Antimicrobial and cytotoxic activity of (thio)alkyl hexopyranosides, nonionic glycolipid mimetics

Article abstract of DOI:10.1016/j.carres.2019.107905

A series of 19 synthetic alkyl and thioalkyl glycosides derived from D-mannose, D-glucose and D-galactose and having C₁₀–C₁₆ aglycone were investigated for cytotoxic activity against 7 human cancer and 2 non-tumor cell lines as well as for antimicrobial potential on 12 bacterial and yeast strains. The most potent compounds were found to be tetradecyl and hexadecyl β-D-galactopyranosides (18, 19), which showed the best cytotoxicity and therapeutic index against CCRF-CEM cancer cell line. Similar cytotoxic activity showed hexadecyl α-D-mannopyranoside (5) but it also inhibited non-tumor cell lines. Because these two galactosides (18, 19) were inactive against all tested bacteria and yeast strains, they could be a target-specific for eukaryotic cells. On the other hand, β-D-glucopyranosides with tetradecyl (11) and hexadecyl (12) aglycone inhibited only Gram-positive bacterial strain Enterococcus faecalis. The studied glycosides induce changes in the lipid bilayer thickness and lateral phase separation at high concentration, as derived from SAXS experiments on POPC model membranes. In general, glucosides and galactosides exhibit more specific properties. Those with longer aglycone show high cytotoxicity and therefore, they are more promising candidates for cancer cell line targeted inhibition.

Full text of DOI:10.1016/j.carres.2019.107905

Journal Pre-proof
Antimicrobial and cytotoxic activity of (thio)alkyl hexopyranosides, nonionic glycolipid
mimetics
Petr Džubák, Soňa Gurská, Kateřina Bogdanová, Daniela Uhríková, Nina Kanjaková,
Sophie Combet, Tomáš Klunda, Milan Kolář, Marian Hajdúch, Monika Poláková
PII:
S0008-6215(19)30585-3
DOI:
https://doi.org/10.1016/j.carres.2019.107905
CAR 107905
Reference:
To appear in: Carbohydrate Research
Received Date: 7 October 2019
Revised Date: 21 November 2019
Accepted Date: 23 December 2019
Please cite this article as: P. Džubák, Soň. Gurská, Kateř. Bogdanová, D. Uhríková, N. Kanjaková, S.
Combet, Tomáš. Klunda, M. Kolář, M. Hajdúch, M. Poláková, Antimicrobial and cytotoxic activity of
(thio)alkyl hexopyranosides, nonionic glycolipid mimetics, Carbohydrate Research (2020), doi: https://
doi.org/10.1016/j.carres.2019.107905.
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Antimicrobial and cytotoxic activity of (thio)alkyl hexopyranosides, nonionic glycolipid
mimetics
Petr Džubáka, Soňa Gurskáa, Kateřina Bogdanováa, Daniela Uhríkovác, Nina Kanjakovác,
Sophie Combetd, Tomáš Klundab, Milan Kolářa, Marian Hajdúcha , Monika Polákováb
a Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry,
Palacky University Olomouc, Hnevotinska 5, 779 00 Olomouc, Czech Republic
b Institute of Chemistry, Center for Glycomics, Slovak Academy of Sciences, Dubravska cesta
9, SK-845 38 Bratislava, Slovakia
c Department of Physical Chemistry of Drugs, Faculty of Pharmacy, Comenius University in
Bratislava, Odbojarov 10, 832 32 Bratislava, Slovakia
dLaboratoire Léon Brillouin (CEA/CNRS), CEA Saclay, 91191 Gif-sur-Yvette Cedex, France
Corresponding author: Phone: +421-2-59410272.
E-mail address: marian.hajduch@upol.cz, Monika.Polakova@savba.sk
1

Keywords: alkyl glycoside; glycolipid mimetic; cytotoxicity; antimicrobial activity; cell
cycle; SAXS
2

Abstract
A series of 19 synthetic alkyl and thioalkyl glycosides derived from D-mannose, D-glucose
and D-galactose and having C10-C16 aglycone were investigated for cytotoxic activity against
7 human cancer and 2 non-tumor cell lines as well as for antimicrobial potential on 12
bacterial and yeast strains. The most potent compounds were found to be tetradecyl and
hexadecyl β-
therapeutic index against CCRF-CEM cancer cell line. Similar cytotoxic activity showed
hexadecyl α- -mannopyranoside (5) but it also inhibited non-tumor cell lines. Because these
two galactosides (18, 19) were inactive against all tested bacteria and yeast strains, they could
be a target-specific for eukaryotic cells. On the other hand, β- -glucopyranosides with
D-galactopyranosides (18, 19), which showed the best cytotoxicity and
D
D
tetradecyl (11) and hexadecyl (12) aglycone inhibited only Gram-positive bacterial strain
Enterococcus faecalis. The studied glycosides induce changes in the lipid bilayer thickness
and lateral phase separation at high concentration, as derived from SAXS experiments on
POPC model membranes. In general, glucosides and galactosides exhibit more specific
properties. Those with longer aglycone show high cytotoxicity and therefore, they are more
promising candidates for cancer cell line targeted inhibition.
3

1. Introduction
Glycolipids are cell membrane amphiphilic components present in all living organisms. The
hydrophilic carbohydrate portion is composed of one or more monosaccharide units linked by
a glycosidic bond while the hydrophobic lipid moiety anchors them to the membrane. It is
known that these compounds fulfill a large variety of functions that are important for many
biological processes, such as recognition, cell adhesion and signaling that are influenced by
membrane properties [1].
The glycolipid ability to be incorporated into the lipid bilayer may lead to either disruption or
modification of the membrane structure. Many of glycolipids are also able to dissolve the
lipid membrane yielding a disruption of the cells. Therefore, the surface tension of the
membranes reduces, allowing water to flow into the cell and finally results in cell lysis. To
enhance the efficiency of this action, a tuned balance between the hydrophilic and
hydrophobic parts of the glycolipid is essential [2].
Among others, the sugar core, its stereochemistry and the structure of hydrophobic aglycone
are the factors that determine the physical and chemical properties of the glycolipids [3].
The ability of the glycolipids to destabilize biological membrane determines their
biomedicinal application as antifungal and antibacterial agents. Due to their antiviral and
cytotoxic properties, they are a subject of interest as therapeutics and pharmaceutics.
Their low toxicity is an additional benefit, important for applications in agro, food and
cosmetic industries [4].
The large potential of these compounds was an impulse for synthesis and evaluation of a
series of synthetic glycolipid mimetics [5]. Alkyl glycosides is one group of such mimetics.
The synthesis of such sugar-based amphiphiles is rapid [6] and, often, it requires just a few
reaction steps. Moreover, these non-ionic surfactants are environmentally friendly,
biocompatible and biodegradable.
4

