Novel Near-Infrared Fluorescent Integrin-Targeted DFO Analogue

Novel Near-Infrared Fluorescent Integrin-Targeted DFO Analogue

Yunpeng Ye, Sharon Bloch, Baogang Xu, and Samuel Achilefu*

Department of Radiology, Washington University, 4525 Scott Avenue, St. Louis, Missouri 63110. Received August 9, 2007

Desferrioxamine (DFO), a siderophore initially isolated from Streptomyces pilosus, possesses extraordinary metalbinding properties with wide biomedical applications that include chelation therapy, nuclear imaging, andantiproliferation. In this work, we prepared a novel multifunctional agent consisting of (i) a near-infrared (NIR)fluorescent probe-cypate; (ii) an integrin Rv�3 receptor (ABIR)-avid cyclic RGD peptide, and (iii) a DFO moiety,DFO-cypate-cyclo[RGDfK(∼)] (1, with ∼ representing the cypate conjugation site at the side chain of lysine; fis D-phenylalanine). Compound 1 and two control compounds, cypate-cyclo[RGDfK(∼)] (2) and cypate-DFO(3), were synthesized by modular assembly of the corresponding protected RGD peptide cyclo[R(Pbf)GD(OBut)fK]and DFO on the dicarboxylic acid-containing cypate scaffold in solution. The three compounds exhibited similarUV–vis and emission spectral properties. Metal binding analysis shows that DFO as well as 1 and 3 exhibitedrelatively high binding affinity with Fe(III), Al(III), and Ga(III). In contrast to Ga(III), the binding of Fe to 1 and3 quenched the fluorescence emission of cypate significantly, suggesting an efficient metal-mediated approach toperturb the spectral properties of NIR fluorescent carbocyanine probes. In vitro, 1 showed a high ABIR bindingaffinity (10-7 M) comparable to that of 2 and the reference peptide cyclo(RGDfV), indicating that both DFO andcypate motifs did not interfere significantly with the molecular recognition of the cyclic RGD motif with ABIR.Fluorescence microscopy showed that internalization of 1 and 2 in ABIR-positive A549 cells at 1 h postincubationwas higher than 3 and cypate alone, demonstrating that incorporating ABIR-targeting RGD motif could improvecellular internalization of DFO analogues. The ensemble of these findings demonstrate the use of multifunctionalNIR fluorescent ABIR-targeting DFO analogues to modulate the spectral properties of the NIR fluorescent probeby the chelating properties of DFO and visualize intracellular delivery of DFO by receptor-specific peptides.These features provide a strategy to explore the potential of 1 in tumor imaging and treatment as well as somemolecular recognition processes mediated by metal ions.

INTRODUCTION

Recent research efforts have demonstrated that molecularimaging is a powerful tool for localizing, identifying, andcharacterizing a host of disease-specific targets. Examplesinclude imaging molecular interactions, cell trafficking, tumorvitality, cell proliferation, apoptosis, angiogenesis, and responseto treatment. In particular, optical molecular imaging holds greatpromise to accelerate drug discovery and development, includingcompound screening, lead discovery, optimization, pharmaco-kinetic studies, and clinical trials (1–3).

An attractive feature of optical imaging is its ability to unravelthe molecular basis of cancers and other diseases, therebyproviding vital information for diagnosing, staging, and moni-toring therapeutic response (4–6). These applications emanatefrom numerous advantages of optical methods, including highdetection sensitivity, real-time monitoring, convenience, andaccessibility. In contrast to other established imaging methodssuch as radionuclear and X-ray-computed tomography, the useof low-energy (nonionizing) radiation for optical imagingprovides additional benefit, especially in longitudinal studies.Over the past few years, tremendous progress has been madein the development of optical contrast agents for in vivo tumoroptical imaging. For instance, we and others have developedNIR fluorescent probes such as cyanine dyes and their biocon-jugates, including somatostatin and RGD peptide analogues, asoptical contrast agents for in vitro and in vivo tumor opticalimaging (7–13). These results have inspired us to further applyoptical imaging and related optical contrast agents in the

discovery and development of novel, multifunctional, andtargeted oncologic drugs. For example, the biological propertiesof therapeutic agents such as desferrioxamine (DFO) can bealtered by linkage to a target-specific NIR fluorescent probeand the intracellular transport visualized by optical methods.

