Oligocarbamate molecular transporters: Design, synthesis, and biological evaluation of a new class of transporters for drug delivery

Article abstract of DOI:10.1021/ja0275109

Molecular transporters have the ability to deliver drugs and probe molecules into cells and tissues irrespective of their physical properties. We now report the design, synthesis, and biological evaluation of a new family of molecular transporters, guanid

Full text of DOI:10.1021/ja0275109

Published on Web 10/18/2002
Oligocarbamate Molecular Transporters: Design, Synthesis, and Biological
Evaluation of a New Class of Transporters for Drug Delivery
Paul A. Wender,*,† Jonathan B. Rothbard,‡ Theodore C. Jessop,† Erik L. Kreider,‡ and
Bryan L. Wylie‡
Department of Chemistry, Stanford UniVersity, Stanford, California 94305-5080, and CellGate Inc.,
Del Rey AVenue, SunnyVale, California 94085
Received June 28, 2002
Scheme 1. Synthesis of Oligocarbamate Transportersa
The effectiveness of drugs and probes aimed at intracellular
targets is significantly determined by their ability to breach
biological barriers. Polar molecules generally diffuse poorly through
the nonpolar bilayer of a cell, while nonpolar compounds ex-
hibit limited solubility in aqueous formulations and extracell-
ular fluids. As a consequence, most drugs must be both water and
lipid soluble, that is, conform to a narrow log P range. In contrast,
certain naturally occurring molecules, irrespective of their log P,
enter cells through a facilitated transport pathway mediated by a
receptor or by a change in physical properties arising from cell
surface interactions.1-3 An exceptional example of such a mole-
cule in this rapidly emerging field of molecular transporters is
the nuclear transcription activator protein (Tat) encoded by HIV
type 1 (HIV-1).4,5 The sequence responsible for the cellular up-
take of HIV-1 Tat is a highly basic region of nine amino acids
(Tat49-57: RKKRRQRRR).6 Structure-function studies have shown
that either truncation of the Tat 9-mer or replacement of any of its
cationic residues with an alanine leads to significantly decreased
cellular uptake. In contrast, replacement of the lysine and glutamine
residues with arginine affords increased rates of uptake, leading to
the identification of 9-mers of D- and L-arginine and related
guanidine rich peptoids as superior transporters.7-9 Conjugates of
these transporters and various probes (e.g., fluorescein) or drugs
(e.g., cyclosporin A) are highly water soluble and rapidly enter cells
and tissues,8,10-12 enabling their advancement into human trials.
Our previous work has shown that guanidine-rich peptides and
peptoids with 1,4-spaced side chains are superior to Tat49-57 in
cellular uptake.8 The current study sought to explore whether the
amide backbone common to peptide and peptoid transporters could
be replaced with a carbamate and whether the resultant increased
spacing (1,4 to 1,6) would enhance uptake into cells and tissues.
A series of novel guanidine-rich oligocarbamates was targeted
for preparation. Our synthesis (Scheme 1) was designed to produce
an oligoamine which upon perguanidinylation would be converted
to the desired oligoguanidine. The required monomer 1 (Scheme
1) was efficiently synthesized by the reduction of Fmoc-Orn(Boc)-
OH (1,1′-carbonyldiimidazole in THF with NaBH4, H2O, 92%
yield),13 followed by activation of the resulting amino alcohol as
the p-nitrophenyl carbonate (96%).14
a Fluor ) Fluorescein-NHCS-aminohexanoic acid; Biotin ) D-biotin-
aminohexanoic acid.
the resin and purification by RP-HPLC afforded oligocarbamates
2-5 in 34-53% yield. Perguanidinylation8,15,16 with 1H-pyrazole-
1-carboxamidine hydrochloride17 and Na2CO3 converted oligo-
amines 2-5 to the corresponding guanidine-rich oligomers 6-9
in 59-72% yield after purification by RP-HPLC (perguanidinyla-
tion was complete (MS), >95% pure by analytical HPLC).
The ability of the oligocarbamates to enable cellular uptake was
determined by FACS analysis of Jurkat cells that had been incubated
with fluoresceinated oligomers for 3 min at 23 °C. In this assay,
fluorescein itself does not enter cells (control not shown), while
the pentacarbamate and pentaarginine conjugates of fluorescein
(Figure 1: 6 and D-arg5, respectively) display very limited cellular
uptake. In striking contrast, the hepta- and nonacarbamate conju-
gates of fluorescein (7 and 8) rapidly entered virtually all cells at
each of the concentrations analyzed. The rate of uptake increased
with guanidine content (8 > 7) and concentration as observed with
other Tat-related transporters.8,9,18,19 Representing one of the best
guanidine-based transporters studied to date, carbamate 9-mer 8
translocated into cells 2.3 times faster than D-arg9, which is taken
8
The oligocarbamate 5-, 7-, and 9-mers were synthesized on Rink
amide resin by iterative coupling with monomer 1 on a PE
Biosystems 433A automated peptide synthesizer using modified
FastMoc chemistry. Incorporation of an aminohexanoic acid spacer
and a fluorescein thiourea or biotin label followed by cleavage from
up into cells 100 times faster than Tat49-57.
The cellular uptake of the carbamate transporters and other
guanidine-rich transporter molecules with nonnatural back-
bones8,18,19 (guanidine-rich peptoids, â-Tat, â-Arg9, D-Tat49-57) is
a further indication that the backbone of the transporter molecule
is simply a scaffold from which the transport enabling guanidinium
functionalities are displayed. Previous studies showed that homo-
oligomers of arginine efficiently cross the cell membrane, whereas
lysine, ornithine, and histidine oligomers do not.7 In agreement with
* To whom correspondence should be addressed. E-mail: wenderp@
stanford.edu.
† Stanford University.
‡ CellGate Inc.
9
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10.1021/ja0275109 CCC: $22.00 © 2002 American Chemical Society

