Polycationic lipophilic-core dendrons as penetration enhancers for the oral administration of low molecular weight heparin

Article abstract of DOI:10.1016/j.bmc.2005.08.004

Two polycationic lipophilic-core carbohydrate-based dendrons 2a-b and five polycationic lipophilic-core peptide dendrons 3-6, containing four arginine or lysine terminal residues, were synthesized and then tested in rats as penetration enhancers for the o

Full text of DOI:10.1016/j.bmc.2005.08.004

Bioorganic & Medicinal Chemistry 14 (2006) 143–152
Polycationic lipophilic-core dendrons as penetration enhancers
for the oral administration of low molecular weight heparin
Patricia Y. Hayes, Benjamin P. Ross, Bradley G. Thomas and Istvan Toth*
Discipline of Chemistry, School of Molecular and Microbial Sciences, and School of Pharmacy,
The University of Queensland, Brisbane, Queensland 4072, Australia
Received 23 June 2005; revised 1 August 2005; accepted 1 August 2005
Available online 16 September 2005
Abstract—Two polycationic lipophilic-core carbohydrate-based dendrons 2a–b and five polycationic lipophilic-core peptide den-
drons 3–6, containing four arginine or lysine terminal residues, were synthesized and then tested in rats as penetration enhancers
for the oral delivery of low molecular weight heparin. Better results were obtained with dendrons containing terminal lysine residues
than terminal arginine. A significant anti-factor Xa activity was obtained when low molecular weight heparin was coadministered
with dendron 5.
ꢀ 2005 Elsevier Ltd. All rights reserved.
1. Introduction
acid;9 and therefore it exhibits poor oral bioavailability.
Consequently, it must be delivered via the less desirable
parenteral route, which is expensive, inconvenient, and
limits use by outpatients.10–12 In this paper, we describe
the synthesis and in vivo evaluation of a series of penetra-
tion enhancers13,14 designed to improve LMWH oral
bioavailability.
Unfractionated heparin (UFH, avg. MW ca. 12,000 Da)
is a heterogeneous mixture of polysaccharides compris-
ing alternating 1-to-4 linked sulfated monosaccharide
residues of D-glucosamine and a uronic acid (90% L-idu-
ronic acid, 10% D-glucuronic acid) (Fig. 1A).1–3 The pen-
tasaccharide binding site (Fig. 1B) for antithrombin
occurs in approximately one-third of heparin chains.2,3
UFH is used for the prevention and treatment of throm-
botic disorders and in the treatment of acute coronary
syndromes.4,5 Formed from the chemical or enzymatic
digestion of UFH, low molecular weight heparin
(LMWH) has replaced UFH as the drug of choice for
the surgical prophylaxis of deep vein thrombosis and
the management of acute coronary syndromes.5 LMWH
was recently approved for the treatment of thrombotic
disorders and is being developed for therapeutic applica-
tions in cancer, transplantation, and immunology.5
LMWH has an average molecular weight of ꢀ4500 Da,
and compared to UFH (ꢀ12,000 Da), it has improved
pharmacokinetic properties and a decreased toxicity pro-
file.4,6,7 Nevertheless, LMWH is a high molecular weight,
hydrophilic polyanion, which can be degraded in the gas-
trointestinal tract by bacteria8 and to a lesser extent by
Intensive effort has focused on developing an oral hepa-
rin formulation. Ubrich et al.15 reviewed the literature
prior to October 2002, pertaining to formulations
designed to increase heparin intestinal absorption via
the oral, intragastric, intraduodenal, or rectal routes of
administration. Ross and Toth16 examined the litera-
ture, mostly published between 2000 and 2005, focusing
on delivery systems that improved the gastrointestinal
absorption of heparin. These review articles noted that
promising delivery agents included microspheres,
Carbopolꢁ, chitosan, deoxycholic acid, fatty acids and
surfactants, saponins, and polymeric micro- and
nanoparticles.
The most advanced studies relating to the development
of oral heparin are attributed to Emisphere Technologies
Inc., which used sodium N-[8-(2-hydroxybenzoyl)ami-
no]caprylate (SNAC) and sodium N-[10-(2-hydroxy-
benzoyl)amino]decanoate (SNAD), as penetration
enhancers for the oral delivery of UFH and LMWH.
SNAC and SNAD were identified as leads from a series
of compounds according to their ability to enhance
Keywords: Low molecular weight heparin; Oral delivery; Dendrons;
Carbohydrates.
*
Corresponding author. Tel.: +61 7 3346 9892; fax: +61 7 3365
4273; e-mail: i.toth@uq.edu.au
0968-0896/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.08.004

144
P. Y. Hayes et al. / Bioorg. Med. Chem. 14 (2006) 143–152
A
CH2OSO3-
O
O
COO-
OH
O
OH
O
O
OSO3-
NHSO3-
n
D-glucosamine
L-iduronic acid
B
CH2OSO3-
CH2OSO3-
CH2OSO3-
COO-
O
OSO3-
O
O
O
O
COO-
OH
OH
OH
O
O
OH
O
O
O
O
NHSO3-
OSO3-
NHSO3-
OH
NHSO3-
C
H
N
O
)
(
R
S
14
CH2
S
O
R
N
H
O
O
)
O
H
H
N
N
NH2
S
O
R
N
N
N
H
H
H
R
O
O
S
O
O
N
H
(
CH2
14
2a: R = L-Lysine
2b: R = L-Arginine
(
)
n
R
CH2
Lys
Lys
Dendron
n
R
O
R
H
N
Lys
3
14 L-Lysine
10 L-Lysine
10 L-Arginine
8
6
NH2
N
N
4a
4b
5
R
H
H
O
O
L-Lysine
L-Lysine
R
6
(
)
CH2
n
Figure 1. Heparin is a heterogeneous mixture of polysaccharides comprising alternating 1-to-4-linked sulfated monosaccharide residues. (A) The
most common type of disaccharide unit. (B) The pentasaccharide binding site for antithrombin. (C) Polycationic lipophilic-core dendrons 2–6. The
lipophilic-core moiety is enclosed by the box. (Lys = L-Lysine.) [(A) and (B) were modified from chromogenix monograph series: heparin;2 Rang
et al.;3 and Samama et al.67].