DFO, a siderophore initially isolated from Streptomycespilosus, is a selective iron chelator (14). This siderphore andsome analogues have been used in chelation therapy to treatiron-overload diseases, such as thalassemia, sickle-cell anemia,and hemochromatosis (15–17). DFO has also been shown topossess antiproliferative activities, and clinical studies havedemonstrated its potential as an adjunct to chemotherapyregimens (18–21). Its radiosensitizing activities have beenutilized in radiation oncology (22). DFO has also served as agood chelator for radioactive metals in cancer nuclear imagingas demonstrated by the use of radioactive 67Ga/68Ga-[DFO]-octreotide to image somatostatin receptor-positive tumors bypositron emission tomography (23). Despite its lack of oralbioavailability and difficulties in patient compliance, the con-tinued use of DFO in clinical settings strongly attests to its greatpotential in biomedical applications. It further suggests thepotential use of DFO as a lead compound for discovering anddeveloping novel DFO analogues and synthetic mimetics withimproved biological properties and activities (24–27). However,the biological activity of DFO is not targeted to a specificmolecular process and its hydrophilic nature limits its internal-ization in cells. For these reasons, conjugation of DFO with areceptor-specific ligand such as RV�3 integrin receptor (ABIR)-avid RGD peptide could overcome these problems.

Integrins are heterodimeric cell-surface receptors that mediatecell-cell and cell-matrix adhesion, metastasis, and angiogenesisprocesses (28–31). In particular, the overexpression of ABIR

* Corresponding author. Phone: 314-362-8599. Fax: [email protected].

Bioconjugate Chem. 2008, 19, 225–234 225

10.1021/bc7003022 CCC: $40.75 2008 American Chemical SocietyPublished on Web 11/27/2007

on activated endothelial cells in the neovasculature of tumorssuch as breast cancer, lung carcinomas, melanomas, andglioblastomas has been correlated with tumor growth, invasion,and metastasis. Therefore, ABIR is an attractive molecular targetfor tumor imaging, early diagnosis, and therapy (32–37). Diverseligands, including peptides and peptidomimetics have beenfound to interact with ABIR. Some of the high ABIR-bindingpeptides have been successfully used to elucidate the structureand mechanisms as well as targeted delivery of NIR fluorescentprobes, radionuclides, and anticancer agents (38–44).

In this study, we report the synthesis and in vitro evaluationof 1 and its two analogues, that is, cypate-[RGDfK(∼)] (2) andcypate-DFO (3) for targeting ABIR (Figure 1). The RGD peptidein the novel multifunctional NIR fluorescent RGD-bearing DFO(1) enhanced the cellular uptake of DFO. We also found thatthe metal-chelating properties of DFO can modulate the NIRfluorescence emission of cypate.

EXPERIMENTAL PROCEDURES

General. All reagents and solvents were purchased fromcommercial sources. Solvents and chemicals were reagent gradeand used without further purification. Cypate was prepared aspreviously reported (45). Analytical (Vydac C-18 column; 250× 4.6 mm) and semipreparative (Vydac C-18 column; 25 ×2.2 cm) HPLC were performed at a flow rate of 1.0 mL/minand 9.5 mL/min, respectively. HPLC solvents consisted ofsolvent A (water containing 0.05% trifluoroacetic acid (TFA))and solvent B (acetonitrile (ACN) containing 0.05% TFA).Linear gradient elution protocol for analytical HPLC startedfrom 90% A to 30% A in B for 20 min. The gradient forsemipreparative HPLC ranged from 90% A to 30% A over 15min and was held at 30% for 30 min. The elution profile wasmonitored by UV/vis at 214 and 254 nm. Mass spectra (ES-MS) were obtained using a Shimadzu mass spectrometer(LCMS-2010A) in the positive electrospray mode.

Solid Phase Peptide Synthesis. All the peptides wereassembled manually on resin using conventional Fmoc chemistry

(46). The coupling reactions were carried out by adding apreactivated solution of N-R-Fmoc-protected amino acid (3equiv), 1-hydroxybenzotriazole (HOBt, 3 equiv), 2-(1H-ben-zotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate(HBTU, 3 equiv), and N,N-diisopropylethylamine (DIEA, 6equiv) in anhydrous N,N-dimethylformamide (DMF, 3–5 mL/gresin) to the resin (1 equiv) and mixing for 2 h. The progressof the coupling was monitored by the Kaiser test. The Fmoc-protecting group was removed by treating the product with asolution of 20% piperidine in DMF for 10 min (2×). The resinwas washed with methanol (1 min, 3 mL, 2×) and DMF (1min, 3 mL, 6×) and used in subsequent reactions.

