Why are certain modifications only available as NHS esters?
Some modifications (such as azidobutyrate or certain rhodamine dyes) are not available or preferred as phosphoramidites. This is either due to chemical instability, incompatibility with standard phosphoramidite coupling conditions, or the modification may not survive even mild oligonucleotide deprotection conditions.
For example, azide phosphoramidites are unstable because the azide group can be reduced by the P(III) center of the phosphoramidite via the Staudinger reaction. On the other hand, rhodamines (TAMRA and ROX) are sensitive to base and experience degradation during oligonucleotide deprotection and cleavage. These dyes are comprised of two structural elements: (1) the modified rhodamine dye and (2) a 6-carboxyl aryl linker. The extent of degradation depends on the fluorophore and may be managed. TAMRA has been prepared as a phosphoramidite and solid support but UltraMild conditions are strongly advised. A phosphoramidite version of ROX is not available. Post-synthesis labeling techniques are often preferred for installing these dyes into an oligonucleotide.
In other cases, there may be a phosphoramidite version available but the NHS ester is preferred. This is particularly true for modifications that are very sensitive to coupling and/or deprotection conditions, such as sulfoCyanine 5 NHS Ester and Methylene Blue NHS Ester. Post-synthetic labelling with the NHS ester precludes the need for using UltraMild conditions.
Relevant products:
Azidobutyrate NHS Ester (50-1904)
sulfoCyanine 5 NHS Ester (50-5915)
Methylene Blue NHS Ester (50-1960)
Can one use the Glen Gel-Pak™ columns for counterion exchanges?
Glen Gel-Pak columns are not designed to exchange counterions during the purification. These columns are mainly designed for the desalting of oligonucleotides, clean-up of conjugation reactions, and the removal of deprotection solution including the released protecting groups.
The Glen Gel-Pak desalting protocol calls for a buffer during the elution step of the desired product. Based on the nature of the buffer, some counterion exchange can occur, however, this is not sufficient to exchange all of the counterions. Therefore, we recommend using other more efficient methods such as ethanol precipitation to exchange the counterions.
Relevant products:
Glen Gel-Pak™ 1.0 Desalting Column (61-5010)
