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Luminescent rhenium(I) polypyridine fluorous complexes as novel trifunctional biological probes.
ARTICLE
pubs.acs.org/IC
Luminescent Rhenium(I) Polypyridine Fluorous Complexes as Novel
Trifunctional Biological Probes
Man-Wai Louie, Tommy Tsz-Him Fong, and Kenneth Kam-Wing Lo*
DepartmentofBiologyandChemistry,CityUniversityofHongKong,TatCheeAvenue,Kowloon,HongKong,P.R.China
ABSTRACT: We present the synthesis, characterization, and
photophysical properties of three luminescent rhenium(I) poly-
pyridine fluorous complexes [Re(Me bpy)(CO) (L)](PF )
2 3 6
0 0
(Me bpy=4,4-dimethyl-2,2-bipyridine;L=3-amino-5-(N-((3-
2
perfluorooctyl)propyl)aminocarbonyl)pyridine(py-Rf-NH )(1),
2
3-isothiocyanato-5-(N-((3-perfluorooctyl)propyl)aminocarbonyl)
pyridine(py-Rf-NCS) (2),3-ethylthioureidyl-5-(N-((3-perfluoro-
octyl)propyl)aminocarbonyl)pyridine (py-Rf-TU-C H ) (3)).
2 5
Theisothiocyanatecomplex2hasbeenusedtolabelbovineserum
albumin(BSA)andglutathione(GSH).Thephotophysicalproper-
tiesoftheresultantbioconjugateshavebeenstudied.Theisolation
of the luminescent fluorous rhenium(cid:1)GSH conjugate from a
mixtureof20aminoacidshasbeendemonstratedusingfluoroussolid-phaseextraction(FSPE).Additionally,thecytotoxicityofcomplexes
1and3towardHeLacellshasbeenexaminedbythe3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazoliumbromide(MTT) assay.The
cellularuptakepropertiesofcomplex3havealsobeeninvestigatedbylaser-scanningconfocalmicroscopy.
’INTRODUCTION labeling reagents are an important addition to the family of
Thefluorouschemistryhascontributedtothedevelopmentof rhenium(I)-based biological probes as they will render the
fluorous-labeledbiomoleculestopossessluminescenceproperties,
newbiphasecatalysisthatallowsthefacileseparation,recovery,
and reuse of catalysts and reagents.1 Recently, this technology which could be applied in the design of new bioassays and
has been implemented to biological applications; for example, imaging experiments. Also, the extent of fluorous labeling of
the use of fluorous affinity tags provides a platform for the biomacromolecules can be readily determined by spectro-
isolation,enrichment,andmassspectrometriccharacterizationof fluorometric methods. Additionally, these reagents allow the
peptides,2a,b oligonucleotides,2c and glycosphingolipids.2d This isolationofthelabeledbioconjugatesbyFluorousSolid-Phase
methodishighlyselectiveandhasovercomemanylimitationsof Extraction(FSPE).
traditionalbioaffinity-basedenrichmentstrategies.Ithasalsoled Hereinwe report three luminescent rhenium(I) polypyridine
tothedevelopmentofsimplesolid-phaseextractionprocedures fluorous complexes [Re(Me 2 bpy)(CO) 3 (L)](PF 6 ) (Me 2 bpy =
involvinginexpensivesolventssuchasmethanolandwater.For 4,4 0 -dimethyl-2,2 0 -bipyridine;L=3-amino-5-(N-((3-perfluorooc-
these reasons, fluorous labeling reagents targeting different tyl)propyl)aminocarbonyl)pyridine (py-Rf-NH ) (1), 3-isothio-
2
functional groups of biomolecules have been designed.3 cyanato-5-(N-((3-perfluorooctyl)propyl)aminocarbonyl)pyridine
Although the development of fluorous chemistry has attracted (py-Rf-NCS)(2),3-ethylthioureidyl-5-(N-((3-perfluorooctyl)
muchattention,theincorporationofperfluorinatedalkylchains propyl)aminocarbonyl)pyridine(py-Rf-TU-C H )(3))(Chart1).
2 5
into emissive organic or inorganic compounds has only been Theisothiocyanatecomplex2hasbeenusedtolabelbovineserum
reported recently.4 Studies of luminescent transition metal albumin(BSA)andglutathione(GSH).Thephotophysicalproper-
complexesappendedwithperfluorinatedalkylchains,tothebest
tiesoftheresultantbioconjugateshavebeenstudied.Theisolation
ofourknowledge,arestillverylimited.4a,c,f of the luminescent fluorous rhenium(cid:1)GSH conjugate from a
On the basis of the rich photophysical properties of lumi- mixture of 20 amino acids has been demonstrated using FSPE.
nescentrhenium(I)polypyridinecomplexes,4a,5(cid:1)15
wehavea Additionally, the cytotoxicity of complexes1 and 3 toward HeLa
longstandinginterestintheuseofthesecomplexesasbiolog- cells has been examined by the 3-(4,5-dimethyl-2-thiazolyl)-2,5-
ical labeling reagents14 and noncovalent probes for biomole- diphenyltetrazolium bromide (MTT) assay. The cellular uptake
cules and ions.15 The advantages of using rhenium(I) com- properties of complex 3 have also been investigated by laser-
plexesoverotherd6transitionmetalcomplexesaretheirease
scanningconfocalmicroscopy.
