Chirality recognition in solvent-free solid-state crystallization: chiral adduct formation by bis-beta-naphthol derivatives and benzoquinone crystals

New adduct crystals were obtained by simply mixing/grinding component crystals of bis-beta-naphthol (BN) derivatives with benzoquinone (BQ) under solvent-free conditions. Chiral recognition was found to operate during this process and either a racemic or a chiral crystal of a BN derivative produced an adduct crystal with BQ by solid-state crystallization. The chirality preference changed subtly according to the molecular structure of the BN derivative. Even in circumstances in which no adduct was formed, addition of a third component, such as crystals of naphthalene, to the grinding mixture yielded an adduct crystal. Remarkably, these adduct crystals were found to decompose spontaneously with time and revert to the starting crystal of the BN derivative by losing BQ molecules from the crystal lattice. Local melting of crystals by the grinding pressure was found unlikely to be the mechanism of adduct formation. Overall, these results demonstrate that molecules in the solid state could change their relative location and hydrogen bonding partners, thereby exerting chiral discrimination.     

Simple protocols for NMR analysis of the enantiomeric purity of chiral primary amines

A simple three-component chiral derivatization protocol for determining the enantiopurity of chiral primary amines by 1H NMR spectroscopic analysis is described here. The method involves condensation of the amines with 2-formylphenylboronic acid and enantiopure 1,1'-bi-2-naphthol. This approach affords a mixture of diastereoisomeric iminoboronate esters whose ratio can be determined by the integration of well-resolved diastereotopic resonances in their 1H NMR spectra, thus enabling the enantiopurity of the parent amine to be determined easily. The protocol, as described, takes less than 90 min to complete.     

Chromatographic enantioseparation by cycloalkylcarbamate derivatives of cellulose and amylose

Cyclopentyl and (+/-)-exo-2-norbornylcarbamates of cellulose and amylose were prepared and their chiral recognition abilities as chiral stationary phases for high-performance liquid chromatography (HPLC) were evaluated. Among these carbamates, cellulose tris(cyclopentylcarbamate) and amylose tris((+/-)-exo-2-norbornylcarbamate) showed particularly high chiral recognition, which is comparable to that of several well-known phenylcarbamate derivatives. The chiral recognition mechanism of cellulose tris(cyclohexylcarbamate), which was previously found to be an effective chiral stationary phase for HPLC, was investigated using NMR spectroscopy. The derivative dissolved in chloroform exhibited the chiral discrimination of several enantiomers in NMR as well as in HPLC. For example, the 1,1'-bi-2-naphthol enantiomers were distinctly discriminated in the (1)H, (13)C, and 2D-NOESY spectra.     

Role of latent heat in chiral symmetry breaking transition in the crystallization of 1,1'-binaphthyl

The influence of latent heat dissipated by the crystallization of 1,1'-binaphthyl in its supercooled molten state on the chiral symmetry breaking transition was investigated. Temperature change in the crystallization system was monitored by infrared thermocamera. Temperature rise due to the dissipation of latent heat in the growing front of polycrystalline aggregate was about 2 degrees C in an unstirred crystallization system. The melting point of racemic mixture and racemic compound of 1,1'-binaphthyl is 145 degrees C and 158 degrees C, respectively. The latent heat generated by the crystallization could thus change the crystallization behavior when the initial temperature of the melt was slightly lower than 145 degrees C. The temperature change in both unstirred and stirred crystallization systems was monitored. In the stirred crystallization system, even in the case when the initial temperature of the melt was about 2 degrees C lower than 145 degrees C, the temperature rose by about 4 degrees C immediately after the onset of crystallization. This indicates that the role of stirring as the critical parameter for the chiral symmetry breaking transition is not only to clone the chiral crystals but also to enhance the dissipation of latent heat due to secondary nucleation.     

