Synthesis and preliminary biological studies of hemifluorinated bifunctional bolaamphiphiles designed for gene delivery

Article abstract of DOI:10.1039/b513944a

The multistep synthesis of a new series of dissymmetric hemifluorocarbon bolaamphiphiles designed for gene transport is described. The dissymmetric functionalization of diiodoperfluoroctane leads to bolaamphiphile molecules composed of a partially fluorocarbon core end-capped with a glycoside and an ammonium salt derived from histidine or lysine. Initial biological results indicate that one of the bolaamphiphile - end-capped with a lysine and a lactobionamide residue - induces a remarkably low cytotoxicity on COS-7 cells and, when self-assembled with DNA plasmid, generates a significant in vitro transfection efficiency without the addition of any fusogenic lipid. the Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2006.

Full text of DOI:10.1039/b513944a

View Article Online / Journal Homepage / Table of Contents for this issue
PAPER
www.rsc.org/njc | New Journal of Chemistry
Synthesis and preliminary biological studies of hemifluorinated
bifunctional bolaamphiphiles designed for gene delivery
a
a
b
b
´
´
Severine Denoyelle, Ange Polidori,* Melanie Brunelle, Pascal Y. Vuillaume,
Sylvette Laurent,b Yousef ElAzharyb and Bernard Pucci*a
Received (in Montpellier, France) 3rd October 2005, Accepted 13th January 2006
First published as an Advance Article on the web 9th February 2006
DOI: 10.1039/b513944a
The multistep synthesis of a new series of dissymmetric hemifluorocarbon bolaamphiphiles
designed for gene transport is described. The dissymmetric functionalization of
diiodoperfluoroctane leads to bolaamphiphile molecules composed of a partially fluorocarbon
core end-capped with a glycoside and an ammonium salt derived from histidine or lysine. Initial
biological results indicate that one of the bolaamphiphile—end-capped with a lysine and a
lactobionamide residue—induces a remarkably low cytotoxicity on COS-7 cells and, when self-
assembled with DNA plasmid, generates a significant in vitro transfection efficiency without the
addition of any fusogenic lipid.
Introduction
Applying drug controlled release technology to DNA delivery
has become one of the major challenges of biomedical re-
search.1 For the purposes of gene therapy and DNA vaccines,
oligonucleotides or DNA plasmids must be targeted at the
nucleus of the cells where gene expression can take place. On
the other hand, transfection efficiency depends, among other
things, on the intake by the cells of the DNA–vector complex,
Fig. 1 Schematic representations of dissymmetric bolaamphiphiles.
the stability of the complex in the cytosol and its unpackaging.
So far, viral vectors are the most efficient carriers in terms of
transfection efficiency but their potential immunogenicity and
oncogenicity seriously restrict their use for in vivo experi-
ments.1,2 On the other hand, synthetic delivery systems are
safe and versatile when appropriately designed but remain
obviously much less sophisticated and efficient than viruses. In
this context, the electrostatically self-assembled complexes
based on DNA and (poly)cationic lipids or synthetic surfac-
tants have emerged as a credible alternative because of their
capacity to transfect cells. However, complexes based on these
types of cationic species are prone to serious limitations such
as the low stability of the complexes in the serum and the
cytotoxicity which both affect the level of gene delivery.3 High
cationic content4,5 is the cause of the hepatic toxicity.6 Other
amphiphilic cationic species7,8 such as gemini surfactants and
bolaamphiphiles deserve particular attention for gene delivery
because such species may also display a high level of function-
ality. These amphiphilic compounds are also capable of sig-
nificant gene transfection especially when co-formulated with
the presence of a helper fusogenic lipid that generally favours
the fusion of the cationic amphiphiles with the endosomal
membranes via a phase transition sensitive to the pH varia-
tions.9
Herein, we report the rational design, synthesis and the
characterization of a new series of dissymmetric bolaamphi-
philes.10 The five novel bolaamphiphiles whose two different
polar head groups are connected together via an apolar
fluorinated or hydrocarbon segment are schematically de-
picted in Fig. 1.
In the following, we will also describe preliminary biological
investigations. The biological data will show that bolaamphi-
philes with an appropriate design can generate significant in
vitro transfection efficiency without inclusion of helper lipid
low cytotoxicity.
Results and discussion
Design and synthesis
In the present case, the designed bolaamphiphiles possess one
or two apolar fragments connected with two polar heads. One
of the two polar heads is a cationic ammonium head group
derived from either lysine or histidine. This part of the
molecule should be involved in the formation of bolaamphi-
phile–DNA complexes (bolaplexes) due to interactions with
the phosphate groups of nucleic acids.
a
`
´
Laboratoire de chimie biorganique et des systemes moleculaires
vectoriels, Faculte´ des sciences d’Avignon, 33 rue Louis Pasteur, F-
84000 Avignon, France. E-mail: bernard.pucci@univ-avignon.fr;
ange.polidori@univ-avignon.fr.; Fax: þ33 490144449;
Tel: þ33 490144442
b
´
´
´
´
´ ´
Universite de Montreal, Faculte de medecine veterinaire, Institut de
´ ´
biotechnologie veterinaire et alimentaire, 3200 rue Sicotte, Saint-
´
Hyacinthe, Quebec, Canada J2S 2M2. Tel: þ1 450 773 8521
ꢀc
This journal is the Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2006
New J. Chem., 2006, 30, 629–646 | 629

View Article Online
Fig. 2 Chemical structure of hybrid dissymmetric bolaamphiphiles A–E.
The other polar head group is neutral and derived from
galactose or lactobionamide and is expected to provide the
required hydrosolubility to the bolaplexes and avoid the
formation of bulky polyaggregates.11,12 By preventing the
presence of electric charges at the outer surface of the bola-
plexes, global electric neutrality may also reduce cytotoxicity.
The apolar segment links together end to end a hydrocarbon
and a fluorocarbon segment and forms the hydrophobic core
of the molecule. A second hydro or perfluorocarbon chain is
grafted onto the main chain through a lysine or a histidine
residue. The position of the perfluorocarbon segment within
the main chain has been adjusted in order to provide a series of
four different bolaamphiphiles (Fig. 2). The perfluorooctyl
segments are located close to either the cationic part (A and
B) or the sugar polar head (C and D) whereas the hydro or
perfluorinated nature of the lateral chain is always the same as
the facing hydrophobic segment of the main chain.
great extent with hydrocarbon chains,13 a partial or full
segregation of hydrocarbon and fluorocarbon surfactants in
aqueous solution is expected, thus forming two kinds of
micelles, one rich in hydrocarbon, the other in fluorocarbon
surfactants.15 In the designed hemifluorocarbon bolaamphi-
philes, the presence of this central fluorocarbon segment
We also synthesized a fully hydrocarbon bolaamphiphile
(E) (see Fig. 2) in order to evaluate the impact of the
fluorinated segment on DNA complexation by hemifluori-
nated bolaamphiphiles and transfection.
It is well known that fluorocarbon chains self-organize in
water due to their high hydrophobicity13 and usually lead to
stable and highly tidy supramolecular systems. For the for-
mation of the bolaplexes, the impact on their organization and
stability should be notable. The stability enhancement of the
packaging brought about by hydrophobic interactions be-
tween the fluorocarbon chains may avoid the use of poly-
cationic polar heads (derived for instance from spermine) that
generate irreversible complexation of the nucleic acid14 and
increase cytotoxicity of the carrier. The hydrophobic core may
also favour the stacking of the molecules by increasing the
cationic charge density around nucleic acids through hydro-
phobic interactions. Moreover, considering the strong ten-
dency of the perfluorocarbon chains not to interact to a
Scheme 1 Synthesis of hemifluorinated central segment.
ꢀc
630 | New J. Chem., 2006, 30, 629–646 This journal is the Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2006

View Article Online
should avoid the hydrophobic chain refolding. The hydrocar-
bon and fluorocarbon chain demixing should induce the
formation of a monolayer exhibiting both polycationic and
non-ionic polar surfaces. This possible dissymmetric organiza-
tion should lead to one level of DNA complexation, restricting
the size of the DNA–bolaamphiphile complex and favouring
the global neutrality of its outer surface.
undecenoic acid was grafted onto the perfluorocarbon chain
by using zinc dust as radical initiator. This monofunctionali-
zation provided the compound 1 in 70% yield. The chemical
conditions used did not lead to the compound resulting from a
double condensation. Thus, radical addition of Z-allylamine
or Boc-allylamine on compound 1 was carried out in good
conditions in the presence of sodium dithionite.17 It has to be
underlined that Huang’s method allowed isolation of com-
pounds 2a–b in 65–80% yield, generally higher than those
obtained by using Commeyras’ procedure (40–60% yield). The
diadduct so obtained was treated with tributyltin hydride in
According to this chemical design, the central hydrofluor-
ocarbon segment was synthesized from 1,8-diiodoperfluo-
rooctane as starting material (Scheme 1). Following the
methodology developed by Commeyras,16 one molecule of
Scheme 2 Synthetic pathway to bolaamphiphiles A and B.
ꢀc
This journal is the Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2006
New J. Chem., 2006, 30, 629–646 | 631

View Article Online
Scheme 3 Synthetic pathway to bolaamphiphiles C and D.
the presence of AIBN to remove the iodine atoms and to give
compounds 3a–b in 60% yield.
treatment with TFA in methylene chloride led to the depro-
tected amine that was then grafted, in the presence of TBTU,
onto the carboxylic function of histidine or (Ne-Z)-lysine
derivatives 5a–b to give compounds 8a–b. Compounds 5a–b
were obtained by grafting the pentafluorophenyl ester 4 of the
2H,2H,3H,3H-perfluoroundecanoic acid onto (Ne-Z)-lysine or
2-amino-3-(N-tritylimidazolyl)propionic acid in an acetone–
water mixture. After purification of the compounds 8a–b by
successive size exclusion chromatography and silica gel col-
umn chromatography, the sugar moieties were deacetylated by
Amphiphiles A and B were derived from compound 3a as
new starting material (Scheme 2) following a convergent
synthetic pathway. In a first sequence, galactosylation of
Z-ethanolamine following the Helferich procedure provided
compound 6.18 After hydrogenolysis of the benzyloxycarbonyl
group, the condensation of this glycosidic amine onto the
hemifluorinated compound 3a was performed in the presence
of BOP as coupling reagent to provide compound 7. Thus,
ꢀc
632 | New J. Chem., 2006, 30, 629–646 This journal is the Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2006

View Article Online
Aggregation behavior of bolaamphiphiles
When directly sonicated in water at a concentration of B4 ꢁ
10ꢂ3 M, bolaamphiphiles A–E formed turbid suspensions. The
poor stability of the suspensions prepared from bolaamphi-
philes A and B [rapid sedimentation at room temperature
(B15 min), detected by the naked eye] probably results from
their greater fluorine content. In contrast, when compared to
the other (A–D) bolaforms, bolaamphiphile E showed a better
dispersibility and stability. TEM observation of bolaform E
revealed the presence of vesicles (B85 nm) and fiber-like
aggregates (Fig. 3c and 3d), such two-dimensional growth of
the aggregates testifying to a rather good solubility. Never-
theless, it was not possible to rely on quasi-elastic light
scattering measurement (QELS) data given the non spherical
aspect of the objects. Sedimentation was also noticed for
bolaamphiphile D but to a lesser extent compared with
bolaamphiphiles A and B. Finally, bolaamphiphile C appeared
much more stable than the other fluorinated compounds when
dispersed in aqueous conditions. Its greater stability was
evidenced by storage in a cold (4 1C) place for at least 12 h.
As expected, these observations suggest that both the presence
of lysine (much more hydrophilic than histidine) and lacto-
bionamide residues confer a greater hydrophilic character on
the bolaamphiphile C and, therefore, a greater stability on the
suspensions than the other bolaamphiphiles.
In more dilute conditions (B10ꢂ6 M, concentration at
which complexation is carried out), QELS performed on
suspensions made from bolaamphiphile C indicated the ab-
sence of significant evolution of the particle size over a period
of 1 h. The suspension was composed of a large proportion of
particles with small size [238 nm (intensity), 95%; 115 nm
(number), 100%] and also of a small fraction of large size
[B5.4 mm (intensity), 5%] that indicated the occurrence of
some aggregation. The bolaamphiphile C was examined by
transmission electron microscopy (TEM) in the same (dilute)
conditions. The observations revealed the presence of vesicles
whose sizes were in the range 100–160 nm (Fig. 3a) Bola-
amphiphile C also presented a better capacity for complexing
DNA than the other bolaamphiphiles (A, B, D). For all the
reasons mentioned above, bolaamphiphile C was used to
demonstrate the transfection capacities of asymmetric fluori-
nated bolaamphiphiles without the addition of a helper lipid.
A detailed comparison of the physico-chemical and biological
properties of the bolaamphiphiles A–D is out of the scope of
this paper and will be detailed elsewhere.19
Fig. 3 Representative TEM images showing the morphology of (a)
bolaamphiphile C (ꢁ150 K), (b) bolaplex C at N/P = 5 (ꢁ300 K), (c)
bolaamphiphile E (ꢁ86 K), (d) bolaplex E at N/P = 1.75 (ꢁ86 K)
(arrows indicate the bolaplex with its internal ordering).
transesterification in a MeONa–MeOH mixture. Then, the
protective groups of amino acid moieties were removed either
by hydrogenolysis in acidic medium (lysine derivative) or acid
hydrolysis (histidine derivative) to provide compounds A and
B which were purified by size exclusion chromatography
(Sephadex LH20).
Compounds C and D were prepared from dodecylamine as
starting material (Scheme 3). Z-(Ne-Boc)-lysine and 2-Fmoc-
amino-3-(N-tritylimidazolyl)propionic acid were coupled with
dodecylamine in the presence of DCC–HOBT to give com-
pounds 9 and 10. After removal of the a-amino protective
groups, 9 and 10 were coupled with compound 3b in the
presence of TBTU to give derivatives 11a and 11b in good
yields (77–84%). The hydrogenolysis of the benzyloxycarbo-
nyl group supported by their propylamine residue allowed
their condensation with the lactobionolactone in boiling
methanol. To carefully purify these derivatives through chro-
matography on silica gel column, their sugar residue was
temporarily acetylated with an acetic anhydride–pyridine
mixture to give compounds 12a and 12b in 58–68% yields (2
steps). Removal of the acetyl groups with sodium methylate
and the amino acid protective groups with TFA finally
afforded compounds C and D.
Compound E was prepared in 58% yield from mercaptoun-
decanoic acid as starting material (Scheme 4). Compound 14
was obtained following a substitution reaction of benzyl
bromoundecanoate by mercaptoundecanoic acid in DMF with
NaH as a strong base. Compound 6 was deprotected by
hydrogenolysis and the amine obtained was then coupled with
compound 14 in the presence of TBTU and DIEA to provide
15, the median segment of compound E. The amino group of
compound 10 was deprotected by hydrogenolysis, then grafted
in the presence of TBTU onto compound 15 previously
submitted to a catalytic hydrogenation in order to hydrolyse
its acidic function. Removal of protecting groups with tri-
fluoroacetic acid and sodium methylate provided finally com-
pound E which was submitted to a last purification by size
exclusion chromatography (LH20).
Preliminary biological results
Bolaamphile–DNA complexation. The ability of bolaamphi-
philes A–E to complex plasmid DNA was tested using agarose
gel shift assay that allowed the separation of macromolecules
on the basis of both charge and size. It must be stressed that all
of the bolaamphiphiles were able to complex DNA due to the
electrostatic interactions between the ammonium and the
phosphate groups of the two oppositely charged partners (data
not shown). The proportion of bolaamphiphiles required to
immobilize DNA varies with their chemical structure; for
instance bolaamphiphiles with histidine head groups appear
less efficient in complexing DNA. This is not surprising given
ꢀc
This journal is the Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2006
New J. Chem., 2006, 30, 629–646 | 633

