A Rapid Method for the Functionalisation of Polymer Supports for Solid Phase Oligonucleotide Synthesis

Abstract

A rapid method is described for the covalent anchoring of appropriately protected 2′-deoxyribonucleoside-3′-O-succinates to LCAA-CPG, widely used support for solid phase oligonucleotide synthesis. The method involves the reaction of nucleoside-3′-O-succinates with aminoalkyl functions of the support in the presence of improved and commercially available condensing reagent, TBTU or TPP-DTNP to generate fully functionalised polymer supports with excellent nucleoside loadings.     

5'-Fluoro-5'-deoxyaristeromycin

5'-Fluoro-5'-deoxyaristeromycin (2) has been prepared via a Mitsunobu coupling of (1S,2S,3R,4S)-2,3-(cyclopentylidenedioxy)-4-fluoromethylcyclopentan-1-ol with N6-bis-boc protected adenine. This procedure is adaptable to preparing a number of 5'-fluoro-5'-deoxycarbocyclic nucleoside analogs with diversity in the heterocyclic base. Antiviral analysis found promising activity for 2 toward measles but no other viruses. No cytotoxicity was observed for 2.     

Novel strategies towards construction of non-ionic and achiral backbones in antisense oligonucleotides.

A convenient synthesis of a thymidine (T) nucleoside dimer (T-3'-CH2-O-NCH3-CH2-4'-T) 12 has been accomplished via a nucleoside coupling reaction. An alternative synthesis of 3'-deoxy-3'-C-hydroxymethyl-thymidine is described. The new dimer and methodology is useful for the development of backbone-modified antisense oligonucleosides.     

Synthesis, characterization and some properties of dideoxynucleoside analogs of cytidine diphosphate diacylglycerol.

Phospholipid conjugates of antiretroviral nucleoside analogs have been proposed to have several advantageous features when compared to the parent drugs (Hostetler, K.Y. et al. (1990) J. Biol. Chem. 265, 6112-6117). Here we report on the synthesis of one such type of lipid conjugates, i.e., nucleosides diphosphate diacylglycerols. The syntheses of 3'-azido-3'-deoxythymidine diphosphate diacylglycerol, 3'-deoxythymidine diphosphate diacylglycerol and 2',3'-dideoxycytidine diphosphate diacylglycerol (with different acyl chains) were performed starting from phosphatidic acid and the antiviral nucleoside. A high-performance liquid chromatography procedure for a single step purification of the compounds is presented. The compounds were characterized biochemically, using rat liver enzymes and chemically by phosphorus, fatty acid, ultraviolet, IR and 1H-NMR analyses. Preliminary data on the behaviour in aqueous solution of some of the compounds are presented.     

Rapid esterification of nucleosides to solid-phase supports for oligonucleotide synthesis using uronium and phosphonium coupling reagents

Nucleosides can be esterified to solid-phase supports using uronium or phosphonium coupling reagents and a coupling additive, such as 1-hydroxybenzotriazole (HOBT), 7-aza-1-hydroxybenzotriazole (HOAT), N-methylimidazole (NMI), or 4-(dimethylamino)pyridine (DMAP). However, DMAP was far superior to other additives and high nucleoside loadings (up to 60 micromol/g) and rapid coupling reactions (< or = 10 min) were possible. Hydroxyl-derivatized CPG was attached to nucleosides with 3'-succinyl or 3'-hydroquinone-O, O'-diacetic acid (HQDA or Q-Linker) carboxyl groups through a primary ester linkage. Alternatively, supports derivatized with succinic acid or the Q-Linker were attached directly to the 3'-OH group of nucleosides through a secondary ester linkage. Uronium reagents (HATU or HBTU) gave the best results with the HQDA linker arm, while the bromophosphonium (BrOP or PyBrOP) reagents were best with the succinyl linker arm. In all cases, the coupling reactions were much faster than previous methods using carbodiimide coupling reagents. The ease and speed of the reaction make this support derivatization procedure suitable for automated in situ couplings on DNA synthesizers.     

