Synthesis of glycolipopeptidic building blocks for carbohydrate receptor discovery

Article abstract of DOI:10.1016/j.carres.2011.03.019

A class of glycolipopeptides for use as building blocks for a new type of dynamic combinatorial library is reported. The members of the library consist of a variable carbohydrate moiety, coded amino acids, and lipoamino acids in order to convert them into amphiphiles. Glycolipopeptidic amphiphiles interact through non-covalent bonding when mixed together in aqueous phase and form micelles in dynamic close-packed fluid mosaic arrays. The head groups of amphiphiles are exposed on the micelle surface, providing entities which could be screened in biological assays to find the most potent combination of building blocks in order to identify new bioactive carbohydrate ligands.

Full text of DOI:10.1016/j.carres.2011.03.019

Carbohydrate Research 346 (2011) 1439–1444
Contents lists available at ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Synthesis of glycolipopeptidic building blocks for carbohydrate
receptor discovery
Zyta M. Ziora a,b, Norbert Wimmer a, Roger New c, Mariusz Skwarczynski a, Istvan Toth a,b,
⇑
a The University of Queensland, School of Chemistry and Molecular Biosciences, Brisbane, Qld 4072, Australia
b The University of Queensland, Centre for Integrated Preclinical Drug Development, CIPDD, TetraQ-Pharmaceutics, Brisbane, Qld 4072, Australia
c Proxima Concepts Ltd, The London Bioscience Innovation Centre, 2 Royal College Street, London NW1 OTU, United Kingdom
a r t i c l e i n f o
a b s t r a c t
Article history:
A class of glycolipopeptides for use as building blocks for a new type of dynamic combinatorial library is
reported. The members of the library consist of a variable carbohydrate moiety, coded amino acids, and
lipoamino acids in order to convert them into amphiphiles. Glycolipopeptidic amphiphiles interact
through non-covalent bonding when mixed together in aqueous phase and form micelles in dynamic
close-packed fluid mosaic arrays. The head groups of amphiphiles are exposed on the micelle surface,
providing entities which could be screened in biological assays to find the most potent combination of
building blocks in order to identify new bioactive carbohydrate ligands.
Received 31 January 2011
Received in revised form 8 March 2011
Accepted 9 March 2011
Available online 15 March 2011
Dedicated to Professor András Lipták on the
occasion of his 75th birthday
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
Lipoamino acids
Glycolipopeptides
Self-assembly
Dynamic combinatorial library
Micellization
1. Introduction
reversible bonds to one or more components in the combinatorial
ensemble. The head groups must cover the geometrical and func-
More than 50% of all human proteins are glycosylated.1,2 Cell
surface carbohydrates existing as glycoconjugates (glycolipids, gly-
coproteins, and proteoglycans) play crucial roles in both physiolog-
ical and pathological events, such as cell–cell communication,
tumor metastasis, inflammatory responses, and viral infection.3,4
In past decades, carbohydrate-labeled polymers, nanoparticles
and liposomes have been investigated for their ability to deliver
drugs to specific cells and tissues.5
The synthesis of glycopeptides requires a combination of syn-
thetic methods from both carbohydrate and peptide chemistry.6
Thus, it is fundamental to develop convenient synthetic pathways
to connect carbohydrates to amino acids, leading to glycosylated
building blocks and efficient glycopeptide synthesis. Such building
blocks, especially when readily available, could be of great value
for the combinatorial chemistry application. Dynamic combinato-
rial chemistry (DCC), an alternative to classical combinatorial
chemistry, relies on the spontaneous assembly of a given number
of building blocks by reversible reactions.7 All building blocks from
dynamic combinatorial library (DCL) must have the ability to form
tional space of potential target sites for the best recognition and
interaction with the biomolecules at the binding site.8 Glycopep-
tide DCLs are of considerable interest because both peptides and
glycosides offer potential for recognition of glycoproteins.9,10
A novel combinatorial screening system incorporating glyco-
lipopeptides as building blocks was designed. The glycolipopep-
tides consisted of a peptidic moiety, together with lipoamino
acids, and a carbohydrate, giving rise to a molecule which had
amphiphilic properties. Compounds containing lipoamino acids,
which combine the properties of amino acids with those of lipids
had several proven positive outcomes including improved biologi-
cal stability without loss of biological activity.11–13 These lipophilic
compounds have the capacity to form particulate systems either in
their own right, or when they were incorporated into other lipidic
vehicles.14 The carbohydrate head groups were linked to the lipoa-
mino acids via a serine-glycine dipeptide spacer. The Ser-Gly
sequence occurs in most of proteoglycans and, in some cases, is
involved in biosynthetic recognition with regard to the carbohy-
drate acceptors.15–17 These glycolipopeptidic building blocks are
designed (a) to come together (self-assemble) without the use of
covalent linkages and (b) to create three-dimensional mosaic
structures, capable of receptor recognition, on the surface of the
micelles formed. The head groups are brought together sufficiently
⇑
Corresponding author. Tel.: +61 7 33469892; fax: +61 7 33654273.
