Precise Synthesis of GSH Specific Fluorescent Probe for

Subscriber access provided by Stockholm University Library

Biological and Medical Applications of Materials and Interfaces

Precise Synthesis of GSH Specific Fluorescent Probe for Hepatotoxicity Assessment Guided by Theoretical Calculation Jiao Chen, Zesi Wang, Mengyao She, Mengdi Liu, Zebin Zhao, Xi Chen, Ping Liu, Shengyong Zhang, and Jianli Li ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.9b08522 • Publication Date (Web): 19 Aug 2019 Downloaded from pubs.acs.org on August 20, 2019

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 29 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

ACS Applied Materials & Interfaces

Precise Synthesis of GSH Specific Fluorescent Probe for Hepatotoxicity Assessment Guided by Theoretical Calculation Jiao Chen,1,‡ Zesi Wang,1,‡ Mengyao She,1,2 Mengdi Liu,1 Zebin Zhao,1 Xi Chen,1 Ping Liu,*,1 Shengyong Zhang,1 Jianli Li*,1

1Ministry

of Education Key Laboratory of Synthetic and Natural Functional Molecule

Chemistry, College of Chemistry & Materials Science, Northwest University, Xi’an, Shaanxi province, 710127, P. R. China 2Ministry

of Education Key Laboratory of Resource Biology and Modern Biotechnology in

Western China, The College of Life Sciences, Northwest University, Xi'an, Shaanxi Province, 710069, P. R. China.

KEYWORDS: precise synthesis, fluorescence imaging, hepatotoxicity assessment, GSH, BODIPY, photo-induced electron transfer

ACS Paragon Plus Environment

1

ACS Applied Materials & Interfaces 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

Page 2 of 29

ABSTRACT

Drug-induced hepatotoxicity is the main cause of acute liver injury, and its early diagnosis is indispensable in pharmacological and pathological studies. As a hepatotoxicity indicator, the GSH distribution in the liver could reflect the damage degree in-situ. In this work, we have provided a theoretical design strategy to determine the generation of PET mechanism and achieve high selectivity for the target. After that, we precisely synthesized a novel near-infrared fluorescent probe BSR1 to specifically monitor endogenous GSH and hepatotoxicity in bio-system with a moderate fluorescent quantum yield (Ф=0.394) and low detection limit (83 nM) under this strategy. Moreover, this mapping method for imaging GSH depletion in vivo to assay hepatotoxicity may provide a powerful molecular tool for early diagnosis of some diseases and contribute to assay hepatotoxicity for the development of new drugs. Importantly, this theoretical calculation guided design strategy may provide an effective way for the precise synthesis of the target-specific fluorescent probe, and change this research area from “trial-and-error” to concrete molecular engineering.


2

Page 3 of 29 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


INTRODUCTION

Liver is one of the most important metabolic and detoxifying organs in the body. The assessment of preclinical hepatotoxicity is an indispensable and even an essential procedure during drug discovery due to the acute liver injury caused by various metabolites of drugs.1-6 Presently, the hepatotoxicity evaluation method in the clinic is to analyze the liver enzymes in serum (such as alanine aminotransferase(ALT), aspartate aminotransferase, alkaline phosphatase, etc.) which are biomarkers of liver injury.7 In fact, it is imprecise to evaluate hepatotoxicity by detecting these indicators in the serum, because the liver enzymes in plasma are not only generated by liver damage. For instance, skeletal muscle injury could also arouse the fluctuation of ALT in the blood.8 Therefore, an accurate method, focusing on the liver area to visualize the distribution and concentration of the indicators, is desperately needed to assay hepatotoxicity with high instantaneity, authenticity, and reliability. Among many indicators, GSH seems to be very sensitive and facile to be captured to reflect the states of liver.9-11 Recently, many efforts have been made for the detection of


3


Page 4 of 29

GSH in vitro and in vivo employing fluorescent imaging technology with high sensitivity, real-time detection and low damaged to bio-samples.12-15 And most of these researches were focused on the strong nucleophilicity of the thiol group, involved in nucleophilic substitution,16 Michael addition,17-23 cleavage of disulfide,24-27 SNAr reaction,28-41 , etc. However, it is empirical to figure out which substrate could trigger the recognition reaction based on these similar chemical mechanisms. Therefore, it remains a tremendous challenge to accurately differentiate GSH from its analogs (Hcy, Cys, RSeH).42 Accordingly, a quantified reactivity is imperative to assist scientists in predicting the recognition specificity, decreasing trial-and-error costs, and determining which analyte could specifically drive the chemical process ahead of synthesis. In the past decades, many powerful tools have been developed to elucidate the chemical reaction mechanism or predict properties of chemicals based on quantum chemistry.43, 44 It follows that the establishment of a theoretical probe model would be a practical and reliable approach to analyze recognition mechanism and provide guidance for the optimal route for the synthesis of GSH specific probe.