The discovery of anticancer activity of fractions containing mixtures of diverse glycolipids
isolated from edible plants [7] motivated the preparation of a series of synthetic decyl to
hexadecyl α- and β-D-galactopyranosides. Some of them were moderately cytotoxic against 4
cancer cell lines with IC50 values at µM level. However, in some cases, equimolar mixtures of
both anomers were even more cytotoxic towards several cancer cell lines than single anomers
[8]. This was the case of the mixture of tetradecyl α/β-D-galactopyranosides, which
accentuates apoptosis of multiple myeloma cells by inducing DNA damage [9]. These are the
only report on cytotoxicity of sugar-based amphiphiles.
Reports on antimicrobial activity of alkyl glycosides are rare. Nevertheless, the synthesis of
a series of alkyl 2 , 3 , 4 deoxy, 2,3 and 3,4 dideoxy glycosides was reported [10,11].
Among them, octyl and dodecyl 2-deoxy-β-
Enterococcus faecalis. This Gram-positive species is also highly sensitive to dodecyl 2,6-
dideoxy α- -arabino-hexopyranoside, which is particularly active against another Gram-
D-arabino-hexopyranosides inhibit the growth of
L
positive bacterium, Bacillus sp.[10]. Additionally, Hu et al. have shown that triazole
glycolipids can reverse the bacterial MRSA phenotype and restore sensitivity to beta-lactam
antibiotics [12]. Such activity may be important for the eradication of resistant bacteria in
cystic fibrosis or, more generally, in the therapy of nosocomial infections.
In our previous study, we showed that antimicrobial activity of 14 synthetic alkyl and
thioalkyl α-D-mannopyranosides, with aglycone from hexyl to eicosyl, against 3 species
(Gram-positive Staphylococcus aureus, Gram-negative Escherichia coli and yeast Candida
albicans), depends on the aglycone length and on the type of glycosidic linkage (O- vs. S-
mannosides). Mannosides having (thio)dodecyl aglycones are the most potent antimicrobials,
with IC50 at the micromolar level [13].
The eco-friendly nature of alkyl glycosides offers a broad potential for their study. We
evaluated a broad variety of synthetic alkyl D-glycosides (glucosides, galactosides and
5

mannosides), which include aglycone of selected length from decyl to hexadecyl. Moreover,
because (thio)dodecyl mannosides are very active [13], both anomers of the corresponding
glucoside and galactoside analogs were screened in this study.
This paper reports the investigation of the inhibitory potential of these synthetic sugar based
amphiphiles against several Gram-positive (including methicillin-resistant Staphylococcus
aureus), Gram-negative bacteria and yeasts, and 7 human cancer and 2 non-tumor lines. Due
to the amphiphilic character of these molecules, the lipid bilayer of the cell membrane is a
target of their biological activity. We examined structural changes of the model membrane
palmitoyl-oleoyl-phosphatidylcholine (POPC) induced by selected glycosides, by Small angle
X-ray scattering (SAXS).
2. Results and discussion
2.1. Synthesis
Tetradecyl α-D-mannopyranoside (4) was prepared by a procedure previously utilized in the
synthesis of various alkyl mannosides, including 1[13], 2[6], 3[13] and 5[6]. This 2 steps
sequence led to the exclusive formation of 4 in 45% overall yield.
The synthesis of another neutral (thio)alkyl α- and β- -glycopyranosides bearing hydrocarbon
D
chains of different length (C10-C16) was performed by a glycosylation reaction between the
corresponding alcohol or thiol as an acceptor with peracetylated sugar donor upon activation
with BF3·OEt2 at room temperature (Scheme 1, Experimental section). When the reaction was
proceeded for 5h in DCM, desired decyl glycosides 6a and 13a were obtained in moderate
yields (~30%). Similar yields of all glycosides having longer, tetradecyl and hexadecyl
aglycones, were achieved after 22 h in 1,2-dichloroethane.
6

α- and β-anomers of (thio)dodecyl glucoside and galactoside were separated from the
corresponding anomeric mixtures. While dodecyl α/β glycosides were formed in
approximately equimolar ratio after 24h, in the case of thio analogs, β-anomers were observed
as major products within 5h. The purified isomers were identified based on a typical chemical
shift for H-1. For dodecyl glycosides (7a, 8a and 14a, 15a), the H-1 signal of α-anomers was
shifted more downfield by 0.6-1.1 ppm than that of β-anomers. The same trend in chemical
shifts of the anomeric H-1 was observed for the corresponding thio analogs 9a, 10a and 16a,
17a. In addition, for β-anomers of all (thio)dodecyl glycosides, C-1 atom resonated at higher
ppm than that of the corresponding α-anomer.
Finally, deacetylation of 6a-19a under Zemplen conditions (sodium methoxide in methanol)
gave deprotected glycosides 6-19 with good yields.
2.2. Biological assay
2.2.1. Cytotoxicity
Compounds 1-19 (Figure 1) were tested in vitro for their cytotoxic activity on seven cancer
cell lines: A549 (human lung adenocarcinoma), CCRF-CEM (T-lymphoblastic leukemia),
CEM-DNR (T-lymphoblastic leukemia, daunorubicin resistant), K562 (acute myeloid
leukemia), K562-TAX (acute myeloid leukemia, overexpressing the P-glycoprotein), HCT116
(human colorectal cancer with wild-type p53), HCT116p53-/- (human colorectal
cancer with deleted p53), and on the two non-malignant cell lines BJ (human fibroblast) and
MRC-5 (human lung fibroblasts). Cytotoxic activities are presented in Table 1 and are
expressed as IC50 values.
7

Figure 1. Structure of the nonionic glycosides used in this study
The tested compounds showed IC50 values between 9.4µM and more than 100 µM.
The acute lymphoblastic leukemia CCRF-CEM cell line was the most sensitive to tested
glycosides, particularly to 5, 11, 18 and 19 (IC50 in the range of 9.4 - 20.3 µM) bearing
tetradecyl or hexadecyl aglycone, what implies correlation between aglycone length and
cytotoxic activity. All the compounds were less active against its daunorubicin resistant CEM-
DNR counterpart.
However, in the case of K562 and the corresponding drug-resistant K562-TAX lines, the
cytotoxic activities of glucosides and galactosides are similar. A more significant difference
was observed for decyl α-
D
-mannoside (1) having 2.6 times higher cytotoxicity against K562
-mannoside
than K562-TAX (IC50 27.7 µM vs. 69.4 µM). On the other hand, hexadecyl α-
D
(5) was 2.9 times more potent against resistant K562-TAX than K562 cell line (IC50 19.7 µM
vs. 57.3 µM). These results indicate that for the resistance are responsible other mechanisms
than P-glycoprotein, which is common for both cell lines.
8

Table 1. Cytotoxic activity of derivatives 1-19 on human malignant cell lines different tissue
origin and drug resistance profile.
Except for decyl β-D-glucopyranoside (6) and decyl β-D-galactopyranoside (13), all other
compounds are active against human lung adenocarcinoma A549. Only a slight increase in
cytotoxic activity is observed with an elongation of alkyl lipid chain. Moreover, the impact of
anomeric configuration is not significant. α-Linked dodecyl O- and S-glucosides 7 and 9, as
well as galactosides 14 and 16, were slightly more active than their β-analogs 8, 10, 15 and
17. The most potent glycolipid is tetradecyl α-D-mannoside (4) (IC50 45.9 µM).
Cytotoxicity of all glycosides tested against HCT116 and HCT116p53-- were similar. The
most efficient were mannosides 2, 4 and 5, with IC50 below 50 µM in the case of HCT116.
Conversely, any effect of the aglycone length on the cytotoxicity of the glycosides against
non-tumor lines BJ and MRC-5 was not observed. The most toxic are mannosides, in
particular hexadecyl α-D-mannoside (5) (IC50 < 35.1 µM). Some of the glucosides and
galactosides inhibit these lines only weakly. Therefore, these glycosides are more suitable as
anticancer drugs with favorable therapeutic index (TI).
TI value is the ratio between the average IC50 value of noncancer cell lines (MRC-5 and BJ)
and the IC50 value of a given cancer cell line.
In general, glycolipids have the best selectivity (TI in a range from 9.1 to 2.27) for
hematological CCRF-CEM (T-lymphoblastic leukemia), K562 (acute myeloid leukemia) and
CEM-DNR (T-lymphoblastic leukemia, daunorubicin resistant) cell lines. The highest TI
value among all tested compounds is observed for tetradecyl β-D-galactoside (18) (TICCRF-CEM
9.10). Tetradecyl β- -glucoside (11) also showed high TI (TICCRF-CEM 5.36) against the same
D
9