C O M M U N I C A T I O N S
(control) were separately applied (0.5 mM) to the back of a nude
mouse, and the residual solution was removed after 30 min. After
a 2 h interval, dissected sections of the treated areas of skin were
exposed to fluorescein-labeled streptavidin. While biotin alone did
not penetrate the skin (Figure 2, panel A), visualization of skin
treated with biotin-transporter 9 (panel C) revealed highly efficient
penetration across the cornified layer of the epidermis and into all
layers of the skin. Nuclear localization (consistent with the known
polycationic nuclear localization signal) was dramatic in the
intensely stained epidermis, and the various cells of the dermis were
also highly stained (detail in Supporting Information).
In conclusion, the oligocarbamate transporters are found to be
among the most efficient molecular transporters studied to date.
They enable exceptionally efficient uptake into cells of a cargo
that by itself does not enter cells. Significantly, they also enable
uptake into skin of a probe molecule that by itself does not penetrate
skin. Structurally these are the first nonamide linked oligoguanidine
transporters and the first with a 1,6-spacing of side chains. The
relationship of these structural features to transport efficiency and
mechanism and the utility of these agents in drug delivery are
subjects of ongoing studies.
Figure 1. FACS determined cellular uptake of oligocarbamate transporters
6-8 and the corresponding fluoresceinated 5-, 7-, and 9-mers of D-arginine
(D-arg5, D-arg7, D-arg9). Jurkat cells were incubated with transporter for 3
min at 23 °C.
Acknowledgment. Support of this work by grants from the
National Institutes of Health (CA 31841, CA 31845) and a CellGate
Fellowship to T.C.J. is gratefully acknowledged.
Supporting Information Available: Experimental data for the
synthesis and biological evaluation of oligocarbamate transporters
(PDF). This material is available free of charge via the Internet at
http://pubs.acs.org.
Figure 2. Uptake into mouse skin. Biotin and biotin-transporter conjugate
9 visualized with streptavidin-FITC (panels A and C). Skin architecture
visualized with propidium iodide counter stain (panels B and D).
References
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this earlier result and further underscoring the importance of the
guanidinium group, nonacationic amine 4 exhibits poor uptake into
cells (Supporting Information), supporting the generality of our
hypothesis that multiple guanidinium groups and not just charge
are required for uptake.
While the mechanism by which arginine-rich transporters cross
the cellular membrane remains unknown, short arginine-rich
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These results are consistent with an energy dependent but nonen-
docytotic mechanism. While further investigation of this pathway
is in progress, it is clear that the oligocarbamates exhibit exceptional
cellular penetration.
To further evaluate oligocarbamate transporters as potential
clinical agents for drug delivery, we sought to determine whether
these agents could transport a drug or probe molecule into the
formidable barrier represented by the skin. While most drug classes
cannot penetrate the skin, intradermal drug delivery of therapeutic
agents for the skin is especially attractive as it minimizes or
eliminates systemic side effects and reduces the amount of drug
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