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Rhenium(I) polypyridine biotin isothiocyanate complexes as the first luminescent biotinylation reagents: synthesis, photophysical properties, biological labeling, cytotoxicity, and imaging studies.

PMID: 18088115
Inorg.Chem.2008, 47, 602- 611 Rhenium(I) Polypyridine Biotin Isothiocyanate Complexes as the First Luminescent Biotinylation Reagents: Synthesis, Photophysical Properties, Biological Labeling, Cytotoxicity, and Imaging Studies Kenneth Kam-Wing Lo,* Man-Wai Louie, Ka-Shing Sze, and Jason Shing-Yip Lau Department of Biology and Chemistry, City UniVersity of Hong Kong, Tat Chee AVenue, Kowloon, Hong Kong, People’s Republic of China ReceivedAugust25,2007 Wereportherethedesignofthefirstclassofluminescentbiotinylationreagentsderivedfromrhenium(I)polypyridine complexes. These complexes [Re(N- N)(CO)(py-biotin-NCS)](PF) (py-biotin-NCS ) 3-isothiocyanato-5-(N-((2- 3 6 biotinamido)ethyl)aminocarbonyl)pyridine; N- N ) 1,10-phenanthroline (phen) (1a), 3,4,7,8-tetramethyl-1,10- phenanthroline(Me-phen)(2a),4,7-diphenyl-1,10-phenanthroline(Ph-phen)(3a)),containingabiotinunitandan 4 2 isothiocyanatemoiety,havebeensynthesizedfromtheprecursoraminecomplexes[Re(N- N)(CO)(py-biotin-NH)]- 3 2 (PF) (py-biotin-NH ) 3-amino-5-(N-((2-biotinamido)ethyl)aminocarbonyl)pyridine; N- N ) phen (1c), Me-phen 6 2 4 (2c),Ph-phen(3c)).Toinvestigatetheamine-specificreactivityoftheisothiocyanatecomplexes1a- 3a,theyhave 2 been reacted with a model substrate ethylamine, resulting in the formation of the thiourea complexes [Re(N- N)- (CO)(py-biotin-TU-Et)](PF)(py-biotin-TU-Et)3-ethylthioureidyl-5-(N-((2-biotinamido)ethyl)aminocarbonyl)pyridine; 3 6 N- N ) phen (1b), Me-phen (2b), Ph-phen (3b)). All the rhenium(I) complexes have been characterized, and 4 2 their photophysical properties have been studied. The avidin-binding properties of the thiourea complexes 1b- 3b havebeenexaminedbythe4¢-hydroxyazobenzene-2-carboxylicacid(HABA)assay.Titrationresultsindicatedthat thecomplexesexhibitedemissionenhancementbyca.1.4- 1.5-folduponbindingtoavidin,andthelifetimeswere elongatedtoca.0.8- 2.0(cid:237)s.Additionally,wehavebiotinylatedbovineserumalbumin(BSA)withtheisothiocyanate complexes.Alltheresultantrhenium- BSAbioconjugatesdisplayedintenseandlong-livedorange-yellowtogreenish- yellow emission upon irradiation in aqueous buffer under ambient conditions. The avidin-binding properties of the bioconjugateshavebeeninvestigatedusingtheHABAassay.Furthermore,thecytotoxicityofthethioureacomplexes 1b- 3btowardtheHeLacellshasbeenexaminedbythe3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazoliumbromide (MTT)assay.TheIC valuesweredeterminedtobeca.17.5- 28.5(cid:237)M,whicharecomparabletothatofcisplatin 50 (26.7 (cid:237)M) under the same conditions. The cellular uptake of complex 3b has been investigated by fluorescence microscopy, and the results showed that the complex was localized in the perinuclear region after interiorization. Introduction tion; this is illustrated in Chart 1. Also, biotinylated bio- moleculescanbepurifiedwithaffinitychromatographyand Theavidin-biotinsystemisausefultoolinimmunology, detected by ELISA and blotting techniques. These applica- histochemistry, and in situ hybridization.1 For example, tionsrelyonthefactsthat(1)avidincanbereadilymodified antibodies multiply labeled with biotin can increase the withfluorescenttags2andenzymes3withoutlosingitsaffinity sensitivity of heterogeneous bioassays by signal amplifica- tobiotinand(2)thebiologicalcharacteristicsandproperties *To whom correspondence should be addressed. E-mail: bhkenlo@ (2) (a)Al-Hakiem,M.H.H.;Landon,J.;Smith,D.S.;Nargessi,R.D. cityu.edu.hk.Fax: (+852)27887406.Tel(+852)27887231. Anal.Biochem.1981,116,264-267.(b)Barbarakis,M.S.;Smith- (1) (a)Green,N.M.AdV.ProteinChem.1975,29,85-133.(b)Wilchek, Palmer,T.;Bachas,L.G.;Chen,S.-Y.;VanDerMeer,B.W.Talanta M.;Bayer,E.A.Anal.Biochem.1988,171,1-32.(c)Green,N.M. 1993,40,1139-1145. MethodsEnzymol.1990,184,51-67.(d)Wilchek,M.;Bayer,E.A. (3) (a) Guesdon, J.-L.; Ternynck, T.; Avrameas, S. J. Histochem. MethodsEnzymol.1990,184,123-138.(e)Mock,D.M.;Horowitz, Cytochem.1979,27,1131-1139.(b)Berti,E.;Monti,M.;Cavicchini, P.MethodsEnzymol.1990,184,234-240. S.;Caputo,R.Arch.Dermatol.Res.1983,275,134-138. 602 InorganicChemistry, Vol.47,No.2,2008 10.1021/ic701675cCCC:$40.75 ©2008AmericanChemicalSociety PublishedonWeb12/19/2007 Rhenium(I)PolypyridineBiotinIsothiocyanateComplexes Chart1. UseoftheBiotin-AvidinSystemToIncreasetheSensitivity Chart2. ComponentsofaLuminescentBiotinylationReagent ofHeterogeneousBioassaysbySignalAmplificationa,b groupforbioconjugationandabiotinmoietyforrecognition by avidin (Chart 2), have never been explored. There are three major reasons for the development of these reagents. First,theyrenderthebiotinylatedproteinsorDNAtopossess luminescencepropertiesthatcouldleadtonewinvitroand invivobioassaydesigns.Second,theextentofbiotinylation of biomacromolecules can be directly determined by more sensitive spectrofluorometric methods. Finally, they can be employedtobiotinylatesmallmolecularsubstratesandallow the isolation and purification of the specific biological receptors, for example, by affinity chromatography. Also, biological uptake of the biotinylated compounds may be followed by luminescence spectroscopy and microscopy. Isothiocyanate (-NdCdS) is a useful functional group forbioconjugationbecauseitreactsreadilywiththe(cid:15)-amine group of lysine residues and the N-terminal of proteins to form a stable thiourea moiety.5b We have reported (1) luminescent transition metal polypyridine isothiocyanate aAntigenrecognizedbyimmobilizedandlabeledantibodies.bAntigen recognizedbyimmobilizedandbiotinlyatedantibodies;thelatterisdetected complexesasbiologicallabelingreagents7and(2)lumines- bylabeledavidin. centbiotincomplexesasnoncovalentprobesforavidin.8In view of the rich photophysical properties of rhenium(I) ofcommonbiomoleculesareusuallyretainedafterbiotiny- polypyridinecomplexes,7a,8a,b,f,h,9-28weanticipatethatanew lation. A number of biotinylation reagents that are reactive classofspecificluminescentbiotinylationreagentscouldbe towarddifferentfunctionalgroupsofbiomoleculeshavebeen designed.4 To optimize avidin-biotin assays and to ensure achieved by functionalizing luminescent rhenium(I) poly- labeling reproducibility, the extent of biotinylation of the (7) (a)Lo,K.K.