Gas phase formation of carbon cluster (fullerenes and graphenes)/prebiotic sugar complexes.

Among the constituent molecular classes of proteins and nucleic acids, the presence of Ribose and deoxy-Ribose in space remains unclear. Here, we provide experimental evidence of astronomically related sugar derivatives - carbon cluster (fullerenes and graphenes)/prebiotic sugar complexes - and study their formation processes in the gas phase. The results show that, with PAH cations (dicoronylene, DC, C48H20+)/(2-deoxy-d-Ribose, dR, C5H10O4, and dehydrated 2-deoxy-d-Ribose, DedR, C5H8O3) and fullerene cations (C60+)/(dR and DedR) as the initial molecular precursors, two series of graphene-prebiotic sugar cluster cations (graphene/dR and graphene/DedR, e.g., (dR)Cn+ and (DedR)Cn+) and two series of fullerene-prebiotic sugar cluster cations (fullerene/dR and fullerene/DedR, e.g., (dR)(DedR)2Cn+, (DedR)3Cn+, and (dR)2(DedR)Cn+) are formed through an ion-molecule reaction pathway under the influence of a strong radiation field. The structures of the newly formed complexes and the binding energies of these formation reactions are initially theoretically calculated. These laboratory studies attest to the importance of ion-molecule reaction synthesis routes for the chemical complexity in space, demonstrating that the gas phase interstellar materials could directly lead to the formation of large and complex sugar derivatives in a bottom-up growth process. The chemical evolution in space in which single molecules are transformed into complex molecules produces a wide variety of organic compounds (e.g., carbon cluster (fullerenes and graphenes)/prebiotic sugar complexes). For their astrobiological implications, this opens up aromatic based biogenic chemistry that is available to the parent of PAHs or fullerenes in the interstellar environments.

Impact of the 2'- and 3'-Sugar Hydroxyl Moieties on Gas-Phase Nucleoside Structure.

Modified nucleosides have been an important target for pharmaceutical development for the treatment of cancer, herpes simplex virus, and the human immunodeficiency virus (HIV). Amongst these nucleoside analogues, those based on 2',3'-dideoxyribose sugars are quite common, particularly in anti-HIV applications. The gas-phase structures of several protonated 2',3'-dideoxyribose nucleosides are examined in this work and compared with those of the analogous protonated DNA, RNA, and arabinose nucleosides to elucidate the influence of the 2'- and combined 2',3'-hydroxyl groups on intrinsic structure. Infrared multiple photon dissociation (IRMPD) action spectra are collected for the protonated 2',3'-dideoxy forms of adenosine, guanosine, cytidine, thymidine and uridine, [ddAdo+H]+, [ddGuo+H]+, [ddCyd+H]+, [ddThd+H]+, and [ddUrd+H]+, in the IR fingerprint and hydrogen-stretching regions. Molecular mechanics conformational searching followed by electronic structure calculations generates low-energy conformers of the protonated 2',3'-dideoxynucleosides and corresponding predicted linear IR spectra to facilitate interpretation of the measured IRMPD action spectra. These experimental IRMPD spectra and theoretical calculations indicate that the absence of the 2'- and 3'-hydroxyls largely preserves the protonation preferences of the canonical forms. The spectra and calculated structures indicate a slight preference for C3'-endo sugar puckering. The presence of the 3'- and further 2'-hydroxyl increases the available intramolecular hydrogen-bonding opportunities and shifts the sugar puckering modes for all nucleosides but the guanosine analogues to a slight preference for C2'-endo over C3'-endo. Graphical Abstract.

Polymorphism of G4 associates: from stacks to wires via interlocks.

We examined the assembly of DNA G-quadruplexes (G4s) into higher-order structures using atomic force microscopy, optical and electrophoretic methods, NMR spectroscopy and molecular modeling. Our results suggest that parallel blunt-ended G4s with single-nucleotide or modified loops may form different types of multimers, ranging from stacks of intramolecular structures and/or interlocked dimers and trimers to wires. Decreasing the annealing rate and increasing salt or oligonucleotide concentrations shifted the equilibrium from intramolecular G4s to higher-order structures. Control antiparallel and hybrid G4s demonstrated no polymorphism or aggregation in our experiments. The modification that mimics abasic sites (1',2'-dideoxyribose residues) in loops enhanced the oligomerization/multimerization of both the 2-tetrad and 3-tetrad G4 motifs. Our results shed light on the rules that govern G4 rearrangements. Gaining control over G4 folding enables the harnessing of the full potential of such structures for guided assembly of supramolecular DNA structures for nanotechnology.

