Nucleotides. Part LXXII

The synthesis of various N‐methylated nucleosides (m⁶A, m³C, m⁴C, m³U) is described. These minor nucleosides can be obtained by simple methylation with diazomethane of [2‐(4‐nitrophenyl)ethoxy]carbonyl(npeoc)‐protected nucleosides. These methylated compounds are easily further derivatized to fit into the scheme of the [2‐(dansyl)ethoxy]carbonyl (dnseoc) approach for RNA synthesis (dansyl=[5‐(dimethylamino)naphthalen‐1‐yl]sulfonyl). Various oligoribonucleotides containing N⁶‐methyladenosine were synthesized, underlining the usefulness of the dnseoc approach, especially for the synthesis of natural tRNA‐derived oligoribonucleotide sequences.     

Direction of glycosylation of 5-substituted 4-chloro-1,2,3-triazoles

The structures of previously obtained nucleosides of 5-substituted 4-chloro-1, 2,3-triazoles were refined by means of high-resolution mass spectrometry and ¹³C NMR spectroscopy. It is shown that fusion of 5-substituted 4-chloro,1,2,3-triazoles with tetra-0-acylribofuranoses in the presence of di(p-nitrophenyl) phosphate leads to the formation of 2-nucleosides of the corresponding triazoles. The signals of the carbon atoms in the ¹³C NMR spectra of the 4,5-di-substituted triazoles and their nucleosides were assigned.Translated from Khimiya Geterotsiklicheskikh Soedinenii, No. 7, pp. 937–940, July, 1987.     

Pyrrolo-dC oligonucleotides bearing alkynyl side chains with terminal triple bonds: synthesis, base pairing and fluorescent dye conjugates prepared by the azide-alkyne "click" reaction

5-(Octa-1,7-diynyl)-2'-deoxyuridine was converted into the furano-dU derivative 7 by copper-catalyzed cyclization; the pyrolodC-derivative 3 was formed upon ammonolysis. The bicyclic nucleosides 3 and 7 as well as the corresponding non-cyclic precursors 4 and 6 all containing terminal C[triple bond]C bonds were conjugated with the non-fluorescent 3-azido-7-hydroxycoumarin 5 employing the copper(I)-catalyzed Huisgen-Sharpless-Meldal cycloaddition "click reaction". Strongly fluorescent 1H-1,2,3-triazole conjugates (30-33) are formed incorporating two fluorescent reporters-the pyrdC nucleoside and the coumarin moiety. Oligonucleotides incorporating 6-alkynyl and 6-alkyl 7H-pyrrolo[2,3-d]pyrimidin-2(3H)-one nucleosides (3 and 2f) have been prepared by solid-phase synthesis using the phosphoramidite building blocks 10 and 13 ; the pyrrolo-dC oligonucleotides are formed during ammonia treatment. The duplex stability of oligonucleotides containing 3 and related derivatives was studied. Oligonucleotides with terminal triple bonded nucleosides such as 3 are more stabilizing than those lacking a side chain with terminal unsaturation; open-chain derivatives (4) are even more efficient. The click reaction was also performed on oligonucleotides containing the pyrdC-derivative and the fluorescence properties of nucleosides, oligonucleotides and their coumarin conjugates were studied.     

Three-bond sugar-base couplings in purine versus pyrimidine nucleosides: a DFT study of Karplus relationships for (3)J(C2/4-H1') and (3)J(C6/8-H1') in DNA

(3)J(C2/4-H1') and (3)J(C6/8-H1') scalar spin-spin coupling constants have been calculated for deoxyadenosine, deoxyguanosine, deoxycytidine, and deoxythymidine as functions of the glycosidic torsion angle chi by means of density functional theory. Except for deoxythymidine, (3)J(C2/4-H1') depends little on the base type. On the contrary, (3)J(C6/8-H1') follows the usual trans to cis ratio ((3)J(C-H(cis)) < (3)J(C-H(trans))) for purine nucleosides, but reveals the opposite relation ((3)J(C-H(cis)) > (3)J(C-H(trans))) for pyrimidine nucleosides. Our results compare well with the experiment for deoxyguanosine and predict a novel trend in the case of pyrimidine bases for which no NMR results are available in the syn region. A breakdown of the key Fermi contact part of (3)J(C6/8-H1') into MO contributions rationalizes this trend in terms of an unusual coupling mechanism in the syn orientation that is very effective for pyrimidine nucleosides and considerably weaker for purine nucleosides.     

