Fluorous base-pairing effects in a DNA polymerase active site

We describe selective "fluorous" effects in the active site of a DNA polymerase, by using nucleotide analogues whose pairing edges are perfluorinated. The 5'-triphosphate deoxynucleotide derivatives of DNA base analogues 2,3,4,5-tetrafluorobenzene ((F)B) and 4,5,6,7-tetrafluoroindole ((F)I), as well as hydrocarbon controls benzene (B) and indole (I), were synthesized and studied as substrates for the DNA Polymerase I Klenow fragment (KF exo-). Modified nucleotides were present in the DNA template or were supplied as nucleoside triphosphates in studies of the steady-state kinetics of single nucleotide insertion. When supplied opposite the non-natural bases in the template strand, the hydrophobic nucleoside triphosphates were incorporated by up to two orders of magnitude more efficiently than the natural deoxynucleoside triphosphates. The purine-like fluorinated indole nucleotide ((F)I) was the most efficiently inserted of the four hydrophobic analogues, with the most effective incorporation occurring opposite the pyrimidine-like tetrafluorobenzene ((F)B). In all cases, the polyfluorinated base pairs were more efficiently processed than the analogous hydrocarbon pairs. A preliminary test of polymerase extension beyond these pairs showed that only the (F)B base is appreciably extended; the inefficient extension is consistent with recently published data regarding other nonpolar base pairs. These results suggest the importance of hydrophobicity, stacking, and steric interactions in the polymerase-mediated replication of DNA base pairs that lack hydrogen bonds. These findings further suggest that the enhanced hydrophobicity of polyfluoroaromatic bases could be employed in the design of new, selective base pairs that are orthogonal to the natural Watson-Crick pairs used in replication.     

2'-Desoxytubercidin: synthese des o-3'-phosphoramidites und kondensation zu 2'-desoxytubercidylyl(3' → 5')-2'-desoxytubercidin

The phosphoramidite of 2'-deoxytubercidin (2) has been prepared by phosphitylation of compound 3b with chlorodiisopropylaminomethoxyphosphane. The intermediate 3b was obtained from 2'-deoxytubercidin (1) by N⁴-benzoylation and subsequent O-5'-dimethoxytritylation. Condensation of compound 2 with O-3'-silylated 3e gave the fully protected dimer 4b, which was converted into 2'-deoxytubercidylyl (3' → 5')-2'-deoxytubercidin(4a). Compound 4a isoster with 2'-deoxyadenosylyl(3' → 5')-2'-deoxyadenosine (d(ApA)) exhibits a hypochromicity of 11% (270 nm) due to strong stacking interactions. Cleavage of the dinucleoside monophosphate 4a with nuclease S₁ occurs five times faster than that of d(ApA). This shows that the single strand specific enzyme recognises the geometry of the stacked nucleobases.     

High-throughput automated DNA sequencing facility with fluorescent labels at the European Molecular Biology Laboratory.

One of the aims of the facility is to develop and push the automated on-line DNA sequencing gel technology to its limit in sequence throughput, which may be somewhere around 100 kilobases of sequence per device per day. Key new developments were initiated and applied in operation on the European Molecular Biology Laboratory (EMBL) automated sequencer and its commercial version A.L.F. (Pharmacia). Sequencing speed was increased by a factor of 10-20, up to 1500 bases per hour per clone on ultrathin (about 100 microns) gels, while the resolution and reading length were extended to 1000 bases on gels with 50 cm separation length, using fluorescein-15-*dATP as the internal label. With our sequencing strategy, closing about 40% of the sequence with "walking" primers and F-15-*dATP as internal label, we sequenced both strands of a cosmid insert of 38.5 kb in length, each strand twice, in only 430 sequencing reactions and with average reading of 380 bases per reaction.     

