Enhanced Aqueous Suzuki–Miyaura Coupling Allows Site-Specific Polypeptide 18F-Labeling

The excesses of reagents used in protein chemistry are often incompatible with the reduced or even inverse stoichiometries used for efficient radiolabeling. Analysis and screening of aqueous Pd(0) ligand systems has revealed the importance of a guanidine core and the discovery of 1,1-dimethylguanidine as an enhanced ligand for aqueous Suzuki–Miyaura cross-coupling. This novel Pd catalyst system has now allowed the labeling of small molecules, peptides, and proteins with the fluorine-18 prosthetic [18F]4-fluorophenylboronic acid. These findings now enable site-specific protein 18F-labeling under biologically compatible conditions using a metal-triggered reaction.


General considerations
All NMR spectra were recorded on Bruker DPX200, DPX300, DPX400, DQX400 or AVN400 spectrometers. Proton and carbon-13 NMR spectra are reported as chemical shifts (δ) in parts per million (ppm) relative to the solvent peak as the internal standard ( 1 H NMR: D 2 O = 4.79,; DMSO-d 6 = 2.50 and 13 C NMR: DMSO-d 6 = 39.5). Fluorine-19 NMR spectra are referenced relative to CFCl 3 in CDCl 3 . Coupling constants (J) are reported in units of hertz (Hz). The following abbreviations are used to describe multiplicities -s (singlet), d (doublet), t (triplet), q (quartet), quin (quintet), m (multiplet) brs (broad singlet). High resolution mass spectra (HRMS, m/z) were recorded on a Bruker MicroTOF spectrometer using positive electrospray ionization (ESI+/ESI-). Infrared spectra were recorded either as the neat compound or in a solution using a Bruker Tensor 27 FT-IR spectrometer. Absorptions are reported in wavenumbers (cm -1 ) and only peaks of interest are reported. Melting points of solids were measured on a Griffin apparatus and are uncorrected. IUPAC names were obtained using the ACD/I-Lab service. Solvents were purchased from Fisher, Rathburn or Sigma-Aldrich. When dry solvents were required they were purified by expression through an activated alumina column built according to the procedures described by Pangborn and Grubbs.1 Chemicals were purchased from Acros, Alfa Aesar, Fisher, Fluorochem, Sigma-Aldrich and used as received. Reactions were monitored by thin-layer chromatography (TLC) carried out on Merck Kiesegel 60 F254 plates, silica gel column chromatography was performed over Merck silica gel C60 (40-60 µm). Deionized water was used for chemical reactions and Milli-Q purified water for protein manipulations. Protein concentrations were determined by BCA (bicinchoninic acid) assay (Pierce) and/or OD280. SDS-PAGE gels were run using pre-cast gels purchased from Invitrogen (NuPAGE 10 % Bis-Tris gel), and stained using InstantBlueTM (Expedeon).

