Synthesis and DNA incorporation of an ethynyl-bridged cytosine C-nucleoside as guanosine surrogate

As a guanosine mimic that lacks the preference for syn or anti conformation a cytosine C-nucleoside was synthesized connecting the nucleobase at the anomeric center by an ethynyl linker. The key step was a Sonogashira cross coupling of 5-iodocytosine with 1'-ethynyl-2'-deoxyribose. The new C-nucleoside incorporated into G/C-alternating oligonucleotides emerged as guanosine substitute, however, with reduced duplex stability. B-Form DNA was strongly stabilized by the new surrogate even in typically Z-DNA forming sequences and in Z-form inducing environment.     

Experimental and structural evidence that herpes 1 kinase and cellular DNA polymerase(s) discriminate on the basis of sugar pucker

Two isomers of methanocarba (MC) thymidine (T), one an effective antiherpes agent with the pseudosugar moiety locked in the North (N) hemisphere of the pseudorotational cycle (1a, N-MCT) and the other an inactive isomer locked in the antipodean South (S) conformation (1b, S-MCT) were used to determine whether kinases and polymerases discriminate between their substrates on the basis of sugar conformation. A combined solid-state and solution conformational analysis of both compounds, coupled with the direct measurement of mono-, di-, and triphosphate levels in control cells, cells infected with the Herpes simplex virus, or cells transfected with the corresponding viral kinase gene (HSV-tk), suggests that kinases prefer substrates that adopt the S sugar conformation. On the other hand, the cellular DNA polymerase(s) of a murine tumor cell line transfected with HSV-tk incorporated almost exclusively the triphosphate of the locked N conformer (N-MCTTP), notwithstanding the presence of higher triphosphate levels of the S-conformer (S-MCTTP).     

An alpha-D-configured bicyclic nucleoside restricted in an E-type conformation: synthesis and parallel RNA recognition

An alpha-D-arabino configured bicyclic nucleoside strongly restricted in an E-type conformation by a 2'-3'-fused oxetane ring is synthesized. Several synthetic strategies toward the target compound are described, and the successful preparation from a D-xylose derivative is based on a ruthenium-mediated cleavage of a double bond, an S(N)2-inversion at the 2-position to give an arabino-configuration, nucleobase coupling, and finally ring closure to give the oxetane ring. The E-type conformation is confirmed by molecular modeling and NMR. The nucleoside is incorporated into short alpha-DNA sequences. In a mixed pyrimidine context, these recognize complementary parallel RNA-sequences with mainly increased affinity and complementary parallel DNA-sequences with decreased affinity. The present bicyclic analogue represents the first conformationally restricted alpha-DNA-analogue to improve nucleic acid recognition in mixmers with alpha-DNA monomers.     

Stabilization of zwitterions in solution: GABA analogues

The solution-phase structures of a number of conformationally restricted gamma-aminobutyric acid (GABA) analogues are investigated at the MP2/6-31+G* level of theory, using both explicit water molecules and the conductor-like screening solvation model (COSMO) to model solvation. GABA analogues constrained in a cis conformation by either a double bond or cyclopropane ring have the potential to attain either folded, intramolecularly hydrogen-bonded, or partially folded conformations in solution. Systems constrained in a cis conformation by a cyclopentane or cyclopentene ring are more conformationally restricted and exist only in a folded, intramolecularly hydrogen-bonded form. GABA analogues constrained in a trans conformation by either a double bond or cyclopropane ring have the potential to adopt either partially folded or fully extended conformations in solution. Due to a lack of conformational flexibility, analogues that are constrained in a trans conformation by a cyclopentane or cyclopentene ring attain only partially folded conformations. Like GABA, conformationally flexible GABA analogues possess a large number of stable rotamers, and may exist in any or all of these conformations in aqueous solution. The structures of these analogues provide an essential foundation for subsequent structure-activity analysis of ligand binding at GABA receptors and transporters. This work is therefore expected to facilitate the design and development of new biologically active GABA analogues to treat GABA-related neurological disorders.     

