Structure Property Analysis of Pentamethine Indocyanine Dyes

70

Bioconjugate Chem. 2004, 15, 70−78

Structure Property Analysis of Pentamethine Indocyanine Dyes: Identification of a New Dye for Life Science Applications Oliver Mader,§ Knut Reiner,# Hans-Joachim Egelhaaf,† Rainer Fischer,§ and Roland Brock*,§ Institute for Cell Biology, University of Tu¨bingen, Auf der Morgenstelle 15, 72076 Tu¨bingen, Germany, and Institute of Physical and Theoretical Chemistry, University of Tu¨bingen, Auf der Morgenstelle 8, 72076 Tu¨bingen, Germany, and FEW Chemicals GmbH, ChemiePark Bitterfeld Wolfen, Areal A Technikumstrasse 1, 06766 Wolfen, Germany. Received October 16, 2003; Revised Manuscript Received November 27, 2003

A collection of nine pentamethine indocyanine dyes was synthesized, and the photophysical characteristics relevant to applications in cell biology and single molecule detection were analyzed in detail. Substituents at the aromatic system covering the auxochromic series and substitutions in the polymethine chain were investigated with respect to absorption and emission spectra, fluorescence lifetimes, fluorescence quantum yields, and fluorescence autocorrelations. Substitutions in the polymethine chain increased the nonradiative energy dissipation of the excited singlet state and decreased the fluorescence quantum yield, relative to the unsubstituted compound. For substituents at the aromatic rings the fluorescence quantum yield negatively correlates with the position of the substituents in the auxochromic series -SO3-, -H, -F, -CH3. Compounds with sulfonic acid groups or halogen atoms attached to the indolenine systems had the highest fluorescence quantum yields. The compound S0387 had nearly 70% of the quantum yield of Cy5 and comparable photostability. The free carboxylic acid of S0387 was attached to peptides in high yield and purity by established procedures of solid-phase synthesis. The dye-labeled peptides did not aggregate or bind to tissue culture cells and proteins unspecifically. The indocyanine dye S0387 is therefore an attractive new fluorophore for in vitro and cell-based detection of receptor ligand interaction at nanomolar concentrations by flow cytometry, fluorescence correlation spectroscopy, and laser scanning microscopy.

INTRODUCTION

Over the past decade, fluorescence has become one of the key detection principles in life sciences and drug discovery. The sensitivity of fluorescence-based detection systems has enabled the miniaturization of assay formats in high-throughput screening for new drug candidates, greatly reducing the consumption of costly assay reagents (1). Spectrally resolved detection modalities, such as fluorescence anisotropy decay, detection of fluorescence lifetimes, and simultaneous detection of different wavelengths (2), have greatly increased the information per assay. Depending on the fluorophore used, the spectral characteristics provide valuable information on the chemical environment of a fluorophore (3), molecular interactions (4), or enzymatic turnover (5). The suitability of a fluorophore for life science applications is determined by its photophysical characteristics and the chemical compatibility with the respective biological application. The key photophysical parameters of a fluorophore are the compatibility of the excitation and emission spectra with available instrumentation, the * To whom correspondence should be addressed. Roland Brock, Institute for Cell Biology, University of Tu¨bingen, Auf der Morgenstelle 15, 72076 Tu¨bingen, Germany. Ph.: +49-70712977629. Fax: +49-7071-295359. E-mail: roland.brock@ uni-tuebingen.de, [email protected]. § Institute for Cell Biology, University of Tu ¨ bingen. † Institute of Physical and Theoretical Chemistry, University of Tu¨bingen. # FEW Chemicals GmbH.

fluorescence quantum yield, the dependence of the photophysical characteristics on the chemical environment, and its photostability. Moreover, the chemical stability of the fluorophore to labeling conditions, minimum interference with biological activity of the labeled molecules, and the solubility in aqueous buffers are essential prerequisites for applications (6). Detection techniques based on fluorescence emitted from single molecules are gaining significance in miniaturized drug screening and biophysical research. Fluorophores employed for such detection modalities should be free of any tendency to aggregate. In addition, the quantum yield for the transition into electronic states with long lifetimes, such as triplet state transitions, should be minimal for techniques based on the timeresolved analysis of fluorescence fluctuations. Applications of fluorophores in life sciences include the labeling of small synthetic molecules and of large biomolecules such as proteins in screening applications for drug development, of antibodies for immunofluorescence (7), of nucleotides in DNA sequencing (8), and of oligonucleotides or proteins as probes in DNA and protein microarrays (9). The requirements of chemical stability depend on the molecules to be labeled. To label proteins, an active ester or maleimido group is usually attached to a free carboxyl group of the fluorophore (10, 11). These groups react with primary amines or thiol groups in aqueous buffers and at mildly basic pH, conditions affecting neither the biomolecule nor the dye. In solidphase synthesis (SPS)1 (12), the in situ activation of carboxylic acids provides a more efficient and cheaper way to label compounds (13) than preactivation. Using

10.1021/bc034191h CCC: $27.50 © 2004 American Chemical Society Published on Web 01/21/2004

Structure Property Analysis of Pentamethine Dyes

Bioconjugate Chem., Vol. 15, No. 1, 2004 71

Figure 2. Processes contributing to the dissipation of energy from the excited singlet state. The fluorescence rate constant kF describes the dissipation by emission of light, kISC intersystem crossing to the triplet state, kIC internal conversion to the ground state, and kPERP the transition to the partially twisted transition state from where the isomerization or the dissipation to the ground state occurs with efficiencies ηISO and ηBACK ) 1 - ηISO, respectively.

