Simultaneous determination of 8‐oxo‐2′‐deoxyguanosine and 8‐oxo‐2′‐deoxyadenosine in DNA using online column‐switching liquid chromatography/tandem mass spectrometry

Sensitive and reliable methods are required for the assessment of oxidative DNA damage, which can result from reactive oxygen species that are generated endogenously from cellular metabolism and inflammatory responses, or by exposure to exogenous agents. The development of a liquid chromatography/tandem mass spectrometry (LC/MS/MS) selected reaction monitoring (SRM) method is described, that utilises online column‐switching valve technology for the simultaneous determination of two DNA adduct biomarkers of oxidative stress, 8‐oxo‐7,8‐dihydro‐2′‐deoxyguanosine (8‐oxodG) and 8‐oxo‐7,8‐dihydro‐2′‐deoxyadenosine (8‐oxodA). To allow for the accurate quantitation of both adducts the corresponding [¹⁵N₅]‐labelled stable isotope internal standards were synthesised and added prior to enzymatic hydrolysis of the DNA samples to 2′‐deoxynucleosides. The method required between 10 and 40 µg of hydrolysed DNA on‐column for the analysis and the limit of detection for both 8‐oxodG and 8‐oxodA was 5 fmol. The analysis of calf thymus DNA treated in vitro with methylene blue (ranging from 5 to 200 µM) plus light showed a dose‐dependent increase in the levels of both 8‐oxodG and 8‐oxodA. The level of 8‐oxodG was on average 29.4‐fold higher than that of 8‐oxodA and an excellent linear correlation (r = 0.999) was observed between the two adducts. The influence of different DNA extraction procedures for 8‐oxodG and 8‐oxodA levels was assessed in DNA extracted from rat livers following dosing with carbon tetrachloride. The levels of 8‐oxodG and 8‐oxodA were on average 2.9 (p = 0.018) and 1.4 (p = 0.018) times higher, respectively, in DNA samples extracted using an anion‐exchange column procedure than in samples extracted using a chaotropic procedure, implying artefactual generation of the two adducts. In conclusion, the online column‐switching LC/MS/MS SRM method provides the advantages of increased sample throughput with reduced matrix effects and concomitant ionisation suppression, making the method ideally suited when used in conjunction with chaotropic DNA extraction for the determination of oxidative DNA damage. Copyright © 2008 John Wiley & Sons, Ltd.     

Tualang honey protects keratinocytes from ultraviolet radiation-induced inflammation and DNA damage(†)

Malaysian tualang honey possesses strong antioxidant and anti‐inflammatory properties. Here, we evaluated the effect of tualang honey on early biomarkers of photocarcinogenesis employing PAM212 mouse keratinocyte cell line. Keratinocytes were treated with tualang honey (1.0%, v/v) before a single UVB (150 mJ cm^?2) irradiation. We found that the treatment of tualang honey inhibited UVB‐induced DNA damage, and enhanced repair of UVB‐mediated formation of cyclobutane pyrimidine dimers and 8‐oxo‐7,8‐dihydro‐2′‐deoxyguanosine. Treatment of tualang honey inhibited UVB‐induced nuclear translocation of NF‐κB and degradation of IκBα in murine keratinocyte cell line. The treatment of tualang honey also inhibited UVB‐induced inflammatory cytokines and inducible nitric oxide synthase protein expression. Furthermore, the treatment of tualang honey inhibited UVB‐induced COX‐2 expression and PGE2 production. Taken together, we provide evidence that the treatment of tualang honey to keratinocytes affords substantial protection from the adverse effects of UVB radiation via modulation in early biomarkers of photocarcinogenesis and provide suggestion for its photochemopreventive potential.     

