Novel Water-Soluble Near-Infrared Cyanine Dyes: Synthesis, Spectral

Bioconjugate Chem. 2007, 18, 1303−1317

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Novel Water-Soluble Near-Infrared Cyanine Dyes: Synthesis, Spectral Properties, and Use in the Preparation of Internally Quenched Fluorescent Probes Ce´dric Bouteiller,†,‡,§ Guillaume Clave´,†,‡,§ Aude Bernardin,†,‡ Bertrand Chipon,†,‡ Marc Massonneau,‡ Pierre-Yves Renard,*,† and Anthony Romieu*,† IRCOF, Equipe de Chimie Bio-Organique, UMR 6014 CNRS, INSA de Rouen et Universite´ de Rouen, 1, rue Lucien Tesnie`res, 76131 Mont-Saint-Aignan Cedex, France, and QUIDD, Technopoˆle du Madrillet, 50, rue Ettore Bugatti, 76800 Saint-Etienne du Rouvray, France. Received January 23, 2007; Revised Manuscript Received May 13, 2007

In this paper, we describe the synthesis and the photophysical properties of two novel near-infrared (NIR) cyanine dyes (NIR5.5-2 and NIR7.0-2) which are water soluble potential substitutes of the commercially available Cy 5.5 and Cy 7.0 fluorescent labels respectively. For each one of these cyanine dyes, the synthetic strategy relies on the postsynthetic derivatization of a cyanine precursor in order to introduce the key functionalities required for bioconjugation of these NIR fluorophores. For NIR5.5-2, a reactive amino group was acylated with an original trisulfonated linker for water solubility. For NIR7.0-2, a vinylic chlorine atom was derivatized through a SRN1 reaction for the introduction of a monoreactive carboxyl group for labeling purposes. Unexpectedly, when these two fluorophores were closely associated within a peptidic architecture, mutual fluorescence quenching between NIR5.5-2 and NIR7.0-2 was observed both at 705 (NIR5.5-2) and 798 nm (NIR7.0-2). On the basis of this property, a novel internally quenched caspase-3-sensitive NIR fluorescent probe was prepared.

INTRODUCTION During the past decade, intensive research efforts have been devoted to the design and synthesis of novel water-soluble fluorescent organic dyes for life science applications (1). Indeed, despite the multitude of commercially available fluorophores, new fluorophoric systems, especially those with spectral properties in the near-infrared (NIR) region, are hotly sought for emerging and more challenging biotechnology and biomedical applications such as genetic analysis, DNA sequencing, in ViVo imaging, and proteomics (2, 3). Thus, numerous longwavelength fluorophores belonging to the BODIPY (4), rhodamine (5, 6), oxazine (7), or cyanine dyes (8-10) have been recently reported. Because of their large molar extinction coefficients, moderate-to-high fluorescence quantum yields, and a broad wavelength tunability, cyanine dyes have received considerable attention and can be considered as the main class of NIR fluorescent probes for biological applications at present (11, 12). Cy 5.5 1 and Cy 7.0 2 (Figure 1) are two of these NIR fluorescent cyanine dyes, widely used for such bioanalytical purposes and commercially available from GE Healthcare (formerly Amersham-Biosciences). Despite its good optical properties in the NIR range, the sulfobenzoindocyanine 1 is prepared through a low-yield (15% as described) and poorly reproducible synthetic route using 6-amino-1,3-naphthalenedisulfonic acid as starting material (13). Major synthetic difficulties are related to the poor solubility of this latter amino-naphthalene derivative in organic media. This synthetic bottleneck and the drastic reaction conditions required explain why only two bioconjugatable derivatives (i.e., amine-reactive succinimidyl ester and thiol-reactive maleimide) of this fluorescent marker * Author to whom correspondence should be addressed. E-mail: [email protected] or [email protected]. Phone: +33-2-35-52-24-14 (or 24-15). Fax: +33-2-35-52-29-59. † IRCOF, Equipe de Chimie Bio-Organique. ‡ QUIDD. § These authors contributed equally to this work.

are available at present. As far as the heptamethine cyanine dye 2 (14) is concerned, its poor chemical stability restrains its use as fluorescent marker in numerous site-specific biolabeling strategies, especially those requiring additional purification steps by RP-HPLC. To overcome this limitation, numerous studies have shown that incorporating a rigid cyclohexenyl (or cyclopentenyl) ring in the polymethine chain increases the stability of the resulting Cy 7.0 analogue (15-17). Furthermore, the central cyclohexenyl (or cyclopentenyl) group offers a reactive site for labeling biomolecules Via substitution of its mesochlorine atom by nucleophiles (metal alcoholates, amines, and thiols) bearing an additional functional group (amine or carboxylic acid) through a SRN1 mechanism. Thus, a variety of water-soluble heptamethine cyanine dyes containing aryl-ether, aryl-thioether and alkyl-ether linkages have been prepared using this route (18-21). However, the susceptibility of most of these dyes to form nonfluorescent aggregates (18) limits their use as probes in biological analysis. With the goal in mind to develop new bright, monofunctional, water-soluble NIR fluorophores suitable for bioconjugation and easily accessible through high-yielding synthetic routes, we have explored an original approach to prepare Cy 5.5 and Cy 7.0 analogues based on the postsynthetic derivatization of cyanine precursors (“convertible cyanine dyes”) readily available through standard cyanine synthesis protocols. This synthetic methodology has been already applied to the chemical derivatization of heptamethine cyanine dyes but never extended to cyanine dyes bearing a shorter polyene chain. We thought that the introduction of the key functionalities of a bioconjugatable NIR fluorophore (for instance hydrophilic moieties for water solubility or functional reactive groups for covalent attachment to biomolecules) in the last step of the synthetic process should improve its synthesis because the time-consuming handling and RPHPLC purification of highly polar materials will be reduced only to the final dye product. Furthermore, both the level of byproducts resulting from interference of the functional reactive groups with the reagents currently used to achieve the condensa-

10.1021/bc0700281 CCC: $37.00 © 2007 American Chemical Society Published on Web 06/21/2007

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Figure 1. Structures of cyanine dyes Cy 5.5 and Cy 7.0 and their novel analogues studied in this work. An unsymmetrical derivative of cyanine dye Cy 5.5 1 bearing the ethyl and carboxypentyl substituents is also commercially available, but its synthesis is not described in the literature.

tion reaction leading to the cyanine dye, and the limitation associated with drastic reaction conditions on those fragile polyconjugated scaffolds, should be minimized and consequently the overall yield of the synthesis significantly improved. We describe here the synthesis and properties of two such novel NIR cyanine fluorophores NIR5.5-2 and NIR7.0-2 which are analogues of Cy 5.5 1 and Cy 7.0 2, respectively. These two dyes display absorption and emission maxima in the NIR region, large extinction coefficients, excellent solubility, and no tendency to self-aggregate in aqueous solutions. Their conversion into thiol- and amine-reactive derivatives is also described. The utility of these latter fluorescent labeling reagents is illustrated by the preparation of an internally quenched fluorescent probe of caspase-3 protease.

EXPERIMENTAL PROCEDURES Chemicals and Reagents. Column chromatography was performed on silica gel (40-63 µm) from SdS. Reversed-phase column flash-chromatography was performed on octadecylfunctionalized silica gel (mean pore size 60 Å) from Aldrich or Whatmann. TLC was carried out on Merck DC Kieselgel 60 F-254 aluminum sheets and visualized by employing a short wavelength UV lamp (i.e., λ ) 254 nm). DMF was dried by distillation over BaO. DIEA, pyridine, and triethylamine were distilled from CaH2 and stored over BaO. Chloro-substituted heptamethine sulfocyanine dye 12 was prepared from 2,3,3trimethyl-3H-indole by using the multistep synthetic procedure developed by Strekowski et al. (20). Cyanine-based amino acid

6 was prepared in five steps from 1,1,2-trimethyl-1H-benz[e]indole by using the convergent synthetic scheme recently reported by us (22, 23). Malonaldehyde dianilido hydrochloride, sodium 2-bromoethanesulfonate, N-succinimidyl[4-iodoacetyl]aminobenzoate (SIAB), sulfocyanine dye Cy 5.0, 1,1,2-trimethyl-1H-benz[e]indole and its derivatives N-(3-sulfonatopropyl)1,1,2-trimethylbenzindolinium, inner salt 3, and N-(γ-carboxypentyl)-1,1,2-trimethylbenzindolinium, bromine salt 4, were prepared by using literature procedures (14, 24-29). Hexapeptide Ac-Cys(StBu)-Asp-Glu-Val-Asp-Lys-NH2 16 was synthesized on an automated peptide synthesizer (ABI 433A, Applied Biosystems) using the stepwise solid-phase synthesis method and Fmoc amino acids, as previously described (30). Indocyanine green (ICG or IR-125, laser grade) was purchased from Acros. Recombinant human caspase-3 enzyme (5.52 U/mg) and Bovine Serum Albumin (BSA, g98%) were purchased from Sigma. The HPLC grade solvents (CH3CN and MeOH) were obtained from Acros. Aqueous buffers for HPLC were prepared using water purified with a Milli-Q system. Triethylammonium acetate (1.0 M) (TEAA) and triethylammonium bicarbonate (1.0 M) (TEAB) buffers were prepared from distilled triethylamine and glacial acetic acid or CO2 gas. Instruments. 1H and 13C NMR spectra were recorded on a Bruker DPX 300 (Bruker, Wissembourg, France). Chemical shifts are expressed in parts per million (ppm) from D2O (δH ) 4.79) or DMSO-d6 (δH ) 2.54, δC ) 40.45) (31). J values are expressed in Hz. Analytical HPLC was performed on a Thermo Electron Surveyor instrument equipped with a PDA

