Two-color fluorescent L-amino acid mimic of tryptophan for probing peptide-nucleic acid complexes

Article abstract of DOI:10.1021/bc300464u

Non-natural amino acids are important tools for site-selective probing of peptide properties and interactions. Here, for the first time a fluorescent l-amino acid, exhibiting excited-state intramolecular proton transfer (ESIPT) and hydration-sensitive dual emission, was synthesized. It is an analogue of l-tryptophan bearing a slightly larger 2-(2-furyl)-3-hydroxychromone aromatic moiety instead of indole. This new amino acid was incorporated through solid-phase synthesis into NC(11-55), the zinc finger domain of the HIV-1 nucleocapsid protein, that exhibits potent nucleic acid chaperone properties. It was substituted for the Trp37 and Ala30 residues, located in the distal finger motif and the linker between the fingers of NC(11-55), respectively. Though the highly conserved Trp37 residue plays a key role in NC(11-55) structure and activity, its substitution for the new fluorescent analogue preserved the folding, the nucleic acid binding and chaperone activity of the peptide, indicating that the new amino acid can conservatively substitute Trp residues. In the presence of oligonucleotides, the Trp37-substituted peptide, but not the Ala30 variant, showed strong changes of the dual emission corresponding to local dehydration. The results are in line with NMR data, suggesting that the fluorescent amino acid interacts similarly to Trp37 with the nucleobases and is thus screened from water. Due to the exceptional sensitivity of its ESIPT fluorophore to hydration in highly polar environment, the new amino acid appears as a promising tool for substituting Trp residues and site-selectively investigating peptide-nucleic acid complexes.

Full text of DOI:10.1021/bc300464u

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Article
Two-color fluorescent L-amino acid mimic of
tryptophan for probing peptide-nucleic acid complexes
Aleksandr V Strizhak, Viktoriia Y Postupalenko, Volodymyr V Shvadchak, Nelly
Morellet, Eric Guittet, Vasyl G Pivovarenko, Andrey S Klymchenko, and Yves Mély
Bioconjugate Chem., Just Accepted Manuscript • Publication Date (Web): 16 Nov 2012
Downloaded from http://pubs.acs.org on November 19, 2012
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Bioconjugate Chemistry
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Twoꢀcolor fluorescent Lꢀamino acid mimic of
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tryptophan for probing peptideꢀnucleic acid complexes
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‡,§
§,
⊥
Aleksandr V. Strizhak,‡,§,† Viktoriia Y. Postupalenko, Volodymyr V. Shvadchak, Nelly Morellet,
∥
Eric Guittet,⊥ Vasyl G. Pivovarenko,†§ Andrey S. Klymchenko,*,§ Yves Mély,*,§
§Laboratoire de Biophotonique et Pharmacologie, UMR 7213 CNRS, Université de Strasbourg, Faculté
de Pharmacie, 67401 Illkirch, France
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†Department of Chemistry, National Taras Shevchenko University of Kyiv, 01601 Kyiv, Ukraine
⊥CNRS UPR 2301, Chimie et biochimie structurales, Institut de Chimie des Substances Naturelles,
Centre de recherche de GifꢀsurꢀYvette, 91190 GifꢀsurꢀYvette, France
RECEIVED DATE (to be automatically inserted after your manuscript is accepted if required
according to the journal that you are submitting your paper to)
‡ AVS and VYP equally contributed to this work
*CORRESPONDING AUTHOR FOOTNOTE: andrey.klymchenko@unistra.fr ; yves.mely@unistra.fr;
Tel: +33 368 854255, +33 368 854263. Fax: +33 368 854313.
RUNNING TITLE: fluorescent Lꢀamino acid
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ABSTRACT
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Nonꢀnatural amino acids are important tools for siteꢀselective probing of peptide properties and
interactions. Here, for the first time a fluorescent Lꢀamino acid, exhibiting excitedꢀstate intramolecular
proton transfer (ESIPT) and hydrationꢀsensitive dual emission, was synthesized. It is an analogue of Lꢀ
tryptophan bearing a slightly larger 2ꢀ(2ꢀfuryl)ꢀ3ꢀhydroxychromone aromatic moiety instead of indole.
This new amino acid was incorporated through solid phase synthesis into NC(11ꢀ55), the zinc finger
domain of the HIVꢀ1 nucleocapsid protein, that exhibits potent nucleic acid chaperone properties. It
was substituted for the Trp37 and Ala30 residues, located in the distal finger motif and the linker
between the fingers of NC(11ꢀ55), respectively. Though the highly conserved Trp37 residue plays a key
role in NC(11ꢀ55) structure and activity, its substitution for the new fluorescent analogue preserved the
folding, the nucleic acid binding and chaperone activity of the peptide, indicating that the new amino
acid can conservatively substitute Trp residues. In the presence of oligonucleotides, the Trp37ꢀ
substituted peptide, but not the Ala30 variant, showed strong changes of their dual emission
corresponding to local dehydration. The results are in line with NMR data, suggesting that the
fluorescent amino acid interacts similarly to Trp37 with the nucleobases and is thus screened from
water. Due to the exceptional sensitivity of its ESIPT fluorophore to hydration in highly polar
environment, the new amino acid appears as a promising tool for substituting Trp residues and siteꢀ
selectively investigating peptideꢀnucleic acid complexes.
