Chemo- and regioselective 18F-labelling of peptides bearing free amino- and sulfhydryl-groups

Abstract

The radioactive prosthetic group 6-[18F]fluoronicotinic acid ([18F]FNA) has been conjugated with numerous biomolecules using its activated esters. All reported conjugations had resulted in N-acylated products, except the conjugation with peptide ACooP (H-ACGLSGLGVA-NH2) in 2024, as reported by Dillemuth et al. ACooP is a variant of the nonapeptide CooP (H-CGLSGLGVA-NH2), which has been seen in recent studies to be a brain tumor-homing peptide that targets fatty acid-binding protein 3 (FABP3), which is also known as a heart-type fatty acid-binding protein or a mammary-derived growth inhibitor. Of the many challenges posed by synthetic chemistry, the achievement of both high-level chemo- and regioselectivity in various biomolecules such as proteins, peptides, and some macromolecules serves as one of the toughest challenges posed to the study. Lately, Dillemuth and coworkers synthesised radiolabelled CooP and ACooP using the prosthetic chemical [18F]FNA 4-nitrophenyl ester to develop radiotracers for positron emission tomography (PET). The conjugation yielded the products [18F]FNA-N-CooP and [18F]FNA-S-ACooP. The disparity in acylation prompted the notion that intramolecular transfer or acyl migration transpired in CooP rather than ACooP, owing to unfavourable circumstances in the transition state of conjugation with ACooP. To verify this hypothesis, studies were conducted utilising FNA and four new peptides C@1-K (H-CGLSGLGVAK-NH₂), C@3 (H-AGCLSGLGVA-NH₂), C@4 (H-AGLCSGLGVA-NH₂), and C@5 (H-AGLSCGLGVA-NH₂). The aim of this study was to radiosynthesize and understand the mechanism underlying the chemoselectivity and regioselectivity of the [18F]FNA with the variants of the ACooP peptides, in this case four new peptides. The radiosynthesis was carried out using two of the peptides C@5 and C@1-K. In the radiosynthesis of C@5, a radiochemical purity of 98.5 % ± 1.3 (n=3) and a decay-corrected radiochemical yield of 14.4 ± 7.4% were obtained. Two syntheses were carried out for C@1-K, with an average radiochemical purity of 98.6% (n = 2) and a decay-corrected radiochemical yield of 6.4% (n = 2). The non-radioactive products of the four peptides were analysed with liquid chromatography-mass spectroscopy (LC-MS)/MS for confirmation of the products and side products. We proceeded with characterisation and structural confirmation of the conjugated product of the four products (C@1-K, C@3, C@4, and C@5) In the LC-MS/MS study for two of the peptides, specifically C@1-K and C@3, certain side products were identified with additional modifications at various other sites. These were also observed during high-performance liquid chromatography purification. The side products were isolated during purification; however, upon drying for nuclear magnetic resonance (NMR) analysis, they yielded minimal to nearly negligible quantities of products that did not allow accurate analysis. The NMR results after analysing the four peptides confirmed the following: C@3, C@4, and C@5 were S-acylated, while C@1-K was N-acylated at the cysteine residue. The regioselectivity was also confirmed with the NMR, with conjugation occurring at the cysteine site. Both commercial and in-house prepared FNA-N-C@1-K were found to be impure. Still, we found that N-acylation of cysteine was observed in the product, so the other part of the product that makes it impure is a question that needs investigating. The results confirmed the hypothesis proposed. Further study would be essential to aid the synthesis of S-acylated tracers when needed.