Discrimination Between 8‐Oxo‐2′‐Deoxyguanosine and 2′‐Deoxyguanosine in DNA by the Single Nucleotide Primer Extension Reaction with Adap Triphosphate

The adenosine derivative of 2‐oxo‐1,3‐diazaphenoxazine (Adap) exhibits a superb ability to recognize and form base pairs with 8‐oxo‐2′‐deoxyguanosine (8‐oxo‐dG) in duplex DNA. In this study, the triphosphate of Adap (dAdapTP) was synthesized and tested for single nucleotide incorporation into primer strands using the Klenow Fragment. The efficiency of dAdapTP incorporation into 8‐oxo‐dG‐containing templates was more than 36‐fold higher than with dG‐containing templates, and provides better discrimination than does the incorporation of natural 2′‐deoxyadenosine triphosphate (dATP). The selective incorporation of dAdapTP into 8‐oxo‐dG templates was therefore applied to the detection of 8‐oxo‐dG in human telomeric DNA sequences extracted from H₂O₂‐treated HeLa cells. The enzymatic incorporation of dAdapTP into 8‐oxo‐dG‐containing templates may provide a novel basis for sequencing oxidative DNA damage in the genome.     

Protective Effect of Tropical Highland Blackberry Juice (Rubus adenotrichos Schltdl.) Against UVB‐Mediated Damage in Human Epidermal Keratinocytes and in a Reconstituted Skin Equivalent Model

Solar ultraviolet (UV) radiation, particularly its UVB (280–320 nm) spectrum, is the primary environmental stimulus leading to skin carcinogenesis. Several botanical species with antioxidant properties have shown photochemopreventive effects against UVB damage. Costa Rica's tropical highland blackberry (Rubus adenotrichos) contains important levels of phenolic compounds, mainly ellagitannins and anthocyanins, with strong antioxidant properties. In this study, we examined the photochemopreventive effect of R. adenotrichos blackberry juice (BBJ) on UVB‐mediated responses in human epidermal keratinocytes and in a three‐dimensional (3D) reconstituted normal human skin equivalent (SE). Pretreatment (2 h) and posttreatment (24 h) of normal human epidermal keratinocytes (NHEKs) with BBJ reduced UVB (25 mJ cm^?2)‐mediated (1) cyclobutane pyrimidine dimers (CPDs) and (2) 8‐oxo‐7,8‐dihydro‐2′‐deoxyguanosine (8‐oxodG) formation. Furthermore, treatment of NHEKs with BBJ increased UVB‐mediated (1) poly(ADP‐ribose) polymerase cleavage and (2) activation of caspases 3, 8 and 9. Thus, BBJ seems to alleviate UVB‐induced effects by reducing DNA damage and increasing apoptosis of damaged cells. To establish the in vivo significance of these findings to human skin, immunohistochemistry studies were performed in a 3D SE model, where BBJ was also found to decrease CPDs formation. These data suggest that BBJ may be developed as an agent to ameliorate UV‐induced skin damage.     

5‐Hydroxyalkyl derivatives of tert‐butyl 2‐oxo‐2,5‐dihydro‐1H‐pyrrole‐1‐carboxylate: diastereoselectivity of the Mukaiyama crossed‐aldol‐type reaction

