Thymine Monomer Research Articles

Acid-Mediated Cleavage oF Oligonucleotide P3′ → N5′ Phosphoramidates Triggered by Sequence-Specific Triplex Formation

The P-N bond in oligonucleotide P3′ → N5′ phosphoramidates (5′ -amino-DNA) is known to be chemoselectively cleaved under mild acidic conditions. We prepared homopyrimidine oligonucleotides containing 5′ -amino-5′ -deoxythymidine (5′ -amino-DNA thymine monomer) or its conformationally locked congener, 5′ -amino-2′,4′ -BNA thymine monomer, at midpoint of the sequence. The effect of triplex formation with homopurineohomopyrimidine dsDNA targets on acid-mediated hydrolysis of the P3′ → N5′ phosphoramidate linkage was evaluated. Very interestingly, it was found that the triplex formation significantly accelerates the P-N bond cleavage.

Synthesis Of The Bicyclo-[4.3.0]-Thymidyl-Nucleoside Via Pd(Ii)-Mediated Ring Expansion Chemistry

A variety of modified nucleosides to improve antisense oligodeoxynucleotide properties such as target affinity, nuclease resistance, and pharmacokinetics were developed in the last two decades. In the context of conformational restriction we present here the synthesis of the [4.3.0]-bicyclo-DNA thymine monomer via Pd(II)-mediated ring expansion of an intermediate of the tricyclo-DNA synthesis.

Characterization of the DNA repair spore photoproduct lyase enzyme from Clostridium acetobutylicum: A radical-SAM enzyme

Abstract Spore photoproduct lyase (SPL) is a “Radical-SAM” repair enzyme which catalyzes the cleavage of spore photoproduct (SP, 5-thyminyl-5,6-dihydrothymine), a specific lesion found in bacterial spore DNA, to thymine monomers by a free-radical mechanism. The enzyme requires S-adenosyl- l -methionine (SAM) and a [4Fe–4S] cluster for activity. SPL from Bacillus subtilis has been difficult to isolate and characterize due to problems with the solubility and stability of the overexpressed protein in Escherichia coli and the lability of the [Fe–S] cluster, even if the protein was purified under strictly anaerobic conditions. In order to overcome these problems we searched for another SPL enzyme and we found that the recombinant SPL enzyme from Clostridium acetobutylicum, isolated either aerobically or anaerobically from overexpressing E. coli, behaves more stably than the B. subtilis one. We report here a complete spectroscopic and biochemical characterization of this enzyme. In particular we show for the first time that, using HYSCORE spectroscopy, SAM binds to the cluster as observed in the case of other members of the “Radical-SAM” enzyme family such as the activases of pyruvate formate lyase and ribonucleotide reductase.

Infrared Laser Spectroscopy of Uracil and Thymine in Helium Nanodroplets: Vibrational Transition Moment Angle Study

Vibrational spectra are reported in the N-H stretching region for uracil and thymine monomers in helium nanodroplets. Each monomer shows only a single isomer, the global minimum, in agreement with previous experimental and theoretical studies. The assignment of the infrared vibrational bands in the spectra is aided by the measurement of the corresponding vibrational transition moment angles (VTMAs) and ab initio frequency calculations. The ambiguity in the VTMA assignment of the N3H band for the uracil monomer is explained by the presence of dimer bands, which are overlapped with the monomer band.

Template-Enhanced Ring-Opening Metathesis Polymerization

The template-enhanced ring-opening metathesis polymerization (ROMP) of a norbornene-based thymine monomer was examined. The template, based on diaminopyridine functionalized norbornenes that are designed to recognize thymine substrates with high fidelity, was synthesized via ROMP. The resulting template was used to harness the polymerization of the thymine monomer producing a bis-poly(norbornene) complex. Using 1H NMR spectroscopy, we determined that the polymerization conditions do not disrupt the hydrogen bonding. In addition, the template enhances the rate of the polymerization by inducing an increase in local monomer concentration. To examine whether the polymerization is controlled, we synthesized a solid-supported diaminopyridine template. After the polymerization off this solid-supported template, we extracted the daughter polymer from the support. Detailed analysis of the daughter template proved that the templated polymerization was controlled and that the supported template produces a well-defined daughter polymer.

