A TAT peptide-based ratiometric two-photon fluorescent probe for detecting biothiols and sequentially distinguishing GSH in mitochondria
Graphical abstract
Introduction
Growing attention is given to the bioassay of common biothiols, such as cysteine (Cys), homocysteine (Hcy) and reduced glutathione (GSH), due to their vital roles in a lot of pathological, physiological and pharmacological processes. The intracellular concentration of Hcy is determined to be closely associated with many diseases. A general molar concentration level of Hcy in cell is about 5–12 μM, and a tiny increase of Hcy can result in hyperhomocysteinemia [1], neural tube defects, and cardiovascular disease [2]. As a precursor of GSH, the normal concentration of Cys is 30–200 μM in cell [3], and abnormalities in Cys levels is closely related with the decreased hematopoiesis neurotoxicity, slowed growth, weakness, skin lesions and AIDS [4,5]. The most abundant GSH in cells normally has a high intracellular concentration of 1–10 mM [6]. GSH plays a vital role in controlling redox state, heterogeneous metabolism, which indicates for the corresponding enzyme activity and gene regulation in cells.
Mitochondria are important organelles found in most eukaryotic cells. In addition to energizing cells, mitochondria are involved in processes such as cell differentiation, cell signaling, and apoptosis, and have the ability to regulate cell growth and cell cycle etc. [7,8]. Thus, the development of sensitive mitochondria-targeted biothiol probes has far-reaching implications for studying the effects of biothiols and redox states on diagnosing many diseases and researching physiological metabolism in many physiological processes [9]. Based on its importance, more fluorescent probes of biothiols have been designed and synthesized in recent years [[10], [11], [12], [13], [14]]. However, most probes are turn-on probes based on fluorescent intensity with low water solubility and biocompatibility, and have the indistinguishable nature for biothiols. These drawbacks limit their further application in real physiological and pathological conditions. Interestingly, a ratiometric two-photon fluorescence system potentially is an ideal sensor. It produces two different wavelengths which act as an internal reference and analyte indicator for one target, thereby minimizing the undesirable interference of external factors and improving the accuracy of analytical studies [[15], [16], [17]]. Other than this, this kind of two-photon fluorescent probe has the advantages of strong penetrability, low phototoxicity, high signal-to-noise ratio, and etc. [18,19]. In general, ratio analysis can be achieved by constructing an energy resonance transfer system [[20], [21], [22]].
In addition, a large amount of peptide-based molecular probes have been used to detect different biomarkers because of their great biocompatibility, water solubility, simplicity of synthesis, inherent biodegradability, favorable targeting specificity, and tissue or cells permeability [[23], [24], [25], [26], [27], [28], [29]]. For example, Yi's group designed and synthesized a peptide-based probe to detect nuclear H2O2 [30], and the peptide (VQRKRQKLMP-NH2) was used as an effective nuclear transport carrier. Recently, Cho et al. also used a peptide sequence (SDYQRL) as the Golgi-apparatus-targeting moiety and detected Golgi apparatus in live tissue [31]. What's more, a number of previous reports had shown that cell penetrating peptide TAT (RRQRRKKRG) was capable to deliver efficiently a variety of macromolecular substances (such as peptides, proteins, and nucleic acids) into cells and mitochondria [[32], [33], [34], [35], [36]]. Native chemical ligation (NCL) method is widely used in the connection of longer peptide sequences, for which the selection of the connection site is in Cys [[37], [38], [39]]. The mechanism of the reaction is that the C-terminal thioester of a peptide exchanged with the N-terminal Cys, after which the peptides automatically formed natural peptide bond by moved from S to N [40]. Although, this mechanism has been utilized to design biothiol probes [41,42], a novel peptide-based probe is still missing for mitochondrial targeted detection of biothiols. Addition, there are many thiol-containing proteins on the cell membranes. The arginine-rich TAT peptide has a strong propensity of interacting with cell surface membranes, and this process can promote its reaction with cell-surface thiols [[44], [45], [46]].
Based on the above research background, we reasonably designed and synthesized a cell-penetration peptide TAT-modified ratiometric two-photon fluorescent probe through a solid phase synthesis method, which consists of the cell penetrating peptide (TAT) and a Förster resonance energy transfer (FRET) system constructed by naphthalimide and rhodamine B fluorophore through thioester bond (Scheme 1). Firstly, the TAT-probe can recognize biothiols by a ratiometric change in green and red fluorescence at 404/820 nm excitation about 20 min. Other than this, GSH can be distinguished by using the TAT-probe through a red fluorescent emission at 585 nm with excitation of wavelength of 545 nm. Overall, the TAT-probe can not only simultaneously detect biothiols and sequentially distinguish GSH with the limit of detection (LOD) of 0.865 μM, 6.51 μM and 5.15 μM for Cys, Hcy and GSH, respectively, but also enter mitochondria faster and have lower toxicity. To our knowledge, the TAT-probe is the first peptide-based fluorescent probe designed based on the NCL reaction mechanism of peptides.
Section snippets
Synthesis of the TAT-probe
Synthesis of involved Compounds in this work were detailed in Supplementary material and the related NMR or Mass spectrum is shown in Figs. S2-8. The solid-phase peptide synthesis method (SPPS) was utilized to synthesis the TAT-probe (Fig. S9) and the detailed synthetic steps were described in Supplementary material. The purified product was proved by Matrix-assisted laser desorption (MALDI) ionization-time of flight (TOF)/TOF, MS: calculated for TAT peptide [(M + H)+]: 1617.94; found
Measurement of the fluorescence quantum yield (ΦPL) and the two-photon action cross sections (Φσ) of TAT-probe
The fluorescence quantum yield ΦPL was measured in HEPES buffer solution (pH = 7.4). The measurement method is carried out in reference and calculated was 12%. The two-photon action cross sections (Φσ) of TAT-probe measured by comparison method pulsed laser in the range of 780–900 nm with rhodamine B as a two-photon reference excitation (TPEF) [43]. The following equation was used for the calculation of Φσ,where superscripts ‘ref’ designate TPEF, is the two-photon
Conclusions
In conclusion, we have designed and synthesized a peptide (TAT)-modified ratiometric fluorescent probe utilizing the native chemical ligation (NCL) reaction mechanism, which can simultaneously distinguish and sequentially detect biothiols using two different excitation and emission channels. When excited at 404 nm, the TAT-probe showed a change in the ratio of fluorescence intensities (I520/I585). When excited at 545 nm, the TAT-probe displayed a red fluorescence (λem = 585 nm) towards GSH and
Author statement
Prof. Yu Tang and Jing Cao: Contributed the central idea and they both revised the manuscript. Pingru Su: Designed the probe, performed the research, analyzed data, and wrote the original draft of the paper. Zhanwu Zhu: Conducted biological experiments and collected important background information. Yihong Tian: Provided the help of biological experiment in the revised article. Bo Cheng: Analyzed the conclusions of biological experiments and revised the manuscript. Lijuan Liang, Wenyu Wu and
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgements
We acknowledge the National Natural Science Foundation of China (Projects 21871121, 21601074, 31771447 and 31970624), the Fundamental Research Funds for the Central Universities (Project lzujbky-2018-ot01, lzujbky-2019-sp01, and lzujbky-2019-kb12, lzujbky-2018-k05), the Foundation of the Ministry of Education Key Laboratory of Cell Activities and Stress Adaptations grantlzujbky-2018-kb05, and Special Fund Project of Guiding Scientific and Technological Innovation Development of Gansu Province (
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P. R. Su and Z. W. Zhu contributed equally to this work.