DOI: 10.1002/cmdc.201000027

Electrocyclization-Based Labeling Allows Efficient In Vivo Imaging ofCellular Trafficking

Katsunori Tanaka,*[a] Kaori Minami,[a] Tsuyoshi Tahara,[b] Yohei Fujii,[a] Eric R. O. Siwu,[a] Satoshi Nozaki,[b]

Hirotaka Onoe,[b] Satomi Yokoi,[c] Koichi Koyama,[c] Yasuyoshi Watanabe,[b] and Koichi Fukase*[a]

Covalent chemical labeling of living cells has garnered signifi-cant attention in the fields related to molecular imaging[1] dueto direct and easy operations, and broad and general applica-bility to both in vitro and in vivo studies, as well as the smallersize of the chemical labels.[2] Undesirable modification of keyfunctions on cell surfaces should be avoided in order to retainthe native functions of the cell. Therefore, bioorthogonal ap-proaches, which can be combined with biological techniques,have actively been investigated.[3] Successful examples includeBertozzi’s Staudinger ligation[4–6] and the strain-acceleratedHuisgen 1,3-cycloaddition reaction.[7–10] These methods havebeen applied to the labeling of cells expressing oligosacchar-ides with azido-containing sugar residues on the cell surfacesthrough biosynthetic pathways,[8] which subsequently reactedwith the methoxycarbonyl phenyldiphenylphosphine or cyclo-octyne derivatives.

In contrast, as a “non-bioorthogonal approach”, we recentlydeveloped a new lysine-based technique for the labeling ofpeptides and proteins (antibodies), which is based on a rapid6p-azaelectrocyclization.[11, 12] We have used this method to effi-ciently and selectively introduce both DOTA (1,4,7,10-tetraaza-cyclodecane-1,4,7,10-tetraacetic acid), as a metal chelatingagent (either for magnetic resonance imaging (MRI), positronemission tomography (PET), or other radiopharmaceutical pur-poses), and fluorescent groups to lysine residues via a reactionwith unsaturated aldehyde probes (such as probes 1 a and 1 bin Scheme 1) at very low concentrations (~10nm) within ashort time (10–30 min) at room temperature. Furthermore,DOTA-labeled somatostatin and glycoproteins have subse-quently been radiometalated with 68Ga, and their receptor-mediated accumulation successfully visualized by PET.[12]

Our new method precisely controls the introduction ofDOTA or fluorescence labels onto lysine residues in target pro-teins by adjusting the reaction concentration so that the activi-ty of the biomolecules is retained. The efficiency of our rapid

azaelectrocyclization protocol depends on the steric accessibili-ty of the primary amino groups. Reactions with internal lysineresidues of the tertiary protein structures, as well as the N-ter-minal amine (amine with a substitution on the a-carbon), arevery slow (>5 h at 24 8C),[11e] while lysines at protein surfacesreact rapidly (10–30 min at 24 8C); therefore, labeling occurspreferentially at these positions.[11b, 12] Site-selective labeling ofthe target protein has also been achieved by directing reactivegroups (unsaturated aldehydes) to a specific site using a small-molecule ligand of the protein[13] so that the decrease in activi-ty is suppressed to a minimum when lysines, which are criticalfor receptor binding, are situated at the most accessible site ofa target protein. Dihydropyridines as the electrocyclizationproducts, which retain the cationic charges as those of the in-herent lysines, might also contribute to the retention of theprotein activity.

Based on previous results, we speculated that our azaelec-trocyclization chemistry could covalently and selectively labelmost of the accessible amino groups on cell constituents via aone-step procedure under mild and physiological conditions,i.e. , lysines of membrane proteins and/or ethanol amine deriv-atives with minimal interference with their native functions.Herein, we report direct labeling of a whole cell through azae-lectrocyclization chemistry under extremely diluted concentra-tions. While the conventional chemical approach, such as thesuccinimidyl ester method, causes a significant internalizationof the reagents leading to cytotoxicity, the present azaelectro-cyclization protocol tightly anchors the fluorescence labels onthe cell surface. An investigation of the in vivo imaging of thefluorescence-labeled lymphocytes was also performed as anapplication of the present protocol; the trafficking of the cellsinto the immune-related organs was clearly visualized withmarkedly high image contrast.

