Accumulation of DNA damage resulting from reactive oxygen species was proposed to cause neurological and degenerative disease in patients, deficient in nucleotide excision repair (NER) or its transcription-coupled subpathway (TC-NER). Here, we assessed the requirement of TC-NER for the repair of specific types of oxidatively generated DNA modifications. We incorporated synthetic 5′,8-cyclo-2′-deoxypurine nucleotides (cyclo-dA, cyclo-dG) and thymine glycol (Tg) into an EGFP reporter gene to measure transcription-blocking potentials of these modifications in human cells. Using null mutants, we further identified the relevant DNA repair components by a host cell reactivation approach. The results indicated that NTHL1-initiated base excision repair is by far the most efficient pathway for Tg. Moreover, Tg was efficiently bypassed during transcription, which effectively rules out TC-NER as an alternative repair mechanism. In a sharp contrast, both cyclopurine lesions robustly blocked transcription and were repaired by NER, wherein the specific TC-NER components CSB/ERCC6 and CSA/ERCC8 were as essential as XPA. Instead, repair of classical NER substrates, cyclobutane pyrimidine dimer and N-(deoxyguanosin-8-yl)-2-acetylaminofluorene, occurred even when TC-NER was disrupted. The strict requirement of TC-NER highlights cyclo-dA and cyclo-dG as candidate damage types, accountable for cytotoxic and degenerative responses in individuals affected by genetic defects in this pathway.

Synthesis and study of RotA, an inhibitor of glycogen phosphorylase (GP), which when bound at the active site of GP, produces a strong fluorescence signal, allowing utilization of RotA as a probe that brings GP “to light” in the cellular milieu.

In this study, (E)-2-cyano-3-(6-(dimethylamino)naphthalen-2-yl)-N-(β-d-glucopyranosyl)acrylamide, a β-d-glucopyranosyl analogue of the widely used molecular rotor julolidine, was synthesized and studied photochemically. The new compound is a fluorescent inhibitor of rabbit muscle glycogen phosphorylase with properties of a molecular rotor. Fluorescence measurements in solutions of increasing viscosity determined that the fluorescence intensity increases with the viscosity of the medium, indicating that the new compound exhibits molecular rotor characteristics. Although the compound fluoresces negligibly in an aqueous buffer solution, in the presence of increasing amounts of rabbit muscle glycogen phosphorylase, we observed an increase in fluorescence intensity, which was attributed to the formation of an inhibitor–enzyme complex. In-vitro cellular studies were also undertaken, yielding promising preliminary results for the use of the new compound as a fluorescent probe.

Dysregulation of glycogen phosphorylase, an enzyme involved in glucose homeostasis, may lead to a number of pathological states such as type 2 diabetes and cancer, making it an important molecular target for the development of new forms of pharmaceutical intervention. Based on our previous work on the design and synthesis of 4-arylamino-1-(β-d-glucopyranosyl)pyrimidin-2-ones, which inhibit the activity of glycogen phosphorylase by binding at its catalytic site, we report herein a general synthesis of 2-substituted-5-(β-d-glucopyranosyl)pyrimidin-4-ones, a related class of metabolically stable, C-glucosyl-based, analogues. The synthetic development consists of a metallated heterocycle, produced from 5-bromo-2-methylthiouracil, in addition to protected d-gluconolactone, followed by organosilane reduction. The methylthio handle allowed derivatization through hydrolysis, ammonolysis and arylamine substitution, and the new compounds were found to be potent (μM) inhibitors of rabbit muscle glycogen phosphorylase. The results were interpreted with the help of density functional theory calculations and conformational analysis and were compared with previous findings.

In the case of type 2 diabetes, inhibitors of glycogen phosphorylase (GP) may prevent unwanted glycogenolysis under high glucose conditions and thus aim at the reduction of excessive glucose production by the liver. Anomeric spironucleosides, such as hydantocidin, present a rich synthetic chemistry and important biological function (e.g., inhibition of GP). For this study, the Suárez radical methodology was successfully applied to synthesize the first example of a 1,6-dioxa-4-azaspiro[4.5]decane system, not previously constructed via a radical pathway, starting from 6-hydroxymethyl-β-d-glucopyranosyluracil. It was shown that, in the rigid pyranosyl conformation, the required [1,5]-radical translocation was a minor process. The stereochemistry of the spirocycles obtained was unequivocally determined based on the chemical shifts of key sugar protons in the 1H-NMR spectra. The two spirocycles were found to be modest inhibitors of RMGPb.

A fluorescence study of N1-(β-D-glucopyranosyl)-N4-[2-acridin-9(10H)-onyl]-cytosine (GLAC), the first fluorescent potent inhibitor of Glycogen Phosphorylase (GP), in neutral aqueous solution, is presented herein. Quantum chemistry (TD-DFT) calculations show the existence of several conformers both in the ground and first excited state. They result from rotation of the acridone and cytosine moieties around an NH bridge which may lead to the formation of non-emitting charge transfer states. The fingerprint of various conformers have been detected by time-resolved fluorescence spectroscopy (fluorescence upconversion and time-correlated single photon counting) and identified using as criteria their energy, polarization and relative population resulting from computations. Such an analysis should contribute to the design of new GP inhibitors with better fluorescence properties, suitable for imaging applications.

The reaction between 1-aminosugars and trimethylisocyanate (TMSNCO) was optimised as a one-step synthetic strategy for the synthesis of sugar biurets. This protocol was successfully applied to a number of 1-aminosugars, which exclusively provided the corresponding biurets in 67–99% yields. The new method- ology was applied in the de novo synthesis of N1-(2-deoxy-α/β-D-erythro-pentofuranosyl)biuret (dfBU) and N1-(2-deoxy-α/β-D-erythro-pentopyranosyl)biuret (dpBU), two known DNA lesions arising from the hydroxyl radical induced decomposition of 2’-deoxycytidine (dCyd).

