2'-Spiro ribo- and arabinonucleosides: synthesis, molecular modelling and incorporation into oligodeoxynucleotides

We have synthesized four conformationally restricted bicyclic 2'-spiro nucleosides via 2'-C-allyl nucleosides as key intermediates. The ribo-configured 2'-spironucleosides 9b and 14b were obtained by a convergent strategy starting from 2-ketofuranose 1 whereas the arabino-configured 2'-spironucleosides 21 and 27 were obtained by a linear strategy with a 2'-ketouridine derivative as starting material. The furanose ring of 9b/14b adopts N-type conformations whereas the furanose ring of 21/27 exists as an N<==>S equilibrium. These compounds showed no anti-HIV-1 activity or cytotoxicity. Incorporation of the four 2'-spironucleosides (as monomers X4 and X5) into oligodeoxynucleotides was accomplished using the phosphoramidite approach on an automated DNA synthesizer. Irrespective of monomeric configuration, hybridization studies revealed that these 2'-spironucleotide monomers (X4 and X5) induce decreased duplex thermostabilities compared with the corresponding DNA:DNA and DNA:RNA duplexes. Molecular modelling indicated that steric constraints are a possible reason for the lowered binding affinities of the modified oligodeoxynucleotides towards complementary single-stranded DNA and single-stranded RNA complements.     

Synthesis of a 2'-Se-uridine phosphoramidite and its incorporation into oligonucleotides for structural study

[chemical reaction: see text]. We report here the synthesis of the 5'-[benzhydryloxybis(trimethylsilyloxy)]silyl-2'-methylseleno-2'-deoxyuridine phosphoramidite and its incorporation into oligonucleotides by solid-phase synthesis. The coupling yield of this phosphoramidite into oligonucleotides is higher than 99%. We also demonstrate that this 2'-methylselenophosphoramidite is compatible with the 5'-silyl-2'-ACE chemistry, for longer Se-RNA solid-phase synthesis. Our preliminary NMR study on the synthesized 2'-Se-DNA has revealed a U(Se)-A base pair and a duplex structure formation when its complementary strand was present.     

Synthesis of a 2'-Se-thymidine phosphoramidite and its incorporation into oligonucleotides for crystal structure study

[reaction: see text] To investigate nucleic acids with selenium derivatization for crystallography, we report the first synthesis of 2'-methylseleno-thymidine phosphoramidite and its incorporation into DNAs and RNAs by solid-phase synthesis with over 99% coupling yield. The d(GT(Se)GTACAC)2 crystal structure was also determined at 1.40 A resolution using Se phasing, revealing that this Se derivatization did not cause significant structure perturbation, consistent with our UV melting study. In addition, we observed that the Se modification largely facilitated the crystallization.     

Synthesis of 2'-C-difluoromethylribonucleosides and their enzymatic incorporation into oligonucleotides

[Chemical reaction: See text] Nucleosides bearing a branched ribose have significant promise as therapeutic agents and biotechnological and biochemical tools. Here we describe synthetic entry into a new subclass of these analogues, 2'-C-beta-difluoromethylribonucleosides. We constructed the glycosylating agent 4 in three steps from 1,3,5-tri-O-benzoyl-alpha-D-ribofuranose 1. The key steps included nucleophilic addition of difluoromethyl phenyl sulfone to 2-ketoribose 2 followed by mild and efficient reductive desulfonation. Ribofuranose 4 glycosylated bis(trimethylsilyl)uracil directly, giving difluoromethyluridine 7 efficiently after deprotection. Conversion of 4 to the corresponding ribofuranosyl bromide allowed efficient access to C, A, and G analogues. A related approach starting from methyl D-ribofuranose offered synthetic entry into the diastereomeric manifold, 2'-C-alpha-difluoromethyl-arabino-alpha-pyrimidine. To incorporate 2'-C-beta-difluoromethyluridine into an oligodeoxynucleotide we converted 7 to the bisphosphate and carried out successive ligation reactions using T4 RNA ligase and T4 DNA ligase. Analogous to natural RNA linkages, the resulting oligonucleotide undergoes hydroxide-catalyzed backbone scission at the difluoromethyluridine residue via internal transphosphorylation.     

Facile synthesis of a cis-syn thymine dimer building block and its incorporation into oligodeoxynucleotides

The synthesis of a building block containing the photobiologically relevant cis-syn thymine cyclobutane photoproduct and its incorporation into oligonucleotides by the phosphoramidite-based solid-phase synthesis is reported. Compared to previous syntheses, this route is extremely short and allows such modified oligonucleotides to be easily available for biological studies.     

