Rational design of a water-soluble, lipid-compatible fluorescent probe for Cu(i) with sub-part-per-trillion sensitivity

Knowledge-driven optimization of the ligand and fluorophore architectures yielded an ultrasensitive Cu(i)-selective fluorescent probe featuring a 180-fold fluorescence contrast and 41% quantum yield.


Synthetic Procedures
Materials and Reagents. Ethenesulfonyl fluoride (ESF), 1 thietane 6, 2 6-bromobenzothiazolin-2-one, 3 and benzothiazolin-2-one-6-carboxaldehyde 4 21 were prepared as previously described. All other starting materials were commercially available and used without further purification. NMR: Spectra were recorded at 400 MHz ( 1 H, ppm vs. internal TMS, referenced directly or indirectly via the known residual proton signal of the solvent), 376 MHz ( 19 F, ppm vs. internal CCl 3 F), and 100 MHz ( 13 C, ppm vs. TMS, referenced to CDCl 3 (77 ppm) or CD 3 OD (49 ppm) chemical shifts). Spectra were recorded at ambient temperature (20-23°C) unless stated otherwise. For 1 H spectra, the abbreviation "ad" denotes an apparent doublet with additional partially resolved coupling (AA'XX' or AA'MM' spin system); only the largest (first order) coupling constant is given for these systems. In cases where the product as isolated contained a substantial amount of solvent, the solvent content was calculated from the initial 1 H NMR integrals and a second 1 H NMR spectrum was acquired after removal of solvent by repeated dissolution in CDCl 3 followed by concentration to dryness. MS: Spectra were acquired by the Georgia Tech Mass Spectrometry Facility. Column chromatography: Flash chromatography on Sorbent Technologies, general purpose silica gel (60 Å pore size, 250 mesh).

