Synthesis of 9-[(2-hydroxy-1-phosphonylethoxy)ethyl]guanine, 1-[(2-hydroxy-1-phosphonylethoxy)ethyl]cytosine and 9-[(2-hydroxy-1-phosphonylethoxy)ethyl] adenine

The acyclic nucleoside phosphonate analogues, 9-[(2-hydroxy-1-phosphonylethoxy)ethyl]guanine 6 , 1-[(2-hydroxy-1-phosphonylethoxy)ethyl]cytosine 7 and 9-[(2-hydroxy-1-phosphonylethoxy)ethyl]adenine 8 , have been prepared by the coupling of a tosylate of the phosphonate side chain 12 with a purine or pyrimidine base followed by deprotection of the blocking groups.     

Janus-type AT nucleosides: synthesis, solid and solution state structures

Novel Janus-type nucleoside analogues (1a-d) were synthesized. Their pyrimido[4,5-d]pyrimidine base moiety has one face with a bidentate Watson-Crick donor-acceptor (DA) H-bond array of adenine and the other face with an acceptor-donor (AD) H-bond array of thymine. These nucleosides may self-associate through the self-complementary base pair. Indeed, in the solid state, compound 6d displayed a honeycomb-like supramolecular structure with tetrameric membered cavities formed through the combination of reverse Watson-Crick base pairs and aromatic stacking, in which the solvent molecules were accommodated. The result of temperature-dependent CD studies showed that the free nucleosides can form higher order chiral structures in aqueous solution.     

New Nucleoside Analogues for the Treatment of Hemorrhagic Fever Virus Infections

Eight different compounds, all nucleoside analogues, could presently be considered as potential drug candidates for the treatment of Ebola virus (EBOV) and/or other hemorrhagic fever virus (HFV) infections. They can be considered as either (i) adenine analogues (3‐deazaneplanocin A, galidesivir, GS‐6620 and remdesivir) or (ii) guanine analogues containing the carboxamide entity (ribavirin, EICAR, pyrazofurin and favipiravir). All eight owe their mechanism of action to hydrogen bonded base pairing with either (i) uracil or (ii) cytosine. Four out of the eight compounds (galidesivir, GS‐6620, remdesivir and pyrazofurin) are C‐nucleosides, and two of them (GS‐6620, remdesivir) also contain a phosphoramidate part. The C‐nucleoside and phosphoramidate (and for the adenine analogues the 1′‐cyano group as well) may be considered as essential attributes for their antiviral activity.     

Synthesis and Anti-HIV Activity of Triazolo-fused 3',4'-Cyclic and 4'-Spiro Nucleoside Analogues

Triazolo-fused 3',4'-cyclic nucleoside 4'-spiro nucleoside analogues were synthesized by an intramolecular 1,3-dipolar cycloaddition of 4'-azido nucleoside derived azido-alkynes in a regio- and stereo-specific manner. The thymine nucleoside base in these target compounds was transformed into the corresponding 5-methyl cytosine component. The synthesized com­pounds were examined in an MAGI(multinuclear-activation galactosidase indicator) assay for exploring the anti-HIV activity and in a H9 T(human T lymphocytes H9) assay for measuring the cell toxicity.     

Preparation and antiviral properties of new acyclic, achiral nucleoside analogues: 1- or 9-[3-hydroxy-2-(hydroxymethyl)prop-1-enyl]nucleobases and 1- or 9-[2,3-dihydroxy-2-(hydroxymethyl)propyl]nucleobases

Acyclic, achiral nucleoside derivatives 1b-e of adenine, cytosine, 5-methylcytosine, and guanine, containing a 3-hydroxy-2-(hydroxymethyl)prop-1-enyl group on N-1 or N-9, have been prepared analogously to the previously described thymine derivative 1a. In contrast to the adenine and guanine derivatives, the cytosine derivative 9 was unstable, and was obtained in a low yield due to side reactions. These include cleavage of the propenyl group from the base, and the formation of a bicyclic compound. The thymine derivative, although stable under neutral conditions, likewise underwent a reversible cyclization reaction (Michael addition) in the presence of acids or bases. The 5-methylcytosine derivative was stable under neutral and basic conditions. Four other nucleoside derivatives 26a-d containing a 2,3-dihydroxy-2-(hydroxymethyl)propyl group on N-1 or N-9, three of which are new, have likewise been prepared. All compounds were evaluated as antiviral agents against HIV-1 and HSV-1 but were devoid of antiviral activity.     

