Synthesis and Properties of Sulfhydryl-Reactive Near-Infrared Cyanine Fluorochromes for Fluorescence Imaging

Abstract

Near-infrared fluorochromes (NIRF) are useful compounds for diverse biotechnology applications and for in vivo biomedical imaging. Such NIRF must have high quantum yield, be biocompatible, and be conjugatable to a wide variety of proteins, peptides, and other affinity ligands. Here, we describe the synthesis of four new nonsymmetrical sulfhydryl-reactive cyanine NIRF with excellent optical and chemical properties. Each fluorochrome was designed to contain an iodoacetamido group that reacts specifically with sulfhydryl-containing molecules. The synthesized fluorochromes were used to label model peptides and sulfhydryl-containing biomolecules.

Introduction

Organic fluorochromes have become one of the most versatile reporter probes for many biotechnology and biomedical applications. Examples of their widespread use include gene chip technology, fluorescence-activated cells sorting (FACS), fluorescence microscopy, in situ hybridization, and, more recently, in vivo imaging [1]–[4]. Most applications utilize fluorochromes that fluoresce in the visible range because of their ease of use, ready detection, good quantum yield, stability, and well-developed chemistries. However, two biomedical needs have pushed the development of fluorochromes in the near-infrared (NIR) range: (a) the need to expand the “color” spectrum for multiwavelength detection of multiple targets, and (b) the need of reporters for medical in vivo imaging. One of the key strategies for imaging deeper tissues (i.e., more than a few millimeters) has been to use NIR light, because hemoglobin, water, and lipids, the major absorbers of visible and infrared light, respectively, have their lowest absorption coefficient in the NIR region around 650–900 nm. Imaging in the NIR region has also added the advantage of minimizing tissue auto fluorescence, which can further improve target to background ratios.

Ideal near-infrared fluorochromes (NIRF) for biomedical use must have high quantum yield, be biocompatible, and be conjugatable to a wide variety of proteins, peptides, and other affinity ligands. A number of fluorochromes in the 600–800 nm range have recently been described, including sulfobenzindocyanine fluorochromes [5]–[7] or nonsymmetrical cyanine fluorochromes [8]. Conjugation of these NIRF to biomolecules of interest is most commonly achieved through conversion of the NIRF into its hydroxysuccimide ester or isothiocyanates reactive with primary amines. Examples of such bioconjugates have included receptor-targeted fluorescent probes [6,7,9], enzyme-activatable beacons [10,11], and NIRF-tagged antibodies [12,13]. For many biological applications, however, it is preferable to attach NIRF to sulfhydryl groups, either in primary cysteines or to site-directed neo-sulfhydryl groups.

Sulfhydryl-reactive derivatives of visible light fluorochromes, for example, fluorescein, rhodamine, Texas Red, and pyrene, are available either as iodoacetamino or maleimide conjugates [3]. Recently, two groups have reported on the synthesis of sulfhydryl-reactive cyanine derivatives. Gruber et al. [14] described the synthesis of sulfhydryl-reactive fluorochrome starting from commercially available Cy5 fluorochrome in which maleimide or pyridyldithio were introduced to Cy5 through an ethylene diamine spacer as sulfhydryl-reactive end groups. More recently, Toutchkine et al. [15] demonstrated the preparation of Cy3 and Cy5 derivatives having cysteine-selective iodoacetamide end group. The goal of the current work was to introduce a novel nonsymmetrical, sulfhydryl-reactive cyanine fluorochromes with further red shifts and which contain a monoiodoacetamido moiety for labeling and imaging. The synthesis and characterization of these NIRF is described.

Materials and Methods

Chemicals

6-Amino-1,3-naphthalene disulfonic acid disodium salt, 1,4-butanesultone, 3-methyl-2-butanone, iodoethane, and 1,3,3-trimethyl-2-methyleneindoline were purchased from Aldrich (Milwaukee, WI). Glutaconaldehyde dianil hydrochloride and malconaldehyde dianil hydrochloride were purchased from TCI America (Portland, OR). All other reagents and solvents were purchased either from Aldrich or Fisher and were used as received.