E-mail address: i.toth@uq.edu.au (I. Toth).
0008-6215/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2011.03.019

1440
Z. M. Ziora et al. / Carbohydrate Research 346 (2011) 1439–1444
closely to enable them to form structures capable of binding to
carbohydrate receptors, in a manner similar to that of oligosaccha-
rides. The method presented here could overcome disadvantages of
classical combinatorial approaches, where production of carbohy-
drate ligands is time-consuming, the compounds are expensive
to make and purify, and the quantities available for testing are of-
ten very limited.
O
O
O
R1
O
H
N
H
N
H
N
H
N
R2
H2N
N
H
N
H
N
H
O
O
O
O
HO
2. Results and discussion
3a: R1 =
3b: R1 =
3c: R1 =
3d: R1 =
3e: R1 =
3f: R1 =
3g: R1 =
3h: R1 =
R2 =
R2 =
R2 =
R2 =
R2 =
R2 =
R2 =
R2 =
Peptides containing a glycosyl unit and lipoamino acid residues
combined with representatives of coded amino acids were
designed and synthesized. The lipoamino acid (LAA) is an alpha
amino acid containing a long hydrocarbon side chain. In all of
the glycolipopeptides constructed, two lipoamino acids were
introduced to modulate lipophilicity and to assist microsphere
formation via a self-assembly process.14,18,19 The glycosyl azides
1a–g were synthesized according to known procedures.20–27 The
glycosyl units 2a–h were obtained by the acylation and in situ
hydrogenation28 of 1a–h (Scheme 1).
Building blocks 3a–k and 4a–g (Fig. 1) were synthesized by
applying conventional solid phase peptide synthesis (SPPS) with
a 4-methyl benzhydrylamine (MBHA) resin. Couplings were per-
formed with Boc-protected amino acids activated by 2-(1H-benz-
triazole-1yl)-1,2,2-tetramethyluronium hexafluoro phosphate
(HBTU) and N,N-diisopropylethylamine (DIPEA). Glycosyl units
2a–h were incorporated into the N-termini of peptides in the final
step of chain elongation on a solid support.
The amphiphiles synthesized were designed to aggregate
spontaneously to form micelles when mixed together in aqueous
solution. Their ability to form supra-molecular assemblies was
examined by isothermal titration calorimetry (ITC), and the size
of micelles then was determined by dynamic light scattering
(DLS) and confirmed by transmission electron microscopy (TEM).
ITC results are presented in Table 1. The low CMC value for 4a–g
suggested a good aptitude of these amphiphiles to self-assemble.
The self-assembly was thermodynamically favored as the negative
OH
NH2
O
HO
HO
OH
N
H
OH
O
OH
HO
HO
OH
O
OH
O
O
OH
O
HO
HO
OH
3i: R1 =
3j: R1 =
3k: R1 =
R2 =
R2 =
R2 =
O
OH
NH
OH
OH
OH
OH
O
HO
HO
N
NH2
H
NAc
O
HO
HO
O
HO
OH
HO
O
O
O
H
N
H
H
H2N
N
N
R1
values of Gibbs free energy
DG were found for all of the com-
N
N
N
H
O
H
H
pounds examined. The process was entropy-driven with an endo-
thermic micellization enthalpy. The similarities in CMC and
thermodynamics values among the compounds suggested that car-
bohydrates diversity had very low impact on the behavior of the
whole construct.