Synthesis and hybridization properties of oligonucleotides containing polyamines at the C-2 position of purines: a pre-synthetic approach for the incorporation of spermine into oligodeoxynucleotides containing 2-(4,9,13-triazatridecyl)-2'-deoxyguanosine

We have developed a synthesis of spermine-containing oligonucleotides (ODN-sper) which allows incorporation of multiple polyamine residues. This approach was based on the pertrifluoroacetylated 5'DMT-dGsper phosphoramidite synthon. Its coupling yield with resin-bound ODN decreased dramatically when close to the 3'-end. Optimization of the coupling conditions allowed 22-mer ODNs containing up to six spermine residues to be synthesized. Several ODNs of different sequences with 1-4 pendent spermines could be purified and their hybridization properties were evaluated. Duplex melting temperatures increased linearly with the number of polyamine residues (deltaTm/sper = 3.0 +/- 0.2 degrees C in 100mM NaCl). This compares very favorably with values reported for duplexes of similar initial stability containing other cation-substituted bases. Moreover, the stability increase was neither sequence nor position-dependent, and even contiguous spermine residues did not cross-talk. Extrapolation based on these findings leads to the conclusion that a duplex formed with a 22-mer oligonucleotide containing seven spermine residues would be as stable as genomic DNA, which highlights its potential for DNA strand invasion.     

Synthesis of enantiopure azetidine 2-carboxylic acids and their incorporation into peptides

Enantiopure azetidine 2-carboxylic acids were prepared by hydrolysis of the corresponding 2-cyano azetidines, without ring cleavage of the azetidine or epimerization. The produced amino acids, which are conformationally constrained analogues of phenylalanine, can be cleanly debenzylated and used for the synthesis of tripeptides. In the course of the synthesis of new enantiopure 2-cyano azetidines through intramolecular alkylation of a metallated amino nitrile, it was found that the involved anionic cyclisation can be thermodynamically controlled, thus enhancing its diastereoselectivity.     

Synthesis and triplex forming properties of pyrrolidino pseudoisocytidine containing oligodeoxynucleotides

Pyrrolidino pseudo-C-nucleosides are isosteres of natural deoxynucleosides which are protonated at the pyrrolidino ring nitrogen under physiological conditions. As constituents of a triplex forming oligodeoxynucleotide (TFO), the positive charge is expected to stabilise DNA triple helices via electrostatic interactions with the phosphodiester backbone of the target DNA. We describe the synthesis of the pyrrolidino isocytidine pseudonucleoside and the corresponding phosphoramidite building block and its incorporation into TFOs. Such TFOs show substantially increased DNA affinity compared to unmodified oligodeoxynucleotides. The increase in affinity is shown to be due to the positive charge at the pyrrolidino subunit.     

Synthesis of a novel bicyclic nucleoside restricted to an S-type conformation and initial evaluation of its hybridization properties when incorporated into oligodeoxynucleotides.

The phosphoramidite (1S,3R,4S)-3-(2-cyanoethoxy(diisopropylamino)phosphinoxymethyl)-5-N-(4-monomethoxytrityl)-1-(uracil-1-yl)-5-aza-2-oxabicyclo[2.2.1]heptane 18 of a novel bicyclic nucleoside structure was synthesized from the known 1-(3'-deoxy-beta-D-psicofuranosyl)uracil 3. Conformational analysis of its structure verified its expected S-type furanose conformation, and the secondary amino group in the 4'-position allowed for incorporation into oligonucleotides using 5' --> 3' directed oligonucleotide synthesis as previously described for phosphoramidates. Thermal denaturation studies showed rather large decreases in duplex stabilities of -4.3 and -2.7 degrees C per modification toward complementary DNA and RNA, respectively.     

Synthesis and hybridization properties of oligodeoxynucleotides incorporating 2-N-carbamoylguanine derivatives as guanine analogs

2-N-Carbamoyldeoxyguanosine and its derivatives were synthesized and incorporated into ODNs. The T_m analyses revealed higher selective base recognition ability of 2-N-carbamoylguanine than that of guanine. The new guanine analog must be useful for the development of functional oligodeoxynucleotides capable of precise base recognition.     