A number of environmental pollutants and endogenous oxidation agents form 1-(2-deoxy-β-D-ribofura- nosyl)-5-hydroxy-5-methylhydantoin (HydT), an important DNA lesion resulting from thymidine oxi- dation. In this paper, two intermediates, postulated in the formation of HydT, have been characterised for the first time. The first, N1-formyl-N3-pyruvoylurea intermediate, was produced by the ozonolysis reaction of 2’,3’,5’-tri-O-acetylribo-, 3’,5’-di-O-TBS- and N3,O3’,O5-tribenzyl-protected thymidines and was shown to produce, upon decomposition and depending on the protecting group and the conditions, HydT alone, or together with protected-β-D-ribofuranosyl-N1-formylurea and formamide products. In addition, the second and long sought, open-chain-pyruvoylurea intermediate, was produced through de novo synthesis in protected β-D-ribofuranosyl-, 2-deoxy-β-D-ribofuranosyl- and 2-deoxy-β-D-ribo- pyranosyl systems. The conditions that induce the cyclization to the hydantoin ring of HydT have been determined. The chemistry utilised in the de novo synthesis is suitable for generating isotopically labelled HydT, as a reference in isotope-dilution-aided quantification of DNA damage.

Abstract Spectroscopic methods such as absorption spectroscopy in ultraviolet (UV), visible (Vis), near-infrared (NIR), or mid-infrared (MIR) or their combinations have been used for nondestructive, fast, real-time, off/online monitoring of olive oil quality parameters, and for detection and determination of olive oil adulterants as well as geographic origin. These methods have also been used in the industrial sector to analyze the quality of olive fruit with respect to oil content, moisture, color, and fatty acid content. This chapter considers several applications of UV-Vis-NIR or/and MIR spectroscopy in off/on-line monitoring of olive oil functional compounds and oxidation status in combination with appropriately used spectral preprocessing and data analysis (chemometrics). The UV-Vis-NIR and MIR spectroscopic methods have the advantages of minimal or no sample preparation and the ability of a very simple and rapid simultaneous analysis of several constituents. They constitute one of the most promising and useful on/in-line detection methods for olive oil quality control.

Glycogen phosphorylase (GP) plays a key role in glucose regulation. The design and synthesis of a new potent inhibitor of GP is described. By exploiting its optical properties, it is shown that, due to an extended hydrogen bonding network, the local pH in the GP catalytic site is higher than 12. For the full story, see the Communication by A. Venturini, E. D. Chrysina, D. Markovitsi, T. Gimisis et al. on page 8800 ff.

© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim The design and synthesis of a glucose-based acridone derivative (GLAC), a potent inhibitor of glycogen phosphorylase (GP) are described. GLAC is the first inhibitor of glycogen phosphorylase, the electronic absorption properties of which are clearly distinguishable from those of the enzyme. This allows probing subtle interactions in the catalytic site. The GLAC absorption spectra, associated with X-ray crystallography and quantum chemistry cal culations, reveal that part of the catalytic site of GP behaves as a highly basic environment in which GLAC exists as a bis-anion. This is explained by water-bridged hydrogen-bonding interactions with specific catalytic site residues.

© 2015 Elsevier Ltd. All rights reserved.A number of leaving groups, including arylsulfonates, triazoles, 3-nitrotriazoles, and tetrazoles, have been studied for the substitution reaction by aryl and alkyl amines at the 4-position of $\beta$-D-glucopyranosyluracils. Examination of the stability, ease of purification and reactivity in the substitution reaction led to a number of optimized conditions with the most convenient involving substitution of triazole derivatives under microwave conditions in the presence of silica gel. Under these conditions, a number of N4-aryl-substituted $\beta$-D-glucopyranosylcytosines were prepared as potential inhibitors of glycogen phosphorylase, a molecular target for type-2 diabetes mellitus.

abstract extra virgin olive oil contains significant quantities of polar phenolic ingredients. The large majority is made up of esters of tyrosol or hydroxytyrosol with secoiridoid derivatives from oleuropein or ligstroside. in the current study we describe a number of new or incompletely characterized forms of ligstroside and oleuropein aglycons. two of them which are stable enolic forms are described for the first time as real olive oil ingredients although their presence in olive oil had been postulated. to minimize the confusion with the complicated names of the agly-con isomers we propose the names oleokoronal and oleomissional for the two ingredients. after screening 2000 samples of olive oil from most major varieties we were able to identify samples of olive oil in which oleokoronal and oleomissional were the major phenolic ingredients and could be used as starting material for their isolation. interestingly, during normal or reversed phase chromatography both compounds were transformed to the known forms of monoaldehydic closed ring aglycons, which offers an explanation as to why those compounds had not been identified so far. Their real presence in olive oil was confirmed by direct nMr observation without the use of any solvent.

Abstract This article focuses on the chemistry of sugar radicals in the context of oxidative damage in nucleic acids. The seven sp3-hybridized CH bonds in 2-deoxyribose (six in ribose) are attacked by reactive oxygen species, generated either by the cell's internal oxygen cycle or by environmental insults, by chemical nucleases including enediyne antibiotics and reactive metal complexes, to generate sugar nucleosidyl radicals. For each position on the 2-deoxyribose or ribose moiety, the factors leading to the generation of the specific sugar radical, together with its fate under aerobic or anaerobic conditions in the nucleoside, nucleotide, single-stranded oligonucleotide, or double helix DNA, are discussed. Furthermore, the model systems that have been constructed for the photochemical-independent generation of each nucleosidyl radical or DNA lesion are described, together with the biochemical studies that have been performed in order to elucidate the stability, reparability, and mutagenic potential of each lesion. Other biologically important events involving modification of nearby nucleobases and the formation of DNA–DNA and DNA–protein cross-links are also discussed.

