Synthesis of a regioregular zwitterionic conjugated oligoelectrolyte, usable as an optical probe for detection of amyloid fibril formation at acidic pH

Article abstract of DOI:10.1021/ja045835e

Changes of the optical properties of conjugated polyelectrolytes have been utilized to monitor noncovalent interactions between biomolecules and the conjugated polyelectrolytes in sensor applications. A regioregular, zwitterionic conjugated oligoelectroly

Full text of DOI:10.1021/ja045835e

Published on Web 01/25/2005
Synthesis of a Regioregular Zwitterionic Conjugated
Oligoelectrolyte, Usable as an Optical Probe for Detection of
Amyloid Fibril Formation at Acidic pH
Anna Herland,† K. Peter R. Nilsson,† Johan D. M. Olsson,‡ Per Hammarstro¨m,‡
Peter Konradsson,*,‡ and Olle Ingana¨s†
Contribution from the Department of Physics and Measurement Technology,
Biology and Chemistry, Linko¨pings UniVersity, SE-581 83 Linko¨ping, Sweden
Received July 12, 2004; E-mail: petko@ifm.liu.se
Abstract: Changes of the optical properties of conjugated polyelectrolytes have been utilized to monitor
noncovalent interactions between biomolecules and the conjugated polyelectrolytes in sensor applications.
A regioregular, zwitterionic conjugated oligoelectrolyte was synthesized in order to create a probe with a
defined set of optical properties and hereby facilitate interpretation of biomolecule-oligoelectrolyte
interactions. The synthesized oligoelectrolyte was used at acidic pH as a novel optical probe to detect
amyloid fibril formation of bovine insulin and chicken lysozyme. Interaction of the probe with formed amyloid
fibrils results in changes of the geometry and the electronic structure of the oligoelectrolyte chains, which
were monitored with absorption and emission spectroscopy.
Introduction
physical properties with chemical structure. Series of well-
defined π-conjugated oligomers have been studied in detail in
Phenomenona such as misfolding and amyloid fibril formation
are driving forces in the development of materials able to detect
conformation changes in biomolecules. Conjugated polyelec-
trolytes have been used as probes for biomolecular interactions,
through alternations of conditions for photoinduced charge
transfer or excitation transfer1-6 and through conformational
changes of the polyelectrolyte chains.7-9 Lately it has been
shown that conjugated polymers (CPs) can be optical probes to
monitor conformation changes of synthetic peptides10,11 and
calcium-induced conformation changes in calmodulin.12 In
photonic and electronic applications, oligomers have been used
as model compounds for π-conjugated polymers.13,14 Oligomers
with a defined system offer the possibility to directly correlate
order to correlate increasing chain length with changes in
quantities such as optical adsorption maximum or oxidation
potential.13-16 Likewise, in biosensing applications where
interactions between biomolecules and conjugated polyelectro-
lytes results in an alternation of the polymer backbone geometry,
a change in absorption and emission characteristics of the
polyelectrolyte would be observed. To facilitate the interpreta-
tion of the relation of such changes, it would be of great interest
to use conjugated oligoelectrolytes in detection of biospecific
interactions. Furthermore, a defined chain length makes detailed
studies of the biomolecule/polyelectrolyte complex with X-ray
crystallography and NMR an appealing task.
Folding of proteins to their functional, highly compact, native
state is one of the most remarkable examples of biological self-
assembly. However, under destabilizing conditions, proteins can
aggregate into fibrillar assemblies, amyloid fibrils.17,18 In
addition to the loss of the original biological function of the
protein, amyloid fibrils and the precursors thereof are being
increasingly recognized as the cause of disease states, including
Alzheimer’s disease and spongiform encephalopathies.17,19-23
Although no structural or sequential homology of amyloidogenic
† Department of Biomolecular and Organic Electronics.
‡ Department of Chemistry.
(1) Wang, J.; Wang, D.; Miller, E. K.; Moses, D.; Bazan, G. C.; Heeger, A. J.
Macromolecules 2000, 33, 5153-5158.
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A. J. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 49-53.
(3) Liu, B.; Gaylord, B. S.; Wang, S.; Bazan, G. C. J. Am. Chem. Soc. 2003,
125, 6705-6714.
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(16) Frere, P.; Raimundo, J. M.; Blanchard, P.; Delaunay, J.; Richomme, P.;
Sauvajol, J. L.; Orduna, J.; Garin, J.; Roncali, J. J. Org. Chem. 2003, 68,
7254-7265.
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D.; Leclerc, M. Angew Chem. Int. Ed. 2002, 41, 1548-1551.
(8) Ho, H. A.; Leclerc, M. J. Am. Chem. Soc. 2004, 126, 1384-1387.
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U.S.A. 2003, 100, 10170-10174.
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C. C. J. Mol. Biol. 1997, 273, 729-739.
(19) Lansbury, P. T.; Kelly, J. W. Amyloid 1994, 1, 186-205.
(20) Sacchettini, J. C.; Kelly, J. W. Nat. ReV. Drug DiscoV. 2002, 1, 267-275.
(21) Dobson, C. M. Nature 2003, 426, 884-890.
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U.S.A. 2004, 101, 11197-11202.
