An efficient, scalable synthesis of the molecular transporter octaarginine via a segment doubling strategy.

Article abstract of DOI:10.1021/ol0161108

[reaction: see text]. Short oligomers of arginine function as remarkably efficient molecular transporters of drugs and probe molecules into cells and tissue. Currently, these compounds are prepared on resin through a unidirectional solid-phase synthesis.

Full text of DOI:10.1021/ol0161108

ORGANIC
LETTERS
2001
Vol. 3, No. 21
3229-3232
An Efficient, Scalable Synthesis of the
Molecular Transporter Octaarginine via
a Segment Doubling Strategy†
Paul A. Wender,* Theodore C. Jessop, Kanaka Pattabiraman, Erin T. Pelkey, and
Christopher L. VanDeusen
Department of Chemistry, Stanford UniVersity, Stanford, California 94305-5080
wenderp@leland.stanford.edu
Received May 11, 2001 (Revised Manuscript Received September 11, 2001)
ABSTRACT
Short oligomers of arginine function as remarkably efficient molecular transporters of drugs and probe molecules into cells and tissue.
Currently, these compounds are prepared on resin through a unidirectional solid-phase synthesis. To extend the utility of these compounds
for therapeutic and research applications, a scalable solution-phase synthesis of Arg8 (1) has been developed on the basis of a segment
doubling strategy that proceeds in 13 steps and 28% overall yield from 4, including a novel one-step perdeprotection−perguanidinylation
reaction.
While considerable structural diversity is found among drugs
and probe molecules, the physical properties of most of these
agents with intracellular targets are limited to a narrow log
P range to ensure solubility in the polar extracellular milieu
and passive diffusion through the nonpolar lipid bilayer of
the cell. Agents falling outside of this range must often be
tuned through reiterative analogue synthesis to achieve the
optimum balance of water solubility and passive membrane
transport. A promising new approach directed at improving
or enabling the cellular uptake of drugs or drug candidates
possessing a wider range of physical properties involves the
use of peptide-based molecular transporters to carry these
agents actively into cells.1-7 Representative of this approach,
† During review a concern was raised about our original preference for
the use of “bidirectional” to describe this strategy. It is noted in reviews
(see Poss, C. S.; Schreiber, S. L. Acc. Chem. Res. 1994, 27, 9-17 and
Magnuson, S. R. Tetrahedron 1995, 51, 2167-2213) that both “simulta-
neous” and “sequential bidirectional” syntheses are possible and that the
second is of lesser importance because such a strategy putatively offers no
step-saving advantage over a linear (unidirectional) synthesis. However,
this restricted definition did not anticipate the work described herein as it
is a sequential bidirectional synthesis and it does offer significant step
savings. The term “segment doubling” was introduced as an alternative name
for this strategy. Abbreviations: Boc ) tert-butoxycarbonyl; Z ) benzyl-
oxycarbonyl; Fmoc ) 9-fluorenylmethoxycarbonyl; Mtr ) 4-methoxy-2,3,6-
trimethylbenzenesulfonyl; Pmc ) 2,2,5,7,8-pentamethylchroman-6-sulfonyl;
Pbf ) 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl; NMM ) N-
methylmorpholine; DMAP ) 4-(dimethylamino)pyridine; RP-HPLC )
reverse phase high performance liquid chromatography; TFA ) trifluoro-
acetic acid.
(1) Wender, P. A.; Mitchell, D. J.; Pattabiraman, K.; Pelkey, E. T.;
Steinman, L.; Rothbard, J. S. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 13003-
13008. For a perspective, see: Rouhi, M. Chem. Eng. News 2001, 79, 49-
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Rothbard, J. B. J. Peptide Res. 2000, 55, 318-325.
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10.1021/ol0161108 CCC: $20.00 © 2001 American Chemical Society
Published on Web 09/21/2001

