Design, synthesis, and membrane-translocation studies of inositol-based transporters

Article abstract of DOI:10.1002/anie.200600312

(Figure Presented) Delivery vehicles: Novel guanidine-containing "transporters" constructed on a dimeric inositol scaffold show significant translocation across the cell membrane and the blood-brain barrier, as well as unique in vitro and in vivo distributions. Doxorubicin was efficiently delivered to mouse-brain tissue by conjugating the compound with such a transporter (see the fluorescence microscopy image).

Full text of DOI:10.1002/anie.200600312

Angewandte
Chemie
Cell-Penetrating Molecules
DOI: 10.1002/anie.200600312
Design, Synthesis, and Membrane-Translocation
Studies of Inositol-Based Transporters**
Kaustabh K. Maiti, Ock-Youm Jeon, Woo Sirl Lee,
Dong-Chan Kim, Kyong-Tai Kim, Toshihide Takeuchi,
Shiroh Futaki, andSung-Kee Chung*
Cell membrane systems represent formidable physical bar-
riers for the trafficking of unintended molecules. Only those
molecules with an appropriate range of molecular size,
polarity, and charge are allowed to pass through cell
membranes. Many potential drug molecules have to over-
come these barriers, and a variety of chemical and physical
methods have been proposed as means of accomplishing this
challenging task. A number of peptides have been reported to
[*] Dr. K. K. Maiti, O.-Y. Jeon, W. S. Lee, Prof. Dr. S.-K. Chung
Department ofChemistry
Pohang University ofScience and Technology
Pohang 790-784 (Korea)
Fax: (+82)54-279-3399
E-mail: skchung@postech.ac.kr
D.-C. Kim, Prof. Dr. K.-T. Kim
Department ofLief Science
Pohang University ofScience and Technology
Pohang 790-784 (Korea)
T. Takeuchi, Prof. Dr. S. Futaki
Department ofBiouf nctional Chemistry
Institute for Chemical Research
Kyoto University
Uji, Kyoto 611-0011 (Japan)
and
PRESTO
Japan Science and Technology Corporation (JST)
Kawaguchi, Saitama 332-0012 (Japan)
[**] We thank Dr. Hyun Soo Kim for technical assistance with cell
cultures, MTT tests, and operation ofthe conof cal laser scanning
microscope at Postech. Financial support from Postech, Posco
(Postech Biotech Center), BK-21, and MOST (National Frontier
Research Program administered through KRICT/CBM), and Grants-
in Aid for Scientific Research from the Ministry of Education,
Culture, Sports, Science, and Technology ofJapan is grateuf lly
acknowledged.
Supporting information for this article is available on the WWW
under http://www.angewandte.org or from the author.
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translocate themselves across cell membranes. They are
an excellent platform to build a molecular diversity by
appending desired structural residues around the scaffold. A
number of research groups have previously attempted to
design various peptidomimetics on the carbohydrate scaf-
folds.[24]
We prepared a number of synthetic transporter molecules
by utilizing myo- and scyllo-inositol dimers as the scaffolds
(Figure 1). Although myo-inositol is most abundant in nature
known as cell-penetrating peptides or protein transduction
domains, and are capable of delivering exogenous molecules
into cells. The first cell-membrane-penetrating property was
observed with HIV-1 Tat protein (Tat-86) and then with the
antennapedia (Antp) protein of Drosophila and others.
