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Synthesis, Emission Characteristics, Cellular Studies, and Bioconjugation Properties of Luminescent Rhenium(I) Polypyridine Complexes with a Fluorous Pendant
Article
pubs.acs.org/Organometallics
Synthesis, Emission Characteristics, Cellular Studies, and
Bioconjugation Properties of Luminescent Rhenium(I) Polypyridine
Complexes with a Fluorous Pendant
*
Man-Wai Louie, Alex Wing-Tat Choi, Hua-Wei Liu, Bruce Ting-Ngok Chan, and Kenneth Kam-Wing Lo
Institute of Molecular Functional Materials (Areas of Excellence Scheme, University Grants Committee (Hong Kong)) and
Department of Biology and Chemistry, City University of Hong Kong, Tat Chee Avenue, Kowloon, Hong Kong,
People's Republic of China
*
S Supporting Information
ABSTRACT: Inthisarticle,wereportthephotophysicalandbiologicalpropertiesofnewluminescentrhenium(I)polypyridine
fluorous complexes. The complexes [Re(N ∧ N)(CO) (py-Rf-NH )](PF ) (py-Rf-NH = 3-amino-5-(N-((3-perfluorooctyl)-
3 2 6 2
∧
propyl)aminocarbonyl)pyridine; N N = 1,10-phenanthroline (phen) (1a), 3,4,7,8-tetramethyl-1,10-phenanthroline (Me -phen)
4
(2a),4,7-diphenyl-1,10-phenanthroline(Ph -phen)(3a))containafluorouspendantandaprimaryamineformodification.They
2
∧
have been converted to the isothiocyanate complexes [Re(N N)(CO) (py-Rf-NCS)](PF ) (py-Rf-NCS = 3-isothiocyanato-5-
3 6
(N-((3-perfluorooctyl)propyl)aminocarbonyl)pyridine; N ∧ N = phen (1b), Me -phen (2b), Ph -phen (3b)) with thiophosgene.
4 2
The isothiocyanate complexes have been reacted with ethylamine, a model substrate, yielding the thiourea complexes [Re-
(N ∧ N)(CO) (py-Rf-TU-Et)](PF ) (py-Rf-TU-Et = 3-ethylthioureidyl-5-(N-((3-perfluorooctyl)propyl)aminocarbonyl)pyridine;
3 6
N ∧ N=phen(1c),Me -phen(2c),Ph -phen(3c)).Uponirradiation,allofthesefluorouscomplexesexhibitedintenseandlong-
4 2
livedgreen-to-yellowtripletmetal-to-ligandcharge-transfer(3MLCT)(dπ(Re)→π*(N ∧ N))emissioninfluidsolutionsat298K
andinrigidglassat77K.Thelipophilicity,cellularuptakeproperties,andcytotoxicityoftheamineandthioureacomplexeshave
been studied. Results of MTT assays showed that the less lipophilic fluorous complexes displayed higher cytotoxicity. The
isothiocyanate complexes 1b−3b have been used to label glutathione (GSH), bovine serum albumin (BSA), branched
poly(ethyleneimine)(bPEI,averageM
w
=25kDa),andpoly-L-lysine(Poly-K,M
w
>30kDa);thephotophysicalpropertiesofthe
resultant conjugates have been examined. Additionally, we have demonstrated that the GSH conjugates can be readily isolated
and purified with fluorous solid phase extraction. The uptake of complex 3c and the conjugate BSA-3c by HeLa cells has also
been studied by laser-scanning confocal microscopy. Furthermore, the DNA-binding and polyplex-formation properties of the
conjugates bPEI-3c and Poly-K-3c have been investigated.
■
INTRODUCTION synthesis,3 and labeling of specific peptide subsets for enrichment
An interesting characteristic of highly fluorinated compounds (or using FSPE and analysis by mass spectrometry.4 Additionally,
fluorouscompounds)istheirhighfluorophilicity,whichoriginates various glycosphingolipids,5 nicotinamide adenine dinucleotides,6
from the strong and selective fluorine−fluorine interactions. This andsurfactants7havebeenmodifiedbyafluorousmoietyforsolid-
importantpropertyhasledtothedevelopmentofseparationtech- phase isolation and fluorous solvent extraction. The fluorous tech-
niques, including fluorous liquid−liquid extraction and fluorous nology has also been utilized for biochemical applications, such as
solid phase extraction (FSPE), which can be utilized to separate
fluorous-labeledspeciesfromunlabeledcomponents.1−4Thefluo-
Special Issue: Organometallics in Biology and Medicine
roustechnologyhasbeenutilizedinbiologicalapplications,suchas
thepurificationandisolationofsyntheticDNAfragments,2termina-
Received: May2, 2012
tionofpeptidechainsresultingfromincompletesolid-phasepeptide Published: July2, 2012
©2012AmericanChemicalSociety 5844 dx.doi.org/10.1021/om3003575|Organometallics2012,31,5844−5855
Organometallics Article
the fabrication of microarrays in the studies of protein−ligand Chart 1. Structures of the Rhenium(I) Polypyridine
binding interactions and enzyme activity; for example, different Complexes
carbohydratesappendedwithafluoroustaghavebeenspottedona
fluorous-modifiedglassslideandscreenedwithfluorescentlylabeled
carbohydrate-bindingproteins.8Fluorous-coatedslidesimmobilized
with fluorous-tagged biotin have also been used to probe Cy5-
labeled avidin.9 Additionally, fluorous-based peptide microarrays
havebeenconstructedtoexaminethepeptide-cleavagepropertiesof
serine proteases.10
Despite the above studies, only a limited number of reports on
fluorouscompoundswithfluorescencepropertieshaveappearedin
the literature.11−14 Most of these studies are confined to organic
fluorophores11 and lanthanide chelates,12 whereas related work on
luminescent transition-metal polypyridine fluorous complexes is
very limited.13,14 There are three reasons for the derivatization of
metalcomplexeswithafluorouspendant.First,thependantrenders
the complexes to be more hydrophobic, which may perturb their
photophysical, biomolecular binding and labeling, and cellular up-
take properties. Second, biomolecules modified with luminescent
transition-metal fluorous complexes can be isolated and purified
by fluorous liquid−liquid extraction and FSPE. Third, the labeled
biomolecules can be readily studied by various optical methods as
wellas19F-NMRspectroscopy,whoseapplicationsinchemicalbio-
logy have been focused with increasing interest.15 Recently, we
have reported new fluorous bioconjugation reagents derived from
luminescentiridium(III)andrhenium(I)polypyridinecomplexes.14
We have now extended the study of the rhenium(I) system and
investigatedtheeffectsofthefluorouspendantsanddiimineligands
onthephotophysicalandbiologicalpropertiesofaseriesofrelated
∧
complexes. The complexes [Re(N N)(CO)(py-Rf-NH)](PF)
3 2 6
(py-Rf-NH = 3-amino-5-(N-((3-perfluorooctyl)propyl)amino-
2
∧
carbonyl)pyridine;N N=1,10-phenanthroline(phen)(1a),3,4,7,-
8-tetramethyl-1,10-phenanthroline (Me-phen) (2a), 4,7-diphenyl-
4
1,10-phenanthroline (Ph-phen) (3a)) contain a fluorous pendant
2
and a primary amine for modification (Chart 1). They have been
∧
convertedtotheisothiocyanatecomplexes[Re(N N)(CO)(py-Rf-
3
NCS)](PF) (py-Rf-NCS = 3-isothiocyanato-5-(N-((3-perfluoroo-
6
∧
ctyl)propyl)aminocarbonyl)pyridine;N N=phen(1b),Me-phen
4
(2b),Ph 2 -phen(3b))withthiophosgene.Theisothiocyanatecom- 3-(perfluorooctyl)propylamine with 5-aminonicotinic acid N-
plexeshavebeenreactedwithethylamine,amodelsubstrate,yield- hydroxysuccinimidyl ester14b,16a in DMF at room temperature.
