A bioorthogonal quadricyclane ligation

Abstract

New additions to the bioorthogonal chemistry compendium can advance biological research by enabling multiplexed analysis of biomolecules in complex systems. Here we introduce the quadricyclane ligation, a new bioorthogonal reaction between the highly strained hydrocarbon quadricyclane and Ni bis(dithiolene) reagents. This reaction has a second-order rate constant of 0.25 M(-1) s(-1), on par with fast bioorthogonal reactions of azides, and proceeds readily in aqueous environments. Ni bis(dithiolene) probes selectively labeled quadricyclane-modified bovine serum albumin, even in the presence of cell lysate. We have demonstrated that the quadricyclane ligation is compatible with, and orthogonal to, strain-promoted azide-alkyne cycloaddition and oxime ligation chemistries by performing all three reactions in one pot on differentially functionalized protein substrates. The quadricyclane ligation joins a small but growing list of tools for the selective covalent modification of biomolecules.

Figure 1

(A) A generic [2 + 2 + 2] reaction between quadricyclane and a π electrophile to yield a norbornene cycloaddition product. (B) 7-Acetoxyquadricyclane (1) reacts with bis(dithiobenzil)nickel(II) (2) to form complex 3. Complex 3 undergoes photodegradation to 2 and 7-acetoxynorbornadiene (4) unless diethyldithiocarbamate (5) is present.

Scheme 1. Water-Soluble Ni Bis(dithiolene) Complexes

Figure 2

Ni bis(dithiolene) reagents selectively label quadricyclane-modified BSA. (A) Modification of BSA’s solvent-exposed lysine residues with compound 16 and subsequent labeling with biotinylated Ni bis(dithiolene) reagent 15. (B, C) Ni bis(dithiolene) 15 displays (B) time- and (C) dose-dependent labeling of QC-BSA. BSA (−) or QC-BSA (+) (5 μg) was treated with (B) 50 μM 15 for various amounts of time or (C) various concentrations of 15 for 30 min. The reactions were quenched with 1 and 5, and the presence of product was detected by Western blot using α-biotin-HRP. (D) Reaction of 15 with QC-BSA can be prevented by pretreatment with tetrasulfonated Ni bis(dithiolene) 17. BSA (−) or QC-BSA (+) (5 μg) was treated with various amounts of 17 for 30 min followed by 50 μM 15 for 30 min. The reactions were quenched with 1 and 5, and the samples were analyzed by Western blot probing with α-biotin-HRP. (E) QC-BSA can be selectively labeled in a mixture of proteins. To 25 μg of lysate in the presence of 1 mM K₃Fe(CN)₆ was added no BSA or reagent (lane 1), BSA (1.5 μg) and 50 μM 15 (lane 2), and QC-BSA (1.5 μg) and 50 μM 15 (lane 3). After 30 min, the reactions were quenched with 1 and 5 and analyzed by Western blot. The BSA monomer and dimer bands are visible in the QC-BSA-treated sample. The bands denoted with asterisks represent a biotinylated E. coli protein. In (B–E), equal protein loading was verified by Ponceau stain.

Figure 3

The quadricyclane ligation is orthogonal to Cu-free click chemistry and the oxime ligation. (A) A mixture of QC-BSA, Az-DHFR, and CHO-MBP (8 μg each) was treated with 15 (150 μM), DIMAC-fluor (250 μM), and H₂NO-FLAG (1 mM) for 3 h at 37 °C, pH 4.5. This mixture was basified with 850 mM tris buffer and quenched with excess 1, 5, and 2-azidoethanol. (B) The mixture was separated into three portions, and each portion was analyzed by Western blot probing with a different antibody: α-biotin-HRP (quadricyclane ligation), α-fluorescein-HRP (Cu-free click chemistry), or α-FLAG-HRP (oxime ligation). The Ponceau stain indicates that all three proteins were present. Oligomer bands were observed for BSA and DHFR.(38)