2¡¥CHEMISTRY

  1. ESR Spin Trapping Studies on the Reactions of Hydroperoxides with Cu(¢£) Complex
  2. Synthesis of (-)-Anisomycin Derivative from (S)-Pyroglutamic Acid Derivative
  3. Synthesis of Dithiocarbamate Derivatives and Spin Trapping of Nitric Oxide In Vivo with Their Iron Complexes



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1¡¥ ESR Spin Trapping Studies on the Reactions of Hydroperoxides with Cu(¢£) Complex

Jun-ichi Ueda and Toshihiko Ozawa

Keywords: copper (¢£) complex, tert-butyl hydroperoxide, cumene hydroperoxide, ESR, spin trapping


¡¡Free radicals such as peroxyl (ROOŽ¥) and alkoxyl (ROŽ¥) radicals generated from lipid hydroperoxides with iron, have been recognized as mediators of tumor initiation and promotion. Copper has received less attention than iron, but is known to be more reactive than iron in stimulating the decomposition of hydrogen peroxide and hydroperoxide. Therefore, biological damage such as DNA damage, protein modification, and oxidation of low-density lipoproteins may be caused by the reactions of hydroperoxides with Cu(¢£) ion. Although Cu(¢£) ion exists as a complex in living organisms, there have been few reports about the reactions of Cu(¢£) complexes with hydroperoxides. Then, we studied the reactions of a Cu(¢£) complex, Cu(CyHH)£² {CyHH: cyclo(L-histidyl-L-histidyl)}, with lipid hydroperoxide model compounds such as tert-butyl hydroperoxide (tBuOOH) and cumene hydroperoxide (Cumene-OOH) using some spin traps. ¡¡Fig.A) shows an ESR spectrum obtained from the reaction of Cu(CyHH)£² (0.25 mM) with tBuOOH (25 mM) in the presence of 5,5-dimethyl-1-pyrroline N-oxide (DMPO) (25 mM) at pH 7.4. This spectrum consists of two ESR signals. One with hyperfine splitting constants (hfsc) of a£Î(1)¡á1.49 mT and a£È(1)¡á1.62 mT is assigned to the DMPO adduct of tert-butoxy radical (Ž¥OBut) based on close agreement to previously reported hfsc. The other [a£Î(1)¡á1.44 mT, a£È(1)¡á1.05 mT and a£È(1)¡á0.14 mT] is assigned to the DMPO adduct of tert-butyl peroxyl radical (Ž¥OOBut), again based on the reported hfsc agreement. Fig.B) shows an ESR spectrum observed from the reaction of Cu(CyHH)£² (0.25 mM) with £ôBuOOH (25 mM) in the presence of PBN (25 mM) at pH 7.4. This ESR signal [a£Î(1)¡á1.50 mT and a£È(1)¡á0.33 mT] is assignable to the PBN adduct of methoxy radical (OCH£³Ž¥), because of the reported hfsc agreement. Fig9.C) shows an ESR spectrum obtained from the reaction of Cu(CyHH)£² (0.25 mM) with tBuOOH (25 mM) in the presence of POBN (25 mM) at pH 7.4. This ESR signal [a£Î(1)¡á1.59 mT and a£È(1)¡á0.27 mT] is assignable to the POBN-methyl radical (CH£³Ž¥) adduct because of the reported hfsc agreement. In order to ascertain the carbon-centered radical assignment, the water-soluble nitroso spin trap, 3,5-dibromo-4-nitrosobenzenesulfonate (DBNBS), was used. Figure D) shows an ESR spectrum obtained from the reaction of Cu(CyHH)£² (0.25 mM) with tBuOOH (25 mM) in the presence of DBNBS (12.5 mM) at pH 7.4. This spectrum consists of two ESR signals. One major signal [one nitrogen (a£Î(1)¡á1.38 mT), three magnetically equivalent protons (a£È(3)¡á1.35 mT), and two magnetically equivalent meta-protons (a£È(2)¡á0.07 mT)] can be assigned to the DBNBS-CH£³ adduct. Similar results are obtained from the reaction of Cu(CyHH)£² with Cumene-OOH. ¡¡In conclusion, the formation of ROOŽ¥ and ROŽ¥ radicals from the reaction of tBuOOH or Cumene-OOH with Cu(CyHH)£² was confirmed by ESR using DMPO as a spin trap. On the other hand, neither ROOŽ¥ nor ROŽ¥ was observed when PBN, POBN, or DBNBS was used in place of DMPO. But, mainly CH£³Ž¥, probably generated by ¦Â-scissions of ROŽ¥, was trapped by these spin traps. Rapid unimolecular decomposition of tert-butoxyl radical to CH£³Ž¥ and acetone is a well established reaction. 
[Publications]
1)Ueda, J., Shimazu, Y. and Ozawa, T.: Free Radic. Biol. Med., 18, 929-933, 1995.
2)Ueda, J., Hanaki, A. and Nakajima, T.: Chem. Pharm. Bull., 43, 359-361, 1995.
	Fig9. ESR spectra obtained from the reactions of Cu(CyHH)£² (0.25 mM) with tBuOOH (25 mM) in the presence of (A) DMPO (25 mM), (B) PBN (25 mM), (C) POBN (25 mM), and (D) DBNBS (12.5 mM) at pH 7.4.