ofemissioncolor-tuningandlonger-livedexcitedstates,which
are useful in the development of multicolor probes for time-
resolved applications such as fluorescence lifetime imaging Received: May28,2011
microscopy (FLIM).16 We anticipate that luminescent fluorous Published: August30,2011
r2011AmericanChemicalSociety 9465 dx.doi.org/10.1021/ic201143f|Inorg.Chem.2011,50,9465–9471
InorganicChemistry ARTICLE
Chart 1. StructuresoftheRhenium(I)Polypyridine petroleumetheraffordedthecomplexasyellowcrystals.Yield:216mg
FluorousComplexes (49%). 1H NMR (300 MHz, acetone-d, 298 K, Si(CH)): δ 9.22
6 3 4
0 0
(d,J=5.7Hz,2H,H6andH6 ofMebpy),8.61(s,2H,H3andH3 of
2
Mebpy),8.06(d,J=2.4Hz,1H,H6ofpyridine),8.03(cid:1)7.97(m,2H,H2
2
ofpyridine,NH),7.82(d,J=5.7Hz,2H,H5andH50ofMebpy),7.53
2
(t,J=1.5Hz,1H,H4ofpyridine),5.54(s,2H,NH),3.44(q,J=6.3Hz,
2
2H,NHCHCHCH),2.63(s,6H,CH ofMebpy),2.42(cid:1)2.20(m,2H,
2 2 2 3 2
NHCHCHCH),1.88(cid:1)1.82(m,2H,NHCHCHCH);13CNMR(75
2 2 2 2 2 2
MHz, acetone-d, 298 K, Si(CH)): δ 196.09, 192.20, 163.65, 155.84,
6 3 4
154.47, 153.35, 147.15, 139.80, 138.65, 133.64, 129.74, 125.66, 121.56,
38.88,20.87;IR(KBr)ν/cm (cid:1)1:3403(br,N—H),2030(s,CtO),1924
(s,CtO),1623(m,CdO),1241(m,C—F),1210(m,C—F),1149
(m, C—F), 1030 (m, C—F), 848 (s, PF (cid:1) ); positive-ion ESI-MS ion
’EXPERIMENTALSECTION clustersatm/z1052[M(cid:1)PF]+;analcalcd 6 (%)forC H NOPF Re:
6 32 24 5 4 23
C,32.12;H,2.02;N,5.85;found:C,32.35;H,2.26;N,5.83.
MaterialsandSynthesis.Allsolventswereofanalyticalreagentgrade [Re(Me 2 bpy)(CO) 3 (py-Rf-NCS)](PF 6 ) (2). A mixture of complex 1
andpurifiedaccordingtostandardprocedures.175-Aminonicotinicacid,N-
(0.16mmol)andfinelycrushedCaCO (64mg,0.64mmol)wasstirred
3
hydroxysuccinimide,AgCFSO,KPF,CaCO,thiophosgene,andethyl-
3 3 6 3 inacetone(20mL)atroomtemperatureunderaninertatmosphereof
aminewerepurchasedfromAcros.Me 2 bpyandRe(CO) 5 Clwereobtained nitrogen,andthiophosgene(26μL,0.34mmol)wasaddedslowlytothe
from Aldrich. BSA was purchased from Calbiochem. 3-(Perfluorooctyl)
mixture.Thesuspensionwasstirredinthedarkatroomtemperaturefor
propylamineandFSPEcolumns(2g,8mLtube)werepurchasedfrom
5 min. The suspensionwas filtered, and the filtrate was evaporatedto
Fluorous Technologies Inc. The 20 amino acids used in the FSPE
drynesstogiveayellowsolid.Recrystallizationofthecrudeproductfrom
purificationincludingalanine,arginine,asparagine,asparticacid,cysteine, CHCl/petroleumetheraffordedthecomplexasyellowcrystals.Yield:
2 2
glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, 200mg(89%).1HNMR(300MHz,acetone-d ,298K,Si(CH)):δ
6 3 4
methionine,phenylalanine,proline,serine,threonine,tryptophan,tyrosine, 9.31 (d, J = 5.7 Hz, 2H, H6 and H6 0 of Mebpy), 8.74 (s, 1H, H6 of
2
andvalinewerepurchasedfromAldrichorAcros.Allthesechemicalswere pyridine),8.70(d,J=1.2Hz,1H,H2ofpyridine),8.62(s,2H,H3andH30
usedwithoutfurtherpurification.5-AminonicotinicacidN-hydroxysucci- ofMebpy),8.42(s,1H,H4ofpyridine),8.29(br,1H,NH),7.84(d,J=5.7
2
nimidyl ester14c and [Re(Me 2 bpy)(CO) 3 (CH 3 CN)](CF 3 SO 3 )8a were Hz, 2H, H5 and H50 of Me 2 bpy), 3.49 (t, J = 6.3 Hz, 2H, NH-
preparedasdescribedpreviously.Allbuffercomponentswereofmolecular CHCHCH), 2.62 (s, 6H, CH of Mebpy), 2.40(cid:1)2.18 (m, 2H,
2 2 2 3 2
