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Phosphoric acid diester and preparation method thereof

A technology of phosphodiester and cyanoborane phosphate, which is applied in the field of phosphodiester and its preparation, can solve the problems that have not yet been achieved at the same time, and achieve the effects of good nuclease hydrolysis and large membrane permeability

Inactive Publication Date: 2010-06-16
石平
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0011] However, antisense oligonucleotides are used as a method for treating diseases, and there are still several problems to be solved: (1) transfection efficiency: whether it is the antisense RNA expressed by the vector or the antisense oligonucleotide synthesized in vitro Only when it enters the target cell, can it bind to the homologous gene and play a regulatory role, and the regulation of antisense RNA has obvious dose-dependence
In fact, there are currently no drugs that meet both requirements

Method used

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  • Phosphoric acid diester and preparation method thereof
  • Phosphoric acid diester and preparation method thereof
  • Phosphoric acid diester and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] DNA cyanoboranyl phosphate analog: double thymidine cyanoboranyl phosphate (Tp BH2CN T or Tp CB T) synthesis:

[0044] Dinucleoside cyanoborane phosphate (Tp BH2CN T or Tp CB The synthetic route of T) is as figure 1 shown.

[0045] a. Nucleoside coupling:

[0046] At room temperature, protected thymidine phosphoramidites ( 1 ) (0.5mmol, purchased from U.S. Chemgenes) reacted with acetylthymidine (0.5mmol) in dimethylformamide DMF (3ml) for 1 hour under the catalysis of tetrazolium (0.5mmol), to obtain phosphorous acid ester( 2 ).

[0047] b, cyanoborane substitution:

[0048] At 68°C, phosphite ( 2 ) reacted with aniline-cyanoborane complex (4mmol) in tetrahydrofuran (12ml) for 2 hours to obtain phosphite-cyanoborane ( 3 ).

[0049] C. Deprotection:

[0050] The compound ( 3 ) After half an hour, the protected dinucleoside cyanoborane phosphate ( 4 ). After removing the solvent with low boiling point by rotary evaporation under reduced pressure, the compo...

Embodiment 2

[0057] RNA cyanoboranyl phosphate analogue: Uridine adenine cyanoboranyl phosphate (Up BH2CN A or Up CB A) Synthesis.

[0058] Uridine adenine cyanoborane phosphate (Up BH2CN A or Up CB A) the synthetic route such as Figure II shown.

[0059] a. Nucleoside coupling:

[0060] At room temperature, the protected uridine phosphoramidite ( 6 ) (0.5mmol, purchased from U.S. Chemgenes Company) reacted with diacetyl adenine nucleoside (0.5mmol) in dimethylformamide DMF (6ml) for 2 hours under the catalysis of tetrazolium (0.5mmol), to obtain Phosphite ( 7 ).

[0061] b, cyanoborane substitution:

[0062] At 68°C, phosphite ( 7 ) reacted with aniline-cyanoborane (4mmol) in tetrahydrofuran (16ml) for 3 hours to obtain phosphite-cyanoborane ( 8 ).

[0063] c. Deprotection:

[0064] Phosphite-cyanoborane ( 8 ) After 1 hour, the protected dinucleoside cyanoborane phosphate ( 9 ). After removing the low-boiling solvent by rotary evaporation under reduced pressure, the protec...

Embodiment 3

[0071] Basic properties of cyanoborane phosphates.

[0072] 1. Acid-base hydrolysis stability.

[0073] Cyanoborane phosphate is very stable in acid-base hydrolysis. At 37° C., under the condition of pH=3 or 11, 100 mM acetic acid / ammonia water was used to treat double thymidine cyanoborane phosphate ( 5 ) for 24 hours, no hydrolysis or degradation products were detected by high performance liquid chromatography. High performance liquid chromatography conditions: 80% 100 mM triethylammonium acetate (Triethylammonium Acetate, TEAA) and 20% acetonitrile as the eluent; the elution flow rate is 1 ml / min.

[0074] 2. Resistance to nuclease hydrolysis.

[0075] The cyanoborane phosphate linkage is very stable to hydrolysis by snake venom phosphodiesterase (SVPDE) and bovine spleen phosphodiesterase (BSPDE). The process of resistance to nuclease hydrolysis was monitored by high performance liquid chromatography. High performance liquid chromatography conditions: the eluent is 80...

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Abstract

The invention discloses a phosphoric acid diester and a preparation method thereof, wherein the phosphoric acid diester is cyanogroup boron hydride organic phosphate. The preparation method comprises the following processes: a. nucleoside coupling, b. cyanogroup boron hydride replacement, c. deprotection, d. purification. Compared with the prior art, the invention provides a brand-new phosphoric acid diester- cyanogroup boron hydride organic phosphate, wherein one non- bridging oxygen atom of the phosphoric acid diester group is replaced by cyanogroup boron hydride. Compared with natural DNA, the phosphoric acid diester can stably and well resist nuclease hydrolysis in a wide rang of pH value; meanwhile, based on capryl alcohol partition experiment (namely lipotropism measurement), cyanogroup boron hydride organic phosphate can demonstrate greater membrane permeability than conventional oligonucleotides.

Description

technical field [0001] The invention belongs to phosphodiester and its preparation method, in particular to phosphodiester used in the field of antisense nucleic acid and gene probe and its preparation method. Background technique [0002] Nucleotides are compounds connected by bases (mainly derivatives of purine and pyrimidine bases), pentose sugars (ribose or deoxyribose) and phosphates. Oligonucleotides are a class of compounds with only about 20 base pairs. The general term for short chains of nucleotides (nucleotides including deoxyribonucleic acid DNA or ribonucleic acid RNA), oligonucleotides can be easily linked to their complementary pairs, so they are often used as probes to determine DNA or RNA The structure is often used in gene chip, electrophoresis, fluorescence in situ hybridization and other processes. [0003] In recent years, the research on the application of oligonucleotides, especially antisense oligonucleotides in the treatment of various diseases has ...

Claims

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Application Information

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IPC IPC(8): C07H23/00C07H1/02A61K48/00A61P35/00
Inventor 石平
Owner 石平
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