Efficient (18)F-labeling of large 37-amino-acid pHLIP peptide analogues and their biological evaluation.
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.
Department of Radiology, Memorial Sloan-Kettering Cancer Center, New York, NY 10065, USA.
Pierre
P
Daumar
Cindy A
CA
Wanger-Baumann
NagaVaraKishore
N
Pillarsetty
Laura
L
Fabrizio
Sean D
SD
Carlin
Oleg A
OA
Andreev
Yana K
YK
Reshetnyak
Jason S
JS
Lewis
D000595:Amino Acid Sequence; D000818:Animals; D002208:Caproates / Q000737:chemistry; D045744:Cell Line, Tumor; D057930:Click Chemistry; D004355:Drug Stability; D005462:Fluorine Radioisotopes; D006801:Humans; D007553:Isotope Labeling; D008297:Male; D008565:Membrane Proteins / Q000097:blood / Q000737:chemistry* / Q000493:pharmacokinetics; D051379:Mice; D008969:Molecular Sequence Data; D049268:Positron-Emission Tomography; D011854:Radiochemistry
D002208:Caproates; D005462:Fluorine Radioisotopes; D008565:Membrane Proteins; C533201:pHLIP protein; C037652:hexanoic acid