Molecular Engineering of Aqueous Soluble ... - ACS Publications

Dec 4, 2015 - Key Laboratory of Photochemical Conversion and Optoelectronic Materials, Technical Institute of Physics and Chemistry Chinese. Academy o...
1 downloads 0 Views 556KB Size
Download PDF
Subscriber access provided by UNIV OF NEBRASKA - LINCOLN

Article

Molecular Engineering of Aqueous Soluble Triarylboron-CompoundBased Two-Photon Fluorescent Probe for Mitochondria H2S with AnalyteInduced Finite Aggregation and Excellent Membrane Permeability Jun Liu, Xudong Guo, Rui Hu, Xinyang Liu, Shuangqing Wang, Shayu Li, Yi Li, and Guoqiang Yang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.5b04248 • Publication Date (Web): 04 Dec 2015 Downloaded from http://pubs.acs.org on December 14, 2015

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 free 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 accessible to all readers and 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.

Analytical Chemistry 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 8

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

Analytical Chemistry

MolecularEngineeringofAqueousSolubleTriarylboronǦCompoundǦ BasedTwoǦPhotonFluorescentProbeforMitochondriaH2Swith AnalyteǦInducedFiniteAggregationandExcellentMembranePerǦ meability JunLiu,[a]XudongGuo,[a]RuiHu,[a]XinyangLiu,[b]ShuangqingWang,[a]ShayuLi*[a],YiLi*[b] andGuoqiangYang*[a] a

BeijingNationalLaboratoryforMolecularSciences,KeylaboratoryofPhotochemistry,InstituteofChemistry, ChineseAcademyofSciences,Beijing100190(China).

b

KeyLaboratoryofPhotochemicalConversionandOptoelectronicMaterials,TechnicalInstituteofPhysicsand ChemistryChineseAcademyofSciences,Beijing100190(China).

 ABSTRACT:Hydrogensulfide(H2S)isamultifunctionalsignalingmoleculethatparticipatesinmanyimportantbiologiǦ calprocesses.Herein,byfunctionalizingtriarylboronwithcyclenanddiphenylamine,wesynthesizedTABǦ1,TABǦ2and TABǦ3forH2Srecongnizationbyrationaldesignofmolecularstructures.Amongthem,aqueoussolubleTABǦ2possesses excellentproperties,includinglargetwoǦphotonactioncrosssection,membranepermeabilityandcaneffectivelycomplex with Cu2+. The complex of TABǦ2ǦCu2+ can selectively detect H2S with an instant response and mitochondria targeted. Moreover,theH2SǦinducedfiniteaggregationofindicatorsenhancestheirphotostabilityandcausesvariationofthefluoǦ rescencelifetime.TABǦ2ǦCu2+hasalsobeensuccessfullyappliedfortheMitochondriaH2SimaginginNIH/3T3fibroblast cellsbyTPMandFLIM.

INTRODUCTION Hydrogen sulfide (H2S), a wellǦknown toxic gas with the odor of rotten eggs, is regarded as the third gaseous transmitterfollowingnitricoxide(NO)andcarbonmonǦ oxide(CO).Asasignalmolecule,H2Splaysanimportant role in various biological processes, including regulation ofneurotransmission,mediationofcellgrowth,inhibition ofinsulinsignaling,modulationofredoxstatusandantiǦ inflammation.1Ǧ3Somestudieshavealsorevealedthatthe maladjustmentofH2Sisassociatedwiththesymptomsof Alzheimer’sdisease,Down’ssyndrome,diabetes,andliver cirrhosis.4Ǧ7 Therefore, detection of the H2S level in vitro and in vivo is of important to the realization of related biologicalprocessesanddiagnosisofsomediseases. Severalmethodshavebeendevelopedforthedetection ofH2S,includingsulfideǦselectiveelectrodes,gaschromaǦ tographyandthemostcommonmethyleneblueassay.8Ǧ10 However, these methods are often limited by low temǦ poralresolution,destructionofsample,laborǦcostsample preparation and poor compatibility with live cells. FluoǦ rescentprobesreceivesconsiderableattentionbecauseof their high specificity, sensitivity and nonǦdestruction in live cells or tissues, and some of them have been develǦ opedcapableofdetectingH2Sinlivecells.11Ǧ18TherecogniǦ tionproceedsmostlythrougheitherH2SǦspecificMichael reactions 11Ǧ13 or the reduction reaction of azide with H2S

