Synthetic routes to 18F-labelled gemcitabine and related 2’-fluoronucleosides

Gemcitabine (2’,2’-difluoro-2’-deoxycytidine, dFdC) is an established chemotherapeutic agent used in the treatment of various carcinomas such as lung, breast, bladder and especially pancreatic cancer. However, its general application and bioavailability is compromised due to both poor cell uptake and rapid metabolism by gut and liver cytidine deaminase (CDA). A 18F-gemcitabine positron emission tomography (PET) probe could enable biodistribution studies and the imaging of gemcitabine pharmacodynamics in vivo. However, the potential and clinical relevance of a 18F-gemcitabine PET probe would have to be evaluated using appropriate PET tumour models. In order to approach the synthetic target molecule 18F-dFdC, proof-of-principle studies on more straightforward synthetic targets including 18F-FAU and 18FFAC (see figure below) were carried out first. An appropriate precursor was synthesised for 2’-stereoselective late-stage radiofluorination based on previously developed conditions. First, the 2’-[18F]fluorinated arabino nucleoside 18F-FAU, which was considered as a rapidly accessible 2’-fluorinated uracil-based dFdC analogue was successfully synthesised in our radiochemical laboratory. Subsequently, this procedure was used as a template method to obtain the cytidine analogue 18F-FAC via a novel synthetic route in moderate radiochemical yield (4.3-5.5%, decay-corrected), high specific activity (1700 mCi/μmol) and purity (98%) after a synthesis time of 168 min

Radiochemical synthesis of 2′-[18F]-labelled and 3′-[18F]-labelled nucleosides for positron emission tomography imaging

This review article considers 2′‐labelled and 3′‐labelled nucleosides, which are of great importance as positron emission tomography (PET) probes in clinical diagnostics and PET research. Although the radiochemical preparation of several [18F]‐labelled nucleosides such as [18F]fluorothymidine or [18F](fluoroarabinofuranosyl)cytosine has been accomplished within the last two decades, a number of potentially interesting nucleoside‐based biomarkers are not yet available for automated good manufacturing practice production due to the lack of fast and efficient synthetic methods for late‐stage [18F]‐introduction. In order to meet recent demands for new PET‐based biomarkers in various clinical applications, appropriate precursors that can easily be fluorinated and deprotected need to be developed

A novel radiochemical approach to 1-(2 '-deoxy-2 '-[F-18] fluoro-beta-D-arabinofuranosyl)cytosine (F-18-FAC)

18F-FAC (1-(2'-deoxy-2'-[18F]fluoro-β-D-arabinofuranosyl)-cytosine) is an important 2'-fluoro-nucleoside-based positron emission tomography (PET) tracer that has been used for in vivo prediction of response to the widely used cancer chemotherapy drug gemcitabine. Previously reported synthetic routes to 18F-FAC have relied on early introduction of the 18F radiolabel prior to attachment to protected cytosine base. Considering the 18F radiochemical half-life (110 min) and the technical challenges of multi-step syntheses on PET radiochemistry modular systems, late-stage radiofluorination is preferred for reproducible and reliable radiosynthesis with in vivo applications. Herein, we report the first late-stage radiosynthesis of 18F-FAC. Cytidine derivatives with leaving groups at the 2'-position are particularly prone to undergo anhydro side-product formation upon heating because of their electron density at the 2-carbonyl pyrimidone oxygen. Our rationally developed fluorination precursor showed an improved reactivity-to-stability ratio at elevated temperatures. 18F-FAC was obtained in radiochemical yields of 4.3–5.5% (n = 8, decay-corrected from end of bombardment), with purities ≥98% and specific activities ≥63 GBq/µmol. The synthesis time was 168 min

The ribosome assembly factor Nep1 responsible for Bowen–Conradi syndrome is a pseudouridine-N1-specific methyltransferase

