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.     

Major and modified nucleosides in tRNA hydrolysates by high-performance liquid chromatography.

We describe a high-performance liquid chromatographic analytical method that can be readily placed in operation, and which is particularly well suited to scientists investigating tRNA structure, biosynthesis, and function, and for the determination of major and modified nucleosides of tRNA. The method is characterized by the following features: (1) Sensitivity at the nanogram level; (2) High chromatographic resolution and selectivity; (3) Direct measurement of nucleosides with accuracy and precision; (4) Analysis is non-destructive and the high capacity of this chromatographic system allows easy isolation of pure nucleosides for further characterization; (5) Rapid separation and measurement in ca. 1 h after hydrolysis to nucleosides; and (6) Quantitation without use of radiolabeled compounds; however, labeled compounds are readily isolated and measured.     

Synthesis of 2'-O-[2-[(N,N-dimethylamino)oxy]ethyl] modified nucleosides and oligonucleotides.

A versatile synthetic route has been developed for the synthesis of 2'-O-[2-[(N,N-dimethylamino)oxy]ethyl] (abbreviated as 2'-O-DMAOE) modified purine and pyrimidine nucleosides and their corresponding nucleoside phosphoramidites and solid supports. To synthesize 2'-O-DMAOE purine nucleosides, the key intermediate B (Scheme 1) was obtained from the 2'-O-allyl purine nucleosides (13a and 15) via oxidative cleavage of the carbon-carbon bond to the corresponding aldehydes followed by reduction. To synthesize pyrimidine nucleosides, opening the 2,2'-anhydro-5-methyluridine 5 with the borate ester of ethylene glycol gave the key intermediate B. The 2'-O-(2-hydroxyethyl) nucleosides were converted, in excellent yield, by a regioselective Mitsunobu reaction, to the corresponding 2'-O-[2-[(1,3-dihydro-1,3-dioxo-2H-isoindol-2-yl)oxy]ethyl] nucleosides (18, 19, and 20). These compounds were subsequently deprotected and converted into the 2'-O-[2-[(methyleneamino)oxy]ethyl] derivatives (22, 23, and 24). Reduction and a second reductive amination with formaldehyde yielded the corresponding 2'-O-[2-[(N,N-dimethylamino)oxy]ethyl] nucleosides (25, 26, and 27). These nucleosides were converted to their 3'-O-phosphoramidites and controlled-pore glass solid supports in excellent overall yield. Using these monomers, modified oligonucleotides containing pyrimidine and purine bases were synthesized with phosphodiester, phosphorothioate, and both linkages (phosphorothioate and phosphodiester) present in the same oligonucleotide as a chimera in high yields. The oligonucleotides were characterized by HPLC, capillary gel electrophoresis, and ESMS. The effect of this modification on the affinity of the oligonucleotides for complementary RNA and on nuclease stability was evaluated. The 2'-O-DMAOE modification enhanced the binding affinity of the oligonucleotides for the complementary RNA (and not for DNA). The modified oligonucleotides that possessed the phosphodiester backbone demonstrated excellent resistance to nuclease with t(1/2) > 24 h.     

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.     

Nucleotides. Part XLII. The 2-dansylethoxycarbonyl (= 2-{[5-(dimethylamino)naphthalen-1-yl]sulfonyl}ethoxycarbonyl; dnseoc) group for protection of the 5′-hydroxy function in oligoribonucleotide synthesis

The 2-dansylethoxycarbonyl (Dnseoc) group was employed for protection of the 5′-hydroxy function in oligoribonucleotide synthesis by the phosphoramidite approach using the acid-labile tetrahydro-4-methoxy-2H-pyran-4-yl (Thmp) group for 2′-protection. The syntheses of monomeric building blocks, both phosphoramidites and nucleoside-functionalized supports, are described for the four common nucleosides adenosine, guanosine, cytidine, and uridine, and for the two modified minor nucleosides ribothymidine (= ribosylthymine) and pseudouridine.     

