Cyanine-Labeling Reagents: Sulfobenzindocyanine Succinimidyl

The labeling reagents are water soluble, and their fluorescence is not sensitive .... William H. White , III , Masayuki Shibata , Shunichi Fukuzumi , ...
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Bioconjugate Chem. 1996, 7, 356−362

356

Cyanine-Labeling Reagents: Sulfobenzindocyanine Succinimidyl Esters Swati R. Mujumdar,‡ Ratnakar B. Mujumdar,*,† Charsetta M. Grant,† and Alan S. Waggoner‡ Department of Biological Sciences and Center for Light Microscope Imaging and Biotechnology, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213, and Amersham Life Science, Harmarville, 955 William Pitt Way, Pittsburgh, Pennsylvania 15238. Received September 15, 1995X

A synthetic method for shifting the absorption and emission wavelengths of cyanine dye labels by 15-30 nm to the red has been developed. This step significantly increases the potential for preparing fluorescent probes for multiparameter analysis in cytometry and diagnostics. The new sulfobenzindocyanine dyes contain succinimidyl esters as reactive groups and can be readily conjugated to antibodies, avidin, modified DNA, and other amino group-containing materials. The labeling reagents are water soluble, and their fluorescence is not sensitive to pH. One of the reagents, Cy3.205, can be optimally excited with the 568 nm Kr laser line and is useful for confocal microscopy and flow cytometry. Another dye, Cy5.205, can be excited with the 633 He-Ne or 647 nm Kr laser lines available with many flow cytometers and laser-scanning microscopes. New laser diodes emitting near 660690 nm should also be excellent excitation sources for Cy5.205.

INTRODUCTION

Bright and water soluble fluorescent labeling reagents are important for sensitive detection with fluorescent antibodies and DNA probes. A group of such probes that emit in different regions of the spectrum, and can be independently detected, provide a powerful tool for correlating multiple antigenic or genetic parameters in individual cells and tissues. Cyanine dyes have an advantage for designing multicolor probes because they can be tuned to desired wavelengths by altering the heterocyclic nucleus and the number of double bonds in the polymethine chain. So far, we have focused our development on the dyes using indolenine as a heterocyclic base, but this approach has left several gaps in the visible and near-infrared regions of the spectrum (1-4). The photographic industry literature describes cyanine dyes containing benzindolenine base that absorb 30 nm toward longer wavelength than the corresponding cyanine dyes of the indolenine base (5). While our investigation was in progress, Narayanan and Patonay (6) published the syntheses and use of benzindolenine-based heptamethinecyanines as nearinfrared (NIR) fluorescent labels. However, they lack water solubility required to avoid dye-dye interaction and nonspecific binding. Using the successful sulfoindocyanine labels as models, we have prepared benzindolenine cyanine active esters with two or four aryl sulfonate groups at various positions on the heterocyclic rings to increase water solubility. EXPERIMENTAL PROCEDURES

Chemicals, Antibodies, and Media. 6-Amino-2naphthalenesulfonic acid, 5-amino-1-naphthalenesulfonic acid, and 6-amino-1,3-naphthalenedisulfonic acid disodium salt were purchased from TCI American, Portland, * Please send correspondence to Ratnakar B. Mujumdar, Science and Technology Center, Carnegie Mellon University, 4400 5th Avenue, Pittsburgh, PA 15213. Telephone: (412) 2683462. Fax: (412) 268-6571. † Carnegie Mellon University. ‡ Amersham Life Science. X Abstract published in Advance ACS Abstracts, May 1, 1996.

