Monofunctional Near-Infrared Fluorochromes for Imaging Applications

Publication Date (Web): August 26, 2005 ... Design and Synthesis of Polymer-Functionalized NIR Fluorescent Dyes–Magnetic Nanoparticles for Bioimagin...
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Bioconjugate Chem. 2005, 16, 1275−1281

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Monofunctional Near-Infrared Fluorochromes for Imaging Applications Scott A. Hilderbrand, Kimberley A. Kelly, Ralph Weissleder, and Ching-Hsuan Tung* Center for Molecular Imaging Research, Massachusetts General Hospital, Harvard Medical School, Room 5406, 149 13th Street, Charlestown, Massachusetts 02129. Received June 21, 2005; Revised Manuscript Received August 9, 2005

In this report, the development of a new class of monocarboxylate functionalized cyanine derivatives using improved synthetic procedures is detailed. The employed synthetic strategy relies on efficient nucleophilic attack of alkyl-thiols on cyanine dyes bearing chloro-substituted polymethinic linkers. Monocarboxylate derivatized fluorochromes (CyTE dyes) can be prepared in one step in greater than 90% yield without the need for additional purification. Several of the fluorochromes synthesized by this route show no tendency to aggregate in aqueous solution and have excitation and emission maxima greater than 800 nm. The potential utility of the CyTE fluorochromes was demonstrated through direct labeling of phage displaying a vascular cellular adhesion molecule-1 (VCAM-1) targeting peptide. Endothelial cell internalization of the VCAM-1 targeted phage was monitored via near-infrared fluorescence microscopy.

INTRODUCTION

In recent years, there has been significant interest in the development of monofunctional, water-soluble nearinfrared fluorochromes for use in a myriad of imaging applications. The development of new bright, watersoluble near-infrared (NIR) fluorochromes that are synthesized readily and are suitable for bioconjugation is necessary for the preparation of improved NIR probes for in vivo imaging. Fluorochromes with absorption and emission maxima between 650 and 900 nm are ideally suited for imaging in tissue due to the minimal absorption coefficients from hemoglobin, water, and lipids over this range (1). A variety of carboxylic acid derivatized cyanine dyes emitting in the NIR have been prepared (2-11). Many commonly used monofunctional dyes are asymmetric with a single carboxylic acid group attached to one of the indoleninium or benzindoleninium moieties (3, 4, 6, 9). The condensation reactions to prepare these fluorochromes proceed in a stepwise fashion resulting in formation of the undesired symmetric dye as a byproduct with overall yields typically less than 10%. Although these dyes are components of a variety of receptor targeted (9, 12-15) and fluorogenic (16-18) probes for optical imaging in vivo, the difficulty in preparation of large quantities of the purified fluorochromes may limit their widespread utility. New high-yield routes for the synthesis of NIR fluorochromes suitable for imaging applications are needed. One strategy that provides access in 90% or greater yields to fluorochromes that can be conjugated is via nucleophilic attack on chloro-substituted cyanine dyes (19). A variety of water-soluble dyes containing aryl-ether, arylthioether, and alkyl-ether linkages have been prepared using this route (2, 7, 10, 11, 20). However, none of these dyes are optimal for imaging applications. Although they are water-soluble, cyanine fluorochromes containing an additional aryl group, such as an aryl-ether-modified * Corresponding author. Tel: (617) 726-5779. Fax: (617) 7265708.

cyanine dye, show an increased tendency to aggregate in aqueous solution (2), and the aryl-ether linkage in some of these fluorochromes is susceptible to cleavage (21). Several aryl-ether-, aryl-thioether-, and alkyl-etherfunctionalized fluorochromes have been reported but there are only a few reports of their alkyl-thioether congeners (22, 23). In this report, we detail general procedures for the synthesis of a variety of alkyl-thioether-containing cyanine fluorochrome derivatives (CyTE dyes). Some of these fluorochromes may be prepared in one step with excellent yields starting from commercially available dyes, show no tendency to aggregate in aqueous solution, and display absorption and emission maxima above 800 nm. To assess the utility of the CyTE fluorochromes, phage displaying the vascular cellular adhesion molecule-1 (VCAM-1) targeting peptide (VHSPNKK) (24) on the pIII coat protein of the phage were labeled with fluorochrome and imaged in live cells. Cell uptake experiments with the fluorochrome-labeled phage demonstrate the potential of the synthetically accessible CyTE dyes for use in NIR fluorescence imaging. MATERIALS AND METHODS

General Considerations. All chemicals were purchased from Aldrich (Milwaukee, WI) or TCI America (Portland, OR) except PEG7 thiol propionic acid, which was purchased from Polypure AS (Oslo, Norway), and were used as received. The dyes IR-783, IR-806, and IR820 were purchased from Aldrich. All solvents including anhydrous dimethylformamide (DMF) and anhydrous dimethyl sulfoxide (DMSO) were obtained from Aldrich. High-performance liquid chromatography (HPLC) was performed on a Hitachi D-7000 instrument equipped with a L-7455 diode array detector. Nuclear magnetic resonance (NMR) data were collected on a Bruker DPX-400 spectrometer at ambient temperature and referenced to tetramethylsilane (TMS) or 2,2-dimethyl-2-silapentane5-sulfonate sodium salt (DSS) internal standards. Absorption spectra were obtained on a Varian Cary 50-Bio UV/visible spectrophotometer (Palo Alto, CA). All extinc-

