Triazole-containing BODIPY dyes as novel fluorescent probes for soluble oligomers of amyloid Aβ1-42 peptide

Article abstract of DOI:10.1016/j.bbrc.2009.12.091

A straightforward functionalization of BODIPY dyes via incorporation of a triazole moiety produced fluorescent dyes that were capable of distinguishing between secondary structure conformations of soluble oligomeric species of Aβ1-42 peptide. Small concen

Full text of DOI:10.1016/j.bbrc.2009.12.091

Biochemical and Biophysical Research Communications 391 (2010) 1455–1458
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications
journal homepage: www.elsevier.com/locate/ybbrc
Triazole-containing BODIPY dyes as novel fluorescent probes for soluble
oligomers of amyloid Ab1–42 peptide
*
Nicholas W. Smith, Annabel Alonso, Christopher M. Brown, Sergei V. Dzyuba
Department of Chemistry, Texas Christian University, Fort Worth, TX 76129, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 7 December 2009
Available online 23 December 2009
A straightforward functionalization of BODIPY dyes via incorporation of a triazole moiety produced fluo-
rescent dyes that were capable of distinguishing between secondary structure conformations of soluble
oligomeric species of Ab1–42 peptide. Small concentrations of the dyes, relative to Ab1–42, provided up
to an 8-fold and 35-fold fluorescence increase in the presence of the unordered and ordered, b-sheet-rich
conformations of soluble Ab1–42 oligomers, respectively. These triazole-containing dyes could prove to
be useful probes for monitoring amyloid conformational transitions in vitro.
Keywords:
Alzheimer’s disease
Amyloid aggregation
Circular dichroism spectroscopy
Fluorescent probes
Click-chemistry
Ó 2009 Elsevier Inc. All rights reserved.
BODIPY
Introduction
Due to superior and tuneable properties, BODIPY dyes (Fig. 1)
have become versatile fluorescent probes for examining biological
Soluble oligomers of amyloid peptides (Ab) are recognized as
one of the main neurotoxic species responsible for impairment of
neuronal function [1]. A number of in vivo and in vitro studies dem-
onstrated that soluble Ab oligomers inhibit hippocampal long-term
potentiation, thus retarding memory formation and learning [2,3].
The nature of these soluble oligomers remains to be clarified [4],
and spectroscopic detection of secondary structure conformations
of soluble amyloid species using small molecular probes currently
presents a challenge.
processes [9,10]. Although several recent studies demonstrated
that BODIPY dyes could be used as fluorophores for amyloid aggre-
gates, when attached to a pharmacophore [11,12], the ability of
BODIPY dyes to act as fluorophoric/pharmacophoric probes for
amyloid self-assembly has been unexplored. Here, we report on
the novel ability of triazole-containing BODIPY dyes to distinguish
between the unordered and ordered, b-sheet-rich conformations of
soluble Ab1–42 oligomers.
A fluorescence dye binding assay is a convenient technique to
probe the amyloid self-assembly processes. The dye has to exhibit
distinct spectral characteristics upon binding to the amyloid
assembly as compared to the unbound state. Thioflavin T, ThT
(Fig. 1), is a standard for monitoring amyloid-related aggregation
processes [5,6]. Despite wide utility, ThT has several major draw-
backs: (i) ThT does not bind to soluble forms of amyloid peptides,
i.e., a substantial increase of ThT fluorescence occurs only in the
presence of b-sheet-rich conformations of amyloid peptides, such
as protofibrils and fibrils [5]; (ii) aggregation of Ab peptides and
the evaluation of the degree of Ab aggregation using ThT are done
under different conditions [6]; (iii) in general, an access of the dye,
relative to Ab peptides is required to achieve high fluorescence
enhancement [5–8]. All these factors might disrupt the integrity
of the Ab self-assembly. Thus, more versatile and more efficient
fluorescent probes are required.