The title compounds, rac‐(1′R,2R)‐tert‐butyl 2‐(1′‐hydroxyethyl)‐3‐(2‐nitrophenyl)‐5‐oxo‐2,5‐dihydro‐1H‐pyrrole‐1‐carboxylate, C₁₇H₂₀N₂O₆, (I), rac‐(1′S,2R)‐tert‐butyl 2‐[1′‐hydroxy‐3′‐(methoxycarbonyl)propyl]‐3‐(2‐nitrophenyl)‐5‐oxo‐2,5‐dihydro‐1H‐pyrrole‐1‐carboxylate, C₂₀H₂₄N₂O₈, (II), and rac‐(1′S,2R)‐tert‐butyl 2‐(4′‐bromo‐1′‐hydroxybutyl)‐5‐oxo‐2,5‐dihydro‐1H‐pyrrole‐1‐carboxylate, C₁₃H₂₀BrNO₄, (III), are 5‐hydroxyalkyl derivatives of tert‐butyl 2‐oxo‐2,5‐dihydropyrrole‐1‐carboxylate. In all three compounds, the tert‐butoxycarbonyl (Boc) unit is orientated in the same manner with respect to the mean plane through the 2‐oxo‐2,5‐dihydro‐1H‐pyrrole ring. The hydroxyl substituent at one of the newly created chiral centres, which have relative R,R stereochemistry, is trans with respect to the oxo group of the pyrrole ring in (I), synthesized using acetaldehyde. When a larger aldehyde was used, as in compounds (II) and (III), the hydroxyl substituent was found to be cis with respect to the oxo group of the pyrrole ring. Here, the relative stereochemistry of the newly created chiral centres is R,S. In compound (I), O—H...O hydrogen bonding leads to an interesting hexagonal arrangement of symmetry‐related molecules. In (II) and (III), the hydroxyl groups are involved in bifurcated O—H...O hydrogen bonds, and centrosymmetric hydrogen‐bonded dimers are formed. The Mukaiyama crossed‐aldol‐type reaction was successful when using the 2‐nitrophenyl‐substituted hydroxypyrrole, or the unsubstituted hydroxypyrrole, and boron trifluoride diethyl ether as catalyst. The synthetic procedure leads to a syn configuration of the two newly created chiral centres in all three compounds.     

Luminescence properties of three structures built from 3‐cyano‐4‐dicyanomethylene‐5‐oxo‐4,5‐dihydro‐1H‐pyrrol‐2‐olate and alkaline metals (Na,K and Rb)

The structures of three salts of 3‐cyano‐4‐dicyanomethylene‐5‐oxo‐4,5‐dihydro‐1H‐pyrrol‐2‐olate with alkali metals (Na, K and Rb) are related to their luminescence properties. The Rb salt, rubidium(I) 3‐cyano‐4‐dicyanomethylene‐5‐oxo‐4,5‐dihydro‐1H‐pyrrol‐2‐olate, Rb⁺·C₈HN₄O₂⁻, is isomorphous with the previously reported potassium salt. For the Na compound, sodium(I) 3‐cyano‐4‐dicyanomethylene‐5‐oxo‐4,5‐dihydro‐1H‐pyrrol‐2‐olate dihydrate, Na⁺·C₈HN₄O₂⁻·2H₂O, two independent sodium ions, located on inversion centers, are coordinated by four water molecules each and additionally by two cyano groups for one and two carbonyl groups for the other. The luminescence spectra in solution are unaffected by the nature of the cation but vary strongly with the dielectric constant of the solvent. In the solid state, the emission maxima vary with structural features; the redshift of the maximum luminescence varies inversely with the distance between the stacked anions.     

Role of Nitrite on Nitration of 2′‐Deoxyguanosine by Nitryl Chloride

Nitryl chloride and peroxynitrite are reactive nitrogen species generated by activated phagocytes against invading pathogens during infections and inflammation. In our previous report, formation of 8‐nitroxanthine and 8‐nitroguanine was observed in reaction of 2′‐deoxyguanosine or calf thymus DNA with nitryl chloride generated by mixing hypochlorous acid (HOCl) with nitrite (NC₂^?). The present study investigates factors control ling the yields of 8‐nitroxanthine and 8‐nitroguanine formation in nitration of 2′‐deoxyguanosine by nitryl chloride. We found that the yields of 8‐nitroxanthine and 8‐nitroguanine in reaction of 2′‐deoxyguanosine with nitryl chloride were highly dependent on the ratio of NO₂^? versus HOCl concentration. The yields of 8‐nitroxanthine and 8‐nitroguanine reached a plateau when the ratio of NC₂^? versus HOCl concentration was higher than 2. A possible mechanism was postulated to explain this observation. While 8‐nitroguanine is not stable in the presence of peroxynitrite, 8‐nitroxanthine is sensitive to HOCl. The stability of these two nitrated ad ducts might be a factor on their final yields in this reaction. Since HOCl is produced by neutrophils at sites of inflammation where the level of NC₂^? is elevated, it is conceivable that nitryl chloride contributes to DNA base nitration in vivo, forming 8‐nitroxanthine and 8‐nitroguanine.     