Figure 1. Structures of the pentamethine indocyanine dyes investigated in this work.

dye and coupling reagents in excess, complete formation of the labeled products can be achieved. Unreacted dye can be removed easily by washing the solid-phase resin. However, relatively harsh chemical conditions may be encountered during further on-resin labeling of compounds, such as strong acid for the cleavage of these compounds from the resin. Thus, the reactivity of a fluorophore to the conditions encountered in a particular SPS protocol needs to be evaluated in detail. Compounds based on the indocyanine scaffold are widely used as fluorophores for labeling biomolecules (14-18). The spectral characteristics of these compounds depend primarily on the length of the polymethine chain linking the two aromatic systems. As a rule-of-thumb, the wavelength of the excitation and emission maxima shifts to the red by 100 nm with every vinylene unit, rendering the n ) 0 dyes (n is the number of vinylene units in the polymethine chain) compatible with excitation with argon-ion lasers at 488 nm, the n ) 1 dyes compatible with excitation by helium-neon lasers at 543 nm, and the n ) 3 dyes compatible with excitation by helium-neon lasers at 633 nm (19, 20). Dyes with n ) 2 or n ) 3 have maximum quantum yields (21). A major factor reducing the fluorescence quantum yield of the polymethine dyes is the photoinduced trans-cis isomerization of the polymethine chain. This process, which occurs via a twisted excited singlet state (22), leads to a nonradiative dissipation of absorbed energy (Figure 2) (23). Efforts to improve the photophysical characteristics of fluorophores based on a number of different scaffolds have identified sulfonic acid groups and halogen atoms as substituents increasing water solubility (17) and fluorescence quantum yield and decreasing the depen1 Abbreviations: Aca, aminocaproic acid; ACN, acetonitrile; Boc2O, di-tert-butyl dicarbonate; DCM, dichloromethane; Dde, 1-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl; DIC, N,N′-diisopropyl carbodiimide; DIPEA, N,N′-diisopropylethylamine; DMF, N,N′-dimethylformamide; DMSO, dimethyl sulfoxide; FCS, fluorescence correlation spectroscopy; Fmoc, 9-fluorenylmethoxycarbonyl; fwhm, full width at half-maximum; HBS, HEPES-buffered saline; HeNe, helium-neon; HEPES, N-2hydroxyethylpiperazine-N′-2-ethanesulfonic acid; HOBt, 1-hydroxybenzotriazol; HPLC, high-performance liquid chromatography; MALDI-MS, matrix-assisted laser desorption ionizationmass spectrometry; MeOH, methanol; MHC, major histocompatibility complex; NMR, nuclear magnetic resonance; PBS, phosphate buffered saline; RP, reversed phase; SPPS, solid-phase peptide synthesis; SPS, solid-phase synthesis; tBuOH, tert-butyl alcohol; TFA, trifluoroacetic acid; TIS, triisopropylsilane.

dence of the fluorescence on the chemical environment. The results of these efforts are exemplified by the difluorinated fluorescein derivatives Oregon Green 488 and Oregon Green 514 (24). Early analyses of the structure property relationship of substituted polymethine dyes in benzene solution demonstrated a sensitive dependence of the photophysical properties on the substituents on the aryl rings (25). Groups with strong -I effects, e.g., trifluoromethyl groups, increase the fluorescence quantum yield compared with the unsubstituted analogues (25). To assess the effect of substitutions on the photophysical characteristics of pentamethine indocyanines in more detail and to identify new fluorophores for life science applications, a collection of nine pentamethine indocyanine dyes was synthesized and the photophysical properties were determined in aqueous buffers, methanol, and glycerol. Two of these compounds bear a methyl or a chlorine substituent in the polymethine chain to investigate the effect of such substitutions on the tendency to undergo photoinduced trans-cis isomerization. Absorption, excitation, and emission spectra were recorded and fluorescence lifetimes and quantum yields were measured for each of the new derivatives. Fluorescence correlation spectroscopy (FCS) was employed to assess the suitability of these compounds for single molecule detection. In FCS, information on molecular interactions and molecular concentrations is derived from temporal autocorrelations of time-resolved fluorescence measurements (26, 27). The autocorrelation function responds sensitively to the presence of fluorescent aggregates. Moreover, photophysical transitions into longlived dark states manifest themselves in the autocorrelation function (28). A detailed analysis of the photoinduced trans-cis isomerization of Cy5 by FCS was presented in 2000 (23). The compound S0387 had about 70% of the quantum yield of Cy5, had comparable photostability, and did not show any aggregation. For this reason, this compound was further characterized with respect to life science applications. A major histocompatibility complex (MHC) class I binding epitope (SIINFEKL) (29, 30) was labeled with this fluorophore and tested for MHC binding by confocal laser scanning microscopy and flow cytometry. In another experiment, the interaction of an S0387 labeled c-myc peptide (31) with a peptide binding antibody was detected by FCS (32). MATERIALS AND METHODS

Synthesis of Fluorophores. S0387. (A) 2-(4-Acetanilino-1,3-butadienyl)-3,3-dimethyl-1-(4-sulfobutyl)-indolium inner salt: A mixture of 1-(4-sulfobutyl)-2,3,3-trimethylindolium inner salt (4.75 g, 50 mmol) and malonaldehyde dianil hydrochloride (13.2 g, 51 mmol) in 80 mL of acetic acid anhydride was allowed to react for 1 h at a temperature of 120 °C. After cooling to room temperature, the

