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.     

Synthesis of a benzotriazole azo dye phosphoramidite for labelling of oligonucleotides

The synthesis of a benzotriazole azo dye phosphoramidite and the subsequent use in solid phase synthesis of oligonucleotides is reported. The azo dye is shown as a surface enhanced resonance Raman label for oligonucleotides that is capable of immobilisation of the oligonucleotide on metal surfaces such as silver nanoparticles.     

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 and properties of 2-azidodeoxyadenosine and its incorporation into oligodeoxynucleotides

2-Azidodeoxyadenosine (7) was conveniently synthesized from deoxyguanosine (2) by use of a combined reagent of TMSN₃–BuONO. The structure of the tautomer of the azido derivative was determined by ¹H NMR. Reaction of 7 with iPr₂NP(OEt)₂ gave an intermediate 10 of the Staudinger reaction. Incorporation of 7 into a DNA 13mer resulted in a significant decrease of the T_m value of the DNA duplex upon hybridization with the complementary strand. The thermal stability was discussed based on the hydrogen bond energy and electrostatic repulsion.     

Toward catalytically active oligonucleotides: synthesis of a flavin nucleotide and its incorporation into DNA

[reaction--see text] The synthesis of a vitamin B(2)-derived flavin-nucleotide is described. A combined H-phosphonate/phosphoramidite protocol was developed for the first incorporation of flavin coenzymes into a DNA stack. The coenzyme-DNA is predicted to have novel biosensing and catalytic properties.     

Synthesis of 5'-terminal capped oligonucleotides using O-N phosphoryl migration of phosphoramidite derivatives

Trivalent phosphoramidite derivatives could be readily converted by reacting with 1-hydroxy-7-azabenzotriazole to phosphotriester intermediates; these intermediates reacted smoothly with phosphorylated compounds to give pyrophosphate derivatives. This new phosphorylation approach enabled a facile and rapid synthesis of 5'-adenylated DNA oligomers (A(5')ppDNA) on resins using a silyl-type linker. Our new approach could be applied to the synthesis of a 2'-OMe-RNA oligomer containing the 5'-terminal 2,2,7-trimethylguanosine cap structure.     

Efficient synthesis of [2'-18O]uridine and its incorporation into oligonucleotides: a new tool for mechanistic study of nucleotidyl transfer reactions by isotope effect analysis

Lack of sufficient quantities of isotopically labeled materials has precluded the use of heavy atom isotope effects to investigate mechanisms of nucleotidyl transfer reactions in nucleic acids. Here we achieve regioselective opening of 2,2'-cyclouridine with [(18)O2]benzoic acid/potassium hydride, allowing an efficient "one-pot" synthesis of [2'-18O]uridine in 88% yield. Conversion to the corresponding phosphoramidite enables solid-phase synthesis of [2'-(18)O] RNA substrates for isotope effect studies with nucleotidyl transferases and hydrolases.     

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 the phosphoramidite derivative of 2'-deoxy-2'-C-beta-methylcytidine

2'-Deoxy-2'-C-beta-methylnucleosides elicit interest as potential therapeutic agents and as analogues for the analysis of nucleic acid structure and function. An efficient route for the synthesis of 2'-deoxy-2'-C-methyluridine (11), 2'-deoxy-2'-C-methylcytidine (12), and the phosphoramidite derivative of 2'-deoxy-2'-C-beta-methylcytidine (10, 46% overall yield) from 1,2,3,5-tetra-O-benzoyl-2-C-beta-methylribofuranose (1) is described.     

Design, synthesis, and incorporation of fluorous 5-methylcytosines into oligonucleotides

A palladium-catalyzed Negishi coupling reaction has been developed to synthesize fluorous 5-methylcytosines. These fluorous nucleosides are incorporated into the oligonucleotides that correspond to part of the promoter region of Oct4, a master gene that undergoes dynamic DNA demethylation during cellular reprogramming. The separation of the fluorous oligonucleotides from its nonfluorous analogues has been achieved through solid-phase extraction over fluorous silica, suggesting its potential use in probing DNA demethylation.     

