COMMUNICATION pubs.acs.org/bc
Synthesis and Evaluation of a Bimodal CXCR4 Antagonistic Peptide Joeri Kuil,#,† Tessa Buckle,#,† Hushan Yuan,‡ Nynke S. van den Berg,† Shinya Oishi,§ Nobutaka Fujii,§ Lee Josephson,‡ and Fijs W. B. van Leeuwen*,† †
Division of Diagnostic Oncology, The Netherlands Cancer Institute, Antoni van Leeuwenhoek Hospital, 1066 CX Amsterdam, The Netherlands ‡ Center for Molecular Imaging Research, Massachusetts General Hospital and Harvard Medical School, Building 149, 13th Street, Charlestown, Massachusetts 02129, United States § Department of Bioorganic Medicinal Chemistry, Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto, Japan
bS Supporting Information ABSTRACT:
The antagonistic Ac-TZ14011 peptide, which binds to the chemokine receptor 4, has been labeled with a multifunctional single attachment point reagent that contains a DTPA chelate and a fluorescent dye with Cy5.5 spectral properties. Flow cytometry and confocal microscopy showed that the bimodal labeled peptide gave a specific receptor binding that is similar to monofunctionalized peptide derivatives. Therefore, the newly developed bimodal peptide derivative can be used in multimodal imaging applications.
T
he chemokine receptor 4 (CXCR4) is a G-protein-coupled membrane receptor which is present in various cell types.1,2 Besides its expression in normal tissues, a 5.5-fold upregulation of CXCR4-expression was found in breast cancer tissue,3 and CXCR4 upregulation is reported for at least 22 other types of cancer.4,5 CXCR4 has also been shown to play a critical role in (breast) cancer progression and metastatic spread.6,7 Because of its prominent role in oncology, overexpression of CXCR4 has been suggested to be of value in imaging applications.8,9 For molecular imaging of cancer-related biomarkers, several modalities are in use, including SPECT, PET, MRI, and fluorescence imaging.10 Each modality has its own strengths and weaknesses, and therefore, often a combination of modalities is needed during the clinical trajectory.1114 Recently, we have shown the added value of multimodal imaging agents in surgical guidance.1517 In this application, combined pre- and intraoperative imaging is achieved, whereby surgical planning is based on the radioactive signal and optical surgical guidance is achieved via the fluorescent component. This application can be expanded to tumor targeting peptides.13 For an imaging agent to be detectable in multiple modalities, it needs to contain at least two different diagnostic labels. As we r 2011 American Chemical Society
recently summarized, the combination of radiolabels for SPECT or PET, and fluorescent labels in particular, has potential in peptide-based molecular imaging.13 This combination benefits from the high sensitivity of nuclear imaging and the high spatial resolution of fluorescence imaging14 and allows the use of stoichiometric 1:1 labels.13 Several CXCR4 antagonistic peptides have been reported.18,19 One of the most potent peptides, T140, is a 14 amino acid containing disulfide-bridged peptide with a rigid β-hairpin conformation.20 Residues 2, 3, 5, and 14 of T140 are mainly responsible for CXCR4 binding, whereas the residues 710 in the type II0 β-turn do not have direct contacts with CXCR4 within 3.0 Å.21 Recently, a crystal structure of CXCR4 with the horseshoe crab peptide polyphemusin analogue CVX15, which is similar to T140, confirmed that residues 2, 3, 5, and 14 form important interactions with the receptor.22 T140 has been optimized to Ac-TZ14011 for better CXCR4 binding, higher in vivo stability, and easier functionalization, i.e., in Ac-TZ14011 Received: February 21, 2011 Revised: March 16, 2011 Published: April 12, 2011 859
dx.doi.org/10.1021/bc2000947 | Bioconjugate Chem. 2011, 22, 859–864
Bioconjugate Chemistry
COMMUNICATION
Scheme 1. Conjugation of the MSAP Label (2) to Ac-TZ14011 (1) and Subsequent Indium Labeling
