Toward intrinsically fluorescent proteomimetics: Fluorescent probe response to alpha helix structure of poly-γ-benzyl-l-glutamate

Article abstract of DOI:10.1016/j.bmcl.2006.07.050

A fluorescent probe (1), developed for recognition of alpha helical secondary structure, shows a large fluorescence change upon titration with the synthetic protein PBLG. Compared to fluorophores of similar size and shape, 1 displayed the smallest dissoci

Full text of DOI:10.1016/j.bmcl.2006.07.050

Bioorganic & Medicinal Chemistry Letters 16 (2006) 5436–5438
Toward intrinsically fluorescent proteomimetics: Fluorescent probe
response to alpha helix structure of poly-c-benzyl-L-glutamate
Kenji Hutt, Randy Hernandez and Michael D. Heagy*
New Mexico Institute of Mining and Technology, Chemistry, 801 Leroy Avenue, Socorro, NM 87801, USA
Received 16 June 2006; revised 14 July 2006; accepted 17 July 2006
Available online 4 August 2006
Abstract—A fluorescent probe (1), developed for recognition of alpha helical secondary structure, shows a large fluorescence change
upon titration with the synthetic protein PBLG. Compared to fluorophores of similar size and shape, 1 displayed the smallest
dissociation constant (K = 80 lM) when titrated with PBLG. These preliminary studies are directed toward developing small
D
molecule proteomimetics that have intrinsic fluorescence and are specific for helical-protein binding-sites.
Ó 2006 Elsevier Ltd. All rights reserved.
Currently, there is considerable interest in developing
molecular scaffolds that can effectively mimic protein
secondary structures. Because b-sheets and a-helices
mediate a myriad of protein-protein interactions, a num-
ber of small-molecule mimetics have been designed to
compete as sequence-selective bioactive analogs. As a-
helices comprise over 40% of polypeptide amino acids
in natural proteins, they represent the most common
2
protein secondary structure. Therefore, the potential
at kmax of 350 nm, its photophysical features lie within
the absorption and emission range of the proteins under
investigation. To derive binding affinity information
8
between the targeted helical peptide and the proteomi-
metic, fluorescent labeling of the peptide has provided
evidence via (1) fluorescence polarization using a fluo-
rescein-tagged protein to observe displacement of the
probe, or (2) titration of a terphenyl proteomimetic with
a dansylated heterodimer complexed with the targeted
1
4
,6
clinical applications for fluorescent biomarkers designed
to have a specific affinity for helical surfaces are far
reaching given the number of protein-protein interac-
helix. Both cases require modification of the native
protein.
3
tions involving a-helices. The development of such
To allow for direct observation of a proteomimetic’s
interaction with a protein surface, an intrinsically fluo-
rescent ter-aryl system would obviate the requirement
of protein labeling and potentially serve as a fluorescent
marker for optical detection of clinically important
proteins. An intrinsically fluorescent proteomimetic
removes additional fluorophores from the protein/prote-
omimetic interaction; a current problem in imaging
small-molecule biomarkers would hold distinct advanta-
ges over current methods involving immunoassays
where antibody incubation time, cross-reactions, and
4
mixed epitope regions can hamper clinical findings.
Recent findings from Hamilton’s group have highlighted
the use of terphenyl derivatives as structural and func-
tional mimics of extended regions of a-helices. Their
work demonstrated the similarities in structural motif
9
technology. Potential applications for probes with these
features are envisioned in the rapid detection of proteins
with exposed helical surfaces such as cardiac troponin I
0
00
of tris-functionalized 3,2 ,2 -terphenyls to the i, i + 3,
and i + 7 side chain residues along one face of the a-he-
4
released at the onset of cardiac arrest.
5
–7
lix.
The critical interactions of a-helical side chains
0
00
compare well with the 3,2 ,2 -substituents when the
terphenyl is in a staggered conformation with dihedral
angles of 68° between aromatic rings. Because the
N-Arylnaphthalimides represent a diverse class of fluo-
rescent compounds which bear a close structural resem-
blance to terphenyl compounds. Their photophysical
properties, in particular the fluorescence emission, have
been found to be very sensitive to substituent groups on
terphenyl system absorbs at k
of 260 nm and emits
max
1
0
the naphthalene ring. As fluorescent biomarkers, such
features are especially useful in providing an optical
signal beyond the blue emission of autofluorescence.
Keywords: Fluorescent proteomimetics; PBLG, Binding affinities.
*
Corresponding author. E-mail: mheagy@nmt.edu
0
960-894X/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.07.050

