Novel Cyanine Dyes with Vinylsulfone Group for Labeling Biomolecules

550–850 nm) were designed and synthesized for fluorescent labeling of .... symmetrical and unsymmetrical cyanine dyes: an experimental and theoretic...
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Novel Cyanine Dyes with Vinylsulfone Group for Labeling Biomolecules Jin Woo Park,†,‡ YoungSoo Kim,† Kee-Jung Lee,‡,* and Dong Jin Kim*,† †

Center for Neuro-Medicine, Brain Science Institute, Korea Institute of Science and Technology, L2317, 39-1 Hawolgok-dong, Sungbuk-ku, Seoul, Korea 136-791 ‡ Department of Chemical Engineering, Hanyang University, 17 Haengdang-dong, Seongdong-gu, Seoul, Korea 133-791 S Supporting Information *

ABSTRACT: Novel vinylsulfone cyanine dyes (em. 550−850 nm) were designed and synthesized for fluorescent labeling of biomolecules via 1,2-addition reaction in aqueous conditions. Due to the virtue of chemical structures of both fluorophore and reactive group, these dyes could be significantly stable and reactive in various aqueous/organic conditions. A wide variety of pH, temperature, buffer concentration, and protein were tested for the optimal labeling condition.

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INTRODUCTION Optical imaging based on fluorescence probes is one of the most famous and widely used tools in life sciences due to the power of visualization. 1−3 Fluorescence probes enable researchers to detect particular components of complex biomolecular assemblies with their exquisite sensitivity and selectivity.4−7 Labeled target molecules are then analyzed via imaging instruments such as confocal microscopes, fluorescence scanners, microarray readers, flow cytometers, capillary electrophoresis apparatus, DNA sequencers, and microfluidic devices.8 Among the various fluorescent dyes, cyanine compounds have been used in a wide range of chemical, biological, and artistic applications, from recording media to genomic/ proteomic microarrays.9 Cyanine is a family of dyes containing polymethine group linking two heterocyclic rings containing nitrogen.9,10 CyDye Fluors (GE Healthcare UK Limited, U.K.) are well-known cyanine dyes for their water solubility, stability, sharp fluorescence band, high sensitivity, and ease of modification of reaction site.11,12 These molecules have reactive groups on either one or both heterocyclic rings so that they can be chemically conjugated to either nucleic acids or protein molecules.11−13 The most popular forms among these series of dyes are amine-reactive cyanine dyes such as N-hydroxysuccinimidyl (NHS) esters, which are widely used in the conjugation to proteins, peptides, ligands, synthetic oligonucleotides, and other biomolecules.14 However, NHS ester is unstable and has a short lifetime in aqueous solution, and N-hydroxysuccinimide is released as a byproduct along with the labeled molecules via nucleophilic substitution.14−16 In order to eliminate this undesired byproduct, we searched for various functional groups that have been used for dyeing animal fibers, comprising proteins. Among various reactive groups such as NHS ester, isothiocyanine, monochlorotriazine, dichlorotriazine, methylsulfonyl pyrimidine, dichloroquinoxaline, chlropyrimidine, and vinylsulfone, it © 2012 American Chemical Society

was found that the azo dyes containing vinylsulfone (VS) group17−21 have been utilized in dyeing natural fabrics such as wool and cellulose in various temperatures since the 1960s.22 Moreover, the dyeing process of VS dye is run in an aqueous solution,17,24,25 and even at a medium temperature 35 °C.17,22−24 Also, 1,2-addition as its dyeing mechanism does not allow any byproduct (Scheme 1).16,23 Herein we report the synthesis and evaluations of novel VS cyanine dyes having various emission wavelengths with improved stability.

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EXPERIMENTAL PROCEDURES Apparatus and Reagents. All chemicals and organic solvents were purchased from Sigma-Aldrich Co. and Tokyo Chemical Industry (TCI, Japan). CyDye Fluors dyes were purchased from GE Healthcare UK Ltd., bovine serum albumin protein from TCI. Molecular weight size marker was bought from GE Healthcare UK Ltd. and Takara Bio Inc. (Japan). For the normal phase flash chromatography, kiesel gel 60 (230−400 mesh, Merck, Germany) was used. Silica gel 60 RP18 F254S (0.25 mm, glass type, Merck) was used in reverse thin layer chromatography (TLC), and reverse column chromatography was carried out in an apparatus for medium pressure liquid chromatography system (MPLC, Buchi, Switzerland) equipped with a Lichroprep RP-18 reverse phase column (40−63 μm, Merck) and a Fraction Collector R-660. High performance liquid chromatography (HPLC) analysis was carried out using Bondapak C18 10 μm 125A (Waters Corp., MA) and Gemini 5u C6-Phenyl 110A (Phenomenex, Inc., CA) with 1100 series (Agilent Technologies, CA). FT-NMR spectra data were obtained with Avance 300 and Avance 400 (Bruker, Received: May 4, 2011 Revised: January 16, 2012 Published: January 16, 2012 350

dx.doi.org/10.1021/bc200232d | Bioconjugate Chem. 2012, 23, 350−362

Bioconjugate Chemistry

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Scheme 1. Dyeing Mechanism for VS Cyanine Dye with the Nucleophilesa

a

m = 1, 2, 3; n = 1, 2, 4; R: methyl, ethyl, propyl, butyl; Nu: nucleophiles.

Scheme 2. Synthetic Scheme for VS Cyanine Dyes

25 °C, filtered through a paper filter. The residue was washed with ethyl acetate and dried under reduced pressure to provide the sulfonic acid as a pink powder (11.3 g, 89%), which was used in the next step without further purification. A solution of crude sulfonic acid (5.1 g, 21.2 mmol) in methanol (35 mL) was added dropwise to a stirred solution of potassium hydroxide (1.4 g, 25.4 mmol) in propanol (35 mL). The resulting mixture was stirred at 25 °C for 24 h and filtered through a paper filter. The residue was dried under reduced pressure to provide the crude 2 (5.4 g, 90%). To obtain spectroscopic data, a small portion of crude 2 was purified with reverse phase chromatography (RP-C18, 15% acetonitrile− water). Rf = 0.68 (RP-C18, 20% acetonitrile−water); 1H NMR (300 MHz, D2O): δ 7.60 (s, 1H), 7.58 (d, 1H, J = 8.3 Hz), 7.32 (d, 1H, J = 7.9 Hz), 2.08 (s, 3H), 1.06 (s, 6H). 3H-Indolium, 1-Methyl-2,3,3-trimethyl-5-sulfonate (3a). A slurry of crude 2 (18.0 g, 64.8 mmol) in iodomethane (70 mL, 1.0 mol) under N2 was heated to reflux for 24 h and cooled down to 25 °C. The liquid phase was decanted, and the residue was washed with acetone (3 × 50 mL), filtered with a paper filter, and dried under reduced pressure at 40 °C to afford the crude 3a as a pink solid (13.5 g, 82%). To obtain spectroscopic data, small portion of crude 3a was purified with reverse phase chromatography (RP-C18, 15% acetonitrile−water). Rf = 0.13 (RP-C18, 20% acetonitrile−water); 1H NMR (400 MHz, DMSO-d6): δ 7.38−7.34 (m, 2H), 6.58−6.56 (m, 1H), 3.88 (s,

Germany) and molecular mass was measured by LC/MS 1200 L Quadrupole (Varian, CA) and Voyager MALDI-TOF DE. The absorption spectra and wavelength data were measured by Lambda 45 UV/vis spectrophotometer (Perkin-Elmer, MA) and fluorescent spectra and wavelength data were obtained from LS-55 fluorescence spectrophotometer (Perkin-Elmer, MA). The apparatus for gel electrophoresis was PowerPac Basic Power Supply (Catalog No. 164−5050, BIO-RAD Laboratories, Inc., CA) coupled to an SE 260 mini-vertical gel electrophoresis unit (Amersham Biosciences, Sweden). PAGEr Gold Precast Gels (Polyacrylamide gels for protein electrophoresis, 10−20% Tris-Glycine gels, Catalog No. 59506, Lonza, ME) were used for this appratus. Geliance 600 (PerkinElmer, MA) was used to observe the labeled biomolecules and measure fluorescence intensity. Light sources used herein were Geliance Blue Epi (Catalog No. L7110027). The measurement was carried out using Geliance Blue Light Filter (550−600 nm) as filters. General Synthesis. Synthesis of cyanine dyes with carboxylic acid was followed from the literature.10−12,23−31 3H-Indole-2,3,3-trimethyl-5-sulfonic Acid, Potassium Salt (2). A stirred solution of p-hydrazinobenzenesulfonic acid (1) (10.0 g, 53.0 mmol) in acetic acid (30 mL) was treated with 3methyl-2-butanone (17 mL, 160.0 mmol) at 25 °C. The reaction mixture was heated to reflux for 4 h, cooled down to 351