To prove the concept that building blocks can self-assemble into
mixed micelles, amphiphiles 4a–g (4/H2O) were combined in an
equimolar ratio and ITC experiments were performed. As antici-
pated, CMC values and thermodynamic parameters were almost
identical to those obtained for each component self-assembled
individually. This suggested that building blocks are mixed in a
random manner during micelle formation. If the compounds 4a–
g were not able to form mixed micelles, the CMC value for 4/H2O
would be sevenfold lower than that observed (as the molar contri-
O
O
O
OH
O
4a: R1 =
4b: R1 =
4c: R1 =
4d: R1 =
HO
HO
OH
OH
OH
HO
HO
O
HO
4e: R1 =
4f: R1 =
4g: R1 =
O
HO
OH
OH
OH
O
O
OH
HO
HO
OH
OH
OH
OH
O
O
HO
HO
HO
NAc
OH
Figure 1. Structure of building blocks 3a–k and 4a–g.
AcO
AcO
O
O
O
H
N
succinic anhydride
DMAP, H2, Pd-C
N3
OH
bution of each compound in the mixture is 1/7). These experiments
were repeated with the compounds dispersed in the phosphate
saline buffer (4/PBS) to mimic physiological conditions. No signif-
icant differences in the self-assembly process were observed
regardless of the solvent used (Table 1).
O
1a-g
2a - (Ac)4-β-D-Glcp
2b - (Ac)4-β-D-Galp
2c - (Ac)3-α-L-Arap
2d - (Ac)3-β-L-Fucp
2e - (Ac)3-β-L-Rhap
2f - (Ac)4-β-D-Manp
The micelles formed during the ITC experiments were subse-
quently analyzed using dynamic light scattering. In the case of all
4a–g compounds, size distribution was quite broad with particles
sizes in the range of 20–80 nm but larger aggregates were also ob-
2g - (Ac)3-β-D-GlcNAcp
Scheme 1. Synthesis of glycosyl units 2.
served (ꢀ1
lm). In case of both types of mixed dispersions, mainly

Z. M. Ziora et al. / Carbohydrate Research 346 (2011) 1439–1444
1441
Table 1
(N2, H2, Ar) were supplied by BOC Gases (Brisbane, Qld, Australia).
Silica for flash chromatography (Silica Gel 60, 230–400 mesh) was
obtained from Lomb Scientific (Taren Point, NSW, Australia). Deu-
terated solvents (CDCl3-d1 and DMSO-d6) were manufactured by
Cambridge Isotope Laboratories Inc. (Andover, MA, USA). All other
reagents were purchased in analytical grade or higher purity from
Sigma–Aldrich (Castle Hill, Vic., Australia) or Merck Pty (Kilsyth,
Vic., Australia). Solvents were freshly distilled and dried prior to
use and all moisture-sensitive reactions were carried out under in-
ert atmosphere (N2/Ar) using oven-dried glassware.