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Introduction
Nonꢀnatural amino acids are important tools to investigate peptides and proteins, since they can be
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incorporated at any position, and thus, serve as local probes for siteꢀselective monitoring of protein
properties and functions. Probably, the most popular ones are those bearing fluorine (1-4) and dyes (5-7)
used with NMR and fluorescence techniques, respectively. Fluorescent amino acids are particularly
attractive, due to the ultimate sensitivity of fluorescenceꢀbased methods. Since the natural fluorescent
amino acid tryptophan suffers from poor fluorescence properties, continuous efforts have been done to
design amino acids with improved fluorophores (5-7). Of special interest in this respect are
environmentꢀsensitive (or solvatochromic) fluorophores, which change their emission properties in
response to changes in their environment (7, 8). Biomolecular interactions commonly decrease the
polarity at the labeling site due to screening from water and thus, can be readily monitored by
environmentꢀsensitive fluorescent amino acids (7). For instance, a Prodanꢀbased amino acid was used
for monitoring the interaction of Sꢀpeptide with ribonuclease S (9) and δꢀopioid receptor with
antagonists (10) and to estimate the local dielectric constant of the B1 domain of the staphylococcal
protein G (5). Moreover, an amino acid based on 4ꢀdimethylaminophthalimide (4ꢀDMAP) fluorophore
was applied to sense binding of labeled octapeptides to the 14ꢀ3ꢀ3bp protein (11). Finally, improved
analogues of 4ꢀDMAP, such as 6ꢀdimethylaminonaphthalimide, were recently applied to investigate the
SH2 phosphotyrosine binding domains (12), major histocompatibility complexes (MHC) at the cell
surface (7) and peptideꢀcalmodulin interactions (13). Despite the importance of environmentꢀsensitive
fluorescent amino acids in biomolecular research, their examples are limited.
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Moreover, most examples are focused on proteinꢀprotein interactions (7), while applications of
fluorescent amino acids for sensing peptideꢀoligonucleotide (ODNs) interactions have been poorly
explored (Figure 1A). To address this problem, we selected 3ꢀhydroxychromone (3HC) fluorophores
which undergo excitedꢀstate intramolecular proton transfer (ESIPT) (14), resulting in the emission of
both a normal (N*) excited state and an ESIPT product tautomer (T*) (Figure 1B). The dual emission of
3HC dyes is highly sensitive to polarity and Hꢀbonding interactions (8, 15-23). The 2ꢀ(2ꢀfuryl)ꢀ3ꢀ
hydroxychromone (FHC) label is particularly interesting due to the high sensitivity of its dual emission
to polar environments (15, 17, 24, 25). Being attached to the Nꢀterminus of peptides and oligocations, it
shows strong changes in its dual emission upon interaction with ODNs (26, 27), and allows
quantification of water content in its surrounding (28). This prompted us to develop an Lꢀamino acid
based on the FHC fluorophore.
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Figure 1. Monitoring of peptide/oligonucleotide interaction using solvatochromic fluorescent amino
acid. (A) Principle: the interaction of the labeled peptide (in green) with oligonucleotides changes the
fluorescence intensity and color of the label. (B) ESIPT reaction in 2ꢀ(2ꢀfuryl)ꢀ3ꢀhydroxychromone
dye.
As a target protein for labeling, we selected the nucleocapsid protein (NC) of the Human
Immunodeficiency Virus, type 1 (HIVꢀ1), which plays a crucial role in the viral life cycle (29, 30). NC
is a small (55 amino acids) basic protein, characterized by two rigid zinc fingers connected by a flexible
basic linker and flanked by poorly folded Nꢀ and Cꢀterminal basic domains (31, 32). NC binds both
specifically (33-38) and nonꢀspecifically (35, 39-41), to a large range of nucleic acid sequences.
Specific binding is mainly mediated through the folded finger motifs, with a key role being played by
the hydrophobic residues forming a hydrophobic platform at the top of the folded fingers (34, 36, 38).
This hydrophobic plateau plays also a key role in the nucleic acid chaperone properties of NC, which
enable to direct the structural rearrangement of ODNs into their most stable conformation and promote
the annealing of complementary ODN sequences through specific pathways (42-44). Within this
hydrophobic plateau, the Trp37 residue plays a particularly important role, since its substitution by a
nonꢀaromatic residue leads to a dramatic decrease in ODN affinity (35, 40, 45), a complete loss in the
highly specific ODN destabilization component of the NC chaperone properties (40, 42) as well as a
complete loss of HIVꢀ1 infectivity (46). In the present work, we synthesized an Lꢀamino acid analogue
based on 2ꢀ(2ꢀfuryl)ꢀ3ꢀhydroxychromone dye through an original route from Lꢀtyrosine. It was then
incorporated at two different positions of the NC(11ꢀ55), inside and outside of its fingers, which
allowed us to siteꢀselectively investigate its interaction with ODNs. Remarkably, substitution of Trp
residue for the new amino acid marginally alters the folding as well as the nucleic acid binding and
chaperone activity of the peptide, indicating that this new amino acid could be used as a Trp mimic. The
ODNs selected for binding (Figure S1, Supporting Information) to the labeled peptides were SL2 and
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Bioconjugate Chemistry
SL3, two stemꢀloops of the HIVꢀ1 RNA encapsidation sequence (33, 35, 38) and ꢀP(ꢀ)PBS, the cDNA
copy of the primer binding site, deleted of its 5’ overhang (36). These three ODNs bind NC with high
affinity and 1:1 stoichiometry, and their structures in complex with NC are known (34, 36, 38). The
results showed that this amino acid allows probing protein sites responsible for DNA binding.
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Materials and methods
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All the solvents and chemicals were purchased from SigmaꢀAldrich Chemical Company. For
absorption and fluorescence studies, the solvents were of spectroscopic grade. ꢀP(ꢀ)PBS DNA, SL2 and
SL3 RNAs were synthesized and HPLCꢀpurified by IBA GmbH (Germany). Concentrations of the
ODNs were calculated from their absorbance using the molar extinction coefficients at 260 nm provided
by the supplier.
NMR spectra were recorded on a 400 MHz and 500 MHz BrukerAvance III, BBFO+ at room
temperature. Mass spectra were obtained on a Bruker HCT Ultra and Agilent Technologies Accurateꢀ
Mass QꢀTOF LC/MS 6520 mass spectrometers.
Synthesis of Fmoc-3HCaa. The synthesis of the compounds 1 and 2 (Scheme S1) was performed as
described previously (47, 48).
