino cyclohexane react at the gamma-position in conjugate addition reactions with alpha, beta-unsaturated carbonyl compounds such as cyclopentenone, 4-(H)-furanone, pyrroline-2-one, and cinnamates to give functionalized adducts. Addition to imines is also possible. The adducts can be transformed into enantiopure or enriched carbocyclic and heterocyclic compounds bearing useable functionality.     

Hemin (Fe(3+))-- and heme (Fe(2+))--smectite conjugates as a model of hemoprotein based on spectrophotometry

Hemin (Fe(3+)) was adsorbed onto synthetic smectite (clay mineral) in acetone to form a hemin-smectite conjugate. The hemin-smectite conjugate became soluble in water to form a transparent colloidal solution with a dark brown color. Its absorption spectrum in water showed a sharp Soret band at 398 nm with the molar extinction coefficient as epsilon(398nm) = 11.6 x 10(4) M(-1) cm(-1), which is in good agreement with epsilon(398nm) = (12.2 +/- 3) x 10(4) M(-1) cm(-1) of monomeric hematin (1). Hemin (Fe(3+))-smectite conjugate had a peroxidase-like activity in the presence of hydrogen peroxide (a hydrogen acceptor) and guaiacol (a hydrogen donor) in aqueous solution and its activity was higher than that of hematin. Hemin (Fe(3+))-smectite conjugate in water was reduced by adding sodium dithionite to form a heme (Fe(2+))-smectite conjugate which is also a transparent colloidal solution in water. Its absorption spectrum in aqueous solution was surprisingly in close agreement with that of oxyhemoglobin. Its peak positions of alpha, beta, and Soret bands were located in only a 9--3 nm shift to shorter wavelengths in comparison with those of oxyhemoglobin. Therefore, heme (Fe(2+))-smectite conjugate was bound to O(2) to form O(2)-heme (Fe(2+))-smectite conjugate. The addition of carbon monoxide, CO, to O(2)-heme (Fe(2+))-smectite conjugate caused the formation of CO-heme (Fe(2+))-smectite conjugate with a similar absorption spectrum of carboxyhemoglobin (HbCO) accompanied by shifting 8--10 nm to shorter wavelength. Therefore, the transformation of O(2)-heme (Fe(2+))-smectite conjugate to CO-heme (Fe(2+))-smectite conjugate was accompanied by shifting of 7, 4, and 3 nm to shorter wavelengths in the alpha, beta, and Soret bands respectively, which are similar to the spectral change from oxyhemoglobin to carboxyhemoglobin. Also the ratio (1:1.6) of the molar extinction coefficient of Soret band of O(2)-heme (Fe(2+))-smectite conjugate and CO-heme (Fe(2+))-smectite conjugate was surprisingly agreement with ratio (1:1.5) of oxyhemoglobin and carboxyhemoglobin. The phenomenon shown above was unexpectedly found during the course of study of bioconjugate of a bioactive substance, hemin (Fe(3+)) or heme (Fe(2+)), and a clay mineral, smectite, in place of the protein of globin in hemoglobin.     

The number and distances of positive charges of polyamine side chains in a series of perylene diimides significantly influence their ability to induce G-quadruplex structures and inhibit human telomerase

We have synthesized eight polyamine perylene diimides to conjugate the efficiency of perylene derivatives in stabilizing G-quadruplex structures and the polyamines' biological activity, due to specific interactions with different DNA domains. Our study was carried out by investigating the ability of these derivatives to induce inter- and intramolecular G-quadruplex structures by polyacrylamide gel electrophoresis (PAGE) and to inhibit telomerase in a modified TRAP assay. The two properties appear to be satisfactorily correlated and they show that the number and distances of positive charges in the side chains dramatically influence both these features. Although our previous studies on perylene derivatives with mono-positively charged side chains indicated that self assembly in aqueous solution leads to a major efficiency, the result observed with the spermine derivative suggests that a too strong aggregation is unfavourable, because it determines a lower solubility of the compounds.     

