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The dosimetric accuracies of CATPhan 504 and CIRS 062 have been evaluated using the kV-CBCT of Varian TrueBeam linac and Eclipse TPS. The assessment was done using the kV-CBCT as a standalone tool for dosimetric calculations towards Adaptive replanning. Dosimetric calculations were made without altering the HU-ED curves of the planning computed tomography (CT) scanner that is used by the Eclipse TPS. All computations were done using the images and dataset from kV-CBCT while maintaining the HU-ED calibration curve of the planning CT (pCT), assuming pCT was used for the initial treatment plan. Results showed that the CIRS phantom produces doses within ±5% of the CT-based plan while CATPhan 504 produces a variation of ±14% of the CT-based plan. [ABSTRACT FROM AUTHOR]

A new potential quality assurance (QA) method is explored (including assessment of depth dose, dose linearity, dose rate linearity and beam profile) for clinical electron beams based on imaging Cerenkov light. The potential of using a standard commercial camera to image Cerenkov light generated from electrons in water for fast QA measurement of a clinical electron beam was explored and compared to ionization chamber measurements. The new method was found to be linear with dose and independent of dose rate (to within 3%). The uncorrected practical range measured in Cerenkov images was found to overestimate the actual value by 3 mm in the worst case. The field size measurements underestimated the dose at the edges by 5% without applying any correction factor. Still, the measured field size could be used to monitor relative changes in the beam profile. Finally, the beam-direction profile measurements were independent of the field size within 2%. A simulation was also performed of the deposited energy and of Cerenkov production in water using GEANT4. Monte Carlo simulation was used to predict the measured light distribution around the water phantom, to reproduce Cerenkov images and to find the relation between deposited energy and Cerenkov production. The camera was modelled as a pinhole camera in GEANT4, to attempt to reproduce Cerenkov images. Simulations of the deposited energy and the Cerenkov light production agreed with each other for a pencil beam of electrons, while for a realistic field size, Cerenkov production in the build-up region overestimated the dose by +8%. [ABSTRACT FROM AUTHOR]

Abstract: HIV integrase (IN) catalyzes the insertion of proviral DNA into the host cell chromosome. While IN has strict sequence requirements for the viral cDNA ends, the integration site preference has been shown to be very diverse. Here, we mapped the HIV IN strand transfer reaction requirements using various short oligonucleotides (ON) that mimic the target DNA. Most double stranded DNA dodecamers served as excellent IN targets with variable integration efficiency depending mostly on the ON sequences. The preferred integration was lost with any changes in the geometry of the DNA double helical structures. Various hairpin-loop-forming ONs also served as efficient integration targets. Similar integration preferences were also observed for ONs, in which the nucleotide hairpin loop was replaced with a flexible aliphatic linker. The integration biases with all target DNA structures tested were significantly influenced by changes in the resulting secondary ON structures. [Copyright &y& Elsevier]

Retroviral integrase participates in two catalytic reactions, which require interactions with the two ends of the viral DNA in the 3'processing reaction, and with a targeted host DNA in the strand transfer reaction. The 3'-hydroxyl group of 2'-deoxyadenosine resulting from the specific removing of GT dinucleotide from the viral DNA in the processing reaction provides the attachment site for the host DNA in a transesterification reaction. We synthesized oligonucleotides (ONs) of various lengths that mimic the processed HIV-1 U5 terminus of the proviral long terminal repeat (LTR) and are ended by 2'-deoxyadenosine containing a 3'-O-phosphonomethyl group. The duplex stability of phosphonomethyl ONs was increased by covalent linkage of the modified strand with its complementary strand by a triethylene glycol loop (TEG). Modified ONs containing up to 10 bases inhibited in vitro the strand transfer reaction catalyzed by HIV-1 integrase at nanomolar concentrations. [ABSTRACT FROM AUTHOR]

The synthesis of modified nucleoside analogs is an attractive area of medicinal research. Here, we have developed a synthetic route leading to a new class of dideoxynucleoside analogs, the phosphanucleosides containing 1-hydroxymethylphospholane 1-oxide rings. The preparation of these compounds consisted of a multistep synthesis of phospholane scaffold using a ring-closing metathesis and stereoselective hydroboration reaction. Subsequent nucleobase construction afforded the phosphanucleosides bearing all four nucleobases. The racemic phosphanucleosides were easily resolved on reverse phase using N -acetyl- l -tryptophan as a derivatizing agent. [ABSTRACT FROM AUTHOR]

We have developed a simple system for the analysis of the affinity of anti-bromodeoxyuridine antibodies. The system is based on the anchored oligonucleotides containing 5-bromo-2'-deoxyuridine (BrdU) at three different positions. It allows a reliable estimation of the reactivity of particular clones of monoclonal anti-bromodeoxyuridine antibodies with BrdU in fixed and permeabilized cells. Using oligonucleotide probes and four different protocols for the detection of BrdU incorporated in cellular DNA, we identified two antibody clones that evinced sufficient reactivity to BrdU in all the tested protocols. One of these clones exhibited higher reactivity to 5-iodo-2'-deoxyuridine (IdU) than to BrdU. It allowed us to increase the sensitivity of the used protocols without a negative effect on the cell physiology as the cytotoxicity of IdU was comparable with BrdU and negligible when compared to 5-ethynyl-2'-deoxyuridine. The combination of IdU and the improved protocol for oxidative degradation of DNA provided a sensitive and reliable approach for the situations when the low degradation of DNA and high BrdU signal is a priority. [ABSTRACT FROM AUTHOR]

