Carbohydrate-protein interactions at interfaces: Comparison of the binding of Ricinus communis lectin to two series of synthetic glycolipids using surface plasmon resonance studies

Article abstract of DOI:10.1039/b306784j

Two C-lactosyl lipids and the related C-galactosyl lipids have been synthesised and their binding to RCA₁₂₀ plant lectin was compared with a second series of thiolactosylethoxyalkanes. The interactions were measured quantitatively in real time by surface plasmon resonance (BIAcore) at a range of concentrations and temperatures from 5 to 30°C. The C-galactosyl lipid (1,3-dimethyl-5-[β-D-galactopyranosyl]-5-(4-octadecyloxybenzyl)pyrimidine-2 ,4,6-trione) bound much more weakly with a K_A = 8.86 × 10⁵ than the corresponding C-lactosyl lipid (1,3-dimethyl-5-[β-D-galactopyranosyl-(1 → 4)-β-D-glucopyranosyl]-5-(4-octadecyloxybenzyl)pyrimidine-2,4,6-trione) (K_A = 2.31 × 10⁷). The influence of the linker region of the two different series of lactosyl lipids was clearly demonstrated by the differences in the binding to RCA₁₂₀ lectin. The changes in kinetic values and in the enthalpic and entropic contribution to the free energy of binding reflected the importance of the linker and the hydrocarbon anchor holding the synthetic glycolipids in the neomembrane.

Full text of DOI:10.1039/b306784j

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Carbohydrate–protein interactions at interfaces: comparison of the
binding of Ricinus communis lectin to two series of synthetic
glycolipids using surface plasmon resonance studies†
P. Critchley* and G. J. Clarkson
Department of Chemistry, University of Warwick, Coventry, UK CV4 7AL.
E-mail: lonpobty@supanet.com
Received 18th June 2003, Accepted 18th September 2003
First published as an Advance Article on the web 16th October 2003
Two C-lactosyl lipids and the related C-galactosyl lipids have been synthesised and their binding to RCA120
plant lectin was compared with a second series of thiolactosylethoxyalkanes. The interactions were measured
quantitatively in real time by surface plasmon resonance (BIAcore) at a range of concentrations and temperatures
from 5 to 30 ЊC. The C-galactosyl lipid (1,3-dimethyl-5-[β--galactopyranosyl]-5-(4-octadecyloxybenzyl)pyrimidine-
2,4,6-trione) bound much more weakly with a KA = 8.86 × 105 than the corresponding C-lactosyl lipid (1,3-dimethyl-
5-[β--galactopyranosyl-(1
4)-β--glucopyranosyl]-5-(4-octadecyloxybenzyl)pyrimidine-2,4,6-trione) (KA =
2.31 × 107). The influence of the linker region of the two different series of lactosyl lipids was clearly demonstrated
by the differences in the binding to RCA120 lectin. The changes in kinetic values and in the enthalpic and entropic
contribution to the free energy of binding reflected the importance of the linker and the hydrocarbon anchor holding
the synthetic glycolipids in the neomembrane.
and the synthesis of the thiolactosyl lipids has been described
Introduction
previously.11 In the first series of compounds a C-glycoside
Specific molecular recognition is a fundamental mechanism
involved in the hour to hour and day to day processes within
living organisms, from viruses and bacteria to homo sapiens. In
a large proportion of cases the first stage of this recognition
involves comparatively weak binding forces between specific
proteins and the carbohydrate moieties of glycoproteins or
bond links the saccharide to a dimethyl barbituric acid spacer
whereas in the second series the linkage is a thio bond to a
hydrophilic linker of ethylene glycol groups (Fig. 1). The bind-
ing experiments were designed to investigate some of the minor
differences in structure, particularly in the linker region of the
molecules, and the effect of temperature on the specific molecu-
glycolipids, often located on the membrane of the cell surface.1,2
lar interactions between the same carbohydrates immobilised at
Synthesis of appropriate saccharide analogues capable of con-
a neomembrane interface and the same lectin in solution. The
trolling these interactions would provide pharmacological
binding events at a synthetic membrane were followed using a
agents useful for the treatment of various infections and other
diseases including cancer.
Much of the literature on carbohydrate–protein interactions
has concentrated on the differences in structure of related sac-
BIAcore 2000 instrument. The kinetic values and the thermo-
dynamic parameters calculated showed that both the linker and
the hydrocarbon chain anchor played an important role in
determinimg the amount of complex formed and its stability.
charides binding to one or more lectins.
C-glycosides are potentially useful tools in glycobiological
studies as they represent hydrolytically stable analogues of O-
glycosides. Moreover, they have equivalent, if not improved,
properties as glycosidase inhibitors,3–5 substrates for glycoside
transferases3,6 and in the increased induction of protein expres-
sion.7 These factors and advances made in their synthesis,8
especially in aqueous media,6,9,10 have promoted recent research
work on C-glycosides. This paper describes the synthesis of two
glycolipids (1,3-dimethyl-5-[β--galactopyranosyl-(1
4)-β--
glucopyranosyl]-5-(4-octadecyloxybenzyl)pyrimidine-2,4,6,-
trione and the N,NЈ-dimethyl malonamide open ring form
based on the C-glycoside of lactose and two analogous
C-galactose glycolipids and a comparison of the binding of
these structures with that of a second series of thiolactosyl-
ethoxyalkanes (thiolactosyl lipids). The glycolipids and thio-
lactosyl lipids were incorporated into a synthetic membrane
(called a neomembrane composed of dipalmitoylphosphatidyl
choline and between 5–10 mol% of the chosen glycolipid). The
single layer neomembrane was deposited on the surface of an
HPA hydrophobic chip and binding to a Ricinus communis
agglutinin (RCA120) was studied at a range of concentrations
and at different temperatures. The SPR model used in this work
Fig. 1 (a) Cartoon of C-glycolipid structure (compound 11) and
(b) the thioglycolipids (compounds 22–27) that shows the ligand, the
linker and the anchor regions of the molecules.
There are many factors that influence: (a) whether specific
molecular recognition can occur at an interface, and (b)
whether the amount of binding is sufficient to trigger a relevant
† The authors would like to dedicate this paper to Professor D. H. G.
Crout on his retirement in September 2003 without whose encourage-
ment the work would not have been published.
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biological cascade such as host defence mechanisms, fertilis-
ation, cell division, or differentiation.
hydrogen bonding. Water as the solvent has a major effect on
interactions and it is the loss of favourable interactions between
solutes and water that is compensated by the favourable inter-
actions between solutes when a complex is formed. Many
organic compounds dissolved in water produce an energetically
unfavourable pertubation to the structure of water (the hydro-
phobic interaction). When a ligand–lectin complex is formed at
least part of this unfavourable pertubation of water is relieved
and the free energy of the bound system is consequently lower
than that of the unbound state because solvated water mole-
cules are returned to the bulk phase.24 Water molecules can be
trapped when a complex is formed and this water can provide
additional hydrogen bonds that stabilize the complex and can
account for the large enthalpic contribution to the free energy
of binding (Ϫ90 kJ molϪ1).27 The vibrational states of both free
and bound molecules are influenced particularly by temper-
ature that changes the collision rate. Moreover, as the chemical
environment changes the activation energies of the reacting
molecules are influenced. The chemical environment can be
influenced experimentally by comparing different chemical
structures in a series and by factors such as pH, temperature
and solvent.28
Some of the factors involved in binding are specific whereas
others are not. Equilibrium association constants for the bind-
ing of simple carbohydrates to proteins in a 1 : 1 complex in
solution are weak (KA = 104
M
Ϫ1). Specific recognition is
enhanced by cluster formation where a group of glycolipid
ligands can aggregate within a membrane to provide a multiple
binding site for a polyvalent protein or particle with several
copies of the binding domain.12,13 This assumes that the ligand
molecules are not rigidly bound to the surface by short covalent
linkers that may make them inaccessible to a protein. The
resultant multivalent binding is strong (KA of 106 to 109 MϪ1
)
and this is very necessary for adherence where dynamic flow
takes place and hydrodynamic shear could remove proteins
from membranes where weak binding of <104 was involved.
Further in a second phase of binding, stronger associations are
formed through protein–protein interactions or by fusion of a
protein or a particle (e.g. a virus) with the host cell mem-
brane.14,15 Whilst clustering of molecules within a membrane by
lateral diffusion can allow specific multivalent interactions to
occur between molecules this is not the only way that strong
binding can be achieved. Some of the more important factors
that play a role in binding are as follows. The concentration of
glycolipid ligand in a membrane, the composition of the sur-
rounding lipids, the length of the spacer holding the glycoside
away from the lipid membrane allowing rotation and deform-
ation of the head group to occur, and the effects of pH and
temperature.16 The way that a ligand is presented at an interface
can influence the strength of interaction with an appropriate
lectin on a scale from 0–100%.17 There are many unexplained
examples in the biological literature of the effect of different
lipid structures, that are either part of a glycolipid or are part of
the phospholipid microenvironment, changing the degree of
interaction between the glycoside (bound to the lipid) and a
lectin16,18 or enzyme.19
Results and discussion
Two different series of novel glycolipid have been synthesised
and used as models to examine the influence of the linker
unit on the binding to the lectin Ricinus communis agglutinin
120 kDa (RCA120). The linker is present between the lactose
residue and the long-chain hydrocarbon that acts as an anchor
designed to hold the glycolipid in a neomembrane (Fig. 1).
Chemical synthesis
The first set of compounds was synthesised by a simple five
stage sequence of reactions (Scheme 1). The anchor part of the
molecule was formed by alkylation of 4-hydroxybenzaldehyde 1
with 1-bromooctadecane29 followed by mild reduction of the
aldehyde group 2 with sodium borohydride.30 Treatment of the
phenyl methanol compound 3 obtained with thionyl chloride
gave the 1-chloromethyl-4-octadecyloxybenzene 4.31 Galactose
or lactose was added to N,NЈ-dimethylbarbituric acid 5
under mild alkaline conditions32 (Scheme 2). The galactosyl
barbiturate 6 and the lactosyl barbiturate 7 were reacted with
1-chloromethyl-4-octadecyloxybenzene 4 to give the required
compounds 10 and 11 (Scheme 3). The cellobiosyl dimethyl-
barbituric acid derivative (compound 8) and the maltosyl di-
methylbarbituric acid 9 were prepared in a similar way and
alkylated with 4 to produce the C-glycosyl lipids 12 and 13.
These compounds were used as negative controls in the binding
experiments to check that the dimethyl barbituric acid linker
and octadecylbenzyl anchor showed no binding to RCA120
lectin. The barbituric acid ring can be cleaved under mild
alkaline conditions.33,34 Treatment of the galactosyl or lactosyl
compound with alkali opened the barbituric acid ring to yield
N,NЈ-dimethylmalonamide derivatives (compounds 14 and 15)
(Scheme 4). The sugar protons resonated over a very small ppm
range in the 1H NMR and it was difficult to obtain an elemental
A further complication of examining binding events at inter-
faces is that non-specific binding of proteins occurs at apolar
sites. Many proteins have hydrophobic regions that will bind
non-specifically to hydrophobic surfaces20 and some bind to
charged surfaces of membranes.21 Recent work has shown that
the introduction of ethylene glycol oligomers or other polar
molecules at an interface can significantly reduce the non-
specific binding and facilitate specific interactions.22
A number of papers have been published to explain the high
binding at interfaces compared with that measured by methods
that use solution systems for binding measurements.16,23 The
explanations offered in the literature do not fit all of the obser-
vations and it seems likely that some of the numbers for binding
constants may result from artefacts introduced by the model
chosen and the experimental technique used to make the meas-
urements.24 Most of the early work was done using agglutin-
ation techniques with cells or liposomes. These techniques
measured equilibrium situations after many minutes to hours
and often used markers such as fluorescent labels that may
modify a response.16 More recently techniques such as titration
microcalorimetry,25 and surface plasmon resonance26 have been
used to follow binding reactions in real time. Surface plasmon
resonance in the form of a BIAcore instrument is particularly
useful to study molecular interactions because it allows one to
follow a reaction over a period of many seconds. No label is
required so native molecules can be used without modification.
