Design, synthesis, and delivery properties of novel guanidine-containing molecular transporters built on dimeric inositol scaffolds

Article abstract of DOI:10.1002/chem.200600898

We have developed a novel class of synthetic molecular transporters that contain eight residues of guanidine with an inositol dimer as the scaffold. The dimers were prepared by connecting two units of myo- or scyllo-inositol via a carbonate or amide linkage, and the multiple units of the guanidine functionality were constructed on the inositol scaffold by means of peracylation with ω-aminocarboxylate derivatives of varying length. Bioassays based on confocal laser scanning microscopy and fluorescence-activated cell sorter analyses indicated that these transporters display a varying degree of membrane translocating ability, and the intracellular localization and mouse-tissue distribution studies strongly suggested that these transporters undergo substantially different mechanistic processes from those of peptide transporters reported to date. It was also shown that doxorubicin, an anticancer antibiotic, can be efficiently delivered into mouse brain by aid of this type of transporter.

Full text of DOI:10.1002/chem.200600898

DOI: 10.1002/chem.200600898
Design, Synthesis, and Delivery Properties of Novel Guanidine-Containing
Molecular Transporters Built on Dimeric Inositol Scaffolds
Kaustabh K. Maiti, Ock-Youm Jeon, Woo Sirl Lee, and Sung-Kee Chung*[a]
Abstract: We have developed a novel
class of synthetic molecular transport-
ers that contain eight residues of guani-
dine with an inositol dimer as the scaf-
fold. The dimers were prepared by con-
necting two units of myo- or scyllo-ino-
sitol via a carbonate or amide linkage,
and the multiple units of the guanidine
functionality were constructed on the
inositol scaffold by means of peracyla-
tion with w-aminocarboxylate deriva-
tives of varying length. Bioassays based
on confocal laser scanning microscopy
and fluorescence-activated cell sorter
analyses indicated that these transport-
ers display a varying degree of mem-
brane translocating ability, and the in-
tracellular localization and mouse-
tissue distribution studies strongly sug-
gested that these transporters undergo
substantially different mechanistic pro-
cesses from those of peptide transport-
ers reported to date. It was also shown
that doxorubicin, an anticancer antibi-
otic, can be efficiently delivered into
mouse brain by aid of this type of
transporter.
Keywords: drug delivery · guani-
dine · inositol · membrane translo-
cation · molecular devices
Introduction
viral proteins, for example, HIV-1 Tat protein (Tat-86), An-
tennapedia (Antp) protein of Drosophila, and herpes sim-
plex virus type 1 (HSV-1) VP22. Furthermore, their mem-
brane penetrating ability has been found to be associated
with relatively short peptide fragments, for example, 9 basic
amino acid residues for the Tat protein (residues 49–57; Tat
peptide, RKKRRQRRR), 16 amino acid residues for the
Antp protein (residues 43–58; penetratin, RQI-
KIWFQNRRMKWKK) that have a potentially amphipathic
and basic helical structure, and 12 hydrophobic amino acid
residues for the Kaposi sarcoma fibroblast growth factor
(KFGF) MTS (AAVLLPVLLAAP).[1–4] Although the un-
derlying mechanisms of the cellular uptake of these peptides
could be multiple and have not been clearly defined, there
have been many attempts to utilize these peptides as deliv-
ery vectors in order to improve the pharmacology of poorly
bioavailable drugs including small molecules,[5] proteins,[6–10]
nucleotides, and genes.[11–13] More recently, there have been
several reports on the development of synthetic molecular
transporters mimicking natural and unnatural peptides and
their structural analogues, especially in the area of polycat-
ionic peptide mimetics, such as peptoids, oligocarbamates,
b,b-peptides, peptide nucleic acids, guanidinoglycosides, and
heterocyclic guanidinium salts.[14–23]
Therapeutic efficiency of a drug or a drug candidate de-
pends on its ability to overcome biological barriers and
reach the desired tissue and intracellular target sites. It is
generally understood that cellular membrane barriers allow
only those molecules with an appropriate range of molecular
size, polarity, and charge to pass through them. As a result,
many drug candidates with promising in vitro activities fail
to be developed into clinically useful agents. Thus, develop-
ing molecular transporters that enable or enhance cellular
uptake of a drug candidate or molecular probe is a highly
desirable and challenging goal in the field of drug research
and development. A number of peptides are known to
translocate across cell membranes. They are variously
known as cell-penetrating peptides (CPPs), protein trans-
duction domains (PDTs), and membrane translocating se-
quences (MTSs), and they are capable of delivering exoge-
nous molecules into cells as covalent conjugates. The cell-
membrane penetrating property was initially observed in
[a] Dr. K. K. Maiti, O.-Y. Jeon, W. S. Lee, Prof. Dr. S.-K. Chung
Department of Chemistry, Division of Molecular and Life Sciences
Pohang University of Science & Technology, Pohang 790-784 (Korea)
Fax : (+82)54-279-2103
The CPPs commonly show good in vitro efficiency as mo-
lecular transporters but their structural motifs and physico-
chemical properties are too diverse to reveal a common
translocation mechanism. It is quite possible that different
E-mail: skchung@postech.ac.kr
Supporting information for this article is available on the WWW
under http://www.chemeurj.org/ or from the author.
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CPPs may use different mechanistic processes for transloca-
tion and even the same peptide may use alternative path-
ways depending on the conditions of administration, concen-
tration, and the nature of the cargo.[10,24] A number of stud-
ies have shown that in the basic CPPs the guanidine head
group is important, because arginine oligomers enter into
cells more rapidly than the corresponding oligomers of
lysine, histidine, or ornithine, and various arginine oligomers
show membrane permeability similar to that of the Tat pep-
tide. It has been found that the optimum number of guani-
dine residues is about eight or nine, and the linking chain
length also plays a substantial role in the case of peptoid
transporters.[14–18]
We envisaged that modifications of the structure and
physicochemical properties of even the known molecular
transporters could generate novel transporters that have dif-
ferent in vivo and in vitro spectra, as well as different trans-
location efficiencies. Additionally, the in vivo efficacy, the
susceptibility to various endogenous peptidases, the synthet-
ic cost, and the observed neurotoxicity liability of the
known arginine-rich peptides are often considered as poten-
tial problems for development. Keeping all these technically
and practically important issues in mind, we have developed
a novel class of cell-penetrating transporters as potential de-
livery vectors. In the molecular design of novel transporters,
we decided to utilize carbohydrate and inositol structures as
the backbone scaffold, because they have the highest func-
tionality density among organic compounds with hydroxyl
groups in diverse stereochemical variations, and they are
highly water soluble and largely free of any noticeable toxic-
ity. Previously, several groups
Results and Discussion
We employed two inositol stereoisomers (myo- and scyllo-)
as the initial backbone starting material. myo-Inositol is the
most abundant inositol in nature and is readily available,
but substitution at any position other than the C-2 and C-5
generates a diastereomeric mixture. On the other hand,
scyllo-inositol is less readily available but has a higher
degree of symmetry with all hydroxyl groups in the equato-
rial orientation. Thus, we decided to use both inositols. To
prepare suitably protected dimeric inositol scaffolds, we syn-
thesized the requisite myo- and scyllo-inositol derivatives as
follows. myo-Inositol was converted to 2,3:5,6-di-O-isopro-
pylidine-myo-inositol (1),[26] and the 1-OH and 4-OH groups
were regioselectively protected as tert-butyldimethylsilyl
(TBDMS) and benzyl (Bn) ethers, respectively, to give 2[27]
and 3. Removal of the TBDMS group in 3 yielded 4.[28] Sim-
ilarly, 1 was converted to the p-methoxybenzyl (PMB)-pro-
tected 5,[29] which was then treated with CaH2 and carbonyl-
diimidazole to give 6. Compounds 4 and 6 were coupled to
give a diastereomeric mixture of 7 (87% yield) in the pres-
ence of 1,8-diazobicycloACHTRE[UGN 5.4.0]undec-7-ene (DBU) in toluene
(Scheme 1). Next, myo-inositol was transformed to scyllo
compound 8 by inversion of the C-2 stereochemistry by
means of the Mitsunobu reaction.[30] Formation of bisaceto-
nide groups followed by regioselective handling of the two
hydroxyl groups gave the suitable intermediates 12 and 15.
Treatment of 12 with carbonyldiimidazole and CaH2 provid-
ed 13, which was coupled with 15 in the presence of NaH in
THF (instead of DBU in toluene as was used in Scheme 1)
have attempted to design vari-
ous peptidomimetics on carbo-
hydrate scaffolds in order to
take advantage of the structur-
al characteristics.[25] Herein, we
describe the synthesis and
properties of the initial version
of such molecular transporters.
The architecture of these syn-
thetic molecular transporters is
based on dimeric inositol scaf-
folds and eight units of guani-
dine functionality, which are
attached to the scaffold as w-
aminocarboxylate derivatives
of varying chain length. More-
over, two additional functional
groups are reserved on the
scaffold as handles on which a
cargo (drug or diagnostic
device) and/or
a fluorescent
probe (or targeting ligand)
may be conjugated.
Scheme 1. i) 2,2-Dimethoxypropane, p-TSA, DMF; ii) BzCl, pyridine; iii) NaOMe, MeOH, overall yield 62%;
iv) TBDMSCl, imidazole, DMF, 70%; v) BnBr, Ag2O, TBAI, 97%; vi) TBAF, THF, quant.; vii) PMBCl, NaH,
DMF, 76%; viii) CaH2, carbonyldiimidazole, toluene, 94%; ix) DBU, toluene, 87%.
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S.-K. Chung et al.
to give 16 in a 62% yield (Scheme 2). Now the two synthetic
scaffolds 7 and 16, which are built with two units of inositol
linked through a carbonate bridge, were ready for further
structural elaborations such as octaguanidinylation and con-
jugations involving a fluorescent probe and drug cargo. The
acid-catalyzed removal of the acetonide protecting groups in
7 with p-toluenesulfonic acid (p-TSA) in CH2Cl2 and MeOH
gave 17 in an 83% yield, which was then exhaustibly acylat-
ed with w-N-Boc-aminocarboxylic acids of varying chain
lengths in the presence of 1-(3-dimethylaminopropyl)-3-eth-
ylcarbodiimide hydrochloride
groups from the guanidine moiety were cleaved by using tri-
fluoroacetic acid (TFA) and dichloromethane to afford,
after rigorous purification, the TFA salts 33, 34, and 35 as
potential molecular transporters (Scheme 3). The target
compounds were thoroughly characterized by using NMR
spectroscopy and matrix-assisted laser desorption/ionization
time-of-flight (MALDI-TOF) mass spectral analyses. The
same synthetic protocols carried out on dimer 16 gave the
octaguanidinylated scyllo-inositol dimeric transporter 72
(see Supporting Information, Scheme 1). In this case, only
(EDC) and 4-dimethylamino-
pyridine (DMAP) in DMF to
provide 18–20 in 60–85%
yields after purifying by means
of column chromatography on
silica gel. The PMB protecting
group of 18–20 was removed
by using ammonium ceriu-
m(IV) nitrate (CAN) in aceto-
nitrile and toluene, and dansy-
lated to give 24–26. After re-
moval of the Boc group from
the aminocarboxylate residues,
perguanidinylation
reactions
were carried out on 27–29 by
using N,N’-di-Boc-N’’-trifluoro-
methanesulfonylguanidine[31]
and triethylamine (TEA) in
DMF to afford the octaguani-
dine products 30–32 in 55–
65% yields after purification
by means of column chroma-
tography. Finally, the N-Boc
Scheme 3. i) p-TSA, CH2Cl2/MeOH 1:9, 83%; ii) EDC, DMAP, N-Boc-protected 4-aminobutyric acid (n=3),
aminocaproic acid (n=5), w-aminocaprylic acid (n=7), DMF, 60–85%; iii) CAN, CH3CN/toluene 3:1, 55–
78%; iv) dansyl-Cl, CH3CN, DMAP, 55–85%; v) HCl in EtOAc, quant.; vi) N,N’-di-Boc-N’’-trifluoromethane-
g
sulfonylguanidine, TEA, DMF, 55–65%; vii) TFA/CH2Cl2 1:1, 85–90%.
Scheme 2. i) 2-Methoxypropene, p-TSA, DMF, 9 (30%), 10 (51%); ii) BnBr, Ag2O, TBAI, CH2Cl2, 80%; iii) NaOMe, MeOH, 97%; iv) CaH2, carbonyl-
diimidazole, toluene, 88%; v) PMBCl, NaH, DMF, 74%; vi) NaOMe, MeOH, 97%; vii) NaH, THF, 62%.
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Molecular Transporters
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one transporter molecule with an N-Boc-aminocaproic acid
linker chain (n=5) was prepared for comparison with the
myo dimer (34) in bioassays.
2) guanidinylation of the free amino groups. In all steps rea-
sonably good yields were obtained after the usual purifica-
tion procedures. Then, a fluorescent tag was attached by re-
acting fluorescein-5-isothiocyanate (FITC-I) with the free
amino groups of 54–56 to afford the fluorescein-labeled
compounds 57–59 in 45–65% yields after purification by
using flash column chromatography. Although the dansyl
group as a fluorophore is easier to handle synthetically, fluo-
rescein has a higher quantum efficiency and is better suited
for confocal laser scanning microscopy (CLSM) detection.
The final octaguanidinylated transporters 60–62 were ob-
tained as TFA and HCl salts by deprotection of the guani-
dine with TFA and ethyl acetate saturated with HCl, respec-
tively. The deprotection yields are generally higher for TFA
salt formation, but the HCl salts have better aqueous solu-
bility (Scheme 5).
In the last phase of the synthetic work we investigated an
experimental protocol for the transporter–doxorubicin con-
jugation. Doxorubicin (Adriamycin) is a potent anticancer
drug, which is used clinically and is fluorescent. Compound
62 was chosen for the conjugation among the various syn-
thetic transporters, because of its presumed higher hydrolyt-
ic stability and flexibility relative to the carbonate-linked
transporters, and also because of its excellent membrane
translocating ability. Compound 56 and carbobenzoxy
(Cbz)-protected l-serine were first coupled under the EDC
coupling conditions, and the hydroxyl group of the serine
moiety of 63 was further coupled with the primary amino
group in the sugar residue of
Considering the possible hydrolytic instability and rigidity
associated with the carbonate functionality used in linking
two units of the inositol skeleton, we next investigated the
amide linkage for the formation of a dimeric scyllo-inositol
scaffold. Thus, compound 9 was treated with allyl bromide
in the presence of Ag2O and tetrabutylammonium iodide
(TBAI) to give 36 in a 94% yield. Hydrolysis of the ben-
zoate of 36, ozonolysis at À788C in CH2Cl2/MeOH (6:1)
buffered with sodium bicarbonate (1.5 equiv) followed by a
reductive workup, and reductive amination with dibenzyla-
mine and NaBHACHTREUNG(OAc)3 at 08C gave 39 in a reasonable over-
all yield. The hydroxyl group of 39 was coupled with methyl
bromoacetate to give 40 in a 94% yield. Debenzylation of
40 under hydrogenation conditions with 10% Pd/C afforded
the free amine compound 41, whereas hydrolysis of the
methyl ester group in 40 gave carboxylate 42 in a 92%
yield. Coupling between 41 and 42 was performed by using
EDC and DMAPin DMF to give the amide-linked dimeric
scyllo-inositol 43 in a 65% yield after purification by means
of column chromatography (Scheme 4). Similar structural
elaborations were carried out on 43 as described for 16 to
prepare the octaguanidinylated transporter molecules 51–53,
namely, 1) exhaustive acylation reactions with three differ-
ent w-N-Boc-aminocarboxylate derivatives after the acid-
catalyzed hydrolysis of the acetonide protecting groups, and
doxorubicin through a carba-
mate bond to give 64.[32] In the
last step, all Boc groups in the
guanidine moieties of 64 were
removed by using gaseous HCl
to yield the covalent conjugate
65 as an HCl salt (Scheme 6).
The target product in the salt
form was purified by means of
reverse-phase
sure liquid chromatography
(MPLC) on C-8 column
medium-pres-
a
(H2O/CH3CN, 1:1–1:2 contain-
ing 0.1% TFA), and its purity
was evaluated by analytical
high-pressure liquid chroma-
tography (HPLC) analysis. The
key intermediates and target
compounds were also satisfac-
torily characterized by using
NMR
spectroscopy
and
MALDI-TOF mass spectral
analyses.