An unique set of 5′-O- and 3′-O-phosphonomethyl derivatives of four natural 2′-deoxyribonucleosides, 1-(2-deoxy-β-D-threo-pentofuranosyl)thymine, 5′-O- and 2′-O-phosphonomethyl derivatives of 1-(3-deoxy-β-D-erythro-pentofuranosyl)thymine, and 1-(3-deoxy-β-D-threo-pentofuranosyl)thymine has been synthesized as a pool of monomers for the synthesis of modified oligonucleotides. The phosphonate moiety was protected with 4-methoxy-1-oxido-2-pyridylmethyl ester group, serving also as an intramolecular catalyst in the coupling step. [ABSTRACT FROM AUTHOR]

The synthesis of monomers (S)-1, (R)-1 and 2 derived from (5′S)-, (5′R)-2′-deoxythymidine-5′-C-phosphonic acids and 2′,5′-dideoxythymidine-5′-C-phosphonic acids was elaborated. The protection of the 5′-hydroxyl by the methoxycarbonyl group was a key step of the synthesis. Prepared monomers were used for the solid-phase assembly of several types oligothymidylate 15-mers (S)-3, (S)-4, (S)-5, (R)-4 and (R)-5 containing the chiral 3′-O-P-CH(OH)-5″ internucleotide linkage. Their hybridization properties with dA15 and rA15 were studied as well as their resistance against nuclease cleavage. [ABSTRACT FROM AUTHOR]

Adenine-based, regioisomeric nucleoside phosphonates with ribo, xylo and arabino configuration were synthesized in the protected form suitable for the phosphotriester-like, solid-phase synthesis of oligonucleotides. Phosphonate moiety was protected by 4-methoxy-1-oxido-2-picolyl group and the furanose hydroxyl by the dimethoxytrityl group. [ABSTRACT FROM AUTHOR]

Abstract: Straightforward, high-yielding syntheses of 3′-deoxy-3′,4′-didehydronucleoside-5′-aldehydes and 3′-deoxy-3′,4′-didehydronucleosides starting from 2′,3′-O-orthoester derivatives of ribonucleosides are described. [ABSTRACT FROM AUTHOR]

Abstract: Cellular uptake and intracellular distribution of the synthetic antisense analogue of dT15 oligonucleotide (homogenously containing 3′-O–P–CH2–O-5′ internucleotide linkages and labeled with tetramethylrhodamine dye) was studied on B16 melanoma cell line by fluorescence micro-imaging and time-resolved microspectrofluorimetry. By using amphotericin B 3-dimethylaminopropyl amide as an enhancer molecule for the uptake process, homogenous staining of the cells with rather distinct nucleoli staining was achieved after 4h of incubation. Two spectral components of 2.7 and 1.3ns lifetime, respectively, were resolved in the emission collected from the cell nucleus. The way of staining and the long-lived component differed from our previous experiments demonstrating complexity of the intracellular oligonucleotide distribution and in particular of the binding inside the nucleus. [Copyright &y& Elsevier]

Cellular 5′‐nucleotidases, enzymes regulating nucleotide/nucleoside pools, are capable of dephosphorylating phosphomonoesters of important nucleoside analogue drugs, thus decreasing their therapeutic efficacy. Human cytosolic (cdN) as well as mitochondrial (mdN) variants of this enzyme represent interesting targets for development of inhibitory compounds. In this work, bisphosphonate nucleoside derivatives were designed by using a structure‐based approach. A second phosphonate group was attached onto a base moiety and by optimization of the linker an increased inhibitor potency towards mdN and cdN was attained. The best compound exhibited inhibition of both enzymes in a nanomolar range, making it the most potent inhibitor of these enzymes prepared to date. In addition, the compounds showed selectivity towards the cdN variant. A series of crystal structures were solved for several inhibitors in the complex with mdN or cdN that provided a structural basis for understanding the inhibition profile of bisphosphonate compounds. By using a structure‐based approach, bisphosphonate nucleoside derivatives have been designed that act as potent inhibitors of human cytosolic and mitochondrial 5′‐nucleotidases. [ABSTRACT FROM AUTHOR]