Further, the concentration of the analyte is constant and a rel-
atively large volume of analyte, compared with the volume of
the reaction cell, is passed over the low concentration of ligand
bound to a surface. Therefore, a bimolecular second order reac-
tion behaves as if it were a pseudo-first order reaction simplify-
ing the mathematics. The forces involved in specific molecular
interactions in aqueous solution include dipole–dipole inter-
actions (including induced dipoles), dispersion interactions and
Scheme
1
Synthesis of the anchor part of the C-glycolipids
compound 4. Reagents and conditions: (i) BrC18H37/K2CO3/DMF,
(ii) NaBH4/MeOH, (iii) SOCl2
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Scheme 2 Synthesis of the ligand and linker part of the C-glycolipids showing the reaction of N,N Ј-dimethylbarbituric acid 5 with (i) galactose,
(ii) lactose, (iii) cellobiose and (iv) maltose, in NaHCO3 (aq) to form compounds 6, 7, 8 and 9.
Scheme 3 Coupling of the C-galactosyl/linker 6 and C-lactosyl/linker 7 molecules to the anchor 1-chloromethyl-4-octadecylbenzene (4) in DMSO.
Scheme 4 Hydrolysis of the dimethylbarbituric acid linker of 10 and 11 with NaOH/THF/H2O to form the dimethylmalonamides 14 and 15,
respectively.
analysis for the compounds with free hydroxyl groups (10–15).
Therefore the compounds were fully characterised as their
peracetates 16–21.
The synthesis of the second series of compounds (22–27) has
been described.11 In brief, α,ω-bifunctional dimers and trimers
of ethylene glycol with a monochloro end-group were converted
to the 2-(2-benzylsulfanylethoxy)ethanol or 2-[2-(2-benzyl-
sulfanylethoxy)ethoxy]ethanol by a base catalysed reaction
with benzyl thiol.37 Reaction of the products with C10, C12 and
C18 1-bromoalkanes in the presence of basic sodium iodide and
a phase transfer catalyst gave the alkylated products.38 Removal
of the protecting benzyl group with sodium and liquid ammo-
nia liberated the thiol group39 for condensation with octa-O-
acetyllactose. The condensation reaction was enabled with
It is known that by replacing the normal glycosidic oxygen
bond with a carbon bond could change the conformation of the
molecule and this may result in a different orientation of the
sugars at the neomembrane surface.35,36 The C-glycosides had a
hydrophilic malonamide or a more hydrophobic barbituric acid
group33b present adjacent to the sugar and this produced struc-
tures that were significantly different from the thioglycolipids
with ethylene glycol spacers.
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Table 1 Binding of Ricinus communis agglutinin (RCA120) at 298 K to the two series of synthetic glycolipids incorporated into a dipalmitoyl-
phosphatidyl choline neomembrane
Glycolipid
ka MϪ1 sϪ1
kd sϪ1
KA MϪ1
KD Mc
10
11
14
15
9.21 × 103
1.03 × 105
NBa
1.04 × 10Ϫ2
4.45 × 10Ϫ3
NB
8.86 × 105
2.31 × 107
NB
1.13 ( 0.22) × 10Ϫ7
4.33 ( 0.42) × 10Ϫ8
NB
1.78 × 105
6.25 × 10Ϫ3
2.84 × 107
3.52 ( 0.31) × 10Ϫ8
22b
23b
24
5.58 × 103
1.76 × 103
8.28 × 103
7.94 × 103
8.81 × 103
6.49 × 104
1.16 × 10Ϫ3
1.77 × 10Ϫ3
9.64 × 10Ϫ3
2.69 × 10Ϫ3
3.64 × 10Ϫ3
6.30 × 10Ϫ3
4.81 × 106
9.94 × 105
8.59 × 105
2.95 × 106
2.42 × 106
1.03 × 107
2.08 × 10Ϫ7
1.01 × 10Ϫ6
1.16 ( 0.12) × 10Ϫ6
3.39 ( 0.21) × 10Ϫ7
4.13 ( 0.29) × 10Ϫ7
9.73 ( 0.34) × 10Ϫ8
25
26
27
a NB No binding detected. b Compound 22 and 23 both failed to remain on the membrane and therefore the kinetic analysis is not valid for these two
compounds. c Standard deviations for KA are given in Table 2. The S.D. values for KD are given in parentheses.
boron trifluoride etherate in dichloromethane to yield the pro-
tected glycolipids.40 Treatment of each of the six acetates with
sodium methoxide gave the deprotected lactosylthioethoxy-
alkane products (compounds 22–27) that were purified separ-
ately by flash chromatography on silica. Some similar com-
pounds have been described with a normal O-glycosidic link-
age.41 The effect of the linker and the length of the hydrocarbon
chain in holding the model glycolipids on the surface of the
sensor chip and any influence that different lengths of hydro-
carbon chain may exert on the binding of the lectin was
explored.
Biological results
Fig. 2 Sensorgram of RCA120 binding to compound 11 showing the
association phase (15–75 s), the dissociation phase (76–286 s) and
the regeneration of the neomembrane with dilute alkali (287–384 s).
We chose to design the experiment with C-glycolipid or
thioglycolipid on the surface of an HPA chip to avoid the
potential difficulty of aggregation of the glycolipid in solution
(which would have made the interpretation of data that requires
an accurate concentration term for the analyte) very diffi-
cult. Also, BIAcore recommend that the best results on the
model 2000 are obtained with analytes of more than 10 kDa
molecular weight. Advantages of the HPA chip over the
CM5 chip (where molecules are covalently linked) and over the
SA chip (where biotinylated analytes are deposited onto a
streptavidin surface) are that the HPA chip allows mobility
of ligands on the surface so that clusters can form by
lateral difusion. Moreover, the chip is easily regenerated and
the lipid/glycolipid ligands can be changed whereas the very
tightly held biotinylated ligands and those covalently-linked to
a CM5 chip cannot be exchanged. Details of the method of
preparation of the glycolipid layers on the HPA chip surface
and the development of the model have been described
previously.11
In BIAcore terms [AB] = the concentration of bound analyte
A that is proportional to RA the resonance response of a
sensorgram at any given time t.
The free ligand concentration [B] is the difference between
the total and bound ligand
[Bt] = [Bt = 0 Ϫ ABt]
Therefore the total concentration of active immobilized
ligand is obtained indirectly as it is saturated with analyte. The
maximum response due to analyte binding Rmax is proportional
to total ligand concentration [Bt 0]. Also, (Rmax Ϫ RAt) is pro-
=
portional to the free ligand concentration [Bt]. The concen-
tration of complex [ABt] formed and of [Bt] the free ligand
remaining can be expressed in terms of analyte response in the
equation
In solution interactions RCA120 binds two galactosides, one
on each face. In our experiments with galactolipids immobilised
at a lipid interface there was no evidence of the second binding.
Neomembranes could be used for several weeks with repeated
regenerations with sodium hydroxide to remove any bound lec-
tin. A 10 mol% of glycolipid ligand concentration in dipal-
mitoylphosphatidyl choline and low concentrations of RCA120
analyte (25–200 nM) were used under carefully controlled con-
ditions for kinetic studies. Binding of RCA120 to the dipal-
mitoylphosphatidylcholine control was negligible and this was
subtracted from all test runs. Neither C-cellobiosyl nor C-mal-
tosyl lipids 12, 13 showed any binding to RCA120. Previous
experiments showed that mass transfer was not a problem with
d[AB]/dt = ka[A][B] Ϫ kd[AB]
In BIAcore terms this becomes
dRA/dt = kaC(Rmax Ϫ RA) Ϫ kdRA
(1)
(2)
C is the concentration of the injected analyte in moles (M).42
At equilibrium dRA/dt = 0 and the equilibrium constant
KA = ka/kd.
Therefore
RA/C = KARmax Ϫ KARA
flow rates of 20–30 µl minϪ1 11
.
A plot of RA/C against RA is a Scatchard plot with a slope of
Ϫ 1/KD (Fig. 3)
We can rearrange eqn. (2) to give
A typical sensorgram for the C-lactosyl lipid (compound 11)
is shown in Fig. 2. A comparison of the rates of association KA
and of dissociation kd and the equilibrium constants KA and KD
is given in Table 1.
The reaction may be expressed as A ϩ B
AB.
dRA/dt = kaCRmax Ϫ (kaC ϩ kd)RA
(3)
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Table 2 Thermodynamic parameters calculated at 298 K for both series of synthetic glycolipid
Compound
KA MϪ1
∆GЊ kJ molϪ1
∆H Њ kJ molϪ1
∆S Њ J molϪ1 KϪ1
10
11
15
8.86 ( 0.30) × 105
2.31 ( 0.12) × 107
2.84 ( 0.13) × 107
Ϫ33.9
Ϫ41.9
Ϫ42.5
Ϫ62.3
Ϫ7.64
Ϫ10.8
Ϫ95
ϩ115
ϩ107
24
25
26
27
8.59 ( 0.26) × 105
2.95 ( 0.25) × 106
2.42 ( 0.12) × 106
1.03 ( 0.17) × 107
Ϫ33.8
Ϫ36.9
Ϫ36.4
Ϫ39.9
Ϫ17.3
Ϫ49.3
Ϫ59.5
Ϫ35.7
ϩ55
Ϫ42
Ϫ77
ϩ14
The values in parentheses after the KA are the standard deviation for 12 measurements.
When the injection of analyte finishes only buffer flows over
the chip surface and the complex AB starts to dissociate. eqn.
(3) can be rearranged to give
dRA/dt = kaC(Rmax Ϫ RA) Ϫ kdRA
Since there is no analyte flowing (C
simplified to
0) the equation can be
dRA/dt = Ϫ kdRA
and values of kd can be obtained from the dissociation
phase of the sensorgram using the rate equation ln(RA1/Rn) =
kd(tn Ϫ t1) where RA1 is the resonance at time t1 when dissoci-
ation starts, Rn and tn are values along the dissociation curve.