4

Page 5 of 29 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


In this work, we have selected the most common recognition mechanism, photoinduced electron transfer (PET),45 as our investigating object, and employed frontier molecular orbital theory to evaluate the PET implementability between fluorophore (thiophene vinyl boron-dipyrromethene, a newly synthesized fluorophore based on boron dipyrromethene(BODIPY) for its excellent fluorescence properties of BODIPY dyes)46 and six quenching/recognition groups. Subsequently, molecular dynamics (Fukui function) and thermodynamics (bond dissociation energies)47 were performed to speculate the recognition specificity of the designed probes for GSH compared with other analytes. The optimized near-infrared probe BSR1 proposed by the simulation results has been synthesized, which exhibited remarkable selectivity for endogenous GSH along with moderate fluorescence quantum yield and low detection limit both in vitro and in vivo. Furthermore, visual monitoring of typical antipyretics drug acetaminophen (APAP) induced acute liver injury could be achieved by BSR1 conveniently and efficiently (Figure 1).


5


Page 6 of 29

Figure 1. The GSH specific fluorescent probe for hepatotoxicity assessment guided by theoretical calculation.

EXPERIMENTAL SECTION 1. Chemicals and Instruments

All chemicals and solvents were purchased from commercial suppliers and they were directly used without further purification. The related instruments used in this work were listed in the Supporting Information.

2. Theoretical Calculation

All calculations were performed by the Gaussian 09 program. The geometry optimization of ground states was computed with density functional theory (DFT) at the B3LYP/6-311G** levels. The HOMO and LUMO were used for investigation of PET


6

Page 7 of 29 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


(photo-induced electron transfer) mechanism according to references.45,

48

All

calculations are performed in water using polarizable continuum model (PCM).

3. HPLC Testing All samples were analyzed by HPLC using a gradient elution program which (0-6 min 80 % methanol/acetonitrile (7/3, v/v) and 20 % H2O was used as mobile phase, and the 6-30 min 100 % methanol/acetonitrile (7/3, v/v) was used as mobile phase) at a flow rate of 1.0 mL/min with detection wavelength at 600 nm, and the sample size was 5 μL. 4. In vivo and Tissues Imaging.

Kunming mice were chosen to assay the capability of imaging endogenous GSH in vivo by BSR1. The mice were divided into four groups(a-d). a: Blank; b: pretreated with NEM (5 mM, 200 μL) for 30 min and then injected with BSR1 (100 μM, 200 μL); c: treated with BSR1 (100 μM, 200 μL); d: pretreated with α-thioctic acid (5 mM, 200 μL) for 30 min and then injected with BSR1 (100 μM, 200 μL). And fluorescence intensity was acquired at 0, 2, 5, 10, 15, 20 min by IVIS Lumina LT Series III from PerkinElmer.


7


Page 8 of 29

The liver tissues were divided into three groups. The first group was blank tissue without any treatment. The second group was pretreated with NEM (5 mM) for 30 min and then incubated with BSR1(100 μM) for 30 min. The last group was incubated with BSR1(100

μM) for 30 min. Afterward washed with PBS for three times. RESULTS AND DISCUSSION

1. Design of GSH specific probe guided by theoretical calculation As shown in Figure 2a, a brand new near-infrared BS was employed as the reporter, and six classical moieties (R1-R6) were selected for masking the signal of fluorophore BS via PET mechanism and achieving the recognition performance for thiols through deprotection. The frontier molecular orbital energies of each moiety were calculated using Density Functional Theory (DFT) in Gaussian 09 program to evaluate the ability of these moieties for achieving PET effect with BS. Among these combinations, BS+R1 and BS+R5, could match with the essential condition that leads to a d-PET effect (an excited state electron transfer process from the LUMO of excited fluorophore to the LUMO of recognition moiety), by reason of their appropriate energies (LUMO of R1 = -3.3976 eV,


8

Page 9 of 29 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


LUMO of R5 = -3.2001 eV) which are right between HOMO and LUMO energies of BS (HOMO = -4.9683 eV, LUMO = 2.8836 eV). It’s reasonable to deduce that nucleophilic ability of the analytes upon the recognition site and the dissociation of the recognition moiety control the sensitivity and selectivity of this probe according to the reported studies of this type of probes. Then, their bond dissociation energies and Fukui function were further calculated by B3LYP (6-311G**) (Figure 2b). The Fukui function results showed that Fukui+ of the attacked carbons are BSR1 (0.1110) and BSR5 (0.1190), respectively, which means that BSR1 might have better selectivity for GSH because of its stronger nucleophilic ability compared with Cys and Hcy. Besides, the bond dissociation energy of BSR5 is almost 73.0627 kcal/mol, while this parameter for BSR1 is as low as 29.9357 kcal/mol. It is manifested that the C–S bond of BSR1 is easier to dissociate than the C–O bond of BSR5 when thiol attacks the probe. Based on the above calculation results, we inferred that BSR1 might be an excellent candidate with satisfying fluorescence performance. Subsequently, we successfully synthesized the probe BSR1~BSR4 and BSR6. As shown in Figure S2, the BSR1 exhibited almost no fluorescence because of PET process,


9


Page 10 of 29

while BSR2/BSR3/BSR4/BSR6 showed strong fluorescence (NO-PET) under the same conditions and parameters. These results coincide with the predicted performance by theoretical simulation, and confirmed the validity of our probe design strategy.

