PNA-DNA Chimeras Containing 5-Alkynyl-pyrimidine PNA Units. Synthesis,Binding Properties,and Enzymatic Stability

Abstract

Three chimeric dimer synthons (oeg_t^NHT, oeg_up^NHT and oeg_uh^NHT) containing thymine (t), 5-(l-propynyl)-uracil (up) and 5-(1-hexyn-1-yl)-uracil (uh) PNA units with N-(2-hydroxyethyl)glycine (oeg) backbone were synthesized in solution and incorporated into T₂₀ oligonucleotide analogues, using standard P-amidite chemistry. Insertion of dimer blocks led to destabilization of duplexes with dA₂₀ target. The smallest T _m drops were found for chimeras containing oeg_up^NHT dimers. Incorporation of the chimeric synthons into the 3′-end of T₂₀ brought about growing resistance to 3′-exonucleolytic (SV PDE) cleavage in the order of oeg_t^NHT < oeg_up^NHT < oeg_uh^NHT. Due to different endonuclease activities of 3′- and 5′-exonucleases applied, placing of five consecutive dimers at the 5′-terminus resulted in a relatively smaller, but also side-chain dependent, stabilization towards the hydrolysis by 5′-exonuclease (BS PDE). Neither exonucleases (SV and BS PDE) nor an endonuclease (Nuclease P₁) could hydrolyse the unnatural phosphodiester bond linking the 3′-OH of thymidine to the terminal OH of N-(2-hydroxyethyl)glycine PNA backbone.     

Nucleosides and Nucleotides. 125. Synthesis and Biological Evaluation of 2′,3′-Dideoxy-3′-fluoro-2′-methylidene Pyrimidine Nucleosides

Abstract

Reaction of 2′-deoxy-2′-methylidene-5′-O-trityluridine (1) with diethylamino-sulfur trifluoride (DAST) in CH₂Cl₂ resulted in the formation of a mixture of (3′R)-2′,3′-dideoxy-3′-fluoro-2′-methylidene derivative 3 and 2′,3′-didehydro-2′,3′-dideoxy-2′-fluoromethyl derivative 4 (3:4 = 1:1.5) in 65% yield. A similar treatment of 1-(2-deoxy-2-methylidene-5-O-trityl-β-D-threo-pentofuranosyl)uracil (19) with DAST in CH₂Cl₂ afforded (3′S)-2′,3′-dideoxy-3′-fluoro-2′-methylidene derivatives 20 and 4 in 38% and 17% yields respectively. Transformation of the uracil nucleosides 4, 12, and 20 into cytosines followed by deprotection furnished the corresponding cytidine derivatives 29, 18, and 25, respectively. The corresponding thymidine congener 27 was also synthesized in a similar manner. All of the newly synthesized nucleosides were evaluated for their inhibitory activities against HIV and for their antiproliferative activities against L1210 and KB cells.     

Synthesis and anticancer activity of 3′-[4-fluoroaryl-(1,2,3-triazol-1-yl)]-3′-deoxythymidine analogs and their phosphoramidates

Abstract

A series of novel 4-chlorophenyl N-alkyl phosphoramidates of 3′-[4-fluoroaryl-(1,2,3-triazol-1-yl)]-3′-deoxythymidines (20–49) was synthesized by means of phosphorylation of 3′-[4-aryl-(1,2,3-triazol-1-yl)]-3′-deoxythymidines (7–11) with 4-chlorophenyl phosphoroditriazolide (14), followed by a reaction with the appropriate amine. The synthesized compounds 7–11 and 20–49 were evaluated along with four known anticancer compounds for their cytotoxic activity in human cancer cell lines: cervical (HeLa), nasopharyngeal (KB), breast (MCF-7), osteosarcoma (143B) (only selected compounds 20, 24, 28, 32–36, 38, 40, 46) and normal human dermal fibroblast cell line (HDF) using the sulforhodamine B (SRB) assay. Among 3′-[4-aryl-(1,2,3-triazol-1-yl)]-3′-deoxythymidines (7–11) the highest activity in all the investigated cancer cells was displayed by 3′-[4-(3-fluorophenyl)-(1,2,3-triazol-1-yl)]-3′-deoxythymidine (9) (IC₅₀ in the range of 2.58–3.61?μM) and its activity was higher than that of cytarabine. Among phosphoramidates 20–49 the highest activity was demonstrated by N-n-propyl phosphoramidate of 3′-[4-(3-fluorophenyl)-(1,2,3-triazol-1-yl)]-3′-deoxythymidine (35) in all the cancer cells (IC₅₀ in the range of 0.97–1.94?μM). Also N-ethyl phosphoramidate of 3′-[4-(3-fluorophenyl)-(1,2,3-triazol-1-yl)]-3′-deoxythymidine (33) exhibited good activity in all the used cell lines (IC₅₀ in the range of 4.79–4.96?μM).     

