Synthesis of new, base-modified PNA monomers

A number of N-Boc-protected peptide nucleic acids (PNA) monomers containing 5-aryl- and 5-alkynyl-uracil bases have been synthesized using different palladium-catalyzed cross-coupling reactions. Starting from the base-unprotected 5-iodo-uracil PNA monomer, only the Stille couplings were accomplished successfully, while Suzuki couplings with boronic acids containing the same aryl groups failed. During Sonogashira couplings with terminal alkynes, significant amounts of unrequired furano[2,3-d]pyrimidine by-products were formed. Protection of the lactam function by p-methoxybenzylation prevented the opportunity for intramolecular cyclization as well as formation of a negative charge on the 4-O atom, making it possible to reach almost quantitative yields at Sonogashira couplings and acceptable conversions in Suzuki reactions.     

Synthesis of 2-5As possessing base-modified adenosines and their activities to human recombinant RNase L.

The unique 2',5'-oligoadenylate (2-5A) acts as a potent inhibitor of translation in vertebrate cells through the activation of a constituent latent 2-5A-dependent endoribonuclease (RNase L). This 2-5A system plays a major role in the interferon natural defense mechanism against viral infection. We report the syntheses of base-modified adenosine-substituted 2-5A derivatives, their interaction with recombinant human RNase L and their biological stability.     

1-Deazaadenosine: synthesis and activity of base-modified hammerhead ribozymes.

The incorporation of 1-deazaadenosine (c1A, 1b) into a hammerhead ribozyme and the resulting catalytic activity is described. For this purpose the phosphoramidite 2a and the 3'-phosphonate 2b as well as Fractosil-linked 1-deazaadenosine (3b) were prepared. The methoxyacetyl group was used for the 6-amino group protection and the triisopropylsilyl residue was introduced as the 2'-OH protecting group. Replacement of residues A14and A15.1 of the hammerhead ribozyme by 1-deazaadenosine resulted in a significantly reduced catalytic activity. Substitution of the A6, A9 and A13 residues has only a minor influence. The findings observed on ribozymes modified with 1-deazaadenosine were compared with those containing other adenosine analogues.     

Novel imidazo[1,2-c]pyrimidine base-modified nucleosides: synthesis and antiviral evaluation

The preparation of a series of novel 6-(beta-D-ribofuranosyl)-2-alkyl/aryl-6H-imidazo[1,2-c]pyrimidin-5-one nucleosides and the 2-nitrile nucleosides, 6-(beta-D-ribofuranosyl)-5-oxo-5,6-dihydro-imidazo[1,2-c]pyrimidine-2-carbonitrile and 2R and 2S isomers of 6-(beta-D-ribofuranosyl)-5-oxo-2,3,5,6-tetrahydro-imidazo[1,2-c]pyrimidine-2-carbonitrile, is described using two synthetic approaches. The nucleoside mimetics described were evaluated against a wide range of viral types and strains in cell culture. With the exception of one nucleoside, which displayed anti-CMV activity at toxic concentrations, none of the compounds showed antiviral activity most likely due to a lack of substrate recognition by viral and/or cellular nucleoside kinases.     

Cyclic monophosphate prodrugs of base-modified 2'-C-methyl ribonucleosides as potent inhibitors of hepatitis C virus RNA replication

A new series of heterobase-modified 2'-C-methyl ribonucleosides was synthesized and tested as inhibitors of hepatitis C virus (HCV) RNA replication. The nucleosides showed a weak inhibitory activity in a HCV replicon system (EC(50)=92 microM) and did not exhibit any cytotoxicity (CC(50)>300 microM). Cyclic monophosphate (cMP) prodrugs of the same nucleosides were synthesized and also tested in the HCV replicon system. Prodrugs exhibited strong potency (EC(50)=0.008 microM) without significant cytotoxicity (CC(50)>50 microM).     

Solid-phase synthesis of oligo(2'-deoxyxylonucleotides) and PCR amplification of base-modified DNA fragments.