Suitably protected (R)‐ and (S)‐HPEP {9‐[3‐hydroxy‐2‐(phosphonoethoxy)propyl]} derivatives containing adenine and thymine nucleobases were prepared as monomers for the introduction of acyclic nucleotide units into oligonucleotides. The solid phase synthesis of a series of ribo and 2′‐deoxyribo nonamers containing (R)‐HPEP and (S)‐HPEP units was successfully performed in both 5′ → 3′ and 3′ → 5′ directions. The measurement of thermal characteristics of the complexes of modified nonamers with complementary DNA and RNA strands revealed a general destabilizing effect of the introduced acyclic units on the thermal stability, but in several cases [modif.RNA*RNA and modif.DNA*RNA with (S)‐HPEPA units], the duplexes were characterized in terms of Tm values. A solid‐phase synthesis afforded a series of ribo and 2′‐deoxyribo nonamers containing acyclic nucleotide units. A general destabilizing effect of the introduced acyclic units on the thermal stability of complexes of modified nonamers with the complementary DNA and RNA strands was observed, but in cases with (S)‐HPEPA units, the duplexes were characterized in terms of Tm values. [ABSTRACT FROM AUTHOR]

We report on a new analogue of locked cyclohexane nucleic acids that contains an adenine nucleobase at an alternative position and which resembles homo-DNA. The crucial locked carbocyclic nucleoside monomer was prepared with high enantioselectivity and was subsequently incorporated into a pair of modified oligonucleotides that were subjected to hybridization studies. [ABSTRACT FROM AUTHOR]

Taurine (2-aminoethanesulfonic acid) is a representative substructure in biologically active compounds, but can raise difficulties in direct coupling reactions. In this study, the synthesis of taurine-derived sulfonic acids and analogous aromatic sulfonic acids was accomplished by a performic acid promoted oxidation of corresponding symmetrical disulfide precursors. In some of the products, the taurine or sulfanilic acid nitrogen atom is incorporated in a heterocyclic scaffold or part of a peptide bond in dipeptide mimetics. [ABSTRACT FROM AUTHOR]

Purine and pyrimidine 4′-alkoxy-2′-deoxynucleosideswere efficiently prepared from nucleoside 4′-5′-enolacetates in three steps by N-iodosuccinimide promotedalkoxylation, hydrolysis, and reduction followed by conversion tophosphoramidite monomers for the solid-phase synthesis of the oligonucleotides.Fully modified 4′-alkoxyoligodeoxynucleotides, which are characterizedby a prevalent N-type (RNA-like) conformation, exhibitedsuperior chemical and nuclease resistance as well as excellent hybridizationproperties with a strong tendency for RNA-selective hybridization,suggesting a potential application of 4′-alkoxy-oligodeoxynucleotidesin antisense technologies. [ABSTRACT FROM AUTHOR]

[Display omitted] The oligoadenylate synthetase-ribonuclease L pathway is a major player in the interferon-induced antiviral defense mechanism of cells. Upon sensing viral dsRNA, 5′-phosphorylated 2′,5′-oligoadenylates are synthesized, and subsequently activate latent RNase L. To determine the influence of 5′-phosphate end on the activation of human RNase L, four sets of 5′-phosphonate modified oligoadenylates were prepared on solid-phase. The ability of these 5′-modified oligoadenylates bearing shortened, isosteric and prolonged phosphonate linkages to activate RNase L was explored. We found that isosteric linkages and linkages prolonged by one atom were in general well tolerated by the enzyme with the EC 50 values comparable to that of the natural activator. In contrast, linkages shortened by one atom or prolonged by two atoms exhibited decrease in the activity. [ABSTRACT FROM AUTHOR]

Abstract: A series of conformationally constrained uridine-based nucleoside phosphonic acids containing annealed 1,3-dioxolane and 1,4-dioxane rings and their “open-structure” isosteres were synthesized and evaluated as potential multisubstrate-like inhibitors of the human recombinant thymidine phosphorylase (TP, EC 2.4.2.4) and TP obtained from peripheral blood mononuclear cells (PBMC). From a large set of tested nucleoside phosphonic acids, several potent compounds were identified that exhibited K i values in the range of 0.048–1 μM. The inhibition potency of the studied compounds strongly depended on the degree of conformational flexibility of the phosphonate moiety, the stereochemical arrangement of the sugar-phosphonate component, and the substituent at position 5 of the pyrimidine nucleobase. [Copyright &y& Elsevier]

Abstract: Novel phosphanucleosides containing a 3-hydroxy-1-hydroxymethylphospholane 1-oxide ring were synthesized as compounds with potential biological activity. A double Arbuzov reaction was employed to prepare a phospholene precursor, which was then converted into an epoxide and subsequently into racemic phosphanucleoside via nucleobase construction. [Copyright &y& Elsevier]

Abstract: Oligonucleotides conduct electric charge via various mechanisms and their characterization and understanding is a very important and complicated task. In this work, experimental (temperature dependent steady state fluorescence spectroscopy, time-resolved fluorescence spectroscopy) and theoretical (Density Functional Theory) approaches were combined to study charge transfer processes in short DNA/DNA and RNA/DNA duplexes with virtually equivalent sequences. The experimental results were consistent with the theoretical model — the delocalized nature of HOMO orbitals and holes, base stacking, electronic coupling and conformational flexibility formed the conditions for more effective short distance charge transfer processes in RNA/DNA hybrids. RNA/DNA and DNA/DNA charge transfer properties were strongly connected with temperature affected structural changes of molecular systems — charge transfer could be used as a probe of even tiny changes of molecular structures and settings. [Copyright &y& Elsevier]

