Oxyluciferin Derivatives: A Toolbox of Environment-Sensitive

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Oxyluciferin Derivatives: A Toolbox of Environment-Sensitive Fluorescence Probes for Molecular and Cellular Applications Avisek Ghose, Oleg V. Maltsev, Nicolas Humbert, Lukas Hintermann, Youri Arntz, Pance Naumov, Yves Mély, and Pascal Didier J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.6b12616 • Publication Date (Web): 24 Jan 2017 Downloaded from http://pubs.acs.org on January 27, 2017

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The Journal of Physical Chemistry

Oxyluciferin Derivatives: A Toolbox of Environment-Sensitive Fluorescence Probes for Molecular and Cellular Applications Avisek Ghosea, Oleg V. Maltsevb, Nicolas Humberta, Lukas Hintermannb, Youri Arntza, Panče Naumovc, Yves Mélya and Pascal Didiera*

a

Laboratoire de Biophotonique et Pharmacologie, UMR 7213 du CNRS, Faculté de

Pharmacie, Université de Strasbourg, 74, Route du Rhin, 67401 Illkirch Cedex, France, b

Department Chemie, Technische Universität München, Lichtenbergstr. 4, 85748

Garching bei München, Germany. c

New York University Abu Dhabi, P.O. Box 129188, Abu Dhabi, United Arab Emirates

*Corresponding author (PD): Phone : +33(0)368854115, Fax : +33(0)368854313 Email : [email protected]

Current affiliation of AG: J.H Institute of Physical Chemistry, Dolejskova 2155/3, 18223 Prague-8, Czech Republic

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Abstract In this work, we used firefly oxyluciferin (OxyLH2) and its polarity-dependent fluorescence mechanism as a sensitive tool to monitor biomolecular interactions. The chromophores, OxyLH2 and its two analogues 4-MeOxyLH and 4,6′DMeOxyL, were modified trough carboxylic functionalization and then coupled to the N-terminus part of Tat and NCp7 peptides of Human Immunodeficiency Virus type-1 (HIV-1). The photophysical properties of the labelled peptides were studied in live cells as well as in complex with different oligonucleotides in solution. By monitoring the emission properties of these derivatives we were able, for the first time, to study in-vitro biomolecular interactions using oxyluciferin as a sensor. As an additional

application,

cyclopropyl-oxyluciferin

(5,5-Cpr-OxyLH)

was

site-

specifically conjugated to the thiol group (Cys-232) of the human protein α-1 antytripsin to investigate its interaction with porcine pancreatic elastase. Our data demonstrate that OxyLH2 and its derivatives can be used as fluorescence reporters for monitoring biomolecular interactions.


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Introduction The familiar flashing light produced by fireflies is an outstanding example of bioluminescence in living organisms.1 Bio(chemi)luminescence is a natural process by which living organisms convert chemical energy into light. In the fireflies, the bioluminescence reaction involves oxidation of the photoactive substrate, luciferin, catalyzed by an enzyme, luciferase, in presence of Mg2+ and adenosine triphosphate (ATP)2, 3 to form the emitting molecule, oxyluciferin (OxyLH2), in its first excited state.4, 5, 6 De-excitation of OxyLH2* is accompanied by emission of visible light, a phenomenon observed in the firefly bioluminescence. Later on, the oxyluciferin is enzymatically regenerated into luciferin.7, 8 Because of its high fluorescence quantum yield3 and excellent signal-to-noise ratio, the firefly bioluminescence stands out as a promising candidate in bioassays.9, 10, 11, 12, 13, 14, 15, 16 The potential of the photoproduct of the enzymatic reaction has been recognized for sensitive in-vivo bio-imaging applications,13 where the major limitation of conventional chemiluminescence reaction for analytical applications, namely its low fluorescence quantum yield (in general, 3‒5%, very rarely up to 10%)17, could be overcome. During the last two decades, extensive studies were performed to unravel the color tuning mechanism1 18 19 20 21 13 22 23

24

. Indeed, despite the fact that the reaction chemistry

and structure of the emitter are identical for all known beetle luciferase, the emission can vary between 536 and 638 nm17

23

. Several experiments were thus dedicated to the

identification of the species involved in this complex emission process. Recently, we investigated the emission mechanism of the photoproduct OxyLH2 in aqueous buffer by using

steady-state

and

time-resolved

spectroscopy

combined

with

multivariate

chemometric analytical approach. To get a better understanding of the underlying processes, we used several synthetic derivatives of oxyluciferin to mimic all the possible