only one amine group is present (D-Lys8).2325 The binding of dye- and chelate-labeled Ac-TZ14011 peptides to CXCR4 has already been validated in vitro and in vivo;13,2325 the specificity of Ac-TZ14011 for CXCR4 has been underlined by the fact that a scrambled peptide and a peptide with inversed stereocenters could not label CXCR4 expressing cells.26 However, a multimodal derivative of this CXCR4-targeting peptide has not yet been reported. Here, we report the conjugation of a multimodal imaging label (also referred to as multifunctional single-attachmentpoint, MSAP, reagent27,28) to Ac-TZ14011. We have performed in vitro validation studies to examine the influence of the MSAP label on the specificity and receptor affinity and we report initial in vivo experiments. The bimodal peptide derivative was generated using the MSAP reagent (2), which bears a DTPA and a CyAL-5.5b fluorochrome reporter and a reactive NHS ester.2729 The synthesis of the MSAP reagent 2 is described in the Supporting Information. Via the active NHS ester, it was selectively coupled to the D-Lys8 amine functionality on Ac-TZ14011 (1; see Scheme 1). The resulting peptide derivative Ac-TZ14011-MSAP (3) was purified with preparative HPLC and characterized using mass spectrometry. To study the possible influence of chelation of a metal ion in the DTPA moiety, a portion of Ac-TZ14011-MSAP (3) was coordinated to (nonradioactive) indium, yielding Ac-TZ14011-MSAP-In 4 (Scheme 1). The absorption spectra of 3 and 4 were analogous and also the emission spectra were very similar (see Supporting Information Figure S1). In both cases, the absorption maximum was found at 680 nm and the emission maximum at 704 nm. Moreover, the
Table 1. Quantitative Analysis of Cellular Uptake of MDAMB231 and MDAMB231CXCR4þ Cells Using a CXCR4 Antibody and the Peptides 3 and 4a compound
a
MFIR ( SD
MFIR ( SD
ratio of
MDAMB231
MDAMB231CXCR4þ
MFIRs 4.4
12G5-PE antibody
1.116 ( 0.054
4.920 ( 1.46
3
10.94 ( 1.62
45.27 ( 2.19
4.1
4
18.24 ( 1.93
56.67 ( 2.84
3.1
MFIR = mean fluorescence intensity ratio.
fluorescent signal intensity was not reduced when indium was bound in the DTPA-chelate. The ability of peptides 3 and 4 to discriminate between basal and CXCR4 overexpressing tumor cells was quantitatively examined with flow cytometry. As a reference “golden standard”, the commercially available phycoerythrin (PE) labeled anti-CXCR4 antibody CD184, clone 12G5 (12G5-PE), was used. The 12G5PE antibody showed that the higher CXCR4 expressing cells, MDAMB231CXCR4þ, had a 4.4 upregulation of CXCR4 compared to the basal cell line MDAMB231 (Table 1). This ratio of 4.4 is an intrinsic property of the two cell lines, and therefore, a peptide-based probe which can also obtain this ratio performs as well as the antibody. The ratio of the fluorescent signals found for peptide derivative 3 was 4.1, which nicely corresponds to the ratio obtained with the antibody (Figure 1 and Table 1). This indicates that this peptide is equally suited to detect CXCR4-expression levels. The mean fluorescence intensity ratios (MFIRs) for AcTZ14011-MSAP (3) were approximately 10 times higher than 860
dx.doi.org/10.1021/bc2000947 |Bioconjugate Chem. 2011, 22, 859–864
Bioconjugate Chemistry
COMMUNICATION
Ac-TZ14011-FITC (6), and Ac-TZ14011 (1) with competition experiments (Figure 2B,C). Incubation of MDAMB231CXCR4þ cells with different concentrations of Ac-TZ14011-MSAP (3) yielded a binding curve that appears to exist out of two components (Figure 2A). The nonlinear component of the binding curve can be ascribed to specific receptor binding and the linear component to nonspecific cell binding. By using this binding model, it was not needed to determine the nonspecific binding directly. The KD of AcTZ14011-MSAP (3) derived from this curve was 186.9 nM (Table 2). The indium labeled derivative Ac-TZ14011-MSAP-In (4) again gave somewhat higher fluorescent values (see Figure 1 and Figure 2A) and a KD of 177.1 nM (Table 2). The similarity in the CXCR4-receptor affinities of 3 and 4 indicates that indium binding does not affect the interaction with CXCR4. To compare the receptor affinities of 3 and 4 with the other peptide derivatives, different amounts of Ac-TZ14011 (1), Ac-TZ14011-DTPA (5), or Ac-TZ14011-FITC (6) were added in the presence of 250 nM of Ac-TZ14011-MSAP (3) (Figure 2B,C). All three peptide derivatives could compete with Ac-TZ14011-MSAP (3) for CXCR4 binding, confirming the specific CXCR4 binding of Ac-TZ14011-MSAP (3). The KD values, obtained by fitting the inhibition curves with a competition model, of the monolabeled peptides Ac-TZ14011-DTPA (5) and Ac-TZ14011-FITC (6) were in the order of the KD values of 3 and 4 (Table 2). These results illustrate that the