Critical micelle concentration and thermodynamic parameters of the self-assembly
process for compounds 4a–g individually and their mixtures in water (4/H2O) and PBS
(4/PBS)
Compound
CMC (
l
M)
D
H (kJ molÀ1
)
T
D
S (kJ molÀ1
)
D )
G (kJ molÀ1
4a
4b
4c
4d
4e
4f
4g
4/H2O
4/PBS
4.5 0.4
5.7 0.3
5.6 0.5
3.7 0.5
5.0 0.5
4.9 0.1
5.0 0.4
4.0 0.2
4.9 0.1
32.2 2.9
26.2 1.0
29.3 2.1
56.5 6.2
41.9 4.2
37.7 3.8
37.7 3.8
43.9 2.1
32.6 2.9
74.3 3.1
67.7 0.2
70.8 2.4
99.1 6.8
83.7 4.4
79.5 3.8
79.5 4.0
86.3 2.2
74.5 2.9
À42.1 0.2
À41.5 0.1
À41.5 0.3
À42.6 0.6
À41.8 0.2
À41.8 0.1
À41.8 0.2
À42.4 0.1
À41.9 0.1
1H NMR spectra were recorded at 297 °K with a Bruker 400 and
500 MHz instrument operating at 400 and 500 MHz for 1H using
CDCl3, CD3OD, or acetone-d6 as solvent and tetramethylsilane as
an internal standard, unless stated otherwise. Coupling constants
are given in Hertz. 13C spectra were measured at 100.62 and
125.77 MHz and referenced to CDCl3 (77.0 Hz), CD3OD (49.0 Hz),
or acetone-d6 (30.5 Hz). Homo- and heteronuclear 2D NMR spec-
troscopy was performed with Bruker standard software.
TLC was performed on Merck pre-coated aluminium sheet (Sil-
ica Gel 60 F254); spots were detected by spraying with H2SO4.
Amine derivatives were detected by ninhydrin spray, followed by
heating the sheets. Column chromatography was performed on sil-
ica gel columns (size A, 28 Â 2; B, 30 Â 2.5; and C, 43 Â 4 cm; silica
gel 0.040–0.063 mm).
small sizes were observed; 20–80 nm for 4/H2O, and 30–50 nm for
4/PBS.
TEM experiments confirmed the results obtained from the DLS
measurements and demonstrated the formation of supra-molecu-
lar structures. TEM images of the mixture 4/H2O showed nanopar-
ticles being in a size range from 15 to 70 nm, and images of the
mixture 4/PBS showed nanoparticles of sizes ranging from 20 to
60 nm (Fig. 2).
3. Conclusion
Analytical RP-HPLC was performed on a Shimadzu instrument
(LC-10AT liquid chromatograph, SCL-10A system controller, SPD-
6A UV detector, a SIL-6B auto injector with a SCL-6B system con-
troller, and a C18-HPLC-column using a CH3CN/water/0.1% TFA
gradient as well as isopropanol/water/0.1% TFA gradient. HPLC
purification was done on a Waters HPLC system (Model 600 con-
troller, 490E UV detector, F pump, and 0.46 Â 15 cm Vydac RP-
C18 column with 0.005 mm particle size) using a CH3CN/water/
0.1% TFA gradient. He-gas was employed for degassing of HPLC-
solvents.
Mass spectra were obtained on a quadrupole-electrospray-MS
(Perkin Elmer API 3000 instrument) in the positive-ion mode. Con-
centration of solutions was performed at reduced pressure at tem-
peratures <40 °C. High-resolution mass spectrometry (HRMS) data
were obtained on a Qstar Pulsar instrument (Applied Biosystems)
operating in positive-ion electrospray mode.
A library comprising members consisting of a new class of
amphiphilic glycolipopeptides were synthesized and character-
ized. These glycolipopeptide building blocks, when dispersed in
aqueous media, were able to associate to form supra-molecular
assemblies. This library was developed to address the structural
diversity of natural carbohydrate ligands. Biological evaluation of
these ligands will be reported elsewhere. Glycolipidic building
blocks employed in a dynamic self assembling mode could become
valuable and inexpensive tools in the development of therapeutics
based on carbohydrates.
4. Experimental section
4.1. Chemicals and equipment
The thermodynamic interactions were measured and calculated
using a MicroCal VP-ITC Microcalorimeter (Northampton, MA,
USA) with ORIGIN 5.0 software and VPViewer 2000.
The particle size measurements were done by using a Zetasizer
Nano ZP instrument (Malvern Instruments, UK) with DTS software.
