Synthesis of Fluorogenic Substrates for Continuous Assay of

The phosphatidylinositol specific phospholipase C (PI-. PLC) family of enzymes plays an important role in cellular signaling (1-4). These enzymes clea...
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Bioconjugate Chem. 2001, 12, 307−313

307

Synthesis of Fluorogenic Substrates for Continuous Assay of Phosphatidylinositol-Specific Phospholipase C Tatiana O. Zaikova,† Aleksey V. Rukavishnikov,† G. Bruce Birrell,‡ O. Hayes Griffith,*,†,‡ and John F. W. Keana*,† Department of Chemistry and Institute of Molecular Biology, University of Oregon, Eugene, Oregon 97403. Received September 19, 2000; Revised Manuscript Received December 21, 2000

An improved synthesis of fluorogenic substrate analogues for phosphatidylinositol-specific phospholipase C (PI-PLC) is described. The water-soluble substrates, which are derived from fluorescein, are not fluorescent until cleaved by the enzyme, and provide a convenient means to continuously monitor PI-PLC activity. The improvement in the synthesis lies in the method used to protect the hydroxyl groups of the inositol portion of the substrate molecule and allows a milder deprotection procedure to be used. The result is a much more reproducible synthesis of the substrate. The improved procedure has been employed to synthesize a series of fluorogenic substrates, which differ in the length of the aliphatic tail attached to the fluorescein portion of the molecule. The length of the tail was found to have a significant effect on the rate of cleavage of these substrates.

INTRODUCTION

The phosphatidylinositol specific phospholipase C (PIPLC) family of enzymes plays an important role in cellular signaling (1-4). These enzymes cleave phosphatidylinositol and its phosphorylated analogues, forming inositol phosphates and diacylglycerol, which can act as intracellular second messengers in eukaryotes, ultimately leading to a variety of cellular responses. Bacterial PI-PLCs are of interest as model systems for the mammalian signal transduction isozymes, as possible virulence factors, and for their ability to cleave glycosylphosphatidylinositol (GPI)-anchored proteins (5). PIPLC activity is most often measured in a discontinuous assay using radiolabeled phospholipids as substrates, with the assays requiring the separation of product from uncleaved substrate before enzyme activities can be determined (6). The discontinuous nature of these assays has led to a search for water-soluble substrates for PIPLC which would also allow continuous monitoring of PIPLC activity (7). Being able to monitor assays continuously is especially advantageous when carrying out studies of enzyme kinetics (8). We previously reported the synthesis of the first fluorescein-containing substrate for the continuous fluorescence assay of PI-PLC (9). This compound 1 (Scheme 1) is the only reported fluorogenic substrate that is cleaved by both the bacterial and mammalian forms of PI-PLC (10). Three specific properties make this compound especially attractive as a fluorogenic probe: (a) the high fluorescence quantum yield of fluorescein compounds, (b) the nonfluorescence of the substrate 1 itself, and (c) the relatively low pKa (∼4.8) of the fluorescent product 3, ensuring that a high percentage of the product is deprotonated, and therefore fluorescent, at physiological pH. * To whom correspondence should be addressed. (J.F.W.K) E-mail: [email protected]. Phone: (541) 346-4609. Fax: (541) 346-0487. (O.H.G) E-mail: [email protected]. Phone: (541) 346-4634. Fax: (541) 346-5891. † Department of Chemistry. ‡ Institute of Molecular Biology.

The useful properties of probe 1 prompted us to investigate its closest analogues by varying the aliphatic tail attached to the fluorescein part of the molecule. The different substituents on the aromatic ring can have a significant impact on certain physicochemical characteristics of the substrate, e.g., lipophilicity and water solubility. The availability of a set of such probes would provide a better optimization of the particular substrate to a specific assay experiment. However, as indicated below, problems with reproducibility were encountered with the synthetic scheme previously described for compound 1. In this paper, we describe a general procedure that overcomes these difficulties and permits the reproducible preparation of high quality fluorogenic substrates for assay of PI-PLC activity. EXPERIMENTAL PROCEDURES

