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Cin,, A wlvtkaf kmtir’y. 21NJ5. ,. IS’. ills Is’
Anion SensingIsirig Quinolinium Based Boronic Acid Probes
RamachandrainBadtigti1 Joseph K. Lakowicf I and hris a Gedde?
Ccnter for FluorescenceSpeclrri.ieopy. fleparfmeol of Blor/trmisny 0,4 Mceccdar Bin/o, &/rdico/
StotecntsIogvCenter, fnh’ersitv efMw,I:nd Srhoo/ ol frdcfrie 15 rVcf Lcsbard Sr. Bolrimore. MD, 2/201,JS4
2lnstiwre WF/aorescence.Jabtai-cs,o,yflsrAdsnrncedFfurs,-eereneeSpcciroseop;MedddotBislIec/,nu?oCenterVol rs10 r rvJa,tj morai-MoIogr Jnnirute. 725 YostLombard Si. Bukimore. MD, 2201. USA
AbsEnce: In liii a paper we mvicw a series oF water-anlubic uinol iii him boronic acid ft reacentsells]nyc to ha I idn and cystide an ionn Wc have inter] igent]y coinbitied the well- know,t tsaridr. in paiticularMonde, hrorntde slid iodide. sensing capabilily si It psi no maw nucletes itlt the fluoride or eyaoiUc on
hi nil lag boror.ic acid noiciy. to atThrd a range or aler-aiaJtab Ic prot sensItive Ill 0 iaotide, chloride. broniid a.iodide and cy asde aniona rhese n cw probes afTer the float a] Lractive capabii ily of senti FLg all importantha] ides at pltys lojagi cal coneatarrallom, coupledwith a cyanide responseSI lie lethal Irealtold let’s r of abo
20-10 M So me of the probessuch as he BAQ BAS show apeetial ih na and intend y changes iii Iltepreacncaoh’ fluoride and c1,anidc n a wavclcngth-r*thoineoicaid ct oiiaueaicmatInee. entl t g th a dcc lionof thesearsi ‘an concntairalionsa’ vfsihie waveleitgths. Al though he aeesingmechatai,mis different for II nor idand cyan ide as cornpa’cd to the other halides chloride, bronide and iodide, die results cues! chat these
probesare In leer potential cand dales towardsthe sensing of the all the halides and cyanide. A Ian we baseronatrucred I lie correspondingcoil] rol toni pe IlIad, I hat have no boron i c acid a i ety and are thereforei naensitivcto IIuo de and cyanidebut are ni tidly acusidveto chloride, brianide and iodide, similar to iltal
the horonic actdprobes
Key.ords; Anion sensing. boronie acid based probes,stern-volmerequalion,chargeslahiliralioolneutralizittlon, ha]ides,tyaqt4e, retiometnesensing,lilriinie based sansing,fluotide, chloride, bromide, iajide
- INTRO!H :crloN
I he d esign ani d evelopmei* cf diemoornsnrsfor thedetection of anal1es,5 uch as anions, call cans. saccharides,etc., is an ni p0 flint topic of clirrern rencarebII] ‘Has masonfor this in torts is rho i rnporlance of the detectionandLJtJanii rtcalion of mcli asIa]yr in discip lines sticli as the lifesciencesand ensirotunencalbi tilclccm airy Ant ong the an ionsensorsdeveloped, iltose displaying an optical sigtialI transduction arc or specia] i nicrest ]2,3]. Dining the lastfew years we have madeconsiderablepro, in thedevelopmentof anion fluorosc’nsnrs for the detectionorcyanide, halides. ete, using boronic acid containingfluorophoers[4-97. SubsequentlyIn this resiewariiclt weprimarily tocusour artenstiataon the amon sensing ability ofa few hearonic acid probes developed recently in ourlaboratory, in conastto their conantnnrole and applicationin glucose/mononsacliaridedetemilnation 10.131.
The imponanceof Euliitc delectionand qumitificalion canqutte simply be tudgcd by the van amount of Fieralurepublished over the last 21] or so years 114.181. A clearsummary on the halide abundancemid the importanceofcachhalide in ana’ytical. envIronmentaland physiologicalsciences describedin a recent revico ariide 1141- Mnbiological fluids an complicated mixtures, which incitide
no rganl c elecolytes such as Itali da i nus. In plasma andmuscle cc1 Is, electrolyte composition is normally fairly
constanthut n gastrtipancreuliejuices, sweal, 55] i Va andurine it may vary considerably,particularly when affectedbyillness, The elevated or reduced concentrations of eachanalyte might affect he normal body fiancfion. A goodexample ni this is Ci.sticfilarnxis, which is caused becauseof high concentrationof chloride in patent’ssweat or salivaas comparedto hat or non-cystic fabrasispateat& Fluorideis presentin biological Onids said lisanesaid eceia!ly inboneand tooth. Fluoride is easily absorbedbut is slowlyexcreted from the body, which can result iii chronic
pOiSORing acute gashic end kidney disorders,dental and,keletal floorosis and evendeath [14,1 5]. mc phvsaologtcalsignificanceof the fluoride, chloride and iodide i’s elik’noasm. ho’ bromide is sti I consideredeither as a Ionessentialtraceelemenl or a truceclement aith an unknownfunction [I9. Iodine is however,unlike bronitoe an easenI i a Itrace clement in the human body [20J. The hmnan bodycontainsau avera of 14mg of iodine, concentratedniostlyin lie lily cold gland, Iodine contalnitig hi tramtoes I thyrox iiiand tthodoiorairie sirontrly influence a range ofhitilogicalreactions and iodine deficiency rca ults ut thyroid disease110].