PNA-DNA Chimeras Forming Quadruplex Structures

Abstract

¹H-NMR, CD, and UV spectroscopy have been used to investigate the structure of PNA/DNA chimeras forming quadruplex structures. In particular, we synthesized ^5′TGGG^3′-t (1) and ^5′TGG^3′-gt (2), where lower and upper case letters indicate PNA and DNA residues, respectively. CD spectrum and all NMR data of (1) are typical of quadruplexes involving four parallel strands. UV melting profile of (1) indicates that its thermal stability is quite similar to that observed for the reference structure [d(TGGGT)]₄. ¹H-NMR spectrum for ^5′TGG^3′-gt (2) shows that this oligonucleotide is not able to fold into a single, well-defined species.     

Synthesis of (2′,5′)-Oligoadenylate Antisense Chimeras Targeting Steroid 5α-Reductase

Abstract

2–5A antisense chimeras have been synthesized which target human steroid 5α-reductase mRNA. To enhance the stability of the chimera towards degradative enzymes the terminal phosphodiester bond was isomerized from 3′,5′ to 3′,3′ and the 5′-phosphate group was thiolated.     

Synthesis of 2′,3′-Dideoxy-2′-fluoro-3′-thioarabinothymidine and Its 3′-Phosphoramidite Derivative

Abstract

An efficient method for the synthesis of 5′-O-monomethoxytrityl-2′,3′-dideoxy-2′-fluoro-3′-thioarabinothymidine [^5′-MMTaraF-T_3′SH, (5)] and its 3′-phosphoramidite derivative (6) suitable for automated incorporation into oligonucleotides, is demonstrated. A key step in the synthesis involves reaction of 5′-O-MMT-2,3′-O-anhydrothymidine (4) (Eleuteri, A.; Reese, C.B.; Song, Q., J. Chem. Soc. Perkin Trans. 1 1996, 2237 pp.) with sodium thioacetate to give ^5′-MMTaraF-T_3′SAc (5) (Elzagheid, M.I.; Mattila, K.; Oivanen, M.; Jones, B.C.N.M.; Cosstick, Lönnberg, H. Eur. J. Org. Chem. 2000, 1987–1991). This nucleoside was then converted to its corresponding phosphoramidite derivative, 6, as described previously ((a) Sun, S.; Yoshida, A.; Piccirilli, J.A. RNA, 1997, 3, 1352–1363; (b) Matulic-Adamic, J.; Beigelman, L. Helvetica Chemica Acta 1999, 82, 2141–2150; (c) Fettes, K.J.; O’Neil, I.; Roberts, S.M.; Cosstick, R. Nucleosides, Nucleotides and Nucl. Acids 2001, 20, 1351–1354).     