(12) Nilsson, K. P.; Ingana¨s, O. Macromolecules 2004, 37, 9109-9113.
(13) Martin, R. E.; Diederich, F. Angew Chem. Int. Ed. 1999, 38.
(14) Mu¨llen, K.; Wegner, G. Electronic materials: the oligomer approach;
Wiley-VCH: Weinheim, 1997.
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(23) Pepys, M. B. In The Oxford Textbook of Medicine, 3rd ed.; Weatherall, D.
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Oxford, 1996; Vol. 2, pp 1512-1524.
9
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J. AM. CHEM. SOC. 2005, 127, 2317-2323
2317

A R T I C L E S
Herland et al.
Scheme 1. (i) NBS, CHCl3/AcOH (1:1), 91 %; (ii) TsCl, Pyridine, CHCl3, 87 %; (iii) N-t-Boc-L-Ser, K2CO3, DMF, 35 °C, 54 %; (iv) Pd(OAc)2,
KF, DMF, 31 %; (v) CH2Cl2/TFA (4:1), Quant.; (vi) FeCl3, TBA-OTf, CHCl3, 32 %
proteins, amyloid fibril structure appear generic showing linear
filaments with a diameter of approximately 10 nm.24 Fiber
diffraction studies have revealed a cross-â structure as charac-
teristic of amyloid fibrils, i.e., the â-strands are perpendicular
to the fibril axes forming axially aligned extended â-sheets.18
Insulin, a 51-residue hormone, has a primarily helical native
structure.25 Insulin exists in equilibrium as a mixture of
monomers, dimers, hexamers, and possibly higher-order oligo-
mers, but the physically predominant form is a zinc-coordinated
hexamer.26 Insulin amyloid fibril formation is a well-studied
phenomenon, which in vitro is known to occur at high protein
concentrations, acidic pH, elevated temperatures, and exposure
to hydrophobic surfaces.27,28 Intermolecular hydrophobic forces
have been suggested as the driving forces of the insulin fibril
formation process.29 Nielsen et al. have proposed a model for
insulin fibril formation where dissociation of the native associ-
ated states (hexamer, tetramer, and dimer) into monomers is a
prerequisite for fibrillation.30 The monomer undergoes partial
unfolding into an intermediate state, which oligomerizes to form
the nucleus from which the amyloid fibrils grow.26,30,31 Insulin
fibril formation cause a variety of problems in handling soluble,
therapeutic insulin.
Amyloid fibril formation of insulin has been studied with
several spectroscopy and microscopy techniques31-33 and
through staining with small molecule dyes such as Congo Red
and thioflavin T.30,34 Herein we report the synthesis and optical
characterization of a regioregular zwitterionic conjugated oli-
goelectrolyte, poly((3,3′′-di[(S)-5-amino-5-carbonyl-3-oxapen-
tyl]-[2,2′;5′,2′′])-5,5′′-terthiophenylene hydrochloride), PONT
(Scheme 1), usable in a novel optical method to monitor amyloid
fibril formation of bovine insulin and chicken lysozyme (CL)
at acidic pH. The method is based on conformation changes of
the conjugated oligoelectrolyte upon binding. The method has
the advantage of being based on noncovalent assembly between
the oligothiophene and the protein and the possibility to either
add the probe after the amyloid fibril formation has occurred
or with the probe present during the process.
Experimental Section
General Methods. Normal workup means drying the organic phase
with MgSO4(s) and filtration and evaporation of the solvent in vacuo
at ∼45 °C. All dry solvents were collected onto 4-A predried molecular
sieves (Merck). Thin-layer chromatography (TLC) was carried out on
0.25-mm precoated silica gel plates (Merck silica gel 60 F254), detected
by UV absorption (254 nm) and/or by charring with PAA-dip (ethanol/
sulfuric acid/p-anisaldehyde/acetic acid 90:3:2:1) followed by heating
to ∼250 °C. FC means flash-column chromatography using silica gel
(24) Cohen, A. S.; Shirahama, T.; Skinner, M. In Electron Microscopy of Protein;
Harris, I., Ed.; Academic Press: London, 1981; Vol. 3, pp 165-205.
(25) Blundell, T. L.; Cutfield, J. F.; Dodson, G. G.; Dodson, E.; Hodgkin, D.
C.; Mercola, D. Biochem. J. 1971, 125, 50P-51P.
(26) Brange, J.; Andersen, L.; Laursen, E. D.; Meyn, G.; Rasmussen, E. J.
Pharm. Sci. 1997, 86, 517-525.
(27) Waugh, D. F. J. Am. Chem. Soc. 1946, 68, 247-250.
(28) Nielsen, L.; Frokjaer, S.; Carpenter, J. F.; Brange, J. J. Pharm. Sci. 2001,
90, 29-37.
(29) Waugh, D. F.; Wilhelmson, D. F.; Commerford, S. L.; Sackler, M. L. J.
Am. Chem. Soc. 1953, 75, 2592-2600.
(32) Nettleton, E. J.; Tito, P.; Sunde, M.; Bouchard, M.; Dobson, C. M.;
Robinson, C. V. Biophys J. 2000, 79, 1053-1065.