Molecular structure of free canonical 2′-deoxyribonucleosides: a density functional study

The molecular structure of isolated canonical 2′-deoxyrinobucleosides was calculated using the density functional theory. It was demonstrated that the geometry of the base unit (BU) is almost unchanged compared to free nucleobases. Only slight out-of-plane deformation of the pyrimidine ring in deoxy-cytidine is observed. The conformation of the furanose ring strongly depends on the nature and orientation of the nucleobase. All nucleosides possess different conformations of this ring. Significant influence of the steric repulsion between the nucleobase and the sugar unit (SU) on puckering of the furanose ring and variation of the C–O and glycosyl bond lengths was demonstrated. The C(3′)-endo conformer of the furanose ring is more stable at the anti-orientation BU with respect to SU. An opposite trend is observed for the syn-orientation which is additionally stabilized by an intramolecular hydrogen bond with participation of the C(5′)OH group.     

Conformational properties of shape modified nucleosides--fleximers

A detailed (1)H NMR conformational study complemented with ab initio computations was performed in solution on fleximer nucleosides 1, 3, and 5 in relation to their natural counterparts. The substitution of the purine nucleobase found in the natural nucleosides with a more flexible two-ring heterocyclic system strongly increased the population of anti conformation around the glycosidic bond. This was accompanied by a large shift toward a north-type sugar conformation, which was explained by the interplay of anomeric, gauche, and steric effects. The formal separation of the bicyclic purine base into its imidazole and pyrimidine moieties allows for formation of a hydrogen bond between the NH(2) and 2'-OH groups and facilitates favorable conjugation between the two heterocyclic rings. Our results show that the interplay of stereoelectronic effects, combined with the flexibility of the nucleobase and possible conjugation effects within the nucleobase, plays a crucial role in the search for shape-mimic nucleosides that will interact with flexible binding sites.     

Application of capillary electrophoresis in the analysis of novel synthetic dideoxynucleoside analogues with potential anti-HIV activity.

The aim of this study was the development of a capillary electrophoretic method for the analysis of a series of novel synthetic dideoxynucleoside analogues with potential anti-HIV activity. These analogues consist of a tetrahydrofuranyl or a tetrahydropyranyl ring as the pseudosugar part and bear a hydroxyethyl side-chain and a nucleobase of the pyrimidine (eg thymine or uracil) or the purine (adenine) type with cis or trans configuration. Analysis of these derivatives was performed by capillary zone electrophoresis using 25 mM phosphate pH 3.00 and 4.00 as operating buffers for pyrimidine and purine analogues, respectively, and detection of separated species at 254 nm.     

Exploring nucleoside hydrolase catalysis in silico: molecular dynamics study of enzyme-bound substrate and transition state

The mechanism of action of inosine-uridine nucleoside hydrolase has been investigated by long-term molecular dynamics (MD) simulation in TIP3P water using stochastic boundary conditions. Five MD studies have been performed with enzyme substrate complex (E.S), enzyme substrate complex with protonated His241 (EH.S), enzyme transition state complex (E.TS), enzyme transition state complex with protonated His241 (EH.TS), and His241Ala transition state complex E(H241A).TS. Special attention has been given to the role of His241, which has been considered as the general acid catalyst to assist departure of the leaving nucleobase on the basis of its location in the active site in the X-ray crystal structure (). Yet on the basis of the location in the active site, Tyr229 is closer to the aniline ring of pAPIR as compared to His241. On initiation of MD simulations, His241 does not approach the nucleobase in the structures of EH.S, E.S, EH.TS, and E.TS. In the solvated enzyme, Tyr229, which is a member of the hydrogen bonding network inosine O2'.Asp14.His241.Tyr229.inosine N7, serves as a proton source to the leaving nucleobase. The loss of significant activity of His241Ala mutant is shown to be related to the disruption of the above hydrogen bonded network and the distancing of Tyr229 from inosine N7. The structures of the enzyme complexes with substrate or TS are not visibly altered on protonation of His241, a most unusual outcome. The bell-shaped pH dependence upon pK(app)'s of 7.1 and 9.1 may be attributed to the necessity of the dissociation of Asp10 or Asp15 and the acid form of Tyr229, respectively. In TS, the residue Ile81 migrated closer, whereas Arg233 moved away from the nucleobase. The probability of ribooxocarbenium ion stabilization by Asn168 and Asp14 is discussed. The Asp14-CO(2)(-) is hydrogen bonded to the ribose 2'-OH for 96% of the MD simulation time. Nucleophilic addition of water138 to ribooxocarbenium ion is suggested to be assisted by the proton shuttle from water138 --> Asp10 --> Asp15 --> water pool. An anticorrelation motion between Tyr229-OH and Asn168-OD1 in EH.S and E.S is observed. The relationship of this anticorrelated motion to mechanism, if any, deserves further exploration, perhaps the formation of a near attack conformation.     

