Highly efficient (R-X-R)-type carbamates as molecular transporters for cellular delivery

Article abstract of DOI:10.1021/ja210026m

The (R-X-R) motif-containing arginine-rich peptides are among the most effective cell-penetrating peptides. The replacement of amide linkages in the (R-X-R) motif by carbamate linkages as in (r-ahx-r)₄ or (r-ahx-r-r-apr-r)₂ increases

Full text of DOI:10.1021/ja210026m

Communication
pubs.acs.org/JACS
Highly Efficient (R-X-R)-Type Carbamates as Molecular Transporters
for Cellular Delivery
Kiran M. Patil,†,§ Rangeetha J. Naik,‡,§ Rajpal,‡ Moneesha Fernandes,† Munia Ganguli,*,‡
and Vaijayanti A. Kumar*,†
†Organic Chemistry Division, National Chemical Laboratory, Dr. Homi Bhabha Road, Pune 411 008, India
‡Lab No. 203, Institute of Genomics and Integrative Biology, Mall Road, Delhi 110 007, India
S
* Supporting Information
amino acid-containing peptides.10 The peptide linkages at
ABSTRACT: The (R-X-R) motif-containing arginine-rich
peptides are among the most effective cell-penetrating
peptides. The replacement of amide linkages in the (R-X-
R) motif by carbamate linkages as in (r-ahx-r)4 or (r-ahx-r-
r-apr-r)2 increases the efficacy of such oligomers several-
fold. Internalization of these oligomers in mammalian cell
lines occurs by an energy-independent process. These
oligomers show efficient delivery of biologically active
plasmid DNA into CHO-K1 cells.
arginine sites were still amenable to cleavage for the (R-X-R)4
peptides and caused toxicity.11 Peptidomimetics such as
peptoids,12 oligoureas,13 oligocarbonates,14 and oligocarba-
mates15 as transporters have been explored with an aim to
increase the stability of the oligomers in vivo. Among the
carbamate analogues, the oligoarginine sequences were
synthesized and were found to have enhanced uptake
properties.15
In this Communication, we report the design, synthesis, and
uptake properties of carbamate-linked (r-x-r)-motif oligomers
based on their (R-X-R)-type amide-linked counterpart. It was
envisaged that the (r-x-r)-motif would exhibit synergistic
properties when carbamate linker is used in tandem with
appropriate spacer (-x-) for optimizing the interaction of two
consecutive guanidine groups as in R-X-R (X = Ahx/Apr) with
the cell membrane, leading to favorable uptake. The carbamate
linkage adds O−CH2 to the amide NH−CO (Figure 1), thus
adding flexibility and hydrophobicity and reducing the
possibility of hydrogen-bonded secondary structures.16 This
would probably leave the oligomers more amenable for
adopting structural features better suited to interact with lipid
membranes. Aminohexanol (ahx) and aminopropanol (apr)
linkers were used to separate selected adjacent guanidine
functionalities and to optimize the effect of chain lengths on
guanidinium display.
The carboxyfluorescein (CF)-tagged (R-Ahx-R)4_amide_CF
(I) and unlabeled oligomer (R-Ahx-R)4_amide_Phe (IV) were
synthesized employing standard Boc chemistry and used as
controls to compare the cell uptake and cargo delivery
capability of designed oligocarbamates (Table 1). The
carbonate monomers for the synthesis of oligocarbamates
were synthesized according to Scheme 1. The ester function in
Nα-Boc-L-Arg(Mts)-methyl ester 1a was reduced to yield the
corresponding alcohol 2a. The hydroxyl groups in 2a, N-Boc-
aminohexanol 2b, and N-Boc-aminopropanol 2c were each
activated as their p-nitrophenyl carbonates by treating with p-
nitrophenyl chloroformate in pyridine and dichloromethane to
get the N-Boc-protected monomers 3a, 3b, and 3c, respectively,
for solid-phase carbamate synthesis. The oligocarbamates (r-
ahx-r)4_carbamate_CF (II), (r-ahx-r-r-apr-r)2_carbamate_CF
(III), and (r-ahx-r)4_carbamate_Phe (V) (Table 1) were
ell-penetrating peptides (CPPs) have proved very useful
C
as carriers for the intracellular delivery of several types of
small molecular drugs as well as of peptide/oligonucleotide
cargos for therapeutic applications. A significant number of
CPPs are cationic in nature and contain at least six amino acids
such as lysine or arginine which are positively charged at
physiological pH. The repertoire of naturally derived CPPs
such as Tat-peptide,1 penetratin,2 and pAntp3 was later
augmented by synthetic homopolymers of cationic α-amino
acids.4 It was demonstrated that the guanidine headgroup of
arginine in polyarginine peptides was the critical component for
this efficiency.4,5
The arginines separated by ω-amino acids (-X-) with variable
chain lengths as in the (R-X-R) peptide motif were shown to be
far better than the natural α-amino acid-containing peptides,6
probably due to their amphipathic nature. The arginine
peptides interspersed with ε-aminohexanoic acid (Ahx) (Figure
1) and γ-aminopropionic acid (Apr) were found to be among
the most effective synthetic CPPs for highly efficient cargo
transportation.7,8 The mechanism of uptake of CPPs is still
under debate, and it is postulated that it could be operating in
multiple modes.9 The (R-X-R)-type of peptides were found to
be comparatively more stable to peptidases than the natural α-
Received: October 25, 2011
Published: April 11, 2012
Figure 1. Representation of (R-Ahx-R) amide- and carbamate-linked
oligomers chemically illustrating their backbone differences.