Subsequent studies have demonstrated that the membrane-
penetrating ability is clearly associated with relatively short
peptide sequences in these proteins;
for example, nine basic amino acid
residues for the Tat protein (residues
49–57), 16 amino acids for the Antp
protein (residues 43–58) that have a
potentially amphipathic and basic
helical structure, and 12 hydrophobic
amino acids from Kaposiꢀs sarcoma
fibroblast growth factor membrane-
translocating
sequence
(KFGF-
MTS).[1–4] Despite the lack of a clear
understanding of the membrane-
translocation mechanisms, many
research efforts have been extended
toward their applications as delivery
vectors in order to improve the phar-
macology of poorly bioavailable
drugs, such as, small molecules,[5] pro-
teins,[6–10] nucleotides, and genes.[11–13]
Reports have also appeared on the
development of membrane-translo-
cating transporter molecules that
mimic natural and non-natural
amino acids and their structural ana-
logues, especially in the area of poly-
cationic peptide mimetics.[14–23]
Although these peptides com-
monly share a high efficiency as
molecular transporters, the structural
motifs and physicochemical proper-
Figure 1. Structures ofthe synthetic molecular transporters 1–3 and a covalent conjugate of 3c
with doxorubicin, 4. Cbz=carbobenzyloxy.
ties of these cell-penetrating peptides are quite diverse. It
appears likely that different peptides may use variant
mechanisms of translocation and that even the same peptide
can use alternative pathways depending on conditions of
administration and the nature of cargo, as was previously
suggested.[10] Therefore, the modifications of the structures
and physicochemical properties of even the known molecular
transporters may yield novel transporters that exhibit differ-
ent spectra in vivo and in vitro, as well as different
translocation efficiencies. In addition, the in vivo efficacy,
stability to various endogenous enzymes, processing cost, and
long-term safety still remain practical concerns. With these
considerations in mind, we have developed a novel class of
molecular transporters as a potential delivery vector which
consist of multiple units of the guanidine residue attached to
the dimeric inositol molecule as a scaffold. Carbohydrate and
inositol structures reveal the highest density of functionality
among organic compounds per unit weight in the form of
multiple hydroxy groups with diverse stereochemical varia-
tions. Also, they occur naturally and are largely devoid of any
noticeable toxicity. Carbohydrates and inositols may provide
and readily available, substitution at sites other than the C2
and C5 positions tends to generate a diastereomeric mixture,
whereas scyllo-inositol, albeit less readily available, has a
higher degree of symmetry with all equatorial hydroxy
groups. We initially developed synthetic protocols with myo-
inositol as the scaffold to give 1, and then prepared the
transporter 2 based on the scyllo-inositol scaffold (see
Supporting Information for details of the syntheses of 1 and
2).[25,26] Compounds 3a–c were prepared as shown in
Scheme 1. Compound 5, prepared from 1-O-Bz-2,3:5,6-di-
O-isopropylidene-scyllo-inositol[26] (see Supporting Informa-
tion), was converted into 6 and 7, and these two components
were coupled using EDC, HOBT, and DMAP in DMF to give
8 in 65% yield. The compound 9, obtained from 8 by removal
of the isopropylidene protecting groups, was exhaustively
acylated with N-Boc-protected 4-aminobutyric (n = 3), 6-
aminocaproic (n = 5), and 8-aminocaprylic (n = 7) acids,
respectively, under EDC coupling conditions to give three
dimeric products, 10a–c, with varying chain lengths (see
Scheme 1). Removal of the N-Boc protecting groups of 10a–c
exposed the terminal amino groups, which were guanidiny-
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purification by medium-pressure liquid chroma-
tography (MPLC) on reverse-phase (C8) silica
gel.
For the preparation of a transporter–cargo
conjugate, Cbz-protected l-serine was attached
through an amide bond to the primary amine of
compound 13c under the EDC coupling con-
ditions. The hydroxy group of the serine moiety
was then coupled with the primary amino group
of doxorubicin through a carbamate bond.[27]
Finally the N-Boc protecting groups of the
guanidine moieties were removed with gaseous
HCl to yield the covalent conjugate 4 as its HCl
salt (see Figure 1). All key synthetic intermedi-
ates as well as the final target compounds were
satisfactorily characterized by NMR spectrosco-
py and MALDI-TOF mass spectrometry.
