The products of these ring-opening reactions feature multiple functional handles for further elaboration, including the 2-(hydroxymethyl) and amine substituents. We aimed to showcase the utility of this elaboration potential by synthesizing a new class of cysteine-reactive, stereopure 2,3-disubstituted azetidine acrylamides. Such azetidine "stereoprobes" can identify ligandable cysteines on proteins in native cellular environments using the chemical proteomic method activity-based protein profiling (ABPP). 4 Such ligandable cysteines are defined as showing stereoselective reactivity with the azetidine acrylamide stereoprobes, which provides evidence of specific interactions between the small molecules and proteins in cells. Current routes to azetidine stereoprobes use C-H arylation reactions to functionalize enantioenriched azetidine-2-carboxylic acid derivatives, 5a,c delivering azetidine acrylamides featuring exclusively 3-aryl substitution (e.g. MY-11/12 and MY-1/3, referred to hereafter as 3-aryl stereoprobes or the 3-aryl chemotype). We hypothesized that ABPP experiments performed with a structurally distinct set of azetidine acrylamide stereoprobes generated using the newly developed synthetic approach may identify additional ligandable cysteines in the human proteome. Stereoprobe synthesis commenced with nucleophilic opening of enantio-and diastereopure ABBs with an aniline derivative. The C2-carbinol was deprotected and functionalized via SnAR chemistry, and the product of this reaction was subjected to sequential amide coupling reactions to install a pentynyl or methyl amide and an acrylamide on the anilino and azetidino nitrogens, respectively. A total of eight "3-amino stereoprobes" were generated in this fashion, four of which contain an alkyne group (36A-D, termed alkyne probes) and four of which contain no alkyne (37A-D, termed competitor probes). Each set of four includes all four unique stereochemical permutations.