72 Bioconjugate Chem., Vol. 15, No. 1, 2004

reaction mixture was diluted with 300 mL of ethyl acetate. The crude solid was boiled with acetonitrile. Yield: 19.4 g. (B) 1-(5-Carboxypentyl)-2,3,3-trimethyl-indolium-5-sulfonate: The sodium salt of 2,3,3-trimethyl-indoleninium5-sulfonate (2.61 g, 10 mmol) and 6-bromohexanoic acid (2.1 g, 11 mmol) were mixed and heated at 110 °C for 2 h with constant stirring. The reaction mixture was cooled to room temperature, and the residue was boiled with acetone until a powder was obtained. Yield: 2.58 g. 1H NMR (DMSO-d ): δ (ppm) 7.94 (s, 1H, arom. CH), 6 7.65 (dd, 1H, arom. CH), 7.46 (d, 1H, arom. CH), 4.43 (t, 2H, NCH2), 2.83 (s, 3H, CH3), 2.21 (t, 2H, CH2), 1.84 (b, 2H, CH2), 1.54(s, 3H, CH3), 1.32-1.61 (b, 4H, CH2) (C) 2-[5-[1-(5-Carboxypentyl)-1,3-dihydro-3,3-dimethyl5-sulfo-2H-indol-2-ylidene]-penta-1,3-dienyl]-3,3-dimethyl1-(4-sulfobutyl)-3H-indolium inner salt, sodium salt: Compound A (2.33 g, 5 mmol) and compound B (1.77 g, 5 mmol) were dissolved in 20 mL of ethanol. After sodium acetate (0.82 g, 10 mmol) was added to the sample, the mixture was heated for 20 min to reflux. The solution was cooled to 5-8 °C, and the crude product was collected by filtration and washed with ethyl acetate. After airdrying the crude dye was chromatographed on silica gel (Kieselgel 60, Merck, Darmstadt, Germany) using a methanol/chloroform (4:9, v/v) mixture as eluent. Yield: 0.81 g, λmax (methanol): 646 nm, ) 187 000 M-1 cm-1. 1H NMR (DMSO-d ): δ (ppm) 8.35 (m, 2H, CH), 7.81 6 (d, 1H, arom. CH), 7.64 (d, 2H, arom. CH), 7.48(m, 1H, arom. CH), 7.30 (m, 2H, arom. CH), 6.62 (t, 1H, CH), 6.36 (q, 2H, CH), 4.08 (b, 4H, NCH2), 2.55 (b, CH2), 2.14 (t, 2H, CH2), 1.75 (b, CH2), 1.69 (s, 12H, CH3), 1.55 (b, CH2), 1.37 (b, CH2), HPLC: 97.3%. S0693. (D) 2,3,3,5-Tetramethyl-1-(4-sulfobutyl)-indolium hydroxide inner salt: 2,3,3,5-tetramethylindolenine (8.65 g, 0.05 mol) and 1,4-butanesulfone (9.0 g, 0,06 mol) were mixed and heated to 120 °C for 2 h. The reaction mixture was cooled and triturated with acetone until a purple colored powder was obtained. Yield: 8.1 g. 1H NMR (DMSO-d ): δ (ppm) 7.91 (d, 1H, arom. CH), 6 7.66 (s, 1H, arom. CH), 7.43 (d, 1H, arom. CH), 4.76 (t, 2H, NCH2), 2.83 (s, 3H, CH3), 2.54 (m, 2H, CH2), 2.45 (s, 3H, CH3), 1.98 (m, 2H, CH2), 1.73 (m, 2H, CH2), 1.52 (s, 6H, CH3). (E) 2-(4-Acetanilino-1,3-butadienyl)-3,3,5-trimethyl-1(4-sulfobutyl)-indolium inner salt: This compound was prepared by reacting compound D with malonaldehyde dianil hydrochloride following the procedure for compound A. Yield: 15.7 g. (F) 2-[5-[1-(5-Carboxypentyl)-1,3-dihydro-3,3-dimethyl5-sulfo-2H-indol-2-ylidene]-penta-1,3-dienyl]-3,3,5-trimethyl-1-(4-sulfobutyl)-3H-indolium inner salt, sodium salt: This dye was prepared by condensing intermediates E and B following the procedure for compound C. Yield: 0.58 g, λmax (methanol): 651 nm, ) 163000 M-1 cm-1. 1H NMR (DMSO-d ): δ (ppm) 8.33 (m, 2H, CH), 7.80 6 (d, 1H, arom. CH), 7.63 (d, 1H, arom. CH), 7.48 (m, 1H, arom. CH), 7.39 (d, 1H, arom. CH), 7.26 (m, 2H, arom. CH), 6.59 (t, 1H, CH), 6.43 (d, 1H, CH), 6.24 (d, 1H, CH), 4.10 (b, 4H, NCH2), 2.60 (b, CH2), 2.39 (s, 3H, CH3), 2.22 (t, 2H, CH2), 1.76 (b, CH2), 1.69 (s, 12H, CH3), 1.56 (b, CH2), 1.39 (b, CH2), HPLC: 95.8%. S0694. (G) 5-Fluoro-2,3,3,-trimethyl-1-(4-sulfobutyl)indolium hydroxide inner salt: The compound was prepared following the procedure for compound D. Yield: 13.7 g. 1 H NMR (DMSO-d6): δ (ppm) 8.23 (q, 1H, arom. CH), 7.86 (d, 1H, arom. CH), 7.50 (t, 1H, arom. CH), 4.51 (t,

Mader et al.