S1043-1802(96)00021-3 CCC: $12.00

OR. Sheep γ-globulin (IgG) was purchased from Sigma Chemical Co., St. Louis, MO. Dowex-50 (8% cross-link, 100-200 mesh, moisture content of 50%), disuccinimidyl carbonate (DSC), triethyl orthoformate, 6-bromohexanoic acid, and dry solvents diethyl ether and ethyl acetate were purchased from Aldrich Chemical Co., Milwaukee, WI. Trimethoxypropene was purchased from Eastman Kodak, Rochester, NY. Texas Red was purchased from Molecular Probes, Eugene, OR. All other solvents were purchased from Fisher Scientific, Pittsburgh, PA. Monoclonal antibodies were purchased from Becton Dickinson, Mountainview, CA. Hank’s balanced salt solution (HBSS) buffer solution was purchased from Gibco, Long Island, NY. Purification and Spectroscopic Analysis. Purification of the dyes for spectroscopic analysis was performed on a Spectra-Physics model SP8700 analytical high-performance liquid chromatography (HPLC) unit equipped with a C8-RP column. Purification could also be achieved by conventional or flash column chromatography on a C18-RP powder (Analtech, Newark, DE). Water/methanol mixtures were used for elution in all experiments. Dyes were recovered from the fractions on a rotary evaporator at 60-70 °C without appreciable loss. Although these purification procedures isolate the fluorophore from various intermediates and precursors, these multi-ionic dyes typically associate with alkali metal cations, halide anions, and water of hydration in nonstoichiometric amounts. Therefore, it is essential to obtain the final product in a form that is chemically welldefined for elemental and mass spectrometric analysis. This was accomplished by passing the dye with unknown counterion composition through a Dowex-50W hydrogen, strongly acidic cation exchange column that had been previously washed with 0.1 N sulfuric acid and then distilled water. For example, a pure sample of Cy5.205. OH (X) was obtained by ion-exchange chromatography for molecular weight determination. Mass spectral determination was made at the Midwest Center for Mass Spectrometry at the University of NebraskasLincoln, Lincoln, NE, with partial support from NSF Grant DIR9017262. © 1996 American Chemical Society

Sulfobenzindocyanine Labeling Reagents

Figure 1. Structures of sulfonaphthylamines and 1,1,2-trimethylsulfobenzindoles used in the cyanine dye synthesis.

Ultraviolet-visible spectra were measured on a HewlettPackard HP 8452 diode array spectrophotometer. The proton NMR spectra were obtained on an IBM 300 FTNMR spectrometer. NMR signals are described by use of s for singlet, d for doublet, t for triplet, q for quartet, and m for multiplet and are expressed in δ with TMS as internal standard. Fluorescence measurements were performed using a Spex Fluorolog 2 system. Quantum yields were determined as previously described (3, 4). Extinction coefficients in Table 2 were determined from absorbance values of HPLC-purified, ion-exchange-purified, weighted samples of dried material and their molecular weights. Flow cytometry was performed on a dual laser FACS 440 instrument (Becton Dickinson) equipped with an Ar+ and a Kr+ laser. General Syntheses. Three new pentamethine cyanines (VII, VIII, and X) and one trimethine cyanine (IX)

Bioconjugate Chem., Vol. 7, No. 3, 1996 357

were synthesized using sulfonated naphthylamines (IIII, Figure 1) as starting materials. The general synthesis of the reactive dye involves five steps with slight modifications of methods described previously (1-4). Following are the representative procedures for the syntheses of disuccinimidyl esters, Cy3.205.OSu (IX), and Cy5.205.OSu (X). The reaction sequence and the conversion of carboxylic acids to succinimidyl esters are shown in Figure 2. (a) Preparation of 6-Hydrazinonaphthalene 1,3-Disulfonate. A suspension of naphthylamine (III) (10 g, 0.033 mol) in 200 mL of 50% hydrochloric acid was stirred and maintained at 0-5 °C. A solution of sodium nitrite (2.5 g, 0.036 mol in 10-20 mL of water) was added dropwise. The rate of addition was controlled to keep the temperature from rising above 5 °C. Cooling was continued while a solution of stannous chloride (8.2 g, 0.036 mol in 10 mL of concentrated HCl) was added dropwise to the stirred mixture at a rate to keep the temperature at or below 5 °C. After the addition of stannous chloride, stirring was continued for 1 h. The product was concentrated on a rotary evaporator and triturated with hot 2-propanol until free powder was obtained. λmax (water): 279, 353 nm. 1H NMR (DMSO-d6): δ 8.72 (d, 1H, J ) 9.2 Hz, 8-H), 8.1 (d, J ) 1.2 Hz, 1H, 4-H), 7.95 (s, 1H, 2-H), 7.3 (s, 1H, 5-H), 7.15 (d, 1H, J ) 9.2 Hz, 7-H). Rf: 0.83 (RP-C18, water). The disodium salt of 6-amino-1,3-naphthalenedisulfonic acid (III) has a very poor solubility in concentrated hydrochloric acid. Sodium free base was obtained by ionexchange chromatography. Cation-exchange resin, Dowex50 (8% cross-link, 100-200 mesh, moisture content of 50%), was washed in a beaker with 0.1 M sulfuric acid (3 × 350 mL), packed in a glass column (25 × 1.5 cm), and eluted with distilled water (approximately 1000 mL) until eluent was neutral. 6-Amino-1,3-naphthalenedisulfonic acid disodium salt (25 g in 200 mL of water) was loaded onto the column and eluted with distilled water (450 mL) at the rate of 2 mL/min. The first fraction of

Figure 2. Scheme showing the structures and methods of preparation of disuccinimidyl esters of sulfonated benzindocyanines.