colonic absorption of UFH in rats.17,18 Subsequent stud-
ies in rats and monkeys demonstrated the effectiveness of
SNAC for the oral delivery of UFH.19 The UFH and
dendrimers) have been a topic of extensive research
since they were first prepared by Tomalia et al.25
and Newkome et al.,26 in the 1980s. Some of the most
promising applications of these molecules lie in the
fields of drug and gene delivery.27–30 For example,
dendrons structurally similar to 3–6 (Fig. 1C) medi-
ated the transfection of Cos-731 and BHK-21 cells32
with plasmid DNA. Recently, human cells (D-407)
were successfully transfected with an oligodeoxynu-
cleotide in vitro, using a dendron related to 5
(Fig. 1C), as a vector.33 In these examples, the poly-
cationic nature of the dendrons was designed to enable
ion-pair formation with polyanionic DNA. The forma-
tion of ion-paired complexes was studied by examining
the dendron mediated exclusion of ethidium bromide
(EB) from DNA–EB complex,34 and also the electro-
phoretic retardation of DNA–dendron complexes in
SNAC formulation was successful in phase I and II clin-
20,21
ical trials;
however, phase III trials were negatively
influenced by poor patient compliance associated with
the taste of the liquid formulation.22,23 SNAD was found
to be about twice as effective as SNAC for facilitating
oral LMWH absorption in rats.24 In monkeys, SNAD
was ꢀ10-fold more effective than SNAC for oral LMWH
delivery.24 The bioavailability of a SNAD and LMWH
combination, administered orally to dogs as a solid dose
form, relative to subcutaneous injection, was 3%.24
In this study, we examined polycationic lipophilic-core
dendrons (2–6) as penetration enhancers for the oral
delivery of LMWH. Dendrimers and dendrons (partial

P. Y. Hayes et al. / Bioorg. Med. Chem. 14 (2006) 143–152
145
agarose gel electrophoresis.31,32 The lipophilic-core
2. Results and discussion
moieties [the moiety of 2–6 enclosed by the box
(Fig. 1C)], which contained lipoamino acids35 (LAAs),
were designed to increase the lipophilicity of the com-
plex and thereby facilitate membrane partitioning.
LAAs are versatile compounds, which have been
incorporated into penetration enhancers,36,37 and poor
absorbed drugs,38–40 to enhance membrane permeabil-
ity and reduce enzymatic degradation. LAAs, in the
form of the lipophilic-core moiety, have also found
application in experimental vaccines.41,42 An advan-
tage of LAAs is that they are bifunctional molecules
that are amenable to solid-phase synthesis techniques
(e.g., Scheme 2).
2.1. Synthesis
The carbohydrate cores of 2a and 2b were synthesized,
as shown in Scheme 1. The b-galactosyl azide 7 was syn-
thesized from b-D-galactose pentaacetate following a
literature procedure.49 The tetraallyl derivative 8 was
obtained by reaction of 7 with allyl bromide/NaH in
DMF50 in 60% yield. Selective reduction of the azido
group was achieved in 52% yield using tin (II) chloride
in methanol. The corresponding amine 951 was quickly
purified and coupled with adipic acid mono benzyl ester
using HBTU/DIPEA activation to afford 10 in 62%
yield. Irradiation of 10 at 254 nm for 4 h52 in dried
and degassed 1,4-dioxane in the presence of 4 equiv of
tert-butyl-N-(2-mercaptoethyl) carbamate afforded the
tetra-Boc-protected derivative 11 in good yield, which
was then saponified with aqueous lithium hydroxide
solution to give the free acid 12 in 88% yield.
We postulated that polycationic lipophilic-core den-
drons 2–6 could be used as penetration enhancers, for
the oral delivery of LMWH, by forming lipophilic
ion-pairs with the polyanionic LMWH. Lipophilic ion-
pairing has previously been examined as a method to
enhance heparin bioavailability. Complexation with a
diamine improved the intraduodenal absorption of
UFH and LMWH, in the rat.43 Monoamines, and a
more hydrophilic diamine, were ineffective. In dogs,
the oral absorption of UFH was enhanced by coadmin-
istration with a diamine, using enteric-coated pellets.44
Di- and triamines, and to a lesser extent monoamines,
were effective at improving the intraduodenal absorp-
tion of UFH in rabbits.45,46
The tetra-Boc-protected acid 12 is a substrate suitable
for solid-phase peptide synthesis and the synthesis of
the carbohydrate-based polycationic lipophilic-core den-
drons 2a and 2b is illustrated in Scheme 2. Compound
14 was obtained using solid-phase synthesis by coupling
Boc-glycine to MBHA resin using HBTU/HOBt/DIPEA
coupling reagents in DMF, followed by removal of the
N-terminal Boc group with trifluoroacetic acid (TFA).
Two 2-tert-butoxycarbonylamino-octadecanoic acid53
residues were then coupled following the same protocol.
The Boc-protected tetraamine 12 was then attached to
the solid phase (HBTU/HOBt/DIPEA in DMF) and
after removal of the Boc groups with TFA, Boc-
Lys(ClZ) (2a) or Boc-ArgTsOH (2b) residues were cou-
pled to the solid phase. Removal of the Boc groups
(TFA) and cleavage of the peptides from the resin using
HF methodology (10% of cresol as scavenger at 0 ꢂC for
2 h) afforded the dendrons 2a and 2b in 61% and 47%
yields, respectively, after purification by preparative
RP-HPLC. These two dendrons were characterized by
electrospray ionization mass spectrometry (ES-MS).
The ability of heparin to bind with polycationic mol-
ecules is exemplified by its interaction with many pro-
teins, a topic that was reviewed by Capila and
Linhardt.47 They stated that the most prominent type
of interaction between heparin and a protein is ionic,
whereby clusters of positively charged basic amino
acids on proteins form ion-pairs with negatively
charged sulfo or carboxyl groups on the heparin
chain. Recently, Kasai et al.48 synthesized two argi-
nine-rich (polycationic) dendrimers, which were de-
signed to act as antiangiogenic agents via a heparin-
binding mechanism.
The synthesis and in vivo evaluation of the series of den-
drons 2–6 (Fig. 1C) as penetration enhancers for the oral
delivery of LMWH, are described below.
Dendrimers 3–6 were synthesized in 35% to 68% yields
using standard solid-phase synthesis techniques33,54
and purified by preparative RP-HPLC.
O
O
O
O
O
OH
O
O
O
O
O
OH
HO
d
a
c
H
b
O
N
NH
N
N
3
2
3
O
O
O
OBn
O
O
O
OH
O
O
7
8
9
10
BocHN
BocHN
BocHN
BocHN
S
S
O
O
S
S
e
O
O
O
H
O
O
N
S
S
OH
O
O
OBn
N
BocHN
BocHN
S
S
O
O
H
O
BocHN
BocHN
O
11
12
Scheme 1. Reagents and conditions: (a) allylbromide, NaH, DMF, 60%; (b) SnCl2Æ2H2O, MeOH, 52%; (c) HBTU, DIPEA, THF, adipic acid
monobenzyl ester, 62%; (d) Boc-Cysteamine, 1,4-dioxane, 254 nm, 4 h, 90%; (e) LiOHÆH2O in aqueous MeOH, pH 13, 88%.

