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.
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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.
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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.
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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.
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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
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Recei ed: Ma ch 21, 2017
Ve sion o eco d online June 12, 2017
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