Bioconjugate Chem. 2002, 13, 605−610
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Novel Near-Infrared Cyanine Fluorochromes: Synthesis, Properties, and Bioconjugation Yuhui Lin, Ralph Weissleder, and Ching-Hsuan Tung* Center for Molecular Imaging Research, Massachusetts General Hospital, Harvard Medical School, Charlestown, Massachusetts 02129. Received October 29, 2001; Revised Manuscript Received January 31, 2002
Recently, near-infrared (NIR) fluorescence light has been applied to image various biological events in vivo, because it penetrates tissue more efficiently than light in the visible spectrum. Compounds exhibiting fluorescent properties in the NIR range are key elements for this upcoming optical imaging technology. In this paper, we report the synthesis of four new, water-soluble NIR cyanine fluorochromes which have superior chemical stability and optical properties. Each fluorochrome was designed with a monoreactive carboxyl group for labeling purposes. When multiple fluorochromes were attached to a single macromolecule, fluorescence quenching was observed. On the basis of this property, a novel autoquenched enzyme sensitive NIR fluorescence probe was prepared.
INTRODUCTION
Noninvasive imaging with light photons represents an intriguing avenue for extracting biological information from living subjects. While light in the visible range is routinely used for intravital microscopy, imaging of deeper tissues (>500 µm to cm) requires the use of nearinfrared (NIR) light, as hemoglobin and water, the major absorbers of visible and infrared light, respectively, have their lowest absorption coefficient in the NIR region (650-900 nm). Light photons can be used to measure different native parameters of tissue through which they travel, such as absorption, scattering, polarization, spectral characteristics, and fluorescence. Near-infrared fluorescence imaging in particular is expected to have a major impact in biotechnology and medicine because of its high sensitivity and the fact that no radiation is necessary for acquisition and the versatility of different reporter probes. The recent descriptions of targeted NIR fluorochromes (1, 2), activatable NIR fluorochromes (3), redshifted fluorescent proteins (4), and bioluminescent probes (5) has lent further credence to the field. Conversely, a number of reflectance (6) and tomographic imaging systems have recently been developed to detect NIR fluorescence in deep tissues (7) including patients (8, 9). At the heart of NIR imaging lies the need for biocompatible fluorochromes. While indocyanine green (ICG) has been used clinically for over 20 years with few side effects (10), its use in designing targeted agents is limited primarily because of the nonavailability of monoderivatized activated precursors. Additional disadvantages are its hydrophobicity, high albumin binding, nonlinear fluorescence, and other intrinsic properties (11). Recently, several new derivatives such as tricarbocyanines (2) and cyptates (12) have been described and synthesized by different groups. General problems with the synthesis of NIR fluorochromes compared to visible light fluorochromes are (1) * To whom correspondence should be addressed. Ching H. Tung, Ph.D., Center for Molecular Imaging Research, Massachusetts General Hospital, 149 13th St., Rm. 5406, Charlestown, MA 02129. (tel): (617) 726-5779. (fax): (617) 726-5708. e-mail:
[email protected].
significant spectral broadening as the wavelength increases, (2) low quantum yield, (3) photoinstability, (4) chemical instability with increasing red-shift, and (5) the tendency to aggregate because of hydrophobicity. The ideal NIRF reporters for in vivo imaging should have the following characteristics: (1) a peak fluorescence close to “700-900 nm”, (2) high quantum yield, (3) narrow excitation/emission spectrum, (4) high chemical and photostability, (5) nontoxicity, (6) good biocompatibility, biodegradability and excretability, (7) availability of monofunctional derivatives as platform technology, and (8) commercial viability and scalable production for large quantities required for human use. We describe here the synthesis of a number of highly stable NIR fluorochromes designed with the above prerequisites in mind. The agents contain multiple hydrophilic groups and have been prepared as monohydroxy succinimide esters for binding to biomolecules such as peptides, metabolites, proteins, and other affinity ligands. MATERIALS AND METHODS
