Potassium‐ion‐selective fluorescent sensors to detect cereulide, the emetic toxin of B. cereus, in food samples and HeLa cells

Po assium-Ion-Selec i eFluo escen Senso s To De ec Ce eulide, he Eme ic Toxin o B. ce eus,inFood Samples and HeLa Cells Jos8Ga c&a-Cal o,[a] Sa u nino Ibeas,[a] E a-Cla a An jn-Ga c&a,[a] Tom#sTo oba,*[a] Ge a do Gonz#lez-Aguila ,[b] Wilson An unes,[c] Elo&sa Gonz#lez-La ado,[d] and Mjnica L. Fana aga*[d] This pape isdedica ed o he memo y o he la e D .S e ano Ma caccini 1. In oduc ion Ce eulide is an ionopho e oxin p oduced by speci ic s ains o Bacillus ce eus.[1] B. ce eus causes wo di e en ypes o ood disease:dia heal and eme ic synd ome.[2] Thedia heal o m is de elopeda e he inges ion o ood con amina ed wi h spo es o ege a i ecells able o p oduce en e o oxins du ing ege a i eg ow h in he small in es ine. The eme icsynd ome is usually associa ed wi h he inges ion o oods u s con ami- na ed wi h he p e o med oxin, ce eulide, p oduced du ing he g ow h o bac e ia in ood;[3] he e o e, in oxica ionwi h ce eulide could be p e en ed by he ea ly de ec ion o he oxin in ood. B. ce eus can p oduce highly esis an s uc u es called endospo es, which a e capable o su i ing ocooking empe a u es. Bacillus endospo es p esen in oods u s can apidly ge mina e and p oduce hei oxins,especially du ing ood chilling o ood hea ing.[4] B. ce eus is aubiqui ous agen in na u e, and hei spo es can be ound as ana u alcon ami- nan in se e al ood p oduc s,especially mea s, ege ables, and milk.[5] The eme icsynd ome is gene ally associa edwi h ice o o he s a ch- ich p oduc s such as pas a and po a o- based p oduc s.[6] The eme ic oxin is acyclic depsipep ide[7] wi h he s uc u e [d-O-Leu-d-Ala-l-O-Val-l-Val]3,which is close- ly ela ed o he s uc u e o alinomycin, ano he na u ally oc- cu ing cyclic depsipep ideand po assium-ca ion ionopho e wi h he e ame s uc u e d-a-hyd oxyiso ale yl-d- alyl-l- lac yl-l- alyl [d-O-Hyi-d-Val-l-O-Lac-l-Val]3(Figu e 1).[8] The ce - We epo he de elopmen o new chemical p obes o ce eu- lide, a oxic me aboli e p oduced by speci ic s ains o Bacillus ce eus, h ough displacemen o po assium ca ions om ap e- o med speci ic complex and asubsequen change in he luo- escence emission. Fo his pu pose, we designed luo escen p obes o po assium ca ions ha we e sui able o displace- men assays wi h ce eulide om o ganic ex ac s. The luo es- cence de ec ion o na u al ce eulide in ice samples was ach- ie ed by using syn he ic ce eulide as a e e ence and apo assi- um luo escen epo e ,and his was ound o be use ul as apo able and as me hod o he in si u de ec ion o ce eu- lide in ood ex ac s.Tos udy he a e o ce eulide in li e cells, we designed ap ocedu e ha was sui able o li e-cell mic os- copy imaging o HeLa cells by compa ing he cellula loca ion o he po assium luo ogenic p obe, which s ained in acellula endolysosomes, in he absence and p esence o ce eulide;we concluded ha in he p esence o ce eulide, he luo escence o he p obe was dec easedbecause o complexa ion o he po assiumions by ce eulide. Figu e 1. Chemical s uc u eso ce eulide and alinomycin. [a] J. Ga c&a-Cal o, D .S.Ibeas,E.-C. An jn-Ga c&a, P o . T. To oba Depa men o Chemis y,Facul y o Science Uni e si y o Bu gos, 09001Bu gos (Spain) E-mail: o [email protected] [b] D .G.Gonz#lez-Aguila Cen e o Applied Pho onics, INESC TEC Rua do Campo Aleg e, Dp o. F&sica 4169-007 Po o (Po ugal) [c] D .W.An unes Labo a j iodeB oma ologia edeDe esa Bioljgica (LBDB) do Ex8 ci o A .D . Al edo Bensafflde, 1849-012 Lisboa (Po ugal) [d] E. Gonz#lez-La ado, P o . M. L. Fana aga G upo de Nanomedicina-IDIVAL Uni e sidad de Can ab ia, San ande 39011(Spain) E-mail:[email protected] Suppo ing In o ma ion and he ORCID iden i ica ion numbe (s) o he au ho (s) o his a icle can be ound unde h ps://doi.o g/10.1002/ open.201700057. T2017 The Au ho s. Published by Wiley-VCH Ve lag GmbH &Co. KGaA. This is an openaccessa icleunde he e mso he C ea i e Commons A ibu ion-NonComme cial-NoDe i s License, which pe mi s useand dis ibu ion in any medium, p o ided he o iginal wo k is p ope ly ci ed, he use is non-comme cial and no modi ica ions o adap a ions a e made. Chemis yOpen 2017,6,562 –570 T2017 The Au ho s. PublishedbyWiley-VCH Ve lag GmbH &Co. KGaA, Weinheim562 DOI:10.1002/open.201700057 eulide oxin is usually iden i ied by liquid ch oma og aphy and mass spec ome y,which equi e dedica ed acili ies.