Dendrimeric molecular transporters: Synthesis and evaluation of tunable polyguanidino dendrimers that facilitate cellular uptake

Article abstract of DOI:10.1021/ol051496y

(Chemical Equation Presented) Nine fluorescently labeled structurally varied polyguanidino dendrimers based on diamino acid monomeric units were individually synthesized in an efficient, scalable sequence using a trifluoroacetamide protecting group - perg

Full text of DOI:10.1021/ol051496y

ORGANIC
LETTERS
2005
Vol. 7, No. 22
4815-4818
Dendrimeric Molecular Transporters:
Synthesis and Evaluation of Tunable
Polyguanidino Dendrimers That
Facilitate Cellular Uptake
Paul A. Wender,*,† Erik Kreider,‡ Erin T. Pelkey,† Jonathan Rothbard,* and
Christopher L. VanDeusen*,†
Department of Chemistry and Department of Molecular Pharmacology,
Stanford UniVersity, Stanford, California 94305-5080, and Cellgate, Inc.,
552 Del Rey AVenue, SunnyVale, California 94086
wenderp@stanford.edu
Received June 27, 2005 (Revised Manuscript Received September 14, 2005 )
ABSTRACT
Nine fluorescently labeled structurally varied polyguanidino dendrimers based on diamino acid monomeric units were individually synthesized
in an efficient, scalable sequence using a trifluoroacetamide protecting group perguanidinylation strategy. While the dendrimers varied
significantly in their ability to enter a human lymphocyte cell line, the best transporters out-performed an oligoarginine reference standard.
−
The identification of molecular transporters that enable
or enhance cellular uptake of drug candidates and probes
that cannot enter cells or do so only poorly is a challenge
of great significance in chemistry, biology, and medi-
cine.1,2 HIV-Tat-derived molecules, most notably Tat49-57
(RKKRRQRRR), have proven to be important leads in this
area, serving as transporters for the uptake of a wide range
of cargos.2 Structure-function studies from our laboratories
(P.A.W., J.R.) have shown that oligomers of L-arginine
outperform Tat49-57 in many uptake assays.2a While the
number of arginines in an oligomer influences uptake up to
a point, 8-mers (RRRRRRRR) offer the best match of
performance and cost of goods. D-Oligomers of arginine work
as well or better than L-isomers due presumably to protease
resistance. The first peptoids corresponding to the arginine
oligomers (e.g., N-arg9) were also synthesized and found to
exhibit superior uptake into cells.3 We subsequently showed
that interdigitation of the arginine backbone with other R,
(2) (a) Rothbard, Jonathan B.; Kreider, Erik; Pattabiraman, Kanaka;
Pelkey, Erin T.; VanDeusen, Christopher L.; Wright, Lee; Wylie, Bryan
L.; Wender, Paul A. In Cell-Penetrating Peptides: Processes and Applica-
tions; Langel, U¨ ., Ed.; CRC Press: Boca Raton, FL, 2202; pp 141-160.
(b) Frankel, A. D.; Pabo, C. O. Cell 1988, 55, 1189-1193. (c) Langel, U¨ .
Cell-Penetrating Peptides; CRC Press LLC: Boca Raton, FL, 2002. (d)
Futaki, S.; Suzuki, T.; Ohashi, W.; Yagami, T.; Tanaka, S.; Ueda, K.;
Sugiura, Y. J. Biol. Chem. 2001, 276, 5836-5840.
† Stanford University.
(3) Wender, P. A.; Mitchell, D. J.; Pattabiraman, K.; Pelkey, E. T.;
Steinman, L.; Rothbard, J. B. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 13003-
13008.
‡ Cellgate, Inc.
(1) Snyder, E. L.; Dowdy, S. F. Expert Opin. Drug Del 2005, 2, 43-51.
10.1021/ol051496y CCC: $30.25
© 2005 American Chemical Society
Published on Web 09/27/2005