358 Bioconjugate Chem., Vol. 7, No. 3, 1996

100 mL was discarded (colorless neutral solution). The second fraction (300 mL, yellow color, strongly acidic) was collected and dried on a rotary evaporator, yielding a pale yellow solid of the free acid almost quantitatively. (b) Preparation of 1,1,2-Trimethylbenzindoleninium 1,3-Disulfonate (VI). A mixture containing the hydrazine (6.2 g, 0.02 mol), methyl isopropyl ketone (6.4 mL, 0.06 mol), potassium acetate (5.88 g, 0.06 mol), and glacial acetic acid (35 mL) was refluxed at 135 °C for 24 h. Acetic acid was removed on a rotary evaporator under vacuum and the solid stirred with 2-propanol until free powder was obtained. Gray powder was filtered off and dried (1.95 g, 21%). λmax (water): 254 nm. 1H NMR (D2O): δ 8.8 (d, 1H, J ) 7 Hz, 4-H), 8.7 (s, 1H, 9-H), 8.45 (s, 1H, 7-H), 7.9 (d, 1H, J ) 7 Hz, 5-H), 2.4 (s, 3H, CH3), 1.5 (s, 6H, (CH3)2). Rf: 0.46 (RP-C18, water). (c) Preparation of N-(γ-Carboxypentynyl)-1,1,3-Trimethylbenzindoleninium 6,8-Disulfonate (Structure A in Figure 2). The dipotassium salt of 1,1,2-trimethylbenzindoleninium-6,8-disulfonic acid (VI) (4.6 g, 0.01 mol) and 6-bromohexanoic acid (4.9 g, 0.025 mol) in 1,2-dichlorobenzene was heated at 145 °C for 24 h in a nitrogen atmosphere. The mixture was cooled, 1,2-dichlorobenzene was decanted, and the solid was washed with ether, until free powder was obtained (3.91 g, 75%). λmax (water): 202, 226, 252 nm. 1H NMR (D2O): δ 8.9 (s, 1H, 9-H), 8.8 (d, 1H, J ) 7 Hz, 4-H), 8.45 (s, 1H, 7-H), 7.7 (d, 1H, J ) 7 Hz, 5-H), 4.2 (t, 2H, J ) 7.5 Hz, R-CH2), 2.25 (t, 2H, J ) 7.5 Hz, -CH2), 1.99 (m, 2H, β-CH2), 1.351.66 (m, 10H, γ-CH2, δ-CH2 with a sharp singlet at 1.61 for (CH3)2). Rf: 0.60 (RP-C18, 10% methanol in water). (d) Preparation of Cy3.205.OH (IX). A mixture of quaternized product A (2 g, 3.8 mmol) and triethyl orthoformate (0.3 g, 2 mmol) in 5 mL of pyridine was heated to reflux for 2 h. The mixture was cooled and diluted with diethyl ether (100 mL). The precipitated product was filtered and dried (1.9 g). The crude product was dissolved in water (10 mL) and chromatographed on a reversed-phase C18 column (30 g) using a 5 to 20% gradient of methanol in water to yield 0.34 g (16%) of IX. The reversed-phase (C18) TLC showed a single spot. Rf: 0.6 (17% methanol in water). λmax (water): 586 nm ( ) 120 000 L mol-1 cm-1). λmax (PBS): 578 nm ( ) 125 000 L mol-1 cm-1). 1H NMR of the ion-exchange purified dye was recorded in deuterated water: δ 8.68.9 (m, 5H, 9-H, 9′-H, 4-H, 4′-H, β-H of the bridge), 8.45 (s, 2H, 7-H, 7′-H), 7.8 (d, 2H, J ) 7 Hz, 5-H, 5′-H), 6.2 (d, 2H, J ) 7 Hz, R-H, R′-H of the bridge), 4.2 (m, 4H, N-CH2, N′-CH2), 1.3-2.5 (singlet at 2.2 for two (CH3)2 is merged in a broad multiplet for eight CH2 groups). Preparation of Cy5.205.OH (X). A mixture of quaternized product A (8.0 g, 0.015 mol) and pyridine (10 mL) was heated to reflux. 1,3,3-Trimethoxypropene (1 mL) was added every 30 min over 2.5 h (total of 4 mL). The mixture was cooled and diluted with diethyl ether. The precipitated product was filtered and dried at reduced pressure to yield the dye (4.8 g). The crude product was dissolved in water (25 mL) and chromatographed on a reversed-phase C18 column (100 g) using a 5 to 20% gradient of methanol in water to yield 0.6 g (7%) of X. The reversed-phase TLC showed a single spot, Rf: 0.74 (3/1 water/methanol). Low-resolution FAB (LRFAB) gave m/e of 1003 (C45H50N2O16S4 requires 1003.13) using triethanolamine as matrix. λmax (PBS): 676 nm ( ) 190 000 L mol-1 cm-1). λmax (methanol): 680 nm ( ) 195 000 L mol-1 cm-1). The absorption and emission spectra of the dye remained unchanged in 0.1 N HCl and 0.1 N NaOH solutions, indicating the dye is insensitive to pH. 1H NMR (D2O): δ 8.85 (d, 2H, J ) 7 Hz, 4-H, 4′H), 8.7 (s, 2H, 9-H, 9′-H), 8.35 (s, 2H, 7-H, 7′-H), 8.2 (t,