Glycogen phosphorylase (GP) is a promising molecular target for the treatment of Type 2 diabetes. The design of potential inhibitors for the catalytic site of the enzyme is based on the high affinity for -D-glucopyranose and the pres- ence of a -cavity that extends from the sugar anomeric position forming a 15 x 7.5 x 10 Å available space. This review is focused on our efforts towards the design and synthesis of various families of potential inhibitors, including N--D- glucopyranosyl oxamic acid esters and oxamides, N--D-glucopyranosylaminocarbonyl L-aminoacids and peptides, as well as glucose-derived purine and pyrimidine nucleosides, spiro- and other bicyclic derivatives. Kinetic and crystallo- graphic study of the interactions of these inhibitors with GP has increased our understanding of the importance of the various functional groups within the catalytic site and has pointed the way towards the in silico prediction and design of potent inhibitors, which are both synthetically viable and pharmacologically relevant.

8-Oxo-7,8-dihydroguanine (8-oxo-G) is the major lesion of oxidatively generated DNA damage. Despite two decades of intense study, several fundamental properties remain to be defined. Its isoelectronic 8-aminoguanine (8-NH 2-G) has also received considerable attention from a biological point of view, although its chemistry involving redox processes remains to be discovered. We investigated the one-electron oxidation and one-electron reduction reactions of 8-oxo-G and 8-NH2-G derivatives. The reactions of hydrated electrons (eaq-) and azide radicals (N 3•) with both derivatives were studied by pulse radiolysis techniques, and the transient absorption spectra were assigned to specific tautomers computationally by means of time-dependent DFT (TD-B3LYP/6-311G**//B1B95/6-31+G**) calculations. The protonated electron adducts of 8-NH2-G and 8-oxo-G showed a substantial difference in their absorption spectra, the unpaired electron being mainly delocalized in the imidazolyl ring and in the six-membered ring, respectively. On the other hand, the deprotonated forms of one-electron oxidation of 8-NH2-G and 8-oxo-G showed quite similar spectral characteristics. In a parallel study, the one-electron reduction of 8-azidoguanine (8-N3-G) afforded the same transient of one-electron oxidation of 8-NH2-G, which represents another example of generation of one-electron oxidized guanine derivatives under reducing conditions. Moreover, the fate of transient species was investigated by radiolytic methods coupled with product studies and allowed self- and cross-termination rate constants associated with these reactions to be estimated. © 2009 American Chemical Society.

8-Oxo-7,8-dihydroguanine (8-oxo-G) is the major lesion of oxidatively generated DNA damage. Despite two decades of intense study, several fundamental properties remain to be defined. Its isoelectronic 8-aminoguanine (8-NH2-G) has also received considerable attention from a biological point of view, although its chemistry involving redox processes remains to be discovered. We investigated the one-electron oxidation and one-electron reduction reactions of 8-oxo-G and 8-NH2-G derivatives. The reactions of hydrated electrons (eaq−) and azide radicals (N3•) with both derivatives were studied by pulse radiolysis techniques, and the transient absorption spectra were assigned to specific tautomers computationally by means of time-dependent DFT (TD-B3LYP/6-311G**//B1B95/6-31+G**) calculations. The protonated electron adducts of 8-NH2-G and 8-oxo-G showed a substantial difference in their absorption spectra, the unpaired electron being mainly delocalized in the imidazolyl ring and in the six-membered ring, respectively. On the other hand, the deprotonated forms of one-electron oxidation of 8-NH2-G and 8-oxo-G showed quite similar spectral characteristics. In a parallel study, the one-electron reduction of 8-azidoguanine (8-N3-G) afforded the same transient of one-electron oxidation of 8-NH2-G, which represents another example of generation of one-electron oxidized guanine derivatives under reducing conditions. Moreover, the fate of transient species was investigated by radiolytic methods coupled with product studies and allowed self- and cross-termination rate constants associated with these reactions to be estimated.

One-electron oxidized guanine is an important reactive intermediate in the formation of oxidatively generated damage in DNA and a variety of methods have been utilized for the abstraction of a single electron from the guanine moiety. In this study, an alternative approach for the site specific, independent generation of the guanine radical, utilizing N-hydroxypyrid-2(1H)-one as a photolabile modifier of guanine, is proposed. Novel photolabile 6-[(1-oxido-2-pyridinyl)oxo]-6-deoxy- and 2′,6-dideoxy-guanosine derivatives capable of generating the neutral guanine radical (G(-H) •) upon photolysis were synthesized and characterized. The generation of G(-H) proceeds through homolysis of the N-O bond and was confirmed through continuous photolysis product analysis and trapping studies, as well as laser flash photolysis experiments. © The Royal Society of Chemistry 2009.

Two tautomeric forms of one-electron oxidized 2-aminoadenosine (2AA) have been produced by reactions of hydrated electrons (eaq-) with 8-bromo-2-aminoadenosine (8-Br-2AA) at natural pH, whereas only one tautomer is formed by oxidation of 2AA. Tailored experiments by pulse radiolysis and time-dependent DFT (TD-B3LYP/6-311G**//B1B95/6-31+G**) calculations allowed the definition of the reaction mechanism in some detail. The electron adducts of 8-Br-2AA protonated at C8 eject Br- and produce the two short-lived tautomers (8 and 9). The first observable species decays by first-order kinetics to produce the second intermediate, which is also obtained by oxidation of 2AA by SO4•-. The rate of tautomerization (ktaut = 4.5 × 104 s -1) is strongly accelerated by phosphate and is retarded in D 2O (kinetic isotope effect 7). B1B95/6-31+G** calculations showed that the tautomerization is a water-assisted process. In acidic or basic solutions, the "instantaneous" formation of one-electron oxidized 2AA or its deprotonated forms has been produced by reactions of eaq- with 8-Br-2AA. $\gamma$-Radiolysis of 8-Br-2AA in aqueous solutions followed by product studies led to the formation of 2AA as a single product. © 2008 American Chemical Society.