Substitution of Cytosine with Guanylurea Decreases the Stability of i-Motif DNA.

Both 5-aza-2'-deoxycytidine (decitabine) and its primary breakdown product, 2'-deoxyriboguanylurea (GuaUre-dR), have been shown to act as mutagens and epimutagens that cause replication stress and alter both DNA methylation and gene expression patterns. As cytosine analogues, both are expected to be preferentially incorporated into regions of GC skew where runs of cytosine residues are sequestered on one strand and guanine residues on the other. Given that such regions have been identified as sites with the potential for effects on gene expression and replication stress linked to formation of alternative DNA secondary structures, it is of interest to determine the influence that these base analogues might have on the stability of structures of this kind. Here we report that incorporation of GuaUre-dR into an i-motif-forming sequence decreases both the thermal and pH stability of an i-motif despite the apparent ability of GuaUre-dR to base pair with cytosine.

The synthesis, conformation and hydrolytic stability of an N,S-bridging thiophosphoramidate analogue of thymidylyl-3',5'-thymidine.

A 3'-N,5'-S-bridging thiophosphoramidate analogue of thymidylyl-3',5'-thymidine was synthesised under aqueous conditions. (1)H NMR conformational measurements show that the 3'-N-substituted deoxyribose ring is biased towards the 'north', RNA-like conformation. Rate constants for hydrolysis of the analogue were measured at 90 °C in the pH range 1.3-10.9. The pH-log kobs profile displays a pH-independent region between approximately pH 7 and 10 (t1/2 ∼13 days). Under acidic conditions, kobs displays a first order dependence on [H3O(+)].

Enzymatic transhalogenation of dendritic RGD peptide constructs with the fluorinase.

The substrate scope of fluorinase enzyme mediated transhalogenation reactions is extended. Substrate tolerance allows a peptide cargo to be tethered to a 5'-chloro-5'-deoxynucleoside substrate for transhalogenation by the enzyme to a 5'-fluoro-5'-deoxynucleoside. The reaction is successfully extended from that previously reported for a monomeric cyclic peptide (cRGD) to cargoes of dendritic scaffolds carrying two and four cyclic peptide motifs. The RGD peptide sequence is known to bind upregulated αVß3 integrin motifs on the surface of cancer cells and it is demonstrated that the fluorinated products have a higher affinity to αVß3 integrin than their monomeric counterparts. Extending the strategy to radiolabelling of the peptide cargoes by tagging the peptides with [(18)F]fluoride was only moderately successful due to the poor water solubility of these higher order peptide scaffolds although the strategy holds promise for peptide constructs with improved solubility.

Practical and Reliable Synthesis of 1,2-Dideoxy-d-ribofuranose and its Application in RNAi Studies.

We developed a practical and reliable method for synthesizing an abasic deoxyribonucleoside, 1,2-dideoxy-d-ribofuranose (dR(H)) via elimination of nucleobase from thymidine. To synthesize oligonucleotides bearing dR(H) by the standard phosphoramidite solid-phase method, dR(H) was converted to the corresponding phosphoramidite derivative and linked to a solid support (controlled pore glass resin). Chemically modified small interfering RNAs (siRNAs) possessing dR(H) at their 3'-overhang regions were synthesized. Introducing dR(H) to the 3'-end of the antisense strand of siRNA reduced its knockdown effect.

Development of 2-Deoxy-2-[(18)F]fluororibose for Positron Emission Tomography Imaging Liver Function in Vivo.

Life-threatening acute liver failure can be triggered by a variety of factors, including common drugs such as acetaminophen. Positron emission tomography (PET) is rarely used to monitor liver function, in part because of a lack of specific imaging agents for liver function. Here we report a new PET probe, 2-deoxy-2-[(18)F]fluororibose ([(18)F]-2-DFR), for use in imaging liver function. [(18)F]-2-DFR was synthesized and validated as a competitive substrate for the ribose salvage pathway. [(18)F]-2-DFR was prepared through an efficient late stage radiofluorination. The desired selectivity of fluorination was achieved using an unorthodox protecting group on the precursor, which could withstand harsh SN2 reaction conditions with no side reactions. [(18)F]-2-DFR accumulated preferentially in the liver and was metabolized by the same enzymes as ribose. [(18)F]-2-DFR could distinguish between healthy liver and liver damaged by acetaminophen. [(18)F]-2-DFR is expected to be a useful PET probe for imaging and quantifying liver functions in vivo, with likely significant clinical utility.