â, γ, and δ amino acids (RXRXRXRXRXRXRXRXR)4 or
even replacement of the peptidic backbone with an oligo-
carbamate improved cellular uptake relative to the corre-
sponding arginine oligomers.5 Collectively, these results
suggest there is not a single optimal position for arginine
along the linear chain, but rather that the number of
guanidinium headgroups in a transporter is the principal
determinant of cellular uptake. An adaptive translocation
mechanism accommodating these and other observations has
recently been proposed.6 Of special note, we found that these
arginine oligomers with attached drugs also penetrate tissue
including human skin, enabling their entry into human
clinical trials.3,7 Concurrent with these studies in which
guanidinium groups are attached to a linear backbone, we
started several years ago to investigate the synthesis and
performance of transporters based on a dendrimeric backbone
to examine the effect of branching on cellular uptake.8 Our
initial studies of these new transporters are reported herein.
arm first generation product. Repetition of this cycle would
then lead to an eight-arm system capable of incorporating
eight guanidinium groups, the number that has worked well
for cellular uptake using a linear backbone.2a Typically,
dendrimers branch out symmetrically from a central point
(i.e., PAMAM).10 This architecture precludes covalent at-
tachment of a drug or probe. However this problem could
be circumvented through the use of “arboreal” dendrimers
based a triamine building block.11 An alternative architecture
based on amino triol subunits has been independently
reported by Goodman and co-workers.12 Arboreal dendrimers
(named for their tree-like structure) possess an orthogonal
core in which the dendrimer branches out from one end.11
This results in a structure whose branches could accom-
modate guanidinium groups while a cargo could be attached
to its trunk.11 One of the more exciting arboreal dendrimers
to come out in the past few years in terms of ease of synthesis
are the triamine based dendrimers. These dendrimers consist
simply of a triamine core that has been used to open an acid
anhydride to generate a diamino acid.13 These monomers are
analogous to lysine but lack the stereochemistry and are
synthetically much more readily varied.13c Recently, they
have been used as templates for the assembly of fluorescent
probes as well as saccharides.13a,c,14 Herein, we report the
use of subunit tunable, triamine based diamino acids to
readily form polyguanidinylated dendrimers that rapidly enter
cells.
Scheme 1. Synthesis of Tetrameric Dendrimers
Theoretically, there are two ways to assemble an arboreal
dendrimer. In the first approach, the terminal generation is
assembled first and attached to cores with fewer and fewer
sites of functionality in a convergent synthesis. In the second
approach, the dendrimer is synthesized from an orthogonal
core and symmetrical branches attached with each successive
generation containing a greater number of terminal functional
groups in a divergent synthesis. We chose the divergent
approach to provide an orthogonally derivatized core.
Shown in Schemes 1 and 2 for clarity is the representative
(4) Rothbard, J. B.; Kreider, E.; VanDeusen, C. L.; Wright, L.; Wylie,
B. L.; Wender, P. A. J. Med. Chem. 2002, 45, 3612-3618.
(5) Wender, P. A.; Rothbard, J. B.; Jessop, T. C.; Kreider, E. L.; Wylie,
B. L. J. Am. Chem. Soc. 2002, 124, 13382-13383.
(6) (a) Rothbard, J. B.; Jessop, T. C.; Lewis, R. S.; Murray, B. A.;
Wender, P. A.J. Am. Chem. Soc. 2004, 126, 9506-9507. (b) Rothbard, J.
B.; Jessop, T. C.; Wender, P. A. AdV. Drug Del. ReV. 2005, 57, 495-504.
(7) (a) Rothbard, J. B.; Garlington, S.; Lin, Q.; Kirschberg, T.; Kreider,
E.; McGrane, P. A.; Wender, P. A.; Khavari, P. A. Nat. Med. 2000, 6,
1253-1257.
(8) VanDeusen, C. L. Ph.D. Thesis, Guanidinium-Rich Molecular
Transporters for Drug Delivery, Department of Chemistry, Stanford
University, 2003.
(9) Wender, P. A.; Jessop, T. C.; Pattabiraman, K.; Pelkey, E. T.;
VanDeusen, C. L.Org. Lett. 2001, 3, 3229-3232.
(10) Tomalia, D. A.; Naylor, A. M.; Goddard, W. A., III. Angew. Chem.,
Int. Ed. Engl. 1990, 29, 138-175.
(11) Newkome, G. R.; Yao, Z. Q.; Baker, G. R.; Gupta, V. K. J. Org.
Chem. 1985, 50, 2003-2004.
(12) (a) Chung, H. H.; Harms, G.; Min Seong, C.; Choi, B. H.; Min, C.;
Taulane, J. P.; Goodman, M. Biopolymers 2004, 76, 83-96. (b) Goodman,
M.; Seong, C. M.; Harms, G.; Min, C.; Choi, B. H.; Chung, H. H. Patent
WO 2004009666.
(13) (a) Adamczyk, M.; Fishpaugh, J.; Heuser, K. Org. Prep. Proced.
Intl. 1998, 30, 339-348. (b) Adamczyk, M.; Fishpaugh, J.; Mattingly, P.
G.; Shreder, K. Bioorg. Med. Chem. Lett. 1998, 8, 3595-3598. (c) Llinares,
M.; Roy, R. Chem. Commun. 1997, 2119-2120.
(14) (a) Veprek, P.; Jezek, J. J. Pept. Sci. 1999, 5, 5-23. (b) Veprek,
P.; Jezek, J. J. Pept. Sci. 1999, 5, 203-220.
Dendrimers represent an attractive transporter scaffold that
offers the advantage of step economical assembly of octa-
guanidinium transporters through a variant (Schemes 1 and
2) of a segment-doubling strategy (2 f 4 f 8) that we
previously reported for the synthesis of octaarginine trans-
porters.9 Specifically, each arm of a two-arm dendrimer core
could be extended with a two-arm segment to give a four-
4816
Org. Lett., Vol. 7, No. 22, 2005