N-(tert-butoxycarbonyl)-3-[(2E)-3-(2-furyl)prop-2-enoyl]-L-tyrosine (3).6.425 g (19.89 mmol) of 3ꢀ
acetylꢀNꢀ(tertꢀbutoxycarbonyl)ꢀLꢀtyrosine 2 were dissolved in 48 ml of ethanol upon stirring under Arꢀ
atmosphere. To the reaction mixture, 48 ml of degassed 25% solution of sodium hydroxide was added
upon cooling in cold water bath. Then, 2.409 g of furfural (25.09 mmol, 1.26 eq.) were added and the
mixture was stirred for 12 h at RT under Ar. Additional 0.5 g of furfural was added and the mixture was
stirred overnight. The reaction mixture was acidified with HCl and diluted twice with water. The formed
precipitate was filtered and washed with diluted ethanol and water, dried and used in the next step
without further purification. Yield 7.62 g (95%) of chalcone 3 as a yellow powder. 1HꢀNMR (500 MHz,
MeOD) δ 1.33 (s, 9H), 2.87ꢀ2.92 (dd, J = 13.9 Hz, J = 8.85 Hz, 1H), 3.16ꢀ3.19 (dd, J = 13.9 Hz, J =
4.43 Hz, 1H), 4.31ꢀ4.34 (dd, J = 8.85 Hz, J = 4.43 Hz, 1H), 6.61ꢀ6.62 (dd, J = 3.16 Hz, J = 1.89 Hz,
1H), 6.87ꢀ6.89 (d, J = 8.21 Hz, 1H), 6.94 (d, J = 3.8 Hz, 1H), 7.39ꢀ7.41 (dd, J = 8.22 Hz, J = 1.9 Hz,
1H), 7.64ꢀ7.71 (dd, J = 15.16 Hz, J = 4.42 Hz, 2H), 7.71 (d, J = 1.9 Hz, 1H), 7.86ꢀ7.87 (d, J = 1.9 Hz,
1H). 13CꢀNMR (500 MHz, MeOD) δ 28.66, 38.11, 55.49, 80.47, 114.01, 118.33, 118.84, 119.02,
120.97, 129.45, 131.65, 132.31, 138.58, 147.17, 152.98, 157.68, 163.09, 175.69, 194.74 (two carbon
signals are absent due to incidental equivalence). HRMS (EI) m/z calcd for C21H23NO7 401.14745,
found: 401.147.
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N-(tert-butoxycarbonyl)-3-[2-(2-furyl)-3-hydroxy-4-oxo-4H-chromen-6-yl]-L-alanine
(4).7.62
g
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(19 mmol) of chalcone 3 was dissolved in 75 ml of ethanol, cooled in ice bath. Then, 75 ml of 1M
solution of sodium hydroxide was added with stirring. Next, 4.73 ml (41.8 mmol) of 30% hydrogen
peroxide was added and the mixture was stirred in ice bath for 4 h. The reaction was monitored by TLC
(silica, EtOAc/MeOH 9:1). After completing the reaction, the mixture was acidified and the formed
precipitate was filtered. Yield of the crude 3ꢀhydroxychromone 4 was 3.56 g (45%). It was purified to
about 95% purity by crystallization several times from toluene and then from acetonitrile. Final yield ~
1.5 g (20%) of yellow crystals. 1HꢀNMR (500 MHz, MeOD) δ 1.33 (s, 9H), 2.98ꢀ3.03 (dd, J = 13.9 Hz,
J = 9.48 Hz, 1H), 3.26ꢀ3.30 (m, J = 5.05 Hz, 1H), 4.36ꢀ4.39 (dd, J = 9.48 Hz, J = 5.05 Hz, 1H), 6.67 (m,
1H), 7.33 (d, J = 3.16 Hz, 1H), 7.51ꢀ7.53 (d, J = 8.85 Hz, 1H), 7.60ꢀ7.62 (d, J = 8.85 Hz, 1H), 7.78 (s,
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1H), 7.97 (d, J = 1.27 Hz, 1H). CꢀNMR (500 MHz, MeOD) δ 28.6, 38.27, 56.26, 80.52, 113.58,
116.91, 119.29, 122.83, 126.26, 135.77, 135.97, 138.44, 141.29, 145.86, 146.08, 155.09, 157.77,
173.81, 175.11 (two carbon signals are absent due to incidental equivalence). HRMS (EI) m/z calcd for
C21H21NO8 415.12672, found: 415.12659.
3-[2-(2-furyl)-3-hydroxy-4-oxo-4H-chromen-6-yl]-L-alanine hydrochloride (5). 944 mg of 3ꢀ
hydroxychromone 4 was dissolved in 10 ml of dioxane. Then, 10 ml of conc. HCl was added to the
reaction mixture in cold water bath. After 5ꢀ10 min, the bath was removed and the mixture was stirred
for 2ꢀ3 h. The reaction mixture was concentrated under reduced pressure to give the desired product 5 as
a dark yellow powder, yield 0.769 g (96%). 1HꢀNMR (500 MHz, MeOD) δ 3.28ꢀ3.34 (dd, J = 14.56 Hz,
J = 7.78 Hz, 1H), 3.42ꢀ3.48 (dd, J = 14.56 Hz, J = 5.77 Hz, 1H), 4.34ꢀ4.37 (dd, J = 7.53 Hz, J = 5.77
Hz, 1H), 6.7ꢀ6.72 (dd, J = 3.52 Hz, J = 1.76 Hz, 1H), 7.37ꢀ7.38 (d, J = 3.51 Hz, 1H), 7.63ꢀ7.65 (d, J =
8.79 Hz, 1H), 7.68ꢀ7.71 (dd, J = 8.79 Hz, J = 2.26 Hz, 1H), 7.81ꢀ7.82 (d, J = 1.00 Hz, 1H), 8.05ꢀ8.06 (d,
J = 1.76 Hz, 1H). 13CꢀNMR (500 MHz, MeOD) δ 36.74, 54.95, 113.70, 117.23, 120.29, 123.34, 126.81,
132.56, 135.79, 138.64, 141.67, 145.83, 146.33, 155.71, 171.00, 173.74. HRMS (EI) m/z calcd for
C16H13NO6 315.07429, found: 315.07419.
