New Methods for Labeling RGD Peptides with Bromine-76

Area of Science:

• Radiochemistry
• Medical Imaging
• Organic Synthesis

Background:

• Direct bromination of tyrosine residues with bromine-76 (Br-76) is established for peptide and antibody PET imaging probes.
• Peptides lacking tyrosine require prosthetic groups for Br-76 labeling via amine conjugation.

Purpose of the Study:

• To develop novel strategies for Br-76 labeling of small peptides, accommodating both direct and prosthetic group approaches.
• To synthesize and evaluate a new prosthetic agent, N-succinimidyl-3-[(76)Br]bromo-2,6-dimethoxybenzoate ([(76)Br]SBDMB), for cyclic RGD peptide labeling.
• To investigate the use of N-succinimidyl-2,6-dimethoxybenzoate for pre-attaching a 2,6-dimethoxybenzoyl (DMB) moiety for subsequent Br-76 labeling.

Main Methods:

• Preparation of [(76)Br]SBDMB and DMB-conjugated peptides.
• Competitive cell binding assays to assess integrin receptor affinity.
• Positron Emission Tomography (PET) imaging in U87MG glioblastoma xenograft mice.
• Ex vivo biodistribution studies.
• Computational investigation of bromination reaction mechanisms using DFT (SCRF-B3LYP/6-31G*).

Main Results:

• Direct labeling yields exceeded 50%, while [(76)Br]SBDMB labeling yield was over 60%.
• Conjugation yields using the prosthetic group were over 70%, with pre-conjugated peptide labeling yields over 60%.
• Brominated peptides retained binding affinity for integrin receptors.
• PET imaging demonstrated high tumor uptake of [(76)Br]-BrE[c(RGDyK)](2) and [(76)Br]-BrDMB-E[c(RGDyK)](2) in U87MG tumors.
• Computational studies revealed a lower energy barrier for bromination on the dimethoxybenzoyl group compared to tyrosine.

Conclusions:

• The developed N-succinimidyl-2,6-dimethoxybenzoate molecule serves as a versatile precursor for both one-step pre-conjugation and indirect labeling via a Br-76 prosthetic group.
• Radiobromination on the dimethoxybenzoyl moiety exhibits selectivity over tyrosine residues, offering advantages for complex labeling strategies.
• The novel labeling strategies facilitate the development of effective Br-76 PET imaging agents for cancer diagnostics.