Click chemistry is a versatile technique that can be used for the synthesis of a variety of conjugates. Virtually it can be applied to any biomolecules.

„It just says click – and the molecules are coupled together“ (1)

With respect to oligonucleotide labeling with small molecules, such as fluorescent dyes, biotin, and alike, click chemistry opens doors to a whole "new world" of exciting and so far unimaginable or de facto commercially rather unrealizable molecule specifications.

The concept of "Click Chemistry" has been coined by Nobel Laureate K. Barry Sharpless around 2000 to identify a reliable and straightforward form of chemistry in which reactions take place fast with the absence of undesired by-products.

A little later, and independently of each other, Morten Meldal and Barry Sharpless described the [3+2] cycloaddition between alkynes and azides (2).

Although around 1960 Rolf Huisgen first reported this typical "click" reaction, the 1,3-dipolar cycloaddition (3, 4), it was only Sharpless and Meldal who subsequently described it as a copper-catalyzed process (Figure 1, 5, 6). The two components - azido and alkyne groups - are nearly never encountered in natural biomolecules. Hence, conjugation reactions arrive at a remarkable degree of selectivity and specificity (7).

Figure 1. The click reaction.

Applying this concept on, and further developing it in the field of Nucleic Acid modification, Thomas Carell's work has been turning click chemistry into a very hot topic for oligonucleotide labeling (Figure 2, 8).

Figure 2. Oligonucleotide labeling via click chemistry. The further development of the concept to modification of nucleic acids by Thomas Carell's working group not only concerns the labelling of individual oligonucleotides with regard to the introduction of multiple modifications, but also the attachment of terminal labels to oligos that exceed a critical length.

Respective citations have been, are, and will be growing rapidly.

Prof. Dr. Thomas Carell is one of the founders of baseclick GmbH, headquartered in Tutzing/Munich. Turning his expertise into a successful business idea, baseclick GmbH was granted the basic patent on copper-catalyzed click reactions on nucleic acids by the European Patent Office in 2010. metabion has been offered a license for the respective labeling processes including an extended collaboration, which we happily assumed.

Always striving to be at the forefront of technical improvements in order to serve our customers best, metabion has secured a worldwide license for employing this interesting chemistry on producing modified oligonucleotides from baseclick GmbH.

Additionally and by using the same technology platform, click chemistry also allows for multiple labeling of polymerized DNA strands via incorporation of Alkyne-dNTPs (triphosphates) followed by post-synthetic click reactions at a so far unmatched efficiency level (Figure 3, 8, 9).

Figure 3. Multiple labelling of probes. Through click chemistry, unprecedented levels of labelling can be achieved: the incorporation of alkyne-labelled dNTPs by nick translation or PCR allows for customised ready-to-use/fully labelled oligonucleotides.

For living systems, unfortunately, the copper catalyst itself is toxic, but an innovative solution to this problem emerged in 2004, when scientist Carolyn Bertozzi found and published an alternative to copper ions to carry out the azide-alkaline reaction in cells (10). It all started when, in the early 1990s, she was attempting to map a glycan that attracted immune cells to lymph nodes. During a seminar, Carolyn Bertozzi listened to a German scientist explaining how he had managed to get cells to produce an unnatural variant of sialic acid, and thought of applying a similar method to get cells to incorporate a modified sialic acid with a type of chemical manipulation, e.g. a fluorescent molecule, into different glycans so that one could map hidden glycans within the cell (Figure 4, 10, 11).

Figure 4. Graphical representation of bio-orthogonal reactions in the cell (© Johan Jarnestad, 1).

Bertozzi removed the copper catalyst from the reaction by inserting the alkyne in a ring structure, which advances the reaction by exploiting the ring strain produced by the cyclically constrained molecules. In this way, such reactions that occur "in parallel" with the chemical environment of the cell, bio-orthogonal reactions, can proceed in cells without disrupting their normal chemistry (Figure 4, 10, 11). This strain-mediated click reaction allowed the glycans to be followed within the cell (Figure 5, 12).

