2′-Deoxy-β-D-ribofuranosides of N6-Methylated 7-Deazaadenine and 8-Aza-7-deazaadenine: Solid-phase synthesis of oligodeoxyribonucleotides and properties of self-complementary duplexes

The syntheses of 7-deaza-N⁶-methyladenine N⁹-(2′-deoxy-β-D -ribofuranoside) ( 2 ) as well as of 8-aza-7-deaza-N⁶-methyladenine N⁸? and N⁹?(2′-deoxyribofuranosides) ( 3 and 4 , resp.) are described. A 4,4′-dimeth-oxylritylation followed by phosphitylation yielded the methyl phosphoramidites 12–14 . They were employed together with the phosphoramidite of 2′-deoxy-N⁶v-methyladenosine ( 15 ) in automated solid-phase oligonucleotide synthesis. Alternating or palindromic oligonucleotides derived from d(A-T)₆ or d(A-T-G-C-A-G-A*-T-C-T-G-C-A) but containing one methylated pyrrolo[2,3-d]pyrimidine or pyrazolo[3,4-d]pyrimidine moiety in place of a N⁶-methylaminopurine (A*) were synthesized. Melting experiments showed that duplex destabilization induced by a N⁶-Me group of 2′-deoxy-N⁶-methyladenosine is reversed by incorporation of 8-aza-7-deaza-2′-deoxy-N⁶-meihyladenosine, whereas 7-deaza-2′-deoxy-N⁶-methyladenostne decreased the T_m value further. Regiospecific phosphodiester hydrolysis of d(A-T-G-C-A-G-m⁶A-T-C-T-G1-C-A) by the endodeoxyribonuclease Dpn I, yielding d(A-T-G-C-A-G-m⁶A) and d(pT-C-T-G-C-A), was prevented when the residue c⁷m⁶A_d ( 2 ), c⁷m⁶z⁸A_d ( 3 ), or c⁷m⁶z⁸A_d′ ( 4 ) replaced m⁶A_d ( 1 ) indicating that N(7) of N⁶-methyladenine is a proton-acceptor site for the endodeoxyribonuclease.     

A novel and facile reaction to N6‐alkylated adenosine via benzotriazole as a synthetic auxiliary

The reaction of benzotriazole with aliphatic, aromatic or heteroaromatic aldehyde and adenosine leads to a benzotriazole adduct which is reduced with sodium borohydride to the corresponding N⁶‐alkylated adenosine derivatives. This procedure is also utilized in a new route to N⁶‐(3‐iodobenzyl)adenosine‐5′‐N‐methyluronamide (IB‐MECA) which is considered an important adenosine agonist at A₃ adenosine receptors.     

A chemical labeling of N6-formyl adenosine（f6A） RNA

N⁶-methyl adenosine（m⁶A） is an eminent epigenetic mark in m RNAs that affects a broad range of biological functions in diverse species. However, the chemically inert methyl group prevents a direct labeling of this modification for subsequent detection and sequencing. Therefore, most current approaches for the labeling of m⁶A still have limitations of relying on the utilization of corresponding methyltransferases,which resulted in the lacking of efficiency. Here w...     

Nucleosides. Part LXI. A Simple Procedure for the Monomethylation of Protected and Unprotected Ribonucleosides in the 2′-O- and 3′-O-Position Using Diazomethane and the Catalyst Stannous Chloride

Intensive studies on the diazomethane methylation of the common ribonucleosides uridine, cytidine, adenosine, and guanosine and its derivatives were performed to obtain preferentially the 2′-O-methyl isomers. Methylation of 5′-O-(monomethoxytrityl)-N²-(4-nitrophenyl)ethoxycarbonyl-O⁶-[2-(4-nitrophenyl)ethyl]-guanosine ( 1 ) with diazomethane resulted in an almost quantitative yield of the 2′- and 3′-O-methyl isomers which could be separated by simple silica-gel flash chromatography (Scheme 1). Adenosine, cytidine, and uridine were methylated with diazomethane with and without protection of the 5′ -O-position by a mono- or dimethoxytrityl group and the aglycone moiety of adenosine and cytidine by the 2-(4-nitrophenyl)ethoxycarbonyl (npeoc) group (Schemes 2–4). Attempts to increase the formation of the 2′-O-methyl isomer as much as possible were based upon various solvents, temperatures, catalysts, and concentration of the catalysts during the methylation reaction.     