Three different fluoro- or chloro-substituted 1'-deoxy-1'-phenyl-ß-D-ribofuranoses.

Crystal structures are reported for three fluoro- or chloro-substituted 1'-deoxy-1'-phenyl-ß-D-ribofuranoses, namely 1'-deoxy-1'-(2,4,5-trifluorophenyl)-ß-D-ribofuranose, C11H11F3O4, (I), 1'-deoxy-1'-(2,4,6-trifluorophenyl)-ß-D-ribofuranose, C11H11F3O4, (II), and 1'-(4-chlorophenyl)-1'-deoxy-ß-D-ribofuranose, C11H13ClO4, (III). The five-membered furanose ring of the three compounds has a conformation between a C2'-endo,C3'-exo twist and a C2'-endo envelope. The ribofuranose groups of (I) and (III) are connected by intermolecular O-H···O hydrogen bonds to six symmetry-related molecules to form double layers, while the ribofuranose group of (II) is connected by O-H···O hydrogen bonds to four symmetry-related molecules to form single layers. The O···O contact distance of the O-H···O hydrogen bonds ranges from 2.7172 (15) to 2.8895 (19) Å. Neighbouring double layers of (I) are connected by a very weak intermolecular C-F···π contact. The layers of (II) are connected by one C-H···O and two C-H···F contacts, while the double layers of (III) are connected by a C-H···Cl contact. The conformations of the molecules are compared with those of seven related molecules. The orientation of the benzene ring is coplanar with the H-C1' bond or bisecting the H-C1'-C2' angle, or intermediate between these positions. The orientation of the benzene ring is independent of the substitution pattern of the ring and depends mainly on crystal-packing effects.

DNA polymerase λ inactivation by oxidized abasic sites.

Base excision repair (BER) plays a vital role in maintaining genomic integrity in mammalian cells. DNA polymerase λ (Pol λ) is believed to play a backup role to DNA polymerase ß (Pol ß) in base excision repair. Two oxidized abasic lesions that are produced by a variety of DNA-damaging agents, including several antitumor antibiotics, the C4'-oxidized abasic site following Ape1 incision (pC4-AP), and 5'-(2-phosphoryl-1,4-dioxobutane) (DOB), irreversibly inactivate Pol ß and Pol λ. The interactions of DOB and pC4-AP with Pol λ are examined in detail using DNA substrates containing these lesions at defined sites. Single-turnover kinetic experiments show that Pol λ excises DOB almost 13 times more slowly than a 5'-phosphorylated 2-deoxyribose (dRP). pC4-AP is excised approximately twice as fast as DOB. The absolute rate constants are considerably slower than those reported for Pol ß for the respective reactions, suggesting that Pol λ may be an inefficient backup in BER. DOB inactivates Pol λ approximately 3-fold less efficiently than it does Pol ß, and the difference can be attributed to a higher K(I) (33 ± 7 nM). Inactivation of Pol λ's lyase activity by DOB also prevents the enzyme from conducting polymerization following preincubation of the protein and DNA. Mass spectral analysis of GluC-digested Pol λ inactivated by DOB shows that Lys324 is modified. There is inferential support for the idea that Lys312 may also be modified. Both residues are within the Pol λ lyase active site. When acting on pC4-AP, Pol λ achieves approximately four turnovers on average before being inactivated. Lyase inactivation by pC4-AP is also accompanied by loss of polymerase activity, and mass spectrometry indicates that Lys312 and Lys324 are modified by the lesion. The ability of DOB and pC4-AP to inactivate Pol λ provides additional evidence that these lesions are significant sources of the cytotoxicity of DNA-damaging agents that produce them.

Structural flexibility of DNA-like conformers of canonical 2'-deoxyribonucleosides.

