Esterification and etherification by silver oxide-organic halide reaction gas chromatography.

In an attempt to separate a number of methylated nucleoside constituents reportedly present in various mammalian RNA preparations, a comparison of the migration of selected nucleoside standards was assessed via thin-layer chromatographic techniques with respect to three solvent systems and an adsorbent of varying proportions of silica gel and microcrystalline cellulose. The results of these studies revealed that a mixture of seventeen methylated constituents could be separated following two-dimensional development on an adsorbent containing 60% cellulose and 40% silica gel. Constituents separated included the 2'-O-methylated derivatives of adenosine, guanosine, cytidine and uridine, 1-, N6- and N6,N6-methyladenosines, 1-methylinosine, 1-, 7-, N2- and N2,N2-methylguanosines, 3-, 4- and 5-methylcytidines and 3- and 5-methyluridines. In addition, all of the above constituents were separated from their respective non-methylated nucleosides and bases.     

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.     

Reversed-phase high-performance liquid chromatographic investigation of mucosal nucleosides and bases and urinary modified nucleosides of gastrointestinal cancer patients

Reversed-phase high-performance liquid chromatography (HPLC) was used to determine the levels of nucleosides, bases and their metabolites in perchloric acid extracts of gastrointestinal mucosa. By comparing the levels of these compounds in the normal portion with the neoplastic portion of mucosa resected from malignant cancer patients, it was found that there was significant elevation of the uracil level in the neoplastic mucosa of all eight patients with colorectal cancer (2.7-fold in normal mucosa), but only in the neoplastic mucosa of one out of four patients with gastric cancer. The levels of hypoxanthine and uridine in the colorectal cancer mucosa samples and the inosine in gastric cancer samples were also significantly higher than those in normal mucosa. The urinary modified nucleosides were prefractionated with a boronate affinity gel column, and their levels were determined by the same HPLC method. There was no significant difference in the concentrations of pseudouridine, 1-methylguanosine N2-methylguanosine and N2,N2-dimethylguanosine between urine samples taken before and after surgery from eight patients with malignant colorectal cancer. Contrary to other reports, no significant differences in modified nucleoside levels were observed between urine samples from patients with colorectal cancer and those from normal subjects.     

A novel polymer supported approach to nucleoside modification

Polymer-supported O(6)-(benzotriazol-1-yl)inosine derivatives (Pol-I and Pol-dI) have been synthesized reasonably effectively via reaction of nucleoside phosphonium salts with polymer-linked HOBt (Pol-HOBt). In constast to solution chemistry, use of polymer-supported BOP (Pol-BOP) did not lead to efficient nucleoside loading. Presence of the nucleosides on the support could be readily detected by MAS NMR. Exposure of the polymer-supported nucleosides, Pol-I and Pol-dI, to alcohol, phenol, thiol and amine nucleophiles caused cleavage from the support leading directly to the C-6 modified nucleoside analogues. To our knowledge, these are the first examples of the application of such technology for nucleoside modification. Where possible, results of reactions with the polymer-supported nucleosides are compared to those from solution chemistry, providing insight into the differences between the two techniques. These new polymer-supported nucleosides can be conveniently utilized for diversity-oriented synthesis.     

Alkylsulfanylphenyl derivatives of cytosine and 7-deazaadenine nucleosides, nucleotides and nucleoside triphosphates: synthesis, polymerase incorporation to DNA and electrochemical study

Aqueous Suzuki–Miyaura cross‐coupling reactions of halogenated nucleosides, nucleotides and nucleoside triphosphates derived from 5‐iodocytosine and 7‐iodo‐7‐deazaadenine with methyl‐, benzyl‐ and tritylsufanylphenylboronic acids gave the corresponding alkylsulfanylphenyl derivatives of nucleosides and nucleotides. The modified nucleoside triphosphates were incorporated into DNA by primer extension by using Vent(exo‐) polymerase. The electrochemical behaviour of the alkylsulfanylphenyl nucleosides indicated formation of compact layers on the electrode. Modified nucleotides and DNA with incorporated benzyl‐ or tritylsulfanylphenyl moieties produced signals in [Co(NH₃)₆]³⁺ ammonium buffer, attributed to the Brdi?ka catalytic response, depending on the negative potential applied. Repeated constant current chronopotentiometric scans in this medium showed increased Brdi?ka catalytic response, which suggests the deprotection of the alkylsulfanyl derivatives to free thiols under the conditions.     

