Functional group (FG), an atom or a group of atoms that has similar chemical properties whenever it occurs in different compounds, is a cornerstone concept in fundamental organic chemistry. In medicinal chemistry and related areas, FGs usually play several important roles, being responsible not only for the chemical transformations of organic molecules, but also for establishing specific ligand-target interactions and improving physicochemical parameterskey desirable features in drug design. Examples of the classical FGs most often occurring in medicinal chemistry include the derivatives of carboxylic acids (i.e., amides and esters), ethers, amines, or halogens. For the last decade, increased attention to fluorinated moieties was observed due to the unique properties of the Fluorine atom. Among them, one should mention enhanced resistance to metabolic degradation, a striking effect on pKa and LogP, or amenability for establishing weak interactions with N-H, C-H, and C=O fragments. The type of fluorination pattern has a profound impact on the overall effect of the Fluorine-containing substituent, therefore design and implementation of novel fluorinated moieties is of special significance to modern drug discovery. Recently, an overwhelmingly increased interest to potential drug substances enriched in sp -hydbrid carbon atoms the led to increased exploitation of small cycloalkyl groups and their heterocyclic analogs in medicinal chemistry. Oxetane, an oxa-analog of the cyclobutane ring, was unfairly omitted from the drug discovery campaigns until early 2000s, when its application as hydrophilic gem-dimethyl or metabolically stable carbonyl surrogate was demonstrated. Since then, a large (and still growing) number of oxetane-centered studies have been reported in the literature, revealing a special place of this ring as a promising core for bioisosteric replacements. A major bias that suspended the application of oxetanes in drug discovery is connected with its potential liability under certain conditions. Refuting this statement, recent studies demonstrated the enhanced tolerance of 3,3-disubstituted oxetanes to the external conditions despite several reported exclusions related to the specific structural environment. With increased interest in 2-substituted oxetanes, application of the Fluorine-containing substituents aimed at modulation of the stability and/or other molecular properties could be regarded as a promising approach to the development of novel oxetane-based functional groups (Figure , bottom). In this work, we have turned our attention to 3,3-difluorooxetane (3,3-diFox) that potentially resembles many common FGs (e.g., ester or amide, acetal, small (cyclo)alkyl, etc.) and hence has a great potential for bioisosteric replacements. In view of that, we developed an approach to the multigram synthesis of 3,3-diFoxcontaining building blocks and studied numerous physicochemical (i.e. acidity/basicity, lipophilicity, Hammett constant), structural, and electronic properties, as well as chemical stability of model derivatives. Finally, we demonstrated feasibility of bioisosteric replacement of tert-butyl group with 3,3-difluorooxetane fragment by preparation and evaluation of 3,3-diFox-substituted Tenovin-6 analog.