Internally Quenched Fluorescent Probes

detector. Semipreparative HPLC was performed on a Finnigan SpectraSYSTEM liquid chromatography system equipped with a UV-Visible 2000 detector. UV-visible spectra were obtained on a Varian Cary 50 scan spectrophotometer. Fluorescence spectroscopic studies were performed either in a semi-micro fluorescence cell (Hellma, 104F-QS, 10 × 4 mm, 1400 µL) or in an ultra-micro fluorescence cell (Hellma, 105.51-QS, 3 × 3 mm, 45 µL) with a Varian Cary Eclipse spectrophotometer. Mass spectra were obtained with a Finnigan LCQ Advantage MAX (ion trap) apparatus equipped with an electrospray source. The purified peptides were characterized by MALDI-TOF mass spectrometry on a Voyager DE PRO in the reflector mode with CHCA as a matrix. HPLC Separations. Several chromatographic systems were used for the analytical experiments and the purification steps. Each one of these systems was optimized in order to improve separation conditions. System A: RP-HPLC (Thermo Hypersil GOLD C18 column, 5 µm, 4.6 × 150 mm) with CH3CN and 0.1% aq trifluoroacetic acid (aq TFA, 0.1%, v/v, pH 2.0) as eluents, at a flow rate of 1 mL/min, with the following gradients: A1: [80% TFA (5 min), linear gradient from 20 to 40% (5 min) and 40 to 100% (50 min) of CH3CN]. Triple UV-visible detection was achieved at 260, 650, and 675 nm. A2: [80% TFA (5 min), linear gradient from 20 to 30% (5 min) and 10 to 90% (50 min) of CH3CN]. Triple UV-visible detection was achieved at 260, 650, and 675 nm. A3: [80% TFA (5 min), linear gradient from 20 to 60% (30 min) of CH3CN]. Dual UV-visible detection was achieved at 260 and 750 nm. A4: [100% TFA (5 min), linear gradient from 0 to 60% (40 min) of CH3CN]. Dual UV-visible detection was achieved at 260 and 750 nm. System B: RP-HPLC (Thermo Hypersil GOLD C18 column, 5 µm, 4.6 × 150 mm) with CH3CN and aq triethylammonium acetate buffer (TEAA, 0.1 M, pH 7.0) as eluents [100% TEAA (10 min), linear gradient from 0 to 60% (30 min) of CH3CN] at a flow rate of 1 mL/min. Dual UV detection was achieved at 260 and 330 nm. System C: RP-HPLC (Varian Kromasil C18 column, 10 µm, 21.2 × 250 mm) with CH3CN and aq triethylammonium bicarbonate buffer (TEAB, 50 mM, pH 7.5) as eluents [100% TEAB (20 min), linear gradient from 0 to 30% (60 min) of CH3CN] at a flow rate of 20 mL/min. Dual UV detection was achieved at 255 and 335 nm. System D: RP-HPLC (Thermo Hypersil GOLD C18 column, 5 µm, 10 × 250 mm) with CH3CN and 0.1% aq trifluoroacetic acid (aq TFA, 0.1%, v/v, pH 2.0) as eluents, at a flow rate of 5 mL/min, with the following gradients: D1: [90% TFA (5 min), linear gradient from 10 to 30% (10 min) and 30 to 70% (55 min) of CH3CN]. UV detection was achieved at 260 nm. D2: [90% TFA (5 min), linear gradient from 10 to 40% (15 min) and 40 to 70% (40 min) of CH3CN]. UV detection was achieved at 260 nm. System E: RP-HPLC (Varian Kromasil C18 column, 10 µm, 21.2 × 250 mm) with CH3CN and 0.1% aq TFA as eluents [95% TFA (5 min), linear gradient from 5 to 25% (5 min) and 5 to 75% (70 min) of CH3CN] at a flow rate of 20 mL/min. UV-visible detection was achieved at 780 nm. System F: RP-HPLC (Waters XTerra MS C18 column, 5 µm, 7.8 × 100 mm) with CH3CN and 0.1% aq TFA as eluents, at a flow rate of 2.5 mL/min, with the following gradients: F1: [100% TFA (5 min), linear gradient from 0 to 36% (18 min) and 36 to 60% (24 min) of CH3CN]. UV detection was achieved at 260 nm.

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F2: [100% TFA (5 min), linear gradient from 0 to 30% (15 min) and 30 to 60% (60 min) of CH3CN]. Triple UV-visible detection was achieved at 260, 675 and 778 nm. F3: [100% TFA (5 min), linear gradient from 0 to 30% (15 min), 30 to 35% (10 min) and 35 to 60% (125 min) of CH3CN]. Dual UV-visible detection was achieved at 675 and 778 nm. Preparation of NIR5.5-1. (a) Hemicyanine Intermediate 5. A solution of N-(3-sulfonatopropyl)-1,1,2-trimethylbenzindolinium, inner salt 3 (1 g, 3.0 mmol) and malonaldehyde dianilido hydrochloride (0.88 g, 3.42 mmol) in a mixture of acetic acid (5 mL) and acetic anhydride (5 mL) was heated under reflux. The reaction was checked for completion by UV-visible spectroscopy (the absorption maximum of 3 at λ ) 380 nm has declined whereas the absorption maximum of hemicyanine intermediate 5 has appeared at λ ) 455 nm). After 90 min, the reaction mixture was evaporated under reduced pressure without heating above 40 °C (to avoid the formation of some symmetrical dye). The resulting residue was dissolved in water and lyophilized. The crude hemicyanine intermediate was used without further purification in the next reaction. (b) Preparation of Cyanine Dye. A solution of crude hemicyanine intermediate 5 (1.44 g, 2.77 mmol) and N-(γcarboxypentyl)-1,1,2-trimethylbenzindolinium, bromine salt 4 (1.11 g, 2.77 mmol) in a mixture of acetic anhydride (5 mL) and dry pyridine (5 mL) was heated under reflux for 30 min. The reaction was checked for completion by UV-visible spectroscopy (the absorption maximum of 5 at λ ) 455 nm has disappeared whereas the absorption maximum of cyanine dye NIR5.5-1 has appeared at λ ) 679 nm). The reaction mixture was evaporated under reduced pressure, dissolved in water, and lyophilized to give a blue solid (2.86 g). One gram of this crude product was purified by chromatography on a silica gel column (40 g) with a step gradient of MeOH (0-5%) in dichloromethane as the mobile phase. After drying under vacuum, NIR5.5-1 was obtained as a blue solid (0.67 g, 0.97 mmol, yield 80%). Rf ) 0.35 (CH2Cl2/MeOH 9/1, v/v). 1H NMR (300 MHz, DMSO-d6): δ 8.52 (t, 2H, J ) 13.2 Hz), 8.29 (d, 2H, J ) 8.7 Hz), 8.11 (m, 4H), 7.86 (d, 1H, J ) 9.0 Hz), 7.77 (d, 1H, J ) 9.0 Hz), 7.71 (t, 2H, J ) 7.5 Hz), 7.54 (t, 2H, J ) 7.5 Hz), 7.32 (s, 1H, NH), 7.15 (s, 1H, NH), 6.98 (s, 1H, NH), 6.65 (t, 1H, J ) 12.4 Hz), 6.55 (d, 1H, J ) 13.9 Hz), 6.41 (d, 1H, J ) 13.6 Hz), 4.43 (m, 2H), 4.27 (m, 2H), 2.67 (t, 2H, J ) 6.4 Hz), 2.25 (t, 2H, J ) 6.8 Hz), 2.11-2.05 (m, 2H), 2.01 (s, 6H, Me), 1.99 (s, 6H, Me), 1.81-1.46 (m, 6H). HPLC (system A1): tR ) 20.5 min, purity >95%. UV-visible (DMSO, 25 °C): λmax ) 688 nm (147 000 M-1 cm-1). UV-visible (EtOH, 25 °C): λmax ) 681 nm (156 000 M-1 cm-1). ΦF (DMSO, 25 °C): 0.27. ΦF (EtOH, 25 °C): 0.17. MS (ESI, positive mode): m/z 691.40 [M + H]+, 713.47 [M + Na]+, 1381.40 [2M + H]+, 1404.53 [2M + Na]+, calcd exact mass for C42H46N2O5S 690.91. Trisulfonated Linker (9). A mixture of 3,5-diaminobenzoic acid (0.25 g, 1.6 mmol), sodium 2-bromoethanesulfonate (1.39 g, 6.6 mmol), KI (0.29 g, 1.8 mmol), and NaOH (64 mg, 1.6 mmol) in deionized water (3 mL) was heated under reflux for 4 days. After 24 and 48 h, further amounts of sodium bromoethanesulfonate (2 × 1.34 g, 2 × 6.4 mmol) and NaOH (2 × 0.64 mL of an aq 5 N solution, 2 × 3.2 mmol) were added. The reaction was checked for completion by RP-HPLC (system B) and purified by RP-HPLC (system C, 12 injections). The product-containing fractions were twice lyophilized (to remove excess of TEAB salts) to give 9 as a highly hygroscopic white powder (yield 36%). 1H NMR (300 MHz, D2O): δ 6.78 (s, 1H), 6.71 (s, 1H), 6.32 (s, 1H), 3.77 (m, 6H, N-CH2-CH2SO3-), 3.11 (m, 30H, TEA & NCH2CH2SO3-), 1.21 (t, 36H, TEA, J ) 7.2 Hz). 13C NMR (75.5 MHz, D2O): δ 175.1, 148.8, 147.7, 138.7, 104.9, 104.2, 100.8, 49.8 (2C), 48.3 (4C),


