Synthesis of Thiol-Reactive, Long-Wavelength Fluorescent

Bioconjugate Chem. 2006, 17, 387−392

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Synthesis of Thiol-Reactive, Long-Wavelength Fluorescent Phenoxazine Derivatives for Biosensor Applications Douglas B. Sherman, J. Bruce Pitner, Arounaguiry Ambroise, and K. Joseph Thomas* BD Technologies, 21 Davis Drive, Research Triangle Park, North Carolina 27709. Received October 24, 2005; Revised Manuscript Received January 10, 2006

Two environmentally sensitive, long-wavelength fluorescent phenoxazine derivatives, INR and IANR, were synthesized with linkers for conjugation to the thiol group of cysteine in binding proteins. The linkers were designed based on the attachment sites at two different positions on the phenoxazine, which were chosen in order to study the orientation of the dye with respect to the binding protein. Conjugation of the dyes to the S337C maltose binding protein (MBP) mutant provided conjugates of these dyes that are capable of detecting maltose with different sensitivities. The dye INR gave a 3-fold (+200%) change in fluorescence intensity upon maltose binding when conjugated to S337C MBP with a binding constant (Kd) of 435 µM. The fluorescence change for IANR was only 20% and the Kd was 1.4 mM. Conformational analysis of the dyes by molecular modeling suggested that the linker in IANR imparted greater conformational freedom to the dye, resulting in little change in environment between the open and the closed-form conformations. The linker in INR, on the other hand, showed restricted motion, which placed the dye in different environments in the open and closed forms of the protein. Thus, design and placement of the linker play a critical role in the performance of these dyes as environmentally sensitive probes.

INTRODUCTION Long-wavelength fluorescent dyes that are thiol-reactive and environmentally sensitive can play an important role in biosensor chemistry (1, 2). Particularly, when dealing with biological interferences during the in vivo measurement of metabolites such as glucose or lactate, dyes that fluoresce above 600 nm are preferred since tissue and serum fluorescence are minimal, and background scattering is limited to its lowest value in the longwavelength window (3, 4). This allows the measurement of fluorescence signal from a sensor dye with greater accuracy, and therefore the long-wavelength region is superior to visible spectral regions for fluorescence-based diagnostic applications. Although there are several dyes known to emit in the longwavelength region (5-7), to our knowledge none are commercially available that are both environmentally sensitive and have a linker for specific conjugation to an engineered cysteine on a binding protein. Binding proteins offer an alternative approach for sensing a variety of analytes such as maltose, glucose, lactate, and other metabolites (8-11). In contrast to enzymatic sensors, fluorophore-labeled binding proteins require no cofactors and produce no enzymatic byproducts, a source of potential biosensor degradation during longer implant times. Binding proteins can be specific to molecules of biological interest and can reversibly bind these ligands, properties that are desirable for constructing continuous biosensors. The most important property of these receptors is they all share a substantial conformational change upon ligand binding, typically a hinge-type motion centered around the ligand binding site that brings two protein domains toward each other. To take advantage of these receptor properties, the binding protein must be coupled to a reporter moiety that can reliably provide a quantitative signal corresponding to the concentration of the desired ligand. A reliable approach for protein biosensor design is to place an environmentally sensitive fluorescent dye near the binding * Corresponding author. Telephone: (919) 597-6617. Fax: (919) 597-6402. E-mail: [email protected].

pocket (12). The fluorescence properties of such dyes can be correlated to changes in microenvironment associated with the binding of analytes to protein. Keeping this in mind, we have developed two long-wavelength fluorescent dyes suitable for bioconjugation that possess significant sensitivity to their microenvironment and emit in the 600-700 nm region. The two dyes are derivatives of Nile Red, a phenoxazine that is known to possess a very high environmental sensitivity. Various research groups have used Nile Red for probing the polarity of organic and micro-heterogeneous media. The absorption and emission maxima are in the range of 550 and 600 nm, respectively, with emission maxima shifting to as much as 650 nm when the dye interacts with the hydrophobic surface of tubulin, for example (13-15). Although Nile Red derivatives have been reported that are amine-reactive (16-18), these would likely have little or no site-specificity when conjugated to proteins such as MBP that contain numerous lysines. A thiol-reactive moiety would provide site-specificity for protein attachment through a native or an engineered cysteine (11). The mutant S337C provides a cysteine location in MBP that has been previously shown to provide a significant signal with the environmentally sensitive visible fluorophore IANBD (N-[2-(iodoacetoxy)ethyl]-N-methylamino7-nitrobenz-2-oxa-1,3-diazole) (8, 9). One of our further objectives was to identify the optimal location on the phenoxazine dye for introducing a thiol-reactive linker. In the first design, the linker was placed on the dialkylamino group, and in the second, the linker was placed on the aromatic chromophore (Schemes 1 and 2). Herein we report the performance of these dyes for maltose detection on S337C MBP and examine the differences with respect to linker design and placement of the dyes.

