Studies of Luminescent Conjugated Polythiophene ... - ACS Publications

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Bioconjugate Chem. 2007, 18, 1860–1868

Studies of Luminescent Conjugated Polythiophene Derivatives: Enhanced Spectral Discrimination of Protein Conformational States Andreas Åslund,†,§ Anna Herland,‡,§ Per Hammarström,† K. Peter R. Nilsson,†,| Bengt-Harald Jonsson,† Olle Inganäs,‡ and Peter Konradsson*,† Chemistry, IFM, Linköping University, SE-581 83 Linköping, Sweden, and Biomolecular and Organic Electronics, IFM, Linköping University, SE-581 83 Linköping, Sweden. Received May 18, 2007; Revised Manuscript Received July 20, 2007

Improved probes for amyloid fibril formation are advantageous for the early detection and better understanding of this disease-associated process. Here, we report a comparative study of eight luminescent conjugated polythiophene derivates (LCPs) and their discrimination of a protein (insulin) in the native or amyloid-like fibrillar state. For two of the LCPs, the synthesis is reported. Compared to their monomer-based analogues, trimer-based LCPs showed significantly better optical signal specificity for amyloid-like fibrils, seen from increased quantum yield and spectral shift. The trimer-based LCPs alone were highly quenched and showed little interaction with native insulin, as seen from analytical ultracentrifugation and insignificant spectral differences from the trimerbased LCP in buffered and native protein solution. Hence, the trimer-based LCPs showed enhanced discrimination between the amyloid-like fibrillar state and the corresponding native protein.

INTRODUCTION Several human disorders, including Alzheimer’s disease, are known to be associated with protein misfolding events and amyloidogenesis. Both in ViVo deposits and in Vitro formed amyloid fibrils have a β-sheet-rich fibrillar structure. Although no structural or sequential homology of amyloidogenic proteins is present, the amyloid fibril structure appears generic, showing linear filaments with a diameter of approximately 10 nm (1). For early detection and better understanding of the processes involved in amyloid fibril formation, a sensitive method is fundamental. Traditionally, Congo Red (CR) provides the most standardized way of staining amyloid plaques, showing green birefringence in cross-polarized light (2). The binding of CR to amyloid is considered specific but requires good control and experience to be reliable (3). The specificity of CR binding to in Vitro amyloid fibrils has, however, been questioned (4), where it was demonstrated that Congo red binds to native, partially folded conformations and amyloid fibrils of several proteins. This finding indicates that CR must be used with caution as a diagnostic test for the presence of amyloid fibrils in Vitro. Thioflavin T (ThT) and the analogue thioflavin S (ThS) are other small molecules well established for use in analysis of aggregated amyloid proteins formed in Vitro. The binding of ThT to amyloid is somewhat weaker than CR binding, but upon binding, the dye increases its fluorescence more than 1000-fold (5). ThT is a bluegreen-emissive dye, causing interference with autofluorescence from tissue, which is a recurring problem. On the basis of the affinity of CR and ThT, several compounds have been synthesized to increase affinity and contrast, and remove metabolic and toxic liabilities for in ViVo applications as well as increasing blood- brain barrier permeability (6–9). A * Phone: +46- (0)13-28 17 28. Fax: +46- (0)13- 28 13 99. E-mail: [email protected]. † Chemistry. ‡ Biomolecular and Organic Electronics. § These authors contributed equally to this work. | Current address: UniversitätsSpital Zürich, Institute of Neuropathology, Department of Pathology, Schmelzbergstrasse 12, CH-8091 Zürich, Switzerland.

more novel approach to designing an in ViVo amyloid imaging probe is the synthesis of a dithienylethenyl compound, on the basis of the empirical experiences of dyes with affinity for amyloid structures performed by Nesterov et al. (8). Luminescent, conjugated polymers (LCPs) have been demonstrated as conformational sensitive optical probes to detect conformal changes in synthetic peptides (10, 11) and calcium induced conformal changes in calmodulin (12). More recently, we have shown that LCPs can be used to monitor amyloid fibril formation in Vitro (13, 14), for the histological labeling of amyloid deposits in ex ViVo tissue samples (15), and conformal mapping of amyloid deposits (16). The electronic structure and thereby the optical processes of LCPs is directly correlated to the conformation of the polymer chains and their organization. If the polymer is governed by a biomolecule capable of folding into several possible conformational states or quaternary organizations, then the optical properties of the polymer will be a spectroscopic signature of the state of the biomolecule. The chain length of π-conjugated polymers is directly correlated with physical properties such as optical absorption maximum or oxidation potential (17–20). Regioregular oligomers of well-defined chain length have been used as model compounds to facilitate the interpretation of physical properties in photonic and electronic applications (18, 20). Likewise, in biosensing, a defined conjugated oligomer can give a more specific interaction with the biomolecule, and the correlation between this interaction and the optical properties of the conjugated oligomer would be more easily interpreted. We have reported the use of a number of polythiophenes with diverse side chain funtionalization for biomolecular interactions (10, 12–16, 21), and more recently, we have synthesized trimer-based analogues of these. In this work, we demonstrate two examples of the synthesis of thiophene-based LCPs, PTT, and tPTAA (see Figure 1 and Scheme 1). These two LCPs complete a series of eight, which we have used in this study for structure/functionality evaluation of the properties influencing the interaction between LCPs and amyloid-like fibrils. The LCPs were designed to have different hydrophobic/hydrophilic properties as well as alterations in steric congestion. Interaction with native and fibrillar insulin in solution was studied and evaluated