bifunctional MSAP label does not hamper receptor binding more than the smaller FITC and DTPA labels. However, the unlabeled Ac-TZ14011 peptide 1 gave an approximately 20 higher receptor affinity than derivatives 3 and 4 (Table 2). Since the affinity of Ac-TZ14011-MSAP-In (4) remains in the nanomolar range, this decrease is not likely to be an obstruction for using 4 in molecular imaging applications.32 The previously reported scrambled peptide TR14010 (7) showed no significant binding; KD > 5000 nM (Table 2, see Supporting Information for the structure of TR14011).26 Molecular modeling studies showed that D-Lys8 in T140 has no interactions with CXCR4.21 Furthermore, the crystal structure of CVX15, which is similar to T140, with CXCR4 also showed no crucial interactions.22 However, we found that D-Lys8 functionalization in Ac-TZ14011 still influences binding affinity. This influence might be explained by looking at the different effects modifications of the peptide can have on (CXCR4) receptor binding. A modification on the peptide (i) can directly change a receptor interaction (e.g., a hydrogen bond), (ii) can change the 3D structure of the peptide, and/or (iii) can hinder the phamacophore to bind properly to the receptor via steric hindrance.21 Here, the third effect is most likely to cause the drop in affinity. A logical chemical modification to reduce the steric hindrance would be the incorporation of a larger spacer between the imaging label and the pharmacophore. Although it has been shown that incorporation of a 6-aminocaproic acid spacer does not yield a higher affinity,24 it is possible that longer spacers will be more effective. However, this influence of the spacer still is under investigation. Next to validating the receptor affinity of peptide 3, also its cellular distribution needs to overlap with the locations where the G-protein-coupled receptor CXCR4 is most likely to be expressed, namely, the cell membranes.1,2 Confocal microscopy imaging of MDAMB231CXCR4þ cells incubated with 3 indeed revealed clear cell membrane staining (Figure 3). The incubation
Figure 1. Flow cytometry analysis of the peptides 3 and 4 using (A) MDAMB231 cells or (B) MDAMB231CXCR4þ cells. Both graphs show untreated cells (trace filled with gray), 1 μM of 3 (black trace), and 1 μM of 4 (gray trace).
that of the antibody (Table 1). This difference is probably caused by the fact that the concentration used for the antibody was significantly lower than that of the peptides, 6.4 nM and 1 μM, respectively. Moreover, due to the lower autofluorescence with the flow cytometer settings used with the MSAP peptides, the voltage of the photomultiplier detector could be set higher when the MSAP peptides were measured, compared to the PE-labeled antibody. For Ac-TZ14011-MSAP-In (4), the obtained uptake ratio for the MDAMB231CXCR4þ and MDAMB231 cells was somewhat lower, namely, 3.1 (Table 1). Since both cell types had a higher MFIR (Table 1), this is perhaps caused by an increase in nonspecific binding of 4 to the cells (Figure 1). The increased nonspecific binding most likely is a charge-related effect. The DPTA chelate in peptide 3 is mainly 2 charged (four carboxylates and two protonated nitrogen atoms), and indium-bound DTPA in 4 has a charge of 1 (four carboxylates, no protonated nitrogen atoms and In3þ),30,31 increasing the hydrophobicity of the imaging label. In general, labels with increased hydrophobicity drive (nonspecific) cellular uptake.13 In general, conjugation of a bifunctional imaging label to a peptide reduces the receptor affinity.13 Therefore, we have thoroughly studied the influence of our MSAP-label in peptides 3 and 4 on receptor binding and compared it to that of monofunctionalized derivatives and the parental peptide. Initially, we determined the dissociation constants (KD) of 3 and 4 by a saturation binding assay (Figure 2A). The KD value of 3 was used to determine the KD values of Ac-TZ14011-DTPA (5), 861
dx.doi.org/10.1021/bc2000947 |Bioconjugate Chem. 2011, 22, 859–864
Bioconjugate Chemistry
COMMUNICATION
Figure 2. (A) Determination of affinity of constructs 3 and 4 with saturation binding experiments. (B) Competition experiments with three other AcTZ14011 peptides in the presence of 250 nM of 3. (C) Structures of the peptides used for competition experiments.