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Bioconjugate Chemistry

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2H), 2.49 (s, 3H), 1.26 (s, 6H); LC/MS m/z 253.04 (C12H15NO3S+ requires 253.08). 3H-Indolium, 1-Ethyl-2,3,3-trimethyl-5-sulfonate (3b). Compound 3b was prepared from compound 2 (20.0 g, 72.1 mmol) and iodoethane (110 mL, 1.4 mol) by the procedure utilized for compound 3a (18.4 g, 95%). Rf = 0.18 (RP-C18, 20% acetonitrile−water); 1H NMR (400 MHz, D2O): δ 7.99 (s, 1H), 7.88 (d, 1H, J = 8.2 Hz), 7.80 (d, 1H, J = 8.4 Hz), 4.43 (m, 2H), 1.52−1.40 (m, 12H); LC/MS m/z 268.16 (C13H18NO3S+ requires 268.10). 3H-Indolium, 1-Propyl-2,3,3-trimethyl-5-sulfonate (3c). Compound 3c was prepared from compound 2 (20.0 g, 72.1 mmol) and iodopropane (107 mL, 1.1 mol) by the procedure utilized for compound 3a (19.8 g, 98%). Rf = 0.45 (RP-C18, 20% acetonitrile−water); 1H NMR (400 MHz, MeOD-d4): δ 8.15 (s, 1H), 8.02 (s, 2H), 4.55 (t, 2H, J = 7.5 Hz), 2.04 (q, 2H, J = 7.5 Hz), 1.68 (s, 6H), 1.09 (t, 3H, J = 7.2 Hz); LC/MS m/z 282.30 (C14H20NO3S+ requires 282.12). 3H-Indolium, 1-Butyl-2,3,3-trimethyl-5-sulfonate (3d). Compound 3d was prepared from compound 2 (18.0 g, 64.8 mmol) and iodobutane (37 mL, 0.2 mol) by the procedure utilized for compound 3a (5.7 g, 96%). Rf = 0.58 (RP-C18, 20% acetonitrile−water); 1H NMR (400 MHz, DMSO-d6): δ 8.01 (s, 1H), 7.89 (d, 1H, J = 8.9 Hz), 7.80 (d, 1H, J = 8.9 Hz), 4.47−4.40 (m, 2H), 1.94−1.89 (m, 2H), 1.52 (s, 6H), 1.45− 1.39 (m, 2H), 0.99−0.89 (br m, 3H); LC/MS m/z 296.04 (C15H22NO3S+ requires 296.13). 3H-Indolium, 1-(4-Carboxybutyl)-2,3,3-trimethyl-5-sulfonate (5a). A slurry of the crude 2 (11.0 g, 39.7 mmol) in 1,2-dichlorobenzene (15 mL) under N2 was treated with 5bromovaleric acid (9.1 g, 50 mmol). The reaction mixture was heated to reflux for 12 h, cooled down to 25 °C, and liquid phase was decanted. The residue was treated with 2-propanol (50 mL), and the resulting mixture was filtered through a paper filter. The residue was dried under reduced pressure to afford the crude 5a as a pink solid (10.2 g, 64%). To obtain spectroscopic data, small portion of crude 5a was purified with reverse phase chromatography (RP-C18, 15% acetonitrile− water). Rf = 0.13 (RP-C18, 20% acetonitrile−water); 1H NMR (400 MHz, DMSO-d6): δ 7.37−7.34 (m, 2H), 6.56 (t, 1H, J = 8.0 Hz), 3.90 (d, 2H, J = 12.4 Hz), 2.25 (t, 2H), 1.66−1.22 (br m, 13H). 3H-Indolium, 1-(5-Carboxypentyl)-2,3,3-trimethyl-5-sulfonate (5b). Compound 5b was prepared from compound 2 (2.8 g, 10.0 mmol) and 6-bromohexanoic acid (2.3 g, 12.0 mmol) by the procedure utilized for compound 5a (2.7 g, 75%). Rf = 0.08 (RP-C18, 20% acetonitrile−water); 1H NMR (400 MHz, D2O): δ 8.00 (s, 1H), 7.90 (d, 1H, J = 8.8 Hz), 7.77 (d, 1H, J = 8.4 Hz), 4.37 (t, 2H, J = 7.4 Hz), 2.25 (t, 2H, J = 7.0 Hz), 1.85 (m, 2H), 1.57−1.26 (br m, 13H); LC/MS m/z 354.18 (C17H24NO5S+ requires 354.14). 3H-Indolium, 1-(7-Carboxyheptyl)-2,3,3-trimethyl-5-sulfonate (5c). Compound 5c was prepared from compound 2 (1.7 g, 6.0 mmol) and 8-bromooctanoic acid (1.3 g, 6.0 mmol) by the procedure utilized for compound 5a (1.9 g, 81%). Rf = 0.20 (RP-C18, 20% acetonitrile−water); 1H NMR (400 MHz, DMSO-d6): δ 7.36−7.32 (m, 2H), 6.53 (t, 1H, J = 8.0 Hz), 3.87 (d, 2H, J = 7.4 Hz), 2.08 (t, 2H), 1.51−1.42 (m, 19H); LC/MS m/z 381.12 (C19H27NO5S+ requires 381.16). 3H-Indolium, 2-[3-[1-(4-Carboxybutyl)-1,3-dihydro-3,3-dimethyl-5-sulfo-2H-indol-2-ylidene]-1-propen-1-yl]-1-ethyl3,3-dimethyl-5-sulfonate (6a). A slurry of crude 3b (3.4 g, 12.5 mmol) and N,N′-diphenylformamidine (2.8 g, 14.1 mmol)

in acetic acid (15 mL) under N2 was heated to reflux for 4 h, cooled down to 25 °C, and agitated a heterogeneous mixture by addition of ethyl acetate (20 mL). The resulting mixture was filtered through a paper filter, and the residue was dried under reduced pressure to provide the crude 4b (4.6 g, 96%). A stirred solution of the crude 4b (4.2 g, 10.0 mmol) in pyridine (20 mL) and acetic anhydride (20 mL) under N2 was treated with the crude 5a (3.4 g, 10.0 mmol) at 25 °C. Reaction mixture was heated to 110 °C for 4 h, cooled down to 25 °C, and agitated a heterogeneous mixture by addition of ethyl acetate (80 mL). Resulting mixture was filtered through a paper filter, and the residue was dried under reduced pressure and purified with flash chromatography (Lichroprep RP-18, 15% acetonitrile−water) to afford 6a as a red powder (1.47 g, 24%). Rf = 0.71 (RP-C18, 30% acetonitrile−water); 1H NMR (300 MHz, DMSO-d6): δ 8.35 (t, 1H, J = 13.5 Hz), 7.79 (s, 2H), 7.68 (d, 2H, J = 8.22 Hz), 7.41 (m, 2H), 6.57 (q, 2H, J = 5.0 Hz), 4.18−4.11 (m, 4H), 2.05 (t, 2H), 1.69−0.83 (br m, 19H); LC/MS m/z 615.28 (C30H35N2O8S2− requires 615.18). 3H-Indolium, 2-[3-[1-(5-Carboxypentyl)-1,3-dihydro-3,3dimethyl-5-sulfo-2H-indol-2-ylidene]-1-propen-1-yl]-1-methyl-3,3-dimethyl-5-sulfonate (6b). Compound 6b was prepared from compound 3a (1.4 g, 5.6 mmol) and compound 5b (2.0 g, 5.6 mmol) by the procedure utilized for compound 6a (0.94 g, 27%). Rf = 0.80 (RP-C18, 30% acetonitrile−water); 1H NMR (300 MHz, D2O): δ 7.95 (m, 1H), 7.84−7.62 (m, 4H), 7.46 (d, 2H, J = 7.8 Hz), 6.51 (d, 2H, J = 8.2 Hz), 4.41 (t, 2H, J = 8.0 Hz), 3.56 (s, 3H), 1.90 (m, 2H), 1.70−1.24 (br m, 18H); MALDI-TOF M/S m/z 617.53 (C30H37N2O8S2+ requires 617.2). 3H-Indolium, 2-[3-[1-(5-Carboxypentyl)-1,3-dihydro-3,3dimethyl-5-sulfo-2H-indol-2-ylidene]-1-propen-1-yl]-1-ethyl3,3-dimethyl-5-sulfonate (6c). Compound 6c was prepared from compound 3b (0.64 g, 2.4 mmol) and compound 5b (0.86 g, 2.4 mmol) by the procedure utilized for compound 6a (0.37 g, 24%). Rf = 0.70 (RP-C18, 30% acetonitrile−water); 1H NMR (300 MHz, D2O): δ 8.38 (t, 1H, J = 13.5 Hz), 7.78 (s, 2H), 7.73 (t, 2H, J = 7.4 Hz), 7.23 (q, 2H, J = 7.9 Hz), 6.24 (q, 2H, J = 4.7 Hz), 3.97 (m, 4H), 2.23 (t, 2H, J = 7.2 Hz), 1.73− 1.20 (br m, 21H); LC/MS m/z 631.31 (C31H39N2O8S2+ requires 631.21). 3H-Indolium, 2-[3-[1-(5-Carboxypentyl)-1,3-dihydro-3,3dimethyl-5-sulfo-2H-indol-2-ylidene]-1-propen-1-yl]-1-propyl-3,3-dimethyl-5-sulfonate (6d). Compound 6d was prepared from compound 3c (6.5 g, 23 mmol) and compound 5b (8.1 g, 23 mmol) by the procedure utilized for compound 6a (3.1 g, 21%). Rf = 0.49 (RP-C18, 30% acetonitrile−water); 1H NMR (300 MHz, DMSO-d6): δ 8.34 (t, 1H, J = 13.2 Hz), 7.78 (s, 2H), 7.65 (d, 2H, J = 8.0 Hz), 7.39 (m, 2H), 6.56 (q, 2H, J = 13.4 Hz), 4.10 (m, 4H), 1.88 (t, 2H, J = 6.8 Hz), 1.77−1.38 (br m, 21H), 0.96 (t, 3H, J = 7.2 Hz); LC/MS m/z 643.29 (C32H39N2O8S2− requires 643.22). 3H-Indolium, 2-[3-[1-(5-Carboxypentyl)-1,3-dihydro-3,3dimethyl-5-sulfo-2H-indol-2-ylidene]-1-propen-1-yl]-1-butyl3,3-dimethyl-5-sulfonate (6e). Compound 6e was prepared from compound 3d (2.1 g, 7.1 mmol) and compound 5b (2.5 g, 7.1 mmol) by the procedure utilized for compound 6a (1.09 g, 23%). Rf = 0.61 (RP-C18, 30% acetonitrile−water); 1H NMR (400 MHz, DMSO-d6): δ 8.34 (t, 1H, J = 13.4 Hz), 7.86 (s, 2H), 7.79 (d, 2H, J = 7.7 Hz), 7.38 (d, 2H, J = 6.1 Hz), 6.56 (q, 2H, J = 13.6 Hz), 4.11 (m, 4H), 1.92 (t, 2H, J = 6.9 Hz), 1.76− 1.22 (br m, 25H), 0.92 (t, 3H, J = 7.2 Hz); LC/MS m/z 657.28 (C33H41N2O8S2− requires 657.23). 352

