S13?). without generating any toxic side products. We demonstrated that the use of this self-immolative linker to conjugate the anticancer drug doxorubicin to a cell-penetrating peptide or an antibody enabled targeted, controlled delivery of the drug to cells. Our results suggest that the linker can be used with a broad range of carriers, such as cell-penetrating peptides, proteins, antibodies, and amine-functionalized polymers, and thus will find a wide range of practical applications. Introduction In drug design and development, prodrug strategies are widely used to improve the pharmacokinetic properties of drugs, especially targeted delivery.1,2 A prodrug is generally constructed by Bikinin conjugation of a drug molecule to a carrier a linker containing a trigger moiety. After delivery of the prodrug to the target cells or tissues, the active drug is released cleavage of the linker, either by an endogenous stimulus such as a pH change,3,4 a redox reaction,5,6 or an enzyme,7,8 or by an exogenous stimulus such as light9C11 or a small-molecule trigger.2,12 The key to achieving efficient, controlled drug release is to choose an appropriate linker.13,14 In some cases, the close proximity of the drug and the carrier impairs linker cleavage by the stimulus. This problem can be overcome by introducing an additional linker, referred to as a self-immolative linker, between the Shh trigger and the drug.15,16 Removal or cleavage Bikinin of the trigger by an appropriate stimulus induces a cascade of disassembly reactions that ultimately lead to drug release. So far, only two types of self-immolative linkers have gained wide acceptance, and both types undergo self-immolative elimination, cyclization, or both to release the conjugated drug.16,17 However, disassembly of these self-immolative linkers can generate toxic side products such as quinone methides, which can have unwanted side effects.18 Therefore, the development of biocompatible self-immolative linkers has attracted considerable attention.11,19,20 We and others have shown that primary amines can catalyze DNA cleavage at C4-oxidized abasic sites (C4APs, which are formed by abstraction of the C4CH from 2-deoxyribose; Fig. 1A).21C26 Specifically, addition of a primary amine to the C1 of a C4AP Schiff base formation leads to sequential elimination of the C3 and C5 phosphates, generating the 5-methylene pyrrolone (5MP) derivative of the primary amine.23 On the basis of these additionCelimination cascade reactions, we designed a photocaged C4AP (PC4AP, Fig. 1B) as a novel light-responsive, self-immolative linker for controlled drug delivery peptide- and protein-drug conjugates. Open in a separate window Fig. 1 Design of a photocaged C4-oxidized abasic site (PC4AP) as a light-responsive, self-immolative linker for controlled drug delivery peptide- and protein-drug conjugates. (A) Primary-amine-catalyzed DNA cleavage at the C4-oxidized abasic site (C4AP). (B) Principle of PC4AP-based drug delivery peptide- and protein-drug conjugates. Results and discussion Design of the PC4AP linker The PC4AP linker is constructed by protection of the C1COH and C4COH of the C4AP with photolabile a carbamate or carbonate bond, and the C5COH of the PC4AP linker is conjugated to a carrier peptide or protein an alkyl chain. Upon photodecaging, the C4AP moiety undergoes an intramolecular addition reaction with any nearby amine Bikinin on the carrier, and a subsequent elimination reaction leads to cleavage of the carbamate or carbonate bond and concomitant release of the active drug, along with nontoxic CO2. Because C5COH is linked to the carrier a stable OCC bond, cleavage of the linker departure of C5COC is disfavored. Therefore, the linker remains attached to the carrier, and a cyclic Bikinin species is generated. Synthesis of a doxorubicin prodrug based on the PC4AP linker Doxorubicin (DOX) is a cytotoxic anthracycline antibiotic and anticancer drug. Because of its natural fluorescence, DOX is widely used as a model cytotoxin for cellular delivery studies.30C32 Here, in a proof-of-principle experiment, DOX was employed to examine the efficacy of controlled drug delivery by means of the PC4AP linker. To this end, we designed and synthesized Mal-PC4AP-DOX (9, Scheme 1), in which DOX and a maleimide (Mal) moiety are bridged by the PC4AP linker. The purpose of the.