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 pK of 6, making this an attractive
a
target for the biological pH drop of PTD-mediated macropinocy-
totic delivery. The objective of this study was to develop a pH-
3
sensitive PTD delivery peptide (NTA -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
NTA -PTD peptide was able to deliver functional Cre recombinase
3
in an in vivo mouse model.
Intracellular delivery of functional macromolecules using peptide
transduction domains is an exciting technology with both bench
1-3
and bedside applications. Recent data
indicate that transduction
3
Figure 1. Cargo delivery via pH-sensitive NTA-PTD. (A) NTA -PTD
forms a coordinate covalent bond with metal ions and the imidazole ring
on histidine residues. (B) Fmoc-protected lysine derivative. (C) pH-sensitive
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,
3
release of 6xHis-rhodamine via NTA -PTD.
sensitive noncovalent link between NTA and the PTD TAT for
intracellular delivery of 6xHis-tagged proteins.
(
2) inducing cellular uptake, and (3) dissociating from the cargo
To facilitate synthesis of NTA-PTD, we synthesized monomeric
Fmoc-protected NTA-derivatized aspartic acid (Figure 1B and
1
6
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.
Supporting Information) by ring-opening condensation of (N-(9-
1
7
fluorenyl)methoxycarbonyl (Fmoc)-aspartic anhydride with an
1
4
NTA derivative of lysine. 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
Intracellular transduction via PTDs occurs via macropinocytosis,
1
-6
a specialized form of fluid-phase endocytosis.
bind the cell surface and become internalized within macropino-
somes. Once internalized, these endosomes undergo an intracel-
Cationic PTDs
3,6
7
,8
lular pH drop to pH ∼4.5. Previous studies have utilized NTA,
9
an aminopolycarboxylic acid capable of sequestering all metal ions,
support. The sequence of the NTA
(NTA)-G-(NTA)-GGSGG-RKKRRQRRRG.
To assess the pH-sensitivity of NTA -PTD, 2.2 nmol of 6xHis-
rhodamine and NTA -PTD plus 3 equiv of NiSO (1:1 molar ratio
with single NTA -PTD peptide) were mixed and added to washed
3
-PTD peptide is (NTA)-G-
1
0-14
in a variety of bioengineering.
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. 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
In the presence of metal ion,
3
3
4
1
5
3
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
4
the plasma membrane. Therefore, we sought to create a pH-
1
0680 9 J. AM. CHEM. SOC. 2010, 132, 10680–10682
10.1021/ja1040418 2010 American Chemical Society