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 .     

N2‐Substituted 2′‐Deoxyguanosine Triphosphate Derivatives as Selective Substrates for Human DNA Polymerase κ

N²‐Alkyl‐2′‐deoxyguanosine triphosphate (N²‐alkyl‐dGTP) derivatives with methyl, butyl, benzyl, or 4‐ethynylbenzyl substituents were prepared and tested as substrates for human DNA polymerases. N²‐Benzyl‐dGTP was equal to dGTP as a substrate for DNA polymerase κ (pol κ), but was a poor substrate for pols β, δ, η, ι, or ν. In vivo reactivity was evaluated through incubation of N²‐4‐ethynylbenzyl‐dG with wild‐type and pol κ deficient mouse embryonic fibroblasts. CuAAC reaction with 5(6)‐FAM‐azide demonstrated that only cells containing pol κ were able to incorporate N²‐4‐ethynylbenzyl‐dG into the nucleus. This is the first instance of a Y‐family‐polymerase‐specific dNTP, and this method could be used to probe the activity of pol κ in vivo.     

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.     

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.     

Enzymatic Synthesis of 7′,5′‐Bicyclo‐DNA Oligonucleotides

The selection of artificial genetic polymers with tailor‐made properties for their application in synthetic biology requires the exploration of new nucleosidic scaffolds that can be used in selection experiments. Herein, we describe the synthesis of a bicyclo‐DNA triphosphate (i.e., 7′,5′‐bc‐TTP) and show its potential to serve for the generation of new xenonucleic acids (XNAs) based on this scaffold. 7′,5′‐bc‐TTP is a good substrate for Therminator DNA polymerase, and up to seven modified units can be incorporated into a growing DNA chain. In addition, this scaffold sustains XNA‐dependent DNA synthesis and potentially also XNA‐dependent XNA synthesis. However, DNA‐dependent XNA synthesis on longer templates is hampered by competitive misincorporation of deoxyadenosine triphosphate (dATP) caused by the slow rate of incorporation of 7′,5′‐bc‐TTP.     

Independent Generation and Time‐Resolved Detection of 2′‐Deoxyguanosin‐N2‐yl Radicals

Guanine radicals are important reactive intermediates in DNA damage. Hydroxyl radical (HO^.) has long been believed to react with 2′‐deoxyguanosine (dG) generating 2′‐deoxyguanosin‐N1‐yl radical (dG(N1‐H)^.) via addition to the nucleobase π‐system and subsequent dehydration. This basic tenet was challenged by an alternative mechanism, in which the major reaction of HO^. with dG was proposed to involve hydrogen atom abstraction from the N2‐amine. The 2′‐deoxyguanosin‐N2‐yl radical (dG(N2‐H)^.) formed was proposed to rapidly tautomerize to dG(N1‐H)^.. We report the first independent generation of dG(N2‐H)^. in high yield via photolysis of 1 . dG(N2‐H)^. is directly observed upon nanosecond laser flash photolysis (LFP) of 1 . The absorption spectrum of dG(N2‐H)^. is corroborated by DFT studies, and anti‐ and syn‐dG(N2‐H)^. are resolved for the first time. The LFP experiments showed no evidence for tautomerization of dG(N2‐H)^. to dG(N1‐H)^. within hundreds of microseconds. This observation suggests that the generation of dG(N1‐H)^. via dG(N2‐H)^. following hydrogen atom abstraction from dG is unlikely to be a major pathway when HO^. reacts with dG.     

