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Click Chemistry


The term „click chemistry“ describes a fast and thermodynamically favoured reaction which enables an efficient and selective linkage of two molecules. In a more specific sense the click reaction is a cycloaddition between an azide and an alkyne either under copper catalyzed or copper free reaction conditions. Due to the fact that such click reactions work efficiently in aqueous media, they are very much suitable for modifying biomolecules or linking different biomolecules together. Furthermore the azide and alkyne reaction partners do not interfere with other functional groups like e.g. amino or carboxy which opens an additional degree of freedom for orthogonal coupling strategies.

Copper catalyzed click reaction

Copper catalyzed azide alkyne cycloaddition

 

The copper catalyzed azide alkyne cycloaddition (CuAAC)1,2 is an improvement of the classical Huisgen cycloaddition3. In both cases a terminal alkyne reacts with an azide to form a 1,2,3-triazole. In comparison to the original Huisgen reaction, which is non-regioselective and only works at elevated temperature, the copper catalyzed version is several times faster, runs efficiently at room temperature and selectively gives the 1,4-regioisomer of the triazole.


copper catalyzed click chemistry


The following clickable modifications are offered by biomers.net:

A reactive 5'-alkine is achieved by coupling of an appropriate cyclohexyl-linker phopshoramidite (1) to the oligonucleotide; for 3'-modifikation an alkine-prolinol linker (2) is used.
Pyrimidine C8-Alkyne-dU (4) und C8-Alkyne-dC (3) from baseclick (www.baseclick.eu) are available for internal oligo modification at the heterocyclic nucleobase. With 2‘-O-Propargyl-RNA building blocks (5), alkine functions at the sugar moiety can be introduced.
Terminal as well as internal azide modifications are introduced by reaction of azidobutyric acid with amino modified oligos (5'-Aminolink or internal Amino-dT) (6,7,8,9).
 

modification  5' 3' internal
Alkine (2) (1) (3),(4),(5)
Azide (6), (7), (8) (10) (9)



             alkyne azid modification


Alkine and azide modified oligos are purified by HPLC and quality checked by MALDI mass spectrometry.



Literature:
1. Peptidotriazoles on solid phase: [1,2,3]-Triazoles by regiospecific Copper(I)-catalyzed 1,3-dipolar cycloadditions of terminal alkynes to azides. Tornøe CW, Christensen C, Meldal MJ; Org. Chem. (2002), 67, 3057-3064.

2. A stepwise Huisgen cycloaddition process: Copper(I)-catalyzed regioselective ligation of azides and terminal alkynes. Rostovtsev VV, Green LG, Fokin VV, Sharpless KB; Angew. Chem. Int. Ed. Engl. (2002), 41, 2596-2599.

3. 1,3-Dipolar cycloadditions. Huisgen R; Proc. Chem. Soc. (1961), 357-396.
 

Copper free click reaction

Copper free click reaction

 

Due to toxicity of copper catalysts for bacteria and mammalian cells, reactions under physiological conditions have been developed for click chemistry. These work as fast and straightforward as the CuAAC but without involving any metal ions. A short review of Schubert et al.1 gives a good overview on click chemistry reactions.
The fast reaction of azides with variously substituted cyclooctynes is called "Strain-Promoted Azide-Alkyne Cycloaddition (SPAAC)" 2 and is greatly facilitated due to the unfavorable ring strain in the cyclooctyne ring3.


copper free click reaction



DBCO (Dibenzocyclooctyne) or ADIBO (Azadibenzocyclooctyne):

Using copper-free click reactions, there are various cyclooctyne derivatives differing greatly in terms of their reaction kinetics and hydrophility 4,5. DBCO (dibenzocyclooctyne), also known as ADIBO (azadibenzocyclooctyne) or DIBAC (dibenzoazacyclooctyne), is increasingly used as click reactant.
In many environmental conditions (in vitro and in vivo), DBCO reacts rapidly and selectively with azides forming the corresponding triazole compounds. The copper-free DBCO-azide click chemistry is a good orthogonal strategy compared to conventional coupling reactions, and thus allowing selective multiple labeling of biomolecules.

