Synthesis and Biological Evaluation of Some Acyclic 4,6-Disubstituted 1H-Pyrazolo[3,4-d]pyrimidine Nucleosides

Abstract

The chemical synthesis and biological evaluation of some acyclic α-[6-(1′-carbamoylalkylthio)-1H-pyrazolo[3,4-d]pyrimidin-4-yl]thioalkylamide nucleosides are described.     

Chemo-enzymatic Synthesis of 3-Deoxy-β-D-ribofuranosyl Purines and Study of Their Biological Properties

Abstract

9-(3-Deoxy-β-d-erythro-pentofuranosyl)-2,6-diaminopurine (2) was synthesized by an enzymatic transglycosylation of 2,6-diaminopurine using 3′-deoxycytidine (1) as a donor of the sugar moiety. Nucleoside 2 was transformed to 3′-deoxy guanosine (3), 9-(3-deoxy-β-d-erythro-pentofuranosyl)-2-amino-6-oxopurine (3′-deoxyisoguanosine; 4), and 9-(3-deoxy-β-d-erythro-pentofuranosyl)-2-fluoroadenine (5). Compounds 2–5 were evaluated for their anti-HIV activity.     

Direct adenylation from 5′-OH-terminated oligonucleotides by a fusion enzyme containing Pfu RNA ligase and T4 polynucleotide kinase

5′-Adenylated oligonucleotides (AppOligos) are widely used for single-stranded DNA/RNA ligation in next-generation sequencing (NGS) applications such as microRNA (miRNA) profiling. The ligation between an AppOligo adapter and target molecules (such as miRNA) no longer requires ATP, thereby minimizing potential self-ligations and simplifying library preparation procedures. AppOligos can be produced by chemical synthesis or enzymatic modification. However, adenylation via chemical synthesis is inefficient and expensive, while enzymatic modification requires pre-phosphorylated substrate and additional purification. Here we cloned and characterized the Pfu RNA ligase encoded by the PF0353 gene in the hyperthermophilic archaea Pyrococcus furiosus. We further engineered fusion enzymes containing both Pfu RNA ligase and T4 polynucleotide kinase. One fusion enzyme, 8H-AP, was thermostable and can directly catalyze 5′-OH-terminated DNA substrates to adenylated products. The newly discovered Pfu RNA ligase and the engineered fusion enzyme may be useful tools for applications using AppOligos.     

Comparison of Hi-C results using in-solution versus in-nucleus ligation

BackgroundChromosome conformation capture and various derivative methods such as 4C, 5C and Hi-C have emerged as standard tools to analyze the three-dimensional organization of the genome in the nucleus. These methods employ ligation of diluted cross-linked chromatin complexes, intended to favor proximity-dependent, intra-complex ligation. During development of single-cell Hi-C, we devised an alternative Hi-C protocol with ligation in preserved nuclei rather than in solution. Here we directly compare Hi-C methods employing in-nucleus ligation with the standard in-solution ligation.ResultsWe show in-nucleus ligation results in consistently lower levels of inter-chromosomal contacts. Through chromatin mixing experiments we show that a significantly large fraction of inter-chromosomal contacts are the result of spurious ligation events formed during in-solution ligation. In-nucleus ligation significantly reduces this source of experimental noise, and results in improved reproducibility between replicates. We also find that in-nucleus ligation eliminates restriction fragment length bias found with in-solution ligation. These improvements result in greater reproducibility of long-range intra-chromosomal and inter-chromosomal contacts, as well as enhanced detection of structural features such as topologically associated domain boundaries.ConclusionsWe conclude that in-nucleus ligation captures chromatin interactions more consistently over a wider range of distances, and significantly reduces both experimental noise and bias. In-nucleus ligation creates higher quality Hi-C libraries while simplifying the experimental procedure. We suggest that the entire range of 3C applications are likely to show similar benefits from in-nucleus ligation.

Electronic supplementary material

The online version of this article (doi:10.1186/s13059-015-0753-7) contains supplementary material, which is available to authorized users.     

