Site-specific incorporation of quadricyclane into a protein and photocleavage of the quadricyclane ligation adduct

Abstract

The quadricyclane (QC) ligation is a bioorthogonal reaction between a quadricyclane moiety and a nickel bis(dithiolene) derivative. Here we show that a QC amino acid can be incorporated into a protein site-specifically using the pyrrolysine-based genetic code expansion platform, and subsequently used for ligation chemistry. Additionally, we exploited the photolability of the QC ligation product to render the adduct cleavable with a handheld UV lamp. We further developed a protein purification method that involves QC ligation of biotin to a protein of interest, capture on streptavidin resin, and finally release using only UV light. The QC ligation thus brings novel chemical manipulations to the realm of bioorthogonal chemistry.

Keywords: Bioorthogonal; Photocleavage; Photolysis; Protein purification; Pyrrolysine; Quadricyclane; Unnatural amino acid.

Figure 1. Site-specific incorporation of QC amino acid into a protein

A) QC amino acid 6 was synthesized in a 2-step procedure from p-nitrophenyl carbonate 5: (1) N-Fmoc-L-lysine, DMF, rt, 16 h. (2) piperidine, DMF, rt, 2 h. B) N-ε-Boc-L-lysine (7), a known substrate of WT PylRS. C) Expression of full-length sfGFP-FLAG in the presence of 6 or 7. E. coli cells were transformed with plasmids pBKPylRS and psfGFP150TAGFLAGPylT (+RS+T) or with psfGFP150TAGFLAGPylT only (−RS+T) (see Supplementary data for plasmid maps and sequences) and cultures were supplemented with 1 mM 6, Boc-lysine (7), or vehicle (−). Protein was expressed for 3.5 h, after which lysates were analyzed by western blot probing with α-GFP and α-FLAG antibodies. D) High-resolution electrospray ionization (ESI) mass spectrometry (MS) analysis of purified QC-sfGFP-FLAG. Red represents the mass of the full-length protein and the green represents the mass of the full-length protein minus methionine. Black numbers represent neutral loss of H₂O, which may occur during fragmentation using ESI-MS. QC-sfGFP-FLAG observed mass: 28147 Daltons, calculated mass: 28147.61 Daltons.

Figure 2. Labeling QC-sfGFP-FLAG via the quadricyclane ligation

A) Schematic showing labeling QC-sfGFP-FLAG with 8. B) Structure of QC acetate (QCOAc, 9). C) Purified QC-sfGFP-FLAG or purified Boc-sfGFP-FLAG was incubated with 200 μM 8 in PBS with 1 mM K₃Fe(CN)₆ (+8) or with 1 mM K₃Fe(CN)₆ in PBS (−8) for 1 h at rt in the dark. The reaction was quenched with QCOAc (100 equiv. relative to 8) followed by treatment with sodium diethyldithiocarbamate (Na-DEDTC). Protein was analyzed by western blot with an α-biotin-HRP antibody. D) Various quantities of purified QC-sfGFP-FLAG or Boc-sfGFP-FLAG (5.0, 2.5, 1.0, 0.5, or 0 μg) were added to 10.0 μg bacterial lysate. The mixtures were incubated with 8 (200 μM) in K₃Fe(CN)₆ (1 mM) in PBS for 1 h at rt, quenched via addition of QCOAc (100 equiv. relative to 8), and treated with Na-DEDTC. Mixtures were analyzed via western blot with an α-biotin-HRP antibody.

Figure 3. Photocleavage of the QC/nickel bis(dithiolene) adduct

A) Reaction scheme for the model photocleavage. Adduct 10 was dissolved in CDCl₃ to a concentration of (5 mM) and placed in a borosilicate glass tube. The solution was placed in a foil-lined container and irradiated with 365 nm light from a handheld lamp for 0-25 min. B) Representative ¹H NMR spectra from Figure 3A at the indicated times. Diagnostic peaks are labeled with colors corresponding to the stars in Figure 3A. C) The progress of photocleavage was determined by comparing the integration of individual peaks of 10 to 11. Each measurement is the average of three separate experiments. Error bars represent one standard deviation.

Figure 4. Photocleavage of the QC/nickel bis(dithiolene) adduct on a protein

A) Lysines on bovine serum albumin (BSA) were capped via reaction with p-nitrophenyl quadricyclane carbonate 5. The resulting QC-BSA and untreated BSA (negative control) were treated with nickel bis(dithiolene) biotin 8 (50 μM in PBS) for 30 min to form adduct 13 or 8-treated BSA. The reactions were quenched with QCOAc and protein was purified from excess small molecule via centrifugal filtration with an Amicon Ultra 10 kDa molecular weight cutoff filter. 13 and 8-treated BSA in PBS were irradiated with a 365 nm handheld UV lamp for 0, 10, 30, or 60 min to cleave the adduct, providing 14 and 8. Protein products were separated from free 8 via centrifugal filtration as described above. C) Protein from Figure 4A was analyzed via western blot with an α-biotin-HRP antibody. Q = 13, B = 8-treated BSA.

Figure 5. QC photocleavage as a method for protein purification

A) Schematic showing general protein purification procedure using QC-BSA and cell lysate. QC-BSA in a mixture with cell lysate was incubated with 8 for 14 h in the dark and subsequently quenched with QCOAc. The samples were transferred to Amicon Ultra centrifugal filters and washed with PBS three times. The concentrated samples were incubated with Pierce™ Streptavidin Agarose for 30 min. The resin was washed with PBS by spin filtration seven times (Washes 1-7). Following the washes, the resin was suspended in PBS and irradiated with 365 nm light by a handheld lamp for 5 min and the supernatant was eluted (Elution 1). The irradiation was repeated three more times with additional PBS each time (Elutions 2-4). The beads were then stripped of all remaining protein with 8 M guanidine HCl (pH = 1.5, 4 washes) and this was concentrated on an Amicon Ultra centrifugal filter (Elution B). Washes 1; 2,3; 6,7 and Elutions 1; 2; B were separated by gel electrophoresis and visualized with Coomassie staining. B) The general procedure from Figure 5A was followed with the following quantities of reagents: QC-BSA or BSA (100 μL in PBS, 1 mg/mL), 8 (100 μL, 50 μM in PBS), QCOAc (2.5 μL, 200 mM in DMSO), PBS rinse (200 μL, then twice with 350 μL), Pierce Streptavidin Agarose (100 μL), PBS washes (50 μL each, lanes Wash 1; 2,3; 6,7), elutions (50 μL PBS each, lanes Elution 1 and 2), guanidine HCl (50 μL washes, Elution B). C) Protein from Wash 1 and Elution 1 of BSA and QC-BSA samples from Figure 5B were subjected to ESI intact mass analysis. Unmodified BSA: BSA with no QC modifications. The numbers represent the number of QC moieties attached to BSA. D) The general procedure from Figure 5A and 5B was performed in the presence of bacterial cell lysate (10 μL, 4.94 g/mL) and K₃Fe(CN)₆ (10 μL, 22 mM). K₃Fe(CN)₆ and lysate were added sequentially to the solutions of QC-BSA or BSA before addition of 8. After QC ligation with 8, the capture, wash, and elutions sequence depicted in Figure 5A was performed. Lanes are as described in Figure 5B.

Scheme 1. The quadricyclane (QC) ligation

The QC ligation is a formal [2σ + 2σ + 2π] reaction between QC (1) and nickel bis(dithiolene) (2) to form adduct 3. This work describes the photocleavage of 3 with UV light to produce norbornadiene (4) and the original nickel bis(dithiolene) 2 applied in a biological context.