Analysis of urinary nucleosides. V. Identification of urinary pyrimidine nucleosides by liquid chromatography/electrospray mass spectrometry

Modified urinary nucleosides are potentially invaluable in cancer diagnosis, as they reflect altered RNA turnovers. High-performance liquid chromatography (HPLC) was combined with full-scan mass spectrometry, tandem mass spectrometry, MS(n) analysis and accurate mass measurements in order to identify pyrimidine nucleosides purified from urine. Potential nucleosides were assessed by their evident UV absorbance in the HPLC chromatogram and then further examined by the various mass spectrometric techniques. In this manner numerous pyrimidine nucleosides were identified in the urine samples from cancer patients including pseudouridine, cytidine, two methylcytidines and an acetylcytidine. Furthermore, a number of novel modified pyrimidine nucleosides were tentatively identified via critical interpretation of the combined mass spectrometric data.     

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.     

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.     

Solid-phase synthesis of dinucleoside and nucleoside-carbohydrate phosphodiesters and thiophosphodiesters

Unprotected nucleosides (ROH) were reacted with two polymers bound to N,N-diisopropylamino-1,3,2-oxathiaphospholane in the presence of 1H-terazole. Oxidation with tert-butyl hydroperoxide or sulfurization with Beaucage's reagent, followed by the 1,3,2-oxathiaphospholane ring opening with unprotected nucleosides or carbohydrates (R'OH) in the presence of DBU, afforded nucleoside-(5'-5')-nucleoside or nucleoside-carbohydrate phosphodiester and thiophosphodiester derivatives through the elimination of polymer-bound ethylene episulfide. This strategy offers the advantages of facile isolation of final products and monosubstitution of unprotected nucleosides and carbohydrates.     

Polarographic properties and potential carcinogenicity of some natural nucleosides and their synthetic analogues

The polarographic reduction and the index of potential carcinogenicity tg alpha determined polarographically in aprotic conditions and in the presence of alpha-lipoic acid of nine naturally occurring and synthetic pyrimidine and six synthetic 1,3,5-triazine (5-aza) nucleosides was compared to the reduction of eight synthetic 1,3,6-triazine (6-aza) nucleosides. Nucleosides are of interest because of their key role in the nucleic acid structure and because of the antimetabolite and cytotoxic/antileukemia properties of their synthetic analogues. It was shown that polarographic reduction of the studied compounds is achieved at gradually increased potentials in the order of 6-aza < 5-aza < pyrimidine nucleosides. On other hand, the potential carcinogenicity of studied compounds increases usually in the order of pyrimidine < 6-aza < 5-aza nucleoside. The only compounds with remarkable potential carcinogenicity identified at this study were those ones from the 5-aza (1,3,5-triazine) antimetabolite series-arabinosyl-5-azacytosine (0.275), 5-aza-cytidine (0.295) and 5-aza-uracil (0.400)-and 2,2'-anhydrouridine (0.260). The relation of the data obtained to biological activity of nucleosides included in the study is discussed.     

Synthesis of 5-fluoroalkylated pyrimidine nucleosides via Negishi cross-coupling

5-Fluoroalkylated pyrimidine nucleosides (1) have potential as therapeutic agents and molecular imaging agents targeting HSV1-tk suicide gene therapy. Thus, straightforward preparation of 5-fluoroalkylated nucleoside derivatives has been developed. Reported herein are the first examples of Pd-catalyzed Negishi cross-coupling of 3-N-benzoyl-3',5'-di-O-benzoyl-5-iodo-2'-deoxyuridine (2a) and 3-N-benzoyl-3',5'-di-O-benzoyl-5-iodo-2'-deoxy-2'-fluoroarabinouridine (2b) with unactivated Csp(3) fluoroalkylzinc bromides. This paper demonstrates the first synthesis of six 5-fluoroalkyl-2'-deoxypyrimidine nucleoside derivatives with three to five methylene chain lengths (5). Furthermore, this methodology has been extended to create a series of 13 5-alkyl-substituted nucleosides, including the target nucleosides 5 and 5-silyloxypropyl and 5-cyanobutyl derivatives.     

2'-C-branched ribonucleosides. 2. Synthesis of 2'-C-beta-trifluoromethyl pyrimidine ribonucleosides

[structure: see text]. The first synthesis of 2'-C-beta-trifluoromethyl pyrimidine ribonucleosides is described. 1,2,3,5-Tetra-O-benzoyl-2-C-beta-trifluoromethyl-alpha-D-ribofuranose (3) is prepared from 1,3,5-tri-O-benzoyl-alpha-D-ribofuranose (1) in three steps and converted to 3,5-di-O-benzoyl-2-C-beta-trifluoromethyl-alpha-D-1-ribofuranosyl bromide (5). The 1-bromo derivative (5) is found to be a powerful reaction intermediate for the synthesis of ribonucleosides. The reaction of silylated pyrimidines with (5) in the presence of HgO/HgBr2 affords exclusively the beta-anomers (6-8). Deprotection of (6-8) with ammonia in methanol yields the 2'-C-beta-trifluoromethyl nucleosides (9-11).     