Definitive Solution Structures for the 6-Formylated Versions of 1-(βD-Ribofuranosyl)-, 1-(2′-Deoxy-β-D-Ribofuranosyl)-, and 1-β-D-Arabinofuranosyluracil,and of Thymidine

Abstract

ROESY and NOESY NMR spectroscopic analyses of the ribofuranosyl (1a), 2′-deoxyribofuranosyl (1b), and arabinofuranosyl (1c) derivatives of 6-formyluracil in (CD₃)₂SO and D₂O solutions have established that each exclusive 7,05′-cyclic hemiacetal diastereomer of 1a,b and the major 7,O2′-cyclic hemiacetal diastereomer of 1c possess the 7R configuration. In addition, (7R)-1c has been shown to be thermodynamically more stable than (7S)-1c, contrary to our previous indication. A new, higher yielding synthetic route to 1a has been developed, 1b has been obtained for the first time in crystalline form, the route to 1c has been modified to better accommodate large scale preparations, and a new, fourth member of this class, 6-formylthymidine (1d), has been synthesized and its solution structures in (CD₃)₂SO, D₂O, and CD₃OD have been determined. Antitumor and antiviral evaluations of 1a-c have revealed no significant levels of activity.     

A novel cadmium(II) complex of bipyridine derivative: synthesis,X-ray crystal structure,DNA-binding and antibacterial activities

Abstract

A mononuclear cadmium(II) complex of formula [Cd(5,5′-dmbipy)₂(OAc)₂]·2H₂O (5,5′-dmbipy = 5,5′-dimethyl-2,2′-bipyridine and OAc?=?acetato ligand) has been synthesized and characterized by FT-IR, UV–Vis, ¹H-NMR, elemental analysis and single-crystal X-ray structure analysis. The molecular structure of the complex shows a distorted tetragonal antiprism CdN₄O₄ coordination geometry around the cadmium atom, resulting in coordination by four nitrogen atoms from two 5,5′-dmbipy ligands and four oxygen atoms from two acetate anions. The interaction of this complex to FS-DNA (fish sperm DNA) has also been studied by electronic absorption, fluorescence and gel electrophoresis techniques. Binding constant (K_b), Stern–Volmer constant (K_sv), number of binding sites (n) and bimolecular quenching rate constant (k_q) have been calculated from these spectroscopic data. These results have revealed that the metal complex can bind effectively to FS-DNA via groove binding. The calculated thermodynamic parameters (ΔH°, ΔS° and ΔG°) show that hydrogen bonding and van der Waals forces have an important function in the Cd(II) complex–DNA interaction. The antibacterial effects of the synthesized cadmium complex have also been examined in vitro against standard bacterial strains: one Gram-positive (Staphylococcus aureus, ATCC 25923) and one Gram-negative (Escherichia coli, ATCC 25922) bacteria, using disk diffusion and macro-dilution broth methods. The obtained results show that the Cd(II) complex exhibits a marked antibacterial activity which is significantly better than those observed for its free ligand and metal salt for both Gram-positive and Gram-negative bacteria. However, this metal complex is a more potent antibacterial agent against the Gram-positive than that of the Gram-negative bacteria.

Communicated by Ramaswamy H. Sarma     

Synthesis and Biological Properties of 2-Amino-3-fluoro-2,3-Dideoxy-D-Pentofuranosides of Natural Heterocyclic Bases

Abstract

The oxidation of methyl 5–0-benzyl-3-deoxy-3-fluoro-α-D-arabi-nofuranoside (1) with DMSO/Ac₂o afforded a ～ 2:1 mixture of 2-keto derivatives with erythro and threo configuration resulting from isomerization at C3. Successive treatment of the above mixture with MeONH₂, LiA1H₄, and S-ethyl trifluoroacetate followed by silica gel chromatography afforded methyl 5–0-benzyl-2, 3-dideoxy-3-fluoro-2-(trifluoroacetamido)-α-D-ribofuranoside (6b) and its lyxo isomer 7b in a total yield of 25% and 5%, respectively. The arabino analogue 25 was prepared from 6b. Compounds 6b, 7b and 25 were converted to the corresponding 5–0-benzoyl derivatives 8a, 9 and 26. A series of 2′-amino-2′, 3′-dideoxy-3′-fluoro-β-D-ribo- and-α-D-lyxofuranosides of natural heterocyclic bases have been synthesized starting from 8a and 9. None of the test compounds had any antiviral activity. 3′-Fluoro-2′-amino-2′, 3′-dideoxycytidine (16) was the only compound showing inhibition of murine L1210 and human Molt/4F cell proliferation (50% effective concentration: 39–42μg/m1).     

