Synthesis and Characterization of Novel Spin-Labeled Photoaffinity

Bioconjugate Chem. 2000, 11, 725−733

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Synthesis and Characterization of Novel Spin-Labeled Photoaffinity Nonnucleoside Analogues of ATP as Structural and EPR Probes for Myosin Xiaoru Chen,† Jean Grammer,† Roger Cooke,‡ Edward Pate,§ and Ralph G. Yount*,† School of Molecular Biosciences, Department of Pure and Applied Mathematics, Department of Chemistry,Washington State University, Pullman, Washington 99164, and Department of Biochemistry and Biophysics & CVRI, University of California, San Francisco, California 94143. Received March 21, 2000

Two new spin-labeled photoreactive nonnucleoside ATP analogues, 1-(4-azido-2-nitrophenyl)amino3-(1-oxyl-2,2,5,5-tetramethylpyrrolidinyl-3-carbamido)-2-propyl triphosphate (SL-NANTP) and 2-(4azido-2-nitrophenyl)amino-2,2-(1-oxyl-2,2,6,6-tetramethyl-4-piperidylidene)di(oxymethylene) ethyl triphosphate (SSL-NANTP), were synthesized and characterized. This study aims to develop a second generation of NANTP-based analogues containing immobile spin labels that can be used to monitor conformational changes in myosin during the contractile cycle of muscle. Previous studies have shown that both a photoaffinity nonnucleoside ATP analogue, 2-[(4-azido-2-nitrophenyl)amino] ethyl triphosphate (NANTP) [Nakamaye et al. (1985) Biochemistry 24, 5226-5235], and a photoaffinity ATP analogue, 3′(2′)-O-4-[4-oxo-(4-amino-2,2,6,6-tetramethyl-piperidino-1-oxyl)-4-benzoyl] benzoyl adenosine 5′-triphosphate (SL-Bz2ATP) [Wang et al. (1999) J. Muscle Res. Cell Motil. 20, 743-753], behave like ATP in their interactions with myosin. Remarkably, photolabeled myosin recovers all of its normal enzymatic properties after treatment with actin in the presence of MgATP [Luo et al. (1995) Biochemistry 34, 1978-1987]. For SL-NANTP, the spin label moiety is attached to NANTP via an aminomethyl side chain. In SSL-NANTP, attachment is via a restricted spiro ring. The two new probes interact with myosin subfragment-1 (S1) in a manner analogous to ATP, and after photoincorporation, labeled S1 recovers full activity after treatment with actin and MgATP. The electron paramagnetic resonance (EPR) spectrum resulting from S1 photolabeled with SL-NANTP shows a very high degree of probe mobility. However, the EPR spectrum of S1 photolabeled with SSL-NANTP shows that the probe is highly immobilized with respect to S1, constrained to move within a cone of angle 52° (fullwidth, half-max). Unlike the parent, NANTP, which photolabels on the 23 kDa tryptic fragment of S1, SSL-NANTP photolabels on the 20 kDa fragment. Its highly immobile nature means that it is potentially a useful reporter group to monitor cross-bridge motion in muscle fibers.

INTRODUCTION 1

Electron paramagnetic resonance (EPR) has been a frequently employed method to monitor possible conformational changes of myosin and actin that may occur during active contraction of muscle (reviewed in refs 1 and 2). The most commonly used target for EPR spin probes is the reactive thiol on the heavy chain of myosin termed SH1 (Cys-707 in rabbit skeletal myosin) (3-7). SH1 on skeletal myosin can be modified with N-(1-oxyl2,2,6,6-tetramethyl-4-piperidinyl)iodoacetamide (IASL) to provide an immobile probe that can monitor changes in the orientation of myosin heads (8). A significant disadvantage to this approach is that chemical modification of SH1 changes the properties of myosin (8, 9) and potentially affects the active tension of muscle fibers (5, 10). Moreover, SH1 is known from the crystal structure of myosin subfragment 1 (S1) to lie in a region between the globular motor domain and the light chain binding domain (11). Recently, it was reported that a large and distinct rotation of the myosin light chain domain occurs * To whom correspondence should be addressed at the Department of Chemistry. E-mail: [email protected]. Phone: (509) 335-3442. Fax: (509) 335-9688. † School of Molecular Biosciences. ‡ Department of Biochemistry and Biophysics & CVRI. § Department of Pure and Applied Mathematics.

upon muscle contraction when Cys-108 of the regulatory light chain (RLC) was labeled with a cysteine-specific spin probe, 3-(5-fluoro-2,4-dinitroanilino)-2,2,5,5-tetramethyl-1-pyrrolidinyloxy (12). This indicates that movement of the tail portion of myosin heads is important in force generation. Similar probes to monitor possible 1 Abbreviations: EPR, electron paramagnetic resonance; IASL, N-(1-oxyl-2,2,6,6-tetramethyl-4-piperidinyl)iodoacetamide; RLC, regulatory light chain; FDNASL, 3-(5-fluoro-2,4dinitroanilino)-2,2,5,5-tetramethyl-1-pyrrolidinyloxy; S1, myosin subfragment 1; ATP, adenosine triphosphate; Bz2ATP, 3′-O-(4benzoyl)benzoyl-ATP; SL-Bz2ATP, 3′(2′)-0-4-[4-oxo-(4-amino2,2,6,6-tetramethyl-piperidino-1-oxyl)-4-benzoyl] benzoyl adenosine 5′-triphosphate; 4-amino-TEMPO, 4-amino-2,2,6,6-tetramethyl-l-piperidinyloxy; NANTP, 2-[(4-azido-2-nitrophenyl)amino]ethyl triphosphate; SL-NANTP, 1-(4-azido-2-nitrophenyl)amino-3-(1-oxyl-2,2,5,5-tetramethylpyrrolidinyl-3-carbamido)-2propyl triphosphate; SSL-NANTP, 2-(4-azido-2-nitrophenyl)amino-2,2-(1-oxyl-2,2,6,6-tetramethyl-4-piperidylidene)di(oxymethylene) ethyl triphosphate; SPC, salicylphosphorochloridite; Tris base, 2-amino-2-(hydroxymethyl)-1,3-propanediol; MALDI-MS, matrix-assisted laser desorption/ionization mass spectroscopy; SL-ester, 1-oxyl-2,2,5,5-tetramethylpyrrolidinyl-3-carboxylic acid N-hydroxysuccinimide ester; TEAB, triethylammonium bicarbonate; AcSSL-NANTP, 2-(4-azido-2-nitrophenyl)amino-2,2-(acetoxy-2,2,6,6-tetramethyl-4-piperidylidene)di(oxymethylene) ethyl triphosphate; SL-ATP, 2′, 3′-O-(1-oxyl-2, 2,6,6-tetramethyl4-piperidylidene)adenosine 5′-triphosphate.