10.1021/bc0501799 CCC: $30.25 © 2005 American Chemical Society Published on Web 08/26/2005

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tion coefficient measurements in phosphate-buffered saline (PBS), pH 7.4, 10 mM phosphate, 2.7 mM potassium chloride, and 137 mM NaCl were performed in triplicate. For determination of the extinction coefficients, fresh stock solutions of HPLC-purified dye were prepared for each trial by dissolution of 2-3 mg portions of the dye, weighed on a Mettler AT201 analytical balance with an error of (0.01 mg, in PBS using a 10 mL volumetric flask. The typical standard deviation for the extinction coefficient measurements is less than 5%. Fluorescence data were collected on a Horiba Jobin Yvon Fluorolog-3 spectrofluorometer (Edison, NJ) and were corrected for the detector sensitivity. Quantum yield measurements, performed in triplicate following published procedures (25), were collected using samples with absorbance values under 0.10 OD in PBS using indocyanine green (ICG) in DMSO (ΦF ) 0.106) (26) as a standard. All samples used for the quantum yield determinations were excited at 775 nm. High-resolution electrospray ionization (ESI) mass spectra were collected on a Bruker Daltonics APEXII 3 T Fourier transform mass spectrometer in the Department of Chemistry Instrumentation Facility (DCIF) at the Massachusetts Institute of Technology. CyTE-777 (1). To a solution of IR-783 (225 mg, 0.30 mmol) in 4 mL of anhydrous DMF was added 3-mercaptopropionic acid (30.5 µL, 0.35 mmol) and triethylamine (TEA) (49 µL, 0.35 mmol). The green solution was allowed to stir in the dark. After 15 h, the reaction was complete as monitored by HPLC. Crude 1, which has sufficient purity for routine work, was isolated in 93% yield as a green solid by precipitation with tert-butyl methyl ether (TBME) and was washed with TBME (3 × 10 mL). Compound 1 was further purified by reverse-phase C-18 chromatography (Waters Sep-Pak C18 cartridge) with a mixture of 25% CH3CN, 75% H2O, and 0.1% TFA followed by 30% CH3CN, 70% H2O, and 0.1% TFA to yield pure 1 (204 mg, 83%). 1H NMR (400 MHz, CD3OD): δ 8.89 (d, 2H, J ) 14.2 Hz), 7.49 (d, 2H, J ) 7.4 Hz), 7.41 (t, 2H, J ) 7.6 Hz), 7.34 (d, 2H, J ) 7.8 Hz), 7.25 (t, 2H, J ) 7.5 Hz), 6.32 (d, 2H, J ) 14.3 Hz), 4.19 (t, 4H, J ) 6.7 Hz), 3.06 (t, 2H, J ) 7.0 Hz), 2.90-2.87 (m, 4H), 2.70 (t, 4H, J ) 5.9 Hz), 2.56 (t, 2H, J ) 6.9 Hz) 2.00-1.92 (m, 10H), 1.76 (s, 12H). λmax (PBS, pH 7.4): 777 nm ( ) 130 000 M-1 cm-1). ΦF (PBS, pH 7.4): 0.02. HRMS-ESI [M]+ m/z calcd for C41H53N2O8S3 797.2959, found 797.2976. Intermediate (2). 1-(4-Sulfonatobutyl)-2,3,3-trimethylindoleninium-5-sulfonate (5) (1.25 g, 3.0 mmol), N-[(3(anilinomethylene)-2-chloro-1-cyclohexen-1-yl)methylene]aniline monohydrochloride (540 mg, 1.5 mmol), and potassium acetate (295 mg, 3.0 mmol) were dissolved in 20 mL of a 2:1:1 solution of MeOH, acetic anhydride, and pyridine. The solution was sealed in a thick-walled glass pressure reactor and heated at 140 °C for 25 min giving a dark blue solution containing a dark blue precipitate. After the mixture cooled to room temperature, the blue solid was isolated by filtration. Purification of the crude material by reverse-phase C-18 chromatography (Waters Sep-Pak C18 cartridge) with a mixture of 15% CH3CN, 85% H2O, and 0.1% TFA yields pure 2 (450 mg, 30%) as a green solid. 1H NMR (400 MHz, D2O): δ 8.16 (d, 2H, J ) 13.8 Hz), 7.85 (s, 2H), 7.74 (d, 2H, J ) 8.3 Hz), 7.27 (d, 2H, J ) 8.4 Hz), 6.13 (d, 2H, J ) 13.9 Hz), 4.10 (broad singlet, 4H), 2.96 (t, 4H, J ) 6.6 Hz), 2.41 (broad singlet, 4H), 1.89-1.79 (m, 10H), 1.63 (s, 12H). λmax (PBS, pH 7.4): 781 nm ( ) 248 000 M-1 cm-1). ΦF (PBS, pH 7.4): 0.02. HRMS-ESI [M - 2H]- m/z calcd for C38H46ClN2O12S4 885.1628, found 885.1567; [M - 3H]2- m/z calcd for C38H45ClN2O12S4 442.0777, found 442.0760.