146
P. Y. Hayes et al. / Bioorg. Med. Chem. 14 (2006) 143–152
BocHN
BocHN
S
S
O
O
( )15
O
H
O
a, b
O
H2N
N
+
P
Gly
O
N
S
OH
O
P
H
N
BocHN
( )15
O
S
O
H
MBHA resin
O
BocHN
12
13
c
H2N
H2N
S
S
O
O
O
( )15
O
O
H
H
O
S
N
N
O
N
H
H2N
N
S
O
H
P
O
H2N
( )15
O
14
d, e
R
R
HN
HN
S
S
O
O
( )15
O
O
O
H
N
H
N
O
O
S
O
N
H
R
HN
R
N
NH2
S
H
O
HN
( )15
O
2a-b,R = Arginine or Lysine residue
Scheme 2. Reagents and conditions: (a) i—2-amino-octadecanoic acid, HBTU, DIPEA, DMF, rt, 30 min; ii—TFA, rt, 2· 1 min; (b) i—2-Amino-
octadecanoic acid, HBTU, DIPEA, DMF, rt, 30 min; ii—TFA, rt, 2· 1 min; (c) i—12, HBTU, DIPEA, DMF, rt, 30 min; ii—TFA, rt, 2· 1 min; (d)
Boc-Lys(ClZ) (2a) or Boc-ArgTsOH (2b), HBTU, DIPEA, DMF, rt; ii—TFA, rt, 2· 1 min; (e) HF-cleavage.
2.2. Microcalorimetry
Fig. 2 shows the DH versus c plot for the injection of ali-
quots of a solution of dendron into the microcalorimeter
sample cell, which contained a solution of LMWH. The
initial portion of the curves (molar ratio < 5:1) indicates
Isothermal titration microcalorimetry (ITC)55,56 was
used to determine the optimal ratio of dendron:LMWH.
Figure 2. Isothermal titration calorimetry curve for the complex formation between LMWH and (A) 2b; (B) 4a.

P. Y. Hayes et al. / Bioorg. Med. Chem. 14 (2006) 143–152
147
complex formation between the dendron and LMWH,
which is an exothermic process. The complex formation
was complete at
dron:LMWH), since beyond that point only an endo-
thermic enthalpy of dendron dilution was observed. A
similar process was used by Wimmer et al.33 to deter-
mine the optimum ratio of dendron:oligodeoxynucleo-
tide, for the purpose of facilitating in vitro cell
transfection.
0.18
0.16
0.14
0.12
0.10
0.08
0.06
0.04
0.02
0.00
A
2a
Control
2b
a
molar ratio of ꢀ7:1 (den-
2.3. Pharmacodynamic study
The ability of the polycationic lipophilic-core dendrons
to enhance the oral absorption of LMWH was examined
in vivo, using rats. The rat is commonly used in studies
of oral LMWH absorption.24 LMWH (7500 IU/kg) was
administered alone or admixed with a penetration
enhancer (7 mol equiv). The doses were delivered as sus-
pensions, via oral gavage, using a vehicle of propylene
glycol and water (1:1, 1 mL) (Propylene glycol was used
as a cosolvent in formulations containing SNAC and
SNAD.).24 The mean plasma anti-factor Xa activities
as a function of time are shown in Figures 3 and 4,
and the pharmacodynamic parameters are listed in
Table 1.
0
30
60
90
120
150
180
Time (min)
0.18
0.16
0.14
0.12
0.10
0.08
0.06
0.04
0.02
0.00
B
4a
4b
Control
Initial work examined the influence of the branching
moiety (either polylysine or a monosaccharide) and the
cationic group (either lysine or arginine) on the effective-
ness of the penetration enhancers. The coadministration
of LMWH and dendrons bearing terminal arginine moi-
eties (2b and 4b) did not improve the plasma anti-factor
Xa activity when compared to control (Fig. 3, Table 1).
However, the dendrons with terminal lysines (2a and 4a)
increased the Cmax and AUC values, relative to control
(Fig. 3, Table 1). Considering the results of Fromm
et al.57 who found that arginine bound more tightly than
lysine to heparin, it is possible that once absorbed, the
arginine dendrons may not release bound heparin as
readily as lysine dendrons, and consequently they are
less effective as delivery agents. Of the compounds 2a–
b and 4a–b, the largest Cmax (0.13 0.02 IU/mL) and
AUC (19.9 3.0 IU min/mL) occurred when LMWH
was coadministered with the peptide dendron 4a. There-
fore, peptide dendrons containing terminal lysines were
examined further, as penetration enhancers.
0
30
60
90
120
150
180
Time (min)
Figure 3. Plasma anti-factor Xa activity following oral administration
of LMWH (7500 IU/kg) with or without penetration enhancer
[7 mol equiv; (A) 2a and 2b; (B) 4a and 4b]. Each data point is the
means SEM of 1–5 rats. Compound 4a is also illustrated in Figure 4.
ranged from 15 to 120 min, and 5 and 3 (the enhancers
with significantly increased Cmax values) had tmax values
of 30 and 15 min, respectively. Although variation of the
LAA moieties in the lipophilic-core did alter penetration
enhancer effectiveness, there was no apparent correla-
tion between the LAA alkyl chain length and penetra-
tion enhancer effectiveness (which was measured as an
increase in either Cmax and/or AUC).
A series of dendrons (3, 4a, 5, and 6) which contained
a variety of LAAs was synthesized to determine
whether the LAA alkyl chain length influenced pene-
tration enhancer effectiveness. Figure 4 illustrates the
plasma anti-factor Xa activity following oral adminis-
tration of LMWH (7500 IU/kg) alone and in combina-
tion with the penetration enhancers 3, 4a, 5, or 6
(7 mol equiv). A statistically significant (P < 0.05) in-
crease in the Cmax value was observed when LMWH
was administered with the penetration enhancer 3 or
5 (Fig. 4 and Table 1).
The highest Cmax value of 0.24 0.11 IU/mL (attained
using 5) was a significant (P < 0.01) 3.4-times greater
than the control Cmax of 0.07 0.01 IU/mL. Bianchini
et al.58 reported that a plasma anti-factor Xa activity
of 0.12 IU/mL was necessary to induce a 50% anti-
thrombotic effect in rats, and concentrations exceeding
0.2 IU/mL always afforded an evident antithrombotic
effect. Therefore, a therapeutically relevant anti-factor
The largest AUC value (24.5 4.6 IU min/mL) occurred
when LMWH was coadministered with 5 and represent-
ed a 2-fold improvement in AUC when compared to the
control AUC (12.3 2.5 IU min/mL). The tmax values

148
P. Y. Hayes et al. / Bioorg. Med. Chem. 14 (2006) 143–152
0.35
0.30
self-associate to form supramolecular aggregates such
as vesicles.62,63 Therefore, the dendrons could act as sur-
factants and enhance drug absorption by perturbing
membrane lipids and proteins.64
5
3
4a
6
0.25
0.20
Control
In vivo studies of LMWH absorption may be classified
into two groups: those that examine oral absorption
(i.e., administration via oral gavage)19,24 and those that
examine in situ intestinal absorption (e.g., intraduodenal
administration).43,45,65,66 It has been stated65 that hepa-
rin is unstable under acidic conditions such as in the
stomach, and this has been used as one reason to reject
oral gavage and to examine only in situ intestinal admin-
istration.43 Nevertheless, the current ꢀgold standardꢁ
penetration enhancers for oral heparin delivery are
SNAC and SNAD, which were studied in numerous ani-
mal models19,24 and in clinical trials,20,21 using the oral
route of administration. Additionally, the literature
indicates that in oral studies the detrimental effects of
the oral route (e.g., degradation and dilution) are typi-
cally compensated for; by using a heparin dose at least
one order of magnitude greater than that used for
in situ intestinal studies. Therefore, we chose the oral
route of delivery, and comparison to the penetration
enhancers SNAC and SNAD is appropriate. Such com-
parison with the literature reveals that although den-
drons can enhance LMWH absorption, they are
approximately one order of magnitude less effective than
SNAC or SNAD, for oral LMWH delivery.24
0.15
0.10
0.05
0.00
60
90
120
150
180
0
30
Time (min)
Figure 4. Plasma anti-factor Xa activity following oral administration
of LMWH (7500 IU/kg) with or without penetration enhancer (3, 4a, 5
and 6; 7 mol equiv). Each data point is the means SEM of 2–5 rats.