N-[(9H-fluoren-9-ylmethoxy)carbonyl]-3-[2-(2-furyl)-3-hydroxy-4-oxo-4H-chromen-6-yl]-L-alanine
(6). 614 mg (1.744 mmol) of amino acid hydrochloride 5 was dissolved in a solution of 500 mg (5.95
mmol) of sodium bicarbonate in 10 mL of water. Then, 30 mL of acetonitrile and 588 mg (1 eq.) of
FmocOSu were added and the mixture was stirred for 24 h. The reaction mixture was filtered and the
precipitate was dissolved in hot water (about 100 mL per 1 g) with further acidification with HCl. Yield
900 mg (96%) of yellow powder. 1HꢀNMR (500 MHz, DMSOꢀd6) δ 3.00ꢀ3.05 (m, 1H), 3.22ꢀ3.26 (dd, J
= 14.04 Hz, J = 4.27 Hz, 1H), 4.12ꢀ4.19 (m, 3H), 4.26ꢀ4.30 (m, 1H), 6.77ꢀ6.78 (dd, J = 3.05 Hz, J =
1.83 Hz,1H), 7.21ꢀ7.25 (m, 2H), 7.27ꢀ7.28 (d, J = 3.05 Hz, 1H), 7.34ꢀ7.37 (t, J = 7.32 Hz, 2H), 7.57ꢀ
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7.58 (d, J = 7.32 Hz, 2H), 7.59ꢀ7.61 (d, J = 8.55 Hz, 1H), 7.68ꢀ7.70 (d, J = 8.55 Hz, 1H), 7.75ꢀ7.76 (d, J
= 8.55 Hz, 1H), 7.82ꢀ7.83 (d, J = 7.32 Hz, 2H), 8.01 (s, 1H), 8.04 (s, 1H), 9.88 (s, 1H), 12.76 (br s, 1H).
13CꢀNMR (500 MHz, DMSOꢀd6) δ 35.80, 46.54, 55.39, 65.64, 112.82, 115.27, 118.03, 120.04, 121.56,
124.95, 125.11, 125.18, 126.98, 127.55, 134.68, 137.20, 139.19, 140.63, 143.67, 144.09, 145.17,
152.96, 156.01, 171.78, 173.07(six carbon signals are absent due to incidental equivalence). HRMS (EI)
m/z calcd for C31H23NO8 537.14237, found: 537.14187.
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Proof of optical purity. To test the enantiomeric purity of the obtained chromoneꢀamino acid
derivative, we prepared the dibenzyl etherꢀester of the amino acid, which was acylated by two
enantiomers of Mosher’s acid chloride to give compounds 9 and 10 (Scheme S1). The NMR spectra of
the diastereomeric Mosher amides were then compared. No signal of the second diastereomer was found
in each spectrum.
Benzyl
3-[3-(benzyloxy)-2-(2-furyl)-4-oxo-4H-chromen-6-yl]-N-[(2S)-3,3,3-trifluoro-2-methoxy-2-
phenylpropanoyl]-L-alaninate (9).1HꢀNMR (400 MHz, CDCl3) δ 3.14ꢀ3.19 (dd, J = 14.24 Hz, J = 7.12
Hz, 1H), 3.20 (s, 3H), 3.24ꢀ3.29 (dd, J = 14.24 Hz, J = 5.60 Hz, 1H), 4.89ꢀ4.94 (dd, J = 14.25 Hz, J =
6.62 Hz, 1H), 5.05ꢀ5.08 (d, J = 12.21 Hz, 1H), 5.11ꢀ5.14 (d, J = 12.21 Hz, 1H), 5.24 (s, 2H), 6.48ꢀ6.49
(dd, J = 3.56 Hz, J = 1.52 Hz,1H), 7.18ꢀ7.44 (m, 17H), 7.59 (d, J = 1.01 Hz, 1H), 7.96 (d, J = 1.53 Hz,
1H).19FꢀNMR (376 MHz, CDCl3) δ ꢀ69.5 (9), ꢀ72.03 (Mosher acid).
Benzyl
3-[3-(benzyloxy)-2-(2-furyl)-4-oxo-4H-chromen-6-yl]-N-[(2R)-3,3,3-trifluoro-2-methoxy-2-
phenylpropanoyl]-L-alaninate (10).1HꢀNMR (400 MHz, CDCl3) δ 3.02ꢀ3.07 (dd, J = 14.05 Hz, J = 7.53
Hz, 1H), 3.16ꢀ3.21 (dd, J = 14.05 Hz, J = 5.52 Hz, 1H), 3.35 (d, J = 1.50 Hz, 3H), 4.94ꢀ5.00 (m, J =
5.52 Hz, J = 7.28 Hz,1H), 5.07ꢀ5.10 (d, J = 12.05 Hz, 1H), 5.14ꢀ5.17 (d, J = 12.05 Hz, 1H), 5.20ꢀ5.23
(d, J = 10.54 Hz, 1H), 5.23ꢀ5.26 (d, J = 10.54 Hz, 1H), 6.49ꢀ6.51 (dd, J = 3.52 Hz, J = 1.76 Hz,1H),
7.01ꢀ7.04 (dd, J = 8.79 Hz, J = 2.26 Hz, 1H), 7.05ꢀ7.07 (d, J = 8.53 Hz, 1H), 7.17ꢀ7.32 (m, 15H), 7.42ꢀ
7.45 (dd, J = 8.29 Hz, J = 1.76 Hz, 1H), 7.60 (dd, J = 1.76 Hz J = 0.75 Hz, , 1H), 7.84 (d, J = 2.26 Hz,
1H). 19FꢀNMR (376 MHz, CDCl3) δ ꢀ69.41(10), ꢀ72.03 (Mosher acid).
Peptide synthesis. The labeled NC(11ꢀ55) peptides were synthesized by solid phase peptide synthesis
on a 433A synthesizer (ABI, Foster City, CA), according to a previously established protocol (49). The
synthesis was performed at a 0.1 mmol scale using the standard fluorenylmethoxycarbonyl (Fmoc)ꢀ
amino acidꢀcoupling protocol starting from 0.44 mmol/g Wang LL resin. At selected positions of the
peptide, the fluorescent amino acid analogue was incorporated by the following procedure. In a flask, 2ꢀ
4 mole equivalents of the Fmocꢀ3HCaa were mixed with 4 eq. of HBTU/HOBt coupling solution (in
DMF) and 5 eq. of DIEA. This mixture was immediately added to the peptidylresin and shaked at 37°C
for 12 h. Then, the resin was washed with 1ꢀmethylꢀ2ꢀpyrrolidone (NMP) and peptide synthesis was
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continued on the synthesizer. At the end of the synthesis, the Fmocꢀdeprotected peptidylresin was
isolated and washed with NMP, methanol and dichloromethane.