Reagents and General Procedures. Reagent-grade solvents were used without further purification unless otherwise noted. All reagents were purchased from Aldrich Chemical Co. and were used as received. 1H NMR spectra were recorded on a Varian INOVA spectrometer at 300 MHz. Chemical shifts are in δ units (ppm) referenced to residual proton signals of the deuterated solvents. Coupling constants (J) are reported in hertz (Hz). NMR splitting patterns are designated as follows: s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; and br, broad. Column chromatography was carried out on Davisil silica gel (60-200 mesh). Analytical TLC was performed on Kieselgel 60 F254, and spots were rendered visible by exposing the plate to UV light. Radial chromatography (RC) was performed on a Chromatotron 7924T using 1, 2, and 4 mm disks covered with silica gel 60PF254 containing gypsum. High-resolution FAB mass spectrometry was performed by Dr. Brian Arbogast (mass spectroscopy facility, Oregon State University) on a Kratos MS-50TC mass spectrometer (Manchester, England). Elemental analyses were obtained from Robertson Microlit Laboratories, Madison, NJ. 1. Alkyl-Substituted Fluoresceins, 14a-d. General Procedure. To a solution of NaOH (2.00 g, 50.0 mmol) in

10.1021/bc0001138 CCC: $20.00 © 2001 American Chemical Society Published on Web 02/28/2001

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Scheme 1

methanol (200 mL) was added fluorescein 11 (8.30 g, 25.0 mmol). The mixture was stirred for 30 min or until all of the fluorescein dissolved. The resulting solution was evaporated and the residue was dried in a vacuum to give the disodium salt of fluorescein (9.40 g, quantitative). It was powdered and used for the alkylation step. In a typical experiment, the disodium salt 12 (9.40 g, 25.0 mmol) was suspended in DMF (200 mL) containing the alkyl bromide or iodide (200 mmol). The reaction mixture was stirred at RT (for methyl iodide) or at a higher temperature that did not exceed the boiling temperature of the alkyl bromide used. After stirring for 48-60 h the mixture was diluted with 5% NaHCO3 (200 mL) and extracted with ether (2 × 100 mL). The combined extracts were washed with brine (50 mL), dried over Na2SO4, and evaporated to give the corresponding ester-ether 13 as a brown oil. This substance was dissolved in methanol (150 mL) and mixed with a 2 M aqueous solution of NaOH (50 mL). The resulting mixture was stirred for 2 h at RT, concentrated under vacuum and diluted with water (80 mL). Impurities were extracted with ether (2 × 50 mL), and then the aqueous phase was acidified with concentrated HCl to pH about 2. The mixture was extracted with ether (3 × 80 mL), and the combined extracts were washed with brine (50 mL), dried over Na2SO4, and evaporated to give the crude alkylation product 14 which was finally purified by chromatography on silica gel using a gradient of ether in hexanes (from 1:1 to 3:1). Evaporation of appropriate fractions gave the alkylation product 14 (50-60% yield) which was pure by TLC and NMR. 14a, 1H NMR (CDCl3) δ 3.83 (s, 3H), 6.54-6.79 (m, 6H), 7.16 (d, J ) 7.2, 1H), 7.59-7.69 (m, 2H), 8.01 (d, J ) 7.5, 1H). 14b, 1H NMR (CDCl3) δ 1.05 (t, J ) 7.5, 3H), 1.84 (sextet, J ) 6.9, 2H), 3.98 (t, J ) 6.6, 2H), 6.606.95 (m, 6H), 7.17 (d, J ) 6.6, 1H), 7.60-7.70 (m, 2H), 8.07 (d, J ) 7.2, 1H). 14c, 1H NMR (CDCl3) δ 0.98 (t, J ) 7.2, 3H), 1.46 (sextet, J ) 7.2, 2H), 1.78 (m, 2H), 3.99 (t, J ) 6.3, 2H), 6.50-6.70 (m, 5H), 6.79 (dd, J ) 2.4, 11.7, 1H), 7.16 (d, J ) 6.9, 1H), 7.25-7.70 (m, 2H), 8.03 (d, J ) 6.6, 1H). 14d, 1H NMR (CDCl3) δ 0.91 (t, J ) 6.6, 3H), 1.32 (m, 6H), 1.77 (m, 2H), 3.96 (t, J ) 6.7, 2H), 6.68-6.72 (m, 2H), 6.76-6.72 (m, 2H), 7.16 (d, J ) 7.2, 1H), 7.66 (m, 2H), 8.02 (d, J ) 6.9, 1H). 2. Phosphate Triesters, 17a-d. General Procedure. To a suspension of alkylated fluorescein 14 (1.75 mol) in methylene chloride (10 mL) was added diisopropylethylamine (2.60 mmol) followed by addition of N,N-diisopropylmethylphosphonamidic chloride (2.60 mmol). The reaction mixture was stirred for 20 min at RT, diluted with ether (50 mL), washed with 5% NaHCO3 (10 mL), brine (10 mL), dried over Na2SO4, and evaporated to dryness. The residue was chromatographed on a short silica gel column with 50:50:1 ether-hexanes-Et3N mixture. The appropriate fractions were combined and evaporated, and the residue was reevaporated twice from toluene to give the corresponding phosphorimidite 15 as a clear oil. This was dissolved in methylene chloride (20