A new method of the light microscopy detection of BrdU-labeled DNA in situ is described. It is based on the oxidative attack at the deoxyribose moiety by copper(I) in the presence of oxygen, which leads to the abstraction of hydrogen atom from deoxyribose culminating in the elimination of the nucleobase, scission of the nucleic-acid strand and formation of frequent gaps. The gaps allow the reaction of the antibodies with the commonly used markers of replication (e.g. 5-bromo-29- deoxyuridine), which are otherwise masked. The method developed makes it possible to detect nuclear and mitochondrial DNA replication efficiently. In most cases, it does not inhibit effective protein detections and in addition enables simultaneous localization of newly-synthesized RNA. The alternative presently-used methods result in protein denaturation and/or extensive DNA cleavage followed by the DNA-bound proteins peeling off. [ABSTRACT FROM AUTHOR]

5-Bromo-2'-deoxyuridine (BrdU) and 2'-deoxy-5-ethynyluridine (EdU) are widely used as markers of replicated DNA. While BrdU is detected using antibodies, the click reaction typically with fluorescent azido-dyes is used for EdU localisation. We have performed an analysis of ten samples of antibodies against BrdU with respect to their reactivity with EdU. Except for one sample all the others evinced reactivity with EdU. A high level of EdU persists in nuclear DNA even after the reaction of EdU with fluorescent azido-dyes if the common concentration of dye is used. Although a ten-time increase of azido-dye concentration resulted in a decrease of the signal provided by anti-BrdU antibodies, it also resulted in a substantial increase of the non-specific signal. We have shown that this unwanted reactivity is effectively suppressed by non-fluorescent azido molecules. In this respect, we have tested two protocols of the simultaneous localisation of incorporated BrdU and EdU. They differ in the mechanism of the revelation of incorporated BrdU for the reaction with antibodies. The first one was based on the use of hydrochloric acid, the second one on the incubation of samples with copper(I) ions. The use of hydrochloric acid resulted in a significant increase of the non-specific signal. In the case of the second method, no such effect was observed. [ABSTRACT FROM AUTHOR]

RNase L, a key enzyme in the host defense system, is activated by the binding of 2′–5′-linked oligoadenylates (2–5A) to the N-terminal ankyrin repeat domain, which causes the inactive monomer to form a catalytically active homodimer. We focused on the structural changes of human RNase L as a result of interactions with four different activators: natural 2–5 pA 4 and three tetramers with 3′-end AMP units replaced with ribo- , arabino- and xylo- configured phosphonate analogs of AMP (pA 3 X). The extent of the RNase L dimerization and its cleavage activity upon binding of all these activators were similar. A drop-coating deposition Raman (DCDR) spectroscopy possessed uniform spectral changes upon binding of all of the tetramers, which verified the same binding mechanism. The estimated secondary structural composition of monomeric RNase L is 44% α-helix, 28% β-sheet, 17% β-turns and 11% of unordered structures, whereas dimerization causes a slight decrease in α-helix and increase in β-sheet (ca. 2%) content. The dimerization affects at least three Tyr, five Phe and two Trp residues. The α–β structural switch may fix domain positions in the hinge region (residues ca. 336–363) during homodimer formation. [ABSTRACT FROM AUTHOR]

A complete series of pyrrolidine nucleotides, (3R)- and (3S)-3-(guanin-9-yl)pyrrolidin-1-N-ylcarbonylphosphonic acids and (3S,4R)-, (3R,4S)-, (3S,4S)-, and (3R,4R)-4-(guanin-9-yl)-3-hydroxypyrrolidin-1-N-ylcarbonylphosphonic acids, were synthesized and evaluated as potentialinhibitors of purine nucleoside phosphorylase (PNP) isolated fromperipheral blood mononuclear cells (PBMCs) and cell lines of myeloidand lymphoid origin. Two compounds, (S)-3-(guanin-9-yl)pyrrolidin-1-N-ylcarbonylphosphonic acid (2a) and (3S,4R)-4-(guanin-9-yl)-3-hydroxypyrrolidin-1-N-ylcarbonylphosphonic acid (6a), were recognizedas nanomolar competitive inhibitors of PNP isolated from cell lineswith Kivalues within the ranges of 16–100and 10–24 nM, respectively. The low MESGKiand PiKivalues of both compounds for PNP isolated from PBMCs suggest thatthese compounds could be bisubstrate inhibitors that occupy both thephosphate and nucleoside binding sites of the enzyme. [ABSTRACT FROM AUTHOR]