Preliminary
membrane
translocating properties of the
synthetic transporters (33–35,
72, 60–62) were examined by
means of confocal laser scan-
Scheme 4. i) Allyl bromide, Ag2O, TBAI, CH2Cl2, 94%; ii) NaOMe, MeOH, 92%; iii) NaHCO3, CH2Cl2/
MeOH 6:1, O3, À788C, PPh3, 86%; iv) Bn2NH, NaBH
ACHTER(UNG OAc)3, DCE, 76%; v) BrCH2CO2Me, Ag2O, TBAI,
CH2Cl2, 94%; vi) 10% Pd/C, H2 (1 atm), CH2Cl2/MeOH 1:9, quant.; vii) NaOH, MeOH, then AcOH, 92%;
viii) 41, 42, EDC, HOBT, TEA, DMF, 65%.
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S.-K. Chung et al.
cells, the cellular uptake of the
transporters was visually com-
pared with arginine nonamer
(dansyl- and Fl-R9). The trans-
porters based on the myo-ino-
sitol dimer linked with carbon-
ate (33–35) displayed qualita-
tively similar cellular uptake
properties to dansyl-R9 in the
COS cell assays, although the
transporters with longer chain
lengths, 34 and 35, showed
somewhat better translocation
than 33 (Figure 1A). The trans-
porters built on the myo- and
scyllo-inositol scaffolds (34 and
72) showed qualitatively simi-
lar translocation ability in
RAW264.7 cells (Figure 1B).
With HeLa cells the transport-
ers based on the scyllo-inositol
dimer linked with amide (60–
62) again showed good translo-
cation, and the longer chains
seem to contribute to a better
uptake, that is, 62>61>60
(Figure 1C). The internaliza-
tion efficiency of 60–62 was
more precisely compared with
that of fluorescein-labeled oc-
taarginine (Fl-R8) through
Scheme 5. i) p-TSA, CH2Cl2/MeOH (9:1), quant.; ii) EDC, DMAP, N-Boc-protected 4-aminobutyric acid (n=
3), aminocaproic acid (n=5), w-aminocaprylic acid (n=7), DMF, 65–80%; iii) HClg in EtOAc, quant.; iv)
N,N’-di-Boc-N’’-trifluoromethanesulfonylguanidine, Et3N, dioxane/H2O 5:1, 55–75%; v) 10% Pd/C, H2
(1 atm), MeOH/CH2Cl2 9:1, 80–90%; vi) FITC-I, Et3N, THF, absolute EtOH, 52–65%; vii) HClg in EtOAc,
76–80% or TFA/CH2Cl2 1:1, 86–94%.
fluorescence-activated
cell
sorter (FACS) analysis. The
analysis showed that with
HeLa cells at 378C for 1 h, 61
and 62 (10 mm) internalized 1.8
and 2.5 times as much as Fl-
R8, respectively, whereas the
amount of internalized 60 was
about half of that for Fl-R8. A
kinetic study carried out on
the most efficient transporter
(62) in comparison with Fl-R8
revealed that the total cellular
uptake of 62 was more than
three times higher than that of
Fl-R8. These results suggest
that transporters with this par-
ticular structural framework
are helped by an increased
chain length due to either the
increased hydrophobicity or
the higher flexibility for the in-
teraction with the cell mem-
Scheme 6. i) 56, N-Cbz-l-serine, EDC, HOBT, DMF, 72%; ii) p-NC, pyridine, CH2Cl2, RT, 3 h, then TEA, dox-
orubicin–HCl, DMF, 77%; iii) HClg in EtOAc, 76%.
ning microscopy (CLSM) with three cell lines: simian
kidney COS-7, mouse macrophage RAW264.7, and HeLa
cells (Figure 1). After 3–5 min exposure to the cultured
brane.[14] The intracellular localization pattern studies on 62
with HeLa cells in the presence of tetramethylrhodamine-la-
beled transferrin, tetramethylrhodamine-labeled Tat peptide,
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Molecular Transporters
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delivery of doxorubicin or a higher toxicity of the conjugate.
The in vivo experiments with mice convincingly showed that
conjugate 65 was also extensively distributed in the cortex
region of the mouse brain 20 min after ip injection, whereas
very little doxorubicin translocated into the brain cortex
during the same time frame. These results suggest that the
uptake of doxorubicin through the BBB is indeed very inef-
ficient, and that conjugation to the transporter significantly
increases the uptake by the brain as well as the intercellular
permeation of doxorubicin in the brain tissue.[33]
Conclusion
We have successfully designed and synthesized novel trans-
porter molecules based on dimeric inositol scaffolds and we
have demonstrated that these transporters possess some in-
teresting cellular uptake characteristics together with unique
spectra of in vitro and in vivo distributions. More specifical-
ly, the amide-linked dimeric inositol transporters 61 and 62
have been found to be superior to arg-8-mer (R8) and arg-
9-mer (R9) in terms of the uptake efficiency and amount.
The novel transporters also display interesting intracellular
and tissue distribution patterns that are different from those
of Tat-related or arginine-rich peptides, thus suggesting that
the exciting possibility of designing highly sophisticated, or-
ganellar- and tissue-selective transporters may be feasible by
varying structural parameters, such as the backbone scaffold
and linker, and the associated physicochemical parameters,
such as charge density and hydrophobicity. In addition, the
novel synthetic transporters may have practical value as
drug-delivery vehicles in terms of better absorption, distri-
bution, metabolism, and excretion (ADME) characteristics
and stability toward endogenous enzymes. Studies on the
next version of molecular transporters with different and im-
proved delivery characteristics are currently underway.
Figure 1. Fluorescence microscope images: A) a) dansyl-R9 (10 mm), b)
33 (10 mm), c) 34 (10 mm), and d) 35 (10 mm) in COS 7 cells incubated for
3 min at RT; B) a) dansyl-R9 (10 mm), b) 34 (10 mm), and c) 72 (10 mm) in
RAW 264.7 cells incubated for 3 min at RT; C) a) Fl-R9 (10 mm), b) 60
(10 mm), c) 61 (10 mm), and d) 62 (10 mm) in HeLa cells incubated for
15 min at 378C.
MitoTracker, and LysoTracker at 37 and 48C showed few
similarities and co-localization, thus strongly suggesting that
62 may use different mechanisms of internalization and cel-
lular localization from the arginine-rich transporters report-
ed to date.[33]
Compound 62 also showed a unique in vivo distribution
pattern compared with the Tat-related peptides, which were
previously reported to show an even distribution in liver,
kidney, lung, heart muscle, and spleen tissues.[23,34] When in-
jected intraperitoneally (ip) into eight-week-old mice, signif-
icantly lower extents of internalization of 62 were observed
in the liver, kidney, and spleen, whereas the heart, lung, and
brain tissues showed much higher distributions of 62. At
present the reasons for the uneven distribution have not
been determined. In the repeated tissue distribution experi-
ment of 62 with mice, the brain cortex region showed strong
fluorescence 20 min after the ip injection, which suggests
that the transporter clearly crossed the blood–brain barrier
(BBB) rapidly and efficiently. Development of vectors to
help cross the BBB is one of the major challenges in drug
delivery, and the above observations should be highly rele-
vant to developing organ-selective delivery technologies.
Doxorubicin hydrochloride (Adriamycin) is extensively used
clinically for the treatment of a variety of neoplastic diseases
including leukemia, and breast, ovarian, and solid cancers,
but not brain cancer because it does not overcome the
BBB.[35] Doxorubicin has good UV/Vis absorption bands
and is also highly fluorescent. Cellular uptake studies of
conjugate 65 with HeLa cells at both 10 and 30 mm concen-
trations showed a much-enhanced translocation of the con-
jugate into the cytoplasm, effectively killing cells at 10 mm, a
concentration at which neither doxorubicin nor carrier 62
caused significant cell damage. At present it is not clear
whether the efficient cell killing is due to cleaved doxorubi-
cin or the conjugate, nor whether it is due to more efficient
Experimental Section
General methods: All nonhydrolytic reactions were carried out in oven-
dried glassware under an inert atmosphere of dry argon or nitrogen. All
commercial chemicals were used as received except for solvents, which
were purified and dried by means of standard methods prior to use. Ana-
lytical thin-layer chromatography (TLC) was performed on Merck 60
F254 silica gel plates (0.25 mm thickness), analytical reverse-phase TLC
on Merck RP-8 F254s plates; visualization was carried out be using UV
light (l=254 and 365 nm) or by spraying the plates with a 5% solution
of phosphomolybdic acid or ninhydrine solution followed by charring
with a heat gun. Column chromatography was performed on Merck 60
silica gel (70–230 or 230–400 mesh), and MPLC was performed on Fluka
100 C8 reversed-phase silica gel. Melting points were determined on a
Thomas-Hoover apparatus and are uncorrected. NMR spectra were re-
corded on Bruker DPX 300 (1H NMR at 300 MHz; 13C NMR at 75 MHz)
and Bruker DRX 500 (1H NMR at 500 MHz; 13C NMR at 125 MHz)
spectrometers. Tetramethylsilane was used as the reference for 1H NMR
spectra. The abbreviations “app.” and “dist.” signify an apparent peak or
set of peaks and a distorted peak, respectively. Analytical HPLC was per-
formed by using an Agilent 1100-HPLC Chemstation with a BU-300 ana-
lytical column (30 nm pore size and 10 mm spherical, 4.6 mm i.d.”25 cm).
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S.-K. Chung et al.
Low-resolution mass spectra were determined on a Micromass PLAT-
FORM ll (EI and FAB) instrument. High-resolution mass spectra were
determined on a JMS-700 instrument, and MALDI-TOF mass spectra on
a Voyager-DE STR system at the Korea Basic Science Support Center.
The standard extraction work-up procedure consisted of pouring the re-
action mixture into a large amount of water, extracting with the organic
solvent indicated, washing the combined extracts successively with water
and with brine, drying the extract over anhydrous Na2SO4 or MgSO4, and
evaporating the solvent.
1H; imidazole-H5), 8.12 ppm (s, 1H; imidazole-H2); 13C NMR (CDCl3):
d=26.2, 27.1, 27.3, 28.1, 55.6, 72.0, 73.4, 76.2, 77.3, 78.9, 79.3, 111.3, 113.5,
114.2, 117.6, 129.8, 130.5, 131.0, 137.5, 148.2, 159.9 ppm; MS (FAB): m/z:
475.13 [M+H]+; HRMS (FAB): m/z calcd for C24H31N2O3: 475.2102;
found: 475.2080 [M+H]+.
(+)-4’-O-Benzyl-1’-O-[(1-O-p-methoxybenzyl-2’,3’:5’,6’-di-O-isopropyli-
dene-myo-inositol)carbonyloxy]-2,3:5,6-di-O-isopropylidene-myo-inositol
(7): DBU (0.05 mL, 0.34 mmol) was added to a solution of 6 (1.62 g,
3.4 mmol) and 4 (1.8 g, 5 mmol) in toluene (21 mL), and the mixture was
stirred at RT for 18 h. The standard extraction work-up procedure fol-
lowed by recrystallization from n-hexane/EtOAc (5:1) gave 7 as a color-
less solid (2.23 g, 86.7%). Rf =0.44 (EtOAc/nHex 1:2, eluted twice); m.p.
225–2268C; 1H NMR (CDCl3): d=1.10–1.72 (m, 24H; 4CMe2), 3.35 (t,
J=9.3 Hz, 2H), 3.49 (t, J=9.3 Hz, 2H), 3.72–3.77 (m, 2H), 3.81 (s, 3H),
4.00–4.39 (m, 4H), 4.69 (t, J=3.2 Hz, 2H), 4.82 (brs, 4H), 4.95–5.06 (m,
1H), 5.09–5.17 (m, 1H), 6.89 (d, J=8.7 Hz, 2H), 7.24–7.42 ppm (m, 7H);
13C NMR (CDCl3): d=26.1, 26.3, 26.4, 27.28, 27.32, 28.1, 71.9, 72.5, 73.5,
73.7, 73.9, 74.7, 74.8, 74.9, 75.1, 75.2, 76.6, 77.16, 77.21, 79.0, 79.1, 79.3,
79.7, 80.1, 81.5, 110.6, 110.9, 113.0, 113.1, 114.2, 128.0, 128.4, 128.6, 130.1,
130.4, 138.4, 154.0, 159.9 ppm; IR (KBr): n˜ =1748 cmÀ1; MS (FAB): m/z:
779.32 [M+Na]+; HRMS (FAB): m/z calcd for C40H53O14: 757.3457;
found: 757.3443 [M+H]+; m/z calcd for C40H52O14Na: 779.3357; found:
779.3241 [M+Na]+.
(+)-2,3:5,6-Di-O-isopropylidene-myo-inositol (1) was prepared according
to a literature procedure.[26]
(+)-1-tert-Butyldimethylsilyl-2,3:5,6-di-O-isopropylidene-myo-inositol (2)
was prepared from 1 according to a literature procedure.[27]
(+)-4-O-Benzyl-2,3:5,6-di-O-isopropylidene-1-O-tert-butyldimethylsilyl-
myo-inositol (3): Silver(I) oxide (10.7 g, 46 mmol), benzylbromide
(5.47 mL, 46 mmol), and TBAI (0.55 g, 1.5 mmol) were added to a solu-
tion of 2 (5.7 g, 15 mmol) in CH2Cl2 (100 mL). The mixture was stirred at
RT for 2 h, and filtered through Celite. The filtrate was washed with
CH2Cl2, and concentrated under vacuum. The crude product was purified
by using column chromatography on silica gel to afford 3 as a colorless
solid (4.91 g, 97%). Rf =0.3 (EtOAc/nHex 1:10); m.p. 898C; 1H NMR
(CDCl3): d=0.12 (s, 6H; SiMe2), 0.90 (s, 9H; CMe3), 1.31, 1.35, 1.40, 1.41
(4s, 3H each; 2CMe2), 3.31 (t, J=9.7 Hz, 1H; H-3), 4.07 (t, J=5.6 Hz,
1H; H-1), 4.27 (t, J=5.6 Hz, 1H; H-6), 4.80 (s, 2H; PhCH2), 7.21–
7.40 ppm (m, 5H; Ph); 13C NMR (CDCl3): d=18.9, 26.1, 26.3, 27.3, 27.4,
27.5, 28.4, 71.0, 72.3, 77.0, 79.1, 79.3, 80.3, 80.4, 110.0, 112.1, 128.4, 128.7,
127.8, 138.8 ppm; MS (FAB): m/z: 465.36 [M+H]+; HRMS (FAB): m/z
calcd for C25H41O6Si: 465.2694; found: 465.2672 [M+H]+.
1-O-Benzoyl-2,3:5,6-di-O-isopropylidene-scyllo-inositol (9) and 1-O-ben-
zoyl-2,3:4,5-di-O-isopropylidene-scyllo-inositol (10): 2-Methoxypropene
(20 mL, 211.2 mmol) was added over 30 min to a solution of 8 (6 g,
21.11 mmol)[30] and p-TSA (400 mg, 2.1 mmol) in dry DMF (100 mL) at
RT. After 24 h, the reaction mixture was quenched with aqueous
NaHCO3. The standard extraction work-up procedure using EtOAc gave
the crude product, from which two regioisomeric products, 9 (2.1 g, 30%)
and 10 (4.3 g, 51%), were isolated as colorless solids by using column
chromatography on silica gel. Compound 9: Rf =0.26 (EtOAc/nHex 1:2);
m.p. 284–2868C;[30] 1H NMR (CDCl3): d=1.44, 1.48 (2s, 6H each;
2CMe2), 2.46 (d, J=2.8 Hz, 1H; OH), 3.76 (app. t, J=9.4 Hz, 2H; H-3,
H-5), 3.88 (app. t, J=9.4 Hz, 2H; H-2, H-6), 4.13 (dt, J=2.8, 8.8 Hz, 1H;
H-4), 5.70 (t, J=9.3 Hz, 1H; H-1), 7.27–8.1 ppm (m, 5H; Ph). Compound
10: Rf =0.34 (EtOAc/nHex 1:2); m.p. 206–2078C;[29] 1H NMR (CDCl3):
d=1.47, 1.49 (2s, 6H each; 2CMe2), 2.88 (d, J=4.1 Hz, 1H; OH), 3.67–
3.90 (m, 4H; H-2, H-3, H-4, H-5), 4.13 (m, 1H; H-6), 5.43 (dd, J=7.9,
10.5 Hz, 1H; H-1), 7.45–8.11 ppm (m, 5H; Ph).
(+)-4-O-Benzyl-2,3:5,6-di-O-isopropylidene-myo-inositol (4): Tetrabuty-
lammonium fluoride (TBAF) (1.0m solution in THF, 29.7 mL) was added
to a solution of 3 (4.6 g, 9.9 mmol) in THF (35 mL), and the solution was
stirred at RT for 7 h. The resulting mixture was concentrated, diluted
with EtOAc and washed with water and with brine. The organic extract
was dried and concentrated to give the crude product, which was recrys-
tallized from n-hexane to obtain 4 as a colorless solid (3.7 g, quant.). Rf =
0.21 (EtOAc/nHex 1:5); m.p. 1318C (lit. 132–1348C);[28] 1H NMR
(CDCl3): d=1.33, 1.36, 1.43, 1.46 (4s, 3H each; 2CMe2), 2.40 (d, J=
8.4 Hz, 1H; OH), 3.39 (t, J=9.6 Hz, 1H; H-5), 3.65 (dd, J=6.4, 10.4 Hz,
1H; H-4), 3.78 (t, J=9.8 Hz, 1H; H-1), 3.94–4.02 (m, 1H; H-3), 4.18 (t,
J=5.6 Hz, 1H; H-2), 4.24 (t, J=4.9 Hz, 1H; H-6), 4.80 (s, 2H; PhCH2),
7.22–7.40 ppm (m, 5H; Ph).