mL) and racemic pentaprotected inositol 16 [0.300 g, 0.930 mmoL, prepared as described (11)] was added to the above solution followed by the addition of tetrazole (0.190 g, 2.70 mmol) dissolved in acetonitrile (5 mL, needs warming to dissolve). The reaction mixture was stirred for 3 h at RT, then a 3 M solution of tert-butyl hydroperoxide in isooctane (0.7 mL, 2.1 mmol) was added to the solution, and the mixture was stirred for 1 h. Then it was diluted with methylene chloride (25 mL) and washed with 5% NaHCO3 (15 mL). The solution was dried over anhydrous MgSO4 and evaporated to give the crude phosphate 17 which was purified on silica gel twice; first with hexanes-ether mixture (from 4:1 to pure ether) then with a methylene chloride-ethyl acetate mixture (from 50:1 to 50:4). The corresponding phosphate 17 was a yellow foam, which was pure by TLC and NMR. 17a, 60%, 1H NMR (CDCl3-py-d5 50:1) δ 1.10-1.60 (m, 18H), 3.22-3.40 (m, 1H), 3.42-3.60 (m, 1H), 3.60-3.78 (m, 1H), 3.75-3.90 (m, 6H), 4.00-4.18 (m, 1H), 4.405.05 (m, 5H), 6.45-6.80 (m, 4H), 6.83-7.00 (m, 1H), 7.10-7.20 (m, 1H), 7.58-7.70 (m, 2H), 7.99 (m, 1H). 17b, 56%, 1H NMR (CDCl3-py-d5 50:1) δ 0.80-1.45 (m, 21H), 1.70 (sextet, J ) 7.5, 2H), 3.20-4.00 (m, 11H), 4.37 (brs, 1H), 4.72 (brs, 1H), 4.87 (m, 1H), 6.30-6.60 (m, 5H), 6.79 (brs, 1H), 6.98 (brs, 1H), 7.45-7.60 (m, 2H), 7.90 (d, J ) 6.9, 1H). 17c, 45%, 1H NMR (CDCl3-py-d5 50:1) δ 0.96 (t, J ) 7.5, 3H), 1.15-1.60 (m, 20H), 1.70-1.82 (m, 2H), 3.29-3.40 (m,1H), 3.50-3.60 (m, 1H), 3.65-3.75 (m, 1H), 3.80-4.15 (m, 8H), 4.45-4.65 (m, 1H), 4.75-4.85 (m, 1H), 4.95-5.05 (m, 1H), 6.50-6.75 (m, 4H), 6.85-6.98 (m, 1H), 7.10-7.15 (m, 1H), 7.25-7.36 (m, 1H), 7.55-7.70 (m, 2H), 8.01 (d, J ) 6.6, 1H). 17d, 69%, 1H NMR (CDCl3-py-d5 50:1) δ 0.88 (t, J ) 6.9, 3H), 1.10-1.60 (m, 24H), 1.76 (quintet, J ) 8.4, 2H), 3.29-3.40 (m, 1H), 3.45-3.60 (m, 1H), 3.65-3.75 (m, 1H), 3.85-3.98 (m, 5H), 4.00-4.13 (m, 3H), 4.44-4.62 (m, 1H), 4.50-4.82 (m, 1H), 4.945.04 (m, 1H), 6.56-6.74 (m, 4H), 6.85-6.96 (m, 1H), 7.08-7.14 (m, 1H), 7.30-7.35 (m, 1H), 7.56-7.68 (m, 2H), 8.00 (d, J ) 6.6, 1H). 3. Phosphate Diesters, 18a-d. General Procedure. To a solution of phosphate 17 (0.260 mmol) in acetone (5 mL) was added LiI (0.035 g, 0.26 mmol). The resulting mixture was stirred at reflux for 1 h. After that, the acetone was evaporated and the residue was chromatographed using a Chromatotron (2 mm silica gel disk). The product was eluted with a methylene chloride-methanol mixture (from 50:1 to 50:10). The eluate was evaporated to give the appropriate phosphate diester 18 as a viscous oil, which was pure by TLC and NMR. 18a, 89%, 1H NMR (CDCl3-py-d5 50:1) δ 0.80-1.60 (m, 18H), 3.20-4.30 (m, 9H), 4.49 (brs, 1H), 4.81 (brs, 1H), 4.92 (brs, 1H), 6.30-6.65 (m, 3H), 6.87 (brs, 1H), 7.03 (brs, 1H), 7.26 (brs, 2H), 7.50-7.70 (m, 2H), 7.96 (d, J ) 6.0, 1H). 18b, 85%, 1H NMR (CDCl3-py-d5 50:1) δ 0.93 (t, J ) 7.5, 3H), 1.00-1.50 (m, 18H), 1.71 (m, 2H), 3.30 (brs, 1H), 3.50 (m, 1H), 3.62 (t, J ) 7.8, 1H), 3.77 (m, 1H), 3.91 (m, 4H), 4.40 (brs, 1H), 4.75 (brs, 1H), 4.91 (m,