Figure 2. a) Theoretical calculation of PET process for the fluorophore and six selected candidates. b) Bond dissociation energy and Fukui function of BSR1 and BSR5

2. Synthesis and characterization of a serials probe


10

Page 11 of 29 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


Scheme 1. Synthesis of Probe BSR1

O 2N S CHO

ClO2S

AcOH, Piperidine CH3CN

BODIPY-OH

NO2

DCM, 0°C  rt

BS Strongly Red Fluorescence

Green Fluorescence

BSR1 None Fluorescence

BODIPY-OH was synthesized according to the reported method,49 and then BS was obtained by conjugating two units thiophene formaldehyde to BODIPY skeleton via Knoevenagel condensation, and BSR1 was successfully synthesized and characterized by 1H NMR,

13C

NMR, and HRMS. Besides, its crystallographic data was obtained (see

supporting information). It can be seen from the spatial structure of the crystal BSR1, the dihedral angle between the conjugate plane α and the axial plane β is θαβ =73.91°, instead of completely vertical (Scheme 1). We speculated that this special spatial structure might imply some hidden information related to fluorescence properties, such as promoting photo-induced transfer progress. Other synthesis procedures and characteristic data of BSR2~BSR4 and BSR6 are in the supporting information. 3. Investigation of optical properties of BSR1


11


Page 12 of 29

To verify the property of BSR1 as expected, the selectivity of BSR1 was investigated in CH3CN/PBS = 4/6 (v:v), which was selected as the best binary solvent system throughout the whole optical testing because high fluorescence intensity was measured in this solvent system (Figure S3). To our delight, the pure probe solution is non-fluorescence and then displayed a significant fluorescence enhancement (45 folds) in the presence of GSH (200 μM) at 663 nm with a moderate fluorescent quantum yield (Ф = 0.394) (Equation S1, supporting information), while slight fluorescence was observed upon the addition of other selected species (Figure 3a). Meanwhile, this exclusive response exhibited good anti-interference ability in the presence of Na2SO4, Na2SO3, NaNO3, NaClO, H2O2, NaSH, Val, His, Ala, Gly, Arg, Met, Lys, Ser, Tyr, Hcy, Cys, PhSH (Figure S4). Subsequently, the kinetic experiments revealed that the fluorecence intensity increased rapidly within 20 minutes and reached a steady state at 45 minutes with almost no longer decays (Figure 3b), which manifested that this probe could be used for the detection of GSH with good stabililty. Importantly, quantitative detection capability, pH stability, and photostability are indispensable parameters to assess the application performance of a probe. Therefore,


12

Page 13 of 29 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


we performed the fluorescence titration experiment of BSR1 upon different concentrations of GSH (0-200 μM). The result exhibited an excellent linear correlation (R2=0.9944) of the fluorescence intensity at 663 nm with the concentration of GSH in the range of 0-100 μM (insert of Figure 3c), and the increasing fluorescence intensity plateaued when GSH concentration reached 200 μM. The limit of detection was as low as 83 nM for GSH calculated by the Equation S2, which means it is suitable for quantitative analysis of low concentration of GSH. Subsequently, pH stability test was evaluated, and the result indicates that the BSR1 has high stability in a wide pH scope (2.0-12.0). In particular, BSR1 showed remarkable fluorescent performance on detecting GSH at physiological pH (6.0 - 9.0), whereas the fluorescence intensity decreased under acidic or basic conditions (Figure S5). And the photostability test results showed that the fluorescence intensity of BSR1 and BSR1+GSH had only a slight decrease after six days illumination with total illumination of 1.44×106 lux·hr (Figure S6). All these data established the foundation for quantitative detecting GSH in physiological condition by imaging techniques.


13


Page 14 of 29

Figure 3. a) Fluorescence spectra of BSR1 (10 μM) in the presence of NaSH, Na2SO4, Cys, Hcy, GSH, PhSH, H2O2, NaClO (200 μM) in CH3CN : PBS (4:6, v/v) at pH=7.4. b) Fluorescence intensity of BSR1 (10 μM) in absence or presence of GSH (20 eq.) at 663 nm in 0 min to 100 min at 25 ℃. c) Fluorescence and d) Ultraviolet absorbance spectra of BSR1 (10 μM) in the presence of a different concentration of GSH (0-200 μM). The


14

Page 15 of 29 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


insert of (c) showed the emission intensity at 663 nm as a function of GSH concentration. λex = 600 nm.

