DD273064A1 - PROCESS FOR THE PREPARATION OF OLIGONUCLEOTIDES - Google Patents

Abstract

The invention relates to a process for the preparation of oligonucleotides, deoxy and ribonucleotides according to the Phosphotriestermethode, wherein the condensation step for internucleotidic phosphate binding is made by means of novel specific reagents. The invention relates to a process for the synthesis of oligonucleotides by the phosphotriester method, in which chlorides or azolides of the thiols, in particular boron sulfo-1-oxy-6-nitrobenzo-1,2,3-triazole, are used as condensation reagents for the preparation of the internucleotidic phosphate bond.

Description

Furthermore, according to the invention it was found that comparably good results are obtained if, instead of the combination of sulphurous sulphonyl chloride / 1-methylurididol, 1-oxy-6-nitrobenzo-1,2,3-triazolide of the thernesulphonic acid is used as condensing agent (Formula 3) ). The 1-hydroxy-6-nitrobenzo-1,2,3-triazole required for this purpose is distinguished from the other azoles used in the nucleotide synthesis, such as 1,2,4-triazole, 3-nitro-1,2,4-triazole, Tetrazole and tlucrmethylierten and higher nitrated 1-hydroxy-benzo-1,2,3-ti iazolen characterized by the fact that it provides a highly active specific condensing agent especially in combination with Durensulfonsäuren, the Zudsm has a good durability. Another advantage of the Durensulfo-1 -oxy-6-nitro-benzo-i, 2,3-triazoles is that its azole component can be conveniently and safely prepared from 2,4-dinitrochlorobenzene, unlike most other azoles used.

Ausfuhrungsbelspfele Abbreviations used: Bz = Ben.ioyl

Mb = 4-methoxybenzyl Cp = 2-chlorophenyl Dmtr = dimethoxytrityl Tea ⁺ = triethylammonium Ce = cyanoethyl

Starting materials:

- Durensulfochlorid

Mp: 97 ^° C (Lit .: 98-99 ⁰ C)

- 1-hydroxy-6-nitro-benzo-i, 2,3-triazole m.p .: 206 ⁰ C (dec.) (Lit ,: 206 ⁰ C [dec.])

- Durensulfo-i-oxy-e-nitro-benzo-I ^ .S-triazole m.p .: 158 ⁰ C (dec.)

C ₁₆ H ₁₈ N ₄ O ₅ S (376.39)

Bar .: C: 51.05% H: 4.28% N: 14.89% Found: C: 51.07% H: 4.22% N: 14.97%

- Durensulfonyltetrazol m.p .: 91 ⁰ C (dec.) C ₁₁ H ₁₄ N ₄ O ₂ S (266.32)

F: C: 49.62% H: 5.39% N: 20.83%

- nucleotide derivatives

nucleotide derivative

( ⁰ C) R _f ^a '

(ppm) elemental analysis (#)

Ber .: Gef .:

, 3s -OKb (1)

DmtrO-O-PO "Tea ⁺ NI. OCp

95-96

0.10 -6.2 C: 64.17 H: 5.76 N: 7.74

64.06 5.91 7.65

DmtrO

, Bz -OMb (2)

0 -OP-0 Tea ^H

102-104 0.27 -5.7

OCp C: 64.49 H: 5.58 N: 5.28

64.52 5.72 5.37

DmtrO

-OLIb 0 -O-P-0

Il

OCp

(3)

118-120 0.06 -8.3 C: 62.66 63.03 H: 5.99 6.19 N: 4.38 4.50

KO

, Bz

-OBs (4) 140-142 0.19

-OBz C: 62.52 62.16 H: 4.20 4.31 IJ: 11.76 11.47

a: CH ₂ Cl ₂ / MeOH (95: 5, v / v) on silica gel 60 F _2M (Merck) b: measured in CHCl against H ₁ PO ₄ (85%) as external standard

example 1

From the Rausteinen (1), (2) and (4), the protected oligonucleotide (5) is built according to the stated principles of Nucleotidtriestörsynthese, but using Durensulfonyltetrazoi at the individual coupling steps.

DmtrO

, Bz

-OMb 0

Il

-0-P-0

OCp

., Bz -OMb

Il

-0-P 0th

Il \

OCp>

Bz OBz

-OBz

In the respective coupling steps yields of> 80% were achieved and obtained pure chromatographic products.

Mp .: 72-74 ⁰ C FV ¹ 0.49

R, ^b) 14.31 min UV / VIS (MeOH): A _nllJI .225nm, 277nm

^ "255 nm

a: CH ₂ C! ₂ / MeOH (95: 5, v / v) on silica gel 60 F _2H (Merck)

b: HPLC: RP 18 column (100 x 2.1 mm), pressure: 40 x 10 ³ kPa at 40 ° C. flow 0.4 ml / min, eluant: 0.05M NH ₄ -acetate sol. pH 7 against a MeOH gradient.

Example 2

Using the building blocks (1M4) with the use of Durensulfo-1-oxy-6-nitro-benzo-1,2,3-triazole as coupling reagent

First, the protected dinucleotides (6) and (7) are built up.

"Bz

, Bz

DmtrO

-O-P-0 with OCp

, Bz

-OMb 0

Il

Lo-P-OCe

DmtrO

-OMb

-0-P-O. OCp

-OBz -OBz

(6) (7)

These are then condensed in the same way in high yield to the protected, chromatographically pure tetranucieotide (8).

DmtrO.