Tris-chelated complexes of nickel(II) with bipyridine derivatives: DNA binding and cleavage,BSA binding,molecular docking,and cytotoxicity

Abstract

Two nickel(II) complexes with substituted bipyridine ligand of the type [Ni(NN)₃](ClO₄)₂, where NN is 4,4′-dimethyl-2,2′-bipyridine (dimethylbpy) (1) and 4,4′-dimethoxy-2,2′-bipyridine (dimethoxybpy) (2), have been synthesized, characterized, and their interaction with DNA and bovine serum albumin (BSA) studied by different physical methods. X-ray crystal structure of 1 shows a six-coordinate complex in a distorted octahedral geometry. DNA-binding studies of 1 and 2 reveal that both complexes sit in DNA groove and then interact with neighboring nucleotides differently; 2 undergoes a partial intercalation. This is supported by molecular-docking studies, where hydrophobic interactions are apparent between 1 and DNA as compared to hydrogen bonding, hydrophobic, and π–π interactions between 2 and DNA minor groove. Moreover, the two complexes exhibit oxidative cleavage of supercoiled plasmid DNA in the presence of hydrogen peroxide as an activator in the order of 1?>?2. In terms of interaction with BSA, the results of spectroscopic methods and molecular docking show that 1 binds with BSA only via hydrophobic contacts while 2 interacts through hydrophobic and hydrogen bonding. It has been extensively demonstrated that the nature of the methyl- and methoxy-groups in ligands is a strong determinant of the bioactivity of nickel(II) complexes. This may justify the above differences in biomolecular interactions. In addition, the in vitro cytotoxicity of the complexes on human carcinoma cells lines (MCF-7, HT-29, and U-87) has been examined by MTT assay. According to our observations, 1 and 2 display cytotoxicity activity against selected cell lines.

Communicated by Ramaswamy H. Sarma     

Improved Synthesis of Adenosine Cyclic 3′, 5′-Phosphoramidates via Anhydrides. Preparation of Adenosine Cylic 3′, 5′-(Rp) - and (p)-N-Methylphosphoramidate

Abstract

The two-step method for the preparation of adenosine cyclic 3′,5′-phosphoramidate diastereoisomers, which involves the activation of adenosine cyclic 3′,5′-monophosphate (1) with an acid chloride and in situ aminolysis of the anhydride intermediate (Bentrude, W.G.; Tomsaz, J. Synthesis 1984, 27; Bottka, S.; Tomasz, J. Tetrahedron Lett. 1985, 24, 2909), has been improved. The best yields were attained when 1 was reacted with 4.4 molar equivalents of phosphorus oxychloride in trimethyl phosphate at O°C for 3 h, and the solution of phosphorus oxychloride in trimethyl phosphate was pretreated with 0.5 molar equivalent of water at room temperature for 20 min. R _p and S _p diastereoisomers of adenosine cyclic 3′,5′-N-methyphosphoramidate and N,N-dimethylphosphoramidate have been synthesized under these experimental conditions.     

