Nucleoside phosphoramidite

The phosphoramidite synthesis method, a biochemical to produce DNA sequences of nucleoside phosphoramidites. It is a method of artificial DNA synthesis.

Principle

Phosphoramidites are used as nucleotide analogs in the synthesis of oligonucleotides. To avoid side reactions at other reactive nucleophilic groups (e.g., hydroxyl or amino groups ), this can be provided with protective groups. Two nucleophilic groups (usually two hydroxyl groups) are not protected, but used to generate the phosphoramidite. By phosphoramidite synthesis, nucleic acids, nucleic acid analogues, LNA, morpholino, nucleotides with modifications at the 2'- position ( for example, methoxy -protected amines, fluoride ), nucleotides with non-natural nucleobases (e.g., hypoxanthine, xanthine ), tricyclic nucleobases such as the G -clamp, selectively reactive groups or fluorescent Nukleinbasederivate.

Preparation of the phosphoramidites

Phosphoramidites are generated using three methods. Most the free hydroxy group is activated with Phosphorodiamidit in the presence of a weak acid. Since some Bisamidite not thermostable, usually 2-cyanoethyl -N, N, N ', N'- Tetraisopropylphosphorodiamidit is used which can be prepared in two steps and a vacuum distillation.

Phosphoramidites with protective groups may also be generated with phosphorochloridite in the presence of an organic base, generally N, N -diisopropylethylamine ( Hunigsche base).

As a third method, the protected nucleobases are first treated with chloro -N, N, N ', N'- Tetraisopropylphosphordiamidit in the presence of an organic base such as Hunigsche base, to obtain a protected nucleoside Phosphordiamidit. Followed by the addition of a corresponding one of the Phosphitschutzgruppe alcohol ( e.g., 2- cyanoethanol ) and a weak acid.

Generated nucleoside phosphoramidites are purified by silica gel column chromatography. To maintain the stability of the phosphoramidites triethylamine is used in the buffer between three and five percent. Purity can be determined inter alia, by 31P NMR. The chiral center at the P (III ) atom has two maxima at about 149 ppm, corresponding to the two diastereomers. Unwanted phosphite triester have a shift 138-140 ppm and H- phosphonates have two maxima at 8 and 10 ppm.

Properties of the phosphoramidites

Nucleoside phosphoramidites are reasonably stable in water and oxygen-free powder form at 4 ° C and in basic solutions. However, they decompose under acidic conditions. The half-life of 2- cyanoethyl - 5'-O- (4,4 '- dimethoxytrityl ) thymidine -3' -O-( N, N- diisopropylamino ) phosphite in 95% aqueous acetonitrile at 25 ° C for 200 hours

The most important feature of the phosphoramidites is the ready and rapid reaction with nucleophiles in the presence of the azole - catalysts, such as 1H- tetrazole, 2- ethylthiotetrazole, 2- Benzylthiotetrazol, 4,5- dicyanoimidazole, or similar materials. The coupling of the phosphoramidites leads to epimerization at the chiral center. When water is used as the nucleophile, the product is an H-phosphonate diester, and therefore, a reaction with water is a common undesirable side reaction in the synthesis of oligonucleotides.

Phosphoramidites are easily oxidized to phosphoramidates, e.g., with aqueous iodine in the presence of weak bases, or with hydrogen peroxide. Furthermore phosphoramidites react with chalcogens. Upon contact me sulfur - containing substances Phosphorthioamidate be formed. The reaction with selenium leads to Phosphorselenoamidaten. Here, the configuration is obtained.

Nucleoside phosphoramidites are converted by the Michaelis- Arbuzov reaction in phosphonamidates. The reaction is stereoselective at room temperature with retention of configuration. At 55 ° C, the racemization occurs.

Similar to phosphines and tertiary phosphites a Staudinger reaction can take place.

Protection groups

The naturally occurring nucleotides ( nucleoside 3' - or nucleoside -5'- phosphate ) and their phosphodiester analogues of low reactivity for a high yield. By 3'-O- (N, N -diisopropyl phosphoramidite ) derivatives bond formation is significantly accelerated in the phosphite - triester method. To prevent undesired side reactions all unnecessary for coupling the reactive groups are provided with protective groups. After completion of the DNA sequence, the protection groups are removed. Phosphoramidites are often protected at the 5'- hydroxyl group with 4,4 '- dimethoxytrityl. Thymine and uracil have no free amino groups and therefore do not require protecting groups. However, adenine, cytosine and guanine are protected at the amino group, since it is otherwise reactive. Although these nucleobases can also be used unprotected, base labile protecting groups to be used and maintained until the last cycle, mostly.

Two different types of protective group used in the phosphoramidite synthesis. The first type comprises benzoyl protecting groups on the N4 atom of the cytosine and the N6 atom of adenine (A, dA, C, dC). Guanine (G and dG) be protected with an isobutyryl group. Cytosine can also be protected by an acetyl group. The second type includes isobutyryl protecting group of adenine (A and dA ) or Phenoxyacetylgruppen (PAC). Cytosines carry an acetyl group and Guanine are with 4- Isopropylphenoxyacetylgruppen (i- Pr -PAC) or Dimethylformamidinogruppen ( dmf ) protected. Protecting groups of the second type can be split off faster, but are generally less stable in solutions.

The phosphite group is protected by a base-labile 2- cyanoethyl group. Upon binding of the phosphoramidite to the growing oligonucleotide, and the conversion of the Phosphitanteils to P (V), the presence of the phosphate protecting group is not necessary for additional coupling steps.

In RNA synthesis, the 2'- hydroxyl group with t- butyldimethylsilyl ( TBDMS ) or with tri -iso- propylsilyloxymethylgruppen (TOM) protected .. Both protecting groups are removed with fluoride.

The phosphite also carries a diisopropylamino group ( iPr2N ), which is reactive under acidic conditions. After activation, the diisopropylamino group is a leaving group in the binding to the growing oligonucleotide.

In most cases, the reaction product is purified after synthesis, eg by precipitation or by high performance liquid chromatography. By use of a polymerizable molecule ( with methacrylic acid group) in the final coupling step of the phosphoramidite correctly synthesized peptides can be separated from the acetylated and erroneous coupling products by a subsequent polymerization.