1-O-Benzoyl-4-O-benzyl-2,3:5,6-di-O-isopropylidene-scyllo-inositol (11):
BnBr (0.19 mL, 1.64 mmol) was added to
a solution of 9 (200 mg,
(+)-1-O-p-Methoxybenzyl-2,3:5,6-di-O-isopropylidene-myo-inositol (5):
p-Methoxybenzylchloride (3.2 mL, 24 mmol) was added to a solution of 1
(5.59 g, 20 mmol) and NaH (0.96 g, 40 mmol) in DMF (90 mL) at 08C,
and the mixture was stirred at RT. After 10 h, the reaction mixture was
quenched with EtOAc and water at 08C. The standard extraction work-
up procedure using CH2Cl2 gave the crude product, which was subjected
to chromatography on silica gel to afford 5 as a colorless solid (6.2 g,
76%). Rf =0.13 (EtOAc/nHex 1:2); m.p. 153–1548C (lit. 158–1608C);[29]
1H NMR (CDCl3): d=1.32, 1.44, 1.46, 1.52 (4s, 3H each; 2CMe2), 2.27
(d, J=2.5 Hz, 1H; OH), 3.24 (d, J=9.7 Hz, 1H; H-5), 3.75 (dd, J=4.3,
10.1 Hz, 1H; H-1), 3.79 (s, 3H; OCH3), 3.83–3.92 (m, 2H; H-3, H-6),
4.79 (d, J=12.6 Hz, 1H; PhCHaCHb), 4.91 (d, J=12.5 Hz, 1H;
PhCHaCHb), 6.86 (d, J=8.6 Hz, 2H; Ph), 7.32 ppm (d, J=8.6 Hz, 2H;
Ph).
0.549 mmol), Ag2O (381 mg, 1.64 mmol), and TBAI (40 g, 0.109 mol) in
dry CH2Cl2 at RT. After 2 h, the reaction mixture was filtered through
Celite and washed with CH2Cl2. The filtrate was washed with aqueous
NaHCO3 and with brine, and the organic layer was separated, dried, and
concentrated to give the crude product, which was purified by using
column chromatography on silica gel to give 11 as a colorless, foamy
1
solid (200 mg, 80%). Rf =0.49 (EtOAc/nHex 1:9); H NMR (CDCl3): d=
1.44, 1.46 (2s, 6H each; 2CMe2), 3.81–3.90 (m, 5H; H-2, H-3, H-4, H-5,
H-6), 4.86 (s, 2H), 5.58 (app. t, J=9.3 Hz, 1H; H-1), 7.25–7.56 (m, 7H),
8.06 ppm (d, J=8.7 Hz, 2H); 13C NMR (CDCl3): d=27.0 (4C; 2CMe2);
70.1, 72.4, 74.5 (2C), 78.7, (2C), 80.6 (2C) (inositol ring carbon atoms);
113.7 (2C), 127.3, 128.0, 128.4, (2C), 128.8 (2C), 130.1 (2C), 130.4 (2C),
133.6, 138.2, 165.9 ppm (COPh); MS (FAB): m/z: 455.18 [M+H]+;
HRMS (FAB): m/z calcd for C26H31O7: 455.2118; found: 455.2073
[M+1]+.
(+)-4-O-(1’-Imidazolylcarbonyloxy)-2,3:5,6-di-O-isopropylidene-1-O-p-
methoxybenzyl-myo-inositol (6): CaH2 (0.59 g, 13.3 mmol) was added in
portions to a solution of 5 (2.2 g, 5.8 mmol) and carbonyldiimidazole
(2.35 g, 15.4 mmol) in toluene (40 mL), and the mixture was stirred for
11 h at RT. The mixture was filtered and the filtrate was evaporated to
give the crude product, which was purified by using column chromatogra-
phy on silica gel to obtain 6 as a colorless solid (2.6 g, 94.2%). Rf =0.42
(EtOAc/nHex 2:1); m.p. 144–1458C; 1H NMR (CDCl3): d=1.35, 1.44,
1.46, 1.63 (4s, 3H each; 2CMe2), 3.49 (dd, J=9.3, 11.4 Hz, 1H), 3.80 (s,
3H; OCH3), 4.15–4.19 (m, 2H), 4.32 (t, J=4.4 Hz, 1H), 4.74 (d, J=
12.2 Hz, 1H; PhCHaCHb), 4.86 (d, J=12.2 Hz, 1H; PhCHaCHb), 5.35
(dd, J=6.9, 11.4 Hz, 1H; H-6), 6.89 (d, J=8.7 Hz, 2H; Ph), 7.05 (dd, J=
0.6, 1.5 Hz, 1H; imidazole-H4), 7.35 (d, J=8.7 Hz, 2H; Ph), 7.41–7.42 (m,
1-O-Benzyl-2,3:5,6-di-O-isopropylidene-scyllo-inositol (12): NaOMe
(0.04 mL, 0.17 mmol, 25% w/v) was added to a solution of 11 (200 mg,
0.44 mmol) in MeOH (15 mL), and the solution was placed at reflux for
3 h. After cooling, the reaction mixture was filtered through silica gel.
The filtrate was concentrated and the crude product was washed with
5% EtOAc in n-hexane to remove the byproduct, methyl benzoate. The
washed product was dried under vacuum to afford 12 as a colorless solid
(150 mg, 97%). Rf =0.33 (EtOAc/nHex 1:1); m.p. 215–2168C; 1H NMR
(CDCl3): d=1.46 (s, 12H; 2CMe2), 2.41 (d, J=2.7 Hz, 1H; OH), 3.63 (t,
J=9.1 Hz, 2H; H-2,6 or H-3,5), 3.71 (t, J=9.0 Hz, 2H; H-2,6 or H-3,5),
3.87 (t, J=8.9 Hz, 1H; H-4), 4.10 (app. t, J=9.1 Hz, 1H; H-1), 4.83 (s,
768
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Chem. Eur. J. 2007, 13, 762 – 775

Molecular Transporters
FULL PAPER
2H; PhCH2-), 7.26–7.39 ppm (m, 5H; PhCH2-); 13C NMR (CDCl3): d=
26.7 (4C; 2CMe2), 69.4, 72.4, 75.2, 77.0 (2C), 80.6 (2C), 80.7 (2C), 113.5,
126.9, 128.0 (2C), 128.7 (2C), 138.5 ppm; MS (FAB): m/z: 351.47
[M+H]+; HRMS (FAB): m/z calcd for C19H27NO6: 351.1844; found:
351.1812 [M+H]+.
calcd for C40H53O14: 757.3523; found: 757.3435 [M+H]+; m/z calcd for
C40H52O14Na: 779.3423; found: 779.3218 [M+Na]+.
(+)-4’-O-Benzyl-1’-O-[(1-O-p-methoxybenzyl-myo-inositol)carbonyloxy]-
myo-inositol (17):
A solution of 7 (2.21 g, 3.70 mmol) and p-TSA
(286.1 mg, 1.46 mmol) in CH2Cl2/MeOH (50 mL, 1:9) was stirred at RT
for 14 h. The solid was filtered off, and the liquor washed with CH2Cl2/
MeOH (19:1, 100 mL) to remove the p-TSA. The residue was concen-
trated and dried under vacuum to afford 17 as a colorless solid (1.45 g,
83%). Rf =0.15 (CH2Cl2/MeOH 9:1); m.p. 1698C; 1H NMR (CDCl3 with
drops of [D6]DMSO): d=3.11–3.24 (m, 4H), 3.35–3.48 (m, 5H), 3.74 (s,
3H), 3.90–3.97 (m, 3H), 4.25–4.33 (m, 2H), 4.70–4.74 (m, 2H), 4.77–4.81
(m, 2H), 6.89 (d, J=8.6 Hz, 2H; Ph), 7.21–7.35 (m, 5H; Ph), 7.43 ppm
(d, J=7.1 Hz, 2H; Ph); 13C NMR (CDCl3 with drops of [D6]DMSO): d=
55.1, 69.4, 69.5, 69.6, 70.2, 70.3, 70.5, 70.91, 71.86, 72.1, 72.6, 72.8, 74.7,
78.0, 78.2, 79.6, 79.7, 81.6, 113.4, 127.0, 127.6, 127.9, 129.2, 131.1, 139.9,
154.58, 154.62, 158.5 ppm; MS (FAB): m/z: 619.09 [M+Na]+; HRMS
(FAB): m/z calcd for C28H37O14: 597.2205; found: 597.2183 [M+H]+; m/z
calcd for C28H36O14Na: 619.2105; found: 619.1951 [M+Na]+.
4-O-(1’-Imidazolylcarbonyloxy)-2,3:5,6-di-O-isopropylidene-1-O-benzyl-
scyllo-inositol (13): Carbonyldiimidazole (81 mg, 0.49 mmol) was added
to a solution of 12 (70 mg, 0.19 mmol) and CaH2 (21 mg, 0.49 mmol) in
dry toluene (6 mL) at RT. After 18 h, the reaction mixture was filtered
and washed with EtOAc (20 mL). The filtrate was concentrated under
vacuum, and the crude product was purified by using column chromatog-
raphy on silica gel to provide 13 as an off-white solid (78 mg, 88%). Rf =
0.37 (EtOAc/nHex 1:1); m.p. 214–2168C; 1H NMR (CDCl3): d=1.45,
1.47 (2s, 6H each; 2CMe2), 3.80–3.87 (m, 5H), 4.85 (s, 2H; PhCH2-),
5.45 (app. t, J=9.1 Hz, 1H; H-1), 7.07 (s, 1H), 7.29–7.42 (m, 6H),
8.16 ppm (s, 1H); 13C NMR (CDCl3): d=27.0 (2C; 2CMe2); 72.5, 73.8,
74.7, 77.0, 78.1 (2C), 80.7 (2C) (inositol ring carbon atoms); 114.1, 117.7,
128.0, 128.2 (2C), 128.7 (2C), 131.1, 137.6, 138.3, 148.0 ppm; MS (FAB):
m/z: 445.20 [M+H]+; HRMS (FAB): m/z calcd for C23H29N2O7: 445.2021;
found: 445.1976 [M+H]+.
A
representative peracylation with N-Boc-protected aminocarboxylic
acid: A solution of 7 (60 mg, 0.10 mmol), 6-aminocaproic acid (558 mg,
2.41 mmol), EDC (463 mg, 2.41 mmol), and DMAP(18.4 mg, 0.15 mmol)
in DMF (6.5 mL) was stirred at RT for 24 h under N2, treated with
EtOAc, and washed several times with saturated NaHCO3, water, and
brine. The organic phase was dried and concentrated to give the crude
product, which was purified by column chromatography on silica gel to
afford 19 as a sticky solid (190 mg, 82%).
1-O-Benzoyl-4-O-p-methoxybenzyl-2,3:5,6-di-O-isopropylidene-scyllo-in-
ositol (14): PMBCl (0.12 mL, 0.93 mmol) was slowly added to a solution
of 9 (300 mg, 0.85 mmol) and NaH (57.8 mg, 1.44 mmol) in dry DMF
(10 mL) at 08C. A catalytic amount of TBAI was added to the reaction
mixture, and it was allowed to slowly warm up to RT. After 4 h with stir-
ring, the reaction mixture was subjected to the standard extraction work-
up procedure using EtOAc to give the crude product, which was purified
by using column chromatography on silica gel to afford 14 as a colorless
solid (296 mg, 74%). Rf =0.43 (EtOAc/nHex 1:1); m.p. 218–2208C;
1H NMR (CDCl3): d=1.44 (s, 12H; 2CMe2), 3.66–3.85 (m, 8H), 4.78 (d,
J=10.2 Hz, 2H), 5.56–5.69 (m, 1H), 6.88 (dd, J=8.4, 6.0 Hz, 2H), 7.31–
7.56 (m, 5H), 8.08 ppm (d, J=8.8 Hz, 2H); 13C NMR (CDCl3): d=27.1,
27.2 (4C; 2CMe2), 56.7, 70.1, 72.0, 72.1, 74.5, 74.7, 80.69, 80.74, 113.1,
113.6, 114.1, 128.7, 130.0, 130.5, 133.6, 159.6, 165.7 ppm; MS (FAB): m/z
calcd for C27H33O8: 485.2197; found: 485.1520 [M+H]+; m/z calcd for
C27H32O8Na: 507.2097; found: 507.2013 [M+Na]+.
(+)-4’-O-Benzyl-1’-O-{[1-O-p-methoxybenzyl-2,3,5,6-O-(4-Boc-aminobu-
tanoyl)-myo-inositol]carbonyloxy}-2’,3’,5’,6’-O-(4-Boc-aminobutanoyl)-
myo-inositol (18): Colorless, sticky solid (166 mg, 62%); Rf =0.43
(EtOAc/nHex 1:1); 1H NMR (CDCl3): d=1.35 (brs, 72H), 1.58–1.79 (m,
16H), 2.01–2.49 (m, 16H), 2.99–3.01 (m, 16H), 3.50–3.57 (m, 1H), 3.71
(s, 3H), 3.86–3.93 (m, 1H), 4.44–4.55 (m, 4H), 4.75–5.64 (m, 18H), 6.77
(d, J=8.6 Hz, 2H), 7.04 (d, J=8.5 Hz, 2H), 7.10–7.26 ppm (m, 5H);
13C NMR (CDCl3): d=25.35, 25.43, 25.6, 25.8, 25.9, 28.77, 28.82, 31.4,
31.5, 31.7, 31.8, 40.1, 55.6, 67.1, 67.2, 68.3, 68.4, 69.1, 69.4, 69.9, 70.3, 70.7,
71.4, 71.6, 72.0, 73.7, 75.4, 75.5, 77.7, 79.5, 114.4, 127.67, 127.72, 128.2,
128.8, 129.1, 138.1, 138.2, 153.3, 153.4, 156.4, 156.5, 160.1, 172.0, 172.1,
172.3, 172.4, 172.5, 172.7 ppm; MALDI-TOF MS: m/z calcd for
C100H156N8O38Na: 2101.0554; found: 2100.0781 [M+Na]+.
1-O-p-Methoxybenzyl-2,3:5,6-di-O-isopropylidene-scyllo-inositol
(15):
NaOMe (0.04 mL, 0.17 mmol, 25% w/v) was added to a solution of 14
(200 mg, 0.44 mmol) in MeOH (15 mL), and the solution was placed at
reflux for 3 h. After cooling to RT, the reaction mixture was filtered
through silica gel. The filtrate was concentrated and the residue was
washed with 5% EtOAc in n-hexane to remove the byproduct, methyl
benzoate. The product was dried under vacuum to afford 15 as a colorless
solid (150 mg, 97%). Rf =0.33 (EtOAc/nHex 1:1); m.p. 198–2008C;
1H NMR (CDCl3): d=1.47 (s, 12H; 2CMe2), 2.41 (brs, 1H; OH), 3.59–
3.63 (m, 5H), 3.65 (s, 3H; OCH3), 3.80 (app. t, J=9.1 Hz, 1H; H-1), 4.76
(s, 2H), 6.87 (d, J=9.0 Hz, 2H), 7.33 ppm (d, J=9.1 Hz, 2H); 13C NMR
(CDCl3): d=27.1 (4C; 2CMe2), 55.6, 69.3, 72.0, 74.9, 80.6, 80.7, 80.8,
113.7, 129.3, 130.7, 130.8, 159.6 ppm; MS (FAB): m/z: 381.30 [M+H]+;
HRMS (FAB): m/z calcd for C20H29O7: 381.1968; found: 381.1913
[M+H]+.
