Since it was first coined by Sharpless over 20 years ago,1 click chemistry has quickly become an invaluable approach to synthesizing and manipulating organic molecules. The reactions described by click chemistry have been used extensively in materials science, bioconjugation, drug discovery and much more. Click chemistry continues to become more popular and was awarded the 2022 Nobel Prize in Chemistry.
Of the reactions that fall under the click chemistry umbrella, the cycloaddition between an azide and an alkyne, to form a stable 1,2,3-triazole linkage, has been by far the most prevalent. Any entity with an azide can be linked to another entity that has an alkyne. The reaction is specific, high yielding and rapid. In addition, there are also no byproducts generated. Standard alkynes will require a copper (I) catalyst while strained alkynes will react with an azide without any additional inputs beyond solvent.
Figure 1. Azide alkyne cycloaddition. A, copper (I) catalyzed; B, strained alkyne (DBCO).
Azide alkyne cycloadditions are excellent methods for oligonucleotide conjugations,2 and Glen Research has a long list of reagents to facilitate such reactions. There are numerous alkynes for copper (I)-catalyzed conjugation (Figure 1A). There are also several strained alkynes (DBCO) for copper-free conjugation (Figure 1B). What might be harder to find are azide-containing reagents. This is mostly because azides can react with phosphoramidite groups (Staudinger reaction), and as a result, azide-containing phosphoramidites are not very stable. There are a couple of workarounds (Figure 2). Oligonucleotides containing amino modifiers can be post-synthetically labeled with Azidobutyrate NHS Ester (50-1904). Alternatively, the bromide from 5’-Bromohexyl Phosphoramidite (10-1946) can be displaced by azide prior to oligonucleotide deprotection. Both approaches work well, but is there a way to avoid additional post-synthesis manipulations?
Figure 2. Strategies for introducing azides to oligonucleotides
Years ago, researchers at the University of Montpellier demonstrated that a synthesis support functionalized with an azide modifier will survive oligonucleotide synthesis and deprotection.3 Subsequently, another team from the University of Innsbruck extended this concept to an azidonucleoside-loaded support.4 With these developments in mind, we showed that an azide support could be prepared using our 3’-Amino-Modifer C7 CPG (20-2958) and Azidobutyrate NHS Ester (50-1904),5 and customers have since had a third approach for labeling oligonucleotides with azides. To make azido oligonucleotides even more readily available, we are now introducing 3’-Azido-Modifier Serinol CPG (Figure 3), essentially an amalgam of Azidobutyrate NHS Ester and Amino-Modifier Serinol CPG (20-2997).
Figure 3. 3'-Azido-Modifier Serinol CPG
The support works very well. It can be used in the same way as any of our other Serinol supports. For coupling, the support should be used in a manner identical to a normally protected nucleoside support since it contains the DMT group. Deprotection should be carried out as required by the nucleobases. In the past, we have observed a small amount of displacement of the azide by ammonia during deprotection using the bromohexyl approach described earlier. Fortunately, this was not observed for this support, even with an ammonium hydroxide deprotection at 55 °C for 17 hours.
3’-Azido-Modifier Serinol CPG is convenient to use and produces excellent synthesis results. Due to its advantages over other approaches, it is possible that this new support will become the most popular method for preparing azide-labeled oligonucleotides.