For copper-free click chemistry, biomers.net offers oligonucleotides with DBCO (dibenzocyclooctyne) as
5'-and 3´-modification:

DBCO



Literature:
1. Peptidotriazoles on solid phase: [1,2,3]-Triazoles by regiospecific Copper(I)-catalyzed 1,3-dipolar cycloadditions of terminal alkynes to azides. Tornøe CW, Christensen C, Meldal MJ; Org. Chem. (2002), 67, 3057-3064.

2. A stepwise Huisgen cycloaddition process: Copper(I)-catalyzed regioselective ligation of azides and terminal alkynes. Rostovtsev VV, Green LG, Fokin VV, Sharpless KB; Angew. Chem. Int. Ed. Engl. (2002), 41, 2596-2599.

3. 1,3-Dipolar cycloadditions. Huisgen R; Proc. Chem. Soc. (1961), 357-396.

4. Click chemistry beyond metal-catalyzed cycloaddition. Becer CR, Hoogenboom R, Schubert US; Angew. Chem. Int. Ed. (2009), 48, 2-11.

5. A Strain-promoted [3 + 2] Azide−alkyne cycloaddition for covalent modification of biomolecules in living systems. Agard NJ, Prescher JA, Bertozzi CR; J. Am. Chem.Soc. (2004), 126, 15046-15047.

6.  The enabled state of DNA nanotechnology. Linko V, Dietz H; Current Opinion in Biotechnology (2013), 24:1–7.
The enabled state of DNA nanotechnology

Tetrazine TCO Click Ligation

Tetrazine Ligation

 

The tetrazine ligation describes another method of bioorthogonal reaction. Comparable to copper-free click chemistry, TCO click reactions do not need any catalyst, thus in vitro and in vivo studies are possible. Through cycloaddition reaction, s-tetrazine and trans-cyclooctene are converted into a cyclic alkene with extremely fast reactivity1. In this reaction, the tetrazine is the reactive, electron-rich group which can be open-chain or cyclic. With elimination of dinitrogen N2, tetrazine and the substituting group of cyclooctene are linked to an alkene.

Tetracine TCO Click reaction

Compared to the copper-free click chemistry of SPAAC (Strain-Promoted Azide-Alkyne Cycloaddition), this metal-free click variant shows a higher reactivity and provides a high yield in organic solvents, water, cell media or cell lysate2,3. Due to its non-toxic properties, this reaction does not restrict the functionality of cellular enzymes and thus enables in vivo labelling of cellular DNA or proteins, as well as multiplex labelling2. In this way, e.g. lipids or other cellular components can be labelled with special dyes, allowing very high resolution and extended live cell images (3D-confocal or STED microscopy)4.

For the copper-free tetrazine TCO click ligation, biomers.net offers oligonucleotides with TCO as 
5'- or 3´-modification:



Literature:
1. The inverse electron demand Diels-Alder click reaction in radiochemistry. Reiner T, Zeglis BM; (2014), J Labelled Comp Radiopharm., 57 (4): 285-290.

2. The Tetrazine Ligation: Fast Bioconjugation based on Inverse-electron-demand Diels-Alder Reactivity. Blackman ML, Royzen M, Fox JM; (2008), J Am Chem Soc.; 130 (41): 13518-13519.

3. Rapid oligonucleotide-templated fluorogenic tetrazine ligations. Seckute J, Yang J, Devaraj NK; (2013), Nucleic Acids Research, Vol. 41, No. 15.

4. Super-resolution imaging of the Golgi in live cells with a bioorthogonal ceramide probe. Erdmann RS, Takakura H, Thompson AD, Rivera-Molina F, Allgeyer ES, Bewersdorf J, Toomre D, Schepartz A, (2014), Angew. Chem. Int. Ed. England, 53 (38): 10242-6.