Saccharide-functionalized alkanethiols for fouling-resistant self-assembled monolayers: Synthesis, monolayer properties, and antifouling behavior

Article abstract of DOI:10.1021/la202774e

We describe the synthesis of a series of mono-, di-, and trisaccharide-functionalized alkanethiols as well as the formation of fouling-resistant self-assembled monolayers (SAMs) from these. The SAMs were characterized using ellipsometry, wetting measurements, and infrared reflection-absorption spectroscopy (IRAS). We show that the structure of the carbohydrate moiety affects the packing density and that this also alters the alkane chain organization. Upon increasing the size of the sugar moieties (from mono- to di- and trisaccharides), the structural qualities of the monolayers deteriorated with increasing disorder, and for the trisaccharide, slow reorganization dynamics in response to changes in the environmental polarity were observed. The antifouling properties of these SAMs were investigated through protein adsorption experiments from buffer solutions as well as settlement (attachment) tests using two common marine fouling species, zoospores of the green macroalga Ulva linza and cypris larvae of the barnacle Balanus amphitrite. The SAMs showed overall good resistance to fouling by both the proteins and the tested marine organisms. To improve the packing density of the SAMs with bulky headgroups, we employed mixed SAMs where the saccharide-thiols are diluted with a filler molecule having a small 2-hydroxyethyl headgroup. This method also provides a means by which the steric availability of sugar moieties can be varied, which is of interest for specific interaction studies with surface-bound sugars. The results of the surface dilution study and the low nonspecific adsorption onto the SAMs both indicate the feasibility of this approach.

Full text of DOI:10.1021/la202774e

ARTICLE
pubs.acs.org/Langmuir
Saccharide-Functionalized Alkanethiols for Fouling-Resistant
Self-Assembled Monolayers: Synthesis, Monolayer Properties,
and Antifouling Behavior
Timmy Fyrner,† Hung-Hsun Lee,‡ Alberto Mangone,‡ Tobias Ekblad,‡ Michala E. Pettitt,§
Maureen E. Callow,§ James A. Callow,§ Sheelagh L. Conlan,|| Robert Mutton,|| Anthony S. Clare,||
Peter Konradsson,† Bo Liedberg,‡ and Thomas Ederth*,‡
†Division of Chemistry and ‡Division of Molecular Physics, IFM, Link€oping University, SE-581 83 Link€oping, Sweden
§School of Biosciences, University of Birmingham, Birmingham B15 2TT, United Kingdom
School of Marine Science and Technology, Newcastle University, Newcastle upon Tyne NE1 7RU, United Kingdom
S Supporting Information
b
ABSTRACT: We describe the synthesis of a series of mono-, di-,
and trisaccharide-functionalized alkanethiols as well as the
formation of fouling-resistant self-assembled monolayers (SAMs)
from these. The SAMs were characterized using ellipsometry,
wetting measurements, and infrared reflectionꢀabsorption spec-
troscopy (IRAS). We show that the structure of the carbohydrate
moiety affects the packing density and that this also alters the
alkane chain organization. Upon increasing the size of the sugar
moieties (from mono- to di- and trisaccharides), the structural
qualities of the monolayers deteriorated with increasing disorder,
and for the trisaccharide, slow reorganization dynamics in re-
sponse to changes in the environmental polarity were observed. The antifouling properties of these SAMs were investigated through
protein adsorption experiments from buffer solutions as well as settlement (attachment) tests using two common marine fouling species,
zoospores of the green macroalga Ulva linza and cypris larvae of the barnacle Balanus amphitrite. The SAMs showed overall good
resistance to fouling by both the proteins and the tested marine organisms. To improve the packing density of the SAMs with bulky
headgroups, we employed mixed SAMs where the saccharideꢀthiols are diluted with a filler molecule having a small 2-hydroxyethyl
headgroup. This method also provides a means by which the steric availability of sugar moieties can be varied, which is of interest for
specific interaction studies with surface-bound sugars. The results of the surface dilution study and the low nonspecific adsorption onto
the SAMs both indicate the feasibility of this approach.
’ INTRODUCTION
to exploit the properties of sugars are available, and by taking
advantage of the diversity and flexibility offered via carbohydrate
synthesis, many new possibilities have emerged in materials
science2,3 and in particular biomaterials4,5 for bioanalytical
applications,6,7 such as the production of well-defined surfaces
with interesting antifouling properties using polysaccharides,8ꢀ10
oligosaccharide-terminated SAMs,11ꢀ13 or polyols.14ꢀ16
The use of SAMs for studies of proteinꢀsurface interactions is
widespread in antifouling research. As a result of extensive
research, SAMs exposing oligoethylene glycols11,17ꢀ19 are well
known for their protein-resistant properties. In previous studies,12,13
it was observed that galactose-terminated alkanethiols can be used
to form well-defined, protein-resistant SAMs that also perform well
in laboratory marine biofouling assays. This is of interest for several
Sugars play diverse and complex roles in the chemistry of
nature, for example, in immune response and cell recognition, as
cell-protecting physical barriers, and as modulators of proteinꢀ
protein interactions. The detailed determination of glycoconju-
gate structures remains a challenge because the precise structure
of glycans is not genetically coded, and the combinatorial possi-
bilities in the linkage of two monosaccharides exceeds by far
those of, for example, two amino acids or two nucleic acids,
resulting in considerable structural variety. For a long time,
studies of the structure/function relationship of saccharides
lagged behind those of other (biological) macromolecules be-
cause of their complexity and the lack of suitable characterization
methods. However, as such methods have been successively
developed, progress in the area has been rapid, and the biology of
saccharides is now both a large and quickly growing area of
research, with applications in biotechnology, pharmaceuticals,
and biomedicine.1 This also implies that effective means required
Received: July 19, 2011
Revised:
November 1, 2011
Published: November 04, 2011
r
2011 American Chemical Society
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Figure 1. Synthesized carbohydrate- and hydroxyethyl-terminated alkanethiols for interaction studies using model proteins and marine fouling species.
Scheme 1. General Preparation of Glycosyl-Terminated
water. Because of the steric constraints in accessibility of the sugars
in the monolayer, it is unlikely that the observed differences result
from specific interactions with sugar groups on the surface. Because
cell attachment to surfaces is frequently mediated by cell-surface-
bound lectins specifically interacting with sugars on the substrate,
this could be an interesting route to investigate by increasing the
steric availability of sugar moieties on the surface. There are also a
number of examples of how specific interactions with sugars affect
the behavior of marine fouling organisms. (See, for example, section
6.4 of Railkin.21)
Alkanethiols 2ꢀ5 and 7a
To continue our previous studies,13 we set out to investi-
gate the importance of the molecular structure of various mono-
saccharides and the effects of the size of the saccharide moieties
using di- and trisaccharides. Both interactions with model pro-
teins and with the settling (attaching) stages of two marine
fouling species are studied. Thus, herein we describe the synth-
esis of five carbohydrate-based (glucosyl-, rhamnosyl-, xylosyl-,
maltosyl-, and maltotriosyl-) alkanethiols (2ꢀ5,7, Figure 1) in a
four-step manner, starting from the corresponding, easily avail-
able 2-azidoglycosides. Also, the synthesis of a 6,60-dimethylated
maltosyl derivative is described (6, Figure 1). Derivative 1 was
synthesized according to published procedures.12 In all deriva-
tives, an amide-linked ω-functionalized hexadecane alkanethiol
group has been employed; long alkane chains are frequently
used to increase the order and stability in SAMs. The presented
functionalized alkanethiols were used to form SAMs on gold
surfaces, and the structures of these SAMs were characterized
using ellipsometry, contact angle goniometry, tensiometry, and
IRAS. As a means to improve the order and density of the
monolayers, we also investigate how mixing the saccharides with
a hydroxyethyl-terminated filler molecule 8, with a much smaller
headgroup, affects the structure of the layers. The filler molecule
was synthesized according to published methods.22 Surface
dilution of the saccharides is also an approach to exposing the
saccharide moieties, permitting access to proteins, and making possi-
ble studies of specific interaction with the sugar moieties.23,24 To
assess the resistance of these SAMs to protein fouling, we have
a (i) Boc2O, EtOH, 10% Pd/C. (ii) TFA/CH2Cl2. (iii) 9, DIPEA, DMF,
61ꢀ77%. (iv) K2CO3, MeOH, 73ꢀ97%.
reasons: the hydroxyl-rich SAMs show very good inhibition of
protein adsorption, which is reasonably consistent with previous
studies of sugar-terminated SAMs11,14 but inconsistent with earlier
work suggesting that protein-resistant surfaces must not contain
hydrogen bond donors.20 In a marine biofouling study,13 we
compared monosaccharide-terminated SAMs with mixed mono-
layers of methyl- and hydroxyl-terminated alkanethiols, demon-
strating that the interactions of marine organisms with surfaces
having similar wettabilities and exposing similar functional groups
varied depending on differences in the molecular structure and, in
particular, the resulting differences in the structure of interfacial
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performed experiments where model proteins (bovine serum
albumin (BSA), lysozyme, fibrinogen, and pepsin) were adsorbed
from buffer solutions. Furthermore, initial experiments using the
settling (attaching) stages of two common marine fouling organisms
were performed, viz., zoospores of a green macroalga (Ulva linza)
and cypris larvae of a barnacle (Balanus amphitrite), indicating areas
of future interest for studies using these coatings.
in 61ꢀ77% yield over three steps. Per-acetylated compounds
2bꢀ5b and 7b could all be deprotected under mild basic con-
ditions using K2CO3 in MeOH to give target molecules 2ꢀ5 and
7 in 73ꢀ97% yields. 2-Azidoethyl glycoside 7a was synthesized
from maltotriose and 2-azidoethanol,25 using BF3 OEt2 as a
3
promotor, in 44% yield (Scheme 2).
In the synthesis of target molecule 6, glycoside 5a was first
deprotected under Zemplꢀen conditions, followed by selective
protection of the two primary hydroxyl groups using TBDPSCl,
imidazole, and DMAP in DMF. The remaining hydroxyls were
benzylated with BnBr and with NaH in DMF to give the desired
compound 11 in 83% yield over two steps. Deprotection of the
silyl ethers using TBAF in THF followed by methylation using
MeI and NaH in DMF gave the desired 6,60-O-dimethylated
derivative 12 in 86% yield over two steps. Hydrogenation, using
20% Pd(OH)2/C in EtOH/HOAc (9:1), removed the benzyl
groups and unmasked the amine that was subsequently acylated
using N-hydroxysuccinimide ester 9 and DIPEA in DMF to
achieve compound 13 in 65% yield. The thioacetyl group was
finally readily deprotected under mild basic conditions using
K2CO3 in MeOH, resulting in final thiol 6 in an overall yield of
39% from 2-azidoglycoside (5a).
’ RESULTS AND DISCUSSION
Thiol Synthesis. Alkanethiols 2ꢀ5 and 7 were all synthesized
according to the route outlined in Scheme 1.