-W.;Ng,D.C.-M.;Hui,W.-K.;Cheung,K.-K.J.Chem. biomolecules involved must be known. This is routinely Soc.,DaltonTrans.2001,2634-2640.(b)Lo,K.K.-W.;Ng,D.C.- determinedbythe4¢-hydroxyazobenzene-2-carboxylicacid M.;Chung,C.-K.Organometallics2001,20,4999-5001.(c)Lo,K. K.-W.;Chung,C.-K.;Ng,D.C.-M.;Zhu,N.NewJ.Chem.2002,26, (HABA)assay,5whichisbasedonthedecreaseofabsorption 81-88.(d)Lo,K.K.-W.;Chung,C.-K.;Lee,T.K.-M.;Lui,L.-H.; due to the displacement of avidin-bound HABA molecules Tsang, K. H.-K.; Zhu, N. Inorg. Chem. 2003, 42, 6886-6897. (e) ((cid:236) abs ) 500 nm) by the biotinylated species. Additionally, L C o .- , M K .; . Z K h .- u W ,N .; .; H C u h i, eu W ng .-K K . . ; K C . h C u o n o g r , d C . . C -K he .; m T . s R a e n V g . , 2 K 0 . 05 H , . 2 -K 49 .; , N 14 g 3 , 4 D - . twochromogenicbiotinreagents,biotin-X2,4-dinitrophenyl- 1450. X-L-lysine NHS ester6a ((cid:15) 364nm ) 15000 dm3 mol-1 cm-1) (8) 1 (a 2 ) 4 L , o 9 , 34 K 4 . - K 9 .- 3 W 45 .; .( H b u ) i L , o W , . K -K .K .; . N -W g, .; D T . s C an .- g M ,K .J . . H A . m -K . . C O h r e g m a . n S o o m c e . t 2 a 0 ll 0 ic 2 s , and EZ-Link NHS-chromogenic-biotin6b ((cid:15) 354nm ) 29000 2004,23,3062-3070.(c)Lo,K.K.-W.;Chan,J.S.-W.;Lui,L.-H.; dm3 mol-1 cm-1), have been used to label biomolecules, Chung,C.-K.Organometallics2004,23,3108-3116.(d)Lo,K.K.- W.; Lee, T. K.-M. Inorg. Chem. 2004, 43, 5275-5282. (e) Lo, K. allowingspectrophotometricdeterminationofthedegreeof K.-W.; Li, C.-K.; Lau, J. S.-Y. Organometallics 2005, 24, 4594- biotinylation. A fluorometric assay involving the displace- 4601.(f)Lo,K.K.-W.;Hui,W.-K.Inorg.Chem.2005,44,1992- 2002.(g)Lo,K.K.-W.;Chung,C.-K.;Zhu,N.Chem.Eur.J.2006, ment of a quencher-substrate from a fluorescent avidin 12,1500-1512.(h)Lo,K.K.-W.;Tsang,K.H.-K.;Sze,K.-S.Inorg. conjugatebythebiotinylatedspecieshasalsobeendeveloped.6a Chem.2006,45,1714-1722.(i)Lo,K.K.-W.;Lau,J.S.-Y.Inorg. Althoughthisassayoffershighersensitivity,itrequirestwo Chem. 2007, 46, 700-709. (j) Lo, K. K.-W.; Lee, T. K.-M.Inorg. Chim.Acta2007,360,293-302.(k)Lo,K.K.-W.;Hui,W.-K.;Chung, reagentsandthebiotinylatedspeciescannotbereusedafter C.-K.;Tsang,K.H.-K.;Lee,T.K.-M.;Li,C.-K.;Lau,J.S.-Y.;Ng, analysis. To the very best of our knowledge, luminescent D.C.-M.Coord.Chem.ReV.2006,250,1724-1736. (9) (a)Wrighton,M.S.;Morse,D.L.J.Am.Chem.Soc.1974,96,998- biotinylation reagents, which contain a reactive functional 1003.(b)Giordano,P.J.;Wrighton,M.S.J.Am.Chem.Soc.1979, 101,2888-2897. 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(27) (a)Waterland,M.R.;Gordon,K.C.;McGarvey,J.J.;Jayaweera,P. (23) (a)Wei,L.;Babich,J.;Eckelman,W.C.;Zubieta,J.Inorg.Chem. M.J.Chem.Soc.,DaltonTrans.1998,609-616.(b)Lundin,N.J.; 2005, 44, 2198-2209. (b) Banerjee, S. R.; Schaffer, P.; Babich, J. Walsh,P.J.;Howell,S.L.;McGarvey,J.J.;Blackman,A.G.;Gordon, W.;Valliant,J.F.;Zubieta,J.DaltonTrans.2005,3886-3897.(c) K.C.Inorg.Chem.2005,44,3551-3560. Wei,L.;Babich,J.;Zubieta,J.Inorg.Chim.Acta2005,358,3691- (28) (a)Lo,K.K.-W.;Hui,W.-K.;Ng,D.C.-M.;Cheung,K.-K.Inorg. 3700. (d) James, S.; Maresca, K. P.; Babich, J. W.; Valliant, J. F.; Chem. 2002, 41, 40-46. (b) Lo, K. K.-W.; Tsang, K. H.-K.; Hui, Doering,L.;Zubieta,J.BioconjugateChem.2006,17,590-596. W.-K.; Zhu, N.Chem.Commun.2003, 2704-2705. (c) Lo, K. K.- (24) (a) Metcalfe, C.; Webb, M.; Thomas, J. A. Chem. Commun. 2002, W.;Lau,J.S.-Y.;Fong,V.W.-Y.;Zhu,N.Organometallics2004, 2026-2027.(b)Metcalfe,C.;Spey,S.;Adams,H.;Thomas,J.A.J. 23,1098-1106.(d)Lo,K.K.-W.;Tsang,K.H.-K.;Hui,W.-K.;Zhu, Chem.Soc.,DaltonTrans.2002,4732-4739.(c)deWolf,P.;Heath, N.Inorg.Chem.2005,44,6100-6110.(e)Lo,K.K.-W.;Tsang,K. S.L.;Thomas,J.A.Inorg.Chim.Acta2003,355,280-285. H.-K.;Zhu,N.Organometallics2006,25,3220-3227. 604 InorganicChemistry,Vol.47,No.2,2008 Rhenium(I)PolypyridineBiotinIsothiocyanateComplexes Scheme1. SynthesisandStructuresoftheRhenium(I)Polypyridine underreducedpressure,resultinginayellowsolid.Thesolidwas BiotinComplexes washedwith2-propanolandthenair-dried.Yield: 317mg(46%). Positive-ionESI-MSionclusteratm/z236{M+H+}+. 3-Amino-5-(N-((2-biotinamido)ethyl)aminocarbonyl)- pyridine,Py-biotin-NH.Biotinylethylenediamine(508mg,1.77 2 mmol) was dissolved in hot DMF (30 mL) under an inert atmosphere of nitrogen. After the solution was cooled to room temperature, 5-aminonicotinic acid N-hydroxysuccinimidyl ester (417mg,1.77mmol)dissolvedinDMF(15mL)wasadded.The solutionwasstirredundernitrogenatroomtemperaturefor12h. Itwasthenevaporatedtodrynessunderreducedpressuretogive a yellow solid. Recrystallization of the solid from MeOH/diethyl etheryieldedpy-biotin-NH asawhitesolid.Yield: 580mg(81%). 