Twenty-five naturally occurring pentacyclic triterpenes, 15 of which were synthesized in this study, were biologically evaluated as inhibitors of rabbit muscle glycogen phosphorylase a (GPa). From SAR studies, the presence of a sugar moiety in triterpene saponins resulted in a markedly decreased activity (7, 18-20) or no activity (21, 22). These saponins, however, might find their value as potential natural prodrugs which are much more water-soluble than their corresponding aglycones. To elucidate the mechanism of GP inhibition, we have determined the crystal structures of the GPb-asiatic acid and GPb-maslinic acid complexes. The X-ray analysis indicates that the inhibitors bind at the allosteric activator site, where the physiological activator AMP binds. Pentacyclic triterpenes represent a promising class of multiple-target antidiabetic agents that exert hypoglycemic effects, at least in part, through GP inhibition.

Two tautomeric forms of one-electron oxidized 2-aminoadenosine (2AA) have been produced by reactions of hydrated electrons (e(aq)(-)) with 8-bromo-2-aminoadenosine (8-Br-2AA) at natural pH, whereas only one tautomer is formed by oxidation of 2AA. Tailored experiments by pulse radiolysis and time-dependent DFT (TD-B3LYP/6-311G**//B1B95/6-31+G**) calculations allowed the definition of the reaction mechanism in some detail. The electron adducts of 8-Br-2AA protonated at C8 eject Br- and produce the two short-lived tautomers (8 and 9). The first observable species decays by first-order kinetics to produce the second intermediate, which is also obtained by oxidation of 2AA by SO4 center dot-. The rate of tautomerization (k(taut) = 4.5 x 10(4) s(-1)) is strongly accelerated by phosphate and is retarded in D2O (kinetic isotope effect 7). B1B95/631+G** calculations showed that the tautomerization is a water-assisted process. In acidic or basic solutions, the "instantaneous" formation of one-electron oxidized 2AA or its deprotonated forms has been produced by reactions of e(aq)(-) with 8-Br-2AA. gamma-Radiolysis of 8-Br-2AA in aqueous solutions followed by product studies led to the formation of 2AA as a single product.

Several examples of oxidative and reductive reactions of DNA components that lead to single and tandem modifications are discussed in this review. These include nucleophilic addition reactions of the one-electron oxidation-mediated guanine radical cation and the one-electron reduced intermediate of 8-bromopurine 2'-deoxyribonucleosides that give rise to either an oxidizing guanine radical or related 5',8-cyclopurine nucleosides. In addition, mechanistic insights into the reductive pathways involved in the photolyase induced reversal of cyclobutadipyrimidine and pyrimidine (6-4) pyrimidone photoproducts are provided. Evidence for the occurrence and validation in cellular DNA of ̇OH radical degradation pathways of guanine that have been established in model systems has been gained from the accurate measurement of degradation products. Relevant information on biochemical aspects of the repair of single and clustered oxidatively generated damage to DNA has been gained from detailed investigations that rely on the synthesis of suitable modified probes. Thus the preparation of stable carbocyclic derivatives of purine nucleoside containing defined sequence oligonucleotides has allowed detailed crystallographic studies of the recognition step of the base damage by enzymes implicated in the base excision repair (BER) pathway. Detailed insights are provided on the BER processing of non-double strand break bistranded clustered damage that may consist of base lesions, a single strand break or abasic sites and represent one of the main deleterious classes of radiation-induced DNA damage. © Schweizerische Chemische Gesellschaft.

An alternative route for the synthesis of a photoaffinity labelling (PAL) dCTP derivative is reported. This method involves the intermediacy of exo-N-[2-(4-azido-2,3,5,6-tetrafluorobenzamido)ethyl]-dC. The latter is prepared from the coupling of known N-(2-aminoethyl)-4-azido-2,3,5,6-tetrafluorobenzamide, prepared in an improved three-step sequence, with an activated 4-triazolyl derivative of dU, followed by deprotection. 19F NMR spectroscopy proved extremely useful in following the synthetic transformations, and enabled control of any adventitious reduction of the azides. © 2007 Elsevier Ltd. All rights reserved.

Abstract [18156-74-6] C6H12N2Si (MW 140.29) InChI = 1S/C6H12N2Si/c1-9(2,3)8-5-4-7-6-8/h4-6H,1-3H3 InChIKey = YKFRUJSEPGHZFJ-UHFFFAOYSA-N (silylating agent for alcohols and 1,3-dicarbonyl compounds; reaction with esters to give imidazolides; preparation of O-trimethylsilyl monothioacetals; aromatization of the A-ring of steroids) Physical Data: bp 93–94 °C/14 mmHg; fp 5 °C; d 0.956 g cm−3. Form Supplied in: liquid. Handling, Storage, and Precautions: flammable. Harmful by inhalation, in contact with skin, and if swallowed. Possible carcinogen. Use in a fume hood. When using, wear protective gear and clothing. Handle and store under N2. Store in a cool dry place.

(Chemical Equation Presented) The synthesis of the C5′ tert-butyl ketone of thymidine 1a and 2′-deoxyguanosine 2 is achieved by reaction of 5′-C-cyano derivatives with tert-butyl lithium followed by acid hydrolysis. The 5′R configuration is assigned by X-ray crystal structure determination of an opportunely protected derivative of 1a. The (5′S)-isomers of both nucleosides are not stable, and a complete decomposition occurs in the reaction medium. The photochemistry of 1a and 2 effectively produced the thymidin-5′-yl radical and the 2′-deoxyguanosin-5′-yl radical, respectively. In the thymidine system, the C5′ radical is fully quenched in the presence of a physiological concentration of thiols. In the 2′-deoxyguanosine system, the C5′ radical undergoes intramolecular attack onto the C8-N7 double bond of guanine leading ultimately to the 5′,8-cyclo-2′-deoxyguanosine derivative. The cyclization of the 2′-deoxyguanosin-5′-yl radical occurs with a rate constant of ca. 1 × 106 s-1 and is highly stereoselective affording only the (5′S)-diastereomer. © 2007 American Chemical Society.