Starting from the known 2-azidoethyl glycosides25ꢀ27 (2aꢀ5a),
conversion of the azides to the corresponding amines was per-
formed in a two-step sequence starting with hydrogenation using
10% Pd/C in absolute ethanol with Boc2O present, followed by
deprotection of the Boc group using TFA in CH2Cl2. Having
Boc2O present during the reduction of the azido group hinders
the migration of the acyl group. The amine was subsequently
acylated using N-hydroxysuccinimide ester 928 and DIPEA in
DMF achieving saccharide-terminated derivatives 2bꢀ5b and 7b
Scheme 2. (i) (a) Ac2O, NaOAc, Toluene, Reflux; (b)
Contact Angle and Ellipsometry Measurements. The wett-
ability of a surface reflects properties at the outermost few atomic
layers. All prepared SAMs, except the one containing derivative 6,
showed very hydrophilic behavior with nearly complete wetting
(Table 1). The SAM produced from 6 has a less hydrophilic
surface with advancing and receding contact angles of 44 and 21°,
respectively. This hysteresis indicates a certain degree of surface
inhomogeneity, which is attributed to conformational freedom
due to a low degree of molecular packing, as also confirmed by
IRAS spectra. To discriminate the saccharides’ wettabilities, the
2-Azidoethanol, BF3 OEt2, 4 Å MS, CH2Cl2
3
Scheme 3. (i) NaOMe, MeOH; (ii) TBDPSCl, Imidazole, DMAP, DMF; (iii) BnBr, NaH, DMF, 83%; (iv) TBAF, THF; (v) MeI,
NaH, DMF, 86%; (vi) 20% Pd(OH)2/C, EtOH/HOAc (9:1); (vii) 9, DIPEA, DMF, 65%; (viii) K2CO3, MeOH, 83%
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Table 1. Ellipsometric Thickness d, Advancing (θa) and Receding (θr) Contact angles, Amide II Peak Positions, and Integrated
Areas of the Amide II Band (Relative to That of 8), of the Single-Component Saccharide-Terminated Alkanethiol SAMs on Gold
contact angles by
goniometry (deg)
contact angles by
tensiometry (deg)
ellipsometric thickness
amide II peak position
relative amide II
area
sample
1, galactoside
d (Å)
θa ( 1
θr ( 1
θa ( 1
θr ( 2
(cmꢀ1
)
27.7 ( 0.2
27.9 ( 0.2
29.7 ( 0.2
27.5 ( 0.0
29.3 ( 0.2
26.9 ( 0.2
29.3 ( 0.2
24.6 ( 0.4
<10
<10
<10
<10
<10
44
6
4
0
1553
1553
1564
1554
1552
1550
1547
1563
0.57
0.60
0.85
0.60
0.44
0.37
0.33
1
2, glucoside
0
3, xyloside
0
0
4, rhamnoside
5, maltoside
1
0
1
0
6, dimethylated maltoside
7, maltotrioside
8, hydroxyethyl
21
19
45
19
0
<10
27
17 f 9
28
18
Figure 2. Contact angles for the dimethylated maltoside (6, solid lines)
and maltotrioside (7, dotted lines) as determined by the Wilhelmy
method. Three complete wetting/dewetting cycles are shown for each
sample. The receding contact angle for 7 is zero in all cycles, and because
of hysteresis, the advancing angle is zero for the first 2 mm on the second
and third wetting cycles (i.e., between 2 and 4 mm in the diagram).
Figure 3. RA spectra in the CꢀH stretching region of saccharide-
terminated alkanethiolate SAMs.
contact angles were also analyzed by the Wilhelmy method using
a tensiometer. The results for the quantifiable goniometric
contact angles of 6 and 8 are in good agreement with the
tensiometry data (Table 1), and for the monosaccharides and
5, the tensiometric data do not provide much additional informa-
tion even though figures can be assigned to the contact angles.
For 1ꢀ5, the immersion cycles are featureless, with the wetting
and dewetting legs of the cycles virtually indistinguishable. 6 and
8 have significant hysteresis. However, they are well-behaved
in the sense that the repeatability between cycles is very good.
(See the data for 6 in Figure 2.) This is not the case for
maltotrioside 7, where although the receding contact angles are
zero in every dewetting cycle the advancing contact angle is both
decreasing for every additional cycle and changing irregularly
within each immersion, indicating a saturation effect or a slow
rearrangement of the surface, which is not completely reversible
on the timescale of the measurement. This is evidence of
considerable disorder in the maltotrioside layer.
of ∼27.7 Å was observed for 1, 2, and 4 and 29.7 Å for 3. This
difference is due to both the variations in the conformation and
the tilt angle of the alkane chain and improved packing of the
headgroups as a result of reduced steric hindrance from the
relatively planar configuration of the sugar moiety in 3. Although
the increased thickness of disaccharide SAM 5 compared to that
of the monosaccharides is less than 3 Å, the increment due to the
addition of the extra sugar ring is significant. However, dimethy-
lated maltoside 6 shows nearly no increase in thickness compared
to the SAMs containing monosaccharides. This demonstrate
how sensitive the molecular packing is to small changes in the
molecular structure, where a change in headgroup interactions or
steric hindrance from the two additional methyl groups reduces
the molecular packing, increases the SAM disorder, and reduces
the total thickness. The contact angles on the two SAMs show
that 5 is a completely wettable surface whereas 6 is much less
hydrophilic. It is interesting to compare the wettabilities of 6 and
4, where the complete wetting of 4 suggests that its methyl group,
although being located close to the SAM surface, is not suffi-
ciently exposed to the surrounding medium (i.e., shielded by
The contribution to the thickness from a single saccharide unit
is estimated to approximately 3 Å from comparison with 8,
consisting only of a hydroxyethyl group (Table 1). A thickness
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Amide Bands. The amide bond in the molecules form lateral
hydrogen bonds that stabilize the overall SAM structure.32 In
Figure 4, boththe amide I band (predominantly CdO stretching)
and the amide II band (CꢀN stretching combined with CꢀNꢀH
in-plane bending) can be seen at ∼1645 and ∼1550 cmꢀ1
,
respectively. According to the surface selection rule, the infrared
absorption of a vibrational mode of a molecule on a metal surface
is determined by the projection of itstransition dipole moment on
the surface normal. Because of the interaction of the dipole
moment of the molecule with its image charges, dipole moments
parallel to the surface cancel while dipole moments perpendicular
to the surface are amplified. Therefore, the corresponding in-
tensity of a vibrational mode in the spectrum is strongly depen-
dent on the molecular orientation relative to the surface. Among
the investigated SAMs, 3 displaꢀys1 a considerably strong, sharp
amide II absorption at 1564 cm and the near absence of the
amide I absorption (∼1645 cmꢀ1). This observation indicates
that the orientation of the carbonyl group (amide I) in 3 is
predominantly aligned parallel to the gold surface and that the
CꢀN bond (amide II) is close to parallel to the surface normal.
Consequently, the preferential orientation of the alkane chains in
3 is nearly parallel to the surface normal. Comparing this result
with the amide bands of the other SAMs, we find that 3 is oriented
in a more upright direction, resulting in a thicker layer, in
agreement with the ellipsometric data (shown in Table 1). Beside
the tendency of the amide II peak intensity to decrease in
response to increased disorder, there is also a shift of the peak
position to lower frequencies with larger carbohydrate termini.
This shift has contributions from the increased effective mass of
the nitrogen atom coupled to the bulky end group, but it is
Figure 4. RA spectra in the amide and the fingerprint regions of
saccharide alkanethiolate SAMs.
hydroxyl groups), indicating that 6 has a more loosely packed
structure. Furthermore, comparingthewettabilityof4with that of a
monomethylated galactopyranoside (θa = 52°, θr = 22°)13 demon-
strates that a small change in the carbohydrate structure may result
in considerable differences in the wettability, representing differ-
ences in the molecular packing. The thickness of trisaccharide 7 was
found to be the same as that for disaccharide 5 (and monosacchar-
ide 3), which, in view of the contact angle tensiometry results
discussed above, clearly is the result of disorder in the molecular
packing caused by the bulky carbohydrate moiety, as is also
confirmed by the IRAS data discussed below.
Infrared ReflectionꢀAbsorption Spectroscopy. Infrared
reflectionꢀabsorption spectroscopy was used to obtain specific
information about the alkane chain orientation and the molecular
order of the monolayer on the gold surface. Reflectionꢀabsorption
(RA) spectra in the CꢀH stretching region of the saccharide
alkanethiols on gold are presented in Figure 3.
dominated by the strength of the NH OdC hydrogen
3 3 3
bond.33,34 As the strength of the hydrogen bond increases, the
amide II peak shifts to higher frequencies, and because the in-
plane hydrogen bonding of neighboring amides is an indicator of
SAM order, these peak positions are also of interest. The amide II
peak positions are included in Table 1 for the single-component
SAMs, and it is clear that monosaccharide 3 is distinctly different
from all the other saccharides in that the peak position is at least
10 wavenumbers higher and very similar to that of hydroxyethyl-
terminated 8, providing strong support to the hypothesis that 3 is
more ordered than the other monosaccharides and that this is the
cause of the difference in SAM thickness. Beside 3, the data in
Table 1 shows a clear trend toward decreasing frequency of the
peak position as the size of the saccharide moiety increases, again
in agreement with the observed increase in disorder of the SAMs.
Fingerprint Region. The frequencies between 1200 and
950 cmꢀ1 in the RA spectra provide conformational informa-
tion about CꢀO, CꢀC, CꢀH, and OꢀH vibrational modes of
the saccharides. This results in a multitude of partially over-
lapping bands, rendering complex spectra that are very diffi-
cult to interpret in detail (Figure 4). The absorptions observed
at ∼1050, ∼1080, ∼1100, ∼1150, and ∼1170 cmꢀ1 are assigned
to CꢀOꢀC vibrations of the carbohydrate ring; the intensity
and peak position vary with the saccharide structure.12 Further-
more, the weak absorptions of methylene CꢀH scissoring at
∼1467 cmꢀ1 and methyl CꢀH bending at around 1385 cmꢀ1
can also be discerned in the spectra.
CꢀH Stretching Region. The CꢀH stretching region (between
3000 and 2800 cmꢀ1) provides information about the molecular
packing and crystallinity of the alkyl chains in the monolayer. For a
perfectly ordered all-trans arrangement of alkyl chains, the absorp-
tionswill appear at 2918 (dꢀ) and2850 (d+) cmꢀ1 29,30 In Figure 3,
.
the RA spectra show the asymmetric methylene CꢀH stretching
from the alkyl segment at 2919 cmꢀ1 for 1, 2, and 3, shifted to
2920 cmꢀ1 for 4 and 5 and finally located at 2921 cmꢀ1 for 6 and 7.
