Chemical Ligation by Click Chemistry—A “Click” Away from Discovery

By: Matthias Junkers, ChemFiles Volume 1 Article 8

The traditional process of drug discovery based on natural secondary metabolites has often been slow, costly, and laborintensive. Even with the advent of combinatorial chemistry and high-throughput screening in the past two decades, the generation of leads is dependent on the reliability of the individual reactions to construct the new molecular framework.

Click chemistry is a newer approach to the synthesis of druglike molecules that can accelerate the drug discovery process by utilizing a few practical and reliable reactions. Sharpless and co-workers have defined what makes a click reaction: one that is wide in scope and easy to perform, uses only readily available reagents, and is insensitive to oxygen and water. In fact, water is in several instances the ideal reaction solvent, providing the best yields and highest rates. Reaction work-up and purification uses benign solvents and avoids chromatography.1

Of the reactions comprising the click universe, the “perfect” example is the Huisgen 1,3-dipolar cycloaddition of alkynes to azides to form 1,4-disubsituted-1,2,3-triazoles (Scheme 1). The copper(I)-catalyzed reaction is mild and very efficient, requiring no protecting groups and no purification in many cases.2 The azide and alkyne functional groups are largely inert towards biological molecules and aqueous environments, which allows the use of the Huisgen 1,3-dipolar cycloaddition in target guided synthesis3 and activity-based protein profiling,4 or the ligation of biopolymers to probes or surfaces.5 For example, Carell and co-workers demonstrated the labelling of alkyne modified DNA oligomers with fluorescence probes by click chemistry.6

Scheme 1

The triazole has similarities to the ubiquitous amide moiety found in nature. Thus triazole formation was used for the otherwise difficult macrocyclization of a cyclic tetrapeptide analog to a potent tyrosinase inhibitor.7

Additionally triazoles are nearly impossible to oxidize or reduce. This is a main reason why material science has discovered Huisgen cycloadditions as major ligation tools in diverse areas such as polymer science or nanoelectronics.8

Using Cu(II) salts with ascorbate has been the method of choice for the preparative synthesis of 1,2,3-triazoles, but it is problematic in bioconjugation applications. However, tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine, TBTA (Figure 1), has been shown to effectively enhance the copper-catalyzed cycloaddition without damaging biological scaffolds.9

Figure 1

In an extensive study Finn and co-workers only recently showed that tris(2-benzimidazolylmethyl)amines (general structure in Figure 2) are the most promising family of accelerating ligands for the Cu catalyzed azide-alkyne cycloaddition reaction from among more than 100 mono-, bi-, and polydentate candidates.10 Under both preparative (high concentration, low catalyst loading) and dilute (lower substrate concentration, higher catalyst loading) conditions, these tripodal benzimidazole derivatives give substantial improvements in rate and yields, with convenient workup to remove residual Cu and ligand.

Figure 2

A new reagent developed by Carolyn R. Bertozzi and co-workers eliminates the toxicity to living cells that is usually associated with the copper catalyzed Huisgen 1,3-dipolar cycloaddition.11 By using a difluorinated cyclooctyne (Figure 3) instead of the usual terminal alkyne a rapid cycloaddition reaction takes place even without a catalyst. The ring strain and the electron-withdrawing difluoro group activate the alkyne for copper-free click chemistry. This method was used to attach fluorescent labels to cells with azidecontaining sialic acid in their surface glycans. Thus, it was possible to study the dynamics of glycan trafficking in living cells over the course of 24 hours with no indication that the reaction or the labels perturb the process. This is an impressive example of how copper-free click chemistry can be used as a biologically friendly method to label and track biomolecules in living cells.

Figure 3

Sigma-Aldrich® proudly offers a choice of catalysts and ligands for the Huisgen cycloaddition reaction. Later sections in this issue present a comprehensive overview of available organic azides, azide sources, and alkynes that may be applied in click chemistry.

If you want to learn about hot new product additions to the click chemistry universe and other innovative areas of chemical synthesis as soon as they become available, please check our regularly updated product highlights at sigma-aldrich.com/chemicalsynthesis.

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References

  1. For recent reviews, see: (a) Kolb, H. C.; Sharpless, K. B. Drug Discovery Today 2003, 8, 1128. (b) Kolb, H. C. et al. Angew. Chem. Int. Ed. 2001, 40, 2004.
  2. (a) Rostovtsev, V. V.; Green, L.G.; Fokin, V.V.; Sharpless, K.B. Angew. Chem. Int. Ed. 2002, 41, 2596. (b) Tornøe, C. W. et al. J. Org. Chem. 2002, 67, 3057.
  3. (a) Manetsch,R. et al. J. Am. Chem. Soc. 2004, 126, 12809. (b) Lewis, W.G. et al. Angew. Chem. Int. Ed. 2002, 41, 1053.
  4. Speers, A. E. J. Am. Chem. Soc. 2003, 125, 4686.
  5. Wolfbeis,O.S. Angew. Chem. Int. Ed. 2007, 46, 2980.
  6. Gierlich, J.; Burley, G.A.; Gramlich,P.M.E.; Hammond, D.M.; Carell, T. Org. Lett. 2006, 8, 3639.
  7. Bock, V.D.; Perciaccente,R.; Jansen, T.P.; Hiemstra, H.; Maarseveen, J.H. Org. Lett. 2006, 8, 919.
  8. Lutz, J.-F.Angew. Chem. Int. Ed. 2007, 46, 1018.
  9. Chan, T.R. et al. Org. Lett 2004, 6, 2853.
  10. Rodionov, V. O.; Presolski, S. I.; Gardinier, S.; Lim, Y.-H.; Finn, M. G. J. Am. Chem. Soc. 2007, 129, 12696.
  11. Baskin, J.M.; Prescher, J.A.; Laughlin, S.T.; Agard, N.J.;Chang, P.V.; Miller, I.A.; Lo, A.; Codelli, J.A.; Bertozzi, C.R. PNAS 2007, 104, 16793.

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