(30) Nielsen, L.; Khurana, R.; Coats, A.; Frokjaer, S.; Brange, J.; Vyas, S.;
Uversky, V. N.; Fink, A. L. Biochemistry 2001, 40, 6036-6046.
(31) Ahmad, A.; Millett, I. S.; Doniach, S.; Uversky, V. N.; Fink, A. L.
Biochemistry 2003, 42, 11404-11416.
(33) Bouchard, M.; Zurdo, J.; Nettleton, E. J.; Dobson, C. M.; Robinson, C. V.
Protein Sci. 2000, 9, 1960-1967.
(34) Glenner, G. G. Prog. Histochem. Cytochem. 1981, 13, 1-37.
9
2318 J. AM. CHEM. SOC. VOL. 127, NO. 7, 2005

Regioregular Zwitterionic Conjugated Electrolytes
A R T I C L E S
(Merck 60 (0.040-0.063 mm)). 1H and 13C NMR spectra were
performed on a Varian Mercury 300-MHz instrument at 25 °C.
Chemical shifts are given in ppm relative to TMS in CDCl3 (δ 0.00)
(5 × 15 mL). The organic layer was subjected to normal workup, FC
(toluene/EtOAc 1:1), and HPLC (MeOH/H2O 80:20 + 0.2% TFA) to
give 4 (0.053 g, 0.074 mmol, 31%) as an oil. Rf ) 0.60 (toluene/EtOAc
1:3).
for H and 13C or CD3OD (δ 3.31) for H and (δ 49.0) for 13C NMR.
Preparative high-performance liquid chromatography (HPLC) was
performed on a Gynkotek (pump, P580; detector, UVD 170S; software,
Chromeleon) using a Kromasil 100-10-C18 (250 × 20 mm) column.
Optical rotations were recorded at room temperature with a Perkin-
Elmer 141 polarimeter; IR spectra were recorded on a Perkin-Elmer
SPECTRUM 1000 FT-IR spectrometer as KBr pellets and melting
points were recorded with a Gallenkampmelting point apparatus.
Synthesis of 2-(2-Bromo-3-thienyl)-p-toluenesulfonyl ethanol (1).
2-(3-Thienyl)ethanol (3.75 mL, 33.34 mmol) was dissolved in CHCl3/
AcOH (1:1, 90 mL) and cooled to 0 °C. N-Bromosuccinimide (6.26 g,
35.17 mmol) was added to the solution, and after 30 min, the solution
was diluted with H2O (250 mL). The organic layer was washed with
10% KOH (aq) (2 × 70 mL) and H2O (2 × 70 mL) and subjected to
normal workup. FC (T/E 4:1) gave the 2-bromosubstituted product (6.30
g, 30.40 mmol, 91%) as an oil (Rf ) 0.43 (toluene/EtOAc 4:1)). The
brominated product (3.21 g, 15.48 mmol) was dissolved in CHCl3 (60
mL) and cooled to 0 °C; pyridine (2.49 mL, 30.5 mmol) and
p-toluenesulfonyl chloride (4.43 g, 23.24 mmol) were added. After 24
h, the reaction was quenched by adding H2O (20 mL) and diluted with
Et2O (100 mL). The organic layer was washed with 2 M HCl (2 × 40
mL), sat. NaHCO3 (aq.) (2 × 40 mL), and H2O (2 × 40 mL) and
subjected to normal workup. FC (toluene) and crystallization from
EtOAc/hexane afforded 1 (4.89 g, 13.54 mmol, 87%) as white needles.
Rf ) 0.64 (toluene/EtOAc 4:1). M
1
1
[R]D ) -18.3 (c 2.0, CHCl3).
IR νmax cm-1: 801, 835, 1060, 1367, 1506, 1713, 2975, 3107, 3399.
13C NMR (CDCl3) δ: 28.3 (6C), 28.4 (2C), 55.8 (2C), 63.5 (2C),
65.1 (2C), 80.3 (2C), 124.8 (2C), 127.0 (2C), 129.8 (2C), 132.2 (2C),
134.0 (2C), 135.6 (2C), 155.5 (2C), 170.8 (2C).
1H NMR (CDCl3) δ: 1.44 (s, 18 H), 3.19 (t, 4H, J ) 7.0 Hz), 3.82
(dd, 2H, J ) 3.6, 11.1 Hz), 3.88 (dd, 2H, J ) 3.6, 11.1 Hz), 4.42 (m,
6H), 6.98 (d, 2H, J ) 5.1 Hz), 7.11 (s, 2H), 7.24 (d, 2H, J ) 5.1 Hz).
Anal. Calcd for C32H42N2O10S3Br: C, 54.1; H, 6.0; S, 13.5. Found:
C, 54.0; H, 6.2; S, 13.6.
Synthesis of PONT (4). Compound 3 (37 mg, 0.052 mmol) was
dissolved in CH2Cl2/TFA (4:1, 2 mL). The reaction was quenched after
1 h by adding MeOH (1 mL) and co-concentrated with toluene (3 × 3
mL). The ammonium salt and TBA-OTf (52 mg, 0.13 mmol) were
dissolved in dry CHCl3 (1.5 mL), and the solution was cooled to 0 °C.
