Nucleotides. Part LXXV

The reactivity of the 2′‐deoxy‐N⁴‐(phenoxycarbonyl)cytidine derivatives 3 and 4 with aromatic amines was studied to form new types of urea derivatives (see 5 – 10 ). On the same basis, labeling of 3 and 4 with 5‐aminofluorescein ( 14 ) was achieved to give the conjugates 15 and 17 , respectively (Scheme 1). Treatment of 17 with 2‐(4‐nitrophenyl)ethanol in a Mitsunobu reaction led to double protection of the fluorescein moiety (→ 18 ) and desilylation yielded 19 . Dimethoxytritylation (→ 20 ) and subsequent phosphitylations afforded the new building blocks 21 and 22 . Synthesis of the fully protected trimer 28 was achieved by condensation of 21 with 23 to 26 which after detritylation (→ 27 ) was coupled with 25 to give 28 (Scheme 2). Deprotection of all blocking groups was performed with 1,8‐diazabicyclo[5.4.0]undec‐7‐ene (DBU) in one step to give 29 . The synthesis of the decamer 5′‐d(C^FluCCG GCC CGC)‐3′ ( 33 ) started from 30 which was attached to the solid support and then elongated with 31, 32 , and 22 at the 5′‐terminal end (C^Flu=deprotected phosphate derivative of 22 ). Hybridization with the complementary oligomer 5′‐d(G GGC CGG GCG)‐3′ ( 34 ) showed the influence of the fluorescein label on the stability of the duplex.     

Nucleotides. Part LXXIX

Two series of new ribonucleoside 3′‐phosphoramidites (see 36 – 42 ) carrying the photolabile [2‐(2‐nitrophenyl)propoxy]carbonyl group at the 5′‐O‐position were synthesized and characterized as monomeric building blocks for photolithographic syntheses of RNA chips. Base protection was achieved in the well‐known manner by the 2‐(4‐nitrophenyl)ethyl (npe) and the [2‐(4‐nitrophenyl)ethoxy]carbonyl (npeoc) group. The carbohydrate moiety carried in addition the 2′‐O‐(tetrahydro‐4‐methoxy‐2H‐pyran‐4‐yl) group for blocking the 2′‐OH function.     

Nucleotides. Part LXXVI

A series of new fluorescing 8‐(6‐hydroxyhexyl)isoalloxazine (=8‐(6‐hydroxyhexyl)benzo[g]pteridine‐2,4(1H,3H)‐dione) derivatives 4 – 13 were synthesized from 6‐[(6‐hydroxyhexyl)amino]uracil ( 2 ) with 1‐chloro‐4‐nitrosobenzene via 8‐chloro‐10‐(6‐hydroxyhexyl)isoalloxazine ( 3 ) and subsequent substitution of the Cl‐atom of 3 by various amines (Scheme). Analogously, 8‐substituted 10‐{3‐[(2,2‐dimethyl‐1,3‐dioxolan‐4‐yl)methoxy]propyl}isoalloxazines 19, 20 , and 23 – 25 were prepared which yielded on deprotection the corresponding 10‐[3‐(2,3‐dihydroxypropoxy)propyl]alloxazines 21, 22 , and 26 – 28 . Their conversion into the 3″‐O‐(4,4′‐dimethoxytrityl) derivatives 29 – 33 and subsequent transformation into the corresponding 2″‐(2‐cyanoethyl N,N‐diisopropylphosphoramidites) 34 – 38 led to new building blocks for oligonucleotide synthesis. A series of 21‐mer oligodeoxyribonucleotides carrying the fluorescing isoalloxazine 37 in various positions of the chain were assembled in a DNA synthesizer. Combination with the complementary sequence yielded the stable duplexes 40 – 54 showing by the melting temperatures T_m that the fluorophor ( F ) does not harm the stability of the unmodified duplex 39 (Table).     

Structure and Function of Nucleosides and Nucleotides

The nucleosides participating in biological processes consist of a sugar and a heterocyclic nucleobase; the nucleotides, which occur as monomers and as building units of polymeric nucleic acids, contain an additional phosphoester group. The complexity of the molecules leads to a complex stereochemistry with which the present progress report is concerned. Particular attention will be devoted to conformational considerations at the sugar groups, the syn-anti conformation, the position of the C(5′)? O(5′) bond relative to the sugar group, and the conformation of the phosphoester bonds. The article touches upon base pairing and base stacking, as well as forces stabilizing the syn conformation, and also deals with the reaction mechanism of the enzyme pancreatic ribonuclease as established from the stereochemistry of nucleotides and the mechanisms of action of the antileukemia drug 6-azauridine and the antibiotic actinomycin D. Views on the effects of the unusual structures of the “rare” nucleosides 4-thiouridine, isopentenyladenosine, and dihydrouridine on the structure of transfer ribonucleic acid are also presented.     