2H, NCH2), 2.86 (s, 3H, CH3), 2.54 (m, 2H, CH2), 1.99 (m, 2H, CH2), 1.76 (m, 2H, CH2), 1.56 (s, 6H, CH3). (H) 2-(4-Acetanilino-1,3-butadienyl)-5-fluoro-3,3,-dimethyl-1-(4-sulfobutyl)-indolium inner salt: This compound was prepared by reacting compound G with malonaldehyde dianil hydrochloride following the procedure for compound A. Yield: 16.1 g. (I) 2-[5-[1-(5-Carboxypentyl)-1,3-dihydro-3,3-dimethyl5-sulfo-2H-indol-2-ylidene]-penta-1,3-dienyl]-5-fluoro-3,3dimethyl-1-(4-sulfobutyl)-3H-indolium inner salt, sodium salt: This dye was prepared by condensing intermediates H and B following the procedure for compound C. Yield: 0.41 g, λmax (methanol): 646 nm, ) 140 000 M-1 cm-1. 1 H NMR (DMSO-d6): δ (ppm) 8.34 (m, 2H, CH), 7.81 (d, 1H, arom. CH), 7.68 (m, 2H, arom. CH), 7.49 (m, 1H, arom. CH), 7.29 (m, 2H, arom. CH), 6.60 (t, 1H, CH), 6.36 (q, 2H, CH), 4.14 (b, 4H, NCH2), 2.58 (b, CH2), 2.07 (t, 2H, CH2), 1.78 (b, CH2), 1.71 (s, 12H, CH3), 1.56 (b, CH2), 1.35 (b, CH2), HPLC: 88.1%. S0436. (J) 2-[5-[1-Carboxypentyl-1,3-dihydro-3,3-dimethyl-2H-indol-2-ylidene]-penta-1,3-dienyl]-3,3-dimethyl-1-(4-sulfobutyl)-3H-indolium hydroxide inner salt: The dye was prepared according to the synthesis of compound C by the reaction of 1-(5-carboxypentyl)-2,3,3-trimethylindoleninium bromide with compound A; λmax (methanol): 643 nm, ) 160 000 M-1 cm-1. 1 H NMR (DMSO-d6): δ (ppm) 8.35 (m, 2H, CH), 7.63 (d, 2H, arom. CH), 7.39 (m, 4H, arom. CH), 7.23 (m, 2H, arom. CH), 6.60 (t, 1H, CH), 6.33 (q, 2H, CH), 4.09 (b, 4H, NCH2), 2.57 (b, CH2), 2.05 (t, 2H, CH2), 1.76 (b, CH2), 1.67 (s, 12H, CH3), 1.52 (b, CH2), 1.38 (b, CH2), HPLC: 98.2%. S0430. (K) 3-Carboxypentyl-2-[5-(1,3-dihydro-3,3-dimethyl-1-(4-sulfobutyl)-2H-indol-2-ylidene)-penta-1,3-dienyl]-1,1-dimethyl-1H-benzo[e]indolium hydroxide inner salt: The dye was prepared according to the synthesis of compound C by the reaction of 1-(5-carboxypentyl)2,3,3-trimethyl-indoleninium bromide with 2-(4-acetanilino-1,3-butadienyl)-1,1-dimethyl-3-(4-sulfobutyl)-Hbenzo[e]indolium hydroxide inner salt, λmax (methanol): 663 nm, ) 162 000 M-1 cm-1. 1 H NMR (DMSO-d6): δ (ppm) 8.43 (t, 1H, arom. CH), 8.33 (t, 1H, arom. CH), 8.22 (d, 1H, arom. CH), 8.05 (dd, 2H, CH), 7.78 (d, 1H, arom. CH), 7.67 (t, 1H, arom. CH), 7.58 (d, 1H, arom. CH), 7.49 (t, 1H, arom. CH), 7.35 (m, 2H, arom. CH), 7.19 (m, 1H, arom. CH), 6.61 (t, 1H, CH), 6.45 (d, 1H, CH), 6.24 (d, 1H, CH), 4.25 (b, 2H, NCH2), 4.05 (b, 2H, NCH2), 2.57 (b, CH2), 2.04 (t, 2H, CH2), 1.91 (s, 3H, CH3), 1.81 (b, CH2), 1.67 (s, 12H, CH3), 1.55 (b, CH2), 1.35 (b, CH2), 1.27 (b, CH2), HPLC: 95.8%. S0223. (L) 2-[5-[1-(5-Carboxypentyl)-1,3-dihydro-3,3dimethyl-2H-indol-2-ylidene]-penta-1,3-dienyl]-1,3,3-trimethyl-3H-indolium bromide: The dye was prepared according to the synthesis of compound C by the reaction of 1-(5-carboxypentyl)-2,3,3-trimethyl-indoleninium bromide with 2-(4-acetanilino-1,3-butadienyl)-1,3,3-trimethylindolium bromide; λmax (methanol): 640 nm, ) 184 000 M-1 cm-1. 1H NMR (DMSO-d ): δ (ppm) 8.09 (t, 2H, CH), 7.506 7.00 (b, 8H, arom. CH), 6.85 (t, 1H, CH), 6.36 (d, 2H, CH), 6.30 (d, 1H, CH), 4.06 (t, 2H, NCH2), 3.68 (s, 3H, CH3), 2.41 (t, 2H, CH2), 2.08 (t, 2H, CH2), 1.73 (s, 12H, CH3), 1.35 (b, CH2), HPLC: 95.3%. The symmetrical dyes S0438 and S0428 were prepared following the procedures described in earlier publications (33, 34). The unsymmetrical dye S0301 was synthesized using a procedure described in the literature (35). The purity of all dyes was confirmed using a Merck-Hitachi analytical high performance liquid chromatography



Table 1. Photophysical Characteristics of Pentamethine Indocyanine Derivativesa

Cy5 S0693 S0694 S0223 S0301 S0387 S0428 S0430 S0436 S0438

λmax abs [nm] in MeOH

λmax em [nm] in MeOH

(λmax abs) [M-1 cm-1] in MeOH

[ns] τF in PBS

ΦF in PBS

kF [109 s-1]

kNR [109 s-1]

[ns] τR in PBS

r in PBS

648 649b 651 646 640 666 646 653 663 643 647

670 670b 675 668 665 692 669 669 688 667 665

250000b

1.00d

0.27c

0.27

0.73

0.58

0.14

163000 140000 184000 203000 187000 240000 162000 160000 270000

0.45 0.60 0.56 0.50 0.70 0.50 0.50 0.67 0.46

0.10 0.14 0.12 0.08 0.18 0.12 0.08 0.15 0.11

0.22 0.23 0.21 0.16 0.26 0.24 0.16 0.22 0.24

2.00 1.43 1.57 1.84 1.17 1.76 1.84 1.27 1.94

0.62 0.54 0.37 0.56 0.47 0.56 0.50 0.39 0.57

0.22 0.18 0.15 0.20 0.15 0.20 0.19 0.14 0.21

a The molar extinction coefficients were determined for fluorophores dissolved in methanol at a concentration of 10 mg/L. The fluorescence lifetime τF, the fluorescence quantum yield ΦF, the rate constant of fluorescence kF, the nonradiative rate constant kNR, as well as the anisotropy r and the rotational correlation time τR were determined as described in the materials and methods section. b Values were adopted from ref 17. c Value in PBS was obtained from Amersham Bioscience. d Agrees very well with reported lifetime of Cy5 (1.0 ns) (45).