Characterization of a New Thermophilic Spore Photoproduct Lyase from Geobacillus stearothermophilus (SplG) with Defined Lesion Containing DNA Substrates

The Geobacillus stearothermophilus splG gene encodes a thermophilic spore photoproduct lyase (SplG) that belongs to the family of radical S-adenosylmethionine (AdoMet) enzymes. The aerobically purified apo-SplG forms a homodimer, which contains one [4Fe-4S] cluster per monomer unit after reconstitution to the holoform. Formation of the [4Fe-4S] cluster was proven by quantification of the amount of iron and sulfur per homodimer and by UV and EPR spectroscopy. The UV spectrum features a characteristic absorbance at 420 nm typical for [4Fe-4S] clusters, and the EPR data were found to be identical to those of other proteins containing an [4Fe-4S]+ center. Probing of the activity of the holo-SplG with oligonucleotides containing one spore photoproduct lesion at a defined site proved that the enzyme is able to turn over substrate. In addition to repair, we observed cleavage of AdoMet to generate 5'-deoxyadenosine. In the presence of aza-AdoMet the SplG is completely inhibited, which provides direct support for the repair mechanism.

Characterization of an Active Spore Photoproduct Lyase, a DNA Repair Enzyme in the Radical S-Adenosylmethionine Superfamily

The major photoproduct in UV-irradiated Bacillus spore DNA is a unique thymine dimer called spore photoproduct (SP, 5-thyminyl-5,6-dihydrothymine). The enzyme spore photoproduct lyase (SP lyase) has been found to catalyze the repair of SP dimers to thymine monomers in a reaction that requires S-adenosylmethionine. We present here the first detailed characterization of catalytically active SP lyase, which has been anaerobically purified from overexpressing Escherichia coli. Anaerobically purified SP lyase is monomeric and is red-brown in color. The purified enzyme contains approximately 3.1 iron and 3.0 acid-labile S(2-) per protein and has a UV-visible spectrum characteristic of iron-sulfur proteins (410 nm (11.9 mM(-1) cm(-1)) and 450 nm (10.5 mM(-1) cm(-1))). The X-band EPR spectrum of the purified enzyme shows a nearly isotropic signal (g = 2.02) characteristic of a [3Fe-4S]1+ cluster; reduction of SP lyase with dithionite results in the appearance of a new EPR signal (g = 2.03, 1.93, and 1.89) with temperature dependence and g values consistent with its assignment to a [4Fe-4S]1+ cluster. The reduced purified enzyme is active in SP repair, with a specific activity of 0.33 micromol/min/mg. Only a catalytic amount of S-adenosylmethionine is required for DNA repair, and no irreversible cleavage of S-adenosylmethionine into methionine and 5'-deoxyadenosine is observed during the reaction. Label transfer from [5'-3H]S-adenosylmethionine to repaired thymine is observed, providing evidence to support a mechanism in which a 5'-deoxyadenosyl radical intermediate directly abstracts a hydrogen from SP C-6 to generate a substrate radical, and subsequent to radical-mediated beta-scission, a product thymine radical abstracts a hydrogen from 5'-deoxyadenosine to regenerate the 5'-deoxyadenosyl radical. Together, our results support a mechanism in which S-adenosylmethionine acts as a catalytic cofactor, not a substrate, in the DNA repair reaction.

Synthesis of a Novel Pyrrolidine-based Peptide Nucleic Acid that Contains Tertiary Amines in the Main Chain and Its Internalization into Cells

Abstract A novel thymine monomer of pyrrolidine-based peptide nucleic acid that contains tertiary amines in the main chain (pyrrolidine-based amino peptide nucleic acid = PAPNA) was synthesized. A mixed oligomer of two PAPNA(T) units and seven units of pyrrolidine-based oxypeptide nucleic acid (POPNA), was synthesized by a solid-phase method. The mixed oligomer was internalized into CHO cells more readily than the previous version of POPNA oligomers.