Mujumdar et al.

2H, J ) 13 Hz, β-H, β′-H of the bridge), 7.7 (d, 2H, J ) 7 Hz, 5-H, 5′-H), 6.15 (t, 1H, J ) 13 Hz, γ-H of the bridge), 6.1 (d, 2H, J ) 13 Hz, R-H, R′-H of the bridge), 4.2 (broad triplet, 4H, N-CH2, N′-CH2), 2.25 (broad m, 4H, -CH2, ′-CH2), 1.99 (m, 4H, β-CH2, β′-CH2), 1.3-2.2 (m, 24H, six CH2 and two (CH3)2). (e) Preparation of Disuccinimidyl Ester. In a typical experiment, a dye was dissolved in a mixture of dry DMF (2 mL per 100 mg of dye) and dry pyridine (0.1 mL per 100 mg of dye). Disuccinimidyl carbonate (DSC) (3 equiv) was added, and the mixture was stirred at 55-60 °C for 90 min, under a nitrogen atmosphere. After dilution of the mixture with dry ethyl ether, the supernatant was decanted. The product was washed repeatedly with dry ether, filtered under a nitrogen atmosphere, and dried. Nearly quantitative yields of active esters were obtained. The formation of the active ester was confirmed by its reaction with benzylamine in dry DMF. For example, Cy5.205.OSu was dissolved in DMF (1 mg per 100 µL), and the aliquot (10 µL) was allowed to react with 10 µL of benzylamine in DMF (10%) for 30 min. The conjugate was precipitated with dry ethyl ether and centrifuged. A reversed-phase C18 plate was spotted with conjugate, succinimidyl ester, and hydrolyzed carboxylate product for comparison and developed with 25% methanol in water. Rf values for benzylamine conjugate and free dye Cy5.205.OH are 0.1 and 0.7, respectively. Since activation was sometimes incomplete, reversed-phase HPLC was also used to determine the percentage of the fluorochrome in the active ester form. The sample was eluted through an Alltech Econosphere 250 mm × 4.6 mm C18 RP column using a mixture of 25% acetonitrile/75% water containing 0.1% trifluoroacetic acid. The percentages of the activated and unactivated fluorophores were determined by integration of the absorbance signals from a Varian UV/vis detector. Alternatively, the percentages can be determined by TLC. For example, the diester Cy5.205.OSu (X) was dissolved in water containing 0.1% trifluoroacetic acid (1 mg per 100 µL). A small portion (25 µL) was loaded as a small strip on a C18 reversedphase, 5 × 10 cm TLC plate. The plate was developed using a mixture of acetonitrile/water (2.5/7.5) containing 0.1% trifluoroacetic acid. The bands containing free acid, monosuccinimidyl ester and disuccinimidyl ester were scrapped from the TLC plate and extracted in equal volumes of methanol (5 mL). The absorbance of each extract was measured at 680 nm, and the fractions of the free acid and active esters were calculated from the relative absorbances of the extracts. The Rf values for Cy5.203 acid and the mono- and disuccinimidyl esters are 0.8, 0.3, and 0.15, respectively. General Protein-Labeling Procedures. Stock solutions of the sulfobenzindocyanine succinimidyl esters were made in dry DMF (1 mg per 100 µL) and were stable for days when stored at 4 °C in a desiccator. The esters are also stable in distilled water for several hours, provided the pH of the solution is not basic. The aqueous solution of the dyes can be used for labeling antibodies if use of DMF is not suitable for certain antibody labeling. The concentration of the reactive dye in a stock solution was determined by measuring the absorbance of an aliquot of the appropriately diluted stock solution in phosphate-buffered saline (PBS) and using the extinction coefficient of the dye (Table 1). Generally, antibodylabeling reactions were carried out in 0.1 M carbonate/ bicarbonate buffer (pH 9.4) for 15 min at room temperature. One milligram of sheep IgG (6.45 × 10-6 mM) was dissolved in 0.25-1 mL of buffer solution, and the desired amount of dye (e.g. 5-25 µL of the 1 mg per 100 µL of stock solution) was added during vigorous vortex mixing.

