IMPROVED HIGH-PERFORMANCE LIQUID CHROMATOGRAPHIC ANALYSIS OF 8-METHOXYPSORALEN MONOADDUCTS and CROSS-LINKS IN POLYNUCLEOTIDE, DNA, and CELLULAR SYSTEMS: ANALYSIS OF SPLIT-DOSE PROTOCOLS

Abstract— The distribution of 8-methoxypsoralen-thymidine photoadducts from polynucleotides, calf thymus DNA and mammalian cells treated with [³H]8-methoxypsoralen under a variety of irradiation conditions was determined using high-performance liquid chromatography and scintillation analysis. The split-dose protocol, with samples treated with 8-methoxypsoralen and low doses of long-wavelength UV radiation to generate monoadducts, washed to remove unreacted 8-methoxypsoralen, then irradiated further to convert the monoadducts to cross-links, was examined. The photoadduct distribution in the first step is dependent upon the UVA dose and the wavelength of the radiation, but it is relatively independent of 8-methoxypsoralen concentration. Low fluence and longer wavelengths generate mainly 4',5'-monoadducts, whereas higher fluences and shorter wavelengths yield more cross-links. The second irradiation step converts the 4',5'-monoadducts to cross-links as well as to 3,4-monoadducts. The overall yield of cross-links after the second irradiation step is not dependent upon the wavelength used in the first step. Cellular studies demonstrated that the split-dose protocol is applicable to mammalian systems. These results may afFect the interpretation of mutagenesis studies based on the split-dose protocol, because the second step can convert 4',5'-monoadducts to both 3,4-monoadducts, the expected cross-links. Therefore, interpretations that link increases in mutagenicity after the second step in a split-dose study solely to cross-link formation may need re-examination.     

METHYLALLOPSORALEN-THYMINE 3,4- and 4'',5''-MONOADDUCTS FORMED IN THE PHOTOREACTION WITH DNA

Abstract— Two new allopsoralens, i.e. 4,7.5'-trimethylallopsoralen and 4,7,4'-trimethylallopsoralen have been irradiated (365 nm) in the presence of DNA. The DNA so treated was hydrolyzed and among the products of its hydrolysis new 3,4- and 4',5'-monocycloadducts between the two furocoumarins and thymine have been isolated. The monoadducts have been characterized on the basis of their spectroscopic properties, of their capacity to undergo photoreversion forming the parent compounds and of the NMR data. A cis-syn conformation has been suggested for both 3,4- and both 4',5'-monoadducts.     

4'-AMINOMETHYL-4,5',8-TRIMETHYLPSORALEN PHOTOCHEMISTRY THE EFFECT OF CONCENTRATION AND UVA FLUENCE ON PHOTOADDUCT FORMATION IN POLY(dAdT) AND CALF THYMUS DNA

Abstract-The photochemistry of 4'-aminomethyl-4,5',8-trimethylpsoralen (AMT) with poly(dA-dT) and calf thymus DNA was studied. The extent of photoadduct formation and the distribution of photoadducts (3,4– and 4',5'-monoadducts and crosslinks) were determined by liquid scintillation analysis and HPLC, respectively. The adducts were characterized on the basis of their UV absorption spectra and mass spectral analysis. The high DNA binding constant for AMT (1.5 x 10⁵ M⁻¹ ) led to a high fraction of intercalated molecules, which contributed to the high level of AMT photoadduct formation, as many as 102 adducts per kilobase pair. In addition, there is a distinct difference in the adduct distribution compared to the previously studied 8-methoxypsoralen (8-MOP). Under the conditions employed for the photochemical studies, virtually all of the AMT molecules in solution are intercalated, occupying 25% of the base pair sites. Under similar conditions, 8-MOP molecules occupied 10 times fewer sites. Thus, for AMT, DNA base pair sites other than 5'TA, the well-characterized strong binding for psoralens in general, are an additional target for photomodification, which results in the formation of a higher percentage of monoadducts. The proportion of photoadducts formed was virtually independent of AMT concentration and UVA (320–400 nm radiation) fluence.     