Cyclo[Asp(OBut)-phe-Lys-Arg(Pbf)-Gly]. The resin-boundprotected peptide (H-Asp(OBut)-phe-Lys-Arg(Pbf)-Gly resin)was assembled manually from H-Gly-2-chlorotrityl resin (1.0g, 0.54 mmol/g) as described above. The side-chain-protectedpeptide was cleaved by mixing the resin with 1% TFA indichloromethane (DCM) for 5 min. The filtrate was added to asolution of pyridine (5 mL)/methanol (10 mL). and the resinwas washed with methanol and dichloromethane. The cleavageprocedure was repeated 5 times. The combined filtrates wereconcentrated and washed with water to afford the desiredprotected RGD pentapeptide H-Asp(OBut)-phe-Lys-(Dde)-Arg-(Pbf)-Gly-OH; 561 mg, 95% yield; ES-MS: [MH]+/[MH]2+

1094.5/547.8).The linear peptide obtained was dissolved in DMF (10 mL)

and added dropwise into a solution of benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate (PyBOP, 520mg, 1.0 mmol), HOBT (135 mg, 1.0 mmol), and DIEA (207mg, 1.6 mmol) in DMF (80 mL)/DCM (720 mL). The mixturewas stirred overnight, concentrated, and washed with water togive the crude product cyclo[Asp(OBut)-phe-Lys(Dde)-Arg-(Pbf)-Gly] (ES-MS: [MH]+ 1076.3). The solid was dissolvedin ACN (47.5 mL)/water (2.5 mL), followed by the addition ofhydrazine (2.5 mL). The mixture was stirred for 15 min,concentrated, washed with water, and dried. The product,cyclo[Asp(OBut)-phe-Lys-Arg(Pbf)-Gly], was further purified

Figure 1. Structures of DFO-cypate-[RGDfK(∼)] (1) and the related compounds.

226 Bioconjugate Chem., Vol. 19, No. 1, 2008 Ye et al.

by column chromatography (silica gel, methanol/dichlo-romethane) to afford the desired compound (295 mg, 60% yield;ES-MS: [MH]+ 912.3).

Cypate-cyclo[phe-Lys(∼)-Arg(Pbf)-Gly-Asp(OBut)] andCypate-cyclo[phe-Lys(∼)-Arg-Gly-Asp] (2). The protectedcyclic peptide obtained above (50 mg, 0.05 mmol) was swirledwith a mixture of cypate (141 mg, 0.2 mmol), HOBT (13 mg,0.1 mmol), and N-(3-dimethylaminopropyl)-N′-ethylcarbodiim-ide hydrochloride (EDCI, 29 mg, 0.15 mmol) in DMF (2.0 mL)overnight. The mixture was concentrated to afford the crudeproduct (ES-MS: [MH2]2+ 759.8) and split into two portions,one of which was dissolved in a mixture of TFA/water (95:5)and deblocked for 4 h. The TFA mixture was added to coldmethyl tert-butyl ether (MTBE, 10 mL). Finally, the solidprecipitate was collected by filtration and purified by HPLC toafford the desired product cypate-cyclo[phe-Lys(∼)-Arg-GlyAsp] (9.0 mg, 13% yield; ES-MS: [MH]+/[MH2]2+ 1210.3/606.0).

DFO-cypate-cyclo[phe-Lys(∼)-Arg(Pbf)-Gly-Asp(OBut)]and DFO-cypate-cyclo[phe-Lys(∼)-Arg-Gly-Asp] (1). Amixture of DFO (106 mg, 0.15 mmol) and DIEA (19.4 mg,0.15 mmol) in DMSO (3 mL) was sonicated to obtain a clearsolution. The solution was added to cypate-cyclo[phe-Lys(∼)-Arg(Pbf)-Gly-Asp(OBut)] (80 mg, 0.05 mmol) in DMF (3 mL),followed by the addition of EDCI (29 mg, 0.15 mmol) andHOBT (21 mg, 0.15 mmol). The mixture was stirred for 3 hand concentrated under high vacuum. Addition of the residueto water (5 mL) precipitated the product, which was collectedand purified by HPLC to obtain DFO-cypate-cyclo[phe-Lys(∼)-Arg(Pbf)-Gly-Asp(OBut)] (27 mg, 25% yield; ES-MS: [MH]2+

1031.6). The resultant solid (20 mg) was dissolved in TFA (1mL) and kept at room temperature for 2 h. The TFA solutionwas concentrated and purified by HPLC to afford DFO-cypate-cyclo[phe-Lys(∼)-Arg-Gly-Asp] (7 mg, 40% yield; ES-MS:[MH]2+/[MH2]3+ 877.2/585.1).

Cypate-DFO (3). Deferoxamine mesylate salt (131 mg, 0.2mmol) was dissolved in DMSO (3 mL) in the presence of DIEA(26 mg, 0.2 mmol) by sonication, followed by the addition ofHOBT (27 mg, 0.2 mmol), cypate (141 mg, 0.2 mmol), andEDCI (38 mg, 0.2 mmol). The mixture was stirred at roomtemperature for 2 h and added with stirring to ether. Theprecipitate was collected by filtration, one-third of which waspurified by HPLC to afford cypate-DFO (25 mg, 30%; ES-MS:[MH]+/[MH]2+ 1167.1/584.4).