K. Hutt et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5436–5438
5437
sents the current aromatic scaffold for peptide recogni-
tion, displayed erratic change in its 300 nm
fluorescence (see Supplementary data) when titrated
with PBLG. Of the six systems used in our investigation,
Figure 2 shows dramatic quenching of the probe’s
O
O
O
O
N
N
420 nm emission upon titration of fluorescent probe 1
with PBLG (MW 20,000) solution in dichloromethane.
The emission spectrum of 1 is more highly structured
than the absorption spectrum indicating that the rings
N
O
N
O
O
O
8
become more coplanar in the excited state. In this case
as both synthetic protein and probe absorbed at 260 nm,
increased emission at 300 nm indicates higher concentra-
tions of the PBLG.
1
2
3
Following Benesi–Hildebrand analysis of this data set, a
K value of 80 lM was obtained based on a 1:1 binding
O
D
1
5
isotherm (Table 1). In comparison, the other isomor-
phous probes 2 and 3 displayed smaller changes in their
fluorescence response. In case the biphenyl component
of 1 was responsible for the peptide recognition, 5 was
examined for its fluorescence response. Although 5 dis-
played more significant fluorescence quenching than
probes 2–4, its affinity for the synthetic protein was less
N
4
5
6
0
0
Figure 1. Structures 1–6, respectively, N-4,4 -biphenyl-1,8-naphtha-
than 1 (K = 130 lM) and its bluer emission at 300 nm
D
0
0
lenedicarboximide, N-1-(phthalimido)-N -4-(1 ,8 -naphthalimido)ben-
zene, N-phenyl-1,8-naphthalene dicarboximide, p-terphenyl, biphenyl,
and Prodan.
was less desirable. To assess the possibility that probe 1
is displaying changes solely due to environmental polar-
ity, Prodan, a well-known probe for micropolarity mea-
surements, was selected for titration with PBLG. Here,
only modest changes were observed with increasing
amounts of the synthetic peptide and indicate that probe
In addition, these compounds display large changes in
fluorescence intensity with solvents of different polarity.
In this regard, their photophysical properties resemble
Prodan, a widely used fluorescent probe for measuring
1’s ter-aryl morphology plays a role in protein
recognition.
1
1
micropolarity. By extending the N-arylnaphthalimide
core to an N-biarylnaphthalimide platform, a prototype
for a terphenyl analog is conceived that is intrinsically
fluorescent and highly sensitive to solvent polarity.
Fluorescent probe 1 was readily prepared with 1,8-naph-
thalenedicarboxylic anhydride and 4-aminobiphenyl.
Figure 1 indicates the structural similarities between p-
terphenyl and compound 1 along with structurally relat-
ed probes such as Prodan, N-phenyl-1,8-naphthalic car-
boximide, and biphenyl used in our initial study with a
helical peptide.
In the present work, these fluorescent studies were limit-
ed to PBLG due to the solubility properties of the
probes under investigation. Attempts to include water-
soluble a-helical proteins such as polyleucine as well as
the non-helical polyisoleucine met with precipitation of
the fluorescent probe. Synthesis of water-soluble ver-
sions of the probe is currently underway. Nevertheless,
these in vitro studies on 1 should not preclude its poten-
tial value in biological systems as hydrophobic pockets
are commonly found features in protein-protein interac-
tions. The red-shifted emission (120 nm) of 1 relative to
To assess the fluorescence response of these probes with
an a-helical secondary structure, the well-studied syn-
thetic polypeptide, poly-c-benzyl-L-glutamate, was
selected for its rigid coil morphology and co-solubility
5
indicates participation of the naphthalimide ring in the
photophysics as well as the fluorescence quenching upon
recognition of the helical secondary structure.
1
2
with probes 1–6 in ordinary organic solvents. PBLG
has attracted considerable attention as a building block
In short, this new fluorescent platform sets the stage for
additional luminescent proteomimetics of a-helical
structure. Despite dissociation constants in the micro-
molar region compared to Hamilton’s terphenyl systems
where nanomolar binding is observed, a preliminary
analysis of the data suggests that the shape and surface
area of 1 correlates well with the p–p stacking arrays
presented by the poly-c-benzyl-L-glutamate. Future ver-
sions of this probe are planned that include recognition
1
3
for the development of self-assembled materials. More
recently, such synthetic peptides have found use as bio-
1
4
foul-resistant polymers for medical devices. Terphenyl,
which when decorated with recognition groups repre-
1
H
Mp 237 °C (dec); UV–vis kmax = 257 nm Emission = 415 nm,
NMR (300 MHz, CDCl
J = 4.12 Hz, 2H), 8.29 (t, J = 10.53 Hz, 2H) 7.92 (m, 4H), 7.64 (d,
3
) 8.66 (d, J = 7.93 Hz, 2H) 8.63 (d,
0
components for the naphthalimide system along the 3
0
1
3
and 2 positions of the biphenyl for other biologically
J = 8.23 Hz, 2H), 7.43 (m, 3H). C (75 MHz, DMSO-d
6
164.5,
relevant proteins. Such functionality should significantly
improve the KD values relative to the parent hydrocar-
bon system observed with compound 1. Finally, the
naphthalimide chromophore can be readily attenuated
1
1
61.1, 135.9, 135.3, 135.4, 133.1, 131.9, 131.4, 130.3, 128.1, 127.7,
ꢀ1
25.9, 124.0, 123.1, 119.5. IR m/cm : 1733, 1364, 1229, 1216, 1204.
15NO : C, 82.52%, H, 4.29%, N 4.01%. Found:
C, 82.41%, H, 4.28%, N, 3.83%.
Anal. Calcd for C24
H
2