[9] Ce eu- lide ac s as apo assium-ca ion ionopho eand is able o dis- up he ansmemb ane po en ial in mi ochond ia o euka y- o ic cells; his leads o mi ochond ial degene a ion and subse- quen ly o cell dea h.[10] Owing o ce eulide oxici y, apid and po able de ec ion me hods o sc een he p esence o p e- o med oxin in oods u s a e equi ed o p e en he occu - ence o ood-bo ne ou b eaks by eme ic oxin. 2. Resul s and Discussion Fluo escen p obes a e agood al e na i e o he de ec ion o oxins o chemical h ea s.[11] Owing o he abili y o ce eulide o complex po assium ca ions, asui able me hod o i s de ec- ion om ex ac s o biological o ood samples should be adisplacemen mechanismo he po assium ca ion om ap e- o med speci ic complex ha would gi e ise o achange in he luo escenceemission.Following his idea, we designed a luo escen p obe o po assium ha is sui able o displace- men assays wi h ce eulide in o ganic sol en s. Because ce eu- lide is no soluble in wa e ,a equisi e o he luo ogenic p obe is ha i ha e good pe o mance in o ganic o mixed o - ganic–aqueous sol en s. The e o e, we selec ed ape ylenemo- noimideas he luo escen epo e [12] and aK +-selec i e phe- nylaza[18]c own-6-la ia -e he [13] o a iazac yp and[14] as he ecogni ion uni s and bonded bo h moie ies in e e y case by Suzuki coupling (Scheme 1). Wi h he simple and mo e accessible JG76 luo escen p obe,wedesigned he luo escencedisplacemen assays on he basis o Figu e 2. The displacemen assays we e ini ially pe o med by using comme cial alinomycin ins ead o less- a ailablece eulide. In his way, he JG76 p obe was weakly lu- o escen ine hanol, bu in he p esence o apo assium chlo- ide solu ion, ad ama ic inc ease in he luo escencewas ob- se ed owing o po assium-ion complexa ion. As expec ed, by he addi ion o a alinomycin solu ion, he p e ioussolu ion becameweakly luo escen .The di e ences in luo escencea e desc ibed in Figu e 2b. On he o he hand, p obe JG103 showed e y low solubili y in e hanol and a e y small inc ease in he luo escencein he p esence o po assium ca ions in all o he es ed sol en s; he e o e, we conside ed ha i s u ili y o he de ec ion o ce - eulide was e y limi ed and i s s udy was discon inued. Po assi- um ca ionsa e usually mixed wi h o he ions in biological sam- ples, so we checked ca e ully he ac ion o di e se ca ions and anions ha could in e e ein he displacemen assays wi h JG76. Wi h espec oalkali-me al and alkaline-ea h me al ca - ions, he JG76 p obe showed ala ge inc easein luo escence in he p esence o po assium ca ions wi h o al selec i i y wi h espec o commonly ound physiological ca ions(i.e. Li+,Na +, Mg2+,Ca 2+,and NH4+)aswell as less-common, albei physio- logical ca ions (i.e. S 2+,Rb +,Cs +)bu also showedala gein- c ease in luo escencein he p esence o he highly oxic Be2+ and Ba2+ca ions, which a e no no mally ound in biological luids (Figu e 3) excep in oxicological assays, o which he JG76 p obe could be o in e es . The assays we e pe o med by dissol ing compound JG76 in E OH o aconcen a ion o 50 mmand he ca ions in wa e o aconcen a ion o 5mm; hen, he ca ion solu ion (10 mL) was added o he JG76 solu ion (0.5 mL), and changes in he colo and luo escencewe e s udied. Wi h espec o hea y-me al ca ions,someacidic ca ions such as Sn2+and Pb2+showed ahigh inc ease in luo escencein he p esence o JG76, and o alesse ex en ,some o he ca ions,including Zn2+,Cu 2+,Fe 3+, Scheme1.Syn hesis o JG76 and JG103. Figu e 2. a) Quali a i e andb)quan i a i e luo escence displacemen assays o JG76,po assium ca ions, and alinomycin. Chemis yOpen 2017,6,562 –570 www.chemis