Two new anti-plasmodial flavonoid glycosides from Duranta repens

The CHCl₃-soluble fraction of the whole plant of Duranta repens showed anti-plasmodial activity against the chloroquine-sensitive (D6) and chloroquine-resistant (W2) strains of Plasmodium falciparum, with IC₅₀ values of 8.5?±?0.9 and 10.2?±?1.5?μg/mL, respectively. From this fraction, two new flavonoid glycosides, 7-O-α-d-glucopyranosyl-3,4′-dihydroxy-3′-(4-hydroxy-3-methylbutyl)-5,6-dimethoxyflavone (1) and 7-O-α-d-glucopyranosyl(6′′′-p-hydroxcinnamoyl)-3,4′-dihydroxy-3′-(4-hydroxy-3-methylbutyl)-5,6-dimethoxyflavone (2), along with five known flavonoids, 3,7,4′-trihydroxy-3′-(4-hydroxy-3-methylbutyl)-5,6-dimethoxyflavone (3), 3,7-dihydroxy-3′-(4-hydroxy-3-methylbutyl)-5,6,4′-trimethoxyflavone (4), 5,7-dihydroxy-3′-(2-hydroxy-3-methyl-3-butenyl)-3,6,4′-trimethoxyflavone (5), 3,7-dihydroxy-3′-(2-hydroxy-3-methyl-3-buten-yl)-5,6,4′-trimethoxyflavone (6), and 7-O-α-d-glucopyranosyl-3,5-dihydroxy-3′-(4′′-acetoxy-3′′-methylbutyl)-6,4′-dimethoxyflavone (7), have been isolated as anti-plasmodial principles. Their structures were deduced by spectroscopic analysis including 1D and 2D NMR techniques. The compounds (1–7) showed potent anti-plasmodial activities against D6 and W2 strains of Plasmodium falciparum, with IC₅₀ values in the range of 5.2–13.5?μM and 5.9–13.1?μM, respectively.     

Design and Synthesis of Triazole-Linked xylo-Nucleoside Dimers

Three triazole-linked nonionic xylo-nucleoside dimers T^L-t-T^xL, T^L-t-A^BzxL and T^L-t-C^BzxL have been synthesized for the first time by Cu(I) catalyzed azide-alkyne [3 + 2] cycloaddition reaction (CuAAC) of 1-(3′-azido-3′-deoxy-2′-O,4′-C-methylene-β-D-ribo-furanosyl)thymine with different alkynes, i.e., 1-(5′-deoxy-5′-C-ethynyl-2′-O,4′-C-methylene-β-D-xylofuranosyl)thymine, 9-(5′-deoxy-5′-C-ethynyl-2′-O,4′-C-methylene-β-D-xylo-furanosyl)-N6-benzoyladenine and 1-(5′-deoxy-5′-C-ethynyl-2′-O,4′-C-methylene-β-D-xylofuranosyl)-N4-benzoylcytosine in 90%–92% yields. Among the two Cu(I) reagents, CuSO₄.5H₂O-sodium ascorbate in THF:^tBuOH:H₂O (1:1:1) and CuBr.SMe₂ in THF used for cycloaddition (click) reaction, the former one was found to be better yielding than the latter one.     

dC-dG Alternating Oligonucleotides: Thermodynamic and Kinetic Aspects of the B-Z Transformation

Abstract

The alternating cytosine-guanine oligodeoxyribonucleotides (dCdG)_n, (dGdC)_n, (dCdG)_n, (dCdG)_ndC (n=3,4), (dGdC)₇ and dG(dCdG)₃ have been studied by UV and CD spectroscopy at different temperatures and NaCl concentrations. The analysis of the melting data, assuming an all-or-none model, reveals that in the B-conformation the 5′G/C3′ stacking interactions are enthalpically favoured with respect to the 5′C/G3′ one. The CD investigation of the B-Z equilibrium shows that the Z-conformation is enthalpically stabilized, while the B-conformation is entropically favoured, in the range of NaCl concentration considered (1 to 5M). The kinetic data for the B-Z transformation, obtained with a salt-jump technique for the hexamer (dCdG)₃, support a mechanism by which (he Watson-Crick hydrogen bonds are broken before the bases flip over separately and eventually stack, reforming the H-bonds, in the new helix.     

Preparation and Properties of a New Type of Acyclic,Achiral Nucleoside Analogue

Abstract

Preparation of the nucleoside analogues 1 and incorporation of 1, B = T, in deoxyribooligonucleotides by the phosphoramidite method is described. A two-step deprotection procedure was developed to reduce cleavage of the modified allylic unit. The binding properties of the modified oligonucleotides towards complementary DNA and RNA has been evaluated by T_m measurements showing a ΔT_m of ?2 to ?6.5°C per modification. An oligonucleotide with two modifications at the 3′-end showed considerable resistance towards cleavage by a 3′-exonuclease. No antiviral activity against HIV-1 or HSV-1 was found for 1, B = G or T, or for any of the trihydroxy derivatives 5.     