Absorption and Emission Spectral Analysis. Absorptionand fluorescence spectra were recorded on a DU 640 UV–visiblespectrophotometer and Fluorolog-3 fluorometer, respectively.Stock solutions (1.0 mM) were prepared by dissolving eachsample in 80% aq DMSO, unless otherwise stated. UV–vis andfluorescence measurements were carried out by sequentiallyadding 5.0 µL aliquots of the stock solutions via a micropipetteinto 3 mL of 20% aq DMSO solution in a quartz cuvette. Thefluorescence quantum yields of 1, 2, 3, cypate, and ICG inDMSO were determined according to an established protocoland referenced to ICG in DMSO (Q.Y. ) 0.13). The absorbanceof five serial dilutions of each compound was measured in theconcentration range between 0.1 and 1.0 µM. The quantum yield(Φ) was determined according to the following equation (47):

Φs )Φr(Grads) ⁄ (Gradr)(ηs2 ⁄ ηr

2)(qs ⁄ qr)

where the subscripts s and r refer to sample and reference ICG(obtained by HPLC purification of commercially available ICGfrom Aldrich), respectively. η represents the refractive indexof the solvent, and q represents a correction factor for theexcitation wavelength used. qs/qr was assumed to be 1 undersimilar excitation conditions.

Metal Binding Studies. Stock solutions (1 mM) of com-pounds 1, 3, and DFO were first prepared with 50% aq ACN.We also prepared stock solutions (1 mM) of the following metalsalts in distilled water: Fe(NO3)3, FeSO4, Al2(SO4)3, Ga2(SO4)3,InCl3, GdCl3, CuSO4, ZnSO4, Co(NO3)2, NiCl2, MnCl2, CaCl2,MgCl2, and MgSO4. All test solutions (100 µL each) containing100 µM ligand and 200 µM metal ion were prepared by mixingcompounds 1, 3, or DFO (10 µL, 1.0 mM) and a metal ion (20µL, 1.0 mM) in 70 µL of 50% aq ACN. After the solutionswere stirred for 2 h, 20 µL of the resulting solutions weresubjected to ESI-MS analysis. Each solution (30.0 µL) wasadded to 2970.0 µL of 20% aq DMSO for UV–vis andfluorescence emission analysis.

ABIR Binding Affinity. Receptor binding assays wereperformed by using human-purified ABIR protein from Chemi-con International, Inc. (Temecula, CA) using the methodreported previously (48). Briefly, assays were carried out in theMillipore Duropore membrane 96-well plates and the MilliporeMultiScreen system (Bedfore, MA). 125I-echistatin was pur-chased from Amersham Biosciences (Piscataway, NJ). Thespecific activity of the radiolabeled peptide was ∼2000 Ci/mmol.Radiochemical purity (90%) was determined by reverse-phaseHPLC. The 96-well membrane plate was blocked with 0.1%polyethylenimine blocking solution overnight at 4 °C. 125I-echistatin (50 nmol/L) was added to the binding buffer (50 mMTris-HCl, pH 7.4, 100 mM NaCl, 2 mM CaCl2, 1 mM MgCl2,1 mM MnCl2, and 1% BSA) containing integrin ABIR protein(50 ng per well in 96-well membrane plate) and ligands in atotal volume of 200 µL per well. The concentration range ofligand was between 0.01 µM and 100 µM. The mixtures wereincubated for 2 h at room temperature, filtered by centrifugationat 1500g, and washed with 0.20 mL of ice-cold binding buffer(3×). The filters containing radioactivity-bound ABIR wereremoved using a punch apparatus and counted using a PackardCobra II Autogamma counter (Meriden, CT). Nonspecificbinding of 125I-echistatin was between 5 to 10% of the totalbinding. The 50% inhibitory concentrations (IC50) were calcu-lated by nonlinear regression analysis using GraphPad Prism 4data fitting software (GraphPad Software, Inc., San Diego, CA).Three independent binding assays were performed in triplicateper sample.

Cell Culture. The human nonsmall cell carcinoma cell lineA549 is widely used to study the role of integrins in normaland pathophysiological processes. These cells were purchasedfrom ATCC and grown in 75 cm tissue culture flasks or onLaboratory-Tek chambered slides in Ham’s F12K medium with2 mM L-glutamine supplemented with 1.5 g/L sodium bicarbon-ate, 10% fetal calf serum, 100 units/mL penicillin, and 100 units/mL streptomycin under humidified atmosphere containing 5%CO2 at 37 °C.