2 1H NMR (300 MHz, DMSO-d, 298 K, TMS): (cid:228) 8.67 (s, 1 H, 6 py-3-CONH),8.13(s,1H,C H-NH-biotin),7.99(s,1H,H2 2 4 of pyridine), 7.81 (s, 1 H, H6 of pyridine), 7.25 (s, 1 H, H4 of pyridine),6.44(s,1H,NHofbiotin),6.37(s,1H,NHofbiotin), 5.51 (s, 2 H, NH ), 4.30-4.26 (m, 1 H, NCH of biotin), 4.12- 2 4.08(m,1H,NCHofbiotin),3.06(d,1H,J)10.8Hz,SCHof biotin),2.83-2.77(m,1H,SCHofbiotin),2.55(d,1H,J)12.5 Hz, SCH of biotin), 2.04-2.03 (m, 2 H, COCHCH of biotin), 2 3 6 1.55-1.26(m,6H,COCHCH ofbiotin).IR(KBr)(cid:238)/cm-1: 3288 2 3 6 (br, NH), 1701 (s, CdO), 1648 (s, CdO). Positive-ion ESI-MS ionclusteratm/z408{M+H+}+. [Re(N-N)(CO)(py-biotin-NH)](PF) (N-N ) Phen (1c), 3 2 6 Me-phen (2c), Ph -phen (3c)). A mixture of [Re(N-N)(CO)- 4 2 3 (CHCN)](CFSO)8f(0.30mmol)andpy-biotin-NH (122mg,0.30 3 3 3 2 mmol)inTHF(30mL)wasrefluxedunderaninertatmosphereof nitrogen for 12 h. The yellow solution was then evaporated to dryness, resulting in a yellow solid. The complex was converted tothehexafluorophosphatesaltbymetathesiswithKPF inMeOH 6 and then purified by column chromatography on alumina. The desiredproductwaselutedwithCHCN/MeOH(10:1,v/v).Upon 3 recrystallization of the crude product from CH Cl/diethyl ether, 2 2 the complex formed as yellow crystals. Complex 1c. Yield: 161 mg(50%).1HNMR(300MHz,acetone-d,298K,TMS): (cid:228)9.90 6 (d,2H,J)4.7Hz,H2andH9ofphen),9.10(d,2H,J)8.2Hz, H4andH7ofphen),8.38-8.33(m,4H,H3,H5,H6andH8of phen),8.14(s,1H,H2ofpyridine),8.07(s,1H,py-3-CONH), 8.03 (s, 1 H, H6 of pyridine), 7.55 (s, 1 H, CH-NH-biotin), 2 4 withoutpurification.Biotinylethylenediamine,29[Re(N-N)(CO) 3 - 7.41(s,1H,H4ofpyridine),5.90(s,1H,NHofbiotin),5.70(s, (CH 3 CN)](CF 3 SO 3 ),8f [Re(Me 4 -phen)(CO) 3 (py-NCS)](PF 6 ) (2d) 1H,NHofbiotin),5.57(s,2H,NH 2 ),4.50-4.46(m,1H,NCH (py-NCS)3-isothiocyanatopyridine),7aand[Re(Me 4 -phen)(CO) 3 - of biotin), 4.29-4.25 (m, 1 H, NCH of biotin), 3.44-3.36 (m, 4 (py-biotin)](PF 6 ) (2e) (py-biotin ) 3-(N-((2-biotinamido)ethyl)- H, C 2 H 4 -NH-biotin), 3.19-3.07 (m, 1 H, SCH of biotin), 2.66 aminocarbonyl)pyridine)8fwerepreparedasdescribedpreviously. (d,1H,J )12.6Hz,SCHofbiotin),2.22(t,2H,J)7.3Hz, gem All buffer components were of molecular biology grade. PD-10 COCHCH ofbiotin),1.70-1.30(m,6H,COCHCH ofbiotin). 2 3 6 2 3 6 columns and YM-30 centricons were purchased from Pharmacia IR(KBr)(cid:238)/cm-1: 3448(br,NH),2032(s,CtO),1919(s,CtO), and Amicon, respectively. Human cervix epithelioid carcinoma 1650(m,CdO),842(s,PF-).Positive-ionESI-MSionclusterat 6 (HeLa)cellswereobtainedfromAmericanTypeCultureCollection. m/z 857 {M - PF}+. Anal. Calcd for C H NOSPFRe(cid:226)HO: 6 33 34 8 6 6 2 Dulbecco’smodifiedEagle’smedium(DMEM),fetalbovineserum C,38.86;H,3.56;N,10.99.Found: C,39.11;H,3.64;N,12.20%. (FBS),trypsin-EDTA,andpenicillin/streptomysinwerepurchased Complex2c.Yield: 178mg(51%).1HNMR(300MHz,acetone- from Invitrogen. The growth medium for cell culture contained d,298K,TMS): (cid:228)9.66(s,2H,H2andH9ofMe-phen),8.47 6 4 DMEMwith10%FBSand1%penicillin/streptomysin. (s,2H,H5andH6ofMe-phen),8.22(d,1H,J)2.3Hz,H2of 4 5-AminonicotinicAcidN-HydroxysuccinimidylEster.5-Ami- pyridine),8.11(s,1H,py-3-CONH),8.07(d,1H, J)1.5Hz, nonicotinicacid(409mg,2.96mmol)wasdissolvedinhotDMF H6ofpyridine),7.58(s,1H,C H-NH-biotin),7.45(s,1H,H4 2 4 (40mL)underaninertatmosphereofnitrogen.Afterthesolution ofpyridine),5.88(s,1H,NHofbiotin),5.75(s,1H,NHofbiotin), wascooledtoroomtemperature,aDMF(10mL)solutionofNHS 5.54 (s, 2 H, NH), 4.50-4.45 (m, 1 H, NCH of biotin), 4.24- 2 (450mg,3.91mmol)andN,N¢-dicyclohexylcarbodiimide(670mg, 4.20 (m, 1 H, NCH of biotin), 3.45-3.31 (m, 4 H, C H-NH- 2 4 3.25mmol)wasadded.Awhitesolidappeared,andthesuspension biotin),3.03-2.99(m,1H,SCHofbiotin),2.94(s,6H,CH at 3 was stirred under nitrogen at room temperature for 12 h. The C4andC7ofMe -phen),2.81(s,6H,CH atC3andC8ofMe - 4 3 4 mixture was filtered, and the filtrate was evaporated to dryness phen),2.67(d,1H,J )12.6Hz,SCHofbiotin),2.20(t,2H, gem J ) 6.7 Hz, COCH C H of biotin), 1.61-1.21 (m, 6 H, 2 3 6 (29) Garlick,R.K.;Giese,R.W.J.Biol.Chem.1988,263,210-215. COCH 2 C 3 H 6 ofbiotin).IR(KBr)(cid:238)/cm-1: 3448(br,NH),2030(s, Inorganic Chemistry, Vol. 47, No. 2, 2008 605 Lo et al. CtO), 1919 (s, CtO), 1655 (m, CdO), 844 (s, PF -). Positive- ofpyridine),8.33-8.24(m,6H,H3,H5,H6andH8ofPh-phen, 6 2 ion ESI-MS ion cluster at m/z 914 {M - PF}+. Anal. Calcd for H4ofpyridineandpy-3-CONH),7.81-7.67(m,11H,CH of 6 6 5 C H NOSPFRe(cid:226)HO: C,41.30;H,4.12;N,10.41.Found: C, Ph-phen and CH-NH-biotin), 4.71-4.68 (m, 1 H, NCH of 37 42 8 6 6 2 2 2 4 41.28; H, 4.42; N, 10.27%. Complex 3c. Yield: 198 mg (64%). biotin),4.50-4.42(m,1H,NCHofbiotin),3.44-3.37(m,4H, 1HNMR(300MHz,acetone-d,298K,TMS): (cid:228)9.99-9.97(dd, CH-NH-biotin),2.97(s,1H,SCHofbiotin),2.21-2.18(m,2 6 2 4 2H,J)5.4and3.1Hz,H2andH9ofPh -phen),8.31(dd,2H, H, COCHCH of biotin), 1.74-1.32 (m, 6 H, COCHCH of 2 2 3 6 2 3 6 J)5.3and3.5Hz,H3andH8ofPh-phen),8.26(s,2H,H5and biotin).IR(KBr)(cid:238)/cm-1: 3423(br,NH),2121(m,NdCdS),2034 2 H6ofPh-phen),8.17(s,2H,H2ofpyridineandpy-3-CONH), (s,CtO),1919(s,CtO),1655(s,CdO),842(s,PF -).Positive- 2 6 8.04(s,1H,H6ofpyridine),7.75-7.69(m,10H,C H ofPh- ionESI-MSionclusteratm/z1050{M-PF}+.Anal.Calcdfor 6 5 2 6 phen), 7.57 (s, 1 H, C H-NH-biotin), 7.48 (s, 1 H, H4 of C H NOSPFRe(cid:226)HO: C,45.50;H,3.49;N,9.23.Found: C, 2 4 46 40 8 6 2 6 2 pyridine),5.92(s,1H,NHofbiotin),5.73(s,1H,NHofbiotin), 45.35;H,3.50;N,9.52%. 