yopen.o g T2017 The Au ho s. PublishedbyWiley-VCH Ve lag GmbH &Co. KGaA, Weinheim563 Sc3+,Al 3+,Hg 2+,Au 3+,and Pd2+,also showed an inc easein luo escenceunde he same condi ions (Figu e 4) and in he p esenceo ala ge excess amoun o s ong acids (pH<5) o oxidan s. None o he common anions [i.e. F@,Cl @,B @,I @, BzO@(Bz=benzoyl), NO3@,H 2PO4@,HSO4@,AcO@,CN @,SCN@] ga e asigni ican change in colo o luo escence(see he Sup- po ing In o ma ion, Figu e S98). In apH7 bu e ed solu ion [JG76,25mmin E OH/H2O(7:3 / ), 20 mm4-(2-hyd oxye hyl)-1-pipe azinee hanesul onic acid (HEPES)],mos o he in e e en ca ions including Fe3+,Sn 2+, Be2+,and Cu2+(5 equi ) did no p omo e achange in he luo- escenceo JG76, bu he sensi i i y o K+,Ba 2+,and Pb2+ e- mained unchanged,and hey showedahigh inc ease in he luo escenceo JG76; he e o e, he only eal in e e en s we e he nonphysiological Ba2+and Pb2+ca ions (Figu e5). Job’s plo analysis, by ep esen ing XJG76(Fo@F) e sus XJG76 (whe e XJG76 =mole ac ion o JG76), a o ded he s oichiome- y o he complex, om he maximumposi ion a XJG76 =0.5, as a1:1 complex. The calcula ed quan um yield o he JG76 p obe in e hanol is F[JG76] (E OH)=0.04:0.01 and o he co espondingpo assium complex, F[JG76–K+](E OH)= 0.17:0.01; he inc ease is F/F0=4, whichissu icien o he expe imen al assays. The li e imeo JG76inE OH was ound o be 3.6 ns, and he e was no changein he li e ime o he po assium complex. The limi o de ec ion o K+,ob ained by linea eg ession o a i a ion o a5mmsolu ion o JG76 in E OH and K+by inc easing he K+concen a ion om 0.01 o 0.2 mmand by measu ing he inc easein he luo escence emission, was calcula ed as 0.06 mm,wi h ap obabili y o alse posi i e and alse nega i e lowe han 5%.The JG76–K+com- plex was s udied by p epa inga2mmsolu ion o K(CF3SO3)in E OH and by inc easing he concen a ion o JG76 om 0 o 10 mmwi hou changing he concen a iono K +in solu ion. The equilib ium cons an (K)o he JG76–K+complex was cal- cula ed by i ing he luo escence i a ion plo in e hanol by nonlinea leas -squa e eg ession; he esul s we e compa ed by pe o ming i s he i a iono K +wi h JG76 and hen ha o JG76 wi h K+,and he same esul s we e ob ained. The i - ing calcula ion o he complexa ion cons an s was epea ed h ee imes o gi e logK(JG76+K+)=6.34:0.04. To alida e he esul s, Job’s plo analysisand he equilib ium cons an o he JG76–K+complex we e also s udied andcalcula ed in benzylalcohol, BnOH, and compa able esul swe eob ained: a1:1 complex and logK(JG76–K+)=6.11:0.03 (Figu e 6). We nex checked he sui abili y o he sys em o he p ac ical de- ec ion o alinomycin by calcula ing he limi o de ec ion o alinomycin in e hanol. The e o e, 0.75 equi alen s o K+ CF3SO3@was added o a5mmsolu ion o JG76 in e hanol. Then, he concen a iono alinomycin was inc eased by suc- cessi e addi ions, and he luo escencespec um was egis- e ed. Reg ession o he i a ion plo o 5 mmJG76and 3.75 mmK+in E OH wi h alinomycin by dec easing he luo- escence emission ga e ade ec ion limi o 0.54 mm,wi h Figu e 3. Colo and luo escence assays o JG76 and alkali-me al and alka- line-ea hme al ca ions (1 equi ). Figu e 4. Colo and luo escence assays o JG76 and selec ed hea y-me al ca ions(1equi ). Figu e 5. a) Colo and luo escence assays o JG76 and selec ed ca ions in abu e ed pH 7solu ion. b) Quan i a i e luo escence assays o JG76 and se- lec edca ions in e hanol(le ) and abu e ed pH 7solu ion ( igh ). Figu e 6. Fi ed i a ion plo o a2mmsolu ion o K+and JG76 by luo es- cenceemission in E OH and BnOH.Inse :Job’s plo o JG76and K+by luo- escenceemission in E OH and BnOH. Chemis yOpen 2017,6,562 –570 www.chemis yopen.o g T2017 The Au ho s. PublishedbyWiley-VCH Ve lag GmbH &Co. KGaA, Weinheim564 ap obabili y o alse posi i eand alse nega i e lowe han 5% (see Figu e S123). The JG76 luo escen p obe was hen eady o i a ionwi h syn he ic ce eulide, which was p epa ed acco ding o he p o- cedu e o Bies a-Pe e s e al.