Oligonucleotide Analogues with a Nucleobase‐Including Backbone,Part 5, 2′‐Deoxy‐8‐(hydroxymethyl)adenosine‐ and 2′‐Deoxy‐6‐(hydroxymethyl)uridine‐Derived Phosphoramidites: Synthesis and Incorporation into 14‐Mer DNA Strands

The pairing propensity of new DNA analogues with a phosphinato group between O−C(3′) and a newly introduced OCH₂ group at C(8) and C(6) of 2′‐deoxyadenosine and 2′‐deoxyuridine, respectively, was evaluated by force‐field calculations and Maruzen model studies. These studies suggest that these analogues may form autonomous pairing systems, and that the incorporation of single modified units into DNA 14mers is compatible with duplex formation. To evaluate the incorporation, we prepared the required phosphoramidites 3 and 4 from 2′‐deoxyadenosine and 2′‐deoxyuridine, respectively. The phosphoramidite 5 was similarly prepared to estimate the influence of a CH₂OH group at C(8) on the duplex stability. The modified 14‐mers 6 – 9 were prepared by solid‐phase synthesis. Pairing studies show a decrease of the melting temperature by 2.5° for the duplex 13 ⋅ 9 , and of 6 – 8° for the duplexes 10 ⋅ 6 , 11 ⋅ 6 , 13 ⋅ 7 , and 14 ⋅ 8 , as compared to the unmodified duplexes.     

Recording the Reaction Process of Loop‐Mediated Isothermal Amplification (LAMP) by Monitoring the Voltammetric Response of 2′‐Deoxyguanosine 5′‐Triphosphate

We describe here a novel strategy for recording the reaction process of loop‐mediated isothermal amplification (LAMP) by monitoring the voltammetric response of 2′‐deoxyguanosine 5′‐triphosphate (dGTP). Unlike the other three kinds of reactive substrates for DNA synthesis in LAMP reaction, dGTP exhibits sensitive voltammetric response at the carbon nanotube array electrode. When the LAMP reaction occurs, the concentration of dGTP decreases accordingly, bringing forth the decrease of the anodic peak current (i_pa). In inversion, the decrease of the i_pa of dGTP was used to characterize the reaction process of LAMP. The relationships among the LAMP reaction time, the initial quantity of template DNA and the value change of the i_pa were studied. The results indicate that the protocol integrated LAMP and voltammetric techniques can be used for not only qualitative gene discrimination but also quantitative gene assay in a wide range. The malB gene extracted from common strains of Escherichia coli cells was tested as a model. The detecting results of LAMPs obtained by voltammetric method were in good agreement with those by optical‐based methods (gel electrophoresis and fluorescent dye).     

Silver Ions in Non‐canonical DNA Base Pairs: Metal‐Mediated Mismatch Stabilization of 2′‐Deoxyadenosine and 7‐Deazapurine Derivatives with 2′‐Deoxycytidine and 2′‐Deoxyguanosine

Novel silver‐mediated dA?dC, dA*?dC, and dA*?dG base pairs were formed in a natural DNA double helix environment (dA* denotes 7‐deaza‐dA, 7‐deaza‐7‐iodo‐dA, and 7‐cyclopropyl‐7‐deaza‐dA). 7‐Deazapurine nucleosides enforce silver ion binding and direct metal‐mediated base pair formation to their Watson–Crick face. New phosphoramidites were prepared from 7‐deaza‐dA, 7‐deaza‐7‐iodo‐dA, and 7‐cyclopropyl‐7‐deaza‐dA, which contain labile isobutyryl protecting groups. Solid‐phase synthesis furnished oligonucleotides that contain mismatches in near central positions. Increased thermal stabilities (higher T_m values) were observed for oligonucleotide duplexes with non‐canonical dA*?dC and dA?dC pairs in the presence of silver ions. The stability of the silver‐mediated base pairs was pH dependent. Silver ion binding was not observed for the dA?dG mismatch but took place when mismatches were formed between 7‐deazaadenine and guanine. The specific binding of silver ions was confirmed by stoichiometric UV titration experiments, which proved that one silver ion is captured by one mismatch. The stability increase of canonical DNA mismatches might have an impact on cellular DNA repair.     