Transmission electron microscopy photographs were taken
on a JEOL-1010 microscope operating at an accelerating voltage
of 100 kV. The sample images were analyzed using the AnalySISÒ
Peptide synthesis solvent, DMF, and reagents, trifluoroacetic
acid (TFA), and DIPEA, were purchased from Auspep (Melbourne,
Vic., Australia). HBTU and di-tert-butyl dicarbonate were obtained
a
from GL Biochem Ltd (Shanghai, China). N -Boc-protected amino
acids and pMBHA resin were supplied by Novabiochem (Läufelfin-
gen, Switzerland). Palladium (10 wt % on carbon) was purchased
from Lancaster Synthesis (Lancashire, England). Ultra pure gases
Figure 2. TEM images of the mixture 4/H2O (a) and 4/PBS (b), scale bar 200 nm.

1442
Z. M. Ziora et al. / Carbohydrate Research 346 (2011) 1439–1444
software (Soft Imaging Systems, Megaview III, Munster,
Germany).
4.2.1.6. 4-Oxo-4-[(2,3,4,6-tetra-O -acetyl-b-D-mannopyranosyl)-
amino]-butanoic acid (2f).
C18H25O12N, 447.40; ESI-MS, m/z:
448.2 [M+H]+, 470.1 [M+Na]+, 895.6 [2M+H]+, 917.8 [2M+Na]+.
NMR data correspond to those reported in the literature.31
4.2. Synthesis
2-Aminododecanoic acid and its Boc-protected form were syn-
thesized according to published method.29
4.2.1.7. 4-Oxo-4-[(2-(acetylamino)-3,4,6-tri-O-acetyl-2-deoxy-b-
D
-glucopyranosyl) amino]-butanoic acid (2g). C18H26O11N2,
446.41; ESI-MS, m/z: 447.5 [M+H]+, 469.4 [M+Na]+, 893.8
[2M+H]+, 915.6 [2M+Na]+. 1H NMR (CDCl3):
7.45 (d, 1H,
4.2.1. General procedure for synthesis of glycosyl units (2a–h)
Glycosyl azide (1 mmol), succinic anhydride (2 mmol), and
DMAP (0.1 mmol) were dissolved in dry THF (4 mL), degassed,
and stirred vigorously with Pd–C (50 mg) under H2-atmosphere
for 16 h. The mixture was filtered over Celite and concentrated un-
der reduced pressure. The organic residue was dissolved in EtOAc/
EtOH (20:1, 50 mL), ice-cold aqueous HCl-solution (5%, 3Â, each
25 mL), satd NaHCO3-solution (3 Â 25 mL), brine (3 Â 20 mL), and
dried (MgSO4). The residue was purified on a column of silica gel,
(1:5?1:1 EtOH/EtOAc, containing 1% acetic acid for 2h; 1:5?hex-
ane/EtOAc, containing 1% acetic acid for 2e–f) and co-evaporated
with toluene (30 mL), to afford glycosyl succinates. Glycosyl-succi-
nates of 4a–d were obtained pure after recrystallization from
EtOH. The compounds were synthesized according to the litera-
ture,28,30,31 except for 2c–e and 2g. All compounds were applied
to the SPPS without any additional purification.
d
JNH,1 = 8.8 Hz, NH), 7.11 (d, 1H, JNH,2 = 9.1 Hz, NHAc), 5.17 (br dd,
2H, H-1, H-3), 5.02 (t, 1H, J3,4 = 9.8 Hz, H-4), 4.23 (dd, 1H,
J6,6 = 12.4 Hz, J6,5 = 4.5 Hz, H-6a), 4.15 (br dd, 1H, H-2), 4.04 (br
dd, 1H, H-6b), 3.80–3.75 (m, 1H, H-5), 2.73–2.60 (m, 2H, CH2(A)),
2.57–2.43 (m, 2H, CH2(B)), 2.04, 2.01, 1.99 and 1.92 (4s, each 3H,
4Ac). 13C NMR (CDCl3): d 175.41, 173.21, 172.54, 170.72, 170.51,
169.30, 79.17, 73.05, 72.79, 68.33), 61.82, 52.58, 30.54, 28.55,
20.42, 20.38, 20.31.
