H-phosphonate Approach Research Articles

Convergent synthesis of phosphorodiamidate morpholino oligonucleotides (PMOs) by the H-phosphonate approach

Phosphorodiamidate morpholino oligonucleotides (PMOs) are a promising type of antisense oligonucleotides, but their challenging synthesis makes them difficult to access. This research presents an efficient synthetic approach for PMOs using the H-phosphonate approach. The use of phosphonium-type condensing reagents significantly reduced coupling times compared with the current synthetic approach. Furthermore, phosphonium-type condensing reagents facilitated the fragment condensation of PMO, synthesizing up to 8-mer containing all four nucleobases with remarkable coupling efficacy. This is the first report on the convergent synthesis of PMOs. This approach would facilitate the large-scale synthesis of PMOs and accelerate their popularity and accessibility as a next-generation therapy.

Photo-induced chemistry for the design of oligonucleotide conjugates and surfaces.

A photocaged diene is introduced at the 5'-end of oligonucleotides using the H-phosphonate approach. The photoenol-functionalized DNA is subsequently employed for the conjugation to a protein and the spatially controlled immobilization onto surfaces using a light-induced Diels-Alder cycloaddition. Fully functional protein-DNA conjugates and patterned DNA surfaces are obtained under mild irradiation conditions.

Phospholipid derivatives of cladribine and fludarabine: Synthesis and biological properties

Phospholipid derivatives of anticancer nucleosides cladribine and fludarabine (F-ara-A) bearing 1,2- and 1,3-diacylglycerol moieties have been prepared by the H-phosphonate approach using 1,1,3,3-tetraisopropyldisiloxane-1,3-diyl protecting group for cladribine and a combination of tert-butyldimethylsilyl and levulinyl protecting groups for 2-fluoroadenine nucleosides. The synthesized conjugates exhibited lower in vitro antiproliferative activity against human tumor cell lines in comparison with the same concentrations of the parent cladribine and fludarabine phosphate. In the course of biokinetic study, it was found that intragastric administration of phospholipid F-ara-A derivatives to Wistar rats and ICR outbred male mice led to a slow release of F-ara-A into the bloodstream, a smooth increase in nucleoside concentration, and prolonged serum circulation of liberated nucleoside. The oral bioavailability of F-ara-A from 1,2-dimyristoylglycerophosphate derivative 29 was similar to its oral bioavailability from fludarabine phosphate.

Synthesis and hydrolytic properties of thymidine boranomonophosphate

Detailed synthetic procedures of thymidine 5′-O-(P-borano)monophosphate (TMPB) via an H-phosphonate approach are reported. The hydrolytic properties of TMPB in pH 1.8 and 6.76 buffers at 22 or 37°C were studied using LC–MS. One nucleoside product, thymidine, was observed, supporting cleavage at the P–O ester bond to release nucleoside and boranophosphate (O3P-BH3−3). Higher temperatures and acidic conditions increase the hydrolysis rates.

A BH3 masked H-phosphonate for coupling in oligonucleotide synthesis

A method of masking 3′-H-phosphonate group for the solution-phase synthesis of ribonucleotide by H-phosphonate approach was described. The phosphonic acid group was masked by bis-(2-cyanoethyl) boranophosphate during the reactions. After a successive demasking treatment by triethylamine, trityl cation, and triethylamine, the triethylammonium 3′-H-phosphonate nucleotide can be obtained efficiently ready for the coupling cycle in synthesis of oligonucleotide.

Synthesis of Lipid A and Inner Core LPS ligands containing 4-amino-4-deoxy-l-arabinose units.