Protein mass spectrometry
Liquid chromatography-mass spectrometry (LC-MS) was performed on a Micromass LCT (ESITOF-MS) coupled to an Agilent 1100 Series HPLC using a Phenomenex Jupiter 5 µm C4 column (100 × 4.6 mm). Water:acetonitrile, 95:5 (solvent A) and acetonitrile (solvent B), each containing 1% formic acid by volume, were used as the mobile phases at a flow rate of 1.2 mL/min. The gradient was programmed S3 as follows: 5% B for 1 min to desalt and then a linear gradient to 95% B over 10 min followed by 95% B for an additional 3 min. A linear gradient over 3 minutes back to 5% B followed by 3 minutes 5%B to re-equilibrate the column. The electrospray source was operated with a capillary voltage of 3.2 kV and a cone voltage of 25 V. Nitrogen was used as the nebulizer and desolvation gas at a total flow of ca 600 L/hr. For reaction analysis, the mass spectra for all protein material in the spectra were combined using MassLynx software (v. 4.1 from Waters). Mass spectra were then calibrated using a calibration curve constructed from a minimum of 15 matched peaks from the multiply charged ion series of equine myoglobin obtained at a cone voltage of 25V. The calibrated, combined ion series was deconvoluted using a maximum entropy (MaxEnt 1) algorithm that is preinstalled on the MassLynx software. For SBL-ArI, the output Mass Ranges were set to 20,000-30,000, damage mode was set to Uniform Gaussian width at half height as 0.6 Da. These MaxEnt 1 settings gave clear final deconvoluted mass spectrometry and helped to resolve the product (SM -32) from side peaks when the conversion is low. 1,1-Dimethylguanidine sulfate (L3(H 2 SO 4 ) 0.5 , 13.6 mg, 0.10 mmol) was mixed with NaOH (0.1 M, 2 mL) and water (1 mL). Palladium acetate (11.2 mg, 0.050 mmol) was then added and the suspension was stirred at 65°C for 45 minutes, vortexing intermittently to give an orange-brown solution. After this period the solution was cooled and diluted with water to a total volume of 5 mL to give a 10 mM catalyst stock solution. 1,1,3,3-Tetramethylguanidine (L4, 11.5 mg, 0.10 mmol) was mixed with NaOH (0.1 M, 1 mL) and water (2 mL). Palladium acetate (11.2 mg, 0.050 mmol) was then added and the suspension was stirred at 65°C for 45 minutes and vortexed intermittently to give a brown solution, which was diluted with water to a total volume of 5 mL to give a 10 mM catalyst stock solution.  Scheme S1 Synthesis of AXAVNTANST peptide (X = p-I-Phe)

Peptide synthesizer settings
Fmoc Deprotection. The resin-bound Fmoc peptide (initial weight 446 mg, 0.25 mmol) was treated with 20% piperidine in NMP (V/V) for 15 min (twice). The resin was washed with DMF and DCM.

Cleavage from Resin and Deprotection of t Bu Group.
Starting from 565 mg of crude material, the peptide was cleaved off the resin and deprotected in TFA/TIPS-H/water (95/2.5/2.5, V/V, 14 mL) at RT for 3 hours. After filtration and washing with TFA (3 × 3 mL), the filtrate was concentrated to c.a. 4 g, then treated with cold diethyl ether (24 mL). After filtration and washing with diethyl ether (3 × 3 mL), the filtrate was concentrated again to 1 g. Diethyl ether (3 mL) was then added to the residue. The precipitate was filtered and washed with diethyl ether (3 × 3 mL). The combined solids (150 mg) were dissolved in 3% MeOH-water containing 0.1% TFA (100 mL) and loaded on to a Biotage RP 100 g column for purification (2-30% MeCN-water with 0.1% TFA). The fractions were analyzed by HPLC and the product collected by rotavapor concentration. The residue was re-suspended in deionized water (3 mL) and lyophilized to give the final product (121 mg in total, 0.11 mmol, yield: 43% from Thr-resin

MS-MS analysis
All the peptide fragmentation b and y ions were found by MS-MS analysis.  2.4 SMC of decamer peptide: reference compound preparation The iodo-decamer peptide (4, 11.2 mg, 0.010 mmol) and 4-fluorophenylboronic acid (1, 14.0 mg, 0.10 mmol) were sonicated in pH 8.0 phosphate buffer (50 mM, 48 mL) at 50 °C to give a clear solution. After cooling down to 37 °C, (L3) 2 Pd(OAc) 2 (10 mM, 1.0 mL, 0.2 mM in reaction solution) was added and the reaction mixture was incubated at 37 °C for 30 min. Additional (L3) 2 Pd(OAc) 2 (10 mM, 1.5 mL) was added. The resulting reaction mixture was incubated for another 30 min before the addition of 3-mercaptopropionic acid (5 µL in 1 mL of water, 57 mM, 1 mL). The reaction mixture was loaded on to a 12-g C18 column and eluted with 2-30% acetonitrile in water (+0.1% formic acid). The product containing fractions (monitored by LCMS) were combined and subjected to another 30-g C18 column and eluted with 5-80% MeOH in water (+0.1% formic acid). The product was concentrated to dryness and re-suspended in deionized water (1 mL), lyophilized to give 12 as a white powder (  3-mercaptopropionic acid (57 mM, 5.7 eq to Pd) for 5 minutes. The crude reaction mixture was then analyzed by LCMS and the conversion was measured by the mass intensity ratio between the product and the starting material.
The yield based on the peptide conversion is given in the table below. (37 °C, 30 min)

