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Click Chemistry

 
 

Click chemistry describes a thermodynamically favored reaction which enables quantitative and selective linkage of two different biomolecules. More precisely the click reaction is a cycloaddition between an azide and an alkyne moiety either under copper catalyzed or copper free reaction conditions. The technology is reliable and stable which makes it an ideal oligonucleotide labeling method.

The overall benefits of click chemistry include:

  • Reaction occurs in aqueous solution and at room temperature
  • Stability toward H2O, O2, and most organic synthesis conditions
  • Robust catalytic process
  • No side reactions and lack of functional group interference
  • Remarkable level of specificity for syntheses which require covalent linkage between two biochemical moieties.

Overview

Click chemistry is a modern synthetic approach for the modular assembly of more complex molecules from “simpler” building blocks [1,2]. In the click reaction an azide funtionalized component is coupled to an alkynyl functionalized building block to form a “bifunctional” conjugate. Since click chemistry is compatible with biomolecules in aqueous environment, this type of reaction is particularly useful to attach molecular probes to DNA [3,4]. The key advantage of the click reaction is its bioorthogonality1, since both reaction partners the azide and the alkyne group are not present in natural systems.

 

In the context of custom-made synthetic DNA oligonucleotides the click approach is often used to conjugate an azide functionalized molecular probe to an oligonucleotide containing an alkynyl residue (or vice versa).

Due to the unique characteristics of the click reaction, DNA and RNA oligonucleotides specifically labelled at multiple sites are now synthetically accessible. Hence click chemistry has opened a door to a new world of modified oligonucleotides which were so far virtually impossible to synthesize.

The most frequently applied type of click reaction uses Cu(I) as catalyst (copper assisted cycloaddition; CuAAC). A copper-free alternative has been developed for cases where the presence of copper is (potentially) harmful to your system. To avoid copper in the DNA probes, the more reactive alkyne residue dibenzocyclooctyne (DBCO) is applied to the click reaction [5-8]. 

The importance of the click reaction in the field of oligonucleotides is still growing. If you want to learn more about this exciting technology, please see references below or the links in the right column.

 

References:

  1. El-Sagheer AH, Brown T. Chem Soc Rev. Chem. Soc. Rev., 2010,39, 1388-1405
  2. V.V. Rostovtsev, Green, L.G., Fokin, V.V. and Sharpless, K.B., Angew Chem Int Ed, 2002, 41, 2596-2599.
  3. R. Huisgen, Angew Chem Int Ed, 1963, 2, 565-598. 
  4. Y.H. Zhang, et al., Tetrahedron, 2007, 63, 6813-6821. 
  5. I.S. Marks, et al., Bioconjugate Chemistry, 2011, 22, 1259-1263. 
  6. Becer CR, Hoogenboom R, and Schubert US, Angew. Chem. Int. Ed. 2009, 48, 2–11 
  7. J. Gierlich, G. A. Burley, P. M. E. Gramlich, D. M. Hammond, T. Carell, Org. Lett. 2006, 8, 3639-3642. F. Seela, V.  R. Sirivolu, Chem. Biodiversity 2006, 3, 509-514.
  8. P. M. E. Gramlich, S. Warncke, J. Gierlich, T. Carell, Angew. Chem. 2008, 120, 3491 - 3493; Angew. Chem. Int.  Ed. 2008, 47, 3442– 3444.

 

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1 Bioorthogonal is defined as: no interference of functional groups existing in biomolecules with the desired reaction. Hence no side products are formed with DNA, peptides, or other biomolecules.

Modifications

Type of Modification
Position
Synthesis Scale [µmol]
Purification
5’
3’
Int.
0.04
0.2
1.0
Des
HPLC
PAGE
Azide-dT
 
 
X
 
X
X
 
X
X
3' Alkyne
 
X
   
X
X
 
X
X
5' Azide
X      
X
X
 
X
X
3' Azide
  X    
X
X
 
X
X
5' DBCO
X      
X
X
 
X
X
5' Hexynyl
X      
X
X
 
X
X

Azide-dT
Microsynth's Azide modification uses an NHS Ester functional group to attach an azide moiety at the 5', 3' or any internal position in an oligo. This azide moiety may subsequently be used to attach alkyne modified groups through the click reaction. The internal version of this modification is attached to the oligo through a dT base. Incorporation of the internal version will add a dT nucleotide at that position. To avoid adding an extra nucleotide, replace an existing T nucleotide in your sequence with the required modification.

3' Alkyne
The alkyne group is used for copper assisted cycloaddition click chemistry.

5' Azide
The 5' azide group is used for copper assisted cycloaddition click chemistry.

3' Azide
The 3' azide group is used for copper assisted cycloaddition click chemistry.

5' DBCO
The 5' DBCO is cyclooctyne-based copper-free click reagent.

5' Hexynyl
The 5' Hexynyl group is used for copper assisted cycloaddition click chemistry. Oligonucleotides prepared using 5'-Hexynyl Phosphoramidite are stable to standard deprotection conditions and exhibit a slightly increased retention time on RP HPLC.

How to Order

 
  • Enter our webshop
  • Click on DNA in the blue “DNA/RNA Synthesis" domain
  • Select Normal Entry in order to type or copy/paste the desired sequence information
  • Follow the further instructions

Important: New customers are invited to send their request (info@microsynth.ch) by indicating sequence & modifications demands as well as foreseen application. Microsynth would then check your inquiry and come back to you with a customized quote suggesting you a reasonable and cost-effective synthesis approach for the desired biomolecule.