biologygrade.PD-10size-exclusioncolumnsandYM-30centriconswere NHCHCHCH), 1.90(cid:1)1.83 (m, 2H, NHCHCHCH); 13C NMR
2 2 2 2 2 2
purchasedfromGEHealthcareandMillipore,respectively.GSHandMTT (75MHz,acetone-d,298K,Si(CH)):δ195.56,191.98,155.94,154.61,
6 3 4
(Sigma)wereusedwithoutfurtherpurification.HeLacellswereobtained 153.63,150.55,149.33,137.43,132.23,129.89,127.77,125.83,20.95;IR
from American Type Culture Collection. Dulbecco’s modified Eagle’s (KBr)ν/cm (cid:1)1:3423(br,N—H),2111(m,NdCdS),2036(s,CtO),
medium (DMEM), MitoTracker Deep Red FM, fetal bovine serum 1931(s,CtO),1671(m,CdO),1241(m,C—F),1209(m,C—F),1149
(FBS),phosphatebufferedsaline(PBS),trypsin(cid:1)EDTA,andpenicillin/ (m, C—F), 1032 (m, C—F), 847 (s, PF (cid:1) ); positive-ion ESI-MS ion
6
streptomycinwerepurchasedfromInvitrogen.Thegrowthmediumforcell clustersatm/z1095[M(cid:1)PF]+;analcalcd(%)forC H NOSPF -
6 33 22 5 4 23
culturecontainedDMEMwith10%FBSand1%penicillin/streptomycin. Re31.5CHCl:C,30.33;H,1.84;N,5.47;found:C,30.31;H,2.08;N,5.34.
2 2
3-Amino-5-(N-((3-perfluorooctyl)propyl)aminocarbonyl)pyridine, [Re(Me
2
bpy)(CO)
3
(py-Rf-TU-C
2
H
5
)](PF
6
)(3).Amixtureofcomplex2
py-Rf-NH
2
.Amixtureof5-aminonicotinicacidN-hydroxysuccinimidyl (0.08mmol)andethylamine(10μL,0.16mmol)wasstirredinacetone
ester (700 mg, 3 mmol), triethylamine (1.67 mL, 12 mmol), and (15mL)atroomtemperatureunderaninertatmosphereofnitrogenfor
3-(perfluorooctyl)propylamine(1.40g,3mmol)inDMF(7mL)was 2h.Thesolutionwasevaporatedtodrynesstogiveayellowsolid,which
stirredunderaninertatmosphereofnitrogenatroomtemperaturefor was purified by column chromatography on alumina. The desired
12 h. The mixture was then evaporated to dryness under reduced productwaselutedwithCH Cl /MeOH(20:1,v/v).Recrystallization
2 2
pressure, resulting in a reddish brown solid. The crude product was of the crude product from CH Cl /petroleum ether afforded the
2 2
purified by column chromatography on silica gel with the eluent complexasyellowcrystals.Yield:86mg(83%).1HNMR(300MHz,
changing from hexane/ethyl acetate (1:4, v/v) to CH Cl /MeOH acetone-d ,298K,Si(CH ) ):δ9.31(s,1H,H2ofpyridine),9.21(d,J=
2 2 6 3 4
(10:1,v/v).Thefractionscontainingtheproductwerecollectedand 5.4Hz,2H,H6andH60ofMe bpy),8.60(cid:1)8.53(m,4H,H4andH6of
2
0
thesolventwasremovedbyrotaryevaporationtogiveawhitesolid.Yield: pyridine,H3andH3 ofMe bpy),8.36(s,1H,NH),7.78(d,J=4.8Hz,
2
1.02 g (57%). 1H NMR (300 MHz, acetone-d 6 , 298 K, Si(CH 3 ) 4 ): 2H, H5 and H5 0 of Me 2 bpy), 3.47(cid:1)3.45 (m, 4H, CH 2 of C 2 H 5 ,
δ8.27(d,J=1.8Hz,1H,H6ofpyridine),8.13(d,J=2.7Hz,1H,H2 NHCH
2
CH
2
CH
2
),2.59(s,6H,CH
3
ofMe
2
bpy),2.39(cid:1)2.13(m,2H,
of pyridine), 7.92 (br, 1H, NH), 7.42 (t, J = 2.4 Hz, 1H, H4 of NHCH 2 CH 2 CH 2 ),1.88(cid:1)1.82(m,2H,NHCH 2 CH 2 CH 2 ),1.40(t,J=
pyridine), 5.07 (br, 2H, NH 2 ), 3.49 (t, J = 7.2 Hz, 2H, NHCH 2 - 7.5 Hz, 3H, CH 3 of Et); 13C NMR (75 MHz, acetone-d 6 , 298 K,
CH 2 CH 2 ),2.39(cid:1)2.18(m,2H,NHCH 2 CH 2 CH 2 ),1.91(p,J=6.9Hz, Si(CH 3 ) 4 ): δ195.97, 192.50, 162.97, 155.87, 154.34, 153.50, 152.51,
2H, NHCH CH CH ); 13C NMR (75 MHz, acetone-d , 298 K, 145.84, 132.77, 129.91, 128.30, 125.81, 124.48, 39.04, 20.93, 20.58,
Si(CH ) ): δ 2 166 2 .22, 2 144.41, 139.31, 136.50, 130.84, 118. 6 92, 38.75, 13.43, 12.28; IR (KBr) ν/cm (cid:1)1: 3391 (br, N—H), 2034 (s, CtO),