Fluorescent oligonucleotide incorporating 5-(1-ethynylpyrenyl)-2-deoxyuridine: sequence-specific fluorescence changes upon duplex formation

We describe the design and properties of a pyrene-labeled deoxyuridine that can be inserted efficiently into oligodeoxynucleotides using phosphoramidite chemistry. An oligonucleotide incorporating the pyrene-labeled deoxyuridine is a sensitive fluorescence probe that can discriminate between perfect and single-base-mismatched pairing by changes in its fluorescence intensity.     

Analysis of nucleosides and monophosphate nucleotides from mushrooms with reversed‐phase HPLC

The retention characteristics of five stationary phases were tested by using a selection of 5′‐mononucleotides and nucleosides with the aim to develop a simple, rapid and sensitive reversed‐phase liquid chromatography method without ion‐pair reagent usage. The method was optimized by changes in temperature, pH and ionic strength on a column showing a superior performance. The mobile phase consisted of a mixture of 0.05 M phosphate buffer and methanol, delivered at a flow rate of 0.4 mL/min and based on a gradient program. UV detection was used at a 254 nm wavelength. The method was validated for a quantitative analysis of 5′‐mononucleotides and nucleosides in wild edible mushrooms. For all nucleosides and nucleotides, the LOD and LOQ were less than 0.02 and 0.07 μg/mL, respectively. Validation parameters yielded recovery rates between 68.6 and 98.2%, with a precision expressed as a relative standard deviation of 7.6–15.3%. The content of 5′‐mononucleotides and nucleosides was determined for 10 samples of wild edible mushrooms found in Croatia and, accordingly, the equivalent umami concentrations were calculated.     

Oligodeoxynucleotides Containing 2′‐Deoxy‐1‐methyladenosine and Dimroth Rearrangement

2′‐Deoxy‐1‐methyladenosine was incorporated into synthetic oligonucleotides by phosphoramidite chemistry. Chloroacetyl protecting group and controlled anhydrous deprotection conditions were used to avoid Dimroth rearrangement. Hybridization studies of intramolecular duplexes showed that introduction of a modified residue into the loop region of the oligonucleotide hairpin increases the melting temperature. It was shown that modified oligonucleotides may be easily transformed into oligonucleotides containing 2′‐deoxy‐N⁶‐methyladenosine.     

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 and Anti‐HIV Activity of Triazolo‐Fused 2′,3′‐Cyclic Nucleoside Analogs Prepared by an Intramolecular Huisgen 1,3‐Dipolar Cycloaddition

Triazolo‐fused 2′,3′‐cyclic nucleoside analogs were synthesized by an intramolecular 1,3‐dipolar cycloaddition of nucleoside‐derived azido alkynes in a regio‐ and stereospecific manner. The uracil base in these target compounds was successfully transformed to the corresponding cytosine. The synthesized compounds were examined in a MAGI assay for their anti‐HIV activities, and in a H9 T lymphocytes assay for their cell toxicities.     

Synthesis,Crystal Structures and Fluorescent Properties of Two 2,2′‐Biquinoline‐4,4′‐dicarboxylate‐based Cadmium(II) and Cobalt(II) Complexes

Two metal‐organic coordination polymers with one‐dimensional infinite chain motif, [Cd(bqdc)(phen)₂]_n ( 1 ) and [Co(bqdc)(phen)(H₂O)₂]_n ( 2 ) (H₂bqdc = 2,2′‐biquinoline‐4,4′‐dicarboxylic acid, phen = 1,10‐phenanthroline), have been synthesized under similar solv/hydrothermal conditions and fully structural characterized by elemental analysis, IR, and single‐crystal X‐ray crystallography. Their thermal stability and photoluminescence properties were further investigated by TG‐DTA and fluorescence spectra. In both complexes, the adjacent metal ions (Cd^II for 1 and Co^II for 2 ) are linked together by dicarboxylate groups of bqdc dianions in chelating bidentate and monodentate modes, respectively, generating a zigzag chain for 1 and linear chain for 2 . The relatively higher thermal stability up to 324 °C for 1 and strong fluorescence emissions jointly suggest that they are good candidates for luminescent materials.     

Use of Adsorptive Transfer Stripping Voltammetry for Analyzing Variations of Cytosine Methylation in DNA

The electrochemical behavior and thermal stability of double stranded oligonucleotides containing 5‐methyl‐cytosine and 7‐deaza‐guanosine as nucleotide analogues, as well as of Jurkat genomic DNAs methylated to different degree were studied by ACV and SWV and by thernal denaturation analysis. ACV and SWV combined with thermal denaturation analysis of the natural and modified oligonucleotides gave information regarding the presence of methylation and the concomitant conformational changes. ACV and SWV of Jurkat DNA mixtures methylated to different degrees revealed a decrease of the peak heights with increasing methylation, indicating an increase of structural rigidity, in agreement with the thermal denaturation data. These results verify the, possibly local, conformational changes introduced by DNA methylation. The results obtained in all cases were reproducible.     