1‐(β‐d‐Erythrofuranosyl)adenosine

The title compound, also known as β‐erythroadenosine, C₉H₁₁N₅O₃, (I), a derivative of β‐adenosine, (II), that lacks the C5′ exocyclic hydroxymethyl (–CH₂OH) substituent, crystallizes from hot ethanol with two independent molecules having different conformations, denoted (IA) and (IB). In (IA), the furanose conformation is ^OT₁–E₁ (C1′‐exo, east), with pseudorotational parameters P and τ_m of 114.4 and 42°, respectively. In contrast, the P and τ_m values are 170.1 and 46°, respectively, in (IB), consistent with a ²E–²T₃ (C2′‐endo, south) conformation. The N‐glycoside conformation is syn (+sc) in (IA) and anti (−ac) in (IB). The crystal structure, determined to a resolution of 2.0 Å, of a cocrystal of (I) bound to the enzyme 5′‐fluorodeoxyadenosine synthase from Streptomyces cattleya shows the furanose ring in a near‐ideal ^OE (east) conformation (P = 90° and τ_m = 42°) and the base in an anti (−ac) conformation.     

Heteronuclear multiple magnetic resonance studies of quaternary ammonium salts derived from heterocyclic bases

¹⁴N tickling experiments performed with simultaneous decoupling of substituent protons are used to show that ²J(¹⁴N? H_ortho) and ³J(¹⁴N? H_meta) are both positive in the N-methyl pyridinium ion and related species. Long range coupling extending over as many as five bonds is observed between N-methyl protons and ring protons in ¹⁴N-decoupled spectra. Triple resonance decoupling is used to permit an analysis of the AA′MM′ spin system given by the ring proton of N-methyl pyrazinium iodide.     

Transfer ribonucleic acids

Transfer ribonucleic acids (tRNAs)

1 Abbreviations used according to IUPAC-IUB convention: tRNA = transfer ribonucleic acid; tRNA_yeast = mixture of tRNAs from yeast; tRNA^Phe = phenylalanine specific tRNA; Phe-tRNA = tRNA esterified (“charged”) with Phe; mRNA = messenger RNA; DNA = deoxyribonucleic acid; U = uridine; A = adenosine; C = cytidine; G = guanosine; pA = 5′-adenylic acid; Ap or A- = 3′-adenylic acid; m²′G = 2′-O-methyl guanosine; m⁷G = 7-methyl guanosine; mG = N(2)-dimethyl guanosine; other methylated nucleosides are abbreviated analogously; abbreviations of other odd nucleosides are given with Fig. 2; p or – signifies phosphate; RNase = ribonuclease; DEAE = diethylaminoethyl; fMet = N-formayl methionine.

occur in all living organisms. In biological protein synthesis they accept activated amino acids which are then transferred to growing peptide chains. With molecular weights lying between 25000 and 30000, tRNAs are easily within the reach of today's physical, chemical, and biochemical methods. The primary structures of several tRNAs as well as some relationships between structure and function have been elucidated. Three-dimensional structure, specificity, and mechanism of action are the subjects of present research efforts.     

