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Ethylthiotetrazole (ETT)

Ethylthiotetrazole (ETT)

CAS No.:89797-68-2

A tetrazole-like product used in the activation step of the oligo synthesis cycle, supplied as a solid.
  • 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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Product information

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.


  1. 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.
  2. (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.
  3. The synthesis of 2'-O-[(triisopropylsilyl)oxy

Applicable Products

LK0234 BTT (Crystalline)
LK0237 ETT (Crystalline)
LK3140/2 ETT (0.25M)
LK3145/6 ETT (0.5M)
LK3160/2 BTT (0.3M)

Traditionally, tetrazole was used as the activator during oligonucleotide synthesis. Although this was chemically efficient for DNA synthesis, there were several drawbacks. Tetrazole has a tendency to precipitate from aceonitrile solution due to its low solubility. This is especially true in winter months, where it was not uncommon for tetrazole to precipitate on the synthesiser overnight resulting in blocked lines. Coupling efficiency during RNA synthesis, or while using other sterically hindered phosphoramidites, was greatly reduced in comparison to DNA synthesis. Finally, tetrazole is classed as an explosive which makes transportation of the product, even in solution, costly or impossible.

There are many alternative activators available today, the most common used being 0.25M ETT (LK3140/LK3142), 0.3M BTT (LK3160/LK3162) and DCI (LK3150). All of these are much more soluble than tetrazole in acetonitrile thus resolving the crystallisation problem. In terms of coupling efficiency in DNA synthesis, they are all comparable with tetrazole. However, they are much more efficient when used with sterically hindered phosphoramidites such as 2’-OTBDMS RNA monomers. In fact, using 0.3M BTT has been reported to reduce the coupling time of 2’-OTBDMS protected RNA amidites to 3min rather than the 12-15min required when using tetrazole. In RNA synthesis, using BTT as an activator has brought coupling efficiencies in line with DNA synthesis, i.e >99%. The same applies to the use of 0.5M ETT (LK3145/LK3146).

One drawback is using ETT or BTT rather than tetrazole is that they are more acidic (pKa: ETT 4.3; BTT 4.1; tetrazole 4.89). This can be problematic when synthesising on a larger scale (>10-15μmol) or for very long oligos. In this case DCI (pKa 5.2) can be used to avoid depurination or loss of trityl groups during coupling (please note that we no longer offer DCI).

Preparation of BTT & ETT Solutions

Rather than use pre-mixed solutions, some customers prefer to prepare their own activator solutions. This gives the flexibility of making only the quantity required for synthesis. There is also the added advantage that shipping the solids does not incur a hazardous charge.

Crystalline BTT (LK0234) and ETT (LK0237) are provided in three pack sizes (1g, 10g and 25g) for dilution to customers’ required concentrations in whatever synthesiser bottle is appropriate. Typical dilution information is given below (these examples equate to routine bottle sizes and concentrations, however, obviously a range of pack sizes is provided to give customers flexibility in this).


Mol. Wt.



BTT (0.3M) 192.24 11.53g 25.95g
ETT (0.25M) 130.17 6.51g 14.64g
ETT (0.5M) 130.17 13.02g 29.29g

To prepare the solution simply weigh out the desired amount of product into your instrument bottle and dilute by adding filtered, anhydrous acetonitrile (Diluent, LK4050) as indicated. Ensure that the solid is entirely dissolved prior to use. This is essential to avoid blockages on the synthesiser. Use end line filters on the activator bottle position, again to prevent any blockages.

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