Although we previously established a preparation methodfor unsaturated aldehyde probes, such as 1 a and 1 b(Scheme 1), via the oxidation of stable and therefore storablealcohol precursors 2 a, b on a 5–20 mg scale in a reactionflask,[12a] such a specialized procedure can only be performedin synthetic laboratories. Hence, for a broader and more gener-al application of labeling reagents to biology-directed research,it is desirable to develop an easy-to-use oxidation (activation)method, such as a kit-like procedure,[14] which enables the insitu generation of the unstable aldehydes that are otherwisedifficult to handle, and that can be performed even on a mi-crogram scale. Therefore, an Eppendorf tube protocol was es-tablished as shown in Scheme 1 (see the Experimental Sectionfor details). Using a simplified Eppendorf tube procedure,probes such as 1 a and 1 b, as well as other unsaturated alde-hyde probes previously developed in our laboratory,[12a] can be

[a] Dr. K. Tanaka, K. Minami, Y. Fujii, Dr. E. R. O. Siwu, Prof. Dr. K. FukaseDepartment of Chemistry, Graduate School of Science, Osaka University1-1 Machikaneyama-cho, Toyonaka-shi, Osaka 560-0043 (Japan)Fax: (+ 81) 6-6850-5419E-mail : ktzenori@chem.sci.osaka-u.ac.jp

koichi@chem.sci.osaka-u.ac.jp

[b] Dr. T. Tahara, Dr. S. Nozaki, Dr. H. Onoe, Prof. Dr. Y. WatanabeRIKEN Center for Molecular Imaging Science6-7-3 Minatojima-minamimachi, Chuo-ku, Kobe-shi, Hyogo 650-0047(Japan)

[c] S. Yokoi, K. KoyamaKishida Chemical Co. , Ltd.14-10 Technopark, Sanda-shi, Hyogo 669-1339 (Japan)

Supporting information for this article is available on the WWW underhttp://dx.doi.org/10.1002/cmdc.201000027.

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conveniently and reproducibly prepared in both organicchemistry and biology laboratories.

We initially treated C6 glioma cells,[15] which were eithercoated on a dish or as a suspension, with a phosphate-buf-fered saline (PBS) solution of TAMRA (carboxytetramethylrhod-amine)–1 a (10mm) at 37 8C for 10 min (Scheme 1). After thecells were washed with a culture medium a few times, theywere fixed with formaldehyde. The fluorescence-labeled cellswere further treated with 4’,6-diamino-2-phenylindole (DAPI), ablue dye to visualize the nuclei, and analyzed by confocal laserscanning microscopy (Figure 1). In the control experiments, C6glioma cells were also treated with the succinimidyl ester ofTAMRA at the same concentration, a reagent generally used tochemically label the amino groups of peptides and proteins.Red fluorescence derived from the TAMRA dye (excitation =

525 nm, emission = 555 nm) was observed on the surface ofthe glioma cells treated with 1 a (Figure 1 a), whereas weakerfluorescence was distributed over the entire cell when theTAMRA succinimidyl ester was used, i.e. , not localized on thecell membrane (Figure 1 b). These results clearly show thatTAMRA–1 a probe rapidly reacts with the amino groups of thelysines and/or other amino-containing cell membrane compo-nents, such as phosphatidyl ethanolamines, to anchor the fluo-rescence label on the cell surface. On the other hand, the suc-cinimidyl ester agent was internalized in the cell before react-ing with the amino groups on the cell membrane due to the

much lower reactivity of thisagent compared with 1 a undersuch mild labeling conditions.

The above conclusion thatprobe 1 a labeled the aminogroups selectively on the cellmembrane is also supported byco-staining with the fluores-cence-labeled anti-N-cadherinantibody (see Supporting Infor-mation); although the TAMRAfluorescence of 1 a does notoverlapped with that of N-cad-herin, these are both found atthe membrane regions of thecells, as judged by two-dimen-sional and three-dimensionalfluorescence microscopy as wellas the cross sections of a labeledcell. Furthermore, when theTAMRA-labeled cells (either byprobe 1 a or the succinimidylester reagent) were treated withTriton X-100, the fluorescence in-tensity of the 1 a-labeled cellgradually decreased in accord-ance with the increase in deter-gent concentration, whereas thefluorescence intensity was re-tained in cells treated with succi-nimidyl ester agent (see Sup-

porting Information).[16] These results also indicated that label-ing by probe 1 a proceeded on the cell surface, whereasTAMRA-NHS was internalized into the cells.