The shift to higher frequencies of the peak of this band indicates a
slight increase in the number of gauche defects along the alkyl chain
due to the increasing size and bulkiness of the saccharide-termini,
causing molecular chain packing disorder. For the same reason, the
corresponding frequencies of the symmetric methylene CꢀH
vibration exhibit a shift from 2850 to 2852 cmꢀ1. The increasing
disorder with increasing complexity (size) of the terminal sacchar-
ide groups is also reflected in an overall broadening of the CꢀH
vibrational peaks. In the SAMs of 4 and 6, there are additional
vibrations in this region originating from methyl CꢀH stretching in
the saccharide moieties. They appear at 2984 (rꢀ), 2817 (r+), and
ca. 2900 cmꢀ1 in 6 and at 2978 (rꢀ) cmꢀ1 in 4, depending on the
substituent to which the methyl group is attached.31
Mixed SAMs. To improve the control of the interfacial proper-
ties of the SAMs and also to increase the availability of the sugar
moieties, we dilute the saccharide-functionalized thiols with a
hydroxyethyl-functionalized filler molecule (8). Thus, for the
SAMs where the bulkiness of the headgroup is observed to
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Figure 5. RA spectra of the fingerprint region of mixed xyloside (3)/
hydroxyethyl filler (8) SAMs.
Figure 6. RA spectra of the fingerprint region of mixed rhamnoside
(4)/hydroxyethyl filler (8) SAMs.
reduce the molecular packing in the SAM, mixed monolayers are
expected to improve the alkyl chain order and for some range of
dilutions (depending on the saccharide headgroup) also improve
the molecular packing of the sugars simply by bringing the bulky
saccharide termini further apart. We examine mixtures of the
xyloside (3), rhamnoside (4), and maltotriose (7) molecules
with filler molecule 8 in different molar ratios. It is anticipated
that the saccharide moieties of these molecules pack differently in
the monolayer because of differences in steric hindrance. The
order in the xyloside monolayer (3) is not expected to be greatly
improved by dilution with filler molecules because the xylose ring
is relatively planar. This is in contrast to the rhamnoside (4),
which has a markedly nonplanar character, and the maltotriose
(7) headgroup whose bulkiness is clearly a severe constraint to
the packing in the SAM. The RA spectra of the amide and finger-
print regions of these mixed SAMs are shown in Figures 5ꢀ7. In
the CꢀH stretching region, a shift from 2919 to 2918 cmꢀ1 of
the CꢀH asymmetric stretching band is observed when mixing 3
with 20% or more of the filler molecule (not shown, available as
Supporting Information). As mentioned previously, the position
of this absorption is a signature of an all-trans arrangement of the
alkane chains, which indicates a slight improvement in methylene
packing with >20% 8 in this SAM. The amide II peak positions
for different dilutions are shown in Figure 8a, and it is seen for
mixtures of 3 with 8 that there is an initial decrease in the peak
frequency and a minimum between the single-component com-
positions. Although this is not in complete agreement with the
shift in the asymmetric CꢀH stretching, the effects are relatively
small. For the mixed systems of 4 and 7, filler molecule 8 needs to
be added to at least 50% in order to obtain an all-trans
arrangement of the alkane chain in the mixed system (as deter-
mined from the CꢀH stretching peak positions; see Supporting
Information). Amide II peak shifts with increasing dilution by 8
show a clear, continuous trend toward increasing alkane chain
order as the fraction of 8 is increased in the SAM (Figure 8a).
Changes in the surface coverage of the carbohydrate moiety in
the mixed SAM systems were obtained by area integration over
the frequencies between 1200 and 950 cmꢀ1. From Figure 8b, it
Figure 7. RA spectra of the fingerprint region of mixed maltotrioside
(7)/hydroxyethyl filler (8) SAMs.
can be inferred that to reduce the surface coverage of saccharides
to half the original value the addition of between 70 and 80% filler
molecule to the mixtures must be made, although it should be
mentioned that the orientation of the terminating carbohydrate
moiety also affects the calculated intensities so that the change in
orientation of the saccharide with surface coverage introduces an
additional uncertainty into these numbers. It is also notable that
the integrated intensity decreases continuously for 3 as 8 is
added, showing that as the SAM is diluted with filler molecule 8
so also are the saccharide moieties, as would be a consequence of
dilution of an already well-packed layer. This is not the case for 4
and 7, where the surface coverage of sugar groups is relatively
constant up to filler fractions of approximately 50%, which means
that up to a 50% mixture the filler appears to fill up the alkane part
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Figure 9. Ellipsometric thicknesses of the adsorbed protein layers. In
each case, the protein solutions were 1 mg/mL protein in PBS. Error
bars show the standard deviation.
drawn from the integrated areas in the fingerprint region. At filler
fractions of up to 70%, the order of the alkane chain layer is
improved as available space is filled beneath the sterically
constrained layer of saccharide moieties. At higher filler fractions,
we start diluting the saccharide moieties, and further increases in
the filler fraction do not improve the packing or the order in the
alkane chain layer.
Protein Adsorption Results. The proteins used in the protein
resistance tests were chosen to cover a range of sizes from 14 to
340 kDa for lysozyme and fibrinogen, respectively, globular (e.g.,
BSA) versus nonglobular (fibrinogen) proteins, and surface
charge properties from the pI 2.9 of pepsin to lysozymes’ pI
11. Albumin and fibrinogen are among the most prevalent
plasma proteins, and the latter is widely studied because of its
role in coagulation and platelet adhesion.35 The results of the
protein adsorption tests are shown in Figure 9. There was very
little adsorption of bovine serum albumin (BSA), lysozyme, or
pepsin to any of the surfaces. Fibrinogen behaved differently,
adsorbing to several of the SAMs in significant amounts. How-
ever, there were clear differences between the surfaces in this
respect. A very dramatic trend in decreasing fibrinogen adsorp-
tion can be seen for the monosaccharide series 1 > 2 > 3 > 4.
However, we do not see any correlation between the variation in
fibrinogen adsorption with any of the physicochemical para-
meters that have been determined for the surfaces, be it thick-
ness, wettability, wetting hysteresis, or the molecular order in the
alkyl chain or headgroup regions. It is also notable that the other
tested proteins were unaffected by these variations. The fact that
fibrinogen behaves so differently from the other proteins is not
surprising; it is well known that fibrinogen has a higher propen-
sity to adsorb to surfaces in greater quantities and to adhere more
strongly than most other proteins.36,37 Although we cannot
exclude that specific interactions between fibrinogen and these
monosaccharides are responsible for the high adsorption, this is
very unlikely because of the limited steric availability of the sugar
moieties in the close-packed monolayer. In any case, rhamnose-
terminated 4 appears to be very protein-resistant under these con-
ditions, which is also true for the tested disaccharides (5 and 6)
and the trisaccharide (7). These SAMs have in common that they
are more disordered than surfaces 1ꢀ3 and 8, where fibrinogen
adsorption is non-negligible. The result for 8 is in agreement with
what is known about fibrinogen adsorption onto OEG-terminated
SAMs; terminating EG1 and EG2 units are too short to provide
protein-resistant surfaces, whereas this is achieved with SAMs
having EG3 or longer termination.38,39 For the monosaccharides,
the situation is more difficult; we have demonstrated in previous
Figure 8. (a) Amide II peak positions, (b) saccharide surface coverage,
as inferred from area integration of the IRAS spectra in the fingerprint
region (1200ꢀ950 cmꢀ1), and (c) ellipsometric thicknesses for the
mixed monolayers where the rhamnoside (3, O), xyloside (4, ꢁ), and
maltotrioside (7, b) have been mixed in various ratios with the
hydroxyethyl (EG1)-terminated filler molecule (8). For clarity, lines
connect data points for each sample.
of the monolayer and that the surface coverage of saccharides is
still limited by packing constraints in the saccharide layer over
this concentration range. This is supported by the ellipsometric
thicknesses of the mixed SAMs, shown in Figure 8c. For 3, there
is a decrease in thickness as filler molecules dilute the layer. For 4,
the thickness is relatively constant up to about 70% dilution, but
for 7, there is a significant increase in the total thickness as the
fraction of the filler molecule is increasing, with a maximum
thickness of 33.1 Å at 70% 8 content compared to 29.3 for the
single-component 7 layer. The thickness variation is more
pronounced for 7 than for 4 because with the former it can be
expected that the initial degree of disorder in the alkane chain
layer is significantly higher. The change in amide band intensities
is also of interest for the characterization of the change in
structure upon dilution with the filler molecule. The spectrum
for the single-component 8 SAM (Figure 5) has a strong amide II
intensity and a barely visible contribution from the amide I band,
demonstrating that the alkane chains have a nearly upright
conformation and that the amide carbonyl is oriented parallel
to the gold substrate. The difference between the amide band
structures of 3 and 8 is small, confirming our expectation that the
xyloside functionalities do not severely impede the packing of the
alkane chains in the monolayer. However, in looking at the RA
spectra for the mixture of 8 with either 4 or 7 (Figures 6 and 7),
we find that the situation is different; the amide II intensity for
pure 4 SAMs is weak and that for pure 7 SAMs is even weaker,
but in both cases, the intensity approaches that of the pure filler
SAM at about 70%, in good agreement with the conclusions
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Figure 10. Results of the Ulva assay for the seven saccharide SAMs and
a Nexterion glass slide used as an internal laboratory control. N = 90,
error bars = (2 ꢁ standard error.
Figure 11. Barnacle assay results for the seven SAMs and for a
polystyrene surface used as an internal laboratory standard to verify
the viability of the cypris larvae and that the settlement behavior was
normal. The results are shown as the percentage of settled cyprids, with
95% confidence intervals.
studies that monosaccharide SAMs may show very good
protein12 and marine biofouling13 resistance. However, these
studies also show that small variations in the structure of the
sugar moieties may be decisive for the antifouling behavior. A
related observation was published recently, where protein ad-
sorption onto chiral polyol SAMs was studied, and it was found
that adsorption onto either of two enantiomers was higher than
adsorpion to the racemic mixture; this difference was speculated
to be caused by variations in water solvation at the interfaces.16
We do not currently have detailed-enough information about
the monosaccharide SAMs to make any claims about the cause of
these differences, so we have to leave this to future work.
In comparing 2, 5, and 7, which consist of one, two, and three
glucose units, respectively, we at least note that there is a clear
trend toward increasing resistance to fibrinogen adsorption with
a greater number of glucose units in the chain. The main effect
that this variation has on the monolayers is a decrease in order in
the SAMs whereas the total layer thickness changes only margin-
ally. Despite the differences in the size of the headgroup, the
change upon going from monosaccharide 2 to disaccharide 5 is
only 1.4 Å but di- and trisaccharides 5 and 7, respectively, have
the same thickness, and it appears that the increased disorder of
the layers could be of relevance. An early study on the protein
resistance of OEG-terminated alkylthiols diluted with methyl-
terminated thiols concluded that protein resistance was pre-
served at higher fractions of methyl-terminated thiols if the OEG
chains were longer, thus suggesting that the surface coverage of
the EG units is critical.40 It appears that a similar relation between
coverage and resistance holds for the set 2, 5, and 7. In either case,
we suggest that oligosaccharide-terminated SAMs will be inter-
esting for further investigations of antifouling properties.