we have recently shown that homooligomers (7-9 mers) of
L-arginine upon conjugation to various probe molecules (e.g.,
fluorescein) or drugs (e.g., cyclosporin A (CsA)) provide
highly water-soluble conjugates that rapidly enter cells (e.g.,
human Jurkat).1,2 In addition, drug conjugates of these
arginine transporters have been shown to exhibit novel and
significant penetration into human skin and to release their
drug cargo in targeted T cells.8
phase segment doubling synthesis13-15 of the same octamer
would require only 9 steps (three coupling and six depro-
tection steps). In the specific case of arginine-based peptides,
solution-phase synthesis offers the additional advantage of
avoiding expensive protecting groups for the guanidinium
subunit (e.g., Mtr,16 Pmc,17 and Pbf18) required in solid-phase
synthesis. We report now the first segment doubling synthesis
of the arginine oligomer 1 that is both cost-effective and
scalable.
The enormous potential of arginine-based molecular
transporters is limited for several applications only by their
availability and cost. Such homooligopeptides are usually
prepared using solid-phase peptide synthesis.1,2,9-11 Although
this approach is readily automated and allows for the
synthesis and purification of long peptides, it suffers
drawbacks including high cost, limited scalability, and the
need for resin attachment and cleavage. In contrast, solution-
phase synthesis avoids the scale and cost restrictions of resins
(the cost of the resin-based synthesis is more than an order
of magnitude greater than that of the solution-phase synthe-
sis) and, in the particular case of certain oligomers, can be
conducted using a step-saving segment doubling strategy
(Figure 1).12
Our first strategy directed at the synthesis of 1 involved
coupling arginine monomers (Figure 2). Of the commercially
Figure 2. Retrosynthetic analysis.
available arginine monomers that contain differentially
protected functionalities, FmocNH-Arg(Pbf)-CO2Me and
BocNH-Arg(NO2)-CO2Me, we chose to utilize the latter
because of the ease of Boc deprotection in solution and the
stability of the intermediate amine salts (which are easy to
handle and less susceptible to intramolecular lactamization).
The Boc group can be deprotected using either HCl or TFA,19
the methyl ester of the carboxyl terminus is base-labile
(NaOH),20 and the nitro protecting group of the guanidine
can be removed by hydrogenation.21 Isobutyl chloroformate
was used for coupling commercially available BocNH-Arg-
(NO2)-CO2H (2) and HCl‚NH2-Arg(NO2)-CO2Me (3) as the
reagent can be removed in vacuo and has been used to couple
Figure 1. Step count comparison between solid-phase and solution-
phase segment doubling strategies.
Illustrative of the latter point, the unidirectional synthesis
of an octamer employing solid-phase synthesis requires 17
steps (one coupling and deprotection step for each added
monomer and one resin cleavage step), whereas a solution-
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Powell, D. R.; Gellman, S. H. J. Am. Chem. Soc. 1999, 121, 7574-7581.
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1229-1238.
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A.; Reiboldt, A.; Siman, R. Bioorg. Med. Chem. Lett. 1996, 6, 287-290.
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3230
Org. Lett., Vol. 3, No. 21, 2001