Instrumental conditions: microwave power, 10 mW; modulation amplitude, 0.079 mT; amplitude, 2x100; time constant, 0.03 s; scan time, 2 min.  Spectra were recorded 60 s after starting the reaction.

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2¡¥ Synthesis of (-)-Anisomycin Derivative from (S)-Pyroglutamic Acid Derivative

Nobuo Ikota

Keywords : polyhydroxylated pyrrolidine, (-)-anisomycin, antibiotic, (S)-pyroglutamic acid, cis-dihydroxylation, chiral ligand


¡¡Polyhydroxylated pyrrolidines show interesting biological activities such as glycosidase inhibitory activity. In continuation of our work on the synthesis of chiral polyhydroxylated amines, we describe here (Fig.1) the facile synthesis of (-)-anisomycin derivative (9) from (S)-pyroglutamic acid derivative. ¡¡Dihydroxylation of trans-¦Á,¦Â-unsaturated methyl ester (2), prepared from 1 by hydrolysis with aqueous lithium hydroxide, methylation, and deselenenylation in 80¡ó yield, with potassium osmate (0.04 equiv.) using hydroquinine 9-phenanthryl ether (0.15 equiv.) as a chiral ligand in the presence of K£³Fe(CN)£¶ (3 equiv.) and K£²CO£³ (3 equiv.) in tert-BuOH-H£²O (1:1) at 0¡î for 24 h gave 3a and 4a in a ratio of 19:81 in 71¡ó yield. The ratio of 3a and 4a was determined by high performance liquid chromatographic (hplc) analysis after their conversion of 3a and 4a into the coresponding diacetate (3b and 4b) (pyridine, acetic anhydride). The mixture of 3a and 4a was converted into the corresponding TBS ether (TBS chloride, imidazole, dimethylformamide (DMF)) and the diastereoisomers (3c and 4c) were separated by column chromatography. The major isomer (4c) was reduced with LiBH£´ in the presence of lithium triethylborohydride in ether to provide an alcohol(5), which was then converted to the pyrrolidine derivative (6a) via mesylate (MsCl, TEA, CH£²Cl£²; then tert-BuOK, THF) in 40¡ó yield. The configuration of 6a was confirmed by converting it into the known pyrrolidine derivative (7). Thus, the removal of TBS group in 6a with tetrabutylammonium fluoride in THF followed by di-O-benzylation (NaH, DMF-THF, then BnBr) gave 6b in 65¡ó yield. Cleavage of tert-butoxycarbonyl and trityl groups in 6b with acidic conditions (MeOH:10¡ó HCl¡á1:1, 70¡î) followed by N-benzylation with benzyl bromide in the presence of K£²CO£³ in acetone gave 7 in 32¡ó yield. Oxidation of 7 by the method of Swern followed by reaction with 4-methoxyphenylmagnesium bromide in ether gave 8 as the sole diastereomer, which was then treated with triethylsilane in the presence of trifluoroacetic acid and trifuluoromethansulfonic acid in CH£²Cl£² to afford 8b in 31¡ó yield. In this reaction, without addition of trifuluoromethansulfonic acid 8b was not obtained. N-Benzyloxycarbonyl-3,4-dihydroxy-2-(4-methoxyphenyl)pyrrolidine (9) was obtained in 60¡ó yield after debenzylation of 8a (10¡ó palladium carbon, 99¡ó HCOOH, EtOH) followed by N-benzyloxycarbonylation. Compound 9 was easily converted into anisomycin in good yield. 
[Publications]
1)Kanai, M.,  Muraoka, A.,  Ikota N.  and  Tomioka, K.: Tetrahedron Lett., 36, 9349-9352, 1995.
2)Hanaki, A., Nagai, A., and Ikota, N.: Chemistry Lett., 611-612, 1995.
3)Ikota, N.: Heterocycles, 41, 983-994, 1995..
4)Hanaki, A. Saito, M. and Ikota, N.: Nippon Kagaku Kaishi, 388-393, 1995.
5)Tomioka, K., Kanai, M. and Ikota, N.: Heterocycles,  42, 43-45, 1996. 
6)Ikota, N. and Hama-Inaba, H.: Chem. Pharm. Bull., in press.

