Tetra-arylborate lipophilic anions as targeting groups

TPB lipophilic anions deliver cargoes to lysosomes and are excluded from mitochondria.


Overview of Synthesis
The general synthetic strategy to make the TPB compounds involved preparing TPB-amine 5 as a universal precursor TPB head group, which could then be coupled with a range of carboxylic acids to attach the desired cargo (Scheme 1). We followed a similar approach to Franzke and Pfaltz in the construction of tetraarylborates with one aryl group differing from the other three 1-2 . The sulfonamide precursor 1, prepared from commercially available N-Boc piperidine and 4-bromosulfonyl chloride, was converted into methyl carbamate derivative 2 so that the protecting group could be removed using base rather than acid once the borate was installed. Miyaura borylation 3 was followed by conversion to the potassium aryltrifluoroborate salt 3 under the conditions of Lennox and Lloyd-Jones 4 , with further removal of the pinacol side product with 50% aqueous MeOH and repeated evaporation-dissolution cycles 5 . Reaction with excess phenylmagnesium chloride completed the tetraarylborate head group and ion exchange to the tetrabutylammonium salt 4 ensured good solubility in organic solvents to assist purification. Removal of the carbamate protecting group then gave the TPB head group 5 bearing an amino group for attachment of any cargo. A carboxylic acid derivative of the chromanol group of a-tocopherol, 2-(6'-hydroxy-2',5',7',8'-tetramethylchroman-2'-yl)acetic acid, was prepared by the route of Scott et al. 6 and coupled with TPB-amine 5 to give TPBE using HBTU. The BODIPY carboxylic acid precursor, 3-(4,4-Difluoro-1,3,5,7-tetramethyl-4bora-3a,4a-diaza-s-indacene-8-yl)-propionic acid, was prepared by the method of Thivierge et al. 7 and was coupled with amine 5 to give TPBBODIPY. The coumarin carboxylic acid was synthesised by the method of Tateishi et al 8 and was coupled to amine 5 to give TPBCoumarin.
TPBM was prepared in a similar way to amine 5, but using morpholine instead of N-Boc piperidine. Stock solutions of compounds were made up in ethanol, flushed with argon, and stored as aliquots at -20 °C before use. While developing the TPB compounds we also generated a number of other tetraphenylborate head groups that were less biologically effective that the sulfonamide derivative of TPB. The synthesis and characterisation of these will be described elsewhere.

General Information on Synthetic Procedures
All reactions under an inert atmosphere were carried out using oven-dried glassware and solvents were added via syringe. Reagents were obtained from commercial suppliers and used without further purification. Dry solvents were collected from a Puresolv solvent purification system or obtained from commercial suppliers. 1 H, 13 C, 11

1'ylsulfonyl]phenyl}triphenylborate 4
PhMgCl (3.2 mL, 6.4 mmol, 5 eq.) was added slowly dropwise to a stirred cooled solution (0 °C) of potassium trifluoroboronate salt 3 (500 mg, 1.28 mmol, 1 eq.) in dry THF (5 mL) under an argon atmosphere and stirred for 30 min. The reaction mixture was allowed to warm up to RT and heated under reflux for 16 h under an argon atmosphere. The reaction mixture was allowed to cool down to RT and was added slowly into an aqueous solution of Na2CO3 (3.31 g in 67 mL of H2O). The reaction mixture was stirred vigorously for 60 min at RT and extracted with EtOAc. The organic layer was washed with H2O and brine, dried over Na2SO4, filtered and concentrated under reduced pressure to give a crude product as an orange oil (732 mg). The crude product was dissolved in dry CH2Cl2 (10 mL) and tetrabutylammonium bromide (490 mg, 1.54 mmol, 1.2 eq.) dissolved in dry CH2Cl2 (7 mL) and was added slowly to this stirring solution. The reaction mixture was stirred for 30 min at RT, filtered and the filtrate was concentrated under reduced pressure to give a crude product as an orange oil (990 mg

Black Lipid Membrane Experiments
Planar Black Lipid Membranes (BLM) were formed from a 10 mg/ml solution of 1,2-dioleoyl-

Submitochondrial Particle Experiments
Bovine heart mitochondria were prepared from Bos taurus heart tissue as described previously 11 and stored as ~5 g pellets at -20˚C. To prepare submitochondrial particles (SMPs), a pellet of bovine heart mitochondria was thawed overnight at 4˚C, resuspended in 10 mM Tris-SO4, 250 mM sucrose at pH 7.0 (final volume 120 ml, pH corrected at 20 ˚C, buffer A), and refrozen.
Then 40 ml of the suspension was thawed at 4˚C, centrifuged (11,300 × g, 12 min, 4˚C), the dark red supernatant discarded, and the pellet was resuspended in buffer A to 40 ml. The pH was adjusted to 9 on ice by the dropwise addition of 2.5 M Tris, and the sample was incubated on ice for 15 min then recentrifuged (37,900 × g, 12 min, 4˚C). The supernatant was discarded, and the pellet was twice resuspended in buffer A to 40 ml and recentrifuged (11, (Fig S2), indicating concentration ranges that can be used safely. For analysis of the uptake of TPBBODIPY the cells were prepared as described above and then placed in 5% CO 2 at 37˚C imaging incubator for acquisition with a Dragonfly Spinning Disk imaging system (Andor Technologies Ltd.) composed by a Nikon Ti-E microscope, Nikon 100x TIRF ApoPlan and an Ixon EMCCD camera. 3D images were acquired every 5 sec using a 488 nm or 568 nm laser and corresponding filters, after a few images the compounds were added at 100 nM. Images were analysed and videos mounted at 7 fps rate using the Imaris v.9.1.2 software (Bitplane AG, Zurich, Switzerland).

Confocal Microscopy
For video analysis of the uptake of TPBCoumarin and TPBBodipy C2C12 cells were prepared the cells were prepared as described above and placed in a Zeiss LSM880 confocal system equipped with a Zeiss Plan-Achromat 100x/1.4NA oil immersion objective. Images were acquired every 10 sec for 20 min using a 405 nm laser and corresponding filters (Coumarin S 20

Movie Legends
Movie S1. TPBCoumarin uptake in C2C12 cells. The cells were exposed to 100 nM of TPBcoumarin at the 2-s mark on the video and incubated for a further 15 min. The video is shown at 5 fps and each frame is 10 s apart in real time.
Movie S2. TPBBodipy uptake in C2C12 cells. TPBBodipy uptake in C2C12 cells. The cells were exposed to 100 nM of TPBBodipy after 15 s and incubated for a further 5 min. The video is shown at 7 fps and each frame is 5 s apart in real time.