C8-alkynyl- and alkylamino substituted 2′-deoxyguanosines: a universal linker for nucleic acids modification

Incorporation of modified nucleosides with a flexible universal linker is of great value for post-synthetic modification of nucleic acids. Thus, C8-alkynyl- and alkylamino substituted 2′-deoxyguanosines were synthesized for the first time and incorporated into short oligonucleotide sequences. The preference for syn conformation of these C8-substituted 2′-deoxyguanosines and the stability of the duplexes were discussed. The stabilizing effect of Z-DNA has also been examined.     

Oligonucleotides Containing Disaccharide Nucleosides

Disaccharide nucleosides with 2′‐O‐(D ‐arabinofuranosyl), 2′‐O‐(L ‐arabinofuranosyl), 2′‐O‐(D ‐ribopyranosyl), 2′‐O‐(D ‐erythrofuranosyl), and 2′‐O‐(5‐azido‐5‐deoxy‐D ‐ribofuranosyl) substituents were synthesized. These modified nucleosides were incorporated into oligonucleotides (see Table). Single substitution resulted in a ΔT_m of +0.5 to −1.4° for DNA/RNA and a ΔT_m of −0.8 to −4.7° for DNA/DNA duplexes. These disaccharide nucleosides can be well accommodated in RNA/DNA duplexes, and the presence of a NH₂−C(5″) group has a beneficial effect on duplex stability.     

A short and novel synthesis of carbocyclic nucleosides and 4′-epi-carbocyclic nucleosides from 2-cyclopenten-1-ones

Carbocyclic nucleoside analogues remain interesting target molecules having the potential to combine biological activity with greater metabolic stability than their sugar counterparts. This paper describes a rapid and versatile synthetic approach to such compounds based on commercial cyclopentenones (e.g., 1) that has been developed in our laboratory. Carbocyclic nucleosides like 2′-methyl-aristeromycin 6 were synthesized in racemic form in 5 steps via key intermediate 4. The procedure was also adapted to the preparation of 4′-epi-carbocyclic nucleosides using epoxide 17 instead of 4 and employing the same methodology.     

Synthesis and NMR studies of some imidazo[4,5-d]pyridazine nucleosides

Unlike imidazo[4,5-d]pyridazin-4(5H)-one ( 1a ), which undergoes ribosylation at N-6 in the Vorbruggen procedure for nucleoside synthesis, the 5-benzyloxymethyl derivative 12 undergoes ribosylation at N-1 and N-3 to give a separable mixture of 14 and 15 . Removal of the N-5 blocking groups from 14 and 15 by treatment with boron trichloride at −78° affords the intermediates 16 and 17 , which were debenzoylated to give the 4-oxo nucleosides 5 and 6 . Thiation of 16 and 17 , followed by S-methylation and ammonolysis leads to the 4-amino nucleosides 2 and 3 . The glycosylation sites of these nucleosides were assigned by using a combination of ¹H and ¹³C nmr data, especially measurements of the spin-lattice relaxation times (T₁) of the base protons. Using these techniques, it is shown that a nucleoside previously reported to be 3 is in fact the N-6 isomer.     

Kinetics and mechanism of the defluorination of 8-fluoropurine nucleosides in basic and acidic media

For investigating the stability of C(8)-fluorine bond in 8-fluoropurine nucleosides some protected 8-fluoroguanosine, 8-fluoroinosine and 8-fluoroadenosine derivatives were prepared by direct fluorination of acetyl-protected purine nucleosides with elemental fluorine in solvents such as chloroform, acetonitrile and nitromethane. Fluorination reactions conducted in chloroform medium gave better yields of 8-fluoropurines. The fluorination yields were slightly lower when acetonitrile or nitromethane was used as solvent, but the product purification was found to be much easier. When the synthesized, protected fluoronucleosides were subjected to standard basic (NH₃ in methanol or 2-propanol) and acidic (HCl in methanol) deprotection conditions relevant to nucleoside chemistry, an efficient defluorination reaction took place. The kinetics of these defluorination reactions were conveniently followed, under pseudo-first-order reaction conditions, using ¹⁹F NMR spectroscopy. ¹H NMR, LC-MS and mass spectroscopy identified the products of the kinetic reaction mixtures. The defluorination reaction rate constants (k_obs) in basic media depended upon the electron density at C(8) while the k_obs data in acidic medium were determined by the pK_a of N⁷. An addition-elimination based mechanism (S_NAr) has been proposed for the defluorination reactions of these 8-fluoropurine nucleosides.     