Fluorogenic Substrates for Assay of PI-PLC

1H), 6.40-6.65 (m, 5H), 6.87 (brs, 1H), 7.02 (brs, 1H), 7.56 (m, 2H), 7.94 (d, J ) 6.3, 1H). 18c, 96%, 1H NMR (CDCl3-py-d5 50:1) δ 0.94 (t, J ) 7.3, 3H), 1.00-1.60 (m, 20H), 1.75 (m, 2H), 3.20-4.00 (m, 8H), 4.30 (brs, 1H), 4.75 (brs, 1H), 4.95 (brs, 1H), 6.40-6.60 (m, 4H), 6.88 (brs, 1H), 7.02 (brs, 1H), 7.22 (m, 1H), 7.56 (m, 2H), 7.94 (d, J ) 6.3, 1H). 18d, 98%, 1H NMR (CDCl3-py-d5 50:1) δ 0.88 (t, J ) 6.3, 3H), 0.90-1.50 (m, 24 H), 1.74 (quintet, J ) 7.5, 2H), 3.30-4.00 (m, 8H), 4.45 (brs, 1H), 4.80 (brs, 1H), 4.93 (brs, 1H), 6.30-6.70 (m, 4H), 6.85 (brs, 1H), 7.02 (brs, 1H), 7.20 (brs, 1H), 7.57 (brs, 1H), 7.95 (brs, 1H). 4. Deprotected Phosphate Diesters, 19a-c and 1. General Procedure. Phosphate 18 (268 mmol) was suspended in the mixture of 8 mL of water and 2 mL of acetic acid. The reaction mixture was stirred for 16 h at RT and then extracted with ether (3 × 20 mL). The aqueous solution was diluted with ethanol and evaporated to dryness (bath temperature did not exceed 40 °C). The residue was re-evaporated from ethanol to remove acetic acid and dissolved in water (about 1 mL). Then ethanol (30 mL) was added to the solution and the resulting solid was filtered. The solid was washed subsequently with ethanol and ether and then it was dried in a vacuum to give the desired phosphate as a white powder, pure by NMR. 19a, 60%, 1H NMR (CD3OD-D2O 10:1) δ 3.28 (t, J ) 9.3, 1H), 3.46 (dd, J ) 2.7, 9.0, 1H), 3.65 (t, J ) 9.3, 1H), 3.80 (t, J ) 9.9, 1H), 4.08 (dt, J ) 3.0, 8.4, 1H), 4.23 (t, J ) 2.7, 1H), 6.60-6.75 (m, 3H), 6.92 (d, J ) 2.1, 1H), 7.01 (d, J ) 8.7, 1H), 7.19 (d, J ) 7.6, 1H), 7.30 (s, 1H), 7.70-7.90 (m, 2H), 8.05 (d, J ) 7.2, 1H). Anal. calcd for C27H24LiO13P‚H2O: C, 52.95; H, 4.28. Found: C, 53.21; H, 4.05. HRMS (FAB) calcd for C27H26O13P: 589.10996. Found: 589.11026 (M + H)+. 