Analytical grade reagents were purchased from Sigma-Aldrich or Fisher, except for 1H-benzotriazolium 1-[bis(dimethylamino)methylene]-5-chlorohexafluorophosphate (1-),3-oxide (HCTU), benzotriazole-1-yloxytrispyrrolidinophosphonium hexafluorophosphate (PyBop), amino acids, and TGR resins, which were obtained from NovaBiochem. Amine-reactive fluorophores were obtained from Aldrich or Invitrogen. The SH2 ligand, Ac-pTyr-Glu-Glu-Ile-Glu, was purchased from Bachem. UV-vis spectra were measured using a Biochrome Libra S22 spectrophotometer. Emission and excitation spectra were obtained using a Horiba Jobin Yvon Fluorolog 3 spectrofluorometer at 23 °C. Quantum yields were measured using indodicarbocyanine iodide as an internal standard. Solutions were not deaerated because control experiments showed that oxygen did not quench the fluorescence of these dyes, due perhaps to their short lifetimes. Mass spectra were obtained on an Applied Biosystems API 2000 LC/MS/MS system. See Supporting Information
for Mass Spectral data. 1
H and 13
C NMR spectra were recorded on Bruker DRX-300 spectrometer. See Supporting Information
for NMR data. All operations with dyes were performed under dim light. Flasks containing dyes were wrapped with aluminum foil. Chemical names for compounds were obtaining using ChemDraw Ultra 7. Human CML cells (imatinib-sensitive Myl and imatinib-resistant MylR) were a generous gift from Dr. Hideo Tanaka (Dept. of Haematology and Oncology, Hiroshima University, Hiroshima, Japan). Cells were cultured in RPMI 1640 medium (Invitrogen) supplemented with 10% fetal bovine serum (FBS; Atlanta Biologicals) and 1% penicillin/streptomycin (P/S; Invitrogen). HEK 293 cells were acquired from ATCC. Cells were cultured in DMEM medium (Invitrogen) supplemented with 10% FBS and 1% P/S. Cells were grown in a humidified incubator at 37°C with 5% CO2
. The Lyn A and Lyn B expression constructs were a generous gift from Dr. Juan Rivera (Mol. Immunol. and Inflammation Branch, NIH, Bethesda, MD). Constructs were cloned from the pLenti6/V5 vector (Invitrogen) into the pCDNA3.1 vector (Invitrogen). Point mutations were made in the Lyn A (K275E, Y508F) and Lyn B (K254E, Y487F) constructs using the Quickchange Site-directed Mutagenesis Kit (Stratagene) according to the manufacturer’s instructions. Expression vectors were introduced into HEK 293 cells by Lipofectamine transfection (Invitrogen) according to the manufacturer’s instructions. 36 h post-transfection cells were washed with PBS and pelleted; cells were stored at −80 °C until experimentation.
Chemical synthesis. 4-(7-methoxy-2,2,4-trimethyl-2H-quinolin-1-yl)-butyric acid ethyl ester (2)
A 500 mL round-bottom flask was set up with a magnetic stir bar, a reflux condenser, and a nitrogen line. The flask was charged with 7-methoxy-2,2,4-trimethyl-1,2-dihydro-quinoline 1
) (43 g, 0.21 mol), ethyl 4-bromobutyrate (88 g, 0.44 mol), sodium iodide (11 g, 0.073 mol), acetonitrile (150 mL, 2.9 mol), and sodium carbonate (52 g, 0.48 mol). The flask was heated at reflux with stirring for 72 h. The reaction mixture was filtered and the acetonitrile was removed with a rotary evaporator. The unreacted starting compound was removed from the product by vacuum distillation using an oil pump. The recovery of 7-methoxy-2,2,4-trimethyl-1,2-dihydro-quinoline was 7 g (16%, b.p. 120–125°C, 0.7 Torr). The viscous yellow residue in the distillation flask was the pure product. The yield was 53.0 g as an oil (79% yield).