Novel binuclear chiral zirconium catalysts used in enantioselective strecker reactions

A novel binuclear chiral zirconium catalyst was successfully used in enantioselective Strecker reactions. The catalyst was readily prepared from zirconium t-butoxide (Zr(OtBu)4), (R)-6,6'-dibromo-1, 1'-bi-2-naphthol ((R)-6-Br-BINOL), and (R)-3,3'-dibromo-1, 1'-bi-2-naphthol ((R)-3-Br-BINOL) to form unique binuclear structure. It was revealed that a combination of (R)-6-Br-BINOL and (R)-3-Br-BINOL was essential in these asymmetric reactions and that much lower selectivities were obtained by using other combinations. Two-component (an imine and hydrogen cyanide (HCN)) and three-component (an aldehyde, an amine, and HCN) Strecker reactions proceeded smoothly in the presence of a catalytic amount of the chiral zirconium catalyst to afford the corresponding alpha-amino nitrile derivatives in high yields with high enantioselectivities.     

Supercritical fluid chromatography comparison of the poly(trans-1,2-cyclohexanediyl-bis acrylamide) (P-CAP) column with several derivatized polysaccharide-based stationary phases

The poly(trans-1,2-cyclohexanediyl-bis acrylamide) (P-CAP) column has so far been primarily used with normal phase and polar organic mobile phase chromatography. Its use in supercritical fluid chromatography (SFC) was investigated via the analysis of 40 commercial and 100 proprietary compounds using a 12-min gradient with methanol as a modifier. Results were then compared against those obtained from the popular derivatized polysaccharide-based chiral stationary phases (CSPs) such as Chiralpak AD-H and Chiralpak AS-H as well as Chiralcel OD-H and Chiralcel OJ-H columns. P-CAP demonstrated separation of 25% of the 140 total compounds, while each of the derivatized polysaccharide-based CSPs separated at least 46%. A study that compared the loading of 1,1'-bi-2-naphthol with P-CAP and Chiralpak AS columns indicated a similar trend in resolution vs. amount injected, though AS appeared capable of allowing a greater loading of material. The P-CAP column was found to be beneficial in the separation of a complex mixture of enantiomers and achiral impurities, where the derivatized polysaccharide-based columns did not show as desirable of a separation. A key advantage of this type of chiral stationary phase is the fact that it is available in both enantiomeric forms, allowing manipulation of elution order of enantiomers, which is especially helpful for preparative applications. P-CAP also demonstrated that it could resolve an achiral impurity from the desired compound in a different mixture, while the same impurity co-eluted on the Chiralpak AD-H column. Overall, the synthetic polymer-based P-CAP showed less chiral discrimination power compared to the derivatized polysaccharide-based CSPs under the conditions explored in this study.     

Central-to-axial chirality transfer revealed by liquid crystals: a combined experimental and computational approach for the determination of absolute configuration of carboxylic acids with an α chirality centre

The conversion into 6,7-dihydro-5H-dibenz[c,e]azepine (DAZ) N-protected amides is a viable route for the determination of the absolute configuration of chiral 2-substituted carboxylic acids. The biphenyl moiety of DAZ, besides being a probe of chirality for the electronic circular dichroism (ECD) spectroscopy, makes these systems suitable for configuration assignment by exploiting the chirality amplification which occurs in nematic liquid crystals. To assess the reliability of the liquid crystal method in detecting the absolute stereochemistry of chiral amides bound to a biphenyl group, we measured the helical twisting power of a series of DAZ-N-protected amides and compared these data with the results obtained from ECD measurements. We will show that the liquid crystal method, corroborated by HTP predictions, is trustworthy with our biphenyl derivatives, even when ECD spectra are ambiguous for the presence of aryl moieties displaying strong UV absorptions in the same range of the biphenyl chromophore.     

Single diastereomers of unsymmetrical tris-spirocyclic cyclotriphosphazenes based on 1,1'-bi-2-naphthol--synthesis and structures

