Rapid and robust cysteine bioconjugation with vinylheteroarenes

Methods for residue-selective and stable modification of canonical amino acids enable the installation of distinct functionality which can aid in the interrogation of biological processes or the generation of new therapeutic modalities. Herein, we report an extensive investigation of reactivity and stability profiles for a series of vinylheteroarene motifs. Studies on small molecule and protein substrates identified an optimum vinylheteroarene scaffold for selective cysteine modification. Utilisation of this lead linker to modify a number of protein substrates with various functionalities, including the synthesis of a homogeneous, stable and biologically active antibody–drug conjugate (ADC) was then achieved. The reagent was also efficient in labelling proteome-wide cysteines in cell lysates. The efficiency and selectivity of these reagents as well as the stability of the products makes them suitable for the generation of biotherapeutics or studies in chemical biology.


General experimental
All solvents and reagents were used as received unless otherwise stated. Ethyl acetate, methanol, dichloromethane, acetonitrile and toluene were distilled from calcium hydride.
Diethyl ether was distilled from a mixture of lithium aluminium hydride and calcium hydride.
Petroleum ether refers to the fraction between 40-60 °C upon distillation. Tetrahydrofuran was dried using Na wire and distilled from a mixture of lithium aluminium hydride and calcium hydride with triphenylmethane as indicator.
Non-aqueous reactions were conducted under a stream of dry nitrogen using oven dried glassware. Temperatures of 0 °C were maintained using an ice-water bath. Room temperature (rt) refers to ambient temperature.
Yields refer to spectroscopically and chromatographically pure compounds unless otherwise stated. Reactions were monitored by thin layer chromatography (TLC) or liquid chromatography mass spectroscopy (LC-MS). TLC was performed using glass plates precoated with Merck silica gel 60 F254 and visualized by quenching of UV fluorescence (λmax = 254 nm) or by staining with potassium permanganate or para-anisaldehyde. Retention factors (Rf)  Reverse-phase flash column chromatography was carried out using a Combiflash Rf200 automated chromatography system with Redisep® reverse-phase C18-silica flash columns (20-40 μm).
Proton and carbon nuclear magnetic resonance (NMR) were recorded using an internal deuterium lock on Bruker DPX-400 (400 MHz,101 MHz), Bruker Avance 400 QNP (400 MHz,101 MHz) and Bruker Avance 500 Cryo Ultrashield (500 MHz,126 MHz). In proton NMR, chemical shifts (δH) are reported in parts per million (ppm), to the nearest 0.01 ppm and are referenced to the residual non-deuterated solvent peak (CHCl3: 7.26,CHD2OD: 3.31,HOD: 4.79 Protein LCMS was performed on a Xevo G2-S TOF mass spectrometer coupled to an Acquity UPLC system using an Acquity UPLC BEH300 C4 column (1.7 μm, 2.1 × 50 mm). H2O with 0.1% formic acid (solvent A) and 95% MeCN and 5% water with 0.1% formic acid (solvent B), were used as the mobile phase at a flow rate of 0.2 mL/min. The gradient was programmed as follows: 95% A for 0.93 min, then a gradient to 100% B over 4.28 min, then 100% B for 1.04 minutes, then a gradient to 95% A over 1.04 min. The electrospray source was operated with a capillary voltage of 2.0 kV and a cone voltage of 40 or 150 V. Nitrogen was used as the desolvation gas at a total flow of 850 L/h. Total mass spectra were reconstructed from the ion series using the MaxEnt algorithm preinstalled on MassLynx software (v4.1 from Waters) according to the manufacturer's instructions.
Non-reducing Tris-Glycine SDS-PAGE with 12% acrylamide with 4% stacking gel was performed as standard. Broad range molecular weight marker (10-200 kDa, New England BioLabs) was run in all gels. Samples were prepared by mixing with loading dye and heated to 90 °C for 5 minutes. Loading dye containing β-mercaptoethanol was used to prepare samples under reducing conditions. Gels were run at constant voltage (160 V) for 70 min to 90 min in ×1 Laemmli running buffer. All gels were stained with Coomassie brilliant blue dye and imaged on a Syngene gel imaging system.
Monoclonal antibodies were deglycosylated and reduced prior to LCMS analysis. This was typically performed by adding 0.1 µL of peptide:N-glycosidase F (PNGase F; New England BioLabs Catalogue number P0704S) to a solution of antibody (10 µL at 1 μM) and was left to stand at rt for 15 h. To this solution, TCEP•HCl (1 µL, 5 mM in H2O) was added and was left to stand at rt for 10 minutes before analysis.
UV-visible (UV-vis) spectrums were obtained using a NanoDrop™ One spectrophotometer (ThermoFisher). Raw data was plotted using GraphPad Prism software (version 8). The following equation 1,2 was used to calculate fluorophore-to-antibody ratio for AlexaFluor488containing antibodies, where ε280 = 215380 M -1 cm -1 is the molar extinction coefficient trastuzumab at 280 nm; ε495 = 71000 M -1 cm -1 is the molar extinction coefficient for AlexaFluor488 at 495 nm; Abs495 and Abs280 are absorbance at 495 nm and 280 nm, respectively. A correction factor of 0.11 was used to account for AlexaFluor488 absorbance at 280 nm. After the first 1 H NMR spectrum (with water suppression) was acquired, subsequent measurements were taken every ~10 minutes for all nucleophile-vinylheteroarene combinations, apart from Table S1, Entries 2-3 and 11-15, where measurements were taken every ~15 seconds. The vinyl peaks at 5.52 ppm, 5.75 ppm, and 5.96 ppm were integrated to determine the concentration of substrates 1, 2 and 3, respectively.
The following equations were used to determine second order rate constants k2. 2.2. Cysteine reactivity with vinylheteroarenes in CD3OD/NaPi (pH 8, 50 mM) Figure S1. Kinetic data used to calculate second order rate constants for the reaction of Boc-Cys-OMe with vinylpyridine 1 in 3:7 CD3OD/NaPi (pH 8, 50 mM in D2O). Two measurements were taken (plots a and b) and averaged.       was prepared. This solution was transferred to an NMR tube, and the atmosphere was purged with argon gas before the tube was sealed. The samples were placed in a water bath at 37 °C for 10 days. 19 F NMR spectra was acquired every day, and the integral of 5, 6 or 7 were compared with that of an internal standard (sodium trifluoroacetate) in order to determine the amount of starting material. The CD3CN co-solvent was required to ensure solubilisation of substrates in an aqueous solution. 3.2. Stability data for pyrimidine 5 Figure S31. Representative 19 F NMR spectra for the stability study of pyrimidine 5. The stacked spectra are offset by an angle of 10°. 3.3. Stability data for triazine 6 Figure S32. Representative 19 F NMR spectra for the stability study of triazine 6 The stacked spectra are offset by an angle of 10°. 3.4. Stability data for succinimide 7 Figure S33. Representative 19 F NMR spectra for the stability study of succinimide 7. The stacked spectra are offset by an angle of 10°. A number of new fluorinated peaks emerged over the ten-day incubation period.

