Molecular Engineering of Î±-Substituted Acrylate Ester Template for

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Molecular Engineering of #-Substituted Acrylate Ester Template for Efficient Fluorescence Probe of Hydrogen Polysulfides JingRu Guo, Sheng Yang, Chongchong Guo, Qinghai Zeng, Zhihe Qing, Zhong Cao, Jishan Li, and Ronghua Yang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b03755 • Publication Date (Web): 06 Dec 2017 Downloaded from http://pubs.acs.org on December 6, 2017

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Analytical Chemistry

Molecular Engineering of α-Substituted Acrylate Ester Template for Efficient Fluorescence Probe of Hydrogen Polysulfides Jingru Guo,† Sheng Yang,†,* Chongchong Guo,†Qinghai Zeng,§ Zhihe Qing,† Zhong Cao,† Jishan Li,‡ and Ronghua Yang†,‡,* †

School of Chemistry and Biological Engineering,Hunan Provincial Key Laboratory

of Materials Protection for Electric Power and Transportation, Changsha University of Science and Technology, Changsha, 410114, P. R. China ‡

State Key Laboratory of Chemo/Biosensing and Chemometrics, College of

Chemistry and Chemical Engineering, Hunan University, Changsha, 410082, P. R. China §

Department of Dermatology, Third Xiangya Hospital, Central South University,

Changsha, 410013, P. R. China

*To whom correspondence should be addressed:

E-mail: [email protected]; [email protected] Fax: +86-731-8882 2523

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ABSTRACT: In this article, hydrogen polysulfide (H2Sn)-mediated Michael addition/cyclization cascade reactions toward acrylate ester analogues had been exploited and utilized to construct novel and robust H2Sn-specific fluorescence probe for the first time. Through rational molecular engineering of the α-substituted acrylate ester

template,

the

trifluoromethyl-substituted

optimal acrylate

candidate ester

group

probe as

FP-CF3 recognition

containing unit

and

3-benzothiazol-7-hydroxycoumarin dye BHC as signal reporter can highly selectively detect H2Sn over other reactive sulfur species, especially biothiols including cysteine (Cys), homocysteine (Hcy)/glutathione (GSH), with a rapid and significant turn-on fluorescence response (less than 60 secondsfor response time and over 44-fold for signal-to-background ratio). The fast response and high selectivity of FP-CF3 for H2Sn could be attributed to a kinetically and spatially favored pentacyclic addition produced by the dual nucleophilic reaction of H2Sn with the CF3-substituted acrylate group. The big off-on fluorescence response is due to the pentacyclic intermediate results in the release of the highly fluorescent BHC. Moreover, it has been successfully applied in imaging of endogenous H2Sn fluctuation in living cells.


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INTRODUCTION Reactive sulfur species (RSS) are sulfur-containing molecules that exert crucial regulatory functions in biological systems.1-3Among of them, hydrogen sulfide (H2S) has been deeply studied and recognized as an important endogenous gasotransmitter in the past decade.4 In contrast, as one member of RSS family and H2S derivates, hydrogen polysulfides (H2Sn, n>1) have always attracted much less attention. Nevertheless, emerging evidences imply that H2Sn might act as the actual mediators in some physiological and/or pathological processes associated with H2S.5,6 It was also reported that H2Snhad higher potency than H2S during activating ion channels, tumor suppressors, and transcription factors.7 Although the significances of H2Sn molecules in redox biology have been acknowledged recently, their biological roles and detailed mechanisms of action are still poorly understood. Therefore, it is critical to develop effective tools for detection of H2Sn fluctuation in biological systems. Due to its advantages of high sensitivity, non-invasiveness, and spatiotemporal resolution capability, fluorescence molecular imaging is the promising approach for real time and in situ monitoring biologically important species of living organisms.8 Since the initial report by Xian et al,9 several fluorescent H2Sn probes have been designed by exploiting 2-fluoro-5-nitrobenzoic ester template as recognition unit.10-18 Although these probes proved to be highly selective, relatively high probe loading may be required during bioimaging applications due to the inevitable probe consumption by the competing reaction between template and intracellular biothiols. To address this issue, other H2Sn fluorescence probes have appeared based on some



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H2Sn-mediated specific reactions, including nucleophilic ring-opening reaction and benzodithiolone formation.19-23Despite of the fact that considerable efforts have been made as above, some limitations including low sensitivity, prolonged response times, and possible toxicity of relatively large recognition moiety are still present, hampering their further application to tackle biological issues. In response, novel sensing molecules that use easily accessible and biologically compatible recognition units with improved sensitivity and response times are needed. It is previous reported that acrylateester containing α,β-unsaturated carbonyl group is a powerful recognition template for biothiols Cys and Hcy via the conjugate addition/cyclization sequence.24 On the basis of this biothiols-triggered cascade reaction, numerous Cys fluorescent probes have been developed over the past few years.25-32 Inspired by this approach and taking into consideration of bis-nucleophilic feature of H2Sn, we envisioned that acrylate ester analogues should also be able to trap H2Sn to give out pentacyclic leaving compound, resulting in the release of the masked fluorophore and then fluorescence recovery as shown in Scheme 1. Moreover, taking the

advantage

of

steric

hindrance

and

electronic

effects,

conquest

of

biothiols-interference and improvement of response performances would be achieved through rational molecular engineering of substituted acrylate ester analogues. Toward this end, we initiated a program to study the reaction between α-substituted acrylate analogues and H2Sn for constructing novel and efficient H2Sn-specific recognition template for the first time. We herein report the design, synthesis, and evaluation of a series of new H2Sn fluorescent probes, namely FP Series, based on


