Radiolabelling of isopeptide Nε-(γ-glutamyl)-l-lysine by conjugation with N-succinimidyl-4-[18F]fluorobenzoate
Introduction
Transglutaminases, also referred to as nature's biological glues, are widely occurring enzymes responsible for the post-translational modification of proteins by the formation of inter- and intramolecular isopeptide bonds (Griffin et al., 2002). This enzyme-catalysed cross-linking is characterized by the formation of ε-(γ-glutamyl)lysine bonds. The resulting cross-linked proteins, often of high molecular mass, have been shown to be highly resistant to mechanical and thermal challenge and proteolytic degradation. Transglutaminases of animal origin need Ca2+ for their activity, and they play an important physiological role in blood clotting, wound healing and in the general maintenance of tissue integrity. Based on the use of a recently described Ca2+-independent microbial transglutaminase (Nonaka et al., 1989), the enzyme-catalysed cross-linking of proteins by the formation of isopeptide bonds has also been proposed as an interesting tool in food biotechnology and food science to modify rheological properties of food, such as certain milk and meat products (Zhu et al., 1995; Lauber et al., 2001). However, up to now little information is available concerning the exact biological pathways of isopeptide metabolism in the human body (Nielsen, 1995). Understanding the potential link between the ingestion of isopeptides as quantitatively important components in food items and their biological pathways requires tools for the metabolic characterization of isopeptides in vivo.
In this context, the radiolabelling of isopeptides with the short-lived β+-emitter 18F (t1/2=109.8 min) and subsequent in vivo studies by means of positron emission tomography (PET) represents a promising approach for the radiopharmacological characterization of isopeptides in vivo. Several 18F-labelled bioactive peptides have been shown to be useful PET radiotracers with great clinical and research potential (Bergmann et al., 2002; Fredrickson et al., 2001; Okavi, 2001; Shai et al., 1989). The incorporation of 18F into peptides, proteins or antibodies usually requires the use of prosthetic groups, also referred to as bifunctional labelling agents. A large number of 18F-labelled prosthetic groups have been developed which can be attached to biomolecules via acylation (Block et al., 1988; Herman et al., 1994; Lang and Eckelman, 1994; Vaidyanathan and Zalutsky, 1994; Wester et al., 1996), amidation (Shai et al., 1989), imidation (Kilbourn et al., 1987), alkylation (Kilbourn et al., 1987), photochemical conjugation (Wester et al., 1996) and solid-phase synthesis (Sutcliffe-Goulden et al., 2002). Every method has its own advantages and limitations, but the acylation approach with N-succinimidyl-4-[18F]fluorobenzoate ([18F]SFB) was shown to be the most suitable and versatile 18F-labelling method in terms of in vivo stability and radiochemical yield. Moreover, the coupling with [18F]SFB can be performed under mild conditions of pH and temperature compatible with peptides and proteins. However, [18F]SFB synthesis requires a laborious three-step procedure, and further improvements of the [18F]SFB synthesis are desirable.
In this report we describe an efficient preparation of [18F]SFB based on the convenient one-pot microwave-assisted synthesis of 4-[18F]fluorobenzoic acid ([18F]FBA) to further reduce total synthesis time. [18F]SFB was used for labelling the isopeptide Nε-(γ-glutamyl)-l-lysine 4 through [18F]fluorobenzoylation of the isopeptide 4 α-amino groups. Special attention was paid to the optimization of the labelling conditions required for an efficient coupling with [18F]SFB.
Section snippets
General
1H-NMR spectra were recorded on a Varian Inova-400 at 400 MHz. Chemical shifts (δ) were determined relative to the solvent and converted to the TMS scale. Elemental analyses were performed on a LECO CHNS 932 elemental analyser. Melting points were determined on a Galen III (Cambridge Instruments) melting point apparatus (Leica, Vienna, Austria) and are uncorrected. Low resolution mass spectra were recorded on a MarinerTM mass spectrometer (Biospectrometry, Applied Biosystems, Forster City, USA).