Synthesis of an amphiphilic poly(gamma-glutamic acid)-cholesterol conjugate and its application as an artificial chaperone

A poly(gamma-glutamic acid) (gammaPGA)-cholesterol conjugate was synthesized and the properties of an aqueous solution were evaluated. The conjugate showed amphiphilic nature derived from the hydrophilic gammaPGA backbone and the hydrophobic cholesterol side chain. The conjugate spontaneously formed nanoparticles in the aqueous solution of the low concentration, and the high concentration resulted in the formation of the physical gel. By utilizing the self-aggregating properties of the conjugate in water, an artificial chaperone was developed. The complex of protein with the nanoparticles of the conjugate was formed and the protein was released upon the dissociation of the nanoparticles by the addition of beta-cyclodextrin. For denatured carbonic anhydrase, the activity was recovered in the artificial chaperone of the nanoparticle conjugate.     

Free radical generation by ultrasound in aqueous solutions of nucleic acid bases and nucleosides: an ESR and spin-trapping study

Direct evidence for the detection of intermediate radicals of nucleic acid constituents induced by ultrasound in argon-saturated aqueous solution is presented. The method of spin trapping with 3,5-dibromo-4-nitrosobenzene sulphonate, which is a water-soluble, non-volatile, aromatic nitroso spin trap, combined with ESR, was used for the detection of sonochemically induced radicals. Spin adducts were also generated by OH radicals produced by UV photolysis of aqueous solution containing H2O2. ESR spectra observed from these photolysis experiments were identical to those after sonolysis. The ESR spectra of the spin adducts suggest that the major spin-trapped radical of thymine and thymidine was the 5-yl radical, and that of cytosine, cytidine, uracil, and uridine was the 6-yl radical. To compare the radicals induced by sonolysis and photolysis, the decay of the ESR spectra of the thymine and thymidine spin adducts was investigated. The decay curves of thymine and thymidine after sonolysis indicated biphasic decay. However, after photolysis the spin adducts from both compounds showed very little decay. These results suggest that the observed spin adducts in the sonolysis of pyrimidine bases and nucleosides were formed by OH radical and H atom addition to the 5,6 double-bond.     

The synthesis of a cobalt(II) tetracarboxyphthalocyanine- deoxyribooligonucleotide conjugate as a reagent for the directed DNA modification

The cobalt(II) tetracarboxyphthalocyanine-deoxyribonucleotide pd(TCTTCCCA) conjugate was synthesized. The phthalocyanine N-succinimide ester prepared from phthalocyanine using DCC was mixed in DMF with an aqueous solution of the oligonucleotide bearing a 1,3-diaminopropane linker at the 5'-phosphate. The resulting conjugate was tested in the intraduplex reaction with target 14-mer and 22-mer oligonucleotides containing conjugate-complementary sequences. In the presence of O2 and a thiol (2-mercaptoethanol or DTT) as a coupled reducer or H2O2, sequence-specific DNA modification was observed that caused the cleavage of the target upon treatment with piperidine.     

Reactions of linoleic acid peroxyl radicals with phenolic antioxidants: a pulse radiolysis study

Linoleic acid peroxyl radicals (LOO.) can be viewed as model intermediates occurring during lipid peroxidation processes. Formation and reactions of these species were investigated in aqueous alkaline solution using the technique of pulse radiolysis combined with kinetic spectroscopy. Irradiation of linoleic acid in N2O/O2-saturated solutions leads to a mixture of peroxyl radical isomers, whereas reaction of 13-hydroperoxylinoleic acid (13-LOOH) with azide radicals in N2O-saturated solution produces 13-LOO. radicals specifically. These peroxyl radicals cannot be observed directly, but their reactions with the two flavonols, kaempferol and quercetin, acting as radical-scavenging antioxidants, produced strongly absorbing aroxyl radicals (ArO.). The same aroxyl radicals were generated by .OH and N3. with rate constants exceeding 10(9) dm3 mol-1 s-1. Applying a reaction scheme that includes competing generation and decay reactions of both LOO. and ArO. radicals, we derived individual rate constants for LOO. reactions with the phenols (greater than 10(7) dm3 mol-1 s-1), with the aroxyl radicals to form covalent adducts (greater than 10(8) dm3 mol-1 s-1), as well as for their bimilecular decay (3.0 X 10(8) dm3 mol-1 s-1). These results demonstrate the high reactivity of both fatty acid peroxyl radicals and the flavone antioxidants in aqueous solution.     