line and a similar TI (TICCRF-CEM 4.20) was calculated for another galactoside 19 having two
more carbon atoms in the aglycone chain. The β-glucoside 11 is the only compound with TI >
2 against some of the multidrug-resistant cell lines (TICEM-DNR 2.27). It exhibits the same TI
value for the K562 cell line (TIK562 2.27). Higher TI values for the K562 cell line were
calculated for α-D-mannosides 1 and 2 (TI K562 3.76 and 2.88, respectively) having shorter
aglycones. Some selectivity on the K532 line (TIK562 2.17) also showed dodecyl α-
D-
glucoside (7).
In a recent report [8], the cytotoxic activity of decyl to hexadecyl galactosides was assayed
against 4 cancer cell lines, from which A549 and HCT116 cell lines were also used in this
study. Cytotoxic activity of dodecyl galactosides 14 and 15 against the A549 cell line
determined in the present study is very similar to those reported, while IC50 values for
galactoside 19 were rather different. In case of HTC116 cell line, the IC50 values for these 3
galactosides differed, but there was a similar trend, elongation of the aglycone chain resulted
in a decrease in cytotoxicity.
2.2.2. Cell cycle analysis
The effect of the most active tetradecyl β-D-galactoside (18) on the cell cycle was analyzed on
CCRF-CEM cell line at 1 x / 5 x IC50 concentrations (9.39 / 46.95 µM) with 24-hour
treatment (Table 2). Accumulation of cells in the G0/G1 and reduction in the S phase of the
cell cycle was observed, which was supported by the decrease of the DNA and RNA synthetic
activity and increase of apoptotic cells percentage following the 5x IC50 treatment.
10

Table 2. Summary of the cell cycle, apoptosis (sub G1), mitosis (pH3), and DNA (BrDU+)
and RNA (BrU+) synthesis analyses for tetradecyl β- -galactopyranoside (18). Data are
D
expressed as a percentage of the total cellular population.
2.2.3. Antimicrobial activity
Antimicrobial activity of the glycolipid mimetic 1-19 were tested against Gram-positive
(Enterococcus faecalis CCM 4224, Staphylococcus aureus CCM 3953, methicillin-resistant S.
aureus (MRSA) 4591 and fluoroquinolone-resistant S. haemolyticus A/16568) and Gram-
negative (Escherichia coli CCM 3954, E. coli C/16702, Pseudomonas aeruginosa CCM
3955, P. aeruginosa A/16575) bacterial strains and yeasts (Candida albicans, C. krusei, C.
tropicalis and C. parapsilosis).
Inhibitory activity of the glycolipids (Table 3) was determined and is expressed as minimal
bactericidal concentration (MBC) and minimal inhibitory concentration (MIC).
All tested glycolipids were inactive against Gram-negative bacterial strains. These species
(Escherichia coli and Pseudomonas aeruginosa) represent two of the most frequently Gram-
negative pathogens isolated in humans. Moreover, none of the decyl glycosides inhibited any
of the other 10 microorganisms tested.
All tested Candida strains were sensitive only to (thio)dodecyl β-glucosides, α-galactosides
and α-mannosides.
The best inhibitors of C. albicans are α-thioglycosides, mannoside 3 and galactoside 16 (both
having MBC 50 µM), which were more bactericidal than their O-counterparts 2 and 14. β-
Glucosides 8 and 10 exhibited weaker activity.
The same inhibition pattern is observed for C. krusei. Dodecyl glycosides 8 and 14 are less
potent than their thio-counterparts 10 and 16. The latter compound is the most efficient C.
krusei inhibitor, along with mannosides 2 and 3, all showing the same MBC 50µM.
11

C. parapsilosis showed an inhibitory pattern identical to that of C. krusei, but the potency of
the glycosides is slightly weaker. C. parapsilosis is the most sensitive to thiomannoside 3,
which showed bactericidal activity equal to that against the two Candida sp. strains (C.
albicans, C. krusei) mentioned above.
C. tropicalis is sensitive to the same glycosides as C.albicans, but it is the weakest inhibited
strain among the tested Candida sp. strains. The most potent glycosides (2, 3 and 16) out of 6
active ones have the same bactericidal activity (MBC 100 µM).
In general, S-glycosides were more efficient Candida sp. strains inhibitors than their O-
counterparts.
Table 3. Antimicrobial activity of derivatives 1-19 on bacterial strains
The Gram-positive strain E. faecalis is the most susceptible strain, since it was inhibited by
ten glycosides. Comparing two active α-galactosides, S-glycoside 16 (MBC 25 µM) is more
efficient than its O-analogue 14, thus resembling the inhibitory pattern of all Candida sp.
strains. Five out of seven tested glucosides possess bactericidal activity which increases with
the elongation of the aglycone hydrocarbon chain. Hexadecyl β-
µM) is the most bactericidal. It was inactive against all other microbes. Similarly, tetradecyl
β- -glucoside 11, as well as hexadecyl α- -mannoside 5 (both MBC 50µM), are selective
D-glucoside 12 (MBC 25
D
D
inhibitors of E. faecalis. However, (thio)dodecyl mannosides 2 and 3 are more bactericidal
glycosides (MBC 25 µM), reaching the efficiency of 16.
Another Gram-positive strain, represented by the more virulent S. aureus, is sensitive to the
same (thio)dodecyl glycosides as Candida sp. strains, and S-glycosides showed again better
inhibitory potential. Among 19 tested glycosides, tetradecyl α-D-mannoside (4) selectively
12

inhibits this strain. Together with another mannoside 3, they were the most bactericidal
(MBC 25 µM). On the other hand, reduced potential of glucosides was observed. S. aureus
was affected only by two of them.
4 (Thio)dodecyl glycosides (2, 3, 10 and 14) exhibit a bactericidal activity against MRSA at a
level similar to that observed against S. aureus.
S. haemolyticus 16568 was the least sensitive to the tested glycolipids. Only two of them
show some activity, but both bactericidal activity of dodecyl α-
D
-mannoside (2) (MBC 200
µM) and bacteriostatic activity of dodecyl α-D-galactoside (14) (MBC 200 µM) are quite
weak.
The study reveals that alkyl glycosides as nonionic glycolipid mimetics can induce a
bactericidal action. In general, glycosides were the most efficient inhibitors of E. faecalis,
which is sensitive to ten of them. On the other hand, the mannosides as the broadest spectrum
antimicrobials are able to inhibit 8 out of 12 tested microbial strains. Next, the length of the
aglycone affects inhibitory activity. Decyl glycosides were completely inactive, while those
with (thio)dodecyl aglycone were efficient on the majority of the tested strains. In addition,
further elongation of glycolipid aglycone chain leads to selective inhibition of some strains
(namely E. faecalis (compounds 5, 11 a 12) and S. aureus (comp. 4)). These conclusions are
in agreement with our previous paper reporting on a brief study of antimicrobial properties of
a series of α-D-mannosides. Nonionic alkyl glycosides presented here affect S. aureus, E.
faecalis and Candida sp. strains, but also show inactivity against E. coli and P. aeruginosa.
The bactericidal activity of the most active glycolipids mimetics (MBC 25µM) demonstrate
their promising antimicrobial potential against some Gram-positive bacterial strains and yeast.
13

