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Bioconjugate Chem. 1998, 9, 160−167
A Homobifunctional Rhodamine for Labeling Proteins with Defined Orientations of a Fluorophore John E. T. Corrie,* James S. Craik, and V. Ranjit N. Munasinghe National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, U.K. Received September 22, 1997; Revised Manuscript Received December 15, 1997
The synthesis and characterization of a bifunctional rhodamine dye bearing 2-(iodoacetamido)ethyl substituents on the 3′- and 6′-nitrogen atoms is described. Aspects of the conversion of chloroacetamides to iodoacetamides are discussed, including a remarkably mild dehalogenation of an aromatic haloacetamide in the presence of NaI and camphorsulfonic acid. The bifunctional rhodamine has been designed for two-site, 1:1 labeling of proteins that contain two suitably disposed cysteine residues and is intended to constrain the orientation of the rhodamine absorption and emission dipoles in a predictable relationship to the protein structure.
INTRODUCTION
We have previously described (1) the synthesis and characterization of the 5- and 6-isomers of iodoacetamidotetramethylrhodamine 1 and 2, and these compounds
have been used to label Cys 707 (SH-1) on the myosin heavy chain of rabbit skeletal muscle and Cys 108 on the regulatory light chain of chicken gizzard myosin. Fluorescence polarization measurements with sub-millisecond time resolution on these labeled proteins in skeletal muscle fibers have provided strong evidence that the regulatory domain of myosin changes orientation during the power stroke of muscle contraction (2a) while the catalytic domain does not (2b). The probe on Cys 707 of the catalytic domain gave evidence for coupling between * Address correspondence to Dr. J. E. T. Corrie, National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, U.K. Phone: 011 44 181 959 3666 (extn 2276). Fax: 011 44 181 906 4419. E-mail:
[email protected].
the actin-myosin and the myosin-nucleotide interaction sites (2b). Despite the success of these biophysical studies, there have been residual uncertainties of interpretation because the orientation of the reporter fluorophore relative to the protein might change during the contractile cycle (3). More importantly, we envisaged that new information would become available if the rhodamine fluorophore could be linked to muscle proteins, particularly to the 20 kDa myosin regulatory light chain (RLC), by two bonds which would constrain the dye to particular orientations fixed by the termini of the two links to the protein. With the probe dipole fixed in the coordinate frame of the RLC, which is itself contained in the ordered filament lattice of muscle cells, it should be possible to measure molecular coordinates in real time in a working muscle cell and thus to bridge the gap between static crystal structures and cellular function. Other workers have used homo- and heterobifunctional reagents as immobilized or oriented probes, with particular activity in the synthesis and use of EPR probes. Gaffney et al. (4) described a series of spin-labeled R,ωalkanedioic acids and studied their interaction with lipid bilayers. A membrane-spanning orientation was found only when the total chain length matched the bilayer thickness. A similar strategy has been used to locate a photolabile diazirene moiety deep in a lipid bilayer, thereby providing a reagent with which to identify transmembrane domains of membrane-spanning proteins (5). In a series of papers, Beth and co-workers have used bis(N-sulfosuccinimidyl) esters of spin-labeled pimelic and azelaic acids to link near-neighbor lysine groups on the extracellular domains of membrane proteins (6). Two-site linkage was demonstrated, and tight immobilization of the probe relative to the protein was found for both reagents. Heterobifunctional spin-labeled reagents have been described (7) which react first with a thiol group on the protein to provide a single attachment site and are then linked nonspecifically to a second site by photolysis of an aryl azide. Rigidification of the label following formation of the second link was explicitly demonstrated. Examples of bifunctional fluorescent probes include crabescein, a fluorescein derivative which has been used specifically to label a reduced disulfide bond in immu-
