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Oligodeoxynucleotides incorporating internucleotide phosphoroselenolate linkages have been prepared under solid-phase synthesis conditions using dimer phosphoramidites. These dimers were constructed following the high yielding Michaelis-Arbuzov (M-A) reaction of nucleoside H-phosphonate derivatives with 5'-deoxythymidine-5'-selenocyanate and subsequent phosphitylation. Efficient coupling of the dimer phosphoramidites to solid-supported substrates was observed under both manual and automated conditions and required only minor modifications to the standard DNA synthesis cycle. In a further demonstration of the utility of M-A chemistry, the support-bound selenonucleoside was reacted with an H-phosphonate and then chain extended using phosphoramidite chemistry. Following initial unmasking of methyl-protected phosphoroselenolate diesters, pure oligodeoxynucleotides were isolated using standard deprotection and purification procedures and subsequently characterised by mass spectrometry and circular dichroism. The CD spectra of both modified and native duplexes derived from self-complementary sequences with A-form, B-form or mixed conformational preferences were essentially superimposable. These sequences were also used to study the effect of the modification upon duplex stability which showed context-dependent destabilisation (-0.4 to -3.1 °C per phosphoroselenolate) when introduced at the 5'-termini of A-form or mixed duplexes or at juxtaposed central loci within a B-form duplex (-1.0 °C per modification). As found with other nucleic acids incorporating selenium, expeditious crystallisation of a modified decanucleotide A-form duplex was observed and the structure solved to a resolution of 1.45 A. The DNA structure adjacent to the modification was not significantly perturbed. The phosphoroselenolate linkage was found to impart resistance to nuclease activity.

International audience; The labelling of DNA oligonucleotides with signalling groups that give a unique response to duplex formation depending on the target sequence is a highly effective strategy in the design of DNA-based hybridisation sensors. A key challenge in the design of these so-called base discriminating probes (BDPs) is to understand how the local environment of the signalling group affects the sensing response. The work herein describes a comprehensive study involving a variety of photophysical techniques, NMR studies and molecular dynamics simulations, on anthracene-tagged oligonucleotide probes that can sense single base changes (point variants) in target DNA strands. A detailed analysis of the fluorescence sensing mechanism is provided, with a particular focus on rationalising the high dependence of this process on not only the linker stereochemistry but also the site of nucleobase variation within the target strand. The work highlights the various factors and techniques used to respectively underpin and rational-ise the BDP approach to point variant sensing, which relies on different responses to duplex formation rather than different duplex binding strengths.

Novel nucleoside analogues containing photoswitchable moieties were prepared using 'click' cycloaddition reactions between 5'-azido-5'-deoxythymidine and mono- or bis-N-propargylamide-substituted azobenzenes. In solution, high to quantitative yields were achieved using 5 mol% Cu(I) in the presence of a stabilizing ligand. 'Click' reactions using the monopropargylamides were also effected in the absence of added cuprous salts by the application of liquid assisted grinding (LAG) in metallic copper reaction vials. Specifically, high speed vibration ball milling (HSVBM) using a 3/32″ (2.38 mm) diameter copper ball (62 mg) at 60 Hz overnight in the presence of ethyl acetate lead to complete consumption of the 5'-azido nucleoside with clean conversion to the corresponding 1,3-triazole.

Using vibration ball milling, 5'-chloro-5'-deoxyadenosine (CldA) reacts cleanly with 4-methoxybenzyl mercaptan (MobSH), under basic conditions, to the corresponding thioether (MobSdA), which is isolated following precipitation and trituration. Under acidic conditions, in a one-pot, two-step process, MobSdA is transformed into 5'-deoxy-5'-(5-nitropyridyl-2-disulfanyl)-adenosine (NPySSdA). Michaelis-Arbuzov (M-A) reaction of NPySSdA with tris(trimethylsilyl) phosphite proceeds to completion within 30 min as determined by 31 P NMR, and the persilylated M-A product thus formed can be stored in solution under anhydrous conditions at room temperature for several days (in contrast, the anionic phosphorothiolate monoester is labile to hydrolysis). Following evaporation, mechanochemical mixing of the crude M-A product with the nucleotide donor adenosine 5'-monophosphomorpholidate under acidic activation in the presence of additional water gives rapid hydrolytic desilylation and phosphate coupling, so that essentially complete reaction is observed after 90 min and dASppA isolated following C-18 reversed phase HPLC and desalting (>99% pure as determined by monitoring at 260 nm). © 2017 by John Wiley & Sons, Inc.