Cyanide is hiowu, as thesixth halide and is often ltrnnedc’ponogen The elecisonicstructureand chemical pro ,ctlica cifcyanide are very sinsliar to thai of the halogens.I it isever,ma Ike the ha]i dos,cyant dc is a nutoritt ocly toxic malolL itttoxici of its sails hasbeenexploited for many lion tired ofyears. It was not until 1782 thai cyantde itselt ssasidentified, isolated by the Ssedish Citentiat Scileele. ho
* A do cii cotxcspandaaceto this ataihui a th Csntcr foe F Ia. eeiertirrSprrtrn.scopy, Depunit tilt tar Biosinn istry ard Mnrcsnlat Biology,kl,o,cai Si aterhaology Cenic: Uaiasriity of Maland Scbi alMoJ’rtrr, 125 Weei Lostband Si, Be emote,MI, IL 101, USA; h-nail:5Sdr5I.i,s]Sinai,d’s
I ri-a ‘t ia’ais SSLtO+.Ol C 2055 tenthais&untr i’i}bi,sbers Li
155 2095, VOL 3, N 2
later himsclr died front cyanide poisoning [2]]. More precise sensors fur both laboratory and environirteotalrecenTly cyanide was unsuccessfully used as a chemicalwarfare agent in World War I, primarily bcuause of the wayit wa delivered ]21 I, and also thought to have been usedagainst the irtabitants of the Kurdish city of Hams, Inq[22], and in Shahabad, Iran, dun rig the Inn-Iraq war [231.Based on rocent cyanide history, acute cyanide poisoningcontinues to constitute a Ireat for nilirary forces in Mureconventional and unconvenhonal conflicts [2tj.
Cy snide is also rc4ctily used in indusny in the making ofplastics, in the recoveryof gold and silver from ores,and inthe electropla:ingof metals, such as silver, golf platinumand copper [1 . however, while cyanide is used in bothmiLitary and indusural applications, cyanide poison,ng isnot common, but can more surprisingly occur froni swokeinhalahon from residentiaj and industhal fires [2124,25].where the combustion of synthetic products that containcarbon and nitrogen, such as plasocs and synthetic fibers.releasecyanide Cigarene smoke also coniniuis eyaiiide. thenonsmokerpictlIy averagesabout 0.06 agimL 2.31 tMof cyanide in blood, where oa a smokerptoally averages0.17 tg’tuL 6.5 xMl [16.
The mechanismof cyanidepoisoning is by absorption.rIough the GI uaclc, akin and lungs Cyanide’s toxicity liesin its abtltty to inhibit oxygen utili,atson by cells, bindingto the active site of c4ochsome oxidne [27.281, hence thetissues with the highest oxygen resiuiiement brain, heart andlungs are the most affected by acute poisoning. lucre havebeen numerous studies of fire victims to artscs the lethallevels of cyanide ]2 1,14,25,291. Fire survivors have beenround to have < 20 sM cyanide in blood, while victimswere &iund to have levels greater than 20-30 Al and iiitone cases as much as 00 sM cyanide [21.29]. Theestimated intravenousdose that is lethal to 50 ‘4-of theexposed population Lass OfHCN for a man is 1.0 m&g,and the estimatedLD55 ttr liquid on the skirt is aboat 100mg/kg [Ill. Hence any cyanide monitoring analyticaltechnique would need a cyanide dynamic range from onlyfw ftM i,l ‘30 rM to ensurephiaIog/ca mfegsard.
Numerous chemical and physioelwinical methods or thedetection and determination or cyanide, fluoride and lieotlter Ii alides such as pole rut iometric, chrotitatograph icapectrophotometric. flow Injeutuon and clccirocheiiiicalanalysis. to name hut just a lb w, are available 130-54].I hurwcver. most of these systems are not cheap. portable andindeed ticist deployable, most requiring the benefits of ananalyfical laborstory 1311-54].
It is well recognized that fluorescence Iechniqties forsensing, such as Itfetone, tntensily and wavcleiigih-
rat i ontetnic sensIng [55 M offer many advantag in thedevelopment of to rui at it ri zed. cheap, remote. accurate and
sensing [55.56]. However, one constraint with Iluoreseencebased sensing to date, has been the developntent of statableprobes that show appropriate changes in theur fluorescentproperties at the required concentration range of a pa* cut sranalyte, ni the prcscnt case cyanide a’td Fualude ani otis.
Hal ides are haown in be qL’cncliers of common orgair icII sorophores [551. Fluorescence quenching [quinine it di lutesulfuric acid by the addition of hydrochluric acid wea firstreported in I S6 by George Stokes, anti ts now conmtonlyatthbuted to the dynamic queitching by aqueous chlorideions [14J Subsequently the quenching ability of tlte lualidesis daty utilized as a sensing meclunisiss, and many halidesensitive prob have subsequently bccn derived from
mrne derivatives, which disp lay a ui ode it sensr nvr ly forchloride due to their relatively long lifetimes whenqoatemised. 7iilc ihe un-protonated quinoline nucleus isonly sparingly water soluble, its qualernised products arereadily water-solobte and have subsequently been littransduction elements in many ehlcirido/lialide sensors [141.
It is nol just the physiological signr icanuc of chloridethat drives workers to mostly report the chloride sensitivityof sonic fluorescent probes, but because tire quenching ofIluorescenix is not a selective proces and any fitlorophorequenclued by chloride is also quenched by bromide to a
greater extent and also by iodide to an even grealer exielil.‘ft ci e fore, for dynani ic quenching, lb suit vuty offluorophores to halide is well knowii tohe I’> Br> Cl.The explanation of this effect lies in the flict that theefficiency of intersysteni crossing to the excited tilpiet state,pronioted by spits-orbit coupling of the excited singletfluorophore and halide upa, contact, depcads on the mai.s ofthe quencher atom, hence the eMpression ‘iecrus-olons itJicf’is sometimes used 114,55. It is for this reason lay thesrnallcst-halide. tluotide. does not typically quench thefluorescence. As such. traditional halide sensitive probes arcnot very sensitive to fluoride and are therefore not suited fordetecting fluoride c 50 mM 41. Only a tcw fluorescentprobes can be found in the literanat which are sensitive 10fluonde, all based on either the benzcne or neplitlialenabackbone mid therefore slao’ing absorption in the deep UV
170 nni. which is not practical for many sensingapplCcalioius ‘14,55,57]. Allhoogh water soluble setisorsystenus are deemed important, a few systems based on ureaand duourea based lluorophoc were reported recently whichshow an optical response tow aids fluoride to ergaiti c media
IS g,59]. The new sets of probe’s descrihes! in thj paper ayereadily water ,othble and indeed show a selective tluondercg,onse Similar to fluorid cyanide is not readib’ knnstias a dynamic quench Cr and 1ic net n iost of the probes used hrhalide sensing re not serts it luic Lu the c.yanide ats,i I
Ohyx
Fig. I. lucIa ilibri airs i ivolvrd in lw nielac lois between the tuor,iri IC 3C id group .,uu i fluoride or cya aide, where de note5 eithercyanide or LI uotide.