to amine.16Ǧ18 However, most of these reactions need sevǦ eraltensofminutesorevenhourstocompletethedetecǦ tionprocessduetoslowreactionrate,andsomeofthem alsoshowpoorselectivityforH2S.AnewclassoffluoresǦ cent probes for H2S based on the fast reaction between sulfide anion and copper (II) ion resolve the problem of slow detection process, but most of them show poor seǦ lectivity.19Ǧ21 Nagano et al developed an azamacrocylicǦ fluorescein complex with Cu2+ showed high selectivity and sensitivity for H2S, but the poor membrane permeaǦ bility of this complex limits its practical application in biological system.19 Besides these drawbacks in detection of H2S, conventional fluorescent probes also suffer from the common challenge of photostability under continual irradiation, especially with a laser excitation source, and photostabilityisasageneralrequirementforfluorescence liveǦcellimagingaswellasalowǦconcentrationofprobes. How to develop good fluorescent probes for H2S with avoidingthesedrawbacks?Recently,ourgroupdeveloped an attractive strategy, named analyteǦinduced finite agǦ gregation,forinvivoATPimaging.22Inthisapproach,the luminophore molecules form finite aggregates upon inǦ teraction with nonchromophoric analyte ATP, in which the luminophore molecules are isolated by surrounding nonchromphoric analytes, suppressing the aggregationǦ causedquenching(ACQ)effect.24,25 Moreover,theaggreǦ gates characteristic of multiluminophore provide excelǦ

ACS Paragon Plus Environment

Analytical Chemistry

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

lentphotostability.22,23AnothersolutiontosolvethephoǦ photobleachingproblemisusingtwoǦphotonmicroscopy (TPM).26Ǧ29 TPM uses nearǦinfrared (NIR) photons as the excitation source, which can not only decrease the photodamage, but also increase the penetration depth, minimizethebackgroundemissionandweakenthelight scattering,andrecently,Kim,H.M.andcoǦworkershave reported a twoǦphoton fluorescent probes for in vivo imaging H2S.29 Furthermore, fluorescence lifetime microscopy(FLIM)canavoidtheeffectoftheexperiment conditionsonthefluorescenceintensity,whichmakesthe quantitative measurement of H2S possible. To obtain good fluorescent probes for H2S, we attempt to take advantages of the analyteǦinduced finite aggregation, TPM and FLIM to create novel fluorescent probes for in vitroandinvivoH2Simaging. ɕǦConjugated triarylboron compounds containing electron donor groups exhibit intramolecular charge transfer (ICT) property and a large dipole moment as a result of strong electron deficiency of boron, endowing them large twoǦphoton absorption cross section.30Ǧ33 It also has been demonstrated that ɕǦconjugated triarylboron compounds can be fluorescent probes in divers research fields with high fluorescence quantum yield and excellent photostability.22, 32 Considering outstanding features of ɕǦconjugated triarylboron derivatives and the fast reaction capability between sulfide anion and copper (II) ion, “turnǦon” twoǦphoton fluorescent probes based on triarylboron compounds functionalized with different numbers of cyclen and diphenylaminesubstituentsattheparapositionofphenyl groups TABǦ1, TABǦ2 and TABǦ3 were designed and synthesizedinthiswork.Thecomplexationofcyclenwith Cu2+ inhibits the ICT process and Cu2+ acts as a paramagnetic center, 19 resulting in fluorescence quenching. The reaction of H2S with Cu2+ releases the fluorescent probes and restores their fluorescence, accomplishingthedetectionofH2S.Moreover,additionof H2S can induce the formation of the finite aggregates of fluorescent probes through hydrogen bonding with cyclens of different probe molecules. The coǦaggregation ofH2Swithprobemoleculespreventstheapproachofthe probe molecules and thus avoids ACQ, and endows the aggregates excellent photostability. The microenviroenment of the aggregates causes variation of thefluorescencelifetimeoftheprobe,givingpossibilityof usingFLIMtodetectH2S(Scheme1). ThedesignofTABǦ1,TABǦ2andTABǦ3isbasedonthe following considerations: 1) Strong electron donor substituents of the cyclen and diphenylamine groups are selected attaching to the triarylboron core to ensure the effective luminescence character of ICT compounds. 2) The formation of the complex of cyclen with Cu2+ can silence the emission of probes through both the inhibitionoftheICTprocessandtheintramolecularPET process from diphenylamine to the complex, and the complex acts as a receptor to react with H2S selectively andsensitively,makingtheemissionofprobesrestored.3) The cell membrane permeability is regulated and

Page 2 of 8

controlled by varying the ratio of the hydrophilic cyclen and the hydrophobic diphenylamine in each probe molecule. The synthesis and characterization of TABǦ1, TABǦ2 and TABǦ3 are described in the Supporting Information. TABǦ1 and TABǦ2 bearing three and two cyclengroupsexhibitexcellentwaterǦsolubility,andTABǦ 3 can hardly be dissolved in water because of less hydrophiliccomponentofcyclen.

Scheme1.The structuresofTABǦ1,TABǦ2 andTABǦ3 and the schematic representation of the H2S detectionprocesses.