10.1021/bc700180g CCC: $37.00  2007 American Chemical Society Published on Web 10/17/2007

Luminescent Conjugated Polythiophene Derivatives

Bioconjugate Chem., Vol. 18, No. 6, 2007 1861

Figure 1. (A) Structures of the eight LCPs: 1 (tPOWT), 2 (tPOMT), 3 (tPTT), 4 tPTAA, 5 (POWT), 6 (POMT), 7 (PTT), and 8 (PTAA). (B) Normalized absorption spectra of LCPs at concentration 25 µg/mL. Filled circles indicate the trimer-based analogue. POWT (green), tPOWT (green), POMT (red), tPOMT (red), PTT (blue), and tPTT (blue) in 50 mM HCl. PTAA (black) and tPTAA (black) are in 100 mM Na2CO3. Photograph of the LCPs 1 mg/mL in H2O; from top to bottom, left row, LCP 1–4; right row, LCP 5–8.

by means of their optical properties. The interaction between native/fibrillar insulin and the well-defined trimer-based tPTAA and tPOWT was investigated in more detail.

EXPERIMENTAL PROCEDURES Polymer Synthesis. Synthesis of PTAA (22), POWT (23), POMT (21), and tPOWT (13) has been described elsewhere, and recently, Nilsson et al. (16) reported the synthesis of tPOMT and tPTT. General Methods. Organic extracts were dried over anhydrous magnesium sulfate, filtered, and concentrated in vacuo at 40 °C. 3-Thiopheneethanol, 3-thiopheneacetic acid, and PEPPSI-IPr are commercially available from Sigma-Aldrich Co. Microwave reactions were carried out on a SmithCreator 482 microwave reactor. NMR-spectra were recorded on a Varian 300 MHz instrument. Chemical shifts are given in ppm relative to TMS in CDCl3 (δ 0.00) for 1H and 13C or CD3OD (δ 3.31) for 1H and CD3OD (δ 49.0) for 13C NMR. TLC was carried out on Merck precoated 60 F254 plates using UV-light and charring with ethanol/sulfuric acid/p-anisaldehyde/acetic acid 90:3:2:1 for visualization. Flash column (FC) chromatography was performed using silica gel 60 (0.040–0.063 mm, Merck). Gradient HPLC-MS was performed on a Gilson system (column, Phenomenex C-18 250 × 15 mm and Phenomenex C-18 150 × 4.6 mm for preparative and analytical runs, respectively;

pump, Gilson gradient pump 322; UV/vis-detector, Gilson 155; MS detector, Thermo Finnigan Surveyor MSQ; Gilson Fraction Collector FC204) using acetonitrile with 0.1% formic acid and deionized water with 0.1% formic acid as mobile phase. MALDI-TOF MS was recorded in linear positive mode with R-cyano-4-hydroxycinnamic acid matrix (CHCA) or 2,5-dihydroxy benzoic acid (DHB) as matrix. (2-Iodo-thiophen-3-yl)-acetic Acid Methyl Ester (9). Acetyl bromide (11.7 mL, 158 mmol) was added slowly to a solution of 3-thiopheneacetic acid (7.51 g, 52.8 mmol) in methanol (60 mL, 0 °C). After 4 h, the mixture was diluted with chloroform (70 mL) and washed twice with NaHCO3 (100 mL, sat, aq.) and once with H2O (100 mL). After concentration, the product (6.08 g, 39.0 mmol) was dissolved in chloroform/acetic acid (30 mL, 1:1) and cooled to 0 °C, and N-iodosuccinimide (11.0 g, 48.8 mmol) was added. Eighteen hours later, water was added to the reaction, and the organic phase was washed twice with KOH (50 mL, 10%, aq.) and H2O (50 mL). The crude product was purified by vacuum distillation to afford 9 in 43% yield. 13 C-NMR(CDCl3) δ: 35.9, 52.4, 123.1, 126.0, 128.6, 139.0, 170.9. 1 H-NMR(CDCl3) δ: 3.63 (s, 2H), 3.78 (s, 3H), 6.93 (d, 1H, J ) 5.48 Hz), 7.57 (d, 1H, J ) 5.48 Hz). HRMS calcd. for C7H7IO2S: [M]+ 281.9211; Found: 281.9209.