Purification and Spectroscopic Analysis

Purification of fluorochromes 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 channel detector was set at 240 and 360 nm. Fluorochromes were collected, and the solvent was removed using a Speed-vac concentrator (Savant, Holbrook, NY). Absorbance spectra were measured on a U-3000 spectrophotometer (Hitachi, San Jose, CA). Fluorescence spectra were recorded using an F-4500 fluorophotometer (Hitachi). The proton NMR spectra were recorded in D₂O/CD₃CN in a 400-MHz FT-NMR spectrometer. MALDI-TOF mass spectrum was performed on a PerSeptive Voyager-DE Biospectrometry Workstation using α-cyano-4-hydroxycinnnamic acid as matrix.

2,3,3-Trimethyl-1-(4-sulfonatobutyl)-3H-benzindolinium-5,7-disulfonate 2b. Into a 250-mL round-bottomed flask were placed 2.2 g of 1,1,2-trimethyl-benzindolenium 1,3-disulfonate 1 [5], 1–4 mL of 1,4-butanesultone, and 20 mL of 1,2-dichlobenzene. The reaction mixture was heated under argon atmosphere at 125 °C for 24 hr. After being cooled to room temperature, the solvent was decanted and the solid was washed three times with acetone. The solid was filtered off and dried under vacuum to yield 1.82 g of crude product 2.

2-{(1E,3E)-4-[N-Acetyl-N-phenylamino]buta-1, 3-dienyl}-3,3-dimethyl-1-ethyl-3H-benzindolium-5, 7-disulfonate (intermediate 3) and 2-{(1E, 3E, 5E)-6-[N-acetyl-N-phenylamino]hexa-1,3,5-trienyl}-3,3-dimethyl-1-ethyl-3H-benzindolium-5,7-disulfonate (intermediate 4) were synthesized as previously published [8].

(2-{(1E,3E)-4-[N-Acetyl-N-phenylamino]buta-1,3-dienyl}-3,3-dimethyl-1-(4-sulfonatobutyl)-3H-benzindolium-5,7-disulfonate (Intermediate 5). In a 200-mL round-bottomed flask were placed 0.90 g of 2b, 0.49 g of malonaldehyde dianil hydrochloride, 10 mL of acetic anhydride, and 4 mL of acetic acid. The mixture was heated at 120 °C for 3 hr, then it was precipitated from ethyl acetate upon cooling. After filtration, the solid was washed twice with ethyl acetate and dried under vacuum to yield 1.08 g of crude intermediate 5. UV (H₂O): 485 nm.

(2-{(1E,3E,5E)-4-[N-Acetyl-N-phenylamino]hexa-1,3,5-trienyl}-3,3-dimethyl-1-(4-sulfonatobutyl)-3H-benzindolium-5,7-disulfonate (Intermediate 6). In a 200-mL round-bottomed flask were placed 0.98 g of 2b, 0.56 g of glutaconaldehyde dianil hydrochloride, 10 mL of acetic anhydride, and 4 mL of acetic acid. The mixture was heated at 125 °C for 3 hr. The mixture was then precipitated from ethyl acetate upon cooling. After filtration, the solid was washed twice with ethyl acetate and dried under vacuum to yield 1.12 g of crude intermediate 6. UV (H₂O): 495 nm.

2-{(1E,3E,5Z)-5-(5-Chloroacetylamino)-1,3,3-trimethyl-1,3-dihydro-2H-indol-2-ylidene)penta-1,3-dienyl]-1-ethyl-3H-indolium-5,7-disulfonate (SNIR1-Cl). A mixture of 121 mg of intermediate 3, 49 mg of 5-chloroacetamidomethyl-1,3,3-trimethyl-2-methyleneindoline 7 [16], 5 mL of acetic anhydride, 2 mL of glacial acid, and 110 mg of potassium acetate were stirred and heated at 120 °C under argon atmosphere for 20 min. After cooling to room temperature, the mixture was poured into 80 mL of ethyl acetate. The precipitate was collected by centrifugation and dried to result in 100 mg of crude SNIR1-Cl. The crude product was then purified by reversed phase HPLC with a yield of 14% of pure product (based on the crude product). ¹H-NMR (D₂O/CD₃CN, 2:1): 8 1.24 (3H, s), 1.43 (6H, s), 1.71 (6H, s), 3.45 (3H, broad s), 3–91 (2H, s), 4.03 (2H, broad q), 5-94-6.03 (2H, broad m), 6.31 (1H, broad m), 7.11 (1H, d), 7.44 (1H, dd), 7.49 (1H, s), 7.75–7.95 (2H, m), 8.21 (1H, s), 8.55 (1H, s), 8.79 (1H, d).