(+)-4’-O-Benzyl-1’-O-{[1-O-p-methoxybenzyl-2,3,5,6-O-(6-Boc-amino-
hexanoyl)-myo-inositol]carbonyloxy}-2’,3’,5’,6’-O-(6-Boc-aminohexanoyl)-
myo-inositol (19): Colorless, sticky solid (190 mg, 82%); Rf =0.62
(EtOAc/nHex 1:1); 1H NMR (CDCl3): d=1.13–1.32 (m, 16H), 1.36 (brs,
72H), 1.45–1.57 (m, 32H), 2.06–2.15 (m, 16H), 2.95–3.02 (m, 16H), 3.76
(s, 3H), 3.85–3.91 (m, 1H), 4.22 (dd, J=1.9, 9.3 Hz, 1H), 4.48 (d, J=
11.8 Hz, 2H), 4.55 (d, J=18.4 Hz, 2H), 4.60–5.68 (m, 18H), 6.76 (d, J=
8.7 Hz, 2H), 7.05 (d, J=8.4 Hz, 2H), 7.10–7.26 ppm (m, 5H); 13C NMR
(CDCl3): d=24.6, 24.7, 25.1, 26.3, 26.4, 28.8, 28.9, 30.1, 30.3, 33.8, 34.1,
34.2, 34.4, 40.7, 55.6, 66.7, 68.1, 69.1, 70.2, 71.3, 71.9, 72.2, 73.6, 73.7, 74.5,
75.1, 75.4, 79.28, 79.34, 114.2, 127.3, 127.5, 128.1, 128.8, 129.4, 129.8,
153.5, 156.4, 156.5, 159.8, 172.2, 172.3, 172.5, 172.7, 172.8, 172.9,
173.1 ppm; MALDI-TOF MS: m/z calcd for C116H188N8O38Na: 2325.3058;
found: 2324.6021 [M+Na]+.
4’-O-Benzyl-1’-O-[(1-O-p-methoxybenzyl-2,3:5,6-di-O-isopropylidene-
scyllo-inositol)carbonyloxy]-2’,3’:5’,6’-di-O-isopropylidene-scyllo-inositol
(16): A solution of 15 (43 mg, 0.11 mmol) and NaH (2.7 mg, 0.11 mmol)
in dry THF (2.5 mL) at 08C was stirred for 30 min, and then added to a
solution containing 13 (50 mg, 0.11 mmol) in dry THF (2.1 mL). After
stirring for 1 h at 0–58C, the reaction mixture was diluted with CH2Cl2
(25 mL) and washed with a saturated solution of NH4Cl. The organic
layer was dried, filtered, and concentrated to give the crude product,
which was purified by using column chromatography on silica gel to
obtain 16 as a colorless solid (53 mg, 62%). Rf =0.46 (EtOAc/nHex 1:2);
(+)-4’-O-Benzyl-1’-O-{[1-O-p-methoxybenzyl-2,3,5,6-O-(8-Boc-aminooc-
tanoyl)-myo-inositol]carbonyloxy}-2’,3’,5’,6’-O-(8-Boc-aminooctanoyl)-
myo-inositol (20): Colorless, sticky solid (220 mg, 82%); Rf =0.55
(EtOAc/nHex 1:1); 1H NMR (CDCl3): d=1.14–1.21 (m, 48H), 1.35 (s,
72H), 1.48–1.56 (m, 32H), 2.03–2.37 (m, 16H), 2.95–3.00 (m, 16H), 3.70
(s, 3H), 4.53 (dd, J=11.7, 17.4 Hz, 4H), 4.63–5.67 (m, 20H), 6.73 (d, J=
8.7 Hz, 2H), 7.03 (d, J=8.7 Hz, 2H), 7.08–7.24 ppm (m, 5H; Ph);
13C NMR (CDCl3): d=24.85, 24.93, 25.2, 25.4, 26.9, 27.0, 28.8, 29.2, 29.3,
29.4, 29.7, 30.0, 30.4, 33.8, 34.2, 34.3, 34.4, 41.0, 55.6, 66.8, 67.9, 68.4, 69.1,
69.8, 70.7, 72.7, 75.5, 76.2, 113.4, 115.4, 119.5, 123.1, 124.7, 126.9, 127.57,
127.64, 128.4, 129.3, 130.5, 132.66, 132.72, 138.9, 151.5, 153.7, 158.0, 159.7,
162.4, 170.7, 171.4, 172.0, 172.4, 172.7 ppm; MALDI-TOF MS: m/z calcd
for C132H220N8O38Na: 2549.5562; found: 2548.3932 [M+Na]+.
1
m.p. 244–2468C; H NMR (CDCl3): d=1.43, 1.47 (2s, 6H each; 2CMe2),
3.66–3.88 (m, 13H), 4.47 (s, 2H), 4.83 (s, 2H), 5.12 (t, J=9.3 Hz, 2H; H-
6, H-6’), 6.88 (d, J=8.7 Hz, 2H), 7.27–7.42 ppm (m, 7H); 13C NMR
(CDCl3): d=27.1 (4C; 2CMe2); 55.7, 69.4, 72.1, 74.1, 74.6, 74.9, 75.0,
75.3, 77.0, 80.5 (inositol ring carbon atoms); 80.6, 80.8, 113.4, 114.2,
127.87, 127.94 (2C), 128.2 (2C), 128.6 (2C), 129.9, 130.6, 138.5, 153.3,
159.7, 171.4 ppm; MS (FAB): m/z: 757.43 [M+H]+; HRMS (FAB): m/z
A representative procedure for the removal of the PMB protecting
group: CAN (20.2 mg, 0.37 mmol) was added to a solution of 19 (170 mg,
Chem. Eur. J. 2007, 13, 762 – 775
ꢀ 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
769

S.-K. Chung et al.
0.07 mmol) in a mixture of CH3CN, toluene, and water (18:5:5), and the
mixture was stirred at 08C for 2 h and at RT for 10 h. The reaction mix-
ture was diluted with EtOAc and subjected to the standard extraction
work-up procedure to give the crude product, which was purified by
using column chromatography on silica gel to afford 22 as a colorless,
foamy solid (126 mg, 78%).
156.4, 156.5, 171.6, 171.8, 172.4, 172.5, 172.7 ppm; MALDI-TOF MS: m/z
calcd for C120H191N9O39SNa: 2438.9024; found: 2438.1826 [M+Na]+.
(+)-4’-O-Benzyl-1’-O-{[1-O-(5-dimethylamino-a-naphthalenesulfonyl)-
2,3,5,6-O-(8-Boc-aminooctanoyl)-myo-inositol]carbonyloxy}-2’,3’,5’,6’-O-
(8-Boc-aminooctanoyl)-myo-inositol (26): Light-yellowish, sticky solid
1
(162 mg, 66%); Rf =0.33 (EtOAc/nHex 1:1); H NMR (CDCl3): d=1.11–
(+)-4’-O-Benzyl-1’-O-{[2,3,5,6-O-(4-Boc-aminobutanoyl)-myo-inositol]-
carbonyloxy}-2’,3’,5’,6’-O-(4-Boc-aminobutanoyl)-myo-inositol (21): Off-
white, foamy solid (89 mg, 58%); Rf =0.14 (EtOAc/nHex 1:1); 1H NMR
(CDCl3): d=1.36 (brs, 72H), 1.56–1.81 (m, 16H), 2.02–2.54 (m, 16H),
2.98–3.18 (m, 16H), 3.80–3.94 (m, 2H), 4.56 (dd, J=12.3, 18.0 Hz, 2H),
4.73–5.57 (m, 18H), 7.11–7.28 ppm (m, 5H; Ph); 13C NMR (CDCl3): d=
24.4, 24.7, 25.1, 25.3, 26.3, 26.6, 28.6, 30.1, 33.8, 34.0, 34.1, 34.5, 40.7, 68.0,
68.9, 70.0, 70.5, 71.2, 72.8, 73.7, 75.2, 79.6, 127.3, 127.5, 128.8, 138.1, 153.5,
156.5, 172.3, 172.49, 172.54, 172.9, 173.2 ppm; MALDI-TOF MS: m/z
calcd for C92H148N8O37Na: 1980.9979; found: 1980.0722 [M+Na]+.
1.16 (m, 48H), 1.32 (brs, 104H), 1.80–2.22 (m, 16H), 2.79 (s, 6H), 2.97
(brs, 16H), 3.83 (t, J=9.8 Hz, 1H), 4.56 (d, J=12.1 Hz, 1H), 4.60 (d, J=
12.6 Hz, 1H), 4.60–5.48 (m, 19H), 7.05–7.20 (m, 5H), 7.24 (s, 1H), 7.42
(dd, J=8.3, 16.3 Hz, 2H), 7.49 (d, J=8.6 Hz, 1H), 8.10 (d, J=7.3 Hz,
1H), 8.49 ppm (d, J=8.5 Hz, 1H); 13C NMR (CDCl3): d=24.4, 24.8, 25.0,
25.5, 28.8, 29.1, 29.3, 29.5, 30.0, 33.5, 33.7, 34.2, 34.4, 41.0, 45.7, 67.9, 68.0,
69.1, 70.5, 70.9, 73.9, 74.0, 74.5, 75.3, 75.4, 77.7, 79.2, 115.8, 119.3, 123.5,
127.5, 128.1, 128.7, 130.1, 130.2, 132.3, 138.1, 153.3, 153.4, 156.4, 171.4,
172.1, 172.3, 172.6 ppm; MALDI-TOF MS: m/z calcd for
C136H223N9O39SNa: 2662.3277; found: 2662.2875 [M+Na]+.
(+)-4’-O-Benzyl-1’-O-{[2,3,5,6-O-(6-Boc-aminohexanoyl)-myo-inositol]-
carbonyloxy}-2’,3’,5’,6’-O-(6-Boc-aminohexanoyl)-myo-inositol (22): Col-
orless, foamy solid (126 mg, 78%); Rf =0.33 (EtOAc/nHex 1:1); H NMR
(CDCl3): d=1.17–1.26 (m, 16H), 1.37 (brs, 72H), 1.41–1.53 (m, 32H),
2.07–2.41 (m, 16H), 2.94–3.05 (m, 16H), 3.86–3.92 (m, 2H), 4.31–5.56 (m,
20H), 7.11–7.27 ppm (m, 5H; Ph); 13C NMR (CDCl3): d=24.9, 25.2, 25.7,
25.8, 28.8, 29.6, 30.4, 31.0, 31.5, 31.7, 32.2, 34.1, 40.1, 68.2, 69.4, 70.7, 71.8,
72.6, 73.6, 75.6, 79.5, 80.1, 126.3, 127.5, 128.7, 128.8, 138.1, 153.4, 156.5,
156.6, 172.0, 172.3, 172.95, 173.00 ppm; MALDI-TOF MS: m/z calcd for
C108H180N8O37Na: 2205.2483; found: 2204.8119 [M+Na]+.
A representative procedure for the N-Boc deprotection: Compound 25
(120 mg, 0.054 mmol) was added to a solution of gaseous HCl in saturat-
ed EtOAc (5 mL) at 08C, and the solution was stirred for 3 h. The precip-
itate was separated and dried under vacuum to give the colorless hydro-
chloride salt 28 (80 mg, quant.).
1
(+)-4’-O-Benzyl-1’-O-{[1-O-(5-dimethylamino-a-naphthalenesulfonyl)-
2,3,5,6-O-(4-aminobutanoyl
hydrochloride)-myo-inositol]carbonyloxy}-
2’,3’,5’,6’-O-(4-aminobutanoyl hydrochloride)-myo-inositol (27): Off-
white solid (48 mg, quant.); 1H NMR (CD3OD): d=1.77–1.90 (m, 16H),
2.18–2.62 (m, 16H), 2.95–3.13 (m, 16H), 3.28–3.30 (m, 1H), 3.47 (s, 6H),
3.81 (t, J=9.7 Hz, 1H), 4.50 (dd, J=12.1, 17.5 Hz, 2H), 4.95–5.62 (m,
11H), 7.23–7.34 (m, 5H; Ph), 7.93 (m, 2H), 8.19 (brs, 1H), 8.52 (d, J=
7.2 Hz, 1H), 8.57 (d, J=7.8 Hz, 1H), 8.97 ppm (d, J=8.4 Hz, 1H);
13C NMR (CD3OD): d=22.3, 22.7, 22.8, 23.2, 29.5, 30.2, 30.5, 30.8, 31.0,
39.0, 39.2, 46.7, 68.0, 69.0, 69.3, 69.7, 69.9, 71.4, 73.9, 75.2, 75.7, 78.0,
119.9, 126.6, 127.3, 128.5, 129.1, 131.9, 153.3, 153.4, 171.3, 171.7, 171.8,
172.1, 172.4 ppm; MALDI-TOF MS: m/z calcd for C64H95N9O23SNa:
1412.6262; found: 1412.6842 [M+Na]+.
(+)-4’-O-Benzyl-1’-O-{[2,3,5,6-O-(8-Boc-aminooctanoyl)-myo-inositol]-
carbonyloxy}-2’,3’,5’,6’-O-(8-Boc-aminooctanoyl)-myo-inositol (23): Col-
orless, foamy solid (132 mg, 70%); Rf =0.11 (EtOAc/nHex 2:1, three
times); 1H NMR (CDCl3): d=1.15–1.21 (m, 48H), 1.36 (brs, 72H), 1.49–
1.59 (m, 32H), 2.07–2.40 (m, 16H), 2.96–3.02 (m, 16H), 4.54 (dd, J=12.0,
17.7 Hz, 2H), 4.63–5.54 (m, 20H), 7.09–7.24 ppm (m, 5H; Ph); 13C NMR
(CDCl3): d=24.6, 25.0, 25.4, 26.9, 28.8, 29.5, 30.0, 33.7, 34.0, 34.4, 40.9,
68.7, 68.9, 69.1, 70.5, 71.1, 72.3, 73.8, 79.3, 127.5, 128.1, 128.7, 138.1, 153.5,
156.4, 172.4, 172.5, 172.8, 173.0, 173.2 ppm; MALDI-TOF MS: m/z calcd
for C124H212N8O37Na: 2429.4987; found: 2429.0366 [M+Na]+.
(+)-4’-O-Benzyl-1’-O-{[1-O-(5-dimethylamino-a-naphthalenesulfonyl)-
2,3,5,6-O-(6-aminohexanoyl hydrochloride)-myo-inositol]carbonyloxy}-
2’,3’,5’,6’-O-(6-aminohexanoyl hydrochloride)-myo-inositol (28): Colorless
solid (80 mg, quant.); 1H NMR (CD3OD): d=1.31–1.78 (m, 48H), 2.23–
2.38 (m, 16H), 2.94–3.18 (m, 16H), 3.28–3.30 (m, 1H), 3.44 (s, 6H), 4.71
(brs, 2H), 4.95–5.62 (m, 11H), 7.21–7.30 (m, 5H), 7.87–7.98 (m, 2H),
8.14 (brs, 1H), 8.53 (dd, J=8.4, 15.0 Hz, 2H), 8.95 ppm (t, J=8.4 Hz,
1H); 13C NMR (CD3OD): d=23.9, 24.2, 24.9, 25.9, 26.0, 26.1, 27.2, 29.4,
29.6, 33.2, 33.6, 33.8, 39.6, 39.7, 46.5, 68.0, 69.3, 69.4, 70.4, 72.1, 74.9, 76.0,
77.7, 119.2, 124.7, 126.1, 127.5, 127.7, 128.5, 128.9, 129.7, 133.1, 138.5,
153.5, 153.6, 171.78, 171.83, 172.2, 172.7, 172.9 ppm; MALDI-TOF MS:
m/z calcd for C80H127N9O23SNa: 1636.8766; found: 1636.4102 [M+Na]+.