1862 Bioconjugate Chem., Vol. 18, No. 6, 2007

Åslund et al.

Scheme 1a

a Reagents and conditions: (i) 1. Bromo acetate, methanol; 2. NIS, chloroform/acetic acid (1:1); (ii) K2CO3, PEPPSI-IPr, toluene/methanol (1:1); (iii) 1. NaOH, H2O, dioxane; 2. FeCl3, TBA-triflate, chloroform. (iv) 1. p-TsCl, chloroform/pyridine (7:1); 2. N-t-Boc-Thr, K2CO3, DMF; (v) 1. TFA, chloroform; 2. FeCl3, TBA-triflate, chloroform.

(3″-Methoxycarbonylmethyl-[2,2′;5′,2″]terthiophen-3-yl)acetic Acid Methyl Ester (11). Compounds 9 (0.185 g, 0.656 mmol), 10 (0.113 g, 0.336 mmol), and K2CO3 (0.120 g, 0.868 mmol) were dissolved in degassed toluene/methanol (5 mL, 1:1), and Ar (g) was bubbled for 10 min. PEPPSI-IPr (0.006 mg) was added, and the reaction was subjected to microwave conditions for 10 min at 100 °C. The mixture was diluted with toluene (20 mL) and washed with HCl (10 mL, 1 M, aq.) and H2O (20 mL). The crude product was further purified by FC (toluene f toluene/ethyl acetate (40:1)) to give the trimer (11) (87 mg, 66%) as a pale yellow oil. 13 C-NMR(CDCl3) δ: 34.7, 52.3, 124.9, 127.5, 130.5, 130.6, 133.0, 135.9, 171.5. 1 H-NMR(CDCl3) δ: 3.72 (s, 6H), 3.79 (s, 4H), 7.05 (d, 2H, J ) 5.21), 7.14 (s, 2H), 7.25 (d, 2H, J ) 5.21). HRMS calcd. for C18H16O4S3: [M]+ 392.0211; Found: 392.0228. tPTAA (4). To a solution of the trimer (11) (0.086 g, 0.218 mmol) in dioxane (10 mL), NaOH (5.5 mL, 0.1 M, aq.) was added slowly. When no starting material was seen by TLC, the solution was transferred to an extraction flask, and ethyl acetate (30 mL) was added. The organic phase was washed with HCl (50 mL, 1 M, aq.) subjected to normal workup and used directly in the next step. To a solution of the deprotected trimer and tetrabutylammonium-trifluoromethanesulfonate (0.171 g, 0.436 mmol) in chloroform (5 mL, dry), FeCl3 (0.158 g, 0.981 mmol) was added at 0 °C. After 18 h, methanol was added and the product precipitated as red flakes. The red flakes were collected, dissolved in NaOH (3 M, aq.), neutralized with HCl (conc.), and precipitated with methanol to give tPTAA (4) as red crystals (17 mg, 20%). (2S,3R)-2-tert-Butoxycarbonylamino-3-(2-thiophen-3-yl-ethoxy)-butyric Acid (12). 3-Thiopheneethanol (7.52 g, 58.7 mmol) and p-toluenesulfonic chloride (190.7 g, 16.8 mmol) were added to a solution of chloroform/pyridine (80 mL, 7:1) at 0 °C. After one day, the solution was diluted with diethyl ether and washed twice with HCl (2 M, aq.), twice with NaHCO3 (5%, aq.), and once with H2O. FC (toluene f toluene/ethyl acetate 3:1) and recrystallization from ethyl acetate/hexane afforded 2-(3-thienyl)ethanol tosylate (16.1 g, 97%) as white crystals. The 1H NMR data was in accordance with that previously reported (24). The tosylated product (1.38 g, 4.90 mmol), N-t-Boc-Thr (2.15 g, 9.81 mmol), and K2CO3 (2.03 g, 14.7 mmol) were added to DMF (90 mL) and heated to 40 °C. After one day, the mixture was poured over HCl/ice (2 M, aq.