5.62(d,2H,J)4.1Hz,NH 2 ),4.50-4.46(m,1H,NCHofbiotin), [Re(N-N)(CO) 3 (py-biotin-TU-Et)](PF 6 )(N-N)Phen(1b), 4.28-4.24 (m, 1 H, NCH of biotin), 3.44-3.37 (m, 4 H, C 2 H 4 - Me 4 -phen (2b), Ph 2 -phen (3b)). A mixture of [Re(N-N)(CO) 3 - NH-biotin),3.09-3.06(m,1H,SCHofbiotin),2.98-2.97(m,1 (py-biotin-NCS)](PF) (0.17 mmol) and ethylamine (0.17 mmol) 6 H,SCHofbiotin),2.65(d,1H,J gem )12.6Hz,SCHofbiotin), inacetone(30mL)wasstirredatroomtemperatureunderaninert 2.19(t,2H,J)6.9Hz,COCH 2 C 3 H 6 ofbiotin),1.63-1.26(m,6 atmosphere of nitrogen for 12 h. The solution was evaporated to H,COCH 2 C 3 H 6 ofbiotin).IR(KBr)(cid:238)/cm-1: 3433(br,NH),2032 dryness, forming an orange solid, which was then purified by (s,CtO),1919(s,CtO),1655(m,CdO),843(s,PF 6 -).Positive- columnchromatographyonalumina.Thedesiredproductwaseluted ionESI-MSionclusteratm/z1009{M-PF 6 }+.Anal.Calcdfor withacetone/MeOH(10:1,v/v).Uponrecrystallizationofthecrude C 45 H 42 N 8 O 6 SPF 6 Re(cid:226)H 2 O: C,46.11;H,3.78;N,9.56.Found: C, productfromCH 2 Cl 2 /diethylether,thecomplexformedasorange 46.35;H,3.99;N,9.68%. crystals.Complex1b.Yield: 122mg(66%).1HNMR(300MHz, [Re(N-N)(CO)(py-biotin-NCS)](PF) (N-N ) Phen (1a), acetone-d, 298 K, TMS): (cid:228) 9.96 (s, 1 H, NH of pyridine), 9.87 3 6 6 Me-phen(2a),Ph-phen(3a)).Thiophosgene(26(cid:237)L,0.34mmol) (d, 2 H, J ) 3.8 Hz, H2 and H9 of phen), 9.73 (s, 1 H, H4 of 4 2 wasaddedtoamixtureof[Re(N-N)(CO)(py-biotin-NH)](PF) pyridine),9.09(d,2H,J)8.2Hz,H4andH7ofphen),8.70(s, 3 2 6 (0.17 mmol) and finely crushed CaCO (64 mg, 0.64 mmol) in 1H,H2ofpyridine),8.38-8.32(m,4H,H3,H5,H6andH8of 3 acetone (10 mL) under an inert atmosphere of nitrogen. The phen),8.05(s,1H,Et-NH),7.95(s,1H,py-3-CONH),7.81(s, suspension was stirred in the dark at room temperature for 4 h. 1H,H6ofpyridine),7.61(s,1H,C H-NH-biotin),6.89(s,1 2 4 Themixturewasfiltered,andthefiltratewasevaporatedtodryness H,NHofbiotin),6.18(s,1H,NHofbiotin),4.61-4.58(m,1H, togiveayellowsolid.Subsequentrecrystallizationofthecomplex NCH of biotin), 4.39-4.36 (m, 1 H, NCH of biotin), 3.79-3.74 fromacetone/diethyletherresultedintheformationofthecomplex (m,2H,CH ofEt),3.42-3.39(m,4H,CH-NH-biotin),3.17 2 2 4 asyellowcrystals.Complex1a.Yield: 109mg(63%).1HNMR (s, 1 H, SCH of biotin), 2.68 (d, 1 H, J ) 12.6 Hz, SCH of gem (300MHz,acetone-d,298K,TMS): (cid:228)9.99(s,2H,H2andH9 biotin),2.27(s,2H,COCHCH ofbiotin),1.73-1.24(m,9H, 6 2 3 6 ofphen),9.53-9.50(m,1H,NHofbiotin),9.10(s,2H,H4and COCHCH of biotin and CH of Et). IR (KBr) (cid:238)/cm-1: 3432 2 3 6 3 H7 of phen), 9.00-8.95 (m, 1 H, NH of biotin), 8.81-8.77 (m, (br,NH),2033(s,CtO),1919(s,CtO),1686(m,CdO),1234 2H,H2andH6ofpyridine),8.39-8.35(m,5H,H3,H5,H6and (m, CdS), 846 (s, PF-). Positive-ion ESI-MS ion cluster at m/z 6 H8ofphenandH4ofpyridine),8.21(s,1H,py-3-CONH),7.57 943 {M - PF}+. Anal. Calcd for C H NOSPFRe(cid:226)HO: C, 6 36 39 9 6 2 6 2 (s,1H,CH-NH-biotin),4.81(s,1H,NCHofbiotin),4.60(s, 39.06; H, 3.73; N, 11.39. Found: C, 38.97; H, 3.97; N, 11.12%. 2 4 1H,NCHofbiotin),3.41-3.31(m,4H,CH-NH-biotin),2.72- Complex2b.Yield: 106mg(51%).1HNMR(300MHz,acetone- 2 4 2.71(m,1H,SCHofbiotin),2.27-2.25(m,2H,COCHCH of d, 298 K, TMS): (cid:228) 9.93 (s, 1 H, NH of pyridine), 9.78 (s, 1 H, 2 3 6 6 biotin), 1.64-1.29 (m, 6 H, COCHCH of biotin). IR (KBr) H4ofpyridine),9.62(s,2H,H2andH9ofMe -phen),8.68(s,1 2 3 6 4 (cid:238)/cm-1: 3422(br,NH),2110(m,NdCdS),2035(s,CtO),1919 H,H2ofpyridine),8.47(s,2H,H5andH6ofMe -phen),8.06(s, 4 (s, CtO), 1655 (m, CdO), 843 (s, PF -). Positive-ion ESI-MS 1H, Et-NH), 7.97 (s, 1 H, py-3-CONH), 7.81 (s, 1 H, H6 of 6 ionclusteratm/z899{M-PF}+.Anal.CalcdforC H NOS- pyridine), 7.62 (s, 1 H, CH-NH-biotin), 6.82 (s, 1 H, NH of 6 34 32 8 6 2 2 4 PFRe(cid:226)HO: C, 38.45; H, 3.23; N, 10.55. Found: C, 38.24; H, biotin),6.17(s,1H,NHofbiotin),4.59-4.56(m,1H,NCHof 6 2 3.15; N, 10.42%. Complex 2a. Yield: 108 mg (61%). 1H NMR biotin),4.38-4.32(m,1H,NCHofbiotin),3.80-3.71(m,2H, (300MHz,acetone-d,298K,TMS): (cid:228)9.78(s,2H,H2andH9 CH of Et), 3.44-3.39 (m, 4 H, C H-NH-biotin), 3.18-3.12 6 2 2 4 ofMe-phen),9.69-9.65(m,1H,NHofbiotin),9.21(s,1H,NH (m, 1 H, SCH of biotin), 2.68 (d, 1 H, J ) 12.9 Hz, SCH of 4 gem ofbiotin),8.85(d,2H,J)5.6Hz,H2andH6ofpyridine),8.48 biotin),2.30-2.23(m,2H,COCHCH ofbiotin),1.66-1.20(m, 2 3 6 (s,3H,H5andH6ofMe-phenandH4ofpyridine),8.25(s,1H, 9H,COCHCH ofbiotinandCH ofEt).IR(KBr)(cid:238)/cm-1: 3432 4 2 3 6 3 py-3-CONH),8.03(s,1H,CH-NH-biotin),4.71-4.66(m,1 (br,NH),2031(s,CtO),1919(s,CtO),1686(m,CdO),1245 2 4 H, NCH of biotin), 4.49-4.45 (m, 1 H, NCH of biotin), 3.42- (m, CdS), 846 (s, PF-). Positive-ion ESI-MS ion cluster at m/z 6 3.40 (m, 4 H, CH -NH-biotin), 3.24-3.14 (m, 2 H, SCH of 1001{M-PF]}+.Anal.CalcdforC H NOSPFRe(cid:226)HO: C, 2 4 6 40 49 9 6 2 6 2 biotin),2.82(s,6H,CH atC3andC8ofMe-phen),2.76-2.72 41.23; H, 4.41; N, 10.82. Found: C, 41.53; H, 4.65; N, 11.03%. 3 4 (m,1H,SCHofbiotin),2.61(s,1H,SCHofbiotin),2.28-2.26 Complex3b.Yield: 126mg(85%).1HNMR(300MHz,acetone- (m,2H,COCHCH ofbiotin),1.64-1.30(m,6H,COCHCH d,298K,TMS): (cid:228)9.95(s,1H,NHofpyridine),9.90(d,2H, 2 3 6 2 3 6 6 ofbiotin).IR(KBr)(cid:238)/cm-1: 3423(br,NH),2116(m,NdCdS), J)5.1Hz,H2andH9ofPh-phen),9.49(s,1H,H4ofpyridine), 2 2035 (s, CtO), 1919 (s, CtO), 1655 (s, CdO), 843 (s, PF -). 8.93(s,1H,H2ofpyridine),8.28-8.26(m,4H,H3,H5,H6and 6 Positive-ion ESI-MS ion cluster at m/z 955 {M - PF}+. Anal. H8ofPh-phen),8.17(s,1H,Et-NH),7.95(s,1H,py-3-CONH), 6 2 CalcdforC H NOSPFRe(cid:226)HO: C,40.82;H,3.79;N,10.02. 7.78-7.68(m,12H,CH ofPh-phen,H6ofpyridineandC H- 38 40 8 6 2 6 2 6 5 2 2 4 Found: C,40.62;H,4.02;N,9.73%.Complex3a.Yield: 154mg NH-biotin),6.84(s,1H,NHofbiotin),6.19(s,1H,NHofbiotin), (76%). 