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A Homobifunctional Thiol-Reactive Rhodamine
noglobulin G (8), and bifunctional cyanine dyes (9) which label -amino groups of lysine residues. The attachment site of crabescein can be predicted with some accuracy when, as in immunoglobulin G, the site of the most readily reduced disulfide is known, but for the lysinedirected cyanines, there is little opportunity to control the site of labeling. Nevertheless, results with each of these probes confirmed a reduction in independent probe mobility resulting from the two-site attachment (8, 10). The studies reviewed above indicate that two-site attachment of spectroscopic probes can provide significant information gain. It is particularly important that 1:1 probe-protein stoichiometry can be demonstrated and achieved with reasonable yields. These factors should not be taken for granted, since formation of a very large macrocycle is an implicit feature of the concept. The protein scaffold, which holds the two attachment sites on the protein in a restricted spatial distribution, evidently makes a major contribution to the experiments described above (8, 10). A key part of our strategy was the possibility that the dye should react with its target protein at chosen sites. We anticipated this would most readily be achieved with a reagent which would alkylate two cysteine residues on the protein, and for our particular application we planned to use the known three-dimensional structures of myosin proteins (11) to select suitable sites at which to introduce these cysteine residues by site-directed mutagenesis. Thus, we hoped to create a set of labeled regulatory light chains which could carry the fluorophore with differing three-dimensional orientations into the ordered actomyosin filament lattice of muscle fibers. The emission dipole of xanthene dyes is coincident with the long axis of the xanthene (12), and we anticipated that fluorescence polarization measurements on these labeled fibers would be able to map the orientations of the dye and hence of their carrier protein in both static and dynamic experimental protocols. This paper deals with the synthesis of the bis-iodoacetamide 3, which was chosen with the expectation that the linker arms would substantially constrain the movement of the fluorophore. These linker arms are as short as is practical, consistent with chemical stability and maintaining the rhodamine chromophore. In accordance with our previous convention (1), all rhodamine compounds are drawn and named as their nonfluorescent tautomeric lactone form. MATERIALS AND METHODS
Microanalyses were carried out by the Chemical Analysis Centre (University of Kent, Canterbury, U.K.) or MEDAC Ltd. (Brunel University, Uxbridge, U.K.). Lowresolution electrospray mass spectra were determined on a VG Platform instrument, and high-resolution FAB spectra were determined on a VG ZAB-SE instrument. 1H NMR spectra were determined in deuteriochloroform on JEOL FX90Q, Bruker WM400, and Varian Unity 500Plus spectrometers with tetramethylsilane as the internal standard unless otherwise stated. J values are given in hertz. The concentration-dependent UV-vis spectra of rhodamine derivatives were recorded on a Cary 3E spectrophotometer. Buffer solutions were prepared from boric acid or potassium dihydrogen phosphate at the molarities specified and adjusted to the required pH value by addition of solid KOH, followed by 4 M aqueous KOH for final adjustment. Merck 9385 silica gel was used for flash chromatography. Trimethylsilyl polyphosphate was from
