The addition of S or N-terminal nucleophiles (signal 14-17, Figure ) on the prodrug conjugates reverses the quaternary nitrogen back to the neutral tertiary amine, releasing and thereby activating the drug molecule. Importantly, this results in traceless drug release. The substitution of the nucleophile on quaternary amine salts likely proceeds via conjugate addition followed by elimination of the allylic leaving group from the enolate, mechanisms that are related to E1cB type eliminations and the MBH-reaction. As signals, we employ a broad range of biological nucleophiles, including N-acetyl cysteine (signal 14), GSH (signal 15), L-proline (signal 16) or L-adrenaline (signal 17). To examine whether our linker design would allow traceless release of a tertiary amine upon reaction with a nucleophile signal, we combined prodrug precursor 1 with tertiary amine DABCO (7) as model drug to form prodrug 13 (Figure ). Here, prodrug 13 was treated with thiol signals (14 and 15) and secondary amine signals (16 and 17) and followed with 1 H NMR spectroscopy over time. Exposure of prodrug 13 (39 mM in 1:9 DMSO-d6/phosphate buffer (pH = 7.4, 0.1 M)) to 14 and 15 (1.0 eq.) led to thiolmediated nucleophilic substitution and generation of free DABCO within minutes (Supplementary Figure and). As a result of the substitution with various nucleophiles (14-17), nucleophile products 18 -20 are formed in the reaction. Similarly to previous research , we found that the reactivity of Nacetyl cysteine (14) and GSH (15) was approximately equal with their half-life being 4.5 and 1.5 min, respectively. In contrast, N-terminal nucleophiles 16 and 17 (1.0 eq., Supplementary Figure and), showed much slower release kinetics compared to thiols with conversions reaching 53 and 35% after 160 hours and half-life of 62 and 247 hours, respectively (Figure and insert). These kinetic differences are attributed to the difference in nucleophilicity between thiols and secondary amines and confirm our previous findings 31, . After confirming the nucleophile-triggered model drug release of free DABCO from prodrug 13, we studied the release of amine drug variations from prodrugs using 16 (Figure ). Initial trials showed that prodrug-candidates 11 and 12 were not soluble using our standard protocol with DMSO-d6/phosphate buffer = 3:7 and required a too large fraction of non-aqueous solvent for dissolution, reducing the use of release experiment data. However, prodrug 8, 9 and 10 (including atropine 4, ofloxacine 3 and lidocaine 2) were soluble and could be studied further in the small molecule release experiments. Upon introduction of 16 to the system (prodrug:signal = 1:1.2), we observed the complete release of amine drugs, alongside the formation of nucleophile product 20 within 65 hours (Figure ) with their half-life being t1/2 = 4.6, 3.3 and 1.3 hours for prodrugs 10, 8 and 9, respectively. We assume that these differences in their release kinetics are related to the drugs aqueous solubility, as seen in other drug delivery systems. 40