Hilderbrand et al.

CyTE-783 (3). To a solution of 2 (100 mg, 0.10 mmol) in 5 mL of anhydrous DMSO was added 3-mercaptopropionic acid (17.5 µL, 0.20 mmol) and TEA (28 µL, 0.20 mmol). The reaction was completed in 15 h as determined by HPLC. The crude product was precipitated by addition of 25 mL of toluene. This blue-green solid was collected by centrifugation and was washed with toluene (2 × 25 mL) followed by diethyl ether (2 × 25 mL) to give crude 3 (92 mg, 86%), which is sufficiently pure for routine work. The product was further purified by reverse-phase C-18 chromatography (Waters Sep-Pak C18 cartridge) with a mixture of 10% CH3CN, 90% H2O, and 0.1% TFA followed by 15% CH3CN, 85% H2O, and 0.1% TFA to yield pure 3 (76 mg, 71%) as a green solid. 1H NMR (400 MHz, DMSO-d6): δ 8.70 (d, 2H, J ) 14.1 Hz), 7.75 (d, 2H, J ) 1.5 Hz), 7.63 (dd, 2H, J ) 8.2 Hz, J ) 1.5 Hz), 7.37 (d, 2H, J ) 8.3 Hz), 6.36 (d, 2H, J ) 14.2 Hz), 4.17 (broad singlet, 4H), 2.98 (t, 2H, J ) 6.9 Hz), 2.67 (t, 4H, J ) 5.7 Hz), 2.49-2.44 (m, 6H), 1.84-1.63 (m, 22H). λmax (PBS, pH 7.4): 783 nm ( ) 174 000 M-1 cm-1). ΦF (PBS, pH 7.4): 0.02. HRMS-ESI [M]+ m/z calcd for C41H53N2O14S5 957.2095, found 957.2125. CyTE-822 (4). To a solution of IR-820 (255 mg, 0.30 mmol) in 4 mL of anhydrous DMF was added 3-mercaptopropionic acid (30.5 5 µL, 0.35 mmol) and TEA (30.5 µL, 0.35 mmol). The green solution was allowed to stir in the dark. After 15 h, the reaction was complete as monitored by HPLC. Crude 4 (246 mg, 89%) was isolated by precipitation from the reaction by addition of 10 mL of TBME and was washed with TBME (3 × 10 mL). Reverse-phase C-18 column chromatography (Waters Sep-Pak C18 cartridge) of the crude material eluting with 35% CH3CN, 65% H2O, and 0.1% TFA afforded 4 (143 mg, 52%) as a green solid. 1H NMR (400 MHz, DMSOd6): δ 8.83 (d, 2H, J ) 14.0 Hz), 8.30 (d, 2H, J ) 8.4 Hz), 8.09-8.02 (m, 4H), 7.80 (d, 2H, J ) 8.9 Hz), 7.65 (t, 2H, J ) 7.4 Hz), 7.51 (t, 2H, J ) 7.4 Hz), 6.40 (d, 2H, J ) 13.7 Hz), 4.33 (br s, 4H), 3.06 (t, 2H, J ) 6.6 Hz), 2.72 (br s, 4H), 2.65-2.55 (m, 6H), 1.97 (s, 12H), 1.89-1.78 (m, 10H). λmax (PBS/20% DMSO, pH 7.4): 822 nm ( ) 128 000 M-1 cm-1). HRMS-ESI [M]+ m/z calcd for C49H57N2O8S3 897.3272, found 897.3259. CyTE-823 PEG (5). To a solution of IR-820 (255 mg, 0.30 mmol) in 4 mL of anhydrous DMF was added PEG7 thiol propionic acid (183 mg, 0.40 mmol) and TEA (56 µL, 0.40 mmol). The green solution was allowed to stir in the dark for 15 h. A green solid was isolated by precipitation with 10 mL of TBME and was washed with 35 mL of TBME. The crude material was purified by reverse-phase C-18 chromatography (Waters Sep-Pak C18 cartridge) with a mixture of 25% CH3CN, 75% H2O, and 0.1% TFA, followed by 30% CH3CN, 70% H2O, and 0.1% TFA, and finally 35% CH3CN, 65% H2O, and 0.1% TFA to yield pure 5 (212 mg, 56%) as a green solid. 1H NMR (400 MHz, DMSO-d6): δ 8.89 (d, 2H, J ) 14.1 Hz), 8.30 (d, 2H, J ) 8.6 Hz), 8.10-8.02 (m, 4H), 7.81 (d, 2H, J ) 9.0 Hz), 7.65 (t, 2H, J ) 7.3 Hz), 7.51 (t, 2H, J ) 7.6 Hz), 6.41 (d, 2H, J ) 14.3 Hz), 4.34 (br s, 4H), 3.64 (t, 2H, J ) 6.4 Hz), 3.58 (t, 2H, J ) 6.4 Hz), 3.53-3.41 (m, 28H), 3.04 (t, 2H, J ) 6.5 Hz), 2.72 (br s, 4H), 2.64 (t, 4H, J ) 7.3 Hz), 2.43 (t, 2H, J ) 6.3 Hz), 1.99 (s, 12H), 1.88-1.78 (m, 10H). λmax (PBS/20% DMSO, pH 7.4): 823 nm ( ) 116 000 M-1 cm-1). HRMS-ESI [M]+ m/z calcd for C65H89N2O16S3 1249.5369, found 1249.5345. CyTE-807 (6). To a solution of IR-806 (221 mg, 0.30 mmol) in 4 mL of anhydrous DMF was added 3-mercaptopropionic acid (30.5 µL, 0.35 mmol) and TEA (49 µL, 0.35 mmol). After being stirred for 15 h, the reaction was complete as monitored by HPLC. Crude 6 (223 mg, 93%)