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.     

Phosphoramidite building blocks for efficient incorporation of 2′-O-aminoethoxy(and propoxy)methyl nucleosides into oligonucleotides

A simple and efficient method for the preparation of eight phosphoramidite building blocks for incorporation of 2′-O-(2-aminoethoxymethyl)ribonucleosides and 2′-O-(3-aminopropoxymethyl)ribonucleosides into synthetic oligonucleotides has been developed. The synthetic routes are maximally convergent and provide sufficient amounts of phosphoramidites for several solid-phase synthesis coupling reactions. Using acyclic derivatives 17a,b the overall yields of phosphoramidites 2 and 3 were increased up to 50% for pyrimidine nucleosides and up to 30% for purine derivatives with substantial decrease of total reaction steps. The 2′-O-substituent was found to be stable during oligonucleotide synthesis. The resulting oligonucleotides are of particular interest for post-synthetic functionalization and conjugation.     

Synthesis of oligoribonucleic acid conjugates using a cyclooctyne phosphoramidite

The conjugation of a ribonucleic acid 16-mer with the cationic amphiphilic peptide penetratin and an anionic hyaluronan tetrasaccharide by means of Cu-free "click" chemistry is reported. The alkyne-functionalized 16-mer was prepared by automated solid-phase synthesis, using a newly developed strained cyclooctyne phosphoramidite in the final coupling. Cycloaddition of the alkyne functionalized RNA to the azide containing biomolecules led to a clean conversion into the corresponding nucleic acid conjugates.     

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.     

A novel phosphoramidite method for automated synthesis of oligonucleotides on glass supports for biosensor development

Two protocols for functionalization of glass supports with hexaethylene glycol (HEG)-linked oligonucleotides were developed. The first method (standard amidite protocol) made use of the 2-cyanoethyl-phosphoramidite derivative of 4,4′-dimethoxytrityl-protected HEG. This was first coupled to the support by standard solid-phase phosphoramidite chemistry followed by extension with a thymidylic acid icosanucleotide. Stepwise addition of the linker phosphoramidite graduated at 1% (relative to the total sites available) perstep at 50°C resulted in an optimal yield of immobilized oligonucleotides at a density of 2.24 × 10¹⁰ strands/mm². This observed loading maximum lies well below the theoretical maximum loading owing to nonspecific adsorption of HEG on the glass and subsequent blocking of reactive sites. Surface loadings as high as 3.73 × 10¹⁰/mm² and of excellent sequence quality were achieved with a reverse amidite protocol. The support was first modified into a 2-cyanoethyl-N,N-diisopropylphosphoramidite analog followed by coupling with 4,4′-dimethoxytrityl-protected HEG. This protocol is conveniently available when using a conventional DNA synthesizer. The reverse amidite protocol allowed for control of the surface loading at values suitable for subsequent analytical applications that make use of immobilized oligonucleotides as probes for selective hybridization of sample nucleic acids of unknown sequence and concentration.     

Promotion of stable triplex formation by partial incorporation of 2',5'-phosphodiester linkages into triplex-forming oligonucleotides

Pentadecamer homopyrimidine oligonucleotides containing three or more 2',5'-phosphodiester linkages in different modes were prepared and used to evaluate the ability as a triplex-forming oligonucleotide (TFO), and it was found that discontinuous replacement of the 3',5'-phosphodiester linkages in TFO by 2',5'-linkages significantly stabilizes parallel-motif triplexes.     

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.     

Synthesis of fluorous tags for incorporation of reducing sugars into a quantitative microarray platform

Carbohydrate microarrays can map out key interactions of carbohydrates and proteins in a high-throughput manner, but require the inclusion of a range of sugars for their optimal use. Here we present the synthesis and use of a new hydroxylamine-modified fluorous tag that allows the facile incorporation of reducing sugars into a noncovalent fluorous-based microarray after simple purification by fluorous solid-phase extraction (FSPE). The microarray supports quantitative screening against carbohydrate-binding proteins.