A Polymeric Support for Chemical and Enzymatic Nucleic Acid Synthesis (1)

Abstract

A non-swellable, highly porous support material -CPG 3000 - was used in building up covalently bound nucleic acids by combined chemical and enzymatic methods. Bases are optimal accessible for hybridization and enzymatic reactions because they are not involved in the linking procedure.     

CNS delivery of l-dopa by a new hybrid glutathione–methionine peptidomimetic prodrug

Parkinson’s disease (PD) is a neurodegenerative disorder associated primarily with loss of dopamine (DA) neurons in the nigrostriatal system. With the aim of increasing the bioavailability of l-dopa (LD) after oral administration and of overcoming the pro-oxidant effect associated with LD therapy, we designed a peptidomimetic LD prodrug (1) able to release the active agent by enzyme catalyzed hydrolysis. The physicochemical properties, as well as the chemical and enzymatic stabilities of the new compound, were evaluated in order to check both its stability in aqueous medium and its sensitivity towards enzymatic cleavage, providing the parent LD drug, in rat and human plasma. The radical scavenging activities of prodrug 1 was tested by using both the DPPH–HPLC and the DMSO competition methods. The results indicate that the replacement of cysteine GSH portion by methionine confers resistance to oxidative degradation in gastric fluid. Prodrug 1 demonstrated to induce sustained delivery of DA in rat striatal tissue with respect to equimolar LD dosages. These results are of significance for prospective therapeutic application of prodrug 1 in pathological events associated with free radical damage and decreasing DA concentration in the brain.     

Synthesis and anti-HCMV activity of novel cyclopropyl phosphonic acid nucleosides

A simple synthetic route for novel acyclic phosphonate nucleosides is described. The characteristic cyclopropyl moiety 8 was constructed employing the Simmons-Smith reaction as key step starting from simple acyclic 2-butene-1,4-diol. The condensation of the mesylate 11 with natural nucleosidic bases (A,C,T,U) under nucleophilic substitution conditions (K₂CO₃, 18-Crown-6, DMF) and hydrolysis afforded the target nucleosides 16, 17, 18, and 19. In addition, the antiviral evaluations against various viruses were performed.     

The diaphragm is better protected from oxidative stress than hindlimb skeletal muscle during CLP-induced sepsis

Objectives: The aim of this study was to determine whether non-lethal sepsis induced by cecal ligation and puncture (CLP) modulates oxidative damage and enzymatic antioxidant defenses in diaphragm and hindlimb skeletal muscles (soleus and Extensor Digitorus Longus (EDL)).

Methods: Female Wistar rats were divided into four experimental groups: (1) control animals, (2) animals sacrificed 2?hours or (3) 7 days after CLP, and (4) sham-operated animals. At the end of the experimental procedure, EDL, soleus, and diaphragm muscles were harvested and 4-hydroxynonenal (HNE)-protein adducts and protein carbonyl contents were examined in relation to superoxide dismutase and catalase expression and activities.

Results: We observed that both non-respiratory oxidative (i.e. soleus) and glycolytic skeletal muscles (i.e. EDL) are more susceptible to sepsis-induced oxidative stress than diaphragm, as attested by an increase in 4-HNE protein adducts and carbonylated proteins after 2?hours of CLP only in soleus and EDL.

Discussion: These differences could be explained by higher basal enzymatic antioxidant activities in diaphragm compared to hindlimb skeletal muscles. Together, these results demonstrate that diaphragm is better protected from oxidative stress than hindlimb skeletal muscles during CLP-induced sepsis.     

Synthesis and Enzymatic Studies of Modified Oligonucleotides with Abiological Monomer Units

Abstract

The synthesis and the enzymatic studies of modified oligonucleotides containing a PNA modified PNA-DNA dimer block and a new acyclic racemic serinol nucleoside is described. We show that both, the PNA-DNA dimer block¹ and the modified PNA-spacer (acyclic serinol nucleoside)² can be used as modified templates for the enzymatic generation of single stranded DNA. Degradation studies of the oligonucleotides containing the PNA-DNA dimer block with snake venom phosphodiesterase show that the modified oligonucleotides are stable towards exonucleolytic degradation.     