Monofunctional Near-Infrared Fluorochromes

was isolated by precipitation from the reaction by addition of 10 mL TBME and was washed with TBME (3 × 10 mL). The crude material, which is sufficiently pure for routine work, was further purified by reverse-phase C-18 chromatography (Waters Sep-Pak C18 cartridge) with a mixture of 25% CH3CN, 75% H2O, and 0.1% TFA, followed by 30% CH3CN, 70% H2O, and 0.1% TFA to yield pure 6 (194 mg, 80%) as a green solid. 1H NMR (400 MHz, DMSO-d6): δ 8.03 (d, 2H, J ) 14.0 Hz), 7.60 (d, 2H, J ) 7.3 Hz), 7.46-7.39 (m, 4 H), 7.25 (d, 2H, J ) 7.4 Hz), 6.21 (d, 2H, J ) 14.1 Hz), 4.18 (t, 4H, J ) 6.7 Hz), 3.10 (t, 2H, J ) 6.7 Hz), 2.94 (s, 4H), 2.60-2.52 (m, 6H), 1.831.73 (m, 8H), 1.66 (s, 12H). λmax (PBS, pH 7.4): 807 nm ( ) 120 000 M-1 cm-1). ΦF (PBS, pH 7.4): 0.02. HRMSESI [M]+ m/z calcd for C40H51N2O8S3 783.2802, found 783.2821. CyTE-777 Succinimide Ester (7). To a solution of 1 (100 mg, 0.122 mmol) in 6 mL of anhydrous DMF was added N-hydroxysuccinimide (NHS) (1.40 g, 12.2 mmol), N,N′-diisopropylcarbodiimide (DIC) (1.91 mL, 12.2 mmol), and N-methylmorpholine (268 µL, 2.44 mmol). The resulting suspension was stirred at room temperature for 45 min. HPLC analysis of the crude reaction indicated over 90% conversion to the succinimide ester. The reaction was filtered to remove excess solid NHS and 20 mL of Et2O was added to give a green oil. The ether layer was decanted, and the green oil was redissolved in 1 mL of DMF. This solution was precipitated by addition of 20 mL of Et2O to yield a dark green paste. After this step was repeated one additional time, a free flowing green solid was isolated. The crude solid was subjected to preparative HPLC chromatography using a gradient of 22.5% CH3CN to 90% CH3CN in H2O with 0.1% TFA to yield analytically pure 7 (47 mg, 42%). 1H NMR (400 MHz, DMSO-d6): δ 8.67 (d, 2H, J ) 14.0 Hz), 7.59 (d, 2H, J ) 7.3 Hz), 7.47-7.39 (m, 4H), 7.26 (t, 2H, J ) 7.3 Hz), 6.37 (d, 2H, J ) 14.2 Hz), 4.20 (t, 4H, J ) 6.7 Hz), 3.12 (t, 2H, J ) 7.1 Hz), 3.01 (t, 2H, J ) 6.3 Hz), 2.79 (s, 4H), 2.68 (br , 4H), 2.53 (t, partially overlapping the solvent peak, 4H, J ) 7.3 Hz), 1.82-1.71 (m, 10H), 1.68 (s, 12H). HRMS-ESI [M]+ m/z calcd for C45H56N3O10S3 894.3122, found 894.3155. Phage Preparation and Labeling. Phage containing the VCAM-1 targeting sequence VHSPNKK (VHS-peptide) were isolated and amplified in Escherichia coli, and their titer was determined as described previously (24). Following isolation and titer determination, the phage were labeled directly with the activated ester of CyTE777 using a modified literature procedure (27). CyTE777 succinimidyl ester (5 µL at 5 µg/µL in DMSO) was added to 1 × 1012 plaque forming units (pfu) of VHSpeptide-displaying phage (VHS-phage) in 50 µL of Dulbecco’s phosphate-buffered saline (DPBS) supplemented with 1 mM Ca2+ and 10 mM Mg2+. The volume was increased to 100 µL with 0.1 M NaHCO3 buffer, pH 8.3, and allowed to incubate for 1 h in the dark at room temperature while shaking. After incubation, the phage was precipitated by addition of 20 µL of a PEG 8000/2.5 M NaCl solution (40% w/v) and allowed to stand on ice for 1 h. The fluorochrome-labeled phage was pelleted by centrifugation at 10 000 rpm for 15 min, and the supernatant was discarded. The light green pellet was resuspended in 400 µL of the supplemented DPBS and subjected to an additional round of PEG precipitation to minimize residual free fluorochrome and to remove bacterial protein contaminants. The degree of labeling was determined by using the extinction coefficient of CyTE-777 in PBS.

