Direct One-Step¹⁸F-Labeling of Peptides via Nucleophilic Aromatic Substitution

Abstract

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.

18F-Labeled Bombesin Analog for Specific and Effective Targeting of Prostate Tumors Expressing Gastrin-Releasing Peptide Receptors

“…Schirrmacher et al reported the direct radiolabeling of an organosilicon-modified peptide in an isotope exchange reaction (32). Several 1-step approaches for the facile 18 F labeling of peptides have been developed in our laboratories (19,31,33). In the present study, 18 F labeling of BAY 86-4367 was achieved in a single step via aromatic nucleophilic substitution.…”

“…The binding affinities of the nonradioactive 19 The inhibitor used for displacement was either nonradioactive 19 F-bombesin (1b) or neuromedin B (Sigma). The test compounds were dissolved in DMSO to produce 1 mM stock solutions and further diluted in incubation buffer to 2 pM-1,000 nM.…”

“…Figure 1 shows the sequences of 1a and 1b and the structures of the nonnatural amino acid derivatives. The synthesized bombesin precursor and reference standard were characterized by mass spectrometry: Direct 1-step 18 F labeling via aromatic nucleophilic substitution of 18 F for 1 N(CH 3 ) 3 -developed previously in our laboratories-was successfully applied to the bombesin peptide analog (19). Using DMSO as the solvent resulted in a higher level of 18 F incorporation than using N,Ndimethylformamide at all of the tested reaction temperatures.…”

“…The distribution coefficient of the nonradioactive 19 F-bombesin derivative (1b) was lower than 23.9. The direct labeling of peptides with 18 F in a single step was recently described by various groups (19,(31)(32)(33). Schirrmacher et al reported the direct radiolabeling of an organosilicon-modified peptide in an isotope exchange reaction (32).…”

“…Potent 99m Tc-based (demobesin-1) and 111 In-based (RM-1) bombesin analogs that were designed for GRPr-based targeting and tested in the PC-3 xenograft model showed high absolute tumor uptake in animals (13,14). Few publications have reported on 18 F labeling of bombesin peptides (15)(16)(17)(18)(19). Our previous study indicated that the negatively charged bombesin derivative 3-cyano-4-18 F-fluoro-benzoyl-Ala(SO 3 H)-Ava-Gln-TrpAla-Val-NMeGly-His-Sta-Leu-NH 2 had much higher prostate tumor uptake than the corresponding positively charged analog (3-cyano-4-18 F-fluoro-benzoyl-Arg-Ava-Gln-Trp-AlaVal-NMeGly-His-Sta-Leu-NH 2 ) (20).…”