Fluorine-18 labeling and simultaneous glycosylation of the model peptide demobesin 1 by the novel prosthetic group, keto-[¹⁸F]FDG

Abstract

With the development of keto-[¹⁸F]FDG in this work a new powerful prosthetic group for labeling of carbonyl reactive compounds has been added to the tools available for labeling biomelecules with the radionuclide fluorine-18. The main feature that distinguishes this prosthetic group from other existing fluorine-18 labeled carbonyl compounds is the starting material. Whereas most of the existing compounds start with [¹⁸F]-fluoride as primary labeling precursor, keto-[¹⁸F]FDG starts with 2 [¹⁸F]fluorodeoxyglucose ([¹⁸F]FDG) as the radionuclide i.e. fluorine 18 bearing precursor. In terms of availability this circumstance has no restrictions as both [¹⁸F]fluoride as well as [¹⁸F]FDG are readily available to every site that is equipped with a medical cyclotron for the purpose of producing and supplying PET drugs. To the contrary [¹⁸F]FDG is even more widely available as it includes all [¹⁸F]FDG customer sites and [¹⁸F]FDG is the major workhorse of PET. However, in terms of chemical and biochemical properties of the resulting labeled compounds, using [¹⁸F]FDG as precursor leads itself to glycosylated biomolecules of interest. As such keto-[¹⁸F]FDG might be of use only in selected cases where glycosylation may result in improved biochemical behavior of the labeled compound of interest. In this context it is important to note that the use of keto [¹⁸F]FDG, in comparison to [¹⁸F]FDG by itself as glycosylation agent, proved to be far superior with respect to its chemical reactivity. For example a low concentration of only 0.05 mg/ml O benzylhydroxylamine when reacted with keto [¹⁸F]FDG was able to give the same high radiochemical yield of oxime as compared to a 5 mg/ml O benzylhydroxylamine concentration needed for [¹⁸F]FDG to obtain the same high radiochemical yield and this even at lower reaction temperature of 60 oC (in case of keto [¹⁸F]FDG) compared to 80 oC (in case of [¹⁸F]FDG). Keto-[¹⁸F]FDG also offers the additional advantage that it may be purified from accompanying stable keto-glucose by means of a simple SPE based purification procedure which was also developed in the course of this work and was able to remove the keto glucose to non detectable levels as measured by HPLC, while purification of [¹⁸F]FDG from glucose is to our knowledge only possible using much more elaborate HPLC methods. This has important consequences with respect to the apparent specific activity of the final product and also for the absolute amount of biomolecules needed to achieve high radiochemical yields. Another interesting aspect is that keto-[¹⁸F]FDG modified molecules in contrast to [¹⁸F]FDG will retain the carbohydrate moiety in its ring closed form while using [¹⁸F]FDG alone will lead to a ring opened structure.<br /> Using keto-[¹⁸F]FDG or [¹⁸F]FDG as carbonyl group bearing prosthetic groups for labeling of biomolecules requires of course a carbonyl reactive moiety on the side of the biomolecule. The targetted group of biomolecules to be [¹⁸F]-fluoroglycosylated in this work were bioactive peptides i.e. the bombesin analog demobesin 1. As peptides including demobesin 1 do usually not consist of the strongly carbonyl reactive moieties such as hydrazides or oxyamines these have to be attached to the peptides before the actual radiolabeling step can be performed. Therefore a series of bifunctional agents bearing boc-protected oxyamines or hydrazines on one end and an activated ester on the other end and as such acting as “linker” between the peptides amino groups and the carbonyl group of keto-[¹⁸F]FDG was synthesized and tested first by using simple amines, then the model peptide RC160 and finally the bombesin analog demobesin 1.<br /> Two derivatives were synthesized and radiolabeled. One was obtained by simple aminooxyacetylation while the second derivative was reacted with a special linker introducing not only the aminooxy moiety but also an additional lysine residue in order to add another potential positive charge to the peptide.<br /> Within the group of simple amines they were easily modified by the synthesized linkers and reacted readily to form the expected oximes upon reaction with keto [¹⁸F]FDG. Within the group of peptides RC160 was chosen as a first test example. RC160 was modified at the lysine group of the peptide by both linker and reacted with keto-[¹⁸F]FDG to a radiochemical yield of 40±2 % at a peptide concentration of 0.5 mg/ml. Demobesin 1 was also modified at D penylalanine (D Phe) residue of the peptide by both linker and reacted with keto-[¹⁸F]FDG. The high radiochemical yield of up to 70 % at a peptide concentration as low as 0.25 mg/ml. <br /> In summary, keto-[¹⁸F]FDG proved to be a very useful and highly reactive [¹⁸F] fluoroglycosylation agent. It can be easily prepared in one step from [¹⁸F]FDG with high radiochemical yields of > 90 % in 15 minutes. If desired additional purification from the keto-glucose analog is possible by using a simple SPE based method. The high radiochemical yields of with the bombesin analog demobesin 1 at a low peptide concentration show the high potential of this method for glycosylation and simultaneous fluorine-18 labeling of complex biomolecules.