Sodium [1-(3-ethoxycarbonyl-propyl)-7-methoxy-2,2-dimethyl-1,2-dihydro-quinolin-4-yl]-methanesulfonate (3)
A 500 mL round-bottom, three-necked flask was set up with a nitrogen line, a mechanical stirrer, and a thermometer. The flask was immersed in a salt-ice cooling bath and 4-(7-methoxy-2,2,4-trimethyl-2H-quinolin-1-yl)-butyric acid ethyl ester (2, 30 g, 0.09 mol) was placed in the flask and the stirrer was started. Sulfuric acid (30 mL, 0.6 mol) was added slowly to the flask over the course of about 5 min. The reaction mixture was then stirred for additional 30 min with cooling. When the temperature inside the flask was about −10°C, fuming sulfuric acid (17 mL, 0.18 mol, 30% of free SO3) was added quickly to the flask. The stirring was continued and the flask was allowed to slowly warm up to room temperature while in cooling bath. The reaction mixture was stirred for another 48 h at room temperature. The reaction mixture was poured into a 2 L beaker containing 500 g of ice and the beaker was immersed in a salt-ice cooling bath. The acid was neutralized with 10% solution of sodium hydroxide in water until the pH of the reaction mixture was about 7. The temperature of the reaction mixture was kept below 10 °C during the neutralization process, at the end of which the product crystallized. The crystals were filtered using a Büchner funnel, washed with 50 mL of ice-cold water, and air-dried. The crystals were placed in 2 L flask equipped with reflux condenser and 1 L of 100% ethanol was added. The mixture was stirred with heating until the ethanol started to boil and some of the solid material dissolved. The mixture was filtered while still hot using a Buchner funnel. The crystals (Na2SO4 hydrate) were washed with three portions of 100 mL of hot ethanol. A combined filtrate solution was evaporated using a rotary evaporator and the resultant white solid was dried under high vacuum overnight. The yield was 26 g (66% yield).
Sodium [1-(3-ethoxycarbonyl-propyl)-7-methoxy-2,2-dimethyl-1,2-dihydro-quinolin-4-yl]-methanesulfonate (4)
Sodium [1-(3-ethoxycarbonyl-propyl)-7-methoxy-2,2-dimethyl-1,2-dihydro-quinolin-4-yl]-methanesulfonate (3, 22 g, 0.052 mol), methanol (250 mL, 6.2), and Pd on carbon (10%, 1.0 g) were added to a 1 L high pressure, heavy wall Parr hydrogenation bottle. The bottle was attached to a high-pressure Parr hydrogenation apparatus and the system was evacuated using a water aspirator and then filled with hydrogen gas. The evacuation/filling procedure was repeated three more times. Then the system was filled with hydrogen at 50 psi pressure and the shaker was activated. The bottle was shaken at room temperature for 4 h until hydrogen consumption stopped. The remaining pressure was carefully released and the catalyst was removed by vacuum filtration through a Celite pad. The solvent was removed under vacuum to give the final product as a white solid. The yield was 20 g (90% yield).
Disodium 4-(7-methoxy-2,2-dimethyl-4-(sulfonatomethyl)-3,4-dihydroquinolin-1(2H)-yl)butanoate) (5)
A 500 mL round-bottomed, one-necked flask was set up with a magnetic stir bar and a reflux condenser. The flask was charged with sodium [1-(3-ethoxycarbonyl-propyl)-7-methoxy-2,2-dimethyl-1,2-dihydro-quinolin-4-yl]-methane-sulfonate (4, 20 g, 0.05 mol), sodium hydroxide (2.09 g, 0.0522 mol), and 100 mL water. The mixture was stirred with heating (50°C) for 1 h and then cooled to room temperature. The solvent was removed under vacuum and the solid was stirred with 100 mL of MeOH at reflux for 30 min. The hot suspension was filtered, the solid was washed with hot methanol, and dried. The yield of the final product (white solid) was 20 g (100% yield).
Disodium 4-[7-hydroxy-2,2-dimethyl-4-(sulfonatomethyl)-3,4-dihydroquinolin-1(2H)-yl]butanoate (6)
A 250 mL round-bottomed, one-necked flask was set up with a magnetic stir bar, a reflux condenser, and a nitrogen line. The flask was charged with disodium 4-(7-methoxy-2,2-dimethyl-4-(sulfonatomethyl)-3,4-dihydroquinolin-1(2H)-yl)butanoate) (5, 12 g, 0.029 mol), sodium iodide (10 g, 0.07 mol), and a solution of 9.0 M hydrogen bromide in water (70 mL). The flask was flushed with nitrogen and then was heated at 105 °C (oil bath) with stirring for 18 h. The reaction mixture was cooled to room temperature and the excess hydrobromic acid was removed using rotavap. To ensure a complete removal of the hydrobromic acid, 100 mL of water was added to the residue and the solvent was removed. The yellow solid was dissolved in 50 mL of water and the solution was neutralized by addition of solid sodium bicarbonate. The solvent was removed by vacuum and the solid was refluxed with stirring with 500 mL of acetone overnight under nitrogen to remove sodium iodide and sodium bromide that are soluble in acetone. The mixture was cooled to room temperature and filtered. The solid was dried was washed with acetone and dried. The yield was 9.3 g (80% yield) as a tan solid.