Marine Fouling Assays. To test the resistance of these SAMs
to marine biofouling, we performed experiments with two model
organisms: the zoospores of the alga Ulva linza and cypris larvae
of the barnacle Balanus amphitrite. In both assays, the number of
spores or larvae attached to the test surfaces was quantified. The
adults of these two species are common macrofoulers in marine
environments.
surfaces until the water contact angle was >50°. Similar results
were also found in relation to the increasing wettability of
hexa(ethylene glycol) SAMs with different alkoxyl end-group
terminations.44
Barnacle Assays. All SAM surfaces resulted in very small, or
negligible, settlement of barnacle cyprids (Figure 11). That this is
not a result of defective larvae is demonstrated by the included
result for an internal laboratory polystyrene (PS) standard, evaluated
in parallel with the SAMs. As well as settlement, a note was made
of the numbers of “dead” cyprids and the percentage mortality
was calculated; no abnormal behavior was noted.
The variation in cyprid settlement among samples 1ꢀ7 is
small, and statistical analysis does not show any significant dif-
ferences in the group (p < 0.05). This is also in agreement with the
previous observation that saccharide SAMs with widely varying
properties (e.g., water contact angles ranging from <10 to 76°)
were equally unappealing to cypris larvae.13 It is tempting to
speculate that there is some optical property of the gold substrate
that universally deters cyprids from the surfaces, though because
the settlement assays are performed in the dark it is difficult to see
what this could be. An analysis of the exploratory behavior of
cypris larvae on the saccharide surface may reveal differences in
the pattern of searching as shown for two settlement-inhibiting
zwitterionic surfaces.45
’ CONCLUSIONS
An efficient strategy for the synthesis of diverse glycosyl-
terminated alkanethiols from often readily available 2-azidogly-
coside in a four-step manner has been explored. The outlined
route shows promise in multigram-scale syntheses because of the
relatively low complexity in the used reaction methods. We have
also established an effective route to derive 6,60-O-dimethylated
maltosyl analogue 6.
Ulva Assays. The density of spores attached to Nexterion glass
and seven oligosaccharide SAM surfaces is shown in Figure 10.
Nested one-way ANOVA showed no significant difference be-
tween the oligosaccharide surfaces (p = 0.05). Further statistical
analysis showed that the density of spores on all of the oligosac-
charide surfaces was significantly lower than on the glass, demon-
strating that spores did not settle indiscriminately (Dunnett’s
posthoc test41). The data concurs with previous data for hydro-
philic surfaces, including mixtures of (OH/CH3)-terminated
SAMs42 and xerogels43 where spore settlement was low on all
The structure of SAMs formed on gold substrates from the
produced thiols was investigated, and it was demonstrated that the
SAM structure, particularly evaluated as order in the alkyl chain
layer, was dependent on the conformation of the monosaccharide
moieties. The relatively planar structure of the xyloside (3)
permits close packing and results in a well-ordered layer struc-
ture, and the markedly nonplanar rhamnoside (4) constrains the
packing and results in a less-ordered SAM. As the headgroups
become more bulky, as in the case of di- and trisaccharides, the
structural qualities are further reduced, resulting in increasing
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disorder in both the headgroup and alkyl chain regions as
compared to that in the monosaccharide SAMs.
wells were filled with the thiol solutions immediately after removal from
the evaporator.
Self-Assembled Monolayer Preparation. All SAMs were pre-
pared from solutions with a total thiol concentration of 50 μM in ethanol
(99.5%, Kemetyl, Sweden). Solutions for mixed SAMs were prepared by
volumetrically mixing 50 μM solutions of the individual components to
the desired molar ratio. (All molar ratios refer to solution concentra-
tions. Note that the molar ratio in the mixed SAM does not necessarily
agree with the solution ratio.) All solutions were stored at room
temperature in polypropylene jars (Nalgene) that had been previously
rinsed and ultrasonicated for 15 min in hexane (Merck) and 15 min in
ethanol. Prior to SAM adsorption, gold substrates were TL1 cleaned
twice, rinsed with water, and dried with nitrogen. Optical characteristics
of the gold substrates were obtained by ellipsometry immediately after
cleaning. The substrates were incubated for at least 20 h in the thiol
solution (exactly 24 h for mixed SAMs). They were then removed from
the thiol solutions, rinsed with 99.5% ethanol, ultrasonicated for 5 min in
ethanol in order to remove any physisorbed thiols, dried with nitrogen,
and immediately analyzed using ellipsometry and/or contact angle
measurements and infrared spectroscopy.
Saccharide-terminated SAMs have previously shown promis-
ing antifouling properties in both protein adsorption studies and
in marine biofouling laboratory assays, and we thus subjected the
SAMs described in this article to similar tests to evaluate their
antifouling properties. We note that the SAMs are overall very
resistant to the tested proteins, with the exception of fibrinogen.
Fibrinogen adsorption varies considerably for the four tested
monosaccharides in a way that we are not able to correlate with
any of the studied physicochemical properties of the SAMs.
Including also the di- and trisaccharide SAMs in a comparison of
fibrinogen adsorption, we note that there is a trend for less-
ordered monolayers to be more resistant to fibrinogen adsorp-
tion. The attachment of both algal zoospores and barnacle cypris
larvae was low on all of the tested SAMs. Attachment was
significantly lower on the SAMs than on the laboratory standards
(glass and polystyrene), but differences among the SAMs were
not statistically significant.
Thiols with bulky sugar moieties produce disordered mono-
layers. For these, the packing of the alkyl chain layer can be
improved by dilution with a filler molecule having a small
headgroup, whereas the total number of saccharide moieties on
the surface is kept relatively unchanged for surface dilution up to
approximately 50%. We also note that further dilution of the
saccharides on the surface is a useful strategy for improving the
accessibility and exposure of the saccharide headgroups to the
environment and thus also for optimizing specific interactions of
proteins or cells with surface-bound sugars. The good resistance
to the nonspecific fouling of our SAMs supports the viability of
this approach and also suggests that further work with oligosac-
charide SAMs could be of considerable interest in understanding
fouling resistance and the development of antifouling coatings.
Ellipsometry. Ellipsometry was performed using an automatic
Rudolph Research AutoEL ellipsometer (using a HeꢀNe laser, λ =
632.8 nm, set at a 70° angle of incidence). The measurements were
carried out using an automatic program that measured five different
points on each sample and used the average. The necessary optical
constants were obtained from the measurements of the gold substrates
immediately after cleaning, as described above. A three-phase air/
organic film/gold model was used to calculate the thickness of the
SAMs, assuming an isotropic, transparent organic layer. The refractive
index of the organic film (SAM as well as protein) was assumed to be 1.5.
Contact Angle Goniometry. A semiautomatic optical contact
angle meter (KSV CAM 200) was used to determine the advancing and
receding contact angles of water on the SAMs. A manual dispenser was
used to expand or retract a droplet on the sample. As the droplet was
expanded, images were acquired for the evaluation of advancing contact
angles; similarly, receding contact angles were determined from images
acquired as water was withdrawn from the droplet into the syringe.
During the measurements, the syringe needle was kept inside the center
of the drop, and a high-speed CCD camera was used for image capture.
The images were analyzed using the software supplied with the
instrument.
Contact Angle Tensiometry. For more accurate measurements
of these very hydrophilic SAMs, contact angles of single-component
SAMs were determined using a Wilhelmy balance (Sigma 70, KSV). The
glass coverslips were suspended from the balance, and the movement of
the water reservoir was set to a speed of 5 mm/min and a 10 mm
maximum immersion depth. Three cycles were carried out for each
sample, cycling only the lowest 8 mm of each sample, and shown values
of the contact angles are the averages of the three cycles, ignoring the
values obtained over the first 2 mm after each turning point. The
instrument control program calculates the wetting force and the contact
angle during the instrument operation, using the known dimensions and
density (2.29 g/cm3) of the glass coverslips (including the thickness in
the periphery) and a measured value of the water surface tension. This
was determined immediately before the contact angle measurement
using a platinum Wilhelmy plate (19.6 mm wide and 0.1 mm thick). Ten
immersion cycles were used for each surface tension data point;
measured values always exceeded 72.2 mN/m.
’ EXPERIMENTAL SECTION
Gold Substrate Preparation. Silicon (100) wafers with native
oxide were cut into pieces (10 ꢁ 19 mm2 for protein adsorption
experiments or 20 ꢁ 40 mm2 for IRAS); 12.5 ꢁ 20 ꢁ 0.15 mm3 glass
coverslips were used for Wilhelmy measurements. All substrates were
TL1 cleaned (immersed in a 5:1:1 mixture of water, 30% H2O2 (Merck),
and 25% NH3 (Merck) for 10 min at 85 °C). After being cleaned, they
were carefully rinsed with water (all water was Milli-Q water
(Millipore)) and moved to a laminar flow cabinet, dried with nitrogen
gas, and mounted in a UHV electron-beam evaporation system (Balzers
UMS 500 P) operating at a base pressure of 10ꢀ9 mbar and an
evaporation pressure of about 10ꢀ7 mbar. A 25 Å titanium adhesion
layer (Balzers, Liechtenstein, 99.9%) was deposited at a rate of 2 Å/s,
followed by a 2000 Å gold layer (Nordic High Vacuum AB, Sweden,
99.9%) at 10 Å/s. Glass coverslips (12.5 ꢁ 20 mm2) for Wilhelmy plate
measurements of contact angles were coated on both sides.
Samples for Ulva assays were prepared on cleanroom-cleaned 76 ꢁ
25 mm2 Nexterion Glass B slides (Schott) and treated like other
samples, except for the metal coating that was carried out in a resistively
heated vacuum evaporation system with a base pressure of <4 ꢁ 10ꢀ6
mbar using a 25 Å chromium adhesion layer (at 0.4ꢀ0.6 Å/s) and then
2000 Å of gold (at 5ꢀ10 Å/s). Samples for barnacle assays were
prepared in 24-well cell culture plates, the wells of which were coated
on the inside by mounting them at a variable angle on a rotating sample
holder, and then evaporated in the resistively heated system under
the conditions described above while gradually varying the angle of the
plate so as to cover both the side and the bottom of the wells. The plates
were sterile when mounted, and to avoid the TL1 cleaning steps, the
Infrared ReflectionꢀAbsorption Spectroscopy. Reflectionꢀ
absorption (RA) spectra were recorded on a Bruker IFS 66 system
equipped with a grazing angle (85°) infrared reflection accessory and a
liquid-nitrogen-cooled MCT detector. The sample chamber was con-
tinuously purged with nitrogen gas during the measurement. All spectra
were acquired at 2 cmꢀ1 resolution between 4000 and 700 cmꢀ1 as a
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summation of 3000 scans. A three-term BlackmannꢀHarris apodization
was applied to the interferograms before Fourier transformation. The
background spectrum (R0) was taken using a deuterated hexadeca-
nethiolate [HS(CD2)15CD3] SAM on gold. This ensures that the
surface is free of hydrocarbons, which otherwise tend to contaminate
clean gold surfaces quickly and interfere with the studied CꢀH
stretching bands. The sample spectrum (R) was acquired under identical
equipment conditions and compared to the background spectrum to
obtain ꢀlog(R/R0) versus reciprocal centimeters. Prior to the IRAS
measurement, the monolayer sample was removed from the incubation
solution, thoroughly rinsed, ultrasonicated, rinsed again with ethanol,
and finally dried in flowing nitrogen gas.
