Use of [4,6-Di-14C]-5′-DMT-thymidine Phosphoramidite Reagent for the Radiolabeling of Synthetic Oligonucleotides

Abstract

A novel synthesis of 5′-radiolabeled oligonucleotides is described. The labeling is carried out by the phosphoramidite method with the aid of building block 1. The feasibility of the method is demonstrated by preparation of 5′-radiolabeled 3′-phosphorylated dodecathymidylate phosphorothioate.     

Assay of Δ1-piperideine-2-carboxylate and synthesis of l-[14C]pipecolate from dl-[14C]pipecolate

Four assay methods were tested for the measurement of Δ¹-piperideine-2-carboxylate, a proposed alicyclic ketimino acid intermediate in the pathway of lysine metabolism to l-pipecolate, and the product of d-amino acid oxidase on d-pipecolate. The method using Δ¹-piperideine-2-carboxylate reductase from Pseudomonas putida was found to be most sensitive and specific. Measurement of Δ¹-piperideine-2-carboxylate by reduction with NaBH₄ and ninhydrin assay of the resultant pipecolate, by direct acidic ninhydrin assay, and by o-aminobenz-aldehyde assay were less desirable because of lower sensitivity and specificity. Two synthetic methods for preparing l-[¹⁴C]pipecolate from the racemic dl-[¹⁴C]pipecolate were investigated. Incubation of dl-[¹⁴C]pipecolate with a combination of d-amino acid oxidase and Δ¹-piperideine-2-carboxylate reductase or d-amino acid oxidase and NaBH₄ totally inverted the d-isomer to the l-isomer, with $\text{Δ}{}^1\text{-[}{}^{14}\text{C]piperideine-2-carboxylate}$ as an intermediate in each cycle of interconversion. No purification except desalting through a Dowex 50 (H⁺) column was necessary in order to recover l-[¹⁴C]pipecolate in pure form. The yield was 95–97% compared to <50% in the conventional method.     

A convenient synthesis of 5′-deoxyribonucleosides

A facile, two-step synthesis has been devised for the chemical preparation of 5′-deoxyribonucleosides from the parent nucleosides via the 5′-chloro-5′-deoxy-nucleosides. Treatment of 5′-chloro-5′-deoxynucleosides with tributyltin hydride and α,α′-azobis(isobutyronitrile) in dry tetrahydrofuran yields the corresponding 5′-deoxy-nucleosides. Dechlorination of 5′-chloro-5′-deoxythymidine with tributyltin hydride gives 1-(2,5-dideoxy-β-D-erythro-pentofuranosyl)thymine (5′-deoxythymidine) in good yield. Similarly, dechlorination of 9-(3,5-dichloro-2,3,5-trideoxy-β-D-threo-pentofuranosyl)adenine and 1-(3,5-dichloro-2,3,5-trideoxy-β-D-threo-pentofuranosyl)thymine yields the corresponding two trideoxynucleosides.     

Synthesis of 2′-Deoxy[5′-13C]Ribonucleotides and HMQC-Noesy NMR Study of the Dickerson's Dodecamer with 13C-Labeling at the 5′ Positions

Abstract

In addition to the synthesis of 2′-deoxy[5′-¹³C]ribonucleosides (6) via the D-[5-¹³C]ribose derivative (4), the construction of the corresponding dodecanucleotide with the Dickerson's sequence and its HMQC-NOESY NMR analysis are described.     

Interaction of α-cyano[14C]cinnamate with the mitochondrial pyruvate translocator

The binding of α-cyanocinnamate to rat-heart mitochondrial membrane was investigated using α-cyano[¹⁴C]cinnamate. The binding was correlated to the inhibition of pyruvate transport. The results obtained demonstrate that both these functions reach saturation at the same titre of the inhibitor. Quantitative parameters of α-cyano[¹⁴C]cinnamate binding have been determined. The binding can be prevented by pyruvate and other substrates of the carrier but not by acetate. Pyruvate decreases the affinity of α-cyanocinnamate binding, leaving the maximum number of binding unchanged. It is concluded that rat-heart mitochondria contain a specific site at which α-cyanocinnamate binds which is directly involved in the inhibition of pyruvate transport.     