A representative dansylation procedure: DMAP(14 mg, 0.114 mmol)
and
5-dimethylamino-a-naphthalenesulfonyl
chloride
(31 mg,
0.114 mmol) were added to a solution of 22 (125 mg, 0.057 mmol) in
CH3CN (4 mL). The reaction mixture was stirred for 15 h at RT and
quenched with EtOAc and then saturated NH4Cl. The standard extrac-
tion work-up procedure gave the crude product, which was purified by
using column chromatography on silica gel to afford the dansylated com-
pound 25 as a light-yellowish, sticky solid (122 mg, 88%).
(+)-4’-O-Benzyl-1’-O-{[1-O-(5-dimethylamino-a-naphthalenesulfonyl)-
2,3,5,6-O-(4-Boc-aminobutanoyl)-myo-inositol]carbonyloxy}-2’,3’,5’,6’-O-
(4-Boc-aminobutanoyl)-myo-inositol (24): Light-yellowish, sticky solid
(+)-4’-O-Benzyl-1’-O-{[1-O-(5-dimethylamino-a-naphthalenesulfonyl)-
1
(110 mg, 52%); Rf =0.23 (EtOAc/nHex 1:1); H NMR (CDCl3): d=1.36–
2,3,5,6-O-(8-aminooctanoyl
hydrochloride)-myo-inositol]carbonyloxy}-
1.40 (m, 72H), 1.49–1.67 (m, 16H), 1.85–2.34 (m, 16H), 2.84 (s, 6H),
2.92–2.98 (m, 16H), 3.88 (t, J=9.7 Hz, 1H), 4.54 (dd, J=12.1, 16.0 Hz,
2H), 4.74–5.49 (m, 19H), 7.10–7.24 (m, 6H; aromatic), 7.49 (dd, J=7.5,
14.1 Hz, 2H), 8.00 (d, J=8.4 Hz, 1H), 8.15 (d, J=7.2 Hz, 1H), 8.58 ppm
(d, J=6.9 Hz, 1H); 13C NMR (CDCl3): d=24.7, 25.1, 25.2, 25.6, 25.8,
28.6, 29.6, 30.4, 31.2, 31.7, 33.1, 40.0, 45.8, 68.0, 69.1, 69.6, 70.4, 71.2, 72.2,
72.8, 73.8, 75.2, 79.3, 79.6, 127.3, 128.1, 128.8, 138.1, 153.5, 156.5, 172.3,
172.4, 172.5, 172.8, 173.2, 173.7 ppm; MALDI-TOF MS: m/z calcd for
2’,3’,5’,6’-O-(8-aminooctanoyl hydrochloride)-myo-inositol (29): Colorless
solid (105 mg, quant.); H NMR (CD3OD): d=1.63–2.03 (m, 80H), 2.41–
1
2.58 (m, 16H), 3.19–3.26 (m, 16H), 3.57 (s, 6H), 4.07–4.20 (m, 1H), 4.81–
4.90 (m, 2H), 5.11–5.84 (m, 11H), 7.10–7.19 (m, 5H), 7.68–7.92 (m, 4H),
8.24 (d, J=7.8 Hz, 2H), 8.66 ppm (d, J=8.4 Hz, 1H); 13C NMR
(CD3OD): d=22.7, 24.3, 24.5, 25.0, 25.1, 25.2, 26.4, 27.1, 27.46, 27.52,
28.9, 29.0, 33.4, 33.9, 39.8, 39.9, 68.0, 68.2, 68.6, 69.6, 69.9, 70.7, 70.8, 74.7,
75.1, 77.7, 119.4, 125.2, 126.3, 127.8, 128.4, 128.9, 131.4, 134.9, 153.4,
153.6, 168.3, 172.0, 172.3, 172.6, 172.89, 172.94, 173.0 ppm; MALDI-TOF
MS: m/z calcd for C96H159N9O23SNa: 1862.1303; found: 1861.3469
[M+Na]+.
C
104H159N9O39SNa: 2214.0490; found: 2214.1921 [M+Na]+.
(+)-4’-O-Benzyl-1’-O-{[1-O-(5-dimethylamino-a-naphthalenesulfonyl)-
2,3,5,6-O-(6-Boc-aminohexanoyl)-myo-inositol]carbonyloxy}-2’,3’,5’,6’-O-
(6-Boc-aminohexanoyl)-myo-inositol (25): Light-yellowish, sticky solid
(122 mg, 88%); Rf =0.29 (EtOAc/nHex 1:1); 1H NMR (CDCl3): d=1.21
(s, 16H), 1.38 (brs, 72H), 1.53–1.59 (m, 32H), 1.99–2.33 (m, 16H), 2.83
(s, 6H), 2.94–3.04 (m, 16H), 3.89 (t, J=9.77 Hz, 1H), 4.56 (dd, J=11.8,
18.6 Hz, 2H), 4.62 (d, J=12.5 Hz, 1H), 4.69–5.54 (m, 19H), 7.11–7.29 (m,
6H), 7.50 (dd, J=8.4, 16.5 Hz, 2H), 8.03 (d, J=7.5 Hz, 1H), 8.18 (d, J=
7.2 Hz, 1H), 8.61 ppm (brs, 1H); 13C NMR (CDCl3): d=24.2, 24.5, 24.7,
26.2, 26.3, 28.8, 29.7, 30.1, 30.3, 34.1, 34.2, 40.7, 41.8, 46.2, 67.9, 68.4, 69.2,
69.7, 72.1, 73.8, 74.7, 77.7, 79.3, 127.3, 128.1, 128.8, 130.6, 138.1, 153.4,
A
representative procedure for guanidinylation: Et3N (0.16 mL,
1.15 mmol) and N,N’-di-Boc-N’’-trifluoromethanesulfonylguanidine
(454 mg, 1.15 mmol) were sequentially added to a solution of 28 (78 mg,
0.048 mmol) in dry DMF (4.5 mL), and the reaction mixture was stirred
at RT for 3 d. The mixture was diluted with EtOAc, and washed with 1n
NaHSO4, saturated NaHCO3, and brine. The organic layer was separated,
dried, and concentrated to give the crude product, which was purified by
using column chromatography on neutral alumina to afford 31 as a color-
less, foamy solid (105 mg, 61%).
770
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Chem. Eur. J. 2007, 13, 762 – 775

Molecular Transporters
FULL PAPER
(+)-4’-O-Benzyl-1’-O-{[1-O-(5-dimethylamino-a-naphthalenesulfonyl)-
2,3,5,6-O-(N,N’-bis-Boc-N’’-aminobutanoylguanidine)-myo-inositol]carbo-
nyloxy}-2’,3’,5’,6’-O-(N,N’-bis-Boc-N’’-aminobutanoylguanidine)-myo-ino-
sitol (30): Colorless, foamy solid (71 mg, 56%); Rf =0.64 (EtOAc/nHex
2:1, two times); 1H NMR (CDCl3): d=1.72 (brs, 144H), 1.84–1.96 (m,
16H), 2.20–2.58 (m, 16H), 2.81 (s, 6H), 3.31 (m, 16H), 3.92 (brs, 1H),
4.52–5.49 (m, 13H), 7.13–7.24 (m, 6H; aromatic), 7.52 (m, 2H), 7.99 (d,
J=8.1 Hz, 1H), 8.18 (d, J=7.2 Hz, 1H), 8.28 (brs, 8H), 8.55 (d, J=
7.8 Hz, 1H), 11.44 ppm (brs, 8H); 13C NMR (CDCl3): d=24.3, 24.4, 24.6,
25.1, 28.17, 28.21, 28.7, 29.5, 31.3, 32.2, 40.3, 45.7, 67.8, 69.7, 69.9, 70.5,
71.2, 72.2, 72.7, 73.0, 73.5, 75.6, 75.9, 79.4, 79.8, 83.3, 83.7, 116.0, 119.4,
127.8, 128.1, 128.2, 128.5, 128.9, 129.4, 132.2, 132.6, 138.1, 153.2, 153.9,
156.5, 157.1, 171.7, 171.8, 172.0, 172.1, 172.4 ppm.
2’,3’,5’,6’-O-(aminohexanoylguanidinium)-myo-inositol·8TFA (34): Light-
brownish, glassy solid (17.1 mg, 89%); UV (H2O): lmax (e)=336.40 nm
(2700 cmÀ1 mÀ1); 1H NMR (CD3OD): d=1.13–1.35 (m, 16H), 1.42–1.57
(m, 32H), 2.13–2.43 (m, 16H), 2.88 (s, 6H), 3.02–3.16 (m, 16H), 4.01 (t,
J=9.6 Hz, 1H), 4.64 (dd, J=11.9, 18.7 Hz, 2H), 5.01–5.61 (m, 11H),
7.16–7.28 (m, 6H; aromatic), 7.52–7.66 (m, 2H), 8.00 (d, J=8.6 Hz, 1H),
8.27 (d, J=7.3 Hz, 1H), 8.64 ppm (d, J=8.5 Hz, 1H); 13C NMR
(CD3OD): d=24.0, 24.2, 24.4, 24.7, 26.0, 28.47, 28.54, 28.6, 33.2, 33.3,
33.7, 41.3, 44.8, 68.1, 68.6, 69.2, 70.0, 70.2, 72.2, 74.1, 75.6, 77.6, 115.3,
115.8, 119.1, 123.1, 123.5, 126.3, 127.5, 128.4, 129.0, 130.3, 130.4, 132.3,
138.4, 152.6, 153.6, 157.8, 161.9, 162.4, 172.0, 172.1, 172.3, 172.6,
172.9 ppm; MALDI-TOF MS: m/z calcd for C88H143N25O23SNa:
1973.0509; found: 1972.8712 [M+Na]+.
(+)-4’-O-Benzyl-1’-O-{[1-O-(5-dimethylamino-a-naphthalenesulfonyl)-
2,3,5,6-O-(N,N’-bis-Boc-N’’-aminohexanoylguanidine)-myo-inositol]car-
bonyloxy}-2’,3’,5’,6’-O-(N,N’-bis-Boc-N’’-aminohexanoylguanidine)-myo-
inositol (31): Colorless, foamy solid (105 mg, 61%); Rf =0.53 (EtOAc/
nHex 2:1, developed three times); 1H NMR (CDCl3): d=1.13–1.65 (m,
192H), 1.60–2.31 (m, 16H), 2.82 (s, 6H), 3.24–3.36 (m, 16H), 3.88 (t, J=
9.9 Hz, 1H), 4.56–5.51 (m, 13H), 7.11–7.29 (m, 6H), 7.51 (dd, J=8.4,
16.2 Hz, 2H), 7.99 (d, J=8.7 Hz, 1H), 8.17 (d, J=7.5 Hz, 1H), 8.22–8.28
(m, 8H), 11.46 ppm (brs, 8H); 13C NMR (CDCl3): d=24.3, 24.4, 24.6,
25.2, 26.6, 26.9, 28.5, 28.7, 29.1, 29.2, 33.9, 34.0, 34.2, 35.9, 41.0, 45.7, 67.7,
68.0, 68.4, 69.1, 69.5, 70.0, 70.5, 72.4, 73.7, 74.8, 75.4, 75.9, 77.6, 79.5,
83.29, 83.34, 115.8, 119.5, 123.5, 127.0, 127.5, 128.1, 128.8, 129.2, 132.4,
132.5, 138.1, 152.3, 153.4, 153.7, 156.4, 171.2, 171.2, 172.1, 172.2,
172.4 ppm; MALDI-TOF MS: m/z calcd for C169H271N25O55SNa:
3574.8932; found: 3572.6201 [M+NaÀ2]+.
(+)-4’-O-Benzyl-1’-O-{[1-O-(5-dimethylamino-a-naphthalenesulfonyl)-
2,3,5,6-O-(N,N’-bis-Boc-N’’-aminooctanoylguanidine)-myo-inositol]carbo-
nyloxy}-2’,3’,5’,6’-O-(N,N’-bis-Boc-N’’-aminooctanoylguanidine)-myo-ino-
sitol (32): Colorless, foamy solid (107 mg, 64%); Rf =0.41 (EtOAc/nHex
2:1, two times); 1H NMR (CDCl3): d=1.21–1.49 (m, 224H), 2.05–2.18
(m, 16H), 2.88 (s, 6H), 3.42 (brs, 16H), 3.94 (t, J=9.7 Hz, 1H), 4.57–5.63
(m, 13H), 7.17–7.30 (m, 6H), 7.50–7.60 (m, 2H), 8.05 (d, J=8.3 Hz, 1H),
8.22 (d, J=7.3 Hz, 1H), 8.41 (brs, 8H), 8.61 (d, J=8.5 Hz, 1H),
11.51 ppm (brs, 8H); 13C NMR (CDCl3): d=21.2, 23.0, 23.3, 24.5, 24.7,
24.9, 26.97, 27.00, 27.08, 27.12, 28.4, 28.7, 29.1, 29.2, 29.3, 29.6, 30.4, 31.6,
32.2, 33.9, 34.0, 34.2, 34.3, 34.4, 41.2, 41.3, 45.7, 53.7, 65.8, 67.9, 68.3, 69.2,
69.4, 70.5, 72.3, 73.9, 75.3, 75.4, 77.6, 78.2, 79.1, 83.5, 115.7, 119.6, 123.4,
127.2, 128.1, 128.7, 129.1, 131.1, 138.1, 152.3, 153.7, 156.4, 164.0, 171.7,
172.0, 172.2, 172.4, 172.6 ppm; MALDI-TOF MS: m/z calcd for
C184H303N25O55SNa: 3800.1469; found: 3798.7681 [M+NaÀ2]+.
(+)-4’-O-Benzyl-1’-O-{[1-O-(5-dimethylamino-a-naphthalenesulfonyl)-
2,3,5,6-O-(aminooctanoylguanidinium)-myo-inositol]carbonyloxy}-
2’,3’,5’,6’-O-(aminooctanoylguanidinium)-myo-inositol·8TFA (35): Light-
brownish, foamy solid (21.7 mg, 87%); UV (H2O): lmax (e)=360 nm
(3100 cmÀ1 mÀ1); 1H NMR (CD3OD): d=1.25–1.35 (m, 48H), 1.50 (brs,
32H), 2.17–2.37 (m, 16H), 2.89 (s, 6H), 3.14 (brs, 16H), 3.40 (brs, 1H),
4.62–4.64 (m, 2H), 5.13–5.60 (m, 11H), 7.21–7.26 (m, 5H; aromatic),
7.47–7.64 (m, 3H), 8.02 (d, J=7.1 Hz, 1H), 8.26 (brs, 1H), 8.64 ppm (d,
J=7.6 Hz, 1H); 13C NMR (CD3OD): d=23.1, 23.2, 23.9, 25.1, 27.4, 27.5,
27.6, 27.8, 28.0, 31.9, 32.1, 32.5, 43.5, 67.8, 68.0, 68.2, 69.5, 70.9, 72.7, 73.6,
74.3, 76.3, 113.9, 114.5, 117.4, 126.1, 126.5, 127.6, 128.6, 129.9, 130.8,
130.9, 137.0, 151.1, 152.1, 152.2, 156.5, 159.9, 160.4, 170.8, 171.0, 171.2,
171.3, 171.5, 171.7 ppm; MALDI-TOF MS: m/z calcd for
C104H175N25O23SNa: 2198.3047; found: 2198.9236 [M+Na]+.
4-O-Allyl-1-O-benzoyl-2,3:5,6-di-O-isopropylidene-scyllo-inositol
(36):
Ag2O (2.22 g, 9.61 mmol), allyl bromide (0.84 mL, 9.61 mmol), and a cat-
alytic amount of TBAI (150 mg) were added to a solution of 8 (1.4 g,
3.84 mmol) in CH2Cl2 (30 mL). The reaction mixture was stirred for 3 h
at RT, and then filtered through a short bed of Celite. The filtrate was
concentrated and purified by using column chromatography on silica gel
to obtain 36 as a colorless solid (1.46 mg, 94.5%). M.p. 214–2168C; Rf =
0.34 (EtOAc/nHex 1:4); 1H NMR (CDCl3): d=1.44 (s, 12H; 2CMe2),
3.75–3.88 (m, 5H), 4.31 (dd, J=6.9, 1.2 Hz, 2H), 5.22 (dd, J=1.5,
10.1 Hz, 1H), 5.35 (dd, J=1.5, 18.1 Hz, 1H), 5.57 (t, J=8.7 Hz, 1H),
5.92–6.02 (m, 1H), 7.47 (t, J=9.0 Hz, 2H), 7.57 (t, J=6.0 Hz, 1H),
8.08 ppm (d, J=9.0 Hz, 2H); 13C NMR (CDCl3): d=27.08 (2C; CMe2),
27.11 (2C; CMe2), 70.1, 71.7, 74.8, 78.8, 80.7, 113.6, 118.0, 126.3, 128.7,
130.1, 130.4, 133.6, 134.9, 165.7 ppm; MS (FAB): m/z: 405.19 [M+H]+;
HRMS (FAB): m/z calcd for C22H29O7: 405.1901; found: 405.1909
[M+H]+.