Chemicals. 6-Amino-1,3-naphthalene disulfonic acid disodium salt, 1,4-butanesultone, 4-hydrazinobenzoic acid, 3-methyl-2-butanone, iodoethane, diisopropylcarbodiimide (DIPCDI), and N-hydroxysuccinimide (NHS) were purchased from Aldrich (Milwaukee, WI). Glutaconaldehyde dianil hydrochloride, malconaldehyde dianil hydrochloride, and p-hydrazinobenzenesulfonic acid were purchased from TCI America (Portland, OR). All other reagents and solvents were purchased from Aldrich and were used as received. Purification and Spectroscopic Analysis. Purification of dyes was performed on a Rainin preparative HPLC instrument (Woburn, MA) using a C18-RP preparative column (Vydac, Hesperia, CA; flow rate ) 6 mL/ min; eluant A, water with 0.1% TFA; eluant B, 90% of acetonitrile and 10% of eluant A; starting at 90% A for 5 min and then a linear gradient over 40 min to 50% A). The dual HPLC detector was set at 240 and 360 nm. Dyes were collected, and solvent was removed by Speed-vac concentrator (Savant, Holbrook, NY). Absorbance spectra were measured on a U-3000 spectrophotometer (Hitachi, San Jose, CA). Fluorescence spectra were recorded using
10.1021/bc0155723 CCC: $22.00 © 2002 American Chemical Society Published on Web 04/17/2002
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a F-4500 fluorophotometer (Hitachi). The proton NMR spectra were recorded in D2O in a 400 MHz FT-NMR spectrometer. Synthesis of Cyanine Dyes. 1,1,2-Trimethylbenzindoleninium 1,3-disulfonate (13), 5-carboxy-1-(4-sulfobutyl)-2,3,3-trimethyl-3H-indolenine 2 (14), and 1-(4sulfonatobutyl)-2,3,3-trimethylindoleninium-5-sulfonate 5 (15) were synthesized according to the literature. All compounds were used in crude form because conventional chromotagraphy and/or recrystalization proved to be difficult for the highly hydrophilic compounds. N-Ethyl-2,3,3-trimethylbenzindoleninium 5,7-Disulfonate 1. In a 50 mL round-bottomed flask were placed 4.7 g of 1,1,2-trimethylbenzindoleninium 1,3disulfonate dipotassium salt, 8 mL of ethyl iodide, and 50 mL of 1,2-dichlorobenzene. The mixture was heated under argon atmosphere at 90 °C for 12 h and then at 125 °C for another 10 h. After being cooled to room temperature, 1,2-dichlobenzene was decanted and the solid was washed three times with an acetone/ether mixture. The solid was filtered off and dried under vacuum to result in 4.1 g of crude 1. Intermediate 3. In a 50 mL round flask were placed 1.92 g of 1, 1.12 g of glutaconaldehyde dianil hydrochloride, 20 mL of acetic anhydride, and 5 mL of glacial acetic acid. The mixture was heated at 120 °C for 3 h and was then precipitated from ethyl acetate upon cooling. After filtration, the solid was washed twice with ethyl acetate and dried under vacuum to yield 2.2 g of crude intermediate 3. NIR1. A mixture of 0.60 g of intermediate 3, 0.33 g of 5-carboxy-1-(4-sulfobutyl)-2,3,3-trimethyl-3H-indolenine 2, 0.49 g of potassium acetate, 12 mL of acetic anhydride, and 5 mL of glacial acetic acid was stirred and heated at 120 °C under argon atmosphere for 30 min. After being cooled to room temperature, the mixture was poured into 200 mL of ethyl acetate and the precipitate was collected by centrifugation and then dried to result in 0.86 g of crude NIR1. The crude product was further purified by reverse phase HPLC to give 14% (based on the crude product) of pure NIR1. 1H NMR (D2O): δ 1.24 (6H, s), 1.28 (3H, t), 1.54 (6H, s), 1.80 (4H, broad m), 2.90 (2H, broad t), 3.86 (2H, broad), 4.12 (2H, broad q), 5.76 (1H, broad), 6.03 (2H, broad), 7.04 (2H, broad), 7.21 (1H, d), 7.33 (1H, broad), 7.53 (1H, broad), 7.58 (1H, s), 7.65 (1H, d), 7.73 (1H, d), 8.19 (1H, s), 8.52 (1H, s), 8.71 (1H, d). Intermediate 4. In a 50 mL round flask were placed 1.70 g of 1, 0.93 g of malonaldehydedianil hydrochloride, 20 mL of acetic anhydride, and 5 mL of glacial acetic acid. The mixture was then heated at 120 °C for 3 h. Upon being cooled, the mixture was precipitated in ethyl acetate. After filtration the solid was washed twice with ethyl acetate and dried under vacuum to yield 1.5 g of crude intermediate 4. NIR2. A mixture of 0.58 g of intermediate 4, 0.35 g of 5-carboxy-1-(4-sulfobutyl)-2,3,3-trimethyl-3H-indolenine 2, 0.49 g of potassium acetate, 12 mL of acetic anhydride, and 5 mL of glacial acid was stirred and heated at 120 °C under argon atmosphere for 30 min. After being cooled to room temperature, the mixture was poured into 200 mL of ethyl acetate. The precipitate was collected by centrifugation and dried to result in 0.9 g of crude NIR2. The crude product was then purified by reversed phase HPLC to give 21% (based on the crude product) of pure NIR2. 1H NMR (D2O): δ 1.19 (3H, t), 1.24 (6H, s), 1.52 (6H, s), 1.80 (4H, broad m), 2.90 (2H, broad t), 3.90 (2H, broad m), 4.02 (2H, broad q), 5.82 (1H, broad d), 5.85 (1H, broad d), 6.14 (2H, t), 7.08 (1H, d), 7.56 (1H, s), 7.617.77 (4H, m), 8.19 (1H, s), 8.51 (1H, s), 8.70 (1H, d).