48.1 (4C), 46.9 (12C), 39.9 (2C), 8.5 (12C). HPLC (system B): tR ) 7.0 min. MS (ESI, negative mode): m/z 157.93 [M 3H]3-, 237.33 [M - 2H]2-, 475.20 [M - H]-, calcd for C13H20N2O11S3 (acid form) 476.50. Preparation of Trisulfonated Linker, Succinimidyl Ester (10). A mixture of trisulfonated linker 9 (50 mg, 56.7 µmol) and N-hydroxysuccinimide (7.2 mg, 62.0 µmol) was dissolved in dry DMF (100 µL). DCC (12.8 mg, 62.0 µmol) was added, and the resulting reaction mixture was stirred at room temperature for 20 h. After 3 h, further amounts of N-hydroxysuccinimide (5.8 mg, 50.8 µmol) and DCC (10.7 mg, 50.8 µmol) were added. The reaction was checked for completion by RP-HPLC (system B), and the resulting succinimidyl ester 10 was used without further purification. HPLC (system B): tR ) 17.8 and 18.6 min (broad peak, compared to tR ) 7.0 min for the free acid 9). Two peaks are observed due to a counterion effect with the triethylammonium cations of HPLC eluent. NIR5.5-2. A solution of cyanine-based amino acid 6 (39.5 mg, 45.5 µmol) in dry NMP (1 mL) was added to the solution of crude succinimidyl ester 10 (56.7 µmol) and DIEA (57.6 µL, 330 µmol). The resulting reaction mixture was protected from light and stirred at room temperature for 24 h. The reaction was checked for completion by RP-HPLC (system A1). Finally, the reaction mixture was quenched by dilution with aq TFA 0.1% and purified by RP-HPLC (system D1, 6 injections). Two products identified as the Cy 5.5 analogue NIR5.5-2 (tR ) 27.0-28.8 min) and its corresponding succinimidyl ester 11 (29.4-31.2 min) were isolated. The product-containing fractions were lyophilized to give NIR5.5-2 and 11 as blue amorphous powders (combined yield 70%). 1H NMR (300 MHz, DMSOd6 + TFA): δ 8.56 (m, 2H), 8.47 (t, 1H, J ) 12.8 Hz), 8.27 (m, 2H), 8.11 (m, 4H), 7.84-7.68 (m, 4H), 7.53 (m, 2H), 7.28 (bs, NH), 7.07 (bs, 1H, NH), 7.01 (bs, 1H, NH), 6.89 (bs, 1H, NH), 6.72 (t, 1H, J ) 12.5 Hz), 6.44 (dd, 2H), 4.28 (m, 4H), 3.75 (m, 6H, NCH2CH2SO3-), 2.83 (m, 2H), 2.72 (m, 6H, NCH2CH2SO3-), 2.24 (t, 2H, J ) 7.2 Hz), 1.99 (s, 12H), 1.831.01 (m, 10H). All spectroscopic characterizations are reported only for the free acid NIR5.5-2 except for the HPLC and MS analyses. HPLC (system A1): tR ) 17.8 min (NIR5.5-2), 18.9 min (11). UV-visible (PBS, pH 7.4, 25 °C): λmax ) 680 nm (197 000 M-1 cm-1). UV-visible (DMSO, 25 °C): λmax ) 688 nm (147 000 M-1 cm-1). ΦF (PBS, pH 7.4, 25 °C): 0.05. ΦF (DMSO, 25 °C): 0.05. MS (MALDI-TOF, positive mode, CHCA matrix): m/z 1098.89 [M + H]+. MS (ESI, negative mode): m/z 400.93 [M - 3H+TEA]3-, 601.87 [M 2H+TEA]2-, calcd for C56H66N5O12S3 (NIR5.5-2, acid form) 1097.37. MS (MALDI-TOF, positive mode, CHCA matrix): m/z 1195.93 [M + H]+, calcd for C60H70N6O14S3 (11, acid form) 1195.45. Preparation of Thiol-Reactive NIR5.5-2 Derivative (15). (a) Preparation of NIR5.5-2 Carboxylic Acid, Succinimidyl Ester 11. Free carboxylic acid dye NIR5.5-2 (10.5 mg, 9.6 µmol) was introduced into a Reacti-Vial and dissolved in 140 µL of dry NMP. A 20 µL amount of a solution of TSTU reagent in dry NMP (2.88 mg, 9.6 µmol) and 11.7 µL of DIEA (67.2 µmol) were added, and the resulting reaction mixture was protected from light and stirred at room temperature for 1 h. The reaction was checked for completion by RP-HPLC (system A1) and the resulting succinimidyl ester 11 was used without further purification. HPLC (system A1): tR ) 18.8 min (compared to tR ) 17.8 min for NIR5.5-2 carboxylic acid, Vide supra). (b) Synthesis of NIR5.5-2 Amine 14. Ethylenediamine dihydrochloride (150 mg, 1.152 mmol) was dissolved in a mixture of deionized water (0.9 mL) and DMF (10 mL). The crude reaction mixture containing the succinimidyl ester 11 and a 10% solution of DIEA in DMF (0.9 mL, 537 µmol) were sequentially

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added, and the resulting reaction mixture was protected from light and stirred at room temperature for 1 h. The reaction was checked for completion by RP-HPLC (system A1), and the mixture was evaporated to dryness. The resulting residue was purified by RP-HPLC (system D2, 2 injections). The productcontaining fractions were lyophilized to give the NIR5.5-2 amine 14 as a blue amorphous powder (8.0 mg, yield 73%). HPLC (system A1): tR ) 16.6 min, purity >95%. MS (MALDITOF, positive mode, CHCA matrix): m/z 1140.98 [M + H]+, calcd exact mass for C58H73N7O11S3 1140.46. (c) Preparation of NIR5.5-2 SIAB DeriVatiVe 15. NIR5.5-2 amine 14 (3.3 µmol) was dissolved in NMP (200 µL). NaHCO3 (2.7 mg, 32.1 µmol) and a solution of SIAB reagent (2.5 mg, 6.3 µmol) in NMP (200 µL) were sequentially added. Thereafter, 0.1 M NaHCO3 buffer (pH 8.5, 1 mL) was added, and the resulting reaction mixture was protected from light and stirred at room temperature for 2 h. The reaction was checked for completion by RP-HPLC (system A2). Finally, the reaction mixture was quenched by dilution with aq TFA 0.1% and purified by RP-HPLC (system D2, 2 injections). The productcontaining fractions were lyophilized to give the thiol-reactive NIR5.5-2 derivative 15 as a blue amorphous powder (3.8 mg, yield 80%). HPLC (system A2): tR ) 23.2 min (broad peak). ΦF (PBS, pH 7.4, 25 °C): 0.056. ΦF (DMSO, 25 °C): 0.145. MS (MALDI-TOF, positive mode, CHCA matrix): m/z 1299.96 [(M - I)+H]+, 1427.85 [M + H]+, calcd exact mass for C67H79IN8O13S3 1427.52. NIR7.0-2. A mixture of crude chloro-substituted heptamethine sulfocyanine dye 12 (220 mg, 0.31 mmol) and 3-mercaptopropionic acid (107 µL, 1.22 mmol) in dry DMF (15 mL) was heated under reflux for 40 min. The reaction was checked for completion by RP-HPLC (system A3), and the mixture was evaporated to dryness. The resulting residue was purified by RP-HPLC (system E, three injections, tR ) 24.5-26.5 min). The product-containing fractions were lyophilized to give NIR7.0-2 as a green amorphous powder (187 mg, 0.24 mmol, yield 80%). 1H NMR (300 MHz, DMSO-d6 + TFA-d1): δ 8.70 (d, 2H, J ) 13.9 Hz), 7.58 (d, 2H, J ) 7.5 Hz), 7.49-7.37 (m, 4H), 7.24 (t, 2H, J ) 7.5 Hz), 6.48 (d, 2H, J ) 13.9 Hz), 4.34 (bt, 4H, N+CH2CH2CH2SO3-), 2.99 (t, 2H, J ) 6.8 Hz), 2.662.49 (m, 10H), 2.05-1.80 (m, 6H), 1.68 (s, 12H). HPLC (system A3): tR ) 20.6 min, purity > 95%. HPLC (system A4): tR ) 34.6 min, purity >95%. UV-visible (PBS, pH 7.4, 25 °C): λmax ) 777 nm (120 000 M-1 cm-1). UV-visible (DMSO, 25 °C): λmax ) 802 nm (146 000 M-1 cm-1). UV-visible (EtOH, 25 °C): λmax ) 790 nm (171 000 M-1 cm-1). ΦF (PBS, pH 7.4, 25 °C): 0.025. ΦF (DMSO, 25 °C): 0.092. MS (ESI, positive mode): m/z 769.60 [M + H]+, MS (ESI, negative mode): m/z 767.80 [M - H]-, calcd exact mass for C39H48N2O8S3 769.02. Preparation of NIR7.0-2 Carboxylic Acid, Succinimidyl Ester (13). NIR7.0-2 carboxylic acid (4.12 mg, 5.36 µmol) was introduced into a Reacti-Vial and dissolved in 50 µL of dry NMP. A 50 µL amount of a solution of TSTU reagent in dry NMP (1.94 mg, 6.45 µmol) and 1.86 µL of DIEA (10.72 µmol) were added, and the resulting reaction mixture was protected from light and stirred at room temperature for 35 min. The reaction was checked for completion by RP-HPLC (system A4), and the resulting succinimidyl ester 13 was used without further purification. HPLC (system A4): tR ) 35.6 min (compared to tR ) 34.6 min for NIR7.0-2 carboxylic acid, Vide supra). Synthesis of Ac-Cys(StBu)-Asp-Glu-Val-Asp-Lys(NIR7.02)-NH2(17). A 5.66 mg amount of crude peptide Ac-Cys(StBu)Asp-Glu-Val-Asp-Lys-NH2 16 (5.95 µmol, weighed in a 1.0 mL Eppendorf tube) was dissolved in 100 µL of dry NMP, and 4.14 µL of DIEA (23.76 µmol) was added. After complete solubilization by vortexing, the resulting solution was added to the crude reaction mixture containing the succinimidyl ester of



