Luminescent rhenium(I) polypyridine complexes appended with an α-D-glucose moiety as novel biomolecular and cellular probes.

DOI:10.1002/chem.201101399 Luminescent Rhenium(I) Polypyridine Complexes Appended with d an a- -Glucose Moiety as Novel Biomolecular and Cellular Probes Man-Wai Louie, Hua-Wei Liu, Marco Ho-Chuen Lam, Yun-Wah Lam, and Kenneth Kam-Wing Lo*[a] Glucose is the most impor- tant carbohydrate in cellular metabolism and an energy source for the growth of cells.[1] One of the most characteristic phenotypes of rapidly growing cancer cells is their propensity to catabolize glucose at high rates, possibly due to the over- expression of glucose transport- ers (GLUTs).[2] Thus, the in vitro and in vivo monitoring of glucose utilization in cancer cells has attracted much atten- tion. Different reporting and therapeutic units such as radio- active labels,[3a] IRDye 800CW,[3b] organic fluorophor- es,[3c,d] and two-photon dyes[3e] have been conjugated to glu- Scheme1.Structuresoftherhenium(I)polypyridinecomplexes. cose or 2-deoxyglucose for the diagnosis andtreatment ofvari- ous tumors or cancers. Despite the development of these reagents, the possibility of using 4,7-diphenyl-1,10-phenanthroline (Ph-phen) (3)) and their 2 luminescent transition-metal glucose conjugates as glucose- glucose-free counterparts [ReACHTUNGTRENNUNG(N^N)(CO)ACHTUNGTRENNUNG(py-3-Et)]- 3 uptake tracers and cancer cell imaging reagents has not ACHTUNGTRENNUNG(CFSO) (py-3-Et=3-(ethylthioureidyl)pyridine, N^N= 3 3 been explored.[4] With our ongoing interest in luminescent phen (1a), Me-phen (2a), Ph-phen (3a)) (Scheme1). The 4 2 rhenium(I) polypyridine complexes as biological probes,[5] glucose complexes were synthesized from the addition reac- weenvisagethatmodificationofthesecomplexeswithana- tion of the isothiocyanate complexes [ReACHTUNGTRENNUNG(N^N)(CO)ACHTUNGTRENNUNG(py-3- 3 d-glucose pendant will generate useful luminescent probes NCS)]ACHTUNGTRENNUNG(PF)[5a]withAcO-glu-C-NH inacetone,followedby 6 6 2 forbiomoleculesandcancercells. deacetylation (see the Supporting Information, Schemes S1 Herein we report three rhenium(I) polypyridine glucose and S2). All the complexes were characterized by 1HNMR complexes [ReACHTUNGTRENNUNG(N^N)(CO)ACHTUNGTRENNUNG(py-3-glu)]ACHTUNGTRENNUNG(PF) (py-3-glu=3- spectroscopy, positive-ion ESI-MS, and IR spectroscopy and 3 6 (N-(6-(N’-(4-(a-d-glucopyranosyl)phenyl)thioureidyl)hex- gave satisfactory elemental analyses (see the Supporting In- yl)thioureidyl)pyridine, N^N=1,10-phenanthroline (phen) formation). Upon irradiation, the complexes exhibited in- (1), 3,4,7,8-tetramethyl-1,10-phenanthroline (Me-phen) (2), tense and long-lived green-to-yellow triplet metal-to-ligand 4 charge-transfer (MLCT; dp(Re)!p*ACHTUNGTRENNUNG(N^N)) emission (see the Supporting Information, Table S1).[6] The structured [a] M.-W.Louie,Dr.H.-W.Liu,M.H.-C.Lam,Dr.Y.-W.Lam, Dr.K.K.-W.Lo emission band and very long lifetime of complex 2 in alco- DepartmentofBiologyandChemistry holglassat77Kareprobablyduetotheinvolvementof3IL CityUniversityofHongKong,TatCheeAvenue (p!p*)(Me-phen)characterinitsemissivestate.[7] 4 Kowloon,HongKong(P.R.China) Since the lectin concanavalinA (ConA) binds a-d-man- Fax:(+852)3442-0522 nopyranosideanda-d-glucopyranoside,[8]thepossibleuseof E-mail:bhkenlo@cityu.edu.hk the glucose complexes 1–3 as a luminescent sensor for this SupportinginformationforthisarticleisavailableontheWWW underhttp://dx.doi.org/10.1002/chem.201101399. lectin has been investigated.