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hydroxycoumarin fluorophore scaffold decorated with different acrylate derivates as recognition unit. One of them, FP-CF3 bearing trifluoromethyl-substituted acrylate group,showed selective and fast response toward H2Sn with high sensitivity and was successfully used for intracellular H2Sn imaging.

EXPERIMENTAL SECTION Synthesis of FP Series. The molecular structures and synthetic procedures of FP Series were depicted in Figure 1. 3-benzothiazol-7-hydroxycoumarin BHC was firstly synthesized following the literature method.33Subsequently, these probe molecules can be readily prepared from BHC with corresponding acrylic acid through only one step esterification reaction. Details for synthesizes and characterizations are attached to the Supporting Information. Spectrophotometric Experiments.Stock solutions of each probe (5 mM) in THF were prepared in a glovebox. A PB buffer solution (10 mM, pH 7.4) with THF as the cosolvent (H2O/THF = 1:1, v/v) was used for performing all spectroscopic measurements.Test solutions were prepared by blendingFP Series and appropriate analyte stock into a tube and then diluting the solution to 500 µL with the PB buffer solution.After incubation at room temperature for 5 min, the absorption or fluorescence spectra measurements were then performed. The fluorescence spectra were recorded at emission wavelength range from 470 to 580 nm with excitation wavelength of 460 nm. Fluorescent Imaging in Living Cells.For imaging of exogenous H2Sn in living cells, the HeLa cells were stained with 2.0 µM FP-H andFP-CF3 for 20 min before washed



three times with PBS, then incubated with 100 µM Na2S4 solution for another 10 min. In order to monitor the intracellular endogenous H2Sn levels upon lipopolysaccharides (LPS) stimulation, RAW264.7 cellswere stained with 5.0 µM FP-CF3 for 20 min under different conditions: (a) Cells only (control); (b–e) The cells were induced by LPS (2 mg/mL) for 0, 4, 8, and 12 h, respectively; (f) The cells were pretreated with DL-propargylglycine (200 µM) for 30 min, and then treated as (e).After rinsing with PBS buffer, these cells were exposed to imaging experiment on the two-photon confocal microscope. Two-photon imaging patterns were obtained from the green channel (480-560 nm) with two-photon excitation wavelength of 800 nm.

RESULTS AND DISCUSSION Design Strategy and Mechanism Verification. As we known, coumarin derivates are frequently-used fluorophores with excellent characteristics including tunable emission and easy functionalization and small molecular size,34 from which various bioimaging probes have been derived over the past years.35-39 Moreover, it is recently reported that some coumarin derivates possess admirable two-photon properties,40-42 which should be beneficial for bioimaging due to the advantages of two-photon microscopy (TPM) such as reduced photo-bleaching of fluorophores, minimal autofluorescence background, improved penetration depth, and less photodamage to biological samples.43-45Hence, 3-benzothiazol-7-hydroxycoumarinBHC was chosen as the fluorophore scaffold for constructing these designed probes by decorating with four different acrylate derivates, as depicted in Figure 1A. We anticipated that incorporation of these strong electron-withdrawing groups with BHC would


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significantly diminish the fluorescence via the PET and ICT mechanisms.46In contrast, tandem addition of H2Sn into β-carbon and carbonyl position of acrylate template would generate the two-photon excitable and highly fluorescent BHC. In order to verify our design strategy, photophysical properties of BHC and probe FP-H were firstly explored after synthesis and structural characterization. Indeed, as expected, FP-H is essentially non-fluorescent (ФF = 0.006) in the aqueous buffer solution, whereas BHC is highly fluorescent (ФF= 0.50) with maximum emission peak around 490 nm under the same conditions. Significant fluorescence differences between FP-H and BHC imply the possibility of realizing the “switch-on” fluorescent sensing approach. We then proceeded to investigate the response capability of FP-H toward H2Sn. Figure 2A illustrated that treatment of FP-H with Na2S4 solutionas H2Sn donor elicited a dramatic fluorescence enhancement in pH 7.4 buffer solution at room temperature, accompanied with an obvious green color fluorescence appearance under a hand-held UV lamp. Notably, real-time reaction kinetics of FP-H with H2Sn in Figure 2B revealed that rapidly increased fluorescence intensity of FP-H reached a plateau in less than 1 min upon the addition of Na2S4 solution, indicating that acrylate ester template possesses the capture ability for H2Sn to quickly release the masked fluorophore.As a matter of fact, the response time of FP-H is shorter than that of most aforementioned H2Sn probes, which usually fulfill the response in the range of 10-30 min. It demonstrates that acrylate template would be benefit for the real-time detection of H2Sn. In order to provide some insight into the mechanism of H2Sn-induced fluorescence



