In vitro and in vivo characterization of novel 18F-labeled bombesin analogues for targeting GRPR-positive tumors.

The gastrin-releasing peptide receptor (GRPR) is overexpressed on a number of human tumors and has been targeted with radiolabeled bombesin analogues for the diagnosis and therapy of these cancers. Seven bombesin analogues containing various linkers and peptide sequences were designed, synthesized, radiolabeled with (18)F, and characterized in vitro and in vivo as potential PET imaging agents. Binding studies displayed nanomolar binding affinities toward human GRPR for all synthesized bombesin analogues. Two high-affinity peptide candidates 6b (K(i) = 0.7 nM) and 7b (K(i) = 0.1 nM) were chosen for further in vivo evaluation. Both tracers revealed specific uptake in GRPR-expressing PC-3 tumors and the pancreas. Compared to [(18)F]6b, compound [(18)F]7b was characterized by superior tumor uptake, higher specificity of tracer uptake, and more favorable tumor-to-nontarget ratios. In vivo PET imaging allowed for the visualization of PC-3 tumor in nude mice suggesting that [(18)F]7b is a promising PET tracer candidate for the diagnosis of GRPR-positive tumors in humans.

Direct one-step 18F-labeling of peptides via nucleophilic aromatic substitution.

Methods for the radiolabeling molecules of interest with [18F]-fluoride need to be rapid, convenient, and efficient. Numerous [18F]-labeled prosthetic groups, e.g., N-succinimidyl 4 [18F]-fluorobenzoate ([18F]-SFB), 4-azidophenacyl-[18F]-fluoride ([18F]-APF), and 1-(3-(2-[18F]fluoropyridin-3-yloxy)propyl)pyrrole-2,5-dione ([18F]-FpyMe), for conjugating to biomolecules have been developed. As the synthesis of these prosthetic groups usually requires multistep procedures, there is still a need for direct methods for the nucleophilic [18F]-fluorination of biomolecules. We report here on the development of a procedure based on the trimethylammonium (TMA) leaving group attached to an aromatic ring and activated with different electron-withdrawing groups (EWGs). A series of model compounds containing different electron-withdrawing substituents, a trimethylammonium leaving group, and carboxylic functionality for subsequent coupling to peptides were designed and synthesized. The optimal model compound, 2-cyano-4-(methoxycarbonyl)-N,N,N-trimethylbenzenaminium trifluoromethanesulfonate, was converted to carboxylic acid and coupled to peptides. The results of the one-step [18F]-fluorination of tetrapeptides and bombesin peptides show that the direct 18F-labeling of peptides is feasible under mild conditions and in good radiochemical yields.

Propagation of melting cooperativity along the phosphodiester backbone of DNA.

The role of the DNA phosphodiester backbone in the transfer of melting cooperativity between two helical domains was experimentally addressed with a helix-bulge-helix DNA model, in which the bulge consisted of a varying number of either conformationally flexible propanediol or conformationally constrained bicyclic anucleosidic phosphodiester backbone units. We found that structural communication between two double helical domains is transferred along the DNA backbone over the equivalent of ca. 12-20 backbone units, depending on whether there is a symmetric or asymmetric distribution of the anucleosidic units on both strands. We observed that extension of anucleosidic units on one strand only suffices to disrupt cooperativity transfer in a similar way as if extension occurs on both strands, indicating that the length of the longest anucleosidic inset determines cooperativity transfer. Furthermore, conformational rigidity of the sugar unit increases the distance of coopertivity transfer along the phosphodiester backbone. This is especially the case when the units are asymmetrically distributed in both strands.