EPR detection of lipid-derived free radicals from PUFA, LDL, and cell oxidations

We have used the spin trap 5,5-dimethyl-pyrroline-1-oxide (DMPO) and EPR to detect lipid-derived radicals (Ld*) during peroxidation of polyunsaturated fatty acids (PUFA), low-density lipoprotein (LDL), and cells (K-562 and MCF-7). All oxygen-centered radical adducts of DMPO from our oxidizable targets have short lifetimes (<20 min). We hypothesized that the short lifetimes of these spin adducts are due in part to their reaction with radicals formed during lipid peroxidation. We proposed that stopping the lipid peroxidation processes by separating oxidation-mediator from oxidation-substrate with an appropriate extraction would stabilize the spin adducts. To test this hypothesis we used ethyl acetate to extract the lipid-derived radical adducts of DMPO (DMPO/Ld*) from an oxidizing docosahexaenioc acid (DHA) solution; Folch extraction was used for LDL and cell experiments. The lifetimes of DMPO spin adducts post-extraction are much longer (>10 h) than the spin adducts detected without extraction. In iron-mediated DHA oxidation we observed three DMPO adducts in the aqueous phase and two in the organic phase. The aqueous phase contains DMPO/HO* aN approximately aH approximately 14.8 G) and two carbon-centered radical adducts (aN1 approximately 15.8 G, aH1 approximately 22.6 G; aN2 approximately 15.2 G, aH2 approximately 18.9 G). The organic phase contains two long-chain lipid radical adducts (aN approximately 13.5 G, aH approximately 10.2 G; and aN approximately 12.8 G; aH approximately 6.85 G, 1.9 G). We conclude that extraction significantly increases the lifetimes of the spin adducts, allowing detection of a variety of lipid-derived radicals by EPR.     

13C-NMR studies of the effects of the carcinogen acetylaminofluorene on the conformation of dinucleoside monophosphate

13C-NMR spectra are obtained in aqueous solution of dinucleoside monophosphates (ApG and GpA) and of their adducts formed by the addition of the carcinogen acetylaminofluorene (AAF) to the C8 position of the guanine. The base and sugar carbons of all dimers and adducts are assigned. The task of assigning base and carbohydrate resonances was accomplished using a series of reference compounds. Significant changes in many of the carbon resonances of the adducts are observed suggesting three general conformational changes, namely: (1) chemical shift changes are noted in base carbon atom resonances as a function of temperature and adduct formation which are indicative of stacking effects; (2) large upfield shifts of the furanose C2' resonance of the guanosine-adduct indicate a shift to higher populations of the syn conformation. Other shifts of carbohydrate resonances are indicative of a change in conformation of the carbohydrate itself. (3) Large temperature effects on linewidth of several fluorine and furanose resonances indicate interconversion of various conformers in the dimer adduct.     

Synthesis of phosphine and antibody-azide probes for in vivo Staudinger ligation in a pretargeted imaging and therapy approach

