Versatile synthesis of fluorescent, cross-linked peptides as biological probes with the advantage of high helix content

Article abstract of DOI:10.1007/s11164-012-0651-5

A variety of fluorophores were introduced at the N-termini of short peptides for use as biological-probes. The fluorescent peptides were cross-linked with a diacetylenic cross-linking agent between the amino acid side chains of ornithine (Orn) residues to produce peptides with high helix content.

Full text of DOI:10.1007/s11164-012-0651-5

Res Chem Intermed (2013) 39:311–319
DOI 10.1007/s11164-012-0651-5
Versatile synthesis of fluorescent, cross-linked peptides
as biological probes with the advantage of high helix
content
•
Kazuhisa Fujimoto Masaoki Kajino
Masahiko Inouye
•
Received: 13 October 2011 / Accepted: 29 November 2011 / Published online: 3 July 2012
Ó Springer Science+Business Media B.V. 2012
Abstract A variety of fluorophores were introduced at the N-termini of short
peptides for use as biological-probes. The fluorescent peptides were cross-linked
with a diacetylenic cross-linking agent between the amino acid side chains of
ornithine (Orn) residues to produce peptides with high helix content.
Keywords Short helical peptide ꢀ Fluorophore ꢀ Fluorescence labeling ꢀ
Biological probe
Introduction
Most intractable diseases involve abnormal protein expression. For instance, tumor
cells overexpress specific proteins which elaborately interact with one another.
Practical molecular probes that competitively bind to specific proteins are essential
to enable cancer diagnosis at earlier stages. Such probes have to fulfill three
requirements—low molecular weight, easy synthesis, and stability for metabolic
enzymes. However, during the design of practical probes, low-molecular-weight
molecules seem to barely cover the large area of the surfaces or the active sites of
the protein–protein interactions targeted. It is also extremely difficult, de novo, to
design and synthesize probes with a moderate molecular weight that can cover the
large areas by organic synthesis only. Thus, peptide-based probes with stable
K. Fujimoto (&)
Department of Applied Chemistry and Biochemistry, Kyushu Sangyo University,
Fukuoka 813-8503, Japan
e-mail: kazuf@ip.kyusan-u.ac.jp
M. Kajino ꢀ M. Inouye (&)
Graduate School of Pharmaceutical Sciences, University of Toyama, Toyama 930-0194, Japan
e-mail: inouye@pha.u-toyama.ac.jp
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a-helical structures are regarded as powerful candidates for the aforementioned
purpose because a-helices are of key importance in protein interactions.
Recently, we succeeded in stabilization of the helices of short peptides by
bridging their amino acid side chains with cross-linking agents [1, 2].1 DNA-
binding cross-linked peptides were first designed and synthesized on the basis of the
binding sites for protein–DNA interactions, and the peptides were then labeled at
their N-termini with Tokyo green [6] to evaluate the binding efficiency of the
peptides to DNAs [7]. The fluorescent, cross-linked peptides retained stable helical
structures and had high binding affinity with substrate specificity for DNAs. The
cross-linked peptides were found to be more stable for protease than non-cross-
linked peptides, and their molecular weights were approximately 3,000. From
different perspectives, fluorescent helical peptides have potential use as specific
probes for specific proteins of clinical importance. Therefore, taking into account
our previous findings, we prepared fluorescent, cross-linked peptides by using a
variety of fluorophores and revealed their high helix content by CD analysis.