Attachment of 4-amino-4-deoxy-l-arabinose to phosphates or sugar hydroxyl groups of lipopolysaccharide contributes to bacterial resistance against common antibiotics. For a detailed study of antigenic properties and binding interactions, Ara4N-containing inner core ligands related to Burkholderia and Proteus LPS have been synthesized in good yields. Glycosylation at position 8 of allyl glycosides of oct-2-ulosonic acids (Ko, Kdo) has been accomplished using an N-phenyltrifluoroacetimidate 4-azido-4-deoxy-l-arabinosyl glycosyl donor followed by azide reduction and global deprotection. The β-l-Ara4N-(1→8)-α-Kdo disaccharide was further extended into the branched β-l-Ara4N-(1→8)[α-Kdo-(2→4)]-α-Kdo trisaccharide via a regioselective glycosylation of a protected triol intermediate. Synthesis of Ara4N-modified lipid A - part structure occurring in the LPS of Burkholderia, Pseudomonas and Klebsiellla strains was accomplished using the H-phosphonate approach. The stereocontrolled assembly of the phosphodiester linkage connecting glycosidic centres of two aminosugars was elaborated employing an anomeric H-phosphonate of cyclic silyl-ether protected 4-azido-4-deoxy-β-l-arabinose which was coupled to the hemiacetal of the lipid A GlcN-disaccharide backbone. Conditions for global deprotection which warrant the integrity of "double anomeric" phosphodiester linkage were successfully developed. Introduction of thiol-terminated spacer at the synthetic ligands allows both coupling to BSA and immobilization on gold nanoparticles as well as generation of glycoarrays.

Synthesis of 9-Fluorenemethyl Boranophosphonodiphosphate Via an H-Phosphonate Approach

9-Fluorenemethyl boranophosphonate 6 and its boranophosphonodiphosphate 7 were synthesized via an H-phosphonate approach. The method is efficient for the synthesis of acyclic compounds 6 & 7 , and can be explored for the synthesis of nucleoside 5′-deoxy boranophosphonodiphosphate.

Synthesis of 3’,5’-cyclic diguanylic acid (cdiGMP) Using 1-(4-chlorophenyl)-4-ethoxypiperidin-4-yl as a Protecting Group for 2’-hydroxy Functions of Ribonucleosides

We herein report a convenient synthesis of 3′,5′-cyclic diguanylic acid via the modified H-phosphonate approach. The 1-(4-chlorophenyl)-4-ethoxypiperidin-4-yl (Cpep) group was used as protecting group for the 2′-hydroxy functions of ribonucleosides. Complete unblocking of the fully protected 3′,5′-cyclic diguanylic acid gave cdiGMP as a homogeneous compound in an excellent yield.

Synthesis of mono- and di-sialophospholipids via the H-phosphonate approach

To overcome their inherent instability, stable modified mono- and di-sialophospholipids of the group C meningococcal polysaccharide were synthesized. Stability was achieved by introducing a spacer between the sialic acid residue and the phospholipid component, and by replacing the native ester linkages to the lipid by ether linkages. Mono- and di-hydroxylethylenesialosides were coupled to phosphoglyceroldietherlipid using H-phosphonate chemistry to give the anomerically pure sialophospholipids in good yields.Key words: polysialic acid, glycolipid, H-phosphonate, meningococcus.

MODEL SYNTHESIS OF NUCLEOSIDE BORANOPHOSPHORAMIDATE WITH AMINO ACID FOR PRODRUG PURPOSE

A model synthesis of a nucleoside boranophosphoramidate prodrug with (L)-tryptophan methyl ester was accomplished in a one-pot reaction via an H-phosphonate approach. This new type of compound is expected to possess the potent antiviral and anticancer advantages conferred by boranophosphates and normal nucleoside amino acid phosphoramidate.

Synthesis of Nucleoside Boranophosphoramidate Prodrugs Conjugated with Amino Acids

[structure: see text] Nucleoside boranophosphates and nucleoside amino acid phosphoramidates have been shown to be potent antiviral and anticancer agents with the potential to act as nucleoside prodrugs. A combination of these two types of compounds results in a boranophosphoramidate linkage between the nucleoside and amino acid. This new class of potential prodrugs is expected to possess advantages conferred by both types of parent compounds. Two approaches, specifically the H-phosphonate and oxathiaphospholane approaches, are described here to synthesize nucleoside boranophosphoramidate prodrugs conjugated with amino acids. The H-phosphonate approach involves a key intermediate, silylated nucleoside amino acid phosphoramidite 6, prepared from a series of reactions starting from nucleoside H-phosphonate in the presence of condensing reagent DPCP. Due to the lengthy procedure and the difficulties in removing DPCP from the final products, we switched to the oxathiaphospholane approach in which the DBU-assisted oxathiaphospholane ring-opening process constituted a key step for the generation of nucleoside amino acid boranophosphoramidates 24. We demonstrate that this key step did not cause any measurable C-racemization of boranophosphorylated amino acids 22. Diastereomers of compounds 24a-f were separated by RP-HPLC. An "adjacent"-type mechanism is proposed to explain the diastereomer ratio in the final products obtained via the oxathiaphospholane approach. A tentative assignment of configuration for the diastereomers was carried out based on the mechanism, molecular modeling, and (1)H NMR. Conclusively, the oxathiaphospholane methodology proved to be more facile and efficient than H-phosphonate chemistry in the preparation of the nucleoside amino acid boranophosphoramidate analogues that are promising as a new type of antiviral prodrugs.