1
2
3
Cleavage of the peptidylresin and deprotection were performed for 2 h using a 10 mL trifluoroacetic
acid (TFA) solution containing water (5%, v/v), phenol (2%, w/v), thioanisole (5%, v/v),
triisopropylsilane (2.5%, v/v) and ethanedithiol (2.5%, v/v). The peptide was precipitated using iceꢀcold
diethyl ether and pelleted by centrifugation. The pellet was airꢀdried for approx. 15 minutes, solubilized
with aqueous TFA (0.05 %, v/v) and lyophilized. Purification by HPLC was carried out on a C8 column
(uptisphere 300A, 5ꢁm; 250Х10, Interchim, France) in water/acetonitrile mixture containing 0.05%
TFA with a linear gradient 10 to 35% of acetonitrile for 30 min and monitored at 210 and 360 nm (3HC
dye absorption). Obtained peptides were characterized by ESIꢀMS analysis. NC(11ꢀ55)ꢀW37ꢀ3HCaa
peptide: calculated M = 5245.40, found [M+7H]7+ = 750.64 corresponding to M = 5245.38 after
deconvolution; NC(11ꢀ55)ꢀA30ꢀ3HCaa peptide: calculated M = 5360.43, found [M+7H]7+ = 767.06
corresponding to M = 5360.37 after deconvolution.
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
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30
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32
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34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Preparation of Zn-bound peptides: Lyophilized labeled peptides were dissolved in water (≈ 0.5 mg
in 500 ꢁL). Then, about 10 ꢁL of this solution was used to determine the peptide concentration using an
extinction coefficient of 15,000 Mꢀ1×cmꢀ1 at 350 nm. Next, 2.2 molar equivalents of ZnSO4 were added
to the peptide and pH was raised to its final value, by adding buffer. This last step was done only at the
end to prevent peptide oxidization. Noticeably, a large excess of Zn2+ ions should be avoided since this
ion could affect the 3HC fluorescence.
NMR structural studies: The two NC(11ꢀ55) conjugates at 0.5 mM concentration in neat water, pH
6.5 at 293 K were studied on an Avance Bruker spectrometer operating at 600.13 MHz equipped with a
cryoprobe. A mixing time of 200 ms and a water gate pulse program for water signal suppression in
NOESY experiments were used.
Spectroscopic measurements: Unless otherwise indicated, the experiments were performed in 10
mM phosphate buffer, pH 6.5, 30 mM NaCl, at 20°C. This low pH was used to prevent deprotonation of
the 3ꢀhydroxy group of the 3HC amino acid. Absorption spectra were recorded with a Cary 4000 UVꢀ
visible spectrophotometer (Varian). Fluorescence spectra were recorded on FluoroMax3 and FluoroLog
spectrofluorimeters (Jobin Yvon) equipped with thermostated cell compartments. Fluorescence spectra
were corrected for Raman scattering. Quantum yields were calculated using quinine sulphate in 0.5 M
sulphuric acid (quantum yield, φ = 0.577) as a reference (50). Excitation wavelength was 350 nm for
3HCaa.
To determine the affinity of the labeled peptides for the ODNs, fixed amounts of the ODN were
titrated with peptides by monitoring the twoꢀband fluorescence of the labeled peptides. For each data
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Bioconjugate Chemistry
point, the emission of the same concentration of labeled peptide in buffer was subtracted from the signal
measured in the presence of the ODN. Affinity constants were determined from direct fitting of the
corrected signal to the rewritten Scatchard equation:
1
2
3
4
5
6
7
8
(1+ (P + nNt )Ka ) − (1+ (P + nNt )Ka )2 − 4PnNt Ka2
(I0 − It )
t
t
t
(1)
I = I0 −
x
Nt
2Ka
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
where I and It are the signal at a given and a saturating peptide concentration, respectively, I0 is the
signal in the absence of peptide, Nt is the total ODN concentration, Pt is the total concentration of
peptide, Ka is the apparent affinity constant, n is the number of binding sites. The parameters were
recovered from nonꢀlinear fits of equation (1) to experimental datasets using the MicrocalOriginTM 6.0
software.
Peptide activity tests. The ability of the labeled peptides to destabilize the secondary structure of
ODNs was tested using cTAR DNA, the complementary sequence of the transactivation response
element, involved in the minus strand DNA transfer during reverse transcription. To this end, we used a
cTAR sequence labeled at its 3’ and 5’ ends by 5(6)ꢀcarboxytetramethylrhodamine (TMR) and 4ꢀ(4′ꢀ
methylaminophenylazo) benzoic acid (Dabcyl), respectively. In the absence of NC, cTAR is mainly in a
nonꢀfluorescent closed form where the TMR and Dabcyl labels are close together, giving excitonic
coupling (51). The destabilization was measured through the fluorescence intensity increase of TMR in
the labeled cTAR as a function of NC(11ꢀ55) concentration (43, 52).
The ability of the labeled peptides to promote the annealing of cTAR (55 base hairpin DNA) with its
complementary dTAR sequence was compared to that of the unlabeled NC peptide (53). The kinetic
measurements were performed under pseudoꢀfirstꢀorder conditions by using unlabeled dTAR at a
concentration which was 30ꢀfold higher than the concentration of cTAR labeled with
carboxytetramethylrhodamine (TMR) at the 5′ end and with 5/6ꢀcarboxyfluorescein (Fl) at the 3′ end
(53). Excitation and emission wavelengths were 480 and 520 nm, respectively, for monitoring the Fl
fluorescence. All reported concentrations correspond to those after mixing. To avoid high local
concentrations during mixing, both reactants were mixed at the same volume. Peptides were added to
each reactant separately at a peptide:ODN ratio of 3:1, and then, the reaction was initiated by mixing the
peptideꢀcoated ODNs together. Experiments were performed in 25 mM Tris, 30 mM NaCl, 0.2 mM
MgCl2, pH 7.5 at 20 ºC.