∧
i ( n p g y- t R h f e -T t U hi - o E u t re = a 3 c - o e m th p yl l t e h x i e o s ur [ e R id e( y N l-5- N (N )( -( C ( O 3- ) p 3 e ( r p fl y u - o R r f o -T oc U ty -E l) t p ) r ] o ( p P y F l 6 ) ) - R ph e e a n ct , io M n e o -p f h [ e R n e , ( P N h ∧ N -p ) h ( e C n) O) w 3 i ( t C h H p 3 y C -R N f- ) N ]( H CF i 3 n SO re 3 fl ) u 16 xin ( g N ∧ T N HF = ,
∧ 4 2 2
aminocarbonyl)pyridine; N N = phen (1c), Me 4 -phen (2c), Ph 2 - followedbyanionexchangewithKPF 6 andpurificationbycolumn
phen (3c)). All of the complexes displayed intense and long-lived chromatography, afforded the luminescent rhenium(I) poly-
3MLCT (dπ(Re) → π*(N ∧ N)) emission upon irradiation. The pyridine fluorous amine complexes 1a−3a in moderate yields.
lipophilicity,cellularuptakeproperties,andcytotoxicityoftheamine Thesecomplexeswereconvertedtotheisothiocyanatecomplexes
andthioureacomplexeshavebeendetermined.Theresultsrevealed 1b−3b with thiophosgene, which were further reacted with a
that the less lipophilic complexes showed higher cytotoxicity and model substrate ethylamine, yielding the thiourea com-
higher cellular uptake efficiencies. The isothiocyanate complexes plexes1c−3c(SchemeS1,SupportingInformation).Allthecom-
1b−3b havebeenusedtolabelglutathione(GSH),bovineserum plexes were characterized by 1H NMR, positive-ion ESI-MS, and
albumin (BSA), branched poly(ethyleneimine) (bPEI, average IR spectroscopy and gave satisfactory elemental analyses. The IR
M w =25kDa),andpoly-L-lysine(Poly-K,M w >30kDa)toafford spectra of these complexes showed absorption bands from ca.
luminescent conjugates. Additionally, we have demonstrated that 1200to1100cm −1,whichhavebeenassignedtoC−Fstretching
theGSHconjugatescanbereadilyisolatedandpurifiedwithFSPE. ofthefluorouspendant.Allthefluorouscomplexeswereyellowin
Laser-scanning confocal microscopy experiments indicated that color and displayed good solubility in common organic solvents,
complex3candtheconjugateBSA-3cwereinternalizedintoHeLa suchasCHCl,acetone,andMeOH.Thefluorinecontentsofthe
cellsefficiently.Furthermore,wehavefoundthattheDNA-binding
complexesra
2
ng
2
edfrom25.1to30.8%,whicharegenerallyconsidered
andpolyplex-formationpropertiesofbPEIandPoly-Kwereretain- aslightfluorouscompoundsthatcanbepurifiedbyFSPEusingcom-
e■d after modificationwith complex 3b. mon organic solvents.17 The fluorous-free amine complexes [Re-
∧
(N N)(CO)(py-Et-NH)](PF) (py-Et-NH = 3-amino-5-(N-
RESULTS AND DISCUSSION 3 2 ∧ 6 2
ethylaminocarbonyl)pyridine; N N = phen (1d), Me-phen (2d),
4
Synthesis. The fluorous pyridine amine ligand py-Rf-NH Ph-phen(3d)) (Chart1)havealsobeenpreparedforcomparison
2 2
was obtained from the nucleophilic substitution reaction of studies.18
5845 dx.doi.org/10.1021/om3003575|Organometallics2012,31,5844−5855
Organometallics Article
Table 1. Photophysical Data of the Rhenium(I) Polypyridine Complexes
complex medium(T/K) λ /nma τ/μsa Φ b complex medium(T/K) λ /nma τ/μsa Φ b
em o em em o em
1a CHCl (298) 532 2.57 0.30 2c CHCl (298) 493sh,511 3.52 0.11
2 2 2 2
CHCN(298) 545 1.29 0.10 CHCN(298) 513 1.73 0.055
3 3
glassc(77) 507,532sh 11.91 glassc(77) 466(max),499, 142.60(34%),
1b CHCl (298) 527 1.91 0.11 533,577sh 43.39(66%)
2 2
CH 3 CN(298) 540 1.13 0.039 2dd CH 2 Cl 2 (298) 488sh,512 11.33 0.44
glassc(77) 466sh,498 10.20 CH 3 CN(298) 486sh,514 9.16 0.28
1c CHCl (298) 529 1.84 0.055 glassc(77) 467(max),501, 41.92(22%),
2 2 539sh 152.17(78%)
CHCN(298) 542 1.21 0.042
3 3a CHCl (298) 544 6.60 0.29
glassc(77) 466sh,498 12.27 2 2
CHCN(298) 559 3.31 0.14
1dd CHCl (298) 530 2.65 0.30 3
2 2 glassc(77) 507,533sh 38.51
CHCN(298) 547 1.26 0.18
3 3b CHCl (298) 537 6.38 0.20
glassc(77) 502 11.19 2 2
CHCN(298) 550 2.11 0.074
2a CHCl (298) 495sh,513 4.48 0.31 3
2 2 glassc(77) 507,531sh 37.48
CHCN(298) 515 3.04 0.13
3 3c CHCl (298) 539 6.66 0.24
glassc(77) 468,502(max), 133.48(33%), 2 2
534sh 25.33(67%) CH 3 CN(298) 553 3.04 0.088
2b CHCl (298) 496sh,511 1.33 0.068 glassc(77) 505,531sh 38.10
2 2
CH 3 CN(298) 513 0.63 0.023 3dd CH 2 Cl 2 (298) 545 8.00 0.33
glassc(77) 466(max),498, 153.52(37%), CH 3 CN(298) 559 3.97 0.20
534sh,577sh 46.22(63%) glassc(77) 510,533sh 20.49
aλ =355 nm.bλ = 455 nm, A = 0.1.cIn n-butyronitrile glass. dFromref 18.
ex ex 455nm
Electronic Absorption and Emission Properties. The ofafluorouschainintherhenium(I)polypyridinecomplexesdid
electronic absorption spectral data of the complexes are sum- not substantially affect their emission energies but reduced the
marizedinTableS1(SupportingInformation),andtheelectronic quantum yields and lifetimes, as revealed by a comparison of the
absorption spectra of the amine complexes 1a−3a in CHCl emissiondataforthefluorousaminecomplexes1a−3awiththeir
2 2
at 298 K are shown in Figure S1 (Supporting Information). All fluorous-freecounterparts1d−3d (Table1).Thisisattributedto
the complexes exhibited intense spin-allowed intraligand (1IL) the more efficient nonradiative vibrational relaxation associated
(π→π*)(diimineandpyridineligands)absorptionbandsatca. with the fluorous pendant.
248−341nmandmoderatelyintensespin-allowedmetal-to-ligand Lipophilicity. The lipophilicity of a biological probe is a
charge-transfer (1MLCT) (dπ(Re) → π*(N ∧ N)) absorption veryimportantparameterbecauseitnotonlyusuallyaffectsthe
shoulders at ca. 371−400 nm.19−29 Upon irradiation, the com- cellularuptakepropertiesoftheprobebutalsoplaysadecisive
plexes exhibited intense and long-lived green-to-yellow 3MLCT role on its subsequent intracellular locations.30 We have deter-
(dπ(Re) → π*(N ∧ N)) emission in fluid solutions at 298 K and minedthelipophilicityofthefluorousaminecomplexes1a−3a
in rigid glass at 77 K.19−29 The photophysical data are listed in and the thiourea complexes 1c−3c by reversed-phase HPLC,
Table1,andtheemissionspectraofcomplexes1a−3aindegassed and the results (log P values) are listed in Table 2. The
o/w
CHCN at 298 K are shown in Figure 1. The structural features
3
Table 2. Lipophilicity, Cellular Uptake, and IC Values of
50
the Rhenium(I) Polypyridine Complexes and Cisplatin
complex logP intracellularamount/fmola IC /μMb
o/w 50
1a 5.14 4.51±0.50 4.04±0.14
1c 6.50 0.56±0.037 12.9±0.23
1dc 0.81 0.27±0.03 15.0±4.8
2a 6.69 2.64±0.57 6.10±0.37
2c 7.38 0.25±0.079 16.6±0.36
2dc 1.09 0.82±0.02 5.0±0.4
3a 8.19 0.76±0.070 10.7±0.53
3c 8.74 0.14±0.003 39.9±1.76
3dc 2.04 2.99±0.07 3.6±0.4
cisplatin −2.30d N.A. 34.0±3.30
Figure1.Emissionspectraofcomplexes1a(green),2a(blue),and3a aAmount of rhenium associated with an average HeLa cell upon
(red)in CH 3 CN at 298 K. incubation with the complexes (10 μM) at 37 °C for 2 h as
determined by ICP-MS. bHeLa cells; incubation time = 48 h. cFrom
and exceptionally long emission lifetimes of the Me-phen com- ref 18. dFromref 49.