Model studies of the (6–4) photoproduct photoreactivation: efficient photosensitized splitting of thymine oxetane units by covalently linked tryptophan in high polarity solvents

Three covalently linked tryptophan-thymine oxetane compounds used as a model of the (6-4) photolyase-substrate complex have been prepared. Under 290 nm light, efficient splitting of the thymine oxetane with aromatic carbonyl compounds gives the thymine monomer and the corresponding carbonyl compounds by the covalently linked tryptophan via an intramolecular electron transfer, and exhibits a strong solvent dependence: the quantum yield (Phi) is ca. 0.1 in dioxane, and near 0.3 in water. Electron transfer from the excited tryptophan residue to the oxetane unit is the origin of fluorescence quenching of the tryptophan residue, and is more efficient in strong polar solvents. The splitting efficiency of the oxetane radical anion within the tryptophan.+-oxetane.- species is also solvent-dependent, ranging from ca. 0.2 in dioxane to near 0.35 in water. Thus, the back electron transfer reaction in the charge-separated species would be suppressed in water, but is still a main factor causing low splitting efficiencies in the tryptophan-oxetane systems. In contrast to the tryptophan-oxetane system, fast nonradiation processes are the main causes of low efficiency in the flavin-oxetane system. Hence, nonradiative processes of the excited FADH-, rather than electron transfer to oxetane, may be an important factor for the low repair efficiency of (6-4) photolyase.

Synthesis and RNA-selective hybridization of α- l- ribo- and β- d- lyxo-configured oligonucleotides

Three α- l-ribofuranosyl analogues of RNA nucleotides (α- l-RNA analogues) have been synthesized and incorporated into oligonucleotides using the phosphoramide approach on an automated DNA synthesizer. The 4′- C-hydroxymethyl-α- l-ribofuranosyl thymine monomer was furthermore synthesized. Relative to the unmodified duplexes, incorporation of a single α- l-RNA monomer into a DNA strand leads to reduced thermal stability of duplexes with DNA complements but unchanged thermal stability of duplexes with RNA complements, whereas incorporation of more than one α- l-RNA monomer lead to moderately decreased thermal stability also of duplexes with RNA complements. Efficient hybridization with an RNA complement and no melting transition with a DNA complement were observed with stereoregular chimeric oligonucleotides composed of a mixture of α- l-RNA and affinity enhancing α- l-LNA monomers (α- l- ribo-configured locked nucleic acid). Furthermore, duplexes formed between oligodeoxynucleotides containing an α- l-RNA monomer and complementary RNA were good substrates for Escherichia coli RNase H. RNA-selective hybridization was also achieved by the incorporation of 1-(4- C-hydroxymethyl-β- d-lyxofuranosyl)thymine monomers into a DNA strand, whereas stable duplexes were formed with both complementary DNA and RNA when these monomers were incorporated into an RNA strand.

Synthesis of a newly peptide nucleic acid that contains tertiary amino groups

A thymine monomer of pyrrolidine-based peptide nucleic acid that contains a tertiary amino group in the backbone has been synthesized for use in the synthesis of novel peptide nucleic acid.

Unusual rate enhancement in the thymine assisted ATRP process of adenine monomers

Thymine and adenine monomers were synthesized and polymerized using ATRP in a controlled fashion. In addition a thymine functionalized block copolymer was prepared using ATRP, starting from a poly(ethylene glycol) macro initiator. Polymerization of adenine monomer in DMSO-d6 showed a significant increase in polymerization rate when polymeric thymine template was present, while thymine monomer did not show a polymerization rate enhancement. Varying the template to monomer ratio demonstrated that the polymerization rate increased even further if an excess of template was applied. Surprisingly, the addition of monomeric complementary moieties resulted in an even greater rate enhancement. These findings led to our conclusion that non-covalent interaction between adenine and thymine in DMSO-d6 protects the adenine monomer from interaction with the copper catalyst, thus resulting in a faster polymerization.

Synthesis of pyrrolidine-based oxy-peptide nucleic acids that contain four bases and their properties

Adenine, guanine, thymine and cytosine monomers of oxy-peptide nucleic acid that contain a pyrrolidine ring in the main chain have been synthesized for use in the synthesis of novel peptide nucleic acid.

(1S,2R/1R,2S)-cis-cyclopentyl PNAs (cpPNAs) as constrained PNA analogues: synthesis and evaluation of aeg-cpPNA chimera and stereopreferences in hybridization with DNA/RNA.