The data for the rate constants and the equilibrium constants
at 25 ЊC are summarised in Table 1 and represent averages of
between 5 and 14 separate experiments on each compound
using five concentrations of lectin in each experiment. Standard
deviations for KA values are given in Table 2. The association
constants KA are orders of magnitude higher than the binding
constant for free lactose16 to RCA120 where KA =3.8 × 103 MϪ1
A comparison of the KA figures shows that the binding of the
C-galactosyl lipid (compound 10) is 26 times weaker than the
corresponding C-lactosyl lipid (compound 11). A small favour-
able change in binding resulted when the dimethylbarbituric
acid ring of the lactosyl compound was opened to form the
N,NЈ-dimethyl malonamide 15. The lactosyl lipid-RCA120 lectin
complex with the open ring was more stable, ie it had a lower
value for KD that resulted largely from an increased rate of
association. However, no binding was detected with the N,NЈ-
dimethyl malonamide attached to galactose (14), contrary to
our expectations. The concentrations of RCA120 used in all
experiments, the pH of the buffer, the temperature range used
in the experiments and the phospholipid used to make up the
bulk of the neomembrane were the same. Moreover, the
amount of ligand available in the neomembranes was very simi-
lar. The ligands represented a small fraction of the neomem-
brane (10 mol%) and therefore the environment around each
ligand and its structure would determine the extent to which
they were exposed at the membrane surface to allow binding to
occur with RCA120 lectin (see similar examples in refs. 16, 18
and 19).
Fig. 3 A Scatchard plot of Requilibrium/concentration of analyte against
Requilibrium for compound 11. The gradient = Ϫ1/KD = KA = 2.33 × 107;
compares with 2.31 × 107 average of 12 experiments (see Table 2 for the
standard deviations).
The total response R = RI a refractive index component ϩ RL
immobilised ligand component ϩ RA component for analyte
bound and is a complex term. However, the derivatives dRI/dt
and dRL/dt = 0 provided that ligand is not lost from the chip
surface during the reaction. Therefore dR/dt = dRA/dt
Substituting into eqn. (3) we obtain
dR/dt = constant Ϫ(kaC ϩ kd)R
This equation can be used with several concentrations of
analyte to find the kobs values that are the gradient of a plot of
dR/dt against R.
As kobs = kaC ϩ kd. a further plot of kobs against concen-
tration of analyte gives a straight line with a slope of ka, the rate
constant for association (Fig. 4).
In the second series of compounds the thiolactosyl lipids
there were suprising differences in the KD resulting from
changes in linker or spacer (between the thiolactose and the
hydrocarbon chain) from three units down to two units of
ethylene glycol. A comparison between two pairs of com-
pounds (24, 25 and 26, 27) showed that removal of an ethylene
glycol group reduced the KD by more than an order of magni-
tude. Earlier work had shown that self-assembled monolayers
terminated by three ethylene glycol groups showed less conform-
ational variation than those terminated by six ethylene glycol
groups.43 There was a noticeable difference in the solubility of
the compounds particularly those with octadecyl hydrocarbon
chains as anchors. The compounds with three ethylene glycol
groups were more easily formulated into liposomes than the
Fig. 4 A plot of kobs against concentration of RCA120 for compound
11 (kobs 11) and compound 15 (kobs 15) The gradient ka 11 is the association
rate constant = 9.76 × 104 and the intercept is the dissociation rate
constant kd = 7.08 × 10Ϫ3. Therefore KA = ka/kd = 2.08 × 107. When the
kd from the sensorgram is used = 4.45 × 10Ϫ3, then the KA = 2.19 × 107
that is closer to the average value in Table 1. The ka = 1.74 × 105 value
for compound 15 with an intercept kd = 7.08 × 10Ϫ3 giving a KA = 2.46 ×
107. The value from the desorption sensorgram was kd = 6.25 × 10Ϫ3
giving a KA = 2.78 × 107 much closer to the average value in Table 1.
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ones with two ethylene glycol groups that were less soluble. The
exception to the change in KD was the pair of compounds 22, 23
with a decyl hydrocarbon tail. In these compounds the short
hydrocarbon chain failed to hold the compounds sufficiently
tightly in the membrane and at higher concentrations of
RCA120 (>75 nM), the thiolactosyl lipid was removed from the
surface as a ligand-lectin complex. This loss of thioglycolipid
resulted in a lower signal at the end of an experiment compared
to that at the start and the baseline decreased when experiments
were repeated. The “kinetic data” found for these compounds
has been included as an illustration of the artifacts that can be
created by analysing sensorgrams that are not kinetically valid.
The values of KA for each compound were measured at 5 ЊC
intervals between 5 and 30 ЊC and analysed graphically by
applying the van’t Hoff isochore that relates variation in
the equilibrium constant (K) with changes in the absolute
temperature.
similar enthalpy-entropy compensation.24 However, in our case
some values for the entropy term (∆S Њ) were positive because
the free energy (∆GЊ) was sometimes greater than the enthalpy
term (∆H Њ). Other authors observed positive entropy values
when they measured the interaction of concanavalin A and pea
lectin with various mannosides44 and the binding of galacto-
sides and lactose to Erythrina corallodendron lectin.45 Such dif-
ferences may reflect the difference in the technique of measur-
ing the changes (see below). The values in the published work
were calculated from titration microcalorimetry data that is
known to include processes that do not contribute to the free
energy of binding.24 In contrast, SPR data is derived from much
more dilute solutions than calorimetric data and it has been
claimed that it includes only the direct interaction at the
interface.46,47 A comparison of thermodynamic parameters
calculated from stopped flow, BIAcore and isothermal micro-
calorimetry on the same thrombin-inhibitor binding reaction
showed significant differences. The range of values found were
ϩ100 > ∆H Њ kJ molϪ1 > Ϫ100 for enthalpy changes and 100 >
∆S Њ J molϪ1 KϪ1 > Ϫ100 for entropy changes.47
Thus, dlnK/dt = Ϫ∆H Њ/RT2 where ∆H Њ is the enthalpy
change and R is the gas constant. Integrating the equation gives
Many workers who study carbohydrate–lectin interactions
agree that they are dominated by solvation/desolvation pro-
cesses of the participating solutes and that the contribution of
an interaction between solute–water (as solvent) is greater than
for a solute A–solute B interaction.24,27 The hydrogen bond
network of water is strong and this network is perturbed
unfavourably when organic compounds are dissolved/solvated
because they disrupt the normal hydrogen bonded tetrahedral
structure of water. This energetically unfavourable pertubation
in the structure of water derives from an unfavourable entropy
term concerned with rotational entropy of water. It can be
reduced or eliminated when solute A binds to solute B. A
comparison of thermodynamic parameters measured in water
and D2O supported the view that a large part of the bind-
ing enthalpy change is derived from solvent reorganisation
(a hydrophobic effect).25 Enthalpy changes involve hydrogen
bonding, electrostatic and van der Waals interactions that can
be identified and calculated. The sources of entropy changes are
much more difficult to identify and to attribute whether a con-
tribution to the free energy of reaction will be positive or
negative.
lnK2/K1 = ∆H Њ/R(T2 Ϫ T1/T1T2)
A plot of logK against 1/T gives a straight line of slope
Ϫ∆H Њ/2.303R (Fig. 5)
In the literature the entropic contributions have been divided
into four sub-groups;48 thus
Fig. 5 A van’t Hoff plot of log KA against 1000/T. The almost parallel
upper curves K1 and K2 represent compounds 11 and 15, respectively;
K3 represents the galactosyl lipid compound 10 and K4 and K5 represent
the thiolactosyl glycolipids compounds 26 and 27, respectively. The
curves with the lower gradient (compounds 11, 15 and 26) are the ones
with a positive entropic contribution to the free energy of binding.
∆S = ∆Stranslational ϩ ∆Srotational ϩ ∆Svibrational ϩ ∆Sconformational
The vibrational changes are claimed to be very small and for
any rigid particles the conformational entropy is zero. An alter-
native summation of entropy changes that is more readily
applied to carbohydrate–protein complex formation involves
the inclusion of a solvation entropy change rather than the
vibrational entropy term that is very small.24 Thus ∆S = ∆ST ϩ R
ϩ ∆Ssolv ϩ ∆Sconf
The average enthalpy change for each compound measured
over the 25 ЊC range of temperature was used to find the change
in free energy (∆GЊ) and the entropy (∆S Њ) of the reactions
using the standard thermodynamic expressions:
The term ∆ST R is greatly reduced in water compared with
ϩ
∆GЊ = ϪRTlnK = ∆H Њ Ϫ T∆S Њ
the gas phase and will be further reduced in our study as the
carbohydrate ligands are part of a membrane structure that will
limit the translocation and rotation of the molecules mainly to
two dimensions rather than three dimensions.
A summary of the thermodynamic results is shown in Table 2
The van’t Hoff plot (Fig. 5) shows the relationship between
the equilibrium constant for association and the absolute tem-
perature. The reactions were all exothermic with an increase in
KA as the temperature was lowered. The gradient of each line is
proportional to the change in enthalpy (∆H Њ) and therefore as
the gradient increases the enthalpic contribution to the free
energy (∆GЊ) of reaction is increased. Conversely, as the gradi-
ent decreases the enthalpic contribution is decreased. In the
case of three of the compounds 10, 25 and 26 the change in the
entropic contribution is compensated for by the change in
enthalpy. Similar thermodynamic parameters for the inter-
action of many different carbohydrates and lectins have been
reviewed recently and in most of these examples there was a
The freedom of rotation about the bond between thiolactose
and the aglycone is greater for the C–S–C bond than a C–O–C
ether bond. The thio bond is longer than the C–O bond and has
a higher torsional entropy.49 Therefore, binding of the sacchar-
ide to lectin will reduce this freedom of rotation and the trans-
lational entropy even more than any reduction that resulted
from the immobilization of the ligand in the neomembrane.
In the case of the C-glycosides the saccharides are attached
to the dimethylbarbituric acid via a quaternary carbon and
one would expect the torsional entropy to be low, particularly
where intramolecular hydrogen bonding can occur between the
saccharide and the barbituric acid. Therefore, a major source of
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negative entropy on binding has been removed and the entropic
contribution to the free energy of association is positive. On
binding to RCA120 lectin there will be a loss of conformational
entropy. X-Ray studies were performed on the solid structure
20, the tetra O-acetate of compound 14 (Fig. 6). The malon-
amide occupies the β-orientation at C-1 of the galactose ring,
confirming the orientation derived from 1H NMR coupling
constants (∼10 Hz).33 The aliphatic chain (not shown) is in an
extended all trans conformation roughly linear with the mean
plane of the galactose ring and the malonamide is orthoganal
to this plane. One malonamide carbonyl (O31) is hydrogen
bonded to the NH (N29) of the other amide, while the other
NH (N32) is hydrogen bonded to the pyranose oxygen (O52) of
the galactose. It is expected that a similar situation is present in
the lactosyl malonamide 15, and that this hydrogen bonding
pre-orientates or reduces the free rotation of the lactose mole-
cule leading to a smaller loss of entropy on binding to the
lectin. There maybe other plausible explanations involving the
loss of steric hindrance from the larger barbiturate ring or
intermolecular hydrogen bonding from the secondary amides
effecting the orientation or aggregation of the C-glycolipid in
the neomembrane.
compounds depends on the orientation of these molecules in
the membrane. Long octadecyl hydrocarbon chains align with
dipalmitoyl chains of the phospholipid in a largely trans con-
figuration that holds the thioglycolipid molecules in a more or
less vertical position with respect to the neomembrane sur-
face.50 Therefore, the degree of exposure of the lactose at the
membrane surface and its ability to form a stable complex with
the RCA120 lectin will depend on the length and rigidity of the
ethylene glycol linker (Fig. 7(a)). The kinetic data show that
three units of ethylene glycol (compound 27) allow better com-
plex formation (KA = 1.03 × 107) and more stability (KD = 9.73 ×
10Ϫ8) than for compound 26 that differs by only one less ethyl-
ene glycol unit (KA = 2.42 × 106, KD = 4.13 × 10Ϫ7). In the case
of compounds 24 and 25 the short dodecyl hydrocarbon chain
has a less favourable interaction with the dipalmitoyl chains of
the phospholipid and the dodecyl chains will have more gauche
bonds so they will show less interdigitation with the phos-
pholipid. Moreover, the thioglycolipids may phase separate
from the phospholipids into islands51,52 (Fig. 7). This will make
complex formation less efficient and less stable because of
unfavourable steric factors. Compound 25 has the longer more
hydrophilic three-ethylene glycol linker (compared with com-
pound 24) that will help to lift the lactose ligand out of the
membrane surface. The binding of this compound to lectin is
similar to that of compound 26 that has only two ethylene
glycol units but it has the octadecyl hydrocarbon chain (KD
=
3.39 × 10Ϫ7). It seems that the presence of an extra ethylene
glycol group can counteract the lower stability of the complex
caused by the shorter dodecyl hydrocarbon chain. Compound
24 has a decyl hydrocarbon chain and only two ethylene glycol
Fig. 6 The solid state stucture of compound 20. For clarity, only the
benzyloxy portion of the major component of the disordered aliphatic
tail is shown. Selected interatomic distances (Å); N29 ؒ ؒ ؒ O31
2.627(15), N32 ؒ ؒ ؒ O52 2.640(17).