[9] Upon addition of ConA to a 8304 (cid:2)2011Wiley-VCHVerlagGmbH&Co.KGaA,Weinheim Chem.Eur.J.2011,17,8304–8308 COMMUNICATION Table1. Results of emission titrations of complexes 1–3 with ConA in Table2. Lipophilicity,cellularuptake,andIC valuesofcomplexes1–3, 50 potassium phosphate buffer (10mm) at pH8.5/methanol (9:1, v/v) with 1a–3a,andcisplatin. CaCl (0.1mm)andMnCl (0.1mm)at298K. 2 2 Complex LogP Amountof IC o/w 50 Complex I/I[a] t t K n [c] complex[fmol][a] [mm][b] o o a H [ms][b] [ms][b] ACHTUNGTRENNUNG[m(cid:3)1][c] 1 0.57 1.10(cid:4)0.09 >150 1 1.8 0.39 1.15(19%), 4.5(cid:3)105 2.0 2 2.73 2.15(cid:4)0.03 90.0(cid:4)7.6 0.22(81%) 3 3.44 3.02(cid:4)0.10 68.9(cid:4)2.3 2 2.0 0.64 4.32(44%), 5.8(cid:3)105 2.3 1a 0.77 1.64(cid:4)0.13 22.8(cid:4)5.2 0.93(56%) 2a 2.94 4.73(cid:4)0.22 7.7(cid:4)0.6 3 3.2 0.40 2.04(33%), 4.4(cid:3)105 2.5 3a 4.03 6.61(cid:4)0.33 2.8(cid:4)0.4 0.40(67%) cisplatin (cid:3)2.30[c] N.A. 27.6(cid:4)1.8 [a]I andIaretheemissionintensitiesofthecomplex(5mm)inthepres- [a]AmountsofrheniumassociatedwithanaverageHeLacelluponincu- o ence of 0 and 5.5mm ConA, respectively. [b]t and t are the emission bationwiththecomplexes(100mm)inaglucose-freemediumat378Cfor o lifetimes of the complex (5mm) in the presence of 0 and 5.5mm ConA, 5min as determined by ICP-MS. [b]HeLa cells, incubation in high glu- respectively.[c]BindingconstantsandHillcoefficientsasdeterminedby cose Dulbecco’s modified Eagle’s medium (DMEM) for 48h. [c]See theHillequation. ref.[24]. buffer solution of the complexes, the emission intensities lowedtheorders:1<2<3and1a<2a<3a(Table2),which were enhanced by (cid:2)1.8 to 3.2 fold (Table1) and the emis- areinaccordancewiththehydrophobic characterofthedii- sionmaximawereblueshiftedby(cid:2)5to15nm.Theemission mine ligands (phen<Me-phen<Ph-phen). Interestingly, 4 2 decay became biexponential with components of 0.22 to the glucose moiety reduced the logP values of complexes o/w 0.93ms and 1.15 to 4.32ms (Table1). The emission titration 1–3 by (cid:2)0.20 to 0.59 units with respect to the glucose-free curvesforcomplexes3and3awithConAareshowninFig- analogues 1a–3a. The somewhat high lipophilicity of com- ureS1 (see the Supporting Information). Since the glucose- plexes 1–3 despite the polar glucose unit is probably a con- free complexes 1a–3a did not give similar observations, it is sequenceofthehydrophobicC6spacer-arm.Wehaveinves- likely that the changes exhibited by the glucose complexes tigated the cellular uptake properties of the complexes originatedfrom the increasedhydrophobicity and rigidity of ([Re]=100mm with HeLa cells at 378C for 5min) and their the local environment of the complexes after binding to cytotoxicity by ICP-MS and MTT assays, respectively. The ConA. The binding has been analyzed by the Hill equation amountsofrheniumtakenupbyanaverageHeLacellwere and the binding constants (K =4.4(cid:3)105 to 5.8(cid:3)105m(cid:3)1, inthefemtomolescale(Table2),whichiscomparabletothe a Table1) are comparable to that of a ruthenium(II) glucose results from other cellular uptake studies.[13] The intracellu- complex L-[Ru(a-Glc-3-bpy)]Cl (K =9.5(cid:3)105m(cid:3)1)[8b] but lar amounts in both series of rhenium complexes followed 3 2 a one order of magnitude larger than that of p-nitrophenyl-a- the orders: 1<2<3 and 1a<2a<3a, which are in accord- d-glucopyranoside (K =1(cid:3)104m(cid:3)1).