10.1021/bc000032b CCC: $19.00 © 2000 American Chemical Society Published on Web 08/31/2000

726 Bioconjugate Chem., Vol. 11, No. 5, 2000

movement of the motor domain have depended on spinlabeled ATP analogues (13-15). The disadvantage to this approach is that a large and sharp spectral signal arises from unbound ATP analogue and overlaps with the desired spectral components from the probe bound at the active site of myosin. This problem potentially can be overcome by use of spin-labeled ATP photoaffinity analogues. As reported previously, it is possible to specifically photolabel rabbit skeletal myosin at Ser-324 with a photoreactive ATP analogue, Bz2ATP (16) and then reactivate the enzyme by treatment with actin and MgATP (17). This approach then when combined with a spin label attached to the photoreactive ATP analogue means it should be possible to introduce a site specific label at the active site. As a first step, we have synthesized and studied the spin-labeled photoaffinity ATP analogue, SL-Bz2ATP (18). A photoreactive p,p′-dicarboxybenzophenone moiety is attached to the 2′(3′) hydroxyls of ATP via an ester linkage, and the spin label 4-amino-TEMPO is amidated to the second carboxyl group. SL-Bz2ATP is a substrate for S1 and myosin in muscle fibers. It can be covalently incorporated at the active site with high specificity and yield, and the photolabeled myosin retains characteristic enzymatic properties. However, the EPR spectrum of SL-Bz2ATP photolabeled muscle fibers showed that this probe is too mobile to provide orientational information about myosin heads during contraction (18). A second synthetic approach utilizing the smaller nonnucleoside ATP analogue based on NANTP (19) was initiated. NANTP is a photoreactive ATP analogue, 2-[(4azido-2-nitrophenyl)amino]ethyl triphosphate that is a substrate for myosin, Fl ATPase and kinesin (Pate et al., unpublished data). It behaves like ATP when it reacts with myosin (19) and supports active tension and shortening of muscle fibers (20). Interestingly, it specifically photolabels Trp-130 in the 23 kDa of rabbit skeletal S1 (21). After treatment, with actin to weaken analogue binding, NANDP-labeled S1 has normal ATPase activity in the presence of MgATP (17). With these considerations in mind, we set out to design and synthesize new analogues of NANTP containing a nitroxide spin probe in addition to its nitrophenyl azide photoaffinity group. The synthesis and characterization of 1-(4-azido-2-nitrophenyl)amino-3-(1-oxyl-2, 2,5,5-tetramethylpyrrolidinyl-3-carbamido)-2-propyl triphosphate (SL-NANTP) and 2-(4-azido-2-nitrophenyl)amino-2,2-(1 -oxyl-2,2,6,6-tetramethyl-4-piperidylidene)di(oxymethylene) ethyl triphosphate (SSL-NANTP) are described here. These two new nonnucleoside ATP analogues are substrates for myosin, and the respective NANDP products are stably trapped at the active site by the phosphate analogues AIF4- or vanadate. While the EPR spectrum of photoincorporated SL-NANDP showed the probe to be too mobile to act as a reporter group, comparable labeling of S1 with SSL-NANDP gives a highly immobile spectrum, suitable to monitor conformational changes in myosin heads. In addition, SSL-NANDP was found to photolabel the carboxyl terminal 20 kDa tryptic fragment of S1 rather than the 23 kDa amino terminal fragment labeled by NANDP (21), showing that both of these major tryptic fragments, 23 and 20 kDa, function to make up part of the nucleotide binding site of myosin. This use of photoreactive spin-labeled substrates then represents a second method to do site-specific spin labeling that potentially can be used to report myosin head movement even in complex systems such as glycerinated muscle fibers.

Chen et al. MATERIALS AND METHODS

Microanalyses were carried out by Desert Analytics (Tucson, AZ) with a Perkin-Elmer 240C Elemental Analyzer. UV spectra were recorded on a Varian 2200 spectrometer. 1H NMR spectra were recorded on a Bruker AMX 300 and referenced to HOD (4.70 ppm) or TMS (0.0 ppm). 31P NMR spectra were obtained on a Varian VXR500 spectrometer by using 85% phosphoric acid as an external standard. EPR spectra of synthesized compounds were obtained on a Varian E-3 spectrometer using an ER 3200 cavity. Low-resolution mass spectra of the compounds without phosphate were recorded on a 7070 EHF VG analytical mass spectrometer. Low-resolution mass spectra of the compounds with phosphate were recorded on a matrix-assisted laser desorption/ionization mass spectrometer (MALDI) from PerSeptive Biosystems. High-resolution mass spectra of the final probes with phosphate were obtained at the Mass Spectrometry Center (University of Washington, Seattle, WA) on a PE Biosystems (Foster City, CA) Mariner electrospray-TOF run in negative ion mode with a mobile phase of 1:1 CH3CN/H2O. Thin-layer chromatographic analyses (TLC) were done on aluminum backed silica gel 60 F254 thinlayer chromatography plates (EM Industries, Inc.) using solvent A [isobutyric acid/H2O/concentrated NH4OH (66: 33:1)] or as described. Analyses of total phosphate and acid-labile phosphate were done as described previously (22). Flash chromatography used TLC silica gel (Aldrich, average particle size ) 2-25 gm) in a 4.2 (w) × 5.2 (h) cm sintered glass funnel. HPLC analysis was performed on a Spherisorb C18 column (100 × 4.6 mm). Eluant A, 100 mM triethylammonium acetate, pH 6.2; eluant B, 100 mM triethylammonium acetate, pH 6.2, in 90% acetonitrile; gradient 0 to 50% over 50 min at a flow rate of 1 mL/min. Capillary electrophoresis (CE) was performed on a Pace 5500 (Beckman) using a 50 cm × 75 µm uncoated capillary. Buffer: 20 mM sodium phosphate, pH 7.5; 20 kV, normal polarity; 20 °C; 215 nm. 4-Oxo-TEMPO, chymotrypsin, and Li3ADP were from Sigma Chemical Co.; 4-fluoro-3-nitrophenyl azide was synthesized from 4-fluoro-3-nitroaniline (Aldrich) as described by Guillory and Jeng (23). Other reagents not from Aldrich were BCA protein assay reagents (Pierce); sodium orthovanadate and Tris base (Fischer); Ultimagold scintillation Fluid (Packard); [32P]phosphoric acid (NEN). The boiling point of the petroleum ether used was 35-60 °C. Bis(tri-n-butylammonium) pyrophosphate was prepared as described by Moffatt (24) as follows: the solution of tetrasodium pyrophosphate decahydrate (4.46 g, 10 mmol) in cold water (100 mL) was applied to Dowex 50WX8 in the H+-form. This column was eluted with water at 4 °C. The fractions with pH below 5 (pH paper) were pooled (approximately 200 mL) and added into a vigorously stirred solution of tributylamine (3.70 g, 20 mmol) in ethanol (40 mL) in an ice bath. The solvents were removed by rotary evaporation and then coevaporated twice with ethanol (20-30 mL each time) at 30 °C. The last traces of water/ethanol were removed by coevaporating twice with anhydrous dimethylformamide (DMF) dried over molecular sieves. The residue was dissolved in DMF (20 mL) to form a light yellowish solution which was stored in a brown bottle under argon at -20 °C until used. Myosin subfragment 1 (S1) (115 000 g/mol) was prepared by a modification of the method of Okamoto and Sekine (25) as previously described (26) and stored in 50