Development and validation of an LC–MS/MS quantitative method for endogenous deoxynucleoside triphosphates in cellular lysate

The endogenous deoxynucleoside triphosphate (dNTP) pool includes deoxyadenosine triphosphate (dATP), deoxycytidine triphosphate (dCTP), deoxyguanosine triphosphate (dGTP) and thymidine triphosphate (TTP). The endogenous dNTP pool is regulated by complex enzymatic pathways that can be targeted by drugs, such as antimetabolites. In addition, these components compete with antiviral nucleos(t)ide analog triphosphates, contributing to the mechanism of pharmacologic action. This communication describes the development and validation of a sensitive method to quantify the intracellular dNTP pool in cellular lysates. The extraction process was optimized for dNTPs using an indirect strategy – the separation of mono‐, di‐ and triphosphate moieties by strong anion exchange, dephosphorylation of target fractions to molar equivalent nucleosides – followed by sensitive quantitation using liquid chromatography–tandem mass spectrometry. The validated analytical range was 50–2500 fmol/sample for each dNTP. The assay was used to quantify dNTPs in peripheral blood mononuclear cells from 40 clinical research participants (n = 279 samples). Median (interquartile range) concentrations were 143 (116, 169) for dATP, 737 (605, 887) for dCTP, 237 (200, 290) for dGTP and 315 (220, 456) for TTP, in femtomoles per million cells. This method allows for future studies of endogenous dNTP disposition in subjects receiving antimetabolites or nucleos(t)ide analogs, or for other clinical scenarios.     

Naphthalenyl- and anthracenyl-ethynyl dT analogues as base discriminating fluorescent nucleosides and intramolecular energy transfer donors in oligonucleotide probes

Fluorescent thymidine analogues functionalised in the 5-position with the moieties naphthalenylethynyl (NeT), anthracenylethynyl (AeT) and anthracenylbuta-1,3-diynyl (AeeT) have been incorporated into oligonucleotides. The modified oligonucleotides undergo significant emission enhancement when hybridised to fully complementary strands and a decrease in fluorescence emission when the modified thymine is paired with guanine. Thus these analogues are potentially useful as base discriminating fluorescent nucleosides (BDFs). When a fluorescein dT monomer is incorporated into the same oligonucleotide strand as the modified base, energy transfer enhances the fluorescein emission, particularly upon duplex formation. These dual-labelled probes may be useful for genetic analysis to detect point mutations and SNPs and could provide multiplexing capability.     

7‐Aryl‐7‐deazaadenine 2′‐Deoxyribonucleoside Triphosphates (dNTPs): Better Substrates for DNA Polymerases than dATP in Competitive Incorporations

A series of 7‐substituted 7‐deazaadenine and 5‐substituted cytosine 2′‐deoxyribonucleoside triphosphates (dNTPs) were tested for their competitive incorporations (in the presence of dATP and dCTP) into DNA by several DNA polymerases by using analysis based on cleavage by restriction endonucleases. 7‐Aryl‐7‐deazaadenine dNTPs were more efficient substrates than dATP because of their higher affinity for the active site of the enzyme, as proved by kinetic measurements and calculations.     

Target-driven DNA association to initiate cyclic assembly of hairpins for biosensing and logic gate operation

A target-driven DNA association was designed to initiate cyclic assembly of hairpins, which led to an enzyme-free amplification strategy for detection of a nucleic acid or aptamer substrate and flexible construction of logic gates. The cyclic system contained two ssDNA (S1 and S2) and two hairpins (H1 and H2). These ssDNA could co-recognize the target to produce an S1–target–S2 structure, which brought their toehold and branch-migration domains into close proximity to initiate the cyclic assembly of hairpins. The assembly product further induced the dissociation of a double-stranded probe DNA (Q:F) via toehold-mediated strand displacement to switch the fluorescence signal. This method could detect DNA and ATP as model analytes down to 21.6 pM and 38 nM, respectively. By designing different DNA input strands, the “AND”, “INHIBIT” and “NAND” logic gates could be activated to achieve the output signal. The proposed biosensing and logic gate operation platform showed potential applications in disease diagnosis.     

A divergent synthesis of - and -carbocyclic 4′-fluoro-2′,3′-dideoxynucleosides as potential antiviral agents

- and -Carbocyclic 4′-fluoro-2′,3′-dideoxynucleosides have been synthesized from 2, which can be conveniently prepared from 1,2:5,6-di-O-isopropylidene- -mannitol 1 in eight steps. Ruthenium-catalyzed ring-closing metathesis has been employed in the synthesis of -nucleosides, whereas the -series have been obtained through an intramolecular nucleophilic substitution reaction. The Mitsunobu condensation was used as a general tool for the synthesis of both purine and pyrimidine nucleosides.     

