Theranostics 2011; 1:341-353. doi:10.7150/thno/v01p0341

Research Paper

New Methods for Labeling RGD Peptides with Bromine-76

Lixin Lang1, Weihua Li1, 2, Hong-Mei Jia3, De-Cai Fang3, Shushu Zhang1, Xilin Sun2, Lei Zhu1, Ying Ma1, Baozhong Shen2, Dale O. Kiesewetter1, Gang Niu1, 4, Xiaoyuan Chen1 ✉

1. Laboratory of Molecular Imaging and Nanomedicine (LOMIN), National Institute of Biomedical Imaging and Bioengineering (NIBIB), National Institutes of Health (NIH), Bethesda, Maryland, 20892, USA
2. Department of Medical Imaging and Nuclear Medicine, the Fourth Affiliated Hospital, Harbin Medical University, Harbin 150001, China
3. Key Laboratory of Radiopharmaceuticals, College of Chemistry, Beijing Normal University, Beijing 100875, China
4. Imaging Sciences Training Program, Radiology and Imaging Sciences, Clinical Center and National Institute Biomedical Imaging and Bioengineering, NIH, Bethesda, Maryland, 20892, USA


Direct bromination of the tyrosine residues of peptides and antibodies with bromine-76, to create probes for PET imaging, has been reported. For peptides that do not contain tyrosine residues, however, a prosthetic group is required to achieve labeling via conjugation to other functional groups such as terminal α-amines or lysine ε-amines. The goal of this study was to develop new strategies for labeling small peptides with Br-76 using either a direct labeling method or a prosthetic group, depending on the available functional group on the peptides. A new labeling agent, N-succinimidyl-3-[76Br]bromo-2,6-dimethoxybenzoate ([76Br]SBDMB) was prepared for cyclic RGD peptide labeling. N-succinimidyl-2, 6-dimethoxybenzoate was also used to pre-attach a 2, 6-dimethoxybenzoyl (DMB) moiety to the peptide, which could then be labeled with Br-76. A competitive cell binding assay was performed to determine the binding affinity of the brominated peptides. PET imaging of U87MG human glioblastoma xenografted mice was performed using [76Br]-BrE[c(RGDyK)]2 and [76Br]-BrDMB-E[c(RGDyK)]2. An ex vivo biodistribution assay was performed to confirm PET quantification. The mechanisms of bromination reaction between DMB-c(RGDyK) and the brominating agent CH3COOBr were investigated with the SCRF-B3LYP/6-31G* method with the Gaussian 09 program package. The yield for direct labeling of c(RGDyK) and E[c(RGDyK)]2 using chloramine-T and peracetic acid at ambient temperature was greater than 50%. The yield for [76Br]SBDMB was over 60% using peracetic acid. The conjugation yields for labeling c(RGDfK) and c(RGDyK) were over 70% using the prosthetic group at room temperature. Labeling yield for pre-conjugated peptides was over 60%. SDMB conjugation and bromination did not affect the binding affinity of the peptides with integrin receptors. Both [76Br]Br-E[c(RGDyK)]2 and [76Br]BrDMB-E[c(RGDyK)]2 showed high tumor uptake in U87MG tumor bearing mice. The specificity of the imaging tracers was confirmed by decreased tumor uptake after co-administration of unlabeled dimeric RGD peptides. The energy barrier of the transition state of bromination for the dimethoxybenzoyl group was about 9 kcal/mol lower than that for the tyrosine residue. In conclusion, the newly developed N-succinimidyl-2, 6-dimethoxybenzoate molecule can be used either for one step labeling through pre-conjugation or as the precursor for a Br-76 labeled prosthetic group for indirect labeling. Radiobromination on a dimethoxybenzoyl group has selectivity over radiobromination on tyrosine. The energy barrier difference of the transition states of bromination between the dimethoxybenzoyl group and the tyrosine residue may account for the reaction selectivity when both groups are present in the same molecule.

Keywords: Bromine-76, Arg-Gly-Asp (RGD) peptides, positron emission tomography (PET), N-succinimidyl-2, 6-dimethoxybenzoate

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How to cite this article:
Lang L, Li W, Jia HM, Fang DC, Zhang S, Sun X, Zhu L, Ma Y, Shen B, Kiesewetter DO, Niu G, Chen X. New Methods for Labeling RGD Peptides with Bromine-76. Theranostics 2011; 1:341-353. doi:10.7150/thno/v01p0341. Available from