4. Response Mechanism To verify the recognition reaction is, as we initially proposed, following the process of PET, the theoretical calculation was first performed (Figure 4a), which indicates that the off-on mechanism of probe BSR1 complies with d-PET process. Furthermore, HPLC has also substantiated the recognition mechanism, owing to the vanishment of BSR1 signal at 12.6 min and the appearance of BS signal at 10.5 min (Figure 4b). Meanwhile, highresolution mass spectrum analysis was performed on the reaction mixtures of BSR1 and GSH. As demonstrated in Figure 4c, peak 472.0763 and 527.1231 belong to the signal of R1-GSH and BS, respectively, which illustrates the termination of d-PET. These results indicate that the fluorescence enhancement of BSR1 for GSH resulted from the release of compound BS.


15


Page 16 of 29

Figure 4. a) Proposed sensing mechanism of BSR1 and theoretical calculation of PET. b) HPLC chromatograms of BSR1 without treatment of GSH (black), with GSH (100 equiv) treatment for 30 min as indicated at 25℃ (blue), and BS only (red). c) High-resolution mass spectrum analysis of reaction mixtures of BSR1 and GSH in CH3CN : PBS (4:6, v/v) at pH=7.4 for 40 min.


16

Page 17 of 29 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


5. Biological applications

5.1 Fluorescence Imaging of Endogenous GSH in Living Organisms.

The detection performance of BSR1 in vivo was preliminary evaluated upon a series of cell experiments. Cytotoxicity was first examined via standard MTT assays (Figure S7) to determine whether this probe is suitable for endogenous GSH detection in cells or not. The results demonstrated that BSR1 had almost no cytotoxicity to living cells even when the concentration of the probe reached up to 100 μM. The flow cytometry experiment also corroborated with this result and then further proved that intracellular GSH could induce the fluorescence changes of BSR1 (Figure S8). Based on the above results, the fluorescent imaging performance was investigated upon NCI-H1975 cell model which were divided into four groups (Figure 5a, b). Group A was control, and it is clear that the experiment group C exhibited an obvious fluorescence signal, while the experimental group B (pretreated with N-Ethylmaleimide, NEM) showed almost no fluorescence and experimental group D (pretreated with α-thioctic acid, α-LPA) presented significant fluorescence enhancement. The magnified image showed that the GSH in the cytoplasm


17


Page 18 of 29

was caputured primarily by BSR1. Additionally, in order to embody the application of BSR1 for evaluating hepatotoxicity, the imaging experiment in HepG2 cells were carried out, and the results were basically consistent with that in NCI-H1975 cells (Figure S9). Furthermore, BSR1 was also used to monitoring the GSH level in Kunming mice model via subcutaneous injection with its physiological saline solution (200 μL, 100 μM). As shown in Figure 5c, the fluorescence signal of mice after injection of BSR1 was clearly observed, and seen from the semiquantitative analysis Figure 5e, the changes in different group were similar to that in living cells imaging. Besides the living cells and animals studies, the ability of BSR1 to detect GSH in living tissues was also examined. The experiment was divided into three groups. The first group was control experiment, the liver tissues were incubated only with PBS, and its fluorescent imaging exhibited almost no fluorescence. The second group which pretreated with NEM (5 mM, 200 μL) for 30 min and then incubated with BSR1 (100 μM, 200 μL) for 30 min exhibited a slight fluorescence. While the third group which incubated with BSR1 (100 μM, 200 μL) for 30 min was observed a significant fluorescence enhancement (Figure 5d). All these positive


18

Page 19 of 29 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


results indicate that BSR1 is suitable for mapping endogenous GSH in living cells, animals and tissues.

Figure 5. a) Confocal microscope images of NCI-H1975 cells treated with BSR1 (10 μM, 1 h, λex=633 nm) in presence/abscence of NEM or α-thioctic acid. Left to right are fluorescence image of (A) Blank, (B) Cells pretreated with NEM (100 μM) for 1 h and incubated with probe BSR1 (10 μM) for 1 h, (C) Cells incubated with BSR1 (10 μM) for 1 h, (D) Cells pretreated with α-LPA (100 μM) for 1 h and incubated with BSR1 (10 μM) for


19


Page 20 of 29

1 h. Top to bottom is bright-field, dark-field, overlay bright-field and dark-field. b) Semiquantitative calculation of average fluorescence intensity in cells incubated with BSR1 conducted by ImageJ software. c) Imaging of endogenous GSH in Kunming Mice. a: Blank; b: pretreated with NEM and then injected with BSR1; c: treated with BSR1 only; d: pretreated with α-thioctic acid before injected with BSR1. d) Fluorescence images of BSR1 in normal liver tissue (left was blank, and middle was pretreated with NEM before incubated with BSR1, right was only incubated with BSR1. e) Semiquantitative analysis of fluorescence intensity in mice of Figure 5c).