3 4
20.81;positive-ionESI-MSionclustersatm/z598[M+H]+. 1925(s,CtO),1654(m,CdO),1241(m,C—F),1210(m,C—F),
[Re(Me 2 bpy)(CO) 3 (py-Rf-NH 2 )](PF 6 ) (1). A mixture of [Re(Me 2 bpy) 1149(m,C—F),1031(m,C—F),848(PF 6 (cid:1) );positive-ionESI-MSion
(CO) 3 (CH 3 CN)](CF 3 SO 3 )(0.35mmol)andpy-Rf-NH 2 (209mg,0.35 clustersatm/z1140[M(cid:1)PF 6 ]+;analcalcd(%)forC 35 H 29 N 6 O 4 SPF 23 Re:
mmol) was refluxed in THF (20 mL) under an inert atmosphere of C,38.47,H,2.01,N,4.98;found:C,38.43;H,2.23,N,4.73.
nitrogenfor12h.Thesolutionwasevaporatedtodrynesstogiveayellow Instrumentation and Physical Measurements. 1H and
Thecomplexwasthenconvertedtothehexafluorophosphatesaltbyanion 13CNMRspectrawererecordedonaVarianMercury300MHzNMR
exchangewithKPF inMeOHandthenpurifiedbycolumnchromatog- spectrometerat298K.Positive-ionESImassspectrawererecordedona
6
raphyonalumina.ThedesiredproductwaselutedwithCHCl/MeOH Perkin-Elmer Sciex API 365 mass spectrometer. IR spectra were
2 2
(20:1, v/v). Recrystallization of the crude product from CHCl/ recorded on a Perkin-Elmer 1600 series FT-IR spectrophotometer.
2 2
9466 dx.doi.org/10.1021/ic201143f|Inorg.Chem.2011,50,9465–9471
InorganicChemistry ARTICLE
ElementalanalyseswerecarriedoutonaVarioELIIICHNelemental Table1. ElectronicAbsorptionSpectralDataofComplexes
analyzer.Electronicabsorptionandsteady-stateemissionspectrawere 1(cid:1)3inCH Cl at298K
2 2
recorded on a Hewlett-Packard 8453 diode array spectrophotometer
complex λ /nm(ε/dm3mol(cid:1)1cm(cid:1)1)
and a SPEX FluoroLog 3-TCSPC spectrophotometer, respectively. abs
Emission lifetimes were measured in the Fast MCS mode with a 1 254(25230),308sh(12050),318(12305),337sh(7405),
NanoLED N-375 as the excitation source. Unless specified, all the
393sh(1960)
solutionsforphotophysicalstudiesweredegassedwithnofewerthan
2 281(19815),303sh(14320),317(11220),355sh(3765),
four successive freeze(cid:1)pump(cid:1)thaw cycles and stored in a 10 cm3
379sh(2130)
round-bottomed flask equipped with a side arm 1 cm fluorescence
3 256(24390),279sh(20780),318sh(9895),337sh(5420)
cuvette and sealed from the atmosphere by a Rotaflo HP6/6 quick-
releaseTeflonstopper.Luminescencequantumyieldsweremeasured 385sh(2045)
by the optically dilute method with an aerated aqueous solution
of [Ru(bpy) ]Cl (Φ = 0.028, λ = 455 nm) as the standard 1h,themediumwasremovedandthecelllayerwaswashedwithPBS
3 2 em ex
solution.18,19 (1mL(cid:3)5).Thecoverslipwasmountedontoasterilizedglassslideand
Labeling of BSA with Complex 2. Complex 2 (1.48 mg, 1.2 thenimagedusingaLeicaTCSSPEconfocalmicroscopewithanoil
μmol) in anhydrous DMSO (50 μL) was added to BSA (10 mg, immersion63(cid:3)objectiveandanexcitationwavelengthat405nm.The
151 nmol) in 50 mM carbonate buffer (450 μL) at pH 9.7. The emission was measured using a long-pass filter at 532 nm. In the
suspensionwasstirredfor12hinthedarkatroomtemperature,and colocalization imaging experiments involving MitoTracker Deep Red
thesolidresiduewasremovedbycentrifugation.Thesupernatantwas FM, the excitation wavelength was 633 nm and the emission was
thendilutedto1.0mLwith50mMpotassiumphosphatebufferatpH measuredat>640nm.
7.4andloadedontoaPD-10columnequilibratedwiththesamebuffer.