Synthesis of Morphology Processable α‐AlPO4 Nanoparticles,Nanowires, and Multi‐strand Nano‐ropes

In this paper we present aluminum phosphate nanocrystals, prepared by a hydrothermal reaction, using amphiphilic triblock copolymer F127 [(EO)₁₀₆(PO)₇₀(EO)₁₀₆] as a morphology‐directing template. By verifying the pH from 10 to 12, the morphology progression of AlPO₄ nanocrystals from nanoparticles to nanoparticle‐aggregated nanowires, and finally to multi‐strand nano‐ropes, was successfully demonstrated. The most influential factors in the morphology process were the initial pH level, the participation of surfactant‐template F127, and the change in pH during the reaction. We proposed a pH‐dependent model to illustrate both the growth of AlPO₄ nanocrystals inside F127 amphiphilic domains and the chemical driving force that aggregated the nanoparticles into chain‐shaped nanowires. The incorporation of water molecules as H‐bonding linkers, to combine single nanowires into multi‐strand nano‐ropes, is also discussed in this model. Powder X‐ray diffraction (XRD) patterns of the nanoparticle‐aggregated nanowires and multi‐strand nano‐ropes were consistent with a mixed phase of berlinite and cristobalite structures, corresponding to the low‐temperature form (a‐form), while the AlPO₄ nanoparticles showed a pure berlinite phase only.     

Synthesis of 2-deoxy-α-DAH based on diazo chemistry by insertion reactions of 2-diazo-3-deoxy- -arabino-heptulosonate derivatives mediated by rhodium(II)

The synthesis of 2-deoxy-α-DAH (2) is described based on the use of diazo chemistry as previously reported by us during the synthesis of the corresponding 2-deoxy-KDO. Initially, the -arabino-aldehydo sugar derivative 3 was reacted with ethyl diazoacetate, using diethyl zinc as promoter. The corresponding β-hydroxy-α-diazo ester 4, obtained in very good yield, was transformed into the corresponding 2-diazo-3-deoxy-heptulosonate derivative 10, which was subjected to the action of rhodium(II). However, the major compound obtained in this reaction was the C-glycofuranoside 12 by the interaction of the benzyl protecting group employed at the OH of C-4 with the carbenoid generated at C-2. To avoid this undesired insertion reaction, aldehyde 13 was selected as a suitable starting material and, following the same chemistry than for 3, diazo 19 was efficiently synthesized. Finally, the intramolecular OH insertion mediated by rhodium(II) of 19 provided the targeted 2-deoxy-DAH derivative 21 in a reasonable good yield, which was finally transformed into the potassium salt of 2-deoxy-α-DAH 2.     

Synthesis and Some Biochemical Properties of a Novel 5,6,7,8‐Tetrahydropyrimido[4,5‐c]pyridazine Nucleoside

A novel nucleoside analogue is described based on the pyridazine ring system. The nucleoside was successfully incorporated into DNA via both its phosphoramidite and 5′‐triphosphate derivatives. Enzymatically, the analogue behaves essentially as thymidine: it is a good substrate for the DNA polymerases Taq and exonuclease‐free Klenow fragment, leading to full‐length products when present in either the primer or template strands. In hybridisation studies, the nucleoside displays ambiguous base‐pairing properties, including universal base properties.     