1:1) and extracted twice with toluene. The combined extracts were washed twice with H2O and purified twice on FC (toluene/ ethyl acetate 4:1 and toluene/ethyl acetate 6:1) to give 12 (1.16 g, 72%). 13 C NMR (CD3OD) δ: 20.3, 28.7, 30.4, 60.8, 66.4, 68.5, 80.8, 122.7, 126.5, 129.3, 139.4, 158.3, 172.7. 1 H NMR (CD3OD) δ: 1.18 (d, 3H, J ) 6.23 Hz), 1.46 (s, 9H), 3.00 (t, 2H, J ) 6.78 Hz), 4.11 (d, 1H, J ) 2.93), 4.22 (dq, 1H, J ) 2.93, 6.23 Hz), 4.34 (t, 2H, J ) 6.78), 7.02 (dd, 1H, J ) 1.47, 4.95 Hz), 7.15 (dd, 1H, J ) 1.47, 2.93 Hz), 7.33 (dd, 1H, J ) 2.93, 4.95). HRMS calcd. for C15H24NO5S: [M + H]+ 330.1375; Found: 330.1303. PTT (7). Compound 13 (0.818 g, 2.48 mmol) was added to a solution of dichloromethane/TFA (11 mL, 1:1). After 1 h, the solution was quenched with methanol (3 mL) and coevaporated with toluene three times. The deprotected product and tetrabutylammonium-trifluoromethanesulfonate (1.26 g, 3.22 mmol) were dissolved in chloroform (32 mL, dry) at 0 °C under a N2 atmosphere. FeCl3 (1.81 g, 11.2 mmol) was added to the solution. After one day, the solution was extracted three times with H2O. The aqueous solution was diluted with acetone, and conc. HCl (4 mL) was added. When the product had precipitated, it was centrifuged (6 min, 3600 rpm). The collected precipitate was dissolved in water (4 mL) and precipitated from acetone/HCl (40 mL, 25:1). The procedure was repeated twice to afford PTT (7) (224 mg, 39%) as red crystals. BoVine Insulin Preparation. Bovine insulin (Sigma-Aldrich) was dissolved in 2 M guanidine hydrochloride and was dialyzed versus three rounds of 25 mM HCl at 4 °C. The insulin stock solutions (0.5 e 2.0 mM) were stored at 4 °C and were stable for several weeks. Absorbance at 280 nm was employed for concentration determinations using ε280 ) 5840 M-1cm for insulin. Amyloid-Like Fibril Formation. A stock solution containing 320 µM bovine insulin in 25 mM HCl was prepared. The solution was placed in a water bath at 65 °C for at least 13 h to induce amyloid fibril formation. The fibrillated samples were ultra sonicated for at least 10 min prior to optical measurements to avoid sedimentation of large fibril aggregates. Optical Measurements. Stock solutions of each LCP at a concentration of 1 mg/mL in deionized water were prepared. For fluorescence measurements, 2.5 µL of the LCP solution was diluted with the buffer used: 50 mM HCl and 100 mM Na2CO3 at pH 10, to a final volume of 912.5 µL. In the samples where



native and fibrillar insulin were studied, 2.5 µL of the LCP solution was mixed 15 µL of the bovine insulin solution (25 mM HCl) diluted with buffer to a final volume of 912.5 µL. In titration experiments, the concentration of LCP was kept constant, and the concentration of sonicated insulin fibrils was varied between 0 and 105 µM. For the absorption measurements, 5 µL of the LCP solution was diluted to a total volume of 200 µL with buffer. The samples were incubated for 5 min at RT, and the excitation and emission spectra were recorded with an ISA Jobin-Yvon spex FluoroMax-2 apparatus. For the absorption measurements and the titration experiments, a Tecan Saphire2 plate reader was used. Analytical Ultracentrifugation. Weight average molecular weights were determined by analytical ultracentrifugation in Beckman Coulter XL-A/XL-I with an An50Ti rotor and a photoelectric scanner. Equilibrium sedimentation experiments were run at 3000, 7000, 11,000, 15,000, 25,000, 32,000, 40,000, and 45,000 rpm until equilibrium was reached at each speed (20 h) at 20 °C. Five microliters of stock LCP solution was mixed with 30 µL of native or fibrillated bovine insulin solution (320 µM in 25 mM HCl) or 30 µL 25 mM HCl and diluted to 200 µL in buffer. Data was collected at 280 and 446 nm and evaluated using the self-association model in the XL-I, XL-A data analysis software. The solvent density was calculated to be 0.99913 g/cm3 and 1.00915 g/cm3 for 50 mM HCl and 100 mM Na2CO3, respectively. The partial specific volume (ν) of insulin used was 0.735. ν of the LCPs was determined densiometrically in Anton Paar DMAC02 Precision Density Meter.