Spin-Labeled Photoaffinity Nonnucleoside Analogues of ATP

mM Tris, 100 mM KCI, pH 8.0, at 4 °C (S1 buffer). F-actin was purified from rabbit skeletal muscle as described (27). Analytical Procedures. Protein concentrations were determined by the BCA protein assay or Coomassie Blue (Pierce) using unmodified S1 as the standard as previously described (28); 2801% ) 7.5 cm-l (29). ATPase assays were performed as described previously (30), except that release of inorganic phosphate was measured at 2 and 8 min after the addition of S1 to the assay mixture. 1-Oxyl-2,2,5,5-tetramethylpyrrolidinyl-3-carboxylic acid N-hydroxysuccinimide ester (SL-ester). SLester was synthesized by the general procedures of Hoffman et al. (31). 1-Oxyl-2, 2,5,5-tetramethylpyrrolidinyl-3-carboxylic acid (3-carboxy-proxyl) (0.53 g, 2.85 mmol) and N-hydroxysuccinimide (0.331 g, 2.88 mmol) were dissolved in the mixed solvent of ethyl acetate (10 mL) and DMF (3 mL) and cooled in an ice bath. A solution of N,N′-dicyclohexylcarbodiimide (0.595 g, 2.89 mmol) in ethyl acetate (4 mL) was added dropwise with stirring. After 2 h, the reaction mixture was taken out of the ice bath and stirred overnight at room temperature. Solvents were removed by rotary evaporation at 30 °C and the residue was fractionated by reduced pressure flash column chromatography using the solvents ethyl acetate/ petroleum ether (1:3, 1:2, and then 1:1, v/v) as eluents. A light yellowish solid (0.63 g) was obtained from the eluent of ethyl acetate/petroleum ether (1:1, v/v). TLC: one spot, Rf ) 0.42 (ethyl acetate/petroleum ether, 1:1, v/v). Yield: 78%. Melting point (mp): 185-186 °C. Electron ionization mass spectroscopic (EI-MS) analysis m/e (M+) found 283 (calcd, C13H19O5N2, 283). The EPR spectrum of the pure solid sample was recorded at 9.46 GHz and showed a typical nitroxyl radical spectrum with a singlet centered at 3381.0 G. 1-Amino-3-(4-azido-2-nitrophenyl)amino-2-propanol (1). 1,3-diamino-2-propanol (3.0 g, 33.34 mmol) was dissolved in 100 mL of 80% DMF/20% H2O and cooled in an ice bath. A solution of 4-fluoro-3-nitrophenyl azide (1.82 g, 10 mmol) in DMF (25 mL) was added dropwise with stirring in a period of 20-30 min. A deepred solid was precipitated from the solution. After 0.5 h, the reaction mixture was taken out of the ice bath and stirred overnight at room temperature. The deep-red solid was harvested by filtration and washed several times with methanol (10 mL each time). TLC of the solid product in solvent A showed one spot (Rf ) 0.58). The filtrate was concentrated to dryness by rotary evaporation at 30 °C, and the residue recrystalized from methanol to produce the second portion of pure product 1. Product: 2.02 g, mp 81-82 °C. Yield 80%. EI-MS m/e (M+) found 252 (calcd, C9H12O3N6, 252). 1H NMR (CD3COCD3): δ 1.17-1.27 (br, 2 H, NH2), 2.74 (br s, 1 H, OH), 2.90-3.25 (m, 2 H, CH2), 3.34-3.52 (m, 2 H, CH2), 4.104.13 (m, 1 H, CH), 7.13-7.25 (m, 2 H, phenyl H), 7.70 (d, 1 H, phenyl H), 8.10 (br s, 1 H, NH). Anal. (C9H12N6O3) C, H, N. 1-(4-Azido-2-nitrophenyl)amino-3-(1-oxyl-2,2,5,5tetramethylpyrrolidinyl-3-carbamido)-2-propanol (2). To a solution of 1-amino-3-(4-azido-2-nitrophenyl)amino-2-propanol (0.504 g, 2 mmol) in DMF (20 mL) was added dropwise SL-ester (0.566 g, 2 mmol) dissolved in 10 mL of ethyl acetate through a dropping funnel with a pressure-equalization arm at room temperature. After stirring overnight, the solvents were removed by rotary evaporation at 30 °C, and the residue isolated by reduced pressure flash column chromatography using acetone/ petroleum ether (1:3 and then 1:2, v/v) as eluents. Product: deep-red amorphous solid, 0.70 g, mp 137-138