The 4-nitrophenyldiazenylphenol 7
Step 1. p-Nitroaniline (0.546 g, 0.00395 mol) was added to a 100 mL round-bottomed, one-necked flask equipped with a magnetic stir bar and an addition funnel. 10 mL of 10% HCl was added to the flask. The mixture was stirred at room temperature until a clear solution formed, and then the flask was immersed in an ice-water bath. A solution of sodium nitrite (0.273 g, 0.00395 mol) in 5 mL of water was added drop-wise to the flask with stirring. When the addition was complete, the mixture was stirred for another 30 min at 0°C. Step 2. Disodium 4-[7-hydroxy-2,2-dimethyl-4-(sulfonatomethyl)-3,4-dihydroquinolin-1(2H)-yl]butanoate, 6) (1.59 g, 0.00395 mol) was added to a 250 mL conical flask, followed by 10 mL of 10% HCl. The flask was immersed in an ice-water cooling bath and stirring was started. The solution of p-nitrophenyldiazonium salt (from step 1) was added to the flask in small portions (1–2 mL) over the course of 10 min. The reaction mixture was stirred for 1 h at 0°C. The precipitated red solid was filtered, washed with water (3×15 mL). The red solid was placed in a 500 mL round-bottomed, one-necked flask equipped with a magnetic stirrer. Water (150 mL) was added to the flask and the stirring was started. Solid sodium bicarbonate (1.125 g) was added to the flask in five portions to avoid excessive foaming. The solvent was removed using a rotary evaporator. The solid residue was re-dissolved in methanol. The solution was filtered to remove inorganic salts and the methanol was removed using a rotary evaporator to give a red solid. The residue was re-dissolved in 25 mL of water, and the volume was reduced to 10 mL using the rotary evaporator (this operation was necessary to remove traces of methanol). The solution was frozen and lyophilized overnight. The yield of red solid product was 1.38 g (66% yield).
Sodium (1,11-bis(3-carboxypropyl)-2,2,10,10-tetramethyl-2,3,4,8,9,10-hexahydro-1H-dipyrido[3,2-b:2',3'-i]phenoxazine-11-ium-4,8-diyl)dimethanesulfonate (8)
(E)-(7-hydroxy-1-(3-carboxypropyl)-2,2-dimethyl-6-((4-nitrophenyl)diazenyl)-1,2,3,4-tetra-hydroquinolinium-4-yl)methanesulfonate) (7, 0.671 g, 0.00132 mol), disodium 4-[7-hydroxy-2,2-dimethyl-4-(sulfonatomethyl)-3,4-dihydroquinolin-1(2H)-yl]butanoate) (6, 0.532 g, 0.00132 mol), and acetic acid (25 mL, 0.44 mol) were added to a 100 mL round-bottomed, one-necked flask set up with a magnetic stir bar and a reflux condenser. The reaction mixture was heated at reflux for 24 h. TLC showed complete consumption of starting material and formation of deeply blue-colored product (SiO2, eluent: acetone-water (1:1), Rf product = 0.1, Rf starting material = 0, and 0.4). The reaction mixture was evaporated and the residue was dissolved in 50 mL of a mixture of acetone-water (4:1). The solution was mixed with 10 g silica and the solvent was removed under vacuum. The dye/silica mixture was loaded onto a silica column prepared in acetone. The column was eluted with an acetone-water gradient (0 – 10%). The dye containing fractions were combined and the solvent was removed using a rotary evaporator. NMR analysis showed the presence of impurities, and the dye was further purified on a preparative RP HPLC column, which was eluted with water and acetonitrile. (Program: 0 – 60 min 100% water, 60 – 120 min 10% acetonitrile/90% water, 120 – 150 min 50% acetonitrile/50% water, 150 – 180 min 80% acetonitrile/20% water, 180 – 200 min 100% water.) The dye eluted at 105–115 min. The dye-containing fractions were combined and reduced in volume from about 120 mL to 10 mL using a rotary evaporator. The dye solution was frozen and lyophilized. The yield of pure dye (blue solid) was 100 mg (10% yield).
