Cell penetrable peptoid carrier vehicles: synthesis and evaluation.

Article abstract of DOI:10.1039/b306438g

Using a highly efficient solid-phase route a series of fluorescein conjugated peptoid oligomers were synthesised and observed to display remarkable cell penetrating properties, offering the possibility of highly efficient cellular targeting.

Full text of DOI:10.1039/b306438g

Cell penetrable peptoid carrier vehicles: synthesis and evaluation†
Ilaria Peretto,a Rosario M. Sanchez-Martin,b Xui-hong Wang,b John Ellard,b Stifun Mittoob and Mark
Bradley*b
a NiKem Research S.r.l., Via Zambelletti, 25, 20021 Baranzate di Bollate (MI), Italy
b Department of Chemistry, University of Southampton, Highfield, Southampton, UK SO17 1BJ.
E-mail: mb14@soton.ac.uk; Fax: +44 (0)23 80596766; Tel: +44 (0)23 80593598
Received (in Cambridge, UK) 6th June 2003, Accepted 25th July 2003
First published as an Advance Article on the web 11th August 2003
Using a highly efficient solid-phase route a series of
fluorescein conjugated peptoid oligomers were synthesised
and observed to display remarkable cell penetrating proper-
ties, offering the possibility of highly efficient cellular
targeting.
carbon (aminohexanoic acid) spacer unit (Scheme 2) to enable
efficient coupling.
Resin bound peptoids 3 were cleaved from the solid support
by treatment with a mixturre of TFA : TIS (95 : 5) for 3 h. The
crude materials were precipitated in cold diethyl ether and
purified by semi-preparative RP-HPLC ( > 95% purity).10 The
use of the sulfonamide during the construction of peptoids 3
(Scheme 2) allowed selective deprotection (2-mercaptoethanol)
Cellular access to foreign compounds, be they probes of a
diagnostic nature or potential drug candidates, is generally
severely limited by the solubility properties of the material in
question. The properties must be such that the substances are
sufficiently polar to dissolve in biological fluids yet not so polar
that they are unable to cross the relatively non-polar cell bilayer.
It has been demonstrated that cellular penetration may be
accomplished by the HIV Tat peptide which has been
successfully used as a delivery vehicle into cells, this ability
being attributed to the relatively short, but highly basic Tat
(49–57) sequence (RKKRRQRRR).1 Reports detailing the use
of structurally analogous cationic b-peptide or peptoid oligo-
mers based on lysine or arginine residues of the Tat sequence
have been reported and potentially represent an efficient means
of translocation across cell membranes.2–6 Equally, poly-
-
L
Scheme 1 Synthesis of monomer units 1 and 2. Reagents and conditions: i,
Boc2O (0.13 eq.), dioxane, 24 h, 91%; ii, 2-nitrobenzenesulfonyl chloride
(0.13 eq), dioxane, 24 h, 87%; iii, ethyl bromoacetate (1 eq.), Et3N (3 eq.),
THF, 24 h (R = Boc, 55%; R = O2NC6H4SO2, 42%); iv, NaOH (1 eq.),
CH3OH : H2O : dioxane (3 : 1 : 8); v, Fmoc-OSu (1 eq.), H2O : CH3CN (1
: 1), pH = 8.5–9.0, 45 min (R = Boc, 40%; R = O2NC6H4SO2, 60% over
two steps).
lysine has been shown to effectively increase the uptake of
small organic molecules7 and arginine rich molecules have been
developed to successfully aid the administration of otherwise
poorly absorbed drugs.8
The aims of this study were the efficient synthesis and
evaluation of a number of peptoid based, polycationic, cell
penetrable moieties. A solid phase route was developed to
enable firstly efficient synthesis, without having to resort to the
more classical methods of peptoid synthesis, and secondly to
enable subsequent derivatisation of the peptoid thus allowing
cellular access. Efforts were concentrated on peptoid deriva-
tives of lysine because cellular penetration has been reported to
be independent of the stereochemistry of polycationic polymers
tested and peptoids are known to possess a greater stability to
proteolytic degradation than peptides.9
Cell penetrable peptoids were therefore synthesised and
conjugated to fluorescein and their cellular uptake assayed on 2
cell lines. The peptoids were constructed on solid phase using
monomer units 1 and 2. These were prepared by reacting ethyl
bromoacetate with mono-Boc or mono-2-nitrobenzenesulfonyl
protected 1,6-hexanediamine (Scheme 1). Saponification of the
resultant ester followed by N-Fmoc protection with Fmoc-
succinimide made available large quantities of monomers 1 and
2 for solid phase oligomer synthesis.
The peptoids were assembled on polystyrene aminomethyl
resin functionalised with the Rink amide linker. In each of the
coupling steps the Fmoc-protected monomer, 1 or 2, and
bromo-tris(pyrrolidino)phosphonium
hexafluorophosphate
(PyBrOP) were added to the resin in a two-fold molar excess
(0.08 M) (PyBrOP was necessary to enable efficent coupling of
the secondary amine). Deprotection of the Fmoc group (20%
piperidine in DMF) and repeated coupling gave oligomers of
the required length. A fluorescein group was then covalently
attached to the N-terminus of each oligomer, separated by a six
Scheme 2 Synthesis of peptoid oligomers 3 (n = 3, 5, 7). Reagents and
conditions: i, 20% piperidine/DMF; ii, 1 or 2 (2 eq.), PyBrOP (2 eq.); N,N-
diisopropylethylamine (DIPEA) (4 eq.), CH2Cl2; iii, N-Fmoc-aminohex-
anoic acid (2 eq.), PyBrOP (2 eq.); DIPEA (4 eq.), CH2Cl2; iv,
5(6)-carboxyfluorescein (3 eq.), 1-hydroxybenzotriazole (HOBt) (3 eq.),
N,NA-diisopropylcarbodiimide (DIC) (3 eq.), DMF : CH2Cl2 (1 : 1); v, TFA
: triisopropylsilane (TIS) (95 : 5); vi, 2-mercaptoethanol (0.3 M)/DBU (0.3
M) in DMF (3 3 45 min).
† Electronic supplementary information (ESI) available: experimental
details. See http://www.rsc.org/suppdata/cc/b3/b306438g/
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CHEM. COMMUN., 2003, 2312–2313
This journal is © The Royal Society of Chemistry 2003