Anhydrous FeCl3 (37 mg, 0.23 mmol) was added to this solution under
an Ar atmosphere. After the solution was stirred for 10 h at room
temperature, the reaction was quenched with H2O (2 mL) and diluted
with CHCl3 (3 mL). The organic layer was washed with H2O (3 × 2
mL). The aqueous solution was diluted with acetone (25 mL), and
concentrated HCl was added dropwise until the polymer precipitated.
After 2 h, the mixture was centrifuged (4 min/2500 rpm), and the red
salt was washed with acetone twice, dissolved in H2O (2.5 mL), and
reprecipitated from acetone/concentrated HCl. The washing procedure
was repeated twice to give 4 (10 mg, 0.017 mmol, 32%) as a dark red
powder.
IR νmax cm-1: 793, 821, 1061, 1229, 1503, 1741, 2952, 3190.
1H NMR (CD3OD) δ: 3.26 (s) (peak partly hidden in MeOH
(δ: 3.31), 3.97 (s, 12H), 4.16 (s, 14H), 4.56 (s, 12H), 7.12 (d, 2H,
J ) 5.5 Hz), 7.23 (s, 4H), 7.31 (s, 6H), 7.42 (d, 2H, J ) 5.5 Hz).
Protein Preparation. Bovine insulin was obtained from Sigma-
Aldrich. The lyophilized protein was dissolved in 2 M guanidine
hydrochloride and was dialyzed vs three rounds of 25 mM HCl at 4
°C. The insulin solutions (0.5-2.0 mM) were stored at 4 °C and were
stable for several weeks. Lyophilized lysozyme from hen egg white
was obtained from Sigma and was dissolved in dH2O at a concentration
of 0.7 mM. The protein was dialyzed vs three rounds of 25 mM HCl
at 4 °C or vs 10 mM Na phosphate buffer, pH 7.5. Filtered (0.45 µm)
stock solutions were made at concentrations of 0.61 mM. Collagen
from rabbit, type I, acid soluble was obtained from Sigma and dissolved
in 0.5 M acetic acid at a concentration of 5 mg/mL.
Amyloid Fibril Formation. A stock solution containing 320 µM
bovine insulin in 25 mM HCl was prepared. A solution containing 320
µM bovine insulin and 390 µM PONT (on a 9-monomer basis) in 25
mM HCl was prepared. The solutions were placed in a water bath with
a temperature of 65 °C to induce the amyloid formation. Samples were
taken and analyzed during a time period of 3 days. Lysozyme fibrils
were made through incubation of the protein in 25 mM HCl at 65 °C
for 220 h.
Transmission Electron Microscopy (TEM) Experiments. Aliquots
collected at different time points during fibril formation were diluted
in 25 mM HCl and applied to carbon-coated grids for 2 min. The grids
were washed and negatively stained with uranyl acetate 2% (wt/vol)
in water and air dried before being examined in a Phillips EM400
transmission electron microscope at an accelerating voltage of 120 kV.
Optical Measurements. Stock solutions containing 1.5 mg mL-1
PONT in deionized water was prepared. The oligoelectrolyte solution
(10 µL) was diluted with deionized water or the buffer in question, 25
mM HCl, 20 mM MES pH 5.9, and 20 mM Na carbonate pH 10, to a
final volume of 1500 µL. In the samples where insulin/lysozyme fibril
formation was studied, 10 µL of the oligoelectrolyte solution was mixed
with aliquots of protein samples taken at different time points of
p: 38-39 °C (from EtOAc/hexane).
IR νmax cm-1: 552, 663, 713, 773, 902, 971, 1171, 1186, 1359, 1595.
13C NMR (CDCl3) δ: 21.9, 29.4, 68.9, 111.1, 126.1, 128.0 (2C),
128.7, 130.1 (2C), 133.1, 135.9, 145.0.
1H NMR (CDCl3) δ: 2.43 (s, 1H), 2.93 (t, 2H, J ) 6.9 Hz), 4.17 (t,
2H, J ) 6.9 Hz), 6.76 (d, 1H, J ) 6.0 Hz), 7.17 (d, 1H, J ) 6.0 Hz),
7.30 (d, 2H, J ) 8.3 Hz), 7.71 (d, 2H, J ) 8.3 Hz).
Anal. Calcd for C13H13BrO3S2: C, 43.2; H, 3.6; S, 17.8. Found: C,
43.4; H, 3.8; S, 17.5.
Synthesis of (S)-3-[2-(2-Bromo-3-thienyl)-ethoxy]-2-tert-butoxy-
carbonylamino-propionic acid (2). N-t-Boc-L-Ser (3.04 g, 14.80
mmol) and 1 (2.97 g, 8.22 mmol) were dissolved in dry DMF (150
mL) under N2 atmosphere. K2CO3 (3.41 g, 24.66 mmol) was added,
and the solution was heated to 35 °C. After 30 h, H2O (100 mL) was
added, and the mixture was poured over cold 2 M HCl (100 mL) and
washed with toluene (3 × 100 mL). The organic layer was washed
with H2O (80 mL), subjected to normal workup, and purified by FC
(toluene/EtOAc 4:1) to give 2 (1.75 g, 4.44 mmol, 54%) as a colorless
syrup. Rf ) 0.54 (toluene/EtOAc 1:1).