[15] The las s ep, consis ing o he cycliza ion o he linea H-(l-Val-d-O-Leu-d-Ala-l-O-Val-l-Val-d- O-Leu-d-Ala-l-O-Val-l-Val-d-O-Leu-d-Ala-l-O-Val)-OH depsipep- ide, is shown in Scheme 2. Pu i iedce eulide was checked by NMR spec oscopy and HRMS (MALDI), and he esul s we e iden ical o he epo ed da a o ce eulide. We i s checked he sui abili y o he sys em o he p ac ical de ec iono syn he icce eulide by calcula ing he limi o de ec ion o ce eulide in e hanol. In an expe imen simila o ha p e iously explained, K+CF3SO3@(0.75 equi ) was added o a5mmsolu ion o JG76 in e hanol. Then, he concen a ion o ce eulide was inc eased by successi e addi- ions, and he luo escencespec um was egis e ed. Reg es- sion o he i a ion plo o 5 mmJG76 and 3.75 mmK+in E OH wi h ce eulide by dec easing he luo escence emission ga e ade ec ion limi o 0.21 mm,wi h ap obabili y o alse posi i e and alse nega i e lowe han 5% (Figu e 7);in his case, he de ec ion limi o ce eulide was lowe han he alue ob ained o alinomycin. Acomple e desc ip ion o he sys em was hen pe o med by calcula ing he equilib ium cons an s be ween alinomycin/ ce eulide and he po assium ca ions in compe i i e equilib ia wi h JG76, o which we p e iously ob ained he binding con- s an s.Equilib ium cons an swe e i s measu ed in E OH solu- ion by inc easing he concen a ion o he p obe in he p es- ence o he analy e. Wepe o med i a ions o cons an con- cen a ionso 2mmK+and 20 mm alinomycin o ce eulide wi h JG76 (0 o 20 mm). In he case o alinomycin, he expe i- men s a ed wi h solu ions o alinomycin (V) and po assium ca ions (K) in e hanol so ha he complex (VK) was p oduced in he equilib ium. Upon adding he JG76p obe (abb e ia ed S), i o med acomplex wi h ee K+and c ea ed anew com- plex (SK) and eplaced he p e ious complex (VK). In his way, he concen a ion o VK dec eased, whe eas he concen a ion o Vinc eased. The luo escencein ensi y only depended on JG76 and he JG76–po assium complex. To sol e he equa ions co esponding o he sys em, apossible app oxima ion could be done, CV@[VK]&CV,inwhich CVis he concen a ion o ali- nomycin, and his simpli ica ion is mo e ealis ic i he ini ial a io o V/K is as high as possible. In his way, an equa ion o desc ibe he equilib ia was ob ained [Eqs. (1)–(5)]: SþKK1 K! KSK ð1Þ VþKK2 K! KVK ð2Þ K1¼½SKA ½SA½KA¼½SKA ðCS@½SKAÞðCK@½SKA@½VKAÞð3Þ K2¼½VKA ½VA½KA¼½VKA ðCV@½VKAÞðCK@½SKA@½VKAÞð4Þ ½SKA¼ CSþCKþ1þK2CV K1 0/ @ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi CSþCKþ1þK2CV K1 0/ 2@4CSCK 2ð5Þ in which CS,CK,and CVa e he concen a ions o JG76, K+,and alinomycin, espec i ely. By aking in o accoun he mass and luo escence balances o all equilib ia and pe o ming he es s se e al imes wi h di e en ini ial a ios o V/K (1:1, 1:0.25, and1:0.1), he bes i - ing o he ob ained equa ion was ound a a1:0.1 a io, a which he app oxima ion was mos alid. By al e na ing he concen a ions o alinomycin and ce eulide in se e al expe i- men s, i ed i a ion plo swe e ob ained o bo h alinomy- cin and ce eulide in E OH and BnOH (Figu e 8). F om he a e ageo he h ee i a ions, we ob ained he ollowing complexa ion equilib ium cons an s: logK( alinomy- cin–K+,E OH)=5.97:0.01, logK( alinomycin–K+,BnOH)= 4.98:0.01, logK(ce eulide–K+,E OH)=5.99:0.01, K(ce eu- lide–K+,BnOH)=5.01:0.01. Wi h he alues o K2and K1, he concen a ion o [SK]eq could be de e mined, and wi h hese da a he concen a ion o he specieswas calcula ed by using Equa ions (6)–(9). Wi h he da a p e iously ob ained, he amoun so eagen s and he p opo ion o he complex exis - ing du ing he i a ioncouldbe ep esen ed (Figu e 9). Scheme2.Ce eulide syn hesis. Figu e 7. Linea eg ession o he i a ion plo o 5 mmJG76 and 3.75 mmK+ in E OH wi h inc easing amoun so ce eulide by dec easing he luo es- cence emission o he sys em. Chemis yOpen 2017,6,562 –570 www.chemis yopen.o g T2017 The Au ho s. PublishedbyWiley-VCH Ve lag GmbH &Co. KGaA, Weinheim565 ½SAeq ¼CS@½SKAeq ð6Þ ½VKAeq ¼ CVþCK@½SKAeq þ1 K2ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi CVþCK@½SKAeq þ1 K2 0/ 2@4CVðCK@½SKAeqÞ 2 ð7Þ ½VAeq ¼CV@½VKAeq ð8Þ ½KAeq ¼CK@½SKAeq @½VKAeq ð9Þ To alida e he me hod,wecalcula ed he binding cons an o alinomycin wi h po assium ca ionsbyci cula dich oism ollowing he epo ed me hodology.