(HPLC) unit, equipped with a Purospher RP18e column (Agilent, Waldbronn, Germany) with acetonitrile/water (1:1, v/v) as eluent. Peptide Synthesis. The SIINFEKL (MHC class I epitope) and EQKLISEEDL (myc-tag) peptides were synthesized using solid-phase Fmoc-chemistry on an automated peptide synthesizer for multiple peptide synthesis (RSP5032, Tecan, Hombrechtlikon, Switzerland). Fmoc-protected amino acids were purchased from Novabiochem (Laüfelfingen, Switzerland). Standard chemicals for peptide chemistry were obtained from Fluka (Deisenhofen, Germany) and Merck (Darmstadt, Germany), solvents were p. a. grade. The SIINFEKL peptide was synthesized on Fmoc-Leu preloaded 2-chloro trityl resin (capacity 0.5 mmol/g; Senn Chemicals, Dielsdorf, Switzerland) using Fmoc-Lys(Dde)-OH as an orthogonally deprotectable building block. To protect the N-terminus from the reaction with dye, the resin bound peptide SIINFEK(Dde)L (15 µmol) was reacted with Boc2O (39.3 mg, 180 µmol) and DIPEA (31 µL, 180 µmol) in DMF for 32 h. The Dde protecting group at the -amino group of lysine was cleaved off using 2% hydrazine hydrate in DMF (3 × 5 min). S0387 was covalently attached to the deprotected -amino group using 5 equiv of DIC (11.7 µL, 75 µmol), 5 equiv of HOBt (11.5 mg, 75 µmol) and 1.5 equiv of S0387 (15.9 mg, 22,5 µmol) in DMF twice for 16 h. The peptide was cleaved off the resin by treatment with 92.5% TFA, 2.5% TIS, 2.5% ethanedithiol, and 2.5% H2O twice for 2 h, each. The crude peptide was precipitated by adding cold diethyl ether (-20 °C). Finally, the peptide was dissolved in tBuOH/H2O 4:1 and lyophilized. The c-myc peptide Aca-EQKLISEEDL-Aca-K-NH2 was synthesized on Rink amide resin (Rapp Polymere, Tu¨bingen, Germany). The dye S0387 was attached to the N-terminus. Coupling conditions as well as cleavage off the resin were performed as described for the SIINFEKL peptide. The identity and purity of the peptides was determined using Maldi-TOF-MS (G2025A, Hewlett-Packard, Waldbronn, Germany) and by analytical RP-HPLC using a H2O (0.1% TFA, solvent A)/ACN (0.1% TFA, solvent B) gradient on a Waters 600 System (Eschborn, Germany) with detection at 214 nm. The samples were analyzed on an analytical column (Nucleosil 100, 250 × 2 mm, C18 column, 5 µm particle diameter; Grom, Herrenberg, Germany), using a linear gradient from 10% B to 100% B within 30 min (flow rate: 0.3 mL/min). The calculated masses were 1631.1 and 2222.1 Da, experimental masses

1631.8 and 2222.8 Da [M+H+] for SIINFEK(S0387)L and S0387-Aca-EQKLISEEDL-Aca-K-NH2 respectively. The respective purities were >63 and >66%. The peptides were purified using preparative RP-HPLC (Nucleosil 300 C18 column, 10 µm particle diameter, 250 × 20 mm; Grom, Herrenberg, Germany) using H2O (0.1% TFA)/ ACN (0.1% TFA) gradient on a Waters 600 Multisolvent Delivery System (HPLC: >98%). Photophysical Characterization. Fluorescence spectra were recorded on an LS50B (Perkin-Elmer, RodgauJu¨gesheim, Germany) and a SPEX Fluorolog 222 spectrofluorometer (Jobin Yvon Horiba, Edison, NJ). The spectra were corrected for the sensitivity of the detection system. Absorption spectra were measured with an Ultrospec 2000 (Pharmacia Biotech, Cambridge, UK). Fluorescence lifetimes were measured on a SPEX Fluorolog 112 fluorometer. Fluorescence was excited by a stabilized HAMAMATSU C4725 diode laser, which provides pulse widths (full width at half-maximum, fwhm) of 64 ps at the excitation wavelength 653 nm. Fluorescence was detected with an R928 photomultiplier tube (Hamamatsu Photonics, Shizuoka, Japan). The resulting signals were fed into an ORTEC 457 time-toamplitude converter via an ORTEC 454 timing filter amplifier and an ORTEC 937 picosecond timing discriminator (AMETEK, Meerbusch, Germany). The fwhm of the resulting instrument response was ∆t ) 600 ps. Fluorescence decay times were obtained by fitting the decay curves to single exponential decays. Fitting to sums of exponentials did not improve the quality of the fits. The quantum yields Φsample of the dyes in PBS were determined using eq 1

Isample [1 - 10-Ereference] K Φsample ) Φreference Ireference [1 - 10-Esample]

(1)

where Esample and Eref are the absorbencies of sample and reference, respectively. Isample and Iref are the integral fluorescence intensities. K is a correction factor accounting for the difference in the steady-state fluorescence anisotropies of sample and reference, rsample and rref, respectively (Table 1). K is given by eq 2, where G is the ratio of the sensitivities of the detection system for vertically and horizontally polarized light, G ) Sv/Sh. A is the ratio of the quantum fluxes of horizontally and vertically polarized components in the exciting light, A ) Iexc,h/Iexc,v.


K)

Mader et al.

- (G + 1)] + (G + 1) [1 3G +A - (G + 1)] + (G + 1) [1 3G +A

rref

(2)

rsample

Cy5 in PBS served as the reference with Φreference ) 0.27 (Amersham Bioscience Europe, Freiburg, Germany). Measurements were performed at room temperature (20 °C) under magic angle conditions (polarizer vertical, analyzer at 54.7° from vertical), i.e., A ≈ 0 and G ) 2G′, where G′ ) 0.30 is the value obtained without polarizer in the detection system. Relative photostabilities were determined by irradiating 10 nM dye solutions in closed capillary tubes using the 633 nm HeNe Laser of a ConfoCor2 fluorescence correlation microscope (Carl Zeiss, Jena, Germany). The fluorescence quantum yields of Cy5 and S0387 in glycerol were measured immediately after preparation of the solutions, because both absorbance and fluorescence intensity of the solutions decreased quickly, even in the dark. Fluorescence Correlation Spectroscopy. FCS measurements were carried out on a ConfoCor2. For excitation, the light of a 633 nm HeNe-laser was reflected into the sample via an HFT633 main beam-splitter, and fluorescence was detected using an LP650 emission filter. The dyes were dissolved in methanol to a concentration of 300 µM and further diluted to working concentrations of 10 nM either in methanol or PBS, pH 7.4. Twenty autocorrelation functions were recorded over 10 s each. The prebleach time was set to zero to include initial photobleaching in the count-trace and the laser power was 84 kW/cm2. Autocorrelation functions not affected by initial photobleaching were fitted from autocorrelation times of 1 µs to 3.4 s with an algorithm describing one diffusing molecule (28) using the ConfoCor2 software (eq 3).