SMC reference compound preparation
The iodo-octamer peptide (5, 9.9 mg, 0.010 mmol) and 4-fluorophenylboronic acid (1, 7.0 mg, 0.05 mmol) were sonicated in pH 8.0 phosphate buffer (50 mM, 48 mL) at 60 °C to give a clear solution. After cooling down to 37 °C, (L3) 2 Pd(OAc) 2 (10 mM, 1.0 mL, 0.2 mM in reaction solution) was added and the reaction mixture was incubated at 37 °C for 30 min. A freshly made dithiothreitol (DTT) solution (1 M, 60 µL) was then added. The resulting reaction mixture was incubated for 10 min before the reaction mixture was loaded on to a 12-g C18 column and eluted with 2-20% acetonitrile in water (+0.1% formic acid). The product containing fractions (monitored by LCMS) were combined and subjected to semi-prep HPLC purification (Synergi 4u Fusion-RP 80A, 0-30% acetonitrile in water (+0.1% formic acid). The product containing fractions were concentrated to dryness and re-suspended in deionized water (1 mL), then lyophilized to give 13 as a white powder (

Optimization of small molecular SMC with reversed stoichiometry (non-radioactive)
General method: The palladium catalyst (10 mM, up to 10 µL) was added to a solution (200 µL) containing 4-fluorophenylboronic acid (1, 0.05 mM) and 3 (0.10 mM). The mixture was stirred at the assigned temperature for the assignedtime before HPLC analysis. The yield was obtained from the UV integration of the product 11 using a standard calibration curve.  Commercial mCPBA (4.7 g, 77% max, from Sigma-Aldrich) and anhydrous sodium sulphate (10 g) were suspended in DCM (20 mL). The liquid was decanted and the solid was washed with DCM (2×15 mL). The combined DCM solutions were filtered through an anhydrous sodium sulphate pad (1 g). The filtrate obtained was titrated (excess NaI, then sodium thiosulphate). This mCPBA solution (0.278 M) was stored in a -18°C freezer and no obvious decomposition was observed after 4 weeks. Crystallisation occurs when cool. It is necessary to warm up the solution to RT and dissolve all the crystals before use.
A 100-mL RB flask was charged with diiodobenzene (3.63 g, 11 mmol), DCM (10 mL) and a solution of mCPBA in DCM (0.278 M, 37 mL, 10 mmol) under N 2 . The solution was cooled in an ice-water bath and triflic acid (0.80 mL, 9 mmol) was added dropwise over 2 min. Once the addition was complete, the ice bath was removed and the reaction mixture was stirred at RT for 45 min. The resulting yellow-green suspension was cooled to 0°C. Triflic acid (1.85 mL, 2.1 mmol) was added, followed by the addition of iodobenzene (1.23 mL, 11 mmol). The mixture was vigorously stirred at 0°C for 45 min, then RT for 10 min before dilution with DCM (30 mL) and stirred until most of the solid was dissolved. The reaction mixture was filtered through celite (3 g) under N 2 and washed with DCM (2×5 mL). The filtrate was concentrated to give 12.8 g of brown solid, which was suspended in diethyl ether (50 mL) in an ultrasound bath for 3 min, then subjected to centrifugation (20°C, 3000 G, 5 min). The supernatant was then discarded. The sonication-centrifugation ether wash was repeated twice (30 mL, then 10 mL). The solid was dried in vacuo to give a grey solid 2.9 g, crude yield 43%. Further recrystallization (1.33 g) from isopropanol (29 mL) gave the product (0.97 g). Overall yield 31%. 1 Figure S3 1 H NMR of Di(4-iodophenyl)iodonium triflate (5). 5