Bisphosphonate derivatives of nucleoside antimetabolites: hydrolytic stability and hydroxyapatite adsorption of 5'-beta,gamma-methylene and 5'-beta,gamma-(1-hydroxyethylidene) triphosphates of 5-fluorouridine and ara-cytidine

Kinetics of the hydrolytic reactions of four bisphosphonate derivatives of nucleoside antimetabolites, viz., 5-fluorouridine 5'-beta,gamma-(1-hydroxyethylidene) triphosphate ( 4), 5-fluorouridine 5'-beta,gamma-methylene triphosphate ( 5), ara-cytidine 5'-beta,gamma-(1-hydroxyethylidene) triphosphate ( 6), and ara-cytidine 5'-beta,gamma-methylene triphosphate ( 7), have been studied over a wide pH range (pH 1.0-8.5) at 90 degrees C. With each compound, the disappearance of the starting material was accompanied by formation of the corresponding nucleoside 5'-monophosphate, the reaction being up to 2 orders of magnitude faster with the beta,gamma-(1-hydroxyethylidene) derivatives ( 4, 6) than with their beta,gamma-methylene counterparts ( 5, 7). With compound 7, deamination of the cytosine base competed with the phosphate hydrolysis at pH 3-6. The measurements at 37 degrees C (pH 7.4) in the absence and presence of divalent alkaline earth metal ions (Mg (2+) and Ca (2+)) showed no sign of metal ion catalysis. Under these conditions, the initial product, nucleoside 5'-monophosphate, underwent rapid dephosphorylation to the corresponding nucleoside. Hydrolysis of the beta,gamma-methylene derivatives ( 5, 7) to the corresponding nucleoside 5'-monophosphates was markedly faster in mouse serum than in aqueous buffer (pH 7.4), the rate-acceleration being 5600- and 3150-fold with 5 and 7, respectively. In human serum, the accelerations were 800- and 450-fold compared to buffer. In striking contrast, the beta,gamma-(1-hydroxyethylidene) derivatives did not experience a similar decrease in hydrolytic stability. The stability in human serum was comparable to that in aqueous buffer (tau 1/2 = 17 and 33 h with 4 and 6, respectively), and on going to mouse serum, a 2- to 4-fold acceleration was observed. To elucidate the mineral-binding properties of 4- 7, their retention on a hydroxyapatite column was studied and compared to that of zoledronate ( 1a) and nucleoside mono-, di-, and triphosphates.     

Novel synthesis of nucleoside 5'-phosphoramidates through reaction of nucleoside triphosphates with amines mediated by trimethylsilyl chloride

Reaction of nucleoside triphosphates (NTPs) with amines in pyridine mediated by trimethylsilyl chloride produced nucleoside 5'-phosphoramidates in moderate yields without any preprotection of nucleosides and amino acid methyl esters. The reaction pathway is very similar to the mechanism of the RNA capping reaction, DNA or RNA ligation reaction, and catalysis of hydrolases and nucleases involving the formation of covalent enzyme-NMP (nucleoside 5'-monophosphate) intermediates in biological systems, which could provide a valuable clue for the enzymatic reactions.     

Analysis of modified nucleosides in the urine of patients with malignant cancer by liquid chromatography/electrospray ionization mass spectrometry

As modified nucleosides reflect altered tRNA turnover which seems to be impaired in the body of cancer patients, they have been evaluated as potential tumor markers. High-performance liquid chromatography/electrosprary ionization quadrupole time-of-flight mass spectrometry (HPLC/ESI-Q-TOFMS) was used to identify nucleosides purified from urine in positive ionization mode. Potential nucleosides were assessed by their evident UV absorbance in HPLC and then further examined by mass spectrometric techniques. In this manner, 21 nucleosides were detected in the urine of a patient with lymphoid cancer including three modified nucleosides 5'-dehydro-2-deoxyinosine, N1,N2,N7-trimethylguanosine and N1-methyl-N2-ethylguanosine, which had never been reported previously.     

Conformation of nucleoside antibiotics

Minor modifications or substitutions in the sugar or in the base part of pyrimidine and purine nucleosides have a profound effect on their biological activity. These modified nucleosides usually become antiviral, antibacterial, or cancerostatic agents and they are collectively called nucleoside antibiotics. The conformational properties of some of these nucleoside antibiotics have been studied by the PCILO method. The results obtained from such study indicate that the conformational preferences of these nucleoside antibiotics are very similar to those of their parent nucleosides and especially so in the situations that occur in aqueous solutions. The important biological significance of these results is that these nucleoside antibiotics can easily get incorporated into growing chains of DNA and RNA by mimicking their parent nucleosides and can interfere with the protein synthesis of RNA or DNA synthesis.     