2.3. Structural study of the model membrane.
As reported above, some of the studied glycosides are biologically active. These nonionic
amphiphiles interact readily with the phospholipid bilayer of the biological membrane.
We studied the effect of selected glycosides (4, 11, 12, 18 and 19) on the lipid bilayer formed
by zwitterionic phosphatidylcholine (POPC), intending to better understand the mechanism of
their biological activity.
Hydrated POPC forms a liquid-crystalline lamellar phase (Lα) in our studied temperature
range (25-60 °C) [14]. Small angle X-ray scattering (SAXS) experiments were performed to
follow structural changes of bilayers induced by glycoside (GL) at two molar ratios
(GL:POPC = 0.2 and 0.5 mol/mol). Figure 2 depicts typical SAXS patterns. Fully hydrated
POPC shows two peaks (L1 and L2) characterizing a lamellar phase (Figure 2, A bottom)
with repeat distance d = 65.30±0.01Å (at 25 oC), a value that agrees with the value d = 64.3 Å
reported for the same POPC in 200 mmol/l of NaCl [15]. All studied mixtures show lamellar
phases. Table S1 (see Supplementary material) summarizes the repeat distances (d) of
GL/POPC mixtures.
Glycosides inserted into the POPC bilayer decrease d : ∆d=dPOPC-dGL/POPC ~ 0.4 - 3.3 Å
(Figure 2, C). Figure 2A shows SAXS patterns of 11/POPC, where the change of d is the
highest one: ∆d=dPOPC-d11/POPC = 65.3 - 62.0 = 3.3 Å at 11/POPC = 0.2 mol/mol. However,
the highest content of 11 in POPC bilayer (11/POPC=0.5 mol/mol) induces a smaller change
of d, ∆d=2.5 Å. Generally, d values of zwitterionic phosphatidylcholines in the La state do not
exceed ~65-67 Å [16]. d is the sum d = dL + dw, where dL is the thickness of the lipid bilayer
and dw is the thickness of the water layer between two neighboring lipid bilayers. For
zwitterionic phosphatidylcholines, the thickness dw ~1.8-2 nm is the result of the balance
between repulsive interbilayer interactions (steric, hydration and fluctuations) and attractive
van der Waals forces [16]. Thus, changes of both thicknesses, the lipid bilayer (dL) as well as
14

the water layer (dw), can contribute to the detected ∆d. Detailed inspection of data in Table S1
reveals that ∆d induced by the glycoside alkyl aglycone (n = number of carbon atoms in
aglycone alkyl chain, n = 14 or 16) is larger for n = 14. Actually, induced changes can be
quantified in the order glucosides > galacto ≈ manno (as it is summarised in Figure 2, C).
POPC molecule is formed by two acyl chains, one saturated palmitic acid (n=16), the other
monounsaturated oleic acid (n=18, 1 double bond). The length of the glycoside alkyl aglycone
with n=16 fits well inside the hydrophobic POPC region, creating smaller disturbance than the
glycoside with n=14. The studied glycosides are nonionic compounds and their molecules
differ in the stereochemical arrangement of hydroxyl groups attached to a tetrahydropyrane
ring. In the bilayer, the hydrophilic part of the glycoside, localized in the polar region of
POPC, causes a lateral expansion of the bilayer that, in synergy with the hydrophobic
mismatch between POPC and tetradecyl aglycone, explains the decrease of the lipid bilayer
thickness (dL). Thus, we attribute ∆d by changes of the lipid bilayer thickness (dL). Our results
indicate small differences in localization of glycoside in the POPC bilayer due to the
stereochemical arrangement of hydroxyl groups. The asymmetry of the peak (second order
reflection, L2) in the pattern of 18/POPC=0.5 mol/mol (Figure 2, A, marked by an arrow)
indicates a phase separation in the mixture. Another lamellar phase was detected with repeat
distance d = 64.0±0.22 Å. Similar structural changes were observed in mixtures with the
highest amount of 4 in the bilayer (0.5 mol/mol) (Figure 2 B). Higher temperature promotes a
phase separation as it is clearly seen for 4/POPC=0.5 mol/mol at 60 °C. The pattern was fitted
assuming the superposition of two coexisting lamellar phases. Dashed lines show individual
peaks (L1a, L1b, etc.). Two phases (La and Lb) with periodicities da = 65.3 ± 0.13 Å and db =
61.5 ± 0.23 Å indicate absence of ideal mixing of the glycoside with the lipid. It was
identified that La is a phase rich of POPC, likely with a low content of 4 (da ~ dPOPC), while a
phase Lb, has a higher amount of 4 (db < dPOPC). d of fully hydrated POPC bilayers increases ~
15

2 Å in the temperature range 25-60 °C [14]. Glycosidic surfactants with alkyl aglycone (C14-
C16) and their interactions with lipid membranes are rarely discussed. In accord with our
findings, Carion-Taravella et al. [17] reported the reduction of the lipid bilayer thickness of
saturated dipalmitoylphosphatidylcholine (DPPC) due to its interaction with dodecyl-β-D-
glucopyranoside, and coexistence of lamellar phases.
Figure 2. SAXS patterns of POPC and GL/phospholipid bilayer mixtures: A) POPC and
selected glycosides (18, 11) + POPC at 25 °C. The asymmetry of L2 peak is marked by an
arrow; B) 4:POPC=0.5 mol/mol at different temperatures. Dashed lines represent the fit of
individual peaks of two lamellar phases (La, Lb). Full line represents the fitted curve of two
phases. C) ∆d=dPOPC-dGL/POPC as a function of the length n of alkyl aglycone of GL: gluco-
(full circles); galacto- (empty squares) and manno- (full triangles).
16

In summary, we did not observe changes of the long-range organization of POPC bilayer in
presence of glycosides, meaning that the lamellar phase was preserved in all studied mixtures.
Instead, SAXS experiments revealed that glycosides affect the structure of the POPC
membrane, changing the lipid bilayer thickness. High glycoside content induces lateral phase
separation resulting in two phases (La, Lb) with slightly different periodicities, although
without macroscopic phase separation. The small difference between the two repeat distances
supports the model of coexistence of two phases within one supramolecular structure, which
can be due to non-ideal mixing of compounds. Lipid components of biological membranes are
not uniformly distributed in the membrane, they create domains. Experiments have shown
that glycosides can alter this distribution by their clustering with neutral phospholipids, main
component of the membrane. This can be toxic for the cell either by preventing the interaction
of lipids with other membrane components, or by disrupting existing natural domains. Finally,
we found that even minor amounts of glycosides affect the lipid bilayer thickness which plays
a crucial role in the activity of transmembrane proteins [18].
3. Conclusions.
It is known that lipophilicity of the compounds plays an important role in their penetration
into cells and in cell permeability. The length of aglycone is a factor determining lipophilicity
of the investigated nonionic alkyl glycosides. The most lipophilic ones show the highest
cytotoxicity, but this dependence was not clear for all tested cancer cell lines. In addition, the
most lipophilic mannosides also do affect negatively non-tumor lines, what is a serious
limitation for their use as anticancer drugs. The most lipophilic glycosides are not the
strongest antibacterials, but, in some cases, their selectivity against different bacterial strains
depends on the length of the aglycone chain.
17