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Intermediate 6. In a 50 mL round flask were placed 0.80 g of 1-(4-sulfonatobutyl)-2,3,3-trimethylindoleninium5-sulfonate 5, 0.50 g of glutaconaldehyde dianil hydrochloride, 10 mL of acetic anhydride, and 5 mL of glacial acetic acid. The mixture was then heated at 120 °C for 3 h. Upon cooling, the mixture was precipitated from ethyl acetate. After filtration the solid was washed twice with ethyl acetate and dried under vacuum to yield 0.88 g of crude intermediate 6. NIR3. A mixture of 0.88 g of intermediate 6, 0.55 g of 5-carboxy-1-(4-sulfobutyl)-2,3,3-trimethyl-3H-indolenine 2, 0.85 g of potassium acetate, 12 mL of acetic anhydride, and 5 mL of glacial acetic acid was stirred and heated at 120 °C under argon atmosphere for 30 min. After being cooled to room temperature, the mixture was poured into 200 mL of ethyl acetate. The precipitate was collected by centrifugation and dried to result in 0.9 g of crude NIR3. The crude product was then purified by reverse phase HPLC to give 6.7% (based on the crude product) of pure NIR4. 1H NMR (D2O): δ 1.36 (6H, s), 1.42 (6H, s), 1.56-1.81 (6H, broad m), 1.81-1.89 (2H, broad m), 2.84-2.90 (4H, m), 3.92 (2H, broad t), 4.06 (2H, broad t), 5.85 (1H, broad d), 6.10 (2H, broad), 6.28 (1H, broad t), 7.17 (2H, broad m), 7.26 (1H, d), 7.45 (1H, broad), 7.53 (1H, broad), 7.67-7.77 (4H, m). Intermediate 7. In a 50 mL round flask were placed 0.80 g of 1-(4-sulfonatobutyl)-2,3,3-trimethylindoleninium5-sulfonate 5, 0.45 g of malonaldehydedianil hydrochloride, 10 mL of acetic anhydride, and 5 mL of glacial acetic acid. The mixture was then heated at 120 °C for 3 h. Upon cooling, the mixture was precipitated from ethyl acetate. After filtration the solid was washed twice with ethyl acetate and dried under vacuum to yield 0.74 g crude intermediate 7. NIR4. A mixture of 0.74 g of intermediate 7, 0.45 g of 5-carboxy-1-(4-sulfobutyl)-2,3,3-trimethyl-3H-indolenine 2, 0.70 g of potassium acetate, 12 mL of acetic anhydride, and 5 mL of glacial acid were stirred and heated at 120 °C under argon atmosphere for 30 min. After being cooled to room temperature, the mixture was poured into 200 mL of ethyl acetate. The precipitate was collected by centrifugation and dried to result in 0.9 g of crude NIR4. The crude product was then purified by reverse phase HPLC to give 8.1% (based on the crude product) of pure NIR3. 1H NMR (D2O): δ 1.29 (6H, s), 1.39 (6H, s), 1.711.78 (6H, broad m), 1.84-1.88 (2H, broad m), 2.79-2.87 (4H, m), 3.92 (2H, broad t), 4.04 (2H, broad t). 5.82 (1H, d), 6.06 (1H, d), 6.23 (1H, t), 7.20 (1H, d), 7.25 (1H, d), 7.60-7.75 (6H, m). Determination of the Extinction Coefficients and Fluorescence Quantum Yield. All NIR dyes were purified by preparative HPLC twice as described above. The K+ ions were replaced with H+ by ion-exchange chromatography in deionized water (cation-exchange resin, Dowex-50, 8% cross-link, 100-200 mesh). About 20 mg of a dye was dissolved in 100 mL of deionized water. The absorbance was measured individually in three dilutions of the stock solution in deionized water or in ethanol (95%). The fluorescence emission maxima and intensities of the dyes were obtained using dilute solutions in water and exciting at both the main absorption peak as well the short-wavelength shoulder of the main absorption peak. In cases of NIR2 and NIR4, the quantum yields were calculated relative to a standard solution of Cy5.5 (Amersham-Pharmacia, Piscataway, NJ) with quantum yield of 0.29, whereas in cases of NIR1 and NIR3. The calculations were performed relative to a standard solution of Cy7 (Amersham-Pharmacia) with a quantum yield of 0.28.