A representative procedure for the N-Boc deprotection of the guanidine
moiety: A TFA/CH2Cl2 mixture (1:1, 2 mL) was added to a solution of
compound 31 (35 mg, 0.01 mmol) in CH2Cl2 (1 mL) at RT, and the solu-
tion was stirred for 5 h. Samples from the reaction were analyzed by
using TLC, and after the starting-material spot had completely disap-
peared, the solution was concentrated. The residue was washed with a
mixture of diethyl ether and MeOH (20:1), and thoroughly dried under
vacuum. The residue was dissolved in deionized water, filtered through a
polytetrafluoroethylene (PTFE) syringe filter, and lyophilized to give the
TFA salt 34 as a light-brownish, foamy solid (17.1 mg, 89%).
1-O-Allyl-2,3:5,6-di-O-isopropylidene-scyllo-inositol
(37):
NaOMe
(0.32 mL, 1.38 mmol) was added to a solution of 36 (1.40 g, 3.46 mmol) in
MeOH (35 mL), and the reaction mixture was placed at reflux for 3 h.
After cooling to RT, the mixture was filtered through silica gel. The fil-
trate was concentrated, and the residue was washed with 5% EtOAc in
n-hexane, and dried under vacuum to give 37 as a colorless solid (957 mg,
92%). M.p. 200–2028C; Rf =0.35 (EtOAc/nHex 1:1); 1H NMR (CDCl3):
d=1.46 (s, 12H; 2CMe2), 2.45 (d, J=2.7 Hz, 1H; OH), 3.57–3.68 (m,
4H), 3.83 (t, J=8.1 Hz, 1H), 4.05 (t, J=7.9 Hz, 1H), 4.28 (dd, J=7.0,
1.2 Hz, 2H), 5.21 (d, J=10.2 Hz, 1H), 5.33 (dd, J=1.5, 18.1 Hz, 1H),
5.95 ppm (m, 1H); 13C NMR (CDCl3): d=27.1 (4C; 2CMe2), 69.4, 71.7,
75.0, 77.0, 80.5, 80.7, 113.5, 118.0, 134.9 ppm; MS (FAB): m/z: 301.17
[M+H]+; HRMS (FAB): m/z calcd for C15H25O6: 301.1700 found
301.1650 [M+H]+.
(+)-4’-O-Benzyl-1’-O-{[1-O-(5-dimethylamino-a-naphthalenesulfonyl)-
2,3,5,6-O-(aminobutanoylguanidinium)-myo-inositol]carbonyloxy}-
2’,3’,5’,6’-O-(aminobutanoylguanidinium)-myo-inositol·8TFA (33): Light-
brownish, glassy solid (11.2 mg, 83%); UV (H2O): lmax (e): 336.80 nm
(3460 cmÀ1 mÀ1); 1H NMR (CD3OD): d=1.71–1.78 (m, 16H), 2.20–2.48
(m, 16H), 2.87 (s, 6H), 3.15–3.27 (m, 16H), 3.92–4.34 (m, 3H), 4.92–5.22
(m, 11H), 7.21–7.29 (m, 6H; aromatic), 7.55–7.62 (m, 2H), 8.02 (d, J=
8.1 Hz, 1H), 8.18–8.22 (m, 1H), 8.65 ppm (d, J=7.9 Hz, 1H); 13C NMR
(CD3OD): d=24.8, 25.0, 25.3, 26.5, 26.6, 29.1, 29.8, 33.8, 41.4, 41.5, 44.8,
67.9, 68.05, 68.07, 69.1, 69.5, 70.7, 72.1, 75.1, 75.57, 75.60, 75.8, 115.7,
119.3, 123.5, 127.4, 128.4, 130.0, 130.1, 130.5, 132.2, 138.4, 152.4, 157.6,
161.7, 162.2, 172.2, 172.5, 172.9, 173.0 ppm; MALDI-TOF MS: m/z calcd
for C72H111N25O23SNa: 1748.8005; found: 1748.5223 [M+Na]+.
1-O-(2-Oxoethyl)-2,3:5,6-diisopropylidene-scyllo-inositol (38): A solution
of 37 (940 mg, 3.13 mmol) and NaHCO3 (395 mg, 4.7 mmol) in CH2Cl2/
MeOH (6:1, 210 mL) was treated with ozone at À788C until the blue
color persisted. Nitrogen was bubbled through the solution until the blue
color was discharged. Triphenylphosphine (928 mg, 3.54 mmol) was
added and the solution was stirred at RT for 12 h. The reaction mixture
was concentrated and purified by using column chromatography on silica
gel to give 38 as a colorless solid (814 mg, 86%). M.p. 145–1478C; Rf =
0.33 (EtOAc/nHex 2:1); 1H NMR (CDCl3): d=1.46 (s, 12H; 2CMe2),
2.45 (brs, 1H; OH), 3.60–3.79 (m, 7H), 3.87 (t, J=6.2 Hz, 1H), 9.72 ppm
(s, 1H; CHO); 13C NMR (CDCl3): d=26.1 (4C; 2CMe2), 69.6, 70.4, 72.0,
(+)-4’-O-Benzyl-1’-O-{[1-O-(5-dimethylamino-a-naphthalenesulfonyl)-
2,3,5,6-O-(aminohexanoylguanidinium)-myo-inositol]carbonyloxy}-
Chem. Eur. J. 2007, 13, 762 – 775
ꢀ 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
771

S.-K. Chung et al.
75.2, 76.8, 80.2, 89.3, 112.7 ppm; MS (FAB): m/z: 303.13 [M+H]+;
HRMS (FAB): m/z calcd for C14H23O7: 303.1530; found: 303.1444
[M+H]+.
(5 mL) at RT under N2. After stirring for 22 h, the reaction mixture was
diluted with EtOAc (35 mL) and washed with aqueous NH4Cl (1”
10 mL), saturated NaHCO3 (2”10 mL), and water (3”15 mL). The stan-
dard extraction workup gave the crude product, which was purified by
using column chromatography on silica gel (EtOAc/nHex 1:2 to 1:1) to
afford 43 as a colorless, foamy solid (109 mg, 65%). Rf =0.36 (EtOAC/
1-O-(N,N-Dibenzylaminoethyl)-2,3:5,6-diisopropylidene-scyllo-inositol
(39): Dibenzylamine (0.76 mL, 3.97 mmol) was added to a solution of 38
(800 mg, 2.65 mmol) in dry dichloroethane (DCE; 45 mL) at 08C. After
1
nHex 1:1); H NMR (CDCl3): d=1.40, 1.43 (2s, 12H each; 4CMe2), 2.72
10 min at 08C, NaBHACHTREUNG(OAc)3 (898 mg, 4.23 mmol) was added and the stir-
(t, J=6.1 Hz, 2H), 3.35–3.88 (m, 25H), 4.23 (s, 2H), 4.36 (s, 2H), 7.02 (t,
J=6.0 Hz, 1H; CONH), 7.19–7.39 ppm (m, 10H; aromatic); 13C NMR
(CDCl3): d=27.1 (4C; 2CMe2), 27.2 (4C; 2CMe2), 39.3, 52.2, 53.1, 59.0,
67.7, 69.1, 69.6, 70.2, 75.5, 76.2, 76.9, 79.8, 79.9, 80.4, 80.6, 113.5, 113.6,
127.2, 128.5, 129.2, 140.2, 170.1, 171.1 ppm; MS (FAB): m/z: 899.48
ring was continued for 12 h at RT. The mixture was subjected to the stan-
dard extraction work-up procedure using CH2Cl2 to give the crude prod-
uct, which was purified by using column chromatography on silica gel to
yield 39 as a colorless solid (972 mg, 76%). M.p. 144–1458C; Rf =0.42
(EtOAc/nHex 2:1); 1H NMR (CDCl3): d=1.44 (s, 12H; 2CMe2), 2.70
(brs, 1H; OH), 2.74 (t, J=6.0 Hz, 2H), 3.54–3.59 (m, 4H), 3.64 (s, 4H),
3.73–3.77 (m, 1H), 3.87 (t, J=6.2 Hz, 2H), 4.0 (t, J=6.0 Hz, 1H), 7.19–
7.39 ppm (m, 10H); 13C NMR (CDCl3): d=27.2 (4C; 2CMe2), 42.6, 53.2,
59.0, 62.3, 69.0, 75.8, 80.6, 113.0, 127.3, 128.5, 128.6, 129.0, 129.2,
140.3 ppm; MS (FAB): m/z: 484.26 [M+H]+; HRMS (FAB): m/z calcd
for C28H38NO6: 484.2738; found: 484.2701 [M+H]+.
[M+H]+, 921.42 [M+Na]+; HRMS (FAB): m/z calcd for C47H67N2O15
:
899.4534; found: 899.4530 [M+H]+; m/z calcd for C47H66N2O15Na:
921.4534; found: 921.4488 [M+Na]+.
4-O-{(2-N,N-Dibenzylaminoethyl)-4’-O-[(methoxycarbonylmethyl)-
scyllo-inositol]-1’-O-ethyl-1-O-methylamido}-scyllo-inositol (44): p-TSA
monohydrate (21 mg, 0.116 mmol) was added in portions to a solution of
43 (90 mg, 0.097 mmol) in CH2Cl2 and MeOH (9:1, 3.3 mL) at RT under
N2. After 10 h, the solution mixture was concentrated. The residue was
washed several times with a mixture of CH2Cl2 and MeOH (19:1) to
remove excess p-TSA, and dried to give 44 as a colorless, sticky solid
(72 mg, quant.). Rf =0.22 (CH2Cl2/MeOH 3:1); 1H NMR (CD3OD): d=
2.91–3.11 (m, 4H), 3.22–3.38 (m, 15H), 3.82 (dist. t, J=6.8 Hz, 2H), 4.05
(t, J=7.2 Hz, 2H), 4.24 (s, 2H), 4.35 (s, 2H), 4.30–4.44 (m, 4H), 7.39–
7.36 ppm (m, 10H; aromatic); 13C NMR (CD3OD): d=39.7, 52.3, 57.60,
66.0, 69.3, 71.3, 71.5, 74.1, 74.2, 74.3, 83.0, 83.4, 84.2, 84.8, 126.0, 126.3,
128.9, 129.5, 129.7, 130.2, 131.5, 140.7, 142.6, 172.9, 173.8 ppm; MS
(FAB): m/z: 739.42 [M+H], 761.42 [M+Na]+; HRMS (FAB): m/z calcd
for C35H50N2O15Na: 761.3211; found: 761.2807 [M+Na]+.
4-O-(2-Dibenzylaminoethyl)-2,3:4,5-diisopropylidene-1-O-(methoxycar-
bonylmethyl)-scyllo-inositol (40): Ag2O (1.0 g, 4.32 mmol), methyl bro-
moacetate (0.41 mL, 4.32 mmol), and TBAI (135 mg) were sequentially
added to a solution of 39 (950 mg, 1.96 mmol) in CH2Cl2 (30 mL). After
stirring at RT for 12 h, the reaction mixture was filtered through Celite
and washed with CH2Cl2 (150 mL). The filtrate was concentrated and pu-
rified by using column chromatography on silica gel to give 40 as a color-
less solid (1.02 g, 94%). M.p. 130–1328C; Rf =0.38 (EtOAc/nHex 1:1);
1H NMR (CDCl3): d=1.41 (s, 12H; 2CMe2), 2.72 (t, J=6.3 Hz, 2H), 3.56
(t, J=9.3 Hz, 2H), 3.63–3.89 (m, 13H), 4.36 (s, 2H), 7.21–7.40 ppm (m,
10H); 13C NMR (CDCl3): d=27.1 (4C; 2CMe2), 52.2, 53.2, 59.0, 67.7,
69.1, 75.6, 77.0, 79.9, 80.6, 113.5, 127.3, 128.5, 129.29, 140.3, 171.1 ppm;
MS (FAB): m/z: 556.19 [M+H]+; HRMS (FAB): m/z calcd for
C31H42NO8: 556.2922; found: 556.2913 [M+H]+; m/z calcd for
C31H41NO8Na: 578.2822; found: 578.2794 [M+Na]+.
4-O-{(2-N,N-Dibenzylaminoethyl)-4’-O-[(methoxycarboxylmethyl)-
2’,3’:5’,6’-O-(4-Boc-aminobutanoyl)-scyllo-inositol]-1’-O-ethyl-1-O-meth-
ylamido}-2,3:5,6-O-(4-Boc-aminobutanoyl)-scyllo-inositol (45): Colorless,
sticky solid (100 mg, 55%); Rf =0.29 (EtOAc/nHex 1:1); 1H NMR
(CDCl3): d=1.42 (brs, 72H), 1.68–1.70 (m, 16H), 2.10–2.25 (m, 18H),
3.11 (brs, 16H), 3.55–3.71 (m, 14H), 4.01–4.19 (m, 5H), 4.99–5.32 (m,
16H), 6.98 (brs, 1H; CONH), 7.22–7.34 ppm (m, 10H; aromatic);
13C NMR (CDCl3): d=24.7, 24.8, 26.7, 28.8, 30.1, 34.2, 34.2, 40.8, 53.2,
59.1, 60.7, 68.5, 71.2, 71.7, 71.8, 72.2, 72.4, 77.0, 78.0, 78.8, 79.3, 127.3,
128.6, 129.0, 139.8, 156.36, 156.40, 168.9, 169.7, 172.4, 172.5, 172.8 ppm;
MALDI-TOF MS: m/z calcd for C107H170N10O39Na: 2243.1660; found:
2243.1539 [M+Na]+.
4-O-(2-Aminoethyl)-1-O-(methoxycarbonylmethyl)-2,3:5,6-diisopropyli-
dene-scyllo-inositol (41): A solution of 40 (400 mg, 0.719 mmol) in a
mixed solvent (CH2Cl2/MeOH 1:9, 30 mL) was hydrogenated (40 psi) at
RT over 10% Pd/C (100 mg). After 3 h, the catalyst was filtered and the
filtrate was evaporated. The crude product was recrystallized from meth-
anol/dichloromethane to give 41 as an off-white solid (270 mg, quant.).
M.p. 193–1958C; Rf =0.22 (CH2Cl2/MeOH 9:1); 1H NMR (CD3OD): d=
1.40 (s, 12H; 2CMe2), 3.10 (t, J=5.9 Hz, 2H), 3.27 (dd, J=6.5, 1.8 Hz,
2H), 3.69–3.90 (m, 11H), 4.28 ppm (s, 2H); 13C NMR (CD3OD): d=27.1
(4C; 2CMe2), 46.2, 52.8, 58.8, 67.9, 68.8, 75.6, 76.8, 77.0, 79.7, 80.5, 82.9,
113.7, 127.4, 128.6, 129.4, 139.5, 162.1, 173.4 ppm; MS (FAB): m/z: 376.26
[M+H]+; HRMS (FAB): m/z calcd for C17H30NO8: 376.2012; found:
376.1987 [M+H]+.
4-O-{(2-N,N-Dibenzylaminoethyl)-4’-O-[(methoxycarboxylmethyl)-
2’,3’:5’,6’-O-(6-Boc-aminohexanoyl)-scyllo-inositol]-1’-O-ethyl-1-O-meth-
ylamido}-2,3:5,6-O-(6-Boc-aminohexanoyl)-scyllo-inositol (46): Colorless,
sticky solid (189 mg, 81%); Rf =0.35 (EtOAc/nHex 1:1); 1H NMR
(CDCl3): d=1.28–1.59 (m, 120H), 2.15–2.31 (m, 16H), 2.51 (brs, 2H),
3.07 (brs, 16H), 3.36 (brs, 2H), 3.62–3.81 (m, 12H), 4.04–4.21 (m, 5H),
4.84–5.39 (m, 16H), 6.98 (brs, 1H; CONH), 7.21–7.30 ppm (m, 10H; aro-
matic); 13C NMR (CDCl3): d=24.2, 26.2, 28.3, 31.3, 34.1, 53.1, 59.0, 67.1,
71.8, 71.9, 72.4, 76.1 78.4, 79.5, 127.1, 128.4, 129.3, 156.2, 159.3, 169.9,
170.7, 172.2, 173.4 ppm; MALDI-TOF MS: m/z calcd for
C123H202N10O39Na: 2467.9636; found: 2467.5435 [M+Na]+.