ISO Ce,nniA.&WCkety, 206I Fez
Tahtr I Photophyc.I oss For BMQBAa, BMOQSAI. RAQftA. and CorrespondingContral Conapoundain Watt, at RosinTom F era ii
BM000L’ *.-HMOQBA p-BMOQBA RPoitlt
A451 iiIisIiots ItS. 46 310341 315.346
--
450 450
17.3
oBMQA OMQBA BMQ
so, 319 322 322 312
427 L 421 -- -- 413
ON] 0.25 I -. 12.1123
4.51 3.12 3.15
I504 0.03S - 1P36
2A1 2.35 2.3* .
0.045
2.39
::004’
2.48
- ors... 12.13 a6i oepe0tioIy. flae IiSi.
preacned in Table I - rypi ii absorpiion and eaisslonband maximum of the probescan be seen a, 319 and 427tim I he !are tokes-sht ed Ii uoreseenee- 1nt salon band of
‘00 pm is ideal for fluorescence sensing, allong easydiserirninattonof the excitation wavelengths I 55]. All probeswest found to he readily wir soluble and have both modestquollium yielL and lifetimes 112.13,611.
10
// / ‘en. / Eo.Sp.
02/ Sp EtatazOOfls 02
300 310 400 450 550 0 eWas.PThinn
Fig. 4. AblorpLion arol ttn!3s5on speln of o-RMQBA in320 ont The spectra oro rrprcscntau r ol he reapective
sonidc phcny I boronlc sold contthning luorophores asd thcontottI "‘} [iS 20 itnd.
Represenlattve emission spectrsol u-BMQBA in vale,with increasiog concentrations of NaCI and thecotresponding Sterot-Volnier phls La water with throes,id,ujm halidesSal shornaon ‘Fm. SI. BMQ and an- and pHMQRic 41mw a ve similar respetasttowards the anions.The Ksv onstanEs oh Lao ted using the re lati on
I -K1,4t] for the concrol conapoaindBMQ and BMQBAsair presenledin table 2 whereland Jo are #50 fluorescenceintensity It, the presenceand absenceof quencher Q,respaaiveiy.As ii canhe Wej] from hefigure about a 6-folddeas,aqeis observedm o-DMQI3A fluorescenceintensityftc o4lditior, of 100 niM NaCI. The ehiontlc responseof alllie probes is ry sinsi lar Table . S tubsegtiently theseprobesshow a greater responsein ihe presesiceol br,tmodeas d an even groer responseto todide due to the q tuen cliability tinIer of the Its I ides asdiscnosed in the i otrodsuciiu it.
Accordi ruly. the fluorescence q ocne]si ng noecloatitant is
simply aitsibuted Lu mostly haltde collisional upcmhing.We also measuitdthe lifettnoe for DMQ and the I3MQBAsin water, in the presenceof all threehid ide . The I th ‘at neilKsv datairon, the respectiveStern-Volmer ploLs. is 3111w
in Table 2. The Ksv values obtained from Jtfetnitemeatureltients are sm ill Cr than that of Intensity basedmeasurementsindicating a small hi it sign,fi cans s tatiqtencbutgcomponentof the probesby the halides. Thisheliaviorhaspreviously beepobservedfor quinolinum basedprobes-]l4]. Aeeord1ngly,BMQ and BMQRA5 arepotentialcandidaias For the sensingnIphysiologicalchloride.
8.2. flIt *0 aod BMOQ]$A Probes
Fig. 6 shows the absorptionand Iluoreseeneeenoissionspeotni for a-BMOQRA in water. The other iwo isoioteranandp-BMOQBAs and he control compound BMOQ showvery similar speciral features. The pholophysical data ciBIsIOQ and OMOQBA5 is shown in Table I. Typicalabsorption and omission band maximum of the probescostbe seen at 31 and 345 and -450 tam respectivelyfor these
U
U
.trerng &eg QI,ss &ed 8,ir .4ridFbn C,isVA4SdCemLsa, less. IeL L Na 2 LII
probes. The adsh tmnal absorptionbandfor theseconipoumisat 350 nni comparedto that of the BMQiA probes isattributed to the n-+x’ iransitcoit of the oxygen 601 TheexcItation independc±i,c emission hand at 450 nm indicatesonly one ground-state p.cies is present [or both ela5ss ofprobes. the large Stokes-shifted tliaoresce’ice emission bandof t 05 nn similar l,i the BMQBA probes is dat] forfluorescence sensing, allowing easy discriininion of theexcitation wavelengThs 1S5}. Additionally the relativelyred-shifted absorpton hand or these eompoimds compared tothat of the BMQ derivahves, has allowed us to use readilyivailable cheap light sources [or tine-resolvedacasoremems smb as LEDs. All probes were fomsl to be
readily water sn[iz]ile. Unlike BMQ and the BMQRAs.IIMOQ and the BMOQBA5 have much higher quantumyields 0.5, and much longer Ii rbcimes of arccmd 25 ns inseater 112,13]. lntcreiingly, keauso ofilte longer Iclelime ofthese probes, the ,Lienching ability towards halides isespected to be tilore pronnuiteed than the BMQBA probes.uhsequently] we tested the halide sensiug abib’ of theI3NIOQ and SMOQEA probes in water, in the presence ofhalidn. The representative emission speen of o-RMDQBAcii waler ith inaeasung coneersfrations ofHaCl is shown it,rig. 7. The fluorescence ititonsity of the pro decreaseswith iiioreaxing conoeolr&ion ofhalides. Fig. 7 also showsthe c orres poll ding Stern- V°l mcr piots obtained for o-BMOQBA in waler with halide. The Ksv data for BMOQand BMOQBA5 is presented in Table 2. ‘The halideresponse of the other two isomers m-BMOQA and pBMOQBA and the control compound BMOQ is verysimilar to that ofo-BMOQeA As we can see, shots a 3’fold decreasehi o -BMQ BA Ilipi rescencC ntensity isobserved by the addition of 100 mM 4aCl. As comparedtothe BMQ and the }3MQDA probes, these probes show amore pronounced response Table 2.