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Sulfobenzindocyanine Labeling Reagents Table 1. Spectral Properties of Sulfobenzindocyanine Dyes dye

solvent

λmax (nm)

Emmax (nm)

 (L mol-1 cm-1)

QY (φ)

Cy5.203

PBS MeOH PBS MeOH PBS MeOH PBS MeOH

680 686 682 686 578 586 674 680

702a 708a 707a 708a 593a 602a 694b 704b

190 000 195 000 197 000 194 000 120 000 125 000 190 000 195 000

0.10c 0.21c 0.10c 0.23c 0.14d 0.28d 0.23c 0.24c

Cy5.204 Cy3.205 Cy5.205

a Excited at 632 nm. b Excited at 514 nm. c Based on Di-S-C2(5) as standard. d Based on rhodamine-6G as standard.

Unconjugated dye was separated from the labeled protein by gel permeation chromatography over Sephadex G-50 (0.7 × 20 cm column) using pH 7 buffer solution as eluant. A series of labeling reactions were set up as described in a procedure above for different dye/protein ratios (5-25 mol of dye per mole of protein). Absorption spectra of the labeled antibody solutions were recorded. The dye/protein ratio for each sample was determined by using the equation below with measured values of absorbance of the labeled dye (Cy3.205 at 586 nm or Cy5.205 at 676 nm) and the absorbance of protein at 280 nm.

AdyeEprot D ) P (A280 - XAdye)Edye The factor X in the denominator accounts for dye absorption at 280 nm which is a percent of the absorption of the dye at its maximum absorption (Adye). The value of X is 0.13 for Cy5.205 and 0.23 for Cy3.205. This value is only 0.05 for sulfoindolenine-based cyanine dyes (1). The significant high absorbance at 280 nm for these dyes and blue fluorescence when excited in the UV region indicate that the properties of the benzindole moiety are retained to a greater extent. The average number of dye molecules bound per antibody molecule obtained with bis reactive Cy5.205.OSu under these labeling reaction conditions is shown in Figure 4. The efficiency of labeling under these conditions (pH 9.4) is about 35-40%. Cell Preparation and Flow Cytometry. Flow cytometry was performed on a dual laser FACS 440 instrument equipped with an Ar+ and a Kr+ laser. Lymphocyte populations were selected on the basis of forward and side scatter characteristics, and total T cell populations were identified using a biotinylated monoclonal antibody and streptavidin labeled with either Texas Red (TR) or Cy3.205.OSu. Mononuclear leukocytes were obtained from the whole blood of healthy volunteers using histopaque separation medium (Sigma). Antibody was added to 1 × 106 cells per 100 mL of Hank’s balanced salt solution containing 2% fetal bovine serum and 0.1% sodium azide (monoclonal wash) and incubated at 4 °C for 45 min. Optimal concentrations of streptavidin were determined by titration. After primary and secondary labeling, the cells were washed twice with monoclonal wash. Prior to analysis on the FACS, cell preparations were fixed with 1% paraformaldehyde in HBSS. The argon laser was emitting 400 mW at 488 nm, and the Krypton laser was emitting 200 mW at 568 nm. Texas Red and Cy3.205 fluorescence was collected using the delayed fluorescence signal from the Krypton laser. Data was collected and analyzed using the Consort 30 VAX and Disp4 programs. Information was collected logarithmically.