Materials and methods
All chemicals and solvents were from commercial sources (Sig-
ma–Aldrich, Acros or Matrix Scientific), they were of highest grade
possible, and were used as received. Ab1–42, NaOH-treated prepa-
ration, was from Recombinant Peptide, Inc. (Athens, GA). Milli-Q
system was used to purify water that was utilized throughout this
work. Stock solutions of the BODIPY dyes for spectroscopic mea-
surements were prepared in EtOH (0.214 mM) and subsequently
diluted into the aqueous buffer. For time-dependent measure-
ments the concentration of the EtOH-stock solutions was
0.0428 mM. Details on the synthesis and characterization of tria-
zole-containing BODIPY dyes are provided in Supporting
Information.
Absorbance and fluorescence measurements were performed
on Agilent 8453 UV–vis instrument and Shimadzu RF-5301PC,
respectively, using 1 cm quartz cells with a resolution of 1 nm.
Fluorescence measurements were carried out as follows: excitation
and emission width slits were 3 mm and 3 mm. Samples were
excited at the absorption maxima. Spectra were smoothed using
* Corresponding author. Fax: +1 817 257 5851.
E-mail address: s.dzyuba@tcu.edu (S.V. Dzyuba).
0006-291X/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved.
doi:10.1016/j.bbrc.2009.12.091

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N.W. Smith et al. / Biochemical and Biophysical Research Communications 391 (2010) 1455–1458
Fig. 1. Structures of fluorescent dyes.
manufacture provided software. CD spectra were acquired on Jasco
J-815 using 0.1 cm quartz cells; spectra were recorded at room
temperature and 1 nm resolution with a scan rate of 100 nm/
min; four scans were acquired and averaged for each sample;
raw data were manipulated by subtraction of appropriate back-
ground spectra, followed by smoothing using manufacturer pro-
vided software, and the data expressed as CD intensity, mdeg.
Preparation of Ab1–42 oligomers. Soluble oligomers of 25 lM
Ab1–42 solution were prepared according to literature procedure
[13].
Basic media was required to obtain an unordered conformation of
Ab1–42, and was consistent with literature data [14].
The ability of BODIPY dyes 1 and 2 (Fig. 1) to recognize distinct
conformations of soluble Ab1–42 oligomers was investigated next.
Titration of 1 and 2 into solutions containing unordered and b-
sheet-rich Ab oligomers revealed that in the presence of the unor-
dered conformation of Ab1–42, the emission intensity of dye 1 was
almost unchanged, while that of 2 is almost doubled (Fig. S1). In
view of self-quenching of dye 1, the concentration of this dye
was decreased 10-fold, as compared to dye 2, to obtain a measur-
able titration curve. As one might expect, both dyes exhibited a
higher fluorescence enhancement in the presence of the ordered,
b-sheet-rich conformation of Ab1–42. In the presence of the or-
dered, b-sheet-rich conformation of Ab1–42 dye 1 exhibited ca.
3-fold fluorescence increase, and dye 2 afforded a 5-fold increase.
Although the increases in the fluorescent intensity are not particu-
larly large, these are the first evidence that BODIPY dyes could rec-
ognize soluble amyloid oligomers.
In order to enhance the recognition ability of BODIPY dyes to-
wards amyloid oligomers, we decided to modify the structure of
BODIPY dyes. Despite the fact a number of syntheses for BODIPY
dyes are known [9,10], they are rarely modular in nature. Thus,
we prepared BODIPY scaffolds, i.e., 3 and 4 that should allow for
a facile and modular access to various BODIPY-based fluorescent
dyes (Scheme 1). Subsequently, these alkyne-containing dyes were
elaborated into the triazole-containing dyes 5 and 6 via a widely
used copper-catalyzed alkyne–azide cycloaddition reaction, com-
monly referred to as ‘‘click-chemistry” [15,16]. This reaction is
known for high efficiency, mild conditions and high tolerance to
many functional groups [15], and in many cases it is biocompatible
[16].
Time-dependent fluorescence monitoring of Ab1–42 aggregation. A
freshly prepared solution of 25
temperature. Periodically, the solution was mixed by dispensing it
with a pipette tip, and 50 M aliquots were withdrawn and placed
into a fluorescence cell. A dye stock solution was added (final con-
centration 0.26 M), gently mixed with the pipette and the emis-
lM Ab1–42 was incubated at room
l
l
sion spectra were recorded. The respective emission spectra of
the dyes in the same buffer, but in the absence of Ab1–42, were ta-
ken at the same time as a control.