TRIPLET EXCITED STATES OF 4'',5''-PHOTOMONOADDUCTS OF 3- CARBETHOXYPSORALEN and 8-METHOXYPSORALEN WITH DNA NUCLEOSIDES

Abstract— Triplet absorption spectra, triplet extinction coefficient and intersystem crossing for 4',5'-monocycloadducts of 3-carbethoxypsoralen (3-CPs) with thymidine (dThd) and uridine (dUrd) in ethanol have been investigated in order to elucidate whether their triplet state properties could be the limitating step for a further photoreaction of 3-CPs monoadducts with DNA nucleosides. The comparison between the triplet characteristics of 4',5'-monoadducts of 3-CPs and those of 8-methoxypsoralen (8-MOP) shows that the quantum yield is much higher in the case of 3-CPs than for 8-MOP. The monofunctionality of 3-CPs cannot therefore be ascribed to the triplet excited states properties of its monoadducts. It is likely that steric hindrance introduced by the bulky carbethoxy group remains a reasonable explanation.     

New aspects of the repair and genotoxicity of psoralen photoinduced lesions in DNA.

Several approaches are described aiming at a better understanding of the genotoxicity of psoralen photoinduced lesions in DNA. Psoralens can photoinduce different types of photolesions including 3,4- and 4',5'-monoadducts and interstrand cross-links, oxidative damage (in the case of 3-carbethoxypsoralen (3-CPs)) and even pyrimidine dimers (in the case of 7-methylpyrido(3,4-c)psoralen (MePyPs)). The characterization and detection of different types of lesions has been essential for the analysis of their possible contributions to genotoxicity. For example, oxidative damage photoinduced by 3-CPs can be detected by the formamidopyrimidine glycosylase (FPG) protein. Furthermore, it is shown how the presence of MePyPs induced monoadducts may interfere with the photoreactivation of concomitantly induced pyrimidine dimers, how the ratio of monoadducts and interstrand cross-links (CL) affects the occurrence of double-strand breaks during the repair of photolesions and genotoxicity. In vitro treatment of yeast plasmids, followed by transformation, also indicates that the repair of photoadducts on exogenous DNA differs for 8-methoxy-psoralen (8-MOP) induced mono- and diadducts and for monoadducts alone. The recombinational rad52 dependent pathway is not needed for the repair of 8-MOP induced monoadducts. The results obtained suggest that the genotoxic effects of psoralens are conditioned by the nature, number, ratio and sequence distribution of the photolesions induced in DNA.     

CELL MEMBRANE DNA: A NEW TARGET FOR PSORALEN PHOTOADDUCT FORMATION

The effects of 8-methoxypsoralen plus long wavelength ultraviolet radiation on cell membrane DNA were examined. Treatment of human lymphocytes with 100 ng/ml 8-methoxypsoralen and 5 J/cm2 UVA led to the formation of 7.1 +/- 3.8 photoadducts per million bases. A monoclonal antibody, specific for 8-methoxypsoralen 4',5'-monoadducts, was used to detect photoadducts in the cell membrane DNA of human lymphocytes and three lymphoblastoid cell lines. Treatment of lymphocytes with 8-MOP and UVA reduced the lymphocyte DNA binding capacity by 56%. Cell membranes of normal lymphocytes were shown to contain three high affinity DNA binding proteins of 28, 59, and 79 kDa, respectively.     

PHOTOPHYSICAL BEHAVIOUR OF NEW PYRROLOCOUMARIN DERIVATIVES

Abstract— The photophysical behaviour of new pyrrolocoumarins with different substituents on the nitrogen are reported. The photophysical properties of these pyrrolocoumarins are generally in agreement with those of the psoralens: a strong absorption (240–400 nm), a weak fluorescence (400–680 nm) characterized by a short singlet lifetime, and a rather strong phosphorescence at 77 K (480–600 nm). The absorption and fluorescence properties were investigated in several solvents. The shift of the fluorescence maximum is interpreted on the basis of the solvatochromic parameters π*, α and β. The triplet-triplet absorption spectra also depend on the nature of the solvent, while the triplet excited state has a lifetime of a few microseconds at room temperature (concentration 2.5 × 10⁻⁴ M ). Some absorption and fluorescence characteristics of the 4',5'-dihydropyrrolocoumarins, which are suitable models for the 4',5'-monoadducts to pyrrolocoumarins are reported.     

Structure of new carotenoids with a 3,4-dihydroxy-beta-end group from the oyster Crassostrea gigas

Three new carotenoids with a 3,4-dihydroxy-beta-end group were isolated from the oyster Crassostrea gigas. These structures were determined to be 3,4,3',8'-tetrahydroxy-beta,kappa-caroten-6'-one (1), 3,4-dihydroxy-3',6'-epoxy-1',2',5',6'7',8'-hexahydro-6'-methyl-16'-nor-beta,-carotene-1',8'-dione (2), and 3,6-epoxy-5,3',4'-trihydroxy-12',13',20'-trinor-beta,beta-caroten-19,11-olide (3) based on chemical and spectral data.     