Protein Adsorption Experiments. Phosphate-buffered saline
(PBS; 10 mM sodium hydrogen phosphate Na2HPO4, 10 mM potas-
sium dihydrogen phosphate KH2PO4, and 150 mM NaCl in water) was
adjusted with NaOH to pH 7.4. The protein solutions were prepared by
dissolving 1 mg/mL of the respective protein in PBS. Shortly before
incubation in protein solutions, the SAMs were rinsed with water and
dried with nitrogen, and the SAM thickness was determined by
ellipsometry. After this measurement, the samples were incubated in
7 mL of the protein solutions at room temperature. After 30 min,
samples were removed from the protein solutions, placed in a beaker
with PBS for 10 min, rinsed with water, and dried with nitrogen. New
ellipsometry measurements were then performed, following the pre-
viously described procedure, and the thickness increments (Δ thickness)
were calculated.
Ulva Assay. Reproductive thalli of green macroalga Ulva linza were
collected from Llantwit Major beach, Glamorgan, Wales (52° 230 N, 3°
300 W). Zoospores were released into artificial seawater (ASW) at pH
8.0 and 32% (“Tropic Marin”, Aquarientechnik GmbH) and prepared
for assay as described previously.46 SAMs were stored and shipped under
nitrogen, assayed within 24 h of receipt, and in contact with air for less
than 5 min before the assay. Settlement assays followed the principles
outlined in Callow et al.46 and Finlay et al.47 In brief, each surface was
placed in a separate compartment of a Quadriperm dish (Greiner Bio-
one Ltd.) to which 10 mL of a suspension containing 1.5 ꢁ 106 mLꢀ1
zoospores was added. Zoospores were allowed to settle for 1 h in the
dark before the residual suspension was aspirated and the slides were
gently washed. The motility of the aspirated zoospores was assessed by
virtue of their negative phototaxis; viable spores retain their motility.
Slides were then fixed in 2.5% (v/v) glutaraldehyde, washed, and air-
dried. The density of settled (adhered) spores was determined using an
epifluorescence microscope with an image analysis system (Zeiss
Kontron 3000).48 Thirty fields of view were counted at 1 mm intervals
along the length of each of three replicates slides.
Barnacle Assay. The 24-well plates were rinsed gently with artificial
seawater (ASW) to remove any remaining solvents and then placed into
ASW for 1 h before the assay was started. Briefly, 10 3-day-old cyprids
were introduced into each well contained within 2 mL of ASW. The
plates were incubated at 28 °C for 24 h. After this period, each plate was
inspected to obtain the percentage settlement. After another period of
24 h, the plates were again inspected to determine the settlement after 48
h. A blank 24-well plate (polystyrene) was included in the assay as an
internal laboratory standard to gauge the settlement behavior of the
cyprids and to act as a control. The results are expressed as the
percentage of the settlement, with 95% confidence intervals. The data
were analyzed statistically using the KruskalꢀWallis test with post hoc
comparisons of treatment means made with the Dunn’s multiple
comparison test.
[EtOH (95%, v/v)/H2SO4/p-anisaldehyde/acetic acid 90:3:2:1] fol-
lowed by heating to ∼250 °C. Flash chromatography (FC) was
performed with silica gel (Sigma-Aldrich, 0.040ꢀ0.063 mm). NMR
spectra were recorded on a Varian Mercury 300 (1H 300 MHz and 13
C
75.4 MHz) instrument at 25 °C in CDCl3, MeOH-d4, or DMSO-d6.
Chemical shifts are given in ppm relative to solvents peaks49 in CDCl3
1
(δ 7.26 for 1H and δ 77.16 for 13C), MeOH-d4 (δ 3.31 for H and δ
49.00 for 13C), and DMSO-d6 (δ 2.50 for 1H and δ 39.52 for 13C). FT-
IR spectra were recorded as KBr pellets (solids) on a Perkin-Elmer
Spectrum 1000 FT-IR spectrometer. Optical rotations were recorded at
room temperature with a Perkin-Elmer 41 polarimeter. MALDI-TOF
mass spectra were recorded with a Voyager-DE STR Biospectrometry
workstation, in positive mode, using a α-cyano-4-hydroxycinnamic acid
matrix. High-resolution mass spectra were recorded by iNovacia AB,
Stockholm. Filler molecule 8 was synthesized as described previously.22
(S-Acetyl)-N-(16-mercapto-palmitoyl)-2-aminoethyl-2,3,
4-tri-O-acetyl-α-L-rhamnopyranoside (4b). A mixture of per-
acetylated 2-azidoethyl-α-rhamnoside (4a) (0.275 g, 0.765 mmol) and
Boc2O (0.250 g, 1.148 mmol) was dissolved in absolute EtOH (3 mL)
and hydrogenated for 22 h using palladium on carbon (50 mg, 10%)
under vigorously stirring and H2 (1 atm). The suspension was filtered
and concentrated. The obtained syrup was treated with TFA (1 mL) in
dry CH2Cl2 (5 mL) for 4 h at ambient temperature, quenched with
MeOH (1 mL), and coevaporated with toluene. Crude amino sugar, 9
(0.425 g, 0.995 mmol) and DIPEA (0.150 mL, 0.842 mmol) were
stirred in dry DMF (10 mL) for 6 h and coevaporated with toluene. FC
(toluene/EtOAc 1:1) gave 4b (0.325 g, 0.503 mmol, 66%) as a white
solid. Rf = 0.36 (toluene/EtOAc 2:1). [α]D = ꢀ30 (c 1.0, CHCl3). IR
νmax cmꢀ1: 3315, 2921, 2849, 1748, 1695, 1647, 1558, 1468, 1373,
1247, 1224. 1H NMR (300 MHz, CDCl3): δ 1.21 (d, 3 H, J = 6.3 Hz),
1.23ꢀ1.35 (m, 22 H), 1.50ꢀ1.57 (m, 2 H), 1.59ꢀ1.64 (m, 2 H), 1.98
(s, 3 H), 2.03 (s, 3 H), 2.14 (s, 3 H), 2.19 (t, 2 H, J = 7.7 Hz), 2.30 (s, 3
H), 2.85 (t, 2 H, J = 7.3 Hz), 3.35ꢀ3.43 (m, 1 H), 3.45ꢀ3.51 (m, 2 H),
3.72ꢀ3.78 (m, 1 H), 3.82 (dq, 1 H, J = 6.3, 9.6 Hz), 4.72 (d, 1 H, J = 1.1
Hz), 5.05 (dd, 1 H, J = 9.6, 9.6 Hz), 5.28ꢀ5.23 (m, 2 H), 5.86 (bs, 1 H).
13C NMR (75.4 MHz, CDCl3): δ 17.5, 20.8, 20.9, 21.0, 25.9, 28.9, 29.2,
29.3, 29.5ꢀ29.7 (several peaks), 30.7, 36.9, 39.0, 66.7, 67.3, 69.2, 69.8,
71.1, 97.7, 170.0, 170.2, 170.3, 173.4, 196.1. HRMS: [M + H]+ calcd for
C32H55NO10S, 646.3619; found, 646.3632.
N-(16-Mercapto-palmitoyl)-2-aminoethyl-α-L-rhamno-
pyranoside (4). To a solution of 4b (0.112 g, 0.173 mmol) in
MeOH (7 mL) was added K2CO3 (3 mg, 0.022 mmol). After 4 h, the
mixture was neutralized with DOWEX-H+, filtered, and concen-
trated. FC (CHCl3/MeOH 9:1) gave 4 (67 mg, 0.140 mmol, 81%) as
a white solid. Rf = 0.25 (CHCl3/MeOH 9:1). [α]D = ꢀ36 (c 1.0,
1
MeOH). IR νmax cmꢀ1: 3311, 2920, 2849, 1635, 1561, 1462. H
NMR (300 MHz, DMSO-d6): δ 1.12 (d, 3 H, J = 6.3 Hz), 1.23ꢀ1.32
(m, 22 H), 1.44ꢀ1.57 (m, 4 H), 2.04 (t, 2 H, J = 7.3 Hz), 2.46 (q, 2 H,
J = 7.4 Hz), 3.14ꢀ3.24 (m, 4 H), 3.33ꢀ3.55 (m, 4 H), 4.53 (d, 1 H,
J = 0 Hz), 7.78 (bs, 1 H). 13C NMR (75.4 MHz, DMSO-d6): δ 17.8,
23.7, 25.2, 27.7, 28.4, 28.6, 28.7, 28.9ꢀ30.0 (several peaks), 33.3,
35.3, 38.3, 65.2, 68.4, 70.4, 70.6, 71.9, 99.9, 172.1. HRMS: [M + H]+
calcd for C24H47NO6S, 478.3197; found, 478.3202.
(S-Acetyl)-N-(16-mercapto-palmitoyl)-2-aminoethyl-2,3,
4-tri-O-acetyl-β-D-xylopyranoside (3b). A mixture of 2-azi-
doethyl-2,3,4-tri-O-acetyl-β-D-xylopyranoside (3a) (0.200 g, 0.579 mmol)
and Boc2O (0.218 g, 0.868 mmol) was dissolved in absolute EtOH
(2 mL) and hydrogenated for 21 h using palladium on carbon (50 mg,
10%) under vigorous stirring and H2 (1 atm). The suspension was
filtered and concentrated, and the obtained syrup was treated with
TFA (1 mL) in dry CH2Cl2 (5 mL) for 4 h at ambient temperature,
quenched with MeOH (1 mL), and coevaporated with toluene.
