Synthesis of 4'-Thionucleoside Analogues Bearing a C2' Stereogenic All-Carbon Quaternary Center.

The design of novel 4'-thionucleoside analogues bearing a C2' stereogenic all-carbon quaternary center is described. The synthesis involves a highly diastereoselective Mukaiyama aldol reaction, and a diastereoselective radical-based vinyl group transfer to generate the all-carbon stereogenic C2' center, along with different approaches to control the selectivity of the N-glycosidic bond. Intramolecular SN2-like cyclization of a mixture of acyclic thioaminals provided analogues with a pyrimidine nucleobase. A kinetic bias favoring cyclization of the 1',2'-anti thioaminal furnished the desired ß-D-4'-thionucleoside analogue in a 7:1 ratio. DFT calculations suggest that this kinetic resolution originates from additional steric clash in the SN2-like transition state for 1',4'-trans isomers, causing a significant decrease in their reaction rate relative to 1',4'-cis counterparts. N-glycosylation of cyclic glycosyl donors with a purine nucleobase enabled the formation of novel 2-chloroadenine 4'-thionucleoside analogues. These proprietary molecules and other derivatives are currently being evaluated both in vitro and in vivo to establish their biological profiles.

Isolation of Adult Mouse Cardiac Fibroblasts.

Fibroblasts are dynamic cells of mesenchymal origin that regulate tissue homeostasis, extracellular matrix production, and acute wound healing. Fibroblasts respond to tissue injury and inflammation by differentiating into myofibroblasts and secreting extracellular matrix proteins. Fibroblasts are the principal mediators of the fibrotic response in all tissues and organs. Adult primary fibroblasts represent an essential tool for in vitro studies. Although they lack surface markers, fibroblasts are relatively easy to obtain and culture; primary fibroblasts are sensitive heterogeneous cell subpopulations with limited expansion potential and increased differentiation capacity. Adult primary fibroblasts fail to maintain an undifferentiated state ex vivo for long periods and quickly differentiate into myofibroblasts in culture, which necessitates the utilization of these cells either directly after isolation or after a few passages. Herein, we describe a detailed protocol for enzymatic isolation of primary cardiac fibroblasts from adult mouse hearts and their culture and expansion in a serum-containing culture medium. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Preparation of primary adult cardiac fibroblasts for culture and single-cell analysis.