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3¡¥ Synthesis of Dithiocarbamate Derivatives and Spin Trapping of Nitric Oxide In Vivo with Their Iron Complexes

Hidehiko Nakagawa, Nobuo Ikota and Toshihiko Ozawa

Keywords: dithiocarbamate, iron complex, nitric oxide, ESR, spin trapping, L-proline


¡¡Nitric oxide (NO) is suggested to be an endogenous radical compound and to play an important role in inflammation, neurotransmission and vasodilation. A few iron complexes of dithiocarbamate derivatives have been used for spin trapping of nitric oxide both in vivo and in vitro. In this report, we describe the synthesis of a series of dithiocarbamate derivatives which have the L-proline moiety and the detection of nitric oxide in septic shock model mouse. ¡¡A solution of L-proline in aqueous ammonia was mixed with a small excess of carbon disulfide in an equal volume of ethanol at 4¡î, and then lyophilized to yield dithiocarboxy-L-proline (1, DTCP) as a slightly yellow powder. In the same manner, 4-trans-hydroxy-L-proline and 4-trans-(methoxymethyl)oxy-L-proline, which was derived from 4-trans-hydroxy-L-proline with methoxymetyl chloride, were converted to DTCHP (2) and DTCMP (3), respectively. ¡¡For the preparation of iron complex (DTCX-iron complex), each dithiocarbamate was mixed with a half equivalent of ferrous sulfate in 40mM Tris-HCl (pH 7.4) under an anaerobic condition. The prepared complex solutions were anaerobically stored at 4¡î until use. ¡¡Synthesized dithiocarbamate iron complexes were used to examine the trapping ability of nitric oxide in 40mM Tris buffer. The NO-adduct signals were detected by ESR spectroscopy and increased as a function of nitric oxide concentration. In these experiments, the three synthesized complexes were suggested to have almost the same affinity for nitric oxide. ¡¡Septic shock model mice were obtained by a treatment of lipopolysaccharide (LPS, E.coli 026:B6) to the tail vein of ddY female mice. The complexes were injected into the septic shock model mice intravenously and a certain amount of blood was collected in a heparinized capillary 15 min after injection. The ESR spectra of the blood in the capillary were measured within 6 min. ¡¡The relative amount of NO-adduct of each complex was measured at 3, 5 and 7 hours after LPS treatment. Using DTCP-iron and DTCMP-iron complexes as spin traps, NO-adducts were detected in mouse blood, and the adduct amounts increased with time after the LPS treatment. By contrast, NO-adducts of DTCHP-iron complex were detected only at noise level. The relative amount of detected NO-adducts was larger for DTCP-iron complex than DTCMP-iron complex. Although these complexes were suggested to have same affinity for nitric oxide in an in vitro experiment, it was shown that these complexes had different signal intensities from the NO-adducts in vivo. These results suggested that the complexes had different properties or behavior in the mouse body, and that they were distributed differently in the body and were trapped by nitric oxide in different places. The distribution and metabolism of these complexes or their NO-adducts in mice remain to be studied. 
[Publications]
Fig.1 Structures of synthesized dithiocarbamate derivatives and their iron complexes.

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