Accepting that the main contribution to the energy changes
on binding of carbohydrate to lectin are derived from the
hydrophobic effect then one can explain the observations made
in this paper. Compounds 11, 15 and 24 all have a low enthalpic
contribution to the free energy of association (∆H Њ < ∆GЊ) and
they have a high positive entropic contribution (ϩ∆S Њ). Key
hydrogen bonds and dipolar interactions are satisfied by solute–
water interactions prior to association but are lost during the
binding producing an unfavourable contribution to the overall
free energy. To some extent the thermodynamic parameters cal-
culated for compound 27 can be explained by similar changes.
In contrast, compounds 10, 25 and 26 all show high changes in
enthalpy (∆H Њ > ∆GЊ) and negative entropic contributions to
the free energy of association. In the case of these molecules the
interactions lost to solvent are replaced in the complex and so
the net favourable free energy that derives from the desolvation
of non-polar surface area drives the formation of the complex.
The KA values for the thiolactosyl lipids, compounds 24 and
26 with two ethylene glycol groups, were lower than those with
three ethylene glycol groups, compounds 25 and 27. The former
were possibly too inflexible and hydrophobic to facilitate opti-
mum placement of the recognition epitope of the lectin. In the
case of the molecules with three ethylene glycol groups one
would expect a more organised and flexible helical phase in the
linker region including tightly bound water.43 An hypothesis
that may explain both the kinetic values and the thermo-
dynamic parameters calculated for the thioglycolipid series of
Fig. 7 The upper cartoon (a) is a diagram of compound 27 in a
neomembrane of dipalmitoylphosphatidyl choline on an HPA chip
surface. The octadecyl hydrocarbon chain aligned with the membrane
lipids and the hydrated helix of three ethylene glycol units that project
the ligand well into the aqueous domain. The dark ovals represent the
galactose units, the lighter ovals represent the glucose and the clear
shapes represent the zwitterionic heads of the phospholipids. The lower
cartoon (b) is a diagram of compound 22 that has a shorter dodecyl
hydrocarbon chain and two ethylene glycol groups. The short ethylene
glycol fails to hold the ligand away from the phospholipid surface and
therefore reduces binding.
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units in the linker. Binding is significantly compromised (KD
=
ring. The two positions were occupied in a 7 : 3 ratio and the
main component was allowed to refine anisotropically.
CCDC reference number 213138.
See http://www.rsc.org/suppdata/ob/b3/b306784j/ for crystal-
lographic data in CIF or other electronic format.
1.16 × 10Ϫ6) because the thiolactose ligand is too close to
the membrane surface to facilitate stable complex formation.
The changes in the kinetics of the reactions are reflected in the
thermodynamic parameters with compound 27 having the
highest ∆GЊ = Ϫ39.9 kJ molϪ1 of this series of compounds and
compound 24 having the lowest ∆GЊ = Ϫ33.8 kJ molϪ1
.
Sodium 1,3-dimethyl-5-[ꢀ-D-galactopyranosyl]barbiturate (6)32
The C-galactosyl lipid (compound 10) is the exception in
both series of compounds because it has only a galactosyl resi-
due available for complex formation with RCA120 lectin. It has
been shown that with lactosyl derivatives under favourable con-
ditions for complex formation both the galactosyl residue and
to a lesser extent the glucosyl residue are involved in binding to
the RCA120 lectin.52 Therefore, the maximum potential for bind-
ing the galactosyl lipid (compound 10) will be less than the
corresponding C-lactosyl lipid (compound 11). The experi-
ments described here show that the KA value (2.31 × 107) for the
C-lactosyl lipid was more than two orders of magnitude greater
than that for the C-galactosyl lipid (8.86 × 105) and the KD
value (1.13 × 10Ϫ7 cf. 4.33 × 10Ϫ8) was more than one order
of magnitude greater, confirming the weaker binding of the
C-galactosyl compound (10).
In this paper we have shown that molecules with the same
lactosyl ligand and an octadecyl hydrocarbon chain have quite
different affinities for RCA120 depending on the linker used.
Moreover, the length of the hydrocarbon chain and the phos-
pholipid environment around the chain can affect the binding
of ligand to lectin at the membrane surface. These differences
are reflected in the kinetics of binding and in the different
values for the enthalpic and entropic contribution to the free
energy of association. The importance of the linker region pre-
senting a saccharide at the surface of a membrane should not
be underestimated.
Galactose (10 g, 55.5 mmol) and 1,3-dimethylbarbituric acid 5
(10 g, 64.0 mmol) were dissolved in water (120 cm3) and
NaHCO3 (5.60 g) was added in small portions to moderate the
effervescence. The mixture was then heated at 80 ЊC for 5 h and
was monitored by TLC (EtOAc–MeOH–H2O, 40 : 15 : 3 v/v) till
complete conversion was achieved. The solvent was reduced to
a small volume and the solution was precipitated into rapidly
stirred methanol (400 cm3). The white powdery solid was
collected by filtration and recrystallized by dissolving in the
minimum amount of hot water and diluting with four times the
volume of methanol. On standing, large colourless hexagons of
product 6 were produced (14.46 g, 82%), mp 223–224 ЊC;
δH (400 MHz; D2O) 3.20 (6H, s, NCH3), 3.58 (1H, dd, J 3.5, 9.7,
3-H), 3.66 (3H, br s, 5-H, 6-HH1), 3.93 (1H, d, J 3.5, 4-H), 4.40
(1H, d, J 9.7, 1-H), 4.50 (1H, t, J 9.7, 2-H); δC (100 MHz, D2O)
28.1, 61.4, 67.8, 70.2, 75.5, 77.0, 79.1, 86.7, 154.7, 164.7; m/z
340 (Mϩ).
General procedure for the synthesis of the disaccharide
barbiturate salts
The disaccharide (10 g, 29.2 mmol) and 1,3-dimethylbarbituric
acid 5 (4.76 g, 30.5 mmol) were dissolved in water (120 cm3) and
NaHCO3 (2.80 g) was added in small portions to moderate the
effervescence. After addition the mixture was heated at 80 ЊC
for 5 h. The solvent was reduced to a small volume and the
solution was precipitated into rapidly stirred isopropanol (300
cm3). The product was isolated by filtration and washed with a
little isopropanol and diethyl ether to give the compounds
below.
Experimental
Melting points are uncorrected. 1H NMR data was recorded on
a Brucker DPX-400 (400 MHz) spectrometer (δ values are in
ppm using tetramethylsilane as internal standard, solvents as
given). For the compounds containing disaccharides, signals for
the hydrogen atoms of the sugar that are not attached directly
to the barbituric acid are designated by primes, as are the
second hydrogen atom of diastereotopic methylenes. 13C NMR
data was recorded on a Bruker DPX-400 (100 MHz). Mass
spectra were recorded by the mass spectrometry service at the
University of Warwick using a Waters Micromass Autospec
instrument. Elemental analysis was performed by Warwick
Analytical service. TLC was performed on aluminium-backed
silica gel plates and visualised under UV and then charred with
conc. H2SO4 (5%) in ethanol. All compounds were acquired
from Lancaster Synthesis unless otherwise stated. RCA120 and
dipalmitoylphosphatidylcholine were obtained from Sigma.
Binding studies were monitored in real time by surface plasmon
resonance using an HPA chip in a BIAcore 2000 automatic
instrument. The following abbreviations were used: Ac2O:
acetic anhydride, DCM: dichloromethane, DMSO: dimethyl
sulfoxide, THF: tetrahydrofuran, DMAP: 4-dimethylamino-
pyridine, DMF: dimethylformamide.
Sodium 1,3-dimethyl-5-[ꢀ-D-galactopyranosyl-(1
glucopyranosyl]barbiturate (7)
4)-ꢀ-D-
Compound 7 (11.6 g, 79%) was obtained as a white powdery
solid; mp 192–194 ЊC; δH (400 MHz, D2O) 3.16 (6H, s, NCH3),
3.60–3.48 (3H, m, 3-H, 5-H and 2Ј-H), 3.62 (1H, dd, J 3.3, 9.9,
3Ј-H), 3.74–3.66 (3H, m, 5Ј-H and 6Ј-HHЈ), 3.77 (1H, t, J 9.6,
H-4), 3.82 (2H, d, J 2.4, 6HHЈ), 3.88 (1H, d, J 3.3, 4Ј-H), 4.35
(1H, t, J 9.8, 2-H), 4.46 (1H, d, J 7.8, 1Ј-H), 4.49 (1H, d, J 9.8,
1-H); δC (100 MHz, D2O) 28.0, 60.5, 61.42, 69.0, 69.6, 71.4,
73.0, 75.7, 76.2, 77.1, 78.5, 78.8, 86.4, 103.3, 154.7, 165.6; m/z
502 (Mϩ).
Sodium 1,3-dimethyl-5-[ꢀ-D-glucopyranosyl-(1
glucopyranosyl]barbiturate (8)
4)-ꢀ-D-
Compound 8 (12.5 g, 85%) was obtained as a white powdery
solid; mp 202–204 ЊC; δH (400 MHz, D2O) 3.16 (6H, s, NCH3),
3.28 (1H, dd, J 8.0, 8.7, 2Ј-H), 3.38 (1H, t, J 9.3, 4Ј-H), 3.46
(2H, m, 3Ј-H and 5Ј-H), 3.52 (1H, m, 5-H), 3.57 (1H, t, J 9.5,
3-H), 3.69 (1H, dd, J 5.5, 12.1, 6Ј-H), 3.75 (1H, dd, J 9.5, 9.8,
4-H), 3.82 (2H, br s, 6-HHЈ), 3.87 (1H, br d, J 12.1, 6Ј-HЈ), 4.33
(1H, dd, J 9.5, 10.1, 2-H), 4.49 (1H, d, J 10.1, 1-H), 4.52 (1H, d,
J 8.0, 1Ј-H); δC (100 MHz, D2O) 28.0, 60.5, 61.0, 69.7, 69.9,
73.6, 75.9, 76.2, 76.4, 77.0, 78.8, 78.9, 86.4, 102.9, 154.7, 165.5;
m/z 502 (Mϩ).