Effects of base pairing on the one-electron reduction rate of cytosine

Using nucleoside derivatives, which are soluble in dichloromethane, we have experimentally demonstrated that the reduction potential of cytosine is lowered by base pairing with guanine.     

Theoretical study of tautomeric and ionizing effects of guanine,cytosine, and their methyl derivatives

The study of pre-translational effects (ionization, tautomerization) and post-translational effects (methylation) of guanine and cytosine has only recently been the focus of some studies. These effects can potentially help regulate gene expression as well as potentially disrupt normal gene function. Because of this wide array of roles, greater insight into these effects in deoxyribonucleic acids (DNA) are paramount. There has been considerable research of each phenomenon (tautomerization, methylation and ionization) individually. In this work, we attempt to shed light upon the pre- and post-translational effects of guanine and cytosine by investigating the electron affinities (EAs) and ionization potentials (IPs) of the major and minor tautomers and their methyl derivatives. We performed all calculations using the density functional theory B3LYP functional accompanied with 6-311G (d,p), 6-311+G (d,p), and 6-311++G (df,pd) basis sets. Our results reveal that the cytosine tautomer has a higher EA and IP than the guanine tautomers. The higher EA suggest that an electron that attaches to the GC base pair would predominately attach to the cytosine instead of guanine. The higher IP would suggest that an electron that is removed from the GC base pair would be predominately removed from the guanine within the base pair. Understanding how tautomerization, ionization, and methylation differences change effects, discourages, or promotes one another is lacking. In this work, we begin the steps of integrating these effects with one another, to gain a greater understanding of molecular changes in DNA bases.     

Synthesis of 3'-deoxynucleosides with 2-oxabicyclo[3.1.0]hexane sugar moieties: addition of difluorocarbene to a 3',4'-unsaturated uridine derivative and 1,2-dihydrofurans derived from D- and L-xylose1

Syntheses of 3'-deoxy analogues of adenosine, cytidine, and uridine with a 2,2-difluorocyclopropane ring fused at C3'-C4' are described. Treatment of a 2',5'-protected-3',4'-unsaturated derivative of uridine with difluorocarbene [generated from (CF3)2Hg and NaI] gave a diastereomeric mixture of the 3',4'-difluoromethylene compounds (alpha-L-arabino/beta-D-ribo, approximately 5:4). The limited stereoselectivity for addition at the beta face results from competitive steric hindrance by an allylic 4-methoxybenzyloxy group at C2' on the alpha face and a homoallylic nucleobase at C1' on the beta face. Protected uracil derivatives were converted into their cytosine counterparts via 4-(1,2,4-triazol-1-yl) intermediates. Treatment of 1,2-dihydrofurans derived from D- and L-xylose with difluorocarbene resulted in stereospecific addition at the beta face (anti to the 1,2-O-isopropylidene group on the alpha face). Glycosylations with activated enantiomeric sugar derivatives with the fused difluorocyclopropane ring on the beta face gave protected adenine nucleosides, whereas attempted glycosylation with an alpha-fused derivative gave multiple products. Removal of base- and sugar-protecting groups gave new difluoromethylene-bridged nucleoside analogues.     

Molecular recognition of cytosine- and guanine-functionalized nucleolipids in the mixed monolayers at the air-water interface and Langmuir-Blodgett films

Molecular recognition of mixed nucleolipids of 1-(2-octadecyloxycarbonylethyl)cytosine and 7-(2-octadecyloxycarbonylethyl)guanine in the monolayers at the air-water interface and Langmuir-Blodgett (LB) films has been investigated in detail using surface pressure/potential-area isotherms, infrared reflection-absorption spectroscopy (IRRAS), and Fourier transform infrared (FTIR) transmission spectroscopy, respectively. Prior to molecular recognition, the cytosine moieties in the monolayer were hydrogen bonded with an almost flat-on orientation, the alkyl chains were uniaxially oriented with respect to the film normal, the guanine moieties in the monolayer were stacked probably through pi-pi interaction with an end-on orientation, and the C-C-C planes of the alkyl chains were preferentially oriented parallel to the water surface. In the monolayer of equimolar mixture, molecular recognition between the cytosine and guanine moieties occurred together with the ring planes of base pairing and the C-C-C planes of the alkyl chains favorably oriented parallel to the water surface. The guanine moieties underwent an orientation change from an end-on mode before molecular recognition to a flat-on one after molecular recognition. The base pairing between the cytosine and guanine moieties in the monolayers was achieved since the N7-substituted guanine derivatives suppressed the formation of guanine tetramers. Both the IRRAS spectra of the monolayers and the FTIR spectra of the LB films presented the exact sites in the cytosine and guanine moieties for the formation of triple hydrogen bonds. The base pairing resulted in a change in molecular orientation and interaction, and the corresponding LB film exhibited a different phase transition behavior from a typical crystal transition for the cytosine-functionalized nucleolipids and an analogous glass transition for the guanine-functionalized nucleolipids. The thermal stability of the mixed LB film was improved in comparison to the LB films of pure components.     