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Bioconjugate Chem., Vol. 13, No. 3, 2002 607
Figure 1. Synthetic scheme of NIR1 and NIR2.
Figure 2. Synthetic scheme of NIR3 and NIR4.
Activation of Cyanine Dyes. In a typical experiment, 10 mg of dye, 30 µL of diisopropylcarbodiimide, 50 µL of N-methylmorpholine, 22.0 mg of NHS, and 0.5 mL of dry DMF were placed in a small round-bottomed flask under argon atmosphere. The mixture was stirred at room temperature for 3 h and then precipitated in ether. After centrifugation, the ether was decanted and the solid was washed four more times with ether and dried in a vacuum. The HPLC result indicated that more than 98% of dye was converted to active ester. Imaging of NIR Dyes. Imaging was performed using a previously developed NIRF reflectance imaging system (6). Briefly, the system consisted of a light-tight chamber equipped with a halogen white light source and excitation band-pass filters providing the excitation wavelengths of 610-650 nm or 750-770 nm (Omega Optical, Brattlebore, VT). Equalmolar NIR dyes and ICG were loaded to individual wells (0.16 nmol in 200 µL) in a clear-bottom 96-well plate (Corning, Corning, NY). Fluorescence was detected by a 12-bit monochrome CCD camera (Kodak, Rochester, NY) equipped with a 12.5-75 mm zoom lens and emission band-pass filters at 680-720 nm or 800820 nm (Omega Optical, Brattlebore, VT). Exposure time was 10 s per image. Images were analyzed using commercially available software (Kodak Digital Science 1D software, Rochester, NY). Synthesis of Enzyme Sensitive Probe with Various Amounts of NIR2. The enzyme sensitive probes
were synthesized similar to the described protocol (3). Briefly, partially PEGylated polylysine (0.1 mg, MW ) 500000 Da. (16)) was reacted with various amounts of NIR2-NHS ester, whose concentration was 0.4, 2, 4, 8, 20, 40, or 80 µM in 20 mM NaHCO3, at room temperature for 3 h. The NIR2-labeled polymers were then separated from excess low molecular weight reagents using a 50 kDa cutoff microconcentrators (Amicon, Beverly, MA). Based on NIR2 absorption measurement at 662 nm, the average numbers of NIR2 fluorochrome per PGC are 0.2, 0.8, 1.4, 2.4, 4.3, 5.7, and 7.0, respectively. Trypsin Activation of NIR2-PGC Probes. The activation of the NIRF probe was done in a 96-well plate with various NIR2-PGC probes. In each well, NIR2PGC (40 pmole) in 200 µL of PBS was incubated with 10 µL of trypsin solution (0.05% trypsin, 0.53 mM EDTA, Mediatech, Herndon, VA). The reactions were monitored using a fluorescence microplate reader (Spectramax, Molecular Devices, Sunnyvale, CA) with excitation and emission wavelength at 662 and 684 nm, respectively. The reactions were purformed in duplicate. RESULTS AND DISCUSSION
Synthesis of Nonsymmetrical Cyanine Dyes. The synthetic pathways for the four new cyanine dyes are outlined in Figures 1 and 2. The synthesis of NIR1 and NIR2 was carried out starting from 1,1,2-tri-
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methylbenzindoleninium 1,3-disulfonate dipotassium salt, which was converted to 1 by treating with ethyl iodide. Reaction of 1 with malconaldehyde dianil hydrochloride or glutaconaldehyde dianil hydrochloride resulted in the intermediates 3 and 4. Intermediates 3 and 4 were stable at room temperature and in aqueous solution, and no significant decomposition could be observed over more than two weeks. The asymmetrical dyes NIR1 and NIR2 were assembled by reacting 3 or 4 with 5-carboxy-1-(4sulfobutyl)-2,3,3-trimethyl-3H-indolenine 2. Similarly, the synthesis of NIR3 and NIR4 was started with 1(4-sulfonatobutyl)-2,3,3-trimethylindoleninium-5-sulfonate 5, which was converted to intermediates 6 or 7 by the reaction with malconaldehyde dianil hydrochloride or glutaconaldehyde dianil hydrochloride. Like intermediates 3 and 4, intermediates 6 and 7 were also stable at room temperature. Reaction of intermediate 6 or 7 with 5-carboxy-1-(4-sulfobutyl)-2,3,3-trimethyl-3H-indolenine 2 yielded the asymmetrical dyes NIR3 and NIR4, respectively. The multistep synthesis of the described compounds showed several peculiarities. First, the intermediateries were difficult to purify, leading us to use mixtures for subsequent reactions followed by final HPLC purification. We assume that purification of the intermediaries was difficult because of their extremely high water solubility. Irrespective