Abstract: To determine the influence of internucleotide linkage and sugar ring conformation, and the role of 5′-terminal phosphate, on the activation of human RNase L, a series of 2′- and 5′-O-methylphosphonate-modified tetramers were synthesized from appropriate monomeric units and evaluated for their ability to activate human RNase L. Tetramers pAAApcX modified by ribo, arabino or xylo 5′-phosphonate unit pcX activated RNase L with efficiency comparable to that of natural activator. Moreover, incorporation of phosphonate linkages ensured the stability against cleavage by nucleases. The substitution of 5′-terminal phosphate for 5′-terminal phosphonate in tetramer pcXAAA afforded tetramers with excellent activation efficiency and with complete stability against cleavage by phosphomonoesterases. [Copyright &y& Elsevier]

INTRODUCTION Intensity-modulated radiotherapy (IMRT) improves planning target volume (PTV) coverage and organ at risk (OAR) avoidance in the brain compared to three-dimensional conformal radiotherapy (3-D CRT). However, IMRT is time consuming with considerable resource implications. Volumetric modulated arc therapy (VMAT) offers a quicker method of delivering an IMRT plan and requires less monitor units. Planning studies have shown VMAT compares well to IMRT in the brain but there is little data regarding clinical experience, particularly in fractionated schedules. METHODS Prospectively collected data (treatment details, delivery parameters, dosimetry, time, toxicity, and quality assurance) from the first 10 patients treated with RapidArc (a VMAT product) for brain tumors at our institution was analyzed in an observational study to establish whether the theoretical advantages of VMAT in the treatment of brain tumors translate into clinical practice. RESULTS Patients were selected for RapidArc due to the proximity of PTV to OAR and complex geometric tumor volumes. These included a range of tumor sites, histology, treatment volumes, and dose schedules. In all cases the PTV received >95% prescribed dose and dose to OAR was within tolerance. One patient required a second noncoplanar arc. Quality assurance parameters were easily met. Treatment time and monitor units were approximately half that taken to deliver IMRT. Patients tolerated treatment well and experienced minimal alopecia. CONCLUSION In practice RapidArc provides excellent radiotherapy plan conformity for a wide range of brain tumors with reduced treatment times and monitor units compared to IMRT. Our experience suggests patients treated with RapidArc may suffer minimal alopecia. [ABSTRACT FROM AUTHOR]

A complete series of the 2 '-5 ' and 3 '-5 ' regioisomeric types of r(ApA) and 2 '-d(ApA) analogues with the α-hydroxy-phosphonate C3 '-O-P-CH(OH)-C4 '' internucleotide linkage, isopolar but non-isosteric with the phosphodiester one, were synthesized and their hybridization properties with polyU studied. Due to the chirality on the 5 '-carbon atom of the modified internucleotide linkage bearing phosphorus and hydroxy moieties, each regioisomeric type of ApA dimer is split into epimeric pairs. To examine the role of the 5 '-hydroxyl of the α-hydroxy-phosphonate moiety during hybridization, the appropriate r(ApA) analogues with 3 '(2 ')-O-P-CH2-C4 '' linkage lacking the 5 '-hydroxyl were synthesized. Nuclear magnetic resonance (NMR) spectroscopy study on the conformation of the modified sugar-phosphate backbone, along with the hybridization measurements, revealed remarkable differences in the stability of complexes with polyU, depending on the 5 '-carbon atom configuration. Potential usefulness of the α-hydroxy-phosphonate linkage in modified oligoribonucleotides is discussed. [ABSTRACT FROM AUTHOR]

Abstract: Epimeric 5′-(RS) dialkyl 3′-deoxy-3′,4′-didehydro-5′-C-phosphonates were prepared by nucleophilic addition of various dialkyl phosphites to 3′-deoxy-3′,4′-didehydronucleoside-5′-aldehydes. Whereas direct NMR configuration assignment for the C5′ atom bearing the phosphoryl and hydroxy groups using the J (P,H4′) and J (H5′,H4′) coupling constants is impossible due to the absence of the H4′ atom, successful separation, crystallisation and X-ray crystallographic analysis of a pair of epimeric 5′-C-phosphonates, followed by correlation with a series of NMR parameters, led to efficacious configuration assignment of individual epimers in the mixtures. [Copyright &y& Elsevier]

New isoelectronic, non-isosteric phosphonate analogues of nucleoside 5'-phosphates featuring the phosphorus moiety directly attached on the sugar ring in the C4' position are described. The analogues were synthesised by a nucleosidation reaction from tetrofuranosyl phosphonate synthons and silylated nucleobases. The pyrimidine compounds with erythro and threo configuration in both D- and L-series were prepared, and the structures were assigned by NMR spectroscopy. The results of NMR conformational studies show that all calculated conformers have a maximum pucker in the range typical for nucleosides. In all compounds, the S-type conformer is preferred and is more significant in α-D-threo-compounds. Studies on inhibition of thymidine phosphorylase revealed that one of the prepared phosphonic acids was a competitive inhibitor of the enzyme (Ki = 4 μM). [ABSTRACT FROM AUTHOR]