Two Zinc(II) Coordination Polymers Based on Terphenyl‐2,2′,4,4′‐tetracarboxylate and Dipyridyl Ligands: Syntheses,Crystal Structures,and Luminescent Properties

Two coordination polymers, {[Zn₂(L)(bpy)] · 2H₂O}_n ( 1 ) and [Zn₂(L)(bpe)]_n ( 2 ) [H₄L = terphenyl‐2,2′,4,4′‐tetracarboxylic acid, bpy = 4,4′‐bipyridine, and bpe = 1,2‐bis(4‐pyridyl)ethane], were hydrothermally synthesized under similar conditions and characterized by elemental analysis, IR spectroscopy, TGA, and single‐crystal X‐ray diffraction analysis. Compound 1 has a 3D framework containing Zn–O–C–O–Zn 1D chains. Compound 2 exhibits a 3D framework, which features tubular channels. The channels are occupied by bpe molecules. The differences in the structures demonstrate that the auxiliary dipyridyl‐containing ligand has a significant effect on the construction of the final framework. Additionally, the fluorescent properties of the two compounds were also studied in the solid state at room temperature.     

Influence of the Size of Aromatic Chelate Ligands on the Structures of Cadmium(II) Tetracarboxylates Polymers: From 2D Layered Network to 3D Metal‐Organic Framework

To determine the influence of the size of the aromatic chelate ligands on the frameworks of metal tretracarboxylate polymers, two new coordination polymers [Cd(btc)_0.5 (2,2′‐bpy)] ( 1 ) and [Cd(btc)_0.5(phen)]·H₂O ( 2 ) (H₄btc = biphenyl‐3,3′,4,4′‐tetracarboxylic acid, 2,2′‐bpy = 2,2′‐bipyridine, phen = 1,10‐phenanthroline) have been synthesized under similar hydrothermal conditions. In complex 1 , the dimeric Cd₂ units are linked by bridging btc^4? ligand to form a 2D layered network, whereas complex 2 possesses a 3D metal‐organic framework consisting of the dimeric Cd₂ units. The differences of two metal‐organic frameworks demonstrate that the size of the rigid aromatic chelate ligands have an important effect on the structures of their complexes. Additionally, the two complexes show strong fluorescence in the solid state at room temperature.     

Oligonucleotide Analogues with Integrated Bases and Backbones. Part 26

The self‐complementary aminomethylene‐linked A*[n] U* dinucleosides 23 – 26 were prepared by reductive coupling of aldehyde 10 and azide 8 . The U*[n] A* sequence isomers 19 – 21 were similarly prepared from aldehyde 14 and azide 3 . The substituents at C(6/I) of 23 – 26 and at C(8/I) of 19 – 21 strongly favour the syn‐conformation. The A*[n] U* dinucleoside 23 associates more strongly than the sequence‐isomeric U*[n] A* dinucleoside 19 . The A*[n] U* dinucleosides 23 and 24 associate more strongly than the analogues devoid of the substituent at C(6/I), while the U*[n] A* dinucleoside 19 associates less strongly than the analogue devoid of the substituent at C(8/I). While 23 and 24 form cyclic duplexes mostly by Watson–Crick‐type base pairing, 25 only forms linear associates. The U*[n] A* dinucleoside 19 forms mostly linear duplexes and higher associates, and 21 forms cyclic duplexes showing both Watson–Crick‐ and Hoogsteen‐type base pairing. The cyclic duplexes of the aminomethylene‐linked dinucleosides show both the gg‐ and gt‐orientation of the linker, with the gg‐orientation being preferred.     

Oligonucleotides Containing 7‐Deaza‐2′‐deoxyinosine as Universal Nucleoside: Synthesis of 7‐Halogenated and 7‐Alkynylated Derivatives,Ambiguous Base Pairing,and Dye Functionalization by the Alkyne–Azide ‘Click’ Reaction