Scheme 1

Scheme 2

NIR7.0-2. The resulting reaction mixture was protected from light and stirred at room temperature for 1 h. The reaction was checked for completion by RP-HPLC (system A4). Finally, the reaction mixture was quenched by dilution with 1 mL of aq TFA 0.1% and purified by RP-HPLC (system F1, 2 injections, tR ) 27.0-28.5 min). The product-containing fractions were lyophilized to give the peptide-NIR7.0-2 conjugate 17 as a green amorphous powder. Quantification was achieved by UV-visible measurements at λmax ) 777 nm of NIR7.0-2 by using the value 120 000 M-1 cm-1 (yield estimated after RP-HPLC purification: 60%). HPLC (system A4): tR ) 35.0 min, purity 91%. MS (MALDI-TOF, positive mode, CHCA matrix): m/z 796.78 [M + 2H]2+, 1590.03, [M + H]+, 1625.99 [M + K]+. MS (ESI, positive mode): m/z 825.00 [M + Na + K]2+, 1609.60 [M + Na]+, 1625.67 [M + K]+, calcd exact mass for C72H102N10O20S5 1587.99. Removal of tert-butylthio Group. Peptide Ac-Cys(StBu)Asp-Glu-Val-Asp-Lys(NIR7.0-2)-NH2 17 (5.7 mg, 3.6 µmol) was introduced into a Reacti-Vial and dissolved in 0.1 M NaHCO3 buffer (pH 8.5, 250 µL). A solution of DTT (8.03 mg in 500 µL, 52.1 µmol) in 0.1 M NaHCO3 buffer (pH 8.5) was added. The reaction mixture was protected from light and stirred at room temperature for 2 h. The reaction was checked for completion by RP-HPLC (system A4). Finally, the reaction mixture was quenched by dilution with aq TFA 0.1% (1 mL) and purified by RP-HPLC (system F1, 2 injections, tR ) 26.527.5 min). The product-containing fractions were lyophilized to give the free sulfhydryl containing peptide-NIR7.0-2 conjugate 18 as a green amorphous powder. Quantification was achieved by UV-visible measurements at λmax ) 777 nm of NIR7.0-2 by using the value 120 000 M-1 cm-1 (estimated quantitative yield after RP-HPLC purification). HPLC (system A4): tR ) 33.8 min, purity >95%. MS (ESI, positive mode):

m/z 752.13 [M + 2H]2+, 1502.67 [M + H]+; MS (ESI, negative mode): m/z 1500.60 [M - H]-, calcd exact mass for C68H94N10O20S4 1499.82. Synthesis of Ac-Cys(NIR5.5-2)-Asp-Glu-Val-Asp-Lys(NIR7.0-2)-NH2 (19). Peptide Ac-Cys-Asp-Glu-Val-Asp-Lys(NIR7.0-2)-NH2 18 (2.3 mg, 1.56 µmol) was introduced into a Reacti-Vial and dissolved in 0.1 M NaHCO3 buffer (pH 8.5, 200 µL). A 300 µL amount of a solution of iodoacetyl derivative 15 (0.75 mg, 0.52 µmol) in 0.1 M NaHCO3 buffer (pH 8.5) was added. The reaction mixture was protected from light and stirred at room temperature for 2 h. The reaction was checked for completion by RP-HPLC (system F2). Finally, the reaction mixture was quenched by dilution with aq TFA 0.1% (1 mL) and purified by RP-HPLC (system F3, 2 injections, tR ) 45.0-46 min). Because of the small difference in polarity between the peptide 19 and iodoacetamido derivative 15, it is essential to use a slow linear gradient of increasing acetonitrile (0.2%/min) in aq TFA to get peptide 19 in a pure form. The product-containing fractions were lyophilized to give the peptide Ac-Cys(NIR5.5-2)-Asp-Glu-Val-Asp-Lys(NIR7.02)-NH2 19 as a blue-green amorphous powder. Stock solution of this fluorogenic caspase-3 substrate was prepared in HPLC grade water and UV-visible quantification was achieved in DMSO at λmax of NIR7.0-2 by using the value 146 000 M-1 cm-1 (yield after RP-HPLC purification: 25%). HPLC (system F2): tR ) 39.9 min, purity >95%. UV-visible (water, 25 °C): λmax ) 649, 676 and 800 nm. UV-visible (DMSO, 25 °C): λmax ) 689 and 799 nm. MS (ESI, positive mode): m/z 1059.20 [M + 2H]2+; calcd exact mass for C99H130N16O28S4 2120.49. Optical Properties of Water-Soluble NIR Cyanine Dyes. The absorption spectra of NIR cyanine dyes NIR5.5-1, NIR5.52, and NIR7.0-2 were recorded (220-900 nm) in deionized


Bouteiller et al.

Scheme 3

water, DMSO, and PBS (concentration: 1.0-5.0 µM) at 25 °C. Emission spectra were recorded under the same conditions after excitation at a wavelength 10 nm below the corresponding λmax (excitation and emission slit ) 5 nm). Relative quantum yields were measured in DMSO and/or PBS at 25 °C by a relative method using either sulfocyanine dye Cy 5.0 (for NIR5.5-1 and NIR5.5-2, ΦF ) 0.20 in PBS (14)) or ICG (for NIR7.0-2, ΦF ) 0.106 in DMSO (32)) as a standard. Steady-State Fluorescence Quenching Experiments of NIR5.5-2 SIAB Derivative 15 by NIR7.0-2. A Stern-Volmer plot for NIR7.0-2 was obtained at 25 °C as follows. A solution of NIR5.5-2 SIAB derivative 15 in PBS (0.72 µM) was treated with an increased amount of NIR7.0-2 from 0 to 7.2 µM in PBS. The integrated fluorescence intensity F (area under the corrected emission curve in the range 670-900 nm after excitation at λ ) 660 nm) was measured, and the relative fluorescence intensity F0/F was plotted versus NIR7.0-2 concentration. A straight line was obtained in the concentration range. The following equation was used to derive the SternVolmer quenching constant:

F0/F ) 1 + Ksv[NIR7.0-2] where F0 is the integrated unquenched fluorescence intensity of 15 and Ksv is the static Stern-Volmer quenching constant (KsV ) 67500 M-1, R2 ) 0.983).

In Vitro Peptide Cleavage by Recombinant Human Caspase-3. A 1.0 µM solution of peptide 19 was prepared in 50 µL of caspase-3 buffer (100 mM NaCl, 40 mM HEPES, 10 mM DTT, 1 mM EDTA, 10% (w/v) sucrose and 0.1% (w/v) CHAPS, pH 7.4) and transferred into an ultra-micro fluorescence cell. A 2 µL amount of human recombinant caspase-3 (3.2 × 10-3 U) was added, and the resulting mixture was incubated at 37 °C. After excitation at 670 and 775 nm (excitation slit ) 5 nm), fluorescence emission at 705 and 805 nm (emission slit ) 5 nm) was monitored over time with measurements recorded every 5 s. Control experiment with BSA was performed under the same conditions by adding 2 µL of 1% (w/v) aq solution of this protein.

RESULTS AND DISCUSSION Synthesis of the Cy 5.5 Analogues. Sulfobenzoindocyanine dye Cy 5.5 1 is probably one of the most widely used NIR dyes as fluorescent reporter group in numerous molecular probes designed to facilitate the detection of molecular processes in solutions and living tissues (33-36). Indeed, this compound fulfils the most requirements expected for an ideal biomarker (i.e., narrow excitation and emission peaks in the NIR range, high quantum yield, high chemical and photostability, reasonable aqueous solubility, and a reactive carboxyl functional group for bioconjugation). However, as mentioned in Introduction, the multistep synthesis reported in 1996 by Mujumdar et al. (13) is not suitable for a convenient preparation of Cy 5.5 dye at a



Scheme 4

gram scale in good yields. We started the study related to the development of improved methods for the synthesis of cyanine dyes in our laboratory with the preparation of the Cy 5.5 analogue NIR5.5-1. Despite the presence of a single watersolubilizing sulfonated group on the cyanine core, we thought that the covalent association of NIR5.5-1 with some biomolecules could lead to NIR fluorescent molecular probes suitable for various biomedical applications. Although this dye is

commercially available (FEW Chemicals GmBH (www.few. de)), there is no detailed experimental procedure for its efficient synthesis. Thus, we have developed a practical high-yield synthesis of NIR5.5-1 from relatively inexpensive precursors (Scheme 1). The nitrogen atom of 2,3,3-trimethyl-3H-indole was alkylated with propanesultone in dry toluene under reflux for 24 h to afford compound 3 in 89% yield, which was treated with malonaldehyde dianilido hydrochloride in a 1:1 mixture


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Scheme 5

Table 1. Optical Properties of NIR Cyanine Dyes compound solvent 5.5a

Cy NIR5.5-1 NIR5.5-2 NIR5.5-2 Cy 7.0a NIR7.0-2 NIR7.0-2

PBS DMSO DMSO PBS PBS DMSO PBS

λmax,abs λmax,em Stokes shift (nm) (nm) (nm) (M-1 cm-1) 674 688 688 680 750 802 777

694 711 714 710 777 818 798

20 23 26 30 27 16 21

195000 147000 147000 197000 200000 146000 120 000

ΦF b 0.23 0.27 0.05 0.05 0.09 0.025

a Values reported by Mujumdar et al. (13, 14). b Determined at 25 °C by using either sulfocyanine dye Cy 5.0 (for NIR5.5-1 and NIR5.5-2, ΦF ) 0.20 in PBS (14)) or ICG (for NIR7.0-2, ΦF ) 0.106 in DMSO (32)) as a standard.

