We have previously described the DNA strand-breaking activity 1,2) and other characteristics (e.g., generation of radical species 3,4) and high chemical reactivity 5-7) ) of dihydropyrazines (DHPs) in vitro. More recently, we have examined the biological effects of DHPs, such as induction of apoptosis 8) and mutagenesis, 9) in vivo. The various phenomena caused by DHPs cannot be explained on the basis of one particular reaction mechanism. In this paper, we attempt to illustrate the relationship of the chemical structure of DHPs on DNA strand scission. ExperimentalSynthesis of Dihydropyrazine Derivatives The dihydropyrazine derivatives ( Fig. 1) employed were synthesized by the condensation of diketones and diamines. 2,3-Dihydro-5,6-dimethylpyrazine (1), 2,3-dihydro-2,5,6-trimethylpyrazine (2), 2,3-dihydro-2,2,5,6-tetramethylpyrazine (3), cis-2, 3-dimethyl-5,6,7,8,9,10-hexahydroquinoxaline (4-cis) and trans-2,3-dimethyl-5,6,7,8,9,10-hexahydroquinoxaline (4-trans) were synthesized by the method of Yamaguchi et al.1) Their derivatives (3a, 7) 2a, 7) 1c, 7) 1b 5) ) and dihydrofructosazine 10) were synthesized according to literature procedures, and 1a was synthesized from aminoacetone hydrochloride 11) by dehydrochlorination.10) Similar methods were also used to replace the methyl group of 1 and 4-trans by a phenyl group to generate 2,3-dihydro-5-methyl-6-phenylpyrazine (5) 12) and trans- 2-methyl-3-phenyl-5,6,7,8,9,10-hexahydroquinoxaline (6), 12) respectively (Table 1), and the mixture (7) 13) of 2, 3,5,6,7,2,3,5,6,.Assay of DNA Strand-Breaking Activity The method to assess the DNA strand-breakage activity of DHPs, using a covalently closed circular duplex DNA (ccc-DNA) of plasmid pBR322 was described previously. Dihydropyrazine, a compound derived from sugars, possesses DNA strand-breakage activity. The relationship between the activity as assayed using pBR 322 ccc-DNA and the chemical structures of derivatives of dihydropyrazine (DHPs) has been investigated. The addition of Cu 2؉ enhanced the activity remarkably. The introduction of a methyl or phenyl group onto the DHP ring or a cyclohexyl group fused onto the DHP ring also increased the activity. These properties indicated that the activity was due to the facility of electron release from the DHP ring, followed by radical generation. The determination of ionization potential and electrostatic potential values, and bond dissociation energy via semi-empirical MO calculations suggested strongly that the activity is induced by a DHP ring structure that contains a configuration suitable for hyperconjugation. 1) Semi-empirical MO Calculations

Reactive oxygen species (ROS) can cause oxidative damage to DNA, proteins, and lipids in all living organisms, and the resulting oxidative damage may play roles in several human disorders as well as the aging process.1) Dihydropyrazine (DHP) is produced as a Maillard reaction intermediate by condensation of two molecules of D-glucosamine, 2) and some DHP derivatives generate various radical species and ROS that cause DNA strand cleavage in vitro.3) Since many pyrazine derivatives have been detected in various foods 4) and human urine, 5) we speculated that DHPs, which are presumed to be precursors of pyrazines, would be responsible for certain diseases. However, there are few reports regarding the biological and physiological roles of DHPs.Previously, we reported that methyl-substituted dihydropyrazines (Me-DHPs), such as 2,3-dihydro-5,6-dimethylpyrazine, had DNA strand-breaking activity, inhibited growth and induced mutagenesis in Escherichia coli.6,7) Although phenyl-substituted dihydropyrazines (Ph-DHPs) have much higher DNA strand-breaking activities and produce higher levels of radical species than Me-DHPs, 8,9) DNA repairdeficient bacteria are less sensitive to Ph-DHPs than to Me-DHPs.10,11) Recently, cyclohexyl-DHP, which was designed according to our hypothesis of a relationship between the chemical structures of DHP derivatives and their DNA strand-breaking activities, 12) was shown to possess a stronger DNA strand-breaking activity than Me-DHPs and PhDHPs.13) Moreover, cyclohexyl-DHP can potentially generate superoxide anions, and not only cause breakage of chromosomal DNA leading to mutagenic lesions but also induce damage to cellular proteins. 14) In the present study, we examined enzyme inactivation by DHPs using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a convenient model protein, since GAPDH is known to be highly sensitive to oxidative damage. 15) Our observations reveal that DHPs inactivate GAPDH in vitro and in vivo. MATERIALS AND METHODSSynthesis of DHP Derivatives 2,3-Dihydro-5,6-dimethylpyrazine (Me-DHP-1), 2,3-dihydro-5-methyl-6-phenylpyrazine (Ph-DHP-1), and a mixture of two isomers of cyclohexyl-DHP (Fig. 1), namely 2,3,5,6,7,8-hexahydroquinoxaline (endo-type) and 1,2,3,5,6,7-hexahydroquinoxaline (exo-type), were prepared using a previously described method. 3)Materials Rabbit muscle GAPDH was obtained from MP Biomedicals. DL-glyceraldehyde-3-phosphate was purchased from Sigma. Nicotinamide adenine dinucleotide (NAD) was obtained from Wako. B-PER Bacterial Protein Extraction Reagent was purchased from Pierce.Bacterial Strain The E. coli Dihydropyrazine (DHP), which is produced during the Maillard reaction, generates radicals that not only cause breakage of chromosomal DNA leading to mutagenic lesions but also induce oxidative damage to cellular proteins. In the present study, we show that three DHP derivatives, which generated superoxide anions, caused inhibition of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). SH-compounds, such as cysteine, dithiothreitol (DTT), 2-mercap...