[R]D ) -4.9 (c 2.0, CHCl3).
IR νmax cm-1: 641, 723, 1061, 1164, 1367, 1510, 1711, 2977, 3430.
13C NMR (CDCl3) δ: 28.5 (3C), 28.9, 56.0, 62.9, 64.5, 80.3, 110.7,
126.1, 128.6, 137.1, 156.1, 171.1.
1H NMR (CDCl3) δ: 1.45 (s, 9H), 2.96 (t, 2H, J ) 6.8 Hz), 3.81
(dd, 1H, J ) 3.5, 11.1 Hz), 3.91 (dd, 1H, J ) 3.5, 11.1 Hz), 4.35 (m,
3H), 6.85 (d, 1H, J ) 5.6 Hz), 7.23 (d, 1H, J ) 5.6 Hz).
Anal. Calcd for C14H20BrNO5S: C, 42.7; H, 5.1; S, 8.1. Found: C,
42.7; H, 5.2; S, 8.1.
Synthesis of 3,3′′-di[(S)-5-tert-Butoxycarbonylamino-5-carbonyl-
3-oxapentyl]-2,2′;5′,2′′-terthiophene (3). Compound 2 (0.195 g, 0.49
mmol), thiophene-2,5-bis-pinacolboronate35 (0.080 g, 0.24 mmol), KF
(0.083 g, 1.428 mmol), and Pd(OAc)2 (0.005 g, 0.024 mmol) were
added to an oven-dried flask. After a flush with Ar, dry DMF (3 mL)
was added. After the solution was stirred for 6 h in an Ar atmosphere,
the mixture was diluted with H2O (15 mL) and washed with toluene
(35) Parakka, J. P.; Jeevarajan, J. A.; Jeevarajan, A. S.; Kispert, L. D.; Cava,
M. P. AdV. Mater. 1996, 8, 54-59.
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A R T I C L E S
Herland et al.
incubation under fibril formation conditions, i.e., 25 µL of the bovine
insulin solution (25 mM HCl) or 13.1 µL of the chicken lysozyme
solution (25 mM HCl), and diluted with 25 mM HCl to a final volume
of 1500 µL containing 5 µM insulin/ lysozyme. In the samples where
collagen was studied, 50 µL of the collagen solution (0.5 M acetic
acid) was mixed with 50 µL 20 mM Na acetate pH 4.2, 6.7 µL of the
oligoelectrolyte solution was added, and the sample was diluted in 25
mM HCl to a final concentration of 1000 µL.
In the samples where PONT were present during the amyloid fibril
formation, 25 µL of the bovine insulin and PONT solution was diluted
with 25 mM HCl to a final volume of 1500 µL. The samples were
incubated for 5 min at room temperature, and the emission spectra were
recorded with an ISA Jobin-Yvon spex FluoroMax-2 apparatus. A
Perkin-Elmer Lambda 9 UV/VIS/NIR spectrophotometer was used for
the absorption measurements, and the circular dichroism (CD) spectra
were recorded with an ISA Jobin-Yvon CD6 (5-mm quartz cell).
Results and Discussion
Figure 1. MALDI-TOF-MS spectra of PONT recorded in a linear positive
Synthesis of PONT. 2-(3-Thienyl)ethanol was brominated
with NBS and tosylated into derivative 1. The tosyl group was
displaced by a Boc-protected amino acid, N-t-Boc-Ser, to afford
2. Palladium-catalyzed cross coupling of 2 and thiophene-2,5-
bispinacolboronate35 gave a regioregular terthiophene, 3, in
acceptable yield. The Suzuki-type coupling turned out to be
crucial. Several Pd reagents, both with different ligands as well
as “ligandsless” Pd(OAc)2, and bases were tried under different
conditions (solvent, temperature, etc.). The choice of activator,
base, and solvent proved to be essential for reaction. Using Pd-
(OAc)2 with KF as base in DMF gave the best yield of
compound 3. The low yield of product was partly explained by
hydrolytic deboronation to a monosubstituted thiophenedimer,
an earlier detected problem in palladium-catalyzed coupling of
electron-rich systems.36 The Boc groups were removed in CH2-
Cl2/TFA to give the corresponding ammonium salt readily for
polymerization. Because of the functional groups of the ter-
thiophene, the polymerization was performed by a method
reported by Sugimoto et al.37,38 using chemical oxidation with
anhydrous FeCl3 in CHCl3. The water-soluble oligomer was then
precipitated by adding acetone and concentrated hydrochloric
acid, washed with acetone, and dried to achieve 4 as a red
powder (Scheme 1).
Matrix Assisted Laser Desorption/Ionization Time-of-
Flight Mass Spectroscopy (MALDI-TOF-MS). The length of
the regioregular thiophene backbone in the oligomer was further
elucidated by MALDI-TOF-MS. An aqueous solution (1 µL)
of the oligomer and 1 µL of the matrix (R-cyano-4-hydroxy-
trans-cinnamic acid (CHCA) in 0.1% TFA/acetonitrile (1:1))
were coevaporated on a target plate, and the spectra were
recorded in linear positive mode. The MALDI-TOF spectra of
the product obtained from polymerization of the terthiophene
3 with FeCl3 in CHCl3 in the presence of TBA-OTf gave a major
peak with a mass of 1529 (Figure 1). This peak corresponds to
an oligomer containing nine thiophene rings in the backbone
(Scheme 1). Minor traces of peaks with masses of 2037 (less
than 15% intensity) and 2546 (less than 5% intensity) were also
found in the spectra, these peaks correspond to oligomers of
12 and 15 thiophene units in the backbone, respectively.
mode using CHCA as matrix.