of acetic acid and acetic anhydride under reflux for 90 min to obtain hemicyanine intermediate 5 in a quantitative yield. Reaction of 5 with the carboxypentyl indole derivative 4 in a 1:1 mixture of acetic anhydride and pyridine under reflux for 30 min furnished NIR5.5-1 which was isolated in good yield (80%) by silica gel chromatography. All spectroscopic data, especially NMR and mass spectrometry, were in agreement with the structure assigned. However, preliminary experiments on bioconjugation of NIR5.5-1 to hydrophilic peptides have shown that the resulting fluorescent conjugates are poorly soluble in water and related aqueous buffers (see Supporting Information). We wished to improve the water solubility of this Cy 5.5 analogue by increasing the number of sulfonated groups on the backbone and to be able to modulate this solubility by varying the nature and the number of solubilizing moieties. Yet possibilities offered for such a further modification of NIR5.5-1 dye were limited, and modulation of the solubility required starting again the synthesis from the beginning. In an effort to develop an improved and flexible synthetic methodology enabling an easy access to numerous analogues of this NIR fluorophore, we have explored the postsynthetic derivatization

of the cyanine-based amino acid 6 (22, 23). This amino acid offers two amenable groups; thus, the derivatization could be undertaken either on the amino group (for instance through selective acylation reactions) or on the carboxylic acid, and derivatizations or bioconjugations could be led successively on the two reactive functions. Moreover, the introduction of watersolubilizing groups after completion of the cyanine synthesis seems to be an interesting approach because it avoids the use of a highly polar naphthalene derivative (for instance, as described for the synthesis of Cy 5.5 itself, 6-amino-1,3naphthalenedisulfonic acid) as starting material whose the restricted solubility in organic solvents made synthesis and purification of cyanine dye and its intermediates tricky. The backbone of this fluorescent amino acid 6 (two naphthalene rings and a pentamethine chain) has been chosen to get the same spectral properties as that of cyanine dye Cy 5.5. Furthermore, it is conveniently prepared using a five-step reaction (overall yield 42%) and could be purified by flash-chromatography on RP-C18 silica gel column at gram quantities. Therefore, we first tried to directly convert 6 into the sulfonated derivatives 7 and 8 by successive alkylations of the amino group with 1,3propanesultone or sodium 2-bromoethanesulfonate. As described in a preliminary report (22, 23), all our attempts to get 7 or 8 failed (Scheme 2). The amino acid 6 is either completely recovered or converted into decomposition products which were not characterized. Since the sulfonation of aniline derivatives is well described in the literature (37, 38), we have explored the synthesis of an original polysulfonated linker derived from cheap, commercially available 3,5-diaminobenzoic acid and its subsequent covalent attachment to the aminobutyl arm of 6 (Scheme 3). Reaction of 3,5-diaminobenzoic acid with a large excess of sodium 2-bromoethanesulfonate and KI in aq NaOH under reflux for 4 days gave the trisulfonated derivative 9 as the major compound. This highly polar linker was purified by semipreparative RP-HPLC. For this latter chromatographic



Scheme 6

purification, it was essential to use a volatile aqueous buffer such as triethylammonium bicarbonate buffer (TEAB, 50 mM, pH 7.5) as eluent both (1) to get a good separation between 9 and the other residual sulfonated species and (2) to avoid after lyophilization its contamination with salts which may further react with the reagents used for the conversion into active ester in the next steps. Thereafter, benzoic acid 9 was quantitatively converted into the corresponding N-hydroxysuccinimidyl ester 10 by treatment with DCC and N-hydroxysuccinimide (NHS) in dry DMF. This reaction was found to be very slow (20 h) and required the use of an excess of reagents (2 equiv) to go to completion. Finally, acylation of amino acid 6 with this crude active ester 10 in near stoichiometric amounts in dry NMP in the presence of DIEA (7 equiv) gave a 1:1 mixture of dye NIR5.5-2 and its NHS ester 11 which was readily purified by semipreparative RP-HPLC (overall yield 70%). Their structures were confirmed by detailed measurements, including MALDITOF mass spectrometry and NMR analyses. As expected, NIR5.5-2 was found to be perfectly soluble in water and related

aqueous buffers in the concentration range (1 µM to 5 mM) suitable for biomolecular labeling applications. Synthesis of the Cy 7.0 Analogue. A variety of water-soluble Cy 7.0 analogues with a functional reactive group at cyclohexenyl bridge included in the heptamethine chain have been already reported in the literature (15-20). Most of them have been obtained from readily available chloro cyanine dye 12. However, most of these dyes are not optimal for applications as biological markers. Although they are water-soluble, cyanine dyes containing an additional aryl group in the meso position through an ether or a thioether bridge show an increased tendency to aggregate in aqueous solution (18), and the aryl(thio)ether linkage in some of these fluorophores is fragile and thus susceptible to cleavage (39). To circumvent these problems and further optimize the chemical and photochemical properties of these NIR fluorophores, Lee et al. have recently reported the preparation of heptamethine cyanine dyes with a robust C-C bond at the central position of the chromophore by an efficient Suzuki-Miyaura palladium-catalyzed cross-coupling reaction


Figure 2. Normalized absorption (s) and emission (---) spectra of NIR5.5-2 (Ex. λ ) 600 nm) and NIR7.0-2 (Ex. λ ) 760 nm) in PBS at 25 °C.

(40). However, their spectral properties were investigated in MeOH or 20% aq DMSO, and no data about the fluorescence efficiency and aggregation tendency under physiological conditions are reported. Alternately, we have explored the postsynthetic derivatization of the precursor chloro cyanine dye 12 with a mercaptoalkyl carboxylic acid through an effective SRN1 reaction of its meso-chlorine atom (Scheme 4a). Indeed, the introduction of a short alkyl linker between the reactive carboxyl group and the heptamethine chain should prevent the π-π stacking interactions observed with the aryl linkers and so lead to fluorescent derivatives that are less likely to aggregate in aqueous solutions than their aryl-thioether congeners. As a starting point, we have developed a synthetic procedure that allows for the facile synthesis of dye NIR7.0-1. Reaction of the chloro-substituted heptamethine sulfocyanine dye 12 (20) with an excess of thioglycolic acid (3 equiv) in dry DMF gave the carboxylic acid functionalized NIR7.0-1 in nearly quantitative yield. However, this dye proved to not be suitable for bioconjugation applications since its conversion into the Nhydroxysuccinimidyl ester or its direct activation with various peptide coupling reagents (i.e., phosphonium and uronium reagents) failed. Therefore, we have turned our attention to the carboxylic acid dye NIR7.0-2 bearing a propanoic acid-based linker. [The Cy 7.0 analogue bearing an hexanoic acid basedbased linker has also been also synthesized (by SRN1 reaction between 12 and 6-mercaptohexanoic acid). These optical properties and its bioconjugation efficiency are similar to those reported for NIR7.0-2.] The synthesis of this new derivative was achieved by using the same synthetic procedure but required an additional heating of the reaction mixture (i.e., 12 and 3-mercaptopropionic acid in dry DMF) under reflux. Pure NIR7.0-2 was obtained by RP-HPLC in 80% yield; its structure was confirmed by detailed measurements, including ESI mass spectrometry and NMR analyses. In addition to these characterizations, we have checked the reactivity of the free carboxylic acid functional group. Its activation was accomplished by treatment of NIR7.0-2 with TSTU uronium salt in the presence of DIEA. Complete conversion to the N-hydroxysuccinimidyl ester 13 was observed by RP-HPLC. It is important to note that during the course of our work, a similar monofunctional NIR fluorophore namely CyTE-777 was reported by Hilderbrand et al. (10). CyTE-777 was obtained by postsynthetic derivatization of the commercially available dye IR-783 through the same SRN1 reaction with 3-mercaptopropionic acid as described above (Scheme 4b). However, in this case, the use of a stoichiometric amount of triethylamine has enabled us to perform this reaction at room temperature. Optical Properties. As expected, the two novel water-soluble cyanine fluorophores NIR5.5-2 and NIR7.0-2 display wavelength absorption and emission bands in the NIR range. The

Bouteiller et al.

optical properties of these dyes are summarized in Table 1, and their absorption and emission spectra are displayed in Figure 2. NIR5.5-2 has absorption and emission bands that match closely those of Cy 5.5 in PBS, which has an absorption maximum of 674 nm. There is no indication for dye-dye aggregation in PBS, which would manifest itself as a second absorption band at approximately 50 nm shorter wavelength (13). Indeed, the short wavelength shoulder visible in the absorption spectrum of NIR5.5-2 is typical of cyanines and related polymethine dyes and attributed to transitions from different vibrational levels of the excited-state of fluorophore (i.e., vibronic shoulder) (14). The formation of dye-dye dimers and other aggregates is detrimental, because they frequently result in intermolecular quenching fluorescence emission. Thus, it appears that the three sulfonate groups introduced onto the 3,5-diaminobenzoic acid linker are sufficient to prevent dyedye aggregation. The quantum yield of NIR5.5-2 is significantly lower than that reported for Cy 5.5. This lower value may be explained by the presence of the trisulfonated aryl linker 9 because aromatic amines are known to quench the excited-state of some fluorophores through the photoinduced electron transfer (PET) process (41, 42). This explanation was supported by the higher value of quantum yield determined for the Cy 5.5 analogue NIR5.5-1. Indeed, this dye contains a single sulfonatopropyl substituent which has no electron-donating ability for such PET to occur. However, the brightness or fluorescence intensity per dye molecule (i.e., the product of the fluorescence quantum yield and molar absorptivity) of NIR5.5-2 is compatible with its use as a donor in NIR molecular probes relying on the fluorescence resonance energy transfer (FRET) process (43). Concerning the long wavelength dye NIR7.0-2, its absorption spectrum shows a characteristic band broadening, which is typical of heptamethine dyes with the absorption maxima in the range of 770-800 nm. As with NIR5.5-2, the Cy 7.0 analogue NIR7.0-2 does not show any tendency to aggregate in aqueous solution. This indotricarbocyanine dye has a 20 nm Stokes shift of the fluorescence emission maximum and a quantum yield of 0.025 in PBS and 0.09 in DMSO. These latter values are comparable to those recently reported for other heptamethine cyanine dye derivatives (17, 40, 44). Furthermore, as illustrated in Figure 2, there is a good spectral overlap between the absorption of NIR7.0-2 and emission of NIR5.52. Thus, NIR7.0-2 might serve as an efficient acceptor for the fluorophores with emission around 700 nm in a FRET configuration. Peptide Labeling. To demonstrate the general applicability of these novel water-soluble NIR cyanine dyes, NIR5.5-2 and NIR7.0-2 were converted into thiol- and amine-reactive derivatives for use in peptide labeling. As these two NIR fluorophores may act as an efficient FRET pair, their sequential and selective introduction within the same peptide architecture seems to be an interesting way to validate the labeling strategy leading to original NIR molecular probes relies on FRET process. We thus chose to couple our FRET pair to the caspase-3 cleavable peptide substrate Ac-Cys-Asp-Glu-Val-Asp-Lys-NH2 16 (cleavage site after the aspartic acid residue at the C-terminal side). Caspases are Cysteine-ASpartic-acid-ProteASES that play a critical role as mediators for apoptotic cell death (45). Caspase-3 has been specifically identified as being a key mediator of apoptosis in mammalian cells (46): activation of caspase-3 indicates that the apoptotic pathway has progressed to an irreversible stage. There is thus a growing interest in identifying caspase inhibitors to minimize cell death in pathological conditions (neurodegenerative diseases for instance) but also for inducing caspase activation in cancer cells (47, 48). In addition, caspase-3 is widely used for monitoring apoptosis induction for general cytotoxicity screening. This interest for



