Chemoselective hydrazone formation between HYNIC-functionalized peptides and ¹⁸F-fluorinated aldehydes

Introduction: Since the demand for ¹⁸F-fluorinated peptides for quantitative in vivo receptor imaging using PET has increased, a new chemoselective two-step ¹⁸F-labeling strategy based on hydrazone formation between an unprotected hydrazine-functionalized peptide and an ¹⁸F-labeled aldehyde was developed. Methods: First, 4-[ ¹⁸F]fluorobenzaldehyde ([¹⁸F]FB-CHO) was prepared from 4-formyl-N,N,N-trimethylanilinium triflate via direct no-carrier-added ¹⁸F-fluorination (dimethyl sulfoxide, 90°C, 5 min) and purified by RP-HPLC. Hydrazone formation between [¹⁸F]FB-CHO and 6-hydrazinonicotinic acid (HYNIC) and the unprotected HYNIC-functionalized peptides (HYNIC-d-Phe¹)-Tyr³-Thr⁸-octreotide and (HYNIC-Arg¹)-substance P was evaluated with respect to the dependence of radiochemical yield on pH, precursor concentration and temperature. The stability of [¹⁸F]FB-CH=N-HYNIC-Tyr ³-Thr⁸(NH₂)-octreotide in aqueous solution at various pH (4.0, 5.5 and 7.5) as well as the in vivo stability of [ ¹⁸F]FB-CH=N-HYNIC-Tyr³-Thr⁸-octreotide in mouse blood (30 min p.i.) was investigated. Results: Yields of the hydrazone formation were independent of pH between pH 0.5 and 5.5. Optimal labeling yields of 85% were obtained with a precursor concentration of 2.1 mM at 70°C for 10 min. The labeling products were stable at pH 7.5 at 37°C, while in more acidic media (pH 4.0) the product slowly decomposed to form up to 31±2% [¹⁸F]FB-CHO within 5 h. Metabolite studies showed no detectable degradation of [¹⁸F]FB-CH=N-HYNIC-Tyr³-Thr ⁸-octreotide in mouse blood (30 min p.i.). Conclusions: In conclusion, chemoselective hydrazone formation between unprotected HYNIC-functionalized peptides and [¹⁸F]FB-CHO is a fast and straightforward radiolabeling method leading to high yields under mild acidic conditions. In addition, it represents a powerful and versatile radiolabeling strategy that is applicable to a variety of radionuclides and peptide precursors already available for ^99mTc labeling.