The Michaelis–Arbuzov reactions of benzylselenocyanate and 5′-deoxythymidine-5′-selenocyanate with thymidine H -phosphonate proceeded rapidly in the presence of a neutral silylating agent and 2,6-lutidine to give the corresponding Se-alkyl phosphoroselenolate triesters. Deprotection under mild conditions enabled the isolation of phosphoroselenolate diesters which were fully characterised.

Modified DNA strands undergo a reversible light-induced reaction involving the intramolecular photodimerization of two appended anthracene tags. The photodimers exhibit markedly different binding behavior toward a complementary strand that depends on the number of bases between the modified positions. By preforming the duplex, photochromism can be suppressed, illustrating dual-mode gated behavior.

Single nucleotide polymorphisms within a sequence of a gene associated with prostate cancer were identified using oligodeoxynucleotide probe sequences bearing internal anthracene fluorophores proximal to the SNP site. Depending upon the nature of the synthesised target sequences, probe-target duplex formation could lead to enhanced or attenuated fluorescence emission from the anthracene, enabling detection of a proximal base-pair as either matching or mismatching.

Michaelis–Arbuzov reactions of S-aryl disulfide derivatives of 3′-thiothymidine or 5′-thioadenosine with tris(trimethylsilyl) phosphite proceeded in high yields to the corresponding phosphorothiolate monoesters. Subsequent hydrolytic desilylation and phosphate coupling were effected in one-pot using liquid-assisted grinding in a vibration ball mill. Novel 3′,5′- and 5′,5′-pyrophosphorothiolate-linked dinucleoside cap analogues were thereby prepared.

Solution-phase conditions and vibration ball milling to effect the preparation of triazole-linked photoswitchable nucleosides like (III) are compared.

A novel phosphoramidite, N , N -diisopropylamino-2-cyanoethyl-9-anthracenemethyl phosphoramidite 1 , was prepared and coupled with the terminal 5′-hydroxyl of support-bound T 10 and the putative phosphite triester intermediate was subsequently reacted with iodine in the presence of either water or a series of primary and secondary amines. The reactivity of 1 compared to a previously reported benzyl phosphoramidite 2 was also investigated: oxidation of the product of coupling 2 with CPG-T 10 -5′OH under aqueous conditions resulted in greater than 30% of the benzyl moiety being retained. In contrast, essentially complete loss of the 9-anthracenemethyl group was observed using 1 under the same conditions. Oligonucleotides modified with a terminal phosphate monoester, lipophilic, fluorescent or cationic groups were thus prepared.

Ionic liquids are shown to be good solvents for elemental sulfur, selenium, phosphorus and tellurium, and can be designed to maximise the solubility of these elements. The presence of the [S(3)](*-) radical anion in diluted solutions of sulfur in some ionic liquids has been confirmed, and is the origin of their intense blue colour (cf. lapis lazuli).

Michaelis-Arbusov chemistry was used to prepare O,S-dialkyl 3′-O-nucleosidyl phosphorothiolate triesters in solution and attached to CPG. The support-bound nucleoside was utilised in the synthesis of a pentaribonucleotide that was fully deprotected on the support. Subsequent treatment with a buffered solution of iodine cleaved the RNA from the CPG with concomitant formation of a terminal 2′,3′-cyclic phosphate.