Synthesis of (2′S)‐2′‐Deoxy‐2′‐C‐methylpurine Nucleosides and Their Phosphoramidites

We describe the stereoselective synthesis of (2′S)‐2′‐deoxy‐2′‐C‐methyladenosine ( 12 ) and (2′S)‐2′‐deoxy‐2′‐C‐methylinosine ( 14 ) as well as their corresponding cyanoethyl phosphoramidites 16 and 19 from 6‐O‐(2,6‐dichlorophenyl)inosine as starting material. The methyl group at the 2′‐position was introduced via a Wittig reaction (→ 3 , Scheme 1) followed by a stereoselective oxidation with OsO₄ (→ 4 , Scheme 2). The primary‐alcohol moiety of 4 was tosylated (→ 5 ) and regioselectively reduced with NaBH₄ (→ 6 ). Subsequent reduction of the 2′‐alcohol moiety with Bu₃SnH yielded stereoselectively the corresponding (2′S)‐2′‐deoxy‐2′‐C‐methylnucleoside (→ 8a ).     

Major‐Groove‐Halogenated DNA: The Effects of Bromo and Iodo Substituents Replacing H−C(7) of 8‐Aza‐7‐deazapurine‐2,6‐diamine or H−C(5) of Uracil Residues

Oligonucleotides containing halogenated `purine' and pyrimidine bases were synthesized. Bromo and iodo substituents were introduced at the 7‐position of 8‐aza‐7‐deazapurine‐2,6‐diamine (see 2b , c ) or at the 5‐position of uracil residues (see 3b , c ). Phosphoramidites were synthesized after protection of 2b with the isobutyryl residue and of 2c with the benzoyl group. Duplexes containing the residues 2b or 2c gave always higher T<sub>m</sub> values than those of the nonmodified counterparts containing 2′‐deoxyadenosine, the purine‐2,6‐diamine 2′‐deoxyribonucleoside ( 1 ), or 2a at the same positions. Six 2b residues replacing dA in the duplex 5′‐d(TAGGTCAATACT)‐3′ ( 11 )⋅5′‐d(AGTATTGACCTA)‐3′ ( 12 ) raised the T<sub>m</sub> value from 48 to 75° (4.5° per modification (Table 3)). Contrary to this, incorporation of the 5‐halogenated 2′‐deoxyuridines 3b or 3c into oligonucleotide duplexes showed very little influence on the thermal stability, regardless of which `purine' nucleoside was located opposite to them (Tables 4 and 5). The positive effects on the thermal stability of duplexes observed in DNA were also found in DNA⋅RNA hybrids or in DNA with parallel chain orientation (Tables 8 and 9, resp.).     

Different nucleobase orientations in two cyclic 2′,3′‐phosphates of purine ribonucleosides: Et3NH(2′,3′‐cAMP) and Et3NH(2′,3′‐cGMP)·H2O

The crystal structures of triethyl­ammonium adenosine cyclic 2′,3′‐phosphate {systematic name: triethyl­ammonium 4‐(6‐amino­purin‐9‐yl)‐6‐hydroxy­methyl‐2‐oxido‐2‐oxoperhydro­furano[3,4‐c][1,3,2]dioxaphosphole}, Et₃NH(2′,3′‐cAMP) or C₆H₁₆N⁺·C₁₀H₁₁N₅O₆P⁻, (I), and guanosine cyclic 2′,3′‐phosphate monohydrate {systematic name: triethyl­ammonium 6‐hydroxy­methyl‐2‐oxido‐2‐oxo‐4‐(6‐oxo‐1,6‐dihydro­purin‐9‐yl)perhydro­furano[3,4‐c][1,3,2]dioxaphosphole monohydrate}, [Et₃NH(2′,3′‐cGMP)]·H₂O or C₆H₁₆N⁺·C₁₀H₁₁N₅O₇P⁻·H₂O, (II), reveal different nucleobase orientations, viz. anti in (I) and syn in (II). These are stabilized by different inter‐ and intra­molecular hydrogen bonds. The structures also exhibit different ribose ring puckering [₄E in (I) and ³T₂ in (II)] and slightly different 1,3,2‐dioxaphospho­lane ring conformations, viz. envelope in (I) and puckered in (II). Infinite ribbons of 2′,3′‐cAMP⁻ and helical chains of 2′,3′‐cGMP⁻ ions, both formed by O—H⋯O, N—H⋯X and C—H⋯X (X = O or N) hydrogen‐bond contacts, characterize (I) and (II), respectively.     