Oligonucleotides containing 7‐deaza‐2′‐deoxyinosine derivatives bearing 7‐halogen substituents or 7‐alkynyl groups were prepared. For this, the phosphoramidites 2b – 2g containing 7‐substituted 7‐deaza‐2′‐deoxyinosine analogues 1b – 1g were synthesized (Scheme 2). Hybridization experiments with modified oligonucleotides demonstrate that all 2′‐deoxyinosine derivatives show ambiguous base pairing, as 2′‐deoxyinosine does. The duplex stability decreases in the order C_d>A_d>T_d>G_d when 2b – 2g pair with these canonical nucleosides (Table 6). The self‐complementary duplexes 5′‐d(F⁷c⁷I‐C)₆, d(Br⁷c⁷I‐C)₆, and d(I⁷c⁷I‐C)₆ are more stable than the parent duplex d(c⁷I‐C)₆ (Table 7). An oligonucleotide containing the octa‐1,7‐diyn‐1‐yl derivative 1g , i.e., 27 , was functionalized with the nonfluorescent 3‐azido‐7‐hydroxycoumarin ( 28 ) by the Huisgen–Sharpless–Meldal cycloaddition ‘click’ reaction to afford the highly fluorescent oligonucleotide conjugate 29 (Scheme 3). Consequently, oligonucleotides incorporating the derivative 1g bearing a terminal C?C bond show a number of favorable properties: i) it is possible to activate them by labeling with reporter molecules employing the ‘click’ chemistry. ii) Space demanding residues introduced in the 7‐position of the 7‐deazapurine base does not interfere with duplex structure and stability (Table 8). iii) The ambiguous pairing character of the nucleobase makes them universal probes for numerous applications in oligonucleotide chemistry, molecular biology, and nanobiotechnology.     

Effect of Metal Ions on the Structures of Coordination Polymers Based on Biphenyl‐2,2′,4,4′‐Tetracarboxylate

Two new coordination polymers, {[Cd₂(btc)(2,2′‐bpy)₂] · H₂O}_n ( 1 ) and [Zn₂(btc)(2,2′‐bpy)(H₂O)]_n ( 2 ) (H₄btc = biphenyl‐2,2′,4,4′‐tetracarboxylic acid, 2,2′‐bpy = 2,2′‐bipyridine), were synthesized hydrothermally under similar conditions and characterized by elemental analysis, IR spectra, TGA, and single‐crystal X‐ray diffraction analysis. In complexes 1 and 2 , the (btc)^4– ligand acts as connectors to link metal ions to give a 2D bilayer network of 1 and a 3D metal‐organic framework of 2 , respectively. The differences in the structures are induced by diverging coordination modes of the (btc)^4– ligand, which can be attributed to the difference metal ions in sizes. The results indicate that metal ions have significant effects on the formation and structures of the final complexes. Additionally, the fluorescent properties of the two complexes were also studied in the solid state at room temperature.     

Synthesis and characterization of di‐n‐butyltin(IV) complexes with 4′/2′‐nitrobiphenyl‐2‐carboxylic acids: X‐ray crystal structures of [{(n‐C4H9)2Sn(OCOC12H8NO2−4′)}2O]2 and (n‐C4H9)2Sn{OCOC12H8NO2−4′}2

Reactions of di‐n‐butyltin(IV) oxide with 4′/2′‐nitrobiphenyl‐2‐carboxylic acids in 1 : 1 and 1 : 2 stoichiometry yield complexes [{(n‐C₄H₉)₂Sn(OCOC₁₂H₈NO₂?4′/2′)}₂O]₂ ( 1 and 2 ) and (n‐C₄H₉)₂Sn(OCOC₁₂H₈NO₂?4′/2′)₂ ( 3 and 4 ) respectively. These compounds were characterized by elemental analysis, IR and NMR (¹H, ¹³C and ¹¹⁹Sn) spectroscopy. The IR spectra of these compounds indicate the presence of anisobidentate carboxylate groups and non‐linear C? Sn? C bonds. From the chemical shifts δ (¹¹⁹Sn) and the coupling constants ¹J(¹³C, ¹¹⁹Sn), the coordination number of the tin atom and the geometry of its coordination sphere have been suggested. [{(n‐C₄H₉)₂Sn(OCOC₁₂H₈NO₂?4′)}₂O]₂ ( 1 ) exhibits a dimeric structure containing distannoxane units with two types of tin atom with essentially identical geometry. To a first approximation, the tin atoms appear to be pentacoordinated with distorted trigonal bipyramidal geometry. However, each type of tin atom is further subjected to a sixth weaker interaction and may be described as having a capped trigonal bipyramidal structure. The diffraction study of the complex (n‐C₄H₉)₂Sn(OCOC₁₂H₈NO₂?4′)₂ ( 3 ) shows a six–coordinate tin in a distorted octahedral frame containing bidentate asymmetric chelating carboxylate groups, with the n‐Bu groups trans to each other. The n‐Bu? Sn? n‐Bu angle is 152.8° and the Sn? O distances are 2.108(4) and 2.493(5) Å. The oxygen atom of the nitro group of the ligand does not participate in bonding to the tin atom in 1 and 3 . Crystals of 1 are triclinic with space group P1 and of that of 3 have orthorhombic space group Pnna. Copyright © 2003 John Wiley & Sons, Ltd.