Results and discussion
In order to evaluate the ability of BODIPY dyes to interact with
soluble oligomers of Ab peptides, we prepared distinct conforma-
tions of Ab1–42 oligomers following our previously developed pro-
tocol [13]. Time-dependent circular dichroism (CD) monitoring of
25 lM Ab1–42 confirmed a transition, with an isosbestic point at
ca. 207 nm, from an unordered to an ordered, b-sheet-rich confor-
mation as evident by increasing the minima at ca. 214 nm (Fig. 2).
Here, 5 and 6 were prepared as proof-of-principle dyes, and
their design was governed by the ease of synthetic accessibility
and structural simplicity. Although several triazole-containing
BODIPY dyes have been reported [17], albeit structurally distinct
from 5 and 6, neither was exploited as a probe for conformational
changes of biomolecules.
The emission spectra revealed the tendency of both triazole-
containing BODIPY dyes to aggregate in the aqueous buffer. A
shoulder at ca. 600 nm for 5 and an additional transition around
612 nm for 6 were observed in the aqueous buffer (Fig. S2). A single
transition was noted for both dyes in EtOH, which matched the
absorption spectra, thus suggesting the absence of aggregates.
Spectroscopic properties of BODIPY dyes are known to be pH
insensitive [9,10]. Accordingly, the triazole-containing dyes also
appeared to be insensitive to pH variations. For example, the emis-
sion spectra of dye 6 were similar in a pH 7.4 buffer (10 mM phos-
phate, 0.135 M NaCl, 2.7 mM KCl) and in pH 2.0 solution (0.1 M
NaCl, HCl) (Fig. S2). The solubility of 5 and 6 in both aqueous and
organic media was decreased as compared to 1 and 2, yet it was
still sufficient to provide final lM-range concentrations.
Next, the potential of triazole-containing BODIPY dyes 5 and 6
to interact with various conformations of soluble oligomers of
Ab1–42 peptide was investigated. Both triazole-containing dyes of-
Fig. 2. Time-dependent CD spectra of a conformational transition of 25 lM Ab1–42
in 10 mM Tris/NH4OH buffer (1.2% EtOH, v/v), pH 8.6; 0 h (dashed line), 80 h (thick
line), intermediate time points (thin lines).

N.W. Smith et al. / Biochemical and Biophysical Research Communications 391 (2010) 1455–1458
1457
Scheme 1. Synthesis of triazole-containing BODIPY dyes: (a)—(i) TFA, CH2Cl2, rt, (ii) DDQ, CH2Cl2, rt, (iii) BF3ÁEt2O, Et3N, CH2Cl2, rt; (b) BnN3, CuSO4, sodium ascorbate, DMSO/
H2O, rt (See Supporting Information for details).
fered much improved sensitivity in the recognition of the unor-
dered amyloid oligomers as compared to dyes 1 and 2. Signifi-
cantly, a marked enhancement of the emission intensity of dyes
5 and 6 in the presence of an unordered conformation of soluble
Ab1–42 oligomers (Fig. 3). Dye 6 appears to be superior to dye 5,
in terms of recognizing the unordered conformation of Ab1–42 sol-
uble oligomers, as an 8-fold increase of fluorescence intensity was
observed. Similar to 1 and 2, the triazole-containing BODIPY dyes
offered a much higher fluorescence enhancement in the presence
of ordered b-sheet-rich Ab1–42 soluble oligomers (Fig. 3). About
a 25-fold and 35-fold emission increase was detected for 5 and 6,
respectively.
Notably, small concentrations of 5 and 6, with respect to Ab
peptide, i.e., 25–50 molar excess of Ab1–42, provided a significant
enhancement of the fluorescent signal. Arguably, this should as-
sure that the integrity of the amyloid self-assembly is retained
upon addition of the dyes. Thus, triazole-containing BODIPY dyes
are favorably distinguished from widely used thioflavin T dyes,
which usually require an excess of the dye that could perturb the
aggregation state of the peptide.