Ring opening of 4',5'-epoxynucleosides: a novel stereoselective entry to 4'-C-branched nucleosides

Stereoselective synthesis of 4'-alpha-carbon-substituted nucleosides has been accomplished through epoxidation of 4',5'-unsaturated nucleosides with dimethyldioxirane (DMDO) and successive SnCl(4)-promoted ring opening of the resulting 4',5'-epoxynucleosides with organosilicon reagents. [reaction: see text]     

Nucleic-Acid Analogues with Constraint Conformational Flexibility in the Sugar-Phosphate Backbone (‘Bicyclo-DNA’). Part 1. Preparation of (3S,5′R)-2′-Deoxy-3′,5′-ethano-αβ-D-ribonucleosides (‘Bicyclonucleosides’)

We describe the synthesis of 2′-deoxy-3′,5′-ethano-D -ribonucleosides 1 – 8 (= (5′,8′-dihydroxy-2′-oxabicyclo-[3.3.0]oct-3′-yl)purines or -pyrimidines) of the nucleobases adenine, thymine, cytosine, and guanine. They differ from natural 2′-deoxyribonucleosides only by an additional ethylene bridge between the centers C(3′) and C(5′). The configuration at these centers (3S,5′R) was chosen as to match the geometry of a repeating nucleoside unit in duplex DNA as close as possible. These nucleosides were designed to confer, as constituents of an oligonucleotide chain, a higher degree of preorganization of a single strand for duplex formation with respect to natural DNA, thus leading to an entropic advantage for the pairing process. The synthesis of these ‘bicyclonucleosides’ was achieved by construction of an enantiomerically pure carbohydrate precursor 18 / 19 (Schemes 1), which was then converted to the corresponding nucleosides by known methods in nucleoside synthesis (Schemes 2 and 3). In all cases, both anomeric forms of the nucleosides were obtained in pure crystalline form, the relative configuration of which was established by ¹H-NMR-NOE spectroscopy. A conformational analysis of the nucleosides with β-configuration at the anomeric center by means of X-ray and ¹H-NMR (including NOE) spectroscopy show the furanose part of the molecules to adopt uniformly a 1′exo-conformation with the base substituents preferentially in the anti-range in the pyrimidine nucleosides (anti/syn ca. 2:1) distribution in the purine nucleosides (in solution).     

Synthesis of novel apio carbocyclic nucleoside analogues as selective a(3) adenosine receptor agonists

On the basis of the biological activity of neplanocin A and apio-dideoxyadenosine (apio-ddA), novel apio-neplanocin A analogues 5a-d, combining the properties of two nucleosides, were stereoselectively synthesized. The apio moiety of the target nucleosides 5a-d was stereoselectively introduced by treating lactol 10 with 37% formaldehyde in the presence of potassium carbonate. The carbasugar moiety of neplanocin A was successively built by exposing diene 12 on a Grubbs catalyst in methylene chloride. The final nucleosides 5a-d were synthesized from the condensation of the glycosyl donor 14 with nucleic bases under the standard Mitsunobu conditions. Similarly, apio-aristeromycin 6 and (N)-apio-methanocarbaadenosine 7 were derived from the common intermediate 13 using catalytic hydrogenation and Simmons-Smith cyclopropanation as key steps. All of the final nucleosides 5a-d, 6, and 7 did not show significant inhibitory activity against S-adenosylhomocysteine hydrolase (SAH) up to 100 muM, maybe due to the absence of the secondary hydroxyl group at the C3'-position, which should be oxidized by cofactor-bound NAD(+). However, apio-neplanocin A (5a) showed potent and highly selective binding affinity (K(i) = 628 +/- 69 nM) at the A(3) adenosine receptor without any binding affinity at the A(1) and A(2A) adenosine receptors. In conclusion, we have first developed novel carbocyclic nucleosides with unnatural apio-carbasugars using stereoselective hydroxymethylation and RCM reaction and also discovered a new template of human A(3) adenosine receptor agonist, which play a great role in developing new A(3) adenosine receptor agonist as well as in identifying the binding site of the receptor.     