Cold optimization of borylation
Borylation was optimized with a reaction time of up to 20 min by varying solvent and base. All reactions were carried out with 6 (0.40 mmol), 7 (0.60 mmol) and base (0.80 mmol) in 4 mL of solvent. The crude reaction mixture was heated or irradiated with microwave at the 60-90°C for 5-90minutes before diluted in methanol (to 50 mL) for HPLC analysis.
A typical mass spectrum is given below.  At higher [Pd] higher product/sm ratios were obtained. However, high Pd loadings (4 mM) caused substantial protein degradation /precipitation. The SDS Page gel analysis suggested the degradation of protein under such conditions. The relative concentration of the remained protein was estimated against an SBL-ArI sample by gel-densitometry and used to correct all the yields/conversions of the SMC. A maximum 10% protein conversion was obtained with 2 mM palladium catalyst.

Protein concentration
In this experiment, boronic acid 1/protein 2 ratio was set to 2 while [2] varies. The maximum achievable protein concentration is between 0.26 -0.3 mM, so 0.2 mM was set as the optimal protein concentration. 6

S18
packed bed of a PD SpinTrap G25 equilibrated with pH 8.0 50 mM phosphate buffer (a stacker volume of 7.6 µL was applied for the sample volume to reach 140 µL). The sample was eluted by centrifugation at 800 × G for 2 minutes. The PD SpinTrap purification was repeated 4-6 times (including the initial purification). A sample of the collected product (100 µL) was mixed with distilled nitric acid (200 µL) and diluted with deionised water to 10.0 mL for palladium content analysis. Palladium was analysed at 340.458 nm using a Perkin Elmer Optima 5300 DV ICP-OES (analysed in axial view, Argon flow rates: Plasma 15 L/min; Aux 0.45 L/min; Neb 0.75 L/min; RF power 1400W). HPLC analysis was performed with a Dionex Ultimate 3000 dual channel HPLC system equipped with shared autosampler, parallel UV-detectors and LabLogic NaI/PMT-radiodetectors with Flowram analog output. Radio-TLC was performed on Merck Kiesegel 60 F254 plates. Analysis was performed using a plastic scintillator/PMT detector. All isolated radiochemical yields quoted are decay corrected. Radiochemical yields are calculated from activity isolated from SPE purificatioin relative to the activity used in the reaction. For small molecule and peptide SMC labeling, the radiochemical yields are calculated from the HPLC radiotrace. The product fraction was collected and re-injected to determine the radiochemical purity (RCP).

Radiosynthesis of [ 18 F]1
The solution of [ 18 F]7 in DMSO was transferred into a 5-mL vial containing KOAc (7 mg), B 2 (OH) 4 (7, 4.5 mg) and Pd(dppf)Cl 2 (0.7 mg) and heated at 90°C for 20 min 8 then cooled down for 1 min. The reaction mixture was diluted with water (4.6 mL) and transferred through two successive Sep-Pak plus C18 cartridges (360 mg each, activated with 5 mL of methanol and 10 mL of water) over 2 min. The cartridges were rinsed with water (5 mL) and 20% methanol/water (12 mL), dried by passing 5 mL of air through. Methanol (1.4 mL) was slowly injected to the air-dried cartridges and the filtrate was discarded. Another 0.9 mL of methanol was passed through the cartridges and the product [ 18 F]1 was collected. The product was concentrated at 90°C with a stream of nitrogen and resolubilized in 50 mM pH 8.0 phosphate buffer (200 µL) for further SMC study. Radiochemical yield 28-39% (typically 70 -140 MBq).
To determine the specific activity, the above [ 18 F]1 (~ 1 MBq, 20 µL) was injected to the HPLC and the chemical concentration was determined using the UV integration at 217 nm. The above protocol gave a specific activity of 9.7 GBq/µmol (ranging 3.1-14.4 GBq/µmol).

SMC of [ 18 F]1 with peptides
The decamer 4 or octamer 5 peptide stock solution (0.10-0.60 mM) was prepared in pH 8.0 buffer and stored at 37 °C prior to use.