Abstract: Structurally diverse, sugar-modified, thymine-containing nucleoside phosphonic acids were evaluated for their ability to inhibit thymidine phosphorylase (TP, EC 2.4.2.4) purified from spontaneous T-cell lymphomas of an inbred Sprague-Dawley rat strain. From a large set of tested compounds, among them a number of pyrrolidine-based derivatives, 10 nucleotide analogues with IC50 values below 1μM were selected. Out of them, four compounds strongly inhibited the enzyme with IC50 values lying in a range of 11–45nM. These most potent compounds might be bi-substrate analogues. [Copyright &y& Elsevier]

Abstract: Novel reagents for the synthesis of 1-substituted uracil and thymine derivatives have been developed. The aminolysis of 2- or 4-nitrophenyl 3-ethoxyacryloylcarbamate and 3-ethoxy-2-methylacryloylcarbamate with a variety of primary amino derivatives proceeded smoothly under very mild reaction conditions yielding almost quantitatively the 1,3-disubstituted urea derivatives. Their subsequent cyclization provided the 1-substituted uracil and thymine compounds, in almost quantitative yield. [Copyright &y& Elsevier]

Abstract: N-Phosphonoalkyl-trans-3,4-dihydroxypyrrolidine derivatives were synthesized and exploited as synthons for the preparation of hydroxypyrrolidine nucleoside phosphonic acids, the 3′-deoxynucleoside 5′-phosphate analogues. Simultaneously, an alternative route, the N-phosphoalkylation of the preformed pyrrolidine nucleosides employing Mannich- and Michael-type reactions, was investigated to obtain desired nucleotide analogues. In contrast to the latter approach, the former resulted in the formation of two diastereoisomers very likely due to the existence of two possible SN2 transition states during a nucleophilic displacement. The stereochemistry of the prepared nucleotide analogues was studied by NMR spectroscopy. [Copyright &y& Elsevier]

Understanding the molecular mechanism of HIV-1 integrase (IN) activity is critical to find functional inhibitors for an effective AIDS therapy. A robust, fast, and sensitive method for studying IN activity is required. In this work, an assay for real-time label-free monitoring of the IN activity based on surface plasmon resonance was developed. This assay enabled direct monitoring of the integration of a viral doubled-stranded (ds) DNA into the host genome. The strand transfer reaction was detected by using two different DNA targets: supercoiled plasmid (pUC 19) and short palindrome oligonucleotide. The effect of the length of the DNA target on the possibility to monitor the actual process of the strand transfer reaction is discussed. The surface density of integrated ds-DNA was determined. IN binding to the oligonucleotide complexes and model DNA triplexes in the presence of various divalent ions as metal cofactors was investigated as well. The assay developed can serve as an important analytical tool to search for potential strand transfer reaction inhibitors as well as for the study of compounds interfering with the binding of ds long terminal repeats–IN complexes with the host DNA. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

Abstract: trans-4-Hydroxy-l-proline has been used as a starting material for the synthesis of prolinol-based nucleotide analogues with an N-phosphonomethyl moiety attached to the prolinol ring nitrogen atom. The synthetic methodology based on the inversion of configuration at both 1- and 4-position led to all diastereoisomeric O-protected 4-mesyloxyprolinol-N-phosphonates. Alkylation of nucleobases using the synthons in the l-series afforded the nucleotide analogues corresponding to α-l- and β-l-nucleotide. The NMR-based conformational study of these compounds in aqueous solution performed at two different pH values, showing either N-fully protonated or deprotonated forms, revealed the occurrence of the same mostly populated conformer in both cases. All final l-prolinol-based nucleoside phosphonic acids were tested for cytotoxic and antiviral properties, but no significant activity was found. [Copyright &y& Elsevier]

Abstract: Two types of DNA oligonucleotide modifications, namely lengthening of the internucleotide linkage by insertion of CH2 methylene group and substitution of deoxyribose by deoxyxylose, were studied with respect to their possible impact on the oligonucleotide binding specificity. Structural effects of the modifications at the mismatched site of DNA/RNA hybrid duplex were monitored via Raman spectroscopy by using a molecular model system consisting of a DNA nonamer (5′-GTG ATA TGC-3′) with the modification at the site of the central thymidine and of RNA counterparts with altered central nucleotide (5′-GCA UNU CAC-3′, N =A or U). A novel approach based on single and double difference Raman spectra allowed extraction and analysis of spectroscopic signatures introduced by chemical modification and mismatched basepairs in NA duplexes. The double difference spectra revealed that substitution of 2′-deoxy-d-deoxyribose by 2′-deoxy-d-deoxyxylose supports bending of the modified dA-dT-dA segment in the mismatched duplex, while lengthening of the internucleotide linkage by insertion of the CH2 methylene group induces irregular distortion of this track. The modifications do not induce a better stacking interaction of the mismatched uracil and thus do not increase the stability of the T·U mismatched duplexes. [Copyright &y& Elsevier]