19b, 68%, 1H NMR (CD3OD-D2O 10:1) δ 1.07 (t, J ) 7.2, 3H), 1.83 (sextet, J ) 6.9, 2H), 3.28 (t, J ) 9.3, 1H), 3.44 (dd, J ) 2.4, 9.9, 1H), 3.55-3.75 (m, 1H), 3.85 (t, J ) 9.6, 1H), 4.02 (t, J ) 6.3, 2H), 4.10 (brt, J ) 9.6, 1H), 4.24 (brs, 1H), 6.65-6.75 (m, 3H), 6.88 (d, J ) 2.1, 1H), 7.05 (d, J ) 8.7, 1H), 7.22 (d, J ) 7.5, 1H), 7.34 (s, 1H), 7.73-7.90 (m, 2H), 8.06 (d, J ) 7.2, 1H). Anal. calcd for C29H28LiO13P‚H2O: C, 54.38; H, 4.72. Found: C, 53.98; H, 4.44. HRMS (FAB) calcd for C29H30O13P: 617.14240. Found: 617.14351 (M + H)+. 19c, 49%, 1H NMR (CD3OD-D2O 10:1) δ 0.96 (t, J ) 7.5, 3H), 1.48 (sextet, J ) 7.5, 2H), 1.76 (quintet, J ) 7.5, 2H), 3.27 (t, J ) 9.3, 1H), 3.45 (dd, J ) 2.4, 9.9, 1H), 3.65 (t, J ) 9.6, 1H), 3.81 (t, J ) 9.3, 1H), 4.04 (t, J ) 6.3, 2H), 4.02-4.11 (m, 1H), 4.28 (t, J ) 2.4, 1H), 6.626.72 (m, 3H), 6.87 (d, J ) 1.8, 1H), 7.02 (d, J ) 8.7, 1H), 7.19 (d, J ) 7.2, 1H), 7.30 (t, J ) 1.8, 1H), 7.72-7.86 (m, 2H), 8.04 (d, J ) 7.2, 1H). Anal. calcd for C30H30LiO13P‚ H2O: C, 55.05; H, 4.93. Found: C, 53.39; H, 4.57. HRMS (FAB) calcd for C30H32O13P: 631.15805. Found: 631.15794 (M + H)+. 1, 53%, 1H NMR (CD3OD-D2O 10:1) δ 0.90 (t, J ) 6.9, 3H), 1.20-1.50 (m, 6H), 1.77 (quintet, J ) 7.8, 2H), 3.26 (t, J ) 9.3, 1H), 3.44 (dd, J ) 2.7, 9.9, 1H), 3.65 (t, J ) 9.3, 1H), 3.81 (t, J ) 9.6, 1H), 4.03 (t, J ) 6.3, 2H), 4.00-4.10 (m, 1H), 4.23 (t, J ) 2.4, 1H), 6.626.74 (m, 3H), 6.87 (d, J ) 1.8, 1H), 7.02 (d, J ) 8.7, 1H), 7.19 (d, J ) 7.8, 1H), 7.31 (s, 1H), 7.70-7.88 (m, 2H), 8.04 (d, J ) 7.2, 1H). Anal. calcd for C32H34LiO13P‚H2O: C, 56.31; H, 5.32. Found: C, 56.58; H, 5.44. HRMS (FAB) calcd for C32H34O13P, 657.17370. Found: 657.17368 (MH)-. 5. Acid, 9. Phosphate diester 5 [10 mg, prepared by the earlier method (9)] was dissolved in 1 mL of water and the resulting solution was mixed with 875 mL of 0.0286 M aqueous NaOH. The solution was kept for 2 h