SYNTHESIS AND REACTIVITY OF NUCLEOSIDE SULFILIMINES

Treatment of 3′- and 5′-protected deoxycytidine and deoxyadenosine with bis-[α,α-bis(trifluoromethyl)benzyloxy]diphenylsulfur resulted in the formation of the corresponding 4-N- and 6-N-diphenylsulfilimine derivatives in good yields. The 4-N- and the 6-N-dimethylsulfilimines of these nucleosides were obtained using DMSO/trifluoroacetic anhydride. These dimethyl- and diphenylsulfilimine derivatives were found to have characteristic UV absorbances at 284-296 nm. Stability tests of the nucleoside dimethylsulfilimine and diphenylsulfilimine under the conditions commonly used in oligonuclotide chemistry by the H-phosphonate approach were executed, and possible applications of these compounds have been discussed.

Solution Phase Synthesis of ISIS 2922 (Vitravene) by the Modified H‐Phosphonate Approach.

The synthesis of Vitravene, a 21-mer oligonucleotide phosphorothioate (d[Gp(s)Cp(s)Gp(s)Tp(s)Tp(s)Tp(s)Gp(s)Cp(s)Tp(s)Cp(s)Tp(s)Tp(s)Cp(s)Tp(s)Tp(s)Cp(s)Tp(s)Tp(s)Gp(s)Cp(s)G]) by the modified H-phosphonate approach in solution is reported. The synthetic strategy adopted involves the use of only four different trimer building blocks. The dimer and trimer blocks are prepared by a novel four component procedure in which the coupling agent (diphenyl phosphorochloridate 5b) and the sulfur-transfer reagent (N-[(2-cyanoethylsulfanyl)succinimide] 19) are added at the same time. Yields both of the building blocks and in the block coupling reactions are very satisfactory. The fully-protected 21-mer 38 is unblocked by a five-step procedure to give Vitravene, which is isolated as its sodium salt.

Side Reactions in the H-Phosphonate Approach to Oligonucleotide Synthesis: A Kinetic Investigation on Bisacylphosphite Formation and 5′-O-Acylation

A kinetic study on the reactions of pivaloyl chloride with a nucleoside H-phosphonate (bisacylphosphite formation), or a protected nucleoside with a free 5′-OH (5′-O-pivaloylation) has been carried out in the presence of differently substituted pyridines. The bisacylphosphite formation is a result of acylation of the mixed carboxylic-phosphonic acid anhydride to give the corresponding bispivaloylphosphite derivative. The rate of this reaction is dependent on the concentration of pyridine derivative and pivaloyl chloride. The 5′-O-pivaloylation reaction is also dependent on the concentration of pyridine derivative and pivaloyl chloride. The rate of both reactions is dependent on the basicity of the pyridine derivative, displaying a higher rate with more basic pyridines. The dependence of log k vs. pKa of pyridine is linear but the slopes are quite different for the two reactions (0.84 for bisacylation and 0.21 for 5′-O-pivaloylation). For both reactions considerably lower rates in the presence of sterically hindered pyridines suggest the existence of nucleophilic catalysis on pivaloyl chloride in reactions with non-hindered pyridines.

An efficient oxidizing reagent for the synthesis of mixed backbone oligonucleotides via the h-Phosphonate approach

The mixture of carbon tetrachloride, N-methyl morpholine (NMM), pyridine and water in acetonitrile has been exploited for the oxidation of dinucleoside H-phosphonate diesters to the corresponding phosphates. The system is found to be inert to the phosphoramidate (P-N) and the phosphorothioate (P-S) linkages and has successfully been applied to the solid phase synthesis of mixed-backbone oligonucleotides (MBOs).

