Antonio Manetto, Simon Warncke and Thomas Frischmuth
Bahnhofstrasse 9 – 15
82327 Tutzing, Germany
Note: All products of baseclick described in this article are patent protected and available from Glen Research Corporation, 22825 Davis Drive, Sterling, VA 20164, USA, email: [email protected], in collaboration with baseclick.
In 2010, we published an article in The Glen Report, Volume 22, No 1, describing the technology that baseclick has offered for Click Chemistry. In the present article, we review advances since that time and specifically highlight our new Oligo-Click Kits, which are designed to make conventional Click reactions much more user-friendly. At the same time, we position these techniques in comparison to Cu-Free Click.
The copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) is the most prominent example of a group of reactions named click-reactions, as shown below.
These reactions are characterized by high yields, mild reaction conditions, and by their tolerance of a broad range of functional groups.1 Typically, the reactions require simple or no workup, or purification of the product. The most important characteristic of the CuAAC reaction is its unique bio-orthogonality, as neither azide nor terminal alkyne functional groups are generally present in natural systems.
The use of this method for DNA modification has been somewhat delayed by the fact that copper ions damage DNA, typically yielding strand breaks.2 As these problems have now been overcome by the use of copper(I)-stabilizing ligands (e.g., tris(benzyltriazolylmethyl)amine, TBTA3), Carell et al. and Seela et al. discovered that the CuAAC reaction can be used to functionalize alkyne-modified DNA nucleobases with extremely high efficiency.4
In comparison to the common post synthetic labeling methods of oligonucleotides like amine/NHS-ester, thiol/iodoacetamide or maleimide labeling, modification of oligonucleotides with Click Chemistry is providing by far the highest conjugation efficiency.6
Single and multiple labeling can be performed with as little as two equivalents of label-azides resulting in complete conversion and high yields of labeled oligo. In addition, the marker azides used for click functionalization are stable to hydrolysis which allows storage in solution (in contrast to sensitive NHS esters and maleimides). Excess amounts can even be recovered after the click reaction.
It has been shown that the 5-position of pyrimidine and the 7-position of 7-deazapurine nucleosides are the ideal positions to introduce functionalities, as these sites lie in the major groove of the DNA providing steric freedom. In order to enable efficient Click Chemistry labeling of alkyne modified oligonucleotides, our nucleosides provide a 5-(octa-1,7-diynyl) side chain. Phosphoramidites of nucleosides 1-4 (Figure 1) were shown to be incorporated into DNA oligomers by solid-phase synthesis with excellent coupling efficiency (e.g., 1: > 99 %). Another feature of the octadiynyl side chain is its stabilizing effect on DNA duplexes (e.g., 1: Tm increase of 1-2 °C).
Since alkyne-modified nucleoside phosphoramidites are incorporated into DNA strands during solid-phase synthesis in excellent yields and even stabilize the DNA-duplexes, Glen Research offers the dC and dT analogues, shown in Figure 2 on the following page, under license from baseclick.
|Figure 2: Structures of dC and dT Click Phosphoramidites|
Purified oligonucleotides bearing a single alkyne moiety are usually modified with 2-5 equivalents of the corresponding marker-azide (e.g., fluorescent-dye azides). After the addition of precomplexed Cu(I), complete conversion to the labeled oligo is observed in a time span of between 30 minutes and 4 hours. After a simple precipitation step, labeled oligonucleotides can be recovered in near quantitative yields.
The Cu(I)-catalyzed Huisgen reaction enables the multiple post synthetic labeling of alkyne modified DNA as well. Complete high-density functionalization of several alkyne moieties can be achieved without the formation of by-products.
For the attachment of up to three different labels, phosphoramidites with the alkyne groups protected with triisopropylsilyl (TIPS) and trimethylsilyl (TMS) protecting groups have been developed.5
In order to modify oligonucleotides with two sensitive molecules, two alkyne nucleosides, one with no alkyne protection and the second with TIPS protection, are incorporated into DNA strands using standard phosphoramidite chemistry. The first click reaction yields the singly modified oligonucleotide with full retention of the TIPS protecting group. For the second click, the TIPS protecting group is cleaved with tetrabutylammonium fluoride (TBAF) without causing any damage to the DNA. The second click reaction in solution yields the doubly modified oligonucleotides in excellent yields (60–90% over three steps).