1-(2'-Deoxy-beta-D-threo-pentofuranosyl)thymine (xTd) and -adenine (xAd) were converted into their appropriately protected 3'-phosphonates 1a, 2a as well as their 2-cyanoethyl phosphoramidites 1b, 2b. These compounds were used for solid-phase syntheses of the oligo(2'-deoxy-beta-D-xylonucleotides) 5-8. Structural properties and behavior against nucleases is described. Apart from oligo(2'-deoxyxylonucleotides) the PCR-amplification of a pUC18 DNA fragment with Taq polymerase was studied in the presence of the 7-deazapurine derivatives of dGTP, dATP, and dITP. The incorporation efficiency of the modified compounds was compared with those of the parent nucleotides. 7-Deaza-2'-deoxyguanosine protected the DNA-fragment from hydrolysis by the restriction endodeoxyribonuclease Eco RI, Pst I, Bam HI, and Sma I if the nucleoside was located within the recognition site.     

Preparation of base-modified nucleosides suitable for non-radioactive label attachment and their incorporation into synthetic oligodeoxyribonucleotides.

A very mild and efficient procedure has been developed for the preparation of C-5 substituted deoxyuridines. The substituent carries a masked primary aliphatic amino group. These compounds are readily converted into their phosphoramidites and can be used to prepare oligonucleotides carrying one or more aliphatic amino groups. Fluorescein isothiocyanate coupled to these compounds gives oligonucleotide probes carrying multiple fluorescein labels. These compounds have a free 5'-hydroxy group enabling additional 5'- end radioactive labelling for evaluation of their hybridization characteristics. It was found that oligonucleotides carrying a long (11 atom) linker arm to the fluorescein hybridize more efficiently to mRNA than those carrying a short (4 atom) arm. The long linker arm derivatives are comparable to underivatized oligonucleotides in hybridizing to mRNA.     

Mismatch discrimination of base-modified nucleic acids and their constituents: non-Watson-Crick base pairing induced by tautomerization

The effect of tautomerism on the mismatch discrimination was studied on 7-deazapurine and 8-aza-7-deazapurine analogues of isoguanosine. 7-Halogenated 7-deaza-2'-deoxyisoguanosines show better base pair discrimination than 2'-deoxyisoguanosine due to the more favored keto tautomer formation. 8-Aza-7-deazaisoguanosine and its 7-halogeno derivatives also show higher keto tautomer population than that of isoguanosine, but the 7-halogens do not bias the tautomeric equilibrium significantly as it is observed for the 7-deaza-2'-deoxyisoguanosine derivatives.     

Triplex-forming properties and enzymatic incorporation of a base-modified nucleotide capable of duplex DNA recognition at neutral pH

The sequence-specific recognition of duplex DNA by unmodified parallel triplex-forming oligonucleotides is restricted to low pH conditions due to a necessity for cytosine protonation in the third strand. This has severely restricted their use as gene-targeting agents, as well as for the detection and/or functionalisation of synthetic or genomic DNA. Here I report that the nucleobase 6-amino-5-nitropyridin-2-one (Z) finally overcomes this constraint by acting as an uncharged mimic of protonated cytosine. Synthetic TFOs containing the nucleobase enabled stable and selective triplex formation at oligopurine-oligopyrimidine sequences containing multiple isolated or contiguous GC base pairs at neutral pH and above. Moreover, I demonstrate a universal strategy for the enzymatic assembly of Z-containing TFOs using its commercially available deoxyribonucleotide triphosphate. These findings seek to improve not only the recognition properties of TFOs but also the cost and/or expertise associated with their chemical syntheses.     