The application of intact monoclonal antibodies (mAbs) as targeting agents in nuclear imaging and radioimmunotherapy is hampered by the slow pharmacokinetics of these molecules. Pretargeting with mAbs could be beneficial to reduce the radiation burden to the patient, while using the excellent targeting capacity of the mAbs. In this study, we evaluated the applicability of the Staudinger ligation as pretargeting strategy using an antibody-azide conjugate as tumor-targeting molecule in combination with a small phosphine-containing imaging/therapeutic probe. Up to 8 triazide molecules were attached to the antibody without seriously affecting its immunoreactivity, pharmacokinetics, and tumor uptake in tumor bearing nude mice. In addition, two (89)Zr- and (67/68)Ga-labeled desferrioxamine (DFO)-phosphines, a (177)Lu-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)-phosphine and a (123)I-cubyl phosphine probe were synthesized and characterized for their pharmacokinetic behavior in nude mice. With respect to the phosphine probes, blood levels at 30 min after injection were <5% injected dose per gram tissue, indicating rapid blood clearance. In vitro Staudinger ligation of 3.33 μM antibody-azide conjugate with 1 equiv of radiolabeled phosphine, relative to the azide, in aqueous solution resulted in 20-25% efficiency after 2 h. The presence of 37% human serum resulted in a reduced ligation efficiency (reduction max. 30% at 2 h), while the phosphines were still >80% intact. No in vivo Staudinger ligation was observed in a mouse model after injection of 500 μg antibody-azide, followed by 68 μg DFO-phosphine at t = 2 h, and evaluation in blood at t = 7 h. To explain negative results in mice, Staudinger ligation was performed in vitro in mouse serum. Under these conditions, a side product with the phosphine was formed and ligation efficiency was severely reduced. It is concluded that in vivo application of the Staudinger ligation in a pretargeting approach in mice is not feasible, since this ligation reaction is not bioorthogonal and efficient enough. Slow reaction kinetics will also severely restrict the applicability of Staudinger ligation in humans.     

Detection and identification of mutagens and carcinogens as their adducts with guanosine derivatives

For use in screening for environmental mutagens and carcinogens, a highly fluorescent derivative of guanosine, 2'-deoxy-2'-(2",3"-dihydro-2",4"-diphenyl-2"-hydroxy-3"-oxo-1"-pyrrol yl) guanosine (FG), was synthesized. When incubated with FG in aqueous solution, mutagens form adducts that can be analyzed with an HPLC-fluorescence detector-system. By this method, mutagens such as glyoxal, methylglyoxal, 2-(2-furyl)-3-(5-nitrofuryl) acrylamide and 4-nitroquinoline-N-oxide, used as model compounds, were detected rapidly with high sensitivity. Reaction with isopropylideneguanosine (IPG), followed by isolation and characterization of the mutagen-IPG-adduct was found to be a useful method for identifying unknown mutagens in crude samples. This method was successfully applied in identification of the mutagens in heated glucose (200 degrees C, 20 min); glyoxal-IPG and 8-hydroxy-IPG were identified in the reaction mixture.     

Synthesis of organosoluble chitosan derivatives with polyphenolic side chains

A one-pot synthesis was used to produce chitosan derivatives with polyphenolic side chains via a regioselective phenolic coupling reaction. Under Mannich reaction conditions, treatment of chitosan with formaldehyde and methyl 2,4-dihydroxybenzoate gave N-(2,6-dihydroxy-3-methoxycarbonylphenyl)methylated chitosan in good yield (87%). Formation of a CC bond occurred regioselectively at the C(3) position of methyl 2,4-dihydroxybenzoate. Chitosan derivatives having various phenolic compounds as a side chain were easily synthesized in a similar manner. The chitosan derivatives showed good biodegradability and improved their solubility in methanol (9.8mgmL(-1)) and 2-methoxyethanol (> 10mgmL(-1)). The UV protection provided by the derivatives with phenolic benzophenone side chain was evaluated using UV spectra of polyethylene terephthalate and poly(vinyl butyral-co-vinyl alcohol-co-vinyl acetate) films coated with the derivatives and the derivatives absorbed effectively in the UV-A region (<60%). Self-aggregation of the chitosan derivatives with the phenolic side chain was observed by using a fluorescent probe in aqueous solution.     