A facile synthesis of 3‐Substituted 5‐Oxo‐1,2,4‐thiadiazoles from amidoximes

The reaction of amidoximes 1 with 1,1′‐thiocarbonyldiimidazole (TCDI) followed by treatment with silica gel or boron trifluoride diethyl etherate (BF₃·OEt₂) provided 3‐substituted 4,5‐dihydro‐5‐oxo‐1,2,4‐thiadiazoles 2 in moderate yields. The Lewis acids are considered to promote the rearrangement of the thioxocarbamate intermediates 5 to the thiol carbarn ate intermediates 7 , which cyclize to afford 4,5‐dihydro‐5‐oxo‐1,2,4‐thiadiazoles 2 .     

Luminescent properties of three structures built from 3‐cyano‐4‐dicyanomethylene‐5‐oxo‐4,5‐dihydro‐1H‐pyrrol‐2‐olate and cadmium

Yellow–orange tetraaquabis(3‐cyano‐4‐dicyanomethylene‐5‐oxo‐4,5‐dihydro‐1H‐pyrrol‐2‐olato‐κN³)cadmium(II) dihydrate, [Cd(C₈HN₄O₂)₂(H₂O)₄]·2H₂O, (I), and yellow tetraaquabis(3‐cyano‐4‐dicyanomethylene‐5‐oxo‐4,5‐dihydro‐1H‐pyrrol‐2‐olato‐κN³)cadmium(II) 1,4‐dioxane solvate, [Cd(C₈HN₄O₂)₂(H₂O)₄]·C₄H₈O₂, (II), contain centrosymmetric mononuclear Cd²⁺ coordination complex molecules in different conformations. Dark‐red poly[[decaaquabis(μ₂‐3‐cyano‐4‐dicyanomethylene‐5‐oxo‐4,5‐dihydro‐1H‐pyrrol‐2‐olato‐κ²N:N′)bis(μ₂‐3‐cyano‐4‐dicyanomethylene‐1H‐pyrrole‐2,5‐diolato‐κ²N:N′)tricadmium] hemihydrate], [Cd₃(C₈HN₄O₂)₂(C₈N₄O₂)₂(H₂O)₁₀]·0.5H₂O, (III), has a polymeric two‐dimensional structure, the building block of which includes two cadmium cations (one of them located on an inversion centre), and both singly and doubly charged anions. The cathodoluminescence spectra of the crystals are different and cover the wavelength range from UV to red, with emission peaks at 377 and 620 nm for (III), and at 583 and 580 nm for (I) and (II), respectively.     

The Equally Important Role of Adenine Derivatives to That of Pyrimidine Derivatives in Near‐0 eV Electron‐Induced DNA Lesions