protonated arginine.22 Unfortunately, low yields of the dimer
were obtained (∼30%) and all attempts to deprotect the
methyl ester of the dimer unexpectedly resulted in decom-
position. This strategy was set aside therefore in favor of a
more attractive plan based on the use of ornithine monomers.
The conversion of oligoamines into oligoguanidines using
a perguanidinylation reaction has been previously demon-
strated by us (guanidinylated peptoids)1 and others (guani-
dinoglycosides).23,24 As such, the arginine octamer 1 could
in principle be prepared from an ornithine octamer through
a late stage perguanidinylation reaction. Orthogonally pro-
tected ornithine monomers that are commercially available
include BocNH-Orn(Z)-CO2H (4) and HCl‚NH2-Orn(Z)-CO2-
Me (5). Thus the orthogonal protecting group strategy for
ornithine utilized an acid-labile Boc group on the R-amine,
a hydrogenation-labile Z group on the δ-amine, and a base-
labile methyl ester on the carboxyl terminus (Figure 2). This
strategy yielded promising results at the outset (initial
couplings and subsequent deprotections). However, the
Z-protected ornithine tetramers proved to have limited
solubility in organic solvents, representing a significant
problem for scale-up procedures.
To improve the solubilities of the ornithine oligomers, a
new protection strategy was required. Previous experience
in our group had demonstrated that trifluoroacetamide-
protected oligoamines were readily soluble in ethyl acetate.
Thus, our synthesis was revised to incorporate the base-labile
trifluoroacetamide protecting group on the δ-amine of
ornithine. In addition to R-amine Boc protection, the remain-
ing orthogonal protecting group was a hydrogenation-labile
benzyl ester on the carboxyl terminus. The requisite ornithine
monomers needed to pursue the synthesis of 1, BocNH-Orn-
(COCF3)-CO2H (6) and HCl‚NH2-Orn(COCF3)-CO2Me (7),
were prepared from 4 (Scheme 1). Protecting group inter-
and DMAP (20 mol %) to give 8 in quantitative yield.
Finally, removal of the Boc group with HCl gave acid 7 in
98% yield.
Our revised synthesis was then initiated by the coupling
of acid 6 with amine 7 using isobutyl chloroformate for
activation of 6 and NMM as a base (Scheme 2). This reaction
proceeded smoothly to give the fully protected ornithine
dimer 9 in 97% yield with sufficient purity after extractive
workup to be utilized directly in subsequent reactions.
Ornithine dimer 9 was divided into two equal portions. The
first part was hydrogenated, giving 11 in quantitative yield,
while the second part was treated with HCl, giving the amine
hydrochloride salt 10 in 83% yield. Both compounds were
of sufficient purity after workup to be utilized directly in
the subsequent coupling.
The ornithine dimers 10 and 11 were subsequently coupled
with isobutyl chloroformate and NMM and upon extractive
workup and purification through a short plug of silica gel
gave the ornithine tetramer 12 in 83% yield. As anticipated,
12 was readily soluble in ethyl acetate on a multigram (4 g)
scale. The fully protected tetramer 12 was then divided into
two equal portions, and each was subjected to the appropriate
conditions for the preparation of the free acid 14 and the
amine hydrochloride salt 13, respectively. Coupling 13 and
14 in the usual fashion (isobutyl chloroformate and NMM)
proceeded smoothly to give the fully protected ornithine
octamer 15 in 83% yield and in sufficient purity to be utilized
in subsequent reactions. At this point, attempts to convert
the trifluoroacetamides into guanidines via a two-step
procedure, involving the initial deprotection of the trifluoro-
acetamides, gave significant quantities of lactamization at
the carboxyl terminus (involving condensation of the free
amine onto the benzyl ester). Thus, deprotection of the benzyl
ester was deemed necessary before proceeding with the
synthesis. Therefore, 15 was hydrogenated to remove the
benzyl ester, giving the free acid 16 in quantitative yield.
At this stage, we envisioned that a novel perdeprotection-
perguanidinylation process could be achieved in one opera-
tion to yield perguanylated 18. Since aqueous sodium
carbonate has previously been utilized to effect the depro-
tection of trifluoroacetamides26 and also as one of the
reagents in the guanidinylation of amines,1,27 we reasoned
that it would be possible to perform both processes in one
operation. This hypothesis proved to have merit, as treatment
of the octaornithine derivative 16 (Scheme 3) with sodium
carbonate and pyrazole-1-carboxamidine hydrochloride (17)
in aqueous methanol gave the octaarginine derivative 18 in
51% isolated yield after purification by RP-HPLC (99+%
purity, with no observed unreacted amine products observed
by MS) and lyophilization. Significantly, eight trifluoro-
acetamides were converted to eight guanidines in one step
(16 transformations overall) under mild conditions. Attempts
Scheme 1. Monomer Preparationa
a Conditions: (a) (i) Pd/C, MeOH, H2; (ii) EtO2CCF3, Et3N
(>99%). (b) (i) ClC(O)OBn, NMM, THF -15 °C; (ii) DMAP
(>99%). (c) HCl, EtOAc (98%).
conversion of the Z group of 4 to the corresponding
trifluoroacetamide of 6 was accomplished in quantitative
yield by hydrogenation followed by treatment with ethyl
trifluoroacetate. Esterification of 6 was accomplished using
a known procedure25 by treatment with benzyl chloroformate
(23) Luedtke, N. W.; Baker, T. J.; Goodman, M.; Tor, Y. J. Am. Chem.
Soc. 2000, 122, 12035-12036.
(24) Feichtinger, K.; Zapf, C.; Sings, H. L.; Goodman, M. J. Org. Chem.
1998, 63, 3804-3805.
(25) Kim, S.; Lee, J. I.; Kim, Y. C. J. Org. Chem. 1985, 50, 560-565.
(26) Boger, D. L.; Yohannes, D. J. Org. Chem. 1989, 54, 2498-2502.
(27) Bernatowicz, M. S.; Wu, Y. L.; Matsueda, G. R. J. Org. Chem.
1992, 57, 2497-2502.
(22) Plapinger, R. E.; Nachlas, M. M.; Seligman, M. L.; Seligman, A.
M. J. Org. Chem. 1965, 30, 1781-1785.
Org. Lett., Vol. 3, No. 21, 2001
3231