and subsequent functionalisation of specific amine groups while
still attached to the solid phase.
have also indicated a greater uptake rate the larger the
oligomer.2,5 In all cases, free fluorescein as control showed no
marked cellular uptake stipulating that the carrier molecules
were playing a vital transportation role.
The ability of the peptoid oligomers to enter cells was
assayed by fluorescence microscopy and flow cytometry on
HEK293T (human embryonic kidney) and B16F10 (mouse
melanoma) cells. The cells were cultured in Dulbecco’s
Modified Eagle’s Medium (DMEM) supplemented with 10%
FCS and 4 mM glutamine and the labelled oligomers were
tested in parallel (in triplicate), using free fluorescein as a
control.
The peptoids 3a,b,c (n = 3, 5, 7) were incubated with the
HEK293T cells at 37 °C for 3 h at concentrations of 0.1, 1, 10
and 100 mM. Results indicated that the order of uptake of the
peptoids was dependent on the number of monomer units of the
oligomer. Higher oligomers yielded greater cellular penetration
(3c (n = 7) > 3b (n = 5) > 3a (n = 3)) as evidenced by a
greater population of fluorescently labelled cells. Quantification
by laser scanning cytometry (Table 1 and Fig. 1) showed
oligomer 3a (n = 3) at 10 mM resulted in 99% of the cell
population being labelled after 4 h and this labelling was
equalled by 3c (n = 7) at an equivalent concentration. Further
examination of the peptoids by fluorescence microscopy
revealed that cellular uptake was concentration dependent,
fewer cells exhibiting fluorescence at lower concentrations,
although in all cases cell permeability was evident. A similar
result was observed with the B16F10 cell line, though it was
noticed that cellular penetration with peptoid 3a (n = 3) was
only observed at concentrations as high as 50 mM. These
findings are complementary to reports by other groups who
For both cell types, treatment of the cells with 0.5% sodium
azide for 30 min prior to exposure to the peptoids (10 mM)
resulted in complete inhibition of uptake, indicating that the
transport mechanism of these cationic homopolymers is energy
dependent. In addition incubation of 3 (n = 3, 5, 7, at c = 10
mM) at varying temperatures (4, 20 and 37 °C) revealed that
lowering the temperature greatly affected the rate of uptake. At
4 °C no internalisation was observed, whereas at 20 °C only
slightly fewer cells showed green fluorescence compared to 37
°C where virtually all the cells were fluorescently labelled
(Table 1). These temperature-dependent results may offer an
insight into the mechanism of uptake, suggesting possible
endocytosis pathways which have been implicated in the
transport of small organic molecules covalently bound to
structurally similar poly-
-lysine7,11 though other transport
L
pathways cannot be completely dismissed.
Finally none of the peptoids 3 (n = 3, 5, 7) assayed were
found to be toxic at any of the concentrations tested, as verified
by a MTT12 toxicity and a trypan blue assay13 to ascertain cell
viability.
A versatile and efficient solid phase approach has been
developed for the synthesis of these cell permeable peptoid
oligomers. A series of fluorescein conjugates were constructed
and fluorescence microscopy and FACS analysis indicated that
these materials possessed the ability to successfully penetrate
the membrane of cells with internal localisation observed in the
cytosol and the nucleus, with concentration in the nucleoli. Of
the peptoid oligomers tested 3c (n = 7) exhibited maximum
internalisation ( > 99% of cells labelled) and enabled efficient
cellular access.
Table 1 FACS analysis on HEK293T cells with oligomer 3
Oligomer 3a
% Incorporation
n = 3
n = 5
n = 5
n = 7
99
93b
97
99
This work was supported by a grant from the MRC
(ROPA).
Notes and references
a c = 10 mM, 37 °C, 4 h. b c = 10 mM, 20 °C, 4 h.
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Fig. 1 FACS analysis of HEK293T cells: (a) untreated cells, (b) incubation
of cells with peptoid 3 (n = 7).
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