Solution phase synthesis of ISIS 2922 (Vitravene) by the modified H-phosphonate approach

The synthesis of Vitravene, a 21-mer oligonucleotide phosphorothioate (d[Gp(s)Cp(s)Gp(s)Tp(s)Tp(s)Tp(s)Gp(s)Cp(s)Tp(s)Cp(s)Tp(s)Tp(s)Cp(s)Tp(s)Tp(s)Cp(s)Tp(s)Tp(s)Gp(s)Cp(s)G]) by the modified H-phosphonate approach in solution is reported. The synthetic strategy adopted involves the use of only four different trimer building blocks. The dimer and trimer blocks are prepared by a novel four component procedure in which the coupling agent (diphenyl phosphorochloridate 5b) and the sulfur-transfer reagent (N-[(2-cyanoethylsulfanyl)succinimide] 19) are added at the same time. Yields both of the building blocks and in the block coupling reactions are very satisfactory. The fully-protected 21-mer 38 is unblocked by a five-step procedure to give Vitravene, which is isolated as its sodium salt.

The H-phosphonate approach to oligonucleotide synthesis. An investigation on the mechanism of the coupling step

A kinetic study on the pivaloyl chloride promoted H-phosphonate condensation step in the presence of differently substituted pyridines has been carried out. The kinetics of the system follow second order kinetics with a 1 ∶ 1 ratio of nucleoside 3′-H-phosphonate and nucleoside component (with free 5-OH). Irrespective of the pyridine derivative used, the reaction rate is dependent on the concentration of this as well as of the hydroxy component and H-phosphonate. A reactive intermediate is indirectly identified from the kinetic evidence. This intermediate is suggested to be a pyridinium adduct formed by attack of the pyridine derivative on the initially formed mixed phosphonic carboxylic anhydride. Considerably lower rates of condensation in the presence of sterically hindered pyridines further support the existence of nucleophilic catalysis in the reactions with non-hindered pyridines. In addition, the rate of reaction in the presence of pyridines with pKa values above ∼4.8 is enhanced by an increase in pivaloyl chloride concentration. The main reason for this enhancement is most likely the fact that pivaloyl chloride removes pivalate ion, which retards the reaction by influencing the equilibrium between the mixed anhydride and the pyridinium intermediate. Although the observed rate constants are composed of several constants, their temperature dependence gives some indication of the nature of the transition-state of the rate-limiting step. Entropies of activation are estimated to be slightly positive, suggesting a transition state arising from attack of the hydroxy component on the pyridinium intermediate but involving a fair degree of bond breakage to the leaving group, i.e. the pyridine.

Large scale, liquid phase oligonucleotide synthesis by alkyl H-phosphonate approach

A new approach to the liquid phase synthesis of oligonucleotide is described, it is based on oxidative coupling using alkyl H-phosphonate synthon and polyethylene glycol (PEG5000) as a soluble support. Nucleoside alkyl H-phosphonate undergoes oxidative coupling in presence of NBS. The use of polyethylene glycol as a soluble polymeric support preserves some convenient features of the solid phase synthesis with new interesting advantages. This liquid phase method appears effective in terms of speed and coupling yield and can be evaluated for the production of large amount of oligonucleotide (100μM).

Synthesis of diuridine 3′,5′-boranophosphate: H-phosphonate approach

Diuriine 3′,5′-boranophosphate, the RNA analogue of boranophosphate nucleic acids, was synthesized by a new approach via the H-phosphonate. Two diastereomers of diuridine 3′,5′-boranophosphate were separated by reverse phase HPLC.

A New H-Phosphonate Approach Using N-Unprotected Monomers and Phosphonium Condensing Reagents

Oligodexyribonucleotides were synthesized by using N-unprotected H-phosphonate monomers and a phosphonium-type of new condensing reagent. In the present H-phosphonate approach, N-sulfonyloxaziridine derivatives were successfully employed as new reagents for oxidation of the H-phosphonate linkages in the presence of silylating reagents.