Preliminary evaluations of the synthetic
transporters were carried out for their ability
to translocate into the cytoplasm with three cell
lines: COS-7, RAW264.7, and HeLa cells. After
exposure of the cultured cells to the transporters
for 3–5 minutes, cellular uptake of the four
compounds 1, 2, 3b, and 3c, without fixing, was
found to be more efficient than with the
reference compounds, dansyl- and fluorescein-
labeled nona-arginine (R9-Fl), on the basis of
confocal laser scanning microscopy observa-
tions; compound 3a appeared to be approxi-
mately as efficient as the reference standard.
Thus, more detailed studies were carried out on
HeLa cells with the more stable amide com-
pounds 3a–c. The internalization efficiencies of
the transporters 3a–c were compared with that
of fluorescein-labeled octaarginine (R8-Fl), a
representative cell-penetrating peptide. The
HeLa cells were treated with the respective
transporter (10 mm) at 378C for 1 h. Fluores-
cence-activated cell sorter (FACS) analysis of
the transporters demonstrated that 3b and 3c
were internalized 1.8 and 2.5 times as much as
R8-Fl, whereas the amount of 3a internalized
was about half that of R8-Fl under the same
conditions (Figure 2A). A more precise kinetics
study of internalization was performed with the
most efficient transporter 3c in comparison with
R8-Fl (Figure 2B). In contrast to the observa-
tion that the amount of R8-Fl internalized
reached a plateau in 3 h, the internalization of
Scheme 1. Synthetic route to compounds 3a–c. Reagents and conditions: a) 10% Pd/
C, H2 (1 atm), CH2Cl2/MeOH (1:9), quant.; b) NaOH, MeOH, then AcOH, 92%; c) 6,
7, EDC, HOBT, DMAP, DMF, 65%; d) p-TSA, CH2Cl2/MeOH (9:1), quant.; e) EDC,
DMAP, N-Boc-protected acids: 4-aminobutyric acid (n=3), 6-aminocaproic acid
(n=5), or 8-aminocaprylic acid (n=7), DMF, 65–80%; f) HCl(g), EtOAc, quant.;
g) N,N’-di-Boc-N’’-triflylguanidine, Et3N, dioxane/H2O (5:1), 55–76%; h) 10% Pd/C,
H2 (1 atm), MeOH/CH2Cl2 (9:1), 75–80%; i) FITC-I, Et3N, THF, absolute EtOH, 52–
65%; j) HCl(g), EtOAc, 76–80%; or TFA/CH2Cl2 (1:1), 86–94%. Bn=benzyl; EDC=1-
ethyl-3-(3-dimethylaminopropyl)-carbodiimide; HOBT=hydroxybenzotriazole;
DMAP=4-(dimethylamino)pyridine; DMF=N,N-dimethylformamide; p-TSA=p-tolue-
nesulfonic acid; Boc=tert-butoxycarbonyl; FITC=fluorescein isothiocyanate; TFA=
trifluoroacetic acid.
lated with a large excess of N,N’-di-Boc-N’’-trifluorometha-
nesulfonylguanidine and triethylamine to give three target
molecules, 12a–c, with the Boc-protected guanidine moiety.
After removal of the N-benzyl protecting group in the side
chain of 12a–c, the fluorescein tag was attached to the
primary amino group of 13a–c using fluorescein-5-isothio-
cyanate (FITC-I) and triethylamine to give 14a–c. The Boc
groups at the guanidine moieties in compounds 14 were
removed with either trifluoroacetic acid or gaseous HCl to
yield three molecular transporters, 3a–c, as salts after
3c kept increasing and did not reach a plateau in 6 h. The total
cellular uptake of 3c was almost three times higher than that
of R8-Fl. However, no significant toxicity was evident for
these cells.