Application of solid-phase extraction on anion-exchange cartridges to quantify 5'-nucleotidase activity.

The enzyme 5'-nucleotidase (5'-ribonucleotide phosphohydrolase, EC 3.1.3.5) catalyzes a critical reaction in intermediary metabolism, the phosphohydrolysis of nucleoside 5'-monophosphates to their corresponding nucleosides. We have evaluated solid-phase extraction on pre-packed anion-exchange cartridges as a chromatographic technique with which 5'-nucleotidase activity may be detected and quantified. Chromatographic conditions were established whereby substrate nucleotide was rapidly and completely separated from its corresponding nucleoside by solid-phase extraction. Both analytes were recovered quantitatively, without loss or degradation. This chromatographic system was integrated into a discontinuous radiochemical assay for 5'-nucleotidase which enabled both substrate utilization and product formation to be assessed simultaneously. Enzyme reaction samples could be analyzed directly for 5'-nucleotidase activity without any pre-chromatography preparation. The high capacity of the solid-phase cartridges and the inability of 5'-nucleotidase to enter the packing bed during analyte elution facilitated termination of the enzyme reaction by applying the entire reaction mixture to the cartridge. Loaded cartridges could then be stored at 4 degrees C prior to chromatography and subsequently batch-eluted. The excellent resolution between substrate and product in solid-phase extraction and the sensitivity of radioisotopic counting enabled detection/quantification of low tissue levels of 5'-nucleotidase in conjunction with ancillary assays for secondary enzyme reactions with the potential to elicit the artifactual loss of 5'-nucleotidase substrate/product when crude biological preparations are examined for 5'-nucleotidase activity. Our results demonstrate that solid-phase extraction on anion-exchange cartridges with elution solvents of appropriate pH offers several unique advantages for 5'-nucleotidase determination.     

Separation of synthetic cycloalkylated bases, nucleosides and nucleotides by reversed-phase high-performance liquid chromatography

Reversed-phase high-performance liquid chromatography was used to determine the elution profiles of a series of synthetic cycloalkylated bases, nucleosides, and their corresponding 5'-monophosphates. A 70% aqueous methanol solution proved to be the most efficient solvent system for the separation of a mixture of the bases, all of which were eluted in times ranging from 3.3 to 4.8 min at a flow-rate of 0.8 ml/min. Subsequently, the same percentage of methanol solvent, at 0.8 ml/min, eluted the nucleoside mixture as well, with retention times ranging from 3.3 to 5.0 min. Optimum separation and resolution were achieved with 70% methanol at a flow-rate of 0.6 ml/min for a mixture of the base and nucleoside series. A phosphate buffer, containing acetonitrile-tetrabutylammonium ion, was used to analyze the 5'-monophosphate derivatives. Elution times ranged from 2.6 to 6.1 min at a flow-rate of 1.0 ml/min. Three variables were considered in order to determine optimum conditions for separation and resolution: (a) the percentage of methanol in the solvent; (b) flow-rate of solvent; and (c) the size of the cycloalkylated group of each synthetic analogue. The procedures and conditions described herein have potential use as a monitoring system to detect modified nucleic acid derivative which are prevalent in the body fluids of patients with certain metabolic disorders.     

Correlation between the conformation of nucleoside antibiotics and their biological activity

Nucleoside antibiotics, which result as a consequence of minor modifications in pyrimidine and purine nucleosides, exhibit a wide variety of antiviral, antibacterial, antitumor, and cancerostatic properties. The conformational properties of a number of these antibiotics have been investigated by using the quantummechanical PCILO method, and the results indicate that the nucleoside antibiotics and their parent nucleosides have very similar conformational preferences. This similarity is strikingly marked in the situations which prevail in an aqueous medium. As a result, these antibiotics easily get incorporated in growing chains of RNA and DNA by mimicking their parent nucleosides and then bring about the inhibition of protein, RNA, or DNA syntheses. The experimental observations corroborate these deductions, and thus a correlation has been obtained between the conformation and the biological activity of nucleoside antibiotics; it is the striking conformational similarity between the nucleoside antibiotics and their parent nucleosides which gives rise to their biological activity. The PCILO investigations carried out on two 3-deazapurine nucleosides demonstrate that the converse of the above correlation also holds true.     