-
We also measured the lifetimes of BMOQ and theBMOQBAs it, water, in the presence of halide. the obtainedKsv values shown in ITable 2. ma Ksy vatties obtainedfrom lifetime measmtnienLs are again relatively smallerthanthat of intensity based mensLaremonis, indicating a small butsignirteant static Ilnoreseence quenching component [141. Itis uiiteres tog to compare the hal ide seics rig abi Ii ty of thetwo sets of c nrts Pt, urids disc ssed here, Because of thesigtuñcautly long lifetinic of he BMOQ and theDMOQBAs, then these probes ore more sensitive to thehalides.
2.3, BAQ and RAQBA Probes
After h.ivcng sLiccessiully testing the halide sensingabti ity 01’ the BMQ. and IIMOQ series, discussed insections 2,1 and 2.2, toe niov our atlentiun towards BAQtIer’ satkies, Fig 3}. Tlitse derivatives arc constructed usinghe stronger electron donating 6-amino substitueni as
compared to the 6-methyl or 6-niethoxy siabstituent as iii theUMQ or BMOQ classes of probes, respecfively. Due to thenew subs ci tuen t, BA Q d en vat’s Cs show uniq ac spectralproçrties.
The ahcoep t ion and ens issioo spectra of these probes isconscderably red-shified, Table I. The typical abtompt5oaand cr.i,ssi,,tt band maximum of the probes are about 390
565 nm enabi iiig the use of many light soure C S [lieprobes are readily soluble in water mid show moderatequsntwii yields
*5
I 0.0mM
f.1,
SI
:‘
W.v.Iangth I lint
Fig. 5. luoreseenc. enhissuuui spectra of o-nMQBA in wet rrwith increasing eoneent,sitou, of NaCI InP. A5 320 muss. Therorreapoiiduag SLen,.Votnar plots for e-BMQBA with ihnchalides bottoui.
Subsequently, we lasted the response of BAQ .ittdBAQBAa toward aqueous chloride, bromide and ,ndide.Fig. 8 shows the fluorescence en, ission spectra of -BAQSA in water ,th incresaing oonecstrutions of sodiumiodide. ‘the botton, panel of the figors shows the SternVolmer plots for o-FIAQI3A for all three halides in water Asexpected the steady-slate Sleni-Volmer constant for Iodidewas the largest. Key 34 M1, wiih bromide and chloridevery similar and substantially smaller than for iodide. Iand 1.0 M1 respectively. A very smittar respoulac isobserved mr BAQ. and or- andp-I3AQBAS in the presenceofhalide. The K51 data for the probes is preseisted in Table2. We also measured the lifetime/s 0tH AQBA probes inflie presence of halide to determine the dynamic quenchingcomponents. Interestingly the dynamic K5p values wereslightly smaller, 2’, OA and 0. NV’ for l, Br mid CVarspec ii ‘e ly, suggesting a Sm alt, but meas table, staticquenching component. A very similar fuitdirig that is noticedvth other systems discussed in the pres ions seci ions.
0 IS 30 45
NaX]/mM105
162 l*.,:nnA Iv6eeI fla.Jans. 29J. VoL 0, tWt 2
Table 2. Sreru.Volmer Cncnuts ksv, M’ oftlw Prob.s Sthdied With ihlitles in Water!
Li
Br F
j !t!sth. I I I niaLny I LfdLnef_ Iflus,stw L Lfttime
573 - J32 237
rn-LlMOoB.. I1S j_Irn 4J3
p-Ea100HA I7 I tti 7Th 23] 95 231
H N 74 U I S21 32 - - 362 - H520 __j_ 425
LrQI± r ill! - ±p-EIMQHA ET.!.i IL :1.5 42.5 I 420 25.9
I EIMe I 20.0 mIt I liii I 5025
-
-. .
Ia
o4no
,_ 0i C-- . a >
/1
.-:-
04 /Ao Sp, tSp
02. -.
--1011 tn * CO 000
c%velsngth rim
F1g t1. .&baorprinn and ethibsiOn spcLtra of o.8MOQBA in
‘sater The arcs [I re rd prc 66111212 of the rcwectove eomcricpile fl5 Lhoronic acid co 1113i flOg fi looto hnree and the cnnoI
11120undo.
Out Ii nte-rsstttved atu tic’ ii ave revealvsl hat n-PA QI3Aht ‘cspoiIrnd7i ,, Miihlwre ner i4Lh hftthi,c of 1 £7
and 01 no, with 2311 pl j toUrs of Q52 and OA S rCOptCtI clv -
Botit thc niean and annp]j Lode weighted ifetim:s were Ibundto he r: slid 2J 415 rcsetHvcIy, Intercsiingty. the lower
eSponse of BAQUA to warda apieous halide can bcalinhuted to its reduced me-oil Ii!tthe as copazedto other
quinoLinntnh floe tluehrophores [11J, noti’ig Ilic weaLrnspci,setowardsaqueousC1, which courn be panlicoilarlydvainges when wsng BAQSA In p&y oiggicld tluids fordetennningeither aqueousUijoddc or cyonide levels,
3, FLUORIOE AND CYAMBE SF’SINC
frIQ, BMOQ and IAQ laYing no ThIQndd or cyanideintdi h oroni c acid moiaty. 5C 3066115 hive towardsthese
Lii oils of ill Lee est Aceordi ngly lie speetal propertiesofthose threeprobesare unaltered ni hit pren6nceoftiuonde or
cyIide. We gi s-c inure comparativespettraic id e]hCeiii thisregard Nb iii’ discussing the respoon0 .,1 be boron’ scodprobestowards fluonde or cyanide
.10
-2
j I-
I FIh
Br . -
Ii 22 20 40 tO
P0aOJm
rig. ?lunivscenen eadssh,n 3rrcll of n-L3MOQI0.- In aIer
with tncrnin concenmitioons of Nsa itoi 1 H5 ott un
coaenpon Lag Skin Vol Inter pioth for c-li MiQi I’ ID n6athr liii
three nodininn haLida bDttnm.