EXPERIMENTAL PROCEDURES Materials. The starting materials for the syntheses were either purchased from Sigma-Aldrich or from another commercial vendor as indicated. S337C MBP was produced by methods

10.1021/bc050309d CCC: $33.50 © 2006 American Chemical Society Published on Web 02/21/2006

388 Bioconjugate Chem., Vol. 17, No. 2, 2006 Scheme 1. Synthesis of INR (1)

Scheme 2. Synthesis of IANR (2)

described by Gilardi et al. (8). Absorption spectra were recorded on a Cary 50 Bio UV-Vis spectrophotometer, and fluorescence measurements were taken on a PTI QM-4/2003 SE spectrofluorimeter. The NMR spectra were obtained on either a Varian Mercury 400 MHz or on a Bruker (400 MHz) AMX 400 spectrometer. Mass spectra were recorded on a Finnigan-MAT 212 (FAB) or on a Sciex 150 Electrospray spectrometer. Synthesis of INR (1). Intermediate 1b: N-Phenyl-N-methylethanolamine 1a (50 mmol) was suspended in concentrated HCl (28 mL) and was cooled to 5 °C. To this solution was added dropwise a sodium nitrite solution (6.67 g in 10 mL water) over a period of 40 min. After the addition, the reaction was kept stirring for 2 h more. The product was then filtered, washed with 0.5 M HCl, and dried in vacuo to give the nitroso compound 1b (yield 40%). 1H NMR (D2O) δ ppm: 3.59 (s, CH3, 3H); 3.90 (t, CH2, 2H); 4.05 (t, CH2, 2H); 7.22-7.30 (m, aromatic, 2H); 7.50 (d, aromatic, 1H); 7.77 (d, aromatic, 1H). 13C NMR (D O) δ ppm: 42.44, 57.92, 58.72, 120.29, 122.52, 2 125.93, 140.56, 149.93, 163.21. Mol wt calculated for C9H12N2O2 is 180 (M+), found 181 (M + 1) (FAB). Intermediate 1d: 1,3-Dihydroxynaphthalene 1c (5 mmol) was suspended in ethanol (25 mL) and was brought to reflux while stirring. To the refluxing solution was added intermediate 1b (5 mmol) in fractions over a period of 45 min. After the addition, the reaction mixture was maintained at reflux for 4 h more and then cooled. The solvent was evaporated, and the product dye 1d was purified by flash column chromatography over silica gel using methanol and chloroform (1:9) as eluent (yield 50%). 1H NMR (CDCl ) δ ppm: 2.98 (s, 3H); 3.48 (t, 2H); 3.83 (t, 3 2H); 6.20 (s, 1H); 6.77 (d, 1H); 7.10 (d, 1H); 7.45 (s, 1H); 7.60-7.75 (m, 2H); 7.72 (m, 1H); 8.08 (m, 1H). 13C NMR (CDCl3) δ ppm: 39.2, 55.6, 60.3, 102.2, 110.2, 113.6, 124.7, 126.3, 126.6, 126.8, 130.6, 132.3, 133.7, 135.1, 145.8, 148.2, 152.4, 182.5, 183.8. Mol wt calculated for C19H16N2O3 is 320 (M+), found 321 (M + 1) (FAB). Compound 1 (INR): The intermediate dye 1d was dissolved in anhydrous acetonitrile (10 mL), and p-(dimethylamino)pyridine (3 mg) was added, followed by iodoacetic anhydride

Sherman et al.