Crude amino sugar, 9 (0.322 g, 0.753 mmol) and DIPEA (0.111 mL,
0.637 mmol) were stirred in dry DMF (5 mL) for 5 h and coevaporated
General Synthesis Methods. Organic phases were dried over
MgSO4 (s), filtered, and concentrated in vacuo at 40 °C. Thin layer
chromatography (TLC) was performed on silica gel plates (Merck F254
plates) with detection by charring with AMC [ammonium molybdate
10 g, cerium(IV) sulfate 2 g, dissolved in 10% H2SO4 (200 mL)] or PAA
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with toluene. FC (toluene/EtOAc 1:1) gave 3b (0.281 g, 0.445 mmol,
77%) as a white solid. Rf = 0.22 (toluene/EtOAc 1:1). [α]D = ꢀ31 (c 1.0,
CHCl3). IR νmax cmꢀ1: 3358, 2917, 2849, 1750, 1686, 1651, 1538, 1472,
1377, 1254, 1234. 1H NMR(300 MHz, CDCl3): δ 1.23ꢀ1.49 (m, 22 H),
1.51ꢀ1.61 (m, 4 H), 2.01 (s, 3 H), 2.02 (s, 3 H), 2.03 (s, 3 H), 2.13 (t, 2
H, J = 7.6 Hz), 2.29 (s, 3 H), 2.84 (t, 2 H, J = 7.3 Hz), 3.33 (dd, 1 H, J =
9.1, 11.5 Hz), 3.39ꢀ3.44 (m, 2 H), 3.60ꢀ3.68 (m, 1 H), 3.75ꢀ3.82 (m, 1
H), 4.08 (dd, 1 H, J = 5.2, 11.7 Hz), 4.45 (d, 1 H, J = 7.1 Hz), 4.89 (dd, 1
H, J = 7.1, 9.1 Hz), 4.95 (ddd, 1 H, J = 5.2, 5.2, 9.1 Hz), 5.16 (dd, 1 H, J =
9.1Hz), 5.85 (bs, 1 H). 13C NMR(75.4 MHz, CDCl3): δ 20.8, 20.8, 20.8,
25.8, 28.9, 29.2, 29.3, 29.4ꢀ29.7 (several peaks), 30.7, 36.9, 39.2, 62.5,
69.0, 71.3, 71.7, 101.2, 169.6, 169.9, 170.1, 173.3, 196.1. HRMS: [M +
Na]+ calcd for C31H53NO10S, 654.3282; found, 654.3284.
N-(16-Mercapto-palmitoyl)-2-aminoethyl-β-D-xylopyra-
noside (3). To a solution of 3b (0.159 g, 0.251 mmol) in MeOH
(7 mL) was added K2CO3 (17 mg, 0.126 mmol). After 4 h, the mixture
was neutralized with DOWEX-H+, filtered, and concentrated. FC
(CHCl3/MeOH 4:1) gave 3 (0.113 g, 0.243 mmol, 97%) as a white
solid. Rf = 0.54 (EtOAc/MeOH 4:1). [α]D = ꢀ16 (c 1.0, MeOH). IR
νmax cmꢀ1: 3327, 2919, 2848, 1652, 1631, 1560, 1539, 1472, 1465. 1H
NMR (300 MHz, DMSO-d6): δ 1.17ꢀ1.35 (m, 22 H), 1.44ꢀ1.57 (m,
4 H), 2.04 (t, 2 H, J = 7.4 Hz), 2.44 (t, 2 H, J = 7.3 Hz), 2.93ꢀ3.13 (m,
4 H), 3.13ꢀ3.30 (m, 3 H), 3.39ꢀ3.46 (m, 1 H), 3.63ꢀ3.71 (m, 2 H),
4.09 (d, 1 H, J = 7.4 Hz), 7.73 (bs, 1 H). 13C NMR (75.4 MHz, DMSO-
d6): δ 23.7, 25.2, 27.7, 28.5, 28.6, 28.8, 28.9ꢀ29.0 (several peaks),
33.4, 35.3, 38.5, 65.7, 67.8, 69.5, 73.2, 76.4, 103.8, 172.2. HRMS: [M +
H]+ calcd for C23H45NO6S, 464.3040; found, 464.3040.
(S-Acetyl)-N-(16-mercapto-palmitoyl)-2-aminoethyl-2,3,4,
6-tetra-O-acetyl-β-D-glucopyranoside (2b). Peracetylated 2-azi-
doethyl-β-glucoside (2a) (0.200 g, 0.479 mmol) and Boc2O (0.157 g,
0.719 mmol) were dissolved in absolute EtOH (3 mL) and hydrogenated
for 25 h using palladium on carbon (50 mg, 10%) under vigorous
stirring and H2 (1 atm). The suspension was filtered and concentrated.
The obtained syrup was treated with TFA (1 mL) in dry CH2Cl2 (5 mL)
for 2 h at ambient temperature, quenched with MeOH (1 mL), and
coevaporated with toluene. Crude amino sugar, 9 (0.266 g, 0.623 mmol)
and DIPEA (0.091 mL, 0.527 mmol) were stirred in dry DMF (5 mL) for
20 h and coevaporated with toluene. FC (toluene/EtOAc 2:1 f EtOAc)
gave 2b (0.235 g, 0.334 mmol, 70%) as a white solid. Rf = 0.27 (toluene/
EtOAc 2:1). [α]D = ꢀ5 (c 1.0, CHCl3). IR νmax cmꢀ1: 3343, 2917, 2849,
1747, 1686, 1639, 1540, 1465, 1370, 1233. 1H NMR (300 MHz, CDCl3):
δ 1.21ꢀ1.36 (m, 22 H), 1.53ꢀ1.63 (m, 4 H), 2.00 (s, 3 H), 2.02 (s, 3 H),
2.04 (s, 3 H), 2.08 (s, 3 H), 2.15 (t, 2 H, J = 7.6 Hz), 2.31 (s, 3 H), 2.85 (t,
2 H, J = 7.3 Hz), 3.40ꢀ3.47 (m, 2 H), 3.66ꢀ3.73 (m, 2 H), 3.80ꢀ3.86
(m, 1 H), 4.14 (dd, 1 H, J = 2.1, 12.4 Hz), 4.25 (dd, 1 H, J = 4.9, 12.4 Hz),
4.50 (d, 1 H, J = 8.0 Hz), 4.97 (dd, 1 H, J = 8.0, 9.3 Hz), 5.06 (dd, 1 H, J =
9.3, 9.6 Hz), 5.20 (dd, 1 H, J = 9.3, 9.6 Hz), 5.9 (bs, 1 H). 13C NMR (75.4
MHz, CDCl3): δ 20.7 (2 C), 20.8 (2 C), 25.8, 28.9, 29.2, 29.2, 29.5ꢀ29.7
(several peaks), 30.7, 36.8, 39.2, 61.9, 68.3, 69.5, 71.4, 72.0, 72.7, 101.0,
169.5 (2 C), 170.3, 170.7, 173.3, 196.2. HRMS: [M + H]+ calcd for
C34H57NO12S, 704.3674; found, 704.3703.
N-(16-Mercapto-palmitoyl)-2-aminoethyl-β-D-glucopyr-
anoside (2). To a solution of 2b (0.066 g, 0.094 mmol) in MeOH
(3 mL) was added K2CO3 (8 mg, 0.059 mmol). After 2 h, the mixture
was neutralized with DOWEX-H+, filtered, and concentrated. FC
(CHCl3/MeOH 9:1) gave 2 (39 mg, 0.079 mmol, 84%) as a white
solid. Rf = 0.40 (CHCl3/MeOH 9:1). [α]D = ꢀ7 (c 1.0, MeOH). IR
νmax cmꢀ1: 3311, 2916, 2849, 1639, 1555, 1472. 1H NMR (300 MHz,
MeOD-d4), δ 1.30ꢀ1.42 (m, 22 H), 1.54ꢀ1.62 (m, 4 H), 2.20 (t, 2 H,
J = 7.4 Hz), 2.50 (t, 2 H, J = 7.14 Hz), 3.17ꢀ3.22 (m, 1 H), 3.27ꢀ3.32
(m, 3 H), 3.33ꢀ3.39 (m, 1 H), 3.43ꢀ3.51 (m, 1 H), 3.62ꢀ3.66 (m, 1
H), 3.67ꢀ3.69 (m, 1 H), 3.85ꢀ3.95 (m, 2 H), 4.27 (d, 1 H, J = 7.7 Hz,
H-1). 13C NMR (75.4 MHz, MeOD-d4): δ 25.0, 27.0, 29.4, 30.2, 30.3,
30.5, 30.6ꢀ30.7 (several peaks), 35.2, 37.1, 40.6, 62.7, 69.7, 71.6, 75.1,
78.0 (2 C), 104.5, 176.4. HRMS: [M + H]+ calcd for C24H47NO7S,
494.3146; found, 494.3146.
(S-Acetyl)-N-(16-mercapto-palmitoyl)-2-aminoethyl-2,20,
3,30,40,6,60-hepta-O-acetyl-β-D-maltoside (5b). A mixture of
peracetylated-2-azidoethyl-β-maltoside (5a) (0.400 g, 0.566 mmol)
and Boc2O (0.186 g, 0.850 mmol) was dissolved in absolute EtOH
(5 mL) and hydrogenated for 18 h using palladium on carbon (50 mg,
10%) under vigorous stirring and H2 (1 atm). The suspension was
filtered and concentrated. The obtained syrup was treated with TFA
(1 mL) in dry CH2Cl2 (5 mL) for 2 h at ambient temperature,
quenched with MeOH (1 mL), and coevaporated with toluene. Crude
amino sugar, 9 (0.315 g, 0.736 mmol) and DIPEA (0.108 mL, 0.623
mmol) were stirred in dry DMF (10 mL) for 1 h and coevaporated with
toluene. FC (toluene/EtOAc 1:1 f EtOAc) gave 5b (0.342 g, 0.345
mmol, 61%) as a white solid. Rf = 0.10 (toluene/EtOAc 1:1). [α]D = 50
(c 1.0, CHCl3). IR νmax cmꢀ1: 3376, 2919, 2849, 1751, 1689, 1657,
1
1528, 1472, 1367, 1245. H NMR (300 MHz, CDCl3): δ 1.18ꢀ1.24
(m, 22 H), 1.50ꢀ1.65 (m, 4 H), 1.99 (s, 3 H), 2.00 (s, 3 H), 2.01 (s, 3
H), 2.02 (s, 3 H), 2.03 (s, 3 H), 2.09 (s, 3 H), 2.13 (s, 3 H), 2.15 (t, 2 H,
J = 7.6 Hz), 2.31 (s, 3 H), 2.85 (t, 2 H, J = 7.3 Hz), 3.37ꢀ3.51 (m, 2 H),
3.65ꢀ3.73 (m, 2 H), 3.76ꢀ3.83 (m, 1 H), 3.93ꢀ4.00 (m, 2 H),
4.05 (dd, 1 H, J = 2.3, 12.4 Hz), 4.21 (dd, 1 H, J = 4.6, 12.4 Hz),
4.25 (dd, 1 H, J = 3.9, 12.4 Hz), 4.50ꢀ4.55 (m, 1 H), 4.52 (d, 1 H,
J = 7.9 Hz), 4.81 (dd, 1 H, J = 7.9, 9.5 Hz), 4.85 (dd, 1 H, J = 4.0, 10.5
Hz), 5.05 (dd, 1 H, J = 9.5, 10.1 Hz), 5.24 (dd, 1 H, J = 8.9, 9.5 Hz), 5.35
(dd, 1 H, J = 9.5, 10.5 Hz), 5.40 (d, 1 H, J = 4.0 Hz), 5.83 (bs, 1 H). 13C
NMR (75.4 MHz, CDCl3): δ 20.5ꢀ20.8 (7 C), 25.6, 28.7, 29.0, 29.1,
29.3, 29.4ꢀ29.5 (several peaks), 30.5, 36.5, 39.1, 61.5, 62.6, 68.0, 68.5,
69.2, 69.4, 70.0, 75.1, 76.7, 77.2, 77.6, 95.5, 100.5, 169.3, 169.6, 169.8,
170.0, 170.3, 170.4, 170.4, 173.2, 195.9. HRMS: [M + H]+ calcd for
C46H73NO20S, 992.4519; found, 992.4536.
