A Dual-Enzyme Cleavable Linker for Antibody-Drug Conjugates

recorded with a Micromass Q-TOF mass spectrometer or a Waters LCT Premier Time of Flight mass spectrometer. Mass values are reported within the error limits of ±5 ppm mass units. ESI refers to the electrospray ionisation technique


General Experimental Details
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 (PE) refers to the fraction between 40-60 °C upon distillation. Tetrahydrofuran (THF) 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 pre-coated with Merck silica gel 60 F254 and visualised by quenching of UV fluorescence (λmax = 254 nm) or by staining with potassium permanganate. Retention factors (Rf) are quoted to 0.01.
High resolution mass spectrometry (HRMS) measurements were recorded with a Micromass Q-TOF mass spectrometer or a Waters LCT Premier Time of Flight mass spectrometer. Mass values are reported within the error limits of ±5 ppm mass units. ESI refers to the electrospray ionisation technique.
Protein LC-MS 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 × 50.5 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 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. Trastuzumab samples were deglycosylated with PNGase F (New England Biolabs) prior to LC-MS analysis.

SDS Page Analysis
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 (10 μL of 0.4 mg/mL) were prepared with reducing loading dye (10 μL, containing β-mercaptoethanol) and heated to 70 °C for 2 min. Gels were run at constant voltage (180 V) for 60 min in 1 × Laemmli running buffer (LRB). All gels were stained with Coomassie dye and imaged on a Syngene gel imaging system. Figure S2: SDS-PAGE analysis of (A) ADC 1 and ADC 2 in 12% acrylamide gel. Lane markings: Tras (NR) = trastuzumab, non-reduced (loaded with no β-mercaptoethanol and not heated), Tras (R) = trastuzumab, reduced.