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chemical forms of OxyLH2 in aqueous buffer. On the basis of our results, we proposed a detailed photoluminescence pathway where the contribution of both enol and keto forms were clearly identified. In parallel, we characterized, for the first time, absorption and emission spectra of the six common chemical forms of firefly oxyluciferin in aqueous buffer, some of which being involved in the actual emission process, as well as the equilibrium constants of firefly oxyluciferin in the ground state and in the excited state.23 Although these results do not directly apply to the luciferase protein where the active site is considered to be of low polarity, they provide support to the hypothesis that the excitedstate potential energy surface and the related dynamics are affected by the microenvironment of the active site. Most of the compounds, used to decipher the complex OxyLH2 emission mechanism, display pH and polarity-dependent optical properties opening the way to their use as probes to monitor biomolecular interactions. Indeed, fluorescence techniques are considered as ideal tools to visualize and characterize biological processes at a molecular and/or cellular level.25 With appropriate instrumentations, fluorescence based methods are non-invasive, very sensitive and quantitative. Through a proper labelling they are thus perfectly suited for monitoring biomolecular interactions. In addition, using environment-sensitive fluorescent probes, it is possible to characterize site-specific interactions between different biomolecules (e.g., DNA, RNA, protein, peptide etc.).25, 26, 27, 28 For instance, fluorophores exhibiting excitedstate proton transfer (ESPT) have notably been largely used as environment-sensitive probes14, 15, 18, 22, 25, 29, 30, 31, 32, 33 for monitoring biomolecular interactions. In this work, we thus investigated the possibility to use OxyLH2 derivatives as environment-sensitive dyes for monitoring biomolecular interactions. In particular, we selected three derivatives: OxyLH2, 4-MeOxyLH and 5,5-Cpr-OxyLH (Figure 1) that display pH and polarity dependent optical properties.23 In addition, 4,6′-DMeOxyL


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(Figure 1), a derivative whose photophysical properties are not influenced by the environment was also used as a negative control.

Figure 1. Chemical structures of the firefly oxyluciferin, presented here in in its neutral enol form, and its derivatives used in this study. OxyLH2 was fused to a HIV-1 Trans activator (Tat)34,

35, 36

peptide that is able to

penetrate in living cells. By using this labelled peptide and Fluorescence Lifetime Imaging Microscopy (FLIM), it was possible to monitor the intracellular distribution of pH values. Moreover, 4-MeOxyLH, which shows an environment-sensitive dual emission in physiologically relevant pH (7.4), was used to monitor the interaction of the HIV-1 Nucleocapsid protein (NCp7) with oligonucleotides through ratiometric measurements. Finally, we used 5,5-Cpr-OxyLH, the cyclopropyl derivative that mimics the keto form of OxyLH2, to label the cysteine residue in position 232 of the Human Alpha1-Antytrypsin (α1-AT), a single chain glycoprotein, commonly found in eukaryotic organisms, plants and viruses. The emission property of this probe was used to characterize the interaction of α1AT with Porcine Pancreatic Elastase (PPE), a serine protease.37 Taken together, our data clearly demonstrate that OxyLH2 and its environment sensitive derivatives can be used as fluorescence reporters for monitoring biomolecular interactions.



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Materials and Methods Synthesis and 1HNMR study of oxyluciferin derivatives The synthesis and 1HNMR study of modified analogues of oxyluciferin derivatives reported herein have already been published in other forms.21, 23, 38 Their structures are deposited as figure S1 in the Supplementary Information. Oxyluciferin derivatives (OxyLH2, 4MeOxyLH and 4,6’-DMeOxyL) were further modified at their thiazole moiety to couple them with the N-terminus part of Tat and/or NCp7 peptides (refer Figure 2, Table 1 and Figure S1 for detailed chemical structure. Also refer section S3 in ESI for synthesis procedure).

Figure 2. Scheme of oxyluciferin derivatives coupled to HIV-1 peptides. Table 1. HIV-1 peptides labelled with oxyluciferin-derived chromophores Chromophores

Conjugate

R1

R2

R3

Molecular mass* (gm/mol) 2654.0 (th) 2659.4 (ex)

OxyLH2H H -(CH2)2SCH2COx Tat(44-61) 4,6’2608.0 (th) 4,6’-DMeOxyL DMeOxyLCH3 -CH2COx H 2613.4 (ex) Tat(44-61) 4-MeOxyLH5535.3 (th) 4-MeOxyLH H -CH2COx H NC(11-55) 5540.5 (ex) *Molecular mass of the peptide-chromophore conjugate (th: theoretical mass, ex: mass observed by ESI Mass Spectrometry); x denotes N-terminus of the peptide. Molecular mass of probes are mentioned in section S1 of ESI. OxyLH2

HIV-1 Peptide Synthesis and oxyluciferin-CPP conjugation The Tat(44-61) and NC(11-55) peptides39,

40, 41, 42

(Figure 3) were synthesized by solid

phase peptide synthesis chemistry using a 433A synthesizer (ABI, Foster City, CA) as previously described.40,