Diastereoselective synthesis and characterization of chiral unsymmetrical tris-spirocyclic cyclotriphosphazenes based on chiral 1,1'-bi-2-naphthol (BINOL) are reported. Specifically, the chiral compounds (-)N(3)P(3)[1,1'-O(2)(C(10)H(6))(2)](O-2,2'C(6)H(4)-C(6)H(4)O)Cl(2) [(-)-4] and (-)N(3)P(3)[1,1'-O(2)(C(10)H(6))(2)](OCH(2)CH(2)NMe)(2) [(-)-5] are prepared by starting with the chiral mono-spiro compound (-)N(3)P(3)[1,1'-O(2)(C(10)H(6))(2)]Cl(4) [(-)-3]. Synthesis of four other chiral spirocyclics, N(3)P(3)[1,1'-O(2)(C(10)H(6))(2)](OCH(2)CH(2) NMe)(O-2,2'C(6)H(4)-C(6)H(4)O)[(-)-6 and (+)-6], N(3)P(3)[1,1'-O(2)(C(10)H(6))(2)](NMe(2))(4) [(-)-7], N(3)P(3)[1,1'-O(2)(C(10)H(6))(2)](O-2,2'C(6)H(4)-C(6)H(4)O)(NMeCH(2)CH(2)OH)(2) [(-)-8 and (+)-8], and N(3)P(3)[1,1'-O(2)(C(10)H(6))(2)](O-2,2'C(6)H(4)-C(6)H(4)O)[NHCH(2)CH(2)CH(2)Si(OEt)(3)](2) (9) is also reported herein. Compounds 4-6 are obtained in the solid state diastereoselectively and their X-ray structures have been determined and discussed. The diastereoselectivity is also shown by structural characterization of two distinct isomers in the case of 6 [(-)-6 and (+)-6, respectively] by starting with precursor of 3 having (R) or (S)-BINOL residue. The (1)H NMR spectra of 7 and 8 exhibit doublets with virtual coupling for the methyl protons, consistent with the chiral nature of the binaphthoxy residue. The potential of 9, which hydrolyzes readily in CDCl(3) solution, as a useful precursor for chiral polymer applications is highlighted.     

Discrimination of chiral guests by chiral channels: variable temperature studies by SXRD and solid state 13C NMR of the deoxycholic acid complexes of camphorquinone and endo-3-bromocamphor

3alpha,12alpha-dihydroxy-5beta-cholan-24-oic acid (deoxycholic acid DCA) is able to discriminate between the R- and S-enantiomers of camphorquinone and endo-(+)-3-bromocamphor and select only the S-enantiomers from a racemic mixture. DCA forms novel well ordered 1:1 adducts with (1S)-(+)-camphorquinone and (1S)-endo-(-)-3-bromocamphor, both of which have been characterized by single crystal X-ray diffraction (SXRD). When DCA is cocrystallized with (RS)-camphorquinone and (RS)-endo-3-bromocamphor, 1:1 adducts of the S-enantiomers are produced together with crystals of the free racemic guest. In contrast, in the absence of (1S)-(+)-camphorquinone, DCA forms a 2:1 adduct with (1R)-(-)-camphorquinone. In this 2:1 adduct the guest is disordered at ambient temperature and undergoes a phase change in the region 160-130 K similar to that observed for the ferrocene adduct, but with only partial ordering of the guest. The SXRD structure of the low temperature form and the variable temperature (13)C CP/MAS NMR are reported. Cocrystallizing DCA with (1R)-endo-(+)-3-bromocamphor gives the free guest and a glassy solid.     

Two-photon absorption circular-linear dichroism on axial enantiomers

We report on the experimental-theoretical analysis of degenerated two-photon absorption circular-linear dichroism (2PA-CLD) in (S)-(-)-1,1'-bi(2-naphthol) and (R)-(+)-1,1'-bi(2-naphthol) dissolved in THF. 2PA-CLD reveals similarities and differences between optical polarization states for the different enantiomers. These results unveil the potential applications of 2PA-CLD for the study and identification of chiral molecules with active and pivotal role in biological systems and the systematic design of chiral photonic structures.     

Effect of geometric isomerism in dinuclear platinum antitumour complexes on the rate of formation and structure of intrastrand adducts with oligonucleotides.