N6-(Carbamoylmethyl)-2′-deoxyadenosine,a rare DNA Constituent: Phosphoramidite Synthesis and Properties of Palindromic Dodecanucleotides

N⁶-(Carbamoylmethyl)-2′-deoxyadenosine ( 1 ), a modified nucleoside occurring in bacteriophage Mu, was synthesized by two different routes. Glycinamide was introdued by nucleophilic displacement of(2,4,6,-triisopro-pylphenyl)sulfonyloxy or ethylsulfinyl groups at C(6) of the purine moiety. Compound 1 was converted into the protected phosphoramidite 6b and employed in solid-phase synthesis of the self-complementary oligonucleotides 7–14 . Replacement of 2′-deoxyadenosine by 1 led to a strong decrease of the T_m values of the oligomers d(A-T)₆ ( 7 ) and d(A-T-G-A-A-G-C-T-T-C-A-T)( 10 ), respectively. As the oligemer 10 contains the recognition site d(A-A-G-C-T-T) of the endodeoxyribonuclease Hind III, it was subjected to sequence-specific hydrolysis experiments. Replacement of the first or second A_d by 1 prevented enzymatic phosphodiester hydrolysis (results with 11 and 12 ). In contrast, slow hydrolysis was observed if the less bulky N⁶-methyl-2′-deoxyadenosine replaced the second A _d residue (results with 14 ).     

RNA Ligation for Mono and Dually Labeled RNAs

Labeled RNAs are invaluable probes for investigation of RNA function and localization. However, mRNA labeling remains challenging. Here, we developed an improved method for 3′-end labeling of in vitro transcribed RNAs. We synthesized novel adenosine 3′,5′-bisphosphate analogues modified at the N6 or C2 position of adenosine with an azide-containing linker, fluorescent label, or biotin and assessed these constructs as substrates for RNA labeling directly by T4 ligase or via postenzymatic strain-promoted alkyne-azide cycloaddition (SPAAC). All analogues were substrates for T4 RNA ligase. Analogues containing bulky fluorescent labels or biotin showed better overall labeling yields than postenzymatic SPAAC. We successfully labeled uncapped RNAs, NAD-capped RNAs, and 5′-fluorescently labeled m⁷Gp₃A_m-capped mRNAs. The obtained highly homogenous dually labeled mRNA was translationally active and enabled fluorescence-based monitoring of decapping. This method will facilitate the use of various functionalized mRNA-based probes.     

Synthesis of New Nucleosides by coupling of chloropurines with 2- and 3-deoxy derivatives of N-methyl-D-ribofuranuronamide

The synthesis of new deoxyribose nucleosides by coupling chloropurines with modified D -ribose derivatives is reported. The methyl 2-deoxy-N-methyl-3-O-(p-toluoyl)-α-D -ribofuranosiduronamide (α-D - 8 ) and the corresponding anomer β-D - 8 were synthesized starting from the commercially available 2-deoxy-D -ribose ( 1 ) (Scheme 1). Reaction of α-D - 8 with the silylated derivative of 2,6-dichloro-9H-purine ( 9 ) afforded regioselectively the N⁹-(2′-deoxyribonucleoside) 10 as anomeric mixture (Scheme 2), whereas β-D - 8 did not react. Glycosylation of 9 or of 6-chloro-9H-purine ( 17 ) with 1,2-di-O-acetyl-3-deoxy-N-methyl-β-D -ribofuranuronamide ( 13 ) yielded only the protected β-D -anomers 14 and 18 , respectively (Scheme 3). Subsequent deacetylation and dechlorination afforded the desired nucleosides β-D - 11 , β-D - 12,15 , and 16 . The 3′-deoxy-2-chloroadenosine derivative 15 showed the highest affinity and selectivity for adenotin binding site vs. A₁ and A_2A adenosine receptor subtypes.     

Novel chiral swallow-tailed amide liquid crystals possessing antiferroelectricity

Novel chiral swallow-tailed amide materials, N,N-dipropyl-(S)-2-{6-[4-(4-alkoxyphenyl)benzoyloxy]-2-naphthyl}propionamides, DPmPBNPA (m=9-13), have been designed and synthesized for the investigation of mesomorphic properties. The materials DPmPBNPA (m=9-11) display a monotropic phase sequence of I-SmA*-SmC_A*-Cr. The antiferroelectric SmC_A* phase for the materials was characterized by microscopic texture, switching behaviour, dielectric permitivity and electro-optical response. The measured maximum P _s values in the SmC_A* phase of the materials are in the range 80–87 nC cm^-2.     