Protein 18 F-labelling and purification (with control)
A mixture of SBL-ArI (7.3 mg/mL, 72.4 µL, 20 nmol, ~0.2 mM in reaction mixture of a total volume 97.2-112.2 µL), (L3) 2 Pd(OAc) 2 (10 mM, 20 µL, 200 nmol) and [ 18 F]1 (5~20 µL, ~15 MBq) was mixed and shaken at 37°C for 30 min. Then 3-MPrAc (0.5% in water, 20 µL, 1.2 µmol) 10 was added to dissolve the precipitation. After a short incubation time (1 minute), the reaction mixture sample was applied slowly in the middle of the packed bed of a PD SpinTrap equilibrated with pH 8.0 buffer (a stacker volume was applied for sample volume to reach 140 µL). The sample was eluted by centrifugation 800 × G for 2 minutes. The collected product was subjected to HPLC analysis after activity measurement (0.2-0.5 MBq). Radioactivity in the trap was also measured to work out the radiochemical yield. The decay corrected RCY obtained were 2-5%. The control experiment was carried out same as the protein labelling method except the Palladium catalyst was replaced with pH 8.0 buffer. The fluorophenylboronic acid was the only radioactive component detectable, which showed negligible non-specific FPBA binding.

Preparation of [ 18 F]1 without purification of intermediate 7
The purification of the radiolabelling intermediate [ 18 F]7 requires two C18 cartridges for purification and formulation. This takes 20-30 minutes in a typical experiment. We also tested the preparation of [ 18 F]1 without such purification. Thus, the crude fluorination mixture was transferred directly in to the borylation mixture (B 2 (OH) 4 , Pd(dppf)Cl 2 and KOAc). After heating at 90°C for 20 min and SPE purification, [ 18 F]1 was obtained in a 5-20% radiochemical yield with 59-80% radiochemical purity.

Method 2
A solution of the bis(4-iodophenyl)iodonium triflate (6, 5 mg) in anhydrous DMF (200 µL) was added to the dry [ 18 F]fluoride. The reaction mixture was heated at 145°C for 20 min, then cooled down for 1 min. The reaction mixture was transferred to a 5-mL vial containing KOAc (7 mg), B 2 (OH) 4 (8, 4.5 mg) and Pd(dppf)Cl 2 (0.7 mg) and heated at 90°C for 20 min then cooled down for 1 min. The reaction mixture was then diluted with water (4.6 mL) and purified through three successive Sep-Pak plus C18 cartridges (360 mg each, activated with 5 mL of methanol and 10 mL of water) over 2 min. The cartridges were rinsed with water (5 mL) and 20% methanol/water (20 mL) and dried by passing 5 mL of air through. 50% methanol/water (2 mL) was slowly injected in to the air-dried cartridges and the filtrate was discarded. Another 2 mL of 50% methanol/water was passed through the cartridges and the product S27 [ 18 F]1 was collected. The product was analyzed by HPLC, giving a 80% RCP and decay corrected RCY of 5% over 2 steps.
The product was concentrated at 90°C with a stream of nitrogen and resolubilized in 50 mM pH 8.0 phosphate buffer (200 µL) for use in SMC reactions.

Review of Prior Metal-Mediated Methods.
Many currently employed Cu(I)-catalyzed triazole-forming protocols used for introducing 18 F to small peptides still use great excesses and often elevated temperatures (60 ˚C and higher) that are not compatible with many proteins. As a result perhaps of these difficulties and those outlined in the main text, effective site-selective applications of this method to proteins are rare. For example, to the best our knowledge, there is only one example of direct, site-selective 18 F prosthetic incorporation into proteins using the Cu(I)-catalyzed triazole reaction 11 and a second example that is two-step process that relies initially on Cys-modification chemistry 12 and then a second triazole-forming step. The single direct siteselective Cu(I) triazole method 11 is an overall less efficient process than the Pd-mediated method that we present here. Other methods have been used to label e.g. lysines indiscriminately and without site control (thus generating a mixed population of proteins) but the goal of this work is to create pure proteins with control of site and label.