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, 25 (9), 1730-8

Two Rapid Catalyst-Free Click Reactions for in Vivo Protein Labeling of Genetically Encoded Strained Alkene/Alkyne Functionalities

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Two Rapid Catalyst-Free Click Reactions for in Vivo Protein Labeling of Genetically Encoded Strained Alkene/Alkyne Functionalities

Yadagiri Kurra et al. Bioconjug Chem.

Abstract

Detailed kinetic analyses of inverse electron-demand Diels–Alder cycloaddition and nitrilimine-alkene/alkyne 1,3-diploar cycloaddition reactions were conducted and the reactions were applied for rapid protein bioconjugation. When reacted with a tetrazine or a diaryl nitrilimine, strained alkene/alkyne entities including norbornene, trans-cyclooctene, and cyclooctyne displayed rapid kinetics. To apply these “click” reactions for site-specific protein labeling, five tyrosine derivatives that contain a norbornene, trans-cyclooctene, or cyclooctyne entity were genetically encoded into proteins in Escherichia coli using an engineered pyrrolysyl-tRNA synthetase-tRNA(CUA)(Pyl) pair. Proteins bearing these noncanonical amino acids were successively labeled with a fluorescein tetrazine dye and a diaryl nitrilimine both in vitro and in living cells.

Figures

Scheme 1
Scheme 1. (A) Tetrazine-Alkene/Alkyne IEDDAC and (B) NADC Reactions
Scheme 2
Scheme 2. Structures of Four Strained Alkene/Alkyne Molecules and a Fluorescein Tetrazine Dye
Figure 1
Figure 1
Characterization of FITC-TZ reactions with (A) NOR, (B) DS1, (C) DS2, and (D) COY. All reactions were carried out in PBS buffer at pH 7.4. The fluorescence emission was detected at 515 nm with excitation at 493 nm. For A–D, each presents the fluorescence change as a function of time at a given concentration shown in the top left corner. The insets show the linear dependence of the pseudo-first-order rate constants of a reaction on dienophile concentrations.
Scheme 3
Scheme 3. Structure of a Hydrozonoyl Chloride, HZCL
Figure 2
Figure 2
Characterization of HZCL reactions with (A) DS1, (B) DS2, and (C) COY. All reactions were carried out in PBS/acetonitrile (1:1) at pH 7.4. HZCL was provided at 1 μM. The fluorescence emission was detected at 480 nm with excitation at 318 nm. The detected fluorescence data for 37.5 μM DS1 and 25 μM DS2 are presented in A and B. C shows data at two conditions, one with only acrylamide and the other with both acrylamide and COY. The insets show the linear dependence of the pseudo-first-order rate constants on the dipolarophile concentrations.
Scheme 4
Scheme 4. Structures of O-Alkylated Tyrosine Derivatives That Contain Strained Alkene/Alkyne Functionalities
Figure 3
Figure 3
Site-specific incorporation of NCAAs 1, 2, and 46 into sfGFP at its 2 position. Proteins were expressed in E. coli BL21 cells transformed with pEVOL-pylT-Y306A/N346A/C348A/Y384F and pET-pylT-sfGFP2TAG. Cells were grown in LB medium supplemented with 1 mM of a NCAA for 8 h. Supplementing LB with 2 mM of 3 or 7 did not yield sfGFP expression.
Figure 4
Figure 4
Selective labeling of sfGFPs that contained site-specifically incorporated NCAAs with strained alkene/alkyne functionalities with (A) FITC-TZ and (B) HZCL. In A and B, the top panels show denaturing SDS-PAGE analysis of proteins stained with Coomassie blue and the bottom panels are from fluorescent imaging of the same gels before Coomassie blue staining. In A, the fluorescent image was captured by a digital camera and displayed was real color of the emitting light. In B, the fluorescent image was captured by a Bio-Rad ChemiDoc XRS system.
Figure 5
Figure 5
Selective labeling of sfGFPs that contained site-specifically incorporated NCAAs with strained alkene/alkyne functionalities with (A) FITC-TZ and (B) HZCL in E. coli cells. Cells were labeled with FITC-TZ and HZCL for 3 h and then washed with PBS buffer 3 times before undertaking epifluorescent and differential interface contrast (DIC) imaging.

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