The role of adenine (A) derivatives in DNA damage is scarcely studied due to the low electron affinity of base A. Experimental studies demonstrate that low‐energy electron (LEE) attachment to adenine derivatives complexed with amino acids induces barrier‐free proton transfer producing the neutral N₇‐hydrogenated adenine radicals rather than conventional anionic species. To explore possible DNA lesions at the A sites under physiological conditions, probable bond ruptures in two models—N₇‐hydrogenated 2′‐deoxyadenosine‐3′‐monophosphate (3′‐dA(N7H)MPH) and 2′‐deoxyadenosine‐5′‐monophosphate (5′‐dA(N7H)MPH), without and with LEE attachment—are studied by DFT. In the neutral cases, DNA backbone breakage and base release resulting from C_3′?O_3′ and N₉?C_1′ bond ruptures, respectively, by an intramolecular hydrogen‐transfer mechanism are impossible due to the ultrahigh activation energies. On LEE attachment, the respective C_3′?O_3′ and N₉?C_1′ bond ruptures in [3′‐dA(N7H)MPH]^? and [5′‐dA(N7H)MPH]^? anions via a pathway of intramolecular proton transfer (PT) from the C_2′ site of 2′‐deoxyribose to the C₈ atom of the base moiety become effective, and this indicates that substantial DNA backbone breaks and base release can occur at non‐3′‐end A sites and the 3′‐end A site of a single‐stranded DNA in the physiological environment, respectively. In particular, compared to the results of previous theoretical studies, not only are the electron affinities of 3′‐dA(N7H)MPH and 5′‐dA(N7H)MPH comparable to those of hydrogenated pyrimidine derivatives, but also the lowest energy requirements for the C_3′?O_3′ and N₉‐glycosidic bond ruptures in [3′‐dA(N7H)MPH]^? and [5′‐dA(N7H)MPH]^? anions, respectively, are comparable to those for the C_3′?O_3′ and N₁‐glycosidic bond cleavages in corresponding anionic hydrogenated pyrimidine derivatives. Thus, it can be concluded that the role of adenine derivatives in single‐stranded DNA damage is equally important to that of pyrimidine derivatives in an irradiated cellular environment.     

Electrochemical Oxidation of 8‐Oxo‐2′‐Deoxyguanosine on Glassy Carbon,Gold, Platinum and Tin(IV) Oxide Electrodes

Cyclic voltammetry was used to investigate the oxidation of 8‐oxo‐2′‐deoxyguanosine (8‐oxo‐dG) on the glassy carbon (GC), platinum, gold and SnO₂ electrodes over a range of the sweep rate, 8‐oxo‐dG concentration and the solution pH. Reaction mechanism that is common to all these electrodes involves the two‐electron two‐proton charge transfer step followed by the irreversible chemical reaction(s). Rate of the charge transfer reaction decreases with the increasing solution pH (GC, Pt, Au), and depends on the nature of the electrode material following the sequence GC>Pt, Au>>SnO₂. These effects can be related to the degree of oxidation of the electrode surface (Pt, Au, SnO₂), or to the density of the active surface sites (GC). Any of these electrodes can be used for the fabrication of an amperometric detector for 8‐oxo‐dG .     

A novel copper(II) complex with 1,10‐phenanthroline and ciprofloxacin

The X‐ray structure analysis of the title compound, chloro[1‐cyclopropyl‐6‐fluoro‐1,4‐dihydro‐4‐oxo‐7‐(piperazin‐4‐ium‐1‐yl)‐3‐quinolinecarboxylate‐κ²O³,O⁴](1,10‐phenanthroline‐κ²N,N′)copper chloride dihydrate, [CuCl(C₁₇H₁₈FN₃O₃)(C₁₂H₈N₂)]Cl·2H₂O or [CuCl(cfH)(phen)]Cl·2H₂O, where cfH is 1‐cyclopropyl‐6‐fluoro‐1,4‐dihydro‐4‐oxo‐7‐(piperazin‐4‐ium‐1‐yl)‐3‐quinolinecarboxylate and phen is 1,10‐phenanthroline, shows that the geometry around the Cu ion is a slightly distorted square pyramid. Two O atoms of the carbonyl and carboxyl groups of ciprofloxacin and two N atoms of 1,10‐phenanthroline are coordinated to the metal centre in the equatorial plane, and a Cl⁻ ion is coordinated at the apical position. Extensive intermolecular hydrogen bonding produces a supramolecular structure that consists of alternating six‐ and 12‐membered rings.     