Among the 19 tested sugar based amphiphiles, we identified those specific for eukaryotes.
Tetradecyl β- -galactopyranoside (18) shows the most promising cytotoxicity and the highest
D
TI (9.1) against CCRF-CEM cancer cell line. At higher concentrations (5 x IC50), we observe
the accumulation of the cells in G0/G1 cell phase and inhibition of DNA / RNA synthesis,
which can be a non-specific phenotype of the pre-/apoptotic cells. Along with hexadecyl β-
galactopyranoside (19) having TI (4.2, CCRF-CEM), these two glycolipids have no
antimicrobial activity. Therefore, they could be a target specific for eukaryotic cells.
D-
Regarding prokaryotes, tetradecyl and hexadecyl β-D-glucopyranosides (11) and (12) are
selective inhibitors of E. faecalis. The Candida sp. strains were sensitive only to glycosides
having (thio)dodecyl aglycone, which are the broadest spectrum antimicrobials inhibiting
enterococci as well as staphylococci.
The structural study reveals small but not negligible changes in the lipid bilayer from
zwitterionic POPC induced by glycosides selected for these pilot experiments. However, the
detected "mild effect" can generate lateral disorder in membrane components resulting in
malfunction of the cell. On the other hand, long-range order in lamellar phase is preserved up
to rather high content of the additive in the lipid bilayer. Therefore, the antimicrobial activity
is not due to membrane fragmentation and formation of non-lamellar phases, as often
observed with antimicrobial peptides [19]. On another hand, one can take advantage of this
"mild effect" when designing a lipid-based carrier for glycosides at targeted drug delivery.
The results presented in this study reveal an interesting potential of nonionic glycosides. This
is in line with recent demands for bioactive compounds, which are strongly preferred because
based on natural origin, contrary to synthetic compounds. The tested glycosides fulfill these
trends for bioprotection, since the combination of saccharides with lipids, two biodegradable
components, fully meet the requirements for nature-like composition. Therefore, the active
18

glycolipid mimetic should become an environmentally friendly alternative to common
products.
4. Experimental
4.1. General
TLC was performed on aluminum sheets precoated with silica gel 60 F254 (Merck). Flash
column chromatography was carried out on silica gel 60 (0.040-0.060 mm, Merck) with
distilled solvents (hexanes, ethyl acetate, methanol). Dry dichloromethane and dry 1,2-
dichloroethane were purchased from Aldrich. POPC (1-palmitoyl-2-oleoyl-glycero-3-
phosphocholine) was purchased from Avanti Polar Lipids, Inc., USA. All reactions containing
sensitive reagents were carried out under an argon atmosphere. 1H NMR and 13C NMR
spectra were recorded at 25 ºC with Bruker AVANCE III HD 400 spectrometer. Chemical
shifts are referenced to either TMS (δ 0.00, CDCl3 for 1H ) or HOD (δ 4.87, CD3OD for 1H),
and to internal CDCl3 (δ 77.00) or CD3OD (δ 49.00) for 13C. Optical rotations were measured
on a Jasco P2000 polarimeter at 20 °C. High-resolution mass determination was performed by
ESI-MS on a Thermo Scientific Orbitrap Exactive instrument operating in positive mode.
All the tested compounds used in biological tests were lyophilized before the use.
Peracetylated donors were synthesized according to published procedure [20].
4.2. General procedure for glycosylation
A stirred solution containing peracetylated donor (1 g, 2.56 mmol, 1 eq) in dry solvent (10
mL) was cooled down on an ice bath and BF3 OEt2 was added. After being stirred for 10 min,
the corresponding alcohol (1.5 eq) was added dropwise. Then the resulting mixture was
stirred at rt. The reaction mixture was then diluted with solvent (50 mL) and poured into ice
cold satd NaHCO3 (100 mL) under stirring. The organic phase was separated, washed with
19

water (100 mL), dried with anhydrous Na2SO4, filtered and concentrated. The crude product
was purified by flash chromatography (Hexane:EtOAc 7:1→3:1).
The conditions (catalyst, solvent, reaction time) used in the synthesis of acetylated glycosides:
β-C10 glycosides 6a and 13a: BF3 OEt2 (5eq), DCM, 5h; α/β C12 glycosides 7a, 8a, 14a,
15a: BF3 OEt2 (5eq), DCM, 24h; α/β SC12 glycosides 9a, 10a, 16a, 17a: BF3 OEt2 (1.5 eq),
DCE, 5h; β-C14 glycosides 11a and 18a: BF3 OEt2 (3eq), DCE, 22h; β-C16 glycosides 12a
and 19a: BF3 OEt2 (3eq), DCE, 22h.
4.3. General procedure for deprotection
To a solution of peracetylated glycoside (1eq) in MeOH (9.5 mL) MeONa (1M, 0.50 mL) was
added. The reaction mixture was stirred for 16h, neutralized with Dowex 50 H+-form and
filtered. After removal of the solvent, the residue was purified by column chromatography
(hexane:EtOAc 1:9 →EtOAc→EtOAc:MeOH 10:1).
Scheme 1. Synthesis of target nonionic glycolipid mimetics 6-19.
Reagents and conditions. a) BF3 OEt2, alcohol, solvent, 5-24h, rt; b) MeONa, MeOH, 16 h, rt.
Synthesis and analytical data for compounds 1a[13], 2a[6], 3a[13] and 5a[6] were reported.
20

4.4.1. Tetradecyl 2,3,4,6-tetra-O-acetyl-α- -mannopyranoside (4a). Synthesis was carried
D
out as reported previously [6]. Yield 0.82 g, 59%, clear oil. [α]D + 23.3 (c 0.5, CHCl3).1H
NMR (400 MHz, CDCl3): δ 5.37 (dd, 1H, J2,3 = 3.4 Hz, H-3), 5.29 (dd, 1H, J3,4 = 9.9 Hz, J4,5 =
9.8 Hz, H-4), 5.23 (dd, 1H, H-2), 4.81 (d, 1H, J1,2 = 1.7 Hz, H-1), 4.29 (dd, 1H, J5,6b = 5.3 Hz,
J6a,6b = 12.1 Hz, H-6b), 4.11 (dd, 1H, J5,6a = 2.4 Hz, H-6a), 3.99 (ddd, 1H, H-5), 3.68 (dt, 1H,
J = 6.7 Hz, J = 9.6 Hz, OCH2(CH2)12CH3), 3.45 (dt, 1H, J = 6.5 Hz, J = 9.5 Hz,
OCH2(CH2)12CH3), 2.15, 2.10, 2.04, 1.99 (each s, each 3H, 4x CH3CO), 1.64-1.24 (m, 24H,
OCH2(CH2)12CH3), 0.88 (t, 3H, J = 6.5 Hz, O(CH2)13CH3). 13C NMR (100 MHz, CDCl3): δ
170.6, 170.1, 170.0, 169.8 (4x CH3CO), 97.6 (C-1), 69.9 (C-2), 69.2 (C-3), 68.6, 68.5 (C-5,
OCH2(CH2)12CH3), 66.4 (C-4), 62.6 (C-6), 32.0, 29.8(3x), 29.7(2x), 29.6(2x), 29.4, 29.3,
26.1, 22.7 (OCH2(CH2)12CH3), 20.9, 20.7(3x) (4x CH3CO), 14.2 (O(CH2)13CH3). HRMS
(ESI) m/z: calcd for C28H48O10Na [M+Na]+: 567.3140; found: 567.3135.
4.4.2. Decyl 2,3,4,6-tetra-O-acetyl-β- -glucopyranoside (6a). Yield 0.39 g, 31%, clear oil.
D
[α]D - 16.5 (c 1.1, CHCl3). Analytical data, [α]D - 15.0 (c 1.19, CHCl3) [21], 1H NMR [22] and
13C NMR [21] data were reported previously. HRMS (ESI) m/z: calcd for C24H40O10Na
[M+Na]+: 511.2514; found: 511.2518.
4.4.3. Dodecyl 2,3,4,6-tetra-O-acetyl-α- -glucopyranoside (7a). Yield 0.45 g, 34%, clear
D
oil. [α]D + 44.5 (c 0.2, CHCl3). 1H NMR (400 MHz, CDCl3): δ 5.48 (t, 1H, J3,4 = 9.8 Hz, H-3),
5.08-5.03 (m, 2H, H-1, H-4), 4.85 (dd, 1H, J1,2 = 3.7 Hz, J2,3 = 10.2 Hz, H-2), 4.26 (dd, 1H,
J5,6b = 4.5 Hz, J6a,6b = 12.4 Hz, H-6b), 4.09 (dd, 1H, J5,6a = 2.3 Hz, H-6a), 4.02 (m, 1H, H-5),
3.67 (dt, 1H, J = 6.6 Hz, J = 9.9 Hz, OCH2(CH2)10CH3), 3.43 (dt, 1H, J = 6.5 Hz, J = 9.9 Hz,
OCH2(CH2)10CH3), 2.09, 2.06, 2.03, 2.01 (each s, each 3H, 4x CH3CO), 1.59-1.26 (m, 20H,
OCH2(CH2)10CH3), 0.88 (t, 3H, J = 6.5 Hz, O(CH2)11CH3). 13C NMR (100 MHz, CDCl3): δ
21