Synthesis of Chlorodideoxynucleosides Using Tris(2,4,6-tribromophenoxy)-dichlorophosphorane (BDCP)

Abstract

5′-Chloro-5′-deoxy-N,3′-O-dibenzoylthymidine (3a), 5′-chloro-5′-deoxy-N⁴, 3′-O-dibenzoyldeoxycytidine(3b), 5′-chloro-5′-deoxy-N⁶,3′-O-dibenzoyldeoxyadenosine(3c), N-benzoyl-1-(3-chloro-2,3-dideoxy-5-O-trityl-ß-D-xylofuranosyl)thymine (5a) and N⁶-benzoyl-9-(3-chloro-2,3-dideoxy-5-O-trityl-ß-D-xylofuranosyl)adenine (5b) have been synthesized in very high yields using a new efficient reagent, tris(2,4,6-tribrom-ophenoxy)dichlorophosphorane (BDCP). The reaction time was greatly reduced to 5–8 min. NOE data suggested an inversion of configuration at C₃-position and thus an SN2 mechanism has been proposed for the chlorination reaction.

     

Inhibition of 5-α-Reductase (TYPE-II) Expression by Antisense 3′-Deoxy-(2′-5′) Oligonucleotide Chimeras

Abstract

ABSTRACT: 3′-Deoxy-(2′-5′) oligonucleotides bind selectively to complementary RNA but not to DNA. 3′-Deoxy-(2′-5′) phosphorothioate ODN chimeras embedded with a short stretch of 3′-5′ phosphorothioate cassette are potent inhibitors of steroid 5-α-reductasc expression with significantly less non-specific interactions in cell culture.     

The effect of monovalent cations on the association behavior of guanosine 5'-monophosphate, cytidine 5'-monophosphate, and their equimolar mixture in aqueous solution

¹H- and ³¹P-nmr spectroscopy have been used to investigate the self-association of M₂(5′-CMP) [M = Li⁺, Na⁺, K⁺, Rb⁺, or (CH₃)₄ N⁺; 5′-CMP = cytidine 5′-monophosphate], the self-association of Li₂(5′-GMP) (5′-GMP = guanosine 5′-monophosphate), and the heteroassociation of 5′-GMP and 5′-CMP (1 : 1 mole ratio) in aqueous solution as a function of the nature of the monovalent cation. Proton spectral differences for the different 5′-CMP salts exhibit a cation-size dependence and have been ascribed to a change in the stacking geometry. An average stacking association constant of 0.63 ± 0.24M^?1 at 1°C, consistent with the weak stacking interactions of the cytosine bases, was determined for the 5′-CMP salts. Heteroassociation of 5′-GMP and 5′-CMP follows the reverse of the cation order for the formation of ordered aggregates of 5′-GMP. Heteroassociation occurs in the presence of Li⁺, Na⁺, and Rb⁺ ions, but only self-association occurs for the K⁺ nucleotides. Li₂(5′-GMP), which does not form ordered species, self-associates to form disordered base stacks with a stacking constant of 1.63 ± 0.11M^?1 at 1°C.     

Synthesis and DNA Binding Properties of DNA-PNA Chimeras

Abstract

A systematic study to evaluate the ability of 5′-DNA-3′-p-(N)-PNA-(C) chimeras to form triple helix structures has been undertaken. Preliminary results carried out on a 16-mer chimera with three PNA monomers at the 3′-end showed the formation of a stable DNA-PNA/DNA/DNA triplex, having similar conformational behaviour to a canonical DNA/DNA/DNA triplex.     

The Synthesis of 2–5A Antisense Chimeras with Various Non-nucleoside COMPONENTS

Abstract

We have synthesized a series of 2–5A chimeras in which the nature of the oligoadenylate-antisense linkage and the length of the 2′,5′-oligoadenylate were varied. In addition, a branched linker was introduced to relocate the 2′, 5′-oligoadenylate with respect to the antisense domain.