Routes for the preparation of 2′-silyl protected phosphoramidites of the modified nucleosides O2-methyluridine and O4-methyluridine are described. Methodology for the site-specific incorporation of these phosphoramidites into a 25 nucleotide long oligoribonucleotide sequence by solid-phase synthesis is reported.

Photoresponsive oligonucleotides (ONs) incorporating isoxazole-linked azobenzene (AB) moieties were prepared by resin-supported nitrile oxide-alkyne cycloaddition (NOAC) chemistry. The thermal and photochromic properties of the modified ONs were significantly influenced by the extent of π-conjugation between the isoxazole and the AB modules.

A novel phosphoramidite; N,N-diisopropylamino-2-cyanoethyl-ortho-methylbenzylphosphoramidite 1 , was prepared. The reaction of 1 with DMTrT and subsequent derivatisation of the phosphite triester product under solution-phase, Michaelis–Arbuzov conditions was investigated. Coupling of 1 with the terminal hydroxyl groups of support-bound oligodeoxyribonucleotides and subsequent reaction with an activated disulfide yielded oligonucleotides bearing a terminal, phosphorothiolate-linked, lipophilic moiety. The oligomers were readily purified using RP-HPLC. Silver(I)-mediated cleavage of the phosphorothiolate linkage and desalting of the oligonucleotides were performed readily in one step to yield cleanly the corresponding phosphate monester-terminated oligomers.

The 5′-O-monomethoxytrityl-3′-S-(aryldisulfanyl)-3′-deoxythymidines 7 and 8 have been prepared by the reaction of 5′-O-monomethoxytrityl-3′-thiothymidine with the appropriate arenesulfenyl chloride. These disulfides undergo a Michaelis–Arbusov reaction with simple trialkyl phosphites to yield 5′-O-monomethoxytrityl-3′-thiothymidin-3′-yl O,O-dialkyl phosphorothiolates. More interestingly, 3′-deoxy-3′-S-(2, 4-dinitrophenylsulfanyl)-5′-O-monomethoxytritylthymidine 8 reacts with a variety of thymidin-5′-yl dialkyl phosphites to give dithymidine phosphorothiolate triesters with the phosphorothiolate group protected with either a methyl or a 2-cyanoethyl group.3′-O-(tert-Butyldimethylsilyl)thymidin-5′-yl triethylammoniumphosphonate 17 is converted into the corresponding bis-(O-trimethylsilyl) phosphite by treatment with bis(trimethylsilyl)trifluoroacetamide. in situ Reaction of this phosphate with disulfide 8 gives, after work-up, the dithymidine phosphorothiolate diester directly. Methylation of compound 17 with methyl chloromethanoate, followed by silylation and subsequent reaction with disulfide 8, gives the methyl-protected dithymidine phosphorothiolate triester.

The yields of the presented approach are equal to or greater than conventional DMF-based methods.

International audience; A fluorescent anthracene-tagged DNA probe has been shown to respond to various DNA sequences by changes to its emission signal upon duplex formation. The fluorescence response for duplexes containing a single mismatch near the anthracene site has been found to be very sensitive to its composition, with the emission signal increasing for a CA mismatch and decreasing for CT and CC mismatches.

Efficient procedures are described for the synthesis of dinucleoside phosphorothiolates using either a Michaelis-Arbusov-type reaction or a phosphotriester approach.

Efficient procedures are described for the synthesis of dinucleoside phosphorothiolates using either a Michaelis-Arbusov-type reaction or a phosphotriester approach.

Solid-phase oligonucleotide conjugation by nitrile oxide–alkyne click cycloaddition chemistry has been successfully demonstrated; the reaction, compatible with all nucleobases, requires no metal catalyst and proceeds under physiological conditions.

Persilylation of nucleoside hydroxyls was effected in quantitative yields under solvent-free conditions using a ball mill. In addition, one-pot persilylation and acylation of cytidine was performed as an exemplar reaction demonstrating the utility of solvent-free approaches to nucleoside chemistry.

