Radical Species in DNA Strand-Cleavage Caused by Dihydropyrazines

In a recent paper, 2) we demonstrated the biological effects, such as growth inhibition and mutagenesis in Escherichia coli, of dihydropyrazines (DHPs). The DHPs revealed single strand-cleavage 3) on the covalently closed circular DNA of plasmid pBR322, especially in the presence of Cu 2ϩ . Since the participation of Cu 2ϩ enhanced the cleaving activity, it is likely that the effects of DHPs on E. coli also would be influenced. Here, we examined the effects of DHPs on E. coli upon the addition of Cu 2ϩ . Pyrazine derivatives have been detected in the urine of humans with diabetes mellitus, 4) which suggests that DHPs, which are precursors of pyrazines, might be involved in diabetes mellitus, although this remains unconfirmed. DHPs also are produced from the accumulation of the heme precursor, 5-aminolevulinic acid (ALA), in lead poisoning and some types of porphyries. 5,6) We have reported that DHPs induce apoptosis of cultured cells, 7) and, therefore investigated other potential biological effects caused by DHPs. The sugar origin of DHPs and their prevalence in the human body suggests that these compounds may be involved in various internal injuries in vivo. 7) MATERIALS AND METHODS Synthesis of DHPsThe DHPs (Fig. 1) were synthesized by condensation of diketones and diamines. 2,3-Dihydro-5,6-dimethylpyrazine (DHP-1), 2,3-dihydro-2,5,6-trimethylpyrazine (DHP-2), and 3-hydro-2,2,5,6-tetramethylpyrazine (DHP-3) were prepared by the method of Yamaguchi et al. 3)Bacterial Strains E. coli K-12 strains used include the following. AB1157 [ara-14 argE3 galK2 his-4 lacY1 leuB6 mtl-1 proA2 rpsL31 supE44 thi- Survival Assay E. coli cultured overnight were diluted 100-fold in fresh LB medium and grown at 37°C until the OD 600 was approximately 0.1. Cells were challenged with DHPs for 1 h, incubated with shaking at 37°C, plated on LB agar, and incubated overnight at 37°C. Survival rates were calculated as the percentage of cells surviving after challenge by DHP-1, DHP-2 or DHP-3. All experiments were conducted at least four times.Enzymes and Chemicals Bovine erythrocyte superoxide dismutase (5000 units/mg), bovine liver catalase (11500 units/mg), dimethyl sulfoxide (DMSO), and 2-mercaptoethylamine hydrochloride (MEA) were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). RESULTSThe effect of DHPs in the presence of Cu 2ϩ was examined through the measurement of the E. coli viability. A wild-type strain, recA, and uvrB strain were incubated with or without Cu 2ϩ using three DHP types as shown in Fig. 1, which revealed in vitro DNA strand-cleaving activity of the order DHP-3ϾDHP-2ϾDHP-1.3) As shown in Fig. 2, the effect of DHPs in all strains of E. coli was accelerated greatly by addition of Cu 2ϩ . This is definitive evidence that the recA and uvrB strains possess greater sensitivity to DHPs upon addition of Cu 2ϩ compared to the wild-type strain. The effect in the recA strain was obvious, even without Cu 2ϩ . Therefore, this effect on E. coli must involve specific DNA damage caused by radical species generat...

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“…3) The high recovery of survival rate by MEA indicated that the main reactive species is a C-radical, 8) supported by (OH and · O 2…”

Section: Fig 2 Survival Rate Of E Coli Mutants Exposed To Dhpsmentioning

confidence: 93%

“…2). 8) In the recA strain, the lethal effect of DHPs was observed even without Cu 2ϩ . Two DNA repair-deficient mutant strains, the recA and uvrB strains, possessed higher sensitivity to DHPs upon addition of Cu 2ϩ compared to the wild-type strain (Fig.…”

Section: Fig 2 Survival Rate Of E Coli Mutants Exposed To Dhpsmentioning

confidence: 99%

The Role of Dihydropyrazines in Accelerated Death of Escherichia coli on Addition of Copper(II)

Takeda

Takechi

Katoh

et al. 2005

Biological & Pharmaceutical Bulletin

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confidence: 99%

“…It is thought that all these phenomena originate from the two natural characteristics of DHPs, i.e., their high chemical reactivity and radical generation ability. [4][5][6][7][8][9] As exemplified in Chart 1, less substituted DHPs such as 5,6-dimethyl-2,3-dihydropyrazine exhibit high chemical reactivity and readily transform to dimeric heterocyclic compounds via aldol condensation or pericyclic ene reaction (Chart 1). 5,6) In order to clarify the inherent chemical reactivity of DHPs, we studied the cycloaddition behavior of DHPs as diazadienes or imines towards ketenes.…”

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confidence: 99%

Chemical Reactivity of Dihydropyrazine Derivatives. Cycloaddition Behavior toward Ketenes

Nakahara

Yamaguchi

Yoshitake

et al. 2009

CHEMICAL & PHARMACEUTICAL BULLETIN

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Dihydropyrazines (DHPs), which are derived from aminosugars, exhibit various properties such as specific DNA strand-breakage activity, [1][2][3] facile dimerization, [4][5][6] unique ESR spectral behavior, [7][8][9] in vitro, induction of apoptosis 10) and mutagenesis 11,12) in vivo. It is thought that all these phenomena originate from the two natural characteristics of DHPs, i.e., their high chemical reactivity and radical generation ability. [4][5][6][7][8][9] As exemplified in Chart 1, less substituted DHPs such as 5,6-dimethyl-2,3-dihydropyrazine exhibit high chemical reactivity and readily transform to dimeric heterocyclic compounds via aldol condensation or pericyclic ene reaction (Chart 1). 5,6) In order to clarify the inherent chemical reactivity of DHPs, we studied the cycloaddition behavior of DHPs as diazadienes or imines towards ketenes. The results are discussed in detail on the basis of molecular orbital (MO) calculations on the cycloaddition pathways and the single crystal X-ray analyses of the cycloadducts. Results and DiscussionFirst, reactions of relatively stable 2,3-diphenyl DHP derivatives (1a-c) 13) with ketenes (2a-c) were performed (Chart 2).The reaction of 1a with 2a provided the 1 : 1 adduct (3aa) and two stereoisomeric 1:2 adducts (anti 4aa and syn 4aa). The yields and product ratios for the reactions with 1a-c and 2a-c are summarized in Table 1. ; 4-22-1 Ikeda, Kumamoto 860-0082, Japan. Received April 9, 2009; accepted May 7, 2009; published online May 12, 2009 The cycloaddition behavior of dihydropyrazines toward ketenes was investigated using single-crystal X-ray structures of the cycloadducts and density functional theory (DFT) calculation data. The reaction proceeds via a stepwise pathway involving an orientation complex prior to formation of the betaine intermediate. This is followed by electrocyclization to afford the 1 : 1 and 1 : 2 adducts bearing b b-lactam ring(s).

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“…We previously described the DNA strand-breaking activity [1][2][3] of dihydropyrazines (DHPs) in vitro, as well as other characteristics such as the generation of radical species 4,5) and high chemical reactivity. [6][7][8] We also investigated the biological effects of DHPs, such as induction of apoptosis 9) and mutagenesis.…”

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confidence: 99%