Published on Web 07/21/2010 Synthesis of a pH-Sensitive Nitrilotriacetic Linker to Peptide Transduction Domains To Enable Intracellular Delivery of Histidine Imidazole Ring-Containing Macromolecules Ronald K. June, Khirud Gogoi, Akiko Eguchi, Xian-Shu Cui, and Steven F. Dowdy* Howard Hughes Medical Institute and Department of Cellular & Molecular Medicine, UniVersity of California San Diego School of Medicine, 9500 Gilman DriVe, La Jolla, California 92093-0686 Received May 11, 2010; E-mail: sdowdy@ucsd.edu Abstract: Intracellular delivery of functional macromolecules using peptide transduction domains (PTDs) is an exciting technol- ogy with both experimental and therapeutic applications. Recent data indicate that PTD-mediated transduction occurs via ﬂuid- phase macropinocytosis involving an intracellular pH drop to ∼5. Nitrilotriacetic acid (NTA)-coordinated metals avidly bind hexa- histidine-tagged macromolecules, including peptides and proteins. Histidine’s imidazole ring has a pKa of 6, making this an attractive target for the biological pH drop of PTD-mediated macropinocy- totic delivery. The objective of this study was to develop a pH- sensitive PTD delivery peptide (NTA3-PTD). We demonstrate the in vitro function of this novel peptide by delivering ﬂuorescently labeled peptides (1.6 kDa) and functional enzymes, -galactosi- dase (119 kDa) and Cre recombinase (37 kDa). Furthermore, the NTA3-PTD peptide was able to deliver functional Cre recombinase in an in vivo mouse model. Intracellular delivery of functional macromolecules using peptide transduction domains is an exciting technology with both bench and bedside applications. Recent data1-3 indicate that transduction occurs via ﬂuid-phase endocytosis involving an intracellular pH drop. Nitrilotriacetic acid (NTA) is a pH-sensitive moiety capable of binding hexahistidine (6xHis)-tagged proteins via a coordinate covalent bond for pH >6. The objective of this study was to develop a pH-sensitive peptide transduction domain (NTA-PTD, Figure 1A) capable of (1) forming a noncovalent bond with 6xHis-tagged cargo, (2) inducing cellular uptake, and (3) dissociating from the cargo following internalization via the intracellular endosomal pH drop. We demonstrate the in Vitro function of this novel peptide by delivering ﬂuorescently labeled peptides (1.6 kDa) and functional enzymes ( -galactosidase and Cre recombinase, 119 and 37 kDa, respectively.) Furthermore, this peptide was able to deliver func- tional Cre recombinase in an in ViVo mouse model. Intracellular transduction via PTDs occurs via macropinocytosis, a specialized form of ﬂuid-phase endocytosis.1-6 Cationic PTDs bind the cell surface and become internalized within macropino- somes.3,6 Once internalized, these endosomes undergo an intracel- lular pH drop to pH ∼4.5.7,8 Previous studies have utilized NTA, an aminopolycarboxylic acid capable of sequestering all metal ions,9 in a variety of bioengineering.10-14 In the presence of metal ion, a coordinate bond can be formed between one molecule of NTA and two imidazole rings on adjacent histidine residues in a protein or other macromolecular cargo.15 Thousands of recombinant proteins have been constructed for puriﬁcation using 6xHis tags. However, for the vast majority of these proteins, intracellular bioavailability is limited due to size restrictions for diffusion through the plasma membrane.4 Therefore, we sought to create a pH- 10680 9 J. AM. CHEM. SOC. 2010, 132, 10680–10682 Figure 1. Cargo delivery via pH-sensitive NTA-PTD. (A) NTA3-PTD forms a coordinate covalent bond with metal ions and the imidazole ring on histidine residues. (B) Fmoc-protected lysine derivative. (C) pH-sensitive release of 6xHis-rhodamine via NTA3-PTD. sensitive noncovalent link between NTA and the PTD TAT for intracellular delivery of 6xHis-tagged proteins. To facilitate synthesis of NTA-PTD, we synthesized monomeric Fmoc-protected NTA-derivatized aspartic acid16 (Figure 1B and Supporting Information) by ring-opening condensation of (N-(9- ﬂuorenyl)methoxycarbonyl (Fmoc)-aspartic anhydride17 with an NTA derivative of lysine.14 This NTA fragment was incorporated into the PTD using Fmoc solid-phase peptide synthesis with a Symphony Quartet peptide synthesizer (Rainin) by SPPS using L-amino acids with Fmoc protection on an amidated support (EMD). Crude products were puriﬁed by reversed-phase HPLC and conﬁrmed by MALDI mass spectroscopy (Supporting Information). Rhodamine-labeled 6xHis peptide (Rhod-GGSGG-HHHHHH-G) was prepared by reaction with tetramethylrhodamine while on solid support. The sequence of the NTA3-PTD peptide is (NTA)-G- (NTA)-G-(NTA)-GGSGG-RKKRRQRRRG. To assess the pH-sensitivity of NTA3-PTD, 2.2 nmol of 6xHis- rhodamine and NTA3-PTD plus 3 equiv of NiSO4 (1:1 molar ratio with single NTA3-PTD peptide) were mixed and added to washed ion-exchange S-Beads. Bead-peptide mixtures were washed three times and aliquoted into separate tubes before addition of 10 mM HEPES with 400 mM NaCl, pH 5-7. Beads were pelleted via centrifugation, and supernatant ﬂuorescence was measured. We 10.1021/ja1040418  2010 American Chemical Society