4
plexes2a−2cinlow-temperatureglass(Table1)shouldbedueto
the involvement of 3IL (π → π*) (Me 4 -phen) character in their isothiocyanate complexes 1b−3b have been omitted in these
emissive states.28 The amine complexes displayed lower emission measurements due to their relatively lower stability in aqueous
energiesthantheisothiocyanateandthioureaanalogues(Table1), solution. We found that the lipophilicity of the complexes
which is a consequence of the electron-donating nature of the followstheorders1a<2a<3aand1c<2c<3c,whicharein
aminegroupthatdestabilizesthedπ(Re)levels.Theincorporation accordance with the hydrophobic character of the diimine
5846 dx.doi.org/10.1021/om3003575|Organometallics2012,31,5844−5855
Organometallics Article
ligands(phen<Me -phen<Ph -phen).Thisillustratesthatthe thatpossessesbothhydrophilicandhydrophobicsurfaces.bPEI
4 2
lipophilicity ofthese complexes canbe readilycontrolled using andPoly-Karepolyaminesthatcanbeusedastransfectionreagents
variousdiimineligands.ThelogP valuesofthe aminecom- formammaliancells.Allofthesebiomoleculescontainatleastone
o/w
plexes1a−3a(from5.14to8.19)aresmallerthanthoseofthe primary amine group, which would allow modification by the iso-
thiourea complexes 1c−3c (from 6.50 to 8.74) by ca. 1.36− thiocyanatecomplexes1b−3b.Wehavedenotedtheresultantcon-
0.55units,whichareascribedtothepolaramineandrelatively jugatesasGSH-nc,BSA-nc,bPEI-nc,andPoly-K-nc (n =1−3)
lesspolarethylthioureidylgroup.Asexpected,allofthefluorous
in view of their structural similarity to the corresponding thiourea
complexesaresubstantiallymorelipophilicthantheirfluorous-free complexes 1c−3c. The GSH conjugates were purified by FSPE,
counterparts, complexes 1d−3d (log P ranged from 0.81 to whereas the BSA, bPEI, and Poly-K conjugates were isolated and
o/w
2.04, Table 2),18 which clearly highlights the hydrophobic chara- purifiedbysize-exclusionchromatographyandmembranefiltration.
cter of the fluorous pendant. All the conjugates displayed green-to-orange emission in
Cellular Uptake Properties. The intracellular amounts of degassedaqueoussolutionuponirradiation.Thephotophysical
rhenium associated with HeLa cells incubated with the fluorous dataoftheconjugatesarelistedinTable3.Theelectronicabsorp-
amine and thiourea complexes (10 μM at 37 °C for 2 h) have
beendeterminedbyICP-MSmeasurements.Theresultsshowed Table 3. Photophysical Data of the Conjugates at 298 K
thattheamountsoftherheniuminanaverageHeLacellwerein
thefemtomoleandsubfemtomolescales(Table2).Interestingly, conjugate λ em /nma τ o /μsa
unlike general inorganic and organometallic transition-metal GSH-1cb 556 0.98
complexes,31 the less lipophilic complexes in this work revealed GSH-2cb 527 1.96
more efficient cellular uptake; for example, the dependence of GSH-3cb 567 1.49
the uptake efficiencies on the diimine ligands is as follows: BSA-1cc 536 0.33(58%),1.25(42%)
1a > 2a > 3a and 1c > 2c > 3c. Also, the intracellular concen- BSA-2cc 524 0.35(50%),2.29(50%)
trationsofthelesslipophilicaminecomplexes1a−3aarehigher BSA-3cc 558 0.27(72%),1.31(28%)
than those of the more lipophilic thiourea complexes 1c−3c by bPEI-1cd 554 0.11
ca. 5.4−10.6fold. These results rule out passive diffusion asthe bPEI-2cd 526 0.18
soleuptakepathwayforthesefluorouscomplexes.Althoughthe bPEI-3cd 578 0.14
originforthesefindingsisunknown,onepossiblereasonisthat Poly-K-1cd 539 0.12
thesehighlylipophilicfluorouscomplexesexhibitacertainextent Poly-K-2cd 524 0.19
of self-aggregation in aqueous solution due to intermolecular Poly-K-3cd 566 0.17
fluorous−fluorous interactions,1−4 which may lower the cellular aλ = 355 nm. bIn 60% aqueous MeOH. cIn potassium phosphate
ex
uptake efficiencies. This argument is supported by the expected buffer(50 mM,pH 7.4). dIn Tris-Cl buffer(50mM, pH 7.4).
positive lipophilicity−uptake efficiency relationship observed for
the fluorous-free complexes (Table 2), with the more lipophilic
complexesdisplayingmoreefficientuptake,thatis,1d<2d<3d.18
Thus, our results show that both the diimine ligands and the sub-
stituents on the pyridine ligand play an important role on the
cellular uptake of the complexes.
Cytotoxicity. The cytotoxicity of the fluorous amine and
thioureacomplexestowardHeLacellshasbeenassessedbythe
MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide) assay.32 The survival of HeLa cells upon exposure
to the complexes at various concentrations for 48 h has been
investigated and expressed in terms of IC values. The results
50
are summarized in Table 2, and the relationships between the
viability of HeLa cells and concentration of complexes 1c−3c
areshowninFiguresS2−S4(SupportingInformation). Allthe
Figure 2. Electronic absorption (blue) and emission (red) spectra of
complexes exhibited potent in vitro cytotoxic activity, which is
theconjugateBSA-3cin50mMphosphatebufferatpH7.4at298K.
comparabletoorhigherthanthatofcisplatin.Thecytotoxicity
ofthecomplexesfollowstheorders1a>2a>3aand1c>2c>3c,
and the amine complexes 1a−3a are more cytotoxic than their tion and emission spectra of the conjugate BSA-3c in phosphate
thioureacounterparts,complexes1c−3c.However,thetrend bufferareshowninFigure2asanexample.Theemissionofallthe
of the cytotoxicity of the fluorous-free complexes is reversed: conjugateshasbeenassignedtoa3MLCT(dπ(Re)→π*(N ∧ N))
1d<2d<3d.Alloftheseobservationsareinaccordancewiththe excitedstate,whichmaybemixedwithsome3IL(π→π*)(Me-
4
cellular uptake efficiencies of the complexes. It is likely that the phen) character for the conjugates labeled by complex 2b. Since
cytotoxicityofthecomplexesoriginatesfromtheirlocalizationatthe the emission energies of related rhenium(I) polypyridine com-
mitochondria (videinfra),causing dysfunctionandsubsequentcell plexes are strongly dependent on the polarity of solvents (lower
death. Similar results have been observed in other inorganic and
emissionenergyinmorepolarsolvents),19−29theycanbeutilized
organometallic transition-metal complexes.33 asareporteroftheirlocalenvironments.Theemissionenergiesof
Fluorous-Labeling Properties. The isothiocyanate com- the conjugates follow the order GSH ≈ bPEI < BSA ≈ Poly-K
plexes1b−3bhavebeenusedtolabelarangeofbiomolecules, (Table3).Theseobservationsshouldbedueto(1)theexposure
including GSH, BSA, bPEI, and Poly-K. GSH is a tripeptide of the rhenium(I) complex to the polar aqueous buffer for the
with antioxidant properties and plays an important role in GSH conjugates, (2) the high positive charges of bPEI, and
cellularmetabolism.BSAiscommonlyusedasamodelprotein (3)thehydrophobicnatureofBSAandPoly-K.TheBSAconjugates
5847 dx.doi.org/10.1021/om3003575|Organometallics2012,31,5844−5855
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exhibitedbiexponentialdecay,whichisinagreementwithprevious foundthattheflow-throughfractionwasnotemissiveuponirradia-
reportsonbiomacromoleculeslabeledwithluminescenttransition- tion.However,changingthemobilephaseto60%aqueousMeOH
metal complexes,34 and is attributable to heterogeneous micro- ledtotheelutionofaluminescentspecieswhoseemissionspectrum
environments of the complex on the protein. The shorter-lived showed a band at 556 nm (Figure S5, Supporting Information),
component of the decay is likely to be a result of emission quen- which is similar to that of the conjugate GSH-1c (Table 3). The
chingoftheexcitedrhenium(I)complexesbynearbytyrosineand identityofthisspecieswasconfirmedbyapeakatm/z =1397in
tryptophan residues of the protein.19c All the bPEI and Poly-K theESImassspectrumofthesample(Figure3c).Interestingly,this
conjugates displayed monoexponential decay, which is ascribed
peakwasabsentinthemassspectrumoftheflow-throughfraction
to the similar microenvironments experienced by the rhenium(I) (Figure 3b). Thus, the unlabeled amino acids were washed out in
polypyridinecomplexes.Overall,despitetheirrigidandbulkyfluo- theflow-throughfraction,whereasthefluorousconjugateGSH-1c
rouspendants,theisothiocyanatecomplexes1b−3bcanfunctionas was retained on the FSPE column due to the highly selective
luminescentfluorous-labeling reagents forarangeof biomolecules. fluorous−fluorous interactions between the fluorous chain of the
Fluorous Solid Phase Extraction. We have investigated
conjugateandtheperfluoroalkylstationaryphase.Importantly,allthe
solvents used in this purification process are mixtures of water and
the use of FSPE to isolate small biomolecules labeled with the
isothiocyanatecomplexes.4 Asanexample,theconjugateGSH-1c methanol, which are inexpensive and highly compatible to mass
spectroscopicanalysisoftheisolatedaminoacidsandpeptides.Thus,
wasspikedintoamixtureof20aminoacids,andtheconjugatewas
isolatedandpurifiedusinganFSPEcolumn.TheESImassspectra complexes 1b−3b not only enable fluorous labeling of amine-
containing biomolecules for purification and MS identification but
of (i) the initial mixture of GSH-1c and the 20 amino acids, (ii)
flow-through fraction with 40% aqueous MeOH, and (iii) elution alsoconferthemwithinterestingluminescencepropertiesfordetec-
tionpurposes.