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NIR Fluorescence Cellular Imaging. Murine cardiac endothelial cells (MCECs) constitutively expressing VCAM-1 were isolated and prepared as described previously (28, 29). The cells were incubated with 1 × 1012 pfu of the CyTE-777-labeled VHS-phage in 400 µL of supplemented DPBS for 1 h at 37 °C, 5% CO2. Following incubation, the cells were washed twice with DPBS/0.01% Tween 20, fixed with 2% paraformaldehyde supplemented with 10 mM Mg2+ and 1 mM Ca2+, and then mounted to preserve fluorescence using VectaShield (Vector Laboratories, Burlingame, CA) containing the nuclear stain 4′,6-diamidino-2-phenylindole (DAPI). The cells were imaged on a Nikon Eclipse 80i fluorescence microscope with a 40× objective equipped with a Photometrics Cascade 512B CCD camera using excitation and emission filters from Chroma Technology. The nuclear stain DAPI was imaged using a 365 ( 5 nm band-pass filter for excitation and a 400 ( 5 nm cutoff filter for emission. The NIR fluorochrome CyTE-777 was imaged using a 775 ( 25 nm band-pass filter for excitation and a 845 ( 28 nm band-pass filter for emission. Images with CyTE-777 were acquired using a 2 s exposure time. RESULTS AND DISCUSSION

Synthesis. In an effort to develop improved methods for the synthesis of cyanine NIR fluorochromes suitable for bioconjugation, new paths to generate symmetric monocarboxylic acid derivatives were explored. The ability to prepare a symmetric monofunctional dye streamlines the synthetic methodology by removing the need for the stepwise synthesis used to prepare asymmetric cyanine fluorochromes. One route to these symmetric fluorochromes, first reported in 1992, relies on the reaction of nucleophiles with cyanine dyes containing an activated chloro-substituent on the polymethinic linker (19). With this approach, it is possible to prepare a variety of symmetric cyanine dyes with several different nucleophiles. However, when ethylthiolate, prepared by deprotonation of mercaptoethane with sodium hydride, was used as the nucleophile, only the dehalogenated cyanine dye was obtained. We have developed a modified procedure using anhydrous DMF and triethylamine as a base that allows for the facile synthesis of symmetric alkylthioether derivatives. Conversion of the commercially available dye IR-783, which contains two alkyl sulfonate moieties, under these conditions with a slight excess of 3-mercaptopropionic acid gives the symmetric carboxylic acid functionalized CyTE-777 in nearly quantitative yield (Scheme 1). The crude product from this reaction is sufficiently pure for subsequent transformation to the activated succinimide ester or for direct activation and use in solid-phase synthesis (unpublished results). Pure CyTE-777 may be obtained by reverse-phase C-18 column chromatography in 83% yield. To evaluate the effects of the degree of sulfonation on the properties of the fluorochromes, the tetrasulfonate analogue of CyTE-777 was prepared. The synthesis of this derivative, CyTE-783, begins with the preparation of the tetrasulfonate intermediate 2. This chloro-containing intermediate was prepared by the condensation of 2 equiv of 1-(4-sulfonatobutyl)-2,3,3-trimethylindoleninium5-sulfonate with N-[(3-(anilinomethylene)-2-chloro-1-cyclohexen-1-yl)methylene]aniline monohydrochloride in the presence of methanol, TEA, and acetic anhydride. The methanol improves the solubility of the highly polar indoleninium precursor in the reaction mixture, thus enabling a shorter reaction time. The reaction was performed with heating to 140 °C for 25 min in a sealed pressure reactor. After the reaction mix cooled, column

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Scheme 1

Figure 1. Normalized absorption (solid line) and emission (dashed line) spectra of 10 µM solutions of (A) CyTE-777 and (B) CyTE-807 in PBS. Table 1. Optical Properties of the CyTE Dyes in PBS, pH 7.4 CyTE-777 CyTE-783 CyTE-822a CyTE-823 PEGa CyTE-807 a

chromatography of the dark blue solid obtained from the reaction gives pure 2 in moderate yield. Due to the high degree of sulfonation on 2, it is only moderately soluble in DMF; thus for the nucleophilic displacement with 3-mercaptopropionic acid, anhydrous DMSO was used as the solvent. As with the synthesis of CyTE-777, the reaction to generate CyTE-783 (Scheme 1) is complete in less than 15 h as indicated by complete consumption of the starting chloro-functionalized dye. The purity of CyTE-783 from the crude reaction is adequate for further work, and it can be purified rigorously by reverse-phase C-18 chromatography in 71% yield. In an effort to prepare fluorochromes with absorption and emission greater than 800 nm, commercially available IR-820 was investigated as a precursor. The conditions used for generation of CyTE-822 are similar to those used for CyTE-777 (Scheme 1) and give a dark green solid in 52% yield after purification. In an effort

λmax,abs (nm)

λmax,em (nm)

 (M-1 cm-1)

777 783 822 823 807

812 814 845 847 839

130 000 174 000 128 000 116 000 120 000

In PBS, pH 7.4, with 20% DMSO.

