Non-Covalent Attachment of BODIPY-caged Amines to Graphene and their Localized Photocleavage

We describe the synthesis of a novel boron-dipyrromethene (BODIPY)- caged amine capable of adhering to a pristine graphene surface via non-covalent bonding and demonstrate the release of the amine with...


Methods
Deposition on Graphene: Chemical-vapor-deposition-grown pristine graphene coated in PMMA was transferred onto a Pd-Ti grid on SiO2 via flotation in a water bath and then dried in a vacuum for 24 h.PMMA was then removed via acetone rinse, and the graphene was annealed for 2 h at 300 °C in an Ar (400 sccm) and H2 (approximately 20 sccm) atmosphere to improve adhesion to the SiO2 substrate and to remove most remaining PMMA residue.
Afterwards, the graphene-coated chips were stored in a closed container under standard atmospheric conditions until use.
To functionalize the graphene with BODIPY derivative 5, droplets of a 10 mM solution of 5 in ethanol (99.5%, Altia) were added to cover the entire surface of the sample chip and left for 1 h under ambient conditions.The sample was then rinsed with ethanol (2x) and isopropanol (1x) and dried under N2 flow.
Raman Measurement: Raman measurements were carried out using a Thermo Scientific DXR Raman Microscope equipped with a 532 nm laser at 10 mW of power and a 10x microscope objective, resulting in a 2 µm spot size as reported by the Raman instrument software.Because the laser used in the Raman system is within the photocleavage range, repeated measurements were taken at the exact same spot throughout photocleavage, allowing the system to both cleave and record the Raman spectra at the same time.Because of this, the initial Raman measurement was after 5.5 s of exposure.

ATR-FTIR:
FTIR measurements were carried out on a Bruker Alpha FTIR spectroscope using the solid form of 5. Measurements were carried out in the 400-4000 cm -1 spectral range at a resolution of 2 cm -1 .

Synthesis and characterization
All the reactions were carried out in a nitrogen atmosphere in oven-dried glassware, except for the hydrolysis reaction.Dry dichloromethane and THF were obtained by passing deoxygenated solvents through activated alumina columns (MBraun SPS-800 series solvent purification system).Pyridine was distilled and dried over molecular sieves (3 Å).All other reagents and solvents were used as received from the supplier.N,N-Diisopropylethylamine (DIPEA), acetoxyacetyl chloride, boron trifluoride diethyl etherate and triethylamine were purchased from Sigma Aldrich, 2,4-dimethyl-3-ethyl-pyrrole from Acros organics, p-nitrophenyl chloroformate from Tokyo Chemical Industry (TCI), 4-phenyl-1-butylamine from BLD pharm, lithium hydroxide monohydrate (LiOH) from Honeywell Fluka, pyridine, sodium hydroxide and sodium hydrogen carbonate from VWR chemicals and ammonium chloride from J. T.

Baker.
All reactions (except for the 1 st step) were monitored by TLC performed on Merck silica gel 60 F254 (230-400 mesh) plates and analyzed with UV light.The compounds (dyes) could be observed without staining.Silica gel flash chromatography (Combiflash next gen 300) was used for chromatographic purification with silica gel columns (20 -40 µm).
The 1 H and 13 C NMR spectra were recorded in CDCl3 on Bruker Avance 300 or 500 MHz spectrometers.The NOESY NMR spectrum was recorded in CDCl3 on Bruker Avance III HD 800 MHz spectrometer.The chemical shifts are reported in ppm relative to CHCl3 (δ = 7.26 ppm for the 1 H NMR spectra and δ = 77.16ppm for the 13 C NMR spectrum).High-resolution mass spectrometric data was measured on Agilent 6560 mass spectrometer, and melting points were determined in open capillaries on Stuart Scientific Melting Point Apparatus SMP3.