Figure 3. Structures of new sulfobenzindocyanines. RESULTS

Fluorescent cyanine dye-labeling reagents developed in this work are shown in Figure 3. Following the naming scheme for reactive polymethine dyes used in the previous papers (1-4) of this series, these dyes will be referenced as Cy3.205.OSu (IX) and Cy5.205.OSu (X). In these names, the Cy portion indicates that they are cyanine dyes, while OSu indicates that the compounds have succinimidyl ester reactive groups, and OH means that the compounds are in the carboxylic acid form. The number immediately after the Cy indicates the number of bridge carbons (i.e. Cy3, m ) 1; Cy5, m ) 2). The number following the decimal point indicates a unique dye structure, which is determined by the heterocyclic rings and the substituents which are introduced for water solubility and reactivity. The labels VII and VIII have two sulfonate groups, and labels IX and X have four sulfonate groups to increase their water solubility and two alkylcarboxyl groups that can be activated to form succinimidyl esters. In the synthesis, generally, a symmetrical benzindocyanine sulfonate is formed when a quaternized benzindoleninium sulfonate (IV-VI), which contains an alkylcarboxyl group for later activation of the fluorophore, is linked to a second identical benzindoleninium sulfonate nucleus through a polymethine chain of three or five conjugated methine carbon atoms. The key intermediates, benzindoleninium sulfonates, required to produce new groups of labeling reagents are prepared from corresponding sulfonaphthylamines (I-III). Many sulfonaphthylamines are commercially available. In our initial preparations, we chose two monosulfonated naphthylamines (I and II) and converted them to respective

360 Bioconjugate Chem., Vol. 7, No. 3, 1996

Mujumdar et al.

Figure 4. Plot of the ratio of moles of covalently attached Cy5.205 per mole of protein in a labeled protein vs the ratio of moles of Cy5.205.OSu per mole of protein originally present in the labeling mixture.

Figure 6. Normal peripheral blood lymphocytes (gated population) labeled with CD3-B followed by a streptavidin-labeled second reagent: (A) unstained control, (B) streptavidin/Cy3.205, and (C) streptavidin/Texas Red.

Figure 5. (a) Absorption spectra of Cy5.203 acid (s) and its disuccinimidyl ester (- - -) in water. The diester showed absorption maxima at 768 nm due to J-aggregate (see arrow). (b) Absorption spectra of Cy5.203 acid (s) and its antibody conjugate (- - -) in PBS. The conjugate showed substantial absorption at 632 nm due to dimer and no absorption for J-aggregate. (c) Absorption spectra of Cy5.205 acid (s) and its antibody conjugate (- - -) in PBS solution.

benzindoles (IV and V) using the Fisher indole synthesis (7). This approach is better than direct sulfonation of benzindoles. Heseltine and Lincoln (8) have synthesized several styryl dyes derived from benzindoleninium sulfonates. However, these sulfonates were obtained by direct sulfonation of the benzindole nucleus. In such a procedure, the exact orientation of the newly introduced sulfonate group(s) was not established. It is our experience that the position of the sulfonate group is very important to increase water solubility and the brightness of a fluorophore. Following the reaction path shown in Figure 2, we have synthesized two pentamethine cyanines, Cy5.203.OH (VII) and Cy5.204.OH (VIII). These two dyes have identical spectroscopic and labeling properties, showing no effect due to change in the position of the sulfonate group. Their succinimidyl esters tend to form J-aggregates in water or PBS solution. However, their absorption spectra in methanol and alkaline buffer (pH

