Efficient (18)F-labeling of large 37-amino-acid pHLIP peptide analogues and their biological evaluation

Abstract

Solid tumors often develop an acidic microenvironment, which plays a critical role in tumor progression and is associated with increased level of invasion and metastasis. The 37-residue pH (low) insertion peptide (pHLIP) is under study as an imaging platform because of its unique ability to insert into cell membranes at a low extracellular pH (pH(e) < 7). Labeling of peptides with [(18)F]-fluorine is usually performed via prosthetic groups using chemoselective coupling reactions. One of the most successful procedures involves the alkyne-azide copper(I) catalyzed cycloaddition (CuAAC). However, none of the known "click" methods have been applied to peptides as large as pHLIP. We designed a novel prosthetic group and extended the use of the CuAAC "click chemistry" for the simple and efficient (18)F-labeling of large peptides. For the evaluation of this labeling approach, a D-amino acid analogue of WT-pHLIP and an L-amino acid control peptide K-pHLIP, both functionalized at the N-terminus with 6-azidohexanoic acid, were used. The novel 6-[(18)F]fluoro-2-ethynylpyridine prosthetic group, was obtained via nucleophilic substitution on the corresponding bromo-precursor after 10 min at 130 °C with a radiochemical yield of 27.5 ± 6.6% (decay corrected) with high radiochemical purity ≥98%. The subsequent Cu(I)-catalyzed "click" reaction with the azido functionalized pHLIP peptides was quantitative within 5 min at 70 °C in a mixture of water and ethanol using Cu-acetate and sodium L-ascorbate. [(18)F]-D-WT-pHLIP and [(18)F]-L-K-pHLIP were obtained with total radiochemical yields of 5-20% after HPLC purification. The total reaction time was 85 min including formulation. In vitro stability tests revealed high stability of the [(18)F]-D-WT-pHLIP in human and mouse plasma after 120 min, with the parent tracer remaining intact at 65% and 85%, respectively. PET imaging and biodistribution studies in LNCaP and PC-3 xenografted mice with the [(18)F]-D-WT-pHLIP and the negative control [(18)F]-L-K-pHLIP revealed pH-dependent tumor retention. This reliable and efficient protocol promises to be useful for the (18)F-labeling of large peptides such as pHLIP and will accelerate the evaluation of numerous [(18)F]-pHLIP analogues as potential PET tracers.

Figure 1

Examples of [¹⁸F]-labeled prosthetic groups for the CuAAC “click” reaction with functionalized peptides and proteins.

Figure 2

HPLC chromatograms with radio and UV-traces (UV traces were recorded at 254 nm and retention times are given with respect to the radiotrace): (A) co-injection of compound 3 and [¹⁸F]-3 with t_R = 10.4 min on column 1; (B) purification of [¹⁸F]-4a and −4b after 5 min at 70 °C with t_R = 18.7 min and t_R = 20.2 min, respectively; (C) purification of [¹⁸F]-5 after 5 min reaction at 70 °C (t_R = 15.8 min). HPLC chromatograms were recorded utilizing column 1 applying elution conditions A.

Figure 3

Representative coronal PET images of mice bearing LNCaP (Right) and PC-3 (Left) tumors injected with [¹⁸F]-4 (A) and [¹⁸F]-5 (B).

Figure 4

Combined histological/autoradiographic analysis obtained after [¹⁸F]-4 injection (4h p. i.). Data from a single 10 μm frozen section obtained from a LNCaP tumor. H&E staining (panel A), the vascular perfusion marker Hoechst 33342 (blue, panel B) and autoradiograph of radioactivity distribution (panel C) are shown. Panel D shows co-registration of autoradiograph (pseudo-color green) overlaid with Hoechst 33342 (blue). Panel E contains a high-magnification region from the image shown in Panel D. Scale bar = 1mm.

Figure 5

Biodistribution of [¹⁸F]-4 and [¹⁸F]-5 at 2 and 4 h p. i. with uptake values expressed as %ID/g.

Scheme 1

Synthetic route yielding the non-radiolabeled reference compounds 3 to 5.

Scheme 2

Radiosynthesis yielding radiolabeled products [¹⁸F]-3 to [¹⁸F]-5.