[10] The higher binding ancewiththeirlipophilicityandcytotoxicity(Table2).Since a affinity has been attributed to the relatively hydrophobic the glucose complexes 1–3 showed lower cytotoxicity com- rhenium(I)polypyridineunits. pared to their glucose-free counterparts complexes 1a–3a The FimH adhesin of E. coli type 1 pili is able to bind d- (Table2), the incorporation of a glucose unit renders the mannosidesandd-glucosidesbyvirtue ofareceptor-binding complexesmorebiocompatible. domain.[11] Two E. coli strains ORN178 and ORN208 have To study the possible role of GLUTs on the internaliza- been used in this work to study the possible binding of the tionoftheglucosecomplexes,thecellularuptakeproperties glucose complexes to FimH. The ORN178 strain expresses of complexes 3 and 3a towards two transformed cell lines, wild-type type 1 pili that exhibit monosaccharide-binding HeLa andhuman breast adenocarcinoma (MCF-7), andtwo properties, whereas the ORN208 strain is deficient of the non-transformed cell lines, human embryonic kidney cells FimH gene and expresses abnormal type 1 pili that do not (HEK293T) and mouse embryonic fibroblast (NIH/3T3), showsimilarbehavior.[12]BothE.colistrainswereincubated have been studied. In general, the intracellular amounts of with the glucose complex 3 for 3h and then imaged with theglucosecomplex3werelowerthanthoseoftheglucose- laser-scanning confocal microscopy. The emission intensity free complex 3a among the cell lines studied (Table3), of the ORN178 strain was 7.7 times that of ORN208 (n= which is attributable to the lower lipophilicity of the former 30)(SeetheSupportingInformation,FigureS2).Thisobser- complex. Interestingly, the uptake of complex 3 by HeLa vationhasbeenascribedtothebindingoftheglucosepend- Table3. CellularuptakeandIC valuesofcomplexes3and3atowards ant of the complex to the lectin expressed on the ORN178 50 differentcelllines. strain. Both strains were also stained by the glucose-free Cellline Amount[fmol][a] IC [mm][b] complex 3abuttheemission intensitieswereveryweakand 50 Complex3 Complex3a Complex3 Complex3a indistinguishable (See the Supporting Information, Fig- HeLa 2.92(cid:4)0.36 6.29(cid:4)0.06 62.9(cid:4)1.7 3.20(cid:4)0.70 ureS2), which further supports the specific binding of the MCF-7 2.33(cid:4)0.37 4.30(cid:4)0.31 20.3(cid:4)2.0 2.94(cid:4)0.14 glucose moiety of complex 3 to FimH on the ORN178 HEK293T 0.86(cid:4)0.04 3.73(cid:4)0.68 40.9(cid:4)1.6 4.94(cid:4)0.54 strain. NIH/3T3 0.33(cid:4)0.03 4.43(cid:4)0.08 >150 2.80(cid:4)0.56 The lipophilicity (logP ) of all the complexes has been [a][Re]=100mm, incubation in glucose-free DMEM at 378C for 5min. o/w measured by reversed-phase HPLC. The logP values fol- [b]IncubationinhighglucoseDMEMfor48h. o/w Chem.Eur.J.2011,17,8304–8308 (cid:2)2011Wiley-VCHVerlagGmbH&Co.KGaA,Weinheim www.chemeurj.org 8305 K.K.-W.Loetal. andMCF-7cellswasatleast2.7timesthatofHEK293Tand NIH/3T3cells.Thisispossiblyaresultoftheoverexpression of GLUTs in the transformed cell lines (HeLa and MCF-7) rather than the non-transformed cell lines (HEK293T and NIH/3T3).