A universal nucleoside with strong two-band switchable fluorescence and sensitivity to the environment for investigating DNA interactions

With the aim of developing a new tool to investigate DNA interactions, a nucleoside analogue incorporating a 3-hydroxychromone (3HC) fluorophore as a nucleobase mimic was synthesized and incorporated into oligonucleotide chains. In comparison with existing fluorescent nucleoside analogues, this dye features exceptional environmental sensitivity switching between two well-resolved fluorescence bands. In labeled DNA, this nucleoside analogue does not alter the duplex conformation and exhibits a high fluorescence quantum yield. This probe is up to 50-fold brighter than 2-aminopurine, the fluorescent nucleoside standard. Moreover, the dual emission is highly sensitive to the polarity of the environment; thus, a strong shielding effect of the flanking bases from water was observed. With this nucleoside, the effect of a viral chaperone protein on DNA base stacking was site-selectively monitored.     

Structural characterization of isoxazolidinyl nucleosides by fast atom bombardment tandem mass spectrometry.

4'-Aza-2',3'-dideoxyerythrofuranosyl derivatives of thymine (AdT, 1) and uracil (AdU, 2) are analogues of 2',3'-dideoxyribofuranosyl thymine (ddT, 3) and uracyl (ddU, 4). Compounds 1 and 2 are representative of a new class of antiviral agents where the sugar moiety is replaced by an isoxazolidine ring. The increasing importance of isoxazolidinyl nucleosides has encouraged the exploitation of simple mass spectrometric rules for unambiguously assigning their structure. The species 1, 2, 5 and 6 were therefore synthesized in order to evaluate the role of the basic centre of the modified sugar moiety in their gas-phase chemistry. The tandem mass spectra of these compounds are similar to those of the wild-type nucleosides and display fragment ions corresponding to [B + 2H](+),[M - BH](+) and [B + 27](+) species, where B is the nucleobase. The last species derives from a retrocycloaddition process which is less evident in 2'-deoxyribosides. This behaviour is consistent with protonation of the analytes at the pyrimidine rings. Model isoxazolidines, in which the nucleobase was replaced by a phenyl or a naphthyl moiety, displayed the expected behaviour of species with a localized charge on the N-O moiety of the isoxazolidine ring.     

Optimization of unnatural base pair packing for polymerase recognition

As part of an effort to expand the genetic alphabet, we have been examining the ability of predominately hydrophobic nucleobase analogues to pair in duplex DNA and during polymerase-mediated replication. We previously reported the synthesis and thermal stability of unnatural base pairs formed between nucleotides bearing simple methyl-substituted phenyl ring nucleobase analogues. Several of these pairs are virtually as stable and selective as natural base pairs in the same sequence context. Here, we report the characterization of polymerase-mediated replication of the same unnatural base pairs. We find that every facet of replication, including correct and incorrect base pair synthesis, as well as continued primer extension beyond the unnatural base pair, is sensitive to the specific methyl substitution pattern of the nucleobase analogue. The results demonstrate that neither hydrogen bonding nor large aromatic surface area is required for polymerase recognition, and that interstrand interactions between small aromatic rings may be optimized for replication. Combined with our previous results, these studies suggest that appropriately derivatized phenyl nucleobase analogues represent a promising approach toward developing a third base pair and expanding the genetic alphabet.     

