Relative performance of alkynes in copper-catalyzed azide-alkyne cycloaddition

Abstract

Copper-catalyzed azide-alkyne cycloaddition (CuAAC) has found numerous applications in a variety of fields. We report here only modest differences in the reactivity of various classes of terminal alkynes under typical bioconjugative and preparative organic conditions. Propargyl compounds represent an excellent combination of azide reactivity, ease of installation, and cost. Electronically activated propiolamides are slightly more reactive, at the expense of increased propensity for Michael addition. Certain alkynes, including tertiary propargyl carbamates, are not suitable for bioconjugation due to copper-induced fragmentation. A fluorogenic probe based on such reactivity is available in one step from rhodamine 110 and can be useful for optimization of CuAAC conditions.

Figure 1

Performance of various alkyne substrates in the ligand-accelerated (LA) CuAAC process under bioconjugation conditions. (Top) Reaction scheme and structures of alkynes used. (Bottom) Time required to reach 50% and 90% of maximum fluorescence under various conditions.

Figure 2

Competition experiment under typical organic conditions.

Figure 3

(A) CuAAC reaction of propiolamide PyrPRA with azidohomoalanine. (B) Emission spectra (excitation at 348 nm) before (PyrPRA) and after (PyrTRZ) CuAAC reaction with AHA. (C) Time course of polyvalent Qβ nanoparticle-azide labeling with PyrPRA (excitation at 348 nm, emission at 394 and 488 nm).

Figure 4

(Top) Cu(I)-induced cleavage of alkynyl carbamate releases fluorescent Rhodamine 110. (Bottom) Kinetic traces of Ynoc₂Rho cleavage in the presence of indicated amount of catalyst (5:1 THPTA:Cu⁺) at 37°C. In the absence of THPTA, the reaction is more than 100 times slower (Supporting Information).