Reduction of human serum albumin
The purity of commercially available recombinant human serum albumin (HSA; purchased from Sigma Aldrich; product code A9731) was determined by protein LCMS. This revealed a mixture of the free Cys-34 form (66445 Da) and the cysteinylated form (i.e. disulfide bonded with cysteine; 66566 Da).

Reduction of cysteine-engineered antibody mAb1
Based on a literature protocol, 5

SDS-PAGE analysis of mAb1 conjugates
SDS-PAGE analysis of mAb1 and mAb1 conjugates under non-reducing (NR) and reducing (R) conditions. Analysis of mAb1-8, mAb1-12 and mAb1-13 reveals that conjugates are found predominantly as the "full antibody" forms (~146 kDa), with all native interchain disulfide bonds present. In-gel fluorescence of Alexa Fluor 488 conjugate mAb1-8-13 under reducing conditions revealed the linker modification to be on the heavy chain (~51 kDa), consistent with mass spectrometry analysis; fluorescence was not observed for the light chain (~23 kDa). Figure S47. SDS-PAGE analysis with 12% acrylamide gel under non-reducing (NR) and reducing (R) conditions. SDS-PAGE was analysed by in-gel fluorescence and coomassie brilliant blue staining. MW=molecular weight marker.
An analogous stability study was also conducted using human serum instead of human plasma ( Figure S52).   For cysteine blockade studies, MCF7 cell lysate (50 µL, 1 mg/mL) was pre-incubated with iodoacetamide (20 mM) at rt for 1 h, prior to the addition of vinylpyrimidine-alkyne 8.