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Nucleotide analogues

A technology for deoxynucleoside triphosphates and compounds, which is applied in the field of nucleotide analogs, and can solve the problems of no disclosure of synthetic methods, no disclosure of deprotection conditions and enzymatic binding data, etc.

Active Publication Date: 2019-03-15
奇根科学有限责任公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For the nitrate group, neither the synthetic method nor the deprotection conditions and enzymatic conjugation data are disclosed

Method used

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Examples

Experimental program
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Effect test

Embodiment 1

[0288] Synthesis of 3'-O-(methylthiomethyl)-5'-O-(tert-butyldimethylsilyl)-2'-deoxythymidine (2)

[0289] In a 250 mL round bottom flask, 5'-O-(tert-butyldimethylsilyl)-2'-deoxythymidine (1) (2.0 g, 5.6 mmol) was dissolved in DMSO (10.5 mL), acetic acid (4.8 mL) and acetic anhydride (15.4 mL) were stirred at room temperature for 48 hours. Then saturate by adding K 2 CO 3 solution quenches the mixture until gaseous CO 2 Release stops. The mixture was then extracted with EtOAc (3X100 mL) using a separatory funnel. Then with saturated NaHCO 3 solution (2X150mL), the combined organic extracts should be washed with a distribution funnel, washed with Na 2 SO 4 Dry the organic layer. The organic portion was concentrated by rotary evaporation. The reaction mixture was finally purified by silica gel column chromatography (Hex:EtOAc / 7:3-1:1), see Figure 8. 3'-O-(methylthiomethyl)-5'-O-(tert-butyldimethylsilyl)-2'-deoxythymidine (2) was obtained as white powder in 75% yield (...

Embodiment 2

[0291] Synthesis of 3'-O-(ethyldithiomethyl)-2'-deoxythymidine (4)

[0292] Dissolve in 20 mL of anhydrous CH 2 Cl 2 Add Et to compound 2 (1.75g, 4.08mmol) in 3 N (0.54 mL, 3.87 mmol) and 5.0 g molecular sieve-3A were stirred under Ar atmosphere for 30 minutes. The reaction flask was then placed on an ice bath to bring the temperature below zero and 1.8eq 1M SO was slowly added 2 Cl 2 CH 2 Cl 2 The solution (1.8 mL) was stirred at the same temperature for 1.0 hr. The ice bath was then removed to allow the flask to come to room temperature and a solution of potassium thiotosylate (1.5 g) in 4 mL of anhydrous DMF was added and stirred at room temperature for 0.5 hours.

[0293] Then 2eq EtSH (0.6mL) was added and stirred for another 40 minutes. Then with 50mL CH 2 Cl 2 The mixture was diluted and filtered through Celite-S in a funnel. with sufficient amount of CH 2 Cl 2 Samples were washed to ensure product was filtered out. Then concentrate CH 2 Cl 2 The extract...

Embodiment 3

[0296] Synthesis of 3'-O-(ethyldithiomethyl)-2'-deoxythymidine triphosphate (5)

[0297] To compound 4 (0.268 g, 0.769 mmol) was added proton sponge (210 mg) equipped with a rubber stopper in a 25 mL flask. The samples were dried overnight under high vacuum. This material was dissolved in 2.6 mL (MeO) under argon atmosphere 3 PO. Place the flask equipped with an Ar-gas supply on an ice bath and stir until the temperature reaches subzero. Then immediately add 1.5 equivalents of POCl with a syringe 3 And stirred at the same temperature under an argon atmosphere for 2 hours. The ice bath was then removed to prepare a mixture of tributylammonium-pyrophosphate (1.6 g) and Bu 3 A mixture of N (1.45 mL) in anhydrous DMF (6 mL). The whole mixture was added at once and stirred for 10 minutes. The reaction mixture was then diluted with TEAB buffer (30 mL, 100 mM) and stirred at room temperature for an additional 3 hours. The crude product was concentrated by rotary evaporation a...

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Abstract

The present invention provides methods, compositions, mixtures and kits utilizing deoxynucleoside triphosphates comprising a 3'-O position capped by a group comprising methylenedisulfide as a cleavable protecting group and a detectable label reversibly connected to the nucleobase of said deoxynucleoside. Such compounds provide new possibilities for future sequencing technologies, including but notlimited to Sequencing by Synthesis.

Description

[0001] Cross References to Related Applications [0002] This application claims the benefit of U.S. Provisional Patent Application No. 62 / 251,884, filed November 6, 2015, and U.S. Provisional Patent Application No. 62 / 327,555, filed April 26, 2016, which are hereby incorporated by reference. field of invention [0003] The present invention provides methods, compositions, mixtures and kits utilizing deoxynucleoside triphosphates comprising a 3' capped with a group comprising methylene disulfide as a cleavable protecting group. -O position and a detectable label reversibly attached to the nucleobase of said deoxynucleoside. Such compounds provide new possibilities for future sequencing technologies, including but not limited to, sequencing by synthesis. Background of the invention [0004] DNA sequencing is one of the most important analytical methods in modern biotechnology. A detailed review of current sequencing technologies is provided in M.L. Metzker, Nature Reviews 2...

Claims

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Application Information

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IPC IPC(8): C07H19/14C07H19/10C07F7/18C07C323/12C07D207/46C12Q1/68
CPCC07C323/12C09B11/24C09B23/083C07C2603/18C07F7/1804C07H19/10C07H19/14C07D207/46C07C323/60C07D207/40C09B23/16C12P19/34C12Q1/6869Y02P20/55C09B1/00C09B5/2436C09B23/166C12Q1/6876C12Q1/6811
Inventor M·S·玛尔玛J·欧乐尼克I·科尔伯克
Owner 奇根科学有限责任公司
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