Bacterial carotenoids. XLVIII. Total syntheses of carotenes of the 1,2-dihydro series.

The total syntheses of 1,2,7,8,1',2',7',8'-octahydro-psi,psi-carotene (1), 1,2,7,8-tetrahydro-psi,psi-carotene (2), 1,2,1',2'-tetradehydro-psi,psi-carotene (3), 1,2-dihydro-psi,psi-carotene (4), 1,2-dihydor-3,4-didehydro-psi,psi-carotene (5), and 1,2,1',2'-tetrahydro-3,4,3',4'-tetrahydro-psi,psi-carotene (6) are described. The properties of products and intermediates, including the three new apocarotenals 1,2,7,8-tetrahydro-12'-apo-psi-caroten-12'-al (20), 1,2-dihydro-8'-apo-pse-caroten-8'-al (25), and 1,2-dihydro-3,4-didehydro-8'-apo-psi-carotene-8'-al (32), are reported. A fragment ion at M68 on electron impact appears to be characteristic for carotenoids with a 1,2,7,8-tetrahydro end-group.     

PHOTOREACTIONS OF ALLOPURINOL AND HYPOXANTHINE WITH 2-PROPANOL

Abstract— Ultraviolet irradiation of allopurinol with 2-propanol, either in aqueous solution at 254 nm or in aqueous acetone (used as a photosensitizer) at > 290 nm, gave 2-(3'-hydroxy-2',3'-dimethylbutan-2'-yl)pyrazolo[3,4-d]pyrimidin-4(5H)-one (3) and 6-(2'-hydroxypropan-2'-yl)pyrazolo[3,4-d]pyrimidin-4(5H)-one (4) as major products. In addition, two minor photoproducts, which were not characterized, were detected by thin layer chromatography. Under similar irradiation conditions, hypoxanthine yielded 8-(2'-hydroxypropan-2'-yl)-2-(propan-2'-yl)hypoxanthine (5), 8-(3'-hydroxy-2',3'-dimethylbutan-2'-yl)hy-poxanthine (6) and 8-(2'-hydroxypropan-2'-yl)hypoxanthine (7) together with two unidentified minor photoproducts. Compounds 5 and 6 appear to be secondary photoproducts derived from 7. Initiation of the reaction by photodecomposition of di- t -butyl peroxide led to specific formation of 4 from allopurinol and of 7 from hypoxanthine. The allopurinol and hypoxanthine photoproducts were characterized by their ¹H-NMR, UV, and mass spectra.     

Acid-promoted reaction of the stilbene antioxidant resveratrol with nitrite ions: mild phenolic oxidation at the 4'-hydroxystiryl sector triggering nitration, dimerization, and aldehyde-forming routes

In 0.1 M phosphate buffer, pH 3.0, and at 37 degrees C, resveratrol ((E)-3,4',5-trihydroxystilbene, 1a), an antioxidant and cancer chemopreventive phytoalexin, reacted smoothly at 25 microM or 1 mM concentration with excess nitrite ions (NO2(-)) to give a complex pattern of products, including two novel regioisomeric alpha-nitro (3a) and 3'-nitro (4) derivatives along with some (E)-3,4',5-trihydroxy-2,3'-dinitrostilbene (5), four oxidative breakdown products, 4-hydroxybenzaldehyde, 4-hydroxy-3-nitrobenzaldehyde, 3,5-dihydroxyphenylnitromethane, and 3,5-dihydroxybenzaldehyde, two dimers, the resveratrol (E)-dehydrodimer 6 and restrytisol B (7), and the partially cleaved dimer 2. The same products were formed in the absence of oxygen. 1H,15N HMBC and LC/MS analysis of the crude mixture obtained by reaction of 1a with Na (15)NO2 suggested the presence of 3,4',5,beta-tetrahydroxy-alpha-nitro-alpha,beta-dihydrostilbene (8) as unstable intermediate which escaped isolation. Under similar conditions, the structurally related catecholic stilbene piceatannol ((E)-3,3',4,5'-tetrahydroxystilbene, 1b) gave, besides (E)-3,3',4,5'-tetrahydroxy-beta-nitrostilbene (3b), 3,4-dihydroxybenzaldehyde and small amounts of 3,5-dihydroxybenzaldehyde. Mechanistic experiments were consistent with the initial generation of the phenoxyl radical of 1a at 4'-OH, which may undergo free radical coupling with NO2 at the alpha- or 3'-position, to give eventually nitrated derivatives and/or oxidative double bond fission products, or self-coupling, to give dimers. The oxygen-independent, NO2(-)-mediated oxidative fission of the double bond under mild, physiologically relevant conditions is unprecedented in stilbene chemistry and is proposed to involve breakdown of hydroxynitro(so) intermediates of the type 8.     