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Article

3H-Indolium, 2-[3-[1-(7-Carboxyheptyl)-1,3-dihydro-3,3dimethyl-5-sulfo-2H-indol-2-ylidene]-1-propen-1-yl]-1-ethyl3,3-dimethyl-5-sulfonate (6f). Compound 6f was prepared from compound 3b (0.72 g, 2.27 mmol) and compound 5c (0.93 g, 2.27 mmol) by the procedure utilized for compound 6a (0.31 g, 25%). Rf = 0.6 (RP-C18, 25% acetonitrile−water); 1H NMR (400 MHz, DMSO-d6): δ 8.35 (t, 1H), 7.80 (s, 2H), 7.68−7.66 (m, 2H), 7.40−7.38 (m, 2H), 6.32−6.29 (m, 2H), 4.20−4.10 (m, 4H), 1.98 (t, 2H), 1.80−1.05 (br m, 25H); LC/ MS m/z 656.9 (C33H41N2O8S2− requires 657.23). 3H-Indolium, 2-[3-[1-(7-Carboxyheptyl)-1,3-dihydro-3,3dimethyl-5-sulfo-2H-indol-2-ylidene]-1-propen-1-yl]-1-propyl-3,3-dimethyl-5-sulfonate (6g). Compound 6g was prepared from compound 3c (2.8 g, 10.0 mmol) and compound 5c (3.8 g, 10.0 mmol) by the procedure utilized for compound 6a (2.09 g, 31%). Rf = 0.52 (RP-C18, 25% acetonitrile−water); 1H NMR (400 MHz, DMSO-d6): δ 8.35 (t, 1H), 7.79 (s, 2H), 7.66 (d, 2H), 7.43−7.37 (m, 2H), 6.60−6.45 (m, 2H), 4.15−4.07 (m, 4H), 1.97 (t, 2H), 1.80−1.20 (br m, 24H), 0.96 (t, 3H); LC/ MS m/z 671.04 (C34H43N2O8S2− requires 671.25). 3H-Indolium, 2-[5-[1-(5-Carboxypentyl)-1,3-dihydro-3,3dimethyl-5-sulfo-2H-indol-2-ylidene]-1,3-pentadien-1-yl]-1methyl-3,3-dimethyl-5-sulfonate (6h). A stirred mixture of crude 3a (2.9 g, 11.5 mmol) and malonaldehyde dianil hydrochloride (3.7 g, 13.9 mmol) in a mixture of acetic acid (60 mL) and acetic anhydride (60 mL) under N2 was heated to reflux for 4 h, cooled down to 25 °C, and agitated a heterogeneous mixture by addition of ethyl acetate (120 mL). The resulting mixture was filtered through a paper filter, and the residue was dried under reduced pressure to provide the crude 4e (4.7 g, 96%). A stirred solution of the crude 4e (4.1 g, 9.6 mmol) in pyridine (100 mL) under N2 was treated with crude 5b (3.4 g, 9.6 mmol) at 25 °C. Reaction mixture was stirred at 60 °C for 4 h, cooled down to 25 °C, and agitated a heterogeneous mixture by addition of ethyl acetate (100 mL). Resulting mixture was filtered through a paper filter, and the residue was dried under reduced pressure and purified with flash chromatography (Lichroprep RP-18, 25% acetonitrile− water) to afford 6h as a blue powder (1.35 g, 22%). Rf = 0.63 (RP-C18, 30% acetonitrile−water); 1H NMR (300 MHz, DMSO-d6): δ 7.91 (t, 2H, J = 13.7 Hz), 7.74−7.69 (m, 4H), 7.24 (t, 2H, J = 8.4 Hz), 6.45 (t, 1H, J = 12.5 Hz), 6.16−6.11 (m, 2H), 3.98 (t, 2H, J = 5.9 Hz), 3.49 (s, 3H), 2.11 (t, 2H, J = 7.0 Hz), 1.73−1.34 (br m, 18H); LC/MS m/z 641.27 (C32H37N2O8S2− requires 641.2). 3H-Indolium, 2-[5-[1-(5-Carboxypentyl)-1,3-dihydro-3,3dimethyl-5-sulfo-2H-indol-2-ylidene]-1,3-pentadien-1-yl]-1ethyl-3,3-dimethyl-5-sulfonate (6i). Compound 6i was prepared from compound 3b (3.9 g, 14.6 mmol) and compound 5b (5.1 g, 14.6 mmol) by the procedure utilized for compound 6h (2.1 g, 22%). Rf = 0.58 (RP-C18, 30% acetonitrile−water); 1 H NMR (400 MHz, DMSO-d6): δ 8.34 (t, 2H, J = 13.2 Hz), 7.80 (s, 2H), 7.63 (d, 2H, J = 8.1 Hz), 7.30 (q, 2H, J = 2.8 Hz), 6.58 (t, 1H, J = 12.2 Hz), 6.30 (q, 2H, J = 8.6 Hz), 4.13−4.06 (m, 4H), 1.98 (t, 2H, J = 6.8 Hz), 1.72−1.18 (br m, 21H); LC/ MS m/z 655.24 (C33H39N2O8S2− requires 655.22). 3H-Indolium, 2-[5-[1-(5-Carboxypentyl)-1,3-dihydro-3,3dimethyl-5-sulfo-2H-indol-2-ylidene]-1,3-pentadien-1-yl]-1propyl-3,3-dimethyl-5-sulfonate (6j). Compound 6j was prepared from compound 3c (5.5 g, 19.7 mmol) and compound 5b (6.9 g, 19.7 mmol) by the procedure utilized for compound 6h (2.2 g, 16%). Rf = 0.49 (RP-C18, 30% acetonitrile−water); 1H NMR (400 MHz, DMSO-d6): δ 8.34

(t, 2H, J = 12.8 Hz), 7.79 (s, 2H), 7.61 (d, 2H, J = 7.8 Hz), 7.31 (t, 2H, J = 9.2 Hz), 6.59 (t, 1H, J = 12.1 Hz), 6.30 (q, 2H, J = 3.5 Hz), 4.05 (m, 4H), 1.97 (t, 2H, J = 6.9 Hz), 1.72−1.22 (br m, 21H), 0.92 (t, 3H, J = 7.2 Hz); LC/MS m/z 669.30 (C34H41N2O8S2− requires 669.23). 3H-Indolium, 2-[5-[1-(5-Carboxypentyl)-1,3-dihydro-3,3dimethyl-5-sulfo-2H-indol-2-ylidene]-1,3-pentadien-1-yl]-1butyl-3,3-dimethyl-5-sulfonate (6k). Compound 6k was prepared from compound 3d (2.1 g, 7.1 mmol) and compound 5b (2.5 g, 7.1 mmol) by the procedure utilized for compound 6h (1.2 g, 24%). Rf = 0.42 (RP-C18, 30% acetonitrile−water); 1 H NMR (400 MHz, DMSO-d6): δ 8.34 (t, 2H, J = 12.0 Hz), 7.80 (s, 2H), 7.62 (d, 2H, J = 7.2 Hz), 7.32 (m, 2H), 6.59 (t, 1H, J = 11.5 Hz), 6.31 (m, 2H), 4.08 (m, 4H), 2.04 (t, 2H), 1.77−1.21 (br m, 25H), 0.90−0.79 (m, 3H); LC/MS m/z 683.33 (C35H43N2O8S2− requires 683.25). 3H-Indolium, 2-[5-[1-(7-Carboxyheptyl)-1,3-dihydro-3,3dimethyl-5-sulfo-2H-indol-2-ylidene]-1,3-pentadien-1-yl]-1ethyl-3,3-dimethyl-5-sulfonate (6l). Compound 6l was prepared from compound 3b (1.6 g, 6.0 mmol) and compound 5c (2.3 g, 6.0 mmol) by the procedure utilized for compound 6h (0.90 g, 22%). Rf = 0.48 (RP-C18, 30% acetonitrile−water); 1H NMR (400 MHz, DMSO-d6): δ 8.29 (t, 2H, J = 13.6 Hz), 7.75 (s, 2H), 7.58 (d, 2H, J = 8.4 Hz), 7.26 (d, 2H, J = 8.4 Hz), 6.55 (t, 1H, J = 12.2 Hz), 6.30−6.23 (m, 2H), 4.07−4.03 (m, 4H), 1.86 (t, 2H), 1.70−1.10 (br m, 25H); LC/MS m/z 682.9 (C35H43N2O8S2− requires 683.25). 3H-Indolium, 2-[7-[1-(5-Carboxypentyl)-1,3-dihydro-3,3dimethyl-5-sulfo-2H-indol-2-ylidene]-1,3,5-heptatrien-1-yl]1-methyl-3,3-dimethyl-5-sulfonate (6m). A stirred mixture of crude 3a (3.2 g, 12.5 mmol) and glutaconaldehyde dianil hydrochloride (4.3 g, 15.0 mmol) in anhydrous acetic acid (15 mL) under N2 was heated to 100 °C for 1 h, cooled down to 25 °C, and agitated to a heterogeneous mixture by addition of ethyl acetate (15 mL). The resulting mixture was filtered through a paper filter, and the residue was dried under reduced pressure to provide the crude 4i (4.3 g, 76%). A stirred solution of the crude 4i (7.9 g, 12.5 mmol) in pyridine (80 mL) under N2 was treated with the crude 5b (4.4 g, 12.5 mmol) at 25 °C. The reaction mixture was heated to 40 °C for 1 h, cooled down to 25 °C, and agitated to a heterogeneous mixture by addition of ethyl acetate (85 mL). Resulting mixture was filtered through a paper filter, and the residue was dried under reduced pressure and purified with flash chromatography (Lichroprep RP-18, 30% acetonitrile−water) to afford 6m as a green powder (1.33 g, 16%). Rf = 0.43 (RP-C18, 30% acetonitrile−water); 1H NMR (300 MHz, DMSO-d6): δ 7.80−7.62 (m, 5H), 7.33 (t, 2H, J = 13.2 Hz), 7.17 (t, 2H, J = 7.3 Hz), 6.34 (t, 2H, J = 12.2 Hz), 6.06 (q, 2H, J = 5.2 Hz), 3.92 (m, 2H), 3.44 (s, 3H), 2.10 (t, 2H, J = 7.4 Hz), 1.90−1.20 (br m, 18H); LC/MS m/z 667.33 (C34H39N2O8S2− requires 667.22). 3H-Indolium, 2-[7-[1-(5-Carboxypentyl)-1,3-dihydro-3,3dimethyl-5-sulfo-2H-indol-2-ylidene]-1,3,5-heptatrien-1-yl]1-ethyl-3,3-dimethyl-5-sulfonate (6n). Compound 6n was prepared from compound 3b (4.7 g, 17.5 mmol) and compound 5b (5.94 g, 17.5 mmol) by the procedure utilized for compound 6m (2.7 g, 23%). Rf = 0.38 (RP-C18, 30% acetonitrile−water); 1H NMR (400 MHz, DMSO-d6): δ 7.87 (t, 2H, J = 12.8 Hz), 7.72 (m, 3H), 7.50 (d, 2H, J = 11.9 Hz), 7.28 (d, 2H, J = 8.2 Hz), 6.58−6.49 (m, 2H), 6.36 (d, 2H, J = 13.7 Hz), 4.10−4.03 (m, 4H), 1.98 (t, 2H), 1.70−1.22 (br m, 21H); LC/MS m/z 681.28 (C35H41N2O8S2− requires 681.23). 353