Compound 4a is also illustrated in Figure 3.
Table 1. Pharmacodynamic parameters
a,b
c
b,d
AUC180
Penetration
enhancer
Cmax
tmax
(IU/mL)
(min)
(IU min/mL)
Control
0.07 0.01
0.06 0.01
90
60
12.3 2.5
2b
4b
6
8.40 1.44
12.3 1.9
12.4 3.6
0.08 0.00
0.12 0.02
120
30
2a
4a
3
0.10 0.01
0.13 0.02
0.19 0.02e (2.7)f
0.24 0.11g (3.4)f
60
90
14.0 0.9
19.9 3.0
3. Conclusion
15
30
23.6 4.7
24.5 4.6h (2.0)i
5
Several polycationic lipophilic-core dendrons 2–6 were
synthesized and screened as penetration enhancers for
the oral administration of LMWH. The results obtained
show that the dendrons with terminal lysine were more
effective than the dendrons with terminal arginine resi-
dues. Dendrons 3 and 5 were the most effective for the
oral absorption of LMHW in rats. An important factor
limiting the absorption of the dendron–LMWH com-
plex may be poor aqueous solubility, which meant that
the compounds in this study were administered as sus-
pensions. Continuing efforts in this research are aimed
at solubilizing the dendron/LMWH complex.
a Maximum plasma anti-factor Xa activity.
b Values are means SEM.
c Time to reach Cmax
.
d Area under the curve.
e P < 0.05 cf. control.
f Cmax/control Cmax
.
g P < 0.01 cf. control.
h P < 0.1 cf. control.
i AUC/AUCcontrol
.
Xa activity was attained by coadministration of LMWH
with the penetration enhancer 5. However, the concen-
tration was only maintained above 0.2 IU/mL for
ꢀ20 min, which is a shorter time period than that
observed in some studies.24
4. Experimental
4.1. Synthesis
The precise mechanism by which polycationic lipophilic-
core dendrons improve LMWH absorption is unknown.
The compounds were designed to act as penetration
enhancers by forming lipophilic ion-pairs with the poly-
anionic LMWH. This ion-pair model of absorption as-
sumes that the dendrons are absorbed as a complex
with LMWH. Ion-pairing has been used as a means to
enhance the absorption of various drugs,59–61 and di-
and triamines were previously used to enhance heparin
bioavailability.43–46 The dendrons examined in this
study are amphipathic molecules, which are likely to
possess surfactant properties. Similar dendrons were
found to adsorb at the air/water interface62 and to
Mass spectra were run on a Perkin-Elmer API 3000 LC/
MS/MS electrospray instrument or with a VG Analyti-
cal ZAB-SE instrument using fast atom bombardment
1
(FAB) techniques (20 kV Cs+ ion bombardment). H
and 13C NMR spectra were recorded on a Bruker AM
500 instrument. Preparative separations were carried
out by column chromatography using Merk silica gel
(230–400 mesh). Analytical RP-HPLC were performed
on a LC-10AT Shimadzu liquid chromatograph (SCL-
10A system controller, SPD-6A UV detector, and a
SIL-6B auto injector with a SCL-6B system controller)
and C4 or C18 columns (Vydac, 25 cm, with 5 lm pore

P. Y. Hayes et al. / Bioorg. Med. Chem. 14 (2006) 143–152
149
size and 4.6 mm internal diameter), using an acetonitrile/
water gradient. Preparative RP-HPLC were performed
using a Waters HPLC system (Model 600 controller,
490E UV detector, F pump, and TSK-GEL C4 column,
25 cm, with 10 lm pore size and 22 mm internal diame-
ter) using an acetonitrile/water gradient.
residue was dissolved in ethyl acetate (200 mL). The
organic phase was washed with 5% HCl solution (2·
150 mL), saturated NaHCO3 solution (2· 150 mL),
and brine (100 mL). After drying over MgSO4, the sol-
vent was removed under vacuum and the residue was
purified by flash chromatography (Silica, hexane/ethyl
acetate 3:2) to give 10 as a colorless oil (2.3 g, 62%).
ES-MS (m/z): 558 (MH+), 575 (M+NH4)+, 580
4.1.1. 2,3,4,6-Tetra-O-(2-propenyl)-b-D-galactopyranosyl
azide (8). Sodium hydride (60% in dispersion oil, 7.76 g,
194 mmol) was slowly added to a solution of b-D-galac-
topyranosyl azide 7 (7.91 g, 38.6 mmol) at 0 ꢂC in dry
DMF (250 mL). After 45 min, allyl bromide (19.8 mL,
234 mmol) was added dropwise and the temperature
was raised to 50 ꢂC. After 4 h, the reaction was cooled
to room temperature and the excess NaH was quenched
by addition of methanol (80 mL). The solvent was then
removed under vacuum and the residue was then dis-
solved in DCM (300 mL) and washed with water (3·
150 mL). After drying over MgSO4, the organic layer
was filtered and then concentrated in vacuo. The residue
was then purified by flash chromatography [Silica, Ethyl
acetate–hexane gradient (2–20%)] to yield the tetra allyl
ether (8.52 g, 60%) as a colorless oil. ES-MS (m/z): 366
(MH+), 383 (M+NH4)+, 388 (M+Na)+. 1H NMR
(500 MHz, CDCl3): 3.34 (dd, J 3.0, 7.7, 1H), 3.51 (dd,
J 9.5, 1H), 3.58–3.64 (m, 3H), 3.80 (d, J 2.7, 1H),
3.99–4.28 (m, 8H), 4.50 (d, J 8.5, 1H), 5.13–5.33 (m,
8H), 5.91 (m, 4H). 13C NMR (125 MHz, CDCl3): 68.2,
71.6, 72.5, 73.0, 73.9, 74.2, 75.4, 78.3, 82.0, 90.4, 116.8,
116.9, 117.1, 134.3, 134.7, 134.8, 135.2.