Thefirstelutionbandwith stronggreenemissionwascollected. The
’RESULTSANDDISCUSSION
resultantbioconjugateRe-BSAwaswashedsuccessivelywithpotassium
phosphatebufferusingaYM-30centricon,concentratedto1.5mL,and
storedat4(cid:1)C. ComplexDesign.Thedesignoftherhenium(I)polypyridine
fluorouscomplexesisbasedontheuseof5-aminonicotinicacid;
LabelingofGSHwithComplex2.Complex2(1.05mg,0.85
μmol)inanhydrousDMSO(200μL)wasaddedtoGSH(21.5mg,70 thecarboxylgroupofwhichcanbefunctionalizedwithafluorous
μmol)inamixtureofH O(1.8mL)andtriethylamine(20μL).The chain, the amine group can be readily converted to the amine-
2
specific isothiocyanate group, and the pyridine can be coordi-
suspensionwasincubatedfor5minatroomtemperature,andthesolid
residue was removed by centrifugation. The supernatant was loaded natedtotherhenium(I)center.Thus,theligandpy-Rf-NH 2 was
ontoanFSPEcolumn,whichhadbeenactivatedbyDMF(5mL)and obtained from the reaction of 3-(perfluorooctyl)propylamine
preconditioned with H O. The column was operated under gradient with5-aminonicotinicacidN-hydroxysuccinimidylesterinDMF
2
elution(H Oto40%aqueousMeOH).Finally,theresultantbioconju- at room temperature. The amine complex 1 was synthesized
2
gateRe-GSHwaselutedusing60%aqueousMeOH. fromthereactionof[Re(Me 2 bpy)(CO) 3 (CH 3 CN)](CF 3 SO 3 )
IsolationofRe-GSHfromaMixtureofAminoAcidswith withpy-Rf-NH inrefluxingTHF,followedbyanionexchange
2
FSPE.AmixtureofRe-GSH(0.7mM)andthe20aminoacids(eachat with KPF and column chromatographic purification. The iso-
6
0.1mg/mL)inwater(2mL)wasloadedontoanFSPEcolumn(2g, thiocyanate complex 2 was prepared from the reaction of
8mLtube),whichhadbeenactivatedbyDMF(5mL)andprecondi- complex 1 with thiophosgene in acetone. The amine reactivity
tionedwithH O.Whenthecolumnwasoperatedundergradientelution ofcomplex2wasinvestigatedbyreactingwithamodelsubstrate
2
(H 2 Oto40%aqueousMeOH),aluminescentbandremainedatthetop ethylamine,yieldingthethioureacomplex3.Allthecomplexes
andtheelutedsolution(flow-throughfraction)wascollected.Whenthe werecharacterizedby1Hand13CNMR,positive-ionESI-MS,IR
mobile phase was changed to 60% aqueous MeOH, the luminescent spectroscopy and gave satisfactory elemental analyses. The IR
band was eluted and collected (elution fraction). The initial mixture, spectra of these complexes showed absorption bands from ca.
flow-throughfraction,andelutionfractionwereallanalyzedbyESI-MS. 1240to1100cm (cid:1)1,whichhavebeenassignedtoC—Fstretch-
MTTAssays.Cellswereseededina96-wellflat-bottomedmicro-
ingofthefluorousmoiety.Theisothiocyanategroupofcomplex
plate (ca. 10000 cells per well) in growth medium (100 μL) and 2 was associated with an absorption peak at ca. 2110 cm (cid:1)1,
incubated at 37 (cid:1)C under a 5% CO 2 atmosphere for 24 h. The typicalofNdCdSstretching.Themoderatelyintenseabsorption
rhenium(I)polypyridinecomplexwasdissolvedinthegrowthmedium
bandatca.850cm
(cid:1)1hasbeenassignedtotheP—Fstretching
with1%DMSO,andthesolutionswereaddedtothewells.Afterthe (cid:1)
of the PF ion. All three fluorous complexes were yellow
microtiterplatewasincubatedfor48h,MTTinPBS(5mgpermL, 6
in color and very soluble in common organic solvents such
10μL)wasaddedtoeachwell.Themicroplatewasincubatedforanother
as CH Cl , CH CN, and alcohols but sparingly soluble in
3 h. The medium was removed carefully and isopropanol (200 μL) 2 2 3
aqueoussolution.