Table 2. Dissociations Constants (KD) of the Constructs construct Ac-TZ14011-unlabeled (1)
KD (nM) 8.61 ( 1.42
Ac-TZ14011-MSAP (3)
186.9 ( 52.4
Ac-TZ14011-MSAP-In (4) Ac-TZ14011-DTPA (5)
177.1 ( 37.2 124.4 ( 23.9
Ac-TZ14011-FITC (6)
203.5 ( 67.1
TR14010 (7)
>5000
was performed at 4 °C to minimize the initial metabolic activity. During imaging at 37 °C, minor staining in vesicular structures was detected. Vesicular internalization is common for liganded membrane receptors, and this form of signal accumulation in the cells proves the potential of using CXCR4 as a target in molecular imaging applications. Initial in vivo studies were performed to evaluate the tumor targeting. Therefore, mice with CXCR4 positive “spontaneous” MIN-O tumors were used instead of the MDAMB231CXCR4þ cell line, because the CXCR4 expression in MDAMB231CXCR4þ cells decreases to basal levels in vivo. Mice were injected with 111In-labeled Ac-TZ14011-MSAP, and after 24 h, the mice were imaged with SPECT/CT and fluorescence imaging (Figure 4). Both imaging modalities showed high tumor uptake. For biodistribution studies, we again used mice with CXCR4 positive MIN-O tumors and mice with CXCR4 negative 4T1 tumors as negative control (both tumors had a murine background to prevent xenograph effects) (Table 3). The tumor-tomuscle ratio for the MIN-O tumor was 4.55 ( 0.68, and for the 4T1 tumor, it was 1.20 ( 0.12 (p < 0.01), indicating that
Figure 3. Confocal microscopy of Ac-TZ14011-MSAP (3) using MDAMB231CXCR4þ cells.
Ac-TZ14011-MSAP can be used to discriminate CXCR4 positive from CXCR4 negative tumors in vivo. In conclusion, we report the first bifunctional CXCR4 antagonistic peptide, containing a Cy5.5-like dye and a DTPA chelate. Despite the functionalization with a relatively large MSAP label, in vitro the Ac-TZ14011 derivative retains its specificity and has a high CXCR4-affinity. Moreover, indium binding to the chelate does not affect the fluorescent properties. The initial in vivo 862
dx.doi.org/10.1021/bc2000947 |Bioconjugate Chem. 2011, 22, 859–864
Bioconjugate Chemistry
COMMUNICATION
(3) Kang, H., Watkins, G., Douglas-Jones, A., Mansel, R. E., and Jiang, W. G. (2005)The elevated level of CXCR4 is correlated with nodal metastasis of human breast cancer. Breast 14, 360–367. (4) Kulbe, H., Levinson, N. R., Balkwill, F., and Wilson, J. L. (2004) The chemokine network in cancermuch more than directing cell movement. Int. J. Dev. Biol. 48, 489–496. (5) Balkwill, F. (2004)The significance of cancer cell expression of the chemokine receptor CXCR4. Semin. Cancer Biol. 14, 171–179. (6) Teicher, B. A., and Fricker, S. P. (2010)CXCL12 (SDF-1)/ CXCR4 pathway in cancer. Clin. Cancer Res. 16, 2927–2931. (7) Lazennec, G., and Richmond, A. (2010)Chemokines and chemokine receptors: new insights into cancer-related inflammation. Trends Mol. Med. 16, 133–144. (8) Nimmagadda, S., Pullambhatla, M., Stone, K., Green, G., Bhujwalla, Z. M., and Pomper, M. G. (2010)Molecular imaging of CXCR4 receptor expression in human cancer xenografts with [64Cu]AMD3100 positron emission tomography. Cancer Res. 70, 3935–3944. (9) Han, Y., Yin, D., Zheng, M., Zhou, W., Lee, Z., Zhan, L., Ma, Y., Wu, M., Shi, L., Wang, N., Lee, J., Wang, C., Lee, Z., and Wang, Y. (2010) Synthesis and preliminary evaluation of a novel 125I-labeled T140 analog for quantitation of CXCR4 expression. J. Radioanal. Nucl. Chem. 284, 279–286. (10) Mather, S. (2009)Molecular imaging with bioconjugates in mouse models of cancer. Bioconjugate Chem. 20, 631–643. (11) Jennings, L. E., and Long, N. J. (2009)’Two is better than one’ probes for dual-modality molecular imaging. Chem. Commun. 