Crystal structure determination of N,NЈ-dimethyl-2-[2,3,4-tetra-
O-acetyl-ꢀ-D-galactopyranosyl]-2-(4-octadecyloxybenzyl)-
malonamide (16)
Crystal data. C44H70N2O12, M = 819.02, orthorhombic,
a = 8.6168(8), b = 9.3641(9), c = 56.595(3) Å, U = 4566.6(7) Å3,
T = 180(2) K, space group P212121 (no. 19), Z = 4, µ(Mo-Kα) =
Sodium 1,3-dimethyl-5-[ꢁ-D-glucopyranosyl-(1
glucopyranosyl]barbiturate (9)
4)-ꢀ-D-
0.086 mmϪ1, 24773 reflections measured, 5957 unique (Rint
=
0.065) which were used in all calculations. The final wR(F 2) was
0.1276 (all data). The aliphatic tail was disordered over two
positions. This disorder was traced right back to the benzene
Compound 9 (11.7 g, 80%) was obtained as a white powdery
solid; mp 219–220 ЊC; δH (400 MHz, D2O) 3.17 (6H, s, NCH3),
3.37 (1H, t, J 9.1, 4Ј-H), 3.51 (1H, m, 5Ј-H), 3.54 (1H, dd, J 3.8,
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9.9, 2Ј-H), 3.66 (1H, dd, J 9.1, 9.9, 3Ј-H), 3.70–3.85 (7H,
m, 3-H, 4-H, 5-H, 6-HHЈ and 6Ј-HHЈ), 4.34 (1H, dd, J 9.0,
10.0, 2-H), 4.48 (1H, d, J 10.0, 1-H) 5.38 (1H, d, J 3.8, 1Ј-H);
δC (100 MHz, D2O) 28.0, 60.9, 61.1, 67.8, 69.7, 72.3, 73.0,
73.4, 76.3, 77.5, 78.6, 79.1, 86.5, 100.2, 154.7, 165.5; m/z 502
(Mϩ).
was analysed by TLC (EtOAc–MeOH–H2O, 20 : 20 : 4 v/v) to
ensure complete conversion. The DMSO was then removed
under high vacuum and the solid mixture tritriated with water
to remove salts. The solid was placed on a column of silica
and eluted with EtOAc then with EtOAc containing an increas-
ing concentration (0–20%) of MeOH–water (5 : 1 v/v). Evapor-
ation of the fractions that contained product under reduced
pressure gave a white powder that was recrystallised from
EtOAc to give 10 (0.98 g, 57%) as tiny needles; mp 144–147 ЊC;
δH (400 MHz, d6-DMSO) 0.86 (3H, t, J 6.9, ArOCH2-
CH2(CH2)15CH3), 1.24 (28H, br s, ArOCH2CH2CH2(CH2)14-
CH3), 1.37 (2H, m, ArOCH2CH2CH2(CH2)14CH3), 1.65 (2H,
qnt, J 6.5, ArOCH2CH2CH2(CH2)14CH3), 2.95 (3H, s, NCH3),
3.00 (3H, s, NCH3), 3.25–3.37 (4H, m, 3-H, 5-H, CHH1Ar),
3.46 (2H, m, C6HH1), 3.58 (1H, ddd, J 5.9, 9.1, 9.6, 2-H),
3.66 (1H, br t, J 3.9, 4-H), 3.75 (1H, d, J 9.6, 1-H), 3.86 (2H,
t, J 6.5, ArOCH2CH2CH2(CH2)14CH3), 4.28 (1H, d, J 3.9,
4-OH), 4.52 (1H, t, J 5.6, 6-OH), 4.70 (1H, d, J 6.0, 3-OH), 5.50
(1H, d, J 5.9, 2-OH), 6.78 (2H, d, JAB 8.7, ArH), 6.82 (2H,
d, JAB 8.7, ArH); δC (100 MHz, d6-DMSO) 14.3, 22.5, 25.9,
28.2, 28.3, 29.0, 29.1, 29.2, 29.4 (10 carbons unresolved under
here), 31.7, 40.0, 60.7, 60.8, 67.6, 68.6, 68.7, 75.4, 80.4,
83.7, 114.5, 127.2, 130.5, 150.8, 158.0, 170.0, 170.1; m/z 699
(M ϩ Naϩ).
4-Octadecyloxybenzaldehyde (2)29
4-Hydroxybenzaldehyde 1 (10 g, 81.8 mmol), 1-bromooctade-
cane (26 g, 80.0 mmol) and K2CO3 (12.6 g) were dissolved in
DMF (100 cm3). The mixture was heated at 80 ЊC for 24 h. The
solution was precipitated into rapidly stirred water (800 ml).
The solid was collected by filtration and dried. It was recrystal-
lised from methanol to furnish a white powdery solid 2 (19.85 g,
65%); δH (400 MHz, CDCl3) 0.86 (3H, t, J 6.9, ArOCH2CH2-
CH2(CH2)14CH3), 1.24 (28H, br s, ArOCH2CH2CH2(CH2)14-
CH3), 1.37 (2H, m, ArOCH2CH2CH2(CH2)14CH3), 1.65 (2H,
qnt, J 6.5, ArOCH2CH2CH2(CH2)14CH3), 4.05 (2H, t, J 6.5,
ArOCH2CH2CH2(CH2)14CH3), 7.01 (2H, d, JAB 6.8, ArH), 7.85
(2H, d, JAB 6.8, ArH), 9.90 (1H, s, OCH); δC (100 MHz, CDCl3)
14.5, 23.1, 26.4, 29.5, 29.7, 29.9, 30.0, 30.1 (9 carbons unresol-
ved under here), 32.3, 68.8, 115.2, 130.1, 132.4, 165.0, 192.0;
m/z 374 (Mϩ)
(4-Octadecycloxyphenyl)methanol (3)30
1,3-Dimethyl-5-[ꢀ-D-galactopyranosyl-(1
4)-ꢀ-D-gluco-
4-Octadecyloxybenzaldehyde 2 (10 g, 26.70 mmol) were dissol-
ved in methanol-THF (40 ϩ 100 cm3). NaBH4 (1 g, 26.43
mmol) was added to the mixture in small portions. The mixture
was stirred at room temperature for 1 h and was analysed by
TLC (Et2O–light petroleum (bp 60–80 ЊC), 1 : 4 v/v) to ensure
complete conversion. The solution was precipitated into rapidly
stirred ice-cold water (800 cm3). The white precipitate was fil-
tered off and recrystallized from methanol to give the product 3
(9.18 g, 90.9%) as needles; mp 84–85 ЊC (lit.30 84–85 ЊC); δH (400
MHz, CDCl3) 0.86 (3H, t, J 6.9, ArOCH2CH2CH2(CH2)14CH3),
1.24 (28H, br s, ArOCH2CH2CH2(CH2)14CH3), 1.37 (2H, m,
ArOCH2CH2CH2(CH2)14CH3), 1.65 (2H, qnt, J 6.5, ArOCH2-
CH2CH2(CH2)14CH3), 3.98 (2H, t, J 6.5, ArOCH2CH2(CH2)15-
CH3), 4.63 (2H, s, ArCH2OH), 6.92 (2H, d, JAB 6.8, ArH), 7.30
(2H, d, JAB 6.8, ArH); δC (100 MHz, CDCl3) 14.5, 23.1, 26.5,
29.7(3), 29.7(7), 29.8, 29.9, 30.0(1), 30.0(7), 30.2 (7 carbons
unresolved under here), 32.3, 65.5, 68.5, 115.0, 129.0, 133.0,
158.2; m/z 376 (Mϩ).
pyranosyl]-5-(4-octadecyloxybenzyl)pyrimidine-2,4,6-trione (11)
Lactose barbituric salt 7 (1 g, 1.99 mmol) and 1-chloromethyl-
4-octadecyloxybenzene 4 (1 g, 2.58 mmol) were dissolved in
DMSO (20 cm3) and the reaction mixture was heated and
stirred at 70 ЊC for 3 h. The reaction mixture was analysed by
TLC (EtOAc–MeOH–H2O, 40 : 20 : 4 v/v) to ensure complete
conversion. The DMSO was then removed under high vacuum
and the solid was dissolved in boiling water (20 cm3) and
allowed to cool. The solid was filtered off and air dried then
placed on a column of silica and eluted with EtOAc, then with
EtOAc containing an increasing concentration (0–20%) of
MeOH–water (5 : 1 v/v). Evaporation of the fractions that con-
tained product under reduced pressure gave a white powder that
was recrystallized from acetone as a white powdery product 11
(0.91 g, 55%); mp 143–145 ЊC;. δH (400 MHz, d6-DMSO) 0.86
(3H, t, J 6.9, ArOCH2CH2CH2(CH2)14CH3), 1.25 (28H, br s,
ArOCH2CH2CH2(CH2)14CH3), 1.37 (2H, m, ArOCH2CH2-
CH2(CH2)14CH3),1.66(2H,qnt,J6.9,ArOCH2CH2CH2(CH2)14-
CH3), 2.94 (3H, s, NCH3), 3.00 (3H, s, NCH3), 3.20 (2H, m,
5Ј-H, CHHЈAr), 3.24–3.34 (6H, m, 2-H, 3-H, 4-H, 5-H, 2Ј-H
and 3Ј-H), 3.45–3.55 (3H, m, 6-H, 6Ј-HHЈ), 3.45 (1H, d, JAB
13.4, CHHЈAr), 3.63 (1H, br s, 4Ј-H), 3.79 (1H, m, 6-HЈ), 3.87
(3H, m, 1-H, ArOCH2CH2CH2(CH2)14CH3), 4.21 (1H, d, J 7.2,
1Ј-H), 4.53 (2H, br s, OH), 4.64 (1H, br s, OH), 4.74 (1H, br s,
OH), 4.79 (1H, br s, OH), 5.12 (1H, br s, OH), 5.49 (1H, br d,
J 5.5, 2-OH), 6.77 (2H, d, JAB 8.9, ArH), 6.81 (2H, d, JAB 8.9,
ArH); δC (100 MHz, d6-DMSO) 14.3, 22.5, 25.8, 28.2, 28.3,
29.0, 29.1, 29.1, 29.3(4), 29.3(6), 29.3(7) (8 carbons unresolved
under here), 29.4, 31.7, 40.6, 60.2, 60.8, 61.1, 67.7, 68.5, 70.9,
71.6, 73.6, 75.9, 76.7, 80.3, 81.1, 82.4, 104.1, 114.6, 126.8, 130.4,
150.6, 158.1, 169.8, 170.1; m/z 861 (M ϩ Naϩ)
1-Chloromethyl-4-octadecyloxybenzene (4)
(4-Octadecycloxyphenyl)methanol 3 (1.25 g, 3.32 mmol) was
dissolved in DCM (20 cm3) and to this was added SOCl2 (0.5
cm3, 6.8 mmol). The reaction mixture was stirred at room tem-
perature for 24 h and was analysed by TLC (DCM) to ensure
complete conversion. The solvent was removed at reduced pres-
sure to furnish a white powder. The solid was recrystallized
from acetonitrile to furnish 4 (1.1 g, 84%) as white needles and
used without further purification, δH (400 MHz, CDCl3) 0.86
(3H, t, J 6.9, ArOCH2CH2CH2(CH2)14CH3), 1.24 (28H, br s,
ArOCH2CH2CH2(CH2)14CH3), 1.37 (2H, m, ArOCH2CH2-
CH2(CH2)14CH3), 1.65 (2H, p, J 6.5, ArOCH2CH2CH2(CH2)14-
CH3), 3.98 (2H, t, J 6.5, ArOCH2CH2(CH2)15CH3), 4.61 (2H, s,
ArCH2Cl), 6.90 (2H, d, JAB 6.8, ArH), 7.32 (2H, d JAB 6.8,
ArH); δC (100 MHz, CDCl3) 14.5, 23.1, 26.4, 29.6, 29.8, 29.9,
30.0, 30.1 (9 carbons unresolved under here), 32.3, 46.8, 68.5,
115.1, 130.0, 130.4, 160.0; m/z 394/396 (Mϩ).