(2′‐O‐Methyl‐RNA)‐3′‐PNA Chimeras: A New Class of Mixed Backbone Oligonucleotide Analogues with High Binding Affinity to RNA

The automated on‐line synthesis of DNA‐3′‐PNA chimeras 1 – 4 and (2′‐O‐methyl‐RNA)‐3′‐PNA chimeras 5 – 8 is described, in which the 3′‐terminal part of the oligonucleotide is linked to the N‐terminal part of the PNA via N‐(ω‐hydroxyalkyl)‐N‐[(thymin‐1‐yl)acetyl]glycine units (alkyl=Et, Ph, Bu, and pentyl). By means of UV thermal denaturation, the binding affinities of all chimeras were directly compared by determining their T_m values in the duplex with complementary DNA and RNA. All investigated DNA‐3′‐PNA chimeras and (2′‐O‐methyl‐RNA)‐3′‐PNA chimeras form more‐stable duplexes with complementary DNA and RNA than the corresponding unmodified DNA. Interestingly, a N‐(3‐hydroxypropyl)glycine linker resulted in the highest binding affinity for DNA‐3′‐PNA chimeras, whereas the (2′‐O‐methyl‐RNA)‐3′‐PNA chimeras showed optimal binding with the homologous N‐(4‐hydroxybutyl)glycine linker. The duplexes of (2′‐O‐methyl‐RNA)‐3′‐PNA chimeras and RNA were significantly more stable than those containing the corresponding DNA‐3′‐PNA chimeras. Surprisingly, we found that the charged (2′‐O‐methyl‐RNA)‐3′‐PNA chimera with a N‐(4‐hydroxybutyl)glycine‐based unit at the junction to the PNA part shows the same binding affinity to RNA as uncharged PNA. Potential applications of (2′‐O‐methyl‐RNA)‐3′‐PNA chimeras include their use as antisense agents acting by a RNase‐independent mechanism of action, a prerequisite for antisense‐oligonucleotide‐mediated correction of aberrant splicing of pre‐mRNA.     

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.     

Structural and theoretical characterization of a new twisted 4′‐substituted terpyridine compound: 4′‐(isoquinolin‐4‐yl)‐2,2′:6′,2′′‐terpyridine

4′‐Substituted derivatives of 2,2′:6′,2′′‐terpyridine with N‐containing heteroaromatic substituents, such as pyridyl groups, might be able to coordinate metal centres through the extra N‐donor atom, in addition to the chelating terpyridine N atoms. The incorporation of these peripheral N‐donor sites would also allow for the diversification of the types of noncovalent interactions present, such as hydrogen bonding and π–π stacking. The title compound, C₂₄H₁₆N₄, consists of a 2,2′:6′,2′′‐terpyridine nucleus (tpy), with a pendant isoquinoline group (isq) bound at the central pyridine (py) ring. The tpy nucleus deviates slightly from planarity, with interplanar angles between the lateral and central py rings in the range 2.24 (7)–7.90 (7)°, while the isq group is rotated significantly [by 46.57 (6)°] out of this planar scheme, associated with a short H_tpy…H_isq contact of 2.32 Å. There are no strong noncovalent interactions in the structure, the main ones being of the π–π and C—H…π types, giving rise to columnar arrays along [001], further linked by C—H…N hydrogen bonds into a three‐dimensional supramolecular structure. An Atoms In Molecules (AIM) analysis of the noncovalent interactions provided illuminating results, and while confirming the bonding character for all those interactions unquestionable from a geometrical point of view, it also provided answers for some cases where geometric parameters are not informative, in particular, the short H_tpy…H_isq contact of 2.32 Å to which AIM ascribed an attractive character.     