4-O-(2-N,N-Dibenzylaminoethyl)-1-O-(methoxycarboxyl)-2,3:5,6-diiso-
propylidene-scyllo-inositol (42): NaOH (136 mg, 3.4 mmol) was added to
a solution of 40 (540 mg, 0.971 mmol) in methanol (25 mL). After being
stirred at RT for 12 h the reaction mixture was concentrated under
vacuum and the residue was dissolved in water (25 mL). The aqueous so-
lution was washed with diethyl ether (2”10 mL) to remove the nonpolar
impurities, and then carefully acidified with 10% aqueous AcOH to
pH 6.0–6.5. The standard extraction workup with EtOAc gave 42 as col-
orless, foamy solid (485 mg, 92%). Rf =0.32 (CH2Cl2/MeOH 9:1);
1H NMR (CDCl3): d=1.41, 1.44 (2 s, 6H each; 2CMe2), 2.77 (t, J=
5.9 Hz, 2H), 3.35–3.83 (m, 10H), 3.89 (t, J=5.9 Hz, 2H), 4.37 (s, 2H),
7.20–7.40 ppm (m, 10H; aromatic); 13C NMR (CDCl3): d=25.9 (2C),
26.0 (2C; 2CMe2), 40.0, 51.3, 66.6, 67.2, 76.2, 76.4, 79.8, 113.3, 171.6 ppm;
MS (FAB): m/z: 542.30 [M+H]+, 564.25 [M+Na]+; HRMS (FAB): m/z
calcd for C30H40NO8: 542.2778; found: 542.2749 [M+H]+; m/z calcd for
C30H39NO8Na: 564.2778; found: 564.2702 [M+Na]+.
4-O-{(2-N,N-Dibenzylaminoethyl)-4’-O-[(methoxycarboxylmethyl)-
2’,3’:5’,6’-O-(8-Boc-aminooctanoyl)-scyllo-inositol]-1’-O-ethyl-1-O-meth-
ylamido}-2,3:5,6-O-(8-Boc-aminooctanoyl)-scyllo-inositol (47): Colorless,
sticky solid (186 mg, 76%); Rf =0.39 (EtOAc/nHex 1:1); 1H NMR
(CDCl3): d=1.25–1.59 (m, 152H), 2.21–2.23 (m, 16H), 2.56 (brs, 2H),
3.09–3.15 (m, 16H), 3.33 (brs, 2H), 3.50–3.67 (m, 14H), 4.05–4.20 (m,
5H), 4.71–5.11 (m, 8H), 5.16–5.41 (m, 8H), 6.95 (brs, 1H; CONH), 7.21–
7.30 ppm (m, 10H; aromatic); 13C NMR (CDCl3): d=21.3, 24.9, 24.95,
25.00, 27.0, 28.8, 29.30, 29.34, 29.37, 29.41, 30.4, 34.3, 34.4, 41.0, 52.0, 53.2,
59.1, 60.6, 71.0, 71.6, 71.8, 72.3, 78.1, 79.2, 79.4, 127.3, 128.5, 128.9, 139.7,
156.3, 156.4, 168.8, 169.7, 172.46, 172.52, 172.8 ppm; MALDI-TOF MS:
m/z calcd for C139H234N10O39Na: 2691.6668; found: 2691.6768 [M+Na]+.
4-O-[(2-N,N-dibenzylaminoethyl)-4’-O-{(methoxycarbonylmethyl)-
2’,3’:5’,6’-di-O-isopropylidene-scyllo-inositol}-1’-O-ethyl-1-O-methylami-
do]-2,3:5,6-di-O-isopropylidene-scyllo-inositol
(43):
Triethylamine
(0.04 mL, 0.276 mmol), HOBT (27.4 mg, 0.202 mmol), and EDC
(38.9 mg, 0.202 mmol) were sequentially added to the equimolar solution
of 41 (69.3 mg, 0.184 mmol) and 42 (100 mg, 0.184 mmol) in dry DMF
4-O-{(2-N,N-Dibenzylaminoethyl)-4’-O-[(methoxycarbonylmethyl)-
2’,3’:5’,6’-O-(4-aminobutanoyl hydrochloride)-scyllo-inositol]-1’-O-ethyl-
1-O-methylamido}-2,3:5,6-O-(4-aminobutanoyl hydrochloride)-scyllo-ino-
772
www.chemeurj.org
ꢀ 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2007, 13, 762 – 775

Molecular Transporters
FULL PAPER
sitol (48): Colorless solid (HCl salt, 54 mg, quant.); 1H NMR (CD3OD):
d=1.23–1.26 (m, 16H), 2.53–2.61 (m, 16H), 2.93–3.00 (m, 16H), 3.41–
3.88 (m, 14H), 4.10–4.23 (m, 5H), 5.11–5.38 (m, 8H), 7.28–7.43 ppm (m,
10H; aromatic); 13C NMR (CD3OD): d=24.4, 26.0, 27.1, 31.3, 39.7, 49.2,
57.2, 67.7, 72.2, 72.8, 78.2, 79.8, 127.2, 128.7, 129.0, 131.7, 171.3, 172.9,
173.0 ppm; MALDI-TOF MS: m/z calcd for C67H108N10O23Na: 1443.7589;
found: 1443.9495 [M+Na]+.
was hydrogenated by using a balloon (1 atm). After stirring for 22 h at
RT, the catalyst was filtered and the filtrate was evaporated to give the
crude product, which was recrystallized from a MeOH/CH2Cl2 mixture to
afford 55 as a colorless, sticky solid (88 mg, 74%).
4-O-{(2-Aminoethyl)-4’-O-[(methoxycarbonylmethyl)-2’,3’:5’,6’-O-(N,N’-
bis-Boc-N’’-aminobutanoylguanidine)-scyllo-inositol]-1’-O-ethyl-1-O-
methylamido}-2,3:5,6-O-(N,N’-bis-Boc-N’’-aminobutanoylguanidine)-
scyllo-inositol (54): Colorless, sticky solid (50 mg, 83%); Rf =0.19
(CH2Cl2/MeOH 9:1); 1H NMR (CD3OD); d=1.49–1.54 (m, 160H), 1.92
(brs, 16H), 2.46 (brs, 16H), 3.22–3.79 (m, 30H), 4.02–4.23 (m, 5H), 5.13–
5.33 ppm (m, 8H); MALDI-TOF MS: m/z: calcd for C141H240N26O55
3176.6726; found: 3176.4531 [M+H]+; calcd for C141H239N26O55Na:
3198.6626; found: 3198.8276 [M+Na]+.
4-O-{(2-N,N-Dibenzylaminoethyl)-4’-O-[(methoxycarbonylmethyl)-
2’,3’:5’,6’-O-(6-aminohexanoyl hydrochloride)-scyllo-inositol]-1’-O-ethyl-
1-O-methylamido}-2,3:5,6-O-(6-aminohexanoyl hydrochloride)-scyllo-in-
ositol (49): Colorless solid (HCl salt, 142 mg, quant.); 1H NMR
(CD3OD): d=1.32–1.68 (m, 48H), 2.21–2.45 (m, 16H), 2.93 (brs, 16H),
3.19–3.80 (m, 14H), 4.02–4.36 (m, 5H), 5.15–5.36 (m, 8H), 7.48–7.53 ppm
(m, 10H; aromatic); 13C NMR (CD3OD): d=19.9, 24.3, 25.3, 27.2, 33.8,
39.6, 48.6, 48.9, 57.3, 60.5, 71.9, 72.3, 74.1, 78.6, 79.0, 129.5, 130.2, 131.7,
131.9, 172.0, 172.4, 173.0, 173.5 ppm; MALDI-TOF MS: m/z calcd for
C83H140N10O23Na: 1668.00; found: 1667.50 [M+Na]+.
4-O-{(2-Aminoethyl)-4’-O-[(methoxycarbonylmethyl)-2’,3’:5’,6’-O-(N,N’-
bis-Boc-N’’-aminohexanoylguanidine)-scyllo-inositol]-1’-O-ethyl-1-O-
methylamido}-2,3:5,6-O-(N,N’-bis-Boc-N’’-aminohexanoylguanidine)-
scyllo-inositol (55): Colorless, sticky solid (88 mg, 74%); Rf =0.22
4-O-{(2-N,N-Dibenzylaminoethyl)-4’-O-[(methoxycarbonylmethyl)-
2’,3’:5’,6’-O-(8-aminooctanoyl hydrochloride)-scyllo-inositol]-1’-O-ethyl-
1-O-methylamido}-2,3:5,6-O-(8-aminooctanoyl hydrochloride)-scyllo-ino-
sitol (50): Colorless solid (HCl salt, 143 mg, 99%); 1H NMR (CD3OD):
d=1.27–1.64 (m, 80H), 2.37 (brs, 16H), 2.93–2.96 (m, 16H), 3.41–3.88
(m, 14H), 4.11 (brs, 5H), 4.22 (s, 2H), 5.27–5.48 (m, 8H), 7.54–7.63 ppm
(m, 10H; aromatic); 13C NMR (CD3OD): d=24.7, 24.9, 26.4, 26.5, 27.49,
27.51, 28.95, 29.03, 29.1, 34.1, 39.8, 39.9, 48.7, 51.7, 57.3, 60.6, 71.8, 72.1,
72.4, 78.8, 129.6, 130.3, 131.8, 170.5, 171.9, 172.9, 173.0, 173.1, 173.3 ppm;
MALDI-TOF MS: m/z calcd for C99H170N10O23Na: 1891.2472; found:
1891.2677 [M+Na]+.
1
(CH2Cl2/MeOH 9:1); H NMR (CD3OD): d=1.30–1.61 (m, 192H), 2.35–
2.37 (m, 16H), 3.28–3.33 (m, 32H), 4.01–4.29 (m, 5H), 5.24–5.38 ppm (m,
8H); MALDI-TOF MS: m/z calcd for C154H270N26O55Na: 3424.9649;
found: 3424.2172 [M+Na]+.
4-O-{(2-Aminoethyl)-4’-O-[(methoxycarbonylmethyl)-2’,3’:5’,6’-O-(N,N’-
bis-Boc-N’’-aminooctanoylguanidine)-scyllo-inositol]-1’-O-ethyl-1-O-
methylamido}-2,3:5,6-O-(N,N’-bis-Boc-N’’-aminooctanoylguanidine)-
scyllo-inositol (56): Colorless, sticky solid (78 mg, 89%); Rf =0.23
(CH2Cl2/MeOH 9:1); 1H NMR (CD3OD): d=1.36–1.57 (m, 224H), 2.35
(brs, 16H), 2.81 (brs, 2H), 3.05 (dist. t, 2H), 3.33–3.43 (m, 26H), 3.89–
4.20 (m, 8H), 5.24–5.35 ppm (m, 8H); MALDI-TOF MS: m/z calcd for
4-O-{(2-N,N-Dibenzylaminoethyl)-4’-O-[(methoxycarbonylmethyl)-
2’,3’:5’,6’-O-(N,N’-bis-Boc-N’’-aminobutanoylguanidine)-scyllo-inositol]-
1’-O-ethyl-1-O-methylamido}-2,3:5,6-O-(N,N’-bis-Boc-N’’-aminobutanoyl-
guanidine)-scyllo-inositol (51): Colorless, foamy solid (68 mg, 51%); Rf =
0.38 (EtOAc/nHex 1:1); 1H NMR (CDCl3): d=1.48 (brs, 144H), 1.74–
1.85 (m, 16H), 2.33–2.36 (m, 16H), 3.36–3.68 (m, 30H), 4.08–4.15 (m,
5H), 4.93–5.22 (m, 8H), 7.26–7.37 (m, 10H; aromatic), 8.34 (brs, 8H),
11.48 ppm (brs, 8H); 13C NMR (CDCl3): d=24.6, 24.8, 28.2, 28.36, 28.44,
28.7, 30.1, 31.8, 36.9, 40.3, 77.8, 83.6, 83.8, 86.2, 86.4, 117.5, 121.8, 128.8,
149.3, 151.8, 152.4, 153.6, 156.6, 163.0, 163.9 ppm; MALDI-TOF MS: m/z
calcd for C155H250N26O55Na: 3378.7565; found: 3376.4721 [M+NaÀ2]+.
4-O-{(2-N,N-Dibenzylaminoethyl)-4’-O-[(methoxycarbonylmethyl)-
2’,3’:5’,6’-O-(N,N’-bis-Boc-N’’-aminohexanoylguanidine)-scyllo-inositol]-
1’-O-ethyl-1-O-methylamido}-2,3:5,6-O-(N,N’-bis-Boc-N’’-aminohexanoyl-
guanidine)-scyllo-inositol (52): Off-white, foamy solid (129 mg, 67%);
Rf =0.44 (EtOAc/nHex 1:1); 1H NMR (CDCl3): d=1.25–1.53 (m, 192H),
2.24–2.30 (m, 16H), 2.34 (app. t, J=6.7 Hz, 2H), 3.36–3.50 (m, 18H),
3.64–3.89 (m, 15H), 4.10 (brs, 2H), 4.22 (brs, 2H), 5.14–5.43 (m, 8H),
6.90 (brs, 1H), 7.27–7.34 (m, 10H; aromatic), 8.29 (brs, 8H), 11.58 ppm
(brs, 8H); 13C NMR (CDCl3): d=24.7, 24.8, 24.9, 26.79, 26.83, 28.2, 28.5,
28.7, 29.2, 34.2, 34.4, 41.1, 71.7, 72.2, 77.9, 79.5, 83.3, 83.4, 127.4, 128.7,
129.0, 139.7, 153.7, 156.5, 164.0, 172.35, 172.42, 172.6, 172.7 ppm;
MALDI-TOF MS: m/z calcd for C171H282N26O55Na: 3604.0103; found:
3603.1566 [M+Na]+.
C
173H302N26O55Na: 3625.1668; found: 3625.2019 [M+Na]+.
A representative procedure for the FITC-I attachment: Fluorescein-5-
isothiocyanate (1.2 mg, 0.03 mmol) and TEA (10 mL, 0.07 mmol) were
added to a solution of 55 (80 mg, 0.023 mmol) in a mixed solvent of THF
and absolute ethanol (3:2, 4 mL). The reaction mixture was stirred for
24 h at RT in the dark, and then concentrated. The crude product was pu-
rified by means of column chromatography by using a long, thin column
of flash silica gel to afford 58 as a light-greenish-yellow, sticky mass
(56 mg, 63%).
4-O-{[2-(N-Fluoresceinyl-5-thioureido)-ethyl]-4’-O-[(methoxycarbonyl-
methyl)-2’,3’:5’,6’-O-(N,N’-bis-Boc-N’’-aminobutanoylguanidine)-scyllo-in-
ositol]-1’-O-ethyl-1-O-methylamido}-2,3:5,6-O-(N,N’-bis-Boc-N’’-amino-
butanoylguanidine)-scyllo-inositol (57): Light-greenish-yellow, sticky
solid (26 mg, 46%); Rf =0.32 (CH2Cl2/MeOH 9:2); 1H NMR (CDCl3):
d=1.48–1.60 (m, 160H), 2.31–2.34 (m, 18H), 3.40–4.20 (m, 35H), 5.24–
5.39 (m, 8H), 6.62–6.80 (m, 6H), 7.23–7.33 (m, 2H), 8.35 (brs, 9H),
11.47 ppm
162H249N27O60SNa: 3588.7017; found: 3587.6681 [M+Na]+.
4-O-{[2-(N-Fluoresceinyl-5-thioureido)ethyl]-4’-O-[(methoxycarbonyl-
(brs,
8H);
MALDI-TOF
MS:
m/z
calcd
for
C
methyl)-2’,3’:5’,6’-O-(N,N’-bis-Boc-N’’-aminohexanoylguanidine)-scyllo-
inositol]-1’-O-ethyl-1-O-methylamido}-2,3:5,6-O-(N,N’-bis-Boc-N’’-ami-
nohexanoylguanidine)-scyllo-inositol (58): Light-greenish, sticky solid
(56 mg, 63%); Rf =0.39 (CH2Cl2/MeOH 9:2); 1H NMR (CDCl3): d=
1.25–1.55 (m, 192H), 2.27 (brs, 16H), 3.38–3.40 (m, 16H), 3.49–3.69 (m,
14H), 4.01–4.21 (m, 5H), 5.11–5.30 (m, 8H), 6.68–6.89 (m, 5H), 7.29
(brs, 2H), 8.30–8.39 (m, 9H), 11.56 ppm (brs, 8H); MALDI-TOF MS:
m/z: calcd for C178H282N27O60S 3790.9621; found: 3790.6613 [M+H]+;
calcd for C178H281N27O60SNa: 3812.9521; found: 3812.3281.