The dinuclear platinum complexes [[trans -PtCl (NH3)2]2[mu]-[NH2(CH2) n NH2]](NO3)2[1,1/t,t ( n = 4,6)] and [[cis-PtCl(NH3)2]2[mu];-[NH2(CH2) n NH2](NO3) 2[1,1/c,c ( n = 4,6)] exhibit antitumour activity comparable with cisplatin. 1,1/c,c complexes do not form 1,2 GG intrastrand adducts, the major adduct of cisplatin, with double-stranded DNA. This 1H NMR spectroscopy study shows that, in the absence of a complementary strand, 1,1/c,c ( n = 4,6) form a 1,2 GG (N7, N7) intrastrand adduct with r(GpG), d(GpG) and d(TGGT). Initial binding to r(GpG) (and also reaction with GMP) at 37 degrees C was slower for 1,1/c,c compared with 1,1/t,t, whereas the second binding step (adduct closure) was faster for 1,1/c,c. However, the 1H NMR spectra of the 1,1/c,c adducts at 37 degrees C show two H8 signals, one of which is broad and becomes sharper on increasing the temperature, indicating restricted rotation around the Pt-N7 bond. For the d(GpG)-1,1/c,c ( n = 4) adduct, 2D NMR spectroscopy assigned the broad H8 signal to the 3' G, which has syn base orientation and 60% S-type/40% N-type sugar conformation. The 5' G has anti base orientation and S-type sugar conformation. Apart from the restricted rotation around the 3' G, the structure is similar to that of 1,2 GG intrastrand adducts of 1,1/t,t. This steric hindrance may explain the inability of 1,1/c,c complexes to form 1,2 GG intrastrand adducts with sterically more demanding double-stranded DNA.     

Glutathione depletion and cytotoxicity by naphthalene 1,2-oxide in isolated hepatocytes

The ability of naphthalene 1,2-oxide to diffuse across intact cellular membranes, the subsequent biotransformation of this epoxide and its potential to produce losses in cellular viability have been examined in incubations of isolated hepatocytes. Addition of 1R,2S- or 1S,2R-naphthalene oxide enantiomers (15, 30 and 60 microM) to isolated hepatocytes resulted in a rapid depletion of intracellular glutathione. Depletion of glutathione was concentration dependent and maximal at 5-15 min. Addition of either of the enantiomeric oxides at 60 microM resulted in the loss of more than 20 nmol glutathione/10(6) cells (1 ml cells); thus more than a third of the added epoxide was available for conjugation with intracellular glutathione. The time course and concentration dependence of glutathione depletion corresponded to the rapid, concentration-dependent formation of naphthalene oxide glutathione conjugates. The levels of glutathione adduct were highest 1 min after addition of naphthalene oxide and declined to 25% of this level after 30 min. Loss of glutathione conjugates from incubations correlated with the formation of N-acetylcysteine adducts. In contrast, the levels of glutathione adducts added exogenously to hepatocytes were relatively stable over a 120-min incubation suggesting that although further metabolism of naphthalene oxide glutathione adducts formed intracellularly is possible, extracellular glutathione adducts cannot penetrate the hepatocellular membrane. Small amounts of radiolabel from [3H]naphthalene 1,2-oxide were bound covalently to macromolecules in hepatocytes; the rate of this binding slowed rapidly after the first minute of incubation. Severe blebbing of the surface of the hepatocytes was noted in cells incubated for 30 min with 480 microM naphthalene oxide. Many of the cells were vacuolated at 60 min and progressed to frank necrosis with pyknotic nuclei and inability to exclude trypan blue. Cells incubated with 1-naphthol responded in a qualitatively similar fashion to those cells incubated with epoxide; however, hepatocytes incubated with 1-naphthol progressed to frank cellular necrosis at a slower rate. In hepatocytes partially depleted of glutathione by pretreatment with buthionine sulfoximine, addition of 1S,2R-naphthalene oxide at a rate of 1 nmol/min/10(6) cells resulted in significant losses in cell viability. In contrast, no losses in cell viability were observed with the enantiomer, 1R,2S-naphthalene oxide. Both epoxides produced similar losses in cellular glutathione levels.(ABSTRACT TRUNCATED AT 400 WORDS)     

Chiral crystallization of glutamic acid on self assembled films of cysteine

In this article, we describe the preparation and use of chiral surfaces derived from enantiomerically pure crystals of amino acids. For this purpose, we chose to employ a self-assembly process to grow nanoscale chiral films of (+)-L or (-)-D cysteine, onto gold surfaces. We utilized those chiral films as resolving auxiliaries in the crystallization of enantiomers from solutions. To demonstrate the chiral discriminating ability of the chiral surfaces in crystallization processes, we investigated the crystallization of rac-glutamic acid onto the chiral films. Our study demonstrates the potential application of chiral films to control chirality throughout crystallization, where one enantiomer crystallizes on the chiral surfaces with relatively high enantiomeric excess. In addition, crystallization of pure glutamic acid enantiomers, and its racemic compound on to chiral films resulted in crystal morphology modification with preferred crystal orientation, which assists in the interpretation of the ability of our chiral surfaces to function as chiral selectors.     

