4-Guanidino-2-pyrimidinone Nucleobases: Synthesis and Hybridization Properties

Abstract

N-Alkylated 4-guanidino-2-pyrimidinone-containing nucleosides, in which the guanidine group mimics the double hydrogen bond donor pattern of protonated cytosine, were introduced in polypyrimidine sequences to explore their triple-helix forming capabilites. UV and CD melting experiments showed that strands containing these base analogues did not form triplex complexes.     

Desulfurization of 2-thiouracil nucleosides: conformational studies of 4-pyrimidinone nucleosides

4-Pyrimidinone ribofuranoside (H(2)o(4)U) and 4-pyrimidinone 2'-deoxyribofuranoside (dH(2)o(4)U) were synthesized by the oxidative desulfurization of parent 2-thiouracil nucleosides with m-chloroperbenzoic acid. The crystal structures of H(2)o(4)U and dH(2)o(4)U and their conformations in solution were determined and compared with corresponding 2-thiouracil and uracil nucleosides. The absence of a large 2-thiocarbonyl/2-carbonyl group in the nucleobase moiety results in C2'-endo puckering of the ribofuranose ring (S conformer) in the crystal structure of H(2)o(4)U, which is not typical of RNA nucleosides. Interestingly, the hydrogen bonding network in the crystals of dH(2)o(4)U stabilizes the sugar moiety conformation in the C3'-endo form (N conformer), rarely found in DNA nucleosides. In aqueous solution, dH(2)o(4)U reveals a similar population of the C2'-endo conformation (65%) to that of 2'-deoxy-2-thiouridine (62%), while the 62% population of the S conformer for H(2)o(4)U is significantly different from that of the parent 2-thiouridine, for which the N conformer is dominant (71%). Such a difference may be of biological importance, as the desulfurization process of natural tRNA 2-thiouridines may occur under conditions of oxidative stress in the cell and may influence the decoding process.     

The synthesis of 2-pyrimidinone nucleosides and their incorporation into oligodeoxynucleotides.

The synthesis of 1-(beta-D-2'-deoxyribosyl)-2-pyrimidinone (dK) and its 5-methyl derivative (d5) from 2'-deoxycytidine or 2'-deoxythymidine, respectively, via silver oxide oxidation of 4-hydrazinopyrimidines is described. The necessary hydrazine substituted pyrimidine nucleosides have been prepared by transamination of a protected cytidine derivative or by addition/elimination reactions to an O4-sulfonated thymidine derivative. Oxidation of the 4-hydrazino pyrimidines was complicated by a competing hydrolytic reaction which generated 2'-deoxyuridine or 2'-deoxythymidine. However, in the presence of an organic base such as triethylamine, oxidation became the predominant reaction. After suitable protection and formation of the 3'-phosphoramidite derivatives, these modified nucleosides were incorporated into seven self-complementary oligodeoxynucleotides by chemical synthesis using phosphite triester methodology. Oligodeoxynucleotides were prepared such that dA-dT and dG-dC base pairs were substituted by dA-d5 or dG-dK base pairs, respectively. Both circular dichroism spectra and thermal denaturation studies were used to characterize the modified oligodeoxynucleotides.     

Attachment of cholesterol to amino-LNA: synthesis and hybridization properties

Here, we present our synthesis of amino-LNA with a C6-linker and hybridization studies of these. A cholesterol moiety was attached at the end of the C6-linker. This resulted in drastic drops against DNA of the modified oligonucleotide.     

The synthesis, physicochemical properties and immunological activity of 5-amino-3-methylisoxazolo[5,4-d]4-pyrimidinone derivatives

A series of 5-amino-3-methylisoxazole[5,4-d]4-pyrimidinone derivatives were obtained by reacting substituted 5-amino-3-methylisoxazol-4-carboxylic acid hydrazide with ethyl ortho-formate. The compounds were tested using the models of in vivo cellular and humoral immune response in mice and pokeweed mitogen-induced (PWM-induced) polyclonal antibody production in a culture of human peripheral blood mononuclear cells (PBMC). The compounds exhibited differential inhibitory activities in the described models, depending on the character and location of the substituted groups. We suggest that the compounds affect the early stages of the immune response.     

