Mengyao
She‡
ab,
Zhaohui
Wang‡
a,
Tianyou
Luo
a,
Bing
Yin
a,
Ping
Liu
a,
Jing
Liu
b,
Fulin
Chen
*b,
Shengyong
Zhang
a and
Jianli
Li
*a
aMinistry of Education Key Laboratory of Synthetic and Natural Functional Molecule Chemistry, College of Chemistry & Materials Science, Northwest University, Xi'an, Shaanxi 710127, PR China. E-mail: lijianli@nwu.edu.cn
bKey Laboratory of Resource Biology and Modern Biotechnology in Western China, Ministry of Education, Northwest University, 229 TaiBai North Road, Xi'an, Shaanxi Province 710069, PR China. E-mail: chenfl@nwu.edu.cn
First published on 25th September 2018
Glutathione (GSH) plays an important role in the body's biochemical defense system, and the detection of GSH in a physiological system is an important tool for understanding redox homeostasis. Protection–deprotection strategies have proven to be the most reliable, among existing detection methods. However, the understanding of how various electronic and steric effects influence a probe's ability to recognize a substrate is still lacking. In this study, we have analyzed various substituent effects on a GSH probe template via theoretical calculations and constructed the performance regulation and control strategy for this kind of probe. We then developed a series of guided probes using eighteen different acrylic ester derivatives to mask the fluorescence of fluorescein. The optical performance differences between the guided probes strongly supported the applicability of our proposed guiding strategy. Moreover, the positively guided probes are excellent for imaging GSH distribution in living cells and mice.
Strategies for detecting biological thiols have undergone rapid development during the past few years and will continue for the foreseeable future.10–14 Protection–deprotection of signal groups has proven to be an effective and reliable method to detect biological thiols, among numerous detection mechanisms.15 Countless fluorescent probes for biological thiol detection based on this strategy have appeared during the past decade.16–19 However, due to the similar chemical characteristics of each probe, they cannot distinguish one specific thiol from another in the complicated physiological environment of the cells. Thus, there is a critical need for improved probes that bind to specific thiols.
It is well known that GSH is at a higher concentration than Cys or Hcy in living organisms during physiological processes, especially in cell growth, metabolism, oxidative stress, and even cancer therapy.20–23 Construction of fluorescent probes to detect GSH has drawn a lot of attention in the past few years, and it is acknowledged that acrylic esters could act as powerful modular moieties for GSH via Michael addition reactions.24,25 However, there are still no universal strategies to regulate the performance of this type of probe during its design, synthesis, and application, and the detailed mechanisms of action influenced by substituent groups are still poorly understood.26–29
Based on the typical mechanism of a protection–deprotection probe, we can reasonably infer that introducing an electron donor/acceptor or sterically hindered groups on an α,β-unsaturated ketone substituted moiety could regulate the effect of GSH detection. And choosing suitable substituent groups may increase the selectivity and sensitivity of the probe.
To verify this conjecture and achieve the objective of constructing a GSH-specific probe, we established a theoretical template for a protection–deprotection probe, using fluorescein as the signal scaffold, and by decoration with different acrylic ester derivatives (Fig. 1). The structural characteristics of these elaborately designed probes have been analyzed via DFT calculations, and their reaction activities with GSH were predicted by energy calculation, frontier orbital theory, and the Fukui function, which have direct significance to the design of a GSH-specific probe.
Fig. 1 The general template for GSH probes based on protection–deprotection of fluorescein by acrylic ester derivatives. |
Based on the simulation results we proposed a practical regulation and control strategy for GSH-specific probes and synthesized the relevant probes that were contained in the probe template. The optical detection performance of our GSH probes strongly demonstrates the viability of this design strategy. Moreover, the positively guided probes revealed excellent applicability for imaging GSH distribution in living cells and mice.
To further clarify these substituent effects from a theoretical perspective, a series of electrostatic charges and bond energies were calculated and are shown in Table 1. As shown by the preliminary estimation, the electron-donating group could apparently enhance the electron cloud density around the enone β carbon, lower the reactivity, and promote the selectivity to the strong nucleophile GSH. These effects are completely converse when the electron-withdrawing group takes effect. Furthermore, the bond dissociation energies (BDES) of ester bonds dwindled significantly when the strong electron-donating group, methoxyl, was introduced into the molecular skeleton. In contrast, the electron-withdrawing group exhibited the opposite effect, which manifested the inert reactivity of probes Fl-2/3/4OMe. Methyl substituents at α/β sites caused slight BDES reductions, suggesting the instability of Fl-α/βOMe to alkalescence. In addition, the formation enthalpy of a C–S bond in the recognition intermediate probe-GSH unequivocally demonstrated that the electron-donating group is capable of further passivating esterolytic action after being stabilized by a benzene ring, and endowing the probe with a unique selectivity for GSH.
Probe | Electrostatic charges | BDES | Formation enthalpy |
---|---|---|---|
Fl-H | −0.122 | 273.38 | −192.15 |
Fl-αMe | −0.14 | 267.96 | — |
Fl-βMe | 0.097 | 268.01 | — |
Fl-2OMe | −0.136 | 275.00 | −186.45 |
Fl-3OMe | −0.132 | 274.77 | −182.93 |
Fl-4OMe | −0.14 | 273.87 | −179.91 |
Fl-2NO2 | −0.13 | 265.34 | — |
Fl-3NO2 | −0.122 | 212.64 | — |
Fl-4NO2 | −0.118 | 225.74 | — |
The HOMO–LUMO energies, gaps and spatial distributions for each probe were determined and are displayed in Fig. 2. All the LUMOs of these probes were located on the recognition part, where the typical nucleophilic addition reaction would proceed. The HOMOs of probes Fl-α/βMe and Fl-2/3/4NO2 were distributed on the xanthene moieties, and were separated from the distributions of their LUMOs. However, for probes Fl-2/3/4OMe, both LUMOs and HOMOs are distributed in the recognition part of the probe. More notably, as electron acceptors, the LUMO energies of Fl-2/3/4NO2 are much lower than those of other probes, which means that these probes are more susceptible to attack by the HOMOs of GSH, –OH or –HS (calculated energies, −6.8329 eV, −5.4993 eV, −5.4448 eV, respectively). These structural characteristics might be the reason for their instability.