NMR analysis of a series of 1,2‐bis(1‐R‐5‐oxo‐2,3‐dihydro‐1H‐pyrrol‐4‐ylidene)ethanes and X‐ray crystal structure analysis of the R = mesityl compound

Four tetramethyl 4,4′‐(ethane‐1,2‐diylidene)bis[1‐R‐5‐oxo‐4,5‐dihydro‐1H‐pyrrole‐2,3‐dicarboxylate] compounds, denoted class (1), are a series of conjugated buta‐1,3‐dienes substituted with a heterocyclic group. The compounds can be used as dyes and pigments due to their long‐range conjugated systems. Four structures were studied using ¹H NMR, ¹³C NMR and mass spectroscopy, viz. with R = 2,4,6‐trimethylphenyl, (1a), R = cyclohexyl, (1b), R = tert‐butyl, (1c), and R = isopropyl, (1d). A detailed discussion is presented regarding the characteristics of the three‐dimensional structures based on NMR analysis and the X‐ray crystal structure of (1a), namely tetramethyl 4,4′‐(ethane‐1,2‐diylidene)bis[5‐oxo‐1‐(2,4,6‐trimethylphenyl)‐4,5‐dihydro‐1H‐pyrrole‐2,3‐dicarboxylate], C₃₆H₃₆N₂O₁₀. The conjugation plane and stability were also studied via quantum chemical calculations.     

Interaction of singlet oxygen and superoxide radical anion with guanine and formation of its mutagenic modification 8‐oxoguanine

Formation of 8‐oxoguanine (8OG) from guanine in biological systems is known to cause lethal mutation and cancer. It has been suggested earlier, on the basis of experimental studies, that the oxygen molecule in its lowest singlet excited state (¹O₂) plays an important role in the formation of 8OG. In order to understand the possible mechanisms in this context, B3LYP/6‐31+G* and MP2/6‐31+G* calculations were carried out on the structures and stabilities of different molecules and complexes involved in the formation of 8OG. All the molecules, complexes, and transition states studied in the present report were solvated in aqueous media. Guanine has been found to make a strong complex with ¹O₂ with the latter species located above the imidazole ring plane, and the complex of guanine with ³O₂ is much weaker than that with ¹O₂. Transition state calculations were carried out to study formation of 7,8‐dihydro,8‐hydroxyguanine (8OHG) and 2‐oxo‐imidazole. It has been shown that 8OG can be formed in two different ways: (i) due to interaction of the radical cation of guanine with O where 8OHG complexed with ¹O₂ would occur as an intermediate, and (ii) due to interaction of guanine with ¹O₂ leading to the formation of guanine hydroperoxide that would react with a water molecule in the presence of two ¹O₂ molecules serving as a source of energy to overcome the barrier. It is shown that because the interaction strengths of ³O₂ and ¹O₂ with other molecules, e.g., guanine, are very different, a crossing of their potential energy surfaces takes place in both gas phase and aqueous media, as a result of which the lifetime of ¹O₂ is strongly decreased. © 2004 Wiley Periodicals, Inc. Int J Quantum Chem, 2005     

The Pseudo‐Michael Reaction of 2‐Hydrazinylidene‐1‐Arylimidazolidines with Diethyl Ethoxymethylenemalonate

The pseudo‐Michael reaction of 2‐hydrazinylidene‐1‐arylimidazolidines with diethyl ethoxymethylenemalonate (DEEM) was investigated. The reaction yields the chain adduct, namely diethyl{[2‐(1‐arylimidazolidin‐2‐ylidene)hydrazinyl]methylidene}propanedioates. This is contrary to the pseudo‐Michael reaction of DEEM with 1‐aryl‐4,5‐dihydro‐1H‐imidazol‐2‐amines that does not allow isolation of chain derivatives and leads to cyclic imidazo[1,2‐a]pyrimidine derivatives while even at thermodynamic control. At first cyclization of diethyl{[2‐(1‐arylimidazolidin‐2‐ylidene)hydrazinyl]methylidene}propanedioates leads to ethyl 1‐aryl‐5(1H,8H)oxo‐2,3‐dihydro‐imidazo[2,1‐c][1,2,4]triazepine‐6‐carboxylates. 1,5‐Sigmatropic shift, following the cyclization, caused isomerization of 5(1H,8H)oxo‐2,3‐dihydro‐imidazo[2,1‐c][1,2,4]triazepine‐6‐carboxylates to ethyl 1‐aryl‐5(1H)hydroxy‐2,3‐dihydroimidazo[2,1‐c][1,2,4]triazepine‐6‐carboxylates. Presence of both isomers in the reaction product was detected in the NMR spectra. The structure of all the compounds was confirmed with spectroscopic studies (¹H NMR and MS). The structure of diethyl{[2‐(1‐phenylimidazolidin‐2‐ylidene)hydrazinyl]methylidene}propanedioate was also confirmed by X‐ray crystallography. In the addition reaction, thermodynamics and HOMO–LUMO orbitals of the reactants were studied by using quantum chemical calculations.     