Condensed isoquinolines. 37.* Heterocyclization using 3-(arylamino)- and 3-(hetarylamino)isoquinolin-1(2H)-ones

The reaction of 3-NHR-isoquinolin-1(2H)-ones (R = Ar) with aromatic aldehydes in the presence of Me3SiCl or in acetic acid leads to the formation of derivatives of dibenzo[b,f][1, 8]naphthyridin-5(6H)- one and benzo[f]isoquino[3,4-b][1, 8]naphthyridine-5,9(6H,7H)-dione. The reaction for R = Het in the presence of Me3SiCl gives derivatives of 5H-pyrido[1',2':1,2]pyrimido[4,5-c]isoquinolin-5-one, benzo[f]isoquinoline[3,4-b][1,8]naphthyridine-5,9[6H,7H]-dione, and derivatives of new heterocyclic systems, 5H-pyrazino[1',2':1,2]pyrimido[4,5-c]isoquinolin-5-one, 5H-[1,3]thiazolo[3',2':1,2]pyrimido- [4,5-c]isoquinolin-5-one, 5-H-benzo[f]pyrazolo[3,4-b][1,8]naphthyridin-5-one, and isoquino[3,4-b]- [1,5]naphthyridin-5(6H)-one. The effect of the structure of substituent R and nature of the substituent in the benzaldehydes on the structure of the reaction products was studied.     

A novel 8.O.6'-neolignan from Taxillus theifer

A new 8.O.6'-neolignan, threo-(7R,?8R)-7-acetoxy-3',4'-dimethoxy-3,4-dimethoxy-Δ-(8')-8.O.6'-neolignan (1) along with two known lignans (2) and (3), was isolated from the leaves and stems of Taxillus theifer. Structural elucidation was carried out by 1-D and 2-D-NMR spectroscopic methods, and the absolute configurations of C-7 and C-8 in 1 were determined on the basis of circular dichroism. Compound 2 is threo-7-acetoxy-3'-methoxy-3,4-dimethoxy-Δ-(7')-8.O.4'-neolignan obtained from a natural source for the first time, previously derived from a synthesis study of 8,4'-oxyneolignans.     

A FLOW LINEAR DICHROISM STUDY OF THE ORIENTATION OF 4'',5''-PSORALEN-DNA PHOTOADDUCTS

Abstract— The flow linear dichroism properties of covalent adducts derived from the photochemical binding of various psoralen derivatives to salmon sperm DNA were investigated. The psoralens studied include bifunctional derivatives (8-methoxypsoralen,5-methoxypsoralen, tetrahydropyrido [3,4: 4',5'] psoralen) and monofunctional derivatives (pyrido [3,4-c] psoralen, 7-methylpyrido [3,4-c] psoralen, 3-carbethoxypsoralen). The orientation of the psoralen moieties (furan-side monoadducts) relative to the orientation of the DNA bases was determined. All of the furan-side monoadducts are characterized by a similar orientation, with mean angles between the psoralen moiety and the normals of the planes of the DNA bases ranging between 70° and values close—but not equal—to 90°. The results are consistent with a pseudo-intercalative adduct geometry, most probably involving stacking interactions with the DNA bases.     

Antiangiogenic polyketides from Peperomia dindygulensis Miq

Two new polyketides: 2Z-(heptadec-12-enyl)-4-hydroxy-3,4,7,8-tetrahydro-2H-chromen-5(6H)-one (1) and 2-(heptadec-12-enyl)-5-hydroxy-5,6,7,8-tetrahydrochromen- 4-one (2), together with eleven known compounds: 4-hydroxy-2-[(3,4-methylenedioxy- phenyl)tridecanoyl] cyclohexane-1,3-dione (3), oleiferinone (4), 4-hydroxy-2-[(3,4- methylenedioxyphenyl)undecanoyl]cyclohexane-1,3-dione (5), 4-hydroxy-2-[(11-phenyl- undecanoyl)cyclohexane-1,3-dione (6), proctorione C (7), surinone C (8), 5-hydroxy- 7,8,4'-trimethoxyflavone (9), 5-hydroxy-7,8,3',4'-tetramethoxyflavone (10), 5-hydroxy- 7,3',4'-trimethoxyflavone (11), 5,8-dihydroxy-7,3',4'-trimethoxyflavone (12) and cepharanone B (13) were isolated from the whole plant of Peperomia dindygulensis Miq. Their structures were elucidated by spectroscopic methods, including 2D-NMR techniques. Compounds 2, 3, 5 and 8 inhibited human umbilical vein endothelial cell (HUVEC) proliferation and compounds 5 and 8 sharply suppressed HUVEC tube formation.     