Results and discussion
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To obtain the Lꢀamino acid bearing the 3HC fluorophore, we started with Lꢀtyrosine that was
converted into corresponding acetophenone 1 using a described procedure (47, 48) based on Fries
reaction (Scheme 1). Then, the amino group was protected with the Boc group, and the product 2 was
condensed with furaldehyde in the presence of a base. The obtained chalcone 3 was further converted
into a 3HC derivative (Bocꢀ3HCaa) using AlgarꢀFlynnꢀOyamada reaction. Then, the Boc group was
removed to obtain the desired amino acid (3HCaa). Finally, it was protected with the Fmoc group,
which is required for solid phase peptide synthesis. We checked that the basic conditions used in the
synthesis do not alter the optical purity of the obtained amino acid. For this purpose, the obtained Bocꢀ
protected amino acid was first benzylated, then the amino group was deprotected and finally modified
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
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24
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26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
19
with (R)ꢀ and (S)ꢀMosher agents (Scheme S2, Supporting Information). The FꢀNMR spectra of the
two products 9 and 10 showed only one fluorine peak corresponding to pure diastereomers, indicating
that the synthesis did not produce any racemization. This conclusion was confirmed by 1HꢀNMR spectra
of these two diastereomers.
Scheme 1. Synthesis of the fluorescent amino acid and the labelled peptides. The substituted positions
of the NC(11ꢀ55) peptide are shown by blue arrows.
Then, the spectroscopic properties of the new amino acid (Bocꢀ3HCaa) were characterized in different
solvents (Figure 2, Table S1, Supporting Information). The absorption and emission maxima of Bocꢀ
3HCaa were remarkably close to those reported previously for the basic fluorophore FHC (Table S1)
(24), indicating that 6ꢀsubstitution in this fluorophore did not change its spectroscopic properties. The
dual emission of Bocꢀ3HCaa was found to depend strongly on solvent polarity, showing an increase of
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Bioconjugate Chemistry
the relative intensity of the N* band in more polar solvents (Figure 2A) due to an inhibition of the
ESIPT reaction (8). However, it should be noted that the ratio of the two emission bands of Bocꢀ3HCaa
in polar protic solvents is considerably lower than that of the parent fluorophore, indicating that the
Bocꢀprotected amino acid group screens partially the fluorophore from polar solvents. Therefore, to
estimate the local water concentration, we used the calibration curve log(N*/T*) vs water concentration
previously reported for the parent FHC dye (Figure 2B) (28).
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
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31
32
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34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
0.5
0.0
Dioxane
DMF
Lin. fit
2.1
1.8
1.5
1.2
0.9
0.6
0.3
0.0
A
B
-0.5
0
10 20 30 40 50 60
[H2O], M
Buffer
MeOH
EtOH
MeCN
400
450
500
550
600
650
Wavelength, nm
Figure 2. Fluorescence spectra of the Bocꢀ3HCaa derivative in different solvents (A) and dependence of
log(N*/T*) of 2ꢀ(2ꢀfuryl)ꢀ3HC (FHC) on water concentration in organic solvents based on data from
(28) (B). In (A), 10 mM phosphate buffer, 30 mM NaCl, pH = 6.5 was used. All the spectra were
normalized at the T* band. Excitation wavelength was 350 nm. Concentration of Bocꢀ3HCaa was 1 ꢁM.
Using the Fmocꢀprotected 3HCaa, we then synthesized two labeled NC(11ꢀ55) peptides: NC(11ꢀ55)ꢀ
A30ꢀ3HCaa and NC(11ꢀ55)ꢀW37ꢀ3HCaa, where the fluorescent amino acid substituted Ala30 and
Trp37 residues, located in the linker and the distal zinc finger, respectively (Scheme 1). Importantly, the
3HCaa was compatible with the standard protocols of peptide synthesis and purification. The NOESY
spectra show that the folding of the zinc fingers is maintained in the two labeled NC(11ꢀ55) peptides
(Figure S2, supporting information), since similar medium and long distance NOEs were found as for
the native peptide. Thus, the incorporated amino acid analogue 3HCaa does not significantly affect the
native folding in the two labeled peptides.
Next, we compared the chaperone activity of the labeled peptides with that of the native peptide. First,
we checked the ability of the peptides to promote destabilization of DNA stemꢀloops, such as for
instance the cTAR sequence involved in the first strand transfer of reverse transcription (40). This test
is particularly important, because substitution of Trp37 in NC(11ꢀ55) for a nonꢀaromatic amino acid
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results in the loss of this component of NC chaperone activity (40). Remarkably, NC(11ꢀ55)ꢀW37ꢀ
3HCaa showed high destabilization activity, nearly identical to that of the native peptide, while NC(11ꢀ
55)ꢀA30ꢀ3HCaa was less active (Figure 3). Secondly, we tested the ability of the labeled peptides to
promote the annealing of two complementary sequences. Both labeled peptides exhibited similar
activities comparable to that of the native peptide (Figure S3, supporting information), indicating that
this component of NC chaperone activity is also preserved. The preserved chaperone activity of NC(11ꢀ
55)ꢀW37ꢀ3HCaa shows that the new amino acid does not affect the peptide folding and can functionally
substitute the Trp37 residue. We could explain this outstanding feature of the new amino acid by the
two factors. Firstly, similarly to Trp, 3HCaa bears a flat and relatively small aromatic moiety. Secondly,
the 3HC moiety, due to its compact structure, can easily intercalate within the nucleobases, as it was
shown in different recent reports (26, 27, 54). This property allows 3HC moiety to mimic the interaction
of Trp37 indole moiety with nucleobases in the complexes of the peptide with nucleic acid sequences
(34-36, 38).
1
2
3
4
5
6
7
8
9
10
11
12
13
14
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48
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50
51
52
53
54
55
56
57
58
59
60
30
25
20
15
10
5
Q
Q
TMR
TMR
0
0
3
6
9
12
15
NC/cTARbeacon
Figure 3. Destabilization of doublyꢀlabelled cTAR DNA by NC(11ꢀ55) (squares), NC(11ꢀ55)ꢀW37ꢀ
3HCaa (disks) and NC(11ꢀ55)ꢀA30ꢀ3HCaa (triangles). Titration of 100 nM Dabcylꢀ5’ꢀcTARꢀ3’ꢀTMR
by NC peptides was performed in 25 mM TRIS (pH 7.5), 30 mM NaCl and 0.2 mM MgCl2. To monitor
TMR fluorescence, excitation wavelength was set at 520 nm.