of this step, we showed that the final products were >98% pure as determined by HPLC. Clean NMR spectra were obtained, except for the integration of one or two bridged methine protons, which might be due to the resonance structure of the cyanine dyes. Indeed similar finding about NMR spectra of cyanine dyes have been reported (17). Unlike previously described and commercially available near-infrared cyanine dyes (2, 13, 18), the ones reported here bear two different heterocyclic ring systems including a 3-ring and a 2-ring heterocyclic system. The rationale for this design is severalfold, including the ability to fine-tune spectral properties for imaging compounds and the improvement in the typically mitigating self-aggregation of large planar dyes. The latter is of particular concern since self-aggregating fluorochromes may be poorly soluble, as is the case for ICG. To further improve solubility, we chose to also include a constant and large number of sulfonate groups in all dyes. Spectral Properties of Dyes. The absorption spectra of NIR1-4 are shown in Figure 3A. As in other cyanine dyes, the difference in absorbance maximum between indodicarbocyanine dye and indotricarbocyanine dye was about 100 nm. The terminal ring system contributes very little to the absorbance maximum compared to that of the bridging methine unit. The difference in absorbance maximum between 3/2 ring systems (e.g., NIR2) compared to 2/2 homocycles (e.g., NIR4) was only 10 nm. Excitation and emission spectra of NIR1 and NIR2 are shown in Figure 3B. Indodicarbocyanine dyes had a 20 nm Stokes shift of the fluorescence emission maximum, while indotricarbocyanine dyes exhibited a 30 nm Stokes shift of the fluorescence maximum. The dyes had high molar extinction coefficient above 250000, similar as the range published for other compounds. Quantum yields of the different fluorochromes varied from 0.23 to 0.43. Optical properities of the compounds were summarized on Table 1. Activation of Dyes. The cyanine dyes were converted to reactive N-hydroxysuccinimide esters by DICPDI and NHS in the presence of N-methylmorpholine in DMF (Figure 4). An almost quantitative yield (typically >98%) was observed using reversed phase HPLC (Figure 4). As
Lin et al.
Figure 3. Absorption (A) and fluorescence (B) spectra of NIR fluorochromes. Table 1. Optical Properties of NIR Fluorochromes λmax,abs λmax,em compd solvent (nm) (nm) NIR1 NIR2 NIR3 NIR4
H2O EtOH H2O EtOH H2O EtOH H2O EtOH
761 769 662 667 750 756 650 654
Stokes shift (nm) (L mol-1 cm-1)
QY
796
35
268000
0.23
684
22
250000
0.34
777
27
275000
0.28
671
21
260000
0.43
seen in Figure 4, the elution times for NIR2 and its active ester were 27.1 and 29.0 min, respectively. The active ester was remarkably stable. Even in water, less than 10% of the active ester was hydrolyzed over a period of 20 days (4 °C), based on HPLC analysis. A test reaction was done by reacting NIR2-NHS active ester with benzylamine. The resultant NIR2-benzylamine conjugate showed an elution time of 32.1 min (HPLC results not shown). Imaging of NIR Dyes. In subsequent studies we utilized a home-built imaging system (6) to image the newly synthesized NIR dyes and compared them to ICG. The images were acquired with two sets of filters. One has 610-650 nm excitation and 680-720 nm emission; the other has 750-770 nm excitation and 800-820 nm emission. As shown in Figure 5, the fluorescence signals of NIRs are well resolved in this fluorescence imaging system. At 700 nm, only NIR2 and NIR4 were detectable, while NIR1 and NIR 3 were detectable at 800 nm. Moreover, the NIR1 and NIR3 showed significantly better optical property than ICG. The signal intensity of NIR1 and NIR3, as compared to ICG, were 7- and 12-fold higher, respectively. Enzyme Activatable NIRF Probe. Recently, we have developed a panel of biocompatible molecular probes for the in vivo detection of specific protease activity, particularly for those proteases that play key roles in different aspects of cancer growth, metastases formation, and angiogenesis (3, 19, 20). In those studies, a commercially available fluorochrome (Cy5.5) was used as the fluorescence reporter. In this study the newly synthesized
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Figure 6. Fluorescence measurement of NIR-PGC probes before (white bars) and after (black bars) cleavage by trypsin for 3 h.
