18^{18}F-Markierung hochaffiner all-D-Peptide zur in vivoin \ vivo Diagnostik von β\beta-Amyloid-Plaques

For the Development of new selective radiotracers which can be used for $\textit{in vivo}$ imaging of $\beta$-Amyloid plaques in the context of Alzerheimer’s Disease by positron-emission-tomography this study aimed at the application of various strategies for $^{18}$F-labelling of the all-d peptide D3 (sequence: RPRTRLHTHRNR). In previous reports D3 showed promising results for the affinity to $\beta$-Amyloid. The labelling reaction with acylation agents required an extension of the original peptide with a lysine residue at the C-terminus. The prosthetic group succinimidyl-4-[$^{18}$F]fluorobenzoate could be produced with a radiochemical yield of 41 - 60 %. While conjugation with various model compounds gave good results the coupling with the D3-derivative was not possible.Through the extension of D3 with cysteine instead of lysine it was possible touse thiol-reactive prosthetic groups. For this 4-[$^{18}$F]fluorobenzylmaleimide was preparedby two different synthetic routes. But neither one of them lead to a radiochemicalyield above 5% so further use was neglected. The preparation of 1-[3-(2-[$^{18}$F]fluoropyridine-3-oxyl)propyl]pyrrol-2,5-dione succeeded by a three-step radiosynthesis within 110 minutes and lead to a radiochemical of 2-20 %. The development of a new strategy via the protection of the maleimide function allowed toperform the radiosynthesis in only two steps within 60 minutes and an overall radiochemicalyield of about 20 ± 5%. The following coupling with the all-d peptidecould be done with yields of about 95% within 15 minutes. This, the synthesis ofthe radiofluorinated D3-derivative including all purification steps could be achievedwithin 120 minutes.The successfully $^{18}$F-labelled D3-derivative was used for further preclinical $\textit{in vitro}$investigation by performing autoradiography of mouse brain slices which expressed$\beta$-Amyloid plaques. Optimisation of the parameters of incubation were done in orderto reduce the affinity of the compounds to the glass surface. It could be determinedthat the l-enantiomer of the radiofluorinated D3-derivative showed no affinity to thetissue. However the all-d peptide showed an increased uptake at some tissue regionswith enrichment of $\beta$-Amyloid

TSLP-activated dendritic cells induce human T follicular helper cell differentiation through OX40-ligand.

T follicular helper cells (Tfh) are important regulators of humoral responses. Human Tfh polarization pathways have been thus far associated with Th1 and Th17 polarization pathways. How human Tfh cells differentiate in Th2-skewed environments is unknown. We show that thymic stromal lymphopoietin (TSLP)-activated dendritic cells (DCs) promote human Tfh differentiation from naive CD4 T cells. We identified a novel population, distinct from Th2 cells, expressing IL-21 and TNF, suggestive of inflammatory cells. TSLP-induced T cells expressed CXCR5, CXCL13, ICOS, PD1, BCL6, BTLA, and SAP, among other Tfh markers. Functionally, TSLP-DC-polarized T cells induced IgE secretion by memory B cells, and this depended on IL-4Rα. TSLP-activated DCs stimulated circulating memory Tfh cells to produce IL-21 and CXCL13. Mechanistically, TSLP-induced Tfh differentiation depended on OX40-ligand, but not on ICOS-ligand. Our results delineate a pathway of human Tfh differentiation in Th2 environments

New strategy of a two-step radiosynthesis of [F-18]fluoropyridine-based maleimide-containing prosthetic groups for labelling of peptides and proteins

Objectives The use of thiol-reactive groups allows the introduction of fluorine-18 in a peptide or protein with cysteine residues, and different radiofluorinated maleimide-containing prosthetic groups have been described in the literature. All compounds were, however, prepared by a two- or three-step synthesis with an overall reaction time of at least 70 minutes. The aim of this work was to improve the radiosynthesis of maleimide-containing compounds with reduced reaction steps by using a new synthetic route via protection of the maleimide function. This was first done here with the example of 1-[3-(2-[18F]fluoropyridin-3-oxy)propyl]pyrrol-2,5-dione ([18F]FPyME) which is usually prepared in three steps [1]. Methods The first step for the preparation of the precursor was a Williamson reaction to form the ether from 3-hydroxy-2-nitropyridine with 1,3-dibromopropane. In a parallel process maleimide was protected with 2,5-dimethylfurane via a Diels-Alder-reaction. These two products were combined by N-alkylation to form the precursor. The radiosynthesis includes the introduction of fluorine-18 by nucleophilic substitution of the nitro group followed by deprotection of the maleimide function (see fig. 1). The reaction steps were optimized with regard to temperature, time and solvents. Results The optimal conditions for the n.c.a. radiofluorination were identified at a temperature of 80 °C in DMSO and a reaction time of 5 minutes, resulting in a radiochemical yield of about 29 ± 3 %. At higher temperatures the deprotection of the precursor and the labelled compound come to the fore and thereby the decomposition of the unprotected maleimide occurs due to the basic reaction conditions. The deprotection step was quantitatively carried out within 15 minutes. [18F]FPyME was isolated by HPLC to provide the pure prosthetic group which was directly used for effective peptide labelling. The overall synthesis time was about 60 minutes and the overall radiochemical yield was about 20 %. Conclusions The described synthetic route provides the possibility to gain a variety of further [18F]fluoropyridine-based maleimide-containing compounds in two steps only. In addition, this method offers to be performed as one-pot synthesis. Acknowledgements References [1] de Bruin B. et al. (2006) Bioconjugate Chem., 16, 406-420