[16] S a ing wi h aconcen- a ion o 30 mmo alinomycin in E OH, he concen a ion o he po assium ca ions was g adually inc eased up o 3.75 equi alen s. This concen a ion o alinomycin was selec - ed because o he op imal concen a ion o ollow changes in he dich oism signal, albei i is oo high o pe o m sa e meas- u emen s wi h oxic ce eulide.Bychecking he i ing a di e - en wa eleng hs, he alue o he cons an appea ed o be di - e en depending on he alue o he wa eleng h selec ed. Wi hin he l=230–250nm ange (a l=238 nm he ob ained logK=5.82 alue was simila o ha p e iously epo ed), he calcula ed ange o alues o he complexa ion cons an was logK( alinomycin–K+,E OH)=5.8–6.0, which is compa ible wi h he alues ob ainedby luo escenceassays.The e o e, he de eloped me hodology was alida ed as ameans o s udy he luo escencede ec ion o ce eulide om na u al samples. To accomplish his objec i e, he p oposed me hod in ol ed he compe i ion be ween ce eulide and he JG76p obe o po- assium.Ex ac ion o na u al ce eulide was pe o med on cul- u es o B. ce eus F4810/72 by ollowing he me hodology de- eloped o cooked ice,[17] which consis ed o inocula ion o ice wi h 300 C u (a e age alue ound in ice dishes). C u was de e mined a se e al ime poin s, and ce eulide p oduc ion was checked a se e al ime poin s by MS (UPLC-TOF). We measu ed he change in luo escencein he p esence o acon- s an concen a iono he p obe, acons an concen a ion o po assium, and an unknown concen a iono he ce eulide sample (usually wi hin he ange o 0.2 o 3.5 mmin ace oni- ile), which we i a ed wi h syn he ic ce eulide. Ini ial ace oni- ile ex ac s we e e apo a ed, he esidue was ex ac ed in o dichlo ome hane/wa e and concen a ed,and he ob ained solid was dissol ed in E OH so ha he in e e ence o wa e - soluble in e e en s was p e en ed. The E OH solu ion was checked by MS (UPLC-TOF) by s udying he elu ion ime o syn he ic ce eulide, and he LC–MSo he ex ac ed ce eulide samples we e measu ed and calib a ed wi h syn he ic ce eu- lide a di e en concen a ions. In his way, he concen a ion o he ce eulide samples om ice emained be ween1.2 and 1.6 mm[as de e mined by MS (UPLC)].We hen measu ed he backg ound luo escence due o he ma ix o he sample. We pe o med ap elimina y i a ion o he sample wi h JG76 o assu e ha he backg ound luo escence was acons an alue independen o he amoun o JG76 (Figu e 10). Wi h he ex ac ed ce eulide samples we measu ed a ia- ions in he emission signal by sub ac ing he backg ound luo escenceo he ma ix,measu ed be o e adding JG76, and i a ed he samples wi h syn he ic ce eulide in he p esence o JG76 (2 mm)and K+(0.75 mm). Wi h his i a ion we ob- Figu e 8. Fi ed i a ion plo s by luo escence emission o 2 mmsolu ions o K(CF3SO3)and a20mmsolu ion o alinomycin o ce eulide wi h JG76in E OH and BnOH:a)K +, alinomycin, and JG76 in E OH;b)K +, alinomycin, and JG76 in BnOH;c)K +,ce eulide, and JG76 in E OH;d)K +,ce eulide, and JG76 in BnOH. Figu e 9. Concen a ions in equilib ium in E OHsolu ion. Top) V( alinomy- cin), VK ( alinomycin–K+), K(K +), and SK (JG76–K+)inequilib ium.Bo om) compa ison o he concen a ions o he complexes co esponding o alino- mycin and ce eulide and CK (ce eulide–K+)inequilib ium. Chemis yOpen 2017,6,562 –570 www.chemis yopen.o g T2017 The Au ho s. PublishedbyWiley-VCH Ve lag GmbH &Co. KGaA, Weinheim566 se ed ha he i s poin s o he ice sample i a ionde ia ed om hose o he e e ence due o he quan i y o na u al ce - eulide p esen in he sample; he e o e, om he ini ial alues o luo escenceweob ained he o iginal concen a iono na - u al ce eulide in he sample by compa ison wi h he e e ence plo om syn he ic ce eulide (Figu e 11). The esul o epea - ing he es h ee imes was (1.0:0.2) mm o ice samples compa ed o he concen a ion o 1.2 mmob ained by mass spec ome y.In he case o asample spikedwi h 1.75 mmce - eulide, he esul o he luo escence i a ion was in acco d- ance wi h he expec ed alue (Figu e 12). I he e wasnoini ial ce eulide in he sample, he cu es we e coinciden (Figu e 13). Indeed, he measu emen s we e mo e p ecise o he sam- ples spiked wi h syn he ic ce eulide han o he samples con- aining na u alce eulide, which is due o he ac ha na u al samples ha e mo e han one ype o ce eulide.