Stable adducts of nerve agents sarin,soman and cyclosarin with TRIS,TES and related buffer compounds—Characterization by LC-ESI-MS/MS and NMR and implications for analytical chemistry

Buffering compounds like TRIS are frequently used in chemical, biochemical and biomedical applications to control pH in solution. One of the prerequisites of a buffer compound, in addition to sufficient buffering capacity and pH stability over time, is its non-reactivity with other constituents of the solution. This is especially important in the field of analytical chemistry where analytes are to be determined quantitatively. Investigating the enzymatic hydrolysis of G-type nerve agents sarin, soman and cyclosarin in buffered solution we have identified stable buffer adducts of TRIS, TES and other buffer compounds with the nerve agents. We identified the molecular structure of these adducts as phosphonic diesters using 1D ¹H-³¹P HSQC NMR and LC-ESI-MS/MS techniques. Reaction rates with TRIS and TES are fast enough to compete with spontaneous hydrolysis in aqueous solution and to yield substantial amounts (up to 20–40%) of buffer adduct over the course of several hours. A reaction mechanism is proposed in which the amino function of the buffer serves as an intramolecular proton acceptor rendering the buffer hydroxyl groups nucleophilic enough for attack on the phosphorus atom of the agents. Results show that similar buffer adducts are formed with a range of hydroxyl and amino function containing buffers including TES, BES, TRIS, BIS-TRIS, BIS-TRIS propane, Tricine, Bicine, HEPES and triethanol amine. It is recommended to use alternative buffers like MOPS, MES and CHES when working with G-type nerve agents especially at higher concentrations and over prolonged times.     

Reversibility of the covalent reaction of CC-1065 and analogues with DNA.

Covalent DNA adducts of the antitumor antibiotic CC-1065 and its analogues undergo a retrohomologous Michael reaction in aqueous/organic solvent mixtures to regenerate the initial cyclopropylpyrroloindole (CPI) structure and, presumably, intact DNA. This reaction, which at higher temperatures competes with depurination of the N3-alkylated adenine, also occurs to a significant extent at 37 degrees C in neutral aqueous solution. Tritium-labeled adozelesin, covalently bonded to a 3-kilobase DNA restriction fragment which was exhaustively extracted to remove unbonded drug, was efficiently transferred to a 1-kilobase fragment upon coincubation for 20 h at 37 degrees C in aqueous buffer. Covalent adducts of adozelesin, but not CC-1065, on calf thymus DNA were cytotoxic to L1210 cells after incubation for 3 days at 37 degrees C, indicating that reversal of DNA alkylation can mediate potent cellular effects for simplified CC-1065 analogues.     

Sugar adducts with alkaline earth metal ions. Interaction of L-arabinose with Sr(II) and Ba(II) ions and the effects of metal ion binding on the sugar anomeric configurations

The reaction between L-arabinose and hydrated Sr(II) or Ba(II) halide salts has been studied in H2O solution and adducts of the type M(L-arabinose)X(2).4H(2)O, where M = Sr(II) or Ba(II) and X = Cl- or Br- have been isolated and characterized by means of Fourier transform infrared spectroscopy, 1H-NMR spectroscopy, molar conductivity and X-ray powder diffraction measurements. Due to the marked spectral similarities with those of the structurally known Ca(L-arabinose)X2 . 4H2O (X= Cl- or Br-) compounds, the Sr(II) and the BA(II) ions are eight-coordinated, binding to two l-arabinose molecules via O1, O5 of the first and O3, O4 of the second sugar moiety and to four H2O molecules. 1H-NMR spectroscopy indicated that the free L-arabinose has the beta-anomer configuration in aqueous solution, whereas the alpha-anomer isomer is preferred by Mg(II), Ca(II), Sr(II) and Ba(II) ions, on complexation.     

N-(1-pyrene)maleimide: a fluorescent cross-linking reagent.