1H NMR (300 MHz, acetone-d, 298 K, TMS): (cid:228) 10.09 4.60-4.55(m,1H,NCHofbiotin),4.37-4.34(m,1H,NCHof 6 (d,2H,J)5.4Hz,H2andH9ofPh -phen),10.00-9.93(m,1 biotin),3.74-3.65(m,2H,CH ofEt),3.44-3.37(m,4H,CH- 2 2 2 4 H,NHofbiotin),8.93(s,1H,H2ofpyridine),8.89(s,1H,H6 NH-biotin), 3.19-3.14 (m, 1 H, SCH of biotin), 2.66 (d, 1 H, 606 InorganicChemistry,Vol.47,No.2,2008 Rhenium(I)PolypyridineBiotinIsothiocyanateComplexes J )12.8Hz,SCHofbiotin),2.28-2.25(m,2H,COCHCH replaced with medium/DMSO (99:1, v/v) containing the thiourea gem 2 3 6 ofbiotin),1.70-1.20(m,9H,COCHCH ofbiotinandCH of complex3b(10(cid:237)M).Afterincubationfor24h,themediumwas 2 3 6 3 Et). IR (KBr) (cid:238)/cm-1: 3426 (br, NH), 2032 (s, CtO), 1919 (s, removed,andthecelllayerwaswashedgentlywithPBS(1mL(cid:2) CtO),1686(m,CdO),1235(m,CdS),843(s,PF-).Positive- 3).Thecoverslipwasmountedontoaglassslide,imagedusinga 6 ionESI-MSionclusteratm/z1097{M-PF}+.Anal.Calcdfor Carl Zeiss Axioplan 2 imaging fluorescence microscope with the 6 C H NOSPFRe(cid:226)HO: C,45.78;H,3.92;N,10.01.Found: C, excitation wavelength in the range of 420-490 nm, and the 48 47 9 6 2 6 2 46.02;H,4.14;N,9.89%. emissionmeasuredusinga545nmlong-passfilter. PhysicalMeasurementsandInstrumentation.Theinstruments usedforthecharacterizationandphotophysicalmeasurementshave Results and Discussion been described previously.8b Luminescence quantum yields were Synthesis. The design of the luminescent amine-specific measured by the optically dilute method30 using an aerated acetonitrilesolutionof[Re(phen)(CO)(pyridine)](CFSO)(…) biotinylation reagents, complexes 1a-3a, is based on the 3 3 3 0.18,excitationwavelengthat355nm)asthestandardsolution.15b useofatrifunctionalcompound,5-aminonicotinicacid.The ThemethodsbywhichtheHABAassay,emissiontitrations,and pyridine may then be coordinated to the rhenium(I) center, determination of avidin-binding parameters of the thiourea com- the carboxyl group functionalized with an amine-biotin plexes 1b-3b were undertaken have also been previously derivative, and the amine group readily activated by thio- described.8f,31 phosgene to form the amine-specific isothiocyanate group. BiotinylationofBSAwiththeIsothiocyanateComplexes1a- The resultant pyridine-biotin-isothiocyanate ligand, to- 3a. The isothiocyanate complex (1.2 (cid:237)mol) in anhydrous DMSO gether with the use of various diimines, leads to the (50 (cid:237)L) was added to BSA (1.23 mg, 18.6 nmol) in 50 mM production of luminescent rhenium(I) complexes as biotin- carbonate buffer (450 (cid:237)L) at pH 9.7. The suspension was stirred ylation reagents with tunable emission colors. The isothio- for12hinthedarkatroomtemperature,andthesolidresiduewas cyanatecomplexes1a-3awerepreparedfromthereaction removed by centrifugation. The supernatant was then diluted to oftheprecursoraminecomplexes1c-3cwiththiophosgene 1.0 mL with 50 mM potassium phosphate buffer at pH 7.4 and inacetoneatroomtemperature(Scheme1).Toexaminethe loadedontoaPD-10columnequilibratedinthesamebuffer.The reactivity of the isothiocyanate complexes toward aliphatic first elution band with strong orange-yellow or greenish-yellow amines,theywerereactedwithamodelsubstrate,ethylamine, luminescencewascollected.Finally,thebioconjugatesBSA-1b- which resulted in the formation of the thiourea complexes BSA-3bwerewashedsuccessivelywithpotassiumphosphatebuffer 1b-3b (Scheme 1). All the complexes were characterized usingaYM-30centricon,concentratedto1.5mLandstoredat4 (cid:176) C. by 1H NMR, positive-ion ESI-MS, IR, and microanalyses. DigestionoftheBioconjugatesBSA-1b-BSA-3bbyPronase. Electronic Absorption and Emission Properties. Thebioconjugatein50mMpotassiumphosphatebufferatpH7.4 The electronic absorption spectral data of the com- (1.5mL)washeatedat80(cid:176) Cfor30min.Afterthesolutionwas plexes are summarized in Table 1, and the electronic cooledtoroomtemperature,pronase(2mg)inwater(200(cid:237)L)was absorption spectrum of complex 1b in CH Cl at 2 2 added. The mixture was maintained at 37 (cid:176) C for 24 h prior for 298 K is shown in Figure 1. With reference to previous analysisbytheHABAassay. studies on related rhenium(I) polypyridine complex- CytotoxicityAssays.Cytotoxicityassayswereconductedin96- es,7a,8a,b,f,h,9,10a,b,11a,c,12-14,15a,b,d-f,16,17a,c,d,18-20,21a,c,22b,c,23a,b,24-28 well, flat-bottomed microtiter plates. The supplemented culture the intense absorption bands at ca. 248-300 nm with medium(100(cid:237)L)withca.10000cellsperwellwasincubatedat extinction coefficients on the order of 104 dm3 mol-1 cm-1 37(cid:176) Cundera5%CO 2 atmospherefor24h.Thethioureacomplexes have been assigned to spin-allowed intraligand (1IL) ((cid:240) f 1b-3bweredissolvedintheculturemediumwith1%DMSOand (cid:240)*) (N-N and pyridine ligands) transitions. Additionally, the solutions added to the wells. The concentrations of the the absorption shoulders at ca. 322-397 nm with smaller complexesrangedfrom2to23(cid:237)M.Supplementedmediawith1% extinction coefficients have been assigned to spin-allowed DMSO(100(cid:237)L)wasusedasacontrol.Afterthemicrotiterplate metal-to-ligandcharge-transfer(1MLCT)(d(cid:240)(Re)f(cid:240)*(N- was incubated for 48 h, 10 (cid:237)L of MTT in PBS (5 mg/mL) was N)) transitions. The Ph -phen complexes 3a-3c showed addedtoeachwell.Themicroplatewasincubatedforanother3h. 2 lower-energy 1IL absorption bands due to the electron- Solubilizationsolution(100(cid:237)L)containing10%SDSin2-propanol/ withdrawing phenyl substituents of the diimine ligand. 0.04 M hydrochloric acid (1:1, v/v) was added to each well, and theplatewasincubatedfor24h.Alltheassayswereruninparallel All the complexes exhibited intense and long-lived withanegativecontrol(i.e.,vehiclecontrol)andapositivecontrol, orange-yellow to greenish-yellow luminescence in fluid inwhichcisplatinwasusedasacytotoxicagent.Theabsorbance solutions at 298 K upon photoexcitation. The emission ofallthesolutionsat570nmwasmeasuredwithaSPECTRAmax spectrum of complex 1b in CH Cl is shown in Figure 1. 