G(τ) ) 1 + 1 - T + Te-τ/τtr 1 ‚ ‚ NApp (1 - T)

1

( )x ( ) τ 1+ τD

(3)

ωxy 2 τ 1+ ω z τD

NApp is the apparent number of molecules affected by scattered light, τD is the diffusional autocorrelation time, and ωxy and ωz are the radii of the detection volume in the optical plane and along the optical axis, respectively (defined by the isocontour of e-2 detection efficiency relative to the center of the confocal detection volume). T denotes the equilibrium molar fraction of fluorophores in a transient nonfluorescent (“dark”) state (assumed, to a first approximation, equal for bound and unbound dyelabeled peptide (see below)) and τT is the triplet lifetime of the dark state. In this work, T and τT account for the sum of all intramolecular deactivation processes which lead to a dark state. Peptide antibody interactions were measured with 8.5 nM c-myc peptide (S0387-Aca-EQKLISSEDL-Aca-KNH2) and 260 nM of the monoclonal anti-c-myc antibody (clone 9E10, Dianova, Hamburg, Germany) in HBS using 384-well glass bottom microplates (MMI, Glattburg, Switzerland). To prevent unspecific binding of peptide and antibody, before use every well was blocked with 1% BSA (Sigma, Taufkirchen, Germany) in HBS for 12 h on ice followed by washing with HBS containing 0.2% BSA. The concentration of the antibody was derived from information provided by the supplier. The FCS data

obtained for peptide antibody interactions were fitted with an algorithm describing independent species of freely diffusing molecules, assuming that all fluorescent molecules have the same fluorescence efficiency (eq 4).

G(τ) ) 1 +

1 NApp

‚

1 - T + Te-τ/τtr

(

(1 - T)

‚ 1

∑j Yj

(

1+

τ

)x ( )

τD,j

1+

ωxy

2

τ

)

(4)

ωz τD,j

Yj is the contribution of the jth fluorescent molecule and τD,j is the respective diffusional autocorrelation time. MHC Peptide Binding. Murine RMA-S cells expressing the H-2kb MHC class I molecule (obtained from S. Stevanovic´) (36) were grown in a 5% CO2 humidified atmosphere at 37 °C in RPMI 1640 medium (PAN Biotech, Aidenbach, Germany), supplemented with 10% fetal calf serum (PAN Biotech), 100 U/mL penicillin, and 0.1 mg/mL streptomycin (Biochrom, Berlin, Germany). Cells were passaged every third to fourth day by a 1:5 dilution with fresh medium. To determine peptide binding to MHC class I molecules, cells were seeded at a density of 40 000 per well in 96-well plates (Nunc, Wiesbaden, Germany) and kept 16 h at room temperature (25 °C) in RPMI 1640 medium containing fetal calf serum to allow the accumulation of peptide free MHC class I molecules on the plasma membrane (37, 38). Cells were then incubated for 1 h with the S0387-labeled SIINFEKL peptide (SIINFEK(S0387)L) at concentrations of 1 and 10 nM and, alternatively, with a mixture of the labeled peptide and a 100fold excess of unlabeled peptide in serum free RPMI medium. Cells were washed four times with PBS followed by confocal laser scanning microscopy and flow cytometry. Confocal fluorescence images were recorded on an inverted LSM510 laser scanning microscope (Carl Zeiss, Go¨ttingen, Germany). S0387 was excited with a heliumneon laser at 633 nm and fluorescence detected with an LP650 long-pass filter. Flow cytometry was performed on a FACSCalibur flow cytometer (Becton Dickinson, Heidelberg, Germany) using a 633 nm HeNe-laser for excitation. Vital cells were gated based on forward light and side scatter. Cells, not incubated with any peptide, were used to set the sensitivity of the fluorescence detector to the appropriate level (histogram peak in the first decade of the 4 decade log scale). RESULTS AND DISCUSSION

Synthesis and General Characterization. The synthesis of the substituted pentamethine indocyanines (Figure 1) followed published protocols with modifications. A quaternary indolium salt having an acidic methyl group was reacted with malonaldehyde dianil hydrochloride followed by condensation of the intermediate with a second indolium salt under basic conditions in ethanol. Two of the compounds were symmetrical (S0428 and S0438), while the remaining dyes were unsymmetrical with respect to the substituents of the indolenine groups. For the asymmetric indocyanines, the second step resulted in the formation of a mixture of the symmetric indocyanine and the desired product which was purified by column chromatography on silica gel. The purity of all compounds was confirmed by analytical HPLC, and



the structures of the dyes were verified using 1H NMR spectroscopy. Except for S0694 (purity, 88%), the purity of all other compounds exceeded 95%. All compounds except S0223 were readily soluble in methanol and no precipitation occurred upon dilution of the methanol stock solutions into water or PBS. To render these compounds compatible with the labeling of biomolecules, each derivative contained at least one carboxyl group for covalent attachment to primary amines. All compounds except S0223 were substituted with at least one sulfonic acid group to increase water solubility. The compounds S0387, S0436, S0693, and S0694 were synthesized to assess the dependence of the photophysical characteristics on substitutions of the aromatic system with substituents of the auxochromic series. Compounds S0428 and S0438 were prepared to address the effect of substitutions at the meso-position of the pentamethine chain on isomerization. Rotation around one of the bonds of the pentamethine chain is known as an effective mechanism of radiationless loss of excitation energy of the pentamethine compounds. With S0301 and S0430 the effect of extensions of the aromatic systems were investigated. Photophysical Characteristics. Absorption, Excitation, and Emission Spectra. The absorption maxima of the dyes were in a range of 640-666 nm around the absorption maximum of Cy5 at 648 nm (Table 1). Replacement of both sulfonic acid groups on the aryl rings by hydrogen atoms (S0436) led to a hypsochromic shift of 5-8 nm compared to Cy5. Substitution of only one sulfonic acid group by hydrogen (S0387) or fluorine (S0694) caused a weak hypsochromic shift by 2 nm, while substitution by a methyl group (S0693) led to a small bathochromic shift by 3 nm. Introduction of a methyl group at the X3 position in the polymethine chain (S0438) caused a slight hypsochromic shift, while a significant bathochromic shift was observed for a chlorine substituent (S0428). The extension of the aromatic system shifted the absorption and emission maxima to the red by about 15 nm (S0301 and S0430). The Stokes shifts of all compounds were in the range between 16 and 26 nm, the smallest values being obtained for the dyes that carry a substituent in the X3 position. Fluorescence Quantum Yields and Lifetimes. After excitation of the indocyanine dyes to the excited singlet state, deactivation by fluorescence competes with nonradiative dissipation of energy (Figure 2). Internal conversion to the ground state with the rate constant kIC, intersystem crossing to the triplet state (with rate constant kISC), and transition into a partially twisted transition state with rate constant kPERP (39) from where trans-cis isomerization occurs are possible mechanisms for nonradiative deactivation (23, 40) that occurs with the rate constant kNR (eq 5). kISC is negligible in carbocya-