[2]Inthecaseofcomplex3a,thedifferenceincel- lular uptake efficiency between the transformed and non- transformed cell lines was much smaller (Table3) and the intracellular amount of rhenium in HeLa cells was slightly higherthanthoseintheotherthreecelllines.Also,thecyto- toxicity of complexes 3 and 3a did not show any depend- enceonthecelltypes(Table3). Glucose derivatives entering the cells through a GLUT- mediated glucose-uptake pathway are competitively inhibit- ed by d-glucose but not by l-glucose.[3c–e] The possible in- volvement of GLUTs in the cellular uptake of complex 3 has been studied. HeLa cells were incubated with this com- plex (100mm) for 5min in the absence or presence of 5 to 50mm d-glucose or l-glucose in a glucose-free medium and the intracellular rhenium associated with an average HeLa cell has been determined. The addition of d-glucose to the mediumledtoadecreaseofcellularuptakeofthecomplex, but l-glucose gave no effects (Figure1, top and middle), which supports the argument that the internalization of this complex occurred by a GLUT-mediated uptake pathway. This is also in accordance with the finding that the cellular uptake of complex 3 by HeLa cells decreased with increas- ing concentration of 2-deoxyglucose in the medium (Figure1, bottom) since 2-deoxyglucose is transported into the cells in a similar manner to d-glucose.[3a] On the contra- ry, the cellular uptake of the glucose-free complex 3a did notshowsignificantchangesinthepresenceofd-glucose,l- glucose, or 2-deoxyglucose (Figure1). All these results re- vealed that the glucose-dependent cellular uptake of com- plex 3 originated from the specific recognition of the d-glu- cose moiety of the complex by the cells. The possible in- volvement of GLUTs in the cellular uptake properties of complex 3 has been further examined. Since the expression of GLUTs in HeLa cells can be upregulated by addition of Figure1.Relativecellularuptakeofrheniumassociatedwithanaverage HeLa cell upon incubation with complexes 3 (shaded) and 3a (empty) hypoxia-mimetic agents such as cobalt(II) chloride,[14] we (100mm)at378Cfor5mininaglucose-freemediumcontainingvarious have incubated HeLa cells with this salt (250mm) in the concentrations of d-glucose (top), l-glucose (middle), and 2-deoxyglu- growth medium for 2h prior to treatment with complex 3 cose(bottom)(n=3). (100mm) for 5min. The results showed that the cellular uptake was (cid:2)1.5 fold higher than that of the control in which cobalt(II) chloride was absent. Additionally, incuba- theglucose-freecomplex 3arevealed adecreaseofintracel- tion of HeLa cells with two glucose-uptake inhibitors fasen- lularrheniumby(cid:2)1.6 fold,mostlikelyduetotheinhibition tin(80mm)andcytochalasinB(10mm)[15]for1hreducedthe of endocytic pathways by sodium azide through cellular intracellular amounts of rhenium by (cid:2)1.4 and 1.7 fold, re- energy depletion.[16] In sharp contrast, HeLa cells treated spectively. In contrast, complex 3a did not show significant with sodium azide before incubation with the glucose com- changes in uptake efficiency upon addition of cobalt(II) plex 3 led to an increase of the intracellular rhenium by chloride, fasentin, or cytochalasin B. These results support a (cid:2)2.5 fold. Since exposure of cells to azide is well known to GLUT-mediatedtransportpathwayfortheglucosecomplex. cause an immediate inhibition of oxidative phosphorylation Incubation of HeLa cells with complexes 3 and 3a in a andadeclineincellATPcontent resultinginarapid stimu- glucose-free medium at 48C resulted in reduction of the in- lation of glucose transport,[14] the increased uptake strongly tracellular rhenium by (cid:2)50%. Thus, the complexes appa- suggests that the internalization of complex 3 occurred rently entered the cells by an energy-dependent endocyto- throughaglucose-specifictransportsystem. sis-like pathway.