The Copper(I) Catalyzed Azide-Alkyne Cycloaddition (CuAAC) is the most recognizable example of click reactions.1 However, the presence of copper may not be favorable in oligonucleotide research or biological samples due to its known toxicity. To circumvent this issue, strain-assisted labels have been developed to facilitate click reactions without requiring any catalyst. We are happy to introduce two new products towards this effort: 3′-DBCO-Serinol CPG (20-2998) and 5′-TCO C6 Phosphoramidite (10-1943).
3′-DBCO-Serinol CPG
Dibenzocyclooctyne (DBCO) is no stranger to the Glen Research catalog and is a popular choice when looking for copper-free click chemistry between an alkyne and an azide. This is referred to as Strain-Promoted Azide-Alkyne Cycloaddition (SPAAC). Copper-free click chemistry offers several advantages to users, including stability in solution on the synthesizer, stability to standard deprotection conditions, and efficiency in click reaction performance.3
With this new addition, we now offer five DBCO products: 5′-DBCO-TEG Phosphoramidite (10-1941), DBCO-dT-CE Phosphoramidite (10-1539), DBCO-Serinol Phosphoramidite (10-1998), DBCO-sulfo-NHS Ester (50-1941), and our new 3′-DBCO-Serinol CPG. Various applications that have used our DBCO products have been described previously.2
3′-DBCO-Serinol CPG is a solid support version of our DBCO-Serinol Phosphoramidite (Figure 1). The introduction of this 3′-DBCO-Serinol CPG allows researchers to place the DBCO modifier at the 3′-end of their oligonucleotide without the need for multiple synthesis reagents. Previously, to incorporate DBCO at the 3′-end, one needed to use the phosphoramidite with universal support or the NHS ester with a 3′-amino modifier.
Figure 1. 3′-DBCO-Serinol Products
Recommended Protocols for 3′-DBCO-Serinol CPG
DBCO is susceptible to damage from standard iodine oxidation and our mild oxidizer, CSO in anhydrous acetonitrile, is required. This is especially crucial since the DBCO is at the 3′-end and must survive several rounds of coupling and oxidation. 3′-DBCO-Serinol CPG is compatible with standard phosphoramidites, including dmf-dG but not iBu-dG. DBCO-oligonucleotides are stable to ammonium hydroxide deprotection for 2 h at 65 °C or overnight at room temperature. If iBu-dG is used, deprotection with AMA for 2 h at room temperature may slightly degrade the cyclooctyne.3 UltraMild deprotection conditions are also compatible with 3′-DBCO-Serinol CPG.
5′-TCO C6 Phosphoramidite
Bioorthogonal reactions used in imaging, diagnostics, and therapy applications require rapid kinetics and high specificity to optimize the time required and amount of labeling agent needed to achieve high coupling yields.4 TCO phosphoramidite is a new addition to our line-up and offers our customers another approach to conjugation chemistry (Figure 2). TCO is a strained alkene that reacts with tetrazines in an inverse electron-demand Diels-Alder cycloaddition (iedDA) reaction. TCO-tetrazine ligation is ultrafast, selective, and works well in mild conditions (e.g. room temperature, neutral pH, and aqueous solutions).5, 6 DBCO and TCO reactions occur very rapidly in practice. In the literature, it has been reported that DBCO derivatives typically display second-order rate constants in the 1-2 M-1s-1 range while TCO-based click reactions exhibit very high bimolecular rate constants (103-105 M-1s-1).4-6 Rate constants depend on both components that are part of the click reaction and conditions of the reaction. For example, in a separate study, DBCO and TCO exhibited more comparable rate constants, 0.1-1 M-1s-1.7
Figure 2. 5′-TCO C6 Phosphoramidite
In the iedDA cycloaddition, an electron-poor diene, commonly 1,2,4,5-Tetrazine, reacts with an electron-rich dienophile, such as TCO, via a normal [4+2] Diels-Alder cycloaddition and then immediately undergoes a subsequent, irreversible retro Diels-Alder step, which releases a molecule of nitrogen (Figure 3).4-7 The reaction can be observed spectroscopically by the disappearance of the visible absorption band around 510-550 nm, which comes from the tetrazine chromophore, as long as the tetrazine label does not interfere with this absorption range.4
Figure 3. Tetrazine Diels-Alder cycloaddition with trans-cyclooctene
Applications
Certain tetrazine fluorophores that are only fluorogenic upon cycloaddition improve signal-to-noise ratio for fluorescence microscopy.7 The tetrazine-TCO cycloaddition has been used to fluorescently label live cells,4 radiolabel biomolecules,5 and in triggered conjugation reactions, activate the tetrazine moiety enzymatically.7, 8
Specificity is obviously important when it comes to labeling DNA probes. Selectively coupling different labels site-specifically to a single oligonucleotide is commonly achieved through two different approaches: (1) two different conjugation reactions are carried out sequentially with or without purification steps or (2) protecting groups must be removed between successive conjugation steps to direct reactivity. The tetrazine-TCO cycloaddition is fully orthogonal to copper-catalyzed azide-alkyne reaction and both conjugations can take place in a one-pot reaction without cross reactivity.9 The iedDA cycloaddition was even shown to be orthogonal to the strain-promoted alkyne-azide cycloaddition (SPAAC) between an azide and DBCO, albeit requiring a more careful choice of reactants based on their reactivity to prevent interference.10 This can be particularly useful in designing oligonucleotide FRET probes.7, 9-10
The tetrazine-TCO conjugation strategy was also employed to ligate modified oligonucleotides into a fully functional sgRNA for CRISPR. The oligonucleotides contained nucleobase modifications, including N1-methyladenosine (m1A), N6-methyladenosine (m6A),
and 4-thiouridine (s4U), all modifications that we also offer, to evaluate the impact of RNA modifications of CRSIPR activity.11
Recommended Protocols for 5′-TCO C6 Phosphoramidite
One major benefit to TCO over DBCO-containing oligonucleotides is its relative compatibility with iodine oxidation. Prolonged exposure to iodine does induce some degradation of TCO. However, this is a 5′-modifier and must only be capable of surviving a single oxidation cycle. In our hands, we found either CSO or iodine oxidation (0.02 M) yielded equivalent coupling efficiency and oligonucleotide purity using 5′-TCO C6 Phosphoramidite. A 10 min coupling time is recommended and the TCO modification is compatible with standard deprotection conditions.
TCO reacts readily with modified tetrazines in organic solvents or aqueous solutions. It is worth mentioning that highly substituted and sterically hindered tetrazines may have difficulty reacting with TCO.5 In our hands, we coupled FAM-tetrazine to a 5′-TCO oligonucleotide using the following procedure.
For a 0.2 µmole synthesis of a TCO-modified oligonucleotide:
Summary
These new additions benefit researchers taking advantage of copper-free click reactions for their oligonucleotide conjugations. DBCO and TCO react readily and selectively with their respective substrates. The biocompatibility of the two types of click reactions (SPAAC and iedDA) has the potential to lead to research where multiple click reactions take place on the same oligonucleotide. We’re excited to see where our customers can go with this.
References
Product Information