Nonpolar isosteres of damaged DNA bases: effective mimicry of mutagenic properties of 8-oxopurines

A substantial fraction of mutations that arise in the cell comes from oxidative damage to DNA bases. Oxidation of purine bases at the 8-position, yielding 8-oxo-G and 8-oxo-A, results in conformational changes (from anti to syn) that cause miscoding during DNA replication. Here we describe the synthesis and biophysical and biochemical properties of low-polarity shape mimics of 8-oxopurines, and we report that these new analogues exhibit remarkable mimicry of the mutagenic properties of the natural damaged bases. A 2-chloro-4-fluoroindole nucleoside (1) was designed as an isosteric analogue of 8-oxo-dG, and a 2-chloro-4-methylbenzimidazole nucleoside (2) as a mimic of 8-oxo-dA. The nucleosides were prepared by reaction of the parent heterocycles with Hoffer's chlorodeoxyribose derivative. Structural studies of the free nucleosides 1 and 2 revealed that both bases are oriented syn, thus mimicking the conformation of the oxopurine nucleosides. Suitably protected phosphoramidite derivatives were prepared for incorporation into synthetic DNAs, to be used as probes of DNA damage responses, and 5'-triphosphate derivatives (3 and 4) were synthesized as analogues of damaged nucleotides in the cellular nucleotide pool. Base pairing studies in 12-mer duplexes showed that 1 and 2 have low affinity for polar pairing partners, consistent with previous nonpolar DNA base analogues. However, both compounds pair with small but significant selectivity for purine partners, consistent with the idea that the syn purine geometry leads to pyrimidine-like shapes. Steady-state kinetics studies of 1 and 2 were carried out with the Klenow fragment of Escherichia coli DNA Pol I (exo-) in single-nucleotide insertions. In the DNA template, the analogues successfully mimicked the mutagenic behavior of oxopurines, with 1 being paired selectively with adenine and 2 pairing selectively with guanine. The compounds showed similar mutagenic behavior as nucleoside triphosphate analogues, being preferentially inserted opposite mutagenic purine partners. The results suggest that much of the mutagenicity of oxopurines arises from their shapes in the syn conformation rather than from electrostatic and hydrogen-bonding effects. The new analogues are expected to be generally useful as mechanistic probes of cellular responses to DNA damage.     

A Convenient Synthesis of 4,6-Dichloro-5-benzylthiopyrimidine

A practical and convenient two-step synthesis of the title compound 4,6-dichloro-5-benzylthiopyrimidine (3) from 4,6-dihydroxypyrimidine (1) is described.     

Regulation of one-electron oxidation rate of guanine by base pairing with cytosine derivatives

Effects of base pairing on the one-electron oxidation rate of guanine derivatives, guanine, 8-bromoguanine, and 8-oxo-7,8-dihydroguanine have been studied. The one-electron oxidation rate of guanine derivatives was determined by triplet-quenching experiments, using N,N'-dibutylnaphthaldiimide (NDI) in the triplet excited state (3NDI*) and fullerene (C(60)) in the triplet excited state ((3)C(60*)) as oxidants. In all three guanine derivatives studied here, acceleration of the one-electron oxidation was observed upon hydrogen bonding with cytosine, which demonstrates lowering of the oxidation potential of guanine derivatives by base pairing with cytosine. When a methyl or bromo group was introduced to the C5 position of cytosine, acceleration or suppression of the one-electron oxidation relative to the guanine:cytosine base pair was observed, respectively. The results demonstrate that the one-electron oxidation rate of guanine in DNA can be regulated by introducing a substituent on base pairing cytosine.     

Synthesis of the G-C DNA base hybrid with a functional tail

Molecules which possess the hydrogen bonding codes of both guanine and cytosine ("G-C DNA base hybrids") are known to organize in a hexagonal array both in solution and the solid state. Including an easily derivatizable functional group in the molecule allows the co-organization of virtually any species in the hexagonal periphery. Simple 5- and 6-step procedures are described for the synthesis of DNA base hybrids with tail groups which are terminated by electrophilic (primary bromide) and nucleophilic (primary alcohol) functions, respectively.     