4.2.2. General protocol for the synthesis of peptides (SPPS
method)
MBHA resin (substitution ratio: 0.54 mmol/g, 0.65 mmol scale
for 3a–d; 0.54 mmol/g, 0.5 mol scale for 3e–k; and 0.54 mmol/g,
0.48 mol scale for 4a–g) was swollen in DMF in a sintered glass
peptide synthesis vessel for 90 min. Each amino acid coupling cy-
cle consisted of Boc-deprotection with neat TFA (2 Â 1 min), a 1-
min DMF flow wash, followed by 20 min coupling with the pre-
activated amino acid. An activation mixture consisting of Boc-
amino acid (3 equiv per mol amino-group), HBTU 0.5 M in DMF,
3 equiv), and DIPEA (0.442 mL, 4 equiv) was shaken for 12 min.
Coupling efficiency was monitored by quantitative ninhydrin test-
ing. (P99.8%).32 Upon completion of synthesis and removal of the
terminal Boc groups, the resin was washed with DMF, CH2Cl2, and
MeOH. The resin was dried to constant weight over KOH in vacuo.
The peptides were cleaved from the resin using HF, and p-cresol
as scavenger. The cleaved peptides were precipitated, filtered,
and washed thoroughly with ice-cold Et2O. After lyophilization
the crude peptide was obtained as an amorphous powder.
200 mg of each crude peptide was separated on Sephadex LH-20
(70 Â 3 cm) using CH3CN–water (1:1) as solvent. Peptide-positive
fractions were identified on silica gel by spraying with ninhydrin-
reagent. Analytical RP-HPLC (C4, 25 cm Vydac C4, C18 column
with 5 nm pore size and 4.6 mm internal diameter) was per-
formed using two different solvent systems in order to check
the peptides’ purity. Solvent system 1: Solvent A (H2O, 0.1%
TFA), Solvent B (90% CH3CN, 10% H2O, 0.1% TFA); Solvent system
2: Solvent A (H2O, 0.1% TFA), Solvent B (90% MeOH, 10% H2O,
0.1% TFA); Gradient: 0–100% B within 20 min; flow rate: 1 mL/
min; wave length: 214 nm; the purity of all peptides analyzed
by both systems on HPLC was over 95%. Peptides were character-
ized as well by ESI-MS and HRMS. The resulting peptides were
used as diastereomeric mixtures.
4.2.1.1. 4-Oxo-4-[(2,3,4,6-tetra-O -acetyl-b-D-glucopyranosyl)-
amino]-butanoic acid (2a).
C18H25O12N, 447.40; ESI-MS, m/z:
448.3 [M+H]+, 470.1 [M+Na]+, 895.5 [2M+H]+, 917.7 [2M+Na]+.
NMR data correspond to those reported in the literature.30,31
4.2.1.2. 4-Oxo-4-[(2,3,4,6-tetra-O -acetyl-b-D-galactopyranosyl)-
amino]-butanoic acid (2b).
C18H25O12N, 447.40; ESI-MS, m/z:
448.2 [M+H]+, 470.2 [M+Na]+, 895.5 [2M+H]+, 917.7 [2M+Na]+.
NMR data correspond to those reported in the literature.28
4.2.1.3. 4-Oxo-4-[(2,3,4-tri-O -acetyl-
a-L-arabinopyranosyl)-
amino]-butanoic acid (2c). 15H21O10N, 375.34; ESI-MS, m/z:
C
376.3 [M+H]+, 398.1 [M+Na]+, 773.2 [2M+Na]+. 1H NMR (CDCl3):
d 6.70 (d, 1H, JNH,1 = 7.7 Hz, NH), 5.29 (br d, 1H, H-4), 5.13–5.09
(m, 3H, H-1, H-2, H-3), 3.94 (dd, 1H, J5a,5b = 13.4 Hz, J5a,4 = 1.5 Hz,
H-5a), 3.75 (br dd, 1H, H-5b), 2.70–2.58 (m, 2H, CH2), 2.49–2.41
(m, 2H, CH2), 2.11, 2.02, and 1.98 (3s, each 3H, 3Ac). 13C NMR
(CDCl3): d 175.58, 172.23, 171.22, 170.04, 169.70, 78.71, 70.60,
68.44, 68.18, 65.77, 30.54, 28.55, 20.65, 20.49, 20.40.