Synthesis and in vitro cytotoxicity of 5-substituted 2-cyanoimino-4-imidazodinone and 2-cyanoimino-4-pyrimidinone derivatives

A series of 5-substituted 2-cyanoimino-4-imidazodinone and 2-cyanoimino-4-pyrimidinone derivatives were synthesized and their anticancer cytotoxicity were evaluated in in vitro assay. It was found that the bulky aryl functionality in the 5-position of the 2-cyanoimino-4-imidazolidinone compounds was essential for the cytotoxicity of these heterocyclic compounds. Some of the derivatives exhibited modest cytotoxicity against a variety of cancer cell lines. One of the derivatives, [1-[6-(4-chlorophenoxy)hexyl]-5-oxo-4-phenyl-3-(4-pyridyl)tetrahydro-1H-2-imidazolyliden]aminomethanenitrile (Compound 11), exhibited the most potent cytotoxic activity with IC(50) in the nanomolar range. The cytotoxicity of these derivatives was selection with no apparent toxic effect toward normal fibroblasts.     

4'-C-modified nucleosides: synthesis and antiviral properties

The synthetic methods for 4'-C-modified nucleosides as well as structure activity relationship of obtained compounds towards hepatitis C virus are reviewed.     

A convenient synthesis of a novel nucleoside analogue: 4-(alpha-diformyl-methyl)-1-(beta-D-ribofuranosyl)-2-pyrimidinone

The nucleoside analogue 4-(alpha-diformyl-methyl)-1-(beta-D-ribofuranosyl)-2-pyrimidinone (5) was prepared from the corresponding 4-methyl pyrimidinone nucleoside by means of the Vilsmeier reaction. The unprotected nucleoside can be phosphorylated directly with phosphorus oxychloride in triethyl phosphate.     

G/C-modified oligodeoxynucleotides with selective complementarity: synthesis and hybridization properties.

Modified oligodeoxyribonucleotides (ODNs) that have unique hybridization properties were designed and synthesized for the first time. These ODNs, called selective binding complementary ODNs (SBC ODNs), are unable to form stable hybrids with each other, yet are able to form stable, sequence specific hybrids with complementary unmodified strands of nucleic acid. To make SBC ODNs, deoxyguanosine (dG) and deoxycytidine (dC) were substituted with deoxyinosine (dI) and 3-(2'-deoxy-beta-D-ribofuranosyl)pyrrolo-[2,3-d]-pyrimidine-2-(3H)-one (dP), respectively. The hybridization properties of several otherwise identical complementary ODNs containing one or both of these nucleoside analogs were studied by both UV monitored thermal denaturation and non-denaturing PAGE. The data showed that while dI and dP did form base pairs with dC and dG, respectively, dI did not form a stable base pair with dP. A self-complementary ODN uniformly substituted with dI and dP acquired single-stranded character and was able to strand invade the end of a duplex DNA better than an unsubstituted ODN. This observation implies that SBC ODNs should effectively hybridize to hairpins present in single-stranded DNA or RNA.     

Simple chemical synthesis and hybridization properties of non-radioactive DNA probes

A simple chemical method for the synthesis of non-radioactive DNA probes is described: triazolyl-containing sequences were built by incorporation of 4-triazolylpyrimidin-2-ones instead of cytidines during oligodeoxyribonucleotide synthesis. The activating triazolyl groups were then displaced by a diamine which was further derivatized by a label, such as biotin. Synthesized DNA probes were oligonucleotides complementary to a cloned human antithrombin III DNA sequence. These probes, containing the same label at different positions of the sequence, were hybridized to their target DNA immobilized on nitrocellulose. Their hybridization specificity and stability were studied. Hybrid detection was performed either colorimetrically by the streptavidin-alkaline phosphatase-based system or by autoradiography after 5'-32P labeling of the probes: 15 fmol (0.05 microgram) of complementary sequence could be visualized in the two cases.     