[16] Additionally, treatment of the cells with Taxolisagenotoxinandamitoticinhibitorthatisusedto sodium azide (3mm) for 30min at 378C before addition of treat several types of cancers including ovarian, breast, and 8306 www.chemeurj.org (cid:2)2011Wiley-VCHVerlagGmbH&Co.KGaA,Weinheim Chem.Eur.J.2011,17,8304–8308 Rhenium(I)PolypyridineComplexesAppendedwithana-d-Glucose COMMUNICATION non-small cell lung cancers.[17] Since genotoxic reagents are known to lower the cellular metabolic rate and reduce the glucose uptake in cancer cells,[18] the effects of taxol on the cellular uptakeof complexes 3 and 3a have been investigat- ed. HeLa cells were incubated with taxol at various concen- trations in the growth medium for 5h, followed by treat- ment with the glucose complex 3 or the glucose-free com- plex 3a (100mm) for 5min before the determination of cel- lular uptake efficiency (Figure2). By using (cid:5)125nm of F in i c g u u b re at 3 io . n La s s u e c r c - e sc s a si n v n e i l n y g w c i o th nf M oc i a to l T m r i a c c r k o e s r co D p e y e i p m R ag e e d s F o M fa ( H 10 e 0 L n a m c , e 2 ll 0 u m po in n , taxol, the intracellular amount of complex 3 in an average l =633nm) and complex 3 (100mm, 5min, l =405nm) in a glucose- ex ex HeLa cell was reduced to (cid:2)50% of that of the control (in freemediumat378C. pendant of complex 3 plays a role in the accumulation of the complex in mitochondria. Although a possible explana- tion is the binding of the glucose complex to hexokinases (the major proteins that phosphorylate glucose),[19] which strongly associate with mammalian mitochondria,[20] we did not observe phosphorylation of complex 3 in both in vitro and in vivo experiments involving the isolated enzyme and E. coli.[21] This is reasonable given the extremely rigid struc- ture requirement of the enzyme for its substrates.[22] Never- theless, other rhenium(I) polypyridine complexes have been reported to show similar mitochondria-targeting proper- ties.[23] Figure2.Relativecellularuptakeofrheniumassociatedwithanaverage HeLa cell upon incubation with 100mm of complexes 3 (solid squares) Finally, we have examined the photostability of complex and3a(emptycircles)at378Cfor5minafterexposuretotaxolatvari- 3. The 2-deoxyglucose analogue 2-(N-(7-nitrobenz-2-oxa- ousconcentrationsfor5h(n=3). 1,3-diazol-4-yl)-amino)-2-deoxy-d-glucose (2-NBDG) is a fluorescent indicator for direct glucose-uptake measure- ments and has been applied in tumor imaging and the ex- whichtaxolwasabsent).However,thepresence oftaxoldid amination of GLUT-related cell metabolism.