5.2 Drug-induced Hepatotoxicity Evaluation in Mouse Models Hepatotoxicity diagnosability of BSR1 was evaluated by in vivo treatment of mice with drug-induced liver injury. Acetaminophen (APAP) was used as an effective antipyretic analgesic,50 which could be metabolically activated by cytochrome P450 enzymes to form a reactive metabolite (NAPQI).51 However, the overdose APAP may cause massive NAPQI, which would deplete GSH in the liver, bound to protein, and further cause serious liver damage.52 In vivo imaging showed that the mouse pretreated with overdose APAP


20

Page 21 of 29 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


by intraperitoneal injection (300 mg/kg) had lower fluorescence intensity than that of normal mice (Figure 6a). After in vivo imaging, the two mice were dissected, and the heart, liver, spleen, lung, and kidney were photographed under 633 nm exciting light. We observed that the fluorescence intensity of these organs from excess APAP pretreated mouse was significantly decreased than of the control group, especially for the liver (Figure 6b). The distribution of BSR1 in vivo after intraperitoneal administration was studied by HPLC (Figure S10, Table S3), and the results confirmed that the content of BSR1 in liver was higher than that in other organs, which was consistent with in vivo fluorescence images. As a result, the organ imaging experiments showed the good sensitivity and deep penetration of BSR1 in evaluating drug-induced hepatotoxicity.


21


Page 22 of 29

Figure 6 a) In vivo images of a mouse injected with probe BSR1 (100 μM) or acetaminophen (APAP, 300 mg/kg in 200 μL of PBS) via intraperitoneal injection for 20 min. (A) The mouse injected with BSR1 (BSR1 only) and (B) The mouse injected with BSR1 after being pretreatment with acetaminophen (APAP + BSR1). b) In vivo images of the organ (including heart, liver, spleen, lung, and kidney) which was dissected from a mouse injected with BSR1 (BSR1 only) and the mouse injected with BSR1 after being preinjected with acetaminophen (APAP + BSR1).

CONCLUSION

In summary, we have successfully presented a practical design strategy for GSH specific probe under the guidance of theoretical calculation. A novel thiophene vinyl BODIPY near-infrared fluorescence probe BSR1 based on this strategy was synthesized and exhibited excellent performance for the detection of GSH both in vitro and in vivo. More importantly, this probe could be used for visual monitoring drug-induced acute liver injury. We prospect that this theoretical calculation guided design strategy will provide an


22

Page 23 of 29 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


effective way for the precise synthesis of target-specific fluorescent probe, and change this research area from “trial-and-error” to concrete molecular engineering. ASSOCIATED CONTENT

Supplemented materials such as synthesis procedure details, characteristic data (1H NMR,

13C

NMR, high-resolution mass spectrum, and crystallographic data), and some

other data are presented in Supporting Information.

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]

*E-mail: [email protected]

Author Contributions ‡J.C.

and Z.W. contributed equally to this work.

Notes

The authors declare no competing financial interest.


23


Page 24 of 29

ACKNOWLEDGEMENTS

This work was supported by the National Natural Science Foundation of China (Grant Nos. 21572177, 21673173, and 21807087), China Postdoctoral Science Foundation (2017M623225), Key Research and Development Program of Shaanxi (2019KWZ-07), the Key Science and Technology Innovation Team of Shaanxi Province (2017KCT-37), Natural Science Basic Research Plan in Shaanxi Province of China (2016JZ004, 2018JQ3038),

and

the

Xi’an

City

Science

and

Technology

Project

(2017085CG/RC048(XBDX004)).

REFERENCES (1) Peng, J.; Samanta, A.; Zeng, X.; Han, S.; Wang, L.; Su, D.; Loong, D. T.; Kang, N. Y.; Park, S. J.; All, A. H.; Jiang, W.; Yuan, L.; Liu, X.; Chang, Y. T. Real-Time In Vivo Hepatotoxicity Monitoring through ChromophoreConjugated Photon-Upconverting Nanoprobes. Angew. Chem. Int. Ed. 2017, 56, 4165-4169. (2) Cheng, D.; Peng, J.; Lv, Y.; Su, D.; Liu, D.; Chen, M.; Yuan, L.; Zhang, X. De Novo Design of Chemical Stability Near-Infrared Molecular Probes for High-Fidelity Hepatotoxicity Evaluation In Vivo. J. Am. Chem. Soc. 2019, 141, 6352-6361. (3) Cheng, D.; Xu, W.; Yuan, L.; Zhang, X. Investigation of Drug-Induced Hepatotoxicity and Its Remediation Pathway with Reaction-Based Fluorescent Probes. Anal. Chem. 2017, 89, 7693-7700. (4) Liu, X.; Fan, N.; Wu, L.; Wu, C.; Zhou, Y.; Li, P.; Tang, B. Lighting Up Alkaline Phosphatase in Drug-Induced Liver Injury Using a New Chemiluminescence Resonance Energy Transfer Nanoprobe. Chem. Commun. 2018, 54, 12479-12482.