(250 mg). The reaction was stirred for 2 h. The product 1 was separated by evaporation of the solvent and then purified by repeated precipitation from methylene chloride and hexane (yield 25%). 1H NMR (CDCl3) δ ppm: 3.14 (s, 3H); 3.66 (s, 2H); 3.74 (t, 2H); 4.38 (t, 2H); 6.40 (s, 1H, ArH); 6.54 (d, 1H, ArH); 6.72 (d, 1H, ArH); 7.64-7.75 (m, 3H, ArH); 8.29 (d, 1H, ArH); 8.65 (d, 1H, ArH). 13C NMR (CDCl3) δ ppm: -6.1, 39.6, 50.9, 63.0, 97.5, 106.4, 110.1, 124.1, 125.6, 126.0, 130.6, 131.2, 131.7, 131.98, 132.0, 141.5, 146.5, 151.8, 152.2, 169.0, 184.1. Exact Mass Calculated for C21H17IN2O4 is 489.0317, found 489.0317 (HRMS). Synthesis of IANR (2). Intermediate 2b: 9-Diethylamino2-hydroxy-5H-benz[a]phenoxazin-5-one 2a (50 mg, 0.15 mmol), N-bromoethylphthalimide (50 mg, 0.20 mmol), and potassium carbonate (60 mg, 0.09 mmol) were combined in DMF (15 mL) under argon with stirring. The reaction proceeded at reflux for 4.5 h. Additional bromoethylphthalimide (25 mg) was added at 4.5 h and at 6.5 h (15 mg). The temperature was lowered to 115 °C, and the reaction proceeded overnight. DMF was removed in vacuo, and the residue was dried in vacuo. Column chromatography over silica gel (2% MeOH/CH2Cl2 as mobile phase) afforded the product 2b in 85% yield. 1H NMR (CDCl3) δ ppm: 1.22 (t, 6H); 3.40 (q, 4H); 4.19 (t, 2H); 4.40 (t, 2H); 6.23 (s, 1H); 6.36 (d, 1H); 6.59 (dd, 1H); 7.09 (d, 1H); 7.52 (d, 1H); 7.70 (m, 2H); 7.84 (m, 2H); 7.96 (d, 1H); 8.13 (d, 1H). 13C NMR (CDCl ) δ ppm: 12.8, 37.5, 45.2, 65.3, 96.3, 105.2, 3 106.5, 109.8, 118.6, 123.5, 124.9, 126.0, 127.9, 131.3, 132.1, 134.2, 139.6, 146.8, 150.7, 152.2, 161.0, 162.8, 168.4, 183.3. Mol wt calculated for C30H25N3O5 is 507, found 508 (MH+) (FAB-MS). Intermediate 2c: Intermediate 2b (30 mg, 0.06 mmol) was dissolved in anhydrous MeOH (8 mL) and was kept under an atmosphere of argon. Next, methylamine (2 M in MeOH, 4 mL, 8 mmol) was added. The reaction was allowed to proceed for 5 min at RT and 2.5 h at reflux. Flash chromatography was performed over silica gel using 10% MeOH/CH2Cl2 as the mobile phase to remove fast moving impurities and then with 30% MeOH/CH2Cl2 to elute the product 2c. Solvent was removed on a rotary evaporator, and the residue was dried in vacuo (70% yield). 1H NMR (CDCl3) δ ppm: 1.25 (t, 6H); 3.17 (t, 2H); 3.45 (q, 4H); 4.20 (t, 2H); 6.27 (s, 1H); 6.43 (d, 1H); 6.63 (dd, 1H); 7.15 (m, 1H); 7.56 (d, 1H); 8.02 (m, 1H); 8.18 (d, 1H). 13C NMR (CDCl3) δ ppm: 12.8, 41.6, 45.3, 63.9, 96.4, 105.4, 106.6, 109.9, 118.4, 124.9, 125.9, 127.4, 128.0, 131.3, 134.3, 147.1, 151.0, 152.3, 161.7, 183.5. Mol wt calculated for C22H23N3O3 is 377, found 378 (MH+) (FAB-MS). Compound 2 (IANR): Intermediate 2c (11 mg, 0.03 mmol) was dissolved in CH2Cl2 (5 mL). Iodoacetic anhydride (21 mg, 0.06 mmol) was then added and allowed to react for 40 min. An additional 25 mL of CH2Cl2 was added, and the reaction mixture was transferred to a separation funnel. The organic phase was washed with 10% Na2CO3 (10 mL each) twice, dried over anhydrous MgSO4, and filtered. After the solvent was removed on a rotary evaporator, the final product 2 was dried under vacuum and precipitated from CH2Cl2/hexane (yield 87%). 1H NMR (CDCl3) δ ppm: 1.17 (t, 6H); 3.35 (q, 4H); 3.69 (t, 2H); 3.78 (s, 2H); 4.14 (t, 2H); 6.12 (s, 1H); 6.27 (s, 1H); 6.48 (dd, 1H); 6.91 (d, 1H); 7.34 (d, 1H); 7.74 (s, 1H); 7.95 (s, 1H). 13C NMR (CDCl3) δ ppm: 12.9, 29.9, 40.1, 45.3, 66.9, 96.3, 105.2, 106.7, 109.8, 118.0, 124.9, 125.9, 127.8, 131.3, 134.1, 139.4, 147.0, 151.0, 152.2, 161.1, 168.1, 183.3. Exact mass calculated for C24H24IN3O4 is 546.0889, found 546.0889 (HRMS). Quantum Yield Measurements. Relative fluorescence quantum yields were determined using optically dilute solutions with rhodamine 101 in ethanol (quantum yield 1.0) (19). Typically, the absorption was matched to 0.05 at the excitation wavelength