kNR ) kIC + kPERP + kISC

(5)

nines without heavy atoms (ΦISC < 10-3) (40, 41). The dyes with extended aromatic systems (S0301 and S0430) had the lowest fluorescence quantum yields (ΦF ) 0.08), and S0387 and Cy5 had the highest ones with ΦF ) 0.18 and ΦF ) 0.27, respectively (Table 1). The latter two compounds also had the longest fluorescence lifetimes τF of 0.70 and 1.0 ns. To get a deeper insight into the relationship of structure and photophysics of the dyes, the radiative rate constants kF and the rate constants of nonradiative deactivation kNR were calculated from the fluorescence quantum yields ΦF and the fluorescence lifetimes τF

Figure 3. Radiative and nonradiative rate constants for the cyanine dyes in PBS. Experimental values were obtained from fluorescence quantum yields and lifetimes (Table 1) using eqs 6 and 7. Calculated values were obtained from absorption spectra using the Strickler-Berg equation (eq 8).

according to eqs 6 and 7, respectively (Table 1).

ΦF τF

(6)

1 - ΦF τF

(7)

kF ) kNR )

A plot of kF vs kNR revealed that the differences in fluorescence quantum yields and decay times between the dyes were mainly due to the variations in kNR, while kF varied only very little (Figure 3). The values of kNR span a range between 0.73 × 109 and 2.0 × 109 s-1, with Cy5 showing the lowest nonradiative rate constant of all dyes followed by S0387. To identify the specific nonradiative process leading to this variation in kNR, the effect of solvent viscosity on fluorescence quantum yields and lifetimes of Cy5 and S0387 was investigated. The increase of the viscosity from 1 cPoise in PBS to 1340 cPoise in glycerol should strongly reduce kPERP, the rate constant of photoinduced twisting about the bonds in the polymethine chain (42, 43). In glycerol, the fluorescence quantum yields increased to ΦF ) 0.60 and ΦF ) 0.59 for Cy5 and S0387, respectively. For the fluorescence lifetimes τF ) 1.93 ns and τF ) 1.90 ns were obtained. While the radiative rate constants increased less than 20% with respect to the values obtained in PBS (Table 1), the nonradiative rate constants decreased by more than 70% to kNR ) 0.21 × 109 s-1 and kNR ) 0.22 × 109 s-1 for Cy5 and S0387, respectively. From the almost identical values of kNR for both dyes in glycerol, we concluded that the difference in kNR of Cy5 and S0387 in PBS was mainly due to the different rates kPERP of bond twisting in the excited state. Assuming that kPERP in glycerol is negligible for both dyes, values of kPERP ) 5.3 × 108 s-1 and kPERP ) 9.6 × 108 s-1 were calculated for Cy5 and S0387 in PBS, respectively. This result has important consequences for the application of these dyes. In environments of high viscosity, as they are encountered for fluorophores conjugated to proteins, the differences between the photophysical properties of Cy5 and S0387 are negligible. In summary, our results provide information on the effect of substitution pattern on kNR for three types of substitutions: (i) kNR increases in the series Cy5 < S0387 < S0436 < S0694 < S0693. Obviously, kNR increases with the position of X1- and X5-substituents in the auxochromic


Mader et al.

series along -SO3- < -H < -F < -CH3. This finding agrees with the results obtained on indocarbocyanine derivatives by Murphy and Schuster (25), who found decreasing kPERP values with increasing -M-effect of the terminal substituents as quantified by their Hammett parameters. Originally sulfonic acid groups were introduced into cyanine dyes to increase the water solubility of these compounds (17). Our results show that in addition to increasing the solubility these substituents also improve the fluorescent properties by decreasing kNR. (ii) Extension of the aromatic system by annellation of benzene rings increases the nonradiative rate constants. (iii) Introduction of substituents into the polymethine chain at the X3 position (S0428, S0438) resulted in relatively high nonradiative rate constants of kNR g 1.76 × 109 s-1, independent of the auxochromic effect of the substituent. This increase in kNR is propably caused by the enhancement of bond torsion due to steric crowding as described by Khimenko et al. (40). The radiative rate constants kF of most of the dyes were in the relatively small range between 0.21 × 109 s-1 and 0.27 × 109 s-1 (Figure 3). Only for the aryl substituted dyes S0430 and S0301 significantly smaller values of kF ) 0.16 × 109 s-1 were obtained. To elucidate the reasons for this anomaly, the radiative rate constants kF (calc) were calculated from the absorption spectra using the Strickler-Berg relationship (eq 8) and compared with the experimental values.