RESULTS AND DISCUSSION Synthesis. PTT. 3-Thiopheneethanol was tosylated in chloroform/pyridine, and the tosylate was substituted with N-t-BocThr in K2CO3 and DMF. Interestingly, the substitution of the tosylate with the secondary alcohol N-t-Boc-Thr gave a higher yield of 13 (72%) than that with the primary alcohol N-t-BocSer previously reported (57%) (23) (see Scheme 1). The functionalized thiophene monomer was subsequently deprotected and polymerized to give PTT (7) in 39% yield. tPTAA (4). Monohalogenated thiophenes are generally hard to separate from the nonhalogenated thiophene and the dihalogenated one using conventional flush chromatography. Therefore 3-thiopheneacetic acid was protected as a methylester in acetyl bromide and methanol before it was iodinated with NIS in chloroform and acetic acid. This product could be purified by distillation to give 9 in 43% over two steps. The diboronic ester (10) used to assemble 11 was made from thiophene with a protocol described by Parakka et al. (25). The coupling of 9 and 10 was performed under microwave conditions for 10 min at 100 °C using PEPPSI-IPr as catalyst to afford the trimer (11) in 66% yield. Finally, the esters were hydrolyzed under basic conditions, and the deprotected trimer was polymerized using iron(III)chloride to give tPTAA in 20% yield. Matrix Assisted Laser Desorption/Ionization Time-ofFlight Mass Spectroscopy (MALDI-TOF MS). MALDI-TOF MS has become increasingly important as a convenient method to characterize polymers even though the result often does not reliably show the actual chain length distribution (26). PTT (0.5 µL, 1 mg/mL) was mixed with CHCA in 0.1% TFA/acetonitrile (0.5 µL, 1:1) as the matrix. The spectra of the product showed polymers mainly in the range 10 to 18 monomer units following ion series corresponding to the monomer unit [227n]+, [227n + 35]+, and [227n + 70]+, where +35 and +70 correspond to covalent end termination of the polymer by one and two chlorines, respectively (see Figure 2A). There are few references (27) in the literature to negatively charged polymers character-

Figure 2. MALDI spectra of (A) PTT and (B) tPTAA.

ized by MALDI-TOF MS, and even the tPTAA-analogue PTAA, synthesized some time ago (28), has yet to be characterized this way. Therefore, a new protocol had to be used. tPTAA (0.5 µL) was mixed with DHB (0.5 µL, 10 mg/mL in 10% TFA). The spectra showed two main peaks corresponding to [362n]+, where n corresponds to 4 or 5 units (Figure 2B). The chain distribution of the LCPs varies to some extent depending on the chemical structure of the LCP. However, it seems that the trimer-based LCPs (1–4) generally have the same molecular weight (around 1500 Da) regardless of the substituent (13). The same trend can be observed for the monomer-based LCPs (5–7), but the molecular weight is higher (around 3000 Da) (21, 29). Optical Characterization of LCPs. The optical properties, measured by absorption, fluorescence, and circular dichroism (CD), of the LCPs in Figure 1 are highly pH-dependent because of the different degree of protonation of the carboxyl and amine groups on the side chains (13, 30). The degree of protonation will influence the propensity of the polymer chains to aggregate in water-based solutions. Aggregation will enhance interchain interactions, leading to a red shift in absorption and fluorescence and lower fluorescence intensity due to higher probability of nonradioactive de-excitation pathways. The trimer-based LCPs (compounds 1–4; Figure 1) showed more red-shifted spectra and lower fluorescence intensity (see Table 1 and Figure 1) compared to their monomer-based analogues (compounds 5–8; Figure 1) in the same buffer solution. These differences in optical properties are likely to arise from the lower degree of side-chain substitution along the polymer backbone. The lower


Åslund et al.

Table 1. Optical properties of LCP values from Figures 1 and 3a abs

fluor. λmax, Imax

LCP

λmax

buffer

native ins.

fibrill. ins.

1 5 2 6 3 7 4 8

445 405 456 449 453 430 478 438

585.5; 8 552.5; 18 585; 21 576.5; 57 587.5; 13 576; 52 587; 13 561.5; 35

585; 9 556.5; 28 586; 21 578; 86 588; 15 579; 48 586; 12 565.5; 57

555.5; 84 563; 50 579.5; 95 581.5; 95 571; 42 575; 58 571; 53 573.5; 100

(tPOWT) (POWT) (tPOMT) (POMT) (tPTT) (PTT) (tPTAA) (PTAA)

fluor. λmax, Imax

fluor. λmax, Imax

a The maximum fluorescence intensity was normalized, and the highest intensity of the spectrum PTAA with fibrillar insulin corresponds to 100.