1
(M+Na)+. H NMR (500 MHz, CDCl3): 1.63–1.70 (m,
4H), 2.24–2.30 (m, 2H), 2.36 (t, J 6.9, 2H), 3.43 (dd, J
2.8, 9.4, 1H), 3.46–3.53 (m, 4H), 3.58–3.64 (m, 1H),
3.80 (br s, 1H), 3.93–4.33 (m, 8H), 5.05 (t, J 9.1, 1H),
5.08 (s, 2H), 5.13–5.33 (m, 8H), 5.78–5.97 (m, 4H),
6.06 (d, J 9.2, 1H), 7.28–7.36 (m, 5H). 13C NMR
(125 MHz, CDCl3): 24.3, 24.6, 33.8, 36.2, 66.1, 67.7,
71.3, 72.3, 73.1, 73.7, 73.9, 74.5, 79.0, 79.1, 82.7, 116.7,
116.8, 117.0, 117.3, 128.1, 128.5, 134.3, 134.7, 135.0,
135.3, 136.0, 172.4, 173.1.
4.1.4. 6-[(2,3,4,6-Tetra-O-(2-propenyl)-b-D-galactopyr-
anosylamino]-6-oxohexanoic acid benzyl ester (11). A
mixture of 10 (200 mg, 0.36 mmol) and tert-butyl-N-(2-
mercaptoethyl) carbamate (Boc-cysteamine) (1.02 g,
5.5 mmol, 16 equiv) in dried and degassed 1,4-dioxane
(3 mL) was irradiated at 254 nm for 4 h. The solvent
was then removed under vacuum and the residue was
purified by flash chromatography (ethyl acetate/hexane
3/2, 5% Et3N) to afford 400 mg (88%) of 11 as a colorless
oil. ES-MS (m/z): 1266 (MH+), 1283 (M+NH4)+, 1288
1
(M+Na)+. H NMR (500 MHz, CDCl3) 1.44 (s, 36H),
1.62–1.69 (m, 4H), 1.74–1.83 (m, 8H), 2.20–2.26 (m,
2H), 2.32–2.35 (m, 2H), 2.53–2.63 (m, 8H), 2.70–2.76
(m, 8H), 3.19–3.28 (m, 8H), 3.35–3.41 (m, 8H), 3.42–
3.90 (m, 6H), 5.01 (t, 5H), 5.10 (s, 2H), 6.03 (br s,
1H), 7.26–7.32 (m, 5H). 13C NMR (125 MHz, CDCl3):
23.1, 23.9, 24.5, 24.6, 24.8, 28.3, 28.4, 28.5, 28.6, 29.1,
29.8, 30.3, 30.5, 31.0, 31.2, 31.9, 32.2, 32.3, 32.4, 34.1,
36.3, 38.9, 40.0, 40.1, 40.2, 66.3, 68.3, 68.8, 69.0, 69.7,
71.1, 71.5, 72.5, 74.3, 74.5, 77.4, 78.7, 79.3, 79.5, 79.6,
83.7, 128.3, 128.4, 128.7, 128.9, 136.2, 155.8, 155.9,
156.0, 156.1, 173.0, 173.3. HRMS (TOF): Calculated
for C59H104N5O16S4 (M+H)+: 1266.6361, Found:
1266.6328.
4.1.2. 2,3,4,6-Tetra-O-(2-propenyl)-b-D-galactopyranosyl
amine (9). A solution of SnCl2Æ2H20 (14.91 g, 66 mmol)
in MeOH (100 mL) with a few drops of deionized water
was added to a flask containing azide 8 (2.42 g,
6.6 mmol) and the mixture was stirred overnight at
room temperature under an inert atmosphere. The sol-
vent was then removed under vacuum and the residue
was diluted with ethyl acetate (200 mL) and basified
by addition of saturated NaHCO3 solution (2·
200 mL). The combined layers were filtered to remove
the solid precipitate. The organic phase was isolated
and dried over MgSO4 and evaporated. The crude prod-
uct was then purified by flash chromatography (50%
ethyl acetate in hexane in the presence of 1% of triethyl-
amine) to yield the desired amine 9 as a colorless oil
(2.34 g, 52%). ES-MS (m/z): 340 (MH+), 357
(M+NH4)+, 362 (M+Na)+. 1H NMR (500 MHz,
CDCl3): 3.34 (dd, J 3.0, 7.7, 1H), 3.53 (dd, J 9.5, 1H),
3.58–3.64 (m, 3H), 3.80 (d, J 2.7, 1H), 3.99–4.28 (m,
8H), 4.26 (dt, J 1.28.5, 1H), 5.13–5.33 (m, 8H), 5.91
(m, 4H), 5.91 (m, 4H). 13C NMR (125 MHz, CDCl3):
69.1, 71.8, 72.6, 74.0, 74.3, 74.4, 80.5, 83.0, 86.6, 116.7,
117.0, 117.3, 117.7, 134.6, 135.2, 134.5, 135.7.
4.1.5. 6-[(2,3,4,6-Tetra-O-(2-propenyl)-b-D-galactopyr-
anosylamino]-6-oxohexanoic acid (12). A solution of 11
(380 mg, 0.3 mmol) in H2O/methanol mixture (20/80)
in which the pH had been adjusted to 13 by addition
of solid LiOH.H2O was stirred at room temperature
overnight. The solvent was then removed under vacuum
and the residue was dissolved in ethyl acetate (150 mL).
The organic layer was washed with cold 0.01 M citric
acid solution (2· 100 mL). After drying over MgSO4,
the solvent was removed and the residue was washed
via a plug of silica gel using 10% MeOH in CHCl3 as
eluent to give the pure acid 12 (312 mg, 88%) as a color-
less oil. ES-MS (m/z): 1176 (MH+), 1193 (M+NH4)+,
4.1.3.
6-(2,3,4,6-Tetra-O-(2-propenyl)-b-D-galactopyr-
anosylamino)-6-oxohexanoic acid benzyl ester (10). Adip-
ic acid monobenzyl ester (1.88 g, 8.07 mmol), HBTU
(3.04 g, 8.07 mmol), and DIPEA (1.38 mL, 8.07 mmol)
in dry THF (50 mL) were added to a stirred solution
of 9 (2.25 g, 6.61 mmol) in THF (150 mL). The reaction
mixture was then stirred overnight under an inert atmo-
sphere. The solvent was removed under vacuum and the
1
1198 (M+Na)+. H NMR (500 MHz, CDCl3) 1.44 (s,
36H), 1.62–1.69 (m, 4H), 1.74–1.83 (m, 8H), 2.20–2.26
(m, 2H), 2.32–2.35 (m, 2H), 2.53–2.63 (m, 8H), 2.70–
2.76 (m, 8H), 3.19–3.28 (m, 8H), 3.35–3.41 (m, 8H),
3.42–3.90 (m, 6H), 5.01 (t, 5H), 6.03 (br s, 1H). 13C
NMR (125 MHz, CDCl3): 23.1, 23.9, 24.5, 24.6, 24.8,

150
P. Y. Hayes et al. / Bioorg. Med. Chem. 14 (2006) 143–152
28.3, 28.4, 28.5, 28.6, 29.1, 29.8, 30.3, 30.5, 31.0, 31.2,
31.9, 32.2, 32.3, 32.4, 34.1, 36.3, 38.9, 40.0, 40.1, 40.2,
66.3, 68.3, 68.8, 69.0, 69.7, 71.1, 71.5, 72.5, 74.3, 74.5,
77.4, 78.7, 79.3, 79.5, 79.6, 83.7, 155.8, 155.9, 156.0,
156.1, 173.0, 173.3. HRMS (TOF): Calculated for
C52H98N5O16S4 (MH)+: 1176.5891, Found: 1176.5861.