EXPERIMENTSECTION General information. All chemical reagents were purchased from J˂K (Beijing, China) and used without furtherpurification.Absorptionspectrawererecordedon HitachiUVǦ3010.Thefluorescencespectrawereobtained on Hitachi FǦ7000. Dynamic light scattering (DLS) was performed on ALV/DLS/SLSǦ5022F. Cells were analyzed usingaconfocalmicroscope(OLYMPUSFV1000ǦIX81for singleǦphoton excited fluorescence, Leica TCS SP8 for twoǦphoton excited fluorescence). 1H NMR spectra were obtained on BrukerAvance III 400 H (400 MHz) spectrometers. A detailed description of the synthesis of TBBT, TDBT andTBDTisprovidedintheSupportingInformation. SynthesisofCompoundTABǦ1 Compound TBBT (174 mg, 0.1mmol) was dissolved in CH2Cl2 (5ml) and trifluoroacetic acid (0.2 ml, 2.8mmol) was added. The reaction mixture was stirred for 24 h (untilnostartingcompoundwasindicatedbyTLC(SiO2, MeOH), evaporated, and dried under vacuum to yield a viscous solid. Then the solid was dissolved in MeOH (10ml) and poured onto the column of strongly basic anion exchanger(Ion exchanger III; Merck).The column was washed with 100 ml MeOH. The solution was dried under vacuum to give 83mg (99%) of TABǦ1 as yellow solid. 1HNMR(400MHz,CD3OD)Ɉ:1.86(s,18H),1.91(s, 6H), 2.53Ǧ2.74 (m, 36H), 3.26 (m, 12H) , 6.50 (s, 6H). 13C

ACS Paragon Plus Environment

Page 3 of 8

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

Analytical Chemistry

NMR(75MHz,CD3OD)Ɉ=150.7,141.9,138.4,131.0,128.5, 122.3,116.5,116.0,65.3,60.1,48.2,45.1,30.3,22.4,20.2,19.5, 18.7ppm.MALDIǦTOF(m/z):Calcd.ForC48H81BN12836.7, found837.9. SynthesisofCompoundTABǦ2andTABǦ3 Compound TDBT (144 mg, 0.1mmol) was dissolved in CH2Cl2:CH3OH (5:2) (7ml) and hydrochloric acid (2 ml) wasadded.Thereactionmixturewasstirredfor48h(unǦ til no starting compound was indicated by TLC (SiO2, MeOH), evaporated, and dried under vacuum to yield yellowsolid.ThenthesolidwasdissolvedinMeOH(10ml) and poured onto the column of strongly basic anion exǦ changer (Ion exchanger III; Merck). The column was washedwith100mlMeOH.Thesolutionwasdriedunder vacuum to give 82mg (99%) of TABǦ2 as yellow solid.1H NMR(400MHz,CD3OD)Ɉ:1.84(s,6H),1.94(s,6H),2.01 (s, 6H), 2.62Ǧ2.82 (m, 24H), 3.34 (m, 8H), 6.56 (m, 6H), 6.98(m,6H),7.22(m,4H). 13CNMR(75MHz,CD3OD)Ɉ = 151.3, 148.6, 147.6, 142.1, 141.8, 128.9, 124.4, 122.7, 121.9, 116.6, 51.1, 22.4, 22.0 ppm. MALDIǦTOF (m/z): Calcd. For C52H72BN9833.6,found834.8. SynthesisofCompoundTABǦ3 Compound TABǦ3 was synthesized according to the same procedure as that of TABǦ2. Compound TBDT (115 mg,0.1mmol),TABǦ3(87mg,99%)wasobtainedasYellow solid. 1H NMR (400 MHz, DMSO) Ɉ: 1.89 (s, 6H), 1.97 (s, 6H), 2.07 (s, 6H), 2.68Ǧ2.92 (m, 12H), 3.48 (m, 4H), 6.56Ǧ 6.61 (m, 6H), 6.97Ǧ7.06(m, 12H), 7.24Ǧ7.28(m, 8H). 13C NMR(75MHz,CD3OD)Ɉ=149.4,143.7,131.0,125.4,123.8, 119.0,116.5,53.0,47.3,46.9,24.0ppm.MALDIǦTOF(m/z): Calcd.ForC56H63BN6830.5,found831.7. PreparationofTABǦ1ǦCu2+andTABǦ2ǦCu2+ 10ɑM TABǦ1ǦCu2+ and TABǦ2ǦCu2+ in PBS were preǦ pared by titrating with 2.4eq and 1.6eq Cu2+, then the complextioncanbefurtherusedforH2Sdetection. Cell culture and viability assay. Mousefibroblastcells (NIH/3T3) were cultured in Dulbecco’s Modified Eagle Medium (DMEM) with glucose (4.5 g/L), LǦglutamine, sodiumpyruvate,and 10% fetal bovine serum (FBS). The cellswereplatedonglassbottomeddishesat37°Cunder 5% CO2 atmosphere before imaging. Cell images were obtained using a confocal microscope FV1000ǦIX81 and were analyzed with FV10ǦASW software. NIH/3T3 cells, preǦwashed twice, were incubated with 10ɑM in cultured medium without FBS at 37°C under 5% CO2 for certain time. Then the cells were washed with PBS to remove unboundedprobesforsixtimesbeforeinsituimagingby OlympusFV1000ǦIX81confocallaserscanningmicroscopy using oil objective, with excitation by 405nm laser or 760nm,and500Ǧ550nmemissionlightwascollected.Cell viability was measured by MTT assay. Briefly, NIH/3T3 cells were cultured on a 96Ǧwell plate at a density of 10,000 cells in each well. After 24 h of incubation, the medium was replaced with 200 ɑL of fresh medium containing varied concentrations of TABǦ2ǦCu2+ (from 0uM to 40uM) and the cells were cultured another 24h. The medium was replaced with fresh medium (200ɑL) containing MTT (0.5mg/mL) and incubated for 4 h. The