2 2 340microplatereader(MolecularDevicesCorporation,California). The photophysical data are listed in Table 2. This The IC values of the complexes were evaluated based on the 50 emission has been attributed to a triplet metal-to-ligand percentagecellsurvivalinadose-dependentmannerrelativetothe charge-transfer (3MLCT) (d(cid:240)(Re) f (cid:240)*(N-N)) excited controls. state.7a,8a,b,f,h,9,10a,b,11a,c,12-17,18a,b,d,19,20a,c,21,22,23a,b,d,24,25a,c,26-28Sup- Cellular Uptake Studies. HeLa cells in growth medium (ca. 100000 cells/mL) were seeded on a sterilized coverslip in a 35 porting this assignment is the observation that the isothio- mm tissue culture dish and grown at 37 (cid:176) C under a 5% CO cyanatecomplexes1a-3aemittedatslightlyhigherenergy 2 atmospherefor48h.Theculturemediumwasthenremovedand than their thiourea 1b-3b and amine 1c-3c counterparts (Table 2). We attributed this to the electron-withdrawing (30) Demas,J.N.;Crosby,G.A.J.Phys.Chem.1971,75,991-1024. isothiocyanatemoietyrenderingthemetalcenterlesselectron- (31) Marek, M.; Kaiser, K.; Gruber, H. J. Bioconjugate Chem. 1997, 8, 560-566. rich and hence increasing the 3MLCT emission energy. Inorganic Chemistry, Vol. 47, No. 2, 2008 607 Lo et al. Table1. ElectronicAbsorptionSpectralDataoftheRhenium(I)PolypyridineBiotinComplexesat298K complex medium (cid:236)abs/nm((cid:15)/dm3mol-1cm-1) 1a CH2Cl2 259sh(16040),276(19700),294sh(12660),333sh(3910),374sh(2515) CH3CN 258sh(17305),274(20170),300sh(11415),328sh(6165),370sh(3185) 1b CH2Cl2 256sh(28055),274sh(28000),298sh(12945),332sh(6870),389sh(2265) CH3CN 255sh(26495),271(27365),297sh(12750),330sh(6675),382sh(2085) buffera 328sh(7690),382sh(2385) 1c CH2Cl2 260(30425),277sh(27050),298sh(11990),335(7960),389sh(3100) CH3CN 259(26530),268(26875),296sh(13655),327(9305),375sh(3675) 2a CH2Cl2 250(27230),281(33575),327sh(11150),376sh(2948) CH3CN 250sh(30755),280(38545),326sh(12105),370sh(3260) 2b CH2Cl2 253(41670),279(41635),328sh(13790),376sh(3395) CH3CN 249(33350),278(31735),325sh(10895),372sh(2545) buffera 322sh(11540),372sh(2795) 2c CH2Cl2 254(29595),281(29705),338sh(9895),371sh(3790) CH3CN 248(37245),280(35875),330sh(12945),370sh(3955) 3a CH2Cl2 265sh(20370),290(30140),342sh(9570),394sh(4805) CH3CN 262(28895),288(46170),334sh(15500),387sh(6335) 3b CH2Cl2 270(41035),287(45580),341sh(17920),397sh(6470) CH3CN 261(35370),287(40110),335sh(16270),389sh(5580) buffera 336sh(12920),392sh(4565) 3c CH2Cl2 267(28895),292(33545),342sh(15300),395sh(6385) CH3CN 263(32245),292(39245),337sh(17440),387sh(6755) a50mMpotassiumphosphateatpH7.4containing30%DMSO(DMSOwasusedtoincreasecomplexsolubility). 1b-3btoanavidin-HABAsolutionresultedinadecrease of absorbance, indicative of the specific binding of the complexes to avidin. Interestingly, the plots of -¢A 500 nm versus [Re]/[avidin] show that the equivalence points oc- curredat[Re]/[avidin])ca.4.5,5.2,and4.8,forcomplexes 1b-3b, respectively. As avidin may only bind up to four biotin molecules, the occurrence of the equivalence points at [Re]/[avidin] > 4 suggests that the binding affinities of these complexes are not substantially higher than that of HABA. Emission Titrations. The binding of the thiourea com- Figure 1. Electronic absorption (s) and emission (---) spectra of plexes 1b-3b to avidin has been studied by emission complex1binCH2Cl2at298K. titrations using the complexes as titrants. Two control Nonetheless,theenergydifferenceisrelativelysmallbecause experiments, in which (1) avidin was absent or (2) avidin theelectrondensityoftherhenium(I)centerisonlyremotely was presaturated with excess biotin, were also performed. controlled by the substituents on the pyridine ligand. The Similar to other luminescent transition metal biotin com- longeremissionlifetimesoftheMe -phencomplexesinfluid plexes we have reported,8 the thiourea complexes 1b-3b 4 solutionsatroomtemperature(Table2)suggestthepresence showedenhancedemissionintensities(ca.1.4-1.5-fold)in of substantial triplet intraligand 3IL ((cid:240) f (cid:240)*) (Me 4 -phen) the presence of avidin (Table 3). The titration curves for character in their emissive states.8b,f,14a,15b,c,e,16a,b,c,22c,28b This complex 2b are shown in Figure 2. Since no changes were is also reflected by the very similar emission wavelengths observedinthecontrolexperiments,theemissionenhance- of complexes 2a-2c in different solvents. The emission of mentmustresultfromthespecificbindingofcomplexes1b- all the complexes in alcohol glass at 77 K showed a 3btoavidin.Theemissionlifetimesofthecomplexeswere significantblue-shiftowingtotherigidochromiceffect(Table elongatedfromca.0.4-1.1to0.8-2.0(cid:237)suponthebinding 2),whichiscommonlyobservedinluminescentrhenium(I) event(Table3).Thesechangesofphotophysicalproperties polypyridinecomplexes.7e,8b,h,9,11a,12a,13b,14d,15a,d-f,16a-c,19b,21,28b-e were ascribed to the increase in the hydrophobicity and HABA Assay. The thiourea complexes 1b-3b can be rigidity of the local environment of the complexes upon considered as models for biomolecules biotinylated by the isothiocyanate complexes 1a-3a. Thus, the avidin-binding binding to avidin.8 The first dissociation constants (K d ) of properties of complexes 1b-3b in buffer have been exam- the avidin adducts of the thiourea complexes 1b-3b were ined by the HABA assay.5 The avidin-HABA adduct is estimatedtobe7.9(cid:2)10-8,1.2(cid:2)10-7,and5.6(cid:2)10-7M, knowntodisplayanintenseabsorptionbandat500nm.As respectively. These values are up to 3 orders of magnitude the affinity of biotin to avidin (K ) ca. 10-15 M) is much higher than those of other rhenium(I) polypyridine biotin d higher than that of HABA (K ) 6 (cid:2) 10-6 M), addition of complexes,8a,b,f,hprobablyduetothesterichindranceofthe d biotintoasolutionoftheavidin-HABAadductwilldisplace thiourea moiety and the lack of a long spacer-arm in the the bound HABA molecules, leading to a decrease of current complexes. The avidin-binding affinity of complex absorbance at 500 nm. In this work, addition of complexes 1bishigherthanthoseofcomplexes2band3b,whichmay 608 InorganicChemistry,Vol.47,No.2,2008 Rhenium(I)PolypyridineBiotinIsothiocyanateComplexes Table2. PhotophysicalDataoftheRhenium(I)PolypyridineBiotinComplexes complex medium(T/K) (cid:236)em/nm (cid:244)o/(cid:237)s … 1a CH2Cl2(298) 527 2.92 0.28 CH3CN(298) 546 1.43 0.12 glass(77)a 462sh,509 9.58 1b CH2Cl2(298) 