4-O-{(2-N,N-Dibenzylaminoethyl)-4’-O-[(methoxycarbonylmethyl)-
2’,3’:5’,6’-O-(N,N’-bis-Boc-N’’-aminooctanoylguanidine)-scyllo-inositol]-
1’-O-ethyl-1-O-methylamido}-2,3:5,6-O-(N,N’-bis-Boc-N’’-aminooctanoyl-
guanidine)-scyllo-inositol (53): Colorless, foamy solid (125 mg, 72%);
Rf =0.42 (EtOAc/nHex 1:1); 1H NMR (CDCl3): d=1.24–1.53 (m, 224H),
2.22–2.30 (m, 18H), 2.34 (app. t, J=7.3 Hz, 2H), 3.00 (m, 2H), 3.36–3.89
(m, 32H), 5.11–5.43 (m, 8H), 7.27–7.29 (m, 10H; aromatic), 8.29 (brs,
8H), 11.50 ppm (brs, 8H); 13C NMR (CDCl3): d=21.3, 24.95, 25.02, 25.1,
27.1, 28.2, 28.5, 28.7, 28.8, 29.4, 30.0, 34.3, 34.5, 41.3, 52.1, 53.2, 59.1, 60.7,
71.2, 71.8, 71.9, 72.3, 79.4, 83.3, 126.3, 127.3, 128.6, 129.0, 139.7, 153.7,
156.5, 164.0, 168.8, 169.6, 171.3, 172.5, 172.7 ppm; MALDI-TOF MS: m/
z: calcd for C187H315N26O55: 3807.6353; found: 3807.2341 [M+H]+; calcd
for C187H314N26O55Na: 3829.6353; found: 3829.7262 [M+Na]+.
4-O-{[2-(N-Fluoresceinyl-5-thioureido)ethyl]-4’-O-[(methoxycarbonyl-
methyl)-2’,3’:5’,6’-O-(N,N’-bis-Boc-N’’-aminooctanoylguanidine)-scyllo-in-
ositol]-1’-O-ethyl-1-O-methylamido}-2,3:5,6-O-(N,N’-bis-Boc-N’’-amino-
octanoylguanidine)-scyllo-inositol (59): Light-yellowish, sticky solid
(35 mg, 58%); Rf =0.38 (CH2Cl2/MeOH 9:2); 1H NMR (CDCl3): d=
1.26–1.53 (m, 224H), 2.24 (brs, 18H), 3.67 (m, 16H), 3.88–4.21 (m, 22H),
5.54–5.71 (m, 8H), 6.62–6.80 (m, 6H), 8.33 (brs, 9H), 11.44 ppm (brs,
8H); MALDI-TOF MS: m/z: calcd for C194H314N27O60SNa: 4014.2002;
found: 4013.0459 [M+H]+; calcd for C194H313N27O60SNa: 4036.1992;
found: 4035.8321 [M+Na]+.
A representative procedure for debenzylation of the terminal dibenzyla-
mine moiety: A mixture of 52 (125 mg, 0.035 mmol) and 10% Pd/C
(65 mg, 10 mol%) in a mixed solvent of CH2Cl2 and MeOH (4 mL, 1:9)
Chem. Eur. J. 2007, 13, 762 – 775
ꢀ 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
773

S.-K. Chung et al.
A representative procedure for the N-Boc deprotection from the guani-
dine moiety: EtOAc (4 mL) saturated with gaseous HCl was added to a
solution of 58 (30 mg, 0.008 mmol) in EtOAc (1 mL) at RT. After being
stirred for 20 h, the solution was concentrated and the residue was
washed with a mixture of diethyl ether and MeOH (20:1) to remove less
polar impurities. The residue was dried and purified by using MPLC on
reverse-phase C-8 silica gel (H2O/CH3CN 1:1 to 1:2 with 0.1% TFA).
The purified product was dissolved in deionized water, filtered through a
PTGE syringe filter, and lyophilized to give 61 as a light-greenish-yellow,
foamy solid (HCl salt, 12.6 mg, 73%).
for 3 h, and concentrated under high vacuum. To the residue dissolved in
dry DMF (2 mL) and TEA (3.5 mL, 0.023 mmol), doxorubicin-hydrochlo-
ride (3.1 mg, 0.0056 mmol) was added, and the resulting solution was stir-
red at RT under dark for 24 h. The reaction mixture was concentrated
and purified by using column chromatography on silica gel (CH2Cl2/
MeOH 10:1 to 5:1) to afford 64 as a reddish, sticky solid (23.6 mg, 77%).
Rf =0.33 (CH2Cl2/MeOH 10:1); 1H NMR (CDCl3): d=1.25–1.68 (m,
224H), 2.26 (brs, 20H), 3.37–3.73 (m, 26H), 4.76–4.86 (m, 4H), 4.90–5.45
(m, 12H), 7.24–7.26 (m, 5H), 7.60–7.68 (m, 1H), 7.72–7.81 (m, 1H),
7.88–7.94 (brs, 1H), 8.28 (brs, 8H), 11.48 ppm (brs, 8H); 13C NMR
(CDCl3): d=24.3, 25.1, 25.3, 27.5, 28.2, 28.8, 29.1, 30.3, 31.9, 33.8, 41.0,
43.6, 49.0, 65.4, 71.2, 76.9, 80.3, 83.9, 123.2, 125.3, 127.8, 128.1, 128.7,
153.5, 155.3, 165.6, 171.9, 172.4, 172.5, 182.3 ppm.
4-O-{[2-(N-Fluoresceinyl-5-thioureido)ethyl]-4’-O-[(methoxycarbonyl-
methyl)-2’,3’:5’,6’-O-(aminobutanoylguanidinium)-scyllo-inositol]-1’-O-
ethyl-1-O-methylamido}-2,3:5,6-O-(aminobutanoylguanidinium)-scyllo-
inositol·8HCl (60): Light-yellowish, foamy solid (HCl salt, 9.6 mg, 76%);
UV (H2O): lmax (e)=495 nm (15700 cmÀ1 mÀ1); 1H NMR (CD3OD): d=
1.86 (brs, 16H), 2.14–2.16 (m, 2H), 2.44 (brs, 16H), 3.22–3.31 (m, 23H),
3.68–4.28 (m, 10H), 5.20–5.34 (m, 8H), 6.65–7.01 (m, 6H), 7.19–7.22 (m,
2H), 8.30 ppm (brs, 1H); MALDI-TOF MS: m/z calcd for
C82H121N27O28SNa: 1989.08; found: 1989.03 [M+Na]+; analytical HPLC
(BU-300): tR =3.79 min (flow rate: 1 mLminÀ1; UV: l=215 nm; CH3CN/
H2O 40:60), purity 95+ %.
HCl salt of the doxorubicin conjugate 65: Light-reddish, glassy solid
(11.6 mg, 76%); UV (H2O): lmax (e): 490 nm (10000 cmÀ1 mÀ1); 1H NMR
(CD3OD): d=1.30–1.60 (m, 80H), 2.38 (brs, 16H), 3.19 (brs, 16H), 3.21–
342 (m, 14H), 3.53–3.72 (m, 18H), 3.78 (brs, 5H), 3.89–4.21 (m, 10H),
5.18–5.34 (m, 8H), 7.31–7.59 ppm (m, 3H); MALDI-TOF MS: m/z: calcd
for C132H213N28O39 2814.56; found: 2815.93 [M+H]+; calcd for
C132H212N28O39Na: 2836.55; found: 2835.67 [M+Na]+; analytical HPLC
(BU-300): tR =3.77 min (flow rate: 1 mLminÀ1; UV: l=215 nm; CH3CN/
H2O 40:60), purity 95+ %.
4-O-{[2-(N-Fluoresceinyl-5-thioureido)ethyl]-4’-O-[(methoxycarbonyl-
methyl)-2’,3’:5’,6’-O-(aminohexanoylguanidinium)-scyllo-inositol]-1’-O-
ethyl-1-O-methylamido}-2,3:5,6-O-(aminohexanoylguanidinium)-scyllo-
inositol·8HCl (61): Light-yellowish, foamy solid (HCl salt, 12.6 mg,
73%); UV (H2O): lmax (e)=490 nm (19400 cmÀ1 mÀ1); 1H NMR
(CD3OD): d=1.39–1.61 (m, 48H), 2.35–2.45 (m, 18H), 3.16–3.31 (m,
24H), 3.67–4.28 (m, 11H), 5.28–5.44 (m, 8H), 6.65–6.93 (m, 6H), 7.20–24
(m., 2H), 8.26 ppm (brs, 1H); 13C NMR (CD3OD): d=24.6, 24.9, 26.3,
27.3, 28.7, 34.1, 41.4, 61.3, 67.9, 70.2, 72.3, 126.3, 127.9, 128.1, 130.7, 157.6,
162.1, 171.6, 172.0, 173.1, 175.8 ppm; MALDI-TOF MS: m/z: calcd for
C98H154N27O28S: 2190.50; found: 2190.39 [M+H]+; calcd for
C98H153N27O28SNa: 2212.49; found: 2212.53 [M+Na]+; analytical HPLC
(BU-300): tR =3.69 min (flow rate:1 mLminÀ1; UV: l=215 nm; CH3CN/
H2O 40:60), purity 99+ %.
Cellular uptake studies
Peptide synthesis and fluorescence labeling: Dansyl-Arg9 and Fl-Arg9
were custom synthesized by PEPTRON, Inc. Daejeon, South Korea, and
satisfactorily characterized by means of HPLC and mass spectrometric
analyses.
Cell culture: COS-7 cells were cultured in Dulbeccoꢁs modified Eagleꢁs
medium (DMEM) and 10% (v/v) fetal bovine serum without antibiotics.
The subculture was conducted every 3–4 d using the cells grown to sub-
confluence. RAW264.7 cells were cultivated according to a literature pro-
cedure.[17] HeLa cells derived from human cervical cancer cells were cul-
tured as exponentially growing subconfluent monolayers on 60 mm
dishes in alpha-minimum essential medium (a-MEM) supplemented with
10% (v/v) calf serum. A subculture was performed every 3–4 d.
d,l-4-O-{[2-(N-Fluoresceinyl-5-thioureido)ethyl]-4’-O-[(methoxycar-
bonyl-methyl)-2’,3’:5’,6’-O-(aminooctanoylguanidinium)-scyllo-inositol]-
1’-O-ethyl-1-O-methylamido}-2,3:5,6-O-(aminooctanoylguanidinium)-
scyllo-inositol·8HCl (62): Light-yellowish, foamy solid (HCl salt, 13.2 mg,
80%); UV (H2O): lmax (e)=488 nm (16600 cmÀ1 mÀ1); 1H NMR
(CD3OD): d=1.30–1.61 (m, 96H), 2.30–2.43 (m, 16H), 2.86 (brs, 2H),
3.20–3.39 (m, 14H), 3.67–4.18 (m, 10H), 5.20–5.34 (m, 8H), 6.65–6.79 (m,
6H), 7.11 (brs, 2H), 8.30 ppm (brs, 1H); 13C NMR (CD3OD): d=25.1,
25.4, 26.2, 27.0, 27.9, 28.7, 29.4, 34.8, 42.2, 44.6, 46.2, 62.1, 68.5, 69.0, 70.7,
72.4, 121.3, 121.9, 126.3, 126.6, 127.2, 128.3, 130.4, 157.6, 162.1, 162.6,
171.0, 172.1, 172.5, 173.2 ppm; MALDI-TOF MS: m/z calcd for
C114H186N27O28S: 2414.92; found: 2414.72 [M+H]+; analytical HPLC
(BU-300): tR =3.57 min (flow rate: 1 mLminÀ1; UV: l=215 nm; CH3CN/
H2O 40:60), purity 95+ %.
Confocal laser scanning microscopy (CLSM): Each kind of cells was
placed into 35 mm glass-bottomed dishes and cultured for 48 h. After re-
moving the medium, the COS-7 cells were washed with PBS (”1). The
cells were incubated for 3 min at RT in the presence of dansyl-R9 and
compounds 33, 34, and 35 at 10 mm concentration in DMEM. After incu-
bation, the cells were washed with PBS (”1), and CLSM was performed
by using a Carl Zeiss LSM 510 Meta Confocal Microscope with a 40”
objective lens. FITC fluorescence was excited with the l=458 nm line of
an argon laser (Figure 1A). After removing the medium, the RAW 264.7
cells were washed with PBS (”1). The cells were incubated for 3 min at
RT in the presence of dansyl-R9 and compounds 34 and 72 at a concen-
tration of 10 mm in DMEM. After incubation, the cells were washed with
PBS (”1), and then ethanol. CLSM was performed in the same manner
as described above (Figure 1B). After removing the medium, the HeLa
cells were washed with PBS (”2). The cells were incubated for 15 min at
378C in the presence of R9-Fl and compounds 60, 61, and 62 at a concen-
tration of 10 mm in DMEM. After incubation, the cells were washed with
cold PBS (”5), and CLSM was performed by using a Carl Zeiss LSM 510
Meta Confocal Microscope with a 40” objective lens. FITC fluorescence
was excited with the l=488 nm line of an argon laser (Figure 1C).
4-O-{[2-(N-Cbz-l-serineamido)ethyl]-4’-O-[(methoxycarbonylmethyl)-
2’,3’:5’,6’-O-(N,N’-bis-Boc-N’’-aminooctanoylguanidine)-scyllo-inositol]-
1’-O-ethyl-1-O-methylamido}-2,3:5,6-O-(N,N’-bis-Boc-N’’-aminooctanoyl-
guanidine)-scyllo-inositol (63): EDC (3.2 mg, 0.0168 mmol) and HOBT
(2.3 mg, 0.0168 mmol) were added to
a solution of 56 (51 mg,
0.014 mmol) and N-Cbz-protected l-serine (4 mg, 0.0168 mmol) in dry
DMF (2 mL) at RT under N2. After stirring for 22 h, the reaction mixture
was subjected to the standard extraction workup using EtOAc to give the
crude product, which was purified by using column chromatography on
silica gel (CH2Cl2/MeOH 10:1 to 6:1) to afford 63 as a colorless, sticky
solid (39 mg, 72%). Rf =0.38 (CH2Cl2/MeOH 10:1); 1H NMR (CDCl3):
d=1.32–1.49 (m, 224H), 2.26 (brs, 16H), 2.81 (brs, 2H), 3.39 (brs, 16H),
3.69–3.89 (m, 20H), 5.13–5.16 (m, 10H), 7.28–7.37 (m, 5H), 8.29 (brs,
8H), 11.5 ppm (brs, 8H); 13C NMR (CDCl3): d=23.1, 25.2, 27.2, 28.5,
28.7, 28.9, 29.4, 30.1, 32.3, 34.4, 41.3, 72.1, 77.0, 79.6, 83.4, 128.5, 128.7,
129.0, 153.7, 156.5, 164.0, 172.1, 172.7, 173.1 ppm.
Protocols for the organ biodistribution study were previously de-
scribed.[33]
Acknowledgements
We thank Dr. Hyun Soo Kim for technical assistance with cell cultures,
MTT tests, and operation of the confocal laser scanning microscope at
Postech. Financial support of this work by grants from Postech, Posco
(Postech Biotech Center), BK-21 and MOST (National Frontier Re-
search Program administered through KRICT/CBM) is gratefully ac-
knowledged. We also highly value the productive collaborations with
Doxorubicin conjugate 64: Pyridine (18.2 mL, 0.0224 mmol) was added to
a solution of 63 (28 mg, 0.0045 mmol) and p-nitrochloroformate (2.7 mg,
0.0131 mmol) in CH2Cl2 (2.5 mL) at 08C. The solution was stirred at RT
774
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Chem. Eur. J. 2007, 13, 762 – 775

Molecular Transporters
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Received: June 23, 2006
Published online: November 6, 2006
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