CD Measurements. Amyloid fibril formation at low pH and
elevated temperature has previously been studied with CD.31,33
Native BI (nBI) in 25 mM HCl shows a characteristic R-helical
spectrum (see Supporting Information, Figure S1) in the far UV
region, with strong negative peaks at 208 and 222 nm, which
is consistent with the crystal structure of the insulin hexamer.18
Fibrillation in BI at 65 °C (320 µM, in 25 mM HCl) was
followed by CD over a period of 10 h, when a â-sheet-rich
structure was obtained (see Supporting Information, Figure S1).
This conversion of the BI from an R-helical-rich to a more
â-sheet-rich structure is a clear indication of amyloid fibril
formation of the protein. Our results are in good agreement with
an earlier CD and FTIR study of the amyloid formation in BI,33
although the concentration of BI was much higher in that study
than the concentration used herein. CD spectra of nBI/PONT
solutions (data not shown) were very similar to the results for
the pure nBI solutions, showing that the interaction between
PONT and BI has no major impact on the secondary structure
of nBI.
TEM Measurements. The conditions, acidic pH and elevated
temperature, used in this procedure are highly favorable for
aggregation and fibril formation.27 The electron micrograph
taken after 2 h after the initiation of heating show spherical
aggregates (Figure 2a), which is consistent with studies made
under similar solution conditions.26,32,33 Figure 2b shows an
electron micrograph of the sample heated for 3.5 h. A few fibrils
can clearly be seen, but there are also numerous spherical
aggregates. In the period of 4-10 h, a network of fibrils appears
followed by a decrease of aggregates. After 10 h, both smooth
fibrils with curvature, indicative of flexibility, and more mature
twisted fibrils with an average width of 7-10 nm can be seen.
The amount of spherical aggregates has substantially declined.
The EM studies agree very well with the lag phase (0-4 h
incubation), the exponential growth phase (4-7 h), and the
plateau phase (from 7 h) observed in a study where the amyloid
formation of BI was monitored by Congo red and an anionic
conjugated polythiophene.39
Optical Characterization of PONT. The absorption spectra
of PONT at different pH and in deionized water are shown in
Figure 3. The oligoelectrolyte undergoes a pH-induced orange
(36) Martina, S.; Enkelmann, V.; Schlueter, A. D.; Wegner, G. Synth. Metals
1991, 41, 403-406.
(37) Sugimoto, R.; Takeda, S.; Gu, H. B.; Yoshino, K. Chem. Exp. 1986, 1,
635.
(38) Yoshino, K.; Hayash, S.; Sugimoto, R.-I. Jpn. J. Appl. Phys. 1984, 23,
(39) Nilsson, K. P.; Herland, A.; Hammarstro¨m, P.; Ingana¨s, O. Biochemistry
2005, in press.
L899.
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Regioregular Zwitterionic Conjugated Electrolytes
A R T I C L E S
Figure 4. Emission spectra of 6.5 µM PONT-HCl (on a chain basis) in
25 mM HCl (×), 25 mM HCl with 5.0 µM of nBI (4), 25 mM HCl with
5.0 µM fBI after 4.5 h in 65 °C (+), 25 mM HCl with 5.0 µM fBI after 6
h in 65 °C ()), and 25 mM HCl with 5.0 µM fBI after 10 h in 65 °C (0).
An aliquot was withdrawn, and after addition of PONT, the samples were
diluted with 25 mM HCl (23 °C) prior to emission measurements. Emission
spectra of 6.5 µM PONT-HCl (on a chain basis) present during heat
incubation in 25 mM HCl with 5.0 µM fBI after 10 h in 65 °C (9). The
emission spectra were recorded with excitation at 400 nm.
Figure 2. (a) 2-h incubation. (b) 3.5-h incubation. (c) 6-h incubation. (d)
10-h incubation. The scale bar represents 200 nm.
conformation, observed as a red shift of the absorption
maximum. Interestingly, the planarization of the backbone is
more significant at alkaline pH, indicating that the oligoelec-
trolyte backbone adopts different conformations in acidic and
alkaline environment. The oligoelectrolyte is probably adopting
the extended rod-shaped conformation due to electrostatic
repulsion forces between the oligoelectrolyte side chains,40,41
or hydrogen bonding42 and hydrophobic assembly between
nearby PONT chains, as these molecular forces are highly
influenced by the charge of the oligoelectrolyte side chains.