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Synthesis of Thiol-Reactive Phenoxazines Table 1. Absorption and Fluorescence Properties of INR and IANR in Different Solvents INR (1)a solvent toluene ethyl acetate CHCl3 CH2Cl2 acetone acetonitrile DMF 2-propanol ethanol methanol

abs.

maxc

(nm)

fl.

500 506 512 515 516 517 528 529 532 534

maxd

(nm)

IANR (2)b quantum

556 585 577 585 600 605 615 617 623 627

yielde

abs.

1.00 0.84 0.78 0.78 0.66 0.53 0.42 0.39 0.33 0.25

maxc 526 523 540 539 533 536 541 543 546 553

(nm)

fl. maxd (nm)

quantum yielde

568 590 592 603 609 615 618 622 628 632

0.97 0.96 0.82 0.83 0.63 0.59 0.43 0.46 0.33 0.25

a Extinction coefficient 14 000 M-1 cm-1. b Extinction coefficient 29 000 M-1 cm-1. c Absorption maximum. d Fluorescence maximum. e Quantum yield was measured using rhodamine 101 in ethanol as the reference.

and adjustments were made for minor changes in absorption at the excitation wavelength in fluorescence quantum yield measurements. Spectroscopic grade solvents were used for fluorescence measurements. Bioconjugation of INR and IANR to MBP. Dyes were conjugated to the binding proteins in the following manner. To S337C MBP (4 nmol) in PBS (200 µL) was added dithiothreitol (8 nmol), and the reaction proceeded for 30 min at room temperature. INR or IANR were prepared as stock solutions (approximately 1 mg in 100 µL DMSO). An aliquot containing 40 nmol of the dye was added to the protein solution and was stirred for 4 h at room temperature in the dark. The reaction mixture was then passed through a NAP-5 size exclusion column (Amersham-Pharmacia, Uppsula, Sweden), with the product eluting in the second (0.5 mL) fraction. The percent change in fluorescence intensity was measured on a PTI fluorimeter using 1 µM protein with either 0 or 100 mM maltose. Determination of Binding Constants. A 0.1 µM solution of S337C MBP/dye conjugate was titrated on the fluorimeter against maltose concentrations from 0 to 100 mM. The binding constant (Kd) was calculated using relationships shown below that were derived from similar calculations by Pisarchik and Thompson (20).