Unraveling the Degradation Mechanism of Purine Nucleotides Photosensitized by Pterins: The Role of Charge‐Transfer Steps

Photosensitized reactions contribute to the development of skin cancer and are used in many applications. Photosensitizers can act through different mechanisms. It is currently accepted that if the photosensitizer generates singlet molecular oxygen (¹O₂) upon irradiation, the target molecule can undergo oxidation by this reactive oxygen species and the reaction needs dissolved O₂ to proceed, therefore the reaction is classified as ¹O₂‐mediated oxidation (type II mechanism). However, this assumption is not always correct, and as an example, a study on the degradation of 2′‐deoxyguanosine 5′‐monophosphate photosensitized by pterin is presented. A general mechanism is proposed to explain how the degradation of biological targets, such as nucleotides, photosensitized by pterins, naturally occurring ¹O₂ photosensitizers, takes place through an electron‐transfer‐initiated process (type I mechanism), whereas the contribution of the ¹O₂‐mediated oxidation is almost negligible.     

Synthesis of 2‐Aryl‐5‐oxo‐4‐[2‐(phenylmethylidene)hydrazino]‐2,5‐dihydro‐1H‐pyrrole‐3‐carboxylates by the Reaction between Hydrazones,Acetylenedicarboxylates, and 1‐Aryl‐N,N′‐bis(arylmethylidene)methanediamines

An efficient approach for the preparation of functionalized 2‐aryl‐2,5‐dihydro‐5‐oxo‐4‐[2‐(phenylmethylidene)hydrazino]‐1H‐pyrroles is described. The four‐component reaction between aldehydes, NH₂NH₂?H₂O, dialkyl acetylenedicarboxylates, and 1‐aryl‐N,N′‐bis(arylmethylidene)methanediamines proceeds in EtOH under reflux in good‐to‐excellent yields (Scheme 1). The structures of 4 were corroborated spectroscopically (IR, ¹H‐ and ¹³C‐NMR, and EI‐MS, and, in the case of 4f , by X‐ray crystallography). A plausible mechanism for this type of reaction is proposed (Scheme 2).     

Facile green one‐pot synthesis of novel thiazolo[3,2‐a]pyrimidine derivatives using Fe3O4@l‐arginine and their biological investigation as potent antimicrobial agents