fraction with 60% aqueous MeOH are shown in Figure 3. We
Live-Cell Confocal Imaging. The cellular uptake and
intracellular localization properties of the thiourea complex 3c,
whichcanbeconsideredasamodelofbiomoleculeslabeledwith
the isothiocyanate complex 3b, have been investigated by laser-
scanning confocal microscopy. The microscopy images of HeLa
cellsincubatedwithcomplex3c at37°Cundera5%CO atmo-
2
spherefor2hareillustratedinFigure4.Wefoundthatthecom-
plexaccumulatedintheperinuclearregionofthecellswithintense
punctate staining and negligible emission from the nucleus.
Incubation of HeLa cells at 4 °C before treatment with the
complex resulted in diminished cellular uptake (data notshown),
indicative of energy requirements. It appears that the staining is
due to localization of the complex in mitochondria that are
prevalent around the nucleus.35 To confirm this, HeLa cells
pretreated with complex 3c (10 μM, 2 h, λ = 405 nm) were
ex
coincubated with MitoTracker Deep Red FM (100 nM, 20 min,
λ = 633 nm), whose spectral properties do not interfere with
ex
each other. It can be seen in Figure 5 that the complex was
significantly colocalized with the MitoTracker dye with a
colocalization coefficient of 78.0%, confirming that the complex
was enriched in the mitochondria. The mitochondrial staining
properties of the complex should be closely related to its formal
cationic charge and highly hydrophobic nature.33 Under the
experimentalconditionsweemployed,thetreatedHeLa cellsstill
remained viable because the MitoTracker dye only stains mito-
chondriaoflivingcells.36Uponfixationwithmethanol,thestaining
ofcomplex-treatedcellsbecamemorehomogeneous,andboththe
nucleiandthecytoplasmdisplayedsignificantemission(Figure6).
Redistribution of the complex in the cells is not unexpected in
viewofitshighsolubilityinmethanol.Thenuclearstainingshould
beaconsequenceofacompromisednuclearmembraneuponcell
fixation.37 The microscopy images also showed that some subnu-
clearstructuresweresignificantlystained.38Thesestructureswere
clearlyvisibleinthedifferentialinterferencecontrastmode(Figure6,
middle)andarepossiblynucleolithatarethemajorsitesforRNA
synthesis.39 The staining may be a result of the binding of the
complex to RNA molecules and nucleolar proteins. Emission
titrationexperimentsrevealedthatthephotophysicalpropertiesof
complex 3c did not show any changes upon addition of RNA,
suggestingalackofinteraction.Incontrast,thecomplexdisplayed
Figure 3. ESI mass spectra of (a) a mixture of GSH-1c and the 20 emission enhancement (ca. 1.2 fold) in the presence of a model
aminoacids,(b)flow-throughfractionwith40%aqueousMeOH,and protein human serum albumin (HSA), which should originate
(c)elutionfractionwith60%aqueousMeOHfromanFSPEcolumn. from noncovalent binding interactions. Thus, we believe that the
5848 dx.doi.org/10.1021/om3003575|Organometallics2012,31,5844−5855
Organometallics Article
Figure 4. Fluorescence (left), differential interference contrast (middle), and overlaid (right) laser-scanning confocal microscopy images of HeLa
cells incubated with complex3c (10 μM) at 37 °Cfor 2h.
Figure 5. Laser-scanning confocal microscopy images of HeLa cells treated successively with complex 3c (10 μM, 2 h, λ = 405 nm, left) and
ex
MitoTracker Deep RedFM(100 nM, 20 min, λ = 633 nm,middle) at 37 °C. The overlaid confocal images are shown on the right.
ex
Figure 6. Fluorescence (left), differential interference contrast (middle), and overlaid (right) laser-scanning confocal microscopy images of HeLa
cells incubated with complex3c (10 μM) at 37 °Cfor 2h aftersubsequent fixation with MeOH. The nucleoli are indicated with arrows.
observednucleolistaininginthefixedcellswasduetobindingof top).Thecondensationefficiencieswereverysimilartothoseof
the complex to nucleolar proteins, probably in a nonspecific unmodified bPEI and Poly-K, respectively (Figures 8 and 9,
manner.38 We have also studied the uptake of the protein bottom). In other words, both bPEI-3c and Poly-K-3c can
conjugate BSA-3c by HeLa cells. Similar to complex 3c, the condenseDNAeffectively,leadingtotheformationofpolyplexes.
proteinconjugatewasalsolocalizedintheperinuclearregionupon This also implies that modification of bPEI with the rhenium(I)
uptake, but the intensity of the punctate staining was much lower polypyridine fluorous complexes did not hamper their DNA-
(Figure7).Asexpected,thestainingpatternwasverysimilartothat binding and condensation behavior.
of the fluorescein-BSA conjugate, which is internalized into Thezetapotentialsandmeanhydrodynamicdiametersofthe
eukaryotic cells by endocytosis and is commonly used to monitor polyplexes bPEI-3c/pRL-TK and Poly-K-3c/pRL-TK, and their
earlyendosomalmembranedynamics.40 controls bPEI/pRL-TK and Poly-K/pRL-TK (with unmodified
DNA-Binding Studies. Polycationic bPEI and Poly-K are bPEIandPoly-K,respectively),withdifferentN/Pratioshavebeen
wellknowntobindplasmidDNA(pDNA),leadingtotheforma- examined by dynamic light-scattering (DLS) measurements. The
tion of polyplexes, which can be efficiently delivered into eukary- zetapotentialsofthepolyplexesbPEI-3c/pRL-TKincreasedfrom
oticcells.41,42Inthiswork,wehaveinvestigatedtheDNA-binding −27.1 to +26.5 mV with increasing N/P ratios (Table 4). At
properties of the conjugates bPEI-3c and Poly-K-3c by agarose N/P ≥8.0,thepolyplexesacquiredapositivezetapotential,which
gel retardation assays. Mixtures of the bPEI-3c and Poly-K-3c, isinagreementwiththegelelectrophoresisresults(Figure8,top).
respectively, and the pDNA pRL-TK with a range of N/P ratios In the case of unmodified bPEI, similar changes of zeta potentials
(thenumberofnitrogenatomsofPEIperDNAphosphate)have werealsoobserved(Table4).Thezetapotentialsofthepolyplexes
been prepared and analyzed. We found that the pDNA bands Poly-K-3c/pRL-TK changed from −23.3 to +11.7 mV with
were retarded with increasing N/P ratios (Figures 8 and 9), increasing N/P ratios (Table 5). At N/P ≥ 1.0, the polyplexes
indicative of the charge neutralization of the pDNA by the posi- becamepositiveincharge,whichisalsoinagreementwiththegel
tively charged bPEI-3c and Poly-K-3c. The bands were com- electrophoresisresults(Figure9,top).Also,thechangeofthezeta
pletely retarded at N/P ratios ≥ 8.0 for bPEI-3c (lanes e−g in potentialsisverysimilartothecaseofthepolyplexesPoly-K/pRL-
Figure8, top),and≥ 1.0 for Poly-K-3c (lanes f−h inFigure 9, TK (Table 5).
5849 dx.doi.org/10.1021/om3003575|Organometallics2012,31,5844−5855
Organometallics Article
Figure 7. Fluorescence (left), differential interference contrast (middle), and overlaid (right) laser-scanning confocal microscopy images of HeLa
cells incubated with BSA-3c (10 μM) at 37 °Cfor 2h.