FR258900 has been discovered as a novel inhibitor of human liver glycogen phosphorylase a and proved to suppress hepatic glycogen breakdown and reduce plasma glucose concentrations in diabetic mice models. To elucidate the mechanism of inhibition, we have determined the crystal structure of the cocrystallized rabbit muscle glycogen phosphorylase b-FR258900 complex and refined it to 2.2 angstrom resolution. The structure demonstrates that the inhibitor binds at the allosteric activator site, where the physiological activator AMP binds. The contacts from FR258900 to glycogen phosphorylase are dominated by nonpolar van der Waals interactions with Gln71, Gln72, Phe196, and Val45' (from the symmetry-related subunit), and also by ionic interactions from the carboxylate groups to the three arginine residues (Arg242, Arg309, and Arg310) that form the allosteric phosphate-recognition subsite. The binding of FR258900 to the protein promotes conformational changes that stabilize an inactive T-state quaternary conformation of the enzyme. The ligand-binding mode is different from those of the potent phenoxy- phthalate and acyl urea inhibitors, previously described, illustrating the broad specificity of the allosteric site.

The synthesis of the C5' tert-butyl ketone of thymidine 1a and 2'-deoxyguanosine 2 is achieved by reaction of 5'-C-cyano derivatives with tert-butyl lithium followed by acid hydrolysis. The 5'R configuration is assigned by X-ray crystal structure determination of an opportunely protected derivative of 1a. The (5'S)-isomers of both nucleosides are not stable, and a complete decomposition occurs in the reaction medium. The photochemistry of 1a and 2 effectively produced the thymidin-5'-yl radical and the 2'-deoxyguanosin-5'-yl radical, respectively. In the thymidine system, the C5' radical is fully quenched in the presence of a physiological concentration of thiols. In the 2'-deoxyguanosine system, the C5' radical undergoes intramolecular attack onto the C8-N7 double bond of guanine leading ultimately to the 5',8-cyclo-2'-deoxyguanosine derivative. The cyclization of the 2'-deoxyguanosin-5'-yl radical occurs with a rate constant of ca. 1 x 10(6) s(-1) and is highly stereoselective affording only the (5'S)-diastereomer.

The photochemistry of N-hydroxypyridine-2(1H)-thione (NHPT), inserted as a photolabile modifier at the 6-position of 2′-deoxyguanosine or guanosine, has been evaluated. In particular, 6-[(1-oxidopyridin-2-yl)sulfanyl]- (1a) and 6-[(pyridin-2-yl)sulfanyl]-2′,6-dideoxyguanosine (2a), novel photolabile derivatives of the natural nucleosides, were synthesized and characterized. The observed photolysis products of 1a in organic solvents could only be rationalized by assuming a rapid equilibrium with the corresponding 6-[(2-thioxopyridin-1(2H)-yl)oxy] analogue 3a (Scheme 5). Transient spectroscopy of 1a indicated a strong triplet-excited state suitable for triplet → triplet energy transfer or singlet-oxygen generation. The NHPT function was stable enough for (slightly modified) automated solid-phase oligonucleotide synthesis. The utility of the above compounds is discussed, as well as their potential use in photosensitization of reactive oxygen species in DNA. © 2006 Verlag Helvetica Chimica Acta AG.

Five oxalyl derivatives of $\beta$-d-glucopyranosylamine were synthesized as potential inhibitors of glycogen phosphorylase (GP). The compounds 1-4 were competitive inhibitors of rabbit muscle GPb (with respect to $\alpha$-d-glucose-1-phosphate) with Ki values of 0.2-1.4 mM, while compound 5 was not effective up to a concentration of 10 mM. In order to elucidate the structural basis of their inhibition, we analysed the structures of compounds 1-4 in complex with GPb at 1.93-1.96 {\AA} resolution. The complex structures reveal that the inhibitors can be accommodated at the catalytic site at approximately the same position as $\alpha$-d-glucose and stabilize the T-state conformation of the 280s loop by making several favourable contacts to Asp283 and Asn284 of this loop. Comparison with the lead compound N-acetyl-$\beta$-d-glucopyranosylamine (6) shows that the hydrogen bonding interaction of the amide nitrogen with the main-chain carbonyl oxygen of His377 is not present in these complexes. The differences observed in the Ki values of the four analogues can be interpreted in terms of subtle conformational changes of protein residues and shifts of water molecules in the vicinity of the catalytic site, variations in van der Waals interactions, conformational entropy and desolvation effects. © 2006 Elsevier Ltd. All rights reserved.

Five oxalyl derivatives of beta-D-glucopyranosylamine were synthesized as potential inhibitors of glycogen phosphorylase (GP). The compounds 1-4 were competitive inhibitors of rabbit muscle GPb (with respect to alpha-D-glucose-l-phosphate) with K-i values of 0.2-1.4 mM, while compound 5 was not effective up to a concentration of 10 mM. In order to elucidate the structural basis of their inhibition, we analysed the structures of compounds 1-4 in complex with GPb at 1.93-1.96 angstrom resolution. The complex structures reveal that the inhibitors can be accommodated at the catalytic site at approximately the same position as alpha-D-glucose and stabilize the T-state conformation of the 280s loop by making several favourable contacts to Asp283 and Asn284 of this loop. Comparison with the lead compound N-acetyl-beta-D-glucopyranosylamine (6) shows that the hydrogen bonding interaction of the amide nitrogen with the main-chain carbonyl oxygen of His377 is not present in these complexes. The differences observed in the Ki values of the four analogues can be interpreted in terms of subtle conformational changes of protein residues and shifts of water molecules in the vicinity of the catalytic site, variations in van der Waals interactions, conformational entropy and desolvation effects. (c) 2006 Elsevier Ltd. All rights reserved.