View Article Online
Scheme 4 Synthetic pathway to bolaamphiphile E.
that a large proportion of the free amines of the imidazole
groups is probably not protonated (pKa is close to the
physiological pH) under electrophoresis buffering conditions
(see Experimental section).20
retardation was only evidenced at higher ratios, namely in the
N/P range 2.5–5.0. Contrasting with C–bolaplexes, a marked
fluorescence was observed in the wells even when high
amounts of bolaform E were used. This indicates that ethi-
dium bromide can diffuse within the DNA strands. Therefore,
Gel mobility shift assays performed at various N/P [number
of amine/number of phosphate] charge ratios for C–DNA
bolaplexes are illustrated in Fig. 4a. In the left lane, naked
DNA was used as a negative control. At an N/P ratio of 0.50
and 0.75 (lanes 2 and 3), the visible band can be reasonably
associated with the free plasmid when compared with the
negative control (lane 1). These formulations were also char-
acterized by a negative z-potential (ꢂ21.4 ꢃ 6.2 mV). At
higher N/P ratios (N/P > 1.0), all of the bands disappeared,
indicating the complete neutralization of DNA plasmid (lanes
4–6).
On the other hand, a complete DNA neutralization by the
non fluorinated bolaamphiphile E was much more difficult to
achieve. Indeed at similar ratios, i.e. at N/P E 1.0, bolaam-
phiphile E failed to retain DNA (Fig. 4b). Complete DNA
Fig. 4 Representative gel electrophoresis shift assays of naked DNA
(lane 1), (a) C–bolaplexes at N/P ratios of 0.5, 0.75, 1, 1.5, 1.75; (lane
2–6) and (b) E–bolaplexes at N/P ratios of 0.75, 1, 1.5, 1.75, 2.5, 5
(lane 2–7).
ꢀc
634 | New J. Chem., 2006, 30, 629–646 This journal is the Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2006

View Article Online
bolaform E probably fails to efficiently compact DNA. The
above comparison between the two bolaamphiphiles (C and E)
would suggest that fluorine segments plays a major role in the
mechanism of bolaplex formation, reducing the hydrophilic
character of the conjugate and allowing for the increase of the
driving force needed for DNA compaction.
Size and morphology of bolaplexes. Size is a crucial factor for
particles internalization by non-specific endocytosis. It is well
documented that internalization is efficient when particle size
is less than 200 nm.21,22 Therefore, the size of bolaplexes
formed from bolaamphiphile C was investigated by QELS
for different N/P ratios. For bolaplexes formed with an excess
of bolaamphiphile C (i.e. for N/P ratios of 2.5 and 5) or with a
stoichiometric ratio (N/P = 1) QELS revealed the presence of
a bimodal distribution that consists in a fraction of small
particle size averaging 225–300 nm (intensity) [130–250 nm
(number), 100%] and a significant proportion of aggregates
[0.7–1.2 mm (intensity), 27–59%]. For N/P ratios of 1.17 and
1.35, larger particles were observed with a mean diameter of
B835 nm (intensity, B100%) [430 nm (number), 100%]. Such
a noticeable increase in the size of the particles is generally
related to the instability of electroneutral DNA–lipid lipo-
plexes.
Fig. 5 (a) In vitro transfection data of C/pBudCE4.1LacZ/CAT
bolaplexes and PEI control data. Standard deviation bars are shown.
(b) Relative cell viability (%) as a function of bolaamphiphile C
concentration (K). Arrows indicate the optimal ratios. Minima and
maxima values are represented as bars. m: PEI control data.
bolaamphiphile testified to the notably low cytotoxicity for a
high amount of bolaamphiphiles.
To estimate the transfection efficiency of bolaplexes formed
from bolaamphiphile C or E, b-Gal protein (encoded by LacZ
gene) expressed in COS-7 cells was quantified. The assays were
performed in the absence of any helper lipid or co-formulated
multifunctional (bola)amphiphile that commonly enhances in
vitro transfection.25 As illustrated on Fig. 5a, the transfection
efficiency as a function of N/P ratios followed an asymmetric
bell-shaped profile. The maximal level of transfection was
observed at an N/P ratio of 1.75 but gene expression was still
significant at higher N/P ratios (2.5 and 3.75). In fact, despite
the fact that the transfection level was significantly lower than
PEI (B45 times less efficient than PEI–DNA polyplexes),
C–bolaplexes generated a significant transfection level, typi-
cally higher than three orders of magnitude than naked DNA.
This result contrasted with the relatively low transfection
efficiency generally obtained with (fluorinated) bolaamphi-
philes when used alone, i.e. without the addition of any helper
lipids27f or other functional (bola)amphiphile.27d A plausible
reason to explain the relative difference measured between the
PEI–polyplexes and the C–bolaplexes could be the ability of
PEI–polyplexes to easily leave the vacuoles of lysosomes.26 In
the case of lipoplexes, efficient release in the cytoplasm would
originate from a lamellar to a fusogenic inverted hexagonal
phase in the acidic environment of the endosomal compart-
ments.9 TEM observations revealed the lamellar-like packing
of the DNA–bolaamphiphile complexes but so far we have no
evidence for such a phase transition. It is rather uncommon
that such pH phase sensitive behaviour is an intrinsic property
of a surfactant.9 In fact, DNA–surfactant complexes generally
appear to undergo the appropriate structural change when
helper lipids are incorporated in the formulation.
The morphology of the particles has also an impact on the
transfection efficiency,23 in particular on the nuclear traffick-
ing.24 At a ratio N/P of 1.7, TEM observations indicated the
presence of distinct and relatively spherical particles with a
contrasted outline and a diameter of around 65–140 nm [see
Fig. 3b]. These particles were assumed to correspond to
bolaplexes. In fact, the propensity of C–bolaplexes to aggre-
gate was also confirmed by TEM at other N/P ratios. At high
N/P ratios (N/P > 1.35), a mixture of membrane debris and
well-defined particles was also observed. A closer examination
of Fig. 3b also revealed the internal lamellar ordering of the
bolaplexes.
TEM examinations of E–bolaplexes (Fig. 3d) revealed, on a
large range of ratios (1 o N/P o 5), the persistence of a fiber-
like morphology, reminiscent of that observed for the uncom-
plexed bolaamphiphile E. DNA appeared to be aggregated
according to a heterogeneous fashion onto the surface of the
fibers, confirming the inefficient complexation process occur-
ring between the non fluorinated bolaamphiphile and DNA.
Transfection and cytotoxicity. In order to evaluate the gene
delivery potential of bolaamphiphile C and its hydrocarbo-
nated analogous compound E, the in vitro cytotoxicity as well
as the transfection efficiency of bolaamphiphiles C and
E–pBudCE4.1LacZ–CAT based-bolaplexes were performed
on COS-7 cell lines, as described in the Experimental section.
Cell viability tests were carried out in the presence of bolaam-
phiphiles C or E and compared with those obtained from 25
kDa branched poly(ethylenimine) (PEI) which is known to be
a very potent transfectant but with an acute cellular cytotoxi-
city (Fig. 5b). The LC50 of PEI was 9 mg mLꢂ1 whereas at the
same dose of bolaamphiphiles C and E (data not shown), the
cell viability was B89%. The profile in the form of a plateau
of the cell viability as a function of the increasing weight of
In closing, it is worth noting that transfection experiments
performed with the E–bolaplexes were characterized by the
absence of any protein expression. The difference in the
transfection efficiencies observed between bolaform C and E
may be due in part to the inability of the non-fluorinated
bolaform to bind and condense efficiently DNA. This last
ꢀc
This journal is the Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2006
New J. Chem., 2006, 30, 629–646 | 635

View Article Online
result suggests that the partial fluorination of the hydrophobic
core of these bolaamphiphiles seems to increase not only the
self association and DNA complexation abilities of these
compounds but also their DNA transfection efficiency.
tus. Elemental analyses were performed by the service central
de microanalyses of the CNRS (Vernaison, France).
Sterile water (DNase free, molecular biology reagent; PBS,
PEI (Mw 25 000 g molꢂ1, branched), and ethidium bromide
were obtained from Sigma-Aldrich (Oakville, ON). Cell cul-
ture plastics were purchased from Corning Costar (Corning,
NY). Zeocin, pBudCE4.1/LacZ/CAT (8433 bp), Escherichia
coli Top 10 chemically competent cells were from Invitrogen
(Burlington, ON). Cell proliferation kit MTT [3-(4,5-di-
methylthiazol-2-yl)-2,5-diphenylpyrazolium)bromide)] assay
and b-gal ELISA kit were purchased from Roche Diagnostics
GmbH (Laval, PQ). DMEM and OPTI-MEM I reduced
serum medium and FBS were purchased from GIBCO
Invitrogen Corporation (Burlington, ON) and Medicorp
Conclusion
The synthesis and the characterization of a new series of
fluorinated asymmetric bolaamphiphiles, designed for gene
delivery, were reported. We demonstrated that fluorinated
bolaamphiphiles were capable of binding and condensing
DNA in nanoparticles whose sizes were, according to TEM
observations, in the range 65–150 nm. Preliminary biological
data based on transfection and cytotoxicity assays indicated
the strong potential of these bolaplexes as a DNA delivery
system. Indeed bolaamphiphiles can present a low cytotoxicity
and a significant in vitro transfection efficiency without the
addition of helper lipids. In this context, post-functionaliza-
tions of bolaamphiphiles with a translocation peptide are
currently under way with the aim of enhancing gene delivery
through a co-formulation process.
(Montreal, PQ), respectively. Endotoxin-free Giga Kit was
´
from QIAgen (Mississauga, ON). COS-7 cell line (Simian
virus 40-transformed kidney cells of an African green monkey)
was from American Type Culture Collection (ATCC)
(Manassas, VA).
DNA preparation
pBudCE4.1/lacZ/CAT plasmid encoding b-galactosidase un-
der the control of the CMV promoter was used. One Shot
TOP10 chemically competent Escherichia coli grown in Luria
Broth (LB) medium containing Zeocin (50 mg mLꢂ1) were
transformed with the plasmid pBudCE4.1/lacZ/CAT. The
plasmid was isolated and purified using the endotoxin-free
Giga Kit and then dissolved in PBS (pH 7.3). The DNA
concentration was measured by UV absorption at 260 nm,
and the purity was confirmed with A260/A280 and by 0.8%
agarose gel electrophoresis [Tris–acetate–EDTA buffer (TAE,
pH 8.0)]. DNA bands were visualized by UV illumination after
coloration by ethidium bromide. DNA restriction analysis was
used to confirm the nature of the plasmid. The DNA aliquots
were stored at ꢂ20 1C prior to use.
Experimental
Abbreviations
AcOEt, ethyl acetate; AIBN, 2,20-azobisisobutyronitrile; Ar,
argon; Boc, tert-butoxycarbonyl; BOP, (benzotriazol-1-yloxy)
tris(dimethylamino)phosphonium
hexafluorophosphate;
DCC, dicyclohexylcarbodiimide; DCM, methylene dichloride;
DIEA, diisopropylethylamine; cHex, cyclohexane; Fmoc, 9-
fluorenylmethoxycarbonyl, His(Tr), 2-amino-3-(N-trityl-imi-
dazolyl)propionic acid; HOBT, 1-hydroxybenzotriazole;
PFP, pentafluorophenol, Pyr, pyridine; TBTU, O-benzotria-
zol-1-yl-N,N,N00,N00-tetramethyluronium tetrafluoroborate;
TEA, triethylamine; TFA, trifluoroacetic acid; THF, tetra-
hydrofuran; Tr, trityl; Z, benzyloxycarbonyl; PBS, phosphate
buffered saline; PEI, polyethylenimine; DMEM, Dulbecco’s
modified Eagle’s medium; FBS, foetal bovine serum; BCA,
bicinchonic acid; CMV, cytomegalovirus; QELS: quasi-elastic
light scattering; TEM, transmission electron microscopy.
Bolaamphiphiles and bolaplexes preparations
Bolaamphiphiles A–E were sonicated in H2O for 2 h at 60 1C
and pH was adjusted at 7 at a final concentration of 5 mg
mLꢂ1 (B4 ꢁ 10ꢂ3 M). Only bolaamphiphiles C and E were
found to be stable as observed by the naked eye (no sedimen-
tation detected after a period at room temperature of at least
4 h). Bolaplexes were prepared in H2O by directly mixing equal
volumes of diluted bolaamphiphiles and DNA solution (20 mg
Materials and methods
All solvents were purchased from Acros Organics. DCM was
distilled from P2O5 and THF from sodium. Other solvents
were used without further purification. All chemicals were
purchased from Sigma-Aldrich or Acros Organics and were
used without further purification. Reactions were monitored
by thin layer chromatography using Merck precoated 60F254
silica gel plates. Purifications were achieved by column chro-
matography over silica gel (Merck 60). Melting points were
measured on an electrothermal apparatus and have not been
corrected. 1H NMR and 13C NMR spectra were recorded on a
Bruker AC 250 spectrometer and processed using
XWINNMR (Bruker). Chemical shifts are given in ppm
relative to tetramethylsilane using the deuterium signal of
mLꢂ1 DNA). Final DNA concentration was 10 mg mLꢂ1
.
Gel electrophoresis shift assay
Bolaamphiphile C–DNA or E–DNA complexes were formed
at various nitrogen (N)/phosphate (P) ratios >N/P of 0.5,
0.75, 1, 1.5, 1.75, 2.5 and 5]. 40 mL of freshly prepared bolaplex
solutions (0.4 mg of DNA) were loaded with bromophenol blue
in glycerol into 0.8% agarose gel in buffer TAE (pH 8.0) and
electrophoresis performed at 90 V for 1 h. Gel stained with
ethidium bromide solution (0.5 mg Lꢂ1) was transilluminated
in ultraviolet light to visualized the DNA and photographed.
the solvent as a heteronuclear reference for H, 13C and 19F.
1
Mass spectra were recorded on a APT III Plus Sciex appara-
ꢀc
636 | New J. Chem., 2006, 30, 629–646 This journal is the Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2006