Bioconjugate Chem., Vol. 9, No. 2, 1998 161
Fluka (Gillingham, Dorset, U.K.). Other reagents were from Aldrich (Gillingham, Dorset, U.K.). Petroleum ether was the fraction boiling at 40-60 °C. Procedures involving rhodamines were performed under subdued light. Organic extracts were dried over anhydrous sodium sulfate and evaporated under reduced pressure. 3′,6′-Bis(1-pyrrolidinyl)spiro[isobenzofuran1(3H),9′-[9H]xanthen]-3-one (5). A suspension of 3′,6′dichlorospiro[isobenzofuran-1(3H),9′-[9H]xanthen]-3one 4 (13) (0.074 g, 0.2 mmol), anhydrous ZnCl2 (0.136 g, 1 mmol), and pyrrolidine (0.142 g, 2 mmol) in dry chlorobenzene (1 mL) was heated at reflux under nitrogen for 4 h and then cooled to room temperature. The reaction mixture was diluted with chloroform (50 mL), and the solution was washed with 2 M aqueous KOH (50 mL) and dried. Silica gel (0.5 g) was added, and the solvent was removed under reduced pressure. The silica gel containing the adsorbed reaction products was added to the top of a flash chromatography column containing silica gel (40 mL), and the column was successively eluted with chloroform (50 mL), methanol-chloroform (1:9 v/v, 100 mL), methanol-chloroform (1:4, 100 mL), and methanol-chloroform (1:3, 200 mL) to afford the rhodamine 5 as a purple glass(0.067 g, 76%): low-resolution electrospray mass spectroscopy (ES-MS) m/e (M + H) calcd 439, found 439; 1H NMR (90 MHz) δ 1.86-2.06 (m, 8H, NCH2CH2), 3.17-3.34 (m, 8H, NCH2CH2), 6.23 (dd, 2H, J ) 9 and 2.5, 2′,7′-H), 6.34 (d, 2H, J ) 2.5, 4′,5′-H), 6.60 (d, 2H, J ) 9, 1′,8′-H), 7.07-7.20 (m, 1H, 7-H), 7.407.63 (m, 2H, 5,6-H), 7.86-8.03 (m, 1H, 4-H). This compound has previously been reported as its hydrochloride salt (14a) and characterized by its visible absorption spectrum and elemental analysis. Ethyl N-[(2-Methylamino)ethyl]carbamate (7). Aqueous methylamine (40%, 100 mL) was added to a solution of ethyl N-(2-bromoethyl)carbamate 6 (25) (3.9
g, 20 mmol) in methanol (20 mL), and the mixture was stirred for 2 h at room temperature. The solvents were removed under reduced pressure, and the residue was partitioned between 5% aqueous NaHCO3 (50 mL) and dichloromethane (50 mL). The organic extract was dried and concentrated under reduced pressure to afford the carbamate 7 as a clear liquid (1.9 g, 65%) which was used without further purification as it decomposed on distillation: 1H NMR (90 MHz) δ 1.23 (t, 3H, J ) 7, CH3), 2.40 (s, 3H, NCH3), 2.69 (t, 2H, J ) 6, MeNHCH2), 3.23 (q, 2H, J ) 6, CONHCH2), 4.08 (q, 2H, J ) 7, OCH2), 5.31 (br s, 2H, 2 × NH). The derived ethyl N-({2-[N′(3,5-dinitrobenzoyl)-N′-methyl]amino}ethyl)carbamate had a melting point of 125-126 °C (EtOAc-petroleum ether). Anal. (C13H16N4O7) C, H, N. 3-Methoxy-N-methylaniline (10). The preparation of the starting amine was modified from the procedure of Amery and Corbett (16) to give a convenient method with minimal manipulation. Benzenesulfonyl chloride (86 g, 487 mmol) was added over the course of 0.5 h to a mechanically stirred mixture of freshly distilled manisidine (50 g, 406 mmol) and 2 M aqueous KOH (1100 mL). The mixture was stirred for 1.25 h at room temperature and then heated on a steam bath for 1 h.
162 Bioconjugate Chem., Vol. 9, No. 2, 1998