Results and discussion
Several procedures are known for the synthesis of [18F]SFB (Fredrickson et al., 2001; Vaidyanathan and Zaluktsky, 1994; Wester et al., 1996). All methods comprise laborious three-step syntheses based on the preparation of [18F]fluorobenzoic acid [18F]FBA starting from no-carrier-added [18F]fluoride. The fast, convenient and high-yielding access to [18F]FBA is therefore a crucial and important step in the [18F]SFB synthesis.
We have synthesized [18F]SFB based on the convenient microwave-assisted
Acknowledgements
The authors wish to thank S. Preusche for radioisotope production, and M. Grote, T. Kniess and T. Krauss for technical assistance.
References (19)
- et al.
Biodistribution and catabolism of 18F-labelled neurotensin(8-13) analogs
Nucl. Med. Biol.
(2002) - et al.
The use of pentafluorophenyl derivatives for the 18F labelling of proteins
Nucl. Med. Biol.
(1994) - et al.
One-step synthesis of 18F labelled [18F]-N-succinimidyl 4-(fluoromethyl)benzoate for protein labelling
Appl. Radiat. Isot.
(1994) - et al.
A comparative study of n.c.a. fluorine-18 labeling of proteins via acylation and photochemical conjugation
Nucl. Med. Biol.
(1996) - et al.
N.c.a. 18F-fluoroacylation via fluorocarboxylic acid esters
J. Labelled Compd. Radiopharm.
(1988) - et al.
Preparation of n.c.a, [17-18F]-fluoroheptadecanoic acid in high yields via aminopolyether supported nucleophilic fluorination
J. Labelled Compd. Radiopharm.
(1986) - et al.
Labeling of human C-peptide by conjugation with N-succinimidyl-4-[18F] fluorobenzoate
J. Labelled Compd. Radiopharm.
(2001) - et al.
Transglutaminasesnature's biological glues
Biochem. J.
(2002) - et al.
Synthesis of 4-[18F]fluorobenzoyl octreotide and biodistribution in tumour-bearing Lewis rats
J. Labelled Compd. Radiopharm.
(1999)
Cited by (74)
Protease-activated receptor 2 (PAR2)-targeting peptide derivatives for positron emission tomography (PET) imaging
2023, European Journal of Medicinal ChemistryCitation Excerpt :The serum half-life was determined to be > 24 h, indicating a more than sufficient time-length for future use as a potential in vivo PET imaging agent and to continue pursuing this peptide as our lead candidate for 18F-labelling (SI, Fig. S2). The overall synthesis of [18F]29 made use of known radiochemistry and is outlined in Scheme 1 [50,51]. The first two steps in the synthesis involved preparation of the prosthetic group precursor, 32.
Evaluating Radioactive Analogs of Doxorubicin to Quantify ChemoFilter Binding and Whole-Body Positron Emission Tomography/Magnetic Resonance Imaging for Drug Biodistribution
2022, Journal of Vascular and Interventional RadiologyPET Radiochemistry
2021, Molecular Imaging: Principles and PracticePeptidomimetic growth hormone secretagogue derivatives for positron emission tomography imaging of the ghrelin receptor
2018, European Journal of Medicinal ChemistryCitation Excerpt :The lysine residue was found to be unimportant for G-7039-GHS-R1a binding owing to an unfavourable interaction energy between its side-chain amino group and polar Glu197/Arg199 residues [35]. This led us to select the lysine side-chain for 18F-radioisotope insertion in the peptidic (GHRP-1, GHRP-2 and GHRP-6) and peptidomimetic (G-7039, [1-Nal4]G-7039 and ipamorelin) GHSs using the [18F]FB prosthetic group via the pre-activated N-succinimidyl-4-[18F]fluorobenzoate ([18F]SFB) [36]. This modification was first tested by coupling a non-radioactive [18F]FB mimic ([19F]FBA) to the lysine side-chain of each individual GHS class using the orthogonal allyloxycarbonyl (Alloc) protecting group by standard Fmoc-solid phase peptide synthesis (Fmoc-SPPS).
Small Prosthetic Groups in <sup>18</sup>F-Radiochemistry: Useful Auxiliaries for the Design of <sup>18</sup>F-PET Tracers
2017, Seminars in Nuclear Medicine<sup>18</sup>F-nanobody for PET imaging of HER2 overexpressing tumors
2016, Nuclear Medicine and Biology