Despite a few decades of intense study, a full description of tautomers of one-electron-oxidized guanine remains to be achieved. Here we show that two of these tautomers are produced by the protonation of an 8-haloguanine electron adduct. The rate constants for the reactions of hydrated electrons (e aq-) with a variety of 8-substituted guanine derivatives have been measured by a pulse radiolysis technique and correlated with both inductive and resonance components of the substituents. The fate of electron adducts was investigated by radiolytic methods coupled with product studies and addressed computationally by means of time-dependent DFT (TD-B3LYP/6- 311G**//B1B95/6-31+G**) calculations. The reaction of eaq- with 8-haloguanosine or 8-halo-2′- deoxyguanosine produces the first observable transient species that decay unimolecularly (k = 1 × 105 s-1 at 22 °C) to give the one-electron oxidized guanosine or 2′-deoxyguanosine. Theory suggests that the electron adducts of 8-bromoguanine derivatives protonated at C8 form a $π$-complex, with the Br atom situated above the molecular plane, that is prompt to eject Br. The two short-lived intermediates, which show a substantial difference in their absorption spectra, are recognized to be the two purine tautomers (i.e., iminic 7 and aminic 3 forms). The spin density distributions of the two tautomers are quite different at the O6 and N10 positions, whereas they are very similar at the N3, C5, and C8 positions. The resonance structures of the two tautomers are discussed in some detail. B1B95/6-31+G** calculations show also that the tautomerization from the iminic (7) to the aminic (3) arrangement is a water-assisted process. © 2006 American Chemical Society.

Oxidatively produced DNA damage is a physiological process that has been associated with many human conditions. Of the four DNA bases, guanine is the most easily oxidized and exhibits a large number of oxidation products. The focus of this Microreview is on the structure of the proposed intermediates and final products of guanine oxidation, as determined by spectroscopic methods and independent synthetic studies. The major and minor primary and secondary oxidative DNA lesions that arise from the oxidation of guanine under a variety of conditions, as well as associated structures, are described and all the relevant information regarding the isolation, characterization, and independent synthesis or generation of these lesions in synthetic oligonucleotides is presented. Finally, information on the biological studies that have been advanced, based on the basic molecular information obtained by the aforementioned studies, is provided. © Wiley-VCH Verlag GmbH {&} Co. KGaA, 2006.

Despite a few decades of intense study, a full description of tautomers of one-electron-oxidized guanine remains to be achieved. Here we show that two of these tautomers are produced by the protonation of an 8-haloguanine electron adduct. The rate constants for the reactions of hydrated electrons (e(aq)(-)) with a variety of 8-substituted guanine derivatives have been measured by a pulse radiolysis technique and correlated with both inductive and resonance components of the substituents. The fate of electron adducts was investigated by radiolytic methods coupled with product studies and addressed computationally by means of time-dependent DFT (TD-B3LYP/6-311G**//B1B95/6-31+G**) calculations. The reaction of e(aq)(-) with 8-haloguanosine or 8-halo-2'-deoxyguanosine produces the first observable transient species that decay unimolecularly (k = 1 x 10(5) s(-1) at 22 degrees C) to give the one-electron oxidized guanosine or 2'-deoxyguanosine. Theory suggests that the electron adducts of 8-bromoguanine derivatives protonated at C8 form a pi-complex, with the Br atom situated above the molecular plane, that is prompt to eject Br-. The two short- lived intermediates, which show a substantial difference in their absorption spectra, are recognized to be the two purine tautomers (i.e., iminic 7 and aminic 3 forms). The spin density distributions of the two tautomers are quite different at the O6 and N10 positions, whereas they are very similar at the N3, C5, and C8 positions. The resonance structures of the two tautomers are discussed in some detail. B1B95/6-31+G** calculations show also that the tautomerization from the iminic (7) to the aminic (3) arrangement is a water-assisted process.

The thermal reaction (at 140°C) of various 1′,2′-didehydro- 2′-deoxynucleosides afforded the corresponding furanyl nucleosides in good yields. The reaction kinetics were monitored by 1H NMR and the mechanism in terms of 'four-center complex fission' is discussed. © 2004 Elsevier Ltd. All rights reserved.

Novel anomeric spironucleosides and 1′-cyano-2′,3′- didehydro-2′,3′-dideoxyuridine, a structural analogue of known anti-HIV agents, were prepared by nucleophilic addition of organolithium reagents to 1′-cyano-2′-deoxy- and 1′-cyano-2′-deoxy- 2′$\beta$-bromo-uridine derivatives, respectively. The yield and distribution of products depended on the reaction conditions, which were studied in detail. Although none of the compounds exhibited antiviral activity, two compounds displayed cytostatic activity against both murine leukemia and human T-lymphocyte cells.

Novel anomeric spironucleosides and 1'-cyano-2',3'-didehydro-2',3'-dideoxyuridine, a structural analogue of known anti-HIV agent, were prepared by nucleophilic addition of organolithium reagents to 1'-cyano-2'deoxy- and 1'-cyano-2'-deoxy-2'beta-bromouridine derivatives, respectively. The yield and distribution of products depended on the reaction conditions, whichwere studied in detail. Although none of the compounds exhibited antiviral activit, twocompounds displayed cytostatic activity against both murine leukemia and human T-lymphocyte cells.