© 2012 American Chemical Society
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Scheme 1. Synthesis of Carbonate Monomers for
a
Oligocarbamate Synthesis
Figure 2. Partitioning of oligomers I (A), II (B), and III (C) in
water−octanol without added sodium laurate (1) and with 3.0 (2) and
6.0 equiv (3) of sodium laurate/guanidinium group.
a
Reagents and conditions: (i) NaBH4:LiCl (1:1) (2.5 equiv),
THF:EtOH (3:4) (2a, 89%); (ii) p-nitrophenyl chloroformate (1.2
equiv), dry pyridine (2.5 equiv), CH2Cl2 (3a, 75%; 3b, 82%; 3c, 86%).
a
Table 1. MALDI-TOF Analysis of the Oligomers
MALDI-TOF mass
oligomer
calcd
obsd
(R-Ahx-R)4_amide_CF (I)
(r-ahx-r)4_carbamate_CF (II)
(r-ahx-r-r-apr-r)2_carbamate_CF (III)
(R-Ahx-R)4_amide_Phe (IV)
(r-ahx-r)4_carbamate_Phe (V)
r8_carbamate_CF15 (VI)
2076.21
2406.33
2322.24
1907.25
2237.36
1833.95
1664.98
2077.35
2408.40
2324.81
1907.33
2238.33
1834.49
1666.71
Figure 3. CD spectra of the oligoamide I and oligocarbamates II and
III (10 μM) in water.
r8_carbamate_Phe (VII)
where replacement of a relatively rigid amide bond by the more
flexible carbamate led to a loss of secondary structure.16
Thus, as per the design expectations, the oligocarbamates II
and III were shown to be amphipathic in nature, similar to the
oligoamide I, but less stuctured. It would be interesting to see if
these attributes could prove to be advantageous in the case of
the oligocarbamates, enabling them to easily adopt a
conformation suited for membrane interactions and cellular
uptake as envisaged. The oligocarbamates, endowed with better
amphipathic, unstructured backbones and eight guanidine
functionalities, were evaluated in this study to gauge their
ability to enter cells in comparison with the control
oligoamides. CF-labeled oligomers I−III and VI were used in
the cellular uptake experiments. Oligomers IV, V, and VII were
used for cargo delivery, in which Phe replaces the CF-tag in I,
II, and VI, respectively.
Cellular uptake was determined by fluorescence-activated cell
sorting (FACS) analysis in CHO-K1 cells after incubation at 37
°C for 1 h at 5 μM concentration. CF itself did not enter cells
in this assay. The uptake at 5 μM of I and II is depicted in
Figure 4. The oligomer II showed excellent uptake in nearly
100% of cells, compared to the 70% of cells showing uptake of
control oligomer I. The mean fluorescence intensity for
oligomer II was also about 9 times greater than that for the
control oligoamide I. The uptake of II was also better than for
the known r8-carbamate15 VI. An additional experiment with
mild acid wash, known to effectively quench cell membrane-
bound fluorescence,18 was carried out to ensure that only the
internalized fluorescence was assayed. The relative results of
cellular uptake did not show any change (data not shown);
however, high cellular toxicity was observed due to the acid
wash.
a
R and Ahx denote amino acid residues; the lowercase r, ahx, and apr
denote amino alcohol residues; CF denotes carboxyfluorescein. CF or
Phe is at the N-terminus of oligomers.
synthesized manually on solid phase (MBHA resin) using 3a,
3b, and 3c appropriately and by following repetitive cycles of
deprotection, neutralization, and coupling. They were cleaved
from the solid support using TFA-TFMSA, purified by RP-
HPLC, and characterized by MALDI-TOF mass spectrometric
analysis. The known r8_carbamate_CF15 (VI) and unlabeled
r8_carbamate_Phe (VII) were also synthesized for comparison.
The oligocarbamates displayed a higher retention time on
reversed-phase HPLC columns than the oligoamide (Support-
ing Information, Figure S22), demonstrating that the carbamate
linkage indeed endowed the oligomers with increased hydro-
phobicity.
Further, partitioning experiments in model octanol−water
system were performed to estimate the affinity of the oligomers
for the lipid membrane.17 The partitioning of the amide (I) and
oligocarbamates (II and III) was monitored using CF-labeled
oligomers. The oligomers were highly water-soluble and
remained completely in the aqueous phase. Upon addition of
sodium laurate, complete migration of amide I and carbamate
II into the octanol phase was observed, whereas carbamate III
was found in aqueous as well as octanol phases. The
observation is indicative of the fact that the linker length
indeed affects an oligomer's affinity for water as well as lipid and
consequently its amphipathic nature. This partitioning could be
monitored visually by the naked eye and is shown in Figure 2.