2-{(1E,3E,5E,7Z)-7-(5-Chloroacetylamino)-1,3,3-trimethyl-1,3-dihydro-2H-indol-2-ylidene)hepta-1,3,5-trienyl]-1-ethyl-3H-indolium-5,7-disulfonate (SNIR2-Cl). A mixture of 170 mg of intermediate 4, 36 mg of intermediate 7, 5 mL of acetic anhydride, 2.5 mL of glacial acid, and 140 mg of potassium acetate were stirred and heated at 115 °C under argon atmosphere for 18 min. After cooling to room temperature, the mixture was poured into 80 mL of ethyl acetate. The precipitate was collected by centrifugation and dried to result in 120 mg of crude SNIR2-Cl. The crude product was further purified by reverse phase HPLC to give 7% (based on the crude product) of pure product. ¹H-NMR (D₂O/CD₃CN, 1:1): 51.31(3H, t),1.59(6H, 6),1.88(6H, s), 3.47 (3H, s), 4.11 (2H, broad q), 4.16(2H, s), 6.13–6.19 (2H, m), 6.43–6.49 (2H, broad), 7.17 (1H, d), 7.47 (2H, d), 7.65 (2H, d), 7.77–7.90 (2H, m), 8.2 (1H, s), 8.60 (1H, s), 8.82 (1H, d).

Figure 1.

Synthetic scheme of the sulfhydryl-reactive NIR fluorochromes.

2-{(1E,3E,5Z)-5-(5-Chloroacetylamino)-1,3,3-trimethyl-1,3-dihydro-2H-indol-2-ylidene)penta-1,3-dienyl]-1-(4-sulfonatobutyl)-3H-indolium-5,7-disulfonate (SNIR3-Cl). A mixture of 150 mg of intermediate 5, 30 mg of intermediate 7, 5 mL of acetic anhydride, 2 mL of glacial acid, and 100 mg of potassium acetate were stirred and heated at 125 °C under argon atmosphere for 25 min. After cooling to room temperature, the mixture was poured into 80 mL of ethyl acetate. The precipitate was collected by centrifugation and dried to result in 120 mg of crude SNIR3-Cl. The crude product was then purified by reversed phase HPLC to yield 9% of pure product (based on the crude product). ¹H-NMR (D₂O/CD₃CN, 2:1): δ 1.84 (6H, s), 2.08 (6H, s), 2.10 (4H, broad m), 3.11 (3H, broad t), 3.78 (2H, broad s), 4.27 (2H, s), 4.32 (2H, broad), 6.36 (1H, broad d), 6.45 (1H, broad d), 6.64–6.71 (1H, broad m), 7.46 (1H, d), 7.68 (1H, dd), 7.84 (1H, s), 7.96 (1H, d), 8.19–8.35 (2H, broad m), 8.47 (1H, s), 8.86 (1H, s), 9.05 (1H, d).

2-{(1E,3E,5E,7Z)-7-(5-Chloroacetylamino)-1,3,3-trimethyl-1,3-dihydro-2H-indol-2-ylidene)hepta-1,3,5-trienyl]-1-(4-sulfonatobutyl)-3H-indolium-5,7-disulfonate (SNIR4-Cl). A mixture of 160 mg of intermediate 6, 34 mg of intermediate 7, 5 mL of acetic anhydride, 2.5 mL of glacial acid and 108 mg of potassium acetate were stirred and heated at 125 °C under argon atmosphere for 25 min. After cooling to room temperature, the mixture was poured into 80 mL of ethyl acetate. The precipitate was collected by centrifugation and dried to result in 110 mg of crude SNIR4-Cl. The crude product was then purified by reversed phase HPLC to yield 6% of pure product (based on the crude product). ¹H-NMR (D₂O/CD₃CN, 2:1): 8 1.80 (6H, s), 2.04 (6H, s), 2.09 (4H, broad m), 3.10 (3H, broad t), 3.78 (2H, broad s), 4.22 (2H, broad) 4.27 (2H, s), 6.40–6.55 (1H, broad d), 6.55–6.75 (2H, broad), 7.46 (1H, broad), 7.67 (1H, dd), 7.75–7.84 (1H, broad), 7.85 (1H, s), 7.90 (1H, d), 7.92–8.10 (2H, broad m), 8.44 (1H, s), 8.83 (1H, s), 9.02 (1H, d).