1H‐Benzotriazole as Synthetic Auxiliary in a Facile Route to N6‐(Arylmethyl)‐2′‐deoxyadenosines: DNA Intercalators Inserted into Three‐Way Junctions

The 2′‐deoxy‐N⁶‐(naphthalen‐1‐ylmethyl)‐ ( 5a ) and N⁶‐(pyren‐1‐ylmethyl)adenosine ( 5b ) were synthesized in two steps from 2′‐deoxyadenosine and the adequate arenecarbaldehyde with 1H‐benzotriazole as a synthetic auxiliary (Scheme). When the N⁶‐(arylmethyl)‐2′‐deoxyadenosines were inserted into the junction region of a DNA three‐way junction, its thermal stability increased.     

Synthesis and conformational properties of 2′-deoxy-2′-methylthio-pyrimidine and -purine nucleosides: Potential antisense applications

A convenient and shorter synthesis of 2′-deoxy-2′-methylthiouridine analogs 5 , ?5-methyluridine 6 , -cyti-dine 15 , ?5-methylcytidine 16 , -adenosine 27 and -guanosine 34 was accomplished. Successful conversion of ribonucleosides (5-methyl U, U, A, G) into the corresponding 2′-substituted nucleosides involves nucleophilic displacement (S_N2) of an appropriate leaving group at the 2′-position by methanethiol, a soft nucleophile. Reaction between 2,2′-anhydrouridine and methanethiol in the presence of N¹,N¹,N³,N³-tetramethylguani-dine in N,N-dimethylformamide gave 5 , in 75% yield. Preparation of 6 by a similar route was described. Acylated 5 and 6 were transformed into their triazole derivatives, which on ammonolysis furnished 15 and 16 , respectively in good yield. Similarly, tetraisopropyldisiloxanyl (TIPS) protected 2′-O-aratriflates- of-adenosine and -guanosine reacted with methanethiol in the presence of 1,8-diazabicyclo[5.4.0]undec-7-ene at - 25°, followed by deblocking of the TIPS protecting group furnished 27 and 34 , respectively. The confor-mational flexibility (N/S equilibrium) of the sugar moiety in nucleosides 5 , 15 , 27 and 34 was studied utilizing nmr spectroscopy, suggesting that the 2′-methylthio group influenced the sugar conformation to adopt a rigid S-pucker in all cases. The extra stiffness of the sugar moiety in these analogs is believed to be due to the electronegativity of the substituent and the steric bulk. The usefulness of these nucleosides to prepare uniformly modified 2′-deoxy-2′-methylthio oligonucleotides for antisense therapeutics is proposed.     

A simple one pot synthesis of condensed 1,2,4‐triazines by using the tandem aN‐SNipso and SNH‐SNipsa reactions

A new synthetic approach to condensed 1,2,4‐triazines based on using the tandem A_N‐S_N^ipso and S_N^H‐S_N^ipso reactions has been developed. 5‐Methoxy‐3‐penyl‐1,2,4‐triazine and its N₁‐methyl quaternary salt were found to react with C,N‐, C,O‐ and N,N'‐bifunctional nucleophiles (m‐phenylenediamine, resor‐cinol, semicarbazide and ureas) into triazacarbazoles, benzofuro[2,3‐e][1,2,4]‐triazines, and 6‐azapurine derivatives. In all cases nucleophiles attack first the unsubstituted C‐6 carbon of the triazine ring, while the final stage is replacement of the methoxy group affording cyclization products.     