N-(16-Mercapto-palmitoyl)-2-aminoethyl-β-D-maltoside
(5). To a solution of 5b (0.192 g, 0.194 mmol) in MeOH (10 mL) was
added K2CO3 (13 mg, 0.097 mmol). After 3 h, the mixture was neutra-
lized with DOWEX-H+, filtered, and concentrated. FC (CHCl3/MeOH
4:1) gave 5 (0.108 mg, 0.165 mmol, 85%) as a white solid. Rf = 0.49
(toluene/EtOH/H2O 10:9:1). [α]D = 46 (c 1.0, MeOH). IR νmax cmꢀ1
:
3329, 2920, 2848, 1621, 1554, 1469. 1H NMR (300 MHz, DMSO-d6): δ
1.19ꢀ1.28 (m, 22 H), 1.43ꢀ1.53 (m, 4 H), 2.04 (t, 2 H, J = 7.4 Hz), 2.42
(t, 2 H, J = 7.0 Hz), 2.99ꢀ3.09 (m, 2 H), 3.14ꢀ3.29 (m, 6 H), 3.32ꢀ3.39
(m, 1 H), 3.42ꢀ3.62 (m, 5 H), 3.67ꢀ3.74 (m, 2 H), 4.16 (d, 1 H, J = 8.0
Hz), 5.00 (d, 1 H, J = 3.6 Hz). 13C NMR (75.4 MHz, DMSO-d6): δ 24.2,
25.8, 28.2, 28.9, 29.1, 29.3, 29.4ꢀ29.5 (several peaks), 33.6, 35.9,
38.5ꢀ39.0 (1 C, overlap with solvent peak), 61.1, 61.2, 68.6, 70.2,
72.7, 73.4, 73.6, 73.8, 75.5, 76.6, 79.9, 101.2, 103.3, 173.9. HRMS: [M + H]+
calcd for C30H57NO12S, 656.3674; found, 656.3677.
2-Azidoethyl-2,20,3,30,40-penta-O-benzyl-6,60-di-O-tert-
butyl-diphenylsilyl-β-D-maltoside (11). To a stirred solution
of peracetylated 2-azidoethyl-β-maltoside 5a (4.00 g, 5.67 mmol) in
MeOH (50 mL) was added NaOMe (0.306 g, 5.67 mmol). Rf = 0.32
(toluene/EtOH/H2O 10:9:1). After 4 h, the reaction was neutralized
with DOWEX-H+, filtered, and concentrated. Without further pur-
ification, the crude solid was dissolved in dry DMF, whereupon
imidazole (6.58 g, 43.659 mmol), DMAP (3.81 g, 31.185 mmol), and
TBDPSCl (6.34 mL, 24.381 mmol) were added. After 5 h, the
reaction was quenched with EtOH, diluted with CH2Cl2, washed
sequentially with 0.1 M HCl and NaHCO3 (sat aq), dried, filtered,
and concentrated. Rf = 0.80 (toluene/EtOH/H2O 10:9:1). To the
obtained solid and BnBr (4.05 mL, 34.02 mmol) was added NaH
(1.48 g, 34.02 mmol, 55% dispersion in oil) dissolved in dry DMF
(20 mL) at 0 °C under an inert atmosphere. After 5 h, additional BnBr
(4.05 mL, 34.02 mmol) was added and stirred overnight. The mixture
was quenched with MeOH, diluted with toluene, washed with
NaHCO3 (sat aq) and H2O, dried, filtered, and concentrated. FC
15044
dx.doi.org/10.1021/la202774e |Langmuir 2011, 27, 15034–15047

Langmuir
ARTICLE
(toluene f toluene/EtOAc 9:1) gave 11 (6.30 g, 4.706 mmol, 83%)
as a colorless oil. Rf = 0.54 (toluene/EtOAc 19:1); [α]D = 24 (c 1.2,
CHCl3). IR νmax cmꢀ1: 3067, 3030, 2929, 2856, 2104, 1589, 1497,
1472, 1453, 1361, 823, 739, 700. 1H NMR (300 MHz, CDCl3): δ 1.00
(s, 9 H), 1.02 (s, 9 H), 3.39ꢀ4.04 (m, 16 H), 4.46 (d, 1 H, J = 7.7 Hz),
4.55ꢀ4.73 (m, 4 H), 4.77 (d, 1 H, J = 11.7 Hz), 4.78 (d, 1 H, J = 10.7
Hz), 4.87 (d, 1 H, J = 10.7 Hz), 4.90 (d, 1 H, J = 10.8 Hz), 4.95 (d, 1 H,
J = 11.8 Hz), 4.96 (d, 1 H, J = 11.1 Hz), 5.74 (d, 1 H, J = 3.8 Hz),
7.12ꢀ7.41 (m, 37 H), 7.57ꢀ7.68 (m, 8 H). 13C NMR (75.4 MHz,
CDCl3): δ 19.4, 19.5, 27.0, 27.0, 51.2, 62.6, 64.0, 67.9, 72.4, 72.6,
73.4, 73.6, 74.8, 75.3, 75.7, 75.8, 77.8, 80.0, 82.0, 82.3, 84.9, 96.0,
103.4, 126.9, 127.3, 127.7ꢀ127.8 (several peaks), 128.0, 128.1, 128.2,
128.4ꢀ128.5 (several peaks), 128.9, 129.6, 129.7, 129.7, 133.3, 133.4,
133.9, 133.9, 135.5, 135.7, 135.9, 136.0, 138.2, 138.6, 138.7, 138.9,
139.0. HRMS: [M + Na]+ calcd for C81H91N3O11Si2, 1360.6084;
found, 1360.6369.
FC (EtOAc/CH2Cl2/MeOH/H2O 20:2:2:1) gave 6 (37 mg, 0.054
mmol, 83%) as a white solid. Rf = 0.16 (EtOAc/MeOH/H2O 20:2:1).
[α]D = 44 (c 0.8, MeOH). IR νmax cmꢀ1: 2919, 2849, 1643, 1551, 1468.
1H NMR (300 MHz, MeOD-d4): δ 1.29ꢀ1.42 (m, 22 H), 1.54ꢀ1.62
(m, 4 H), 2.20 (t, 2 H, J = 7.6Hz), 2.49(t, 2 H, J = 7.1Hz), 3.22ꢀ3.27 (m,
2 H), 3.34ꢀ3.39 (m, 1 H), 3.38 (s, 3 H), 3.39 (s, 3 H), 3.42ꢀ3.72 (m, 11
H), 3.75ꢀ3.81 (m, 1 H), 3.84ꢀ3.91 (m, 1 H), 4.29 (d, 1 H, J = 7.7 Hz),
5.10 (d, 1 H, J = 3.6 Hz). 13C NMR (75.4 MHz, MeOD-d4): δ 25.0, 27.0,
29.4, 30.2, 30.3, 30.4, 30.6ꢀ30.7, 35.2, 37.1, 40.5, 59.3, 59.5, 69.8, 71.5,
72.4, 73.4, 73.5, 74.1, 74.5, 75.0, 75.3, 77.5, 81.8, 103.1, 104.4, 176.5.
HRMS: [M + H]+ calcd for C32H61NO12S, 684.3987; found, 684.3989.
2-Azidoethyl-2,20,200,3,30,300,400,6,60,600-undeca-O-acetyl-β-
D-maltotrioside (7a). A solution of maltotriose (5.00 g, 9.91 mmol),
NaOAc (0.10 g, 1.22 mmol), and Ac2O (10 mL, 10.80 g, 0.105 mol) in
toluene (25 mL) was heated to reflux. After 2 h, H2O was added, and the
solution was neutralized with 1 M NaOH(aq), dried, filtered, and
concentrated. Crude peracetylated maltotriose, 2-azidoethanol (3.45 g,
39.64 mmol), and 4 Å MS were stirred in dry CHCl2 (20 mL),
2-Azidoethyl-2,20,3,30,40-penta-O-benzyl-6,60-di-O-methyl-
β-D-maltoside (12). To a stirred solution of 11 (2.01 g, 1.500 mmol)
in dry THF (20 mL) was added TBAF (1.42 g, 4.500 mmol). After 5 h,
the reaction mixture was diluted with toluene, washed with NaHCO3
(sat aq) and H2O, dried, filtered, and concentrated. Rf = 0.33 (toluene/
EtOAc 1:1). To the obtained solid and MeI (0.56 mL, 9.00 mmol)
dissolved in dry DMF (50 mL) was added NaH (0.262 g, 6.00 mmol,
55% dispersion in oil) at 0 °C under an inert atmosphere with stirring
overnight. The reaction mixture was quenched with EtOH (10 mL),
diluted with toluene, washed with NaHCO3 (sat aq) and H2O, dried,
filtered, and concentrated. FC (toluene f toluene/EtOAc 1:1) gave 12
(1.146 g, 1.288 mmol, 86%) as a colorless oil. Rf = 0.48 (toluene/EtOAc
4:1). [α]D = 26 (c 1.0, CHCl3). IR νmax cmꢀ1: 3063, 3031, 2885, 2104,
whereupon BF3 OEt2 (3.73 mL, 4.22 g, 29.74 mmol) was added and
3
stirred overnight. The mixture was filtered through Celite 545, diluted
with CH2Cl2, washed sequentially NaHCO3(s) and H2O, dried, filtered,
and concentrated. FC (petroleum ether (60ꢀ80)/EtOAc 1:1 f 1:2)
gave 7a (4.38 g, 4.01 mmol, 44%) as a colorless solid. Rf = 0.62
(petroleum ether (60ꢀ80 °C)/EtOAc 1:2). [α]D = 73 (c 1.0, CHCl3).