supernatantwasremoved,and100ɑLofDMSOwasadded to each well to dissolve the formed formazan and the absorbance of the solution was measured to assess the relative viability of the cells.The absorbance values (A) were read at a wavelength of 490 nm. Relative cell viability was expressed as: A/A0 × 100%, where A0 is the absorbance of the experimental group and A0 is the absorbanceofthecontrolgroup.

RESULTSANDDISSCUSSION The absorption and emission spectra of TABǦ1, TABǦ2 and TABǦ3 were measured in different polarity solvents (Figures S1), and the significant bathochromic shifts of the fluorescence spectra with increasing solvent polarity indicatethatthesecompoundsshowtypicalICTcharacter asthetriarylboroncompoundsweinvestigatedbefore.30Ǧ33 The absorption and fluorescence spectra of these comǦ pounds in DMSO are shown in Figure 1. The absorption and emission maxima of the synthesized triarylboron compounds show slightly bathochromic shift with the increased number of diphenylamine in each molecule, which can be rationalized to the crossǦconjugation beǦ tweendiphenylamineandthetriarylboroncore.ThefluoǦ rescence quantum yield of TABǦ1, TABǦ2 and TABǦ3 in DMSOweredeterminedtobe0.501,0.501,and0.565bya Hamamatsu absolute PL quantum yield spectrometer C11347, indicating all these compounds maintain the inǦ tenseemissioneveninhighpolarenvironment.ThetwoǦ photon action cross section (ȶɈ) of TABǦ1, TABǦ2 and TABǦ3 was further determined by their TPǦexcited fluoǦ rescence measurements with fluorescein as the reference (Figure S2).  ȶɈmax increases with the number of the diǦ phenylamine substituent in molecular structure, which can also be ascribed to the enhance crossǦconjugation contributedbydiphenylamine.

 Figure 1. (a) UV absorption and (b) fluorescence spectra of TABǦ1,TABǦ2andTABǦ3inDMSO(10ɑM).

ThecomplexationofthewatersolubleTABǦ1andTABǦ 2 with Cu2+ and their further reaction with sulfide ion werefirstinvestigatedinvitro.A10mMPBSphysiological buffer (pH = 7.4) was used in these measurements. The absorption and fluorescence spectra of TABǦ1 and TABǦ2 with differentamount of Cu2+ are shown in Figure 2 and FigureS3.UponadditionofCu2+,theabsorptionbandat the longer wavelength region and the fluorescence emission decrease monotonously, implying the combination of cyclen with Cu2+ (Figures 2a). Further addition of sulfide into the solution, the absorption and fluorescence spectra of TABǦ1 and TABǦ2 are restored

ACS Paragon Plus Environment

Analytical Chemistry

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

gradually because the reaction of sulfide ion with Cu2+ makes TABǦ1and TABǦ2 comeback (Figures 2d and S3d), giving a measurement of sulfide. Moreover, the addition of sulfide causes obvious background absorption with typical character of Rayleigh scattering, indicating the formation of aggregates, which was validated by the experimentsofdynamiclightscattering(DLS)(FigureS4). The origin of aggregation may be ascribed to the hydrogen bonding of sulfide with cyclen of different molecules,15whichisfurthervalidatedbytheH2SǦinduced aggregateformationofTABǦ1andTABǦ2intheabsenceof Cu2+ (Figures S5 and S6). The coǦaggregation of nonchromophoric sulfide and fluorescent molecules prevents the interaction between fluorescent molecules, thus avoiding the ACQ effect in the aggregates. The emission maximum of TABǦ2ǦCu2+ shows a slightly hypsochromicshiftupontheadditionofH2S(FigureS3d), indicating a lower polarity of the aggregates than that of thephysiologicalbuffer,whichcanberationalizedtothe extrusion of water and electrolytes during aggregation. The emission maximum of TABǦ1ǦCu2+ does not shift much during the addition of H2S, which can be ascribed to the less polar sensitivity of TABǦ1 than TABǦ2. The microenvironment should also affect the fluorescence lifetime of probes, therefore, the measurements of the fluorescencelifetimeofTABǦ1ǦCu2+andTABǦ2ǦCu2+upon addition of H2S will give a new readout signal. Figure S7 displays fluorescence decay traces of the TABǦ1ǦCu2+ and TABǦ2ǦCu2+systemsinthepresenceofdifferentamounts ofH2S.AdditionofH2Sshortensthefluorescencelifetime, which can be attributed to the reaction Cu2+Ǧcomplexes withH2SandtheH2SǦinducedfiniteaggregateformation. Moreover, the decrement of the fluorescence lifetime is relatedtotheaddingamountsofH2S,indicatingthatthe formedfiniteaggregatesarenotaltogethersimilarinthe presence of different amount of H2S. Obviously, the fluorescence lifetime can be a new measure for the detection of H2S, which is validated by the FLIM experiments. The detection limit of TABǦ2ǦCu2+ by the means of fluorescence and lifetime is  calculated 47nM and123nM,respectively.