[18] The exis- ence o amix u e o na u alce eulides (isoce eulides) in odu- ces some unce ain y in he expe imen al measu emen s, be- cause hei po assium-ion-complexa ioncons an s a e no known.The luo escencede ec ion o na u al ce eulide in ice samples by using syn he ic ce eulide and apo assium luo es- cen epo e is he e o e ause ul, po able, and as me hod o he in si u de ec ion o ce eulidebysimple ex ac ion and luo escence i a ion o he sample. In he case o posi i ede- ec ion, he me hod can be complemen ed by s anda dme h- odology in ol ing LC–MS o mo e accu a e analysis. Taking in o accoun ha aLC–MS (Q-TOF)ins umen isno acheap o po able de ice ela i e o abench op luo ome e , he e- po edme hod may easily p e en he occu ence o ood- bo ne ou b eaks by eme ic oxin by in si u de ec iono ce eulide. Ano he in e es ing aspec o ce eulide de ec ion is isualiza- ion o he ac ion o ce eulide in li e cells. Fo his pu pose, we pe o med cellula localiza ion s udies in HeLa cells (human ce ical ca cinoma cells) wi h he JG76 luo ogenic p obe. HeLa cells, cul u ed unde s anda d condi ions,[19] we e incu- ba ed wi h he p obe (18 mmin 1% / DMSO/cul u e medium). Cells we e ixed wi h 4% pa a o maldehyde be o e aking images. The nuclei o he ixed cellswe e s ained wi h Hoechs dye (bisbenzimide) be o e high- esolu ion con ocal mic oscopy imaging was pe o med. All con ocalcell images we e pseudocolo ed (Figu e 14). A e exposu e o 12 h, he p obe s ained in acellula esicula s uc u es ha esembled endolysosomes (Figu e 14, op). A e exposu e o 24 h, he Figu e 10. Compa ison be ween e hanol and ice sample solu ions uponin- c easing he JG76 concen a ion. The backg ound luo escence was acon- s an alue o 77 au. Figu e 11. Fluo escence emission o aco ec ed sample e sus he e e ence by i a ion wi h inc easing quan i ies o ce eulide. The concen a iono JG76 was 2 mm,and he concen a iono K +was 0.75 mm.The concen a ion o ce eulide in he ice sample was 0.95 mm. Figu e 12. Fluo escence emission o a ice sample spiked wi h 1.75 mmce - eulide e sus he e e enceby i a ion wi h inc easing quan i ies o ce eu- lide. The concen a ion o JG76 was 2 mm,and he concen a ion o K+was 0.75 mm. Figu e 13. Fluo escence emission o a ice sample wi h no ce eulide e sus he e e ence by i a ion wi h inc easingquan i ies o ce eulide. The con- cen a ion o JG76 was 2 mm,and he concen a ion o K+was 0.75 mm. Chemis yOpen 2017,6,562 –570 www.chemis yopen.o g T2017 The Au ho s. PublishedbyWiley-VCH Ve lag GmbH &Co. KGaA, Weinheim567 JG76 p obe displayed an endosomal-RER pa e n clea ly local- ized wi hin he cy oplasmic and endosomal memb anes (a ows) (Figu e 14, middle). A e 120 ho s aining, he JG76 p obe wasalso localized in he cy oplasmic memb ane (g een a ow) (Figu e 14, bo om). HeLa cells did no display de ec a- ble signs o oxici y i g own in he p esence o he po assium- ion JG76 luo escen p obe o up o 120 h. Then, HeLa cells, cul u ed unde s anda d condi ions, we e incuba ed wi h he p obe (0.1 mgmL@1) o 2h.Samples o cells we e ixed wi h 4% pa a o maldehyde be o e images we e aken. The nuclei o he ixed cells we es ainedwi h Hoechs dye (bisbenzimide) be o e high- esolu ion con ocal mic oscopy imaging was pe o med. All con ocal cell images we e pseudocolo ed(exci a ion a l=488 nm and emission in he g een/ ed/nea - ed egion) (Figu e 15). Simila o he p e i- ous expe imen , he p obe s ained in acellula esicula s uc- u es (Figu e 15, op le ). A e exposu e o JG76 o 2h, he cells we e exposed o syn he ic ce eulide (0.2 mgmL@1) o an- o he 2h(Figu e 15, op igh ). A e exposu e o JG76 and ce - eulide o 2h, he nea - ed emission o he endosomes was di- minished (mo e