4.2.1.4. 4-Oxo-4-[(2,3,4-tri-O -acetyl-b-L-fucopyranosyl)amino]-
butanoic acid (2d). 16H23O10N, 389.36; ESI-MS, m/z: 390.4
C
[M+H]+, 412.4 [M+Na]+, 801.4 [2M+Na]+. 1H NMR (CDCl3): d 6.52
(d, 1H, JNH,1 = 9.0 Hz, NH), 5.25 (dd, 1H, J4,5 = 1.0 Hz, J3,4 = 3.0 Hz,
H-4), 5.19 (br t, 1H, H-1), 5.14–5.02 (m, 2H, H-2, H-3), 3.91 (br
ddd, 1H, J5,methyl = 6.4 Hz, H-5), 2.76–2.54 (m, 2H, CH2), 2.48–2.42
(m, 2H, CH2), 2.14, 2.02, and 1.96 (3s, each 3H, 3Ac), 1.15 (d, 3H,
CH3). 13C NMR (CDCl3): d 176.65, 171.89, 171.54, 170.42, 169.81,
78.32, 71.23, 70.88, 70.36, 68.45, 30.56, 20.65, 20.53, 20.50, 15.98.
4.2.2.1. Compound 3a. HPLC, system A, Rt, (min): 17.42 and
17.52; system B: 23.01; ESI-MS, m/z: 1021.9 [M+H]+; HRMS calcd
for C50H84O14N8Na 1043.5999, found: 1043.5577.
4.2.1.5. 4-Oxo-4-[(2,3,4-tri-O -acetyl-b-
L-rhamnopyranosyl)-
4.2.2.2. Compound 3b. HPLC, system A, Rt, (min): 16.22 and
16.45; system B: 21.02 and 21.43; ESI-MS, m/z: 1003.1 [M+H]+;
HRMS calcd for C47H88O14N9 1002.6445, found: 1002.6472.
amino]-butanoic acid (2e). 16H23O10N, 389.36; ESI-MS, m/z:
C
390.5 [M+H]+, 412.4 [M+Na]+, 801.3 [2M+Na]+. 1H NMR (CDCl3):
d 6.92 (d, 1H, JNH,1 = 9.3 Hz, NH), 5.45 (br dd, 1H, H-1), 5.29 (dd,
1H, J1,2 = 1.1 Hz, J2,3 = 3.2 Hz, H-2), 4.95 (t, 1H, J3,4 = 10.1 Hz, H-4),
3.65–3.56 (m, 1H, H-5), 2.67–2.59 (m, 2H, CH2), 2.49–2.43 (m,
2H, CH2), 2.15, 2.01, and 1.92 (3s, each 3H, 3Ac), 1.19 (d, 3H,
CH3). 13C NMR (CDCl3): d 175.41, 171.66, 170.79, 170.03, 170.00,
75.80, 72.19, 71.49, 70.18, 70.11, 30.60, 28.92, 20.73, 20.67,
20.46, 17.35.
4.2.2.3. Compound 3c. HPLC, system A, Rt, (min): 19.41; system
B: 22.66; ESI-MS, m/z: 1060.8 [M+H]+, 1083.0 [M+Na]+; HRMS calcd
for C52H85O14N9Na 1082.6108, found: 1082.6111.
4.2.2.4. Compound 3d. HPLC, system A, Rt, (min): 16.74 and
16.89; system B: 22.06 and 22.36; ESI-MS, m/z: 1038.3 [M+H]+,

Z. M. Ziora et al. / Carbohydrate Research 346 (2011) 1439–1444
1443
and 1059.9 [M+Na]+; HRMS calcd for C50H84O15N8Na 1059.5948,
found: 1059.5930.