Page 4 of 8

Figure 2. (a) UV absorption and (b) fluorescence spectra changes of TABǦ1 (10uM) upon addition of Cu2+. (c) UV absorptionand(d)fluorescencespectrachangesofTABǦ1Ǧ Cu2+ in the presence of different amount of H2S. ɐexc = 345nm,PH=7.4,10mMPBS.

TheselectivityandthephotostabilityofTABǦ1ǦCu2+and TABǦ2ǦCu2+ for H2S were examined in vitro. TABǦ1ǦCu2+ and TABǦ2ǦCu2+ show the best selectivity for H2S over other biologically relevant sulfur (glutathione, homocysteineandcysteine),inorganicsulfurcompounds (NaSCN, Na2SO3, Na2S2O3 and Na2S2O8), and a reducing condition (sodium ascorbate) (Figure 3a). Moreover, exposing TABǦ1ǦCu2+ and TABǦ2ǦCu2+ to a series of biologically reactive oxygen species (ROS) and reactive nitrogen species (RNS), including H2O2, tertǦbutyl peroxide (tBuOOH), hypochlorite (OCl‫)ޤ‬, hydroxyl radicals(ȉOH),andsuperoxide(O2‫)ޤ‬,doesnottriggerany fluorescenceenhancementtothesameextentasexposure toH2S.TABǦ1ǦCu2+andTABǦ2ǦCu2+alsodisplaynegligible responses to the metal ions Ca2+, K+, Na+, Mg2+, which is commonly existed in vivo. Meanwhile, the photostability ofTABǦ1andTABǦ2inthepresenceofH2Sisbetterthan that in the absence of H2S and no obviously improve in the presence of sodium ascorbate, which can roule out the affect of H2S as a reducing agent ((Figure 3c, Figure S9bandFigureS10).Therefore,thegoodphotostabilitybe ascribedbytheH2SǦinducedformationofaggregates.22





Figure 3. (a) Fluorescence responses of TABǦ2ǦCu2+ to variousionsatPH=7.4.1)K+(1mM),2)Ca2+(1mM),3)Na+ (1mM), 4) Mg2+ (1mM), 5) F‫( ޤ‬1mM), 6) ClǦ (1mM), 7) Br‫ޤ‬ (1mM), 8) I‫( ޤ‬1mM), 9) ClO‫( ޤ‬500ɑM), 10) O2‫( ޤ‬1mM), 11) •OH (1mM), 12) H2O2 (10mM), 13) ONOO‫( ޤ‬50ɑM), 14) sodium ascorbate (10mM), 15) SCN‫( ޤ‬1mM), 16) SO32‫ޤ‬ (1mM),17)S2O32‫(ޤ‬1mM),18)S2O82‫(ޤ‬1mM),19)Cys(100ɑM), 20) Hcy (100ɑM), 21) GSH (10mM), 22) S2‫( ޤ‬100ɑM). (b) PhotographofTABǦ2ǦCu2+inthepresenceandabsenceof H2S(10ɑM).(c)Signalloss(%)offluorescenceemissionof TABǦ2 in the absence (black) and presence (red) of H2S (100ɑM)withincreasingtheexposuredose(ɐ=365nm).