8-aza-7-deazaadenine and 7-deazaguanine: synthesis and properties of nucleosides and oligonucleotides with nucleobases linked at position-8

The 8-aza-7-deazaadenine (pyrazolo[3,4-d]pyrimidin-4-amine) N8-(2'-deoxyribonucleoside) (2) and the 7-deazaguanine (pyrrolo[3,4-d]pyrimidine-2-amin-(3H)-4-one) C8-(2'-deoxyribonucleoside) (4) were synthesized and incorporated in oligonucleotides employing phosphoramidite chemistry. Oligonucleotides carrying compound 2 are able to form base pairs with the four canonical DNA constituents without significant structural discrimination. The nucleoside 4 was obtained from the corresponding ribonucleoside by deoxygenation. Oligonucleotides containing compound 4 showed similar base pairing properties as those with 2'-deoxyisoguanosine.     

Oligo(2'-O-tetrahydropyranylribonucleotides): hybridization properties and nuclease resistance

Oligo(2'-tetrahydropyranylribonucleotides) and their analogues containing a 3'-3'-internucleotide bond at the 3'-terminus are nuclease-resistant and possess rather high affinity toward RNA, the main target in the antisense approach.     

Solid-phase synthesis and hybridization properties of DNA containing sulfide-linked dinucleosides.

Oligodeoxyribonucleotides incorporating non-hydrolyzable dialkyl sulfide linked thymidine dimers (TsT) were synthesized chemically by the solid-phase approach. The sulfide dimer TsT was stable to degradation by snake-venom phosphodiesterase, calf spleen phosphodiesterase, Nuclease P1 and Nuclease S1. Thermal denaturation analysis indicated that the incorporation of TsT dimers into DNA weakened, but did not prevent, binding to complementary DNA and RNA over a wide range of salt concentrations (10 mM to 2 M NaCl).     

Investigation of some properties of oligodeoxynucleotides containing 4'-thio-2'-deoxynucleotides: duplex hybridization and nuclease sensitivity.

The thermal stabilities of the duplexes formed between 4'-thio-modified oligodeoxynucleotides and their DNA and RNA complementary strands were determined and compared with those of the corresponding unmodified oligodeoxynucleotides. A 16mer oligodeoxynucleotide containing 10 contiguous 4'-thiothymidylate modifications formed a less stable duplex with the DNA target (deltaTm/modification -1.0 degrees C) than the corresponding unmodified oligodeoxynucleotide. However, when the same oligodeoxynucleotide was bound to the corresponding RNA target, a small increase in Tm was observed (deltaTm/modification +0.16 degrees C) when compared with the unmodified duplex. A study to identify the specificity of an oligodeoxynucleotide containing a 4'-thiothymidylate modification when forming a duplex with DNA or RNA containing a single mismatch opposite the modification found the resulting Tms to be almost identical to the wild-type duplexes, demonstrating that the 4'-thio-modification in oligodeoxynucleotides has no deleterious effect on specificity. The nuclease stability of 4'-thio-modified oligodeoxynucleotides was examined using snake venom phosphodiesterase (SVPD) and nuclease S1. No significant resistance to degradation by the exonuclease SVPD was observed when compared with the corresponding unmodified oligodeoxynucleotide. However, 4'-thio-modified oligodeoxynucleotides were found to be highly resistant to degradation by the endonuclease S1. It was also demonstrated that 4'-thio-modified oligodeoxynucleotides elicit Escherichia coli RNase H hydrolysis of the RNA target only at high enzyme concentration.     

4'-alpha-C-Branched N,O-nucleosides: synthesis and biological properties

The synthesis of 4'-alpha-C-branched N,O-nucleosides has been described, based on the 1,3-dipolar cycloaddition of nitrones with vinyl acetate followed by coupling with silylated nucleobases, The obtained compounds have been evaluated for their activity against HSV-1, HSV-2, HTLV-1. Cytotoxicity and apoptotic activity have been also investigated: compound 10c shows moderate apoptotic activity in Molt-3 cells.     

