Boranophosphates

In this context, it is interesting to note that the first synthesis of 2, 3 -0,0-cyclic phosphorothioate 22a was reported by Eckstein in 1968 [25], He also isolated pure Rp diastereomer by fractional crystallization of the triethylammonium salts [26] and used it as reference to determine the absolute configurations of the other phosphorothioate analogues [27], 2, 3 -0,0-Cyclic H-phosphonate 20a was used as a key substrate for the synthesis of uridine 2, 3 -0,0-cyclic boranophosphate 27. Silylation of H-phosphate 20a gave the phosphite triester 25 (two diastereomers). Its boronation, with simultaneous removal of the trimethylsilyl group, was achieved by its reaction with borane-A.A-diisopropylethylamine complex (DIPEA-BH3). 

The formed 5 -protected 2, 3 -(9,(9-cyclic boranophosphate 26 was finally deprotected by acid treatment and isolated in 70% overall yield by ion-exchange chromatography (Scheme 10) [24], The two pure P-diasteromers of 27 were separated by HPLC. 

Scheme 10 Synthesis of 5 -protected 2 ,3 -0,0-cyclic boranophosphate 26 and its deprotection...

Isosteric analogues of glycosyl phosphates phosphorothioates, boranophosphates, phosphoroamidates... 

According to the latter approach, reducing sugars 128 were reacted with triethylammonium dimethyl boranophosphate in the presence of bis(2-oxo-3-oxazolidinyl)phosphinic chloride as condensing reagent, 3-nitro- 1,2,4-triazole as nucleophilic catalyst and A,A-diisopropyl-A-ethyl-amine to provide boranophosphate triester derivatives 129 as anomeric... 

Shaw has reviewed the various approaches to the synthesis of organophos-phate-oligonucleosides and has further commented on their chemical and biophysical properties along with their interactions with various enzymes such as DNA-polymerases, and compared them to other members of the family of phosphorus modified nucleic acids. She also reported the synthesis of P-tyrosinyl(P-0)-5 -P-nucleosidyl boranophosphates (23a,b), as antiviral and anticancer prodrug candidates. The P-boranophosphates were prepared by reacting a phosphoramidite intermediate obtained from protected tyrosine and the protected nucleoside in the presence of IH-tetrazole, followed by in situ borona-tion of the phosphite triester intermediate. The two diastereomers were then separated by reverse-phase HPLC. ... 

The first synthesis of a dinucleotide analogue combining phosphorothioate and boranophosphate features has been reported. To prepare dithymidine boranophosphorothioate (71), Fmoc-protected thymidine was phosphitylated by bis(diisopropylamino)-chlorophosphine in the presence of DMAP. The resulting phosphoramidite was treated in situ with a 3 -protected thymidine and tetrazole and was then converted to a phosphite triester with nitrophenol. The phosphite triester was subsequently treated with BH3-SMe2 followed by LiS2 in the presence of 18-crown-6 to yield (71). This novel type of dinucleoside... 

Phosphorothioate and boranophosphate linkages introduced into DNA or RNA using nucleoside 5 -[a-thio]triphosphates and nucleoside 5 -[a-borano]tri-phosphates are more resistant to exo- and endonucleases than normal 3, 5 phosphodiester linkages. Combination of the two modifications yielded a novel non-bridging-disubstituted chiral a-triphosphate nucleoside, namely the thymidine 5 -[a-P-borano, a-P-thio] triphosphate (119). The synthesis was the modification of a procedure developed for the synthesis of nucleoside triphosphates and previously reported by the group. 

Hall AH, Wan J, Shaughnessy EE, Ramsay SB, Alexander KA (2004) RNA interference using boranophosphate siRNAs structure-activity relationships. Nucleic Acids Res 32(20) 5991-6000... 

The 2 -deoxynucleoside 5 -a-[P-borano]-triphosphates 118 have been used for PCR-based DNA sequencing.The method relies on the resistance of borano-phosphate linkages to nucleases, thus the positions of the boranophosphates can be revealed by exonuclease digestion, thereby generating a set of fragments that defines the DNA sequence. An abstract has also described the use of fluorescently labelled 2 -deoxynucleoside 5 -a-[P-borano]-triphosphates for DNA sequencing. 