dx.doi.org/10.1021/bc200232d | Bioconjugate Chem. 2012, 23, 350−362

Bioconjugate Chemistry

Article

dene]-1-propen-1-yl]-1-ethyl-3,3-dimethyl-5-sulfonate (7c). Red powder (106.4 mg, 82%); Rf = 0.65 (RP-C18, 30% acetonitrile−water); 1H NMR (400 MHz, DMSO-d6): δ 8.32 (t, 1H, J = 13.4 Hz), 8.02 (t, 1H, J = 4.9 Hz), 7.80 (s, 2H), 7.67 (m, 2H), 7.39 (q, 2H, J = 3.0 Hz), 6.96 (q, 1H, J = 9.9 Hz), 6.53 (q, 2H, J = 4.3 Hz), 6.23 (m, 2H), 4.17−4.09 (m, 4H), 3.21 (m, 2H), 2.04 (t, 2H, J = 6.9 Hz), 1.69−1.28 (br m, 23H); LC/MS m/z 746.27 (C35H44N3O9S3− requires 746.22). 3H-Indolium, 2-[3-[1-[6-[(2-(Vinylsulfonyl)ethyl)amino]-6oxohexyl]-1,3-dihydro-3,3-dimethyl-5-sulfo-2H-indol-2-ylidene]-1-propen-1-yl]-1-propyl-3,3-dimethyl-5-sulfonate (7d). Red powder (114.6 mg, 84%); Rf = 0.55 (RP-C18, 30% acetonitrile−water); 1H NMR (400 MHz, DMSO-d6): δ 8.35 (t, 1H, J = 13.4 Hz), 7.99 (m, 1H), 7.79 (s, 2H), 7.66 (d, 2H, J = 7.8 Hz), 7.39 (q, 2H, J = 8.6 Hz), 6.96 (q, 1H, J = 9.9 Hz), 6.51 (t, 2H, J = 9.2 Hz), 6.23 (m, 2H), 4.09 (m, 4H), 3.21 (m, 2H), 2.05 (m, 2H), 1.76−1.24 (br m, 23H), 0.96 (t, 3H, J = 6.9 Hz); LC/MS m/z 760.30 (C36H46N3O9S3− requires 760.24). 3H-Indolium, 2-[3-[1-[6-[(2-(Vinylsulfonyl)ethyl)amino]-6oxohexyl]-1,3-dihydro-3,3-dimethyl-5-sulfo-2H-indol-2-ylidene]-1-propen-1-yl]-1-butyl-3,3-dimethyl-5-sulfonate (7e). Red powder (108.7 mg, 81.9%); Rf = 0.60 (RP-C18, 30% acetonitrile−water); 1H NMR (400 MHz, DMSO-d6): δ 8.36 (t, 1H), 8.02 (t, 1H), 7.81 (s, 2H), 7.68 (d, 2H), 7.40 (d, 2H), 6.97 (d, 1H), 6.53 (d, 2H), 6.23 (m, 2H), 4.12 (m, 4H), 3.22 (t, 2H), 2.05 (t, 2H), 1.75−1.18 (br m, 27H), 0.93 (t, 3H); LC/MS m/z 774.53 (C37H48N3O9S3− requires 774.26). 3H-Indolium, 2-[3-[1-[8-[(2-(Vinylsulfonyl)ethyl)amino]-8oxooctyl]-1,3-dihydro-3,3-dimethyl-5-sulfo-2H-indol-2-ylidene]-1-propen-1-yl]-1-ethyl-3,3-dimethyl-5-sulfonate (7f). Red powder (71.1 mg, 53%); Rf = 0.55 (RP-C18, 30% acetonitrile−water); 1H NMR (400 MHz, DMSO-d6): δ 8.35 (t, 1H), 7.96 (t, 1H), 7.79 (s, 2H), 7.69−7.65 (m, 2H), 7.40− 7.38 (m, 2H), 6.99−6.93 (m, 1H), 6.51 (d, 2H), 6.25−6.21 (m, 1H), 4.17−4.10 (m, 4H), 3.62−3.55 (m, 2H), 3.20−3.09 (m, 2H), 2.01 (t, 2H), 1.70−1.10 (br m, 25H); LC/MS m/z 774.00 (C37H48N3O9S3− requires 774.26). 3H-Indolium, 2-[3-[1-[8-[(2-(Vinylsulfonyl)ethyl)amino]-8oxooctyl]-1,3-dihydro-3,3-dimethyl-5-sulfo-2H-indol-2-ylidene]-1-propen-1-yl]-1-propyl-3,3-dimethyl-5-sulfonate (7g). Red powder (63.2 mg, 46%); Rf = 0.45 (RP-C18, 30% acetonitrile−water); 1H NMR (300 MHz, DMSO-d6): δ 8.35 (t, 1H), 7.96 (t, 1H), 7.80 (s, 2H), 7.66 (d, 2H), 6.99−6.93 (m, 2H), 6.54−6.49 (m, 2H), 6.25−6.20 (m, 2H), 4.15−4.05 (m, 4H), 3.65−3.57 (m, 2H), 3.26−3,20 (m, 2H), 2.01 (t, 2H), 1.80−1.17 (br m, 24H), 0.97 (t, 3H); LC/MS m/z 788.27 (C38H50N3O9S3− requires 788.27). 3H-Indolium, 2-[5-[1-[6-[(2-(Vinylsulfonyl)ethyl)amino]-6oxohexyl]-1,3-dihydro-3,3-dimethyl-5-sulfo-2H-indol-2-ylidene]-1,3-pentadien-1-yl]-1-methyl-3,3-dimethyl-5-sulfonate (7h). Blue powder (107.9 mg, 81%); Rf = 0.63 (RP-C18, 30% acetonitrile−water); 1H NMR (400 MHz, DMSO-d6): δ 8.34 (t, 2H, J = 13.5 Hz), 8.00−7.94 (m, 3H), 7.79 (s, 2H), 7.61 (t, 2H, J = 8.0 Hz), 7.30 (q, 2H, J = 3.0 Hz), 6.97 (q, 1H, J = 10.5 Hz), 6.57 (t, 1H, J = 12.2 Hz), 6.31−6.21 (m, 4H), 4.06 (m, 2H), 3.58 (s, 3H), 3.21 (t, 2H, J = 6.6 Hz), 2.02 (t, 2H, J = 7.0 Hz), 1.67−1.24 (br m, 20H); LC/MS m/z 758.47 (C36H44N3O9S3− requires 758.22). 3H-Indolium, 2-[5-[1-[6-[(2-(Vinylsulfonyl)ethyl)amino]-6oxohexyl]-1,3-dihydro-3,3-dimethyl-5-sulfo-2H-indol-2-ylidene]-1,3-pentadien-1-yl]-1-ethyl-3,3-dimethyl-5-sulfonate (7i). Blue powder (107.3 mg, 79%); Rf = 0.58 (RP-C18, 30% acetonitrile−water); 1H NMR (300 MHz, DMSO-d6): δ 8.35

3H-Indolium, 2-[7-[1-(5-Carboxypentyl)-1,3-dihydro-3,3dimethyl-5-sulfo-2H-indol-2-ylidene]-1,3,5-heptatrien-1-yl]1-propyl-3,3-dimethyl-5-sulfonate (6o). Compound 6o was prepared from compound 3c (5.3 g, 18.7 mmol) and compound 5b (6.6 g, 18.7 mmol) by the procedure utilized for compound 6m (2.60 g, 20%). Rf = 0.37 (RP-C18, 30% acetonitrile−water); 1H NMR (300 MHz, DMSO-d6): δ 7.92− 7.73 (m, 5H), 7.62 (d, 2H, J = 8.1 Hz), 7.30 (t, 2H, J = 7.6 Hz), 6.50 (t, 2H), 6.38 (q, 2H, J = 4.0 Hz), 4.04 (m, 4H), 2.01 (t, 2H, J = 6.9 Hz), 1.75−1.23 (br m, 21H), 0.94 (t, 3H, J = 7.3 Hz); LC/MS m/z 695.29 (C36H43N2O8S2− requires 695.25). 3H-Indolium, 2-[7-[1-(5-Carboxypentyl)-1,3-dihydro-3,3dimethyl-5-sulfo-2H-indol-2-ylidene]-1,3,5-heptatrien-1-yl]1-butyl-3,3-dimethyl-5-sulfonate (6p). Compound 6p was prepared from compound 3d (2.1 g, 7.1 mmol) and compound 5b (2.5 g, 7.1 mmol) by the procedure utilized for compound 6m (1.26 g, 25%). Rf = 0.35 (RP-C18, 30% acetonitrile−water); 1 H NMR (400 MHz, DMSO-d6): δ 7.79−7.73 (m, 5H), 7.61 (d, 2H, J = 6.8 Hz), 7.29 (q, 2H, J = 3.5 Hz), 6.56 (t, 2H, J = 12.1 Hz), 6.36 (d, 2H, J = 13.6 Hz), 4.04 (m, 4H), 2.01 (t, 2H), 1.50−1.33 (br m, 25H), 0.90 (t, 3H, J = 7.1 Hz); LC/MS m/z 709.34 (C37H45N2O8S2− requires 709.26). General Synthesis of Cyanine Dyes with Vinylsulfone. A stirred solution of 6a−p (0.174 mmol) in DMF (22 mL) under N2 was heated to 55 °C and treated with a solution of disuccinimdyl carbonate (DSC, 135.0 mg, 0.527 mmol) in a mixture of pyridine (0.11 mL) and DMF (2.5 mL). Resulting mixture was stirred at 55 °C for 1 h, cooled down to 25 °C, and agitated a heterogeneous mixture by the addition of ethyl acetate (30 mL). Resulting mixture was filtered through a paper filter, and the residue was washed with ethyl acetate and diethyl ether, dried under reduced pressure, and redissolved in DMF (20 mL). The solution was treated with N,N-diisopropylethylamine (0.30 mL) and a solution of 2-(2-chloroethylsulfonyl)ethylamine hydrochloride (36 mg, 0.18 mmol)32 in DMF (1 mL). The reaction mixture was stirred at 25 °C for 24 h under N2 and quenched with distilled water (20 mL). The aqueous phase was washed with dichloromethane (3 × 50 mL) and concentrated under reduced pressure. Flash chromatography provided (RP-C18, 15% acetonitrile−water) VS cyanine dye, 7a−p. 3H-Indolium, 2-[3-[1-[5-[(2-(Vinylsulfonyl)ethyl)amino]-5oxopentyl]-1,3-dihydro-3,3-dimethyl-5-sulfo-2H-indol-2-ylidene]-1-propen-1-yl]-1-ethyl-3,3-dimethyl-5-sulfonate (7a). Red powder (93.6 mg, 75.8%); Rf = 0.70 (RP-C18, 30% acetonitrile−water); 1H NMR (400 MHz, DMSO-d6): δ 8.34 (t, 1H, J = 13.7 Hz), 8.07 (m, 1H), 7.80 (s, 2H), 7.67 (d, 2H, J = 7.6 Hz), 7.39 (q, 2H, J = 3.9 Hz), 6.94 (q, 1H, J = 10.1 Hz), 6.51 (d, 2H, J = 13.3 Hz), 6.21 (m, 2H), 4.08 (m, 4H), 3.21 (t, 2H, J = 6.7 Hz), 2.04 (t, 2H, J = 6.8 Hz), 1.68−1.23 (br m, 20H); LC/MS m/z 734.07 (C34H44N3O9S3+ requires 734.22). 3H-Indolium, 2-[3-[1-[6-[(2-(Vinylsulfonyl)ethyl)amino]-6oxohexyl]-1,3-dihydro-3,3-dimethyl-5-sulfo-2H-indol-2-ylidene]-1-propen-1-yl]-1-methyl-3,3-dimethyl-5-sulfonate (7b). Red powder (107.9 mg, 84%); Rf = 0.71 (RP-C18, 30% acetonitrile−water); 1H NMR (300 MHz, DMSO-d6): δ 8.31 (t, 1H), 8.06 (t, 1H, J = 5.8 Hz), 7.64 (s, 2H), 7.65 (m, 2H), 7.38 (q, 2H, J = 4.3 Hz), 6.97 (q, 1H, J = 9.9 Hz), 6.46 (q, 2H, J = 5.2 Hz), 6.23 (m, 2H), 4.08 (m, 4H), 3.64 (s, 3H), 3.21 (t, 2H, J = 6.7 Hz), 2.04 (t, 2H, J = 6.8 Hz), 1.68−1.23 (br m, 20H); LC/MS m/z 732.47 (C34H42N3O9S3− requires 732.21). 3H-Indolium, 2-[3-[1-[6-[(2-(Vinylsulfonyl)ethyl)amino]-6oxohexyl]-1,3-dihydro-3,3-dimethyl-5-sulfo-2H-indol-2-yli354