F ) Finf +

F0 - Finf 1 + x/Kd

where F is fluorescence, Finf and F0 are fluorescence at infinity and at the initial value, and Kd is the binding constant. The free concentration of maltose ([Mal]free) is denoted by x and is determined by the relationship: [Mal]free ) [Mal]tot - [Prot]tot - Kd + x([Mal]tot - [Prot]tot - Kd)2 + 4 × [Mal]tot × Kd 2

where [Mal]tot and [Pro]tot are total maltose and protein concentrations, respectively. If [Mal]tot . Kd and [Mal]tot . [Pro]tot, then the equations simplify to the following form:

F ) F0 + [(Fconst × x)/(1 + x/Kd)] where Fconst ) (Finf - F0)/Kd. Molecular Modeling. Models of INR and IANR were constructed in Sybyl (Tripos, St. Louis, MO). X-ray crystal structures of MBP in the open (1OMP.pdb) and closed (1ANF.pdb) forms were obtained from the Protein Data Bank (21). After residue S337 was modified to Cys, the dyes were attached to form the models of the bioconjugates. Genetic algorithm (GA) conformational searches were then performed using the method provided in Sybyl. For INR, the eight bonds between the Cys alpha-carbon and the dialkylamino nitrogen were defined as

rotatable. In the IANR linker, the amide bond was fixed in the trans conformation, and the bond between the aryl oxygen and the aromatic ring was defined as rotatable, which also produced a total of eight rotatable bonds. Default settings in the GA search were employed, except the duplicate window was set to 10. The results of the search were up to 20 conformers within a 20 kcal/mol energy window.

RESULTS Synthesis. Nile Red derivative INR (1) was synthesized according to Scheme 1. Briefly, intermediate 1b was synthesized by reacting N-phenyl-N-methylethanolamine 1a with sodium nitrite at 5 °C in the presence of concentrated HCl, and the resulting product was dried to give 1b. The parent dye 1d was prepared by the cyclization reaction of 1b with 1,3-dihydroxynaphthalene (1c) at reflux in ethanol followed by purification. Reaction of 1d with iodoacetic anhydride in the presence of (dimethylamino)pyridine afforded the thiol-reactive product INR (1). The synthesis of derivative IANR (2) was carried out in three steps according to Scheme 2. Starting material 9-diethylamino2-hydroxy-5H-benz[a]phenoxazin-5-one 2a was reacted with N-bromoethylphthalimide in DMF in the presence of potassium carbonate to produce the intermediate 2b. Intermediate 2c was then prepared by the deprotection of the amine in 2b with methylamine at refluxing temperatures in methanol. The final thiol-reactive compound IANR (2) was synthesized by reaction of the amine derivative 2c with iodoacetic anhydride. Absorption and Fluorescence Properties. The sensitivity of INR and IANR toward surrounding microenvironment was tested by studying their spectral properties in solvents of varying dielectric constants. Table 1 summarizes the absorption, emission, and quantum yield of these dyes in a range of polar and nonpolar solvents. The absorption and emission spectra of INR and IANR showed a bathochromic shift with increase in polarity of the medium. For example, the absorption maximum of INR shifted from 500 nm in toluene to 534 nm in methanol, and the fluorescence emission maximum shifted from 556 to 627 nm. IANR gave the same trends, although the absorption maximum shifted 27 nm from toluene to methanol versus 34 nm for INR. The shift in fluorescence maximum was 71 nm for INR and 64 nm for IANR. When conjugated to S337C MBP (not shown in Table 1), the absorption maxima of the dyes in PBS shifted to 580 nm for INR and 550 nm for IANR. Likewise, the fluorescence emission maxima shifted significantly to 640 nm for INR and 653 nm for IANR, placing the emission maxima in the desired long-wavelength range. In a nonpolar solvent such as toluene, the quantum yield of fluorescence was found to be 1.0 for INR and 0.97 for IANR. High fluorescence quantum yields were also observed in solvents such as chloroform and methylene chloride. With increasing solvent polarity the quantum yield decreases significantly. For

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S337C-INR MBP, on the other hand, returned 20 conformers in the open form that were within 2 kcal/mol of each other and that formed a single family, as shown in Figure 3c. The search on S337C-INR MBP also returned a single family of 20 conformers (Figure 3d). Here the dye was parallel to the surface of the upper domain in the protein.

DISCUSSION

Figure 1. Fluorescence intensity change of S337C-INR MBP between 0 (dotted line) and 100 mM (solid line) maltose.