Oligonucleotides incorporating 8-aza-7-deazapurines: synthesis and base pairing of nucleosides with nitrogen-8 as a glycosylation position

Oligonucleotides incorporating the unusually linked 8-aza-7-deazapurine N8-(2'-deoxyribonucleosides) 3a,b (purine and 6-amino-2-chloropurine analogues) were used as chemical probes to investigate the base pairing motifs of the universal nucleoside 8-aza-7-deazapurin-6-amine N8-(2'-deoxyribofuranoside) 2 (adenine analogue) and that of the 2,6-diamino compound 1. Owing to the absence of an amino group on the nucleoside 3a the low stability of oligonucleotide duplexes incorporating this compound opposite to the four canonical DNA-constituents indicate hydrogen bonding and base pairing for the universal nucleosides 1 and 2 which form much more stable duplexes. When the 6-amino-2-chloro-8-aza-7-deazapurine nucleoside 3b replaces 1 and is located at the same positions, two sets of duplexes are formed (i) high Tm duplexes with 3b located opposite to dA or dC and (ii) low Tm duplexes with 3b located opposite to dG or dT. These results are due to the steric clash of the 2-chloro substituent of 3b with the 2-oxo group of dT or the 6-oxo group of dG while the 2-halogeno substituents are well accommodated in the base pairs formed with dA or dC. For comparison duplexes incorporating the regularly linked nucleosides 4-6a,b containing the same nucleobases as those of 1-3a,b were studied.     

Properties of a new fluorescent cytosine analogue, pyrrolocytosine

Pyrrolocytosine is a novel, environment sensitive, fluorescent base that can be used in place of cytosine as a fluorescent marker in nucleic acids. In this work the results of a detailed computational investigation into the hybridization and photochemical properties of the base are reported. The interaction energy of the base pair formed between pyrrolocytosine and guanine, calculated at the MP2/6-31G(d,0.25)//HF/6-31G(d,p) level, was found to be -27.2 kcal mol(-1), comparing very favorably with the value calculated for the cytosine and guanine base pair, -25.8 kcal mol(-1). The wavelengths for the vertical transitions of pyrrolocytosine and cytosine were determined using both the configuration interaction technique, with singly excited configurations (CIS) and time-dependent density functional theory using the B3LYP functional (TDB3LYP). It was found that the spacing between the first pipi state and the first npi state was significantly larger in the case of pyrrolocytosine than cytosine, providing a rationale for the higher fluorescence quantum yield of the former. Hydrogen bonding of pyrrolocytosine to guanine did not affect the predicted fluorescence properties of pyrrolocytosine whereas stacking guanine above pyrrolocytosine, in a manner appropriate to B-form DNA, significantly reduced the predicted fluorescence. Calculations on the two base systems using the TDB3LYP method produced low-lying charge-transfer states which are not predicted when the CIS method is used and are not thought to be physically meaningful.     

At nonzero temperatures, stacked structures of methylated nucleic acid base pairs and microhydrated nonmethylated nucleic acid base pairs are favored over planar hydrogen-bonded structures: a molecular dynamics simulations study

The dynamic structure of all ten possible nucleic acid (NA) base pairs and methylated NA base pairs hydrated by a small number of water molecules (from 1 to 16) was determined by using molecular dynamics simulations in the NVE microcanonical and NVT canonical ensembles with the Cornell force field (W. D. Cornell, P. Cieplak, C. I. Bayly, I. R. Gould, K. M. Merz, D. M. Ferguson, D. C. Spellmeyer, T. Fox, J. E. Caldwell, P. Kollman, J. Am. Chem. Soc. 1995, 117, 5179). The presence of one water molecule does not affect the structure of any hydrogen-bonded (H-bonded) nonmethylated base pair. An equal population of H-bonded and stacked structures of adenine...adenine, adenine...guanine and adenine... thymine pairs is reached if as few as two water molecules are present, while obtaining equal populations of these structures in the case of adenine...cytosine, cytosine...thymine, guanine... guanine and guanine...thymine required the presence of four water molecules, and in the case of guanine...cytosine, six. A comparable population of planar, H-bonded and stacked structures for cytosine...cytosine and thymine... thymine base pairs was only obtained if at least eight water molecules hydrated a pair. Methylation of bases changed the situation dramatically and stacked structures were favoured over H-bonded ones even in the absence of water molecules in most cases. Only in the case of methyl cytosine...methyl cytosine, methyl guanine...methyl guanine and methyl guanine...methyl cytosine pairs were two, two or six water molecules, respectively, needed in order to obtain a comparable population of planar, H-bonded and stacked structures. We believe that these results give clear evidence that the preferred stacked structure of NA base pairs in the microhydrated environment, and also apparently in a regular solvent, is due to the hydrophilic interaction of a small number of water molecules. In the case of methylated bases, it is also due to the fact that the hydrogen atoms most suitable for the formation of H-bonds have been replaced by a methyl group. A preferred stacked structure is, thus, not due to a hydrophobic interaction between a large bulk of water molecules and the base pair, as believed.     