1,3-Dimethyl-5-[ꢀ-D-glucopyranosyl-(1
4)-ꢀ-D-glucopyrano-
syl]-5-(4-octadecyloxybenzyl)pyrimidine-2,4,6-trione (12)
The same procedure was followed as for the alkylation of
sodium 1,3-dimethyl-5-[β--galactopyranosyl-(1
4)-β--glu-
copyranosyl]barbiturate 11 above. Evaporation of the fractions
that contained product under reduced pressure gave a white
powder that was recrystallized from ethyl acetate as a white
powdery product 12 (1.13 g, 68%); mp 146–149 ЊC from EtOAc.
δH (400 MHz, d6-DMSO) 0.86 (3H, t, J 6.9, ArOCH2CH2CH2-
(CH2)14CH3), 1.25 (28H, br s, ArOCH2CH2CH2(CH2)14CH3),
1.37 (2H, m, ArOCH2CH2CH2(CH2)14CH3), 1.66 (2H, qnt,
1,3-Dimethyl-5-[ꢀ-D-galactopyranosyl]-5-(4-octadecyloxy-
benzyl)pyrimidine-2,4,6-trione (10)
Galactose barbituric salt 6 (1 g, 2.94 mmol) and 1-chloro-
methyl-4-octadecyloxybenzene 4 (1 g, 2.55 mmol) were dissol-
ved in DMSO (20 cm3) and the reaction mixture was heated and
stirred at 70 ЊC for 3 h. After this time the reaction mixture
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 4 1 4 8 – 4 1 5 9
4156

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J 6.9, ArOCH2CH2CH2(CH2)14CH3), 2.95 (3H, s, NCH3), 3.00
(3H, s, NCH3), 2.95–3.10 (2H, m, 2Ј-H and 3Ј-H), 3.15–3.45
(9H, m, 2-H, 3-H, 4-H, 5-H, 4Ј-H, 5Ј-H, 6Ј-H, CHHЈAr), 3.55
(1H, m, 6-H), 3.75 (2H, m, 6-HЈ, 6Ј-HЈ), 3.87 (3H, m, 1-H,
ArOCH2CH2CH2(CH2)14CH3), 4.26 (1H, d, J 7.9, 1Ј-H), 4.56
(1H, br t, J 5.9, 6Ј-OH), 4.58 (1H, br t, J 5.2, 6-OH), 4.70 (1H,
br s, 4Ј-OH), 5.00 (1H, br d, J 4.8, 3-OH), 5.03 (1H, br d, J 4.8,
3Ј-OH), 5.26 (1H, br d, J 4.8, 2Ј-OH), 5.49 (1H, br d, J 5.7,
2-OH), 6.77 (2H, d, JAB 8.6, ArH), 6.81 (2H, d, JAB 8.6, ArH);
δC (100 MHz, d6-DMSO) 14.3, 22.5, 25.9, 28.2, 28.3, 29.0, 29.1,
29.3(4), 29.3(5), 29.3(6) (8 carbons unresolved under here),
29.4, 31.7, 40.6, 60.2, 61.0, 61.4, 67.6, 70.4, 71.5, 73.6, 76.8,
76.9, 77.1, 80.3, 80.9, 82.4, 103.5, 114.6, 126.8, 130.4, 150.6,
158.1, 169.9, 170.1; m/z (M ϩ Naϩ) 862
N,NЈ-Dimethyl-2-[ꢀ-D-galactopyranosyl-(1
pyranosyl]-2-(4-octadecyloxybenzyl)malonamide (15)
4)-ꢀ-D-gluco-
1,3-Dimethyl-5-[β--galactopyranosyl-(1
4) -β--gluco-
pyranosyl]-5-(4-octadecyloxybenzyl)pyrimidine-2,4,6-trione 11
(0.5 g, 0.59 mmol) was dissolved in THF (10 cm3) and 1 M
NaOH (2 cm3, excess) was added dropwise over 5 min to the
stirred solution. The reaction mixture was stirred at room tem-
perature for 1 h until the starting material was consumed (TLC:
EtOAc–MeOH–H2O, 40 : 10 : 2 v/v). The reaction mixture was
neutralised with 1 M HCl. Water (20 cm3) was added and the
THF was removed under reduced pressure. The solution was
chilled in ice and the resulting solid was collected by filtration
and dried to give the product 15 as a powder (0.25 g, 52%); mp
210–211 ЊC; δH (400 MHz, d6-DMSO) 0.86 (3H, t, J 6.9, ArO-
CH2CH2CH2(CH2)14CH3), 1.24 (28H, br s, ArOCH2CH2CH2-
(CH2)14CH3), 1.38 (2H, m, ArOCH2CH2CH2(CH2)14CH3), 1.67
(2H, qnt, J 6.9, ArOCH2CH2CH2(CH2)14CH3), 2.49 (3H, d,
J 4.5, NHCH3), 2.65 (3H, d, J 4.5, NHCH3), 3.02 (1H, t, J 9.1,
2-H), 3.10 (1H, d, JAB 13.5, CHHЈAr), 3.20–3.40 (6H, m, 3-H,
4-H, 2Ј-H, 3Ј-H, 5Ј-H, CHHЈAr), 3.51–3.60 (3H, m, 4-H,
6Ј-HHЈ), 3.64 (1H, br s, 4Ј-H), 3.76 (2H, m, 6-HHЈ), 3.87 (3H,
m, 1-H, ArOCH2CH2CH2(CH2)14CH3), 4.19 (1H, d, J 6.5,
1Ј-H), 4.71 (7H, br s, 7 × OH), 6.72 (2H, d, JAB 8.5, ArH), 6.93
(2H, d, JAB 8.5, ArH), 7.65 (1H, q, J 4.5, NHCH3), 9.00 (1H, q,
J 4.5, NHCH3); δC (100MHz, d6-DMSO) 14.3, 22.5, 25.9, 26.0,
26.3, 29.1(0), 29.1(5), 29.3(5), 29.3(7) (9 carbons unresolved
under here), 29.4, 31.7, 39.9, 59.6, 59.9, 60.7, 67.5, 68.5, 70.9,
72.3, 73.6, 75.8, 76.6, 79.4, 79.9, 80.4, 104.2, 113.8, 129.1, 130.9,
157.6, 170.9, 172.1; m/z 835 (M ϩ Naϩ).
1,3-Dimethyl-5-[ꢁ-D-glucopyranosyl-(1
4)-ꢀ-D-glucopyrano-
syl]-5-(4-octadecyloxybenzyl)pyrimidine-2,4,6-trione (13)
The same procedure was followed as for the alkylation of
sodium 1,3-dimethyl-5-[β--galactopyranosyl-(1
4)-β--glu-
copyranosyl]barbiturate 11 above. Evaporation of the fractions
that contained product under reduced pressure gave a white
powder that was recrystallised from acetone as a white powdery
product 13 (0.98 g, 59%); mp 142–144 ЊC from EtOAc;. δH (400
MHz, d6-DMSO) 0.86 (3H, t, J 6.9, ArOCH2CH2CH2(CH2)14-
CH3), 1.25 (28H, br s, ArOCH2CH2CH2(CH2)14CH3) 1.37 (2H,
m, ArOCH2CH2CH2(CH2)14CH3), 1.66 (2H, qnt, J 6.9, ArO-
CH2CH2CH2(CH2)14CH3), 2.95 (3H, s, NCH3), 2.99 (3H,
s, NCH3), 3.08 (1H, m, 4Ј-H), 3.15–3.55 (11H, m, 2-H, 3-H,
4-H, 5-H, 6-H, 2Ј-H, 3Ј-H, 5Ј-H, 6Ј-H, CHHЈAr), 3.63 (1H,
m, 6-HЈ), 3.71 (1H, dd, J 5.3, 11.5, 6Ј-HЈ), 3.87 (3H, m, 1-H,
ArOCH2CH2CH2(CH2)14CH3), 4.45 (1H, t, J 5.6, 6-OH), 4.54
(1H, t, J 5.6, 6Ј-OH), 4.90 (2H, m, 2 × OH), 5.00 (1H, d, J 3.2,
1Ј-H), 5.42 (1H, d, J 5.5, OH), 5.52 (2H, m, 2 × OH), 6.77 (2H,
d, JAB 8.6, ArH), 6.81 (2H, d, JAB 8.6, ArH); δC (100 MHz,
d6-DMSO) 14.3, 22.5, 25.8, 28.2, 28.3, 28.9, 29.0, 29.1, 29.4
(10 carbons unresolved under here), 31.5, 40.1, 60.3, 61.1, 61.3,
67.6, 70.2, 71.2, 72.7, 73.6, 73.8, 78.5, 80.2, 80.6, 82.8, 101.2,
114.6, 126.8, 130.4, 150.6, 158.1, 169.9, 170.1; m/z 862 (M ϩ
Naϩ).
General procedure for the acetylations
The sugar derivative (0.5 g), acetic anhydride (5 cm3) and a
catalytic amount of DMAP (50 mg) were dissolved in pyridine
(5 cm3). The reaction mixture was stirred at room temperature
for 24 h. Water (20 cm3) was added to the reaction mixture and
it was stirred for 30 min. The mixture was extracted with DCM
(3 × 20 cm3). The combined DCM fractions were washed with
saturated NaHCO3 (2 × 25 cm3) followed by 1 M HCl (2 ×
25 cm3). The combined DCM fractions were dried with MgSO4,
filtered and the solvent removed under reduced pressure to give
the required compounds.