Relaxed force constants (RFC) and vibrational root-mean-square (VRMS) deviations are used for comparative characterization of mechanical properties of canonical 2'-deoxyribonucleosides (2DRs) and 1,2-dideoxyribose molecule, their model sugar residue. It has been shown that RFC and VRMS should be preferred over traditional force constants when one needs to obtain the quantitative measure of the 'collective' parameter flexibility (furanose sugar pseudorotation phase P in particular) and compare it with classical torsion angles (ß, γ, ε, χ). It has been found that torsions ε and ß determining the 2DRs backbone hydroxyl orientations are as soft as the pseudorotation phase P with RFC values within 1-10 kcal mol(-1) rad(-2) depending on conformation. Torsion γ is the most rigid one with RFC 15-30 kcal mol(-1) rad(-2), while the glycosidic torsion χ is characterized by intermediate values of RFC (typically 5-10 kcal mol(-1) rad(-2)) and its RFC changes by 10 times, depending on the furanose sugar conformation (K(χ)≈ 3 kcal mol(-1) rad(-2) in B- vs. K(χ)≈ 21 kcal mol(-1) rad(-2) in A-DNA-like conformation of 2'-deoxycytidine). Quantum zero-point motion of the nuclei makes the dominant contribution to VRMS deviations of molecules structural parameters: 9-22° for ß, ε and P, 5-7° for γ and χ at the temperature of 0 K, and 15-38° for ß, ε and P, 9-26° for γ and χ at the room temperature (298.15 K). Obtained results can be used in constructing simple dynamical models of the DNA fragments.

Stereoselective N-glycosylation of 2-deoxythioribosides for fluorescent nucleoside synthesis.

An efficient method for the N-2-deoxyribosylation of modified nucleobases by 2-deoxythioriboside donors is reported. In the presence of an in situ silylated nucleobase, thioglycosides can be activated with NIS/HOTf to give nucleosides in high yields and with good ß-selectivity. By tuning the protecting groups on the C3 and C5 hydroxyls, α/ß ratios ranging from 1.0:4.0 to 4.5:1.0 can be obtained. This strategy is applicable to the synthesis of various nucleosides, including ring-expanded pyrimidine derivatives containing sulfur that have previously been reported in low yields. The utility of this approach is further demonstrated by the synthesis of fluorescent nucleosides analogues such as quinazoline and oxophenothiazine that should find broad utility in DNA-folding and recognition studies.

2'-Deoxyriboguanylurea, the primary breakdown product of 5-aza-2'-deoxyribocytidine, is a mutagen, an epimutagen, an inhibitor of DNA methyltransferases and an inducer of 5-azacytidine-type fragile sites.

5-Aza-2'-deoxycytidine (5azaC-dR) has been employed as an inhibitor of DNA methylation, a chemotherapeutic agent, a clastogen, a mutagen, an inducer of fragile sites and a carcinogen. However, its effects are difficult to quantify because it rapidly breaks down in aqueous solution to the stable compound 2'-deoxyriboguanylurea (GuaUre-dR). Here, we used a phosphoramidite that permits the introduction of GuaUre-dR at defined positions in synthetic oligodeoxynucleotides to demonstrate that it is a potent inhibitor of human DNA methyltransferase 1 (hDNMT1) and the bacterial DNA methyltransferase (M.EcoRII) and that it is a mutagen that can form productive base pairs with either Guanine or Cytosine. Pure GuaUre-dR was found to be an effective demethylating agent and was able to induce 5azaC-dR type fragile sites FRA1J and FRA9E in human cells. Moreover, we report that demethylation associated with C:G → G:C transversion and C:G → T:A transition mutations was observed in human cells exposed to pure GuaUre-dR. The data suggest that most of the effects attributed to 5azaC-dR are exhibited by its stable primary breakdown product.

Synthesis and biological properties of chemically modified siRNAs bearing 1-deoxy-D-ribofuranose in their 3'-overhang region.

To elucidate the role of the sugar moiety in the two natural nucleotides of the 3'-overhang region of small interfering RNA (siRNA), we synthesized siRNAs that incorporated two abasic nucleosides, 1-deoxy-D-ribofuranose (R(H)). We improved the method for preparing an O-protected abasic nucleoside, 1-deoxy-2,3,5-tri-O-benzoyl-ß-D-ribofuranose, via the reductive cleavage of the anomeric position of 1-O-acetyl-2,3,5-tri-O-benzoyl-ß-D-ribofuranose. To incorporate R(H) into oligonucleotides by the standard phosphoramidite solid phase method, R(H) was converted into its phosphoramidite derivative and the solid support linked to a controlled pore glass resin. Chemically modified RNAs possessing R(H) at the 3'-overhang region were easily prepared in good yields. siRNAs containing R(H) showed moderate nuclease-resistance and a desirable knockdown effect.

An expedient biocatalytic procedure for abasic site precursors useful in oligonucleotide synthesis.