to devise derivatives that are less likely to aggregate in aqueous solution than CyTE-822, which is structurally similar to indocyanine green, a dye known to aggregate in aqueous media, functionalization of IR-820 with monodisperse PEG7 thiol propionic acid was probed. Nucleophilic attack of the polyethylene glcyol (PEG) thiol on the chloro-substituted polymethinic linker of IR-820 gives CyTE-823 PEG (Scheme 1). Despite incorporation of the large PEG acid, purification of the fluorochrome is straightforward giving high-purity product in 56% yield. A final long wavelength fluorochrome, CyTE-807, was prepared through use of IR-806 in 80% yield following purification (Scheme 1). With this dye, the cyclic sixmembered ring containing linker present in the previous CyTE fluorochrome derivatives is replaced by a more constrained five-membered ring. The removal of one methylene carbon from the cyclic ring results in significantly altered optical properties in comparison to the related CyTE-777. Optical Properties. All of the alkyl-thioether-functionalized fluorochromes display long wavelength absorption and emission bands. The optical properties of these dyes are summarized in Table 1. CyTE-777 and CyTE783 have absorption and emission bands that match closely those of ICG in water, which has an absorption maximum of 780 nm. Therefore these two dyes are well suited for imaging using currently available filter sets optimized for ICG. The absorption and emission spectra of CyTE-777 are shown in Figure 1a. With CyTE-777, there is no indication for aggregation in PBS, which

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Monofunctional Near-Infrared Fluorochromes Scheme 2

Figure 2. Absorption spectra of 1 µM solutions of CyTE-823 PEG in PBS (dashed line) showing the dimer at 750 nm, while in PBS with 20% DMSO (solid line), only the monomer is observed.

would manifest itself as a second absorption band approximately 70 nm to shorter wavelength (30). The short wavelength shoulder visible in the absorption spectrum of CyTE-777 is a common feature of virtually all cyaninebased fluorochromes and is observed in less polar solvents such as DMSO and MeOH where there is no tendency for aggregation. The formation of dimers and other aggregates is undesirable because they frequently result in intermolecular quenching of fluorescence emission (30). The only significant difference in the optical properties between the disulfonate CyTE-777 and the tetrasulfonate CyTE-783 is their extinction coefficients, where a 25% increase is observed in PBS with the tetrasulfonate CyTE-783. In contrast to the increased extinction coefficient of CyTE-783 when compared to CyTE-777, there are only minor differences in the absorption and emission maxima and quantum yields of the two dyes. The long wavelength dye CyTE-822 with benzindoleninium end groups is structurally similar to ICG; however, the alkyl-thioether moiety in the polymethinic linker results in a significant bathochromic shift of the absorption and emission maxima in comparison to ICG. As with ICG, CyTE-822 shows a tendency to aggregate in aqueous solution. This aggregation is evident even at concentrations as low as 1 µM in PBS. The free monomer is only obtained in PBS with 20% DMSO. Aggregation of this derivative limits its potential utility for use as a fluorescent label for imaging applications. To reduce the tendency for aggregation in aqueous solution, CyTE-822 was modified with a hydrophilic poly(ethylene glycol) chain. This modified dye, CyTE-823 PEG, retains the long wavelength excitation and emission properties of CyTE-822; however, as with CyTE822, it shows a tendency to aggregate in PBS (Figure 2). This indicates that the short PEG7 chain is not suf-

ficiently long to increase the solvent-dye affinity, which would result in diminished dye-dye aggregate formation. As an alternative for the preparation of a monocarboxylic acid derivatized fluorochrome with long wavelength absorption and emission, IR-806 was investigated as a building block. The alkyl-thioether containing CyTE807 has an absorption maximum of 807 nm in PBS and an emission maximum of 839 nm (Figure 1b). It also behaves like CyTE-777 in PBS with no observable dyedye aggregation. When compared to the structurally related CyTE-777, the absorption and emission bands shift 30 and 27 nm to longer wavelength, respectively. In addition to the wavelength shifts induced by the steric strain imposed by the cyclopentyl-containing polymethinic linker of CyTE-807, a slight decrease in the extinction coefficient from 130 000 M-1 cm-1 for CyTE777 to 120 000 M-1 cm-1 is observed. CyTE-807 as well as other potential dyes containing a cyclopentyl-based polymethinic linker hold promise for use in imaging applications. Coupled with the proper filter sets, it may be possible to add an 800+ nm NIR fluorescence imaging channel to the currently used shorter wavelength channels. Phage Labeling. To demonstrate the general applicability of the CyTE fluorochromes, CyTE-777 was activated for use in phage labeling. Activation was

Figure 3. Fluorescence microscopy of MCECs incubated with CyTE-777-labeled VHS-phage for 1 h: (A) nuclear staining with DAPI; (B) staining of the CyTE-777 conjugated phage; (C) the merged image indicating internalization of the labeled phage.