View Article Online
Size distribution and zeta (f) potential measurement
DMEM. Bolaamphiphiles were directly dissolved in OPTI-
MEM I at different concentrations (mg mLꢂ1) [related to the
transfection experiments]. Untreated cells were used as a
positive control and PEI cells treated for comparison model.
Cells were grown for 24 h in the presence of complexes and
then immediately tested for metabolic activity. MTT reagent
(10% v/v) was added to each well (final concentration 0.5 mg
mLꢂ1). After 4 h of incubation at 37 1C, the purple insoluble
salt was dissolved by adding 100 mL of solubilization solution.
The plate was incubated in the dark at 37 1C for 24 h.
Absorbance was measured at 550 nm using a reference wave-
length of 650 nm. The results were expressed as a relative
percentage of cell viability related to the control (untreated
Size distribution of bolaamphiphile C and E was obtained
from quasi-elastic light scattering (QELS) using Zetasizer
(Malvern, NanoZS ZEN3600). The instrument was equipped
with a monochromatic coherent helium neon laser (633 nm) as
a light source. The scattered light was recorded at an angle of
90o, and the analysis of the autocorrelation function was
performed automatically to yield the diffusion coefficient,
DT, taking the values 1.33 and 25 1C for refractive index and
temperature, respectively. Size distributions by intensity or
number (see text for details) were given relative to the hydro-
dynamic diameter using a bimodal distribution. For bola-
plexes, both size and z potential measurements were carried
out at different N/P ratios with the disposable cell (folded
capillary cell DTS 1060). z potential was calculated by using
the Schmoluchowsky approximation.
cells). Cell viability (%) = (OD550(sample)/OD550(negative control)
)
ꢁ 100. Experiments were performed in triplicate.
Syntheses
Transmission electron microscopy (TEM)
10, 19-Diiodo-12, 12, 13,13,14,14,15,15,16,16,17,17,18,18,19,
19 hex adecafluorononadecanoic acid (1). Undecylenic acid
(0.289 g, 1.53 mmol), 1,8-diiodoperfluorooctane (2 g, 3.06
mmol) and zinc dust (0.4 g) were dissolved in freshly distilled
and degassed DCM (10 mL) under argon. The mixture was
refluxed for 2 h. The mixture was diluted with DCM (20 mL)
and filtered over Celite. The solvent was removed from the
filtrate in vacuo. The crude product was purified by flash
chromatography on silica gel (cHex–AcOEt 9 : 1) to yield
900 mg (1.07 mmol, 70%) of monoadduct intermediate 1 as a
white solid. Rf: 0.5 (cHex–AcOEt 7 : 3), mp: 66–68 1C. 1H
NMR (CDCl3, 250 MHz): d 1.29–1.45 (10H, s, CH2 chain), 1,66
(2H, m, HO2C-CH2-CH2), 1.80 (2H, m, CH2-CH2-CHI), 2.38
(2H, t, HO2C-CH2, J = 7.5 Hz), 2.85 (2H, m, CH2-CF2), 4.34
(1H, m, CHI). 13C NMR (CDCl3, 62.86 MHz): d 20.9 (HO2C-
CH2-CH2), 24.6 (CHI), 28.4–29.4 (CH2 chain), 34.0 (HO2C-
CH2), 40.0 (CH2-CHI), 41.9 (CH2-CF2), 106.9–117.9 (CF2),
180.3 (CO). 19F NMR (CDCl3, 235.19 MHz): d ꢂ60 (2F, s,
CF2I), ꢂ111.0 to ꢂ115.3 (4F, m, CH2-CF2, CF2-CF2I),
ꢂ120.8 to ꢂ123.5 (10F, m, 5 CF2).
Bolaamphiphile C and E and C or E–bolaplexes prepared at
different N/P ratios were analyzed for their morphological
characteristics. 10 mL of the sample was added onto a 150
mesh copper grid covered with Formvar. The excess of solu-
tion was gently dried with filter paper and the preparation was
negatively stained with a droplet of 1% uranyl acetate (aqu-
eous solution at a pH of 4.5) for 2 min. After removal of the
stain the grid was air-dried. Samples were observed under
transmission electron microscope Philips EM 410 operating at
80 kV.
Transfection protocol
COS-7 cells were used for transfection and cell viability tests.
Cells (2.7 ꢁ 105 per well) were grown to 90% confluence 24 h
after plating in six-well tissue culture plates using complete
medium [90% (v/v) DMEM containing l-glutamine, 10%
FBS] at 37 1C in a humidified atmosphere containing 5%
CO2 (v/v). Immediately before the initiation of b-galactosidase
transfection, the medium was removed, the cells were washed
twice with PBS, and 1 mL of OPTI-MEM I serum-free
medium was added before the addition of the fresh bolaplex
solution (500 mL, 5 mg of DNA per well). Serum-free transfec-
tion mixtures were incubated for 4 h, and replaced by complete
medium without antibiotics for an additional 18 h. b-Galac-
tosidase quantity was assayed using a commercially available
kit (Roche). Results were expressed as pg of b-galactosidase
per mg of total protein using the BCA assay (Pierce). Control
experiments were performed with naked DNA plasmid (5 mg
per well) and PEI/DNA polyplexes at weight ratios of 1.29
[equivalent to a N/P ratio of 10].26 Experiments were per-
formed in triplicate.
22-N-(tert-Butyloxycarbonylamino)-10, 21-diiodo-12,12, 13,
13,14,14,15,15,16,16,17,17,18,18,19,19-hexadecafluorodocosa-
noic acid (2a). Compound 1 (2 g, 2.38 mmol) and Boc-
allylamine (1.5 g, 9.55 mmol) were dissolved in 15 mL freshly
distilled acetonitrile. Sodium dithionite (0.414 g, 2.38 mmol)
and sodium hydrogencarbonate (0.4 g, 4.76 mmol) dissolved
in 5 mL of distilled water were added to the mixture. After
stirring for 16 h at room temperature, the mixture was
acidified to pH = 3 with 2 M HCl. Acetonitrile was removed
in vacuo and the mixture was extracted with ethyl acetate. The
organic layers were washed with 1 M HCl then water and
dried over anhydrous Na2SO4. The solvent was removed
in vacuo. The crude product was purified by flash chromato-
graphy on silica gel (cHex–AcOEt 8 : 2) to yield 1.9 g (1.9
mmol, 80%) of compound 2a as a white solid. Rf: 0.51 (cHex–
Cytotoxicity
Cytotoxicity of bolaamphiphiles was evaluated by measuring
the viability of treated cells as a function of the concentration
of bolaamphiphile (mg mLꢂ1). The viability of metabolically
viable COS-7 cells was measured by using the MTT Cell
Proliferation Assay kit, 24 h after incubation with the bo-
laamphiphile. Cells were grown in 96-well plates 24 h before at
an initial seeding density of 1.9 ꢁ 104 cells per well in 100 mL of
1
AcOEt 6 : 4), mp: 93–951C. H NMR (CDCl3, 250 MHz): d
1.27–1.40 (10H, s, CH2 chain), 1.47 (9H, s, C(CH3)3), 1.63 (2H,
m, HO2C-CH2-CH2), 1.79 (2H, m, CH2-CH2-CHI), 2.35 (2H,
t, HO2C-CH2, J = 7.5 Hz), 2.83 (4H, m, 2 CH2-CF2), 3.58
(2H, m, CH2-NH), 4.37 (2H, m, 2 CHI), 5.09 (1H, s, NH).
13C NMR (CDCl3, 62.86 MHz): d 18.8 (CHI-CH2-NH),
ꢀc
This journal is the Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2006
New J. Chem., 2006, 30, 629–646 | 637

View Article Online
20.9 (HO2C-CH2-CH2), 24.6 (CHI-CH2-CF2), 26.9–33.9
(CH2 chain, C(CH3)3), 33.9 (HO2C-CH2), 38.6 (CF2-CH2),
40.3 (CH2-CHI), 41.7 (CH2-CF2), 49.0 (CH2-NH), 81.1
(C(CH3)3), 106.1–121.9 (CF2), 155.7 (NH-CO), 179.1
(CO2H). 19F NMR (CDCl3, 235.19 MHz): d ꢂ112.2 to
ꢂ114.1 (4F, m, 2 CH2-CF2), ꢂ120.8 to ꢂ123.5 (12F, m, 6
CF2).
white powder after purification by flash chromatography on
silica gel (cHex–AcOEt 8 : 2) and recrystallization from
heptane. Rf: 0.36 (cHex–AcOEt 6 : 4), mp: 97–98 1C. 1H
NMR (CDCl3, 250 MHz): d 1.32 (12H, m, CH2chain), 1.63 (4H,
m, CH2-CH2-CO2H, CH2-CH2-CF2), 1.86 (2H, m, CH2-CH2-
NH), 2.07 (4H, m, 2 CH2-CF2), 2.35 (2H, t, HO2C-CH2, J3 =
7.5 Hz), 3.31 (2H, t, CH2-NH, J3 = 6 Hz), 4.88 (1H, s, NH),
5.14 (2H, s, CH2-F), 7.36 (5H, s, CHarom.). 13C NMR (CDCl3,
62.86 MHz): d 20.1–30.6 (CH2chain, 2 CH2-CF2, CH2-CH2-
NH(Z)), 34.0 (HO2C-CH2), 40.2 (CH2-NH(Z)), 66.9 (CH2-F),
106.5–123.1 (CF2), 128.1–128.6 (CHarom.), 136.1 (Carom.),
156.9 (NH-CO-O), 179.4 (HO2C). 19F NMR (CDCl3, 235.19
MHz): d ꢂ114.3 (4F, s, 2 CH2-CF2), ꢂ121.8 (8F, m, 4 CF2),
ꢂ123.5 (4F, s, 2 CF2).
22-N-(tert-Butyloxycarbonylamino)-12,12,13,13,14,14,15,15,
16,16,17,17,18,18,19,19-hexadecafluorodocosanoic acid (3a).
Compound 2a (1.85 g, 1.86 mmol) was dissolved in 20 mL
of anhydrous and degassed acetonitrile with argon. The solu-
tion was refluxed and then Bu3SnH (1.10 mL, 4.09 mmol) and
AIBN (0.34 g, 2.04 mmol) dissolved in 2 mL of dry acetonitrile
were added slowly via a syringe. The mixture was refluxed for
16 h under argon. The solvent was removed in vacuo and the
crude product was purified by flash chromatography on silica
gel (cHex–AcOEt 8 : 2) to yield 830 mg (1.11 mmol, 60%) of
compound 3a as a white solid. Rf: 0.53 (cHex–AcOEt 6 : 4),
mp: 99–1001C. 1H NMR (CDCl3, 250 MHz): d 1.23–1.39
(12H, m, CH2 chain), 1.47 (9H, s, C(CH3)3), 1.61 (4H, m,
HO2C-CH2-CH2, CH2-CH2-CF2), 1.80 (2H, m, CH2-CH2-
NH(Boc)), 2.15 (4H, m, 2 CH2-CF2), 2.35 (2H, t, HO2C-
CH2, J = 7.5 Hz), 3.24 (2H, m, CH2-NH), 4.67 (1H, s, NH).
13C NMR (CDCl3, 62.86 MHz): d 20.1–21.3 (CH2-CH2-NH,
HO2C-CH2-CH2), 24.8–31.2 (CH2 chain, C(CH3)3, 2 CH2-
CF2), 34.0 (HO2C-CH2), 39.6 (CH2-NH), 79.6 (C(CH3)3),
106.5–122.4 (CF2), 156 (NH-CO), 179.7 (CO2H). 19F NMR
(CDCl3, 235.19 MHz): d ꢂ114.27 (4F, s, 2 CH2-CF2), ꢂ121.86
to ꢂ123.59 (12F, m, 6 CF2).
Pentafluorophenyl 4,4,5,5,6,6,7,7,8,8,9,9,10,10,11,11,11-hep-
tadecafluoroundecanoate (4). 4,4,5,5,6,6,7,7,8,8,9,9,10,10,11,
11,11-Heptadecafluoroundecanoic acid (3 g, 6.1 mmol) and
pentafluorophenol (1.23 g, 6.7 mmol) were dissolved in 20 mL
of freshly distilled DCM. The mixture was cooled to 0 1C and
DCC (1.63 g, 7.9 mmol) was added. After stirring for 12 h at
room temperature the solvent was removed in vacuo and the
crude product purified by flash chromatography on silica gel
(cHex–AcOEt 9 : 1) then recrystallized from AcOEt–Hexane
to yield 3.9 g (5.92 mmol, 97%) of compound 4 as white
crystals. Rf: 0.44 (cHex–AcOEt 7 : 3), mp: 48–50 1C. 1H NMR
(CDCl3, 250 MHz): d 2.55 (2H, m, CH2-CF2), 3.05 (2H, t,
CH2CO, J3 = 7.5 Hz). 13C NMR (CDCl3, 62.86 MHz): d
25.09 (CH2-CO), 26.5 (CF2-CH2), 106.6–121.7 (CF2), 136.0 (2
CFPFP), 138.7 (CFPFP), 143.1 (2 CFPFP), 167.5 (CO). 19F
NMR (CDCl3, 235.19 MHz): d ꢂ80.9 (3F, s, CF3), ꢂ114.8
(2F, s, CF2-CH2), ꢂ121.8 to ꢂ123.5 (10F, m, 5 CF2), ꢂ126.2
(2F, s, CF3-CF2), ꢂ152.8 (2F, s, 2 CFPFP), ꢂ157.3 (1F, s,
CFPFP), ꢂ162.1 (2F, s, 2 CFPFP).
22-N-(Benzyloxycarbonylamino)-10, 21-diiodo-12, 12, 13, 13,
14,14,15,15,16,16,17,17,18,18,19,19-hexadecafluorodocosanoic
acid (2b). The same procedure as used for compound 2a was
applied to 2b. From compound 1 (2 g, 2.386 mmol), Z-
allylamine (1.37 g, 7.158 mmol), sodium dithionite (0.415 g,
2.39 mmol) and sodium hydrogencarbonate (0.4 g, 4.77
mmol), 1.6 g (1.55 mmol, 65%) of compound 2b was obtained
as a white powder after purification by flash chromatography
on silica gel (cHex–AcOEt 9 : 1), Rf: 0.32 (cHex–AcOEt 8 : 2),
mp: 74–76 1C. 1H NMR (CDCl3, 250 MHz): d 1.25–1.55 (10H,
m, CH2 chain), 1.66 (2H, m, CH2-CH2-CO2H), 1.82 (2H, m,
CH2-CH2-CHI), 2.37 (2H, t, HO2C-CH2, J3 = 7.5 Hz), 2.86
(4H, m, 2 CH2-CF2), 3.55 (1H, m, CHI), 3.66 (1H, m, CHI),
4.38 (2H, m, CHI-CH2-NH), 5.16 (2H, s, CH2-F), 7.27 (1H, s,
NH), 5.39 (5H, s, CHarom.). 13C NMR (CDCl3, 62.86 MHz): d
18.4 (CHI-CH2-NH), 20.9 (HO2C-CH2-CH2), 24.6 (CHI-
CH2-CF2), 28.4–29.5 (CH2chain), 34.0 (HO2C-CH2), 38.7
(CF2-CH2), 40.3 (CH2-CHI), 41.7 (CH2-CF2), 49.3 (CH2-
NH), 67.3 (CH2-F), 106.3–121.8 (CF2), 128.4 (CHarom.),
146.2 (CHarom.), 156.3 (NH-CO), 179.1 (CO2H). 19F NMR
(CDCl3, 235.19 MHz): d ꢂ111 to ꢂ115.1 (4F, m, 2 CH2-CF2),
ꢂ121.7 (8F, s, 4 CF2), ꢂ123.2 (4H, s, 2 CF2).
Na-(4,4,5,5,6,6,7,7,8,8,9,9,10,10,11,11,11-Heptadecafluorou-
ndecanoyl)-Ne-(benzyloxycarbonyl)lysine (5a). Lys(Z)OH (0.5
g 1.78 mmol) and HOBT (0.12 g, 0.89 mmol) were dissolved in
10 mL of acetone–distilled water (1 : 1). The solution was
basified to pH = 8–9 with TEA. Compound 4 (1.29 g, 1.96
mmol) dissolved in 2 mL of acetone was added dropwise. After
stirring for 12 h at room temperature the mixture was acidified
to pH = 2 with 3 M HCl solution and the solvent removed in
vacuo. The aqueous layer was extracted with ethyl acetate
three times. The organic layer was washed with 1 M HCl
solution and water, dried over sodium sulfate and concen-
trated in vacuo. The crude product was purified by flash
chromatography on silica gel (cHex–AcOEt 5 : 5 to cHex–
AcOEt 3 : 7) to yield 834 mg (1.10 mmol, 62%) of 5a as a
white solid. Rf : 0.46 (AcOEt), mp: 112–1141C, [a]2D0: þ3.94 (c
1, CH2Cl2). 1H NMR (CDCl3, 250 MHz): d 1.31–1.52 (4H, m,
CH2-(CH2)2-CH2), 1.83 (2H, m, CH-CH2), 2.46–2.57 (4H, m,
CH2-CH2- CF2), 3.19 (2H, m, CH2-NH(Z)), 4.57 (1H, m, CH-
CH2), 5.09 (2H, s, CH2-F), 5.25 (1H, s, NH), 7.29 (5H, s,
CH arom.), 7.87 (1H, s, NH). 13C NMR (CDCl3, 62.86 MHz):
d 22.1–30.9 (CH2-CH2-CF2, CH-(CH2)3), 40.1 (CH2-NH(Z)),
52.4 (CH), 67.3 (CH2-F), 106.5–122.2 (CF2), 126.5–128.6
(CHarom.), 138.6 (Carom.), 151.8 (NH-CO-O), 169.5 (NH-CO-
CH2), 173.1 (HO2C). 19F NMR (CDCl3, 235.19 MHz): d
22 -N - (Benzyloxycarbonylamino) - 12, 12, 13, 13,14,14,15,15,
16,16,17,17,18,18,19,19-hexadecafluorodocosanoic acid (3b).
Deiodination was carried out with the same procedure used
for compound 3a. From compound 2b (1.5 g, 1.46 mmol),
Bu3SnH (863 mL 3.21 mmol) and AIBN (0.263 g 1.60 mmol),
610 mg (0.78 mmol, 54%) of compound 3b was obtained as a
ꢀc
638 | New J. Chem., 2006, 30, 629–646 This journal is the Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2006