pink, less g een in pseudocolo ), and he s ain- ing o he cy oplasmic memb ane also diminished. The cy osol appea ed o be mo e s ainedwi h he p obe. A 24 ha e he addi ion o ce eulide o he cells exposed o JG76, he cellula iabili y dec eased signi ican ly and se e al cells appea ed w inkled and showed memb ane blebbing and cy osol acuoli- za ion. F om he images i is clea ha he ini ial luo escenceo JG76, complexed wi h po assium ions in he po assium- ich s uc u es o he cells, is quenched by ce eulide in HeLa cells h ough po assium-ion-displacemen complexa ion, and his lea es only he esidual luo escenceo JG76 in he mem- b aneso he cells as e idence o he ac ion o ce eulide. E en mo e, mo phological changes in he HeLa cells, such as he o ma ion o la ge acuoles andmemb ane blebbing, as a esul o he ac ion o ce eulide wi h ime a e easily ollowed by he esidual luo escenceo JG76 on he memb anes; his p o es he e iciency o he JG76 p obe as ace eulide chemical senso by po assium-ion-complexa ion displacemen .The e- o e, he JG76 luo escen p obe can be conside ed as ause ul ool o he isualiza ion o ce eulideinli e cells and o he lo- Figu e 14. Top) Con ocal mic oscopy p ojec ion images o he JG76 p obe in HeLa cells 12 ha e s aining; igh ) Z-la e al p ojec ion images o he loca- ions o he s ained s uc u es inside ixed HeLa cells 12 ha e s aining. Nuclei a e s ained wi h Hoechs dye (blue channel). Middle) Con ocal mic os- copy p ojec ion imageso he JG76 p obe in HeLa cells 24 ha e s aining ob ained by exci ing he p obesequen ially wi h l=488, 562, and 638 nm lase s. Di e en luo opho e emissions a e pseudocolo edin hei espec i e wa eleng hs (g een:500–550 nm; ed:570–620 nm, pu ple:662–737 nm). Bo om)Con ocal mic oscopy p ojec ionimages o he JG76 p obe in HeLa cells 120 ha e s aining. Figu e 15. Tople ) Con ocal mic oscopy p ojec ion imageso he JG76 p obeinHeLa cells 2ha e s aining ob ained by exci ing he p obe se- quen ially wi h a l=488 nm lase .Nuclei a e s ained wi hHoechs dye (blue channel). Top igh ) Con ocal mic oscopy p ojec ion images o he JG76 p obeinHeLa cells 2ha e he addi ion o ce eulide o he p e ious sample. Bo om le ) Con ocalmic oscopy p ojec ion image o he JG76 p obeinHeLa cells 24 ha e he addi ion o ce eulide o he i s sample showing la ge da k acuoles. Bo om igh ) Fluo escencemic oscopy image o HeLa cells 12 ha e he addi ion o ce eulide, used as a e e ence. Nuclei a e s ained wi h Hoechs dye (bluechannel). Chemis yOpen 2017,6,562 –570 www.chemis yopen.o g T2017 The Au ho s. PublishedbyWiley-VCH Ve lag GmbH &Co. KGaA, Weinheim568 caliza ion o highly pola po assium- ich s uc u es,incompa i- son o low-pola i y memb ane s uc u es, om li e cells. 3. Conclusions In conclusion, we de eloped a luo ogenic p ocedu e ha was able o de ec ce eulide, a oxic me aboli e p oduced by spe- ci ic s ains o Bacillusce eus,in ice samples h ough displace- men o po assium ca ions om ap e o med speci ic complex wi h asubsequen changein he luo escenceemission.The designed luo escen p obe o po assium ca ionswas sui able o displacemen assays wi h ce eulide om o ganic ex ac s so ha he luo escencede ec ion o na u al ce eulide in ice samples was achie ed,and his was p o en o be apo able and as me hod o he in si u de ec ion o ce eulide in ood ex ac s.Tos udy he a e o ce eulide in li e cells, we de- signed ap ocedu e sui able o li e-cell mic oscopy imaging o HeLa cells by compa ing he cellula loca ion o he po assium luo ogenic p obe, which s ainedin acellula esicula s uc- u es ha esembled endolysosomes, in he absence and p es- ence o ce eulide.Wewe e able o conclude ha in he p es- ence o ce eulide he luo escenceo he p obe was dec eased because o complexa ion o he po assium ions by ce eulide. Expe imen al Sec ion Syn hesis o JG76 Pd(PPh3)4(10 mg, 5mol%) was added o asolu ion o 16-[4- b omo-2-(2-me hoxye hoxy)phenyl]-1,4,7,10,13-pen aoxa-16-azacy- clooc adecane (JG70;100 mg, 0.16 mmol) in oluene/nBuOH (15 mL/4 mL) unde ani ogen a mosphe e in a100 mL Schlenk