Figure9.GelelectrophoresisofpolyplexesPoly-K-3c/pRL-TK(top)
andPoly-K/pRL-TK(bottom).ThelanescorrespondtodifferentN/P
ratios:(a)DNAonly,(b)0.06,(c)0.13,(d)0.25,(e)0.5,(f)1.0,(g)
2.0, and (h) 4.0.
Figure 8. Gel electrophoresis of polyplexes bPEI-3c/pRL-TK (top)
and bPEI/pRL-TK (bottom). The lanes correspond to different N/P Poly-K/pRL-TKalsodisplayedasimilarincreaseofhydrodynamic
diameter with an abrupt expansion at N/P = 1.0 (Table 5), and
ratios:(a)DNAonly,(b)1.0,(c)2.0,(d)4.0,(e)8.0,(f)16.0,and(g)
32.0. thesubsequentsizewasca.3600nm,whichishigherthanthatof
Poly-K-3c/pRL-TK. The observation of relatively large hydro-
The hydrodynamic diameters of the polyplexes bPEI-3c/ dynamic diameters is most likely due to clustering of polyplexes,
pRL-TKrangedfromca.440to919nmwithN/Pfrom1.0to which is very common to related Poly-K polyplexes.45 Taken
4.0 (Table 4); beyond this ratio, the polyplexes started shrink- together, all the results showed that the DNA-binding and
ingandthediameterbecameca.410nmatN/P=32.0.These polyplex-formation properties of bPEI and Poly-K were both
results indicate that the positively charged polymer bPEI-3c r■etained after modification with complex 3b.
bound and condensed the negative pDNA efficiently and
formedmorecompactpolyplexesatN/P>4.0.43,44Thecontrol SUMMARY
polyplexes bPEI/pRL-TK also showed similar expansion and A family of luminescent rhenium(I) fluorous complexes with
subsequent shrinking in size (Table 4). The hydrodynamic variousdiimineandpyridineligandshavebeensynthesizedand
diametersofthepolyplexesPoly-K-3c/pRL-TKincreasedfrom characterized.Irradiationofallthecomplexesledtointenseand
ca. 334 to 976 nm with N/P from 0.13 to 1.0 (Table 5). long-lived 3MLCT/3IL emission in fluid solutions at room
Interestingly, the diameter showed a further and more significant temperature and in low-temperature rigid glass. The pre-
increase to ca. 2750 nm at N/P ≥ 2.0. The control polyplexes sence of the fluorous pendant did not substantially change the
5850 dx.doi.org/10.1021/om3003575|Organometallics2012,31,5844−5855
Organometallics Article
Table4.ZetaPotentialsandMeanHydrodynamicDiametersofthePolyplexesbPEI-3c/pRL-TKandbPEI/pRL-TKinTris-Cl
Buffer (50 mM, pH 7.4) at Various N/P Ratios
zetapotential/mV meanhydrodynamicdiameter/nm
N/Pratio bPEI-3c/pRL-TK bPEI/pRL-TK bPEI-3c/pRL-TK bPEI/pRL-TK
1 −27.1±3.32 −33.3±8.5 440.1±11.8 225.5±12.8
2 −29.4±0.07 −25.5±1.7 552.7±7.42 234.2±3.6
4 −13.1±1.77 −7.8±2.0 919.0±146.4 815.5±148.6
8 +4.39±1.64 +3.4±2.1 584.0±76.1 713.0±64.3
16 +18.0±3.75 +28.3±1.7 217.9±14.4 234.9±16.4
32 +26.5±0.92 +37.9±2.3 410.4±36.9 248.5±20.9
Table5.ZetaPotentialsandMeanHydrodynamicDiametersofthePolyplexesPoly-K-3c/pRL-TKandPoly-K/pRL-TKinTris-
Cl Buffer (50 mM, pH 7.4) at Various N/P Ratios
zetapotential/mV meanhydrodynamicdiameter/nm
N/Pratio Poly-K-3c/pRL-TK Poly-K/pRL-TK Poly-K-3c/pRL-TK Poly-K/pRL-TK
0.13 −23.3±13.7 −25.5±2.62 344.0±13.2 471.2±29.4
0.25 −31.5±4.80 −31.2±2.97 280.2±13.7 649.7±49.9
0.5 −27.6±3.89 −30.9±3.46 639.3±42.7 757.5±76.7
1.0 +1.73±0.53 +5.83±0.22 976.0±178.2 2966.0±264.5
2.0 +8.51±0.63 +9.39±4.04 2743.3±235.7 3702.0±912.1
4.0 +11.7±0.07 +13.4±1.51 2761.0±326.6 3675.0±138.7
emissionenergiesofthecomplexes,butreducedtheirquantum
Py-Rf-NH14band[Re(N∧N)(CO)(CHCN)](CFSO)16wereprepared
2 3 3 3 3
yieldsandlifetimes.Variousexperimentsrevealedthatthemore as described previously. Autoclaved Milli-Q water was used for the pre-
lipophilic complexes showed lower cellular uptake efficiencies paration for the aqueous solutions. All buffer components were of
molecularbiologygradeandwereusedasreceived.PD-10size-exclusion
and lower cytotoxic activity. We argue that it is a consequence
ofself-aggregationofthelipophilicfluorouscomplexesinaque- columnsandYM-30centriconswerepurchasedfromGEHealthcareand
ous solution. The isothiocyanate complexes 1b−3b have been M Cu il l l t i u p r o e re C , o re ll s e p c e ti c o t n iv . el H y. ig H h e g L l a uc c o e s l e ls D w u e l r b e ec o c b o t ’ a s in m ed od f i r fi o e m d E A a m gl e e r ’ i s ca m n e T di y u p m e
used to label a range of biomolecules, including GSH, BSA,
(DMEM),MitoTrackerDeepRedFM,fetalbovineserum(FBS),phos-
bPEI, and Poly-K, yielding conjugates that displayed green-to- phatebufferedsaline(PBS),trypsin-EDTA,penicillin/streptomycin,and
orange 3MLCT/3IL emission in aqueous solutions under UltraPureagarosewerepurchasedfromInvitrogen.Thegrowthmedium
ambient conditions. Importantly, the photophysical data can for cell culture contained DMEM with 10% FBS and 1% penicillin/
reflect the local environment of the complexes on these bio- streptomycin.ThepDNApRL-TK(4.0kb)wasobtainedfromPromega
molecules.WehavedemonstratedthatthelabeledGSHcanbe
andamplifiedinE.coliandpurifiedbyaHiPureFilterPlasmidKit,andits
readily isolated and purified by FSPE. Laser-scanning confocal concentrationsweremeasuredspectrophotometrically.
[Re(N∧N)(CO) (py-Rf-NH )](PF ) (N∧N = phen (1a), Me -phen
m BS ic A ro -3 s c co w p e y re re e su ffi lt c s ie s n h t o ly w i e n d te t r h n a a t li c ze o d m i p n l t e o x H 3c eL an a d ce t l h ls e . c A o d n d j i u t g io a n te - (2a), Ph 2 -phen 3 (3a)). A 2 mixtur 6 e of [Re(N∧N)(CO) 3 (CH 4 3 CN)]-
ally,gelretardationassaysandDLSexperimentsconfirmedthat (
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the DNA-binding and polyplex-formation properties of both
Thesolutionwasevaporatedtodrynesstogiveayellowsolid.Thecomplex
bPEIandPoly-K wereretainedafter labeling with complex 3b. was then converted to the hexafluorophosphate salt by metathesis with
In conclusion, we have examined the effects of the fluorous KPF inMeOHandthenpurifiedbycolumnchromatographyonalumina.
6
pendant on the photophysical and biological properties of ThedesiredproductwaselutedwithCHCl/MeOH(20:1v/v).Recry-
2 2
luminescent rhenium(I) polypyridine complexes. The bio- stallizationofthecrudeproductfromCHCl/petroleumetheraffordedthe
2 2
conjugation,FSPE,andcellularuptakestudieshavehighlighted complexasyellowcrystals.
the potential biological applications of these complexes.