View Article Online
ꢂ80.7 (3F, s, CF3), ꢂ114.6 (2F, s, CF2-CH2), ꢂ121.9 to
ꢂ126.1 (12F, m, 6 CF2).
product was dissolved in 5 mL of freshly distilled DCM.
Compound 3a (713 mg, 0.96 mmol) and BOP reagent (508
mg, 1.15 mmol) were added. The mixture was basified to pH
= 8 with DIEA. After stirring for 12 h under argon, the
solvent was removed in vacuo and the crude product was
purified by flash chromatography on silica gel (cHex–AcOEt
5 : 5 to cHex–AcOEt 2 : 8) to yield 514 mg (0.46 mmol, 48%)
of compound 7 as a white solid. Rf: 0.6 (AcOEt), mp: 45–47
Na-(4,4,5,5,6,6,7,7,8,8,9,9,10,10,11,11,11-Heptadecafluorou-
ndecanoyl)-Nim-(trityl)histidine (5b). The procedure described
for the synthesis of compound 5a was applied to 5b. From
His(Trt)OH (0.640 g, 1.61 mmol), HOBT (0.109 g, 0,81 mmol)
and compound 4 (1.165 g, 1.77 mmol) and after purification by
flash chromatography on silica gel (AcOEt–cHex 9 : 1 to
AcOEt–MeOH 9 : 1) and recrystallization from AcOEt–n-
heptane 1.15 g of compound 5b was obtained (1.32 mmol,
82%) as white crystals. Rf: 0.33 (AcOEt–MeOH 9 : 1), mp: 109
–111 1C. [a]2D0: þ14.03 (c 1, CH2Cl2). 1H NMR (DMSO-d6, 250
MHz, 45 1C): d 2.34–2.51 (4H, m, CH2-CH2-CF2), 2.88 (2H,
m, CH2-CH), 4.52 (1H, m, CH-CH2), 6.9–7.4 (17H, m,
CHarom., CHhis), 8.04 (1H, s, OH), 8.37 (1H, d, NH, J3 = 8
Hz). 13C NMR (DMSO-d6, 62.86 MHz): d 26.1–28.8 (CO-
CH2-CH2-CF2), 39.6 (CH-CH2), 52.2 (CH-CH2), 76.4 (C(F)3),
105.8–119.3 (CF2, CF3), 120.6 (N–CHQChis), 128.8–130.1
(CHarom.), 134 (Chis), 137.6 (N–CQNhis), 141.7 (Carom.),
169.7 (NH-CO), 172.9 (CO-OH). 19F NMR (DMSO-d6,
235.19 MHz): d ꢂ80.2 (3F, s, CF3), ꢂ113.3 (2F, s, CH2-
CF2), ꢂ121.4 to ꢂ122.9 (10F, m, 5 CF2), ꢂ125.5 (2F, s,
CF2-CF3).
1
1C. H NMR (CDCl3, 250 MHz): d 1.35 (12H, m, CH2 chain),
1.46 (9H, s, C(CH3)3), 1.61 (4H, m, NH-CO-CH2-CH2, CH2-
CH2-CF2), 1.76 (2H, m, CF2-CH2-CH2-CH2-NH(Boc)), 1.96–
2.30 (18H, m, CH3CO, 2 CH2-CF2, NH-CO-CH2), 3.15 (2H, t,
CH2-NH(Boc), J3 = 6.5 Hz), 3.37 (2H, m, CH2NH), 3.70 (1H,
m, OCH2), 3.86 (1H, m, OCH2), 4.15 (3H, m, CH2(6), CH(5)),
4.68 (1H, d, CH(1), J = 7.25 Hz), 4.91–5.51 (4H, m, CH(2)
,
CH(3), CH(4), NH). 13C NMR (CDCl3, 62.86 MHz): d 19.1–
20.7 (CH3-CO, CH2-CH2-NH(Boc), NH-CO-CH2-CH2), 25.6
–29.1 (CH2 chain, 2 CH2-CF2, C(CH3)3, NH-CO-CH2), 35.7
(CH2-NH(Boc)), 38.9 (OCH2-CH2-NH), 61.2 (CH2(6)), 67.4–
71 (OCH2-CH2-NH, CH(2)
, CH(3), CH(4), CH(5)), 78.7
(C(CH3)3), 100.8 (C1b), 105.3–122.5 (CF2), 157.1 (COBoc),
170.1–170.6 (CO-CH3), 175.1 (NH-CO-CH2). 19F NMR
(CDCl3, 235.19 MHz): d ꢂ115.3 (4F, s, 2 CH2-CF2), ꢂ122.8
to ꢂ124.5 (12F, m, 6 CF2).
O-Benzyl N-[2-((2,3,4,6-tetra-O-acetyl)-b-D-galactopyrano-
syloxy)ethyl]carbamate (6). O-Benzyl-N-hydroxyethylcarba-
mate (4 g, 20.51 mmol), Hg(CN)2 (7.77 g, 30.76 mmol) and
molecular sieves (3 A) (2 g) were stirred in 50 mL of anhydrous
and degassed acetonitrile under argon. After stirring for
15 min, 2,3,4,6-tetra-O-acetyl-a-D-galactopyranosyl bromide
(12.64 g, 30.76 mmol) was added and the reaction mixture was
stirred for 1 d at room temperature. The resulting mixture was
filtered and the solvent was removed from the filtrate in vacuo.
The crude product was dissolved in ethyl acetate (100 mL) and
the organic layer washed successively with 10% NaHCO3
solution, 10% KI solution, 10% sodium thiosulfate solution.
The solution was dried over sodium sulfate, concentrated in
vacuo and the crude product was purified by flash chromato-
graphy on silica gel (cHex–AcOEt 6 : 4) to yield 6.78 g (12.91
mmol, 63%) of compound 6 as a white solid. Rf: 0.46 (cHex–
N - [2 - ((2, 3, 4, 6 - Tetra -O-acetyl)-b-D-galactopyranosyloxy)
ethyl]-22-[2-(4, 4, 5, 5, 6, 6, 7, 7, 8, 8, 9,9, 10,10,11,11,11-heptade-
cafluoroundecanamido)-6-(benzyloxycarbonylamino)hexanami-
do]-12,12,13,13,14,14,15,15,16,16,17,17,18,18,19,19-hexadeca-
fluorodocosanamide (8a). Compound 7 (200 mg, 0.179 mmol)
was dissolved in 5 mL of TFA–CH2Cl2 2 : 8 mixture After
stirring for 1 h the solvent was removed in vacuo. The crude
product was dissolved in 10 mL of ethyl ether and the solvent
removed in vacuo. The operation was repeated twice until total
removal of TFA. The crude product was dissolved in 5 mL of
freshly distilled DCM. Compound 5a (162 mg, 0.215 mmol)
and TBTU (75 mg, 0.233 mmol) were added. The mixture was
basified to pH = 8–9 with DIEA. After stirring for 16 h under
argon in boiling DCM (CH2Cl2), the solvent was removed in
vacuo, the crude product was purified by flash chromatogra-
phy on silica gel (AcOEt–cHex 8 : 2) and filtration by size
exclusion chromatography using LH20 (MeOH–CH2Cl2 1 : 1)
to yield 220 mg (0.12 mmol, 70%) of fully protected com-
pound 8a as a white solid. Rf: 0.46 (AcOEt), mp: 100–101 1C.
[a]2D0: ꢂ4.17 (c 1, CH2Cl2), 1H NMR (DMSO-d6, 250 MHz): d
1.21–1.72 (24H, m, CH2 chain, CH2-CH2-CH2-NH(Z), CF2-
CH2-CH2-CH2-NH, CH-CH2), 1.90–2.11 (18H, m, CH3CO, 2
CH2-CF2, NH-CO-CH2), 2.39 (4H, m, C8F17-CH2-CH2), 2.97
(2H, m, CH2-NH-(Z)), 3.17 (4H, m, CF2-(CH2)2-CH2-NH,
OCH2-CH2-NH), 3.51 (1H, m, OCH2), 3.64 (1H, m, OCH2),
4.04 (3H, m, CH-CH2, CH2(6)), 4.18 (1H, m, CH(5)), 4.82 (1H,
d, CH(1), J3 = 8 Hz), 4.90–5.00 (3H, m, CH(2), CH2-F), 5.15
(1H, dd, CH(3), J3 = 3.25 Hz, J3 = 10.5 Hz), 5.26 (1H, m,
CH(4)), 7.25 (1H, m, NH), 7.29 (5H, s, CHarom.), 7.78 (1H, m,
NH), 7.99 (1H, m, NH), 8.25 (1H, m, NH-CH). 13C NMR
1
AcOEt 4 : 6), mp: 46–48 1C. [a]2D0: þ20.7 (c 1, CH2Cl2). H
NMR (CDCl3, 250 MHz): d 1.95–2.13 (12H, m, CH3CO), 3.38
(2H, m, CH2-NH), 3.89 (2H, t, O-CH2, J3 = 6 Hz), 4.11 (3H,
m, CH(5), CH2(6)), 4.45 (1H, d, CH(1), J3 = 7,5 Hz), 4.96–5.49
(6H, m, CH(2), CH(3), CH(4), CH2-F, NH), 7.31 (5H, s,
CHarom.). 13C NMR (CDCl3, 62.86 MHz): d 20.6 (CH3CO),
61.3–70.6 (CH2-F, CH(2), CH(3), CH(4), CH(5), CH(6)), 101.5
(C1b) 128.3 (CHarom.), 136.4 (Carom.), 156.4 (CO-CH2-F),
169.7–170.5 (CO-CH3).
N - [2 - ((2, 3, 4, 6 - Tetra-O-acetyl) - b-D-galactopyranosyloxy)
ethyl] - 22 - (N - tert-butyloxycarbonylamino)-12,12,13,13,14,14,
15, 15, 16, 16, 17, 17, 18, 18, 19, 19-hexadecafluorodocosanamide
(7). Compound 6 (604 mg, 1.15 mmol) was dissolved in 10 mL
of ethanol. The solution was stirred and cooled at 0 1C and
palladium on charcoal (69 mg, 60 mg mmolꢂ1) was added
portionwise. The mixture was stirred under hydrogen pressure
(8 bars) for 12 h at room temperature. The mixture was filtered
over Celite and the solvent removed in vacuo. The crude
(DMSO-d6, 62.86 MHz): d 20.0–31.3 (CO-CH3, CH2
,
chain
CF2-CH2-CH2-CO, CH2-CF2, CF2-(CH2)2-CH2-NH, CH-
CH2-CH2-CH2), 35.7–41.0 (OCH2-CH2-NH, CF2-(CH2)2-
CH2-NH, CH2-NH(Z)), 54.2 (CH-CH2), 61.7 (CH2(6)), 65.5–
ꢀc
This journal is the Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2006
New J. Chem., 2006, 30, 629–646 | 639