Peptide synthesis. Lyn Sensor Peptide (9)
The peptide-resin Ac-Glu(tBu)-Lys(Mtt)-Glu(tBu)-Ile-Tyr(tBu)-Gly-Glu(tBu)-Ile-Glu(tBu)-Ala-amide-resin was synthesized using standard Fmoc peptide synthesis protocol [amino acids 5 eq, HCTU 5 eq, DIPEA (N,N-diisopropylethylamine) 10 eq] as previously described.(21
) The side chains of Glu and Tyr were protected with t-Bu. The side chain of Lys was protected with the acid sensitive 4-methyltrityl (Mtt) group. Tetramethylfluoroform-amidinium (TFFH) (5 eq) and DIPEA (10 eq) were used to ensure successful coupling of Fmoc-Lys(Mtt)-OH (5 eq) at the Y-3 position. After synthesis of the peptide on the resin and subsequent acetylation of the N-terminus, the Mtt on Lys was selectively removed with 2% TFA. The side chain deprotected Lys amine was reacted with the doubly activated oxazine diacid 8
(oxazine 1 eq, PyBop 10 eq, DIPEA 20 eq) in DMF. The peptide was cleaved with TFA:H2
O:TIS (triisopropylsilane) in a ratio of 95:2.5:2.5, and purified via HPLC.
Abl Sensor Peptide (11)
NH2-Gly-Gly-Ile-Tyr(tBu)-Ala-Ala-Pro-Phe-Lys(Boc)-Lys(Boc)-Lys(Boc)-Ala-amide-resin was synthesized via a standard Fmoc peptide synthesis protocol using a Prelude automatic peptide synthesizer from Protein Technology (amino acids 5 eq, HCTU 5 eq, DIPEA 10 eq). The free N-terminus was reacted with Cascade Yellow succinimidyl ester (2 eq) and DIPEA (4 eq) in DMF. The peptide was cleaved with TFA:H2O:TIS (triisopropylsilane) in a ratio of 95:2.5:2.5, and purified via HPLC.
Protein kinase assays
Assays were generally performed following a previously reported protocol (22
) but with a total volume of 25 µl from the following stock solutions: 10 µl H2
O, 2.5 µL 0.1 mM peptide stock solution, 2.5 µL 1M Tris (pH 7.5), 2.5 µL 200 mM MgCl2
, 2.5 µL 0.1 mg mL−1
BSA, 0.5 µL 50 mM dithiothreitol (DTT), 2 µL 0.3 µM kinase and 2.5 µL 50 mM ATP. Final concentration of the assay solution was: 100 mM Tris (pH 7.5), 0.01 mg mL−1
BSA, 1 mM DTT, 24 nM kinase, 20 mM MgCl2
, 5 mM ATP and 10 µM peptide sensor. Assays were initiated either by adding ATP or peptide and the fluorescence was monitored using a Gemini EM fluorescence microplate reader from Molecular Devices with 384-well microplates at 30°C. Peptide 9
= 665 nm; λem
= 705 nm); Peptide 11
= 400 nm; λem
= 525 nm). Dual monitoring of Abl and Lyn kinase activity was performed in an analogous fashion, but in the presence of both 9
, and either Abl or Lyn alone or in combination. IC50
determinations were performed as described above with varying concentrations of imatinib or dasatinib. Cell lysate assays were likewise performed as described above using the following conditions: 100 mM Tris (pH 7.5), 5 mM ATP, 20 mM MgCl2
, 1/100 (volume) protease inhibitor cocktail (P8340, Sigma-Aldrich), 1/20 phosphatase inhibitor cocktail 2 (P5726, Sigma-Aldrich), 10 µM peptide sensor. Protein concentration was determined using Bradford reagent (Thermo Scientific), and the concentration of each sample was equilibrated to 2.5 µg µL−1
by addition of lysis buffer.