Conformationally constrained chiral PNA analogues were designed on the basis of stereospecific imposition of a 1,2-cis-cyclopentyl moiety on an aminoethyl segment of aegPNA. It is known that the cyclopentane ring is a relatively flexible system in which the characteristic puckering dictates the pseudoaxial/pseudoequatorial dispositions of substituents. Hence, favorable torsional adjustments are possible to attain the necessary hybridization-competent conformations when the moiety is imposed on the conventional PNA backbone. The synthesis of the enantiomerically pure 1,2-cis-cyclopentyl PNA monomers (10a and 10b) was achieved by stereoselective enzymatic hydrolysis of a key intermediate ester 2. The chiral (1S,2R/1R,2S)-aminocyclopentylglycyl thymine monomers were incorporated into PNA oligomers at defined positions and through the entire sequence. Hybridization studies with complementary DNA and RNA sequences using UV-Tm measurements indicate that aeg-cpPNA chimera form thermally more stable complexes than aegPNA with stereochemistry-dependent selective binding of cDNA/RNA. Differential gel shift retardation was observed on hybridization of aeg-cpPNAs with complementary DNA.

Synthesis and conformation of a novel bridged nucleic acid having a trans-fused 3,5,8-trioxabicyclo65.3.09decane structure

A novel bridged nucleic acid analogue, 2′-deoxy-trans-3′,4′-BNA thymine monomer, was successfully synthesized. An ab initio calculation and X-ray structure analysis revealed that the trans-fused bicyclo[5.3.0]decane structure of the 2′-deoxy-trans-3′,4′-BNA effectively constrained the sugar puckering in C2′-endo with appropriate γ, δ and χ angles.

Synthesis and conformation of a novel bridged nucleic acid having a trans-fused 3,5,8-trioxabicyclo[5.3.0]decane structure

A novel bridged nucleic acid analogue, 2′-deoxy-trans-3′,4′-BNA thymine monomer, was successfully synthesized. An ab initio calculation and X-ray structure analysis revealed that the trans-fused bicyclo[5.3.0]decane structure of the 2′-deoxy-trans-3′,4′-BNA effectively constrained the sugar puckering in C2′-endo with appropriate γ, δ and χ angles.

Synthesis and evaluation of (1S,2R/1R,2S)-aminocyclohexylglycyl PNAs as conformationally preorganized PNA analogues for DNA/RNA recognition.

Conformationally constrained cis-aminocyclohexylglycyl PNAs have been designed on the basis of stereospecific imposition of 1,2-cis-cyclohexyl moieties on the aminoethyl segment of aminoethylglycyl PNA (aegPNA). The introduction of the cis-cyclohexyl ring may allow the restriction of the torsion angle beta in the ethylenediamine segment to 60-70 degrees that is prevalent in PNA(2):DNA and PNA:RNA complexes. The synthesis of the optically pure monomers (10a and 10b) is achieved by stereoselective enzymatic hydrolysis of an intermediate ester 2. The chiral PNA oligomers were synthesized with (1S,2R/1R,2S)-aminocyclohexylglycyl thymine monomers in the center and N-terminus of aegPNA. Differential gel shift retardation with one or more units of modified monomer units was observed as a result of hybridization of PNA sequences with complementary DNA sequences. Hybridization studies with complementary DNA and RNA sequences using UV-T(m) measurements indicate that PNA with (1S,2R)-cyclohexyl stereochemistry enhances selective binding with RNA over DNA as compared to control aegPNA and PNA with the other (1R,2S) isomer.

α‐CycloPNA: 1‐Aminocylopentane‐1‐carboxylic Acid‐Derived Peptide Nucleic Acid

AbstractFor Abstract see ChemInform Abstract in Full Text.

α-cycloPNA: 1-Aminocylopentane-1-carboxylic Acid-Derived Peptide Nucleic Acid

All four diastereoisomers of 3-thymine-1-(tbutoxycarbonyl)aminocyclopentane- 1-carboxylic acid have been synthesised from (S)-dimethyl malate and thymine monomer 12 has been incorporated into an α-cycloPNA oligomer.

Methylphosphonate LNA: A Locked Nucleic Acid with a Methylphosphonate Linkage.

AbstractFor Abstract see ChemInform Abstract in Full Text.