Pyrromethene compound 2
Compound 2 was synthesized according to the literature procedure 1 with few modifications.
2,4-dimethyl-3-ethyl-pyrrole 1 (4.4 ml, 32.0 mmol, 2.0 eq) was dissolved in dry DCM (180 ml) at RT under an N2 atmosphere.Acetoxyacetyl chloride (1.7 ml, 16.0 mmol, 1.0 eq) was added to the solution dropwise within 10 minutes while stirring under an N2 atmosphere.The reaction mixture was left to stir for 2 h at 50 ˚C in the dark.The reaction mixture was cooled down to 0 ˚C, after which dry triethylamine (10.6 ml, 76.0 mmol, 4.8 eq) was added with stirring and keeping the temperature at 0 ˚C.The mixture was stirred for 30 minutes at RT in the dark, followed by the addition of boron trifluoride diethyl etherate (13.6 ml, 110.0 mmol, 6.9 eq).The reaction mixture was stirred for 1 h at 50 ˚C in the dark.
The reaction mixture was extracted with deionized water 180 ml and the water phase with DCM.The combined organic phases were washed with 2 M NaHCO3 (180 ml) and brine (180 ml), and water phases were extracted with DCM between each washing.The combined organic phases were dried with MgSO4 and evaporated under reduced pressure.The crude product was purified by column chromatography (eluent: 30 -95 % DCM in petroleum ether (bp 40 -60 ˚C)).The residue was recrystallized from methanol at -20 ˚C overnight and dried under vacuum, yielding product 2 as a dark purple-green powder (2.03 g, 33.7 %).
Recorded 1 H NMR spectrum corresponded to the literature 1 .Synthesis was done according to the literature procedure 2 , except for the purification step.
Compound 2 (999 mg, 2.66 mmol, 1.0 eq) was dissolved in THF (53 ml) under an N2 atmosphere to yield a 0.05 M solution.A solution of lithium hydroxide monohydrate (560 mg, 13.3 mmol, 5.0 eq) in deionized water (53 ml) was added to the reaction mixture.The reaction was left to stir for 2 h at RT in the dark while monitored by TLC (20 % EtOAc in n-hexane).
Upon completion, the reaction mixture was diluted with 53 ml of ethyl acetate, and the organic phase was separated.The organic phase was washed with saturated NHCl4 solution and brine (2 x back extraction of water phases between each washing).The combined organic phases were dried with MgSO4 and evaporated under reduced pressure.The crude was dried overnight in a vacuum and purified twice by flash chromatography (eluent: 5-20% EtOAc in n-hexane), yielding product 3 as a red solid with brown flakes (330 mg, 36.0 %).Recorded 1 H NMR spectrum corresponded to the literature 2 .

EtBODIPYPNP (4)
Synthesis was carried out as reported earlier 3 except for the purification.Success of the reaction is dependent on the purity of the starting material 3: the success of the reaction was most likely when using extremely pure compound 3 (twice purified by chromatography)..
p-Nitrophenyl chloroformate (241.01 mg, 1.20 mmol, 4.0 eq) was added to the solution, followed by the addition of pyridine (122 µl, 1.50 mmol, 5.0 eq).The reaction mixture was stirred for 2 h at RT in the dark while monitored by TLC (eluent: 20 % EtOAc in n-hexane).
Upon completion, the reaction was diluted with ethyl acetate (15 ml) and washed with saturated NHCl4 solution (1 x 20 ml) and brine (1 x 20 ml).The organic phase was dried with MgSO4 and evaporated under reduced pressure.The crude product was purified by flash chromatography (eluent: 0 -20 % EtOAc in hexane), and the solvents were evaporated.The residue was dissolved in ethyl acetate (10 ml) and washed with 5 % NaOH solution (18 x 10 ml) and deionized water (1 x 10 ml).The organic phase was dried with Na2SO4 and evaporated.
The residue was dissolved in MeOH with slight heating and recrystallized at RT.The precipitate was separated from the recrystallization solvent by pipetting and dried under vacuum, yielding product 4 as a pink-red solid (68.5 %, 102.3 mg).
Recorded 1 H NMR spectrum corresponded to the literature 3 .

Photocleavage
Photocleavage in solution: As a preliminary test of the photochemical activity and photocleaving properties of the BODIPY compound 5, fluorescence and absorption spectra were measured in 1 µM acetonitrile-water (7:3) solution.Purity of acetonitrile purchased from Honeywell Riedel-de Haen was 99.9%.The measurements were performed using a Varian Cary Eclipse fluorescence spectrometer (excitation wavelength 510 nm), and for absorption measurements, a Varian Cary Conc100 spectrometer using quartz-crystal cuvettes.
Fluorescence and absorption spectra were recorded in sequence, with each absorption measurement taken immediately after each fluorescence measurement to match the fluorescence to a given absorption spectrum.Between each set of measurements, the cuvette was exposed to a 520 nm LED (50 mW, 1 mm distance) for varying exposure times to promote photocleavage of 5.After each exposure, cuvettes were lightly stirred by vortex motion before being placed back in the fluorescence spectrometer.The absorption spectrum of the uncaged amine (5a), prior to any chemical conjugation, was also measured, and can be seen in Fig. S13.In both cases, some degree of 5 remains bound to the surface and photoreactive, even after 2 and 6 weeks of storage in a standard atmosphere.As these are the samples that had other areas exposed via Raman previously, this qualitatively demonstrates that the photodegradation is indeed spatially selective.

Possible orientations of 5 on graphene
In theory, also the BODIPY part may interact with graphene (Figure S22), but we believe this is less likely due to the charges on the BODIPY molecule and the other moieties attached to the benzene ring.Also, based on solution state H-H NOESY NMR studies, there is no evidence of the interaction between the benzene ring and BODIPY core, which could interfere the assembly on graphene (ESI Fig. S10).
Figure S22.Other possible orientations of 5 on graphene.

Figure
Figure S1. 1 H NMR spectrum of 2 (300 MHz, CDCl3).Impurities are marked with * (they do not affect the next reactions).

Figure S10 .
Figure S10.H-H NOESY spectrum of 5 (800 MHz, CDCl3).NOE-correlation peaks related to the other conformation (syn) of 5 are marked with * and red boxes in the spectrum.

Figure S21 :
Figure S21: Raman measurements from different graphene samples a) 2 weeks after and b) 6 weeks after conjugation.