Preparation of Oligodeoxynucleotides Containing 6-Methylthiopurine Residues by Chemical Synthesis or Specific Methylation

Abstract

Two methods (chemical synthesis and specific methylation) are described for the preparation of oligodeoxynucleotides containing 6-methylthiopurine residues. 6-Methylthiopurine phosphoramidite (6) is prepared and incorporated into oligomers. Methylation with methyl iodide of 6-thiopurine (or 6-thioguanine) in oligonucleotides also leads to exclusive production of 6-methylthiopurine (or 6-methylthioguanine) oligomers.     

Efficient synthesis of exomethylene- and keto-exomethylene-d-glucopyranosyl nucleoside analogs as potential cytotoxic agents

A novel series of exomethylene- and keto-exomethylene-d-glucopyranonucleosides with thymine, uracil, and 5-fluorouracil as heterocyclic bases have been designed and synthesized. Wittig condensation of the 3-keto glucoside 1 gave the corresponding 1,2:5,6-di-O-isopropylidene-3-deoxy-3-methylene-d-glucofuranose (2), which after hydrolysis and acetylation led to the precursor 1,2,4,6-tetra-O-acetyl-3-deoxy-3-methylene-d-glucopyranose (4).Compound 4 was condensed with silylated thymine, uracil, and 5-fluorouracil, respectively, deacetylated and acetalated to afford 1-(3′-deoxy-4′,6′-O-isopropylidene-3′-methylene-β-d-glucopyranosyl)pyrimidines 7a–c. Oxidation of the free hydroxyl group in the 2′-position of the sugar moiety led to the formation of the labile 1-(3′-deoxy-4′,6′-O-isopropylidene-3′-methylene-β-d-glucopyranosyl-2′-ulose)pyrimidines 8a–c. Finally, deisopropylidenation of the resulted derivatives 8a–c afforded the diol nucleosides 9a–c. The target keto-exomethylene analogs 9a–c were more cytostatic against a variety of tumor cell lines than the corresponding saturated-hydroxy-exomethylene derivatives 6. In particular, the 5-fluorouracil derivative 9c was highly cytostatic at an IC₅₀ (50% inhibitory concentration) ranging between 0.56 and 9.4 μg/mL, which was comparable to the free parental 5-fluorouracil base.     

Synthesis and Anti-HIV Activity of β-D-3′-Azido-2′,3′-unsaturated Nucleosides and β-D-3′-Azido-3′-deoxyribofuranosylnucleosides

Since the discovery of 3′-azido-3′-deoxythymidine (AZT) and 2′,3′-didehydro-2′,3′-dideoxythymidine (d4T) as potent and selective inhibitors of the replication of human immunodeficiency virus (HIV), there has been a growing interest for the synthesis of 2′,3′-didehydro-2′,3′-dideoxynucleosides with electron withdrawing groups on the sugar moiety. Here we described an efficient method for the synthesis of such nucleoside analogs bearing structural features of both AZT and d4T. The key intermediate, 3-azido-1,2-bis-O-acetyl-5-O-benzoyl-3-deoxy-D-ribofuranose, 5 was synthesized from commercially available D-xylose in five steps, from which a series of pyrimidine and purine nucleosides were synthesized in high yields. The resultant protected nucleosides were converted to target nucleosides using appropriate chemical modifications. The final nucleosides were evaluated as potential anti-HIV agents.     

Discovery of a nanomolar inhibitor of lung adenocarcinoma in vitro

Efficient methods for the preparation of 5′-substituted 5′-amino-5′-deoxy-N⁶-ureidoadenosine derivatives are described. Compounds were screened for antiproliferative activity against a panel of murine and human cell lines (L1210, CEM, and HeLa) and/or against the NCI-60. The most potent derivative inhibited the lung adenocarcinoma cell line NCI-H522 at low nanomolar concentrations (GI₅₀ = 9.7 nM).     

Chemico-enzymatic method of synthesis of mixed oligonucleotides

Oligo (ribodeoxyribo)nucleotide d(TGTGTAT)r(GCCAU) was synthesized from oligonucleotide blocks by chemical ligation, the oligoribonucleotide and oligodeoxynucleotide blocks being prepared by enzymatic and chemical synthesis, resp. A general procedure of the hybride duplex strand separation is described.     