The hot solution was filtered, and the filtrate was cooled in an ice bath. Dimethyl sulfate (42.5 mL, 447 mmol; Caution: potent alkylator) was added, and the mixture was allowed to warm to room temperature and stirred for 1.5 h, and then kept overnight. Concentrated aqueous ammonia (50 mL) was added, and the mixture was stirred for 0.5 h and then extracted with ether (4 × 150 mL). The ether solution was washed with 2 M aqueous HCl (2 × 100 mL), dried, and evaporated under reduced pressure to give the crude N-methylsulfonamide (54 g). This material was refluxed with 6 M aqueous HCl (350 mL) for 7 h, then cooled to room temperature, neutralized by addition of solid KOH, and extracted with ether. The ether extract was dried and evaporated, and the residue was distilled to give the title compound as a pale yellow oil (20.55 g, 37% overall): bp 98 °C at 1.6 mmHg [lit. (17) 131 °C at 17 mmHg]; 1H NMR (400 MHz) δ 2.76 (s, 3H, NCH3), 3.74 (s, 3H, OCH3), 6.13 (t, 1H, J ) 2.2, 2-H), 6.18-6.27 (m, 2H, 4,6-H), 7.06 (t, 1H, J ) 8, 5-H). The N-acetyl derivative was crystallized from petroleum ether, with a melting point of 64-65 °C [lit. (17) 64 °C]. Ethyl N-(3-Methoxyphenyl)-N-methylglycinate (11). A solution of the amine 10 (17.0 g, 124 mmol) and ethyl bromoacetate (16.5 mL, 149 mmol) in acetonitrile (175 mL) together with NaHCO3 (10.9 g, 130 mmol) was stirred and refluxed under nitrogen for 4 h and allowed to cool. Triethylamine (9.8 mL) was added, and the mixture was stirred for 16 h at room temperature and then filtered and evaporated. The residue was dissolved in ether (250 mL), washed with water (3 × 150 mL), dried, evaporated, and distilled to give the ester 11 as a clear liquid (24.0 g, 86%): bp 130-132 °C at 0.6 mmHg; low-resolution ES-MS m/e (M + H) calcd 224, found 224; IR (film) 1745 cm-1; 1H NMR (400 MHz) δ 1.23 (t, 3H, J ) 7.15, CH2CH3), 3.04 (s, 3H, NCH3), 3.76 (s, 3H, OCH3), 4.02 (s, 2H, NCH2), 4.16 (q, 2H, J ) 7.15, CH2CH3), 6.23 (br s, 1H, 2-H), 6.28-6.32 (m, 2H, 4,6-H), 7.12 (t, 1H, J ) 8.2, 5-H). 2-[N-(3-Methoxyphenyl)-N-methylamino]ethanol (12). A suspension of sodium borohydride (7.1 g, 187 mmol) and lithium bromide (16.3 g, 187 mmol) in dry THF (188 mL) was refluxed under nitrogen for 1 h. A solution of the ester 11 (22.0 g, 98.7 mmol) in dry THF was added over the course of 10 min, and the mixture was refluxed for a further 5 h. The solvent was evaporated, and the residue was treated slowly with 1 M aqueous KH2PO4 (500 mL) and stirred until hydrogen evolution ceased (ca. 1 h), and then extracted with dichloromethane (5 × 100 mL). The organic extracts were washed with saturated aqueous NaHCO3 (150 mL), dried, and evaporated. The residue was distilled to give the alcohol 12 as a colorless viscous oil (15.1 g, 84%): bp 127-128 °C at 0.7 mmHg; low-resolution ES-MS m/e (M + H) calcd 182, found 182; IR (film) 3350 cm-1; 1H NMR (90 MHz) δ 2.40 (br s, 1H, OH), 2.86 (s, 3H, NCH3), 3.37 (t, 2H, J ) 5, OCH2CH2N), 3.71 (s, 3H, OCH3) superimposed on 3.70 (t, 2H, J ) 5, OCH2CH2N), 6.17-6.45 (m, 3H, Ar-H), 7.06 (t, 1H, J ) 8, 5-H). The derived 2-[N-(3-methoxyphenyl)-N-methylamino]ethyl 3,5-dinitrobenzoate was crystallized from EtOAc-petroleum ether as red needles, with a melting point of 152-153 °C. Anal. (C17H17N3O7) C, H, N. N-{2-[N′-(3-Methoxyphenyl)-N′-methylamino]ethyl}phthalimide (13). A mixture of the alcohol 12 (7.88 g, 43.5 mmol), triphenylphosphine (12.3 g, 47 mmol), 4 Å molecular sieves (4.0 g), and phthalimide (6.8 g, 46.3 mmol) in dry THF (230 mL) and dry acetonitrile (38 mL) was stirred at -78 °C under nitrogen. Diethyl
Corrie et al.
azodicarboxylate (8.1 g, 46.6 mmol) was added dropwise over a period of 5 min, and the solution was stirred for 3 h at -78 °C and then allowed to warm to room temperature and stirred overnight. The solvent was evaporated under reduced pressure, and the residue was dissolved in 1 M aqueous sulfuric acid (100 mL) and washed with ether (200 mL). The organic layer was back-extracted with 1 M aqueous sulfuric acid (5 × 50 mL), and the combined aqueous solution was stirred vigorously and basified to pH 8.5 by slow addition of 2 M aqueous KOH and solid NaHCO3. The resulting suspension was extracted with ether (4 × 100 mL), dried, and evaporated. The residue was filtered through a short column of silica gel (flash chromatography grade) in EtOAc-petroleum ether (1:4), and the eluate was evaporated and triturated with diisopropyl ether to give the phthalimide 13 (8.37 g, 62%) as yellow needles: mp 81 °C from diisopropyl ether; IR (Nujol) 1770, 1705 cm-1; 1H NMR (400 MHz) δ 2.98 (s, 3H, NCH3), 3.60 (t, 2H, J ) 7, CH2N), 3.76 (s, 3H, OCH3), 3.88 (t, 2H, J ) 7, CH2N), 6.15 [dd, 1H, J ) 8 and 1.9, 4(6)-H], 6.33 (t, 1H, J ) 1.9, 2-H), 6.37 [dd, 1H, J ) 8 and 1.9, 6(4)-H], 7.03 (t, 1H, J ) 8, 5-H), 7.647.69 (m, 2H, phthaloyl 4- and 5-H), 7.76-7.80 (m, 2H, phthaloyl 3- and 6-H). Anal. (C18H18N2O3) C, H, N. N-{2-[N′-(3-Acetoxyphenyl)-N′-methylamino]ethyl}acetamide (14). A solution of the phthalimide 13 (11.2 g, 36.1 mmol) in concentrated aqueous hydrobromic acid (325 mL) was refluxed under nitrogen for 2 h and allowed to cool. The solution was evaporated to dryness in vacuo (dry ice condenser), and the crude amine hydrobromide was dissolved in 2 M aqueous potassium borate (pH 9.3, 360 mL) under nitrogen and cooled in an ice bath. A solution of acetic anhydride (27 mL, 286 mmol) in dichloromethane (180 mL) was added over the course of 20 min with vigorous stirring. The aqueous phase was maintained at pH 9.0-9.5 by addition of KOH pellets as required, and after 15 min, a further portion of acetic anhydride (9 mL) in dichloromethane (18 mL) was added. The solution was stirred for 2 h with ice cooling, while the pH was maintained as described above, and then allowed to warm to room temperature and treated with a final portion of acetic anhydride (4 mL) in dichloromethane (10 mL). After a further period of 0.5 h at pH 9-9.5, the organic phase was separated and the aqueous phase was extracted with dichloromethane (3 × 100 mL). The combined organic extracts were washed with saturated aqueous sodium hydrogen carbonate (2 × 100 mL), dried, and concentrated to give a pale gum which solidified upon trituration with ether and was recrystallized from EtOAc-petroleum ether to afford the acetamide 14 (6.65 g, 74%) as clear slabs: mp 87 °C; UV (EtOH) nm ( in M-1cm-1) 256 (17 700), 310 (3100); IR (Nujol) 3270, 1740, 1630 cm-1; 1H NMR (400 MHz) δ 1.91 (s, 3H, NCOCH3), 2.29 (s, 3H, OCOCH3), 2.93 (s, 3H, NCH3), 3.42-3.47 (m, 4H, CH2CH2), 5.74 (br s, 1H, NH), 6.41-6.43 (m, 2H, Ar-H), 6.60 (1H, dd, J ) 8.5 and 2, Ar-H), 7.23 (t, 1H, J ) 8.5, 5-H). Anal. (C13H18N2O3) C, H, N. 2-{4′-[N-(2-Acetamidoethyl)-N-methylamino]-2′hydroxybenzoyl}benzoic Acid (15). The acetamide 14 (1.00 g, 4 mmol) was dissolved in methanol (10 mL) containing KOH (0.5 g) and stirred at room temperature under nitrogen for 25 min. The solvent was removed under reduced pressure, and the residue was mixed with 2 M aqueous potassium phosphate (pH 8.5, 50 mL) and extracted with dichloromethane (4 × 35 mL). The combined organic extracts were dried and evaporated, and the residual gum was azeotroped with dry toluene (3 × 5 mL) to remove traces of water to leave the crude
A Homobifunctional Thiol-Reactive Rhodamine
phenol 9 (0.83 g), which was dissolved in dry dichloromethane (5 mL) and transferred to a glass tube (150 × 16 mm). The solvent was removed under a current of nitrogen and finally by evacuation (∼1 mmHg). Freshly sublimed phthalic anhydride (0.65 g, 4.4 mmol) was added, and the tube was flushed with nitrogen, sealed, and then heated at 135 °C for 4 h. During the first 1 h of heating, the tube was inverted each 15 min to ensure mixing of its contents. After cooling to room temperature, the contents of the tube were dissolved in a mixture of hot ethanol (200 mL) and ethylenediamine (2 mL) to quench excess phthalic anhydride. The solution was concentrated to ∼5 mL, diluted with EtOAc (200 mL), and washed with 2 M hydrochloric acid (2 × 70 mL). The aqueous washings were back-extracted with EtOAc (2 × 50 mL), and the combined organic layers were extracted with 10% aqueous NaHCO3 (5 × 50 mL). The combined aqueous extracts were acidified (pH