Complex1a:Yield:274mg(66%).1HNMR(300MHz,methanol-
■ d,298K,relativetoTMS):δ=9.70(d,2H;J=3.9Hz,H2andH9
4
of phen), 8.94 (d, 2H; J = 6.6 Hz, H4 and H7 of phen), 8.24−8.17
EXPERIMENTAL SECTION
(m,4H;H3,H5,H6andH8ofphen),7.86(d,1H;J=2.4Hz,H2of
Materials and Synthesis. All solvents were of analytical reagent pyridine),7.79(d,1H;J=1.8Hz,H6ofpyridine),7.20(t,1H;J=2.1
g p d r h ic a e y d n c e , lo P a h n h e d 2 x - y p p lc h u a e r r n i b fi , o e 5 d d - i a im a m c i c i d n o e o r , d n i i A n co g g C ti t n F o ic S s O t a a c n i , d d , a K r N P d - F h p y , r d o C r c o e a x d C y u s O r u e c s , c .4 i t n 6 h im i p o h i p d e h e n o , , s N g M e ,N n e e 4 ′ - - , H p 20 p z 3 m , 6 H ( ( 4 s m , o , C f 2 p H yr O ; id ) N in , H 1 e) 9 C , 2 2 H 9 .2 2 ( C 3 s − , H C 2 2 . C 08 H O ( 2 m ) ) , . , 1 I 2 6 R H 37 ; (K N (m B H r , ) C C : H ν 2 C O = H ) 3 , 2 4 C 1 0 2 H 9 4 2 3 ) ( , b ( 1 m r . , 8 , 9 N C − − − 1 H . F 7 ) ) 1 , ,
ethylamine,andcisplatinwerepu 3 rcha 3 sedfro 6 mAcros. 3 Re(CO)Cland 1210 (m, C−F), 1149 (m, C−F), 1028 (m, C−F), 846 cm−1
b (P P e E r I flu ( o a r v o e o r c a t g y e l)p M ro w py = lam 2 i 5 ne k a D n a d ) F w S e P r E e c o o b lu ta m in n e s d (2 fro g m , 8 A m ld L r 5 ich tu . be 3 ) - P (m F 6 , −] P + F . 6 − E ) l . em P e o n si t t a i l ve a -i n o a n lys E is SI- c M alc S d io ( n %) clu fo s r ter C s 32 a H t 2 m 0 N / 5 z O 1 4 P 0 F 48 23 R [ e M : C − ,
werepurchasedfromFluorousTechnologiesInc.The20aminoacids 32.23; H,1.69; N, 5.87. Found: C, 32.17; H, 1.82; N, 5.99.
usedintheFSPEpurificationexperiments,includingalanine,arginine, Complex2a:Yield:232mg(53%).1HNMR(300MHz,methanol-
asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, d, 298 K, relative to TMS): δ = 9.38 (s, 2H; H2 and H9 of Me-
4 4
histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, phen),8.39(s,2H;H5andH6ofMe-phen),7.83(d,1H;J=2.1Hz,
4
serine, threonine,tryptophan, tyrosine,and valine, were purchased from H2ofpyridine),7.80(d,1H;J=1.5Hz,H6ofpyridine),7.20(t,1H;
AldrichorAcros.GSH,Poly-K(M >30kDa),MTT,andtorulayeast J=2.1Hz,H4ofpyridine),2.88(s,6H;CH atC4andC7ofMe-
w 3 4
RNAwereobtainedfromSigma.Allofthesechemicalswereusedwith- phen),2.73(s,6H;CH atC3andC8ofMe-phen),2.25−2.08(m,
3 4
outfurtherpurification.BSAandHSAwerepurchasedfromCalbiochem. 2H; NHCHCHCH), 1.89−1.71 ppm (m, 2H; NHCHCHCH).
2 2 2 2 2 2
5851 dx.doi.org/10.1021/om3003575|Organometallics2012,31,5844−5855
Organometallics Article
IR (KBr): ν = 3442 (br, N−H), 2032 (s, CO), 1925 (s, CO), stirred in acetone (15 mL) at room temperature under an inert atmo-
1636(m,CO),1247(m,C−F),1210(m,C−F),1153(m,C−F), sphereofnitrogenfor2h.Thesolutionwasevaporatedtodrynesstogive
1031(m,C−F),847cm−1(s,PF−).Positive-ionESI-MSionclusters ayellowsolid,whichwaspurifiedbycolumnchromatographyonalumina.
6
at m/z 1104 [M − PF−]+. Elemental analysis calcd (%) for ThedesiredproductwaselutedwithCHCl/MeOH(20:1v/v).Recry-
6 2 2
C H NOPF Re·(CH)CO: C, 35.84; H, 2.62; N, 5.36. Found: stallization of the product from acetone/petroleum ether afforded the
46 28 5 4 23 3 2
C, 35.80; H, 2.39; N, 5.66. complexasyellowcrystals.
Complex3a:Yield:289mg(61%).1HNMR(300MHz,methanol- Complex1c:Yield:75mg(73%).1HNMR(300MHz,methanol-
d,298K,relativetoTMS):δ=9.74(d,2H;J=5.4Hz,H2andH9of d,298K,relativetoTMS):δ=9.69(d,2H;J=5.4Hz,H2andH9of
4 4
Ph-phen),8.19(s,2H;H5andH6ofPh-phen),8.15(d,2H;J=5.4 phen),9.06(s,1H;H2ofpyridine),8.93(d,2H;J=8.1Hz,H4and
2 2
Hz,H3andH8ofPh-phen),8.00(d,1H;J=2.1Hz,H2ofpyridine), H7 of phen), 8.65 (s, 1H; H6 of pyridine), 8.25 (s, 2H; H5 and H6
2
7.93(d,1H;J=1.5 Hz,H6ofpyridine), 7.72−7.63(m,10H;Phof of phen), 8.21 (m, 2H; H3 and H8 of phen), 7.81 (s, 1H; H4
Ph-phen), 7.28 (t, 1H; J = 1.8 Hz, H4 of pyridine), 2.28−2.09 (m, of pyridine), 3.44−3.38 (m, 4H; CH of Et, NHCH CH CH ),
2 2 2 2 2
2H; NHCHCHCH), 1.86−1.74 ppm (m, 2H; NHCHCHCH). 2.32−2.10 (m, 2H; NHCH CH CH ), 1.91−1.86 (m, 2H;
2 2 2 2 2 2 2 2 2
IR (KBr): ν = 3438 (br, N−H), 2034 (s, CO), 1928 (m, CO), NHCHCHCH), 1.11 ppm (t, 3H; J = 7.2 Hz, CH of Et). IR
2 2 2 3
1630(m,CO),1244(m,C−F),1212(m,C−F),1151(m,C−F), (KBr):ν=3447(br,N−H),2036(s,CO),1928(s,CO),1650
1030(m,C−F),844cm−1(m,PF−).Positive-ionESI-MSionclusters (m,CO),1240(m,C−F,CS),1211(m,C−F),1148(m,C−F),
at m/z 1200 [M − PF−]+. Eleme 6 ntal analysis calcd (%) for C H - 1030(m,C−F),849cm−1(m,PF−).Positive-ionESI-MSionclusters
NOPF Re: C, 39.30; 6 H, 2.10; N, 5.21. Found: C, 39.60; H, 44 2.2 2 6 8 ; at m/z 1135 [M − PF−]+. E 6 lemental analysis calcd (%) for
5 4 23 6
N, 5.30. C H NOSPF Re·0.5(CH)CO: C, 33.50; H, 2.16; N, 6.42.
35 25 6 4 23 3 2
[Re(N∧N)(CO) (py-Rf-NCS)](PF )(N∧N=phen(1b),Me -phen Found: C, 33.80; H, 2.44; N, 6.72.
(2b), Ph -phen 3 (3b)). A mixture o 6 f [Re(N∧N)(CO)(py-Rf- 4 NH)]- Complex2c:Yield:54mg(51%).1HNMR(300MHz,methanol-
2 3 2
(PF) (0.16 mmol) and finely crushed CaCO (64 mg, 0.64 mmol) d, 298 K, relative to TMS): δ = 9.38 (s, 2H; H2 and H9 of Me-
6 3 4 4
was stirred in acetone (20 mL) at room temperature under an inert phen),9.17(s,1H;H2ofpyridine),8.63(s,1H;H6ofpyridine),8.39
atmosphereofnitrogen,andthiophosgene(26μL,0.34mmol)wasadded (s,2H;H5andH6ofMe-phen),7.76(s,1H;H4ofpyridine),3.48−
4
tothemixture.Thesuspensionwasstirredinthedarkatroomtemperature 3.37 (m, 4H; CH of Et,NHCHCHCH), 2.89(s, 6H; CH atC4
2 2 2 2 3
for5min.Thesuspensionwasfiltered,andthefiltratewasevaporatedto andC7ofMe-phen),2.74(s,6H;CH atC3andC8ofMe-phen),
4 3 4
dryness to give a yellow solid. Recrystallization of the product from 2.30−2.05 (m, 2H; NHCH CH CH ), 1.98−1.85 (m, 2H;
2 2 2
acetone/petroleumetheraffordedthecomplexasyellowcrystals. NHCHCHCH), 1.11 ppm (t, 3H; J = 6.9 Hz, CH of Et). IR
2 2 2 3
Complex1b:Yield:156mg(79%).1HNMR(300MHz,acetone- (KBr):ν=3426(br,N−H),2033(s,CO),1926(s,CO),1656
d,298K,relativetoTMS):δ=9.97(d,2H;J=4.8Hz,H2andH9 (m,CO),1240(m,C−F),1208(m,C−F),1151(m,C−F),1026
6
of phen), 9.09 (d, 2H; J = 8.4 Hz, H4 and H7 of phen), 8.81−8.76 (m, C−F), 848 cm−1 (m, PF−). Positive-ion ESI-MS ion clusters at
6
(m,2H;H2andH6ofpyridine),8.41−8.30(m,6H;H3,H5,H6and m/z 1191 [M − PF −]+. Elemental analysis calcd (%) for
6
H8 of phen, NH, H4 of pyridine), 3.47−3.39 (m, 2H; C H NOSPF Re·1.5(CH)CO: C, 36.72; H, 2.97; N, 5.91.