The cellular (organellar) localizations of the transporter
3c were examined next without fixing. First, HeLa cells were
treated with 3c (10 mm) at 378C for 1 h in the presence of
tetramethylrhodamine-labeled transferrin (Rho-Tf) as
a
marker of clathrin endocytosis (25 mgmLÀ1). Considerable
fractions of both labels in punctate structures showed a
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arginine-rich transporter. HeLa cells were treated with 3c in
the presence of tetramethylrhodamine-labeled Tat peptide
(Rho-Tat; 10 mm each) at 378C for 3 h. Also, little colocaliza-
tion of the signals of 3c and Rho-Tat was observed. The
majority of the signals from Rho-Tat were observed in the
peripheries of the nucleus, whereas the wider spread signals of
3c were observed in the cytosolic region. Moreover, as was
previously observed, the Tat-positive structures were often
considerably larger than those for 3c (Figure 2D). We further
examined whether 3c was delivered into lysosomes or
mitochondria by co-staining with specific dyes for lysosomes
(LysoTracker) and mitochondria (MitoTracker), respectively
(Figure 2E). No significant colocalization of the signals of 3c
with MitoTracker (250 nm) was observed (Figure 2E, part a),
and the majority of 3c did not reach the lysosome in 1 h
(lysosomes were stained with 50 nm LysoTracker; see Fig-
ure 2E, part b). These results strongly suggest that 3c may use
different mechanisms of internalization and cellular local-
ization from those transporters reported to date, even though
it comprises a cluster of guanidinium moieties albeit on a
dimeric inositol scaffold.
Next, we examined if compound 3c also showed a unique
in vivo biodistribution pattern. Thus, 3c (HCl salt, 77 mgkgÀ1)
was dissolved in sterile distilled water and injected into 8-
week-old mice (C57BL/6) intraperitoneally. After 20 minutes,
the treated mice were perfused with 4% paraformaldehyde in
phosphate buffer solution (PBS; pH 7.4), and the organs,
including the heart, spleen, liver, kidneys, and lungs, cut into
15 mm sections, were incubated overnight in a solution of 0.5m
sucrose in PBS. After drying at 378C, the sections were
washed with PBS and treated with 0.3% Triton X-100 at room
temperature and analyzed by fluorescence microscopy. The
Tat peptides were previously reported to show predominant
tissue distribution in the liver, kidneys, lungs, heart muscle,
and spleen.[23,28] In contrast to these observations, significantly
lower extents of internalization of 3c into the liver, kidney,
and spleen were observed; 3c was located only in the
peripheries of these organs. However, much higher distribu-
tions of 3c were seen in heart, lung, and brain tissues
(Figure 3). Because of its potential implications on the
possible selective delivery into the central nervous system,
we specifically repeated the tissue distribution experiment of
compound 3c in the mouse brain at a dosage level of
57 mgkgÀ1 for 20 minutes. The brain cortexregion showed
strong fluorescence, suggesting that the transporter indeed
Figure 2. Cellular uptake studies ofcompound 3c using HeLa cells.
A) FACS analysis ofthe compounds 3a–c taken up by HeLa cells. The
cells were treated with compounds 3a–c (10 mm each) in serum-
containing minimum essential medium (MEM) at 378C for 1 h and
were analyzed by FACS. B) Kinetics study ofthe amount ofcompound
3c taken up by the cells in comparison with that ofthe lfuorescein-
labeled octaarginine (R8-Fl). The cells were incubated with compound
3c or R8-Fl (10 mm each) at 378C for different time intervals and
analyzed by FACS. C) Observation by confocal microscopy of the cells
treated with 3c (10 mm) in the presence oftetramethylrhodamine-
labeled transferrin (Rho-Tf; 25 mgmLÀ1) at 378C for 1 h. D) The cells
were treated with 3c and tetramethylrhodamine-labeled Tat peptide
(Rho-Tat; 10 mm each) at 378C for 1 h. E): a) No significant colocaliza-
tion ofthe signals of 3c with MitoTracker (250 nm) is seen; b) the
majority of 3c did not reach the lysosome in 1 h (lysosomes were
stained with 50 nm LysoTracker); c) no significant internalization was
observed either for the cells treated with 3c (10 mm) for 1 h at 48C.