43, 44, 45

The synthesis was performed at 0.1 mmol scale using

standard side-chain protected fluorenylmethyloxycarbonyl (Fmoc)-amino acids and


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HBTU/HOBt coupling protocol.43 Fmoc-Gly-Wang resin LL (Novabiochem, 0.38 mmol/g reactive group concentrations) or Fmoc-Asn(trt)-Wang resin (Activotec, 0.52 mmol/g reactive group concentration) were used as a solid support for coupling the functionalized oxyluciferin to the N-terminus of Tat(44-61) or NC(11-55) peptides. After completion of the synthesis, peptidylresins were isolated and washed twice with MeOH and CH2Cl2. OxyLH2 and 4,6’-DMeOxyL were coupled to Tat(44-61) and 4-MeOxyLH was coupled to NC(11-55). A β-alanine (3-aminopropanoic acid) derivative was used as a spacer between the peptide and the label.46 Three to five equivalents of the oxyluciferin derivative were dissolved in 500 µL of DMF and mixed with six equivalents of HBTU/HOBt coupling solution (in DMF) and further added to the Fmoc deprotected peptidyl resin swelled in 500 µL of DMF. After a few minutes of gentle shaking, six equivalents of DIEA solution were added and the reaction mixture was stirred overnight at 37°C. Afterwards, the peptidyl resins were ﬁltered and washed with MeOH and CH2Cl2. Cleavage and deprotection of labelled Tat(44-61) peptidylresin were performed by addition of 10 mL Triﬂuoroacetic Acid (TFA) solution containing 5% (v/v) water and 5% (v/v) TIS(iPr)3SiH for more than 2 hours. In addition to the previous protocol, 1% (w/v) phenol, 5% (v/v) thioanisole and 2.5% (v/v) ethanedithiol were added to the mixture for the cleavage of labelled NC(11-55) peptidylresin. The peptides were precipitated by using cold diethyl ether and then pelleted by centrifugation at 3000 rpm for 10 min. Then the pellets were dried at room temperature. The labelled peptides were finally solubilized with aqueous TFA (0.05% v/v) and were lyophilized under vacuum. All labelled peptides were purified by High Performance Liquid Chromatography (HPLC) using a C18 column (Nucleosil 100A, 5 µm; 250x10, Macherey-Nagel) in water-acetonitrile mixture containing 0.05% TFA with linear gradients [15 to 70% of acetonitrile during



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90 min for the labelled Tat(44-61) peptides and 20 to 50% acetonitrile during 90 min for the labelled NC(11-55) peptides] and monitored at 220 or 370 nm. All purified labelled peptides were analyzed by ESI Mass Spectrometry. The molecular mass obtained experimentally was in good agreement with the theoretical molecular mass of the labelled peptides (Table 1). Lyophilized peptides were stored at –20°C. All HPLC grade chemicals used for the synthesis were purchased from Sigma-Aldrich or Fluka, unless mentioned otherwise. The concentrations of labelled Tat(44-61) peptides were determined from their absorbance at 375 nm using a molar extinction coefficient Ɛ375 = 4.80×104 M-1.cm-1 for 4,6’-DMeOxyL and Ɛ375 = 2.30×104 M-1.cm-1 for OxyLH2. The zinc-bound form of NC(1155) labelled with 4-MeOxyLH was prepared by reacting the peptide with a 2.5 fold molar excess of zinc sulphate in 25 mM Tris-HCl-30 mM NaCl-0.2 mM MgCl2 pH 7.4 at 20°C buffer. The pH was adjusted only after the addition of zinc to avoid oxidation of the zincfree peptide. The peptide concentrations were determined by using an Ɛ280 = 5.70×103 M1

.cm-1.47 The solutions of peptide were stored at –20°C in small aliquots.

Figure 3. Amino acid sequences of the HIV-1 peptides used in this study: Tat(44-61) and NC(11-55)28, 40.