N,NЈ-Dimethyl-2-[ꢀ-D-galactopyranosyl]-2-(4-octadecyloxy-
benzyl)malonamide (14)
1,3-Dimethyl-5-[β--galactopyranosyl]-5-(4-octadecyloxyben-
zyl)pyrimidine-2,4,6-trione 10 (0.5 g, 0.74 mmol) was dissolved
in THF (10 cm3) and 1 M NaOH (2 cm3, excess) was added
dropwise over 5 min to the stirred solution. The reaction mix-
ture was stirred at room temperature for 2 h until the starting
material was consumed (TLC: EtOAc–MeOH–H2O, 40 : 5 : 1
v/v). The reaction mixture was neutralised with 1 M HCl. Water
(20 cm3) was added and the THF was removed at reduced pres-
sure. The solution was chilled in ice and the resulting solid col-
lected by filtration and dried. The solid was recrystallised from
ethyl acetate to furnish a white powder 14 (0.39 g, 81%); δH (400
MHz, d6-DMSO) 0.86 (3H, t, J 6.7, ArOCH2CH2CH2(CH2)14-
CH3), 1.25 (28H, br s, ArOCH2CH2CH2(CH2)14CH3), 1.39
(2H, m, ArOCH2CH2CH2(CH2)14CH3), 1.67 (2H, qnt, J 6.4,
ArOCH2CH2CH2(CH2)14CH3), 2.50 (3H, d, J 4.5, NHCH3),
2.63 (3H, d, J 4.5, NHCH3), 3.11 (1H, d, JAB 13.4, CHHЈAr),
3.26 (1H, d, JAB 13.4, CHHЈAr), 3.28 (1H, dd, J 2.8, 8.90,
3-H), 3.40 (2H, m, 2-H, 5-H), 3.56 (1H, dd, J 5.1, 11.3, 6-H),
3.63 (1H, dd, J 6.9, 11.3, 6-HЈ), 3.70 (1H, d, J 2.8, 4-H), 3.83
(1H, d, J 9.6, 1-H), 3.88 (2H, t, J 6.4, ArOCH2CH2CH2(CH2)14-
CH3), 4.25 (4H, br s, 4 × OH), 6.72 (2H, d, JAB 8.6, ArH),
6.94 (2H, d, JAB 8.6, ArH), 7.67 (1H, q, J 4.5, NHCH3), 8.88
(1H, q, J 4.5, NHCH3); δC (100MHz, d6-DMSO) 14.3, 22.5,
25.9, 26.0, 26.2, 29.1(0), 29.1(5), 29.2, 29.3 (9 carbons unresol-
ved under here), 29.4, 31.7, 39.9, 59.9, 61.3, 67.5, 68.8, 69.4,
75.4, 79.7, 81.1, 113.8, 129.3, 131.0, 157.6, 171.0, 172.8; m/z 651
(M ϩ Hϩ).
1,3-Dimethyl-5-[2,3,4,6-tetra-O-acetyl-ꢀ-D-galactopyranosyl]-5-
(4-octadecyloxybenzyl)pyrimidine-2,4,6-trione (16)
Compound 16 (0.52 g, 83%); δH (400 MHz, CDCl3) 0.81 (3H, t,
J 6.7, ArOCH2CH2CH2(CH2)14CH3), 1.19 (28H, br s, ArO-
CH2CH2CH2(CH2)14CH3),1.35(2H,m,ArOCH2CH2CH2(CH2)14-
CH3), 1.67 (2H, qnt, J 6.8, ArOCH2CH2CH2(CH2)14CH3), 1.90
(6H, s, 2 × OCOCH3), 1.94 (3H, s, OCOCH3), 2.10 (3H, s,
OCOCH3), 3.00 (3H, s, NCH3), 3.07 (3H, s, NCH3), 3.09 (1H,
d, JAB 12.9, CHHЈAr), 3.27 (1H, d, JAB 12.9, CHHЈAr), 3.80
(3H, m, 5-H, ArOCH2CH2CH2(CH2)14CH3), 3.97 (2H, m,
6-HHЈ), 4.29 (1H, d, J 9.9, 1-H), 4.96 (1H, dd, J 3.0, 9.7, 3-H),
5.30 (1H, d, J 3.0, 4-H), 5.67 (1H, dd, J 9.7, 9.9, 2-H), 6.64 (2H,
d, JAB 8.3, ArH), 6.80 (2H, d, JAB 8.3, ArH); δC (100 MHz,
CDCl3) 14.5, 20.9 (2 carbons unresolved under here), 21.0(4),
21.0(7), 23.0, 26.4, 28.6, 28.7, 29.6, 29.7, 29.8, 29.9, 30.0
(9 carbons unresolved under here), 32.3, 40.7, 60.9, 61.9, 67.5,
67.8, 68.3, 73.3, 75.3, 81.2, 114.8, 125.6, 130.7, 150.5, 159.2,
169.2, 169.4, 169.9, 170.4, 170.5, 170.6; m/z 845 (M ϩ Hϩ).
Found: C, 63.81, H, 8.10, N, 3.23%. C45H68N2O13 requires C,
63.96, H, 8.11, N, 3.32%.
1,3-Dimethyl-5-[2,3,4,6-tetra-O-acetyl-ꢀ-D-galactopyranosyl-
(1
4)-2,3,6-tri-O-acetyl-ꢀ-D-glucopyranosyl]-5-(4-octadecyl-
oxybenzyl)pyrimidine-2,4,6-trione (17)
Compound 17 (0.56 g, 83%); δH (400 MHz, CDCl3) 0.85 (3H, t,
J 6.7, ArOCH2CH2CH2(CH2)14CH3), 1.22 (28H, br s, ArO-
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 4 1 4 8 – 4 1 5 9
4157

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CH2CH2CH2(CH2)14CH3), 1.39 (2H, m, ArOCH2CH2CH2-
(CH2)14CH3), 1.70 (2H, qnt, J 6.8, ArOCH2CH2CH2(CH2)14-
CH3), 1.93 (3H, s, OCOCH3), 1.96 (3H, s, OCOCH3), 1.99 (3H,
s, OCOCH3), 2.00 (3H, s, OCOCH3), 2.01 (3H, s, OCOCH3),
2.05 (3H, s, OCOCH3), 2.12 (3H, s, OCOCH3), 3.02 (3H, s,
NCH3), 3.04 (1H, d, JAB 12.8, CHHЈAr), 3.10 (3H, s, NCH3),
3.23 (1H, d, JAB 12.8, CHHЈAr), 3.50 (1H, m, 5-H), 3.69 (1H, t,
J 9.4, 4-H), 3.83 (3H, m, 5Ј-H, ArOCH2CH2CH2(CH2)14CH3),
3.91 (1H, dd, J 5.2, 12.0, 6-H), 4.08 (2H, m, 6Ј-HHЈ), 4.30 (1H,
d, J 10.0, 1-H), 4.46 (1H, d, J 7.9, 1Ј-H), 4.48 (1H, dd, J 2.0,
12.0, 6-HЈ), 4.93 (1H, dd, J 3.4, 10.4, 3Ј-H), 5.07 (1H, dd, J 7.9,
10.4, 2Ј-H), 5.14 (1H, t, J 9.4, 3-H), 5.31 (1H, d, J 3.4, 4Ј-H),
5.52 (1H, dd, J 9.4, 10.0, 2-H), 6.66 (2H, d, JAB 8.6, ArH), 6.81
(2H, d, JAB 8.6, ArH); δC (100 MHz, CDCl3) 14.5, 20.8, 20.9,
21.0 (2 carbons unresolved under here), 21.1(0), 21.1(5), 21.2,
23.1, 26.4, 28.6(5), 28.6(8), 29.6, 29.7, 29.8, 29.9(5), 29.9(8),
30.1 (8 carbons unresolved under here), 32.3, 40.4, 61.1, 61.2,
61.6, 67.0, 68.4, 69.5, 70.3, 71.1, 71.3, 75.2, 76.2, 77.5, 80.7,
101.4, 114.9, 125.6, 130.7, 150.5, 159.2, 169.4, 169.5, 169.6,
170.0, 170.3, 170.4(1), 170.4(6), 170.5, 170.75; m/z 1133 (Mϩ).
Found: C, 60.71, H, 7.59, N, 2.41%. C57H84N2O21 requires C,
60.41, H, 7.47, N, 2.47%.
(1H, dd, J 8.8, 9.8, 2-H), 6.62 (2H, d, JAB 8.6, ArH), 6.78 (2H, d,
JAB 8.6, ArH); δC (100 MHz, CDCl3) 14.5, 20.9 (3), 20.9 (9),
21.0, 21.1, 21.4, 23.1, 26.4, 28.6(4), 28.6(8), 29.6, 29.7(1),
29.7(7), 29.9(3), 29.9(5), 30.1 (10 carbons unresolved under
here), 32.3, 40.3, 61.1, 61.8, 62.3, 68.3, 68.4, 68.9, 69.7, 70.4,
70.7, 72.7, 77.0, 77.7, 80.6, 96.1, 114.9, 125.5, 130.7, 150.5,
159.2, 169.5, 169.6, 169.7, 170.0, 170.2, 170.3, 170.8 (2 carbons
unresolved under here), 170.9; m/z 1156 (M ϩ Naϩ). Found: C,
60.44, H, 7.39, N, 2.41%, C57H84N2O21 requires C, 60.41, H,
7.47, N, 2.47%.
N,NЈ-Dimethyl-2-[2,3,4,6-tetra-O-acetyl-ꢀ-D-galactopyranosyl]-
2-(4-octadecyloxybenzyl)malonamide (20)
Compound 20 (0.49 g, 78%); mp 137–138 ЊC (from Et2O–light
petroleum (bp 60–80 ЊC)); δH (400 MHz, CDCl3) 0.80 (3H, t,
J 6.7, ArOCH2CH2CH2(CH2)14CH3), 1.18 (28H, br s, ArOCH2-
CH2CH2(CH2)14CH3), 1.36 (2H, m, ArOCH2CH2CH2(CH2)14-
CH3), 1.67 (2H, qnt, J 6.7, ArOCH2CH2CH2(CH2)14CH3), 1.81
(3H, s, OCOCH3), 1.87 (3H, s, OCOCH3), 2.00 (3H, s,
OCOCH3), 2.10 (3H, s, OCOCH3), 2.52 (3H, d, J 4.7, NHCH3),
2.85 (3H, d, J 4.6, NHCH3), 3.02 (1H, d, JAB 13.3, CHHЈAr),
3.14 (1H, d, JAB 13.3, CHHЈAr), 3.82 (2H, t, J 6.7, ArOCH2-
CH2CH2(CH2)14CH3), 3.95 (1H, dd, J 4.9, 11.1, 6-H), 4.01 (1H,
dd, J 4.9, 7.5, 5-H), 4.39 (1H, dd, J 7.5, 11.1, 6-HЈ), 4.59 (1H, d,
J 10.2, 1-H), 4.97 (1H, dd, J 3.0, 9.9, 3-H), 5.24 (1H, dd, J 9.9,
10.2, 2-H), 5.40 (1H, d, J 3.0, 4-H), 6.66 (2H, d, JAB 8.6, ArH),
6.83 (2H, d, JAB 8.6, ArH), 7.20 (1H, q, J 4.6, NHCH3), 9.23
(1H, q, J 4.7, NHCH3); δC (100 MHz, CDCl3) 13.1, 19.4, 19.5,
19.6, 19.8, 21.7, 24.9, 25.0, 25.1, 28.3(0), 28.3(4), 28.4, 28.6,
28.6(5), 28.7 (8 carbons unresolved under here), 30.9, 40.9, 57.1,
60.5, 65.3, 66.8, 66.9, 71.4, 74.4, 79.0, 113.0, 126.0, 129.5, 157.3,
168.1, 168.6, 168.8, 168.9, 169.8, 171.3; m/z 819 (M ϩ Hϩ).
Found: C, 64.44, H, 8.60, N, 3.42%, C44H70N2O12 requires C,
64.52, H, 8.61, N, 3.42%.