, Bz

-OMb 0

Il

-0-P-O

OCp

, 3z -OMb

-Grandpa

OCp ^N

-OMb

-O-P-0, H \

OCp ^N

, Bz

-OBz

-03z

Mp: 65-68TR, ¹¹ 0.44

R, ^bl 14.35 min UV / VIS (MeOH): X _m , 226nm, 265nm A _m in. 243nm

a: Information as in a) Example b: Information as in b) Example

Example 3

Starting from nem Dinucleotidderivat (6) and the Ti inucleotidderival (5) is obtained using Ourensulfochlorid / 1-methylimidazole as coupling the protected pentanucleotide (9) in chromatographically pure form and in a yield of> 90%.

DnrfcrO.

-OMb

0 ii

-0-P-O II OCp

-OMt "

-0-P-O. OCp

'\ J

, Bz -OLIb

-0-P-0

OCp

, Bz -OMb

-0-P-O.

Bz OBz

-OBz

(9)

Mp .: 69-72 ⁰ C

R ( ^bl 14.64 min

UV / VIS (MeOH): X _1111x . 230nm, 275nm

X _m , _n .250nm

a: Information as in a) Example 1

 b: details as in b) Example 1.

equation

5 ¹

O (ORS)

-Ρ · 0 I X

VY

9 cor)

Cl

Dmtr = dimethoxytrityl

X = arenesulfonyl Y e.g. = Acyl R e.g. = Tetrahydropyranyl

DmtrO

HX

FORMULA 1

NQ ₂

N-

-N

N-SO

0 = P-0 (5 ') - R 0 0 0

CH.

 f Cf-

FO RM E ". 2

CH ₃ CH ₃

NO-

FORMJLUL

Claims (2)

Process for the preparation of oligonucleotides, deoxy- and ribonucleotides according to the phosphotriester method, characterized in that chlorides or azolides, e.g. TetrazoI, 1-hydroxybenzo-1,2,3-triazole and their substitution products derived from 2,3,5,6-tetramethylbenzenesulfonic acid (durene sulfonic acid) can be used as condensing agents for the preparation of the internucleotidic phosphate bond. 2. The method according to claim 1, characterized in that comes for the purpose Durensulfo-1-oxy-6-nitro-benzo-1,2,3-triazole used. For this 2 pages formulas Field of application of the invention The invention relates to a process for the preparation of deoxyribo- and ribonucleotides according to the phosphotriester method, wherein the condensation step for internucleotidic phosphate binding is carried out by means of novel specific reagents. Characteristic of the hitherto known technical solutions For the construction of deoxyribo- or ribonucleotides the phosphotriester method is a proven principle of operation in which the 3'-OH group of a protected Nucleosides transferred to do fully masked 3'-phosphotriester derivative of the same after exposure and activating a phosphate-OH group the internucleotidic bond with the 5'-OH group of a second Nucleosidbaustei.ies is prepared (Principle Formula 1). For the activation of the phosphate component (X in formula 1) and their condensation with the 5'-OH-free nucleoside component have been preferred mesitylene sulfochloride (MS) or 2,4,6-Triisopropylbenzensulfochlorid (TPS) in the presence of substituted imidazoles such. B. 1 -Methylimidazol used as a catalyst. Alternatively, the 1,2,4-triazolides, tetrazolides, 1-hydroxy-benzo-1,2,3-triazolides, as well as 3-nitro-1,2,4-triazolides of the sulfonic acids mentioned have also proven themselves (principle of formula 2). The mixed anhydride-type reactive intermediates formed in dabai then either react directly with the 5'-OH component to form the nucleotide or first disproportionate to pyrophosphates, which then, on their own, form the internucleotidic bond. An essential prerequisite for this activation method is that sulfenic acid derivatives of highly alkylated arenes be used, whereby a cleavage of the P-O, but not the S-O bond of the M η hydride is ensured and thus side reactions are avoided at the 5'-OH component. However, the two arensulfonic acids used to date for the aforementioned activation principle have the disadvantage that they are either relatively expensive to produce or do not optimally take into account the stated requirements. It is therefore desirable to seek other arene sulfonic acid derivatives suitable for the condensation step in nucleotide synthesis. Object of the invention The aim of the invention is to develop a method which makes it possible to produce the internucleotidic phosphate binding in oligonucleotide syntheses according to the Triestermethoda by means of novel, condensed condensation reagents in high yield and free of by-products. Presentation of the essence of the invention The object of the invention is to develop new, readily available reagents arensulfonic acid type, which make it possible to produce the condensation step in oligonucleotide syntheses by the triester method in high yield and to form uniform end products. In accordance with the invention, the object has been achieved by using activated derivatives of the sulfonic acid (2,3,5,6-tetramethylbenzene-1-sulfonic acid). Duren as a starting material is a cheap product of coal tar processing and can be converted with the simplest Mittein in the sulfonic acid. A particular advantage of this alkyl arensulfonic acid type is also that the activated intermediate reacts exclusively and rapidly in the desired direction by the electron donating action and strong steric shielding of the four methyl groups according to formula 2 and no by-products are formed on the 5'-OH component. In the same way, it is possible to build up the intornucleotide phosphine bond very specifically and in high yield. Durene sulfonic acid can be used in accordance with the known procedures both ice chloride in the presence of 1-methylimidazole as a catalyst as well as in the form of one of the common azolides with very good results for the synthesis of deoxyribo · as well as ribonucleotides.