Internalization and Fluorescence Microscopy. A549 cellswere grown on Laboratory-Tek slides. The culture medium wasreplaced, and the cells were incubated for different times up to24 h at 37 °C in the presence of the cypate-labeled DFO probes(1 µM in 0.02% DMSO). After treatment, cells were washed(3×) with PBS containing 1 mM CaCl2 and mounted withProlong Gold containing DAPI mounting medium and cover-slipped (Invitrogen). For fluorescence microscopy, cells werevisualized with an Olympus FV1000 microscope using a 60X/1.20M, 0.13–0.21 NA water immersion objective. Fluorescenceemission was detected from 805–830 nm using a 780 nm laserat 100% transmission for excitation. Images of each group ofslides were acquired with the same microscope settings duringa single imaging session, allowing for qualitative comparisonof the relative fluorescence. The relative fluorescence intensityof five regions of interest for each image was determined withSlidebook software from Olympus and the results averaged. The

Fluorescent Integrin-Targeted DFO Analogue Bioconjugate Chem., Vol. 19, No. 1, 2008 227

results were normalized to the average relative fluorescence ofDFO-cypate treated cells.

For mitochondrial staining, 5 nm Mitotracker Green (Invit-rogen) was added during the last 15 min of incubation. Forlysosomal staining, cells were stained with anti-LAMP1 anti-body as described in the literature (49). Briefly, cells were fixedfor 15 min with 4% paraformaldehyde for 15 min, after whichthey were washed 3× with PBS containing 1 mM CaCl2. Slideswere incubated for 1 h at room temperature with 10% rabbitserum to block nonspecific binding after which they wereincubated overnight at 4 °C with 2 µg/mL anti-LAMP1 goatpolyclonal IgG (Santa Cruz). After washing 3× with PBS, slideswere incubated for 1 h at room temperature with 10 µg/mLCy3 conjugated rabbit antigoat IgG (Zymed). After washing3× with PBS, slides were mounted with Prolong Gold andcoverslipped. Cells were visualized with an Olympus FV1000microscope using a 60X/1.20M, 0.13–0.21 NA water immersionobjective. Fluorescence emission was detected by using a 488nm laser at 5% transmission and 500–600 nm emission(Mitotracker green) or by using a 543 nm laser at 15%transmission and 555–655 nm emission (LAMP-1).

RESULTS AND DISCUSSION

Molecular Design. Previously, we reported the synthesisand applications of a dicarboxylic acid-containing carbocya-nine fluorophore (cypate) (10, 45). The facile synthesis ofcypate and its excellent NIR spectral properties have allowedus to conveniently label diverse peptides and other bioactivemolecules with cypate in solution and on solid support.Particularly, the two carboxylic acid groups of cypate havefacilitated its use as an optical scaffold to construct diversebioactive NIR fluorescent molecular probes, such as mono-meric,dimeric,macrocyclic,andmultivalentanalogues(8,45,50,51).Conjugation of cypate with small peptides that target specificcell-surface receptors, such as ABIR and the somatostatinreceptor, allows site-specific targeting of diseased tissues foroptical imaging (9, 10, 51). In particular, previous reportshave shown that cyclic RGD pentapeptide cyclo(RGDfV) andsome of its analogues bind to ABIR with high affinity andselectivity. Further modification of the biological propertiesof cypate-labeled RGD peptides could be obtained byincorporating another bioactive molecule such as DFO.

The metal-binding properties and the related biologicalactivities of DFO have been ascribed to its three hydroxamategroups (26, 52). Therefore, the amino group of DFO providesa desirable site for its structural modification. In this work,we used cypate to construct a three-pronged architecturebearing both the DFO motif and ABIR-targeting RGD peptide(cyclo[RGDfK(∼)] (Figure 1). We anticipated that the newNIR fluorescent RGD-bearing DFO compound (1) wouldserve as a multifunctional platform for biomedical applica-tions. For example, the RGD peptide motif could be used

for the targeted delivery of DFO to ABIR positive tumors,while cypate serves as the optical antenna to track, visualize,and quantify the fate of 1 in cells and in vivo by NIRfluorescence imaging. In addition, the integration of DFOand RGD peptide into cypate could cooperatively enhancethe related biological activities. Finally, the metal-bindingDFO could modify the structure, function, and regulation ofdivalent, cation-dependent ABIR (28, 38). Together, thesepotential applications of NIR fluorescent DFO compoundsmotivated the design, synthesis, and evaluation of thecompounds described in this article.