In addition, dyes 5 and 6 proved suitable for monitoring time-
dependent changes of the amyloid peptide conformation
(Fig. S3). The emission intensity of both 5 and 6 was steadily
increasing as the b-sheet-content of the Ab1–42 oligomers was
increasing. The time-dependent fluorescence data were in correla-
tion with the CD time-dependent spectra (Fig. 2) over the same
time period. Notably, no ‘‘lag” phase was observed. The ‘‘lag” phase
is characteristic for ThT assays [5–8], due to the fact that early-
stage, soluble oligomers are not recognized by ThT. This suggests
that these triazole-containing BOIDPY dyes can indeed recognize
all soluble conformations of Ab1–42 oligomers.
Although the role of the triazole moiety requires further inves-
tigation, our data highlight its importance. Noteworthy, a number
of Alzheimer’s disease related compounds feature a triazole moiety
[18–20], yet to the best of our knowledge, there are no precedents
that indicate that a triazole group is the major contributor in the
recognition of amyloid oligomers. Tentatively, we can propose that
a triazole group, which is iso-structural and iso-electronic to a pep-
tide bond [21–23], might be responsible for the enhanced recogni-
tion of amyloid oligomers by providing an amino acid–amino acid
type of interaction.
In conclusion, we demonstrated that the introduction of the tri-
Fig. 3. Emission spectra of 5 (top, kex 520 nm) and 6 (bottom, kex 529 nm), 1.0 lM
azole moiety onto the BODIPY scaffolds afforded dyes that allowed
for an unambiguous differentiation between an unordered confor-
mation, which is mostly a benign form, and an ordered conforma-
tion, which is largely a neurotoxic form, of Ab1–42 soluble
each, in the presence of unordered oligomeric (open symbols), b-sheet-rich
oligomeric (closed symbols) and in the absence (solid line) of Ab1–42. Insets:
titration of the dyes into the solutions of Ab1–42; data are the average of two
independent preparations of Ab1–42 SD.

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N.W. Smith et al. / Biochemical and Biophysical Research Communications 391 (2010) 1455–1458
[8] R. Eisert, L. Felau, L.R. Brown, Methods for enhancing the accuracy and
reproducibility of Congo red and thioflavin T assays, Anal. Biochem. 353 (2006)
144–146.
[9] A. Loudet, K. Burgess, BODIPY dyes and their derivatives: synthesis and
spectroscopic properties, Chem. Rev. 107 (2007) 4891–4932.
oligomers. Triazole-containing BODIPY dyes could serve as suitable
in vitro probes for monitoring conformational transitions of amy-
loid species.
[10] G. Ulrich, R. Ziessel, A. Harriman, The chemistry of fluorescent Bodipy dyes:
versatility unsurpassed, Angew. Chem. Int. Ed. 47 (2008) 1184–1201.
[11] K. Parhi, M.-P. Kung, K. Ploessl, H.F. Kung, Synthesis of fluorescent probes
based on stilbenes and diphenylacetylenes targeting b-amyloid plaques,
Tetrahedron Lett. 49 (2008) 3395–3399.
[12] A. Ojida, T. Sakamoto, M.-a. Inoue, S.-h. Fujishima, G. Lippens, I. Hamachi,
Fluorescent BODIPY-based Zn(II) complex as a molecular probe for selective
detection of neurofibrillary tangles in the brains of Alzheimer’s disease
patients, J. Am. Chem. Soc. 131 (2009) 6543–6548.
Acknowledgments
This work was supported in part by the Donors of the Petroleum
Research Fund (administered by the American Chemical Society,
47965-G7) and TCU (RCAF 60486 and TCU-JFSR award to S.V.D.).
We would like to thank Prof. D.E. Minter for assistance with acquir-
ing NOE spectra. A.A. is a McNair scholar. N.W.S. acknowledges the
ACS Division of Medicinal Chemistry for a predoctoral fellowship
sponsored by Wyeth.
[13] N.W. Smith, O. Annunziata, S.V. Dzyuba, Amphotericin B interactions with
soluble oligomers of amyloid Ab1–42 peptide, Bioorg. Med. Chem. 17 (2009)
2366–2370.
[14] M. Bartolini, C. Bertucci, M.L. Bolognesi, A. Cavalli, C. Melchiorre, V. Andrisano,
Insight into the kinetic of amyloid
elucidation of inhibitors’ mechanism of action, Chembiochem
2152–2161.
b (1–42) peptide self-aggregation:
Appendix A. Supplementary data
8 (2007)
[15] M. Meldal, W. Christian, Cu-catalyzed azide–alkyne cycloaddition, Chem. Rev.