(2 mmol each time) using the same protocol followed,
by TFA deprotection, and DMF flow-wash. Boc2Lys
(692 mg, 2 mmol) was coupled on the resin following
the same protocol (shaking time: 30 min, TFA deprotec-
tion and DMF flow-wash). This step was repeated for
the coupling of the second lysine residue (using 4 mmol
of Boc2Lys). Finally, Boc-Lys(ClZ) (8 mmol) or Boc-
ArgTsOH (8 mmol, for 4b) was coupled on the resin fol-
lowing the same protocol with 1 h shaking time resulting
in a negative ninhydrin test. The 4 Boc groups were re-
moved by treatment with neat TFA (2· 1 min), and the
resin was washed well with DMF, then MeOH, and
finally DCM, and then dried under vacuum to give the
loaded resin. The peptides (1 mmol) were then cleaved
from the resin using anhydrous hydrofluoric acid using
10% cresol as scavenger at 0 ꢂC for 1 h. The products
were precipitated with diethyl ether and then lyophilized
from water/acetonitrile to give the peptides as a white
powder. Purification by preparative HPLC (using an
acetonitrile/water gradient) afforded the purified pep-
tides after lyophilization as white powder. 3 (64%).
ES-MS: 1535 (M+H), 768, 512, 254. Compound 4a:
(35%). ES-MS: 1422 (M+H), 711, 285. Compound 4b
(40% yield). ES-MS: 1534 (M+H), 767, 512. 5 (55%).
ES-MS: 1366 (M+H), 683,456, 194, 170. Compound 6
(68%). ES-MS: 1310 (M+H), 655, 437.
4.1.6. Dendron 2a. MBHA resin (926 mg, 0.25 mmol))
was swollen in DMF in a solid-phase peptide synthesis
reaction vessel for 90 min. This resin was washed with
DIPEA (500 lL) in DMF by shaking the resin 2·
1 min. The resin was thoroughly washed with DMF,
and then the resin was shaken for 15 min with Boc-gly-
cine (350.2 mg, 2 mmol), HBTU (4 mL of a 0.5 M solu-
tion in DMF), and DIPEA (460 lL), resulting in a
negative ninhydrin test. After washing well the resin
with DMF, the Boc group was removed by treatment
with neat TFA (2· 1 min), and then the resin was again
washed well with DMF. 2-tert-Butoxycarbonylamino-
octadecanoic acid was coupled twice (239 mg, 0.6 mmol
each time) on the resin following the same protocol. The
sugar derivative 12 (705 mg, 0.6 mmol) was coupled on
the resin following the same protocol (shaking time 2 h
and 30 min). Finally, Boc-Lys(ClZ) (831.4 mg, 2 mmol,
twice) was coupled on the resin following the same pro-
tocol with 1 h shaking time, resulting in a negative nin-
hydrin test.
4.2. Microcalorimetry
The four Boc groups were removed by treatment with
neat TFA (2· 1 min), and the resin was washed well with
DMF, then MeOH, and finally DCM, and then dried
under vacuum to give 1.128 g of the loaded resin. The
peptide was then cleaved from the resin using anhydrous
hydrofluoric acid using 10% cresol as scavenger at 0 ꢂC
for 1 h. The product was precipitated with diethyl ether
and then lyophilized from water/acetonitrile to give the
peptide as a white powder. The crude peptide was then
purified by preparative HPLC using an acetonitrile/
water gradient. The purified peptide was then lyophi-
lized from water/acetonitrile to give the pure glycoden-
drimer as a white powder (244 mg, 17%). ES-MS: 1907
(M+H), 954, 637, 478, 382.
Isothermal titration microcalorimetry (ITC) was per-
formed using a MicroCal VP-ITC titration calorimeter
(MicroCal, Northampton, MA). A solution of LMWH
(1.70 lm) was placed in the sample cell (1.4395 mL)
and a solution of dendron 2b (62.5 lm) was placed in
the syringe. The cell temperature was maintained at
30 ꢂC and dendron 2b was added to the solution of
LMWH using 1· 5 lL and then 29· 8 lL injections,
each injection being 4 min apart. The experiment was
performed in triplicate and the software Origin (version
5.0, Microcal) was used for data analysis.
4.3. Pharmacodynamic study
4.1.7. Dendron 2b. This dendron (1 mmol) was synthe-
sized, cleaved, and purified, following the same protocol
as that for dendron 2a. The purified peptide was then
lyophilized from water/acetonitrile to give the pure gly-
codendrimer as a white powder (275 mg, 27.5%). ES-
MS: 2020 (M+2H), 1010, 674, 506, 405.
LMWH (sodium salt, from porcine intestinal mucosa,
produced by oxidative depolymerization, avg.
MW = 4125 Da, anti-factor Xa activity = 105 IU/mg)
was purchased from MP Biomedicals (Seven Hills, Aus-
tralia; catalog #194113). Male Sprague–Dawley rats
(200–300 g, Herston Medical Research Centre, Bris-
bane, Australia) were fasted for 12 h prior to dosing.
Each rat was dosed by oral gavage, using a blunt-tipped
feeding needle, while the animal was under light anesthe-
sia (O2/CO2, 1:1). The doses consisted of LMWH
(7500 IU/kg) alone or in combination with a penetration
enhancer (7 mol equiv). The delivery vehicle was propyl-
ene glycol and water (1:1, 1 mL), and LMWH alone was
a clear solution, whereas doses containing a penetration
enhancer were suspensions. Two blood samples were
collected in series from each rat using cardiac puncture,
while the animal was anesthetized (O2/CO2, 1:1). After
the second blood collection, the anaesthetized animal
was promptly euthanized by cervical dislocation. Blood
samples (450 lL) were collected in tubes containing
4.1.8. General procedure for the synthesis of dendrons 3–
6. MBHA resin (1.85 g, 1 mmol)) was swollen in DMF
in a solid-phase peptide synthesis reaction vessel for
90 min. This resin was washed with DIPEA (500 lL)
in DMF by shaking the resin 2· 1 min. The resin was
thoroughly washed with DMF, and then the resin was
shaken for 15 min with Boc-Glycine (700 mg, 4 mmol),
HBTU (8 mL of a 0.5 M solution in DMF), and DIPEA
(920 lL), resulting in a negative ninhydrin test. After
washing the resin with DMF well, the Boc group was re-
moved by treatment with neat TFA (2· 1 min), and then
the resin was again washed well with DMF. The appro-
priate LAA, C10 to C18 was coupled twice on the resin

P. Y. Hayes et al. / Bioorg. Med. Chem. 14 (2006) 143–152
151
9. Jandik, K. A.; Kruep, D.; Cartier, M.; Linhardt, R. J.
J. Pharm. Sci. 1996, 85, 45–51.
10. Therapeutic Guidelines Antibiotic, 11th ed.; Therapeutic
Guidelines Limited: North Melbourne, 2000.