Synthesis of 2′-Deoxyisoguanosine 5′-Triphosphate and 2′-Deoxy-5-methylisocytidine 5′-Triphosphate

The syntheses of the 5′-triphosphates of 2′-deoxyisoguanosine (=p₃isoG_d) and 2′-deoxy-5-methylisocytidine (=p₃me⁵isoC_d), two new bases for the genetic alphabet, are described. The triphosphates were synthesized from the corresponding nucleosides using a transient-protection procedure. The introduction of a methyl group at the 5-position of 2′-deoxyisocytidine remarkably improved the stability of the triphosphate. Characterization of the triphosphates included enzymatic incorporation opposite the complementary base in a template oligonucleotide.     

Oligonucleotides Containing Novel 4′‐C‐ or 3′‐C‐(Aminoalkyl)‐Branched Thymidines

The synthesis of four novel 3′‐C‐branched and 4′‐C‐branched nucleosides and their transformation into the corresponding 3′‐O‐phosphoramidite building blocks for automated oligonucleotide synthesis is reported. The 4′‐C‐branched key intermediate 11 was synthesized by a convergent strategy and converted to its 2′‐O‐methyl and 2′‐deoxy‐2′‐fluoro derivatives, leading to the preparation of novel oligonucleotide analogues containing 4′‐C‐(aminomethyl)‐2′‐O‐methyl monomer X and 4′‐C‐(aminomethyl)‐2′‐deoxy‐2′‐fluoro monomer Y (Schemes 2 and 3). In general, increased binding affinity towards complementary single‐stranded DNA and RNA was obtained with these analogues compared to the unmodified references (Table 1). The presence of monomer X or monomer Y in a 2′‐O‐methyl‐RNA oligonucleotide had a negative effect on the binding affinity of the 2′‐O‐methyl‐RNA oligonucleotide towards DNA and RNA. Starting from the 3′‐C‐allyl derivative 28 , 3′‐C‐(3‐aminopropyl)‐protected nucleosides and 3′‐O‐phosphoramidite derivatives were synthesized, leading to novel oligonucleotide analogues containing 3′‐C‐(3‐aminopropyl)thymidine monomer Z or the corresponding 3′‐C‐(3‐aminopropyl)‐2′‐O,5‐dimethyluridine monomer W (Schemes 4 and 5). Incorporation of the 2′‐deoxy monomer Z induced no significant changes in the binding affinity towards DNA but decreased binding affinity towards RNA, while the 2′‐O‐methyl monomer Z induced decreased binding affinity towards DNA as well as RNA complements (Table 2).     

Efficient Synthesis of Novel Coumarin‐3‐carboxamides (=2‐Oxo‐2H‐1‐benzopyran‐3‐carboxamides) Containing Lipophilic Spacers

The novel coumarin‐3‐carboxamides (=2‐oxo‐2H‐1‐benzopyran‐3‐carboxamides) 5a – 5g containing lipophilic spacers were synthesized through the Ugi‐four‐component reaction (Scheme 1). The reactions of aromatic aldehydes 1 , 4,4′‐oxybis[benzenamine] or 4,4′‐methylenebis[benzenamine] as diamine 2 , coumarin‐3‐carboxylic acid (=2‐oxo‐2H‐benzopyran‐3‐carboxylic acid; 3 ), and alkyl isocyanides 4 lead to the desired substituted coumarin‐3‐carboxamides 5a – 5g at room temperature with high bond‐forming efficiency. These novel coumarin derivatives exhibit brilliant fluorescence at 544 nm in CHCl₃.     