The pH-induced conformational changes of the oligoelectro-
lyte chains will also alter the emission spectra for the oligo-
electrolyte solutions (Figure 3). At pH ) 5.9 (pI for serine)
and in deionized water, light with a shorter wavelength is emitted
and the emission maximum is approximately 555 nm. Interest-
ingly, as the net charge of the oligoelectrolyte side chains
become more negative or positive, PONT emits light with a
decreased intensity and longer wavelengths. At pH 10, the
oligoelectrolyte emits light with a wavelength around 615 nm,
and in 25 mM HCl (pH 1.6), an emission maximum of 600 nm
is seen (Figure 3), indicating that the deprotonation and
protonation of the amino and the carboxyl groups are inducing
a planarization of the oligoelectrolyte backbone and aggregation
of oligoelectrolyte chains. Similar results have been seen for a
zwitterionic conjugated polyelectrolyte, POWT, and the de-
creased intensity of the emitted light is due to de-excitation
events created by contact between polymer chains.43,44
Figure 3. Absorption spectra (top) and emission spectra (bottom) of PONT
after 10 min of incubation in deionized water (black), pH 1.6 (blue), pH
5.9 (red), and pH 10 (green). All the emission spectra were recorded with
excitation at 400 nm.
Detection of Amyloid Fibril Formation. Upon addition of
5 µM nBI (pH 1.6) to PONT, the emission maximum remains
at 600 nm (Figure 4), but the intensity of the emission has
slightly increased, suggesting that a minor separation of poly-
to red color (a shift of the absorption maximum from 434 to
484 nm) alteration, indicating that deprotonation and protonation
of the amino and the carboxyl groups have an influence on the
coil-to-rod (nonplanar to planar) conformation transition of the
oligoelectrolyte backbone. The nonplanar conformation seems
to be most abundant at pH 5.9 (pKa for serine) and in deionized
water. As the side chains become charged, either positively or
negatively, the oligoelectrolyte chains adopt a more planar
(40) Kim, B.; Chen, L.; Gong, J.; Osada, Y. Macromolecules 1999, 32, 3964-
3969.
(41) Fa¨ıd, K.; Leclerc, M. J. Am. Chem. Soc. 1998, 120, 5274-5278.
(42) McCullough, R. D.; Ewbank, P. C.; Loewe, R. S. J. Am. Chem. Soc. 1997,
119, 633-634.
(43) Berggren, M.; Bergman, P.; Fagerstrom, J.; Inganas, O.; Andersson, M.;
Weman, H.; Granstrom, M.; Stafstrom, S.; Wennerstrom, O.; Hjertberg,
T. Chem. Phys. Lett. 1999, 304, 84-90.
(44) Nilsson, K. P. R.; Andersson, M. R.; Ingana¨s, O. J. Phys.: Condens. Matter
2002, 14, 1-10.
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A R T I C L E S
Herland et al.
electrolyte chains occurs. This separation of the polyelectrolyte
chains is probably due to the interaction between PONT and
nBI. Furthermore, in Figure 4, selected spectra from the different
phases of the fibril formation process are shown. Upon addition
of BI incubated for 4.5 h at 65 °C, a further increase in emission
intensity can be seen, signifying a stronger interaction between
the insulin and PONT. A major difference in the emission
spectrum can be seen after a 6-h incubation of BI. There are
two clear emission peaks in the spectrum, the previously
observed at 600 nm and a more blue-shifted peak at 560 nm,
and the intensity has increased several orders of magnitude. The
peak at shorter wavelengths indicates that the polymer backbone
adopts a more twisted conformation upon interaction with
amyloid fibrils.9,43 The noteworthy enhancement of the emission
intensity suggests that a separation of the oligoelectrolyte chains
occur due to the interaction between PONT and fBI. Upon
addition of BI heated for 10 h, these phenomena are even more
apparent, especially the emission peak at 560 nm, which is
increased compared to the peak at 600 nm, and the emission
spectrum resembles the spectra seen for PONT in deionized
water and at pH 5.9. Hence, the binding of PONT to fBI will
induce a more twisted conformation of the oligoelectrolyte
backbone and separation of the oligoelectrolyte chains. The
optical changes show that the formation of â-sheet-containing
amyloid fibrils in fBI are influencing the conformation and the
electronic structure of the polyelectrolyte backbone when it binds
the fibrils. The geometric alteration of the polyelectrolyte chains
might be a result of the conversion, from R-helices to â-sheets,
of the secondary structure in the BI molecule. Interestingly, in
a similar study with another polymer probe, poly(thiophene
acetic acid), PTAA, the formation of amyloid fibrils can be
followed, but upon addition of fBI to PTAA, a red shift of the
emission maximum is seen.39
Figure 5. Kinetics of the insulin fibril formation, monitored by PONT
fluorescence. The fibril formation was induced by incubation of 320 µM
BI at pH 1.6 and 65 °C. An aliquot was withdrawn, and after addition of
PONT, the samples were diluted with 25 mM HCl (23 °C) prior to emission
measurements.
tion). The lower emission intensity of the spectrum with fCL
compared to the spectrum with nBI is likely due to the large
size of the CL fibrils that had been incubated for 220 h,
providing lower bulk fluorescence intensity. The fCL spectrum
is more blue-shifted and has one well-defined maximum at 550
nm along with shoulders at 520 and 600 nm. These differences
can be caused by slightly different amyloid fibril morphology.