O-tert-Butyldimethylsilylimidazolyl aminals are N,O-acetals that form readily from aldehydes, and although they function as aldehyde stabilizing and protecting groups under various conditions, we report here that they react with organolithium reagents similarly to the parent aldehydes. The mechanism involves the intermediate formation of a 2-imidazolyl anion as is exemplified by the isolation of 2-TBDMS-imidazole. Substitution of the imidazolyl moiety at the 2-position renders these aldehyde derivatives stable to organolithium reagents, thus allowing for the tuning of their reactivity.

O-tert-Butyldimethylsilylimidazolyl aminals are N,O-acetals that form readily from aldehydes, and although they function as aldehyde stabilizing and protecting groups under various conditions, we report here that they react with organolithium reagents similarly to the parent aldehydes. The mechanism involves the intermediate formation of a 2-imidazolyl anion as is exemplified by the isolation of 2-TBDMS-imidazole. Substitution of the imidazolyl moiety at the 2-position renders these aldehyde derivatives stable to organolithium reagents, thus allowing for the tuning of their reactivity.

The development of new hydrogen selective neutral carriers based on 3-hydroxypicolinic acid derivatives, and its application to pH selective liquid membrane ISE based on these compounds is described. The optimization of the sensor has been based on the use of different plasticizers, additives, and mole ratios of the picolinate derivatives. Based on these results it is shown that the membrane composition can be adjusted for the measurement of pH at very low pH values (down to pH -0.5) and up to at least pH 10. The ability of the presented carriers to measure hydrogen activities up to 3N gives new directions in the use of these sensors for a variety of critical applications.

Relative kinetic data were determined for the 5-endo-trig cyclization of radical 12 compared to hydrogen abstraction from (TMS) 3SiH in the temperature range of 344-430 K, which allows for the estimation of a rate constant of 2 × 10 4 s -1 at 298 K with an activation energy of ca. 9 kcal/mol for the cyclization process. The 5-endo-trig cyclization of a variety of radicals that afford five-membered nitrogen-containing heterocycles was addressed computationally at the UB3LYP/6-31G* level. The 5-endo vs 4-exo mode of cyclication and the effect of delocalization of the unpaired electron in the transition state were investigated. Because the ring formed during cyclization contains five sp 2 centers, electrocyclization via a pentadienyl-like resonance form was also considered. For comparison, similar calculations were performed for 4-penten-1-yl and related radicals. The factors that affect the activation energies of homolytic 5-endo-trig cyclization were determined. In the absence of steric or conformational effects, the endo cyclization to form the five-membered ring was strongly favored over exo cyclization to form the four-membered ring not only on thermodynamic grounds but also kinetically. When a substituent on the double bond was able to delocalize the unpaired electron in the transition state of the 4-exo path, the two modes of cyclization became kinetically comparable. These results have an important bearing on the generalization of the Baldwin-Beckwith rules, which classified the 5-endo-trig radical cyclization as a "disfavored" process.

Relative kinetic data were determined for the 5-endo-trig cyclization of radical 12 compared to hydrogen abstraction from (TMS)(3)SiH in the temperature range of 344-430 K, which allows for the estimation of a rate constant of 2 x 10(4) s(-1) at 298 K with an activation energy of ca. 9 kcal/mol for the cyclization process. The 5-endo-trig cyclization of a variety of radicals that afford five-membered nitrogen-containing heterocycles was addressed computationally at the UB3LYP/6-31G* level. The 5-endo vs 4-exo mode of cyclication and the effect of delocalization of the unpaired electron in the transition state were investigated. Because the ring formed during cyclization contains five sp(2) centers, electrocyclization via a pentadienyl-like resonance form was also considered. For comparison, similar calculations were performed for 4-penten-1-yl and related radicals. The factors that affect the activation energies of homolytic 5-endo-trig cyclization were determined. In the absence of steric or conformational effects, the endo cyclization to form the five-membered ring was strongly favored over exo cyclization to form the four-membered ring not only on thermodynamic grounds but also kinetically. When a substituent on the double bond was able to delocalize the unpaired electron in the transition state of the 4-exo path, the two modes of cyclization became kinetically comparable. These results have an important bearing on the generalization of the Baldwin-Beckwith rules, which classified the 5-endo-trig radical cyclization as a "disfavored" process.

Efficient methodologies based on radical cascade reactions for the preparation of anomeric spironucleosides of general structure 3 and 4 are reported. The reactions were performed on modified uridine and 2'-deoxyuridine substrates. The protected derivatives 6 and 28 afforded the anomeric spironucleosides 7 and 29, respectively, in a stereospecific manner and in moderate yields (3-50{%}). In the 2'-deoxyribo series, the efficiency increased considerably (yields higher than 70{%}) with a concomitant decrease in stereoselectivity. In fact, the protected derivatives 13 and 21 gave mixtures of the anomeric products 11/14 and 22/23, respectively. Chemical transformations of some of these spironucleosides were successfully performed. The circular dichroism spectra of the anomeric spironucleosides displayed some striking features which can be attributed to the restricted rotation of the glycosidic bond. The reaction mechanism, which has been studied in some detail, comprises of a cascade of radical reactions in which the key step is the 1,5-radical translocation from an alkoxyl or vinyl radical. conveniently situated on the base moiety in the vicinity of the anomeric position. After the translocation, the alkoxyl radical 15, generated photolytically from an in situ prepared hypoiodite. afforded spironucleosides which possess an unusual orthoamide structure at the anomeric position. Alternatively, the vinyl radical 30, generated by the reaction of vinyl bromides with tributyltin radical, undergoes a 5-endo-trig cyclization followed by a bromine atom elimination alter the 1.5-radical translocation step.

The reaction of the 1'-C-cyano-2'-deoxyuridine derivative 1 with organolithium reagents can be favorably tuned to give a new class of anomeric spironucleosides.