Development of PCR-based assays for allelic discrimination in maize by using the 5′-nuclease procedure

We describe the development of non-electrophoresis-based PCR assays for the allelic discrimination at two linked loci flanking an important QTL controlling days to pollen shed in maize. The assays are based on the fluorogenic 5-nuclease procedure (TaqMan), which allows for the direct detection of the PCR product by the release of a fluorescent reporter dye as a result of DNA amplification. The assays were developed after sequencing the alleles at both loci, by designing suitable primers and probes based on single nucleotide or insertion/deletion polymorphisms. The TaqMan procedure allowed for a fast and highly reproducible analysis directly in the PCR vials. The lack of any fragment separation step allows for an almost complete automation of the process thus making this technique particularly valuable for the large-scale screening required for map-based cloning projects and for marker-assisted selection, particularly when the results are needed in a short time.     

A practical post-modification synthesis of oligodeoxynucleotides containing 4,7-diaminoimidazo[5′,4′:4,5]pyrido[2,3-d]pyrimidine nucleoside

We describe herein the practical post-modification synthesis of oligodeoxynucleotides (ODNs) containing 4,7-diaminoimidazo[5′,4′:4,5]pyrido[2,3-d]pyrimidine nucleoside (ImN^N). Since the ImN^N nucleoside unit possessing tribenzoyl groups on its exocyclic amino groups as the protecting group was quite unstable under acidic conditions, cleavage of its glycosidic linkage in ODN has been suggested throughout the conditions of solid-phase synthesis. As an alternative approach, we investigated a post-modification synthesis of the desired ODNs containing the ImN^N unit. Starting with protected 4-amino-7-chloro-1-(2-deoxy-β-d-ribofuranosyl)imidazo[5′,4′:4,5]pyrido[2,3-d]pyrimidine derivative 1, conversion into the corresponding phosphoramidite unit was examined. The p-bromobenzoyl group (p-BrBz) was the best protecting group of 4-amino group of 1 to give the phosphoramidite unit 9 for the post-modification synthesis. After carrying out the ODN synthesis linked to the controlled pore glass (CPG) support, the support was treated with ammonium hydroxide at 55 °C to remove the protecting groups, detach the ODN form the CPG support, and convert the 7-chloro group into a desired amino group. As a result, the desired ODNs containing ImN^N were obtained in good yield.     

Disposition of [4-14C]oestradiol-l7β in the isolated perfused brain of the rat

[4-¹⁴C] Oestradiol-17β was perfused through isolated brains of male and ovariectomized female rats. Two different perfusion media were used. The uptake of oestradiol-17β was higher in female brains, the highest concentrations being found in the hypophysis and hypothalamus. Oestradiol-17β was metabolized to a greater extent by female brains, the most important metabolite being oestrone. Additionally, 2-hydroxyoestradiol-17β, 6ζ-hydroxyoestradiol-17β, and 7α-hydroxyoestradiol-17β were found; 7α-hydroxyoestrone and another polar metabolite could not be definitely identified. Quantitatively, 2-hydroxylation was no more important than hydroxylation at C atom 6 or 7.     

Molecular recognition in the P2Y14 receptor: Probing the structurally permissive terminal sugar moiety of uridine-5′-diphosphoglucose

The P2Y₁₄ receptor, a nucleotide signaling protein, is activated by uridine-5′-diphosphoglucose 1 and other uracil nucleotides. We have determined that the glucose moiety of 1 is the most structurally permissive region for designing analogues of this P2Y₁₄ agonist. For example, the carboxylate group of uridine-5′-diphosphoglucuronic acid proved to be suitable for flexible substitution by chain extension through an amide linkage. Functionalized congeners containing terminal 2-acylaminoethylamides prepared by this strategy retained P2Y₁₄ activity, and molecular modeling predicted close proximity of this chain to the second extracellular loop of the receptor. In addition, replacement of glucose with other sugars did not diminish P2Y₁₄ potency. For example, the [5′′]ribose derivative had an EC₅₀ of 0.24 μM. Selective monofluorination of the glucose moiety indicated a role for the 2′′- and 6′′-hydroxyl groups of 1 in receptor recognition. The β-glucoside was twofold less potent than the native α-isomer, but methylene replacement of the 1′′-oxygen abolished activity. Replacement of the ribose ring system with cyclopentyl or rigid bicyclo[3.1.0]hexane groups abolished activity. Uridine-5′-diphosphoglucose also activates the P2Y₂ receptor, but the 2-thio analogue and several of the potent modified-glucose analogues were P2Y₁₄-selective.     