Figure 3. ESI mass spectrum of the fluorogenic substrate of caspase-3 protease 19, in the positive mode, [M + 2H]2+: m/z: calcd for C99H130N16O28S4: 1061.24, found 1059.20. Loss of thiol NIR dye 21 occurred during the ionization process. Structures of peptide 20 and thiol NIR dye 21.

caspase-3 resulted in the development of several assays using a variety of formats and amenable to high throughput screening (49-54). Hexapeptide 16 has thus been chosen as a suitable target in order to get a novel fluorogenic substrate useful for detecting apoptosis in whole cells and for cell-based high throughput screening assays for apoptosis inhibitors or inducers.(For other examples of FRET-based probes used for caspase-3 detection, see refs 55-58.) To the core caspase-3 substrate sequence, cysteine and lysine residues were incorporated at Nand C-termini, respectively, as handles for the attachment of fluorophores. The use of a cysteine side-chain protecting group removable under mild reducing conditions (i.e., tert-butylthio group, StBu) has enabled the selective introduction of the two fluorescent labels within this peptide, thus obtaining the target FRET substrate through an efficient solution-phase labeling method. First, NIR5.5-2 was converted into the thiol-reactive iodoacetyl derivative 15 using a conventional three-step method (Scheme 5). NIR5.5-2 N-hydroxysuccinimidyl ester 11, generated by reaction with TSTU/ DIEA in dry NMP, was reacted with ethylenediamine under the conditions reported by Gruber et al. (59) to give NIR5.5-2 amine 14 in 73% yield. Finally, acylation of the primary amino group of 14 with the heterobifunctional cross-linker reagent succinimidyl 4-[(iodoacetyl)-

amino]benzoate (i.e., SIAB) in a mixture of NMP and aq sodium bicarbonate buffer (pH 8.5) provided, after RP-HPLC purification, the thiol-reactive Cy 5.5 analogue 15 (yield 80%). Its structure was confirmed by ESI mass spectrometry. Site-specific fluorescent labeling of caspase-3-cleavable peptide substrate started with the reaction between Ac-Cys(StBu)-Asp-Glu-ValAsp-Lys-NH2 16 and the N-hydroxysuccinimidyl ester of NIR7.0-2 13 in dry NMP in the presence of DIEA (Scheme 6). Purification by RP-HPLC provided the NIR7.0-2-labeled peptide Ac-Cys(StBu)-Asp-Glu-Val-Asp-Lys(NIR7.0-2)-NH2 17 in 60% yield. The removal of the StBu protecting group from the cysteine residue was achieved by treatment with an excess of dithiothreitol (DTT) in aq sodium bicarbonate buffer (pH 8.5). Purification of the free sulfhydryl-containing peptide-NIR7.0-2 conjugate 18 was achieved by RP-HPLC. Finally, iodoacetamido-activated NIR5.5-2 15 was attached on the free cysteine of peptide 18 by using a 3:1 molar excess (18/15) in aq sodium bicarbonate buffer (pH 8.5). The caspase-3 substrate 19 was obtained in a pure form after purification by RP-HPLC (yield 25%, purity >95%). When this fluorogenic substrate was analyzed by ESI mass spectrometry (Figure 3), the expected molecular ion [M + H]+ around m/z 2800.00 Da was not observed. The mass spectrum shows a single peak at m/z 1059.20 Da corresponding to the positive ion [M + 2H]2+ of


Bouteiller et al.

Figure 4. Absorption and emission (Ex. λ ) 660 nm) spectra of probe 19 at 25 °C (concentration 2.6 µM). (a,b) In PBS. (c,d) In DMSO. The efficiency of this energy transfer (E) is typically measured using the relative fluorescence intensity of the donor (NIR5.5-2), in the absence (FD) and presence (FDA) of acceptor (NIR7.0-2): E ) 1 - (FDA/FD). For our fluorescent probe, this was accomplished by comparing the NIR5.5-2 emission (i.e., area under the corrected emission curve in the range 670-760 nm after excitation at λ ) 660 nm) for the iodoacetyl derivative 15 and peptide 19. From these emission spectra, a value of FDA/FD close to 0.45 was found.

NIR5.5-2 derivative 20. The formation of this single fluorescently labeled peptide was explained by the elimination of thiol NIR7.0-2 derivative 21 (i.e., retro-Michael reaction) followed by the 1,4-addition of a water molecule onto the newly formed Michael olefin. This fragmentation pathway was also observed when the fluorogenic probe was subjected to a MALDI-TOF mass spectrometry analysis and observed with other FRET probes bearing the same NIR cyanine dye as energy acceptor (see Supporting Information). Furthermore, as the photophysical characterization (see below) has confirmed the presence and integrity of the two fluorophores NIR5.5-2 and NIR7.0-2 within the hexapeptide, the hydrolytic release of 21 obviously occurs during the ionization process. Photophysical Characterization and in Vitro Cleavage by Caspase-3 of the Fluorogenic Peptide. The absorption and emission spectra of the probe 19 recorded in PBS and DMSO are shown in Figure 4. In DMSO, two major absorption peaks at 689 and 800 nm, indicating the presence of NIR5.5-2 and NIR7.0-2, respectively, were observed. Surprisingly, a new peak at 649 nm, which is blue-shifted by about 30 nm from the absorption peak of the free NIR5.5-2 dye, was observed for the probe dissolved in aqueous buffered solution. As the absorption peak of NIR7.0-2 dye is also red-shifted by about 20 nm, it is clear that this probe displays significant dye-dyeaggregation leading to the formation of an intramolecular heterodimer (60). Further evidence for the formation of an H-aggregate within the dual-labeled peptide 19 was given by the fluorescence emission spectrum (recorded after excitation of the donor NIR5.5-2 at 660 nm). Indeed, no significant emission at 798 nm corresponding to the NIR7.0-2 fluorescence (see Supporting Information) or a minor emission at 705 nm (diminished by 3 times compared to the fluorescence of free NIR5.5-2 dye at the same concentration) corresponding to the remaining untransferred NIR5.5-2 fluorescence was observed. This quenching was not observed in the fluorescence emission spectrum recorded in DMSO, where an energy transfer efficiency close to 55% was found. Thus, NIR7.0-2 acts as an efficient quencher for NIR5.5-2. As this quenching effect is also observed with the probe bearing the less polar Cy 5.5

Figure 5. Static bimolecular Stern-Volmer plot for determining quenching efficiency of NIR7.0-2 toward the idoacetyl derivative of NIR5.5-2 in PBS. F0 and F are fluorescence intensity of 15 (0.72 µM) in the absence and in the presence of NIR7.0-2, respectively.

analogue NIR5.5-1 as donor (see Supporting Information) but not observed with the fluorogenic Cy 5.0/NIR7.0-2 hexapeptide, dye-dye aggregation may result from strong π-π stacking interactions between the benz[e]indole and indole units. To assess the quenching efficiency of the NIR5.5-2/NIR7.0-2 pair, a Stern-Volmer plot was obtained from steady-state fluorescence intensity measurements of the donor NIR5.5-2 with or without the acceptor NIR7.0-2 (Figure 5). Since the structural features of the linker connecting the Cy 5.5 analogue to the peptide core may influence the quenching, this study was performed with iodoacetyl derivative 15 instead of the free carboxylic acid dye. The Stern-Volmer plot displays an upward curvature; this positive deviation from linearity with increasing NIR7.0-2 concentrations indicates that static quenching is much higher than expected for a diffusion-controlled bimolecular reaction. This strongly supports the idea of the formation of a nonfluorescent ground-state complex between the two NIR cyanine dyes in aqueous solutions (61). Interestingly, a comprehensive study of the optical properties of the thiol-reactive Cy 5.5 analogue 15 has revealed that the chemical derivatization of NIR5.5-2 with the SIAB cross-linker significantly increases the quantum yield of our Cy 5.5 analogue especially in DMSO (ΦF ) 0.145 compared to ΦF ) 0.05 for NIR5.5-2). We presume that this increase of fluorescence in 15 as compared with NIR5.5-2 is due to the longer distance between the cyanine core and the aniline nitrogen atoms of the