9.4) solution show no evidence of J-aggregate (Figure 5a). It appears that the one sulfonate group on these benzindoles is not sufficient to reduce dye-dye aggregation. The antibody conjugates of these dyes (VII and VIII) showed substantial dimer formation (Figure 5b) with associated fluorescence quenching. We, therefore, synthesized benzindocyanines with two sulfonate groups on each of the benzindolenine nuclei. The added sulfonate groups would increase the water solubility and dye-dye repulsion. The intermediate 6-amino-1,3-naphthalenedisulfonic acid disodium salt was used for the synthesis of two tetrasulfonated fluorophores, Cy3.205.OH (IX) and Cy5.205.OH (X). After purification of the resulting fluorophores, carboxyl groups were activated to produce reactive dyes as labeling reagents. Their protein conjugates showed no evidence of dye-dye aggregation (Figure 5c) and fluorescence quenching. Table 1 shows absorption and emission properties of the cyanine dyes. Absorption and emission wavelengths are relatively insensitive to solvent polarity. The quantum yields of the tetrasulfonated dyes are somewhat higher in both water and organic solvents. The succinimidyl ester of Cy3.205 was reacted with sheep γ-globulin in 0.1 M carbonate/bicarbonate buffer (pH 9.4). The labeling efficiency of Cy3.205.OSu (IX) was close to 30%. The quantum yield of the fluorophore increased by 2-fold upon conjugation to antibody and was aproximately 0.25. This is consistent with our previous findings on trimethine cyanines which showed higher quantum yields in viscous media (1). Labeling of Antibodies and Cells. Human lymphocytes were prepared and incubated with a biotinylated

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Sulfobenzindocyanine Labeling Reagents Table 2. Peripheral Blood Lymphocytes Labeled with CD3-Biotin followed by Streptavidin Labeled with either Cy3.205 or Texas Red mean linear sample cell unstained cell (control) CD3-biotin + streptavidin/ Cy3.205 CD3-biotin + streptavidin/ Texas Red

negative cells 9.65 12.2 15.98

positive cells 400 97.84

positive fluorescence over negative 32.8 6.12

monoclonal antibody T-cell marker (CD3). Streptavidin labeled with either Texas Red (TR) (Molecular Probes) or Cy3.205.OSu was used as a secondary reagent to mark the CD3-positive cells for analysis by flow cytometry. Samples were excited with 200 mW at the 568 nm Krypton laser line, and fluorescence was collected using a 625/35 nm band-pass filter. Cells labeled with Cy3. 205.OSu gave a signal 6 times brighter than those labeled with Texas Red and showed low nonspecific binding (Table 2). DISCUSSION

Planar and nonpolar dyes which prevail as fluorescent labels for fluorescence detection are particularly susceptible to molecular stacking. On the surface of labeled proteins, dye-dye interactions at high labeling densities generally lead to fluorescence quenching, thus limiting the brightness with labeled antibodies. Our earlier work (1-4) has shown that the addition of charged groups (such as sulfonate) on the indolenine base significantly reduces dye-dye interaction. It is also our experience that the position of the sulfonate group on the heterocyclic base affects the quantum yield and the brightness of the dye-antibody complexes. For example, oxacyanine derived from 6-sulfo-2-methylbenzoxazole is 2 times brighter than its isomeric dye obtained from 5-sulfo-2methylbenzoxazole.1 Bearing this observation in mind, we initially focused the present investigation on the syntheses of cyanines derived from two sulfonaphthylamines (I and II) which have only one sulfonate group. The additional benzene ring fused to sulfoindocyanine dyes shifts the dye absorption to a longer wavelength and increases the quantum yield (9). Our present results showed that the two pentamethine cyanine dyes, Cy5. 203.OH (VII) and Cy5.204.OH (VIII), derived from monosulfonated benzindoles (IV and V) have the same spectral properties. The position of the sulfonate group produced no effect on the spectral properties, and both dyes showed substantial aggregation when bound to antibody. The tendency to form dye-dye aggregates on antibodies appeared to be far greater than that of previously discussed indocyanines. Even more surprisingly, disuccinimidyl esters Cy5.203.OSu (VII) and Cy5.204.OSu (VIII) form J-aggregates very freely when dissolved in water (Figure 5a). However, in pH 9.4 carbonate buffer, the same compound showed no evidence of J-aggregate. The free carboxylate ions in an alkaline solution apparently increase water solubility and reduce dye-dye aggregation. The formation of J-aggregates suggests higher-order dye-dye stacking (four dye molecules or more) (10). It is possible that the sulfonate groups of the monosulfonated naphthylamine-based dyes form tight ion pairs with the quaternary nitrogen of the adjacent dye molecule, giving rise to a ladder and stair structure which favors J-aggregates (9). The absence of J-aggregates in antibody-dye conjugate (Figure 5b) is 1