5，3’，4’—三羟基—6，7—二甲氧基黄酮的另法全合成

从两个易得原料3，4，5—三甲氧基苯甲酸和3，4-二羟基苯甲醛出发，分别合 成出2—羟基4，5，6—三甲氧基苯乙酮和3，4-二苄氧基苯甲酰氯，随后采用相转 移催化法成功地全合成了5，3’，4’—三羟基—6，7—二甲氧基黄酮。     

Neolignans from Piper futokadsura and their inhibition of nitric oxide production

From a MeOH extract of the aerial part of Piper futokadsura, the tetrahydrofuran lignans, futokadsurin A [(7S,8S,7'S,8'R)-3,4,3'-trimethoxy-4'-hydroxy-7,7'-epoxylignan], futokadsurin B [(7R,8R,7'R,8'S)-3,4-dimethoxy-3',4'-methylenedioxy-7,7'-epoxylignan], and futokadsurin C [(7R,8R,7'S,8'S)-3,4-methylenedioxy-3',4'-dimethoxy-7,7'-epoxylignan] were isolated, together with nine known neolignans. In addition, L-tryptophan, pellitorine, phytol, elemicin, and 1,2,4-trimethoxyphenyl-5-aldehyde were isolated. The structures of the new compounds were elucidated using spectroscopic methods. These lignans inhibited nitric oxide production by a murine macrophage-like cell line (RAW 264.7), which was activated by lipopolysaccharide and interferon-gamma.     

Pyrrolopyrimidines. 5. Reaction of 6-Amino-1,3-dimethylpyrrolo[3,4-d]pyrimidine-2,4(1H,3H)-diones with 1,3-Diketones

The reaction of 6-amino-1,3-dimethylpyrrolo[3,4-d]pyrimidine-2,4-dione with 1,3-diketones leads to formation of predominantly pyrimido[4',5':3,4]pyrrolo[1,2-b]pyridazine-2,4(1H,3H)-diones and, to a lesser extent, pyrimido[5',4':3,4]pyrrolo[1,2-b]pyridazine-1,3(2H,4H)-diones. The ease and direction of the cyclization reaction suggests a very -electron rich pyrrole ring in the initial state, especially in the position 7.     

Synthesis of 8-amino derivatives of condensed furo[3,2-<Emphasis Type="Italic">d</Emphasis>]pyrimidines

A method for the synthesis of new 8-amine derivatives of pyrano[4",3":4',5']pyrido[3',2':4,5]furo-[3,2-d]pyrimidines based on 6-oxo derivatives of pyrano[3,4-c]pyridines has been developed.     

Organic thin-film transistors based on carbonyl-functionalized quaterthiophenes: high mobility N-channel semiconductors and ambipolar transport

New carbonyl-functionalized quaterthiophenes, 5, 5' '-diheptanoyl-2,2':5',2' ':5' ',2' '-quaterthiophene (DHCO-4T), 5, 5' '-diperfluorohexylcarbonyl-2,2':5',2' ':5' ',2' '-quaterthiophene (DFHCO-4T), and 2,7-[bis-(5-perfluorohexylcarbonylthien-2-yl)]-4H-cyclopenta[2,1-b:3,4-b']-dithiophen-4-one (DFHCO-4TCO) have been synthesized and characterized. Field-effect transistors fabricated with these materials exhibit high electron mobilities both in a vacuum (up to 0.6 cm2 V-1 s-1) and in air (up to 0.02 cm2 V-1 s-1) and very high Ion:Ioff currents ratios (>107). DHCO-4T is the first organic material exhibiting excellent ambipolar transport (mue/muh up to 0.1/0.01 cm2 V-1 s-1, (Ion:Ioff)e/(Ion:Ioff)h up to 107/108 for the same device) over a broad range of deposition temperatures. These materials are therefore promising for organic complementary circuits.