ACS Paragon Plus Environment

Page 5 of 8

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

Analytical Chemistry

CellǦmembrane permeability is another important factor for evaluating the performance of a biological fluorescent probe, because the probe with poor permeability can only be used through microinjection, electroporation, or scrape loading to load inside cells, whichareharmful.34ThemembranepermeabilityofTABǦ 1, TABǦ2 and TABǦ3 was analyzed by incubating living NIH/3T3 cells with them for different time interval. NIH/3T3 cells incubated with TABǦ2 for only 5 min exhibit strong fluorescence, indicating the excellent permeability of TABǦ2 (Figure S11b). NIH/3T3 cells incubated with TABǦ1 and TABǦ3 even for 1 h present no and very week fluorescence, respectively (Figure S11a, c), demonstratingthepoorpermeabilityofTABǦ1andTABǦ3. The dissimilar permeability of the probes can be rationalized to the ratio of the hydrophilic and hydrophobic substituents. The hydrophilicity of TABǦ1 containing three cycen groups is too strong to penetrate the hydrophobic domains of cellular membrane. The strong hydrophobic TABǦ3 containing three diphenylamine groups needs DMSO (1%) as a coǦsolvent to dissolve it in physiological buffer, and a longer incubation time of >1h is required. The proper ratio of hydrophilicity and lipophilicity of TABǦ2 endows its excellent cellular membrane permeability, making it an ideal candidate of fluorescent probes for in vivo H2S detection. Biocompatibility is also considered as one of the foreǦ mostpropertyforaprobetobepracticallyusedinliving cells.ThecytotoxicityofTABǦ2ǦCu2+onNIH/3T3cellswas evaluated by a standard 3Ǧ(4, 5ǦdimethylǦ2Ǧthiazolyl)Ǧ2,5Ǧ diphenyltetrazolium bromide (MTT) assay. The applicaǦ tion of TABǦ2ǦCu2+ shows no apparent effects on the cell viability,evenatahighconcentrationof40.0ɑM(Figure S12), demonstrating a good biocompatibility of TABǦ2Ǧ Cu2+ with NIH/3T3 cells. Although the heavy metal ion Cu2+ is toxic to organisms, the strong chelating ability of cyclenwithCu2+preventsbindingofCu2+withproteinsin cells,providingTABǦ2ǦCu2+lowcytotoxicity. The reality of for sensing intracellular sulfide was analyzed by confocal fluorescence microscopy experiments.NIH/3T3cellswerefirstincubatedwithNa2S (500ɑM)for30minutesandthenwithTABǦ2ǦCu2+(10μM) for another 5 minutes after removing the extracellular Na2S. Upon singleǦphoton (405 nm) and TP (760 nm) excitation, bright fluorescence images were observed, while only a very weak fluorescence images could be obtained for the cells incubated with only TABǦ2ǦCu2+ (FigureS13).Thefluorescenceimagesofthesamesample under singleǦphoton excitation and TP excitation exhibit the same features, substantiating that TABǦ2ǦCu2+ is a promising“turnǦon”TPfluorescentprobeforH2Sinliving cells.ThecellsincubatedwithTABǦ2ǦCu2+andNa2Swere furtherstainedwithMitoǦTrackerDeepRedFM(MDRF), a commercial mitochondria specific fluorescent dye, for 30minforcolocalizationanalysis.Thefluorescenceimage collected from green channel for TABǦ2 is highly overlapped in cytoplasm with that from red channel for

MDRF (Rr = 0.87, Figure 4), demonstrating that TABǦ2Ǧ Cu2+localizesandstainspreferentiallyinmitochondria.



Figure 4. Confocal fluorescence images of NIH/3T3 cells treated with TABǦ2ǦCu2+(10ɑM) and MDRF (100nM) and thenincubatedwithNa2S(200ɑM).(a)Greenchannelfor TABǦ2ǦCu2+.(b)RedchannelforMDRF.(c)Mergedimage ofaandb. TABǦ2ǦCu2+wasfurtherappliedtomeasureendogenous H2S in mitochondria. Cystathionine ɅǦsynthase (CBS) 35 was chosen to catalyze the in situ H2S production. NIH/3T3cellswerepretreatedwith0.01mg/mLCBSfor8 h at 37 and 20°C, and then with TABǦ2ǦCu2+ 5 min seǦ quently.AbrightTPfluorescenceimagewasobtainedfor the cells incubated at 37°C upon excitation with the femtosecondlaserof760nm,indicatingthatTABǦ2ǦCu2+ candetectendogenousH2S(Figure5a).Incontrast,negliǦ giblefluorescencewasobservedforthecellsincubatedat 20°CbyTPMundersameexperimentalconditions(Figure 5b), which can be attributed to the decreased enzymatic activity of CBS when the temperature is far below the normalbodytemperature(37°C).TheintracellularphotoǦ stabilityofTABǦ2wasalsoexaminedusinga405nmlaser by increasing the number of scans. The fluorescence imǦ agesoflivingNIH/3T3cellsobtainedwith1,10,20,30and 40scansasshowninFigureS14.Evenafter40scanswith laser power of 100 ɑW, the bright florescence image can stillbeobtained.