Bioconjugate Chem., Vol. 11, No. 5, 2000 727

°C. Yield: 83%. TLC: one spot, Rf ) 0.46 (acetone/ petroleum ether, 1:2, v/v). Chemical ionization mass spectroscopic analysis (CI-MS) m/e (M+) found 420 (calcd, C18H26O5N7, 420). The EPR analysis of the pure solid sample was performed at a frequency of 9.46 GHz and showed a typical nitroxide spectrum with a singlet centered at 3381.0 G. The compound was further characterized by reduction to the hydroxylamine as described below. Anal. (C18H26N7O5) C, H, N. To obtain 1H NMR spectrum, the nitroxy group of compound 2 was reduced to the N-hydroxyl derivative as described (32). To a solution of compound 2 (0.1 g, 0.24 mmol) in chloroform (1 mL) 2 mL of 0.2 M phenylhydrazine in chloroform was added. This mixture was stirred for 1 h and then the solvent was removed. The residue was purified by flash chromatography using acetone/ petroleum ether (1:3 and then 1:2, v/v) as eluents. Product: deep-red solid, 50 mg. TLC: one spot, Rf ) 0.46. 1 H NMR (CDCl3): δ 1.12 (s, 3 H, CH3), 1.18 (s, 3 H, CH3), 1.22 (s, 6 H, CH3), 1.74-1.78 (d, 2H, CCH2C), 2.01-2.08 (t, 1 H, CHCO), 2.70 (br s, 1 H, OH), 2.90-3.10 (m, 2 H, CH2), 3.36-3.50 (m, 2 H, CH2), 4.10-4.13 (m, 1 H, CHO), 7.10-7.21 (m, 2 H, phenyl H), 7.65 (d, 1 H, phenyl H), 8.10 (br s, 1 H, NH). CI-MS m/e (M+) found 421 (calcd, C18H27O5N7, 421). 1-(4-Azido-2-nitrophenyl)amino-3-(1-oxyl-2, 2, 5, 5-tetramethylpyrrolidinyl-3-carbamido)-2-propyl triphosphate (SL-NANTP). SL-NANTP was synthesized according to the procedures of Ludwig and Eckstein (33-35) by which nucleoside triphosphates were synthesized. Compound 2 (0.39 g, 0.93 mmol) was dissolved in anhydrous dioxane/pyridine (8 mL, 3/1, v/v) in a rubber septum capped vessel under argon and cooled in an ice bath. A freshly prepared solution of SPC (0.207 g, 1 mmol) in anhydrous dioxane (2 mL) was injected through the septum and the resulting solution vigorously stirred for 10 min in an ice bath and another 10 min at room temperature. A solution of 0.5 M tetra(tri-n-butylammonium) pyrophosphate in anhydrous DMF (4 mL, 2 mmol) was injected into the reaction system cooled in an ice bath. After stirring for 10 min, a solution of 1% iodine in pyridine/water (98/2, v/v) (20 mL, 1.6 mmol) was added to oxidize phosphite to phosphate. After stirring for an additional 10 min in an ice bath and then for 30 min at room temperature, the excess iodine was destroyed by addition of 6 mL of 5% aqueous solution of sodium bisulfite. The reaction mixture was concentrated to dryness by rotary evaporation at 30 °C. The residue was dissolved in H2O (20 mL), and the solution cooled in an ice bath, and a concentrated ammonium hydroxide solution (40 mL) was added to hydrolyze the cyclic triphosphate. After 2 h, the reaction mixture was taken out of the ice bath and stored overnight at 4 °C. Argon was bubbled for 0.5 h to remove ammonia. The solution was evaporated to dryness by rotary evaporation at 30 °C, and the residue was dissolved in H2O (30 mL) (pH 6.95). One-half of this solution was applied to DEAE Sephadex A-25 (Aldrich, 40-120 gm) in a column (3.2 × 50 cm) and eluted with a linear gradient of 0.3 to 0.6 M triethylammonium bicarbonate (TEAB) at 4 °C (flow rate ) 3.5 mL/min, 4.8 min/fraction, total elution time ) 24 h). Pyrophosphate was eluted in fractions 95-141, and the deep-red product in fractions 149-173. A total of two runs of the DEAE Sephadex A-25 column were performed, and the fractions containing product SL-NANTP as determined by TLC (solvent A) were pooled. Water and triethylammonium bicarbonate were removed by rotary evaporation at 30 °C. The product showed a single spot on TLC with solvent A (Rf ) 0.48). HPLC analysis