Synthesis and pairing properties of oligodeoxynucleotides containing bicyclo-RNA and bicyclo-ANA modifications

The synthesis of the ribo(bc-rT)- and arabino(bc-araT)-version of bicyclothymidine (bc-dT) has been achieved. A conformational analysis by X-ray and/or (1)H NMR spectroscopy on the corresponding 3',5'-benzyl-protected nucleosides featured a rigid C(2')-endo conformation for the furanose ring, irrespective of the configuration of the OH group at C(2'). The conformation of the carbocyclic ring in these nucleosides was found to be less defined and thus more flexible. Both nucleosides were converted into the corresponding phosphoramidites and incorporated into oligodeoxynucleotides by standard DNA chemistry. T(m)-data of duplexes with cDNA and RNA revealed that a bc-rT unit strongly destabilized duplexes with cDNA and RNA by 6-8 °C/mod, while bc-araT was almost T(m) neutral. A rationale based on a previous structure of a bc-DNA mini duplex suggests that the strong destabilization caused by a bc-rT unit arises from unfavorable steric interactions of the equatorial 2'-OH group with the sugar residue of the 3'-neighboring nucleotide unit.     

Synthesis of a C-linked glycosylated thymine-based PNA monomer and its incorporation into a PNA oligomer

Backbone modification of peptide nucleic acids (PNAs) by glycosylation has been shown to enhance selective biodistribution and cellular targeting of PNA oligomers based on sugar and cell surface lectin interactions. Here we report the synthesis of a new backbone-glycosylated thymine-based PNA monomer (T(gal)). The sugar residue was attached to the backbone of PNA via a stable carbon-carbon linkage between the sugar and the PNA monomers. Also, incorporation of the modified monomer into a PNA decamer (H-Ala(gal)-G-G-G-T(gal)-C-A-G-C-T(gal)-T-Lys-NH2) was successfully performed. Melting temperature (UV-Tm) of the modified PNA against the complementary DNA was only slightly lower than unmodified PNA.     

Synthesis of 4,4-disubstituted 2-aminocyclopentanecarboxylic acid derivatives and their incorporation into 12-helical beta-peptides

An enantioselective synthetic route is reported for trans-2-aminocyclopentanecarboxylic acids (ACPC) bearing geminal side chain pairs at the 4-position. Beta-peptides containing the 4,4-disubstituted ACPC residues adopt the 12-helical conformation, as demonstrated by 2D NMR analysis in aqueous solution. These 4,4-disubstituted ACPC residues display functional groups, including acidic and hydrogen bond donating groups, in a geometrically defined fashion, which should be useful for the design of beta-peptides for specific applications. [structure: see text]     

Synthesis of 2'-modified oligodeoxynucleotides via on-column conjugation.

Oligodeoxynucleotides modified at the 2'-position of 2'-amino-2'-deoxyuridine or uridine were prepared in high yield and purity using phosphoramidites 2 and 3, respectively. Oligodeoxynucleotide conjugates were prepared on the solid-phase synthesis support following selective unmasking of the nucleophile incorporated in these phosphoramidites. Synthesis of oligodeoxynucleotides modified at the 2'-position of an internal nucleotide provides molecules that are complementary to those previously prepared via a similar approach using C5-substituted pyrimidines. The efficiency of functionalization of the 2'-O-alkylamino-uridine derived from 3 in a protected oligodeoxynucleotide was less susceptible to steric hindrance than the 2'-amino-2'-deoxyuridine in the same polymeric substrate. However, the greater reactivity of the 2'-O-alkylamine containing nucleotide gave rise to undesired acetamide formation resulting from nucleophilic attack on the 5'-terminal acetate in capped failure sequences. This problem was overcome by using 2,2,2-trimethylacetyl anhydride as a capping agent during the automated synthesis cycles. Finally, the efficiency of the photochemical unmasking of the support bound alkylamine on a 1 mumole scale was improved by using two 20 min photolysis cycles, coupled with removing reaction byproducts between cycles.     

Synthesis of L-kedarosamine in protected form and its efficient incorporation into an advanced intermediate to kedarcidin chromophore

An efficient route to the complex L-kedarosamine alpha-glycosidic ether 2, a synthetic precursor to kedarcidin chromophore, is described. Central to the route, which is suitable for the preparation of multigram amounts of material, is a short synthetic sequence from D-threonine to protected L-kedarosamine derivatives and methodology for their alpha-selective coupling with appropriate hydroxyl acceptors.     