I
SM. Sad ai,r ‘th&:li.a. Ra H.....i- I;J P,oa. i-Al’-a-jt 2MS VL 1. t 2 lii
0.0mM
lee mM NM
,-1-
50 tSU 5& 5O
waventh I nm
Br4-.. -_,-_-._. ----
IL rSO 45 50 75 90 a:
NaXI mM
Fig. . A Lep:.5 nLaLi.,r Iloirescence anissiOn al Lii Of -EIAQEtA iii saEer wi Ill nereising conce’,tratuoi Nal lopi. X,
mu, and the corresponding Sreta-Votmer plots with th,esod Elm’ ha] des hotto A vs dm1 lar responca is nleredform-BAQBA oadp-?AQflA with all diree halides.
lab Is 3. Discocialios rnaia ohs r I.e I,rr,lr, i51 Fluorideslid rya aide iii Wa Es
I’ rissr E,ieride IN15 ni M31 ,.idr N0 I mM3l
9t0
ii!- II M tQt 54.0
p 5liiQVk lee 20.5
530 6.7
ue-WlQR sic,
jr.lLEjA 500 IS 9
:.IiAQR.4 LFk. - -
p.BAQBA
31 SMQIA Probes
3.1,1. fluoride Sensingl’}re emission epes_Era of o-BMQBA ill water wilh
iticreasltig concentratsons of fluoride are shown in big. 9.Th other two Isomers m- and p- BMQBA show a very
sunilar spccal response k,wnrds aqueous f]ucrride. The p]otabased on the ‘ormalized fluorescence in ens ty vercus thefluoride concen]xation are sl,om in the bottom pane] ofFig 9. The nonlinear response of the plots shown in diefigure may be indicative of the complex binding interactjirnof fluoride with the horonic acid group The calculateddissociation constants for alt threr probes with flULrIdV towaler arc ahowis i Table 3. The corresponding contra]compound, not having a boronic acid group is nonresponsive to fluoride.
1mM
. ,j__4Iiit NMa ,
W.atenfli rim
42wfl MOM
30* ThB5SA‘ 0-OMOBA
30
a. *
:a
50 iO 150 ItO 250 X0
Fig. fl. FL vorcacance emission spe ins ,,f,-El SQL A ii .aLCrwith hrcreasiirg coliceil mid ,,,rs of Nap top L. - 320 im,F’ oodde rponce plots of all hess aorrrena Lbo I
32. Cys,eideSeirsine,
Thte,nn. Sgsd L.nfrfr Sna/l,,
Fig. 10 shows the emission spectra for the o-BMQBAprobe for licrnst hg ryan ide cn.tecntral i rrns in water. Al]three isonieric boron ic acid prolics chow a notable decreaseha flucrcstntce intensity wrtb riM cyanide cotLeenTrationUI- BMQ is however relalivcly unpertairhod. with a LerlLVotmer 000slasit of 0.8 nM l We ogain plotted tileintensity raliometric type ptots for the data shown, in ]l’ig.to, Fig. 0 shows an t 1-tb Id decrease in fi meescenccIntensity with 30 PM cyanide for m-BMQBA. ideal fhrcyanide physiological ssfegajard monitonng. At the staticsodium cyanide concennfion lie fiuorcence i nttenisfty ofand p-BMQBAs has reduced by factor of’ ‘ and 7,respectivoty. Subsequentty, all three probes saturate at about
aF]IniM
1i4 Cor.ii! 0oOiEaI Ce,th&y, 2505. IaL J. Ms 2 03 3 e! dl.
40 LM NaCN, identinng the procs as tronthil candidatestbr cyanide detecuc!n at jthysiologieal safeguard IevIs 171.
4033 1..:<i *. I
3oo. ..1 - I
/ "-: l0OpMNCNe 2%
= .1’:Es
Fig. lit. itic rcaince aehissiort deir- Os o-SMQIS.4 in tr3 ith i netting concanLmiaoiia sr NaCN ii. p i. 320 mis. Thecyaside response 013 It three isolnelt bolto .
With notable chanues iii he fluoreseen cc intent i L s ofthe probes in he ,resenee oroyanide ‘Sc questloLted whother
chaogos in the mean Iiftt,nir Lif the probe would alsoprovsdc for lifetnie based sensing. Our reasoning ‘Sax based
os abe reaillls obithned wills 6-tniinoqinnolinkjsn probes
AQaAs which show bulb spectral shins and !atenitychanges in the eseace uf cyrsidc ,Mwing Iir oII.
cxci flu on and cmiss Dii ‘S avckngth rstititneftlc cylililoCsensiiog psorc is section 3,3 I] We hate st’bseqeotlymeasured tht lifetimes of the BMQIIA probes USLI]g hewelI-kssowa Time-correlated Sintle Photon I ztrsLisThehoiquc. TCSPC III. Tahle 4 shna die Ititerisity decskinetics otaMQ and o.EflQBA ftc ccntn,1 rompstund
HMQ as found to be mono-exponentialin waxer with alifetime of 239 ns. The presence nf cyaissde rsults In aslight decrease in the II ‘cii axe, the Stem-Vol tier quenchingconstant 04 nMt not unlike thai determined dons theintensity plots. The iniensisy decay of o-BMQBA was fltuitdo be hi-exj,u, neat is I in water, ss ii ft t Etc meat, lifciiiit
decreasing Tom 4 0 t to 3.22 ns in tha presence of 50 kMcyanide, a 25% change in mcdii llfOIIme which can tot beexplained by dynamic cyanEtle qoenchin. an it thereforeathibisied to the Ii Ici me of the cytnide hoticil fonsi.
3.2. MOQRA Frobe,
Fig. II shows he floorca cenee enlist i Lsn spec ira nra-BMOQB A in water wi iii i ic reasin co licent rations
flui,rid. the hottoni panel nt tile figure rprese,iting thefluoride rasponse of the tliscc isonserie OMOQRAs.,Asshown here h Fig. I t-boitom. the other two isomers nandp-BMQFIA show ve simElar specal ilelises towardsaqLseous fluoride. The response plots were again fisted to thefluoride hiodin isotherm yicJdsng he lflssocmtioui clinsirnits
in the rsngt 900-1000 mM3 Table 3’. "he BMQUA
T.hli 4. MuliiexpnneutLsl lniaosity bvay of MQ nO o-EMQBA
4. oe5t s. - -
30 - .44 I 0.1762 I 27 0.5335 iii __l_ Z.051L2140 i.’42 _J_ 0.3311 2.50 -_j I5.O4& 3.21 3.9050 1.51 ll.33 4.53 3.22 _j__.oi H 1,03
- - J. ±?!__J. _J5 2.53 tO ‘__ I I 2.5 I LotL ..,.