kF (calc) ) 2.8825 × 10-9cm2 s-1mol-1 ‚ n2 ‚ 〈ν˜ F-3〉-1 ‚

∫(ν˜ )dlnν˜

(8)

where n is the refractive index of the solution, ν˜ F is a wavenumber of the fluorescence spectrum, and (ν˜ ) is the extinction coefficient. For most compounds the Strickler-Berg equation resulted in radiative rate constants larger than those determined experimentally. For S0387, S0436, and S0694, the deviation was close to or within the margin of error. For S0223 and S0693, the deviation was less than 20%. Significantly larger deviations were found for the dyes with extended aromatic systems (S0301 and S0430), with substituents in the polymethine chain (S0428 and S0438) and for Cy5. The kF values calculated for S0301 and S0430 were in the range of most of the other compounds, whereas the values calculated for S0428 and S0438 exceeded those of all other compounds, due to the large extinction coefficients of the X3-substituted dyes. The most likely explanation for the discrepancies between calculated and experimental kF values are fast conformational changes upon excitation of the dyes, which are not accounted for by eq 8. However, although care was taken to avoid any aggregation and adsorption effects in the determination of the fluorescence quantum yields, the presence of nonfluorescent aggregates as another possible reason for this discrepancy cannot be excluded completely. The large difference between the experimental and calculated rate constants of fluorescence observed for Cy5 may be due to a too large extinction coefficient provided by the supplier. Rotational Diffusion. The rotational correlation times τR were calculated from the fluorescence lifetime τF and the steady-state anisotropy r according to eq 9.

τR )

τF r0 -1 r

(9)

Figure 4. Autocorrelation functions and fits for an S0387 labeled c-myc peptide in the absence (bottom curve, fit for one diffusing species) and presence of an anti-myc-tag antibody (top curve, fit for two diffusing species). Center curve: Fit to the autocorrelation function in the presence of antibody, normalized to the amplitude of the autocorrelation function in the absence in order to better illustrate the shift of the diffusional autocorrelation time. The laser power was 84 kW/cm2.

where the value observed in glycerol, r ) 0.38, was inserted for the limiting anisotropy, r0. The values of τR for the free dyes in PBS were in a narrow range of 0.37 ns for S0223 and 0.61 ns for Cy5. They were mainly determined by the hydrodynamic volume of the substituents in the X1 and X5 positions. Applications in FCS, Fluorescence Microscopy, and Flow Cytometry. Fluorescence Correlation Spectroscopy. To determine the suitability of the dyes for single molecule detection, fluorescence correlation spectroscopy was applied to fluorophores dissolved in methanol and diluted with PBS. For S0223, aggregates interfered with the acquisition of autocorrelation functions. For all other dyes the autocorrelations could be fitted with a function containing one diffusion term and one exponential term, the latter accounting for the formation of nonfluorescent states by all intramolecular deactivation processes, e.g., intersystem crossing (data not shown). Due to the limited temporal resolution of our instrument, no detailed analysis of this fast process was possible as presented by Widengren and Schwille (23). Autocorrelation functions with low noise were obtained for Cy5, S0693, S0694, S0387, S0428, and S0438. In contrast, for S0301, S0430, and S0436 the acquisition of the autocorrelation functions was complicated by the low molecular brightness. An important requirement for the application of fluorescent dyes in fluorescence microscopy and FCS is a high photostability. Therefore, the photostability of S0387 was compared to the photostability of Cy5. Aqueous solutions of both dyes in glass capillaries were illuminated with a focused HeNe laser at 633 nm and fluorescence was recorded over time (data not shown). Both dyes had comparable photostabilities. Due to its favorable photophysical properties, S0387 was further characterized as a reporter group in biochemical and cellular applications. First S0387 was used in the detection of molecular interactions in vitro by FCS measurements of an S0387 labeled c-myc peptide (S0387Aca-EQKLISEEDL-Aca-K-NH2) incubated with an antic-myc antibody (Figure 4). From the autocorrelation curve of S0387 and c-myc-S0387 a diffusion coefficient of 1.3 × 10-6 cm2 s-1 was calculated for the dye-labeled peptide, assuming a diffusion coefficient of 2.5 × 10-6 cm2 s-1 (44)



the Volkswagen-Foundation (Conformational Control of Biomolecular Function, Nachwuchsgruppen an Universita¨ten). LITERATURE CITED

Figure 5. Receptor binding of an S0387 labeled peptide. (A) RMA-S cells were incubated with 10 nM S0387 labeled SIINFEKL for 1 h followed by intensive washing with PBS. Images were acquired by confocal laser scanning microscopy. (B) Cells incubated with 10 nM S0387 labeled SIINFEKL peptide in the presence of a 100-fold excess of unlabeled SIINFEKL peptide. One confocal section is shown in each case.

for the free dye molecule. After incubation of the free c-myc-S0387-peptide with anti-c-myc antibodies, an equilibrium between free and antibody-bound peptide was observed. The antibody bound peptide was detected as a second diffusive component in the autocorrelation function with a longer diffusion time compared to the unbound peptide resulting in a diffusion coefficient of 3.5 × 10-7 cm2 s-1. Flow Cytometry and Fluorescence Microscopy. Nonspecific binding of fluorophores to proteins and cellular components is a major concern in the development of new fluorophores for life science applications. The fluorophore was attached to the MHC class I binding peptide SIINFEKL (SIINFEK(S0387)L) by solid-phase chemistry after selective deprotection of the -amino-group of the lysine side chain. The activation of the carboxyl group was carried out in situ using DIC and HOBt. Complete turnover of the amino group was achieved by coupling twice with only 1.5 times excess of dye. The fluorophore was fully resistant to the acidic conditions during the cleavage of the peptide from the resin and workup procedure. Incubation of RMA-S cells expressing empty MHC class I molecules (H-2kb) on the cell surface with S0387-labeled SIINFEKL resulted in a fluorescence confined to the cell surface (Figure 5). Using flow cytometry fluorescence could be detected from cells incubated in the presence of as little as 1 nM peptide (data not shown). Incubation in the presence of a 100-fold excess of unlabeled competitor peptide reduced the fluorescence to background levels. ACKNOWLEDGMENT

We thank Gu¨nther Jung for excellent facilities in peptide chemistry and instrumental analytics, Nicole Sessler for expert technical assistance in peptide synthesis, Lisa Neumann for proofreading the manuscript, and Manfred Claassen for technical assistance in FCS measurements. R.B. acknowledges financial support from

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BC034191H

Structure Property Analysis of Pentamethine Indocyanine Dyes

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