dx.doi.org/10.1021/bc200232d | Bioconjugate Chem. 2012, 23, 350−362

Bioconjugate Chemistry

Article

2H, J = 8.1 Hz), 7.29 (q, 2H, J = 8.4 Hz), 6.96 (q, 1H, J = 10.1 Hz), 6.54 (t, 2H, J = 12.1 Hz), 6.36 (t, 2H, J = 14.1 Hz), 6.23 (m, 2H), 4.03 (m, 4H), 3.22 (m, 2H), 2.03 (t, 2H, J = 7.2 Hz), 1.74−1.22 (br m, 23H), 0.93 (t, 3H, J = 7.2 Hz); LC/MS m/z 812.35 (C40H50N3O9S3− requires 812.27). 3H-Indolium, 2-[7-[1-[6-[(2-(Vinylsulfonyl)ethyl)amino]-6oxohexyl]-1,3-dihydro-3,3-dimethyl-5-sulfo-2H-indol-2-ylidene]-1,3,5-heptatrien-1-yl]-1-butyl-3,3-dimethyl-5-sulfonate (7p). Green powder (99.6 mg, 69%); Rf = 0.44 (RP-C18, 30% acetonitrile−water); 1H NMR (400 MHz, DMSO-d6): δ 8.02−7.72 (m, 6H), 7.63 (d, 2H), 7.28 (m, 2H), 6.98 (d, 1H), 6.55 (t, 2H), 6.36 (d, 2H), 6.28−6.22 (m, 2H), 4.05 (m, 4H), 3.23 (m, 2H), 2.02 (t, 2H), 1.70−1.21 (br m, 23H), 0.92 (t, 3H); LC/MS m/z 826.80 (C41H52N3O9S3− requires 826.29). Measurement of Optical Properties. The synthesized 7a−7p dye compounds were dissolved in distilled water at 1 mg/100 uL, and then were diluted to 0.01 mM, 0.005 mM, and 0.0025 mM for measurement of absorbance and fluorescence spectrum with UV/vis spectrophotometer and fluorescence spectrophotometer. Beer−Lambert equation was used to determine the molar extinction coefficient of each compound at the maximum absorbable wavelength, and statistical measurements were done to ensure that all values are valid. The maximum absorbable wavelength was set at 0.0025 mM and 25 °C, and the fluorescence spectrum and the maximum fluorescence wavelength were measured. Reactivity Test. Dependence of Spacer. Stock solutions were prepared by dissolving bovine serum albumin (BSA, 10 mg/mL) in 0.1 M sodium phosphate buffer (pH 9.5) and 7a, 7c, 7f, 7g, 7i, 7l, and 7n (10 mg/mL) in distilled water. Each dye solution (7a (11.0 μL), 7c (11.2 μL), 7f (11.6 μL), 7g (11.9 μL), 7i (11.7 μL), 7l (12.1 μL), and 7n (12.1 μL)) was reacted with the BSA solution (1 mL) in equivalent at 25 °C for 6 h. The labeling reactions were then monitored every 1 h via HPLC at 550, 650, and 750 nm (Gemini C6-Phenyl Column, 15−25% acetonitrile−water, 1.5 mL/min). Dependence of pH. Stock solutions (10 mg/mL) were prepared by dissolving BSA in 0.1 M sodium phosphate buffers (pH 8.5, 9, and 9.5), PBS, and distilled water and 7g, 7l, and 7n (10 mg/mL) in distilled water. Each protein solution (1 mL) was reacted with each dye solution (7g (11.9 μL), 7l (12.1 μL), and 7n (12.1 μL)) in equivalent at 25 °C for 6 h. The labeling reactions were then monitored every 1 h via HPLC at 550, 650, and 750 nm (Gemini C6-Phenyl Column, 15−25% acetonitrile−water, 1.5 mL/min). Dependence of Temperature. Stock solutions (10 mg/mL) were prepared by dissolving BSA in 0.1 M sodium phosphate buffer (pH 9.5) and 7g, 7l, and 7n (10 mg/mL) in distilled water. The protein solution (1 mL) was reacted with each dye solution (7g (11.9 μL), 7l (12.1 μL), and 7n (12.1 μL)) in equivalent at 25 and 36.5 °C for 4 h. The labeling reactions were then monitored every 1 h via HPLC at 550, 650, and 750 nm (Gemini C6-Phenyl Column, 15−25% acetonitrile−water, 1.5 mL/min). Dependence of Buffer Concentration. Stock solutions (10 mg/mL) were prepared by dissolving BSA in pH 9.5 sodium phosphate buffers (0.5 M, 0.1 M, 0.01 M, and 0.001 M) and 7g, 7l, and 7n (10 mg/mL) in distilled water. Each protein solution (1 mL) was reacted with each dye solution (7g (11.9 μL), 7l (12.1 μL), and 7n (12.1 μL)) in equivalent at 25 °C for 3 h. The labeling reactions were then monitored every 0.5 h via HPLC at 550, 650, and 750 nm (Gemini C6-Phenyl Column, 15−25% acetonitrile−water, 1.5 mL/min).

(t, 2H, J = 13.0 Hz), 8.00 (t, 1H, J = 5.2 Hz), 7.81 (s, 2H), 7.62 (d, 2H, J = 10.3 Hz), 7.31 (d, 2H, J = 7.5 Hz), 6.98 (q, 1H, J = 9.9 Hz), 6.62 (t, 1H, J = 12.3 Hz), 6.32−6.21 (m, 4H), 4.13− 4.07 (m, 4H), 3.25 (m, 2H), 2.03 (t, 2H, J = 7.1 Hz), 1.68− 1.23 (br m, 23H); LC/MS m/z 774.29 (C37H48N3O9S3+ requires 774.25). 3H-Indolium, 2-[5-[1-[6-[(2-(Vinylsulfonyl)ethyl)amino]-6oxohexyl]-1,3-dihydro-3,3-dimethyl-5-sulfo-2H-indol-2-ylidene]-1,3-pentadien-1-yl]-1-propyl-3,3-dimethyl-5-sulfonate (7j). Blue powder (124.9 mg, 88%); Rf = 0.55 (RP-C18, 30% acetonitrile−water); 1H NMR (300 MHz, DMSO-d6): δ 8.34 (t, 2H, J = 12.6 Hz), 7.97 (t, 1H), 7.80 (s, 2H), 7.62 (d, 2H, J = 7.8 Hz), 7.31 (t, 2H), 6.95 (q, 1H, J = 9.8 Hz), 6.58 (t, 1H, J = 11.9 Hz), 6.33−6.21 (m, 4H), 4.06 (m, 4H), 3.21 (t, 2H, J = 6.8 Hz), 2.03 (t, 2H, J = 6.9 Hz), 1.74−1.24 (br m, 23H), 0.93 (t, 3H, J = 7.2 Hz); LC/MS m/z 786.34 (C38H48N3O9S3− requires 786.26). 3H-Indolium, 2-[5-[1-[6-[(2-(Vinylsulfonyl)ethyl)amino]-6oxohexyl]-1,3-dihydro-3,3-dimethyl-5-sulfo-2H-indol-2-ylidene]-1,3-pentadien-1-yl]-1-butyl-3,3-dimethyl-5-sulfonate (7k). Blue powder (107.3 mg, 77%); Rf = 0.51 (RP-C18, 30% acetonitrile−water); 1H NMR (400 MHz, DMSO-d6): δ 8.35 (t, 2H, J = 12.4 Hz), 8.00 (t, 1H), 7.80 (s, 2H), 7.61 (d, 2H, J = 8.3 Hz), 7.30 (q, 2H, J = 4.2 Hz), 6.96 (m, 1H), 6.58 (t, 1H), 6.31−6.21 (m, 4H), 4.07 (m, 4H), 3.22 (t, 2H, J = 6.6 Hz), 2.03 (t, 2H, J = 7.0 Hz), 1.53−1.22 (br m, 27H), 0.91 (t, 3H, J = 7.2 Hz); LC/MS m/z 800.36 (C39H50N3O9S3− requires 800.27). 3H-Indolium, 2-[5-[1-[8-[(2-(Vinylsulfonyl)ethyl)amino]-8oxooctyl]-1,3-dihydro-3,3-dimethyl-5-sulfo-2H-indol-2-ylidene]-1,3-pentadien-1-yl]-1-ethyl-3,3-dimethyl-5-sulfonate (7l). Blue powder (56.9 mg, 41%); Rf = 0.40 (RP-C18, 30% acetonitrile−water); 1H NMR (400 MHz, DMSO-d6): δ 8.35 (t, 2H), 7.96 (t, 1H), 7.80 (s, 2H), 7.80−7.61 (m, 2H), 7.31 (d, 2H), 6.70−6.93 (m, 1H), 6.57 (t, 1H), 6.32−6.21 (m, 3H), 4.13−4.08 (m, 4H), 3.62−3.58 (m, 2H), 3.28−3.20 (m, 2H), 2.01 (t, 2H), 1.65−1.18 (br m, 25H); LC/MS m/z 800.00 (C39H50N3O9S3− requires 800.27). 3H-Indolium, 2-[7-[1-[6-[(2-(Vinylsulfonyl)ethyl)amino]-6oxohexyl]-1,3-dihydro-3,3-dimethyl-5-sulfo-2H-indol-2-ylidene]-1,3,5-heptatrien-1-yl]-1-methyl-3,3-dimethyl-5-sulfonate (7m). Green powder (117.7 mg, 84%); Rf = 0.57 (RPC18, 30% acetonitrile−water); 1H NMR (400 MHz, DMSOd6): δ 8.00 (m, 1H), 7.88−7.86 (m, 2H), 7.73 (m, 3H), 7.61 (t, 2H, J = 6.6 Hz), 7.28 (q, 2H, J = 8.1 Hz), 7.10−6.90 (m, 1H), 6.53−6.49 (m, 2H), 6.34−6.22 (m, 4H), 4.02 (m, 2H), 3.57 (s, 3H), 3.22 (t, 2H), 2.03 (t, 2H), 1.74−1.22 (br m, 20H); LC/ MS m/z 784.47 (C38H46N3O9S3− requires 784.24). 3H-Indolium, 2-[7-[1-[6-[(2-(Vinylsulfonyl)ethyl)amino]-6oxohexyl]-1,3-dihydro-3,3-dimethyl-5-sulfo-2H-indol-2-ylidene]-1,3,5-heptatrien-1-yl]-1-ethyl-3,3-dimethyl-5-sulfonate (7n). Green powder (103.8 mg, 73%); Rf = 0.50 (RP-C18, 30% acetonitrile−water); 1H NMR (400 MHz, DMSO-d6): δ 8.03 (t, 1H), 7.88−7.60 (m, 7H), 7.29 (q, 2H, J = 3.2 Hz), 6.97 (m, 1H), 6.54 (m, 2H), 6.39−6.22 (m, 4H), 4.10−4.03 (m, 4H), 3.23 (m, 2H), 2.03 (t, 2H, J = 7.0 Hz), 1.62−1.12 (br m, 23H); LC/MS m/z 800.32 (C39H50N3O9S3+, calcd 800.27). 3H-Indolium, 2-[7-[1-[6-[(2-(Vinylsulfonyl)ethyl)amino]-6oxohexyl]-1,3-dihydro-3,3-dimethyl-5-sulfo-2H-indol-2-ylidene]-1,3,5-heptatrien-1-yl]-1-propyl-3,3-dimethyl-5-sulfonate (7o). Green powder (102.9 mg, 70%); Rf = 0.48 (RP-C18, 30% acetonitrile−water); 1H NMR (400 MHz, DMSO-d6): δ 7.98 (m, 1H), 7.87 (t, 2H, J = 10.6 Hz), 7.73 (m, 3H), 7.61 (d, 355