View Article Online
71.1 (CH(2), CH(3), CH(4), CH(5), CH2-F, OCH2-CH2), 100.5
(C1b), 105.9–119.8 (CF2, CF3), 128.2–128.8 (CHarom.), 137.7
(Carom.), 156.5 (NH-CO-O), 169.6–172.8 (CO-CH3, NH-CO-
CH2, NH-CO-CH, CH-NH-CO). 19F NMR (DMSO-d6,
235.19 MHz): d ꢂ80.2 (3F, s, CF3), ꢂ114.3 (6F, s, 3 CF2),
ꢂ121.8 to ꢂ123.4 (22F, m, 11 CF2), ꢂ126 (2F, s, CF2). m/z
(FAB1MS): [M þ H1] 1753, [M þ Na1] 1775.
(CH2)2-CH2-NH, OCH2-CH2-NH, CH(2), CH(3), CH(4),
CH(5), CH2(6)), 4.27–4.30 (2H, m, CH(1), CH-CH2). 13C
NMR (CD3OD, 62.86 MHz): d 22,4–30,7 (CH2 chain, CF2-
CH2-CH2-CO, CH2-CF2, CF2-CH2-CH2-CH2-NH, CH-CH2-
CH2-CH2), 35.7–42.0 (OCH2-CH2-NH, CF2-(CH2)2-CH2-
NH, CH2-NH31), 53.7 (CH-CH2), 61.1 (CH2(6)), 68.2–75.3
(CH(2), CH(3), CH(4), CH(5), OCH2-CH2), 103.7 (C1b), 106.9–
123.2 (CF2, CF3), 171.5–173.1–175.1 (3 NH-CO).19F NMR
(CD3OD, 235.19 MHz): d ꢂ76.8 (3F, s, CF3-CO2ꢂ), ꢂ82.2
(3F, s, CF3), ꢂ115.3 (6F, s, 3 CF2), ꢂ122.6 to ꢂ124.3 (22F, m,
11 CF2), ꢂ127 (2F, s, CF2). m/z (FAB1MS): [M1] 1451
(without CF3COOꢂ). Elemental analysis calculated for
C49H60F36N4O11 ꢄ 2H2O: C: 36.76, H: 4.03, N: 3.50; found C:
37.19, H: 4.14, N: 3.72%.
N - [2-(b-D-Galactopyranosyloxy)ethyl]-22-[2-(4,4,5,5,6,6,7,7,
8, 8, 9, 9, 10, 10,11,11,11-heptadecafluoroundecanamido)-6-(ben-
zyloxycarbonylamino)hexanamido] -12, 12, 13, 13, 14, 14, 15, 15,
16,16,17,17,18,18,19,19-hexadecafluorodocosanamide (deacety-
lated compound 8a). Compound 8a (180 mg, 0.103 mmol) was
dissolved in 10 mL of MeOH. A catalytic amount of sodium
methylate was added. After stirring for 6 h, IRC50 ion
exchange resin (1 g) was added. After 10 min the mixture
was filtered. The solvent was removed in vacuo and the crude
product purified by filtration by size exclusion chromatogra-
phy using LH20 (MeOH) to yield 153 mg (0.096 mmol, 94%)
of deacetylated compound 8a as a white solid. Rf: 0.67 (AcOEt
–MeOH–H2O 7 : 2 : 1). 1H NMR (CD3OD, 250 MHz): d 1.18–
1.85 (24H, m, CH2 chain, CH2-CH2-CH2-NH(Z), CF2-CH2-
CH2-CH2-NH, CH-CH2), 2.08–2.26 (6H, m, 2 CH2-CF2, NH-
CO-CH2), 2.46–2.64 (4H, m, C8F17-CH2-CH2), 3.11–3.97
(14H, m, CH2-NH(Z), CF2-(CH2)2-CH2-NH, OCH2-CH2-
N - [2 - ((2, 3, 4, 6 - Tetra - O-acetyl)-b-D-galactopyranosyloxy)
ethyl] - 22 - [2-(4,4,5,5,6,6,7,7,8,8,9,9,10,10,11,11,11-heptadeca-
fluoroundecanamido) - 3 - (Nim - (trityl)imidazoyl)propanamido]-
12, 12, 13, 13, 14, 14, 15, 15, 16, 16, 17,17,18,18,19,19-hexadeca-
fluorodocosanamide (8b). The same procedure as used for
compound 8a was applied to compound 8b. From compound
7 (226 mg, 0.20 mmol), compound 5b (209 mg, 0.24 mmol) and
TBTU (83.5 mg, 0.26 mmol), 277 mg (0.15 mmol, 73%) of
compound 8b was obtained as a white powder after purifica-
tion by flash chromatography on silica gel (AcOEt) and
filtration by size exclusion chromatography using LH20
(MeOH–CH2Cl2 1 : 1). Rf: 0.7 (AcOEt–MeOH 9.5 : 0.5),
mp: 72.7–74.5 1C. [a]2D0: þ2.76 (c 1, CH2Cl2). 1H NMR
(CDCl3, 250 MHz, 45 1C): d 1.25–1.32 (12H, m, CH2 chain),
1.62 (4H, m, NH-CO-CH2-CH2, CH2-CH2-CF2), 1.82 (2H, m,
CF2-CH2-CH2-CH2-NH), 2.00–2.22 (18H, m, CH3CO, 2 CH2-
CF2, NH-CO-CH2), 2.55 (4H, m, C8F17-CH2-CH2), 2.89 (1H,
m, CH-CH2), 3.05 (1H, m, CH-CH2), 3.31 (2H, m, CF2-
(CH2)2-CH2-NH), 3.50 (2H, m, OCH2-CH2-NH), 3.72 (1H,
m, OCH2-CH2-NH), 3.92 (2H, m, OCH2-CH2-NH, CH(5)),
4.16 (2H, d, CH2(6), J3 = 6.75 Hz), 4.50 (1H, d, CH(1)an, J3 =
7.85 Hz), 4.60 (1H, td, CH-CH2, J3 = 5.15 Hz), 5.05 (1H, dd,
CH(2), J3 = 3.35 Hz, J3 = 10.45 Hz), 5.21 (1H, dd, CH(3), J3
= 2.55 Hz, J3 = 10.4 Hz), 5.43 (1H, m, CH(4)), 5.86 (1H, m,
NH-CO-CH), 6.70 (1H, s, N-CH=Chis), 7.10–7.16 (6H, m, N-
CHQNhis, CHarom.), 7.29–7.38 (10H, m, CH arom.), 7.61 (1H,
d, CH-NH, J3 = 6.25 Hz), 7.69 (1H, s, NH). 13C NMR
(CDCl3, 62.86 MHz): d 20.1–20.8 (CF2-CH2-CH2-CH2-NH,
CH2-CH2-CF2, CH3CO), 25.7–30.9 (CH2 chain, NH-CO-CH2-
CH2, 2 CH2-CF2, C8F17-CH2-CH2, CH-CH2), 36.7 (NH-CO-
CH2), 38.5–39.1 (CF2-(CH2)2-CH2-NH, OCH2-CH2-NH),
53.5 (CH-CH2), 61.3 (CH2(6)), 67.0–70.8 (OCH2-CH2-NH,
CH(2), CH(3), CH(4), CH(5)), 75.5 (C(F)3), 101.5 (C1b), 106.2–
118.9 (CF2, CF3), 119.7 (N-CHQChis), 128.1–129.7 (CHarom.),
136.8 (N-CHQNhis), 138.2 (CH2-Chis), 142.1 (Carom.), 169.7–
NH, CH(2), CH(3), CH(4), CH(5), CH2(6)), 4.26 (2H, m, CH(1)
,
CH-CH2), 5.09 (2H, s, CH2-F), 7.33 (5H, s, CHarom.). 13C
NMR (CD3OD, 62.86 MHz): d 19.9–30.9 (CH2 chain, CF2-
CH2-CH2-CO, CH2-CF2, CF2-CH2-CH2-CH2-NH, CH-CH2-
CH2-CH2), 35.7–40.0 (OCH2-CH2-NH, CF2-(CH2)2-CH2-
NH, CH2-NH-(Z)), 54.1 (CH-CH2), 61.1 (CH2(6)), 65.9–73.3
(CH(2), CH(3), CH(4), CH(5), CH2-F, OCH2-CH2), 103.7 (C1b),
104.5–123.6 (CF2, CF3), 127.3–128.1 (CH arom.), 137 (C arom.),
157.6 (NH-CO-O), 168.5–175.0 (NH-CO-CH2, NH-CO-CH,
CH-NH-CO). 19F NMR (CD3OD, 235.19 MHz): d ꢂ82.2 (3F,
s, CF3), ꢂ115.1 (6F, s, 3 CF2), ꢂ122.6 to ꢂ124.3 (22F, m, 11
CF2), ꢂ127.0 (2F, s, CF2).
N-[2-(b-D-Galactopyranosyloxy)ethyl]-22-[2-(4, 4, 5,5,6,6,7,7,
8, 8, 9, 9, 10, 10, 11,11,11-heptadecafluoroundecanamido)-6-(am-
monio)hexanamido]-12,12,13,13,14, 14,15,15,16,16,17,17,18,
18,19,19-hexadecafluorodocosanamide trifluoroacetate (A).
Deacetylated compound 8a (153 mg, 0.0966 mmol) was dis-
solved in 10 mL of methanol. The solution was stirred and
cooled at 0 1C and palladium on charcoal (20 mg, 200 mg
mmolꢂ1) was added portionwise. TFA (22 mg, 0.193 mmol)
was added and the mixture was stirred under hydrogen
pressure (8 bar) for 48 h. The mixture was filtered over Celite
and the solvent removed in vacuo. The crude product was
dissolved in diethyl ether and the solvent removed in vacuo.
The operation was repeated twice until total removal of TFA
was achieved. The crude product was purified by filtration by
size exclusion chromatography using LH20 (MeOH) to yield
142 mg (0.090 mmol, 94%) of final compound A as a white
solid, mp (dec) > 123 1C. [a]2D0: ꢂ3.07 (c 1, MeOH). 1H NMR
(CDCl3, 250 MHz): d 1.35–1.86 (24H, m, CH2 chain, CH2-CH2-
CH2-NH31, CF2-CH2-CH2-CH2-NH, CH-CH2), 2.07–2.25
173.2 (CH3CO, NH-CO-CH2, NH-CO-CH, CH-NH-CO). 19
F
NMR (CDCl3, 235.19 MHz): d ꢂ80.7 (3F, s, CF3), ꢂ114.2 to
ꢂ114.6 (6F, m, CH2-CF2, CF2), ꢂ121.9 to ꢂ123.5 (22F, m,
CF2), ꢂ126.1 (2F, s, CF2-CF3). m/z (FAB1MS): [M þ H1]
1870, [M þ Na1] 1892.
N - [2-(b-D-Galactopyranosyloxy)ethyl]-22-[2-(4,4,5,5,6,6,7,7,
8, 8, 9, 9, 10,10,11,11,11-heptadecafluoroundecanamido)-3-(Nim-
(trityl)imidazoyl)propanamido]-12,12,13,13,14,14, 15,15,16,16,
17,17,18,18,19,19-hexadecafluorodocosanamide (deacetylated
(6H, m, 2 CH2-CF2, NH-CO-CH2), 2.44–2.67 (4H, m,
1
C8F17-CH2-CH2), 2.90–3.96 (14H, m, CH2-NH3 , CF2-
ꢀc
640 | New J. Chem., 2006, 30, 629–646 This journal is the Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2006

View Article Online
compound 8b). The same procedure as used for the deacetyla-
tion of compound 8a was applied to deacetylated compound
8b. From compound 8a (267 mg, 0.143 mmol), 228 mg of
deacetylated compound 8b (0.13 mmol, 94%) was obtained as
a white powder after purification by filtration by size exclusion
chromatography using LH20 (MeOH). Rf: 0.63 (AcOEt–
CF2), ꢂ127.2 (2F, s, CF2). m/z (FAB1MS): [M1] 1460 (with-
out CF3COOꢂ). Elemental analysis calculated for
C49H55F36N5O11: C: 37.39, H: 3.50, N: 4.45; found C: 37.79,
H: 3.74, N: 4.38%.
N-Dodecyl-[Na-((9H-fluorenyl)methyloxycarbonyl)-Nim-(tri-
tyl)]histidinamide (9). Fmoc-Hist(Trt)OH (3 g, 4.84 mmol),
DCC (1.2 g, 5.81 mmol) and HOBT (0.785 g, 5.81 mmol) were
dissolved in 30 mL of DCM freshly distilled and degassed
under argon. After stirring for 15 min, dodecylamine (0.986 g,
5.33 mmol) was added and the mixture stirred for 2 d. The
mixture was then filtered and the solvent removed from the
filtrate in vacuo. The crude product was purified by flash
chromatography on silica gel (AcOEt–cHex 4 : 6) to yield
3.14 g (3.99 mmol, 82%) of compound 9 as a white solid. Rf:
0.35 (AcOEt–cHex 4 : 6), mp: 59–61 1C. 1H NMR (CDCl3, 250
MHz): d 0.90 (3H, t, CH3, J3 = 6.86 Hz), 1.21–1.30 (20H, m,
CH2 chain), 2.93 (1H, m, CH-CH2 his), 3.18–3.33 (3H, m, CH-
CH2 his, CO-NH-CH2), 4.11–4.33 (3H, m, CH-CH2 Fmoc), 4.65
(1H, m, CH-CH2 hist), 5.79 (2H, m, 2 NH), 6.72 (1H, s, N–CH
QChis), 7.04–7.78 (24H, m, CHTrt., CHFmoc., N–CHQNhis).
13C NMR (CDCl3, 62.86 MHz): d 14.2 (CH3), 22.7–31.9
(CH2 chain, CH-CH2 his), 39.8 (CO-NH-CH2), 47.1 (CH
-CH2 Fmoc), 55.0 (CH-CH2 his), 67.2 (CH-CH2 Fmoc), 76.3
(C(F)3), 119.1–129.6 (N–CHQChis, CHarom.(Trt), CHarom. (Fmoc)),
1
MeOH–H2O 7 : 2 : 1). H NMR (CD3OD, 250 MHz): d 1.31
–1.47 (12H, m, CH2 chain), 1.62 (4H, m, NH-CO-CH2-CH2,
CH2-CH2-CF2), 1.80 (2H, m, CF2-CH2-CH2-CH2-NH), 2.05–
2.25 (6H, m, 2 CH2-CF2, NH-CO-CH2), 2.39–2.49 (4H, m,
C8F17-CH2-CH2), 2.84 (1H, dd, CH-CH2), 2.92 (1H, dd, CH-
CH2), 3.21–3.89 (12H, m, CF2-(CH2)2-CH2-NH, OCH2-CH2-
NH, CH(2), CH(3), CH(4), CH(5), CH2(6)), 4.26 (1H, d, CH(1), J3
= 6.85 Hz), 4.54 (1H, m, CH-CH2), 6.76 (1H, s, N-CHQChis),
7.12–7.16 (6H, m, N–CHQNhis, CHarom.), 7.36–7.41 (10H, m,
CHarom.), 8.54 (3H, m, NH). 13C NMR (CD3OD, 62.86 MHz):
d 19.9 (CF2-CH2-CH2-CH2-NH, CH2-CH2-CF2), 25.6–30.4
(CH2 chain, NH-CO-CH2-CH2, 2 CH2-CF2, C8F17-CH2-CH2,
CH-CH2), 35.7 (NH-CO-CH2), 37.9–39.2 (CF2-(CH2)2-CH2-
NH, OCH2-CH2-NH), 53.9 (CH-CH2), 61.1 (CH2(6)), 68.3–
75.5 (OCH2-CH2-NH, CH(2), CH(3), CH(4), CH(5), C(F)3),
103.7 (C1b), 106.9–119.3 (CF2, CF3), 119.7 (N–CHQChis),
127.9–129.5 (CHarom.), 136.2 (N–CHQNhis), 138.1 (CH2-Chis),
142.2 (Carom.), 171–175 (NH-CO-CH2, NH-CO-CH, CH-NH-
CO). 19F NMR (CD3OD, 235.19 MHz): d ꢂ82.2 (3F, s, CF3),
ꢂ115.1 (6F, s, 3 CF2), ꢂ122.5 to ꢂ124.3 (22F, m, 11 CF2),
ꢂ126.9 (2F, s, CF2).
135.0–137.5 (N–CHQN , CH2-Chis), 141.3–143.9 (Carom. (Trt)
,
his
Carom. (Fmoc)), 156.2 (O-CO-NH), 170.9 (CO-NH-CH2).
N-Dodecyl-[Na-(benzyloxycarbonyl)-Ne-(tert-butyloxycarbo-
N-[2-(b-D-Galactopyranosyloxy)ethyl]-22-[2-(4, 4, 5,5,6,6,7,7,
8,8,9,9,10,10,11,11,11-heptadecafluoroundecanamido)-3-(imida-
zolio)propanamido]-12,12,13,13,14,14,15,15,16,16, 17,17,18,18,
19,19-hexadecafluorodocosanamide trifluoroacetate (B). Deace-
tylated compound 6a (213 mg, 0.125 mmol) was dissolved in 5
mL of ethanol. Et3SiH (15.5 mg, 0.125 mmol) and 15 mL of
DCM–TFA 8 : 2 were added to the mixture. After stirring for
two days, ethanol and TFA were removed in vacuo. The crude
product was dissolved in diethyl ether and the solvent removed
in vacuo. The operation was repeated twice. 175 mg of final
compound B (0.11 mmol, 89%) was obtained as a white
powder after purification by filtration by size exclusion chro-
matography using LH20 (MeOH), mp (dec) > 145 1C. [a]2D0:
nyl)]lysinamide (10). Z-Lys(Boc)OH (1 g, 2.63 mmol), DCC
(0.651 g 3.16 mmol) and HOBT (0.427 g, 3.16 mmol) were
dissolved in 20 mL of freshly distilled DCM. After stirring for
15 min under argon, dodecylamine (0.532 g, 2.89 mmol) was
added and the mixture was stirred for 2 d at room tempera-
ture. The mixture was filtered and the solvent removed from
the filtrate in vacuo. The crude product was purified by flash
chromatography on silica gel (AcOEt–cHex 4 : 6) to yield 1.3 g
(2.3 mmol, 90%) of compound 10 as a white solid. Rf: 0.49
(AcOEt–cHex 4 : 6), mp: 83–85 1C. [a]2D0: þ2.77 (c 1, CH2Cl2).
1H NMR (CDCl3, 250 MHz): d 0.90 (3H, t, CH3, J3 = 6.77
Hz), 1.14–1.37 (20H, m, CH2 chain, CH-CH2-CH2), 1.44 (9H,
m, C(CH3)3), 1.59–1.91 (6H, m, 2 CH2-CH2-NH, CH2-CH),
3.10 (2H, m, CO-NH-CH2), 3.24 (2H, m, CH2-NH(Boc)), 4.15
(1H, m, CH-CH2), 4.65 (1H, s, NH), 5.12 (2H, s, CH2-F), 5.55
(1H, s, NH), 6.19 (1H, s, NH), 7.31 (5H, s, CHarom.). 13C
1
þ2.61 (c 1, CH2Cl2). H NMR (CD3OD, 250 MHz): d 1.23
(12H, m, CH2 chain), 1.50 (4H, m, NH-CO-CH2-CH2, CH2-
CH2-CF2), 1.70 (2H, m, CF2-CH2-CH2-CH2-NH), 2.07–2.13
(6H, m, 2 CH2-CF2, NH-CO-CH2), 2.39–2.46 (4H, m, C8F17-
CH2-CH2), 2.89–3.85 (14H, m, CH-CH2, CF2-CH2-CH2-CH2-
NH, OCH2-CH2-NH, CH(2), CH(3), CH(4), CH(5), CH2(6)),
4.17 (1H, d, CH(1)), 4.52 (1H, m, CH-CH2), 7.21 (1H, s, N–CH
QChis), 8.62 (1H, s, N–CHQNhis). 13C NMR (CD3OD, 62.86
MHz): d 19.9 (CF2-CH2-CH2-CH2-NH, CH2-CH2-CF2), 25.6
–39.1 (CH2 chain, NH-CO-CH2-CH2, 2 CH2-CF2, C8F17-CH2-
CH2, CH-CH2), 35.7 (NH-CO-CH2), 38.0–39.2 (CF2-(CH2)2-
CH2-NH, OCH2-CH2-NH), 52.8 (CH-CH2), 61.1 (CH2(6)),
68.2–75.4 (OCH2-CH2-NH, CH(2), CH(3), CH(4), CH(5)),
103.7 (C1b), 106.1–119.2 (CF2), 117.9 (N–CHQChis), 130.2
(N–CHQNhis), 133.8 (CH2-Chis), 171.0–175.4 (NH-CO-CH2,
NH-CO-CH, CH-NH-CO). 19F NMR (CD3OD, 235.19
MHz): d ꢂ76.8 (3F, s, CF3-CO2ꢂ), ꢂ82.3 (3F, s, CF3),
ꢂ115.4 to ꢂ115.8 (6F, s, CF2), ꢂ122.8 to ꢂ124.6 (22F, m,
NMR (CDCl3, 62.86 MHz):
d 14.1 (CH3), 22.4–31.1
(CH2 chain, CH-CH2-(CH2)2, C(CH3)3), 33.9 (CO-NH-CH2),
39.6 (CH2-NH(Boc)), 49.1 (CH-CH2), 54.9 C(CH3)3),
67.0 (CH2-F), 128.0–128.5 (CHarom.), 132.2 (Carom.), 156.2
(HN-CO-O), 171.5 (CH-CO-NH).
N-[1-(Dodecylcarbamoyl)-4-(tert-butyloxycarbonylamino)bu-
tyl] - 22 -(benzyloxycarbonylamino)-12,12,13,13,14,14,15,15,16,
16,17,17,18,18,19,19-hexadecafluorodocosanamide (11a). Com-
pound 10 (300 mg, 0.548 mmol) was dissolved in 10 mL of
ethanol. The solution was stirred and cooled at 0 1C and
palladium on charcoal (33 mg, 60 mg mmolꢂ1) was added
portionwise. The mixture was stirred under hydrogen pressure
(8 bar) for 6 h at room temperature. The mixture was filtered
over Celite and the solvent removed in vacuo. The crude
ꢀc
This journal is the Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2006
New J. Chem., 2006, 30, 629–646 | 641