was added to each well. The microplate was further incubated for
ElectronicAbsorptionandEmissionSpectroscopy.Theelec-
5min.Alltheassayswereruninparallelwithapositivecontrol,inwhich
tronicabsorptionspectraldataofcomplexes1(cid:1)3inCHCl at298K
cisplatinwasusedasacytotoxicagent.Theabsorbanceofthesolutions 2 2
aresummarizedinTable1.Theabsorptionspectraofthecomplexes
at570nmwasmeasuredwithaSPECTRAmax340microplatereader
arecharacterizedbyhighenergyintraligand(πfπ*)(Mebpyand
(Molecular Devices Corp., Sunnyvale, CA). The IC 50 values of the 2
complexes were determined from dose dependence of surviving cells pyridine) and low-energy metal-to-ligand charge-transfer (MLCT)
afterexposuretothecomplexesfor48h. (dπ(Re) f π*(Me 2 bpy)) features at ca. 250(cid:1)320 and 340(cid:1)
Live-CellConfocalImaging.HeLacellsingrowthmediumwere 390nm,respectively.Irradiationofthecomplexesinfluidsolutions
seededonasterilizedcoverslipina60-mmtissueculturedishandgrown at298Kandn-butyronitrileglassat77Kresultedinintenseandlong-
at37(cid:1)Cundera5%CO atmospherefor48h.Thegrowthmediumwas livedgreentoyellowemission.Thephotophysicaldataarelistedin
2
thenremovedandreplacedwithmedium/DMSO(99:1,v/v)containing Table 2. The emission of these complexes has been assigned to a
the rhenium(I) polypyridine complex (5 μM). After incubation for 3MLCT (dπ(Re) f π*(Mebpy)) excited state.4a,5(cid:1)15 The
2
9467 dx.doi.org/10.1021/ic201143f|Inorg.Chem.2011,50,9465–9471
InorganicChemistry ARTICLE
Table2. PhotophysicalDataofComplexes1(cid:1)3
complex medium(T/K) λ /nm τ/μs Φ
em o
1 CHCl (298) 533 0.70 0.20
2 2
CHCN(298) 546 0.35 0.065
3
glass(77)a 496 4.97
2 CHCl (298) 526 0.68 0.17
2 2
CHCN(298) 539 0.31 0.047
3
glass(77)a 490 4.55
3 CHCl (298) 528 0.79 0.098
2 2
CHCN(298) 538 0.25 0.014
3
glass(77)a 490 4.61
aInn-butyronitrileglass.
Figure2. Electronicabsorption(solidline)andemission(dashedline)
spectraofRe-BSAin50mMphosphatebufferatpH7.4at298K.
Figure1. ElectronicabsorptionspectruminCH Cl (blue)andemis-
2 2
sion spectra of complex 1 in CH CN at 298 K (red) and in n-
3
butyronitrile(green)at77K. Figure 3. Emission spectrum of Re-GSH in degassed 60% aqueous
MeOHat298K.
emissionenergiesofthecomplexesfollowedtheorder:1<2≈3.The whichhasbeenassignedtoa3MLCTexcitedstate.4a,5(cid:1)15 Interest-
occurrence of lower emission energy for complex 1 has been ingly, unlike Re-BSA, the modified glutathione exhibited single
attributedtoitselectron-donatingaminegroup,whichenrichedthe exponentialdecay.Also,theemissionenergyofRe-GSH(565nm)
electron density of the metal center and hence destabilized the was lower than that of its BSA counterpart (523 nm). It is
dπ(Re) levels. The absorption and emission spectra of complex 1 conceivablethatthedoubleexponentialdecayandhigheremission
areshowninFigure1asanexample. energy of Re-BSA are a consequence of the hydrophobic local
LabelingofBSAandGSHwithComplex2.Astheisothio- environmentoftheluminescentcomplex.14
cyanatecomplex2hasbeendemonstratedtobereactivetoward IsolationofRe-GSHfromaMixtureofAminoAcidswith
ethylamine, it was used to label a model protein BSA. The FSPE.Tostudythepotentialuseofthefluorouslabelingreagent
resultantbioconjugateRe-BSAwasisolatedandpurifiedbysize- complex 2 in biomolecular purification and enrichment,2a the
exclusionchromatography andultrafiltration. Uponirradiation, isolation of Re-GSH from a mixture of amino acids has been
the bioconjugate exhibited intense and long-lived green performed. In this experiment, a mixture of Re-GSH and 20
emission in degassed 50 mM potassium phosphate buffer at aminoacidswasloadedontoanFSPEcolumn,whichwasthen
pH 7.4. The electronic absorption and emission spectra of the washedwith40%aqueousMeOH.Asexpected,theflow-through
bioconjugateinbufferareshowninFigure2.Wehaveassigned fractionwasnotemissiveuponirradiation.Importantly,changing
theemissionbandat523 nmto a3MLCTexcited state.4a,5(cid:1)15 themobilephaseto60%aqueousMeOHledtotheelutionofa
Theobservedbiexponentialdecay(τ =0.17μs(60%),τ =0.54μs luminescent species. The ESI mass spectra of (a) the initial
1 2
(40%))oftheemissionisnotuncommonforbiomoleculeslabeled mixture, (b) flow-through fraction with 40% aqueous MeOH,
withluminescenttransitionmetalcomplexes.14Onthebasisofthe and(c)elutionfractionwith60%aqueousMeOHareshownin
spectroscopicdata,therhenium-to-BSAratiowasdeterminedtobe Figure 4. The ESI mass spectrum of the elution fraction
ca. 4.1, which is comparable to those of other rhenium(cid:1)protein (Figure 4c) revealed a peak at m/z = 1401, corresponding to
conjugates (ca. 1.8(cid:1)4.8).14 Thus, despite the rigid and bulky Re-GSH.Thissignalwasabsentinthemassspectrumoftheflow-
fluorouschain,therhenium(I)polypyridineisothiocyanatecomplex through fraction (Figure 4b), meaning that the 20 unlabeled
2 is capable of functioning as a luminescent fluorous labeling aminoacidswerewashedoutintheflow-throughfractionwhereas
reagent.WehavealsotaggedthesmallpeptideGSHwiththesame thefluorousconjugateRe-GSHwasretainedontheFSPEcolumn
complex.TheresultantbioconjugateRe-GSHwaspurifiedbyFSPE duetohighlyselectivefluorous(cid:1)fluorousinteractions.