Dimerisation and complexation of 6-(4′-t-butylphenylamino)naphthalene-2-sulphonate by β-cyclodextrin and linked β-cyclodextrin dimers

This study shows that stereochemical factors largely determine the extent to which 6-(4′-t-butylphenylamino)-naphthalene-2-sulphonate, BNS^?  and its dimer, (BNS^? )₂, are complexed by β-cyclodextrin, βCD, and a range of linked βCD dimers. Fluorescence and ¹H NMR studies, respectively, show that BNS^?  and (BNS^? )₂ form host–guest complexes with βCD of the stoichiometry βCD.BNS^?  (10^? 4 K ₁ = 4.67 dm³ mol^? 1) and βCD.BNS₂ ^2 ?  (10^? 2 K ₂′ = 2.31 dm³ mol^? 1), where the complexation constant K ₁ = [βCD.BNS^? ]/([βCD][BNS^? ]) and K ₂′ = [βCD. (BNS^? )₂]/([βCD.BNS^? ][BNS^? ]) in aqueous phosphate buffer at pH 7.0, I = 0.10 mol dm³ at 298.2 K. (The dimerisation of BNS^?  is characterised by 10^? 2 K _d = 2.65 dm³ mol^? 1.) For N,N-bis((2^AS,3^AS)-3^A-deoxy-3^A-β-cyclodextrin)succinamide, 33βCD₂su, N-((2^AS,3^AS)-3^A-deoxy-3^A-β-cyclodextrin)-N′-(6^A-deoxy-6^A-β-cyclodextrin)urea, 36βCD₂su, N,N-bis(6^A-deoxy-6^A-β-cyclodextrin)succinamide, 66βCD₂su, N-((2^AS,3^AS)-3^A-deoxy-3^A-β-cyclodextrin)-N′-(6^A-deoxy-6^A-β-cyclodextrin)urea, 36βCD₂ur, and N,N-bis(6^A-deoxy-6^A-β-cyclodextrin)urea, 66βCD₂ur, the analogous 10^? 4 K ₁ = 11.0, 101, 330, 29.6 and 435 dm³ mol^? 1 and 10^? 2 K ₂′ = 2.56, 2.31, 2.59, 1.82 and 1.72 dm³ mol^? 1, respectively. A similar variation occurs in K ₁ derived by UV–vis methods. The factors causing the variations in K ₁ and K ₂ are discussed in conjunction with ¹H ROESY NMR and molecular modelling studies.     

Stereoselective Synthesis of Indazole 2′-Deoxy-β-D-ribonucleosides: Glycosylation of the Nucleobase Anion

The glycosylation of indazolyl anions derived from 4a , b with 2-deoxy-3,5-bis-O-(4-methylbenzoyl)-α-D -erythro-pentofuranosyl chloride ( 5 ) is described. The reaction was Stereoselective – exclusive β-D -anomer formation – but regioisomeric N¹- and N²-(2′-deoxy-β-D -ribofuranosides) (i.e. 6a and 7a , resp., and 6b and 7b , resp.) were formed in about equal amounts. They were deprotected to yield 8a , b and 9a , b . Compound 1 , related to 2′-deoxyadenosine ( 3 ), and its regioisomer 2 were obtained from 8b and 9b , respectively, by catalytic hydrogenation. The anomeric configuration as well as the position of glycosylation were determined by 1D NOE-difference spectroscopy. The first protonation site of 1 and 2 was found to be the NH₂ group. The N-glycosylic bond of 1H-indazole N¹-(2′-deoxyribofuranosides) is more stable than that of the parent purine nucleosides. Compound 1 is no substrate for adenosine deaminase.     

Novel drug‐based Fe(III) heterochelates: synthetic,spectroscopic, thermal and in‐vitro antibacterial significance