39 33 6 4 23 3 2
NHCHCHCH), 2.28−2.08 (m, 2H; NHCHCHCH), 1.86−1.80 Found: C, 37.01; H, 2.93; N, 6.21.
2 2 2 2 2 2
ppm (m, 2H; NHCHCHCH). IR (KBr): ν = 3422 (br, N−H), Complex3c:Yield:78mg(68%).1HNMR(300MHz,methanol-
2 2 2
2111 (m, NCS), 2034 (s, CO), 1932 (s, CO), 1667 d,298K,relativetoTMS):δ=9.71(d,2H;J=5.4Hz,H2andH9of
4
(m,CO),1243(m,C−F),1208(m,C−F),1148(m,C−F),1030 Ph-phen),9.00(s,1H;H2ofpyridine),8.86(s,1H;H6ofpyridine),
2
(m, C−F), 848 cm−1 (s, PF−). Positive-ion ESI-MS ion clusters at m/z 8.18(s,2H;H5andH6ofPh-phen),8.10(d,2H;J=5.4Hz,H3and
6 2
1090 [M − PF−]+. Elemental analysis calcd (%) for C H NO- H8 of Ph-phen), 7.80 (s, 1H; H4 of pyridine), 7.72−7.62 (m, 10H;
6 33 18 5 4 2
SPF Re·2.5HO: C, 30.97; H, 1.81; N, 5.47. Found: C, 30.75; H, 1.75; Ph of Ph-phen), 3.46−3.37 (m, 4H; CH of Et, NHCHCHCH),
23 2 2 2 2 2 2
N,5.27. 2.28−2.16 (m, 2H; NHCH CH CH ), 1.91−1.84 (m, 2H;
2 2 2
Complex2b:Yield:146mg(71%).1HNMR(300MHz,acetone- NHCHCHCH), 1.13−1.06 ppm (m, 3H; CH of Et). IR (KBr):
2 2 2 3
d, 298 K, relative to TMS): δ = 9.72 (s, 2H; H2 and H9 of Me- ν=3428(br,N−H),2033(s,CO),1926(s,CO),1655(m,CO),
6 4
phen), 8.86 (s, 1H; H2 of pyridine), 8.73 (s, 1H; H6 of pyridine), 1237(m,C−F,CS),1210(m,C−F),1149(m,C−F),1027(m,C−F),
8.46−8.43(m,3H;H5andH6ofMe-phen,NH),8.32(s,1H;H4of 844 cm−1 (m, PF−). Positive-ion ESI-MS ion clusters at m/z 1287
pyridine),3.48−3.39(m,2H;NHCH 4 CHCH),2.98(s,6H;CH at [M − PF−]+. Elem 6 ental analysis calcd (%) for C H NOSPF Re·
2 2 2 3 6 47 33 6 4 23
C4 and C7 of Me-phen), 2.79 (s, 6H; CH at C3 and C8 of Me- 0.5(CH)CO:C,39.87;H,2.48;N,5.75.Found:C,39.77;H,2.66;N,6.03.
4 3 4 32
phen), 2.36−2.18 (m, 2H; NHCH CH CH ), 1.88−1.78 ppm Instrumentation and Photophysical Measurements. 1H
2 2 2
(m, 2H; NHCHCHCH). IR (KBr): ν = 3446 (br, N−H), 2110 NMR spectra were recorded on a Varian Mercury 300 MHz NMR
2 2 2
(m,NCS),2034(s,CO),1923(s,CO),1668(m,CO), spectrometerat298K.Positive-ionESImassspectrawererecordedon
1243 (m, C−F), 1208 (m, C−F), 1152 (m, C−F), 1030 (m, C−F), a PerkinElmer Sciex API 365 mass spectrometer. IR spectra were
842cm−1(m,PF−).Positive-ionESI-MSionclustersatm/z1146[M− recorded on a PerkinElmer 1600 series FT-IR spectrophotometer.
6
PF−]+.Elementalanalysiscalcd(%)forC H NOSPF Re·2HO:C, ElementalanalyseswerecarriedoutonaVarioELIIICHNelemental
6 37 26 5 4 23 2
33.49;H,2.28;N,5.28.Found:C,33.50;H,2.29;N,5.15. analyzer.Electronicabsorptionandsteady-stateemissionspectrawere
Complex3b:Yield:180mg(81%).1HNMR(300MHz,acetone- recorded on a Hewlett-Packard 8453 diode array spectrophotometer
d,298K,relativetoTMS):δ=10.02(d,2H;J=4.8Hz,H2andH9 andaSPEXFluoroLog3-TCSPCspectrophotometerequippedwitha
6
of Ph-phen), 8.90 (s, 1H; H2 of pyridine), 8.82 (s, 1H; H6 of Hamamatsu R928 PMT detector, respectively, and the emission
2
pyridine), 8.35−8.29 (m, 4H; H3 and H8 of Ph-phen, NH, H4 of spectrawereuncorrectedforPMTresponses.Emissionlifetimeswere
2
pyridine),8.22(s,2H;H5andH6ofPh-phen),7.79−7.75(m,10H; measured inthe FastMCSor aTCSPClifetime modewith aNano-
2
Ph of Ph-phen), 3.49−3.40 (m, 2H; NHCHCHCH), 2.39−2.18 LED N-375 as the excitation source, respectively. All the solutions for
2 2 2 2
(m, 2H; NHCH CH CH ), 1.89−1.78 ppm (m, 2H; photophysical studies were degassed with no fewer than four successive
NHCHCHCH).IR( 2 KBr) 2 :ν= 2 3427(br,N−H),2111(m,NCS), freeze−pump−thaw cycles and stored in a 10 cm3 round-bottom flask
2034(s 2 ,C 2 O) 2 ,1929(s,CO),1666(m,CO),1240(m,C−F), equippedwithasidearm1cmfluorescencecuvetteandsealedfromthe
1208(m,C−F),1150(m,C−F),1030(m,C−F),842cm−1(m,PF−). atmosphere by a Rotaflo HP6/6 quick-release Teflon stopper. Emission
6
Positive-ionESI-MSionclustersatm/z1242[M−PF−]+.Elemental quantumyieldsweremeasuredbytheopticallydilutemethod47awithan
analysiscalcd(%)forC H NOSPF Re·HO:C,38.4 6 7;H,2.01;N, aeratedaqueoussolutionof[Ru(bpy)]Cl (Φ =0.028,λ =455nm)as
45 26 5 4 23 2 3 2 em ex
4.98.Found:C,38.43;H,2.23;N,4.73. the standard solution.47b The errors of emission lifetime and quantum
[Re(N∧N)(CO) (py-Rf-TU-Et)](CF SO ) (N∧N = phen (1c), Me - yield measurements are estimated to be ±10% and ±20%, respectively.
phen (2c), Ph - 3 phen (3c)). A mix 3 ture 3 of [Re(N∧N)(CO)(py-R 4 f- Thelipophilicityofthecomplexesweredeterminedaccordingtoarepor-
2 3
NCS)](PF) (0.08 mmol) and ethylamine (10 μL, 0.16 mmol) was tedmethod.48
6
5852 dx.doi.org/10.1021/om3003575|Organometallics2012,31,5844−5855
Organometallics Article
ICP-MS.HeLacellsingrowthmediumwereincubatedwiththerhenium- microscope.Theexcitationwavelengthwas405nm,andtheemissionwas
(I)polypyridinecomplex(10μM)ina60mmtissueculturedishfor2h. measured using a 532 nm long-pass filter. In the colocalization experi-
The culture medium was then removed and washed thoroughly with ments, after being treated with complex 3c, HeLa cells were incubated
PBS(1mL×5).Thecellsweretrypsinizedandharvested.Theharvested with MitoTracker Deep Red FM (100 nM, λ = 633 nm) in a FBS-
ex
cells,togetherwiththecollectedPBS,weredigestedwith65%HNO at60°C. free medium for 20 min, and the cells were finally washed with PBS
3
The digestedsolutionwasfiltered,andtheconcentrationofrhenium inthe (1 mL × 3). The colocalization coefficient was determined by ImageJ
filtratewasmeasuredusinganElan6100DRC-ICP-MSsystem(PerkinElmer (version1.4.3.67).