4-Hydrazinyl-N-methylbenzenesulfonamide 14.
A solution of 4-fluorobenzenesulfonyl chloride (8.49 g, 43.6 mmol) in dichloromethane (60 mL) was cooled in an ice bath and methylamine solution (7 ml, 40% aqueous, d = 0.9 g/mL, 4 equiv.) was added slowly under stirring. Gentle boiling occurred, and the ice bath was removed once this had subsided. After 15 minutes, the mixture was diluted with crushed ice and carefully acidified with concentrated HCl (10 mL). The organic layer was separated, and the aqueous layer was extracted with dichloromethane (2 x 20 mL). The combined organic layers were dried with Na 2 SO 4 and concentrated. The residue was transferred to a 50 mL round-bottom flask and dissolved in DMSO (12 mL). The flask was sealed under argon, and hydrazine (4.1 mL, 3.0 equiv.) was added. The reaction vessel was vented to an oil bubbler, and the mixture was stirred at 50°C overnight. A further 2 mL (1.5 equiv.) of hydrazine were added, and the mixture was stirred at 60°C for 24 hours. After cooling, the mixture was slowly diluted with cold water (100 mL), and the resulting precipitate was collected by filtration, washed with cold water, and recrystallized from ethanol to give the product 14 as colorless needles. Yield 7.54 g (37.5 mmol, 86%). Mp 140-141°C. 1  Chalcone 18. Acetophenone derivative 7 (480 mg, 2.25 mmol) and 4-N,N-dimethylaminobenzaldehyde (320 mg, 2.14 mmol) were stirred in ethanol (4 mL) at 50°C until completely dissolved, and pyrrolidine (180 µL, 2.14 mmol) was then added. After 1 hour, the deep red solution was cooled under rapid stirring to initiate crystallization of the product, and the resulting orange slurry was stirred overnight at 50°C. After cooling, the product was collected by filtration, washed with cold ethanol, and dried by suction and then under vacuum to give an orange crystalline powder. Yield 583 mg (1.69 mmol, 79%). Mp 153-154°C. 1  Pyrazoline derivative (±)-19. A mixture of chalcone 18 (393 mg, 1.14 mmol), arylhydrazine 14 (321 mg, 1.4 equiv.), PPTS (400 mg, 1.4 equiv.) and methanol (4 mL) was stirred under argon in a sealed vessel at 90°C for 3 hours. The mixture was poured into water (50 mL) and an attempt was made to extract the product with toluene (50 mL). A large amount of insoluble material remained so MTBE (25 mL) and dichloromethane (25 mL) were added, resulting two clear liquid phases after agitation and settling. The organic layer (top) was separated, dried with Na 2 SO 4 , and concentrated. The residue was subjected to column chromatography (DCM-MTBE) to give the product as a yellow glassy solid containing 0.6 molar equiv. of MTBE by 1  Reference triarylpyrazoline (±)-3. Sulfonyl fluoride (±)-20 (74 mg of material containing 8% MTBE, 91 µmol) was stirred in 1 mL of a mixture containing acetonitrile (45%), triethylamine (45%) and water (10 %). After 10 min, TLC (10:1 dichloromethane-MTBE) indicated a mixture of the starting material (Rf 0.8), a small amount of single-elimination product (Rf 0.5) and a trace of double elimination product (Rf 0.2, identical to triarylpyrazoline (±)-19), as well as hydrolysis products (Rf ≈0). After stirring overnight, only the hydrolysis products and double-elimination product remained. The mixture was concentrated to dryness under a stream of argon, and the residue was taken up in methanol (2 mL) + concentrated ammonia (~100 µL) and concentrated again. The residue was dissolved in water (2 mL), and the product was isolated by RP-HPLC using a gradient of 25-33% CH3CN in 0.1% aqueous (NH 4 )HCO 3 to give a yellow glassy solid after repeated evaporation with methanol and vacuum drying. The 1 H NMR spectrum of this material is consistent with a mixed ammonium-triethylammonium salt containing 44 mol% Et 3 NH + , corresponding to a formula weight of 815 g/mol. Yield 74 mg (71 µmol, 78%). 1  Benzothiazolinone 22. A mixture of benzothiazolin-2-one-6-carboxaldehyde 4 (21, 255 mg, 1.42 mmol), iodide 7 (562 mg, 1.25 equiv.), and K 2 CO 3 (600 mg, 3 equiv.) in DMF (5 mL) was stirred at 90°C for 12 hours. The mixture was diluted into a solution of 1 M NaOH in 20% aqueous methanol (100 mL), and the resulting emulsion was extracted with MTBE (100 mL). The extract was washed with a further 100 mL of the aqueous-methanolic NaOH solution, dried with Na 2 SO 4 , and concentrated under reduced pressure to a yellow oily residue. Crystallization from diethyl ether-pentane under stirring gave the product as a slightly tan crystalline powder. Yield 306 mg (833 µmol, 59%). Mp 134-134.5°C. 1

Absorption and Fluorescence Spectroscopy
General. All buffers and probe stock solutions were prepared using JT Baker HPLC-grade water or 18.2 MΩ . cm Mili-Q water. Nevertheless, we frequently observed an increased background fluorescence due to traces of adventitious copper. It was therefore necessary to add a small amount of the high-affinity Cu(I) ligand MCL-1 6 as a sequestrant in most experiments as described in detail below. Deoxygenation, where specified, was achieved by bubbling with argon. Initial detection limit experiments indicated that the brass gas regulator could serve as an additional source of copper contamination, which was prevented by passing the argon through a 5 µM filter. UV-vis absorption spectra were acquired at 25°C with a Varian Cary Bio50 spectrophotometer with constant temperature accessory. Fluorescence spectra were recorded with a PTI fluorimeter equipped with a 75 W xenon arc lamp excitation source and model 814 photomultiplier detection system (PMT voltage 1100 V for all measurements). The fluorescence spectra were corrected for the spectral response of the detection system and for the spectral irradiance of the excitation source (via a calibrated photodiode). The path length was 1 cm for absorbance and fluorescence spectra and 10 cm for absorbance measurement for quantum yield determination. Fluorescence Enhancement Factors and Quantum Yields. Fluorescence enhancement factors were determined at 1-5 µM probe concentration in pH 7.2 MOPS buffer containing 1 µM MCL-1 6 as a sequestrant for traces of background copper and 100 µM sodium ascorbate as a reducing agent. Excitation was provided at 365 nm for CTAP-3 and at 380 nm for probes 4 and 5. Emission spectra were recorded before and after saturation with Cu(I) generated by in situ reduction of CuSO 4 with ascorbate. After background subtraction, spectra were integrated over the range of λ max ± 10 nm, and the ratio of the integrated intensities before and after Cu(I) saturation were taken as the enhancement factor. There was no observable difference in emission maximum between the free and Cu(I)-saturated form of each probe. In each case, complete reversal of the fluorescence response to Cu(I) was observed upon addition of excess MCL-1. Fluorescence quantum yields were determined using norharmane in 0.1 N H 2 SO 4 (Φ f 0.58) 7 as standard. During the optimization process, the quantum yields of the Cu(I)-saturated probes were initially determined via single-point measurement at OD 0.1, and that of CTAP-3 was subsequently verified by a four point measurement over the OD range of 0.1-0.4 (10 cm path length).