Accumulation of M1dG DNA adducts after chronic exposure to PCBs, but not from acute exposure to polychlorinated aromatic hydrocarbons

Oxidative DNA damage is one of the key events thought to be involved in mutation and cancer. The present study examined the accumulation of M1dG, 3-(2'-deoxy-beta-D-erythro-pentofuranosyl)-pyrimido[1,2-a]-purin-10(3H)-one, DNA adducts after single dose or 1-year exposure to polyhalogenated aromatic hydrocarbons (PHAH) in order to evaluate the potential role of oxidative DNA damage in PHAH toxicity and carcinogenicity. The effect of PHAH exposure on the number of M1dG adducts was explored initially in female mice exposed to a single dose of either 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or a PHAH mixture. This study demonstrated that a single exposure to PHAH had no significant effect on the number of M1dG adducts compared to the corn oil control group. The role of M1dG adducts in polychlorinated biphenyl (PCB)-induced toxicity and carcinogenicity was further investigated in rats exposed for a year to PCB 153, PCB 126, or a mixture of the two. PCB 153, at doses up to 3000 microg/kg/day, had no significant effect on the number of M1dG adducts in liver and brain tissues from the exposed rats compared to controls. However, 1000 ng/kg/day of PCB 126 resulted in M1dG adduct accumulation in the liver. More importantly, coadministration of equal proportions of PCB 153 and PCB 126 resulted in dose-dependent increases in M1dG adduct accumulation in the liver from 300 to 1000 ng/kg/day of PCB 126 with 300-1000 microg/kg/day of PCB 153. Interestingly, the coadministration of different amounts of PCB 153 with fixed amounts of PCB 126 demonstrated more M1dG adduct accumulation with higher doses of PCB 153. These results are consistent with the results from cancer bioassays that demonstrated a synergistic effect between PCB 126 and PCB 153 on toxicity and tumor development. In summary, the results from the present study support the hypothesis that oxidative DNA damage plays a key role in toxicity and carcinogenicity following long-term PCB exposure.     

2-O-alpha-D-glucopyranosyl-L-ascorbic acid scavenges 1,1-diphenyl-2-picrylhydrazyl radicals via a covalent adduct formation

The 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical-scavenging mechanism of 2-O-alpha-D-glucopyranosyl-L-ascorbic acid (AA-2G) was studied. We found two undefined products, named X and Y, in the reaction mixture of AA-2G and the DPPH radical under acidic conditions by HPLC analysis. The reaction mixture was further subjected to LC-MS analysis. X was found to be a covalent adduct of AA-2G and the DPPH radical. On the other hand, Y could not be identified, probably because it was a mixture. A time-course study of the radical-scavenging reaction revealed that one molecule of AA-2G scavenged one molecule of DPPH radical to generate an AA-2G radical, which readily reacted with another molecule of the DPPH radical to form a covalent adduct (X). Subsequently, this adduct slowly quenched a third molecule of the DPPH radical, resulting in reaction products (Y). Therefore, one molecule of AA-2G has only one oxidizable -OH group, but can scavenge three molecules of the DPPH radical. The radical-scavenging mechanism of AA-2G elucidated in this study should be useful in understanding the biological roles of AA-2G per se in the food and cosmetic fields.     

