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| Home > Publications database > Synthese des $^{18}$F-markierten Coenzyms Uridindiphosphatglucose als Basis für die $^{18}$F-Glykosylierung von Glykoproteinen |
| Home > Publications database > Synthese des $^{18}$F-markierten Coenzyms Uridindiphosphatglucose als Basis für die $^{18}$F-Glykosylierung von Glykoproteinen |
| Dissertation / PhD Thesis/Book | PreJuSER-44138 |
2001
Forschungszentrum Jülich Gmbh Zenralbibliothek, Verlag
Jülich
Please use a persistent id in citations: http://hdl.handle.net/2128/20001
Report No.: Juel-3902
Abstract: The chemo-enzymatic radiosynthesis of no carrier added (n.c.a.) uridine diphospho-2-deoxy- 2-[$^{18}$F]fluoro-$\alpha$-D-glucose (UDP-[$^{18}$F]FGlc) was developed. In order to overcome the problem of poor regioselectivity when using the commonly strategy to label proteins via $^{18}$F-labelled prosthetic groups, the use of enzyme systems in addition to the corresponding $^{18}$F-labelled coenzymes was shown to be a reliable, regioselective and mild labelling method. With regard to the comparison and evaluation of the stereoselectivity of the phosphorylating agents used in the chemical synthesis of cold uridine diphospho-2-deoxy-2-fluoro-$\alpha$-Dglucose, $^{31}$P-decoupled and $^{1}$H-NMR-studies were successfully realized. Uridine diphospho- 2-deoxy-2-fluoro-$\alpha$-D-glucose was obtained in a 7 step synthesis. Tetrabenzylpyrophosphate was shown to be a highly stereoselective phosphorylating agent for FDG ($\alpha /\beta$=3:1). Moreover, a multienzymatic pathway for the synthesis of uridine diphospho-2-deoxy-2-fluoro-$\alpha$- D-glucose was adopted starting from FDG and four commercially available enzymes. This strategy was adjusted to a mg-scale synthesis providing 35% chemical yield. Within the scope of this procedure, a comparison of the natural substrate $\alpha$-D-glucose-1-phosphate with 2-fluoro-2-deoxy-$\alpha$-D-glucose-1-phosphate indicated that the enzyme activity of UDP-glucose pyrophosphorylase (UDP-Glc PPase) was decreased by a factor of 30. With regard to the adaptability of the multiple enzyme system for the radiosynthesis of n.c.a. uridine diphospho-2-deoxy-2-[$^{18}$F]fluoro-$\alpha$-D-glucose a rapid hexokinase-mediated phosphorylation of [$^{18}$F]FDG utilizing ATP or UTP as phosphate donor was performed. A further enzymatic isomerization of n.c.a [$^{18}$F]FDG-6-phosphate to n.c.a. [$^{18}$F]FDG-1-phosphate was limited due to the formation of [$^{18}$F]FDG-1.6-diphosphate as main product. Experiments using a multiple enzyme system to develop a fully enzymatic synthetic route to UDP-[$^{18}$F]FGlc turned out to be less efficient due to the necessity of carrier added conditions. Thus, a chemo-enzymatic synthesis of n.c.a. UDP-[$^{18}$8F]FGlc has been developed, starting from 1.3.4.6-tetra-O-acetyl-2-[$^{18}$F]fluoro-2-deoxy-D-glucose, which occurs as an intermediate in the [$^{18}$F]FDG synthesis. The chemical phosphorylation via MacDonald reaction and subsequent deprotection led to a radiochemical yield of 55% of [$^{18}$F]FDG-1-phosphate. UDP- [$^{18}$F]FGlc was synthesized enzymatically by condensation of [$^{18}$F]FDG-1-phosphate with UTP in presence of UDP-Glc PPase. In order to overcome the problem of decreased enzyme acitivty the reaction was performed in a minimized reaction volume and optimized UTP-concentration of 0.5 mmol/l leading to an overall radiochemical yield of 20% of UDP-[$^{18}$F]FGlc within 110 min. The $^{18}$F-labelled coenzyme UDP-[$^{18}$F]FGlc was used as a tool for $^{18}$F-glycosylation of N-acetylglucosamine mediated by $\beta$-1.4-galactosyltransferase. The $^{18}$F-glycosylated product was obtained in a radiochemical yield of 56% and was easily isolated by solid phase extraction. In addition to the general availability of [$^{18}$F]FDG worldwide, this new strategy for enzymatic transfer of "activated [$^{18}$F]FDG" has demonstrated its potential as a highly selective and mild $^{18}$F-labelling method of glycosylated biopolymers to study their pharmacokinetics using positron-emission-tomography.
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