Experimental
Synthesis of 3-[4-(7-hydroxycoumarin-3-ylethynyl)phenyl]propanoic acid
To a THF (6 mL) solution of 3-bromo-7-hydroxycoumarin (120 mg, 0.5 mmol),
Pd(PPh3)4 (29 mg, 0.025 mmol), and CuI (9.5 mg, 0.05 mmol,) at 70 °C under an
Ar atmosphere, was added a solution of 3-(4-ethynylphenyl)propanoic acid [8]
(120 mg, 0.85 mmol) in Et3N (10 mL). The reaction mixture was heated under
reflux for 6 h. After removal of the solvent by use of a rotary evaporator, the residue
was dissolved in aqueous 1 M HCl and extracted with AcOEt. The AcOEt extract
was evaporated by use of a rotary evaporator and chromatographed (silica gel;
mobile phase from CH2Cl2 to CH2Cl2–CH3OH 10:1 v/v) to give 3-[4-(7-
hydroxycoumarin-3-ylethynyl)phenyl]propanoic acid: yield 57 % (95 mg); mp
231–233 °C; IR (KBr) v = 3384, 1705, 1610, 1567, 1502, 1302, 1225, 1150,
1120 cm-1 1H NMR (DMSO-d6) d = 2.57 (t, J = 7.6 Hz, 2 H), 2.86 (t, J =
;
7.6 Hz), 6.76 (d, J = 2.0 Hz, 1 H), 6.84 (dd, J = 2.4, 8.8 Hz, 1 H), 7.20 (d, J =
8.0 Hz, 2 H), 7.44 (d, J = 8.0 Hz, 2 H), 7.57 (d, J = 8.4 Hz, 1 H), 8.29 (s, 1 H),
10.83 (s, 1 H), 12.17 (s, 1 H); 13C NMR (DMSO-d6) d = 30.1, 34.6, 84.0, 93.0,
102.1, 106.3, 111.2, 113.7, 119.4, 128.7, 129.9, 131.2, 142.2, 146.4, 154.9, 159.0,
162.1, 173.5; HRMS (ESI): m/z: calcd for C20H14NaO5 [M?Na]? 357.0739; found
357.0732.
Synthesis of fluorescent, cross-linked peptides
The extending reaction of amino acid residues were performed with an automated
peptide synthesizer on an Fmoc-NH-SAL resin (capacity 0.59 mmol/g). After the
automated synthesis, the N-terminal amino groups of the peptides were
1
Other helical peptides and helical mimetics are discussed in Refs. [3–5].
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fluorescence-labeled on the resin by treating with fluorophore-based carboxylic
acids2 [8] (5 equiv), HBTU (10 equiv), HOBt (10 equiv), and N,N0-diisopropylethyl
amine (10 % v/v) in a mixture of DMF and DMSO (1:1) over 8 h at room
temperature. Dde (= 1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl) groups on
the ornithine (Orn) residues of the peptides were selectively removed by use of
hydrazine monohydrate (3 % v/v) in DMF in 1 h 9 2 at room temperature. The
deprotected peptides were cross-linked with the cross-linking agent [3–5] (3 equiv)
over 12 h at room temperature on the resin. Peptide cleavage and side chain
deprotection of amino acids were achieved by treatment under suitable conditions:
TFA–ethanedithiol–thioanisole 18:1:1 for 1.5 h at room temperature. All of the
peptides were purified by reversed-phase HPLC (Cosmosil 5C18-MS-2 column,
10 9 150 mm2, Nacalai Tesque) and eluted with a mixture of 0.1 % TFA buffer
and CH3CN including 0.1 % TFA by use of the following linear CH3CN gradients
at a flow rate of 2.0 mL min-1: 5–45 % (0–40 min) for cA, 15–55 % (0–40 min)
for cAꢀ1 and cAꢀ2, 20–60 % (0–40 min) for cAꢀ3, 20–60 % (0–40 min) for cAꢀ4,
and 10–50 % (0–40 min) for cAꢀ5. The peptide fractions were monitored at 220 nm
with a UV detector, and were identified by MALDI TOF MS.
Mass spectral data for peptides
Aꢀ1: m/z: calcd for [M ? H]?; C124H206N57O22: 2845.7; found: 2846.2
cAꢀ1: m/z: calcd for [M ? H]?; C134H211N57O26: 3035.7; found: 3035.4
Aꢀ2: m/z: calcd for [M ? H]?; C126H210N57O22: 2873.7; found: 2873.7
cAꢀ2: m/z: calcd for [M ? H]?; C136H215N57O26: 3063.7; found: 3063.1
Aꢀ3: m/z: calcd for [M ? H]?; C119H208N57O22: 2787.7; found: 2787.2
cAꢀ3: m/z: calcd for [M ? H]?; C129H213N57O26: 2977.7; found: 2977.7
Aꢀ4: m/z: calcd for [M ? H]?; C123H210N57O22: 2837.7; found: 2837.2
cAꢀ4: m/z: calcd for [M ? H]?; C133H216N57O26: 3027.7; found: 3028.4
Aꢀ5: m/z: calcd for [M ? H]?; C119H206N57O25: 2833.7; found: 2833.8
cAꢀ5: m/z: calcd for [M ? H]?; C129H212N57O29: 3023.7; found: 3023.2.