Notably, the fluorescence labeling by 1 a was effective evenat a concentration of 10 nm, while no fluorescence was detect-ed when cells were treated with TAMRA–NHS ester at thishighly dilute concentration (at 37 8C for 10 min; see Figure 1 cand d). To date, this is the lowest concentration at whichchemistry-based bioconjugation through a covalent bond for-mation has been successful. Interestingly, confocal microscopydetected specific localization of the TAMRA fluorescence onthe cell membrane, such that the fluorescence surrounded thenucleus and then radiated outward (Figure 1 c). Based on previ-ous results on the reactivity of 1 a and other unsaturated alde-hyde probes,[12, 13] azaelectrocyclization might proceed selec-tively at the protein assembly (high concentration of lysine)and/or the other ethanol amine functions on the cell surfaceunder such a diluted concentration of 1 a. The preliminarySDS-PAGE experiments of the lysate of the 1 a-labeled C6glioma cell gave a clear protein band at 40 KDa with thestrong TAMRA fluorescence, which is currently being investi-gated.

Due to the mild labeling conditions used with 1 a, namely,37 8C for 10 min, 95 % of the glioma cells were alive based ontrypan blue tests (labeled cells by 1 a at both 10 mm and10 nm). Therefore, the labeled glioma cells divided over 2–44 h

Scheme 1. “In Eppendorf tube” preparation of unsaturated aldehyde probes and fluorescence labeling of thecells.

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without incident (see Supporting Information). Cell divisionwas successfully observed by fluorescence microscopy; fluores-cence on the cell surfaces was distributed to each dividing cell,and each cell-division cycle was accompanied by a decrease influorescence intensity, which might also be influenced by thedecomposition and/or the metabolism of the TAMRA label.Thus, the excellent reactivity of unsaturated aldehyde 1 a withthe amino functions enables the simple one-step chemical la-beling of living cells under extremely mild conditions.

The whole-cell labeling was applied to the in vivo fluores-cence imaging of the lymphocytes (Figure 2). The lymphocytes,directly extracted from the abdominal cavity of nude mice (seeExperimental Section), were fluorescently labeled by reactingwith Cy5-fluorescence probe 1 b (Scheme 1, excitation =

646 nm, emission = 663 nm) at 10 mm in a PBS buffer solutionat 37 8C for 10 min. Although labeling was also possible atlower concentrations (10 nm), the decay of the fluorescencedue to cell division, decomposition and/or metabolism, duringthe in vivo imaging over one week caused a problem in detec-tion sensitivity (see Figure 2). The Cy5-labeled lymphocytes

were then administrated and the dynamic fluorescence imageswere recorded over a week (Figure 2);[17] the mouse did notshow any rejection response during the injection process. Thelymphocytes gradually accumulated over 6 h in the organs ofthe immune system, namely then spleen and intestinal lymphnodes, after which the fluorescence intensity in the spleen dis-appeared (Figure 2). These results show that cell trafficking,particularly into the spleen, is clearly visualized by chemical la-beling technique with exceptionally high imaging contrastcompared with those reported using the antibody-based label-ing protocols;[18, 12b] thus, this observation highlights the use of“direct” electrocyclization-based labeling to a whole cell-basedin vivo imaging application.

In conclusion, we established chemical labeling of wholecells through rapid azaelectrocyclization. Because 1) the label-ing probes are prepared through an “easy handling” Eppendorfprocedure, and 2) subsequent labeling is achieved at extremelylow concentrations (10 nm), the present electrocyclization pro-tocol offers one of the most direct and convenient chemicalapproaches to labeling living cells. The preliminary investiga-tions into the labeling of lymphocytes and subsequent whole-body imaging were efficiently performed giving high-contrastimages in comparison with the previously used protocols, pre-sumably due to the covalent attachments of the fluorescentlabel on the cell surface under mild conditions without deacti-vating cell functions.

Based on the results described herein, it may be possible tolabel living cells with the metal-incorporated DOTA,[12] whichwould expand the method to include the radiotracer-basedtargeting or imaging of whole cells for PET, MRI,[19] or other ra-

Figure 1. Confocal microscopy of the TAMRA-labeled C6 glioma cells (excita-tion at 525 nm). As a blue dye, DAPI is introduced after fixing the TAMRA-la-beled cells by treatment with paraformaldehyde. Two-dimensional andthree-dimensional images of a cell labeled by a) unsaturated aldehydeprobe 1 a and b) TAMRA–succinimidyl ester at 10 mm (37 8C for 10 min), andlabeling performed at 10 nm (37 8C for 10 min) using c) 1 a and d) TAMRA–succinimidyl ester.