Scheme 2. Synthess of Octamer Dendrimer and Perdeprotection/Perguanidinylation
synthesis of the dendrimers from triamine 1. The exact same
our group reported for the synthesis of arginine oligomers
from the corresponding ornithines.9 Removal of the benzyl
carbamate using trifluoroacetic acid yielded the dendrimer
transporters in a total of 8 steps following purification by
preparative HPLC. This is comparable in step economy to
our previously reported segment doubling strategy for
octaarginine synthesis.9
To study the effect of spacer length in the dendrimer on
cell uptake activity, nine dendrimers EE-HH (Scheme 2,
8-16) were synthesized with varying numbers of methylenes
(diethylene triamine, 3-aminopropylpropane diamine, and
hexamethylene triamine). To study the effect of the terminal
dendrimer generation on activity, each triamine 1-3 (Scheme
1) was used to synthesize the first two generations of
dendrimer. At this stage, each of these core dendrimers was
capped with each of the three corresponding diamino acids,
allowing overall for the parallel synthesis of nine dendrimers,
differing by the spacing between their cores and their
terminal guanidino groups (Scheme 2).
Varying concentrations of the fluorescently labeled den-
drimers, EE-HH in Scheme 2 were dissolved in phosphate
buffered saline pH 7.4 (PBS) and incubated with 3 × 105
transformed human T lymphocytes, Jurkat, for 3 min.
Following this, the fluorescent dendrimer was removed from
the cells by centrifugation, and the resultant cell pellet was
washed with PBS containing 2% fetal calf serum, spun, and
resuspended in PBS containing 2% fetal calf serum. The
fluorescence in the resulting cell suspension was analyzed
by flow cytometry (Figure 1).
Even though all dedrimeric transporters had an identical
number of guanidinium groups, they exhibit a wide range
of uptake into lymphocytes. Several patterns are apparent,
with the most obvious being that the longer and more flexible
the dendrimeric hydrocarbon chains, the better the uptake.
The hexyl hexyl (HH; 16) and the hexyl propyl (HP; 15)
sequence of monomer synthesis and assembly was repeated
for each of the triamines 1-3 to produce the family of nine
dendrimers shown in Scheme 2. The synthesis of the core
began with the selective protection of the primary amines
in 1 with ethyl trifluoroacetate (Scheme 1). Solvent re-
moval followed by reaction of the crude amine with the
isobutyl mixed carbonic anhydride of Z-aminocaproic acid
yielded the core element 4 that was ready for expansion
(Scheme 1).
The synthesis of the acid proceeded with the same primary
trifluoracetamide protection as above but was followed by
reaction of the crude product with succinic anhydride.
Extractive workup yielded the diamine-protected acid 5
(Scheme 1). Attempts to activate this acid in situ (i.e., acid
chloride, DCC, isobutyl chloroformate) to react with the
diamine core during the expansion of the dendrimer led to
complex mixtures of products. Therefore, the N-hydroxysuc-
cinimidyl ester 6 of the acid 5 was synthesized and was found
to provide clean acylation reactions in subsequent steps
(Scheme 1).
The subsequent acylation procedure that was ultimately
developed involved first removing the primary trifluoro-
acetamides of 4 with sodium carbonate in aqueous methanol.
Following removal of the methanol in vacuo, the crude
polyamine solution was reacted using a biphasic acylation
for which the N-hydroxysuccinimidyl ester 6 was introduced
in ethyl acetate. After separation of the organic and aqueous
phases, the product 7, along with unreacted ester can be
obtained by flash chromatography (Scheme 1). This proce-
dure proved to be the most effective, as the isolation of the
polyamines was difficult. To complete the synthesis after
the second expansion of the core (generation three, Scheme
2), the various octatrifluoroacetamides were perguanidiny-
lated using the perdeprotection/perguanidinylation procedure
Org. Lett., Vol. 7, No. 22, 2005
4817