Synthesis. As shown in Scheme 1, synthesis of the multi-functional compound 1 consists of three major steps: (i)synthesis of the protecting RGD peptide, (ii) cypate conjugation,and (iii) DFO conjugation. The resin-bound protected RGDpenta-peptide, H-Asp(OBut)-phe-Lys(Dde)-Arg(Pbf)-Gly-O-Resin (1), was first assembled on a H-Gly-loaded 2-chlorotritylresin using conventional Fmoc chemistry. Cleavage of theprotected peptide with 1% TFA in DCM afforded the desiredprotected RGD pentapeptide, H-Asp(OBut)-phe-Lys(Dde)-Arg-(Pbf)-Gly-OH. This linear peptide was cyclized to form cyclo-[phe-Lys(Dde)-Arg(Pbf)-Gly-Asp(OBut)] in the presence ofPyBOP, HOBT, and DIEA in solution. Removal of the ε-aminolysine-protecting Dde group with hydrazine in aqueous aceto-nitrile afforded cyclo[phe-Lys-Arg(Pbf)-Gly-Asp(OBut)], whichwas further purified by flash column chromatography. Theconjugation of the cyclic peptide (1 equiv) and cypate (3 equiv)was achieved in the presence of EDCI and HOBT in anhydrousDMF. Similarly the crude product, cypate-[Arg(Pbf)-Gly-As-p(OBut)-phe-Lys(∼)], was coupled with DFO, as reported previ-ously (53). Aqueous TFA was used to deblock the resultantproduct, DFO-cypate-[Arg(Pbf)-Gly-Asp(OBut)-phe-Lys(∼)],which was then purified by HPLC to give the desired compound1, DFO-cypate-cyclo[Arg-Gly-Asp-phe-Lys]. The identity andpurity of the compound were confirmed by both HPLC and ES-MS. A combination of solution and solid-phase reactionsfacilitated the preparation of all compounds used in this study.

Spectral Properties. Stock solutions (1 mM) of the DFO-containing compounds were prepared in DMSO, and spectralmeasurements were carried out by adding 5 µL aliquots of thestock solutions via a micropipette into 3 mL of DMSO solutionin a quartz cuvette. All of the compounds showed similarspectral features, with minor spectral shifts in the absorptionand emission maxima (Figure 2). The attachment of cyclo-[RGDfK(∼)] to cypate increased the molar absorptivity (ε) whilethe DFO attachment decreased ε (Table 1). Compound 1containing both DFO and cyclo[RGDfK(∼)] showed similar εwith cypate. Overall, conjugation of the highly hydrophilic DFOto cypate improved the water solubility of the dye without effecton its spectral properties.

Metal Binding Studies. Siderophores such as DFO are goodligands for Fe(III), and the biological activities of DFO are

Scheme 1. Synthesis of DFO-cypate-[RGDfK(∼)] (1)


related to its metal-binding properties (20, 54–56). Previousstudies have shown that DFO and the related hydroxamatecompounds also possess high binding affinities with Al(III) andGa(III) (26, 53). Therefore, we evaluated the metal-bindingproperties of 1 and the related effects on the correspondingoptical properties of DFO-bearing fluorescent compounds.Because of its high sensitivity, we used ES-MS to study metalcoordination with DFO, as described previously (26, 57). Thismethod is suitable for screening the relative metal-bindingselectivity and affinity of some compounds, especially those inlimited supply.

Compounds 1, 3, and DFO (100 µM in 50% aq acetonitrile)were screened for their binding with 13 different metal ions,Fe(III), Al(III), Ga(III), In(III), Gd(III), Cu(II), Zn(II), Co(II),Ni(II), Mn(II), Fe(II), Ca(II), and Mg(II), at 200 µM in water.All the three DFO-bearing compounds exhibited high bindingaffinity and selectivity for Ga(III), Al(III), and Fe(III) comparedto the other metal ions. The ES-MS spectra of 1 and the metalchelates shown in Figure 3 are representative of the trendobserved for these compounds. Compound 1 showed two typicaldoubly and triply charged peaks at 876.7 and 584.9 in its ES-MS spectrum. Upon metal binding, these peaks were replacedby the corresponding doubly charged (903.5, 889.0, and 910.1)and triply charged (602.2, 592.8, and 607.5) peaks of theresulting Ga(III), Al(III), and Fe(III) complexes, respectively.These peaks were correlated with the theoretical masses basedon [(L-3H)MHn]n+, where L and M represent the molecularweight of a ligand and a metal, respectively. The high abundanceof the desired metal chelate peaks with negligible residue fromthe starting DFO ligands strongly suggests the completeformation of 1:1 metal complexes in aq ACN.

We further calculated the relative metal-binding affinity (%)of each ligand as a percentage of the relative abundance of themetal-binding ligand peaks with the total ligand-related peaks

in a positive ES-MS spectrum according to the followingequation, as reported previously (26, 58):

[LFe2 +LFe3] ⁄ [L2 +L3 +LFe2 +LFe3 +L] × 100%

where L2 and LFe2 are the doubly charged peak abundance ofa ligand and its complex, while L3 and LFe3 are the triplycharged peak abundance of a ligand and its complex. The relatedpeaks associated with the addition of sodium ions were alsocalculated. As shown in Figure 4, the ES-MS analysis clearlydemonstrated the relative metal binding selectivity and affinitywith Fe(III), Fe(II), Ga(III), Al(III), and In(III). Despite theirstructural differences, the metal binding selectivity of 1, 3, andDFO was similar, indicating that the chelating property of DFOwas maintained, independent of the molecular design.