Structural Biochemistry 14. Permethylation of Nucleosides

Prominent molecular ions are generally observed in the field ionization (FI) mass spectra of unprotected nucleosides.² In the one exception so far observed, that of guanosine, we found the simple methyl derivative, N²,N²-dimethylguanosine, to afford an easily detectible molecular ion. The electron impact (EI) mass spectrum of N²,N²-dimethylguanosine also displayed a molecular ion and suggested that nucleoside methyl derivatives might be more easily studied by EI methods. For this purpose and the more important objective of developing methods for sequencing small nucleic acid units by computer assisted³ FI-EI mass spectrometry, a program was initiated (1967) to explore permethylation of nucleosides. We believe protection by permethylation to be superior to pertrimethylsilylation and acetylation principally for reasons involving molecular weight and stability. Concurrently it was anticipated that such nucleoside methylation studies would afford routes to partially mathylated nucleosides of value in characterizing such components of virus and cellular DNA and RNA⁴. Subsequently, using variations of the methanol-DCCI,⁵ diazomethane with various Lewis acids,⁶ methyl iodide in, for example, dimethylsulfoxide,⁷ methyl iodide-sodium hydride in dimethylformamide,⁸ methyl iodide-silver oxides⁹ and methyl iodidemethylsulfinyl carbanion in dimethylsulfoxide,¹⁰ techniques were evaluated but none was found to provide permethyl nucleosides in high yield.¹¹ The latter two methods have, however, been applied to methylating nucleosides for EI mass spectral investigations.¹²     

The High-Anti Conformation of 7-Halogenated 8-Aza-7-deaza-2′-deoxy-guanosines: A Study of the Influence of Modified Bases on the Sugar Structure of Nucleosides

The conformation of the 7-bromo- and 7-iodo-substituted 8-aza-7-deazapurine nucleosides 1 and 2 in the solid state and in aqueous solution was studied by single-crystal X-ray analyses and by ¹H-NMR spectroscopy. In the solid state, both compounds display a high-anti conformation around the glycosylic bond, and their 2′-deoxy-β-D -ribofuranose moieties adopt an N-type sugar puckering. The orientation of the exocyclic C(4′)−C(5′) bond was found to be ap in both cases. In D₂O solution, both compounds display i) an 8 – 10% higher N-conformer population than 2′-deoxyguanosine and ii) a preference of the −sc conformation about the C(4′)−C(5′) bond. A comparative study on the influence of modified bases on the sugar structure of nucleosides is made.     

Design and Synthesis of Some New α‐Phenyl Cinnamoyl Derivatives for Selective Protection of Purine Nucleosides

Three new α‐phenylcinnamic acid derivatives [4‐methoxy‐α‐phenylcinnamic acid, α‐(4‐methoxyphenyl)‐cinnamic acid, and 4,4′‐bismethoxy‐α‐phenylcinnamic acid] were synthesized, characterized, and selectively used for protecting the exocyclic amino function of purine nucleosides (2′‐deoxyadenosine and 2′‐deoxyguanosine) via active ester generation. The acids were first activated using p‐nitrophenol, and these activated esters were used subsequently for the selective protection of amino groups. The N‐protected derivatives of 2′‐deoxyguanosine and 2′‐deoxyadenosine have been found to be sufficiently stable toward acids, thus minimizing depurination under oligodeoxyribonucleotide synthesis protocol. The ease of syntheses of N‐protected purine nucleosides, their stability under an acidic environment, and mild deprotection conditions are the key advantages of the new protecting groups.     