170.7, 170.2(2x), 169.7 (4x CH3CO), 95.6 (C-1), 71.0 (C-2), 70.3 (C-3), 68.8, 68.7 (C-4,
OCH2(CH2)10CH3), 67.1 (C-5), 62.0 (C-6), 31.9, 29.7(2x), 29.6(3x), 29.4, 29.3, 26.1, 22.7
(OCH2(CH2)10CH3), 20.8(2x), 20.7(2x) (4x CH3CO), 14.1 (O(CH2)11CH3). HRMS (ESI) m/z:
calcd for C26H44O10Na [M+Na]+: 539.2827; found: 539.2829.
4.4.4. Dodecyl 2,3,4,6-tetra-O-acetyl-β- -glucopyranoside (8a). Yield 0.42 g, 32%, clear
D
oil. [α]D - 16.0 (c 0.5, CHCl3). 1H NMR data were reported [23]. 13C NMR (100 MHz,
CDCl3): δ 170.7, 170.4, 169.5, 169.3 (4x CH3CO), 100.9 (C-1), 73.0 (C-3), 71.8 (C-5), 71.5
(C-2), 70.4 (OCH2(CH2)10CH3), 68.6 (C-4), 62.1 (C-6), 32.0, 29.8, 29.7(3x), 29.5, 29.4(2x),
25.9, 22.8 (OCH2(CH2)10CH3), 20.8, 20.7(3x) (4x CH3CO), 14.2 (O(CH2)11CH3). HRMS
(ESI) m/z: calcd for C26H44O10Na [M+Na]+: 539.2827; found: 539.2823.
4.4.5. Dodecyl 2,3,4,6-tetra-O-acetyl-1-thio-α- -glucopyranoside (9a). Yield 0.16 g, 12%,
D
yellowish oil. [α]D + 120.0 (c 0.25, CHCl3). 1H NMR (400 MHz, CDCl3): δ 5.61 (d, 1H, J1,2 =
5.8 Hz, H-1), 5.32 (dd, 1H, J4,5 = 9.7 Hz, H-4), 5.01 (dd, 1H, J3,4 = 9.7 Hz, H-3), 4.98 (dd, 1H,
J2,3 = 10.2 Hz, H-2), 4.39 (ddd, 1H, J5,6a = 2.3 Hz, J5,6b = 4.7 Hz, H-5), 4.26 (dd, 1H, J6a,6b
=
12.3 Hz, H-6b), 4.02 (dd, 1H, H-6a), 2.55-2.41 (m, 2H, SCH2(CH2)10CH3), 2.04, 2.02, 2.00,
1.97 (each s, each 3H, 4x CH3CO), 1.58-1.19 (m, 20H, SCH2(CH2)10CH3), 0.83 (t, 3H, J = 6.6
Hz, S(CH2)11CH3). 13C NMR (100 MHz, CDCl3): δ 170.7, 170.4, 170.3, 169.9 (4x CH3CO),
81.8 (C-1), 70.6 (C-2), 70.2 (C-4), 68.5 (C-3), 67.3 (C-5), 62.6 (C-6), 30.3
(SCH2(CH2)10CH3), 32.0, 30.0, 29.7(2x), 29.6(2x), 29.5, 29.4, 28.9, 22.7 (SCH2(CH2)10CH3),
20.6(2x), 20.5(2x) (4x CH3CO), 14.2 (S(CH2)11CH3). HRMS (ESI) m/z: calcd for
C26H44O9SNa [M+Na]+: 555.2598; found: 555.2596.
22

4.4.6. Dodecyl 2,3,4,6-tetra-O-acetyl-1-thio-β- -glucopyranoside (10a). Yield 0.70 g, 38%,
D
yellowish oil. [α]D - 28.0 (c 0.15, CHCl3). Analytical data, [α]D20 - 29.2 (c 0.12, CHCl3) [24],
1H and 13C NMR [25] data were reported. HRMS (ESI) m/z: calcd for C26H44O9SNa
[M+Na]+: 555.2598; found: 555.2593.
4.4.7. Tetradecyl 2,3,4,6-tetra-O-acetyl-β- -glucopyranoside (11a). Yield 0.49 g, 35%,
D
white solid. [α]D - 29.4 (c 0.5, CHCl3). Analytical data, [α]D20 - 63.8 (c 0.2, CHCl3) [26], H
and 13C NMR data were reported [27]. HRMS (ESI) m/z: calcd for C28H48O10Na [M+Na]+:
567.3140; found: 567.3143.
1
4.4.8. Hexadecyl 2,3,4,6-tetra-O-acetyl-β- -glucopyranoside (12a). Yield 0.56 g, 38%,
D
white solid. [α]D - 18.0 (c 0.5, CHCl3). 1H and 13C NMR data were reported [27]. HRMS
(ESI) m/z: calcd for C30H52O10Na [M+Na]+: 595.3453; found: 595.3445.
4.4.9. Decyl 2,3,4,6-tetra-O-acetyl-β- -galactopyranoside (13a). Yield 0.40 g, 32%, clear
D
oil. [α]D - 15.6 (c 1.1, CHCl3). Analytical data, [α]D20 - 11.6 (c 1.5, CHCl3) [22], 1H NMR data
were reported [8]. 13C NMR (100 MHz, CDCl3): δ 170.5, 170.4, 170.3, 169.5 (4x CH3CO),
101.5 (C-1), 71.1 (C-3), 70.7 (C-5), 70.5 (OCH2(CH2)8CH3), 69.1 (C-2), 67.2 (C-4), 61.4 (C-
6), 32.0, 29.7(2x), 29.6, 29.5(2x), 26.0, 22.8 (OCH2(CH2)8CH3), 20.9, 20.8(2x), 20.7 (4x
CH3CO), 14.3 (O(CH2)9CH3). HRMS (ESI) m/z: calcd for C24H40O10Na [M+Na]+: 511.2514;
found: 511.2513.
4.4.10. Dodecyl 2,3,4,6-tetra-O-acetyl-α- -galactopyranoside (14a). Yield 0.29 g, 22%,
D
white semisolid. [α]D + 10.0 (c 0.5, CHCl3). 1H NMR data were reported [8]. 13C NMR (100
MHz, CDCl3): δ 170.4, 170.3(2x), 170.0 (4x CH3CO), 96.2 (C-1), 68.7 (OCH2(CH2)10CH3),
23