degree of side-chain substitution can enhance interchain backbone interaction and promote aggregation. Optical Characterization of LCPs Interacting with Native and Fibrillar Insulin. We have shown earlier the characteristic changes in optical properties, in fluorescence–excitation and emission, of LCPs upon interaction with amyloidlike fibrils (13, 14, 16), PTAA and tPOWT interacting with insulin fibrils, and PTAA, POMT, tPOMT, and tPTT interacting with fibrils of Aβ 1–42. To further characterize these, we have made a comparison of the monomer- and trimer-based LCPs in fluorescence emission (Figure 3 and Table 1) and fluorescence–excitation spectra of two of the trimer-based LCPs, tPOWT and tPTAA (Figure 4), upon interaction with insulin in native and fibrillar form. Figure 3 and Table 1 show that there is virtually no difference in the fluorescence emission spectra between buffer and native insulin in the case of the trimer-based LCPs, whereas the monomer-based LCPs show intensity differences and in some cases spectral shifts upon interaction with native insulin. As also seen with fibrillar Aβ (1–42) (16), interaction with fibrillar insulin gives a higher quantum yield (QY) for all LCPs, but the relative difference is significantly larger with trimer-based LCPs. All of the emission spectra have been recorded with excitation at 488 nm, which is near the peak maximum of the fluorescence–excitation spectra shown earlier (16) and in Figure 4. Also, in these spectra, there is an augmented fluorescence response upon interaction with insulin fibrils. The possibility of exciting these LCPs with 488 nm is advantageous considering that the 488 nm laser line of argon is often used in confocal microscopy. The increased fluorescence emission of the LCPs upon interaction with fibrils was most likely due to polymer chain separation caused by the binding event. Hence, LCPs alone in the corresponding buffer associated into clusters, and notably, the clusters were still in solution. In all cases, except for PTAA, fibril interaction was associated with a blue shift in emission, which is associated with chain separation or an increased twist of the polymer backbone compared to the free LCP cluster. The spectral discrimination between a LCP in buffered solution, solution with native insulin, or with fibrillar insulin seems to be governed by both the backbone and the side chain of the LCP. It appears important that the backbone is exposed for maximum hydrophobic interaction with fibrillar insulin, while the side chain must remain hydrophilic to ensure water solubility as well as reducing the interaction with native insulin. The six cationic LCPs (1–3 and 5–7) were all buffered at pH 1 and are therefore easily compared with each other. At pH 1, both the LCPs and the protein in its native and fibrillar state were positively charged. Electrostatic repulsion from the charges is likely to occur, and it is reasonable to assume that a trimerbased LCP with every third cationic substituent removed and thereby with a more exposed backbone more easily interacts with the amyloid fibrils. If the three monomer-based cationic

LCPs 5–7 are compared (Figure 3 and Table 1), there is a relationship between hydrophobicity of the side chain and the spectral discrimination of native and fibrillar insulin. The LCP POWT, substituted with serine, showed a larger shift and higher intensity change than the two methylated species, PTT, methylated at the β-position, and POMT, the methyl ester of serine, when the discrimination between the native and fibrillar state of insulin is compared. Possibly, a more hydrophobic substituent interferes with the fibril/polymer backbone interaction. In the trimer-based LCPs 1–3, the spectral trend as observed with the monomer-based analogues with respect to their side-chain substituents was more pronounced. tPOWT showed almost twice as large a blue-shift and intensity increase as tPOMT and tPTT when comparing spectra from native and fibrillar insulin. Moreover, the spectral difference between the signal of the trimer-based LCPs in buffered solution compared to the signal with native insulin was insignificant. The anionic trimer-based tPTAA (4), which was used in a buffered environment at pH 10, shows the same spectral discrepancy as the LCPs 1–3. Compared to the monomer-based 5–7, PTAA showed larger spectral shifts, although not as distinct as for tPTAA. For the application as a probe for amyloid fibril detection, it is clearly advantageous with a low signal toward native protein and buffer. Besides the substitution pattern, there are other differences between the monomer- and trimer-based LCPs that might influence the contrast. The trimer-based LCPs are completely regioregular because of the 2-fold rotational axis in the center thiophene, whereas the monomer-based LCPs do not contain a rotational axis and therefore when polymerized can bind headto-tail as well as head-to-head. The trimer-based LCPs are also shorter with a more well-defined chain length that could influence the discrimination behavior. To further evaluate the interaction between LCP and fibrillar insulin, tPTAA and tPOWT were more thoroughly studied. The two LCPs showed the best signal discrepancy and emission enhancement between native and fibrillar insulin. Moreover, tPTAA had so far not been further characterized and was also interesting because of the good tissue staining properties published earlier with its analogue PTAA (15). Titration experiments were performed, where the amount of LCP was kept constant, and the fibril concentration was increased (Figure 5). The titration curve was fitted to a Hill plot cooperative model. Y ) Bmax[X]nH ⁄ (Kn1⁄2H + [X]nH)

(1)

In eq 1, Bmax is the maximum recorded signal, nH is the cooperativity value, where a value larger than 1 indicates positive cooperativity, and K1/2 is the value where 50% of the maximum signal is reached. At fibril concentration K1/2, 50% of the LCP chain population was bound to fibrils. In the model, tPOWT had a K1/2 ) 0.2 µM and n ) 1.2, whereas tPTAA had K1/2 ) 2.4 µM and n ) 1. This indicates that tPTAA (at pH 10) had a lower affinity for fibrils than tPOWT at pH 1. Whether the affinity difference is a side-chaininduced effect or a reduced fibril polymer–backbone interaction at the higher pH is unclear. None of the LCPs showed a clear cooperativity in the interaction event since n was not significantly larger than 1. To evaluate the interaction between tPTAA and tPOWT with amyloid-like fibrils, we studied the LCP fluorescence response at a constant total protein concentration with increasing fibril content compared to native protein. In Figure 6, the result is presented as the percentage of amyloid-like fibrils in total protein concentration (5.26 µM) as relative change in the fluorescence ratios 550/600 (tPOWT) and 570/670 (tPTAA). Clearly, a small amount of fibrils could be detected in the presence of native protein. The lowest detection level was reached at 0.5% and