11. Atkinson, H. C.; Chambers, S. T.; McGinlay, A. M. N. Z.
Med. J. 1989, 102, 409–411.
12. Mustafa, F.; Yang, T.; Khan, M. A.; Ahsan, F. J. Pharm.
Sci. 2004, 93, 675–683.
3.8% sodium citrate solution (50 lL). The blood samples
were centrifuged (2000g, 15 min, 20 ꢂC) within 1 h of
collection, and the plasma was harvested and stored fro-
zen (À70 ꢂC) until analysis. Plasma samples were ana-
lyzed for anti-factor Xa activity using a chromogenic
assay (Coatestꢁ LMW Heparin/Heparin, Chromogenix
Instrumentation Laboratory S.p.A., Milan, Italy). All
animal experiments were approved by The University
of Queensland Animal Ethics Committee.
13. Lee, V. H.; Yamamoto, A.; Kompella, U. B. Crit. Rev.
Ther. Drug Carrier Systems 1991, 8, 91–192.
14. Aungst, B. J. J. Pharm. Sci. 2000, 89, 429–442.
15. Ubrich, N.; Hoffart, V.; Vigneron, C.; Hoffman, M.;
Maincent, P. STP Pharma Sci. 2002, 12, 147–155.
16. Ross, B. P.; Toth, I. Curr. Drug Delivery 2005, 2, 277–287.
17. Leone-Bay, A.; Paton, D. R.; Freeman, J.; Lercara, C.;
OꢁToole, D.; Gschneidner, D.; Wang, E.; Harris, E.;
Rosado, C.; Rivera, T.; DeVincent, A.; Tai, M.; Merco-
gliano, F.; Agarwal, R.; Leipold, H.; Baughman, R. A. J.
Med. Chem. 1998, 41, 1163–1171.
18. Leone-Bay, A.; Freeman, J.; OꢁToole, D.; Rosado-Gray,
C.; Salo-Kostmayer, S.; Tai, M.; Mercogliano, F.; Baugh-
man, R. A. J. Med. Chem. 2000, 43, 3573–3576.
19. Rivera, T. M.; Leone-Bay, A.; Paton, D. R.; Leipold, H.
R.; Baughman, R. A. Pharm. Res. 1997, 14, 1830–1834.
20. Baughman, R. A.; Kapoor, S. C.; Agarwal, R. K.; Kisicki,
J.; Catella-Lawson, F.; FitzGerald, G. A. Circulation
1998, 98, 1610–1615.
4.4. Analysis of pharmacodynamic data
The maximum plasma anti-factor Xa activity (Cmax) and
time to reach Cmax (tmax) are observed values taken
directly from analytical data. The area under the plasma
anti-factor Xa activity versus time curve (AUC180) was
estimated using the linear trapezoidal rule for the time
period 0–180 min. The means of the Cmax data were
compared by one-way analysis of variance (ANOVA)
combined with the Tukey multiple comparison proce-
dure. AUC for the treatment group that received den-
dron 5 was compared to control AUC using a two-
tailed unpaired t-test with unequal variance (Welchꢁs
approximate t).
21. Berkowitz, S. D.; Marder, V. J.; Kosutic, G.; Baughman,
R. A. J. Thromb. Haemost. 2003, 1, 1914–1919.
22. Emisphere Technologies Reports Results From Phase III
Oral Heparin PROTECT Trial [News Release]. Emisphere
Technologies, Inc.; 2002 [cited 2004 11 March]; Available
from: URL:http://www.emisphere.com/html/pr.html.
23. Emisphere Technologies Presents First Analysis of PRO-
TECT Trial at the Annual Meeting of the American
Society Of Hematology [News Release]. Emisphere Tech-
nologies, Inc.; 2002 [cited 2004 11 March]; Available from:
URL:http://www.emisphere.com/html/pr.html.
Acknowledgments
We thank Dr. Philip Kearns [The University of Queens-
land (U.Q.)] for assistance with microcalorimetry.
B.P.R. acknowledges U.Q. for a Ph.D. scholarship and
Alchemia Ltd for a Ph.D. studentship. This work was
supported financially by Alchemia Ltd.
24. Leone-Bay, A.; OꢁShaughnessy, C.; Agarwal, R.; Rivera-
Schaub, T.; Rosado-Gray, C.; Gerspach, L.;
Baughman, R. A. Pharm. Technol. North Am. 2002, 26,
38,40,42,44,46.
25. Tomalia, D. A.; Baker, H.; Dewald, J.; Hall, M.; Kallos,
G.; Martin, S.; Roeck, J.; Ryder, J.; Smith, P. Polymer J.
(Tokyo Japan) 1985, 17, 117–132.
Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/
j.bmc.2005.08.004.
26. Newkome, G. R.; Yao, Z.; Baker, G. R.; Gupta, V. K.
J. Org. Chem. 1985, 50, 2003–2004.
References and notes
27. Liu, M.; Frechet, J. M. J. Pharm. Sci. Technol. Today
1999, 2, 393–401.
28. Patri, A. K.; Majoros, I. J.; Baker, J. R. Curr. Opin. Chem.
Biol. 2002, 6, 466–471.
29. Cloninger, M. J. Curr. Opin. Chem. Biol. 2002, 6, 742–748.
30. Sakthivel, T.; Florence, A. T. Drug Delivery Technol. 2003,
3(50), 52–56.
31. Toth, I.; Sakthivel, T.; Wilderspin, A. F.; Bayele, H.;
OꢁDonnell, M.; Perry, D. J.; Pasi, K. J.; Lee, C. A.;
Florence, A. T. STP Pharma Sci. 1999, 9, 93–99.
32. Shah, D. S.; Sakthivel, T.; Toth, I.; Florence, A. T.;
Wilderspin, A. F. Int. J. Pharm. 2000, 208, 41–48.
33. Wimmer, N.; Marano, R. J.; Kearns, P. S.; Rakoczy, E.
P.; Toth, I. Bioorg. Med. Chem. Lett. 2002, 12, 2635–
2637.
1. Linhardt, R. J. J. Med. Chem. 2003, 46, 2551–2564.
2. Heparin [Monograph]. Chromogenix Instrumentation
Laboratory SpA; [cited 2004 16 May]; Available from:
URL:http://www.chromogenix.com.