Results and discussion
Synthesis of fluorescent, cross-linked peptides
Figure 1 shows the amino acid sequence of the peptides used in this study and the
chemical structures of fluorophores tethered at the N-termini of the peptides: the
resulting fluorescent peptides are denoted Aꢀ1–Aꢀ5. The amino acid sequence,
designed according to that of the Rev peptide binding to RRE RNA, includes two
Orn residues as cross-linked moieties. Five fluorophores were introduced at the
N-termini of the Rev-arranged peptide: phenylethynylpyrenes 1 and 2 [8], pyrene 3,
perylene 4, and phenylethynylcoumarin 5. To examine the effect on helix content of
2
Synthesis of the fluorophore-based carboxylic acids of Aꢀ2 and Aꢀ4 is discussed elsewhere [9, 10].
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spacer length between the N-termini and fluorophores, two types of phenylethy-
nylpyrene-based fluorophore (1 and 2) were prepared.
All of the fluorescent peptides Aꢀ1–Aꢀ5 were synthesized in accordance with a
previously published method [7]. The extending reaction of amino acid residues,
fluorophore-labeling reaction, and cross-linking reaction were performed on the
synthetic resin. In the labeling reactions, the fluorophore-based carboxylic acids
were reacted with the deprotected N-termini of the synthesizing peptides in the
presence of HBTU, HOBt, and N,N0-diisopropylamine. When chemical modification
of the peptides on the synthetic resin was complete, the peptides were released from
the resin and purified by HPLC. Fluorescent peptides, Aꢀ1–Aꢀ5, were cross-linked
with a diacetylenic cross-linking agent to yield cAꢀ1–cAꢀ5. The combination of the
diacetylenic agent and orn was the most suitable for stabilizing the helical structures
of such Arg-rich peptides at the i/i ? 11 position for the cross-linking reaction
[1, 2].
UV–visible absorption and excitation/emission spectra of fluorescent peptides
The UV–visible absorption spectra of the fluorescent, cross-linked peptides cAꢀ1–
cAꢀ5 are displayed in Fig. 2. These spectra were measured at concentrations ranging
from 33 to 82 lM. The kmax and e values of the peptides are shown in each
spectrum. The kmax values of cAꢀ1 and cAꢀ2 were found to be shorter than that of
the phenylethynylpyrene fluorophore, which is reportedly ca 380 nm. Furthermore,
their e values were almost half that of cAꢀ3, in which native pyrene was used as the
fluorophore. These findings suggest that, at UV–visible-measurement concentra-
tions, the phenylethynylpyrene moieties of cAꢀ1 and cAꢀ2 self-associate to form
H-aggregates [11]. It is plausible that, during aggregation, the peptide moieties align
Fig. 1 Amino acid sequence of peptides, and chemical structures of the cross-linking agent and
fluorophores 1–5 in this study
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Versatile synthesis of fluorescent, cross-linked peptides
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alternately, as shown in Fig. 2, to minimize the electrostatic repulsion of the Arg
residues.
In the excitation/emission spectra of the fluorescent, cross-linked peptides at
3.8–8.2 lM, excluding cAꢀ1 (Fig. 3), each emission spectrum was almost consistent
with the reflection of the corresponding excitation spectrum in a mirror. The mirror
relationship indicates that the fluorophores on cAꢀ2–cAꢀ5 scarcely aggregate at the
measurement concentrations. The alkyl-spacer of cAꢀ2 retained the mirror
relationship, whereas the relationship was deformed in the spectra of cAꢀ1, with
no spacer. The intensity of vibronic band I was smaller than that of vibronic band III
in the excitation spectrum of cAꢀ1, with no spacer, meaning that the attached
Fig. 2 UV–visible absorption spectra of the fluorescent, cross-linked peptides cAꢀ1–cAꢀ5 dissolved in a
100 mM phosphate buffer (pH 6.6) at 25 °C: [cAꢀ1] = 33 lM, [cAꢀ2] = 77 lM, [cAꢀ3] = 44 lM,
[cAꢀ4] = 76 lM, and [cAꢀ5] = 82 lM. A plausible H-aggregation form of the phenylethynylpyrene
moieties shown as a black rectangle (lower right)
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fluorophore exists in a hydrophobic environment [12]. Moreover, the emission
spectrum of cAꢀ1 was broadened and shifted to longer wavelength than that of alkyl-
spacer-type cAꢀ2. These findings imply that cAꢀ1 self-assembles more easily than
cAꢀ2 to form H-aggregates.