MQ
50 -f 255 : .-C f 5_]_
25I
1::
l3
4,lia 3e,nMg O,sg Qeteeiaiaa Beard &wfr 4th! PeJka s,,flt. Thdjlkei C r&fos 1fl1 Vet !. ale 2 loS
disc ijased ira the las section. show rc ativel y lower K0 cyanide with a I 0_thId listens Fly eiesnge ‘,th as I ‘trIo as 20valisco as *-nparad to the corresponding SMOQBAL Thecontrol to iipotd BMOQ. again having no boronic acidgroup, is non-responsive to the fluodde ajijon.
500mM
f 4o‘
- - - I
200 .F 300mMNBF
I I, -U- lao
$fl_40 x!.1Oi L:-.-v
tM cyanide. Similar to resultsobtainedSalnh RMQBA5 wewcrc able to determhie die cyanide dissociation colittints forthe ortho nero and pane boronic acid probes to hr 129,84.0 and 20.X ‘M ‘ Table 3, noting the units ii M ormel3 dm9 based on the equilibrium shown in ‘Fir Ii.Iliese respoosta arc most encouraging and suest the UL ofthese isomers for physiological cyanide safegua, a Ii,addition, m-BMOQBA may rind applications Thr cyanidedetermination post mortem for fire viLtima whur cvso, IClevel a es coed the 20 aM lethal oncentration thrcarijal2
2’
14-
* p.HMOOnA
I mOMOOSA10 L_oBMOQ6A
!
5’
.
.
a-
50 CO ISO 2 Z50 300
NSFI/niM -
Y. Ii. Ftuorcact,ice emission spatia or o-SMOQBA ml aaer
with mere sing conerniraniosts of Na? top.AaaJ4S un. [heItsonde respea’r of alt three Rn Icr, bono
3.2.2. cyanide Seiisiag
eec
Wavdeswthlnm
‘
rA1SF oOMOOGA
- S -BMOCA
5 a SMOG.
0 iS 30 45 Es; 5 50 105
Fig. 21 shows the emission spetno of o-BMOQBA inwater for inereasing cyanide concentrations, with A 34Snns As the cyanide conceatatjo n inc rases, the emissionband at 45 nm decreases. For tho control compound,RMOQ, we pically observed only a yen slight dettesac in
em lBs OO intc nsify [or increasing cyanide colic cnin lions.which we have attribatcd to lie dynamic tuorescentcquenching by oyaos’de. nonng that BMCX does not P5ScSa boronuc aud group and tlacrefisrc call not bind cyanide aspo5tu1ted tao our retent reports [4,z7]. fly plotting hueIntensIty of I3MCIQ the prescrac of cyanide, noniialszcdby the intensity tn the absence of cyanide we were-
.subsequently stile to determine the Stem-’ elmer quenchingoiistaot to be 3aMC
For the dais shown In Fig. Ui-top, again we Were ableto construct o ensi ty ratiorne rue type plots - i e. themteosiw hi the absenceof cyanide divided hy the intensityin the presenceof cyanide,Pig. tZ-bottons. Illtereslrnglythe m-BMOQBA isomer shows a much strongerresponse to
acNJI iM
Fl. 2. Fluoreseenso en, isaims open s-s of c-SM UQRA in waterwith iucfting concennations oINaCN topi. A 343 cm Thecyanide response ondi fete isomers boltulu.
To understandthe differmit responses of the as omentov,ards cyanide it is islthunative to consider the chargencutra]izaiion-stabilizationmechanism of theae probesdesoñbed n1 lie Fig. 1. Upon binding, the electron deiiityon the boron atom of the probe is increased facilitating thepartial neul’alization of the positively charged quatemary
.,iogen of the qumolinium moie. I lie quaternary sitirugennot only reduces the pX5 of the probe IC-li, hut also
.sonbilisca the horonate-cyarnde complc formed woncyanide addition. The diffrences in cyanide sensitivitybe wean the isomers is e,cplai ned by either their it urotjghspace or Ihroogji-bound interactions with the pnsiiivclycharged nitrogen, the nero thnn of tile probes Thoubt tOinteracl viaboth niecliajuisms NI-?].
1W 450 500 55°
Lob Cr.4LiaJV&l rke,ais,,- 3005. tL 3, .V 2
TaMe S. Multinpoasatial IjiIeasIty Dmeay of BMOQ md o-ISMOQBA
Ccapeixmd CCNH 1±M t, O a IS < 1’’
I
- 35 0.324
45 310
10 0455
I 0 27,31
5 27.04
II 26.14
OMOQ 5 26.53
0.1160 -- Ii.YtTh1 - 23.33 - 25.14
aol - 25.10 tflLS 1i0 14.64
0.0176 75.25 ILflN :.t7i
I.e -. 17.31 2720
- .0 = 111114: :7.44
10 .._ 1614 16.74 -
..- ... 2553 16i3
____
H
1.35
1.4
IAI
L.O
JO
ill
I Ui
172 ! .lii! cII0 4t 12 a.off fiIa DM00 Kr’.. LalO It-I.]. 1 rnn jdk. - din iclajrj &=y ii jn!iiad iLI Ii:
‘I.. 1UiU nf.ilina tiny is.
mc dissociation coastant values for o-SMQBA and o- men positively ckmxged nilrogen center, stabilize the anionicBMOQBA are 16.7 and 52.9 mM3, respectivcly. I-B’Xs] roroed con binding cyanide. yielding the loweredSubsequently. ±16 difference in the cyanide sensing abiIi K0 valuic for the BMQBAS.of the o-BMQBA and o-BMOQBA is explalncd similarly to
rir.e cg.ra VtFhat described it the last parawaph. The re lativoly strongdonating substiment .-0CH3 reduces the positive charge The lifetime of o-BMOQRA was found ELI
density on the qtttnolinwn nhtrogea center more effectively monocxponcntial In water with a Ii letinte of 26,7’ its,than hr - CH1 group does. Accordingly, BMQBA1 with a Table 5. However in the prence of cyanide the intensity
- .,i i, .C -
I
___
Fig. 13 F It Ioresca nc absorption lop .ft and :miMLOI, hop righ I apecifa of c-BAQ 0’ iii watel WLth L.lCrCa tn cenc en I n!Lii. INay 355 om. Die excitation { h.ttn h and cm as tin I, siloin right ratiotuetric lois for all thr ISAQIi As with ri uonde
a,ec Seath Lv Q4a.ian Sara Snk,I&flt deal AMaSS VsL I. N 2 167
decay is biexponentialwith a ouch shottsr componentpo*present, 394S ps This bn the sesolt f resciag themean Ii etime by 4-S % over the range of physiologicalcyanide importance. lnterestittgly these measuretnesits wereundertaken ssir!i a pulsed UV LED with an emissioncentered at 372 nm suggesong the utility of the new probesfor potenrial use in low power. field-deployable poisonsafeguard devices. Similar findings were observed for theother isomers.