108 (2008) 2952–3015.
[16] M.D. Best, Click chemistry and bioorthogonal reactions: unprecedented
selectivity in the labelling of biological molecules, Biochemistry 48 (2009)
6571–6584.
[17] L. Li, J. Han, B. Nguyen, K. Burgess, Syntheses and spectral properties of
functionalized, water-soluble BODIPY derivatives, J. Org. Chem. 73 (2008)
1963–1970.
[18] C. Fischer, B. Munoz, S. Zultanski, J. Methot, H. Zhou, W.C. Brown, Preparation
of triazole derivatives for treating Alzheimer’s disease and related conditions,
PCT Int. Appl., 2008, WO 2008156580 A1 20081224.
[19] T. Kimura, K. Kawano, E. Doi, N. Kitazawa, T. Miyagawa, N. Sato, T. Kaneko, K.
Shin, K. Ito, M. Takaishi, T. Sasaki, H. Hagiwara, Preparation of heterocyclic
type cinnamide compounds for inhibiting amyloid-b production, PCT Int.
Appl., 2007, WO 2007135970 A1 20071129.
[20] S. Bowers, A.W. Garofalo, R.K. Hom, A.W. Konradi, M.N. Mattson, M.L. Neitzel,
C.M. Semko, A.P. Truong, J. Wu, Y.-Z. Xu, Preparation of bridged N-cyclic
sulfonamide compounds as inhibitors of gamma secretase. PCT Int. Appl.,
2007, WO 2007022502 A2 20070222.
[21] H.C. Kolb, K.B. Sharpless, The growing impact of triazole-containing chemistry
on drug discovery, Drug Discov. Today 8 (2003) 1128–1137.
[22] W.S. Horne, C.D. Stout, M.R. Ghadiri, A heterocyclic peptide nanotube, J. Am.
Chem. Soc. 125 (2003) 9372–9376.
[23] V.D. Bock, R. Perciaccante, T.P. Jansen, H. Hiemstra, J.H. van Maarseveen,
Triazole-containing chemistry as a route to cyclic tetrapeptide analogues:
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bbrc.2009.12.091.
References
[1] C. Haass, D.J. Selkoe, Soluble protein oligomers in neurodegeneration: lessons
from the Alzheimer’s amyloid b-peptide, Nat. Rev. Mol. Cell Biol. 8 (2007) 101–
112.
[2] D.M. Walsh, I. Klyubin, J.V. Fadeeva, W.K. Cullen, R. Anwyl, M.S. Wolfe, M.J.
Rowan, D.J. Selkoe, Naturally secreted oligomers of amyloid
b protein
potentially inhibit hippocampal long-term potentiation in vivo, Nature 416
(2002) 535–539.
[3] J.P. Cleary, D.M. Walsh, J. Hofmeister, G.M. Shankar, M.A. Kuskowski, D.J.
Selkoe, K.H. Ashe, Natural oligomers of the amyloid-b protein specifically
disrupt cognitive function, Nat. Neurosci. 8 (2005) 79–84.
[4] A. Frydman-Maron, M. Rechter, I. Shefler, Y. Bram, D.W. Shalev, E. Gazit,
Cognitive-performance recovery of Alzheimer’s disease model mice by
modulation of early soluble amyloidal assemblies, Angew. Chem. Int. Ed. 48
(2009) 1981–1986.
[5] A. Hawe, M. Sutter, W. Jiskoot, Extrinsic fluorescent dyes as tools for protein
characterization, Pharm. Res. 25 (2008) 1487–1499.
[6] S.R. Leliveld, C. Korth, The use of conformation-specific ligands and assays to
dissect the molecular mechanisms of neurodegenerative diseases, J. Neurosci.
Res. 85 (2007) 2285–2297.
synthesis of cylco-[Pro-Val-
W(triazole)-Pro-Tyr], Org. Lett. 8 (2006) 919–
922.
[7] M.R. Nillson, Techniques to study amyloid fibril formation in vitro, Methods 34
(2004) 151–160.