Protein interactions with platinum-DNA adducts: from structure to function

Because of the efficacy of cisplatin and carboplatin in a wide variety of chemotherapeutic regimens, hundreds of platinum(II) and platinum(IV) complexes have been synthesized and evaluated as anticancer agents over the past 30 years. Of the many third generation platinum compounds evaluated to date, only oxaliplatin has been approved for clinical usage in the United States. Thus, it is important to understand the mechanistic basis for the differences in efficacy, mutagenicity and tumor range between cisplatin and oxaliplatin. Cisplatin and oxaliplain form the same types of adducts at the same sites on DNA. The most abundant adduct for both compounds is the Pt-GG intrastrand diadduct. Cisplatin-GG adducts are preferentially recognized by mismatch repair proteins and some damage-recognition proteins, and this differential recognition of cisplatin- and oxaliplatin-GG adducts is thought to contribute to the differences in cytotoxicity and tumor range of cisplatin and oxaliplatin. A detailed kinetic analysis of the insertion and extension steps of dNTP incorporation in the vicinity of the adduct shows that both pol beta and pol eta catalyze translesion synthesis past oxaliplatin-GG adducts with greater efficiency than past cisplatin-GG adducts. In the case of pol eta, the efficiency and fidelity of translesion synthesis in vitro is very similar to that previously observed with cyclobutane TT dimers, suggesting that pol eta is likely to be involved in error-free bypass of Pt adducts in vivo. This has been confirmed for cisplatin by comparing the cisplatin-induced mutation frequency in human fibroblast cell lines with and without pol eta. Thus, the greater efficiency of bypass of oxaliplatin-GG adducts by pol eta is likely to explain the lower mutagenicity of oxaliplatin compared to cisplatin. The ability of these cellular proteins to discriminate between cisplatin and oxaliplatin adducts suggest that there exist significant conformational differences between the adducts, yet the crystal structures of the cisplatin- and oxaliplatin-GG adducts were very similar. We have recently solved the solution structure of the oxaliplatin-GG adduct and have shown that it is significantly different from the previously published solution structures of the cisplatin-GG adducts. Furthermore, the observed differences in conformation provide a logical explanation for the differential recognition of cisplatin and oxaliplatin adducts by mismatch repair and damage-recognition proteins. Molecular modeling studies are currently underway to analyze the mechanistic basis for the differential bypass of cisplatin and oxaliplatin adducts by DNA polymerases.     

Chiral symmetry-breaking transition in growth front of crystal phase of 1,1'-binaphthyl in its supercooled melt

Although the theory of spontaneous chiral symmetry-breaking in open systems was proposed some time ago, experimental realization of this phenomenon has not been achieved. In this article, we note that the crystal growth front of 1,1'-binaphthyl shows many of the characteristics of an open system in which chiral symmetry-breaking has occurred. We studied the temperature profiles of the crystallizing surface and obtained X-ray diffraction data of the crystals grown from the melt under controlled conditions. The data show that, under appropriate conditions, the observed bimodal probability distribution of enantiomeric excess (ee) with maxima approximately 60% is due exclusively to chiral crystals and not due to racemic crystals of 1,1'-binaphthyl that can also form at large supercooling. The mass independence of the ee shows that the growing front maintains a constant ee, which is a clear signature of open systems in steady state. Chirality 16:131-136, 2004.     

Structural basis of protein-bound endogenous aldehydes. Chemical and immunochemical characterizations of configurational isomers of a 4-hydroxy-2-nonenal-histidine adduct

4-Hydroxy-2-nonenal (HNE), a major racemic product of lipid peroxidation, reacts with histidine to form a stable HNE-histidine Michael addition-type adduct possessing three chiral centers in the cyclic hemiacetal structure. In the present study, we characterized configurational isomers of a HNE-N(alpha)-acetylhistidine adduct by NMR spectroscopy and by molecular orbital calculations. In addition, we raised monoclonal antibodies against (R)-HNE-histidine and (S)-HNE-histidine adducts, characterized their specificities, and examined in vivo localizations of each adduct under oxidative stress. To facilitate structural characterization of the configurational isomers of an HNE-histidine adduct, we prepared the (R)-HNE-histidine and (S)-HNE-histidine adducts by incubating N(alpha)-acetylhistidine with each HNE enantiomer, both of which provided two peaks (Ra and Rb from (R)-HNE-histidine and Sa and Sb from (S)-HNE-histidine adducts) in reversed-phase high-performance liquid chromatography. The NMR analysis showed that each peak was a mixture of two diastereomers. In addition, the analysis of the nuclear Overhauser effect enabled the determination of configurations of the eight isomers. The relative amounts of these isomers in the NMR analysis correlated with the relative energies calculated by molecular orbital methods. On the other hand, using (R)-HNE-modified and (S)-HNE-modified keyhole limpet hemocyanins as the antigens, we raised the monoclonal antibodies, mAbR310 and mAbS412, which enantioselectively recognized the (R)-HNE-histidine and (S)-HNE-histidine adducts, respectively. Among the mixtures (Ra, Rb, Sa, and Sb) of diastereomers, mAbR310 showed the highest immunoreactivity to Rb (the mixture of 2R,4S,5R and 2S,4S,5R isomers), whereas mAbS412 preferentially recognized Sa (the mixture of 2R,4S,5S and 2S,4S,5S isomers). The presence of (R)-HNE and (S)-HNE epitopes in vivo was immunohistochemically examined in the kidney of rats exposed to the renal carcinogen, ferric nitrilotriacetate, by which nuclear and cytosolic stainings with mAbR310 and mAbS412, respectively, were detected.     