In contrast to the ES-MS spectra of Ga(III) and Al(III), theaddition of Fe(III) and Cu(II) resulted in complicated ES-MSspectra with multiple peaks around the doubly and triply chargedpeaks. This observation suggests that Cu(II) had weaker bindingwith 1, but might greatly impact the ionization of 1 and thestability of the resulting ions in gas phase. In addition, the lowabundance of the related 1:1 ligand/metal complex indicates thatCu(II) binding might occur between two or more ligands of 1with two or more Cu(II).

The relatively high binding affinity of DFO-bearing com-pounds with Fe(III), Al(III), and Ga(III) makes it possible tofurther functionalize 1 via metal binding. Therefore, we preparedand isolated the Ga(III) complex of 1 to demonstrate thefeasibility of obtaining fluorescent DFO chelates. By mixing 1with excess Ga(III) in aq ACN, the Ga(III) complex wasobtained by HPLC purification and further characterized by ES-MS. The metal complex has a longer retention time than 1,suggesting that the coordination of Ga(III) by the three DFOhydroxamates reduces the hydrophilicity of 1 (Figure 5). Thesuccessful preparation and characterization of the Ga(III)complex provide an exciting dimension for exploring additionalapplications of this compound in biomedicine.

Spectrally, there was no significant change in the greencolor of 1 upon the additions of the metals, with the exceptionof Fe(III), Fe(II), and Cu(II), where a change was observed(Figure 6). Clearly, the addition of Cu(II) and Fe(III)markedly decreased the UV–vis absorption and quenched thefluorescence emission of 1 as shown in Figure 6. On the basisof the ES-MS analysis discussed above, it is likely thatspectral changes by these metals occurred by different

Figure 2. Normalized UV–vis and emission spectra of DFO-cypate-[RGDfK(∼)] (1), cypate-[RGDfK(∼)] (2), and cypate-DFO (3) in DMSO.

Table 1. Optical Properties of the Compounds in DMSO

compds λmax(ab) λmax(em) ε (cm-1 M-1)Φ (excited at

730 nm)

1 793 816 2.0×105 0.102 792 817 2.3×105 0.103 788 815 1.4×105 0.17cypate 791 814 2.1×105 0.12ICG 794 820 1.9×105 0.13


mechanisms. While Cu (II) appears to quench fluorescenceby interacting directly with the cypate motif, the stable Fecomplex may quench fluorescence by the photoinducedelectron transfer mechanism. In contrast to Fe(III) and Cu(II),Ga(III) binding did not significantly alter the UV–visabsorbance of 1. Interestingly, Ga(III) seemed to enhancelight emission to some extent. This finding is in agreementwith a previous study with 3, where a similar enhancementwas observed (53). Considering that a DFO chelate ofradioactive Ga(III) has been used previously for nuclearimaging, the Ga(III)-mediated fluorescence enhancementfavors the use of the Ga(III) complex of compounds 1 and 3to develop a dual optical-nuclear imaging agent. In addition,the contrasting effects of Fe(III) and Ga(III) on the opticalproperties of cypate provide an efficient approach to perturbthe spectral properties of NIR fluorescent carbocyanine probesvia metal binding.

Receptor Binding Assays. Among the diverse RGD pep-tides that have been developed for targeting ABIR, thelactam-based cyclic penta-peptide cyclo(RGDfV) and itsanalogues are well known for their high binding affinity andselectivity with ABIR. In this study, commercially availableABIR-avid peptide cyclo(RGDfV) was used as a referencecompound to study the effect of DFO on the ABIR bindingaffinity of 1 and 2. The IC50 values of these compounds weredetermined by competitive inhibition of Echistatin bindingto purified ABIR, as described previously (48, 59). Echistatinis a polypeptide that binds irreversibly with high affinity andspecificity to the ABIR and the radiolabeled analogue. 125I-echistatin was used as a tracer in the binding assay.Compounds 1, 2, and cyclo(RGDfV) were found to have IC50

values of 1.8 × 10-7 M, 7.7 × 10-8 M, and 4.1 × 10-7 M,respectively. Under similar conditions, the presence of theDFO motif in 1 decreased the ABIR affinity of 2 by a factorof about 2, but the resulting affinity was comparable to thatof the reference cyclic RGD compound. These findingsindicate that the RGD peptide can modulate the biologicalactivities of DFO through receptor-mediated delivery of thisotherwise nonspecific drug to target tissue and cells.