Chemoselective ligation of biologically significant moieties through azide alkyne Click Chemistry has recently received much attention1. The reaction is attractive in that it regioselectively affords stable triazole linked bioconjugated products under mild conditions. However, from the view point of the synthetic oligonucleotide chemist, a significant disadvantage is that the non-thermal reaction requires an in situ generated Cu (I) catalyst. Unwanted Cu (I) mediated chemistry, specifically oxidative degradation etc.

Directed Michaelis–Arbuzov reactions of support-bound internucleotide O-benzyl- or O-methyl-phosphite triesters with meta-phenylazobenzylamine or alkane-/glycol-linked α,ω-diamines were effected in the presence of iodine. The corresponding tritylated phosphoramidate-linked 11-mers were fully deprotected and released from the support under standard conditions and the fast- and slow-diastereoisomers of both the E- and the Z-meta-phenylazobenzyl-appended oligomers were readily resolved by RP-HPLC. The primary amine-functionalised oligonucleotides were either purified, detritylated and then finally treated with N-hydroxysuccinimidyl carboxylic acid ester derivatives of photoswitchable moieties (Route A) or first derivatised and then subsequently purified and detritylated (Route B). This latter route enabled resolution of fast- and slow-isomers of the trityl-on oligomers bearing novel photoswitchable azopyridine or 9-alkoxyanthracene moieties using RP-HPLC, following which the pure diastereoisomers were detritylated and characterised by MALDI-MS.

Protein TrwC is the conjugative relaxase responsible for DNA processing in plasmid R388 bacterial conjugation. TrwC has two catalytic tyrosines, Y18 and Y26, both able to carry out cleavage reactions using unmodified oligonucleotide substrates. Suicide substrates containing a 3'-S-phosphorothiolate linkage at the cleavage site displaced TrwC reaction towards covalent adducts and thereby enabled intermediate steps in relaxase reactions to be investigated. Two distinct covalent TrwC–oligonucleotide complexes could be separated from noncovalently bound protein by SDS–PAGE. As observed by mass spectrometry, one complex contained a single, cleaved oligonucleotide bound to Y18, whereas the other contained two cleaved oligonucleotides, bound to Y18 and Y26. Analysis of the cleavage reaction using suicide substrates and Y18F or Y26F mutants showed that efficient Y26 cleavage only occurs after Y18 cleavage. Strand-transfer reactions carried out with the isolated Y18–DNA complex allowed the assignment of specific roles to each tyrosine. Thus, only Y18 was used for initiation. Y26 was specifically used in the second transesterification that leads to strand transfer, thus catalyzing the termination reaction that occurs in the recipient cell., This work was financed by Grants BFU2005-03477 from the Spanish Ministry of Education and Science, and EU 6th Framework Programme project SHM-CT-2005-019023 to FC and by the School of Chemistry and Chemical Engineering to JSV and LAC.

Uridine-3′-phosphorothiolate triesters bearing lipophilic moieties were prepared via Michaelis-Arbuzov chemistry. Subsequent deprotection of the S-cholesteryl phosphorothiolate triester afforded the corresponding diester which underwent spontaneous Cyclization to cleanly afford uridine 2′,3′-cyclic phosphate. This transesterification reaction could be expedited by treatment with iodine under mild, neutral conditions.

A novel anthracene-tagged oligonucleotide can discriminate between a fully-matched DNA target sequence and one with a single mismatching base-pair through a remarkable difference in fluorescence emission intensity upon duplex formation.

Lights, camera, action! Photoswitchable nucleoside analogues containing o-, m-, or p-azobenzenes can be inserted in the catalytic core of RNA-cleaving 10-23 deoxyribozymes by replacing a nonconserved residue (see picture). Irradiation of the modified deoxyribozymes at 366 nm enhances RNA cleavage rates up to ninefold, thus achieving the rates observed for the unmodified deoxyribozyme.