The lifetime of the control compound BMOQ isoionoe,cponential both in water and in the presence ofcyanide, decreasing from 27.30 -3 250 ns with op to 50tM cyanide. In comparison,the lifetime of o-flMOQA inwatcr was found to be slightly shorter, 26-71 ns. Wecalculated tha dynamic Steni-Volmcr constant to be nM
which is very similar to the value obtained Ikorn theintensity-based neasiirements. Interestingly the presence ofamuch shorter lifetime component for o-t3MOQBA withcyanide. suggests more than a simple collisionai quenchingprocess present Given Otis, and the fact that the iiuensityrapidly decreases in the presence of cyanide, we speculatethat the debocndprobehasbothashort lifetin,e, and asigniOcanily reduced quantum yield as compared In theimbaw4 probe tbtm 71
The flMOQBA’s typically display greater dynamicrange for sensing, with notable changes observed in thecyanide concenatioo rangeS-hi LIM The BMOQOA’s arehighly water-soluble and can be prepared iii a one stepsynthesis [131. The 350 nm absorption hand readilyallows for UV LED ccilation or even 370 / 400 liii laserdiode excitation, which would nor be possible with theBMQBAs. The long lifetime of lie SMOQBA’s 26 asaccounts tin tfr cyanide cothsional qtiencbing. also obsetvedwith the control compound SMOQ, Subsequently, these
Me to be ssasceØbk In ethes inttrft,nccs anthas aqueous chloride or oxygen I
The BMQBA probes also show notable changes inl’ljurescence intensity in the presence of 30 M cyanide, 14to S-fold, for the anise -‘pare isomers respectively. Unlikethe BMOQBA probes, this class of probes shows a hie,tponentinl lifetime in water, and a relatively much shorteran lifetime 4 us thE decreases 13 t aith theaddition of 50 pM cyanide. The lifetime reduction isthought to be due to the cyanide hound inn, &vcn the veryminor changes obsen,ed with the conuol conqtound BMQ.nteresttngly, these probes art not ikely ‘0 be perturbed
much by other collisional quenchers due to heir shortlifetimes. With regard to fluorescence lifetime semis thesechanges are readily detectable using simple and cheapinsthmientation [551. One part:ctilar disadvaniage of theseprobes however. is their requirement for UV excitation at320 nfl, which while possible with LEP5 as shown here,limits their practical use in repid analysis, portable, fielddeployable deces, areas ofactivo rerch ii.62j.
3.3 BAQBA Probes
3.3.1. FIaaride Sensing
Fig. 171 show the absocption and emission spectra oleBAQBA i Mtlctpo,t wakr 9itl wt% tontttbsts
of fluoride As the concetthation of Iltioride increrses, theabsotvion band at 38S ten decreases whMe the band rn342 nm increases. We can also see a significant change it’the 38$ nit band with each 5 mM fluoride increment. Asimilar response with fluoride is observed for mesa- andpam-BAQOA5. ‘1iibscqueit]s, Pig 71 -bottom left showsthe absorption wavelength ratiometric plot of the 342 and388 nm bonds for all tlee BAQBAS with fluoride. A linearresponse to fluoride was typically observed up to shout 00mM liurride. As con be seer,. lie dynamic range for fluorides,sing is impressive where a -Ibld change in A4l1occurs up to 200mM fluoride foro-8AQRA.
Fig. 141- A Photoip Ii or three via Ie containing eq uslconcearatlous ofo-tIAQi9A and 0, 50 end 300 mM fluoride, leftto right respectively B I A photog,apli of Iwo vials containingeqrsj amounts olo-BAQBA in water with 0 and 10mM sodunincyanide. left ed rift rrspectively
The fluorescence emisnioji of o-BAQA shows similarbehavior.Xce 355 am Fir I 1-top right, where theintensity ofthe bend at 54d mn decrei>es while the emissionhand ai 450 om increases, noting the visible emission of liefluoride cojuplexed form, :11 $51 am as compan,tJ to the red-
68 csIChsmm,¾ 265, L 2.Yii 2
shi lied cmi Santo of ‘he u000mp Lexed form at 546 tm Tb iscolorrmernc ncsponse can sue be seen visually as siiot.si th{Fig. where the threa vials concaining 0qu41amoilt,ss ofo-RAQBA Is, wcter with 0. 50 sjid 300 toM floorid thevia] having no flaortdz exhibits a greenish-yellow color andbecomes sIihrly clear by adding 50 mM fluoride rodeventually becomes completely olorIess by the addition of300 mM fluoride. For the dots showo it, Fig, 13 topright, we esslistruc ted lie oorescencc emission taliometneresponse, l’rg, I 3 - botlotti right- Again we were able todetermine he d’s Soc Lll U on 000stants of the probes withfluoride. Table 3. Inwresting]y, die rttomettir responseplots, <Fig 13 -bottom panels. sho, &frercnr dyrtaitijesensing ranges, reflecting Lhe differences in extinctonoclTtc:cnrs an, annim yields of the F a,boei,d aMbe,, od forms respectiveLy
We measured Ll,c Itfetitoes of o-AQBA separatelythrough two long pass filters. 416 and 546 ott,. totavesiigstc tire Iifcttttre cI,rnges duñog Ou&ride
noipE axatie a. Ta Es] 6 hc wa thai ‘Ire Ii lelime of the
4
awomp! exed fluoride probe lbml, iC. Vt ‘tia] ice, us rig theam beg pess Slier, reaisirss b purreraial tiporr fluoride
addition, where the mean lrfrtme changes from 2.32 -+