Scale bars: 20 mm. See text for details.
distinct localization pattern under the experimental condi-
tions (Figure 2C). When the cells were similarly treated with
3c at 48C, no significant internalization was observed
(Figure 2E, part c). These results suggest that 3c internalizes
through an endocytic route that is different from the well-
characterized clathrin-dependent pathway. Interestingly, 3c
may possibly internalize using pathways that are different
even from those of the Tat peptide, another representative
Figure 3. Distribution of 3c (HCl salt) into mouse tissues. Fluores-
cence microscopy images (green fluorescence from FITC) of heart
muscle (A,B), spleen (C,D), liver (E,F), kidney (G,H), and lung (I,J)
tissue sections, and coronal brain sections (K,L) isolated from mice
20 minutes after intraperitoneal injection. Exposure time: 14000 ms
(A,B); 500 ms (C,D); 300 ms (E,F); 500 ms (G,H); 1200 ms (I,J);
1000 ms (K,L); lem =488 nm.
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crossed the blood–brain barrier (BBB) rapidly and efficiently
(see Supporting Information). Development of vectors to
help cross the BBB is one of the major challenges in drug
delivery, and these observations should be useful in the
development of organ-selective delivery technologies.
Last, we investigated the in vitro and in vivo distribution
of the transporter 3c–doxorubicin conjugate 4. Doxorubicin
hydrochloride (adriamycin) is extensively used clinically for
the treatment of a variety of neoplastic diseases, including
leukaemia and breast, ovarian, and solid cancers, but not
brain cancer, as it does not overcome the BBB.[29] Doxor-
ubicin reveals strong UV/Vis absorption bands and is also
highly fluorescent. Compound 4 was examined for its cell-
penetrating ability with HeLa cells without fixing. The
conjugate 4 at concentrations of 10 and 30 mm showed
much-enhanced translocation into the cytoplasm when com-
pared with doxorubicin itself (Figure 4 A). However, the cells
extensively distributed in the cortex region of the mouse brain
(Figure 4B, image c), whereas a very small amount of
doxorubicin translocated into the brain cortex in the same
timeframe (Figure 4B, image b). These in vivo results suggest
that the uptake of doxorubicin into the brain across the BBB
is very inefficient, as expected, and that conjugation to the
transporter 3c significantly increases the uptake as well as the
intercellular permeation of doxorubicin in the brain tissue.
In summary, the novel transporter structures based on
dimeric inositol scaffolds reported here display unique
spectra of distribution in vitro and in vivo. The lack of cell/
organ specificities has generally been considered as one of the
shortcomings of the Tat and oligoarginine-mediated delivery
methods. However, the results shown here strongly suggest a
possibility of designing highly sophisticated transporters by
varying the structure of the backbone scaffold, charge
densities, and perhaps other parameters. Thus, it is suggested
that the structural diversity of sugars may be beneficial as
scaffolds in the design of transporters in terms of not only
aqueous solubility but also selective tissue and organellar
distributions. Why the transporter 3c shows these unique
properties is not clear at this stage. Studies designed to
provide additional information on these issues are in progress.
Received: January 24, 2006
Published online: March 23, 2006
Keywords: bioorganic chemistry · carbohydrates · drug delivery ·
.
fluorescence · membranes
Figure 4. A) Fluorescence microscopy images ofdoxorubicin (green
fluorescent emission) and doxorubicin–3c conjugate, 4: a) doxorubicin
(10 mm), b) doxorubicin–3c conjugate, 4 (HCl salt, 10 mm), c) doxoru-
bicin (30 mm), and d) conjugate 4 (30 mm) in HeLa cells incubated for
15 minutes at 378C. B) Fluorescence microscopy images ofmouse
brain sections (cortex region): a) control (water), b) doxorubicin
(21.3 mgkgÀ1; Mr =580 gmolÀ1), and c) conjugate 4 (HCl salt,
115.8 mgkgÀ1; Mr =4452 gmolÀ1). For part (B), sample treatment
time: 20 minutes; exposure time: 9000 ms; lem =488 nm.
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