Time-resolved Fluorescence Spectroscopy.
Fluorescence decay profiles were acquired at the respective emission maximum of each fluorophore using a single photon counting spectrometer (Edinburgh Instruments, LifeSpec Series) equipped with a pulsed laser diode as the excitation source (372 nm, FWHM = 80 ps, 10 MHz repetition rate, 1024 channel resolution). Sample solutions of the Cu(I)-saturated probes were prepared via in situ reduction of CuSO 4 with ascorbate in deoxygenated pH 7.2 MOPS-K + buffer (10 mM, 25°C). The time decay data were analyzed by non-linear least squares fitting with deconvolution of the instrumental response function using the FluoFit software package. 8  procedure was performed prior to the potentiometric titrations, and the experimental electrode potential and slope were derived as the average of the data from three independent titrations.

Determination of the Protonation Constant of CTAP-3.
A 5 µM solution of CTAP-3 in aqueous 0.1 M KCl was prepared and the solution pH was adjusted to 6.0. To remove interfering dust particles or fibers, the solution was passed through a 0.2 µm membrane filter. A combined potentiometric and fluorimetric titration was carried out in a quartz cuvette with 1 cm pathlength by addition of HCl to adjust the pH between 6.0 and 0.5 (constant temperature accessory set to 25°C). After addition of each aliquot acid, the solution was allowed to equilibrate, the potential was recorded (in mV), and the fluorescence intensity was measured at 455 nm with excitation at 365 nm. The data were analyzed by non-linear least-squares fitting using equation (1) where F is the measured fluorescence intensity at pH c = -log[H 3 O + ], F max and F min are the limiting fluorescence intensities, and K a1 and K a2 are the first and second protonation constants, respectively. ) and the data were analyzed by non-linear least squares fitting to yield the average pK a values of pK H1 = 1.99 ± 0.01, and pK H2 = 1.32 ± 0.01.

Cu(I) Binding
Affinity of CTAP-3. The Cu(I) stability constant for CTAP-3 was determined through a fluorimetric titration using the affinity standard MCL-2 as competing ligand. 6 A solution of [(MCL-2)Cu]Na 3 PF 6 •7.5 H 2 O (5 µM) and CTAP-3 (5 µM) in aqueous buffer (pH 6.0, 10 mM MES, 0.1 M KClO 4 , 25°C) was equilibrated and then titrated with MCL-2 (3 mM stock solution in water) from 10-400 µM. After the addition of each aliquot, a fluorescence emission spectrum was acquired with excitation at 365 nm over the range of 380-700 nm. The data were analyzed by non-linear least squares fitting using Specfit. 10 Based on the MCL-2 formation constant of logβ = 13.08 and a pK a of 8.98 (corrected upward by 0.11 units to account for 0.1 M ionic strength), 11 Figure B shows the fluorescence intensity change and corresponding fit at 455 nm.
Liposome Size Distribution. Liposome diameters were measured based upon the Tunable Resistive Pulse Sensing (TRPS) principle using an Izon qNano particle analyzer (Izon Science Ltd., Burnside, New Zealand). The instrument was calibrated with monodisperse carboxylated polystyrene particles (Izon, 200 nm diameter) in deionized water following the manufacturer's recommendations. Calibration and particle analysis runs were conducted with a 46 mm nanopore stretch (NP150), an electric potential of 0.44 V, and an applied pressure of 10 mm Hg (variable pressure module). Each measurement was performed by detecting a minimum of 1000 particles. The data were analyzed using the Izon Control Suite 2.1 software to determine the mean diameter and size distribution of the liposomes.