Nep1 (Emg1) is a highly conserved nucleolar protein with an essential function in ribosome biogenesis. A mutation in the human Nep1 homolog causes Bowen–Conradi syndrome—a severe developmental disorder. Structures of Nep1 revealed a dimer with a fold similar to the SPOUT-class of RNA-methyltransferases suggesting that Nep1 acts as a methyltransferase in ribosome biogenesis. The target for this putative methyltransferase activity has not been identified yet. We characterized the RNA-binding specificity of Methanocaldococcus jannaschii Nep1 by fluorescence- and NMR-spectroscopy as well as by yeast three-hybrid screening. Nep1 binds with high affinity to short RNA oligonucleotides corresponding to nt 910–921 of M. jannaschii 16S rRNA through a highly conserved basic surface cleft along the dimer interface. Nep1 only methylates RNAs containing a pseudouridine at a position corresponding to a previously identified hypermodified N1-methyl-N3-(3-amino-3-carboxypropyl) pseudouridine (m1acp3-Ψ) in eukaryotic 18S rRNAs. Analysis of the methylated nucleoside by MALDI-mass spectrometry, HPLC and NMR shows that the methyl group is transferred to the N1 of the pseudouridine. Thus, Nep1 is the first identified example of an N1-specific pseudouridine methyltransferase. This enzymatic activity is also conserved in human Nep1 suggesting that Nep1 is the methyltransferase in the biosynthesis of m1acp3-Ψ in eukaryotic 18S rRNAs

2019 ESC Guidelines for the diagnosis and management of acute pulmonary embolism developed in collaboration with the European Respiratory Society (ERS)

Peer reviewe

The Bowen–Conradi syndrome protein Nep1 (Emg1) has a dual role in eukaryotic ribosome biogenesis, as an essential assembly factor and in the methylation of Ψ1191 in yeast 18S rRNA

The Nep1 (Emg1) SPOUT-class methyltransferase is an essential ribosome assembly factor and the human Bowen–Conradi syndrome (BCS) is caused by a specific Nep1D86G mutation. We recently showed in vitro that Methanocaldococcus jannaschii Nep1 is a sequence-specific pseudouridine-N1-methyltransferase. Here, we show that in yeast the in vivo target site for Nep1-catalyzed methylation is located within loop 35 of the 18S rRNA that contains the unique hypermodification of U1191 to 1-methyl-3-(3-amino-3-carboxypropyl)-pseudouri-dine (m1acp3Ψ). Specific 14C-methionine labelling of 18S rRNA in yeast mutants showed that Nep1 is not required for acp-modification but suggested a function in Ψ1191 methylation. ESI MS analysis of acp-modified Ψ-nucleosides in a Δnep1-mutant showed that Nep1 catalyzes the Ψ1191 methylation in vivo. Remarkably, the restored growth of a nep1-1ts mutant upon addition of S-adenosylmethionine was even observed after preventing U1191 methylation in a Δsnr35 mutant. This strongly suggests a dual Nep1 function, as Ψ1191-methyltransferase and ribosome assembly factor. Interestingly, the Nep1 methyltransferase activity is not affected upon introduction of the BCS mutation. Instead, the mutated protein shows enhanced dimerization propensity and increased affinity for its RNA-target in vitro. Furthermore, the BCS mutation prevents nucleolar accumulation of Nep1, which could be the reason for reduced growth in yeast and the Bowen-Conradi syndrome

July 2017 ENCALS statement on edaravone

Peer reviewe

Mendelian randomisation implicates hyperlipidaemia as a risk factor for colorectal cancer.

While elevated blood cholesterol has been associated with an increased risk of colorectal cancer (CRC) in observational studies, causality is uncertain. Here we apply a Mendelian randomisation (MR) analysis to examine the potential causal relationship between lipid traits and CRC risk. We used single nucleotide polymorphisms (SNPs) associated with blood levels of total cholesterol (TC), triglyceride (TG), low-density lipoprotein (LDL), and high-density lipoprotein (HDL) as instrumental variables (IV). We calculated MR estimates for each risk factor with CRC using SNP-CRC associations from 9,254 cases and 18,386 controls. Genetically predicted higher TC was associated with an elevated risk of CRC (odds ratios (OR) per unit SD increase = 1.46, 95% confidence interval [CI]: 1.20-1.79, P=1.68x10−4). The pooled ORs for LDL, HDL, and TG were 1.05 (95% CI: 0.92-1.18, P=0.49), 0.94 (95% CI: 0.84-1.05, P= 0.27), and 0.98 (95% CI: 0.85-1.12, P=0.75) respectively. A genetic risk score for 3-hydoxy-3-methylglutaryl-coenzyme A reductase (HMGCR) to mimic the effects of statin therapy was associated with a reduced CRC risk (OR=0.69, 95% CI: 0.49-0.99, P=0.046). This study supports a causal relationship between higher levels of TC with CRC risk, and a further rationale for implementing public health strategies to reduce the prevalence of hyperlipidaemia. This article is protected by copyright. All rights reserved