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Bioconjugate Chemistry
1
2
3
Table 1. Spectroscopic properties of the 3HCaaꢀlabeled NC(11ꢀ55) peptides in complexes with ODNs.a
4
5
6
7
8
9
Oligoꢀ
λABS λN*
nm nm
364 426
λT*
QY,
%
Peptide
N*/T* [H2O], M
WA
nucleotide
ꢀ
nm
520 0.57
526 0.46
523 0.57
523 0.54
522 0.61
534 0.33
531 0.29
533 0.24
31
27
30
30
32
23
21
18
0.56
0.49
0.54
0.54
0.58
0.41
0.38
0.32
3.7
2.5
2.5
2.5
9.0
5.1
4.0
4.8
ꢂP(ꢀ) PBS 364 425
NC(11ꢀ55)ꢀ
A30ꢀ3HCaa
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
SL3
SL2
ꢀ
363 426
363 426
366 427
ꢂP(ꢀ) PBS 367 425
NC(11ꢀ55)ꢀ
W37ꢀ3HCaa
SL3
SL2
368 427
368 425
a λABS, λN* and λT* are the maxima of absorption, N* and T* emission bands, respectively; N*/T* is
the intensity ratio of the two emission bands measured at the peak maxima (error ±5%); [H2O] is the
water concentration estimated using a method reported elsewhere (error ±1M);(28) WA is water access
coefficient (28); QY is the fluorescence quantum yield. Excitation wavelength was 350 nm.
Measurements were done in 10 mM phosphate buffer, 30 mM NaCl, pH 6.5. Peptide and ODN
concentrations were 1 ꢁM.
Labeled peptides in aqueous solutions (Figure 4) showed dual emission, characteristic of 3HC dyes,
where the shortꢀ and longꢀwavelength bands can be assigned to N* and T* forms, respectively. The
observed intensity ratio of the two emission bands, N*/T*, is close to that for the FHC fluorophore in
ethanol (Tables 1 and S1, Supporting Information), indicating that the 3HCaa fluorophore is partially
screened from water by the peptide in its folded form. Using a recently developed method (28), the local
water concentration [H2O]L of the 3HCaa solvation shell in both peptides was estimated around 31ꢀ32
M (Table 1), which corresponds to a water access coefficient WA for the probe of 0.57 (WA = [H2O]L /
[H2O], where [H2O] = 55.56 M is the concentration of neat water). This estimation indicates that almost
half of the space around the 3HC fluorophore is screened from the bulk water by the peptide backbone.
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1
2
3
4
5
6
Free Peptide
P(-)PBS
+ SL2
+ SL3
1.0
0.5
+
ꢀ
A
7
8
9
0.0
1.0
10
11
12
13
14
15
16
17
18
19
20
21
22
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24
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26
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33
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36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
B
0.5
0.0
400
450
500
550
600
650
Wavelength, nm
Figure 4. Effect of ODN interaction on the fluorescence spectra of the 3HCaaꢀlabeled NC(11ꢀ55)
peptides. Fluorescence spectra of NC(11ꢀ55)ꢀW37ꢀ3HCaa (A) and NC(11ꢀ55)ꢀA30ꢀ3HCaa (B) were
normalized at the T* band. Peptide and ODN concentrations were 1 ꢁM. Measurements were done in 10
mM phosphate buffer, 30 mM NaCl, pH 6.5. Excitation wavelength was 350 nm.
Figure 5. Correlation between the 3D structures of NCꢀODN complexes and the response of the
3HCaaꢀlabeled peptides on ODN binding. 3D structures of NC complexes with SL3 (A) and ∆P(ꢀ)PBS
(B) drawn based on NMR data (34, 36). Only the NC(11ꢀ55) part is shown on the figures. Zn atoms are
represented as yellow spheres, Ala30 and Trp37 residues are in red, and blue, respectively. Fluorescence
intensity ratio N*/T* for NC(11ꢀ55)ꢀA30ꢀ3HCaa (Ala) and NC(11ꢀ55)ꢀW37ꢀ3HCaa (Trp) peptides in
buffer and bound to SL3 or ∆P(ꢀ)PBS (C).
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Bioconjugate Chemistry
On interaction with ODNs SL2, SL3 and ꢀP(ꢀ)PBS, a strong change in the NC(11ꢀ55)ꢀW37ꢀ3HCaa
fluorescence spectrum was observed, with notably a large drop in the N*/T* ratio, a shift of the T*
emission maximum to the red (Figure 5) and a decrease in the fluorescence quantum yield (Table 1).
The drop in the N*/T* ratio can be assigned to a decrease in the local hydration of the 3HCaa
environment, with a water access coefficient WA of the probe dropping to 0.32ꢀ0.41 for the three ODNs
(Table 1). The observed dehydration is likely connected to the interaction of the 3HC fluorophore with
the nucleobases, which leads to an efficient screening of this fluorophore from water (26, 27).
Moreover, this conclusion is substantiated by the observed red shift of the T* band (26, 27) and is in
line with the NMRꢀderived 3D structures of the NCꢀODN complexes (Figure 5), showing that the Trp37
residue strongly interacts with the nucleobases (34-36, 38). Therefore, this observation further confirms
that the 3HCaa can mimic the Trp residue at position 37. In contrast, binding of NC(11ꢀ55)ꢀA30ꢀ3HCaa
to the three ODNs led to either no change (with SL2 and SL3) or a limited decrease (with ꢀP(ꢀ)PBS) in
the N*/T* ratio with a slight decrease in its quantum yield (Table 1) and almost negligible spectral shifts
of the absorption and emission maxima (Table 1). The absence of changes in the N*/T* ratio observed
with the SL2 and SL3 sequences can be rationalized by the large distance between the A30 residue and
the nucleobases in the NCꢀODN complexes (Figure 5). As a consequence, the 3HCaa at position 30 in
the complexes with SL2 and SL3 cannot interact with the ODN and remains highly exposed to water, as
in the free peptide. Finally, the moderate change in the N*/T* ratio observed when NC(11ꢀ55)ꢀA30ꢀ
3HCaa was complexed with ꢀP(ꢀ)PBS is fully consistent with the closer proximity of the Ala30 residue
to the ꢀP(ꢀ)PBS nucleobases, as compared to that in the complexes with SL2 and SL3 (34, 36, 38).