Sulfonamides with the N-alkyl-N′-dialkylguanidine moiety as 5-HT7 receptor ligands

A series of arylsulfonamides containing guanidine incorporated in the structure of secondary amines (piperidine, piperazine) was synthesized on SynPhase Lanterns and evaluated for 5-HT_1A, 5-HT_2A, and 5-HT₇ receptors. The results demonstrated that N-alkyl-N′-dialkylguanidines displayed good 5-HT₇/5-HT_1A selectivity and may be regarded as promising structural core for development of 5-HT₇ ligands.     

Design and synthesis of a novel series of (1′S,2R,4′S)-3H-4′-azaspiro[benzo[4,5]imidazo[2,1-b]oxazole-2,2′-bicyclo[2.2.2]octanes] with high affinity for the α7 neuronal nicotinic receptor

We describe an efficient and convergent synthesis of a series of (1′S,2R,4′S)-3H-4′-azaspiro[benzo[4,5]imidazo[2,1-b]oxazole-2,2′-bicyclo[2.2.2]octanes] displaying potency for the α7 nicotinic acetylcholine receptor (nAChR) and good selectivity vs. the related 5-HT_3A receptor.     

A new synthesis and an antiviral assessment of the 4′-fluoro derivative of 4′-deoxy-5′-noraristeromycin

A synthetic route to (1S,2S,3R,5S)-3-(6-amino-9H-purin-9-yl)-5-fluorocyclopentane-1,2-diol (that is, the 4′-fluoro derivative of 4′-deoxy-5′-noraristeromycin, 3) is described via a fluorinated cyclopentanol, which is in contrast to existing schemes where fluorination occurred once the purine ring was present. Compound 3 was assayed versus a number of viruses. A favorable response was observed towards measles (IC₅₀ of 1.2 μg/mL in the neutral red assay and 14 μg/mL by the visual assay) but this was accompanied by cytotoxicity in the CV-1 host cells (21–36 μg/mL). Among the viruses unaffected by 3 were human cytomegalovirus and the poxviruses (vaccinia and cowpox), which are three viruses that were inhibited by the 4′,4′-difluoro analog of 3 (that is, 2).     

Inhibition of the synthesis of polyamines and macromolecules by 5′-methylthioadenosine and 5′-alkylthiotubercidins in BHK21 cells

5′-Methylthioadenosine and four 5′-alkylthiotubercidins were tested for their ability to inhibit polyamine synthesis in vitro and to decrease polyamine concentration and prevent growth of baby-hamster-kidney (BHK21) cells. 5′-Methylthioadenosine and 5′-methylthiotubercidin decreased the activity of spermidine synthase from brain to roughly the same extent, whereas brain spermine synthase was much more strongly inhibited by 5′-methylthioadenosine compared with 5′-methylthiotubercidin. These nucleoside derivatives also inhibited the growth of BHK21 cells and increased the concentration of putrescine. 5′-Methylthioadenosine decreased cellular spermine concentration, whereas 5′-methylthiotubercidin lowered the concentration of spermidine. The activities of ornithine decarboxylase and S-adenosylmethionine decarboxylase were enhanced in cells grown in the presence of 5′-methylthiotubercidin. The growth inhibition produced by these nucleoside derivatives was not reversed by exogenous spermidine or spermine. 5′-Ethylthiotubercidin, 5′-propylthiotubercidin and 5′-isopropylthiotubercidin did not appreciably inhibit spermidine or spermine synthase in vitro or decrease the cellular polyamine content, but effectively prevented the growth of BHK21 cells. All nucleoside derivatives at concentrations of 0.2–1 mm caused a rapid inhibition of protein synthesis. It is concluded that the growth inhibition produced by 5′-methylthioadenosine and 5′-alkylthiotubercidins was not primarily due to polyamine depletion but other target sites, for instance the cellular nucleotide pool, cell membranes etc. must be considered.     

