A fluorescent photoaffinity probe for formyl peptide receptor 1 labelling in living cells

Fluorescent ligands for G-protein coupled receptors (GPCRs) are valuable tools for studying the expression, pharmacology and modulation of these therapeutically important proteins in living cells. Here we report a fluorescent photoaffinity probe for Formyl peptide receptor 1 (FPR1), a critical component of the innate immune response to bacterial infection and a promising target in inflammatory diseases. We demonstrate that the probe binds and covalently crosslinks to FPR1 with good specificity at nanomolar concentrations in living cells and is a useful tool for visualisation and characterisation of this receptor.


General reagents and equipment
All glassware used was dried in an oven prior to the reaction. Unless otherwise stated all commercial reagents were used as received and reactions were performed in an inert atmosphere using N 2 . All reactions were monitored by analytical thin layer chromatography using aluminium TLC plates coated with silica gel 60F 254 purchased from Merck. TLC plates were visualised by UV light (λ=254 nm) and an aqueous potassium permanganate dip.
Flash column chromatography was performed using silica gel (60 Å, 40 -63 micron) purchased from Merck. 1 H NMR and 13 C NMR spectra were recorded using a Bruker 400 ultra shield spectrometer (400 MHz) in CDCl 3 (reference of 7.26 for 1 H NMR and 77.2 for 13 C NMR) and MeOD (reference of 4.78 and 3.31 for 1 H NMR, and 49.15 for 13 C NMR). All chemical shifts are expressed in parts per million downfield from tetramethylsilane. Peak splittings are noted as singlet (s), doublet (d), triplet (t), quartet (q), pentet (p) and multiple (m) and combinations of the stated J coupling constants are recorded to the nearest 0.5 Hz.
Low resolution electrospray (ES+) ionisation mass spectra were obtained on a Bruker Amazon mass spectrometer. High resolution ES+ mass spectra were obtained on a Bruker MaXis Impact mass spectrometer.
For solid phase peptide synthesis, all amino acids and other reagents were purchased from Novabiochem and Sigma Aldrich and were used without further purification.
Fritted polypropylene tubes (10 mL) were purchased from Biotage and used as the vessels for peptide synthesis. Dissolution of reagents and peptides was achieved by agitation through the use of a Stuart rotator. 2-Chlorotrityl chloride resin (loading 1.33 mmol/g) was used as the stationary phase for peptide synthesis.
Analysis of peptides during SPPS was performed using a Thermo Ultimate 3000 UHPLC, Bruker Amazon Speed ion trap mass spec with a Phenomenex Aeris Peptide XB C18 column (100 x 2.1mm, 2.6um particle size). Gradient from 0.1% TFA/ 2% MeCN (ν/ν) in water to 0.1% TFA/ 98% MeCN (ν/ν) in water with a flow rate of 0.85ml/min. Analysis of final peptides was performed using an Agilent 1290 Infinity with Diode Array Detection with an Ascentis Peptide ES C18 column (100 x 2.1mm, 2.7um particle size). Gradient from 0.1% TFA/ 5% MeCN (ν/ν) in water to 0.1% TFA/ 95% MeCN (ν/ν) in water with a flow rate of 0.5ml/min.  H-Glu(OMe)-OH (10.0 g, 50.6 mmol) was dissolved in dioxane/water (2:1, 150 mL) and cooled to 0 °C. Boc 2 O (13.2 g, 60.7 mmol) and NaHCO 3 (10.6 g, 127 mmol) were added and the reaction mixture was warmed up to room temperature, before being stirred for 12 hr. The dioxane was removed under reduced pressure and the aqueous solution was washed with diethyl ether (50 mL). 1M HCl was added to adjust the pH to 3 and the resulting solution was extracted with ethyl acetate (2 × 50 mL). The organic solution was washed with water (50 mL) and brine (50 mL), and dried over anhydrous Na 2 SO 4 . Removal of the solvent under reduced pressure yielded the product 1 as a pale yellow oil (10.0 g, 38.3 mmol, 76%).