Figure 2. Fluorescence imaging of lymphocytes in mice. Labeled cells wereadministrated intravenously (n = 3, 100 mL per mouse, 104 cells), and thewhole body was scanned from the back side by eXplore Optix, GE Health-care, Bioscience (excitation = 646 nm, emission = 663 nm), 1 h, 2 h, 4 h, 6 h,48 h, 1 week after injection. Data were normalized. SP: spleen; LN; lymphnode of epidermal intestinal tract.

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diopharmaceutical purposes, for clinical applications, such asthe diagnosis of specific tumors, the detection of cancer meta-stasis, and so on. Research in this direction is currently underway in our laboratory.

Experimental Section

The animal studies were performed according to a protocol ap-proved by the Ethics Committee at RIKEN.

General procedure for fluorescence labeling of the cell surfaces : IBX-resin (5.0 mg, 4.0 mmol) was added at room temperature to a solu-tion of allylic alcohol 2 a (60.0 mg, 72.3 nmol) in DMF (25 mL) andCH2Cl2 (25 mL) in an Eppendorf tube. The reaction mixture wasshaken at room temperature for 30 min and then passed througha centrifugal filtration tube (Nacalai Tesque, Cosmospin Filter H,0.45 mm) to remove the IBX-resin. The solvents were removed bycentrifugal concentration at room temperature. Resulting unsatu-rated aldehyde probe 1 a in an Eppendorf tube was dissolved inthe appropriate volume of PBS containing 10 % DMSO to give PBSsolutions of probe 1 a with varying concentrations. The 10 nm solu-tion of 1 a in PBS (0.0001 % DMSO, 1.0 mL) was added to C6glioma cells (1.0 � 105 cells per 1 mL PBS) either by being coatedon the dish or as a suspension.[15] The cells were then incubated at37 8C for 10 min under a carbon dioxide atmosphere. The fluores-cence-labeled cells were then washed three times with culturemedium to remove the excess aldehyde probe.

Preparation of lymphocytes and in vivo fluorescence imaging : Awild-type mouse (BALB/cAJcl, CLEA Japan, Inc.) was killed byexcess anesthetic (isoflurane). After incision into the abdomen,~1 mL of blood was collected from the aorta descendens. Heparinsodium (100 mL, 100 units mL�1) was added to the collected blood,which was then stored at room temperature for 2 h before extract-ing the lymphocytes. A mouse peripheral blood diluted withmedium was added onto the lymphosepar II layer, and the mixturewas centrifuged. The lymphocyte fraction (middle layer of suspend-ed cell culture) was collected and diluted with medium, and againcentrifuged. The resulting pellet was resuspended in cell culturemedium, and the number of cells was measured. By repeating thisprocedure, a total of 3.6 � 106 cells were collected from 3.5 mL ofblood. After adjusting the cell number using culture medium, thecells were immobilized on the antibody-attached plates and cul-tured at 37 8C under 5 % carbon dioxide atmosphere. The lympho-cyte cultures were stored for 2 h at room temperature in a physio-logical salt solution before labeling (procedure described above).The lymphocytes (1 � 105 cells per 1 mL PBS buffer) were labeledby cy5-probe 1 b (incubation concentration: 20 mm).

Eight-week-old mice (BALB/cAJcl-nu/nu, CLEA Japan, Inc.) wereused for in vivo fluorescence imaging. Fluorescence-labeled lym-phocytes (1 � 104 cells per 100 mL in physiological salt solution)were injected via the caudal vein without anesthesia, and a whole-body scan was performed using an eXplore Optix (GE Healthcare)at different time periods up to 1 week after injection. The in vivofluorescence image was taken under inhalation anesthesia with iso-flurane; the concentration of isoflurane was 4 % from 15 min to 1 hafter the injection, and then maintained at 1.5–2 % during the restof the measurements.

Acknowledgements

This work was supported in part by the Japan Society for thePromotion of Science (JSPS ; Grants-in-Aid for Scientific ResearchNo. 19681024 and 19651095), a Collaborative Development of In-novative Seeds grant from the Japan Science and TechnologyAgency (JST), the New Energy and Industrial Technology Develop-ment Organization (NEDO ; project ID: 07A01014a), researchgrants from the Yamada Science Foundation, as well as a Molec-ular Imaging Research Program, Grants-in-Aid for Scientific Re-search from the Ministry of Education, Culture, Sports, Scienceand Technology (MEXT) of Japan.

Keywords: electrocyclic reactions · fluorescent probes ·imaging agents · lysine · positron emission tomography

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Received: January 21, 2010Revised: March 24, 2010Published online on April 20, 2010

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