Figure 3. Fluorescence emission spectra. Excitation was at 488 nm. The filled symbols are monomer-based LCPs and the unfilled ones are the trimer-based analogues. ;0 and ;9 are 2.7 µg/mL LCP in buffer. 4 and 2 are 2.7 µg/mL LCP with 5 µM native insulin. O and b are 2.7 µg/mL LCP with 5 µM fibrillar insulin. (A) POWT and tPOWT in 50 mM HCl, (B) POMT and tPOMT in 50 mM HCl, (C) PTT and tPTT in 50 mM HCl, and (D) PTAA and tPTAA in 100 mM Na2CO3.

1.4% fibrils for POWT and tPTAA, respectively, corresponding to a 20% relative change in the signal. Analytical Ultracentrifugation. Along with the limitations of MALDI-TOF MS (vide supra), it is vital to consider that weakly associated assemblies, for example, linked through hydrogen bonding, can easily break during measurements. Analytical ultracentrifugation (AUC) is a nondisruptive solutionbased technique, well known for the characterization of proteins and protein complexes and also synthetic macromolecules (31). This makes the technique suitable to study the state of LCPs in solutions and the interaction between LCPs and proteins. AUC experiments were run in equilibrium sedimentation mode at 9 velocities, and absorption was measured at 280 and 445 nm. tPTAA and tPOWT were studied in buffer and with native insulin and fibrillar insulin. tPTAA and tPOWT in buffer were fitted to a single species self-association model as shown in Figure 7. The main disadvantages of AUC are related to the difficulty in obtaining accurate partial specific volumes in some cases (31). The partial specific volume νj; is needed to determine the absolute molar mass (M) from the buoyant mass as follows: Mb ) M(1 - νF)

(2)

νj of tPTAA and tPOWT was determined densiometrically at the concentration (25 µg/mL) used in the AUC experiments in order to avoid the formation of different states of interchain interactions. The low concentration made the measurement of νj; difficult, and the obtained value 0.5 for both tPOWT and tPTAA should be seen as a guideline. To our knowledge, there are no values of νj; for polythiophenes in the literature, but our value can be compared with the study of Michaels et al., which determined νj; of a

polyparaphenylene to 0.62, of a polyfluorene to 0.65, and a polydiphenylenevinylene to 0.55 (32). If the sedimentation curves of tPTAA are fitted to a selfassociation model, the average buoyant mass (Mb) is 25600 ( 1600 g/mol, which gives an molecular mass (M) of 12700 ( 800 g/mol. tPOWT gives a value of Mb of 24800 ( 4000 g/mol, which corresponds to an M of 12400 ( 2000 g/mol. Interestingly, if we compare these values with the MALDI-TOF spectra reported by us earlier (13) and in this publication, we can see clear differences since the majority of the tPOWT chains had a mass value of 1509 g/mol and tPTAA had a corresponding value of 1447 g/mol. These values lead us to the conclusion that the studied free LCPs were in a state of small clusters when present in aqueous solution. On average, a cluster of tPTAA consisted of nine polymer chains, and for tPOWT, this value is 8 ( 1. This result could explain the very low fluorescence signal intensity from tPOWT and tPTAA in acidic and basic buffers, respectively, due to intermolecular quenching. To evaluate the interaction between native insulin and the LCP, an equilibrium sedimentation experiment was run with 48 µM native insulin and 16.6 (tPOWT, 25 µg/mL) and 17.2 (tPTAA, 25 µg/mL) µM LCP. There was no significant difference between the sedimentation profiles at A445nm of the pure LCP samples and those where insulin was present (Figure S1, Supporting Information). The sedimentation profiles of pure insulin measured at A280nm were fitted to the self-association model and resulted in an average weight in 100 mM Na2CO3 at pH 10 and in 50 mM HCl at pH 1 of 5700 g/mol and 11500 g/mol, respectively, corresponding to the monomer and dimer weight of insulin. The association state of insulin is highly dependent on pH, ion concentration, and insulin concentration


Figure 4. Fluorescence–excitation spectra. (A) Fluorescence emission at 550 nm from 2.7 µg/mL tPOWT in (;9) 50 mM HCl, (;4) 20 mM HCl with 5 µM native insulin, and (O) 50 mM HCl with 5 µM fibrillar insulin. (B) Fluorescence emission at 580 nm from 2.7 µg/mL tPTAA in (;9) 20 mM NaCaO3, (;4) 20 mM NaCaO3 with 5 µM native insulin, and (O) 20 mM NaCaO3 with 5 µM fibrillar insulin.

