SU1439108A1 - Method of producing carriers for solid-phase synthesis of oligonucleotides - Google Patents

Abstract

The invention relates to the chemistry of polymers and makes it possible to obtain carriers for the solid-phase synthesis of oligonucleotides containing 0.310 - 0.450 mmol of immobilized nucleuside per gram of carrier. Carriers are obtained by sequential processing of silica with 5-16% aqueous solution of D-mannitol, -2% solution of epoxy resin DEG-1 in dioxane and nucleoside, 1 table. (L 4 00; o

Description

one . . one

The invention relates to the chemistry of polymers, namely to a method for producing carriers for the solid phase synthesis of oligonucleotides and can be used in bioorganic chemistry and bio. technology for the synthesis of oligonucleotides in both manual and automatic mode.

The aim of the invention is to increase the amount of immobilizable nucleoside.

Example 1. K1g porous CPG-10 glass (specific surface 17 m / g, pore diameter 2200 A, particle size 120-20 microns) is added with 30 ml of concentrated nitric acid, kept for 2-3 hours, washed with water until neutral pH. Then 10 ml of a 0% aqueous solution of D-mannitol (h, ea) are added, degassed, stirred for 1 h, washed twice on a porous glass filter with 10 ml of water and three times with 10 ml of dioxane. Next, add 20 ml of dioxane, 0.2 1-w O BFg (h), stir for 5 minutes, add 0.2 ml of epoxy resin DEG-i (dynamic viscosity not more than 15-30 cP, mol. Mass 240 - 260, the content of the epoxy group 24%) and vigorously stirred for 40 min at room temperature, then washed with dioxane, water, pyridine. 0.1 mmol of 5 -o-dimethoxytrityl-N-iso-butyrylguanosine-3 -o-succinate (DMTrGiBu), 0.3 mmol of TPSC1, 0.9 gm mol Melm and 0 , 2 ml abs. pyridine, gently intermixed for an hour. After that, the carrier is washed with pyridine, chloroform-1m, acetone and dried.

The nucleoside EjMKOCTb (the amount of immobilized nucleotide) on the carrier is determined spectrophotometrically by dimethoxytrityl carbinol (DMTrOH) after removal from the carrier of the dimethoxytrityl group by treatment with a solution of perchloric acid in ethyl alcohol (3; 2 V / V) at 499 or extinction DMTrOH

71 7.

 mmolsm

The calculation of the content of Nuk teoside on the carrier is carried out according to the formula

WITH

D v

1000 E m. one

082

where C is the content of the nucleoside — the content of the dimethoxytrityl group on the carrier, mmol / g; D total optical density

eluate with DMTrOH; V is the volume of the eluate, ml; E - the extinction coefficient of DMTrOH

at 499 im;

m is the carrier weight, g; 1-1 cm is the optical path length of the cuvette, in which the optical density is measured. Determination of nucleoside content on the resulting carrier. To 0.01 g of the carrier containing the DMTrGiBu group, add 2 ml of a solution of perchloric acid in ethanol, the solution turns orange, hold for 1-2 minutes, filter, repeat the operation, combine the eluates and measure the optical density on a spectrophotometer : D 55.56, V 4 ml, m 0.01, the content of nucleoside C - 0.310 mmol / g. Example 2 To 0.1 g CPG 10 treated with B mannitol and DEG-1, as described in Example I, 0.1 mmol 5 -o-dimethoxytritylthymidine-3 -o-succinate was added. , 0.3 mmol TPSC1, 0.9 N ore Melm, 0.2 ml of abs. Of pyridine, gently stirred for 1 hour. Then washed with pyridine, chloroform, acetone and dried. The content of the nucleoside is determined as in Example 1 „D 80.66, m 0.01 g, V 4 ml, C 0.450 mmol / g with

Example 3, porous glass MPS 2000 (15-20 mg / g, 2000 A, d ,,, 100-300 microns), add 10 ml of a 5% aqueous solution of B-mannitol, degas and mix for 1 hour. Further processing is carried out as in example 1, the content of the nucleoside on the carrier is determined as in example 1, D 54.85, V 4 ml, m 0.01 g, C 0.306 mmol / g.