The Fukui+ function34,35 was also employed to evaluate the likelihood of a nucleophilic attack by GSH/thiols/–OH on each probe (Fig. 3). Collectively, the Fukui+ functions are nearly all concentrated on alkene bonds and carbonyl C atoms, and the Fukui+ function values of the β-sites are much larger than those of other active sites, indicating that the β-site is the first choice for nucleophilic attack by GSH/thiols/–OH.
Fluorescein protected by cinnamic acid (Fl-H) was stabilized by conjugated groups (benzene) and passivated the reaction activity of the β-site of the carbonyl group to ensure that the Michael addition reaction could only be triggered by GSH with its high pKa and nucleophilic attack ability. The introduction of a methyl group into the α/β site of the carbonyl will cause structural torsion and cause the maintenance of coplanarity between the alkene and benzene (dihedral angle: Fl-H = 0.08°; Fl-αMe = 30.42°; Fl-βMe = 33.72°, Fig. S31†) to be challenging, potentially weakening the effect of conjugation. This is reflected in the high activity of carbonyl C to the nucleophile and decreased selectivity of these methyl substituted probes to GSH. An intense + I effect (electron-donating inductive effect) generated by methoxyl can enhance the electron density on the acrylic ester moiety, passivate the activation on the β site of carbonyl, and eliminate the possibility of reaction with other nucleophiles aside from GSH. Although methoxyl could lead to excellent selectivity to GSH, it comes at the expense of the reaction rate and signal intensity. So, in this case, the electron donor group could turn out to be a double-edged sword for the construction and modification of “Michael addition reaction” type probes. In contrast, the strong − I effect (electron-withdrawing inductive effect) of nitryl will break the conjugated system, decrease the protective effect of carbonyl C, and lead to an extreme instability towards various nucleophiles, causing probes to lose their selectivity.
For example, take Fl-H as a representative probe (Fig. 5a), as expected, Fl-H alone was virtually non-fluorescent and showed a dramatic increase in fluorescence at 530 nm when 50 equiv. GSH was added to the system. By contrast, other analytes including potentially interfering thiols, such as Cys, Hcy, and H2S, hardly triggered any fluorescent signal from Fl-H (front bars). The addition of competing thiols, along with GSH, indicated that probe Fl-H was able to differentiate GSH from other thiols at high concentrations (back bars). This is in accordance with the relatively weak reaction activity of FI-H with GSH that was described in the structure–activity analysis.
To further evaluate the performance of Fl-H for the detection of GSH in vitro, we investigated the ability of Fl-H to sense GSH quantitatively (Fig. 5b). An emission peak at 530 nm gradually increased as increasing concentrations of GSH (0–80 equiv.) were added to the solution with probe Fl-H. The linear relationship indicated that Fl-H could quantify GSH in the range of 2–50 equiv. of its own concentration (Fig. 5c and d). The optical properties of Fl-2/3/4OMe were similar to those of Fl-H, and their optical spectra are shown in Fig. S1–S13.†
In order to investigate how different substituent groups on the probes influenced performance, we performed a time response experiment (Fig. 6). Fl-H and Fl-2/3/4OMe are fairly stable in PBS buffer solution (0.1 M, pH = 7.4) and exhibit good performance for GSH detection. It is obvious that Fl-H has a higher intensity than probes with methyl substituents after the fluorescence intensity stabilizes. In fact, their intensities correspond to their substitutional position on the benzene ring (Fl-H > Fl-3OMe > Fl-2OMe ≈ Fl-4OMe). It takes nearly 90 min for the fluorescence intensity of Fl-H/3OMe to stabilize with GSH, and 120 min for Fl-2/4OMe. These probes also have remarkable stability for other thiols. These results demonstrated that the electron donor group could slow down the recognition reaction and provide good selectivity for the detection of GSH, as we predicted in the optimized strategy. Furthermore, Fl-H/2/3/4OMe could withstand a wide pH range of 6–9, indicating potential applications under physiological conditions. In contrast, Fl-α/βMe and Fl-2/3/4NO2 are unstable and non-selective for GSH in PBS buffer solution, since the Michael addition reaction was inhibited by the steric hindrance of a dense electron cloud making the carbonyl C atom the first attacked site. Furthermore, the positively guided GSH specific probes (Fl-H/2/3/4OMe) exhibited good photostability both in vitro and in vivo (Fig. S18–S25†). All of these reaction kinetics and fluorescence intensities essentially agree with theoretical calculation results.
Additionally, probes dually protected by acrylate derivatives were also synthesized and investigated. The fluorescence masking effects of Di-Fl-H and Di-Fl-2/3/4OMe were too strong to be activated by any of all these analytes, including GSH. Di-Fl-α/βMe and Di-Fl-2/3/4NO2 were unstable and non-selective in PBS buffer solution with a much faster rate, at close to 30 min than monosubstituted probes.
Footnotes |
† Electronic supplementary information (ESI) available: Details of synthesis, characterization data and theoretical calculation of all probes; computational details. See DOI: 10.1039/c8sc03421d |
‡ These authors contributed equally to this work. |
This journal is © The Royal Society of Chemistry 2018 |