Abstract: We found that the syntheses of O-monosubstituted 1-N-alkyl-trans-3,4-dihydroxypyrrolidines, normally faces serious obstacles due to poorly reactive hydroxy groups as a consequence of the presence of a highly basic pyrrolidine nitrogen atom, but that they can be obtained easily in high yields by conversion of 1-N-alkyl-trans-3,4-dihydroxypyrrolidines into the corresponding N-oxides. N-Oxidation leads to the loss of the pyrrolidine nitrogen atom basicity and discrimination in the reactivity of the originally equivalent hydroxy groups by at least one order of magnitude. The reaction of N-oxide derivatives with DMTrCl or TBDPSCl then proceeds in an almost quantitative yield, rapidly, and stereospecifically on the hydroxy group which is in a cis-position to the N-oxide oxygen atom. In contrast to the TBDPS derivative, the DMTr derivative could be easily deoxygenated with triphenylphosphine in high yield. The structures of the products obtained were confirmed by 2D NMR experiments, and quantum-chemical calculations were performed to explain the reaction mechanism of the stereospecific course of the reaction. [Copyright &y& Elsevier]

Abstract: An electrophile-promoted addition of dialkyl-hydroxymethylphosphonate to the protected 4′,5′-didehydronucleosides resulted in an epimeric mixture of 4′-dialkylphosphonomethoxy derivatives of 2′,5′-dideoxynucleosides, novel analogues of 2′-deoxynucleoside 5′-monophosphates. Several types of electrophiles (pyridinium tosylate, NIS, NBS, MCPBA and others) were evaluated in addition reactions with 4′,5′-didehydrothymidine. Out of them, pyridinium tosylate was found to have a practical usefulness in these transformations. Its use has led to the preparation of a series of 4′-phosphonomethoxy derivatives of common 2′-deoxynucleosides. On biological screening, these free phosphonic acids exerted no significant cytostatic or antiviral activity. [Copyright &y& Elsevier]

Abstract: The work deals with the synthesis of hydroxypyrrolidine analogs of nucleosides. Starting from the optically pure l- or d-tartaric acid, we improved the synthesis of enantiomeric trans-3,4-dihydroxypyrrolidines and elaborated a procedure for the synthesis of all possible diastereoisomers of 3-hydroxy-4-pyrrolidinyl derivatives of both purine and pyrimidine nucleobases. The prepared compounds were tested for cytostatic and antiviral properties but no significant activity was found. [Copyright &y& Elsevier]

Abstract: We report here an oxidative cleavage of (5R)- and (5S)-ribofuranosyl-5-C-phosphonate derivatives with periodate anion under both strong acidic and neutral conditions. In both cases, only (5R)-configured compound underwent the expected oxidation reaction and afforded the desired (4R)-erythrofuranosylphosphonate, whereas the second epimer, (5S)-ribofuranosyl-5-C-phosphonate did not provide the corresponding (4S)-erythrofuranosylphosphonate derivative. This different behavior of epimers toward oxidative cleavage is an important phenomenon. The obtained (4R)-erythrofuranosylphosphonate was used for the preparation of phosphonate mimic of adenosine 5′-phosphate via classical nucleosidation reaction. Condensation of the protected shortened AMP analogue with adenosine derivatives, however, provided only the 2′,5′-linked ApA analogue. Study on hybridization of the modified 2′-5′ ApA with polyU revealed its ability to form stable triplex-like complex, similar to natural 2′-5′ r(ApA) and 3′-5′ r(ApA). NMR spectroscopy study showed that the erythrofuranose part of the phosphonate nucleotide unit of modified 2′-5′ ApA was predominantly in the C2′-endo conformation, which is characteristic for B-DNA. [Copyright &y& Elsevier]

Abstract: The synthesis of novel 3-pyrrolidinyl derivatives of nucleobases is described. Starting from malic acid, we improved the synthesis of both racemic and optically active N-benzyl-3-hydroxypyrrolidine-2,5-diones, which were transformed in four steps into N-tert-butyloxycarbonyl-3-mesyloxypyrrolidines, the key synthons for the alkylation of purine and pyrimidine nucleobases. Alkylations of cesium salts of purines and sodium salts of pyrimidines with N-tert-butyloxycarbonyl-3-mesyloxypyrrolidines proceeded smoothly, giving high yields of 9-substituted purine derivatives and moderate yields of 1-substituted pyrimidine derivatives. Using (S)-N-tert-butyloxycarbonyl-3-mesyloxypyrrolidine as the same intermediate for the synthesis of both enantiomeric N-Boc-3-pyrrolidinyladenines, and considering the results obtained on chiral HPLC analysis of the products, we proved that nucleophilic displacement of the mesyloxy group proceeded with inversion and not with retention of the configuration. Prepared compounds were tested for cytostatic and antiviral properties, but no significant activity was found. [Copyright &y& Elsevier]

Abstract: We found that various dialkyl phosphites, dialkyl trimethylsilyl phosphites, and tris-trimethylsilyl phosphite reacted smoothly with nucleoside 5′-aldehydes to afford epimeric nucleoside 5′-C-phosphonates in high yields. A number of these compounds in both the 2′-deoxyribo and ribo series were prepared. In the case of 2′-deoxythymidine-5′-aldehyde, a thorough study was made on the influence of the 3′-hydroxyl protecting group, type of phosphite, base, and solvent, on the yield and epimeric ratio of the resulting 5′-hydroxyphosphonates. Partial stereoselectivity in favour of either R or S epimers was observed. An attempt to transform the α-hydroxyl of the phosphonate moiety into a halo or azido moiety was not successful. Only intramolecular substitution reaction of the mesyloxy group for an alkoxy residue of the 2-hydroxyethyl ester took place in a low yield. [Copyright &y& Elsevier]