(>97% pure): tR ) 27.8 min. CE analysis (>97% pure): tR ) 9.9 min. Yield: 0.20 mmol, 21.6% (based on its molar extinction coefficient of 4680 M- 1 cm-1 at 473 nm). Phosphate assays showed that the ratio of total phosphate to acid labile phosphate was 1.49; theory, 1.50. UV spectrum (water, pH 7.20): λmax at 261 and 473 nm. Molar extinction coefficients calculated from total phosphate assays were 24 360 ( 440 M-l cm-1 at 261 nm and 4680 ( 80 M-1 cm-1 at 473 nm. High-resolution negative electrospray MS m/e (M - H)- found 659.0911 (calcd, M - H, C18H28O14N7P3, 659.0907). The EPR spectrum of the solution of SL-NANTP dissolved in methanol recorded at 9.46 GHz showed a typical nitroxide spectrum with a triplet centered at 3378.3 G and a hyperfine splitting of 17.5 G. To obtain NMR spectra free of triethylamine, SLNANTP was precipitated as the sodium salt by addition of NaI acetone solution (1 M) and washed several times with cold acetone as described previously (21). A portion of SL-NANTP (0.2 mL, 23 mM, and pH 7.0) was reduced to the hydroxyamine derivative by addition of 50 µL of 100 mM ascorbic acid (pH 7.0) (13). Negative MALDIMS using R-cyano-4-hydroxy-cinnamic acid as matrix found m/e (M - H)- 660.09 (calcd, C18H29O14N7P3, 660.10) (36). 1H NMR (D2O): δ 1.26 (s, 3 H, CH3), 1.39 (s, 3 H, CH3), 1.42 (s, 3 H, CH3), 1.43 (s, 3 H, CH3), 1.82-1.86 (d, 2 H, CCH2C), 2.10-2.21 (m, 1 H, CHCO), 2.96-3.18 (m, 2 H, CH2), 3.59-3.72 (m, 2 H, CH2), 4.53-4.57 (m, 1 H, CH-O), 7.20-7.30 (m, 2 H, phenyl H), 7.82 (d, 1 H, phenyl H). 31P NMR (D2O): -9.70 (d, γ-P), -11.25 (d, R-P) and - 22.34 (t, β-P). 2-(Hydroxymethyl)-2-(4-azido-2-nitrophenyl)amino-1,3-propanediol (3). To a solution of 4-fluoro-3nitrophenyl azide (7.50 g, 41.0 mmol) dissolved in 26 mL of 80% DMF/20% H2O, 2-amino-2-(hydroxymethyl)-1,3propanediol (Tris base) (4.84 g, 20.0 mmol) was added. After stirring for 8 days, the solvents were removed with rotary evaporation at room temperature. The residue was isolated by flash chromatography using acetone/petroleum ether (1:2 and then 1:1). Product: 0.75 g, orange solid, mp 153-154 °C. Yield 26.5%. TLC showed one spot, Rf ) 0.32 (acetone/petroleum ether, 1:1). 1H NMR (CDCl3): δ 1.48 (br s, 3 H, OH), 3.86 (s, 6 H, CH2), 6.907.28 (m, 2 H, phenyl), 7.70 (d, 1 H, phenyl H), 8.30(s, 1 H, NH). EI-MS m/e (M+) found 283 (calcd, C10H13O5N5, 283). Anal. (C10H13N5O5) C, H, N. 2-(4-Azido-2-nitrophenyl)amino-2,2-(1-acetoxy2,2,6,6-tetramethyl-4-piperidylidene)di(oxymethylene) ethanol (5). 1-Acetoxy-4-methoxy-2,2,6,6-tetramethyl-1,2,5,6-tetrahydropyridine (4) was prepared according to the method of Alessi et al. (32). A total of 2.6 g (11.45 mmol) of compound 4 was added into a solution of compound 3 (0.3 g, 1.06 mmol) in tetrahydrofuran (THF) (5 mL), followed by a catalytic amount of ptoluenesulfonic acid monohydrate. After the reaction solution was stirred for 6 days, THF was removed by rotary evaporation at room temperature. The residue was isolated by flash chromatography using ethyl acetate/ petroleum ether (1:3, 1:2, and then 1:1) as eluents. Product: 0.16 g, orange solid, mp 135-136 °C. Yield 31.6%. TLC: one spot at Rf ) 0.37 (ethyl acetate/ petroleum ether, 1:1). 1H NMR (CDC13): δ 1.01 (s, 6 H, CH3), 1.19 (s, 3 H, CH3), 1.21 (s, 3 H, CH3), 1.50 (br s, 1 H, OH), 1.77-1.86 (t, 2 H, CH2), 1.98-2.06 (m, 5 H, CH2 and CH3CO), 3.76 (m, 6 H, CH2), 7.0-7.39 (m, 2 H, phenyl H), 7.79 (d, 1 H, phenyl H), 8.34 (s, 1 H, NH). CI-MS m/e (M + 1)+ found 479 (calcd, C21H30O7N6, 478). Anal. (C21H30N7O6) C, H, N. 2-(4-Azido-2-nitrophenyl)amino-2,2-(1-acetoxy-

Chen et al.

2,2,6,6-tetramethyl-4-piperidylidene)di(oxymethylene) Ethyl Triphosphate (AcSSL-NANTP). The phosphorylation of the spiroketal intermediate 5 and the subsequent displacement reaction were done as described previously (19, 20). Phosphorus oxychloride (0.15 mL, 1.6 mmol) was pipetted into a solution of N-acetoxy spiro ketal 5 (0.19 g, 0.4 mmol) in trimethyl phosphate (3 mL) in a glovebag under argon at room temperature. The reaction flask was sealed with a septum, an argon balloon was connected to the reaction flask to exclude moisture, and the reaction flask was immersed in an ice-bath immediately. After 8 h with stirring, the reaction solution was injected into a solution of bis(tri-n-butylammonium) pyrophosphate in DMF (6.4 mL, 3.2 mmol) and tri-nbutylamine (1.52 mL, 6.4 mmol) under argon and precooled in an ice bath. After 10 min, 1 M TEAB (25 mL) was added, and this mixture was left overnight at 4 °C. The solvents were removed with rotary evaporation at 30 °C. TEAB was removed by coevaporating with methanol. The residue was dissolved in water (30 mL), and this solution applied to DEAE Sephadex A-25 (Aldrich, 40120 gm) column (3.2 cm × 50 cm). The product was eluted with a linear gradient of 0.3 to 0.6 M TEAB (5 L total volume, pH 7.60) at 4 °C with a flow rate of 3.5 mL/min, and 16.8 mL fractions were collected. The major peak, AcSSL-NANTP, was eluted at 0.46 M TEAB buffer. The product fractions were pooled and taken to dryness by rotary evaporation at 30 °C. The residue was coevaporated three times with methanol to remove TEAB. Product: deep red solid, 0.2 mmol (based on the molar extinction coefficient M of 4100 M-1 cm-1 at 464 nm). Yield: 50.0%. TLC: one spot at Rf ) 0.32 (solvent A). HPLC analysis (>97% pure): tR ) 31.6 min. CE analysis (>97% pure): tR ) 8.4 min. Mass spectroscopic analysis (negative MALDI) found m/e (M - H)- 717.42 (calcd, C2lH32O16N6P3, 717.43) (36). 1H NMR(D2O): δ 1.21 (s, 6 H, CH3), 1.26 (s, 3 H, CH3), 1.39 (s, 3 H, CH3), 1.66-1.72 (t, 2 H, CH2), 2.01-2.12 (m, 5 H, CH2 and CH3CO), 3.64 (m, 6 H, CH2), 7.02-7.30 (m, 2 H, phenyl H), 7.80 (d, 1 H, phenyl H). 31P NMR (D2O): -10.20 (d, γ-p), -11.10 (d, R-p), -22.81 (t, β-p). 2-(4-Azido-2-nitrophenyl)amino-2,2-(1-oxyl-2,2,6,6tetramethyl-4-piperidylidene)di(oxymethylene) ethyl triphosphate (SSL-NANTP). SSL-NANTP was prepared from AcSSL-NANTP by a method modified from Alessi et al. (14). A solution of potassium hydroxide (0.55 g) in methanol (10 mL) was added into a reaction flask containing AcSSL-NANTP (164 µmol), methanol (15 mL), and water (15 mL). After the reaction mixture was saturated with oxygen, it was connected to an oxygen balloon and vigorously stirred for 8 days. A few drops of glacial acetic acid were added to bring the pH to 7.0. This solution was applied to a DEAE Sephadex, A-25 (Aldrich, 40-120 µM) column (3.2 cm × 50 cm) and eluted with a linear gradient of 0.3 to 0.6 M TEAB (5 L total volume, pH 7.60) at 4 °C and a flow rate of 3.5 mL/min. The red fractions containing the product were pooled and concentrated to dryness with rotary evaporation at 30 °C. The last traces of TEAB were removed by coevaporating three times with methanol (50 mL each). The yield was 156 µmol (95%) (based on M 4100 M-1 cm-1 at 464 nm). TLC showed one spot at Rf ) 0.32 (solvent A). HPLC analysis (>97% pure): tR ) 32.3 min. CE analysis (>97% pure): tR ) 10.6 min. High-resolution mass spectroscopy (negative electrospray) m/e (M - H)- found 674.0911 (calcd, C19H29O15N6P3, 674.0904). Phosphate assays showed that the ratio of total phosphate to acid labile phosphate was 1.47; theory, 1.50. UV (water, pH 7.0): λmax at 464 and 262 nm. Molar