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accomplished by treatment of CyTE-777 with NHS in the presence of DIC and N-methylmorpholine (Scheme 2). Greater than 90% conversion to the activated ester was observed by HPLC. To label the VHS-phage, a modified literature procedure (27) was used where 1 × 1012 pfu of phage were treated with 25 µg of the activated fluorochrome for 1 h under slightly basic conditions (Scheme 2). By monitoring the fluorochrome absorbance, we determined the degree of labeling to be 1700 dye molecules per phage. The efficient labeling will facilitate imaging applications by enabling delivery of a large quantity of fluorochrome to the phage target. Conjugation of the dye to the phage was supported by observation of a shift in the absorption band of the conjugated CyTE777 from 777 to 798 nm in DPBS buffer. Bathochromic shifts in the absorption maxima of cyanine dyes are observed frequently in conjunction with a decrease in polarity of the fluorochrome local environment, such as provided by conjugation to the phage. In a similar system, the absorption maximum of ICG was reported to shift from 780 nm in water to 800 nm when bound to albumin (30). Fluorescence Microscopy. To assess the performance of the fluorochrome-labeled VHS-phage for imaging, the probe was incubated with MCECs expressing VCAM-1. Following incubation for 1 h, the cells were monitored by fluorescence microscopy to visualize the distribution of the VCAM-1-specific phage. Dual channel fluorescence microscopy of stained nuclei and phage particles clearly shows phage internalization (Figure 3). The strong fluorescence signal observed is the result of multiple factors. Due to the high degree of fluorochrome labeling, the phage is capable of delivering a large fluorochrome payload, resulting in increased signal. In addition, binding and recognition of the phage by the surface receptors (VCAM-1) is a highly selective event. The VHS-phage have a high affinity for VCAM-1 since the peptides are present in 4-5 copies on the phage pIII coat proteins (31) resulting in multivalency, which increases the affinity through avidity effects (32). Finally, the VHS-phage is internalized by VCAM-1-expressing endothelial cells resulting in increased signal-to-noise ratios by biological amplification. The alkyl-thioether-derivatized NIR fluorochromes hold promise for use in a wide variety of imaging applications. They are easily prepared from commercially available starting materials and require no additional purification prior to bioconjugation. One derivative, CyTE-807, displays absorption and emission maxima greater than 800 nm and does not aggregate in aqueous solution. By conjugation of CyTE-777 to VHS-phage, imaging of VCAM-1-mediated internalization of the phage is possible. The fluorochrome-labeled phage constructs demonstrate a new potential imaging strategy that allows for highly selective, efficient delivery of large fluorochrome payloads. ACKNOWLEDGMENT

This research was supported in part by NIH Grants P50-CA86355, R21-CA114149, and RO1-CA99385. S.A.H. was supported by Grant T32-CA79443. LITERATURE CITED (1) Weissleder, R., and Ntziachristos, V. (2003) Shedding light onto live molecular targets. Nat. Med. 9, 123-128. (2) Flanagan, J. H., Jr., Khan, S. H., Menchen, S., Soper, S. A., and Hammer, R. P. (1997) Functionalized tricarbocyanine

Hilderbrand et al. dyes as near-infrared fluorescent probes for biomolecules. Bioconjugate Chem. 8, 751-756. (3) Lin, Y., Weissleder, R., and Tung, C. H. (2002) Novel nearinfrared cyanine fluorochromes: synthesis, properties, and bioconjugation. Bioconjugate Chem. 13, 605-610. (4) Mader, O., Reiner, K., Egelhaaf, H. J., Fischer, R., and Brock, R. (2004) Structure property analysis of pentamethine indocyanine dyes: identification of a new dye for life science applications. Bioconjugate Chem. 15, 70-78. (5) Mujumdar, R. B., Ernst, L. A., Mujumdar, S. R., Lewis, C. J., and Waggoner, A. S. (1993) Cyanine dye labeling reagents: sulfoindocyanine succinimidyl esters. Bioconjugate Chem. 4, 105-111. (6) Mujumdar, S. R., Mujumdar, R. B., Grant, C. M., and Waggoner, A. S. (1996) Cyanine-labeling reagents: sulfobenzindocyanine succinimidyl esters. Bioconjugate Chem. 7, 356362. (7) Narayanan, N., and Patonay, G. (1995) A new method for the synthesis of heptamethine cyanine dyes: synthesis of new near-infrared fluorescent labels. J. Org. Chem. 60, 23912395. (8) Pham, W., Lai, W. F., Weissleder, R., and Tung, C. H. (2003) High efficiency synthesis of a bioconjugatable near-infrared fluorochrome. Bioconjugate Chem. 14, 1048-1051. (9) Pham, W., Medarova, Z., and Moore, A. (2005) Synthesis and application of a water-soluble near-infrared dye for cancer detection using optical imaging. Bioconjugate Chem. 16, 735740. (10) Strekowski, L., Mason, C. J., Lee, H., Gupta, R., Sowell, J., and Patonay, G. (2003) Synthesis of water-soluble nearinfrared cyanine dyes functionalized with [(succinimido)oxy]carbonyl group. J. Heterocyclic Chem. 40, 913-916. (11) Zhang, Z., and Achilefu, S. (2004) Synthesis and evaluation of polyhydroxylated near-infrared carbocyanine molecular probes. Org. Lett. 6, 2067-2070. (12) Tung, C. H., Lin, Y., Moon, W. K., and Weissleder, R. (2002) A receptor-targeted near-infrared fluorescence probe for in vivo tumor imaging. ChemBioChem 3, 784-786. (13) Tung, C. H., Quinti, L., Jaffer, F. A., and Weissleder, R. (2005) A branched fluorescent peptide probe for imaging of activated platelets. Mol. Pharm. 2, 92-95. (14) Chen, X., Conti, P. S., and Moats, R. A. (2004) In vivo nearinfrared fluorescence imaging of integrin alphavbeta3 in brain tumor xenografts. Cancer. Res. 64, 8009-8014. (15) Zaheer, A., Lenkinski, R. E., Mahmood, A., Jones, A. G., Cantley, L. C., and Frangioni, J. V. (2001) In vivo nearinfrared fluorescence imaging of osteoblastic activity. Nat. Biotechnol. 19, 1148-1154. (16) Law, B., Curino, A., Bugge, T. H., Weissleder, R., and Tung, C. H. (2004) Design, synthesis, and characterization of urokinase plasminogen-activator-sensitive near-infrared reporter. Chem. Biol. 11, 99-106. (17) Weissleder, R., Tung, C. H., Mahmood, U., and Bogdanov, A., Jr. (1999) In vivo imaging of tumors with proteaseactivated near-infrared fluorescent probes. Nat. Biotechnol. 17, 375-378. (18) Xing, B., Khanamiryan, A., and Rao, J. (2005) Cellpermeable near-infrared fluorogenic substrates for imaging beta-lactamase activity. J. Am. Chem. Soc. 127, 4158-4159. (19) Strekowski, L., Lipowska, M., and Patonay, G. (1992) Substitution reactions of a nucleofugal group in heptamethine cyanine dyes. Synthesis of an isothiocyanato derivative for labeling proteins with a near-infrared chromophore. J. Org. Chem. 57, 4578-4580. (20) Flanagan, J. H., Jr., Owens, C. V., Romero, S. E., Waddell, E., Kahn, S. H., Hammer, R. P., and Soper, S. A. (1998) Nearinfrared heavy-atom-modified fluorescent dyes for basecalling in DNA-sequencing applications using temporal discrimination. Anal. Chem. 70, 2676-2684. (21) Zaheer, A., Wheat, T. E., and Frangioni, J. V. (2002) IRDye78 conjugates for near-infrared fluorescence imaging. Mol. Imaging 1, 354-364. (22) Matsui, M., Hashimoto, Y., Funabiki, K., Jin, J.-Y., Yoshida, T., and Minoura, H. (2005) Application of near-infrared absorbing heptamethine cyanine dyes as sensitizers for zinc oxide solar cell. Synth. Met. 148, 147-153.