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at RT and then acidified with 2 M HCl to pH about 2. The mixture was extracted with ether (2 × 50 mL), the combined extracts were washed with brine (20 mL), dried over Na2SO4 and evaporated. The residue was purified using a Chromatotron (1 mm silica gel disk). The product was eluted with 1:1 hexanes-ether mixture. Evaporation of the eluate gave 2 mg of acid 9 as clear viscous oil, pure by TLC and NMR. 1H NMR (CDCl3) δ 0.90 (s, 3H), 1.201.50 (m, 6H), 1.76 (quintet, J ) 7.2, 2H), 3.92 (t, J ) 7.2, 2H), 6.36 (s, 1H), 6.44 (dd, J ) 2.7, 8.7, 1H), 6.51 (dd, J ) 2.4, 8.4, 1H), 6.60-6.63 (m, 2H), 6.89 (d, J ) 8.4, 1H), 6.94 (d, J ) 9.0, 1H), 7.11 (d, J ) 7.5, 1H), 7.18 (t, J ) 6.9, 1H), 7.32 (t, J ) 8.1, 1H), 7.92 (dd, J ) 1.2, 7.8, 1H). HRMS (EI) calcd for C26H26O5: 418.17802. Found: 418.17795 (M+). Enzymatic hydrolysis of 5 was accomplished as described below for PI-PLC assay. The aqueous phase after hydrolysis was extracted with ether (2 × 10 mL) and the combined extract was washed with brine (10 mL), dried over Na2SO4 and concentrated in a vacuum. The extracted product was identical by TLC with the product obtained after hydrolysis of 5 with sodium hydroxide. PI-PLC Assays. Stock solutions of substrate (∼2 mg/ mL) were prepared in deionized water, and accurate concentrations were determined by phosphate assay (12). A stock solution containing 0.5 nM PI-PLC (13) from Bacillus cereus was prepared in 6 mM HEPES, 0.06 mM EDTA, pH 7.0 [containing 0.1% poly(ethylene glycol) (PEG, average molecular weight 8000)]. Enzyme assays were carried out in 100 mM HEPES, 1 mM EDTA, pH 7.0 (containing 0.008% PEG) in a total volume of 0.8 mL in polymethacrylate cuvettes at 26 °C. Fluorescence measurements were made using a Hitachi F-4500 fluorescence spectrometer equipped with a yellow filter to reduce scatter from the excitation peak. Excitation was at 465 nm, emission was measured at 520 nm, and excitation and emission slits were set at 10 nm. After addition of enzyme, the fluorescence emission was recorded for 3 min in time scan mode. The change in fluorescence over the 3 min period was then converted to activity units (µmol product/min/mg enzyme) using a calibration curve of fluorescence vs concentration of product (produced by NaOH-induced cleavage of the substrate) and the concentration of enzyme used in the assay. RESULTS

The search for an alternative synthetic route for compound 1 was stimulated by the fact that some of the subsequent batches prepared by the published procedure exhibited 2-5-fold lower rates of production of fluorescence by PI-PLC. To investigate this phenomenon, a larger (100 mg) batch of substrate was prepared using the published protocol. Surprisingly, this larger batch did not show any rise of fluorescence characteristic of substrate 1 after exposure to PI-PLC under standard conditions. The NMR spectrum of the desired compound 1 differs from the NMR spectra of this freshly made batch of product in the aromatic region, and indicates that concomitant hydrogenolysis of the lactone ring took place during the hydrogenolysis reaction used to remove the benzyl groups in 4 (Scheme 2). The HRMS (FAB) spectra of the resulting reduced compound shows a peak at m/e 659.18979 ([M - H]-, calcd for C32H36O13P, 659.18936) which corresponds to the opened ring form 5. By comparison, the presumptive oxidized compound gives a peak 657.17368 ([M - H]-, calcd for C32H34O13P, 657.17370) corresponding to the lactone form 1.

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Scheme 2

Scheme 3

Scheme 4

A search of the literature revealed that hydrogenolysis of fluorescein 3,6-bis(dibenzyl phosphate) 6 can produce either ring-opened product 7 or fluorescein bisphosphate 8 by using different hydrogenation conditions (Scheme 3). Hydrogenolysis at -5 °C at atmosphere pressure in the presence of palladium on carbon gives mostly fluorescein bisphosphate 8. At temperatures higher than 5 °C the spiro lactone ring underwent hydrogenolysis to give the reduced acid 7 (14). Further experiments showed that keeping the diluted solution of compound 5 at room temperature in the presence of room light resulted in spontaneous conversion to substrate 1 (Scheme 2), which did produce a fluorescent product 3 when exposed to B. cereus PI-PLC (Scheme 1). Cleavage of 5 with either PI-PLC or sodium hydroxide produced the nonfluorescent reduced fluorescein derivative 9 (Scheme 4). We did not explore the mechanism of oxidation of nonfluorogenic compound 5 to fluorogenic substrate 1. It is possible that a photooxidation reaction is responsible for the conversion of 5 to 1. An example of a similar transformation is the known process of photooxidation

