We investigated the complexation between CB and SIE1 by introducing 1-2 equivalents of CB to a solution of SIE1-TFA in D2O. H NMR demonstrated a notable shift in the benzyl signals, while protons on the coumarin motif remained unaffected even at increased CB concentrations (Figure ). This observation suggests that CB binds selectively and efficiently to the benzylamine moiety in SIE1. The formation of supramolecular complexes was further supported by isothermal titration calorimetry (ITC) measurement. The isotherm displayed a sharp transition, consistent with a 1:1 CB:SIE1 binding stoichiometry and revealing a binding affinity Ka = 5.4×10 5 M -1 . With the formation of CB/SIE1 complex confirmed, we then examined the effect of the CB cap on the self-immolative reactivity of SIE1 (Figure ). The introduction of one equivalent of CBreferred to as 1×CB-into a 2 mM solution of SIE1 in pH 6.5 phosphate buffer increased the t1/2 of coumarin release to 1.22 h (Figure ), as compared to t1/2 = 0.45 h for free SIE1. Increasing the stoichiometry of CB to two and four equivalents slightly increased the t1/2 further to 1.58 h and 1.61 h, respectively. This weak dependence on CB stoichiometry is expected as the high binding affinity between CB and SIE1 ensures near-complete complexation across the concentrations studied. Although the increase of t1/2 to 1-2 hours was modest, these proof-of-principle results encouraged us to design the next generation of CBbinding linker SIE2. Accordingly, the remainder of this paper will focus on the SIE2 scaffold.