Nucleosides. CXXXV. Synthesis of some 9-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)-9H-purines and their biological activities

Seven purine nucleosides containing the 2'-deoxy-2'-fluoro-beta-D-arabinofuranosyl moiety were synthesized and tested for their antitumor activity. Direct condensation of 3-O-acetyl-5-O-benzoyl-2-deoxy-2-fluoro-D-arabinofuranosyl bromide (1) with N6-benzoyladenine in CH2Cl2 followed by saponification of the product afforded the adenine nucleoside (I, 2'-F-ara-A). Deamination of I with NaNO2 in HOAc gave the hypoxanthine analogue (II, 2'-F-ara-H). The 6-thiopurine nucleoside (III, 2'-F-ara-6MP) was prepared by condensation of 1 with 6-chloropurine by the mercury procedure followed by thiourea treatment and saponification of the product. Methylation of III gave the 6-SCH3 analogue (IV). Raney Ni desulfurization of III afforded the unsubstituted purine nucleoside (V, 2'-F-ara-P). Condensation of 1 with 2-acetamido-6-chloropurine by the silyl procedure afforded the protected 2-acetamido-6-chloropurine nucleoside which served as the precursor for both the guanine and 6-thioguanine nucleosides (VI, 2'-F-ara-G and VII, 2'-F-ara-TG, respectively). Thus, alkaline hydrolysis of the precursor gave VI. Thiourea treatment prior to alkaline hydrolysis gave VII. The new nucleoside, 2'-F-ara-G (VI) is found to be selectively toxic to human T-cell leukemia CCRF-CEM.     

核苷及其类似物的三磷酸化方法研究进展

三磷酸核苷是一种具有三个磷酸基团的核苷酸,不仅在细胞的能量代谢中起着重要作用,也可用于心脑血管和神经系统等许多疾病的治疗和辅助治疗,随着分子生物学的发展,三磷酸核苷在许多生物检测技术中也应用广泛。新冠疫情爆发以来,抗病毒核苷类药物成为研究的热点,这类药物大多在体外不具有活性,而是在细胞内代谢为三磷酸形式发挥作用,在对这类化合物的抗病毒机理研究中,研究其抗病毒活性、抗病毒机制等都需要将核苷类药物进行三磷酸化,因此开发一种高效的核苷三磷酸化方法是十分必要的。本文根据三磷酸核苷的合成历史进程,综述了迄今为止三磷酸核苷的化学合成方案。旨在为核苷及其类似物的三磷酸化以及相关药物的研发提供参考。     

Highly selective capture of nucleosides with boronic acid functionalized polymer brushes prepared by atom transfer radical polymerization

The nucleoside or modified nucleoside level in biological fluids reflects the pathological or physiological state of the body. Boronate affinity absorbents are widely used to selectively extract nucleosides from complex samples. In this work, a novel functionalized absorbent was synthesized by attaching 4‐mercaptophenylboronic acid to gold nanoparticles on modified attapulgite. The surface of the attapulgite was modified by poly(acryloyloxyethyltrimethyl ammonium chloride) by atom transfer radical polymerization, creating many polymer brushes on the surface. The resultant material exhibited superior binding capacity (30.83 mg/g) for adenosine and was able to capture cis‐diol nucleosides from 1000‐fold interferences. Finally, to demonstrate its potential for biomolecule extraction, this boronate affinity material was used to preconcentrate nucleosides from human urine and plasma.     

Nucleoside Phosphoramidate Analogues with Modification in the Bridging Positions of the Phosphodiester Linkage

Abstract

Phosphoramidate modified DNA oligomersl[1] have recently been shown to have promising properties for antisensdantigene applications. An eficient method, for the preparation of nucleoside P3′ → N5′ and N3′ → P5′ phosphoramidates and their thio analogues 3 was developedl'l. I t consists of the oxidative generation of a pyridine adducr of type 2 of nucleoside metaphosphate from the corresponding nucleoside H-phosphonate, nucleoside H-phosphonothioate or nucleoside H-phosphondithioatc monoester, and its consecutive clean reaction with 5′- or 3′-aminonucleosides in the presence of triethylamine.     