View Article Online
product was dissolved in 5 mL of freshly distilled DCM.
Compound 3b (327.8 mg, 0,422 mmol) and TBTU reagent
(176 mg, 0.548 mmol) were added. The mixture was basified to
a pH of 8 with DIEA. After stirring for 12 h under argon at
room temperature, the solvent was removed in vacuo and the
crude product was purified by flash chromatography on silica
gel (cHex–AcOEt 5 : 5 to cHex–AcOEt 5 : 5) to yield 415.3 mg
(0.35 mmol, 84%) of compound 11a as a white solid. Rf: 0.5
(AcOEt–cHex 5 : 5), mp: 90–91 1C. 1H NMR (CDCl3, 250
MHz): d 0.89 (3H, t, CH3, J3 = 6.82 Hz), 1.17–1.35 (32H, m,
CH2 chain, CH-CH2-CH2), 1.45 (9H, s, C(CH3)3), 1.51–1.70
(10H, m, 3 CH2-CH2-NH, CF2-CH2-CH2, CH2-CH2-CO-
NH), 1.82–2.24 (8H, m, CH-CH2, 2 CH2-CF2, CH2-CO-
NH), 3.09 (2H, m, CH2-NH-CO-CH), 3.18–3.32 (4H, m,
235.19 MHz): d ꢂ114.0 to ꢂ114.3 (4F, d, 2 CH2-CF2),
ꢂ121.7 (8F, m, 4 CF2), ꢂ123.4 (4F, s, 2 CF2).
N-[1-(Dodecylcarbamoyl)-4-(tert-butyloxycarbonylamino)bu-
tyl] - 22 - [(2, 3, 5,6,20,30,40,60-octa-O-acetyl)lactobionamido]-12,
12, 13, 13, 14, 14,15,15,16,16,17,17,18,18,19,19-hexadecafluoro-
docosanamide (12a). Compound 11a (375 mg, 0.320 mmol)
was dissolved in 10 mL of ethanol. The solution was stirred
and cooled at 0 1C and palladium on charcoal (70 mg, 220 mg
mmolꢂ1) was added portionwise. The mixture was stirred
under hydrogen pressure (8 bar) for 48 h at room temperature.
The mixture was filtered over Celite and the solvent removed
in vacuo to obtain the amine. At the same time, lactobionic
acid (168 mg, 4.705 mmol) was dissolved in 20 mL of a mixture
of toluene–2-methoxyethanol 1 : 1. A few drops of TFA were
added and the solvents were removed in vacuo. The operation
was repeated twice and the crude product obtained was added
to the previous amino compound. The mixture was dissolved
in 20 mL of methanol under argon and basified at pH = 8–9
with few drops of TEA. The mixture was refluxed under argon
for 12 h. The solvent was removed in vacuo and a mixture of
acetic anhydride–pyridine 1 : 1 (20 mL) was added. The
reaction was stirred for 12 h at room temperature. The mixture
was poured into cooled 1 M HCl solution and the solution
extracted with ethyl acetate. The organic layer was washed
with saturated sodium hydrogencarbonate solution, then with
brine. The organic layer was dried over sodium sulfate and the
solvent removed in vacuo. The crude product was purified by
flash chromatography on silica gel (AcOEt–cHex 5 : 5 to
AcOEt–cHex 8 : 2) to yield 370 mg (0.21 mmol, 67%) of fully
protected compound 12a as a white solid. Rf: 0.07 (AcOEt–
cHex 5 : 5), mp: 83–84 1C. [a]2D0: þ5.09 (c 1, CH2Cl2). 1H NMR
(CDCl3, 250 MHz): d 0.89 (3H, t, CH3, J3 = 6.85 Hz), 1.28–
1.40 (32H, m, CH2 chain, CH-CH2-CH2), 1.45 (9H, s,
C(CH3)3), 1.49–1.66 (10H, m, 3 CH2-CH2-NH, CF2-CH2-
CH2, CH2-CH2-CO-NH), 1.80–2.21 (32H, m, CH-CH2, 8
CO-CH3, 2 CH2-CF2, CH2-CO-NH), 3.09–3.42 (6H, m,
CH2-NH-CO-CH, CH2-NH(Boc), NH-CH2-(CH2)2-CF2),
CH2-NH(Boc), (Z)NH-CH2), 4.41 (1H, td, CH-CH2, J3
=
6.42 Hz, J3 = 7.37 Hz), 4.71 (1H, s, NH), 5.00 (1H, s, NH),
5.12 (2H, s, CH2-F), 6.43 (1H, d, NH-CH-CH2, J3 = 7.35 Hz),
6.60 (1H, s, NH), 7.35 (5H, s, CHarom.). 13C NMR (CDCl3,
62.86 MHz): d 14.0 (CH3), 20.1–31.1 (CH2 chain, CH-CH2-
CH2, C(CH3)3, 3 CH2-CH2-NH, CH2-CH2-CO-NH, 2 CH2-
CF2), 36.6–40.2 (CH2-NH-CO-CH, CH2-NH(Boc), NH-CH2-
(CH2)2-CF2), 52.3 (CH-CH2), 66.9 (CH2-F), 79.1 (C(CH3)3),
106.9–119.0 (CF2), 128.1–128.6 (CHarom.), 136.4 (Carom.),
156.2 (NH-CO-O), 156.5 (NH-CO-O), 171.7 (CO-NH), 173.4
(CO-NH). 19F NMR (CDCl3, 235.19 MHz): d ꢂ114.1 to
ꢂ114.3 (4F, d, 2 CH2-CF2), ꢂ121.8 (8F, m, 4 CF2), ꢂ123.5
(4F, s, 2 CF2).
N-[1-(Dodecylcarbamoyl)-2-(Nim-(trityl)imidazolyl)ethyl]-22-
(benzyloxycarbonylamino)-12, 12, 13, 13, 14, 14, 15,15,16,16,17,
17,18,18,19,19-hexadecafluorodocosanamide (11b). Compound
9 (607 mg, 0.772 mmol) was dissolved in 10 mL of a mixture of
acetonitrile–DIEA 9 : 1. After stirring for 1 h at room
temperature, the solvent was removed in vacuo. The crude
product was dissolved in 5 mL of dry DCM under argon.
Compound 3b (400 mg, 0.515 mmol) and TBTU reagent (215
mg, 0.669 mmol) were added to the solution. The mixture was
basified to pH = B8–9 with few drops of DIEA and stirred
for 12 h under argon at room temperature. The solvent was
removed in vacuo and the crude product was purified by flash
chromatography on silica gel (AcOEt–cHex 3 : 7 to AcOEt–
cHex 6 : 4) and filtration by size exclusion chromatography
using LH20 (MeOH) to yield 525 mg (0.39 mmol, 77%) of
compound 11b as a white solid. Rf: 0.43 (AcOEt–cHex 5 : 5),
mp: 87–88 1C. 1H NMR (CDCl3, 250 MHz): d 0.89 (3H, t,
CH3, J3 = 6.84 Hz), 1.20–1.50 (32H, m, CH2 chain), 1.53–1.62
(4H, m, CF2-CH2-CH2, CH2-CH2-CO-NH), 1.82 (2H, m, NH-
CH2-CH2-CH2-CF2), 1.87–2.23 (6H, m, 2 CH2-CF2, CH2-CO-
NH), 2.93 (2H, m, CH-CH2), 3.17 (2H, m, CH2-NH-CO), 3.26
(2H, m, NH-CH2-(CH2)2-CF2), 4.68 (1H, td, CH-CH2, J3 =
6.1 Hz, J3 = 6.56 Hz), 5.08 (1H, s, NH), 5.10 (2H, s, CH2-F),
6.68 (1H, s, CQCH–Nhist), 7.09–7.42 (23H, m, CHarom., N–
CHQNhis, 2 NH). 13C NMR (CDCl3, 62.86 MHz): d 14.1
(CH3), 20.1–31.9 (CH2 chain, 2 CH2-CF2, 2 NH-CH2-CH2,
CH-CH2), 36.6–42.0 (CH2-CO-NH, 2 CH2-NH-CO), 53.1
(CH-CH2), 66.7 (CH2-F), 75.6 (C(F)3), 106.0–118.7 (CF2),
119.0 (N–CHQChis), 128.1–129.7 (CHarom.), 136.7–137.9 (N–
CHQNhis, CH2-Chis), 142.1 (Carom.), 156.6 (O-CO-NH), 171.1
–173.3 (CH2-CO-NH, NH-CO-CH). 19F NMR (CDCl3,
0
0
3.93 (1H, m, CH(5 )), 4.06–4.39 (3H, m, CH2(6 ), CH(6)), 4.31–
4.39 (2H, m, CH-CH2, CH(4)), 4.47–4.63 (2H, m, CH(6), NH),
4.70 (1H, d, CH(1 ), J3 = 7.80 Hz), 4.96–5.20 (3H, m, CH(2 )
,
,
0
0
0
0
CH(3 ), CH(5)), 5.39 (1H, m, CH(4 )), 5.56–5.60 (2H, m, CH(2)
CH(3)), 6.20–6.33 (3H, m, NH). 13C NMR (CDCl3, 62.86
MHz): d 14.0 (CH3), 20.4–31.9 (CH2 chain, CH-CH2-CH2,
C(CH3)3, 3 CH2-CH2-NH, CH2-CH2-CO-NH, 2 CH2-CF2,
CO-CH3), 36.6–39.6 (CH2-NH-CO-CH, CH2-NH(Boc), NH-
0
CH2-(CH2)2-CF2), 52.9 (CH-CH2), 60.9 (CH2(6 )), 61.8
0
0
,
(CH2(6)), 66.9–71.6 (CH(2), CH(3), CH(5), CH(2 ), CH(3 )
0
0
0
CH(4 ), CH(5 )), 77.4 (CH(4)), 79.1 (C(CH3)3), 101.6 (C1 b),
106.9–123.0 (CF2), 156.1 (NH-CO-O), 167.4–173.2 (3 CO-
NH, CO-CH3). 19F NMR (CDCl3, 235.19 MHz): d ꢂ114.1
to ꢂ114.4 (4F, d, 2 CH2-CF2), ꢂ121.7 (8F, m, 4 CF2), ꢂ123.4
(4F, s, 2 CF2). m/z (FAB1MS): [M þ 2H1] 1716.
N-[1-(Dodecylcarbamoyl)-4-(tert-butyloxycarbonylamino)bu-
tyl]-22-lactobionamido-12,12,13,13,14,14,15,15,16,16,17,17,18,
18,19,19-hexadecafluorodocosanamide (deacetylated compound
12a). The same procedure as used for the deacetylation of
compound 8a was applied to compound 12a. From compound
12a (320 mg, 0.187 mmol), we obtained after purification by
ꢀc
642 | New J. Chem., 2006, 30, 629–646 This journal is the Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2006