Thiazolopyrimidine derivatives are well known because of their excellent therapeutic properties. In this investigation, an effective one‐pot three‐component method is described for the synthesis of novel 2‐[(Z )‐1‐(substituted phenyl)methylidine]‐7‐methyl‐3‐oxo‐5‐(substituted phenyl)‐2,3‐dihydro‐5H ‐thiazolo[3,2‐a]pyrimidine‐6‐carboxilic acid tert ‐butyl ester derivatives by condensation reaction of 3,4‐dihydropyrimidine‐2(1H )‐thiones, various aromatic aldehydes and chloroacetyl chloride under reflux conditions in the presence of Fe₃O₄@l ‐arginine nanoparticles as a magnetically reusable and eco‐friendly catalyst with short reaction times and moderate yields. The chemical structures of all synthesized compounds were determined using infrared, ¹H NMR and ¹³C NMR spectroscopies. In vitro antimicrobial activities of 3,4‐dihydropyrimidine‐2(1H )‐thiones and newly fused thiazolo[3,2‐a]pyrimidine derivatives were examined using the well diffusion method against diverse pathogenic strains, namely Staphylococcus aureus ATCC 6538, S. epidermidis ATCC 12228, Escherichia coli ATCC 8739 and Pseudomonas aeruginosa ATCC 9027 (bacteria), Candida albicans ATCC 10231 (yeast) and Aspergillus niger ATCC 16404 (fungus). The compounds having 2‐hydroxy, 4‐hydroxy, 2‐chloro and 4‐chloro groups attached to the phenyl ring on the pyrimidine and 4‐CH₃, 4‐OCH₃ and 3‐NO₂ groups attached to benzylidine on the thiazolo moiety showed significant antibacterial activity.     

Formation of Oxygen Radicals in Solutions of Different 7,8‐Dihydropterins: Quantitative Structure‐Activity Relationships

Under certain conditions, 7,8‐dihydroneopterin in aqueous solution promotes hydroxyl‐radical formation. Thus, we investigated the stimulation of hydroxyl‐radical formation by ten different 7,8‐dihydropterins (=2‐amino‐7,8‐dihydropteridin‐4(1H)‐one), i.e., 6‐(1′‐hydroxy) derivatives 1 and 2 , methyl derivatives 3 – 7 , and 6‐(1′‐oxo) derivatives 8 – 10 . All but the 6‐(1′‐oxo) derivatives produced hydroxyl radicals, as measured by the amount of salicylic acid hydroxylation products. This amount was dependent on the stability of the dihydropterin used. In the presence of chelated iron ions, hydroxylation was increased in every case; even 6‐(1′‐oxo) derivatives showed a low hydroxylation of salicylic acid. The degree of increase, however, strongly depended on the side chain of the dihydropterin. The 7,8‐dihydroneopterin ( 2 ) was investigated in more detail. Iron ions influenced both, the stability of 2 and hydroxyl‐radical formation. While iron ions determined the kinetics of the reaction, the amount of 2 was responsible for the amount of hydroxyl radicals formed. Our data establish that promotion of hydroxyl‐radical formation by 7,8‐dihydropterins depends on the oxidizability of the dihydropterins and on their iron‐chelating properties.     

The first 3′:5′‐cyclic nucleotide–amino acid complex: l‐His–cIMP

In the crystal structure of the l ‐His–cIMP complex, i.e.l ‐histidinium inosine 3′:5′‐cyclic phosphate [systematic name: 5‐(2‐amino‐2‐carboxyethyl)‐1H‐imidazol‐3‐ium 7‐hydroxy‐2‐oxo‐6‐(6‐oxo‐6,9‐dihydro‐1H‐purin‐9‐yl)‐4a,6,7,7a‐tetrahydro‐4H‐1,3,5,2λ⁵‐furo[3,2‐d][1,3,2λ⁵]dioxaphosphinin‐2‐olate], C₆H₁₀N₃O₂⁺·C₁₀H₁₀N₄O₇P⁻, the Hoogsteen edge of the hypoxanthine (Hyp) base of cIMP and the Hyp face are engaged in specific amino acid–nucleotide (His...cIMP) recognition, i.e. by abutting edge‐to‐edge and by π–π stacking, respectively. The Watson–Crick edge of Hyp and the cIMP phosphate group play a role in nonspecific His...cIMP contacts. The interactions between the cIMP anions (anti/C3′–endo/trans–gauche/chair conformers) are realized mainly between riboses and phosphate groups. The results for this l ‐His–cIMP complex, compared with those for the previously reported solvated l ‐His–IMP crystal structure, indicate a different nature of amino acid–nucleotide recognition and interactions upon the 3′:5′‐cyclization of the nucleotide phosphate group.