C-1' acylated derivatives of 2'-deoxyuridine were obtained either by revising the existing procedures or by introducing a new methodology.

The reaction of the 1'-C-cyano-2'-deoxyuridine derivative 1 with organolithium reagents can be favorably tuned to give a new class of anomeric spironucleosides. (C) 1999 Elsevier Science Ltd. All rights reserved.

C-1' acylated derivatives of 2'-deoxyuridine were obtained either by revising the existing procedures or by introducing a new methodology.

The synthesis of protected 1',2'-didehydro-2'-deoxyadenosines has been optimized by incorporating a phosphoranylidene protection of the adenine amine function. These unsaturated adenosines have served as substrates for the electrophilic iodopivaloyloxylation leading to new nucleosides modified at the anomeric position. Reaction of halopivaloates 10, 11 and 12 with tributyltin hydride generates indirectly C-1' radicals through a $\beta$- (acyloxy)alkyl rearrangement. Rate constants for these rearrangements have been measured by using free-radical clock methodology and comparison of these data with previous reported results provides structural information about the nature of this important class of radicals.

The attempted intermolecular addition of malonyl radicals to 1',2'- unsaturated nucleosides has led to the unexpected formation of furanones. Thus, only catalytic amounts of ceric(IV) ammonium nitrate (CAN), induce a Ferrier rearrangement. The unsaturated lactone was isolated in good yield and can serve as a precursor for the synthesis of optically active products.

The attempted intermolecular addition of malonyl radicals to 1',2'-unsaturated nucleosides has led to the unexpected formation of furanones. Thus, only catalytic amounts of ceric(IV) ammonium nitrate (CAN), induce a Ferrier rearrangement. The unsaturated lactone was isolated in good yield and can serve as a precursor for the synthesis of optically active products. (C) 1998 Elsevier Science Ltd. All rights reserved.

The synthesis of protected 1',2'-didehydro-2'-deoxyadenosines has been optimized by incorporating a phosphoranylidene protection of the adenine amino function. These unsaturated adenosines have served as substrates for the electrophilic iodopivaloyloxylation leading to new nucleosides modified at the anomeric position. Reaction of halopivaloates 10, 11 and 12 with tributyltin hydride generates indirectly C-1' radicals through a beta-(acyloxy)alkyl rearrangement. Rate constants for these rearrangements have been measured by using free-radical clock methodology and comparison of these data with previous reported results provides structural information about the nature of this important class of radicals. (C) 1997 Elsevier Science Ltd. All rights reserved.

The title C-1' radical, obtained by photolysis of the corresponding tert-butyl ketone in water, was studied spectroscopically by EPR and laser flash photolysis methods and computationally.

The mechanism of 2-deoxyribonolactone formation from the reaction of photogenerated 2'-deoxyuridin-1'-yl radical with molecular oxygen in water has been investigated.

The mechanism of 2-deoxyribonolactone formation from the reaction of photogenerated 2′-deoxyuridin-1′-yl radical with molecular oxygen in water has been investigated.

The title C-1' radical, obtained by photolysis of the corresponding tert-butyl ketone in water, was studied spectroscopically by EPR and laser flash photolysis methods and computationally. (C) 1998 Elsevier Science Ltd. All rights reserved.

A 1,5-hydrogen migration of a conveniently situated alkoxyl radical affords spironucleosides which possess an unusual orthoamide structure at the anomeric position; X-ray crystallography establishes the configuration of the C-1' position.

A number of nucleoside substrates has been reduced under free radical conditions with tris(trimethylsilyl)silane. In one case a novel type of a $\beta$-(acyloxy)alkyl radical rearrangement has been observed, which leads through the generation of a C-1' radical species to the stereoselective preparation of an $\alpha$-ribonucleoside. The rate of the above 1,2-migration has been estimated, and a comparison with previously reported results has been made.

The deoxygenation of secondary alcohols via thionoesters with the use of 5,10-dihydrosilanthrene as the radical reducing agent has been studied in detail. The ability of hydrogen donation of this silane has been measured using the one-carbon ring expansion of 1-(2-oxocyclopentyl)ethyl radical as a timing device.

A number of nucleoside substrates has been reduced under free radical conditions with tris(trimethylsilyl)silane. In one case a novel type of a beta-(acyloxy)alkyl radical rearrangement has been observed, which leads through the generation of a C-1' radical species to the stereoselective preparation of an alpha-ribonucleoside. The rate of the above 12-migration has been estimated, and a comparison with previously reported results has been made.

A five-step synthesis of 3-methylcholanthrene (1) has been achieved starting from 5-methylhomophthalic anhydride and N,N-diethyl-1-naphthamide in 55{%} overall yield. Treatment of a solution of the preformed lithium enolate of 5-methylhomophthalic anhydride (3) with an equimolar solution of 2-lithio-1-naphthamide (4), followed by acid hydrolysis, provides cleanly the spirobislactone 5 in 80{%} overall yield. In addition, the synthesis features a unique, highly selective double Friedel-Crafts cyclization of the aryl diacid 2 with PPA to give rise, after acetylation, to keto acetate 6. © 1993 America Chemical Society.

A five-step synthesis of 3-methylcholanthrene (1) has been achieved starting from 5-methylhomophthalic anhydride and N,N-diethyl-1-naphthamide in 55{%} over all yield. Treatment of a solution of the preformed lithium enolate of 5-methylhomophthalic anhydride (3) with an equimolar solution of 2-lithio-1-naphthamide (4), followed by acid hydrolysis, provides cleanly the spirobislactone 5 in 80{%} overall yield. In addition, the synthesis features a unique, highly selective double Friedel-Crafts cyclization of the aryl diacid 2 with PPA to give rise, after acetylation, to keto acetate 6.