lask. Then, asolu ion o N-[1-(1-adaman yl)e hyl]-8-pinacolylbo o- na epe ylene-3,4-dica boxylmonoimide (JG75;81mg, 0.16 mmol) in oluene/nBuOH/wa e (5 mL:1.5 mL:0.5 mL) was added, ollowed by Na2CO3(174 mg, 1.64 mmol), and he mix u e was hea ed unde e lux o 24 h. The mix u e was hen pou ed in o wa e (100 mL), ex ac ed wi h CH2Cl2(3V100 mL), and wo ked-up, and hen he esidue was pu i ied by column ch oma og aphy (silica gel, CH2Cl2/MeOH 92:8 / ) o gi e N-[1-(1-adaman yl)e hyl]-8-[4- (1,4,7,10,13-pen aoxa-16-azacyclooc adecan-16-yl)-3-(2-me hoxye- hoxy)phenyl]pe ylene-3,4-dica boxylmonoimide (JG76;58mg, 40%) as apu ple solid. M.p. 135–1368C. 1HNMR (300 MHz, CDCl3): d=8.53 (m, 2H,C A H), 8.49–8.33 (m, 3H,C A H), 8.02 (m, 1H,C A H), 7.59–7.54 (m, 2H,C A H), 7.25–6.91 (m, 4H,C A H), 5.10 (q, J=7.1 Hz, 1H,CH), 4.23 (m, 2H,CH 2), 3.80–3.54 (m, 24H, 12CH2), 3.66–3.40 (s, 5H,CH 2+CH3), 1.98 (m, 3H,3CH), 1.85–1.81 (m, 3H,1.5CH2), 1.73–1.62 ppm (m, 12H, 4.5CH2+CH3). 13CNMR (100 MHz, CDCl3): d=173.7 (C=O), 165.7 (CA ), 165.0 (CA ), 136.8 (CA ), 132.6–123.2 (CA +CHA ), 121.9 (CA ), 121.0 (CA ), 120.1 (CA H), 116.5 (CA ), 72.1–67.0 (CH2), 59.3–58.2 (CH +CH3), 40.5 (CH), 38.9 (CH2), 38.2 (Cq), 37.2 (CH), 34.2 (CH), 32.1–29.5 ppm (CH +CH3). IR (KB ): n ˜=2955, 2924, 2848, 1738 (C=O), 1692 (C=O), 1685 (C=O), 1651, 1590, 1571, 1506, 1457, 1384, 1354, 1248, 1122 [email protected] (MALDI): m/z:calcd o C55H63N2O9:895.4528 [M++H]+; ound:895.4535. Syn hesis o T iazac yp and-pe ylenemonoimide JG103 Pd(PPh)3(5.6 mg, 5mol%) was added o asolu ion o b omo iza- c yp and JG101 (59 mg, 0.097 mmol) dissol ed in oluene/nBuOH (10 mL:3.3 mL) unde ani ogen a mosphe e in a100 mL Schlenk lask. Then, bo onic es e JG75 (82 mg, 0.10 mmol) dissol ed in ol- uene/nBuOH (3.5 mL:1 mL) was added d opwise. Then, Na2CO3 (102.2 mg, 0.97 mmol) dissol ed in wa e (3 mL) was added, and he mix u e was s i ed unde e lux o 24 h. The mix u e was pou ed in o wa e (30 mL), and he p oduc was ex ac ed wi h CH2Cl2(3V100 mL). A e wo kup, he solid esidue was pu i ied by column ch oma og aphy (silica gel, CH2Cl2/MeOH 50:4) om which he iazac yp and-pe ylenemonoimide JG103 (48 mg, 42%) was ob ained as apu ple solid. M.p. 193–1958C. 1HN MR (300 MHz, CDCl3): d=8.53 (m, 2H,C A H), 8.49–8.33 (m, 3H,C A H), 8.02 (m, 1H, CA H), 7.59–7.54 (m, 2H,C A H), 7.25–6.91 (m, 4H,C A H), 5.10 (q, J= 7.1 Hz, 1H,CH), 4.23–3.50 (m, 39H, 18CH2+CH3), 2.26–2.20 (m, 6H,2CH3), 1.98 (m, 3H,3CH), 1.85–1.81 (m, 2H,CH 2), 1.73– 1.62 ppm (m, 13H, 6CH2+CH3). 13CNMR (100 MHz, CDCl3): d= 165.8 (CA ), 165.1 (CA ), 153.2 (CA ), 137.3 (CA ), 132.9, 132.3, 132.2, 130.0, 129.6, 128.9, 128.5, 128.2, 127.0, 126.8, 126.7, 123.5, 122.3, 121.3, 120.4, 120.1, 114,8, 114.4, 114.2, and 110.0 (CA +CHA ), 71.4, 71.0, 70.8, 70.5, 69.6, 68.2, and 67.3 (CH2), 59.2 (CH3), 58.2 (CH), 53.6–52.5 (CH2), 40.4 (CH), 38.2 (Cq), 37.1 (CH), 31.7, 31.1, 29.2, and 29.0 (CH +CH2), 21.3 (CH2), 14.3 and 13.3 ppm (CH3). IR (KB ): n ˜= 2955, 2922, 2856, 1736 (C=O), 1696 and 1682 (C=O), 1651, 1592, 1557, 1509, 1456, 1351, 1250, 1170, 1119, 1106, 1049, 959, 812, 750, 721, 697, 667 [email protected] (ESI): m/z:calcd o C73H82N4NaO10: 1197.5923 [M++Na]+; ound:1197.6001. Acknowledgemen s We g a e ully acknowledge inancial suppo om he Minis e io de Econom&ayCompe i i idad, Spain (P ojec s CTQ2015-71353-R and AES-PI16/000496), Jun a de Cas illa yLejn, Conseje &adeEd- ucacijnyCul u a yFondo Social Eu opeo (P ojec BU232U13), and he Eu opean Commission, Se en h F amewo k P og amme (P ojec SNIFFERFP7-SEC-2012–312411). J. G.-C. hanks Minis e io de Econom&ayCompe i i idad o his p edoc o al FPU ellow- ship. Con lic o In e es The au ho s decla e no con lic o in e es . Keywo ds: ce eulide · luo escen p obes ·po assium · senso s · alinomycin [1] a) L. P. S en o s A nesen, A. Fage lund, P. E. G anum, FEMS Mic obiol. Re . 2008,32,579–606;b)E.G anum, T. Lund, FEMS Mic obiol. Le . 1997, 157,223–228. [2] G. Lecking, M. K. 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