A method for the isolation of segments from the 5′ ends of retrovirus RNA

A method for the isolation of segments of any desired length from the 5′ end of retrovirus RNA has been tested. The method is based on selection of 5′-specific segments by hybridizing suitably fragmented genomic (35 S) RNA to mercurated strong stop cDNA followed by chromatography on sulfhydryl-agarose. The method has been shown to be effective for Akv viral RNA by observing the T1 oligonucleotide fingerprints of a 5′-enriched fraction. This fingerprint pattern is of lower complexity than that of total 35 S RNA, contains oligonucleotide spots that have previously been assigned as 5′ specific by conventional fingerprinting methods, and does not overlap with the pattern from 3′-specific RNA.     

A new synthesis of 4′-resveratrol esters and evaluation of the potential for anti-depressant activity

The 4′-ester analog of the disease preventative resveratrol 1 (RV), 4′-acetyl-RV 2 along with 4′-pivaloate 13 and benzoate 14 RV were synthesized. The previously developed palladium catalyzed decarbonylative Heck coupling was used to assemble the stilbene core together with 3,5-dibenzyl protected phenol intermediates that allowed for efficient coupling and deprotection using boron trifluoride etherate. Studies with Long-Evans rats were performed to establish safety, toxicity, and behavioral parameters. In addition, the Porsalt forced-swim test was used to demonstrate anti-depressant activity.     

Incorporation of label from d-β-hydroxy-[14C]butyrate and [3-14C]acetoacetate into amino acids in rat brain in vivo

The metabolism of ketone bodies by rat brain was studied in vivo. Rats starved for 48h were given either d-beta-hydroxy[3-(14)C]butyrate or [3-(14)C]acetoacetate by intravenous injection and killed after 3 or 10min. Total radioactivity in the acid-soluble material of the brain and the specific radioactivities of the brain amino acids glutamate, glutamine, aspartate and gamma-aminobutyrate were determined. A group of fed animals were also given d-beta-hydroxy[3-(14)C]butyrate. In the brains of all animals (14)C was present in the acid-soluble material and the specific radioactivity of glutamate was greater than that of glutamine.     

Incorporation of [14C]glucosamine and [14C]leucine into mouse kidney β-glucuronidase induced by gonadotrophin

Male BALB/C mice were injected intraperitoneally with 2.5 i.u. of gonadotrophin. After the injection, increase of β-glucuronidase activity was first observed in the microsomal fraction. By 36h 45–50% of the total homogenate activity was found in the microsomal fraction compared with 20–25% in the control microsomal fraction. From 36 to 80h not only microsomal β-glucuronidase but also lysosomal β-glucuronidase increased progressively. After 69h stimulation with 2.5 i.u. of gonadotrophin, d-[1-¹⁴C]glucosamine or l-[U-¹⁴C]leucine was injected intraperitoneally. After a further 3h the kidneys were homogenized and five particulate fractions were prepared by differential centrifugation. The β-glucuronidase in the microsomal and lysosomal fractions was released respectively by ultrasonication and by freezing and thawing treatment. The enzyme was purified by organic-solvent precipitation and by sucrose-density-gradient centrifugation. The results demonstrated the incorporation of these two labels into the mouse renal β-glucuronidase. The microsomal β-glucuronidase was much more radioactive than the lysosomal enzyme and approx. 80% of the newly synthesized enzyme appeared in microsomes and approx. 20% of that was found in lysosomes at this period. These results suggest that the mouse renal β-glucuronidase is a glycoprotein and that the newly synthesized enzyme is transported from endoplasmic reticulum to lysosomes.