> HE. If the core spacing is compared, the same trends hold
true, with longer, more lipophillic dendrimers displaying
faster uptake, in the order HH,HP > PP,PH > EH,EP and
HE > PE . EE. A final point that might have mechanistic
implications is that the curve shapes of the more effective
dendrimers are quite different from that of a fluoresceinated
conjugate of nonaarginine (r9). The more effective dendrim-
ers outperform the nonaarginine conjugate at higher con-
centrations (Figure 1). While interpretation of these obser-
vations requires further investigation, these results are
consistent with an adaptive translocation mechanism6a in-
volving transient association of the guanidinium headgroups
with cell surface groups (e.g., phosphates, carboxylates,
sulfates), which serves to enable passage through the
membrane as a transient lipidated ion pair complex.
Given the effective performance of several of these
dendrimers and their expected use for cargo delivery, we
investigated whether the synthesis described above could be
conducted on a larger scale (grams). The synthesis proved
robust providing 14.5 g of the fully protected all propyl
octamer (PP) with no procedural modifications. A 1.5 g
portion of this supply was then perguanadinylated to yield
over a gram of the final product (PP: 12).
Figure 1. Comparison of the mean fluorescence as measured by
flow cytometry of the nine dendrimers (8-16) and a fluorescenated
nonamer of D-arginine (r9: a linear transporter) after a 3-min
incubation with the human T cell line Jurkat. Each point is the
average fluorescence of 10000 live cells (propidium iodide nega-
tive), measured in triplicate. In all but three cases, the viability of
the cells was greater than 95%. Data were not included for HH**
at 25 and 50 µM and at 50 µM for HP* and PH* due to greater
than 20% cell death.
In summary, we report the synthesis and evaluation of
dendrimeric molecular transporters. The transporters were
synthesized in eight steps from inexpensive triamines 1-3
using biphasic acylations to expand the dendridic core.
Fluorescently labeled dendrimers were evaluated for their
cellular uptake kinetics. The longer, more lipophilic, and/or
more flexible spacing in the dendrimers resulted in the most
effective transport into cells exceeding that of even oligo-
arginine transporters at high concentrations.
dendrimers entered the cells most effectively, while the ethyl
ethyl analogue (EE; 8) could not be detected within the cells
during the time course of the experiment. The nine com-
pounds can be placed into five groups, the most effective,
(HH and HP), the second fastest group, (HE, PP, PH),
marginal transporters (EH and EP), a nontransporter (EE),
and an intermediate transporter (PE), less effective than the
second group, but better than the marginal transporters.
These cellular uptake results show some important cor-
relations with our earlier work on peptoid analogues of the
arginine based molecular transporters, in which peptoids with
longer hydrocarbon side chains and greater flexibility
exhibited faster rates of uptake into cells.3 If the dendrimers
are divided into families with identical cores, the dendrimers
with hexyl or propyl spacing at the terminal generation in
all cases are more effective than the ethyl spaced transporters
in entering cells, i.e., EH,EP > EE; PH,PP > PE; HH,HP
Acknowledgment. Support of this work by grants from
the National Institutes of Health (CA31841, CA31845) is
gratefully acknowledged.
Supporting Information Available: Experimental results
and procedures for the synthesis and assays. This material
is available free of charge via the Internet at http://pubs.acs.org.
OL051496Y
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Org. Lett., Vol. 7, No. 22, 2005