1
IR νmax cmꢀ1: 2964, 2109, 1751, 1373, 1237. H NMR (300 MHz,
CDCl3): δ 1.98 (s, 3 H), 1.99 (s, 3 H), 2.00 (s, 3 H), 2.01 (s, 3 H), 2.01
(s, 3 H), 2.02 (s, 3 H), 2.05 (s, 3 H), 2.09 (s, 3 H), 2.15 (s, 3 H), 2.17 (s, 3
H), 3.26 (ddd, 1 H, J = 3.4, 4.9, 13.4 Hz), 3.47 (ddd, 1 H, J = 3.4, 8.2, 13.4
Hz), 3.66ꢀ3.75 (m, 2 H), 3.89ꢀ4.00 (m, 5 H), 4.02 (dd, 1 H, J = 2.3,
12.4 Hz), 4.16ꢀ4.26 (m, 2 H), 4.29 (dd, 1 H, J = 4.2, 12.1 Hz), 4.45 (dd,
1 H, J = 2.2, 12.3 Hz), 4.51 (dd, 1 H, J = 3.0, 12.2 Hz), 4.61 (d, 1 H, J = 7.8
Hz), 4.73 (dd, 1 H, J = 4.1, 10.3 Hz), 4.81ꢀ4.87 (m, 2 H), 5.06 (dd, 1 H,
J = 9.6, 10.1 Hz), 5.23ꢀ5.32 (m, 2 H), 5.35ꢀ5.42 (m, 3 H). 13C NMR
(75.4 MHz, CDCl3): δ 20.4ꢀ20.7 (10 C), 50.3, 61.3, 62.3, 62.7, 67.8,
68.4, 68.5, 68.8, 69.2, 69.3, 70.3, 71.6, 71.8, 72.1, 72.5, 73.6, 75.1, 95.5,
95.6, 100.0, 169.2, 169.5, 169.5, 169.6, 169.9, 170.2, 170.3, 170.3, 170.4,
170.3. HRMS: [M + Na]+ calcd. for C40H55N3O26, 1016.2966; found,
1016.2972.
1
1497, 1453, 1359, 736, 697. H NMR (300 MHz, CDCl3): δ 3.34 (s,
3 H), 3.35 (s, 3 H), 3.38ꢀ3.75 (m, 12 H), 3.78 (dd, 1 H, J = 8.8, 9.1 Hz),
3.92 (dd, 1 H, J = 8.5, 9.6 Hz), 4.02ꢀ4.11 (m, 2 H), 4.45 (d, 1 H, J = 7.7
Hz), 4.52 (d, 1 H, J = 12.1 Hz), 4.60 (d, 1 H, J = 11.8 Hz), 4.62 (d, 1 H,
J = 12.1 Hz), 4.62 (d, 1 H, J = 11.0 Hz), 4.76 (d, 1 H, J = 11.3 Hz), 4.82
(d, 1 H, J = 10.7 Hz), 4.85 (d, 1 H, J = 11.9 Hz), 4.90 (d, 1 H, J = 11.3
Hz), 4.91 (d, 1 H, J = 10.7 Hz), 4.96 (d, 1 H, J = 11.9 Hz), 5.66 (d, 1 H,
J = 3.6 Hz), 7.14ꢀ7.20 (m, 5 H), 7.22ꢀ7.32 (m, 20 H). 13C NMR
(75.4 MHz, CDCl3): δ 51.2, 59.3, 59.3, 68.1, 70.9, 71.0, 71.3, 72.1, 73.4,
74.1, 74.4, 74.8, 75.2, 75.6, 77.7, 79.4, 82.1, 82.3, 84.9, 97.0, 103.7, 126.7,
127.2, 127.6, 127.7, 127.8, 127.8, 127.9, 127.9, 128.0, 128.3, 128.4ꢀ128.4
(several peaks), 128.5, 138.1, 138.4, 138.7, 138.9, 138.9. HRMS: [M + Na]+
calcd for C51H59N3O11, 912.4042; found, 912.4046.
(S-Acetyl)-N-(16-mercapto-palmitoyl)-2-aminoethyl-
2,20,200,3,30,300,400,6,60,600-undeca-O-acetyl-β-D-maltotrioside (7b).
Compound 7a (0.400 g, 0.402 mmol) and Boc2O (0.131 g, 0.603
mmol) were dissolved in absolute EtOH (5 mL, 99.5%) and hydro-
genated for 19 h using palladium on carbon (50 mg, 10%) under
vigorous stirring and H2 (1 atm). The obtained syrup was treated with
TFA (1 mL) in dry CH2Cl2 (5 mL) for 3 h at room temperature,
quenched with EtOH (1 mL), and coevaporated with toluene. Crude
amino sugar, 9 (0.223 g, 0.523 mmol) and DIPEA (0.077 mL, 0.442
mmol) were stirred in dry DMF (10 mL) for 6 h and coevaporated with
toluene. FC (toluene/EtOAc 1:1 f EtOAc) gave 7b (0.351 g, 0.274
mmol, 68%) as a white solid. Rf = 0.21 (toluene/EtOAc 1:1). [α]D = 73
(c 1.0, CHCl3). IR νmax cmꢀ1: 3411, 2926, 2854, 1755, 1691, 1534,
(S-Acetyl)-N-(16-mercapto-palmitoyl)-2-aminoethyl-6,60-
di-O-methyl-β-D-maltoside (13). Compound 12 (0.163 g, 0.183
mmol) was dissolved in EtOH/HOAc (5 mL, 9:1) and hydrogenated for
23 h using palladium hydroxide on carbon (70 mg, 20%) under vigorous
stirring and H2 (1 atm). The suspension was filtered and concentrated.
Crude amino sugar, 9 (0.102 g, 0.239 mmol) and DIPEA (0.035 mL, 0.201
mmol) were stirred in dry DMF (3 mL) for 4 h and coevaporated with
toluene. FC (EtOAc/MeOH/CH2Cl2/H2O 20:2:2:1) gave 13 (87 mg,
0.120 mmol, 65%) as a white solid. Rf = 0.23 (EtOAc/MeOH/H2O
20:2:1). [α]D = 30 (c 1.1, CHCl3). IR νmax cmꢀ1: 2917, 2849, 1696,
1
1369, 1237. H NMR (300 MHz, CDCl3): δ 1.25ꢀ1.38 (m, 22 H),
1
1.54ꢀ1.61 (m, 4 H), 1.99 (s, 3 H), 2.00 (s, 3 H), 2.00 (s, 3 H), 2.02 (s, 3
H), 2.02 (s, 3 H), 2.03 (s, 3 H), 2.05 (s, 3 H), 2.10 (s, 3 H), 2.15 (s, 3
H), 2.17 (s, 3 H), 2.14ꢀ2.19 (m, 2 H), 2.32 (s, 3 H), 2.86 (t, 2 H, J = 7.3
Hz), 3.42ꢀ3.51 (m, 2 H), 3.71ꢀ3.83 (m, 3 H), 3.91ꢀ3.98 (m, 4 H),
4.06 (dd, 1 H, J = 2.0, 12.5 Hz), 4.16ꢀ4.32 (m, 3 H), 4.46ꢀ4.56 (m, 3
H), 4.74 (dd, 1 H, J = 4.1, 10.4 Hz), 4.80 (dd, 1 H, J = 8.0, 9.3 Hz), 4.86
(dd, 1 H, J = 4.0, 10.4 Hz), 5.07 (dd, 1 H, J = 9.8, 9.9 Hz), 5.23ꢀ5.33 (m,
2 H), 5.36ꢀ5.42 (m, 3 H), 5.86 (bs, 1 H). 13C NMR (75.4 MHz,
CDCl3): δ 20.5ꢀ20.8 (10 C), 25.7, 28.8, 29.1, 29.1, 29.4ꢀ29.6
(several peaks), 30.6, 36.6, 39.2, 61.4, 62.3, 62.7, 68.0, 68.5, 69.0, 69.3,
69.5, 70.1, 70.4, 71.7, 72.2, 72.4, 72.5, 73.6, 75.1, 95.6, 95.8, 100.5,
169.4, 169.6, 169.7, 169.8, 170.0, 170.3, 170.4, 170.4, 170.5, 170.6, 173.2,
1646, 1545, 1465, 1215. H NMR (300 MHz, CDCl3): δ 1.18ꢀ1.35
(m, 22 H), 1.49ꢀ1.59 (m, 4 H), 2.16 (t, 2 H, J = 7.0 Hz), 2.30 (s, 3 H),
2.84 (t, 2 H, J = 7.3 Hz), 3.35 (s, 3 H), 3.36 (s, 3 H), 3.29ꢀ3.88 (m, 16
H), 4.30 (d, 1 H, J = 7.7 Hz), 5.07 (d, 1 H, J = 3.1 Hz), 7.12 (bs, 1 H).
13C NMR (75.4 MHz, CDCl3): δ 25.5, 25.9, 29.0, 29.3, 29.6ꢀ29.9
(several peaks), 30.7, 36.5, 39.6, 59.1, 59.3, 69.4, 70.0, 71.4, 72.0 (2 C),
72.5, 73.0, 73.7, 74.0, 76.1, 80.5, 101.8, 102.9, 174.8, 196.2. HRMS: [M
+ H]+ calcd for C34H63NO13S, 726.4093; found, 726.4093.
N-(16-Mercapto-palmitoyl)-2-aminoethyl-6,60-di-O-me-
thyl-β-D-maltoside (6). To a solution of 13 (47 mg, 0.065 mmol) in
MeOH (3 mL) was added K2CO3 (7 mg, 0.051 mmol). After 1 h, the
mixture was neutralized with DOWEX-H+, filtered, and concentrated.
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195.9. HRMS: [M + H]+ calcd for C58H89NO28S, 1280.5365; found,
1280.5373.
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side (7). To a solution of 7b (0.368 g, 0.287 mmol) in MeOH (10 mL)
was added K2CO3 (55 mg, 0.395 mmol). After 3 h, the mixture was
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(CHCl3/MeOH 4:1 f CHCl3/MeOH 3:2) gave 7 (0.172 mg,
0.210 mmol, 73%) as a white solid. Rf = 0.43 (toluene/EtOH/H2O
10:9:1). [α]D = 73 (c 1.0, CHCl3). IR νmax cmꢀ1: 2917, 2849, 1645,
1553, 1467, 1368. 1H NMR (300 MHz, DMSO-d6): δ 1.20ꢀ1.30 (m, 22
H), 1.43ꢀ1.52 (m, 4 H), 2.05 (t, 2 H, J = 7.4 H), 2.43 (t, 2 H, J = 7.0 Hz),
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S
Supporting Information. Ellipsometric, contact angle,
b
and IRAS data for the mixed SAMs. This material is available
free of charge via the Internet at http://pubs.acs.org.
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Corresponding Author
*e-mail: ted@ifm.liu.se.
’ ACKNOWLEDGMENT
The authors acknowledge support from the AMBIO project
(NMP-CT-2005-011827) funded by the European Commis-
sion’s Sixth Framework Programme. The views expressed in this
publication reflect only those of the authors, and the Commis-
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’ NOTE ADDED AFTER ASAP PUBLICATION
This article was published ASAP on November 23, 2011. The
headings of paragraphs 24 and 25 of the Experimental Section
have been modified. The correct version was published on
December 13, 2011.
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