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     

Systematic assignment of NMR spectra of 5‐substituted‐4‐thiopyrimidine nucleosides

Unambiguous characterization of 5‐substituted‐4‐thiopyrimidine nucleosides (ribonucleosides and 2'‐deoxynucleosides) was performed using NMR spectroscopy. Assignments of all proton and carbon signals of 5‐bromo‐4‐thiouridine and related nucleosides were systematically carried out and firmly established by COSY and HMQC techniques. The NMR data of various 4‐thiopyrimidine nucleosides are compared, and the key contributing factors discussed. The approach presented here is applicable to other modified nucleosides and nucleotides, as well as nucleobases. Copyright © 2013 John Wiley & Sons, Ltd.     

Preparation of highly substituted 6-arylpurine ribonucleosides by Ni-catalyzed cyclotrimerization. Scope of the reaction

Transition metal complex catalyzed cocyclotrimerization of protected alkynylpurine ribonucleosides 1 with various diynes 2 gave rise to a series of 6-arylpurine nucleosides 3 that were further deprotected to free nucleosides 4. Generally, the best yields of cyclotrimerizations were obtained with a catalytic system Ni(cod)2/2PPh(3). On the other hand, CoBr(PPh(3))3 proved to be a superior catalyst for cyclotrimerization of 1 with dipropargyl ether 2g. In addition, Ni catalysis is also suitable for direct cyclotrimerization of unprotected alkynylpurine ribonucleosides 5 to the corresponding 6-arylpurinylribosides 4.     

NMR studies on 4‐thio‐5‐furan‐modified and 4‐thio‐5‐thiophene‐modified nucleosides

Systematic NMR characterization of 4‐thio‐5‐furan‐pyrimidine nucleosides or 4‐thio‐5‐thiophene‐pyrimidine nucleosides (ribonucleosides and 2′‐deoxynucleosides) was performed. All proton and carbon signals of 4‐thio‐5‐thiophene‐ribouridine and related analogues were unambiguously assigned. The orientations of the base (4‐thiouridine or its deoxy analogue) relative to the ring (furan or thiophene) are explored by a NMR approach and further supported by X‐ray crystallographic studies. The procedures presented here would be applicable to other modified nucleosides and nucleotides. Copyright © 2016 John Wiley & Sons, Ltd.     

The cyclobutane dimers of 2'-deoxyuridine, 2'-deoxycytidine, 5-methyl-2'-deoxycytidine and 5-bromo-2'-deoxyuridine

Irradiation of DNA and RNA pyrimidine nucleosides with UV light in frozen aqueous solution or in solution with acetone often results in the formation of cyclobutane dimers (CBDs). Many of these photodimers have not been characterized. We present here the results of work designed to achieve the isolation, spectroscopic characterization and determination of the stereochemical nature of a number of little studied or previously unstudied CBDs of four 2'-deoxyribonuclesides. These nucleosides are 2'-deoxyuridine (dUrd), 2'-deoxycytidine (dCyd), 5-methyl-2'-deoxycytidine (5-MedCyd) and 5-bromo-2'-deoxyuridine (5-BrdUrd). In particular, we have isolated and characterized six dUrd CBDs, five dCyd CBDs, five 5-MedCyd CBDs and four 5-BrdUrd CBDs. Photoproducts were studied by UV spectroscopy, mass spectrometry, proton NMR spectroscopy and via chemical approaches. Also presented are results from less definitive studies of a number of (6-4) (or 5-4) photoadducts of these nucleosides. In addition, results from exploratory photochemical studies of other 2'-deoxyribonucleosides in frozen solution, as well as some mixtures of two nucleosides, are given. The latter results indicate that 5-iodo-2'-deoxyuridine (5-IdUrd), 5-bromo-2'-deoxycytidine and 5-iodo-2'-deoxycytidine each form putative CBDs and that 5-BrdUrd is capable of forming putative mixed CBDs and (6-4) and/or (5-4) adducts with thymidine (Thd); 5-IdUrd similarly forms a (6-4) (or (5-4)) adduct with Thd.