24

Page 25 of 29 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


(5) Zhang, J.; Jin, Z.; Hu, X. X.; Meng, H. M.; Li, J.; Zhang, X. B.; Liu, H. W.; Deng, T.; Yao, S.; Feng, L. Efficient Two-Photon Fluorescent Probe for Glutathione S-Transferase Detection and Imaging in Drug-Induced Liver Injury Sample. Anal. Chem. 2017, 89, 8097-8103. (6) Patel, S. J.; Luther, J.; Bohr, S.; Iracheta-Vellve, A.; Li, M.; King, K. R.; Chung, R. T.; Yarmush, M. L. A Novel Resolvin-Based Strategy for Limiting Acetaminophen Hepatotoxicity. Clin. Transl. Gastroenterol. 2016, 7, e153. (7) van Swelm, R. P.; Kramers, C.; Masereeuw, R.; Russel, F. G. Application of Urine Proteomics for Biomarker Discovery in Drug-Induced Liver Injury. Crit. Rev. Toxicol. 2014, 44, 823-841. (8) Nathwani, R. A.; Pais, S.; Reynolds, T. B.; Kaplowitz, N. Serum Alanine Aminotransferase in Skeletal Muscle Diseases. Hepatology 2005, 41, 380-382. (9) Yin, J.; Kwon, Y.; Kim, D.; Lee, D.; Kim, G.; Hu, Y.; Ryu, J. H.; Yoon, J. Cyanine-Based Fluorescent Probe for Highly Selective Detection of Glutathione in Cell Cultures and Live Mouse Tissues. J. Am. Chem. Soc. 2014, 136, 5351-5358. (10) McGill, M. R.; Jaeschke, H. Metabolism and Disposition of Acetaminophen: Recent Advances in Relation to Hepatotoxicity and Diagnosis. Pharm. Res. 2013, 30, 2174-2187. (11) Chan, R.; Benet, L. Z. Evaluation of the Relevance of DILI Predictive Hypotheses in Early Drug Development: Review of In Vitro Methodologies vs BDDCS Classification. Toxicol. Res. 2018, 7, 358-370. (12) Yue, Y.; Huo, F.; Cheng, F.; Zhu, X.; Mafireyi, T.; Strongin, R. M.; Yin, C. Functional Synthetic Probes for Selective Targeting and Multi-Analyte Detection and Imaging. Chem. Soc. Rev. 2019, 48, 4155-4177. (13) Song, A.; Feng, T.; Shen, X.; Gai, S.; Zhai, Y.; Chen, H. Fluorescence Detection of Glutathione S-Transferases in a Low GSH Level Environment. Chem. Commun. 2019, 55, 7219-7222. (14) Xiong, K.; Huo, F.; Chao, J. B.; Zhang, Y.; Yin, C. Colorimetric and NIR Fluorescence Probe with Multiple Binding Sites for Distinguishing Detection of Cys/Hcy and GSH In Vivo. Anal. Chem. 2019, 91, 1472-1479. (15) Zhang, J.; Ji, X.; Zhou, J.; Chen, Z.; Dong, X.; Zhao, W. Pyridinium Substituted BODIPY as NIR Fluorescent Probe for Simultaneous Sensing of Hydrogen Sulphide/Glutathione and Cysteine/Homocysteine. Sens. Actuat. BChem. 2018, 257, 1076-1082. (16) Niu, L. Y.; Guan, Y. S.; Chen, Y. Z.; Wu, L. Z.; Tung, C. H.; Yang, Q. Z. BODIPY-Based Ratiometric Fluorescent Sensor for Highly Selective Detection of Glutathione over Cysteine and Homocysteine. J. Am. Chem. Soc. 2012, 134, 18928-18931.