(33). Kadima et al. found that zinc-free insulin was present as monomers at a concentration of 1.9 mg/mL at pH 10.5 (33). At an insulin concentration of 2 µM at pH 2.0, Nettleton et al. reported that insulin was to be found mostly in monomeric form but also that dimers were present (34). Our results, obtained at pH 1, indicate that the dimerization of insulin increases at lower pH. From the sedimentation curves shown in Figure S1, Supporting Information and the mass determination of native insulin, it can be assumed that the LCPs did not interact with the native protein in the mentioned solution conditions. If one considers the preservation of the fluorescence–excitation and emission properties of the LCP, it is unlikely that the interaction with native insulin led to the disruption of the clusters of LCPs and the formation of a complex of similar mass. The sedimentation of the ultrasonicated insulin fibrils occurred already before equilibrium at the lowest rotation speed (3000 rpm), indicating that the masses of these fibril fragments are >1000000 g/mol. This result shows that the dissolution of insulin fibrils with aqueous ammonia at pH 10.5 showed by Nilsson et al. (35) did not occur in 100 mM Na2CO3 at pH 10. In samples with both LCP and sonicated fibrils, the cosedimentation of both species at low rotation speed was obvious, showing interaction between LCP and fibrils.

CONCLUSIONS From the optical measurements, in absorbance and fluorescence, of the LCP, it was clear that the trimer-based analogues

Åslund et al.

Figure 5. Titration curves of LCP fluorescence. (A) tPOWT (2.7 µg/ mL) in 50 mM HCl (B) tPTAA (2.7 µg/mL) in 100 mM Na2CO3 at pH 10. Sonicated fibrillated insulin in concentrations given as monomer insulin was added. Excitation was at 488 nm.

Figure 6. Detection of fibrillar insulin in the presence of native insulin. tPOWT (black) (2.7 µg/mL) in 50 mM HCl. tPTAA (grey) (2.7 µg/ mL) in 100 mM Na2CO3 at pH 10. The percentage of sonicated fibrillated insulin of the total amount of insulin is shown as the monomer insulin added. Total protein concentration (5.26 µM) was kept constant. Excitation was at 488 nm. The relative change in the emission ratio at 550 nm/600 nm (tPOWT) and 570 nm/670 nm (tPTAA) is shown.

were more quenched in solution, giving lower quantum yields. The trimer-based LCPs showed optical signal specificity for amyloid-like fibrils as seen from increased QY and spectral shift, whereas no spectral changes could be seen with native insulin compared to buffered solution. These results were also supported by the possibility of distinguishing the fibrillar signal in a 70-



presence of native insulin. This information is available free of charge via the Internet at http://pubs.acs.org.

LITERATURE CITED

Figure 7. Analytical ultracentrifuge sedimentation curves of absorbance at 445 nm (A) tPOWT (25 µg/mL) in 50 mM HCl. (B) tPTAA (25 µg/mL) in 100 mM Na2CO3 at pH 10. Rotation speed in rpm is shown in the legend. Lines are fitted to a self-association model.

fold excess of native insulin. Furthermore, AUC demonstrated that the trimer-based tPTAA and tPOWT showed no interaction with native insulin, whereas they cosediment with fibrils, indicating strong noncovalent binding. We also report that tPTAA and tPOWT form small clusters in solution, likely to avoid exposure of the hydrophobic polythiophene backbone. The LCP/amyloid-like fibril interaction seems to be governed by the backbone of the polymer. If one compare the cationic monomerbased polymers with the trimer-based cationic LCPs, it is apparent that the removal of one out of three side chains and thereby increasing the exposure of the backbone, gave rise to a strong signal enhancement upon interaction with amyloid fibrils compared to monomer-based LCPs. The anionic LCPs PTAA and tPTAA showed the same behavior as their cationic counterparts, even though the pH was changed from acidic to basic. This study verifies our hypothesis that trimer-based LCPs have a better potential than monomer-based LCPs as optical probes for amyloid fibril detection. Moreover, LCPs 2, 3, 6, and 8 have been discussed for use in diagnostics of aggregation proteopathies (16), and here, we demonstrate that tPTAA is another potential candidate for these applications.

ACKNOWLEDGMENT We acknowledge partial funding from VINNOVA and CENANO at LiU (A.H.). Support from the Foundation of Strategic Research (P.H., P.K. and A.Å.), the Wenner-Gren Foundations (P.H.), The Knut and Alice Wallenberg Foundation (K.P.R.N), and the Swedish research council (P.H., P.K, O. I., B-H. J., and A.H) are greatly appreciated. Supporting Information Available: Analytical ultracentrifugation curves of tPOWT and tPTAA in buffer and in the

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