extinction coefficients calculated from the total phosphate assay were 4100 ( 30 M-1 cm-l at 464 nm and 25 480 ( 360 M-1 cm-l at 262 nm. The EPR analysis of the solution of the product in methanol recorded at 9.45 GHz showed a typical nitroxide spectrum with a triplet centered at 3375 G and a hyperfine splitting of 16.0 G. To obtain NMR spectra free of triethylamine, SSLNANTP was converted to the sodium salt as described (22). A small portion of the solution of the product in water (2.3 µmol, pH 7.0) was reduced with a solution of sodium ascorbate in water (2.3 µmol, pH 7.30) and then dried with a speed vacuum. The residue was dissolved in D2O for running 31P NMR which showed three sets of peaks: -8.70 (d, γ-p), -10.80 (d, R-p), -22.24 (t, β-p). 1 H NMR (D2O): δ 1.20 (s, 6 H, CH3), 1.24 (s, 3 H, CH3), 1.38 (s, 3 H, CH3), 1.70-1.78 (t, 2 H, CH2), 2.03-2.15 (m, 2 H, CH2), 3.68 (m, 6 H, CH2), 7.01-7.22 (m, 2 H, phenyl H), 7.82 (d, 1 H, phenyl H). Negative MALDI mass spectroscopic analysis found (M - H)- 675.12 (calcd, C19H30O15N6P3, 675.10) (36). Synthesis of 2-(4-Azido-2-nitrophenyl)amino-2,2(1-oxyl-2,2,6,6-tetramethyl-4-piperidylidene)di(oxymethylene) ethyl [β-32P]diphosphate ([32P]SSLNANDP). [32P]SSL-NANDP was prepared by coupling of [32P]phosphate to 2-(4-azido-2-nitrophenyl)amino-2,2(1-oxyl-2,2,6,6-tetramethyl-4-piperidylidene)di(oxymethylene) ethyl monophosphate (SSL-NANMP) as described (19, 21). Yield 76% (based on the molar extinction coefficient of 4100 M-1 cm-1 at 464 nm). Specific activity 63 000 cpm/nmol. After 5 months, a small amount of [32P]SSL-NANDP was reduced by sodium ascorbate to obtain NMR spectra. 1H NMR (D2O) δ 1.10 (s, 6 H, CH3), 1.24 (s, 3 H, CH3), 1.37(s, 3 H, CH3), 1.69-1.75 (t, 2 H, CH2), 2.04-2.12 (m, 2 H, CH2), 3.68 (m, 6 H, CH2), 7.02-7.20 (m, 2 H, phenyl H), 7.81 (d, 1 H, phenyl H). 31P NMR (D2O): -9.62 (d, β-P), -12.20 (d, R-P). Negative MALDI mass spectroscopic analysis found (M - H)- 594.10 (calcd, C19H28O12N6P2, 594.12). SSL-NANMP was prepared by a similar method as for synthesis of SSL-NANTP, except that 1 N NaOH was added to pH 7.30 to hydrolyze the dichlorophospate to the monophosphate instead of addition of pyrophosphate. NMR analysis of SSL-NANMP reduced by sodium ascorbate: 1H NMR (D2O) δ 1.11 (s, 6 H, CH3), 1.21 (s, 3 H, CH3), 1.36(s, 3 H, CH3), 1.681.76 (t, 2 H, CH2), 2.01-2.12 (m, 2 H, CH2), 3.67 (m, 6 H, CH2), 7.03-7.23 (m, 2 H, phenyl H), 7.80 (d, 1 H, phenyl H). 31P NMR (D2O): 0.387 (s). Negative MALDI mass spectroscopic analysis found (M - H)- 514.12 (calcd, C19H27O9N6P, 514.16). Vanadate Trapping and Photoincorporation of SSL-NANDP and SL-NANDP onto Rabbit Skeletal Myosin S1. For SSL-NANTP trapping, S1 (26.1 µM) was incubated with 2 mM NiC12, 0.1 mM [32P]SSL-NANDP, and 1 mM sodium orthovanadate or an equivalent volume of S1 buffer (control) at 25 °C. After 40 min, EDTA (pH 8.0) and ATP were added to 20 and 5 mM, respectively, and the samples purified by Sephadex G-50 spin columns in S1 buffer (50 mM Tris, 100 mM KCI, pH 8.0 at 4 °C). The purified S1‚Ni‚[32P]SSL-NANDP‚Vi complex was irradiated at 0 °C for 16 min using a Hanovia 450 W medium-pressure Hg lamp. To avoid photodamage and nonspecific labeling, a Pyrex filter and a Schott 408 filter were used to remove most of the radiation below 400 nm. To determine the amount of trapping, samples of the complex and the control before irradiation were counted by liquid scintillation and the protein concentrations were measured by use of Coomassie blue. To determine the amount of covalent incorporation, known samples of the complex after ir-