Halo-Exchange Reaction for Cyanine Fluorochromes

In a typical experiment, 10 mg of 5-chloroacetylamino-fluorochrome, 20 mg of sodium iodide, and 5 mL of methanol were placed in a small round-bottomed flask under argon atmosphere. The mixture was heated to reflux for 2.5 hr. After reaction, methanol was evaporated to dryness. The HPLC result indicated that more than 98% of chloroacetyl moiety was converted to the more reactive iodoacetyl moiety.

Figure 2.

Excitation and emission spectra of SNIR1-Cl and SNIR2-Cl.

Determination of the extinction coefficients and fluorescence quantum yield. All NIR fluorochromes were purified by preparative HPLC twice as described above. The K⁺ ions were replaced with H⁺ by ion-exchange chromatography in deionized water/CH₃CN (Dowex-50 cation exchange resin, 8% cross-link, 100–200 mesh). Approximately 10 mg of a given compound was dissolved in 100 mL of deionized water/CH₃CN. The absorbance was measured individually in three dilutions of the stock solution in deionized water/CH₃CN. The fluorescence emission maximal and intensities of the fluorochromes were obtained using dilute solutions in water and exciting at both the main absorption peak as well as the short-wavelength shoulder of the main absorption peak. In cases of SNIR1 and SNIR3, the quantum yields were calculated relative to a standard solution of Cy5-5 (Amersham-Pharmacia, Piscataway, NJ) with a quantum yield of 0.29, whereas in cases of SNIR2 and SNIR4, the calculations were performed relative to a standard solution of Cy7 (Amersham-Pharmacia) with a quantum yield of 0.28.

Labeling a Model Cysteine-Containing Peptide

The peptide, GRRGGGGYC, synthesized by standard solid phase synthesis (3.0 mg) was dissolved in 0.5 mL of 0.1 M aqueous NaHCO₃. To this solution was added 2.0 mg of SNIR1 dissolved in 0.5 mL of EtOH. The mixture was stirred at RT overnight. After reaction, the fluorochrome-peptide conjugate was purified by reversed phase HPLC and analyzed by MALDI-TOF mass spectrum, M + 1: expected = 1545, found = 1548.

Results and Discussion

Synthesis of Fluorochromes

Both maleimide- and iodoacetamide-converted visible light fluorochromes are available for protein labeling. In order to have more stable NIRF, we choose to employ the iodoaceamide group as the sulfhydryl-reactive functionality, because it has certain advantage over maleimide functionality [17]. The synthetic scheme for iodoacetamido-containing cyanine fluorochromes is summarized in Figure 1. Reaction of 1 with iodoethane resulted in compound 2a, which was then treated with malonaldehyde dianil hydrochloride or glutaconaldehyde dianil hydrochloride to yield intermediates 3 and 4, respectively. Intermediates 5 and 6 were prepared similarly using 1.4-butanesultone. The nonsymmetrical, chloroacetamino-containing cyanines SNIR1-Cl to SNIR4-Cl were synthesized by the reaction of intermediates 3–6 with 7, respectively. Purification of cyanine fluorochromes SNIR1-Cl to SNIR4-Cl was performed by reversed phase semipreparative HPLC, and the final products were approximately 98% pure as determined by reversed phase HPLC. The representative excitation and fluorescent emission spectra of SNIR1-Cl and SNIR2-Cl were given in Figure 2.

Figure 3.

Halo-exchange reaction of SNIR1-Cl.

Table 1.