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enhancement of FP-H, we studied the reaction product of FP-H and Na2S4 by mass spectrometry analysis. MS data in Figure S1 revealed that new peaks appeared at m/z 119 and 295, indicating that pentacyclic compound 1,2-dithiolan-3-one was generated through Michael addition/cyclization cascade reaction to afford strongly fluorescent hydroxycoumarin derivate for a turn-on fluorescence response. Furthermore, superposition of thin-layer chromatography (TLC) spot and the normalized fluorescence emission spectra of the BHCand FP-H + Na2S4 also provide complementary proofs that the fluorescent product is the same as BHC (Figure S2,S3). Molecular Engineering for Performance Evolution. As mentioned issue that biothiols-triggered

cascade

reaction

toward

unsubstituted

acrylate

ester,

unsurprisingly, FP-H exhibited poor selectivity toward H2Sn over biothiols, especially Cys, at their physiologically relevant concentrations (Figure S4), consistent with the previously reported resultsby Feng et al.26It implies that, although FP-H has sensing capacity for H2Sn, serious interference from intracellular biothiols would be an annoying shortcoming when bioimaging applications. To deal with such selectivity drawback, we focused on the substituted influence of α-carbon position on the acrylate ester template. Expectantly, increasing the steric crowding around α-carbon by substituted group would hinder the cascade reaction toward sterically demanding biothiols such as GSH, Cys, and Hcy. Nevertheless, for H2Sn with small molecular size, such steric hindrance effect would be out of action. In addition, the electronic perturbation from substituent may also influence response performances of probes by


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changing the electrophilicity of acrylate ester template. To verify above hypothesis, three analogues, FP-CF3 bearing electron-withdrawing substituent of trifluoromethyl in the α-carbon position of acrylate ester template, FP-CH3 bearing electron-donating substituent of methyl, andFP-Ph with phenyl substituent were synthesized. With theseFP series in hand, their response performances toward H2Sn over biothiols were firstly evaluated. As can been seen from Figure 3A that all other nonfluorescent FP series can also be obviously lighted up after treatment with Na2S4 solution in pH 7.4 PB buffer, despite they exhibited inferior fluorescence enhancement ratio than unsubstituted FP-H. More importantly, as our expected, the interference from biothols is significantly suppressed under its biologically relevant concentration for FP-CH3,FP-CF3, and FP-Ph, indicating that substituents play crucial role in improving selectivity of acrylate ester template toward H2Sn via steric hindrance. Real-time reaction kinetics of other FP serieswithH2Sn were then examined by recording the variations in the emission intensity of wavelength at 490 nm. Figure S5 illustrated that all of them possessed fast response characteristic toward H2Sn similar as FP-H. It is noteworthy that electron effect of substituent exerts a certain degree of influence on the response rate of acrylate ester template. As depicted in Figure 3B, the time-dependent processes of their response toward H2Sn follow first-order kinetics with different observed rate constant k,47 the relevant observed rate constants of FP-CF3 bearing electron-withdrawing trifluoromethyl substituent (kFP-CF3 = 6.0 × 10-2 s-1) is about 1.5-fold larger than that of unsubstituted FP-H (kFP-H= 4.0 × 10-2 s-1),



whereas FP-CH3 bearing electron-donating substituents exhibited inferior response rate toward H2Sn than FP-H. It manifests that electron-withdrawing substituent would be beneficial for acrylate ester template to capture H2Sn. To shed light on these substituent effects from a theoretical basis, computational calculations for these probes were carried out (Table 1, Figure S6). To our delight, the introduction of electron-withdrawing trifluoromethyl group increased the positive charge at the enone β-carbon of template (−0.253 eV of FP-CF3 versus −0.371 eV of FP-H), which would make it more electrophilic to handily react with nucleophilic sulfhydryl of H2Sn. In sharp contrast, electron-donating substituent exhibited opposite effect on the electrostatic charges of enone of template, indicative of inert reactivity of FP-CH3relative to that of FP-H. For FP-Ph, the electrostatic charge of enone of template was slight influenced by phenyl substituent due to the mutually offsetting result between electron-donating and conjugated effect of phenyl, which is in good agreement the similar reaction kinetics between FP-Ph and FP-H. In addition, the bond dissociation energies (BDES) of the ester bond gradually magnify from 249.38 to 280.65 kJ mol-1 following the order the trend ofFP-CH3 < FP-Ph

Molecular Engineering of Î±-Substituted Acrylate Ester Template for

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