5438
K. Hutt et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5436–5438
4
3
.00E+06
.00E+06
.00E+06
.00E+06
.00E+00
2
1
0
2
70
320
370
420
470
Wavelength (nm)
0
Figure 2. Stacked fluorescence spectra of N-4,4 -biphenyl-1,8-naphthalenedicarboximide (1) at constant concentration of 10 lM with PBLG in
aliquots of 10 lM using kexc at 260 nm.
Table 1. Comparison of dissociation constants between PBLG and
fluorescent probes
2. Cochran, A. G. Chem. Biol. 2000, 7, R85.
3. Yin, H.; Lee, G.; Sedey, K. A.; Kutzki, O.; Park, H. S.;
Orner, B. P.; Ernst, J. T.; Wang, H.-G.; Sebti, S. M.;
Hamilton, A. D. J. Am. Chem. Soc. 2005, 127, 10191.
Probe
D
K (lM)
1
2
5
80
230
130
4
. Panteghini, M.; Gerhardt, W.; Apple, F. S.; Dati, F.;
Ravkilde, J.; Wu, A. H. Clin. Chem. Lab. Med. 2001, 39,
1
74.
Percent error for K
D
values is ±10%. Probes 3, 4, and 6 did not display
5. Orner, B. P.; Ernst, J. T.; Hamilton, A. D. J. Am. Chem.
Soc. 2001, 123, 5382.
6. Ernst, J. T.; Kutzki, O.; Debnath, A. K.; Jiang, S.; Lu, H.;
Hamilton, A. D. Angew. Chem. Int. Ed. 2002, 41, 278.
7. Kutzki, O.; Park, H. S.; Ernst, J. T.; Orner, B. P.; Yin, H.;
Hamilton, A. D. J. Am. Chem. Soc. 2002, 124, 11838.
significant changes in fluorescence suitable for binding isotherm
analysis.
(
with substituents) to absorb at longer wavelengths
thereby removing protein autofluorescence from the
spectral window.
8
. Birks, J. B. Photophysics of Aromatic Molecules; John
Wiley & Sons: New York, 1979.
9
. Howarth, M.; Takao, K.; Hayashi, Y.; Ting, A. Y. Proc.
Natl. Acad. Sci. U.S.A. 2005, 102, 7583.
Acknowledgment
1
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Biczok, L.; Wintgens, V.; Valat, P.; Kossanyi, J. J. Phys.
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Kossanyi, J.; Demeter, A.; Biczok, L.; Berces, T.
J. Photochem. Photobiol. A: Chem. 1996, 93, 109.
We thank the National Institute of General Medical
Science division of NIH for supporting this research.
Supplementary data
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York, 1999.
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1
1
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Supplementary data associated with this article can be
found in the online version at doi:10.1016/j.bmcl.
2
006.07.050.
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2