25


Page 26 of 29

(17) Yuan, L.; Lin, W.; Yang, Y. A Ratiometric Fluorescent Probe for Specific Detection of Cysteine over Homocysteine and Glutathione Based on the Drastic Distinction in the Kinetic Profiles. Chem. Commun. 2011, 47, 6275-6277. (18) Wang, H.; Zhou, G.; Gai, H.; Chen, X. A Fluorescein-Based Probe with High Selectivity to Cysteine over Homocysteine and Glutathione. Chem. Commun. 2012, 48, 8341-8343. (19) Liu, J.; Sun, Y. Q.; Huo, Y.; Zhang, H.; Wang, L.; Zhang, P.; Song, D.; Shi, Y.; Guo, W. Simultaneous Fluorescence Sensing of Cys and GSH from Different Emission Channels. J. Am. Chem. Soc. 2014, 136, 574-577. (20) Chen, J.; Jiang, X.; Zhang, C.; MacKenzie, K. R.; Stossi, F.; Palzkill, T.; Wang, M. C.; Wang, J. Reversible Reaction-Based Fluorescent Probe for Real-Time Imaging of Glutathione Dynamics in Mitochondria. ACS. Sens. 2017, 2, 1257-1261. (21) Liu, X.-L.; Niu, L.-Y.; Chen, Y.-Z.; Zheng, M.-L.; Yang, Y.; Yang, Q.-Z. A Mitochondria-Targeting Fluorescent Probe for the Selective Detection of Glutathione in Living Cells. Org. Biomol. Chem. 2017, 15, 1072-1075. (22) Wu, Q. H.; Wu, Y. C.; Yu, C. J.; Wang, Z. Y.; Hao, E. H.; Jiao, L. J. A Highly Selective Visible Light Excitable Boron Dipyrromethene Probe for Cysteine over Homocysteine and Glutathione Based on a Michael Addition Reaction. Sens. Actuat. B-Chem. 2017, 253, 1079-1086. (23) Umezawa, K.; Yoshida, M.; Kamiya, M.; Yamasoba, T.; Urano, Y. Rational Design of Reversible Fluorescent Probes for Live-Cell Imaging and Quantification of Fast Glutathione Dynamics. Nat. Chem. 2017, 9, 279-286. (24) Wang, F.; Zhou, L.; Zhao, C.; Wang, R.; Fei, Q.; Luo, S.; Guo, Z.; Tian, H.; Zhu, W. H. A Dual-Response BODIPY-Based Fluorescent Probe for the Discrimination of Glutathione from Cystein and Homocystein. Chem. Sci. 2015, 6, 2584-2589. (25) Kong, F.; Liang, Z.; Luan, D.; Liu, X.; Xu, K.; Tang, B. A Glutathione (GSH)-Responsive Near-Infrared (NIR) Theranostic Prodrug for Cancer Therapy and Imaging. Anal. Chem. 2016, 88, 6450-6456. (26) Gangopadhyay, M.; Mengji, R.; Paul, A.; Venkatesh, Y.; Vangala, V.; Jana, A.; Singh, N. D. P. RedoxResponsive Xanthene-Coumarin-Chlorambucil-Based FRET-Guided Theranostics for "Activatable" Combination Therapy with Real-Time Monitoring. Chem. Commun. 2017, 53, 9109-9112. (27) Liu, Y.; Zhu, S.; Gu, K.; Guo, Z.; Huang, X.; Wang, M.; Amin, H. M.; Zhu, W.; Shi, P. GSH-Activated NIR Fluorescent Prodrug for Podophyllotoxin Delivery. ACS Appl. Mater. Interfaces 2017, 9, 29496-29504. (28) Zhou, W.; Shultz, J. W.; Murphy, N.; Hawkins, E. M.; Bernad, L.; Good, T.; Moothart, L.; Frackman, S.;


26

Page 27 of 29 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


Klaubert, D. H.; Bulleit, R. F.; Wood, K. V. Electrophilic Aromatic Substituted Luciferins as Bioluminescent Probes for Glutathione S-Transferase Assays. Chem. Commun. 2006, 4620-4622. (29) Fujikawa, Y.; Urano, Y.; Komatsu, T.; Hanaoka, K.; Kojima, H.; Terai, T.; Inoue, H.; Nagano, T. Design and Synthesis of Highly Sensitive Fluorogenic Substrates for Glutathione S-Transferase and Application for Activity Imaging in Living Cells. J. Am. Chem. Soc. 2008, 130, 14533-14543. (30) Zhang, J.; Shibata, A.; Ito, M.; Shuto, S.; Ito, Y.; Mannervik, B.; Abe, H.; Morgenstern, R. Synthesis and Characterization of a Series of Highly Fluorogenic Substrates for Glutathione Transferases, a General Strategy. J. Am. Chem. Soc. 2011, 133, 14109-14119. (31) Maity, D.; Govindaraju, T. A Turn-On NIR Fluorescence and Colourimetric Cyanine Probe for Monitoring the Thiol Content in Serum and the Glutathione Reductase Assisted Glutathione Redox Process. Org. Biomol. Chem. 2013, 11, 2098-2104. (32) Chen, W.; Luo, H.; Liu, X.; Foley, J. W.; Song, X. Broadly Applicable Strategy for the Fluorescence Based Detection and Differentiation of Glutathione and Cysteine/Homocysteine: Demonstration in Vitro and in Vivo. Anal. Chem. 2016, 88, 3638-3646. (33) Gong, D.; Han, S. C.; Iqbal, A.; Qian, J.; Cao, T.; Liu, W.; Liu, W.; Qin, W.; Guo, H. Fast and Selective TwoStage Ratiometric Fluorescent Probes for Imaging of Glutathione in Living Cells. Anal. Chem. 2017, 89, 13112-13119. (34) He, L.; Yang, X.; Xu, K.; Yang, Y.; Lin, W. A Multifunctional Logic Gate by Means of a Triple-Chromophore Fluorescent Biothiol Probe with Diverse Fluorescence Signal Patterns. Chem. Commun. 2017, 53, 13168-13171. (35) Li, Z.; Ding, J.; Chen, C.; Chang, J.; Huang, B.; Geng, Z.; Wang, Z. Dual-Target Cancer Theranostic for Glutathione S-Transferase and Hypoxia-Inducible Factor-1α Inhibition. Chem. Commun. 2017, 53, 12406-12409. (36) Liu, X. L.; Niu, L. Y.; Chen, Y. Z.; Yang, Y.; Yang, Q. Z. A Multi-Emissive Fluorescent Probe for the Discrimination of Glutathione and Cysteine. Biosens. Bioelectron. 2017, 90, 403-409. (37) Xiang, H. J.; Tham, H. P.; Nguyen, M. D.; Fiona Phua, S. Z.; Lim, W. Q.; Liu, J. G.; Zhao, Y. An Aza-BODIPY Based Near-Infrared Fluorescent Probe for Sensitive Discrimination of Cysteine/Homocysteine and Glutathione in Living Cells. Chem. Commun. 2017, 53, 5220-5223. (38) Mulay, S. V.; Kim, Y.; Choi, M.; Lee, D. Y.; Choi, J.; Lee, Y.; Jon, S.; Churchill, D. G. Enhanced Doubly Activated Dual Emission Fluorescent Probes for Selective Imaging of Glutathione or Cysteine in Living Systems. Anal. Chem. 2018, 90, 2648-2654.


