A Photoinducible 1,3‐Dipolar Cycloaddition Reaction for Rapid, Selective Modification of Tetrazole‐Containing Proteins

Abstract

Bioorthogonal chemistry provides an exciting new strategy to visualize protein expression, track protein localization, measure protein activity, and identify protein interaction partners in living systems.[1] Two steps are typically involved in this approach: 1) the incorporation of a bioorthogonal group into a protein through either a biochemical pathway or semisynthesis; 2) a site-specific reaction between the protein that carries the bioorthogonal group and a cognate small-molecule probe. Although a plethora of methods have been developed to address the first step, such as non-sense suppression mutagenesis, [2] expressed protein ligation, [3] metabolic engineering, [4] and tagging-via-substrate, [5] only a small number of bioorthogonal reactions are known for the second step. These site-specific reactions include the acid-catalyzed nucleophilic addition of hydrazine to a ketone or aldehyde, [6] Staudinger ligation, [7] Cu I -catalyzed azide-alkyne 1,3-dipolar cycloaddition (click chemistry), [8] strain-promoted azide-alkyne 1,3-dipolar cycloaddition, [9] and the oxidative coupling of aniline.[10] To fully realize the potential of bioorthogonal chemistry in probing protein function, there is an urgent need for the discovery of additional bioorthogonal reactions with robust reaction attributes. Herein, we report a bioorthogonal, photoinducible 1,3-dipolar cycloaddition reaction that allows rapid and highly selective modification of proteins carrying a diaryl tetrazole group in biological media.Forty years ago, Huisgen and co-workers reported a photoactivated 1,3-dipolar cycloaddition reaction between 2,5-diphenyltetrazole and methyl crotonate.[11] A concerted reaction mechanism was proposed, whereby the diaryl tetrazole undergoes a facile cycloreversion reaction upon photoirradiation to release N 2 and generate in situ a nitrile imine dipole, which cyclizes spontaneously with an alkene dipolarophile to afford a pyrazoline cycloadduct (Scheme 1). The photolysis of diaryl tetrazoles was found to be extremely efficient upon UV irradiation at 290 nm, with quantum yields in the range 0.5-0.9.[12] Despite its robust mechanism, this photoactivated reaction has seen very few applications in the past four decades.[13]In our initial studies, we identified an extremely mild photoactivation procedure in the use of a hand-held UV lamp from UVP (UVM-57, 302 nm, 115 V, 0.16 amps). Under these mild conditions, the solvent compatibility, functional-group tolerance, regioselectivity, and yield of the photoactivated 1,3-dipolar cycloaddition reaction were excellent. [14] We then examined the reaction kinetics by incubating a tetrazole peptide with acrylamide in phosphate-buffered saline (PBS) at pH 7.5 under UV light (302 nm; see Figure S1 in the Supporting Information). We found that the photolysis of the tetrazole peptide to generate the nitrile imine intermediate was extremely rapid, with a first-order rate constant k 1 = 0.14 s À1 ; the subsequent cycloaddition with the dipolarophile acrylamide proceeded very efficiently, w...

Reversible and Rewritable Surface Functionalization and Patterning via Photodynamic Disulfide Exchange

“…Proteins, DNA fragments, peptides and antibodies, [1][2][3][4][5][6] as well as hydrogels, [7] have been immobilized and patterned using a number of photochemical methods, such as thiol-yne, [8] thiol-ene, [9] azide-yne (by photoreduction of copper II), [10] terazole-ene, [11] photo-triggered Diels-Alder reaction, [12] Paterno-Buchi reaction, [13] and some other chemistries capable of photo-triggered formation of reactive functional groups. [14][15][16][17] However, most of the existing photochemical methods lead to irreversible permanent surface functionalization, limiting possible applications in the formation of materials and surfaces with dynamic and responsive properties or reusable 2 functionalities.…”

UV‐Induced Tetrazole‐Thiol Reaction for Polymer Conjugation and Surface Functionalization

“…[6] For instance, Lin et al, [7] introduced a photo-click 1,3-dipolar tetrazole-ene reaction based on Huisgen's studies. [8] The tetrazole-ene reaction presents several advantages: simplicity of implementation, fast reaction kinetics, high yields, it is catalyst free, yields inoffensive byproducts (N2) and, therefore, bio-compatible. This and other photo reactions have been implemented in many different applications such as dendrimers synthesis, [9] bioconjugation, [6b, 6d, 6e, 10] in situ bio-labeling, [6c, 11] hydrogels formation, [12] surface functionalization [13] etc.…”

From UV to NIR light, photo-triggered 1,3 dipolar cycloadditions as a modern ligation method in solution and on surface

“…1 H and 13 C NMR spectra were recorded on a 400 MHz spectrometer and referenced to the relevant solvent peak stated in the spectrum caption. The 4 methyl groups α to the protected nitroxide species from the methoxyamine derivatives are not visible in 13 C NMR.…”

“…Methoxy-2,2,6,6-tetramethylpiperidin-4-yl-4-(5-(2-hydroxyethyl)-4,6-dioxo-1-phenyl-1,3a,4,5,6,6a-hexahydropyrrolo[3 Figure 74 13 C NMR (100 MHz, CDCl3) spectrum of 2-methoxy-1,1,3,3-tetramethyl-5-(2-phenyl-2H-tetrazol-5-yl)isoindoline 13.…”

“…A mixture of 11-hydroxyundecyl 4-formylbenzoate 22 (472.6 mg, Figure 97 13 C NMR (100 MHz, CDCl3) spectrum of 11-hydroxyundecyl 4-formylbenzoate 11-hydroxyundecyl 4-(2-(pyren-1-yl)-2H-tetrazol-5-yl)benzoate 23.…”