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Stability Test. Water Stability. Stock solutions of 7g, 7l, and 7n (10 mg/mL in distilled water, 10 μL) were diluted with distilled water (1 mL), and the changes in the absorption intensities were then monitored for 24 h via HPLC at 550, 650, and 750 nm (Bondapak C18 10 μm 125A, 15−25% acetonitrile−water, 1.0 mL/min). pH Stability. Stock solutions of 7g, 7l, and 7n (10 mg/mL in distilled water, 5 μL) were added to 0.1 M sodium phosphate buffer (100 μL) in pH 5, 7.5, and 10 and the changes in absorption intensities were then monitored for 24 h via HPLC at 550, 650, and 750 nm (Bondapak C18 10 μm 125A, 15−25% acetonitrile−water, 1.0 mL/min). Dye Conjugation Test. For the first test, stock solutions (2 mg/mL) were prepared by dissolving BSA in 0.1 M sodium phosphate buffers (pH 9.5) and 7g, 7l, and 7n (10 mg/mL) in distilled water. Each protein solution (1 mL) was reacted with each dye solution (7g (11.9 μL), 7l (12.1 μL), and 7n (12.1 μL)) in equivalent at 25 °C for 4 h. Each resulting mixture was filtered using disposable PD-10 desalting column (GE Healthcare UK Ltd.) and VIVASPIN 500 (5 kDa, Sartorius Stedim Biotech GmbH, Germany). Labeled proteins were diluted in distilled water, and then absorption intensity values (A280, Adye) at each maximum absorption wavelength BSA (280 nm), 7g (280 nm, 549 nm), 7l (280 nm, 647 nm), and 7n (280 nm, 749 nm)) were measured using UV/vis spectrophotometer. Dye/ protein ratio (D/P) was calculated using the equation (D/P = (AdyeEprot)/((A280 − AD.C.Adye)Edye)).12 The extinction coefficient of dye (Edye) or BSA (Eprot) was calculated using Beer− Lambert equation. The denominator factor for dye absorption at 280 nm (AD.C.) was obtained by dividing absorption intensity value at 280 nm by absorption intensity value at maximum absorption wavelength. For the second test, the above BSA solution was mixed with each dye solution at a molar ratio of 1:7 at 25 °C for 4 h. The labeling reactions were then monitored for a period of 1 h, 2 h, and 4 h via HPLC at 550, 650, and 750 nm (Bondapak C18 10 μm 125A, 15−25% acetonitrile−water, 1.0 mL/min). Labeling Test. Dye stock solutions (10 mg/mL) were prepared by dissolving Cy3-NHS in DMF and 7g in distilled water. Protein molecular weight markers (Broad, Code No.3452 available from Takara Bio Inc.), including myosin (200 kDa), β-galactosidase (116 kDa), phosphorylase B (97.2 kDa), serum albumin (66.4 kDa), ovalbumin (44.3 kDa), carbonic anhydrase (29 kDa), trypsin inhibitor (20.1 kDa), lysozyme (14.3 kDa), aprotinin (6.5 kDa), EDTA, NaCl, glycerol, and tris buffer solution, were respectively filtered using VIVASPIN 500 (5 kDa, Sartorius Stedim Biotech GmbH, Germany). Those filtered proteins (1 μL) were placed in each of 10 e-tubes and diluted with 0.1 M sodium phosphate buffer solution (pH 9.5, 18 μL). The stock solutions of Cy3-NHS (0.25 μL, 0.125 μL, 0.0625 μL, 0.025 and 0.0025 μL) and 7g (0.25 μL, 0.125 μL, 0.0625 μL, 0.025, and 0.0025 μL) were added to each of protein solution in ten e-tubes, and solvents (DMF or distilled water) were appropriately added to adjust the reaction volume (20 μL). The mixtures were vigorously mixed using a vortex shaker and each e-tube was incubated in a heating block at 25 °C for 4 h. SDS-PAGE gel electrophoresis (125 V, 2 h) was employed to separate labeled proteins, and then the fluorescent imaging was observed via Geliance 600.

protocol, and then treated with potassium hydroxide to provide 2,3,3-trimethyl-3H-indole-5-sulfonic acid, potassium salt (2) in Scheme 2. The indole compound 2 was converted to its Nalkylated compound 3 with corresponding alkylating agents. The Methyl group on C-2 in 3 was condensed with acetanilidovinyl moiety in the presence of acetic anhydride to produce compound 4. In order to induce the VS reactive group, the indole intermediate 5 that has a fixed length of carbon linkers (spacer) with carboxylic acid group is a prerequisite. Accordingly, the intermediate 5 having five, six, or eight carbons was synthesized in 1,2-dichlorobenzene under refluxing condition. The asymmetrical cyanine dye 6 was prepared by reacting with intermediates 4 and 5 in the presence of pyridine.12,25,26 Finally, the carboxylic group of 6 was activated to ester by using DSC and pyridine, which was subsequently reacted with VS intermediate, 2-(2′-chloroethylsulfonyl)ethylamine hydrochloride,32 under the polar condition of Hünig’s base and DMF to afford the desired VS cyanine dye 7. Especially, during column purification process, alcoholic solvents such as methanol must be excluded, since they can react with activated VS group by nucleophilic attack. Optical Properties. Optical data of the synthesized VS cyanine dyes are shown in Table 1. Absorption and emission Table 1. Optical Data of VS Cyanine Dyes (Absorption Maximum Wavelength (λabs), Extinction Coefficient (εmax), and Emission Maximum Wavelength (λem)) dye no.

m

n

R

λAbs (nm)

εmax (×10 L/ molcm)

λem (nm)

7a 7b 7c 7d 7e 7f 7g 7h 7i 7j 7k 7l 7m 7n 7o 7p

1 1 1 1 1 1 1 2 2 2 2 2 3 3 3 3

1 2 2 2 2 4 4 2 2 2 2 4 2 2 2 2

CH2CH3 CH3 CH2CH3 CH2CH2CH3 CH2CH2CH2CH3 CH2CH3 CH2CH2CH3 CH3 CH2CH3 CH2CH2CH3 CH2CH2CH2CH3 CH2CH3 CH3 CH2CH3 CH2CH2CH3 CH2CH2CH2CH3

549 549 549 549 550 549 549 646 647 648 647 647 746 746 749 749

0.87 0.75 1.03 1.12 1.33 1.07 1.46 1.70 1.41 1.84 2.45 2.72 1.12 2.25 1.86 1.20

567 565 566 568 567 568 570 670 668 672 670 671 779 782 780 779

wavelengths of cyanine dyes depend on the number of double bonds between two indole compounds, and thus, our synthesized VS cyanine dyes were classified into 3 groups (7a−7g, 7h−7l, and 7m−7p). It is shown that variations in the length of the spacer between the fluorophores and VS (n) or the alkyl groups on indoles (R) have relatively little effect on the wavelength of VS cyanine dyes. Among them, the highest extinction coefficient was observed with 7g (m = 1), 7l (m = 2), and 7n (m = 3). In Figure 1, absorbance and fluorescence wavelength spectra are marked with matched maximum absorbance, absorbance at maximum fluorescence wavelength, and fluorescence intensity. All spectra showed approximately 20 nm of Stokes shift. Reactivity. Various spacers, pH, temperature, and buffer concentration were applied to evaluate reactivity of VS cyanine

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RESULTS Synthesis. p-Hydrazinobenzenesulfonic acid (1) was reacted with 3-methyl-2-butanone via Fisher Indole synthesis 356