531 2.40 0.51 CH3CN(298) 546 1.11 0.10 buffer(298)b 546 0.18 0.017 glass(77)a 474sh,492 11.17 1c CH2Cl2(298) 535 2.92 0.56 CH3CN(298) 548 1.15 0.16 glass(77)a 490sh,509 10.03 2a CH2Cl2(298) 485sh,512 11.13 0.44 CH3CN(298) 485sh,514 5.11 0.14 glass(77)a 470(max),503,542sh 84.59(49%),16.79(51%) 2b CH2Cl2(298) 488sh,510 14.41 0.52 CH3CN(298) 482sh,513 7.59 0.39 buffer(298)b 485sh,514 6.28 0.037 glass(77)a 468(max),500,537sh 103.10(56%),21.21(44%) 2c CH2Cl2(298) 489sh,513 14.67 0.45 CH3CN(298) 484sh,515 13.50 0.20 glass(77)a 470(max),501,540sh 111.69(43%),20.84(57%) 3a CH2Cl2(298) 542 8.75 0.29 CH3CN(298) 553 3.77 0.19 glass(77)a 510,536sh 21.28 3b CH2Cl2(298) 543 8.45 0.46 CH3CN(298) 556 2.60 0.14 buffer(298)b 560 2.41 0.010 glass(77)a 507,546sh 21.93 3c CH2Cl2(298) 547 7.64 0.42 CH3CN(298) 558 3.85 0.24 glass(77)a 509,543sh 21.52 aInbutyronitrileglass.b50mMpotassiumphosphateatpH7.4containing5%DMSO.Forquantumyieldmeasurements,50mMpotassiumphosphate atpH7.4containing30%DMSOwasused. Table3. RelativeEmissionIntensitiesandLifetimesoftheThiourea Table4. PhotophysicalDataoftheBioconjugatesBSA-1b-BSA-3bin Complexes1b-3binAeratedBuffer/DMSO(97:3,v/v)at298K Degassed50mMPhosphateBufferatpH7.4at298K complex I((cid:244)/(cid:237)s)a I((cid:244)/(cid:237)s)b I((cid:244)/(cid:237)s)c conjugates (cid:236)em/nm (cid:244)o/(cid:237)s … 1b 1.00(0.44) 1.42(0.77) 1.08(0.44) BSA-1b 543 0.89(9%),0.10(91%) 0.011 2b 1.00(1.14) 1.46(1.96) 1.03(1.11) BSA-2b 488sh,518 7.15(39%),1.02(61%) 0.024 3b 1.00(0.90) 1.51(1.96) 1.11(0.87) BSA-3b 552 2.91(32%),0.25(68%) 0.010 a[avidin])0(cid:237)M,[biotin])0(cid:237)M.b[avidin])3.8(cid:237)M,[biotin])0 (cid:237)M.c[avidin])3.8(cid:237)M,[biotin])380.0(cid:237)M. itywiththecorrespondingthioureacomplexes1b-3b,were purifiedbysizeexclusionchromatographyandultrafiltration. Control experiments using the biotin-free isothiocyanate complex[Re(Me -phen)(CO) (py-NCS)](PF )(2d)7aandthe 4 3 6 isothiocyanate-freebiotincomplex[Re(Me -phen)(CO) (py- 4 3 biotin)](PF )(2e)8fwerealsoperformed.WhereasBSAwas 6 successfully labeled with complex 2d, no luminescent bioconjugate was produced when complex 2e was used, confirming that the bioconjugation originates from the reactionoftheisothiocyanatemoietyofthecomplexeswith BSA.ThebioconjugatesBSA-1b-BSA-3bdisplayedintense andlong-livedorange-yellowtogreenish-yellow3MLCT/3- ILemissionuponirradiationinaqueousbufferunderambient conditions. The photophysical data are listed in Table 4. Figure2. Luminescencetitrationcurvesforthetitrationsof(i)3.8(cid:237)M avidin (b), (ii) 3.8 (cid:237)M avidin and 380 (cid:237)M biotin (2), and (iii) a blank Additionally,theelectronicabsorptionandemissionspectra solution(0)withcomplex2b. ofthebioconjugateBSA-1binphosphatebufferareshown inFigure3.AllthebioconjugatesBSA-1b-BSA-3bshowed be the consequence of the steric demands of the Me -phen 4 biexponential emission decay, which is not uncommon for and Ph -phen ligands of the latter complexes. 2 biomolecules labeled with luminescent transition metal Biotinylation of BSA with the Isothiocyanate Com- plexes 1a-3a. To evaluate the biotinylation properties of complexes.7,12a,b,28a,32 The average lifetimes are longer than theisothiocyanatecomplexes1a-3a,wehaveusedthemto (32) (a)Lo,K.K.-W.;Li,C.-K.;Lau,K.-W.;Zhu,N.DaltonTrans.2003, label a model protein, BSA. The resultant bioconjugates, 4682-4689.(b)Lo,K.K.-W.;Chan,J.S.-W.;Chung,C.-K.;Tsang, denotedbyBSA-1b-BSA-3bduetotheirstructuralsimilar- V.W.-H.;Zhu,N.Inorg.Chim.Acta2004,357,3109-3118. Inorganic Chemistry, Vol. 47, No. 2, 2008 609 Lo et al. receivingincreasingattention.33Tounderstandthepotential cytotoxicity of biomolecules biotinylated with the isothio- cyanatecomplexes,theMTTassaywasemployedtoexamine the cytotoxicity of the thiourea complexes 1b-3b, which can be considered as models for biomolecules biotinylated bycomplexes1a-3a,towardthecervicalepithelioidcarci- nomacellline(HeLa).34TheIC valuesofcomplexes1b- 50 3b have been determined to be 22.7, 17.5, and 28.5 (cid:237)M, respectively. This is comparable to that of cisplatin (26.7 (cid:237)M) and indicates potential anticancer properties. The cytotoxicityofcomplexes1b-3bislowerthantherhenium- (I)diphosphinecomplexes[Re(CO) (diphosphine)Br]33abut 3 Figure3. Electronicabsorption(s)andemission(---)spectraofthe slightlyhigherthantherelatedrhenium(I)diiminecomplex bioconjugateBSA-1bin50mMphosphatebufferatpH7.4at298K. [Re(CO) (2-appt)Cl] (2-appt ) 2-amino-4-phenylamino-6- 3 (2-pyridyl)-1,3,5-triazine) (IC ) ca. 50 (cid:237)M), which has 50 those of the thiourea complexes 1b-3b under the same beenidentifiedasaminor-groovebindertodouble-stranded experimentalconditions,asaresultofthemorehydrophobic DNA.33b local environment associated with the protein mole- CellularUptakeStudies.Thecellularuptakeofrhenium- cules.7,8,12a,b,28a,b,d,e,32Fromacorrelationoftheluminescence (I)complexeshasattractedmuchattentionrecentlybecause intensitiesofthethioureacomplexestotheirconcentrations, of their potential diagnostic and therapeutic applications.35 the biotin/BSA ratios of the bioconjugates BSA-1b-BSA- As noted in the Introduction, one of the reasons for the 3b have been determined to be ca. 1.9, 2.5, and 1.8, development of luminescent biotinylation reagents is that respectively.Sincefluorometricmethodsofferhigherdetec- theycanbeusedtobiotinylatesmallmoleculesandbiological tionsensitivityandlowerlimitsofdetectioncomparedwith uptakeofthelabeledcompoundsmaythenbeexaminedby luminescencespectroscopyandmicroscopy.Thus,wehave absorption methods, the current luminescent biotinylation studiedthecellularinternalizationpropertiesofthethiourea reagents are particularly useful for the detection and quan- complex3b,whichactsasamodelforbiomoleculeslabeled titation of biotinylated