Facile protein conjugation of platinum for light-activated cytotoxic payload release†

The novel Pt(iv) complex trans,trans-[Pt(N3)2(Py)2(OH)(OCO-(PEG)2-NHCSNH-Ph-NCS)] (Pt4) conjugates to the side chain of lysine amino acids in proteins under mild conditions. Reaction with myoglobin generated a bioconjugate that was stable in the dark, but released a Pt(iv) prodrug upon visible light irradiation. A similar procedure was used to conjugate Pt4 to the antibody trastuzumab, resulting in the first photoactivatable Pt(iv)-antibody conjugate, demonstrating potential for highly selective cancer phototherapy.


Materials and Methods
TBTU and formic acid were obtained from Fluka Chemicals and trifluoroacetic acid (TFA) from Fisher Scientific. trans,trans,trans-[Pt(N3)2(Py)2(OH)2] (Pt1, Py = pyridine) was synthesised according to published procedures. 1 Trastuzumab (Herceptin®,Roche,24.12 mg/mL in saline) was obtained from the Pharmacy Department at Guy's and St. Thomas' NHS Trust, London. All other chemicals were obtained from Sigma Aldrich and used without further purification. All H2O used in reactions was MilliQ ultrapure water to minimize unwanted metal contaminants. Silica gel used was technical grade, pore size 60 Å, 230-400 mesh, 40-63 μm particle size.
NMR spectra were recorded on a Bruker Avance III 500 MHz (for 1 H) spectrometer and referenced to the residual solvent signal of the solvent. ESI-HRMS spectra in positive mode were recorded on a Bruker microTOF instrument in the range of 200-3000 m/z. Electronic absorption spectra were recorded on a Varian Cary 300 UV-vis spectrophotometer in a quartz cuvette at 298 K using neat solvent as reference. Platinum content was analysed on an ICP-MS 7500cx (Agilent).
LC-MS experiments were carried out on Bruker Amazon X ion trap instrument connected online with an Agilent 1260 HPLC. An Agilent ZORBAX Eclipse XDB-C18 column (250×4.6 mm, 5 μm) was used with 0.7 mL/min flow rate. The mobile phase contained 0.1% v/v formic acid in H2O (solvent A) and in CH3CN (solvent B). Gradient 1 included 5 min of equilibration at 10% B at the start of the run, then concentration of B increased from 10% (5 min) to 80% (at 30 min, maintained until 35 min) and subsequently decreased to 10% (at 40 min).
FTICR-MS experiments were carried out on a 12 T Bruker solariX instrument.

Synthesis and characterisation data
No problems were encountered during this work. However, heavy metal azides are known to be shock sensitive detonators therefore extra care was taken during handling.
All synthesis, purification and analysis, with the exception of the irradiation studies, were carried out in the dark with minimal light exposure.
Similarly, 2 species were observed for this complex by NMR with a 80:20 ratio, identical coupling pattern, but slightly different chemical shift suggesting they belong to the same complex in different chemical environment. NMR peak assignments below are reported for the major species only.
Purification was monitored by TLC using 10% DCM in MeOH as a mobile phase. Conjugation to myoglobin 700 μL of a 17 μM myoglobin solution in water was treated with a 1 M sodium carbonate solution to reach pH 8.5-9 (pH paper). A 1.7 mM solution of Pt4 in DMSO was then added in 5 μL aliquots to the myoglobin solution in a microcentrifuge tube, vortexing the tube after each addition to ensure full mixing. For the 5 mol. equiv. batch, 34 μL of Pt4 was added in total; for the 1 mol. equiv. batch only 7.5 μL was added. The reaction mixture was then vigorously mixed and left to incubate at room temperature for 4 h, followed by purification by size-exclusion chromatography using a PD10 column (GE Healthcare) preconditioned with 25 mM ammonium acetate.

Size-exclusion HPLC
Size-exclusion HPLC analyses were carried out on an Agilent 1100 HPLC equipped with a DAD detector. An Agilent ZORBAX GF250 4-400 kDa size-exclusion column (250×4.6 mm, 4 μm) was utilised for the analysis, using 50 mM ammonium acetate in water as a mobile phase and a 1 mL/min flow rate. Detecting wavelength was set at 254 nm, reference wavelength at 360 nm.

Conjugation to trastuzumab
A PBS solution of trastuzumab (730 μL, 16.6 μM) was treated with 1 M sodium bicarbonate to reach pH 8.5, then 34 μL of a 1.7 mM DMSO solution of Pt4 (5 mol. equiv.) was added in small aliquots and vortexing the solution between additions to ensure good mixing. After the final addition, the reaction mixture was vortexed and then left to incubate at room temperature for 2 h, followed by purification and buffer exchange to 100 mM ammonium acetate using 8 cycles of ultracentrifugation (Vivaspin MWCO 50,000).

Determination of platinum/protein ratios
Protein concentration for myo-Pt4 samples was determined by UV-vis spectroscopy using (ε408 = 179,000 mol L -1 cm -1 ). 2 Trastuzumab concentration in Trastuzumab-Pt4 samples was determined using a NanoDrop Lite spectrophotometer (Thermo Scientific) using settings for IgG. The concentration of platinum in samples of both myo-Pt4 and Trastuzumab-Pt4 was determined using ICP-MS. Samples were prepared in a 3.6% v/v HNO3 solution and analysed using a no-gas mode.

Dark stability and photodecomposition
Complexes Pt4 and its precursors were dissolved in 5% DMSO 95% water v/v; myo-Pt4 was in 25 mM ammonium acetate. For dark stability, the UV-Vis spectrum was monitored over time while keeping the solution in the dark. For photodecomposition studies, the UV-Vis spectrum was measured at the same time point following irradiation. For blue light irradiation (λ = 420 nm), a LZC-ICH2 photoreactor (Luzchem Research Inc.) was used equipped with a temperature controller and 8 Luzchem LZC-420 lamps without light filtration. For green light irradiation (λ = 517 nm), an LED source was used (BASETech model no. SP-GU10 230 V~50 Hz 1.3-2.1 W).

FT-ICR Mass spectrometry
A 12 T solariX instrument (Bruker Daltonik GmbH, Bremen, Germany) equipped with an infinity cell was used for these experiments. Nano-electrospray ionisation was performed in positive-ion mode using a home-built source. To produce emitters, 1.2-mm thin-walled glass capillaries (World Precision Instruments, Hitching, UK) were pulled in-house with a P97 Flaming/ Brown type micropipette puller (Sutter Instrument Co., Novato, CA, USA) to obtain tips of ca. 1-μm orifice diameter. Data analysis was performed using Bruker Compass