Figure 6. Fluorescence emission time course of probe 19 (concentration 1.0 µM) with recombinant human caspase-3 (3.2 10-3 U, incubation time 180 min) in caspase assay buffer (100 mM NaCl, 40 mM HEPES, 10 mM DTT, 1 mM EDTA, 10% (w/v) sucrose, and 0.1% (w/v) CHAPS, pH 7.2, 37 °C) at 705 nm (Ex. λ ) 670 nm) and 805 nm (Ex. λ ) 770 nm). To avoid overloading of this figure, only the emission at 805 nm (Ex. λ ) 770 nm) is shown for the control reaction without caspase-3. The same straight line was obtained for the emission at 705 nm but at a fluorescence level around 25 a.u.

hydrophilic linker which acts as a quencher (see above). Indeed, strong π-π stacking interactions between the benzene rings of trisulfonated linker 9 and SIAB cross-linker may favor a conformation in which the quencher-fluorophore distance is significantly increased. Thus, this conformational change would reduce the rate of the PET-based quenching process, thereby enhancing the fluorescence emission. With this interesting tool in hand, the internally quenched fluorescent probe 19 was assayed against recombinant human caspase-3. Within 180 min, the fluorescence emission of 19 at 705 and 805 nm has increased about 2- and 8-fold, respectively, while no activation was observed when the peptide 19 was incubated only with the caspase-3 buffer (Figure 6). Furthermore, no fluorescence enhancement was obtained after prolonged incubation of 19 with BSA protein (see Supporting Information). This latter observation asserts that an hypothetical nonspecific binding between peptide 19 and protein (such as caspase-3) cannot induce the fluorescence revealing of both NIR dyes through conformational changes of the probe. Overall, these results confirm that the fluorogenic peptide-based probe 19 is a substrate for recombinant caspase-3 protease. However, fluorescence recovery was significantly lower than expected if complete proteolytic cleavage of 19 had occurred. One possible explanation for this result is the low accessibility of the enzyme cleavage site within the peptide backbone. As the enzyme activity and substrate recognition are usually extremely sensitive to peptide conformation, the formation of the intramolecular heterodimer has probably significantly modified the shape of the peptide recognition unit, and the activity rate was dramatically decreased.

CONCLUSION In this paper, we have described the synthesis and photophysical properties of two novel water-soluble NIR cyanine dyes NIR5.5-2 and NIR 7.0-2 which are potential substitutes of the commercially available Cy 5.5 and Cy 7.0 labels. They are easily prepared in good yields by postsynthetic derivatization of an amino or a chloro cyanine precursor through an efficient acylation and a SRN1 reaction respectively. These two derivatives display absorption and emission maxima in the NIR range and do not self-aggregate in aqueous solution, and their fluorescence efficiency is compatible with their use in a wide variety of optical imaging applications and NIR fluorescence studies.


However, further efforts are required in order to improve the quantum yield of NIR5.5-2 especially by introducing a polysulfonated aminoalkyl linker as water-solubilizing moiety onto the cyanine precursor. Efficient synthetic protocols were also found for conversion of NIR5.5-2 and NIR7.0-2 into thiol- and aminereactive derivatives 13 and 15. These novel fluorescent labeling reagents have been successfully used in solution-phase peptide labeling to provide an original peptide-based NIR probe of caspase-3 protease. Efficient quenching effect between NIR5.5-2 and NIR7.0-2 at 705 and 798 nm was demonstrated. In the caspase-3 model, the probe is able to release fluorescence signal by 8-fold at 805 nm after enzymatic degradation. However, enzyme activity is dramatically decreased compared with other fluorogenic FRET-based probes previously reported by us (30, 62). Thus, further studies aimed at re-engineering the placement of the NIR dye labels and their covalent attachment, such that dimer formation will not alter the hexapeptide secondary structure, are necessary to get an internally quenched fluorescent probe suitable to assay caspase-3 activity both in Vitro and in ViVo. Yet, the observation of an efficient formation of an intramolecular heterodimer yielding a nonfluorescent groundstate complex between the two NIR cyanine dyes in aqueous solutions, even with a very short peptide which is unlikely to fold, opens the door to the use of this efficiently quenched fluorophore pair using coil-containing longer peptidic backbones.

ACKNOWLEDGMENT The contribution of Dr. Je´roˆme Leprince (U 413 INSERM/ IFRMP 23) to MALDI-TOF mass spectrometry measurements and solid-phase synthesis of hexapeptide 16 is greatly acknowledged. We thank Dr. Xavier Franck for helpful discussions concerning the sulfonation reactions of amines. The financial support of the “Re´gion Haute Normandie” is gratefully acknowledged. Supporting Information Available: Synthesis and characterization of thiol-reactive NIR5.5-1 derivative 24, detailed synthetic procedures and photophysical characterization of fluorogenic NIR5.5-1/NIR7.0-2 hexapeptide. Photophysical characterization of fluorogenic NIR5.5-2/NIR7.0-2 hexapeptide 19 and fluorescence emission time course of incubation with BSA. This material is available free of charge via the Internet at http:// pubs.acs.org/ BC.

LITERATURE CITED (1) Haugland, R. P. (2002) Handbook of Fluorescence Probes and Research Products, 9th ed., Molecular Probes, Inc., Eugene, OR. (2) Buschmann, V., Weston, K. D., and Sauer, M. (2003) Spectroscopic study and evaluation of red-absorbing fluorescent dyes. Bioconjugate Chem. 14, 195-204 and references cited therein. (3) Go´mez-Hens, A., and Aguilar-Caballos, M. P. (2004) Longwavelength fluorophores: new trends in their analytical use. Trends Anal. Chem. 23, 127-136. (4) Zhao, W., and Carreira, E. M. (2006) Conformationally restricted aza-BODIPY: highly fluorescent, stable near-infrared absorbing dyes. Chem. Eur. J. 12, 7254-7263. (5) Liu, J., Diwu, Z., Leung, W.-Y., Lu, Y., Patch, B., and Haugland, R. P. (2003) Rational design and synthesis of a novel class of highly fluorescent rhodamine dyes that have strong absorption at long wavelengths. Tetrahedron Lett. 44, 4355-4359. (6) Bandichhor, R., Petrescu, A. D., Vespa, A., Kier, A. B., Schroeder, F., and Burgess, K. (2006) Synthesis of a new water-soluble rhodamine derivative and application to protein labeling and intracellular imaging. Bioconjugate Chem. 17, 1219-1225. (7) Jose, J., and Burgess, K. (2006) Benzophenoxazine-based fluorescent dyes for labeling biomolecules. Tetrahedron 62, 11021-11037.

1316 Bioconjugate Chem., Vol. 18, No. 4, 2007 (8) Lin, Y., Weissleder, R., and Tung, C.-H. (2002) Novel near-infrared cyanine fluorochromes: synthesis, properties, and bioconjugation. Bioconjugate Chem. 13, 605-610. (9) 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. (10) Hilderbrand, S. A., Kelly, K. A., Weissleder, R., and Tung, C.H. (2005) Monofunctional near-infrared fluorochromes for imaging applications. Bioconjugate Chem. 16, 1275-1281. (11) Patonay, G., Salon, J., Sowell, J., and Strekowski, L. (2004) Noncovalent labeling of biomolecules with red and near-infrared dyes. Molecules 9, 40-49. (12) Tung, C.-H. (2004) Fluorescent peptide probes for in vivo diagnostic imaging. Biopolymers 76, 391-403 and references cited therein. (13) Mujumdar, S. R., Mujumdar, R. B., Grant, C. M., and Waggoner, A. S. (1996) Cyanine-labeling reagents: sulfobenzindocyanine succinimidyl esters. Bioconjugate Chem. 7, 356-362. (14) 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, 105111. (15) Narayanan, N., and Patonay, G. (1995) A new method for the synthesis of heptamethine cyanine dyes: synthesis of new nearinfrared fluorescent labels. J. Org. Chem. 60, 2391-2395. (16) Narayanan, N., Strekowski, L., Lipowska, M., and Patonay, G. (1997) Erratum to “A new method for the synthesis of heptamethine cyanine dyes: synthesis of new near infrared fluorescent labels”. J. Org. Chem. 62, 9387. (17) Song, F., Peng, X., Lu, E., Zhang, R., Chen, X., and Song, B. (2004) Syntheses, spectral properties and photostabilities of novel water-soluble near-infrared cyanine dyes. J. Photochem. Photobiol. A. Chem. 168, 53-57. (18) Flanagan, J. H., Jr., Khan, S. H., Menchen, S., Soper, S. A., and Hammer, R. P. (1997) Functionalized tricarbocyanine dyes as nearinfrared fluorescent probes for biomolecules. Bioconjugate Chem. 8, 751-756. (19) Li, C., Greenwood, T. R., Bhujwalla, Z. M., and Glunde, K. (2006) Synthesis and characterization of glucosamine-bound near-infrared probes for optical imaging. Org. Lett. 8, 3623-3626. (20) Strekowski, L., Mason, C. J., Lee, H., Gupta, R., Sowell, J., and Patonay, G. (2003) Synthesis of water-soluble near-infrared cyanine dyes functionalized with [(succinimido)oxy]carbonyl group. J. Heterocycl. Chem. 40, 913-916. (21) Zhang, Z., and Achilefu, S. (2004) Synthesis and evaluation of polyhydroxylated near-infrared carbocyanine molecular probes. Org. Lett. 6, 2067-2070. (22) Chipon, B., Clave´, G., Bouteiller, C., Massonneau, M., Renard, P.-Y., and Romieu, A. (2006) Synthesis and post-synthetic derivatization of a cyanine-based amino acid. Application to the preparation of a novel water-soluble NIR dye. Tetrahedron Lett. 47, 8279-8284. (23) Chipon, B., Clave´, G., Bouteiller, C., Massonneau, M., Renard, P.-Y., and Romieu, A. (2007) Corrigendum to “Synthesis and postsynthetic derivatization of a cyanine-based amino acid. Application to the preparation of a novel water-soluble NIR dye”. Tetrahedron Lett. 48, 501. (24) Jung, M. E., and Kim, W.-J. (2006) Practical syntheses of dyes for difference gel electrophoresis. Bioorg. Med. Chem. 14, 92-97. (25) Marvel, C. S., and Sparberg, M. S. (1930) Sodium 2-bromoethanesulfonate. Organic Syntheses, Coll. Vol. 10, pp 96-97, Wiley, New York. (26) Leone-Bay, A., and Timony, P. E. (1988) A new synthesis of Sulfo-N-succinimidyl p-(iodoacetylamino)benzoate. Synth. Commun. 18, 1637-1640. (27) Portoghese, P. S., Sultana, M., and Takemori, A. E. (1990) Design of peptidomimetic δ opioid receptor antagonists using the messageaddress concept. J. Med. Chem. 33, 1714-1720. (28) Kappe, T., Fruhwirth, F., Roschger, P., Jocham, B., Kremsner, J., and Stadlbauer, W. (2002) Synthesis and reactions of 10,10dimethyl-10H-pyrido[1,2-a]indol-6-ones. J. Heterocycl. Chem. 39, 391-397. (29) Strekowski, L., Gorecki, T., Mason, J. C., Lee, H., and Patonay, G. (2001) New heptamethine cyanine reagents for labeling of