Scheme 2. Segment Doubling Assembly of Ornithine Octamera
a Conditions: (a) (i) acid, i-BuOCOCl, NMM, THF, DMF; (ii) amine, NMM. (b) HCl, EtOAc or dioxane. (c) H2, Pd/C, MeOH.
to use cyanamide to effect the perguanidinylation were
unsuccessful.28 Finally, the synthesis was completed by
treatment of 18 with TFA, which gave the desired octaargi-
nine product 1 in quantitative yield. Octaarginine 1 was
identical in all respects to an authentic sample prepared using
Fmoc-based solid-phase synthesis.
In summary, a solution-phase synthesis of the novel
peptide molecular transporter, octaarginine 1, was completed
in 10 steps and 29% overall yield from protected ornithine
monomers 6 and 7. The choice of appropriate orthogonal
protecting groups proved essential to the success of this
synthesis, and the segment doubling strategy provided a
substantial improvement in the preparation of arginine
homooligomers such as 1 in terms of both cost (>10-fold
lower) and scalability compared to the previously reported
synthesis utilizing a solid-phase strategy.2 The mild conver-
sion of trifluoroacetamides directly to guanidines allows one
to consider a trifluoroacetamide as a masked guanidine, thus
avoiding the need to use expensive guanidine protecting
groups normally required in peptide synthesis. Overall, this
work lays the foundation for the efficient preparation of
greater quantities of 1 to further in vitro, in vivo, and clinical
studies (in progress) of conjugates of 1 with drugs, drug
candidates, and molecular probes.
Scheme 3. Perguanidinylation to Yield 1a
Acknowledgment. Support of this work by grants from
the National Institutes of Health (CA 31841, CA 31845), a
National Institute of Health Fellowship to E.T.P. (CA 80344),
an Eli Lilly Graduate Fellowship to K.P., a Stanford Graduate
Fellowship to C.L.V.D., and a CellGate Fellowship to T.C.J.
is gratefully acknowledged.
Supporting Information Available: Full experimental
details and characterization data for compounds 1, 6-16,
and 18. This material is available free of charge via the
Internet at http://pubs.acs.org.
OL0161108
a Conditions: (a) 17, Na2CO3, aq. MeOH, 55 °C (51%). (b) TFA
(>99%).
(28) Garrigues, B.; Lopez, L.; Mulliez, M. Phosphorus, Sulfur Silicon
Relat. Elem. 1991, 57, 195-202.
3232
Org. Lett., Vol. 3, No. 21, 2001