1,3-Dimethyl-5-[2,3,4,6-tetra-O-acetyl-ꢀ-D-glucopyranosyl-
(1
4)-2,3,6-tri-O-acetyl-ꢀ-D-glucopyranosyl]-5-(4-octadecyl-
oxybenzyl)pyrimidine-2,4,6-trione (18)
Compound 18 (0.59 g, 87%); δH (400 MHz, CDCl3) 0.90 (3H, t,
J 6.9, ArOCH2CH2CH2(CH2)14CH3), 1.28 (28H, br s, ArO-
CH2CH2CH2(CH2)14CH3),1.45(2H,m,ArOCH2CH2CH2(CH2)14-
CH3), 1.76 (2H, qnt, J 6.5, ArOCH2CH2CH2(CH2)14CH3), 2.00
(3H, s, OCOCH3), 2.01 (3H, s, OCOCH3), 2.03 (3H, s,
OCOCH3), 2.04 (6H, s, 2 × OCOCH3), 2.05 (3H, s, OCOCH3),
2.13 (3H, s, OCOCH3), 3.10 (3H, s, NCH3), 3.13 (1H, d, JAB
12.9, CHHЈAr), 3.15 (3H, s, NCH3), 3.28 (1H, JAB 12.9,
CHHЈAr), 3.54 (1H, m, 5-H), 3.68 (1H, m, 5Ј-H), 3.73 (1H, t,
J 9.4, 4-H), 3.88 (2H, t, J 6.5, ArOCH2CH2CH2(CH2)14CH3),
3.96 (1H, dd, J 5.1, 12.0, 6-H), 4.07 (1H, dd, J 1.8, 12.5, 6Ј-H),
4.34 (1H, d, J 9.9, 1-H), 4.40 (1H, dd, J 4.4, 12.5, 6Ј-HЈ), 4.52
(1H, d, J 7.9, 1Ј-H), 4.56 (1H, d, J 12.0, 6-HЈ), 4.95 (1H, dd,
J 7.9, 9.1, 2Ј-H), 5.09 (1H, t, J 9.5, 4Ј-H), 5.17 (2H, br t, J ∼9.1,
3-H, 3Ј-H), 5.59 (1H, dd, J 9.1, 9.9, 2-H), 6.71 (2H, d, JAB 8.6,
ArH), 6.87 (2H, d, JAB 8.6, ArH); δC (100 MHz, CDCl3) 14.5,
20.9 (3 carbons unresolved under here), 21.0(3), 21.0(7), 21.2,
23.1, 26.4, 28.7 (2 carbons unresolved under here), 29.6, 29.7,
29.8, 30.0, 30.1 (10 carbons unresolved under here), 32.3, 40.4,
61.0, 61.5, 61.9, 68.1, 68.4, 70.2, 72.0, 72.4, 73.3, 74.9, 76.4,
77.6, 80.8, 101.2, 114.9, 125.5, 130.7, 150.5, 159.2, 169.3, 169.5
(2 carbons unresolved under here), 169.7, 170.0, 170.3, 170.4,
170.6, 170.9; m/z 1133 (M ϩ Hϩ). Found: C, 60.54, H, 7.49, N,
2.44%. C57H84N2O21 requires C, 60.41, H, 7.47, N, 2.47%.
N,NЈ-Dimethyl-2-[2,3,4,6-tetra-O-acetyl-ꢀ-D-galactopyranosyl-
(1
4)-2,3,6-tri-O-acetyl-ꢀ-D-glucopyranosyl]-2-(4-octa-
decyloxybenzyl)malonamide (21)
Compound 21 (0.48 g, 71%); mp 137–138 ЊC (from aceto-
nitrile); δH (400 MHz, CDCl3) 0.81 (3H, t, J 6.7, ArOCH2CH2-
CH2(CH2)14CH3), 1.19 (28H, br s, ArOCH2CH2CH2(CH2)14-
CH3), 1.36 (2H, qnt, J 7.0, ArOCH2CH2CH2(CH2)14CH3), 1.67
(2H, qnt, J 7.0, ArOCH2CH2CH2(CH2)14CH3), 1.79 (3H, s,
OCOCH3), 1.91 (3H, s, OCOCH3), 1.93 (3H, s, OCOCH3), 2.00
(6H, s, 2 × OCOCH3), 2.08 (3H, s, OCOCH3), 2.09 (3H, s,
OCOCH3), 2.52 (3H, d, J 4.5, NHCH3), 2.78 (3H, d, J 4.3,
NHCH3), 3.03 (1H, d, JAB 13.3, CHHЈAr), 3.11 (1H, JAB 13.3,
CHHЈAr), 3.72 (2H, m, 4-H, 5-H), 3.82 (3H, m, ArOCH2CH2-
CH2(CH2)14CH3, 5Ј-H), 4.01 (1H, dd, J 9.5, 11.1, 6Ј-H), 4.08
(1H, dd, J 5.0, 11.1, 6Ј-HЈ), 4.35 (1H, dd, J 2.6, 12.0, 6-H), 4.47
(3H, m, 1-H, 6-HЈ, 1Ј-H), 4.80 (1H, t, J 9.1, 2-H), 4.91 (1H, dd,
J 3.0, 10.2, 3Ј-H), 5.06 (1H, dd, J 8.7, 10.2, 2Ј-H), 5.15 (1H, m,
3-H), 5.29 (1H, d, J 3.0, 4Ј-H), 6.65 (2H, d, JAB 7.3, ArH), 6.82
(2H, d, JAB 7.3, ArH), 6.87 (1H, q, J 4.5, NHCH3), 9.14 (1H, q,
J 4.3, NHCH3); δC (100 MHz, CDCl3) 14.5, 20.7, 20.9, 21.0(2
carbons unresolved under here), 21.0(5), 21.1, 21.4, 23.1,
26.3, 26.4, 26.7, 29.6, 29.7, 29.8, 29.9(5), 29.9(7), 30.0, 30.1
(7 carbons unresolved under here), 32.3, 42.2, 58.4, 61.1, 62.5,
66.9, 68.2, 69.5, 69.9, 71.1, 71.3, 74.5, 76.9, 78.2, 79.7, 101.1,
114.4, 127.2, 130.9, 158.7, 169.3, 169.7, 169.8, 170.1, 170.4,
170.5, 170.7, 171.4, 172.3; m/z 1107 (M ϩ Hϩ). Found: C, 60.98,
H, 7.90, N, 2.41%, C56H86N2O20 requires C, 60.74, H, 7.83, N,
2.53%.
1,3-Dimethyl-5-[2,3,4,6-tetra-O-acetyl-ꢁ-D-glucopyranosyl-
(1
4)-2,3,6-tri-O-acetyl-ꢀ-D-glucopyranosyl]-5-(4-octadecyl-
oxybenzyl)pyrimidine-2,4,6-trione (19)
Compound 19 (0.55 g, 82%), δH (400 MHz, CDCl3) 0.81 (3H, t,
J 6.8, ArOCH2CH2CH2(CH2)14CH3), 1.18 (28H, m, ArOCH2-
CH2CH2(CH2)14CH3), 1.35 (2H, m, ArOCH2CH2CH2(CH2)14-
CH3), 1.66 (2H, qnt, J 6.6, ArOCH2CH2CH2(CH2)14CH3),
1.90 (3H, s, OCOCH3), 1.93 (3H, s, OCOCH3), 1.94 (3H, s,
OCOCH3), 1.95 (3H, s, OCOCH3), 1.97 (3H, s, OCOCH3), 1.98
(3H, s, OCOCH3), 2.02 (3H, s, OCOCH3), 3.01 (3H, s, NCH3),
3.03 (1H, d, JAB 12.8, CHHЈAr), 3.05 (3H, s, NCH3), 3.18 (1H,
d, JAB 12.8, CHHЈAr), 3.53 (1H, m, 5-H), 3.78 (2H, t, J 6.6,
ArOCH2CH2CH2(CH2)14CH3), 3.83 (1H, t, J 8.8, 4-H), 3.88
(1H, m, 5Ј-H), 3.95 (1H, dd, J 4.6, 12.2, 6-H), 3.98 (1H, dd,
J 2.0, 12.4, 6Ј-H), 4.18 (1H, dd, J 3.8, 12.4, 6Ј-HЈ), 4.33 (1H, d,
J 9.8, 1-H), 4.47 (1H, dd, J 2.3, 12.2, 6-HЈ), 4.79 (IH, dd, J 4.0,
10.5, 2Ј-H), 4.97 (1H, t, J 9.4, 4Ј-H), 5.19 (1H, t, J 8.8, 3-H),
5.27 (1H, dd, J 9.4, 10.5, 3Ј-H), 5.32 (1H, d, J 4.0, 1Ј-H), 5.45
BIAcore experiments
Preparing the sensor chip
An HPA sensor chip (from BIAcore, Sweden) was washed
overnight at 25 ЊC with degassed, ultrafiltered 20 mM HEPES
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 4 1 4 8 – 4 1 5 9
4158

View Article Online
14 N. Stahl, D. R. Borchelt and S. B. Prusiner, Biochemistry, 1990, 29,
buffer pH 7.0 (containing 90 mM NaCl) at a flow rate of 2 µl
minϪ1. The chip was then washed with 40 mM n-octyl β--
glucopyranoside (Sigma, Dorset, UK) dissolved in buffer for 7
min at a flow rate of 5 µl minϪ1 immediately prior to deposition
of the selected lipids. Dipalmitoyl phosphatidylcholine (Sigma,
Dorset, UK) dissolved in chloroform–methanol (1 : 1 v/v) was
mixed with varying amounts of the selected lactosyl thiooxa-
alkane or carboglycolipid in the same solvent to give solutions of
10, 15 and 25 mol% of glycolipid. A control of pure dipalmitoyl
phosphatiylcholine was used. The solutions were evaporated
under reduced pressure, dried for several hours over P2O5 in a
vacuum dessicator, and then suspended in HEPES buffer by
warming to 30 ЊC and vortexing to give a final concentration of
phospholipid of 0.5 mM. Each of the four channels of the
sensor chip was exposed for 3 h to a different concentration of
glycolipid from 0 to 25 mol% at a flow rate of 2 µl minϪ1 and 35
ЊC, to allow the lipids to impregnate the existing thioalkyl layer
attached to the gold surface by the manufacturers.
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The chip surface was washed with a fast flow of buffer (100 µl
minϪ1 for 5 min) to remove loosely adherent lipid, followed by
two washes with 20 mM NaOH (5 µl minϪ1 for 5 min each) and
then with buffer to stabilize the phospholipid monolayer.
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Kinetic measurements (binding constants)
Low concentrations of RCA120 analyte (Sigma, Dorset,UK) 25,
50, 75, 100 and 150 nM were used to minimize bulk refractive
index changes and possible mass transfer effects. Each solution
was injected into all channels and channel 1 (the negative
control of phospholipid) was subtracted from each channel
(10 mol% glycolipid) to remove any bulk refractive index
changes. Experiments were repeated at least five times. The
results in Table 1 are the means of the ka and kd values derived
from the sensorgram and the calculated values for KA and KD
The temperature was changed in random order to obtain the
equilibrium constants for each compound at 5, 10, 15, 20, 25
and 30 ЊC
30 Y-F. Leu and C. K. Lai, J. Chin. Chem. Soc. (Taipei), 1997, 44,
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Acknowledgements
We thank Dr H. D. B. Jenkins, Department of Chemistry,
University of Warwick for his helpful suggestions, particularly
on the kinetic and thermodynamic work, during the prepar-
ation of this manuscript and Mr M. N. Willand who syn-
thesised the thiolactosyl lipids.
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