Dipicolylamine/metal complexes that promotes direct cell-membrane penetration of octaarginine

Yoshimasa Kawaguchi, Shoko Ise, Yusuke Azuma, Toshihide Takeuchi, Kenichi Kawano, Toan Khanh Le, Junko Ohkanda, and Shiroh Futaki
Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/ acs.bioconjchem.8b00691 • Publication Date (Web): 14 Nov 2018
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Bioconjugate Chemistry – A special issue on “Delivery of Proteins and Nucleic
Acids: Achievements and Challenges”.
Article

Dipicolylamine/Metal Complexes that Promotes Direct Cell-Membrane
Penetration of Octaarginine
Yoshimasa Kawaguchi,a† Shoko Ise,a† Yusuke Azuma,a Toshihide Takeuchi,a
Kenichi Kawano,a Toan Khanh Le,a Junko Ohkanda,b and Shiroh Futakia
aInstitute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011, Japan

bInstitute of Agriculture, Shinshu University, Kami-Ina, Nagano 399-4598,
Japan † These authors equally contributed to this study.

*Corresponding author: Shiroh Futaki, Ph. D., Professor Institute for Chemical Research, Kyoto University
[email protected]
ORCID ID: 0000-0002-0124-4002

Abstract

Marked promotion of membrane permeation of a cell-penetrating peptide, octaarginine

(R8), was attained by attachment to a single 2,2′-dipicolylamine moiety (DPA-R8) thatforms complexes with metal ions. Studies using giant
unilamellar vesicle that DPA targets phospholipids and enhances R8 binding to the
membranes in the presence of metal ions. While DPA/Zn(II) complex has been
most frequently employed to chelate formation with phosphates, Ni(II) had the
most prominent effect on membrane binding and penetration of DPA-R8. Facile
cytosolic distribution of DPA-R8 was also attained in a few minutes in the presence of
Ni(II). Analysis of cellular uptake methods of DPA-R8/Ni(II) suggested the
involvement of direct permeation through cell membrane without the use of
endocytosis. The applicability of this system to the intracellular delivery of
bioactive compounds was exemplified using a peptidomimetic farnesyltransferase
inhibitor, FTI277.
Table of Content graphic

Introduction
Intracellular delivery of bioactive molecules using cell-penetrating
peptides (CPPs) has firmly established its importance in chemical biology and
molecular cell biology studies.1-4 CPPs are peptides, with a few to 30 amino
acids, that show cell membrane permeability. Chemical conjugation or stable
complex formation of these CPPs with biofunctional macromolecules, whic
inherently have poor membrane permeability (e.g., peptides/proteins, nucleic acid
derivatives and various nano-particles), leads to their efficient internalization into
cells. This technology has been applied successfully for in-cell imaging/sensing,
and for modulation of in-cell molecular interactions.5 CPPs also have been
gaining research interests due to its therapeutic potentials to deliver
biopharmaceuticals.6
Among CPPs, those being rich in arginine such as the HIV-1 Tat
delivery.3,4 The internalization routes of these arginine-rich CPPs include direct
translocation through cell membranes (i.e., biophysical methods of permeation)
and via endocytosis (i.e., physiological, vesicular transport systems) (Supporting
Information Fig. S1).7-9 The former method may deliver macromolecules of
interest immediately to cytosol to exert the expected in-cell functions. In contrast,
use of the latter pathway needs an extra step of escaping from vesicular
compartments (endosomes) to reach cytosol after engulfment into endosomes.10
Direct penetration is thus more effective. Cell surface accumulation of CPPs is
advocated to facilitate the direct membrane translocation.11 Alternatively,
extensive interaction of CPP-cargo with membranes not only retain them on
either sides of membrane but may also be accompanied by perturbation of
membranes and cytotoixicity.12 We thus hypothesized that employing functional
groups that can appropriately enhance the membrane interaction may facilitate
translocation of R8 (Fig. 1a).

2,2′-Dipicolylamine (DPA) has been reported to form a complex with
Zn(II) and can be exploited as a chemosensor to detect phosphorylated peptides
and proteins.13 Binuclear DPA-Zn(II) complexes have been FTI 277 frequently
employed
to increase the recognition ability to anionic phosphate derivatives.14,15 Oligomers
of a tyrosine derivative bearing a DPA-Zn(II) complex has also been developed
as a CPP.16 Via the chelate formation with cell surface carboxylates and
phosphates, the oligoDPA-Zn(II) CPPs are taken up into cells via endocytosis.