Hydrothermal synthesis, crystal structures, and properties of Co and Ni supramolecular complexes with 2,4,6-trimethyl benzoate and 4,4′-bipyridyl

Two new coordination complexes, viz. [Co(tmb)₂(4,4′-bpy)₂(H₂O)₂](Htmb)₂ (1) and {[Ni(tmb)₂(μ-4,4′-bpy)₂(H₂O)₂](4,4′-bpy)}_n (2), have been hydrothermally synthesized by reaction of the corresponding metal acetate with 2,4,6-trimethylbenzoic acid (Htmb) and 4,4′-bipyridyl (4,4′-bpy). X-ray single-crystal diffraction suggests that complex 1 represents a discrete mononuclear species in which the central metal ion is coordinated by the terminal carboxylate moiety and the 4,4′-bipyridyl ligand. The crystal structure of complex 2 reveals a 1D chain coordination polymer in which the Ni(II) ions are connected by the bridging 4,4′-bipyridyl ligands. In both cases, the coordination arrays are further extended via hydrogen bonding interactions to generate 3D supramolecular networks. Complexes 1 and 2 have also been characterized by spectroscopic (IR and UV/Vis), thermal (TGA) and magnetic susceptibility measurements. In addition, both complexes exhibit antimicrobial activity.     

Synthesis, structure and dynamic stereochemistry of (O → Si)-chelate N-(trifluorosilylmethyl)-[N-(S)-(1-phenylethyl)]acetamide and 1-(trifluorosilylmethyl)-2-oxoperhydroazepine: Retention of the O → Si coordination in the adduct with KF and 18-crown-6

The novel compounds, N-(trifluorosilylmethyl)-[N-(S)-(1-phenylethyl)]-acetamide (1a) and 1-(trifluorosilylmethyl)-2-oxoperhydroazepine (1b) have been prepared from the corresponding NH-compounds using ClCH₂SiCl₃/Et₃N or ClCH₂SiCl₃/(Me₃Si)₂NH followed by methanolysis or hydrolysis of the reaction mixture in the presence of Lewis bases, and then BF₃ etherate. Potassium-(18-crown-6)-(2-oxoperhydroazepinomethyl)tetrafluorosilicate (2) was synthesized by reaction of the trifluoride (1b) with KF in the presence of 18-crown-6. Using ¹⁹F, ²⁹Si NMR and X-ray diffraction techniques it was established that the silicon atom is pentacoordinate in the trifluorides (1a, b) and hexacoordinate in the adduct 2. Thus the internal coordination of the O → Si bond present in the trifluoride (1b) is retained in the adduct 2.The stereochemical non-rigidity of the trifluorides (1a, b) and the N-(trifluorosilylmethyl)-N-methylacetamide (1c) was investigated using dynamic ¹⁹F NMR spectroscopy. The activation barriers for permutational isomerization are in the range 9.5-10 kcal mol⁻¹. Lower values of ΔG^# for permutation of trifluorides (1a-c) compared to the monofluorides with the coordination core OSiC₃F together with small negative values for the activation entropy implies a non-dissociative mechanism. Quantum-chemical analysis suggests a mechanism involving a turnstile rotation.     

Multiple hydrogen bonds-mediated formation of supramolecular polysiloxanes based on oligoamide duplexes

Formation of supramolecular polysiloxane polymers was studied by using oligoamide strands 1a, 2a, and 3a bearing multiple hydrogen bonds as association units. According to SEM and TEM studies one polymeric strand associated into supramolecular spherical assemblies via duplex formation. The results demonstrated the effect of such complementary oligoamides upon constructing main-chain supramolecular polymers.     

Cloning,Sequence Analysis and Three-dimensional Structure Prediction of DNA Pol I from Thermophilic <Emphasis Type="Italic">Geobacillus</Emphasis> sp. <Emphasis Type="Italic">MKK</Emphasis> Isolated from an Iranian Hot Spring

Molecular phylogenetic analysis of a novel thermophilic eubacterium isolated from an Iranian hot spring using 16S rDNA sequence showed that the new isolate belongs to genera Geobacillus. DNA pol I gene from this isolate was amplified, cloned, sequenced, and the three-dimensional (3D) structure of deduced amino acid sequence was predicted. Sequence analysis revealed the gene is 2,631 bp long, encodes a protein of 876 amino acids with a calculated molecular mass of 99 kDa, and belongs to family A DNA polymerases. Comparison of 3′–5′exonuclease domain of Klenow fragment (KF) with corresponding region of newly identified DNA pol I (MF), the large fragment of Bacillus stearothermophilus DNA pol I (BF) and Klentaq1, revealed not only deletions in three regions compared to KF, but that three of the four critical metal-binding residues in KF (Asp355, Glu357, Asp424, and Asp501) are altered in MF as well. Predicted 3D structure and sequence alignments between MF and BF showed that all critical residues in the polymerase active site are conserved.     