Figure 5. Imaging of glycans in the cell by labelling with Cu-free click chemistry: labelled cell-surface glycans (green glow) and cell nucleus (blue, modified from 12).

Hence, bio-orthogonal chemistry has marked a new milestone in click chemistry that leads to a new dimension in which click reactions function within living organisms, enabling researchers to visualize and follow molecules in living cells in their natural state without influencing them.

As a crowning achievement, scientists Carolyn R. Bertozzi, Morten Meldal and K. Barry Sharpless were awarded the 2022 Nobel Prize in Chemistry for their development of click chemistry and bio-orthogonal chemistry (Figure 6, 1).

Figure 6. The Nobel Prize in Chemistry 2022 (© Johan Jarnestad, 1).

Consequently, it is intuitive that the number of areas in which click chemistry and bio-orthogonal reactions have been employed has significantly risen (13). Researchers have enhanced the targeting of anti-cancer drugs, without perturbing healthy cells, which are now undergoing clinical trials (14). Bertozzi herself, through her studies on glycans, is scientific advisor of a company which has developed a pharmaceutical drug, currently in phase II clinical trials on advanced cancer patients (15), capable of breaking down the glycans, present on the surface of cancer cells, which in some cases are able to protect these cells from attack by the immune system (16). Anticorps have also been designed to collimate tumour cells using the click response and then be attacked by a second molecule. Likewise, large quantities of polymers for building materials have been produced sustainably and rapidly.

"Click" in summary...

New, quick and easy reaction in water;
Compatible with many functions, non-solvent sensitive;
Very sensitive products can be connected;
No by-products are generated;
It possesses several applications in biochemistry, polymers, drug discovery…. and the most important: labelling of biomolecules with dyes;
Bio-orthogonal Click for copper-free applications in live cells which makes processes in the cell visible.

Click chemistry and its potential for new drugs and new treatments are still a long way off, but the game is on.

So, what are you waiting for? Check out our Portfolio!

What we offer

“ready-to-use/fully labeled” custom oligonucleotides

Having successfully introduced this additional labeling procedure for synthetic Nucleic Acids and other biomolecules into our custom synthesis operations, we are now able to offer significantly increased flexibility in terms of:

multiple labeling of oligonucleotides (2 or more modifications);
producing modified oligonucleotides exceeding a critical sequence length.

While in the past state-of-the-art chemistry and technologies either did not allow for the above options at all or if anything, not in a commercially reasonable and affordable way, we are happy to provide new solutions for your innovative ideas.

“ready-to-click-it-yourself” custom oligonucleotides

Moreover, we can provide you with alkyne-modified oligonucleotides to be labelled at your end by “clicking” the desired “Azide-modification”. Click here for our Azide-portfolio.

“modimerization” solutions

In line with above´s “click it yourself” approach, we can also supply you with Alkyne Nucleotides (triphosphates) to be incorporated into DNA during polymerization reactions (PCR, reverse transcription, nick-translation, etc.). This prepares the produced DNA strands for post-synthetic “click” conjugations to azide-activated labels – a very elegant and efficient way of “modimerizing” DNA strands.

Applications

Extended means of (multiple) labeling of biomolecules such as Nucleic Acids (DNA/RNA), peptides, proteins, and antibodies will have a vigorous effect on the following areas (making no claim to be exhaustive):

Fluorescence In Situ Hybridization (FISH)
qPCR-Assays
DNA/RNA Microarrays
Bioconjugation to Nanoparticles
Fluorescence Microscopy
Electrophoretic Mobility Shift Assay (EMSA)
Fluorescence Activated Cell Sorting (FACS)
Flow Cytometry (FCM), Cell Tracking and Cell-based Assays
Fluorescence Correlation Spectroscopy (FCS)
... We at metabion use it daily to produce fluorescent DNA for dyes that do not survive/are not available for synthesis.

References