Figure 5. Confocal fluorescence images of NIH/3T3 cells incubatedwithCBSinDMEMat(a)37°Cand(b)25°Cfor 8h and then incubated with TABǦ2ǦCu2+ for 5min. Excitation wavelength: ɐexc =760 nm. Emission wavelength:500–550nm Asdiscussedinthepreviousparagraph,H2Scaninduce the aggregation of fluorescent probes, giving different fluorescence lifetimes. Considering the fluorescence lifeǦ timeismoreindependentoftheexperimentalconditions, FLIM mayprovidemore accurateinformationforthe inǦ tracellular H2S distribution. Therefore, the spatial distriǦ bution of the endogenous H2S produced by CBS was exǦ

ACS Paragon Plus Environment

Analytical Chemistry

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

aminedwiththeFLIMtechniqueusingTABǦ2ǦCu2+asthe fluorescent probe. The FLIM image of intracellular H2S distributionisnearlyinaccordwiththatfromthefluoresǦ cence intensity technique, but more detail information can be received by the FLIM technique (Figure 6). The FLIM image shows that H2S distributes uniformly in miǦ tochondria,whichcannotbeobtainedfromtheintensity image because the fluorescence intensity is strongly afǦ fectedbythedistributionofprobemoleculesevenunder the same experiment condition. The shorter lifetime at theperipheryofcellsmayresultfromlowpolarityofthe lipid cellǦmembrane or strong aggregation caused by the cellǦmembrane.Evidently,FLIMcanprovidemoreprecise intracellularH2SinformationthanthefluorescenceintenǦ sity image because the FLIM technique rules out the efǦ fectofprobedistribution.

Page 6 of 8

We are grateful for funding from the National Basic ReǦ search Program (2013CB834703, 2013CB834505, 2011CBA00905and2009CB930802)andtheNationalNatǦ ural Science Foundation of China (grant nos21233011, 91123033,21273252,21205122,21261160488and21072196).

ASSOCIATEDCONTENT SupportingInformation ThisinformationisavailablefreeofchargeviatheInternetat http://pubs.acs.org/.

AUTHORINFORMATION CorrespondingAuthor *EǦmail:[email protected](G.Y.)˗[email protected] (S.L.);[email protected](Y.L.). 

REFERENCES

 Figure6.ConfocalfluorescenceimagesofliveNIH/3T3cells incubated with CBS in DMEM at 37°C for 8h and then 2+ incubated with TABǦ2ǦCu for 5min a) Fluorescence intensity image. b) Fluorescence lifetime image. Excitation wavelength:ɐexc=760nm.Emissionwavelength:500–550nm.

CONCLUSIONS AseriesoftriarylboronderivativesbasedontheconsiderǦ ationforTPfluorescentprobesforH2SwithhighperforǦ mance were synthesized. The aqueous solubility and the cellǦmembranepermeabilityofthefluorescentprobescan be tuned by regulating the number of the hydrophilic cyclen group and the hydrophobic diphenylamine subǦ stituentwithineachmolecule.ThefluorescenceofTABǦ1 and TABǦ2 is quenched by effective complexation with Cu2+ in aqueous solution. The complexes of TABǦ1ǦCu2+ andTABǦ2ǦCu2+showhighspecificityfordetectionH2Sin vitro. The H2SǦinduced finite aggregation of fluorescent probes endows them with excelent photostability and superior tolerance to environmental electrolytes. The inǦ stantly specific response to H2S, the excellent cellǦ membrane permeability, the low cytotoxicity and the preferentially mitochondria distribution of TABǦ2ǦCu2+ make it an excellent fluorescent probe for the realǦtime imaging H2S in mitochondria. TABǦ2ǦCu2+ has been sucǦ cessfully applied for imaging the H2S distribution in NIH/3T3cellsbyTPMandFLIM,andtheFLIMtechnique canprovidesmoreinformationforthedistributionofH2S in mitochondria than the fluorescence intensity techǦ nique.