Polymer supported synthesis of aminooxyalkylated oligonucleotides,and some applications in the fabrication of microarrays

A new protocol has been described for solid phase preparation of 3′- and 5′-aminooxylalkylated oligonucleotides using commercially available reagents. This involves attachment of linker 4 either with an LCAA-CPG support via succinoylation followed by synthesis (3′-aminooxyalkylated oligomers) or formation of its phosphoramidite 6 followed by coupling with desired oligomer (for generating 5′-aminooxyalkylated oligomers). Both the routes produced modified oligonucleotides in sufficiently high yields and purity (on HPLC) via conventional oligonucleotide synthesis on an automated synthesizer and deprotection step using aqueous ammonia (16 h, 60 °C). Aminooxyalkylated oligonucleotides were used to construct microarrays on glass surface (biochips). The performance of the biochips was evaluated by immobilizing modified oligonucleotides on epoxylated glass microslides under different sets of conditions with respect to pH, temperature and time. Further, the constructed microarrays were successfully used for detection of nucleotide mismatches and bacterial typhoid.     

Synthesis of 5-Substituted 2′-Deoxyuridine-5′- Phosphonate Analogues and Evaluation of their Antiviral Activity

A small series of 5-(hetero)aryl-modified nucleoside phosphonates was synthesized via an 8-step procedure including a Wittig reaction and Suzuki–Miyaura coupling. An unanticipated anomerization during phosphonate deprotection allowed us to isolate both anomers of the 5-substituted 2′-deoxy-uridine phosphonates and assess their antiviral activity against a broad panel of viruses.     

A new and efficient strategy for the synthesis of shimofuridin analogs: 2'-O-(4-O-stearoyl-alpha-L-fucopyranosyl)thymidine and -uridine

Two shimofuridin analogs: 2'-O-(4-O-stearoyl-alpha-L-fucopyranosyl)thymidine (2) and -uridine (3) have been synthesized using D-arabinose, L-fucose, thymine, uracil, and stearoyl chloride as the starting materials. The synthetic procedures involve the facile preparation of 1-(3,5-di-O-benzyl-beta-D-ribofuranosyl)thymine (9) and -uracil (10) by coupling of 1,2-anhydro-3,5-di-O-benzyl-alpha-D-ribofuranose (8) with silylated thymine and uracil, and then stereoselective formation of the 1,2-cis (alpha) interglycoside bonds through condensation of the nucleoside derivatives 9 and 10 with 2-(2,3-di-O-benzyl-4-O-stearoyl-beta-L-fucopyranosylsulfonyl) pyrimidine (18). The 1,2-anhydro-3,5-di-O-benzyl-alpha-D-ribofuranose (8) was prepared by an improved procedure from D-arabinose.     

The broadly selective human Na+/nucleoside cotransporter (hCNT3) exhibits novel cation-coupled nucleoside transport characteristics

The concentrative nucleoside transporter (CNT) protein family in humans is represented by three members, hCNT1, hCNT2, and hCNT3. hCNT3, a Na+/nucleoside symporter, transports a broad range of physiological purine and pyrimidine nucleosides as well as anticancer and antiviral nucleoside drugs, and belongs to a different CNT subfamily than hCNT1/2. H+-dependent Escherichia coli NupC and Candida albicans CaCNT are also CNT family members. The present study utilized heterologous expression in Xenopus oocytes to investigate the specificity, mechanism, energetics, and structural basis of hCNT3 cation coupling. hCNT3 exhibited uniquely broad cation interactions with Na+, H+, and Li+ not shared by Na+-coupled hCNT1/2 or H+-coupled NupC/CaCNT. Na+ and H+ activated hCNT3 through mechanisms to increase nucleoside apparent binding affinity. Direct and indirect methods demonstrated cation/nucleoside coupling stoichiometries of 2:1 in the presence of Na+ and both Na+ plus H+, but only 1:1 in the presence of H+ alone, suggesting that hCNT3 possesses two Na+-binding sites, only one of which is shared by H+. The H+-coupled hCNT3 did not transport guanosine or 3'-azido-3'-deoxythymidine and 2',3'-dideoxycytidine, demonstrating that Na+- and H+-bound versions of hCNT3 have significantly different conformations of the nucleoside binding pocket and/or translocation channel. Chimeric studies between hCNT1 and hCNT3 located hCNT3-specific cation interactions to the C-terminal half of hCNT3, setting the stage for site-directed mutagenesis experiments to identify the residues involved.     