27


Page 28 of 29

(39) Sedgwick, A. C.; Han, H. H.; Gardiner, J. E.; Bull, S. D.; He, X. P.; James, T. D. The Development of a Novel AND Logic Based Fluorescence Probe for the Detection of Peroxynitrite and GSH. Chem. Sci. 2018, 9, 3672-3676. (40) Zhan, C.; Zhang, G. X.; Zhang, D. Q. Zincke's Salt-Substituted Tetraphenylethylenes for Fluorometric Turn-On Detection of Glutathione and Fluorescence Imaging of Cancer Cells. ACS Appl. Mater. Interfaces 2018, 10, 1214112149. (41) Yuan, Z. W.; Gui, L. J.; Zheng, J. R.; Chen, Y. S.; Qu, S. S.; Shen, Y. Z.; Wang, F.; Er, M.; Gu, Y. Q.; Chen, H. Y. GSH-Activated Light-Up Near-Infrared Fluorescent Probe with High Affinity to αvβ3 Integrin for Precise Early Tumor Identification. ACS Appl. Mater. Interfaces 2018, 10, 30994-31007. (42) Yin, C. X.; Xiong, K. M.; Huo, F. J.; Salamanca, J. C.; Strongin, R. M. Fluorescent Probes with Multiple Binding Sites for the Discrimination of Cys, Hcy, and GSH. Angew. Chem. Int. Ed. 2017, 56, 13188-13198. (43) Liu, X. G.; Qiao, Q. L.; Tian, W. M.; Liu, W. J.; Chen, J.; Lang, M. J.; Xu, Z. C. Aziridinyl Fluorophores Demonstrate Bright Fluorescence and Superior Photostability by Effectively Inhibiting Twisted Intramolecular Charge Transfer. J. Am. Chem. Soc. 2016, 138, 6960-6963. (44) Liu, X.; Cole, J. M.; Low, K. S. Solvent Effects on the UV-vis Absorption and Emission of Optoelectronic Coumarins: A Comparison of Three Empirical Solvatochromic Models. J. Phys. Chem. C 2013, 117, 14731-14741. (45) Nagano, T. Development of Fluorescent Probes for Bioimaging Applications. Proc. Jpn. Acad., Ser. B 2010, 86, 837-847. (46) Kowada, T.; Maeda, H.; Kikuchi, K. BODIPY-Based Probes for the Fluorescence Imaging of Biomolecules in Living Cells. Chem. Soc. Rev. 2015, 44, 4953-4972. (47) Blanksby, S. J.; Ellison, G. B. Bond Dissociation Energies of Organic Molecules. Acc. Chem. Res. 2003, 36, 255-263. (48) He, L.; Yang, X.; Xu, K.; Kong, X.; Lin, W. A Multi-Signal Fluorescent Probe for Simultaneously Distinguishing and Sequentially Sensing Cysteine/Homocysteine, Glutathione, and Hydrogen Sulfide in Living Cells. Chem. Sci. 2017, 8, 6257-6265. (49) Wang, M.; Zhang, Y.; Wang, T.; Wang, C.; Xue, D.; Xiao, J. Story of an Age-Old Reagent: An Electrophilic Chlorination of Arenes and Heterocycles by 1-Chloro-1,2-benziodoxol-3-one. Org. Lett. 2016, 18, 1976-1979. (50) Hinson, J. A.; Reid, A. B.; McCullough, S. S.; James, L. P. Acetaminophen-Induced Hepatotoxicity: Role of Metabolic Activation, Reactive Oxygen/Nitrogen Species, and Mitochondrial Permeability Transition. Drug Metab.


28

Page 29 of 29 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


Rev. 2004, 36, 805-822. (51) Toyoda, Y.; Tamai, M.; Kashikura, K.; Kobayashi, S.; Fujiyama, Y.; Soga, T.; Tagawa, Y. AcetaminophenInduced Hepatotoxicity in a Liver Tissue Model Consisting of Primary Hepatocytes Assembling around an Endothelial Cell Network. Drug Metab. Dispos. 2012, 40, 169-177. (52) James, L. P.; Mayeux, P. R.; Hinson, J. A. Acetaminophen-Induced Hepatotoxicity. Drug Metab. Dispos. 2003, 31, 1499-1506.

TOC


29

Precise Synthesis of GSH Specific Fluorescent Probe for

Recommend Documents