Synthesis of Fmoc-photo-Met
(S)-5-(tert-butoxy)-4-((tert-butoxycarbonyl)amino)-5-oxopentanoic acid (Boc-Glu(OMe)-Ot-Bu), 2  A 1M LiOH solution (15.8 mL, 15.8 mmol) was added dropwise to a solution of Boc-Glu(OH)-Ot-Bu (2.5 g, 7.9 mmol) in THF (50 mL) over 30 min. The reaction mixture was left to stir for 2 h before being cooled to 0 °C. 0.1M HCL was added dropwise to the solution until the pH was 5. The organic layer was extracted with ethyl acetate (2 × 50 mL), washed with brine (50 mL) and dried over anhydrous Na 2 SO 4 . Concentrating under reduced pressure yielded the product 3 as a white solid (2.3 g, 7.6 mmol, 96%).  (S)-tert-butyl-2-((tert-butoxycarbonyl)amino)-5-(methoxy(methyl)amino)-5-oxopentanoate (1.07 g, 3.08 mmol) was dissolved in toluene (50 mL) and cooled to −78 °C. Methylmagnesium bromide solution 3.0 M (2.1 mL, 6.34 mmol) was added dropwise over 30 min, after which the solution was warmed to −5 °C and stirred for three hr. The reaction was quenched with 0.1 M HCl (20 mL) and the organic solution extracted with ethyl acetate (3 × 20 mL). The solution was dried over anhydrous Na 2 SO 4 and concentrated under reduced pressure. The crude product was purified via column chromatography (10% ethyl acetate in hexane) to furnish the product 5 as a colourless oil (0.56 g, 1.84 mmol, 64%). The mixture was stirred overnight, being allowed to warm to room temperature. Nitrogen was subsequently blown through the reaction for one h to remove any ammonia. The solution was then filtered, washed with MeOH (20 mL) and concentrated under reduced pressure. The resulting oil was re-dissolved in dry MeOH (10 mL) and cooled to 0 °C. Et 3 N (1.4 mL, 9.9 mmol) was added dropwise to the solution whilst iodine (1.1 g, 4.3 mmol) was crushed and dissolved in dry MeOH (20 mL). The iodine solution was then added dropwise to the reaction solution until the colour stayed dark brown. After 1 h of stirring at 0 °C, EtOAc (10 mL) was added to the reaction mixture. The solution was washed with 1M HCl (10 mL), 10% Na 2 S 2 O 3 (10 mL) and brine (10 mL). The organic solution was dried over MgSO 4 and concentrated under reduced pressure to yield the product 6 as a pale yellow oil (940 mg, 3.00 mmol, 94%).  4M HCl (25 mL) was added to (S)-tert-butyl 2-((tert-butoxycarbonyl)amino)-4-(3-methyl-3H-diazirin-3yl)butanoate (250 mg, 0.798 mmol) in THF (25 mL) and the solution was stirred at room temperature until the starting material was consumed. The solvent was removed under reduced pressure and the resulting yellow powder re-dissolved in water/dioxane (1:2) (12 mL). NaHCO 3 was added to the solution until the pH was basic. FmocOSu (324 mg, 0.960 mmol) was dissolved in dioxane (1 mL) and added dropwise to the reaction, which was then stirred at room temperature for 24 h. The dioxane was removed under reduced pressure and water (5mL) added to the residue. 1M HCl was added until the pH reached 4 and following this the organic solution was extracted with EtOAc (3 × 10 mL). The combined organics were dried over Na 2 SO 4 and concentrated to give the crude product. Purification using column chromatography (20% EtOAc in hexane, 1% TFA) furnished the product 7 as a white solid (220 mg, 0.580 mmol, 73%).  2-Chlorotrityl chloride resin was swollen in DCM for 30 min. Fmoc-Lys(Dde)-OH (1 equiv.) and DIPEA (4 equiv.) were dissolved in dry DCM (2 mL). This solution was added to the swollen resin and rotated for 2 h. The resin was then washed with DCM/MeOH/DIPEA (17:2:1), DCM and DMF sequentially. The lysine residue was deprotected (Fmoc) in 20% v/v piperidine in DMF (2 mL for 3 min  3) and washed with DMF, DCM and DMF sequentially. Fmoc-Tyr(t-Bu)-OH (5 equiv.), Oxyma Pure (5 equiv.) and DIC (5 equiv.) were dissolved in minimal amount of DMF, added to the resin and r for 40 min. The resin was washed sequentially with DMF, DCM and DMF. The remaining amino acids were coupled using the same procedure (Fmoc-Nle, Fmoc-Phe, Fmoc-Leu, Fmoc-NLe). Following Fmoc deprotection of the final amino acid, the N-terminus was formylated: p-Nitrophenyl formate (5 equiv.) and DIPEA (10 equiv.) were dissolved in DCM (2 mL) and added to the resin which was subsequently stirred for 12 h. The resin was washed sequentially with DMF, DCM and DMF. The lysine side chain was deprotected (Dde) in 4% v/v hydrazine monohydrate in DMF (2 mL for 3 min  3) and washed with DMF, DCM and DMF sequentially. FITC (6 equiv.) and DIPEA (10 equiv.) were dissolved in the minimal amount of DMF, added to the resin and rotated for 12 h in darkness. The resin was washed sequentially with DMF, DCM and MeOH, and dried overnight in vacuo. The peptide was cleaved from the resin with 2.5% TIS, 2.5% water and 95% TFA and precipitated in cold diethyl ether. The isolated peptide was then purified using UV-directed HPLC. Gradient from 0.1% TFA/ 5% MeCN (ν/ν) in water to 0.1% TFA/ 95% MeCN (ν/ν) in water over 15 min. The peptide was obtained from lyophilisation as a yellow amorphous solid in 28% yield.  LB-agar media: 25 g L -1 LB freeze-dried powder (Fisher) and 15 g L -1 of Agar powder (Fisher) in H 2 O, sterilised in an autoclave for 20 min at 120 °C.