The present work relates to the synthesis of pyrrolidine nucleoside analogs. Starting from malic acid, we have elaborated a high-yield synthesis of racemic and enantiomeric N -protected 3-pyrrolidinols and their O -mesyl derivatives as key compounds for alkylations of purine and pyrimidine nucleobases. On varying base and solvent, we have found conditions providing both satisfactory N -/ O -regioisomeric ratio and acceptable yield for pyrimidine compounds. [ABSTRACT FROM AUTHOR]

The gene encoding an integrase of Mason–Pfizer monkey virus (M-PMV) is located at the 3′-end of the pol open reading frame. The M-PMV integrase has not been previously isolated and characterized. We have now cloned, expressed, isolated, and characterized M-PMV integrase and compared its activities and primary structure with those of HIV-1 and other retroviral integrases. M-PMV integrase prefers untranslated 3′-region-derived long-terminal repeat sequences in both the 3′-processing and the strand transfer activity assays. While the 3′-processing reaction catalyzed by M-PMV integrase was significantly increased in the presence of Mn2+ and Co2+ and was readily detectable in the presence of Mg2+ and Ni2+ cations, the strand transfer activity was strictly dependent only on Mn2+. M-PMV integrase displays more relaxed substrate specificity than HIV-1 integrase, catalyzing the cleavage and the strand transfer of M-PMV and HIV-1 long-terminal repeat-derived substrates with similar efficiency. The structure-based sequence alignment of M-PMV, HIV-1, SIV, and ASV integrases predicted critical amino acids and motifs of M-PMV integrase for metal binding, interaction with nucleic acids, dimerization, protein structure maintenance and function, as well as for binding of human immunodeficiency virus type 1 and Rous avian sarcoma virus integrase inhibitors 5-CI-TEP, DHPTPB and Y-3. [ABSTRACT FROM AUTHOR]

This work deals with isopolar, phosphonate-based nucleotide analogues containing a bridging P-C bond instead of the ester P-O linkage. Specifically, starting from activated derivatives 1, 2, and 3, a simple process for preparation of mixtures of short oligomers and their analyses were elaborated. [ABSTRACT FROM AUTHOR]

Discusses a study on the hybridization of 4'-branched oligonucleotides. Preparation of 4'-Modified monomers in spite of chemical difficulties and significant decomposition of the compound during the key reaction steps; Details of the enzymatic resistance of the oligonucleotides; Measurement of the nuclear magnetic resonance spectra.

The recently described epimeric nucleosidyl-5′-C-phosphonates (α-hydroxyphosphonates) represent novel nucleotide analogues that can be incorporated into chimeric oligonucleotides by the phosphotriester condensation method. In order to prepare suitable protected monomer(s) we have studied condensation reaction between protected 2′-deoxythymidine and 2′-deoxythymidinyl-5′-C-phosphonate, both as model compounds, in dependence on the nature of the 5′-hydroxyl protecting group. We have found that the O-acetyl group is unstable in the presence of TPSCl or MSNT used as condensing agents for activation of the phosphorus moiety. This instability negatively influences the scope of the condensation process. On the other hand, introduction of the O-methoxycarbonyl group gave excellent results. The O-methoxycarbonyl group does not participate in the condensation process, and its quantitative introduction into the nucleotide analogues is accomplished using a novel acylating agent, methoxycarbonyl tetrazole. [ABSTRACT FROM AUTHOR]

A series of novel, unusual type of acyclic phosphonate-based nucleotide analogues related to well-known antivirals (PMEA and HPMPA) was synthesized using easily available synthon. These compounds, which are distinguished for the presence of phosphonomethyl acetal linkage, form a group of derivatives that contribute to the understanding of structure-activity relationship within the area of acyclic nucleotide analogues. [ABSTRACT FROM AUTHOR]

Investigates HIV-1 integrase inhibition by modified oligonucleotides derived from U5′ long-terminal repeat. Determination of the attachment sites to the host DNA; Basis of binding affinity and inhibition activity; Modification of the furanose ring.

Analogs of nucleosides and nucleotides represent a promising pool of potential therapeutics. This work describes a new synthetic route leading to 2′-deoxy-2′-fluorotetradialdose D-nucleoside phosphonates. Moreover, a new universal synthetic route leading to tetradialdose d -nucleosides bearing purine nucleobases is also described. All new compounds were tested as triphosphate analogs for inhibitory potency against a variety of viral polymerases. The fluorinated nucleosides were transformed to phosphoramidate prodrugs and evaluated in cell cultures against various viruses including influenza and SARS-CoV-2. [Display omitted] [ABSTRACT FROM AUTHOR]