Halogen bonding in DNA base pairs

Halogen bonding (R-X···Y) is a qualitative analogue of hydrogen bonding that may prove useful in the rational design of artificial proteins and nucleotides. We explore halogen-bonded DNA base pairs containing modified guanine, cytosine, adenine and thymine nucleosides. The structures and stabilities of the halogenated systems are compared to the normal hydrogen bonded base pairs. In most cases, energetically stable, coplanar structures are identified. In the most favorable cases, halogenated base pair stabilities are within 2 kcal mol(-1) of the hydrogen bonded analogues. Among the halogens X = Cl, Br, and I, bromine is best suited for inclusion in these biological systems because it possesses the best combination of polarizability and steric suitability. We find that the most stable structures result from a single substitution of a hydrogen bond for a halogen bond in dA:dT and dG:dC base pairs, which allows 1 or 2 hydrogen bonds, respectively, to complement the halogen bond.     

Photophysical characters of rationally designed hetero-ring-expanded guanine analogues and effect of cytosine pairing

We present the results of the CIS and TDB3LYP calculations of the optical absorption and emission spectra of some newly designed guanine (G) analogues and their Watson-Crick base pairs. Compared with natural G, the onset absorption peaks of these newly designed analogues are red-shifted, while the fluorescence peaks are blue-shifted. In general, the first excited singlet states (pipi*) of these analogues are nonplanar for all bases considered here. But, the Stokes shifts for the designed G-analogues are much smaller than that of natural G, suggesting that they have stronger molecular rigidity and higher fluorescence quantum yields than those of natural G. The first excited states of these Watson-Crick base pairs essentially originate from those of their isolated purine moieties, as demonstrated from the S1 geometries of their Watson-Crick base pairs. For G and its analogues, A1 and A2 (they are ring-expanded with one-bond intercalation at the C5 site), the pairing with cytosine reduces the oscillator strengths of both the first absorption peak (by 27%-60%) and the fluorescent emission (by 19%-23%), while for the analogues A3, A4, and xG in which G is ring-expanded with a two-bond intercalation at the C5 site, the pairing, in contrast, increases the oscillator strengths of both the first absorption peak (by 11%-15%) and the fluorescent emission (by 3%-20%). These observations indicate that the pairing with cytosine can quench the fluorescence for G, A1, and A2 but enhance the fluorescence quantum yields for A3, A4, and xG. The significant shifts induced by ring-expansion in the ring-expanded G with a two-bond intercalation at the C5 site reveal a possibility for their fluorescent detections.     

Synthesis of amino- and guanidino-G-clamp PNA monomers

Syntheses of the protected amino- and guanidino-G-clamp PNA monomers, 9a and 9b, respectively, have been accomplished in eight steps from 5-bromouracil. Enhanced stacking interactions and additional hydrogen bonds with guanine should increase the affinity of PNAs incorporating these cytosine analogues for their complementary strands. [reaction: see text]     

Synthesis of oxepane ring containing monocyclic, conformationally restricted bicyclic and spirocyclic nucleosides from D-glucose: a cycloaddition approach

Carbohydrate-derived substrates having (i) C-5 nitrone and C-3-O-allyl, (ii) C-4 vinyl and a C-3-O-tethered nitrone, and (iii) C-5 nitrone and C-4-allyloxymethyl generated tetracyclic isoxazolidinooxepane/-pyran ring systems upon intramolecular nitrone cycloaddition reactions. Deprotection of the 1,2-acetonides of these derivatives followed by introduction of uracil base via Vorbrüggen reaction condition and cleavage of the isooxazolidine rings as well as of benzyl groups by transfer hydrogenolysis yielded an oxepane ring containing bicyclic and spirocyclic nucleosides. The corresponding oxepane based nucleoside analogues were prepared by cleavage of isoxazolidine and furanose rings, coupling of the generated amino functionalities with 5-amino-4,6-dichloropyrimidine, cyclization to purine rings, and finally aminolysis.