Incorporating Duplex Modifications (dI, dU, Me-dC, dX, Amino-dA)

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Applicable Products

2013 dU-CE Phosphoramidite
2016 dI-CE Phosphoramidite
2017 5-Me-dC(Bz)-CE Phosphoramidite
2145 2-Amino-dA-CE Phosphoramidite
2164 2'-Deoxyxanthosine-CE Phosphoramidite
2287 dU SynBase™ CPG 1000/110
2293 dI SynBase™ CPG 1000/110
2323 5-Me-dC SynBase™ CPG 1000/110
2529 5-Me-dC(Ac)-CE Phosphoramidite

Physical & Dilution Data

Dilution volumes (in ml) are for 0.1M solutions in dry acetonitrile (4050). Adjust accordingly for other concentrations. For µmol pack sizes, products should be diluted as 100µmol/ml to achieve 0.1M, regardless of molecular weight.

Item

Mol. Formula

Mol. Wt.

Unit Wt.

250mg

500mg

1g

2013 C39H47N4O8P 730.80 290.17 3.42 6.84 13.68
2016 C40H47N6O7 754.83 314.19 3.31 6.62 13.25
2017 C47H54N5O8P 847.90 303.21 2.95 5.90 11.79
2145 C58H83N10O6P 1047.33 328.22 2.39 4.77 9.55
2164 C56H61N8O12P 1069.12 330.20 2.34 4.68 9.35
2287 - - 290.17 - - -
2293 - - 314.19 - - -
2323 - - 303.21 - - -
2529 C42H52N5O8P 785.88 303.21 3.18 6.36 12.72

Coupling

Couple the phosphoramidites 2013, 2016 and 2017 using the standard method as recommended by synthesiser manufacturer with coupling times as per standard bases. 90s is recommended for 2529. A coupling time of 15min is recommended for 2145. A coupling time of 3min is recommended for 2164. The solid supports are used as per standard nucleoside supports.

Deprotection

For 2013, 2016, 2017 and 2529 standard oligonucleotide deprotection conditions can be applied when deprotecting an oligo containing these modifications, however 2013, 2016 and 2529 are also compatible with AMA deprotection; typically 10min at 55oC.

2145 - Use AMA deprotection in conjunction with Ac-dC, rather than Bz-dC, or transamidation will occur. The supports are cleaved and deprotection carried out using the protocols required by the nucleobases.

2164 - Cleavage from support and primary deprotection is accomplished by treatment with ammonium hydroxide solution at room temperature for 24h.

After removing the solution1, the NPE groups are removed by treatment with 0.3M tetramethylguanidinium 2-nitrobenzaldoximate solution in water/dioxan (1:1) at 70°C for 48h. We find that this extended treatment is necessary to ensure complete removal of both of the NPE protecting groups.

It should be noted that this treatment generates a variety of low molecular weight by-products which are observed in the HPLC. Satisfactory results are obtained by de-salting the deprotection mixture into water using a NAP or G25 column before HPLC, from which the final oligonucleotide product can then be isolated.

Storage & Stability

Refrigerate dry compounds. Stability of phosphoramidites in solution is similar to standard dA, dC, dG and dT monomers.

Note

  1. This is best achieved by removing the deprotection solution by G25 then freeze drying to remove the water. Heat cannot be used or the NPE groups would cleave.
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