SCIEX Instruments) equipped with a peristaltic pump, Meinhard quartz Fixed-Cell Confocal Imaging. The incubation procedures were
nebulizer,cyclonicspraychamber,nickelskimmer,andsamplecones. similartothatoflive-cellconfocalimagingexceptthat,afterwashingwith
MTT Assays. Cytotoxicity assays were conducted in 96-well, flat- PBS,thecoverslipswereincubatedinMeOH/PBS(1:1v/v)for4minand
bottommicrotiterplates.Thesupplementedculturemedium(100μL) then in MeOH for 4 min. The coverslips were finally washed with PBS
with ca. 10000 cells per well was incubated at 37 °C under a 5% CO beforebeingmountedontotheslidesformeasurements.
2
atmospherefor24h.Therhenium(I)polypyridinecomplexwasdissolvedin Agarose Gel Electrophoresis Retardation Assays. Polyplexes
the culture medium with 1% DMSO, and the solutions were added to withdifferentN/PratioswerepreparedbymixingbPEI-3corPoly-K-3c
the wells.Afterthe microtiterplate was incubated for48h,MTT inPBS andpRL-TKinTris-Clbuffer(10μL,50mM,pH7.4).Afterincubation
(5mg/mL,10μL)wasaddedtoeachwell.Themicroplatewasincubatedfor at room temperature for 30 min, the polyplexes were analyzed by
another3h.Themediumwasremovedcarefully,andisopropanol(200μL) electrophoresisona0.9%(w/v)agarosegelcontainingethidiumbromide
wasaddedtoeach well.Allthe assayswere runinparallelwith apositive
withTris-acetatebufferat100Vfor45min.Thegelwasvisualizedusing
control,inwhichcisplatinwasusedasacytotoxicagent.Theabsorbanceof aBio-RadGelDocimager.
all the solutions at 570 nm was measured with a SPECTRAmax 340 Zeta Potentials and Mean Hydrodynamic Diameter Meas-
microplate reader (Molecular Devices Corporation, Sunnyvale, California). urements. A mixture of bPEI-3c or Poly-K-3c and pRL-TK (4 μg)
TheIC valuesofthecomplexeswereevaluatedbasedonthepercentagecell
withdifferentN/P ratiosinTris-Clbuffer(80μL,50mM,pH7.4)was
50
survivalinadose-dependentmannerrelativetothecontrols. incubatedatroomtemperaturefor30min.Themixturewasthendiluted
Labeling of GSH with Complexes 1b−3b. The isothiocyanate 10-fold withthe same buffer.The zeta potentials of the polyplexeswere
complex(0.85μmol)inanhydrousDMSO(200μL)wasaddedtoGSH measured using a Zetasizer Nano ZS (Malvern Instruments). Specifica-
(21.5 mg, 70 μmol) in a mixture of HO (1.8 mL) and triethylamine tions: sampling time, 10−20 s; medium viscosity, 1.0031 cP; dielectric
(20 μL). The suspensionwas incubated 2 for 5 min at room temperature, constant,80.4;temperature,20°C;beam mode F(Ka)= 1.50(Smoluc-
andthesolidresiduewasremovedbycentrifugation.Thesupernatantwas howsky). Particle size was determined with the following specifications:
loadedontoanFSPEcolumn,whichhadbeenactivatedbyDMF(5mL) samplingtime,180s;mediumviscosity,1.0031cP;refractiveindex(RI)
andpreconditionedwithHO.Thecolumnwasoperatedundergradient
medium,1.330;RIparticle,1.450;temperature,20°C.Alltheexperiments
elution (HO to 40% aque
2
ous MeOH). Finally, the resultant conjugates
w■erecarriedoutintriplicate.
2
GSH-1c−GSH-3c were eluted using 60% aqueous MeOH. Positive-ion
ESI-MSionclustersatm/z1397[GSH-1c−PF−]+,m/z1453[GSH-2c ASSOCIATED CONTENT
−PF 6 −]+,andm/z 1549[GSH-3c −PF 6 −]+. 6 * S Supporting Information
Labeling of BSA, bPEI, and Poly-K with Complexes 1b−3b. Electronicabsorptionspectraldataoftherhenium(I)polypyridine
Theisothiocyanatecomplexes(1.2μmol)inanhydrousDMSO(50μL)
fluorous complexes; electronic absorption spectra of complexes
wereaddedtoBSA(10mg,151nmol),bPEI(3mg,120nmol),orPoly- 1a−3a; concentration dependence of surviving HeLa cells after
K(2.5mg,83nmol)in50mMcarbonatebuffer(450μL)atpH9.7.The
exposure of complexes 1c−3c, respectively, for 48 h; emission
suspensionwasstirredfor12hinthedarkatroomtemperature,andthe
spectrumofGSH-1cindegassed60%aqueousMeOHat298K;
solid residue was removed by centrifugation. The supernatant was then
dilutedto1.0mLwith50mMpotassiumphosphatebufferatpH7.4and and synthetic scheme for the rhenium(I) polypyridine fluorous
loadedontoaPD-10columnequilibratedwiththesamebuffer.Thefirst complexes.ThismaterialisavailablefreeofchargeviatheInternet
elution band with a strong green emission was collected. The resultant a■t http://pubs.acs.org.
conjugates were washed successively with potassium phosphate buffer
usingaYM-30centricon,concentratedto1.5mLandstoredat4°C.For AUTHOR INFORMATION
solubilityreasons,thebuffersforbothbPEIandPoly-Kconjugateswere
Corresponding Author
finallyexchangedto50mMTris-ClbufferatpH7.4.Onthebasisofthe
*E-mail: bhkenlo@cityu.edu.hk. Fax: (852) 3442 0522. Tel:
spectroscopic data, the rhenium-to-protein ratios of the BSA conjugates
weredeterminedtobeca.1.8−2.7,whicharecomparabletothoseofBSA (852) 3442 7231.
labeledbyrelatedrhenium(I)isothiocyanatecomplexes(ca.1.8−2.5).16a Notes
Therhenium-to-bPEIandrhenium-to-Poly-Kratiosweredeterminedto T■he authors declare no competing financial interest.
beca.0.8−1.1.
FluorousSolidPhaseExtraction.AmixtureofGSH-1c(0.7mM) ACKNOWLEDGMENTS
andthe20aminoacids(videsupra)(eachat0.1mg/mL)inwater(2mL)
WethanktheHongKongUniversityGrantsCommitteeAreas
wasloadedontoanFSPEcolumn(2g,8mLtube),whichhadbeenactiva-
of Excellence Scheme (AoE/P-03/08), Hong Kong Research
tedbyDMF(5mL)andpreconditionedwithHO.Whenthecolumnwas
2
operated under gradient elution (HO to 40% aqueous MeOH), a Grants Council (CityU 102410), and City University of Hong
luminescentbandremainedatthetop,a 2 ndtheelutedsolution(flow-through Kong(ProjectNo.7008174)forfinancialsupport.M.-W.L.and
fraction) was collected. When the mobile phase was changed to 60% A.W.-T.C.acknowledgeaPostgraduateStudentship,aResearch
aqueous MeOH, the luminescent band was eluted and collected (elution Tuition Scholarship, and an Outstanding Academic Perform-
fraction).Theinitialmixture,flow-throughfraction,andelutionfractionwere
anceAwardadministeredbyCityUniversityofHongKong.We
allanalyzedbyESI-MS.
aregratefultoMr.KennethKing-KwanLau,Mr.MichaelWai-
Live-Cell Confocal Microscopy. HeLa cells in growth medium
LunChiang,andMr.Ho-HangChanfortheirassistanceonthe
were seeded on a sterilized coverslip in a 60 mm tissue culture dish
andgrownat37°Cundera5%CO atmospherefor48h.Theculture c■ellular experiments.
2
mediumwasthenremovedandreplacedwithmedium/DMSO(99:1v/v)
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