Synthesis and spectroscopic characterization of site-specific 2-amino-1-methyl-6-phenylimidazo

The aim of the present study is to determine the chemical structure and conformation of DNA adducts formed by incubation of the bioactive form of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), N-acetoxy-PhIP, with a single-stranded 11mer oligodeoxyribonucleotide. Using conditions optimized to give the C8-dG-PhIP adduct as the major product, sufficient material was synthesized for NMR solution structure determination. The NMR data indicate that in duplex DNA this adduct exists in equilibrium between two different conformational states. In the main conformer, the covalently bound PhIP molecule intercalates in the helix, whilst in the minor conformation the PhIP ligand is probably solvent exposed. In addition to the C8-dG-PhIP adduct, at least eight polar adducts are found after reaction of N-acetoxy-PhIP with the oligonucleotide. Three of these were purified for further characterization and shown to exhibit lowest energy UV absorption bands in the range 342–347 nm, confirming the presence of PhIP or PhIP derivative. Accurate mass determination of two of the polar adducts by negative ion MALDI-TOF MS revealed ions consistent with a spirobisguanidino-PhIP derivative and a ring-opened adduct. The third adduct, which has the same mass as the C8-dG-PhIP oligonucleotide adduct, may contain PhIP bound to the N² position of guanine.     

Spin trapping of polyunsaturated fatty acid-derived peroxyl radicals: reassignment to alkoxyl radical adducts

Polyunsaturated fatty acid (PUFA) peroxyl radicals play a crucial role in lipid oxidation. ESR spectroscopy with the spin-trapping technique is one of the most direct methods for radical detection. There are many reports of the detection of PUFA peroxyl radical adducts; however, it has recently been reported that attempted spin trapping of organic peroxyl radicals at room temperature formed only alkoxyl radical adducts in detectable amounts. Therefore, we have reinvestigated spin trapping of the linoleic, arachidonic, and linolenic acid-derived PUFA peroxyl radicals. The slow-flow technique allowed us to obtain well-resolved ESR spectra of PUFA-derived radical adducts in a mixture of soybean lipoxygenase, PUFA, and the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO). However, interpretation of the ESR spectra was complicated by the overlapping of the PUFA-derived alkoxyl radical adduct spectra. In order to understand these spectra, PUFA-derived alkoxyl radical adducts were modeled by various alkoxyl radical adducts. For the first time, we synthesized a wide range of DMPO adducts with primary and secondary alkoxyl radicals. It was found that many ESR spectra previously assigned as DMPO/peroxyl radical adducts based on their close similarity to the ESR spectrum of the DMPO/superoxide radical adduct, in conjunction with their insensitivity to superoxide dismutase, are indeed alkoxyl radical adducts. We have reassigned the PUFA alkylperoxyl radical adducts to their corresponding alkoxyl radical adducts. Using hyperfine coupling constants of model DMPO/alkoxyl radical adducts, the computer simulation of DMPO/PUFA alkoxyl radical adducts was performed. It was found that the trapped, oxygen-centered PUFA-derived radical is a secondary, chiral alkoxyl radical. The presence of a chiral carbon atom leads to the formation of two diastereomers of the DMPO/PUFA alkoxyl radical adduct. Therefore, attempted spin trapping of the PUFA peroxyl radical by DMPO at room temperature leads to the formation of the PUFA alkoxyl radical adduct.