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
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23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Finally, using the changes in the N*/T* ratio or the quantum yield of 3HCaa as an analytical signal of
interaction, we further measured the binding constant of the three labeled peptides to SL3. The obtained
binding constants matched closely that obtained with the native peptide (Table S2, Supporting
Information) (35, 37), indicating that the incorporated 3HCaa probe did not change the binding affinity
of the peptides and could thus be also used for quantification of peptideꢀODN interaction. Since Trp37
was shown to be critical for the high affinity of NC to its target ODNs (40, 42, 45), this indicates that
the substitution of Trp37 by 3HCaa does not alter the ability of NC to recognize its ODN targets. The
results are in line with the peptide activity tests and NMR structural data, providing a clear background
for considering 3HCaa as new amino acid analogue that can readily substitute Trp even at the places
where it plays a crucial role in the peptide.
Though the N*/T* ratio of 3HCaa is mostly controlled by the environment polarity and hydration, we
could not exclude possible quenching effects, which are frequently observed for dyes interacting with
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nucleobases (35, 41). Indeed, the N*/T* ratio could decrease for instance due to a more efficient
quenching of the N* state compared to the T* state, as a consequence of static or dynamic quenching by
the neighbor nucleobases. However, we expect this effect to be minor in the probe response because the
red shift of the T* band produced by peptideꢀDNA interaction correlates well with the changes in the
N*/T* ratio (Table 1). As this red shift is directly linked to the dehydration of the dye environment (26,
27, 55) and not to quenching, the ratio changes appear mainly driven by the dehydration within the
peptideꢀDNA complex. Moreover, as the N* and T* states are coupled by the ESIPT reaction, the
quenching of the N* state should influence the N*/T* ratio to a less significant extent, compared to the
case where the two states are independent.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Table 2. Spectroscopic properties and response to environment of the new 3HC amino acid in
comparison to other amino acid analogues.a
Fluoroꢀ
phore
λ
abs (nm) ε (Mꢀ1
λem (nm) λem (nm)
ꢀλ
Amino acid
Refs
EtOH
cmꢀ1)
EtOH
Water
(nm)
Indole
FHC
280
5500
342
355
13
(56)
O
O
O
OH
O
HO
360
16000
18400
6500
531
496
534b
427
531
562
ꢀ104
ꢀ
NH2
(5, 9,
57)
Prodan 360
35
28
4ꢀ
(11,
58)
408
DMAP
a λabs and λem – absorption and emission maxima of the fluorophores in the corresponding solvent; ε ꢀ
absorption coefficient of the fluorophore in ethanol; ꢀλ ꢀ shift of the emission maximum in response to
changes in the solvent polarity (water vs ethanol); b data in methanol.
Compared to Trp, the new fluorophore features strongly red shifted absorption and emission (Table
2), allowing more convenient spectroscopic studies in biological samples with less background noise
and photoꢀdamage. Moreover, it preserves its fluorescence on peptide binding to nucleic acids, while
Trp is frequently quenched (35, 41). Moreover, due to ESIPT, it presents much higher sensitivity to the
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environment in highꢀpolarity range, so that it almost switches its fluorescence from dominant T*
emission in ethanol (at 531 nm) to dominant N* emission in water (at 427 nm), while Trp in these
conditions changes its emission maximum only by a few nm (56). This exclusive environment
sensitivity places it in front of all other existing fluorescent amino acid analogues of similar small size,
based on Prodan and 4ꢀDMAP (Table 2). Indeed, for highly polar environments (alcohols and water),
the fluorophore of the new amino acid (FHC) is about 3ꢀfold more sensitive to solvent polarity
(hydration) as compared to Prodan and 4ꢀDMAP. Moreover, the FHC provides an opportunity to
quantitatively evaluate the hydration in the site of interaction between the two biomolecules. These
exclusive properties originate from the strong effect of Hꢀbonding solvents on the ESIPT reaction in
these derivatives (15). As peptides and nucleic acids present rather highly polar microꢀenvironments, the
new amino acid constitutes a unique tool for probing interactions of these biomolecules in a site specific
manner.
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Conclusion
For the first time, a fluorescent Lꢀamino acid, exhibiting ESIPT and hydrationꢀsensitive dual
emission, was synthesized. This amino acid bears as a side chain, the 2ꢀ(2ꢀfuryl)ꢀ3ꢀhydroxychromone
fluorophore, which is flat and close in size to the indole moiety of LꢀTryptophan. It was incorporated at
two different positions of the NC(11ꢀ55) peptide. Remarkably, substitution of the highly conserved
Trp37 residue by this fluorescent amino acid was found to preserve both the native folding and
chaperone activity of the peptide. Interaction of the labeled peptides with target ODNs changed
dramatically the dual emission of the incorporated amino acid and these changes depended on its
position in the peptide and on the ODN sequence. The obtained results established a correlation between
the fluorescence response of the amino acid probe and its proximity to the interacting ODN bases. This
amino acid appears as an attractive tool for probing the binding sites in peptideꢀnucleic acid complexes,
being particularly suitable to substitute tryptophan residues. Compared to tryptophan and its close
fluorescent analogues, this new amino acid presents superior environment sensitivity.
Acknowledgment
This work, VYP and VGP were supported by ANR (ANRꢀ07ꢀBLANꢀ0287), ANR Femtostack, ANRS,
CNRS and Université de Strasbourg. OVS was supported by ARCUS Alsace. We thank to Cyril
Antheaume and Patrick Wehrung for NMR and Mass measurements, respectively.
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Supporting Information Available: Additional characterization data on the fluorescent amino acid and
its peptide conjugates and the studied ODN sequences are presented. This material is available free of
charge via the Internet at http://pubs.acs.org.
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