Buffers for protein and DNA analysis
Lysis buffer: 0.1% (w/v) SDS, 1% (v/v) Triton X-100, 150 mM NaCl, 50 mM Tris (pH 7.5), 1 × EDTAfree protease inhibitors. 300 ng of plasmid was mixed with 1 unit of BamHI and CutSmart buffer (×1) and made to 50 µL with H 2 O. The sample was incubated at 37 °C for 40 min and then stored at -20 °C. Analysis of this digestion was performed with the use of agarose gel electrophoresis using Tris-acetate gels. These were prepared by adding 1% w/v agarose to 40 mL of TAE buffer and heating for 45 s to near boiling. The solution was allowed to cool and 0.4 µL of SYBR stain was added. The solution was poured into a mould with a comb added and allowed to set. The set gel was placed in a gel tank and DNA samples were loaded in loading buffer. Gels were run with TAE buffer (diluted to ×1) at 400 V for 40 min.

Mammalian cell culture, photocrosslinking and lysis
Cells were incubated in a humidified atmosphere at 37 °C containing 5% CO 2. HEK293T cells were cultured in Dulbecco's Modified Eagle Medium (DMEM, Fisher) supplemented with 1% L-glutamine, 10% v/v fetal bovine serum (FBS) and 1% v/v Pen-Strep. Cells were detached using 1  trypsin in PBS.

Transient Transfection
HEK293T cells were seeded in a 6-well plate (2 × 10 5 cells/well) and incubated at 37 °C overnight. The plasmid containing FPR1 gene (0.7 µg/µL stock) and Turbofect (Fisher) were mixed with DMEM (no FBS) and incubated at room temperature for 15 min (1 µg of plasmid and 3 µL of Turbofect in 100 µL of media per well). The solution was added to the plated cells and incubated at 37 °C overnight. For mock transfection, the plasmid was omitted.
For 10 cm plate transfection for lysate preparation, the following modifications were used: 1.6×10 6 cells were seeded per plate; 8 µg plasmid and 24 µL Turbofect were mixed in 800 µL DMEM.

Probe Incubation, crosslinking and lysis on suspended cells
Transfected cells were washed with PBS and incubated in 1 mL of PBS with EDTA (per well) at 37 °C for 3 min to detach. The cells were then transferred into sterile Eppendorf tubes and centrifuged at 2000 rpm for 3 min at 4 °C to form pellets. Subsequently the cells were incubated with 1 mL of 10 nM probe in PBS at 0 °C for 30 min. Following incubation the cells were washed with PBS (1 mL × 2) and irradiated with UV light (365 nm) for 30 s using a UV LED device developed for diazirine crosslinking. 6 Samples were centrifuged at 2000 rpm for 3 min at 4 °C and the PBS solution removed. 200 µL of lysis buffer was added and the lysate chilled for 15 min. Cell debris was pelleted by centrifugation at 13,300 rpm for 15 min at 4 °C. Protein concentration was determined by the DC protein assay (Bio-Rad) using BSA to generate a standard curve. Lysates were kept at -80 °C until required.

Probe incubation and crosslinking experiments on lysates
Transfected cells from 10 cm plates were harvested by scraping in ice-cold PBS and centrifugation at 2000 rpm (500 ×g) for 3 min at 4 °C to form pellets. These were snap frozen and stored at -80 °C until needed. Pellets were thawed, resuspended in 0.5 mL PBS and sonicated 3 × 40% intensity, 10 s, with 30 s on ice in between. Intact cells were removed through centrifugation (500 ×g for 3 min at 4 °C) and the supernatant (total lysate) retained. Crude membrane fractions were prepared by centrifugation (17,000 ×g, 15 min, 4 °C) of the total lysate, removal of the supernatant ("soluble fraction") and resuspension of the pellet ("membrane fraction") in 500 µL PBS. Aliquots were flash frozen.
For competition labelling experiments, 50 µL aliquots of crude membrane fraction were thawed on ice, co-incubated with Probe-TAMRA (50 nM) and various competitors (fMLF, BocMLF or fMLFF at final concentrations of 5 µM, 20 µM, 5 µM respectively) or DMSO for 30 min on ice (final DMSO concentration of 2%). Membranes were pelleted by centrifugation (17,000 ×g, 15 min, 4 °C) and the supernatant discarded. Membranes were resuspended in 25 µL PBS, irradiated for 30 s at 365 nm, deglycosylated as described in section 2.6, and then analysed by SDS-PAGE.