Synthesis of spiro[indoline‐3,3′‐pyrrolizines] by 1,3‐dipolar reactions between isatins,l‐proline and electron‐deficient alkenes

Two spiro[indoline‐3,3′‐pyrrolizine] derivatives have been synthesized in good yield with high regio‐ and stereospecificity using one‐pot reactions between readily available starting materials, namely l ‐proline, substituted 1H‐indole‐2,3‐diones and electron‐deficient alkenes. The products have been fully characterized by elemental analysis, IR and NMR spectroscopy, mass spectrometry and crystal structure analysis. In (1′RS ,2′RS ,3SR ,7a′SR )‐2′‐benzoyl‐1‐hexyl‐2‐oxo‐1′,2′,5′,6′,7′,7a′‐hexahydrospiro[indoline‐3,3′‐pyrrolizine]‐1′‐carboxylic acid, C₂₈H₃₂N₂O₄, (I), the unsubstituted pyrrole ring and the reduced spiro‐fused pyrrole ring adopt half‐chair and envelope conformations, respectively, while in (1′RS ,2′RS ,3SR ,7a′SR )‐1′,2′‐bis(4‐chlorobenzoyl)‐5,7‐dichloro‐2‐oxo‐1′,2′,5′,6′,7′,7a′‐hexahydrospiro[indoline‐3,3′‐pyrrolizine], which crystallizes as a partial dichloromethane solvate, C₂₈H₂₀Cl₄N₂O₃·0.981CH₂Cl₂, (II), where the solvent component is disordered over three sets of atomic sites, these two rings adopt envelope and half‐chair conformations, respectively. Molecules of (I) are linked by an O—H…·O hydrogen bond to form cyclic R ₆⁶(48) hexamers of (S ₆) symmetry, which are further linked by two C—H…O hydrogen bonds to form a three‐dimensional framework structure. In compound (II), inversion‐related pairs of N—H…O hydrogen bonds link the spiro[indoline‐3,3′‐pyrrolizine] molecules into simple R ₂²(8) dimers.     

7‐Halogenated 7‐Deazapurine 2′‐Deoxyribonucleosides Related to 2′‐Deoxyadenosine, 2′‐Deoxyxanthosine,and 2′‐Deoxyisoguanosine: Syntheses and Properties

A series of 7‐fluorinated 7‐deazapurine 2′‐deoxyribonucleosides related to 2′‐deoxyadenosine, 2′‐deoxyxanthosine, and 2′‐deoxyisoguanosine as well as intermediates 4b – 7b, 8, 9b, 10b , and 17b were synthesized. The 7‐fluoro substituent was introduced in 2,6‐dichloro‐7‐deaza‐9H‐purine ( 11a ) with Selectfluor (Scheme 1). Apart from 2,6‐dichloro‐7‐fluoro‐7‐deaza‐9H‐purine ( 11b ), the 7‐chloro compound 11c was formed as by‐product. The mixture 11b / 11c was used for the glycosylation reaction; the separation of the 7‐fluoro from the 7‐chloro compound was performed on the level of the unprotected nucleosides. Other halogen substituents were introduced with N‐halogenosuccinimides ( 11a → 11c – 11e ). Nucleobase‐anion glycosylation afforded the nucleoside intermediates 13a – 13e (Scheme 2). The 7‐fluoro‐ and the 7‐chloro‐7‐deaza‐2′‐deoxyxanthosines, 5b and 5c , respectively, were obtained from the corresponding MeO compounds 17b and 17c , or 18 (Scheme 6). The 2′‐deoxyisoguanosine derivative 4b was prepared from 2‐chloro‐7‐fluoro‐7‐deaza‐2′‐deoxyadenosine 6b via a photochemically induced nucleophilic displacement reaction (Scheme 5). The pK_a values of the halogenated nucleosides were determined (Table 3). ¹³C‐NMR Chemical‐shift dependencies of C(7), C(5), and C(8) were related to the electronegativity of the 7‐halogen substituents (Fig. 3). In aqueous solution, 7‐halogenated 2′‐deoxyribonucleosides show an approximately 70% S population (Fig. 2 and Table 1).