Preparation of abasic site precursors through a divergent chemoenzymatic synthesis has been accomplished. Several biocatalysts and acylating agents were studied furnishing a practical and scalable green method useful for industrial applications. Highly regioselective acylation and deacylation reactions with 1,2-dideoxy-D-ribose are described resulting in excellent yield. A fast, atom-efficient and convenient synthesis of 3-, and 5-O-DMTr-1,2-dideoxyribose 17 and 19 has been achieved. These compounds are useful precursors for the preparation of phosphoramidites required for the assembly of oligonucleotides containing the tetrahydrofuran abasic lesions.

Synthesis and photochemical behavior of the tetrazolo tautomer of 2-azido-4-pyrimidinone-2'-deoxyriboside.

The 2-azido analogue of 2'-deoxyuridine was prepared in three steps from 2'-deoxy-2-thiouridine. The sulfur atom of the 2-thio nucleoside was methylated and then displaced by hydrazine to furnish the corresponding 2-hydrazino derivative. After diazotization, the 2-azido compound that exists as its tetrazolo tautomer was obtained. Upon UV irradiation in aqueous solution, the title compound led to isocytosine.

Synthesis of a fluorescent 2'3'-dideoxycytosine analog, tCdd.

The pathway leading to the preparation of a novel tricyclic 2'3'-dideoxycytosine analog, tCdd (1) is reported. A protected 2'3'-dideoxyribose prepared from l-glutamic acid was coupled to a silylated fluorescent base to yield a mixture of the alpha- and beta-anomers of the 2'3'-dideoxyribonucleoside of 1,3-diaza-2-oxophenothiazine, tCdd (1). The fluorescent base analog retains a high fluorescence emission over a large pH range and should be useful in a variety of probe applications.

A new 1'-methylenedisulfide deoxyribose that forms an efficient cross-link to DNA cytosine-5 methyltransferase (DNMT).

A newly designed 1'-methylenedisulfide deoxyribose probe can efficiently cross-link to DNA cytosine-5-methyltransferases (DNMTs) through the active Cys residue, which provides a new tool to covalently trap DNMT-DNA complexes.

Measurement of very low rates of cell proliferation by heavy water labeling of DNA and gas chromatography/pyrolysis/isotope ratio-mass spectrometric analysis.

DNA replication during S-phase represents a biochemical metric of cell division. We present here a protocol for measuring very low rates of cell proliferation, on the basis of the incorporation of deuterium ((2)H) from heavy water ((2)H(2)O) into the deoxyribose moiety of purine deoxyribonucleotides in DNA of dividing cells, by use of gas chromatography/pyrolysis/isotope ratio-mass spectrometry (GC/P/IRMS). Very low levels of label incorporation (>or=0.002% atom percent excess (2)H) can be quantified by GC/P/IRMS. This protocol thereby permits shorter periods or lower amounts of (2)H(2)O administration than would be required using standard GC/MS techniques for measuring cell proliferation kinetics (see accompanying protocol in this issue). A disadvantage of this approach compared to GC/MS is the requirement for larger numbers of cells (> approximately 10(7)). This protocol enables definitive in vivo studies of the fraction or absolute number of newly divided cells and their subsequent survival kinetics in animals and humans, even when turnover rates are very low. Indolent hematologic malignancies, such as chronic lymphocytic leukemia, and other relatively quiescent cells represent promising areas of application.

Base excision repair intermediates are mutagenic in mammalian cells.

Base excision repair (BER) is the main pathway for repair of DNA damage in mammalian cells. This pathway leads to the formation of DNA repair intermediates which, if still unsolved, cause cell lethality and mutagenesis. To characterize mutations induced by BER intermediates in mammalian cells, an SV-40 derived shuttle vector was constructed carrying a site-specific lesion within the recognition sequence of a restriction endonuclease. The mutation spectra of abasic (AP) sites, 5'-deoxyribose-5-phosphate (5'dRp) and 3'-[2,3-didehydro-2,3-dideoxy-ribose] (3'ddR5p) single-strand breaks (ssb) in mammalian cells was analysed by RFLP/PCR and mutation frequency was estimated by quantitative PCR. Point mutations were the predominant events occurring at all BER intermediates. The AP site-induced mutation spectrum supports evidence for the 'A-rule' and is also consistent with the use of the 5' neighbouring base to instruct nucleotide incorporation (5'-rule). Preferential adenine insertion was also observed after in vivo replication of 5'dRp or 3'ddR5p ssb. We provide original evidence that not only the abasic site but also its derivatives 'faceless' BER intermediates are mutagenic, with a similar mutation frequency, in mammalian cells. Our findings support the hypothesis that unattended BER intermediates could be a constant threat for genome integrity as well as a spontaneous source of mutations.