3. Rang, H. P.; Dale, M. M.; Ritter, J. M. Pharmacology,
fourth ed.; Churchill Livingstone: Edinburgh, 1999.
4. Hirsh, J.; Warkentin, T. E.; Shaughnessy, S. G.; Anand, S.
S.; Halperin, J. L.; Raschke, R.; Granger, C.; Ohman, E.
M.; Dalen, J. E. Chest 2001, 119, 64S–94S.
5. Hoppensteadt, D.; Walenga Jeanine, M.; Fareed, J.; Bick
Rodger, L. Hematol. Oncol. Clin. North Am. 2003, 17,
313–341.
6. Hirsh, J.; Levine, M. N. Blood 1992, 79, 1–17.
7. Majerus, P. W.; Tollefsen, D. M. Anticoagulant Throm-
bolytic and Antiplatelet drugs. In Goodman & Gilmanꢁs
The Pharmacological Basis of Therapeutics; Goodman, L.
S., Gilman, A. G., Hardman, J. G., Limbird, L. E., Eds.;
McGraw-Hill: New York, 2001; pp 1519–1538.
8. Ahn, M. Y.; Shin, K. H.; Kim, D.-H.; Jung, E.-A.; Toida,
T.; Linhardt, R. J.; Kim, Y. S. Can. J. Microbiol. 1998, 44,
423–429.
34. Ramaswamy, C.; Sakthivel, T.; Wilderspin, A. F.; Flor-
ence, A. T. Int. J. Pharm. 2003, 254, 17–21.
35. Gibbons, W. A.; Hughes, R. A.; Charalambous, M.;
Christodoulou, M.; Szeto, A.; Aulabaugh, A. E.; Mas-
cagni, P.; Toth, I. Liebigs Ann. Chem. 1990, 1175–1183.
36. Ross, B. P.; DeCruz, S. E.; Lynch, T. B.; Davis-Goff, K.;
Toth, I. J. Med. Chem. 2004, 47, 1251–1258.

152
P. Y. Hayes et al. / Bioorg. Med. Chem. 14 (2006) 143–152
37. Ross, B. P.; Braddy, A. C.; McGeary, R. P.; Blanch-
field, J. T.; Prokai, L.; Toth, I. Mol. Pharm. 2004, 1,
233–245.
53. The different lipoamino acids (LAAs) used in this study
were synthesized following literature methods described in
Ref. 35.
38. Toth, I.; Hillery, A. M.; Wood, I. P.; Magnusson, C.;
Artursson, P. Int. J. Pharm. 1994, 102, 223–230.
39. Toth, I.; Hughes, R. A.; Dekany, G.; Hillery, A. M.;
Ward, P. Liebigs Ann. Chem. 1994, 685–688.
40. Flinn, N.; Coppard, S.; Toth, I. Int. J. Pharm. 1996, 137,
33–39.
41. Olive, C.; Batzloff, M.; Horvath, A.; Clair, T.; Yarwood,
P.; Toth, I.; Good, M. F. Infect. Immun. 2003, 71, 2373–
2383.
54. Merrifield, R. B. J. Am. Chem. Soc. 1963, 85, 2149–2154.
55. Lobo, B. A.; Rogers, S. A.; Wiethoff, C. M.; Choos-
akoonkriang, S.; Bogdanowich-Knipp, S.; Middaugh, C.
R. Methods Mol. Med. 2001, 65, 319–348.
56. Tame, J. R. H.; OꢁBrien, R.; Ladbury, J. E. Isothermal
titration calorimetry of biomolecules. In Biocalorimetry:
Applications of Calorimetry in the Biological Sciences;
Ladbury, J. E., Chowdhry, B. Z., Eds.; John Wiley and
Sons: Chichester, 1998, pp 27–38.
42. Olive, C.; Batzloff, M. R.; Toth, I. Expert Rev. Vaccines
2004, 3, 43–58.
57. Fromm, J. R.; Hileman, R. E.; Caldwell, E. E. O.; Weiler,
J. M.; Linhardt, R. J. Arch. Biochem. Biophys. 1995, 323,
279–287.
58. Bianchini, P.; Bergonzini, G. L.; Parma, B.; Osima, B.
Haemostasis 1995, 25, 288–298.
59. Quintanar-Guerrero, D.; Allemann, E.; Fessi, H.; Doel-
ker, E. Pharm. Res. 1997, 14, 119–127.
60. Jonkman, J. H. G.; Hunt, C. A. Pharm. Weekbl. Sci. 1983,
5, 41–48.
61. Neubert, R. Pharm. Res. 1989, 6, 743–747.
62. Sakthivel, T.; Toth, I.; Florence, A. T. Pharm. Res. 1998,
15, 776–782.
63. Al-Jamal, K. T.; Sakthivel, T.; Florence, A. T. Int. J.
Pharm. 2003, 254, 33–36.
64. Swenson, E. S.; Curatolo, W. J. Adv. Drug Delivery Rev.
1992, 8, 39–92.
65. Mori, S.; Matsuura, A.; Prasad, Y. V. R.; Takada, K. Biol.
Pharm. Bull. 2004, 27, 418–421.
43. Dal Pozzo, A.; Acquasaliente, M.; Geron, M. R.;
Andriuoli, G. Thromb. Res. 1989, 56, 119–124.
44. Andriuoli, G.; Bossi, M.; Caramazza, I.; Zoppetti, G.
Haemostasis 1990, 20(Suppl 1), 154–158.
45. Zoppetti, G.; Caramazza, I.; Murakami, Y.; Ohno, T.
Biochim. Biophys. Acta 1992, 1156, 92–98.
46. Andriuoli, G.; Caramazza, I.; Galimberti, G.; Zoppetti,
G.; Benedini, F.; Sala, A.; Del Soldato, P. Haemostasis
1992, 22, 113–116.
47. Capila, I.; Linhardt, R. J. Angew. Chem., Int. Ed. 2002, 41,
390–412.
48. Kasai, S.; Nagasawa, H.; Shimamura, M.; Uto, Y.; Hori,
H. Bioorg. Med. Chem. Lett. 2002, 12, 951–954.
49. Broeder, W.; Kunz, H. Bioorg. Med. Chem. 1997, 5, 1–
19.
50. McGeary, R. P.; Jablonkai, I.; Toth, I. Tetrahedron 2001,
57, 8733–8742.
51. Sridhar, P. R.; Prabhu, K. R.; Chandrasekaran, S. J. Org.
Chem. 2003, 68, 5261–5264.
66. Rama Prasad, Y. V.; Minamimoto, T.; Yoshikawa, Y.;
Shibata, N.; Mori, S.; Matsuura, A.; Takada, K. Int. J.
Pharm. 2004, 271, 225–232.
52. Kieburg, C.; Dubber, M.; Lindhorst, T. K. Synlett 1997,
1447–1449.
67. Samama, M.-M.; Gerotziafas, G. T. Thromb. Res. 2003,
109, 1–11.