We here report the preparation of a new 2,6,8-trisubstituted bicyclic tripeptidomimetic scaffold through TFA-mediated cyclization of a linear precursor containing three side chains. The introduction of a triphenylmethyl-protected thiol into carboxylic acid containing building blocks through sulfa Michael additions onto α,β-unsaturated hexafluoroisopropyl esters is described. The stereoselectivity of the bicycle formation was found to be somewhat lower than that previously reported for analogous 3,6,8-trisubstituted scaffolds. Moreover, the configuration of the linear precursor directs the stereochemical outcome of the cyclization differently when the R1 side chain is positioned on C2 in the bicycles (present work) instead of C3 (previous work). Tripeptidomimetic compounds based on the new scaffold were synthesized and evaluated for antagonistic potency toward CXCR4, and one compound (45a) displayed similar activity to earlier reported 3,6,8-tripeptidomimetic bicycles.