68.3, 68.2 (C-3, C-4), 67.7 (C-2), 66.2 (C-5), 61.9 (C-6), 32.0, 29.7(3x), 29.6(2x), 29.4(2x),
26.2, 22.7 (OCH2(CH2)10CH3), 20.8, 20.7(3x) (4x CH3CO), 14.2 (O(CH2)11CH3). HRMS
(ESI) m/z: calcd for C26H44O10Na [M+Na]+: 539.2827; found: 539.2833.
4.4.11. Dodecyl 2,3,4,6-tetra-O-acetyl-β- -galactopyranoside (15a). Yield 0.40 g, 30%,
D
white solid. [α]D - 10.0 (c 0.5, CHCl3). Analytical data, [α]D25 - 14.0 (c 0.8, CHCl3) [28] and
1H NMR data were reported [23]. 13C NMR (100 MHz, CDCl3): δ 170.5, 170.4, 170.2, 169.4
(4x CH3CO), 101.4 (C-1), 71.1 (C-3), 70.76 (C-5), 70.4 (OCH2(CH2)10CH3), 69.1 (C-2), 67.2
(C-4), 61.4 (C-6), 32.0, 29.7(3x), 29.5(2x), 29.4(2x), 25.9, 22.8 (OCH2(CH2)10CH3), 20.8,
20.7(3x) (4x CH3CO), 14.3 (O(CH2)11CH3). HRMS (ESI) m/z: calcd for C26H44O10Na
[M+Na]+: 539.2827; found: 539.2831.
4.4.12. Dodecyl 2,3,4,6-tetra-O-acetyl-1-thio-α- -galactopyranoside (16a). Yield 0.17 g,
D
12%, yellowish oil. [α]D + 144.0 (c 0.5, CHCl3). 1H NMR (400 MHz, CDCl3): δ 5.71 (d, 1H,
J1,2 = 5.2 Hz, H-1), 5.45 (dd, 1H, J4,5 = 1.3 Hz, H-4), 5.26 (dd, 1H, J2,3 = 10.8 Hz, H-2), 5.22
(dd, 1H, J3,4 = 10.8 Hz, H-3), 4.60-4.57 (m, 1H, H-5), 4.14-4.06 (m, 2H, H-6a, H-6b), 2.60-
2.45 (m, 2H, SCH2(CH2)10CH3), 2.14, 2.07, 2.04, 1.99 (each s, each 3H, 4x CH3CO), 1.53-
1.25 (m, 20H, SCH2(CH2)10CH3), 0.87 (t, 3H, J = 6.5 Hz, S(CH2)11CH3). 13C NMR (100
MHz, CDCl3): δ 170.5, 170.3(2x), 170.0 (4x CH3CO), 82.3 (C-1), 68.3, 68.2(2x) (C-2, C-3,
C-4), 66.6 (C-5), 62.0 (C-6), 32.1 (SCH2(CH2)10CH3), 30.0, 29.8(2x), 29.7(2x), 29.5, 29.3,
29.1, 26.1, 22.8 (SCH2(CH2)10CH3), 21.0, 20.8(2x), 20.7 (4x CH3CO), 14.3 (S(CH2)11CH3).
HRMS (ESI) m/z: calcd for C26H44O9SNa [M+Na]+: 555.2598; found: 555.2601.
4.4.13. Dodecyl 2,3,4,6-tetra-O-acetyl-1-thio-β-D-galactopyranoside (17a). Yield 0.50 g,
37%, white solid. [α]D - 8.0 (c 0.5, CHCl3). Analytical data, [α]D20 - 16.0 (c 0.3, CHCl3) [24],
24

1H and 13C NMR data were reported [29]. HRMS (ESI) m/z: calcd for C26H44O9SNa
[M+Na]+: 555.2598; found: 555.2600.
4.4.14. Tetradecyl 2,3,4,6-tetra-O-acetyl-β- -galactopyranoside (18a). Yield 0.39 g, 28%,
D
white solid. [α]D - 35.7 (c 0.5, CHCl3). 1H NMR data were reported [23]. 13C NMR (100
MHz, CDCl3): δ 170.5, 170.4, 170.3, 169.5 (4x CH3CO), 101.5 (C-1), 71.1 (C-3), 70.7 (C-5),
70.5 (OCH2(CH2)12CH3), 69.1 (C-2), 67.2 (C-4), 61.4 (C-6), 32.1, 29.8(4x), 29.6(2x),
29.5(3x), 26.0, 22.8 (OCH2(CH2)12CH3), 20.9, 20.8(2x), 20.7 (4x CH3CO), 14.3
(O(CH2)13CH3). HRMS (ESI) m/z: calcd for C28H48O10Na [M+Na]+: 567.3140; found:
567.3138.
4.4.15. Hexadecyl 2,3,4,6-tetra-O-acetyl-β- -galactopyranoside (19a). Yield 0.29 g, 20%,
D
white solid. [α]D - 22.8 (c 0.5, CHCl3). 1H NMR data were reported [8]. 13C NMR (100 MHz,
CDCl3): δ 170.5, 170.4, 170.3, 169.5 (4x CH3CO), 101.5 (C-1), 71.1 (C-3), 70.7 (C-5), 70.5
(OCH2(CH2)14CH3), 69.1 (C-2), 67.2 (C-4), 61.4 (C-6), 32.1, 29.9, 29.8(4x), 29.7 (2x),
29.6(2x), 29.5(2x), 26.0, 22.8 (OCH2(CH2)14CH3), 20.9, 20.8(2x), 20.7 (4x CH3CO), 14.3
(O(CH2)15CH3). HRMS (ESI) m/z: calcd for C30H52O10Na [M+Na]+: 595.3453; found:
595.3456.
Synthesis and analytical data for compounds 1[13], 2[6], 3[13] and 5[6] were reported.
4.4.16. Tetradecyl α- -mannopyranoside (4). Yield 0.42 g, 77%, white solid. [α]D + 35.6 (c
D
0.5, MeOH), ref [30] [α]D + 50.0 (c 1.0, MeOH). 1H NMR (400 MHz, CD3OD): δ 4.76 (d, 1H,
J1,2 = 1.7 Hz, H-1), 3.86 (dd, 1H, J5,6a = 2.4 Hz, J6a,6b = 11.9 Hz, H-6a), 3.80 (dd, 1H, J2,3 = 3.3
Hz, H-2), 3.77-3.68 (m, 3H, H-3, H-6b, OCH2(CH2)12CH3), 3.63 (t, J3,4 = 9.5 Hz, J4,5 = 9.5
25

Hz, H-4), 3.56 (m, 1H, H-5), 3.44 (dt, 1H, J = 6.3 Hz, J = 9.5 Hz, OCH2(CH2)12CH3), 1.65-
1.29 (m, 24H, OCH2(CH2)12CH3), 0.93 (t, 3H, J = 6.6 Hz, O(CH2)13CH3). 13C NMR (100
MHz, CD3OD): δ 101.5 (C-1), 74.6 (C-5), 72.7 (C-3), 72.3 (C-2), 68.6(2x) (C-4,
OCH2(CH2)12CH3), 62.9 (C-6), 33.1, 30.8(3x), 30.7(3x), 30.6(2x), 30.5, 27.4, 23.7
(OCH2(CH2)12CH3), 14.4 (O(CH2)13CH3). HRMS (ESI) m/z: calcd for C20H40O6Na [M+Na]+:
399.2917; found: 399.2920.
4.4.17. Decyl β- -glucopyranoside (6). Yield 0.20 g, 79%, white powder. [α]D - 35.0 (c 1.1,
D
MeOH). Analytical data, [α]D20 - 25.4 (c 1.15, MeOH) [31], 1H and 13C NMR data were
reported [32]. HRMS (ESI) m/z: calcd for C16H32O6Na [M+Na]+: 343.2091; found: 343.2090.
4.4.18. Dodecyl α- -glucopyranoside (7). Yield 0.27 g, 90%, white powder. [α]D + 54.5 (c
D
0.9, MeOH), ref [31] [α]D20 + 101 (c 1.72, MeOH). 1H NMR (400 MHz, CD3OD): δ 4.79 (d,
1H, J1,2 = 3.7 Hz, H-1), 3.81 (dd, 1H, J5,6a = 2.4 Hz, J6a,6b = 11.9 Hz, H-6a), 3.75 (dt, 1H, J =
6.9 Hz, J = 9.5 Hz, OCH2(CH2)10CH3), 3.69 (dd, 1H, J5,6b = 5.6 Hz, H-6b), 3.66 (t, 1H, J3,4
9.4 Hz, H-3), 3.59 (ddd, 1H, J4,5 = 9.9 Hz, H-5), 3.46 (dt, 1H, J = 6.5 Hz, J = 9.6 Hz,
=
OCH2(CH2)10CH3), 3.40 (dd, 1H, J2,3 = 9.7 Hz, H-2), 3.30 (dd, 1H, H-4), 1.69-1.29 (m, 20H,
OCH2(CH2)10CH3), 0.91 (t, 3H, J = 6.8 Hz, O(CH2)11CH3).13C NMR (100 MHz, CD3OD): δ
100.1 (C-1), 75.1 (C-3), 73.6 (2x) (C-2, C-5), 71.8 (C-4), 69.1 (OCH2(CH2)10CH3), 62.7 (C-
6), 33.1, 30.8(2x), 30.7(3x), 30.6, 30.5, 27.3, 23.7 (OCH2(CH2)10CH3), 14.4 (O(CH2)11CH3).
HRMS (ESI) m/z: calcd for C18H36O6Na [M+Na]+: 371.2404; found: 371.2409.
4.4.19. Dodecyl β- -glucopyranoside (8). Yield 0.24 g, 87%, white powder. [α]D - 26.3 (c
D
0.9, MeOH). Analytical data, [α]D20 - 24.1 (c 1.06, MeOH) [31], 1H and 13C NMR data were
reported [32]. HRMS (ESI) m/z: calcd for C18H36O6Na [M+Na]+: 371.2404; found: 371.2405.
26