Scheme 5

of leuko-fluorescein 10 which gives fluorescein 11, as shown in the Scheme 5 (15). To avoid the problematic hydrogenolysis step, we developed an improved synthesis of fluorogenic substrates 1 and 19a-c (Scheme 6). This scheme makes use of pentaprotected inositol derivative 16 which was previously utilized for the improved synthesis of the chromogenic substrate 4-nitrophenyl myo-inositol-1-phosphate (NPIP) (11). Easily hydrolyzable protective groups in 16 facilitate the final acid-mediated deprotection. The fluorescein-derived phosphorimidites 15a-d were prepared from fluorescein 11

Fluorogenic Substrates for Assay of PI-PLC

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Scheme 6

as described earlier (9). Different substituents on the aromatic ring were introduced by varying the alkylating agent reacting with fluorescein disodium salt 12. The resulting ester-ether intermediates 13a-d were hydrolyzed to provide alkyl substituted fluoresceins 14a-d. Reaction of the alkylated fluorescein intermediates with MeOPClNiPr2 gave 15a-d. Protected inositol intermediate 16 was coupled with fluorescein-derived phosphorimidites 15a-d to form, after oxidation, protected phosphates 17a-d. Demethylation of the phosphate triester was accomplished using lithium iodide in acetone in place of trimethylsilyl bromide, which was used in our earlier synthesis (9). This change resulted in an increase in the yield of demethylated compound 18 from ∼65 to ∼95%. Finally, all inositol-protecting groups were removed at once using aqueous acetic acid. The resulting fluorogenic substrates were purified by precipitation with ethanol from aqueous solution followed by washing of the precipitates with ether and drying under vacuum. The final products 1 and 19a-c are likely mixtures of two racemic diastereomers because of the racemic nature

of the inositol moiety and the chirality center in the fluorescein moiety. The latter is not expected to have a significant effect on the enzyme-catalyzed hydrolysis reaction given that a wide variety of hydrophobic residues have been attached to D-myo-inositol and shown to be enzyme substrates (7). In the present case, the fluorescein moiety and the attached alkyl groups serve as the hydrophobic segment of the substrate. In previous studies of chromogenic substrates (i.e., NPIP) for PI-PLC, we found that racemic phosphoinositols posed no difficulties. One enantiomer (D-NPIP) was a substrate while the other enantiomer (L-NPIP) was neither a substrate nor an inhibitor (16). Other studies demonstrate that PI-PLC cleaves only the D-enantiomer of myo-inositol phosphate esters (17, 18). Thus, 1 and 19a-c should be adequate for most enzymatic and cell biological studies. Fluorogenic substrates 19a-c readily dissolved in water. Substrate 1 dissolves in water with some difficulty, due to the longer hydrophobic tail. All of these solutions exhibited only very low levels of fluorescence, which was due to the small quantity of fluorescent

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Zaikova et al. Table 1. Cleavage of Fluorogenic Substrates by B. cereus PI-PLCa fluorogenic substrate 19a 19b 19c 1

acronym

no. of carbon atoms in side chain

specific activity (µmol min-1 mg-1)

methyl FLIP propyl FLIP butyl FLIP hexyl FLIP

1 3 4 6

27 ( 3 86 ( 2 122 ( 4 110 ( 4

a Determined using 500 µM substrate at 26 °C in 100 mM HEPES, 1 mM EDTA, pH 7.0 (containing 0.008% PEG 8000).

°C to preserve activity). In the biochemical literature, it is common practice to use acronyms for chromogenic and fluorogenic substrates. For this purpose, we suggest the name FLIP (fluorescein myo-inositol phosphate) for this series of substrates by analogy with NPIP for the chromogenic 4-nitrophenyl myo-inositol-1-phosphate. For example, compound 1 is hexyl FLIP. DISCUSSION

Figure 1. (A) Change in fluorescence emission intensity for the cleavage of 50 µM substrate 1 as a function of time. The numbers on the right indicate the amount of PI-PLC added. (B) Correlation between the initial rates and enzyme concentration. The initial rates were obtained from the slopes of the curves in Figure 1A and a calibration plot of fluorescence vs concentration of product. Assays were carried out at 26 °C in 100 mM HEPES, 1 mM EDTA, pH 7.0 [containing 0.008% poly(ethylene glycol) 8000]. Excitation was at 465 nm and emission (in arbitrary units) was measured at 520 nm.

product (e.g., 3;