An oxidative methyl esterification of aldehydes was effectively achieved. The trivalent indium reagent, indium(III) triflate, was revealed to accelerate the reactions in many cases. Aromatic aldehydes with various substituents were subjected to this method, and each produced the corresponding methyl esters in good to excellent yields within a relatively short reaction time.

The Maillard reaction, which comprises a non-enzymatic reaction between reducing sugars and amino acids, produces hundreds of highly reactive compounds. Dihydropyrazine (DHP) is produced by condensation of 2 mol of D-glucosamine, 1) and some DHP derivatives generate various radical species that cause DNA strand cleavage in vitro.2) Since many pyrazine derivatives have been detected in various foods 3) and human urine, 4) we speculated that DHPs, which are presumed to be precursors of pyrazines, would be responsible for certain diseases. However, there are few reports regarding the biological and physiological roles of DHPs.In a previous paper, we reported that methyl-substituted dihydropyrazines (Me-DHPs), such as 2,3-dihydro-5,6-dimethylpyrazine, had DNA strand-breaking activity, inhibited growth and induced mutagenesis in Escherichia coli.5) Although phenyl-substituted dihydropyrazines (Ph-DHPs) have much higher DNA strand-breaking activities and produce higher levels of radical species than Me-DHPs, 6,7) DNA repair-deficient bacteria were less sensitive to Ph-DHPs than to Me-DHPs. 8) Recently, cyclohexyl-DHP, which was designed according to our hypothesis of a relationship between the chemical structures of DHP derivatives and DNA strandbreaking activities, 9) was shown to possess a stronger DNA strand-breaking activity than Me-DHPs and Ph-DHPs. 10)In the present study, we examined the cellular effects of a novel cyclohexyl-DHP with a cyclohexyl ring fused to the DHP. We found that it could strongly generate superoxide anions, and cause not only breakage of chromosomal DNA leading to mutagenic lesions but also induce oxidative damage to cellular proteins. MATERIALS AND METHODS Synthesis of DHPsA mixture of two isomers of cyclohexyl-DHP (Fig. 1), namely 2,3,5,6,7,8-hexahydroquinoxaline (endo-type) and 1,2,3,5,6,7-hexahydroquinoxaline (exotype), was prepared using a previously described method. 11) were obtained from the National BioResource Project (NIG, Japan). CATD [AB1157 katE katG] and SODD [AB1157 sodA sodB] were described in a previous paper. 8)DNA Strand-Breaking Assay Reaction mixtures (20 ml) containing 1 mg of plasmid pUC18 were incubated with DHP in the presence or absence of 1 mM CuCl 2 for 1 h at 37°C. The resulting mixtures were analyzed by 0.8% agarose gel electrophoresis and stained with 0.5 mg/ml ethidium bromide. The DNA bands were visualized with an Epi-LightUV FA500 (Taitec, Japan).Survival Assay Overnight cultures of E. coli were diluted by 100-fold with fresh LB medium and grown at 37°C until the OD 600 was approximately 0.1. Approximately 1ϫ10 8 bacteria were then challenged with DHP in the presence or absence of 1 mM CuCl 2 for 1 h with shaking at 37°C, plated on LB plates after serial dilutions in M9 salts and incubated overnight at 37°C. The survival rates were calculated as the percentages of cells surviving after challenge with DHP. All experiments were conducted at least four times. The Maillard reaction contributes to the complications of diabetes and normal aging. Dihydropyrazine...

Gadolinium(III) ion (Gd(3+)) complexes are widely used as contrast agents in magnetic resonance imaging (MRI), and many attempts have been made to couple them to sensor moieties in order to visualize biological phenomena of interest inside the body. However, the low sensitivity of MRI has made it difficult to develop practical MRI contrast agents for in vivo imaging. We hypothesized that practical MRI contrast agents could be designed by targeting a specific biological environment, rather than a specific protein such as a receptor. To test this idea, we designed and synthesized a Gd(3+)-based MRI contrast agent, 2BDP3Gd, for visualizing atherosclerotic plaques by linking the Gd(3+)-complex to the lipophilic fluorophore BODIPY to stain lipid-rich environments. We found that 2BDP3Gd was selectively accumulated into lipid droplets of adipocytes at the cellular level. Atherosclerotic plaques in the aorta of Watanabe heritable hyperlipidemic (WHHL) rabbits were clearly visualized in T1-weighted MR images after intravenous injection of 2BDP3Gd in vivo.