Figure 2. Plot for the determination of binding constant (Kd) for S337C-INR MBP conjugate.

example, in a moderately polar solvent such as acetonitrile, the quantum yield observed for INR was 0.53, and for IANR it was 0.59. In protic polar solvents the quantum yield was further reduced; in methanol the quantum yield for both INR and IANR was observed as 0.25. This is a 4-fold decrease in quantum yield as compared to toluene. Bioconjugate Performance. Figure 1 shows the fluorescence spectrum of the conjugate S337C-INR MBP in PBS. Upon maltose binding by the protein, the fluorescence intensity underwent a 3-fold enhancement (+200%), whereas S337C-IANR MBP gave only a 20% change in fluorescence intensity. The binding curve for S337C-INR MBP is shown in Figure 2, and the binding constant (Kd) was calculated as described in Experimental Section. The Kd calculated for the S337C-INR MBP conjugate was 435 µM, and the Kd for S337C-IANR MBP was 1.4 mM. These values are higher than the Kd of 62 µM reported for S337C MBP conjugated with IANBD ester (9) and are 100 to 400 times higher than the Kd (3.5 µM) of wild-type MBP (22). Molecular Modeling Analysis. Models of the dyes attached to S337C MBP in the open and closed form protein conformations were prepared, and GA conformational searches were performed as described in Experimental Procedures. Representative conformers are illustrated in Figure 3 for the open (left) and closed (right) forms of the protein. S337C-IANR MBP returned 20 conformers that formed five families in the open form as illustrated by representative structures in Figure 3a. All 20 conformers were within 5 kcal/mol of each other and were distributed in the cleft between the domains or near the surface of one of the domains. In the closed form, the conformational search of S337C-IANR MBP returned four families (Figure 3b).

INR and IANR were synthesized in three steps as outlined in Schemes 1 and 2. Both dyes displayed absorption, emission, and quantum yield properties that demonstrated the environmental sensitivity required in order for these dyes to function as a reporter for binding protein-based reagentless biosensors. As the polarity of the solvent increased, INR and IANR displayed bathochromic shifts in absorption (up to 34 nm) and emission (up to 71 nm) wavelengths. Although both structures were based on the same chromophore, the absorption and emission maxima of INR were blue shifted compared to IANR in any given solvent. In the case of INR, placement of the linker at the dialkylamino group may be affecting the degree of charge transfer, which would in turn lead to a blue shift for INR with respect to IANR. The presence of the linker at the dialkylamino group may also be the reason that the molar extinction coefficient of INR (14 000 M-1 cm-1) is lower than that of IANR (29 000 M-1cm-1). Previous reports in the literature described similar trends for the parent dye Nile Red, which were explained as arising from a twisted intramolecular charge transfer (TICT) (23-25). TICT can occur upon excitation when an electron donor group in a molecule transfers an electron to an acceptor group in the molecule with a concomitant 90° rotation of the bond between the donor and acceptor (26-28). In Nile Red, the diethylamino group would serve as the donor, the ketone moiety would serve as the acceptor, and the single bond between the diethylamino group and the phenoxazine ring would rotate. Later reports of experimental and theoretical calculations (29, 30) on Nile Red suggested that the solvatochromic properties could be adequately explained based on the change in the dipole moment of the dye between the ground and excited state (31), which has been calculated to be in the range of 5.5 to 7.4D (23, 24, 29). Thus, the excited dye molecule could be better stabilized in a polar medium such as methanol or DMF as compared to a nonpolar medium such as toluene. This stabilization leads to lower free energy of the excited state, whereas the ground state remains relatively unaffected from nonpolar to polar medium. The decrease in the free energy of the excited state leads to the reduction in the separation between the S0-S1 state, and thereby a shift toward higher wavelength (bathochromic shift) is observed. The quantum yield of both dyes showed a 4-fold decrease from toluene to methanol. The decrease in quantum yield of fluorescence between hydrophobic and polar solvent environments may also be explained based on changes in the dipole moment of the dyes. In nonpolar solvents, electrostatic interactions are limited with the solvent molecules, which would significantly arrest the nonradiative decay pathways. In toluene, all the nonradiative decay pathways for INR are eliminated, leading to an absolute quantum yield of fluorescence. However, in polar solvents such as methanol, strong electrostatic interactions exist between the dye and solvent molecules, leading to radiationless vibronic relaxations and thereby a significant decrease in fluorescence intensity. INR and IANR displayed considerably different fluorescence responses when conjugated to S337C MBP for detection of maltose. The INR conjugate gave a +200% fluorescence change with a Kd of 435 µM, whereas IANR gave only a +20% fluorescence change with a Kd of 1.4 mM. These results