A Versatile Synthesis of Dihydropyrimidinone C‐Nucleosides

A versatile synthesis of N‐substituted dihydropyrimidinone C‐nucleosides (20–29) is described. Glycosyl amino esters (3–9), obtained by reductive alkylation of glycosyl amino esters 1 and 2, on condensation with different isocyanates afforded respective ureido derivatives (10–19) in good to quantitative yields. The latter on cyclative amidation with a combination of DBU/TBAB (tetrabutylammonium bromide)/4Å molecular sieve gave the corresponding nucleosides (20–29) in good yields.     

Solid-phase Parallel Synthesis of 4-β-D-Ribofuranosylpyrazolo[4,3-d]pyrimidine Nucleosides

The synthesis of pyrazolo[4,3-d]pyrimidine nucleoside library using solid-phase parallel synthesis methodology is described. Glycosylation of the trimethylsilyl (TMS) derivative of 1- and 2-(methyl)-1H and 2H-pyrazolo[4,3-d]pyrimidine-5,7-(4H,6H)-dione (5) with 1-O-acetyl-2,3,5-tri-O-benzoyl-D-ribofuranose in the presence of TMS triflate provided two novel protected nucleosides 6 and 7. The structures of 6 and 7 were assigned by 1H and 2D NMR experiments. Nucleosides 6 and 7 were then transformed to the key intermediates 12 and 15 respectively. Reaction of 12 and 15 with MMTCl resin in the presence of 2,6-lutidine afforded the necessary scaffolds B and C. Different amines (96) were introduced selectively by nucleophilic substitution on scaffolds B and C using solid-phase parallel semi-automated synthesizer. Cleavage of the products from the solid support with 30% HFIP in a parallel fashion yielded nucleoside libraries simultaneously, and they were analyzed and characterized by high-throughput LC-MS.     

Chemical reactions in double-stranded nucleic acids. The nature of the bond formed during chemical ligation using a cis-diol group

Chemical and enzymatic ligation between the 5'-terminal phosphate of one oligonucleotide and the 3'-terminal 2',3'-cis-diol group of the other oligonucleotide on a complementary template was studied. Carbodiimide, imidazolide and N-hydroxybenzotriazole ester methods were used for chemical activation of the phosphate group, and T4 DNA ligase for enzymatic ligation. All the chemical activation methods produced 3',5'- and 2',5'-phosphodiester bonds (40-45 and 55-60%, resp.), whereas enzymatic ligation gave the product only with 3',5'-phosphodiester bond.     

Synthesis and Properties of 2′-Deoxycytidine Triphosphate Carrying C-Myc Tag Sequence

Abstract

The synthesis of 2′-deoxycytidine triphosphate carrying mercaptoethyl groups at position 4 of cytosine is described. This nucleoside triphosphate was reacted with a maleimido-peptide carrying the c-myc tag-sequence to yield a peptide-nucleoside triphosphate chimera. Primer extension studies showed that the nucleoside triphosphate modified with the peptide sequence is incorporated by DNA polymerases opposite guanine.     

Determination of pseudouridine and other nucleosides in human blood serum by high-performance liquid chromatography

A specific, sensitive, and rapid method to measure pseudouridine in human blood serum is described. The method is based on the following steps: (i) deproteinization of serum samples by filtration on membrane cones or by acetonitrile; (ii) purification of nucleosides and concentration of the sample by affinity chromatography on phenylboronate gel followed by lyophilization; and (iii) separation of nucleosides and their quantitation by reserse-phase high-performance liquid chromatography.The pseudouridine mean value in 30 normal subjects was 2.52 ± 0.28 nmol/ml. The procedure also allows the identification of inosine, uridine, guanosine, and adenosine. Nevertheless, the presence in human blood serum of enzymatic activities which convert adenosine to inosine and cytidine to uridine prevents the precise quantitation of these nucleosides. All the compounds were identified by comparing their retention times and absorbance ratios ($\text{A}{}_{280}\text{A}{}_{254}$) with those of pure compounds, as well as by cochromatography.