[3c] However, not cause a similar effect to the glucose-free complex 3a thefastphotobleachingrateofthisorganiccompoundlimits (Figure2).Thus,theincorporationofaglucoseunitintothe its applications in prolonged exposure to the light source or complex rendersits uptake to beregulatedby an anticancer time-lapse imaging experiments. We have compared the reagent. Since the cellular uptake of the luminescent com- photostability of the glucose complex 3 and 2-NBDG. Con- plex can be readily assessed by fluorescence spectroscopy focal microscopy images and irradiation time-dependence and microscopy (see below), these findings could form the emission intensity of HeLa cells treated with complex 3 basisofnewcell-viabilityassays. (100mm, 5min, l =405nm, 25mW) or 2-NBDG (100mm, ex Theintracellularlocalizationof complex 3 upon internali- 5min, l =488nm, 15mW) are illustrated in FigureS4 (see ex zationbyHeLacellshasbeeninvestigatedbylaser-scanning the Supporting Information) and Figure4, respectively. The confocal microscopy. The complex was diffusely distributed emissionintensityof2-NBDGdecreasedmuchmorerapidly in the cytoplasm with punctate staining (see the Supporting comparedto that of the glucose complex uponlaser irradia- Information, FigureS3). The nucleus gave much weaker or no emission, indicative of negligible nuclear uptake. Similar intracellular distribution has been observed for other lumi- nescent rhenium(I) polypyridine complexes.[5c,d] In addition to the perinuclear region, the complex was concentrated in specificcompartmentsofthecells,whichappearedtobethe mitochondria.Thus,HeLacellspretreatedwithMitoTracker Deep Red FM (100nm, 20min, l =633nm), whose spec- ex tral properties do not interfere, were incubated with com- plex 3 (100mm, 5min, l =405nm). The fluorescence stain- ex ing pattern showed that the mitochondria of a typical HeLa cell have been co-stained by the fluorescent dye and the rhenium(I)complex(Figure3).Theintracellularlocalization Figure4.Irradiation time-dependence of the emission intensity of HeLa of the complex 3 is different to that of its glucose-free com- cells upon incubation with complex 3 (solid squares) and 2-NBDG plex 3a, which did not show granular appearance, but a dif- (emptycircles)underexposureto405nm(25mW)and488nm(15mW) fused staining of the whole cytoplasm.[5c] Thus, the glucose laserexcitation,respectively. Chem.Eur.J.2011,17,8304–8308 (cid:2)2011Wiley-VCHVerlagGmbH&Co.KGaA,Weinheim www.chemeurj.org 8307 K.K.-W.Loetal. tion (Figure4). After continuous exposure to 488nm irradi- ChemBioChem 2010, 11, 649–652; b)H.-W. Liu, K.Y. Zhang, ation for 150s, theemission intensity of 2-NBDG decreased W.H.-T.Law,K.K.-W.Lo,Organometallics2010,29,3474–3476. [5] a)K.K.-W. Lo, D.C.-M. Ng, W.-K. Hui, K.-K. Cheung, J. Chem. to only (cid:2)10% of its initial value, whereas the emission in- Soc. Dalton Trans. 2001, 2634–2640; b)K.K.-W. Lo, M.-W. Louie, tensity of the glucose complex was maintained at (cid:2)70% of K.-S. Sze, J.S.-Y Lau, Inorg. Chem. 2008, 47, 602–611; c)M.-W. its initial value after irradiation at a shorter wavelength Louie,H.-W.Liu,M.H.-C.Lam,T.-C.Lau,K.K.-W.Lo,Organome- (405nm) for the same period (Figure4). This much higher tallics 2009, 28, 4297–4307; d)K.K.-W. Lo, M.-W. Louie, K.Y. photostability renders this class of luminescent rhenium(I) Zhang,Coord.Chem.Rev.2010,254,2603–2622. [6] A.Kumar,S.-S.Sun,A.J.Lees,Top.Organomet.Chem.2010,29,1– glucose complexes excellent candidates for time-lapse cellu- 35. larimagingapplications. [7] I.R. Farrell, F. Hartl, S. Z(cid:5)liSˇ, T. Mahabiersing, A. Vlcˇek,Jr., J. In summary, we have designed three luminescent rhe- Chem.Soc.DaltonTrans.2000,4323–4331. nium(I) polypyridine glucose complexes and investigated [8] a)I.J. 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