In comparison to the PONT fluorescence signal bound to
amyloid fibrils (fBI or fCL), binding of PONT to collagen type
I, which has a native fibril state, showed a spectrum very similar
to that of nBI (see Supporting Information, Figure S3), likely
due to the intrinsic polyproline type II helical structure of
collagen. The similarities in the results obtained with fBI and
fCL together with the distinct different spectra of collagen
clearly indicate that PONT is a conformation-sensitive probe.
Changes of the protein secondary and quaternary structure,
associated with the formation of amyloid fibrils, results in
conformational changes of the polyelectrolyte backbone upon
binding to these structures.
When PONT is present during amyloid fibril incubation, the
changes in emission spectra upon insulin fibril formation have
different characteristics (Figure 4). After 10 h of incubation,
one clear peak at 585 nm can be seen, indicating that the insulin
fibril formation induces a somewhat more twisted conformation
of the thiophene backbone compared to PONT in solution (pH
1.6). The increase in emission intensity is a result of separation
of the oligomer chains. This blue shift is not a thermochromic
phenomenon, since it cannot be seen if the pure oligomer is
heated; in fact, if a pure oligomer solution is heated for 10 h,
very strong quenching of the fluorescence occurs. The change
of emission characteristics and the preservation of the optical
properties of the oligomer indicate an incorporation of PONT
into the amyloid fibrils. A conjugated polymer integrated in the
defined nanostructure of amyloid fibrils is a highly interesting
material for the development in self-assembling organic elec-
tronics.
To perform a spectroscopic evaluation of the different
backbone conformations of PONT, induced by the formation
of amyloid fibrils of BI, a kinetic study of the formation of
amyloid fibrils in BI was carried out. PONT was added after
fibrillation, and the emission of the oligoelectrolyte was utilized.
The time plot for the formation of amyloid fibrils of BI is shown
in Figure 5. By following the ratio of the intensity of the emitted
light at 560 and 600 nm, 560/600 nm, the amyloid fibrillation
can clearly be monitored by the geometrical alterations in the
polymer chains.
An initial lag phase, an exponential growth phase, and a final
plateau phase are evident in the graph. The exponential growth
phase for amyloid formation occurs during 4-7 h, which is in
good agreement with the results obtained from similar experi-
ments using a different polymer.39 The fluorescence was also
recorded for samples incubated for 24 and 72 h, and the ratio
of the intensity of the emitted light at 560 and 600 nm was just
slightly altered, compared with the sample incubated for 10 h,
indicating that the plateau phase for the amyloid formation are
reached after 10 h (data not shown). The emission studies agree
very well with the results obtained by the TEM studies and the
CD measurements. The consistence of these results shows how
To further verify the use of PONT as a conformational-
sensitive probe to detect amyloid fibril formation in proteins at
acidic pH, experiments with chicken lysozyme were performed
(see Supporting Information). Interaction between native chicken
lysozyme (nCL) and PONT gives a similar spectrum as the one
obtained with nBI (Figure 4 and Supporting Information).
Likewise, after addition of 5 µM (on a monomer basis) of
amyloid fibrils of lysozyme (fCL) to a PONT solution, the
emission spectrum has similar characteristics as the spectrum
seen for BI amyloid fibrils (Figure 4 and Supporting Informa-
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Regioregular Zwitterionic Conjugated Electrolytes
A R T I C L E S
the degree of amyloid fibril formation can be monitored with
PONT fluorescence.
suggest that the method described herein can be used for a wide
range of proteins, in sensor applications and bioelectronic
devices. Furthermore, we have shown indications of the
possibility to incorporate the semiconducting oligoelectrolytes
into the amyloid fibril structure. The potential of this material
lies in the formation of highly ordered, self-assembling elements
for organic electronic applications on the nanometer scale.
In the fluorescence spectra and the kinetic studies, it is clearly
seen how different states of the amyloid formation can be easily
monitored with the oligoelectrolyte. The possibility to detect at
low pH used in the amyloid formation process is a noteworthy
difference compared to frequently used probes, such as thio-
flavine T and Congo red.
Acknowledgment. We acknowledge partial funding from
VINNOVA and CENANO at LiU (A.H.). Support from the
Foundation of Strategic Research (P.H.), the Wenner-Gren
Foundations (P.H.), and the Swedish research council (P.H. and
J.D.M.O.) are greatly appreciated.
Conclusions
We have reported the synthesis of a regioregular, zwitterionic,
conjugated oligoelectrolyte. Our study shows how this oligo-
electrolyte successfully can be used as an optical probe for
detection of amyloid fibril formation at low pH. As amyloid
fibril formation is a well-known phenomenon associated with
several disease conditions, a simple detection system is highly
desirable. The method is based on a fast, noncovalent interaction
between the protein and the probe. Binding of the oligoelec-
trolyte to amyloid fibrils results in changes of the geometry and
the electronic structure of the oligoelectrolyte chains, which have
been monitored with absorption and emission spectroscopy. We
Supporting Information Available: CD spectra of amyloid
fibrillation, emission spectra of PONT interacting with chicken
lysozyme in native and amyloid fibril form, and emission spectra
of PONT interacting with collagen. This material is available
free of charge via the Internet at http://pubs.acs.org.
JA045835E
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