The syntheses of 5-( Z )-(3-aminoallyl)- and 5-(3-aminopropyl)-substituted 2′-deoxyuridine and 2′-deoxycytidine are reported. These compounds were derived from the corresponding 5-propargylamine derivative. [Hobbs, F. W. J. Org. Chem. 1989 , 52 , 3420.] The catalyst we have employed for these reductions is a NiCl 2 /NaBH 4 system, which we have found to be superior to the more conventional palladium-catalysts previously reported with similar compounds.

The hairpin ribozyme is an example of a small catalytic RNA that catalyses the endonucleolytic transesterification of RNA in a highly sequence-specific manner. We have utilised chemical synthesis of RNA to create mutants of the hairpin ribozyme in which a nucleoside analogue replaces one of the essential pyrimidines in the ribozyme. Individual pyrimidine nucleosides were substituted by 4-thiouridine, O 4 -methyluridine, O 2 -methyluridine or 2-pyrimidinone-1-β- d -riboside. To facilitate the synthesis of oligoribonucleotides containing 4-thiouridine, we have devised a new synthetic route to the key intermediate 5′- O -(4,4′-dimethoxytrityl)-2′- O - tert -butyldimethylsilyl- S -cyanoethyl-4-thiouridine. The ability of the modified ribozymes to support catalysis was studied and the steady-state kinetic parameters were determined for each mutant. The range of analogues used in this study allows the important functional groups of the essential pyrimidines to be identified. The results demonstrate that each pyrimidine (U 41 , U 42 and C 25 ) plays an important role in hairpin ribozyme catalysis. The findings are discussed in terms of the various models that have been proposed for loop B of the hairpin ribozyme.

Stable bisubstrate ligands of phosphoglycerate kinase (PGK) have been synthesised with AMP or ADP conjugated to hydrolytically-stable, symmetrical analogues of 1,3-bisphosphoglycerate and their binding to yeast PGK evaluated. Their Kds decrease with net negative charge, with a penta-anionic analogue 7 showing highest affinity - in accordance with its approximation to the transition state for the reaction catalysed by PGK.

[EN] Two ionic liquids, 1-ethylpyridinium docusate (IL1) and tri-n-butyl(2-hydroxyethyl)phosphonium docusate (IL2), were designed and synthesized with the explicit intention of imparting a combination of plasticization and antimicrobial efficacy when incorporated into medical grade poly(vinyl chloride)s (PVCs). The glass transition (T-g) of PVC can be reduced by >20 degrees C on addition of 15 wt% IL2. Both IL1 and IL2 leached to varying extents from the base PVC resins rendering the surface of the PVCs hydrophilic. The antimicrobial activity of both ILs is related to the presence and concentration of both cationic and anionic component of the ILs leached from the PVC and inversely proportional to the extent of PVC gelation. Blends of the PVCs with IL1 displayed antibacterial activity against almost all Gram-positive bacteria tested, including coagulase-negative Staphylococci (CoNS) and methicillin-resistant Staphylococcus aureus (MRSA), but not with IL2 at low concentration in contrast to our previous study when high concentrations of IL2 were used. The more hydrophilic IL1 when added to PVC retards biofilm formation., S. Y. Choi thanks QUB for funding her studentship. We acknowledge the technical assistance of Gareth Graham, Drs Bronagh Millar, Paula Douglas, Saloni Shukla, Lan Wei, and Colorite Europe Ltd. for supplying the PVC formulations. We thank Dr David Timson (QUB) for helpful discussions.

Strain promoted cycloaddition is presented as a tool for RNA conjugation on the solid phase; RNA-cyclooctyne conjugates are prepared by cycloaddition to both azide (strain-promoted azide-alkyne cycloaddition, SPAAC) and nitrile oxide dipoles (strain-promoted nitrile oxide-alkyne cycloaddition, SPNOAC). The conjugation is compatible with 2'-OMe blocks and with 2'-O-TBDMS protection on the ribose moieties of the sugar. Nitrile oxide dipoles are found to be more reactive click partners than azides. The conjugation proceeds within 10 min in aqueous solvents, at room temperature without any metal catalyst and tolerates dipoles of varying steric bulk and electronic demands, including pyrenyl, coumarin and dabcyl derivatives.

