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- Available as solid for dilution by customer in anhydrous acetonitrile.
- Tested in solution to be free of particulates.
- Assay > 99.9% pure.
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The oligonucleotide synthesis cycle consists of four steps: deblocking (detritylation); activation/coupling; capping; and oxidation (or sulphurisation). Synthesis occurs in the 3’ to 5’ direction; which is opposite to enzymatic synthesis by DNA polymerases.
Conventionally, the 3’ base in the sequence is incorporated by use of a base-functionalised CPG or polystyrene support, although ‘universal’ supports are available. Synthesis initiates with removal (‘deblocking’ or ‘detritylation’) of the 5'-dimethoxytrityl group by treatment with acid (classically 3% trichloroacetic acid in DCM to afford the reactive 5'-OH group. The phosphoramidite corresponding to the second base in the sequence is activated(1) using a tetrazole-like product such as ETT or BTT (see below), then coupled to the first nucleoside via the 5'-OH to form a phosphite linkage.
Solid phase phosphoramidite coupling usually proceeds to around 99% efficiency. If the 1% of molecules remaining with reactive 5'-OH groups are left untreated, unwanted side-products will result. To prevent this, a ‘capping’ step is introduced typically prior to the oxidation to acetylate the unreacted 5'-OH (5). Where sulphurisation is performed, capping must come after this step. This is typically achieved using a solution containing acetic anhydride (Cap Mix A) and the catalyst N-methylimidazole (Cap Mix B). Unless blocked these truncated oligonucleotides can continue to react in subsequent cycles giving near full- length oligonucleotide with internal deletions (species referred to as (N-1)mers).
The unstable trivalent phosphite triester linkage is oxidised, via an iodine-phosphorous adduct, to the stable pentavalent phosphotriester using iodine in a THF/pyridine/water solution. After oxidation, the cycle is repeated, starting with detritylation of the second molecule and so on. The synthesis cycle is repeated until the desired length of oligonucleotide is achieved. At this point the synthesis is complete.
Activators containing tetrazole (traditionally as a 0.45 M solution in anhydrous acetonitrile) have classically been the reagents of choice in routine automated DNA and RNA synthesis. There are, however, two main disadvantages to using this product. Firstly, at lower laboratory temperatures (typically 18 °C), solid tetrazole can crystallise from the near-saturated solution causing blockage of delivery lines. Secondly, the product has become more difficult to obtain because of shipping restrictions due to its classification as an explosive. As a consequence, we do not offer this product.
ETT (5-Ethylthio-1H-tetrazole) can offer more effective activation than tetrazole without crystallisation problems. In particular it has been shown to decrease the coupling times in both RNA synthesis(2) and DNA synthesis.
BTT (5-Benzylthio-1H-tetrazole) activator is particularly useful in RNA synthesis with a view to reducing the longer coupling times, due to the steric effects of protecting the 2'-OH. BTT has been classed as a non-explosive material, and therefore, availability of the product is not restricted. In DNA synthesis, coupling efficiency is routinely at least as good as with tetrazole, and often better. In RNA synthesis, the coupling of TBDMS or TOM monomers with 1H-tetrazole activation conditions can require 12-15 min. Using BTT, 3 min coupling times are recommended, although 90s has been used effectively using 6.5eq of 0.25 M BTT and 6eq of 0.1 M phosphoramidite.(3)methyl (TOM) phosphoramidites of methylated ribonucleosides for the use in automated RNA solid-phase synthesis, C. Höbartner, C. Kreutz, E. Flecker, E. Ottenschläger, W. Pils, K. Grubmayr and R. Micura, Monatchefte für Chemie, 134, 851-873, 2003.] BTT is, in fact, very slightly more acidic than ETT, however, it has been shown that N+1 peaks are no more significant using BTT with shorter coupling times than ETT with a 6 min coupling time or 1H-tetrazole for 12 min.
Note that we also provide crystalline BTT and ETT - these can ship as non-hazardous products thereby reducing costs and removing some regional transportation limitations.
The various capping and oxidizer formulations are generally dependent on the recommendations of the synthesizer manufacturer. For cap mixtures, pyridine is more wider used than lutidine as the base. In the case of oxidizers, most people have moved to 0.02M iodine as it isn’t as harsh on the oligo.
- A description of the mechanism of activation via the phosphorotetrazolide intermediate can be found in Studies on the role of tetrazole in the activation of phosphoramidites, S. Berner, K. Mühlegger and H. Seliger, Nucleic Acids Research, 17, 853-864, 1989.
- (a) Synthesis, deprotection, analysis and purification of RNA and ribozymes, F. Wincott, A. DiRenzo, C. Shaffer, S. Grimm, D. Tracz, C. Workman, D. Sweedler, C. Gonzalez, S. Scaringe and N. Usman, Nucleic Acids Research, 23, 2677-2684, 1995; (b) An efficient method for the isolation and purification of oligoribonucleotides, B. Sproat, F. Colonna, B. Mullah, D. Tsou, A. Andrus, A. Hampel and R. Vinayak, Nucleosides & Nucleotides, 14, 255-273, 1995; (c) Large-scale synthesis of oligoribonucleotides on high-loaded polystyrene (HLP) support, D. Tsou, A. Hampel, A. Andrus and R. Vinayak, Nucleosides & Nucleotides, 14, 1481-1492, 1995.
- The synthesis of 2'-O-[(triisopropylsilyl)oxy