R. B. Mujumdar and A. S. Waggoner, unpublished data.

not a surprise. Free carboxylate ion generated after the hydrolysis of the succinimidyl ester increases water solubility. Besides, the dye-antibody solution is further diluted during Sephadex gel filtration chromatography. We have demonstrated that placement of four sulfonate groups close to the planar ring structures of the chromophore appears to provide more effective repulsion and reduces the dimer formation. The J-aggregates are not observed in the absorption spectra of Cy3.205.OSu and Cy5.205.OSu in water at neutral pH. The dye-dye aggregation is also absent in the dye-antibody conjugates as is evident from Figure 5c. Dyes containing single reactive groups could not lead to complications from the cross-linking reactions that might seem likely to be encountered when bifunctional dyes are used. The actual experience has been that crosslinking of protein does not occur to a significant extent when disuccinimidyl ester of Cy5.205 is used for labeling at a low dye/protein ratio (10-15 dyes/protein). When proteins labeled with a high concentration of disuccinimidyl ester were subjected to sodium dodecyl sulfate (SDS) gel separation, very minimal cross-linking products were seen. This was consistent with our observations with cyanine dyes (1, 4). The new cyanines present an opportunity to take advantage of additional laser lines that are already available or could be made available in fluorescence detection systems. A 568 nm Krypton laser excitation source and a 597 or 600/30 nm emission filter work well for flow cytometry. The longer wavelength Cy5.205 can be excited with new laser diodes emitting near 690 nm, and a 690-710 nm emission filter or Cy3.205 can give a strong fluorescence signal which is easy to detect using the standard Texas Red filter set. ACKNOWLEDGMENT

The authors gratefully acknowledge the assistance of Mr. Marc Wagner in the flow cytometry experiments and Dr. Ken Giuliano for the protein cross-linking experiment. This work was supported in part by grants from the National Science Foundation (MCB 8920118), NIH (NS 19353), and The Western Pennsylvania Advanced Technology Center through Pennsylvania’s Ben Franklin Partnership Program. LITERATURE CITED (1) Mujumdar, R. B., Ernst, L. A., Mujumdar, S. R., Lewis, C. J., and Waggoner, A. S. (1993) Cyanine Dye Labeling Reagents: Sulfoindocyanine Succinimidyl Esters. Bioconjugate Chem. 4, 105-111. (2) Ernst, L. A., Gupta, R. K., Mujumdar, R. B., and Waggoner, A. S. (1989) Cyanine Dye Labeling Reagents for Sulfhydryl groups. Cytometry 10, 3-10. (3) Mujumdar, R. B., Ernst, L. A., Mujumdar, S. R., and Waggoner, A. S. (1989) Cyanine Dye Labeling Reagents containing Isothiocyanate groups. Cytometry 10, 11-19. (4) Southwick, P. L., Ernst, L. A., Tauriello, E. W., Stephen, R. P., Mujumdar, R. B., Mujumdar, S. R., Clever, H. A., and Waggoner, A. S. (1990) Cyanine Dye Labeling reagents Carboxymethylindocyanine Succinimidyl Esters. Cytometry 11, 418-430. (5) Hamer, F. M. (1964) The Cyanine Dyes and Related Compounds, Wiley, New York. (6) Narayanan, N., and Patonay, G. (1995) A New Method for the Synthesis of Heptamine Cyanine Dyes: Synthesis of New Near-Infrared Fluorescent Labels. J. Org. Chem. 60, 23912395. (7) Illy, H., and Funderburk, L. (1968). Fisher Indole SynthesisDirection of Cyclization of Isopropylmethyl Ketone Phenylhydrazone. J. Org. Chem. 33, 4283-4285.

362 Bioconjugate Chem., Vol. 7, No. 3, 1996 (8) Heseltine, D. W., Jones, J. E., and Lincoln, L. L. (1969) Butadienyl Dyes for Photography, U.S. Patent 3,481,927. (9) Sturmer, D. M. (1977) Syntheses and Properties of Cyanine and Related dyes. In Special Topics in Heterocyclic Chemistry (W. T. Weissberger and E. C. Taylor, Eds.) pp 441-587, John Wiley & Sons, New York.

Mujumdar et al. (10) Herz, A. H. (1974) Dye-dye Interactions of Cyanines in Solution and at Silver Bromide Surfaces. Photogr. Sci. Eng. 18, 323-335.

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