Cell Internalization. Uptake and distribution of drugs andcontrast agents in cells provide a key index for understandingthe functions and predicting the in vivo behavior of fluorescentcompounds. Therefore, labeling DFO with fluorescent dyeallows the use of the resultant molecule as molecular probe forimaging DFO distribution in cells. Considering that the ABIRbinding assay revealed the retention of RGD peptide bindingaffinity by 1, we assessed the internalization and subcellulardistribution of 1, 2, and 3 in an ABIR-positive cell line, A549.The incubation of A549 cells with 3 (1 µM) in mediumcontaining 0.02% DMSO showed minimal uptake of thecompound, with normalized fluorescence similar to the back-

Figure 3. ES-M spectra of 1 and its Ga(III), Al(III), and Fe(III) complexes (100 µM) in 50% aq ACN.

Figure 4. Relative binding affinities of 1and DFO with different metalions at a molar ratio of 1:2 as determined by ES-MS.


ground signal (Figure 7). Expectedly, hydrophilic moleculessuch as DFO have difficulty crossing the plasma membranesof cells without an active transport mechanism. In contrast, both1 and 2 internalized rapidly within 1 h post incubation andremained in the intracellular space up to 10 h. The enhancedinternalizations of 1 and 2 could be ascribed to the interactionof the RGD peptide with ABIR in the cell membranes. Thus,the RGD peptide provided an endocytotic pathway for DFO.At 1 h postincubation, the internalization of 1 and 2 in A549cells was similar to each other, but the retention of 1 in thecells was lower at longer incubation times than that of 2 (Figure7D). The differences in the internalization rate appear tocorrelate with their ABIR binding affinity, with 2 having higheraffinity than 1. Once internalized, however, the rates ofsubsequent efflux of the two compounds from the cell weresimilar, with the intracellular fluorescence intensity for bothcompounds approaching background at 24 h post incubation.The fluctuations in fluorescence shown in Figure 7D could beattributed to changes in receptor density as a function of time.

On the basis of the dissimilarity in structural features,ABIR binding affinity, and internalization rates, we expecteddifferences in the intracellular distribution of compounds 1,2, and 3. Interestingly, once inside the cell, all threecompounds appeared to localize in the mitochondria, thelysosomes, and the cytosol (Figures 8 and 9). The absenceof identifiable uptake in the cell nuclei favors using thefluorescent DFO compounds as imaging agents because ofthe need to minimize nuclear translocation of imaging agents,thereby preventing the potential mutagenic effect on the cells.

CONCLUSIONS

We have developed a novel multifunctional molecularprobe consisting of a metal-chelating DFO, a NIR fluorescentcypate, and an ABIR-avid cyclic RGD peptide. Eachcomponent synergistically contributes to the overall propertiesof new compound 1. Cypate provides a detectable signal forimaging purposes; the RGD peptide effectively delivers bothcypate and DFO into cells; and metal-binding DFO efficiently

Figure 5. Proposed metal complex structure of 1.

Figure 6. Effects of Ga(III), Fe(III), and Cu(II) ions on UV–vis and emission spectra of 1 in 20% aq DMSO.

Figure 7. Internalization in A549 cells treated for 10 h with 1 µM of compounds 3 (A), 2 (B), or 1 (C) and quantitation of the internalization ofeach compound as a function of time (D).


modulates the optical properties of cypate. The impressiveABIR-mediated internalization of 1 in cells without permeat-ing the cell nucleus is advantageous for imaging applications,where nuclear uptake can be detrimental to the cells andliving organisms. This work represents our initial efforttoward the discovery and development of novel ABIR-targeting DFO compounds for optical imaging. Specifically,1 provides a multifunctional molecular platform for optimiz-ing the structure, improving biological activities, exploring

new applications, and understanding the mechanisms ofaction. The synthetic approach we used is amenable tocombinatorial chemistry and the parallel synthesis of diverseNIR fluorescent DFO analogues.

ACKNOWLEDGMENT

This study was supported in part by the NIH (R01 CA109754,R33 CA100972, and R21 CA123537).

Figure 8. Subcellular distribution of cells treated for 10 h with 1 µM 3 (A), 2 (B), or 1 (C) in medium containing 0.02% DMSO costained withMitotracker green. (D-F) Inserts at 3× magnification corresponding to A, B, and C, respectively.

Figure 9. Subcellular distribution of cells treated for 10 h with 3 (A), 2 (B), or 1 (C) costained with anti-LAMP1 antibody. (D-F) Inserts at 3×magnification corresponding to A, B, and C, respectively.


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Novel Near-Infrared Fluorescent Integrin-Targeted DFO Analogue