After confirming the CB-SIE host-guest interactions and the cyclization-elimination chemistry of SIE2, we systematically investigated the 'gating' effect of the supramolecular CB protecting group on the rate of self-immolative release. We initiated the self-immolation by adding pH 6.5 PBS buffering salts into solutions of SIE linkers (2 mM) with varying equivalents of CB, and monitored the release of coumarin by NMR (Figure ). SIE1-3 all underwent rapid self-immolation, releasing > 90% of coumarin payloads within 2 h (Figure ). The introduction of 1×CB slowed their release rates to 58%, 33%, and 78% at 2 h, respectively. Further increasing the CB stoichiometry to 2× and 4× only marginally enhanced the suppression of SIE1 and SIE3, as 1×CB already enabled near-complete complexation of those high-affinity self-immolative structures (Figure and Figure ). In contrast, linker SIE2 showed a significantly suppressed reactivity when the CB equivalence was increased from 1× to 2× and then to 4×: the percentage of released coumarin was estimated to be about 33%, 6%, and 3% after 2 h incubation, respectively. By fitting the time-dependent coumarin release results to a first-order rate expression (Figure , left), the introduction of 1×, 2×, and 4×CB extended the t1/2 of coumarin release from SIE2 to 4.8 h, 44.5 h, and 88.4 h, respectively, corresponding to a 14-, 131-, and 260-fold decrease in pseudo-first-order reaction rates compared to free SIE2 (Figure ). The CB-stoichiometry-dependent reactivity of SIE2 is attributed to the dual-binding interactions at both benzyl substituents, characterized by a high binding constant of 6.26 × 10 6 M -1 and a second, more moderate constant of 3.73 × 10 4 M -1 . Equilibrium calculations based on ITC-determined binding constants (see SI for details) indicate that 89% of SIE2 molecules are dual-capped in the presence of 2×CB under our experimental conditions ([SIE2]0 = 2 mM). This percentage increases further to > 99% when 4×CB is used. Although the proof-ofconcept SIE2 requires excess CB for maximized 'gating', our future work is focusing on increasing the binding affinity at both sites to ensure more complete complexation. When both benzyl groups of SIE2 were masked by the CB cages, the resulting steric hindrance around the secondary amine and carbonyl reactive groups essentially prevented the intramolecular cyclization in the dual-binding complex. Note that for the reaction to occur, the SIE2 must undergo a structural rearrangement involving multiple rotations around single bond, which can readily occur in the absence of the CB cages. We calculated the free energy barrier for the reaction of SIE2model without the CB cages to be 17.24 kcal/mol (See SI: Computations). In contrast, the CB cages make the SIE2model rearrangement unfavorable. The structurally rearranged reactant is 25.14 kcal/mol above the reactant in the presence of CB. Therefore, even the structural rearrangement of the reactant requires much higher energy when the CB cages are present.