Phosphoramidite synthesis

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2'-deoxynucleoside phosphoramidites with protecting groups.

The phosphoramidite synthesis is a method of biochemistry for the production of RNA - or DNA sequences from nucleoside -Phosphoramiditen. It is a method for artificial DNA synthesis , the products belong to synthetic DNA .

principle

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

Production of the phosphoramidites

Phosphorodiamidite method. DMT = 4,4'-dimethoxytrityl, B = optionally protected nucleobase, R = protecting group of the phosphate.

Phosphoramidites are produced by three methods. Usually the free hydroxyl group is activated with phosphorodiamidite in the presence of a weak acid. Since some Bisamidite not thermally stable are is mostly 2-cyanoethyl N , N , N ', N ' -tetraisopropylphosphorodiamidit used, which in two steps and a vacuum distillation can be produced.

Phosphorochloridite method.

Phosphoramidites with protective groups can also be produced with phosphorochloridite in the presence of an organic base, usually N, N-diisopropylethylamine ( Hünig's base ).

Chlorine tetraisopropyl phosphoramidite method.

As a third method, the protected nucleic bases are first treated with chlorine- N , N , N ', N ' -tetraisopropylphosphordiamidite in the presence of an organic base such as Hunig's base in order to obtain a protected nucleoside phosphorodiamidite. An alcohol corresponding to the phosphite protective group (e.g. 2-cyanoethanol) and a weak acid are then added.

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

Properties of phosphoramidites

Nucleoside phosphoramidites are reasonably stable in water- and oxygen-free powder form at 4 ° C and in basic solutions. However, they disintegrate 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 is 200 h.

Reaction with nucleophiles. X = O, S, NH.

The most important property of phosphoramidites is their ready and rapid reaction with nucleophiles in the presence of azole catalysts such as 1 H -tetrazole , 2-ethylthiotetrazole, 2-benzylthiotetrazole, 4,5-dicyanoimidazole, or similar substances. The coupling of the phosphoramidites leads to an epimerization at the center of chirality. If water is used as the nucleophile, the product is an H-phosphonate diester, which is why reaction with water is a common undesirable side reaction in the synthesis of oligonucleotides.

Oxidation. X = S, Se.

Phosphoramidites are easily oxidized to phosphoramidates, e.g. B. with aqueous iodine in the presence of weak bases or with hydrogen peroxide . Furthermore, phosphoramidites react with chalcogens . Phosphorothioamidates are formed on contact with sulfur- containing substances. The reaction with selenium leads to phosphorus selenoamidates. The configuration is retained here.

Michaelis-Arbusov reaction.

Nucleoside phosphoramidites are converted to phosphonamidates by the Michaelis-Arbusow reaction . The reaction is stereoselective at room temperature with retention of configuration. At 55 ° C which takes place racemization .

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

(RO) 2 P-N (R 1 ) 2 + R 2 -N 3 + H 2 O → (RO) 2 P (= O) -N (R 1 ) 2 + R 2 -NH 2 + N 2

Protecting groups

The naturally occurring nucleotides (nucleoside-3'- or nucleoside-5'-phosphates) and their phosphodiester analogs are not reactive enough for a high yield. 3'- O - ( N , N -diisopropyl phosphoramidite) derivatives significantly accelerate the bond formation in the phosphite triester method. To avoid undesired side reactions, all reactive groups not required for coupling are provided with protective groups. After the DNA sequence has been completed, the protective groups are removed. Phosphoramidites are often protected on the 5'-hydroxy group with 4,4'-dimethoxytrityl. Thymine and uracil have no free amino groups and therefore do not require any protective groups. On the other hand, adenine , cytosine and guanine are protected on the amino group, since it is otherwise reactive. Although these nucleobases can also be used unprotected, base-labile protective groups are mostly used and retained until the last cycle.

Two different types of protecting groups are used in phosphoramidite synthesis. The first type comprises benzoyl protective groups on the N4 atom of the cytosines and on the N6 atom of the adenines (A, dA, C, dC). Guanines (G and dG) are protected with an isobutyryl group. Cytosines can also be protected by an acetyl group. The second type of protecting group includes isobutyryl groups on adenines (A and dA) or phenoxyacetyl groups (PAC). Cytosines carry an acetyl group and guanines are protected with 4-isopropylphenoxyacetyl groups (i-Pr-PAC) or dimethylformamidino groups (dmf). Protective groups of the second type can be split off more quickly, but are generally less stable in solutions.

The phosphite group is protected by a base-labile 2-cyanoethyl group. After the phosphoramidite has bound to the growing oligonucleotide and the phosphite content has been converted to P (V), the presence of the phosphate protective group is not necessary for further coupling steps.

Phosphoramidites for RNA synthesis. 2'- O -protected ribonucleoside phosphoramidites.