4.4.20. Dodecyl 1-thio-α- -glucopyranoside (9). Yield 0.09 g, 81%, yellowish powder.
D
[α]D20 + 171.8 (c 0.9, MeOH), ref [33] [α]D20 + 179 (c 1, MeOH). 1H NMR (400 MHz,
CD3OD): δ 5.33 (d, 1H, J1,2 = 5.4 Hz, H-1), 3.97 (ddd, 1H, J4,5 = 9.9 Hz, J5,6b = 5.3 Hz, H-5),
3.83 (dd, 1H, J5,6a = 2.4 Hz, J6a,6b = 12.0 Hz, H-6a), 3.75-3.68 (m, 2H, H-2, H-6b), 3.55 (t, 1H,
J2,3 = 9.3 Hz, J3,4 = 9.3 Hz, H-3), 3.33 (dd, 1H, H-4), 2.68-2.55 (m, 2H, SCH2(CH2)10CH3),
1.68-1.32 (m, 20H, SCH2(CH2)10CH3), 0.92 (t, 3H, J = 6.8 Hz, S(CH2)11CH3). 13C NMR (100
MHz, CD3OD): δ 87.2 (C-1), 75.7 (C-3), 73.9 (C-5), 73.2 (C-2), 71.8 (C-4), 62.6 (C-6), 33.1
(SCH2(CH2)10CH3), 31.1, 30.8(3x), 30.7(2x), 30.5, 30.4, 30.0, 23.7 (SCH2(CH2)10CH3), 14.4
(S(CH2)11CH3). HRMS (ESI) m/z: calcd for C18H36O5SNa [M+Na]+: 387.2176; found:
387.2179.
4.4.21. Dodecyl 1-thio-β- -glucopyranoside (10). Yield 0.38 g, 79%, yellowish powder.
D
[α]D20 - 100.4 (c 0.8, MeOH). 1H and 13C NMR data were reported [25]. HRMS (ESI) m/z:
calcd for C18H36O5SNa [M+Na]+: 387.2176; found: 387.2173.
4.4.22. Tetradecyl β-
-glucopyranoside (11). Yield 0.28 g, 82%, white powder. [α]D20 - 10.1
D
(c 0.25, MeOH). Analytical data, [α]D25 - 10.8 (c 0.2, MeOH) [26], 1H and 13C NMR data
were reported [27]. HRMS (ESI) m/z: calcd for C20H40O6Na [M+Na]+: 399.2717; found:
399.2710.
4.4.23. Hexadecyl β-
-glucopyranoside (12). Yield 0.31 g, 73%, white powder. [α]D20 - 64.5
D
(c 0.33, MeOH). 1H and 13C NMR data were reported [27]. HRMS (ESI) m/z: calcd for
C22H44O6Na [M+Na]+: 427.3030; found: 427.3035.
27

4.4.24. Decyl β- -galactopyranoside (13). Yield 0.18 g, 70%, white powder. [α]D - 23.2 (c
D
0.45, MeOH). 1H NMR data were reported [8]. 13C NMR (100 MHz, CD3OD): δ 104.9 (C-1),
76.5 (C-5), 75.0 (C-3), 72.5 (C-2), 70.8 (OCH2(CH2)8CH3), 70.3 (C-4), 62.4 (C-6), 33.1, 30.8,
30.7(2x), 30.6, 30.5, 27.1, 23.7 (OCH2(CH2)8CH3), 14.4 (O(CH2)9CH3). HRMS (ESI) m/z:
calcd for C16H32O6Na [M+Na]+: 343.2091; found: 343.2089.
4.4.25. Dodecyl α- -galactopyranoside (14). Yield 0.17 g, 85%, white powder. [α]D + 104.2
D
(c 0.35, MeOH). 1H NMR (400 MHz, CD3OD): δ 4.78 (d, 1H, J1,2 = 3.3 Hz, H-1), 3.88 (dd,
1H, J3,4 = 2.8 Hz, J4,5 = 1.2 Hz, H-4), 3.80-3.66 (m, 6H, H-2, H-3, H-5, H-6a, H-6b,
OCH2(CH2)10CH3), 3.41 (dt, 1H, J = 6.5 Hz, J = 9.6 Hz, OCH2(CH2)10CH3), 1.67-1.27 (m,
20H, OCH2(CH2)10CH3), 0.88 (t, 3H, J = 6.8 Hz, O(CH2)11CH3). 13C NMR (100 MHz,
CD3OD): δ 100.2 (C-1), 72.2 (C-5), 71.5 (C-3), 71.0 (C-4), 70.2 (C-2), 69.2
(OCH2(CH2)10CH3), 62.6 (C-6), 33.0, 30.8(2x), 30.7(2x), 30.6(2x), 30.5, 27.3, 23.7
(OCH2(CH2)10CH3), 14.5 (O(CH2)11CH3). HRMS (ESI) m/z: calcd for C18H37O6 [M+H]+:
349.2585; found: 349.2584; calcd for C18H36O6Na [M+Na]+: 371.2404; found: 371.2404.
4.4.26. Dodecyl β- -galactopyranoside (15). Yield 0.23 g, 87%, white powder. [α]D - 13.2 (c
D
0.26, MeOH). Analytical data, [α]D25 - 15.8 (c 0.54, MeOH) [34], 1H and 13C NMR data were
reported [35]. HRMS (ESI) m/z: calcd for C18H36O6Na [M+Na]+: 371.2404; found: 371.2407.
4.4.27. Dodecyl 1-thio-α- -galactopyranoside (16). Yield 98 mg, 82%, yellowish powder.
D
[α]D + 217.5 (c 0.23, MeOH). 1H NMR (400 MHz, CD3OD): δ 5.37 (d, 1H, J1,2 = 5.6 Hz, H-
1), 4.18 (m, 1H, H-5), 4.07 (dd, 1H, J2,3 = 10.1 Hz, H-2), 3.90 (dd, 1H, J4,5 = 1.3 Hz, H-4),
3.72-3.70 (m, 2H, H-6a, H-6b), 3.61 (dd, 1H, J3,4 = 3.4 Hz, H-3), 2.65-2.51 (m, 2H,
SCH2(CH2)10CH3), 1.67-1.26 (m, 20H, SCH2(CH2)10CH3), 0.89 (t, 3H, J = 6.7 Hz,
28