Bioconjugate Chem., Vol. 16, No. 5, 2005 1281

Monofunctional Near-Infrared Fluorochromes (23) Song, F., Peng, X., Lu, E., Zhang, R., Chen, X., and Song, B. (2004) Synthesis, spectral properties and photostabilities of novel water-soluble near-infrared cyanine dyes. J. Photochem. Photobiol. A 168, 53-57. (24) Kelly, K. A., Allport, J. R., Tsourkas, A., Shinde-Patil, V. R., Josephson, L., and Weissleder, R. (2005) Detection of vascular adhesion molecule-1 expression using a novel multimodal nanoparticle. Circ. Res. 96, 327-36. (25) Demas, J. N., and Crosby, G. A. (1971) The measurement of photoluminescence quantum yields. A review. J. Phys. Chem. 75, 991-1024. (26) Reindl, S., Penzkofer, A., Gong, S.-H., Landthaler, M., Szeimies, R. M., Abels, C., and Ba¨umler, W. (1997) Quantum yield of triplet formation for indocyanine green. J. Photochem. Photobiol. A 105, 65-68. (27) Jaye, D. L., Geigerman, C. M., Fuller, R. E., Akyildiz, A., and Parkos, C. A. (2004) Direct fluorochrome labeling of phage display library clones for studying binding specificities: applications in flow cytometry and fluorescence microscopy. J. Immunol. Methods 295, 119-127. (28) Allport, J. R., Lim, Y. C., Shipley, J. M., Senior, R. M., Shapiro, S. D., Matsuyoshi, N., Vestweber, D., and Luscin-

skas, F. W. (2002) Neutrophils from MMP-9- or neutrophil elastase-deficient mice show no defect in transendothelial migration under flow in vitro. J. Leukocyte Biol. 71, 821828. (29) Tsourkas, A., Shinde-Patil, V. R., Kelly, K. A., Patel, P., Wolley, A., Allport, J. R., and Weissleder, R. (2005) In vivo imaging of activated endothelium using an anti-VCAM-1 magnetooptical probe. Bioconjugate Chem. 16, 576-581. (30) Philip, R., Penzkofer, A., Ba¨umler, W., Szeimies, R. M., and Abels, C. (1996) Absorption and fluorescence spectroscopic investigation of indocyanine green. J. Photochem. Photobiol. A 96, 137-148. (31) Smith, G. P., and Petrenko, V. A. (1997) Phage display. Chem. Rev. 97, 391-410. (32) Lees, W. J., Spaltenstein, A., Kingery-Wood, J. E., and Whitesides, G. M. (1994) Polyacrylamides bearing pendant R-sialoside groups strongly inhibit agglutination of erythrocytes by influenza A virus: multivalency and steric stabilization of particulate biological systems. J. Med. Chem. 37, 3419-3433.

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