View Article Online
filtration by size exclusion chromatography usingLH20
(MeOH), 237 mg of deacetylated compound 12a (0.17 mmol,
92%). Rf: 0.72 (AcOEt–MeOH–H2O 7 : 2 : 1). 1H NMR
(CD3OD, 250 MHz): d 0.92 (3H, t, CH3, J3 = 6.85 Hz), 1.32–
1.38 (32H, m, CH2 chain, CH-CH2-CH2), 1.45 (9H, s,
C(CH3)3), 1.49–1.62 (10H, m, 3 CH2-CH2-NH, CF2-CH2-
CH2, CH2-CH2-CO-NH), 1.86 (2H, m, CH-CH2), 2.05–2.29
(6H, m, 2 CH2-CF2, CH2-CO-NH), 3.04 (2H, td, CH2-
NH(Boc), 3.19 (2H, td, CH2-NH-CO-CH), 3.33–4.93 (13H,
mide (12b). The same procedure as used for compound 12a
was applied to compound 12b. From compound 11b (373 mg,
0.282 mmol) and lactobionic acid (150 mg, 0.410 mmol), after
acetylation, the crude product was purified by flash chroma-
tography on silica gel (AcOEt–cHex 7 : 3 to AcOEt) and
filtration by size exclusion chromatography using LH20
(MeOH) to yield 296 mg (0.16 mmol, 58%) of compound
12b as a white solid. Rf: 0.41 (AcOEt), mp: 80–82 1C. [a]2D0:
þ8.12 (c 1, CH2Cl2). 1H NMR (CDCl3, 250 MHz): d 0.88 (3H,
t, CH3, J3 = 6.8 Hz), 1.25–1.43 (32H, m, CH2 chain), 1.52–1.59
(4H, m, CF2-CH2-CH2, CH2-CH2-CO-NH), 1.83 (2H, m, NH-
CH2-CH2-CH2-CF2), 1.99–2.25 (30H, m, CO-CH3, 2 CH2-
CF2, CH2-CO-NH), 2.91–3.8 (6H, m, CH-CH2, CH2-NH-CO,
0
0
,
m, NH-CH2-(CH2)2-CF2, CH(4), CH(5), CH2(6), CH(2 ), CH(3 )
0
0
0
CH(4 ), CH(5 ), CH2(6 ), NH), 4.25–4.28 (2H, m, CH-CH2,
CH(2)), 4.38 (1H, m, CH(3)), 4.50 (1H, d, CH(1 ), J3 = 7,23
0
Hz), 6.58 (1H, s, NH), 7.95–8.10 (2H, m, NH). 13C NMR
(CDCl3, 62.86 MHz): d 12.9 (CH3), 20.1–31.7 (CH2 chain, CH-
CH2-CH2, C(CH3)3, 3 CH2-CH2-NH, CH2-CH2-CO-NH, 2
CH2-CF2), 35.9–39.8 (CH2-NH-CO-CH, CH2-NH(Boc), NH-
NH-CH2-(CH2)2-CF2), 3.93–4.33 (5H, m, CH(4)
,
CH(6)
,
CH(5 ), CH2(6 )), 4.53 (1H, dd, CH(6), J2 = 2.25 Hz, J3
=
0
0
12.15 Hz), 4.65 (1H, d, CH(1’), J3 = 7.85 Hz), 4.99–5.19 (4H,
0
CH2-(CH2)2-CF2), 53.3 (CH-CH2), 61.2 (CH2(6 )), 62.4
0
0
0
m, CH-CH2, CH(2 ), CH(3 ), CH(5)), 5.40 (1H, m, CH(4 )), 5.53–
5.58 (2H, m, CH(2), CH(3)), 6.43 (2H, m, NH), 6.78 (1H, s, CQ
CH-Nhis), 7.24–7.44 (17H, m, CHarom., N–CHQNhis, NH).
13C NMR (CDCl3, 62.86 MHz): d 14.1 (CH3), 20.1–20.8 (CO-
CH3), 22.7–31.9 (CH2 chain, 2 CH2-CF2, 2 NH-CH2-CH2, CH-
CH2), 36.4–39.9 (CH2-CO-NH, 2 CH2-NH-CO), 53.3 (CH-
0
0
,
(CH2(6)), 69.0–75.8 (CH(2), CH(3), CH(5), CH(2 ), CH(3 )
0
0
0
CH(4 ), CH(5 )), 79.2 (C(CH3)3), 81.9 (CH(4)), 104.4 (C1 b),
106.9–122.8 (CF2), 157.1 (NH-CO-O), 172.9, 174.1, 174.8 (3
CO-NH). 19F NMR (CD3OD, 235.19 MHz): d ꢂ115.2 (4F, s,
2 CH2-CF2), ꢂ122.7 (8F, m, 4 CF2), ꢂ124.4 (4F, s, 2 CF2).
0
CH2), 60.9 (CH2(6 )), 61.8 (CH2(6)), 66.8–71.6 (CH(2), CH(3)
,
N-[1-(Dodecylcarbamoyl)-4-(ammonio)butyl]-22-(lactobiona-
mido)-12,12,13,13,14,14,15,15,16,16,17,17,18,18,19,19-hexade-
cafluorodocosanamide trifluoroacetate (C). Deacetylated
compound 12a (200 mg, 0.145 mmol) was dissolved in 8 mL
of DCM. 2 mL of TFA was added dropwise at room tem-
perature. After stirring for 48 h, the solvents were removed in
vacuo. The crude product was dissolved in diethyl ether and
the solvent removed in vacuo. The operation was repeated
twice. The crude product was purified by filtration by size
exclusion chromatography using LH20 (MeOH) to yield 184
mg of final compound C (0.13 mmol, 91%) as a white powder,
mp (dec) > 109 1C. [a]2D0: þ1.06 (c 1, MeOH). 1H NMR
(CD3OD, 250 MHz): d 0.92 (3H, t, CH3, J3 = 6.84 Hz), 1.31–
1.35 (32H, m, CH2 chain, CH-CH2-CH2), 1.46–1.73 (10H, m, 3
CH2-CH2-NH, CF2-CH2-CH2, CH2-CH2-CO-NH), 1.83 (2H,
m, CH-CH2), 2.14–2.30 (6H, m, 2 CH2-CF2, CH2-CO-NH),
2.94 (2H, t, CH2-NH31, J3 = 7.60 Hz), 3.19–3.93 (1H, m,
0
0
0
0
CH(5), CH(2 ), CH(3 ), CH(4 ), CH(5 )), 76.5 (C(F)3), 78.8
0
(CH(4)), 101.7 (C1 b), 107.1–118.8 (CF2), 120.7 (N–CHQChis),
127.9–129.6 (CHarom.), 132.7–134.5 (N–CHQNhist, CH2-Chis),
139.5 (Carom.), 167.6–174.0 (3 NH-CO, CO-CH3). 19F NMR
(CDCl3, 235.19 MHz): d ꢂ114.0 to ꢂ114.3 (4F, d, 2 CH2-
CF2), ꢂ121.7 (8F, m, 4 CF2), ꢂ123.4 (4F, s, 2 CF2). m/z
(FABþ): [M þ H]1 = 1866.
N-[1-(Dodecylcarbamoyl)-2-(Nim-(trityl)imidazolyl)ethyl]-22-
(lactobionamido)-12,12,13,13,14,14,15,15,16,16,17,17,18,18,19,
19-hexadecafluorodocosanamide (deacetylated compound 12b).
The same procedure as used for the deacetylation of com-
pound 8a was applied to compound 12b. From compound 12b
(250 mg, 0.134 mmol), we obtained after purification by
filtration by size exclusion chromatography using LH20
(MeOH), 193 mg of deacetylated compound 12b (0.126 mmol,
94%). Rf: 0.69 (AcOEt–MeOH–H2O 7 : 2 : 1). 1H NMR
(CD3OD, 250 MHz): d 0.90 (3H, t, CH3, J3 = 6.74 Hz), 1.24–
1.65 (36H, m, CH2 chain, CF2-CH2-CH2, CH2-CH2-CO-NH),
1.86 (2H, m, NH-CH2-CH2-CH2-CF2), 2.02–2.24 (6H, m, 2
CH2-CF2, CH2-CO-NH), 2.83–3.16 (4H, m, CH-CH2, CH2-
CH2-NH-CO-CH, NH-CH2-(CH2)2-CF2, CH(4)
,
CH(5)
,
0
0
0
0
0
CH2(6), CH(2 ), CH(3 ), CH(4 ), CH(5 ), CH2(6 )), 4.25–4.38
(3H, m, CH-CH2, CH(2), CH(3)), 4.51 (1H, d, CH(1’), J3
=
7.10 Hz). 13C NMR (CD3OD, 62.86 MHz): d 12.7 (CH3), 29.9
–31.7 (CH2 chain, CH-CH2-CH2, 3 CH2-CH2-NH, CH2-CH2-
NH-CO), 3.32–3.97 (12H, m, NH-CH2-(CH2)2-CF2, CH(4)
,
CO-NH, 2 CH2-CF2), 35.4–39.1 (CH2-NH-CO-CH, CH2-
1
0
0
0
0
0
CH(5), CH2(6), CH(2 ), CH(3 ), CH(4 ), CH(5 ), CH2(6 )), 4.26
(1H, m, CH(2)), 4.38 (1H, m, CH(3)), 4.61 (1H, d, CH(1 ), J3 =
NH3
,
NH-CH2-(CH2)2-CF2), 53.0 (CH-CH2), 61.2
0
0
(CH2(6 )), 62.3 (CH2(6)), 69.0–75.8 (CH(2), CH(3), CH(5)
,
7.3 Hz), 4.61 (4H, m, CH-CH2), 6.76 (1H, s, CQCH-Nhis),
7.10–7.39 (16H, m, CHarom., N–CHQNhis, NH). 13C NMR
(CD3OD, 62.86 MHz): d 13.1 (CH3), 19.9–7–31.7 (CH2 chain, 2
CH2-CF2, 2 NH-CH2-CH2, CH-CH2), 35.6–39.1 (CH2-CO-
0
0
0
0
0
CH(2 ), CH(3 ), CH(4 ), CH(5 )), 81.9 (CH(4)), 104.4 (C1 b), 106.8
–122.9 (CF2), 172.7, 174.1, 174.9 (3 CO-NH). 19F NMR
(CD3OD, 235.19 MHz): d ꢂ76.8 (3F, s, ꢂO2C-CF3), ꢂ115.3
(4F, s, 2 CH2-CF2), ꢂ122.7 (8F, m, 4 CF2), ꢂ124.4 (4F, s, 2
CF2). m/z (ESI-MS) [M]1 = 1279.8 (without CF3COOꢂ).
Elemental analysis calculated for C54H87F19N4O15 ꢄ 1H2O: C:
45.96, H: 6.36, N: 3.97; found C: 45.97, H: 6.44, N:
4.20%.
0
NH, 2 CH2-NH-CO), 53.3 (CH-CH2), 61.3 (CH2(6 )), 62.4
0
0
,
(CH2(6)), 69.0–75.8 (CH(2), CH(3), CH(5), CH(2 ), CH(3 )
0
0
0
CH(4 ), CH(5 ), C(F)3), 82.0 (CH(4)), 104.4 (C1 b), 106.5–118.8
(CF2), 119.6 (N–CHQChis), 127.3–129.5 (CHarom.), 136.4–
138.0 (N–CHQNhis, CH2-Chis), 142.3 (Carom.), 172.0–174.1–
175.5 (3 NH-CO). 19F NMR (CD3OD, 235.19 MHz): d ꢂ114.8
(4F, s, 2 CH2-CF2), ꢂ122.4 (8F, m, 4 CF2), ꢂ124.1 (4F, s, 2
CF2).
N-[1-(Dodecylcarbamoyl)-2-(Nim-(trityl)imidazolyl)ethyl]-22-
[(2, 3, 5,6,20,30,40,60-octa-O-acetyl)lactobionamido]-12,12,13,13,
14, 14, 15, 15, 16,16,17,17,18,18,19,19-hexadecafluorodocosana-
ꢀc
This journal is the Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2006
New J. Chem., 2006, 30, 629–646 | 643

View Article Online
N - [1 - (Dodecylcarbamoyl)-2-(imidazolio)ethyl]-22-(lactobio-
namido)-12,12,13,13,14,14,15,15,16,16,17,17,18,18,19,19-hexa-
decafluorodocosanamide trifluoroacetate (D). Deacetylated
compound 12b (170 mg, 0.111 mmol) was dissolved in 5 mL
of ethanol. 15 mL of a mixture of DCM–TFA 8 : 2 and
Et3SiH (13 mg, 0.11 mmol) were added to the solution. After
stirring for 2 d at room temperature solvents were removed in
vacuo and the crude product dissolved in diethyl ether. The
solvent was removed in vacuo and the operation repeated
twice. The crude product was purified by filtration by size
exclusion chromatography using LH20 (MeOH) to yield 139
mg (0.099 mmol, 89%) of final compound D as a white solid,
mp (dec) >101 1C. [a]2D0: þ6.18 (c 1, MeOH). 1H NMR
(CD3OD, 250 MHz): d 0,90 (3H, t, CH3, J3 = 6.84 Hz), 1.29
–1.59 (36H, m, CH2 chain, CF2-CH2-CH2, CH2-CH2-CO-NH),
1.83 (2H, m, NH-CH2-CH2-CH2-CF2), 1.99–2.23 (6H, m, 2
CH2-CF2, CH2-CO-NH), 2.96–3.91 (16H, m, CH-CH2, CH2-
0
The crude product was purified by flash chromatography on
silica gel (AcOEt–cHex 1 : 9) to yield 340 mg (0.487 mmol,
58%) of compound 14 as a white powder. Rf: 0.49 (AcOEt–
cHex 3 : 7), 0.32 (AcOEt–cHex 2 : 8), mp: 78–79 1C. 1H NMR
(CDCl3, 250 MHz): d 1.30–1.45 (24H, m, CH2 chain), 1.53–1.65
(8H, m, HO2C-CH2-CH2, CH2-CH2-CO, 2 CH2-CH2-S), 2.33
–2.39 (4H, m, HO2C-CH2, CH2-CO), 2.54 (4H, t, CH2-S-CH2,
J3 = 7.17 Hz), 5.13 (2H, s, CH2-F), 7.33 (5H, s, CHarom.). 13
C
NMR (CDCl3, 62.86 MHz): d 24.7–25.0 (HO2C-CH2-CH2,
CH2-CH2-CO), 29.9–29.7 (CH2 chain), 32.2 (CH2-CO), 34.1–
34.3 (CH2-S-CH2, HO2C-CH2), 66.1 (CH2-F), 128.2–128.5
(CHarom.), 136.1 (Carom.), 173.8 (CH2-CO), 180.1 (HO2C-
CH2).
Benzyl 22-[2-((2,3,4,6-tetra-O-acetyl)-b-D-galactopyranosy-
loxy)ethylcarbamoyl]-12-thiadocosanoate (15). Compound 6
(960 mg, 1.83 mmol) was dissolved in 10 mL of ethanol. The
solution was stirred and cooled at 0 1C and palladium on
charcoal (109 mg, 60 mg mmolꢂ1) was added portionwise. The
mixture was stirred under hydrogen pressure (8 bar) for 12 h at
room temperature. The mixture was filtered over Celite and
the solvent removed in vacuo to obtain the amine (150) as a
white powder. It was dissolved in 10 mL of DCM freshly
distilled and degassed under argon. Then, compound 14 (750
mg, 1.52 mmol) and TBTU (587 mg, 1.83 mmol) were added
to the reaction mixture. The pH was adjusted to 8 with DIEA.
After stirring for 12 h at room temperature, 596 mg of amine
was added again. After 12 h, the solvent was removed in vacuo.
The crude product was purified by flash chromatography on
silica gel (AcOEt–cHex 4 : 6) to yield 1.05 g (1.22 mmol, 80%)
of compound 15 as a translucent oil. Rf: 0.25 (AcOEt–cHex 5 : 5).
1H NMR (CDCl3, 250 MHz): d 1.28–1.44 (24H, m, CH2 chain),
1.52–1.63 (8H, m, NH-CO-CH2-CH2, CH2-CH2-CO, 2 CH2-
CH2-S), 2.00–2.20 (14H, m, 4 CH3CO, NH-CO-CH2), 2.36
(2H, t, CH2-CO, J3 = 7.43 Hz), 2.50 (4H, t, CH2-S-CH2, J3 =
7.22 Hz), 3.49 (2H, m, CH2-NH-CO), 3.66 (1H, m, OCH2),
3.93 (2H, m, OCH2, CH(5)), 4.05–4.18 (2H, m, CH2(6)), 4.48
NH-CO, NH-CH2-(CH2)2-CF2, CH(4), CH(5), CH2(6), CH(2 )
,
0
0
0
0
CH(3 ), CH(4 ), CH(5 ), CH2(6 )), 4.24–4.50 (3H, m, CH(2)
,
0
CH(3), CH(1 )), 4.68 (4H, m, CH-CH2,), 7.33 (1H, s, CQCH–
N
his), 8.82 (1H, s, N–CHQNhis). 13C NMR (CDCl3, 62.86
MHz): d 13.0 (CH3), 22.9–3–29.4 (CH2 chain, 2 CH2-CF2, 2
NH-CH2-CH2, CH-CH2), 35.4–39.1 (CH2-CO-NH, 2 CH2-
0
NH-CO), 52.1 (CH-CH2), 61.2 (CH2(6 )), 62.4 (CH2(6)), 69.0–
0
75.8 (CH(2), CH(3), CH(5), CH(2 ), CH(3 ), CH(4 ), CH(5 )), 82.3
0
0
0
0
(CH(4)), 104.2 (C1 b), 106.5–119.2 (CF2), 116.5 (N–CHQChis),
130.0 (N–CHQNhis), 133.6 (CH2-Chis), 170.6–174.1–174.9 (3
NH-CO). 19F NMR (CD3OD, 235.19 MHz): d ꢂ76.8 (3F, s,
ꢂO2C-CF3), ꢂ115.3 (4F, s, 2 CH2-CF2), ꢂ122.8 (8F, m, 4
CF2), ꢂ124.4 (4F, s, 2 CF2). m/z (ESI-MS): [M]1 1288.8
(without CF3COOꢂ). Elemental analysis calculated for
C54H82F19N5O15 ꢄ 3H2O: C: 44.54, H: 6.09, N: 4.81; found C:
44.58, H: 5.80, N: 4.77%.
Benzyl 11-bromoundecanoate (13). 11-Bromoundecanoic
acid (3 g, 11.3 mmol) was dissolved in 10 mL of benzyl alcohol.
When the solution was perfectly homogeneous, SOCl2 (0.9
mL, 17.5 mmol) was added. After stirring for 16 h at room
temperature, benzyl alcohol was removed by distillation under
reduced pressure and the crude product was purified by flash
chromatography on silica gel (cHex–AcOEt 8 : 2) to yield 3.2 g
(9.04 mmol, 80%) of compound 13 as a translucent oil. Rf:
0.57 (cHex–AcOEt 5 : 5). 1H NMR (CDCl3, 250 MHz): d 1.32
–1.45 (12H, m, CH2 chain), 1.68 (2H, m, CH2-CH2-CO), 1.88
(2H, qt, CH2-CH2-Br, J3 = 6.80 Hz), 2.39 (2H, t, CH2-CO,
J3 = 7.58 Hz), 3.44 (2H, t, CH2-Br, J3 = 6.85 Hz), 5.16 (2H, s,
CH2-F), 7.40 (5H, s, CHarom.). 13C NMR (CDCl3, 62.86
MHz): d 24.9 (CH2-CH2-CO), 28.2–30.3 (CH2 chain), 32.8
(CH2-CH2-Br), 34.0–34.3 (CH2-CO, CH2-Br), 66.1 (CH2-F),
126.9–128.5 (CHarom.), 136.1 (Carom.), 173.6 (CO).
(1H, d, CH(1), J3 = 7.83 Hz), 5.00–5.48 (5H, m, CH(2), CH(3)
,
CH(4), CH2-F), 5.89 (1H, s, NH), 7.34 (5H, s, CHarom.). 13C
NMR (CDCl3, 62.86 MHz): d 20.6 (CH3CO), 24.9–25.7 (NH-
CO-CH2-CH2, CH2-CH2-CO), 28.9–29.7 (CH2 chain), 32.2
(CH2-CO, NH-CO-CH2), 34.3 (CH2-S-CH2), 39.1 (OCH2-
CH2-NH), 61.3 (CH2(6)), 65.9–70.8 (CH(2), CH(3), CH(4)
,
CH(5), CH2-F, OCH2-CH2-NH), 101.4 (C1b), 128.2–128.5
(CHarom.), 136.1 (Carom.), 169.6–170.4 (CH3CO), 173.2–173.7
(NH-CO-CH2, CH2-CO-O).
N - [1 - (dodecylcarbamoyl) - 5 -(tert-butyloxycarbonylamino)-
pentyl] -22- [2- ((2,3,4,6 - tetra - O- acetyl)-b-D-galactopyranosyl-
oxy)ethylcarbamoyl]-12-thiadocosanamide (16). Compound 10
(220 mg, 0.40 mmol) was dissolved in 10 mL of ethanol. The
solution was stirred and cooled at 0 1C and palladium on
charcoal (24 mg, 60 mg mmolꢂ1) was added portionwise. The
mixture was stirred under hydrogen pressure (8 bar) for 12 h at
room temperature. The mixture was filtered over Celite and
the solvent removed in vacuo to obtain the amine as a white
powder. It was dissolved in 5 mL of anhydrous and degassed
DMF under argon. 208 mg of amine 150 (0,27 mmol, 1 eq) and
103 mg of TBTU (0.32 mmol, 1.2 eq) were added. The pH was
Benzyl 22-carboxy-12-thiadocosanoate (14). Mercaptopro-
pionoic acid (368 mg, 1.69 mmol) was dissolved in 10 mL of
anhydrous and degassed DMF with argon. At the same time,
NaH 60% (135 mg, 3.38 mmol) was rinsed with heptane, dried
under reduced pressure during 10 min and added to the
reaction mixture. Then, compound 13 (300 mg, 0.84 mmol)
dissolved in 5 mL of DMF was added dropwise. After stirring
for 4 h at room temperature, the solvent was removed in vacuo.
ꢀc
644 | New J. Chem., 2006, 30, 629–646 This journal is the Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2006