Conformationally locked nucleosides. Synthesis and hybridization properties of oligodeoxynucleotides containing 2',4'-C-bridged 2'-deoxynucleosides.

A conformationally locked, 2',4'-C-bridged 2'-deoxyribofuranoside2 was condensed with silylated pyrimidines to give 2',4'-C-bridged bicyclonucleosides, which were converted to the phosphoramidites and incorporated into oligodeoxynucleotides (ODNs). The hybridization data of the modified ODNs to DNA and RNA are presented.     

Design,synthesis and antimicrobial evaluation of novel 1-benzyl 2-butyl-4-chloroimidazole embodied 4-azafluorenones via molecular hybridization approach

A series of novel 1-benzyl-2-butyl-4-chloroimidazole embodied 4-azafluorenone hybrids, designed via molecular hybridization approach, were synthesized in very good yields using one pot condensation of 1-benzyl-2-butyl-4-chloroimidazole-5-carboxaldehyde, 1,3-indanedione, aryl/heteroaryl methyl ketones and ammonium acetate. All the synthetic derivatives were fully characterized by spectral data and evaluated for antimicrobial activity by disc diffusion method against selected bacteria and fungal strains. Among the 15 new compounds screened, 4-(1-benzyl-2-butyl-4-chloro-1H-imidazol-5-yl)-2-(furan-2-yl)-5H-indeno[1,2-b]pyridin-5-one(10k) has pronounced activity with higher zone of inhibition (ZoI) against Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella pneumoniae, Aspergillus flavus and Candida albicans. Also 4-(1-benzyl-2-butyl-4-chloro-1H-imidazol-5-yl)-2-(dibenzo[b,d]thiophen-2-yl)-5H-indeno [1,2-b]pyridin-5-one (10n) and 4-(1-benzyl-2-butyl-4-chloro-1H-imidazol-5-yl)-2-(3-tosyl-3H-inden-1-yl)-5H-indeno[1,2-b]pyridin-5-one (10o) showed selective higher inhibitory activity against Aspergillus flavus and Candida albicans. The results demonstrated potential importance of molecular hybridization in the development of 10k as potential antimicrobial agent.     

Biosynthesis of riboflavin: structure and properties of 2,5-diamino-6-ribosylamino-4(3H)-pyrimidinone 5'-phosphate reductase of Methanocaldococcus jannaschii

The pyrimidine reductase of the riboflavin biosynthetic pathway (MjaRED) specified by the open reading frame MJ0671 of Methanocaldococcus jannaschii was expressed in Escherichia coli using a synthetic gene. The synthetic open reading frame that was optimized for expression in E. coli directed the synthesis of abundant amounts of the enzyme with an apparent subunit mass of 25 kDa. The enzyme was purified to apparent homogeneity and was shown to catalyze the conversion of 2,5-diamino-6-ribosylamino-4(3H)-pyrimidinone 5'-phosphate into 2,5-diamino-6-ribitylamino-4(3H)-pyrimidinone 5'-phosphate at a rate of 0.8 micromol min(-1) mg(-1) at pH 8.0 and at 30 degrees C. The protein is a homodimer as shown by sedimentation equilibrium analysis and sediments at an apparent velocity of 3.5 S. The structure of the enzyme in complex with the cofactor nicotinamide adenine dinucleotide phosphate was determined by X-ray crystallography at a resolution of 2.5 Angstroms. The folding pattern resembles that of dihydrofolate reductase with the Thermotoga maritima ortholog as the most similar structure. The substrate, 2,5-diamino-6-ribosylamino-4(3H)-pyrimidinone 5'-phosphate, was modeled into the putative active site. The model suggests the transfer of the pro-R hydrogen of C-4 of NADPH to C-1' of the substrate.