In RNA synthesis, the 2'-hydroxy groups are protected with t -butyldimethylsilyl groups (TBDMS) or with tri- iso- propylsilyloxymethyl groups (TOM). Both protective groups can be removed with fluorides.

The phosphite part also carries a diisopropylamino group ( i Pr 2 N), which is reactive under acidic conditions. After activation, the diisopropylamino group is a leaving group upon binding to the growing oligonucleotide.

Most often the reaction product is purified after synthesis , e.g. B. by precipitation or by high performance liquid chromatography . By using a polymerizable molecule (with a methacrylic acid group) in the last coupling step of the phosphoramidite synthesis, the correctly synthesized oligonucleotides can be separated from the defective and acetylated coupling products after a subsequent polymerization .

Solid phase synthesis

Nucleic acids can be prepared by solid phase synthesis using phosphoramidites. CPG (controlled pore glass) can be used as the carrier material. The nucleic acid is condensed on the surface of the solid phase with the aid of a coupling reagent. The coupling of the nucleic acids takes place by reacting the fused-on nucleic acid with a phosphoramidite derivative of the other nucleic acid. In order to facilitate the separation of unreacted nucleic acids, the free alcohol group of the solid phase is acetylated. This process is known as * capping *. The phosphorus is then oxidized. The nucleic acid can either be purified or reacted further in a further cycle.

Phosphoramidite solid phase synthesis 3.svg

literature

  • Comprehensive Natural Products Chemistry, Volume 7: DNA and Aspects of Molecular Biology. Kool, Eric T .; Editor. Neth. (1999), 733 pp. Publisher: (Elsevier, Amsterdam, Neth.)
  • Beaucage, SL; Iyer, RP Advances in the synthesis of oligonucleotides by the phosphoramidite approach. Tetrahedron 1992 , 48 , 2223-2311.
  • Beaucage, SL; Iyer, RP The functionalization of oligonucleotides via phosphoramidite derivatives. Tetrahedron 1993 , 49 , 1925-1963.
  • Beaucage, SL; Iyer, RP The synthesis of modified oligonucleotides by the phosphoramidite approach and their applications. Tetrahedron 1993 , 49 , 6123-6194.
  • Beaucage, S. L. Oligodeoxyribonucleotides synthesis. Phosphoramidite approach. Methods in Molecular Biology (Totowa, NJ, United States) (1993), 20 (Protocols for Oligonucleotides and Analogs), 33-61.
  • Reese, CB The chemical synthesis of oligo- and poly-nucleotides: a personal commentary. Tetrahedron 2002 , 58 , 8893-8920.
  • Brown T., Brown DJS 1991. In Oligonucleotides and Analogues. A Practical Approach, ed. F Eckstein, pp. 1 - 24. Oxford: IRL

Individual evidence

  1. ↑ Beaucage, SL; Marvin H. Caruthers : Deoxynucleoside phosphoramidites - A new class of key intermediates for deoxypolynucleotide synthesis . In: Tetrahedron Letters . 22, 1981, pp. 1859-1862. doi : 10.1016 / S0040-4039 (01) 90461-7 .
  2. ↑ Lin, K.-Y., Matteucci, MD: A cytosine analog capable of clamp-like binding to a guanine in helical nucleic acids . In: J. Amer. Chem. Soc. . 120, No. 33, 1998, pp. 8531-8532. doi : 10.1021 / ja981286z .
  3. ↑ Nielsen, J .; Marugg, JE; Taagaard, M .; Van Boom, JH; Dahl, O .: Polymer-supported synthesis of deoxyoligonucleotides using in situ prepared deoxynucleoside 2-cyanoethyl phosphoramidites . In: Rec. Trav. Chim. Pays-Bas . 105, No. 1, 1986, pp. 33-34.
  4. ↑ Nielsen, J .; Taagaard, M .; Marugg, JE; Van Boom, JH; Dahl, O .: Application of 2-cyanoethyl N, N, N ′, N′-tetraisopropylphosphorodiamidite for in situ preparation of deoxyribonucleoside phosphoramidites and their use in polymer-supported synthesis of oligodeoxyribonucleotides . In: Nucl. Acids Res. . 14, No. 18, 1986, pp. 7391-7403. doi : 10.1093 / nar / 14.18.7391 .
  5. ↑ Nielsen, J .; Marugg, JE; Van Boom, JH; Honnens, J .; Taagaard, M .; Dahl, O .: Thermal instability of some alkyl phosphorodiamidites . In: J. Chem Res. Synopses . No. 1, 1986, pp. 26-27.
  6. ↑ Nielsen, J .; Dahl, O .: Improved synthesis of 2-cyanoethyl N, N, N ′, N′-tetraisopropylphosphorodiamidite (iPr2N) 2POCH2CH2CN . In: Nucl. Acids Res. . 15, No. 8, 1987, p. 3626. doi : 10.1093 / nar / 15.8.3626 .
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