3511–3524. (12) Louie, A. (2010)Multimodality imaging probes: design and challenges. Chem. Rev. 110, 3146–3195. (13) Kuil, J., Velders, A. H., and Van Leeuwen, F. W. B. (2010) Multimodal tumor-targeting peptides functionalized with both a radioand a fluorescent-label. Bioconjugate Chem. 21, 1709–1719. (14) Culver, J., Akers, W., and Achilefu, S. (2008)Multimodality molecular imaging with combined optical and SPECT/PET modalities. J. Nucl. Med. 49, 169–172. (15) Buckle, T., Chin, P. T., van den Berg, N. S., Loo, C. E., Koops, W., Gilhuijs, K. G., and van Leeuwen, F. W. (2010)Tumor bracketing and safety margin estimation using multimodal marker seeds: a proof of concept. J. Biomed. Opt. 15, 056021. (16) Buckle, T., van Leeuwen, A. C., Chin, P. T. K., Janssen, H., Muller, S. H., Jonkers, J., and van Leeuwen, F. W. B. (2010)A selfassembled multimodal complex for combined pre- and intraoperative imaging of the sentinel lymph node. Nanotechnology 21, 355101. (17) Van der Poel, H. G., Buckle, T., Brouwer, O. R., Valdes Olmos, R. A., and Van Leeuwen, F. W. B. (2011)Intraoperative laparoscopic fluorescence guidance to the sentinel lymph node in prostate cancer patients; clinical proof of concept of an integrated functional imaging approach using a multimodal tracer. Eur. Urol., doi:10.1016/ j.eururo.2011.03.024. (18) Tamamura, H., Omagari, A., Hiramatsu, K., Gotoh, K., Kanamoto, T., Xu, Y., Kodama, E., Matsuoka, M., Hattori, T., Yamamoto, N., Nakashima, H., Otaka, A., and Fujii, N. (2001)Development of specific CXCR4 inhibitors possessing high selectivity indexes as well as complete stability in serum based on an anti-HIV peptide T140. Bioorg. Med. Chem. Lett. 11, 1897–1902. (19) Tamamura, H., Hiramatsu, K., Kusano, S., Terakubo, S., Yamamoto, N., Trent, J. O., Wang, Z., Peiper, S. C., Nakashima, H., Otaka, A., and Fujii, N. (2003)Synthesis of potent CXCR4 inhibitors possessing low cytotoxicity and improved biostability based on T140 derivatives. Org. Biomol. Chem. 1, 3656–3662. (20) Tamamura, H., Sugioka, M., Odagaki, Y., Omagari, A., Kan, Y., Oishi, S., Nakashima, H., Yamamoto, N., Peiper, S. C., Hamanaka, N., Otaka, A., and Fujii, N. (2001)Conformational study of a highly specific CXCR4 inhibitor, T140, disclosing the close proximity of its intrinsic pharmacophores associated with strong anti-HIV activity. Bioorg. Med. Chem. Lett. 11, 359–362. (21) Trent, J. O., Wang, Z. X., Murray, J. L., Shao, W., Tamamura, H., Fujii, N., and Peiper, S. C. (2003)Lipid bilayer simulations of CXCR4 with inverse agonists and weak partial agonists. J. Biol. Chem. 278, 47136–47144.
Figure 4. (A) SPECT/CT (axial) and (B) surgical fluorescence image of a MIN-O tumor bearing mouse.
Table 3. Biodistribution of Ac-TZ14011-MSAP in FVB Mice tissue
uptake (%ID/g)
blood
0.13 ( 0.02
brain lungs
0.02 ( 0.00 0.93 ( 0.12
heart
0.83 ( 0.20
liver
19.46 ( 4.84
kidneys
6.89 ( 1.29
spleen
4.38 ( 1.16
stomach
0.75 ( 0.14
intestines
1.70 ( 0.08
muscle
0.29 ( 0.01
studies showed the potential of Ac-TZ14011-MSAP for image guided surgery applications.
’ ASSOCIATED CONTENT
bS
Supporting Information. Experimental procedures and spectra of 3 and 4. This material is available free of charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION Corresponding Author
*E-mail:
[email protected]. Author Contributions #
These authors contributed equally to this work.