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7g, with the same absorption wavelength at ∼550 nm (m = 1), 7g showed the highest labeling rate. In the case of 7i and 7l, with ∼650 nm (m = 2), 7l with an eight-carbon spacer showed much stronger labeling performance than 7i. 7n with 750 nm of the absorption wavelength (m = 3) reached the saturation point within one hour, which was faster than any other dyes compared here. Compounds with the longest spacers exhibited faster conjugation rate compared to other spacers. Considering that BSA (66 430 Da) is at least 80 times larger in size than VS dyes, flexibility endowed by long spacers may provide better accessibility to the target molecule for those compounds. Dependence of pH. To establish a proper conjugation method of these novel VS cyanine dyes, the previously chosen 7g, 7l, and 7n dyes were used to optimize the reaction time and yield with various pHs. Water was used as the solvent for VS dyes. pH was kept basic, as most commercially available NHS ester dyes are recommended to be used at pH 9.0. Because most commercially available proteins are sold as diluted in PBS, sodium phosphate buffer at pH 9.5, 9.0, and 8.5; PBS; and distilled water were used as solvents. The yields were recorded for comparison every hour for 6 h. The result showed that labeling rates of tested dyes increased in proportion to the pH of labeling solutions (Figure 3). This tendency agreed with the specific characteristics of reactions with the VS group.16,22,23,35 In the case of 7l and 7n, binding reaction was saturated within 2 h at pH 9.5. Dependence of Temperature. This experiment lies in determining the effects of temperature variation, as this is an important factor for conjugation. Depending on manufacturers of proteins and antibodies, products in solid form are to be kept at between 2 °C and room temperature, and −20 °C for buffer diluted ones. Most references indicated room temperature as their optimal conjugation temperature, without any cases above the body temperature. The reactions between BSA and VS reactive dyes are known to take place at 25 °C and pH 9.5.12 We tried to determine the point where reaction time could be shortened while avoiding high temperature. We postulated the body temperature (∼36.5 °C) as the limit. As done in previous experiments, molar equivalent amounts of BSA and dye were used to measure the reaction time of dye consumption rate. The labeling rates of VS cyanine dyes were expected to increase in proportion to the temperature. Protein labeling at the body temperature becomes saturated within 1 h, while at 25 °C relatively slower binding was observed (Figure 4). It is evident that reaction speed usually increases with temperature. However, it was quite remarkable that the reaction continued at high pH 9.5 and at 25 °C. In the case of compound 7g, the reaction process was about 20% slower than that at 25 °C at the 1 h point, but in general, the other three compounds reached the maximum yield after 2 h. The reaction certainly became faster with temperature. However, considering the stability and safety of the conjugation target (protein, antibody, etc), we concluded that room temperature (25 °C) would be more appropriate. Dependence of Buffer Concentration. This experiment was to find the optimal molar concentration of sodium phosphate buffer, because it would have minimal effect on the stability of the dye while achieving a high conjugation yield. The previously optimized reaction temperature of 25 °C was used, and sodium phosphate buffer was made in-house. As seen in previous results, compound 7n showed the fastest reaction time, followed by compounds 7l and 7g. However, in this experiment, the reactions for all three compounds could be

Figure 1. Absorption and emission spectra of 7g, 7l, and 7n in distilled water.

dyes with biomolecules, and BSA was chosen as a target protein.33,34 All tests were analyzed by measuring the relative amounts of conjugated and free dyes via HPLC. Through our various reactivity tests, the conjugation reaction condition of VS dyes was optimized when BSA was dissolved in pH 9.5, 0.5 M sodium phosphate buffer at 36.5 °C for 1 h. Dependence of Spacer. To test the variability of reaction time depending on the length of the spacer where the VS group is incorporated, a cyanine dye with equal wavelength was chosen for comparison. Each dye and BSA were used at the same molar concentration, and HPLC analysis was done at every hour for 6 h. Although the BSA and dye have the same molar equivalent weight, it was difficult to measure how many dye molecules were conjugated to the BSA molecule, because it is possible for big BSA molecules to conjugate to multiple dye molecules. Therefore, dye consumption ratio was calculated by using HPLC analysis data of the dye preconjugation and postconjugation. Here, we observed the trend of dye consumption to see how the length of the spacer affected the reaction time. For the spacer-dependent reactivity test, dyes with the highest extinction coefficients (7g, 7l, and 7n) and those with ethyl groups as R on indole (7a, 7c, 7f, and 7i) were selected. As a result, dyes with longer spacers were observed to have faster binding affinity to BSA (Figure 2). Among 7a, 7c, 7f, and

Figure 2. Spacer-dependent reactivity of VS cyanine dyes. 7a, 7c, 7f, 7g, 7i, 7l, and 7n were mixed with BSA in equivalent at 25 °C for 6 h. Labeling reactions were then monitored every 1 h via HPLC. Dyes with longer spacer (n = 2 or 4) showed faster conjugation with BSA. 357

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Figure 3. pH-dependent reactivity of VS cyanine dyes. 7g (λabs 550 nm, left), 7l (λabs 650 nm, middle), and 7n (λabs 750 nm, right) were mixed with BSA in equivalent at 25 °C for 6 h in various pH solutions. Labeling reactions were then monitored every 1 h via HPLC. Dyes showed the fastest conjugation with BSA in pH 9.5 solution.

Figure 4. Temperature-dependent reactivity of VS cyanine dyes. 7g (λabs 550 nm, left), 7l (λabs 650 nm, middle), and 7n (λabs 750 nm, right) were mixed with BSA in equivalent at 25 °C for 6 h at room temperature (25 °C) and the body temperature (36.5 °C). Labeling reactions were then monitored every 1 h via HPLC. Dyes showed the fastest conjugation with BSA at 36.5 °C.

Figure 5. Buffer concentration-dependent reactivity of VS cyanine dyes. 7g (λabs 550 nm, left), 7l (λabs 650 nm, middle), and 7n (λabs 750 nm, right) were mixed with BSA in various concentrations of sodium phosphate buffer in pH 9.5 (0.5 M, 0.1 M, 0.01 M, and 0.001 M). Labeling reactions were then monitored every 1 h via HPLC. Dyes showed the fastest conjugation with BSA in a 0.5 M solution.

general temperature for storing the dye stock solution, but also at room temperature. In addition, those dyes can be safely stored and used even in water instead of organic solvent such as DMSO or DMF, which provides advantages of avoiding organic solvents in biological assays using fluorescence dyes. Therefore, it can be expected that the dilution factor is controlled easily and the dye is easily retrieved with the use of a freeze−dryer. pH Stability. This experiment was to confirm the stability of the dyes in three conditions: acidic, neutral, and basic conditions. Sodium phosphate buffers of pH 5.0, 7.5, and 10.0 were used, and the experimental method was similar to that of the water stability test. Through HPLC analysis, change in peak level and appearance of new peaks were observed. The HPLC data collected with the dye dissolved in water were used as the standard. All three compounds were the most stable in

finished 30 min earlier with 0.5 M sodium phosphate buffer (Figure 5). Protein labeling in 0.5 M buffer becomes saturated within 30 min showing relatively slower binding in lower concentration buffer. The high concentration buffer might be promoted due to hydrolysis reaction of the VS group; however, unexpectedly high salt concentration of the buffer solution accelerated the labeling reaction.16,23 Stability. Water Stability. In this study, all VS cyanine dyes were dissolved in water. It is common to keep the dye stock solution at −20 °C; however, we tested the stability of the VS dyes at room temperature (25 °C) for their stability in water. HPLC results showed that VS cyanine dyes were highly stable in water by displaying no change at 25 °C for 1 day (Figure 6). Accordingly, the fluorescent dyes with VS reactive groups can be safely stored not only at −20 °C, which is the 358

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Figure 6. Water stability of VS cyanine dyes. 7g (λabs 550 nm, left), 7l (λabs 650 nm, middle), and 7n (λabs 750 nm, right) were dissolved in water for 24 h. Dye solutions were then monitored every 1 h via HPLC. Dyes showed no changes in water during the test.

Figure 7. pH stability of VS cyanine dyes. 7g (λabs 550 nm, left), 7l (λabs 650 nm, middle), and 7n (λabs 750 nm, right) were dissolved in 0.1 M sodium phosphate buffers (pH 5, 7.5, and 9.5). Dye solutions were then monitored every 1 h via HPLC. Dyes showed high stability in various pH conditions.

acidic condition, and especially, compound 7g was the most stable compound in all three conditions as well as pHs. During the first 1 h period, 7l and 7n showed similar decrease. However, after 24 h, 7n showed approximately 10% less unconsumed dye than 7l in all pH conditions. Even in pH 10, the remaining ratio of active VS dyes for 7n was more than 60% after 3 h, and more than 50% of dyes remained in the active state after 24 h (Figure 7). Therefore, VS cyanine dyes are expected to be used when either long-term reaction or labeling the biomolecules is needed, because they are stable even under the severe basic conditions. With this result taken into consideration, VS cyanine dyes are expected to show a good labeling yield in the reaction in an aqueous solution. Dye Conjugation Test. Labeling proteins usually requires large amount of dyes. To determine how many dyes are conjugated to each protein, an equation called D/P ratio is being widely used. D/P ratio utilizes the maximum absorbance wavelength. Here, a correction factor is included in the denominator to normalize the molar extinction coefficient and main absorbance wavelength values of protein, and this correction factor can be obtained with a UV spectrophotometer. The molar extinction coefficient used in this experiment for BSA was 42 665 M−1 cm−1 at 280 nm, similar to literature. For each BSA molecule, the numbers of dye molecules conjugated to 7g, 7l, and 7n were approximately 0.657, 0.594, and 0.901, respectively. In the buffer concentration experiment mentioned previously, 7g, 7l, and 7n showed 70%, 80%, and 83% dye consumption rate, respectively. These numbers were different from the D/P ratio numbers by approximately 5% to 10%. When a UV/vis spectrophotometer was used, the concentration of the protein was about 1/5 lower at 2 mg/mL, and absorbance was measured only with water. However, when HPLC was used, besides the difference in concentrations, two solvents were used as mobile phases for

separation. This may be the culprit of the difference in calculated values. Based on the previous results, HPLC was considered reliable in measuring the strength of conjugation. Thus, we used this method to observe the change in strength of conjugation as the amount of dye increased. The amount of dye was approximately seven times the molar equivalent to the protein. Initial absorbance level of each dye at the maximum absorbance wavelength, and absorbance level at every hour were measured to find out the D/P ratio based on the dye consumption amount. At the first 1 h spot, most samples were found to have 1 or 2 as the calculated values (Figure 8). However, 7g did not show any increase in D/P ratio even after incubation hours, while 7l and 7n were shown to conjugate to three or four dye molecules conjugated after 4 h. These results suggest that VS dyes can continuously be reacted over time. It is predicted that more dye would be consumed over time; however, 7g seemed to have a threshold of a saturation point. Labeling Test. To test affinity of synthesized dyes to various proteins, 7g was selected to stain nine different proteins and compared with commercially available Cy3-NHS dye. To get rid of ingredients (glycerol, EDTA, NaCl, etc.), molecular weight cutoff filters were used, and were diluted with pH 9.5 sodium phosphate buffer. Cy3-NHS dye was dissolved in DMF, and compound 7g in water. The molecular weights of Cy3NHS and 7g are 765.95 and 790.02, respectively; thus, 0.25 μL of Cy3-NHS and 7g contained 0.00326 μmol and 0.00321 μmol, respectively. After SDS-PAGE, significant amount of unreacted dye could be found at the bottom of the gel. Small amount of unreacted dye was present even with just 0.025 μL of dyes. In the literature, as well as the manufacturer’s protocol for labeling of Cy3-NHS, it was recommended that up to 20 times the molar equivalent of dye and protein be used. At least up to land C of the gel, we determined that the comparison 359