molecules. Under the experimental bycomplex3a,anditspossibleuseasabiologicalimaging conditionsemployed,thelimitsofdetectionforthethiourea reagent. Incubation of HeLa cells with the complex at 37 complexes1b-3bwereca.90,10,and120nM,respectively. (cid:176) Cundera5%CO atmospherefor24hresultedincellular 2 These concentrations are comparable with the fluorometric uptake. Upon visible-light irradiation, the cytoplasm of the displacement assay (40-800 nM)6a and about 1-2 orders cellsexhibitedorangeluminescence(Figure4).Interestingly, of magnitude lower than the micromolar range of both the their nuclei displayed much weaker emission, indicative of HABA assay5 and the absorption methods, which use the negligiblenuclearuptakeofthecomplex.Thecomplexwas two chromogenic biotinylation reagents.6 not homogeneously distributed within the cytoplasm but localized in the perinuclear region (Figure 4). From the Theavidin-bindingpropertiesofthebioconjugatesBSA- 1b-BSA-3b have been studied by the HABA assay. Upon images, it appears that the complex binds to the Golgi apparatus,36a,balthoughitmayalsobindtootherhydrophobic addition of the biotinylated proteins to a solution of avidin organellessuchasendoplasmicreticulumandmitochondria.36c and HABA, only a small change in the absorbance at 500 The internalization of the complex appears to occur via nm was observed. This indicated that the avidin-bound energy-requiring processes such as endocytosis since there HABAmoleculeswerenotdisplacedbythebiotinmoieties was no evidence of uptake following incubation at 4 (cid:176) C. of the bioconjugates. It is likely that the biotin groups on When the cells were loaded with the cytoskeletal inhibitor the bioconjugates are sterically restricted by the protein nocodazole,thecomplexwasmoreevenlydistributedinthe matrix and inaccessible to the biotin-binding sites of the avidin molecules. Thus, we used the protease pronase to (33) (a)Zhang,J.;Vittal,J.J.;Henderson,W.;Wheaton,J.R.;Hall,I.H.; digestthebioconjugates(byhydrolysisofthepeptidebonds) Hor,T.S.A.;Yan,Y.-K.J.Organomet.Chem.2002,650,123-132. (b)Ma,D.-L.;Che,C.-M.;Siu,F.-M.;Yang,M.;Wong,K.-Y.Inorg. prior for analysis by the HABA assay. Addition of the Chem.2007,46,740-749. digestionmixturestoanavidin-HABAsolutionresultedin (34) Mosmann,T.J.Immunol.Methods1983,65,55-63. (35) (a)Stephenson,K.A.;Banerjee,S.R.;Besanger,T.;Sogbein,O.O.; adecreaseofabsorbanceat500nm,indicatingthatthebiotin Levadala, M. K.; McFarlane, N.; Lemon, J. A.; Boreham, D. R.; groups of the bioconjugates bound to avidin. Interestingly, Maresca,K.P.;Brennan,J.D.;Babich,J.W.;Zubieta,J.;Valliant,J. F. J. Am. Chem. Soc. 2004, 126, 8598-8599. (b) Amoroso, A. J.; the biotin/BSA ratios were determined to be 2.2, 2.4, and Coogan, M. P.; Dunne, J. E.; Ferna´ndez-Moreira, V.; Hess, J. B.; 1.4 for BSA-1b-BSA-3b, respectively, which are in good Hayes,A.J.;Lloyd,D.;Millet,C.;Pope,S.J.A.;Williams,C.Chem. Commun.2007,3066-3068. agreementwiththeresultsfromtheemissionmeasurements (36) (a)Kobayashi,T.;Arakawa,Y.J.CellBiol.1991,113,235-244.(b) (ca. 1.9, 2.5, and 1.8, respectively). Pagano,R.E.;Martin,O.C.;Kang,H.-C.;Haugland,R.P.J.Cell Biol.1991,113,1267-1279.(c)Haugland,R.P.TheHandbooksA Cytotoxicity Assays. Although relatively unexplored, Guide to Fluorescent Probes and Labeling Technologies, 10th ed.; Molecular Probes, Inc.: Eugene, OR, 2005; Section 12. See http:// cytotoxicitystudiesoftricarbonylrhenium(I)complexesare probes.invitrogen.com/handbook/sections/1200.html. 610 InorganicChemistry,Vol.47,No.2,2008 Rhenium(I)PolypyridineBiotinIsothiocyanateComplexes Figure4. Bright-field(left),overlaid(middle),andfluorescence(right)microscopyimagesofHeLacellsincubatedwithcomplex3b(10(cid:237)M)at37(cid:176) Cfor 24h. Figure5. Bright-field(left),overlaid(middle),andfluorescence(right)microscopyofHeLacellsincubatedwithnocodazole(30(cid:237)M)at37(cid:176) Cfor90min followedbycomplex3b(10(cid:237)M)atthesametemperaturefor24h. perinuclearregion(Figure5),highlightingtheimportantrole fluorescence images that the complex was localized in the of cytoskeleton in the intracellular transportation of the perinuclear region, as a result of possible interactions with complex. hydrophobic organelles such as the Golgi apparatus. These important results reveal that the isothiocyanate complexes Conclusion not only serve as novel biotinylation reagents but may also We have designed a series of rhenium(I) biotin isothio- contribute to the development of luminescent tracers for cyanatecomplexesasthefirstclassofluminescentbiotiny- specific intracellular delivery of biomacromolecules and lation reagents. The photophysical properties of these small molecular substrates including potential anticancer complexes and their amine and thiourea counterparts have drugs. been studied. These isothiocyanate complexes provide the biotinylated biological molecules with rich luminescence Acknowledgment. We thank the Hong Kong Research properties allowing direct determination of the degree of GrantsCouncil(ProjectNos.: CityU101606and8730025) biotinylationbysensitivespectrofluorometricmethods.The forfinancialsupport.K.-S.S.andJ.S.-Y.L.acknowledgethe cytotoxicity of the thiourea complexes toward HeLa cells receipt of a Postgraduate Studentship administered by the has been examined, and the IC values are comparable to 50 City University of Hong Kong. We thank Dr. Shuk-Han that of the anticancer drug cisplatin. The cellular uptake of ChengforherhelpfuldiscussionsandDr.CharlesHarford- the thiourea complex 3b has also been examined. Interest- Cross for reading this manuscript. ingly,theinternalizedcomplexmaintainsitsintenseemission in the cytoplasm of the cell. It can be seen from the IC701675C Inorganic Chemistry, Vol. 47, No. 2, 2008 611