New base pairing motifs. The synthesis and thermal stability of oligodeoxynucleotides containing imidazopyridopyrimidine nucleosides with the ability to form four hydrogen bonds

The synthesis and thermal stability of oligodeoxynucleotides (ODNs) containing imidazo[5',4':4,5]pyrido[2,3-d]pyrimidine nucleosides 1-4 (N(N), O(O), N(O), and O(N), respectively) with the aim of developing two sets of new base pairing motifs consisting of four hydrogen bonds (H-bonds) is described. The proposed four tricyclic nucleosides 1-4 were synthesized through the Stille coupling reaction of a 5-iodoimidazole nucleoside with an appropriate 5-stannylpyrimidine derivative, followed by an intramolecular cyclization. These nucleosides were incorporated into ODNs to investigate the H-bonding ability. When one molecule of the tricyclic nucleosides was incorporated into the center of each ODN (ODN I and II, each 17mer), no apparent specificity of base pairing was observed, and all duplexes were less stable than the duplexes containing natural G:C and A:T pairs. On the other hand, when three molecules of the tricyclic nucleosides were consecutively incorporated into the center of each ODN (ODN III and IV, each 17mer), thermal and thermodynamic stabilization of the duplexes due to the specific base pairings was observed. The melting temperature (T(m)) of the duplex containing the N(O):O(N) pairs showed the highest T(m) of 84.0 degrees C, which was 18.2 and 23.5 degrees C higher than that of the duplexes containing G:C and A:T pairs, respectively. This result implies that N(O)and O(N) form base pairs with four H-bonds when they are incorporated into ODNs. The duplex containing N(O):O(N) pairs was markedly stabilized by the assistance of the stacking ability of the imidazopyridopyrimidine bases. Thus, we developed a thermally stable new base pairing motif, which should be useful for the stabilization and regulation of a variety of DNA structures.     

Synthesis of certain 6-alkylthio-2,2′-anhydro-5-azauridines

β-D-Arabinofurano[1′,2′:4,5]oxazolo-s-triazin-4-one-6-thione ( 7b ) and its t-butyldimethylsilyl protected counterpart 7a were synthesized by treating the appropriate 2-amino-β-D-arabinofurano[1′,2′:4,5]-2-oxazoline with ethoxycarbonyl isothiocyanate. These 2,2′-anhydro-s-triazine nucleosides were then subjected to alkylation under similar reaction conditions. Alkylation of 3′,5′-bis(O-t-butyldimethylsilyl)-β-D-arabinofurano[1′,2′:-4,5]oxazolo-s-triazin-4-one-6-thione ( 7a ) provided the targeted S-alkylated nucleosides, i.e., the C6-SCH₃ ( 9a ), C6-SCH₂-CH = CH₂ ( 10a ), and C6-S-CH₂-C = CH ( 11a ), in reasonable yields. Attempted deprotection of these nucleosides failed. In order to circumvent this problem, 7b was alkylated with the same reagents. In each case, instead of the expected S-alkylated anhydronucleosides, a mixture of the 5-N-alkylanhydro-s-triazine-4,6-dione and 5-N-alkylanhydro-s-triazin-4-one-6-thione derivatives were obtained. The 2,2′-anhydro linkage of 7a was also found to be more stable than the s-triazine ring to mild base. Basic conditions displaced the C6-sulfur substituent and eventually caused ring opening of the s-triazine aglycone.     

Studies on imidazole derivatives and related compounds. 3. Regioselective glycosylation of 4-carbamoylimidazolium-5-olate

In the synthesis of glycosyl derivatives of 4-carbamoylimidazolium-5-olate ( 2 ) by the silyl-Hilbert-Johnson method using trimethylsilyl trifluoromethanesulfonate as catalyst, we obtained N-3 nucleosides 5 as major products and N-1,N-bis-nucleosides 6 as minor ones. The desired N-1 nucleosides 4 were isolated in only low yields. However, the yields of 4 were improved by adding ca. One equivalent of stannic chloride to the silylated 4-carbamoylimidazolium-5-olate ( 3 ). On the basis of nuclear magnetic resonance (¹³C and ²⁹Si) and ultraviolet spectroscopic studies, we verify the formation of σ-complexes between the silylated base 3 and the Lewis acid (stannic chloride or trimethylsilyl trifluoromethanesulfonate), and the propose the structures of these complexes and the reaction mechanism.     