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study was feasible since it seemed that the amount of dye used was more than necessary. As a result (Figure 9), the labeling intensity of both dyes increased in proportion to the usage of dyes. Cy3-NHS tended

Article

DISCUSSION

Labeling protein, antibody, or peptide with NHS ester fluorescent dyes can be quite burdensome, as these dyes are unstable at high temperature or high pH, and hydrolyze in aqueous solutions. The main objective of this research was to develop stable fluorescent dyes to facilitate the labeling of dyes. Among various intermediates reacting with nucleophiles, intermediates with the VS group are known to be very stable in aqueous solution, high pH, and high temperature. Keeping these advantages in mind, we utilized VS groups in place of NHS ester groups of cyanine-based fluorophores, since they are widely used in biochemistry research. Our biggest concerns in constructing a cyanine-based dye were its hydrophobicity and the length of the spacer between the VS group and the main body, as those factors have significant impact on reacting with biological materials. Following the references, cyanine dyes with NHS ester were synthesized. A VS intermediate, 2-(2′-chloroethylsulfonyl)ethylamine hydrochloride, was introduced as a nucleophile in the presence of N,N-diisopropylethylamine at 25 °C. We were able to obtain cyanine dyes with VS groups with this method. These dyes were divided into 550, 650, and 750 nm excitation wavelength ranges, and their emission wavelengths were 570, 671, and 782 nm, respectively. In the functionality standpoint, the fluorescence intensity of these dyes is the most important factor. The general consensus is that the intensity is directly proportional to the molar extinction coefficient of the dyes. According to references,12 the molar extinction coefficients of cyanine dyes at the three wavelengths mentioned above range from 150 000 to 250 000. As seen in Table 1, the VS in cyanine dyes exhibited molar extinction coefficients within that range. To achieve the highest fluorescence intensity, we selected 7g, 7l, and 7n showing 550, 650, and 750 nm, respectively, since they had the highest molar extinction coefficient. The difference in the length of the spacer, and the introduction of methyl, ethyl, propyl, and butyl group for controlling hydrophobicity, did not have any notable impact on the molar extinction coefficients, and we have selected compounds with the highest values. The results suggest that absorbance and fluorescence spectra of the three cyanine-based VS dyes are similar to those of cyanine-based NHS ester dyes reported in the reference.12 We also confirmed that red shift of emission, by 100 nm, occurs depending on the length of the polymethine group. To ensure labeling efficiency, we aimed to determine whether there is any effect from the difference in the length of the spacer. For the 550 nm range, 7a, 7c, 7f, and 7g having five, six, eight, and eight carbon atoms in the spacer, respectively, were chosen. 7g was included for its highest extinction coefficient in the 550 nm wavelength, as well as for its future use as a standard compound. Compound 7g, having 8 carbon atoms, exhibited the fastest labeling time, and had the least amount of unreacted dye after 6 h. Given enough reaction time, dyes at other wavelengths should give approximately 85% labeling efficiency. Both 7f and 7g showed 85% labeling efficiency. However, 7g was found to be quicker in labeling after just 1 h, since its labeling efficiency was about 5% higher than that of 7f. Regarding compounds 7i and 7l, both of which are in the 650 nm wavelength range, compound 7l with two more carbon atoms was more efficient in labeling than 7i when compared at 1 and 6 h. No additional compound was added, since 7n already exhibited 90% efficiency. This result suggests

Figure 8. The number of dyes that conjugated to BSA. Labeling solutions were monitored for 4 h via HPLC.

Figure 9. Comparison of labeling intensity using various proteins. Each protein (1 μL) was diluted with pH 9.5, 0.1 M sodium phosphate buffer (18 μL). Stock solutions of Cy3-NHS (A, 0.25 μL; B, 0.125 μL; C, 0.0625 μL; D, 0.025 μL; and E, 0.0025 μL) and 7g (A, 0.25 μL; B, 0.125 μL; C, 0.0625 μL; D, 0.025 μL; and E, 0.0025 μL) were added to the protein solutions. The mixed protein solutions were incubated at 25 °C for 4 h. Fluorescent molecular weight markers (Takara Bio Inc., Japan) were used.

to stain low molecular weight proteins (less than 29 kDa), and 7g stained high molecular weight proteins (more than 97.2 kDa). Because VS cyanine dye has greater long-term stability and reactivity in basic condition than NHS-type dyes, VS cyanine dyes were more evenly labeled with whole proteins. 360

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that the length of the spacer affects the overall flexibility of the structure of the dyes. According to established labeling protocols and references on dye labeling, the determinant factors for labeling efficiency were pH, solvent, and temperature. Organic solvents such as DMF or DMSO were not used in this study, since the majority of proteins and antibodies in the market are either in solid form or diluted in aqueous solution. 7g, 7l, and 7n compounds as well were water-soluble. Moreover, DMF and DMSO are toxic and can become cumbersome during compound analysis. Reactivity tests showed that dye conjugation is accelerated in basic pH conditions and in body temperature (36.5 °C), where amine groups of targeted proteins can easily react with vinylsulfone. Additionally, 0.1 M or higher phosphate buffer positively affected dye labeling. For further experimentation, reaction conditions of pH 9.5, 25 °C, 0.1 M phosphate buffer were chosen for optimized dye conjugation, considering that common biological experiments prefer 25 °C where there is less than 20% labeling efficacy difference from body temperature, and 0.5 M phosphate buffer tends to have a salting out effect which led decrease in conjugation rate of 7n. Also, to determine the stability of the dye in aqueous solutions, the dye was dissolved in water and underwent HPLC analysis in order to monitor hydrolysis of the VS group. Results indicated that 7g, 7l, and 7n showed no change at 25 °C for at least one day. We predicted that, as long as the pH is not extremely high, the reactivity of dye would not drastically change, in spite of minor changes in the stability of cyanine chromophore (Figure 7). The overall data of 7g, 7l, and 7n suggested that compound 7g exhibited the slowest reaction time but with the best stability, and 7n exhibited the fastest reaction time and was proven to be unstable with the change in reaction conditions. Reaction time and stability can be easily changed depending on the chemical structure of the dyes. Therefore, more intensive research on the relationship of various cyanine compounds with their reaction time and stability is necessary to improve reactivity and stability. When labeling protein, antibody, or peptide with fluorescent dyes, excessive amounts of dye are usually used due to limited labeling conditions including intricate structure of biologics, temperature, solvent, time, and so forth. For more accurate quantification of dye labeling, the D/P ratio equation is being widely used, with unique equations for different wavelengths. This equation can also be used to determine how many dyes should be used for proteins with different sizes. Experimental results suggested that 0.7−0.8 molecule of dye is conjugated to a protein molecule when molar equivalents of both dye and protein were reacted together and when the D/P ratio equation was incorporated for 7g, 7l, and 7n, only with less than 10% error (Table 2). When an excessive amount of dye by 7× molar equivalent was used, a higher number of molecules of each dye was conjugated to a protein molecule with 7l being the most conjugated. However, we cannot jump to the conclusion that this result would be reproducible with other kinds of protein. There are various factors to be considered in protein such as size, structure, affinity to dye, the number and location of amine groups in the protein molecule, and so forth. For the study on labeling feature of VS cyanine dyes to proteins, compound 7g was compared with GE Healthcare’s Cy3-NHS dye. The reaction of Cy3-NHS was carried out under the manufacturer’s suggested conditions, whereas compound 7g was done in its optimal condition stated above. Considering the variability in structures and characteristics of proteins, a

Table 2. Number of Conjugated VS Cyanine Dyes (Absorption Intensity Values (A280, Adye) at Each Maximum Absorption Wavelength (BSA (280 nm), 7g (280 nm, 549 nm), 7l (280 nm, 647 nm), and 7n (280 nm, 749 nm)), BSA Extinction Coefficient (Eprot), BSA Extinction Coefficient (Edye), Denominator Factor for Dye Absorption at 280 nm (AD.C.), and Dye/Protein Ratio (D/P = (AdyeEprot)/(A280 − AD.C.Adye)Edye)) dye no. (conjuated BSA)

AD.C.

Edye (×105 L/mol cm)

Eprot/ Edye

A280

Adye

D/P

7g 7l 7n

0.067 0.023 0.03

1.46 2.72 2.25

0.292 0.157 0.19

0.204 0.132 0.147

0.398 0.461 0.610

0.657 0.594 0.901

marker protein (protein ladder for SDS-PAGE) containing nine different proteins was used. This marker was not labeled with dyes, thus enables us to directly compare how Cy3-NHS and 7g conjugate to the proteins. The conjugation efficiency varied depending on the protein size. When the two dyes were compared, compound 7g was conjugated better with higher molecular weight proteins such as myosin, β-galactosidase, and phosphorylase B. Cy3-NHS showed higher intensity with small molecular weight proteins such as trypsin inhibitor, lysozome, and aprotinin. We determined that compound 7g performed better with high molecular weight proteins. On the other hand, Cy3-NHS performed better with low molecular weight proteins because of the shorter reaction time.

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ASSOCIATED CONTENT

S Supporting Information *

Reactivity test of VS cyanine dye with benzyl amine; dye conjugation study via MALDI-TOF M/S; multiple band analysis using SDS-PAGE. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Kee-Jung Lee: Tel +82-2-2220-0528, Fax +82-2-2298-4101, Email: [email protected]. Dong Jin Kim: Tel +82-2-958-5142, Fax +82-2-958-5189, E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the project No. 10033024 from the Advanced Technology Center (ATC) Support Program of Ministry of Knowledge Economy (Republic of Korea). We thank Kinam Park, Ph.D. (Professor of Pharmaceutics, Purdue University) for support in dye conjugation ratio measurements, Sang Yeul Lee, Ph.D. (Sogang University), and Namho Kim (BioActs Co. Ltd.). The three novel cyanine compounds (7g, 7l, and 7n) are available by BioActs Co. Ltd (Code No. PWA1119, PWA1215, and PWA1308).

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REFERENCES

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