Synthesis of 2'-N-methylamino-2'-deoxyguanosine and 2'-N,N-dimethylamino-2'-deoxyguanosine and their incorporation into RNA by phosphoramidite chemistry

The 2'-hydroxyl groups within RNA contribute in essential ways to RNA structure and function. Previously, we designed an atomic mutation cycle (AMC) that uses ribonucleoside analogues bearing different C-2'-substituents, including -OCH(3), -NH(2), -NHMe, and -NMe(2), to identify hydroxyl groups within RNA that donate functionally significant hydrogen bonds. To enable AMC analysis of the nucleophilic guanosine cofactor in the Tetrahymena ribozyme reaction and at other guanosines whose 2'-hydroxyl groups impart critical functional contributions, we describe here the syntheses of 2'-methylamino-2'-deoxyguanosine (G(NHMe)) and 2'-N,N-dimethylamino-2'-deoxyguanosine (G(NMe(2))) and their corresponding phosphoramidites. The key step in obtaining the nucleosides involved S(N)2 displacement of 2'-β-triflate from an appropriate guanosine derivative by methylamine or dimethylamine. We readily obtained the G(NMe(2)) phosphoramidite and incorporated it into RNA. However, the G(NHMe) phosphoramidite posed a significantly greater challenge due to lack of a suitable -2'-NHMe protecting group. After testing several strategies, we established that allyloxycarbonyl (Alloc) provided suitable protection for 2'-N-methylamino group during the phosphoramidite synthesis and the subsequent RNA synthesis. This work enables AMC analysis of guanosine's 2'-hydroxyl group within RNA.     

Synthesis of new fluorogenic cyanine dyes and incorporation into RNA fluoromodules

A new fluorogenic cyanine dye was synthesized and found to have low fluorescence quantum yield in fluid solution and in the presence of double-stranded DNA but 80-fold enhanced fluorescence in viscous glycerol solution. An RNA aptamer selected for binding to the new dye exhibits K(d) = 87 nM and 60-fold fluorescence enhancement. The dye-aptamer pair is a fluoromodule that can be incorporated into fluorescent sensors and labels.     

Synthesis of protected L-4-[sulfono(difluoromethyl)phenylalanine and its incorporation into a peptide

[reaction: see text] A protected form of L-4-[sulfono(difluoromethyl)]phenylalanine (F(2)Smp), a novel non-hydrolyzable phospho- and sulfotyrosine mimetic, was synthesized via electrophilic fluorination of a benzylic sulfonate followed by a Pd-catalyzed cross-coupling reaction between the fluorinated sulfonate and the zincate of protected iodoalanine. F(2)Smp was incorporated into a peptide using solid-phase peptide synthesis techniques.     

Retraction: Divergent synthesis of 5-substituted pyrimidine 2′-deoxynucleosides and their incorporation into oligodeoxynucleotides for the survey of uracil DNA glycosylases

Retraction of ‘Divergent synthesis of 5-substituted pyrimidine 2′-deoxynucleosides and their incorporation into oligodeoxynucleotides for the survey of uracil DNA glycosylases’ by Ai Tran et al., Chem. Sci., 2020, 11, 11818–11826, DOI: 10.1039/D0SC04161K.

The Royal Society of Chemistry hereby wholly retracts this Chemical Science article due to concerns about the reproducibility of the data.The Royal Society of Chemistry has been contacted by the authors of this article to alert us that recent experiments by their group have shown that some results are not reproducible, especially the yields of several key intermediates. Given the focus on the easy access to epigenetically important nucleosides and related ODNs, unreliable yields significantly impact the confidence in the results. In addition, it was discovered that one of the biological samples provided to the authors was mislabelled. The human stem cells were actually mouse stem cells, hence some discussion and conclusions in the article may no longer apply. Ahead of a thorough re-examination of the entire study, in order to preserve the rigor of the scientific record, the signing authors have chosen to retract the article and they would like to apologise for any inconvenience this may have caused for readers.Signed: Ai Tran, Song Zheng, Dawanna S. White, Alyson M. Curry and Yana CenRetraction endorsed by May Copsey, Executive Editor, Chemical Science     

Triazolylpyrenes: synthesis, fluorescence properties, and incorporation into DNA

Synthesis of 1,6- and 1,8-triazolylpyrenes and their incorporation into oligonucleotides is described. In hybrids, triazolylpyrenes adopt interstrand stacking interactions. Exciton coupling is observed for the duplex containing a pair of the 1,6-isomer indicating a well-defined helical arrangement of the triazolylpyrene building blocks. Triazole substitution results in pronounced red-shifts of monomer as well as excimer fluorescence. Furthermore, quantum yields of the formed excimers are remarkably high.