Helix content of fluorescent peptides
The helix content of the fluorescent, cross-linked peptides cAꢀ1–cAꢀ5 were
estimated on the basis of their CD spectra recorded at 5 °C in accordance with a
Fig. 3 Excitation/emission spectra of fluorescent, cross-linked peptides cAꢀ1–cAꢀ5 dissolved in a
100 mM phosphate buffer (pH 6.6) at 25 °C: [cAꢀ1] = 5.0 lM, [cAꢀ2] = 5.0 lM, [cAꢀ3] = 4.4 lM,
[cAꢀ4] = 3.8 lM, and [cAꢀ5] = 8.2 lM
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Fig. 4 CD spectra of peptides A, Aꢀ1–Aꢀ5, cA, and cAꢀ1–cAꢀ5 dissolved in a 100 mM phosphate buffer
(pH 6.6) at 5 °C: [A] = 75 lM, [Aꢀ1] = 49 lM, [Aꢀ2] = 150 lM, [Aꢀ3] = 81 lM, [Aꢀ4] = 75 lM,
[Aꢀ5] = 75 lM, [cA] = 82 lM, [cAꢀ1] = 33 lM, [cAꢀ2] = 77 lM, [cAꢀ3] = 44 lM, [cAꢀ4] = 76 lM,
and [cAꢀ5] = 82 lM
published procedure [1, 2] (Fig. 4; Table 1). The CD spectra of the non-cross-linked
peptides A and Aꢀ1–Aꢀ5 were also recorded to examine the effect of the cross-linker
on helix stabilization in the presence of the fluorophores. The helix content of the
non-cross-linked A was 20 %, and that of Aꢀ1–Aꢀ5 was below 14 %. The cross-
linked cA without a fluorophore folded into a stable a-helix, as reported in our
previous study; its helix content was 93 % [1, 2]. Values for the fluorescent, cross-
linked peptides ranged from 55 to 76 %, which were smaller than that of cA. The
fluorophores at the N-termini of the cross-linked peptides cAꢀ1–cAꢀ5 can be
regarded as affecting the helical structures. Nevertheless, the helix content of the
cross-linked peptides was much higher than that of their non-cross-linked
counterparts. Comparing the CD spectra of cAꢀ1 and cAꢀ2, we could speculate
that the alkyl spacer between the N-termini and the phenylethynyl groups affect
their higher-order structures. Although fluorophores should be selected with care to
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Table 1 Helix content of peptides A–Aꢀ5 and cA–cAꢀ5 at 5 °C
Helix content (%) of non-cross-linked peptides
A
Aꢀ1
Aꢀ2
Aꢀ3
Aꢀ4
\5
Aꢀ5
20
14
10
11
14
Helix content (%) of cross-linked peptides
cA
cAꢀ1
cAꢀ2
cAꢀ3
cAꢀ4
cAꢀ5
93
76
61
64
55
56
avoid such aggregation, the high helix content observed here confirms that our
fluorescent, cross-linked peptides can be potent peptide-based probes for biological
analysis.
Conclusion
We have synthesized fluorescent, cross-linked peptides with high helix content. A
variety of fluorophores could be easily attached to the N-termini of helical peptides
by use of fluorophore-based carboxylic acids. In the future, fluorescent, cross-linked
peptides that bind strongly to a specific protein overexpressed in a tumor cell are
expected to be developed.
Acknowledgments This work was partially supported by Grants-in-Aid for Scientific Research
(B) (22350072) and for Young Scientists (B) (23750184) from the Ministry of Education, Culture, Sports,
Science, and Technology (MEXT) of Japan.
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