N-(1-Pyrene)maleimide is nonfluorescent in aqueous solution but forms strongly fluorescent adducts with sulfhydryl groups of organic compounds or proteins. The conjugation reactions of N-(1-pyrene)maleimide are relatively fast and can be monitored by the increase in fluorescence intensity of the pyrene chromophore. In cases where primary amino groups are also present in the system, we have observed a red shift of the emission spectra of the fluorescent adducts subsequent to the initial conjugation, as characterized by the disappearance of three emission peaks at 376, 396, and 416 nm, and the appearance of two new peaks at 386 and 405 nm. Model studies with N-(1-pyrene)maleimide adducts of L-cysteine and cysteamine indicate that the spectral shift is the result of an intramolecular aminolysis of the succinimido ring in the adducts. Evidence from both chemical analysis and nuclear magnetic resonance studies of the addition products supports this reaction scheme. N-(1-Pyrene)maleimide adducts of N-acetyl-L-cysteine and beta-mercaptoethanol, which have no free amino group, do not exhibit a spectral shift. Among several protein conjugates only the N-(1-pyrene)maleimide adduct of bovine serum albumin (PM-BSA) shows the spectral shift resembling that of PM-cysteine. N-(1-Pyrene)maleimide reacts with the sulfhydryl group of the single cysteine residue at position 34 in BSA. The finding that the alpha-amino group of the N-terminus in PM-BSA is blocked after the spectral shift is completed strongly suggests that N-(1-pyrene)maleimide cross-links the N-terminus and the cysteine residue in BSA. The relative proximity of the sulfhydryl and amino groups is very critical in the cross-linking as demonstrated by the observation that the spectral shift observed with PM-BSA can be prevented by addition of denaturing reagents such as 1% sodium dodecyl sulfate immediately after labeling, and by the failure of PM-glutathione to undergo the intramolecular aminolysis. Since the intramolecular rearrangement of PM adducts is associated with characteristic fluorescence changes, N-(1-pyrene)maleimide can serve as a fluorescent cross-linking reagent which provides information about the spatial proximity of sulfhydryl and amino groups in proteins.     

Activation of carboxyl group with cyanate: peptide bond formation from dicarboxylic acids

The reaction of cyanate with C-terminal carboxyl groups of peptides in aqueous solution was considered as a potential pathway for the abiotic formation of peptide bonds under the condition of the primitive Earth. The catalytic effect of dicarboxylic acids on cyanate hydrolysis was definitely attributed to intramolecular nucleophilic catalysis by the observation of the ¹H-NMR signal of succinic anhydride when reacting succinic acid with KOCN in aqueous solution (pH 2.2–5.5). The formation of amide bonds was noticed when adding amino acids or amino acid derivatives into the solution. The reaction of N-acyl aspartic acid derivatives was observed to proceed similarly and the scope of the cyanate-promoted reaction was analyzed from the standpoint of prebiotic peptide formation. The role of cyanate in activating peptide C-terminus constitutes a proof of principle that intramolecular reactions of adducts of peptides C-terminal carboxyl groups with activating agents represent a pathway for peptide activation in aqueous solution, the relevance of which is discussed in connexion with the issue of the emergence of homochirality.     

Characterization of bis(levuglandinyl) urea derivatives as products of the reaction between prostaglandin H2 and arginine

Levuglandins are gamma-keto aldehydes formed by rearrangement of prostaglandin (PG) H(2) in aqueous solution. Levuglandins are highly reactive with primary amines. We had previously characterized adducts formed after reaction of levuglandin E(2) (LGE(2)) or PGH(2) with lysine. In this study, we assessed whether reaction of PGH(2) with arginine yielded covalent adducts. Using N(alpha)-acetylarginine and both PGH(2) and synthetic LGE(2), we discovered a novel series of levuglandinyl adducts derived from reaction of two levuglandin moieties with the guanidino group of arginine. Subsequent spontaneous hydrolysis of the adducted amino acid yields bis(levuglandinyl) urea and the corresponding ornithine residue. Using liquid chromatography tandem mass spectrometry, we characterized the molecular structure of these novel adducts and demonstrated their formation after coincubation of PGH(2) with synthetic peptides and proteins. The soluble characteristic of these molecules provides a potential strategy for development of biological markers of lipid modification of proteins following cyclooxygenase activity or lipid peroxidation.