Optical Properties of the Synthesized Sulfhydryl-Reactive Fluorochromes

Compound	Solvent	λmax, abs (nm)	λmax, em (n>m)	Stokes Shift (nm)	ɛ (L mol−1 cm−1)	QY
SNIR1	H2O/CH3CN (1:1)	666	695	29	218,000	0.24
SNIR2	H2O/CH3CN (1:1)	763	803	40	224,000	0.11
SNIR3	H2O/CH3CN (2:1)	667	697	30	245,000	0.24
SNIR4	H2O/CH3CN (2:1)	764	803	39	238,000	0.13

Halo-Exchange Reaction

Although chloroacetylamino can react with sulfhydryl group, the reaction is slow. The more sulfhydryl-reactive iodoacetylamino functionality was easily obtained by a halo-exchange reaction. The iodoacetamino-containing cyanine fluorochromes SNIR1–4 were obtained respectively by the reaction of SNIR1-Cl to SNIR4-Cl with sodium iodide in methanol under reflux condition (Figure 3). The halo-exchange reaction went cleanly, and an almost quantitative yield (typically > 98%) was observed using reversed phase HPLC. The elution times for SNIR1-Cl and SNIR1 were 30.0 and 31.3 min, respectively. The optical property of SNIR1 is the same as that of SNIR1-Cl.

Spectral Properties of Fluorochromes

Unlike previously reported NIRF, the described cyanine fluorochromes SNIR1–4 bear two different heterocyclic rings. This design was deliberate for fine tuning of spectral properties and to facilitate self-quenching due to aggregation of large planar fluorochromes for the construction of enzyme-activatable sensors [10]. As can be seen in Figure 2 and Table 1, the difference in absorbance maximum between indodicarbocyanine and indotricarbocyanine fluorochromes was about 100 nm, similar as in other cyanine NIRF [18]. The indodicarbocyanine fluorochromes had a 30-nm Stokes shift of fluorescence emission maximum, while indotricarbocyanine fluorochromes had a 40-nm Stokes shift in 2:1 water/acetonitrile. The SNIR compounds were stable, had high molar extinction coefficient around 200,000–250,000, and quantum yield ranged from 0.11 to 0.24.

Labeling of Cysteine-Containing Biomolecules with Fluorochromes

The coupling of SNIR fluorochrome with a sulfhydryl group was first tested by reacting SNIR1 with cysteine at pH 8.2. The reaction resulted in a single product as determined by HPLC analysis (data not shown), that is, a cysteine-modified SNIR. The absorbance spectrum of cysteine-fluorochrome conjugate was similar to that of the free fluorochrome, except for the slight peak broadening (Figure 4A). Excitation and fluorescence emission spectra of the conjugate and free SNIR remain the same (Ex = 666 nm; Em = 695 nm).

Figure 4.

(A) Absorbance of SNIR1 and SNIR1-cysteine conjugate; (B) HPLC traces of peptide, GRRGGGGYC, and SNIR1-peptide conjugate; and (C) fluorescence excitation and emission of SNIR1-peptide conjugate.

In subsequent experiments, we used the sulfhydryl-reactive SNIR to label a cysteine containing peptide (GRRGGGGYC) using similar reaction conditions as for the cysteine conjugation. The elution time for the peptide conjugate was 28.8 min, while that of SNIR1 was 31.3 min (Figure 4B). The structure of the SNIR1-peptide conjugate was confirmed by MALDI-TOF mass spectrum. The spectral properties of the peptide-NIRF conjugate were almost identical to those of the free fluorochromes (Figure 4C). These results indicated that iodoacetamido SNIR had high selectivity for sulfhydryl groups and is useful for the specific labeling of sulfhydryl-containing proteins, peptides, and, potentially, other biomolecules.

Conclusion

New nonsymmetrical NIR fluorescent cyanine fluorochromes containing an iodoacetamido moiety have been synthesized and characterized. SNIR compounds are ideally suited for labeling of cysteine-containing biomolecules. We anticipate that the described compounds will be particularly useful for labeling a wide range of sulfhydryl-containing peptides, proteins, and, potentially, other biomolecules for their use as contrast agents in molecular optical imaging.