Investigation of a facile synthetic method for phosphorothioate dimer synthons in oligonucleotide phosphorothioates synthesis

A facile synthetic method of a phosphorothioate dimer block was investigated. Dinucleoside phosphite triester intermediates were obtained in one-pot synthesis by the coupling of a protected nucleoside bearing free 5'-OH and a protected nucleoside bearing free 3'-OH in the presence of phosphorous trichloride (PCl3) and 1,2,4-triazole. The intermediates were easily sulfurized to afford the desired phosphorothioate dimer blocks in 33-64% overall yields.     

DIRECT MALDI-TOF MS ANALYSIS OF OLIGONUCLEOTIDES ON SOLID SUPPORT THROUGH A PHOTOLABILE LINKER

MALDI-TOF mass spectrometry was used to analyze oligonucleotides still anchored to long-chain alkylamine controlled-pore glass (LCAA-CPG) through a photolabile linker. This technique is useful to follow supported chemical reactions in real time and monitor by-products formation.     

Preparation of oligodeoxyribonucleoside phosphorodithioates by a triester method.

A method to prepare thymidine phosphorodithioate dimers (ref. 1) has been extended to allow the preparation of oligo-2'-deoxyribonucleotide phosphorodithioates containing all four bases. The method is suitable for large-scale synthesis and gives phosphorodithioates without phosphorothioate impurities (31P nmr, detection limit 0.5 to 1%). Oligonucleotides up to octamers which contain -0-(PS2-)-0- linkages at all positions have been prepared by block synthesis in solution. The phosphorodithioate linkage is introduced by the reaction of a 5'-O, N-protected nucleoside (or oligonucleotide) with a dithiophosphorylating agent RSP(S)(ODhbt)2, R = 2,4-dichlorobenzyl, Dhbt = 3,4-dihydro-4-oxo-benzotriazin-3-yl, followed by coupling of the product to a 3'-O,N-protected nucleoside (or oligonucleotide). This method gives pure protected oligodeoxyribonucleoside phosphorodithioates, and phosphorothioate linkages are only introduced if contact with conc. aqueous ammonia during or after deblocking is unduly prolonged.     

Nucleoside-3''-phosphotriesters as key intermediates for the oligoribonucleotide synthesis. III. An improved preparation of nucleoside 3''-phosphotriesters, their 1H NMR characterization and new conditions for removal of 2-cyanoethyl group.

An improved procedure for the transformation of 5'-O-monomethoxytrityl-2'-O-acetyl-3'-phosphates of uridine la, inosine ib and 6-N-benzoyladenosine lc into corresponding 3'/2,2,2-trichloroethyl, 2-cyanoethyl/-phosphates iiaic is reported. H NMR characterization of nucleoside 3'-phosphotriesters is presented. New conditions i.e. anhydrous triethylamine-pyridine treatment have been found for the selective removal of 2-cyanoethyl group from nucleoside 3'-phosphotriesters in the presence of neighbouring 2'-O-acetyl one.     

Synthesis of amide-linked [(3')CH2CO-NH(5')] nucleoside analogues of small oligonucleotides

We report syntheses of new amide-linked (di-penta)nucleoside analogues of antisense oligonucleotide components. Solution-phase coupling of 3'-(carboxymethyl)-3'-deoxy- and 5'-amino-5'-deoxynucleoside derivatives provides amide dimers. Activated [3'-(carboxymethyl)-3'-deoxy] units with a 5'-azido-5'-deoxy function provide "masked" 5'-amino-5'-deoxy residues for chain extension, and a 5'-O-DMT-protected unit provides the 5'-terminus for attachment to a phosphodiester linkage.     