View Article Online
adjusted to 8 with DIEA. The mixture was refluxed for 16 h.
Then, the solvent was removed in vacuo. The crude product
was purified by flash chromatography on silica gel (AcOEt–
cHex 8 : 2) and filtered by size exclusion chromatography
using LH20 (MeOH–CH2Cl2 1 : 1) to yield 207 mg (0.178
mmol, 66%) of fully protected compound 16 as a white solid.
Rf: 0.38 (AcOEt), mp: 98–99 1C. [a]2D0: ꢂ5.79 (c 1, CH2Cl2). 1H
NMR (CDCl3, 250 MHz): d 0.88 (3H, t, CH3, J3 = 6.86 Hz),
1.25–1.37 (42 H, m, CH2 chain, CH-CH2-CH2), 1.44 (9H, s,
C(CH3)3), 1.47–1.67 (12H, s, NH-CO-CH2-CH2, CH2-CH2-
CO-NH, 2 CH2-CH2-S, CH2-CH2-NH-CO-CH, CH2-CH2-
NH(Boc)), 1.79 (2H, m, CH-CH2), 1.99–2.16 (12H, m,
CH3CO), 2.19 (4H, m, NH-CO-CH2, CH2-CO-NH-CH),
2.49 (4H, t, CH2-S-CH2, J3 = 7.23 Hz), 3.08 (2H, m, CH2-
NH(Boc)), 3.20 (2H, td, CH2-NH-CO-CH, J3 = 6.33 Hz),
3.46 (2H, m, OCH2-CH2-NH), 3.68 (1H, m, OCH2), 3.91 (2H,
m, OCH2, CH(5)), 4.14 (2H, m, CH2(6)), 4.37 (1H, m, CH-
CH2), 4.47 (1H, d, CH(1), J3 = 7.83 Hz), 4.62 (1H, m, NH),
5.02 (1H, dd, CH(3), J3 = 3.38 Hz, J3 = 10.46 Hz), 5.19 (1H,
dd, CH(2), J3 = 7.82 Hz, J3 = 10.46 Hz), 5.40 (1H, m, CH(4)),
5.87 (1H, s, NH), 6.23 (1H, m, NH), 6.33 (1H, m, NH). 13C
NMR (CDCl3, 62.86 MHz): d 14.5 (CH3), 20.6–29.7 (CH3CO,
CH2 chain, CH-CH2, C(CH3)3), 32.2 (NH-CO-CH2), 35.5
(CH2-S-CH2), 36.6–36.7 (CH2-CO-NH-CH, CH2-NH-CO-
CH), 39.06–39.58 (OCH2-CH2-NH-CO, CH2-NH(Boc)), 52.8
S-CH2, CH2-CO-NH-CH), 39.0–39.2 (OCH2-CH2-NH-CO,
CH2-NH31), 53.0 (CH-CH2), 61.1 (CH2(6)), 68.3–75.3
(CH(2), CH(3), CH(4), CH(5), OCH2-CH2-NH), 105.2 (C1b),
172.6 (OCH2-CH2-NH-CO), 178.7 (NH-CO-CH2, NH-CO-
CH). m/z (ESI-MS): [M þ H1]1 904.1 (without ꢂO2C-CF3),
[M þ H1 þ Na1]21 927.0 (without ꢂO2C-CF3).
Acknowledgements
We are grateful to D. Montpetit for her valuable help in
conducting examination by transmission electron microscopy.
Discussions with M. R. Van Calsteren were highly appre-
ciated. Biological work was supported by Valorisation Re-
cherche Quebec (Gouvernement du Quebec, Project Number:
´ ´
2201-141). PYV also acknowledges the Food Research and
Development Centre (Agriculture and Agri-Food Canada) for
technical support.
References
1 (a) A. K. Pannier and L. D. Shea, Mol. Ther., 2004, 10, 19; (b) S.
Zhang, Y. Xu, B. Wang, W. Qiao, D. Liu and Z. Liu, J. Controlled
Release, 2004, 100, 165.
2 (a) T. Serikawa, N. Suzuki, H. Kikuchi, K. Tanaka and T.
Kitagawa, Biochim. Biophys. Acta, 2000, 1467, 419; (b) A. D.
Miller, Angew. Chem., Int. Ed., 1998, 37, 1768.
(CH-CH2), 61.3 (CH2(6)), 67.0–70.7 (CH(2), CH(3), CH(4)
,
3 (a) O. Zelphati, L. S. Uyechi, L. G. Barron and F. C. Szoka,
Biochim. Biophys. Acta, 1998, 1390, 119; (b) I. Moret, J. E. Peris, V.
M. Guillem, M. Benet, F. Revert, F. Dasi, A. Crespo and S. F.
Alino, J. Controlled Release, 2001, 76, 169; (c) Y. K. Ghosh, S. S.
Visweswariah and S. Bhattacharya, FEBS Lett., 2000, 473, 341; (d)
D. L. Reimer, S. Kong, M. Monck, J. Wyles, P. Tam, E. K. Wasan
and M. B. Bally, J. Pharmacol. Exp. Ther., 1999, 289, 807.
4 D. M. L. Morgan, V. L. Larvin and J. D. Pearson, J. Cell Sci.,
1989, 94, 553.
CH(5), OCH2-CH2-NH), 79.4 (C(CH3)3), 101.4 (C1b), 169.7–
173.4 (CH3CO, NH-CO-CH2, NH-CO-CH, COBoc). m/z
1 1
(FAB1MS): [M þ H1]1 1172.1, [M þ NH4
]
1189.2,
[M þ Na1]1 1194.1, [M þ K1]1 1210.1.
N-[1-(dodecylcarbamoyl)-5-(ammonio)pentyl)]-22-[2-(b-D-ga-
lactopyranosyloxy)ethylcarbamoyl]-12-thiadocosanamide tri-
fluoroacetate (E). Compound 16 (150 mg, 0.128 mmol) was
dissolved in 10 mL of MeOH freshly distilled and degassed
under argon. A catalytic amount of sodium methylate was
added. After stirring for 6 h, IRC50 ion exchange resin (1 g)
was added. After 10 min the mixture was filtered. The solvent
was removed in vacuo and the crude product purified by
filtration by size exclusion chromatography using LH20
(MeOH) to yield 120 mg (0.196 mmol, 93%) of deacetylated
compound. Then, it was dissolved in 9 mL DCM freshly
distilled and degassed under argon. 1 mL of TFA was added
dropwise to the reaction mixture. After stirring for 16 h at
room temperature, the solvent was removed in vacuo and the
product was filtered by size exclusion chromatography using
LH20 (MeOH–CH2Cl2 1 : 1) to yield 95.5 mg (0.159 mmol,
81%) of final compound E as a white powder, mp (dec) > 88 1C.
1H NMR (CDCl3, 250 MHz): d 0.80 (3H, t, CH3, J3 = 6.21
Hz), 1.19–1.35 (42 H, m, CH2 chain, CH-CH2-CH2), 1.36–1.72
(14H, s, NH-CO-CH2-CH2, CH2-CH2-CO-NH, 2 CH2-CH2-
S, CH2-CH2-NH-CO-CH, CH2-CH2-NH31, CH-CH2), 2.06–
2.14 (4H, m, NH-CO-CH2, CH2-CO-NH-CH), 2.39 (4H, t,
CH2-S-CH2, J3 = 7.09 Hz), 2.79 (2H, m, CH2-NH31), 3.09
(2H, m, CH2-NH-CO-CH), 3.19–3.85 (10H, m, OCH2-CH2-
5 I. R. C. Hill, M. C. Garnett, F. Bignotti and S. S. Davis, Biochim.
Biophys. Acta, 1999, 1427, 161, and references therein.
6 (a) J. D. Toussignant, A. K. Gates, L. A. Ingram, C. L. Johnson, J.
B. Nietupski, S. H. Cheng, S. J. Eastman and R. K. Scheule, Hum.
Gene Ther., 2000, 11, 2493; (b) S. Loisel, C. Le Gall, L. Doucet, C.
Ferec and V. Floch, Hum. Gene Ther., 2001, 12, 685.
7 D. Pijper, E. Bulten, J. Smisterova, A. Wagenaar, D. Hoekstra, J.
Engberts and R. Hulst, Eur. J. Org. Chem., 2003, 22, 4406.
8 A. J. Kirby, P. Camilleri, J. Engberts, M. C. Feiters, R. J. M.
Nolte, O. Soderman, M. Bergsma, P. C. Bell, M. L. Fielden, C. L.
G. Rodriguez, P. Guedat, A. Kremer, C. McGregor, C. Perrin, G.
Ronsin and M. C. P. van Eijk, Angew. Chem., Int. Ed., 2003, 42,
1448.
9 P. C. Bell, M. Bergsma, I. P. Dolbnya, W. Bras, M. C. A. Stuart,
A. E. Rowan, M. C. Feiters and J. Engberts, J. Am. Chem. Soc.,
2003, 125, 1551.
10 For a review, see: J.-H. Fuhrhop and T. Wang, Chem. Rev., 2004,
104, 2901.
11 C. Jacopin, M. J. Egron, D. Scherman and J. Herscovici, Bioorg.
Med. Chem. Lett., 2002, 12, 1447.
12 C. Jacopin, H. Hofland, D. Scherman and J. Herscovici, Bioorg.
Med. Chem. Lett., 2001, 11, 419.
13 E. Kissa, Fluorinated Surfactants. Synthesis, Properties, Applica-
tions, Marcel Dekker, New York, 1994, Surfactants Science Series
50.
14 R. I. Mahato, K. Kawabata, T. Nomura, Y. Takakura and M.
Hashida, J. Pharm. Sci., 1995, 84, 1267.
15 (a) M. Kadi, P. Hansson, M. Almgren and I. Furo, Langmuir,
2002, 18, 9243; (b) P. Barthelemy, V. Tomao, J. Selb, Y. Chaudier
´ ´
and B. Pucci, Langmuir, 2002, 18, 2557.
16 N. Requirand, H. Blancou and A. Commeyras, Bull. Soc. Chim.
Fr., 1984, 30, 798.
17 W.-Y. Huang, J. Fluorine Chem., 1992, 58, 1.
18 B. Helferich and J. Zinner, Chem. Ber., 1962, 95, 2604.
NH, CH(2), CH(3), CH(4), CH(5), CH2(6)), 4.14 (1H, d, CH(1)
,
J3 = 7.07 Hz), 4.18 (1H, m, CH-CH2). 13C NMR (CDCl3,
62.86 MHz): d 14.3 (CH3), 22.4–29.4 (CH2 chain, CH-CH2),
31.7 (NH-CO-CH2), 35.6–35.7 (CH2-NH-CO-CH, CH2-
ꢀc
This journal is the Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2006
New J. Chem., 2006, 30, 629–646 | 645

View Article Online
19 M. Brunelle, S. Denoyelle, A. Polidori, B. Pucci, S. Laurent and
P. Y. Vuillaume, to be published.
20 D. Putnam, A. N. Zelikin, V. A. Izumrudov and R. Langer,
Biomaterials, 2003, 24, 4425.
21 R. I. Mahato, A. Rolland and E. Tomlinson, Pharm. Res., 1997,
14, 853.
22 W. Zauner, M. Ogris and E. Wagner, Adv. Drug Delivery Rev.,
1998, 30, 97.
23 P. Y. Vuillaume, M. Brunelle, M. R. Van Calsteren, S. Lewan-
´
dowski, R. Lewandowski, A. Begin, B. Talbot and Y. ElAzhary,
Biomacromolecules, 2005, 6, 1769.
24 H. Pollard, J. S. Remy, G. Loussouarn, S. Demolombe, J. P. Behr
and D. Escande, J. Biol. Chem., 1998, 273, 7507.
25 (a) T. Yoshimura, S. Hasegawa, N. Hirashima, M. Nakanishi and
T. Ohwada, Bioorg. Med. Chem. Lett., 2001, 11, 2897; (b) R.
Iwaura, K. Yoshida, M. Masuda, M. Ohnishi-Kameyama, M.
Yoshida and T. Shimizu, Angew. Chem., Int. Ed., 2003, 42, 1009;
(c) M. L. Miramon, N. Mignet and J. Herscovici, J. Org. Chem.,
2004, 69, 6949; (d) M. A. Eaton, T. S. Baker, C. F. Catterall, K.
Crook, G. S. Macaulay, B. Mason, T. J. Norman, D. Parker, J. J.
B. Perry, R. J. Taylor, A. Turner and A. N. Weir, Angew. Chem.,
Int. Ed., 2000, 39, 4063; (e) V. Weissig and V. P. Torchilin, Adv.
Drug Delivery Rev., 2001, 49, 127; (f) J. Gaucheron, C. Santaella
and P. Vierling, Bioconjugate Chem., 2001, 12, 569; (g) K. Fabio, J.
Gaucheron, C. Di Giorgio and P. Vierling, Bioconjugate Chem.,
2003, 14, 358; (h) T. Ren, G. Zhang and D. Liu, Bioorg. Med.
Chem., 2001, 9, 2969.
26 O. Boussif, F. Lezoualch, M. A. Zanta, M. D. Mergny, D. Scher-
man, B. Demeneix and J. P. Behr, Proc. Natl. Acad. Sci. U. S. A.,
1995, 92, 7297.
ꢀc
646 | New J. Chem., 2006, 30, 629–646 This journal is the Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2006