42, 44

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Steady-state spectroscopy measurements All steady-state spectroscopic measurements were performed in 10 mm path length quartz cuvette purchased from Hellma Analytics. Absorption spectra were measured by using a dual beam Cary-4000 spectrometer (Agilent Technologies), equipped with a thermostated sample holder, at the rate of 100 nm/min. Fluorescence spectra was collected at the rate of 150 nm/min with a Fluorolog spectrofluorometer (Jobin Yvon Horiba), equipped with a Peltier thermostated sample holder, with excitation and emission slits of 2 or 3 nm. The fluorescence spectra were corrected for the fluorescence of the buffer and the wavelength dependence of the optical elements in the emission pathway. While observing time-dependent fluorescence emission, excitation and emission wavelength have been fixed at the point of interest with 2 or 3 nm excitation and emission slits. Two-photon excitation microscopy Two photon excitation fluorescence microscopy was performed using an in-house constructed multi-photon laser scanning system based on an Olympus IX70 inverted microscope with an Olympus 60x 1.2NA water immersion objective.48 Fluorescence Lifetime Imaging Microscopy (FLIM) measurements were performed on this setup, using the multidimensional Time-Correlated Single Photon Counting (TCSPC) technique in which the sample is scanned by a focused beam of 80 MHz pulsed (mode-locked Ti:Sapphire) laser (Tsunami, Spectra Physics). Two-photon excitation was fixed at 780 nm and the laser power was adjusted to give count rates with peaks up to 106 photons.s-1, to avoid pileup effect. Photons were collected using a short pass filter with a cut-off wavelength of 680 nm (F75-680, AHF, Germany). The fluorescence was directed by an optical fiber coupled APD (SPCM-AQR-14-FC, Perkin Elmer), which was connected to a TCSPC module (SPC830, Becker & Hickl, Germany) operating in the reversed start-stop



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mode. Typically, the samples were scanned continuously for about 180 seconds to achieve appropriate photon statistics to analyze the fluorescence decays. Data were analyzed using the commercial software package (SPCImage V4.3.2, Becker & Hickl, Germany), which uses an iterative deconvolution method to recover the lifetimes from the fluorescence decays.49, 50, 51, 52, 53 Cell culture HeLa cells (ATCC CCL-2) were cultured on a 35 mm glass bottom µ-Petridish (Ibidi, Germany) in DMEM (Dulbecco's Modified Eagle Medium) from Gibco, Life Technologies, supplemented with 10% FBS (Fetal Bovine Serum, Gibco) and 0.1% PEN-STREP (Lonza) for 24 h at 37°C in 5% CO2 atmosphere. After incubation of 24 h in aseptic conditions, the cells were washed with PBS and Opti-MEM (Gibco). Then, oxyluciferin-labelled Tat dissolved in water was added at a final concentration of 0.3‒0.7 µg/mL and incubated with the cells for 30‒45 min. After incubation, cells were washed with PBS and Opti-MEM and further incubated in Opti-MEM before being observed under the microscope placed in a thermally incubated chamber at 37°C. Later 30 µM (~20 µg/mL) monensin sodium salt (CAS No. 22373-78-0) was added directly over the cells, three minutes prior observation, in order to neutralize the intracellular pH.54 Oligonucleotide sequences Double HPLC grade purified custom-made oligonucleotide sequences were purchased from IBA GmbH and their stock solution was prepared in deionized water. The oligonucleotide concentrations were calculated from their molar absorption coefficient provided by IBA GmbH.

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Results and Discussion In cellulo Fluorescence Lifetime Imaging Microscopy (FLIM) with oxyluciferin Recently, we have shown that the excited-state lifetime of OxyLH2 is sensitive to pH within the physiological pH range (5‒11).23 In the present work, the OxyLH2 chromophore was functionalized with a carboxylic acid and covalently linked to the HIV-1 Tat(44-61) peptide by using solid-state peptide synthesis. This small peptide which includes six arginine and two lysine residues exhibits strong cell translocation property34,

35

and thus

acts as a cell penetrating peptide (CPP) able to carry a large variety of cargoes ranging from very small molecules (~100 Da) to large biomolecules of ~200 nm diameter.35, 42 The ATP and temperature-independent cellular uptake permitted by this CPP was used for cell delivery of low-molecular weight drugs, small oligonucleotides, peptides and proteins in their active state.35 Uptake and cytoplasmic distribution of Tat(44-61) peptide labelled with either the pH-sensitive OxyLH2 or the pH-insensitive 4,6′-DMeOxyL probes was first monitored in HeLa cells by using two-photon microscopy. Both OxyLH2-Tat(44-61) and 4,6′-DMeOxyLTat(44-61) peptides were added at 0.3-0.7 µg/mL and incubated for 30‒45 min before imaging. As depicted in Figure 4, both labelled Tat(44-61) peptides are efficiently internalized within living cells. Their intracellular distribution perfectly matches with the ones observed earlier using Tat(44-61) peptides labelled with other fluorophores.55, 56, 57 In addition, Figure 4 reveals that the conjugates OxyLH2-Tat(44-61) and 4,6′-DMeOxyLTat(44-61) can be efficiently excited by two-photon excitation. Furthermore, long observation time (3‒6 hr post-incubation) under two-photon excitation showed that the cell morphology was not affected, suggesting marginal toxicity of the labelled peptides under these conditions.

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Figure 4. Two-photon excitation microscopy of HeLa cells incubated with the labelled peptide conjugates OxyLH2–Tat(44-61) (left) and 4,6′-DMeOxyL–TAT(44-61) (right) (λex: 780 nm, average laser power

Oxyluciferin Derivatives: A Toolbox of Environment-Sensitive

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