A series of novel heterochelates of the type [Fe(Aⁿ)(L)(H₂O)₂]^?mH₂O [where H₂Aⁿ = 4,4′‐(arylmethylene)bis(3‐methyl‐1‐phenyl‐4,5‐dihydro‐1H‐pyrazol‐5‐ol); aryl = 4‐nitrophenyl, m = 1 (H₂A¹); 4‐chlorophenyl, m = 2 (H₂A²); phenyl, m = 2 (H₂A³); 4‐hydroxyphenyl, m = 2 (H₂A⁴); 4‐methoxyphenyl, m = 2 (H₂A⁵); 4‐hydroxy‐3‐methoxyphenyl, m = 1.5 (H₂A⁶); 2‐nitrophenyl, m = 1.5 (H₂A⁷); 3‐nitrophenyl, m = 0.5 (H₂A⁸); p‐tolyl, m = 1 (H₂A⁹) and HL = 1‐cyclopropyl‐6‐fluoro‐4‐oxo‐7‐(piperazin‐1‐yl)‐1,4‐dihydroquinoline‐3‐carboxylic acid] were investigated. They were characterized by elemental analysis (FT‐IR, ¹H‐ & ¹³C‐NMR, and electronic) spectra, magnetic measurements and thermal studies. The FAB‐mass spectrum of [Fe(A³)(L)(H₂O)₂]^?2H₂O was determined. Magnetic moment and reflectance spectral studies revealed that an octahedral geometry could be assigned to all the prepared heterochelates. Ligands (H₂Aⁿ) and their heterochelates were screened for their in‐vitro antibacterial activity against Bacillus subtilis, Staphylococcus aureus, Escherichia coli and Serratia marcescens bacterial strains. The kinetic parameters such as order of reaction (n), the energy of activation (E_a), the pre‐exponential factor (A), the activation entropy (ΔS^#), the activation enthalpy (ΔH^#) and the free energy of activation (ΔG^#) are reported. Copyright © 2009 John Wiley & Sons, Ltd.     

Synthesis and characterization of polyimides based on novel tricyclo[6,2,2,02,7] dianhydride

3-Phenyl-tricyclo [6,2,2,0^2,7]dodeca-2,11-ene-5,6,9,10-tetracarboxylic dianhydride was prepared from 1,1-diphenyl ethylene and maleic anhydride in 1 : 2 mole ratio by [4 + 2]π Diels-Alder cycloaddition. The structure of the dianhydride was determined by mass spectroscopy, IR, ¹H-NMR, elemental analyses, and single crystal x-ray diffraction. The monomer was condensed with several diamines in N-methyl pyrrolidone or m-cresol. The polyamic acids and the polyimides synthesized had inherent viscosities in the range of 0.19–0.31 and 0.17–0.25 dL/g, respectively, measured in N-methyl pyrrolidone at 30°C. Both the polyamic acids and the polyimides were found to be soluble in m-cresol, N-methyl pyrrolidone, dimethylacetamide, dimethyl formamide, and dimethyl sulfoxide. The polymides showed a low degree of crystallinity from wide angle x-ray diffraction. Thermal analysis of these polyimides revealed that their glass transition temperatures (T_g) were in the 215–237°C range and they decomposed in two stages. The first-stage decomposition temperatures were almost the same in O₂ or N₂ atmospheres, but the polymers showed a better thermal stability in O₂ rather than in N₂ in the second stage. The mechanism of thermal degradation is discussed.     

8-Aza-7-deazaadenine N8- and N8-(β-D-2′-Deoxyribofuranosides): Building blocks for automated DNA synthesis and properties of oligodeoxyribonucleotides

Oligonucleotides with alternating 8-aza-7-deaza-2′-deoxyadenosine (= c⁷z⁸A_d2) and dT residues (see 11, 14 and 16 ) or 4-aminopyrazolo [3,4-d] pyrimidine N²-(β-D -2′-deoxyribofuranoside) (= c⁷z⁸A′_d¹); ( 3 ) and dT residues (see 12 ) have been prepared by solid-phase synthesis using P(III) chemistry, Additionally, palindromic oligomers derived from d(C-T-G-G-A-T-C-C-A-G) but containing 2 or 3 instead of dA (see 18 – 22 ) have been synthesized. Benzoylation of 2 or 3 , followed by 4,4′-dimethoxytritylation and subsequent phosphitylation yielded the methyl or the cyanoethyl phosphoramidites 8a,b and 9 . They were employed in automated. DNA synthesis. Alternating oligomers containing 2 or 3 showed increase dT_m values compared to those with dA, in particular 12 with an unusual N²-glycosylic bond. The palindromic oligomers 18 - 22 containing 2 or 3 instead of dA outside of the enzymic recognition side reduced the hydrolysis rate, replacement within d(G-A-T-C) abolished phosphodiester hydrolysis.