Radiolabelling of isopeptide N^ε-(γ-glutamyl)-l-lysine by conjugation with N-succinimidyl-4-[¹⁸F]fluorobenzoate

Abstract

The isopeptide N^ε-(γ-glutamyl)-l-lysine 4 was labelled with ¹⁸F via N-succinimidyl-4-[¹⁸F]fluorobenzoate ([¹⁸F]SFB). A modified approach for the convenient synthesis of [¹⁸F]SFB was used, and [¹⁸F]SFB could be obtained in decay-corrected radiochemical yields of 44–53% (n=20) and radiochemical purity >95% within 40 min after EOB. For labelling N^ε-(γ-glutamyl)-l-lysine with [¹⁸F]SFB the effects of isopeptide concentration, temperature, and pH were studied to determine the optimum reaction conditions. The coupling reaction was shown to be temperature and pH independent while being strongly affected by the isopeptide concentration. Using the optimized labelling conditions, in a typical experiment 1.3 GBq of [¹⁸F]SFB could be converted into 447 MBq (46%, decay-corrected) of [¹⁸F]fluorobenzoylated isopeptide within 45 min, including HPLC purification.

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 (t_1/2=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 N^ε-(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.