andcharacterizedbyESI-MS(m/z=1401).Uponirradiation,the CytotoxicActivityandCellularUptake.Thecytotoxic activity
bioconjugateshowedintenseandlong-livedyellowemission(λ = andcellularuptakebehaviorofluminescentrhenium(I)polypyridine
em
565nm,τ =0.43μs)indegassed60%aqueousMeOH(Figure3), complexeshavereceivedmuchattentionrecently.12a,13,14c,15f(cid:1)15hIn
o
9468 dx.doi.org/10.1021/ic201143f|Inorg.Chem.2011,50,9465–9471
InorganicChemistry ARTICLE
Figure 5. Laser-scanning confocal microscopy images of HeLa cells
(pseudocolored)incubatedwithcomplex3(5μM)at37(cid:1)C(left)and
4(cid:1)C(right)for1h.
(5μM)at37(cid:1)Cundera5%CO atmospherefor1hresultedin
2
cellular uptake, as revealed by laser-scanning confocal microscopy
(Figure 5, left). The complex was not homogeneously distributed
within the cytoplasm but localized in the juxtanuclear region with
brightpunctatestaining.Incontrast,thenucleishowedmuchweaker
emission,indicativeofnegligiblenuclearuptake.Incubationat4(cid:1)C
ledtomuchreduceduptakeefficiency(Figure5,right),suggesting
thattheinternalizationoccurredthroughanenergy-requiringpathway
such as endocytosis.22 Most of the punctate emissive dots are
presumably due to staining of mitochondria, which are prevalent
around the nucleus. To confirm this, HeLa cells pretreated with
complex 3 (5 μM, 1 h, λ = 405 nm) were coincubated with
ex
MitoTrackerDeepRedFM(100nM,20min,λ =633nm),whose
ex
spectral properties do not interfere. The significant overlap in the
imageshowninFigure6indicatesthatthecomplexwaspredomi-
nantlyenrichedinmitochondria.Themitochondrial-targetingprop-
erties of the complex can be attributed to its cationic charge
and highly hydrophobic
nature.13,14c,15f(cid:1)h
In view of the fact that
the MitoTracker only stains mitochondria in living cells, the
cells incubated with the complex remained viable under our
experimental conditions. These findings highlight that small
molecules conjugated with the fluorous isothiocyanate complex
2canbereadilyfollowedandexaminedbyconfocalmicroscopy.
Thisstrategyisparticularlyusefulforthosemoleculesthatcannot
Figure4. ESImassspectraof(a)Re-GSHandamixtureof20amino penetratelivingcellsontheirown.
acidsinwater,(b)flow-throughfractionwith40%aqueousMeOH,and
(c)elutionfractionwith60%aqueousMeOHfromanFSPEcolumn.
’CONCLUSION
thiswork,thecytotoxicityoftheaminecomplex1andthethiourea Themainthemeofthisworkisthedevelopmentofrhenium-
complex3hasbeenexaminedbytheMTTassayusingHeLacellsasa (I)complexesasnoveltrifunctionalbiologicalprobesthathave
modelcellline.20Complex2wasomittedinthisstudybecausethe luminescence properties for detection, a fluorous moiety for
isothiocyanategroupisunstableinaqueoussolution.The48-hIC selective isolation and interactions, and a reactive functional
50
valueofcomplex1(ca.8.70μM)wassignificantlylowerthanthatof group for bioconjugation. Since the development of fluorous
cisplatin(ca.17.80μM),indicativeofhighcytotoxicity.Additionally, labeling reagents was reported several years ago,2a there is an
theIC valueofcomplex3(ca.17.02μM)wassimilartothoseof increasinginterestinusingthesereagentsinproteomics,meta-
50
relatedrhenium(I) thioureacomplexes(IC =17.5(cid:1)28.5μM)14c bolomics,andmicroarraystudies.2,3Notonlycanthefluorous-
50
but slightly higher that of complex 1. Thus, the conversion of the taggedmoleculesbeseparated andenrichedonFSPE,butalso
aminegroupofcomplex1tothethioureagrouphasrenderedthis they possess excellent mass spectrometry characteristics, en-
complex more biocompatible. It is important to mention that the ablingefficientcharacterization.Inadditiontotheseproperties,
cytotoxicityofthesecomplexescanbereadilymodifiedwithachoice therhenium(I)complexesinthisworkofferinterestinglumines-
of ligands with different lipophilicity, and the use of poly(ethylene cencebehaviortothelabeledmolecules,whichwouldallownew
glycol) is known to substantially reduce the cytotoxic activity of assaydesign,easyquantitationofdegreeoffluorousmodification
relatedtransitionmetalcomplexes.21 ofthetargetmolecules,andthepossibilityofstudyingthecellular
The cellular uptake and intracellular localization of complex 3, uptakeofthelabeledmoleculesbyconfocalmicroscopy.Indeed,
whichcanbeconsideredasamodeloffluorous-labeledbiomolecules, we have demonstrated that (1) biomolecules can be readily
havebeeninvestigated.IncubationofHeLacellswiththecomplex labeledwiththeisothiocyanatecomplex2toaffordluminescent
9469 dx.doi.org/10.1021/ic201143f|Inorg.Chem.2011,50,9465–9471
InorganicChemistry ARTICLE
Figure6. Laser-scanningconfocalmicroscopyimagesofHeLacells(pseudocolored)uponincubationsuccessivelywithcomplex3(5μM,1h,λ =
ex
405nm,left)at37(cid:1)CandMitoTrackerDeepRedFM(100nM,20min,λ =633nm,middle).Theoverlaidconfocalimagesareshownontheright.