Figure 1.2 Modified nucleotides and nucleosides described in the text. (A) Nucleotides incorporated by polymerases yielding nuclease-resistant oligonucleotides. Top from left to right 2 -fluoro, 2 -amino, 2 -0-methyl. Bottom from left to right boranophosphate, phosphorothioate, 4 -thio. (B) Photosensitive residues. Left to right 5-bromo-U, 2-(2-nitrophenyl)ethyl T, 2-(2-nitrophenyl)propyl T. (C) Amino-imino equilibrium used in 2D-SELEX (see the text).

A previously-reported bicyclic 3 -aminonucleoside has now been converted to the dinucleotide building block 225, which caused a reduction in values when incorporated into oligonucleotides. There has been an extended and expanded account of the preparation of dideoxynucleoside boranophosphates through the intermediacy of H-phosphonate diesters. The conversion of these to the boranophosphates proceeds with retention of configuration. ... 

Boranophosphate linkages have been of interest because they mimic natural DNA, and may have potential in diagnostic and therapeutic applications (boron neutron capture therapy). Boranophosphate-modified siRNA has been shown to possess potent gene-silencing properties compared to the unmodified siRNA. Two novel methods have been reported for the synthesis of boranophosphate oligonucleotides, one using phosphoramidite chemistry, the other boranophosphotriester chemistry. Dinucleoside boranophosphates and a-borano nucleoside triphosphates have also been described. " ... 

The glycosyl boranophosphate triester (20) has been synthesized via bor-anophosphorylation of reducing sugars and used as a versatile, chemically stable precursor of the glycosyl phosphate derivative (21) (Scheme 5). ... 

Stable hybrids with DNA and may enter cells through passive transport mechanisms. Finally, and potentially important for drug delivery, we have shown that the boranophosphates are nuclease resistant (12), like the phosphorothioates and methyIphosphonates. Short boranophosphate oligomers exhibit a high degree of stability to spleen phosphodiesterase and snake venom phosphodiesterase, indicating that they could have long half-lives in the cell. 

The synthetic methods used to prepare short boranophosphate oligo-deoxynucleotides are reviewed below. Approaches to synthesizing longer boron-containing oligomers by solid-phase methods are discussed. 

Tetrazole (0.50 g) is dissolved in freshly dried CH3CN (15 mL) under argon. To this solution, 5 -0-DMT-thymidine phosphoramidite (1.00 g, 95% pure) dissolved in CH3CN (10 mL) is added by a syringe. The amidite vial is rinsed with another 3-4 mL of CH3CN. 3 -Acetyl-thymidine (0.40 g) is added to the reaction mixture, and the mixture is stirred at room temperature. After 15 min, dimethylsulfide borane (470 iL, 3.3 Eq) is added, and the mixture is stirred for 3-4 min. A small portion of the reaction mixture is taken in CDCI3 for P-NMR, which shows complete disappearance of phosphite resonances and, after a large number of accumulations, the appearance of a broad peak at 118 ppm for boranophosphate. After 4 h, solvent is removed from... 

Dimer 1 (40.7 mg, 0.068 mmol) is taken in cone, ammonium hydroxide (10 mL) in a sealed tube. The mixture is shaken overnight at room temperature. The tube is cooled in ice and opened to atmosphere. After allowing the ammonia to escape, the solution is lyophilized to give a white solid. Yield of crude product 42.0 mg. H-NMR of crude product shows product, ammonium acetate, and a small amount of unidentified impurity. A fraction of crude product is purified on reverse-phase HPLC by a stepwise gradient of 0-10% B in 5 min and 10-35% B in 25 min at a flow rate of 9.2 mL/min. Yield 38.6%. iR-NMR (D2O) see Fig. 3 >B-NMR 5 = -40.5 ppm, m, Jb.h and Jb,p are not measured. P-NMR 8 = 93.8 ppm, br. q. peak. FAB mass spectral data m/e 545 (M + 2H) where M is dithymidyl boranophosphate anion. 

The boranophosphate, 2, and the boranophosphate methyl ester, 1, were the two types of boronated TpT dimers prepared. The methyl ester was prepared by reacting 5 -DMT-thymidine phosphoramidite with 3 -acetylthymidine in the presence of tetrazole, which results in the formation of an intermediate phosphite (15). The phosphite is then converted to the dithymidyl boranophosphate methyl ester, 1, by reaction with 3.3 Eq of dimethyl sulfide-borane (Scheme 1). Both reactions can be easily followed with P-NMR. In the first step, the amidite peaks at 148.7 and 148.4 ppm disappear within the time required for recording the spectrum, and are replaced with sharp phosphite peaks at 140.4 and 139.8 ppm. In the second reaction, the phosphite peaks dis-... 

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