radiation were precipitated by addition of perchloric acid to 5%, centrifuged and the pellets were analyzed for radioactivity. For SL-NANTP trapping, S1 (34 µM) was incubated with 2 mM MgCl2 and 0.2 mM SL-NANTP in S1 buffer for 10 min at 25 °C followed by addition of 5 mM NaF and 1 mM AlCl3 or an equivalent volume of S1 buffer (control). After 40 min at 25 °C, EDTA and pyrophosphate were added to 20 and 1.8 mM, respectively, and the samples purified by Sephadex G-50 spin columns in S1 buffer (for ATPase assays) or S1 buffer with NaCl replacing the KCl. The purified S1‚MgSL‚NANDP‚AlF4 complex in NaCl-S1 buffer was irradiated at 0 °C for 4 min using a Hanovia 450 W medium-pressure Hg lamp. To determine the amount of trapping and covalent incorporation, samples of the complex before and after irradiation were precipitated by addition of cold perchloric acid to 5% and microcentrifuged, and the OD261 of the neutralized supernatent was measured. The extinction coefficient at 261 nm for the photolyzed SL-NANDP was determined by measuring the OD261 of a sample of SLNANDP followed by irradiation of the sample and remeasuring the OD261 and was determined to be 11 880 M-1 cm-1. SDS-PAGE Analysis. Myosin S1 samples were analyzed by SDS-PAGE on 18 × 20 cm 12% gels according to the procedure of Laemmli (37). Protein bands were visualized by staining with a solution of Coomassie blue (1.61 g of Coomassie blue in 1 L of methanol, 1 L of water, and 200 mL of glacial acetic acid). Gel strips containing the bands of interest were excised and placed in 20 mL glass vials. Ultimagold (Packard) (10 mL) was added, and the radioactivity was determined. EPR Spectroscopy. After irradiation in the presence of SL-NANTP or SSL-NANTP and Pi analogue, the S1 solution is a mixture of covalently photolabeled S1, unmodified S1, and trapped but unmodified S1. The spectrum of covalently bound diphosphate analogue is desired. Irradiated samples were treated with actin at low ionic strength to release trapped Pi analogue and noncovalently bound SSL-NANDP or SL-NANDP before adding excess ATP to dissociate S1 and photolabeled S1 from the actin as previously described by Luo et al. (17). The samples were then centrifuged, and the supernatant was purified by gel filtration (17). The protein solution was dialyzed into a buffer containing 0.12 M KOAc, 5 mM Mg(OAc)2, 40 mM MOPS, 1 mM EGTA, pH 7.0, and then concentrated to 65 µM S1-SL-NANDP or S1-SSLNANDP by centrifugation through a semipermeable membrane (Centricon, 30 kDa cutoff). A sample in a 50 µL glass capillary was placed in the center of a TE011 cavity. Experiments were done at room temperature, 21-23 °C. EPR measurements were performed with an ER/200D EPR spectrometer from IBM Instruments, Inc. (Danbury, CT). First derivative, X-band spectra were recorded using 50 s, 125 G wide sweeps with the following instrument settings: microwave power, 25 mW; gain, 1.0 × 105-1.0 × 106; center field, 3478-3480 G; time constant, 200 ms; frequency, 9.3 GHz; modulation, 1 gauss at a frequency of 100 kHz. Each spectrum is the average of 25 distinct sweeps from an individual experimental preparation. RESULTS AND DISCUSSION

Synthesis of SL-NANTP. The purpose of this study is to develop a series of second generation spin-labeled photoaffinity nonnucleoside ATP analogues based on NANTP. The plan is to photolabel the residues of myosin


Chen et al.

Scheme 1: Synthesis of SL-NANTP

Scheme 2: Synthesis of SSL-NANTP

specifically near the active site so that motion of the head (or part of the head) can be monitored spectroscopically during the contraction process. The key step in the synthesis of SL-NANTP was phosphorylation of intermediate 2. Phosphorus oxychloride has been used in the past as a phosphorylating reagent for primary alcohols to synthesize ATP analogues (19, 20). Initially, we tried to use phosphorus oxychloride to phosphorylate the secondary alcohol of intermediate 2 without success, possibly because of steric hindrance. Salicylphosphorochloridite (SPC) has been previously employed as a phosphorylating reagent for the preparation of ATP analogues (33-35). This reagent is capable of undergoing three nucleophilic displacement reactions. We have exploited this multifunctionality in the synthesis of SLNANTP. The sequence of the synthetic reactions is shown in Scheme 1. The synthesis proceeds via the phosphorylation of intermediate 2 by SPC. This is followed by displacement of the leaving group with pyrophosphate, oxidation of phosphite by iodine, and hydrolysis of the cyclic meta-triphosphate by concentrated ammonium hydroxide. The total yield from phosphorylation of intermediate 2 to the final product SL-NANTP was around 22%, which compares favorably with other syntheses that used phosphorus oxychloride. The 31P NMR and 1H NMR spectra of SL-NANTP are very broad because of the presence of the nitroxide spin label. Reduction of the nitroxide of SL-NANTP with ascorbic acid yields the hydroxyamine derivative whose 31P NMR and 1H NMR spectra confirmed the predicted structure. Synthesis of SSL-NANTP. The route used to synthesize SSL-NANTP, in which the free radical is attached to NANTP via a spiro ketal group, is shown in Scheme 2. The most important steps in the synthesis of SSLNANTP were the syntheses of the triol intermediate 3 and N-acetoxy spiroketal intermediate 5. Because Tris base is only soluble in water whereas 4-fluoro-3-nitrophenyl azide is not, a mixture of 80% DMF and 20% water was chosen as the reaction solvent. Tris base was still not completely soluble, which resulted in long reaction times (8 days) and relatively low yields (26%). Nevertheless, Tris base was chosen for several reasons. First, Tris base contains three hydroxyl groups and an