Azetidines-Containing Fluorescent Purine Analogs: Synthesis and Photophysical Properties

Analogues of N,N-dimethyladenine exploiting both thieno-and isothiazolo-pyrimidine cores were modified with 3-subsituted azetidines to yield visibly emissive and responsive fluorophores. The emission quantum yields, among the highest seen for purine analogues (0.64 and 0.77 in water and dioxane respectively), correlated with the Hammett inductive constants of the substituents on the azetidine ring. Ribosylation of the difluoroazetidino-modified nucleobase yielded an emissive nucleoside that displayed a substantially lower emission quantum yield in water, compared to the precursor nucleobase. Importantly, high emission quantum yield was restored in deuterium oxide, which highlights the potential impact of the sugar moiety on the photophysical features of fluorescent nucleosides, a functionality usually considered non-chromophoric and photophysically benign.     

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.     

Synthesis of proline-modified analogues of the neuroprotective agent glycyl-l-prolyl-glutamic acid (GPE)

The synthesis of ten proline-modified analogues of the neuroprotective tripeptide GPE is described. Five of the analogues incorporate a proline residue with a hydrophobic group at C-2 and two further analogues have this side chain locked into a spirolactam ring system. The pyrrolidine ring was also modified by replacing the γ-CH₂ group with sulfur and/or incorporation of two methyl groups at C-5.     

Synthesis and inhibition properties of conformational probes for the mutase-catalyzed UDP-galactopyranose/furanose interconversion

UDP-galactose mutase is a flavoenzyme that catalyzes the isomerization of UDP-galactopyranose into UDP-galactofuranose, a key step in the biosynthesis of important bacterial oligosaccharides. Several mechanisms for this unique ring-contraction have been proposed, one of them involving a putative 1,4-anhydrogalactopyranose as an intermediate in the reaction. The purpose of this study was to probe the mutase binding site with conformationally restricted analogues of its substrate. Thus, we describe the straightforward synthesis of two C-glycosidic UDP-galactose derivatives: analogue 1, presenting a galactose moiety locked in a bicyclic (1,4)B boat conformation, and UDP-C-Galf 2, where the galactose residue is locked in the conformation of the mutase substrate. The two molecules were found to be inhibitors of UDP-galactose mutase at levels depending on the redox state of the enzyme. Strong inhibition of the native enzyme, but a low one of the reduced mutase, were observed with UDP-C-Galf 2, whereas 1 displayed intermediate inhibition levels under both native and reducing conditions. These data provide evidence of a significant conformational difference of the mutase binding pocket in the reduced enzyme and in the native one, the enzyme switching from a low Galf-affinity state (reduced enzyme) to a very strong one (native enzyme). It is remarkable that the mutase binds the boat-locked analogue 1 with similar affinities in both its conformational states. These results support a mechanism involving the formation of 1,4-anhydrogalactopyranose as a low-energy intermediate. An alternative explanation would be that the distortion of the galactose moiety during the cycle contraction transiently brings the carbohydrate into a conformation close to a (1,4)B boat.     

Addition of difluorocarbene to 3',4'-unsaturated nucleosides: synthesis of 2'-deoxy analogues with a 2-oxabicyclo[3.1.0]hexane framework

Treatment of protected 2'-deoxy-3',4'-unsaturated nucleosides derived from adenosine and uridine with difluorocarbene [generated from bis(trifluoromethyl)mercury and sodium iodide] gave fused-ring 2,2-difluorocyclopropane compounds. Stereoselective alpha-face addition to the dihydrofuran ring resulted from hindrance by the protected beta-anomeric nucleobases. A protected uracil compound was converted smoothly into the cytosine derivative via a 4-(1,2,4-triazol-1-yl) intermediate. Removal of the protecting groups gave new difluorocyclopropane-fused nucleoside analogues. The solid-state conformation of the nearly planar furanosyl ring in the uracil compound had a shallow 2E pucker, and a more pronounced 1E conformation was present in the furanosyl ring of the cytosine derivative.     