cell (1.5 mL) loaded with water. Sequences were built as follows:
title compound concentration, 0.05 mM; rotation at 450 rpm;
number of injections, 27; injection duration, 29.9 s; injection vol-
4.2.2.5. Compound 3e. HPLC, system A, Rt, (min): 20.22 and
20.38; system B: 22.09 and 22.43; ESI-MS, m/z: 1026.4 [M+Na]+;
HRMS calcd for C46H82O16N8Na 1025.5741, found: 1025.5768.
ume, 10 lL; spacing 760 s. Mixture 4/H2O (the mixture of com-
pounds 4a, 4b, 4c, 4d, 4e, 4f and 4g in water) and mixture 4/PBS
(the mixture of compounds 4a, 4b, 4c, 4c, 4d, 4e, 4f and 4g in
PBS) were prepared at the same concentration, 0.05 mM each
(the compounds from the same mixture were combined at the so-
lid stage and then solubilized in milliQ water finally giving the con-
centration of 0.05 mM). Experiments were performed in triplicate.
Thermodynamic parameters were calculated as described
previously.18
4.2.2.6. Compound 3f. HPLC, system A, Rt, (min): 20.17; system
B: 22.29 and 22.79; ESI-MS, m/z: 1022.2 [M+H]+, and 1044.1
[M+Na]+; HRMS calcd for C50H84O14N8Na 1043.5999, found:
1043.5992.
4.2.2.7. Compound 3g. HPLC, system A, Rt, (min): 20.31 and
20.44; system B: 21.86 and 22.19; ESI-MS, m/z: 988.2 [M+H]+;
HRMS calcd for C46H81O15N8 985.5827, found: 985.5838.
4.3.2. DLS experiments
Sizes were analyzed using a non-invasive back scatter system
with a scattering angle of 173°. DLS analyses were performed after
ITC experiments and using samples from microcalorimeter sample
cell. The samples of compounds 4a–g or the mixtures 4/H2O or 4/
PBS after the self-assembly process were analyzed at concentra-
4.2.2.8. Compound 3h. HPLC, system A, Rt, (min): 21.10; system
B: 20.55 and 23.36; ESI-MS, m/z: 1028.3 [M+Na]+; HRMS calcd
for C50H84O13N8Na 1027.6050, found: 1027.6069.
tion of 7.6 lM. The measurements were conducted at 25 °C using
4.2.2.9. Compound 3i. HPLC, system A, Rt, (min): 20.12 and
20.28; system B: 21.88 and 22.19; ESI-MS, m/z: 989.2 [M+H]+;
HRMS calcd for C46H81O15N8 985.5827, found: 985.5809.
disposable low volume cuvettes. Experiments were performed at
least three times.
4.3.3. TEM experiments
4.2.2.10. Compound 3j. HPLC, system A, Rt, (min): 18.54 and
18.75; system B: 20.66 and 21.04; ESI-MS, m/z: 1072.1 [M+H]+;
HRMS calcd for C49H91O14N12 1071.6772, found: 1071.6812.
The samples were taken from the microcalorimeter sample cell
after self-assembly process. A drop of mixture 4/H2O or 4/PBS solu-
tion was allowed to air-dry onto a 200 mesh copper grid.
4.2.2.11. Compound 3k. HPLC, system A, Rt, (min): 19.59 and
19.70; system B: 24.09 and 24.18; C50H82O15N8, 1034.5900; HRMS
calcd for C50H81O15N8 1033.5827, found: 1033.5819.
Acknowledgments
This work was supported by ARC Linkage Grant LP0347149. The
authors thank Dr. Tri Le and Graham MacFarlane for their advice
and help on NMR acquisition and for accurate mass measurements,
respectively.
4.2.2.12. Compound 4a. HPLC, system A, Rt, (min): 18.47 and
18.67; system B: 21.36 and 21.76; ESI-MS, m/z: 897.2 [M+Na]+;
HRMS calcd for C41H75O13N7Na 896.5315, found: 896.5304.
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4.2.2.13. Compound 4b. HPLC, system A, Rt, (min): 18.48 and
18.70; system B: 21.04 and 21.36; ESI-MS, m/z: 897.2 [M+Na]+;
HRMS calcd for C41H75O13N7Na 896.5315, found: 896.5327.
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