Binding of actinomycin C1 (D) and actinomin to base-modified oligonucleotide duplexes with parallel chain orientation

The binding of actinomycin D (C1, 1) and its analog actinomin (2) was studied on base-modified oligonucleotide duplexes with parallel chain orientation (ps) and with anti-parallel chains (aps) for comparison. Actinomycin D binds not only to aps duplexes containing guanine-cytosine base pairs but also to those incorporating modified bases such as 7-deazaguanine or its 6-deoxo derivative. For this, novel phosphoramidites were prepared. The new building block of 7-deaza-2'-deoxyguanosine is significantly more stable than the one currently used and allows normal oxidation conditions during solid-phase oligonucleotide synthesis. Actinomycin binds weakly to ps duplexes containing guanine-isocytosine base pairs but not to ps-DNA incorporating pairs of isoguanine-cytosine residues. On the contrary, the actinomycin D analog actinomin, which contains positively charged side chains instead of the chiral peptide rings, is strongly bound to both ps- and aps-DNA. Guanines, isoguanine, as well as other 7-deaza derivatives are accepted as nucleobases. Apparently, the pentapeptide lacton rings of actinomycin do not fit nicely into the groove of ps-DNA thereby reducing the binding strength of the antibiotic while the groove size of ps-DNA does not affect actinomin binding notably.     

Molecular basis for methoxyamine initiated mutagenesis. 1H nuclear magnetic resonance studies of base-modified oligodeoxynucleotides.

In order to reach a more detailed understanding of the mechanism of the mutagenic action of methoxyamine and of N4-methoxycytidine and its 2'-deoxyribo-analogue, the solution structures of the self-complementary octanucleotide, d(CGAATTCG) and its analogues, d(CGAATCCG), d(CGAATMCG) and d(CGAATPCG) (designated 8mer-AT, 8mer-AC, 8mer-AM, and 8mer-AP, respectively), were investigated by 1H nuclear magnetic resonance spectroscopy; M is N4-methoxycytosine (mo4C) and P is an analogue, the bicyclic dihydropyrimido[4,5-c][1,2]oxazin-7-one, in which the N-O bond is held in the anti configuration with respect to N3 of the cytosine ring. Correlated spectroscopy and nuclear Overhauser spectroscopy allowed assignment of the base, anomeric and H2'/H2" protons in 8mers-AT, -AM and -AP, and showed that all three had features consistent with a regular B-DNA duplex structure. Duplex-to-coil transition temperatures were determined to be 52(+/- 2) degrees C (8mer-AT), 51(+/- 2) degrees C (8mer-AP), 32(+/- 2) degrees C (8mer-AM); on the chemical shift timescale, the melting transition was fast for 8mer-AT and 8mer-AP, but slow for 8mer-AM. Imino proton spectra were indicative of Watson-Crick base-pairing in 8mers-AT, -AP and -AM. The 8mer-AP duplex had a structure and melting characteristics virtually identical with those of the 8mer-AT duplex. The preferred syn configuration of the methoxyl group in M had a destabilising effect on the 8mer-AM duplex. At low temperatures, the A.M base-pair was in fast equilibrium between Watson-Crick and wobble configurations, with the methoxyl function anti-oriented, but the melting transition was accompanied by isomerization of the methoxyl group to the syn conformation. This syn-anti isomerization was the rate-determining step in the duplex-to-coil transition. The 8mer-AC oligomer did not form a stable duplex.     

DNA recognition by quinoline antibiotics: use of base-modified DNA molecules to investigate determinants of sequence-specific binding of luzopeptin

The luzopeptin antibiotics contain a cyclic decadepsipeptide to which are attached two quinoline chromophores that bisintercalate into DNA. Although they bind DNA less tightly than the structurally related quinoxaline antibiotics echinomycin and triostin A, the molecular basis of their interaction remains unclear. We have used the PCR in conjunction with novel nucleotides to create specifically modified DNA for footprinting experiments. In order to study the influence that removal, addition or relocation of the guanine 2-amino group, which normally identifies G.C base pairs from the minor groove, has on the interaction of luzopeptin antibiotics with DNA. The presence of a purine 2-amino group is not strictly required for binding of luzopeptin to DNA, but the exact location of this group can alter the position of preferred drug binding sites. It is, however, not the sole determinant of nucleotide sequence recognition in luzopeptin-DNA interaction. Nor can the selectivity of luzopeptin be attributed to the quinoline chromophores, suggesting that an analogue mode of DNA recognition may be operative. This is in contrast to the digital readout that seems to predominate with the quinoxaline antibiotics.     