Heterocyclic analogues of cyclohexene: theoretical studies of the molecular structures and ring-inversion processes

The equilibrium geometry, ring-inversion barrier, and pathway for heterocyclic analogues of cyclohexene have been studied using the MP2/6-311G(d,p) level of theory. It is concluded that the replacement of one methylene group in cyclohexene by heteroatom results in significant changes in the character of the potential-energy surface in comparison with cyclohexene. The equilibrium conformation of ring strongly depends on the position of the heteroatom due to the existence of the n-pi conjugation. However, the character of the ring-inversion process is determined by the nature of the heteroatom. In the case of sulfur- and selenium-containing rings, the boat or twist-boat conformation corresponds to an additional minimum on the potential-energy surface. Moreover, the barriers of the conformational transition from this conformer to two different half-chair forms are significantly different. Nitrogen-containing heterocycles possess two pairs of minima corresponding to the different configurations of the nitrogen atom. However, the transition between the two minima with the same configuration of the heteroatom proceeds only in two steps that include ring inversion and nitrogen inversion.     

cADPR analogues: effect of an adenosine 2'- or 3'-methoxy group on conformation

[structure: see text] The 2'-OMe-A (2) and 3'-OMe-A (3) analogues of the calcium release agent cADPR (1) were prepared and their solution structures studied by NMR spectroscopy. Compared to 1, 2 shows a shift in its A ring conformation and changes in its R ring N:S and gammat:gamma+ ratios, while 3 displays a significant change in the conformation of its A ring gamma-bond.     

Lanosterol Biosynthesis: The Critical Role of the Methyl‐29 Group of 2,3‐Oxidosqualene for the Correct Folding of this Substrate and for the Construction of the Five‐Membered D Ring

Lanosterol synthase catalyzes the polycyclization reaction of (3S)‐2,3‐oxidosqualene ( 1 ) into tetracyclic lanosterol 2 by folding 1 in a chair‐boat‐chair‐chair conformation. 27‐Nor‐ and 29‐noroxidosqaulenes ( 7 and 8 , respectively) were incubated with this enzyme to investigate the role of the methyl groups on 1 for the polycyclization cascade. Compound 7 afforded two enzymatic products, namely, 30‐norlanosterol ( 12 ) and 26‐normalabaricatriene ( 13 ; 12 / 13 9:1), which were produced through the normal chair‐boat‐chair‐chair conformation and an atypical chair‐chair‐boat conformation, respectively. Compound 8 gave two products 14 and 15 ( 14 / 15 4:5), which were generated by the normal and the unusual polycyclization pathways through a chair‐chair‐boat‐chair conformation, respectively. It is remarkable that the twist‐boat structure for the B‐ring formation was changed to an energetically favored chair structure for the generation of 15 . Surprisingly, 14 and 15 consisted of a novel 6,6,6,6‐fused tetracyclic ring system, thus differing from the 6,6,6,5‐fused lanosterol skeleton. Together with previous results, we conclude that the methyl‐29 group is critical to the correct folding of 1 , with lesser contributions from the other branched methyl groups, such as methyl‐26, ‐27, and ‐28. Furthermore, we demonstrate that the methyl‐29 group has a crucial role in the formation of the five‐membered D ring of the lanosterol scaffold.     

Click fleximers: a modular approach to purine base-expanded ribonucleoside analogues

The synthesis of nucleoside analogues incorporating 4-(5-pyrimidinyl)-1,2,3-triazole aglycons as expanded purine nucleobase mimics were accessed using the copper-catalyzed azide-alkyne Huisgen cycloaddition between a ribosyl azide and 5-alkynylpyrimidines. Depending on the nature of the alkyne employed, other nucleoside analogues that possess fluorescence or potential metal-binding properties were prepared. Computational studies were undertaken on the purine analogues and indicate that the heterocycles of the unfused nucleobase prefer a coplanar arrangement and the anti-glycosidic conformer is favoured in most instances.