CD spectroscopic studies were then carried out to study the
presence of secondary structures in these oligomers (Figure 3).
The oligoamide I exhibited a significant CD pattern with
prominent signals around 200 and 230 nm, indicating a
structured backbone, whereas no CD pattern emerged for the
oligocarbamates II and III, indicating random backbone
conformations. This is in tune with the literature reports
All energy-dependent pathways such as endocytosis are
known to be inhibited at low temperatures and pre-treatment
of cells with sodium azide.9 To study the effect of temperature,
the cellular uptake of the oligomers I, II (Figure 4), and VI was
compared at 4 and 37 °C using the same protocols as above.
Surprisingly, the oligocarbamate II was taken up by almost
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Figure 4. FACS data of uptake in CHO-K1 cells at 37 and 4 °C.
100% of cells at 4 °C, whereas amide I was taken up in only
about 20% of cells. The ratio of mean fluorescence at 4 °C for
II:I was almost 16:1. Uptake of oligomer VI was found to be
adversely affected at lower temperature (Figure S28) in our
studies.
Confocal microscopy studies were further undertaken in
CHO-K1 cells in order to see the intracellular distribution of
the synthesized oligomers. As can be seen from Figure 5, the
oligomer II (at 5 μM) exhibited strong fluorescence, with
predominantly cytosolic localization. The fluorescence for the
control oligoamide I was observed only at higher concentration
(10 μM) and was found in cytosol as well as near/within the
nucleus. The observed uptake of oligomer III was similar to
that of oligomer II (Figure S25). The entry of the oligomer II
at 4 °C was also checked by confocal microscopy. The majority
of the cells showed significant uptake of the oligomer (Figure
S24), confirming the energy-independent entry pathway, as
indicated by the FACS analysis. Cell uptake studied by confocal
microscopy after pretreating the cells with sodium azide
indicated no apparent inhibition (Figure S27) of uptake and
also showed the residence of oligocarbamate II mainly in the
cytoplasm. These experiments clearly indicate the high
efficiency of oligomer II compared to the control oligomers I
and VI to enter cells by energy-independent mechanisms, most
likely by direct translocation into cytoplasm, thereby reducing
endosomal entrapment. Additional cell uptake experiments
were then undertaken in HeLa cells using oligocarbamate II
along with the control oligoamide I. As in the case of CHO-K1
cells, very high uptake localized in the cytoplasm was observed
also in HeLa cells for the oligocarbamate II as compared to
oligoamide I (Figures S36 and S37).
The cytotoxicity of the synthesized oligomers in CHO-K1
cells was assessed using the Cell Titer-Glo Luminescent Cell
Viability Assay system (Figure S29). The oligocarbamates II
and III showed viability/toxicity comparable with that of the
control oligomer I at both concentrations studied and at
extended time. To assess the potential applicability of
oligocarbamates II, III, and VI, experiments in comparison
with oligomer I in the presence of serum were also performed.
The uptake of oligocarbamates was again found to be better
than that of the oligoamide (Figure S30). To test the
applicability of the oligocarbamates in delivering cargo
molecules in biologically active form, we carried out pMIR-
Report plasmid DNA transfection in CHO-K1 cells. The
Figure 5. Confocal microscopy images of CHO-K1 cells after 4 h
incubation at 37 °C with the oligoamide I (10 μM) and
oligocarbamate II (5 μM). (A) Staining with nuclear stain, Hoechst
33342. (B) Fluorescence image. (C) Merged images. Scale bar: 20 μm.
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oligocarbamate V (unlabeled counterpart of oligocarbamate II
was chosen to avoid any interference of the CF label), after
complexation with plasmid DNA (Figure S34) and transfection,
exhibited efficient expression of the reporter gene (Figure 6)
AUTHOR INFORMATION
Corresponding Author
va.kumar@ncl.res.in; mganguli@igib.res.in
■
Author Contributions
§K.M.P. and R.J.N. contributed equally.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
CSIR-New Delhi is acknowledged for a research grant
(NWP0036A) to V.A.K.; NCL-IGIB Joint Research Initiative
for funding; CSIR-New Delhi for Research Fellowships to
K.M.P. and Rajpal; DBT-New Delhi for Research Fellowship to
R.J.N.; and Manika Vij (IGIB) for help with some FACS
experiments.
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ASSOCIATED CONTENT
* Supporting Information
■
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K. E. J. Leukoc. Biol. 1993, 53, 462.
NMR and mass spectra of 2a and 3a−c; HPLC chromatograms
and MALDI-TOF spectra of oligomers; further confocal
microscopy images, FACS analysis, cell viability assay, and
cargo delivery data in CHO-K1 cells; FACS analysis, confocal
microscopy images, and cell viability assay in HeLa cells; DNA
complexation studies by gel mobility shift assay of pDNA with
oligomers IV and V and cell viability assay of the complexes in
CHO-K1 cells. This material is available free of charge via the
Internet at http://pubs.acs.org.
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