Furan Decorated Nucleoside Analogues as Fluorescent Probes: synthesis, photophysical evaluation and site-specific incorporation

The synthesis and photophysical evaluation of modified nucleoside analogues in which a five-membered heterocycle (furan, thiophene, oxazole, and thiazole) is attached to the 5-position of 2′-deoxyuridine are reported. The furan-containing derivative is identified as the most promising responsive nucleoside of this family due to its emission quantum efficiency and degree of sensitivity to its microenvironment. The furan moiety was then attached to the 5-position of 2′-deoxycytidine as well as the 8-position of adenosine and guanosine. Photophysical evaluation of these four furan-containing nucleoside analogues reveals distinct differences in the absorption, emission, and quantum efficiency depending upon the class of nucleoside (pyrimidine or purine). Comparing the photophysical properties of all furan-containing nucleosides, identifies the furan thymidine analogue, 5-(fur-2-yl)-2′-deoxyuridine, as the best candidate for use as a responsive fluorescent probe in nucleic acids. 5-(Fur-2-yl)-2′-deoxyuridine was then converted to the corresponding phosphoramidite and site specifically incorporated into DNA oligonucleotides with greater than 88% coupling efficiency. Such furan-modified oligonucleotides form stable duplexes upon hybridization to their complementary DNA strands and display favorable fluorescent features.     

Synthesis of bitriazolyl nucleosides and unexpectedly different reactivity of azidotriazole nucleoside isomers in the Huisgen reaction

Novel bitriazolyl nucleosides were synthesized via the Huisgen reaction, starting with 3-azidotriazole nucleoside (1). Surprisingly, its isomer, 5-azidotriazole nucleoside (1') did not yield the corresponding Huisgen reaction products efficiently because it was rapidly reduced to amine in the presence of Cu(II)-ascorbate. The significant differences between the reactivity of these two isomers in Cu(II)-ascorbate mediated reactions are mainly due to differences in their electronic properties and steric congestion as a result of different relative positions of the azido and the ribosyl moieties.     

Upregulated plasma and urinary levels of nucleosides as biological markers in the diagnosis of primary gallbladder cancer

We first detected aberrant nucleoside levels in the plasma, urine, bile, and tissues from cases and controls to explore them as biomarkers in the diagnosis of gallbladder cancer. Reversed‐phase high‐performance liquid chromatography was used to assess the levels of ten nucleosides in these samples from gallbladder cancer patients, gallstone patients, and healthy controls. Plasma and urine samples were collected from patients with gallbladder cancer (n = 202), patients with gallstones (n = 203), and healthy controls (n = 205); bile and tissue samples were collected from 91 gallbladder cancer patients, 93 gallstone patients; and 90 were donated after cardiac death. Of the ten nucleosides analyzed, eight urinary nucleosides, five plasma nucleosides, three bile nucleosides, and one tissue nucleoside were significantly upregulated in the gallbladder cancer patients compared to control groups (p < 0.05). Among these upregulated nucleosides, the sensitivity, specificity, and accuracy of urinary nucleosides in the diagnosis of gallbladder cancer patients were 89.4, 97.1, and 95.7%, respectively, those of plasma nucleosides were 91.2, 95.6, and 94.2%, respectively, those of bile nucleosides were 95.3, 96.4, and 95.1%, respectively, and those of tissue nucleosides were 86.2, 93.8, and 92.6%, respectively. These results suggest that nucleosides may be as useful as biological markers for gallbladder cancer.     

6-bromopurine nucleosides as reagents for nucleoside analogue synthesis.

Surprisingly facile direct substitution reactions with acetyl-protected 6-bromopurine nucleosides are described. Included in the series of bromonucleosides studied is the guanosine derivative N(2)-2',3',5'-tetraacetyl-6-bromopurine ribonucleoside, the synthesis of which is reported here for the first time. Brominated nucleosides had not previously been considered optimal substrates for S(N)Ar reactions given the general reactivity trend for halogenated aromatic systems (i.e. F > Cl > Br > I). However, even weakly nucleophilic aromatic amines give high yields of the substitution products in polar solvents with these 6-bromopurine nucleosides. For primary aromatic amines, secondary aliphatic amines, and imidazole, reaction takes place only at C6, with no effect on the acetyl-protected ribose. In addition, we report the first synthesis of 3',5'-di-O-acetyl-6-bromopurine-2'-deoxyribonucleoside and its reaction with an arylamine in MeOH in the absence of added metal catalyst. Thus, C6-arylamine derivatives of both adenosine and 2'-deoxyadenosine can be prepared via simple S(N)Ar reactions with the corresponding 6-bromo precursor. We also describe high yielding and C6-selective substitution reactions with 6-bromonucleosides using alcohol and thiol nucleophiles in the presence of added base (DBU). Finally, C6-bromonucleosides are shown to be readily hydrogenated to give purine or 2-aminopurine products in good yield. This work increases the arsenal of reactions and strategies available for the synthesis of nucleoside analogues as potential biochemical tools or new therapeutics.