The chemoselective acylation of primary aliphatic amines has been achieved in under ten minutes (and for aromatic amines under 120 min) using vibration ball-milling, avoiding undesirable solvents which are typically employed for such reactions (e.g.DMF). Under optimised conditions, the synthesis of amides in the presence of both primary and secondary alcohol functions was achieved in high to excellent yields (65–94%). Overall, the methods described have significant practical advantages over conventional approaches based upon bulk solvents including greater yields, higher chemoselectivity and easier product separation.

Using a ball mill, rapid, atom-economic coupling between adenosine-5'-phosphoromorpholidate and phosphorylated ribose derivatives as their sodium or barium salts was achieved. Facile purification by reversed-phase HPLC enabled product isolation within hours.

A fluorescent DNA probe containing an anthracene group attached via an anucleosidic linker can identify all four DNA bases at a single site as well as the epigenetic modification C/5-MeC via a hybridisation sensing assay.

ALL catalytic RNAs (ribozymes) require or are stimulated by divalent metal ions, but it has been difficult to separate the contribution of these metal ions to formation of the RNA tertiary structure1 from a more direct role in catalysis. The Tetrahymena ribozyme catalyses cleavage of exogenous RNA2,3 or DNA4,5 substrates with an absolute requirement for Mg2+ or Mn2+ (ref. 6). A DNA substrate, in which the bridging 3′ oxygen atom at the cleavage site is replaced by sulphur, is cleaved by the ribozyme about 1,000 times more slowly than the corresponding unmodified DNA substrate when Mg2+ is present as the only divalent metal ion. But addition of Mn2+ or Zn2+ to the reaction relieves this negative effect, with the 3′ S–P bond being cleaved nearly as fast as the 3′ O–P bond. Considering that Mn2+ and Zn2+ coordinate sulphur more strongly than Mg2+ does7,8, these results indicate that the metal ion contributes directly to catalysis by coordination to the 3′ oxygen atom in the transition state, presumably stabilizing the developing negative charge on the leaving group. We conclude that the Tetrahymena ribozyme is a metalloenzyme, with mechanistic similarities to several protein enzymes9–12.

Oligonucleotides containing a 3'-thiothymidine residue (T3's) at the cleavage site for the EcoRV restriction endonuclease (between the central T and A residues of the sequence GATATC) have been prepared on an automated DNA synthesizer using 5'-O-monomethoxytritylthymidine 3'-S-(2-cyanoethyl N,N-diisopropylphosphorothioamidite). The self-complementary sequence GACGAT3'sATCGTC was completely resistant to cleavage by EcoRV, while the heteroduplex composed of 5'-TCTGAT3'sATCCTC and 5'-GAGGATATCAGA (duplex 4) was cleaved only in the unmodified strand (5'-GAGGATATCAGA). In contrast, strands containing a 3'-S-phosphorothiolate linkage could be chemically cleaved specifically at this site with Ag+. A T3's residue has also been incorporated in the (-) strand of double-stranded closed circular (RF IV) M13mp18 DNA at the cleavage site of a unique EcoRV recognition sequence by using 5'-pCGAGCTCGAT3'sATCGTAAT as a primer for polymerization on the template (+) strand of M13mp18 DNA. On treatment of this substrate with EcoRV, only one strand was cleaved to produce the RF II or nicked DNA. Taken in conjunction with the cleavage studies on the oligonucleotides, this result demonstrates that the 3'-S-phosphorothiolate linkage is resistant to scission by EcoRV. Additionally, the phosphorothiolate-containing strand of the M13mp18 DNA could be cleaved specifically at the point of modification using iodine in aqueous pyridine. The combination of enzymatic and chemical techniques provides, for the first time, a demonstrated method for the sequence-specific cleavage of either the (+) or (-) strand.