ACKNOWLEDGMENT

(1)Abe,K.;Kimura,H.J.Neurosci.1996,16,1066.Kaneko,Y.; (2)Kimura,Y.;Kimura,H.;Niki,I.Diabetes2006,55,1391.Kabil, O.;Banerjee,R.J.Biol.Chem.2010,285,21903. (3)Wang,C.Szab,Proc.Natl.Acad.Sci.U.S.A.2009,106,21972. (4) K. Eto, T. Asada; K. Arima; T. Makifuchi; H. Kimura; BioǦ chem.Biophys.Res.Commun.2002,293,1485; (5) W. Yang; G. Yang; X. Jia; L. Wu; R. Wang; J. Physiol. 2005, 569,519˗ (6) Kamoun, P.; Belardinelli, M.ǦC.; Chabli, A.; Lallouchi, K.; ChadefauxǦVekemans,B.Am.J.Med.Genet.PartA,2003,116,310; (7)S.Fiorucci;E.Antonelli;A.Mencarelli;S.Orlandi;B.Renga; G. Rizzo; E. Distrutti; V. Shah; A. Morelli; Hepatology 2005, 42, 539. (8)Kroll,A.V.;Smorchkov,V.;Nazarenko,A.Y.Sens.Actuators B1994,21,97. (9) Mitchell, T. W.; Savage, J. C.; Gould, D. H. J. Appl. Toxicol. 1993,13,389. (10)Kuban,V.;Dasgupta,P.K.;Marx,J.N.Anal.Chem.1992,64, 36. (11) Qian, Y.; Karpus, J.; Kabil, O.; Zhang, S. Y.; Zhu, H. L.; Banerjee,R.;Zhao,J.;He,C.Nat.Commun.2011,2,495. (12) Liu, C.; Pan, J.; Li, S.; Zhao, Y.; Wu, L. Y.; Berkman, C. E.; Whorton,A.R.;Xian,M.Angew.Chem.Int.Ed.2011,50,10327.; (13) Liu, C.Chen, W.; Shi, W.;Peng, B.; Zhao, Y.; Ma, H.;Xian, M.,J.Am.Chem.Soc.2014,136,7257. (14)Chen,Y.;Zhu,C.;Yang,Z.;Chen,J.;He,Y.;Jiao,Y.;He,W.; Qiu,L.;Cen,J.;Guo,Z.,Angew.Chem.Int.Ed.2013,52,1688. (15) Evans, N. H.; Beer, P. D., Angew. Chem. Int. Ed. 2014, 53, 11716. (16)Lippert,A.R.;New,E.J.;Chang,C.J.J.Am.Chem.Soc.2011, 133,10078. (17) Peng, H.; Cheng, Y.; Dai, C.; King, A. L.; Predmore, B. L.; Lefer,D.J.;Wang,B.Angew.Chem.Int.Ed.2011,50,9672. (18) Chen, S.; Chen, Z.; Ren, W.; Ai, H.ǦW. J. Am. Chem. Soc. 2012,134,9589. (19)Sasakura,K.;Hanaoka,K.;Shibuya,N.;Mikami,Y.;Kimura, Y.; Komatsu, T.; Ueno, T.; Terai, T.; Kimura, H.; Nagano, T., J. Am.Chem.Soc.2011,133,18003; (20)Choi,M.G.;Cha,S.;Lee,H.;Jeon,H.L.;Chang,S.ǦK.,Chem. Commun.2009,7390. (21) Qu, X.; Li, C.; Chen, H.; Mack, J.; Guo, Z.; Shen, Z., Chem. Commun.2013,49,7510. (22)Li,X.;Guo,X.;Cao,L.;Xun,Z.;Wang,S.;Li,S.;Li,Y.;Yang, G.,Angew.Chem.Int.Ed.2014,53,7809. (23)Leung,C.W.T.;Hong,Y.;Chen,S.;Zhao,E.;Lam,J.W.Y.; Tang,B.Z.,J.Am.Chem.Soc.2013,135,62.

ACS Paragon Plus Environment

Page 7 of 8

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

Analytical Chemistry

(24)W.Z.Yuan,P.Lu,S.Chen,J.W.Y.Lam,Z.Wang,Y.Liu,H. S.Kwok,Y.Ma,B.Z.Tang,Adv.Mater.2010,22,2159. (25)C.Zhang,Y.Yuan,S.Zhang,Y.Wang,Z.Liu,Angew.Chem. Int.Ed.2011,123,698. (26)Kim,H.M.;Cho,B.R.Chem.ǦAsianJ.2011,6,58. (27)Kim,H.M.;Cho,B.R.Acc.Chem.Res.2009,42,863. (28)Zhou,L.;Zhang,X.;Wang,Q.;Lv,Y.;Mao,G.;Luo,A.;Wu, Y.;Wu,Y.;Zhang,J.;Tan,W.,J.Am.Chem.Soc.2014,136,9838. (29)Bae,S.K.;Heo,C.H.;Choi,D.J.;Sen,D.;Joe,E.ǦH.;Cho,B. R.;Kim,H.M.,J.Am.Chem.Soc.2013,135,9915. (30) Feng, J.; Tian, K.; Hu, D.; Wang, S.; Li, S.; Zeng, Y.; Li, Y.; Yang,G.,Angew.Chem.Int.Ed.2011,50,8072. (31)Liu,X.;Li,S.;Feng,J.;Li,Y.;Yang,G.,Chem.Commun.2014, 2778. (32)Liu,J.;Guo,X.;Hu,R.;Xu,J.;Wang,S.;Li,S.;Li,Y.;Yang,G., Anal.Chem.2015,87,3694. (33)Guo,X.;Zhang,X.;Wang,S.;Li,S.;Hu,R.;Li,Y.;Yang,G., Anal.Chim.Acta2015,869(0),81. (34)A.Ray,Y.ǦE.KooLee,T.Epstein,G.Kim,R.Kopelman,AnaǦ lyst2011,136,3616. (35)Kimura,H.AminoAcids2011,41,113.



ACS Paragon Plus Environment

Analytical Chemistry

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 8 of 8

ForTOConly



8

ACS Paragon Plus Environment