Phosphoramidites of base-modified 2''-deoxyinosine isosteres and solid-phase synthesis of d(GCI*CGC) oligomers containing an ambiguous base.

Novel phosphoramidites (1,2) of appropriately protected 2'-deoxyinosine isosteres (I*) such as allopurinol 2'-deoxyribofuranoside (4a) and 7-deaza-2'-deoxyinosine (4b) have been synthesized. They were employed together with the phosphoramidite of 2'-deoxyinosine in solid-phase synthesis of d(GCI*CGC) hexamers (12a-d). From thermodynamic data of these alternating hexamers it was shown that allopurinol 2'-deoxyribofuranoside destabilizes such duplexes less strongly than 2'-deoxyinosine. Additionally, the phosphoramidite of 7-deaza-2'-deoxyinosine (2) exhibits an extraordinary stability of the N-glycosylic bond. Since the new phosphoramidites are structurally related to 2'-deoxyinosine, they can be used in the construction of hybridization probes containing an ambiguous base.     

New synthetic route to ethynyl-dUTP: A means to avoid formation of acetyl and chloro vinyl base-modified triphosphates that could poison SELEX experiments

5-Ethynyl-2′-deoxyuridine is a common base-modified nucleoside analogue that has served in various applications including selection experiments for potent aptamers and in biosensing. The synthesis of the corresponding triphosphates involves a mild acidic deprotection step. Herein, we show that this deprotection leads to the formation of other nucleoside analogs which are easily converted to triphosphates. The modified nucleoside triphosphates are excellent substrates for numerous DNA polymerases under both primer extension and PCR conditions and could thus poison selection experiments by blocking sites that need to be further modified. The formation of these nucleoside analogs can be circumvented by application of a new synthetic route that is described herein.     

Kinetic mechanism of nucleotide cofactor binding to Escherichia coli replicative helicase DnaB protein. stopped-flow kinetic studies using fluorescent, ribose-, and base-modified nucleotide analogues

The kinetic mechanism of binding nucleotide cofactors to the Escherichia coli primary replicative helicase DnaB protein has been studied, using the fluorescence stopped-flow technique. The experiments have been performed with fluorescent ATP and ADP analogues bearing the modification on the ribose, MANT-AMP-PNP and MANT-ADP, and on the base, epsilonAMP-PNP and epsilonADP. Association of the DnaB helicase with nucleotide cofactors is characterized by four relaxation times that indicate that the binding occurs by a minimum of four-steps. The simplest mechanism which can describe the data is a four-step sequential process where the bimolecular binding step is followed by three isomerization steps. This mechanism is described by the following equation: [equation in text]. The binding mechanism is independent of the location of the nucleotide cofactor modification and is an intrinsic property of the DnaB helicase-nucleotide system. Quantitative amplitude analyses, using the matrix projection operator technique, allowed us to determine specific fluorescence changes accompanying the formation of all intermediates relative to the fluorescence of the free nucleotide. It shows that the major conformational change of the DnaB helicase-nucleotide complex occurs in the formation of the (H-N)(1). Moreover, the value of the bimolecular rate constant, k(1), is 3-4 orders of magnitude lower than the value expected for the diffusion-controlled reaction. These results indicate that the determined first step includes formation of the collision and an additional transition of the enzyme-nucleotide complex. The obtained results provide evidence of profoundly different conformational states of the ribose and base regions of the nucleotide-binding site in different intermediates. The sequential nature of the mechanism of the nucleotide binding to the DnaB helicase indicates the lack of the existence of a kinetically significant conformational equilibrium of the helicase protomer and the DnaB hexamer prior to the binding. The significance of these results for the functioning of the DnaB helicase is discussed.     

Synthesis of glycolipids

The chemical syntheses of naturally occurring glycolipids derived from sphingosine bases and glycerol derivatives, and the syntheses of polyisoprenoid lipid intermediates and other miscellaneous glycolipids recorded up to the end of 1977 are reviewed.