Synthesis of nucleoside phosphosulfates

We describe an efficient and scalable procedure for the chemical synthesis of nucleoside 5'-phosphosulfates (NPS) from nucleoside 5'-phosphorimidazolides and sulfate bis(tributylammonium) salt. Using this method we obtained various NPS with yields ranging from 70-90%, including adenosine 5'-phosphosulfate (APS) and 2',3'-cyclic precursor of 3'-phosphoadenosine 5'-phosphosulfate (PAPS), which are the key intermediates in the assimilation and metabolism of sulfur in all living organisms.     

Mitochondrial nucleoside diphosphate kinase: Mode of interaction with the outer mitochondrial membrane and proportion of catalytic activity functionally coupled to oxidative phosphorylation

In the present study, we found that ionic interactions are not essential for the binding of nucleoside diphosphate kinase of liver mitochondria outer compartment to outer mitochondrial membrane and that the proportion of the enzyme activity involved in functional coupling with oxidative phosphorylation (we demonstrated the existence of functional coupling earlier) is only 17%. Additional evidence was obtained that functionally coupled activity of nucleoside diphosphate kinase is associated with the outer surface of mitochondria. Dextran (10%) did not increase functional coupling. The physological importance of these effects is discussed. Published in Russian in Biokhimiya, 2008, Vol. 73, No. 3, pp. 395–407.     

Synthesis and incorporation of methyleneoxy(methylimino) linked thymidine dimer into antisense oligonucleosides

A convenient synthesis of a thymidine (T) nucleoside dimer (T-3′-CH₂-O-NCH₃-5′-T) 12 has been accomplished via a nucleoside coupling reaction. An alternative synthesis of 3′-deoxy-3′-C-hydroxymethylthymidine is described. The new dimer and methodology is useful for the development of backbone-modified antisense oligonucleosides.     

Investigation of a Facile Synthetic Method for Phosphorothioate Dimer Synthons In Oligonucleotide Phosphorothioates Synthesis

Abstract

A facile synthetic method of a phosphorothioate dimer block was investigated. Dinucleoside phosphite triester intermediates were obtained in one-pot synthesis by the coupling of a protected nucleoside bearing free 5′-OH and a protected nucleoside bearing free 3′-OH in the presence of phosphorous trichloride (PCl₃) and 1,2,4-triazole. The intermediates were easily sulfurized to afford the desired phosphorothioate dimer blocks in 33-64% overall yields.     

Hypermodified nucleoside carboxyl group as a target site for specific tRNA modification

The free carboxyl group of hypermodified nucleosides N6-methyl-N6-(threoninocarbonyl)adenosine (mt6A37) and 3-(3-amino-3-carboxypropyl)uridine (acp3U20:1) in tRNAmMet (yellow lupine), and N6-(threoninocarbonyl)adenosine (t6A37) in tRNAiMet (yellow lupine) can be converted quantitatively and under very mild conditions into the respective anilides in a reaction with aniline and a water-soluble carbodiimide. The tRNA reactions proceed with rates very similar to that reported previously for t6A nucleoside. Detailed analysis of the products of tRNA modification with [3H]aniline on tRNA (chromatography on BD-DEAE-cellulose), oligonucleotide (polyacrylamide gel electrophoresis) and nucleoside (HPLC on Aminex A6) levels clearly indicates that only the hypermodified nucleoside residues undergo the reaction. The site of modification is confirmed for mono-modified (at mt6A37) and bis-modified (at mt6A37 and acp3U20:1) tRNAmMet, and for mono-modified (at t6A37) tRNAiMet by sequence analysis using 5'end 32P-labeled tRNAs. The modification procedure seems to be universally applicable for all hypermodified nucleosides bearing a free carboxyl group and for different amine reagents designed for the studies on tRNA function.     

Studies on reactions of nucleoside H-phosphonates with bifunctional reagents. Part VI. Reaction with diols

Reactions of nucleoside H-phosphonates with various diols using different types of condensing agents have been studied. Depending on the coupling procedure and the length of a polymethylene chain of the diol, acyclic H-phosphonate diesters or cyclic phosphite triesters were formed. The course of oxidation with iodine to produce cyclic nucleoside alkyl phosphotriesters or hydroxyalkyl nucleoside phosphodiesters can be controlled by the amount of water present in the reaction medium.