Controlling the transmembrane transport of nucleosides

Nucleoside analogues are used as drugs. Due to their hydrophilicity, nucleosides are poorly permeable to membranes and transporter proteins are required for efficient uptake. One approach towards improving membrane permeability of nucleosides is to use synthetic transporters. We describe ways to control transport of nucleosides across a liquid membrane. Hexanoylguanosine 1 selectively extracts and transports cytidines across a CHCl₃ membrane. Transport catalysed by G 1 was influenced by the nucleoside's sugar, with a selectivity of dC 4 > rC 3 > araC 5. Selective transport could be modulated by adding compounds to the aqueous source phase or to the organic phase. Addition of K⁺2,6-DNP^–8 to CHCl₃ containing G 1 switched off transport of rC 3 and dC 4 due to formation of a G-quartet assembly. A lipophilic G 1·C 2 base pair could not transport dC 4, but did catalyse transport of dG 7 across the CHCl₃ barrier. We propose that transport occurs because of formation of a base triple G 1·C 2·dG 7. Addition of Na₂B₄O₇9 to a source phase containing rC 3, dC 4 and araC 5 shuts down transport of rC 3 by G 1, due to formation of borate esters. These results indicate that one can control the selective transport of nucleosides.     

肠癌患者尿中核苷排放的高效液相法研究

 用反相高效液相法测定尿中核苷。通过苯基硼酸亲和法提取尿中核苷,在柱(4 6mmi d ×250mm,5μm)上以25mmol/L磷酸二氢钾溶液(pH4 55)和60%的甲醇水溶液作为流动相进行二元梯度淋洗,于22℃下进行反相分离,260nm处紫外检测。用该法测定了41例肠癌患者和52例正常人尿中15种核苷的含量(用核苷与肌酐的摩尔比表示,下同),结果表明肠癌患者中有12种核苷的含量比正常人显著性增高(P<0 001)。以15种核苷的含量作为参量,结合主成分分析区分正常人和肠癌患者,对癌症病人的识别率达76%(31/41)。     

Alkylating nucleosides. 3. The reaction of 3,5-dimethylpyrazole nucleosides with N-bromosuccinimide

Bromination of 3,5-dimethylpyrazole nucleosides with N-bromosuccinimide gave the corresponding 4-bromo-3,5-dimethylpyrazole, 3-methyl-5-(bromomethyl)pyrazole and 4-bromo-3-methyl-5-(bromomethyl)pyrazole nucleosides. Structural assignments were made on basis of analytical and ¹ H nmr spectral data. All of the bromomethylpyrazole nucleosides described showed cytostatic activity against HeLa cell sultures.     

Design,Synthesis, and Characterization of Photolabeling Probes for the Study of the Mechanisms of the Antiviral Effects of Ribavirin

Ribavirin, the only small molecule available so far for treating hepatitis‐C‐virus infection, was recently used in an emergency context to treat patients with severe acute respiratory syndrome (SARS) in the early stages of the disease. To study the mechanisms responsible for the antiviral effects of ribavirin by using a photolabeling approach, we designed, synthesized, and characterized the azidotriazole nucleosides 1 and 2 as photolabeling probes of ribavirin. These probes were synthesized either by performing nucleophilic substitution of the corresponding bromotriazole nucleoside with NaN₃ (Scheme 2) or by directly coupling the azidotriazole with the protected ribose sugar (Scheme 4). The azidotriazole nucleosides 1 and 2 showed a fast, clear‐cut photochemical reaction, which suggests that they are promising candidates for use in photolabeling studies.     

Recyclable Ruthenium Catalyst for Distal meta-C−H Activation

We disclose the unprecedented hybrid-ruthenium catalysis for distal meta-C−H activation. The hybrid-ruthenium catalyst was recyclable, as was proven by various heterogeneity tests, and fully characterized with various microscopic and spectroscopic techniques, highlighting the physical and chemical stability. Thereby, the hybrid-ruthenium catalysis proved broadly applicable for meta-C−H alkylations of among others purine-based nucleosides and natural product conjugates. Additionally, its versatility was further reflected by meta-C−H activations through visible-light irradiation, as well as para-selective C−H activations.