ex
bioconjugatesand(2)theseparationandpurificationoftheGSH
S.-K; Liu, H.-W.; Lo, K. K.-W. Chem. Commun. 2011, DOI: 10.1039/
bioconjugate can be performed readily on an FSPE column. C1CC11423A.
Although the emission behavior and cellular uptake properties (5) (a) Busby, M.; Gabrielsson, A.; Matousek, P.; Towrie, M.; Di
of many luminescent rhenium(I) complexes have been re-
Bilio,A.J.;Gray,H.B.;Vl(cid:3)cek,A.,Jr.Inorg.Chem.2004,43,4994–5002.
ported in the literature, the fluorous moiety has offered new (b)Gabrielsson,A.;Matousek,P.;Towrie,M.;Hartl,F.;Z(cid:1)ali(cid:3)s,S.;Vl(cid:3)cek,
A., Jr. J. Phys. Chem. A 2005, 109, 6147–6153. (c) Vl(cid:3)cek, A., Jr. Top.
and unique properties to the new complexes in this work,
Organomet. Chem. 2010, 29, 73–114. (d) Blanco-Rodríguez, A. M.;
rendering them novel trifunctional biological probes. The Towrie, M.; S(cid:1)ykora, J.; Z(cid:1)ali(cid:3)s, S.; Vl(cid:3)cek, A., Jr. Inorg. Chem. 2011, 50,
design of related luminescent transition metal fluorous com- 6122–6134.
plexesasahandletoisolateandidentifyintracellularbiologi- (6) (a) Sun, S.-S.; Lees, A. J. Organometallics 2002, 21, 39–49.
calreceptorsisunderway. (b) Sun, S.-S.; Lees, A. J. Coord. Chem. Rev. 2002, 230, 171–192.
(c) Sun, S.-S.; Lees, A. J.; Zavalij, P. Y. Inorg. Chem. 2003, 42,
’AUTHORINFORMATION 3445–3453.(d)Kumar,A.;Sun,S.-S.;Lees,A.J.Top.Organomet.Chem.
2010,29,1–35.
CorrespondingAuthor (7) (a) Metcalfe, C.; Webb, M.; Thomas, J. A. Chem. Commun.
*E-mail: bhkenlo@cityu.edu.hk. Fax: (+852) 3442 0522. Tel: 2002,2026–2027.(b)Foxon,S.P.;Phillips,T.;Gill,M.R.;Towrie,M.;
(+852)34427231. Parker,A.W.;Webb,M.;Thomas,J.A.Angew.Chem.,Int.Ed.2007,46,
3686–3688.
(8) (a)Caspar,J.V.;Meyer,T.J.J.Phys.Chem.1983,87,952–957.
’ACKNOWLEDGMENT (b)Walters,K.A.;Dattelbaum,D.M.;Ley,K.D.;Schoonover,J.R.;
Meyer, T. J.; Schanze, K. S. Chem. Commun. 2001, 1834–1835.
WethankTheHongKongResearchGrantsCouncil(Project
(c) Dattelbaum, D. M.; Martin, R. L.; Schoonover, J. R.; Meyer,T.J.
No.CityU102109)andCityUniversityofHongKong(Project J.Phys.Chem.A2004,108,3518–3526.(d)Patrocinio,A.O.T.;Brennaman,
No.7002679)forfinancialsupport.M.-W.L.acknowledgesthe
M. K.; Meyer, T. J.; Murakami Iha, N. Y. J. Phys. Chem. A 2010, 114,
receipt of a Postgraduate Studentship, a Research Tuition 12129–12137.
Scholarship,andanOutstandingAcademicPerformanceAward (9) (a)Wenger,O.S.;Henling,L.M.;Day,M.W.;Winkler,J.R.;
alladministeredbyCityUniversityofHongKong. Gray,H.B.Inorg.Chem.2004,43,2043–2048.(b)Belliston-Bittner,W.;
Dunn,A.R.;Nguyen,Y.H.L.;Stuehr,D.J.;Winkler,J.R.;Gray,H.B.
J.Am.Chem.Soc.2005,127,15907–15915.
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