232 os by th adthtion sf300 mM NaF, However whet,consider the Lifr&oe ct both bound and utsborn,d farmsusing the 416 Urn long pass filter, we find th intensitydecay data is bat desetibed by a 3-exsonentaal fmscrion witha shoti cotuponclit now evident, which increeses inamplitude ai as the concenttion of fluoride is incrcüaetL‘Thn short compottent, <200 ps is attributed to lIre fluorideoimd form, shown aer 35 tin, steady-state illumitiatton at450 on, in Fig. II As expected ho amplitude vetghtcditfethne chaitges oocabiy, Sm 241 - .R4 i’s, art 25 %change. liii suggests the p. ,,csib i Lily ci Ii let i,u e bsedtIuwrE,k sens’a aIs these yew ,uoycssent probes. Si,nulayresults were tisund for all lraee HAQBA probes,
£12 cyem’dr Stnth,g
Very similar to rh at observed th IitLor dc al threeisomen sisow all excitation and emission wavelengthralt emetic re sp o ‘se towards the cyanide an oil - A
Table 6. MultIeaponentSl Intensity ,Incay of BAQ tad n-BAQRA
co.,,,,.nd J lfltrldtlIiaM ‘t - i ‘:li n, a, - x’
ii ;t IS 0i20 so!_j__7o_L_4 1.05 - jo&
- li I uiso onoj I[Lo14 usISO 0.11 0j593 Lj8 O.3& 281 I 5.470 2$ t.9
200 0.15 t.IbI5 1.55 i_°*_1_j_ 0230 2.42 -59
-- j1 jI stersc4uiso 45 IS] IL06
- - - -22_ lullS - 5.92 flo’o 0 t20 a47 SS t :4
-
-EiAQIt..t I . F- I-
‘.lIIc 45
eQ .93 Ore 3.4L 1.250 - - 2.53 2.34
‘I as pul;,,: Nrri !!,,tt-a!,reId s,tp f,u,a!,,. Sitrit’!!!a it Stfil!t!i Iarie ,-,dt,e-nAOb ptab.i
I
A,,i,s.S,ath,g tJt2 Q.nsn It ,,,j Rs.* .lddPreb Ce,eAjwkeI Car 2ro3 PL .‘, tvs Its.
a,
HI"
I.. ,-asua!a,
o lSs! .
SAn
Fig. IS. Fluorescence thsarp ion lap Sft and emlasiofi tup oiaht apectn 01’ o-SAQJ1A in sea Err seith I nereisiiig cn,,cetunotpons ofNaC N - 3 5 flat The esciratlon bottom left’ and calisajon botto ribt wa,rlcngth-ntjomclnc plots for a I’ three BAQEAswith cyanide
representattveabsnrpdon mid emission spectsaofo-BAQBAin water with macusing concentrationsof cyanide and thecorresponding wavelengih-raiiometric plots obimned with
ma and lta&lo nra are alsown in Fig, IS. Forcornpmison the mtiometrlc plot of the cnnoI compoond isalso included in Fig. 15 The sbsoipt’ois or fluorescenceapectra t* BAQ are unchanged by the addition of thecyanide. However, oil three boronie acid probes okw similarresponse to cyanidc Interestingly, m-BAQBA shows arelatively much stronger response with a greater dynamicsensing range than ro-BAQIIA and oa-BAQBA. Fig. ISbottom paneL Ry conipitrilig Fig. 15-bottom right an4 left,we can see that a greater change is observed for lieratiometric absorption measurements, reflecting thedifference in the cictmcrion coefficient and the quantumyields of the cyanide unhnund and bound forms.respecti V ely. The dissociation constants calC tolated from theplots t,r s,. In- and p-BAOBAs are &33, 5- and 7.14rM 3, rc,peetjvely table 3. The probes satmme below 2011M cyanide, suggesting the probes as potential eandidaLcs tonon i br lethal cyanide levels.
The II AQDA probes also show a colorunetric-typeresponse to cyanide as shown it, Fig. 24. The two vialscoanain equal concentrattons of o-I3AQBA, ith both laoand 10 tM NaCN respectively. B can be read’dy seeni’romthe ligc that the col nr chmige can be easily visualizedsuggesting the use of these BAQI3A probes far simplecolonmetric eyarndtdetermination.
The dual emission hands horse enabled as to clearlyresolve lie litetimc nt’hoth the cynaide bound and unbotaidprobe forms where we concluded that the hound form had atouch shoder lifetime, a few hundred ps, itt camparison tothe unbound form wh’c had a mean lifetime of 2.59 ns.The detai]rd time-resolved data fur o-BAQBA md BAQ inwater with cyanide has been recendy rcpnrted by us f1j
4. CONCUISIONS
In this review article SAC have demonsnated a ratirinole Us,constructing new anion probes based on two differentfiansduction mechanisms of sensing. These new probes areunique in that they couple both die halide quenching ahiliiyof the quinolinium nucleus, SAth the ability to chelate bothcyanidc and flumid which CStfl otherwise be readily senpiby other fluorescence mcams The new probes arc rradibywater-soluble, have high quantum yields and have thedynamic sensing ranges main stream ithin theconecritration ranges of both physiological and industrialimportance To sonic degree, these ranges a also some’}’aLnoosE In
A CKNOWLEDGEMENTS
This won was supported by the NEIl, USA, NationalCenter for Researeh Resources. RR-Og]l 9. Partial oalarysupport to IRt tm UMBI is also gratefully acknowledged
*u’RONVMS AND SYMBOlS
I4AQ - N-Ben,qt-6-animoquitiolinii,’o bromide
UtQ N-Bfl-6-n1ethy]qLamnunJurn hrooniide
BMOQ N.BenzyD-6-incthoxyquinii]in:uot bromide0mm-or N-2-, 3-, or 4-Boronobenzyly6-omioo-p-BAQOA qutnolirtiunt bromide
0-, a,-, cx 3-, Orp-BlQR; qulnolinium bromide
o-, m-, or N-2-, 3_, or 4-l3ioronnhc,izyt l-6-mcthuxy-p-EMOQRA quiutialinititn bromide
55 no
as
-7
P0 A.t*ft Ck,.py. 29*5. VL I.Y 2
cr LiØit kntiftfng Diode
SEQ - .V-<3-cphopro[,v]6ne%hoquinollmum
TCSPC Ine-CoIrEk3 Single Ptton Cowth1g
RIERE NCE S
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