’ ACKNOWLEDGMENT This research is supported, in part, by a KWF-translational research award (Grant No. PGF 2009-4344; FvL), and within the framework of CTMM, the Centre for Translational Molecular Medicine (http://www.ctmm.nl), project Breast CARE (grant 030-104; JK). ’ REFERENCES (1) Furusato, B., Mohamed, A., Uhlen, M., and Rhim, J. S. (2010) CXCR4 and cancer. Pathol. Int. 60, 497–505. (2) Bhandari, D., Robia, S. L., and Marchese, A. (2009)The E3 ubiquitin ligase atrophin interacting protein 4 binds directly to the chemokine receptor CXCR4 via a novel WW domain-mediated interaction. Mol. Biol. Cell 20, 1324–1339. 863
dx.doi.org/10.1021/bc2000947 |Bioconjugate Chem. 2011, 22, 859–864
Bioconjugate Chemistry
COMMUNICATION
(22) Wu, B., Chien, E. Y. T., Mol, C. D., Fenalti, G., Liu, W., Katritch, V., Abagyan, R., Brooun, A., Wells, P., Bi, F. C., Hamel, D. J., Kuhn, P., Handel, T. M., Cherezov, V., and Stevens, R. C. (2010)Structures of the CXCR4 chemokine GPCR with small-molecule and cyclic peptide antagonists. Science 330, 1066–1071. (23) Nomura, W., Tanabe, Y., Tsutsumi, H., Tanaka, T., Ohba, K., Yamamoto, N., and Tamamura, H. (2008)Fluorophore labeling enables imaging and evaluation of specific CXCR4-ligand interaction at the cell membrane for fluorescence-based screening. Bioconjugate Chem. 19, 1917–1920. (24) Oishi, S., Masuda, R., Evans, B., Ueda, S., Goto, Y., Ohno, H., Hirasawa, A., Tsujimoto, G., Wang, Z., Peiper, S. C., Naito, T., Kodama, E., Matsuoka, M., and Fujii, N. (2008)Synthesis and application of fluorescein- and biotin-labeled molecular probes for the chemokine receptor CXCR4. ChemBioChem 9, 1154–1158. (25) Hanaoka, H., Mukai, T., Tamamura, H., Mori, T., Ishino, S., Ogawa, K., Iida, Y., Doi, R., Fujii, N., and Saji, H. (2006)Development of 111 a In-labeled peptide derivative targeting a chemokine receptor, CXCR4, for imaging tumors. Nucl. Med. Biol. 33, 489–494. (26) Nishizawa, K., Nishiyama, H., Oishi, S., Tanahara, N., Kotani, H., Mikami, Y., Toda, Y., Evans, B. J., Peiper, S. C., Saito, R., Watanabe, J., Fujii, N., and Ogawa, O. (2010)Fluorescent imaging of high-grade bladder cancer using a specific antagonist for chemokine receptor CXCR4. Int. J. Cancer 127, 1180–1187. (27) Garanger, E., Aikawa, E., Reynolds, F., Weissleder, R., and Josephson, L. (2008)Simplified syntheses of complex multifunctional nanomaterials. Chem. Commun. 4792–4794. (28) Garanger, E., Blois, J., Hilderbrand, S. A., Shao, F., and Josephson, L. (2010)Divergent oriented synthesis for the design of reagents for protein conjugation. J. Comb. Chem. 12, 57–64. (29) Shao, F., Yuan, H., Josephson, L., Weissleder, R., and Hilderbrand, S. A. (2011)Facile synthesis of monofunctional pentamethine carbocyanine fluorophores. Dyes Pigments 90, 119–122. (30) Moulin, C., Amekraz, B., Steiner, V., Plancque, G., and Ansoborlo, E. (2003)Speciation studies on DTPA using the complementary nature of electrospray ionization mass spectrometry and timeresolved laser-induced fluorescence. Appl. Spectrosc. 57, 1151–1161. (31) Maecke, H. R., Riesen, A., and Ritter, W. (1989)The molecular structure of indium-DTPA. J. Nucl. Med. 30, 1235–1239. (32) Schottelius, M., and Wester, H. J. (2009)Molecular imaging targeting peptide receptors. Methods 48, 161–177.
864
dx.doi.org/10.1021/bc2000947 |Bioconjugate Chem. 2011, 22, 859–864
