FIGURE 1: Structures of TBTA and THPTA
FIGURE 2: RP HPLC Analysis - 10 nmole Reaction with HEX Azide and THPTA, 60 Minutes
FIGURE 3: RP HPLC Analysis - 100 nmole Reaction with FAM Azide, THPTA and TBTA, 15 Minutes
FIGURE 4: RP HPLC Analysis - 1 µmole Aqueous Reaction with FAM Azide and THPTA, 30 Minutes
Click Chemistry has become a mainstay for bioorthogonal conjugation for three simple reasons: it is fast, efficient, and specific.
Classic Click Chemistry uses copper, Cu(I), to catalyze the 1,3-dipolar cycloaddition of an alkyne with an azide to form a 1,2,3-triazole.1,2 Sources of Cu(I) include copper(I) iodide, copper(I) bromide, copper turnings, or copper(II) sulfate (CuSO4) and a reducing agent.1
An improvement in Click Chemistry uses the in situ preparation of Cu(I) from the reduction of CuSO4 with sodium ascorbate and a Cu(I) stabilizing ligand, tris-(benzyltriazolylmethyl)amine (TBTA).3 This leads to a more reliable click reaction by avoiding the oxidation of catalytic Cu(I) by dissolved oxygen. In a typical reaction, copper sulfate is pre-complexed with TBTA to form a brilliant blue solution. This complexed catalyst is mixed with the alkyne labeled oligonucleotide and the azide label, followed by the addition of sodium ascorbate to initiate the click reaction.
TBTA (1) covers most of the practical applications for Click Chemistry except for a completely aqueous conjugation reaction. The benefits of a completely aqueous reaction include the biological labeling of live cells or the labeling of proteins without the concern of denaturing secondary structures. The water-soluble tris(3-hydroxypropyltriazolylmethyl)amine (THPTA) (2) click ligand further simplifies Click Chemistry by allowing the entire reaction to be run in water, affording biological compatibility for Click reactions. The THPTA ligand binds Cu(I), blocking the bioavailability of Cu(I) and ameliorating the potential toxic effects while maintaining the catalytic effectiveness in click conjugations. The THPTA ligand was effectively used to label live cells with high efficiency while maintaining cell viability.4
In our hands, we have found THPTA to be a highly efficient ligand for click chemistry with working ranges from 10nmol up to 1000nmol, in partially organic and completely aqueous reactions. Labeling is complete in as little as 15 minutes at room temperature. The ligand CuSO4 complex exhibits no loss of activity when frozen for at least a month.
All of the oligonucleotides used in this study were 5'-hexynyl modified 12mers. Figure 2 shows the result from the click conjugation of HEX Azide at the 10 nmole scale. In this case, the ligand CuSO4 complex had been stored frozen for 1 month before use. We also compared the results of conjugations at the 100 nmole level using TBTA or THPTA as ligand. At all time points, the levels of conjugation were equivalent. The 15 minute time point is shown in Figure 3. These reactions were done in a mixture of organic and aqueous conditions to ensure the TBTA was in solution. In Figure 4, we illustrate the click conjugation in aqueous conditions using THPTA. The conjugation with 4 equivalents of FAM Azide (dissolved in water at slightly basic pH) was complete in 30 minutes at room temperature.
Our results indicate that THPTA performs at least as well as TBTA under equivalent conditions. However, THPTA can be used in aqueous conjugations where TBTA is insoluble, a distinct advantage. The simple procedure is as follows:
Glen Research offers several options for the incorporation of an alkyne label into an oligonucleotide with a selection of nucleosidic and non-nucleosidic alkynes. We have several nucleosidic alkynes that are ideal for internally labeling oligonucleotides, and, with the use of our protected alkynes, oligos can be labeled with different reporters on the same oligo.5 We also offer several non-nucleosidic alkynes that can be used for terminus or internal labeling using our 5’-Hexynyl Phosphoramidite or Alkyne Serinol Modifiers. A selection of azide labels is also available. Please see our web site for details.
We are now happy to add THPTA to our line of products for Click Chemistry.
1. V.V. Rostovtsev, Green, L.G., Fokin, V.V. and Sharpless, K.B., Angew Chem Int Ed, 2002, 41, 2596-2599.
2. C.W. Tornøe, C. Christensen, and M. Meldal, The Journal of Organic Chemistry, 2002, 67, 3057-3064.
3. T.R. Chan, R. Hilgraf, K.B. Sharpless, and V.V. Fokin, Org. Lett., 2004, 6, 2853-2855.
4. V. Hong, N.F. Steinmetz, M. Manchester, and M.G. Finn, Bioconjugate Chemistry, 2010, 21, 1912-1916.
5. P.M.E. Gramlich, S. Warncke, J. Gierlich, and T. Carell, Angewandte Chemie-International Edition, 2008, 47, 3442-3444.