Gel-and Western blot analysis of samples
Crosslinked cell lysate samples were analysed using gel-based fluorescent imaging. Proteins were separated by SDS-PAGE (180 V, 50 min) with 4% stacking gels and 12% resolving gels on a BioRad Mini-PROTEAN Tetra Cell system with 10 µL All blue standards (BioRad). Fluorescence was measured using a BioRad ChemiDoc MP Imaging System with a Cy3 filter.
For Western blot analysis, samples were separated by SDS-PAGE. A PVDF membrane (BioRad) was soaked in MeOH for 1 min, followed by transfer buffer for 1 min. Two squares of extra thick blot paper (BioRad) and the protein gel were soaked in transfer buffer for 2 min. The transfer sandwich was then prepared in a Trans-Blot Semi-Dry Transfer Cell (BioRad) in the following order: blot paper, PVDF membrane, gel, blot paper. The transfer was run using a BioRad Power PAC 1000 at 15 V for 30 min.
The membrane was soaked in 3% blocking buffer for 1 h at room temperature. The membrane was then incubated with the primary antibody in 10 mL blocking buffer for 2 h at room temperature, washed with 0.05% Tween in PBS (3 × 5 min), incubated with the secondary antibody in 10 mL blocking buffer for 1 h at room temperature and finally washed with 0.05% Tween in PBS (3 × 5 min).

Deglycosylation of FPR1
Cell lysate sample adjusted to 1 µg/ µL (50 µL, 50 µg). K 2 HPO 4 was added to 0.1 M, SDS was added to 1% and NP40 was added to 1%. The sample was agitated for 30 min at room temperature. 20 units of PNGase F was added to the sample and agitated for 1 h at room temperature. A further 20 units of PNGase F was added to the sample and agitated for 1 h at room temperature. Sample loading buffer was added and the sample stored at -20 °C. Deglycosylation was analysed by Western blot as described above.

Anti-FLAG Pull-Down
Cell lysate sample adjusted to 1 µg/ µL (200 µL, 200 µg). 50 µL of suspended anti-FLAG M2 magnetic beads (Sigma Aldrich, M8823) were transferred to an Eppendorf and washed three times with TBS, once with 0.1 M glycine (pH 3.5), three times with TBS and once with lysis buffer. The cell lysate sample was added to the beads and incubated overnight with agitation at 4 °C. The supernatant was removed to a new tube and the beads washed three times with lysis buffer. 40 µL of 3X FLAG-peptide (Sigma Aldrich, F4799) in TBS (600 ng/L) was added to the beads and incubated for 30 min with agitation at 25 °C. The elution mixture was removed to a new tube and the sample analysed by SDS-PAGE and Western blot as above.

Flow Cytometry
Cells were transfected and incubated with the probe as above (section 2.4). The binding of the probes was observed using a CytoFLEX S 4-laser flow cytometer. The cell samples were loaded in a 1.5 mL Eppendorf and 10,000 events were taken per sample at a flow rate of 30 µL/min. For probes containing a FITC group the 488 nm laser with a 525/40 band pass filter was used, for probes containing a TAMRA group the 561 laser with a 585/42 band pass filter was used. The data produced from the flow cytometer was then analysed using Kaluza.
To calculate a K d for Probe-TAMRA binding, FPR1 transfected and mock transfected cells treated with Probe-TAMRA at concentrations of 0, 1, 2, 5, 10, 20, 50, 100 and 200 nM were analysed by flow cytometry as described. This experiment was repeated in biological triplicate. Data were elaborated in Excel and Origin. Background values from mock-transfected samples were subtracted from data values for each concentration. The mean and standard deviation across the three replicates were then calculated for this "specific binding" and data fitted and plotted in Origin using a non-linear logistic fit.

Confocal Microscopy
Cells were plated on a glass plate and transfected as above (section 2.4.1), then incubated with the probe adhered to plate (35 mm glass-bottom plate). The cells were incubated with 1 mL of 10 nM probe in PBS at 0 °C for 30 min. The sample was then warmed to 37 °C and binding and internalisation of the probes was observed using a Zeiss LSM880 + Airyscan inverted confocal microscope. A DPSS 561 nm laser was used to view the TAMRA fluorescence with an objective of 20x. The data produced was then analysed using Zen.