amino group which are necessary to attach the spin label to NANTP via a spiroketal ring. Second, Tris base is the simplest compound suitable for our design, which makes the synthesized probe SSL-NANTP more likely to be a substrate for myosin. And third, Tris base is a convenient and cheap reagent. The N-acetoxy spiroketal intermediate 5 was synthesized by the reaction of the triol intermediate 3 and the enol ether spin label precursor 4 in the presence of the catalyst, F-toluenesulfonic acid monohydrate, by a procedure similar to that of Alessi et al. (14, 32). Because the steric hindrance to the reaction of the two hydroxyl groups of the intermediate 3 with enol ether 4 is large, the reaction proceeded slowly. However, using the chemistry we have described, intermediate 5 could be prepared smoothly. The N-acetoxy triphosphate derivative, AcSSL-NANTP, was prepared by phosphorylation of the intermediate 5 with POCl3 followed by the addition of tributylammonium pyrophosphate (19) or tributylammonium phosphate (38) to form the tri- and diphosphate products, respectively. In our experiments, the yield from the tributylammonium pyrophosphate reaction was much higher (50%) than that from tributylammonium phosphate (10%). To oxidize AcSSL-NANTP to the free radical probe SSL-NANTP, it was helpful to saturate the reaction solution with oxygen and to keep it in an oxygen atmosphere to accelerate the reaction. Alternatively, the final product SSL-NANTP was also prepared by oxidation of the intermediate 5 to the free radical nitroxide followed by phosphorylation with POCl3 and reaction with tributylammonium pyrophosphate, but the yield was lower (36%). SSL-NANTP and SL-NANTP Are Substrates for Myosin. It was first important to verify that SSLNANTP and SL-NANTP bound to myosin in a manner similar to ATP. The hydrolysis studies of SSL-NANTP, SL-NANTP, and ATP by S1 are summarized in Table 1. S1 cleaved SSL-NANTP in the presence of Ca2+ and K+/ EDTA at the rate of 208% and 16% of that with ATP, respectively. The hydrolysis rates for SL-NANTP in the presence of Ca2+ and K+/EDTA were 76% and 1.5% of that with ATP, respectively. In the presence of actin, Vmax



Table 1. Reaction Rates for SSL-NANTP and SL-NANTP as substrates for S1a assay conditions

ATP (µmol of Pi/ mg/min)

SSL-NANTP (µmol of Pi/ mg/min)

SL-NANTP (µmol of Pi/ mg/min)

K+ Ca2+ Mg2+/actin

5.39 ( 0.20 1.18 ( 0.04 5.68 ( 0.14

0.89 ( 0.13 2.45 ( 0.07 2.83 ( 0.02

0.08 ( 0.01 0.90 ( 0.10 not determined

a S1 and actin-activated ATPase activities were determined from liberated Pi assayed using the methods of Wells and Yount (42) and White (43). Assays were measured at 25 °C with the following conditions. Ca2+ATPase: 152 mM KC1, 15.2 mM CaCl2, 152 mM Tris, 7.6 mM SSL-NANTP or SL-NANTP or ATP, 0.35 µM S1, pH 7.70. K+/EDTA ATPase: 50 mM Tris, 5 mM EDTA, 0.6 M KC1, 5 mM SSL-NANTP or ATP, 0.35 µM S1, pH 7.50. Actin-activated Mg2+ATPase: 10 mM Tris, 3 mM MgCl2, 3 mM SSL-NANTP or SL-NANTP or ATP, 5.6 µM S1, pH 7.90, with 4.0-119 µM actin.

Table 2. Trapping and Incorporation of SSL-NANDP in S1 samplea control Vi/Ni2+ trapped a

% trapped % covalent % ATPaseb SSL-NANDP/S1 SSL-NANDP/S1 98.0 31.0

2.5 66.0

13.5

S1‚Ni2+‚SSL-NANDP‚Vi

The complex was prepared and irradiated as described as under Materials and Methods. b The NH4+-EDTA ATPase activity of the purified S1‚Ni.SSL-NANDP‚Vi complex and control sample were determined as previously described (42), except that the release of inorganic phosphate was measured after 2 and 8 min. These were then compared to standard S1. Table 3. Trapping and Incorporation of SL-NANDP in S1 samplea

% ATPaseb

% trapped SL-NANDP/S1

% covalent SL-NANDP/S1

control AlF4 trapped

101.0 22.4

4.4 89.7

38.5

a

S1‚Mg2+‚SL-NANDP‚AlF

The 4 complex was prepared and irradiated as described as under Materials and Methods. b The + NH4 -EDTA ATPase activity of the purified S1‚Mg2+‚SLNANDP‚A1F4 complex and control sample were determined as described in Table 2 and were then compared to standard S1.

for the MgSSL-NANTPase was 50% of MgATPase. These results show that both SSL-NANTP and SL-NANTP are substrates for S1 with properties similar to ATP. Trapping and Covalent Photoincorporation of SSL-NANDP and SL-NANDP into S1. The specificity of labeling is an important consideration in the photoincorporation of ATP analogues in myosin. It has been shown that trapping of nucleotide analogues prior to irradiation is essential to obtain specific photolabeling of the active site of myosin (25, 26, 19). S1 was incubated with [32P]SSL-NANDP and Vi as described in Methods and Materials, and untrapped SSL-NANDP and Vi were then removed by gel centrifugation columns. It was found that 66% of the sites were occupied. After photoirradiation and removal of unbound SSL-NANDP by actin treatment, S1 was covalently labeled at 13.5% of the sites (see Table 2). Here the Ni2+ acts to prevent vanadatepromoted photooxidation of myosin that occurs in the presence of Mg2+ (26). If S1 was treated with SL-NANTP, MgCl2 and AlF4-, almost 90% of the active sites were trapped, and 38% of the active sites were subsequently photolabeled (See Table 3). EPR Spectroscopy of Photoincorporated SLNANTP and SSL-NANTP. Figure 1 shows EPR spectra obtained from S1 photolabeled with SL-NANDP (dashed line) and SSL-NANDP (solid line). The three sharp peaks (P2, P3, and the central large peak) in the SL-NANDP

Figure 1. EPR spectra of SL-NANDP (dashed line) and SSLNANDP (solid line) covalently attached to S1 in solution. The spectra are the average of 25 distinct, 125 mT wide sweeps.

spectrum are indicative of a probe that is covalently attached to the protein. However, the probe is highly mobile with respect to the protein, undergoing rapid, nanosecond rotation. The low-field shoulder at P1 in the SL-NANDP spectrum does indicate a very small, more highly immobilized population of probes. This component is too small to be of use in orientational studies, however. The solid line in Figure 1 is the EPR spectrum obtained from S1-SSL-NANDP. The splitting between the low field peak, P1, and the high field dip, P4, is 65.5 ( 0.5 G (mean ( SEM, 4 obs) indicating a covalently attached probe that is highly immobilized with respect to S1. The analysis of Griffith and Jost (39) indicates the probe constrained to move within a cone of angle approximately 52° (fullwidth, half-max). The small superimposed peaks at P2 and P3 show an additional, small (

Synthesis and Characterization of Novel Spin-Labeled Photoaffinity

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