Figure 4. A. Activation of NIR2. B. HPLC traces before (top) and after modification (bottom).
Figure 5. NIR fluorescence and white light images of probes at equimolar concentration. Well 1 to 5 is NIR1, NIR2, NIR3, NIR4, and indocyanine green, respectively. Note the bright fluorescence of the new dyes compared to ICG.
NIR fluorochromes as alternative reporters was tested. The fluorochromes were attached to a partially peglyated graft coplymer (PGC), which has a polylysine backbone (21). The probes were designed to have minimum fluorescence signal in their native states and become highly fluorescent after enzyme-mediated release of fluorochromes, resulting in signal amplification. To reduce the initial fluorescence signal, a high local concentration of fluorochromes was desired to have significant selfquenching. Since the lysine residues on PGC were only partially pegylated, free amino groups on the unmodified lysine side chain could be used for fluorochrome attachment. Nevertheless, additional free lysine residues were also needed for trypsin recognition. As a consequence, the number of fluorochromes per polymer had to be optimized in order to maximize the fluorescence increase after enzymatic cleavage.
For this purpose, PGC probes were labeled with different amounts of NIR2. Overall, seven conjugates with 0.2, 0.8, 1.4, 2.4, 4.3, 5.7, and 7.0 NIR2 residues per PGC molecule were prepared. The white bars in Figure 6 shows the fluorescent signal of the labeled polymers before trypsin treatment. As expected, there is an increase in the signal from 0.2 to 0.8 dyes/polymer, while at higher dye/polymer ratio, considerable self-quenching is observed. The black bars in Figure 6 show the fluorescence signals obtained after 3 h of tryptic cleavage. Maximum recovery was found for 4.3 NIR2 per PGC. At this ratio, the fluorescence signal increased 5-fold in 3 h and 9-fold in 24 h. Interestingly, recovery was lower when more NIR2 was attached to the backbone, presumably due to the less enzyme-accessible cleavage sites on the backbone. These studies demonstrate the importance of the proposed optimization studies which are required to maximize the dequenching effect upon enzymatic cleavage. The self-quenching phenomenon of NIRF cyanine dyes was not only shown by NIR2, as similar results were obtained when NIR1 was used in the preparation (data not shown). Extensive self-quenching was also reported with Cy5, Cy5.5, and Cy7 (22). The fluorescence signal was not enhanced by attaching multiple labels onto one molecule; on the contrary, it was significantly reduced due to serious self-quenching. We have applied this negative effect in a positive way to design several quenched probes as previously described and used them to imaging tumor-associated protease activity in vivo (3, 19, 20). In summary, we describe the synthesis and properties of a number of nonsymmetrical cyanine dyes. The dyes are characterized by high stability, high quantum yield, and water solubility. Likewise, the monoactivated hydroxysuccinimide esters of the fluorochromes were exceptionally stable, yet fully reactive with primary amines. We are currently testing the use of fluorochrome-tagged molecules for imaging of tumors and other diseases in vivo. It is anticipated that the developed fluorochromes have a range of biotech applications extending beyond imaging. ACKNOWLEDGMENT
This research was supported by NIH CA86355, CA88365, CA79443, CO97065, and NSF BES-0119382. LITERATURE CITED (1) Neri, D., Carnemolla, B., Nissim, A., Leprini, A., Querze, G., Balza, E. et al. (1997) Targeting by affinity-matured
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