Syntheses and X-ray structures of complexes with (η-allyl)Mo-units in oxygen-rich coordination spheres

The reaction of Mo(η³-C₃H₄(CH₃))(CH₃CN)₂(CO)₂Cl with AgBF₄ in THF yields the cationic complex [Mo(η³-C₃H₄(CH₃))(CH₃CN)₂(CO)₂(THF)]⁺[BF₄]⁻, 1, whose X-ray structure has been determined. Oxo nucleophiles are capable of replacing the weakly bound THF molecule in 1 and under simultaneous loss of CH₃CN the resulting complexes aggregate to oligonuclear compounds. Accordingly, the reactions with NaOMe and KOH yield [Na(THF)₄]⁺[(η³-C₃H₄(CH₃))(CO)₂Mo(μ-OCH₃)₃Mo(CO)₂(η³-C₃H₄(CH₃))]⁻, 2 and [K(18-crown-6)]⁺[[Mo(η³-C₃H₄(CH₃))(CO)₂]₃(μ₂-OH)₃(μ₃-OH)]⁻, 3, which were characterized by means of single crystal X-ray diffraction. Due to fluoride abstraction from BF₄⁻ the reaction of 1 with KOH also yields fluorinated derivatives of 3 but incorporation of fluorine in 3 can be avoided if AgO₃SCF₃ rather than AgBF₄ is used to generate the cation of 1. For purposes of comparison the dinuclear complex [K(18-crown-6)]⁺[[Mo(η³-C₃H₄(CH₃))(CO)₂]₂(μ₂-F)₃]⁻, 4, has been prepared, too, showing fluoride bridges and KF bonding. The chemical properties and the structures of these compounds in solution as well as their role as structural models for intermediates during molybdenum oxide catalysed propene oxidation are discussed.     

5'-Substituted 2,2'-anhydrouridines

3',5'-Di-O-acetyl-2'-halogenouridines (1a, 1c and 1d) can be partially deacetylated at C-5' by transesterification with methanol-HCl, providing the 3'-O-acetyl derivatives 2a–2c. These can be converted into the 5'-O-mesyl derivatives 3a–3c, and latter into the 5'-chloro compounds 3d-3f. All 5'-substituted 2'-halogeno compounds give the corresponding 2,2'-anhydrouridine derivatives 4a–4c on treatment with NaOMe. Structures were proved by IR and ¹H-NMR.     

Studies on the Extension of Sequence‐independence and the Enhancement of DNA Triplex Formation

A more elaborate sequence‐independent triple‐helix formation viability study was carried out and extended from a recombination‐like triple‐helical DNA motif of a previous study (J. Mol. Recognition 14, 122–139 (2001)). The intended triple‐helix was formed by mixing one part of a DNA hairpin duplex and one part of a single (or third) strand identical to one of the duplex strands and complementary to the other strand. In contrast to the common purine and pyrimidine motifs in triple‐stranded DNA, the strands of the recombination‐like motif are not monotonously built from pyrimidine only, or purine only, in the sequence. The stability of the recombination‐like motif triplexes with varying sequences was monitored by UV thermal melting curves. The results showed that the order of the stability of the R‐form DNA base triads (J. Mol. Biol., 239, 181–200 (1994)) is G*(G ○ C) > C*(C ○ G) > A*(A ○ T) >T*(T ○ A) (the Watson‐Crick base pair is denoted in the parentheses) in 200 mM NaCl, at pH 7. In an attempt to increase the stability of the triplex in the recombination‐like motif, we replaced cytidine by 5‐methylcytidine (^mC) of the third strand. There is a general trend that ^mC modification stabilizes the complex (<2 °C per ^mC). The complex is furthermore stabilized by Mg²⁺ ion. The Tm increases from 7 to 2 °C from less stable to highly stable triplex by 20 mM Mg²⁺ ion in solution.