Example 4. 1g of porous glass MPS 2000 is treated analogously to example 1, but instead of a 10% solution of D-mannitol a 16% solution is taken. The nucleoside content is determined as in Example 1. D 75.46, V 4 ml, m 0.01 g, C 0.421 mmol /

Example 5. 1 g of porous CPG 10 glass is treated as in Example 1, but instead of 0.2 ml of DEG-1, 0.4 ml of epoxy is added

1A

resin. The content of nucleoside on the support is 0.352 mmol / g.

Example 6 (control), 1 g of porous glass MPS 2000 is treated analogously to example 1, but instead of a 10% solution of D-mannitol, a 2% solution is taken. The content of the nucleoside, as determined similarly

Example 1 0.126 mmol / g.

Example 7 (control). 1 g of porous glass CPG 10 is treated analogously to example 1, but instead of 0.2 ml of deg-1 take 0.1 ml of epoxy resin. The content of the nucleoside O, 150 mmol / g.

084

On the carrier obtained in Example 2, the octadecadequoxoxy-bonucleotide d (AAAATASTCCSSATSCCST) was synthesized using the phospho triether method. The average yield on the DMTrOH stage is 84%, and the total yield after chromatographic separation is 1.20% of the theoretical one. On the carrier obtained in Example 2, the oxyderibe d nucleotide d (CCCSSSTAS) is synthesized using the method. The average yield per stage for DMTrOH is 90%, and the total yield after chromatographic separation is 20% of the theoretical one.

Characteristics of the proposed and known carriers are shown in the table.

Example 8. 1 C-20 15-25 mg / g.

g silochrome d,

1300 A, d

 since then

is clean

 315-500 microns) is treated with nitric acid, washed with water to a neutral pH as in Example 1, then washed with dioxane, added with 20 ml of dioxane, 0.2 ml of boron trifluoride etherate, stirred for 5 minutes, added 0.4 ml of deg-1, intensively stirred for 40 min at room temperature, then washed with dioxane, water, pyridine. A nucleoside is added to the thus treated silica (0.1) in the same manner as described in Example 1. The content of the nucleoside is 0.003 mmol / g.

Formulas

the invention

20

five

0

The method of producing carriers for the solid phase synthesis of oligonucleotides, including the processing of silica with a polymer and ntsleoside, characterized in that, in order to increase the amount of nucleoside to be immobilized, the silica is pretreated with an aqueous solution of D-mannitol and 1- before the application of the nucleoside. 2% solution in dioxane zoxy resin based on polyol and epichlorohydrin in the presence of boron trifluoride.

Claims (1)

The claims * 20 A method of producing carriers for solid-phase synthesis of oligonucleotides, comprising treating silica with a polymer and a nucleoside, comprising 25 and, in order to increase the amount of nucleoside immobilized, silica is pre-treated with a 5-16% aqueous solution of 30 D-mannitol and a 1-2% solution in dioxane of an epoxy resin based on a polyol and epichlorohydrin in the presence of boron trifluoride. Example Matrix d por, A Treatment D-mannitol, Z number DEG-1, mg / g carrier The number of immobilized nucleoside, mmol / g 1 CPG 10 2200 17 10 0.2 0.310 according to DMTrGIBu 2 _ n _ „Η _- 0.450 PMGT 3 MPS 2000 1800-2600 15-20 5 0.2 0.306 TIG GiBu 4 _ · "_ _ and _ _- n _ 16 0.2 0.421 - - 5 CPG 10 . 2200 17 10 0.4 0.352 - ⁿ - 6 (control) Mpc 2ooo 1800-2600 15-20 ' 1 θ, 2 0.126 - - 7 (control) 10 2200 17 10 0.1. 0.150 - - * 8 (control) Silochrome •, C 20 MK 1300 15-25 - 0.4 0.003 - - 9 (famous) Kuselqubr 2000-3000 15-20 Peptide Polydimethylamine 0.08 - 0.2 VNIIPI Order 6038/24 ______. Circulation 348 ______ Subscription. Custom polygr. ave, city of Uzhhorod, st. Project, 4