Synthesis of Thiol-Reactive Phenoxazines

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Figure 3. Results of genetic algorithm conformational searches for IANR (a and b) and INR (c and d) at S337C MBP in the open (left) and closed (right) conformations. Dye structures are in blue and maltose is in red.

suggested that the position and nature of the linker on the dye may have a profound effect on the sensor performance when these dyes are used to construct fluorescent sensors for maltose. Such behavior was previously reported for studies of phosphate binding protein (PBP), in which the coumarin dye N-[2(1-maleimidyl)ethyl]-(diethylamino)coumarin-3-carboxamide (MDCC) gave a +405% fluorescence change when conjugated to mutant A197C of PBP (32). Replacement of the ethyl group between the coumarin carboxamide and the maleimide group with n-propyl (MPrCC) or a p-phenyl group (MPhCC) changed the binding activity to -4% and -22%, respectively. Retention of the ethyl group but replacement of the maleimide group with an iodoacetamide group resulted in no change in fluorescence (33). In the current study, the linker of INR contains a flexible ester compared to the more rigid amide in the linker of IANR. On the other hand, the INR linker is connected to the ring system of the dye through a dialkylamino group that donates electrons to the aromatic ring system as mentioned above, whereas the IANR linker is attached through an aryl ether bond that may allow greater freedom of rotation near the chromophore. To evaluate the effect of the linker on dye orientation with respect to the S337 C MBP protein, molecular modeling studies were performed. The conformational analysis studies by mo-

lecular modeling suggested that IANR appeared to be much more flexible in the bound and unbound states than INR. The added flexibility in the linker may prevent IANR from experiencing a large change in environment between the bound and unbound states. INR, on the other hand, appeared locked into two conformations of distinctly different environments in the bound and unbound states, which would agree with the larger change in fluorescence that was observed for this dye, especially if the dye interacts more with polar residues on the surface of the protein in one conformation over the other (34). Furthermore, IANR was located more deeply and in parallel with the cleft between the two domains of the open form model than INR. It is possible that placement of the dye in this position may help to stabilize the open form of the protein relative to the closed form (35), which in turn would account for the weaker binding affinity for the IANR conjugate relative to the INR conjugate. In conclusion, we have synthesized two long-wavelength fluorescent thiol-reactive derivatives of Nile red for biosensor applications. The thiol reactive pendants were placed at two different locations on the dye in order to understand the effect of linker position on the fluorescence response of the dyes, especially when conjugated to binding proteins. The high environmental sensitivity of these dyes was demonstrated by

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measuring their spectral properties in solvents of varying dielectric constants. Bioconjugation of these dyes to maltose binding protein S337C MBP showed fluorescence changes of up to 200% upon binding of maltose for INR but only 20% for IANR. To a first approximation, the binding event places INR in an environment with relatively fewer interactions with the solvent water molecules, while IANR experiences less of an environmental change. Furthermore, we have observed that the attachment position of the linker to the dye and the flexibility of the linker play a key role in sensor performance, which was supported qualitatively by modeling studies. Placing the dye such that the charge transfer center would be closer to the protein surface may also explain the higher fluorescence performance for the biosensor. We believe that these dyes can be employed with binding proteins for other ligands, which can lead to the development of binding protein-based metabolite sensors that can be used with relatively low interference from the biological media when measured in vivo.

ACKNOWLEDGMENT This work is supported in part by a grant from the Technologies for Metabolic Monitoring (TMM) program, contract number W81XWH-04-1-0076. The authors thank Dr. A. E. G. (Tony) Cass (Imperial College) for providing clones of S337C MBP as well as Colleen Nycz and David Wright of BD Technologies for assistance with protein production.

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