Bouteiller et al. biomolecules with a near-infrared chromophore. Heterocycl. Commun. 7, 117-122. (30) Lapeyre, M., Leprince, J., Massonneau, M., Oulyadi, H., Renard, P.-Y., Romieu, A., Turcatti, G., and Vaudry, H. (2006) Aryldithioethyloxycarbonyl (Ardec): a new family of amine protecting groups removable under mild reducing conditions and their applications to peptide synthesis. Chem. Eur. J. 12, 3655-3671. (31) Gottlieb, H. E., Kotlyar, V., and Nudelman, A. (1997) NMR chemical shifts of common laboratory solvents as trace impurities. J. Org. Chem. 62, 7512-7515. (32) Reindl, S., Penzkofer, A., Gong, S. H., Landthaler, M., Szeimies, R. M., Abels, C., and Baeumler, W. (1997) Quantum yield of triplet formation for indocyanine green. J. Photochem. Photobiol. A. Chem. 105, 65-68. (33) Cheng, Z., Wu, Y., Xiong, Z., Gambhir, S. S., and Chen, X. (2005) Near-infrared fluorescent RGD peptides for optical imaging of integrin Rv. beta.3 expression in living mice. Bioconjugate Chem. 16, 1433-1441. (34) Medarova, Z., Bonner-Weir, S., Lipes, M., and Moore, A. (2005) Imaging β-cell death with a near-infrared probe. Diabetes 54, 17801788. (35) Vera, D. R., Hall, D. J., Hoh, C. K., Gallant, P., McIntosh, L. M., and Mattrey, R. F. (2005) Cy5.5-DTPA-galactosyl-dextran: a fluorescent probe for in vivo measurement of receptor biochemistry. Nucl. Med. Biol. 32, 687-693. (36) Cheng, Z., Levi, J., Xiong, Z., Gheysens, O., Keren, S., Chen, X., and Gambhir, S. S. (2006) Near-infrared fluorescent deoxyglucose analogue for tumor optical imaging in cell culture and living mice. Bioconjugate Chem. 17, 662-669. (37) Ho, N.-H., Weissleder, R., and Tung, C.-H. (2006) Development of water-soluble far-red fluorogenic dyes for enzyme sensing. Tetrahedron 62, 578-585. (38) Jose, J., and Burgess, K. (2006) Syntheses and properties of watersoluble Nile Red derivatives. J. Org. Chem. 71, 7835-7839. (39) Zaheer, A., Wheat, T. E., and Frangioni, J. V. (2002) IRDye78 conjugates for near-infrared fluorescence imaging. Mol. Imaging 1, 354-364. (40) Lee, H., Mason, J. C., and Achilefu, S. (2006) Heptamethine cyanine dyes with a robust C-C bond at the central position of the chromophore. J. Org. Chem. 71, 7862-7865. (41) Sasaki, E., Kojima, H., Nishimatsu, H., Urano, Y., Kikuchi, K., Hirata, Y., and Nagano, T. (2005) Highly sensitive near-infrared fluorescent probes for nitric oxide and their application to isolated organs. J. Am. Chem. Soc. 127, 3684-3685. (42) Sparano, B. A., and Koide, K. (2005) A strategy for the development of small-molecule-based sensors that strongly fluoresce when bound to a specific RNA. J. Am. Chem. Soc. 127, 1495414955. (43) Sapsford, K. E., Berti, L., and Medintz, I. L. (2006) Materials for fluorescence resonance energy transfer analysis: beyond traditional donor-acceptor combinations. Angew. Chem., Int. Ed. 45, 4562-4588. (44) Chen, X., Peng, X., Cui, A., Wang, B., Wang, L., and Zhang, R. (2006) Photostabilities of novel heptamethine 3H-indolenine cyanine dyes with different N-substituents. J. Photochem. Photobiol. A. Chem. 181, 79-85. (45) Denault, J.-B., and Salvesen, G. S. (2002) Caspases: keys in the ignition of cell death. Chem. ReV. 102, 4489-4499. (46) Kothakota, S., Azuma, T., Reinhard, C., Klippel, A., Tang, J., Chu, K., McGarry, T. J., Kirschner, M. W., Koths, K., Kwiatkowski, D. J., and Williams, L. T. (1997) Caspase-3-generated fragment of gelsolin: effector of morphological change in apoptosis. Science 278, 294-298. (47) Talanian, R. V., Brady, K. D., and Cryns, V. L. (2000) Caspases as targets for anti-inflammatory and anti-apoptotic drug discovery. J. Med. Chem. 43, 3351-3371. (48) Nuttall, M. E., Lee, D., McLaughlin, B., and Erhardt, J. A. (2001) Selective inhibitors of apoptotic caspases: implications for novel therapeutic strategies. DDT 6, 85-91. (49) Tawa, P., Tam, J., Cassady, R., Nicholson, D. W., and Xanthoudakis, S. (2001) Quantitative analysis of fluorescent caspase substrate cleavage in intact cells and identification of novel inhibitors of apoptosis. Cell Death Differ. 8, 30-37. (50) Waud, J. P., Bermudez Fajardo, A., Sudhaharan, T., Trimby, A. R., Jeffery, J., Jones, A., and Campbell, A. K. (2001) Measurement


Internally Quenched Fluorescent Probes of proteases using chemiluminescence-resonance-energy-transfer chimaeras between green fluorescent protein and aequorin. Biochem. J. 357, 687-697. (51) Gopalakrishnan, S. M., Karvinen, J., Kofron, J. L., Burns, D. J., and Warrior, U. (2002) Application of micro arrayed compound screening (mARCS) to identify inhibitors of caspase-3. J. Biomol. Screening 7, 317-323. (52) Karvinen, J., Hurskainen, P., Gopalakrishnan, S. M., Burns, D. J., Warrior, U., and Hemmila, I. (2002) Homogeneous time-resolved fluorescence quenching assay (LANCE) for caspase-3. J. Biomol. Screening 7, 223-231. (53) Preaudat, M., Ouled-Diaf, J., Alpha-Bazin, B., Mathis, G., Mitsugi, T., Aono, Y., Takahashi, K., and Takemoto, H. (2002) A homogeneous caspase-3 activity assay using HTRF technology. J. Biomol. Screening 7, 267-274. (54) O’Brien, M. A., Daily, W. J., Hesselberth, P. E., Moravec, R. A., Scurria, M. A., Klaubert, D. H., Bulleit, R. F., and Wood, K. V. (2005) Homogeneous, bioluminescent protease assays: caspase-3 as a model. J. Biomol. Screening 10, 137-148. (55) Mizukami, S., Kikuchi, K., Higuchi, T., Urano, Y., Mashima, T., Tsuruo, T., and Nagano, T. (1999) Imaging of caspase-3 activation in HeLa cells stimulated with etoposide using a novel fluorescent probe. FEBS Lett. 453, 356-360. (56) Komoriya, A., Packard, B. Z., Brown, M. J., Wu, M. L., and Henkart, P. A. (2000) Assessment of caspase activities in intact

apoptotic thymocytes using cell-permeable fluorogenic caspase substrates. J. Exp. Med. 191, 1819-1828. (57) Pham, W., Weissleder, R., and Tung, C.-H. (2002) An azulene dimer as a near-infrared quencher. Angew. Chem., Int. Ed. 41, 36593662. (58) Bullok, K., and Piwnica-Worms, D. (2005) Synthesis and characterization of a small, membrane-permeant, caspase-activatable far-red fluorescent peptide for imaging apoptosis. J. Med. Chem. 48, 5404-5407. (59) Gruber, H. J., Kada, G., Pragl, B., Riener, C., Hahn, C. D., Harms, G. S., Ahrer, W., Dax, T. G., Hohenthanner, K., and Knaus, H. G. (2000) Preparation of thiol-reactive Cy5 derivatives from commercial Cy5 succinimidyl ester. Bioconjugate Chem. 11, 161-166. (60) Johansson, M. K., and Cook, R. M. (2003) Intramolecular dimers: a new design strategy for fluorescence-quenched probes. Chem. Eur. J. 9, 3466-3471. (61) Marme´, N., Knemeyer, J.-P., Sauer, M., and Wolfrum, J. (2003) Inter- and intramolecular fluorescence quenching of organic dyes by tryptophan. Bioconjugate Chem. 14, 1133-1139. (62) Poupart, S., Boudou, C., Peixoto, P., Massonneau, M., Renard, P.-Y., and Romieu, A. (2006) Aminopropargyl derivative of terpyridine-bis(methyl-enamine) tetraacetic acid chelate of europium (Eu (TMT)-AP3): a new reagent for fluorescent labelling of proteins and peptides. Org. Biomol. Chem. 4, 4165-4177. BC0700281

Novel Water-Soluble Near-Infrared Cyanine Dyes: Synthesis, Spectral

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